JP2001329002A - Modified hyaluronic acid gel, its preparing method and medical material containing same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modified hyaluronic acid gel which can be obtained by using no crosslinking agent, is excellent in safety and biocompatibility and has controlled solubility, and to provide a medical material containing the same. SOLUTION: A modified hyaluronic acid gel is made of only a modified hyaluronic acid which is hardly soluble in neutral water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel modified hyaluronic acid gel and a method for producing the same, and more particularly to a medical material having good biocompatibility.

[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 using chicken crest, umbilical cord and the like as an extraction material, purified products are also 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 .

[0003] We have determined that aqueous solutions of hyaluronic acid have a specific pH.
H, and freezing and then thawing the aqueous solution at least once to obtain a hyaluronic acid gel, and found that the dissolution rate of the thus obtained hyaluronic acid gel in water was extremely slow. (Japanese
-117564). In addition, it was also confirmed that various medical materials including an adhesion preventing material can be provided by using this gel.

Until now, various methods have been devised for introducing an optional substituent by chemically modifying hyaluronic acid. As one example, there is disclosed a method for chemically modifying hyaluronic acid utilizing the feature that a tetrabutylammonium salt of hyaluronic acid is dissolved in an organic solvent such as dimethyl sulfoxide (Japanese Patent Application Laid-Open No. 3-105003). Also, the tetrabutylammonium salt of hyaluronic acid is mixed with triethylamine and iodide 2 in dimethyl sulfoxide.
Also disclosed is a method of adding and reacting -chloro-1-methylpyridinium to form an ester bond between a carboxyl group and a hydroxyl group of hyaluronic acid (EP 0341).
745A1). Furthermore, a method of introducing an organic acid such as lactic acid or pyruvic acid through an ester bond has been devised (JP-A-6-016702, JP-A-6-298804).
issue).

[0005]

SUMMARY OF THE INVENTION In the course of studying the above-mentioned hyaluronic acid gel, it was found that even if any modifying group was introduced into hyaluronic acid, gelation by acidic freezing was not prevented. In addition, it has been confirmed that such a modified hyaluronic acid gel composition can be used as a carrier of a drug delivery system by utilizing the poor solubility of the gel. In our previous studies, a gel incorporating a drug was proposed by mixing an arbitrary drug into the system during gelation (Japanese Patent Application Laid-Open No. H11-043287), but the drug was directly bound to hyaluronic acid. The use of the modified hyaluronic acid gel allows the dissolution of the gel to coincide with the release of the drug. In addition, the hyaluronic acid gel produced by binding cinnamic acid to hyaluronic acid has a lower solubility than the gel of hyaluronic acid alone and is stable, and the hyaluronic acid gel is obtained by introducing a specific modifying group. Can be changed. Then, they have found that the solubility of the hyaluronic acid gel can be controlled by introducing a specific modifying group, and have completed the present invention.

[0006]

That is, the present invention provides (1)
Modified hyaluronic acid gel formed of modified hyaluronic acid alone, which is poorly soluble in neutral aqueous solution, (2)
The modified hyaluronic acid gel according to (1), wherein a substituent is introduced into a hydroxyl group of hyaluronic acid, (3) a modified hyaluronic acid having a cinnamic acid as a substituent (2) (4) The modified hyaluronic acid gel according to (3), wherein the rate of introduction of cinnamic acid into hyaluronic acid is 0.05% by mass or more, (5) Neutral 25. The modified hyaluronic acid gel according to any one of (1) to (4), wherein the dissolution rate per day in an aqueous solution at 25 ° C. is 25% or less, and (6) promotion of modified hyaluronic acid. The modified hyaluronic acid solubilized by treating the modified hyaluronic acid gel under acid hydrolysis conditions partially contains a molecular weight fraction having a degree of branching of 0.5 or more (1) to (1). Any one of 5)
The modified hyaluronic acid gel according to item 7, wherein the aqueous solution of modified hyaluronic acid is adjusted to pH 3.5 or less, and the aqueous solution is frozen and then thawed at least once. Method for producing gel, (8)
A modified hyaluronic acid gel is formed by coexisting a modified hyaluronic acid, water having a modified hyaluronic acid concentration of 5% by mass or more, and an acid component in an equimolar amount or more with the carboxyl group of the hyaluronic acid, and maintaining the coexistence state. A method for producing a modified hyaluronic acid gel, (9) coexistence of modified hyaluronic acid, water having a modified hyaluronic acid concentration of 5% by mass or more, and an acid component having an equimolar amount or more of a carboxyl group of the modified hyaluronic acid Under the condition, the coexistence state is −10 ° C. to 30 ° C.
(8) the method for producing a modified hyaluronic acid gel according to (8), wherein the modified hyaluronic acid gel is formed by holding the modified hyaluronic acid gel at a temperature of 5 ° C. And a solution in which a carboxyl group of the modified hyaluronic acid and an equimolar or more acid component are coexisted and the coexisting state is maintained at -10 ° C to 30 ° C to form a modified hyaluronic acid gel, and the gel is used for neutralization. A method for producing a modified hyaluronic acid gel, characterized by treating with (1)
1) The method for producing a modified hyaluronic acid gel according to any one of (7) to (10), wherein the modified hyaluronic acid is cinnamic acid-modified hyaluronic acid, (12) the following (a), A medical material comprising a gel formed of the modified hyaluronic acid alone that satisfies the requirement of (b),
(A) The solubility in one day in a neutral aqueous solution at 25 ° C. is 25
% Or less, the modified hyaluronic acid solubilized by treating the modified hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid has a branched structure, and in the solubilized modified hyaluronic acid, (13) Gel formed of modified hyaluronic acid alone, partially containing a molecular weight fraction having a degree of branching of 0.5 or more, in the form of a sheet, film, crushed, sponge, lump, fibrous, or tube (12) characterized in that it is one selected from the group consisting of a shape.
(14) The medical material according to (13), wherein the medical material is one selected from the group consisting of an adhesion preventing material, a wound dressing material, and a drug carrier intended for a drug delivery system (DDS). It is a medical material as described.

[0007]

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 a fermentation method. The strain used in the fermentation method is a microorganism capable of producing hyaluronic acid, such as Streptococcus sp. Isolated from nature, or JP-A-63-1233.
No. 92, Streptococcus equi FM
-100 (Microtechnical Laboratory No. 9027), JP-A-2-23
A mutant strain that stably produces hyaluronic acid at a high yield, such as Streptococcus equi equi-FM-300 (No. 2319, manufactured by B.I.
Those cultured and purified using the above mutant strains are used.

[0008] The modified hyaluronic acid of the present invention refers to a hyaluronic acid in which a substituent is introduced at an arbitrary site. Hyaluronic acid is composed of β-DN-acetylglucosamine and β-D
-Glucuronic acid, but a substituent may be introduced into either of them, and the site is not limited. The method for synthesizing the modified hyaluronic acid is not particularly limited, and various synthetic methods including esterification and acylation can be applied as long as gelation of the modified hyaluronic acid is not prevented. The introduction ratio of the substituent may be one unit or more modified per one molecule of hyaluronic acid, and the introduction ratio can be increased before gelation of the modified hyaluronic acid is not hindered.

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 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 modified hyaluronic acid gel referred to in the present invention is:
The modified hyaluronic acid gel is characterized by being hardly soluble in a neutral aqueous solution. When the modified hyaluronic acid gel is put into a neutral aqueous solution, the modified hyaluronic acid gel shows a significantly lower solubility than a non-gelled modified hyaluronic acid. Poor solubility is defined by the retention of the gel form in a neutral 25 ° C. aqueous solution and the gel dissolution rate. Here, the neutral aqueous solution is a physiological saline having a buffer capacity adjusted to pH7. The modified hyaluronic acid gel referred to in the present invention also has a feature that when it is put into an alkaline aqueous solution, for example, an alkaline buffered aqueous solution having a pH of 11, it is rapidly dissolved.

The modified 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 is formed by a crosslinked structure of the modified hyaluronic acid.

The modified hyaluronic acid gel referred to in the present invention can be decomposed and solubilized by treating the modified hyaluronic acid gel under the conditions of the accelerated acid hydrolysis reaction of hyaluronic acid. When the solubilized modified hyaluronic acid has a crosslinked structure, it can be distinguished from a modified hyaluronic acid having a branch point from a linear modified hyaluronic acid in polymer solution theory.

The conditions for the accelerated acid hydrolysis reaction of hyaluronic acid referred to in the present invention are as follows: pH 1.5 of aqueous solution, temperature 60 ° C.
Is appropriate. The main chain cleavage reaction due to hydrolysis of the glycosidic bond of hyaluronic acid, compared with neutral aqueous solution,
Significantly promoted in acidic or alkaline aqueous solutions. Further, the acid hydrolysis reaction is promoted at a higher reaction temperature.

The reaction time of accelerated acid hydrolysis of the modified hyaluronic acid gel referred to in the present invention largely depends on the structure of the modified hyaluronic acid gel, for example, the molecular weight and molecular weight distribution of the raw material hyaluronic acid, and the degree of crosslinking. Reaction conditions in which the ratio of solubilized modified hyaluronic acid is large and the degree of branching is large can be selected. When the reaction conditions are weak, the ratio of the solubilized modified hyaluronic acid is small, and when the reaction conditions are strong, the molecular weight of the solubilized modified hyaluronic acid becomes extremely small, so that the measurement of the degree of branching becomes difficult. . Further, the possibility that the branch point itself is decomposed also increases. The reaction conditions are preferably a reaction time in which the modified hyaluronic acid gel that can be visually confirmed almost disappears, or a reaction time in which the ratio of the solubilized modified hyaluronic acid is 50% or more.

The molecular weight of the hyaluronic acid and the modified hyaluronic acid used in the present invention is from about 1 × 10 ⁵ to about 1 × 10 ⁵
Those within the range of ⁷ daltons are preferred. In addition, as long as it has a molecular weight within the above range, a higher molecular weight one and a low molecular weight one obtained by hydrolysis or the like can also be preferably used. The term “hyaluronic acid” and “modified hyaluronic acid” used in the present invention are used in a concept that includes alkali metal salts thereof, for example, sodium, potassium and lithium salts.

The aqueous solution of modified hyaluronic acid used in the present invention is obtained by mixing powder of modified hyaluronic acid and water and stirring the mixture. A concentration of the modified hyaluronic acid of 5.0% by mass or less is convenient for treating an aqueous solution. 2 × 1 molecular weight
When using 0 ⁶ daltons or more modified hyaluronic acid concentration is preferably 2.5 wt% or less.

As the acid used for adjusting the pH of the aqueous solution of the modified hyaluronic acid, any acid can be used as long as the pH can be adjusted to 3.5 or less. 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 pH of the aqueous solution of the modified hyaluronic acid is adjusted to a pH at which the carboxyl groups of the hyaluronic acid are protonated at a sufficient ratio. The equilibrium constant for the dissociation of the acid form of hyaluronic acid is log K ₀ = infinite dilution of hyaluronic acid concentration.
4.25 (Acta Chimica Hungarica-Models in
Chemistry 129 (5) 671-683 1992). The pH to be adjusted 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 concentration of the aqueous solution, the conditions for freezing and thawing, and the strength of the gel to be formed. It is necessary to adjust to less than 0.5. Preferably, the pH is 2.5 or less.

In the freezing and thawing, the prepared aqueous solution of the modified hyaluronic acid is placed in an arbitrary container, frozen at a predetermined temperature, and after the freezing is completed, the operation of thawing at a predetermined temperature is performed at least once. Do. The temperature and time for freezing and thawing are appropriately determined within the range of the temperature and time for freezing and thawing the acidic aqueous solution of the modified hyaluronic acid depending on the size of the container and the amount of the aqueous solution. The above thawing temperature is preferred. Since the freezing and thawing times can be shortened, it is more preferable that the freezing temperature is -5 ° C or less, and 5 ° C or less.
The above thawing temperature is selected. The time is not particularly limited as long as it is equal to or longer than the time at which freezing and thawing is completed at that temperature.

The number of repetitions of the operation of freezing and then thawing the prepared acidic aqueous solution of the modified hyaluronic acid depends on the molecular weight of the modified hyaluronic acid used, the concentration of the aqueous solution, the pH of the aqueous solution, the temperature and time of freezing and thawing, and the production time. It is appropriately determined according to various properties such as the strength of the gel to be formed. Normally, it is preferable to repeat it one or more times. Further, each time the operation of freezing and thawing is repeated, the temperature and time of freezing and thawing may be changed.

The modified hyaluronic acid gel obtained by freezing and thawing the adjusted acidic solution of the modified hyaluronic acid contains components such as an acid used for adjusting the acidity in order to avoid acid hydrolysis of the modified hyaluronic acid. It needs to be neutralized. Neutralization is usually performed with a physiologically or pharmaceutically acceptable solution. There is no particular limitation as long as the function of the modified hyaluronic acid gel is not impaired. For example, physiological saline, phosphate buffer and the like are preferably used.

The neutralization method is not particularly limited.
Usually, a batch method, a filtration method, a method of filling a column or the like and passing the solution, and the like are used. These neutralization conditions may be any conditions as long as the components to be neutralized can be reduced to a target concentration or less, including the amount of the neutralizing solution, the number of times, etc., and can be appropriately selected depending on the form and application of the modified hyaluronic acid gel. It is.

The modified hyaluronic acid gel treated with the solution used for the neutralization may be immersed in a solvent, wet with a solvent, air-dried, dried under reduced pressure or freeze-dried, depending on the purpose of use. It is provided as a medical material in a dry state after processing.

Next, a method for producing the modified hyaluronic acid gel by mixing a solid modified hyaluronic acid and an acid solution so that the concentration becomes 5% by mass or more without freezing and leaving the mixture to stand is described. The origin and molecular weight of the modified hyaluronic acid used, the type of acid used, and the like are exactly the same as those in the case of gelation by freezing / thawing (freezing method). Furthermore, the neutralization and washing methods after forming the gel and the subsequent treatment are not different from those produced by the freezing method.

The mixing of the modified hyaluronic acid and the acid solution is desirably performed at a temperature as low as possible to avoid acid hydrolysis of the modified hyaluronic acid. However, when the mixing is completed in a very short time even at a high temperature, it is possible to obtain a modified hyaluronic acid gel even under such conditions. In addition, a modified hyaluronic acid gel can be obtained as long as a homogeneous mixture of the modified hyaluronic acid and the acid solution can be obtained, even if the mixture is mixed under conditions below the freezing point such that the acid solution freezes. In addition, if the mixing is not uniform, a phenomenon may occur in which the gel is not gelled in the unmixed portion, but the unreacted components are removed by a subsequent washing operation (neutralization) to remove the modified hyaluronic acid gel. It is also possible to prepare. The mixture of the modified hyaluronic acid and the acid solution is allowed to stand at a constant temperature to progress gelation to obtain a modified hyaluronic acid gel, but the standing temperature and the standing time are not defined uniquely. If the standing temperature is low, acid hydrolysis can be suppressed, and the decomposition of the main chain of the modified hyaluronic acid molecule can be suppressed.However, since the gelation rate tends to decrease more, it is determined in consideration of the required physical properties. It is.

The modified hyaluronic acid gel referred to in the present invention can be decomposed and solubilized by treating the modified hyaluronic acid gel under the conditions of accelerated acid hydrolysis reaction of the modified hyaluronic acid. When the solubilized modified hyaluronic acid has a crosslinked structure, it can be distinguished from a modified hyaluronic acid having a branch point from a linear modified hyaluronic acid in polymer solution theory.

The conditions for the accelerated acid hydrolysis reaction of the modified hyaluronic acid referred to in the present invention are as follows: pH 1.5 of the aqueous solution;
0 ° C. is appropriate. The main chain scission reaction by hydrolysis of the glycosidic bond of hyaluronic acid is remarkably promoted in an acidic or alkaline aqueous solution as compared with a neutral aqueous solution.
Further, the acid hydrolysis reaction is promoted at a higher reaction temperature.

In the present invention, the GPC-MALLS method is used,
The molecular weight and the degree of branching of the molecular weight fraction separated by GPC were continuously measured online. In the present invention, the degree of branching is measured using an elution volume method in which the molecular weight of a solubilized modified hyaluronic acid of a fraction having the same elution volume is compared with the molecular weight of a linear modified hyaluronic acid as a control to calculate the degree of branching. Was done. The degree of branching is the number of branch points per polymer chain of the solubilized modified hyaluronic acid, and is plotted against the molecular weight of the solubilized modified hyaluronic acid. The measurement of the degree of branching by the elution volume method using the GPC-MALLS method is described in detail in PCT / JP98 / 03536.

[0029] The solubilized modified hyaluronic acid is obtained by GPC.
The concentration was adjusted by diluting with a solvent, followed by filtration through a 0.2 μm membrane filter and then used for measurement. In the modified hyaluronic acid gel referred to in the present invention, when there is a cross-linked structure stably present even under the accelerated acid hydrolysis conditions of the modified hyaluronic acid,
The branched structure of the solubilized modified hyaluronic acid is confirmed by polymer solution theory. The degree of branching of the modified hyaluronic acid gel referred to in the present invention is 0.5 or more.

Next, a processing method for utilizing the modified hyaluronic acid gel produced as described above for medical use will be described. At the time of preparation, the modified hyaluronic acid gel may be formed into a sheet, film, crushed, sponge, lump, fibrous, or tubular modified hyaluronic acid gel in a desired form at the time of preparation. Can be produced. For example, a film-like or sheet-like form can be obtained by casting and freezing on a plate, or a crushed form can be obtained by freeze-thawing with vigorous mixing and stirring with an organic solvent immiscible with water.

Examples of processing after the preparation of the modified hyaluronic acid gel include fine crushing by mechanical pulverization, film formation by rolling, and spinning. On the other hand, there is a case where a modified hyaluronic acid gel having a desired shape can be obtained by selecting an appropriate preparation condition without particularly performing a processing for molding. For example, a modified hyaluronic acid gel in the form of fine fibers is obtained by freezing and thawing a modified acidic solution having a modified hyaluronic acid concentration of 0.1% or less, preferably 0.05% or less.

The modified hyaluronic acid gel obtained by the present invention can be used without particular limitation as long as it is a field in which general biodegradable medical materials and hyaluronic acid are used. For example, an anti-adhesion agent, a carrier of a pharmacologically active substance, a wound dressing, an artificial skin, a tissue replacement type tissue repair agent, a joint injection, a surgical suture, a hemostatic agent, an artificial organ, an artificial extracellular matrix or an artificial base Use in biomedical products or pharmaceutical compositions such as membranes, medical devices and medical devices used for diagnosis and treatment, and the like.

The various shaped and processed products of the modified hyaluronic acid gel may be used in a single form, or may be mixed or used in combination with different hyaluronic acid gel forms, or further mixed or used in combination with ungelled hyaluronic acid. The effect can be expected to be enhanced by the prescription. For example, the effects of the combined use of a sheet-shaped modified hyaluronic acid gel and a hyaluronic acid solution as an anti-adhesion agent after intraperitoneal surgery can be expected to have a local effect and an entire intraperitoneal effect. The effect of mixing the crushed modified hyaluronic acid gel and the hyaluronic acid solution as a joint injection agent can be expected to have an immediate effect and a delayed effect.

Hereinafter, the usefulness of the modified hyaluronic acid gel obtained in the present invention as a medical material will be described with reference to the use of a pharmacologically active substance as a carrier for sustained release as an example. The modified hyaluronic acid gel obtained in the present invention can be used as a carrier for binding a pharmacologically active substance in its structure to release the pharmacologically active substance slowly. In this case, by controlling the properties and shapes represented by the degradability of the modified hyaluronic acid gel according to the type of the pharmacologically active substance, the form of use, the site of use, and the required duration, to various pharmacologically active substances, It can be applied to various usage forms. Along with the appropriate formulation, a pharmaceutical formulation capable of releasing a pharmacologically active substance according to the purpose can be obtained. Administration methods of the sustained release preparation include oral, transdermal, transmucosal, injection, and implantation into the body.

Next, the adhesion preventing material of the present invention will be described. The adhesion preventing material for the modified hyaluronic acid gel obtained in the present invention is used for a surgical operation in the form of a sheet, a film, a crush, a sponge, a lump, a fiber, a tube, or the like. As a form to be used, it is preferable to apply directly to a surgical site as a film or a sheet.
Alternatively, it is preferably applied as a fine crushed form to a surgical site with a syringe. Alternatively, it is preferable to apply it to a surgical site with a laparoscope as a gel or film. When applied to these medical materials, sterile materials are required from the viewpoint of repelling inflammation and the like. As for the modified hyaluronic acid gel medical material, such a sterile material can be prepared by synthesizing the modified hyaluronic acid in a sterile system such as a bioclean room and gelling the same. Similar materials can be produced by subjecting them to heat sterilization or radiation sterilization after production.

Furthermore, by freeze-thawing with an adjusted acidic solution of the modified hyaluronic acid bound to the physiologically active substance, it is possible to obtain an adhesion preventing material for the modified hyaluronic acid gel containing the physiologically active substance. The anti-adhesion material of the modified hyaluronic acid gel can be applied to any animal in which adhesion occurs, and can suitably prevent adhesion after surgery in mammals, particularly humans.

The place of administration in a living body is abdominal surgery, gynecological surgery, thoracic surgery, orthopedic surgery related to tendons and ligaments, hard tissue related to various organs in the abdominal cavity, thoracic cavity, tendon sheath, skull, nerve, eyeball, etc. It is not particularly limited, such as a neurosurgery involving a membrane.

The modified hyaluronic acid gel obtained in the present invention may be administered at any time at which adhesion can be prevented after the operation, and may be administered during or at the end of surgery.
In particular, it is preferable to administer immediately before the end of the operation.

Next, the wound dressing of the present invention will be described. The clinical application range is assumed to be wounds in the field of plastic surgery such as tissue rupture / defect due to trauma, burn wounds, ulcer wounds, pressure ulcers, trauma in the field of otolaryngology, eardrum perforation due to otitis media, and wounds in blood vessels and nerves Is done. Further, the shape can be selected from a sheet, a film, a crushed shape, a sponge, a lump, a fiber, and a tube according to an application site.

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

[0041]

EXAMPLE 1 80 mg of sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in a mixed solution of 20 ml of water and 10 ml of dioxane. Cinnamic acid chloride 14 with stirring on ice
4 μl and 139 μl of triethylamine were added.
A solution prepared by dissolving 122 mg of dimethylaminopyridine in 2 ml of dioxane was added and reacted at room temperature for 1 hour. After the completion of the reaction, the modified hyaluronic acid was separated and washed by precipitation with ethanol, and this was freeze-dried to obtain a white modified hyaluronic acid powder. Confirmation of the synthesis of the modified hyaluronic acid was evaluated by GPC analysis. That is,
Using a phosphate buffer as a solvent, the sample was eluted using a SHOdex 806 column which is a column for polymer fractionation, and cinnamic acid was detected by a UV detector and hyaluronic acid was detected by an RI detector. Since cinnamic acid has a much lower molecular weight than hyaluronic acid, when analyzing a mixture of hyaluronic acid and cinnamic acid, hyaluronic acid elutes first, and then cinnamic acid elutes. However, analysis of the synthesized modified hyaluronic acid confirmed that both were eluted at the same time, indicating that cinnamic acid-modified hyaluronic acid in which cinnamic acid was chemically bonded to hyaluronic acid was synthesized. It could be confirmed. According to this analysis, the coupling ratio was 0.25% by mass. In addition to GPC, pyrolysis GC / MS (equipment: DX303 manufactured by JEOL Ltd. and PY-2020D manufactured by Frontier Lab, column: DB-1HT, pyrolysis temperature: 590 ° C.) can also be used to obtain ethyl cinnamate as a pyrolysis product. From the detection, the binding of cinnamic acid-modified hyaluronic acid was also evaluated, and it was confirmed that cinnamic acid-modified hyaluronic acid was synthesized.

Example 2 80 mg of sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in a mixed solution of 20 ml of water and 10 ml of dioxane. Cinnamic acid chloride 72 while stirring on ice
μl and 139 μl of triethylamine were added, and a solution prepared by dissolving 122 mg of 4-dimethylaminopyridine in 2 ml of dioxane was added, and the mixture was reacted at room temperature for 1 hour. After the reaction is completed, the modified hyaluronic acid is separated and precipitated by ethanol precipitation.
It was washed and freeze-dried to obtain white cinnamic acid-modified hyaluronic acid powder. The obtained white powder was dissolved in water and subjected to GPC analysis. Substituent with UV detector, R
Hyaluronic acid was quantified using an I detector, and the binding rate was evaluated. The coupling ratio was 0.1% by mass.

Example 3 The modified sodium hyaluronate produced in Example 2 was dissolved in distilled water to prepare a 1% by mass aqueous solution of modified hyaluronic acid. The pH of this aqueous solution is adjusted to p with 1 mmol / l hydrochloric acid.
Adjusted to H1.5. Aqueous solution of modified hyaluronic acid 1
5 ml was placed in a 30 ml glass bottle and placed in a freezer set at -20 ° C. After standing for 65 hours, the mixture was thawed at 25 ° C. As a result, a sponge-like modified hyaluronic acid gel was obtained. Next, this gel was immersed several times in a 100 mmol / l phosphate buffer (pH 6.8) to completely neutralize the gel, then frozen at -20 ° C, freeze-dried and sponge-like modified hyaluronic acid. The acid was obtained. The sponge-like modified hyaluronic acid was used for a solubility test of the modified hyaluronic acid gel.

Example 4 In Example 3, an aqueous solution of modified hyaluronic acid was prepared by changing the concentration of modified hyaluronic acid to 0.1% by mass. Then, the same operation as in Example 3 was performed. As a result, a sponge-like modified hyaluronic acid gel was obtained.

Example 5 In Example 3, an aqueous solution of modified hyaluronic acid was prepared by dissolving modified sodium hyaluronate having a molecular weight of 6 × 10 ⁵ daltons. Adjusted in the same manner as in Example 1;
Put in the freezer set. Freezing for more than 6 hours and 25 ° C
Thawing for 2 hours or more was repeated 5 times. As a result, a sponge-like modified hyaluronic acid gel was obtained.

Example 6 In Example 3, the freezing temperature was set to -10 ° C. 7
After allowing to stand in that atmosphere for 7 hours, it was thawed at 25 ° C. As a result, a sponge-like modified hyaluronic acid gel was obtained.

Example 7 1 g of modified hyaluronic acid was placed in a mortar and 5 ml of 1 mmo
A 1 / l nitric acid solution was added and the modified hyaluronic acid and the acid were mixed by stirring with a pestle for 5 minutes while cooling the vessel with ice. This was transferred to a plastic bag and left at 4 ° C. for 4 days. Thereafter, this was taken out and immersed in 500 ml of a 100 mmol / l phosphate buffer (pH 6.8), and the solution was replaced several times every 6 hours with the same solution to completely neutralize the gel. I got

Comparative Example 1 In Example 3, freezing and thawing without adjusting the pH of the aqueous solution of the modified hyaluronic acid were repeated eight times.
As a result, no change occurred in the aqueous solution of the modified hyaluronic acid. That is, it did not gel.

Comparative Example 2 Using the aqueous solution of the modified hyaluronic acid prepared in Example 3, it was air-dried at 60 ° C. to obtain a cast film having a thickness of about 100 μm. The cast film was used for the solubility test of the modified hyaluronic acid gel.

REFERENCE EXAMPLE 1 A sponge-like hyaluronic acid gel sheet was produced under the same conditions using the unmodified hyaluronic acid in place of the modified hyaluronic acid in Example 3, and used for a solubility test of the hyaluronic acid gel. Using.

Example 8 Measurement of Branching Degree of Modified Hyaluronic Acid Gel The modified hyaluronic acid gel obtained in Examples 3 to 7 was subjected to pH measurement.
It was immersed in 15 ml of 1.5 aqueous hydrochloric acid solution and hydrolyzed at 60 ° C. for 6 hours. The gel was solubilized by hydrolysis, and diluted twice with GPC solvent to a concentration of 0.05.
%, And filtered through a 0.2 μm membrane filter, and then 0.1 ml was injected to measure GPC-MALLS. The degree of branching of each of the modified hyaluronic acid gels obtained in Examples 3 to 7 was 0.5 or more.

Example 9 Solubility Test of Modified Hyaluronic Acid Gel A phosphate buffer component was added to physiological saline at a concentration of 50 mmol / l to prepare pH 7 phosphate buffered physiological saline.
The sponge-shaped modified hyaluronic acid gel obtained in the above example was washed with distilled water and dehydrated on filter paper. The modified hyaluronic acid gel containing 150 mg of modified hyaluronic acid by dry weight was immersed in phosphate buffered saline at a rate of 50 ml of phosphate buffered saline. The modified hyaluronic acid solid of Comparative Example was immersed in a dry weight of 150 mg in 50 ml of phosphate buffered saline. Then, the ratio of the modified hyaluronic acid eluted into the phosphate buffered saline at 25 ° C. was determined from the concentration of the modified hyaluronic acid in the phosphate buffered saline. Therefore, the solubility of the modified hyaluronic acid gel in a neutral aqueous solution at 25 ° C. is defined by the above test.

Measurement of Modified Hyaluronic Acid Concentration The concentration of modified hyaluronic acid in phosphate buffered saline was determined from the peak area of the RI detector using GPC.

As described above, specifically, the solubility test of the modified hyaluronic acid gel of Example 3 and the modified hyaluronic acid solid of Comparative Example 2 was conducted. For reference, a solubility test of the hyaluronic acid gel of Reference Example 1 was also performed. Table 1 shows the results.

[0055]

[Table 1]

For example, in Experiment No. When the dissolution rate of the modified hyaluronic acid gel obtained in Example 3 was examined, the dissolution rate was 0% (below the detection limit) after 1 day, and 4.8 ± 2.0% after 3 days. And after 7 days, 14.3 ± 7.7%. That is,
Even after 7 days, 85.7% of the modified hyaluronic acid gel remained. The sponge-like form was also maintained. On the other hand, in Experiment No. Thickness of about 1 obtained in Comparative Example 2
When the dissolution rate of the 00 μm cast film was examined, the dissolution rate was 100% after one day had elapsed, and the cast film was completely dissolved. Also, after 4 days and 7 days had passed, the state of 100% dissolution was maintained. Furthermore, as compared with the unmodified hyaluronic acid gel (Experiment No. 3), the dissolution rate in water was slow and significantly stable. Therefore, the modified hyaluronic acid solid obtained in the comparative example (Experiment No. 2) has a very high dissolution rate in water, whereas the modified hyaluronic acid gel obtained in the present invention (Experiment No. 1) has a very high dissolution rate. The dissolution rate in water is found to be very slow. This suggests that the modified hyaluronic acid gel obtained by the present invention has a long residence time in a living body.

Example 10 Adhesion Prevention Test in Rat Cecal Scratching Model Adhesion Induction Method Anesthetic (keratan solution) was intramuscularly injected into the lower limb of a rat (SD, female, 9 weeks or older). After disinfecting the abdominal skin, shaving was performed. The abdominal muscle of the rat was opened along the midline, the cecum was removed from the abdominal cavity, and the cecum was fixed with a perforated (φ16 mm) Teflon (registered trademark) sheet. A rotating rod (φ13 mm) covered with gauze was pressed against the cecal portion exposed from the hole and rubbed about 120 times (two places on one side). About 4 cm × 4 cm pieces of a film-like adhesion preventing material prepared using the modified hyaluronic acid gel composition obtained in Example 3 as a raw material were applied to the rubbing part, and the cecum was returned to the original state and sewn with cotton thread. In addition, a control in which the cecum was returned without treatment with the adhesion preventing material was used as a control. These treatments used 10 rats in each experiment, including controls. Necropsy was performed about one week after the operation, and the adhesion preventing effect was evaluated. Table 2 shows the results.

[0058]

[Table 2]

From Table 2, it can be seen that the rate of formation of adhesions without treatment was 10%.
The incidence of the modified hyaluronic acid gel sheet was reduced to 5 out of 9 out of 9 animals. Furthermore, the average value of the adhesion score was also reduced. In addition, no inflammation or the like was observed at the site treated with these modified hyaluronic acid gel sheets.

Example 11 Test of Wound Healing Effect of Gel Wound Dressing Material Using Rat Skin Defect Model A 7-week-old (about 200 g) Wistar strain female rat was shaved on the back and ophthalmic scissors under ether anesthesia. The back skin part was removed in a circular shape with a diameter of 2 cm using a to prepare a complete skin defect wound. An untreated group to which only medical non-woven gauze (40 × 40 mm: two layers) was applied, and a wound sheet coated with a gel sheet (30 × 30 mm) prepared using the modified hyaluronic acid gel obtained in Example 3 A treatment group to which a nonwoven fabric gauze (40 × 40 mm: two sheets) was applied was set. Six rats were used in each group. The medical nonwoven gauze was set with an adhesive bandage and further fixed by taping. The therapeutic effect was compared by measuring the change over time in the wound area. That is, the area ratio with respect to the area of the initial wound surface was determined by the following equation, and the change with time was examined. Table 3 shows the results. Area ratio (%) = {(major axis of the wound surface on observation day × minor axis) / (major axis of the initial wound surface × minor axis)} × 100

[0061]

[Table 3]

From Table 3, it was found that the modified water-insoluble hyaluronic acid gel sheet enhanced the wound treatment effect.

[0063]

As described above, according to the present invention, a modified water-insoluble hyaluronic acid gel can be obtained. Since the solubility of the hyaluronic acid gel is reduced by the cinnamic acid modification, it is useful as a method for controlling the solubility of the hyaluronic acid gel. In particular, the poorly water-soluble modified hyaluronic acid gel of the present invention provides (1) ideal in vivo storage properties and extremely high safety as an anti-adhesion material, and (2) greatly improves the residence time on the wound surface. This makes it possible to remarkably suppress post-operative adhesion. (3) Further, it becomes possible to apply the modified hyaluronic acid gel directly bound with a drug as a carrier of a drug delivery system.

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Claims (14)

[Claims] 1. A modified hyaluronic acid gel formed of modified hyaluronic acid alone, which is poorly soluble in a neutral aqueous solution. 2. The modified hyaluronic acid gel according to claim 1, wherein a substituent is introduced into a hydroxyl group of hyaluronic acid. 3. The modified hyaluronic acid gel according to claim 2, wherein a modified hyaluronic acid having a substituent of cinnamic acid is used. 4. The introduction rate of cinnamic acid into hyaluronic acid is 0.
The modified hyaluronic acid gel according to claim 3, wherein the content is not less than 05% by mass. 5. The modified hyaluronic acid gel according to claim 1, wherein the solubility in a neutral aqueous solution at 25 ° C. in one day is 25% or less. 6. The modified hyaluronic acid which has been solubilized by treating the modified hyaluronic acid gel under accelerated acid hydrolysis conditions for the modified hyaluronic acid partially contains a molecular weight fraction having a degree of branching of 0.5 or more. 2. The method according to claim 1, wherein
6. The modified hyaluronic acid gel according to any one of items 5 to 5. 7. An aqueous solution of the modified hyaluronic acid having a pH of 3.
5. A method for producing a modified hyaluronic acid gel, comprising adjusting the aqueous solution to 5 or less, freezing and then thawing the aqueous solution at least once. 8. Modified hyaluronic acid by coexisting a modified hyaluronic acid, water having a modified hyaluronic acid concentration of 5% by mass or more, and an acid component in an equimolar amount or more with a carboxyl group of hyaluronic acid, and maintaining the coexistence state. A method for producing a modified hyaluronic acid gel, which comprises forming an acid gel. 9. Under the coexistence of a modified hyaluronic acid, water having a modified hyaluronic acid concentration of 5% by mass or more, and an acid component having an equimolar amount or more with respect to the carboxyl group of the modified hyaluronic acid, the coexistence state is from -10 ° C. The method for producing a modified hyaluronic acid gel according to claim 8, wherein the modified hyaluronic acid gel is formed by holding the gel at 30 ° C. 10. Under coexistence of a modified hyaluronic acid, water having a modified hyaluronic acid concentration of 5% by mass or more, and an acid component having an equimolar amount or more with respect to a carboxyl group of the modified hyaluronic acid, the coexistence state is −10 ° C. A method for producing a modified hyaluronic acid gel, comprising forming a modified hyaluronic acid gel at 30 ° C. and treating the gel with a solution used for neutralization. 11. The method for producing a modified hyaluronic acid gel according to claim 7, wherein the modified hyaluronic acid is cinnamic acid-modified hyaluronic acid. 12. A medical material comprising a gel formed of modified hyaluronic acid alone, which satisfies the following requirements (a) and (b): (A) The solubility in one day in a neutral aqueous solution at 25 ° C. is 25
% Or less, the modified hyaluronic acid solubilized by treating the modified hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid has a branched structure, and in the solubilized modified hyaluronic acid, Partially contains a molecular weight fraction having a degree of branching of 0.5 or more. 13. The gel formed of the modified hyaluronic acid alone, wherein the gel is selected from the group consisting of a sheet, a film, a crushed, a sponge, a lump, a fiber, and a tube.
13. The medical material according to claim 12, which is a seed. 14. The medical material may be an adhesion preventing material, a wound dressing,
14. The medical material according to claim 13, wherein the medical material is one selected from the group consisting of a drug carrier for a drug delivery system (DDS).