RU2214256C2 - Agent for stimulation of cells proliferation, composite for stimulation of cells proliferation and method for treatment of wounds, burns and ulcers of different etiology - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to agents promoting proliferation of cells and using these agents in surgery for treatment of wounds of different genesis: ulcers of different etiology, sluggish wounds, among them, wounds formed after burn. Agent for stimulation of cells proliferation contains sterile extract of human brain at the fetus stage of its development as the brain extract. The composite for stimulation of cells proliferation contains an aqueous solution of sterile extract of human brain from at the fetus stage of its development in sterile isotonic saline solution. Method for treatment of wounds, burns and ulcers of different etiology involves treatment of wound surface with composite containing sterile extract of human brain at the fetus stage of its development in sterile isotonic saline solution. Agent and composite based on thereof provide the enhanced effectiveness of stimulation of angiogenesis and treatment of ulcers of different etiology, burns and sluggish wounds. EFFECT: valuable properties of agent, simplified technology for preparing agent and composite. 19 cl, 1 tbl, 5 ex

Description

 The present group of inventions relates to medicine, namely to agents that promote cell proliferation, and to the use of these agents in surgery for the treatment of wounds of various origins: ulcers of various etiologies; long non-healing wounds, including wounds formed after a burn.

 Currently, the following are used to stimulate cell proliferation: growth factors obtained using recombinant DNA technology; as well as embryonic tissues isolated from the tissues of animals or humans by complex purification, their homogenates and extracts from tissues, which are used to treat various pathologies.

 Thus, it is known a means for stimulating cell proliferation in the form of an extract obtained from bovine hypothalamus, pituitary or brain (see Maciag T., Cerundolo J., Ilsley S., Kelley PR, Forand R.-Proc. Natl. Acad. Sci. USA. Vol. 76, .11, pp. 5674-5678, November 1979).

 A serious drawback of the known agent is the narrow scope of its application: it is intended only to stimulate the proliferation of cell cultures in vitro. The use of this tool to stimulate cell proliferation in the body is not possible due to the immune response of the latter to the drug.

 A known composition for stimulating cell proliferation, including human endothelial growth factor, a suitable carrier and heparin (see US patent 5827826, CL A 61 K 038/18, published 10/27/1998).

 A disadvantage of the known composition is the fact that the growth factor is obtained by expensive and complex recombinant DNA technology, consisting of a large number of precision operations. This tool contains only one growth factor that has a selective effect on endothelial cells, which narrows the scope of this tool and excludes its complex effect on the wound process. Recently, it has been shown that a number of growth factors, such as bFGF, have a significantly more pronounced effect on endothelial cells, which casts doubt on the relative effectiveness of this composition. The effectiveness of the use of this composition for tissue regeneration and angiogenesis has not been investigated. It is not intended to include antimicrobials in the known composition, which makes it difficult to use it in conditions of infected wounds.

 A known method of treating burns of the II and III degree AB, including applying a biograft, as a suspension of fetal tissue of a human fetus of 16-20 weeks is applied to the wound (see RF patent 2122416, class A 61 K 35/48, published 11/27/1998 )

 The known method does not provide for the sterilization of the composition used, therefore, strict adherence to the sterility conditions when sampling fetal tissues is required to prevent additional infection of the patient. The application of the known method in conditions of an infected wound can lead to inactivation of the composition of the suspension and increased reproduction of the wound flora, for which the suspension can serve as a good nutrient medium. The problem of storing fetal tissues without loss of activity of the composition is not resolved. In addition, since the extraction of biologically active substances from these substrates is not provided, their content in the suspension is relatively low.

 Known means for stimulating cell proliferation in the form of cultured tissue of the spinal cord of a human embryo 6-7-week development (see RF patent 2090143, CL A 61 17/00, published 09/20/1997).

 Known tool has a narrow scope, as it is intended only for the treatment of springomyelia. The process of tissue cultivation is complex, time consuming and laborious; it significantly increases the cost of application and increases the likelihood of infection of the material. It should also be noted and the invasiveness of the method of introducing cultured tissue.

 A known composition for stimulating tissue regeneration in the treatment of diseases of the central nervous system, containing suspended embryonic brain tissue together with protein-nucleic extracts of this tissue (see RF patent 2129427, class A 61 K 35/48, published 04/27/1999).

 The known composition has a limited scope, as it is intended only for the treatment of diseases of the central nervous system. In addition, the method of introducing the composition into the cerebrospinal fluid of the brain is traumatic and unsafe for the patient.

 A known method of treating eye burns by transplantation of homotissues into the area of damage. A suspension of cultured corneal epithelial cells obtained from human embryos is introduced into the conjunctiva in an amount of 200,000 cells in 0.2 ml of physiological saline. To preserve the biomaterial, the method of low-temperature conservation is used (see RF patent 2140241, class A 61 F 9/007, published October 27, 1999).

 The known method of treatment is very specific and therefore cannot be used for the regeneration of other tissues. The cultivation of corneal epithelial cells is a complex, laborious process, which significantly increases the cost of the method and increases the risk of infection of the material. It should also be noted the problematic nature of low-temperature preservation due to the complexity of the method and the relatively low percentage of viable cell output after thawing.

 A biologically active preparation is known in the form of a freeze-dried homogenizate from brain tissue (see RF patent 2071335, class A 61 K 35/30, published January 10, 1997).

 A well-known drug is intended for use in veterinary medicine, the effectiveness and safety of its use for the treatment of humans has not been investigated. The preparation has a relatively low content of biologically active substances, and the presence of a significant amount of ballast substances causes undesirable side reactions. Obtaining a well-known drug is multistage, which complicates the process of its manufacture.

 A known composition for stimulating cell proliferation containing vascular endothelial growth factor with a polyanionic compound comprising heparin, a heparin derivative oligosaccharide or a heparin-like oligosaccharide (see US patent 5851989, class A 61 K 038/18, published 22.12.1998) .

 The disadvantage of the known composition should include extremely complex technology for its manufacture.

 There is a method of treating long-term non-healing wounds, according to which, after treatment of the wound cavity and surrounding skin with disinfectant and antimicrobial agents, under the chloroethyl local anesthesia, a bed is formed in the muscle layers of the wound, into which cryopreserved neonatal or embryonic tissue of the spleen and thymus is placed in a ratio of 1: 1 with a total weight of 7 -8 mg / kg body weight of the patient (see USSR author's certificate 1711896, class A 61 K 35/26, published 02.15.1992).

 In the known method of treatment, sterilization of the material used is not provided, therefore, the risk of additional infection of the patient is high. The formation of a bed in the layers of the wound carries a significant risk of the spread of the infectious process beyond the wound. The implantation of tissue in a known manner, despite the use of chloroethyl anesthesia, is traumatic for the patient. Due to the fact that extraction of biologically active substances is not used, their content in the preparation used is relatively low, the latter contains many ballast compounds, which can cause adverse reactions in the patient.

 The closest in combination of essential features to the claimed means for cell proliferation and the composition based on it is the prototype tool for stimulating corneal repair in the form of an aqueous solution of a brain extract with a concentration of 2 mg / ml (see USSR author's certificate 1773401, cl. A 61 K 35/30, published on November 7, 1992).

 The known composition has a narrow scope, as it is intended only for repair of the cornea. It should be noted the invasiveness of the introduction of the composition, which makes it necessary to suture the eyeball. When using the brain of an adult, the likelihood of an immune reaction to the drug is high. Due to the fact that sterilization of the drug is not provided, strict adherence to asepsis is necessary when taking the brain and in the manufacture of the composition in order to prevent infection of the patient.

A known method of treating trophic ulcers and long-term healing wounds adopted as a prototype is known, including applying to the wound and surrounding skin with a continuous layer of a wound healing agent, which is applied to the original cheese-like lubricant of the skin of a human fetus, heated to 35-37 ^o C (see USSR author's certificate 1718947 , according to class A 61 K 35/36, published on March 15, 1992).

 The known method of treating trophic ulcers and long non-healing wounds has serious disadvantages. The cheese-like lubricant of the skin of the human fetus is relatively poor in biologically active compounds; in addition, the method does not provide for their isolation from the native substance, therefore, in many cases of the treatment of trophic ulcers, burns and long-healing wounds, this method is not effective enough. Since sterilization of a cheese-based lubricant is not provided, there is a great danger of additional infection of the patient. Due to the lack of antimicrobial agents in the composition, its use in an infected wound can cause activation of the wound flora, for which the composition used can serve as a good nutrient medium.

 The task was to develop such a tool for stimulating cell proliferation and composition based on it, which could be obtained by simple technology and at the same time would provide increased efficiency in stimulating angiogenesis and treatment of ulcers of various etiologies, burns and long-term healing wounds.

 The problem is solved by a group of inventions, united by a single inventive concept.

 Namely, the problem is solved by the fact that in the means for stimulating cell proliferation containing an aqueous solution of the brain extract, as a brain extract, the agent contains a sterile extract of the human brain at the stage of intrauterine development.

The problem is also solved by the fact that in the composition for stimulating cell proliferation containing an aqueous solution of the brain extract, as a brain extract, the composition contains a sterile extract of the human brain at the stage of its intrauterine development in sterile isotonic saline in the following ratio of components, mg / ml :
Sterile extract of the human brain at the stage of its intrauterine development (for protein) - 0.1 - 100
Sterile Isotonic Saline - Rest
The problem is also solved by the fact that in the method of treating wounds, burns and ulcers of various etiologies, including the local use of a wound healing agent, a composition containing a sterile extract of the human brain at the stage of intrauterine development in sterile isotonic saline solution is used as a wound healing agent, in the following ratio components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 0.5 - 20.0
Sterile Isotonic Saline - Rest
As a result of the studies, the authors found that the human brain extract at the stage of intrauterine development effectively stimulates the proliferation of cells of ectodermal and mesodermal origin, as well as hybridomas, including the cultivation of these cells in culture. In contrast to the brain extract of an adult, an extract of the human brain at the stage of intrauterine development has a higher activity with respect to stimulating the proliferation of these cells during their in vitro cultivation. In addition, the human brain tissue at the stage of its intrauterine development practically does not have antigenic activity, which allows you to enter this tissue, even without preliminary treatment, into another organism, without fear of the latter's immune reaction.

 As established by the experiments of the authors, the range of concentrations of the human brain extract at the stage of intrauterine development (protein) in the composition for stimulating cell proliferation is 0.1-100 mg / ml. When the concentration of the human brain extract at the stage of intrauterine development is less than 0.1 mg / ml, the composition was not stimulated by cell proliferation. Compositions with an extract concentration of 100 mg / ml are impractical due to the risk of tissue irritation at the injection site (due to the hyperosmoticity of the composition).

 The authors experimentally determined that the preferred range of concentrations of the human brain extract at the stage of intrauterine development for use in the method of treating wounds, burns and ulcers of various etiologies is 0.5-20.0 mg / ml.

 It is advisable to use an extract of the brain of a human fetus 16-24 weeks as the most affordable.

 A composition for stimulating cell proliferation can be administered heparin. Introduction to the composition of heparin stabilizes the composition and lengthens the stimulating effect of the human brain extract at the stage of intrauterine development on cell proliferation. When the concentration of heparin is less than 0.06 mg / ml, this effect is practically not determined. Compositions with heparin concentrations of more than 15 mg / ml are not advisable to use due to the possible side effect of the drug.

 The preferred range of heparin concentrations when using the composition in the method of treating wounds, burns and ulcers of various etiologies is 0.6-12.0 mg / ml.

 The composition to stimulate cell proliferation can also be entered drugs with antibacterial and antifungal activity. The introduction of these drugs ensures the suppression of bacterial and fungal flora, respectively, and prevents its inactivation of the brain extract. It is not advisable to use drugs with antibacterial and antifungal activity in concentrations less than 0.05 mg / ml due to the absence of this effect. At concentrations of an antibacterial drug of more than 200 mg / ml and an antifungal drug of more than 2 mg / ml, the risk of their side effects is high. The preferred concentration of the drug with antibacterial activity when using the composition in the method of treating wounds, burns and ulcers of various etiologies is 0.05-10.0 mg / ml.

 As an antibacterial drug, miramistin, chlorhexidine bigluconate, dioxidine, gentamicin, cefotaxime (claforan), cefoperazone and other known drugs can be used. As an antifungal drug, amphotericin B, fluconazole (diflucan) and other drugs can be used.

 As a solvent, any isotonic (isosmotic with respect to blood plasma) saline solutions can be used, for example, isotonic sodium chloride solution (the so-called "physiological solution"), Ringer's solution and its modifications, Hanks's solution, Dulbecco's solution with phosphate buffer (Dulbecco's phosphate) buffered saline) and other known isotonic saline solutions.

 In the presence of severe pain, local anesthetics in the amount of 10.0-50.0 mg / ml can be introduced into the composition. For example, lidocaine in an amount of 10-50 mg / ml, trimecain in an amount of 20-50 mg / ml. Concentrations of local anesthetics less than 10 mg / ml do not give the desired effect, and at concentrations more than 50 ml / mg, the likelihood of side effects is high.

The human brain extract at the stage of intrauterine development is obtained as follows. 35 g of fresh brain tissue is homogenized in 50 ml of 0.1 M NaCl with a microcracker for 4 minutes. The resulting suspension is stirred for 8-12 hours using a magnetic stirrer. Then the suspension is centrifuged for 2-2.5 hours at 13000 rpm, the supernatant is separated, 5 mg / ml streptomycin sulfate is added to it to precipitate soluble lipids, stirred until complete dissolution and, if necessary, the pH is adjusted to 7 by adding 1M NaOH. The resulting mixture was incubated for 20-24 hours, then centrifuged for 2.0-4.5 hours. The resulting supernatant is sterilized by filtration through a filter with pores of 0.45 and 0.22 μm or using ultraviolet irradiation and stored further at a temperature of +4 ^o C until use. If necessary, long-term storage of the drug, as well as in order to increase the concentration of biologically active substances in the extract, the latter is subjected to lyophilization. The lyophilisate is stored without loss of activity for one year, provided that it is stored at a temperature of -30 ^o C. The yield of the extract is 350-600 mg of protein at a concentration of 5-10 mg / ml. Protein concentration is determined by the biuret method. All operations are carried out at a temperature of + (4-6) ^o C, deionized water is used to prepare solutions.

 The inventive tool and composition based on it can be useful wherever, in order to achieve the desired effect, stimulation of cell proliferation is required. For example, when growing cell cultures in vitro, to stimulate angiogenesis in diseases accompanied by tissue ischemia, to stimulate tissue regeneration in the treatment of wounds, burns and ulcers of various etiologies, as well as in the treatment of gastrointestinal ulcers. In the latter case, it is desirable to use the inventive composition in combination with antisecretory and antacid drugs. As antisecretory drugs, H2-histamine-blockers and m-anticholinergics can be used, and as antacid drugs - aluminum hydroxide, sucralfate, sodium bicarbonate, phosphalugel and others.

 When using the inventive composition to stimulate the proliferation of cells of new vessels (angiogenesis) in diseases accompanied by tissue ischemia, such as atherosclerosis obliterans of the lower limb arteries, diabetic lower limb angiopathy, coronary heart disease and others; it can be administered both topically (intraarterially, subcutaneously, intramuscularly) and systemically (intravenously). The total amount of injected brain extract can vary from 0.01 mg to 100 mg per 1 kg of body weight per day. Antimicrobials can be added to the composition at the rate of 0.1-1000 mg per 1 kg of body weight per day, for example, cefotaxime (claforan) in an amount of 50 mg / kg of body weight per day or ampiox in an amount of 50 mg / kg of body weight per day .

 Studies of the effect of the claimed composition on angiogenesis in ischemic tissue in mammals were carried out on four groups of rabbits weighing from 3 to 3.5 kg Each group consisted of experimental and control groups, 3 animals in each subgroup. All these animals underwent ligation and excision of the femoral artery (from the external iliac to the popliteal arteries) to create experimental ischemia of the hind limb. Immediately after excision of the femoral artery (during the operation), as well as 10 and 12 days after the operation, the animals of the experimental subgroups were injected with a solution of the human brain extract at the stage of intrauterine development, and the animals of the control subgroups were injected with a solution of a similar composition, but without brain extract.

 I group.

Experimental subgroup 1. Intra-arterial administration of a solution of the following composition (based on 0.3 mg of extract - 3 ml of solution per 1 kg of weight), mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 0.1
Sterile Isotonic Sodium Chloride Solution - Else
Control subgroup 2. Intraarterial administration of a sterile isotonic sodium chloride solution (at the rate of 3 ml of solution per 1 kg of weight).

 II group.

Experimental subgroup 3. Intra-arterial administration of a solution of the following composition (based on 0.3 mg of extract - 3 ml of solution - per 1 kg of weight), mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 0.1
Heparin (5 units) - 0.06
Sterile Isotonic Sodium Chloride Solution - Else
Control subgroup 4. Intraarterial administration of a sterile isotonic sodium chloride solution (at the rate of 3 ml of solution per 1 kg of weight) containing heparin in the amount of 0.06 mg (5 units) in 1 ml of solution.

 III group.

Experimental subgroup 5. Intraarterial administration of a solution of the following composition (based on 5.0 mg of extract - 0.05 ml of solution per 1 kg of weight), mg / ml:
A sterile extract of the human brain at the stage of its intrauterine development (for protein) - 100
Sterile Isotonic Sodium Chloride Solution - Else
Control subgroup 6. Intraarterial administration of a sterile isotonic sodium chloride solution (at the rate of 0.05 ml of solution per 1 kg of weight).

 IV group.

Experimental subgroup 7. Intra-arterial administration of a solution of the following composition (based on 5.0 mg of extract — 0.05 ml of solution per 1 kg of weight), mg / ml:
A sterile extract of the human brain at the stage of its intrauterine development (for protein) - 100
Heparin (1250 units) - 15
Cefotaxime - 200
Sterile Isotonic Sodium Chloride Solution - Else
Control subgroup 8. Intraarterial administration of a solution of the following composition (based on 0.5 ml of solution per 1 kg of weight), mg:
Heparin (1250 units) - 15
Cefotaxime - 200
Sterile Isotonic Sodium Chloride Solution - Else
Intraarterial administration was performed into the internal iliac artery.

 All animals underwent angiographic examination immediately after excision of the femoral artery and 30 days after surgery. On the basis of angiograms taken at the 4th second after the introduction of contrast, a morphological analysis of the development of collateral vessels was carried out according to the following method: a template consisting of 2.5 mm diameter circles arranged in rows at intervals of 5 mm was applied to the angiogram. The number of contrasted arteries in the circles and the total number of circles in the mid-thigh area were counted. An angiographic index was defined as the ratio of the number of arteries in all circles to the total number of circles in a given area. This indicator reflects the density of development of arterial collaterals in the middle of the thigh. The severity of necrotic changes was evaluated macroscopically according to the following three-point system: "0" - no change; "1" - color change; "2" - ulcer or necrosis of a limited area; "3" is a common ulcer or necrosis. On the 30th day of the experiment, after slaughtering rabbits, preparations for histological examination were made from their hind limbs. The research results are shown in the table.

Histological studies revealed an increase in the density of capillaries by 1 mm ² in animals of experimental subgroups: in group I - 2.3 times; in the II group - 3.1, in the III group - 4.8 times and in the IV group - 5.9 times in comparison with the animals of the control subgroups of each group.

A method of treating wounds, burns and ulcers of various etiologies is as follows. The ulcer (wound) surface is cleaned of necrotic masses using the well-known antimicrobial and enzyme therapy. A sterile pad (for example, from gauze) moistened with a sterile composition containing an extract of the human brain at the stage of intrauterine development in a sterile isotonic saline solution is applied to a cleaned ulcer (wound) surface, with the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 0.5 - 20.0
Sterile Isotonic Saline - Rest
The composition can be added heparin in an amount of 0.6-12 mg / ml; a drug having antibacterial activity in an amount of 0.05-10.0 mg / ml; a drug having antifungal activity in an amount of 0.05-2.0 mg / ml, as well as a local anesthetic drug in an amount of 10.0-50.0 mg / ml.

 The gauze pad on top can be covered with a water-impervious film, for example from cellophane, to prevent evaporation of the solution and create a moist environment on the surface of the ulcer (wound). In this case, the film is fixed with a gauze bandage. If it is necessary to create external compression in the case of treating a trophic ulcer, therapeutic compression knitwear (knee socks, stockings), elastic bandages or other known devices, for example, Lebedev’s boot, described in RF patent 2146510, according to cl. A 61 H 9/00, published March 20, 2000. Perform dressings, including the treatment of ulcers (wounds) with antiseptic solutions and changing the pad with the composition 1-2 times a day until healing.

Example 1. Patient C., 72 years old. Diagnosis: varicose disease in the stage of decompensation. Trophic ulcer of the left leg. An ulcer (4.5 cm ² ) exists for 2 years, despite the various treatment methods used (elastic bandaging, daily dressings using iruxol, levomecol, solcoseryl and other drugs). The patient is worried about severe pain in the area of the ulcer (takes 6 tablets of analgin per day).

The patient was discharged Lebedev's boot and treatment began by the claimed method. Every day after treatment of the ulcer with a solution of chlorhexidine bigluconate 0.05%, a gauze pad moistened with a composition containing a sterile extract of the human brain at the stage of intrauterine development in a sterile isotonic solution of sodium chloride, heparin, cefotaxime (claforan), amphotericin B and lidocaine was applied to the ulcer in the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (protein) - 0.5
Heparin - 0.6
Cefotaxime - 10.0
Amphotericin B - 0.2
Lidocaine - 50.0
Sterile Isotonic Sodium Chloride Solution - Else
After 6 days, an increase in granulation, an increase in the bottom of the ulcer, the onset of marginal epithelization were noted, pain in the ulcer area almost disappeared. For dressings began to use a solution of the following composition, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 20.0
Heparin - 12.0
Cefotaxime - 1.0
Amphotericin B - 0.05
Lidocaine - 10.0
Sterile Isotonic Sodium Chloride Solution - Else
A gauze pad moistened with this solution was covered on top with a cellophane film. After 30 days, complete epithelialization of the ulcer surface was observed with minimal scarring.

Example 2. Patient T., 45 years old. Diagnosis: postthrombotic disease. Trophic ulcer of the right lower leg. The ulcer opened 5 years ago after a minor injury. Conducted therapy (elastic bandaging, daily dressings using chlorhexidine bigluconate, levomekol, iruksol, solcoseryl and other drugs) was ineffective. At the time of the first examination, the ulcer area was 15 cm ² , the depth was up to 10 mm, the granulations were sluggish, the edges were necrotic, and there was a profuse purulent discharge.

The patient was assigned a Lebedev boot, a course of treatment with Iruxol was started. 6 days after a significant cleansing of the ulcer surface of necrotic masses, treatment by the claimed method was started. A gauze pad moistened with a sterile composition containing an extract of the human brain at the stage of intrauterine development in a sterile isotonic solution of sodium chloride, heparin, claforan and diflucan was daily applied to the ulcer, with the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (protein) - 0.5
Heparin - 0.6
Claforan - 10.0
Diflucan - 2.0
Sterile Isotonic Sodium Chloride Solution - Else
After 7 days of treatment, a complete disappearance of purulent discharge, a pronounced growth of granulation tissue, marginal epithelization was noted. Then spent daily dressings with a solution of the following composition, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 20.0
Heparin - 12.0
Sterile Isotonic Sodium Chloride Solution - Else
A gauze pad was coated on top with a cellophane film. Ulcer healing occurred after 47 days (complete epithelization of the surface with minimal scarring).

Example 3. Patient M., 30 years old. Diagnosis: postthrombotic disease. Trophic ulcer of the left leg. The ulcer occurred after a minor injury 1 year ago. Despite a variety of treatment methods (troxevasin, compliance, laser therapy, elastic bandaging, daily dressings using placept, iruxol, actovegin), there was no tendency to ulcer healing. At the time of the first examination, the ulcer area was 6 cm ² , the depth was up to 8 mm, the edges were necrotic, profuse purulent discharge.

A course of therapy was carried out using levomekodya (daily dressings together with elastic bandaging). After 7 days, dressings were applied to the cleaned ulcer surface using a composition containing an extract of the human brain at the stage of intrauterine development in a sterile solution of Dulbecco's phosphate buffer (Dulbecco's phosphate buffered saline), heparin, claforan and amphotericin B, in the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of its intrauterine development (for protein) - 2.0
Heparin - 3.6
Claforan - 5.0
Amphotericin B - 0.1
Dulbecco Sterile Phosphate Buffered Solution - Other
For external compression, elastic knee-high socks were used (medical knitwear from ITA-MED, USA). After 5 days, the purulent discharge completely disappeared, there was an active growth of granulation, marginal epithelization. Complete epithelialization of the ulcer surface occurred after 53 days from the start of treatment of the claimed method.

Example 4. Patient O., 15 years old. Diagnosis: burn with boiling water of both thighs of II-IIIA degree-5% of the body surface. Treatment started from 1 day after the injury. Gauze pads moistened with a solution containing a sterile extract of the human brain at the stage of intrauterine development in a sterile isotonic solution of sodium chloride and trimecaine were placed on burned surfaces 1 time per day under a cellophane film, in the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 20.0
Trimecaine - 50.0
Sterile Isotonic Sodium Chloride Solution - Else
Dressings were performed 2 times a day. From the 5th day after the injury, due to a significant reduction in pain for dressings, the following composition began to be used, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 20.0
Sterile Isotonic Sodium Chloride Solution - Else
The course of the wound process is smooth. On the 14th day from the moment of the injury, complete epithelization of the burn surfaces occurred without gross scarring.

Example 5. Patient C., 60 years old. Diagnosis: decompensated type II diabetes mellitus. Diabetic angiopathy of the lower extremities. Condition after amputation of the left lower limb at the level of the upper third of the thigh. In the postoperative period, suppuration of the stump with a divergence of sutures developed. The usual local therapy (daily dressings with Iruxol, Levomekol, Plivacept, etc.), carried out for 2 weeks, was ineffective. In this regard, the claimed method of treatment was applied: a gauze pad moistened with a composition containing a sterile extract of the human brain at the stage of intrauterine development in a sterile isotonic solution of sodium chloride, heparin, lidocaine and miramistin was applied daily to the wound, with the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (protein) - 0.5
Heparin - 0.6
Lidocaine - 10.0
Miramistin - 0.05
Sterile Isotonic Sodium Chloride Solution - Else
After 3 days, the wound was completely cleansed, filled with juicy granulations, along the circumference of the wound - marginal epithelization. Significantly reduced soreness of the wound. From 4 days on, a gauze pad (under a cellophane film) moistened with a composition containing a sterile extract of the human brain at the stage of intrauterine development in a sterile solution of Dulbecco's phosphate buffered saline, heparin and miramistin, was applied with the following ratio of components, mg / ml:
Sterile extract of the human brain at the stage of intrauterine development (for protein) - 20.0
Heparin - 12.0
Miramistin - 0.05
Dulbecco Sterile Phosphate Buffered Solution - Other
On day 8, complete wound healing was observed.

 As can be seen from the above examples, the claimed tool and composition has a strong and complex effect on the processes of tissue regeneration and angiogenesis, does not exhibit antigenic properties, which allows them to be introduced into the patient’s body without side effects. The inventive tool allows long-term storage without loss of its activity.

Claims (16)

 1. An agent for stimulating cell proliferation, characterized in that it contains an extract obtained by homogenizing the human fetal brain at the stage of intrauterine development in isotonic saline, stirring the suspension for 8-12 hours and then centrifuging the suspension for 2- 2.5 hours at 13,000 rpm, separating the supernatant, adding streptomycin sulfate to it to precipitate soluble lipids, neutralizing the resulting mixture and holding it for 20-24 hours, subsequent centrifugation for 2.0-4.5 hours, sterilizing the resulting supernatant and lyophilization.  2. The tool according to claim 1, characterized in that the brain of the human fetus is used for 16-24 weeks as a brain of a human fetus at the stage of its intrauterine development. 3. A composition for stimulating cell proliferation, characterized in that it contains an extract obtained by homogenizing the brain of a human fetus at the stage of intrauterine development in isotonic saline, mixing the resulting suspension for 8-12 hours and then centrifuging the suspension for 2-2 , 5 hours at 13,000 rpm, separating the supernatant, adding streptomycin sulfate to it to precipitate soluble lipids, neutralizing the resulting mixture and holding it for 20-24 hours, followed by cent rifugation for 2.0-4.5 h, sterilization of the obtained supernatant and its lyophilization, and sterile isotonic saline in the following ratio, mg / ml:
Extract (for protein) - 0.1-100
Sterile Isotonic Saline - Rest
4. The composition for stimulating cell proliferation according to claim 3, characterized in that the brain of the human fetus is used for 16-24 weeks as a brain of a human fetus at the stage of intrauterine development.  5. The composition for stimulating cell proliferation according to claim 3, characterized in that as the said saline solution contains sodium chloride solution. 6. The composition for stimulating cell proliferation according to claim 3, characterized in that it further comprises heparin in the following ratio of components, mg / ml:
Extract (for protein) - 0.1-100
Heparin - 0.06-15
Sterile Isotonic Saline - Rest
7. The composition for stimulating cell proliferation according to claim 3 or 6, characterized in that it further comprises a preparation having antibacterial activity in an amount of 0.05-200.0 mg / ml.  8. The composition for stimulating cell proliferation according to claim 3, 6, or 7, characterized in that it further comprises a preparation having antifungal activity in an amount of 0.05-2.0 mg / ml.  9. The composition for stimulating cell proliferation according to claim 3, or 6, or 7, or 8, characterized in that it further comprises a local anesthetic preparation in an amount of 10.0-50.0 mg / ml. 10. A method of treating wounds, burns and ulcers of various etiologies, characterized in that it includes treating the wound surface and surrounding skin with antimicrobial and enzymatic agents, applying a sterile pad moistened with a sterile composition containing the extract obtained by homogenizing the human fetal brain stages of intrauterine development in isotonic saline, stirring the resulting suspension for 8-12 hours and subsequent centrifugation of the suspension during 2-2.5 hours at 13,000 rpm, separating the supernatant, adding streptomycin sulfate to it to precipitate soluble lipids, neutralizing the resulting mixture and holding it for 20-24 hours, followed by centrifugation for 2.0-4, 5 hours, by sterilization of the obtained supernatant and its lyophilization, and sterile isotonic saline in the following ratio, mg / ml:
Extract (for protein) - 0.5-20.0
Sterile Isotonic Saline - Rest
change the said strip 1-2 times a day until epithelialization of the wound surface.  11. The method according to p. 10, characterized in that as the brain of a human fetus, said composition contains an extract of the brain of a human fetus for 16-24 weeks.  12. The method according to claim 10, characterized in that as a sterile isotonic saline solution, said composition contains a solution of sodium chloride. 13. The method according to claim 10, characterized in that heparin is additionally added to said composition in the following ratio of components, mg / ml:
Extract (for protein) - 0.5-20.0
Heparin - 0.6-12.0
Sterile Isotonic Saline - Rest
14. The method according to claim 10 or 13, characterized in that the preparation having antibacterial activity in an amount of 0.05-10.0 mg / ml is additionally introduced into said composition.  15. The method according to 14, characterized in that as a preparation having antibacterial activity, cefotaxime is administered in an amount of 1.0-10.0 mg / ml.  16. The method according to claim 10, or 13, or 14, characterized in that the preparation having antifungal activity is additionally introduced into said composition in an amount of 0.05-2.0 mg / ml.  17. The method according to p. 16, characterized in that as a drug having antifungal activity, amphotericin B is administered in an amount of 0.05-0.2 mg / ml.  18. The method according to p. 10, or 13, or 14, or 16, characterized in that a local anesthetic preparation in an amount of 10.0-50.0 mg / ml is additionally introduced into said composition.  19. A method of treating wounds, burns and ulcers of various etiologies according to claim 18, characterized in that lidocaine is administered as a local anesthetic.

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