N-alkanoylated hyaluronic acid or its salt, and its production method

【0001】

[Technical field to which the invention belongs]

The present invention relates to n-alkanoylated hyaluronic acid or a salt thereof, and a method for producing the same. More specifically, the present invention relates to n-alkanoylated hyaluronic acid or a salt thereof, which has both a hydrophobic part and a hydrophilic part and is useful as a new material in the fields of medical products, foods, cosmetics, etc., and a method for producing the same [0002].

[Conventional technology]

Hyaluronic acid is a linear high molecular weight polysaccharide formed by alternating N-acetyl-D-glucosamine and D-glucuronic acid, and has a large number of free carboxyl groups and a large number of free hydroxyl groups, so it has an affinity for water. It is rich in properties and dissolves in any amount of water to form a highly viscous aqueous solution. It is expected that hyaluronic acid having different physical characteristics can be obtained by blocking the free carboxyl group and the free hydroxyl group. Therefore, hyaluronic acid or a salt thereof (hereinafter, may be referred to as "hyaluronic acid (salt)") is chemically modified for the purpose of using it in the fields of medical products, foods, cosmetics, etc. (for example, , See Patent Documents 1 and 2.). However, all of the conventional reactions are reactions in a system in which water molecules are present, and the organic reactions applicable to the chemical modification of hyaluronic acid (salt) are limited. For this reason, it has been extremely difficult to produce a new type of hyaluronic acid (salt) having both hydrophobic and hydrophilic parts, which is a new material in the fields of medical products, foods, and cosmetics.

【0003】

[Patent Document 1]

Japanese Unexamined Patent Publication No. 7-309902 [Patent Document 2]

Japanese Unexamined Patent Publication No. 8-53501 [0004]

[Problems to be Solved by the Invention]

The present inventors have diligently studied a new type of hyaluronic acid (salt) having both hydrophobic and hydrophilic sites, which is useful as a new material in the fields of medical products, foods, cosmetics, and the like. As a result, the complex of hyaluronic acid (salt) and the cationic compound was dissolved in a non-aqueous solvent, and then the complex was reacted with an acid halide having an n-alkanoyl group to create a hydrophobic region and a hydrophilic region. We have found that a new type of hyaluronic acid (salt) can be obtained together, and completed the present invention based on this finding. As is clear from the above description, an object of the present invention is a new type of hyaluronic acid (salt) having both a hydrophobic part and a hydrophilic part, which is useful as a new material in the fields of medical products, foods, cosmetics, etc., especially non-aqueous type. Since it is manufactured in, there is little contamination of pyrogens and antigenic substances, and since it is metabolized in the body, especially in the pharmaceutical field, for example, moisturizers, lubricants, wound dressings, and DDS (drugs) during surgery. Delivery system) To provide hyaluronic acid (salt) that can be used as a material and a method for producing the same.

【0005】

[Means for solving problems]

The present invention is shown by the following (1) to (10). (1) n-alkanoylated hyaluronic acid represented by the following general formula (1) or a salt thereof.<img file="JP2004123785A_D0001.tif" />(In the formula, R₁, R₂, R₃, And R₄Are hydrogen or linear C, respectively₅~ C₁₅Represents the n-alkanoyl group of R₅Represents hydrogen, a metal cation, or a cationic compound, and n represents an integer from 2 to 7,500. ).

【0006】

(2) The n-alkanoylated hyaluronic acid or a salt thereof according to (1) above, wherein the n-alkanoyl group is an n-alkanoyl group represented by the following general formula (2). CH₃-(CH₂) m-CO- (2) (In the equation, m represents an integer from 4 to 10).

【0007】

(3) The n-alkanoylated hyaluronic acid or a salt thereof according to (1) above, wherein the cationic compound is a quaternary ammonium salt.

【0008】

(4) The n-alkanoylated hyaluronic acid or a salt thereof according to (3) above, wherein the quaternary ammonium salt is a distearyldimethylammonium salt.

【0009】

(5) n-alkanoylated hyaluronic acid represented by the following general formula (3), which comprises reacting a complex of hyaluronic acid or a salt thereof with a cationic compound with a halogenated n-alkanoyl in a non-aqueous solvent. A method for producing an acid or a salt thereof.<img file="JP2004123785A_D0002.tif" />(In the formula, R₁, R₂, R₃, And R₄Are hydrogen or C, respectively₄~ C₁₈Represents the n-alkanoyl group of R₅Represents hydrogen, a metal cation, or a cationic compound, and n represents an integer from 2 to 7,500. ).

【0010】

(6) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to (5) above, wherein the halogenated n-alkanoyl is a halogenated n-alkanoyl represented by the following general formula (4). CH₃-(CH₂) M-CO-A (4) (In the equation, m represents an integer of 3 to 15 and A represents a halogen.).

【0011】

(7) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to (5) above, wherein the halogenated n-alkanoyl is a halogenated n-alkanoyl represented by the following general formula (5). CH₃-(CH₂) M-CO-A (5) (In the equation, m is an integer of 4 to 10 and A represents halogen).

【0012】

(8) The n-alkanoylated hyaluronic acid or a salt thereof according to (5) above, wherein the non-aqueous solvent is one or more solvents selected from DMF, ethanol, methanol, acetone, chloroform, toluene, methylene chloride or heptane. Production method.

【0013】

(9) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to (5) above, wherein the cationic compound is a quaternary ammonium salt.

【0014】

(10) The method for producing n-alkanoylated hyaluronic acid or a salt thereof according to (9) above, wherein the quaternary ammonium salt is a distearyldimethylammonium salt.

【0015】

BEST MODE FOR CARRYING OUT THE INVENTION

The hyaluronic acid (salt) used in the present invention is not particularly limited, and may be derived from various animal tissues such as chicken combs or from microorganisms capable of producing hyaluronic acid (salt). , The hyaluronic acid (salt) used in the present invention is preferably derived from a microorganism. Examples of microorganisms capable of producing hyaluronic acid include Streptococcus pyogenes, Streptococcus equisimilis, Streptococcus equi, Streptococcus equi, Streptococcus epic, Streptococcus dysgalactoccus zooepidemicus) and so on.

【0016】

For example, 20 to 30 g of glucose, 5 g of yeast extract, 3 g of potassium phosphate, 2 g of 2 potassium phosphate, 0.1 g of sodium thiosulfate, 0.1 g of magnesium sulfate 7 culture medium, 0.02 g of sodium sulfite, are produced by microorganisms. Add the prepared inoculum to a medium containing 0.01 g of cobalt chloride, 0.01 g of manganese chloride, and 5 g of foaming agent, pH 6.0 to 8.5, preferably 7.0, culture temperature 25 ° C to 40 ° C, Preferably, the cells are shake-cultured or aerated-cultured at 30 ° C to 35 ° C for 1 to 3 days to generate and accumulate hyaluronic acid (salt) in the culture medium, and the culture medium is centrifuged or filtered to obtain bacterial cells. After removal, the low molecular weight substance is removed by ultrafiltration or dialysis of the filtrate, sodium chloride is dissolved in the filtrate from which the low molecular weight substance has been removed, and then a water-soluble organic solvent is added to precipitate sodium hyaluronate. Then, the precipitate is separated and recovered by centrifugation, and then vacuum-dried. In addition, commercially available reagent-grade hyaluronic acid (salt) manufactured by Wako Junyaku, Nacalai Tesque, Tokyo Kasei, Seikagaku, or Sigma may be used.

【0017】

Further, the hyaluronic acid (salt) used as a raw material in the present invention preferably has an average molecular weight measured by an HPLC method of at least 5,000 to 1,500,000. For the measurement of the average molecular weight by the HPLC, any column suitable for the measurement of the molecular weight of the polysaccharide can be used, and it is preferable to use a column such as Shodex Ionpak KS806 and Ionpak KS-G. In this case, a 0.2 mol / L sodium chloride aqueous solution is used as the eluate, and the solution is flown at a flow rate of 1.0 ml / min, and hyaluronic acid (salt) can be detected at 206 nm. The average molecular weight can be calculated by using a calibration curve prepared with sodium hyaluronate having a known molecular weight, which is determined by the ultimate viscosity.

【0018】

Examples of the cation include alkali metal ion, potassium ion, sodium ion and the like, and sodium ion is preferable in the present invention.

【0019】

Specific examples of the cationic compound used in the present invention include a quaternary ammonium salt, an amino acid having two or more amino groups, a peptide, a polyamino acid salt, and a sugar salt having an amino group. , Preferably a quaternary ammonium salt.

【0020】

The quaternary ammonium salt preferably has at least one alkyl group having 8 or more carbon atoms. Specifically, Distearyldimethylammonium Chloride, Dioleyldimethylammonium Chloride, Cetylpyridinium Chloride, Cetyltrimethylammonium Chloride, Cetyltrimethylammonium Chloride, Cetyltrimethylammonium Chloride, Cetyltrimethylammonium Chloride. , Ditetradecyldimethylammonium Bromide, Didodecyldimethylammonium Bromide, Didecyldimetylammonium Bromide), Octadecyltrimethylammonium Chloride, Normal Octadecyltrimethylammonium Bromide, Tridodecylmethylammonium Chloride, Trioctylmethylammonium bromide, Trioctylmethylammonium phosphatidyl Dioctanoyl L-α-Phosphatidylcholine, dilauroyl, L-alphaphosphatidylcholine, dipalumitoyl D, L-alphaphosphatidylcholine (Dipalmitoyl D, L-α-Phosphatidylcholine) , 1,2-Dimyristoyl-3-Trimethylammonium Propane), 1,2-Dioleoyl-3-Trimethylammonium Propane, 1,2-Dioleoyl-3-Trimethylammonium Propane ), 1,2-Distearoyl-3-Trimethylammonium Propane, Benzalkonium Chloride, Benzethonium Chloride and the like.

【0021】

The complex of hyaluronic acid (salt) and a cationic compound refers to an ionic bond between an anionic moiety (carboxyl group) of hyaluronic acid and a cationic compound, and an example of a preparation method thereof is shown below.

【0022】

&lt;Preparation of complex of hyaluronic acid (salt) and cationic compound&gt; (A) Hyaluronic acid (salt) in distilled water or equivalent pure water at a concentration of 0.01 to 10% by weight, preferably 0.05 to 1 Dissolve to a concentration of% by weight. In the present invention, the pure water corresponding to distilled water means, for example, water purified by continuous ion exchange (Electric Deionization), reverse osmosis (Reverse Osmosis), or the like. (B) On the other hand, a cationic compound that forms a complex with hyaluronic acid (salt), preferably a quaternary ammonium salt, is added to distilled water or pure water and uniformly dispersed.

【0023】

The molar ratio of the cationic group in the aqueous solution of the cationic compound prepared in (B) to the carboxyl group in the aqueous solution of hyaluronic acid (salt) prepared in (A) is 0.5 to 5: 1, preferably 0.7 to. Mix the two aqueous solutions 1.5: 1, eg 1: 1.

【0024】

The temperature at the time of mixing may be room temperature, but preferably, both liquids are heated to a temperature equal to or higher than the gel-liquid crystal transition temperature of the cationic compound to be used.

【0025】

The water-insoluble matter generated by mixing is recovered from the mixed solution by a separation method usually used in chemical experiments, such as centrifugation, suction filtration, and pressure filtration. The recovered water-insoluble matter is washed with distilled water or pure water heated to a gel-liquid crystal transition temperature or higher of the cationic compound, and then subjected to drying. Drying can be carried out by drying means usually used in chemical experiments, for example, atmospheric drying, vacuum drying, freeze drying and the like.

【0026】

The non-aqueous solvent that can be used in the present invention is, for example, DMF, ethanol, methanol, acetone, chloroform, methylene chloride, toluene, heptane, propylene glycol, polyethylene glycol, DMSO, THF, etc., or two or more of them. Mixed solvents are included. DMF, ethanol, methanol, acetone, chloroform, methylene chloride, toluene or heptane, or a mixed solvent thereof is preferable.

【0027】

The concentration of the complex of hyaluronic acid (salt) and the cationic compound dissolved in a non-aqueous solvent is not particularly limited, but is preferably in the range of 1 to 1000 mmol / L.

【0028】

The O-acyllation reaction (n-alkanoylation reaction) performed after the complex is dissolved in a non-aqueous solvent is an O-acyllation method (Schotten) using a halogenated n-alkanoyl in the presence of a basic solvent such as pyridine. -Baumann method) and so on. Here, as the halogenated n-alkanoyl, specifically, n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoyl chloride, n-dodecanoyl chloride, n-stearoyl chloride, etc. Can be mentioned, C₄~ C₁₈, Preferably C₅~ C₁₇, More preferably C₆~ C₁₂Halogenated n-alkanoyl.

【0029】

For example, when the O-acyllation reaction is n-dodecanoylation, that is, the production of n-dodecanoylated hyaluronic acid (salt), the cationic compound complex of hyaluronic acid (salt) obtained as described above is subjected to a nitrogen atmosphere. Below, dissolved in DMF, pyridine and n-dodecanoyl chloride are added and stirred, sodium acetate saturated ethanol is added to the reaction solution, and the precipitated precipitate is washed with acetone / water (9/1), vacuum dried, and n. -Dodecanoylated hyaluronic acid (salt) can be obtained.

【0030】

The use of the n-alkanoylated hyaluronic acid (salt) of the present invention is not particularly limited, but it can be used as various materials used in the fields of pharmaceuticals, foods, cosmetics and the like. In particular, the n-alkanoylated hyaluronic acid (salt) of the present invention is produced in a non-aqueous system, so that it is less likely to be contaminated with pyrogens and antigenic substances, and is metabolized in the body. It can be used as a moisturizer, a lubricant, a wound dressing, and a DDS (drug delivery system) material. Above all, since the decomposition rate in the uterus is expected to show a high correlation with the biorhythm in the human body (uterus), for example, an intrauterine or vaginal implant preparation carrying a therapeutic agent for endometriosis. Extremely effective as a device.

【0031】

[Example]

Hereinafter, the present invention will be described in detail with reference to Examples. Examples of production of various n-alkanoylated hyaluronic acid (salts) are shown below. Example 11. Production of n-butyrylated hyaluronic acid (salt) 1-1. Preparation of hyaluronic acid and distearyldimethylammonium chloride complex Sodium hyaluronate (Chiso CHA, average molecular weight determined from extreme viscosity 50,000, hereinafter, " 1.8 g (described as CHA) was dissolved in 300 ml of pure water, and 2.47 g of distearyldimethylammonium chloride (hereinafter abbreviated as DSC) was turbid in 560 ml of pure water. Both solutions were heated to 45 ° C and mixed with stirring, and stirring was continued for 5 minutes. The generated complex was recovered by centrifugation (5000 rpm, room temperature) and washed with warm water at 45 ° C. After the washing was completed, the mixture was freeze-dried overnight and then vacuum dried at 50 ° C. to obtain a hyaluronic acid and distearyldimethylammonium chloride complex (hereinafter abbreviated as CHA-DSC). Yield 3.3g, yield 85%.

【0032】

1-2. Synthesis of n-butyrylated hyaluronic acid 464 mg of the obtained CHA-DSC was stirred and dissolved in 30 ml of DMF under a nitrogen atmosphere, and the amount of n-butyryl chloride and pyridine required for the desired DS was added at 60 ° C. Stirred for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and DSC was removed. This was washed 5 times with a solution of acetone / water = 9: 1 and then vacuum dried at 60 ° C. to obtain n-butyrylated hyaluronic acid as a white solid.

【0033】

Example 2 Synthesis of n-hexanoylated hyaluronic acid CHA-DSC 464 mg obtained according to 1-1. Of Example 1 was stirred and dissolved in 30 ml of DMF under a nitrogen atmosphere, and the amount of n-hexanoyl required for the target DS was dissolved. Chloride and pyridine were added, and the mixture was stirred at 60 ° C. for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and DSC was removed. This was washed 5 times with a solution of acetone / water = 9: 1 and then vacuum dried at 60 ° C. to obtain n-hexanoylated hyaluronic acid as a white solid.

【0034】

Example 3 Synthesis of n-octanoylated hyaluronic acid CHA-DSC 464 mg obtained according to 1-1. Of Example 1 was stirred and dissolved in 30 ml of DMF under a nitrogen atmosphere, and 16.3 mg of n-octanoyl chloride and 30 ml of pyridine were added. The mixture was stirred at 60 ° C for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and DSC was removed. This was washed 5 times with a solution of acetone / water = 9: 1 and then vacuum dried at 60 ° C. to obtain n-octanoylated hyaluronic acid as a white solid.

【0035】

Example 4 Synthesis of n-decanoylated hyaluronic acid CHA-DSC 464 mg obtained according to 1-1. Of Example 1 was stirred and dissolved in 30 ml of DMF under a nitrogen atmosphere, and 19.1 mg of n-decanoyl chloride and 30 ml of pyridine were added. The mixture was stirred at 60 ° C for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and DSC was removed. This was washed 5 times with a solution of acetone / water = 9: 1 and then vacuum dried at 60 ° C. to obtain n-decanoylated hyaluronic acid as a white solid.

【0036】

Example 5 Synthesis of n-dodecanoylated hyaluronic acid 464 mg of CHA-DSC obtained according to 1-1. Of Example 1 was stirred and dissolved in 30 ml of DMF under a nitrogen atmosphere, and 21.9 mg of n-dodecanoyl chloride and 30 ml of pyridine were added. The mixture was stirred at 60 ° C for 2 hours. After completion of the reaction, a saturated ethanol solution of sodium acetate (60 ml) was added to precipitate a gel and DSC was removed. This was washed 5 times with a solution of acetone / water = 9: 1 and then vacuum dried at 60 ° C. to obtain n-dodecanoylated hyaluronic acid as a white solid.

【0037】

Example 6 Synthesis of n-stearoylated hyaluronic acid CHA-DSC 464 mg obtained according to 1-1. Of Example 1 was dissolved in 30 ml of DMF at 60 ° C under a nitrogen atmosphere, and pyridine was required for the desired DS. A large amount of stearoyl chloride was added and stirred for 2 hr. 60 ml of saturated ethanol sodium acetate was added to the reaction solution, and the precipitated precipitate was washed 5 times with acetone / water (9/1). This was vacuum dried to obtain n-stearoylated hyaluronic acid as a white solid. The DS here refers to the ratio of alkanoyl groups introduced into the four hydroxyl groups present per theoretical hyaluronic acid disaccharide unit when the molar ratio of HA-DSC and acid halide under each reaction condition is used. Represent.

【0038】

Example 7 Evaluation of solubility and gelation of each n-alkanolated hyaluronic acid obtained in Examples 1 to 6 After adding 5 mg of each n-alkanolated hyaluronic acid to 1 ml of each solvent and homogenizing with a test tube mixer. , The state was left for one day at room temperature, the state was observed, and the results are shown in Tables 1 to 3. In each table, the expression of solution characteristics means the following states: Soluble: A state in which it is completely dissolved in a solvent and does not form a gel, etc. Insoluble: A state in which a white solid remains insoluble in a solvent SG (sol-gel): Solvent A state in which a fine gel is formed Non-uniform G (non-uniform gel): Gel that is a gel but lacks uniformity G (gel): Uniform gel [0039]

Table 1 Dissolving properties of various n-alkanoylated hyaluronic acids<img file="JP2004123785A_D0003.tif" /> 【0040】

Table 2 Dissolving properties of n-butyrylized and hexanoylated hyaluronic acid with different DS<img file="JP2004123785A_D0004.tif" /> 【0041】

Table 3 Dissolving properties of different n-stearoylated hyaluronic acid in DS<img file="JP2004123785A_D0005.tif" /> 【0042】

[Effect of the invention]

The n-alkanoylated hyaluronic acid or a salt thereof of the present invention is a new type of hyaluronic acid (salt) having both a hydrophobic region and a hydrophilic region, and is extremely useful as a new material in the fields of medical products, foods, cosmetics and the like. In particular, the n-alkanoylated hyaluronic acid (salt) of the present invention is produced in a non-aqueous system, so that it is less likely to be contaminated with pyrogens and antigenic substances, and is metabolized in the body. It can be used as a moisturizer, lubricant, wound dressing, and DDS (drug delivery system) material. Further, in the method for producing n-alkanoylated hyaluronic acid or a salt thereof of the present invention, a complex of hyaluronic acid (salt) and a cationic compound is dissolved in a non-aqueous solvent, and then the complex is subjected to an n-alkanoyl group. By reacting with the acid halide having, it is possible to easily produce hyaluronic acid (salt) which has both a hydrophobic part and a hydrophilic part and is useful as a new material in the fields of medical products, foods, cosmetics and the like.