WO2025071439A1 - Intranasal composition for the prophylaxis and/or treatment of viral and bacterial infections - Google Patents

Abstract

The invention relates to the field of medicine, and more particularly to pharmacology, and can be used for the prophylaxis and/or treatment of viral and bacterial infections of the nasopharynx and nasal cavity, including coronavirus infection, influenza and other acute viral respiratory infections. An intranasal composition for the prophylaxis and/or treatment of inflammatory infectious diseases of the nasal cavity contains high molecular weight hyaluronic acid, high molecular weight polyvinylpyrrolidone (high molecular weight PVP), poviargol, an antibacterial polypeptide, sodium hypochlorite solution and gelofusine, wherein the components are present in given proportions.

Description

INTRANASAL COMPOSITION FOR THE PREVENTION AND/OR TREATMENT OF INFECTIONS OF VIRAL AND BACTERIAL ETIOLOGY FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to the field of medicine, namely pharmacology, and can be used for the prevention and/or treatment of viral and bacterial infections of the nasopharynx, nasal cavity, including coronavirus infection, influenza, and other acute respiratory viral infections.

LEVEL OF TECHNOLOGY

The increasing role of infectious viral diseases in the structure of general morbidity of the population, the emergence of new dangerous viruses (including previously unidentified ones) contribute to the development and implementation of promising antimicrobial compositions with a broad spectrum of activity and prolonged action.

The nasal cavity is an antimicrobial filter, which is widely represented by air microflora, including fungal spores, bacilli, micrococci, nonpathogenic Neisseria, epidermal staphylococcus, beta-hemolytic streptococcus. Facultative inhabitants of the nose include pneumococci, Klebsiella pneumoniae, Staphylococcus aureus, influenza bacteria, Candida. These species can cause autoinfection: rhinitis, bronchitis, nasopharyngitis, pneumonia, and also be a source of infection for other people. In children, the nasal microflora is even more numerous and diverse.

The prototype of the claimed solution is the invention according to patent RU 2616523 C1 (17.04.2017, IPC A61K9/06, A61K31/22, A61K36/61, A61K36/53, A61P31/04, A61P31/16), which is an intranasal ointment for the treatment and prevention of respiratory infections. According to the data provided in the description, the invention is characterized by a prolonged effect, which is provided by the synergism of the individual components of the drug. The results of using the drug were shown in patients with influenza and acute respiratory viral infections. However, the authors of this invention do not indicate the required frequency and duration of use of the intranasal ointment, which will achieve the stated treatment and prevention.

In addition, the authors do not confirm the claimed antiviral effect due to the synergistic action of individual components of the ointment with their in vitro study results. The authors also did not provide data on the effectiveness of the intranasal ointment against human coronaviruses, influenza virus, other RNA- and DNA-containing viruses, nor did they declare the antibacterial effect of the ointment and its effect against microbial biofilms.

DISCLOSURE OF THE ESSENCE OF THE INVENTION

The objective of the present invention is to solve the problem of the lack of an effective broad-spectrum composition for the prevention and/or complex treatment of infectious and inflammatory diseases of the nasal cavity when administered intranasally.

The technical result of the invention is the creation of a more effective, compared to the prototype, broad-spectrum composition for the prevention and/or complex treatment of infectious and inflammatory diseases of the nasal cavity with intranasal use, possessing prolonged broad antimicrobial activity, in particular, with respect to human coronavirus, influenza virus, other RNA- and DNA-containing viruses and bacteria, possessing the ability to prevent the formation and destroy already formed microbial biofilms.

The problem is solved and the specified technical result is achieved by creating a composition for application to the nasal cavity, characterized by the fact that it contains high-molecular hyaluronic acid, high-molecular polyvinylpyrrolidone (PVP), gelofusin, sodium hypochlorite, poviargol, an antibacterial polypeptide, which is gramicidin C or lysozyme, in the following ratio of components, wt. %:

• High molecular weight hyaluronic acid - 0.1 -2.0;

• High molecular weight PVP - 4.0-6.0;

• Poviargol - 0.8-1.2;

• Antibacterial polypeptide (gramicidin C - 0.03-0.15, or lysozyme - 0.1-18.0);

• Sodium hypochlorite solution with an active chlorine content of 1566 mg/l - 49.0-51.0;

• Gelofusin up to 100.0. The quantitative and qualitative combination of components in the composition proposed for patenting leads to the manifestation of a synergistic effect.

The subject of the invention is also the use of the claimed composition for the prevention and/or treatment (especially during periods of exacerbation of seasonal infectious diseases, epidemics and pandemics) of infectious and inflammatory diseases of the nasal cavity caused by coronaviruses, influenza virus, other RNA- and DNA-containing viruses and bacteria, accompanied by the formation of microbial biofilms, characterized in that the composition is applied to the mucous membrane of the nasal cavity for prophylactic or therapeutic purposes once a day.

The term "high-molecular hyaluronic acid" in the context of the present invention characterizes non-sulfated glycosaminoglycan, which is part of connective, epithelial and nervous tissues. High-molecular hyaluronic acid is one of the main components of the extracellular matrix, is contained in many biological fluids (saliva, synovial fluid, etc.), plays a significant role in the proliferation and migration of cells, is produced by some bacteria (Nair. Streptococcus. The body of a person weighing 70 kg contains on average about 15 grams of hyaluronic acid, a third of which is converted (broken down or synthesized) every day.

One of the important functions of hyaluronic acid in connective tissue is water binding. As a result, the intercellular substance acquires the character of a jelly-like matrix that supports the cells. Water binding and the swelling caused by it determine the biological role of hyaluronic acid in regulating tissue permeability. Hyaluronic acid is the main structure-forming glycosaminoglycan, since it has the ability to concentrate other glycosaminoglycans around itself and form proteoglycan aggregates that have greater hydrophilicity and elasticity compared to free proteoglycans. By binding collagen fibers, other proteins and components of the intercellular substance and even cells into a single system, hyaluronic acid creates a “buffer volume” that determines the strength and elasticity of mechanical tissues, helping them to overcome temporary impact (N.N. Sigaeva, S.V. Kolesov, P.V. Nazarov, R.R. Vildanova. Chemical modification of hyaluronic acid and its use in medicine // Bulletin of the Bashkir University. 2012. Vol. 17. No. 3. Pp. 1220-1241).

The term "high-molecular medical polyvinylpyrrolidone" (High-molecular PVP) in the context of the present invention characterizes binding agent. High-molecular PVP is available as a powder of the "pharmaceutically pure" grade, a free-flowing white or yellowish-white powder with particles of various sizes, a specific odor. The average molecular weight of PVP is 1,000,000-1,500,000 Da [VolkerBuhler. Kollidon®. Polyvinylpyrrolidone for the pharmaceutical industry / Translation from English, edited by Doctor of Philosophy K.V. Alekseev. BASF: 2001. - 310 p.]. An aqueous solution of PVP is a sterile, viscous, transparent, slightly colored liquid with a specific, weak odor.

Despite the wide range of materials for immobilization of medicinal substances, the greatest interest is presented by biodegradable carriers based on high-molecular PVP, which allow for the long-term presence of antibacterial substances in the wound, their local use both for the purpose of prevention in metalloosetting synthesis and for the treatment of surgical infections. The molecular weight of PVP is of great importance in this case. Modification of biologically active substances with high-molecular polymers is carried out for the purpose of targeted change of their properties: reduction of toxicity, improvement of solubility, pharmacokinetics and bioavailability due to complexation [Afinogenov G.E., Panarin E.F. Antimicrobial polymers, 1993. Publishing house "Gipppokrat", St. Petersburg. - 260 p.]. The main function of soluble high-molecular PVP is to prolong the action of medicinal preparations with improved bioavailability.

The authors found that high-molecular PVP, which has sorbent and antitoxic properties, as a polymer carrier helps to ensure a prolonged effect of the active substances in the present composition.

The term "poviargol" in the context of the present invention characterizes highly dispersed metallic silver stabilized by low-molecular medical polyvinylpyrrolidone; it is a light powder from greenish-gray to greenish-brown color. In medical practice, poviargol is used as an aqueous solution for external use, which is prepared immediately before use.

Poviargol is a broad-spectrum antimicrobial agent, active against aerobic and anaerobic microflora, including antibiotic-resistant ones. In concentrations up to 100 μg/ml, it inhibits the growth of most bacteria (staphylococci, streptococci, Pseudomonas aeruginosa and Escherichia coli, Proteus, Shigella, Salmonella, etc.).

The drug in concentrations of 1-3% has an anti-inflammatory effect and stimulates reparative processes in the wound at the stage of epithelialization; it is low-toxic, does not has an irritating effect on the skin and mucous membranes, does not cause allergic reactions and dermatitis. Poviargol is used as an antimicrobial agent for the prevention and treatment of purulent-septic complications of wounds, ulcers, burns and bedsores, including those that do not heal for a long time when treated with antibiotics; in infectious diseases of the upper respiratory tract, ear, throat, nose, eyes and oral cavity; in diseases of the genitourinary system and musculoskeletal system [http://sktb-technolog.ru/poviargolum2; "Poviargol: a new bactericidal agent for the treatment of infected wounds. (Experience of clinical use in traumatology, purulent surgery, burn therapy, gynecology, urology and ophthalmology). Reference manual for doctors / Ed. Corresponding Member of the Russian Academy of Sciences Panarin E.F., Doctor of Medical Sciences, Professor Blagitko E.M. Novosibirsk: Publishing house. 1998. - 66 p.

The term "sodium hypochlorite" in the context of the present invention characterizes an effective antiseptic. The range of its application areas includes disinfection of laboratory equipment, water disinfection, local treatment of wounds, etc. Sodium hypochlorite exhibits its biological activity through reactions with nucleic and amino acids. In reactions with nucleic acids, in particular DNA, it chlorinates bases, preventing the formation of hydrogen bonds, which initiates denaturation of the molecule and, as a consequence, loss of biological activity.

The term "lysozyme" in the context of the present invention characterizes an enzyme of the innate immune system of a large number of different organisms, from prokaryotes to higher eukaryotes. Being 1,4-L-O-N-acetyl muramidase, it is, among other things, capable of exhibiting chitinase activity, hydrolyzing glycosidic bonds of bacterial peptideglycan, activating autolysins and exhibiting antiviral activity, including against HIV. At the same time, the protein has a fairly low toxicity, and even when doses sufficient to achieve a bactericidal effect are exceeded, no significant effect on the patient's body was detected.

The term "gramicidin C" in the context of the present invention characterizes a molecule of organic origin and serves as a natural defense mechanism for the Bacillus brevis bacterium, which begins producing the substance in response to a threat to its vital activity. The antibiotic has a polypeptide structure, has a bacteriostatic (preventing the reproduction of bacteria) and bactericidal (destroying bacteria) effect. The mechanism of action of gramicidin C is associated with an increase in the permeability of the cytoplasmic membrane of the microbial cell, which disrupts its stability and causes cell death. Gramicidin C is cyclic sequence of amino acids, and such a structure allows preventing the development of resistance of microorganisms to this antibiotic, while at the moment, the development of addiction to the drug in various bacterial pathogens has not been described in the available literature, which is important for the effectiveness of treatment.

The term "gelofusine" in the context of the present invention characterizes a 4% solution of succinylated gelatin (also known as modified liquid gelatin) for intravenous administration with an average molecular weight of 23,200 daltons. It has a colloid osmotic pressure of 34 mm Hg. The isoelectric point is reached at pH 4.5. Negative charges arising in the molecule as a result of succinylation lead to an increase in the size of the molecule and, thus, more voluminous protein chains are formed than non-succinylated ones, while maintaining the molecular weight. As a result, Gelofusine has a sufficient volemic effect for 3-4 hours. Gelofusine is used as a colloidal plasma substituting agent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 - Kinetics of interaction of viral RNA with sodium hypochlorite.

FIG. 2 - Changes over time in the absorption spectra of poly AU (C = 0.98*10' ⁵ M a. o.) in the presence of sodium hypochlorite (C _hyp . = 5*10' ⁵ M, or 3.75 mg/l).

FIG. 3 - Changes over time in the absorption spectra of pAU-hypochlorite complexes in the presence of poviargol at λ=260 nm.

FIG. 4 - Absorption spectra of the mixture "sodium hypochlorite - poviargol" at λ = 260 nm.

FIG. 5 - Control of human lung adenocarcinoma cell culture (ATCC® CRL-5800), h600.

FIG. 6 - Control of the cytotoxic effect of coronavirus OC43 on human lung adenocarcinoma cell culture (ATCC® CRL-5800), x900.

FIG. 7 - Morphological picture of the state of the culture of human lung adenocarcinoma cells (ATCC® CRL-5800) in the presence of coronavirus OC43 and a polymer composition of three polymers, xbOO.

FIG. 8 - Absorption spectra of the DNA-hypochlorite complex, Cdna ⁼ 2.2 x 10' ⁵ _{Molecular weight} (1 - 1 min. p/p, 2 - 5 min. p/p, 3 - 15 min. p/p, 4 - 1 day p/p). a) Mixture ⁼ 1 x 10' ⁴ M; b) C _NaCl = 3.5 x 10' ⁴ M; c) C _NaCl = 8.0 x 10' ⁴ M. FIG. 9 - A: Kinetics of the reaction of the ternary system Hyp-DNA-Pov; B: time derivative of the absorption dependence on the reaction time of the ternary system DNA-Hyp-Pov (black squares) and DNA-Hyp (red circles).

FIG. 10 - Relationship between the bactericidal action of an intranasal composition and the time of its exposure to different microorganisms.

FIG. 11 - Bactericidal action of the intranasal composition and the antimicrobial ingredients included in its composition.

FIG. 12 - Visualization of the intranasal composition on the nasal mucosa after 8 hours of application.

FIG. 13 - No visualization of the intranasal composition based on the prototype on the nasal mucosa after 8 hours of application.

FIG. 14 (A) - Control 0.5 MF Staphylococcus aureus.

FIG. 14 (B) - Experimental sample of 0.5 MF Staphylococcus aureus.

FIG. 14 (B) - Prototype 0.5 MF Staphylococcus aureus.

FIG. 15 (A) - Control 0.5 MF Pseudomonas aeruginosa.

FIG. 15 (B) - Experimental sample of 0.5 MF Pseudomonas aeruginosa.

FIG. 15 (B) - Prototype 0.5 MF Pseudomonas aeruginosa.

FIG. 16 (A) - Control 0.5 MF Acinetobacter baumannii.

FIG. 16 (B) - Experimental sample of 0.5 MF Acinetobacter baumannii.

FIG. 16 (B) - Prototype 0.5 MF Acinetobacter baumannii.

FIG. 17 (A) - Control 0.5 MF Klebsiella pneumoniae.

FIG. 17 (B) - Experimental sample of 0.5 MF Klebsiella pneumoniae.

FIG. 17 (B) - Prototype 0.5 MF Klebsiella pneumoniae.

IMPLEMENTATION OF THE INVENTION

The claimed composition for the prevention and/or treatment, alone or as part of a complex therapy of infectious and inflammatory diseases of the nasal cavity with intranasal use, which has prolonged antimicrobial activity against human coronavirus, influenza virus, other RNA- and DNA-containing viruses and bacteria, the ability to prevent the formation and destroy already formed microbial biofilms, includes components in the following ratio, May. %:

• High molecular weight hyaluronic acid - 0.1 -2.0;

• High molecular weight PVP - 4.0-6.0;

• Poviargol - 0.8-1.2; • Antibacterial polypeptide (gramicidin C - 0.03-0.15, or lysozyme - 0.1-18.0);

• Sodium hypochlorite solution with an active chlorine (AC) content of 1566 mg/l - 49.0-51.0;

• Gelofusin up to 100.0.

The amount of high molecular weight hyaluronic acid can also be 0.5-2 wt.%, 0.5-1 wt.%, or 1-2 wt.%.

The amount of high molecular weight PVP can also be 4.5-5.5 wt%.

The amount of poviargol can also be 0.8-1 wt%.

The amount of lysozyme can also be 1-16.0 wt%.

The claimed composition can be used for prevention on its own, as well as as part of complex therapy for infectious and inflammatory diseases of the nasal cavity caused by coronaviruses, influenza viruses, other RNA- and DNA-containing viruses and bacteria, by applying it (application) to the mucous membrane of the nasal cavity.

To obtain the composition, all powdered components (hyaluronic acid, polyvinylpyrrolidone, poviargol, gramicidin C (or lysozyme)) are dissolved sequentially in a sodium hypochlorite solution, observing the above-mentioned quantitative ratios, and a gelofusin solution is added to 100.0 wt.%, mixed until a homogeneous mass is obtained.

The preparation of the claimed composition based on gelofusin, high-molecular hyaluronic acid and polyvinylpyrrolidone provides a prolonged effect due to the formation of a protective barrier film on the mucous surface of the nasal cavity, which contributes to the long-term antimicrobial effect of poviargol and sodium hypochlorite, moisturizing the nasal passages, which, in turn, reduces the risk of developing irritation, dryness of the nasal mucosa, pain reaction, bleeding.

The ratio of the components of the claimed composition is the result of extensive experimental studies and is optimally suited to achieve the above results. At the same time, a significant advantage of the claimed composition is the presence of a prolonged antimicrobial effect against a wide range of RNA- and DNA-containing viruses and bacteria, microbial biofilms, and the absence of side effects.

A special feature of the claimed composition is the formation of a thin polymer barrier film on the mucous surface of the nasal cavity with a single applications, which ensures high sorption of viral and bacterial particles for one hour. In addition, the presence of poviargol during the first 50 seconds accelerates the denaturing effect of sodium hypochlorite on viral RNA or viral and bacterial DNA.

The following experimental data are presented as non-limiting examples.

Example 1.

The antiviral activity against RNA-containing viruses of the proposed intranasal composition of the following composition, wt. %, was studied:

Sodium hypochlorite solution (AX 1566 mg/l) - 49.0

Hyaluronic acid - 1.0

PVP - 5.0

Poviargol - 1.0

Gramicidin C - 0.03

Gelofusine - 43.97.

The following virus strains were used:

- human coronavirus, strain OC43,

- rpnnnaA/PuertoRico/8/34 (H1N1) virus.

To analyze the antiviral activity, 1 ml of the sample of the proposed intranasal composition was mixed in a 1.5 ml test tube with 0.1 ml of the virus-containing liquid (the initial titer of coronavirus OC43 was 6.5 1 g; influenza virus 7.5 lg). Instead of the composition, 0.1 ml of the cell medium was added to the control test tube. The test tubes were incubated at room temperature for 3 minutes, after which the infectious activity of the corresponding virus was determined in the virus-containing liquid. Three analytical parallels were used in each study group.

Table 1. Activity of RNA-containing viruses upon incubation with a sample of the intranasal composition

Note: The significance level was P<0.05.

As follows from the presented data, the proposed intranasal composition reliably reduced the titers of the influenza virus and coronavirus OC43 in cell culture.

The antiviral efficacy against RNA-containing viruses was at least 99.9% within 3 minutes of exposure.

Example 2.

The antiviral activity against RNA-containing viruses of the proposed intranasal composition of the following composition, wt. %, was studied:

Sodium hypochlorite solution (AX 1566 mg/l) - 49.0

Hyaluronic acid - 1.0

PVP - 5.0

Poviargol - 1.0

Lysozyme - 16.0

Gelofusine - 28.0.

The following virus strains were used:

- human coronavirus, strain OC43,

- rpnnnaA/PuertoRico/8/34 (H1N1) virus.

To analyze the antiviral activity, 1 ml of the sample of the proposed intranasal composition was mixed in a 1.5 ml test tube with 0.1 ml of the virus-containing liquid (the initial titer of coronavirus OC43 was 4.7 1 g; influenza virus 5.5 lg). Instead of the composition, 0.1 ml of the cell medium was added to the control test tube. The test tubes were incubated at room temperature for 3 minutes, after which the infectious activity of the corresponding virus was determined in the virus-containing liquid. Three analytical parallels were used in each study group. Table 2. Activity of RNA-containing viruses upon incubation with a sample of the intranasal composition

Note: The significance level was P<0.05.

As follows from the data provided, the proposed intranasal composition reliably reduced the titers of the influenza virus and coronavirus OC43 in cell culture. The effectiveness of the antiviral action against RNA-containing viruses was at least 99% during a 3-minute exposure.

Example 3.

To prove the enhancement of the antiviral effect of the components of the claimed composition, the antiviral activity against the RNA-containing virus (coronavirus) of the ingredients of the proposed intranasal composition was studied in the following concentrations, May %:

Sample No. 1

Gramicidin (polypeptide antibiotic) - 0.03

Sample #2

Poviargol (silver nanoparticles) - 1.0

Sample #3

Gramicidin (polypeptide antibiotic) - 0.03

Poviargol (silver nanoparticles) - 1.0

Sample No. 4

Lysozyme (antibacterial polypeptide) - 16.0

Poviargol (silver nanoparticles) - 1.0

Sample #5

Lysozyme (antibacterial polypeptide) - 16.0 Gramicidin (polypeptide antibiotic) - 0.03

Sample No. 6

Lysozyme (antibacterial polypeptide) - 16.0.

The following virus strains were used:

- human coronavirus, strain OC43.

To analyze the antiviral activity, 1 ml of each sample was mixed in a 1.5 ml test tube with 0.1 ml of virus-containing fluid (the initial titer of coronavirus OC43 was 5.5 1g). Instead of the corresponding sample, 0.1 ml of cell medium (complete RPMI-1640 medium containing 2 mM L-glutamine, 250 mg/l gentamicin, 10% fetal bovine serum) was added to the control tube. The tubes were incubated at room temperature for 3 minutes, after which the infectious activity of the corresponding virus was determined in the virus-containing fluid. Three analytical parallels were used in each study group.

Table 3. Activity of RNA-containing coronavirus when incubated with samples of ingredients of the proposed intranasal composition

Note: The significance level was P<0.05.

For the first time, the presence of gramicidin C's own antiviral activity has been demonstrated, since there is no data on its antiviral action in the available literature. In this case, an additive effect (distinctive feature) of the interaction of gramicidin C with poviargol and lysozyme with poviargol was observed in relation to the coronavirus strain OC43. Such interaction provides an advantage in the intranasal composition of prolonged action, when the initial concentrations of antimicrobial components are gradually reduced.

Example 4.

The ability of poviargol (silver nanoparticles) to accelerate the process of destruction of viral and synthetic RNA by sodium hypochlorite was studied.

The study used human coronavirus OC43 from the collection of viral strains of the Pasteur Research Institute of Epidemiology and Microbiology (St. Petersburg). The original sample of viral RNA (C = 3*10' ⁴ M a. o.) was diluted with 0.00 IM NaCl to achieve the minimum volume required for measurement in a special spectrophotometer cuvette (600 μl). The concentration of viral RNA used in the experiment was C = 1*10' ⁵ M a. o.

The concentration of sodium hypochlorite (molar) in all experiments was 5 times greater than the concentration of nucleotides in the solution, i.e. _Chyp = 5*10' ⁵ M, or 3.75 mg/l.

The results of the interaction of viral RNA and different concentrations of sodium hypochlorite are presented in Figure 1.

The different course of time dependences in different regions of the spectrum indicates the interaction of sodium hypochlorite with various RNA components present in the solution. In this case, it is the wavelength region of 260 nm that can reflect the interaction of sodium hypochlorite with the nitrogenous bases of the polynucleotide.

A similar experiment was carried out with synthetic RNA (poly AU). The results are shown in Figure 2.

The nature of the time dependences of the relative change in absorption and its rate in the region of X = 260 nm, which characterizes the state of nitrogenous bases in polynucleotides, is similar for viral and synthetic RNA. It indicates that the process of interaction of polynucleotides with hypochlorite has at least 2 stages. The first is rapid, expressed in an increase in the intensity of absorption, which is usually associated with denaturation or destruction of the secondary structure of the polynucleotide. At this stage, chlorination of nitrogenous bases occurs. At the second stage, gradual destruction of nitrogenous bases occurs, caused by the products formed at the first stage of interaction. Changes in the absorption spectra of the poly AU + sodium hypochlorite complexes in the presence of poviargol over time are shown at the following component concentrations: Cpdi ⁼ 7.6* 10' ⁵ M, _CNa oci ⁼ 3.8* 10' ⁴ M, _CPOV iargol ⁼ 0.002%. The results are presented in Figure 3.

In the presence of poviargol, the destruction of nucleotides, which occurs in the first 2 minutes of interaction of sodium hypochlorite with the polynucleotide, is significantly enhanced.

The mechanism of this action is that sodium hypochlorite interacts with poviargol. This is evidenced by the absorption spectra of the "sodium hypochlorite - poviargol" mixture (FIG. 4).

The products of this interaction contribute to the destruction of nucleotides in RNA.

The effect of acceleration of the process of RNA destruction by sodium hypochlorite in the presence of poviargol has been identified and described for the first time. The mechanism of this action is that sodium hypochlorite interacts with poviargol. The products of this interaction contribute to more rapid destruction of nucleotides in the RNA.

Example 5.

The sorption activity of polymer combinations of the following composition, wt. %, in relation to RNA-containing viruses was studied:

Sample No. 1

High molecular weight PVP - 5.0

Cell culture medium - up to 100.0.

Sample #2

High molecular weight hyaluronic acid - 1.0

Gelofusin (4% liquid gelatin) - up to 100.0.

Sample No. 3

High molecular weight hyaluronic acid - 1.0

High molecular weight PVP - 5.0

Gelofusin (4% liquid gelatin) - up to 100.0.

The following virus strains were used:

- human coronavirus, strain OC43,

- rpnnnaA/PuertoRico/8/34 (H1N1) virus. 100 μl of virus-containing liquid (initial titer of coronavirus OC43 6.5 1g; influenza virus 7.5 lg) were added to 1 ml of the corresponding polymer combination and the resulting mixtures were incubated for 1 hour at room temperature. After that, the samples were centrifuged for 20 minutes at 3000 rpm and the infectious activity of the virus was determined in the supernatant. In the control samples, cell culture medium was used instead of the polymer combination.

Table 4. Sorption activity of polymer combinations in relation to RNA-containing viruses

Note: The significance level was P<0.05.

As follows from the presented data, during combined pre-incubation for 1 hour, the ability of high-molecular PVP to reduce the titer of both test viruses by 1 lg was demonstrated; a mixture of high-molecular hyaluronic acid with gelatin - by 2.5 lg; a mixture of three polymers - high-molecular PVP and hyaluronic acid on gelofusin - by 3.5 lg.

For the first time, an exceptional antiviral effect of the polymer composition has been established due to the high ability of polymers and their combinations to sorb particles of RNA-containing viruses. This ensures the ability of the studied polymer combination to prevent the attachment of viral particles to the mucous membrane of the nasal cavity, to retain them in the polymer matrix, facilitating the action of antimicrobial agents in the antimicrobial intranasal composition.

Figures 5-7 show the morphological picture of the state of the cell culture in the presence of the test coronavirus OS43 and a polymer composition of three polymers - high-molecular PVP and hyaluronic acid on gelofusin.

As can be seen from Figure 6, coronavirus OC43 has a pronounced cytotoxic effect, completely destroying the cell culture monolayer. As can be seen from Figure 7, the polymer composition has a protective effect on the cell culture monolayer, preventing its destruction due to the cytotoxic effect of the coronavirus.

Example 6.

The ability of poviargol (silver nanoparticles) to accelerate the process of destruction of plasmid DNA of microorganisms by sodium hypochlorite has been studied.

The plasmid DNA of the foc/zerzc/zztzco/z'DHS-Alpha strain was used, the size is 5.5-6 thousand nucleotide pairs, the concentration used in the experiment is C=2.2-10“ ⁵ M _{a o}

To conduct the spectral study, 3 hypochlorite complexes with plasmid DNA were prepared. For each complex, three different solutions with a constant DNA concentration and different sodium hypochlorite concentrations were prepared:

1) C _Na0C i= 1.46-10 ^-4 M;

2) C _NaO ci=3.5-10“ ⁴ M;

3) With _NaO ci=8.0-10“ ⁴ M.

The complexes were prepared by direct mixing. An aqueous solution of sodium chloride NaCl with an ionic strength of 0.015 M was used as a solvent.

Figure 8 shows the absorption spectra of complexes of E. colic plasmid DNA with sodium hypochlorite at different concentrations of NaOCl.

As can be seen from the obtained spectra, regardless of the sodium hypochlorite concentration, during the first minute after mixing, the absorption of the solution increases in the entire absorption region of DNA. In this case, the total spectrum is close to the sum of the spectra of DNA and sodium hypochlorite of the concentrations used.

Noticeable changes in the spectrum shape and absorption intensity at a wavelength of 260 nm were observed after 10-15 minutes.

концентрации Смасю ⁼ 8,0 х 10'⁴ М полоса поглощения гипохлорита натрия сохранялась в течение суток. Это говорит о том, что при данной концентрации антисептик взят в избытке и после взаимодействия остается в растворе. After 24 hours, the absorption band of DNA (260 nm) practically disappears.

concentration of Smasu ⁼ 8.0 x 10' ⁴ M the absorption band of sodium hypochlorite remained for 24 hours. This indicates that at this concentration the antiseptic is taken in excess and after the interaction remains in the solution.

Thus, it has been established that when interacting with sodium hypochlorite, both the secondary structure of microbial DNA is destroyed (denaturation) and the nitrogenous bases are chemically modified, presumably chlorination. The presence of a secondary structure slows down the chemical reaction of sodium hypochlorite with the nitrogenous bases of DNA. The poviargol solution was prepared from a dry powder sample. The solvent was an aqueous solution of NaCl salt with an ionic strength of I = 0.001L4. The concentration of the prepared solution was further reduced by half to achieve the condition of mixing equal volumes of reagents Cp _ov = 0.5%.

The substances used in the work have a characteristic absorption spectrum in the wavelength range from 220 to 500 nm. The spectra were measured on a Shimadzu 1800 spectrophotometer in quartz cuvettes with an optical path length of = 1 cm ₂ = 0.101 cm. The above spectrophotometer operates in a dual-beam mode. In all measurements, the cuvette space for the background solution remained empty, i.e. the measurements were performed relative to atmospheric air, the absorption of which in the range under consideration did not exceed D ^ _r < 0.005. After each measurement, the cuvette with the solution was washed and used to measure the solvent used in the previous measurement. The solvent spectrum was subtracted at the data processing stage using the Origin software package. The solutions were prepared using laboratory electronic scales (accuracy 0.0002 g) and dispensers, stirring was performed using a Vortex.

Analysis of the time derivative of the change in absorption at 260 nm (FIG. 9) shows that the first step of the DNA destruction reaction occurs much faster. This is consistent with the fact that poviargol (silver nanoparticles) induces denaturation of microbial DNA in the presence of sodium hypochlorite, making nitrogenous bases more accessible for the chlorination reaction.

Thus, in the presence of poviargol at the initial stage of interaction, the destruction of the microbial DNA molecule by sodium hypochlorite occurs faster. The reaction products of silver nanoparticles with sodium hypochlorite facilitate the denaturation of the double helix of microbial DNA, which accelerates the reaction of chlorination of nitrogenous bases by sodium hypochlorite.

Thus, the proposed intranasal composition destroys RNA- and DNA-containing viruses and bacteria.

Example 7.

The dependence of the bactericidal action on the time of exposure of the proposed intranasal composition of the following composition, mass %, was studied:

Sodium hypochlorite solution (AX 1566 mg/l) - 49.0

Hyaluronic acid - 2.0

PVP - 5.0

Poviargol - 1.0 Gramicidin C - 0.03

Gelofusine - 42.97.

Bacterial isolates were obtained from loci of patients from different departments of a multidisciplinary healthcare facility, and the proposed antimicrobial composition was tested using the “checkerboard” method.

The results of the assessment of the dependence of the bactericidal effect on the exposure time are presented in Figure 10.

After just 1.5 hours of exposure to the intranasal composition, a complete absence of growth of the tested microorganisms was observed. It should be noted that the antiseptics - sodium hypochlorite, poviargol, included in the intranasal composition, were used in subbactericidal doses, which are hundreds and thousands of times lower than the permitted doses of individual drugs for clinical local use.

Example 8.

The bactericidal action of the proposed intranasal composition of the following composition, wt. %, was studied:

Sodium hypochlorite solution (AX 1566 mg/l) - 49.0

Hyaluronic acid - 1.0

PVP - 5.0

Poviargol - 1.0

Lysozyme - 16.0

Gelofusine - 28.0.

It is known that lysozyme has a very weak antibacterial effect on gram-negative microorganisms [Renata Cegielska-Radziejewska Grzegorz Lesnierowski • Tomasz Szablewski • Jacek Kijowski. Physico-chemical properties and antibacterial activity of modiWedeggwhite — lysozyme // Eur Food Res Technol (2010) 231 :959–964; DOI 10.1007/s00217-010-1347-y; Goncharova A.I., Okulich B.K., Zemko V.Yu., Senkovich S.A. Antimicrobial activity of lysozyme as a factor of non-specific resistance // Bulletin of Vitebsk State Medical University. - 2019. - Vol. 18, No. 4. - pp. 40–45; DOI: https://doi.Org/10.22263/2312-4156.2019.4.40].

The study used clinical multiresistant (resistant to more than 5 groups of antibiotics, including gentamicin) Staphylococcus epidermidis 8242008, isolated from the patient's blood culture.

The clinical strain Klebsiella pneumoniae 5299639, isolated from the wound discharge of a patient with a maxillofacial profile, was used. This isolate was characterized by resistance to 8 groups of antimicrobial drugs, including carbapenems.

The proposed intranasal composition was tested using the “checkerboard” method with an exposure time of 1.5 hours.

The results of the evaluation of the bactericidal action of the intranasal composition and the antimicrobial ingredients included in its composition are presented in Figure 11.

In relation to gram-negative K. pneumoniae, the absence of antimicrobial action of lysozyme was observed; poviargol reduced the level of microbial contamination by 1 1 g after only 1.5 hours of exposure; with the action of the intranasal composition, a decrease in the number of K. pneumoniae by 21 g was observed.

In relation to gram-positive S. epidermidis, a decrease in contamination by 1:1 g was observed in the presence of lysozyme after 1.5 hours of exposure; poviargol reduced the level of microbial contamination of staphylococcus by 2:1 g; when exposed to the intranasal composition, a decrease in the number of S. epidermidis by 3:1 g was observed.

Thus, an increase in the bactericidal effect of lysozyme in the presence of poviargol against pathogenic microorganisms was demonstrated.

Example 9.

Evaluation of the prolonged action of the intranasal composition of the following composition, mass %:

Sodium hypochlorite solution (AX 1566 mg/l) - 49.0

Hyaluronic acid - 1.0

PVP - 5.0

Poviargol - 1.0

Gramicidin C - 0.03

Gelofusine - 43.97.

The studies were conducted on 10 volunteers, men and women aged 18 to 83 years. The composition was preliminarily supplemented with crushed activated carbon to improve visualization. All subjects were given the proposed intranasal composition once in both nasal passages, then the subjects squeezed the wings of the nose with their fingers to distribute the composition evenly over the mucosal surface. Under anterior rhinoscopy conditions using a hardware method (STORZ Karl Storz Endoscope tele pack XLEDTP100 device), the distribution of the intranasal composition over the nasal mucosa was assessed 8 hours after application. In all volunteers, a continuous, uniform distribution of the intranasal composition over the mucous membrane of the anterior sections of the nasal cavity was observed at all times, which indicated the presence of the proposed intranasal composition during the 8 hours of observation (FIG. 12).

When conducting a similar test using an intranasal composition based on the prototype, there was no visualization on the nasal mucosa after 8 hours of application (FIG. 13).

Example 10.

Comparative tests of intranasal composition (hydrogel) and prototype.

When assessing the virucidal activity using the above method, it was found that with joint incubation of the OC43 coronavirus with the prototype ointment for 3 minutes, the decrease in the virus titer was 1.3 1g TCID50 (the virus titer in the control was 5.3 1g).

For comparison, example 1 presented above showed a decrease in the titer of coronavirus OC43 during co-incubation with the intranasal composition for 3 minutes, which was 3 1g (the titer of the virus in the control was 6.5 1g). In example 2, the decrease in the titer of the virus OC43 during incubation with the intranasal composition with lysozyme was 1.5 lg (when the titer of the virus in the control was 4.7 1g).

Therefore, the proposed hydrogel has higher efficiency compared to the prototype.

Example 11.

Comparative evaluation of the bactericidal activity of the intranasal composition (hydrogel) and the prototype depending on the time of joint incubation (according to the method of Example 7, Time-kill assay method - evaluation of the dependence "time - lethal effect") and the dose of test cultures of microorganisms at a dose of 10 ⁵ CFU/ml (Staphylococcus aureus (FIG. 14 (A-B)), Pseudomonas aeruginosa (FIG. 15 (A-B)), Acinetobacter baumannii (FIG. 16 (A-B)), Klebsiella pneumoniae (FIG. 17 (A-B)) with an exposure of 1 hour.

The following results were obtained.

The prototype preparation did not have a bactericidal effect on test cultures of microorganisms. Microbial growth was noted on Petri dishes (FIG. 14 (B), FIG. 15 (B), FIG. 16 (B), FIG. 17 (B)). Moreover, the intranasal composition according to example 7 leads to a decrease in the microbial population by 4-5 1g in relation to all gram-positive and gram-negative microorganisms.

Thus, the claimed composition is more effective than the prototype drug in terms of both virucidal and bactericidal activity.

Claims

CLAUSE OF INVENTION 1. An intranasal composition for the prevention and/or treatment of infectious and inflammatory diseases of the nasal cavity containing high-molecular hyaluronic acid, high-molecular polyvinylpyrrolidone (high-molecular PVP), poviargol, an antibacterial polypeptide, sodium hypochlorite solution, gelofusin, in the following ratio of components, wt. %: High molecular weight hyaluronic acid - 0.1 -2.0; High molecular weight PVP - 4.0-6.0; Poviargol - 0.8-1.2; Antibacterial polypeptide - 0.03-18.0; Sodium hypochlorite solution - 49.0-51.0; Gelofusin up to 100.0. 2. The composition according to claim 1, characterized in that the amount of high molecular weight hyaluronic acid is 0.5-2 wt.%. 3. The composition according to claim 1, characterized in that the amount of high molecular weight hyaluronic acid is 1-2 wt.%. 4. The composition according to I.1, characterized in that the amount of high-molecular PVP is 4.5-5.5 wt.%. 5. The composition according to I.1, characterized in that the amount of poviargol is 0.8-1 wt.%. 6. The composition according to claim 1, characterized in that the antibacterial peptide is gramicidin C or lysozyme. 7. The composition according to I.6, characterized in that the amount of gramcidin C is 0.03-0.15 wt.%. 8. The composition according to I.6, characterized in that the amount of lysozyme is 0.1-18.0 wt.%. 9. The composition according to I.6, characterized in that the amount of lysozyme is 1-16.0 wt.%. 10. The composition according to claim 1, characterized in that infectious and inflammatory diseases of the nasal cavity are caused by coronaviruses, the influenza virus, other RNA- and DNA-containing viruses and bacteria, accompanied by the formation of microbial biofilms. 11. Use of the intranasal composition according to I. 1 for the prevention and/or treatment of infectious and inflammatory diseases of the nasal cavity caused by coronaviruses, influenza virus, other RNA- and DNA-containing viruses and bacteria, accompanied by the formation of microbial biofilms, where the application of the composition to the mucous membrane of the nasal cavity is carried out once a day. 12. Use according to item 11, characterized in that the composition is used independently or as part of complex therapy.