WO2016167229A1 - Cross-linked product of carboxymethyl group-containing modified hyaluronic acid and/or salt of same, and method for producing same - Google Patents

Cross-linked product of carboxymethyl group-containing modified hyaluronic acid and/or salt of same, and method for producing same Download PDF

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Publication number
WO2016167229A1
WO2016167229A1 PCT/JP2016/061733 JP2016061733W WO2016167229A1 WO 2016167229 A1 WO2016167229 A1 WO 2016167229A1 JP 2016061733 W JP2016061733 W JP 2016061733W WO 2016167229 A1 WO2016167229 A1 WO 2016167229A1
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Prior art keywords
hyaluronic acid
salt
modified hyaluronic
carboxymethyl group
containing modified
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PCT/JP2016/061733
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French (fr)
Japanese (ja)
Inventor
諒太 中前
俊一 藤川
秀人 吉田
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キユーピー 株式会社
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Priority to JP2016535197A priority Critical patent/JP6046869B1/en
Publication of WO2016167229A1 publication Critical patent/WO2016167229A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses

Definitions

  • 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.
  • 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.
  • hyaluronic acid has been actively applied to medical materials and beauty materials by utilizing the high biocompatibility, gel swellability, and viscoelasticity.
  • hyaluronic acid is easily decomposed by hyaluronidase in vivo and viscoelasticity is reduced by heat sterilization treatment, hyaluronic acid having higher resistance to enzymatic degradation and higher thermal stability is required.
  • Patent Document 2 Japanese Patent No. 4958368 discloses a method for producing crosslinked hyaluronic acid by adding an acidic solution to a hyaluronic acid aqueous solution and freezing it without using a crosslinking agent. It has been reported. However, the above-mentioned method is not satisfactory in terms of enzyme degradation resistance and thermal stability, and further has low water swellability required for the crosslinked hyaluronic acid gel, so that it can be put into practical use as a medical material or beauty material. There were many issues.
  • an object of the present invention is to provide a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in thermal stability and resistance to enzymatic degradation, and a method for producing the same.
  • the present inventors use a condensing agent to crosslink the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof with an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof.
  • a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in thermal stability and enzyme degradation resistance was obtained.
  • the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has excellent water swellability.
  • the present invention (1) Using a condensing agent, Carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is crosslinked by an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof, A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, (2) In the method for producing a crosslinked product of (1), The condensing agent is at least one selected from a carbodiimide condensing agent or a triazine condensing agent.
  • a method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof (3)
  • the ratio of the condensing agent is 5 parts by mass or more and 400 parts by mass or less with respect to 1,000 parts by mass of the carboxymethyl group-containing modified hyaluronic acid and / or its salt.
  • a method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof (4)
  • a condensing agent is added to a solution of 1% by mass to 80% by mass of the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
  • the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has an average molecular weight of 4,000 to 4,000,000.
  • the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is The carboxymethylation rate for the disaccharide units constituting hyaluronic acid is 10% or more and 200% or less
  • the method for producing the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof Reacting the dissolved raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof in a hydrous solvent having a temperature of 30 ° C.
  • the water-containing solvent is water or a mixed solution of water-soluble organic solvent and water, The ratio of the water-soluble organic solvent in the mixed solution is 60 v / v% or less,
  • the crosslinked product has water swellability, and the degree of swelling is 5 to 250 times (mass ratio).
  • a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in heat stability and enzymatic degradation resistance can be provided, a highly safe hyaluronic acid without using a crosslinking agent can be provided.
  • a cross-linked product it can be used as a component of medical materials, cosmetic materials, and cosmetics.
  • the crosslinked product has excellent water swellability and can form a soft water-swellable gel, it can be suitably used as a medical material / beauty material.
  • parts means “parts by mass” and “%” means “mass%” unless otherwise specified.
  • the method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof uses a condensing agent, Carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is crosslinked by an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof, It is characterized by a method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  • the crosslinked product obtained by the production method of the present invention has excellent thermal stability and resistance to enzymatic degradation. Furthermore, since the crosslinked product has water swellability, a soft water-swelled gel can be formed.
  • 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 more containing at least one disaccharide unit in which the 1-position of ⁇ -D-glucuronic acid and the 3-position of ⁇ -DN-acetyl-glucosamine are bonded, and ⁇ It is basically composed of -D-glucuronic acid and ⁇ -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.
  • 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.
  • organic group refers to a group having a carbon atom.
  • Carboxymethyl group refers to the group represented by "- - CH 2 -CO 2 H” or "-CH 2 -CO 2".
  • the raw material modified hyaluronic acid and / or salt thereof used in the production method of the present invention can be produced by the method described later.
  • “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.
  • a hydroxyl group constituting hyaluronic acid (see the following formula (1)) (in the following formula (1), C-4 position, C-6 position of N-acetylglucosamine constituting hyaluronic acid, And the hydrogen atom of at least a part of the hydroxyl groups of glucuronic acid constituting hyaluronic acid is represented by —CH 2 —CO 2 H and / or —CH 2 —CO 2 — . Group).
  • n a number of 1 to 7,500.
  • the carboxymethyl group-containing modified hyaluronic acid can be, for example, a compound represented by the following formula (2).
  • the salt of carboxymethyl group-containing modified hyaluronic acid 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, An ammonium salt etc. are mentioned.
  • R 1 to R 5 independently represent a hydrogen atom, —CH 2 —CO 2 H, —CH 2 —CO 2 — , or a group having a carbon atom, and n represents 1 or more and 7,500 or less. (However, the case where R 1 to R 5 of the carboxymethyl group-containing modified hyaluronic acid and / or the salt thereof all represent hydrogen atoms is excluded.)
  • an ester bond is formed between the hydroxyl group and carboxyl group of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof by reacting with the condensing agent.
  • the same or different carboxymethyl group-containing modified hyaluronic acid and / or salts thereof are cross-linked by an ester bond, thereby improving the thermal stability and the enzymatic degradation resistance.
  • an ester bond is formed between the hydroxyl group and the carboxyl group in the carboxymethyl group, it is presumed that the thermal stability and the enzyme degradation resistance are further improved.
  • a carboxyl group derived from unmodified hyaluronic acid and a hydroxyl group may form an ester bond.
  • a crosslinked product of hyaluronic acid and / or a salt thereof has a three-dimensional network structure formed by bonding a hydroxyl group and a carboxyl group of the crosslinked product via an ester bond. It is presumed that a water-swellable gel can be formed by incorporating water into.
  • the amount of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is larger in one constituent unit of the hyaluronic acid skeleton than the unmodified hyaluronic acid and / or salt thereof, because of having a carboxymethyl group. Having a carboxyl group. That is, since the number of carboxyl groups that can participate in ester bonds in the one structural unit is greater than that of unmodified hyaluronic acid and / or salts thereof, more ester bonds are formed when reacted with the condensing agent. Therefore, it is presumed that a soft water-swellable gel excellent in water-swellability can be formed.
  • the condensing agent used in the production method of the present invention is to form an ester bond, and in particular, to form an ester bond between a hydroxyl group and a carboxyl group of a carboxymethyl group-containing modified hyaluronic acid and / or salt thereof. It is. Specific examples include carbodiimide condensing agents, triazine condensing agents, imidazole dehydrating condensing agents, phosphonium condensing agents, and uronium condensing agents.
  • carbodiimide condensing agent examples include 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDC), N, N′-dicyclohexylcarbodiimide (DCC), and N, N′-diisopropylcarbodiimide (DIC). Or N-cyclohexyl-N ′-(2-morpholinoethyl) carbodiimide meth-p-toluenesulfohydrochloride (CME-carbodiimide).
  • EDC 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide
  • DCC N′-dicyclohexylcarbodiimide
  • DIC N, N′-diisopropylcarbodiimide
  • CME-carbodiimide N-cyclohexyl-N ′-(2-morpholinoethyl) carbodiimide me
  • 1-hydroxybenzotriazole As an additive for the condensation reaction used in combination with the carbodiimide condensing agent, 1-hydroxybenzotriazole (HOBt), 1-hydroxyazabenzotriazole (HOAt), N-hydroxysuccinimide (NHS) or the like should be used. Can do.
  • Examples of the triazine-based condensing agent include 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium-chloride n hydrate (DMT-MM), or Examples thereof include trifluoromethanesulfonic acid (4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl) dimethylammonium (interface integrated DMT).
  • Examples of the imidazole dehydrating condensing agent include N, N′-carbodiimidazole (CDI).
  • Examples of the phosphonium-based dehydrating condensing agent include 1H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphorus Acid salt, or chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP).
  • uronium condensing agents include ⁇ [(1-cyano-2-ethoxy-2-oxoethylidene) amino] oxy ⁇ -4-morpholinomethylene ⁇ dimethylammonium hexafluorophosphate (COMU), O- (7- Azabenzotriazol-1-yl) -N, N, N ′, N ′, -tetramethyluronium hexafluorophosphate (HBTU), O- (7-azabenzotriazol-1-yl) -N, N , N ', N',-Tetramethyluronium hexafluorophosphate (HATU), O- (N-succinimidyl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TSTU) Or O- (3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl) -N, N, N ′
  • a carbodiimide condensing agent or a triazine condensing agent may be used.
  • EDC is used as a carbodiimide-based condensing agent
  • DMT-MM is used as a triazine-based condensing agent because it easily forms an ester bond between a hydroxyl group and a carboxyl group of a modified carboxymethyl group-containing modified hyaluronic acid and / or salt thereof. It is good to use.
  • the hardness of the water-swellable gel obtained using the crosslinked product of the present invention is adjusted by adjusting the ratio of the condensing agent to the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof.
  • a condensing agent for 1,000 parts by mass of carboxymethyl group-containing modified hyaluronic acid and / or its salt is more easily formed by more easily forming an ester bond to easily obtain a crosslinked product having excellent thermal stability and enzymatic degradation resistance.
  • the ratio is preferably 5 parts by mass or more and 400 parts by mass or less, more preferably 30 parts by mass or more and 300 parts by mass or less, and 60 parts by mass or more and 200 parts by mass or less.
  • the temperature of the reaction solution may be 1 ° C. or higher and 100 ° C. or lower, and may be 1 ° C. or higher and 50 ° C. or lower, 1 ° C. or higher and 30 ° C. or lower.
  • the reaction time may be 30 minutes or more and 168 hours or less, and may be 1 hour or more and 72 hours or less, and 1 hour or more and 48 hours or less.
  • the solvent for dissolving the raw material-modified hyaluronic acid and / or salt thereof can be water or an admixture with a water-soluble organic solvent miscible with water.
  • water-soluble organic solvent miscible with water examples 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 alone or in combination. In particular, water or ethanol is preferably used as the solvent since the purification step of the crosslinked product can be simplified.
  • one of the raw material-modified hyaluronic acid and / or a salt thereof can be formed by more reliably forming an ester bond and improving the thermal stability and enzymatic degradation resistance of the resulting crosslinked product.
  • a condensing agent may be added to a solution of not less than 80% by mass and not more than 80% by mass, and a condensing agent may be further added to a solution of not less than 5% by mass and not more than 70% by mass and not less than 5% by mass.
  • the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof is preferably 4,000 to 4,000,000.
  • the lower limit is preferably 200,000 or more, more preferably 300,000 or more and 800,000 or more.
  • the upper limit is preferably 3 million or less, and is preferably 2.8 million or less and 2.5 million or less.
  • the crosslinked product of the present invention is used for a subcutaneous injection for cosmetic surgery, a gel with a high degree of swelling is easily obtained.
  • the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof is 500,000 or more and 200 It is good if it is 10,000 or less, and it is good if it is 800,000 or more and 1.8 million or less.
  • the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof can be measured by the following method.
  • a gel filtration column 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), and 1200 Series (Agil).
  • Examples of the column that can be used for liquid chromatography analysis include 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", “SUGARKS-805", “SUGAR KS-806”, “SUGARKS-807”, TOSOH size exclusion chromatography column, type The name “TSKgel GMPW” is mentioned.
  • the kinematic viscosity of the raw material-modified hyaluronic acid and / or salt thereof is 1 mm 2 / s to 200 mm 2 / s. Furthermore, it can be set to 30 mm 2 / s or more and 150 mm 2 / s or less.
  • the kinematic viscosity of the raw material modified hyaluronic acid and / or salt thereof can be measured using an Ubbelohde viscometer (manufactured by Shibata Kagaku Kikai Kogyo Co., Ltd.). At this time, a Ubbelohde viscometer having a coefficient such that the number of seconds of flow is 200 seconds or more and 1,000 seconds is selected. The measurement is performed in a constant temperature water bath at 30 ° C. so that there is no temperature change.
  • the kinematic viscosity (unit: mm 2 / s) can be obtained from the product of the number of seconds of flow of the aqueous solution measured by the Ubbelohde viscometer and the coefficient of the Ubbelohde viscometer.
  • carboxymethylation rate (hereinafter also simply referred to as “carboxymethylation rate (CM conversion rate)”) of the disaccharide unit constituting the hyaluronic acid of the modified hyaluronic acid and / or salt thereof having a carboxyl group.
  • carboxymethylation rate (hereinafter also simply referred to as “carboxymethylation rate (CM conversion rate)”) of the disaccharide unit constituting the hyaluronic acid of the modified hyaluronic acid and / or salt thereof having a carboxyl group.
  • carboxymethyl with respect 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.
  • 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.
  • the raw material-modified hyaluronic acid and / or its salt carboxymethyl can be improved in that the thermal stability and enzymatic degradation resistance of the cross-linked product obtained by more reliably forming an ester bond can be improved.
  • the conversion rate is preferably 10% or more and 200% or less.
  • the lower limit is preferably 20% or more, 30% or more, or 50% or more.
  • the upper limit is preferably 150% or less, 100% or less, or 90% or less.
  • the raw material-modified hyaluronic acid and / or salt thereof is reacted with haloacetic acid and / or a salt thereof in a hydrous solvent having a temperature of 30 ° C. or less.
  • the water-containing solvent is water or a mixed solution of water-soluble organic solvent and water, It can be obtained when the ratio of the water-soluble organic solvent in the mixed solution is 60 v / v% or less.
  • the reaction step at least a part of the raw material hyaluronic acid and / or its salt (which may be all or most of the hyaluronic acid and / or its salt), haloacetic acid and / or in the reaction solution (hydrous solvent)
  • the hyaluronic acid and / or salt thereof and the haloacetic acid and / or salt thereof can be reacted with the salt dissolved.
  • the average molecular weight of the raw material hyaluronic acid and / or salt thereof can be 4,000 to 4,000,000 in that carboxymethylation can be carried out smoothly.
  • the lower limit is preferably 200,000 or more, more preferably 300,000 or more and 800,000 or more.
  • the upper limit is preferably 3 million or less, and is preferably 2.8 million or less and 2.5 million or less.
  • the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof can be measured by an intrinsic viscosity method.
  • the concentration of the hyaluronic acid in the reaction solution can be 0.05 g / mL or more and 0.5 g / mL or less.
  • haloacetic acid and / or its salt is used to introduce a carboxymethyl group into raw material hyaluronic acid and / or its salt.
  • the haloacetic acid may be, for example, monohaloacetic acid and / or a salt thereof, more specifically, chloroacetic acid and / or a salt thereof, or bromoacetic acid or a salt thereof.
  • the salt of haloacetic acid may be, for example, an alkali metal salt of chloroacetic acid and / or an alkali metal salt of bromoacetic acid, and may be sodium chloroacetate and / or sodium bromoacetate.
  • the amount of the haloacetic acid and / or salt thereof used can be 10% by mass or more and 500% by mass or less, and further 50% by mass or more and 200% by mass or less based on the amount of the raw material hyaluronic acid and / or salt thereof can do.
  • the water-containing solvent is water or a mixed solution of water-soluble organic solvent and water because the raw material hyaluronic acid and / or salt thereof has high solubility. Good.
  • the solubility of hyaluronic acid can be increased, and the proportion of the water-soluble organic solvent in the mixed solution is 60 v / v% or less. Furthermore, it is good in it being 20 v / v% or more and 40 v / v% or less.
  • water-soluble organic solvent examples 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 monoalcohols having 1, 2 or 3 carbon atoms such as isopropanol and ethanol may be used.
  • the reaction of the raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof is basic in that the nucleophilicity of the hydroxyl group can be increased. It may be carried out under conditions, and the pH of the reaction solution may be 9 or more and 14 or less, and may be 10 or more and 14 or less, or 11 or more and 14 or less.
  • a basic electrolyte in order to adjust the reaction solution of the above reaction to basic, a basic electrolyte can be used in the reaction solution.
  • 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 preferably 0.2 mol / L or more and 10 mol / L or less in that the raw material-modified hyaluronic acid and / or salt thereof can be obtained efficiently. It is good that they are 5 mol / L or more and 8 mol / L or less.
  • the temperature of the reaction solution is 30 ° C. because carboxylation can proceed smoothly and low molecular weight of raw material-modified hyaluronic acid and / or salt thereof can be suppressed. It is good that it is below, and it is good that it is more than 0 degreeC and below 10 degreeC. In particular, when the temperature of the reaction solution is higher than 0 ° C. and lower than or equal to 10 ° C., high-molecular raw material-modified hyaluronic acid and / or a salt thereof having an average molecular weight of 800,000 or more can be easily obtained.
  • reaction time is 30 minutes or more from the viewpoint that carboxylation can proceed smoothly and the molecular weight reduction of raw material-modified hyaluronic acid and / or salt thereof can be suppressed. It is preferable that the time be less than or equal to 60 hours or less and 60 hours or less.
  • the cross-linked product of the present invention is cross-linked by an ester bond formed between a hydroxyl group and a carboxyl group of the same or different carboxymethyl group-containing modified hyaluronic acid and / or salts thereof.
  • the cross-linked product is superior in thermal stability and resistance to enzymatic degradation described later than a cross-linked product of unmodified hyaluronic acid and / or a salt thereof.
  • the crosslinked product of the present invention has excellent heat stability.
  • the gel remaining rate in the thermal stability test described later is 10% or more, and further preferably 15% or more and 100% or less, or 20% or more and 80% or less. If the gel remaining rate in the thermal stability test is less than 10%, the gel is thermally decomposed by the heat sterilization treatment and cannot be used as a medical material or a cosmetic material.
  • the thermal stability test is performed by the following method.
  • A In a 50 mL vial, 1 g of the crosslinked product obtained in the present invention (in terms of dry mass) is dispersed in 50 mL of phosphate buffered saline (pH 7.4), and the concentration of the crosslinked product is 2% by mass.
  • An aqueous solution of a crosslinked product is prepared.
  • B The cross-linked product aqueous solution is autoclaved at 121 ° C. for 20 minutes. More specifically, the temperature is raised to 121 ° C. over 40 minutes at a starting temperature of 25 ° C., autoclaved at 121 ° C.
  • the cross-linked product of the present invention contains a carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, compared to unmodified hyaluronic acid and / or a salt thereof, it contains more carboxyl groups that can participate in an ester bond. Excellent thermal stability.
  • the cross-linked product of the present invention has excellent enzyme resistance (hyaluronidase) degradability. More specifically, the gel remaining rate in the enzyme degradation resistance test described later is 60% or more, and more preferably 60% or more and 95%, or 70% or more and 90% or less. If the gel residual ratio in the enzyme degradation resistance is less than 60%, the gel is easily decomposed by hyaluronidase and the retention is lowered.
  • the enzyme degradation resistance test is carried out by the following method.
  • A 100 mg (in terms of dry mass) of the crosslinked product of the present invention is dispersed in 9.5 mL of 50 mM phosphate buffer to prepare a crosslinked product aqueous solution having a concentration of the crosslinked product of 1% by mass (solid content), Let stand at 25 ° C. for 10 minutes to swell and dissolve.
  • B The aqueous solution of the cross-linked product is stored at 40 ° C. for 15 hours in the presence of hyaluronidase (0.25 unit / per mg of cross-linked product).
  • the cross-linked product of the present invention is excellent in enzyme degradation resistance is that the cross-linked product contains a carboxymethyl group. Specifically, since the cross-linked product contains a carboxymethyl group, hyaluronidase is difficult to recognize the hyaluronic acid skeleton contained in the cross-linked product.
  • water swelling> since the crosslinked product of the present invention has excellent water swellability, a soft water-swellable gel can be formed.
  • water swellability refers to the property of taking water to swell and generally refers to the property of taking water to become a gel.
  • the cross-linked product of the present invention has a three-dimensional network structure formed by ester bonds generated by reaction with a condensing agent, and swells by incorporating water into the three-dimensional network structure to form a gel. be able to.
  • the crosslinked product of the present invention has a degree of swelling with respect to water of 5 to 250 times (mass ratio), more preferably 5 to 200 times in that a softer water-swellable gel can be formed.
  • the “swelling degree” means the mass of the swollen gel relative to the mass of the dry gel (swelled gel / dry gel). If the degree of swelling is less than 5 times, the gel becomes too hard to be used as a medical material / beauty material. On the other hand, if the degree of swelling is more than 250 times, the gel becomes brittle even if it is too soft.
  • the degree of swelling before the thermal stability test is preferably 5 to 50 times (mass ratio), and more preferably 5 to 20 times.
  • the degree of swelling after the thermal stability test is preferably 10 to 250 times (mass ratio), more preferably 15 to 200 times, and 20 to 150 times.
  • the storage elastic modulus G ′ (frequency 1 Hz) is preferably 100 Pa to 100,000 Pa, and more preferably 150 Pa to 50,000 Pa. It may be the following. In the present invention, the storage elastic modulus G ′ can be measured by the method shown in Examples described later.
  • the knee joint injection, the anti-adhesion agent, the subcutaneous injection, or the drug sustained-release agent of the present invention contains the cross-linked product (or water-swellable gel), thereby being excellent in thermal stability and enzyme degradation resistance. Furthermore, since it can be stored as a solid, it has excellent storage stability.
  • the crosslinked product of the present invention When the crosslinked product of the present invention is used as an joint injection / adhesion inhibitor, the crosslinked product has excellent thermal stability and resistance to enzymatic degradation, so that it is not degraded in a living body for a certain period of time. Since it remains and then decomposes in vivo, it is possible to prevent adhesion between tissues, and it is excellent in safety because it does not use a crosslinking agent. Particularly in joint injections, the cross-linked product has excellent water swellability, so that pain after injection can be reduced. Moreover, in the anti-adhesion agent, since the said crosslinked material has the outstanding workability, it can be used with a wide form as a sheet form or a film form.
  • ⁇ Subcutaneous injection> When the cross-linked product of the present invention is used as a subcutaneous injection, not only medical materials but also cosmetically, for example, by injection into the face, head, neck, chest, abdomen, buttocks, back, waist, upper limb, and lower limb. It can be used to produce effects (for example, breast enhancement, beautiful face, beautiful legs, etc., to improve the appearance). Since the water-swellable gel has excellent heat stability, enzyme degradation resistance, and water-swellability, it can maintain a cosmetic effect.
  • the cross-linked product of the present invention When the cross-linked product of the present invention is used as a drug sustained-release agent, the cross-linked product has excellent thermal stability and resistance to enzymatic degradation, so that it remains without being decomposed for a certain period of time in vivo. Thereafter, since it is decomposed in vivo, it has an action of assisting the sustained release of the drug and is excellent in safety.
  • the cosmetic of the present invention contains the cross-linked product, and the cross-linked product has a high water retention effect due to the carboxyl group that constitutes it. More specifically, since the carboxyl group contained in the cross-linked product of the present invention constitutes a hydrogen bond with water, it is presumed that excellent water retention is exhibited due to the carboxyl group. For this reason, it has a high water retention effect in biological tissues such as skin.
  • the cosmetic composition of the present invention contains the water-swellable gel, it has an appropriate viscoelasticity as a gel, so that it can feel a fresh feel unique to the gel, and an active ingredient is blended in the gel. Thus, the active ingredient can be released gradually.
  • the aspect of the cosmetic of the present invention is not particularly limited, and examples thereof include skin cosmetics.
  • skin cosmetics Specifically, for example, facial cleanser, cleansing agent, lotion, cream, milky lotion, serum, pack, cleansing, foundation, lipstick, lip balm, lip gloss, lip liner, blusher, shaving lotion, after sun lotion, deodorant Lotion, body lotion, body oil, soap, bath salt.
  • Example Next, this invention is further demonstrated based on an Example and a comparative example. Note that the present invention is not limited to this.
  • ⁇ Preparation Example 1 Preparation of carboxymethyl group-containing modified hyaluronic acid> After weighing 1.04 g of sodium hydroxide into a 30 mL sample bottle, 12 mL of water was added and dissolved. Next, 2.0 g of hyaluronic acid having an average molecular weight of 1.75 million 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.
  • the precipitate was collected with a 400-mesh filter cloth, and 40 mL of 10% aqueous sodium chloride 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 modified hyaluronic acid containing carboxymethyl group of Preparation Example 1 (raw material) A composition containing (modified hyaluronic acid) was obtained.
  • the carboxymethyl group-containing modified hyaluronic acid obtained in Preparation Example 1 had a molecular weight of 1.70 million and a carboxymethylation rate of 77%.
  • the carboxymethylation rate of the carboxymethyl group-containing modified hyaluronic acid obtained in Preparation Example 1 was determined from the integrated value of 1 H-NMR spectrum by the following method.
  • sample preparation 7 mg of a 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.
  • DSS sodium 4,4-dimethyl-4-silapentanesulfonate
  • Example 1 Preparation of crosslinked product of carboxymethyl group-containing modified hyaluronic acid> 1.
  • HOBt aqueous solution 1-hydroxybenzotriazole aqueous solution
  • 54 mL and 1.21 mL of ion exchange water were added.
  • the concentration of the raw material-modified hyaluronic acid in the reaction solution was 40% by mass.
  • This reaction solution was kneaded by hand and stored at 25 ° C. for 2 hours to swell and dissolve.
  • Example 1 Purification of crosslinked product of carboxymethyl group-containing modified hyaluronic acid> The composition containing the crosslinked product obtained in Example 1 was transferred to a 500 mL beaker, and 250 mL of ion-exchanged water was added. After grinding with Hiscotron (5000 rpm ⁇ 2 minutes), the mixture was stirred for 5 minutes and allowed to stand for 5 minutes to precipitate the cross-linked product and the supernatant was removed by decantation. Subsequently, it was washed twice with 200 mL of ion-exchanged water, once with 200 mL of 60% ethanol, and once with 100 mL of 90% ethanol.
  • the cross-linked product was collected by suction filtration with a 420 mesh nylon strainer and then dried under reduced pressure at 55 ° C. for 4 hours to obtain 1.54 g of a powdery carboxymethyl group-containing modified hyaluronic acid cross-linked product.
  • Examples 2 to 10 and Comparative Examples 1 to 6 Except having changed into the conditions described in Table 1, powdered carboxymethyl group-containing modified hyaluronic acid of Examples 2 to 10 and Comparative Examples 1 to 6 were obtained in the same manner as in Example 1.
  • ⁇ Test Example 1 Thermal stability test> In a 50 mL vial, 1 g of each crosslinked product of carboxymethyl group-containing modified hyaluronic acid obtained in Examples 1 to 7 and Comparative Examples 1 to 10 (in terms of dry mass) was added to phosphate buffered saline (pH 7. 4) The mixture was dispersed in 50 mL to prepare a mixture having a crosslinked product concentration of 2% by mass (solid content). The vial mouth was then fitted with a rubber stopper, covered with an aluminum lid, and the mixture was autoclaved at 121 ° C. for 20 minutes.
  • the treated crosslinked product was placed on a 420 mesh nylon strainer, washed with distilled water, recovered by suction filtration, spread on a weighed petri dish, and dried under reduced pressure at 55 ° C. for 3 hours. 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 cross-linked product and the mass of the residue, the gel remaining rate in the thermal stability test was calculated by the above formula (3).
  • the autoclave treatment specifically, the temperature is raised to 121 ° C. over 40 minutes at a starting temperature of 25 ° C., autoclaved at 121 ° C. for 20 minutes, and allowed to stand for 1 hour after completion of pressurization and heating. The step of taking out the cross-linked product.
  • ⁇ Test Example 2 Enzymatic degradation resistance test> 100 mg (in terms of dry mass) of each of the crosslinked products obtained in Test Example 1 described above was dispersed in 9.5 mL of 50 mM phosphate buffer (pH 6.0) to prepare a mixture having a concentration of 1% by mass (solid content). And allowed to stand at 25 ° C. for 10 minutes to swell and dissolve. Subsequently, after adding 0.25 units (0.5 mL) of hyaluronidase (from Sigma, bovine tests) to the mixture, the mixture was allowed to stand at 40 ° C. for 15 hours. Further, 0.25 unit (0.5 mL) of hyaluronidase was added, and the mixture was allowed to stand at 40 ° C.
  • the crosslinked product was recovered by suction filtration with a 420 mesh nylon strainer, washed with distilled water, spread on a weighed petri dish, and dried under reduced pressure at 55 ° C. for 3 hours. 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 gel remaining rate in the enzyme degradation resistance test was calculated by the above formula (4). Since the crosslinked products obtained in Comparative Examples 1 to 7 and Comparative Example 10 had a gel residual ratio of 0% in the thermal stability test of Test Example 1, the enzyme degradation resistance test of Test Example 2 was not going.
  • Measuring device AR-G2 (manufactured by TA Instruments Japan Co., Ltd.) Gap: 800 ⁇ m Measurement mode: frequency sweep step Measurement temperature: 25 ° C Amplitude frequency: 0.1 to 10 Hz
  • a carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is cross-linked by an ester bond between a hydroxyl group and a carboxyl group of the modified hyaluronic acid and / or salt thereof using a condensing agent. It can be understood that the cross-linked product obtained by the method for producing a cross-linked product of methyl group-containing modified hyaluronic acid and / or a salt thereof is excellent in thermal stability and resistance to enzymatic degradation (Examples 1 to 10).
  • the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has a gel residual ratio of 10% or more in the thermal stability test of Test Example 1 and has excellent thermal stability. Can be understood (Examples 1 to 10).
  • the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof has a gel residual ratio of 60% or more in the enzyme degradation resistance test of Test Example 2 and has excellent enzyme degradation resistance. (Examples 1 and 2, Examples 4-7, and Examples 9 and 10).
  • a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof has a property of forming a water-swellable gel and has a swelling degree of 5 to 250 times (mass ratio). (Examples 1 to 10).
  • Example 11 Anti-adhesion agent> The cross-linked product obtained in Example 3 was rolled into a 1 mm thick film and molded. After sterilization, a sheet-shaped adhesion inhibitor was obtained.
  • Example 12 Subcutaneous injection> The dried product of the crosslinked product obtained in Example 7 (1% in terms of solid content) was swollen with water for injection containing 0.9% NaCl, and aseptically filled into a 1 mL syringe. After sterilization, a subcutaneous injection was used. Obtained.
  • Example 13 Drug sustained-release agent> The dried product of the cross-linked product obtained in Example 5 (2% in terms of solid) was swollen with water for injection containing 0.9% NaCl and 0.001% prostaglandin E1, and after sterilization, a 3 mL syringe. Aseptic filling was carried out to obtain a sustained-release drug.
  • Example 14 knee joint injection> The dried product of the cross-linked product obtained in Example 6 (0.8% in terms of solids) was swollen with water for injection containing 0.9% NaCl, sterilized, and then aseptically filled into a 2 mL syringe. An injection was obtained.

Abstract

Provided is a method for producing a cross-linked product of carboxymethyl group-containing modified hyaluronic acid and/or a salt of the same, having excellent thermal stability, enzymolysis resistance and water-swelling characteristics. The method for producing a cross-linked product of carboxymethyl group-containing modified hyaluronic acid and/or a salt of the same uses a condensing agent to crosslink a carboxymethyl group-containing modified hyaluronic acid and/or a salt of the same by ester bonding between hydroxyl groups and carboxyl groups on the modified hyaluronic acid and/or the salt of the same.

Description

[規則26に基づく補充 16.05.2016]　カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物およびその製造方法[Supplement based on Rule 26 16.05.2016] Cross-linked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof and method for producing the same
　本発明は、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物およびその製造方法に関する。 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.
　ヒアルロン酸は、鶏冠、さい帯、皮膚、軟骨、硝子体、関節液などの生体組織中に広く分布しており、例えば、化粧料、医薬品、食品の成分として広く利用されている。
　特に、ヒアルロン酸の高い生体適合性、ゲル膨潤性、および粘弾性を利用して、医療材料や美容材料への応用が盛んに行われている。しかしながら、ヒアルロン酸は生体内でヒアルロニダーゼによって分解されやすく、さらに加熱滅菌処理により粘弾性が低下してしまうことから、より耐酵素分解性および熱安定性が高いヒアルロン酸が求められている。 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.
Particularly, hyaluronic acid has been actively applied to medical materials and beauty materials by utilizing the high biocompatibility, gel swellability, and viscoelasticity. However, since hyaluronic acid is easily decomposed by hyaluronidase in vivo and viscoelasticity is reduced by heat sterilization treatment, hyaluronic acid having higher resistance to enzymatic degradation and higher thermal stability is required.
　そこで、架橋剤を使用してヒアルロン酸を架橋させた架橋ヒアルロン酸ゲルの製造方法が報告されている（特許文献１：特許第４８７５９８８号公報）
　しかしながら、このような方法で架橋された架橋ヒアルロン酸ゲルは生体内でゲルが分解された後、残存する架橋剤成分が生体にとって異物として認識されてしまい、炎症反応を引き起こしてしまう等の悪影響を及ぼすといった、安全性の面で課題があった。 Then, the manufacturing method of the crosslinked hyaluronic acid gel which bridge | crosslinked hyaluronic acid using the crosslinking agent is reported (patent document 1: patent 4875988).
However, the crosslinked hyaluronic acid gel crosslinked by such a method has an adverse effect such that after the gel is decomposed in the living body, the remaining crosslinking agent component is recognized as a foreign substance by the living body and causes an inflammatory reaction. There was a problem in terms of safety.
　このような安全性を鑑みて、特許文献２（特許第４９５８３６８号公報）では、架橋剤を使用せず、ヒアルロン酸水溶液に酸性溶液を加え、凍結保存することで架橋ヒアルロン酸を製造する方法が報告されている。しかしながら、上述の方法では耐酵素分解性や熱安定性において十分満足できるものではなく、さらに、架橋ヒアルロン酸ゲルに必要とされる水膨潤性も低く、医療材料や美容材料として実用化するには課題が多かった。 In view of such safety, Patent Document 2 (Japanese Patent No. 4958368) discloses a method for producing crosslinked hyaluronic acid by adding an acidic solution to a hyaluronic acid aqueous solution and freezing it without using a crosslinking agent. It has been reported. However, the above-mentioned method is not satisfactory in terms of enzyme degradation resistance and thermal stability, and further has low water swellability required for the crosslinked hyaluronic acid gel, so that it can be put into practical use as a medical material or beauty material. There were many issues.
特許第４８７５９８８号公報Japanese Patent No. 4875988 特許第４９５８３６８号公報Japanese Patent No. 4958368
　そこで、本発明の目的は、熱安定性および耐酵素分解性に優れたカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物およびその製造方法を提供するものである。 Accordingly, an object of the present invention is to provide a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in thermal stability and resistance to enzymatic degradation, and a method for producing the same.
　本発明者等は、縮合剤を用いて、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を、前記修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合によって架橋させることで、熱安定性および耐酵素分解性に優れたカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物が得られることを見出した。 The present inventors use a condensing agent to crosslink the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof with an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof. Thus, it was found that a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in thermal stability and enzyme degradation resistance was obtained.
　さらに、前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物は、優れた水膨潤性を有している。 Furthermore, the crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has excellent water swellability.
つまり、本願発明は、
（１）縮合剤を用いて、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を、前記修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合によって架橋させる、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（２）（１）の架橋物の製造方法において、
前記縮合剤が、カルボジイミド系縮合剤、またはトリアジン系縮合剤から選ばれる少なくとも１種である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（３）（１）または（２）の架橋物の製造方法において、
前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩１，０００質量部に対して前記縮合剤の割合が５質量部以上４００質量部以下である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（４）（１）ないし（３）のいずれかの架橋物の製造方法において、
前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の１質量％以上８０質量％以下の溶液に縮合剤を添加する、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法
（５）（１）ないし（４）のいずれかの架橋物の製造方法において、
前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の平均分子量が４，０００以上４００万以下である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（６）（１）ないし（５）のいずれかの架橋物の製造方法において、
前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩が、
ヒアルロン酸を構成する２糖単位に対するカルボキシメチル化率が１０％以上２００％以下である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（７）（１）ないし（６）のいずれかの架橋物の製造方法において、
前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の製造方法は、
温度が３０℃以下の含水溶媒中で、溶解した原料ヒアルロン酸および／またはその塩をハロ酢酸および／またはその塩と反応させる工程を含み、
前記含水溶媒は、水、または水溶性有機溶媒と水との混合液であり、
前記混合液における水溶性有機溶媒の割合が６０ｖ／ｖ％以下である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法、
（８）同一または異なるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩同士をエステル結合により架橋させた架橋物であって、
前記架橋物は水膨潤性を有し、膨潤度が５倍以上２５０倍以下（質量比）である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
（９）（８）の架橋物において、
熱安定性試験におけるゲル残存率が１０質量％以上である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
（１０）（８）の架橋物において、
耐酵素分解性試験におけるゲル残存率が６０％以上である、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
（１１）（１）ないし（７）のいずれかの製造方法により得られるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
または（８）ないし（１０）のいずれかのカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物を含む、
関節注射剤、癒着防止剤、皮下注射剤、または薬物徐放剤、
（１２）（１）ないし（７）のいずれかの製造方法により得られるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
または（８）ないし（１０）のいずれかのカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物を含む、化粧料、
である。 In other words, the present invention
(1) Using a condensing agent,
Carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is crosslinked by an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof,
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(2) In the method for producing a crosslinked product of (1),
The condensing agent is at least one selected from a carbodiimide condensing agent or a triazine condensing agent.
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(3) In the method for producing a crosslinked product of (1) or (2),
The ratio of the condensing agent is 5 parts by mass or more and 400 parts by mass or less with respect to 1,000 parts by mass of the carboxymethyl group-containing modified hyaluronic acid and / or its salt.
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(4) In the method for producing a crosslinked product according to any one of (1) to (3),
A condensing agent is added to a solution of 1% by mass to 80% by mass of the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
In the method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof (5) (1) to (4),
The carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has an average molecular weight of 4,000 to 4,000,000.
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(6) In the method for producing a crosslinked product according to any one of (1) to (5),
The carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is
The carboxymethylation rate for the disaccharide units constituting hyaluronic acid is 10% or more and 200% or less,
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(7) In the method for producing a crosslinked product according to any one of (1) to (6),
The method for producing the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof,
Reacting the dissolved raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof in a hydrous solvent having a temperature of 30 ° C. or lower,
The water-containing solvent is water or a mixed solution of water-soluble organic solvent and water,
The ratio of the water-soluble organic solvent in the mixed solution is 60 v / v% or less,
A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(8) A crosslinked product obtained by crosslinking the same or different carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof with an ester bond,
The crosslinked product has water swellability, and the degree of swelling is 5 to 250 times (mass ratio).
Carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(9) In the crosslinked product of (8),
The gel remaining rate in the thermal stability test is 10% by mass or more,
Carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(10) In the crosslinked product of (8),
The gel remaining rate in the enzyme degradation resistance test is 60% or more,
Carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
(11) A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof obtained by the production method of any one of (1) to (7),
Or a crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of (8) to (10),
Joint injection, anti-adhesion agent, subcutaneous injection, or drug sustained-release agent,
(12) A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof obtained by the production method of any one of (1) to (7),
Or a cosmetic comprising a crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of (8) to (10),
It is.
　上記製造方法によれば、熱安定性および耐酵素分解性に優れたカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物を提供できることから、架橋剤を使用しない安全性の高いヒアルロン酸の架橋物として、医療材料、美容材料、および化粧料の成分として使用することができる。 According to the above production method, since a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof excellent in heat stability and enzymatic degradation resistance can be provided, a highly safe hyaluronic acid without using a crosslinking agent can be provided. As a cross-linked product, it can be used as a component of medical materials, cosmetic materials, and cosmetics.
　さらに、前記架橋物は優れた水膨潤性を有し、やわらかい水膨潤性ゲルを形成することができることから、医療材料・美容材料として好適に用いることができる。 Furthermore, since the crosslinked product has excellent water swellability and can form a soft water-swellable gel, it can be suitably used as a medical material / beauty material.
　次に、本発明を詳細に説明する。なお、本発明において、格別に断らない限り、「部」は「質量部」を意味し、「％」は「質量％」を意味する。 Next, the present invention will be described in detail. In the present invention, “parts” means “parts by mass” and “%” means “mass%” unless otherwise specified.
＜本発明の特徴－カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法＞
　本発明のカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物（以下、単に「架橋物」ともいう）の製造方法は、縮合剤を用いて、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を、前記修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合によって架橋させる、
カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法に特徴を有する。
　本発明の製造方法で得られる架橋物は、優れた熱安定性および耐酵素分解性を有する。さらに、該架橋物は水膨潤性を有することから、やわらかい水膨潤ゲルを形成することができる。 <Characteristics of the present invention-Method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof>
The method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof (hereinafter also simply referred to as “crosslinked product”) of the present invention uses a condensing agent,
Carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is crosslinked by an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof,
It is characterized by a method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
The crosslinked product obtained by the production method of the present invention has excellent thermal stability and resistance to enzymatic degradation. Furthermore, since the crosslinked product has water swellability, a soft water-swelled gel can be formed.
　＜ヒアルロン酸および／またはその塩＞
　本発明において、「ヒアルロン酸」とは、グルクロン酸とＮ－アセチルグルコサミンとの二糖からなる繰り返し構成単位を１以上有する多糖類をいう。また、「ヒアルロン酸の塩」としては、特に限定されないが、食品または薬学上許容しうる塩であることが好ましく、例えば、ナトリウム塩、カリウム塩、カルシウム塩、亜鉛塩、マグネシウム塩、アンモニウム塩等が挙げられる。 <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.
　ヒアルロン酸は、基本的にはβ－Ｄ－グルクロン酸の１位とβ－Ｄ－Ｎ－アセチル－グルコサミンの３位とが結合した２糖単位を少なくとも１個含む２糖以上のものでかつβ－Ｄ－グルクロン酸とβ－Ｄ－Ｎ－アセチル－グルコサミンとから基本的に構成され、２糖単位が複数個結合したものである。該糖は不飽和糖であってもよく、不飽和糖としては、非還元末端糖、通常、グルクロン酸の４，５位炭素間が不飽和のもの等が挙げられる。 Hyaluronic acid is basically a disaccharide or more containing at least one disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-DN-acetyl-glucosamine are bonded, 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.
　＜修飾ヒアルロン酸および／またはその塩＞
　本発明において、「修飾ヒアルロン酸および／またはその塩」とは、少なくとも一部に有機基が導入されているヒアルロン酸および／またはその塩のことをいい、ヒアルロン酸および／またはその塩とは異なる構造を有する。また、本発明において「有機基」とは、炭素原子を有する基のことをいう。 <Modified hyaluronic acid and / or salt thereof>
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.
＜カルボキシメチル基＞
　本発明において、「カルボキシメチル基」とは、「－ＣＨ２－ＣＯ２Ｈ」または「－ＣＨ２－ＣＯ２ －」で表される基のことをいう。また、本発明の製造方法で使用する原料修飾ヒアルロン酸および／またはその塩は、後述する方法にて製造されたものであることができる。　
　したがって、本発明において、「カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩」とは、少なくとも一部にカルボキシメチル基が導入されているヒアルロン酸および／またはその塩のことをいう。 <Carboxymethyl group>
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 of the present invention can be produced by the method described later.
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.
　より具体的には、例えば、ヒアルロン酸（下記式（１）参照）を構成する水酸基（下記式（１）において、ヒアルロン酸を構成するＮ－アセチルグルコサミンのＣ－４位、Ｃ－６位、ならびに、ヒアルロン酸を構成するグルクロン酸のＣ－２位、Ｃ－３位）の少なくとも一部の水酸基の水素原子が、－ＣＨ２－ＣＯ２Ｈおよび／または－ＣＨ２－ＣＯ２ －で表される基)で置換されていることができる。 More specifically, for example, a hydroxyl group constituting hyaluronic acid (see the following formula (1)) (in the following formula (1), C-4 position, C-6 position of N-acetylglucosamine constituting hyaluronic acid, And the hydrogen atom of at least a part of the hydroxyl groups of glucuronic acid constituting hyaluronic acid is represented by —CH 2 —CO 2 H and / or —CH 2 —CO 2 — . Group).
Figure JPOXMLDOC01-appb-C000001
 ・・・（１）
　（式中、ｎは１以上７，５００以下の数を示す。）
　
Figure JPOXMLDOC01-appb-C000001
 ... (1)
(In the formula, n represents a number of 1 to 7,500.)
　カルボキシメチル基含有修飾ヒアルロン酸は例えば、下記式（２）で表される化合物であることができる。
　また、カルボキシメチル基含有修飾ヒアルロン酸の塩としては、特に限定されないが、食品または薬学上許容しうる塩であることが好ましく、例えば、ナトリウム塩、カリウム塩、カルシウム塩、亜鉛塩、マグネシウム塩、アンモニウム塩等が挙げられる。 The carboxymethyl group-containing modified hyaluronic acid can be, for example, a compound represented by the following formula (2).
Further, the salt of carboxymethyl group-containing modified hyaluronic acid 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, An ammonium salt etc. are mentioned.
Figure JPOXMLDOC01-appb-C000002
 ・・・（２）
　（式中、Ｒ１～Ｒ５は独立して、水素原子、－ＣＨ２－ＣＯ２Ｈ、－ＣＨ２－ＣＯ２ －、または炭素原子を有する基を表し、ｎは１以上７，５００以下の数を示す。ただし、当該カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩全体のＲ１～Ｒ５がいずれも水素原子を表す場合を除く。）
　
Figure JPOXMLDOC01-appb-C000002
 ... (2)
(Wherein R 1 to R 5 independently represent a hydrogen atom, —CH 2 —CO 2 H, —CH 2 —CO 2 — , or a group having a carbon atom, and n represents 1 or more and 7,500 or less. (However, the case where R 1 to R 5 of the carboxymethyl group-containing modified hyaluronic acid and / or the salt thereof all represent hydrogen atoms is excluded.)
　＜本発明の製造方法のメカニズム＞
　本発明の製造方法では、前記縮合剤と反応させることで、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合が形成される。
　その結果、同一または異なるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩同士がエステル結合によって架橋されることにより、熱安定性および耐酵素分解性が高まると推察される。
　さらに、前記水酸基とカルボキシメチル基中のカルボキシル基との間でエステル結合が形成された場合、熱安定性および耐酵素分解性がより高まることが推察される。
　なお、前記架橋物において、未修飾ヒアルロン酸に由来するカルボキシル基と水酸基がエステル結合を形成していてもよい。 <Mechanism of the production method of the present invention>
In the production method of the present invention, an ester bond is formed between the hydroxyl group and carboxyl group of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof by reacting with the condensing agent.
As a result, it is assumed that the same or different carboxymethyl group-containing modified hyaluronic acid and / or salts thereof are cross-linked by an ester bond, thereby improving the thermal stability and the enzymatic degradation resistance.
Furthermore, when an ester bond is formed between the hydroxyl group and the carboxyl group in the carboxymethyl group, it is presumed that the thermal stability and the enzyme degradation resistance are further improved.
In the cross-linked product, a carboxyl group derived from unmodified hyaluronic acid and a hydroxyl group may form an ester bond.
　また、ヒアルロン酸および／またはその塩の架橋物は、該架橋物が有する水酸基とカルボキシル基とがエステル結合を介して結合することにより、３次元網目構造が構築され、この３次元網目構造の中に水を取り込むことにより、水膨潤性ゲルを形成することができると推察される。 In addition, a crosslinked product of hyaluronic acid and / or a salt thereof has a three-dimensional network structure formed by bonding a hydroxyl group and a carboxyl group of the crosslinked product via an ester bond. It is presumed that a water-swellable gel can be formed by incorporating water into.
　特に、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩は、カルボキシメチル基を有している分、未修飾ヒアルロン酸および／またはその塩と比較して、ヒアルロン酸骨格の一構成単位中により多くのカルボキシル基を有する。すなわち、該一構成単位中においてエステル結合に関与することができるカルボキシル基が、未修飾ヒアルロン酸および／またはその塩よりも多いため、前記縮合剤と反応させると、より多くのエステル結合を形成することができるため、水膨潤性に優れたやわらかい水膨潤性ゲルを形成することができると推察される。 In particular, the amount of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is larger in one constituent unit of the hyaluronic acid skeleton than the unmodified hyaluronic acid and / or salt thereof, because of having a carboxymethyl group. Having a carboxyl group. That is, since the number of carboxyl groups that can participate in ester bonds in the one structural unit is greater than that of unmodified hyaluronic acid and / or salts thereof, more ester bonds are formed when reacted with the condensing agent. Therefore, it is presumed that a soft water-swellable gel excellent in water-swellability can be formed.
＜縮合剤＞
　本発明の製造方法に用いる縮合剤は、エステル結合を形成させるものであり、特に、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間にエステル結合を形成させるものである。
　具体的には、例えば、カルボジイミド系縮合剤、トリアジン系縮合剤、イミダゾール系脱水縮合剤、ホスホニウム系縮合剤、またはウロニウム系縮合剤等が挙げられる。
　カルボジイミド系縮合剤としては、例えば、１－［３－（ジメチルアミノ）プロピル］―３－エチルカルボジイミド（ＥＤＣ）、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド（ＤＣＣ）、Ｎ，Ｎ’－ジイソプロピルカルボジイミド（ＤＩＣ）、またはＮ－シクロヘキシル－Ｎ’－（２－モルホリノエチル）カルボジイミドメト－ｐ－トルエンスルホ塩酸（ＣＭＥ－カルボジイミド）等が挙げられる。カルボジイミド系縮合剤と併用して使用する縮合反応用添加剤としては、１－ヒドロキシベンゾトリアゾール（ＨＯＢｔ）、１－ヒドロキシアザベンゾトリアゾール（ＨＯＡｔ）、またはＮ－ヒドロキシスクシンイミド（ＮＨＳ）等を使用することができる。
　トリアジン系縮合剤としては、例えば、４－（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－４－メチルモルホリニウム－クロリドｎ水和物（ＤＭＴ－ＭＭ）、またはトリフルオロメタンスルホン酸（４，６－ジメトキシ－１，３，５－トリアジン－２－イル）－（２－オクトキシ－２－オキソエチル）ジメチルアンモニウム（界面集積型ＤＭＴ）等が挙げられる。
　イミダゾール系脱水縮合剤としては、例えば、Ｎ，Ｎ’－カルボジイミダゾール（ＣＤＩ）が挙げられる。
　ホスホニウム系脱水縮合剤としては、例えば、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ジメチルアミノ）ホスホニウムヘキサフルオロリン酸塩（ＢＯＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリピロリジノホスホニウムヘキサフルオロリン酸塩、またはクロロトリピロリジノホスホニウムヘキサフルオロリン酸塩（ＰｙＣｌｏＰ）等が挙げられる。
　ウロニウム系縮合剤としては、｛｛［（１－シアノ－２－エトキシ－２－オキソエチリデン）アミノ］オキシ｝－４－モルホリノメチレン｝ジメチルアンモニウムヘキサフルオロりん酸塩（ＣＯＭＵ）、Ｏ－（７－アザベンゾトリアゾール－１－イル）－Ｎ,Ｎ,Ｎ’,Ｎ’, －テトラメチルウロニウムヘキサフルオロりん酸塩（ＨＢＴＵ）、Ｏ－（７－アザベンゾトリアゾール－１－イル）－Ｎ,Ｎ,Ｎ’,Ｎ’, －テトラメチルウロニウムヘキサフルオロりん酸塩（ＨＡＴＵ）、Ｏ－（Ｎ－スクシンイミジル）－Ｎ,Ｎ,Ｎ’,Ｎ’－テトラメチ ルウロニウムテトラフルオロほう酸塩（ＴＳＴＵ）、またはＯ－（３，４－ジヒドロ－４－オキソ－１,２,３－ベンゾトリアジン－３－イル）－Ｎ,Ｎ,Ｎ’,Ｎ ’－テトラメチルウロニウムテトラフルオロほう酸塩（ＴＤＢＴＵ）等が挙げられる。
　前記縮合剤が水溶性を有することから、カルボジイミド系縮合剤、またはトリアジン系縮合剤を使用するとよい。
　特に、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間のエステル結合を形成させやすいことから、カルボジイミド系縮合剤としてＥＤＣを、トリアジン系縮合剤としてＤＭＴ－ＭＭを使用するとよい。 <Condensation agent>
The condensing agent used in the production method of the present invention is to form an ester bond, and in particular, to form an ester bond between a hydroxyl group and a carboxyl group of a carboxymethyl group-containing modified hyaluronic acid and / or salt thereof. It is.
Specific examples include carbodiimide condensing agents, triazine condensing agents, imidazole dehydrating condensing agents, phosphonium condensing agents, and uronium condensing agents.
Examples of the carbodiimide condensing agent include 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDC), N, N′-dicyclohexylcarbodiimide (DCC), and N, N′-diisopropylcarbodiimide (DIC). Or N-cyclohexyl-N ′-(2-morpholinoethyl) carbodiimide meth-p-toluenesulfohydrochloride (CME-carbodiimide). As an additive for the condensation reaction used in combination with the carbodiimide condensing agent, 1-hydroxybenzotriazole (HOBt), 1-hydroxyazabenzotriazole (HOAt), N-hydroxysuccinimide (NHS) or the like should be used. Can do.
Examples of the triazine-based condensing agent include 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium-chloride n hydrate (DMT-MM), or Examples thereof include trifluoromethanesulfonic acid (4,6-dimethoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl) dimethylammonium (interface integrated DMT).
Examples of the imidazole dehydrating condensing agent include N, N′-carbodiimidazole (CDI).
Examples of the phosphonium-based dehydrating condensing agent include 1H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphorus Acid salt, or chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP).
Examples of uronium condensing agents include {{[(1-cyano-2-ethoxy-2-oxoethylidene) amino] oxy} -4-morpholinomethylene} dimethylammonium hexafluorophosphate (COMU), O- (7- Azabenzotriazol-1-yl) -N, N, N ′, N ′, -tetramethyluronium hexafluorophosphate (HBTU), O- (7-azabenzotriazol-1-yl) -N, N , N ', N',-Tetramethyluronium hexafluorophosphate (HATU), O- (N-succinimidyl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TSTU) Or O- (3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl) -N, N, N ′, N′-tetramethyluronium tetrafluoroborate (TD BTU) and the like.
Since the condensing agent has water solubility, a carbodiimide condensing agent or a triazine condensing agent may be used.
In particular, EDC is used as a carbodiimide-based condensing agent and DMT-MM is used as a triazine-based condensing agent because it easily forms an ester bond between a hydroxyl group and a carboxyl group of a modified carboxymethyl group-containing modified hyaluronic acid and / or salt thereof. It is good to use.
＜カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩に対する縮合剤の割合＞
　本発明の製造方法において、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩に対する縮合剤の割合を調整することにより、本発明の架橋物を用いて得られる水膨潤性ゲルの硬さを調整することができる。エステル結合をより確実に形成して熱安定性および耐酵素分解性に優れた架橋物を得られやすい点から、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩１，０００質量部に対する縮合剤の割合は、５質量部以上４００質量部以下であるとよく、さらに３０質量部以上３００質量部以下、６０質量部以上２００質量部以下であるとよい。 <Ratio of condensing agent to carboxymethyl group-containing modified hyaluronic acid and / or salt thereof>
In the production method of the present invention, the hardness of the water-swellable gel obtained using the crosslinked product of the present invention is adjusted by adjusting the ratio of the condensing agent to the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof. be able to. A condensing agent for 1,000 parts by mass of carboxymethyl group-containing modified hyaluronic acid and / or its salt is more easily formed by more easily forming an ester bond to easily obtain a crosslinked product having excellent thermal stability and enzymatic degradation resistance. The ratio is preferably 5 parts by mass or more and 400 parts by mass or less, more preferably 30 parts by mass or more and 300 parts by mass or less, and 60 parts by mass or more and 200 parts by mass or less.
＜反応温度および反応時間＞
　本発明の製造方法において、反応液の温度は、１℃以上１００℃以下であればよく、さらに１℃以上５０℃以下、１℃以上３０℃以下であるとよい。
　また、反応時間は、３０分以上１６８時間以下であればよく、さらに１時間以上７２時間以下、１時間以上４８時間以下であればよい。 <Reaction temperature and reaction time>
In the production method of the present invention, the temperature of the reaction solution may be 1 ° C. or higher and 100 ° C. or lower, and may be 1 ° C. or higher and 50 ° C. or lower, 1 ° C. or higher and 30 ° C. or lower.
The reaction time may be 30 minutes or more and 168 hours or less, and may be 1 hour or more and 72 hours or less, and 1 hour or more and 48 hours or less.
　＜溶媒＞
　本発明の製造方法において、原料修飾ヒアルロン酸および／またはその塩を溶解させる溶媒は、水、または水と混和する水溶性有機溶媒との混和物であることができる。 <Solvent>
In the production method of the present invention, the solvent for dissolving the raw material-modified hyaluronic acid and / or salt thereof can be water or an admixture with a water-soluble organic solvent miscible with water.
　水と混和する水溶性有機溶媒としては、例えば、メタノール、エタノール、１－プロパノール、２－プロパノールなどのアルコール系溶媒、アセトン、メチルエチルケトンなどのケトン系溶媒、テトラヒドロフラン、アセトニトリル等を挙げることができ、これらを単独でまたは組み合わせて使用することができる。
　特に、前記架橋物の精製工程を簡易にすることができる点から、前記溶媒は水、またはエタノールを使用するとよい。 Examples of the water-soluble organic solvent miscible with water 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 alone or in combination.
In particular, water or ethanol is preferably used as the solvent since the purification step of the crosslinked product can be simplified.
［原料カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩（原料修飾ヒアルロン酸および／またはその塩）］
＜反応溶液中の原料修飾ヒアルロン酸および／またはその塩の濃度＞
　本発明の製造方法では、エステル結合をより確実に形成して、得られる架橋物の熱安定性および耐酵素分解性を高めることができる点で、前記原料修飾ヒアルロン酸および／またはその塩の１質量％以上８０質量％以下の溶液に縮合剤を添加するとよく、さらに５質量％以上７０質量％以下、５質量％以上５０質量％以下の溶液に縮合剤を添加するとよい。 [Raw material carboxymethyl group-containing modified hyaluronic acid and / or salt thereof (raw material modified hyaluronic acid and / or salt thereof)]
<Concentration of raw material modified hyaluronic acid and / or salt thereof in reaction solution>
In the production method of the present invention, one of the raw material-modified hyaluronic acid and / or a salt thereof can be formed by more reliably forming an ester bond and improving the thermal stability and enzymatic degradation resistance of the resulting crosslinked product. A condensing agent may be added to a solution of not less than 80% by mass and not more than 80% by mass, and a condensing agent may be further added to a solution of not less than 5% by mass and not more than 70% by mass and not less than 5% by mass.
　＜原料修飾ヒアルロン酸および／またはその塩の分子量＞
　本発明の製造方法において、粘弾性に富む水膨潤ゲルを形成する架橋物が得られやすいことから、前記原料修飾ヒアルロン酸および／またはその塩の分子量は４，０００以上４００万以下であるとよい。下限としては、さらに２０万以上であるとよく、３０万以上、８０万以上であるとよい。一方、上限としては、さらに３００万以下であるとよく、２８０万以下、２５０万以下であるとよい。
　特に、本発明の架橋物を美容整形用の皮下注射剤用途に用いる場合、膨潤度の高いゲルが得られ易いことから、前記原料修飾ヒアルロン酸および／またはその塩の分子量は、５０万以上２００万以下であるとよく、さらに８０万以上１８０万以下であるとよい。
　なお、本発明において、原料修飾ヒアルロン酸および／またはその塩の分子量は、以下の方法にて測定することができる。 <Molecular weight of raw material modified hyaluronic acid and / or salt thereof>
In the production method of the present invention, since a crosslinked product that forms a water-swelling gel rich in viscoelasticity is easily obtained, the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof is preferably 4,000 to 4,000,000. . The lower limit is preferably 200,000 or more, more preferably 300,000 or more and 800,000 or more. On the other hand, the upper limit is preferably 3 million or less, and is preferably 2.8 million or less and 2.5 million or less.
In particular, when the crosslinked product of the present invention is used for a subcutaneous injection for cosmetic surgery, a gel with a high degree of swelling is easily obtained. Therefore, the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof is 500,000 or more and 200 It is good if it is 10,000 or less, and it is good if it is 800,000 or more and 1.8 million or less.
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.
　ゲル濾過カラムを用いて、分子量が既知である複数の(精製)ヒアルロン酸（基準物質）を液体クロマトグラフィー分析することで、それらの保持時間より検量線を作成する。同様に、測定対象である原料修飾ヒアルロン酸を液体クロマトグラフィー分析し、前記検量線を用いて分子量を求めることで、原料修飾ヒアルロン酸の分子量を求めることができる。
　前記液体クロマトグラフィー分析に使用することができる液体クロマトグラフィー分析装置としては、例えば、ＷａｔｅｒｓＡｌｌｉａｎｃｅ ２６９０ ＨＰＬＣ Ｓｅｐａｒａｔｉｏｎｓ Ｍｏｄｕｌｅ（Ｗａｔｅｒｓ社製）、Ｗａｔｅｒｓ Ａｌｌｉａｎｃｅ ２６９５ ＨＰＬＣ ＳｅｐａｒａｔｉｏｎｓＭｏｄｕｌｅ（Ｗａｔｅｒｓ社製）、１２００ Ｓｅｒｉｅｓ（Ａｇｉｌｅｎｔ社製）が挙げられる。
　また、液体クロマトグラフィー分析に使用することができるカラムとしては、例えば、ｓｈｏｄｅｘ社製　配位子交換クロマトグラフィー用カラム（配位子交換モード＋サイズ排除モード)、型名「ＳＵＧＡＲ ＫＳ－８０１」、「ＳＵＧＡＲ ＫＳ－８０２」、「ＳＵＧＡＲ ＫＳ－８０３」、「ＳＵＧＡＲ ＫＳ－８０４」、「ＳＵＧＡＲＫＳ－８０５」、「ＳＵＧＡＲ ＫＳ－８０６」、「ＳＵＧＡＲＫＳ－８０７」や、ＴＯＳＯＨ製　サイズ排除クロマトグラフィーカラム、型名「ＴＳＫｇｅｌ ＧＭＰＷ」が挙げられる。 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), and 1200 Series (Agil). Is mentioned.
Examples of the column that can be used for liquid chromatography analysis include 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", "SUGARKS-805", "SUGAR KS-806", "SUGARKS-807", TOSOH size exclusion chromatography column, type The name “TSKgel GMPW” is mentioned.
＜原料修飾ヒアルロン酸および／またはその塩の動粘度＞
　本発明の製造方法において、粘弾性に富む水膨潤ゲルを形成する架橋物が得られやすいことから、前記原料修飾ヒアルロン酸および／またはその塩の動粘度は１ｍｍ２／ｓ以上２００ｍｍ２／ｓ以下とすることができ、さらに３０ｍｍ２／ｓ以上１５０ｍｍ２／ｓ以下とすることができる。本発明において、原料修飾ヒアルロン酸および／またはその塩の動粘度は、ウベローデ粘度計（柴田科学器械工業株式会社製）を用いて測定することができる。この際、流下秒数が２００秒以上１，０００秒になるような係数のウベローデ粘度計を選択する。また、測定は３０℃の恒温水槽中で行ない、温度変化のないようにする。ウベローデ粘度計により測定された前記水溶液の流下秒数と、ウベローデ粘度計の係数との積により、動粘度（単位：ｍｍ２／ｓ）を求めることができる。 <Kinematic viscosity of raw material modified hyaluronic acid and / or salt thereof>
In the production method of the present invention, since a crosslinked product that forms a water-swelling gel rich in viscoelasticity is easily obtained, the kinematic viscosity of the raw material-modified hyaluronic acid and / or salt thereof is 1 mm 2 / s to 200 mm 2 / s. Furthermore, it can be set to 30 mm 2 / s or more and 150 mm 2 / s or less. In the present invention, the kinematic viscosity of the raw material modified hyaluronic acid and / or salt thereof can be measured using an Ubbelohde viscometer (manufactured by Shibata Kagaku Kikai Kogyo Co., Ltd.). At this time, a Ubbelohde viscometer having a coefficient such that the number of seconds of flow is 200 seconds or more and 1,000 seconds is selected. The measurement is performed in a constant temperature water bath at 30 ° C. so that there is no temperature change. The kinematic viscosity (unit: mm 2 / s) can be obtained from the product of the number of seconds of flow of the aqueous solution measured by the Ubbelohde viscometer and the coefficient of the Ubbelohde viscometer.
＜カルボキシメチル化率＞
　本発明において、カルボキシル基を有する修飾ヒアルロン酸および／またはその塩のヒアルロン酸を構成する２糖単位に対するカルボキシメチル化率（以下、単に「カルボキシメチル化率（ＣＭ化率）」ともいう。）は、１Ｈ－ＮＭＲスペクトルにおいて、ヒアルロン酸骨格中のＣ－２位に結合するＮ－アセチル基のメチル基（－ＣＨ３）のプロトンを示すピーク（２ｐｐｍ付近に発現）の積算値に対する、カルボキシメチル基（－ＣＨ２－ＣＯ２Ｈまたは－ＣＨ２－ＣＯ２ －）のメチレン基（－ＣＨ２－）のプロトンを示すピーク（３．８ｐｐｍ以上４．２ｐｐｍ以下の範囲に発現）の積算値の割合（％）で表される。 <Carboxymethylation rate>
In the present invention, the carboxymethylation rate (hereinafter also simply referred to as “carboxymethylation rate (CM conversion rate)”) of the disaccharide unit constituting the hyaluronic acid of the modified hyaluronic acid and / or salt thereof having a carboxyl group. In the 1 H-NMR spectrum, carboxymethyl with respect 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. The integrated value of the peak (expressed in the range of 3.8 ppm to 4.2 ppm) indicating the proton of the methylene group (—CH 2 —) of the group (—CH 2 —CO 2 H or —CH 2 —CO 2 − ) Expressed as a percentage.
　本発明において、「ヒアルロン酸を構成する２糖単位」とは、ヒアルロン酸を構成する、隣り合って結合する２糖（グルクロン酸およびＮ－アセチルグルコサミン）で構成される１単位をいい、「ヒアルロン酸を構成する２糖単位に対するカルボキシメチル化率」とは、該１単位に対する、該１単位に含まれるカルボキシメチル基の数であり、より具体的には、該１単位を１００％とした場合、該１単位に対する、該１単位に含まれるカルボキシメチル基の数の割合（％）をいう。 In the present invention, “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.
　＜原料修飾ヒアルロン酸および／またはその塩のカルボキシメチル化率＞
　本発明の製造方法では、エステル結合をより確実に形成して得られる架橋物の熱安定性および耐酵素分解性を高めることができる点で、前記原料修飾ヒアルロン酸および／またはその塩のカルボキシメチル化率は１０％以上２００％以下であるとよい。下限としては、さらに２０％以上、３０％以上、５０％以上であるとよい。一方、上限としては、さらに１５０％以下、１００％以下、９０％以下であるとよい。 <Carboxymethylation rate of raw material modified hyaluronic acid and / or salt thereof>
In the production method of the present invention, the raw material-modified hyaluronic acid and / or its salt carboxymethyl can be improved in that the thermal stability and enzymatic degradation resistance of the cross-linked product obtained by more reliably forming an ester bond can be improved. The conversion rate is preferably 10% or more and 200% or less. The lower limit is preferably 20% or more, 30% or more, or 50% or more. On the other hand, the upper limit is preferably 150% or less, 100% or less, or 90% or less.
［原料修飾ヒアルロン酸および／またはその塩の製造方法］
　本発明の製造方法において、原料修飾ヒアルロン酸および／またはその塩は、温度が３０℃以下の含水溶媒中で、溶解した原料ヒアルロン酸および／またはその塩をハロ酢酸および／またはその塩と反応させる工程を含み、前記含水溶媒は、水、または水溶性有機溶媒と水の混合液であり、
前記混合液における水溶性有機溶媒の割合が６０ｖ／ｖ％以下であることによって得ることができる。 [Method for producing raw material-modified hyaluronic acid and / or salt thereof]
In the production method of the present invention, the raw material-modified hyaluronic acid and / or salt thereof is reacted with haloacetic acid and / or a salt thereof in a hydrous solvent having a temperature of 30 ° C. or less. Including the step, the water-containing solvent is water or a mixed solution of water-soluble organic solvent and water,
It can be obtained when the ratio of the water-soluble organic solvent in the mixed solution is 60 v / v% or less.
　上記反応させる工程において、反応液（含水溶媒）中に原料ヒアルロン酸および／またはその塩の少なくとも一部（ヒアルロン酸および／またはその塩の全部または大部分であるとよい）とハロ酢酸および／またはその塩とが溶解した状態で該ヒアルロン酸および／またはその塩と該ハロ酢酸および／またはその塩とを反応させることができる。 In the reaction step, at least a part of the raw material hyaluronic acid and / or its salt (which may be all or most of the hyaluronic acid and / or its salt), haloacetic acid and / or in the reaction solution (hydrous solvent) The hyaluronic acid and / or salt thereof and the haloacetic acid and / or salt thereof can be reacted with the salt dissolved.
　＜原料ヒアルロン酸および／またはその塩の平均分子量＞
　原料ヒアルロン酸および／またはその塩の平均分子量は、カルボキシメチル化を円滑に行うことができる点で、４，０００以上４００万以下とすることができる。下限としては、さらに２０万以上であるとよく、３０万以上、８０万以上であるとよい。一方、上限としては、さらに３００万以下であるとよく、２８０万以下、２５０万以下であるとよい。
　なお、本発明において、原料修飾ヒアルロン酸および／またはその塩の分子量は、極限粘度法により測定することができる。 <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 can be 4,000 to 4,000,000 in that carboxymethylation can be carried out smoothly. The lower limit is preferably 200,000 or more, more preferably 300,000 or more and 800,000 or more. On the other hand, the upper limit is preferably 3 million or less, and is preferably 2.8 million or less and 2.5 million or less.
In the present invention, the molecular weight of the raw material-modified hyaluronic acid and / or salt thereof can be measured by an intrinsic viscosity method.
＜含水溶媒における原料ヒアルロン酸および／またはその塩の濃度＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、反応液（含水溶媒）における前記ヒアルロン酸の濃度は０．０５ｇ／ｍＬ以上０．５ｇ／ｍＬ以下とすることができる。 <Concentration of raw material hyaluronic acid and / or salt thereof in hydrous solvent>
In the method for producing raw material-modified hyaluronic acid and / or a salt thereof, the concentration of the hyaluronic acid in the reaction solution (aqueous solvent) can be 0.05 g / mL or more and 0.5 g / mL or less.
　＜ハロ酢酸および／またはその塩＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、ハロ酢酸および／またはその塩は、カルボキシメチル基を原料ヒアルロン酸および／またはその塩に導入するために使用される。 <Haloacetic acid and / or salt thereof>
In the method for producing raw material-modified hyaluronic acid and / or its salt, haloacetic acid and / or its salt is used to introduce a carboxymethyl group into raw material hyaluronic acid and / or its salt.
　ハロ酢酸は例えば、モノハロ酢酸および／またはその塩であるとよく、より具体的には、クロロ酢酸および／またはその塩、または、ブロモ酢酸またはその塩とするとよい。ハロ酢酸の塩は例えば、クロロ酢酸のアルカリ金属塩および／またはブロモ酢酸のアルカリ金属塩であるとよく、クロロ酢酸ナトリウムおよび／またはブロモ酢酸ナトリウムであるとよい。 The haloacetic acid may be, for example, monohaloacetic acid and / or a salt thereof, more specifically, chloroacetic acid and / or a salt thereof, or bromoacetic acid or a salt thereof. The salt of haloacetic acid may be, for example, an alkali metal salt of chloroacetic acid and / or an alkali metal salt of bromoacetic acid, and may be sodium chloroacetate and / or sodium bromoacetate.
　＜ハロ酢酸および／またはその塩の使用量＞
　ハロ酢酸および／またはその塩の使用量は、原料ヒアルロン酸および／またはその塩の使用量に対して１０質量％以上５００質量％以下とすることができ、さらに５０質量％以上２００質量％以下とすることができる。 <Amount of haloacetic acid and / or salt thereof>
The amount of the haloacetic acid and / or salt thereof used can be 10% by mass or more and 500% by mass or less, and further 50% by mass or more and 200% by mass or less based on the amount of the raw material hyaluronic acid and / or salt thereof can do.
　＜含水溶媒＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、原料ヒアルロン酸および／またはその塩の溶解性が高い点から、前記含水溶媒は、水、または水溶性有機溶媒と水との混合液であるとよい。 <Water-containing solvent>
In the method for producing raw material-modified hyaluronic acid and / or salt thereof, the water-containing solvent is water or a mixed solution of water-soluble organic solvent and water because the raw material hyaluronic acid and / or salt thereof has high solubility. Good.
　含水溶媒が水溶性有機溶媒と水との混合液である場合、ヒアルロン酸の溶解性を高めることができる点で、該混合液中における水溶性有機溶媒の割合は６０ｖ／ｖ％以下であるとよく、さらに２０ｖ／ｖ％以上４０ｖ／ｖ％以下であるとよい。 When the water-containing solvent is a mixed solution of a water-soluble organic solvent and water, the solubility of hyaluronic acid can be increased, and the proportion of the water-soluble organic solvent in the mixed solution is 60 v / v% or less. Furthermore, it is good in it being 20 v / v% or more and 40 v / v% or less.
　水溶性有機溶媒としては、例えば、メタノール、エタノール、１－プロパノール、２－プロパノールなどのアルコール系溶媒、アセトン、メチルエチルケトンなどのケトン系溶媒、テトラヒドロフラン、アセトニトリル等を挙げることができ、これらを単独でまたは組み合わせて使用することができる。このうち、イソプロパノール、エタノール等の炭素原子数１、２または３の低級モノアルコールを用いるとよい。 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 monoalcohols having 1, 2 or 3 carbon atoms such as isopropanol and ethanol may be used.
　＜ｐＨ＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、水酸基の求核性を高めることができる点で、前記原料ヒアルロン酸および／またはその塩とハロ酢酸および／またはその塩との反応は塩基性条件下で行われるとよく、反応液のｐＨが９以上１４以下とすることができ、さらに１０以上１４以下、１１以上１４以下とすることができる。 <PH>
In the method for producing raw material-modified hyaluronic acid and / or salt thereof, the reaction of the raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof is basic in that the nucleophilicity of the hydroxyl group can be increased. It may be carried out under conditions, and the pH of the reaction solution may be 9 or more and 14 or less, and may be 10 or more and 14 or less, or 11 or more and 14 or less.
　なお、前記反応の反応液を塩基性に調整するために、塩基性電解質を反応液中で使用することができる。塩基性電解質としては、例えば、水酸化ナトリウム、水酸化カリウム等のアルカリ金属の水酸化物、水酸化カルシウム、水酸化マグネシウム、水酸化バリウム等のアルカリ土類金属の水酸化物が挙げられる。原料修飾ヒアルロン酸および／またはその塩を効率良く得ることができる点で、反応液中の塩基性電解質の濃度は、０．２モル／Ｌ以上１０モル／Ｌ以下であるとよく、さらに０．５モル／Ｌ以上８モル／Ｌ以下であるとよい。 In addition, in order to adjust the reaction solution of the above reaction to basic, a basic electrolyte can be used in the reaction solution. 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 preferably 0.2 mol / L or more and 10 mol / L or less in that the raw material-modified hyaluronic acid and / or salt thereof can be obtained efficiently. It is good that they are 5 mol / L or more and 8 mol / L or less.
　＜反応温度＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、カルボキシル化を円滑に進行でき、かつ、原料修飾ヒアルロン酸および／またはその塩の低分子化を抑制できる点から、反応液の温度は３０℃以下であるとよく、さらに０℃超１０℃以下であるとよい。特に、反応液の温度を０℃超１０℃以下にする方が、平均分子量８０万以上の高分子の原料修飾ヒアルロン酸および／またはその塩を容易に得ることができる。 <Reaction temperature>
In the method for producing raw material-modified hyaluronic acid and / or salt thereof, the temperature of the reaction solution is 30 ° C. because carboxylation can proceed smoothly and low molecular weight of raw material-modified hyaluronic acid and / or salt thereof can be suppressed. It is good that it is below, and it is good that it is more than 0 degreeC and below 10 degreeC. In particular, when the temperature of the reaction solution is higher than 0 ° C. and lower than or equal to 10 ° C., high-molecular raw material-modified hyaluronic acid and / or a salt thereof having an average molecular weight of 800,000 or more can be easily obtained.
　＜反応時間＞
　原料修飾ヒアルロン酸および／またはその塩の製造方法において、カルボキシル化を円滑に進行でき、かつ、原料修飾ヒアルロン酸および／またはその塩の低分子化を抑制できる点から、反応時間は３０分以上１００時間以下とするとよく、さらに６０分以上６０時間以下とするとよい。 <Reaction time>
In the method for producing raw material-modified hyaluronic acid and / or salt thereof, the reaction time is 30 minutes or more from the viewpoint that carboxylation can proceed smoothly and the molecular weight reduction of raw material-modified hyaluronic acid and / or salt thereof can be suppressed. It is preferable that the time be less than or equal to 60 hours or less and 60 hours or less.
　［架橋物］
　＜構造＞
　本発明の架橋物は、同一または異なるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩同士が有する水酸基とカルボキシル基との間に形成されるエステル結合によって架橋されている。該架橋物は、未修飾ヒアルロン酸および／またはその塩の架橋物よりも後述する熱安定性および耐酵素分解性に優れている。 [Crosslinked product]
<Structure>
The cross-linked product of the present invention is cross-linked by an ester bond formed between a hydroxyl group and a carboxyl group of the same or different carboxymethyl group-containing modified hyaluronic acid and / or salts thereof. The cross-linked product is superior in thermal stability and resistance to enzymatic degradation described later than a cross-linked product of unmodified hyaluronic acid and / or a salt thereof.
　＜熱安定性＞
　本発明の架橋物は優れた熱安定性を有する。具体的には、後述の熱安定性試験におけるゲル残存率が１０％以上であり、さらに１５％以上１００％以下、２０％以上８０％以下であるとよい。熱安定性試験におけるゲル残存率が１０％未満であると、加熱滅菌処理のよりゲルが熱分解してしまうため、医療材料や美容材料として使用することができない。 <Thermal stability>
The crosslinked product of the present invention has excellent heat stability. Specifically, the gel remaining rate in the thermal stability test described later is 10% or more, and further preferably 15% or more and 100% or less, or 20% or more and 80% or less. If the gel remaining rate in the thermal stability test is less than 10%, the gel is thermally decomposed by the heat sterilization treatment and cannot be used as a medical material or a cosmetic material.
本発明において、熱安定性試験は以下の方法で行う。
（ａ）５０ｍＬバイアル瓶に、本発明で得られた架橋物１ｇ（乾燥質量換算）をリン酸緩衝生理食塩水（ｐＨ７．４）５０ｍＬに分散させ、該架橋物の濃度が２質量％である架橋物水溶液を調製する。
（ｂ）前記架橋物水溶液を、１２１℃で２０分間オートクレーブ処理する。より具体的には、開始温度２５℃で４０分間かけて１２１℃まで昇温させ、１２１℃で２０分間オートクレーブ処理し、加圧・加熱終了後１時間静置してから、該架橋物を取り出す工程をいう。
（ｃ）前記オートクレーブ処理後の架橋物を４２０メッシュのナイロンストレーナーに載置し、残存物を蒸留水で洗浄後、吸引ろ過する。
（ｄ）前記ろ過残渣の全量を、秤量済みシャーレ上で５５℃で３時間減圧乾燥する。
（ｅ）前記減圧乾燥後のシャーレを含めた質量を秤量し、シャーレの質量の変化分から残留物の質量を算出する。次いで、架橋物の質量および残留物の質量から、熱安定性試験におけるゲル残存率を以下の式（３）により算出する。
 
熱安定性試験におけるゲル残存率（質量％）＝オートクレーブ処理後の架橋物の残留物の質量／オートクレーブ処理前の架橋物の質量×１００・・・（３）
  In the present invention, the thermal stability test is performed by the following method.
(A) In a 50 mL vial, 1 g of the crosslinked product obtained in the present invention (in terms of dry mass) is dispersed in 50 mL of phosphate buffered saline (pH 7.4), and the concentration of the crosslinked product is 2% by mass. An aqueous solution of a crosslinked product is prepared.
(B) The cross-linked product aqueous solution is autoclaved at 121 ° C. for 20 minutes. More specifically, the temperature is raised to 121 ° C. over 40 minutes at a starting temperature of 25 ° C., autoclaved at 121 ° C. for 20 minutes, and left to stand for 1 hour after completion of pressurization and heating, and then the crosslinked product is taken out. Refers to a process.
(C) The autoclaved crosslinked product is placed on a 420 mesh nylon strainer, and the residue is washed with distilled water and suction filtered.
(D) The whole amount of the filtration residue is dried under reduced pressure on a weighed petri dish at 55 ° C. for 3 hours.
(E) The mass including the petri dish after drying under reduced pressure is weighed, and the mass of the residue is calculated from the change in the mass of the petri dish. Next, the gel remaining rate in the thermal stability test is calculated from the mass of the crosslinked product and the mass of the residue by the following formula (3).

Gel residual ratio (mass%) in thermal stability test = mass of residue of crosslinked product after autoclave treatment / mass of crosslinked material before autoclave treatment × 100 (3)
　本発明の架橋物は、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を含むことにより、未修飾ヒアルロン酸および／またはその塩と比較して、エステル結合に関与できるカルボキシル基を多く含むため、熱安定性により優れている。 Since the cross-linked product of the present invention contains a carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof, compared to unmodified hyaluronic acid and / or a salt thereof, it contains more carboxyl groups that can participate in an ester bond. Excellent thermal stability.
　＜耐酵素分解性＞
　本発明の架橋物は優れた耐酵素（ヒアルロニダーゼ）分解性を有する。より具体的には、後述の耐酵素分解性試験におけるゲル残存率が６０％以上であり、さらに６０％以上９５％、７０％以上９０％以下であるとよい。耐酵素分解性におけるゲル残存率が６０％未満であると、ヒアルロニダーゼにより分解されやすく、滞留性が低くなってしまう。 <Enzyme degradation resistance>
The cross-linked product of the present invention has excellent enzyme resistance (hyaluronidase) degradability. More specifically, the gel remaining rate in the enzyme degradation resistance test described later is 60% or more, and more preferably 60% or more and 95%, or 70% or more and 90% or less. If the gel residual ratio in the enzyme degradation resistance is less than 60%, the gel is easily decomposed by hyaluronidase and the retention is lowered.
　本発明において、耐酵素分解性試験は以下の方法で行う。
（ａ）本発明の架橋物１００ｍｇ（乾燥質量換算）を５０ｍＭリン酸緩衝液９．５ｍＬに分散させて、該架橋物の濃度が１質量％（固形分）である架橋物水溶液を調製し、２５℃で１０分間静置して、膨潤溶解させる。
（ｂ）前記架橋物水溶液を、ヒアルロニダーゼ（０．２５単位／架橋物１ｍｇあたり）存在下にて４０℃で１５時間保存する。
（ｃ）さらに、ヒアルロニダーゼ（０．２５単位／架橋物１ｍｇあたり）を追加し、４０℃で２４時間保存する。
（ｄ）前記保存後の架橋物を４２０メッシュのナイロンストレーナーに載置し、残存物を蒸留水で洗浄後、吸引ろ過する。
（ｅ）前記ろ過残渣の全量を、秤量済みシャーレ上で５５℃で３時間減圧乾燥する。
（ｆ）前記減圧乾燥後のシャーレを含めた質量を秤量し、シャーレの質量の変化分から残留物の質量を算出する。次いで、架橋物の質量および残留物の質量から、耐酵素分解性試験におけるゲル残存率を以下の式（４）により算出する。
 
耐酵素分解性試験におけるゲル残存率（％）＝残留物の質量／架橋物の質量（固形分）
×１００　・・・（４）
  In the present invention, the enzyme degradation resistance test is carried out by the following method.
(A) 100 mg (in terms of dry mass) of the crosslinked product of the present invention is dispersed in 9.5 mL of 50 mM phosphate buffer to prepare a crosslinked product aqueous solution having a concentration of the crosslinked product of 1% by mass (solid content), Let stand at 25 ° C. for 10 minutes to swell and dissolve.
(B) The aqueous solution of the cross-linked product is stored at 40 ° C. for 15 hours in the presence of hyaluronidase (0.25 unit / per mg of cross-linked product).
(C) Further, add hyaluronidase (0.25 unit / mg of cross-linked product) and store at 40 ° C. for 24 hours.
(D) The crosslinked product after storage is placed on a 420-mesh nylon strainer, and the residue is washed with distilled water and suction filtered.
(E) The whole amount of the filtration residue is dried under reduced pressure on a weighed petri dish at 55 ° C. for 3 hours.
(F) The mass including the petri dish after drying under reduced pressure is weighed, and the mass of the residue is calculated from the change in the mass of the petri dish. Next, the gel residual ratio in the enzyme degradation resistance test is calculated from the mass of the cross-linked product and the mass of the residue by the following formula (4).

Gel residual ratio (%) in enzyme degradation resistance test = mass of residue / mass of cross-linked product (solid content)
× 100 (4)
　本発明の架橋物が耐酵素分解性に優れている原因のひとつとして、前記架橋物がカルボキシメチル基を含有することが挙げられる。具体的には、前記架橋物がカルボキシメチル基を含有することより、ヒアルロニダーゼが、前記架橋物に含まれるヒアルロン酸骨格を認識しづらくなるため、耐酵素分解性が向上すると推測される。 One of the reasons why the cross-linked product of the present invention is excellent in enzyme degradation resistance is that the cross-linked product contains a carboxymethyl group. Specifically, since the cross-linked product contains a carboxymethyl group, hyaluronidase is difficult to recognize the hyaluronic acid skeleton contained in the cross-linked product.
＜水膨潤性＞
　さらに、本発明の架橋物は、優れた水膨潤性を有することから、やわらかい水膨潤性ゲルを形成することができる。ここで、「水膨潤性」とは、水を取り込んで膨潤する性質のことをいい、一般に、水を取り込んでゲル状になる性質のことをいう。本発明の架橋物は、縮合剤との反応によって生じたエステル結合によって形成される３次元網目構造を有し、該３次元網目構造の中に水を取り込むことにより膨潤して、ゲルを構成することができる。 <Water swelling>
Furthermore, since the crosslinked product of the present invention has excellent water swellability, a soft water-swellable gel can be formed. Here, “water swellability” refers to the property of taking water to swell and generally refers to the property of taking water to become a gel. The cross-linked product of the present invention has a three-dimensional network structure formed by ester bonds generated by reaction with a condensing agent, and swells by incorporating water into the three-dimensional network structure to form a gel. be able to.
　＜膨潤度＞
　本発明の架橋物は、よりやわらかい水膨潤性ゲルを形成することができる点で、水に対する膨潤度が５倍以上２５０倍以下（質量比）であり、さらに５倍以上２００倍以下であるとよい。ここで、「膨潤度」とは、乾燥ゲルの質量に対する、膨潤ゲルの質量（膨潤ゲル／乾燥ゲル）を意味する。
　膨潤度が５倍未満であると、医療材料・美容材料として使用するには硬すぎるゲルになってしまう。一方、膨潤度が２５０倍より大きいと、やわらかすぎてもろいゲルとなってしまう。 <Swelling degree>
The crosslinked product of the present invention has a degree of swelling with respect to water of 5 to 250 times (mass ratio), more preferably 5 to 200 times in that a softer water-swellable gel can be formed. Good. Here, the “swelling degree” means the mass of the swollen gel relative to the mass of the dry gel (swelled gel / dry gel).
If the degree of swelling is less than 5 times, the gel becomes too hard to be used as a medical material / beauty material. On the other hand, if the degree of swelling is more than 250 times, the gel becomes brittle even if it is too soft.
　より具体的には、本発明の架橋物において、熱安定性試験前の膨潤度は、５倍以上５０倍以下（質量比）であるとよく、さらに５倍以上２０倍以下であるとよい。 More specifically, in the crosslinked product of the present invention, the degree of swelling before the thermal stability test is preferably 5 to 50 times (mass ratio), and more preferably 5 to 20 times.
　また、熱安定性試験後の膨潤度は、１０倍以上２５０倍以下（質量比）であるとよく、さらに１５倍以上２００倍以下、２０倍以上１５０倍以下であるとよい。 Further, the degree of swelling after the thermal stability test is preferably 10 to 250 times (mass ratio), more preferably 15 to 200 times, and 20 to 150 times.
＜貯蔵弾性率Ｇ’＞
　本発明の架橋物は、粘弾性が優れた水膨潤性ゲルが得られやすいことから、貯蔵弾性率Ｇ’（周波数１Ｈｚ）が１００Ｐａ以上１０万Ｐａ以下であるとよく、さらに１５０Ｐａ以上５万Ｐａ以下であるとよい。なお、本発明において、貯蔵弾性率Ｇ’は後述の実施例に示される方法により測定することができる。 <Storage elastic modulus G '>
Since the cross-linked product of the present invention is easy to obtain a water-swellable gel having excellent viscoelasticity, the storage elastic modulus G ′ (frequency 1 Hz) is preferably 100 Pa to 100,000 Pa, and more preferably 150 Pa to 50,000 Pa. It may be the following. In the present invention, the storage elastic modulus G ′ can be measured by the method shown in Examples described later.
［架橋物の用途］
　本発明の膝関節注射剤、癒着防止剤、皮下注射剤、または薬物徐放剤は、前記架橋物（または、水膨潤性ゲル）を含むことにより、熱安定性および耐酵素分解性に優れており、さらに固体にて保存が可能であるため、保存安定性に優れている。 [Uses of cross-linked products]
The knee joint injection, the anti-adhesion agent, the subcutaneous injection, or the drug sustained-release agent of the present invention contains the cross-linked product (or water-swellable gel), thereby being excellent in thermal stability and enzyme degradation resistance. Furthermore, since it can be stored as a solid, it has excellent storage stability.
　＜関節注射剤・癒着防止剤＞
　本発明の架橋物を関節注射剤・癒着防止剤として使用する場合、前記架橋物は優れた熱安定性および耐酵素分解性を有していることから、生体内である程度の期間分解されずに残存し、その後、生体内で分解されるため、組織同士の癒着を防止することができる上、架橋剤を使用していないため安全性に優れている。
　特に、関節注射剤においては、前記架橋物は優れた水膨潤性を有しているため、注射後の痛みを低減することができる。また、癒着防止剤においては、前記架橋物が優れた加工性を有していることから、シート状やフィルム状として幅広い形態で使用することができる。　 <Joint injections and anti-adhesion agents>
When the crosslinked product of the present invention is used as an joint injection / adhesion inhibitor, the crosslinked product has excellent thermal stability and resistance to enzymatic degradation, so that it is not degraded in a living body for a certain period of time. Since it remains and then decomposes in vivo, it is possible to prevent adhesion between tissues, and it is excellent in safety because it does not use a crosslinking agent.
Particularly in joint injections, the cross-linked product has excellent water swellability, so that pain after injection can be reduced. Moreover, in the anti-adhesion agent, since the said crosslinked material has the outstanding workability, it can be used with a wide form as a sheet form or a film form.
＜皮下注射剤＞　
　本発明の架橋物を皮下注射剤として使用する場合、医療材料だけでなく、例えば、顔、頭、首、胸部、腹部、臀部、背中、腰、上肢、下肢に注射することにより、美容上の効果（例えば、豊胸、美顔、美脚等、外観をより良くするため）を奏するために使用することができる。前記水膨潤性ゲルは優れた熱安定性および耐酵素分解性、さらに水膨潤性を有するため、美容上の効果を持続させることができる。 <Subcutaneous injection>
When the cross-linked product of the present invention is used as a subcutaneous injection, not only medical materials but also cosmetically, for example, by injection into the face, head, neck, chest, abdomen, buttocks, back, waist, upper limb, and lower limb. It can be used to produce effects (for example, breast enhancement, beautiful face, beautiful legs, etc., to improve the appearance). Since the water-swellable gel has excellent heat stability, enzyme degradation resistance, and water-swellability, it can maintain a cosmetic effect.
　＜薬物徐放剤＞
　本発明の架橋物を薬物徐放剤として使用する場合、前記架橋物は優れた熱安定性および耐酵素分解性を有していることから、生体内である程度の期間分解されずに残存し、その後、生体内で分解されるため、薬物の徐放を補助する作用を有し、かつ、安全性に優れている。 <Slow drug release agent>
When the cross-linked product of the present invention is used as a drug sustained-release agent, the cross-linked product has excellent thermal stability and resistance to enzymatic degradation, so that it remains without being decomposed for a certain period of time in vivo. Thereafter, since it is decomposed in vivo, it has an action of assisting the sustained release of the drug and is excellent in safety.
　＜化粧料＞
　本発明の化粧料は前記架橋物を含み、該架橋物は構成するカルボキシル基に起因して、高い保水効果を有する。より具体的には、本発明の架橋物に含まれるカルボキシル基が水と水素結合を構成するため、該カルボキシル基に起因して、優れた保水力を発揮すると推測される。このため、例えば皮膚等の生体組織において高い保水効果を有する。　
　また、本発明の化粧料が前記水膨潤性ゲルを含む場合、ゲルとして適度な粘弾性を有するため、ゲル特有のみずみずしい触感を実感することができる上、ゲルの中に有効成分を配合することにより、有効成分を徐放させることができる。 <Cosmetics>
The cosmetic of the present invention contains the cross-linked product, and the cross-linked product has a high water retention effect due to the carboxyl group that constitutes it. More specifically, since the carboxyl group contained in the cross-linked product of the present invention constitutes a hydrogen bond with water, it is presumed that excellent water retention is exhibited due to the carboxyl group. For this reason, it has a high water retention effect in biological tissues such as skin.
In addition, when the cosmetic composition of the present invention contains the water-swellable gel, it has an appropriate viscoelasticity as a gel, so that it can feel a fresh feel unique to the gel, and an active ingredient is blended in the gel. Thus, the active ingredient can be released gradually.
　本発明の化粧料の態様は特に限定されないが、例えば、皮膚用化粧料が挙げられる。具体的には、例えば、洗顔料、洗浄料、化粧水、クリーム、乳液、美容液、パック、クレンジング、ファンデーション、口紅、リップクリーム、リップグロス、リップライナー、頬紅、シェービングローション、アフターサンローション、デオドラントローション、ボディローション、ボディオイル、石鹸、入浴剤が挙げられる。 The aspect of the cosmetic of the present invention is not particularly limited, and examples thereof include skin cosmetics. Specifically, for example, facial cleanser, cleansing agent, lotion, cream, milky lotion, serum, pack, cleansing, foundation, lipstick, lip balm, lip gloss, lip liner, blusher, shaving lotion, after sun lotion, deodorant Lotion, body lotion, body oil, soap, bath salt.
　［実施例］
　次に、本発明を実施例および比較例に基づき、さらに説明する。なお、本発明はこれに限定するものではない。 [Example]
Next, this invention is further demonstrated based on an Example and a comparative example. Note that the present invention is not limited to this.
　＜調製例１：カルボキシメチル基含有修飾ヒアルロン酸の調製＞
　３０ｍＬのサンプル瓶に水酸化ナトリウム１．０４ｇを秤り取った後、水１２ｍＬを添加して溶解させた。次に、平均分子量が１７５万のヒアルロン酸２．０ｇを添加し溶解させた後、モノブロモ酢酸３．６２ｇを添加して溶解させて、１℃で１６時間静置した。その後、２００ｍＬビーカーにエタノール８０ｍＬを入れ、該反応液を撹拌しながら添加した。その後、４００メッシュのろ布で沈殿を回収した後、１０％塩化ナトリウム水溶液４０ｍＬを添加して沈殿を溶解させた。さらに、８％塩酸水溶液でｐＨを調製した後、エタノール１００ｍＬで３回洗浄した後、減圧濾過し、５５℃で３時間減圧乾燥することにより、調製例１のカルボキシメチル基含有修飾ヒアルロン酸（原料修飾ヒアルロン酸）を含有する組成物を得た。 <Preparation Example 1: Preparation of carboxymethyl group-containing modified hyaluronic acid>
After weighing 1.04 g of sodium hydroxide into a 30 mL sample bottle, 12 mL of water was added and dissolved. Next, 2.0 g of hyaluronic acid having an average molecular weight of 1.75 million 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% aqueous sodium chloride 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 modified hyaluronic acid containing carboxymethyl group of Preparation Example 1 (raw material) A composition containing (modified hyaluronic acid) was obtained.
　調製例１で得られたカルボキシメチル基含有修飾ヒアルロン酸は分子量が１０７万であり、カルボキシメチル化率が７７％であった。 The carboxymethyl group-containing modified hyaluronic acid obtained in Preparation Example 1 had a molecular weight of 1.70 million and a carboxymethylation rate of 77%.
　＜カルボキシメチル化率の測定および算出＞
　調製例１で得られたカルボキシメチル基含有修飾ヒアルロン酸のカルボキシメチル化率は、以下の方法にて１Ｈ－ＮＭＲスペクトルの積算値より求めた。 <Measurement and calculation of carboxymethylation rate>
The carboxymethylation rate of the carboxymethyl group-containing modified hyaluronic acid obtained in Preparation Example 1 was determined from the integrated value of 1 H-NMR spectrum by the following method.
（試料調製）
　試料７ｍｇと内部標準物質４，４－ジメチル－４－シラペンタンスルホン酸ナトリウム（ＤＳＳ）１ｍｇを重水０．７ｍｌに溶かし、ＮＭＲ試料管に移し入れ、キャップをした。
 
（測定条件）
装置：Ｖａｒｉａｎ　ＮＭＲ　ｓｙｓｔｅｍ　４００ＮＢ型(バリアンテクノロジーズジャパンリミテッド)
観測周波数：４００ＭＨｚ
温度：３０℃
基準：ＤＳＳ（０ｐｐｍ）
積算回数：６４回
 
（解析方法）
　１Ｈ－ＮＭＲスペクトルの２．０ｐｐｍ付近に現れるヒアルロン酸のＮ－アセチル基（ＣＨ３）のピークと、３．８ｐｐｍ以上４．２ｐｐｍ以下の範囲に現れるカルボキシメチル基のメチレン基（－ＣＨ２―）のピークを積分した。積分値から下記の式より、カルボキシメチル化率（ＣＭ化率）を求めた。
　ＣＭ化率＝(３．８ｐｐｍ以上４．２ｐｐｍ以下の範囲に現れるピークの積分値／２)／(２．０ＰＰＭのピークの積分値／３)
　 (Sample preparation)
7 mg of a 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 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 carboxymethylation rate (CM conversion rate) was determined from the following formula.
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)
　＜実施例１：カルボキシメチル基含有修飾ヒアルロン酸の架橋物の調製＞
　１０ｃｍ×７ｃｍのポリエステル製チャック袋に、調製例１で得られた原料修飾ヒアルロン酸１．５ｇを添加し、さらに９０ｍｇ／ｍＬ　１－ヒドロキシベンゾトリアゾール水溶液（以下、「ＨＯＢｔ水溶液」ともいう）２．５４ｍＬおよびイオン交換水１．２１ｍＬを加えた。ここで、反応液における原料修飾ヒアルロン酸の濃度は４０質量％であった。この反応液を手で捏ねて２５℃で２時間保存し、膨潤溶解させた。
　次いで、この反応液に１－エチル－３－（３－ジメチルアミノプロピル) カルボジイミド塩酸塩（以下、「ＥＤＣ」ともいう）を２８８ｍｇ添加した。この反応液を手で捏ねて２５℃で１６時間保存し、膨潤溶解させることで、カルボキシメチル基含有修飾ヒアルロン酸の架橋物を含む組成物を得た。 <Example 1: Preparation of crosslinked product of carboxymethyl group-containing modified hyaluronic acid>
1. Add 1.5 g of the raw material-modified hyaluronic acid obtained in Preparation Example 1 to a 10 cm × 7 cm polyester chuck bag, and further add a 90 mg / mL 1-hydroxybenzotriazole aqueous solution (hereinafter also referred to as “HOBt aqueous solution”). 54 mL and 1.21 mL of ion exchange water were added. Here, the concentration of the raw material-modified hyaluronic acid in the reaction solution was 40% by mass. This reaction solution was kneaded by hand and stored at 25 ° C. for 2 hours to swell and dissolve.
Next, 288 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter also referred to as “EDC”) was added to the reaction solution. This reaction solution was kneaded by hand, stored at 25 ° C. for 16 hours, and swelled and dissolved to obtain a composition containing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid.
＜実施例１：カルボキシメチル基含有修飾ヒアルロン酸の架橋物の精製＞
　５００ｍＬビーカーに実施例１で得られた架橋物を含む組成物を移し、イオン交換水２５０ｍＬを加えた。ヒスコトロン（５０００ｒｐｍ×２分）で粉砕処理後、５分間撹拌、５分間静置し、架橋物を沈殿させて上澄みをデカンテーションにより除去した。
　次いで、イオン交換水２００ｍＬで２回、６０％エタノール２００ｍＬで１回、９０％エタノール１００ｍＬで１回洗浄した。その後、架橋物を４２０メッシュのナイロンストレイナーで吸引ろ過により回収した後、５５℃で４時間減圧乾燥し、粉末状のカルボキシメチル基含有修飾ヒアルロン酸の架橋物１．５４ｇを得た。 <Example 1: Purification of crosslinked product of carboxymethyl group-containing modified hyaluronic acid>
The composition containing the crosslinked product obtained in Example 1 was transferred to a 500 mL beaker, and 250 mL of ion-exchanged water was added. After grinding with Hiscotron (5000 rpm × 2 minutes), the mixture was stirred for 5 minutes and allowed to stand for 5 minutes to precipitate the cross-linked product and the supernatant was removed by decantation.
Subsequently, it was washed twice with 200 mL of ion-exchanged water, once with 200 mL of 60% ethanol, and once with 100 mL of 90% ethanol. Thereafter, the cross-linked product was collected by suction filtration with a 420 mesh nylon strainer and then dried under reduced pressure at 55 ° C. for 4 hours to obtain 1.54 g of a powdery carboxymethyl group-containing modified hyaluronic acid cross-linked product.
＜実施例２ないし１０、および比較例１ないし６＞
　表１記載の条件に変更した以外は、実施例１と同様の方法で、実施例２ないし１０、および比較例１ないし６の粉末状のカルボキシメチル基含有修飾ヒアルロン酸の架橋物を得た。 <Examples 2 to 10 and Comparative Examples 1 to 6>
Except having changed into the conditions described in Table 1, powdered carboxymethyl group-containing modified hyaluronic acid of Examples 2 to 10 and Comparative Examples 1 to 6 were obtained in the same manner as in Example 1.
＜比較例７＞
　２０ｍＬビーカーに未修飾ヒアルロン酸ナトリウム（平均分子量：２２２万）２．０ｇを添加し、さらに１０％ＨＯＢｔ／７０％エタノール溶液を１０ｍＬ、ＥＤＣを１４４ｍｇ添加した。この反応液を２５℃で終夜撹拌し、ヒアルロン酸の架橋物を含む組成物を得た。
　その後、実施例１と同様の精製処理を行い、比較例７の粉末状のヒアルロン酸の架橋物１．９２ｇを得た。 <Comparative Example 7>
To a 20 mL beaker, 2.0 g of unmodified sodium hyaluronate (average molecular weight: 2.22 million) was added, 10 mL of a 10% HOBt / 70% ethanol solution, and 144 mg of EDC were added. This reaction solution was stirred at 25 ° C. overnight to obtain a composition containing a crosslinked product of hyaluronic acid.
Then, the refinement | purification process similar to Example 1 was performed, and the powdery hyaluronic acid crosslinked material 1.92g of the comparative example 7 was obtained.
＜比較例８ないし１０＞
　表１記載の条件に変更した以外は、比較例７と同様の方法で、比較例８ないし１０の粉末状のヒアルロン酸の架橋物を得た。 <Comparative Examples 8 to 10>
Except for changing to the conditions described in Table 1, powdery hyaluronic acid crosslinked products of Comparative Examples 8 to 10 were obtained in the same manner as in Comparative Example 7.
　＜試験例１：熱安定性試験＞
　５０ｍＬバイアル瓶中に、実施例１ないし７、および比較例１ないし１０で得られたカルボキシメチル基含有修飾ヒアルロン酸の架橋物それぞれ１ｇ（乾燥質量換算）を、リン酸緩衝生理食塩水（ｐＨ７．４）５０ｍＬ中に分散させて、架橋物濃度が２質量％（固形分）の混合物を調製した。
　次いで、前記バイアルの口にゴム栓をはめて、さらにアルミニウム製の蓋をし、該混合物を１２１℃で２０分間オートクレーブ処理した。
　処理後の架橋物を４２０メッシュのナイロンストレイナーに載置し、蒸留水で洗浄後、吸引ろ過により回収し、秤量済みのシャーレ上に広げて５５℃で３時間減圧乾燥した。
　その後、シャーレの質量を測定して、シャーレの質量の変化分から残留物の質量を算出した。架橋物の質量および残留物の質量から、熱安定性試験におけるゲル残存率を上述の式（３）により算出した。
　なお、前記オートクレーブ処理としては、具体的には、開始温度２５℃で４０分間かけて１２１℃まで昇温させ、１２１℃で２０分間オートクレーブ処理し、加圧・加熱終了後１時間静置してから、該架橋物を取り出す工程をいう。 <Test Example 1: Thermal stability test>
In a 50 mL vial, 1 g of each crosslinked product of carboxymethyl group-containing modified hyaluronic acid obtained in Examples 1 to 7 and Comparative Examples 1 to 10 (in terms of dry mass) was added to phosphate buffered saline (pH 7. 4) The mixture was dispersed in 50 mL to prepare a mixture having a crosslinked product concentration of 2% by mass (solid content).
The vial mouth was then fitted with a rubber stopper, covered with an aluminum lid, and the mixture was autoclaved at 121 ° C. for 20 minutes.
The treated crosslinked product was placed on a 420 mesh nylon strainer, washed with distilled water, recovered by suction filtration, spread on a weighed petri dish, and dried under reduced pressure at 55 ° C. for 3 hours.
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 cross-linked product and the mass of the residue, the gel remaining rate in the thermal stability test was calculated by the above formula (3).
As the autoclave treatment, specifically, the temperature is raised to 121 ° C. over 40 minutes at a starting temperature of 25 ° C., autoclaved at 121 ° C. for 20 minutes, and allowed to stand for 1 hour after completion of pressurization and heating. The step of taking out the cross-linked product.
　＜試験例２：耐酵素分解性試験＞
　上述の試験例１で得られた架橋物それぞれ１００ｍｇ（乾燥質量換算）を、５０ｍＭリン酸緩衝液（ｐＨ６．０）９．５ｍＬに分散させて濃度１質量％（固形分）の混合物を調製し、２５℃にて１０分間静置して、膨潤溶解させた。
　次いで、該混合物にヒアルロニダーゼ（シグマ社、ｂｏｖｉｎｅ ｔｅｓｔｅｓ由来）０．２５単位（０．５ｍＬ）を添加した後、該混合物を４０℃にて１５時間静置した。さらに、ヒアルロニダーゼ０．２５単位（０．５ｍＬ）を追加し、４０℃にて２４時間静置した。
　この架橋物を４２０メッシュのナイロンストレイナーで吸引ろ過により回収した後、蒸留水で洗浄し、秤量済みのシャーレ上に広げて５５℃で３時間減圧乾燥した。
　その後、シャーレの質量を測定して、シャーレの質量の変化分から残留物の質量を算出した。耐酵素分解性試験におけるゲル残存率を上述の式（４）により算出した。
　なお、比較例１ないし７、および比較例１０で得られた架橋物は、試験例１の熱安定性試験においてゲルの残存率が０％であったため、試験例２の耐酵素分解性試験は行っていない。 <Test Example 2: Enzymatic degradation resistance test>
100 mg (in terms of dry mass) of each of the crosslinked products obtained in Test Example 1 described above was dispersed in 9.5 mL of 50 mM phosphate buffer (pH 6.0) to prepare a mixture having a concentration of 1% by mass (solid content). And allowed to stand at 25 ° C. for 10 minutes to swell and dissolve.
Subsequently, after adding 0.25 units (0.5 mL) of hyaluronidase (from Sigma, bovine tests) to the mixture, the mixture was allowed to stand at 40 ° C. for 15 hours. Further, 0.25 unit (0.5 mL) of hyaluronidase was added, and the mixture was allowed to stand at 40 ° C. for 24 hours.
The crosslinked product was recovered by suction filtration with a 420 mesh nylon strainer, washed with distilled water, spread on a weighed petri dish, and dried under reduced pressure at 55 ° C. for 3 hours.
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 gel remaining rate in the enzyme degradation resistance test was calculated by the above formula (4).
Since the crosslinked products obtained in Comparative Examples 1 to 7 and Comparative Example 10 had a gel residual ratio of 0% in the thermal stability test of Test Example 1, the enzyme degradation resistance test of Test Example 2 was not going.
＜膨潤度の測定＞
　実施例１ないし１０、および比較例１ないし１０で得られた架橋物について、以下の方法により膨潤度を算出した。
　具体的には、架橋物を膨潤させて、水膨潤ゲルを形成させた後、４２０メッシュのナイロンストレイナーで吸引ろ過により回収し、秤量済みのシャーレ上に広げて膨潤ゲルの質量を測定した。次いで、５５℃で３時間減圧乾燥し、乾燥ゲルの質量を測定した。
　膨潤ゲルの質量および乾燥ゲルの質量から、水膨潤ゲルにおける膨潤度を以下の式（５）により算出した。
　　膨潤度（質量比）＝膨潤ゲルの質量／架橋物の質量（固形分）・・・（５）
  <Measurement of swelling degree>
The degree of swelling of the crosslinked products obtained in Examples 1 to 10 and Comparative Examples 1 to 10 was calculated by the following method.
Specifically, after the crosslinked product was swollen to form a water-swollen gel, it was collected by suction filtration with a 420 mesh nylon strainer, spread on a weighed petri dish, and the mass of the swollen gel was measured. Subsequently, it dried under reduced pressure at 55 degreeC for 3 hours, and measured the mass of the dried gel.
From the weight of the swollen gel and the weight of the dried gel, the degree of swelling in the water swollen gel was calculated by the following equation (5).
Swelling degree (mass ratio) = mass of swelling gel / mass of crosslinked product (solid content) (5)
＜貯蔵弾性率Ｇ’の測定＞
　実施例１ないし１０、および比較例１ないし１０で得られた架橋物について、以下の測定条件にて貯蔵弾性率Ｇ’を測定した。 <Measurement of storage elastic modulus G '>
With respect to the crosslinked products obtained in Examples 1 to 10 and Comparative Examples 1 to 10, the storage elastic modulus G ′ was measured under the following measurement conditions.
（測定条件）
測定装置：ＡＲ－Ｇ２（ティー・エー・インスツルメント・ジャパン（株）製）
ギャップ：８００μｍ
測定モード：ｆｒｅｑｕｅｎｃｙ ｓｗｅｅｐ ｓｔｅｐ
測定温度：２５℃　　
振幅周波数：０．１～１０Ｈｚ
  (Measurement condition)
Measuring device: AR-G2 (manufactured by TA Instruments Japan Co., Ltd.)
Gap: 800μm
Measurement mode: frequency sweep step
Measurement temperature: 25 ° C
Amplitude frequency: 0.1 to 10 Hz
Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000003
  
　表１より、縮合剤を用いて、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を、前記修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合によって架橋させる、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法によって得られた架橋物は、熱安定性および耐酵素分解性に優れていることが理解できる（実施例１ないし１０）。 From Table 1, a carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is cross-linked by an ester bond between a hydroxyl group and a carboxyl group of the modified hyaluronic acid and / or salt thereof using a condensing agent. It can be understood that the cross-linked product obtained by the method for producing a cross-linked product of methyl group-containing modified hyaluronic acid and / or a salt thereof is excellent in thermal stability and resistance to enzymatic degradation (Examples 1 to 10).
　具体的には、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物は、試験例１の熱安定性試験におけるゲル残存率が１０％以上であり、優れた熱安定性を有することが理解できる（実施例１ないし１０）。 Specifically, the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has a gel residual ratio of 10% or more in the thermal stability test of Test Example 1 and has excellent thermal stability. Can be understood (Examples 1 to 10).
　また、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物は、試験例２の耐酵素分解性試験におけるゲル残存率が６０％以上であり、優れた耐酵素分解性を有することが理解できる（実施例１および２、実施例４ないし７、ならびに実施例９および１０）。 Further, it is understood that the crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof has a gel residual ratio of 60% or more in the enzyme degradation resistance test of Test Example 2 and has excellent enzyme degradation resistance. (Examples 1 and 2, Examples 4-7, and Examples 9 and 10).
　さらに、カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物は、水膨潤性ゲルを形成する性質を有し、膨潤度が５倍以上２５０倍以下（質量比）であることが理解できる（実施例１ないし１０）。 Furthermore, it can be understood that a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof has a property of forming a water-swellable gel and has a swelling degree of 5 to 250 times (mass ratio). (Examples 1 to 10).
　＜配合例１：美容液＞
　本配合例では、以下に記す処方にて、実施例４で得られたカルボキシメチル基含有修飾ヒアルロン酸の架橋物を配合した美容液を調製した。
　　カルボキシメチル基含有修飾ヒアルロン酸の架橋物　　　　０．８％
　　ヒアルロン酸ナトリウム（平均分子量：２００万）　　　　０．２％
　　加水分解ヒアルロン酸（平均分子量：８０万）　　　　　　０．１％
　　カチオン化ヒアルロン酸　　　　　　　　　　　　　　　　０．１％
　　加水分解ヒアルロン酸アルキル（C12‐C13）グリセリル　　０．１％
　　１，３－ブチレングリコール　　　　　　　　　　　　　　５．０％
　　カルボキシビニルポリマー　　　　　　　　　　　　　　　５．０％
　　エチルヘキシルグリセリン　　　　　　　　　　　　　　　３．０％
　　カプリル酸グリセリン　　　　　　　　　　　　　　　　　３．０％
　　ペンチレングリコール　　　　　　　　　　　　　　　　　５．０％　　
　　グリセリン　　　　　　　　　　　　　　　　　　　　　　１．５％
　　ＰＯＥソルビタンモノステアリン酸エステル　　　　　　　１．０％
　　ソルビタンモノステアリン酸エステル　　　　　　　　　　０．５％
　　キサンタンガム　　　　　　　　　　　　　　　　　　　　０．２％
　　アルギン酸ナトリウム　　　　　　　　　　　　　　　　　０．２％
　　水酸化カリウム　　　　　　　　　　　　　　　　　　　　０．１％
　　オリーブ油　　　　　　　　　　　　　　　　　　　　　　０．２％
　　トコフェロール　　　　　　　　　　　　　　　　　　　　０．１％
　　ＥＤＴＡ－２ナトリウム　　　　　　　　　　　　　　　０．０２％
　　アルギニン　　　　　　　　　　　　　　　　　　　　　０．１５％
　　グリチルリチン酸ジカリウム　　　　　　　　　　　　　０．０５％
　　アルブチン　　　　　　　　　　　　　　　　　　　　　　０．２％
　　パルミチン酸レチノール　　　　　　　　　　　　　　　　０．２％
　　トラネキサム酸　　　　　　　　　　　　　　　　　　　　０．１％
　　エラスチン　　　　　　　　　　　　　　　　　　　　　　０．１％
　　コラーゲン　　　　　　　　　　　　　　　　　　　　　　０．１％
　　リン酸アスコルビン酸マグネシウム　　　　　　　　　　　０．１％
　　クエン酸ナトリウム　　　　　　　　　　　　　　　　　　１．０％
　　クエン酸　　　　　　　　　　　　　　　　　　　　　　　０．１％
　　プロピルパラベン　　　　　　　　　　　　　　　　　　　０．１％
　　メチルパラベン　　　　　　　　　　　　　　　　　　　　０．１５％
　　香料　　　　　　　　　　　　　　　　　　　　　　　　　　　適量
　　精製水　　　　　　　　　　　　　　　　　　　　　　　　　　残量
　　 <Combination Example 1: Essence>
In this blending example, a beauty essence was prepared by blending the carboxymethyl group-containing modified hyaluronic acid cross-linked product obtained in Example 4 with the formulation described below.
Cross-linked product of modified hyaluronic acid containing carboxymethyl group 0.8%
Sodium hyaluronate (average molecular weight: 2 million) 0.2%
Hydrolyzed hyaluronic acid (average molecular weight: 800,000) 0.1%
Cationized hyaluronic acid 0.1%
Hydrolyzed alkyl hyaluronate (C12-C13) glyceryl 0.1%
1,3-butylene glycol 5.0%
Carboxyvinyl polymer 5.0%
Ethylhexylglycerin 3.0%
Glycerol caprylate 3.0%
Pentylene glycol 5.0%
Glycerin 1.5%
POE sorbitan monostearate 1.0%
Sorbitan monostearate 0.5%
Xanthan gum 0.2%
Sodium alginate 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%
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
　＜実施例１１：癒着防止剤＞
　実施例３で得られた架橋物を厚さ１ｍｍのフィルム状に圧延して成型し、滅菌処理後、シート状の癒着防止剤を得た。 <Example 11: Anti-adhesion agent>
The cross-linked product obtained in Example 3 was rolled into a 1 mm thick film and molded. After sterilization, a sheet-shaped adhesion inhibitor was obtained.
　＜実施例１２：皮下注射剤＞
　実施例７で得られた架橋物（固形分換算１％）の乾燥物を、０．９％ＮａＣｌを含む注射用水で膨潤させ、１ｍＬの注射器に無菌充填し、滅菌処理後、皮下注射剤を得た。 <Example 12: Subcutaneous injection>
The dried product of the crosslinked product obtained in Example 7 (1% in terms of solid content) was swollen with water for injection containing 0.9% NaCl, and aseptically filled into a 1 mL syringe. After sterilization, a subcutaneous injection was used. Obtained.
　＜実施例１３：薬物徐放剤＞
　実施例５で得られた架橋物（固形物換算２％）の乾燥物を、０．９％ＮａＣｌ、０．００１％プロスタグランジンＥ１を含む注射用水で膨潤させ、滅菌処理後、３ｍＬの注射器に無菌充填し、薬物徐放剤を得た。 <Example 13: Drug sustained-release agent>
The dried product of the cross-linked product obtained in Example 5 (2% in terms of solid) was swollen with water for injection containing 0.9% NaCl and 0.001% prostaglandin E1, and after sterilization, a 3 mL syringe. Aseptic filling was carried out to obtain a sustained-release drug.
　＜実施例１４：膝関節注射剤＞
　実施例６で得られた架橋物（固形物換算０．８％）の乾燥物を、０．９％ＮａＣｌを含む注射用水で膨潤させ、滅菌処理後、２ｍＬの注射器に無菌充填し、膝関節注射剤を得た。
  <Example 14: knee joint injection>
The dried product of the cross-linked product obtained in Example 6 (0.8% in terms of solids) was swollen with water for injection containing 0.9% NaCl, sterilized, and then aseptically filled into a 2 mL syringe. An injection was obtained.

Claims (12)

  1. 縮合剤を用いて、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩を、前記修飾ヒアルロン酸および／またはその塩が有する水酸基とカルボキシル基との間でエステル結合によって架橋させる、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     Using a condensing agent
    Carboxymethyl group-containing modified hyaluronic acid and / or salt thereof is crosslinked by an ester bond between the hydroxyl group and carboxyl group of the modified hyaluronic acid and / or salt thereof,
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  2. 請求項１記載の架橋物の製造方法において、
    前記縮合剤が、カルボジイミド系縮合剤、またはトリアジン系縮合剤から選ばれる少なくとも１種である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material of Claim 1,
    The condensing agent is at least one selected from a carbodiimide condensing agent or a triazine condensing agent.
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  3. 請求項１または２記載の架橋物の製造方法において、
    前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩１，０００質量部に対して前記縮合剤の割合が５質量部以上４００質量部以下である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material of Claim 1 or 2,
    The ratio of the condensing agent is 5 parts by mass or more and 400 parts by mass or less with respect to 1,000 parts by mass of the carboxymethyl group-containing modified hyaluronic acid and / or its salt.
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  4. 請求項１ないし３のいずれかに記載の架橋物の製造方法において、
    前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の１質量％以上８０質量％以下の溶液に縮合剤を添加する、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material in any one of Claim 1 thru | or 3,
    A condensing agent is added to a solution of 1% by mass to 80% by mass of the carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof,
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  5. 請求項１ないし４のいずれかに記載の架橋物の製造方法において、
    前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の平均分子量が４，０００以上４００万以下である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material in any one of Claims 1 thru | or 4,
    The carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has an average molecular weight of 4,000 to 4,000,000.
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  6. 請求項１ないし５のいずれかに記載の架橋物の製造方法において、
    前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩が、ヒアルロン酸を構成する２糖単位に対するカルボキシメチル化率が１０％以上２００％以下である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material in any one of Claim 1 thru | or 5,
    The carboxymethyl group-containing modified hyaluronic acid and / or salt thereof has a carboxymethylation rate of 10% to 200% with respect to a disaccharide unit constituting hyaluronic acid,
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  7. 請求項１ないし６のいずれかに記載の架橋物の製造方法において、
    前記カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の製造方法は、
    温度が３０℃以下の含水溶媒中で、溶解した原料ヒアルロン酸および／またはその塩をハロ酢酸および／またはその塩と反応させる工程を含み、
    前記含水溶媒は、水、または水溶性有機溶媒と水との混合液であり、
    前記混合液における水溶性有機溶媒の割合が６０ｖ／ｖ％以下である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物の製造方法。     In the manufacturing method of the crosslinked material in any one of Claim 1 thru | or 6,
    The method for producing the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof,
    Reacting the dissolved raw material hyaluronic acid and / or salt thereof with haloacetic acid and / or salt thereof in a hydrous solvent having a temperature of 30 ° C. or lower,
    The water-containing solvent is water or a mixed solution of water-soluble organic solvent and water,
    The ratio of the water-soluble organic solvent in the mixed solution is 60 v / v% or less,
    A method for producing a crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  8. 同一または異なるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩同士をエステル結合により架橋させた架橋物であって、
    前記架橋物は水膨潤性を有し、膨潤度が５倍以上２５０倍以下（質量比）である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物。     A crosslinked product obtained by crosslinking the same or different carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof with an ester bond,
    The crosslinked product has water swellability, and the degree of swelling is 5 to 250 times (mass ratio).
    A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  9. 請求項８記載の架橋物において、
    熱安定性試験におけるゲル残存率が１０質量％以上である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物。     The cross-linked product according to claim 8,
    The gel remaining rate in the thermal stability test is 10% by mass or more,
    A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  10. 請求項８記載の架橋物において、
    耐酵素分解性試験におけるゲル残存率が６０％以上である、
    カルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物。     The cross-linked product according to claim 8,
    The gel remaining rate in the enzyme degradation resistance test is 60% or more,
    A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof.
  11. 請求項１ないし７のいずれかに記載の製造方法により得られるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
    または請求項８ないし１０のいずれかに記載のカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物を含む、
    関節注射剤、癒着防止剤、皮下注射剤、または薬物徐放剤。     A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof obtained by the production method according to claim 1,
    Or a crosslinked product of the carboxymethyl group-containing modified hyaluronic acid and / or salt thereof according to any one of claims 8 to 10.
    Joint injection, anti-adhesion agent, subcutaneous injection, or sustained drug release agent.
  12. 請求項１ないし７のいずれかに記載の製造方法により得られるカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物、
    または請求項８ないし１０のいずれかに記載のカルボキシメチル基含有修飾ヒアルロン酸および／またはその塩の架橋物を含む、化粧料。
          A crosslinked product of carboxymethyl group-containing modified hyaluronic acid and / or a salt thereof obtained by the production method according to claim 1,
    Or cosmetics containing the crosslinked product of the carboxymethyl group containing modified hyaluronic acid and / or its salt in any one of Claims 8 thru | or 10.
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