Vodootvedenie derived glycosaminoglycans and collagen, crosslinked derived glycosaminoglycans and collagen, and methods for their preparation, composition for medical use, the way to prevent cell and tissue adhesion

 

(57) Abstract:

Vodootvedenie derived fractions having a molecular weight 4000-2000000, can be represented by one of formulas: gag-O-CO-R1, gag-OCO-R3-NH-CO-R1, gag-O-CO-R3-O-CO-R1, gag-CO-O-R3-NH-CO-R1, gag-CO-NH-R3-O-CO-R1, gag-CO-NH-R3-NH-CO-R1, gag-CO-O-R1, gag-O-CO-R3-CO-O-R1, gag-CO-O-R3-CO-O-R1, gag-CO-NH-R3-CO-O-R1where gag-O, gag-CO means glycosaminoglycans part of glycosaminoglycan selected from hialuronowy acid, chondroitin, chondroitin sulphate, dermatosurgery, heparin, heparansulfate, keratocanthoma, keratoprosthesis, kolominova acid, or derivatives thereof, R1- photoreactive group having at least one double-bond, capable of forming cyclobutanone ring using the expression; R3-(CH2)n-, -(CH2)pCHY-, -(CH2)m-C6H4-(CH2)l-, where l, m, n, p is a number from 1 to 10, Y is COOH, NH2have a high level of safety and biocompatibility. 8 C. and 7 C.p. f-crystals, 30 tab., 10 table.

The invention relates to fototermin derived fractions, each of which receive chemical binding, is sewn to glycosaminoglycans having a three-dimensional netted structure which is obtained by exposing the above derived expression for the dimerization photoreactive compounds, to processes for their preparation, and, further, to a satisfactory biocompatible materials for medical use that contain them.

Fototerapia resin containing a hydrophobic polymer systems that can be photodimerizable for knitting, still used in lithography, in paints and printing, along with others. In contrast, there are only a few known examples of the implementation of fotoschule hydrophilic polymers.

On the other hand, attempts were made to sew Dadi, who are usually hydrophilic polymers, with aldehydes, epoxy compounds, diphenylsulfone connections, etc. for extension of validity GAG in vivo or to obtain material for medical use in the form of films or powders, for example, to prevent adhesion of the tissue. However, since Dadi are macromolecules made GAG derivatives have a higher molecular weight, and this fact complicates the complete removal of unreacted materials and/or catalysts from cross-linked to harmful effects, and therefore they are not suitable for practical use. In addition, crosslinked GAG derivatives exist as gels or solids, and therefore they are difficult to mould after crosslinking, and therefore they are not suitable for practical purposes. Moreover, with regard to their use as carriers in drugs with a slow or controlled release JP-A-62-129226/ corresponding U.S. patent 5128326/; the term "JP-A" in the sense used here, refers to unexamined published Japanese patent application/, slow release of the active ingredients can only be achieved by taking advantage of the viscosity of the crosslinked GAG derivatives, and this lack makes them unsuitable for practical use. Thus, such methods and sewn GAG derivatives can hardly control the rate of release of drugs.

It is also known that photosensitive materials obtained in the esterification of hydroxyl groups of polysaccharides obtained by microorganisms or plants, such as pullulan, amylose and mannan, cinnamonny groups that are photodimerizable functional groups, as applicable adsorbentami applications /JP-B-56-14969/ corresponds to U.S. patent 3960685 /the term JP-B means the last examination published Japanese patent application/ JP-A-60-219202/.

However, the above photosensitive materials have problems from the point of view of security can hardly control of cell adhesion and have poor biocompatibility in the human body, and therefore not suitable for use as medical materials for direct use in living organisms, in particular, as artificial organs, medical devices, intended to cover wounds or surgery, media, pharmaceutical products, or other materials intended for a method of medical treatment.

Accordingly, the present invention is the creation of fototerapia glycosaminoglycans derivatives and derived crosslinked glycosaminoglycans with high safety and biocompatibility.

Another objective of the present invention is to provide a method of producing fototerapia derived glycosaminoglycans and collagen, which are easily formed, if desired, for example, by casting in a solvent, and in this way you can easily eliminate unreacted substances that can cause harmful side effects, and the manner of obtaining them from Schine, based on the above curable derived GAG or crosslinked GAG and can be widely used for different purposes.

The fourth purpose of the present invention is to provide a non-adhesive materials containing stitched glycosaminoglycan that do not adhere to tissues, but are biodegradable in accordance with the speed of colonization of the Russian Academy of Sciences, and mechanical strength which can be easily adjusted in accordance with the mechanical loads in the place of their use, and non-adhesive materials containing vodootvedenie derived glycosaminoglycans and collagen, in turn sewn of glycosaminoglycan when exposed to light in vivo.

The fifth purpose of the present invention is to provide materials or preparations for the implementation of controlled release drugs that contain custom made of glycosaminoglycan, and ensure the release of drugs with the speed required for this includes medication, prisoner or disseminated in this material, and for the purposes of applying this medicine, as well as materials to carry out a controlled release of drugs, which includes phototherapy of glycosaminoglycan, and that may be the La materials.

The present invention provides fototerapia derived glycosaminoglycans and collagen /hereinafter sometimes referred to for brevity "fototerapia GAG/ that contain glycosaminoglycan and photoreactive connection associated with it, and sewed glycosaminoglycan /hereinafter sometimes for the sake of brevity referred to as "crosslinked GAG/ obtained by introducing a specified fototerapia GAG in response matching the specified photoreactive connection. Specified phototherapy GAG can preferably be obtained by the etherification of hydroxyl or carboxyl groups fractions photoreactivation connection, activation of the carboxyl groups fractions and subjecting the activated carboxyl group of the amidation reaction with photoreactivation connection or subjecting the carboxyl group of glycosaminoglycans and collagen the amidation reaction with photoreactivation compound in the presence of a condensing agent. Sewn of glycosaminoglycan can be obtained by irradiating phototherapy GAG light, thus causing a crosslinking reaction fragments photoreactivation connection with each other. Materials on the basis of these crosslinked GAG suitable for use in honey for use in medicine.

A brief description of the drawings.

Fig. 1 is1H-NMR spectrum, which is used in example 1 for the quantitative determination of bound brown acid.

Fig. 2 is a UV /visible spectrum, illustrating the decrease of absorption at 279 nm due to exposure to light is obtained in the example 1 film crosslinked hyaluronic acid.

Fig. 3 graphically represents the dependence of contact angle on the degree of substitution /DS/ cinnamic acid residues among different films crosslinked hyaluronic acid.

Fig. 4 graphically depicts the dependence of the ability to swell from DS residue of cinnamic acid among different films crosslinked hyaluronic acid.

Fig. 5 is a pictures of cell morphology, which illustrate the differences in the adhesion of endothelial cells, as a result of differences in DS due to the remnants of cinnamic acid among films crosslinked hyaluronic acid.

Fig. 6 graphically represents the degree of gelation /%/ when vodootvedenie of ester chondroitin sulfate-cinnamic acid /lot CS-Cin-2/ his concentration in his PBS solution.

Fig. 7 graphically represents the dependence of hardness flat surface is t to the exposure time to the light of ester chondroitin sulfate-cinnamic acid /lot CS-Cin-3/ PBS.

Fig. 8 graphically represents the degree of gilotinirovaniya when gelation crosslinked chondroitin sulphate and the ability to swell from DS at the expense of the rest of cinnamic acid.

Fig. 9 graphically represents the dependence of the ability to swell from DS films crosslinked chondroitin sulphate.

Fig. 10 graphically represents the dependence of the contact angle from DS residue of cinnamic acid in films crosslinked chondroitin sulphate, obtained in example 8.

Fig. 11 graphically represents the dependence of the percentage of education timedemobog dimer from the time of exposure to UV/UV/ in the process of film forming crosslinked hyaluronic acid during exposure of ON-Thym-I UV rays in example 14.

Fig. 12 graphically represents the relationship between the molar ratio of 1-/2-carboxyethyl/thymine/ HA and DS in the synthesis of HA-Thym in example 16.

Fig. 13 graphically represents the relationship between the molar ratio of 1-/2-carboxyethyl/thymine/CS and DS in the synthesis of CS-Thym in example 17.

Fig. 14 graphically represents the relationship between the change in absorption at 270 nm of thymine and the time of irradiation with the exposure light films CS-Thym in example 18.

Fig. 15 graphically represents the relationship between the degree rela graphically represents the relationship between the degree of gilotinirovaniya /%/ and the time of irradiation with the exposure light another thin film CS-Thym in example 18.

Fig. 17 graphically represents the relationship between the ability to swell and the time of irradiation with the exposure light thin film HA-Thym in example 18.

Fig. 18 graphically represents the relationship between the ability to swell and DS for thin films HA-Thym before and after exposing them to light in example 18.

Fig. 19 is a1H NMR spectrum of ester chondroitin sulfate-/7-coumarelos/acetic acid obtained in example 20.

Fig. 20 graphically represents the relationship between the molar ratio chloranhydride forms of coumarin/chondroitinsulfate hydroxyl groups /CS - OH/ and DS in the resulting composite air.

Fig. 21 graphically depicts the relationship between changes in the absorption at 320 nm coumarin and time of irradiation with the exposing light of a thin film of ester chondroitin sulfate-/7-coumarelos/acetic acid in example 21.

Fig. 22 graphically depicts the relationship between the ability to swell and the time of irradiation with the exposing light of a thin film of ester chondroitin sulfate/7 coumarelos/acetic acid in example 21.

Fig. 23 graphically represents the relationship between the ability to swell and DS at exhibiting light thin the RCM represents the relationship between the degree of gilotinirovaniya /%/ and the time of irradiation with the exposing light of a thin film of ester chondroitin sulfate-/7-coumarelos/acetic acid in example 21.

Fig. 25 graphically represents the relationship between the degree of gilotinirovaniya /%/ and the concentration of ester chondroitin sulfate-/7-coumarelos/acetic acid in the exposed light thin films containing a specified ester in example 21.

Fig. 26 graphically represents the rate of release of the drug from cross-linked under the action of light film HA-Thym /DS = 0,7/ content drug 10% or 50%, as described in example 28, depending on time, and this rate is expressed in units of change of absorption at 269 nm.

Fig. 27 graphically represents the rate of release of drug from the film HA-Thym, crosslinked under the action of light /DS = 2,2/ content medications 10% or 50%, as defined in example 28, depending on time, and this rate is expressed in units of change of absorption at 269 nm.

Fig. 28 graphically represents the rate of drug release from film photolitho CS-Thym /DS = 0,8/ content medications 10% or 50%, as described in example 28, depending on time and the specified speed is expressed in units of change of absorption at 269 nm.

Fig. 29 graphically represents the rate of release preparations is, the time-dependent, and the rate is expressed in units of change of absorption at 269 nm.

Fig. 30 graphically represents the rate of drug release from film photolitho CS-Thym /DS = 1,8/ content medications 10% or 50%, as described in example 28, depending on time, and this rate is expressed in units of change of absorption at 269 nm.

The term "material for use in medicine, in the framework of the present invention includes, among others, the materials for the manufacture of medical devices used for diagnostic or therapeutic purposes, or for artificial organs, artificial blood vessels, artificial heart, artificial leather and so on, the materials constituting the coating for wounds, dentures, non-adhesive materials or devices for controlled release of drugs, and artificial extracellular matrix or artificial membranes that can be used for adhesion and multiplication of endothelial cells, epithelial cells and smooth muscle cells in the formation of hybrid artificial organs.

Phototherapy GAG present invention preferably has a partial G. Under each of these formulas is the appropriate type of reaction, where the gag means a partial structure of any fractions, R1is photoreactive group /a group containing at least vanilinovoi group/, and R1-COOH, R1-NH2and R1-OH are each photoreactive connection.

[A] gag-O-CO-R1) [1]

gag-OH + R1-CCOH ---> essential binding ---> [1]

[B] gag-CO-(NH-R1) [2]

gag-COOH + R1-NH2---> amide linking ---> [2]

[C] gag-CO-(O-R1) [3]

gag-COOH + R1-OH ---> essential binding ---> [3]

[D] gag-O-R2a-R1[4]

R2ais a group derived from a spacer capable of reacting with gag-OH /for example, HOOC-R3-COOH, HOOC-R3-NH2/ and the functional group photoreactive connection.

[D-1] gag-OH + HOOC-R3-COOH,- - - > gag-O-CO-R3-COOH

[D-1-c] gag-O-CO-R3-COOH + R1-OH ---> gag-O-CO-R3-CO-O-R1= [4a]

[D-1-b] gag-O-CO-R3-COOH + R1-NH2---> gag-O-CO-R3-CO-NH-R1= [4b]

[D-2] gag-OH + HOOC-R3-NH2---> gag-O-CO-R3-NH2< / BR>
[D-2-a] gag-O-CO-R3-NH2+ R1-COOH,- - - > gag-O-CO-R3-NH-CO-R1= [4c]

[E] gag-CO-R2bR1[5]

R2bis the gr2H2N-R3-NH2H2N-R3-COOH/ and functional group photoreactive connection.

[E-0]

[E-0-a] N2N-R3-NH2+ R1-COOH,- - - > H2N-R1-NH-CO-R1< / BR>
gag-COOH oC H2N-R3-NH-CO-R1[F-1] ---> gag-CO-NH-R3-NH-CO-R1= [5-1]

[E-0-b] RNH-R3-NH2+ R1-COOH,- - - > RNH-R3-NH-CO-R1[F-2]

RNH-R3-NH-CO-R1[F-2]oC H2N-R3-NH-CO-R1< / BR>
gag-COOHoC H2N-R3-NH-CO-R1[F-1] or

[F-2]

---> gag-CO-NH-R3-NH-CO-R1= [5-1]

[F-2] gag-COOHoC HO R3-COOH,- - - > gag-CO-O-R3-COOH

[E-1-a] gag-CO-O-R3-COOHoC R1-OH ---> gag-CO-O-R3-CO-O-R1= [5a]

[E-1-6] gag-CO-O-R3-COOHoC R1-NH2---> gag-CO-O-R3-CO-NH-R1= [5c]

[E-2] gag-COOHoC HO R3-OH ---> gag-CO-O-R3-OH

[E-2-a] gag-CO-O-R3-OHoC R1-COOH = gag-CO-O-R3-O-CO-R1= [5c]

[E-3] gag-COOHoC HO R3-NH2---> gag-CO-O-R3-NH2< / BR>
[E-3-a] gag-CO-O-R3-NH2oC R1-COOH,- - - > gag-CO-O-R3-NH-CO-R1= [5d]

[E-4] gag-COOH + H2N-R3-OH ---> gag-CO-NH-R3-OH

[E-4-a] gag-CO-NH-R3-OH + R1-COOH,- - - > gag-CO-NH-R3-O-CO-R1= [5e]

[E-5] gag-COOH + H1-NH-CO-R1= [5f]

[E-6] gag-COOH + H2N-R1-COOH,- - - > gag-CO-NH-R3-COOH

[E-6-a] gag-CO-NH-R3-COOH + R1-OH ---> gag-CO-NH-R3-CO-O-R1= [5g]

[E-6-b] gag-CO-NH-R3-COOH + R1-NH2---> gag-CO-NH-R3-CO-NH-R1= [5h]

R3has preferably one of the following values:

R3a: -/CH2/n- /n = 1-10/;

R3b: -/CH2/pGHY - /Y is COOH or NH2; and p = 1-10/;

R3c: -/CH2/m- C6H4- /CH2/l- /m = 1-10, l = 1-10/.

R3has preferably one of the following values:

R3a: -/CH2/n- /n = 1-10/;

R3b: -/CH2/pGHY - /Y is COOH or NH2and p = 1 to 10/;

R3c: /CH2/m-C6H4-/CH2/n- /m = 1-10, l = 1-10/.

Although in the above examples was used only one spacer, you can use a variety of spacers.

The above photoreactive connection /F - 1/, or /F - 2/ can be used instead of R1- NH2upon receipt of the product formula /4b/, /5b/ or /5h/.

In principle, R1that is photoreactive group can be any group, provided that it is able at least to temeritate

In each of the above formula-COOH may take the form of a reactive derivative of /for example, halide, carboxylic acid anhydride, active ether complex/ capable of reacting with a hydroxyl or amino group.

As partially preferred examples of R1-CO-occurring in formulas /1/, /4c/, /5c/ - /5f/ and so forth, you can specify the group of the following formula:

< / BR>
where R4and R5may be the same or different and each is a hydrogen atom, or lower alkyl, or lower alkyloxy, or a nitro-group, or amino group;

< / BR>
where R6is a hydrogen or halogen atom, or lower alkyl, or halogen-nishiuchi group, R7is a hydrogen or halogen atom, or cyano, carboxyla, lower alkoxycarbonyl, lower alkyl or the group, halogen-lower alkyl, and R8is the lowest alkalinous group;

< / BR>
where R9, R10and R11may be the same or different and each independently is a hydrogen atom or a group of lower alkyl, and R12is a group of lower alkylene.

Groups of the above formulas /6/ - /8/ are the most preferred when R1a-CO- /and R1a3aor R3bpreferred as R3. If R3is R3bas Y is preferably COOH.

As the preferred examples of R1-O-, occurring in formulas /3/, /4/, /5/, /5g/ and so forth, you can specify the remains of the following formulas:

< / BR>
where R13is a hydrogen or halogen atom, or lower alkyl, or the group of halogen-lower alkyl, R14is a hydrogen or halogen atom, or cyano, carboxyla, lower alkoxycarbonyl, lower alkyl or the group, halogen-lower alkyl, and R15is a group of lower alkylene;

< / BR>
where R16and R17may be the same or different and each is a hydrogen atom, or lower alkyl, or lower alkoxy, or nitro, or amino group.

Groups represented by the formulas above, for example, can also be used as elements of R1:

< / BR>
Phototherapy GAG by the method of the present invention is capable of intermolecular dimerization during exposure to light, resulting in photoreactive groups i.e. groups containing at least a vinyl group/ contained in the molecules photoreactive connection form p is leaving a two - or three-dimensional mesh structure with the desired degree of crosslinking. In the process of fototerapia may also be included intramolecular dimerization

< / BR>
Thus, in accordance with the present invention biocompatible photosite GAG with the desired degree of crosslinking, and therefore, with the desired physical properties /for example, the mechanical property retention patterns, the ability to retain water, hydrophilicity, lubricity, the rate of release of drugs/ and with the desired biological properties /for example, cell adhesion, the ability to biodegrade - it absorbs, and physiological activity/, which correspond to their intended use, can be obtained by selecting or regulation photoreactive connection and GAG /for example, chemical structure, molecular weight and so on/ and content photoreactive connection /degree of substitution /DS/ photoreactive connection/ along with the others.

The term "degree of substitution /DS/", as used here, is defined as the number of moles photoreactive compounds or groups associated with each of the repeating disaccharide glycosides fragment each fragment contains uronic acid and hexosamine/ GAG. So, for example, hyaluronic acid contains four hydroxyl groups substituted hydroxylase hydroxyl groups gives DS = 4, while the three hydroxyl groups on the disaccharide glycosides fragment of chondroitin sulfate, after its modification photoreactive connection or group, give DS = 3.

The term "glycosaminoglycan" or "GAG" in the sense here used, includes clomidbuy acid, hyaluronic acid /HA/, chondroitin, chondroitin sulfate /CS/, teichuronic acid, dermatooncology, heparin, heparansulfate, keratolytic, Kermanshah, keratohyalin, chitin, chitosan and their derivatives /acylphosphate, polysulfate, products desulfatation, products diallylamine and so on /. The term "lower" means that the carbon chain may be branched or unbranched, and consists of 1 to 6 carbon atoms. The term "halogen atom" includes chlorine, bromine, fluorine and iodine, and the term "halogen" is used to indicate that one or more of the hydrogen atoms has been replaced by such atoms of Halogens.

In the combinations formula /6/ formula /1/, /4c/, /5-1/, /5c/, /5f/ etc., R1CO - is, for example, a residue derived from a substituted or unsubstituted cinnamic acid or its reactive derivative. As the substituted residue of cinnamic acid can be mentioned residues, which contain one or two nauczanie of cinnamic acid, is the most preferred.

In the combinations formula /7/ formula /1/, /4c/, /5-1/, /5c/ - /5f/ etc., R1CO - is, for example, a residue derived from brazilero derivative containing carboxialkilnuyu group as a substituent in position 1, or its reactive derivative, with a hydrogen or halogen atom, or lower alkyl, or the group of halogen-lower alkyl represented by R6in position 5 of the pyrimidine ring, a hydrogen or halogen atom, or cyano, carboxyl, lower alkoxycarbonyl, lower alkyl group or halogen-lower alkyl represented by R7in position 6, and the lower alkalinous group, R8obtained from carboxialkilnuyu group, and forming ester bond with a hydroxyl group oao in position 1. In particular, preferred is R1CO-, derived from the 1-/2-carboxyethyl/thymine.

In combination formulas /8/ formula /1/, /4c/, /5-1/, /5c/ - /5f/ etc., R1CO - is, for example, a residue derived from 7-carboxylicacid-substituted derivative of coumarin or its reactive derivative, and the provisions of articles 5, 6 and 8 of the coumarin ring each independently is a hydrogen atom or a lower alkyl group, afirmou bond with a hydroxyl group oao in position 7. The most preferred R1CO-group is obtained from 7-comparisonvalue acid.

In the combinations formula /10/ formulas /3/, /4/, /5/, /5g/ etc., R1O - is a residue obtained from 1-hydroxyalkyl-substituted brazilero derivative or its reactive derivative, with a hydrogen or halogen atom, or lower alkyl, or the group of halogen-lower alkyl represented by R13in position 5 of the pyrimidine ring, a hydrogen or halogen atom, or cyano, carboxyla, lower alkoxycarbonyl, lower alkyl or the group, halogen-lower alkyl represented by R14in position 6, and the lower alkalinous group, R15derived from a hydroxyalkyl group, and forming an ester bond with a carboxyl group oao in position 1.

In the combinations formula /11/ formula /3/, /4/, /5/, /5g/ etc., R1O - is, for example, a residue derived from a substituted or unsubstituted cinnamic alcohol or its reactive derivative. The remainder of substituted cinnamic alcohol is, for example, one or two lower alkyl, lower alkoxy-, nitro - or amino-groups in any position of the benzene ring.

The source connection

GA application and use either separately, either in the form of mixtures of two or more of them. Although GAG of natural origin, obtained as extracts of organs of animals, products of fermentation, etc. typically use GAG chemically or enzymatically synthesized or synthesized GAG synthetic methods can also be used for the products derived from them by modifying functional groups.

GAG can lead to interaction with photoreactive connection as it is, but it is usually used in the form of salts of alkaline metal such as sodium, potassium, etc., alkaline earth metal, such as magnesium, calcium, etc., tertiary amine, such as tri-n-butylamine, triethylamine, pyridine, etc. etc.

If photoreactive connection that is subject to binding to GAG, not soluble in water, it photoreactive compound is administered in an organic solvent containing GAG for the reaction.

An organic solvent containing GAG, get, for example, treating an aqueous solution of sodium salt GAG kationoobmennikom to obtain a carboxyl radical and sulfate radical, adding to this mix with water, an organic solvent, for example, dimethylformamide /DMF), dimethylsulfoxide is Oli amine GAG and removing water by distillation.

If photoreactive compound is soluble in water, the reaction can be conducted by adding photoreactive connection to aqueous solution GAG /for example, in the form of sodium salt/.

Photoreactive connection

Photoreactive compounds, which form connections with such groups as-OH, -COOH or-NH2GAG or spacer connected or able to contact GAG, as mentioned earlier, have groups such as-OH, -COOH or-NH2.

/1/ If-COOH is a functional group:

Photoreactive connection associated with GAG or with spacer elements groups in the formation of ester in accordance with formulas /1/, /4c/, /5-1/, /5c/, /5d/, /5e/ 5f/ and include compounds with chemical structures in which OH or halogen, for example, is associated with groups of formulas /6/ - /8/, namely substituted or unsubstituted cinnamic acid, 1-carboxyethylidene uracile and 7 carboxyethylidene coumarins /7-Kumaritashvili acids/ and their reactive derivatives. As reactive derivatives can specify galodamadruga /for example, the acid chloride and so on/ and the anhydrides of the acids.

Galodamadruga above 7-comparisonstrojan acids can poluciti acid in the presence of alkali and gidrolizu obtained ester 7-Kumaritashvili acid to obtain 7-Kumaritashvili acid or condensing the corresponding optionally substituted 7-hydroxycoumarin with malodorous acid to obtain 7-Kumaritashvili acid and then interacting thus obtained 7-Kumaritashvili acid corresponding to the formula /8/, which may not necessarily be the Deputy or deputies, with toniguy.com /see, for example, JP-A-3-48674/.

/2/ If-NH2is a functional group:

Photoreactive compounds of the formula /G/, below, can be obtained by the interaction of the above-mentioned substituted or unsubstituted cinnamic acid, 1-carboxyethylidene of orallow or 7-carboxylicacid-substituted coumarins /7-comparisonstrojan of acids and/ or their reactive derivative with the compound of the formula /X-1/, or connection /X-2/, obtained from it by protecting one of the amino groups such aminosidine group as tert.-butoxycarbonyl or benzyloxycarbonyl, with the subsequent removal of the protection if necessary.

H2N-R3-NH2/x-1/

RNH-R3-NH2/x-2/

H2N-R3-NH-CO-R1/F-1/

RNH-R3-NH-CO-R' /F-2/

/3/ If-OH is the functional group:

Reactive derivatives corresponding to the formula /9/ can be used as photoreactive compounds. So, for example, compounds of cyclic esters between positions 1 and 2 of the pyrimidine ring can be used. Specific examples include 1,2-O-ethnoracial, 1,2-O-etnoteam,2-O-ethano-6-trichlorotoluene.

You can also specify a substituted or unsubstituted cinnamic alcohols corresponding to the formula /10/.

/4/ the Preferred photoreactive connection

Although to be used photoreactive connection you should choose among the above, in accordance with the intended use, preferable from a medical point of view are photoreactive compounds derived from compounds that have the least possible adverse effect, even if crosslinked GAG remain in the unreacted form, for example, cinnamic acid, thymine and coumarin.

In the practice of the present invention fototerapia GAG can contain one or more of photoreactive connections associated with the same GAG molecule, or with many different GAG molecules. This applies to the above formulas /1/ - /3/. Therefore, it should be noted that the crosslinked GAG present invention include, within their values, products derived from fotoschule such fototerapia GAG.

Reaction injection photoreactive groups

/1/ the Reaction of esterification with gag-OH

The corresponding salt of the tertiary amine (for example, salt is tri-n-butylamine salt of triethylamine salt of pyridine, etc./ GAG dissolved in with the work of esterification of substituted or unsubstituted cinnamic acid partial structure of the formula /6/ in the amount of from 0.5 to 5 mol per mol of hydroxyl groups of the GAG or its reactive derivative, for example halogenerator, in the presence of a basic catalyst (for example, anhydrous pyridine, etc., or R1COOH partial structure of the formula /7/ or /8/ or their reactive derivatives in the presence of a basic catalyst (for example, 2-chloro-1-methylpyridinium iodide, pyridine and so on/ at a temperature of from 0 to 100oC, preferably from 70 to 90oC for from several tens minutes to several tens of hours and preferably from 1 to 10 hours, to obtain vodootvedenija GAG of the present invention.

The degree of substitution /DS/ each fotootdela GAG can be adjusted, if desired, by changing the reaction conditions. For example, the value of DS can be increased by increasing the molar ratio of R1COOH or its reactive derivative with respect to the initial GAG and/or increasing the reaction time.

After the reaction, to the reaction mixture add ethanol saturated with sodium acetate, ethanol or methanol and the precipitate is collected by filtration, washed with ethanol or methanol, and then dried under reduced pressure, resulting in a gain target product as a white powder.

The reaction for obtaining compounds of formulas /5c/ or /5e/ you can maintain almost fototerapia GAG, in which each disaccharide glycosides fragment substituted by one molecule photoreactive compounds are presented in the following structural formulas fragments. Structural differences that make up the whole of the fragments, for example, fragments that differ by the number of associated photoreactive groups, and/or provisions, which are binding, can occur in the same molecule.

1/ Phototherapy GAG /HA-Cin/, obtained with the introduction of cinnamic acid in hyaluronic acid

< / BR>
2/ Phototherapy GAG /HA-Thym/, obtained by the introduction of 1-/2-carboxyethyl/thymine in hyaluronic acid

< / BR>
3/ Phototherapy GAG, obtained with the introduction of 7-comparisonvalue acid chondroitin sulfate

< / BR>
/2/ the Amidation using gag-COOH /in particular when photoreactive connection spacer elements associated with the group; for example, the formula /5-1/

/2-1/: If R3= R3a< / BR>
If the connection formula /X-1/, or /X-2/ is alkylenediamines /for example, Ethylenediamine/ corresponding salt of tertiary amine (for example, salt is tri-n-butylamine salt of triethylamine salt of pyridine, etc./ GAG dissolved in an appropriate solvent (for example, DMF, pyridine, DMSO, HMPA etc. /, as shown in method /1/, then reviewsin the number of moles with respect to the number of carboxyl groups in GAG, and condensing agent (for example, dicyclohexylcarbodiimide, 1-ethyl-3-/dimethylaminopropyl/carbodiimide and so on/ and the reaction is conducted at temperatures from 0 to 50oC for 1 to 20 hours to obtain a modified GAG activated by its carboxyl groups. Then carry out the amidation reaction at a temperature of from 0 to 50oC for from 30 minutes to 20 hours between modified so GAG and amino group of a compound of the formula /F-1/, or /F-2/ to get vodootvedenija GAG.

Fototerapia GAG can also be obtained using an aqueous solution GAG and carrying out the amidation reaction between the compound of the formula /F-1/, or /F-2/ and carboxyl groups GAG at a temperature of from 0 to 50oC for 1 to 20 hours in the presence of a condensing agent (for example, such a water-soluble carbodiimide, 1-ethyl-3-/dimethylaminopropyl/carbodiimide/.

/2-2/: If R3= R3bY = COOH/:

If the connection formula /X-1/, or /X-2/ is, for example, basic amino acid /for example, L-lysine/, GAG used in the form of an aqueous solution and carry out the amidation reaction between the amino groups of the compounds of formula /F-1/, or /F-2/ and carboxyl groups GAG at a temperature of from 0 to 50oC in Pris is ropel/carbodiimide/ to obtain vodootvedenija GAG.

Fototerapia GAG can also be obtained when carrying out the reaction between GAG in the form of a salt of the corresponding tertiary amine and hydroxyl compounds, such as N-hydroxycinnamic, in a suitable solvent such as DMF in the presence of such a condensing agent, as dicyclohexylcarbodiimide as in method /2-1/ and subjecting the obtained GAG activated by its carboxyl groups, the amidation reaction with amino compounds of the formula /F-1/, or /F-2/.

The reaction described in formulas /2/, /4b,/, /5b/ or /5h/, you can spend almost the same way as mentioned previously.

/3/ Etherification using gag-COOH

Similarly, the above method /1/, the corresponding salt of the tertiary amine (for example, salt is tri-n-butylamine,

salt of triethylamine salt of pyridine, etc./ GAG dissolved in a suitable solvent (for example, DMF, pyridine, DMSO, HMPA and so on/ and carry out the esterification reaction between the reactive derivative brazilero derived formula /9/, for example, cyclic ether, or a substituted or unsubstituted cinnamic alcohol of the formula /10/, or its reactive derivative and the carboxyl groups GAG at a temperature of from 0 to 100oC in the presence/4a/, /5a/ or /5g/, you can maintain almost the same way as shown previously.

Isolation and purification of fototerapia GAG

After the above reactions generated fototerapia GAG can be extracted and cleaned using any of the methods commonly used for isolation and purification GAG, without any restrictions.

For example, fototerapia GAG, namely the target products can be distinguished from unreacted GAG or unreacted photoreactive compounds, and can be cleaned by means of chromatography using anionic or cationic ion exchanger, the method that uses the difference in solubility in organic solvents such as, by way of deposition of alcohol by salting out or by dialysis.

Although such crosslinked glycosaminoglycans, which, as we all know exist in the form of gels or solids, and therefore, unreacted materials, catalysts, impurity microorganisms, pyrogens and other impurities are extracted from them with great difficulty, fototerapia GAG of the present invention are soluble in water and/or organic solvents and are therefore easy to clean. As such, peeled fototerapia GAG when the exposure you can get stitched GAG minimally contaminated with unreacted materials, catalysts, contaminating microorganisms, pyrogens, etc.

Physical properties fototerapia GAG

Physical properties fototerapia GAG, obtained and purified in accordance with the above, may vary depending on the GAG, used as source material, its molecular weight, used photoreactive connections, quantity and other factors, and can be adjusted, if desired, so that they were suitable for the purposes intended. They usually have a molecular weight in the range from 4000 up to 2,000,000, preferably from 10000 to 1000000, and the degree of substitution /DS/ photoreactive groups is in the range of values from 0.1 to 4.0, preferably from 0.1 to 3.0, and they are soluble in water and/or organic solvents. If desired, the solubility can be adjusted. Typically, an increase DS leads to lower solubility in water, but to increase the solubility in such organic solvents as DMF. If photoreactive group is a cinnamic acid derivative of the formula /6/, 7-Kumaritashvili acid formula /8/ or a derivative of cinnamic alcohol of the formula /11/ received fototerapia GAG are relatively hydrophobic. In contrast, e is /7/ or /10/, get phototherapy GAG is relatively hydrophilic.

The preferred interval of DS values may vary depending on the intended use vodootvedenija GAG or crosslinked GAG and GAG used, and photoreactive compounds. To obtain crosslinked GAG not sticking to the cells and use them as material not adhesive to the tissue, for example, the value of DS should preferably be from about 0.1 to 0.5 in the case of esters hialuronowy acid-cinnamic acid /HA-Cin/, from about 0.1 to 3.0 in the case of esters chondroitin sulfate-cinnamic acid /CS-Cin/, from about 0.2 to 1.0 in the case of esters hialuronowy acid derivatives of thymine and from about 0.2 to about 1.0 in the case of ester chondroitinsulfat-derived thymine /CS-Thym/. To enable biological substances, medicines or the like in a three-dimensional netted structure crosslinked GAG and achievements due to this controlled-release preferred DS values from 1.0 to 2.5 for each of the crosslinked GAG listed above.

More specifically, if phototherapy GAG is derived hialuronowy acid formula /1/, the molecular weight is preferably in the range ZnO,1 to 3.0. If phototherapy GAG is fototermin derivative of chondroitin sulfate formula /1/, the molecular weight is preferably in the range from 10000 to 60000 and DS in the range from 0.1 to 0.3.

As medical materials fototerapia GAG and sewn GAG can be used in any form. So, they can be used in various forms, for example, in the form of solutions, gels, solids, etc. Medical materials of the present invention, which is based on the specified fototerapia GAG or crosslinked GAG, can contain various solvents such as water, buffer, PBS, DMF, DMSO, etc./ media /for example, gauze, knitted or woven fabric, non-woven cloth, cotton or chlopromazine materials, thread or yarn, film, porous sponges, resins, plastics, metals, artificial organs, the surface tissues of living organisms, including wounds of living bodies, biological substances /collagen, gelatin, heparin, chondroitin sulfate, hyaluronic acid, dermatologit etc./, medicinal substances, and so forth.

Forming fototerapia GAG /casting from a solvent/ etc.

For use as medical materials fototerapia GAG can Otley is or can be incorporated into other substances or materials, as noted previously. In these cases fototerapia GAG can be cast prior to the expression, dissolving them in water /preferably purified water/ buffer solution /for example, phosphate buffer, carbonate buffer and/ or in an organic solvent (for example, DMF, DMSO/ purity acceptable for medicine, placing or distributing the solution on the surface of the flat plate in a container or the like, made of glass, quartz, polyvinyl chloride, polystyrene, polyurethane or the like/, drying it with an air blower or else to obtain a film thickness of 1 μm to 1 mm

Obtaining crosslinked GAG /photoreactive/

Photosite GAG can be obtained by exposing phototherapy GAG, formed in the above manner, or in the form of solutions, activating rays or radiation to fotoschule in order to cause the reaction photodimerization. Used wavelength or wavelength interval may vary depending on the nature photoreactive groups, but typically lies in the range of from about 260 to 400 nm. More specifically, you can use the light from mercury lamps high pressure /450 W/ exposure /exposure/, with clip light with shorter lengths VLN, temperature and distance from the light source, the preferred conditions under which the reaction may be carried out prior to completion in 30 minutes. The degree of gilotinirovaniya /cure/ can be adjusted by setting the concentration vodootvedenija GAG at any given time of expression. Usually the specified concentration is from 1 to 30% and preferably from about 2 to 20%.

Specific examples of dimeric structures formed as a result of expression fototerapia GAG, the following:

1/ If photoreactive compound is cinnamic acid

< / BR>
2/ If photoreactive compound is 1-/2-carboxyethyl-thymine

< / BR>
3/ If photoreactive compound is 7-numericana acid

< / BR>
Physical properties of crosslinked GAG

Physical characteristics of the thus obtained crosslinked GAG present invention can be adjusted by such factors as the nature of the GAG, nature photoreactive groups of compounds, the concentration vodootvedenija GAG, the degree of substitution /DS/ photoreactive connection, the degree of crosslinking and time of expression. However, they usually show the ability to swell /defined as the weight in the swelled state, minus the weight in the dry state and defined what Icesave in a certain interval.

The ability to swell decreases with increasing the degree of crosslinking and DS due photoreactive groups connection.

They also exhibit a contact angle with water in a certain interval from 10 to 100o/. The contact angle reflects the hydrophobicity /hydrophilicity of the surface crosslinked GAG/. The increase in the rate of crosslinking due to the dimerization photoreactive group leads to an increase in contact angle, that is, to increase the hydrophobicity.

The degree of crosslinking, which is the percentage with which the above photoreactive group undergoes dimerization, can be raised by increasing DS. It can further be adjusted by controlling photoreactive.

Therefore, the biological function of crosslinked GAG can be adjusted by adjusting the degree of crosslinking. For example, these cells, like endothelial cells, tend to stick with crosslinked GAG, demonstrating a high degree of crosslinking, although this trend may differ depending on the nature of the GAG and photoreactive connection. This trend is noticeable for hyaluronic acid, which does not contain sulfate groups /see photo listed in the examples section/, although this trend is less for sulfidogenic GAG /maplesea combination of two or more GAG. Further, this property can be used for differentiation, function, intima /the internal floor/ and adventitia artificial blood vessels, due to the cover intima and adventitia various crosslinked GAG different trends to cell adhesion, which is carried out by choosing the GAG and photoreactive groups and regulation of DS and/or the degree of crosslinking. For example, an intim can make the property not to stick with the cells, thus preventing thrombosis, whereas adventitia can make the adhesion property to the cells, thereby providing an opportunity for the fibroblasts to adhere to it and make it impervious to blood.

Use as medical materials

Fototerapia GAG and sewn GAG present invention can be used as medical materials, including not only materials for the manufacture of intima and adventitia artificial blood vessels, as described earlier, but also the materials to create artificial leather, film or non-adhesive materials to the tissues of materials, accelerating wound healing, hybrid artificial organs, artificial extracellular matrices and artificial bases for membranes. In addition, it becomes possible to vkprimer, heparin, dermatosurgical, heparansulfate, an anticancer agent, an anti-inflammatory agent, cytokine, hormone, growth factor/ or this enzyme, as tissue plasminogen activator, superoxide dismutase, urokinase, or similar enzyme and by photoreactive crosslinking solution as it is, or forms derived from it. So, fototerapia GAG and sewn GAG can also be used as carriers for controlled release of a prisoner in medicinal drug.

Some typical applications are more specifically described below.

The non-adhesive materials

Crosslinked GAG present invention can be used as the non-adhesive material to prevent undesired adhesion of surgical wounds and promote healing. So, for example, films crosslinked GAG can be used by covering the abdominal wall intraperitoneal organ or organs such as liver, etc./ this film, which protects the ulcer /defects/ in the peritoneum and thereby prevent adhesion, and accelerate the healing of wounds. This film can be degraded and absorbed as healing wounds.

Fototerapia R RAS or applied on the wound surface, and then irradiating them with light, causing the formation of cross-linked films or membranes in vivo. In particular, this technique can be used to prevent adhesion and/or filling defects in endoscopic operations.

For use as the non-adhesive material sewn GAG includes GAG sewn in vivo/ should have an appropriate level of strength in order to avoid cracking of the film, and resistance to tissue /cell/ adhesion and Biodegradability in accordance with the speed of wound healing; the products of biodegradation must be practically non-toxic, even if they can be absorbed by living organisms.

These functions can be controlled by choosing fototerapia GAG /GAG samples and photoreactive compounds, and DS/ and irradiation conditions /for example, the distance from the light source, light source type, the intensity, the film thickness and so on/.

Modified release drug

Crosslinked GAG present invention can be used as materials /carriers or excipients for inclusion of medicines in the three-dimensional netted structure to achieve controlled release of drugs. So, vkljuchennogo preparation and method of application, while maintaining the concentration of drugs in a certain interval are needed in the environment in which these drugs are released.

The speed of the controlled release of drugs can be adjusted by choosing phototherapy GAG /sample GAG and photoreactive groups, as well as DS/ and irradiation conditions /for example, time of exposure, distance from the light source, the light source power and so on/. In this case, although the rate of release is usually reduced with increasing molecular weight drugs and electrostatic forces of attraction between drug and sewn GAG specified speed can be adjusted to the desired level by choosing the above phototherapy GAG in combination with medication and taking into account their relative chemical structure /terms of molecular weight, electrical charge/ degree of hydrophilicity or hydrophobicity /and so on/.

In this aspect of the present invention medications immobilized under mild conditions so that they are stabilized in this state, when prevented the degradation and loss of activity compared to previously known methods.

Materials that provide controlled release of drugs, or prostii the nature of the medication and the type of application. For example, they can be manufactured in the form of films /films or coatings/, jellies, gels, creams, suspensions, microcapsules, tablets, granules, powders, etc. Can also be prepared fototerapia GAG, ready for use by immersing the substrate /for example, gauze, bandages, knitted or woven fabric, paper, cotton, nonwoven, film, porous sponge/ composition containing phototherapy GAG and medicine, or the application of specified composition on such a substrate, followed by irradiation to become crosslinked GAG. Moreover, the composition comprising phototherapy GAG and medicine, can be applied on the surface or inside such structures as artificial organ /for example, artificial blood vessel, artificial heart/ and then otvetit /stitching/.

To enable drugs crosslinked GAG of the present invention, the drug is dissolved or suspended in an aqueous solution or an organic solvent (for example, DMF/ containing from about 1 to 30 wt.%, for example, the corresponding vodootvedenija GAG to the concentration of drugs from about 0.001 to 80%, and, after adding various additives, if necessary, the resulting composition is molded, dried, and then irradiated with light. After stitching the first selection of medicines in the form of a solid, semi-solid substances or suspensions.

Materials with controlled release of medicines of the present invention can be used as pharmaceuticals. In this case, the crosslinked GAG containing drugs, can be made either as they are or together with conventional pharmaceutically acceptable additives, such as preservatives, stabilizers, local anesthetic agents, dispersing agents, modifiers forming, dissolving agents, and so forth, in the desired dosage form. For example, after bringing average particle size of from about 0.5 to 40 μm containing drugs crosslinked GAG can be used to obtain aqueous suspensions, together with dispersing agents (for example, Tweeh 80, NS/ Nippon chemicals/, carboxymethylcellulose, sodium alginate, etc./, preservatives (for example, methylparaben, propyl paraben, benzyl alcohol, chlorbutanol etc./ isotonic agents such as sodium chloride, glycerin, sorbitol, glucose and so on/ and other additives, if necessary, or get oily suspensions by dispersing them in such vegetable oils as olive oil, sesame oil, peanut oil, cottonseed oil or corn is because as they are or in a mixture with excipients /for example, starch, calcium carbonate, etc./ linking agents /for example, starch, gum Arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose etc./, lubricating agents such as talc, magnesium stearate, polyethylene glycol 6000 etc., and/ or the like for receiving tablets by molding under pressure or to obtain powders, granules, etc. If in capsule fill powders, granules or the like, receive containing medication capsules. Moreover containing medications GAG can be molded as they are in the film, or immobilizative on any of the other substrates, thereby creating preparations for cutaneous absorption of ophthalmic medications /for example, accelerating wound healing in the cornea/, preparations for the introduction of the living body, or drugs that are subject to introduction into the body cavity /for example, suppositories/. They can be used as bandages for wounds, patches produce medicines /for example, adhesive bandages/ and among other contraceptive devices.

Medications that should be used to obtain such pharmaceutical preparations which contain contain Lakers to enter often to achieve effective concentrations in the blood or effective local concentration, provided that they can be sufficiently retained in the mesh structure crosslinked GAG and can be released orderly manner. The following can be specified in the specific examples:

1. Antipireticescoe analgesic anti-inflammatory agents such as indomethacin, Nevanlinna acid, acemetacin, alclofenac, ibuprofen, termicheskaya, fenbufen, Marisol, salicylic acid and so on/.

2. Such agents against malignant tumors, as methotrexate, fluorouracil, vincristina, mitomycin C, actinomycin C, daunorubicinol etc.

3. Anti-ulcer agents, such as aceglutamide, L-glutamine, para-/TRANS-4-aminomethyltransferase/ phenylpropionic acid hydrochloride, tetracampidae, sulpiride, gefarnate, cimetidine etc.

4. Such preparations of enzymes like chymotrypsin, streptokinase, lysozymal, bromelain, urokinase activator tissue plasminogen etc.

5. Such anti-hypertensive agents, as clonidinegeneric, bonitalakesmall, protoceratopsid, captopril, butanedisulfonate, metoprololtartrate, hydrochlorothiazide methyldopa, etc.

6. Agents for urinary organs, such as Flavell, warfarin, etc.

8. Such antiarteriosclerotic agents, as clofibrate, simfibrate, elastase, nicomol etc.

9. Agents for circulatory organs, such as nordiphenhydramine, nanodimensional, cytochrome C, tocopherylacetate etc.

10. Such steroids like hydrocortisone, prednisolone, dexamethasone, betamethasone, etc.

11. Such promoters of wound healing, as a growth factor, collagen, and so on (see JP-A-60-222425/.

In addition, you can also specify a physiologically active polypeptides, hormones, anti-tuberculosis, anti-diabetic agents, vasodilator agents, antiarrhythmic agents, cardiac, anti-allergic agents, antidepressants, anti-epileptic agents, relaxants of kaskulov, antibiotics and the like.

Materials with controlled release of drugs of the present invention can be used as medical materials for making components of /for example, surfaces/ such structures as artificial organs such as artificial blood vessels, artificial heart and so on/. In this case, they are particularly useful as a medical material comprising the surface, which finding is of Nolita /for example, tissue plasminogen activator, urokinase/ and/or antithrombotics substance can be included in the crosslinked GAG to achieve a controlled release of these substances and make these surfaces antithrombotic properties.

Artificial extracellular matrix and membrane on artificial foundations

Fototerapia GAG of the present invention or a mixture of such proteins with adhesive cells, as collagen, gelatin and fibronectin, or after chemical linking them with proteins can be converted into a crosslinked GAG that can be used as an artificial extracellular matrices or membranes on an artificial basis to promote adhesion and growth of cells /endothelial cells, epithelial cells, smooth muscles, etc. / /see JP-A-1-124465, 61-128974 and 62-270162/. They can be used in a hybrid-type artificial organs, artificial blood vessels, artificial leather and so on/.

The following examples further illustrate the present invention but in no way limit its scope.

In all the examples of some typical physical properties and biological functions determine or estimate the following ways, with approx/visible spectra spectrometer JASCO-Ubest-30 UV/Vis.

The degree of substitution /DS/ for each photoreactive compounds calculated on the basis of data1H NMR and UV/. It also determines, by comparing data on the absorption of UV for low molecular weight model compounds /decomposition GAG/ data related photoreactivity connections.

The ability to swell for each sewn derived GAG is determined as follows.

The ability to swell is determined as follows: Weight /in the swelled state/ Weight /dry/ /Weight/dry, where the Weight /dry/ represents the weight of the dried film crosslinked GAG received by photoablation, and

Weight /in the swelled state/ represents the weight of the film after 24 hours after immersing it in purified water.

As for the contact angle, the contact angles of leakage and ettekanne determined by the method of liquid drops using a goniometer contact angle /static angle meter Kyowa CA-D, Kyowa Kaimen Depending K. K./. If the term "contact angle" is used as it is, it usually means "the angle of contact of leakage".

Experiments on the adhesion of endothelial cells is as follows. Each phototherapy GAG prepared in the form of a film by casting from rest and seeded with endothelial cells, obtained from the aorta of a bull in a sterile environment. Use culture medium Dulbecco modified medium Needle /DMEM/, supplemented by 10 - 15% serum, fetal calf /FCS/. Is there any adhesion or not, judged by the observation through a phase contrast microscope after 24 hours incubation at 37oC. For evaluation of cell growth using the growth rate after 1 - 2 days.

Example 1. Obtaining complex ester hyaluroni acid - cinnamic acid and obtaining a crosslinked hyaluronic acid due to the expression.

/1/ Receipt of ester of hyaluronic acid - cinnamic acid

30 ml of anhydrous pyridine are added to a solution of tri-n-butylamine salt of hyaluronic acid molecular weight 880000/ dimethylformamide /DMF/ 150 mg /35 ml/, followed by the addition 26,64 mg of cinnamic acid chloride acid under vigorous stirring at room temperature. The esterification give the opportunity to proceed at 75oC for 2 hours, then the reaction mixture ethanol saturated with sodium acetate, and the precipitate is collected and washed thoroughly with ethanol to remove unreacted acid chloride of cinnamic acid to obtain a complex ester of cinnamic kiloo data1H NMR/

1H spectrum of the above product is shown in Fig. 1. The characteristic absorption at the following CBM:

6 - 8 MD: refers to the protons of the benzene ring of cinnamic acid and the double bond cinnamic acid.

2 MD: refers to the methyl protons of the N-acetyl group of hyaluronic acid.

Calculate the ratio of the number of protons and used to determine the number of bound cinnamic acid and DS specified earlier.

/2/. Getting utverzhdenii crosslinked film of hyaluronic acid

A solution of 30 mg of the lot HA-Cin-3 in DMF) was placed on a glass plate /24 mm x 24 mm and dried with sterile air, heated to 40oC. the resulting films exhibit irradiation of a mercury lamp high pressure 450 W, through piracy filter filled with water, which is used to cut off wavelengths below 270 nm. So get utverzhdennuyu /that is sewn/ film of hyaluronic acid.

Peak absorption at 279 nm decreases upon irradiation /Fig. 2/.

Exposure continues as long as the percentage of attenuation becomes permanent /exposure time is 30 minutes.

Lot: HA-Cin-3-2

Example 2. Esters of hyaluronic acid cyclothem, what is the quantitative relationship between hyaluronic acid and cinnamic acid are changed as follows in the table. 1 /the number of hyaluronic acid always is 150 mg/. Further cured crosslinked film of hyaluronic acid get by the method of example 1.

Example 3. The contact angles of the films utverzhdenii hyaluronic acid

Each of the hardened hyaluronic films of hyaluronic acid obtained in examples 1 and 2, determined by measuring the contact angles of leakage and ettekanne. The results are shown in Fig. 3. Both the contact angle significantly increase as DS increases at the expense of the rest of cinnamic acid.

The increase of the contact angle reflects the increase in the hydrophobicity of the film surface.

Example 4. The ability to swell to assess hardened films of hyaluronic acid

Each of the hardened hyaluronic films obtained in examples 1 and 2, evaluated by the ability to swell, after they give the ability to swell in purified water for 20 hours. The results are shown in Fig. 4.

As can be seen in Fig. 4, the ability to swell decreases with increasing DS residue of cinnamic acid>Example 5. Adhesion of endothelial cells to utverzhdennym films hyaluronic acid

Adhesion or adherence of endothelial cells to utverzhdennym films of hyaluronic acid obtained in examples 1 and 2 /lots: HA-Cin-3-2 /DS = 0,5/ 4 to 2 /DS = 0,87/, 5 - 2 /DS = 1,28/ 6 - 2 /DS = 2,43// evaluate after 24 hours incubation. The results obtained are shown in Fig. 5 /photo/. As can be seen from Fig. 5, the adhesion of endothelial cells tends to increase with increasing DS due to the remnants of cinnamic acid.

The film HA-Cin-3-2 DS = 0.5 to exhibit a sufficient effect of preventing cell adhesion, and it was found that they have all the basic characteristics required for materials, the non-adhesive to the tissues.

The data obtained are of the value as the fundamental data that can be referenced in the manufacture of artificial extracellular matrices or membranes on artificial substrates using crosslinked GAG of the present invention in combination with sticking to cells and proteins such as collagen, gelatin and fibronectin, and allowing endothelial cells, epithelial cells, smooth muscle cells or similar cells to adhere to and grow on shown the axis of the vessel, artificial leather and so on/.

Example 6. Receipt of ester chondroitin sulfate-cinnamic acid and obtaining crosslinked chondroitin sulphate as a result of its expression

/1/. Receipt of ester chondroitin sulphate-cinnamic acid

30 ml of anhydrous pyridine are added to a solution of tri-n-butylamine salt of chondroitin sulfate /mol.weight = 60000/ DMF /247 mg/15 ml/. To this mixture is added with vigorous stirring at room temperature 19,78 mg of cinnamic acid chloride acid. The reaction is carried out at 75oC for 2 hours. To the reaction mixture add ethanol saturated with sodium acetate, and the precipitate is collected, washed thoroughly with ethanol and dried under reduced pressure.

Lot: CS-Cin-1

Output: 152 mg

Related cinnamic acid 7,52% wt. DS = 0,33

/2/. Obtaining a crosslinked film chondroitin sulphate

Product CS-Cin-1, obtained at the stage /1/ previously dissolved in phosphate buffer to a concentration of 15%, and then you get the gel, irradiation, using the same mercury lamp as in example 1.

Lot: CS-Cin-1-2

Example 7. Esters of chondroitin sulfate-cinnamic acid get, using the same materials and procedure as in example 6, except for the table.2 /the number of chondroitin sulfate remains always 247 mg/, and sewn chondroitinsulfate film receive the same manner as in example 1.

Lot CS-Cin /DS= 0,51/ dissolved in phosphate buffer saline /PBS/ in various concentrations and the resulting solution is irradiated with light for 30 minutes, and then determine the degree of gilotinirovaniya. The results are presented in Fig. 6, the degree of gilotinirovaniya increases with increasing concentration. The degree of gilotinirovaniya /%/ calculated as follows:

The percentage of gilotinirovaniya /%/ = 100 x /A-X/ / /A-B/,

where A = absorption /ODNm/ before irradiation,

B = absorption /ODNm/ measured after sufficient exposure, in which the absorption reaches a constant level, and

X = absorption /ODNm/ after 30 minutes exposure.

15% solution of the lot, CS-Cin-3 /DS=0,65/ PBS irradiated light and the state of the gel is monitored as a function of time. Thus, after each period of exposure resulting gel is immersed in deionized water for 24 hours for deionization and weighed after removal of moisture from the surface of the gel. Then the gel is dried and expect the ability to swell. Determine the degree of gilotinirovaniya. The results obtained are shown in Fig. 7.

The degree of W is 2">

Lots of CS-Cin-1 to 5 are also assessed on their behavior regarding their behavior in PBS and the resulting gels on their ability to swell. Thus was discovered the relation between the degree of gilotinirovaniya or the ability to swell and DS residue of cinnamic acid per molar ratio relative to chondroitin sulphate a constituent disaccharide glycosides of the fragment shown in Fig. 8.

The increase in DS due to the remnants of cinnamic acid leads to an increase in the degree of gilotinirovaniya, with the rapid decline in the ability to swell.

Example 8. Evaluation of the ability to swell hardened chondroitinsulfate films

Esters of chondroitin sulfate-cinnamic acid obtained in examples 6 and 7, formed in fototerapia film, which is then irradiated with light for 30 minutes for crosslinking to obtain a solidified chondroitinsulfate films.

These solidified chondroitinsulfate film evaluated for the ability to swell. The results are shown in Fig. 9.

The obtained results indicate that in contrast to the hardened films of hyaluronic acid cured film chondroitinsulfate not show such a rapid reduction in sung DS due to the remnants of cinnamic acid. This is presumably due to the fact that the cured film of chondroitin sulfate contain sulfate groups, and their hydrophilicity higher than the hydrophilicity of the films of hyaluronic acid.

Example 9. The contact angles of the hardened films of chondroitin sulfate obtained in example 8, measure and lay against DS residue of cinnamic acid. The results obtained are shown in Fig. 10. Graph of Fig. 10 shows that crosslinked chondroitin sulfate gives a smaller contact angles compared with crosslinked hyaluronic acid. This is presumably due to the difference in hydrophilicity.

Example 10. Adhesion of endothelial cells to utverzhdennym films chondroitin sulphate

Adhesion of endothelial cells to utverzhdennym the films obtained in example 8, determined in the same manner as in example 5. Unlike the hardened films of hyaluronic acid, no adhesion was observed regardless DS residue of cinnamic acid.

Example 11. Linking derivative of cinnamic acid with the carboxyl groups of hyaluronic acid and stitching

/1/. Getting amide of cinnamoylcocaine

The acid chloride cinnamic acid /1,666 g/ dissolved in 100 ml of chloro what SUP>oC, the reaction mixture was washed with saturated sodium bicarbonate solution, and then thoroughly washed with water. The layer of organic solvent concentrated under reduced pressure and the residue is recrystallized from ethanol to obtain the target product /1,58 g, hereinafter referred to as compound a/.

/2/. Obtaining NL-cynnamoyl-L-lysine

NL-tert. -butoxycarbonyl-L-lysine /2.58 g/ dissolved in 50 ml of dimethylformamide and the resulting solution was added 30 ml of anhydrous pyridine. To this mixture add a solution of acid chloride of cinnamic acid in chloroform /1,665 g/ 20 ml, and the reaction is carried out at 40oC. the Reaction mixture is concentrated to dryness under reduced pressure, the residue is dissolved in 3.6 n HCl /dioxane/33 ml/ and after 4 hours of maturation resulting solution was concentrated under reduced pressure to obtain the target compound /2,43 g hereinafter referred to as compound B/.

/3/. Linking amide of cinnamoylcocaine with hyaluronic acid

In 30 ml of water was dissolved 150 mg of sodium salt of hyaluronic acid /mol. weight = 1200000/, and then add 42,75 mg of compound A and to 71.9 mg of 1-ethyl-3-/3-dimethylaminopropyl/carbodiimide hydrochloride. The reaction is carried out at room temperature in the course is the temperature for 1 hour, add ethanol saturated with sodium acetate. The obtained white precipitate is collected and washed thoroughly with ethanol.

Lot: HA-CinA-1

Output: 176,2 mg

Related compound A: 16,58% wt. DS=0,5

/4/. Linking NL-cynnamoyl-L-lysine with hyaluronic acid

To a solution of tri-n-butylamine salt of hyaluronic acid /mol. weight = 880000/ dimethylformamide /150 mg/35 ml/ add 1,224 g of N-hydroxysuccinimide and 55 mg of dicyclohexylcarbodiimide and reaction are initially at 0oC for 1 hour and then at room temperature for 10 hours, to activate the carboxyl group of hyaluronic acid. To the reaction mixture was added ether and the resulting precipitate is collected, washed with ether and dried under reduced pressure, to obtain the corresponding complex of the activated ester of hyaluronic acid.

This activated hyaluronic acid is dissolved in dimethylformamide. To this solution add a solution of compound B in dimethylformamide /44 mg/50 ml and the reaction is carried out at room temperature for 20 hours. To the reaction mixture is added saturated sodium acetate ethanol, and the precipitate is filtered off and purified by washing with ethanol.

Lot: HA-CinB-1

Output: 122,1 mg
/5/. Getting the hardened films of hyaluronic acid

Each of the lots HA-CinA-1 and HA-CinB-1 /30 mg/ formed into films in the same way as in example 1, and sew, irradiating light.

Thus obtained cured film of hyaluronic acid /HA lots-CinA-1-2 and HA-CinB-1-2/ demonstrate the ability to swell 1.2 and 1.4 g of H2O/g of gel, respectively.

Example 12. Obtaining a complex ester of hyaluronic acid-/1-/2-carboxyethyl/thymine/ /1/

To a solution of hyaluronic acid /mol. weight 1000000/ /hereinafter referred to as HA100/ dimethylformamide /DMF/ /175 mg/50 ml/ containing MX 0.317 g of 2-chloro-1-methylpyridine-iodide, add 0,245 g 1/2-carboxyethyl/thymine and 0,461 g of triethylamine and the reaction is carried out at 90oC for 4 hours. DMF is removed under reduced pressure and the excess amount of ethanol added, the precipitate collected, washed with ethanol and dried under reduced pressure. The obtained white precipitate is an ester of hyaluronic acid-/1-/2-carboxyethyl/thymine/.

Lot: HA-Thym-1

Output: 160,0 mg

Linked thymine: 9.1 wt.% DS = 0,46

/Value DS is determined based on the ratio between the number of methyl protons of thymine and the number of acetyl is islote- /1-/2-carboxyethyl/thymine/ /2/

Several lots of ester of hyaluronic acid- /1-/2-carboxyethylidene/ receive in the same manner as in example 12, except that use the conditions listed in the table. 3.

Example 14. Obtaining crosslinked hyaluronic acid by expression of ester of hyaluronic acid-/1-/2-carboxyethyl/thymine/ /1/

Prepare a film of the lot HA-Thym-1 /DS = 0,46/ by way of example 1 and irradiated with UV light /UV/ from a xenon lamp. The course of the formation of thymine dimer as a function of time of exposure, is shown in Fig. 11.

Example 15. Getting the hardened films of hyaluronic acid by expression of fototerapia esters of hyaluronic acid-/1-/2-carboxyethyl/thymine/ /2/

Lots HA-Thym-3, 4, 5, 6, 7, 8 and 9 /DS= 0,125, 0,14, 0,26, 0,35, 0,37 and 2,40 respectively/, obtained in example 13, respectively, formed into films by the method of example 1, and these tapes are UV irradiated using a xenon lamp. Thus obtained cured film of hyaluronic acid is designated as lots HA-Thym-3-2, 4-2, 6-2, 8-2 and 9-2, respectively. Next in the table. 4 presents data on the extent of gilotinirovaniya /%/ and the ability to swell.

Example 16. Production of esters hialuronowy over molecular sieves thoroughly dehydrate additional deaeration within 4 hours.

Separately stirred for 30 minutes at room temperature DMF) solution containing 1-/2-carboxyethyl/thymine/ in different molar relationship relative to each hydroxyl group of hyaluronic acid: see Fig. 12/, 2-chloro-1-methylpyridinium /1.2 mm/ triethylamine /1.2 mm/. This solution is added dropwise to the above solution of hyaluronic acid, supplemented with triethylamine /1.2 mm/. The resulting mixture was stirred at 90oC for 3.5 or 8 hours. The reaction mixture was concentrated under reduced pressure and to the residue is added methanol. The precipitate is filtered off, washed with methanol and dried. So get esters of hyaluronic acid -/1-/2-carboxyethyl/thymine/ different DS /HA-Thym; DS= 0,2, 0,4, 0,6, 0,7, 0,9, 1,3, 1,8, 2,2/.

An example of the relationship between the molar ratio used 1-/2-carboxyethyl/thymine and DS shown in Fig. 12.

Example 17. Production of esters chondroitin sulfate- /2-/2-carboxyethyl/thymine/

According to the method of example 16, except that the use of chondroitin sulfate /mol. weight = 60000/ s molar ratio of 1-/2-carboxyethyl/thymine changing relative to each hydroxyl group of chondroitin sulfate, and the fact that rhenium product. If using diethyl ether, the precipitate is thoroughly washed with methanol. So get esters chondroitin sulfate- /1-/2-carboxyethyl/thymine/ different DS /CS-Thym; DS= 0,09, 0,4, 0,8, 0,9, 1,3, 1,7, 1,8, 2,2/.

The link between the molar ratio used 1-/2-carboxyethyl/thymine and DS shown in Fig. 13.

The relationship between DS and solubility /water or DMF/ presented in table. 5.

Example 18. Getting the hardened films of hyaluronic acid and hardened films of chondroitin sulfate using a derivative of thymine

The above film get using esters of 1-/2-carboxyethyl/thymine each GAG obtained in examples 16 and 17.

Thus, each of the esters of hyaluronic acid- /1-/2-carboxyethyl/thymine/ /HA-Thym; DS= 0,2, 0,4, 0,6, 0,7, 0,9, 1,3, 1,8, 2,2/ and esters chondroitin sulfate- /1-/2-carboxyethyl/thymine/ /CS-Thym; DS= 0,09, 0,4, 0,8, 0,9, 1,3, 1,7, 1,8, 2,2/ dissolved in DMF to obtain 5% dissolve and 200 μl of the solution was placed on a glass plate /14 mm diameter) and dried using sterile air at a temperature of 35oC. the Obtained film is irradiated with light from a mercury high-pressure lamps with a capacity of 400 watts through eraksoy the filter with water to obtain films sshi is incorporating a thin dried film of ester chondroitin sulfate-/1-/2-carboxyethyl/thymine/ /CS-Thym/ /DS = 0,09, the thickness of 2 - 3 µm /specified previously by the method shown in Fig. 14. The relation between the degree of gilotinirovaniya /%/ and the exposure time, which in this case is shown in Fig. 15. Film CS-Thym /DS = 0,09/ and of a thickness corresponding to the practical use of /10 - 12 µm/ also irradiated in a similar way; the relationship between the degree of gilotinirovaniya /%%/ and the exposure time is shown in Fig. 16.

The dried film of ester of hyaluronic acid - 1-/2-carboxyethyl/thymine /DS = 0,9, thickness of 10 - 12 mm/ irradiated in the same manner, and communication abilities to swell depending on the exposure time is shown in Fig. 17. The ability to swell HA-Thym films with different DS, determined before and after irradiation /3 hours/ and the data obtained are in the form of graphics from the DS, the obtained curves are shown in Fig. 18.

Data on the contact angles and the ability to swell obtained for several obtained from HA-Thym or CS-Thym photoallergen films obtained in this example and different DS, are presented in table. 6.

Example 19. Adhesion of endothelial cells to a crosslinked film of hyaluronic acid

Endothelial cells grown on a stitched square the Yan of cell adhesion appreciate later, 6 hours after the start of cultivation.

The results showed that adhesion, elongation, proliferation and growth of endothelial cells hardly observed at all crosslinked films of hyaluronic acid with different DS. On the other hand, normal connection, growth, distribution and growth are observed in cells with the control TCPS.

Example 20. Receipt of ester chondroitin sulfate-/7-comparisonvalue acid/

A solution of chondroitin sulfate-tri-n-butylamine salt /of 10.25 mg/ml in DMF) previously dried over molecular sieves and 70 ml of this solution is dried under vacuum with stirring in a three-neck flask with a volume of 100 ml at room temperature. Then the air is replaced with nitrogen gas and add 15 ml of distilled pyridine. To this add a solution of one molar equivalent of 7-kumarasambhava /acid chloride coumarin/ /1.12 g in 5 ml of DMF for each group of chondroitin sulfate. The reaction is carried out at boiling under reflux the mixture in a nitrogen atmosphere at 80oC for 3 hours. Then the reaction mixture was concentrated and added dropwise diethyl ether. The precipitate exhale, dissolved in deionized water and cialiswhat running water for 3 days. The resulting dialysate lyophilize="ptx2">

Output 965,1 mg

DS = 0.021

The ability to swell: 2.8 g H2O/g of dry gel

Range1H NMR shown in Fig. 19.

The above procedure is repeated except that the molar ratio of acid chloride of coumarin to the hydroxyl groups of chondroitin sulfate change, and DS lay molar ratio. The results obtained are shown in Fig. 20.

Example 21. Getting photoallergen film chondroitinsulfate of ether complex chondroitin sulfate-/7-comparisonvalue acid/

Ester chondroitin sulfate-/7-comparisonvalue acid, obtained in example 20 is prepared in the form of a dry film according to the method of example 1 and the film is irradiated with UV light /320 nm to obtain photoallergen film chondroitin sulphate.

The relationship between time of exposure and changes in the absorption spectrum /320 nm/ coumarin shown in Fig. 21. The ability to swell /g H2O/ dry gel/ received films define and lay the exposure time and DS. The results obtained are shown in Fig. 22 and 23, respectively. In addition, the degree of gilotinirovaniya /%/ define and lay on the axis of ordinates depending on time Exponet shown in Fig. 24 and 25, respectively.

Example 22. Crosslinked film of hyaluronic acid/ chondroitin sulphate

HA-Cin-3 /DS=0.5 in/ obtained in example 1 was dissolved in 20% aqueous DMF solution at a final concentration of 5 wt.%. Then this dissolved CS-Cin-4 /DS=1,37/ obtained in example 7, to a final concentration of 5 wt.% to obtain the mixed complex ester of hyaluronic acid-cinnamic acid and ether complex chondroitin sulfate-cinnamic acid. Using this solution, by the method of example 1 is poured and is exposed to the solution, getting crosslinked film of hyaluronic acid/chondroitin sulfate.

Similarly, a mixed solution containing HA-Cin-3 and CS-Cin-4 in the ratio of 2:1 or 1:2 /weight/ get and for each drug determine the effect of naughtynati /description will follow/.

Example 23. /1/ Receipt of ester heparin-cinnamic acid

To a solution of salts of heparin-tri-n-butylamine in dimethylformamide /DMF/ /500 mg/ 125 ml/ add 20 ml of pyridine and the resulting mixture dehydration under reduced pressure. Then with vigorous stirring at room temperature add 69,35 mg of cinnamic acid chloride acid. The reaction is carried out at 75oC for 2 hours and then the reaction mixture is again concentrated di reduced pressure. It is dissolved in 5 ml of DMF and then add 40 ml of PBS, then the solution is mixed well. It cialiswhat through the dialysis membrane to carefully remove low molecular weight compounds, and then lyophilized until the desired ether complex.

Lot: Hep-Cin-1

The amount of bound cinnamic acid /according to1H NMR DS/ and solubility are shown in table. 7.

/2/ Getting stitched films heparin

In DMF dissolving 30 mg lot /Hep-Cin-1/ and the resulting solution was placed on a glass plate, 24 mm x 24 mm, and dried with sterile warm air at 40oC. the resulting film is irradiated with a mercury lamp high pressure output of 450 watts through eraksoy filter filled with water, to obtain a crosslinked film of heparin.

Lot: Hep-Cin-1-2

Contact angles /in-leakage and ettekanne angle/ and the ability to swell in water, and weight change in water are presented in table. 8.

/3/ Using the above materials and procedures, receive esters, and the ratio of heparin to the acid chloride cinnamic acid change Hep-Cin-2, Hep-Cin-3, Hep-Cin-4/, and the number of heparin constantly 500 mg/. The results obtained are also present in the table. 7. In addition, the crosslinked film heparin /Hep-Cin-2-2, Hep-Cin-3-2/ floor is p 24. A solution of tri-n-butylammonium salt of hyaluronic acid /1.5 g corresponding to 10 mmol of OH groups/ in 200 ml of dry DMF is kept under argon and cooled to 0oC. Successively added 4-dimethylaminopyridine /0,305 g, 2.5 mmol/, cinnamic acid anhydride /2,78 g, 10 mmol/ and tri-n-butylamine /4,6 ml, 10 mmol) and the reaction allowed to proceed at room temperature for 24 hours. After cooling to 0oC gradually add 5% NaHCO3in 100 ml of water and the resulting solution was stirred at room temperature for 48 hours. Excess NaHCO3remove the sequential addition of 1M HCl to pH 4 and IM NaOH to pH 7. With stirring, add cold ethanol. After desantirovaniya residue is dissolved in water and the deposition procedure is repeated with ethanol. The precipitate is removed by centrifugation, dissolved in water and passed through a column of Dowex 50 /H+/ s cation exchange resin at 4oC. the Acid is neutralized 1M NaOH and lyophilized.

Example 25. The non-adhesive effect photoallergen film hyaluronic acid

Photoallergen film of ester of hyaluronic acid-cinnamic acid /DS=0.5; the thickness of 30 μm, 20 x 20 mm/ get as a lot HA-Cin-3-2 in example 1, and used as neijia and abdominal cut to to prepare the place, where would exposing the layer of muscles. This place is covered previously prepared non-adhesive material. The implant is removed after 1 week and two weeks after the coating and the degree of adhesion of the fabric to the film surface are examined using a light microscope after coloring the hematoxylin-Sosina/ and electron microscope for evaluation. As a control using rats have not cut cover with foil.

In this experiment, using the non-adhesive materials of the present invention in all samples was barely detectable deposition of fibrin and cellular adhesion after one and two weeks. Observations within two weeks show that starts biodegradation of the film.

On the other hand, in the control experiment, in place of the cut there is a sequential deposition of fibrin, the invasion of phagocyto, fibroblasts, and so on , and collagen formation, and intestinal canal is closely attached to the place of the cut a week later.

The above experiment is repeated except that the use of crosslinked film HA-Cin-3/CS-Cin-4 obtained in example 22. Compared to the above homogeneous HA-Cin-3-film /HA-Cin-3-2/ ability to biodegradation of cells in film, and the progress of decomposition becomes apparent after 2 weeks.

Example 26. The non-adhesive effect photoallergen film chondroitin sulphate when relativeiy in situ

Ester chondroitin sulfate-cinnamic acid lot CS-Cin-3 /Ds=0,65/ obtained in example 7, dissolved in phosphate buffer to a final concentration of 20 wt.% to obtain the non-adhesive material in the form of a solution.

Animal model of the operated organ similar to the one used in example 25, designs and place cut cover above the non-adhesive material. Then this material is irradiated with UV light for 15 minutes to cause gelation in situ. Histological studies confirm the formation of a gel layer in close contact with the surface tissue of the abdominal cavity.

A series of observations designated cut analogously to example 25 shows that, as in example 25, the adhesion does not occur.

Example 27. The effect of preventing adhesion photoallergen crosslinked film of hyaluronic acid and photoallergen crosslinked film of chondroitin sulfate.

Vodootvedenie film of ester of hyaluronic acid-cinnamic acid /HA-Cin/ /DS = 0,1, 0,5/ received on how primerov,8/ and a complex ester of chondroitin sulfate-derived thymine /CS-Thym; DS = 0,4, 0,9/, obtained by the method of example 18, is used as the non-adhesive materials and conduct the following experiments. So, every photoallergen film /14 mm in diameter, 15 - 20 microns/ sterilized by immersing in 70% ethanol for 30 minutes and then leave to swim in sterilized water for 1 hour. As for samples with low DS, they are dried after immersion in water to remove ethanol and before the animal is immersed for 10 minutes in sterile water. The film swelling, absorbs water and forms a hydrogel.

Rats /white, strain Wistar, males weighing 300 g/ analiziruyut ether and support in this state, the air and oxygen prior to surgery. For each sample using the same rat. Spend a vertical dissection of the peritoneum in rats to generate, to expose the liver. The surface of the liver Nude mechanically cut to create a damaged surface up to 1 cm2and cause swelling above photoallergen film. As for the film, which is unable to fix at the injury site, for fixing this film with four of its ends prilasec polyurethane adhesive. Then sutured to the abdominal wall nylon thread. After one and two weeks on the key, and the top part of the excised with surrounding tissues and subjected to histological examination using light microscope. The results obtained are presented in table. 9.

Histological examination photoallergen film HA-Cin /DS=0,1/ DS = 0,1/, for example, 1 week after implantation does not show any cellular adhesion on the film surface, and biodegradation film already by this time had begun, and there is invasion of tissue. If fotoallergiyami film HA-Thym /DS = 0,2/ on the top layer there are flat cells similar to the cells of the peritoneum, and biodegradation is already progressing and remained intact with only a small Central part.

Using the uncovered portion of the wound as a control, 1 week conducting similar studies. As a result, the adhesion cannot be broken with the blunt end of the surgical knife or hand, or even cutting knife /was found between wound surface biscuits and a wall of the peritoneum/.

Example 28. Controlled release of drugs through the use as carriers photoallergen films of hyaluronic acid and chondroitin sulphate

/1/ Controlled release InTouch indomethacin dissolved in 200 μl of the above solution. The resulting solution was placed on a glass plate 15 mm in diameter and dried by air /sterilized warm air, 35oC/. Thus obtained film is irradiated as in example 18, to obtain indometacina crosslinked film of hyaluronic acid. The contents of the medicines control at 10%. In the same way prepare crosslinked hyaluronic film containing drugs 30%, 50% and 73%. Moreover, using CS-Thym variable DS, as were obtained in example 17, in the same way prepare crosslinked film of chondroitin sulfate content medications 10%, 30% and 50%.

The test drug release of each film is as follows. So the test film is suspended in water at 20oC or phosphate bateriafina saline /37oC/ and stirred. After a certain period of time selected samples of the liquid phase and measure the UV absorption at 269 nm.

In table. 10 shows the relationship test data solubility in water for photoallergen films, hyaluronate acid /DS = 0,7, 1,3, 1,8/ content medications 30%, with the DS and the ability to swell. As the test data on the solubility of the use time of the used film in the new knitting and therefore, the harder the film, with a corresponding decline in the ability to swell and the associated decrease in the rate of release of drugs. Is this fact suggests that the ability of the film to absorb water is an important factor in the rate of drug release. As for films with DS values of 1.3 and 1.8, the rate of release has a tendency to decrease with increasing content of the medication.

Vodootvedenie film of hyaluronic acid /DS = 0,7, 2,2/ and vodootvedenie film chondroitin sulphate /DS = 0,8, 1,3, 1,8/ compare the speed of release of the drug in PBS content medicines 10% and 50%. The results obtained are shown in Fig. 26 - 30. From these graphs it is obvious that the controlled release of medicines is possible for films with DS values not less than a certain threshold value /for CS-Thym, DS = 1,3/.

/2/ Controlled allocation of heparin

A 20% aqueous solution of DMF is dissolved ester chondroitin sulfate-cinnamic acid /CS-Cin-4 /DS = 1,37/ CS-Cin-5 /DS = 2,43/ obtained in example 7 at a final concentration of 20 wt.%, as well as a complex ester of hyaluronic acid-cinnamic acid /HA-Cin-3 /DS = 0,50/ HA-Cin-5 /DS = 1,28/ HA-Cin-6 /DS = 2,43/ obtained in examples 1 and 2 PR cover glass plate /10 cm x 10 cm/, and dried at room temperature for 1 hour to obtain a film. This film is irradiated with a mercury lamp high pressure output of 450 watts for 30 minutes. The thickness of each film is approximately 100 microns.

Each of these films deposited on a glass plate, fully immersed in a vessel containing 100 ml of water and stirred at a speed of 60 rpm. /min. and the Amount of released heparin depending on time is determined by a method using a carbazole-sulfuric acid. The results show that all films are suitable for controlled release of heparin.

In addition, each of the above films with controlled release of heparin formed on the inner wall of the test tube. Then, in accordance with JP-A-4-41432 add citrate blood and determine the formation of clots. All samples exhibit antithrombotic activity.

/3/ Controlled release factor release growth hormone

A 20% aqueous solution of DMF is dissolved ester of hyaluronic acid-cinnamic acid /HA-Cin-3 /DS = 0,5// at a final concentration of 10 wt.% and 1 mg factor release growth hormone /GRF, human mol. weight 5039,8/ PR dried at room temperature for 1 hour to obtain a film. This film is irradiated with a mercury lamp high pressure output of 450 watts for 30 minutes. The thickness of this film is 110 μm.

The above-mentioned films, as they applied on a glass plate, immersed in a vessel containing 10 ml of water and stirred at a speed of 60 Rev/min the Rate of release of GRF from time determined using high-performance liquid chromatograph and calculate the cumulative amount of released substances. It was found that can successfully carry out the controlled release GRF.

Example 29. Vascular implants

The inner surface of the artificial blood vessel with a small cavity /inner diameter 3 mm/ cover solution HA-Cin-3 obtained in example 1, using the technique of rotation, and after drying the applied film utverjdayut UV irradiation, using the optical fiber of small caliber, resulting in a gain of vascular prosthesis, the inner surface of which is covered utverzhdenii hyaluronic acid.

/Effect of the invention/

The present invention can easily provide easy-to-clean fototerapia GAG due to the selection of high security and biocompatible raw materials, such totoriai. Further, the present invention provides materials for medical applications due to the exposure of these fototerapia GAG light. These materials have a two-dimensional or three-dimensional mesh structure and are completely safe, biocompatible and bio-degradable/absorbable. The present invention also stitched materials based on the GAG with the desired physical properties required for materials for use in medicine, through the appropriate choice of molecular weights GAG, DS photoreactive connections and other factors. Thus, the invention can widely be used in various fields of medicine.

1. Vodootvedenie derived fractions having a molecular weight from about 4000 to 2000000, which presents one of the following formulas:

gag-O-CO-R1) (1),

gag-O-CO-R3-NH-CO-R1(4c),

gag-CO-O-R3-O-CO-R1(5c),

gag-CO-O-R3-NH-CO-R1(5d),

gag-CO-NH-R3-O-CO-R1(5c) or

gag-CO-NH-R3-NH-CO-R1(5f)

where gag-O and gag-CO means glycosaminoglycans part, and glycosaminoglycan selected from the group consisting of hyaluronic acid, chondroitin, chondroitin sulphate, dermatosurgery, geyh derivatives;

R1is photoreactive group having at least one double-bond, capable of forming cyclobutanone ring using the expression;

R3means - (CH2)n-, -(CH2)pCHY - and (CH2)m-C6H4(CH2)lwhere l, m, n and p represent the number from 1 to 10 and Y represents COOH or NH2,

or vodootvedenie derived glycosaminoglycan represented by the following formula:

gag-CO-(O-R1) (3),

gag-O-CO-R3-CO-O-R1(4a),

gag-CO-O-R3-CO-O-R1(5a) or

gag-CO-NH-R3-CO-O-R1(5g)

where gag-O, gag-CO, R1and R3have the meanings given above.

2. Vodootvedenie derived glycosaminoglycans and collagen under item 1, in which-CO-R1in formulas (1), (4c), (5c), (5d), (5e) and (5f) means one of the following formulas:

< / BR>
where R4and R5independently mean a hydrogen atom, a C1- C6is an alkyl group, a C1- C6-CNS group, nitro or amino group,

< / BR>
where R6means a hydrogen atom, a halogen atom, a C1- C6alkyl - or haloalkyl group;

R7means a hydrogen atom, a halogen atom, a C1- C6-alkyl - or haloalkyl group, C is C2- C6alkylenes group, or

< / BR>
where R9, R10and R11independently mean a hydrogen atom or a C1- C6is an alkyl group;

R12means2- C6-alkylenes group; and-O-R1in formulas (3), (4A), (5A), (5g) means the following formula:

< / BR>
where R13, R14, R15have the same values as in R6, R7, R8respectively, or

< / BR>
where R16, R17have the same values as R4and R5.

3. Vodootvedenie derived glycosaminoglycans and collagen under item 1 or 2, represented by formula (1), in which glycosaminoglycanes part selected from hyaluronic acid or chondroitin sulfate.

4. Vodootvedenie derived glycosaminoglycans and collagen under item 1 or 2, represented by formula (5f), in which glycosaminoglycans part selected from hyaluronic acid or chondroitin sulphate and means R3or -(CH2)n- or-CH2)pCHY, where n, p, and Y are defined as in point 1.

5. Vodootvedenie derived glycosaminoglycans and collagen under item 4, in which the partial structure-NH-R3-NH - is the residue of ethylene diamine or a residue of L-lysine.

6. Composition, characterized the water, buffer solution or an organic solvent that is acceptable in medicine.

7. Stitched derived fractions obtained by irradiation vodootvedenija derived glycosaminoglycans and collagen under item 1 or 2 light having a wavelength in the range of from about 260 to 400 nm.

8. Sewn-derived glycosaminoglycans and collagen under item 7, which includes the following recurring structural units dimeric structure

< / BR>
9. Sewn-derived glycosaminoglycans and collagen under item 7, which contains the following recurring structural units dimeric structure

< / BR>
10. Stitched derived fractions in PP.7 to 9, which are molded and dried in the form of a film, tube or granules.

11. Composition, characterized in that it contains sewn derived fractions in PP.7 - 9 and sterilized water.

12. The method of obtaining vodootvedenija derived glycosaminoglycans and collagen under item 1 or 2, characterized in that conduct the interaction of at least one of glycosaminoglycans and collagen or its salts with an acid anhydride of formula (R1CO)2O or acid chloride of the acid of formula R1COCl at a temperature of from 0 to 100oC in the presence of a basic catalyst, where R1there are thus intermolecular and/or intramolecular stitching specified photoreactive part.

13. A method of obtaining a crosslinked derivative of glycosaminoglycans and collagen in PP.7 to 10, characterized in that the conducting irradiation of at least one vodootvedenija derived fractions in PP.1 - 5 light having a wavelength of from about 260 to 400 nm.

14. The way to prevent cellular and/or tissue adhesion, characterized in that it comprises applying to the wound vodootvedenija derived fractions in PP. 1 to 5 and irradiated with light having a wavelength of from about 260 to 400 nm for education due to this crosslinked glycosaminoglycans and collagen.

15. The way to prevent cellular and/or tissue adhesion under item 14, characterized in that it comprises applying to the wound stitched fractions in PP.7 - 10.

Priority points:

05.02.92 on PP.1 - 5, 7 - 10;

08.07.92 on PP.6, 12, 13;

21.12.92 on PP.14 and 15.

 

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