JP2006111867A - Photoreactive polysaccharide, its photocrosslinking polysaccharide product, and medical material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a photocrosslinking polysaccharide excellent in barrier effect and degradability which are necessary characteristics for medical materials, and to provide a photoreactive polysaccharide for producing the same and a medical material comprising the photo crosslinking saccharide. <P>SOLUTION: This photoreactive polysaccharide includes therein a covalent bonding of a polysaccharide and glycidyl ester expressed by the general formula (1) or (2). [In the formula, R1-CO- or -OC-R2-CO- shows photoreactive residues of a photoreactive compound having carboxyl groups or unsaturated carbon double bonds in molecules for each respectively. However, in the case when the polysaccharide is heparin, R1-CO- is not acryloyl group and methacryloyl group]. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a photoreactive polysaccharide, a photocrosslinked polysaccharide obtained by photocrosslinking it, and a medical material, and more specifically, a glycidyl ester of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond. The present invention relates to a photoreactive polysaccharide, a photocrosslinked polysaccharide obtained by irradiating the polysaccharide with light, and a medical material comprising the photocrosslinked polysaccharide.

  Since polysaccharides are safe polymer substances derived from living bodies, attempts have been made to apply the modified substances to various medical materials. For example, Non-Patent Document 1 describes an example of gelling by cross-linking chondroitin sulfate with a diglycidyl ether cross-linking agent as a polysaccharide cross-linking gel. However, when such a cross-linking agent is used, a cross-linking reaction occurs simultaneously with the reaction between chondroitin sulfate and the cross-linking agent, so that it is difficult to remove unreacted cross-linking agents and by-products from the cross-linked gel after the reaction is completed. There was a problem. On the other hand, when gel-like cross-linked polysaccharide with insufficient removal of cross-linking agent is applied intraperitoneally, for example, as an anti-adhesion material, it has been experimentally confirmed that it causes damage to the liver. It is an important issue. If the cross-linked polysaccharide is in the form of a sponge, it is relatively easy to remove by washing. However, in the case of a gel-like product or a film-shaped product, the cross-linking agent is taken into the inside, so that the removal becomes extremely difficult.

  Therefore, a technique for obtaining a photoreactive polysaccharide that can be easily removed by binding a compound having a photoreactive cross-linking group as a cross-linking agent to the polysaccharide, and capable of becoming a solution, and photocrosslinking the photoreactive polysaccharide. Has been developed, and attempts have been made to use the photocrosslinked polysaccharide thus obtained for various medical materials. For example, after removing unreacted crosslinker by purifying photoreactive glycosaminoglycan obtained by introducing a crosslinker such as cinnamic acid, thymine, coumarin etc. into glycosaminoglycan which is natural polymer Patent Document 1 discloses that a photo-crosslinked product is obtained by UV-crosslinking to obtain a cross-linked glycosaminoglycan and its use as an anti-adhesion material and a carrier for sustained drug release. Patent Documents 2 and 3 disclose a photocrosslinkable hyaluronic acid derivative in which cinnamic acid is bonded to hyaluronic acid via a spacer selected from an amino acid or a derivative thereof, a peptide, an amino alcohol, or a diamine, and a photocrosslink thereof. The body is listed.

  Patent Document 4 discloses a hydrogel having specific physical properties obtained by irradiating ultraviolet rays onto a photoreactive hyaluronic acid derivative in which a photoreactive crosslinking group is chemically bonded to a functional group of hyaluronic acid, and the use of the hydrogel. A medical material having an anti-adhesion effect has been described. Patent Document 5 describes a polysaccharide sponge that can be used for a medical device obtained by photocrosslinking a photoreactive polysaccharide. Patent Document 6 improves hydrophilicity and improves the filterability of an aqueous solution. Photoreactive hyaluronic acid and medical materials are described. Furthermore, it is described in Patent Document 7 that a heparin derivative that is highly useful as an intermediate of a medical polymer material is produced by reacting heparin with glycidyl acrylate or glycidyl methacrylate, and glycidyl acrylate or glycidyl Patent Document 8 discloses a cross-linked product of a mixture with a methacrylate-linked polysaccharide (excluding hyaluronic acid) and hyaluronic acid.

Among medical materials, the essential requirement as an antiadhesive material is to have a sufficient barrier effect and moderate degradability. However, when conventional photocrosslinked polysaccharides are used as anti-adhesion materials, if the barrier performance is to be increased, the in vivo persistence becomes extremely long, while on the other hand, an appropriate barrier property is to be provided. The dilemma of not being effective has been repeated. For example, when a photo-crosslinked polysaccharide gel sheet or sponge having a high barrier effect is used as an adhesion-preventing material, there remains a problem that the in vivo persistence is slightly high. Therefore, it has been desired to develop a medical material, particularly an adhesion-preventing material, having both properties as described above, which is excellent in biodegradability while maintaining sufficient strength as a medical material.
Eur J. Pharm Sci 2002 Mar; 15 (2): 139-48 JP-A-6-73102 JP-A-8-143604 JP-A-9-87236 11-512778 WO02 / 060971 JP2002-249501 JP 56-147802 USP6586493

As a result of diligent research to find a photocrosslinked polysaccharide having a high barrier effect and an appropriate degradability, the present inventor has conducted a photoreaction having a specific crosslinker in the polysaccharide, that is, a carboxyl group and an unsaturated carbon double bond. That the photo-crosslinked polysaccharide obtained by photo-crosslinking a photoreactive polysaccharide to which a glycidyl ester (for example, glycidyl cinnamate) is bonded has a sufficient barrier effect and is excellent in early degradability The inventor has completed the present invention.
An object of the present invention is to provide a photocrosslinked polysaccharide excellent in barrier effect and degradability, which are necessary characteristics as a medical material, a photoreactive polysaccharide for producing the polysaccharide, and a medical material comprising the photocrosslinked polysaccharide. And

This invention solves the said subject, and the summary consists of the item as described in the following.
1. A photoreactive polysaccharide in which a polysaccharide and a glycidyl ester represented by the following general formula (1) or (2) are covalently bonded.

〔式中、Ｒ¹−ＣＯ−または−ＯＣ−Ｒ²−ＣＯ−は、それぞれ、分子中にカルボキシル基及び不飽和炭素二重結合を有する光反応性化合物の光反応性残基を示す。但し、多糖がヘパリンの場合、Ｒ¹−ＣＯ−はアクリロイル基及びメタクリロイル基ではない。〕

[Wherein, R ¹ —CO— or —OC—R ² —CO— represents a photoreactive residue of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond in the molecule, respectively. However, when the polysaccharide is heparin, R ¹ —CO— is not an acryloyl group or a methacryloyl group. ]

2. 2. The photoreactive polysaccharide according to 1 above, wherein the covalent bond is an ester bond.
3. The photoreactive compound is a monocarboxylic acid selected from cinnamic acid, hydroxycinnamic acid, acrylic acid, methacrylic acid, furylacrylic acid, thiophene acrylic acid, cinnamylideneacetic acid and sorbic acid, or a dicarboxylic acid selected from maleic acid and fumaric acid 3. The photoreactive polysaccharide according to item 1 or 2, which is an acid.
4). 4. The photoreactive polysaccharide according to 3 above, wherein the photoreactive compound is cinnamic acid.
5). The above 1 wherein the polysaccharide is glycosaminoglycan, polyuronic acid, polyaminosaccharide, glucan, mannan, fructan, galactan, pectin, vegetable gum, agar, porphyran, carrageenan, fucoidan or a pharmaceutically acceptable salt or derivative thereof. The photoreactive polysaccharide according to item.
6). Glycosaminoglycan is hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate or keratan sulfate, polyuronic acid is alginic acid, glucan derivative is carboxymethylcellulose, polyaminosaccharide or derivative thereof is chitin or 6. The photoreactive polysaccharide according to item 5, which is chitosan.
7). 7. The photoreactive polysaccharide according to 6 above, wherein the glycosaminoglycan is hyaluronic acid.

8). The photoreactive compound is an acid selected from cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, acrylic acid, methacrylic acid, furyl acrylic acid, thiophene acrylic acid, cinnamylidene acetic acid and sorbic acid, and the polysaccharide is 2. The photoreactive polysaccharide according to item 1, which is a polysaccharide selected from hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, chitin, chitosan, alginic acid and carboxymethylcellulose.
9. The photoreactive compound is an acid selected from cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, furylacrylic acid, thiophene acrylic acid, cinnamylideneacetic acid and sorbic acid, and the polysaccharide is heparin, chitin, chitosan 2. The photoreactive polysaccharide according to item 1, which is a polysaccharide selected from alginic acid and carboxymethylcellulose.

10. 10. A photocrosslinked polysaccharide obtained by photocrosslinking the photoreactive polysaccharide according to any one of 1 to 9 above.
11. 10. A gel-like photocrosslinked polysaccharide obtained by irradiating light to the solution of the photoreactive polysaccharide according to any one of 1 to 9 above.
12 10. A sponge-like photocrosslinked polysaccharide obtained by freezing or lyophilizing a solution of the photoreactive polysaccharide according to any one of 1 to 9 above and irradiating the frozen solution or lyophilized product with light.
13. The gel-like product produced by irradiating the photoreactive polysaccharide solution according to any one of 1 to 9 above with light is frozen and irradiated with light while maintaining the frozen state, or the gel-like product A photocrosslinked polysaccharide that combines the properties of a gel and a sponge obtained by lyophilizing and then irradiating light.
14 10. A film-like photocrosslinked polysaccharide obtained by irradiating light to a thin film obtained by drying the solution layer of the photoreactive polysaccharide according to any one of 1 to 9 above.
15. The gel-like product produced by irradiating the photoreactive polysaccharide solution according to any one of the above items 1 to 9 with light is frozen, irradiated with light while maintaining the frozen state, and further dried. Film-like photocrosslinked polysaccharide obtained by irradiating with light.
16. 16. A medical material comprising the photocrosslinked polysaccharide according to any one of 10 to 15 above.
17. 17. The medical material according to the above item 16, which is an adhesion preventing material.
18. 17. The medical material according to the above item 16, which is a base for sustained drug release.
19. Item 17. The medical material according to Item 16, which is a cell culture substrate.

  According to the present invention, photocrosslinking as a photocrosslinking product obtained by photocrosslinking a photoreactive polysaccharide in which a glycidyl ester of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond in the molecule is bonded to the polysaccharide. Polysaccharides can be provided, and the photocrosslinked polysaccharides are excellent in strength and biodegradability, and also retain the excellent characteristics of conventional photocrosslinked glycosaminoglycan gels and sponges. Therefore, by using the photocrosslinked polysaccharide of the present invention, it has become possible to provide medical materials, in particular, postoperative adhesion prevention materials, base materials for sustained drug release, or base materials for cell culture.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention, but is not limited to these descriptions.
The photoreactive polysaccharide of the present invention is one in which the glycidyl ester represented by the above general formula (1) or (2) is covalently bonded to the polysaccharide. The “polysaccharide” constituting this photoreactive polysaccharide includes Specifically, glycosaminoglycans, polyuronic acids, polyamino sugars, glucans, mannans, fructans, galactans, pectin, vegetable gums, agar, porphyrans, carrageenans, fucoidans or pharmaceutically acceptable salts or derivatives thereof. . Of these, glycosaminoglycans or salts and derivatives thereof are particularly preferred in the present invention.

  A glycosaminoglycan (hereinafter sometimes referred to as GAG) is a polysaccharide having a basic skeleton containing an amino sugar and uronic acid (or galactose). Examples include hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, preferably hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate, or keratan sulfate, more preferably hyaluronic acid. It is done. These pharmaceutically acceptable salts include alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium salts, salts with inorganic bases such as ammonium salts, pyridine salts, diethanolamine salts, cyclohexyl salts. Among the salts with organic bases such as amine salts and amino acid salts, pharmaceutically acceptable ones can be mentioned, and sodium salts are particularly preferable.

  “Derivatives” of glycosaminoglycans include sulfated derivatives in which sulfate groups are bound to GAG, desulfated derivatives in which sulfate groups are partially or completely removed from GAGs, and redox derivatives in which GAGs are subjected to redox reactions. And a redox desulfated derivative obtained by desulfating a redox derivative of GAG. Examples of sulfated derivatives include sulfated hyaluronic acid and chondroitin polysulfate, and examples of desulfated derivatives include 6-position desulfated heparin (see WO00 / 06608), 2-position desulfated heparin (Japanese Patent Laid-Open No. 2003-113090). And completely desulfated heparin, and examples of the redox-desulfated derivative include periodate redox-desulfated heparin (see JP-A-11-310602). Of these, desulfated derivatives and redox-desulfated derivatives are preferable, and desulfated derivatives are particularly preferable.

  On the other hand, as polysaccharides other than glycosaminoglycan (sometimes referred to as other polysaccharides), for example, polyuronic acid, polyamino sugar, glucan, mannan, fructan, galactan, pectin, vegetable gum, agar, porphyran, carrageenan , Fucoidan or pharmaceutically acceptable salts and derivatives thereof. Examples of polyuronic acid include alginic acid, examples of polyaminosaccharide and deacetylated derivatives thereof include chitin and chitosan, and examples of glucan and derivatives thereof include amylose, amylopectin, glycogen, cellulose, hydroxymethylcellulose and carboxymethylcellulose. Is mentioned. Examples of other polysaccharide salts are the same as those mentioned as the above-mentioned glycosaminoglycan salts. Examples of other polysaccharide derivatives include the above-mentioned carboxymethyl derivatives, hydroxymethyl derivatives, and deacetylated derivatives. Of these other polysaccharides and their derivatives, chitin, chitosan, alginic acid or carboxymethylcellulose is most preferred.

The molecular weight (weight average molecular weight) of the polysaccharide used in the photoreactive polysaccharide of the present invention is as follows. In the case of polysaccharides other than hyaluronic acid, it is usually 2,000 to 3,000,000, preferably 3,000 to 2,700,000, more preferably 4,000 to 2,500,000, and in the case of hyaluronic acid, usually 20,000 to 3,000,000, preferably 100,000 to 2,000,000, more preferably 200,000. ~ 1,200,000.
The polysaccharide in the present invention may be derived from a natural product, chemically synthesized, or produced by a microorganism such as yeast by a genetic engineering technique. In general, GAG can be prepared by extraction from a partial biological material (chicken crown, umbilical cord, cartilage, skin, small intestine, blood vessel, etc.), and they are preferable.

In the present invention, a carboxyl group and an unsaturated carbon double are formed in the molecule forming the R ¹ —CO— or —OC—R ² —CO— group of the glycidyl ester represented by the general formula (1) or (2). The photoreactive compound having a bond is acrylic acid represented by the following general formula (3) and derivatives thereof, and methacrylic acid.
[Chemical formula 3]
(R ³ ) —CH═C (R ⁴ ) —COOH (3)
[Wherein, R ³ represents a hydrogen atom, a phenyl group, a hydroxyphenyl group, a carboxyl group, a furyl group, a thienyl group, a styryl group, or a 1-propenyl group, and R ⁴ represents a hydrogen atom or a methyl group. However, when R ⁴ is a methyl group, R ³ represents a hydrogen atom. ]

Specific examples of the photoreactive compound include cinnamic acid, hydroxycinnamic acid, acrylic acid, methacrylic acid, furylacrylic acid, thiophene acrylic acid, cinnamylidene acetic acid, sorbic acid and other monocarboxylic acids or maleic acid, fumaric acid, etc. Dicarboxylic acid is mentioned. Among these, carboxylic acid having one unsaturated group such as cinnamic acid and thiophenacrylic acid, and carboxylic acid having two unsaturated groups such as cinnamylideneacetic acid and sorbic acid are preferable. From the aspect, cinnamic acid is most preferable.
A glycidyl ester of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond (hereinafter sometimes referred to as “photoreactive glycidyl ester”) reacts the photoreactive compound with an epihalohydrin, preferably epichlorohydrin. Can be obtained. When the photoreactive compound is a dicarboxylic acid such as maleic acid or fumaric acid, a monoester or diester can be obtained as the glycidyl ester, but a diester is preferred.

The photoreactive polysaccharide of the present invention is obtained by introducing the glycidyl ester of the photoreactive compound into the polysaccharide, and the glycidyl ester epoxy group is ring-opened so that the polysaccharide has a carboxyl group, hydroxyl group or It is covalently bonded to an amino group. The photoreactive polysaccharide is preferably a glycidyl ester of a photoreactive compound ester-bonded to a carboxyl group of a carboxyl group-containing polysaccharide. Examples of the carboxyl group-containing polysaccharide include hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, alginic acid, carboxymethylcellulose, and heparin.
As a preferred photoreactive polysaccharide of the present invention, the photoreactive compound of the glycidyl ester is cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, acrylic acid, methacrylic acid, furylacrylic acid, thiophenacrylic acid, Light selected from cinnamylideneacetic acid and sorbic acid, and the polysaccharide is composed of a polysaccharide selected from hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, chitin, chitosan, alginic acid and carboxymethylcellulose It is a reactive polysaccharide.

As other preferable photoreactive polysaccharides, the photoreactive compounds of the above glycidyl esters are cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, furylacrylic acid, thiophenacrylic acid, cinnamylideneacetic acid and sorbic acid. And the polysaccharide is a photoreactive polysaccharide composed of a polysaccharide selected from heparin, chitin, chitosan, alginic acid and carboxymethylcellulose.
Among the photoreactive polysaccharides, most preferably, the photoreactive compound of the glycidyl ester is cinnamic acid, and the polysaccharide is a photoreactive polysaccharide composed of hyaluronic acid.

The photoreactive polysaccharide of the present invention can be prepared according to the following method.
<Reverse precipitation method>
An organic solvent that mixes with water and does not react with the photoreactive glycidyl ester in an aqueous solution containing 0.1 to 15% by weight of the polysaccharide having a carboxyl group so that the mixing ratio of the aqueous solution with water in the aqueous solution is 0 to 50%. Photoreactive glycidyl ester is added to the added so that the weight concentration is 0.1 to 10%, and the mixture is stirred at 40 to 80 ° C. for 0.5 to 240 hours. Next, salt of 0.5 to 5 times the amount of polysaccharide used is added and poured into 2 to 5 times the amount of ethanol of the reaction solution to precipitate the precipitate. The precipitate is filtered off with a filter, and the precipitate is further thoroughly washed with ethanol and dried to obtain a photoreactive polysaccharide.
<Sequential precipitation method>
An organic solvent that mixes with water and does not react with the photoreactive glycidyl ester in an aqueous solution containing 0.1 to 15% by weight of the polysaccharide having a carboxyl group so that the mixing ratio of the aqueous solution with water in the aqueous solution is 0 to 50%. Photoreactive glycidyl ester is added to the added so that the weight concentration is 0.1 to 10%, and the mixture is stirred at 40 to 80 ° C. for 0.5 to 240 hours. Next, 0.5 to 5 times the amount of sodium chloride used is added and 0.5 to 5 times the amount of ethanol is poured into the reaction solution to precipitate the precipitate. The precipitate is filtered off with a filter, and the precipitate is further thoroughly washed with ethanol and dried to obtain a photoreactive polysaccharide.

  In the photoreactive polysaccharide of the present invention, the amount of the glycidyl ester represented by the general formula (1) or (2) introduced into the polysaccharide, that is, the introduction rate of the photoreactive group (unsaturated carbon double bond) is desired. The cross-linking ratio of the photo-crosslinked polysaccharide is determined depending on the type of the polysaccharide and the photoreactive compound constituting the glycidyl ester. For example, the molecular weight of the polysaccharide and the carboxyl group or hydroxyl group which is a functional group of the polysaccharide Alternatively, it is selected in consideration of the type and number of amino groups and the like, and unsaturated groups of the photoreactive compound. The rate of introduction of the photoreactive group into the polysaccharide is expressed as a percentage of the number of photoreactive groups introduced per saccharide unit of the polysaccharide, for example, the repeating disaccharide unit of glycosaminoglycan, as described in the examples below. It is the value expressed by. The introduction rate of the photoreactive group is usually about 1 to 20%, preferably about 1 to 15%, although it depends on the type of polysaccharide.

The photocrosslinked polysaccharide of the present invention is a crosslinked product obtained by irradiating the photoreactive polysaccharide with light, and has photoreactivity (crosslinking) possessed by a glycidyl ester of a photoreactive compound covalently bonded to the photoreactive polysaccharide. It has a cyclobutane ring formed by crosslinking groups, that is, unsaturated carbon double bonds, and has a network structure.
The light irradiation to the photoreactive polysaccharide is preferably performed under conditions that allow the photoreactive group to efficiently cause a photodimerization reaction. Although the kind of light to irradiate is not specifically limited, When a photoreactive compound is cinnamic acid, an ultraviolet-ray is preferable. As the ultraviolet ray to be irradiated, an ultraviolet ray having a wavelength (for example, 200 to 600 nm) that does not break the glycosidic bond of the photoreactive polysaccharide and causes a photodimerization reaction in the photoreactive group is selected. As the ultraviolet lamp, a high-pressure mercury lamp or a metal halide lamp is preferable. Furthermore, if necessary, it is preferable to remove unnecessary wavelength light of ultraviolet rays generated by these lamps, for example, with a cut filter in order to obtain ultraviolet rays in a desired wavelength region. The amount of light to be irradiated is about 0.01 to 200 J / cm ² and is appropriately selected according to the material shape (gel, sponge, composite material, film) of the object.
The crosslinking rate of the photocrosslinked polysaccharide of the present invention is usually about 1 to 60%, although it varies depending on the rate of introduction of the photoreactive group introduced into the photoreactive polysaccharide and the conditions for photocrosslinking. The crosslinking rate can be determined by the measuring method described in the examples described later.

In the present invention, by changing the method and conditions in the photoirradiation step of the photoreactive polysaccharide, various forms of materials such as gel, sponge, composite having the properties of gel and sponge, or film Can be provided.
In the present invention, the gel-like photocrosslinked polysaccharide is a form containing a hydrogel containing an aqueous medium in the network structure (three-dimensional network structure) of the photocrosslinked polysaccharide. The photocrosslinked polysaccharide gel can be produced by a known method as described in, for example, JP-T-11-512778. Taking the case where the polysaccharide is hyaluronic acid as an example, a solution of photoreactive hyaluronic acid, which is a photoreactive polysaccharide, is prepared, and the solution is made into a mode (shape) that allows easy transmission of ultraviolet rays. The method including the process of irradiating is mentioned. For the details of the production method, the method described in JP-T-11-512778 can be referred to. Examples of the aqueous medium of the photoreactive hyaluronic acid solution at the time of ultraviolet irradiation include water, buffer solution, physiological saline, buffered physiological saline, and the like. Saline and buffered physiological saline are preferred.

  In the present invention, the sponge-like photocrosslinked polysaccharide is a porous substance having closed cells or open cells, has elasticity, is excellent in water absorption and drainage properties, and is insoluble in water and other aqueous media. Specifically, when the sponge-like photocrosslinked polysaccharide is dipped in a liquid medium such as water or other aqueous solvent in a dry state, the liquid medium is quickly absorbed and swollen, and an external pressure is applied after the swelling. It has a property of quickly discharging a liquid medium such as moisture absorbed by an operation such as placing on paper. The porous structure of the sponge varies depending on the type of photoreactive polysaccharide, production conditions, etc., but usually 50% or more of all pores per unit area have a pore diameter of 10 to 50 μm. In addition, the absorption and release properties of the liquid medium are good and the strength is maintained.

  The method for producing a photocrosslinked polysaccharide sponge includes a step (A) of freezing or lyophilizing a solution of a photoreactive polysaccharide and a polysaccharide sponge in which the photoreactive polysaccharide is crosslinked by irradiating the frozen solution or lyophilized product with light. It can be produced by the method described in WO02 / 060971 in more detail. The pore diameter can also be measured by the method described in the publication.

In the present invention, a photocrosslinked polysaccharide having the properties of a gel and a sponge (hereinafter referred to as a photocrosslinked polysaccharide composite) is a combination of the above-described method for preparing a photocrosslinked polysaccharide gel and the method for preparing a sponge. It can be prepared by the method described above, and has the properties of both gel and sponge characteristics. Specifically, the composite material has both the high barrier effect of the photocrosslinked polysaccharide gel and the high strength of the photocrosslinked polysaccharide sponge.
For example, when the polysaccharide is hyaluronic acid, the method for producing the photocrosslinked polysaccharide composite of the present invention prepares a high-concentration hyaluronic acid aqueous solution, and forms the solution into a shape that easily transmits ultraviolet light, for example, a solution layer having a desired thickness. A gel is obtained by irradiating with ultraviolet rays, and the obtained gel is frozen and manufactured by further irradiating with ultraviolet rays while maintaining the frozen state, or lyophilized and then further irradiating with ultraviolet rays. be able to.
The method for producing a photocrosslinked polysaccharide film of the present invention comprises producing a photoreactive polysaccharide solution by irradiating ultraviolet rays onto a thin film obtained by air-drying a coating film applied to a desired film thickness on a substrate, for example. More specifically, it can be produced by the method described in JP-A-6-73102. The photocrosslinked polysaccharide film thus obtained has a very high barrier effect, but may become slippery and brittle when wet. Therefore, for example, a sheet-shaped photocrosslinked polysaccharide composite material having a thickness of a solution layer obtained by the above-described production method is dried to form a thin film, and further irradiated with ultraviolet rays, whereby a barrier higher than that of the photocrosslinked polysaccharide composite material is obtained. A film having an effect and strength (hereinafter referred to as a photocrosslinked polysaccharide composite film) can be obtained.

A conventional method for crosslinking a polysaccharide using diglycidyl ether as a crosslinking agent (for example, the method described in Eur J. Pharm Sci 2002 Mar; 15 (2): 139-48) directly forms a bridge between polysaccharides and physicochemically Compared to the modification of the mechanical properties, the method for producing a photo-crosslinked polysaccharide molded product such as a sponge, composite material, film, composite film as described above of the present invention is significantly different in the following points, and excellent: ing.
First, in the method of the present invention, a molded product is formed during the photocrosslinking step from a photoreactive polysaccharide in which a glycidyl ester of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond is covalently bonded. Excellent in "Able to thoroughly wash or purify photoreactive polysaccharides and remove unreacted substances" and "Able to mold photoreactive polysaccharides to give diversity in material shape" ing. In other words, since no cross-linking occurs in the photoreactive polysaccharide production stage in which the glycidyl ester of the photoreactive compound is introduced into the polysaccharide, the glycidyl ester of the unreacted photoreactive compound must be confined within the photoreactive polysaccharide. However, it can be removed by sufficient washing and purification.

Further, in the present invention, a purified photoreactive polysaccharide is obtained by mixing a reaction solution of a polysaccharide with a glycidyl ester of a photoreactive compound and ethanol by the reverse precipitation method or the forward precipitation method, and precipitating the precipitate. It is possible. The photoreactive compound glycidyl ester is hardly soluble in water and easily soluble in ethanol. On the other hand, the photoreactive polysaccharide as a reaction product is hardly soluble in ethanol. This is because most of them are dissolved in ethanol and are not taken into the precipitate. In addition, by thoroughly washing the resulting photoreactive polysaccharide precipitate with ethanol, the glycidyl ester of the unreacted photoreactive compound adhering to the surroundings can be easily washed away, so an extremely high purity photoreaction Sex polysaccharides can be obtained.
In addition, when introducing a glycidyl ester of a photoreactive compound into a polysaccharide, no other condensing agent or catalyst is required, so that it is possible to avoid mixing of unnecessary substances. Furthermore, since the photoreactive polysaccharide can be formed into a desired form and then irradiated with light to form a crosslinked body, it is possible to obtain a crosslinked body having a material shape suitable for the application.

  The photocrosslinked polysaccharide of the present invention is a “medical material”, for example, a medical material for preventing adhesion between tissues after surgery (adhesion prevention material), a medical material constituting a drug sustained release device (base for drug sustained release) ), Base material for cell culture in cell culture (cell culture base material), medical material for wound protection (wound dressing material), medical material for maintaining space in living body (void) Retaining material), medical materials for filling cavities of connective tissues such as bone (bone filler), artificial body fluids (artificial joint fluid, artificial tears, ophthalmic surgical aids, etc.), moisturizing the surface of living bodies, etc. It can be used as an additive (excipient) to be added in order to maintain a dosage form such as a material (moisturizing material) or a medicine. Particularly useful as an anti-adhesion material, a drug sustained-release base material or a cell culture base material, and further in the form of a sponge, composite material, film, composite material film, etc., while having strength, Since it is excellent in degradability, it is particularly useful as an adhesion preventing material.

  The photocrosslinked polysaccharide of the present invention can be used as a drug sustained-release base material for embedding a drug in its three-dimensional network structure and slowing the drug release. Although it will not specifically limit if it is a chemical | medical agent hold | maintained at the network structure of a photocrosslinking polysaccharide and it is controlled and released, Specifically, the following chemical | medical agents are illustrated.

1. Salicylic acid, aspirin, mefenamic acid, tolfenamic acid, flufenamic acid, diclofenac, sulindac, fenbufen, indomethacin, acemetacin, ampenac, etodolac, felbinac, ibuprofen, flurbiprofen, ketoprofen, naproxen, pranoprofen, fenoprofen acid, thiaprofen acid , Non-steroidal anti-inflammatory analgesics such as oxaprozin, loxoprofen, aluminoprofen, zaltoprofen, piroxicam, tenoxicam, lornoxicam, meloxicam, tiaramid, tolmetine, diflunisal, acetaminophen, fructaphenine, tinolidine, thiaridine hydrochloride, mepyrizole,
2. Antineoplastic agents such as methotrexate, fluorouracil, vincristine sulfate, mitomycin C, actinomycin C, daunorubicin hydrochloride,
3. Anti-ulcer agents such as aceglutamide aluminum, L-glutamine, P- (trans-4-aminomethylcyclohexanecarbonyl) -phenylpropionate hydrochloride, cetraxate hydrochloride, sulpiride, gefarnate, cimetidine,
4). Enzyme preparations such as chymotrypsin, streptokinase, lysozyme chloride, bromelain, urokinase,
5). Antihypertensive agents such as clonidine hydrochloride, bunitrolol hydrochloride, brazocine hydrochloride, captopril, betanidin sulfate, metoprolol tartrate, methyldova,
6). Urological agents such as flavoxate hydrochloride,
7). Anticoagulants such as heparin, dichromal, warfarin,
8). Arteriosclerotic agents such as clofibrate, synfibrate, elastase, nicomol,
9. Circulatory organ agents such as nicardipine hydrochloride, nimodipine hydrochloride, cytochrome C, tocopherol nicotinate,
10. Steroids such as hydrocortisone, prednisolone, dexamethasone, betamethasone,
11. Wound healing promoters such as growth factors and collagen (see JP-A-60-222425),
Other physiologically active polypeptides, hormonal agents, antituberculosis agents, hemostatic agents, antidiabetic agents, vasodilators, arrhythmia agents, cardiotonic agents, antiallergic agents, antidepressants, antiepileptics, muscle relaxants, antitussives Examples include expectorants and antibiotics.

The photocrosslinking polysaccharide of the present invention is sufficiently impregnated with a photocrosslinking polysaccharide impregnated with a medium for culturing cells (including cultured cells and primary cultured cells) and tissues (such as tissue pieces taken from a living body). After that, it is useful as a cell culture substrate used for culturing cells and tissues.
The “cells” and “tissues” applied to the culture substrate of the present invention are not particularly limited as long as they are cells / tissues that can be proliferated in vitro. In particular, cells / tissues derived from mesoderm are exemplified. Preferred examples include cells / tissue derived from connective tissue, and epithelial cells, chondrocytes, hepatocytes, and neuroblasts are particularly preferable.

EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded. In the following examples, the concentration represents% by weight unless otherwise specified.
The analysis method used in this example will be described below.
<Measurement of epoxy equivalent>
It measured according to the epoxy equivalent test method (JIS K7236: 2001) of an epoxy resin.

<Measurement of rotational viscosity>
An E-type rotational viscometer TOKI RE-80H (manufactured by Toki Sangyo Co., Ltd.) was used. In the case of a test substance whose rotational viscosity exceeds 50 Pa · s, a 3 ° cone with a diameter of 14 mm is used at 25 ° C. and 1.0 rpm, and in the case of a test substance with a viscosity of 50 Pa · s or less, a 1 ° cone with a diameter of 24 mm is used at 20 ° C. Each measurement was performed at 1.0 rpm. However, the test substance having a rotational viscosity in the vicinity of 50 Pa · s may be a value measured under either condition.

<Measurement of breaking strength>
Texture Analyzer TA-XT2 (manufactured by Stable Micro Systems) was used. A test substance that has been sufficiently swollen in distilled water in advance is cut into a size of 6 × 2.5 cm and fixed on a stage, and a spherical probe having a diameter of 12.7 mm is pressed against it at a speed of 1 mm / sec. The breaking strength when breaking through the test substance was measured.

<Measurement of introduction rate of photoreactive crosslinking group>
The introduction rate of the photoreactive crosslinking group means a value representing the number of photoreactive crosslinking groups introduced per repeating disaccharide unit of glycosaminoglycan as a percentage. The amount of glycosaminoglycan required for calculating the introduction rate was measured by a carbazole measurement method using a calibration curve, and the amount of the photoreactive crosslinking group was measured by an absorbance measurement method using a calibration curve.

<Measurement of crosslinking rate>
The crosslinking rate was determined by saponifying 1 g of a test substance with 1 ml of 1 mol / L sodium hydroxide aqueous solution for 1 hour, then acidifying the resulting solution with ethyl acetate to produce a photoreactive crosslinking group (monomer, dimer). ) Were extracted, analyzed by high performance liquid chromatography (HPLC), and the amount of dimer was measured by a calibration curve method. The number of moles of the photoreactive cross-linking group that became a dimer with respect to the photoreactive cross-linking group introduced into the glycosaminoglycan was expressed as a percentage.

Reference Example 1: Synthesis of glycidyl ester of photoreactive compound 1 <Synthesis of glycidyl cinnamate>
To 7.5 g of tran-cinnamic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added 3 g of tetraethylammonium bromide (manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mL of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.), and refluxed at 110 ° C. for 3 hours. Distilled water (50 mL) was added, the organic layer was separated, concentrated under reduced pressure at 80 ° C., and then distilled under reduced pressure conditions of 6 mmHg, and the fraction at 130 to 160 ° C. was recovered to obtain 12 g of glycidyl cinnamate ester. . The epoxy equivalent of the glycidyl cinnamate ester was 240.3.

The obtained glycidyl cinnamate was analyzed by gas chromatography.
Gas chromatography uses GC-17A (manufactured by Shimadzu Corporation), column: DB-5 (film thickness 0.5 μm, inner diameter 0.25 mm, length 30 m), column temperature: 100 ° C. (0-5 min) -250 ° C. Measurement was performed at (5 ° C./min temperature increase) (FIG. 1). As a result, the maximum peak was observed at 32.6 min, confirming the formation of glycidyl cinnamate.
Peak epichlorohydrin 3.6min
tran-cinnamic acid 22.5min
Glycidyl cinnamate 32.6min

2 <Synthesis of thiophene acrylate glycidyl ester>
To 2.5 g of trans-3-3-thiopheneacrylic acid (Aldrich), 1 g of tetraethylammonium bromide (Wako Pure Chemical Industries) and 60 mL of epichlorohydrin (Wako Pure Chemical Industries) were added and refluxed at 110 ° C. for 3 hours. Thereafter, 30 mL of distilled water was added, the organic layer was separated, and concentrated under reduced pressure at 80 ° C. to obtain 6.5 g of glycidyl thiophene acrylate. The epoxy equivalent of this thiophene acrylic acid glycidyl ester was 596.8.

3 <Synthesis of cinnamylidene acetic acid glycidyl ester>
To 9.0 g of cinnamylideneacetic acid (manufactured by Lancaster), 3.5 g of tetraethylammonium bromide (manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mL of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.) were added. After refluxing at 110 ° C. for 3 hours, 50 mL of distilled water was added. And the organic layer was separated and concentrated under reduced pressure at 80 ° C. to obtain 17.3 g of cinnamylideneacetic acid glycidyl ester. The epoxy equivalent of this cinnamylideneacetic acid glycidyl ester was 444.3.

4 <Synthesis of glycidyl sorbate ester>
To 1.8 g of sorbic acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added 1 g of tetraethylammonium bromide (manufactured by Wako Pure Chemical Industries, Ltd.) and 60 mL of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.), and after refluxing at 110 ° C. for 3 hours, 30 mL of distilled water is added. In addition, the mixture was separated and concentrated under reduced pressure at 80 ° C. to obtain 3.5 g of glycidyl sorbate ester. The epoxy equivalent of this glycidyl sorbate ester was 367.4.

Example 1: Production of glycidyl cinnamate ester-introduced hyaluronic acid and cross-linked product thereof <1- (1) Synthesis of glycidyl cinnamate ester-introduced hyaluronic acid (reverse precipitation method)>
50 mL of water for injection and 75 mL of 1,4-dioxane were added to 100 mL of an aqueous solution of 1% by weight hyaluronic acid (weight average molecular weight: 800,000), 3 mL of glycidyl cinnamate (epoxy equivalent 240.3) was added, and the temperature was kept constant at 50 ° C. Stir in the bath for 24 hours.
To this solution, 1 g of sodium chloride was added and stirred, and then poured into 800 mL of ethanol to obtain a flocculent precipitate. This precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of glycidyl cinnamate ester-introduced hyaluronic acid (hereinafter also referred to as GLCN-HA). When the introduction rate of glycidyl cinnamate was measured, it was 2.9%.

<1- (2) Synthesis of glycidyl cinnamate ester-introduced hyaluronic acid (forward precipitation method)>
1 g of sodium chloride was added to the solution obtained by reacting in the same manner as in 1- (1) and stirred, and then 500 mL of ethanol was slowly poured to obtain a powdery precipitate. This precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of hyaluronic acid introduced with glycidyl cinnamate ester. When the introduction rate of glycidyl cinnamate was measured, it was 3.5%.

<1- (3) Synthesis of glycidyl cinnamate ester-introduced hyaluronic acid (sequential precipitation method)>
1 g of sodium chloride was added to the solution obtained by reacting in the same manner as 1- (1) except for stirring for 48 hours in a constant temperature bath at 60 ° C. After stirring, about 500 mL of ethanol was slowly poured to form a powdery precipitate. Obtained. This precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of hyaluronic acid introduced with glycidyl cinnamate ester. When the introduction rate of glycidyl cinnamate was measured, it was 10.1%.

<Preparation of photocrosslinked hyaluronic acid gel>
An aqueous solution obtained by dissolving the glycidyl cinnamate ester-introduced hyaluronic acid obtained in 1- (1) in distilled water so as to have a concentration of 3% is placed in a hard glass container having a gap of 1 mm, and a 3 kW metal halide lamp (US When a total of 50.0 J / cm ² ultraviolet irradiation was performed on each side at a rate of 25.0 J / cm ² (measurement wavelength: 280 nm) using an electric company, a gel-like substance was obtained. When the crosslinking rate and isomerization rate of this gel substance were measured, the crosslinking rate was 18.4% and the isomerization rate was 71.4%.
The rotational viscosity of 0.5 mL of this gel substance was measured and found to be 142.0 Pa · s. The rotational viscosity of the glycidyl cinnamate ester-introduced hyaluronic acid aqueous solution that was not irradiated with ultraviolet rays was 83.0 Pa · s.

  The isomerization rate represents the rate of change from trans-cinnamic acid to cis-cinnamic acid. Since cis-cinnamic acid is generated after the trans-cinnamic acid has undergone an excited state necessary for forming a double bond, this value is an index for judging whether the photoreaction has been performed properly. A calibration curve for cis-cinnamic acid is prepared in advance and quantitatively calculated.

<Preparation of photocrosslinked hyaluronic acid film>
5 mL of an aqueous solution obtained by dissolving the glycidyl cinnamate ester-introduced hyaluronic acid obtained in 1- (1) in distilled water to a concentration of 1% was poured into a plastic petri dish having a diameter of 50 mm, and the mixture was placed in a thermostatic chamber at 50 ° C. It was allowed to stand and dry to obtain a transparent film. An 800 W high-pressure mercury lamp (manufactured by Oak Manufacturing) was used and irradiated with ultraviolet rays of 2.5 J / cm ² (measurement wavelength: 280 nm) per side from both sides to obtain a film-like substance insoluble in water. When the crosslinking rate and isomerization rate of this film-like substance were measured, the crosslinking rate was 7.2% and the isomerization rate was 68.5%.
The film-like substance obtained by irradiating with ultraviolet rays immediately swelled when immersed in water, and the shape was maintained even if it was allowed to stand at room temperature for one month. Moreover, it was 29.8g when the breaking strength after swelling of this film-form substance was measured.

<Preparation of photocrosslinked hyaluronic acid sponge (1)>
An aqueous solution prepared by dissolving the glycidyl cinnamate glycidyl ester-introduced hyaluronic acid obtained in 1- (1) in distilled water so as to have a concentration of 3% is filled into a high-density polyethylene pack and sealed. The sample was sandwiched between two hard glass plates and frozen in an ethanol bath at -40 ° C. While maintaining the frozen state, an 800 W high-pressure mercury lamp (manufactured by Oak Manufacturing Co., Ltd.) was used to irradiate each side with ultraviolet rays of 2.0 J / cm ² (measurement wavelength 280 nm), and then melted to form a sponge-like substance (GLCN-HA). Sponge (1)) was obtained. When the crosslinking rate and isomerization rate of this sponge-like substance were measured, the crosslinking rate was 16.7% and the isomerization rate was 68.2%.
In the case where the material was thawed again without being irradiated with ultraviolet rays after freezing, no change in physical properties was observed in the solution. In addition, the breaking strength of the obtained sponge-like substance was measured and found to be 178.7 g.

<Preparation of photocrosslinked hyaluronic acid sponge (3)>
<Photo-crosslinked hyaluron, except that 4% aqueous solution of glycidyl cinnamate ester-introduced hyaluronic acid obtained in 1- (3) was used and UV irradiation was performed on each side at 9.0 J / cm ² (measurement wavelength: 280 nm). Preparation of acid sponge (1)> A sponge-like substance (GLCN-HA Sponge (3)) was obtained in the same manner. When the crosslinking rate and isomerization rate of this sponge-like substance were measured, the crosslinking rate was 39.6% and the isomerization rate was 66.4%.

<Preparation of Photocrosslinked Hyaluronic Acid Composite Sheet (1)>
An aqueous solution prepared by dissolving the glycidyl cinnamate glycidyl ester-introduced hyaluronic acid obtained in 1- (1) in distilled water so as to have a concentration of 3% is filled into a high-density polyethylene pack and sealed. Between the two hard glass plates, UV irradiation was performed on both surfaces using a 800 W high-pressure mercury lamp (manufactured by Oak Manufacturing Co., Ltd.) for a total of 10 J / cm ² (measurement wavelength 280 nm).
Subsequently, it was frozen in an ethanol bath at −40 ° C. as it was. While maintaining the frozen state, each surface was melted after being irradiated with ultraviolet rays of 0.5 J / cm ² (measurement wavelength: 280 nm), and a slightly translucent translucent sheet material (GLCN-HA Sheet ( 1)) was obtained. When the crosslinking rate and isomerization rate of this sheet-like substance were measured, the crosslinking rate was 6.0% and the isomerization rate was 68.5%. Moreover, it was 110.9 g when the breaking strength of this sheet-like composite material was measured.

<Preparation of photocrosslinked hyaluronic acid composite sheet (3)>
Using a 4% aqueous solution of glycidyl cinnamate-introduced hyaluronic acid obtained in 1- (3), using a 3 kW metal halide lamp (USHIO Inc.), 50.0 J / cm ^{2 on} each side for a total of 100.0 J / cm ² (measurement wavelength: 280 nm) UV irradiation is performed, and then irradiation in the frozen state is performed using an 800 W high-pressure mercury lamp (manufactured by Oak Manufacturing Co., Ltd.), and each side is 9.0 J / cm ² (measurement wavelength: 280 nm). A sheet-like substance (GLCN-HA · Sheet (3)) was obtained in the same manner as in the above <Preparation of photocrosslinked hyaluronic acid composite sheet (3)>. When the crosslinking rate and isomerization rate of this sheet-like substance were measured, the crosslinking rate was 48.0% and the isomerization rate was 67.3%.

<Preparation of photocrosslinked hyaluronic acid composite film>
Using the 4% aqueous solution of cinnamate glycidyl ester-introduced hyaluronic acid obtained in 1- (3), using an 800W high-pressure mercury lamp (Oak Seisakusho), 5.0 J / cm ^{2 on} one side (measurement wavelength: 280nm) UV irradiation was performed, then it was frozen in an atmosphere of -20 ° C, and while maintaining the frozen state, UV irradiation was performed on each side by 0.2J / cm ² (measurement wavelength: 280nm) using an 800W high-pressure mercury lamp. . The obtained sheet-like material was dried in a constant temperature dryer at 50 ° C., and further irradiated with ultraviolet rays of 0.2 J / cm ² (measurement wavelength: 280 nm) on one side using an 800 W high-pressure mercury lamp. A film-like material was obtained. When the crosslinking rate and isomerization rate of this film-like substance were measured, the crosslinking rate was 9.8% and the isomerization rate was 67.8%.

Example 2: Preparation of thiophene acrylate glycidyl ester-introduced hyaluronic acid and its crosslinked product <Synthesis of thiophene acrylate glycidyl ester-introduced hyaluronic acid>
50 mL of water for injection and 75 mL of 1,4-dioxane were added to 100 mL of a 1% by weight hyaluronic acid (weight average molecular weight: 800,000) aqueous solution, and 3 mL of thiophenacrylic acid glycidyl ester (epoxy equivalent: 596.8) was added at 50 ° C. It stirred for 24 hours in the thermostat.
To this solution, 1 g of sodium chloride was added and stirred, and then poured into 800 mL of ethanol to obtain a flocculent precipitate. The obtained precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of glycidyl thiophene acrylate introduced hyaluronic acid (GLTA-HA). When the introduction rate of glycidyl thiophene acrylate was measured, it was 1.40%.

<Preparation of photocrosslinked hyaluronic acid gel>
A gel-like substance was obtained in the same manner as the gel preparation method described in Example 1, except that thiophene acrylate glycidyl ester-introduced hyaluronic acid was used.
When the rotational viscosity of 0.5 mL of this gel-like substance was measured, it was 246.5 Pa · s. The rotational viscosity of the thiophene acrylic acid glycidyl ester-introduced hyaluronic acid aqueous solution without ultraviolet irradiation was 90.5 Pa · s.

<Preparation of photocrosslinked hyaluronic acid film>
A film insoluble in water was obtained in the same manner as the film preparation method described in Example 1, except that thiophene acrylate glycidyl ester-introduced hyaluronic acid was used.
The film obtained by irradiating with ultraviolet rays immediately swelled when immersed in water, and the shape was maintained even if left for one month at room temperature. The breaking strength of this film was measured and found to be 76.8 g.

<Preparation of photocrosslinked hyaluronic acid sponge>
A sponge-like substance (GLTA-HA Sponge) was obtained in the same manner as the preparation of the sponge (1) described in Example 1 except that thiophene acrylate glycidyl ester-introduced hyaluronic acid was used. The breaking strength of this sponge-like substance was measured and found to be 92.6 g.
In the case where the solution was melted without being irradiated with ultraviolet rays after freezing, no change in physical properties was observed in the solution.

<Preparation of photocrosslinked hyaluronic acid composite sheet>
Except for the use of thiophene acrylate glycidyl ester-introduced hyaluronic acid, a slightly whitish translucent sheet-like material (GLTA-HA Sheet) in the same manner as the preparation of the composite material sheet (1) described in Example 1. ) The breaking strength of this translucent sheet material was measured and found to be 87.8 g.

Example 3 Preparation of Cinnamylidene Acetate Glycidyl Ester-Introduced Hyaluronic Acid and Cross-Linked Product <Synthesis of Cinnamylidene Acetate Glycidyl Ester-Introduced Hyaluronic Acid>
50 mL of water for injection and 75 mL of 1,4-dioxane were added to 100 mL of a 1 wt% hyaluronic acid (weight average molecular weight: 800,000) aqueous solution, 3 mL of cinnamylideneacetic acid glycidyl ester (epoxy equivalent: 444.3) was added, and then 50 ° C. It stirred for 24 hours in the thermostat. To this solution, 1 g of sodium chloride was added and stirred, and then poured into 800 mL of ethanol to obtain a flocculent precipitate. The obtained precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid (GLCdN-HA). When the introduction rate of cinnamylideneacetic acid glycidyl ester was measured, it was 3.1%.

<Preparation of photocrosslinked hyaluronic acid gel>
A gel-like substance was obtained in the same manner as the gel preparation method described in Example 1, except that cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid was used.
The rotational viscosity of 0.5 mL of this gel substance was measured and found to be 220.0 Pa · s. The rotational viscosity of the cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid aqueous solution that was not irradiated with ultraviolet rays was 61.0 Pa · s.

<Preparation of photocrosslinked hyaluronic acid film>
A water-insoluble film was obtained in the same manner as the film preparation method described in Example 1, except that cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid was used.
The film obtained by irradiating with ultraviolet rays immediately swelled when immersed in water, and the shape was maintained even if left for one month at room temperature.
When the breaking strength after swelling of this film was measured, it was 23.6 g.

<Preparation of photocrosslinked hyaluronic acid sponge>
A sponge-like substance (GLCdN-HA Sponge) was obtained in the same manner as the preparation of the sponge (1) described in Example 1, except that cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid was used. When the breaking strength of this sponge-like substance was measured, it was 70.3 g.
Further, in the case where the material was melted without being irradiated with ultraviolet rays after freezing, no change in physical properties was observed in the solution.

<Preparation of photocrosslinked hyaluronic acid composite sheet>
Except for using cinnamylideneacetic acid glycidyl ester-introduced hyaluronic acid, a slightly whitish translucent sheet-like substance (GLCdN-HA Sheet) in the same manner as the preparation of the composite material sheet (1) described in Example 1 Got.
When the breaking strength of this translucent sheet-like substance was measured, it was 102.2 g.

Example 4: Preparation of glycidyl sorbate ester-introduced hyaluronic acid and its crosslinked product <Synthesis of glycidyl sorbate ester-introduced hyaluronic acid>
50 mL of water for injection and 75 mL of 1,4-dioxane were added to 100 mL of an aqueous solution of 1% by weight hyaluronic acid (weight average molecular weight; 800,000), and 3 mL of glycidyl sorbate (epoxy equivalent: 367.4) was added, followed by constant temperature at 50 ° C. Stir in the bath for 24 hours.
After 1 g of sodium chloride was added to this solution and stirred, it was poured into 800 mL of ethanol to obtain a flocculent precipitate. The obtained precipitate was sufficiently washed with ethanol and dried to obtain 1.1 g of glycidyl sorbate ester-introduced hyaluronic acid (GLSR-HA). When the introduction rate of glycidyl sorbate was measured, it was 1.7%.

<Preparation of photocrosslinked hyaluronic acid gel>
A gel-like substance was obtained in the same manner as in the gel preparation method described in Example 1 except that glycidyl sorbate ester-introduced hyaluronic acid was used.
The rotational viscosity of 1.0 mL of this gel substance was measured and found to be 51.8 Pa · s. The rotational viscosity of a sorbic acid glycidyl ester-introduced hyaluronic acid aqueous solution that was not irradiated with ultraviolet rays was 31.1 Pa · s.

<Preparation of photocrosslinked hyaluronic acid film>
A water-insoluble film was obtained in the same manner as the film preparation method described in Example 1, except that sorbic acid glycidyl ester-introduced hyaluronic acid was used. The breaking strength of this film was measured and found to be 68.9 g.
The film obtained by irradiating with ultraviolet rays immediately swelled when immersed in water, and the shape was maintained even if left for one month at room temperature.

<Preparation of photocrosslinked hyaluronic acid sponge>
A sponge-like substance (GLSR-HA Sponge) was obtained in the same manner as the preparation of the sponge (1) described in Example 1, except that sorbic acid glycidyl ester-introduced hyaluronic acid was used. The breaking strength of this sponge-like substance was measured and found to be 35.5 g.
Further, in the case where the material was melted without being irradiated with ultraviolet rays after freezing, no change in physical properties was observed in the solution.

<Preparation of photocrosslinked hyaluronic acid composite sheet>
Except for using sorbic acid glycidyl ester-introduced hyaluronic acid, a slightly whitish translucent sheet-like substance (GLSR-HA Sheet) in the same manner as the preparation of the composite material sheet (1) described in Example 1. Got.

Reference Example 2: Preparation of aminopropyl cinnamate-introduced hyaluronic acid and its crosslinked product <Preparation of aminopropyl cinnamate-introduced hyaluronic acid>
Preparation was carried out according to the method described in JP-A-2002-249501. That is, after adding 50 mL of distilled water and 75 mL of 1,4-dioxane to 100 mL of a 1 wt% aqueous solution of hyaluronic acid (weight average molecular weight; 800,000), 172 mg of N-hydroxysuccinimide (HOSu), 1-ethyl-3- (3- Dimethylaminopropyl) carbodiimide hydrochloride (EDCl · HCl) 143 mg, cinnamic acid aminopropyl hydrochloride (HCl · H ₂ N (CH ₂ ) ₃ OCO—CH═CH—Ph: —Ph represents a phenyl group) 181 mg After sequentially adding and reacting at room temperature for 3 hours, 0.5 g of sodium bicarbonate was added and stirred overnight, and then 6 g of NaCl was added and then 400 mL of ethanol was poured into the reaction solution to precipitate a precipitate. The precipitate was washed twice with 80% ethanol and collected, and then dried under reduced pressure at 40 ° C. to obtain 1.0 g of aminopropyl cinnamate-introduced hyaluronic acid (hereinafter also referred to as 3APC-HA). The introduction rate of aminopropyl cinnamate was measured and found to be 8.2%.

<Preparation of photocrosslinked 3APC-HA sponge>
A solution in which 3APC-HA is dissolved in distilled water to a concentration of 3% is filled and sealed in a high-density polyethylene pack, and sandwiched between two hard glass plates with a gap of 1 mm. Frozen in an ethanol bath. While maintaining the frozen state, 2.0 J / cm ² (measurement wavelength: 280 nm) was applied to one side of each of the two surfaces and then melted after ultraviolet irradiation to obtain a sponge-like substance. The crosslinking rate of the photocrosslinked 3APC-HA sponge thus obtained was 32.8%.
Further, the breaking strength of this sponge-like substance was measured and found to be 157.8 g.

<Preparation of Photocrosslinked 3APC-HA Composite Sheet>
A solution in which 3APC-HA is dissolved in distilled water to a concentration of 3% is filled and sealed in a high-density polyethylene pack, and sandwiched between two hard glass plates with a gap of 1 mm. Irradiation with ultraviolet rays of 5.0 J / cm ² (measurement wavelength 280 nm) was performed.
Next, it was frozen in an ethanol bath at −40 ° C., and it was melted after being irradiated with ultraviolet rays of 0.5 J / cm ² (measurement wavelength 280 nm) while maintaining the frozen state. Sheet material was obtained.
The crosslinking rate of the photocrosslinked 3APC-HA composite sheet thus obtained was 5.8%. Moreover, it was 181.7g when the fracture strength of this translucent sheet-like substance was measured.

Reference example 3
<Preparation of diglycidyl ether crosslinked hyaluronic acid sponge>
Preparation was performed according to the method described in JP-A-2002-233542. That is, a solution obtained by adding 1 mL of diglycidyl ether (trade name: Denacol EX-313, Nagase Kasei Kogyo) to 100 mL of a 1% by weight hyaluronic acid (weight average molecular weight; 800,000) aqueous solution is made of polypropylene so that the liquid layer becomes about 2 mm. The mixture was poured into a container and allowed to react for 9 hours in a 50 ° C. constant temperature bath, and then the reaction solution was rapidly frozen in a freezer at −80 ° C. and freeze-dried at 10 Pa. The resulting diglycidyl ether crosslinked hyaluronic acid sponge was then carefully washed with a sufficient amount of water for injection. This was lyophilized again to obtain a diglycidyl ether crosslinked hyaluronic acid sponge (DGLE-HA Sponge).

Example 5: Degradability study A strip-like sheet prepared as 2 cm × 1 cm and 1 mm thick of the photocrosslinked polysaccharide shown in FIG. 2 is stirred in a 0.5N NaOH aqueous solution, and the time required for saponification is measured. Then, the decomposability was compared. The result is shown in FIG.
The determination was made visually, and the end time was defined as the point at which no solid matter was observed in the 0.5N NaOH aqueous solution.
In Fig. 2, "GLCN-HA" is glycidyl cinnamate ester-introduced hyaluronic acid, "GLCdN-HA" is cinnamylidene acetate glycidyl ester-introduced hyaluronic acid, and "GLTA-HA" is thiophene acrylate glycidyl ester-introduced hyaluronic acid. "GLSR-HA" means hyaluronic acid with glycidyl sorbate ester introduced, "3APC-HA" means hyaluronic acid with aminopropyl cinnamate introduced, Sheet means sheet material, Sponge means sponge material To do.
As a control, SepraFilm (Genzyme, sold by Kaken Pharmaceutical Co., Ltd.) was used.

From Fig. 2, 3APC-HA has a sponge form of nearly 1 hour for alkali saponification, and the composite sheet form took 57 seconds to disappear, whereas GLCN-HA takes 15 seconds regardless of the form. It was confirmed that it disappeared.
In addition, GLCN-HA (GLCN-HA Sponge (3), GLCN-HA Sheet (3)) with an increased introduction rate and cross-linking rate can be reached within 17 seconds regardless of the form of sponge or sheet. All disappeared. In general, it is known that the degradation rate decreases as the introduction rate and the crosslinking rate increase, but in the present invention, it is confirmed that the degradation rate is stable and stable regardless of the introduction rate and the crosslinking rate. It was.

Example 6: In vivo degradability study Using the following rat abdominal wall-deficient cecal abrasion model, the anti-adhesion effect and degradability were examined.
The rat cecum side was lightly rubbed several times with absorbent cotton, then a 30 × 40 mm defect was created on the abdominal wall side, and the above was coated with a photocrosslinked polysaccharide material described in Table 1 of 4 cm × 5 cm × 0.3 mm. After 7 days, dissection was performed, and the adhesion prevention effect and degradability were examined by determining the presence or absence of adhesion between the abdominal wall and the cecum and the presence or absence of each photocrosslinked polysaccharide material. The results are shown in Table 1 below.
Each photocrosslinked polysaccharide material was prepared according to Example 1, Reference Example 2 and Reference Example 3, and as GLCN-HA, GLCN-HA Sponge (1) and GLCN-HA Sheet (1) were used. .

As a result of the test, adhesion of glycidyl cinnamate ester-introduced hyaluronic acid (GLCN-HA) was not observed in 3 out of 3 sheets. The sheet was completely lost, and no abnormality was observed in the liver or other organs.
The photocrosslinked composite sheet of aminopropyl cinnamate-introduced hyaluronic acid (3APC-HA) did not adhere to any of the 3 cases, but the sheet remained in a solid shape.

As for the photocrosslinked sponge of glycidyl cinnamate ester-introduced hyaluronic acid, no adhesion was observed in 11 of 11 cases. The sponge disappeared completely, and no abnormality was observed in the liver or other organs.
Two of the two photocrosslinked sponges of aminopropyl cinnamate-introduced hyaluronic acid remained in a milky white resin-like state, causing strong adhesion to the wound.
Diglycidyl ether cross-linked hyaluronic acid (DGLE-HA) sponges showed adhesion in 2 of 2 cases. Although the sponge itself disappeared completely, it was recognized that the margin of the liver was enlarged and clouded over a wide area.
As described above, the sponge material prepared with 3APC-HA showed severe adhesion, whereas the sponge material prepared with GLCN-HA showed an adhesion prevention effect in all cases. This is considered to be due to the fact that the photocrosslinking GLCN-HA sponge material is more degradable than the 3APC-HA sponge material.

Example 7: In vivo adhesion prevention performance study Using a rabbit intestinal adhesion model, the adhesion prevention effect of SepraFilm (Genzyme, manufactured by Kaken Pharmaceutical Co., Ltd.) and the photocrosslinked polysaccharide composite sheet of the present invention was compared.
1. Preparation of primary intestinal adhesion model Rabbits are JapanWhite (male, about 20 weeks old, body weight about 3kg), and the abdomen is opened under inhalation anesthesia with halothane (Foren, Takeda Pharmaceutical). Scratching was performed 30 times at a constant speed. Further, the surface of the ileum adjacent to the opposite side of the worn jejunum was similarly abraded. The rubbed area was 15 mm wide and 30 mm long (area 450 mm ² ). Next, both the scraped parts were covered with absorbent cotton containing iodine tincture (3% iodine + 2% potassium iodide). Absorbent cotton was removed after 7 minutes, and after drying for 3 minutes, the abdominal wall and skin were sutured to complete the production of the primary adhesion model. This procedure can form an adhesion between the jejunum and the ileum.

2. Detachment of primary intestinal adhesion and administration of test substance (preparation of evaluation model)
One month after the creation of the primary adhesion model, the abdomen was opened once, the degree of adhesion was observed, and an individual that caused strong adhesion accompanied by the formation of a thin film was selected.
The adhesion site was exfoliated under a surgical microscope, and the area of the site where the adhesion was exfoliated was measured, and then GLCN-HA Sheet (same as used in Example 6) and SepraFilm were administered as test substances. Each test substance was cut in an area 20% larger than the area of the adhesion peeling site and attached to the adhesion peeling site. In addition, nothing was administered to the control group. After administration of the test substance, the abdominal wall and skin were sutured to complete the creation of the evaluation model.

3. Adhesion prevention performance evaluation After preparing the evaluation model, the abdomen was opened in 2 weeks, the presence or absence of re-adhesion was observed, and the area of re-adhesion was measured. The adhesion score was 0, none, 1: weak, 2: medium, and 3: strong.
result:
A strong re-adhesion with a score of 3 was observed in the control group. The primary adhesion area was 350 mm ² , whereas the re-adhesion area was 1200 mm ² and the re-adhesion ratio was 342.9%.
In contrast, in the GLCN-HA Sheet, the primary adhesion area was 1900 mm ² , whereas no re-adhesion was observed and the score was 0. The re-adhesion area was 0 mm ² and the re-adhesion ratio was 0%.
Meanwhile observed adhesions moderate in SepraFilm score 2, whereas the primary adhesion area was 750 mm ^2, re-adhesion area was 1335Mm ^2, re-adhesion ratio was 178.0%%.
From the above, it was clarified that GLCN-HA Sheet exhibits an excellent anti-adhesion effect compared to SepraFilm.

Example 8: Examination of adhesion prevention performance using rat uterine horn adhesion model Since the rat uterine horn adhesion model is known to cause stronger adhesion than the rabbit intestinal re-adhesion model, further adhesion using this model The prevention performance was examined.
1. Model creation:
The rats were Crj: SD strain (SPF.) Female rats (7 weeks old), which were used for this test after preliminary breeding for 1 week before the test.
After trimming the rat abdomen under Nembutal anesthesia, about 4 cm midline incision was made. (A) The right abdominal wall of the rat was cut out to the muscle layer with an ophthalmic trepan, and the muscle layer was peeled off with tweezers. (B) Next, after exposing the uterine horns, transverse incisions were made at intervals of 2 to 3 mm from about 1 cm from the ovary to the cervix, and the wound was hemostatic with an electric knife as needed. (C) About 3-4mm from the end of the uterine horn lateral incision and 3-4mm from the end of the abdominal wall defect, suture with one needle with 8/0 thread, and create by (a) and (b) Each cut was made closer.

2. Administration of test substance A 2.0 cm × 1.0 cm GLCN-HA Sheet (3) and SepraFilm were sandwiched between the abdominal wall defect part and the uterine horn cut part to prepare a test group. In addition, 10 animals were used in each group.
3. Evaluation:
In the evaluation method, 7 days after implantation, the rat was dissected from the carotid artery under ether anesthesia, and then dissected, and the adhesion occurrence site was evaluated by each score system shown below according to the degree of adhesion.
0: No adhesion.
1: Mild adhesions. Easy to peel.
2: Medium adhesion. Peelable.
3: Severe adhesion. Unpeelable.

本癒着モデルにおいてはSepraFilmは10例中全例で癒着を認め全く効果を示さなかったが、本発明のGLCN-HA Sheet(3)は10例中8例で癒着を防止していた。
以上の結果からGLCN-HA Sheetは十分に癒着防止効果を示すことが明らかになった。 4.Result:
The determination results are shown in Table 2 below.

In this adhesion model, SepraFilm showed adhesion in all 10 cases and showed no effect, but GLCN-HA Sheet (3) of the present invention prevented adhesion in 8 of 10 cases.
From the above results, it was revealed that GLCN-HA Sheet exhibits a sufficient anti-adhesion effect.

The measurement result by the gas chromatography of the glycidyl cinnamate ester in the reference example 1 is shown. In the figure, the horizontal axis represents time (min). The examination result of the decomposability | degradation regarding the sheet material form and sponge material form of photocrosslinking polysaccharide in Example 5 is shown.

Claims (19)

A photoreactive polysaccharide in which a polysaccharide and a glycidyl ester represented by the following general formula (1) or (2) are covalently bonded.

[Wherein, R ¹ —CO— or —OC—R ² —CO— represents a photoreactive residue of a photoreactive compound having a carboxyl group and an unsaturated carbon double bond in the molecule, respectively. However, when the polysaccharide is heparin, R ¹ —CO— is not an acryloyl group or a methacryloyl group. ]   The photoreactive polysaccharide according to claim 1, wherein the covalent bond is an ester bond.   The photoreactive compound is a monocarboxylic acid selected from cinnamic acid, hydroxycinnamic acid, acrylic acid, methacrylic acid, furylacrylic acid, thiophene acrylic acid, cinnamylideneacetic acid and sorbic acid, or a dicarboxylic acid selected from maleic acid and fumaric acid The photoreactive polysaccharide according to claim 1 or 2, which is an acid.   The photoreactive polysaccharide according to claim 3, wherein the photoreactive compound is cinnamic acid.   The polysaccharide is a glycosaminoglycan, polyuronic acid, polyaminosaccharide, glucan, mannan, fructan, galactan, pectin, vegetable gum, agar, porphyran, carrageenan, fucoidan or a pharmaceutically acceptable salt or derivative thereof. 2. The photoreactive polysaccharide according to 1.   Glycosaminoglycan is hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate or keratan sulfate, polyuronic acid is alginic acid, glucan derivative is carboxymethylcellulose, polyaminosaccharide or derivative thereof is chitin or The photoreactive polysaccharide according to claim 5, which is chitosan.   The photoreactive polysaccharide according to claim 6, wherein the glycosaminoglycan is hyaluronic acid.   The photoreactive compound is an acid selected from cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, acrylic acid, methacrylic acid, furyl acrylic acid, thiophene acrylic acid, cinnamylidene acetic acid and sorbic acid, and the polysaccharide is The photoreactive polysaccharide according to claim 1, which is a polysaccharide selected from hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, chitin, chitosan, alginic acid and carboxymethylcellulose.   The photoreactive compound is an acid selected from cinnamic acid, hydroxycinnamic acid, maleic acid, fumaric acid, furylacrylic acid, thiophenacrylic acid, cinnamylideneacetic acid and sorbic acid, and the polysaccharide is heparin, chitin, chitosan The photoreactive polysaccharide according to claim 1, which is a polysaccharide selected from alginic acid and carboxymethylcellulose.   A photocrosslinked polysaccharide obtained by photocrosslinking the photoreactive polysaccharide according to any one of claims 1 to 9.   A gel-like photocrosslinked polysaccharide obtained by irradiating the solution of the photoreactive polysaccharide according to any one of claims 1 to 9 with light.   A sponge-like photocrosslinked polysaccharide obtained by freezing or freeze-drying a solution of the photoreactive polysaccharide according to any one of claims 1 to 9, and irradiating the frozen solution or freeze-dried product with light.   The gel-like product produced by irradiating the photoreactive polysaccharide solution according to any one of claims 1 to 9 with light is frozen and irradiated with light while maintaining the frozen state, or the gel-like product is A photocrosslinked polysaccharide that combines the properties of a gel and a sponge obtained by irradiating light after lyophilization.   A film-like photocrosslinked polysaccharide obtained by irradiating light to a thin film obtained by drying the solution layer of the photoreactive polysaccharide according to any one of claims 1 to 9.   The gel-like product produced by irradiating the photoreactive polysaccharide solution according to any one of claims 1 to 9 with light is frozen, irradiated with light while maintaining the frozen state, and further dried, A film-like photocrosslinked polysaccharide obtained by irradiating light.   A medical material comprising the photocrosslinked polysaccharide according to any one of claims 10 to 15.   The medical material according to claim 16, which is an adhesion preventing material.   The medical material according to claim 16, which is a base material for sustained drug release. The medical material according to claim 16, which is a cell culture substrate.

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