RU2742752C1 - Medicinal preparation in form of sol for treating a disease and/or condition characterized by violation of skin integrity, and a method for preparing it - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions refers to medicinal preparations for therapy of diseases and/or conditions characterized by violation of skin integument, including in case of burn injury, and discloses a medicinal preparation in the form of a sol for treating a skin burn caused by a thermal or chemical exposure, as well as a method for production thereof.

EFFECT: preparation is characterized by that it contains titanium dioxide, anionic surfactant and viscosity increasing additive; preparation of titanium dioxide is characterized by manifestation of antibacterial effect, as well as immunostimulating properties, as well as increased rate of wound burns healing on skin and recovery of skin integrity.

7 cl, 5 dwg, 10 tbl

Description

The invention relates to pharmaceuticals, namely to drugs for the treatment of diseases and / or conditions characterized by a violation of the integrity of the skin, including burn injury.

The modern experience of world and domestic medicine makes it possible to use highly effective approaches to the treatment of non-fire, non-penetrating, uncomplicated wounds with primary bacterial contamination [1, 2, 3]; nevertheless, every day new challenges arise that are associated not only with an increase in the number of various skin injuries [ 4], but also with the problem of restoring the original structure of the skin in order to achieve better cosmetic results [5], since in the wound process, the inflammatory response in the alteration phase is characterized by damage to cells and intercellular substance caused by the release of a significant amount of inflammatory mediators, providing an increase in vascular permeability [6 ]. In addition, at the existing rates of adaptation of microbes to antibiotics and antiseptics [7, 8], the role of agents providing the activation of nonspecific, innate immunity increases significantly [9]. The latter in the skin is provided with an antigen-providing function and other types of intercellular cooperation of tissue basophils, dendritic cells, macrophages, and lymphocytes, mediated by the presence of Toll-like receptors on their membranes [10]. Toll-like receptors (TLR) are one of the key receptors of the innate immune system in mammals. Receptors of this family are able to recognize specific highly conserved molecular regions (patterns) in the structure of pathogens, initiating the development of reactions of both innate and acquired immune responses, ultimately leading to the elimination of the pathogen from the body. Moreover, each separate type of TLR is able to bind a wide range of microbial molecules with different chemical properties and structure.

Infection of the wound surface with various opportunistic bacteria initiates the work of various TLRs due to the corresponding molecules in the surface structures of the bacterial cell. Lipoteichoic acid, peptidoglycan and other components of gram-positive bacteria are known to activate TLR2. In turn, the lipopolysaccharide (LPS) of gram-negative bacteria binds to TLR4. In addition to these compounds, flagellin of bacterial flagella activates TLR5, unmethylated regions of CpG DNA, TLR9, and various triacillipopeptides, TLR1. Thus, numerous molecules of microbial origin initiate a whole cascade of responses from the macroorganism, as a result of which antimicrobial immunity is activated in the skin, mucous membranes of the respiratory and gastrointestinal tract.

Managing the immune response by using various nanostructures is a promising approach to the treatment of human diseases. Of particular interest is the development of new drugs that affect the PAMP (Pathogen Associated Molecular Patterns) and DAMP (Danger Associated Molecular Patterns) systems, in particular, Toll-like receptors [11]. Targeted modulation of these receptors using pharmacological drugs is a relatively new direction in the treatment of allergies, autoimmune pathology, and inflammatory processes [12].

It should be noted that insufficient activation will not give the desired therapeutic effect, while TLR hyperactivation under the action of endogenous ligands can lead to the development of an excessive inflammatory response, accompanied by tissue damage and triggering the mechanism of immunopathogenesis [13].

TLR ligands are a group of PAMPs that activate the immune system by binding to a TLR receptor that recognizes pathogenic agents on cells such as monocytes / macrophages, dendritic cells, and neutrophils. Even if nanoparticles are not directly toxic to immune cells, they alter the normal defense response by binding to ligands and subsequent suppression of TLR activation [14].

There is a known experience of using gold nanoparticles as carriers for the delivery of peptide drugs that target Toll-like receptors or block the pathways of intracellular signaling of excessive inflammatory responses, in particular TLR4 [15]. However, synthetic highly porous lipoprotein-like gold nanoparticles bound to LPS, thereby provoking a significant decrease in NFκB activation [16].

Experiments aimed at studying the immunomodulatory effect of cerium dioxide nanoparticles in the presence of lipopolysaccharides (LPS) also showed no significant effect [17].

Similar experiments with iron oxide nanoparticles demonstrated the effect of particles on TLR ligand-induced cytokine production. The levels of cytokines induced by LPS strongly depended on both the LPS concentration and the concentration and size of the particles themselves [18].

The prototype of the proposed solution is currently actively used for the treatment of burn wounds preparations based on silver or its salts, for example, Argosulfan®, which includes silver sulfathiazole as an active substance (ht1ps: //www.rlsnet.ru/tn_index_id_357.htm). This drug is a cream for external use and includes cetostearyl alcohol as auxiliary agents; liquid paraffin; petrolatum; glycerol; sodium lauryl sulfate; methyl parahydroxybenzoate; propyl parahydroxybenzoate; potassium dihydrogen phosphate; sodium hydrogen phosphate; water for injections. However, Argosulfan and similar drugs have a number of disadvantages, which include the complexity of the selection of doses and the duration of the course of treatment, since the use of small doses will not have the desired effect, exceeding the dose can lead to irreversible accumulation of silver in the patient's organs and tissues.

The prototype of the proposed method is a method for producing an anti-inflammatory wound-healing agent, including silver nanoparticles [19]. The method includes the following stages: under aseptic conditions with heating mix 5 wt. % PEO-1500, 4 wt. % stabilized sol of silver nanoparticles, 4 wt. % stabilized sol of iron nanoparticles, 0.70 wt. % chloramphenicol, 3.0 wt. % methyluracil, cooled, adjusted to 100 wt. % PEO-400 is mixed with simultaneous homogenization. However, this method is difficult to implement, and the drugs obtained by it do not have the required efficiency.

Thus, the object of the present invention is to provide a simple and effective method for preparing a medicinal product in the form of a sol for the treatment of diseases and / or conditions characterized by a violation of the integrity of the skin, when using which there are no risks observed when taking silver preparations, when commensurate with or exceeding that for prototype treatment efficacy.

Another object of the present invention is to provide a medicament in the form of a sol for the treatment of diseases and / or conditions characterized by a violation of the integrity of the skin, having an antibacterial effect and stimulating the development of local immunity.

The titanium dioxide preparation proposed in this application is characterized by the fact that it exhibits an antibacterial effect due to the generation of reactive oxygen species (ROS) during photoactivation with ultraviolet light. In addition, the proposed drug is characterized by immunostimulating ability due to increased activation of TLR types.

The set tasks are solved due to the drug in the form of a sol, intended for the treatment of diseases and / or conditions characterized by a violation of the integrity of the skin, which has an antibacterial effect and stimulates the development of local immunity.

The technical result is the creation of a simple effective method for producing a medicinal product of titanium dioxide in the form of a sol, intended for the treatment of diseases and / or conditions characterized by a violation of the integrity of the skin;

The technical result is the proposed preparation of titanium dioxide is characterized by the manifestation of an antibacterial effect, as well as immunostimulating properties. In addition, the drug is characterized by an increased rate of healing of burn wounds on the skin and restoration of the integrity of the skin in comparison with the prototype - an anti-burn drug based on silver sulfathiazole. The drug exhibits improved immunostimulating properties compared to the prototype.

The technical result is achieved by preparing the preparation in the form of a sol by a method characterized by the fact that hydrolysis of titanium isopropoxide in the presence of acid at a molar concentration ratio [Ti] / [H +] - 2-3: 1 accompanied by ultrasonic cavitation with a frequency of 24-42 kHz and heating to 50-90 ° C, while obtaining a titanium dioxide sol, then add an anionic surfactant in a molar ratio of titanium dioxide to surfactant - 1500: 1-1000: 1, then an additive that increases the viscosity is introduced into the sol, in a mass ratio titanium dioxide: additive - 100: 1-1: 1.

The technical result is achieved by using the drug in the form of sols of the following composition, in wt. %:

Active substance Titanium dioxide 95,000-99,900 Excipients Anionic surfactant 0.01000-0.0500 Viscosity increasing additive up to 100%

The essence is illustrated in FIG. 1-5, where on:

FIG. 1, 2 - photographs of the epidermis in the area of the regenerate stained with hematoxylin and eosin.

FIG. 3 - photographs of the vessels of the hemomicrocirculatory bed in the regenerate zone at the level of the projection of the skin dermis.

FIG. 4 - photographs of the vessels of the hemomicrocirculatory bed in the regenerate zone at the border with the underlying tissues.

FIG. 5 - photographs of tissue basophils of the regenerate connective tissue, stained with toluidine blue.

Description of the invention.

Obtaining medicinal preparations of titanium dioxide hydrosol.

Preparation of titanium dioxide hydrosols.

The medicinal preparations according to the invention are modified titanium dioxide hydrosols. Their receipt takes place in several stages.

Obtaining a titanium dioxide sol

TiO ₂ nanoparticles are synthesized by the sol-gel method of hydrolysis of titanium isopropoxide. The colloidal sol is formed by hydrolysis and polymerizing reactions of a precursor, which is usually an inorganic metal salt or an organic compound with a metal, such as an alcoholate. The resulting sol is characterized by high purity, controlled particle size and high crystallinity. Taking into account that the course of the hydrolysis of titanium alkoxides is always accompanied by the formation of a crystalline core (only the crystalline phase is responsible for the induction of the photoactive function, which determines the controlled bactericidal activity), the task of developing an effective method for producing a drug was also in the development of the degree of crystallinity of the dispersed phase. The presence of protonating ions significantly develops the ionic strength of the solution, significantly increasing the growth rate of the crystal core and leveling the content of the "coat" of ligands. For this, various acids with a high dissociation constant in aqueous solutions are used in the technological cycle, which determine the growth of crystallinity and stabilization of the dispersed phase particles in aqueous media.

In the method, the following acids can be used: sulfuric, nitric, hydrochloric and acetic acids. The initial concentration of acids is 0.5 M-15 M, the initial concentration of titanium isopropoxide is 3 M.

The preferred variant is using nitric acid. The choice of the concentration parameter is based on two criteria, the main of which is the formation of a stable colloidal system (hydrosol stability) with minimal doses of the protonating agent. A suitable molar concentration range for Ti (C3H7O) 4 / HNO3 solutions is 3 / 1-2, with the most suitable ratio being 2.6, which provides high specific surface area solids, allowing the hydrosol on the burn wound to provide more effective penetration and protective properties.

An important parameter that maintains the stability of the hydrosol over time is the conditions of ultrasonic cavitation (physical peptization), which effectively stabilizes the particles of the dispersed phase, neutralizing the action of active components (nitric acid) and which is actively used as a stage in many technological industries, including pharmaceuticals. For such activation, indicators of power and frequency of ultrasonic exposure remain extremely important. The preferred frequencies for ultrasonic cavitation are 24 kHz - 42 kHz. The most preferred frequency is 24 kHz. Ultrasonic cavitation power is 100-300 W / cm2.

To achieve the stage of intermolecular dehydration in aqueous solutions, heating is used, which involves activation of the mechanisms of destruction of the "coat" of ligands, in parallel with the stage of chemical peptization and ultrasonic cavitation. It is preferable to heat the system in the range of 50-90 ° C. The most effective option involves heating to 80 ° C.

Selection of auxiliary components to ensure stability of the dosage form.

It is known that anionic surfactants have antimicrobial action, they affect the permeability of cell membranes, and also have an inhibitory effect on the enzymatic systems of microorganisms. Anionic surfactants are characterized by the fact that in an aqueous medium, as a result of electrolytic dissociation, they form surface active anions and adsorptively inactive cations. The main types of anionic surfactants of the greatest practical importance include carboxylic acids and their salts, alkyl sulfates, alkyl sulfonates, and others.

According to the invention, anionic surfactants are added in the required amounts to the sol obtained in the previous stage.

The mass ratio of the components of the reagents titanium dioxide: surfactant is 1500: 1-1000: 1. The preferred weight ratio is 1200: 1.

The following anionic surfactants can be used as auxiliary components: sodium dodecylbenzenesulfonate, dodecyl phosphate, sodium cocoyl isothionate, sodium miret sulfate, sodium disodium laureth sulfosuccinate, sodium dodecyl sulfate, potassium oleate, potassium laurate, sodium lauroyl ammonium ethanolate, sodium lauroyl ammonium ethanolamine, sodium sulfate diarcamine, sodium sulfate

Primary dodecyl sulfate and straight-chain dodecyl benzene sulfonate have optimal surfactant properties. These substances are thermally stable, have low toxicity, do not irritate human skin and are biodegradable. They combine well with other surfactants.

Obtaining preparations of a given viscosity.

The following additives can be used to give the dosage form the desired consistency (viscosity): ethylene glycol, propylene glycol, diethylene glycol. Ethylene glycol is the preferred option. An additive that increases the viscosity is introduced into the stable hydrosol of titanium dioxide nanoparticles coated with a shell of sodium dodecyl sulfate obtained at the previous stage, thus obtaining the target product hydrosol of nanoparticles of titanium dioxide modified with surfactants and an additive that increases the viscosity. Varying the ratio of components in the reaction mixture sol: viscosity-increasing additive occurs in the mass ratio of titanium dioxide: additive - 100: 1-1: 1. The preferred ratio range is 99.9: 1-95: 1.

Based on the proposed method, preparations were made in the form of sols of the following composition, in wt. %:

Active substance Titanium dioxide 95,000-99,900 Excipients Anionic surfactant 0.01000-0.0500 Viscosity increasing additive up to 100%

The most preferred preparations are in the form of sols of the following composition, in wt. %:

Active substance Titanium dioxide 95,000-99,900 Excipients Sodium dodecyl sulfate 0.01000-0.0500 Ethylene glycol up to 100%

A disease and / or a condition characterized by a violation of the integrity of the skin, in the context of the present invention, may, without being limited to specific examples, be a burn injury to the skin caused by thermal or chemical exposure, leading to damage to up to 30% of the skin.

The present invention is supported by the following examples:

Example 1. Obtaining a preparation in the form of a sol based on titanium dioxide.

To carry out the reaction, 11.0 ml of titanium (IV) isopropoxide and 16.0 ml of isopropanol are used. The initial reagents are stirred for 30 minutes and a solution of titanium (IV) isopropoxide in isopropanol is obtained. Next, a nitric acid solution is prepared by mixing 100 ml of deionized water and 7 ml of nitric acid. The next step is the dropping of a solution of titanium isopropoxide in isopropanol to a solution of nitric acid for 3 minutes, the resulting mixture is subjected to ultrasonic irradiation with a power of 300 W / cm3 and stirring is continued for 180 minutes without cooling, as a result of which the temperature of the reaction mixture increases from 25 ° C to 75 ° C. The yield of stable titanium oxide hydrosol is 99% based on the starting material. Then, a weighed portion of 0.0001 kg of sodium dodecyl sulfate is added to the titanium dioxide TiO ₂ hydrosol with constant stirring on a magnetic stirrer. The reaction mixture is stirred at room temperature for 2 hours. Temperature control is carried out using a thermometer. Next, a weighed portion of 0.0013 kg of ethylene glycol is added to the titanium dioxide TiO ₂ hydrosol with sodium dodecyl sulfate with constant stirring on a magnetic stirrer. The reaction mixture is stirred at room temperature for 2 hours. Further, the modified titanium dioxide hydrosol is peptized for 2 days until the required particle size is obtained.

This example is not limiting, but only demonstrates an embodiment of the invention.

An example of a specific composition of a hydrosol, in mg, obtained by the claimed method and selected for further research:

Active substance Titanium dioxide 99.959 Excipients Sodium dodecyl sulfate 0.040 Ethylene glycol 0.001 Sol total volume 100 ml.

Example 2. Study of the effectiveness of the pharmacological action of the preparation in the form of a sol based on titanium dioxide.

The experiments were carried out on 40 male rats of the outbred Wistar drain, weighing 200-220 g, veterinary certificate no. 744816875. Each experimental group consisted of 10 males. The keeping of animals and experiments were carried out in accordance with the order of the Ministry of Health of the USSR No. 755 of 08/12/1977 and the European Convention for the Protection of Vertebrate Animals Used for Experiments or Other Scientific Purposes (Strasbourg, March 18, 1986). Three days before the modeling of the burn injury in the back of the rat, skin areas were shaved with a clipper. Thermal burns were caused by applying 10 sec to the skin of the interscapular region of the back of the hairline heated to 200 C of a copper coin, the contact area of 380 cm2. A day later, at the site of burns, wounds appeared in the form of ulcerations filled with necrotic masses, which corresponds to III AB degree of burns in humans. All manipulations were performed under temporary anesthesia with zoletil. Anesthetic effect is achieved in 6-7 minutes after a single intraperitoneal injection of the drug, at a dose of 30 mg / kg. The duration of the surgical stage of anesthesia is 2-3 hours [19].

The animals were divided into 4 groups: 1 - control group, included 10 males. The control group of animals did not apply any preparations to the surface of the burn, that is, the healing of burn wounds took place on their own, 2 - the comparison drug group, included 10 males, who, after modeling the pathology for therapeutic purposes, were daily applied the comparison drug Argosulfan® to the surface of the wound based on silver sulfathiazole); 3-experimental group No. 1, included 10 males, who, after modeling the pathology, daily with a therapeutic purpose on the surface of the wound, the presented sample of the drug acting on Toll-like receptors 4 and 6 was applied daily at a dose of 0.1 ml / 100 g body weight of the animal; 4 - experimental group No. 2, included 10 males, who, after modeling the pathology, daily with a therapeutic purpose on the surface of the wound was daily applied the presented sample of the drug acting on Toll-like receptors 4 and 6, at a dose of 0.2 ml / 100 g body weight of the animal. The nature of the course of the reparative process was assessed visually by destructive processes in the wound, planimetry was performed (the area of the wound defect), the selected areas of the skin of the burn surface and the border area of the wound defect were studied macroscopically, the weight of the animal was measured weekly, as well as the temperature of the skin around the wound. The degree of contamination was determined by standard methods: microscopy of Gram stained smears; sowing and cultivation on nutrient media (cultural and phenotypic characteristics). The number of colony-forming units (CFU) in 1 ml of wound discharge was established on the 1st, 3rd, 7th and 14th days after modeling by counting macrocolonies grown on agar and semi-agar nutrient media [1].

To assess changes in the behavioral activity of the animals, an "open field" test was performed on days 15 and 21. We used a standard field model for rats - a playpen 80 × 80 cm, divided into 16 squares with a side of 20 cm. On the 15th and 21st days, the state of the animal was studied in the "Open Field" test. In 21 days after the simulation, the animals were removed from the experiment by an overdose of zoletil, after which a histological examination of the regenerate excised with a section of the wound edges was performed. In addition, the following histological examination was carried out: the myocardium of the left ventricle, the cerebral cortex and the cortex of the kidneys. In the future, fixation and histological examination of microscope preparations were carried out. On the 1.7, 14 and 21 days of the experiment, the wound was photographed, with the determination of the area of the wound defect. According to literature data, when studying the effect of drugs for local treatment of wounds on the healing of thermal burns of II-III degree in an experiment on rats, the most indicative are 2-3 weeks after the formation of the burn surface, because It is during these periods that significant differences are observed in the healing of burn injury under the influence of drugs used in clinical practice. Comparison of the effectiveness of the funds a month after the formation of the burn seems ineffective, since it is during these periods that re-epithelialization occurs and complete healing occurs in animals that have not received any therapy [20]. To study the wound site with the regenerate, the following histological stains were used: hematoxylin and eosin, Trichromic stain according to Picro Malory - to identify the structures of connective tissue, toluidine blue - to identify tissue basophils, as well as methylene green and pyronine (stain according to Brachet) to assess the processes transcription in the epithelial layer. All internal organs were stained with hematoxylin and eosin, brain slices were also stained according to Nisl.

Methods used in assessing the specific pharmacological activity of the drug

Histological slides were assessed visually (Levenhuk С1400 NG digital camera, Levenhuk ToupView software). Also, a morphometric study of a number of parameters was carried out on micropreparations of regenerated laboratory rats using ImageJ.

The thickness of the cell layer of the epidermis (up to the stratum corneum) was estimated by 3 measurements in 1 field of view at about. x40, held perpendicular to the basement membrane. One section was examined from each animal in the group.

To determine the geometric mean dimensions of the keratinocyte of the spiny layer, one section was examined from each animal from the group. The measurements were made with a x40 eyepiece. The area of keratinocytes and their nuclei was measured in the field of view, the area of the cytoplasm was determined as the difference between the area of the keratinocyte and the area of its nucleus. The number of measurements in the group was 100 or more. The nuclear-cytoplasmic ratio was calculated using the formula (1):

where Sk is the area of the cell;

Sc is the area of the cell cytoplasm;

Sя is the area of the keratinocyte nucleus.

To determine the area of the nucleolus of the spiny layer keratinocyte, we examined one section from each animal from the group. The measurements were carried out with an immersion magnification of the eyepiece x90, on micropreparations stained with methylene green - pyronine. The number of measurements in the group was 100 or more. The specific proportion of microvasculature vessels in the connective tissue scar at the level of the subepidermal dermis was evaluated. For this, one section stained with hematoxylin and eosin was examined from each animal from the group. Measurements were made in all fields of view, where vessels were present, with a x10 eyepiece. The total area of the vessels of the microvasculature of the subepidermal layer of the dermis was measured, after which the total area of the dermis in the field of view was measured. The calculation was carried out according to the formula (2):

where P is the specific proportion of vessels (%);

Sc is the total area of blood vessels in the field of view;

Sp is the area of the connective tissue scar at the level of the subepidermal layer of the dermis in the field of view.

Determined the cellular density of the dermis with the distribution of cells by shape factor. One section was examined from each animal in the group. Measurements were made in all fields of view with an eyepiece x40 0). The total number of cells in the subepidermal layer of the regenerate was measured, followed by separation according to the shape factor. Cells with a form factor from 0 to 0.6 were considered fibroblastic cells, and from 0.61 to 1.0 - mononuclear (inflammatory infiltrate cells).

The specific gravity and the level of degranulation of tissue basophils were evaluated. For this, one section stained with toluidine blue was examined from each animal from the group. Measurements were made in all fields of view, where dense loose connective tissue was located, replacing the dermis defect of the skin. In each field of view, the number of degranulated, partially degranulated and non-degranulated tissue basophils was separately counted, with subsequent recalculation per unit area (S = 1 mm2). The calculation was carried out according to the formula (3):

where p is the relative density of tissue basophils per cut unit,

Nc - the number of cells in the field of view in the field of view, Sp - the area of the connective tissue scar at the level of the subepidermal layer of the dermis in the field of view.

Mathematical processing of the results was carried out by the methods of biomedical statistics with the determination of the reliability of changes using the Student's t-test.

Experiment Results

When tested in the "open field" in animals of the control group 15 days after the application of the burn, the most characteristic changes can be considered a decrease in defecation compared to the background by 50%, and an increase in grooming by 1.5 times compared to the background, which, apparently , is explained by the desire of animals to keep the skin damaged by the burn in a clean state. In the same period of the experiment, the animals of the comparison group and the experimental groups were characterized by an increase in the frequency of grooming compared with the indicators that did not receive treatment, the animals of the control group (by 20% for the group receiving the comparison drug Argosulfan®, by 25% for the experimental group No. 1 , who received the presented sample of the drug acting on Toll-like receptors 4 and 6, at a dose of 0.1 ml / 100 g of animal body weight, by and 35%, for experimental group No. 2, which received a drug acting on Toll- similar receptors 4 and 6, at a dose of 0.2 ml / 100 g of animal body weight (Table 1).

On the 21st day of the experiment, the experimental groups showed a significant increase in various types of behavioral activity when compared with the control group. The most pronounced was a significant increase in horizontal activity - 4.2 times for experimental group No. 2, which received the presented sample of a drug acting on Toll-like receptors 4 and 6, at a dose of 0.2 ml / 100 g of animal body weight, with comparison with a control group represented by animals that did not receive treatment after simulated burn. 3.9 times compared with the control group for the experimental group No. 1, which received the presented sample of the drug acting on Toll-like receptors 4 and 6, at a dose of 0.1 ml / 100 g of animal body weight. 2.1 times - for the comparison group that received Argosulfan® as a treatment.

Thus, according to the data of the "open field" test, it can be concluded that the behavioral activity of animals in the experimental groups and the comparison group is normalized in relation to the animals that did not receive treatment - the control group. The data obtained indicate an improvement in the general clinical state of animals against the background of the use of the studied samples of the drug acting on Toll-like receptors 4 and 6. As a result of studying the dynamics of wound healing and reparative-regenerative changes on micropreparations in all studied groups of male rats, a complex of changes in epidermis of the wound defect, characterized by progressive restoration of the skin (table 2).

Visual assessment of the micropreparations of the studied internal organs of the animals of the experimental groups that received a sample of the drug acting on Toll-like receptors 4 and 6 did not reveal any deviations from the morphological picture of the micropreparations of the animals of the control group, which indicates the absence of general resorptive toxicity. On micropreparations of the wound site, stained with hematoxylin and eosin, the low level of reparative processes in the control group attracts attention. The stratified squamous epithelium in the center of the wound defect is thin, the thickness of the prickly layer is about 3 layers of corneocytes. The cells are small. The kernels are compact. The relief of the basement membrane is smoothed (Fig. 1A). In the comparison group, the thickness of the epithelial layer increases significantly, which is reliably confirmed by the data of a statistical study (Table 3) The basement membrane becomes tortuous. The thickness of the prickly layer increases; granules are clearly visualized in keratinocytes of the granular layer. The cells become large, the nuclei increase in size, acquire a rounded shape, and the nucleolus is visualized in many cells. The revealed changes are also characteristic for the cells of the basal layer of the epidermis (Fig. 1).

The experimental groups are characterized by even deeper basal outgrowths of the newly formed epidermis, which are characterized in the experimental group No. 2 as hair follicles growing into the center of the wound defect from the edges of the wound (Fig. 1D) and their neoplasm (Fig. 2D).

For both experimental groups, signs of activation of recovery processes in the diferon of keratinocytes are also characteristic. However, the severity of morphological criteria of reparative processes in the epidermis of the experimental groups does not exceed that in the comparison group. This is confirmed by the higher indicators of nuclear-cytoplasmic ratio (0.54 ± 0.03 in the comparison group and 0.59 ± 0.02 in the experimental group No. 1, 0.56 ± 0.03 in the experimental group No. 2) (Table 4 ). The cells of the spinous layer of the comparison group are characterized by the highest level of differentiation, followed by the corneocytes of the experimental group No. 2, No. 1 and only then the control group.

This tendency is also confirmed by the statistical study of micropreparations stained according to Brachet, on which the area of the nucleolus was measured, which serves as an indicator of the activity of the processes of intranuclear transcription (Table 5): the largest area of the nucleus in the comparison group, then in the experimental group No. 2, No. 1, and, the lowest in the control group.

The regenerate replacing the wound defect is a dense fibrous loose connective tissue into which the elements of the hemomicrocirculatory bed penetrate. The vessels of the latter are more numerous in the area of the bottom of the wound, at the level of the hypodermis, and also under the epithelium in the area of the superficial plexus of the skin. In this area, they are usually located parallel to the basement membrane of the epidermis (Fig. 3). In the control group, these vessels are characterized by the presence of morphological signs of venous hyperemia: dilated lumens of veins filled with erythrocytes in a sludge state, as well as arterioles with an empty lumen (Fig. 3A). The comparison group is characterized by a significant decrease in the number of subepidermal vessels of the hemomicrocirculatory bed. However, in the experimental groups their number increases, to a greater extent in the experimental group No. 2, while there are no signs of venous hyperemia.

Similar changes are characteristic of the vessels in the thickness of the connective tissue of the regenerate. In the control group, they look like constricted connective tissue fibers, full-blooded, with parietal adhesion of erythrocytes. In the comparison group, the number of vessels decreases. In the experimental group, their number increased, however, neither spasm of arterioles, nor parietal adhesion of erythrocytes, nor other morphological equivalents of impaired hemomicrocirculation were observed. The visual assessment is confirmed by the data of the statistical study (Table 6). The largest specific proportion of blood vessels in the hemomicrocirculatory bed is characteristic of the experimental group No. 2, followed by the experimental group No. 1, the control group and the comparison group.

It should be noted that if in the control group and the comparison group the course of vessels in the regenerate is usually tangential - in the direction from the hypoderm to the basement membrane of the epidermis, then in the experimental groups not only longitudinal, but also transverse sections of the vessels are often found, which indicates more a branched network of hemomicrocirculation (Fig. 4).

Thus, the complex of morphological rearrangements presented in Figures 5 for animals of the experimental groups includes the formation of a more complete papillary layer of the dermis in the area of the wound defect, more intensive restoration of the skin appendages and the hemomicrocirculatory bed, which in general is the morphological substrate of the plastic effect of the drug sample. acting on Toll-like receptors 4 and 6, at a dose of 0.1 ml / 100 g and, to a greater extent, at a dose of 0.2 ml / 100 g of animal body weight. The bottom of the wound defect in all studied groups is characterized by the presence of a large vascular plexus with dilated vessels of the venous link of the hemomicrocirculatory bed (Fig. 5).

In all the groups under consideration, the mast cell population is actively involved in the response, the cells of which are in a different functional state, depending on the group under study. In the control group, these are numerous cells located perivasally, mainly in the area of the bottom of the wound defect and, to a lesser extent, in the area of its walls. These are mainly tissue basophils, which are in a state of complete and less often partial degranulation, when the granules are located outside the mast cell in the surrounding connective tissue. In the comparison group, tissue basophils are few in number, reduced in size, characterized by the same microtopography as in the control group (Fig. 5A, B). The experimental group is characterized not only by the restoration of the bulk density of tissue basophils (Table 7), a significant decrease in the degree of their dergranulation, but also by a change in their topics (Fig. 5D) In the experimental group No. 2, they are located in typical areas, and subepidermally - in the center of the regenerate. Visually, these are large cells with a large number of intracellularly located metachromatically staining granules.

In the study of contamination of the wound surfaces of the studied groups, a significant decrease in CFU, in comparison with the control group, in animals receiving the comparison drug Argosulfan® on the 3rd day after the experiment, draws attention (Table 8). The performance of the experimental groups was also lower than the performance of the control group. Subsequently, the greatest decrease in CFU in 1 ml of washout from the wound surface was observed in animals receiving the presented sample of the drug acting on Toll-like receptors 4 and 6. The difference gradually increased with increasing time after surgery and reached more than 14 days in the experimental groups. 3-fold decrease in comparison with the control group, which was significantly lower than the indicators of the comparison group - animals receiving the comparison drug Argosulfan®. The changes described above can characterize a gradual increase in the protective effect of the drug acting on Toll-like receptors. Based on the presented dynamics of the degree of contamination of the wound surface, the latter is probably not based on a direct bactericidal effect, in the early stages after modeling the burn defect (on the 3rd day of the experiment); and on a gradual increase in the intrinsic protective properties of the wound surface, including barrier, bactericidal and immune components, which manifests itself in a more intensive decrease in the degree of contamination of the wound surface in the experimental groups, compared with the group of animals receiving Argosulfan®, on the 7th and 14th day of the experiment.

Thus, our data not only reflect not only the staging of the wound process, i.e. clinical course of local changes (inflammation, development of necrosis and its limitation, scarring and wound epithelialization) (Table 9), but also indicates a reduction in the duration of the above processes.

This is observed early (7 days after surgery), to a greater extent, in animals that received the presented sample of the drug acting on Toll-like receptors 4 and 6, at a dose of 0.2 ml / 100 g of animal body weight (Table 10 ).

Findings.

The results of the studies of the proposed drugs allow us to conclude that they have antibacterial and immunostimulating properties that exceed those for the reference drug. The proposed drugs proved to be excellent in the treatment of conditions characterized by a violation of the integrity of the skin, including burn injuries.

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Claims (8)

1. A medicinal preparation in the form of a sol for the treatment of burn injuries of the skin caused by thermal or chemical exposure, which includes the following composition of components, wt. %: Active substance: Titanium dioxide 99.959 Excipients: Sodium dodecyl sulfate as anionic surfactant 0.040 Ethylene glycol as a viscosity increasing additive up to 100% 2. A method of obtaining a medicinal product in the form of a sol according to claim 1, characterized in that hydrolysis of titanium isopropoxide in the presence of acid at a molar concentration ratio [Ti] / [H +] - 2-3: 1, accompanied by ultrasonic cavitation at a frequency of 24 kHz and heating to 50-90 ° C, then add anionic surfactant in a molar ratio of titanium dioxide to surfactant - 1500: 1 - 1000: 1, then an additive that increases the viscosity is introduced into the sol in a molar ratio of titanium dioxide: additive - 100: 1 - 1: 1. 3. A method according to claim 2, wherein the acid is selected from the group of sulfuric, nitric, hydrochloric or acetic acids. 4. The method according to claim 2, wherein the anionic surfactant is sodium dodecylbenzenesulfonate, dodecyl phosphate, sodium cocoylisothionate, sodium miret sulfate, disodium laureth sulfosuccinate, sodium dodecyl sulfate, potassium oleate, potassium laurate, sodium lauroyl ammonium ethanol, sodium diarcamine sulfate or sodium sulfate triethanolamine. 5. The method according to claim 2, wherein the molar ratio of titanium dioxide to surfactant is 1200: 1. 6. The method of claim 2, wherein the molar ratio of titanium dioxide to viscosity-increasing additive is 99.9: 1 to 95: 1. 7. The method of claim 2, wherein the viscosity-increasing additive is ethylene glycol, propylene glycol, or diethylene glycol.

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