RU2398596C2 - Methods of prevention and treatment of diseases caused by a/h5n1 bird influenza virus with using interferon inducer and neuraminidase inhibitor - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. The invention concerns methods of prevention and treatment of virus diseases, particularly caused by the strains subtype H5N1 bird influenza virus, a preparation of interferon inducer exhibiting antiviral action, or preparations of interferon inducer and neuraminidase inhibitor. The method involves introduction of said preparation of interferon inducer or interferon inducer and neuraminidase inhibitor Ozeltamivir. Ridostine is used as a preparation of interferon inducer. The invention can be used in medicine.

EFFECT: ensured higher efficacy, lowered toxicity and good tolerance by using a preparation of interferon inducer based on duplex and single RNA recovered from received from Saccharomyces cerevisiae killer yeast together with neuraminidase inhibitor and development of more optimum regimen.

2 cl, 4 tbl, 4 ex

Description

The invention relates to medicine, namely to virology, immunology, pharmacology, and relates to methods for the prevention and treatment of viral diseases, in particular, caused by strains of the H5N1 subtype of avian influenza virus, an interferon inducer drug having an antiviral effect, or interferon inducer and neuraminidase inhibitor drugs.

The relevance of the development of the proposed method. New epidemic strains of influenza A virus occur every 1-2 years as a result of point mutations in two surface proteins - hemagglutinin (HA) and neuraminidase (NA). In December 2003 and the first quarter of 2004, a new variant of the H5N1 subtype of influenza A virus caused the disease in people in 8 countries in Southeast Asia. During the outbreak in Vietnam and Thailand, 34 people became seriously ill, in more than 60% of cases the disease was fatal. Currently, the disease caused by the H5N1 subtype of the influenza virus has already been registered in 15 countries, and the total number of cases according to WHO as of July 19, 2008 was 385 people, 243 of them (63%) died. Many experts believe that this variant of the influenza virus can become pandemic, and one of the priority tasks of public health at present is the development of effective preventive and therapeutic drugs against the influenza A virus subtype H5N1.

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. - V.363, N 9406. - P.373]. This is due to mutations in the gene encoding the M2 protein that make the virus resistant to this class of compounds [Li K.S., Guan Y., Wang J., et al. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia // Nature. - 2004. - V.430, N 6996. - P.209-213], these mutations occur in about 30% of cases.

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.

Oseltamivir (Tamiflu) is currently considered one of the most effective antiviral drugs. Oseltamivir is characterized by a low ability to select resistant strains of the influenza A virus [Kiso M., K. Mitamura, Sakai-Tagawa Y., et al. Resistant influenza A viruses in children treated with oseltamivir: descriptive study // Lancet. - 2004 - V.364 - N 9436 - P.759-765]. However, the possibility of oseltamivir resistance is not completely ruled out [Menno D. de Jong, Tran Tan Thanh, Truong Huu Khanh et al. Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection // N Engi J Med. - 2005. - N 353. - P.2667-2572], which requires a combination of oseltamivir with other antiviral drugs and / or interferon preparations or their inducers. There are also doubts about the effectiveness of oseltamivir in relation to strains currently circulating [Smee D.F., Wong M.H., Bailey K.W., et al. Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses // Antivir Chem Chemother. - 2006. - V.17, N4. - P.185-192]. All of the above confirms the need for combined treatment of influenza infection, combining drugs of various kinds. In the last decade, the complex treatment of influenza includes immunotropic drugs interferons (IFN) and their inducers (IIFN), which have a combined etiotropic and immunomodulating effect [Kolobukhina L.V. Clinic and treatment of influenza ". // Breast cancer. - 2001. - T.9, No. 16-17. - S.135-136].

A known method of increasing the body's resistance to viral infections by introducing into the body sublingually granules containing an interferon inducer, which was used according to the scheme: 5 granules 1 time per day in the morning 30 minutes before meals for 10 days. As an inducer of interferon, drugs selected from the group: amixin, cycloferon, neovir, ridostin, half-dan, larifan, kagocel, and also from the group: dibazole, papaverine, caffeine, chimes were used. 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, coronaviruses, enteroviruses, rhinoviruses, respiratory syncytial virus, chlamydia, mycoplasma [Patent RF 2191594, A61K 38/21, publ. October 27, 2002].

However, this method only indicates that the content of interferon inducers in sublingual granules is 0.01-5 mg in a granule without a clear dosage recommendation for each drug from the claimed group: amiksin, cycloferon, neovir, ridostin, poludan, larifan, kagocel, and from the group: dibazole, papaverine, caffeine, chimes. This method has the following disadvantages: sublingual granules contain an interferon inducer in small doses, 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 an interferon inducer when used to treat a disease. In this case, the drug exhibits the claimed effect when used together with the drug alpha-2 interferon. In addition, there is no information about the therapeutic effect of the drug in relation to diseases caused by strains of the H5N1 subtype of avian influenza virus.

The known drug Ridostin and its use, which is produced by the Russian pharmaceutical company Dia-Pharm LLC on the basis of a license agreement for the transfer of rights [according to RF Patent No. 2083221, IPC A61K 38/20, publ. July 10, 1997] with the Federal State Institution Scientific Center of the World Bank "Vector" of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare It is known that this drug is recommended for the prevention of influenza and acute respiratory infections, injection at a dose of 8 mg per person 1 time: before the disease or on the day the patient goes to the doctor, and in the treatment of influenza and acute respiratory infections, the drug is prescribed 8 mg per person 2 times: per day the patient’s visit to the doctor and after 2 days.

However, for the prevention and treatment of diseases caused by strains of the H5N1 subtype of avian influenza virus, this drug regimen does not have a reliable effect, especially during treatment, and requires the development of a different scheme for the use of this interferon inducer in combination with other antiviral drugs.

The closest analogue (prototype) is a method for the prevention and treatment of antiviral diseases, for example, bird flu, in particular diseases caused by strain A / Duck / MN / 1525/81, H5N1 [Application US No. 20060035859, IPC A61K 48/00, publ. 02.16.2006]. For prevention and treatment, the drug of the neuraminidase inhibitor oseltavir (Tamiflu) is proposed to be administered orally or sublingually in a dose of up to 100 mu.g / ml together with dsRNA (Ampligen RTM).

However, the antiviral properties of these drugs were studied only on transplantable cultures of Vero or MDCK cells, which is insufficient to substantiate their antiviral effect (in particular, on the strain of avian influenza virus H5N1) when introduced into animals or humans. Moreover, in the prototype, the strain A / Duck / MN / 1525/81 used is not relevant for the current epidemiological situation and does not reflect the resistance of modern strains to chemotherapy drugs, including Tamiflu. In addition, it is known that the dsRNA preparation (Ampligen RTM) is a double-stranded RNA, which is a synthetic polymer consisting of polyriboinosyl and cytidyl chains of various lengths with inclusions of uracil or guanine. It has (see the description of the patent of the Russian Federation No. 2083221) high toxicity and statistically significant mutagenic effect in the form of an increase in the number of chromosomal aberrations by 2-2.5 times in comparison with the control (intact animals).

The technical result of the present invention is the creation of a method for the prevention and treatment of viral diseases caused by modern strains of the H5N1 subtype of avian influenza virus, which has higher efficiency, reduced toxicity and good tolerance, by using an interferon inducer preparation based on double-stranded and single-stranded RNA obtained from Saccharomyces killer yeast cerevisiae, together with an inhibitor of neuraminidase and creating a more optimal scheme for their introduction into the body.

The specified technical result is achieved by the fact that in the method for the prevention and treatment of diseases caused by strains of the H5N1 subtype of avian influenza virus using an interferon inducer and a neuraminidase inhibitor, comprising administering to the body an interferon inducer preparation or together an interferon inducer and an oseltamivir neuraminidase inhibitor according to the invention as the interferon inducer uses the drug Ridostin, which is injected into the body during the prevention of diseases in the dose range from 0.05 to 0.5 m g / kg 1 time per day and then 1 and 3 days after the first injection of Ridostin. In the treatment of diseases, Ridostin is injected into the body in a dose range from 0.05 to 0.5 mg / kg once a day per day of infection and then 1 and 3 days after infection and the neuraminidase inhibitor oseltamivir orally in a dose range from 0.2 up to 2 mg / kg 2 times a day for 5-7 days after infection with the H5N1 influenza virus.

Interferon inducers (IIFN) are more preferable to use due to the shortcomings of IFNov preparations: the need for parenteral use (i / m, s / c or iv) of high doses in the treatment of various diseases. Meanwhile, the parenteral use of IFN drugs (recombinant and natural) in high doses, as a rule, is accompanied by the development of a flu-like syndrome: fever, aches, arthralgia, catarrhal symptoms, headache. In addition, repeated administration of exogenous IFNs leads to the appearance of anti-interferon antibodies that neutralize exogenous interferon. In addition, to date, treatment with IFN drugs remains quite expensive and not all available.

Based on the foregoing, the advantages of IFN inducers aimed at the synthesis of endogenous IFN over exogenous IFN preparations (recombinant natural IFN) are:

- endogenous IFN is produced with the introduction of IFN inducers that do not have antigenicity;

- there are no negative effects inherent in exogenous IFN preparations;

- the synthesis of induced IFN in the body is balanced and undergoes regulatory and control mechanisms (repressor translation), which protect the body from IFN oversaturation;

- a single introduction into the body of the inducer IFN provides a relatively long circulation of endogenous IFN;

- combined with various medications traditionally used in the clinic, providing a synergistic effect when used together [Interferonogens: prospects for clinical use. Manual for Doctors Ed. M.G. Romantseva. - Moscow - St. Petersburg: NTFF "Polisan", 1998].

One of the most effective IIFN is Ridostinum. Ridostin is a high molecular weight inducer of interferon of natural origin, its active principle is double-stranded ribonucleic acid (dsRNA) of killer strains of Saccharomyces cerevisiae yeast. The drug Ridostin is a mixture of sodium salts of double-stranded and single-stranded ribonucleic acid ["Interferon Ridostin Inductor" RF Patent No. 2083221, IPC A61K 38/20, publ. July 10, 1997].

Since the active principle has a physicochemical structure similar to the structure of the genetic material of pathogenic viruses and bacteria, the administration of Ridostin induces a powerful immune response in the body, including interferon induction, stimulation of the phagocytic activity of macrophages and peripheral blood neutrophils, and other processes that counteract infections.

It was shown that with intraperitoneal administration to mice of Ridostin at a dose of 5 mg / kg, an increase in the level of serum interferon begins 3-6 hours later, with a maximum level 8 hours after administration, maintaining an increased up to 24 hours [Masycheva V.I., Ershov F. I., Malinovskaya V.V. The results of clinical testing of the interferon inducer ridostin / B sb. "The use of ridostine for the treatment of viral and bacterial infections and the prospects for its use in diseases of a non-infectious nature." - Berdsk, 1998]. This is especially important, since the infection caused by the H5N1 avian influenza virus is systemic with dissemination of the pathogen to various organs.

Example 1. A method for the prevention of diseases caused by strains of the H5N1 subtype of avian influenza virus

As an inducer of interferon, the drug Ridostin is used, which is injected into the human body in the prevention of diseases in the dose range from 0.05 to 0.5 mg / kg once a day and then 1 and 3 days after the first injection of Ridostin.

Example 2. A method for the treatment of diseases caused by strains of the H5N1 subtype of avian influenza virus

During treatment, Ridostinum is injected into the human body in the form of injections in the dose range from 0.05 to 0.5 mg / kg once a day per day of infection and then 1 and 3 days after infection and the neuraminidase inhibitor oseltamivir orally in the dose range from 0 , 2 to 2 mg / kg 2 times a day for 5-7 days after infection with the H5N1 influenza virus.

Example 3. The study of therapeutic and prophylactic effectiveness of the proposed method in animals

To test the antiviral activity of the preparations, 6-week-old Balb / c mice were used (females weighing 16-18 g) obtained from the vivarium of the FGUN SSC VB Vektor and the influenza virus strain A / chicken / Kurgan / 05/2005 (H5N1) obtained from the Department of Collection of Microorganisms, Federal State Institution of Science and Science, VB Vektor, and passed 1 passage at 10 daily developing chicken embryos (RCE).

Interferon inducer Ridostin was administered intraperitoneally at a dose of 5 mg / kg of mouse weight, and Tamiflu was administered orally at a dose of 5 mg / kg of mouse weight according to a specific scheme for each drug (table 1).

Virus titration was performed on 10-day RCE. The virus concentration in the virus-allantoic fluid (IMPORTANT) was 7.8 lg EID ₅₀ / ml (50% of embryonic infectious doses in ml). The 50% lethal dose (LD ₅₀ ) of the influenza virus for mice, evaluated on outbred mice and expressed as lgEID ₅₀ , was 0.6 lgEID ₅₀ / mouse. Mice were intranasally injected with 100 LD ₅₀ or 10 LD ₅₀ HB in a volume of 40 μl in total in both nostrils.

After infection, animals were monitored for 16 days, the percentage (%) of death and life expectancy (LSS) in the control and experimental groups of animals were evaluated. When determining the average life expectancy (LIF) in each experimental group, the number of animals that lived a certain number of days after infection before death and the number of surviving animals were taken into account. For the maximum value of life expectancy for surviving animals was taken 16 days, i.e. the day after the cessation of the death of infected mice. The protection coefficient (SC) was also determined by the formula:

% death in control -% death in experience.

Statistical processing of results. Statistical data processing was carried out by generally accepted methods for biological research [Ashmarin I.P., Vorobyov A.A. Statistical methods in microbiological research. - Leningrad: “State. Ed. Honey. lit. ", 1962; Lakin G.F. Biometrics. - Moscow: Vysshaya Shkola, 1990], as well as using a package of computer programs for analyzing Microsoft Excel data using the Student t-test and "Statistica 6.0" using the Fisher criterion and Yetz correction for small samples [StatSoft Inc. 1984-2001]. When comparing the indicators between the groups, the probability of error — p and the significance of the difference — P were estimated. The differences were considered significant at p≤0.05, respectively, P≥95%.

Table 1 shows the design of experiments on groups of Balb / c mice with the administration of Ridostin intraperitoneally (ip) and / or Tamiflu orally (p / o) before infection (d / c) or after infection (p / s) with AHP strain A / chicken / Kwgan / 05/2005 (H5N1) at a dose of 100 LD ₅₀ .

When mice were infected with 100 LD ₅₀ VHPs, it was found that a significant advantage in the number of surviving animals compared with the control (0%) was observed in Groups 1, 2, and 5 treated with Ridostin d / z (35.7%), Ridostin and Tamiflu d / s (26.7%), as well as Ridostin and Tamiflu s / s (33.3%) (table 2). In accordance with this, the share (%) of fallen mice in Group 1 was significantly lower than in the Control at p = 0.0170 (with Yetz correction p = 0.0586), in Group 2 - at p = 0.0443 (with correction Yetz p = 0.1417), in Group 5 - at p = 0.0216 (with the Yetz correction p = 0.0715) (table 2).

Table 1 Scheme of animal experiments using Ridostin and / or Tamiflu before and after infection at a dose of 100 LD ₅₀ VGP A / chicken / kurgan / 05/2005 (H5N1) Group No. Preparations The number of mice (female Balb / c) one Ridostin v / b for 4 hours d / s VGP, after 1 and 3 days p / s VG 14 goal 2 Ridostin intravenously for 4 h d / w of VHF, after 1 and 3 days p / s of VH + Tamiflu p / o for 4 h of d / s of VGP, twice a day for 6 days of v / v 15 goal 3 Tamiflu p / o for 4 h d / z VGP, twice a day for 6 days p / z VG 12 goal four Ridostin in / b after 1 h p / s of VGP, after 1 and 3 days p / s of VG 13 goal 5 Ridostinum intravenously after 1 hour p / s of VGP, after 1 and 3 days p / s of VG + Tamiflu p / o after 1 h of p / s of VGP, twice a day for 6 days p / s of VG 15 goal 6 Tamiflu p / o after 1 h p / s VGP, twice a day for 6 days p / s VG 14 goal 7 HCV control without drug administration 13 goal

Nevertheless, it is worth noting that the combined administration of Ridostin and Tamiflu p / s 100 LD ₅₀ AHP in Group 5 contributed to a tendency to increase the survival of mice (33.3%) compared with Group 4 receiving only Ridostin s / s (7 , 7%, p = 0.0991), or with Group 6, who received only Tamiflu s / s (14.3%, p = 0.2310) (table 2).

Table 2 shows the data on the survival in the experimental groups of Balb / c mice treated with Ridostin and / or Tamiflu infected with AH / A / chicken / Kurgan / 05/2005 (H5N1) in a dose of 100 LD ₅₀ .

In terms of life expectancy during infection with 100 LD _{50, the} AHP of the mouse in Groups 1, 2, 3, 5 and 6 was significantly higher than in control Group 7 (table 2). The highest LSS was in Group 5, which received Ridostin and Tamiflu s / s, but it significantly differed only from the values of SJ in the Control and in Group 4, which received only Ridostin s / s (table 2).

Table 3 shows the design of experiments on groups of Balb / c mice with the administration of Ridostin intraperitoneally (ip) and / or Tamiflu orally (p / o) before infection (d / c) or after infection (p / s) with AHP strain A / chicken / Kurgan / 05/2005 (H5N1) at a dose of 10 LD ₅₀

Table 3 Scheme of experiments using a dose of infection of 10 LD ₅₀ VG A / chicken / Kurgan / 05/2005 (H5N1) Group No. Preparations The number of mice (female Balb / c) 1-1 Ridostin v / b for 4 hours d / s AHP, after 1 and 3 days p / s AHP 30 goal 2-1 Ridostinum intravenously for 4 hours d / w AHP, after 1 and 3 days p / s AHP + Tamiflu p / o for 4 hours a / c AHP, twice a day for 6 days p / s AH 30 goal 3-1 Tamiflu p / o for 4 hours d / s AHP, twice a day for 6 days p / s AHP 30 goal 4-1 Ridostin in / b after 1 h p / s AHP, after 1 and 3 days p / s AHP 30 goal 5-1 Ridostinum intravenously after 1 hour p / s AHP, after 1 and 3 days p / s AHP + Tamiflu p / o after 1 h p / s AHP, twice a day for 6 days p / s AHP 30 goal 6-1 Tamiflu p / o after 1 h p / s AHP, twice a day for 6 days p / s AHP 30 goal 7-1 HCV control without drug administration 30 goal

When using 10 LD ₅₀ VGP for infection of mice, it was found that a significant advantage in the number of surviving animals compared with the Control (10%) was observed in mice in Groups 2-1 receiving Ridostin and Tamiflu d / z (33.3%), 3-1, who received only Tamiflu d / z (36.7%), 5-1, who received Ridostin and Tamiflu s / s (63.3%), and 6-1, who received only Tamiflu s / s (33.3 %) (table 4). According to this, the share (%) of fallen mice in Group 2-1 was significantly lower than in the Control at p = 0.0283 (with Yetz correction p = 0.0601), in Group 3-1 - at p = 0.0146 ( with Yetz correction p = 0.0326), in Group 5-1 - at p = 0.0000 (with Yetz correction p = 0.0001), in Group 6-1 - at p = 0.0375 (with Yetz correction p = 0.0791) (table 4).

Moreover, in Group 5-1, the combined administration of Ridostin and Tamiflu p / z 10 LD ₅₀ AHP led to a significant increase in the survival of mice (63.3%) compared with Group 4-1, which received only Ridostin p / s (16.7 %, p = 0.0002, with the Yetz correction p = 0.0006), or with the Group 6-1, who received only Tamiflu s / s (32.1%, p = 0.0175, with the Getz correction p = 0, 0347) (table 4). Table 4 presents the data on the survival in the experimental groups of Balb / c mice treated with Ridostin and / or Tamiflu when infected with the AHP strain A / chicken / Kurgan / 05/2005 (H5N1) at a dose of 10 LD ₅₀ .

Table 4 The results of the experiments described in table 3 No. of group of mice The number of dead mice per day after infection with AHP Number of survivors % of the fallen SPG (days) M ± m 7 8 9 10 eleven 12 13 fourteen fifteen 16 Gr 1-1 Reed d / s (n = 30) 6 8 3 2 one one one 8 73.3 & 10.7 ± 0.7 * # Gr 2-1 Reed + Tami d / s (n = 30) one 10 6 one 2 10 $ $ 66.7 & 11.1 ± 0.7 * # Gr 3-1 Tami d / s (n = 30) 7 7 3 2 11 $ $ 63.3 & 11.6 ± 0.6 * # Gr 4-1 Reed s / s (n = 30) 8 13 2 2 5 83.3 & 9.3 ± 0.6 # Gr 5-1 Reed + Tami s / s (n = 30) one four 2 3 one $ 19 $ 36.7 13.8 ± 0.6 * Gr 6-1 Tami s / s (n = 30) 7 four 3 3 2 one 10 $ $ 66.7 & 11.8 ± 0.6 * # Gr 7-1 Control (n = 30) 10 13 3 one 3 90.0 & 8.6 ± 0.6 # Note: $ - differences from the Control according to the χ ² criterion with a probability of 95%; & - differences from Group 5-1 by the χ ² criterion with a probability of 95%; * - differences from the Control with a probability of at least 95%; # - differences from Group 5-1 with a probability of at least 95%.
Group 1-1 - mice receiving iv Ridostin for 4 hours for infection with 10 LD of ₅₀ AHP, and then after 1 and 3 days a / w of AHP;
Group 2-1 - mice receiving iv Ridostin for 4 h d / z infection with 10 LD ₅₀ VHP, and then after 1 and 3 days s / w VH, and Tamiflu s / o for 4 h d / w infection of VG, and then within 6 days p / s AHP;
Group 3-1 - mice treated with Tamiflu p / o for 4 h d / z infection with 10 LD ₅₀ VGP, and then within 6 days p / z VGP;
Group 4-1 - mice receiving iv Ridostin after 1 hour of infection with 10 LD of ₅₀ AHP, and then after 1 and 3 days of infection with AHP;
Group 5-1 - mice that received Ridostinum intravenously after 1 hour of p / s infection with 10 LD ₅₀ VGP, and then after 1 and 3 days of p / s of VH, and Tamiflu p / o after 1 h of p / s of infection of VG, and then within 6 days p / s AHP;
Group 6-1 - mice treated with Tamiflu p / o after 1 h p / z infection with 10 LD ₅₀ AHP, and then within 6 days p / s AHP;
Group 7-1 - mice that did not receive drugs when infected with 10 LD _{50 of the} virus.

The survival rate at infection of mice with 10 LD ₅₀ AHPs was significantly higher than in the control Group 7-1, only in Groups 1-1, 2-1, 3-1, 5-1 and 6-1 (table 4). The highest life expectancy was in Group 5-1, which received Ridostin and Tamiflu p / s AHP, while it significantly differed from the indicators of life expectancy in all other groups, including Group 4-1, which received only Ridostin p / s AHP, and in Group 6-1, receiving only Tamiflu s / s VGP (table 4). This suggests that the combined administration of Ridostin and Tamiflu p / z AHP has the most favorable effect on the life expectancy and% survival of mice infected with 10 LD ₅₀ AH chicken / chicken / Kurgan / 05/2005 compared with all other administration schemes studied ( table 4).

Moreover, the short-circuit in Group 5-1 with the combined administration of Ridostin and Tamiflu p / s AHP was also the highest and amounted to 53.3%, whereas in Group 4-1, which received only Ridostin p / s AHP, the short-circuit was 6, 7%, and in the Group 6-1, which received only Tamiflu s / s VGP, short-circuit reached no more than 23.3% (table 4).

findings

1. In the control, when mice were infected with 100 LD ₅₀ VHPs, 0% of mice survived; when used for infection with 10 LD ₅₀ VHPs, 10.0% of mice survived.

2. In the group treated with Ridostin and Tamiflu d / z 100 LD ₅₀ AHP, 26.7% of mice survived, while using 10 LD _{50 50} AHP for infection, 33.3% of mice survived, which is significantly higher than the control, but does not differ from the groups receiving only Ridostin d / z or only Tamiflu d / z, and indicates the absence of an increase in the prophylactic effect with the combined use of Ridostin and Tamiflu with influenza.

3. In the group treated with Ridostin and Tamiflu s / s 100 LD ₅₀ AHP, 33.3% of mice survived, while using 10 LD ₅₀ AHP for infection, 63.3% of mice survived, which exceeds the survival rate in the control, as well as in the groups receiving only Ridostinum s / s or only Tamiflu s / s, and indicates an increase in the therapeutic effectiveness of the combined use of Ridostin and Tamiflu with influenza.

Example. 4. Calculations of the ratio of doses of Ridostin and Tamiflu recommended for humans with the introduction of these drugs to mice

Recalculation of the dose ratio of the drug for mouse and human on the basis of the data [Trakhtenberg IM, Sova R.E., Sheftel V.O., Onikienko F.A. The norm problem in toxicology (Modern ideas and methodological approaches, basic parameters and constants) .- Moscow: Medicine, 1991. - 208 p.]:

The dose of the drug administered to the animal is calculated as follows:

D = d × k,

where D is the dose administered to the animal;

d is the dose administered to humans;

k is the ratio of the coefficients of the ratio of the surface of the body and body weight for a person and the studied animal species.

The values of the coefficients:

Person: mass (m _h ) 70,000 g, body surface (S _h ) 18,000 cm ² , S _h / m _h ~ 0.26.

Mouse: mass (m _m ) 20 g, body surface (S _m ) 78 cm ² ; S _m / m _m ~ 3.9.

Coefficient (k) of S / m ratios for mouse and human:

k = 3.9 / 0.26 = 15.

That is, it can be considered that the metabolic rate and metabolic rate of the administered drug in a mouse weighing 18-20 g is approximately 15 times higher than in a person weighing 70-75 kg. Thus, the calculations show that, in any case, a 15-fold excess of the doses of drugs when administered to mice relative to the doses of the same drugs when administered to humans is quite acceptable and adequate to characterize and compare the effects caused by certain drugs in mice and humans.

Ridostin dose ratio calculations

Recommended injection dose of Ridostin for humans: 1 mg per day, 8 mg, two injections with an interval of 2 days. For a person weighing 70-75 kg, a single dose of Ridostin is ~ 0.1 mg / kg. Accordingly, this dose of Ridostin for a mouse weighing 18-20 g can be increased up to ~ 1.5 mg / kg.

In our experiments, Ridostin was used at a dose of 5 mg / kg of mouse weight, which in terms of humans was 3 times the dose recommended in the instructions for use of Ridostin for the treatment of influenza in humans.

Tamiflu dose ratio calculations

The recommended oral dose of Tamiflu for humans: 2 times a day, 75 mg per person for 5 days. For a person weighing 70-75 kg, a single dose of Tamiflu is ~ 1 mg / kg. Accordingly, this dose of Tamiflu for a mouse weighing 18-20 g can be increased up to ~ 15 mg / kg.

In our experiments, Tamiflu was used at a dose of 5 mg / kg of mouse weight, which in terms of humans was 3 times less than the dose recommended in the instructions for use of Tamiflu for the treatment of influenza in humans.

Thus, the claimed method for the prevention and treatment of viral diseases caused by modern strains of the H5N1 subtype of avian influenza virus has higher efficiency, reduced toxicity and good tolerance due to the use of the drug interferon inducer Ridostin in the prevention of diseases and in the treatment of the combined use of Ridostin with a neuraminidase inhibitor Tamiflu and creating a more optimal scheme for their introduction into the body.

Claims (2)

1. A method for the prevention of diseases caused by avian influenza A / H5N1 virus using an interferon inducer, comprising administering an interferon inducer preparation to the body, characterized in that Ridostin is used as an interferon inducer, which is injected into the body in the form of injections in a dose range from 0.05 to 0.5 mg / kg 1 time per day and then 1 and 3 days after the first injection of Ridostin. 2. A method of treating diseases caused by avian influenza A / H5N1 virus using an interferon inducer and a neuraminidase inhibitor, comprising administering an interferon inducer drug and an oseltamivir neuraminidase inhibitor to the body, characterized in that Ridostin is used as an interferon inducer, which is administered to the body in the form of injections in the dose range from 0.05 to 0.5 mg / kg once a day per day of infection and the neuraminidase inhibitor oseltamivir orally in the dose range from 0.2 to 2 mg / kg 2 times per day for 5-7 sous ok after infection with influenza virus A / H5N1.