WO2022108471A1

WO2022108471A1 - Agent for treating and preventing acute viral respiratory infections

Info

Publication number: WO2022108471A1
Application number: PCT/RU2020/000623
Authority: WO
Inventors: Герман Петрович БЕККЕР; Борис Алексеевич НИКОНОВ
Original assignee: Герман Петрович БЕККЕР
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2022-05-27

Abstract

The invention relates to medicine and concerns a medicinal agent for treating and preventing influenza and other acute viral respiratory infections (AVRI) in nasal form. The technical result of the invention is a preparation with high antiviral activity in the form of a spray or drops, which can be taken intranasally. The preparation is a medicinal and prophylactic agent for protecting against AVRIs. It has been demonstrated using, as an example, influenza viruses A and B that the proposed agent has a highly protective effect in relation to acute viral respiratory infections. The essence of the invention is an agent in the form of a nasal spray or drops, comprising the antiviral oligopeptide Alloferon in combination with stabilizers and antiseptics. The agent is used to treat and prevent acute viral respiratory infections by means of intranasal administration.

Description

MEDICINE FOR THE TREATMENT AND PREVENTION OF ACUTE RESPIRATORY VIRAL INFECTIONS

DESCRIPTION

SUBSTANCE: invention relates to medicine and pharmaceutical industry and concerns an antiviral drug for the prevention and treatment of influenza and other respiratory viral infections.

Acute respiratory viral infections (ARVI) are a group of acute inflammatory respiratory diseases of a viral nature. ARVI includes diseases caused by influenza viruses, rhinoviruses, adenoviruses, parainfluenza viruses, etc.

The proximity of the nature of the viruses that cause ARVI and the clinical symptoms of diseases determines the similarity of therapies used in their treatment. Currently, the number of drugs used in ARVI is very small. These are interferons, interferon inducers, as well as drugs that directly inhibit the functions of viral proteins. So adamantane derivatives (amantadine and rimantadine) act on the M2 protein of the influenza virus, which forms ion channels, oseltamivir (Tamiflu) and zanamivir (Relenza) are neuraminidase inhibitors. The high variability of viruses and the development of resistance have led to the fact that all currently circulating strains of viruses are resistant to the action of adamantane derivatives; also appeared strains resistant to the action of neuraminidase inhibitors - oseltamivir and zanamivir. The formation of resistance to existing drugs indicates the need to develop a new generation of antiviral drugs based on the impact on other targets of viruses that cause ARVI.

Entrance gates for respiratory viruses are epithelial cells of the upper respiratory tract - the nose, trachea, bronchi. In these cells, the virus multiplies and leads them to destruction and death. Therefore, it is advisable to deliver an antiviral drug capable of influencing virus replication directly to these entrance gate. The preparation for administration into the nose can be nasal drops, into the trachea or bronchi by a spray. The inner surface of the nasal cavity is rich in blood vessels; therefore, the intranasal application of drugs is practically bioequivalent to the injection route of administration.

However, there are very few antiviral drugs in nasal form, and most of them contain interferons as an active ingredient. These are Grippferon, Nazoferon and other drugs based on interferon-alpha, Ingaron based on interferon-gamma. In addition to them, there are several nasal immunomodulatory drugs used in the treatment of influenza and SARS. These are Timogen, Imunofan, Taktivin.

A family of peptides with antiviral action is known - Alloferons (Chernysh S.I., Kim Su Ying, Becker G.P., Makhaldiani N., Hoffmann J., Bule F., RF Patent No. 2172322 "Alloferons - immunomodulatory peptides"). The mechanism of action of alloferons consists in the induction of the synthesis of endogenous interferons and the activation of the natural killer system. Peptide Alloferon is registered as a pharmaceutical substance (P N002830/02-240510) and is produced by Russian enterprises. Based on it, an antiviral drug "Allokin-alpha, lyophilisate for preparing a solution for subcutaneous administration, 1 mg" (P N002829/01-210610) was created. The drug has found wide application in clinical practice in the treatment of papillomavirus infections and herpes. The mechanism of antiviral action of Alloferon is universal, which should ensure its effectiveness against influenza viruses and other acute respiratory viral infections. Previously, it was experimentally shown that Allokin-alpha is indeed highly effective against SARS, including bird flu (H5N1). However, the fact that it is used in the form of injections significantly limits its use for such massive infections as SARS. These infections require drugs in forms convenient for use by a wide range of ordinary consumers.

Sprays and drops are liquid dosage forms for repeated use. Since nasal preparations are used repeatedly, air and, of course, microorganisms inevitably get into them during use. Due to this circumstance, special requirements are imposed on drugs for intranasal use. niya. One of these is to ensure their stability, both the chemical stability of the active substance and the stability of the composition in relation to microflora. In order to suppress undesirable effects, stabilizers and preservatives (antiseptics) are introduced into nasal preparations. The former provide the chemical stability of the active components, the latter prevent the reproduction of microorganisms. In addition to stabilizers and antiseptics, they may include substances that increase the permeability of the mucous membrane and substances that increase the viscosity of the system.

Sprays and drops based on synthetic and natural peptides are known. The drug Thymogen, which is based on the peptide alpha-glutamyl-tryptophan, is used intramuscularly, as well as in the form of a spray. The drug is a solution of the peptide in 0.9% sodium chloride solution using benzalkonium chloride as a preservative.

The drug Timogen practically does not contain auxiliary components that could increase its effectiveness when administered intranasally.

A more rational solution and a closer analogue is the composition of the drug Grippferon (patent RU2140285 Gaponyuk P. Ya., Markova E. A., Markov I. A. antiviral agent - nose drops "Grippferon", publ.: 27.10.1999.). Essence of the invention: the antiviral agent contains interferon, a biocompatible polymer and a buffer mixture. As an interferon, it contains genetically engineered interferon, and also additionally contains an antioxidant with the following content of components in 1 ml of the buffer mixture: genetically engineered interferon, ME 1000-500000; biocompatible polymer, g 0.005-0.714; antioxidant, g 0.0001-0.0006. In addition, the agent contains alpha, beta or gamma interferon. According to the invention, the product obtained has a viscosity of (1.1-30.0)»10-3. It contains Trilon B as an antioxidant. It contains polyvinylpyrrolidone and/or polyethylene oxide as a biocompatible polymer. Moreover, polyvinylpyrrolidone and polyethylene oxide are taken in a ratio of 1:1-50. The antiviral agent provides prolonged contact with the nasal mucosa and action at the site of the primary introduction and reproduction of the virus, as well as good absorption. The essence of the prototype is also that it includes substances that increase the bioavailability of the active ingredient for the body, namely: disodium edetate (trilon B) as chemical stabilizer and additives that increase the viscosity of the solution.

The technical problem of the prototype is the lack of therapeutic efficacy of the drug due to the limited permeability of interferons through the nasal mucosa. The low bioavailability of interferons with intranasal administration is due to their large molecular weight.

The objective of the invention is to eliminate these problems and create a drug in the form of a spray or nasal drops, which has a pronounced antiviral activity.

The technical result of the invention is a drug with high antiviral activity in the form of a spray or drops applied intranasally. The drug is a medicinal and prophylactic protection against SARS. Using the example of influenza A and B viruses, it has been proved that the proposed agent has a high protective effect against acute respiratory viral infections (ARVI.

The specified technical result is achieved due to the fact that the declared agent for the treatment and prevention of acute respiratory viral infections in the form of a spray or nasal drops, characterized in that it contains Alloferon oligopeptide at a concentration of at least 0.01%, osmotically active substances, acceptable stabilizers and antiseptics.

It is acceptable that as osmotically active substances polyhydric alcohols are used, selected from the range: 1,2-propylene glycol, glycerin, low molecular weight polyethylene glycols (PEG) at a concentration of up to 5%.

It is acceptable that as an antiseptic it contains 0.02% benzalkonium chloride or phenylethyl alcohol.

It is acceptable that it contains 0.001 - 0.03% disodium edetate as a stabilizer.

It is permissible that 0.05% xylometazoline hydrochloride or its analogue, which provide a vasoconstrictive effect, is additionally introduced into its composition.

A method for preparing the developed agent is also claimed, consisting in the fact that the calculated amounts are successively dissolved in purified water with stirring. properties of benzalcolnium chloride (in the form of a 2% solution), disodium edatate and sodium chloride, after their complete dissolution, an osmotically active substance and Alloferon are introduced, purified water is added to a predetermined value, filtered through a filter with a pore size of 0.4 μm; the resulting solution is packaged in glass vials, dosing pumps (spray) or nozzles (drops) are inserted and sealed.

Implementation of the invention

The essence of the invention lies in the fact that the agent contains Alloferon oligopeptide in combination with excipients.

The solution to the problem and the technical result is a composition containing at least 0.01% of Alloferon oligopeptide and excipients. The composition adopted by us contains up to 5% polyhydric alcohols: 1,2-propylene glycol, glycerin or low molecular weight polyethylene glycols (PEG), which, by increasing the permeability of the mucous membrane, perform a transport function for the active substance. In addition, their use increases the viscosity of the composition, which provides better wetting of the nasal cavity and, accordingly, greater availability of Alloferon. Their bacteriostatic action is also known. As a standard preservative, it is permissible to use benzalkonium chloride or phenylethyl alcohol at a concentration of 0.02%. The function of a chemical stabilizer can be performed by disodium edetate.

Respiratory viral infections are usually accompanied by symptoms of acute inflammatory processes in the nose and nasopharynx. To relieve these symptoms, as a rule, vasoconstrictive substances are used that increase vascular tone and relieve swelling of the mucosa in the affected area. The standard vasoconstrictor components used in respiratory diseases are xylometazoline and its derivatives. Their introduction in the amount of 0.05% allows you to achieve the desired result.

Evaluation of the effectiveness of the proposed agent in relation to SARS was performed on the examples of influenza A and influenza B viruses. The assessment was carried out both in therapeutic - use after infection, and therapeutic and prophylactic - use before and after infection, schemes.

The result obtained is confirmed by the following examples. Example 1 Preparation of Composition 1

To prepare 100 ml of solution, proceed as follows.

In 50 ml of purified water, 1 ml of a 2% solution of benzalkonium chloride, 0.015 g of disodium edetate (trilon B) and 0.90 g of sodium chloride are added with stirring. When the components have been completely dissolved, add 5.0 g of propylene glycol (glycerol, PEG-400), and then add 0.01 g of Alloferon. If necessary, 0.05 g xylometazoline hydrochloride may be administered. Bring the volume of the resulting solution with water to 100 ml and filter through a filter with a pore size of 0.4 μm. The resulting solution is packaged in 10 g glass vials, dosing pumps (spray) or nozzles (drops) are inserted and sealed.

The dynamic viscosity of the compositions at a temperature of 20°C in the case of using 1,2-propylene glycol was 1.05 ± 0.02 cP; for glycerin 1.14 ± 0.03 cP, for PEG-400 - 1.31 ± 0.03 cP. The viscosity of the tested compositions was determined by the capillary method using an Ubbelohde viscometer.

Example 2. Preparation of composition 2.

In 50 ml of purified water, 1 ml of a 2% solution of benzalkonium chloride, 0.015 g of disodium edetate (trilon B) and 0.90 g of sodium chloride are added with stirring. When the components have been completely dissolved, add 5.0 g of propylene glycol, and then add 0.05 g of Alloferon. Bring the volume of the resulting solution with water to 100 ml and filter through a filter with a pore size of 0.4 μm. The resulting solution is packaged in 10 g glass vials, dosing pumps (spray) or nozzles (drops) are inserted and sealed.

Example 3. Induction of type 1 interferons.

The induction of type 1 interferons was determined according to the method described in the work of Ershov F.I., Kiselev O.I. Interferons and their inducers (from molecule to drug). M.: "Izd. Geotar - Media", 2005 - p.356, Chemysh et al., 2002.

The compositions were administered to mice once intranasally in a volume of 0.1 ml, which corresponds to 10 μg for composition 1 and 50 μg for composition 2. As a positive control used cycloferon at a dose of 500 mcg administered intraperitoneally. The control group received 0.1 ml of 0.9% sodium chloride solution intranasally. The results are presented in table 1.

Table 1. Mean titers of type 1 interferons in mouse serum

n/a - did not

The results of the study showed that intranasal administration of the compositions caused a significant increase in the concentration of interferons in the blood serum with maxima 6 and 24 hours after the administration of the drugs. Composition 2 showed a particularly high level of induction of interferons. The maximum level of induction occurs 24 hours after their administration. Composition 1 causes a weaker, however, significant increase in the induction of interferons. Both compositions at a dose of 10 to 50 μg cause an induction of interferons in excess of that caused by cycloferon at a dose of 500 μg.

Conclusion: the proposed compositions for intranasal administration are effective inducers of type 1 interferons.

Example 4. Antiviral activity of the drug on the model of experimental lethal influenza infection in white mice caused by influenza A virus. Therapeutic scheme for the use of the drug. To test the antiviral activity of the drug, a model of a lethal influenza infection was used on white outbred mice of both sexes weighing 10-12 g obtained from the Rappolovo nursery. We used a strain of influenza virus A/Aichi/2/68 (H3N2), adapted to white mice in the laboratory and exhibiting high pathogenicity, causing a fatal infection within 5–10 days, depending on the dose of the virus.

The compositions prepared according to examples 1 and 2 were used in the study. The mice were divided into 2 parts of 5 groups each: The first part was intended for the study with a ten-fold lethal dose of the virus 10 LD ₅₀ , the second - with a single lethal dose of 1 LD _S0 . For the experiment, the virus was administered to animals intranasally under light ether anesthesia.

The test compositions were administered to animals according to the general scheme for two parts: 1/ 0.1 ml of composition 1 (dose 10 μg) intranasally once 30 minutes after infection, 2/ 0.1 ml of composition 2 (dose 50 μg) intranasally once after 30 minutes after infection, 3/ composition 1 four times in 30 minutes and then 1, 2 and 3 days after infection, 4/ positive control - subcutaneously four times the drug "Allokin-alpha" in 0.9% sodium chloride solution at a dose of 25 mcg, 5/ control - intranasally 0.1 ml of 0.9% sodium chloride solution.

Animals were observed for up to 15 days; the period during which 100% lethality of animals is observed in experimental influenza. Based on the data obtained in each group, we calculated the percentage of lethality M (the ratio of the number of animals that died over 15 days to the total number of infected animals in the group) and the protection index IP (the ratio of the difference between the percentages of lethality in the control and experimental groups to the percentage of lethality in the control group).

M=N ₁₅ /NX100%, where N15 is the number of animals in the group that died within 15 days after infection,

N is the total number of animals in the group;

IP = Ms-Me/Msx100%, where Ms and Me are lethality expressed as a percentage in the control and experimental groups, respectively.

The protective effect of the study drug and its comparison with the standard sub- skin form of Alloferon (Allokin-alpha) in lethal influenza infection in mice is presented in Table 2.

Table 2. Protective effect of the developed agent on the model of experimental lethal influenza infection in white mice caused by the influenza A virus when using a treatment regimen using a single (1 LD ₅₀ ) and tenfold (10 LD ₅₀ ) lethal dose, respectively.

Presented in table. 2 data show that the claimed compositions with intra- nasal administration in the treatment regimen, provide a high index of animal protection against the influenza A virus. exceeds it.

Example 5. Antiviral activity of the drug on the model of experimental lethal influenza pneumonia in white mice caused by the influenza A virus.

For testing, a model of a lethal influenza infection was used on outbred mice of both sexes weighing 10–12 g obtained from the Rappolovo nursery. We used a strain of influenza virus A/Aichi/2/68 (H3N2), adapted to white mice in the laboratory and exhibiting high pathogenicity, causing a fatal infection within 5–10 days, depending on the dose of the virus.

The mice were divided into 3 groups - two experimental and one control. For the experiment, the virus was administered to animals intranasally under light ether anesthesia at a dose of 1 LD ₅₀ .

In the experiment, compositions 1 and 2 prepared according to examples 1 and 2 were used. In the experimental groups, the compositions were administered to mice intranasally in an amount of 0.1 ml (doses of 10 and 50 μg, respectively, of the composition) according to the following scheme relative to the date of infection: 1/ day before infection , 2/ 30 minutes before infection, 3/ 30 minutes after infection, 4/ then after 1, 2 and 3 days. Animals were observed for up to 15 days; the period during which 100% lethality of animals is observed in experimental influenza. In the control group, mice were injected intranasally with 0.1 ml of 0.9% sodium chloride solution.

The results of the experiment are presented in table 3.

Table 3. The protective effect of the developed agent on the model of experimental lethal influenza infection in white mice caused by the influenza A virus when using a treatment-and-prophylactic regimen using a single lethal dose of 1 LD ₅₀ . influenza A.

Table data. 3 show that the developed compositions have a high protective effect in the therapeutic and prophylactic scheme of application. Moreover, both compositions show effectiveness.

Conclusion: The proposed compositions based on the Alloferon oligopeptide, when administered intranasally, are highly effective against the influenza A virus in both therapeutic and preventive treatment regimens.

Example 6. Antiviral activity of the drug on the model of experimental lethal influenza infection in white mice caused by influenza B virus. Treatment regimen for the use of the drug.

To test the antiviral activity of the drug, a model of lethal influenza infection was used on white outbred mice of both sexes weighing 10-12 g. Mice were infected intranasally with influenza B virus, strain B/Lee/40, taken at doses of the corresponding 3 and 30 LD ₅₀ . The mice were divided into 6 groups. The study used composition 2, prepared according to example 2, which was administered intranasally in an amount of 0.05 ml at a dose of 25 μg according to the scheme: one day before infection, then after 1, 2, 4, 6 and 8 days. Ribavirin at a dose of 30 μg administered intraperitoneally was used as a positive control. The control group received intranasally 0.9% sodium chloride solution.

The results are presented in table 4.

Table 4. Protective effect of the developed agent on the experimental models a lethal influenza infection in white mice caused by the influenza B virus when using a treatment and prophylactic regimen.

The data in Table 4 show that the developed composition at a dose of 25 μg showed a high protective effect against influenza B, and its protective effect significantly exceeds the protective effect of the standard antiviral drug Ribavirin. In the experiment, Ribavirin caused a pronounced protective (protective) effect only at low doses. virus (3 LD ₅₀ ). The studied composition showed high efficiency, both at low and high viral load.

Conclusion. Using the example of influenza A and B viruses, it was proved that the proposed agent based on the Alloferon oligopeptide, when administered intranasally, has a high protective effect against acute respiratory viral infections, both in therapeutic and therapeutic and prophylactic regimens.

Claims

FORMULA

1. An agent for the treatment and prevention of acute respiratory viral infections in the form of a nasal spray or drops, characterized in that it contains Alloferon oligopeptide at a concentration of at least 0.01%, osmotically active substances, acceptable stabilizers and antiseptics.

2. The agent according to claim 1, characterized in that as osmotically active substances polyhydric alcohols are used, selected from the range: 1,2-propylene glycol, glycerin, low molecular weight polyethylene glycols (PEG) at a concentration of up to 5%.

3. The agent according to claim 1, characterized in that it contains 0.02% benzalkonium chloride or phenylethyl alcohol as an antiseptic.

4. The agent according to claim 1, characterized in that it contains 0.001 - 0.03% disodium edetate as a stabilizer.

5. The agent according to claim 1, characterized in that 0.05% xylometazoline hydrochloride or its analogue, which provide a vasoconstrictor effect, is additionally introduced into its composition.

6. A method for preparing an agent for the treatment and prevention of acute respiratory viral infections in the form of a spray or nasal drops, consisting in the fact that calculated amounts of benzalcolnium chloride (in the form of a 2% solution), disodium edatate (trilon B) are successively dissolved in purified water with stirring and sodium chloride, after their complete dissolution, an osmotically active substance according to claim 2 and Alloferon are introduced, purified water is added to a predetermined value, filtered through a filter with a pore size of 0.4 μm; the resulting solution is packaged in glass bottles, dosing pumps (spray) or nozzles (drops) are inserted and sealed

7. The method according to claim 6, characterized in that simultaneously with the addition of Alloferon, 0.05 g of xylometazoline hydrochloride is administered.

8. The use of the agent according to claim 1 for the treatment and prevention of acute respiratory viral infections by intranasal administration.