RU2697869C1 - Pharmaceutical substance for treating infected wounds of various origins - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention refers to pharmaceutics, namely to a pharmaceutical substance for preparing a medicinal agent for treating infected wounds. Substance is characterized by the inclusion of an effective amount of the active ingredients miramistin and chitosan, as well as acetic acid as an adjuvant and water with the following ingredients content, wt. %: Miramistin - 0.05, Chitosan - 1.0, Acetic acid - 0.6, Purified water - up to 100.0. Method of producing a pharmaceutical substance is characterized by the fact that chitosan is mixed with 0.5 % acetic acid solution, continuously mixed and poured water to obtain a suspension, mixed until complete dissolution of chitosan and left to stand until a gel-like solution is obtained, then the antimicrobial preparation Miramistin is immobilized on chitosan at room temperature at a weight ratio of chitosan to miramistin 20:1 and is obtained by lyophilisation at -40 °C chitosan and miramistin substance in the form of a spongy cancellous mass, wherein water is removed from frozen substance by sublimation of ice.

EFFECT: invention enables to obtain a biologically active compound: a chitosan complex with an antimicrobial preparation miramistin (CCM), as well as a wider range of wound healing and antimicrobial agents.

1 cl, 7 dwg, 3 ex

Description

The invention relates to the pharmaceutical industry and is an innovative pharmaceutical substance that can be used both independently and in combination with hydrolase class enzymes capable of cleaving peptides and proteins and / or anesthetic agents for the treatment of infected wounds of various origins.

In surgical practice, a complicated course of the wound process is often found, and suppuration of the wound takes the first place among all possible complications. Suppuration occurs as a result of the development and progression of infection in damaged tissues - with trauma, in postoperative wounds, which is associated with both external and internal causes, as well as in wounds formed after opening various abscesses. The development of purulent infection is determined by the interaction of macro- and microorganisms. The critical level of microorganisms contributing to the development of infection is 10 ⁵ -10 ⁶ bacteria per 1 g of tissue. In wounds with damage to local tissue protection, the infectious process can also develop with a lower level of microorganisms.

The formation of resistance of microorganisms to obsolete drugs widely used in clinics necessitates the development of new drugs with a wide spectrum of activity not only in relation to the aerobic, but also anaerobic component, as well as strictly corresponding to the phase of the wound process.

One of the modern approaches in the development of new-generation drugs is the construction of original forms based on substances of a known spectrum of activity using modern innovative technologies that make it possible to obtain drugs with high therapeutic efficacy and, which is important, with minimal side effects. This approach allows you to develop an innovative drug in a shorter time and with significantly lower economic costs.

As you know, the process of developing, synthesizing or producing a pharmaceutical substance using biotechnological methods is the most labor-intensive and expensive stage of production. In the light of the foregoing, it is urgent and necessary to develop such a pharmaceutical substance that would have complex therapeutic activity, contribute to faster healing of wounds, more efficiently clean the wound surface from purulent-necrotic masses, provide an antimicrobial effect, the production of which at the same time would be less costly.

Many technological solutions are known for combining an active drug or biologically active substance with a matrix. But regardless of the type of interaction, the active center of the substance should be free and the medicine should be able to leave the carrier to act on the substrate, in this case, go out into the wound.

For example, an antimicrobial agent for treating wounds and burns based on collagen and sanguirytrin is known (RF patent 2014089). The tool has antimicrobial and wound healing activity, provides a reduction in the healing time of wounds.

Known glue antiseptic wound healing (RF Patent No. 2185155 from 07.20.2002). The invention relates to medicine and relates to glue, which includes miramistin, ethyl alcohol and glue BF-6 in a certain ratio. The glue has a pronounced antibacterial, antifugal effect, enhances regeneration and does not have an allergic effect on surrounding tissues.

Patent No. 2284824 dated 03/18/2005 “Surgical antiseptic glue ARGACOL” describes an adhesive containing collagen hydrolyzate, sodium salt of alginic acid, catapol, dioxidine, poviargol. The invention provides increased antimicrobial activity and regulation of the rate of biodegradation of the coating, depending on the level of the inflammatory process in the wound, which leads to an acceleration of wound healing.

However, these drugs are not direct analogues of the substance being developed.

To indirect analogues, i.e. to the analogs for the intended purpose, one can include Levomekol ointment, Baneocin powder, Solcoseryl ointment and gel. However, the above preparations do not contain proteolytics, without which it is impossible to completely cleanse the wound from non-viable tissues and exudate, therefore, there are no favorable conditions for the formation of granulations and regeneration. Thus, the development of new complex substances will significantly expand today's arsenal of funds for the care of complex wounds.

As already noted, in the modern pharmaceutical market of Russia there are drugs created for the treatment and healing of infected wounds, including combinations.

For example, in patent RU 2115418 ("Combined chemotherapeutic agent" butol for the treatment of local wound infections and inflammatory diseases "dated 01/23/98) a combined chemotherapeutic agent for the treatment of local wound infections and inflammatory diseases containing an antiseptic - chinosol, characterized in that additionally contains an antibiotic - streptomycin. However, this pharmaceutical composition is made in the form of a solution, which significantly affects the prolonged action of the composition, as well as its bioavailability. In addition, the tool does not meet the above criteria.

Another drug that is actively used in the treatment of infected wounds is Levomekol (Manufacturer OAO Nizhpharm, Russia). This combined topical preparation has anti-inflammatory and antimicrobial effects, is active against gram-positive and gram-negative microorganisms (staphylococci, Pseudomonas aeruginosa and Escherichia coli). It easily penetrates deep into tissues without damaging biological membranes, and stimulates regeneration processes. In the presence of pus and necrotic masses, the antibacterial effect persists. However, the spectrum of action on the pathogenic microflora of the active substances that make up the drug is not wide enough. When applying Levomekol ointment, allergic reactions are possible in cases of hypersensitivity to the components of the drug.

The combined antibacterial drug Baneocin has a bactericidal effect. Neomycin and bacitracin, which are part of the drug, exhibit a synergistic effect. Bacitracin is a polypeptide antibiotic that inhibits the synthesis of the bacterial cell wall, is active against gram-positive microorganisms. Neomycin is an aminoglycoside that inhibits the synthesis of bacterial proteins. The drug with a wide spectrum of antimicrobial action is povidone-iodine, which acts on most gram-positive and gram-negative bacteria, while its effect is longer if compared with inorganic iodine. The mechanism of action is the interaction with the proteins of the cell wall of microorganisms with the formation of iodamines. It is active mainly against fungi and viruses.

An effective medicine for the treatment of wounds is ointment "Stellanin" - a tissue regeneration stimulant, antibacterial and anti-inflammatory agent that has analgesic and wound healing effects. The active component of Stellanin® is 1,3-diethylbenzimidazolium triiodide. The mechanism of the pharmacological activity of the drug lies in the direct regenerative effect of 1,3-diethylbenzimidazolium on damaged skin. Active iodine, which is part of the drug, inactivates the proteins of the bacterial wall and enzyme proteins of bacteria, thereby exerting a bactericidal effect.

Gel "Pronosan" for the treatment and hydration of contaminated wounds of any genesis also has a broad antimicrobial activity. The gel helps to eliminate odor from the wound, the destruction and removal of the biological film. It has pronounced effectiveness in the treatment of wounds with slow epithelization. Compatible with almost any dressing, does not have an allergic and irritating effect, has no color and smell.

Gel for wound healing "APPOLO" is intended for the treatment of wounds of various origins, ulcers, bedsores. The basis of the drug is a hydrogel with the drugs introduced into its composition: the antiseptic miramistin and the anesthetic anilokain. The gel has an analgesic effect for 1.5 hours after application, prevents infection of the wound and, importantly, due to the structure of the gel on the skin there are no cosmetic defects in the form of scars.

Sodium alginate hydrogel compositions are also known. For example, “Kolegel” with dimexide is an antimicrobial, anti-inflammatory and regenerative stimulating therapeutic agent used in surgery to treat long-term non-healing wounds, trophic ulcers and burns.

The biological composition for the treatment of wounds "Collachite" contains collagen crosslinked with glutaraldehyde or glyoxal, and chitosan isolated from crab shell. The composition may further comprise antiseptic preparations, for example furagin, and an anesthetic, for example anilocaine (“Collachite-FA”). The composition "Collachite" is used as a wound dressing in the form of porous sponges and films. Collachite coatings promote the growth of granulation tissue, stimulate epithelization, provide scarless wound healing, have an antimicrobial effect and local anesthetic effect.

Thus, the task of developing new pharmaceutical substances that exceed the existing level of technology, namely, substances and agents with complex proteolytic, antimicrobial and regenerating activity for the local treatment of infected wounds of various origins, remains relevant.

The problem was solved by developing a technology for producing a pharmaceutical substance, which consists of active components and has a therapeutic effect on the wound. Key innovative solutions are reduced to the production of a biologically active compound: a complex of chitosan with the antimicrobial drug miramistin (KXM).

Based on the above urgent problems, in order to achieve the stated objective, a pharmaceutical substance was developed that could potentially consist in combination with hydrolase class enzymes capable of cleaving peptides and proteins and / or anesthetic agents. The substance is an innovative complex of chitosan-miramistin (KXM). The complex is original in composition and method of preparation. No patent data were found on the preparation of other similar compositions with the antimicrobial drug miramistin based on glucosamine derivatives. A preferred form of the final drug is a gel.

To increase stability and provide a prolonged action of miramistin, it is advisable to immobilize it on a carrier with the formation of non-valently bound complexes. Given the features of the introduction (wound surface), the following requirements are imposed on the proposed complex: biocompatibility with human tissues, the absence of allergic reactions, pyrogenic and toxic effects on healthy tissues.

As a result of experimental studies on the selection of the optimal carrier for the preparation of complexes with miramistin and chymopsin, chitosan was chosen, which is the product of chitin deacetylation.

The natural polymer-polysaccharide chitosan has a unique set of properties - biocompatibility, biodegradability, non-toxicity against the background of high biological and sorption activity, which allows us to attribute this aminopolysaccharide to a small group of industrially available, environmentally friendly polymers that are exclusively suitable for medical use.

Upon deacetylation, about 60% of the chitin becomes acid soluble and turns into chitosan. This occurs by protonation of free amino groups at a pH of about below 5.

Functional groups are present in chitosan, such as: —OH, NHCOOCH ₃ , —NH ₂ . Amino groups give chitosan the properties of a cationic polyelectrolyte (pKa≈6.5) with unique properties. Chitosan-based materials have a positive charge of NH ³⁺ groups, due to which they are able to retain on negatively charged surfaces or to hold biomolecules with a low isoelectric point on them, forming interpolyelectrolyte complexes (IPEC). The reaction of the formation of such complexes in aqueous solutions is completely reversible. Two types of sites were noted in the complexes: ordered sites that are formed by oppositely charged units of both polyelectrolytes connected to each other (A), and sites that alternate with defects or loops formed by sequences of disconnected units of polyelectrolytes (B). Due to the presence of hydrophilic units in the defective areas (B), the interpolyelectrolyte complexes swell in water. Hydrophobic ordered sites (A) limit the ability to swell and cause their insolubility in water. Due to its chemical nature, chitosan is capable of various types of interaction with the formation of four main types of bonds: covalent, ionic, hydrogen, hydrophobic, as well as bonds of the type of complexation, in which chitosan acts as a complexing agent.

Thus, the basis of the complex is preferably chitosan, since the wound healing effect of chitosan is proved, which can be explained by mechanisms such as activation of the immune response through stimulation of macrophages and the use of acetylglucosamine as a precursor of mucopolysaccharides, which are directly involved in the creation of biostructures, stimulate fibroblast proliferation, increase isolation of mediators of the immune response. The mechanism of the stimulating effect of chitosan on immunogenesis is associated with the adjuvant effect of polymers, with their ability to influence the processes occurring at the initial stages of immunogenesis, apparently at the stage of antigen capture by macrophages and transmission of antigenic information to B-lymphocytes. An increase in the contact time of antigen with macrophages leads to an increase in the immunological stimulus. In addition to the effect on macrophage-linked immunogenesis, chitosan causes changes in the migration and cooperative interactions of cells involved in the immune response, and the effect of stimulating cell interaction can be mediated through the complement system.

Development of a pharmaceutical substance - a complex of KXM - chitosan - miramistin

KCM has antibiotic resistance, enhances the functional activity of immune cells, stimulating local (non-specific) immunity, promotes antigen uptake by macrophages, its accumulation in the lysosomal fraction, and does not have local irritating effect and allergenic properties.

In the framework of solving the problem of wound healing, the use of drugs that have a powerful antimicrobial effect and at the same time are free from side effects that cause antibiotics plays an important role. In addition to antibiotics, these can be antiseptics or bacteriolytic enzymes, i.e. preparations that have a bactericidal or bacteriostatic effect by lysis of the cell wall of microorganisms.

An experimental selection of the antimicrobial component was made. For this purpose, polymer compositions based on acid-soluble chitosan were obtained: chitosan-miramistin; chitosan-lysozyme and chitosan-chlorhexidine. Antimicrobial preparations were immobilized in different concentrations on a chitosan carrier (lysozyme, chlorhexidine, miramistin). The concentration of chitosan is 1.0%, as was determined during the development of the first FS. The resulting gel formulations were also subjected to freeze drying. After receiving the lyophilisate, the antibacterial effect of the obtained complexes was evaluated. The antibacterial action of chitosan-lysozyme, chitosan-chlorhexidine, chitosan-miramistin was studied on strains of Esherichia coli and Staphylococcus aureus.

Of the antiseptics (for medical use, including for the treatment of wounds), miramistin and chlorhexidine are most widely used, lysozyme is the representative of bacteriolytic enzymes. At the stage of technology development of the second innovative substance, it was decided to compare these drugs and choose the most suitable antimicrobial component of the new substance.

Lysozyme - an enzyme of the class of hydrolases, functions as an antibacterial agent, catalyzing the hydrolysis of the polysaccharide, which is part of the cell walls of a number of bacteria. This polysaccharide is formed by alternating residues of N-acetylglucosamine (NAG) h N-acetylmuramic acid (NAM) connected by a β-1,4-glycosidic bond (polysaccharide chains are linked by short peptide fragments).

The bacteriolytic effect of lysozyme is well known and is confirmed by many years of clinical use. Studies show that gram-positive bacteria can be completely destroyed, while gram-negative bacteria undergo bacteriolysis only when their cell wall is additionally destroyed.

The selective effect of lysozyme on the cell walls of predominantly gram-positive bacteria is associated with differences in the composition of the cell walls of gram-positive and gram-negative microorganisms: gram-positive microorganisms, unlike gram-negative bacteria, do not contain aromatic amino acids -proline, histidine, arginine. The shell of gram-negative bacteria contains more lipopolysaccharides. The biological role of lysozyme is not limited only to antibacterial action, it takes part in the protective, immune reactions of the body, in the processes of regeneration and healing of wounds.

Chlorhexidine bigluconate (mainly used in the form of solutions of various concentrations) is a local antiseptic with a predominantly bactericidal effect. The chemical structure of chlorhexidine bigluconate is close to that of bigumal and is a dichloro-containing derivative of biguanide. The mechanism of action is based on its ability to change the properties of the cell membrane of a microorganism. After dissociation of chlorhexidine salts, the formed cations react with bacterial shells that have a negative charge. In this case, the lipophilic groups of the drug contribute to the disaggregation of the lipoprotein membrane of bacteria, resulting in a violation of the osmotic balance and the loss of potassium and phosphorus from the bacterial cell. Under the influence of the drug, the cytoplasmic membrane of the bacterium is destroyed and its osmotic balance is disturbed, resulting in the death of the bacterium.

The drug is effective against strains of the following microorganisms: Trichomonas vaginalis, Neisseriagonorrhoeae, Chlamidia spp., Bacteroides fragilis, Treponema pallidum, Gardnerella vaginalis. In addition, chlorhexidine bigluconate is active against Ureaplasma spp. and moderately active against certain strains of Proteus spp. and Pseudomonas spp. Viruses (except for the herpes virus), as well as fungal spores, are resistant to the action of the drug.

Miramistin is benzyldimethyl [3- (myristoylamino) propyl] ammonium chloride monohydrate. This compound has a pronounced bactericidal effect against aerobic and anaerobic bacteria, gram-positive (Staphylococcus, Streptococcus, Bacillus subtilis, Bacillus anthracoides) and gram-negative organisms (Shigella, Pseudomonas aeruginosa, Escherichia coli, Salmonella), both in the form of monocultures (Pseudomonas aeruginosa and staphylococcus, Escherichia and staphylococcus), including hospital strains with antibiotic resistance. The drug’s molecules act on the outer shell of the microbial cell, which leads to its destruction and death. Its biological effect is based on a direct effect on the cell membranes of microorganisms, despite the fact that it affects the cells of a macroorganism very weakly. Miramistin is able to inhibit the enzymatic activity of cells of pathogenic microorganisms, which is good with respect to antibacterial activity, but can significantly affect the FA of drugs, as the chemical modification of the protein globule can significantly affect the stability of enzymes. The results of the experiments showed that miramistin does not lead to a decrease in the FA of the enzymes used, both native and immobilized in chitosan gel.

Miramistin provides an antimicrobial and fungicidal effect, enhances the functional activity of immune cells during stimulation of local (non-specific) immunity, accelerates the healing process of wounds, reduces the resistance of pathogenic microorganisms to antibacterial therapy, helps activate protective reactions at the site of application by activating the absorbing and digesting function of phagocytes. It does not damage granulation and viable skin cells, does not inhibit marginal epithelization, and does not have a local irritant effect.

Example 1

The experiment was carried out using a 1.0% solution of chitosan. The solution is prepared by constant stirring at room temperature. After complete dissolution of chitosan, the solution was left for a day at room temperature for further structuring.

Next, immobilization of antimicrobial preparations in different concentrations on a chitosan carrier (lysozyme, chlorhexidine, miramistin) was carried out.

The antiseptics miramistin and chlorhexidine and the bacteriolytic enzyme lysozyme in various amounts: 0.1 mg / ml, 0.5 mg / ml and 1.0 mg / ml were dissolved in a 1.0% chitosan solution (pH 5.5), subjected to stirring on magnetic stirrer at room temperature for two hours.

During the experiment, it was necessary to choose an adequate drying method, the most sparing in relation to biologically active substances in the composition. In the case of drying in air at room temperature, thin brittle films form.

One of the drying options is freeze drying - a soft drying method in which the substance to be dried is frozen and the solvent is sublimated by means of vacuum. Samples after freeze drying easily dissolve, which simplifies laboratory research. This is possible due to the fact that the volume of the dried mixture varies slightly due to the formation of a fine-crystalline structure.

The choice of freeze drying in obtaining experimental substances is due to several advantages:

- due to the low temperature there is no danger of microbiological infection;

- due to the high vacuum there is no danger of oxidation of unstable substances with oxygen;

- less than 1% moisture remains in the dried product, therefore it can be stored for a long time.

In addition to the listed advantages of this method, in this case, the removal of excess acetic acid used to dissolve chitosan takes place. The dried material retains its structural integrity and biological activity to a greater extent than films dried in air. When moistened, the material restores its original properties.

Lyophilization of gel-like solutions of enzymes in chitosan is carried out in a frozen state under vacuum, while water is removed from the frozen substance by sublimation of ice, i.e. turning it into steam, bypassing the liquid phase.

The gel-like solution is packaged in 100 ml containers, frozen in a freezer at a temperature of -40 ° C and placed in a vacuum chamber of a freeze drying apparatus for sublimation of ice. The total volume of loading is 0.5 l (500 g). Two cycles of freeze drying are carried out. Drying time ~ 20-24 hours. A lyophilized substance is obtained in the form of a loose mass (lumps and plates).

After receiving the lyophilisate, the antibacterial effect of the obtained complexes was evaluated. The antibacterial action of chitosan-lysozyme, chitosan-chlorhexidine, chitosan-miramistin was studied on strains of Esherichia coli and Staphylococcus aureus.

The species composition was represented by the following microorganisms:

- Esherichia coli (ATCC 25922);

- Staphylococcus aureus (ATCC 6538-P).

The antibacterial activity of the studied objects was investigated by the "wells" method. Bacteria were cultured on L-broth at a temperature of 37 ° C for 20 hours. Then, using the methods of quantitative accounting of microorganisms, namely the Koch method, the number of cells in 1 ml of the initial suspension was determined. For further research, dilutions were used that gave the seeded medium on the Petri dish 10 ⁴ and 10 ⁶ CFU / ml, which corresponds to the seeded area of the wound at the initial and final stages of the disease.

From the dilutions obtained, which were thoroughly mixed beforehand, they were plated on the surface of the agar plate in Petri dishes with a sterile pipette in an amount of 0.1 ml. The volume of the suspension was distributed over the surface of the medium with a sterile spatula.

In addition to surface growth on an agar plate, a method for the growth of microorganisms in the thickness of the agar layer was applied. To implement this method, flasks with molten L-agar with a temperature not exceeding 40 ° C (pre-autoclaved) were prepared, into which the inoculum of a given concentration was introduced. Next, the contents of the flasks were poured into Petri dishes of 25-30 ml.

Wells method (through)

Previously, a hollow cylinder with a diameter of 8.0 mm was placed on the agar, which made it possible to create wells of a given depth and diameter. After seeding in wells, the test solution with a volume of 100 μl was interrupted. As objects of comparison, sterile distilled water was used. The working temperature of the experiment is 37 ° C, which corresponds to the temperature of the patient. The following materials were used as objects of study:

- chitosan-lysozyme ("square" 1 × 1),

- chitosan-chlorhexidine ("square" 1 × 1),

- chitosan-miramistin ("square" 1 × 1),

- chitosan (“square” 1 × 1).

The experiments were carried out in two series. When working with Xt-L, Xt-Xp and Xt-M samples, the control was a chitosan gel, the mass of the preparation was about 10 mg. Cups were marked on the back.

After placing the samples on the medium seeded with cells, and applying the solution or powder into the wells, as well as simply applying a 1 × 1 “square” on the surface of the agar, the plates were placed in a thermostat, where they were incubated for 20 hours at 37 ° C. At the end of the incubation, growth zone retardation was counted. When measuring the zones, they focused on complete inhibition of visible growth. The Figure 1 illustrates Staphylococcus aureus 104 CFU / ml ("wells"): 1 - chitosan-miramistin, 2 - chitosan-lysozyme, 3 - chitosan-chlorhexidine, 4 - chitosan. The Figure 2 clearly shows E. Coli, 106 CFU / ml ("wells"): 5 - chitosan-miramistin, 6 - chitosan-lysozyme, 7 - chitosan, 8 - chitosan-chlorhexidine.

As can be seen from the data obtained, the studied preparations have biocidal properties in relation to staphylococcus, similar data were obtained by E. Coli. The action of miramistin is more pronounced compared with lysozyme and chlorhexidine. Chitosan does not have biocidal properties in relation to the studied cultures.

Based on the results of the experimental work, it was determined that miramistin is the optimal antimicrobial component of the complex. The choice of miramistin, which can be considered an antiseptic of a new generation of a wide spectrum of action, is explained by the fact that its therapeutic effect will be more effective and complete in comparison with other studied drugs. In addition, preliminary studies show that miramistin and chymopsin in the preferred gel composition have an additive effect in the treatment of experimental purulent wounds. Chlorhexidine may have an inactivating effect on the proteolytic component.

Characterization of a pharmaceutical substance - a complex of chitosan-miramistin (KXM)

According to the state of aggregation, the KXM chitosan-miramistin complex is a lyophilized mass in the form of lumps or plates of white or light yellow color, made on the basis of acid-soluble chitosan.

Example 2. The technology of obtaining a pharmaceutical substance - a complex of chitosan-miramistin (KXM)

The first stage of the technological process is the preparation of a 1.0% solution of chitosan in 0.5% (not more than 1.0%) acetic acid. Process parameters: room temperature; the concentration of acetic acid - 0.5% (not more than 1.0%) of the mass; the concentration of chitosan - 1.0% of the mass.

At the second stage of the technological process, the miramistin antimicrobial drug is immobilized - benzyldimethyl [3- (myristoylamino) propyl] ammonium chloride monohydrate onto the high molecular weight chitosan polysaccharide with the formation of the chitosan-miramistin complex (KCM). The miramistin and chitosan molecules contain functional groups through which hydrogen bonds and hydrophobic contacts are formed, resulting in the formation of polysaccharide conjugates of chitosan with miramistin. Process parameters: room temperature; immobilization time - two hours; the concentration of miramistin is 0.05% of the mass (0.5 mg / ml); pH 4.5-5.5.

As a result of a series of experiments, the following was recorded:

- the optimal method of obtaining a composition with miramistin is a method of physical immobilization followed by freeze drying;

- the optimal polymer to obtain the composition is chitosan;

- the optimal mass ratio of miramistin / chitosan is 1:20.

The preferred composition of the KXX complex is chitosan-miramistin (amount, in% (mass)):

Miramistin - 0.05

Chitosan - 1.0

Acetic acid - 0.6

Purified water - up to 100.0

Example 3. Physico-chemical properties of the polymer composition of the KXM complex (chitosan-miramistin)

As can be seen from the presented picture of the IR spectra of chitosan, miramistin, and chitosan-miramistin, it is impossible to distinguish characteristic peaks of miramistin in a binary mixture (KXM) due to the coincidence of the absorption bands. Figure 3 shows the IR spectrum of chitosan-miramistin, Figure 4 the IR spectrum of chitosan, Figure 5 the IR spectrum of miramistin in native form, and Figure 6 the IR spectra of miramistin, chitosan-miramistin and chitosan. Figure 6 is particularly informative, reflecting the infrared spectra of all components on an enlarged scale.

Spectrophotometric method for the determination of miramistin (UV spectra)

A possible qualitative response to miramistin is the removal of UV spectra. With the help of this technique, a qualitative determination of miramistin in combined medicines by the UV spectrum is performed. The Figure 7 presents the UV absorption spectra of miramistin, chitosan and the chitosan-miramistin complex.

The method is based on comparing the spectrum of an unknown compound with a known one; the identity of the spectra indicates the identity of the structures of the studied compounds. UV spectroscopy measures optical absorption in the wavelength range from 170 nm to 400 nm. For miramistin spectroscopy, UV radiation is used in the range 190-300 nm, since they absorb in this spectral region.

The UV-Vis-SF 2000 spectrophotometer records the UV absorption spectrum of a 0.06% aqueous solution of the test drug, the chitosan-miramistin complex, in the wavelength range from 240 nm to 280 nm in a 10 mm thick cuvette.

For comparison and identification, the UV absorption spectra of 0.06% aqueous solutions of chitosan and miramistin are recorded in the same region in a 10 mm thick cuvette.

The ultraviolet absorption spectrum of a 0.06% aqueous solution of the test substance in the region from 240 nm to 280 nm should have:

- maximums at wavelengths (257 ± 2) nm, (262 ± 2) nm, (268 ± 2) nm;

- minima at wavelengths (25 ± 92) nm, (266 ± 2) nm;

- shoulder at a wavelength of (252 ± 2) nm to (254 ± 2) nm.

Quantification of Miramistin

The method is based on non-aqueous titration of a preparation with a solution of perchloric acid in acetic acid in the presence of a crystalline violet indicator until a blue color appears and establishes a quantitative determination of miramistin in combined medical preparations.

5% solution of mercury oxide acetate. 5 g of mercury oxide acetate is placed in a 100 ml volumetric flask and dissolved in glacial warm acetic acid. After cooling, the volume of the solution was adjusted with glacial acetic acid to the mark (HF XII).

0.1 M solution of perchloric acid (HCl4) in acetic acid. To prepare an acetic acid solution of perchloric acid, the calculated volume of the applied solution of the initial perchloric acid (V cm3) is slowly, with stirring, poured from the burette into a volumetric flask with a capacity of 1 dm ³ with glacial acetic acid, the calculated volume of acetic anhydride (V1 cm ³ ) is added from the burette, mix, bring the volume of the solution with acetic acid to the mark and mix again. The solution is suitable for use for 24 hours.

The volume of perchloric acid solution required to prepare 1 dm ^{3 of an} acetic acid solution of perchloric acid with a concentration of (HClO4) = 0.1 mol / dm ³ , (V) in cubic centimeters is calculated by the formula:

where A is the mass fraction of perchloric acid in the applied solution,%;

ρ is the density of the perchloric acid solution used, g / cm ³ ;

10.046 - 0.1 molar mass of the equivalent of perchloric acid.

Due to the fact that the perchloric acid solution used contains water and when it is introduced into 0.5% acetic acid, a certain amount of water gets in, which interferes with further titration, this amount of water should be bound with acetic anhydride. The required volume of acetic anhydride (Vi) in cubic centimeters is calculated by the formula:

where (100 - A) is the mass fraction of water in the applied perchloric acid solution,%;

A - mass fraction of perchloric acid in the applied solution,%;

V is the volume of the perchloric acid solution used, calculated for the preparation of the titrated solution, cm3;

ρ is the density of the perchloric acid solution used, g / cm3;

ρ1 is the density of acetic anhydride, g / cm3;

102.09 is the relative molecular weight of acetic anhydride;

18.02 is the relative molecular weight of water.

Mass fraction of acetic anhydride in acetic acid should not exceed 0.001%.

About 0.3 g (accurately weighed) of the drug is dissolved in 10 ml of a 0.5% solution of acetic acid, 5 ml of a pre-neutralized solution of mercury oxide acetate (5% solution in acetic acid) are added and titrated with a 0.1 M solution of perchloric acid in acid acetic to blue staining (indicator - 0.05 ml of a solution of crystalline violet). In parallel, a control experiment is carried out.

1 ml of a 0.1 M perchloric acid solution corresponds to 0.04391 g of miramistin. This reaction can be used to determine miramistin as part of a binary mixture - the pharmaceutical substance KXM.

Thus, as a result of experiments, the properties of the claimed pharmaceutical substance, its qualitative and quantitative composition, and the technology for its preparation were developed and confirmed. The developed innovative substance can be used both independently and preferably in combination with hydrolase class enzymes capable of cleaving peptides and proteins, as well as anesthetics, which significantly improves the potential of the developed pharmaceutical substance.

The substance in the formulation can be an ointment, spray, cream or gel. The gel form of the drug for the treatment of wounds, especially complex ones, for a long time not healing, is preferable both due to the use of a water base and other serious advantages: a soft effect on the wound, preservation of a moist environment, lowering the temperature of the wound, anesthetic effect.

The preferred composition and method of obtaining the substance indicates that its receipt does not require high costs and is suitable for industrial production. This opportunity determines the availability, prolongation and versatility of the substance, helps to expand the arsenal of wound healing and antimicrobial agents.

Claims (6)

1. A pharmaceutical substance for the manufacture of a medicament for the treatment of infected wounds, characterized by the inclusion of an effective amount of the active components of miramistin and chitosan, as well as acetic acid as an auxiliary substance and water, with the following ingredients, in% (mass): Miramistin - 0.05 Chitosan - 1.0 Acetic acid - 0.6 Purified water - up to 100.0 2. A method of obtaining a pharmaceutical substance according to claim 1, characterized in that chitosan is mixed with a 0.5% solution of acetic acid, constantly mixed and poured water until a suspension is obtained, stirred until the chitosan is completely dissolved and left to stand until a gel-like solution is obtained, then the miramistin antimicrobial drug is immobilized on chitosan at room temperature at a mass ratio of chitosan and miramistin 20: 1 and obtained by lyophilization at a temperature of -40 ° C, the substance of chitosan-miramistin in the form of loose ubchatoy mass, the water is removed from the frozen substance by the sublimation of ice.

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