RU2523554C1 - Method for body protection against infection caused by influenza a virus subtype h1n1 strains with preparation of human alpha-2 interferon - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: preparation Reaferonum-EC-lipint is administered sublingually or orally in a single dose of 10000 to 30 IU/kg body weight. An infection is prevented by administering the preparation 30 minutes before meals once a day 2 times a week for 1 month in a dose of 20000 IU/kg in adults and children aged 15 years and older and in a dose of 10000 IU/kg in children between 3 to 15 years old. The infection is treated by administering the preparation 30 minutes before meals two times a day daily for 3-5 days in a dose of 30000 IU/kg in adults and children aged 15 years and older and in a dose of 15000 IU/kg in children between 3 to 15 years old.

EFFECT: prevention and effective treatment of influenza caused by virus subtype H1N1 strains in the patients of various age groups with minimal side effects.

4 cl, 2 tbl, 2 ex

Description

The invention relates to medicine, namely to virology, immunology, pharmacology, and relates to a method of protection against infection caused by strains of the H1N1pdm09 subtype of influenza A virus with a drug based on recombinant human alpha-2 interferon in liposomal form, having an antiviral effect.

The problem of the prevention and treatment of influenza caused by strains of the H1N1 subtype is relevant due to the fact that throughout the entire period of monitoring this infection, the H1N1 subtype almost always circulated in the human population. Due to the presence of the NS1 protein, which can function as an interferon antagonist, the influenza virus, when propagated, can inhibit the production of this cytokine [Hale BG, Randall RE, Ortin J, Jackson D: The multifunctional NS1 protein of influenza A viruses. J Gen Virol 2008, 89: 2359-2376]. In this regard, the introduction of endogenous interferon effectively corrects the course of the disease, reducing complications and undesirable consequences of the disease and reducing the severity and risk of death. Thus, diseases caused by new strains of the influenza virus subtype H1N1 with mutations in the NS1 protein that determine resistance to interferons can be corrected by interferon-containing drugs.

In 2009, a pandemic hit the world caused by a new variant of the H1N1 subtype influenza virus. A new variant of the pandemic influenza virus A (H1N1) pdm09 is a triple reassortant of pathogens of classical swine flu (HA, NP, NS genes) of swine flu from the Eurasian line (M gene), bird flu (PB2 and PA genes) and human (PB1).

Due to the fact that earlier strains of the influenza A (H1N1) pdm09 virus did not circulate in the human population, people have no protection against this variant of the influenza virus. The disease is usually severe, and in people at risk can be fatal. According to official statistics for the period of the epidemic (October-December 2009) in Russia, 13.26 million people fell ill with influenza and SARS, which is 5.82 million more than in the same period of 2008, when the flu epidemic was not registered, which is 4.09% of the total population. In a number of regions, the proportion of patients with illness ranged from 6 to 11%. According to official statistics in the Russian Federation in 2009 more than 33.3 million cases of diseases of the acute upper respiratory tract infections of multiple and unspecified localization were registered, in 2010 - more than 28 million cases, in 2011 - more than 30 million cases. The diagnosis of influenza was made in 2009 at 592516 people, in 2010 - at 27346 people, in 2011 - at 309510 people [About the sanitary and epidemiological situation in the Russian Federation in 2009: State report. - M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2010. - 456 p .; On the sanitary and epidemiological situation in the Russian Federation in 2010: State report. - M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2011. - 431 p .; On the state of sanitary-epidemiological well-being of the population in the Russian Federation in 2011: State report. M .: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor, 2012. 316 p.].

In total, in 2009-2010. the number of deaths from pandemic influenza (laboratory-confirmed) in the world amounted to more than 17.4 thousand (according to WHO, ECDC, CDC). The most affected regions are the American (more than 8.1 thousand deaths) and the European (more than 4.6 thousand deaths) [Letter No. 01/5578-10-32 of 04/13/2010 Rospotrebnadzor "On the results of the A / H1N1 influenza pandemic / 09 in the world and the Russian Federation in the epidemic season 2009-2010. and the forecast for the 2010-2011 epidemic season. ”].

A characteristic feature of influenza A (H1N1) pdm09 virus is that even with initially mild symptoms, influenza A (H1N1) pdm09 can very quickly transform into a deadly disease (within 24 hours or less) [Kutsar K. Pandemic influenza (H1N1) 2009 in Europe. EpiNorth. 2010; 11 (3): 73-74]. This feature of the influenza A (H1N1) pdm09 virus was also observed in young people who are practically healthy and are not at risk of a high incidence of SARS. They noted a high risk of morbidity and a severe course of influenza among pregnant women, newborns, and other most vulnerable groups of the population.

Severe influenza A (H1N1) pdm09 is characterized by:

- the formation of acute lung injury syndrome (ARS), respiratory failure, increased dyspnea at rest and with minimal stress, including conversation;

- the development of multiple organ failure (myocarditis, acute renal failure, toxic hepatitis, DIC).

Influenza A (H1N1) pandemic pdm09 2009-2010 and its adverse outcomes were the basis for the analysis and correction of organizational and therapeutic measures aimed at timely diagnosis and treatment of influenza and its complications.

In the schemes of temporary guidelines of the Ministry of Health and Social Development of the Russian Federation dated October 30, 2009, developed for the treatment and prevention of influenza caused by the influenza A (H1N1) pdm09 virus in adults and children, preference is given to domestic medicines, it is recommended to use Arbidol, Kagocel, Interferons, and Oseltamivir and Zanamivir [Letter of the Ministry of Health and Social Development of the Russian Federation No. 24-0 / 10 / 1-4053 of June 30, 2009 “Temporary guidelines“ Treatment and prevention regimens for influenza caused by highly pathogenic type A H1N1 virus for oslyh "]. The range of antiviral drugs for treating influenza A (H1N1) pdm09 is limited because the virus is resistant to the antiviral drugs amantadine and rimantadine.

Osterlund and other authors demonstrated the sensitivity of the influenza A (H1N1) pdm09 virus to interferon [Osterlund P, Pirhonen J, Ikonen N, Ronkko E, Strengell M, Makela SM et al. Pandemic H1N1 2009 influenza A virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons. J Virol. 2010; 84 (3): 1414-22]. Since interferon has an immunomodulating and antiviral effect, the timely administration of dosage forms of interferon helps to eliminate the pathogen, prevents the development of severe infections and the development of complications. The biological effect of interferon is characterized by universality (active against many DNA and RNA-containing viruses), tissue specificity (active only in homologous structures) and aftereffect (viruses are suppressed in the treated cells even after removal of interferon).

According to the results of clinical observations, the presence of complicated premorbid background inhibits the interferon-producing activity of immunocompetent cells of patients with influenza and acute respiratory infections, which often correlates with their reduced antioxidant activity. In the presence of prolonged intoxication, regardless of the absence or presence of complications, a sufficiently low serum IFN-γ content was detected (70, 84, 16 and 71, 45, 52 pg / ml, respectively) [Steel J, Staeheli P, Mubareka S, Garcia-Sastre A, Palese P, Lowen AC. Transmission of pandemic H1N1 influenza virus and impact of prior exposure to seasonal strains or interferon treatment. J Virol 2010; 84 (1): 21-6]. What is a direct indication for interferon therapy.

Known methods of prescribing drugs of interferon inducers. Recommended regimens for use of Kagocel are 72 mg on the first day, 36 mg on the next 3 days in combination with Arbidol [Letter of the Ministry of Health and Social Development of the Russian Federation No. 24-0 / 10 / 1-4053 dated June 30, 2009 “Temporary guidelines“ Treatment and prevention regimens for influenza caused by highly pathogenic H1N1 type A virus for adults ”].

The disadvantage of the described method of treatment using inducers is the delayed action necessary for the production of endogenous interferon and individual differences in the intensity of the interferon response of the body in patients.

Known methods for the appointment of Interferon alfa-2b for the treatment and prevention of influenza caused by the influenza A (H1N1) pdm09 virus in adults and children with varying degrees of severity of the process. According to the recommendations [Lvov D.K., Malyshev N.A., Kolobukhina L.V. et al. Influenza caused by the new pandemic virus A / H1N1 swl: clinic, diagnosis, treatment. Guidelines. M.: Moscow Department of Health, 2009. 18 p.]. Interferon alfa-2b is used to treat mild, moderate and severe forms of the disease, as well as for non-specific prophylaxis according to the scheme: Interferon alfa-2b 1 time per day for 50,000 ME every other day for 10 days.

Known methods for treating pregnant women (starting from the 14th week of pregnancy) with Interferon alfa-2b preparations in suppositories based on the daily dose of 1,000,000 ME (500,000 ME 2 times a day) for 10 days. Then, maintenance therapy of 300,000 ME (150,000 ME 2 times a day) 2 times a week for 3 weeks [Lvov D.K., Burtseva E.I., Schelkanov M.Yu. Distribution of a new pandemic influenza A (H1N1) v virus in Russia // Issues of Virology. 2010.V. 55. Number 3. 4-9].

The main disadvantage of the known treatment and prophylaxis schemes for influenza A (H1N1) pdm09 in adults is the lack of a therapeutic response to monotherapy with interferon alfa and the need for combination with gamma interferon preparations.

It is known to use drugs such as amantadine and remantadine as antiviral prophylactic and therapeutic agents that have low antiviral activity against strains of avian influenza virus [Watts J. Asian nations step up action to curb spread of avian influenza. Lancet, 2004; 363 (9406): 373]. This is due to mutations in the gene encoding the M2 protein that make the virus resistant to this class of compounds [Li KS, Guan Y., Wang J., Smith GJD, Xu KM, Duan L., Rahardjo AP, Puthavathana P., Buranathai C ., Nguyen TD, Estoepangestle ATS, Chaisingh A., Auewarakul P., Long HT, Hanh NTH, Webby RJ, Poon LLM, Chen H., Shortridge KF, Yuen KY, Webster RG, Peiris JSM Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature, 2004; 430 (6996): 209-213], these mutations occur in approximately 30% of cases. A new variant of the influenza A (H1N1) pdm09 virus also contains a mutation of resistance to the action of amantadine series drugs.

A new anti-influenza drug Tamiflu has been developed, which is an inhibitor of the neuraminidase of the influenza A and B viruses, oseltamivir phosphate (oseltamivir), which, when combined with the hydrophobic portion of the active site of the influenza neuraminidase, blocks the ability of the latter to cleave sialic acid residues from the surface of the infected cell, thereby inhibiting the yield her new formed virions. However, data are published in the literature that the influenza virus that caused infection in children treated with oseltamivir may become resistant to oseltamivir [Kiso M., Mitamura K., Sakai-Tagawa Y., Shiraishi K., Kawakami C., Kimura K. , Hayden FG, Sugaya N., Kawaoka Y. Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet 2004; 364 (9436): 759-765], and its further use may be less effective. In addition, there is evidence of the emergence of strains of the influenza A (H1N1) pdm09 virus that are resistant to oseltamivir.

A known method of increasing the body's resistance to viral infections by introducing into the body sublingually granules containing 2000 ME reaferon was used according to the scheme: 5 granules (10000 ME) 1 time per day in the morning 30 minutes before meals for 10 days. The drug is proposed to be used to increase resistance to infection by viruses and other infectious agents, such as influenza viruses, parainfluenza, adenoviruses, herpes simplex, Epstein-Bar, coronoviruses, enteroviruses, rhinoviruses, respiratory syncytial virus, chlamydia, mycoplasma (RF Patent 2191594, A61K 38/21, published on October 27, 2002).

However, this preparation has the following disadvantages: sublingual granules contain recombinant human IFNα2a at a dose of 2000 to 4000 ME / granule, for this reason, as indicated in the patent, even for the prevention of the disease, it is necessary to use up to 5 granules simultaneously and to achieve the effect, take them 1- 3 times a day. There are no data on the effectiveness of sublingual granules containing low doses of recombinant human IFNα2a when used to treat a disease. In addition, the drug exhibits the claimed effect when used together with immunomodulators, ridostin, larifan, half-dan, amiksin, cycloferon, kagocel, neovir, dibazole, chimes and others. In addition, there is no information on the preventive effect of the drug against diseases caused by strains of the H5N1 subtype of influenza virus birds.

The well-known drug Reaferon-EU-Lipint, which is produced by the Russian pharmaceutical company CJSC Vector-Medica on the basis of a license agreement for the transfer of rights (according to RF patent No. 2123328, IPC A61K 38/21, publ. 12/20/1998) with FGUN SSC WB "Vector" Rospotrebnadzor. It is known that this drug is recommended for the prevention of influenza and acute respiratory infections in a dose of 0.5 million ME once a day 2 times a week for 1 month, and in the treatment of influenza and acute respiratory infections, the drug is prescribed at 0.5 million ME daily 2 times a day within 3 days.

There is also known a method for the prevention of disease caused by avian influenza A virus subtype H5N1, including the introduction into the body of the drug alpha-2 human interferon. As a preparation of human alpha-2 interferon, “Reaferon EC-Lipint” is used, obtained by incorporating recombinant alpha-2 interferon into liposomes, which is administered sublingually at a dose of at least 5000-10000 IU / kg of weight once a day after 2 days for 6 days, and then at the same dose once a day after 3-4 days for no more than 3 weeks (RF patent No. 2395295, IPC A61K 38/21, publ. 07.27.2010). For adults and children over 15 years of age, the drug is administered 30 minutes before meals at a dose of 10,000 IU / kg, and for children from 3 to 15 years of age, with the prevention of diseases, the drug is prescribed at a dose of 5,000 IU / kg.

However, to protect the body (prophylaxis and treatment) from infections caused by strains of the H1N1 subtype of the swine flu virus, including the pandemic influenza virus A (H1N1) pdm09, this drug scheme does not give a reliable effect and requires the development of a different scheme for the use of this liposomal drug (Olson F., Hunt C. A. Preparation of liposomes of defined size distribution by extrusion through policarbonate membranes. Biohim. Et Biophys. Acta. 1979, 557: 9-23).

The closest way to protect the body against infection caused by strains of the swine flu virus H1N1 (prototype) is to inhibit the swine flu virus H1N1 (strain A / PR / 8/34 (PR8) virus) using chemically synthesized preparations of oseltamivir (Tamiflu), thiazolides , zanamivir, as well as their use in combination with interferon alpha 2 people (Application US No. 2010330173, IPC A61K 38/21, publ. 30.12.2010).

However, the antiviral properties of the drugs were studied, in particular the strain of avian influenza virus H1N1, only on animal cell cultures Vero or MDCK, which is insufficient to substantiate the antiviral effect of this drug on animals and humans. In addition, back in the period 2004-2006. in a number of patients with avian influenza subtype H5N1 in Southeast Asia, cases of resistance to Tamiflu and Relenza were recorded [Hurt A.C. et al. Susceptibility of highly pathogenic A (H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes // Antivir. Res. - 2007. - V.73. - P.228-231]. In the 2007-2008 season already 24% of human influenza A virus subtypes of the H1N1 subtype isolated in 22 of 30 European countries were resistant to Tamiflu [Ciancio B.C. et al. Oseltamivir-resistant influenza A (H1N1) viruses detected in Europe during season 2007-8 had epidemiologic and clinical characteristics similar to co-circulating susceptible A (H1N1) viruses // Euro Surveill. - 2009. - V.14. - P.19412]. And in the next season of 2008-2009. already 100% of 745 H1N1 influenza A isolates isolated from patients with influenza in Japan were Tamiflu-resistant (> 300-fold decrease in susceptibility to the drug) [Baranovich T. et al. Emergence of H274Y oseltamivir-resistant A (H1N1) influenza viruses in Japan during the 2008-2009 season // J Clin Virol. - 2010 .-- V.47. - P.23-28]. It was found that resistance to Tamiflu is due to a new genetic mutation that leads to the replacement of histidine with tyrosine at the 274th position of the amino acid sequence of viral neuraminidase.

The technical result of the invention is the creation of a cheaper and more effective way to protect the body from infection caused by strains of the H1N1 subtype of influenza A virus, including the pandemic influenza A (H1N1) pdm09 virus, by using a preparation based on recombinant human alpha-2 interferon in liposome form .

The specified technical result is achieved by the fact that in the method of protecting the body against infection caused by H1N1 2009 pdm strains of influenza A virus, comprising administering human alpha-2 interferon to the body, according to the invention, Reaferon EU-Lipint is used as the human alpha-2 interferon preparation "Obtained by the inclusion of recombinant alpha-2 interferon in liposomes, which are administered to the body sublingually or orally in a single dose from 10,000 to 30,000 IU / kg of body weight.

In the prevention of infection, the drug is taken once a day twice a week for 1 month, and in the treatment of infection, the drug is taken 2 times a day after 12 hours daily for 3-5 days, depending on the duration of the disease from the onset of the disease to the start of treatment.

Moreover, the drug is prescribed 30 minutes before meals for adults and children over 15 years of age - for the prevention of diseases at a dose of 20,000 IU / kg, for treatment - at 30,000 IU / kg, and for children from 3 to 15 years old for the prevention of diseases, the drug is prescribed in a dose 10,000 IU / kg and treatment - 15,000 IU / kg.

Characterization of the liposome preparation used in the present method

The drug "Reaferon EC Lipint" is a liposomal form of recombinant alpha-2 interferon in the following quantitative ratio of components, mg / ml:

Phosphatidylcholine 80-100 Cholesterol 12-20

In addition, vitamin E is added to the preparation, and thus, the final composition of the preparation includes interferon alpha-2 human recombinant (250,000, 500,000 or 1,000,000 ME) and excipients: sodium chloride - 8.01 mg, sodium hydrogen phosphate dodecahydrate - 4, 52 mg, sodium dihydrogen phosphate dihydrate - 0.56 mg, lecithin or C100 lipoid - 41.18 mg, cholesterol - 4.53 mg, vitamin E - 0.56 mg, lactose - 91.34 mg

Example 1. A method of protecting (preventing) the body from infection caused by strains of the H1N1 subtype of the virus of "swine" influenza A

The drug Reaferon EU - Lipint is administered orally 30 minutes before meals to children from 3 to 15 years old orally for the prevention of diseases at a dose of 10,000 IU / kg of weight once a day 2 times a week for 1 month.

The drug "Reaferon EU - Lipint" is administered 30 minutes before meals into the body for adults and children over 15 years of age sublingually in the prevention of diseases at a dose of 20,000 IU / kg of weight once a day 2 times a week for 1 month.

Example 2. The method of protection (treatment) of the body from infection caused by strains of the H1N1 subtype of the virus of "swine" influenza A

The drug "Reaferon EU - Lipint" is administered orally to children from 3 to 15 years 30 minutes before meals in the treatment of diseases at a dose of 15,000 IU / kg of weight daily 2 times a day after 12 hours for 5 days.

The drug Reaferon EU - Lipint is administered orally to adults and children over 15 years of age 30 minutes before meals in the treatment of diseases at a dose of 30,000 IU / kg of weight daily 2 times a day after 12 hours for 5 days.

Clinical trials of the drug. A preclinical study of Reaferon-EU-Lipint showed the absence of a toxic, allergenic effect on animals; the drug also did not have an irritating effect on the mucous membranes of the gastrointestinal tract ["Report on the laboratory experimental study of the liposomal form of recombinant alpha-2 interferon" was provided by the Recombinant Drugs Research Department, Head of Ph.D. N.B. Bazhutin, r.p. Koltsovo, 1993]. A study of the absorption of experimental samples showed that liposomal interferon is able to penetrate the intestinal mucosa, enter the blood of experimental animals and circulate in the bloodstream twice as long as free interferon administered intravenously, while completely eliminating from the bloodstream 6 hours after administration. The results of the study allow us to conclude that the drug Reaferon-EU-Lipint is harmless, and therefore it is recommended for clinical trials.

A clinical study “Double-blind, placebo-controlled study of the drug“ Reaferon-EU-Lipint ”liposomal in the complex treatment of patients with influenza and other acute respiratory infections with oral administration” to assess the effectiveness of Reaferon-EU-Lipint in the treatment of acute respiratory viral infections and influenza was conducted on the basis of the Military Medical Academy them. CM. Kirova (St. Petersburg, 2002) [Report on a double-blind, randomized, placebo-controlled clinical trial of Reaferon-EU-Lipint liposome in the complex treatment of patients with influenza and other acute respiratory diseases with the oral route of administration. St. Petersburg, Military Medical Academy]. The number of examined - 80 patients with influenza and acute respiratory infections of moderate severity. It was found that the use of Reaferon-EU-Lipint leads to a statistically significant decrease in the duration of objective signs of general intoxication syndrome, productive cough and a decrease in the frequency of occurrence of cases of the development of complications of influenza and acute respiratory infections (pneumonia, nosebleeds). A significant decrease in the frequency of nosebleeds in a group of patients can be associated with inhibition of the cytopathic effect of influenza viruses on vascular wall cells (antiviral effect).

Clinical studies “A randomized, placebo-controlled study to evaluate the efficacy and safety of the drug“ Reaferon-EU-Lipint lyophilized powder for oral solution (bottles) 500,000 IU / ml ”for the prevention of influenza and other acute respiratory viral diseases in children” was conducted based on the State Research Institute of Influenza RAMS (St. Petersburg, January-February 2005). The study involved 384 healthy children aged 7-15 years who were prescribed Reaferon-EU-Lipint as a prophylactic [Clinical trial report “A randomized, placebo-controlled study to evaluate the efficacy and safety of the drug“ Reaferon-EU-Lipint lyophilized powder for the preparation of an oral solution (vials) of 500,000 IU / ml "for the prevention of influenza and other acute respiratory viral diseases in children." St. Petersburg, Research Institute of Influenza, 2006]. It was found that the prophylactic course helped to reduce the incidence of diseases with influenza (IE = 3.5, p <0.05) and other acute respiratory viral infections among children 7-10 years old compared with the placebo group. The drug is recommended for the prevention of influenza and other acute respiratory viral infections in adults and children.

The rationale for choosing the optimal dose of Reaferon EU Lipint for testing its antiviral activity in mice. When calculating the dose equivalents for mice, the preparations were used using previously published data [Guide to the experimental (preclinical) study of new pharmacological substances / Ed. Khabrieva R.U. M .: Medicine, 2005 .-- 832 p .; Trachtenberg I.M. et al. The Problem of the Norm in Toxicology - 2nd ed. - M .: Medicine, 1991. - 208 p.], Where the use of increased values of such doses in terms of unit mass of small laboratory animals is scientifically justified.

The recommended therapeutic dose of Reaferon EU Lipint for a person is 1 million units. In humans, an average height of 160 cm and an average weight (m) of 60,000 g, the surface of the body (S) is 16,000 cm ^{2, the} coefficient S / m = 0.27. At the same time, in a mouse with a body length of 5 cm and a weight (m) of 15 g, S = 18-20 cm ² , the calculated coefficient S / m = 1.2. Based on the fact that the S / m coefficient in mice is 4 times higher than in humans, we can conclude that the metabolic rate and metabolic rate of the injected substance in mice are 4 times higher than in humans. For this reason, the dose of the drug administered to the mouse per unit weight should be 4 times greater than in humans.

The law of the dependence of metabolism on body weight reflects a tendency to establish a correspondence between heat production and the rate of heat transfer to the surrounding space. The loss of heat per unit mass of the body is the greater, the greater the ratio between the surface and the volume of the body, while the latter ratio decreases with increasing body size. In addition, in small animals, the insulating layer of the body is thinner: the metabolic rate in the mouse per 1 g of body weight is greater than in humans.

Based on the considerations that the optimal dose of Reaferon EU - Lipint for a person with an average body weight of 60 kg is 16,700 units / kg of weight, the optimal dose for a mouse (average body weight of 15 g) will be, taking into account the recommended 4-fold increase in dose , approximately 1000 units / mouse. To test the effectiveness of increased doses of the liposome form of the recombinant alpha-2 human interferon "Lipint" in mice, the indicated dose was increased 3.75 times, i.e. the optimal dose of the drug per mouse was 3750 IU / mouse or 250 IU / g.

Studies of the effectiveness of the drug against strains of influenza A (H1N1) pdm09 virus in experiments in mice

Intranasal infection of mice with influenza virus. Infection of mice with influenza virus was carried out intranasally (and / n) under light ether anesthesia. In this case, using an automatic pipette with a tip, a total dilution of virus-containing fluid (VSL) in a volume of 40 μl was introduced into both nostrils of the mouse. Mice were subjected to cervical dislocation 4 and 5 days after infection, 1% lung homogenates were obtained, and the presence of influenza virus was determined in them. The virus concentration in the VSL and lung homogenates was determined by titration in a culture of MDCK cells and expressed in lgTCD ₅₀ / ml (decimal logarithms of 50% tissue cytopathic doses in ml) according to the Spearman-Kerber method [Sachs L. Statistical evaluation. M .: Statistics, 1976. - 598 p.]. Statistical processing and comparison of data was carried out by generally accepted methods for biological research using a computer program using the Spearman-Kerber method with an assessment of the significance of differences for a 95% confidence level (195) [Sachs L. Statistical estimation. M .: Statistics, 1976. - 598 p.].

Example 1. The use of strains of the pandemic influenza virus A (H1N1) pdm09 and outbred white mice to test the antiviral activity of the drug according to the index of neutralization of the virus. Outbred white mice weighing 15-17 g in the control and in the experiment were infected intranasally with strains of the pandemic influenza virus A (H1N1) pdm09, and were infected with 5 ten-fold dilutions for each of the HB strains in a volume of 40 μl. After 4 days, 50% infectious doses (ID ₅₀ ) of influenza virus strains were determined in lgTCD ₅₀ / head in the combined lung homogenates from 4 mice. Based on the obtained indicators, the neutralization index (IN) of the virus was calculated under the influence of the drug in vivo: IN = ID ₅₀ experience - ID ₅₀ control (lg).

In the experiment, when studying the therapeutic effect, Reaferon-Lipint was administered to mice orally at 250 IU / g in 0.2 ml of distilled water daily 1 time per day or 125 IU / g in 0.2 ml of distilled water daily 2 times a day after 12 hours. 1 hour after infection, the first dose of the drug is administered, and then within 4 days after infection with influenza A (H1N1) pdm09 influenza virus strains. To the control group of mice, distilled water was injected in a similar manner.

The research results are presented in table 1.

Table 1 Therapeutic and prophylactic activity of Reaferon-Lipint in a model of influenza infection caused by strains of influenza A (H1N1) pdm09 virus in mice by neutralization index (IN) Drug for treatment ID ₅₀ strains in lg TCD ₅₀ / goal. (± 195) for mice ID in lg # A / Califomia / 04/2009 (H1N1) v experience 1.8 ± 0.4 * 0.8 * the control 1.0 ± 0.4 A / Califomia / 07/2009 (H1N1) v experience 0.9 ± 0.4 * 0.8 * the control 0.1 ± 0.3 A / Moscow / 225/09 (H1N1) v experience 1.5 ± 0.4 * 0.9 * the control 0.6 ± 0.3 A / Moscow / 226/09 (H1N1) v experience 1.6 ± 0.4 * 1.0 * the control 0.6 ± 0.3 A / Salekhard / 01/2009 (H1N1) v experience 1.2 ± 0.4 @ 0.7 @ the control 0.5 ± 0.3 A / Bishkek / 03/2009 (H1N1) v experience 1.7 ± 0.4 * 1.0 * the control 0.7 ± 0.3 Note:

* - the value of ID ₅₀ with a probability of 95% differs from the control; @ - the value of ID ₅₀ with a probability of 90% differs from the control; # - indicator (in lg) is defined as the difference between ID ₅₀ (in lg TCD ₅₀ / goal) between the experimental and control groups of animals.

The results of the presented studies indicate the presence of a reliable therapeutic and prophylactic effect in the preparation Reaferon-Lipint in infection in mice caused by strains of the influenza A (H1N1) virus pdm09 A / Califomia / 04/2009 (H1N1) v, A / Califomia / 07/2009 ( H1N1) v, A / Moscow / 225/09 (H1N1) v, A / Moscow / 226/09 (H1N1) v and A / Bishkek / 03/2009 (H1N1) v. There was also a positive tendency to the antiviral effect of Reaferon-Lipint upon infection of mice with the influenza virus strain A / Salekhard / 01/2009 (H1N1) v.

Example 2. The use of strains of the pandemic influenza virus A (H1N1) pdm09 and outbred white mice to test the antiviral activity of the drug to suppress the production of the virus in the lungs. Outbred white mice weighing 15-17 g were infected intranasally with strains of the pandemic influenza virus A (H1N1) pdm09 at a dose of 30-50 ID ₅₀ / mouse (50% infectious doses per mouse). The concentration of hepatitis B in the lungs was determined 5 days after infection by titration of the combined lung homogenates from 4 mice in an MDCK cell culture, calculated using the Spearman-Kerber method and expressed in lg TCD ₅₀ / ml (lg 50% tissue cytopathic doses per ml). Based on the difference between virus titers in lung homogenates in mice in the control and in the experiment, the index of inhibition of virus production (PPI) under the influence of the drug in vivo was calculated.

When studying the therapeutic and prophylactic effect, Reaferon-Lipint was administered to mice orally at 250 IU / g in 0.2 ml of distilled water daily 1 time per day or 125 IU / g in 0.2 ml of distilled water daily 2 times a day after 12 hours. 1 hour after infection, the first dose of the drug is administered, and then within 4 days after infection with influenza A (H1N1) pdm09 influenza virus strains. To the control group of mice, distilled water was injected in a similar manner.

The research results are presented in table 2.

table 2 Therapeutic and prophylactic activity of Reaferon-Lipint in a model of influenza infection caused by strains of the influenza A (H1N1) pdm09 virus in mice by the virus production inhibition index (PPI) Drug for treatment Virus titers in mouse lungs lgTCD ₅₀ / ML (± 195) IPPV in lg # A / California / 04/2009 (H1N1) v experience 1.50 ± 0.40 * 2.20 * the control 3.70 ± 0.40 A / California / 07/2009 (H1N1) v experience 2.13 ± 0.72 * 2.17 * the control 4.30 ± 0.43 A / Moscow / 225/09 (H1N1) v experience 2.00 ± 0.40 * 1.90 * the control 3.90 ± 0.40 A / Moscow / 226/09 (H1N1) v experience 1.83 ± 0.72 * 1.67 * the control 3,50 ± 0,40 A / Salekhard / 01/2009 (H1N1) v experience 1.83 ± 0.72 * 2.17 * the control 4.00 ± 0.50 A / Bishkek / 03/2009 (H1N1) v experience 4.75 ± 0.4 * 1.75 * the control 6.50 ± 0.00 Note: * - the value of the titer of the virus in the lungs with a probability of 95% differs from the control; # - indicator (in lg) is defined as the difference between the titers of influenza virus in the lungs (in lgTCD ₅₀ / ml) of the control and experimental groups of animals 5 days after their infection.

The results of the presented studies indicate the presence of a reliable therapeutic and prophylactic effect in the Reaferon-Lipint drug in case of infection in mice caused by all studied strains of the influenza A (H1N1) pdm09 virus, which confirms the effectiveness of the proposed method of protecting (preventing and treating) the body from infection caused by strains subtype of H1N1 virus of the "swine" flu, as well as the achievement of the technical result indicated in the application.

Claims (4)

1. A method of protecting the body from infection caused by strains of the H1N1 subtype of influenza A virus, comprising administering human alpha-2 interferon to the body, characterized in that "Reaferon EC-Lipint" obtained by incorporating recombinant interferon alpha-2 in liposomes, which is administered to the body sublingually or orally in a single dose from 10,000 to 30,000 IU / kg of body weight. 2. The method according to claim 1, characterized in that in the prevention of infection, the drug is taken once a day twice a week for 1 month. 3. The method according to claim 1, characterized in that in the treatment of infection, the drug is taken 2 times a day after 12 hours daily for 3-5 days. 4. The method according to claim 1, characterized in that the drug is prescribed 30 minutes before meals for adults and children over 15 years old - with the prevention of diseases at a dose of 20,000 IU / kg, with treatment - at 30,000 IU / kg, and for children with 3- x up to 15 years in the prevention of diseases, the drug is prescribed at a dose of 10,000 IU / kg and treatment - at 15,000 IU / kg.