Intranasal composition for preventing and / or treating viral and bacterial infections

Abstract

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

Description

Technical Field

[0001] This invention relates to the medical field, namely pharmacology, and to methods for the prevention and / or treatment of nasopharyngeal and nasal infections caused by viruses and bacteria, including coronavirus infections, influenza, and other acute respiratory viral infections. Background Technology

[0002] The increasing prevalence of infectious viral diseases in the overall population, along with the emergence of novel and dangerous viruses (including previously identified viruses), has spurred the development and application of promising antimicrobial compositions with broad-spectrum and long-lasting effects.

[0003] The nasal cavity acts as an antimicrobial filter, harboring a diverse array of airborne microorganisms, including fungal spores, bacilli, micrococci, non-pathogenic Neisseria, Staphylococcus epidermidis, and β-hemolytic streptococci. Potential nasal conditional pathogens include pneumococcus, Klebsiella pneumoniae, Staphylococcus aureus, influenza bacteria, and Candida. These populations can cause autoinfections such as rhinitis, bronchitis, nasopharyngitis, and pneumonia, and can also serve as a source of infection for others. The nasal microbiota in children is even more diverse and abundant.

[0004] The claimed solution is based on an invention according to patent RU 2616523 C1 (April 17, 2017, IPCA61K9 / 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 its description, the invention is characterized by a long-lasting effect, provided by the synergistic effect of the drug's components. Results from using the drug have been demonstrated in patients suffering from influenza and acute respiratory viral infections.

[0005] The authors of this invention have not specified the required frequency and duration of use of the intranasal ointment, which may affect the effectiveness of the claimed treatment and prevention.

[0006] Furthermore, the authors did not use their in vitro studies to confirm that the claimed antiviral effect is due to the synergistic effect of the ointment's components. They also did not provide data on the effectiveness of the intranasal ointment against human coronaviruses, influenza viruses, and other RNA and DNA-containing viruses, nor did they claim protection for the ointment's antibacterial activity or its effect on microbial biofilms. Summary of the Invention

[0007] The purpose of this invention is to address the lack of an effective broad-spectrum composition for the prevention and / or comprehensive treatment of nasal infectious and inflammatory diseases when used intranasally.

[0008] The technical result of this invention is the development of a broad-spectrum composition that, when used intranasally, is more effective than the prototype in preventing and / or comprehensively treating nasal infectious and inflammatory diseases. It has durable broad-spectrum antimicrobial activity, particularly effective against human coronaviruses, influenza viruses, other viruses and bacteria containing RNA and DNA, and is able to prevent the formation of microbial biofilms and disrupt existing microbial biofilms.

[0009] The above-mentioned problems are solved and specific technical results are achieved by developing a composition for application in the nasal cavity, characterized in that the composition comprises high molecular weight hyaluronic acid, high molecular weight polyvinylpyrrolidone (PVP), succinyl gelatin, sodium hypochlorite, poviargol, and an antibacterial polypeptide (which is bacitracin C or lysozyme), having the following component ratios (wt.%): - High molecular weight hyaluronic acid - 0.1-2.0; - High molecular weight PVP - 4.0-6.0; - Povidone Silver - 0.8-1.2; - Antibacterial peptides (bacitracin C - 0.03-0.15, or lysozyme - 0.1-18.0); - Sodium hypochlorite solution with an available chlorine content of 1566 mg / L - 49.0-51.0; - Succinyl gelatin - up to 100.0.

[0010] The quantitative and qualitative combination of the components in the proposed patent composition leads to a synergistic effect.

[0011] Another object of the present invention is to use the claimed composition for the prevention and / or treatment (especially during seasonal infectious diseases, epidemics and pandemics) of nasal infectious and inflammatory diseases caused by coronaviruses, influenza viruses, other RNA and DNA-containing viruses and bacteria and accompanied by microbial biofilm formation, characterized by the fact that the composition is applied to the nasal mucosa once daily for preventive or therapeutic purposes.

[0012] In the context of this invention, the term "high molecular weight hyaluronic acid" refers to unsulfated glycosaminoglycans that are part of connective tissue, epithelial tissue, and nerve tissue. High molecular weight hyaluronic acid is a major component of the extracellular matrix, present in many biological fluids (saliva, synovial fluid, etc.), plays an important role in cell proliferation and migration, and is produced by certain bacteria (e.g., Streptococcus spp.). Streptococcus Hyaluronic acid is produced. An average human body weighing 70 kg contains about 15 grams of hyaluronic acid, one-third of which is converted (broken down or synthesized) every day.

[0013] One of the important functions of hyaluronic acid in connective tissue is water binding. Therefore, the intercellular matrix becomes a jelly-like matrix supporting cells. The bound water and the resulting swelling determine the biological role of hyaluronic acid in regulating tissue permeability. Hyaluronic acid is a major structure-forming glycosaminoglycan because it has the ability to condense other glycosaminoglycans around itself and form proteoglycan polymers, which are more hydrophilic and elastic than free proteoglycans. Hyaluronic acid creates a “buffer volume” by binding collagen fibers, other proteins, and components of the intercellular matrix and even cells into a single system. This buffer volume determines the strength and elasticity of mechanical tissues and helps them overcome temporary effects (NN Sigaeva, SV Kolesov, PV Nazarov, RR Vildanova. Chemical modification of hyaluronic acid and application thereof in medicine / / Bulletin of the Bashkir University. 2012. Vol. 17. No. 3. pp.1220-1241).

[0014] In the context of this invention, the term "high molecular weight medical-grade polyvinylpyrrolidone" (PVP) is characterized as a binder. High molecular weight PVP exists as a pharmaceutical-grade "pharmaceutical-pure" powder, a white or slightly yellowish-white loose powder of varying particle sizes with a characteristic odor. The average molecular weight of PVP is 1,000,000-1,500,000 Da [Volker Buhler. Collidon®. Polyvinylpyrrolidone for the pharmaceutical industry / Translated from English, edited by Dr. KV Alekseev. BASF: 2001. - 310 p.]. Aqueous solutions of PVP are sterile, viscous, transparent, slightly colored liquids with a faint characteristic odor.

[0015] Although a wide variety of materials are available for immobilizing drug substances, biodegradable carriers based on high molecular weight polyphenols (PVPs) have attracted the most attention because they allow antibacterial substances to persist at the wound site for extended periods and can be used for both local prevention of metal-bonded bone grafts and treatment of surgical infections. In this context, the molecular weight of PVP is crucial. Modification of bioactive substances with high molecular weight polymers can directionally alter their properties: reducing toxicity, improving solubility, pharmacokinetics, and bioavailability due to complexation [Afinogenov GE, Panarin EF Antimicrobial polymers, 1993. «Hippocrates» Publishing House, St. Petersburg, 260 p.]. The primary function of soluble high molecular weight PVPs is to prolong the duration of drug action and improve bioavailability.

[0016] The authors found that high molecular weight PVP, with its adsorption and antitoxic properties, served as a polymer carrier, providing a lasting effect on the active substances in the composition.

[0017] In the context of this invention, the term "silver povidone" refers to a highly dispersed metallic silver stabilized with low molecular weight medical-grade polyvinylpyrrolidone; it is a bright greenish-gray to greenish-brown powder. In medical practice, silver povidone is used as an aqueous solution for external application, which requires preparation prior to use.

[0018] Silver povidone is a broad-spectrum antimicrobial agent active against both aerobic and anaerobic microbial communities, including antibiotic-resistant microorganisms. At concentrations up to 100 μg / ml, it inhibits the growth of most bacteria (Staphylococci, Streptococci, Pseudomonas aeruginosa, Escherichia coli, Proteus, Shigella, Salmonella, etc.).

[0019] The drug at a concentration of 1-3% has anti-inflammatory effects and can promote the repair process of wound epithelialization; it has low toxicity, does not irritate the skin and mucous membranes, and will not cause allergic reactions or dermatitis. Poviargol is used as an antimicrobial agent for the prevention and treatment of purulent-septic complications of wounds, ulcers, burns, and pressure sores, including prolonged non-healing during antibiotic treatment; for the prevention and treatment of infections of the upper respiratory tract, ear, throat, nose, eye, and oral cavity; and for the prevention and treatment of diseases of the genitourinary and musculoskeletal systems [http: / / sktb-technolog.ru / poviargolum2; “Poviargol: a newbactericidal agent for the treatment of infected wounds. (Experience of clinical use in traumatology, purulent surgery, burn therapy, gynecology, urology, and ophthalmology). A reference manual for doctors / Edited by RASmember-corr. Panarina EF, MD, Professor. Blagitko EM Novosibirsk: Publishing House. 1998. - 66 p.»].

[0020] In the context of this invention, the term "sodium hypochlorite" is characterized as an effective preservative. Its applications include the disinfection of laboratory equipment, water disinfection, and topical wound treatment. Sodium hypochlorite exerts its biological activity through reactions with nucleic acids and amino acids. In its reaction with nucleic acids (particularly DNA), it chlorinates bases, preventing the formation of hydrogen bonds, which induces molecular denaturation and leads to loss of biological activity.

[0021] In the context of this invention, the term "lysozyme" refers to an enzyme in the innate immune system of a wide variety of organisms, ranging from prokaryotes to higher eukaryotes. It is a 1,4-bDN-acetylmuramidase, and also possesses chitinase activity, capable of hydrolyzing the glycosidic bonds of bacterial peptidoglycans, activating autolysins, and exhibiting antiviral activity, including against HIV. Furthermore, this protein has relatively low toxicity, and even at doses exceeding those sufficient to achieve bactericidal effects, no significant effects on the patient's body have been observed.

[0022] In the context of this invention, the term "short bacitracin C" is characterized as an organically derived molecule and as a molecule derived from Bacillus brevis (…). Bacillus brevis This is a natural protective mechanism of bacteria, which initiates the production of this substance in response to threats to their life activities. The antibiotic has a polypeptide structure and exhibits both antibacterial (inhibiting bacterial growth) and bactericidal (destroying bacteria) effects. The mechanism of action of bacitracin C is associated with increasing the permeability of the cytoplasmic membrane of microbial cells (which disrupts their stability and leads to cell death). Bacitracin C has a cyclic amino acid sequence, a structure that prevents microorganisms from developing resistance to this antibiotic. Currently, no drug-dependent resistance to this drug has been described in existing literature, which is important for effective treatment.

[0023] In the context of this invention, the term "gelofuzin" describes a 4% succinylated gelatin solution (also known as modified liquid gelatin) for intravenous administration, having an average molecular weight of 23,200 Daltons. It has a colloidal osmotic pressure of 34 mm Hg and an isoelectric point of pH 4.5. Succinylation imparts a negative charge to the molecules, leading to an increase in molecular size, resulting in the formation of protein chains that are larger than unsuccinylated protein chains while maintaining their molecular weight. Therefore, succinylated gelatin provides a sufficient volemic effect that lasts for 3-4 hours. Succinylated gelatin is used as a substitute for colloidal plasma. Attached Figure Description

[0024] Figure 1 The kinetics of the interaction between viral RNA and sodium hypochlorite are shown.

[0025] Figure 2 The following is an illustration of sodium hypochlorite (S) hyp. = 5 10 -5 In the presence of M (or 3.75 mg / l), poly-AU (C = 0.98 mg / l) 10 -5 M bp The change of absorption spectrum over time.

[0026] Figure 3 The absorption spectrum of the pAU-sodium hypochlorite complex in the presence of silver povidone at λ=260 nm is shown as a function of time.

[0027] Figure 4 The absorption spectrum of the sodium hypochlorite-povidone silver mixture at λ=260 nm is shown.

[0028] Figure 5 A control group of human lung adenocarcinoma cell culture (ATCC® CRL-5800) is shown, magnified 600 times.

[0029] Figure 6 A control, magnified 900x, shows the cytotoxic effect of OC43 coronavirus on human lung adenocarcinoma cell culture (ATCC® CRL-5800).

[0030] Figure 7 Morphological images of human lung adenocarcinoma cell culture (ATCC® CRL-5800) in the presence of a polymer composition of OC43 coronavirus and three polymers are shown, magnified 600 times.

[0031] Figure 8 The absorption spectrum of the DNA-hypochlorite complex is shown, C DNA = 2.2 x 10 -5 M bp (1 - 1 min.a / a, 2 - 5 min.a / a, 3 - 15 min.a / a, 4 - 1 day a / a). a) С NaClO = 1.46 x 10 -4 M;b)С NaClO = 3.5 x 10 -4 M;c) C NaClO = 8.0 x 10 -4 M.

[0032] Figure 9 A: Reaction kinetics of the ternary Hyp-DNA-Pov system; B: Time derivative of absorbance of the ternary DNA-Hyp-Pov system (black square) and DNA-Hyp (red circle) as a function of reaction time.

[0033] Figure 10 The relationship between the bactericidal effect of the intranasal composition and its exposure time to various microorganisms was shown.

[0034] Figure 11 The bactericidal activity of the intranasal composition and its antimicrobial components was demonstrated.

[0035] Figure 12 Visualization of the intranasal composition on the nasal mucosa 8 hours after application is shown.

[0036] Figure 13 The study showed that the prototype-based intranasal composition was not visualized on the nasal mucosa 8 hours after application.

[0037] Figure 14 (A) shows the 0.5 MF Staphylococcus aureus control.

[0038] Figure 14 (B) shows a 0.5 MF Staphylococcus aureus experimental sample.

[0039] Figure 14 (C) shows the prototype of 0.5 MF Staphylococcus aureus.

[0040] Figure 15 (A) shows the 0.5 MF Pseudomonas aeruginosa control.

[0041] Figure 15 (B) shows a 0.5 MF Pseudomonas aeruginosa experimental sample.

[0042] Figure 15 (C) shows the 0.5 MF Pseudomonas aeruginosa prototype.

[0043] Figure 16 (A) shows the 0.5 MF Acinetobacter baumannii control.

[0044] Figure 16 (B) shows a 0.5 MF Acinetobacter baumannii experimental sample.

[0045] Figure 16 (C) shows the prototype of Acinetobacter baumannii at 0.5 MF.

[0046] Figure 17 (A) shows the 0.5 MF Klebsiella pneumoniae control.

[0047] Figure 17 (B) shows a 0.5 MF Klebsiella pneumoniae experimental sample.

[0048] Figure 17 (C) shows the prototype of 0.5 MF Klebsiella pneumoniae. Detailed Implementation

[0049] The composition claimed in this invention is intended for use intranasally to prevent and / or treat, or as part of a comprehensive therapy, nasal infectious and inflammatory diseases. It possesses durable antimicrobial activity against human coronaviruses, influenza viruses, other RNA and DNA-containing viruses and bacteria, and is capable of preventing the formation of microbial biofilms and disrupting existing microbial biofilms. It comprises the following components in the following ratios (wt.%): - High molecular weight hyaluronic acid - 0.1-2.0; - High molecular weight PVP - 4.0-6.0; - Povidone Silver - 0.8-1.2; - Antibacterial peptides (bacitracin C - 0.03-0.15, or lysozyme - 0.1-18.0); - Sodium hypochlorite solution with an available chlorine content (AH) of 1566 mg / L - 49.0-51.0; - Succinyl gelatin up to 100.0.

[0050] The amount of high molecular weight hyaluronic acid can also be 0.5-2 wt.%, 0.5-1 wt.%, or 1-2 wt.%.

[0051] The amount of high molecular weight PVP can also be 4.5-5.5 wt.%.

[0052] The amount of silver povidone can also be 0.8-1 wt.%.

[0053] The amount of lysozyme can also be 1-16.0 wt.%.

[0054] The claimed composition, when applied to the nasal mucosa, can be used for prevention only or as part of a comprehensive treatment of nasal infectious and inflammatory diseases caused by coronaviruses, influenza viruses, other viruses containing RNA and DNA, and bacteria.

[0055] To obtain the composition, all powdered components (hyaluronic acid, polyvinylpyrrolidone, silver povidone, and bacitracin C (or lysozyme)) were dissolved sequentially in sodium hypochlorite solution at the above-mentioned quantitative ratios. Then, succinyl gelatin solution was added to 100.0 wt.%, and the mixture was stirred until a homogeneous product was obtained.

[0056] The claimed composition, prepared based on succinyl gelatin, high molecular weight hyaluronic acid, and polyvinylpyrrolidone, has a lasting effect due to the formation of a protective barrier film on the surface of the nasal mucosa. This protective barrier film facilitates the long-term antimicrobial activity of silver povidone and sodium hypochlorite, keeps the nasal passages moist, and thus reduces the risk of nasal mucosal irritation, dryness, pain, and bleeding.

[0057] The component ratios of the claimed composition are the result of extensive experimental studies and have been optimized to achieve the aforementioned results. Furthermore, a significant advantage of the claimed composition is its sustained antimicrobial activity against a variety of RNA and DNA-containing viruses, bacteria, and microbial biofilms, without any side effects.

[0058] The claimed composition is characterized by the ability to form a thin polymeric barrier film on the nasal mucosa with a single application, which efficiently adsorbs viral and bacterial particles within one hour. Furthermore, the presence of silver povidone during the initial 50 seconds accelerates the denaturing effect of sodium hypochlorite on viral RNA or viral and bacterial DNA.

[0059] The following experimental data are provided as non-limiting examples.

[0060] Example 1 The antiviral activity of the proposed intranasal composition against RNA-containing viruses was investigated. The intranasal composition comprises the following components (wt.%): Sodium hypochlorite solution (AH 1566 mg / L) - 49.0 Hyaluronic acid - 1.0 PVP - 5.0 Povidone Silver - 1.0 Short-chain bacitracin C - 0.03 Succinyl gelatin - 43.97.

[0061] The following virus strains have been used: - Human coronavirus, OC43 strain - Influenza A virus / PuertoRico / 8 / 34 (H1N1) virus.

[0062] To analyze antiviral activity, 1 ml of the proposed intranasal composition sample was mixed with 0.1 ml of virus-containing fluid (initial titer of 6.5 g for OC43 coronavirus and 7.5 g for influenza virus) in a 1.5 ml vial. 0.1 ml of cell culture medium was added to the control vial instead of the composition. The vials were incubated at room temperature for 3 minutes, after which the infectivity of the corresponding virus in the virus-containing fluid was measured. Triple analyses were performed in each study group.

[0063] Table 1. Activity of RNA viruses during incubation with intranasal composition samples

[0064] Note: The similarity score is P<0.05.

[0065] As the data above show, the proposed intranasal composition may reduce the titers of influenza virus and coronavirus OC43 in cell cultures.

[0066] During a 3-minute exposure period, the efficacy against RNA viruses was at least 99.9%.

[0067] Example 2 The antiviral activity of the proposed intranasal composition against RNA-containing viruses was investigated. The intranasal composition comprises the following components (wt.%): Sodium hypochlorite solution (AH 1566 mg / L) - 49.0 Hyaluronic acid - 1.0 PVP - 5.0 Povidone Silver - 1.0 Lysozyme - 16.0 Succinyl gelatin - 28.0.

[0068] The following virus strains have been used: - Human coronavirus, OC43 strain - Influenza A virus / PuertoRico / 8 / 34 (H1N1) virus.

[0069] To analyze antiviral activity, 1 ml of the proposed intranasal composition sample was mixed with 0.1 ml of virus-containing fluid (initial titer of 4.7 g for OC43 coronavirus and 5.5 g for influenza virus) in a 1.5 ml vial. 0.1 ml of cell culture medium was added to the control vial instead of the composition. The vials were incubated at room temperature for 3 minutes, after which the infectivity of the corresponding virus in the virus-containing fluid was measured. Triple analyses were performed in each study group.

[0070] Table 2. Activity of RNA viruses during incubation with intranasal composition samples

[0071] Note: The similarity score is P<0.05.

[0072] As the data above show, the proposed intranasal composition may reduce the titers of influenza virus and coronavirus OC43 in cell cultures. During a 3-minute exposure, the antiviral efficacy against RNA-containing viruses was at least 99%.

[0073] Example 3 To demonstrate that the components of the claimed composition possess enhanced antiviral activity, the antiviral activity of the components of the proposed intranasal composition against RNA-containing viruses (coronaviruses) was investigated at the following concentrations (wt.%): Sample No. 1 Short-chain bacitracin (peptide antibiotic) - 0.03 Sample No. 2 Povidone-silver (silver nanoparticles) - 1.0 Sample No. 3 Short-chain bacitracin (peptide antibiotic) - 0.03 Povidone-silver (silver nanoparticles) - 1.0 Sample No. 4 Lysozyme (antibacterial peptide) - 16.0 Povidone-silver (silver nanoparticles) - 1.0 Sample No. 5 Lysozyme (antibacterial peptide) - 16.0 Short-chain bacitracin (peptide antibiotic) - 0.03 Sample No. 6 Lysozyme (antibacterial peptide) - 16.0.

[0074] The following virus strains have been used: - Human coronavirus, OC43 strain.

[0075] To analyze antiviral activity, 1 ml of each sample was mixed with 0.1 ml of virus-containing liquid (initial titer of coronavirus OC43 was 5.5 g) in a 1.5 ml vial. In the control vial, 0.1 ml of cell culture medium (complete RPMI-1640 medium containing 2 mM L-glutamine, 250 mg / L gentamicin, and 10% bovine embryonic serum) was added instead of the corresponding sample. The vials were incubated at room temperature for 3 minutes, after which the infectivity of the corresponding virus in the virus-containing liquid was measured. Triple analyses were performed in each study group.

[0076] Table 3. Activity of RNA coronaviruses during incubation with samples containing the proposed intranasal composition components

[0077] Note: The similarity score is P<0.05.

[0078] This study demonstrates the antiviral activity of grammacin C for the first time, as there is currently no data on its antiviral effects in the existing literature.

[0079] In this context, the interactions between bacitracin C and povidone silver, as well as lysozyme and povidone silver, were observed to have an additive effect on the OC43 coronavirus strain (a unique characteristic). This interaction provided an advantage for the sustained-action intranasal composition as the initial concentration of the antimicrobial component gradually decreased.

[0080] Example 4 The ability of povidone-silver (silver nanoparticles) to accelerate the destruction of viruses and the synthesis of RNA by sodium hypochlorite was investigated.

[0081] This study used human coronavirus OC43 from the virus collection of the Pasteur Research Institute of Epidemiology and Microbiology in St. Petersburg. The initial viral RNA sample (C=3) was used... 10 -4 M bp Dilute with 0.001 M NaCl until the minimum volume (600 µl) required for measurement in the cuvette of the dedicated spectrophotometer is reached. The viral RNA concentration used in the experiment was C = 1. 10 -5 M bp .

[0082] In all experiments, the concentration (molar concentration) of sodium hypochlorite was 5 times higher than the concentration of nucleotides in the solution, i.e., S hyp. = 5 10 -5 M, or 3.75 mg / l.

[0083] Figure 1 The results show the interaction between viral RNA and different concentrations of sodium hypochlorite.

[0084] The different time-dependent pathways in different spectral regions indicate that sodium hypochlorite interacts with various RNA components present in the solution. Specifically, the 260 nm wavelength region reflects the interaction between sodium hypochlorite and polynucleotide nucleobases.

[0085] A similar experiment was performed using synthetic RNA (poly-AU). The results were in... Figure 2 As shown in the image.

[0086] In the region of λ = 260 nm, the relative changes in absorbance of viral RNA and synthetic RNA, and the time-dependent patterns of their rates (characterized by the state of the nucleotides in the polynucleotides), are similar. This indicates that the interaction between the polynucleotide and hypochlorite involves at least two phases. The first phase proceeds rapidly, manifested as an increase in absorbance intensity, which is typically associated with denaturation or secondary structure disruption of the polynucleotide. Nucleotides also undergo chlorination during this phase. In the second phase, the products of the interactions from the first phase cause a gradual breakdown of the nucleotides.

[0087] The following component concentrations are shown to illustrate the change in the absorption spectrum of the "poly-AU + sodium hypochlorite" complex over time in the presence of silver povidone: C pAU = 7.6 10 -5 M, C NaOCl= 3.8 10 -4 M, C 聚维酮银 = 0.002%. The result is in Figure 3 As shown in the image.

[0088] The nucleotide destruction that occurs within the first 2 minutes of the interaction between sodium hypochlorite and polynucleotides is significantly enhanced in the presence of silver povidone.

[0089] The mechanism of action involves the interaction between sodium hypochlorite and silver povidone. This is demonstrated by the absorption spectrum of the sodium hypochlorite-silver povidone mixture. Figure 4 )show.

[0090] The product of this interaction contributes to the breakdown of nucleotides in RNA.

[0091] This study identifies and describes for the first time the role of sodium hypochlorite in accelerating RNA destruction in the presence of silver povidone. The mechanism involves the interaction between sodium hypochlorite and silver povidone. The product of this interaction facilitates the faster destruction of nucleotides in RNA.

[0092] Example 5 The adsorption activity of polymer combinations with the following composition (wt.%) for RNA-containing viruses was investigated: Sample No. 1 High molecular weight PVP - 5.0 Cell culture medium - up to 100.0.

[0093] Sample No. 2 High molecular weight hyaluronic acid - 1.0 Succinyl gelatin (4% liquid gelatin) - up to 100.0.

[0094] Sample No. 3 High molecular weight hyaluronic acid - 1.0 High molecular weight PVP - 5.0 Succinyl gelatin (4% liquid gelatin) - up to 100.0.

[0095] The following virus strains have been used: - Human coronavirus, OC43 strain - Influenza A virus / PuertoRico / 8 / 34 (H1N1) virus.

[0096] 100 µl of virus-containing liquid (initial titer of 6.5 g for OC43 coronavirus; initial titer of 7.5 g for influenza virus) was added to 1 ml of the corresponding polymer combination, and the resulting mixture was incubated at room temperature for 1 hour. Afterwards, the samples were centrifuged at 3000 rpm for 20 minutes, and the infectivity of the virus was detected in the supernatant. Cell culture medium was used instead of the polymer combination for the control group samples.

[0097] Table 4. Adsorption activity of polymer combinations for RNA-containing viruses

[0098] Note: The similarity score is P<0.05.

[0099] As shown in the data above, during the simultaneous 1-hour pre-incubation period, high molecular weight PVP reduced the titer of the two test viruses by 1 lg; a mixture of high molecular weight hyaluronic acid and gelatin reduced the titer by 2.5 lg; and a mixture of three polymers (high molecular weight PVP based on succinyl gelatin and hyaluronic acid) reduced the titer by 3.5 lg.

[0100] For the first time, polymer compositions have been found to possess superior antiviral activity due to the high adsorption capacity of polymers and their combinations for RNA-containing virus particles. This enables the studied polymer compositions to prevent virus particles from adhering to the nasal mucosa and retain them within the polymer matrix, which is beneficial for the action of antimicrobial agents in antimicrobial intranasal compositions.

[0101] Figure 5-7 Morphological images of cell culture states in the presence of a polymer composition testing OC43 coronavirus and three polymers (high molecular weight PVP based on succinyl gelatin and hyaluronic acid) are shown.

[0102] As from Figure 6 It is evident that OC43 coronavirus has significant cytotoxic effects and can completely disrupt monolayer cell cultures.

[0103] As from Figure 7 It is evident that this polymer composition has a protective effect on monolayer cell cultures, preventing them from being destroyed by the cytotoxic effects of coronaviruses.

[0104] Example 6 The ability of povidone-silver (silver nanoparticles) to accelerate the destruction of microbial plasmid DNA by sodium hypochlorite was investigated.

[0105] E. coli (used) Escherichiacoli The plasmid DNA of strain DH5-α, with a size of 5.5-6 kilonucleotide pairs, was used in the experiment at a concentration of C = 2.2 × 10⁻⁶. -5 Mbp .

[0106] Three hypochlorite-plasmid DNA complexes were prepared for spectroscopic analysis. For each complex, three different solutions with constant DNA concentrations and different concentrations of sodium hypochlorite were prepared: 1) C NaOCl =1.46×10 -4 M; 2) C NaOCl =3.5×10 -4 M; 3) C NaOCl =8.0×10 -4 M.

[0107] The complex was prepared by direct mixing. An aqueous solution of sodium chloride (NaCl) with an ionic strength of 0.015 M was used as the solvent.

[0108] Figure 8 The absorption spectra of complexes of E. coli plasmid DNA with sodium hypochlorite of different concentrations are shown.

[0109] As can be seen from the obtained spectra, regardless of the sodium hypochlorite concentration, the absorbance of the solution increased throughout the DNA absorption region during the first minute after mixing. The total spectrum is close to the sum of the spectra of the DNA and sodium hypochlorite at the used concentrations.

[0110] After 10-15 minutes, significant changes in spectral shape and absorption intensity were observed at a wavelength of 260 nm.

[0111] One day later, the DNA absorption band (λ) 最大 = 260 nm) basically disappears. In C NaClO Concentration = 8.0 x 10 -4 At concentration M, the absorption band of sodium hypochlorite persists for a full day. This indicates that at a given concentration, the amount of preservative used is excessive and remains in the solution after interaction.

[0112] Therefore, it has been confirmed that interaction with sodium hypochlorite leads to the disruption (denaturation) of the secondary structure of microbial DNA and chemical modification of its nuclei, presumably through chlorination. The presence of secondary structure slows down the chemical reaction between sodium hypochlorite and DNA nuclei.

[0113] The povidone-silver solution is prepared by weighing a dry powder. The solvent has an ionic strength of [missing information]. I = 0.001 M NaCl An aqueous solution of salt. The concentration of the prepared solution is further halved to achieve a mixture of equal volumes of reagents. C Pov = 0.5%.

[0114] The substances used in this work exhibit characteristic absorption spectra in the wavelength range of 220 to 500 nm. A Shimadzu 1800 spectrophotometer was used to measure the absorption spectra at optical path lengths of [missing information]. d 1 = 1 cm and d 2 = 0.101 cm The spectra were measured in quartz cuvettes. The spectrophotometer was operated in dual-beam mode. Throughout all measurements, the cuvette position was kept empty, meaning the measurements were performed relative to atmospheric air, whose absorbance did not exceed D within the considered range. 空气 ≤ 0.005. After each measurement, the cuvette containing the solution was rinsed and used to measure the solvent used in the previous measurement. During data processing, the solvent spectrum was subtracted using the Origin software package. The solution was prepared using a laboratory electronic balance (accuracy 0.0002 g) and a dispenser, and stirred using a vortex mixer.

[0115] Derivative analysis of the absorbance at 260 nm wavelength over time ( Figure 9 This allows us to determine that the first stage of the DNA destruction reaction is much faster. This fact is consistent with the fact that, in the presence of sodium hypochlorite, povidone silver (silver nanoparticles) induces denaturation of microbial DNA, making the nucleobases more susceptible to chlorination.

[0116] Therefore, in the initial stages of the interaction, sodium hypochlorite can more quickly destroy microbial DNA molecules in the presence of silver povidone. The reaction products of silver nanoparticles and sodium hypochlorite promote the denaturation of the microbial DNA double helix, which accelerates the chlorination reaction between the nucleobases and sodium hypochlorite.

[0117] Therefore, the proposed intranasal composition destroys viruses and bacteria containing both RNA and DNA.

[0118] Example 7 The relationship between the bactericidal effect of the proposed intranasal composition and exposure time was studied. The intranasal composition comprises the following components (wt.%): Sodium hypochlorite solution (AH 1566 mg / L) - 49.0 Hyaluronic acid - 2.0 PVP - 5.0 Povidone Silver - 1.0 Short-chain bacitracin C - 0.03 Succinyl gelatin - 42.97.

[0119] Bacteria were isolated from patient sites in different departments of a comprehensive medical institution, and the proposed antimicrobial composition was tested using the checkerboard method.

[0120] Figure 10 The results of the assessment of the relationship between bactericidal effect and exposure time are shown.

[0121] After only 1.5 hours of exposure to the intranasal composition, the tested microorganisms completely ceased to grow. It should be noted that, in this case, the preservatives—sodium hypochlorite and silver povidone—which are components of the intranasal composition, are used at a sub-bactericidal dose, which is hundreds or even thousands of times lower than the permissible dose for clinical topical application of a single drug.

[0122] Example 8 The bactericidal activity of the proposed intranasal composition was investigated. The intranasal composition comprises the following components (wt.%): Sodium hypochlorite solution (AH 1566 mg / L) - 49.0 Hyaluronic acid - 1.0 PVP - 5.0 Povidone Silver - 1.0 Lysozyme - 16.0 Succinyl gelatin - 28.0.

[0123] Lysozyme is known to have very weak antibacterial activity against 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 AI, Okulich VK, Zemko V.Yu., Senkovich SA Antimicrobial activity of lysozyme as a factor of nonspecific resistance / / Bulletin of Vitebsk State Medical University. - 2019. - volume 18, No. 4. -pp. 40-45; DOI: [https: / / doi.org / 10.22263 / 2312-4156.2019.4.40].

[0124] This study used clinically multi-resistant (resistant to more than 5 groups of antibiotics, including gentamicin) Staphylococcus epidermidis isolated from patient blood cultures. Staphylococcus epidermidis )8242008.

[0125] The clinical strain Klebsiella pneumoniae 5299639, isolated from a wound on the patient's maxillofacial contour, was used. This isolate is characterized by resistance to eight groups of antimicrobial agents, including carbapenems.

[0126] The proposed intranasal composition was tested using a checkerboard method with an exposure time of 1.5 hours.

[0127] Figure 11 The results of the evaluation of the bactericidal activity of the intranasal composition and its antimicrobial components are shown.

[0128] No antimicrobial activity of lysozyme was observed against Gram-negative Klebsiella pneumoniae; povidone silver reduced microbial contamination levels by 1 g after 1.5 hours of exposure; and the number of Klebsiella pneumoniae decreased by 2 g upon application of the intranasal composition.

[0129] For Gram-positive Staphylococcus epidermidis, in the presence of lysozyme, a 1 g reduction in contamination level was observed after 1.5 hours of exposure; silver povidone reduced the microbial contamination level of Staphylococcus by 2 g; and the number of Staphylococcus epidermidis decreased by 3 g when the intranasal composition was applied.

[0130] Therefore, it has been shown that the bactericidal effect of lysozyme against pathogenic microorganisms is enhanced in the presence of silver povidone.

[0131] Example 9 The lasting effect of intranasal compositions comprising the following components (wt.%) was evaluated: Sodium hypochlorite solution (AH 1566 mg / L) - 49.0 Hyaluronic acid - 1.0 PVP - 5.0 Povidone Silver - 1.0 Short-chain bacitracin C - 0.03 Succinyl gelatin - 43.97.

[0132] This study was conducted in 10 male and female volunteers aged 18 to 83 years. Ground activated charcoal was added to the composition beforehand to improve visualization. The proposed intranasal composition was applied once to both nasal passages of all subjects, who then squeezed their nostrils with their fingers to ensure even distribution of the composition on the mucosal surface. Distribution of the intranasal composition along the nasal mucosa was assessed 8 hours after application under conditions of anterior rhinoscopy using a hardware method (STORZ Karl Storz endoscope camera system (telepack) XLEDTP100 device).

[0133] A continuous and uniform distribution of the intranasal composition was observed on the anterior nasal mucosa in all volunteers across all time periods, indicating that the proposed intranasal composition was consistently present during the 8-hour observation period. Figure 12 ).

[0134] When a similar test was performed using an intranasal composition based on a nasal mucosa prototype, no visual results were observed 8 hours after application. Figure 13 ).

[0135] Example 10 Comparison test of the intranasal composition (hydrogel) with the prototype.

[0136] When the antiviral activity was evaluated using the above method, it was found that during the period of co-incubation of OC43 coronavirus with the prototype ointment for 3 minutes, the viral titer decreased to 1.3 lg TCID50 (the viral titer in the control was 5.3 lg).

[0137] For comparison, Example 1 above showed that the titer decreased to approximately 3 g during co-incubation of OC43 coronavirus with the intranasal composition for 3 minutes (the viral titer in the control was 6.5 g). In Example 2, the titer decreased by 1.5 g during co-incubation of OC43 virus with the intranasal composition containing lysozyme (while the viral titer in the control was 4.7 g).

[0138] Therefore, the proposed hydrogel has higher efficiency compared to the prototype.

[0139] Example 11 Based on the common incubation time (according to the method in Example 7, the time-killing assay was used to evaluate the "time-lethal effect" relationship) and a dose of 10 5 The bactericidal activity of the intranasal composition (hydrogel) and the prototype was evaluated by exposing the CFU / ml test microbial cultures (Staphylococcus aureus (Fig. 14 (AC)), Pseudomonas aeruginosa (Fig. 15 (AB)), Acinetobacter baumannii (Fig. 16 (AB)) and Klebsiella pneumoniae (Fig. 17 (AB))) to the formulation for 1 hour.

[0140] The following results have been obtained.

[0141] The prototype drug did not have bactericidal activity against the test microbial cultures. Microbial growth was observed on the culture dishes (Fig. 14 (C), Fig. 15 (C), Fig. 16 (C), Fig. 17 (C)).

[0142] Furthermore, the intranasal composition according to Example 7 resulted in a 4-5 g reduction in the microbial population against all Gram-positive and Gram-negative microorganisms.

[0143] Therefore, the claimed composition is more effective than the prototype drug in both antiviral and antibacterial activity. Claims (as amended under Article 19 of the Treaty) 1. A nasal composition for the prevention and / or treatment of infectious and inflammatory diseases of the nasal cavity, comprising hyaluronic acid, polyvinylpyrrolidone, silver nanoparticles, antibacterial peptides, sodium hypochlorite solution, and gelatin, having the following component ratios in wt.%: Hyaluronic acid - 0.1-2.0; Polyvinylpyrrolidone - 4.0-6.0; Silver nanoparticles - 0.8-1.2; Antibacterial peptides - 0.03-18.0; Sodium hypochlorite solution - 49.0-51.0; Liquid gelatin - up to 100.0. 2. The composition according to claim 1, wherein the amount of hyaluronic acid is 0.5-2 wt.%. 3. The composition according to claim 1, wherein the amount of hyaluronic acid is 1-2 wt.%. 4. The composition according to claim 1, characterized in that the amount of polyvinylpyrrolidone is 4.5-5.5% wt.%. 5. The composition according to claim 1, wherein the amount of silver nanoparticles is 0.8-1 wt.%. 6. The composition according to claim 1, wherein the antibacterial peptide is bacitracin C or lysozyme. 7. The composition according to claim 6, characterized in that the amount of bacitracin C is 0.03-0.15 wt.%. 8. The composition according to claim 6, wherein the amount of lysozyme is 0.1-18.0 wt.%. 9. The composition according to claim 6, wherein the amount of lysozyme is 1-16.0 wt.%. 10. The composition according to claim 1, wherein the nasal infectious and inflammatory disease is caused by coronavirus, influenza virus, other viruses and bacteria containing RNA and DNA, and is accompanied by the formation of microbial biofilms. 11. The use of the intranasal composition of claim 1 for the prevention and / or treatment of infectious and inflammatory diseases of the nasal cavity caused by coronaviruses, influenza viruses, other viruses and bacteria containing RNA and DNA and accompanied by the formation of microbial biofilms, wherein the composition is applied to the nasal mucosa once daily. 12. The use according to claim 11, characterized in that the composition is used alone or as part of a combination therapy.

Claims

1. An intranasal composition for the prevention and / or treatment of nasal infectious and inflammatory diseases comprising high molecular weight hyaluronic acid, high molecular weight polyvinylpyrrolidone (high molecular weight PVP), povidone silver, antibacterial polypeptide, sodium hypochlorite solution, succinylated gelatin, with the following ratio of components wt.%: High molecular weight hyaluronic acid - 0.1-2.0; High molecular weight PVP - 4.0-6.0; Povidone silver - 0.8-1.2; Antibacterial polypeptide - 0.03-18.0; Sodium hypochlorite solution - 49.0-51.0; Succinylated gelatin - up to 100.

0.

2. The composition of claim 1, wherein, The amount of high molecular weight hyaluronic acid is 0.5-2 wt.%.

3. The composition of claim 1, wherein, The amount of high molecular weight hyaluronic acid is 1-2 wt.%.

4. The composition of claim 1, wherein, The amount of high molecular weight PVP is 4.5-5.5% wt.%.

5. The composition of claim 1, wherein, The amount of povidone silver is 0.8-1 wt.%.

6. The composition of claim 1, wherein, The antibacterial peptide is gramicidin C or lysozyme.

7. The composition of claim 6, wherein, The amount of gramicidin C is 0.03-0.15 wt.%.

8. The composition of claim 6, wherein, The amount of lysozyme is 0.1-18.0 wt.%.

9. The composition of claim 6, wherein, The amount of lysozyme is 1-16.0 wt.%.

10. The composition of claim 1, wherein, The nasal infectious and inflammatory diseases are caused by coronaviruses, influenza viruses, other RNA- and DNA-containing viruses and bacteria, and are accompanied by the formation of microbial biofilms.

11. Use of the intranasal composition according to claim 1 for the prevention and / or treatment of infectious and inflammatory diseases of the nasal cavity caused by coronaviruses, influenza viruses, other RNA- and DNA-containing viruses and bacteria and accompanied by the formation of microbial biofilms, wherein, The composition is applied to the nasal mucosa once a day.

12. Use according to claim 11, characterized in that, The composition is used alone or as part of a combined therapy.

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