RU2687102C1 - Pharmaceutical substance for treating infected wounds of various origins - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention refers to a pharmaceutical composition for treating infected wounds of various geneses. Pharmaceutical composition contains chymopsin, chitosan, acetic acid and water at the following weight ratio, wt%: chymopsin – 0.2, chitosan – 1.0, acetic acid – 0.6, purified water – up to 100.0.EFFECT: invention provides complex proteolytic, antimicrobial and regenerative activity for local therapy of infected wounds of various geneses.1 cl, 7 dwg, 7 tbl, 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 antiseptic and / or anesthetic agents for the treatment of infected wounds of various origins.

Treatment of wounds, especially complicated ones, is an urgent problem of surgery. Purulent infection is considered the most serious complication, both in mortality and in material costs. The frequency of deaths from purulent-septic diseases and complications in inpatients has not decreased in recent years. The incidence of suppuration can be explained by various factors: the initial infection of the wound, suture material, which can cause aseptic inflammation in the surrounding tissues.

Of great importance are a number of circumstances characterizing the state of the entrance gate of the infection and the general reactivity of the organism. Cross-infection in the structure of hospital purulent-septic infections is 30% and increases the stay of patients in the hospital by an average of 15-18 days.

As you know, the development process, the synthesis or production of a pharmaceutical substance using biotechnology methods is the most time-consuming and expensive production stage. In the light of the above, it is relevant and necessary to develop such a pharmaceutical substance that would have complex therapeutic activity, contribute to faster wound healing, more effectively clean the wound surface from purulent-necrotic masses and at the same time would be less costly to manufacture.

One of the areas of modern pharmacology is the concept of developing "multi-functional drugs". Within this paradigm, the concept of a drug with maximum selectivity, acting on one strictly defined target, is opposed to the breadth of pharmacological action and the ability of a medicinal substance to interact simultaneously with several targets. Multifunctional drugs contain in their structure several pharmacophores, the action of which complements each other. Such drugs have a more predictable pharmacokinetic profile, they have significantly reduced the risk of incompatibility with other drugs.

There are many fundamental methods and techniques for administering a drug to the matrix, but in any case, the idea, the principle of the manifestation of the therapeutic effect is the creation of a depot, in which a solid polymer acts as an active sorbent into which a medicinal or biologically active substance is introduced. From this depot containing one drug or mixture, the latter, released from the depot, pass into the body (in this case, into the wound). The principles of the formation of a depot of medicinal substances may be different (chemical: ionic or covalent bonds, physical sorption on the surface of the carrier, binding in the polymer composition). The most difficult of the three named technologies is the technology associated with the covalent fixation of a drug by a polymer matrix, since it requires either a special polymer containing certain functional groups or its chemical modification. But in any case, the biological activity of the active principle can manifest itself only if it is mobile, mobile, able to diffuse from the depot into the body (wound, as a special case) and exhibit healing properties.

For example, there is a patent №2468814 "wound healing gel" for external use containing chitosan, salicylic acid, a bioregulator isolated from bovine blood serum, which is a fraction of acidic proteins in the range of pH values <3.0, and water taken in a certain ratio.

Also known is patent No. 12140264, in which the external agent “Polimed” is claimed, containing medical phenol, resorcin, boric acid, distilled water, chitosan, dimethyl sulfoxide, potassium iodide, glacial acetic acid, glycerin and anesthetic. For wound lesions, the drug can be used as a means of first aid, since it forms a film on the surface of the affected skin, which serves as a protective coating.

The disadvantages of this tool should include the presence in its composition of toxic and carcinogenic substances such as phenol, boric acid, dimethyl sulfoxide, which can cause allergic reactions from the body in contact with the skin and have a toxic effect when injected into the blood. In addition, the drug is not effective enough due to the lack of an active wound healing agent in its composition.

The closest in technical essence to the claimed means is patent No. 2323748 (dated 21.02.2006) “A medical bandage containing a complex of enzymes from a crab hepatopacreas, and a method for producing it”, which contains chitosan as a carrier and a proteolytic complex.

In the modern pharmaceutical market of Russia there are drugs for the treatment of infected wounds, including combinations. Basically, these are ointments on a hydrophobic and hydrophilic basis. However, all of them are not without some disadvantages. The spectrum of action on the pathogenic microflora of the active substances contained in the preparations is not wide enough. Allergic reactions are also possible in cases of hypersensitivity to the drug components.

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

The task was solved through the development of a technology for the production of a pharmaceutical substance, which includes the active components and has a therapeutic effect on the wound. Key innovative solutions boil down to the production of a biologically active compound: the proteolytic complex of chemopsin with chitosan (CXX).

Based on the above-mentioned actual problems, a pharmaceutical substance was developed to achieve the task that could potentially be combined with antiseptic and / or anesthetic agents. The substance is an innovative complex chitosan - himopsin (KXX). The complex is original in composition and method of preparation. The available patent and literature sources did not find data on the preparation of other similar compositions with the proteolytic enzyme himopsin based on glucosamine derivatives. The preferred form of the final drug is a gel.

To increase the stability and provide a prolonged action of the enzyme complex, it is advisable to immobilize it onto carriers to form non-valence-related complexes. Given the characteristics of the introduction (wound surface), the following requirements are made for the intended 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 production of complexes with miramistin and himopsin, chitosan was chosen, which is a product of chitin deacetylation.

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

Functional groups are present in chitosan, such as: -OH, NHCOOCH ₃ , -NH ₂ . The 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 hold onto negatively charged surfaces or hold biomolecules with a low isoelectric point on themselves, forming an interpolyelectrolyte complex (IPEC). The reaction of the formation of such complexes in aqueous solutions is completely reversible. There are two types of plots in the complexes: ordered plots, which are formed by oppositely charged links of both polyelectrolytes connected to each other (A), and plots that alternate with defects or loops formed by sequences of disconnected polyelectrolyte links (B). Due to the presence of hydrophilic units in the defective areas (B), interpolyelectrolyte complexes swell in water. Hydrophobic ordered areas (A) limit the ability to swell and cause their insolubility in water. Due to its chemical nature, chitosan is capable of various types of interactions with the formation of four main types of bonds: covalent, ionic, hydrogen, hydrophobic, and also bonds by the type of complex formation, in which chitosan acts as a complexing agent.

Thus, the basis of the complex is preferably chitosan. Proven wound-healing effect of chitosan, 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 the proliferation of fibroblasts, increase the release of mediators of the immune response. The mechanism of chitosan's stimulating effect on immunogenesis is associated with the adjuvant effect of polymers, with their ability to influence the processes occurring in the initial stages of immunogenesis, apparently, at the stage of the capture of antigen by macrophages and the transfer of antigenic information to B lymphocytes. Increasing the time of contact of the antigen with macrophages leads to increased immunological stimulus. In addition to affecting the immunogenesis unit associated with macrophages, 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 - chitosan-himopsin complex (CXX)

During the development of substance KXX, a series of experiments was conducted with the aim of choosing a proteolytic component.

The change in the enzymatic activity of the samples in 1/15 M potassium-sodium phosphate buffer (PB) was studied at various temperatures. Simultaneously with the determination of the activity, the UV-Vision spectra of the buffer solutions were taken after keeping the samples of the preparations in them.

The choice of active ingredients, and primarily proteolytic, is based on a theoretical and experimental basis. It is important not only to obtain a drug with a high specific activity, but also to preserve it in accordance with the shelf life of the drug. Preservation of enzymatic activity is an important parameter, which is necessary for its further storage and practical application. Modification of the enzyme includes several factors that affect its structure. This chemical modification of individual groups of the protein molecule, leading to a change in the charge of these groups or their hydrophilicity, as well as cooperative interaction of the protein and the matrix, accompanied by the formation of multiple bonds (often of a different nature), leading to both an increase in the rigidity of the structure and a violation of its native nature . These factors can affect the resistance of the protein to denaturing effects, increasing or decreasing its stability.

The observed change in the stability of the enzyme can be associated either with a change in the activation parameters of denaturation (kinetic stability), or with a change in the equilibrium parameters (thermodynamic stability), or with both. In addition, it is important to know which factors — enthalpy or entropy — make a significant contribution to the change in the activation and equilibrium parameters of denaturation. Modification of the enzyme and the formation of salt or other bonds with the matrix leads to an increase in the stability of the protein due to a significant decrease in the activation entropy, and the observed decrease in the activation enthalpy overlaps with the entropy contribution.

Three main mechanisms of chitosan interaction with proteins can be distinguished: the formation of a polyelectrolyte complex (PEC), the encapsulation of proteins in a chitosan gel and their adsorption on chitosan aggregates (Fig. 1). The path through which the interaction proceeds can be determined by the concentration of chitosan in the medium: at low concentrations of chitosan, PEC is formed, at its increase - encapsulation of proteins into the chitosan gel, and at high concentrations of chitosan, its aggregation is observed, with the result that protein molecules are adsorbed on the surface formed chitosan units.

To select a proteolytic enzyme as an active component, the interaction of chitosan solutions with various enzyme preparations - trypsin, a proteolytic complex of crab hepatopancreas and himopsin was studied.

Trypsin is the most common enzyme related to serine proteinases. Trypsin cleaves only denatured protein, due to which it is able to cleave necrotic tissue and fibrinous formations, thin the viscous secret, exudate, blood clots, without affecting healthy tissue.

PC (proteolytic complex) is a complex of proteolytic enzymes of animal origin, isolated from crab hepatopancreas. The proteolytic complex has a collagenolytic and fibrinolytic action and consists of many individual enzymes: collagenolytic serine proteinases, pancreatic elastase, trypsin, chymotrypsin, ribonuclease, deoxyribonuclease, phosphodiesterase, phosphatase, lipase and β-glucanase.

Himopsin is an enzyme preparation of complex proteolytic action. Active active ingredients are chymotrypsin and trypsin, which have proteolytic activity. The components of himopsin cleave various peptide bonds in the protein molecule: trypsin hydrolyzes peptide bonds containing arginine and lysine residues; chymotrypsin cleaves peptide bonds with aromatic amino acid residues - tyrosine and tryptophan.

Chitosan complexes with proteolytic enzymes are obtained by sequential treatment of chitosan with acetic acid solution, introduction of the enzyme into the resulting gel, and thorough mixing in a reactor, followed by drying.

In addition to the choice of the proteolytic component as the main active ingredient in this composition, the choice of chitosan concentration was made. For this, a series of chitosan solutions was prepared with a concentration of from 0.5 to 1.5 mass%. The enzyme solutions selected for the experiment were also prepared in various concentrations from 0.01 to 0.03%. Take into account the reaction time and the temperature at which it proceeded. The main goal of the experiment is to find the right balance of carrier and active substance.

Example 1

Cooking 0.5%, 1%; 1.5% solution of chitosan with the addition of acetic acid.

Solutions are prepared by stirring on a magnetic stirrer at room temperature. After the chitosan is completely dissolved, the solution is left for a day at room temperature for further structuring. Next is the immobilization of the enzyme in different concentrations on chitosan carrier (trypsin, PC, himopsina).

Proteolytic enzymes - trypsin, the proteolytic complex of the hepatopancreas crab and himopsin in various quantities: 100 mg, 200 mg and 300 mg were dissolved in chitosan solutions of different concentrations, were stirred on a magnetic stirrer at room temperature for two hours.

Before proceeding with the drying of the samples, an intermediate assessment of proteolytic activity was carried out. The data are shown in Table 1.

In the course of the experiment, it was necessary to choose an adequate method of drying, the most gentle with respect to biologically active substances included in the composition.

When dried in air at room temperature, thin brittle films are formed. To obtain Xt-enzyme films, the resulting gel (after complete dissolution of the enzyme) was dropped (usually 100 μl of the solution) onto a polyethylene substrate. To determine the enzymatic activity, a substrate with a dried gel was cut out and placed in tubes with the substrate. In specially conducted experiments it was found that the substrate is inert and does not affect the enzymatic activity of the studied drugs.

One of the drying options is freeze drying - a soft drying method in which the substance being dried is frozen and the solvent is sublimated by vacuum. Samples after freeze drying are easily dissolved, which simplifies laboratory tests. This is possible due to the fact that the volume of the mixture being dried 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 contamination, and the enzymatic cleavage is minimized;

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

- less than 1% of moisture remains in the dried product, so 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 air-dried films. When moistened, the material restores its original properties.

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

The gel solution is packaged in 100 ml containers, frozen in a freezer at -40 ° C and placed in a vacuum chamber of a freeze drying unit for ice sublimation. Total loading 0.5 liters (500 g). Conduct two cycles of freeze drying. Drying time ~ 20-24 hours. It should be noted that increasing the concentration of chitosan somewhat complicates the process of freeze-drying, increasing the process time. A lyophilized mass of the substance is obtained in the form of a powder in the amount of 14.8 g in a container.

Evaluation of proteolytic activity was carried out in films and lyophilisate and recorded in Table 2.

As can be seen from the data in the table, the proteolytic activity of the samples after drying by lyophilization remained almost unchanged.

To select the optimal conditions for obtaining immobilized preparations, the influence of the method of immobilization and storage on the biological activity of the systems created was studied.

The kinetics of enzyme inactivation in the process of immobilization, drying or storage is described by complex exponential dependence characteristic of hydrolases.

The effective inactivation rate constant k _{in was} determined as the slope of the direct dependence of the natural logarithm of the residual activity on time (semi-log coordinates ln (A / A ₀ ) -τ) is similar. In specially conducted experiments, it was found that the percentage of enzyme activity preservation does not affect the number of enzymes in the sample (both for immobilized and unmodified PC or X). The data obtained are shown in Table 3.

In Table 3, as an example, data on the loss of enzymatic activity during drying and storage in air (on a polyethylene substrate) of PC preparations, trypsin, himopsin, Xt-PC gels, Xt-Tr, Xt-X gels at 25 ° C are given.

where k _d is the effective rate constant of inactivation in the drying process (usually within 20 hours), k ₁ - characterizes the labile part of the enzyme, and k ₂ - stable. When comparing the obtained systems, the effective inactivation constants for the obtained preparations were calculated, where the smallest constant corresponds to the minimum loss of enzymatic activity, k ₁ characterizes enzymes bound by weak bonds or included mechanically in the drug (labile part of the drug), k ₂ - bound by strong bonds (stable part drug).

A quantitative measure of stabilization during thermal inactivation is the magnitude of the stabilization effect (Θ _t ) at a given temperature, which is the ratio of the rate constants of inactivation of the native and immobilized enzyme. If Θ _t > 1, then immobilization of enzymes stabilizes them at a given temperature. For immobilized preparations, calculation should be carried out using k ₂ .

The thermostability of the preparations was investigated at various pH values. Test tubes with samples were placed in a pre-heated water thermostat. At a certain time, samples were taken and the residual enzyme activity was measured. The data obtained are shown in table 4.

* Himopsin films were obtained by drying a solution of himopsin on a polyethylene substrate.

** Xt-X films (gel) were prepared by drying the Xt-X gel solution on a polyethylene substrate.

*** Himopsin films on dried Xt were obtained by applying a solution of himopsin on an already dried Xt film.

One of the essential goals that modification (immobilization) of enzymes pursues is their stabilization, which is determined by maintaining activity at a higher temperature or for a longer time of use under given conditions compared with the initial enzyme in solution. Modification of the enzyme includes several factors that affect its structure. This chemical modification of individual groups of the protein molecule, leading to a change in the charge of these groups or their hydrophilicity, as well as cooperative interaction of the protein and the matrix, accompanied by the formation of multiple bonds (often of a different nature), leading to both an increase in the rigidity of the structure and a violation of its native nature . These factors can affect the resistance of the protein to denaturing effects, increasing or decreasing its stability. The observed change in the stability of the enzyme can be associated either with a change in the activation parameters of denaturation (kinetic stability), or with a change in the equilibrium parameters (thermodynamic stability), or with both.

The interaction of chitosan structural units (N-acetyglucosamine, glucosamine) with the enzyme preparations under study was investigated and it was shown that N-acetylglucosamine does not affect the enzymatic activity of the enzymes studied, while glucosamine is able to reduce activity at concentrations greater than 5 mM.

Data on the interaction of chitosan with various enzymes and enzyme complexes: trypsin (Tr), PC, and himopsin are displayed in FIG. 2

Chitosan slightly reduces the enzymatic activity of the studied enzymes (no more than 30%) with respect to the high molecular substrate (casein or azocoll).

The interaction of proteins with chitosan can proceed according to the following scheme, depicted in FIG. 3

The analysis of the zeta potential and the geometric parameters of the systems under study (Table 5) also confirms the proposed variants of interaction between enzymes and chitosan.

Thus, the absence of a strong interaction (formation of ionic or other strong bonds) between Xt and the studied proteins (except PC) and the lack of encapsulation of the enzyme molecules in Xt, apparently, leads to the absence of loss of enzymatic activity when the enzyme dissolves in Xt.

Based on the results of the experimental work, it was found that the optimal proteolytic component of the complex is himopsin. The choice of chemopsin, which is a proteolytic complex, is due to the fact that the combination of two proteolytic enzymes increases the effectiveness of therapy by increasing the therapeutic effect due to the complete (more rapid and complete) hydrolysis of peptide bonds.

Characteristics of the pharmaceutical substance - chitosan-himopsin complex (CXX)

The complex chitosan - chemopsin CXX is a combined pharmaceutical substance of the enzyme preparation of the proteolytic action - himopsin and a high-molecular-weight polysaccharide of natural origin - acid-soluble chitosan.

Example 2. Technology for the production of a pharmaceutical substance - chitosan-himopsin complex (KXX)

1. Preparation of an aqueous solution of chitosan. 10.0 g of chitosan is placed in the reactor, 5.7 ml (6.0 g) of a 0.5% solution of acetic acid (d = 1.05 kg / l) are added, mixed well and the mixture is left to stand for 5-10 minutes until it is moistened and swollen. chitosan. Then slowly, with constant stirring, the purified water is added to 1.0 l. The resulting suspension is vigorously stirred for 1.5-2.0 hours until the chitosan is almost completely dissolved and left to stand at room temperature for 10-12 hours until a homogeneous gel-like solution of chitosan is obtained. The pH of the solution is 4.5-5.5.

2. Immobilization of the enzyme complex of chemopsin on chitosan. In the prepared solution of chitosan in the reactor, add 2.0 g of himopsin, the Mixture is continuously stirred for 0.5-1.0 hours until complete dissolution of himopsin. Immobilization is carried out at room temperature. The mass ratio of chitosan and himopsin is 5: 1, the pH of the solution is 4.5-5.5.

Get 1000 g of a gel-like solution of the complex chitosan-himopsin CXX.

3. Obtaining freeze-dried mass of chitosan-himopsin complex. Substance CXX is obtained by freeze-drying a gel-like solution of CXX in a frozen state under vacuum, and water is removed from the frozen substance by sublimation of ice, i.e. converting it to steam, bypassing the liquid phase. Water vapor is pumped out of the process chamber by a vacuum pump and is condensed on the coils of a low-temperature condenser. In order to avoid losses of the lyophilized mass during packing, the filling of the gel-like solution of KXX is carried out in containers intended for the storage and storage of the KXX substance.

The gel-like solution of KXX is packaged in 100 ml containers, frozen in a freezer at -40 ° C and placed in a vacuum chamber of a freeze drying unit for ice sublimation. Total loading 1.0 liters (1000 g). Conduct two cycles of freeze drying. Drying time ~ 20-24 hours.

Get freeze-dried mass of the substance CXX in the form of plates, powder in the amount of 14.8 g

4. Quality control. In the resulting substance, determine the residual moisture content. Humidity samples of substance KXX should be no more than 10%. (GF X1, p. 176). Conduct a qualitative reaction to chitosan. The test drug should paint a solution of iodine with the addition of potassium iodide in 0.13 M nitric acid in purple color.

A qualitative reaction to himopsin is also carried out. The test drug must be able to curl the milk. The closing time for substance CXX should be no more than 50 sec. These reactions confirm the authenticity of the components.

The quantitative definition includes the definition of a protein according to Lowry-Gartley and the determination of proteolytic activity. Substance CXX should contain - (1.95 ± 0.5) g of protein (himopsin) per 10 mg of the preparation.

The proteolytic activity of the solution of the complex of chemopsin-chitosan is 2.75 PE / ml. The proteolytic activity of substance CXX in the form of a lyophilized mass at the end of the shelf life is at least 0.1 PE / mg.

As a result of a series of experiments in order to select the proteolytic component, select the optimal composition and technological parameters of its preparation, as well as select the concentration of chitosan and enzyme solutions selected for the experiment, the following were recorded:

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

- the optimal polymer for obtaining the composition is chitosan;

- the optimal mass ratio of himopsin / chitosan is 1: 5.

The preferred composition of the complex KXX-chitosan-himopsin (number,% (mass)):

Himopsin - 0,2

Chitosan - 1.0

Acetic acid - 0.6

Purified water - up to 100.0

Properties of the pharmaceutical substance - chitosan-himopsin complex (CXX)

Mandatory step is to conduct qualitative and quantitative reactions that establish authenticity for himopsin.

Example 3. Determination of the conjugating ability of himopsin. Determination of UV spectra, determination of proteolytic activity and determination of the amount of protein by the method of Lowry-Gartley

Determination of soiliness

The method is based on the ability of chemopsin to form milk. Milk solution is prepared immediately before analysis. Prepare 1 M acetate buffer (pH 5.6).

About 12 mg of the drug is dissolved in 4 ml of 0.1 M acetate buffer solution (pH 5.6). In three test tubes with a capacity of 10 ml add 3 ml of milk solution and the tubes are heated for 15 min at (35.5 ± 0.5) ° C. Then in two tubes (successively) add 0.5 ml of the test substance solution, shake and mark the time with a stopwatch. The observation is carried out in transmitted light without removing the tubes from the thermostat. The end of the reaction is noted by the appearance of the first signs of spreading. In the control sample (3rd tube), 0.5 ml of 0.1 M acetate buffer solution (pH 5.6) is added to the milk solution and kept in a thermostat for 60 minutes using a stopwatch. Milk should not be coagulated.

The blocking activity is expressed by the time that has passed since the moment the substance was added until the first signs of curdling milk appeared. The dividing time for substance KXX should be no more than 50 seconds, which was recorded during the experiment.

Determination of the UV spectra of substance KXX

The qualitative reactions to himopsin, which determine its authenticity, also include the UV absorption spectrum of the preparation solution in the range from 240 to 300 nm. Max absorption corresponds to 279 nm.

The UV spectra of himopsin, chitosan and pharmaceutical substance KXX (complex of chemopsin-chitosan) were determined to determine the possibility of this method to reflect the authenticity of this substance.

In the spectra of binary mixtures, there are no new peaks or valleys, which indicates the absence of strong valent formations or a covalent or ion-ion bond between the molecules of chitosan or himopsin (Fig. 4-6).

Determination of proteolytic activity

Proteolytic activity (PA) characterizes the ability of enzymes to catalyze the breakdown of protein to peptides and amino acids and is expressed by the number of units of proteinase in 1 gram of the drug. Methods for determining the activity of proteolytic enzymes in most cases are based on a quantitative account of low-molecular-weight products (amino acids and peptides) or chromogenic substances formed during enzymatic hydrolysis of the substrate.

This method establishes the determination of the proteolytic activity of a protein in combined medical preparations with immobilized enzymes and enzyme complexes, such as himopsin, chymotrypsin, trypsin collagenase, elastase, pronase and other proteases. This technique is a modification of the KunitzM method. et. al. The method is based on the hydrolysis of casein according to Hammersten's studied enzyme preparation to peptides and amino acids, followed by their determination.

Preparation of 0.067 M phosphate buffer solution.

By mixing different volumes of solutions of sodium hydrogen phosphate (Na2HOP4⋅12H2O) and potassium dihydrophosphate (KH2PO4), solutions with different pH values are obtained.

Preparation of a 2% solution of casein according to Hammersten. Hammersten 2.0 g of casein is dissolved in 100 ml of phosphate buffer with pH = 8.0 at room temperature with constant stirring. After dissolution, measure the pH of the solution and, if necessary, bring it to pH = 8.05 M solution of sodium hydroxide. The solution is used freshly prepared.

Preparation of 10% trichloroacetic acid solution. In a volumetric flask with a capacity of 1000 ml pour 200-250 ml of water and make 100.0 g of trichloroacetic acid. After dissolving the acid, the volume of the solution is made up to the mark with water and stirred.

Preparation of solution A. 36.2 mg of tyrosine (precise weighed amount) are placed in a volumetric flask with a capacity of 500 ml, dissolved in 100-150 ml of solution B, and the volume is adjusted to the mark with the same solvent. 1 ml of the resulting solution contains 0.40 μmol of tyrosine.

Preparation of solution B. In a volumetric flask with a capacity of 500 ml, 250 ml of phosphate buffer solution pH = 8.0 are poured and brought to the mark with a 10% solution of trichloroacetic acid. The solution is used freshly prepared.

Build a calibration graph. Solutions A and B are poured into test tubes with ground stoppers in accordance with Table 3.

The solutions are mixed, incubated at room temperature for 10-15 minutes, and the optical density (D) is measured on a spectrophotometer at a wavelength of (280 ± 2) nm in a cell with a layer thickness of 10 mm.

As a reference solution using solution B.

The calibration graph is plotted by plotting the concentration of tyrosine (C) on the abscissa, and the corresponding optical density (D) on the ordinate.

Per unit of proteolytic activity (PE) take the amount of the enzyme, which for 1 min at 37 ° C catalyzes a transition to a non-precipitated state, with a 10% solution of trichloroacetic acid, that amount of casein, which corresponds to 1 µM tyrosine.

Test solution. About 0.2 g (exact weight) of the substance KXX is placed in a 20 ml volumetric flask, 10 ml of distilled water is added, the mixture is stirred until the substance is completely dissolved, and the solution is made up to the mark with distilled water.

Substrate solution. As a substrate, a 2% solution of Hammersten casein (MP Biochemicals, 0210128901) in a 0.067 M phosphate buffer solution, pH 8.0, is used. Before analysis, the pH of the casein solution is adjusted to pH 8.0 with a solution of 30% sodium hydroxide. The substrate solution is stored in the dark at a temperature of 5-7 ° C for no more than 10 days.

To determine take three analytical samples of the test solution with a volume of 0.05 ml - two experimental and one control.

In three test tubes with ground-in stoppers with a capacity of 10 ml, 2.0 ml of 0.067 M phosphate buffer solution with pH = 8.0 and 2.0 ml of 2% casein Hammersen solution are poured into each. The tubes are capped and placed in a water thermostat at a temperature of (37 ± 0.5) ° C. After 10 minutes, the test solution was quickly poured into the test tubes. The reaction is stopped after 25-30 minutes (the exact time is noted by a stopwatch) by adding to a test tube 4 ml of 10% solution of trichloroacetic acid (TCA). After 15 min, the solution is filtered through a medium blue filter paper filter; in the filtrate, the optical density is determined spectrophotometrically at a wavelength of (280 ± 2) nm in a cuvette with a thickness of 10 mm.

To the control tube, first add 4 ml of 10% solution of TCA, and then the test solution, then similarly to the test.

The spectrophotometer is adjusted to a solution consisting of equal volumes of 0.067 M phosphate buffer solution and 10% trichloroacetic acid solution.

Proteolytic activity X is calculated by the formula:

A ₁ - the optical density of the test solution at 280 nm;

And ₂ - the optical density of the control solution at 280 nm;

V ₁ - the volume of the test solution, ml;

V is the volume of the reaction mixture in a test tube, ml;

V ₀ - volume of analytical sample of substance, ml;

a is the weight of the substance, in mg;

CT - tyrosine coefficient - 1.20;

t is the reaction time, min;

Norm: The proteolytic activity of substance KXX must be at least 0.1 PE / gram, which was established as a result of the experiment.

Determining the amount of protein according to Lowry-Gartley

The method establishes the determination of the amount of immobilized protein by the method of Lowry-Gartley, which can be used to determine the total protein in combined medical preparations with immobilized proteolytic enzymes, such as himopsin, chymotrypsin, trypsin and other similar nature.

A. 2 g of 4-water potassium-sodium tartrate and 100 g of anhydrous sodium carbonate are placed in a 1 liter volumetric flask, dissolved in 500 ml of 1 M sodium hydroxide solution, stirred and made up to the mark with distilled water.

B. 2 g of 4-water potassium-sodium tartrate and 1 g of 5-water copper sulfate are dissolved in a 100 ml volumetric flask in 90 ml of distilled water and brought to the mark with 1 M sodium hydroxide solution.

B. Divorced Folin's reagent. 1 volume of concentrated folin's reagent is diluted with 8 ÷ 15 volumes of water (depending on normality). The solution is used freshly prepared.

Take three test tubes (one control, two experimental). To the sample weighed in the test tubes, 1 ml of water is poured, mixed thoroughly, and 0.9 ml of reagent A is added. The tubes are incubated in a water thermostat at 50 ° C for 10 minutes. After cooling to room temperature, 0.1 ml of reagent B is added to the tubes, kept at room temperature for 10 minutes, then 3 ml of diluted Folin's reagent are quickly added and again incubated for 10 minutes at 50 ° C. After cooling to room temperature, add 5 ml of distilled water to each tube, mix thoroughly and measure the color intensity at 650 nm on a UV-Vis-SF2000 spectrophotometer.

A sample of the sample without an enzyme is placed in the control tube, treated in parallel with the same solutions as the samples in the test tubes. The calibration curve is constructed each time using a solution of himopsin with a concentration of 1.0 mg in 10 ml of 0.0001 M HCl.

The graph (Fig. 7) shows the linear dependence of the optical density of the solution on the concentration of himopsin, which is illustrated by the equation obtained for the calculation:

D = 0.0021⋅С,

Where D is the optical density of the solution, opt. ed;

C is the concentration of himopsin, mcg.

Thus, as a result of the experiments, the properties of the declared pharmaceutical substance, its qualitative and quantitative composition, and the technology for its production were recorded and confirmed. The developed innovative substance is capable of being used both independently and preferably in combination with antiseptic preparations, as well as anesthetics, which significantly improves the potential of the developed pharmaceutical substance.

The substance in the composition of the finished dosage form may be an ointment, spray, cream or gel. The gel form of the drug for the treatment of wounds, especially complex, nonhealing, is preferable because of the use of water base, and other serious advantages: a mild effect on the wound, maintaining a moist environment, lowering the temperature of the wound, anesthetic effect.

The preferred composition and method of obtaining the substance indicates that its production does not require high costs and is suitable for industrial production. This possibility determines the availability, prolongation and universality of the substance and contributes to the expansion of the arsenal of wound healing and antimicrobial agents.

Claims (2)

Pharmaceutical composition for the treatment of infected wounds of various origins, containing himopsin, chitosan, acetic acid and water at the following mass ratio, wt.%: Himopsin 0.2 Chitosan 1.0 Acetic acid 0.6 Purified water up to 100.0

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