RU2703284C1 - Bactericidal anti-burn biodegradable agent based on a collagen pharmaceutical composition with lactoferrin, methods of producing (versions) and properties - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical composition, a method for preparing it, an agent in the form of a wound coating based on a pharmaceutical composition and use of such a preparation as a wound healing agent. Pharmaceutical composition for applicative application possesses the wound-healing property. Composition is made of collagen of natural origin, related to type I and III collagen as active component (90.0–97.0 wt%) and lactoferrin (3.0–10.0 wt%).

EFFECT: obtaining a pharmaceutical composition for preparing a wound coating having a wound-healing and bactericidal property, providing a wound-healing period that is easy to use, long-lasting, having no contraindications and biosafety.

10 cl, 2 dwg, 5 ex

Description

The field of technology.

The invention relates to medicine, namely to experimental and clinical surgery, transplantology, dermatology, combustiology and can be used as a wound cover for the treatment of burns and combined wounds. A remedy called “LACTOKOL” is proposed in the form of a wound dressing. For this, a pharmaceutical composition of collagen and lactoferrin is used. Collagen provides reliable shelter of the wound surface from external infection, promotes accelerated wound healing, as the biodegradable material does not require removal of the primary dressing, and lactoferrin, which has bactericidal activity, prevents the development of pathogenic microorganisms, including antibiotic-resistant ones, that got on the wound surface at the time of the traumatic effect . The prior art.

The creation of integumentary material for the treatment of burns of varying degrees and prevalence, as well as combined wounds, has a long history. In this case, the primary task was to protect the affected surface of the skin defect, which is necessary for the effective fight against shock, plasma loss and the development of secondary infection from external infection. However, to date, coatings for the treatment of burns are not effective enough in case of infection of the wound surface at the time of injury.

The number of registered wound dressings is quite large: familiar textile materials (gauze, mesh, knitwear, non-woven fabric), film-forming compositions of artificial materials, sponges, hydrocolloids, gels, powders, pastes, etc. Traditional textile dressings provide only reliable mechanical protection, but, absorbing wound secretion, they become a favorable environment for the development of pathogenic microflora. In addition, they stick to the wound, cause pain when changing dressings, etc.

A fundamentally new method of treating thermal burns was the use of synthetic materials based on various polymers. They are an alternative to textile-based dressings, since they protect the wound well from external influences, are modeled on the wound surface, adhere tightly to it, provide the required steam exchange and are fixed on the wound without the use of special means, in addition, they can be used together with some phytochemicals, or biologically active inorganic substances. The presence of medicinal proteins in their composition is problematic, and the use, in particular, of lactoferrin, is practically impossible for technical reasons. Therefore, we did not consider synthetic dressings during a patent search, with the exception of those that had a biological component, for example, contained collagen.

Biological coatings for burn wounds - this is an independent group of dressings represented by a wide range of biological materials. In the process of patent search, we examined well-known biological burn dressings for any indications of the presence of lactoferrin, including those specifically for dressings created on the basis of collagen.

A biological wound cover is known from de-cellulose pig skin (RF Patent No. 2478403). The technological disadvantages of this biological wound dressing are its unsatisfactory mechanical qualities: poor elasticity, difficulties in modeling the cover on wounds with a complex skin texture, expansion after drying of the skin pores and, as a result, weakening of the transdermal barrier. This invention differs from the claimed composition and method of preparation.

Known wound cover "Collachite" based on a collagen-chitosan complex (Patent RU No. 2108114). This wound cover is different in composition (does not contain lactoferrin), and also differs in execution. The disadvantage of this wound cover is that it does not have the bactericidal and immunomodulating properties that are inherent in lactoferrin.

Another wound dressing based on the collagen-chitosan complex is known (Patent RU No. 2254145). The invention is used to treat skin defects. It is a coating in the form of a sponge, gel, colloidal solution, or film. The invention differs in composition from the claimed and does not contain bactericidal protein lactoferrin.

Known biodegradable wound cover and method for its production (Patent RU No. 2519158). The coating is in the form of a film, contains chitosan, glycerin, ceruloplasmin, L-aspartic acid. The disadvantage is the inability to use for the treatment of infected wounds. This coating differs from the claimed composition and method of preparation.

A known method of local treatment of wounds using a biological dressing containing living cells of the line of diploid human fibroblasts (Patent No. 2526811). The method involves the use of a dressing that contains a polymer base of a hydrophobic perforated organosilicon film coated with a layer of type I collagen and living human diploid cells - fibroblasts of the M-20 line. This wound coverage is different in composition (does not contain lactoferrin), and also differs in execution. The disadvantage of this invention is that this wound cover does not have the bactericidal and immunomodulatory properties that are inherent in lactoferrin.

Known bioabsorbable matrix, consisting of native, unreconstructed collagen I, used to fill defects in soft and hard tissues, as an implant, as well as treatment, repair or tissue replacement (Patent RU No. 2353397). The invention provides the creation of a bioabsorbable collagen matrix consisting of native unreconstructed type I collagen, which has a collagen fiber structure identical to that of natural collagen-containing tissue, has significant strength properties and can serve as a matrix for growth, directed regeneration, improvement of trophism and structure of soft and hard tissues . This invention differs from the claimed use (used not to treat wounds, but to fill defects in soft and hard tissues) and composition (does not contain lactoferrin), as well as the method of preparation.

Known porous sponges (USSR author's certificate No. 561564) for the treatment of wounds, created on the basis of collagen, a mixture of gelatin, chitosan and formaldehyde (China patent No. 1097980); gelatin and formaldehyde with the addition of antibiotics (RF patent No. 2033149); cellulose and chitosan (JP patent 0376029); collagen and chitosan (PCT patent No. 8504413). The disadvantage in using all of these wound dressings is the low clinical efficacy.

It is known to use a transparent polyethylene perforated film with the application of powder on its inner surface containing 95% talc and 5% a mixture of 11 antibiotics and an antiseptic. The mixture could additionally contain a powder based on water-soluble keratin (a protein derived from wool) with an equal ratio of the components of the mixture. This film had very limited use due to the absence of any type of fixation and uneven distribution of the dressing on the wound surface, which led to a violation of the correct location and the need to remove it from the wound along with the wound.

The invention is known (patent RU No. 21266693), in which, in addition to collagen, Cytochrome C, extracted from the hearts of cattle, is included in the composition. The disadvantage of this coating is that it uses a scarce natural material, which cannot be widely used to obtain wound coverings, in addition, it did not show the declared effectiveness in clinical conditions.

Known wound healing coating based on collagen and sea buckthorn oil (patent RU No. 602185), called "Oblekol". In the experiment, under the conditions of modeling wounds and ulcers, Oblekol was more effective than a pure collagen film. However, with all kinds of wounds, especially complicated by infection, with post-burn ulcers, trophic ulcers, bedsores and other chronic damage to the skin, sea buckthorn oil as a bacteriostatic was not effective. When using this traditional medicine, additional antibacterial therapy was required. The purpose of this film according to these indications was considered impractical.

Known wound dressings containing collagen and other biologically active substances. For example, methyluracil stimulating tissue growth (patent RU No. 2226406; Begma A.N. et al., 2014). The use of methyluracil together with collagen in wound surgeons is not widespread. Collagen plates with methyluracil were not recommended for children under 3 years of age, during pregnancy and during lactation. Contraindications to the use of methyluracil were with hypersensitivity to this drug, the presence in the wound of excessive granulation and individual intolerance. They were contraindicated in the complex treatment of superficial injuries in the elderly, when the reproduction of their own collagen was reduced. Then methyluracil began to be released in the form of suppositories (suppositories), tablets and ointments.

Known two-layer film system, which showed good stimulating ability to regenerate new living tissue (US patent US 5994325 A). The material contained as a top layer a thin film of cross-linked polydimethylsiloxane of the Silastic Medical A brand, which has high permeability to water and oxygen. The lower layer of this material consisted of an interpolymer complex based on collagen and glucosaminoglucan-chondroitin-6-sulfate and was obtained by freeze-drying a mixture of polymer solutions. To improve the regeneration process, endothelial cells were introduced into this lower layer. The disadvantage of the coating was the individual intolerance of the individual components.

Known anti-burn dressing COLLOST® - a bioplastic material of collagen origin (patent No. 2353397). It is a combination of collagen obtained from cattle skin. According to the authors, this material is almost identical to the collagen of the human body, therefore, does not cause rejection. KOLLOST® actively participates in the process of regulating the level of proteolytic enzymes, activates osteoclasts. Thanks to this effect on the body, patients regenerative processes improve and accelerate significantly - epithelial, epidermal and endothelial tissues are restored. In the course of studies, it was revealed that the presented biological material contributes to the activation of the synthesis of endogenous collagen.

Unlike the LAKTOKOL anti-burn composition presented in this application, the KOLLOST® anti-burn membrane must undergo a preparatory stage before use (it must be placed in a sodium chloride solution (sterile) to soften). If there are indications for antibiotic therapy, then this solution is replaced with antibiotics or antiseptics - this may be due, for example, to existing purulent processes in the lesion. After the membrane is softened, the specialist must accurately determine its required size - it must not be allowed that healthy skin areas are covered with this material. But, at the same time, it is necessary to adhere to the “2 cm” rule - the edges of the material should “grasp” the borders of the affected area. After applying and fixing the COLLOST® membrane, the wound is covered with a sterile dressing. The dressing should not be completely dry, therefore, a dressing should be applied using any suitable solution - for example, antibiotic, antiseptic. The COLLOST® anti-burn dressing is deprived of the main main advantages of LACTOCOL, which can be used without any preliminary preparation for the treatment of domestic, industrial and military burn injuries and contains lactoferrin protein, which has a universal bactericidal effect against a wide range of pathogenic microorganisms, including including antibiotic resistant.

It is known to use "spraying" on the wound surface of lactoferrin powder, however, it will still require shelter of the wound from external infection, and then traumatic removal of this coating, which can be performed on the basis of collagen or other covering material, in order to ensure the introduction of new servings of lactoferrin, as the development of internal wound infection begins [TakayamaY1, et.al., 2012).

Thus, technical solutions that coincide with the totality of the essential features of the claimed invention from the prior art have not been identified, which allows us to conclude that the claimed invention meets such a patentability condition as “novelty”.

Terms and Definitions

The term "wound cover" means any cover on the wound, traditionally used in routine medical practice, selected from the group not limiting the invention: gels; dressing bags; medical wipes; patches (fixing and integumentary); tampons; plates; medical sponges, including absorbable gelatin, gelatin-starch, collagen, alginate (algipor); other specialized dressings and acceptable applicative forms for use on wound surfaces.

The term "lactoferrin" means a full-sized highly purified protein lactoferrin obtained from natural raw materials (human milk, cow's milk), or "Neolactoferrin" isolated from the milk of genetically engineered goats. In the latter case, it is represented by a complex of goat lactoferrin (10%) and the bio-analog of human lactoferrin (90%).

In the present application for the invention, the term "collagen" means a highly purified collagen protein of natural origin of pharmaceutically acceptable quality, I, III and other types.

Brief description of figures, drawings.

Figure 1. Curve for the release of lactoferrin from collagen material. Figure 2. The growth curve of the strain - clinical isolate Staphylococcus aureus when using collagen plates with different concentrations of lactoferrins (bovine - bLf, or "Neolactoferrin" - hLf)

The basis for the implementation of the invention.

In the previously proposed to cover the lost skin during burns, chemical and mechanical, open and closed injuries, various biological materials were used: allogeneic skin, amniotic membrane of the fetus, animal skin, as well as fragments of the intestines and peritoneum, combined synthetic and biological materials. They have not received wide practical application in a surgical clinic for the treatment of burns.

Such materials do not possess an independent antimicrobial therapeutic effect. Meanwhile, the presence in the coating material of means with a bactericidal effect, which is vital both in the acute period of a burn lesion and in the treatment of difficult to heal, especially open wounds. In this regard, the idea arose of creating a fundamentally new biodegradable integumentary material for the treatment of burns based on collagen and the bactericidal protein lactoferrin.

The team of authors of the participants of this development (Institute of Gene Biology, Russian Academy of Sciences, Sechenov First Moscow State Medical University) at different times carried out research work, which in the submitted application for the invention were combined into the Laktokol fire protection coating.

In addition, in the Burn Center Research Institute of Emergency Medicine. Sklifosovsky, positive experience has been gained in using collagen dressings as a cover material for burn wounds, which under clinical conditions have shown anti-inflammatory effects, stimulation of reparative processes in wounds, accelerated growth and maturation of granulation tissue and epithelization. Based on these studies, two patents were obtained (patents RU No. 2314129, RU No. 2455997).

Despite the relative popularity, collagen itself, unfortunately, does not have bactericidal properties, which significantly reduced the scope of such wound dressings, especially infected burn wounds. These properties have a protein of natural origin lactoferrin. It demonstrates a wide range of biological activities, the most valuable of which are its bactericidal and immunomodulating properties.

The bactericidal properties of lactoferrin are due to the fact that it accumulates iron, which is necessary for the life of pathogenic bacteria (in natural liquids, the lactoferrin's iron content is 12-15%), destroys the polysaccharide membrane of both gram-negative and gram-positive bacteria. At the same time, lactoferrin is also effective against antibiotic-resistant forms of pathogenic microorganisms. Bacteria lack the genetic mechanisms of adaptation to this protein. In addition, lactoferrin significantly enhances the effect of antibiotics on pathogenic microflora (Drago-Serrano et al., 2017; Femandes & Carter, 2017; Giansanti et al., 2016; Bruni et al., 2016).

In our country, the first studies of the biological activities of lactoferrin were begun in the nineties of the last century at the Moscow Cancer Institute. Herzen, where natural human lactoferrin obtained from human milk was studied. It could not have industrial significance due to the shortage of human milk, and is currently banned for clinical use (Appendix 5a to SanPiN 2.3.2.1078-01 approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of November 14, 2001 N 36) the reason for the possibility of transmission of infectious pathology: AIDS, hepatitis. Currently not produced.

Lactoferrin obtained from cow's milk is commercially available in several countries and for this protein a method for its preparation and purification has been developed, making it suitable for external use (patents RU 2579661, RU 2390253, RU 2634859).

At the Institute of Gene Biology of the Russian Academy of Sciences, for the first time in world practice, a pilot industrial technology for the production of the human lactoferrin bioanalogue (patents RU No. 2491343, No. 2553515, No. 2554742) was developed, for which its identity to the natural human protein and its biosafety were shown. Lactoferrin stimulates the cells of the immune system (Chernousov A.D. et al., 2013), which enhances natural immunity. It is known that at the initial stage of the inflammatory wound process, lactoferrin causes an increase in the production of pro-inflammatory cytokines, which enhances the infiltration of neutrophils and macrophages into the foci of affected tissues. At the same time, along with providing an anti-inflammatory effect, the excess natural immune response is neutralized (D. Legrand, 2016). Lactoferrin helps to activate the formation of granulation tissue, angiogenesis and re-epithelialization of a tissue defect by enhancing the proliferation and migration of fibroblasts and keratinocytes. In the last decade, the idea of the physiological role of lactoferrin has significantly replenished, and they began to consider it not only as an anti-infection agent, but also as a multifunctional factor in tissue differentiation and activation of cells. These properties of lactoferrin at the cellular level are realized, at least due to several mechanisms. Firstly, upon direct penetration of lactoferrin into progenitor cells due to the protein part of the receptor for low density lipids (LPR1) (Curran CS et.al., 2006;), secondly, upon the action of lactoferrin on activation kinases (MAPK) and finally, cell activation occurs when lactoferrin interacts with Toll receptors or their functional counterparts, receptors of “hazard molecules” (Oh SM et.al., 2004).

Due to the fact that the activation of lactoferrin affects the proliferation and differentiation of cells of various origins, the biological activity of lactoferrin can be effectively used to provide a complex and dynamic process of wound healing, which requires regulation of the interaction of various types of cells, including neutrophils, macrophages of immune cells, keratinocytes, fibroblasts and endothelial cells (Sadchikov P.E. et al., 2016). The subtle molecular mechanism of the manifestation of the biological activities of lactoferrin has not been fully studied. It is believed that it interacts with specific tissue and cell receptors (Rosa L. et.al., 2017).

At the Institute of Regenerative Medicine of the First Moscow State Medical University. Sechenov gained great experience in creating collagen plates (patents RU 2396984, RU 2127128, RU 2287333, RU 2314129, RU 2455997, RU 2385726, RU 2308960).

Wound healing begins with hemostasis and is accompanied by an inflammatory phase in which the bacteria that have got into the wound are neutralized and it is cleansed of the remnants of the affected cells and extracellular fragments of the skin matrix by means of neutrophil and macrophage infiltration into the wound area. The results of animal studies indicate that local administration of lactoferrin increases the rate of skin wound healing in healthy mice tested with traumatic effects and diabetic ulcers by increasing the production of anti-inflammatory cytokines that enhance the natural infiltration of neutrophils and macrophages into the wound area. From the collapsing neutrophils into the wound, the recipient lactoferrin contained in them is released. The combination of the action of endogenous and exogenous lactoferrin helps to cleanse the wound from bacteria that got into it during an injury, preventing the development of an infectious process. Then begins the proliferation of keratinocytes and their migration to the wound area, ending with the gradual closure of the wound defect (Pattamatta et al., 2013; Takayama & Aoki, 2012; Tang et al., 2010; Weinberg, 2007).

et al., 2015). Показано, что применение лактоферрина на биопленках является удачным вариантом в случае его аппликативного использования (Ammons &

2013).With ulcers - one of the most common devastating complications of diabetes, in the chronic course of the wound healing process, an excessive inflammatory reaction occurs, the bacterial infection joins, which often leads to gangrene and the need for amputation of the foot. Randomized clinical trials have shown that lactoferrin accelerates the healing of a wound defect (Lyons et al., 2007). A similar effect has been reported in other studies (Engelmayer & Varadhachary, 2003; Engelmayer et al. 2008;

et al., 2015). It has been shown that the use of lactoferrin on biofilms is a good option in the case of its applicative use (Ammons &

2013).

The pharmaceutical composition proposed in the present application containing collagen with lactoferrin is a wound dressing that has been established to gradually lysate (biodegradable) and gradually release the lactoferrin included in its composition. Due to this biologically active bactericidal protein, wound healing processes are stimulated and an antibacterial effect is provided. The source of natural lactoferrin is animal milk, or the bioanalogue of human lactoferrin obtained in a biotechnological way. Technologies have been created that allow the production of this protein from biological raw materials in an industrial way, which opens up the possibility of its wide medical use.

Lactoferrin has a complex of biological properties that accelerate the healing process of wounds, and at the same time shows high antibacterial activity. It is not toxic, does not cause morphological and biochemical changes in the blood, does not exhibit embryotoxic and teratogenic effects, which allowed it to be considered biologically safe for use in children and adults.

In order to impart other pharmacological properties, it will be possible to additionally introduce the corresponding biologically active or medicinal substances, additional antiseptics, etc. into the collagen coating composition, due to the absence of contraindications of any possible incompatibility with them.

With extensive surface burns of IIIa degree (burn area more than 20% of the body surface), the course of the wound process is fraught with purulent complications, melting of the scab, intoxication, the development of a burn disease, when skin appendages, sources of expected epithelization, die under the influence of bacterial invasion, which requires other approaches to epithelization treatment of burn wounds.

Known means for treating wounds, the basis of which is collagen, for example, collagen sponges, a variety of dressings based on collagen with the addition of antiseptics (G.I. Nazarenko et al., 2002). However, these dressings are of limited use due to the lack of clinical efficacy, especially in the treatment of burn wounds.

The closest technical solution is a method for the treatment of burns of IIIa degree using dressings from native collagen on perforated film coatings (RD Bodun, Cand. Diss. "The use of biocoating with dermal native collagen in the treatment of burns of II-III degree", Saratov, 2004 ) In the treatment with this dressing, epithelization of burn wounds occurs 15-16 days after the injury and 5-6 days earlier than self-epithelialization of the wound.

Description of the invention

The present invention is the development of a new wound cover, for the purpose of accelerating the healing process by accelerating epithelization and at the same time having bactericidal properties.

As a basis for wound dressing, a pharmaceutical composition is proposed consisting of collagen and lactoferrin in an effective amount in the following ratio (wt.%): Lactoferrin 3.0-10.0%, collagen 90.0-97.0%. This tool (the proposed trade name "LACTOKOL") can be used to treat burns, combined wounds, ulcers. It can protect the open wound from external infection, and the lactoferrin contained in it, gradually going out into the wound, will inhibit the development of the infection process in it, help stimulate the activity of the immune system cells and epithelize the wound defect.

The tool "LACTOKOL" has the following basic properties:

• The physico-chemical parameters of lactoferrin and its initial biological activity are preserved.

• The optimal concentration of lactoferrin used was established.

• The release of bactericidal lactoferrin into the wound occurs gradually, during the first day when “LACTOCOL” gets wet with wound exudate.

• Sealed in a plastic shell, retains its biological properties for at least two years. When stored dry in the temperature range 4-25 ° C.

• Due to their elasticity (flexibility) repeat the shape of the wound surface.

The technical result consists in the fact that for the first time a wound dressing (LACTOKOL agent) was obtained, which has a wound healing and bactericidal property. It will provide a reduction in the healing time of wounds, convenient in practical use, ensures its long-term preservation, has no contraindications and is biosafe. A system of microbiological quality control of LACTOKOL was developed.

The idea to create a coating material for wounds based on collagen and lactoferrin turned out to be revolutionary. It was known that many proteins tend to aggregate with collagen macromolecules, but often these processes are irreversible and depend on the nature of the proteins, their concentration, type of solvent, salts, metal ions, acids, bases, pH, temperature and other factors. In the case of lactoferrin, this did not happen. On the contrary, the possibility of dissociation of lactoferrin was discovered, the release rate of which depends on the rate of resorption of the collagen base in the wound, under the influence of soaking with exudate. The released lactoferrin retains its inherent antimicrobial and antifungal properties.

The inventive features that predetermine the receipt of the above technical result clearly do not follow from the prior art, which allows us to conclude that the claimed invention meets such a patentability condition as "inventive step".

The condition of patentability "industrial applicability" is confirmed by examples of specific studies with which the claimed invention is illustrated, but not exhausted.

Examples of carrying out the invention

Example 1. A method of obtaining a pharmaceutical composition and funds "LACTOKOL" (option 1)

To 1 kg of a 1% solution of dispersed collagen add 3.0-10.0% lactoferrin (300-1000 mg of lactoferrin), dissolved in 100 ml of distilled water. The concentration of lactoferrin 10% is determined by its maximum bactericidal activity, and the lower limit of 3.0% is determined by the minimum bactericidal effect. Stir until a homogeneous mass. The resulting mixture is poured into cuvettes with a layer thickness of 7-9 mm and placed in a freeze-drying chamber.

Next, get the product itself containing the pharmaceutical composition for wound dressing. To do this, freeze the resulting product to a temperature of -25 ° C, followed by freeze-drying under a vacuum of 0.1 mm Hg and a condenser temperature of -60 ° C. The drying time under these conditions is 24 hours, followed by drying at a temperature of 45 ° C. The resulting product in the form of a dry sponge is cut into 90 × 90 mm plates or other acceptable sizes and packaged in double polymer bags, sterilized by irradiation with a dose of 25 kGy.

Sterile collagen sponges with lactoferrin are a wound cover ready for medical use.

Example 2. A method of obtaining a pharmaceutical composition and means "LACTOCOL" (option 2)

To 1 kg of a 1% solution of dispersed collagen add 300 mg of lactoferrin dissolved in 100 ml of distilled water and 50 mg of glutaraldehyde. Mixed. Next, carry out the same technological methods as in example 1.

Structuring a dry sponge with glutaraldehyde followed by removal of unbound aldehydes increases the time for collagen resorption and lactoferrin release. Glutaraldehyde also contributes to the production of a higher density plate, which is conveniently applied to the wound.

Example 3. A method of obtaining a pharmaceutical composition and funds "LACTOKOL" (option 3)

Obtained in Example 1, a dry sponge is placed in formaldehyde vapor and incubated for 4 hours at a temperature of 20 ° C. The sponge is transferred to a vacuum chamber and held for 24 hours until there is no free formaldehyde in the sponges. As in example 2, the structuring of a dry sponge with formaldehyde followed by the removal of unbound aldehydes increases the time of collagen resorption and lactoferrin release.

Example 4. Evaluation of the time of release of lactoferrin from the pharmaceutical composition and means "LACTOKOL"

Obtained as in Examples 1,2 and 3 dry sponges with a size of ~ 1 cm ³ containing 20 mg of lactoferrin are placed in cylindrical tubes with 1 ml of phosphate buffer with the addition of 0.01% tween 20 (detergent) and kept at 37 ° C. Samples are taken after 2, 4, 8, 20 hours of incubation with stirring of the liquid phase during sampling. The protein concentration in the solution is determined by Bradford. Calibration is carried out using pure lactoferrin dissolved in phosphate buffer with the addition of 0.01% Tween 20 (detergent) (Fig. 1).

The rate of release of lactoferrin depends on the rate of resorption of the collagen base, under the influence of getting wet. Under experimental conditions, this time was extended to 24 hours.

Example 5. Evaluation of the antimicrobial and antifungal activity of the pharmaceutical composition and means "LACTOKOL"

Evaluation of antimicrobial activity as an example, is given in relation to the strain - clinical isolate Staphylococcus aureus. Isolation and cultivation of the microorganism strain was carried out in accordance with the well-known standard protocol (Davydova et al., 2013). For the safety of viable microorganisms during their transportation to the laboratory, the M306 Stuart Transport Medium (Himedia, In.) Transport medium was used. Primary seeding was carried out on a nutrient medium M144 (Himedia, In.) With the addition of blood. Crops were placed in a thermostat at 37 ° C for 48 hours and in an anaerostat for 7 days.

After obtaining and identifying pure cultures, in the experimental part, the Reverse-Spinner RTS-1 bioreactor (BioSan, Latvia) was used. This system is designed to cultivate microorganisms and evaluate their growth in real time. The interpretation of the results was carried out by changing the optical density OD at a wavelength of λ = 850 nm. Brain Heart Infusion Broth (M210) manufactured by HiMedia Laboratories Pvt was used to cultivate microorganisms in the bioreactor. Limited (In.). To obtain reproducible results for the bioreactor, 50 ml Falcon tubes with a cap without a membrane were used (BioSan, Latvia).

The sensitivity of the isolated strains of microorganisms was determined using the serial dilution method developed at the Department of Microbiology, Virology, Immunology, Moscow State Medical University named after A.I. Evdokimova, which was as follows. The investigated agents (collagen plates containing lactoferrin) were diluted in 10 ml of culture medium, which was further used for the cultivation of microorganisms. The total volume in each tube was adjusted to 20 ml. Accordingly, the suspension density before the start of the experiment was 102 CFU / ml, which approximately corresponded to an optical density of OD 0.2.

For microorganisms, their bioreactor settings were used:

Staphylococcus aureus (Temperature: 37 ° C; RPM: 1600 min ^-1 ; Measurement freq .: 3 h ^-1 ; Rev. Spin period: 3 sec; Volume: 20 ml λ ,: 850 nm)

50 ml tubes with a membrane filter (TubeSpin®, SW) were used to cultivate microorganisms in the bioreactor.

The calculated parameters of the lactoferrin samples used during the experiment are as follows: four collagen plates containing 0.04 g and 0.08 g of cow milk (bLf) and human lactoferrin bioanalogue from human engineered goats (hLf), respectively in the form of a cube of a collagen plate (V = 1 cm3).

For each experiment, separately, in sterile 15 ml tubes, a bacterial suspension in a total amount of 5 ml was prepared. The optical density of the obtained suspension was measured using a DEN-1B densitometer (BioSan, Latvia), which for each experiment was 2.82 ± 0.3 McF.

In each experiment, cultivation was carried out in five different tubes:

I samples - growth control;

II samples. - microbial suspension of Staphylococcus aureus + 0.04 g of hLF crushed;

III samples. - microbial suspension of Staphylococcus aureus + 0.08 g of hLF crushed;

IV samples. - microbial suspension of Staphylococcus aureus + 0.04 g bLF crushed;

V samples - microbial suspension of Staphylococcus aureus + 0.08 g of bLF crushed.

For cultivation, 20 ml of culture medium was added to each 50 ml test tube and a pre-prepared bacterial suspension was added in an amount of 0.5 ml, then crushed lactoferrin cube was added to test tubes II, III, IV, V and tightly closed with a lid.

The growth dynamics of microorganisms was studied in four parallels, which was reflected in the graphs of the growth curves of bacterial populations in the presence of plates with bLf and hLf. As a control, bacterial growth was assessed, which was characterized by the onset of a short lag phase (up to 2 hours), accelerated growth almost immediately went into the exponential phase, and by the 4th hour of cultivation, the bacterial growth rate increased significantly. In the presence of collagen with bLf (II and III) and hLf (IV and V), the growth rate of bacterial populations significantly decreased. A smooth transition to the stationary phase was noted at 14 hours of cultivation. The total cultivation time for all samples is 36 hours (Fig 1).

Additionally, the bactericidal and antifungal activity of the obtained collagen plates with lactoferrin against Str was evaluated. agalactiae and C. albicans. Similar to the results of the experiment described above, lactoferrin plates also show visible inhibitory activity.

Thus, microbiological analysis confirmed that the lactoferrin released upon exert a noticeable antibacterial effect, which is necessary in the healing process of wounds and burns, especially those infected.

Thus, the analysis of specific examples of the use of the pharmaceutical composition of collagen with lactoferrin according to the claimed technical solution, as well as a comparison of the test results with those of the known technical solutions, suggests that the inventive tool "LACTOKOL" has high antiseptic properties against the vast majority of gram-positive and gram-negative bacteria fungicidal properties against fungi and provides an effective antibacterial effect due to lactose rina released during resorption of the collagen sponge, which is necessary in wound healing and burns, especially infected. Also, the claimed tool "LACTOKOL" is quite affordable for economic reasons.

Sources of information taken into account when preparing the application:

1. Begma A.N. and others. The experience of using collagen dressings and sponges Meturakol in surgical practice. "Breast cancer", 2014, No. 17, p. 1248.

2. Takayama Yl, Aoki R., Roles of lactoferrin on skin wound healing, Biochem Cell Biol. 2012 Jun; 90 (3): 497-503. doi: 10.1139 / o11-054. Epub 2012 Feb 14].

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Claims (12)

1. A pharmaceutical composition having a wound healing property, for applicative use, containing natural collagen belonging to type I or III collagen, as an active component, characterized in that it additionally contains lactoferrin in an effective amount in the following ratio (wt.%): - Lactoferrin 3.0-10.0, - Collagen 90.0-97.0. 2. The pharmaceutical composition according to claim 1, characterized in that “Neolactoferrin” is used as the lactoferrin, which is a complex of goat lactoferrin (10%) and the human analog of human lactoferrin (90%). 3. The method of obtaining the pharmaceutical composition according to p. 1, 2, which consists in the fact that to 1 kg of a 1% collagen solution add from 300 to 1000 mg of lactoferrin dissolved in 100 ml of distilled water, mix until a homogeneous mass is poured into cells the layer thickness of 7-9 mm and placed in a freeze-drying chamber, freeze the resulting product to a temperature of -25 ° C, carry out freeze-drying for 24 hours, followed by drying at a temperature of 45 ° C, under vacuum 0.1 mm Hg and temperature condenser -60 ° С, floor chennoe product as a dry sponge was cut into wafers and packed in double plastic bags and sterilized by irradiation dose of 25 kGy. 4. The production method according to claim 3, characterized in that to 1 kg of a 1% collagen solution add 300 mg of lactoferrin dissolved in 100 ml of distilled water, and 50 mg of glutaraldehyde. 5. The production method according to claim 4, characterized in that the obtained dry sponge is additionally placed in formaldehyde vapor and incubated for 4 hours at a temperature of 20 ° C, the sponge is transferred to a vacuum chamber and incubated for 24 hours until there is no free formaldehyde in the sponges. 6. The tool in the form of a wound dressing based on the pharmaceutical composition according to claims. 1, 2, obtained by the method according to claims. 3-5. 7. The tool according to p. 6, characterized in that it can be made in the form of a coating on a wound selected from the group: gels; dressing bags; medical wipes; patches (fixing and integumentary); tampons; plates; medical sponges, including absorbable gelatin, gelatin-starch, collagen, alginate (algipor) for use on wound surfaces. 8. The tool according to p. 6, characterized in that the release of active lactoferrin occurs within 20 hours. 9. The use of funds for PP. 6-7 as a wound healing agent for long-term non-healing wounds, including burn wounds and diabetic foot. 10. The use of the agent according to claim 9, characterized in that it is intended for use against one or more bacteria or yeasts associated with infection selected from the group consisting of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, coagulase-negative staphylococci (CoNS) , vancomycin-resistant enterococci (VRE), carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, methicillin-resistant Staphylococcus pseudintermedius, MRSP, Malassezia pachydermatimi coli colma coli, monassezia coli campis, ., Candida albicans, Streptococcus agalactiae, S treptococcus uberis, Esherichia coli, Salmonella choleraesuis, Stenotrophomonas maltophilia, Enterococcus faecalis, Proteus mirabilis, Klebsiella spp., Enterobacter spp and Citrobacter spp.

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