JP2000044603A - Hyaluronic acid gel and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a hyaluronic acid gel having extremely slow dissolving rate in water, excellent in transparency and useful as a biocompatible material without using a chemical cross-linking agent and a chemical modifier by using a specific acidic aqueous solution of the hyaluronic acid. SOLUTION: This method for producing a hyaluronic acid gel hardly soluble in neutral aqueous solution comprises using an acidic aqueous solution of the hyaluronic acid having >=5 wt.% hyaluronic acid concentration and containing an acid component in the mols the same as or more than the mols of the hyaluronic acid. For example, the transparent single hyaluronic acid gel having <=50 wt.% dissolution rate per day in the neutral aqueous solution is obtained by aging the acidic aqueous solution at -10 to 30 deg.C before neutralization.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a hyaluronic acid gel and a method for producing the same.

[0002]

2. Description of the Related Art Hyaluronic acid is a linear high molecular polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately bonded. Hyaluronic acid is known to be distributed in connective tissues of mammals and also present in chicken crusts, capsular streptococci, and the like. In addition to chicken crest, umbilical cord and the like being used as an extraction material, purified products have also been prepared from streptococcal cultures. Naturally occurring hyaluronic acid is polydisperse in molecular weight, but has no species or organ specificity and is known to exhibit excellent biocompatibility even when implanted or injected into living organisms . Further, a variety of chemical modifications of hyaluronic acid have been proposed because of their disadvantages derived from the water solubility of hyaluronic acid itself when applied to living bodies, for example, the relatively short residence time in living organisms.

A typical example thereof is a highly swellable crosslinked hyaluronic acid gel obtained by using a bifunctional reagent such as divinylsulfone, bisepoxides or formaldehyde as a crosslinking agent. (U.S. Pat. No. 4,582,865, JP-B-6-37575, JP-A-7-97401, JP-A-60-13)
No. 0601).

Further, a method for chemically modifying hyaluronic acid utilizing the feature that the tetrabutylammonium salt of hyaluronic acid is dissolved in an organic solvent such as dimethyl sulfoxide has been disclosed (JP-A-3-105003). Also, a method in which a tetrabutylammonium salt of hyaluronic acid is added to triethylamine and 2-chloro-1-methylpyridinium iodide in dimethyl sulfoxide and reacted to form an ester bond between a carboxyl group and a hydroxyl group of hyaluronic acid. It has been disclosed (European Patent No. 0341745).
A1).

As a method for insolubilizing hyaluronic acid in water without using a chemical reagent that forms a covalent bond, hyaluronic acid and a polymer compound having an amino group or an imino group are combined with the carboxyl group of hyaluronic acid. And a method of preparing a hyaluronic acid polymer complex by binding an amino group or an imino group of a polymer compound as an ion complex (see JP-A-6-73103).

An aqueous solution of hyaluronic acid is acidified, for example, pH
It is known that when adjusted to a range of 2.0 to 2.7, a gel in a jelly-like state called a patty gel is formed, but when the pH is less than 2.0, a patty gel is not formed. This patty gel dissolves promptly when put into a neutral aqueous solution, and is different from the hyaluronic acid gel referred to in the present invention.

[0007] Further, an aqueous solution of hyaluronic acid is adjusted to pH 2.0.
A method for producing a hyaluronic acid gel characterized by being placed in the presence of a water-soluble organic solvent of from 20 to 80% by weight (see JP-A-5-58881). However, the hyaluronic acid gel obtained by this production method is
It is described in the publication that when coating is not performed, the substance is dissolved when put into water.

[0008]

SUMMARY OF THE INVENTION In order to make the most of the excellent biocompatibility inherent in hyaluronic acid itself, there is no need to use any chemical cross-linking agent or chemical modifying agent, and it is not necessary to use a cationic cross-linking agent. A hyaluronic acid gel that has a very low dissolution rate in water without forming a complex with the above polymer is useful. The present inventors have intensively studied the physicochemical properties of hyaluronic acid itself, and found that the hyaluronic acid concentration was 5%.
It has been found that the above-mentioned hyaluronic acid gel is formed from an aqueous solution of hyaluronic acid in an amount of not less than% by weight. Furthermore, they have found that a transparent hyaluronic acid gel is also formed.

[0009]

That is, the present invention provides (1)
A method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution, characterized by being formed from a hyaluronic acid acidic aqueous solution containing a carboxyl group of hyaluronic acid and an acid component in equimolar or more at a hyaluronic acid concentration of 5% by weight or more; 2) Forming by aging an aqueous hyaluronic acid solution having a hyaluronic acid concentration of 5% by weight or more and containing an acid component having an equimolar amount or more with the carboxyl group of hyaluronic acid at -10 ° C to 30 ° C before the neutralization treatment. (3) A method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution, comprising the steps of: (3) preparing an acidic aqueous solution containing hyaluronic acid and a carboxyl group of hyaluronic acid in an equimolar amount or more with a hyaluronic acid concentration of 5% by weight or more; It is formed from an acidic aqueous solution of hyaluronic acid obtained by mixing, and is poorly soluble in a neutral aqueous solution according to (1) or (2). Method for producing hyaluronic acid gel,
(4) It is formed from an aqueous solution of hyaluronic acid containing an acid component in an equimolar amount or more with a carboxyl group of hyaluronic acid at a concentration of 5% by weight or more of hyaluronic acid obtained by concentrating the aqueous solution of hyaluronic acid. ) Or (2) a method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution; (1) The method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution according to (1) or (2), which is formed from a hyaluronic acid acidic aqueous solution having a hyaluronic acid concentration of 5% by weight or more obtained by the addition.
(6) The solubility in a neutral aqueous solution at 25 ° C. per day is 50
% Or less, and is a gel formed of hyaluronic acid alone, which is characterized by being transparent.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The hyaluronic acid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissues or produced by fermentation. The strain used in the fermentation method is a microorganism having hyaluronic acid-producing ability, such as Streptococcus sp. Isolated from nature, or JP-A-63-1233.
No. 92, Streptococcus equi FM
-100 (Microbial Research Laboratories No. 9027), JP-A-2-2346
No. 89, Streptococcus equii FM
A mutant strain that stably produces hyaluronic acid with high yield, such as -300 (Microtechnological Laboratory No. 2319), is desirable. Those cultured and purified using the above mutant strains are used.

The gel is defined as "a polymer having a three-dimensional network structure insoluble in any solvent and a swollen body thereof" according to the New Edition of the Polymer Dictionary (Asakura Shoten, 1988). According to the Dictionary of Physical and Chemical Sciences (Iwanami Shoten, 4th Edition, 1987), it is defined as "sol (colloidal solution) solidified in a jelly-like form". The hyaluronic acid gel referred to in the present invention is characterized by being hardly soluble in a neutral aqueous solution.When this hyaluronic acid gel is poured into a neutral aqueous solution, it is significantly less soluble than non-gelled hyaluronic acid. Is shown. Poor solubility is defined by the gel dissolution rate in a neutral aqueous solution at 25 ° C. Here, the neutral aqueous solution is a physiological saline having a buffer capacity adjusted to pH7.

The hyaluronic acid gel referred to in the present invention is a polymer having a three-dimensional network structure and a swollen body thereof. The three-dimensional network structure is formed by a cross-linked structure of hyaluronic acid.

The hyaluronic acid gel referred to in the present invention can be decomposed and solubilized by treating the hyaluronic acid gel under the conditions of the accelerated acid hydrolysis reaction of hyaluronic acid. When the solubilized hyaluronic acid retains a crosslinked structure,
Hyaluronic acid having a branch point can be distinguished from linear hyaluronic acid in terms of polymer solution theory. It is known that hyaluronic acid itself is a linear polymer and does not have a branched structure (polysaccharide biochemistry 1 Chemistry, Kyoritsu Shuppan, 1969).

If the hyaluronic acid gel referred to in the present invention has a crosslinked structure which is stably present even under the accelerated acid hydrolysis conditions of hyaluronic acid, the solubilized hyaluronic acid has a branched structure confirmed by polymer solution theory. Be done

As a method for measuring the molecular weight and the degree of branching of the solubilized hyaluronic acid, a differential refractometer and a multi-angle laser light scattering detector (MALLS) are used as detectors in a gel permeation chromatogram (GPC). GPC-MA to use
There is an LLS method.

The hyaluronic acid gel obtained in the present invention is formed of hyaluronic acid alone. The term “hyaluronic acid alone” as used herein means that no chemical crosslinking agent or chemical modifier other than hyaluronic acid is used, and that it is not complexed with a cationic polymer.

The chemical crosslinking agent for hyaluronic acid is a polyvalent compound which forms a covalent bond by reacting with a carboxyl group, a hydroxyl group or an acetamido group of hyaluronic acid, and a polyvalent epoxy compound such as polyglycidyl ether, divinyl sulfone, Examples include formaldehyde, phosphorus oxychloride, a combination use of a carbodiimide compound and an amino acid ester, and a combination use of a carbodiimide compound and a dihydrazide compound. The reaction of hyaluronic acid with the chemical crosslinking agent forms a three-dimensional network.

A chemical modifier for hyaluronic acid is a compound which reacts with carboxyl, hydroxyl and acetamido groups of hyaluronic acid to form a covalent bond. A combination of acetic anhydride and concentrated sulfuric acid, trifluoroacetic anhydride and organic acid And alkyl iodide compounds. Hydrophobizes the hydrophilic groups of hyaluronic acid, reducing the water solubility of hyaluronic acid.

The cationic polymer that forms a complex with hyaluronic acid is a polymer that forms an ionic complex between the carboxyl group of hyaluronic acid and the amino group or imino group of the polymer compound, and includes chitosan, polylysine, Examples include polyvinyl pyridine, polyethylene imine, and polydimethylaminoethyl methacrylate.
Hyaluronic acid and the cationic polymer are insoluble in water by forming a complex.

On the other hand, a substance which is not directly related to the introduction of a crosslinked structure into hyaluronic acid or the insolubilization or insolubilization of hyaluronic acid can be added when forming the hyaluronic acid gel according to the present invention. Like hyaluronic acid, a material having excellent biocompatibility, for example, chondroitin sulfate, carboxymethylcellulose, or the like can be mixed and complexed to form a hyaluronic acid gel, and is not limited at all. When forming a hyaluronic acid gel, a pharmaceutically or physiologically active substance is added,
It can also form a hyaluronic acid gel containing these, and is not limited at all.

The molecular weight of the hyaluronic acid used in the present invention is preferably in the range of about 1 × 10 ⁵ to about 1 × 10 ⁷ daltons. In addition, as long as it has a molecular weight within the above range, a higher molecular weight compound and a low molecular weight compound obtained through hydrolysis treatment or the like can also be used. Incidentally, the hyaluronic acid referred to in the present invention is the alkali metal,
For example, it is used in a concept including salts of sodium, potassium and lithium.

The acidic aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more used in the present invention means an aqueous solution prepared so that the carboxyl group of hyaluronic acid is protonated at a sufficient ratio. The amount of the acid component used for preparing the acidic solution is appropriately determined according to various kinds of properties such as the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the hyaluronic acid concentration, and the strength of the generated gel. The amount of the acid component more than equimolar to the carboxyl group of hyaluronic acid is preferred. As the acid component, any acid can be used as long as it is stronger than hyaluronic acid. In order to reduce the amount of acid used, it is desirable to use a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid and the like.

The acidic aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more used in the present invention requires aging for promoting gelation before neutralizing the resulting hyaluronic acid gel. The temperature and time for this aging are determined as appropriate according to various kinds of properties such as the type of counter ion of the hyaluronic acid salt in the aqueous solution of the hyaluronic acid, the molecular weight of the hyaluronic acid, the hyaluronic acid concentration, and the strength of the generated gel. Is preferably carried out at -10 ° C or more and 30 ° C or less in order to prevent the aqueous solution of hyaluronic acid from freezing and to suppress the decomposition of hyaluronic acid by acid.

The method of preparing the aqueous hyaluronic acid solution having a hyaluronic acid concentration of 5% by weight or more used in the present invention is not limited.
Examples thereof include a method of concentrating a low-concentration aqueous solution of hyaluronic acid to a predetermined concentration, and a method of adding an acid component to a high-concentration aqueous solution of hyaluronic acid.

In the method of mixing the solid hyaluronic acid and the acidic aqueous solution, the form of the solid hyaluronic acid to be mixed is not limited, but the powder, the block-shaped molded product obtained by press-molding the hyaluronic acid powder, and the hyaluronic acid are mixed. A form such as a cast film obtained by dissolving in distilled water to form an aqueous solution and then ventilation drying or a sponge-like hyaluronic acid obtained after freeze-drying is considered. Examples of the mixing method include a method in which solid hyaluronic acid is impregnated with an acidic aqueous solution so as to have a predetermined concentration, and a method in which an acidic aqueous solution is added to solid hyaluronic acid and kneaded.

In the method of concentrating a low-concentration acidic aqueous solution of hyaluronic acid to a predetermined concentration, first dissolve solid hyaluronic acid in distilled water and then add an acid component, or dissolve solid hyaluronic acid directly in the acidic aqueous solution. By doing so, a low-concentration aqueous solution of hyaluronic acid is prepared. Here, the form of the dissolved solid hyaluronic acid does not matter. The term “low concentration” as used herein means lower than the hyaluronic acid concentration of the target hyaluronic acid gel. However, the hyaluronic acid concentration is preferably 5% by weight or less from the viewpoint of easy handling. Examples of the concentration method include ultracentrifugation, ventilation drying, drying under reduced pressure, and freeze drying.

In the method of adding an acid component to a high-concentration aqueous solution of hyaluronic acid, first, a high-concentration hyaluronic acid solution is mixed by mixing solid hyaluronic acid and distilled water, or by concentrating a low-concentration aqueous solution of hyaluronic acid. Prepare an aqueous solution. The form of the solid hyaluronic acid used at this time does not matter. Examples of a method for adding an acid component to the obtained high-concentration aqueous solution of hyaluronic acid include a method of exposing an acid in a gaseous state, for example, an atmosphere of hydrogen chloride, and an acid solution of a poor solvent for hyaluronic acid, for example, ethanol-hydrochloric acid. A method of dipping in a solution may be used.

The hyaluronic acid gel obtained according to the present invention is used to avoid acid hydrolysis of hyaluronic acid, and to form a component such as an acid type hyaluronic acid having a carboxyl group protonated or an acid used for adjusting the acidity. Need to be neutralized. Neutralization is usually performed with an aqueous solvent. There is no particular limitation as long as the function of the hyaluronic acid gel is not impaired. For example, a phosphate buffer, an aqueous solution of sodium hydroxide and the like are used.

The neutralization method is not particularly limited, but usually, a batch method, a filtration method, a method of filling a column or the like and passing the solution, or the like is used. These neutralization conditions are:
Including the number of times, any condition can be used as long as it can neutralize components such as acid-type hyaluronic acid and acid used for adjusting to acidity, and can be appropriately selected depending on the form and use of the hyaluronic acid gel. .

The neutralized hyaluronic acid gel may be immersed in a solvent, wet with a solvent, dried with air, dried under reduced pressure or freeze-dried depending on the purpose of use. Served in.

The processing such as molding of the hyaluronic acid gel is as follows.
At the time of preparation, the sheet, film, crushed, sponge, lump, fibrous, and tube-shaped desired forms of hyaluronic acid gel can be prepared by selecting the container and method of hyaluronic acid and the prepared aqueous solution of hyaluronic acid. Is possible. For example, block-shaped and sheet-shaped forms can be obtained from a product obtained by pressing and molding hyaluronic acid powder. Examples of processing after the production of the hyaluronic acid gel include fine crushing by mechanical pulverization, sponge by freezing and thawing, film formation by rolling, spinning, and the like.

The hyaluronic acid gel obtained in the present invention may be in a transparent state immediately after production or in water after neutralization.

[0033]

The present invention will be described in more detail with reference to the following examples. Note that the present invention is not limited to this.

Example 1 100 mg of sodium hyaluronate powder having a molecular weight of 2 × 10 ⁶ daltons was pressurized at 300 kgf / cm ² for 3 minutes to obtain a rectangular molded article of 8 mm × 8 mm × 2 mm.
This molded product is placed in a square container made of styrene, and 570 mg of 1N hydrochloric acid is used, and the concentration of hyaluronic acid is 15% by weight.
And the container was hermetically sealed, and stored in a refrigerator set at 5 ° C. for 6 days. As a result, a rectangular parallelepiped transparent hyaluronic acid gel was obtained.

Example 2 In Example 1, 1330 mg of 1N hydrochloric acid was impregnated so that the hyaluronic acid concentration became 7% by weight. And
The same operation as in Example 1 was performed. As a result, a rectangular parallelepiped transparent hyaluronic acid gel was obtained.

Example 3 The same operation as in Example 1 was performed using sodium hyaluronate having a molecular weight of 9 × 10 ⁵ daltons, and the mixture was stored in a refrigerator set at 5 ° C. for 17 days. As a result, a rectangular parallelepiped transparent hyaluronic acid gel was obtained.

Example 4 The same operation as in Example 1 was performed using 0.45N hydrochloric acid, and the mixture was stored in a refrigerator set at 5 ° C. for 17 days. As a result, a rectangular parallelepiped transparent hyaluronic acid gel was obtained.

Example 5 The same operation as in Example 1 was performed, and the mixture was stored at 25 ° C. for 3 days. As a result, a rectangular parallelepiped transparent hyaluronic acid gel was obtained.

Example 6 Sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in distilled water to prepare a 1% by weight aqueous solution of hyaluronic acid. The prepared aqueous solution of hyaluronic acid was air-dried on a glass plate at 80 ° C. to obtain a cast film having a thickness of about 200 μm. In Example 1, the same operation was performed using this cast film. As a result, a sheet-shaped transparent hyaluronic acid gel was obtained.

Example 7 100 mg of sodium hyaluronate powder having a molecular weight of 2 × 10 ⁶ daltons was placed in a 50 ml glass bottle, and 1N
Was added so that the concentration of hyaluronic acid became 10% by weight, and mixed with a spatula. Then, after sealing the bottle, the bottle was stored in a refrigerator set at 5 ° C. for 8 days. As a result, a hyaluronic acid gel was obtained.

Example 8 A powder of sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in 1N hydrochloric acid to prepare a 1% by weight aqueous solution of hyaluronic acid. After the prepared hyaluronic acid solution was subjected to ultracentrifugation, the supernatant was removed. As a result, the concentration of hyaluronic acid was increased to 18% by weight. The concentrated hyaluronic acid solution was stored in a refrigerator set at 5 ° C. for 3 days. As a result, a transparent hyaluronic acid gel was obtained. The ultracentrifugation process was performed using a machine of CS120EX manufactured by Hitachi Koki Co., Ltd., a rotor of S100AT5, a sample tube of 4PC tube, and a rotation speed of 99.
000 rpm, the treatment temperature was 5 ° C., and the treatment time was 24 hours.

Comparative Example 1 The same operation as in Example 1 was performed using distilled water instead of 1N hydrochloric acid. As a result, a clear hyaluronic acid gel-like solution was obtained.

Example 9 Solubility Test of Hyaluronic Acid Gel A phosphate buffer component was added to physiological saline at a concentration of 50 mM.
A phosphate buffered saline at pH 7 was prepared. The obtained hyaluronic acid gel was immersed in phosphate buffered saline at a ratio of 50 ml of phosphate buffered saline to a hyaluronic acid gel containing 10 mg of hyaluronic acid by dry weight. The gel solution of hyaluronic acid of Comparative Example 1 was also immersed in a dry weight of 10 mg in 50 ml of phosphate buffered saline. Then, the ratio of hyaluronic acid eluted in phosphate buffered saline under stirring at 25 ° C.
It was determined from the concentration of hyaluronic acid in phosphate buffered saline. Therefore, the solubility of the hyaluronic acid gel in a neutral aqueous solution at 25 ° C. is defined by the above test.

Measurement of Hyaluronic Acid Concentration The concentration of hyaluronic acid in phosphate buffered saline is G
It was determined from the peak area of the differential refractive index detector using a PC.

As described above, the solubility test of the hyaluronic acid gels of Examples 1 to 8 and Comparative Example 1 was specifically performed. Table 1 shows the results. For example, in experiment no. When the dissolution rate of the hyaluronic acid gel obtained in Example 1 was examined, the dissolution rate was 0% after 1 day, 2% after 3 days, and 1% after 7 days. The dissolution rate was 11% after 14 days. That is, even after 7 days, 95% or more of the hyaluronic acid gel remained. On the other hand, in Experiment No. 9, the dissolution rate of the hyaluronic acid-like solution obtained in Comparative Example 1 was 100% after one day.
% Dissolution and complete dissolution. Therefore, the solution of the hyaluronic acid gel obtained in the comparative example (Experiment No. 9) has a very high dissolution rate in water, whereas the hyaluronic acid gel obtained by the production method of the present invention (for example, the experiment It is found that the dissolution rate of Nos. 1 to 8) in water is extremely slow. This suggests that the hyaluronic acid gel obtained by the present invention has a long residence time in a living body.

[0046]

[Table 1]

Example 10 Solubilization Test of Hyaluronic Acid Gel Distilled water was adjusted to pH 1.5 with hydrochloric acid. The hyaluronic acid gel obtained in Example 1 was immersed in the phosphate buffered saline described in Example 9 for 5 days. Next, the hyaluronic acid gel immersed in the phosphate buffered saline and swollen was taken out of the phosphate buffered saline. 15 by dry weight
A hyaluronic acid gel containing mg of hyaluronic acid at pH 1.
5 was immersed in 15 ml of the aqueous solution. This solution was left in an oven set at 60 ° C. One hour, 2.5 hours, and 5 hours later, 0.5 ml of the solution was sampled. After 2.5 hours, the hyaluronic acid gel that could be visually confirmed almost disappeared.

Comparative Example 2 Sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in distilled water to prepare a 0.1% by weight aqueous solution of hyaluronic acid. The pH of this aqueous solution is adjusted to pH with 1N hydrochloric acid.
Adjusted to 1.5. This acidic aqueous solution of hyaluronic acid 15
ml was left in a 60 ° C. oven for 4 hours to effect acid hydrolysis of linear hyaluronic acid.

Example 11 Measurement of Molecular Weight and Degree of Branching of Solubilized Hyaluronic Acid The hyaluronic acid solubilized in Example 10 and the acid hydrolyzate of the linear hyaluronic acid obtained in Comparative Example 2 were dissolved in a GPC solvent. After adjusting the concentration to 0.04% by weight by diluting 2.5 times and filtering through a 0.2 μm membrane filter,
GPC-MALLS was measured by injecting 0.1 ml. One SB806HQ manufactured by Showa Denko KK as a GPC column, and 830-R manufactured by JASCO Corporation as a differential refractive index detector
I and MALLS were measured using DAYNDSP-F manufactured by Wyatt at a 0.2 M aqueous solution of sodium nitrate as a solvent, at a measurement temperature of 40 ° C., at a flow rate of 0.3 ml / min, and at a data acquisition interval of once every 2 seconds. The scattering intensity was measured at a scattering angle of 21.7.
Eight detectors between ° and 90 ° were used. The data processing software is ASTRA Version 4.1 manufactured by Wyatt.
0 was used.

A method for measuring and calculating the molecular weight and the degree of branching of the solubilized hyaluronic acid using the GPC-MALLS method will be described. In the GPC-MALLS method, the molecular weight and the radius of gyration of each fraction separated by GPC can be continuously measured. The measurement of the degree of branching using the GPC-MALLS method is performed by calculating the degree of branching by comparing the molecular weight of the solubilized hyaluronic acid in the fraction having the same elution volume with the molecular weight of the control linear hyaluronic acid. Performed by law.

The degree of branching is the number of branch points present per one polymer chain of solubilized hyaluronic acid, and is plotted against the molecular weight of solubilized hyaluronic acid.

The molecular weight and the radius of gyration of each fraction were calculated using the Jim plot (1) at a finite concentration. The molecular weight was extrapolated to a scattering angle of 0 ° and the radius of gyration was calculated from the angle-dependent initial gradient as follows. Calculated according to the formula:

[0053]

(Equation 1)

Where M is the molecular weight, <S ² > is the mean square radius of inertia, K is the optical constant, R (θ) is the excess reduction scattering intensity at the scattering angle θ, c is the polymer concentration, and P (θ) Is the particle scattering function, λ is the wavelength of the laser beam in the solution, A ₂ is the second virial coefficient, and 0.002 ml for hyaluronic acid.
- it is a mol / g ^2. c is the output of the differential refractometer,
The concentration gradient of the refractive index of the aqueous solution of hyaluronic acid (dn / dc:
0.153 ml / g).

The degree of branching of each fraction by the elution volume method was calculated according to the following equation (2). Assuming that the molecular weight of the branched polymer is M _b and the molecular weight of the linear polymer is M _{1 in} the fractions having the same elution volume, the shrinkage factor g is obtained.

[0056]

(Equation 2)

Here, a is a Mark-Houwink constant, which is 0.78 for hyaluronic acid. e is a clear factor and is set to 1.0. Assuming tetrafunctional long-chain disordered branching, the number B (degree of branching) of branch points on one polymer chain is calculated from the following equation (3).

[0058]

(Equation 3)

The calculation method of the degree of branching by the elution volume method is GP
This is the same as the measurement of the degree of branching by the C-LALLS method, and the details are described in Size Exclusion Chromatography (Kyoritsu Shuppan, 1991).

As described above, the acid hydrolyzate of the hyaluronic acid solubilized in Example 10 and the linear hyaluronic acid obtained in Comparative Example 2 was measured. Table 2 shows the measurement results.

[0061]

[Table 2]

For example, in Experiment No. In the case of sampling in Example 10 with a reaction time of 1 hour, the solubilization rate of the hyaluronic acid gel is small. Experiment No. When the sample is sampled for 12 reaction times of 6 hours, the molecular weight is greatly reduced and the measurement of the degree of branching becomes difficult. Experiment No. 11 reaction time 2.5
When sampled over time, the solubilization rate of the hyaluronic acid gel is large, and the molecular weight distribution is as large as 2.7, reflecting the presence of hyaluronic acid having a branch point.

Experiment No. The solubilized hyaluronic acid sampled in Example 10 of Example 11 at a reaction time of 2.5 hours was used in combination with Experiment No. 11. The GPC chromatogram and the calculation results of the degree of branching of the acid hydrolyzate of linear hyaluronic acid obtained in Comparative Example 2 are shown in FIGS. From FIG. 1, the first embodiment
It can be seen that GPC chromatogram 1 of 0 has a shoulder on the high molecular weight side as compared with GPC chromatogram 2 of Comparative Example 2. Comparing the molecular weights of the fractions with the same elution volume, the elution volume in Example 10 was 8.6 ml or less,
It can be seen that the polymer has a clearly larger molecular weight than Comparative Example 2 in a region where the molecular weight is about 200,000 or more. In Example 10, since a branch point was present, the molecular weight of the fraction having the same elution volume was higher than that of Comparative Example 2.

FIG. 2 shows the relationship between the degree of branching and the molecular weight of Example 10 in which Comparative Example 2 was calculated as a linear hyaluronic acid. That is, the degree of branching was calculated from the molecular weights of both fractions having the same elution volume in FIG.
FIG. 2 shows that the degree of branching in Example 10 rapidly increases from a degree of branching of 0.5 or more in a region having a molecular weight of about 200,000 or more. It can be seen that the hyaluronic acid gel obtained in the present invention contains a crosslinked structure that is stably present even under the conditions of accelerated acid hydrolysis of hyaluronic acid.

Example 12 Test for Degree of Swelling of Hyaluronic Acid Gel The obtained hyaluronic acid gel was subjected to Example 9 against a hyaluronic acid gel containing 10 mg of hyaluronic acid by dry weight.
The indicated phosphate buffered saline was immersed in phosphate buffered saline at a rate of 50 ml. The swelling degree of the hyaluronic acid gel was determined from the ratio of the weight of the hyaluronic acid gel at 25 ° C. to the weight obtained by subtracting the weight of the hyaluronic acid eluted in the phosphate buffered saline determined in Example 9 from the dry weight. Was. Therefore, the shape retention of the hyaluronic acid gel in a neutral 25 ° C. aqueous solution is defined by the above test.

As described above, the swelling degree test of the hyaluronic acid gels of Examples 1 to 3 and Comparative Example 1 was specifically performed. Table 3 shows the results.

[0067]

[Table 3]

For example, in experiment no. When the degree of swelling of the hyaluronic acid gel obtained in Example 14 was examined, the swelling degree was 35 times after 1 day, 49 times after 3 days, and 56 times after 7 days. It was the degree of swelling.
That is, even after 7 days, the hyaluronic acid gel remained while maintaining its form. On the other hand, in Experiment No. As shown in the solubility test results in Table 1, the hyaluronic acid gel-like solution obtained in Comparative Example 1 was 10 days later.
The dissolution rate was 0%, and the degree of swelling could not be measured because it was completely dissolved. Therefore, the hyaluronic acid gel-like solution obtained in the comparative example (Experiment No. 17) has no form retention in water, whereas the production method of the present invention provides excellent form retention in a neutral aqueous solution. It is found that a hyaluronic acid gel having properties (for example, Experiment Nos. 14 to 16) is obtained.

Further, according to Example 12, a test was also performed on the hyaluronic acid gel of Example 7. When observed after 3 days, the hyaluronic acid gel obtained in Example 7 was easily deformed and had fluidity.

Example 13 Transparency Test of Hyaluronic Acid Gel The obtained hyaluronic acid gel and the hyaluronic acid gel in the course of the swelling degree test of Example 12 were cut out or sandwiched between two preparations to increase the thickness. Was 2 mm. However, when the obtained hyaluronic acid gel was a sheet having a thickness of 2 mm or less as in Example 6, for example, the test was performed as it was. The prepared slide or the prepared gel sandwiched between the gels was placed on a newspaper and the lower letters could be read.

As described above, the hyaluronic acid gels of Examples 1 to 6 and 8 and Example 1 after one day after the swelling degree test
When 8 hyaluronic acid gels were tested, all were clear.

[0072]

As described above, according to the present invention, it is possible to produce a hyaluronic acid gel having a very low dissolution rate in water without using any chemical crosslinking agent or chemical modifier. The resulting hyaluronic acid gel avoids adverse effects on biocompatibility due to the use of chemical cross-linking agents and chemical modifiers, and has a long residence time in the living body, which is useful in the field of biocompatible materials. is there. Further, according to the present invention, a transparent hyaluronic acid gel can be obtained.

[Brief description of the drawings]

FIG. 1 is a graph comparing the GPC chromatograms of Example 10 and Comparative Example 2 with the molecular weight of each fraction.

FIG. 2 is a graph showing the relationship between the degree of branching and the molecular weight of Example 10 in which Comparative Example 2 was calculated as linear hyaluronic acid.

[Explanation of symbols]

 1 GPC chromatogram of Example 10 2 GPC chromatogram of Comparative Example 2 3 Molecular weight of each fraction of Example 10 4 Molecular weight of each fraction of Comparative Example 2

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuhiro Oshima 3-5-1 Asahicho, Machida-shi, Tokyo Denka Kagaku Kogyo Co., Ltd. (72) Inventor Osamu Yamamoto 3-chome Asahicho, Machida-shi, Tokyo No. 5-1 F-term in Rikagaku Kogyo Co., Ltd. (Reference) 4C090 AA03 AA07 BA67 BD02 BD14 BD24 BD41 CA04 CA07 CA32 DA10

Claims (6)

[Claims] 1. A hyaluronic acid gel which is hardly soluble in a neutral aqueous solution, wherein the gel is formed from an acidic aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more and an acid component in an equimolar amount or more with the carboxyl group of hyaluronic acid. Manufacturing method. 2. An aqueous solution of acidic hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more and containing an acid component in an equimolar amount or more with a carboxyl group of hyaluronic acid before being neutralized at -10 ° C. to 30 ° C.
A method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution, wherein the hyaluronic acid gel is formed by aging in a neutral solution. 3. An aqueous hyaluronic acid solution obtained by mixing a solid hyaluronic acid and an acidic aqueous solution containing at least an equimolar amount of an acid component with a carboxyl group of the hyaluronic acid at a hyaluronic acid concentration of 5% by weight or more. The method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution according to claim 1 or 2. 4. A method comprising forming an acidic aqueous solution of hyaluronic acid having a concentration of at least 5% by weight of hyaluronic acid obtained by concentrating an aqueous solution of acidic hyaluronic acid and containing an acid component in an equimolar amount or more with a carboxyl group of hyaluronic acid. The method for producing a hyaluronic acid gel according to claim 1 or 2, which is hardly soluble in a neutral aqueous solution. 5. An aqueous solution of hyaluronic acid having a concentration of 5% by weight or more obtained by adding an acid component having an equimolar amount or more to the carboxyl group of hyaluronic acid to an aqueous solution of hyaluronic acid having a concentration of 5% by weight or more. 3. The method for producing a hyaluronic acid gel which is hardly soluble in a neutral aqueous solution according to claim 1 or 2. 6. A gel formed of hyaluronic acid alone, which is transparent at a solubility of 50% or less per day in a neutral aqueous solution at 25 ° C.