Photoallergen gel based on crosslinked hyaluronic acid and method thereof

 

(57) Abstract:

Describes Vodootvodny gel crosslinked hyaluronic acid, which has a storage modulus (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and a tan Delta (G"/G') of from 0.1 to 0.8 in dynamic viscoelasticity measured by the rheometer under the following conditions: measuring method, the oscillation test method, the load control; temperature measuring 37oC; cone size 4 cm; clearance of 800 μm and a frequency of 10 Hz, which is a hydrogel polymer having a density of grid (frequency cross-linking or grid nodes) from 0.01 to 0.5% per 1 mol of constituent disaccharide glycosides level of hyaluronic acid which is a hydrogel obtained by irradiation with ultraviolet rays photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically linked to a functional group of hyaluronic acid and is derived from cinnamic acid, containing a spacer selected from a range that includes groups derived from amerosport, amino acids or peptide, these related photoreactivation crosslinking group specified photoreactivation derived hyaluronic kiwanian thus the mesh, and the gel is a hydrogel in such condition that it can be extracted for injection from the container during extrusion. Also described is a method of obtaining vodootvedenija gel. 4 C. and 11 C.p. f-crystals, 5 tab., 3 Il.

The technical field

The invention relates to biocompatible fototienda gel based on crosslinked hyaluronic acid, which has specific physical properties, the methods of its production and its applications as biomedical materials.

The background to the invention

Hyaluronic acid is present in animal tissues and has biocompatibility and Biodegradability. With regard to physical properties, then it has such excellent properties, such as high water absorption ability, and its aqueous solution has a high viscosity.

The hydrogel obtained by chemical modification of hyaluronic acid, crosslinking of the modified hyaluronic acid some ways with the formation of the mesh structure and the inclusion of the aquatic environment, such as water, in a mesh structure. The hydrogel detects viscoelasticity and viscosity.

Crosslinked hyaluronic acid, in view of the second acid can be adjusted by selection of the degree of crosslinking.

There are different types of stitching. For example, as the implementation type of stitching using hydrophobic binding or ion binding, known crosslinking of hyaluronic acid by introducing into it the nucleophilic reagent (JP-W-3-502704 (corresponding to U.S. patent 4937270), the term "JP-W" as used here means "not passed examination published international patent application"), the binding through hydrophobic binding by the esterification of hyaluronic acid (U.S. patent 4851521) and the binding through ionic binding of polyvalent ions (ER 0507604 A2). As they contacted the weak force binding compared with the strength of covalent binding, they are affected by external changes, such as pH, ionic strength, temperature and the like. In addition, when they are used as biomedical materials, the ability to remain in the living body is short, and it is difficult to adjust properly the ability to remain in the body to provide the physiological effects of hyaluronic acid on the body.

Besides, as the type of stitching by linking the molecules of hyaluronic acid covalent binding of known crosslinking through devesa examination patent publication Japan") and the stitching via epoxide (JP-W-61-502729 (corresponding to U.S. patent 4886787), JP-A-5-140201, the term "JP - a" as used here means "not passed examination published patent application of Japan"). However, crosslinking agents or crosslinking compounds used in these crosslinks are toxic. In addition, the three-dimensional mesh structure is created by stapling at the same time, when diphenylsulfone, epoxide or the like are introduced into hyaluronic acid and educated structured gel based on hyaluronic acid is translated into an insoluble state in a solvent such as water and the like. Unreacted low molecular weight compounds included therefore in the mesh structure, it is difficult to separate and remove.

On the other hand, crosslinking of hyaluronic acid by cross-linking reaction fototerapia when irradiated with ultraviolet rays (JP-A-6-73102 (corresponding to U.S. patent 5462976), ASAIO Journal, 38, M-M (1992)) are also known. This type of linkage has the advantage that photoreactivation derivative of hyaluronic acid that is injected photoreactivation cross-linking group, is soluble in water up to fotoallergiyami stitching and three-dimensional mesh structure is not formed at this time, so the wrong is Oh pure reaction, that gives photoallergen derived crosslinked hyaluronic acid without unreacted low molecular weight compounds, and the resulting crosslinked structure is formed by covalent binding, so that the control ability to remain (in the body) vodootvedenija derived crosslinked hyaluronic acid can be easily carried out by adjusting the degree of crosslinking.

In addition, when the above-described photoallergen derived crosslinked hyaluronic acid suitable for use as biomedical materials, such as anti-adhesive materials, they plenkoobrazovatel form studied (ASAIO Journal, 38, M-M (1992), but they can hardly be used to prevent adhesions in the thin parts of the tissues or organs. There is therefore a need for suitable for injection gel photoallergen crosslinked hyaluronic acid, which can be injected in such a fine place.

However, the hydrogel photoallergen crosslinked hyaluronic acid and methods of its production, as disclosed in this invention, is unknown.

Conventional hydrogel crosslinked hyaluronic acid has difficulty in removing impurities, such as unreacted low is entrusted to create the conditions to obtain a hydrogel, having desirable physical properties.

Description of the invention

The first objective of this invention is to provide Vodootvodny gel crosslinked hyaluronic acid-containing aqueous medium, which is obtained by irradiation with ultraviolet rays photoreactivation derivative of hyaluronic acid that is injected photoreactivation cross-linking group, and dimerization of adjacent photoreactivation crosslinking groups with the formation of CYCLOBUTANE rings and education so the mesh, and how easy access to the above gel.

The second objective of this invention is to provide suitable for injection biomedical material containing Vodootvodny gel crosslinked hyaluronic acid, which is excellent in safety, biocompatibility and ability to biodegrade.

As a result of intensive studies, the authors of this invention have succeeded in achieving the above objectives through the following build.

1) Vodootvodny gel crosslinked hyaluronic acid,

which has a storage modulus (dynamic modulus) (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and tangentor measurements:

oscillation test method, the load control

temperature measurement 37oWITH

geometry measuring 4 cm

a gap of 800 μm

frequency 10 Hz,

and which is a hydrogel obtained by irradiation with ultraviolet rays photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and stitching connecting photoreactivation cross-linking groups.

2) Vodootvodny gel crosslinked hyaluronic acid,

which is the density of the polymer network (frequency cross-linking or grid nodes) from 0.01 to 0.5% per 1 mol of constituent disaccharide glycosides link hyaluronic acid

which is a hydrogel obtained by irradiation with ultraviolet rays photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and by stitching adjacent photoreactivation cross-linking groups.

3) Vodootvodny gel crosslinked hyaluronic acid,

which has a water absorption of from 2000 to 15000% how determine who is a hydrogel, obtained by irradiation with ultraviolet rays photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and by stitching adjacent photoreactivation cross-linking groups.

4) Vodootvodny gel crosslinked hyaluronic acid according to any of the above 1) to 3) where the specified photoreactivation cross-linking group is derived from cinnamic acid, containing the spacer (the separation group), and is chemically bound with a functional group of hyaluronic acid, to provide the specified photoreactivation derived hyaluronic acid;

these related photoreactivation crosslinking group specified photoreactivation derived hyaluronic acid will dimerized by irradiation with ultraviolet rays with the formation of CYCLOBUTANE rings and education so the mesh, and

the specified gel is a hydrogel containing water environment in the specified mesh structure.

5) Vodootvodny gel crosslinked hyaluronic acid according to the above 4), where kazimanyi gel crosslinked hyaluronic acid according to the above 4) or 5), where specified photoreactivation crosslinking group represented by the following formula (1) or (2):

-NH(CR1R2)nOCOCH=CH-Ph (1)

where R1and R2each independently represent a hydrogen atom or altergroup having from 1 to 8 carbon atoms; Ph is panelgroup and n represents an integer from 2 to 18;

-A-NH-Ph-CH=CHCOOR3(2)

where R3is altergroup having from 1 to 8 carbon atoms or arakalgudu; a represents -(NR4R5CO)m- or-NH(CR4R5)hCO-; R4and R5each independently represents a hydrogen atom or altergroup having from 1 to 8 carbon atoms; -Ph - represents a group of para-phenylene; m is an integer from 1 to 6 and h denotes an integer from 1 to 18.

7) Vodootvodny gel crosslinked hyaluronic acid according to any of the above 1) to 6), where the specified photoreactivation crosslinking group introduced in a ratio of from 0.05 to 10% on 1 mol of constituent disaccharide glycosides link.

8) Vodootvodny gel crosslinked hyaluronic acid, which has a storage modulus (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and a tan Delta (G"/G') of from 0.1 to 0.8 in the dynamic viscoelasticity of the control load

temperature measurement 37oWITH

geometry measuring 4 cm

a gap of 800 μm

frequency 10 Hz,

and which is a hydrogel obtained by the irradiation with ultraviolet rays photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and stitching connecting photoreactivation cross-linking groups, and then heat-treated cross-linked product.

9) Vodootvodny gel crosslinked hyaluronic acid,

which has a storage modulus (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and a tan Delta (G"/G') of from 0.1 to 0.8 in dynamic viscoelasticity measured by the rheometer under the following conditions,

method of measurement:

oscillation test method, the load control

temperature measurement 37oWITH

geometry measuring 4 cm

a gap of 800 μm

frequency 10 Hz,

and which is a hydrogel obtained by heat treatment photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and then exposed by the project related photoreactivation cross-linking groups.

10) Vodootvodny gel crosslinked hyaluronic acid,

which has a storage modulus (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and a tan Delta (G"/G') of from 0.1 to 0.8 in dynamic viscoelasticity measured by the rheometer under the following conditions,

method of measurement:

oscillation test method, the load control

temperature measurement 37oWITH

geometry measuring 4 cm

a gap of 800 μm

frequency 10 Hz,

and which is a hydrogel obtained by heat treatment photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and then by irradiation with ultraviolet rays heated photoreactivation derived hyaluronic acid and stitching adjacent photoreactivation crosslinking groups and then by heat treatment cross-linked product again.

11) Vodootvodny gel crosslinked hyaluronic acid according to any of the above 1) to 10), where the concentration of endotoxin in the gel is 0.25 endotoxin units (unit e)/g or less.

12) the Method of obtaining vodootvedenija gel crosslinked hyaluronan the mass photoreactivation derived hyaluronic acid, in which photoreactivation cross-linking group chemically bind with the functional group of hyaluronic acid, and

the formation of intermolecular and intramolecular crosslinking by dimerization of adjacent photoreactivation cross-linking groups to ensure the mesh.

13) the Method of obtaining vodootvedenija gel crosslinked hyaluronic acid according to the above 12), where the heat treatment is carried out before and/or after irradiation with ultraviolet rays of a specified solution of the water environment photoreactivation derived hyaluronic acid.

14) the Method of obtaining vodootvedenija gel crosslinked hyaluronic acid according to the above 13) where the specified heat treatment is carried out at a temperature from 100 to 125oC for from 5 to 30 minutes vapor pressure.

15) biomedical material containing Vodootvodny gel crosslinked hyaluronic acid according to any of the above 1) to 11).

16) a biomedical material according to the above 15), which has anti-adhesive effect.

17. a Set of biomedical material containing a crosslinked gel of hyaluronic acid and a container enclosing the criminal code is Arial according to the above 17), where the specified container is a container that can squeeze the specified gel for injection.

19. a Set of biomedical material containing Vodootvodny gel crosslinked hyaluronic acid as described in any of 1) to 11), and the container comprising a specified gel in such condition that it can be extracted to the outside.

20. a Set of biomedical material according to the above 19) where the specified container is a container that can squeeze the specified gel for injection.

Brief description of drawings

Fig. 1 is a graphical representation of the General idea of photoallergen the stitching in the solution photoreactivation derived hyaluronic acid.

Fig. 2 is a graphical representation of the relationship between DS (degree of substitution) and the degree of water absorption vodootvedenija gel crosslinked hyaluronic acid.

Best mode for carrying out in practice of the invention

This invention will be explained in detail below.

The term "derived photoallergen crosslinked hyaluronic acid", as used in this invention is intended to encompass Pro is one of hyaluronic acid, in which photoreactivation cross-linking group is chemically bound, and dimerization of adjacent photoreactivation cross-linking groups of the crosslinking derivative of hyaluronic acid and education thus a mesh structure. The term "Vodootvodny gel crosslinked hyaluronic acid", as used in this invention, denotes the hydrogels, as a General concept, containing the aquatic environment, such as water, buffer, physiological saline, buffered saline solution, an aqueous solution containing water-soluble organic solvent and the like, as the dispersion medium in the net structure (three-dimensional mesh structure) photoallergen crosslinked hyaluronic acid (hereinafter sometimes simply referred to as "gel of the present invention"). The term "functional group of hyaluronic acid", as used in this invention is meant to include functional groups that are present in the hyaluronic acid and is capable of chemical bonding with photoreactivation cross-linking groups. Typical examples of functional groups are a carboxyl group and a hydroxyl group. The term "lower alkyl" or "n or alkoxygroup, which has from 1 to 8 carbon atoms, preferably from 1 to 4 carbon atoms.

As for the gel of the present invention, first, its physical properties accurately determine from the point of view of viscoelasticity and, secondly, slivaushiesia structure install it from the point of view of the degree of crosslinking.

Photoreactivation cross-linking group in photoreactivation derived hyaluronic acid of the present invention is not a specific group, while the group has vanilinovoi group, which is capable of timeresults under the influence of ultraviolet radiation with the formation of CYCLOBUTANE ring, and the group is derived from cinnamic acid or its substituted derivatives (for example, derivatives of cinnamic acid and the like, where one or two hydrogen atoms in any positions of the benzene ring substituted cinnamic acid group, a lower alkyl (e.g., stands, ethyl, propylene, isopropyl, bootrom, tert-bootrom and the like), lower CNS group (for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy and the like), amino group, hydroxyl group and the like), carboxy-lower alkyl-thymine (nepomucena acid and the like) and the like. Of them, particularly preferred is photoreactivation cross-linking group, which entered the group derived from cinnamic acid or its derivative. In addition, as photoreactivation cross-linking groups are also preferred group derived from compounds in which the spacer is associated with photoreactivation connection, such as cinnamic acid and the like. Preferred spacers include those that have two or more functional groups capable of linking with a functional group photoreactivation compounds, such as cinnamic acid and the like, and a functional group of hyaluronic acid. Specifically, the preferred amino acids or their derivatives, peptides and aminoalcohols, and the like, particularly preferred are aminoalcohols. Photoreactivation cross-linking group may be entered in any functional group components sharidny parts of hyaluronic acid, for example N-acetyl-D-glucosamine and D-glucuronic acid, but particularly preferably specified cross-linking group in the carboxyl group of D-glucuronic acid.

When to use the measures it is preferable photoreactivation derivative of hyaluronic acid with such a structure, when the carboxyl group of cinnamic acid is chemically linked with a hydroxyl group amerosport through ester bonds, and the amino amerosport is chemically bound to a carboxyl group of hyaluronic acid through an amide bond. When using aminacrine acid as photoreactivation crosslinking group, and the amino acid or peptide as spacer preferred photoreactivation hyaluronic acid with such a structure, where the carboxyl group of the spacer is chemically bound with the amino group aminacrine acid through an amide bond, and the amino group of this amino acid or peptide is chemically bound to a carboxyl group of hyaluronic acid through an amide bond.

Specifically, photoreactivation cross-linking groups is associated with the spacer, especially preferred are such, which are represented below by the following formula (1) or (2).

-NH(CR1R2)nOCOCH=CH-Ph (1)

In the formula (1) R1and R2each independently represent a hydrogen atom Il which may be not only the group, expressed as-C6H5- but also by the group, including substituted benzene ring, one or two atoms which any provisions substituted by one or two substituents selected from the group lower alkyl or alkoxyl having from 1 to 4 carbon atoms, amino group, hydroxyl group and the like; n represents an integer from 2 to 18, preferably from 2 to 12.

Photoreactivation crosslinking group represented by the formula (1), chemically linked, for example, with a carboxyl group of hyaluronic acid through an amide bond with the formation of photoreactivation derived hyaluronic acid.

-A-NH-Ph-CH=CHCOOR3(2)

In the formula (2) R3is lower accelgroup, preferably altergroup having from 1 to 4 carbon atoms (e.g. methyl, ethyl or the like) or arakalgudu having from 7 to 20 carbon atoms, preferably benzyl or phenethyl; And is -(NHCR4R5CO)m- or-NH(CR4R5)hCO-; R4and R5each independently represents a hydrogen atom or a lower accelgroup (preferably having from 1 to 4 carbon atoms); -Ph - represents a group of para-phenylene, which may not be thde the hydrogen atom in the ortho - or meta-position in the benzene ring is substituted by lower alkyl or CNS group having from 1 to 4 carbon atoms, amino group, hydroxyl group or the like; m represents an integer from 1 to 6, preferably from 1 to 3; h represents an integer from 1 to 18, preferably from 1 to 12.

Hyaluronic acid for use in this invention has no specific limitation, however, as the source material is usually used hyaluronic acid having srednevekovoy molecular weight of from 10,000 to 5000000. In accordance with the purpose of application can be chosen in different molecular weight. Preferred srednevekovye molecular weight of from 500,000 to 3000000 and more preferred from 800000 to 2500000. The following method of synthesis used is preferably water-soluble salt of hyaluronic acid, such as alkali metal salt (e.g. sodium salt, potassium salt or the like), alkali earth metal salt (e.g. calcium salt, or the like) or the like, but other salts or the free acid can also be used, as they are soluble in the used reaction solvent and do not affect the reaction. The term "hyaluronic acid", as used here, sometimes below, includes a salt thereof.

Photoreactivation proizvodi acid, for example, in water or in an aqueous solution containing miscible with water, an organic solvent (e.g. dioxane, dimethylformamide, N-organic, ndimethylacetamide, alcohol (e.g. methanol, ethanol, pyridine and the like) and the introduction of photoreactivation cross-linking group, for example, carbodiimide method in the presence of water soluble carbodiimide (e.g., the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (commonly referred to hereafter as "EDCHCl"), methiodide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide) and contributing to the condensation agent (for example, N-hydroxysuccinimide, N-hydroxybenzotriazole, and the like).

Purification of the product obtained after the reaction can be carried out in the usual way, for example by precipitation with ethanol or by dialysis. After cleaning and drying the degree of substitution (commonly referred to hereafter as "DS", which is the degree of introduction of photoreactivation cross-linking groups on the constituent disaccharide glycosides link hyaluronic acid) formed photoreactivation derived hyaluronic acid can be measured using the spectrophotometer ponie of 0.25 endotoxin units of e/g or less) photoreactivation derivatives of hyaluronic acid and vodootvedenie gels crosslinked hyaluronic acid can be obtained through the use of sterile or essentially, do not contain endotoxin reagents, water, containers and the like and attention to sterilization of the working environment.

Specific compounds used for the introduction of photoreactivation cross-linking groups in hyaluronic acid include those represented by the following formula (1-1) or (2-1):

-N2H(CR1R2)nOCOCH=CH-Ph (1-1)

where R1, R2Ph and n are the same as defined above;

H-A-NH-Ph-CH=CHCOOR3(2-1)

where A, -Ph -, and R3are the same as defined above.

The compounds (1-1) and (2-1) is preferably used in the form of salts, for example salts with an attached acid, such as hydrochloride, hydrobromide and the like, hydrochloride is particularly preferred.

Specifically, hydrochloride (1-2) of the compound (1-1) can be synthesized according to the following reaction scheme.

The first reaction

R6HN(CR1R2)nOH (3)+XCOCH=CH-Ph (4) --> R6HN(CR1R2)nOSON=CH-Ph (5)

The second reaction

(5) + HCl --> HClH2N(CR1R2)nOSON=CH-Ph (1-2)

where R6is aminosidine group which can be split by acid, such as tert-butoxycarbonyl specifically, the compound (1-2) is synthesized as follows.

An organic solvent (such as chloroform and the like) are added to the compound (3) and organic base (e.g. triethylamine, and the like) add back with ice cooling. Consistently add connection (4) and the catalyst for the main character (for example, 4-dimethylaminopyridine and the like). After stirring the mixture at room temperature organic solvent (e.g. ethyl acetate, and the like) are added to the reaction mixture and the mixture is washed successively with several portions of weakly acidic aqueous solution, water, a couple of servings weakly alkaline aqueous solution, water, saturated aqueous sodium chloride, and the like. The separated layer of organic solvent is dried over anhydrous sodium sulfate or the like. Dessicant (drier) and the like are removed by filtration and the filtrate is dried under reduced pressure to obtain the compound (5).

1-5 M solution of hydrogen chloride in an organic solvent (such as dioxane and the like) are added to the compound (5) under ice cooling, followed by stirring. There also add an organic solvent (for example, a simple diethyl ether and so on and dried under reduced pressure, to obtain the compound (1-2).

Specifically, hydrochloride (2-2) of the compound (2-1) can be synthesized according to the following reaction scheme.

The first reaction

R6-A-OH (6) H2N-Ph-CH=CHCOOR3(7) --> R6-A-NH-Ph-CH=CHCOOR3(8)

The second reaction

(8) + HCl --> HClH-A-HN-Ph-CH=CHCOOR3(2-2)

where R3and R6is as defined above.

In addition, the compound (2-2) specifically synthesized as follows.

Organic solvent (e.g. chloroform or the like) is added to the compound (6), there add the activator (e.g., dimethylphosphorodithioate and the like) in the presence of organic bases (e.g. triethylamine, and the like) under ice cooling, thereby activating the carboxyl group of compound (6). After activating compounds (6) add connection (7) in the presence of organic bases (e.g. triethylamine, and the like) under ice cooling, followed by stirring at room temperature. Organic solvent (e.g. ethyl acetate, and the like) are added to the reaction mixture and the mixture is washed successively with several portions of weakly acidic aqueous solution, water, a few hollow organic solvent is separated and dried over anhydrous sodium sulfate or the like. Dessicant and the like are removed by filtration and the filtrate is dried under reduced pressure to obtain compound (8).

1-5 M solution of hydrogen chloride in an organic solvent (such as dioxane and the like) are added to the compound (8) under ice cooling, followed by stirring. There also add an organic solvent (for example, a simple diethyl ether, and the like), and the precipitated crystals are collected by filtration, washed with an organic solvent and dried under reduced pressure to obtain the compound (2-2).

The presence of the spacer, included in photoreactivation crosslinking group largely contributes to photoreactivation photoreactivation derived hyaluronic acid, and the same time, this improvement depends on the degree of freedom of the spacer and its ability to hydrophobic binding. Enhanced sensitivity in photoreactivation introduced by the spacer makes it possible to achieve photoallergen knitting with such a small degree of introduction photoreactivation cross-linking group in which fototienda stitching had been difficult under such conditions.

The authors of this invention have found that Vodootvodny gel crosslinked hyaluronic acid can be formed by preparing an aqueous solution photoreactivation derived hyaluronic acid of high concentration, to make a more closely spaced adjacent photoreactivation cross-linking group, more frequently, by giving the layer of solution form, allowing ultraviolet rays to pass through this layer with ease, and then irradiated with ultraviolet rays.

According to this invention, when photoreactivation hyaluronic acid is irradiated with ultraviolet rays, Vodootvodny gel crosslinked hyaluronic acid, nieuwesteeg choice of conditions of expression, such as the concentration of the solution of the expression, the irradiation time of ultraviolet rays and the like, or DS.

The preferred concentration of the solution photoreactivation derivative of hyaluronic acid in the aquatic environment, which must be irradiated with light (hereinafter sometimes referred to as "the concentration of expression") is from 0.5 to 10% by weight. When using photoreactivation derivative of hyaluronic acid having a molecular weight of about 1,000,000, more preferred concentration is from 1 to 4% by weight. At lower concentrations isomerization dominates the reaction, as mentioned here above. In contrast, at higher concentrations it is difficult to obtain a homogeneous gel.

When a more dilute aqueous solution of concentration than that specified a certain concentration, is irradiated with ultraviolet rays, there is a tendency to predominant formation of isomers as described above. With continued irradiation with ultraviolet rays itself sahariana chain hyaluronic acid will disintegrate under the influence of ultraviolet rays, resulting in a decrease in molecular weight. From these points of view, the different influences on sharenow chain of hyaluronic acid. Therefore, it is important to prepare an aqueous solution with concentrations defined above. Fig. 1 graphically gives an overview of fototienda the stitching in the aquatic environment, where (a) shows the change photoreactivation derivative of hyaluronic acid in dilute solution under irradiation with ultraviolet rays. Water molecules prevent adjacent photoreactivation cross-linking groups to line up in molecular order, ready for stitching, and the predominant isomerizate. In (b) shows the change photoreactivation derivative of hyaluronic acid in solution at a precise concentration upon irradiation with ultraviolet rays. Hydrophobic photoreactivation cross-linking group, being less susceptible to interference by water molecules than in dilute solution, obviously, attract each other due to the strength of the hydrophobic binding, taking the molecular order, ready for stitching. So photoreactivation crosslinking group timeresults under irradiation, which is accompanied by the inclusion of the aquatic environment, thus ending with stitching. To secure improved photorearrangement cross-linking in solution at exactly Obradovi acid, including photoreactivation cross-linking group containing the above-described spacer, and having a high flexibility.

The above-mentioned concentration of the expression depends on the degree of substitution (DS) photoreactivation crosslinking groups included in hyaluronic acid. DS can be calculated on the basis of the degree (%) introduction photoreactivation cross-linking group comprising disaccharide glycosides link hyaluronic acid. For example, DS photoreactivation derivative of hyaluronic acid with one photoreactivation cross-linking group comprising disaccharide glycosides link or 200 components sharidny links equal 100% or 1% respectively. Under the same conditions of irradiation light lower than DS, the lower the degree of crosslinking.

In this invention DS photoreactivation derived hyaluronic acid to achieve a staple in just a certain concentration the expression mentioned above, can be from about 0.05 to 10%, preferably from about 0.3 to 5% and more preferably about from 0.5 to 3%, in the case of hyaluronic acid having a molecular weight of 500,000 or more, varying depending on the molecular weight of the source of hyaluronic acid.

The solution photoreactivation derived hyaluronic acid is usually prepared by dissolving in water photoreactivation derived hyaluronic acid, freshly isolated and purified from the reaction system synthesis. As a solution photoreactivation derived hyaluronic acid is possible in some cases to use photoreactivation derivative of hyaluronic acid in the reaction system of its synthesis as such or in its concentrated state.

When DS photoreactivation derived hyaluronidases change with change in the concentration limits of the expression, mentioned above, and accordingly change the physical properties of the resulting gel. With increasing concentration of the expression degree of crosslinking, appears to be increasing, and the elastic nature of the gel increases, which is confirmed by the measurement of viscoelasticity vodootvedenija gel crosslinked hyaluronic acid. With increasing degree of crosslinking mesh structure becomes denser. The relative share cyclobutanone ring fototienda gel crosslinked hyaluronic acid can be defined as the density of the polymer network, which is the product of DS and the degree of crosslinking and expressed as molar ratio (%) of dimers on a constituent disaccharide glycosides link hyaluronic acid. The preferred density of the polymer network is in the range from 0.01 to 0.5% per 1 mol of constituent disaccharide glycosides level of hyaluronic acid.

The absorption of water by dry photoallergen gel crosslinked hyaluronic acid (referred to hereafter simply as "dry gel") depends on the degree of crosslinking and therefore becomes a measure of the degree of crosslinking. The water absorption is expressed by the following formula:

Water absorption (%) = weight of absorbed water/mass of dry gel 100

With increasing degree of Slivnica which is reduced by the absorption of water by dry gel. The absorption of water by the dry gel of the present invention typically ranges from about 20 (100%) 150 (100%), preferably from 30 (100%) 120 (100%), more preferably from 40 (100%) 100 (100%), when measured after immersion of the dry gel for 24 hours in a physiological solution (0.9% aqueous solution of sodium chloride) in the quality of the aquatic environment.

The gel of the present invention may be a gel containing from 0.5 to 10% by weight, based on the content of hyaluronic acid, vodootvedenija derived hyaluronic acid. In particular, Vodootvodny the hyaluronic acid gel obtained from photoreactivation derivative of hyaluronic acid having a molecular weight of about 1,000,000, preferably has a content of hyaluronic acid from 1 to 4% by weight in the gel.

The physical properties of the gel due to the viscoelasticity can be expressed in dynamic viscoelasticity, such as storage modulus (G'), loss modulus (G") and tan Delta (tg; G"/G') and the like. High modulus and low modulus losses indicate high elasticity, indicating a solid gel. Conversely, a high loss modulus and low modulus indicate the gel with high viscosity.

Gel dunnage from 100 to 500 PA, the loss modulus (G") of from 10 to 300 PA, preferably from 50 to 150 PA, and tan Delta (tg; G"/G') of from 0.1 to 0.8, preferably from 0.2 to 0.5, the dynamic viscoelasticity at a frequency of 10 Hz.

When exposed to light such as ultraviolet rays, the type of ultraviolet rays in a special way is not limited. Irradiation with light or ultraviolet rays is usually carried out from several seconds to several minutes using a light source that emit light containing wavelengths required for the expression, i.e., from 200 to 450 nm (for example, mercury lamps, high pressure, metallogenesis lamp or the like), while short wavelengths, undesirable for the dimerization, cut off filter ultraviolet rays or the like (for example, glass, Pyrex, Pyrex glass (trade name) or the like). The method of irradiation is not particularly limited, and choose different methods suitable for this purpose. For example, the solution photoreactivation derivative of hyaluronic acid are loaded into a container for delivery as a final commercial product gel of the present invention and irradiated as described hereafter; this solution held between a pair of tape conveerter photoreactivation derived hyaluronic acid serves inside the pipe, made from transparent to UV rays material and irradiated with ultraviolet rays during its passage through it.

In the process of obtaining the gel of the present invention the gel, which meets the requirements described above viscoelasticity can be obtained even when the solution photoreactivation derived hyaluronic acid is subjected to heat treatment vapor pressure at a temperature of from 100 to 125oC for from 5 to 30 minutes (autoclave)

before and/or after irradiation with ultraviolet rays. Such heat treatment may correspond to a sterilization process for medical devices and medicines.

State solution photoreactivation derived hyaluronic acid, which must be irradiated with ultraviolet rays, and the material and shape of the container to receive it for the expression and perception of ultraviolet rays part in a special way is not limited provided that the ultraviolet rays can pass through them. For example, they can be similar to layer, like a tube, similar to a syringe, such ampoule or the like. Taking into account the homogeneity schifosi. The irradiation with ultraviolet rays of a layer shaped as a thin layer, is particularly suitable for obtaining a uniformly cross-linked gel. The container for the expression may have such a form that it could hold the solution photoreactivation derived hyaluronic acid and the formed gel of the present invention in the system of expression, and it is not always necessary that it was a closed container. For example, the container may be in the form of a simple plate.

When the gel of the present invention is used as a biomedical material, such as adhesive material, the container to prevent damage to the gel of the present invention may preferably have a shape with which the formed gel of the present invention can be kept sterile before use and from which the gel can be removed properly when using it. Examples of such a container in which the gel can be kept sterile before use and from which the gel can be easily applied to a designated object or place (when the gel is used as an anti-adhesive material, object, or place is the affected part, which must be protected from adhesions), to include what can be learned and applied, include containers such as vials and the like.

When photoreactivation derived hyaluronic acid load in the container and loaded photoreactivation derived hyaluronic acid is irradiated with ultraviolet rays to expose the expression, as mentioned above, the container material must be selected from such materials that allow ultraviolet rays and not subject to degradation by ultraviolet rays. Further, in the case when the gel of the present invention is intended for application to the human body as a biomedical material, the gel after the expression may preferably be sterilized, for example, high pressure steam (autoclave). When Vodootvodny crosslinked gel loaded in the container is sterilized by autoclave sterilization, the container material may be preferably made of glass, plastics or the like having heat resistance to some extent. The amount of gel loaded in the container is not specifically limited; however, from an operational and economic considerations, and the like, it is, for example, from about 0.5 to 500 ml.

by drying or the like and then transferred in the swollen state by adding the desired quantity of the water environment, to ensure that the gel of the present invention having the above physical properties. In this case, the drying is preferably carried out in a way that has no adverse effect on sharenow chain hyaluronic acid and crosslinked structure.

It is also possible to store or transport Vodootvodny gel crosslinked hyaluronic acid in the form of dry solid gel, as mentioned above, and use it as a re-saturated with a suitable aqueous medium to swell immediately prior to use.

The gel of the present invention are mainly used as a biomedical material. High inherent biocompatibility of hyaluronic acid in combination with the newly added due to the crosslinking properties, such as a prolonged effect in a living organism (an improvement in the ability to stay in place), and improved physico-chemical properties, such as viscoelasticity and the like, are suitable for use as a biomedical material, make the gel of the present invention very suitable for use in the medical field.

Because the gel of the present invention for the most part contains resursele for living organisms.

More specifically, the introduction of a single dose of 100 ml/kg (equivalent to 2000 mg of hyaluronic acid per 1 kg) gel of the present invention administered intraperitoneally to rats does not cause neither death nor serious disorders attributed to gel.

In addition, a test was performed of antigenicity to identify active anaphylaxis, in which Guinea pigs were administered intraperitoneally senzibilizirani 20 mg or 2 mg of the gel of the present invention and then administered intraperitoneally injected with 40 mg of gel. In the anaphylactic reaction was not induced.

When the gel of the present invention is used as a biomedical material, such as adhesive material, it is understood that the increased elasticity of the gel leads to improvement in barrier effect between tissues and the ability to remain in the body, while high viscosity leads to improvement in adhesion to fabric and suitability for injection into the affected part. Therefore, it is desirable that the gel for this application had a well-balanced elasticity and viscosity. If G' is greater than 1500 or tan Delta less than 0.1, the gel becomes super-elastic gel, the so-called hard and brittle gel, and stonewashed 0,8, the gel becomes highly viscous gel, resembling a solution, lacking in providing the desired stiffness and barrier effect, need for anti-adhesive action is lost. That is, the gel of the present invention is seen as having the most suitable physico-chemical properties as an anti-adhesive material.

Spike bodies, for example, which often happens after the operation, which is undesirable from a clinical point of view, and from these positions is desirable to develop effective anti-adhesive material. Desirable characteristics for anti-adhesion material are the following: the material must (1) create a barrier effect between the bodies, which can suffer from adhesions, (2) to have the covering property over the wounded part, (3) not to slow wound healing, (4) to remain in the body during the healing period and preferably be degraded and absorbed in the body after healing, (5) to be harmless, non-toxic, biocompatible, and the like.

Vodootvodny gel crosslinked hyaluronic acid of the present invention, which is obtained photoallergen the stitching and which has excellent biocompatibility and besagni, provides physicochemical properties of cross-linked gel, and the ability to remain in the body is achieved by forming photoallergen stitched mesh structure.

The amorphous gel, for example, allows the gel to be injected through the injection tip or needle, that is, when it is loaded into a syringe and inject into a small affected part (e.g., the wounded part, and the like). In this regard, the gel of the present invention is characterized in that it has a much higher elasticity than the elasticity solution of hyaluronic acid, but its density is such that it retains sufficient softness to pass through the needle of the syringe. The gel can also be injected on the affected part through a tube of small diameter (injection tip) and it is intended for use in laparoscopic surgery and the like.

Briefly, the anti-adhesion material containing gel of the present invention, as described above, is characterized as the following:

(1) having excellent anti-adhesive effect;

(2) which is a hydrogel having physical properties that allow to be injected it into the affected part;

(3) detecting a high adhesiveness to the military part of the period, necessary to prevent adhesions;

(4) without the need to remove, and which is absorbed from the specific location of application, such as the abdominal cavity, and the like, and metabolisable secreted and

(5) having safety: no safety problems were observed in these animals as test toxicity single dose toxicity test repeated doses and testing of antigenicity.

As examples the following applications, when the gel of the present invention are used as anti-adhesive material.

(1) the Area of obstetrics and gynecology

Anti-adhesions accompanying intrapelvic surgery for infertility treatment, surgery on the uterus, surgery on the tubes, surgery on the ovaries, surgery for endometriosis, caesarean section, intrapelvic division of adhesions or the like.

(2) the Area of gastrointestinal surgery

Anti-adhesions accompanying intestinal adhesion after abdominal surgery or the like.

(3) Orthopedic area

Anti-adhesions that accompany surgery on the Achilles (calcaneal) tendon, surgery is the first invention in the medical field as a biomedical material other than antiadhesive material, described above, is shown below.

(1) an excipient in ophthalmic surgery. For example, the gel is injected in front of the camera operations, such as inserting intraocular lenses, total corneal transplantation or the like, or gel is used to ensure intraocular pressure, retinal detachment, and the like, or for replenishment of the vitreous body.

(2) Tools that improve the function of joints. For example, the gel is injected into the joint cavity to alleviate pain, improve limit the mobility of the joint, the normalization of pathological synovial fluid and the like in the treatment of arthritis such as arthritis deformans of the knee joint, periarthritis of the shoulder joint or the like.

(4) the artificial defects in the field of plastic surgery.

(5) Dressings for pressure sores and burns.

(6) Materials or medicines to slow excretion of drugs.

For use as anti-adhesive gel material of the present invention, the amount of gel that should be applied on the affected part cannot be precisely defined because it varies the program. This is usually about 0.5 to 500 ml/place, preferably about 1 to 100 ml/place, more preferably from about 2 to 50 ml/place.

Vodootvodny gel crosslinked hyaluronic acid of the present invention has a three-dimensional mesh structure. The inclusion of medicines in the grid will provide useful preparation for a delayed excretion of drugs. The introduction of the drug in the gel can be carried out by immersing the dry gel in a solution containing the drug. And, as Vodootvodny crosslinked gel does not require cleaning after stapling, the medication may be previously added to the solution photoreactivation derived hyaluronic acid, which must be irradiated. Can be ispolzuya any of the methods described above.

In addition, the drug may be chemically linked to photoreactivation derivative of hyaluronic acid through chemical bonding (covalent binding, ionic binding, and the like) and can then be subjected to the expression for stitching. For example, when the medicine is injected covalent binding, medicine and carboxyl or hydroxyl group photoreactivation derived hyaluronic acid is mowimy rays. And when the medicine is injected ion binding, cation medicine, capable of binding to a carboxyl group of hyaluronic acid by ion binding, can be mixed with photoreactivation hyaluronic acid and then subjected to irradiation with ultraviolet rays. In addition, photoreactivation derivative of hyaluronic acid containing photoreactivation cross-linking group is bound to the drug, may be subject to expression for stitching.

The invention will now be illustrated in more detail with reference to preparation Examples, Examples, Examples and tests, but it should be clear that this invention should not be considered limited to these.

SAMPLE PREPARATION 1

Preparation photoreactivation derived hyaluronic acid (DS 0,53%).

1.5 l of water was dissolved 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; srednevekovaja molecular weight (Mw) 950000) and to the solution was added 750 ml of 1,4-dioxane. To the solution successively added 50 ml of dioxane containing 288 mg (2.5 mmol) of N-hydroxysuccinimide, 50 ml of an aqueous solution containing 124h at 5-minute intervals while cooling with ice. After stirring the mixture at room temperature for 8 hours to it was added an aqueous solution of 10 g of sodium chloride, followed by stirring for 1 hour. The resulting solution was poured into 5 l of ethanol. Thus obtained the desired precipitate is collected by centrifugation (4000 rpm, 15 min), washed with 3 successive portions 80% ethanol and one portion of ethanol and dried to obtain 9,73 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 0,53%; endotoxin of 0.8 PG/mg).

Endotoxin in the Sample preparation and in the following Examples of the preparation was determined by using a set Toxicolor System LS-20, set DIA and the set of Et-1, all available from Seikagaku Corporation (trademark; hereinafter the same).

The content of endotoxin in fototienda gel crosslinked hyaluronic acid is determined by the method described above, then Vodootvodny gel crosslinked hyaluronic acid, as described in the Examples, solubilizers digesting enzyme (for example, chondroitinase ABC, a product of Seikagaku Corporation, and the like).

The content of endotoxin one endotoxin unit (unit e) corresponds 345 PG of endotoxin.

EXAMPLE ptx2">

It is produced in the same manner as in Example of preparation 1, using 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 65 ml of 0.05 M solution of N-hydroxysuccinimide (3.25 mmol) in dioxane, 65 ml of a 0.025 M aqueous solution EDHC1 (1,625 mmol) and 65 ml of a 0.025 M aqueous solution SN2N(CH2)6OSON=h (1,625 mmol). Get 9,74 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 0,75%; endotoxin of 2.5 PG/mg).

EXAMPLE PREPARATION OF 3

Preparation photoreactivation derived hyaluronic acid (DS 0,90%).

It is produced in the same manner as in Example of preparation 1, using 2.0 g (5.0 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 3 ml of an aqueous solution containing 69 mg (0.6 mmol) of N-hydroxysuccinimide, 3 ml of an aqueous solution containing 58 mg (0.3 mmol) EDHC1 and 3 ml of an aqueous solution containing 85 mg (0.3 mmol) NSN2N(CH2)6OSON=h. Obtain 2.1 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 0,90%; endotoxin of 2.4 PG/mg).

EXAMPLE PREPARATION OF 4

Preparation photoreactions is otopleniya 1 using 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 100 ml of 0.05 M N-hydroxysuccinimide (5.0 mmol) in dioxane, 100 ml of a 0.025 M aqueous solution EDHC1 (2.5 mmol) and 100 ml of 0.025 M aqueous solution SN2N(CH2)6OSON=CHPh (2.5 mmol). Get for 9.64 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 1,06%; endotoxin of 3.2 PG/mg).

EXAMPLE PREPARATION OF 5

Preparation photoreactivation derived hyaluronic acid (DS 1,26%).

It is produced in the same manner as in Example of preparation 1, using 5 g (12.5 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 50 ml of an aqueous solution of 288 mg (2.5 mmol) of N-hydroxysuccinimide in dioxane, 50 ml of an aqueous solution of 240 mg (1.25 mmol) EDHC1 and 50 ml of an aqueous solution of 355 mg (1.25 mmol) NSN2N(CH2)6OCOCH= CHPh. Get 4.9g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 1,26%; endotoxin of 1.0 PG/mg).

EXAMPLE 6 PREPARATION

Preparation photoreactivation derived hyaluronic acid (DS 1,29%).

It is produced in the same manner as in Example of preparation 1, using 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (product S(2.5 mmol) and 50 ml of 0.05 M aqueous solution SN2N(CH2)6OSON=h (2.5 mmol). Get 10.0 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 1,29%; endotoxin of 2.5 PG/mg).

EXAMPLE PREPARATION OF 7

Preparation photoreactivation derived hyaluronic acid (DS 1,55%).

It is produced in the same manner as in Example of preparation 1, using 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 150 ml of 0.05 M solution of N-hydroxysuccinimide (7.5 mmol) in dioxane, 150 ml of a 0.025 M aqueous solution EDCHC1 (3.75 mmol) and 150 ml of a 0.025 M aqueous solution SN2N(CH2)6OSON=h (3.75 mmol). Get 9,92 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 1,55%; endotoxin of 1.2 PG/mg).

EXAMPLE OF PREPARATION 8

Preparation photoreactivation derived hyaluronic acid (DS 1,93%).

In 600 ml of water was dissolved 4.0 g (10.0 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000) and to the solution was added 300 ml of 1,4-dioxane. To the solution successively added 10 ml of an aqueous solution containing 230 mg (2.0 mmol) of N-hydroxysuccinimide, 10 ml aqueous solution/SUB>OSON=h at 5-minute intervals while cooling with ice. After stirring the mixture at room temperature for 24 hours there was added an aqueous solution of 2.0 g of sodium chloride, followed by stirring. The resulting solution was poured into 3.0 l of ethanol. Thus obtained the desired precipitate is collected by centrifugation (4000 rpm x 15 min), washed successively three times with 80% ethanol and once with ethanol and dried to obtain 4.1 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 1,93%; endotoxin of 2.1 PG/mg).

SAMPLE PREPARATION 9

Preparation photoreactivation derived hyaluronic acid (DS 2,87%).

It is produced in the same manner as in Example of preparation 1, using 10 g (25 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 50 ml of a solution of 864 mg (7.5 mmol) of N-hydroxysuccinimide in dioxane, 50 ml of aqueous solution of 718 mg (3.75 mmol) EDCHC1 and 50 ml of an aqueous solution of 1.06 g (3.75 mmol) NSN2N(CH2)6OSON=h. Get 10 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 2,87%; endotoxin of 2.8 PG/mg).

EXAMPLE TH>/P>It is produced in the same manner as in Example of preparation 1, using 50 g (125 mmol disaccharide glycosides link) of sodium hyaluronate (a product of Seikagaku Corporation; Mw 950000), 250 ml of an aqueous solution of 3.45 g (30 mmol) of N-hydroxysuccinimide, 250 ml of an aqueous solution 2,88 g (15 mmol) EDHC1 and 250 ml of an aqueous solution of 15 mmol SN2N(CH2)6OSON=h. Get 49 g photoreactivation derivative of hyaluronic acid in the form of a white solid substance (DS 2,28%; endotoxin of 3.2 PG/mg).

EXAMPLE 1

This Example applies to fototienda gel crosslinked hyaluronic acid, which is produced photoallergen the stitching photoreactivation derived hyaluronic acid obtained in Example 6 preparation, in an aqueous solution with subsequent substitution of the environment buffered 1.5 mm phosphate saline solution (pH 7.4).

1.4 wt. % aqueous solution photoreactivation derived hyaluronic acid (DS 1,29%) obtained in Example 6 preparation, held between a pair of plates of glass, Pyrex, each of which has a thickness of 2.5 mm, with a gap between them to 1.0 mm is irradiated with ultraviolet rays (3 kW, metallogenica lamp) for 4 minutes on each side (8 minutescase solution (pH 7.4), bringing the concentration to 2 wt.%. Then, the dry gel is subjected to swelling within 1 day to get Vodootvodny gel crosslinked hyaluronic acid (endotoxin 0,11% e/g).

EXAMPLE 2

This Example applies to photoallergen gels crosslinked hyaluronic acid, which receive photoallergen the stitching photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1, 4 and 7, 1.4 wt.%-nom solution in buffered 1.5 mm phosphate saline solution (pH 7.4).

1.4 wt. % solution of each of photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1, 4 and 7 (DS 0,53%, of 1.06% and 1.55%, respectively), in buffered 1.5 mm phosphate saline solution (pH 7.4) hold between a pair of plates of glass, Pyrex, each of which has a thickness of 2.5 mm, with a gap between them to 1.0 mm is irradiated with ultraviolet rays (3 kW, metallogenica lamp) for 4 minutes on each side (8 minutes in total) to get Vodootvodny gel crosslinked hyaluronic acid.

The content of endotoxin is 0.03, 0.12 and 0.05 in units of e/g gels having a DS of 0.53%, of 1.06% and 1.55%, respectively).

EXAMPLE 3

This Primere photoreactivation derivatives of hyaluronic acid, obtained in preparation Examples 1 to 9, 2.0 wt.%-nom solution in buffered 1.5 mm phosphate saline solution (pH 7.4).

2.0 wt. % solution of each of photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1-9 (DS 0,53%, 0,75%, 0,90%, 1,06%, 1,26%, 1,29%, 1,55%, 1,93% are 2.87%, respectively), in buffered 1.5 mm phosphate saline solution (pH 7.4) hold between a pair of plates of glass, Pyrex, each of which has a thickness of 2.5 mm, with a gap between them to 1.0 mm is irradiated with ultraviolet rays (3 kW, metallogenica lamp) within 4 minutes on each side (8 minutes in total) to get Vodootvodny gel crosslinked hyaluronic acid.

EXAMPLE 4

This Example applies to photoallergen gels crosslinked hyaluronic acid, which receive photoallergen the stitching photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1, 4 and 7, 3.2 wt.%-nom solution in buffered 1.5 mm phosphate saline solution (pH 7.4).

3.2 wt. % solution of each of photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1, 4 and 7 (DS 0,53%, of 1.06% and 1.55% sootvetstvenno, each of which has a thickness of 2.5 mm, with a gap between them to 1.0 mm is irradiated with ultraviolet rays (3 kW, metallogenica lamp) for 4 minutes on each side (8 minutes in total) to get Vodootvodny gel crosslinked hyaluronic acid.

EXAMPLE 5

This Example applies to photoallergen gels crosslinked hyaluronic acid, which is produced by heat treatment photoallergen gels crosslinked hyaluronic acid having a DS of 1.55% and 2.87% of that in Example 3.

Vodootvedenie gels crosslinked hyaluronic acid obtained in Example 3 from photoreactivation derivatives of hyaluronic acid having a DS of 1.55% and 2.87% of each load in 10 ml vials and subjected to heat treatment in an autoclave at 121oC for 8 minutes.

Measurement of physical properties

Conduct measurement of dynamic viscoelasticity (storage modulus G', loss modulus G" and tan Delta tg (G"/G')), dynamic viscosity () and absorption of water solutions photoreactivation derivatives of hyaluronic acid obtained in preparation Examples 1, 4, 5 and 7, having a concentration preparation (solution concentration, as the concentration of hyaluronic acid) 1.4 wt.%, 2.0 wt.% or 3.2 wt.%, foooter is from.%, 2.0 wt.% or 3.2 wt.%, and heat-treated photoallergen gels crosslinked hyaluronic acid obtained in Example 5. The physical properties of a solution of hyaluronic acid prepared so that the concentration of the preparation is the same as that of the above-mentioned vodootvedenija gel crosslinked hyaluronic acid, measured as described above. Measurement of dynamic viscoelasticity and the dynamic viscosity is performed by the rheometer Model CSL-50, manufactured by Carri-Med, a work element of which is the cone shown in Fig.3, rotating with angular velocity rad/sec.

For test was used oscillation method at a controlled load.

Measuring conditions:

Temperature measurement 37oWITH

Cone size 4 cm (2R)

A gap of 800 μm

Frequency 10 Hz

The water absorption is calculated by the method of determining the optical density of ultraviolet rays using Blue Dextran (Blue Dextran), abbreviated hereafter referred to as B. D., described in EP 0205674 A1 as follows. Physiological solution (0.9% aqueous solution of sodium chloride) is used as the aqueous medium.

When the dry gel was placed in the solution B. D., gel absorbs tze solution B. D., the remaining unabsorbed increases compared to the original, depending on the absorbed water. This difference in concentration is determined from the optical densities (610 nm), and then the absorption of water can be calculated according to the equation:

The water absorption ( 100%) = (1 - U1/U2)/1000,

where N1 denotes the optical density at initial concentrations As mg dry gel per gram of 0.1 wt.%-aqueous solution B. D. and N2 denotes the optical density after 24 hour soaking in solution B. D.

Next, the absorption of water by exploring on dry samples photoallergen gels crosslinked hyaluronic acid obtained in Example 3 from photoreactivation derivatives of hyaluronic acid having a DS 0,53%, 0,75%, 0,90%, 1,26%, 1,55% and of 1.93%.

Some of the results of measurement of the dynamic viscoelasticity and the like shown in table. 1 and the relationship between DS and water absorption are shown in Fig.2. In table. 1 detected when analyzing the concentration of the samples is obtained by determining the content of a component of hyaluronic acid in the gel in accordance with the method of the reaction of carbazole-sulfuric acid.

EXAMPLE 6

This Example applies to photoallergen gels crosslinked hyaluronic acid, which kadogo of photoreactivation derivatives of hyaluronic acid, obtained in Example of preparation 10, in buffered 1.5 mm phosphate saline solution (pH 7.4).

Photoreactivation derivative of hyaluronic acid having a DS of 2.28%, which was obtained in Example of preparation 10, dissolved in buffered 1.5 mm phosphate saline solution (pH 7.4) to obtain 2.0 wt. %-aqueous solution. The resulting solution was irradiated with ultraviolet rays in accordance with the following three methods. Before and/or after irradiation with ultraviolet rays solution (or gel) is subjected to heat treatment under the following conditions. The degree of cross-linking density of the polymer network and the characteristics of viscoelasticity is measured.

The conditions of irradiation with ultraviolet rays (UV-irradiation):

(1) The same method as in Examples 2-4. An aqueous solution photoreactivation derivatives of hyaluronic acid holds between a pair of plates of glass, Pyrex, each of which has a thickness of 2.5 mm, with between 1.0 mm and irradiated with ultraviolet rays (3 kW) metallogenesis lamp for 4 minutes on each side (8 minutes in total).

(2) the Solution is held between a pair of plastic films and irradiated with ultraviolet rays of 400 W mercury lamp high is at high pressure, by passing it through a quartz tube of diameter 5 mm

Conditions of heat treatment (method of sterilization (autoclave):

A: After UV irradiation; 121oC, 8 min

Q: Before UV irradiation; 121oC, 8 min

With: Before UV irradiation; 121oC, 8 min after UV irradiation; 100oC, 10 min

D: After UV irradiation; 121oC, 15 min

The measurement results are shown in table. 2, where the degree of crosslinking calculated by the following equation:

The degree of crosslinking (%) = Number of moles dimenisonal cinnamic acid 2/the number of moles introduced cinnamic acid 100

More specifically, cinnamic acid and its dimer chemically otscheplaut and extracted from vodootvedenija gel crosslinked hyaluronic acid. Taking advantage of the differences in molecular mass between cinnamic acid and its dimer, the extract is separated into cinnamic acid and its dimer by gel permeation chromatography (GPC) and each component of quantify to get the corresponding number of moles. Then the degree of crosslinking is calculated on the basis of the above equations.

The density of the polymer network is obtained from the following equation:

The density of the polymer network (%) = DS degree of crosslinking/100

As can be seen from legislate, the density of the polymer network (the product of the degree of crosslinking and DS) can be expressed as a molar ratio (%) of dimers on a constituent disaccharide glycosides link hyaluronic acid.

EXAMPLE TEST 1

The antiadhesive effect on a rat model of uterine horns

This Example tests refers to the antiadhesive effect of the gels obtained in Examples 1, 3, 5, and 6, and for comparison of solutions photoreactivation derived hyaluronic acid before irradiation with ultraviolet rays, as obtained in Examples 1 and 3 (referenced hereafter as "gel unstitched hyaluronic acid), commercially available anti-adhesive material TS (Interceed (trade name), manufactured by Johnson & Johnson), and 3.2 wt.%-aqueous solution of hyaluronic acid in buffered 1.5 mm phosphate saline in a rat model of the horns of the uterus.

1. The tested animal

Seven female rats, Crj:SD (SFF.) gain and feed for 1 week before testing. Each group consists of 5 rats.

2. Test method

2-1. Preparation of rat model of adhesion of the horns of the uterus.

Belly rat shave under anesthesia with Nembutal. The midline doing a bit (treponem) for ophthalmic surgery and muscle layer prepare the chin tongs.

(b) uterine horn naked and doing 4 transverse incision about 1 cm down from the ovary towards the uterus (womb) at intervals of 2-3 mm. Bleeding stop at each section of an electric cautery.

(C) Place about 3-4 mm from the end of the transverse incision in the uterine horn and place 3-4 mm from the end of the defect in the abdominal wall to draw together the only bead 8/0 to bring injury, made in (a) and (b), see above.

2-2. Introduction

The test group

Between the defective part in the abdominal wall and incised part of the uterine horns inject or insert 1 ml photoallergen gels crosslinked hyaluronic acid gels unstitched hyaluronic acid and a solution of hyaluronic acid described above and commercially available TS, with an area of 1,5 x1,5 cm2. More specifically, the above-mentioned gels injected as follows. Each 1 ml of the gel described above, accurately weighed, to take in the syringe 1 ml Terumo Syringe (trade name) to tuberculin, sterilized-rays, the inner tube diameter of about 4 mm, the inner diameter of the tip is about 1 mm) and injected through the tip of the syringe between the defective part in the abdominal wall and the abuse is alasti and uterine horn on the left side of the same animal, which is used for the test group, without the introduction of materials.

3. Assessment

Seven days after implantation in rats killed by exsanguination via the carotid artery under anesthesia with ethyl ether. After opening the side affected by adhesions, scored according to the degree of adhesion according to the following scoring system.

0... No spikes.

1... Weak and easily detachable spike.

2... the Middle and detachable spike.

3... Strong and nonshared spike.

4. Results

The test results are shown in table. 3 below. In table. 3 conditions of irradiation with ultraviolet rays and heat treatment conditions are the same as described in Example 6.

As you can see from the results in table. 3, while the commercially available film TS against adhesions is not effective in this model, spikes, Vodootvodny the hyaluronic acid gel of the present invention is quite effective against adhesions in this model.

Based on the fact that no effect does not appear from unstitched gels or 3.2%-aqueous solution of hyaluronic acid, which has viscoelasticity and viscosity, like potassium g is tion of the invention appears when fototienda the stitching.

EXAMPLE OF TEST 2

This Example tests refers to the antiadhesive effect of the gel of the present invention in the same rat model of adhesion of the horns of the uterus, as in test Example 1, without stopping bleeding defective part of the wall of the abdominal cavity and incised part of the uterine horns.

The antiadhesive effect of the gel of the present invention examine and evaluate in the same way as in test Example 1, except that do not stop bleeding cuts the wall of the abdomen and uterine horns and using 0.5 ml vodootvedenija gel crosslinked hyaluronic acid having a concentration of 2% and a DS of 2.5% and heat treated at 105oC for 10 minutes before irradiation with ultraviolet rays and 121oC for 8 minutes after irradiation with ultraviolet rays (n=10). As a control examined the same animal that is used for the test group, without the introduction of material in the same manner as in test Example 1.

The results obtained are shown in table. 4.

Vodootvodny gel crosslinked hyaluronic acid at a dose of 0.5 ml shows a noticeable effect against spikes on the model without stop bleeding. In other words, the results will TS, which may not be imposed on the affected part, without the necessity to stop the bleeding.

THE TEST EXAMPLE 3

The antiadhesive effect vodootvedenija gel crosslinked hyaluronic acid on rat model adhesions bowel

1. The test substance.

Vodootvodny gel crosslinked hyaluronic acid having a concentration of 2% and a DS of 2.5%, heat-treated at 105oC for 10 minutes before irradiation with ultraviolet rays and 121oC for 8 minutes after irradiation with ultraviolet rays.

2. The tested animal

Seven female rats SD acquire raise within 1 week before the test.

3. Preparation of model adhesions

Serous membrane from the ileum to the colon prepare the chin strip length of 20 cm and a width of 3-4 mm Stop bleeding does not hold.

4. Introduction and grouping

The prescribed amount (0.5 ml, 1.0 ml or 2.0 ml) of gel injected from a syringe with a capacity of 1 ml and applied on the affected area. After applying the intestinal canal return into the abdominal cavity and close the abdominal cavity. Model adhesions without causing gel take as the control group. Models with naneseniami CLASS="ptx2">

5. Evaluation and results

Seven days after implantation in rats killed by exsanguination via the carotid artery under anesthesia with ethyl ether. After opening degree of adhesion is evaluated according to the same scoring system as in test Example 1. The results obtained are shown in table. 5 .

All three subjects groups show a noticeable anti-adhesive effects compared with the control group. The results in table. 5 shows not only the effectiveness of the gels of this invention against the formation of adhesions, but also their superior adhesion to fabric and biocompatibility with tissue, whereas vigorous peristalsis.

In addition, at autopsy in seven days after implantation in animal tests in the Examples, tests 1 and 2 the majority of the entered photoallergen gels crosslinked hyaluronic acid become invisible to the naked eye. Thus found biorthogonal gel of the present invention.

As described and demonstrated above, Vodootvodny gel crosslinked hyaluronic acid can be easily obtained by irradiation with ultraviolet rays is highly concentrated aqueous solution potogonnogo invention exhibits physical properties, such as suitable viscoelasticity, the affinity to fabric, biorthogonal and the like, at the same time, perfectly preserving properties that are characteristic of hyaluronic acid, such as non-toxicity, panthenol, biocompatibility, biorthogonal and the like. Therefore, it is assumed the possibility of its application in various fields as a medical material of high security, such as anti-adhesion material, a carrier for delayed allocation of a medicine or the like. In addition, the gel can be injected into the thin part of the affected areas of the body with a syringe, cannula or the like, and therefore it is assumed for use in microsurgery and the like.

1. Vodootvodny gel crosslinked hyaluronic acid, which has a storage modulus (G') of from 50 to 1500 PA, a loss modulus (G") of from 10 to 300 PA and a tan Delta (G"/G') of from 0.1 to 0.8 in dynamic viscoelasticity measured by the rheometer, under the following conditions: measuring method, oscillation-test method, the load control; temperature measuring 37oC; cone size 4 cm; clearance of 800 μm and a frequency of 10 Hz, which is a hydrogel polymer having a density of mesh from 0.01 to 0.5% to 1 mol Sonia ultraviolet rays photoreactivation derived hyaluronic acid, in which photoreactivation cross-linking group chemically linked to a functional group of hyaluronic acid and is derived from cinnamic acid, containing a spacer selected from a range that includes groups derived from amerosport, amino acids or peptide, these adjacent photoreactivation crosslinking group specified photoreactivation derived hyaluronic acid demonizovana by irradiation with ultraviolet rays with the formation of CYCLOBUTANE ring, with the formation thereby of a mesh structure, and the gel is a hydrogel in such condition that it can be extracted for injection from the container during extrusion.

2. Vodootvodny gel crosslinked hyaluronic acid under item 1, which has a water absorption of from 2000 to 15000%, as defined as follows: water absorption (%) = weight of absorbed water/weight of dried gel x 100%.

3. Vodootvodny gel crosslinked hyaluronic acid in PP. 1 and 2, where this photoreactivation crosslinking group represented by the following formula 1 or 2

-NH(CR1R2)nOCOCH= CH-Ph (1)

where R1and R2each independently represent a hydrogen atom and a is a number from 2 to 18;

-A-NH-Ph-CH= CHCOOR3(2)

where R3is altergroup having from 1 to 8 carbon atoms, or arakalgudu;

A represents - (NHCR4R5CO)m- or-NH(CR4R5)hCO-; R4and R5each independently represents a hydrogen atom or altergroup having from 1 to 8 carbon atoms;

-Ph - refers to a group of paraphenylene;

m denotes an integer from 1 to 6;

h denotes an integer from 1 to 18.

4. Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-3, where the specified photoreactivation crosslinking group introduced in a ratio of from 0.05 to 10% on 1 mol of constituent disaccharide glycosides link.

5. Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-4, where the specified hydrogel is produced by irradiation with ultraviolet rays photoreactivation derivative of hyaluronic acid in water and then heat-treated cross-linked product.

6. Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-4, where the specified hydrogel obtained by heat treatment specified photoreactivation derived hyaluronic acid and then by irradiation with ultraviolet light in voinea conduct further heat treatment.

8. Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-7, where the concentration of endotoxin in the gel is 0.25 endotoxin units (unit e)/g or less.

9. The method of obtaining vodootvedenija gel crosslinked hyaluronic acid on p. 1, including irradiation with ultraviolet rays solution aqueous medium containing from 0.5 to 10 wt. % photoreactivation derived hyaluronic acid, which photoreactivation cross-linking group chemically linked to a functional group of hyaluronic acid and is derived from cinnamic acid, containing a spacer selected from a range that includes groups derived from amerosport, amino acid or peptide; the formation of intermolecular and/or intramolecular crosslinking by dimerization of adjacent photoreactivation cross-linking groups to ensure the mesh.

10. The method of obtaining vodootvedenija gel crosslinked hyaluronic acid on p. 9, where before and/or after irradiation with ultraviolet rays of a specified solution in the aquatic environment photoreactivation derivative of hyaluronic acid are heat treated.

11. The method of obtaining vodootvedenija gel crosslinked hyaluronic is high pressure.

12. Biomedical material containing Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-8.

13. Biomedical material under item 12, which has anti-adhesive effect.

14. Kit biomedical material containing Vodootvodny gel crosslinked hyaluronic acid according to any one of paragraphs. 1-8 and the container comprising a specified gel in such condition that it can be extracted from it.

15. Kit biomedical material on p. 14, where the specified container is a container that can squeeze the specified gel for injection.

 

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