The antiadhesive agent

 

(57) Abstract:

The invention relates to a new anti-adhesion agent. As substances that can prevent or reduce undesirable adhesion of damaged tissue with adjacent or surrounding tissue in healing wounds, applied chitosan and polysaccharide immobilized with him. The polysaccharide is selected from heparin, heparan sulfate and dextran sulfate. New anti-adhesion agent has the ability to improve wound healing by stimulating tissue regeneration. 2 C. and 12 C.p. f-crystals, 2 Il.

The present invention relates to new anti-adhesive agents, i.e. products that can prevent unwanted adhesion of tissues in connection with the healing of wounds. This product also has the ability to improve wound healing by stimulating tissue regeneration.

The invention also includes a method of preventing such undesirable adhesion of the tissue.

Life is the ability to move freely in accordance with our desires and needs. A necessary condition for this is the corresponding function of the musculoskeletal system in close cooperation in basic is such as bone, muscles and tendons to move freely relative to each other. Such actions require a slip zones, minimum friction and maximum freedom of movement. Thus, for optimal performance between, for example, adjacent to each other by muscles and tendons, as well as between the skin and adjacent thereto the tissues slip systems. The same applies to the structure of internal organs, such as gastrointestinal tract, heart, lung, brain and spinal cord. Slip systems are formed from thin plates of loose connective tissue, which in the abdominal cavity, chest cavity, heart handbags and for brain and spinal cord delimited mesothelial cells. Loose connective tissue surrounding the tendon, has a similar structure. These slip systems are very sensitive to inflammation and damage. The rapid formation of scar tissue may lead to the weakening or even loss of function. Adhesion of the tissue formed in the abdominal cavity, such as the formation of fiber and film-like growths adjacent or surrounding structures may even lead to obstruction of the condition, life threatening. After surgery the effect of reconstruction, always formed scars and correspondingly more or less tangible loss "natural and original" slip systems.

Wound healing in the skin and mucous membranes is complicated, on the one hand, the limited ability of connective tissue to regenerate and, on the other hand, the formation of immature granulation tissue, which then limited the ability of maturing to the level of normal tissue. Thus, the skin after damage is not reformed neither children, nor adults, with rare exception. Small and/or surface damage to the skin healed by replacing damaged tissue structures adjacent type and education responsive to stimulation of granulation tissue. In cases of more significant loss of tissue, as, for example, after deep burns, burn wounds of the third degree, and after the loss of part of the skin tissue, while healing occurs inevitably scarring, different, but inevitable loss of tissue and irreversible deformation. Mechanically strong component of scar tissue consists mainly of collagen type III in the form of short fibers and lower the organization and therefore its mechanical properties compared with collagen nomenclautra. Over time, reduced number of blood vessels in relation to their number in normal tissue, as well as changing the distribution and types of vessels. Often there are a wide thick-walled vessels with lower functionality in comparison with the corresponding normal blood vessels, and abnormal lymph-vascular system. Slip systems, thus, replaced the hard fibrous collagen connective tissue.

Another very significant complicating factor arises due to the appearance of myofibroblasts, i.e. "normal" connective tissue cells (fibroblasts) that as part of macrophages have an increased number of cytoplasmic sites of muscle proteins, which creates the possibility of slow and strong compression of the cells and the retention of such compression in a long time. The result can cause contracture, which then distort and limit the functionality of damaged tissue. The increased number of myofibroblasts observed, for example, around breast implants (silicone prostheses implanted to increase or breast reconstruction; more detail C. Lossing & H-A. Hansson article the high frequency distributed around the joints in some rheumatic diseases and can lead to curvature of the fingers, and sometimes also to the displacement of the articular surfaces. This cell is a pathogenic factor causing deformation of the hands, affecting patients suffering from Dupuytren's contracture. Myofibroblast, as well as normal fibroblasts attached to collagen threads with specific heterodimeric receptors, one element of which is always formed 1-integrin. Blocking of integrins leads to the elimination of contractures. Anti-inflammatory remedies can influence the expression of integrins.

Thus, system, slip in loose connective tissue with or without clearly defined sliding surface is restored only in cases of minimal inflammation. The formation of granulation tissue occurs, however, only in connection with the inflammatory process, which per se leads to the formation of immature cells and tissue components. However, the inability of new tissue formed during the healing process, to approach normal levels of differentiation is the reason that scar tissue is tissue of lower quality quantitatively and qualitatively compared with the original Mature, but lost tissue. The maturation of the regenerated tissue requires LASS="ptx2">

Research has been directed at solving the problem of avoiding unwanted adhesion of the tissue in connection with the healing of wounds, such as wounds caused by surgical intervention resulting from accident, inflammation or tumors. A PCT application N US 90/02406 describes the technology associated with this specific problem, and also includes a relatively broad interpretation of the prior art. The methods described in this patent application, based on the use of sandwich structures, including biodegradable bioactive membrane, the opposite surfaces of which have a different composition and therefore different biological functions. However, as it turned out, the products are missing in the market.

The object of the invention, accordingly, is an anti-adhesive substance, which causes only minimal inflammation, which quickly passes, but such substance is biologically acceptable and biodegradable, do not produce hazardous decomposition products.

Another object of the invention is the antiadhesive substance having the ability to cause the surface of the partition and simplify the mechanical and technical manipulirua the way to prevent or significantly reduce unwanted adhesion of the adjacent or surrounding tissues and organs in the healing of wounds.

The next object of the invention is the stimulation of tissue regeneration in connection with the healing of wounds.

For these and other purposes that will be explained below, this invention provides a new use of chitosan and polysaccharide immobilized with him, with the specified polysaccharide selected from heparin, heparan sulfate and dextran sulfate. Using such a composition can be obtained a substance having the ability to eliminate or significantly reduce unwanted adhesion of damaged tissue with adjacent or surrounding tissues in connection with the healing of wounds.

Used the polysaccharide can be immobilized with chitosan mainly in three different ways. Thus, the linking can be done ionic binding, covalent binding and mechanical incorporation in chitosan by deposition from a solution. The way covalent binding of the corresponding polysaccharide to a substrate, which is a carrier of amino groups is described in U.S. patent N 4.613.665.

As polysaccharide is particularly preferable to use heparin or heparan sulfate, these substances are commercially available on the market and are supplied by several manufacturers. M is aktivnosti.

The antiadhesive substance used in the present invention, may be present in various physical forms, such as films or membranes, gels, tubes or sleeves, powders, aerosols or solutions. Proper form, of course, opt for specific damage. In most cases, using film, while tubes or gels can be used in some cases, for example, associated with narrow extensions of organic tissues, such as muscles and tendons.

Chitosan is a linear 1,4-linked polysaccharide consisting of D-glucosamine units. Chitosan receive N-deacetylation of chitin, a polymer forming the shell inter alia insects and mollusks. For commercial purposes chitin is extracted from the shells of crabs and shrimp, which are waste from the fishing industry. By regulating alkaline processing chetinov you can get chitosans with different degrees of N-acetylation. When processing of chitin with concentrated alkali, usually sodium hydroxide, is N-deacetylation, i.e. acetaminop converted into amino groups with the formation of chitosan.

Physical properties of chitosan determine its use, depend on the extent of the sustained fashion system, and secrete lysozyme and other enzymes in body fluids.

In connection with the use of the present invention, preferably, when the chitosan has a degree of N-acetylation maximum of about 90% or more, most preferably about 50%. Particularly preferably, when the degree of N-acetylation of chitosan less than about 25%.

The present invention also provides a method of preventing or significantly reducing unwanted adhesion of the tissue in connection with wound healing. This method includes overlaying the plot selecively wounds substances containing chitosan and polysaccharide immobilized with him and selected from heparin, heparan sulfate and dextran sulfate.

Depending on the nature of the wounds of this substance can be applied in the form of a film, in the form of a gel or in the form of a tube or sleeve. You can easily choose the product for use in connection with, for example, a surgical operation.

Further, the invention is illustrated is not limiting examples. In the examples all films get in Petri dishes having a surface area of 54 cm2.

EXAMPLE 1

Obtaining chitosan films

5 g hydrochloric salt of chitosan (50% is the W and drying in a drying Cabinet at 70oC for 24 hours. Then, the resulting film is neutralized by adding sodium phosphate buffer, 0.2 M, pH of 9.0. The film is kept in a Petri dish in the specified buffer at room temperature for 2-4 h, and then washed 3-4 times with water and allowed to dry.

EXAMPLE 2

Obtaining chitosan films

5 g hydrochloric salt of chitosan (20% degree of acetylation, Pronova) dissolved in 2% acetic acid solution (0.5 l, 1% vol./weight.). The solution is treated in an autoclave for 1 hour at 125oC for sterilization. After cooling the film get in the Petri dish, in this case using 20 ml of solution. Then the film is allowed to dry at room temperature and neutralized by adding sodium phosphate buffer, 0.2 M, pH of 9.0, which is added to the Cup. The film is kept in this buffer for 2-4 h at room temperature, then washed with distilled water 3-4 times and let it dry.

EXAMPLE 3

Attestation decomposition heparin

One gram of heparin dissolved in 300 ml of water. The solution is cooled to 0oC in ice water and keep refrigerated. First, add 10 mg of sodium nitrite (NaNO2). Then to the solution with stirring debbaut at temperatures below 0oC. Obtain 0.7 g of decomposed heparin.

EXAMPLE 4

Periodate oxidation of heparin

The solution is oxidized periodates sodium Matricaria prepared in the following way. One gram of periodate sodium NaIO4dissolve in 200 ml of distilled water. Ten grams of Matricaria added to a solution of periodate sodium and stirred overnight in the dark. The resulting solution after adding 10 ml of glycerol and stirring for two hours cialiswhat with water. Water change every hour. This gives a solution containing oxidized periodata heparin at a concentration of about 19 mg/ml

EXAMPLE 5

Obtaining chitosan films with covalently-bound heparin (end connection)

To the neutralized chitosan film obtained by the method of Example 1, add 20 ml of a solution containing 125 mg nitrite is decomposed heparin, obtained as in Example 3, dissolved in 0.5 l of water containing 4.4 g HaCl. To the solution was added 15 mg of cyanoborohydride sodium. the pH of the solution was adjusted to 3.9 using 0.5 M hydrochloric acid or another acid. The solution containing chitosan film, allow to settle at room temperature for 14 h, then treatments and with covalently-bound heparin (multiple connection)

Neutralized chitosan film obtained by the method of Example 2, was incubated for 24 hours in 20 ml of the following solution: 4.4 g of sodium chloride and 125 mg oxidized periodata heparin, obtained as described in Example 4, dissolved in 0.5 l of water, pH adjusted to 3.9 using 0.5 M hydrochloric acid. To the solution was added 15 mg of cyanoborohydride sodium and incubated for 10 h at room temperature. The treated film is then washed with water 3-4 times and allowed to dry. Regarding details on how covalent binding of heparin, reference the aforementioned U.S. patent N 4.613.665.

EXAMPLE 7

Obtaining chitosan films with ion-associated heparin

Neutralized chitosan film get as in Example 2. Add a solution of heparin (125 g in 0.5 l of water containing 4.4 g of NaCl). After incubation for 3 h at room temperature, the film was washed with 2 x 0.5 l of water and dried.

EXAMPLE 8

Biological test control

The film obtained in accordance with Example 2, is used as an anti-adhesion membrane in the next model of the animal.

The abdominal wall of the rat reveal and on each side of the sagittal LPO Example 2, the piece size is about I mm, while the more damage is left open. The membrane sew surgical suture, using Dexon7-0 so that no seam is not exposed in the abdominal cavity. The result is evaluated after 2 and 4 weeks. In this regard, there is a little adhesion to the membrane in the abdominal cavity, while the uncoated film damage will give a strong adhesion.

Damage to the abdominal cavity under film heals mainly with the formation of scar tissue and there are signs of inflammatory reaction and formation of capsules around the film.

EXAMPLE 9

Biological testing in accordance with the invention

The film obtained by the method of Example 3, used as an anti-adhesion membrane in the next model of the animal.

Open the abdominal cavity of the rat and on both sides of the sagittal line cause surgical wound size 12 x 10 mm

One damage cover film of about I mm, while the more damage is left open. The membrane sew as in Example 8.

The injury area left open, showing several of the adhesion lipani, and in the field, covered with a film of ion-associated heparin

5 g hydrochloric salt of chitosan (54% degree of acetylation, Pronova) dissolved in water (0.5 l, 1% vol./weight.). The solution is sterilized in an autoclave for one hour at 125oC. After cooling the film get in the Petri dish, using 20 ml of solution. Then the film is allowed to dry at room temperature and add a solution of heparin (125 g in 0.5 liters of water). After incubation for 3 hours at room temperature the film is washed 2x0,5 l of water and dried.

EXAMPLE 11

Obtaining chitosan films

5 g hydrochloric salt of chitosan (45% degree of acetylation, Pronova) dissolved in water (0.5 l, 1% vol./weight.). The solution is sterilized in an autoclave for 1 h at 125oC. After cooling the film get in the Petri dish, using 20 ml of solution. The film is then allowed to dry at room temperature and neutralized by adding sodium phosphate buffer, 0.2 M, pH of 9.0, which is added into the Cup. The film is kept in this buffer for 2-4 h at room temperature and then washed with distilled water 3-4 times and let it dry.

EXAMPLE 12

Biological testing in accordance with the invention

Films obtained from chitosan-heparin, as described to enter the W is the wound inflicted on the opposite side of the abdominal cavity and covers the chitosan film, as described in Example 11. The emergence of adhesi evaluate in 2 weeks. The wound is covered with heparin-chitosan film, no adhesion, whereas on the other, covered with just chitosan film, there are small adhesi in small quantities. Light microscopic study of heparin-chitosan film finds improved wound healing, including area coverage mesothelial-like cells, as well as less extensive infiltration of inflammatory cells on the boundary surface between the heparin-chitosan film and injured tissue of the abdominal cavity than in the corresponding area covered just chitosan film.

EXAMPLE 13

Obtaining chitosan-heparin films

5 g hydrochloric salt of chitosan (16% degree of acetylation, Pronova) dissolved in 2% acetic acid solution (0.5 l, 1% vol./weight.). The solution is sterilized in an autoclave for 1 hour at 125oC. After cooling the film get in the Petri dish, using 20 ml of solution. Then the film is allowed to evaporate in an oven at 70oC for 16 hours. The film is treated with 0.1 M NaOH solution for 3 h at room temperature and then washed with distilled water 3-4 times and let it dry in natural heparin (1% wt./about., pork mucosa, Kabivitrum) in 0.2 M phosphate buffer (pH of 6.4). The film was incubated over night at room temperature, then washed with sterilized water and dried in LAF furnace. Four more films prepared by the method described above, with the difference that they are treated with 0.5%, 0,1%, 0,01% 0,00% solution of heparin, respectively. Heparinised film is subjected to elemental analysis, which shows that the films contain respectively 1,2%, 0,9%, 1,3%, 0,23 and 0.007% sulfur. These values correspond to the contents of heparin 9.2 percent, 7.7 percent, 10.8 percent, divided into six groups with ten biopsies 1.9% and 0%, respectively.

EXAMPLE 14

Cooking the Russian Academy of Sciences

Sterilized human skin obtained from masectomy samples. In each experiment using skin from a single donor. Under sterile conditions, cut out circles with a diameter of 6 mm with a tool for excision of skin (Stiefel Laboratories, UK). In the center of each sample on the epidermal side is partially wound using a 3 mm dermatome and the corresponding samples are placed in a 12-hole plate (Costar) epidermal side up. Each well is filled modified by Dulbecco environment of EGL (DMEM) to the epidermal level, supporting the wound on the surface of the section of gas/LM and streptomycin 50 mg/ml).

EXAMPLE 15

Test on heal

In vitro wound described in Example 14, were divided into five groups with 10 biopsies in each group. Each wound was covered with heparinised film as described in Example 13. The medium was changed every day. After seven days, the samples were fixed in 4% neutral buffered formaldehyde solution, dehydrational through an ethanol-xylene series and immersed in paraffin. Section size 10-20 mm in thickness, stained with hematoxylin and eosin and the degree of reepithelialization determined using a light microscope. Only the wound is completely covered by keratinocytes, are healed. As can be seen from Fig. 1, the film containing heparin below 2% not stimulate cell proliferation.

EXAMPLE 16

Getting gel compositions

For the preparation of the following four gel compositions using water containing 0.9% NaCl:

A = 2% methylcellulose

B = 2% methylcellulose + 0.2% Matricaria

C = 0.5% methylcellulose + 1% chitosan (16% acetylation)

D = 0.5% methylcellulose + 1% chitosan (16% acetylation) + 0.2% Matricaria

EXAMPLE 17

Test healing in vitro

In vitro wound described in Example 14, are divided into six groups with ten biopsies in kadoglou group treated with medium (2% FCS). The environment is changing every day. After seven days, the samples fixed in 4% neutral buffered formaldehyde solution, dehydration through ethanol-xylology series and immersed in paraffin. Section size 10-20 mm in thickness, stained with hematoxylin and eosin and the degree of reepithelialization evaluated using a light microscope. Only the wound is completely covered with the keratinocytes, are healed. As can be seen from Fig. 2, gel, consisting of a combination of chitosan with heparin, heals wounds better than the gel containing only the chitosan or heparin.

EXAMPLE 18

Biological testing in accordance with the invention

Repeat the procedure of Example 9 using the film obtained by the method of Example 7.

As is clear from biological experiments described above, using the methods of the present invention can significantly improve the quality of healing by preventing adhesion and growth. The invention is not limited to the examples above, and its volume is limited only by the attached claims.

With regard to the application according to the invention, it should be noted that films and/or membranes, gels or powders obtained to the second cavity; chest; lung; heart atrium; Central vessels; gastrointestinal tract; urinary tract; the meninges; spinal cord; the tendons, nerves, muscles, bones, mucous membrane, cornea, skin, etc.

Products in the form of tubes or sleeves or gels can be used as a guide to stimulate growth and at the same time you can save sliding surface as possible to avoid adhesion with the environment. Such products can be used to damage the nerves, tendons and ligaments, gastrointestinal tract, urinary tract, blood vessels, etc.

Probably more can be done to promote quality of healing by combining the present invention with growth factors.

1. Chitosan and polysaccharide immobilized with him and selected from heparin, heparan sulfate and dextran sulfate as substances that can prevent or reduce undesirable adhesion of damaged tissue with adjacent or surrounding tissue in healing wounds.

2. Application under item 1, characterized in that the polysaccharide immobilized with chitosan using ionic bond.

3. Application under item 1, characterized in that the polysaccharide mctow, characterized in that the polysaccharide is heparin or heparan sulfate.

5. The use according to any one of the preceding paragraphs, characterized in that the substance has the form of a film or membrane.

6. The use according to any one of paragraphs.1 to 4, characterized in that the substance has the form of a tube or sleeve.

7. Application under item 6, characterized in that the substance is in the form of a gel.

8. Application under item 6, characterized in that the substance is in the form of a powder, aerosol or solution.

9. The use according to any one of the preceding paragraphs, characterized in that the chitosan has a degree of N-acetylation most about 90%, and most preferably about 50%.

10. The way to prevent or reduce undesirable adhesion of damaged tissue with adjacent or surrounding tissue in healing wounds, characterized in that region selecively wounds apply a substance containing chitosan and polysaccharide immobilized with him, selected from heparin, heparan sulfate and dextran sulfate.

11. The method according to p. 10, characterized in that the substance is used in the form of a film or membrane.

12. The method according to p. 10, characterized in that the substance used CLASS="ptx2">

14. The method according to p. 10, characterized in that the substance is used in the form of a powder, aerosol or solution.

 

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SUBSTANCE: a sow should be twice injected with oxytocin and, additionally, intramuscularly about 2-4 h after afterbirth detachment one should introduce clathroprostin at the dosage of 1 ml. The innovation suggested is very efficient in preventing metritis-mastitis-agalactia and endometritis in sows, as well.

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1 ex, 1 tbl

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3 ex

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