JP2003252905A - Crosslinked hyaluronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a crosslinked hyaluronic acid causing no affect to biological compatibility due to modification of the molecular structure of the hyaluronic acid, having practical stability and priority in quality control and usable in a biologically compatible material field. <P>SOLUTION: The crosslinked hyaluronic acid is characterized (1) in that the carboxy group of the hyaluronic acid is crosslinked to the hydroxy group of the same hyaluronic acid molecule and/or another hyaluronic acid molecule via an ester bond and (2) in that the carboxy group of the hyaluronic acid is crosslinked to a primary hydroxy group at the sixth position of an N-acetyl- D-glucosamine unit of the same hyaluronic acid molecule and/or another hyaluronic acid molecule via the ester bond. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel crosslinked hyaluronic acid.

[0002]

BACKGROUND OF THE INVENTION Hyaluronic acid is a straight chain formed from disaccharide units in which N-acetyl-D-glucosamine and D-glucuronic acid are alternately linked by β-1,4 bond and β-1,3 bond. It is a high molecular weight polysaccharide. Hyaluronic acid is distributed in the connective tissues of mammals, and is also known to be present in chicken bones, streptococcal capsules, and the like. In addition to chicken umbilical cords and umbilical cords used as extraction materials, purified products have also been prepared from streptococcal cultures.

Hyaluronic acid is polydisperse in molecular weight, but has no species and organ specificity, and is known to exhibit excellent biocompatibility even when transplanted or injected into a living body. . Furthermore, in order to obtain a hyaluronic acid derivative having new physical properties, a wide variety of chemical modifications and chemical crosslinking have been proposed. Many techniques for controlling the water solubility of hyaluronic acid by chemical modification and chemical crosslinking of hyaluronic acid have been reported, and formation of hyaluronic acid gel by three-dimensional crosslinking is also known.

Typical of these are highly swelling crosslinked hyaluronic acid gels obtained by using a bifunctional reagent such as divinylsulfone, bisepoxides and formaldehyde as a crosslinking agent. (U.S. Pat. No. 4,582,865, JP-B-6-37575, JP-A-7-97401, JP-A-60-13
0601).

Also disclosed is a method for chemically modifying hyaluronic acid, which utilizes the feature that the tetrabutylammonium salt of hyaluronic acid is soluble in an organic solvent such as dimethylsulfoxide (Japanese Patent Laid-Open No. 3-105003).

Further, as a method which does not use a chemical reagent for forming a covalent bond, hyaluronic acid and a polymer compound having an amino group or an imino group are used, and a carboxyl group of hyaluronic acid and an amino group or an imino group of the polymer compound are used. Is disclosed as a ionic complex to prepare a hyaluronic acid polymer complex (JP-A-6-7).
3103).

When cross-linking hyaluronic acid using a bifunctional reagent, the hyaluronic acid aqueous solution has a low concentration,
The cross-linking efficiency is significantly reduced. That is, one reactive group of the bifunctional reagent reacts with hyaluronic acid, and the other reactive group remains unreacted. Both reactive groups of the bifunctional reagent are less efficient at reacting with hyaluronic acid to form crosslinks. Finally, a crosslinked product having a high degree of substitution with the structural unit of hyaluronic acid can be obtained. Such cross-linked hyaluronic acid not only significantly reduces the original properties of the molecular structure of hyaluronic acid, but also increases the amount of chemically reactive substances bound to it, thus increasing the risk of toxicity and biocompatibility. In essence, you have to hold it.

A method for obtaining crosslinked hyaluronic acid without going through a crosslinking bridge with a bifunctional reagent has also been proposed. Also disclosed is a method of reacting a tetrabutylammonium salt of hyaluronic acid in dimethylsulfoxide with triethylamine and 2-chloro-1-methylpyridinium iodide and reacting to form an ester bond between the carboxyl group and the hydroxyl group of hyaluronic acid. (European Patent 0341745A
1). In this method, the carboxyl group of hyaluronic acid is reacted with an activator to form an activated ester intermediate, and the activated ester intermediate reacts with a hydroxyl group of hyaluronic acid in an anhydrous environment to form a bridge bridge by a bifunctional reagent. It is said that an internal ester that does not go through is formed. However, it is not clear what structure the ester bond including the lactone bond is formed with the four types of hydroxyl groups of the disaccharide unit constituting hyaluronic acid.

It is known that a biodegradable product of a hyaluronic acid crosslinked product which is crosslinked by an ester bond using a carboxyl group of hyaluronic acid is good. Using a di- or polyfunctional epoxide as the activating reagent, the carboxyl group of hyaluronic acid is reacted with the epoxy group to form an ester bond. Cross-linked hyaluronic acid is obtained by ester linkage via a cross-linking bridge with a bifunctional reagent. It is disclosed that the crosslinkable hyaluronic acid obtained by this method has good degradability in phosphate buffered saline, that is, the hydrolyzability of this ester bond is good (Japanese Patent Publication No. 7-116242). , See Special Publication 3-503551).

[0010]

DISCLOSURE OF THE INVENTION In order to maximize the excellent biocompatibility inherent in hyaluronic acid itself, the degree of substitution of hyaluronic acid with respect to the structural unit is minimized, that is, the crosslinking efficiency is high, A cross-linked hyaluronic acid having a controlled quality-controllable molecular structure and having stability that can be used as a biocompatible medical material has not yet been developed.

The present inventors have extensively studied the physicochemical properties of hyaluronic acid itself in order to achieve the above object. As a result, a novel (1) cross-linked hyaluronic acid which is cross-linked with a carboxyl group and a hydroxyl group of hyaluronic acid by an ester bond, particularly (2) N-acetyl-D-glucosamine of a hyaluronic acid molecule having the same carboxyl group of hyaluronic acid. The crosslinked hyaluronic acid which is crosslinked to the primary hydroxyl group at the 6-position of the unit and / or to the primary hydroxyl group at the 6-position of the N-acetyl-D-glucosamine unit of another hyaluronic acid molecule by an ester bond is hyaluronic acid itself. It has a high cross-linking efficiency that maximizes the excellent biocompatibility characteristics that are inherently possessed, has stability that can be used as a biocompatible medical material, and has a controlled quality controllable molecular structure. It was found to be crosslinked hyaluronic acid.

Further, the modified hyaluronic acid prepared by the conventional method is
Since a chemically reactive substance is used in spite of many efforts, there is a problem that the danger such as toxicity and biocompatibility due to new modification must be inherently held.

For example, a hyaluronic acid derivative obtained by an ionic method using chemical modification / crosslinking and a metal salt is
Even if the storability in the living body can be improved, the modified hyaluronic acid has a structure different from that of natural hyaluronic acid because it encapsulates a cross-linking substance or metal in the molecule of hyaluronic acid by covalent bond or ionic bond. However, it cannot be said that the physiological effects, biocompatibility, and safety including toxicity are essentially equivalent to those of hyaluronic acid, and further, the residual toxicity of these cross-linking agents and the cross-linking contained in the degradation products in vivo. It was difficult to completely avoid the problem of drug safety.

Without using a crosslinking agent such as a bifunctional reagent,
As a method of introducing a crosslinked structure into hyaluronic acid, there is a method of directly bonding a functional group of hyaluronic acid itself, that is, a carboxyl group, a hydroxyl group or an acetamide group. The crosslinked structure obtained by this method is an ester bond and an amide bond. The crosslinked structure formed by the intermolecular ester bond is advantageous in terms of biocompatibility because it is hydrolyzed in vivo to return to the structure of hyaluronic acid itself.

The functional group existing in the disaccharide unit constituting hyaluronic acid is an acetamide group of N-acetyl-D-glucosamine, a secondary hydroxyl group bonded to C-4 and a primary hydroxyl group bonded to C-6, D -The carboxyl group of glucuronic acid,
A secondary hydroxyl group bonded to C-2 and a secondary hydroxyl group bonded to C-3. The ester bond can be formed between one kind of carboxyl group and four kinds of hydroxyl groups. The ester bond between the secondary hydroxyl group directly bonded to the sugar ring and the carboxyl group is more unstable than the ester bond between the primary hydroxyl groups not directly bonded to the sugar ring.

Crosslinked hyaluronic acid, which is crosslinked by an intermolecular ester bond which is unstable to hydrolysis and the like, has a poor storage stability of the final product, a medical material, which is a serious obstacle to practical use. In addition, the mixture of four kinds of intermolecular ester bonds having different stability causes a fatal problem in quality control and becomes a major obstacle in practical use.

The present inventors have found that the crosslinked hyaluronic acid obtained in the present invention has ideal biocompatibility as a medical material,
Moreover, the stability that can be used as a biocompatible medical material,
The inventors have found that the molecular structure is controlled so that quality control is possible, and completed the present invention.

[0018]

Means for Solving the Problems That is, the present invention provides (1)
Crosslinked hyaluronic acid, wherein the carboxyl group of hyaluronic acid is crosslinked to the hydroxyl group of the same hyaluronic acid molecule and / or to the hydroxyl group of another hyaluronic acid molecule by an ester bond, (2) carboxyl group of hyaluronic acid Is an ester at the 6-position primary hydroxyl group of the N-acetyl-D-glucosamine unit of the same hyaluronic acid molecule, and / or at a 6-position primary hydroxyl group of the N-acetyl-D-glucosamine unit of another hyaluronic acid molecule. The crosslinked hyaluronic acid according to (1), which is crosslinked by a bond.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The hyaluronic acid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissue or produced by a fermentation method. 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.
Streptococcus equi FM described in Japanese Patent No. 92
-100 (Microtechnology Research Institute, No. 9027), JP-A-2-2346
Streptococcus equi FM described in Japanese Patent No. 89
A mutant strain capable of stably producing hyaluronic acid in a high yield, such as -300 (Ministry of Industrial Science and Technology No. 2319) is desirable. What is cultivated and purified using the above mutant strain is used.

The crosslinked hyaluronic acid referred to in the present invention has a molecular weight higher than that of the starting hyaluronic acid if the hyaluronic acid retains the crosslinked structure and is soluble in the solvent. The molecular weight can be generally measured using GPC, but the molecular weight of hyaluronic acid that retains a crosslinked structure, that is, has a branched structure, is measured using GPC-MALLS (multi-angle light scattering). It is more accurate to do. By controlling the molecular weight and the degree of crosslinking of hyaluronic acid used as a raw material, a crosslinked hyaluronic acid insoluble in a solvent can also be obtained.

The degree of crosslinking of the crosslinked hyaluronic acid referred to in the present invention,
That is, the amount of intermolecular ester bond to be introduced can be arbitrarily controlled according to the purpose of use and required characteristics of the crosslinked hyaluronic acid. The ester bond amount can be defined as a ratio to the total number of carboxyl groups in hyaluronic acid. Hyaluronic acid is used as an aqueous solution in joint injections and the like. When preparing a water-soluble crosslinked hyaluronic acid for the purpose of improving the viscoelastic properties of an aqueous hyaluronic acid solution, the intermolecular ester bond content to be introduced is 0.5%, although it depends on the molecular weight of the hyaluronic acid used as a raw material. Less than is preferred.

In the case of solvent-insoluble crosslinked hyaluronic acid,
Although it depends on the molecular weight of hyaluronic acid used as a raw material, the amount of intermolecular ester bond to be introduced is preferably less than 1%. When the amount of intermolecular ester bond to be introduced is 1% or more,
It becomes a cross-linked hyaluronic acid with insufficient swelling properties, and it becomes difficult to express the original properties of hyaluronic acid.

The molecular structure of the crosslinked hyaluronic acid referred to in the present invention can be confirmed by using NMR. The molecular structure of hyaluronic acid itself has been analyzed in detail using NMR, including its terminal groups (Carbohydr.Res.Vol245, p113-
128, 1993, Macromolecules Vol29, p2894-2902, 1996).
When the structure of hyaluronic acid is modified by modification or crosslinking of hyaluronic acid, a new peak on the spectrum can be assigned using various techniques such as multidimensional NMR to determine the structure. When the ratio of the modified structure is small and its detection is difficult, it is also possible to concentrate the structure to be detected and then analyze it by utilizing a decomposition reaction having a high recognition property for the molecular structure such as enzymatic decomposition.

There are various types of enzymes that decompose hyaluronic acid, such as hyaluronidase derived from actinomycetes and hyaluronidase derived from ovine testicles. It is known that the decomposition site of the main chain of hyaluronic acid varies depending on the type of enzyme used. The structure of hyaluronic acid oligosaccharides digested with ovine testicular hyaluronidase has been analyzed, including the structure of the resulting terminal groups. It is known that in the enzymatic degradation of hyaluronic acid, transglycosylation occurs depending on the degradation conditions. To avoid transglycosylation,
It is necessary to adjust the pH of the hyaluronic acid aqueous solution to a low level (T
he Journal Biological Chemistry.Vol. 270, p3741-37
47, 1995).

The crosslinked hyaluronic acid referred to in the present invention does not contain a lactone bond. The lactone bond is a kind of ester bond. The lactone bond is an ester bond with strain due to ring closure, and it is known that the stretching vibration of carbonyl observed in the infrared absorption spectrum is shifted to a higher wave number side than the ester bond without strain. .
In the crosslinked hyaluronic acid referred to in the present invention, strained ester bonds are not observed in the infrared absorption spectrum.

The hyaluronic acid used in the present invention can be similarly preferably used as a polymer purified product obtained by animal tissue extraction or fermentation, or a low molecular weight product obtained by further hydrolyzing. The hyaluronic acid referred to in the present invention is used under the concept of including its alkali metal salts, for example, sodium, potassium and lithium salts.

The aqueous solution of hyaluronic acid used in the present invention is obtained by mixing hyaluronic acid powder and water and stirring the mixture.

In the present invention, the esterification reaction selectivity is improved by esterification under mild reaction conditions. Since the activated ester intermediate using an activator such as 2-chloro-1-methylpyridinium iodide has high reactivity, it has no selectivity for the reaction with respect to four kinds of hydroxyl groups of the disaccharide unit constituting hyaluronic acid. . Therefore, even a strained ester bond is formed.

By esterifying under mild reaction conditions, side reactions other than the esterification reaction can be suppressed. Hyaluronic acid is easily reduced in molecular weight by a main chain cleavage reaction. The main chain cleavage reaction due to hydrolysis is significantly promoted by acidity or alkalinity as compared with neutrality. It is also known that the main chain cleavage is significantly promoted by radical decomposition. These reactions are further promoted by heating.
The lowering of the molecular weight due to the side reaction results in a decrease in the crosslinking efficiency.

In the present invention, the aqueous solution of hyaluronic acid is adjusted to be acidic, and the dissociated carboxyl groups are converted to the acid form to obtain crosslinked hyaluronic acid. A method of preparing an aqueous solution of hyaluronic acid to be acidic and heating it is disclosed as a method of preparing low molecular weight hyaluronic acid. (See Japanese Patent Laid-Open No. 1-266102) When an aqueous solution of hyaluronic acid is heated under acid, N-
It is described that a deacetylation reaction of the acetyl-D-glucosamine unit also occurs.

It is necessary to lower the reaction temperature in order to suppress lowering of the molecular weight due to acid hydrolysis, which competes with cross-linking formation due to intermolecular esterification reaction. The reaction temperature for preferentially forming crosslinks is preferably room temperature or lower, and more preferably 10 ° C or lower.

In order to accelerate the intermolecular esterification reaction, it is necessary to increase the concentration of hyaluronic acid. Since the esterification reaction by dehydration condensation is an equilibrium reaction, it is necessary to increase the concentration of hyaluronic acid in order to reduce the amount of water present in the reaction system. Hyaluronic acid concentration is 5% by mass
The above is preferable, and more preferably 10% by mass or more.

Addition of a substance which catalyzes the dehydration condensation reaction to the reaction system is important for promoting the intermolecular esterification reaction. An acidic catalyst is generally used as a catalyst for promoting the dehydration condensation reaction, and sulfuric acid, hydrochloric acid, an aromatic sulfonic acid derivative or the like can be used.

The degree of crosslinking depends on the reaction temperature, hyaluronic acid concentration,
The reaction time can be controlled together with the type and amount of the dehydration condensation catalyst to be added.

The cross-linked hyaluronic acid thus obtained must be free of components such as the acid used for adjusting the acidity in order to avoid acid hydrolysis of hyaluronic acid. In order to remove components such as acids, washing with an aqueous solvent or dialysis is usually performed. There is no particular limitation as long as it does not impair the function of the cross-linked hyaluronic acid. For example, water, physiological saline, phosphate buffer, etc. are used, but physiological saline, phosphate buffer, etc. are preferably used. To be

When acid-type carboxyl groups remain in the washed crosslinked hyaluronic acid, it is necessary to salify into sodium-type or the like in order to avoid acid hydrolysis of hyaluronic acid. The crosslinked hyaluronic acid aqueous solution can be salified by adjusting the pH of the crosslinked hyaluronic acid aqueous solution to 7 with an aqueous sodium hydroxide solution or by immersing the crosslinked hyaluronic acid in physiological saline or phosphate buffered physiological saline.

The washed and chlorinated cross-linked hyaluronic acid may be subjected to treatments such as a solution state, a state immersed in a solvent, a wet state containing a solvent, air drying, reduced pressure drying or freeze drying depending on the purpose of use. It is used as a medical material in a dried state.

For use as a medical material, high biocompatibility is essential, and it is generally necessary to prove that there is no cytotoxicity. The crosslinked hyaluronic acid of the present invention satisfies these conditions sufficiently, and can be used without particular limitation as long as it is a field in which hyaluronic acid is used as a biodegradable medical material. For example, anti-adhesion material, joint injection, soft tissue injection, vitreous substitute,
Examples include use in cosmetics, biomedical products such as medical instruments and medical devices used for diagnosis and treatment, or pharmaceutical compositions.

Cross-linked hyaluronic acid and its molded products are
As a matter of course, the use in a single form can be expected to enhance the effect by mixing or combining with different forms of cross-linked hyaluronic acid, and further combining them by mixing or combining with a hyaluronic acid solution.

For use as a medical material, a material as biocompatible as hyaluronic acid, such as chondroitin sulfate, carboxymethyl cellulose, etc., can be added to the crosslinked hyaluronic acid. These materials can be mixed and compounded in the reaction for forming crosslinked hyaluronic acid, and are not limited in any way. Moreover, a pharmaceutically or physiologically active substance can be added to the crosslinked hyaluronic acid to prepare a crosslinked hyaluronic acid containing these substances, and there is no limitation.

[0041]

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

Example 1 Sodium hyaluronate having a molecular weight of 2.8 × 10 ⁴ daltons was dissolved in distilled water to prepare a 6.0 mass% aqueous solution of hyaluronic acid. The pH of the adjusted aqueous solution of hyaluronic acid was 6.0. The pH of this aqueous solution was adjusted to pH 1.2 with 1N hydrochloric acid. 50 ml of an acidic aqueous solution of hyaluronic acid was placed in a freezer set at -20 ° C. 150
After freezing for a day, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained. An acidic aqueous solution of crosslinked hyaluronic acid was neutralized to pH 7.0 with 0.2N aqueous NaOH solution.
The neutralized hyaluronic acid aqueous solution was freeze-dried to recover crosslinked hyaluronic acid. The molecular weight was 8.7 × 10 ⁴ .

Example 2 Sodium hyaluronate having a molecular weight of 2.0 × 10 ⁶ dalton was dissolved in distilled water to prepare a 1.0 mass% aqueous solution of hyaluronic acid. The pH of the adjusted aqueous solution of hyaluronic acid was 6.0. The pH of this aqueous solution was adjusted to pH 1.5 with 1N nitric acid. 50 ml of an acidic aqueous solution of hyaluronic acid was freeze-dried. The obtained dried product was left in the refrigerator overnight. A crosslinked hyaluronic acid insolubilized in the solvent was obtained. The obtained crosslinked hyaluronic acid was washed with distilled water, and then immersed in phosphate buffered physiological saline adjusted to pH 7 by adding a phosphate buffer component at a concentration of 50 mM to physiological saline, washed, and then distilled. It was washed with water. The dried crosslinked hyaluronic acid was recovered.

Example 3 H ¹ -NMR and C ¹³ -N of the crosslinked hyaluronic acid obtained in Example 1 and sodium hyaluronate used as a raw material
The MR was measured. NMR measurement is JNMα manufactured by JEOL Ltd.
Performed using -500. The measuring solvent is 0.15M N
Heavy water in which aCl and a 20 mM phosphate buffer component (pD = 6.5) were dissolved was used.

H ¹ -NMR and C ¹³ -of crosslinked hyaluronic acid
The NMR spectrum was completely in agreement with the spectrum of sodium hyaluronate used as a raw material. That is, the structure change at the cross-linking point of cross-linked hyaluronic acid and the structure change due to side reaction during the reaction for preparing cross-linked hyaluronic acid have not been detected. The detection sensitivity of NMR measurement for hyaluronic acid is about 1 mol% based on the disaccharide structural unit of hyaluronic acid. In order to detect the structure of a trace amount of cross-linking points existing in cross-linked hyaluronic acid, it is necessary to concentrate the cross-linking points.

Example 4 Enzymatic Degradation of Crosslinked Hyaluronic Acid 2.0 g of the crosslinked hyaluronic acid obtained in Example 1 was added to 0.1 g.
M acetate buffer (pH 5.0, NaCl 0.15M) 40
Dissolved in ml. The solution was aseptically filtered through a 0.2 μm membrane filter. Hyaluronidase (Sigma, type 5, about 3000 units / mg) 80 mg
It was dissolved in 1M acetate buffer and added aseptically. 4 at 40 ° C
After stirring for 0 hour, the mixture was cooled to obtain an enzyme-decomposed product solution.

Comparative Example 1 0.2 g of sodium hyaluronate having a molecular weight of 2.8 × 10 ⁴ daltons for enzymatic decomposition of hyaluronic acid was added to 0.1 M acetate buffer (pH 5.0, N).
aCl 0.15M) 4 ml. The solution was aseptically filtered through a 0.2 μm membrane filter. Hyaluronidase (Sigma, Type 5, about 3000 units /
8 mg) was dissolved in 0.1 M acetate buffer and added aseptically. After stirring at 40 ° C. for 40 hours, the mixture was cooled to obtain an enzymatic decomposition product solution.

Example 5 Hydrolysis of Crosslinked Hyaluronic Acid 25 mg of the crosslinked hyaluronic acid obtained in Example 1
M acetate buffer (pH 5.0, NaCl 0.15M)
It was dissolved in 5 ml. No enzyme was added to this solution at 40 ° C.
It was left for 40 hours and cooled. The molecular weight of crosslinked hyaluronic acid in this solution was 8.4 × 10 ⁴ . That is,
It can be seen that the hydrolysis of crosslinked hyaluronic acid hardly occurs under the enzymatic degradation conditions of Example 4.

Example 6 Measurement of Molecular Size of Enzymatic Degradation Product The molecular size of the enzymatic degradation product obtained in Example 4 and Comparative Example 1 was measured using GPC. Using one SB802.5HQ manufactured by Showa Denko KK as a GPC column, and using 830-RI manufactured by JASCO Corporation as a differential refractive index detector, a 0.2M aqueous solution of sodium nitrate as a solvent, a measurement temperature of 40 ° C.,
The flow rate was measured at 0.6 ml / min. The measurement results are shown in FIG.

From the results shown in FIG. 1, a mixture of hexasaccharides, tetrasaccharides and oligosaccharides having two saccharide units was produced in the enzymatic degradation product of hyaluronic acid, whereas a mixture of hexasaccharides in the enzymatic degradation product of crosslinked hyaluronic acid was produced. It can be seen that oligosaccharides having a larger molecular size than the unit are present. Since the three-dimensional structure of the cross-linking point of cross-linked hyaluronic acid is different from the three-dimensional structure of hyaluronic acid having a linear structure, decomposition by hyaluronidase does not proceed to a molecular size of 6 sugar units or less.

Example 7 Purification of crosslinked hyaluronic acid-derived oligosaccharides having a large molecular size The enzyme hydrolyzate solution obtained in Example 4 was filtered through a 0.2 μm membrane filter. To about 40 ml of this solution, 1.2 g of sodium chloride was added, and further about 150
ml was added dropwise with stirring. The resulting precipitate was filtered off and dissolved in 30 ml of a 0.2 M aqueous solution of sodium nitrate. The insolubilized hyaluronidase was filtered off and the solution was recovered.
Showa Denko SB2002.5HQ as a GPC column
Was used and LC-908 manufactured by Nippon Analytical Industry Co., Ltd. was used to perform preparative purification with a 0.5 M aqueous solution of sodium nitrate as a solvent, room temperature, a flow rate of 2.0 ml / min, and a sample injection amount of 1 ml. The preparative GPC chromatogram is shown in FIG. 19.0
About 20.5 minutes was collected as an oligosaccharide having a large molecular size. The preparative solution was filtered with a 0.2 μm membrane filter. To the fractionated solution, 5 volumes of ethanol were added to precipitate oligosaccharides having a large molecular size. The precipitate was dissolved in heavy water, freeze-dried and collected.

Comparative Example 2 Purification of Hyaluronic Acid Oligosaccharides 23.0-2 of the preparative GPC chromatogram in Example 7.
3.5 minutes was collected as a hyaluronic acid oligosaccharide. The preparative solution was filtered with a 0.2 μm membrane filter. Hyaluronic acid oligosaccharides were precipitated by adding 5 volumes of ethanol to the preparative solution. The precipitate was dissolved in heavy water, freeze-dried and collected.

Example 8 NMR Measurement of Crosslinking Point Component The oligosaccharide having a large molecular size recovered in Example 7 and the hyaluronic acid oligosaccharide recovered in Comparative Example 2 were dissolved in the solvent used in Example 2 and analyzed by NMR. The measurement was performed. The chemical shift was based on TSP. The measurement results are shown in FIG. 3 and FIG.
Shown in. That is, C ^{13 of an} oligosaccharide having a large molecular size
The -NMR spectrum in FIG. 3, showing the C ¹³ -NMR spectrum of hyaluronic acid oligosaccharide in FIG.

From the results of the C ¹³ -NMR spectra of FIGS. 3 and 4, there are many small intensity peaks which are not found in hyaluronic acid oligosaccharides in oligosaccharides having a large molecular size. Here, peaks that are largely shifted from the main peak are peaks of 171 ppm and 67 ppm. The peaks of both of these peaks are the carboxyl group of the D-glucuronic acid unit of hyaluronic acid and 1 of the N-acetyl-D-glucosamine unit.
C-6 of the D-glucuronic acid unit and C of the N-acetyl-D-glucosamine unit shifted from the main peak due to the formation of an ester bond between the primary hydroxyl groups, respectively.
-6. The small intensity peaks other than 171 ppm and 67 ppm are peaks separated from the main peak by the remote effect accompanying the introduction of the ester bond. That is, it is understood that the crosslinked hyaluronic acid of the present invention is crosslinked by the formation of a selective ester bond between the carboxyl group of the D-glucuronic acid unit of hyaluronic acid and the primary hydroxyl group of the N-acetyl-D-glucosamine unit. . C ¹³ -NM in FIG.
The result of R spectrum is Carbohydr. Res. Vol245, p113-
This is in agreement with the C ¹³ -NMR spectrum of the hyaluronic acid oligosaccharide described in 128,1993.

Example 9 The crosslinked hyaluronic acid obtained in Example 2 was placed in the phosphate buffered saline described in Example 2 and allowed to stand at room temperature for 2 days,
Allowed to swell sufficiently. The swollen crosslinked hyaluronic acid was washed with distilled water and swollen with 50 ml of the solvent described in Example 4. 10 mg of sodium azide was added. 30 mg of hyaluronidase described in Example 4 was added, and the mixture was stirred at 40 ° C. for 40 hours and then cooled to obtain a solution of an enzymatic decomposition product. Oligosaccharides having a large molecular size were recovered from this solution by the same method as in Example 7, and C ¹³ -NMR was measured in the same manner as in Example 8. The spectrum of the same pattern as FIG. 3 was obtained. That is, the cross-linked hyaluronic acid obtained in Example 2 was also D- of hyaluronic acid.
It can be seen that cross-linking occurs due to the formation of a selective ester bond between the carboxyl group of the glucuronic acid unit and the primary hydroxyl group of the DN-acetylglucosamine unit.

[0056]

INDUSTRIAL APPLICABILITY As described above, the crosslinked hyaluronic acid obtained according to the present invention avoids adverse effects on biocompatibility due to modification of the molecular structure of hyaluronic acid, and has practical stability and superior quality control. Therefore, it is useful in the field of biocompatible materials.

[Brief description of drawings]

FIG. 1 is a GPC chromatogram of the enzymatic degradation product of crosslinked hyaluronic acid described in Example 4 and the enzymatic degradation product of hyaluronic acid described in Comparative Example 1.

FIG. 2 is a preparative GPC chromatogram of the enzymatic degradation product of crosslinked hyaluronic acid described in Example 4.

FIG. 3 is a C ¹³ -NMR spectrum of the oligosaccharide having a large molecular size recovered in Example 7.

FIG. 4 C of hyaluronic acid oligosaccharide recovered in Comparative Example 2
^{It is a 13-} NMR spectrum.

[Explanation of symbols]

1 G of the enzymatic degradation product of crosslinked hyaluronic acid described in Example 4
PC chromatogram 2 GPC of the enzymatic degradation product of hyaluronic acid described in Comparative Example 1.
Chromatogram

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Claims (2)

[Claims] 1. A crosslinked hyaluronic acid characterized in that a carboxyl group of hyaluronic acid is crosslinked to a hydroxyl group of the same hyaluronic acid molecule and / or to a hydroxyl group of another hyaluronic acid molecule by an ester bond. 2. A carboxyl group of hyaluronic acid is a primary hydroxyl group at the 6-position of an N-acetyl-D-glucosamine unit of the same hyaluronic acid molecule, and / or an N-acetyl-D-glucosamine of another hyaluronic acid molecule. 6th place of unit
The crosslinked hyaluronic acid according to claim 1, wherein the primary hydroxyl group is crosslinked by an ester bond.