JPH0797401A - Crosslinked hyaluoronic acid, sustained-release preparation, and plug - Google Patents

Abstract

PURPOSE:To obtian a water-insoluble, low-swelling, crosslinked hyaluronic acid for a sustained-release preparation and a plug by crosslinking the carboxyl groups of a hyaluronic acid residue with an epoxy-terminated crosslinking agent. CONSTITUTION:The title acid is represented by the formula (wherein R is the residue of an epoxy crosslinking agent) and is formed by crosslinking the carboxyl groups of a hyaluronic acid residue with an epoxy-terminated crosslinking agent (e.g. diglycidyl ether or diglycidyl ester). The degree of crosslinking of the acid is desirably such that the content of the crosslinked carboxyl groups is 0.3% or above based on all the carboxyl groups of the hyaluoronic acid. A sustained-release preparation is formed from a conjugate of the crosslinked hyaluronic acid with a medicine. A desirable example of the sustained-release preparation is an anticancer agent in which the medicine and the crosslinked hyaluronic acid form a conjugate through the ionic interaction of the carboxyl groups of the hyaluronic acid on the medicine. Further, a plug can be formed from the water-insoluble crosslinked hyaluronic acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to crosslinked hyaluronic acid, sustained release preparations and embolic agents using the same, and more particularly to improvement of the crosslinking mechanism.

[0002]

2. Description of the Related Art Hyaluronic acid is a high-molecular-weight in-vivo component and, of course, has high biocompatibility and is excellent in functionality such as water retention, and is used by itself in the fields of cosmetics and the like. It is also expected to be used as a carrier for drug delivery systems due to its complex with components. On the other hand, in recent anti-cancer therapy, anti-cancer drug is distributed only around the affected area of the cancer to prevent side effects on normal cells, and at the same time, a polymeric carrier such as a resin is used to study the administration method and dosage form for sustained drug efficacy. Is being actively carried out. As a so-called sustained-release topical administration method for continuously supplying an effective concentration of a drug only to the affected area for a long time, for example, a carcinostatic agent is put in a capsule or a tablet-formed preparation is embedded in the peripheral area of the cancer affected area. Examples include a method of injecting the drug-encapsulating microsphere into a muscle or a blood vessel and embolizing the local blood vessel so that the drug is leached only into the occluded local blood vessel.

[0003] Conventionally, a polymer coating or matrix made into microspheres for sustained release of a drug has been formulated with ethyl cellulose or wax (JP-A-54-163).
808), polylactic acid microencapsulated preparations (JP-A-54-55517, JP-A-59-33214), and the like. Even if hyaluronic acid is used as a drug carrier, for example, JP-A-62-129226.
JP-A-1-156912 and the like are known. In addition, in the combination of hyaluronic acid and an anti-cancer agent, covalent conjugates of hyaluronic acid and adriamycin (doxorubicin hydrochloride) are POLYMER PREPRINTS, JAPAN VOL142, No.3, 898 (1993).
Etc. have been reported.

[0004]

However, for example, when a microsphere-formulated preparation is administered to an affected area, the drug release rate tends to be very high in the initial period of administration, which may cause side effects or sustained release over a long period of time. It was difficult to obtain a sustained-release effect of supplying a drug at a constant concentration, such as the inability to obtain properties. Further, the release rate of the drug is sensitive to the shape of the particles and the particle size distribution, and a uniform shape and a uniform particle size have been required. However, it is difficult to form such fine and uniform particles, which makes it difficult to control the drug release rate well. In addition, even in a sustained-release preparation using hyaluronic acid as a carrier, hyaluronic acid is water-soluble and the hyaluronic acid in vivo can be absorbed only by chemically or physically adsorbing the drug on the hyaluronic acid. The drug was rapidly released with the dissolution of the acid, and it was difficult to properly obtain the sustained release effect of the drug.

Further, in the case of a covalent conjugate of hyaluronic acid and adriamycin, although there is an advantage that the drug is retained until the hyaluronic acid is enzymatically decomposed, several steps are required for the synthesis and the production is complicated. However, there are drawbacks such as low yield. As described above, various problems remain in sustained-release preparations of microspheres or sustained-release preparations using hyaluronic acid, and it has been desired to devise an appropriate formulation to bring out a more preferable pharmacological effect. It was As a means for solving such a problem, for example, JP-A-60-1306
01, the use of crosslinked hyaluronic acid described in JP-A-61-138601 and the like can be considered. However, since the substance used for cross-linking is generally not an in-vivo component, it is necessary to pay attention to the safety of the cross-linking substance in vivo, and the epoxy compound is preferable in this respect ((Polymer Preprints , Japan Vol.142, No3,938,199
3).

As cross-linked hyaluronic acid using an epoxy compound as a cross-linking agent, those described in JP-A-60-233101 or JP-A-61-164558 are known, but these are water-soluble or water-swellable. There was a problem that the rate was extremely high. That is, when the crosslinked hyaluronic acid is embedded in the living body, it is particularly preferable to use, for example, JP-A-63
When applied as an embolizing agent in a blood vessel as described in JP-A-281660, a certain degree of swelling property is required, but if the swelling rate is too high, the tissue around the application portion may be pressed. However, in this respect, the conventional crosslinked hyaluronic acid prepared by the epoxy compound is not suitable for in vivo application. The present invention has been made in view of the problems of the prior art, the purpose is to examine the cross-linked state of hyaluronic acid, adjust the water solubility and the degree of swelling, further using the cross-linked hyaluronic acid for a long period of time. It is intended to provide a sustained-release preparation capable of supplying a drug at a constant concentration.

[0007]

Means for Solving the Problems As a result of intensive investigations by the present inventors in order to achieve the above object, it was found that crosslinking the carboxyl groups of hyaluronic acid affects the basic structure of hyaluronic acid. However, they have found that their water solubility and their sustained release properties when complexed with a drug can be adjusted, and completed the present invention. That is, the crosslinked hyaluronic acid according to claim 1 of the present application is characterized in that the carboxyl group of the hyaluronic acid residue is crosslinked by an epoxy compound-based crosslinking agent at both ends and is represented by the following general formula 2.

[0008]

[Chemical 2] In the above chemical formula 2, R is the remaining cross-linking of the epoxy compound cross-linking agent. Further, the crosslinked hyaluronic acid according to claim 2 is characterized in that it is water-insoluble and has low swelling in which the crosslinked carboxyl groups are 0.3% or more based on all the carboxyl groups of hyaluronic acid. The sustained-release preparation according to claim 3 is characterized by comprising a complex of the crosslinked hyaluronic acid and a drug. The sustained-release preparation according to claim 4 is characterized in that the drug is an anticancer drug capable of ionically interacting with the carboxyl group of hyaluronic acid to form a complex. The embolic agent according to claim 5 is characterized by comprising the crosslinked hyaluronic acid. The embolic agent according to claim 6 is characterized in that a drug that ionically interacts with the carboxyl group of hyaluronic acid is complexed.

The present inventors can adjust the solubility of hyaluronic acid in water by cross-linking the carboxyl groups of hyaluronic acid with an epoxy compound, and further control the swelling ratio thereof appropriately, and It has been found that the above-mentioned problems can be solved by complexing with a water-insolubilized crosslinked hyaluronic acid as long as it is a drug capable of ionically interacting with the crosslinked hyaluronic acid to form a complex. That is, the present invention comprises a complex of water-insoluble cross-linked hyaluronic acid and a drug, and the drug is released as the hyaluronic acid skeleton is enzymatically decomposed. Or to provide an embolic agent.

The present invention will be described in detail below. The hyaluronic acid used as the starting material of the present invention is usually a sodium salt, but the type of salt is not particularly limited. Also,
The molecular weight is likewise not limited. However, if the molecular weight is too low, the following cross-linking reaction does not proceed well, so a molecular weight of 200,000 or more is preferable. The cross-linking reaction is with hyaluronic acid,
After the epoxy compound having both terminals as a cross-linking agent and the neutral to weakly acidic catalyst are dissolved in pure water, the reaction is terminated by heating to evaporate the water to dryness. The crosslinking mechanism is shown in FIG. As is clear from the figure, under weak acidity, the carboxyl groups of hyaluronic acid are crosslinked by the epoxy compounds at both ends. The concentration of hyaluronic acid at this time is usually 1 to 3% because if the viscosity is too high, it is difficult to form a uniform solution. The amount of the cross-linking agent may be added so that the desired cross-linking rate is obtained, and the rate of decomposition in vivo is determined by controlling the cross-linking rate. That is, the higher the crosslinking rate, the slower the enzymatic degradation in vivo.

In this reaction, the carboxyl group of hyaluronic acid and the epoxy group of the cross-linking agent undergo a cross-linking reaction. However, the reaction proceeds with evaporation of water to dryness, so that the cross-linking reaction proceeds efficiently with an extremely short intermolecular distance. . Further, the carboxyl group having electrostatic repulsion ability participates in the cross-linking point, so that the repulsion ability is blocked, the water swelling property is almost lost, and the compound becomes water-insoluble.
For example, if the cross-linking rate is 100% when all the carboxyl groups are cross-linking points, water-insolubility is obtained at the cross-linking rate of 0.3% or more. On the other hand, the conventional hyaluronic acid crosslinking method (JP-A-60-233101, JP-A-61-13860)
In the case of 1), it exhibits water solubility at such a crosslinking rate. In the conventional method, since the hydroxyl group is cross-linked in a basic aqueous solution, the polymer chains participating in the cross-linking are cross-linked with a long distance, and the carboxyl group that causes electrostatic repulsion remains intact. . Therefore, it becomes water-soluble or water-swellable.

As the epoxy compound at both ends used in the present crosslinking method, diglycidyl compounds such as diglycidyl ether, diglycidyl ester, diglycidyl amine and diglycidyl ammonium salt are desirable. Of course, the number of glycidyl groups may be 3 or more. Of these, glycidyl ether compounds are the most readily available and inexpensive, but specific examples include ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene diglycidyl ether, glycerin diglycidyl ether, and the like. . Further, as described above, it is reported that these epoxy-based cross-linking agents have higher safety in vivo than other cross-linking agents. Suitable catalysts for the reaction between the carboxyl group of hyaluronic acid and the epoxy compounds at both ends include quaternary ammonium salts, tertiary amines, phosphates, imidazole compounds and the like. However, considering the toxicity of the catalyst itself and the fact that the hyaluronic acid chain is easily decomposed even under weak basicity, it is preferable to use a neutral to weakly acidic safe inorganic catalyst.

For example, p of an aqueous solution of primary ammonium phosphate, primary sodium phosphate, primary potassium phosphate, etc.
Phosphates with H = 4-7 are preferred. The concentration as a catalyst should be 2 mg or more with respect to 100 ml of pure water as a solvent. Usually, about 20 mg is sufficient. The cross-linking reaction proceeds while evaporating the water to dryness by heating, and the film-like cross-linked hyaluronic acid obtained in this way is then pulverized to an average particle size of 1 mm or less for complexing with the drug in the next step. Be transferred. At this time, it is not necessary to particularly limit the average particle size, but a smaller particle size is preferable because the compounding with the drug in the next step can be completed in a short time. The drug targeted for conjugation may be anything that ionically interacts with the carboxyl group of cross-linked hyaluronic acid to form a complex, but in particular, the anticancer drug is present in the affected area for a long time. ,
The sustained-release preparation of the present invention, which releases a drug in response to enzymatic degradation, is effective because it is expected to significantly improve the pharmacological effect.

A carcinostatic agent which interacts ionically to form a complex is required to have a basic ion in its chemical structure. Specifically, specifically, nimustine hydrochloride, ancitabine hydrochloride, daunorubicin hydrochloride, Examples include doxorubicin hydrochloride, bleomycin hydrochloride, bleomycin sulfate, peplomycin sulfate, aclarubicin hydrochloride, epirubicin hydrochloride, vinblastine sulfate, vincristine sulfate, vindesine sulfate and the like. The complexing of the anti-cancer agent with the cross-linked hyaluronic acid powder is essentially due to chemisorption by ionic interactions. Therefore, the amount ratio capable of being complexed is directly related to the amount of chemisorption, and varies depending on the type of anticancer agent.

To prepare a complex, first, crosslinked hyaluronic acid is dispersed in an aqueous solution of an anticancer agent, and the mixture is allowed to stand when chemisorption reaches equilibrium, and the precipitated complex is separated by filtration, dried and ground. To obtain a granular or particulate composite. In use, it is necessary to satisfy the conditions required for ordinary injections. Although it depends on the stability of the anticancer drug to be complexed, it is preferable to use a dispersible type preparation which is dispersed in an isotonic solution such as physiological saline. The release of the carcinostatic agent depends on its complexing rate (= carcinostatic agent amount / crosslinked hyaluronic acid amount). The conjugation takes place in pure but the release takes place under physiological conditions.
Therefore, if the adsorption equilibrium under pure water conditions shifts to the adsorption equilibrium under physiological conditions and the chemisorption amount of the anticancer agent in pure water is less than the equilibrium adsorption amount under the physiological conditions, the release of the anticancer agent is complex. If the rate is equal to the enzymatic degradation rate of hyaluronic acid in the body, and conversely high, the anticancer agent corresponding thereto is temporarily released, and then the anticancer agent is released at a rate corresponding to the enzymatic degradation rate. Such a temporary release is used as an effective means for quickly raising the blood concentration to a drug effective concentration.

The administration site of the present preparation may be any place where the hyaluronan degrading enzyme is present, and it can be administered as a safe bioabsorbable preparation having sustained release. In fact, since hyaluronan degrading enzyme is widely distributed in the living body such as liver, testis, lung, kidney, synovial fluid, it can be effectively used in these organs. Thus, this formulation
Since the drug is released by utilizing the decomposition reaction of hyaluronic acid by the enzyme, it is not affected by the particle size and particle size unlike the conventional capsule-type drug.

Further, the sustained-release preparation of the present invention is effective as an embolic agent which also has a sustained release of an anticancer agent depending on the particle size.
The embolization therapy is widely known as a therapy for an unresectable tumor that blocks nutrients by administering an intravascular embolic agent to its controlling artery. It has also been reported that good results have been obtained by maintaining a high concentration of an anticancer agent in a tumor in combination with the anticancer agent (Nippon Kaihatsu, '92, 187, 1).
991). If the sustained release preparation of the present invention is used for the same purpose, sustained release and embolization can be simultaneously achieved with one preparation. Moreover, since the sustained-release preparation of the present invention is bioabsorbable, it is not necessary to take it out after the operation unlike the therapy using a balloon which is partially performed as an embolization therapy. The embolization therapy is not particularly limited as long as it is a tumor in which a catheter can be inserted into the main artery, but the tumor for which the embolization therapy is most often performed at present is unresectable liver cancer. However, current advanced medical technology allows catheters to be inserted into arteries of most organs, and it is expected that the application of this therapy will be further expanded in the future.

[0018]

EXAMPLES Specific examples of the present invention will be described in detail below. The present invention is not limited to these examples. Example 1 0.4% of 10% ammonium phosphate monobasic solution in 200 ml of pure water
Prepare 4 liquids, each containing 2 ml of glycerin diglycidyl ether aqueous solution (20,5,1.67).
After adding 0.62 g, 2 g of hyaluronic acid having a molecular weight of about 2 million was dissolved in each aqueous solution. Next, the solutions were transferred to a petri dish and placed in an air-circulating constant temperature bath at 80 ° C for 10
It was left to stand for a time to cause a crosslinking reaction. The cross-linked hyaluronic acid film thus obtained was washed with a 50% ethanol solution, dried, pulverized, and sieved to 100 μm or less. All of them were insoluble in water, and swelling in water was hardly observed. The cross-linking ratio of these fine cross-linked hyaluronic acid powders was calculated on the assumption that the total amount of the added cross-linking agent reacts with the carboxyl groups of hyaluronic acid.
A degree of crosslinking of 100% means that all carboxyl groups are crosslinking points. With the above amounts added, the crosslinking rates correspond to 59.3, 14.8, 4.9 and 1.8%, respectively.

Four kinds of crosslinked hyaluronic acid fine powders having different crosslinking ratios of Example 1 were put in test tubes in an amount of 10 mg each, and 4 ml of an acetic acid buffer solution of pH 6.0 containing 100 units of hyaluronidase (Amano Pharmaceutical Co., Ltd.). Add 50 while stirring
Hold at ℃. In order to examine the change with time of the amount of free hyaluronic acid (decomposition rate), sampling was performed while filtering with a 0.45 μ membrane filter with time, and the amount of hyaluronic acid dissolved therein was colorimetrically determined by the carbazole / sulfuric acid method. The results are shown in Table 1. From Table 1, it is understood that the higher the cross-linking rate, the more difficult the enzyme is to decompose. It is also suggested that the rate of decomposition can be controlled by controlling the crosslinking rate.

[Table 1] ─────────────────────────────────── Crosslinking ratio 59.3 14.8 4.9 1.8 ─────────────────────────────────── 0.5 hours later 1.0 1.5 2 .2 2.3 1 1.6 2.8 4.5 4.8 2 2.5 6.0 12.2 13.0 4 3.6 12.1 25.6 27.0 7 4.5 19. 7 45.1 40.3 10 5.9 31.3 64.6 60.9 24 12.5 56.0 81.3 91.5 36 17.9 63.5 86.7 90.3 48 26.2 72.5 96.3 98.7 ────────────────────────────────────

Of the crosslinked hyaluronic acid fine powder according to this example, two samples with crosslinking rates of 59.3% and 1.8% were subjected to transplantation test using rabbits. Each sample was dispersed in a 10-fold amount of physiological saline, and 0.5 ml of each dispersion was subcutaneously injected at three points on both sides of the spinal column of a healthy rabbit weighing 2.5 kg. One week later, after the rabbit was anesthetized and exsanguinated,
The tissue surrounding the sample was examined with a magnifying glass. as a result,
In the case of the sample having a cross-linking rate of 59.3%, the injected cross-linked hyaluronic acid fine powder remained almost, but the cross-linking rate of 1.8%.
In the case of, the trace was not recognized and it was absorbed in the living body. In addition, no bleeding or encapsulation was observed in both samples. From this, it is understood that the present crosslinked hyaluronic acid fine powder has high safety and excellent bioabsorbability.

Example 2 To 2 ml of an aqueous solution having a doxorubicin hydrochloride concentration of 200 to 5000 ppm, 5 mg of the crosslinked hyaluronic acid fine powder of Example 1 was added and incubated at 25 ° C. for 3 hours. After centrifuging this, the amount of doxorubicin hydrochloride in the supernatant was adjusted to 479 nm.
The amount of adsorption to the crosslinked hyaluronic acid fine powder was examined from the difference from the initial concentration. The adsorption isotherm curve is shown in FIG. The figure suggests that doxorubicin hydrochloride chemisorbs with crosslinked hyaluronic acid regardless of the crosslinking rate. Furthermore, it was also confirmed that the doxorubicin hydrochloride chemisorbed in this way was desorbed again when the surrounding environment was adjusted to pH 3 or lower.

Example 3 10 mg of doxorubicin hydrochloride was dissolved in 10 cc of pure water, 50 mg of hyaluronic acid fine powder having a cross-linking rate of 4.9% prepared in Example 1 was dispersed therein, chemically adsorbed, and then the supernatant was obtained. Was discarded and dried in vacuum. The dried product was pulverized again and then sieved to have an average particle size of 100 μm or less. Doxorubicin hydrochloride / crosslinked hyaluronic acid complex fine powder 5 thus prepared
0 mg was dispersed in 20 ml of physiological saline and shaken at 37 ° C at 100 strokes / minute. The amount of doxorubicin hydrochloride released at this time was quantified by measuring the absorbance. FIG. 3 shows the time course of the concentration of released doxorubicin hydrochloride. About 1
Equilibrium was reached after 30 minutes, but when 1000 units of hyaluronidase (Amano Pharmaceutical Co., Ltd.) were added, the release started again, and 220 minutes later, the total amount was released.

Example 4 Crosslinked hyaluronic acid prepared by adding ethylene glycol diglycidyl ether to a crosslinking ratio of 2,5,10% was finely powdered in the same manner as in Example 1. Part 10
Each of 00 mg was dispersed in an aqueous solution containing 10 mg of doxorubicin hydrochloride, centrifuged, and dried under vacuum. 100μ
After pulverizing to the following, 50 mg thereof was dispersed in 10 ml of a physiological saline solution containing 500 units of hyaluronidase, and 37
Shake at 100 ° C./minute at 100 ° C. At this time, the amount of doxorubicin hydrochloride released in the supernatant was quantified by measuring absorbance over time. The results are shown in FIG. The released doxorubicin hydrochloride is initially released to an effective concentration at one time, and then maintains a constant release rate. Moreover,
The release rate becomes slower as the crosslinking rate becomes higher. In the control test containing no hyaluronidase, only a temporary drug release was observed in the initial stage, but not after that.

Example 5 A test was conducted in the same manner as in Example 7 except that bleomycin hydrochloride was used instead of doxorubicin hydrochloride in Example 7. The results showed almost the same behavior.

Example 6 0.2 ml of 10% ammonium phosphate monobasic solution in 50 ml of pure water
Eight liquids added each were prepared, and 2% ethylene glycol diglycidyl ether aqueous solution was added to each of 1.76,0.
88, 0.35, 0.15, 0.060, 0.030,
After adding 0.015 and 0.008 g, 0.5 g of hyaluronic acid having a molecular weight of about 1.6 million was dissolved in each aqueous solution.
Next, the solutions were transferred to a petri dish, left in an air-circulating constant temperature bath at 100 ° C. for 5 hours to cause a crosslinking reaction, and then 70
% Aqueous solution of ethanol and then dried to obtain a hyaluronic acid crosslinked film. The cross-linking ratio of the hyaluronic acid film thus obtained was 59, 30, 12, 4.9 in order from the top.
It was 2.0, 1.0, 0.5, and 0.25%, but all were water-insoluble except for the film with a crosslinking rate of 0.25%, so the swelling rate with pure water and saline solution It was subjected to measurement. The measurement method is to first measure 1.5 x 1.5 from each film.
Cut out cm and measure the length x width x film thickness using a reading microscope (manufactured by Olympus) and a film thickness meter (Tokyo Seimitsu Co., Ltd.), then at 25 ° C.
It was dipped in pure water or physiological saline solution until the swelling equilibrium was reached. Next, the film that reached the swelling equilibrium was taken out of the liquid, water droplets on the surface were cut off, and then the length × width × thickness was measured again. Based on this result, the volume swelling ratio before and after immersion was calculated and plotted in FIG. There is no great difference in the swelling ratio between pure water and physiological saline, and the swelling ratio rises as the crosslinking rate decreases. However, it is understood that the swelling ratio does not exceed 4 and the swelling ability is remarkably low even in the case of a film having water-insolubility and a crosslinking rate of 0.5%. As described above, the cross-linked hyaluronic acid according to the present invention is not only water-insoluble but also has a low degree of water swelling, and therefore, even when it is implanted in a living body or used as an embolizing agent, the surrounding tissues are compressed more than necessary. There is nothing to do.

Example 7 For crosslinked hyaluronic acid fine powder having a crosslinking rate of 1.8% and 100% prepared according to Example 1, a hot spring such as adsorption of doxorubicin hydrochloride was examined in the same manner as in Example 2. Results are shown in FIG. Doxorubicin hydrochloride chemisorbs to the crosslinked hyaluronic acid powder having a crosslinking rate of 1.8%, but the crosslinking rate is 10%.
No chemical adsorption was observed on 0% of the powder, and only physical adsorption was observed. This difference is due to the fact that the crosslinking reaction according to the present invention is a crosslinking between the carboxyl groups of hyaluronic acid.
That is, in the case of a powder with a crosslinking rate of 100%, since all carboxyl groups are crosslinking points, no free carboxyl groups remain, and as a result, doxorubicin hydrochloride cannot be chemically adsorbed, and only physical adsorption is observed. It is thought that it was done.

[0027]

As described above, since the crosslinked hyaluronic acid according to the present invention is designed to crosslink carboxyl groups with each other, it can have water insolubility and low swelling property.
When the sustained-release preparation is prepared by complexing the drug with the cross-linked hyaluronic acid, the drug can be gradually released when administered in vivo due to the enzymatic decomposition of hyaluronic acid. Furthermore, when the crosslinked hyaluronic acid according to the present invention is used as an embolic agent, it does not apply unnecessary pressure to other tissues.

[Brief description of drawings]

FIG. 1 is an explanatory view of a crosslinked state of crosslinked hyaluronic acid according to the present invention.

FIG. 2 is a schematic diagram of Example 2 in which pure water is prepared for adriamycin (doxorubicin hydrochloride) crosslinked hyaluronic acid powder.
Adsorption isotherm at 25 ° C.

FIG. 3 is an explanatory diagram showing the change over time in the amount of adriamycin released from the sustained-release preparation according to Example 3.

FIG. 4 is an explanatory diagram showing the change over time in the amount of adriamycin released from the sustained release preparation according to Example 4.

FIG. 5 is an explanatory diagram of the cross-linking rate dependency of the volume swelling ratio of the cross-linked hyaluronic acid film prepared in Example 6 in pure water and physiological saline at 25 ° C.

FIG. 6 Crosslinked hyaluronic acid powder of doxorubicin hydrochloride measured in Example 7 (crosslinking rate 1.8%: ▲, 100%:
● is the adsorption isotherm of pure water against 25 ° C.

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[Procedure amendment]

[Submission date] October 21, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

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[Figure 1]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

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[Fig. 2]

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Chemical 1] In the above chemical formula 1, R is the remaining cross-linking of the epoxy compound cross-linking agent.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

[0008]

[Chemical 2] In the above chemical formula 2, R is the remaining cross-linking of the epoxy compound cross-linking agent. Further, the crosslinked hyaluronic acid according to claim 2 is characterized in that it is water-insoluble and has low swelling in which the crosslinked carboxyl groups are 0.3% or more based on all the carboxyl groups of hyaluronic acid. The sustained-release preparation according to claim 3 is characterized by comprising a complex of the crosslinked hyaluronic acid and a drug. The sustained-release preparation according to claim 4 is characterized in that the drug is an anticancer drug capable of ionically interacting with the carboxyl group of hyaluronic acid to form a complex. The embolic agent according to claim 5 is characterized by comprising the crosslinked hyaluronic acid. The embolic agent according to claim 6 is characterized in that a drug that ionically interacts with the carboxyl group of hyaluronic acid is complexed.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ken Uchida 4-39-21-104 Hikawadai, Nerima-ku, Tokyo

Claims (6)

[Claims] 1. A crosslinked hyaluronic acid represented by the following general formula 1 in which the carboxyl group of a hyaluronic acid residue is crosslinked with an epoxy compound-based crosslinking agent at both ends. [Chemical 1] In the above chemical formula 1, R is the remaining cross-linking of the epoxy compound cross-linking agent. 2. The crosslinked hyaluronic acid according to claim 1, wherein the crosslinked hyaluronic acid has a crosslinked carboxyl group of 0.3% or more based on all the carboxyl groups of hyaluronic acid and is water-insoluble and low swelling. 3. A sustained-release preparation comprising the complex of the crosslinked hyaluronic acid according to claim 1 and a drug. 4. The sustained-release preparation according to claim 3, wherein the drug is an anticancer agent capable of ionically interacting with the carboxyl group of hyaluronic acid to form a complex. 5. An embolic agent comprising the crosslinked hyaluronic acid according to claim 2. 6. The embolic agent according to claim 5, wherein a drug that ionically interacts with the carboxyl group of hyaluronic acid is complexed.