JPH08301903A - Production of cross-linked polysaccharide - Google Patents

Abstract

PURPOSE: To produce a cross-linked polysaccharide free of unreacted substances, high in safety and biocompatibility, and useful as a medical material by irradiating an ionizing radiation-reactive polysaccharide with an ionizing radiation. CONSTITUTION: An ionizing radiation-reactive polysaccharide having a polysaccharide (biocompatible polysaccharide such as glycosaminoglycan is pref.) bonded to at least 1 ionizing radiation-reactive compd. (compd. low in toxicity and little in side effect even when used as a medical material is pref., examples of which include cinnamic acid and coumarin) via covalent bonds is irradiated with an ionizing radiation to bring about a mutual cross-linking reaction of ionizing radiation-reactive compd. residues to produce a cross-linked polysaccharide. The cross-linked polysaccharide free of unreacted substances causative of a side effect, etc., high in safety and biocompatibility, and useful as a medical material is obtd. according to the foregoing procedure.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a crosslinked polysaccharide. Specifically, a crosslinked polysaccharide having good biocompatibility and useful as a medical material, a crosslinked polysaccharide containing a physiologically active substance, and a method for producing a modified medical material containing a crosslinked polysaccharide having good biocompatibility Regarding

[0002]

2. Description of the Related Art Conventionally, electron beams and γ have been applied to polymer compounds.
Irradiation of wires and the like (hereinafter, these may be collectively referred to as ionizing radiation) has been used for crosslinking rubber of tires and electric wires. The ionizing radiation has also been used for sterilization of medical equipment. The cross-linking reaction by ionizing radiation has been mainly made of synthetic polymers. For the purpose of chemically cross-linking bio-derived polymers with aldehyde compounds, epoxy compounds, divinyl sulfone compounds, etc. to maintain the physiologically active action of bio-polymers in vivo, and to prevent adhesion by forming films or powders. There has been an attempt to use it as a medical material (JP-A-60-130601, JP-A-63-50355).
1).

However, the cross-linked polysaccharide produced is a polymer having a higher molecular weight than that of the starting polysaccharide and has a network structure, which makes it difficult to remove unreacted substances and catalysts, and has side effects when administered in vivo or transplanted. It was likely to occur and was not practical. Further, as an example of using a method of irradiating the polysaccharide derivative with ionizing radiation to crosslink, in order to obtain an antithrombotic medical material coated with crosslinked heparin, a carrier and acrylic acid or methacrylic acid in the hydroxyl group of heparin or Contact these derivatives with the ester-bonded derivatives,
A method of crosslinking the heparin derivative by irradiating it with an electron beam is known (Japanese Patent Publication No. 61-57788).

However, acrylic acid and the like are highly toxic and have problems in safety and biocompatibility as medical materials.
Furthermore, a method is known in which a photocrosslinking-reactive compound is bound to a polysaccharide and crosslinked by irradiating ultraviolet rays, but the irradiating time of ultraviolet rays is relatively long (several minutes or more), and the temperature is high during that time Was not suitable for. In addition, cross-linking in solution is difficult, and the morphology of irradiated objects is limited to relatively thin films (artificial organ, 22 (2), p376-37).
9 (1993)).

[0005]

SUMMARY OF THE INVENTION The first object of the present invention is to:
In addition, it is to provide a method for producing a crosslinked polysaccharide which is free from unreacted substances causing side effects and has high safety and biocompatibility and is used as a medical material. Secondly, to provide a method for producing a sustained-release material or preparation of a physiologically active substance having a release rate according to the type of the physiologically active substance and the purpose of use by incorporating the physiologically active substance into the crosslinked polysaccharide. Thirdly, to provide a method for producing a medical material having a crosslinked polysaccharide inside or on the surface thereof.

[0006]

As a result of intensive studies, the inventors of the present application succeeded in solving the problem by the following constitution. That is, the present invention includes: 1) irradiating ionizing radiation to an ionizing radiation-reactive polysaccharide obtained by covalently bonding a polysaccharide and at least one ionizing radiation-reactive compound, thereby covalently bonding to the reactive polysaccharide A method for producing a crosslinked polysaccharide characterized by causing a cross-linking reaction between reactive compound residues, 2) a method for producing a crosslinked polysaccharide according to 1) above, wherein the polysaccharide is a biocompatible polysaccharide, 3) biocompatibility Wherein the polysaccharide is a glycosaminoglycan, the method for producing a crosslinked polysaccharide according to 2) above, 4) the ionizing radiation-reactive polysaccharide according to 1) above containing a physiologically active substance, is irradiated with ionizing radiation,
A method for producing a crosslinked polysaccharide including a physiologically active substance, which comprises causing a crosslinking reaction between ionizing radiation reactive compound residues covalently bound to the reactive polysaccharide, 5) an ionizing radiation reactive polysaccharide The method for producing the crosslinked polysaccharide according to any one of 1) to 4) above, which is in the form of a solution, a gel, or a sheet, 6) the crosslinked polysaccharide, or a crosslink containing a physiologically active substance. 1) to 1) above, wherein the polysaccharide is in the form of a film, granules or gel
5) The method for producing a crosslinked polysaccharide according to any one of 5), 7) An ionizing radiation-reactive polysaccharide obtained by covalently bonding a polysaccharide and at least one ionizing radiation-reactive compound to the surface or inside of a medical material. A method for producing a modified medical material characterized by causing a cross-linking reaction between the reactive compound residues covalently bound to the reactive polysaccharide by irradiating it with ionizing radiation, 8) It is an object of the present invention to provide a method for producing a modified medical material as described in 7) above, wherein the ionizing radiation-reactive polysaccharide is in the form of a solution, gel or sheet.

Hereinafter, the present invention will be described in detail. In the present invention, the ionizing radiation means electron beams and γ rays which can be dimerized by carbon-carbon double bonds used for dimerization (crosslinking) of cinnamic acid, thymine, coumarin or their derivatives. To do. The ionizing radiation-reactive polysaccharide used in the present invention is a polysaccharide derivative in which a polysaccharide and an ionizing radiation-reactive compound are covalently bonded, and an amide bond or an ester bond is added to a carboxyl group, a hydroxyl group, and / or an amino group of the polysaccharide. , And / or a polysaccharide derivative in which an ionizing radiation-reactive compound is bound via a bond represented by —NHCH ₂ —. Particularly, a polysaccharide derivative in which a hydroxyl group of a polysaccharide and an ionizing radiation-reactive compound are ester-bonded, and a polysaccharide derivative in which a carboxyl group of a polysaccharide and an ionizing radiation-reactive compound are amide-bonded are preferable.
These can be produced by using known chemical modification reactions of polysaccharides. At that time, a method that does not involve lowering the molecular weight of the polysaccharide is preferable.

To produce an ionizing radiation-reactive polysaccharide, one or more ionizing radiation-reactive compounds are covalently attached to a single polysaccharide, or a single or multiple ionizing radiation-reactive compounds are attached to a plurality of polysaccharide molecules. Multiple types of ionizing radiation-reactive compounds may be covalently bound. Furthermore, in the present invention, the former ionizing radiation-reactive polysaccharide and the latter ionizing radiation-reactive polysaccharide can be used alone or after mixing, and then subjected to a crosslinking reaction by irradiation with ionizing radiation to produce a crosslinked polysaccharide.

The polysaccharide used in the present invention is preferably a biocompatible polysaccharide. In the present specification, biocompatibility means a property that is safe and nontoxic to a living body, and preferably also has a property that is easily compatible with the living body. Biocompatible polysaccharides include hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate and other glycosaminoglycans, chitin, chitosan, pullulan, starch, mannan, pectic acid, alginic acid, gum arabic. Alternatively, these derivatives (acyl derivative, polysulfate, desulfate, deacylate, etc.) and the like can be mentioned. Glycosaminoglycan is particularly preferable because of its high biocompatibility, and more preferable examples include hyaluronic acid and chondroitin sulfate.

As the polysaccharides, those derived from natural products are used, but if necessary, they may be chemically or enzymatically prepared by synthetic or semi-synthetic methods, and those functional groups are modified. It may be. However, the polysaccharide must remain unmodified with the functional groups necessary to attach the ionizing radiation-reactive compound to the polysaccharide. These may be selected according to the purpose and used alone or in combination of two or more.

The molecular weight of the polysaccharide is not limited, but the larger the molecular weight, the more effective the crosslinking. For example, high molecular weight polysaccharides provide sufficient cross-linking even with few ionizing radiation-reactive compounds attached to the polysaccharide. As for the molecular weight range, the lower limit is 5,000, preferably 30,000, and the upper limit is 10 million. A more preferred molecular weight range for hyaluronic acid is about 50,000 to 5,000,000.

The ionizing radiation-reactive compound used in the present invention is a compound having a functional group capable of covalently bonding with a polysaccharide and capable of dimerizing (crosslinking) at least the compound molecules by irradiation with ionizing radiation. I wish I had it.
The ionizing radiation-reactive compound may also include a spacer for covalent attachment to the polysaccharide. As the raw material of the spacer, diamine, amino alcohol, amino acid, dicarboxylic acid, hydroxy acid and the like are used, and those having low toxicity are preferable. Preferred spacers include amino alcohols. The reactive compound containing a spacer can be produced using a known chemical modification reaction. Preferred ionizing radiation-reactive compounds include cinnamic acid, thymine, coumarin and the like, which are compounds having low toxicity and little side effects even when used as medical materials, or derivatives thereof.

Examples of the thymine derivative include compounds represented by the general formula (1),

[0014]

Embedded image (In the formula, R ¹ represents hydrogen, a lower alkyl group, a halogen atom or a halo lower alkyl group, and R ² represents hydrogen, a cyano group, a carboxyl group, a lower alkoxycarbonyl group, a lower alkyl group, a halogen atom or a halo lower alkyl group. Group, and R ³ represents a lower alkylene group. Examples of the coumarin derivative include compounds represented by the general formula (2) (JP-A-6-73102).

[0015]

Embedded image (In the formula, R ⁴ , R ⁵ and R ⁶ may be the same or different from each other and each independently represent hydrogen or a lower alkyl group,
R ⁷ represents a lower alkylene group. ) More preferably, cinnamic acid, aminocinnamic acid, a cinnamic acid derivative containing a spacer and the like, which are considered to have low toxicity even when dimerized and have few side effects, can be mentioned.

Examples of the cinnamic acid derivative containing a spacer include compounds represented by the general formulas (3) to (5). _{^{H 2 N (CR 8 R 9}} ) n OCOCH = CH-Ph (3) Equation (3), R ⁸ and R ⁹ (preferably having from 1 to 4 carbon atoms) each independently represent a hydrogen atom or a lower alkyl group and Represent Ph represents a phenyl group, but may have a substituent such as a lower alkyl group or an alkoxy group having about 1 to 4 carbon atoms, an amino group or a hydroxyl group. n is 2-18,
Preferably, it represents an integer of 2 to 12.

A—NH—Ph—CH═CHCOOR ¹⁰ (4) In formula (4), R ¹⁰ is an alkyl group, preferably 1 carbon atom.
~ 8 represents an alkyl group. A is H ₂ NCR ¹¹ R ¹² CO-
(NHCR ¹¹ R ¹² CO) _m − or H ₂ N (CR
¹¹ R ¹² ) represents _h CO-. R ¹¹ and R ¹² each independently represent a hydrogen atom or a lower alkyl group (preferably having 1 to 4 carbon atoms). Although -Ph- represents a paraphenylene group, it may have a substituent such as a lower alkyl group or an alkoxy group having about 1 to 4 carbon atoms, or a hydroxyl group. m is an integer of 0 to 5, preferably 0 to 2, and h is 1 to 1.
8, preferably an integer of 1-12.

[0018] _{^{HOOC- (CR 13 R 14) k}} -NH-COCH = CH-Ph (5) formula (5), the R ¹³ and R ¹⁴ each independently represent a hydrogen atom or a lower alkyl group (preferably, the number of carbon atoms 1 to 4). Ph represents a phenyl group, but may have a substituent such as a lower alkyl group or an alkoxy group having about 1 to 4 carbon atoms, an amino group or a hydroxyl group. k is 2-18,
Preferably, it represents an integer of 2 to 12.

In the present invention, the binding amount of the ionizing radiation-reactive compound to the polysaccharide is defined as the number of moles of the ionizing radiation-reactive compound (degr) per mole of the monosaccharide constituting the polysaccharide.
ee of substitution; hereinafter referred to as DS). For example, DS is 3 when the ionizing radiation-reactive compound is covalently bonded to all the hydroxyl groups that do not participate in the glycoside bond of the polysaccharide consisting of glucose only. Further, DS is 0.5 when covalently bonding all the carboxyl groups of hyaluronic acid (including glucuronic acid for each disaccharide unit) with an ionizing radiation-reactive compound capable of covalently bonding only with the carboxyl group.

The DS of the ionizing radiation-reactive polysaccharide can be adjusted at will by adjusting the reaction conditions. For example, the DS can be increased by appropriately combining means such as increasing the molar ratio of the ionizing radiation-reactive compound to the polysaccharide during the reaction, heating, adding a suitable catalyst, or extending the reaction time. . The preferred range of DS varies depending on the purpose, but in the case of obtaining a biocompatible crosslinked polysaccharide, it is preferably 0.01% to 10% of the above-mentioned theoretical maximum value of DS.

A typical method for producing the ionizing radiation-reactive polysaccharide will be specifically described below. The present invention is not limited to this. 1) In the case where an ionizing radiation-reactive compound having an amino group is bound to a polysaccharide having a carboxyl group (1) An activating reagent such as carbodiimides (eg, dicyclohexylcarbodiimide, water-soluble carbodiimide (1) -Ethyl-3- (3-dimethylaminopropyl) carbodiimide etc.) and the like) to form an amide bond by a dehydration condensation reaction. In this case, the reaction efficiency can be increased by adding an N-hydroxy group compound such as N-hydroxysuccinimide as an additive.

(2) A method in which a carboxyl group of a polysaccharide is activated in advance in an organic solvent as an active ester such as pentachlorophenyl ester or a mixed acid anhydride such as dimethylphosphinothioyl oil, and then reacted with an amino group. 2) When esterifying an ionizing radiation-reactive compound having a hydroxyl group with a polysaccharide having a carboxyl group After converting the carboxyl group of the polysaccharide into a quaternary ammonium salt such as tetrabutylammonium, dimethylformamide, dimethylsulfoxide, etc. A method of reacting with a halide of an ionizing radiation-reactive compound in an organic solvent to form an ester bond.

3) In the case of binding an ionizing radiation-reactive compound having a carboxyl group to a polysaccharide having a hydroxyl group After activating the carboxyl group of the ionizing radiation-reactive compound as an acid chloride or an acid anhydride, it is suitable for the hydroxyl group of the polysaccharide. Method of ester bond in various solvents. In this case, pyridine or an acylation catalyst (for example, 4-dimethylaminopyridine) can be added to the solvent to increase the reaction rate.

4) In the case of binding an ionizing radiation-reactive compound having a carboxyl group to a polysaccharide having an amino group (1) After activating the carboxyl group as an acid chloride, an acid anhydride or an active ester, the amino of the polysaccharide A method of forming an amide bond with a group in a suitable solvent. In this case, reaction efficiency can be improved by adding pyridine or an acylation catalyst (for example, 4-dimethylaminopyridine) to the solvent.

5) When binding an ionizing radiation-reactive compound having an aldehyde group to a polysaccharide having an amino group A method of mixing both in a suitable solvent and binding as a Schiff base. In this case, the reaction efficiency can be increased by heating the reaction solution or adding a dehydrating agent such as molecular sieve. Furthermore, by treating the Schiff base with a reducing agent such as sodium cyanoborohydride, a more stable amino bond (—NHCH ₂ —) can be converted.

Of these production methods, from the viewpoints of stability of reaction products, reaction efficiency, etc., the above 1)-
The methods (1) and 3). As the treatment method and purification method after the reaction between the polysaccharide and the ionizing radiation-reactive compound, there are used an alcohol precipitation method, washing on a filter, centrifugation, and a salt, which are usually used for separation, purification and chemical modification of the polysaccharide. Examples include a method of appropriately selecting and combining methods such as precipitation, dialysis, gel filtration, ion exchange method, reduced pressure drying, and freeze drying.

Since the ionizing radiation-reactive polysaccharide used in the present invention is soluble in water or an organic solvent, it can be easily purified. Prior to the crosslinking reaction, a solution of an ionizing radiation-reactive polysaccharide can be prepared and the solution can be purified, and the thus-reacted reactive polysaccharide can be crosslinked by irradiating it with ionizing radiation. Reagents used, water,
By paying attention to the container, it is possible to easily produce a crosslinked polysaccharide substantially free of unreacted substances, catalysts, contaminating microorganisms, pyrogens and the like.

These ionizing radiation-reactive polysaccharides are selected according to the intended use, and the ionizing radiation-reactive groups contained in the ionizing radiation-reactive compound residue are obtained by irradiating with ionizing radiation after selecting one or a mixture of two or more species. (Carbon-carbon double bond) Cleavage and bond between them to form a cyclobutane ring. As a result, at least the reactive polysaccharide molecules are cross-linked with each other, whereby a cross-linked polysaccharide having a three-dimensional network structure can be produced. At this time, the cross-linking ratio (the ratio of cross-linking of the reactive polysaccharide) increases with the increase of DS of the reactive polysaccharide and with the increase of irradiation time and irradiation dose, but by controlling the irradiation conditions of ionizing radiation, It is also possible to control the increase and decrease.

The crosslinking rate can be estimated using the gelation rate described below as an index. The ionizing radiation-reactive polysaccharide can be formed into a desired shape (for example, a film shape, a tubular shape, a granular shape, etc.) before irradiation with ionizing radiation. In this case, for example, a solvent that uniformly dissolves the reactive polysaccharide, such as water (preferably purified water), a buffer solution (phosphate buffer solution,
(A carbonate buffer etc.) or an organic solvent (dimethylformamide, dimethylsulfoxide etc.) with about 0.1 of the reactive polysaccharide.
~ 90% by weight, dissolved on a flat plate, in a container, on the surface of a container, or the like as a solution or gel, or heated at room temperature such that ventilation or ionizing radiation-reactive polysaccharide is not affected ( (Eg, 40 to 50 ° C.), freeze-drying, vacuum drying and the like.

When the ionizing radiation-reactive polysaccharide is present on the surface or inside of the medical material and is irradiated with the ionizing radiation, the medical material (for example, gauze, bandage, woven cloth,
(Paper, non-woven fabric, cotton-like material, filamentous material, film, porous sponge, rubber, plastic, metal support, artificial organs, etc.) is coated with the solution or gel of the ionizing radiation-reactive polysaccharide prepared as described above. It can also be coated, adhered, buried or impregnated.
There is no particular limitation on the material and shape of the medical material.

Furthermore, a physiologically active substance (for example, heparin, dermatan sulfate, heparan sulfate, carcinostatic agent, anti-inflammatory agent, antibacterial agent, cytokine, hormone, growth factor, wound healing promoter is added to a solution of the ionizing radiation-reactive polysaccharide as described above. Agent (JP-A-60-222425), tissue plasminogen activator, superoxide dismutase,
It is possible to embed physiologically active substances by mixing them with enzymes such as urokinase, etc., and then by applying ionizing radiation to crosslink the solution as it is, or after performing the above-mentioned molding and coating. is there. According to this method, even when the physiologically active substance is a polymer, it can be embedded not only in the surface layer of the crosslinked polysaccharide but also in the deep layer. A crosslinked polysaccharide including a physiologically active substance that can be released can be obtained.

The crosslinked polysaccharide containing a physiologically active substance thus obtained can also be used as a pharmaceutical preparation by a known preparation technique. The irradiation conditions of ionizing radiation are the type of ionizing radiation-reactive polysaccharide (polysaccharide as a raw material, ionizing radiation-reactive compound, DS, etc.), the obtained crosslinked polysaccharide, and a crosslinked polysaccharide containing a physiologically active substance,
It is appropriately selected so as to obtain the optimum crosslinking rate and gelation rate in consideration of the use of the medical material containing the crosslinked polysaccharide.

The irradiation dose is generally 0.001 to
A range of 10 Mrad can be mentioned. The range is preferably 0.01 to 2 Mrad, and more preferably 0.01 to 0.5 Mrad. In the case of hyaluronic acid, about 0.01 Mra
At doses below d and above about 0.5 Mrad, the crosslinked hyaluronic acid is not sufficiently water insoluble.

The irradiation dose can be adjusted or repeated irradiation can be performed if necessary. The irradiation time is usually
Since it is extremely short at 0.01 to 10 seconds, preferably 0.1 to 1 second, and the temperature does not rise so much, it is possible to effectively prevent the lowering of the molecular weight of the sugar chain of the obtained crosslinked polysaccharide and the bactericidal effect of the reaction system. Also has the advantage of being superior. The temperature at the time of irradiation can be any temperature, but it is generally advantageous to keep it at room temperature. The reaction atmosphere is not particularly limited, and the crosslinking can be performed in any atmosphere such as air, inert gas, or under reduced pressure.

The form of the ionizing radiation-reactive polysaccharide upon irradiation is not limited as long as it can be crosslinked, and examples thereof include thin films, thick sheets, gels and solutions. May be transparent or colored. In particular, the ionizing radiation used in the present invention has a high energy (permeability), unlike ultraviolet rays which have been conventionally used for crosslinking polysaccharides having a cinnamoyl group, so that a thick sheet of ionizing radiation reactive polysaccharide or the reaction It can be preferably used for a crosslinking reaction of a gel or solution of a natural polysaccharide. The thickness of the sheet is 0.01 to 10 m
m, preferably about 0.05 to 2 mm, and the solution is, for example, an aqueous solution of about 0.1 to 90% by weight. A preferable concentration as an aqueous solution can be selected depending on the kind of the reactive polysaccharide. If the reactive polysaccharide is hyaluronic acid selected as the polysaccharide, 0.1 to 5% by weight is preferable, and if it is chondroitin sulfate, 0.1 to 50% by weight is a preferable concentration of the aqueous solution.

As ionizing radiation, the irradiation time for cross-linking may be short, there is almost no rise in temperature during irradiation, and the effect on the irradiated object (such as lowering the molecular weight of the polysaccharide) is small. Lines are preferred. Examples of the electron beam irradiation device include an ion plasma type electron beam irradiation device and a continuous beam scanning type electron beam irradiation device.

The type of polysaccharide, the molecular weight,
The desired cross-linking is performed by selecting or controlling the type of ionizing radiation reactive compound to be bound, DS, etc. to produce an ionizing radiation reactive polysaccharide, and further selecting or controlling the irradiation conditions of the ionizing radiation to irradiate the reactive polysaccharide. Rate and thus the desired physical properties (eg mechanical strength, water retention, hydrophilic or hydrophobic, lubricity, drug release rate, etc.) and biological properties (eg biodegradability, bioabsorption, in vivo or in vitro). It is possible to produce a cross-linked polysaccharide having long-term stability, cell adhesion, physiological activity (antithrombogenicity, etc.).

For example, an ionizing radiation-reactive polysaccharide having a high DS is easily crosslinked by irradiation with ionizing radiation,
The cross-linked polysaccharide has a high cross-linking rate and a dense three-dimensional network structure.
In addition, the strength is generally higher and the swelling ratio [(weight when swelling-weight when drying) / weight when drying] is lower than that of a coarse three-dimensional structure. Further, the ionizing radiation-reactive polysaccharide containing a spacer can be crosslinked with a DS lower than that of a spacer-free polysaccharide, and a crosslinked polysaccharide having a high swelling rate can be obtained. For example, when hyaluronic acid having a molecular weight of 1 million is used as a raw material and a compound obtained by binding cinnamic acid through a spacer is irradiated with appropriate ionizing radiation, the DS is 0.5 or less.
Even if it is less than or equal to%, it is crosslinked and a high-strength crosslinked polysaccharide is obtained.

Further, the biological function can be controlled by changing the crosslinking rate. For example,
The tendency varies depending on the types of the polysaccharide and the ionizing radiation-reactive compound, but cells such as endothelial cells tend to easily adhere to the crosslinked polysaccharide when the hydrophobicity, DS, and crosslinking rate increase. Utilizing this property, the types of polysaccharides and ionizing radiation-reactive compounds are selected to control cross-linking polysaccharides having different cell adhesion properties by controlling DS and / or cross-linking ratio, and By coating the outer membrane, different functions can be given to the inner membrane and the outer membrane. That is, for example, it is possible to prevent the formation of a thrombus by making the inner membrane non-cell-adhesive, while imparting cell-adhesiveness to the outer membrane to adhere fibroblasts to make it blood-impermeable. it can.

Further, the crosslinked polysaccharide can be formed into a desired shape (membrane, tubular, granular, gel, etc.) as it is before irradiation with ionizing radiation or by further crushing. The cross-linked polysaccharide and the modified medical material containing the cross-linked polysaccharide obtained by the production method of the present invention have good biocompatibility, and therefore, artificial organs (for example, artificial blood vessels, artificial hearts,
In addition to the materials that make up the artificial skin, etc., wound dressing materials, defect filling materials, adhesion prevention materials, materials that make up the sustained-release device for physiologically active substances, endothelial cells, epithelial cells, smooth surfaces when creating hybrid artificial organs. It can be used as an artificial extracellular matrix useful for adhering and proliferating muscle cells, a material for a contact lens or an artificial basement membrane, a medical device used for diagnosis and treatment, and the like.

Further, the crosslinked polysaccharide containing a physiologically active substance can be used as a material or a pharmaceutical preparation capable of sustained-release of the embedded physiologically active substance. Hereinafter, typical uses will be described more specifically. [Anti-adhesion material] The cross-linked polysaccharide obtained by the production method of the present invention is
It can be used as an adhesion preventive material for preventing adhesion of a surgical scar during surgery and for accelerating recovery. In this case, it is preferable that the polysaccharide itself has a wound healing effect such as hyaluronic acid.

For example, a film (film) of a cross-linked polysaccharide (hereinafter sometimes referred to as cross-linked hyaluronic acid) in which hyaluronic acid is selected as the polysaccharide is coated on the abdominal wall or an abdominal organ (liver etc.) By protecting the defect portion, adhesion can be prevented, wound healing can be promoted, and the membrane can be decomposed and absorbed depending on the wound healing rate. It is also possible to enhance the above-mentioned effect by containing an anti-inflammatory agent, a wound healing promoter and the like in the membrane and gradually releasing them.

When the crosslinked polysaccharide obtained by the production method of the present invention is used as an anti-adhesion material, it has an appropriate strength with no membrane cracks, non-adhesiveness to tissues (cells), and a wound healing rate. It is desirable that it has a function of exhibiting biodegradability in accordance with the above, and does not exhibit toxicity even if absorbed into the living body after degradation. These functions can be controlled by selecting the type of ionizing radiation reactive polysaccharide (type of polysaccharide, type of ionizing radiation reactive compound, selection of DS) and the ionizing radiation irradiation condition at the time of ionizing radiation irradiation.

[Controlled Release of Drug] A crosslinked polysaccharide containing a physiologically active substance obtained by the production method of the present invention has a physiologically active substance (hereinafter referred to as a drug) in its three-dimensional network structure. Can be used as a material (carrier) for sustained release of a drug. That is, the drug can be released while maintaining the drug concentration in a certain range required in the environment in which the drug is released, for a period corresponding to the type of drug and the mode of use.

The controlled release rate of the drug is controlled by selection of the type of ionizing radiation-reactive polysaccharide (type of polysaccharide, type of ionizing radiation-reactive compound, selection of DS), ionizing radiation irradiation conditions at the time of ionizing radiation irradiation, Specifically, it can be performed by adjusting the ionizing radiation dose and the like. In this case, generally, the greater the molecular weight of the drug and the greater the electrostatic or hydrophobic attraction between the drug and the cross-linked polysaccharide, the slower the release rate, but the drug and the ionizing radiation-reactive polysaccharide The desired release rate can be controlled by selecting the combination of the respective chemical structures (molecular weight, charge state (degree of hydrophilicity or hydrophobicity), etc.) or devising the dosage form.

The drug sustained-release material or formulation in which the drug is embedded in the cross-linked polysaccharide can be processed into any form depending on the type of drug and the mode of use. For example, it can be processed into a film (film, coating), jelly, gel, cream, suspension, microcapsule, tablet, granule, powder and the like. Also, gauze, bandages, knitted fabrics, paper, cotton,
After impregnating or applying a solution or gel of an ionizing radiation-reactive polysaccharide containing a drug onto a support such as a non-woven fabric, a film, or a porous sponge, it is dried as necessary and irradiated with ionizing radiation to be used as a crosslinked polysaccharide. May be offered.
Furthermore, the ionizing radiation-reactive polysaccharide containing a drug may be applied to the surface or luminal surface of a structure such as an artificial organ (artificial blood vessel, artificial heart, etc.) for crosslinking.

For embedding a drug in the crosslinked polysaccharide of the present invention, for example, an aqueous solution or an organic solvent (eg, dimethylformamide) solution containing about 0.1 to 90% by weight of an ionizing radiation-reactive polysaccharide can be added to about 0.1% by weight. The drug is dissolved or suspended so that the concentration becomes 001 to 80%, various additives are added as necessary, molded, and dried as necessary, and then ionized radiation is irradiated. After cross-linking, the drug sustained-release material in solid, semi-solid or suspension form can be obtained as it is or by pulverizing it as required.

The crosslinked polysaccharide including the drug obtained by the production method of the present invention can be used as a pharmaceutical preparation. In this case, the cross-linked polysaccharide containing the drug is used as it is, or if necessary, a pharmaceutically acceptable preservative, stabilizer, soothing agent, dispersant, additive for adjusting moldability, solubilizing agent, etc. It can be used in any dosage form by known formulation techniques. For example, a cross-linked polysaccharide containing a drug
It can be made into a film as it is or fixed using another support, and then it is percutaneously absorbed, intraocularly administered (eg, corneal wound healing promoter), in vivo implant, or inserted into body cavity (eg, seat). Agent). These can be used as wound dressings, drug release patches (such as bandaids), and contraceptives.

As a drug used when a cross-linked polysaccharide containing a drug is used as a pharmaceutical preparation, it is a drug that is usually forced to be frequently administered to maintain an effective blood concentration or an effective local concentration. A drug that is retained in its structure and is controlled and released is particularly effective, but is not particularly limited. Specifically, the following drugs may be mentioned as examples.

1. 1. Antipyretic and analgesic and anti-inflammatory agents such as indomethacin, mefenamic acid, acemethacin, alclofenac, ibuprofen, tiaramid hydrochloride, fenbuene, mepyrizole and salicylic acid. 2. Anti-neoplastic agents such as methotrexate, fluorouracil, vincristine sulfate, mitomycin C, actinomycin C, daunorubicin hydrochloride, etc. Aceglutamide Aluminum, L-Glutamine, P-
(Trans-4-aminomethylcyclohexanecarbonyl)-
3. Anti-ulcer agents such as phenylpropionate hydrochloride, cetraxate hydrochloride, sulpiride, gefarnate, cimetidine, etc. 4. Enzyme preparations such as chymotrypsin, streptokinase, lysozyme chloride, bromelain, urokinase, etc. 5. Antihypertensive agents such as clonidine hydrochloride, bunitrolol hydrochloride, brazosin hydrochloride, captopril, betanidine sulfate, metoprolol tartrate and methyldova; 6. Agents for urinary organs such as flavoxate hydrochloride 7. 7. Anticoagulants such as heparin, dichromal, warfarin; 8. Arteriosclerotic agents such as clofibrate, synfibrate, elastase and nicomol. 10. Cardiovascular agents such as nicardipine hydrochloride, nimodipine hydrochloride, cytochrome C, tocopherol nicotinate, etc. 10. Steroid agents such as hydrocortisone, prednisolone, dexamethasone and betamethasone; Wound healing accelerators such as growth factors and collagen (see JP-A-60-222425), other polypeptides having physiological activity, hormone agents, antituberculous agents, hemostatic agents, antidiabetic agents, vasodilators, antiarrhythmic agents, Examples include cardiotonic agents, antiallergic agents, antidepressants, antiepileptic agents, muscle relaxants, antitussive agents, antibiotics and the like.

The drug sustained-release material of the present invention is used as a constituent part (surface, etc.) of a structure such as an artificial organ (artificial blood vessel, artificial heart, etc.).
It can be used as a medical material.
In this case, particularly as a medical material constituting the surface that comes into contact with blood, an anticoagulant substance (heparin, heparan sulfate, thrombomodulin, etc.) for imparting antithrombotic properties,
A fibrinolytic activator (tissue plasminogen activator, urokinase, etc.) and an antiplatelet activator can be embedded in a cross-linked polysaccharide to control / release these substances.

[Artificial extracellular matrix, artificial basement membrane]
Ionizing radiation-reactive polysaccharides and cell-adhesive proteins such as collagen, gelatin and fibronectin are mixed and cross-linked to form cross-linked polysaccharides, or those chemically bound to these cross-linked polysaccharides are used as cells (endothelial cells, epithelium). Use as an artificial extracellular matrix or artificial basement membrane for adhering and proliferating cells, smooth muscle cells, etc. (JP-A-1-124465, JP-A-61-128974, JP-A-62-270162, etc.) You can These can be applied to hybrid (type) artificial organs (artificial blood vessels, artificial skin, etc.).

[0053]

The present invention will be described in more detail with reference to the following examples. In the examples, various physical properties and biological functions were measured by the following instruments and methods. UV / Vis spectra were measured using a Jasco Ubest-30 UV / VS spectrometer.

Bonding Degree of Ionizing Radiation Reactive Compound (D
S) was calculated from UV. That is, it was determined by comparison with the UV absorption of a low molecular weight model compound (polysaccharide degradation product) bound with an ionizing radiation reactive compound. The gelation rate of the crosslinked polysaccharide was measured by the following formula. Gelation rate (%) = Wg (swollen) / Wg (dr
y) × 100 Wg (dry) is the weight of the dried cross-linked polysaccharide Wg (swollen) is the dry weight of the cross-linked polysaccharide after soaking the dried cross-linked polysaccharide in purified water for 24 hours Example 1 Hyaluronic acid-cinnamic acid Preparation of ester and preparation of cross-linked hyaluronic acid by electron beam irradiation (1) Preparation of hyaluronic acid-cinnamic acid ester Hyaluronic acid (molecular weight 880,000) 5 g water-dioxane mixed solution (2 l water / 500 ml dioxane)
In addition, cinnamic acid (50 mmol), 4-dimethylaminopyridine (50 mmol), triethylamine (50
Dioxane solution (200 ml) of the mixture (mmol) was added, and the mixture was stirred at room temperature for 2 hours. Ethanol saturated with sodium acetate was added to the reaction solution, and the resulting precipitates were collected, thoroughly washed with ethanol, and dried to obtain a cinnamic acid ester of hyaluronic acid.

Yield: 4.5 g DS: 0.0593 (determined from UV) GPC measurement showed that hyaluronic acid did not have a low molecular weight. (2) Preparation of cross-linked hyaluronic acid An aqueous solution of the above hyaluronic acid-cinnamic acid ester (400
(mg / 80 ml) was placed on a 6 cm × 9 cm plastic square petri dish and dried overnight with sterile 40 ° C. warm air. The obtained colorless and transparent film having a thickness of 50 μm was irradiated with an electron beam of 0.125 Mrad by an ion plasma type electron beam irradiation device.

As a result of irradiation, the film which was readily soluble in water without irradiation became a water-insoluble film. Example 2 Preparation of electron beam-reactive hyaluronic acid using 6-aminohexanoyl-4-aminocinnamic acid methyl ester hydrochloride and preparation of cross-linked hyaluronic acid by electron beam irradiation (1) of electron beam-reactive hyaluronic acid Preparation t-butoxycarbonyl-6-aminohexanoic acid 693
mg (3.0 mmol) was dissolved in 3 ml of chloroform, and 417 μl (3.0 mmol) of triethylamine and 389 μl of dimethylphosphinothioyl chloride (3.
1 mmol of a chloroform solution (0 mmol) was sequentially added under ice cooling. After stirring for 5 minutes at room temperature, triethylamine 4
A chloroform solution of 17 μl (3.0 mmol) and 4-aminocinnamic acid methyl ester 532 mg (3.0 mmol) was added under ice cooling, and the mixture was again stirred at room temperature for one day.
After the reaction solution was concentrated under reduced pressure, 50 ml of ethyl acetate was added,
The layers were separated and washed with a 5% aqueous citric acid solution twice, water, 5% sodium hydrogen carbonate twice, water and a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate and then filtered, the filtrate was concentrated under reduced pressure, and ether was added to precipitate crystals. The obtained crystals were washed with ether and then dried under reduced pressure to obtain 468 mg (yield 40%) of t-butoxycarbonyl-6-aminohexanoyl-4-aminocinnamic acid methyl ester as white crystals.

468 mg (1.2 mmol) of this compound
Under ice cooling, 4 ml of 4N hydrogen chloride-dioxane solution was added to
The mixture was stirred at room temperature for 30 minutes to carry out a deprotection reaction. After confirming the completion of the reaction (deprotection of t-butoxycarbonyl group) by thin layer chromatography (developed with ether), 20 ml of anhydrous ether was added to precipitate crystals. The crystals were washed with ether several times on a glass filter and then dried under reduced pressure.
Aminohexanoyl-4-aminocinnamic acid methyl ester hydrochloride (377 mg, yield 97%) was obtained.

Hyaluronic acid (molecular weight 880,000) sodium salt (400 mg) and 6-aminohexanoyl-4-aminocinnamic acid methyl ester hydrochloride (0.1 mmol) were mixed with water-dioxane (100 ml water / 20 ml dioxane). In addition to the above, N-hydroxysuccinimide (0.2 mmol) and water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (0.1 mmol) were added, and the mixture was stirred at room temperature for 2 Reacted for hours. Precipitates formed by adding ethanol saturated with sodium acetate to the reaction solution were collected, thoroughly washed with ethanol, and dried to obtain electron beam-reactive hyaluronic acid.

Yield: 372 mg DS: 0.0008 (determined from UV) (2) Preparation of crosslinked hyaluronic acid film An aqueous solution of the above electron beam-reactive hyaluronic acid (372 mg /
80 ml) was placed on a 6.2 cm x 9.2 cm plastic square petri dish and dried overnight with sterile 40 ° C warm air. The obtained colorless and transparent film having a thickness of 50 μm was irradiated with an electron beam of 0.125 Mrad by an ion plasma type electron beam irradiation device.

As a result of the irradiation, the film which was easily soluble in water when not irradiated became a water-insoluble and highly swellable film. Gelation rate: 85% (3) Table 1 shows the relationship between the electron beam irradiation dose of the electron beam reactive hyaluronic acid and the shape of the obtained crosslinked hyaluronic acid in water. As for the shape in water, a cross-linked hyaluronic acid film (about 1 x 1 cm) was used in an excess amount of purified water.
It is a film shape after soaking for 4 hours.

[0061]

[Table 1]

Example 3 Preparation of electron beam reactive hyaluronic acid using cinnamic acid (6-aminohexyl) ester hydrochloride and preparation of crosslinked hyaluronic acid by electron beam irradiation (1) Preparation of electron beam reactive hyaluronic acid Preparation t-butoxycarbonyl-6-aminohexanol 2.
085 g (9.6 mmol) of chloroform 9.6 ml
Dissolved in 1.32 ml of triethylamine (9.6 m
mol), cinnamic acid chloride 1.38 ml (9.6 m)
mol) and 4-dimethylaminopyridine 1.173
mg (4.8 mmol) was added under ice cooling. After stirring for 5 minutes, the mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure, 50 ml of ethyl acetate was added, and the mixture was separated and washed with 5% aqueous citric acid solution twice, water, 5% sodium hydrogen carbonate twice, water and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2.974 g (yield 89.1) of cinnamic acid (t-butoxycarbonyl-6-aminohexyl) ester as a white powder. %)Obtained.

2.974 g (8.6 mmo) of this compound
Under ice-cooling, 50 ml of 4N hydrogen chloride-dioxane solution was added to 1), and the mixture was stirred for 2 hours at room temperature to carry out a deprotection reaction. After confirming completion of the reaction (deprotection of t-butoxycarbonyl group) by thin layer chromatography (developed with ether), the reaction solution was concentrated under reduced pressure and ether was added to obtain a precipitate. The precipitate was washed with ether and dried under reduced pressure to give white powder of cinnamic acid (6-aminohexyl) ester hydrochloride (1.974 g).
(Yield 70%) was obtained.

Hyaluronic acid (molecular weight 880,000) sodium salt (400 mg) and cinnamic acid (6-aminohexyl) ester hydrochloride (0.1 mmol) were added to a water-dioxane mixture (100 ml water / 20 ml dioxane). , Further, N-hydroxysuccinimide (0.2 mmo
1) and water-soluble carbodiimide (0.1 mmol) were added, and the mixture was reacted at room temperature for 2 hours. Precipitates formed by adding ethanol saturated with sodium acetate to the reaction solution were collected, thoroughly washed with ethanol, and dried to obtain the subject electron beam-reactive hyaluronic acid.

Yield: 371 mg DS: 0.0022 (determined from UV) (2) Preparation of crosslinked hyaluronic acid film An aqueous solution of the above electron beam-reactive hyaluronic acid (371 mg /
80 ml) was placed on a 9.2 cm × 6.2 cm plastic Petri dish and dried overnight with sterile 40 ° C. warm air. The obtained colorless and transparent film having a thickness of 50 μm was irradiated with an electron beam of 0.125 Mrad by an ion plasma type electron beam irradiation device.

As a result of the irradiation, the film which was easily soluble in water when not irradiated became a water-insoluble and highly swellable film. Gelation ratio: 72% Example 4 Preparation of electron beam reactive chitosan using cinnamoyl-6-aminohexanoic acid and preparation of crosslinked chitosan by electron beam irradiation (1) Electron beam reactive chitosan 6-aminohexanoic acid 1 Dissolve 0.31 g (10 mmol) in 2 ml of water, and add 4M aqueous sodium hydroxide solution 2.5.
ml (10 mmol) was added. Under ice cooling, cinnamic acid chloride 1.58 ml (11 mmol) / dioxane solution 2 m
1 and 4 ml of 3 M sodium hydroxide (3 ml, 12 mmol)
The reaction solution was divided into two parts and slowly added dropwise so that the reaction solution did not become acidic. After stirring overnight at room temperature, dioxane was distilled off under reduced pressure, ethyl acetate was added, and liquid separation washing was performed. The aqueous phase was acidified with citric acid and extracted three times with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a white solid. The obtained solid was recrystallized from ethanol-ether-hexane to give 2.00 as white crystals.
g (77% yield) of cinnamoyl-6-aminohexanoic acid was obtained.

The carboxyl group of this compound was made into a mixed acid anhydride with dimethylphosphinothioyl oil in dimethylformamide (DMF). Triethylamine (0.2 mmol) was added to a suspension aqueous solution of chitosan (Seikagaku Corporation, deacetylation degree 70 to 90%) (290 g), and the mixed acid anhydride was further added. After stirring at room temperature for 2 hours, the reaction solution was mixed with ethanol saturated with sodium acetate and the resulting precipitates were collected, thoroughly washed with ethanol on a glass filter, and dried to obtain the title compound.

Yield: 230 mg (2) Preparation of crosslinked chitosan film An aqueous solution of the above electron beam reactive chitosan in acetic acid (230 mg /
200 ml) was placed on a 4 cm × 7 cm plastic square petri dish and dried overnight with sterile 40 ° C. hot air. The colorless transparent film having a thickness of 50 μm was irradiated with an electron beam of 0.25 Mrad by an ion plasma type electron beam irradiation device.

As a result of irradiation, the film which was fragile and hardly soluble in water when not irradiated became a completely water-insoluble and flexible film. Example 5 Preparation of Crosslinked Polysaccharide Embedding Indomethacin 25 μg of indomethacin was dissolved in 5 ml of a 5% aqueous solution of hyaluronic acid-cinnamic acid ester obtained in (1) of Example 1, and placed on a slide glass to be sterilized. Dried with warm air. The film thus formed was irradiated with an electron beam of 0.25 Mrad using an ion plasma type electron beam irradiation apparatus to obtain a target product.

Example 6 Artificial Blood Vessel The inner surface of a small diameter artificial blood vessel (inner diameter 3 mm) was spin coated with the aqueous solution of the electron beam reactive hyaluronic acid obtained in Example 3 and then dried. Subsequently, an electron beam of 0.5 Mrad is irradiated from the outside of the artificial blood vessel using an electron beam irradiation device,
An artificial blood vessel having an inner surface coated with crosslinked hyaluronic acid was obtained.

As described above, after coating the inner surface of the artificial blood vessel with cross-linked hyaluronic acid, the outer surface was further covered with an ionizing radiation-reactive polysaccharide having a hydrophobic property, a high DS and a high cross-linking degree, and an electron beam was applied from the outside of the artificial blood vessel. When irradiated, the inner membrane is covered with a hydrophilic, low DS and low degree of cross-linking membrane to prevent thrombus formation by making the inner membrane non-cell-adhesive and the outer membrane to be hydrophobic, high DS and By covering with a membrane having a high degree of cross-linking and making it cell-adhesive, fibroblasts can be adhered to produce a blood-impermeable artificial blood vessel.

[0072]

EFFECTS OF THE INVENTION According to the production method of the present invention, by irradiating an ionizing radiation-reactive polysaccharide with ionizing radiation, it has a safety, biocompatibility, biodegradability and absorbability having a two-dimensional and / or three-dimensional crosslinked structure. A high medical material can be provided. In addition, the molecular weight of the polysaccharide, the type of ionizing radiation reactive group, the DS of the ionizing radiation reactive polysaccharide, the crosslinking rate,
By selecting and controlling the gelation rate and the like, it is possible to provide a medical material composed of a crosslinked polysaccharide having desired physical properties, and therefore, the applicability in various medical fields is extremely wide.

Claims (8)

[Claims] 1. An ionizing radiation-reactive polysaccharide obtained by covalently bonding a polysaccharide and at least one ionizing radiation-reactive compound, and the reactive covalently bonded to the reactive polysaccharide by irradiating with ionizing radiation. A method for producing a crosslinked polysaccharide, which comprises causing a crosslinking reaction between compound residues. 2. The polysaccharide is a biocompatible polysaccharide.
A method for producing the crosslinked polysaccharide described. 3. The method for producing a crosslinked polysaccharide according to claim 2, wherein the biocompatible polysaccharide is glycosaminoglycan. 4. A cross-linking reaction between ionizing radiation-reactive compound residues covalently bonded to the reactive polysaccharide by irradiating the ionizing radiation-reactive polysaccharide according to claim 1 containing a physiologically active substance. A method for producing a crosslinked polysaccharide containing a physiologically active substance, which comprises causing 5. The ionizing radiation-reactive polysaccharide is in the form of a solution, a gel or a sheet.
4. The method for producing the crosslinked polysaccharide according to any one of 4 above. 6. The method for producing a crosslinked polysaccharide according to claim 1, wherein the crosslinked polysaccharide or the crosslinked polysaccharide containing a physiologically active substance is in the form of a film, particles or gel. 7. An ionizing radiation-reactive polysaccharide formed by covalently bonding a polysaccharide and at least one ionizing radiation-reactive compound to the surface or inside of a medical material, and irradiating with ionizing radiation to cause the reaction. A method for producing a modified medical material, which comprises causing a cross-linking reaction between the reactive compound residues covalently bound to a sex polysaccharide. 8. The method for producing a modified medical material according to claim 7, wherein the ionizing radiation-reactive polysaccharide is in the form of a solution, gel or sheet.