RU2447082C1 - Method of stimulating skin and mucous lining defect repair and medicinal agent for realising said method - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology. Described is a method of stimulating repair of skin and mucous lining wound defects, involving local application of a prodrug containing an effective amount of bacterial lipopolysaccharides. Disclosed is a prodrug for stimulating repair of skin and mucous lining wound defects, characterised by that it is in form of a gel and contains bacterial lipopolysaccharides, a water-soluble cellulose derivative, a polybasic alcohol, preservatives and treated water, with the following ratio of components in g per 100 g gel: bacterial lipopolysaccharides 0.002-5.00, water-soluble cellulose derivative 1.00-10.00, polybasic alcohol 2.00-15.00, preservatives 0.0001-1.00, treated water - up to 100.00.

EFFECT: invention widens the range of agents and methods which enable to repair wood defects.

10 cl, 7 dwg, 7 ex

Description

A method of stimulating the regeneration of wound defects of the skin and mucous membranes and a drug for its implementation.

The invention relates to medicine and pharmacology, and in particular to methods of stimulating regeneration (the ability to induce and / or accelerate regeneration processes) of the skin and mucous membranes, as well as to medicines having a multifactorial effect on the processes of regeneration and inflammation, and can be used in surgery, including in cosmetic surgery, dermatology, cosmetology, gynecology, proctology, urology, dentistry, otorhinolaryngology.

A known method for the prevention and treatment of infections of the cornea using lipopolysaccharides Pseudomonas aeruginosa (US Patent No. 6984622). The disadvantage of this method is the limited scope (ophthalmology). In addition, the disadvantages of this method include the lack of effectiveness associated with the chemical structure of the lipopolysaccharides of Pseudomonas aeruginosa.

Known drug "Pyrogenal" containing bacterial lipopolysaccharides Salmonella typhi, intended for systemic administration and produced in the form of solutions for injection, as well as rectal suppositories, used for the treatment of chronic liver diseases, chronic prostatitis and urethritis, inflammatory processes of the uterus, infertility , adhesive diseases of the abdominal cavity, psoriasis and other diseases (RF patent 2139043). The mechanism of action of the drug is associated with activation of the innate immune response, which is accompanied by a pyrogenic effect (hyperthermia, fever). In the treatment of wound defects of the skin and / or mucous membranes, a systemic route of administration is not advisable, because it is impossible with systemic administration, given the high metabolic rate of this compound, to achieve therapeutic concentrations of lipopolysaccharides in the wound defect. The disadvantages also include the presence of a pyrogenic effect.

A known method of treating wounds, consisting in the use of albumin-free calf blood extract (US Patent No. 4177261). The well-known gel "Solcoseryl", in which the deproteinized hemodialysate containing low molecular weight components of the cell mass and blood serum of dairy calves is used as the active component (VIDAL Handbook. Medicinal Products in Russia: Handbook. M .: AstraFarmService, 2010. 1728 p. - p. B-1173). It is used as a stimulator of regeneration processes. The disadvantages of this method include the difficulty of standardizing the drug due to the complexity of the composition of the active substances represented by a wide range of various chemical compounds, as well as lack of effectiveness. The disadvantage of this method is the potential insecurity of the drug for patients, which consists in the possible contamination of the drug with pathogens of diseases that may be present in the blood of cattle (for example, spongiform encephalopathy viruses).

The objective of the invention is to increase the effectiveness of the treatment of wound defects of the skin and mucous membranes of various etiologies and localizations, increase the safety of therapy, ensure the standardization of the content of the active substance in the preparation, and also create a highly effective drug from the point of view of bioavailability that has a multi-factor therapeutic effect on inflammatory and regenerative processes of wound defects.

The problem is achieved due to the fact that the method of stimulating the regeneration of wound defects of the skin and mucous membranes is carried out by topical application of a dosage form containing an effective amount of bacterial lipopolysaccharides. The producer of these bacterial lipopolysaccharides is Salmonella typhi, or Escherichia coli. Moreover, the inventive method is used to stimulate the regeneration of wound defects of the skin and mucous membranes of various etiologies and localizations. The concentration of bacterial lipopolysaccharides is in the range of 0.002-5.0 g per 100 g of dosage form. Bacterial lipopolysaccharides and excipients are contained in a pharmaceutically acceptable carrier adapted for topical application. A dosage form containing an effective amount of bacterial lipopolysaccharides is made in the form of a gel and contains bacterial lipopolysaccharides, a water-soluble cellulose derivative, polyhydric alcohol, preservatives and purified water in the following ratio of components in g per 100 g of gel:

Bacterial Lipopolysaccharides   0.002-5.00 Water Soluble Cellulose Derivative 1.00-10.00 Polyhydric alcohol 2.00-15.00 Preservatives 0.0001-1.00 Purified water Up to 100.00

the carboxymethyl cellulose sodium salt is used as a water-soluble cellulose derivative, glycerol or propylene glycol is used as the polyhydric alcohol, and nipagin, or nipazole, or nipagin and nipazole are used as a preservative.

Bacterial lipopolysaccharides and excipients contained in a pharmaceutical carrier can be used in the form of a solution for topical application, a rinse, gel, cream, paste, ointment, liniment, transdermal therapeutic system, patch, powder, powder, glue, powder, lotions , suspensions, lotion, balm, shampoo, aerosol, spray, tablets for resorption in the oral cavity, lozenges for resorption in the oral cavity, vaginal tablets, vaginal suppositories, intranasal, eye and ear capes eh, eye films, ear candles, dressings.

The invention consists in the following.

The proposed method of treatment consists in topical application to the area of a wound defect in the skin or mucous membrane using a therapeutically substantiated dosage regimen, which contains bacterial lipopolysaccharides as the active ingredient. The producer of these bacterial lipopolysaccharides is Salmonella typhi, or Escherichia coli. Moreover, the inventive method is used to stimulate the regeneration of wound defects of the skin and mucous membranes of various etiologies and localizations. Improving the effectiveness of treatment of wound defects of the skin and mucous membranes is achieved by the multifactorial effect of bacterial lipopolysaccharides when applied topically to inflammation, debridement and regeneration. The effects of topical application of bacterial lipopolysaccharides include activation of the innate immune response system, which determines its local antibacterial and antiviral effect, in the healing of wounds from necrotic tissues due to stimulation of the secretion of several enzymes and enhancement of the phagocytosis process, as well as stimulation of regeneration processes by enhancing the secretion of biologically active substances , which determines its regenerative effect. An increase in the safety of therapy is achieved by the absence of animal components in the preparation. The expansion of the scope is achieved through the use of dosage forms for the treatment of wound defects of the skin and mucous membranes of various etiologies and localizations, adapted for local use. The ability to standardize the drug is achieved by using bacterial lipopolysaccharides, similar in chemical structure and biological properties, as an active component.

The tool is made in the form of a gel and contains bacterial lipopolysaccharides, a water-soluble cellulose derivative, polyhydric alcohol (glycerin or propylene glycol), preservatives and purified water in the following ratio of components in g per 100 g of gel:

Bacterial Lipopolysaccharides   0.002-5.00 Water Soluble Cellulose Derivative 1.00-10.00 Polyhydric alcohol 2.00-15.00 Preservatives 0.0001-1.00 Purified water Up to 100.00

Bacterial lipopolysaccharides (LPS) are biomolecules composed of covalently linked lipid and polysaccharide moieties. LPS is the main component of the outer membrane of the cell wall of gram-negative bacteria, ensuring the structural integrity of microorganisms.

LPS induces a complex humoral and cellular response of the macroorganism's immune system mediated by toll-like receptor 4 (TLR-4). According to modern concepts, the mechanism of action of LPS is as follows. After the binding of LPS molecules to the extracellular domains of TLR-4, dimerization of neighboring receptors occurs, which leads to a change in the conformation of intracellular domains. A change in the conformation of the latter leads to the appearance of binding sites for adapter molecules, the recruitment and activation of which triggers a cascade of reactions that ultimately lead to the activation of a number of transcription factors. Transcription factors alter the expression of many genes whose products are involved in the response of a macroorganism to the presence of a microorganism or its structural molecules. It is also known that TLR-4 is involved in the processes of activation and differentiation of dendritic cells, as well as B and T lymphocytes.

Local application of LPS helps to reduce the microbial contamination of wound defects, and also stimulates the healing process. This effect is based on the ability of LPS to alter (increase) the reactivity of the immune system and nonspecific macroorganism defense factors.

The antibacterial effect of LPS is caused by the secretion of macroorganism by cells in response to the use of LPS by endogenous antimicrobial factors, such as defensins (attach to the pathogen’s cell membrane and deepen into it, forming pores), lysozyme (destroys bacterial cell walls by hydrolysis of muramylglucosamine cell wall), perforins ( pore formation in the cytoplasmic membrane of bacteria), lactoferrin (binds iron and deprives the bacterial microflora of the microelement necessary for its growth and vital activity nt), as well as reactive oxygen species and nitric oxide, exhibiting antimicrobial activity and stimulating phagocytosis. LPS activates the complement system, which leads to the stimulation of phagocytosis due to the opsonization of bacteria, as well as to an increase in cytotoxic function.

The antiviral effect of LPS is associated with the induction of secretion of endogenous β and γ interferons, which inhibit the synthesis of viral proteins and inhibit virus replication. In addition, interferon γ stimulates the cellular immune response.

The effect of wound debridement from necrotic tissues when using LPS is associated with the secretion of a number of enzymes, such as collagenase and elastase, providing enzymatic destruction of necrotic tissues, as well as with the stimulation of the phagocytosis process associated with the absorption and elimination of cellular detritus.

The regenerative effect of LPS is mediated by stimulation of the secretion of a number of factors that regulate the processes of regeneration and development of connective tissue:

the main fibroblast growth factor (bFGF) stimulates the processes of angiogenesis in the wound defect, proliferation and differentiation of various types of cells in the wound, chemotaxis of fibroblasts and keratinocytes, and also stimulates the production of extracellular matrix components (fibronectin and collagen) by fibroblasts;

vascular endothelial growth factor (VEGF) stimulates the process of angiogenesis in a wound defect (proliferation, migration of endothelial cells and the formation of new blood vessels);

transforming growth factor β (transforming growth factor beta, TGF-β) stimulates the proliferation, differentiation and apopotosis of various types of connective tissue cells, stimulates chemotaxis of fibroblasts and their production of collagen and fibronectin;

epidermal growth factor (EGF) stimulates the proliferation and differentiation of various epidermal and epithelial cells, including fibroblasts and keratinocytes, is a chemoattractant for these types of cells, stimulates the process of angiogenesis;

as well as a number of other factors involved in the processes of proliferation, differentiation, migration and apoptosis of various cells during the regeneration of damaged tissues.

Thus, the local use of LPS has a complex therapeutic effect on the wound process, consisting in reducing microbial contamination; stimulation of the process of cleaning the wound from necrotic tissue; stimulation of regeneration.

As therapeutic concentrations of bacterial lipopolysaccharides in the dosage form, a range of 0.002-5.00 g per 100 g of the form is selected. The range is determined on the basis of experimental data and literary information (Kraitserov B.V. Conceptual approach to pyrotherapy from the perspective of stress theory. M: Labyrinth, 2001. - 560 p.).

For the treatment of wound defects, mainly wounds during the second phase of the wound process, it is proposed to use a gel on a hydrophilic basis, containing LPS as an active component. As a hydrophilic base, it is proposed to use water-soluble cellulose derivatives, for example, sodium salt of carboxymethyl cellulose (sodium carmellose) (Monograph 01/2008: 0472 European Pharmacopoeia European Pharmacopoeia 6.0, Strasbourg, 2008. Volume 2. Pages 1423-1424).

The advantages of these basics:

- have a wound healing effect;

- create a film on the surface of the wound that protects the wound from environmental factors, including from microbial contamination;

- have a weak osmotic effect;

- do not interfere with gas exchange;

- hypoallergenic.

For the proposed composition of the dosage form, taking into account the nature and properties of its constituent ingredients, a preparation technology has been developed consisting of the following successive stages: dissolving the LPS in purified water; the introduction of preservatives into the resulting solution, for example, nipagin-methyl parahydroxybenzoate, nipagin (Monograph 01/2008: 0409 European Pharmacopoeia European Pharmacopoeia 6.0, Strasbourg, 2008. Volume 2. Pages 231-2392) and nipazole-propyl parahydroxybenzoate, nipazole (Monograph 01/2008: 0431 European Pharmacopoeia European Pharmacopoeia 6.0, Strasbourg, 2008. Volume 2. Pages. 272-2773). Nipagin and nipazole are well-known preservatives that are approved for use in the pharmaceutical industry in the manufacture of dosage forms (State Pharmacopoeia of the USSR. 11th edition. M: Medicine, 1990. Issue 2, p. 162) - (solution A); gel preparation of a water-soluble cellulose derivative; mixing and homogenizing solution A, gel and polyhydric alcohol, for example glycerol (Monograph 01/2008: 0496 European Pharmacopoeia 6.0, Strasbourg, 2008. Volume 2. Pages. 1987-1988), or propylene glycol (Monograph 01/2008: 0430 European Pharmacopoeia European Pharmacopoeia 6.0, Strasbourg, 2008. Volume 2. Pages. 273-2774).

1st stage. LPS is dissolved in purified water with stirring. Preservatives are added to the resulting solution with stirring, stirring is continued until the preservatives are completely dissolved (solution A).

2nd stage. A gel of a water-soluble cellulose derivative is prepared. For this purpose, a sample of a water-soluble cellulose derivative is added to purified water heated to 50-70 ° C, left to swell (1.5-2 hours), and then thoroughly mixed.

3rd stage. The gel of the water-soluble cellulose derivative is combined with solution A, polyhydric alcohol is added and homogenized to obtain a uniform gel.

Example 1. Obtaining a gel.

0.20 g of LPS is dissolved in 50 g of purified water with stirring. 0.08 g of nipagin and 0.02 g of nipazole are introduced into the resulting solution with stirring, stirring is continued until the preservatives are completely dissolved (solution A). To 30 g of purified water heated to 50-70 ° C, add 5.00 g of sodium salt of carboxymethyl cellulose and leave for 1.5-2 hours until swelling, then mix thoroughly. The gel of the sodium salt of carboxymethyl cellulose is combined with solution A, 10.00 g of glycerol and 4.70 g of purified water are added and homogenized to obtain a uniform gel.

The resulting gel remains stable for three years in a number of indicators: rheological and microbiological stability, specific activity, etc.

A number of studies have established that the lipopolysaccharides of bacteria of the genus Salmonella have biological activity that is almost equivalent to the biological activity of E. coli lipopolysaccharides (Structure and function of lipopolysaccharides, review article, p. 846, “Structure and function of lipopolysaccharides” Erridge C., Bennett-Guerrero E. , Poxton IR // Microbes and Infection. 2002 Jul; 4 (8): 837-851.)

Further studies were performed using lipopolysaccharides of bacteria of the genus Salmonella.

Example 2. Evaluation of the effectiveness of the proposed method. Biomechanical tests.

The strength of a wound defect is an indicator characterizing the overall degree of wound healing, indirectly characterizing the processes of fibroblast proliferation and collagen formation.

The experiment used ICR mouse outbreds, males weighing 26-30 g, in an amount of 95 individuals. Animals were acclimatized within 10 days. Mechanical depilation was performed with an animal trimmer Oster Mark II (USA). Animals were anesthetized with zoletil-xylazine anesthesia (45 mg / kg zoletil and 7.5 mg / kg xylazine intramuscularly). The surgical field was treated with 70% ethanol. On the back of the animals along the ridge line, the epidermis, dermis and subcutaneous tissue were cut with a scalpel to the fascia so as to create a 4 cm incision. The wound defect was sutured with two sutures using the Polycon polyamide suture material. After the operation, animals were randomly assigned to groups and placed in cages individually. Animals were given free access to feed and water. Therapy was started on the first postoperative day and consisted of daily local application to the wound area of 150 μl of the corresponding drug.

Groups of animals participating in the experiment:

control groups, including: C1 - conditionally intact animals (all the above manipulations were performed with animals, except for surgical operations and therapy), the number of animals was 5; group C2 - all the above manipulations were performed with animals, with the exception of therapy, the number of animals - 15 group C3 - all the above manipulations were performed with animals, placebo therapy (with the gel of the above composition and manufacturing method, but without LPS), the number of animals - 15; group C4 - all the above manipulations were performed with animals; therapy with the reference drug - Solxoseryl gel dosage form manufactured by Valeant Pharmaceuticals Switzerland GmbH, Switzerland;

test groups, including: T1 - all the above manipulations were performed with animals, gel therapy containing LPS Salmonella typhi at a concentration of 0.2 mg / g dosage form; T2 - all the above manipulations were performed with animals, gel therapy containing LPS Salmonella typhi at a concentration of 2 mg / g dosage form; T3 - all the above manipulations were performed with animals; gel therapy containing LPS Salmonella typhi at a concentration of 10 mg / g of the dosage form.

Five animals from each group (with the exception of group C4) were withdrawn from the experiment on the 7th, 14th, and 21st days of the experiment. Animals of group C4 were withdrawn from the experiment on the 7th day.

Biomechanical tests were carried out according to the methodology presented in The effect of topical tretinoin on tissue strength and skin components in a murine incisional wound model. Muehlberger T., Moresi J.M., Schwarze H., Hristopoulos G., Laenger F., Wong L. // Journal of the American Academy of Dermatology, 2005 Apr 52 (4): 583-588.

For comparative tests were used: placebo, the inventive gel containing bacterial lipopolysaccharides, the dosage form "Solcoseryl gel". The obtained biomechanical test data are presented in figure 1, where H is the force required to rupture a wound defect. The data are presented for all groups in the form of a histogram, and for groups C3, C4 and T3 - also in the form of a graph. The figure shows the values in the format: "average ± standard deviation." * p <0.05 compared with the placebo group, calculated using Student's criterion.

The skin of animals containing a wound defect was cut into strips 3 cm long and 0.5 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. Strips of skin were placed in a phosphate buffered isotonic solution containing antibiotics for 12 hours prior to testing. The tests were carried out on an Instron 5848 Microtester installation (England) using a 50 N sensor. The distance between the clamps was 20 mm, the speed of the lower clamp was 1 cm / min, while the upper clamp remained motionless. The force required to break the strip of skin along the wound defect was fixed.

The data presented indicate an increase in the force required to rupture a wound defect during therapy according to the proposed method (gel at a dosage of 2 mg / g and 10 mg / g) at all experimental times compared to placebo therapy and Solcoseryl gel dosage form therapy.

Example 3. Evaluation of the effectiveness of the proposed method. Assessment of inflammation in a wound defect.

Inflammation is a necessary step in the healing process, but prolonged inflammation can lead to a delay in the regeneration of wound defects.

The experimental conditions are shown in example 2.

The inflammation was evaluated using the method described in the publication “The effect of topical tretinoin on tissue strength and skin components in a mouse model of cut wounds.” The effect of topical tretinoin on tissue strength and skin components in a murine incisional wound model. Muehlberger T. , Moresi JM, Schwarze H., Hristopoulos G., Laenger F., Wong L. // Journal of the American Academy of Dermatology, 2005, 52 (4): 583-588.

The skin of animals of the middle part of the wound was cut into strips 3 cm long and 1 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. The material was subjected to histological examination. Skin strips were fixed in 10% formalin. Posting of samples was carried out according to standard methods. In the manufacture of paraffin blocks, the material was oriented so that the cut surface was perpendicular to the length of the wound defect. Histological examination of the samples was carried out on paraffin sections with a thickness of 4 μm, stained with hematoxylin and eosin according to standard methods. A histological examination evaluated the process of inflammation in a wound defect.

A week after wounding in groups C2 and C3, pronounced inflammatory phenomena were observed. The presence of ulcerative defects of the epidermis with a bottom formed by necrotic detritus, delimited from the surrounding tissue by a leukocyte shaft, was noted. A significant amount of histiocytes and neutrophilic leukocytes was noted in the forming scar tissue. In groups C4, T1, T2, T3, a smaller number of histiocytes and the absence of neutrophilic leukocytes in the scar tissue were observed. After two weeks, inflammatory changes were present only in individual animals of groups C2 and C3, in groups C4, T1, T2, T3 there were no inflammatory phenomena. By the third week, there was a lack of inflammation in all groups.

Representative photographs of histological sections are presented in figure 2 - assessment of the process of inflammation in a wound defect. The time point is the first week of the experiment. Hematoxylin-eosin stain. 40x magnification. Where A - without therapy, B - placebo therapy, C - Solcoseryl gel therapy, G - claimed gel therapy 0.2 mg / g LPS, D - declared gel therapy 2 mg / g LPS, E - declared gel 10 mg / g LPS

The data presented indicate that the use of therapy according to the proposed method (gel at a dosage of 0.2 mg / g, 2 mg / g and 10 mg / g) contributes to the rapid completion of the inflammation process.

Example 4. Evaluation of the effectiveness of the proposed method. Assessment of the process of angiogenesis in a wound defect.

The experimental conditions are shown in example 2.

Angiogenesis is necessary for wound healing in the early stages of the wound healing process. The resulting capillaries provide the wound area with blood, oxygen and nutrients.

The evaluation of the process of angiogenesis was carried out according to the methodology presented in the publication “A synthetic analogue of uric acid accelerates the healing of skin wounds in mice”. A synthetic uric acid analog accelerates cutaneous wound healing in mice. Chigurupati S., Mughal M.R., Chan S.L., Arumugam T.V., Baharani A., Tang S.C., Yu Q.S., Holloway H.W., Wheeler R., Poosala S., Greig N.H., Mattson M.P. // PLoS One. 2010 6; 5 (4): e10044.

The skin of animals of the middle part of the wound was cut into strips 3 cm long and 1 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. Skin strips were fixed in 10% formalin. The material was subjected to histological examination. Posting of samples was carried out according to standard methods. In the manufacture of paraffin blocks, the material was oriented so that the cut surface was perpendicular to the length of the wound defect. Histological examination of the samples was carried out on paraffin sections with a thickness of 4 μm, stained with hematoxylin and eosin according to standard methods. Histological examination evaluated the following criteria: the number of capillaries in one field of view at a magnification of × 400. A blood vessel was not considered as a capillary if it had an elastin membrane, smooth muscle cells, or more than three red blood cells in the lumen.

The data obtained are presented in figure 3 - assessment of the process of angiogenesis in a wound defect. The data are presented for all groups in the form of a histogram, and for groups C3, C4 and T3 - also in the form of a graph. The figure shows the values in the format: "average ± standard deviation." * p <0.05 compared with the placebo group, calculated using Student's criterion.

According to the data obtained, the use of therapy (gel in a dosage of 2 mg / g and 10 mg / g) according to the proposed method contributes to the process of angiogenesis in the early stages of wound healing, while using the comparison drug and placebo therapy the maximum number of capillaries in a wound defect observed only two weeks after wounding.

Example 5. Evaluation of the effectiveness of the proposed method. Assessment of the fibroplasia process in a wound defect.

Fibroplasia is necessary in the early stages of the wound healing process. Activated fibroblasts are involved in the process of collagen formation, and also secrete a number of regulatory molecules. At the same time, the presence of fibroblasts in the later stages is undesirable, as this indicates a delay in regeneration and is associated with the formation of a rough scar.

The experimental conditions are shown in example 2.

The fibroplasia process was evaluated according to the methodology presented in the publication “Therapeutic effects of topical use of ozone on the healing of skin wounds”. Therapeutic effects of topical application of ozone on acute cutaneous wound healing. Kim M.S., Noh S.U., Han Y.W., Kim K.M., Kang H, Kim H.O., Park Y.M. Journal of Korean medical science. 2009, 24 (3): 368-374.

The skin of animals of the middle part of the wound was cut into strips 3 cm long and 1 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. Skin strips were fixed in 10% formalin. The material was subjected to histological examination. Posting of samples was carried out according to standard methods. In the manufacture of paraffin blocks, the material was oriented so that the cut surface was perpendicular to the length of the wound defect. Histological examination of the samples was carried out on paraffin sections with a thickness of 4 μm, stained with hematoxylin and eosin according to standard methods. A histological examination evaluated the following criterion: the total number of fibroblasts and fibrocytes in one field of view at a magnification of × 400.

The data obtained are presented in figure 4 - assessment of the process of fibroplasia in a wound defect. The data are presented for all groups in the form of a histogram, and for groups C3, C4 and T3 - also in the form of a graph. The figure shows the values in the format: "average ± standard deviation." * p <0.05 compared with the placebo group, calculated using Student's criterion.

According to the data obtained, the use of therapy according to the proposed method (gel at a dosage of 2 mg / g and 10 mg / g) contributes to the process of fibroplasia in the early stages of wound healing (first week), while the number of fibroblasts and fibrocytes decreases sharply in the second and third weeks. When compared with a comparison drug, this effect is less pronounced. When using a placebo, maximum fibroplasia occurs in the second week, while the number of fibroblasts and fibrocytes remains high by the end of the third week, which may indicate a delay in the regeneration process.

Example 6. Evaluation of the effectiveness of the proposed method. Assessment of the process of collagen formation in a wound defect.

The process of collagen formation is a key process of wound healing.

The experimental conditions are shown in example 2.

Assessment of the process of collagen formation was carried out according to the methodology presented in the publication “Therapeutic effects of topical use of ozone on the healing of skin wounds”. Therapeutic effects of topical application of ozone on acute cutaneous wound healing. Kim H.S., Noh S.U., Han Y.W., Kim K.M., Kang H., Kim H.O., Park Y.M. Journal of Korean medical science. 2009, 24 (3): 368-374.

The skin of animals of the middle part of the wound was cut into strips 3 cm long and 1 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. Skin strips were fixed in 10% formalin. The material was subjected to histological examination. Posting of samples was carried out according to standard methods. In the manufacture of paraffin blocks, the material was oriented so that the cut surface was perpendicular to the length of the wound defect. Histological examination of the samples was carried out on paraffin sections with a thickness of 4 μm, stained according to Masson according to the standard method. A histological examination evaluated the following criteria: morphology and color intensity of collagen fibers.

In samples of the first week, the color intensity of collagen fibers was reduced for all operated groups. On the samples of the second week for groups T2 and T3, the appearance of large in diameter, parallel to the surface of the epidermis of collagen fibers with a more intense Masson coloration, compared with the other operated groups. The samples of the third week during Masson staining showed the presence of collagen fibers having the same color intensity and thickness as the dermis fibers outside the wound defect. These changes are most pronounced in groups T2 and TK.

Representative photographs of histological sections are presented in figure 5. and Fig.6.

5 is an assessment of the process of collagen formation in a wound defect. Masson stain. Magnification × 100, time point - the second week of the experiment. Where A - without therapy, B - placebo therapy, C - Solcoseryl gel therapy, G - claimed gel therapy 0.2 mg / g LPS, D - declared gel therapy 2 mg / g LPS, E - declared gel 10 mg / g LPS

6. Assessment of the process of collagen formation in a wound defect.

The time point is the third week of the experiment. Masson stain. Magnification × 100. Where A - without therapy, B - placebo therapy, C - Solcoseryl gel therapy, G - claimed gel therapy 0.2 mg / g LPS, D - declared gel therapy 2 mg / g LPS, E - declared gel 10 mg / g LPS

The data presented indicate that the use of therapy (gel in a dosage of 2 mg / g and 10 mg / g) according to the proposed method stimulates the processes of collagen formation in a wound defect.

Example 7. Evaluation of the effectiveness of the proposed method. Assessment of the process of epithelization of a wound defect.

The dynamics of the epithelialization process, consisting in the formation of the epithelium at the site of damage to the skin or mucous membrane, is an important characteristic of the rate of regeneration.

The experimental conditions are shown in example 2.

The assessment of the epithelialization process was carried out according to the methodology presented in the publication “The effect of topical tretinoin on tissue strength and skin components in a mouse model of cut wounds.” The effect of topical tretinoin on tissue strength and skin components in a murine incisional wound model. Muehlberger T., Moresi JM, Schwarze H., Hristopoulos G., Laenger F., Wong L. // Journal of the American Academy of Dermatology, 2005, 52 (4): 583-588.

The skin of animals of the middle part of the wound was cut into strips 3 cm long and 1 cm wide, while the wound defect was located in the center of the strip perpendicular to the largest size. The material was subjected to histological examination. Skin strips were fixed in 10% formalin. Posting of samples was carried out according to standard methods. In the manufacture of paraffin blocks, the material was oriented so that the cut surface was perpendicular to the length of the wound defect. Histological examination of the samples was carried out on paraffin sections with a thickness of 4 μm, stained with hematoxylin and eosin according to standard methods. A histological examination evaluated the following criterion: the thickness of the epidermis in the area of the wound defect.

A week after wounding, groups C4, T1, T2, T3 showed complete epithelization of the wound defect with an acanthetically thickened epidermis, while ulcer defects of the epidermis were observed in groups C2 and C3. Two weeks later, in the groups C2, C3 and C4, the presence of an acanthetically thickened epidermis was noted, while in the groups T1, T2, T3 the number of layers of the epidermis was 2-3 layers, which corresponds to the thickness of the epidermis of intact animals (group C1). By the third week, the number of epidermis layers in all groups was 2-3 layers, which corresponds to the epidermis thickness of intact animals (group C1).

The data presented indicate that the use of therapy according to the proposed method (gel at a dosage of 0.2 mg / g, 2 mg / g and 10 mg / g) stimulates the processes of epithelization of a wound defect.

Example 8. Safety assessment of the proposed method.

The safety assessment of the proposed method was carried out using generally accepted methods (Guidelines for the experimental (preclinical) study of new pharmacological substances ”M: OJSC“ Publishing House “Medicine”, 2005. 832 pp.). For this study, mice were selected as the species generally accepted for safety studies. CD1 outbred mice were used. The number of animals in each group was 10 individuals in order to assess the nature and frequency of the manifestation of toxic effects and to subject the experimental results to statistical processing. Groups of animals participating in the experiment: A - application of a gel containing LPS Salmonella typhi at a concentration of 10 mg / g dosage form; B - application of a gel containing LPS Salmonella typhi at a concentration of 50 mg / g dosage form; B - applying a placebo (gel of the above composition and manufacturing method, but without LPS content). The application of drugs was carried out on pre-shaved skin of mice once a day for 14 days. Daily recorded body weight of animals, as well as the consumption of feed and water. A clinical examination of each animal was carried out during the first hour after the first application of the drug and then daily to register signs of intoxication, assess the behavior and general condition of the animals. With necropsy, macroscopic and gravimetric (weighing) studies of the following organs were performed: adrenal glands, brain, appendages of the testes, heart, kidneys, liver, lungs, spleen, testes / ovaries, thymus. Organs and tissues (liver, kidneys, heart, thymus, spleen, lymph nodes, place of application of the preparation (skin)) were taken from each opened animal and recorded in 10% formalin. Subsequently, histological sections of these organs and tissues were stained with hematoxylin and eosin. The material was subjected to histological examination.

According to the results obtained, no significant deviations of the experimental and control group indices by the studied parameters were found. In accordance with the data of histological studies, pathological changes in the studied organs / tissues of animals treated with LPS gel at a concentration of 10 mg / g and 50 mg / g were not detected.

7. Safety assessment of skin application of LPS gel with a concentration of 50 mg / g. Photographs of representative histological sections of the skin of mice.

The time point is 14 days. Hematoxylin-eosin stain. Magnification × 40. Where A is a placebo application, B is the claimed gel therapy with 50 mg / g LPS.

The data presented indicate the safety of using therapy by the proposed method.

According to a number of publications, the structure of lipopolysaccharides characteristic of E. coli and bacteria of the genus Salmonella is recognized to the greatest extent by the mammalian immune system, and a full spectrum of biological activity is manifested. At the same time, almost all known structures that differ from the indicated have a reduced biological activity. It is known that Pseudomonas aeruginosa lipopolysaccharides have significantly lower biological activity than E. coli lipopolysaccharides and bacteria of the Salmonella genus (“Structure and function of lipopolysaccharides.” Structure and function of lipopolysaccharides. Erridge C., Bennett-Guerrero E., Poxton IR // Microbes and Infection. 2002 Jul; 4 (8): 837-851. In connection with this, the use of biologically highly active lipopolysaccharides makes it possible not only to reduce the microbiological contamination of the wound defect, but also to effectively influence the regeneration of the wound and its debridement from necrotizing Rowan fabrics.

The invention can be used to treat wounds of various etiologies and localization, including punctured, gunshot, lacerated, chopped, chopped, crushed, bitten, mixed, wounds after surgery; diabetic wounds; radiation damage to the skin and mucous membranes; ulcers, including trophic ulcers; erosion; burns, including chemical burns and sunburn; frostbite, dermatitis, pressure sores, cracks in the skin and mucous membranes, insect bites, stomatitis, gingivitis and periodontitis.

Bacterial lipopolysaccharides and excipients contained in a pharmaceutical carrier can also be used in the form of a solution for topical application, a solution for rinsing, gel, cream, paste, ointment, liniment, transdermal therapeutic system, patch, powder, powder, glue, powder, lotions, suspensions, lotion, balm, shampoo, aerosol, spray, oral tablets, lozenges for oral absorption, vaginal tablets, vaginal suppositories, intranasal, ophthalmic and ear x drops, eye films, ear candles, dressings.

In the proposed method, in comparison with the prototype, the drug cannot be contaminated with pathogens of diseases that may be contained in the albumin-free calf blood extract (for example, spongiform encephalopathy viruses), as in the prototype. Accordingly, the safety of the claimed drug is ensured in comparison with the prototype.

Thus, the objective of the invention, namely, increasing the effectiveness of the treatment of wound defects of the skin and mucous membranes of various etiologies and localizations, increasing the safety of therapy, making it possible to standardize the content of the active substance in the preparation, as well as creating a highly effective drug with multifactorial therapeutic in terms of bioavailability action on the inflammatory and regenerative processes of wound defects, implemented in the claimed invention.

Compared with other drugs, for example pyrogenal, firstly, with local administration, as in the claimed method, it is possible to achieve therapeutic concentrations of lipopolysaccharides in the wound defect, which is impossible with systemic administration, given the high metabolic rate of this compound. Secondly, local application allows you to influence the regeneration processes in the absence of a pyrogenic effect, since in this case the drug does not enter the systemic circulation. Thirdly, the local use of the drug in the form of a therapeutically acceptable dosage form (for example, in the form of a gel, ointment, etc.) increases the effectiveness of the drug also due to the fact that a film is created on the surface of the wound that protects the wound from environmental factors , including from microbial contamination; outflow of exudate is provided, etc.

Claims (10)

1. A method of stimulating the regeneration of wound defects of the skin and mucous membranes, which consists in topical application of a dosage form containing an effective amount of bacterial lipopolysaccharides. 2. The method according to claim 1, where the producer of these bacterial lipopolysaccharides is Salmonella typhi. 3. The method according to claim 1, where the producer of these bacterial lipopolysaccharides is Escherichia coli. 4. A dosage form for stimulating the regeneration of wound defects of the skin and mucous membranes, characterized in that it is made in the form of a gel and contains bacterial lipopolysaccharides, a water-soluble cellulose derivative, polyhydric alcohol, preservatives and purified water in the following ratio of components in grams per 100 g of gel:
Bacterial Lipopolysaccharides 0.002-5.00 Water Soluble Cellulose Derivative 1.00-10.00 Polyhydric alcohol 2.00-15.00 Preservatives 0.0001-1.00 Purified water Up to 100.00 5. The dosage form according to claim 4, characterized in that the sodium salt of carboxymethyl cellulose is used as a water-soluble cellulose derivative. 6. The dosage form according to claim 4, characterized in that glycerol is used as the polyhydric alcohol. 7. The dosage form according to claim 4, characterized in that propylene glycol is used as the polyhydric alcohol. 8. The dosage form according to claim 4, characterized in that nipagin is used as a preservative. 9. The dosage form according to claim 4, characterized in that nipazole is used as a preservative. 10. The dosage form according to claim 4, characterized in that nipagin and nipazole are used as a preservative.

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