RU2790481C1 - Antibacterial composition based on endolysins and drugs in form of gel or spray, using it - Google Patents

Abstract

FIELD: medicine; pharmaceutical industry.

SUBSTANCE: invention relates to medicine, namely to the pharmaceutical industry, and concerns dosage forms (DF) in the form of gel and spray for the treatment or prevention of wound and hospital infections. A drug for the treatment or prevention of hospital and wound infections contains a mixture of recombinant endolysins LysECD7, LysAm24, and LysAp22, at a concentration of 0.03-0.15 wt.%, and auxiliary components. At the same time, the drug in the form of gel contains a gel-forming agent, PEG 1500 or PEG 3000, and a buffer solution with pH of 7.0-8.0. The drug in the form of spray contains Poloxamer 338 or Poloxamer 407, PEG 1500 or PEG 3000, and a buffer solution with pH of 7.0-8.0.

EFFECT: drugs provide a local antibacterial effect by applying them to a wound surface or administering in the abscess cavity, have a wide range of action, without affecting representatives of normal flora of the gastrointestinal tract.

2 cl, 4 dwg, 9 tbl, 6 ex

Description

The invention relates to medicine, namely to the pharmaceutical industry, and concerns dosage forms (LF) in the form of a gel and spray containing recombinant endolysins belonging to the classes of muramidase and peptidase, and a method for their preparation.

Currently, the development of healthcare-associated infections (HAIs) is a serious problem both in the Russian Federation and abroad. Of great importance are surgical infections (up to 230 cases per 1000 operations), the main causative agents of which are gram-negative bacteria, among which Escherichia coli, Klebsiella pneumoniae, Enterobacte spp. and Pseudomonas aeruginosa are more common. These pathogens cause purulent-septic complications that can develop even with proper care of surgical wounds.

In addition, there is a trend towards an increase in the number of multiresistant pathogens, which makes antibiotic therapy ineffective.

There are many ways to combat bacterial infections, among which phage therapy is gaining popularity due to the high efficiency of bacteriophages against antibiotic-resistant pathogens. Despite this, phage therapy has several disadvantages, including:

узкий спектр литической активности;

narrow spectrum of lytic activity;

трудоемкость технологии получения (их сложно выделять из бактериальных культур с требуемой степенью чистоты);

the complexity of the production technology (it is difficult to isolate them from bacterial cultures with the required degree of purity);

необходимость постоянного определения и нормирования их фармакокинетических характеристик;

the need for constant determination and regulation of their pharmacokinetic characteristics;

культивирование необходимо проводить на близких к патогену видах и штаммах, для которых отсутствуют эффективные стандартные протоколы культивирования, и применяются высокие требования к безопасности;

cultivation must be carried out on species and strains close to the pathogen, for which there are no effective standard cultivation protocols, and high safety requirements apply;

умеренные бактериофаги могут переносить гены бактериальных токсинов;

temperate bacteriophages can carry genes for bacterial toxins;

низкое, но вероятное появление к ним устойчивости;

low but likely emergence of resistance to them;

иммуногенность, которая снижает эффективность их повторного применения.

immunogenicity, which reduces the effectiveness of their repeated use.

The above disadvantages are deprived of endolysins - enzymes that are synthesized and contribute to the lysis of a bacterial cell at the end of the reproductive cycle of a bacteriophage, destroying the cell wall.

Endolysins are divided into several classes, which determine the sites of their action on peptidoglycan, namely:

1. Muramidases:

a. Lysozyme-like enzymes

b. Lytic transglycosylases;

2. N-acetyl-β-D-glucose aminidases;

3. N-acetylmuramoyl-L-alanine amidases;

4. peptidases.

Endolysins are obtained biotechnologically using producer strains containing a plasmid that carries the gene encoding endolysin.

After cultivation and isolation of the product by ultrasonic destruction of cells, a stage of chromatographic purification at low temperatures (+4°C) follows by metal-chelate affinity chromatography, followed by gel exclusion chromatography with the addition of stabilizing substances (150 mM NaCl) at the final stages, which allows us to obtain active stable preparations of proteins.

Recombinant endolysins, unlike bacteriophages, have a wide spectrum of action. Thus, it was found that the recombinant endolysin of the bacteriophage ECD7 has lytic activity against strains of Campylobacter jejuni, Enterobacter spp., Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae, while the bacteriophage ECD7 is active only against E. coli .

In addition, it was found that when using endolysins in the composition of LF in the form of a gel and spray, no formation of antibodies to recombinant endolysins is observed, which indicates the possibility of repeated use of these drugs without reducing efficiency.

Based on the above facts, it can be concluded that recombinant endolysins have a number of advantages over bacteriophages, namely:

Широкий спектр литической активности в отношении антибиотикорезистентных возбудителей раневых инфекций;

A wide range of lytic activity against antibiotic-resistant pathogens of wound infections;

Отсутствие иммунного ответа при применении;

Lack of immune response when applied;

Простота производства и контроля качества. Известна антибактериальная композиция, содержащая ковалентно сшитые литический фермент бактериофага (эндолизин) КРР10 и модифицированный фрагмент миелоидного антимикробного пептида овцы SMAP-29, проявляющая активность в отношении бактерий Pseudomonas aeruginosa (патент РФ №2703043).

Ease of production and quality control. Known antibacterial composition containing covalently cross-linked bacteriophage lytic enzyme (endolysin) KPP10 and a modified fragment of myeloid antimicrobial peptide sheep SMAP-29, showing activity against bacteria Pseudomonas aeruginosa (RF patent No. 2703043).

Known endolysin OBPgpLYS, which has a broad spectrum of action on gram-negative bacteria, the effectiveness of which increases when used in conjunction with EDTA, which enhances the permeability of the outer membrane of bacteria (US patent No. 8846865).

Known endolysin EL188, showing activity against bacteria Pseudomonas aeruginosa, while the activity is manifested only in the presence of substances that increase the permeability of the outer membranes (WO2015071437).

The difference between the present invention and those presented above is the possibility of including in the composition of the antibacterial composition both one and a cocktail of recombinant endolysins belonging to the classes of muramidase and peptidase, which makes it possible to expand the spectrum of lytic activity of the resulting composition, as well as the development of formulations of two finished dosage forms in the form of a gel and spray for topical use in the treatment or prevention of wound and hospital infections caused by antibiotic-resistant pathogens.

The objective of the present invention is to develop compositions of two finished dosage forms, namely a gel and a spray, containing as an active pharmaceutical substance recombinant endolysins belonging to the class of muramidase and peptidase, with an individual concentration of each endolysin of 100-10000 μg per ml of the dosage form, for the treatment or prevention of wound and hospital infections.

The technical result of the claimed invention is the fact that, for the first time in the world, gel and spray formulations for topical use for the treatment or prevention of healthcare-associated infections based on one or more recombinant endolysins have been developed, which have the necessary antibacterial activity and a local mechanism of action in the treatment wound and hospital infections. This will further serve as the basis for the creation of a new method of antibiotic therapy for the treatment of healthcare-associated infections, including those caused by drug-resistant bacterial strains.

Preclinical trials of the claimed dosage forms, conducted on laboratory animals (mice and rabbits), confirm the safety and effectiveness of the developed formulations. When used in the cavity of the abscess, the claimed dosage forms are not immunogenic, do not have a toxic and irritating effect and have the necessary therapeutic effect, which is confirmed by a significant increase in the life expectancy of the experimental groups of animals compared to the control ones. In addition, recombinant endolysins, unlike bacteriophages, have a wide spectrum of action without affecting the normal flora of the gastrointestinal tract, which suggests the creation of a new approach to antibiotic therapy and prevention of purulent-septic postoperative complications and community-acquired wound infections caused by multiresistant pathogens.

Thus, the technical result achieved in the implementation of the invention is to provide local antibacterial action of the compositions by applying them to the wound surface or introducing into the abscess cavity.

To achieve this goal, biotechnologically using specialized producer strains, subsequent purification, which was carried out at low temperatures (+4°C) by metal chelate affinity chromatography, followed by size exclusion chromatography with the addition of stabilizing substances at the final stages (150 mM NaCl ), which made it possible to obtain active stable preparations of proteins, and by lyophilization, substances of recombinant endolysins belonging to the classes of muramidase and peptidase were obtained, auxiliary components were selected, and ready-made dosage forms were designed - gel and spray, which have antibacterial activity that persists after injection into the abscess cavity, which was confirmed in tests on an infectious model on laboratory animals.

When developing a gel and spray based on recombinant bacteriophage endolysins, microbiological, molecular genetic, biotechnological methods and preclinical testing procedures were used.

The essence of the claimed invention is as follows. Two antibacterial compositions are proposed in the form of a gel and a spray, which contain a mixture of recombinant endolysins belonging to the classes of muramidase and peptidase, with an individual concentration of each endolysin of 100-10,000 μg per ml of LF.

As a hydrophilic gelling agent, it is proposed to use polymers from the group of cellulose derivatives: hydroxyethylcellulose or hydroxymethylpropylcellulose, or sodium carboxymethylcellulose, or methylcellulose, or hydroxypropylcellulose in the concentration range from 0.5 to 3.0%. To increase the stability of the gel structure and increase the bioadhesive properties, it is proposed to introduce polyethylene glycol 1500 (PEG 1500) or polyethylene glycol 3000 in the concentration range from 0.5 to 5.0%. As a solvent, a suitable buffer solution with a pH of 7.0-8.0 is proposed in the following ratio of components per 100 g, wt. %:

Mixture of recombinant endolysins 0.03-0.15% Gelling agent - cellulose derivative 0.5-3.0% PEG 1500 or 3000 0.5-5.0% Buffer solution with pH=7.0-8.0 91.85-98.97%

The composition of the spray is proposed to include Poloxamer 338 or Poloxamer 407, polyethylene glycol 1500 or polyethylene glycol 3000, as well as a solvent - a buffer solution with a pH of 7.0-8.0 in the following ratio of components per 100 g, wt. %:

Spray composition example:

Mixture of recombinant endolysins 0.03-0.15% Poloxamer 338 or 407 0.75-3.0% PEG 1500 or 3000 0.75-3.0% Buffer solution with pH=7.0-8.0 93.85-98.47%

The use of concentrations of excipients (AE) below those proposed does not provide consumer and technological properties of LF, and the use of higher concentrations of these leads to a decrease in the lytic activity of endolysins.

A method for preparing the above antibacterial compositions is also proposed, in which, first, dry excipients are dissolved in 20 mmol/l Tris-HCl buffer with pH=7.5, heated to 50°C, and sterilized by autoclaving at 120°C for 12 minutes, and then concentrated solutions of recombinant endolysins are introduced into the resulting base with constant stirring.

In connection with the task, an algorithm was developed for the creation of new drugs based on recombinant endolysins, which includes the following steps:

1. Evaluation of the effectiveness of pharmaceutical substances of endolysins on cultures of susceptible bacteria in vitro,

2. Development of compositions and technology of dosage forms of endolysins, determination of quality indicators of FPP,

3. Verification of the effect of developed drugs (PM) on an animal model,

4. Study of the immunogenicity of endolysins,

5. Evaluation of the effectiveness of pharmaceutical substances of endolysins on pathogenic bacterial strains in vitro,

6. Study of the effect of endolysins on representatives of the normoflora in vitro and in vivo,

7. Development of technology for obtaining medicines in the form of a gel and spray,

8. Preclinical trials of dosage forms in the form of a gel and spray based on a cocktail of recombinant endolysins on laboratory animals (rabbits), confirming the presence of a therapeutic effect.

1. Evaluation of the effectiveness of pharmaceutical substances of endolysins on cultures of susceptible bacteria in vitro.

For the study, 20 strains of bacteria Salmonella enterica, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Campylobacter jejuni, 10 strains of Acinetobacter baumannii and Enterobacter spp., as well as a strain of Fusobacterium necrophorum were selected. The study was carried out according to the previously developed method [Antonova N.P., Balabanyan V.Y., Tkachuk A.P., Makarov V.V., Gushchin V.A. Physical and chemical properties of recombinant KPP10 phage lysins and their antimicrobial activity against Pseudomonas aeruginosa. // Bulletin of Russian State Medical University. - 2018. - no. 1. - P. 21-27. Doi: 10.24075/brsmu.2018.010] with minor modifications. The methods are presented below:

Suspensions of bacterial cells of strains K. Pneumoniae, P. Aeruginosa, E. Coli, S. Enterica, A. Baumannii, Enterobacter spp. Prepared from overnight broth cultures obtained in meat-peptone broth (MPB) in a volume of 4.5-5 ml. Overnight cultures were diluted with fresh MPB and grown at +37°C in a thermostat TS-1/80 SPU (JSC Smolensk SKTB SPU, Russia) until a McFarland turbidity of 0.5 units was reached. Turbidity was measured using a Densi-La-Meter II densitometer (ErbaLachemas.ro, Czech Republic). The resulting cultures were centrifuged in a volume of 1 ml at 6000×g for 10 minutes, the supernatant was removed. The pellet was resuspended in PBS until a McFarland turbidity of 0.5 was reached. The bacterial cell suspension was diluted 100 times with 20 mM Tris-HCl buffer at pH=7.5 to obtain a working suspension, which was used for no more than 30 minutes to determine sensitivity to endolysins. A working suspension of bacterial cells in a volume of 100 μl was mixed with a working solution of endolysin in a volume of 100 μl in a sterile flat-bottomed 96-well polystyrene plate (Eppendorf, Germany). Plates with studied samples of suspensions of bacteria and endolysins were incubated at +37°C for 30 minutes with continuous shaking at 200 rpm on a ShakerS-3 shaker (ELMI, Latvia). After incubation, tenfold dilutions in PBS were prepared from the samples. From dilutions of 10 ^-1 and 10 ^-2 , samples in a volume of 100 μl were applied to the surface of Muller-Hinton agar (HiMedia, India) in Petri dishes and triturated with a Drygalsky glass spatula until completely dry on the surface of the nutrient medium. The plates were incubated at +37°C overnight, after which the colonies were counted. The sensitivity of bacterial strains was assessed in comparison with control samples containing 20 mMTris-HCl buffer at pH=7.5 instead of the working solution of endolysin. The study of experimental and control samples was carried out in two independent repetitions.

Suspensions of bacterial cells of C. Jejuni strains were prepared from daily cultures obtained on the surface of Colombian agar (Conda, Spain) with the addition of 10% sterile defibrinated blood in Petri dishes during incubation in a _CO2 incubator NB-203 (N-BIOTEK, Korea) at +37°С and 5% carbon dioxide content in the atmosphere. Cultures from solid nutrient medium were suspended in 20 mM Tris-HCl buffer at pH=7.5 until a McFarland turbidity of 0.5 units was reached. The bacterial cell suspension was diluted 100-fold with 20 mM Tris-HCl buffer to obtain a working suspension, which was used for no more than 30 minutes to determine sensitivity to endolysins. A working suspension of bacterial cells in a volume of 100 μl was mixed with a working solution of endolysin in a volume of 100 μl in a sterile flat-bottomed 96-well polystyrene plate (Eppendorf, Germany). The plates with the studied samples of suspensions of bacteria and endolysins were incubated at +37°C and the content of carbon dioxide in the atmosphere of 5% for 30 minutes with continuous shutellation at 200 rpm. After incubation, tenfold dilutions in PBS were prepared from the samples. From dilutions of 10 ^-1 and 10 ^-2 , samples in a volume of 100 μl were applied to the surface of blood agar (BA) (GRM-agar base, SRC PMB, Russia) with the addition of 5% sterile defibrinated blood in Petri dishes and rubbed with a Drygalsky glass spatula until completely drying on the surface of the nutrient medium. The plates were incubated at +37°C and 5% carbon dioxide in the atmosphere for two days, after which the colonies were counted. The sensitivity of bacterial strains was assessed in comparison with control samples containing 20 mM Tris-HCl buffer at pH=7.5 instead of the working solution of endolysin. The study of experimental and control samples was carried out in two independent repetitions.

Suspensions of bacterial cells of the F. Necrophorum strain were prepared from two-day cultures obtained on the surface of Colombian agar (Conda, Spain) with the addition of 10% sterile defibrinated blood in Petri dishes during incubation in an anaerobic station (the test culture is an anaerobic microorganism) Bactron 300-2 (Sheldon Manufacturing Inc., USA) at +37°C and 10% carbon dioxide, 10% hydrogen, and 80% nitrogen. The culture was washed in 1.5 ml of 20 mM Tris-HCl buffer at pH=7.5 and resuspended until uniform turbidity was obtained. Before using Tris-HCl, the buffer was heated at a temperature of +80°C for 10 minutes, followed by cooling in cold water. This manipulation was used to minimize the presence of oxygen in the buffer. The culture from the cup was washed in 1.5 ml of 20 mm Tris-HCl buffer at pH=7.5 and resuspended until a uniform turbidity was obtained. Subsequently, 100 µl of the resulting suspension in buffer was transferred into control and test tubes. Each endolysin and control was done in duplicate. 100 μl of Tris buffer were added to the control tubes, and 100 μl of each endolysin or their mixture, respectively, were added to the test tubes.

Subsequently, after adding all the components and resuspension, the eppendorfs were placed in an anaerostat at 37°C for 30 minutes. After the end of the incubation, 100 μl of the contents of each eipendorf were transferred to a Petri dish with Columbia agar with 10% horse blood and the suspension was rubbed into the agar with a spatula or loop. The plates were cultured for 48 hours at 37°C in an anaerobic station, after which the result was taken into account by counting the colonies.

The lytic activity of endolysins was determined by the formula:

where X - lytic activity, %

A - the average number of colonies in the experiment, CFU

B - the average number of colonies in the control, CFU

II. Determination of the action of recombinant endolysins on representatives of the normoflora in vitro and in vivo.

A. Cultures of lactobacilli and bifidobacteria, when grown in semi-liquid media, poured in a high column, form single characteristic colonies, which can be counted in test tubes of a number of tenfold limiting dilutions, and the titer (CFU / ml) can be quantitatively determined using the formula (MUK 4.2.999-00 Definition number of bifidobacteria in fermented milk products - Collection of methodological documents necessary to ensure the Federal Law of June 12, 2008 No. 88-FZ "Technical regulations for milk and dairy products. - M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2010. - 79 With.). This made it possible to assess the degree of influence of the studied substances on the population of normoflora cultures.

B. Preparation of seed culture

Ampoules with freeze-dried strains were opened under aseptic conditions and rehydrated with the culture medium.

To work with bifidobacteria, we used a medium for growing and isolating bifidobacteria, dry, produced by the State Scientific Center for Applied Microbiology, Obolensk (BS), for lactobacilli - MRS-2, prepared in the laboratory of nutrient media of the FBUN MNIIEM them. G.N. Gabrichevsky but to the attached instructions.

For rehydration of the cultures, 1 ml of sterile medium was added to the ampoule, the contents were evenly mixed with circular movements of the pipette, the resulting suspension was taken and added to the first test tube of a series of tenfold dilutions containing 9 ml of semi-liquid media. Limiting tenfold dilutions were prepared by mixing the contents of the tubes 12-15 times by pipetting, avoiding aeration of the suspension, and transferring 1 ml of the suspension from the previous tube to the next one up to 10 ^-10 . The crops were incubated for 24 hours at a temperature of +37.5±1°C, thus obtaining the first generation.

The grown crops were examined, a test tube with well-formed 100-150 colonies was selected, its contents were mixed by pipetting, and 10% of the inoculum was added to the volume of the medium (10 ml per 100 ml of the medium). Incubated for 24 hours at a temperature of +37.5±1°C, while receiving the second generation.

The third generation was obtained similarly to the second generation.

At all stages of the work, the absence of growth of foreign microflora was controlled by seeding on Sabouraud and MPA media.

The fourth generation was grown on the surface of agar media in an anaerostat with the creation of an atmosphere using Anaero gas generating packages. Cultures were removed with a microbiological loop and a suspension was prepared in sterile saline according to the turbidity standard 5 MakFarland. 2 ml were transferred into 110 ml of sterile 20 mM Tris-HCl buffer at pH=7.5 G.N. Gabrichevsky.

The resulting material was poured into 12 sterile test tubes with plastic stoppers, 9 ml each. From the remaining 2 ml of the suspension, rows of tenfold dilutions were prepared to determine the titer of the working material.

1 ml of solutions of endolysins with a concentration of 1 mg/ml were added to the prepared test tubes, and the concentration of the studied substances in the suspension was 0.1 mg/ml (100 μg/ml). A solution of each endolysin was prepared in a volume of 11±0.1 ml, necessary for the study of 11 strains.

The control was an inoculum in a volume of 9 ml with the addition of 1 ml of 20 mm Tris-HCl buffer.

Samples were kept for 30 minutes at +37.5±1°C, then 1 ml were taken to prepare tenfold series of limiting dilutions to determine the effect of endolysins on cultures and 5 ml each to determine titratable acidity.

Rows of tenfold dilutions were incubated for 48 hours at a temperature of +37.5±1°C. The preliminary count of the grown colonies was carried out after 24 hours, the final one - after 48 hours.

The studies were carried out in duplicate. Strains B. longum OV-20, L. Helveticus NK-1 were studied in 4 replicates.

B. Determination of titratable acidity

Titratable acidity was measured in Turner degrees (°T). In accordance with GOST 3624, titratable acidity showed the number of cubic centimeters of a decinormal (0.1 N) alkali solution used to neutralize 100 ml or 100 g of the product in the presence of the phenolphthalein indicator [OFS.1.7.2.0008.15 Determination of the concentration of microbial cells. - State Pharmacopoeia of the Russian Federation, XIV edition]. The end point of the titration was considered to be the appearance of a slightly pink color that did not disappear within 1 minute.

10 ml of a well-mixed sample was taken with a pipette into a thin beaker or flask with a capacity of 100 ml, 3 drops of an indicator (1% solution of phenolphthalein in alcohol) were added, the mixture was stirred and titrated with 0.1 N sodium hydroxide solution until a faint pink color appeared, which did not disappear during 1 minute. The amount of caustic soda used for titration was multiplied by 10. The result obtained expressed the acidity in

D. Determination of the number of colony forming units Colony forming unit, CFU - a standard indicator indicating the number of bacteria forming colonies in 1 ml of medium. Tenfold dilutions were prepared. To do this, 1 ^cm3 of the test suspension was added to the first test tube with 9 cm ³ of sterile BS and carefully mixed thoroughly by pipetting 10-15 times without bubbles. 1 ml of the resulting suspension was taken and transferred to the next test tube. The second dilution was mixed with a new sterile pipette, as described earlier, and 1 ml of the resulting suspension was transferred to the third tube of the row. Thus, dilutions up to 10 ^-10 were prepared. The crops were incubated at a temperature of +37±1°C in a thermostat for 48 hours, and characteristic colonies were counted in test tubes with single colonies every 24 hours. The calculation results were calculated by the formula:

X \u003d a × 10 ^n-1 ,

where X is the number of colony-forming units of bacteria in 1 ml of the sample;

a is the number of colonies in the considered dilution.

E. To assess changes in the composition of the normoflora when using endolysins, a study was conducted on healthy outbred white mice weighing 22-27 g. The experiment was planned taking into account the "Guidelines for conducting preclinical studies of drugs (Immunobiological drugs)" (Guidelines for conducting preclinical studies of drugs ( Immunobiological drugs), part two, Moscow: Grif i K, 2012-536 p.).

In vivo tests were performed with each of the candidate endolysins in 5 experimental and 2 control mice. The drug was administered intraperitoneally to experimental mice daily for 5 days, 0.5 ml of endolysin per day once, at a concentration of 500 μg (0.5 mg)/animal. The control group was injected with a buffer solution (20 mm Tris-HCl, pH=7.5). Before the introduction of endolysins on the 5th and 7 days, feces were taken from each mouse and bacterial culture was performed (assessment of the translucent microflora). At the end point (2 days after the cessation of administration), the animals were sacrificed and inoculated from the intestine washed under pressure with a syringe (assessment of the parietal microflora). Microbiological cultures were carried out taking into account OST 91500.11.0004-2003.

2. Development of compositions and technology of dosage forms of endolysins, determination of quality indicators of FPP

As excipients for the development of dosage forms, polymers were considered, which, according to the literature data, have a high compatibility with immunobiological substances. Hydroxyethylcellulose Natrosol 250 HHX (ASHLAND) was used as a gelling agent in the technology of soft dosage forms. Cellulose derivatives are the most indifferent gelling agents used in dosage forms for all types of applications, including parenteral. Hydroxyethylcelluloses of various commercial grades are distinguished by the simplicity of technology, the absence of the need for heating, resistance to sterilization, a wide range of viscosities, and a neutral pH value. According to preliminary studies, Natrosol 250 HHX, among other hydroxyethylcelluloses, is distinguished by increased mucoadhesion, due to which this brand was chosen as the most promising for the development of bases for gels.

Poloxamers Kolliphor P 338 and Kolliphor P 407 (BASF) were used as viscosity modifiers for sprays and as a mucoadhesive component in their composition. Poloxamers have hydrophilic properties due to the presence of two hydrophilic tails in the molecule. The use of hydrophilic polymers lengthens the retention time of the dosage form on the mucous membrane, which leads to a gradual release of the pharmaceutical substance and improved patient tolerance. In addition, poloxamers 338 and 407 have surface activity, which will improve the wettability and distribution of the solution over the wound surface and stabilize the droplets of the solution in the aerosol jet.

To vary the stability indicators, rheological and bioadhesive properties of the gel and spray compositions, polyethylene glycol with a molecular weight of 1500 (Khimmed, Russia) was used. Being osmotically active excipients, polyethylene glycols are recommended for introduction into the composition of dosage forms intended for application to the wound surface, as they will facilitate the outflow of purulent contents and clean the wound.

A. Gel preparation method

In a weighed and measured amount of Natrosol 250 NIH and PEG add 20 mm Tris-HCl (pH=7.5), pre-thermostated for 2 hours at a temperature of +50°C, thermostated at a temperature of +50°C for 15 minutes. Mix thoroughly. The resulting gel base is sterilized at a temperature of +120°C for 12 minutes. Endolysins are introduced into the sterilized base for the gel in the form of concentrated solutions with a concentration of 1-20 mg/ml. When preparing a solution of excipients (BB), the amount of solvent (Tris-HCl buffer) is reduced by the volume of injected endolysins.

B. Spray manufacturing method

20 mM Tris-HCl (pH=7.5), pre-thermostated for 2 hours at +50°C, is added to the weighed and measured amount of poloxamer and PEG, and thermostated at +50°C for 15 minutes. Mix thoroughly. The resulting spray base is sterilized at +120°C for 15 minutes. Endolysins are introduced into the sterilized spray base in the form of concentrated solutions with a concentration of 1-20 mg/ml. When preparing a solution of excipients (BB), the amount of solvent (Tris-HCl buffer) is reduced by the volume of injected endolysins.

B. Definition of technological indicators

The pH of manufactured dosage forms was determined in accordance with GPM.1.2.1.0004.15 "Ionometry". The viscosity of the gel was determined by rotational viscometry on a rotational viscometer of the “cylinder in cylinder” type, and the viscosity of the spray was determined by the method of capillary viscometry on an Ostwald viscometer in accordance with GPM.1.2.1.0015.15 “Viscosity” and GPM.1.2.1.0014.15 “Density” .

D. Definition of mucoadhesion

The characteristics of mucoadegysia of dosage forms used on mucous membranes determine the degree of their targeted effect on the affected area, the time of exposure to the mucous membrane, and the therapeutic efficacy of the dosage form.

Bioadhesive properties were studied by measuring the pull-off force and retention time of a dosage form sample on a substrate treated with an aqueous solution of type II porcine gastric mucin with a bound salicylic acid content of 0.5% (SIGMA, Sigma-Aldrich, USA, cat. No. M 2378).

The peel force was evaluated at a temperature of +20°C using the lever mechanism shown in Fig. 1. To the lower side of the movable part of the lever (6) with a glass plate fixed on it (1) and to the fixed plate (4) fixed on the tabletop (5), a non-woven spunbond material (SPANLAB, Russia) was attached as a substrate. A 20% (weight-volume concentration) aqueous solution of porcine stomach mucin type II (3) was applied to the surface of the fixed plate (4), and a test sample weighing 1.0 g (2), evenly distributed over the area, was applied to the lower part of the plate (1). working surface (1820 mm ² ) of the plate (1) with a spatula. A load (7) was placed on the second arm of the lever. The breakaway force was calculated as the product of the mass of the load, at which the upper arm (6) with a fixed plate (1) was torn off from the plate (4) fixed on the table top (5), by the free fall acceleration (g=9.81 m/s ² ) .

E. Determination of lytic activity

The bacterial strains listed in Table 1 were used to determine the lytic activity of the endolysins selected for the composition.

Also, when determining the activity of endolysins, the test strain Acinetobacter baumannii Ts 50-16 was used.

An overnight culture (1-2 ml) was diluted and grown to an optical density OD ₆₀₀ =0.6 (exponential growth phase of bacteria). The resulting culture (1 ml) was pelleted by centrifugation (3000×g, 10 min.), the cells were resuspended in the same volume of PBS (the turbidity of the obtained suspension corresponded to the McFarland turbidity standard of 0.5). The bacterial culture was diluted 100 times with Tris-HCl buffer, this suspension was used for 30 min. After cooking. The following mixtures were prepared in a 96-well plate:

1. 100 µl of bacterial suspension and 100 µl of LF with the required concentration of endolysins (experiment);

2. 100 µl of a suspension of bacteria and 100 µl of an IV solution without the addition of endolysins (control).

The resulting mixtures were incubated at 37°C for 30 min at 200 rpm. Then 10 and 100-fold dilutions were made in PBS and 100 μl of each dilution was applied to Petri dishes with LB agar medium. Colonies on plates were counted after overnight incubation at 37°C.

3. Verification of the effect of developed drugs (drugs) on an animal model

A. Laboratory animals

For the experiment, rabbits of the "Soviet chinchilla" breed weighing 2 kg were used. Only females were used in the experiments. Rabbits are a standard subject of drug efficacy studies, especially in reproducing Fusobacterium necrophorum wound infections. For the preliminary experiment, 2 rabbits were used: Rabbit No. 1 after infection was treated with drugs in the form of a gel, rabbit No. 2 was treated with drugs in the form of a spray. During the main experiment, 26 rabbits were used:

- experimental group No. 1 consisted of 10 rabbits (No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 17), which were treated with drugs in the form of a gel with DV;

- control group No. 1, consisting of 3 rabbits (No. 1, 14, 16) were treated with placebo in the form of a gel;

- experimental group No. 2 consisted of 10 rabbits (No. 11, 12, 13, 15, 18, 19, 21, 22, 24, 25), which were treated with drugs in the form of a spray with DV;

- control group #2 consisted of 3 rabbits (#20, 23, 26) treated with a placebo spray.

Rabbits used for scientific purposes have been kept in accordance with the Guide for the Care and Use of Laboratory Animals, as well as with Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals. During the experiment, all animals were housed in individual isolator cages. All cages were covered with steel lattice covers with a stern recess and a place for a drinker. The vivarium maintains standard microclimate conditions: relative air humidity 30-70%, air temperature 22-24°C, natural lighting, illumination -110 Lux at a distance of 1 meter from the floor. Each cell was labeled, with the label indicating: the number of the animal, the number of the infecting strain, the date of infection, data on the dose and method of administration of the test samples of the agents. The animals were fed with granulated extruded feed for rodents, corresponding to GOST R 50258-92 “Complete feed for laboratory animals. Specifications". Food was given to adlibitum rabbits. Water was given to adlibitum in standard bottles with screw caps and steel drinking spouts. The water used for drinking complies with SanPiN 2.1.4.1074-01 “Drinking water. Hygienic requirements for water quality of centralized drinking water supply systems. Quality control". Sterile sawdust was used as bedding. The care and manipulation of rabbits was carried out in accordance with the SOPs adopted by the organization of the performer, and the principles of humane treatment of animals.

Before the experiment, the rabbits were kept in the vivarium for 8 days (the animals were quarantined before the start of the tests) in accordance with SP 2.2.1.3218-14 "Sanitary and epidemiological requirements for the arrangement, equipment and maintenance of experimental biological clinics (vivariums)". Animals were subjected to daily veterinary examination. During the quarantine period, during daily inspections, no animals with health deviations were found that could affect the results of the experiment.

During the tests, the general methods of clinical research were used: examination, palpation and thermometry. A general clinical study was carried out: determination of habitus, examination of visible mucous membranes, skin, lymph nodes, measurement of body temperature, as well as system-by-system studies of the respiratory apparatus, cardiovascular system, digestive tract, genitourinary apparatus and nervous system.

B. Assessment of biochemical parameters of blood

Blood biochemical parameters were determined on a ChemWell+ biochemical analyzer (USA) using Vital reagents (Russia) in blood serum without traces of hemolysis. The following indicators were evaluated: Aminotransferases (AlAT and AcAT) optimized enzymatic kinetic method; total protein - biuret method.

B. Assessment of hematological parameters of blood

Hematological studies of whole blood were performed on a hematological analyzer BC-3200 (Myndrey). The principle of determination is based on the impedance method, which consists in determining the number and size of cells depending on the change in electrical resistance when a particle (cell) in a conductive liquid passes through a small aperture. Each cell in this case causes a change in the impedance of the conductive suspension of blood cells. These changes are recorded as an increase in voltage between the electrodes. The number of pulses determines the number of cells. The pulse amplitude is proportional to the volume of the cell. Pulses are counted only within limits that are between pre-set lower and upper limits (discriminators).

The principle of hemoglobin measurement. Hemoglobin is determined in a 1:196 lysed dilution by the cyanmethemoglobin method. The reagent lyses red blood cells, releasing hemoglobin. Hemoglobin iron oxidizes from ferrous to trivalent, forming methemoglobin, which reacts with potassium cyanide to form stable cyanmethemoglobin or hemoglobin cyanide. The concentration of this product is then measured photometrically.

D. Infection model for evaluating the therapeutic efficacy of the developed compositions

The rationale for choosing a bacterial agent - Fusobacterium necrophorum to reproduce a wound infection in laboratory animals is the well-studied characteristics of the strain, its sensitivity to the tested drugs, as well as the possibility of obtaining a time-controlled infection. This strain is used by a number of domestic biological enterprises in the control of the immunogenic activity of inactivated vaccines against necrobacteriosis, in the test of seroprotection, as a "punch" strain. Subcutaneous administration of a daily suspension of Necrophorum strain F, actively growing in Kitt-Tarozzi medium at a concentration of 3 billion microbial cells in a volume of 1 cm ³ , causes the death of infected animals within 10-14 days. At the same time, the planned period of observation of the animals was 30 days or until the moment of their death. The injection was carried out in the area of the withers. The choice of this localization for the reproduction of wound infection was due to the fact that the animals would thus not be able to disrupt the rubber drainage installed to ensure the outflow of purulent masses. All animals were infected at the same time using the same infectious suspension. The correctness of the manipulation was confirmed by the formation of an abscess at the injection site on days 4-7. Treatment of an infection with the formation of wound and abscessing processes requires surgical intervention (OS) by opening an abscess to remove exudate, providing oxygen access, washing the wound and laying the drug. To remove exudate from the wound, two small incisions were made surgically so that one incision was below the localization of the second. This approach made it possible to ensure the outflow of exudate. When performing EA, a passive type of drainage was used, which is explained by the localization of the abscess in a shallow localization (subcutaneous tissue). Small strips from sterile latex medical gloves were used as drainage.

E. Doses, scheme and method of administration of the test agent During the testing of drugs, each test form was administered to animals after preliminary opening of the abscess, installation of active drainage, douching of the abscess cavity with sterile saline in order to wash it from purulent masses. The gel was introduced from a syringe (without a needle) into the wound passage, the hole of which is located above the lower hole. This manipulation should contribute to the spontaneous outflow of purulent masses, in contrast to cases where both surgical openings would remain in the same horizontal plane. A similar approach was used when using drugs in the form of a spray. The exception was that the spray was applied to the wound channel for a longer time than the gel. An important condition during the experiment was the need to maintain a low temperature of the tested forms of drugs. So, syringes and spray bottles were constantly in a thermal container with ice packs. The funds were removed from the container immediately before their use. An indicator that both forms of drugs were successfully and correctly applied was that this form should have flowed in a small amount from the descending wound hole. This suggests that the wound passage is completely filled with drugs. In both cases, regardless of the form of the drug, the volume of drugs for a single use per head was 5 cm ³ . Washing the wound and introducing a new dose of the drug was carried out twice a day for 5 days in a row. Thus, 10 doses were prepared for each animal.

During the study, each animal was examined daily. The examination included an assessment of the general behavior and general condition of the animals, thermometry, blood sampling for clinical and biochemical analyzes, as well as for serological diagnostics. To assess the effectiveness of the use of drugs in the treatment of wound infection, the following indicators were used: the safety of animals in the group; time of death of animals; appearance and activity of animals; results of a biochemical blood test; results of a hematological blood test. Blood in rabbits was taken from the marginal vein of the ear in the amount necessary for the study. Before blood sampling, the ear was wiped with a swab moistened with xylene. Blood for biochemical parameters and complete blood count were taken before infection, on the 11th and 20th day after infection.

The killing of animals, if necessary, was to be carried out by a responsible person in accordance with the requirements adopted in the laboratory, by decapitation after the application of ether anesthesia without causing suffering to the animals in a special vivarium room. The corpses of the dead rabbits were removed from the cages immediately after the discovery and declaration of death by the responsible person. The corpses were examined visually, after which they were opened and the possible causes of death were examined. Laboratory diagnostics of necrobacteriosis was carried out in order to confirm the cause of death of the animals used in the experiment. Thus, to confirm the cause of death of animals, they were immediately autopsied in order to determine the pathoanatomical changes typical for necrobacteriosis, as well as to select sectional material to isolate the previously introduced infectious agent. So, inoculations were carried out from the blood of the heart and liver of corpses in test tubes with MPPB under vaseline oil with 0.2% glucose and for control in test tubes with MPA and MPB. The inoculated media were incubated in a thermostat at a temperature of +37 ± 1°C under aerobic conditions for 24 hours. The implementation of this work was carried out in accordance with MP: "Laboratory methods for diagnosing necrobacteriosis of agricultural animals" dated 06/01/1987, as well as "Methodological recommendations for the diagnosis, treatment and prevention of necrobacillosis, digital dermatitis and diseases of the hooves of cattle of non-contagious etiology” of the Ministry of Agriculture of the Russian Federation, 2017

Macroscopic examination of organs and tissues was carried out according to the "Guidelines for the pathomorphological diagnosis of diseases of animals, birds and fish in veterinary laboratories." The autopsy of the dead and forcedly killed rabbits was carried out by the method of complete evisceration. Direct autopsy was carried out according to the scheme:

- external inspection;

- internal inspection;

- evaluation of the obtained results.

The experimental animals were dissected in the dorsal position. The evaluation of the results obtained was carried out in accordance with the methodology described by Zharov A.V.

Preclinical tests on laboratory animal species (rabbits of the Soviet Chinchilla breed) were carried out in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals (Strasbourg, 1986) and Article No. 11 of the Federal Law of April 12, 2010 No. 61-FZ "On circulation of medicines” (as amended on 04.06.2018).

All stages of work were carried out in accordance with the regulatory and technical documentation:

- Sanitary and epidemiological rules SP 2.2.1.3218-14 "Sanitary and epidemiological requirements for the arrangement, equipment and maintenance of experimental biological clinics (vivariums)" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of August 29, 2014 No. 51);

- Guidelines for conducting preclinical studies of drugs. Part one. - M.: Grif and K, 2012. - 944 p. (FGBU NTsESMP of the Ministry of Health of the Russian Federation);

- Guidelines for conducting preclinical studies of drugs. Part two. - M.: Grif and K, 2012. - 536 p. (FGBU NTsESMP of the Ministry of Health of the Russian Federation);

- Guidelines for the examination of medicines. Volume I. - M.: Grif and K, 2013. - 328 p. (FGBU NTsESMP of the Ministry of Health of the Russian Federation);

- Guidelines for the examination of medicines. Volume II. - M.: Grif and K, 2013. - 280 p. (FGBU NTsESMP of the Ministry of Health of the Russian Federation);

- Federal guidelines for the use of medicines (formulary system). Issue XV. - M.: Ekho, 2013. - 1020 p.;

- GOST R ISO 20776-1-2010. Clinical laboratory studies and in vitro diagnostic test systems. Investigation of the sensitivity of infectious agents and evaluation of the functional characteristics of products for the study of sensitivity to antimicrobial agents;

- Clinical guidelines: determination of the sensitivity of microorganisms to antimicrobial drugs Version-2015-02, approved at the Expanded Meeting of the Interregional Association for Clinical Microbiology and Antimicrobial Chemotherapy (Moscow, 22.05.2015);

- Clinical and Laboratory Standards Institute (2006), Performance Standards for Antimicrobial Susceptibility Testing, 16 ^th edn. Informational Supplement M100-S16, Wayne, PA;

- ISO/TS 34/SC 9 - Microbiology;

- Carol Kilkenny, William J. Browne, Innes C. Cuthill, Michael Emerson and Douglas G. Altman. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J PharmacolPharmacother. 2010 Jul-Dec; 1(2): 94-99;

- Guide to laboratory animals and alternative models in biomedical technology. N.N. Karkishchenko and S.V. Grachev. Moscow, 2010;

- ISO/IEC 17025-99 "General requirements for the competence of testing and calibration laboratories";

- SP 1.3.2322-08 "Safety of working with microorganisms of III-IV groups of pathogenicity (danger) and pathogens of parasitic diseases".

4. Study of the immunogenicity of endolysins

A. Preparation of rabbit polyclonal antiserum to endolysin cocktail.

To obtain rabbit polyclonal antiserum to the endolysin cocktail, a cycle of intramuscular immunizations of rabbits was carried out, consisting of 4 injections with the corresponding cocktail. For a guaranteed immune response, a high dose of antigen was taken for immunization (2-2.2 mg of each endolysin per immunization). The contents of vials with endolysins were dissolved in 20 mM Tris-HCl buffer (pH=7.5).

Immunization consisted of a cycle of intramuscular injections of antigen mixed with Freund's adjuvant at four points (in equal volume in four paws of a rabbit) and was carried out according to the following scheme.

первая иммунизация: 1,2 мл АГ+1,2 мл полного адъюванта Фрейнда;

first immunization: 1.2 ml of antigen + 1.2 ml of complete Freund's adjuvant;

вторая иммунизация через 10 дней: 1,22 мл АГ+1,22 мл неполного адъюванта Фрейнда;

second immunization 10 days later: 1.22 ml of antigen+1.22 ml of incomplete Freund's adjuvant;

третья иммунизация через 20 дней: 0,51 мл АГ+0,6 мл неполного адъюванта Фрейнда;

third immunization after 20 days: 0.51 ml of antigen+0.6 ml of incomplete Freund's adjuvant;

четвертая иммунизация через 30 дней: 0,9 мл АГ+0,9 мл полного адъюванта Фрейнда;

fourth immunization after 30 days: 0.9 ml of AG + 0.9 ml of complete Freund's adjuvant;

забор крови через 37 дней. Полученная кроличья антисыворотка была проанализирована иммунохимическими методами (преципитация по Оухтерлони, иммуноэлектрофорез).

blood sampling after 37 days. The resulting rabbit antiserum was analyzed by immunochemical methods (Ouchterlony precipitation, immunoelectrophoresis).

B. Designing an enzyme immunoassay for the detection of anti-endolysin IgG antibodies and conducting enzyme immunoassay

Schematic diagram of the enzyme immunoassay for the detection of IgG antibodies to endolysins is as follows. The antigen (endolysin cocktail) is adsorbed into the wells of the polystyrene tablet in the selected concentration. After incubation, the contents of the wells are shaken out and the plate is washed with washing solution (5 washing cycles, which are also repeated after incubation with the test samples and with the conjugate). Then the test sample of blood serum is introduced, and if there are IgG antibodies to endolysins in the sample, they interact with the adsorbed antigen. Several wells must be assigned for the introduction of positive and negative controls. Serum known to contain antibodies to endolysins (rabbit polyclonal antiserum to a cocktail of endolysins in a selected dilution) is used as a positive control. Phosphate-buffered saline with tween (PBS-T), as well as blood serum known to be free of antibodies to endolysins (for example, blood serum of an intact rabbit) are used as negative controls. An additional control of the specificity of the enzyme immunoassay is the "empty" wells, i.e. wells in which the antigen was not sorbed, while the remaining components of the reaction will be introduced. interact with IgG of many mammalian species, in particular humans and rabbits) in combination with the enzyme peroxidase. At the final stage, a substrate (hydrogen peroxide) is added to the wells along with a reaction witness tetramethylbenzidine (TMB). The reaction is stopped by adding a stone reagent (acid) and the optical density of the contents of the wells is measured on a plate photometer. In the case when all components of the reaction are present (antigen, IgG antibodies, conjugate and substrate), a color reaction develops in the well, the intensity of which depends on the amount of IgG antibodies in the test sample.

At the first stage of designing an ELISA test system for detecting IgG antibodies to endolysins, the concentration of the antigen (endolysin cocktail) was selected for sorption in the wells of the tablet. To do this, a series of trial sorptions of each of the endolysins separately was initially carried out at various concentrations (from 100 μg to 0.01 pg with a multiplicity of 10). Antigens (endolysins) were adsorbed in the wells of a polystyrene plate, then the resulting rabbit antiserum to endolysins was added to the wells in dilutions from 1/5 to 1/10000 to establish the necessary dilution of the antiserum as a positive control. Antigen-bound IgG antibodies were detected using the Protein A-peroxidase conjugate (Protein A-Peroxidase, Sigma, USA) or the conjugate of affinity-purified polyclonal goat antibodies to rabbit immunoglobulins with horseradish peroxidase (Polygoatanti-rabbitHR, Bialeksa, Russia). ), and the enzymatic reaction with the added substrate (BCM Diagnostics, USA) was inhibited by acid (stop reagent). Registration of optical density in the wells was carried out at 450 nm against air.

The selection of the conditions for the enzyme immunoassay also included the selection of the dilution of the conjugate, the development of incubation conditions at various temperature conditions (from +35°C to +44°C), the reaction time (from 30 to 90 minutes), the need or absence of shaking.

Conjugate "Protein A-peroxidase" in the amount of 1 mg (the contents of one vial) was dissolved in 0.5 ml of 0.01 M nitrate buffer (pH 6.4) with the addition of 0.5 ml of glycerol (glycerin prevents the reagent from freezing). The resulting solution with a conjugate concentration of 1 mg/ml was stored at -20°C, periodically used. To select the optimal dilution of the conjugate during ELISA, its dilutions were prepared in PBS-T in the range of 1/16000-1/128000. The Polygoatanti-rabbitHR conjugate was available in liquid form at a concentration of 0.7 mg/mL. The studies used a different range of dilution of this conjugate in PBS-T - 1/2000-1/8000.

C. Studies to detect anti-endolysin antibodies in the blood of rabbits after treatment with an endolysin cocktail

Blood for ELISA was taken before infection on days 14 and 19 after the start of treatment in rabbits from the marginal vein of the ear in the amount necessary for the study. Before blood sampling, the ear was wiped with a swab moistened with xylene.

5. Evaluation of the effectiveness of pharmaceutical substances of endolysins on pathogenic bacterial strains in vitro

In order to evaluate the effectiveness of pharmaceutical substances of endolysins and determine the spectrum of their lytic activity, 121 strains of various types of pathogens obtained from various sources were selected. The list of strains used is presented in Table 2.

The study included 5 candidate recombinant endolysins of bacteriophages ECD7, Am24, Ap22, Si3, and St11 - typical representatives of the main classes of endolysins - muramidases and peptidases. As can be seen from the results presented in Fig. 2, recombinant endolysins of bacteriophages HCD7, Am24, and Ap22 had the widest spectrum of lytic activity.

6. Study of the effect of endolysins on representatives of normal flora in vitro and in vivo

The same candidate bacteriophage endolysins ECD7, Am24, Ap22, Si3, and St11 were selected to study the effect on the normoflora.

An in vitro study of the normoflora showed that LysSi3 endolysin had a minimal inhibitory effect on the B. longum B379M population, a slight decrease in the activity of the B. Breve OV-12 strain was detected after treatment of the population with LysAM24 and LysECD7, which does not exceed changes in the activity of the culture in the control. A decrease in the number of viable colony-forming units of B. infantis 73-15 under the influence of LysSi3 and a decrease in the number of cfu/ml of B. bifidum 791 treated with LysSt11 were noted. A decrease in the number of CFU B. longum OV-20 was noted after exposure to LysSi3.

The greatest growth inhibition was found for L. helveticus NK-1 culture after exposure to LysSi3 endolysins for 30 minutes.

There was no effect of endolysins on B. Bifidum OV-19, B. Adolescentis GO-13, L. Casei KNM-12, L. Caisei K3Sh24.

An in vivo study revealed the effect of the following endolysins:

- EysSt11 increased the number of coagulase-negative staphylococci and reduced the number of enterococci in the parietal microflora by the end of the experiment, and also increased the titer of fungi in the translucent microflora on the fifth day of the experiment compared with the control group of animals;

- LysSi3 reduced the titer of bacteria of the genus Enterococcus in the luminal microflora compared with the control group on the fifth (P<0.001) and seventh (P<0.05) days of the experiment.

Against the background of the use of LysAM24. LysAP22 and EysECD7 showed no statistically significant changes in the seeded luminal and parietal microflora.

7. Technology for obtaining drugs in the form of a gel and spray

Gel making

The preparation of a gel containing a cocktail of recombinant endolysins belonging to the classes of muramidase and peptidase consists of three stages.

Stage 1 - obtaining a sterile base of the gel. In a weighed and measured amount of explosives (Natrosol 250 ННХ and PEG 1500), add the required volume of 20 mmol/l Tris-HCl buffer with pH=7.5, pre-thermostated for 2 hours at a temperature of +50°C, taking into account the subsequent introduction of concentrated endolysin solutions. Thermostat at a temperature of +50°C for 15 minutes. Mix thoroughly. The resulting gel base is sterilized at a temperature of +120°C for 12 minutes.

Stage 2 - obtaining concentrated solutions of endolysins. The required volume of sterile 20 mmol/l Tris-HCl buffer with pH=7.5 is added to the lyophilized protein to obtain a concentration of 1-20 mg/ml, gently mixed until complete dissolution.

Step 3 - obtaining a drug in the form of a gel. The required volume of concentrated solutions of endolysins is introduced into the sterilized and cooled gel base until the desired concentration is obtained, and thoroughly mixed.

Spray making

The manufacture of a spray containing a cocktail of recombinant endolysins belonging to the classes of muramidase and peptidase consists of three stages.

Stage 1 - obtaining a sterile base of the spray. In a weighed and measured amount of explosives (Poloxamer 338 or 407 and PEG1500) add the required volume of 20 mmol / l Tris-HCl buffer with pH = 7.5, pre-thermostated for 2 hours at a temperature of +50 ° C, taking into account the subsequent introduction of concentrated solutions endolysins. Thermostat at a temperature of +50°C for 15 minutes. Mix thoroughly. The resulting gel base is sterilized at a temperature of +120°C for 12 minutes.

Stage 2 - obtaining concentrated solutions of endolysins. The required volume of sterile 20 mmol/l Tris-HCl buffer with pH=7.5 is added to the lyophilized protein to obtain a concentration of 1-20 mg/ml, gently mixed until complete dissolution.

Step 3 - obtaining a drug in the form of a gel. The required volume of concentrated solutions of endolysins is introduced into the sterilized and cooled gel base until the desired concentration is obtained, and thoroughly mixed.

Determination of the lytic activity of recombinant endolysins in the composition of drugs

Studies of the lytic activity of endolysins in the composition of LF were carried out on samples containing a cocktail of three recombinant endolysins of bacteriophages ECD7, Am24 and Ap22 with an individual concentration of each endolysin of 150 μg/ml, using the bacterial strains presented in Table 1. The results of determining lytic activity are presented in Table 3 .

8. Preclinical trials of dosage forms in the form of a gel and spray based on a cocktail of recombinant endolysins on laboratory animals (rabbits), confirming the presence of a therapeutic effect

Two LFs were included in the study, namely a gel and a spray based on a cocktail of recombinant endolysins of bacteriophages ECD7, Am24 and Ap22 with individual concentrations of 150 µg/ml.

Survival of rabbits of experimental and control groups The results of determining the survival of animals of experimental and control groups from the moment of infection to the death of animals are shown in Table 4. The day of death of animals was determined depending on the time of detection of dead rabbits. So, if the discovery occurred in the morning, while rigor mortis and low carcass temperature were clearly recorded, then the date of the case was the previous day. If, during the morning examination, the carcass of a dead rabbit was warm, and there was no pronounced rigor mortis, then the current day was recognized as the time of death.

Biochemical parameters of blood (ACT, ALT, total protein) of rabbits of experimental and control groups

Table 5 presents statistically processed data on the level of ACT, ALT and total protein in rabbits of the experimental and control groups, which showed statistically significant differences (p<0.05).

A statistically significant difference was observed only in the ALT index on the 11th day after infection of rabbits in the treatment with the gel, the remaining indicators, as well as all indicators in the group treated with the spray, did not show statistically significant differences.

Clinical analysis of blood of rabbits of experimental and control groups Table 6 presents the results of clinical analysis of blood of rabbits of experimental and control groups, which showed statistically significant differences.

A statistically significant difference was observed only with a change in the level of segmented neutrophils on the 11th day after infection of rabbits during gel treatment. The remaining parameters, as well as all parameters in the group treated with a spray, did not show statistically significant differences.

The results of the thermometry of rabbits and the area of abscesses on days 9 and 12 after infection.

Statistically processed data on rabbit thermometry throughout the entire period of the experiment are shown in Table 7.

Table 8 shows the average abscess area in the combined (gel and placebo spray), control and experimental groups before infection, on the day of treatment, on days 9 and 12 of infection.

An analysis of the data on the survival of experimental and control rabbits from the day of their infection to the day of their death (inclusive) allows us to indicate that the average life expectancy of rabbits in the control groups is 13.67 ± 0.82 days, the average life expectancy of rabbits in the experimental group treated with the help of drugs in the form of a gel - 22±2.18 days, the average life expectancy of rabbits of the experimental group treated with drugs in the form of a spray - 20.44±2.24 days. When comparing the data obtained from animals in the treatment with gel and spray, no significant differences were found (p> 0.05), although the average values for these groups of animals differ by more than 1.5 days.

An indirect confirmation of the greater effectiveness of the gel with endolysins can be data on the area of abscesses formed on animals: in the group where the gel with endolysins was used, the abscess area was significantly larger before the start of treatment and on the 4th day of treatment, which should have led the animals to faster death . Additionally, in rabbits treated with drugs in the form of a gel, body temperature was significantly lower on the 9th day after infection (3-4 days after the start of treatment) compared with the control. This may indicate the antibacterial properties of the test agent, preventing the generalization of the infection. A similar phenomenon was recorded in the group of rabbits who were treated with drugs in the form of a spray, but poured on the 12th day after infection.

A statistically significant decrease in the level of leukocytes on the 11th day of infection (the next day after the end of treatment) in the group of rabbits treated with endolysin gel, compared with the control, may indicate the effectiveness of a local bactericidal action that reduces the systemic inflammatory response.

Analysis of the immunogenicity of endolysins in the cocktail Analysis of antiserum in the Ouchterlony precipitation test revealed the presence of antibodies to all endolysins used. In FIG. 3 presents the results of the analysis. In the central well antiserum (rabbit blood serum immunized with a cocktail of endolysins), peripheral wells contain antigens (endolysins LysAP22, LysAM24 and LysECD7). The precipitation lines indicate an antigen (endolysin)-antibody interaction reaction.

Analysis of the obtained rabbit antiserum by immunoelectrophoresis confirmed the formation of antibodies to each endolysin cocktail (Fig. 4). The upper well and the upper trench are human blood serum and antiserum to human blood serum proteins, respectively (to control electrophoresis), a cocktail (mixture) or individual endolysins is added to the remaining wells, and the studied rabbit antiserum is in the trenches. In the case of LysECD7 lysin, the presence of two precipitation lines was revealed, which indicates the formation of antibodies to two separate epitopes of endolysin.

Thus, using intramuscular immunization of a producer rabbit, a polyclonal antiserum against the endolysin cocktail was obtained, containing antibodies to all three components of the cocktail (LysAP22, LysAM24, and LysECD7).

Carrying out enzyme immunoassay

After a series of experiments, the optimal conditions for ELISA were chosen:

содержание каждого сорбированного эндолизина в лунке - 10-20 пг;

the content of each sorbed endolysin in the hole - 10-20 pg;

длительность сорбции антигена в лунках 18-20 ч;

the duration of antigen sorption in the wells is 18-20 hours;

температура сорбции+4 -+8°С;

sorption temperature +4 - +8°C;

температура проведения реакции+37°С;

reaction temperature+37°C;

разведение конъюгата «Белок А-пероксидаза» 1/64000;

dilution of the conjugate "Protein A-peroxidase"1/64000;

разведение конъюгата «Poly goat anti-rabbit HR» 1/8000;

dilution of the conjugate "Poly goat anti-rabbit HR"1/8000;

реакция проводится без встряхивания. Результаты исследования по выявлению антиэндолизиновых антител в крови кроликов после лечения коктейлем эндолизинов

the reaction is carried out without shaking. The results of a study on the detection of anti-endolysin antibodies in the blood of rabbits after treatment with an endolysin cocktail

As a result of the research, the following results were obtained (Table 9). A pronounced color reaction develops only in wells with a positive control, and even at a dilution of 1/1600. The negative control wells have an optical density of about 0.3. On average, the optical density was also about 0.3 in wells with sera from other animals, and about 0.35 was optical density in wells with intact rabbit serum. In the "empty wells" the reaction did not develop.

The results were recorded using the cut-off count. To cut off possible false-positive results, we chose a cut-off value equal to twice the optical density of the negative control (buffer solution). The assessment of the presence of antibodies to endolysins was carried out using an indicator - the coefficient of positivity, which is the ratio of the optical density of the sample to the cut-off value. In all the studied blood serum samples, the presence of antibodies to endolysins was not recorded (Kp is significantly less than 1).

The presence of antibodies in all blood serum samples of the examined rabbits was not recorded, which may indicate that when using these forms of endolysins, antibodies are not produced during these periods.

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1

Gel for local treatment and prevention of postoperative purulent-septic complications (AIMP).

The composition of the gel per 100 ml: liquid substance containing recombinant endolysin of the bacteriophage ECD7, belonging to the class of peptidases, with an individual concentration of 20 mg/ml - 50 ml; Natrosol 250 HHX (hydroxyethylcellulose) - 1.0 g; PEG 1500-1.5 g; 20 mmol/l Tris-HCl buffer pH 7.5-50 ml.

The method for preparing the gel is as follows: 20 mM Tris-HCl (pH=7.5), pre-thermostated for 2 hours at a temperature of +50°C, is added to a weighed and measured amount of Natrosol 250 HHX and PEG, thermostated at a temperature of +50° C for 15 minutes. Mix thoroughly. The resulting gel base is sterilized at a temperature of +120°C for 12 minutes. Endolysin is introduced into the sterilized base for the gel in the form of a concentrated solution with a concentration of 20 mg/ml. Mix thoroughly.

Example 2

Spray for local treatment and prevention of postoperative purulent-septic complications (AIMP).

The composition of the spray per 100 ml: a liquid substance containing recombinant endolysin of the bacteriophage ECD7, belonging to the class of peptidases, with an individual concentration of 1 mg / ml - 15 ml; a liquid substance containing recombinant endolysin of the bacteriophage Am24, belonging to the class of muramidase, with an individual concentration of 1 mg/ml - 15 ml; Kolliphor P 338 (Poloxamer 338) - 1.0 g; PEG 1500 - 1.0 g; 20 mmol/l Tris-HCl buffer pH 7.5-70 ml.

The spray manufacturing method is as follows: 20 mM Tris-HCl (pH=7.5), pre-thermostated for 2 hours at a temperature of +50°C, is added to a weighed and measured amount of poloxamer and PEG, thermostated at a temperature of +50°C in within 15 minutes. Mix thoroughly. The resulting spray base is sterilized at +120°C for 15 minutes. Endolysin is introduced into the sterilized spray base in the form of a concentrated solution with a concentration of 1 mg/ml. Mix thoroughly.

Example 3

Gel for the treatment and prevention of wound infections (community-acquired).

The composition of the gel per 100 ml: liquid substance containing recombinant endolysin of the bacteriophage ECD7, belonging to the class of peptidases, with an individual concentration of 1 mg/ml - 10 ml; liquid substance containing recombinant endolysin of the bacteriophage Am24, belonging to the class of muramidase, with an individual concentration of 1 mg/ml

- 10 ml; a liquid substance containing recombinant endolysin of the bacteriophage Ap22, belonging to the class of muramidase, with an individual concentration of 1 mg/ml - 10 ml; Natrosol 250 HHX (hydroxyethyl cellulose) -1.0 g; PEG 1500 - 1.5 g; 20 mmol/l Tris-HCl buffer pH 7.5-70 ml.

Example 4. The method of preparing the gel is as follows: In a weighed and measured amount of Natrosol 250 HHX and PEG, add 20 mm Tris-HCl (pH = 7.5), pre-thermostated for 2 hours at a temperature of +50 ° C, thermostated at a temperature +50°C for 15 minutes. Mix thoroughly. The resulting gel base is sterilized at a temperature of +120°C for 12 minutes. Endolysins are introduced into the sterilized base for the gel in the form of concentrated solutions with a concentration of 1 mg/ml. Mix thoroughly.

Example 5

Spray for the treatment and prevention of wound infections (community-acquired).

The composition of the spray per 100 ml: liquid substance containing recombinant endolysin of the bacteriophage ECD7, belonging to the class of peptidases, with an individual concentration of 1 mg / ml - 10 ml; a liquid substance containing recombinant endolysin of the bacteriophage Am24, belonging to the class of muramidase, with an individual concentration of 1 mg/ml - 10 ml; a liquid substance containing recombinant endolysin of the bacteriophage Ap22, belonging to the class of muramidase, with an individual concentration of 1 mg/ml - 10 ml; Kolliphor P 407 (Poloxamer 407) - 1.0 g; PEG 1500-1.0 g; 20 mmol/l Tris-HCl buffer pH 7.5-70 ml.

Example 6

The spray manufacturing method is as follows: 20 mM Tris-HCl (pH=7.5), pre-thermostated for 2 hours at a temperature of +50°C, is added to a weighed and measured amount of poloxamer and PEG, thermostated at a temperature of +50°C in within 15 minutes. Mix thoroughly. The resulting spray base is sterilized at +120°C for 15 minutes. Endolysins are introduced into the sterilized spray base in the form of concentrated solutions with a concentration of 1 mg/ml. Mix thoroughly.

As a result of the study, we announced the receipt of dosage forms of recombinant endolysins related to peptidoglycan hydrolases belonging to various classes of hydrolases, in the form of a gel and a spray. In the claimed formulations that are optimal in terms of technology and physical parameters (defined in the GF), containing one or more recombinant endolysins belonging to the classes of muramidase and peptidase, the minimum individual concentration of endolysins in the LF is 100-10000 μg/ml.

Claims (10)

1. A drug for the treatment or prevention of hospital and wound infections containing endolysins belonging to the classes of muromidases and peptidases, characterized in that it is made in the form of a gel and consists of the following components per 100 g, wt.%: A mixture of recombinant endolysins LysECD7, LysAm24 and LysAp22 - 0.03-0.15%; Gelling agent selected from hydroxyethylcellulose, hydroxymethylpropylcellulose, sodium carboxymethylcellulose, methylcellulose and hydroxypropylcellulose - 0.5-3.0%; PEG 1500 or PEG 3000 - 0.5-5.0%; Buffer solution with pH 7.0-8.0 - 91.85-98.97%. 2. A drug for the treatment or prevention of hospital and wound infections containing endolysins belonging to the classes of muromidases and peptidases, characterized in that it is made in the form of a spray and consists of the following components per 100 g, wt.%: A mixture of recombinant endolysins LysECD7, LysAm24 and LysAp22 - 0.03-0.15%; Poloxamer 338 or Poloxamer 407 - 0.75-3%; PEG 1500 or PEG 3000 - 0.75-3.0%; Buffer solution with pH 7.0-8.0 - 93.85-98.47%.

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