RU2283126C1 - Agent for prophylaxis and treatment of viral avian influenza - Google Patents

Abstract

FIELD: veterinary, in particular agents for prophylaxis and treatment of viral avian influenza.

SUBSTANCE: invention relates to application of birch bark extract as agent for prophylaxis and treatment of viral avian influenza.

EFFECT: antiviral agent of increased effectiveness.

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Description

Technical field

The invention relates to the field of veterinary medicine, and more particularly to the search for funds for the prevention and treatment of avian influenza

State of the art

Type A influenza viruses (VH) infect various species of birds and mammals, including humans, horses, pigs, whales, seals, etc. They are characterized by high antigenic heterogeneity of the surface proteins of hemagglutinin (HA) and neuraminidase (NA) and are presented according to the nomenclature of 15 subtypes of HA and 9 NA. The natural hosts of influenza A viruses are birds of the aquatic and near-water complexes. Viruses with all known combinations of surface proteins are isolated from birds. Despite the antigenic heterogeneity of influenza viruses, only four HA subtypes: H1-H3 and H5 and two NA (N1-N2) circulated among people. An outbreak of human influenza in Hong Kong in 1997, caused by the A / H5N1 avian influenza virus, showed for the first time that natural-bird cross-species barriers can be overcome. This was the first case of direct transmission of the virus from birds to humans. Of the 18 hospitalized patients, 6 died; transmission of the virus from person to person was not detected. In 2003-2004, the number of people infected in different countries of Southeast Asia increased to 53, with 43 passing away. The situation is complicated by the possibility of the formation of a new pathogenic reassortant between avian influenza viruses (chickens in this case) and human influenza epidemic viruses.

Currently, no specific prophylaxis and treatment for avian influenza virus is known.

SUMMARY OF THE INVENTION

Lack of specific prophylaxis means, i.e. the impossibility of preparing the vaccine in the traditional way on chicken embryos due to the pathogenicity of the A / H5N1 virus for chickens and chicken embryos, makes the task of searching for substances with activity against influenza A viruses relevant. The authors of the present invention established the antiviral activity of birch bark extract (white part of birch bark ) for avian influenza viruses.

Disclosure of invention

The antiviral activity of birch bark extract was studied in vitro and in vivo using the Birch Bark Extract Company (Russia, Moscow) “Dry Birch Bark Extract” (BES) and used as a raw material in the production of biologically active additives.

I. In vitro studies

1.1. Materials and methods

Viruses were cultured in the allantoic cavity of 10-day-old chicken embryos (CE). Infectious and hemagglutinating activities were determined according to the methods recommended by the World Health Organization.

Cytotoxic effect. MDSK cells were cultured in Costar 96 and 24 well panels in MEM supplemented with 10% fetal calf serum (Gibco), 10 mM glutamine and antibiotics.

A monolayer of MDCK cells was cultured in the presence of a BAS preparation added to the MEM growth medium at concentrations from 1 to 500 μg / ml for 48-72 hours at 37 ° C. Then the cells were washed 2 times with phosphate buffer (PBS), stained with the addition of a neutralroth solution at a concentration of 33 mg / ml. After incubation for 3 hours at 37 ° C, the solution was removed and the neutralrot was extracted from live stained cells with a 50% alcohol solution of Na ₂ PO ₄ . The optical density was determined at a wavelength of 525 nm. The concentration of the drug, inhibiting the value of OD by 50% compared with the cell control, was taken as a 50% cytotoxic dose (CD ₅₀ ).

The antiviral activity of the studied drugs with respect to hepatitis A / H5 was taken into account by reducing the infectious titer of the virus in the MDSC cell culture and lowering the level of optical density suppression (OD ₄₅₀ ) in the test system based on enzyme-linked immunosorbent assay (ELISA) in multi-cycle experiments.

Before infection with the virus, the MDSK cells were washed 2 times with serum-free medium to reduce a possible non-specific reaction. Then, preparations were added at the test concentration in 100 μl of MEM medium and incubated with cells for 1.5 hours. Infection was performed with 10-fold dilutions of viruses in MEM medium supplemented with trypsin (TRSC treated, Sigma) at a concentration of 2 μg / ml. Virus adsorption was carried out for 40 min at 37 ° C. Non-absorbed virus was removed by 3-fold washing with serum-free medium. To the cell monolayer, various concentrations of the studied preparations were added in MEM medium with trypsin at a concentration of 2 μg / ml. Virus and cell controls were cultured in the same medium. Next, the plates were incubated in a CO ₂ thermostat for 72 hours at 37 ° C. Analysis was performed after 72 hours.

When studying the activity of antiviral drugs by ELISA, MDSK cells were grown in 96-well plates. Before infection with the virus, the MDSK cells were washed 2 times with serum-free medium to reduce a possible non-specific reaction. The test preparations were added to the cells at a double concentration in 100 μl of MEM medium. 100 μl of medium was added to the virus control, and 200 to the cell control. After incubation of the cells with the studied drugs for 1.5–2 hours at 37 ° C, 100 μl of the virus in MEM medium was added to the wells, excluding the cell control. The multiplicity of infection was 0.1-0.01 TTsID ₅₀ per cell. All procedures were performed on MEM medium supplemented with trypsin (TRSC treated, Sigma) at a concentration of 2 μg / ml. Next, the plates were incubated in a CO ₂ thermostat for 20 hours at 37 ° C. After incubation, the cells were examined under an inverted microscope to detect the absence of cytotoxic and cytopathic changes in them. The medium was removed and the cells were fixed with 80% acetone in PBS for 15 minutes, dried well and then washed 3 times with phosphate-buffered saline (PBS, PBS with 0.05% TWEEN 20). These and all further washing procedures were performed with the indicated solution. Then, 100 μl of PBS solution with 1% fetal serum and 0.05% TWEEN 20 were added to the cells, incubated at 37 ° C for 30 min. After removal of the solution, 100 μl of monoclonal antibodies (MCA) to the influenza A virus NP protein were added to the cells at a concentration of 10 μg / ml. After incubation with antibodies for 1 hour at 37 ° C, 100 μl of rabbit IgG against mouse IgG labeled with horseradish peroxidase at a dilution of 1: 1000 were added to the wells. After 4-fold washing, ligated peroxidase was detected by adding 100 μl of TMB to the wells (33'55 'Tetramethylbenzidine - substrate buffer). The reaction was taken into account by optical density at 450 nm in a Biocom spectrophotometer. Each dilution of the virus was investigated in 4 repetitions, for which the average value of OD-450 was calculated. The percentage of inhibition was determined as the ratio of OP-450 experience / OP-450 virus control, multiplied by 100%.

1.2. Cytotoxic effect of drugs

When studying the toxic effect of BAS in the cell culture of MDSK, the following results were obtained. By visual assessment of the state of control uninfected cells cultured with the test drug for 72 hours, it was found that 50% of the cytotoxic dose for the standard commercial preparation of remantadine is 60 μg / ml, for BAS also 60 μg / ml.

The results of a study of the cytotoxic effect of drugs by staining cells with neutralrotome showed that remantadine and BAS equally inhibit the value of OP-525 by 50% compared with the cell control at a drug concentration of 62.5 μg / ml. (Table 1)

1.3. The study of the antiviral activity of BAS against influenza viruses A / H5 and A / H7 in a culture of MDS cells depending on the duration of contact with the drug.

The antiviral activity and the action of BAS in relation to HB A / H5 and A / H7 were studied in a culture of MDS cells.

The results of the effect of various concentrations (BAS) on the reproduction of influenza A / H5 and A / H7 are presented in Table 2. (BAS) in various concentrations from 10 to 50 μg / ml has a weaker effect on the reproduction of influenza A / H5 and A / H7 compared with remantadine, which inhibited the replication of the influenza viruses A / duck / Altai / 1285/91 (H5N3) and A / FPV / Rostok / (H7N1) in the MDSC cell culture, as evidenced by the OD ₄₅₀ value. At a concentration of remantadine 5 μg / ml is 65% and 72%, respectively.

The influence of the contact time of the BAS preparation (24 hours, 6 hours, 3 hours and 1 hour) at various concentrations with MDS cells on the reproduction of influenza viruses A / H5 and A / H7 was studied. From the data presented in table 3, it is seen that BAS in various concentrations from 10 to 50 μg / ml had an insignificant effect on the reproduction of influenza viruses A / duck / Altai / 1285/91 (H5N3) and A / FPV / Rostok / (H7N1 ), suppressing the magnitude of the OP-450 by 8-20%.

1.4. The effect of BAS on the reproduction of avian influenza A viruses in chicken embryos.

When studying the effect of BES on the reproduction of various strains of influenza A viruses, 10-day-old chicken embryos infected 0.2 ml of viruses diluted in 10-fold increments (10 ^-1 -10 ^-10 EID / 0.2 ml). The drug was added to each embryo at a concentration of 20-250 μg / embryo or placebo (physiological saline). After incubation at 37 ° C for 48 hours, the presence of the virus in the hemagglutination reaction (RGA) was determined. The virus titer is presented in lg EID ₅₀ / 0.1 ml.

As a result of the studies, it was found (Table 4) that BAS in concentrations of 20-250 μg / ml does not have a significant virus-specific effect on the reproduction of influenza viruses A / duck / Altai / 1285/91 (H5N3), A / ICP / Rostock / 34 (H7N1). The infectious titer of the virus with simultaneous administration of the compound in CE did not decrease. A significant change in the functional properties of the virus, namely the ability to agglutinate red blood cells, was noted. Hemagglutinating activity at a concentration of 50-100 μg / ml decreased by 2-4 log ₂ in comparison with the control. At higher concentrations of BAS (150 and 250 μg / ml), the hemagglutinating activity remained at the level of the virus control, and the infectious titer of the virus increased slightly (by 0.25-0.5 lgEID ₅₀ / 0.1 ml) in the case of the virus (H5N3) )

1.5. The virucidal effect of BAS on avian influenza viruses A: H5 and H7.

Virucidal action, i.e. the binding of the drug to intact viral particles, which prevents the further development of the infectious process in the cell, plays a significant role in the manifestation of the antiviral activity of the drugs.

We studied the virucidal effect of BAS on strains of avian influenza A virus: A / duck / Altai / 1285/91 (H5N3), A / VChP / Rostock / 34 (H7N1). As an additional control, the virucidal effect of remantadine was studied under similar conditions.

Virus-containing liquid in the absence and in the presence of drugs was incubated for 17 hours at 37 ° C, after which the infectious titer was determined by infection with TBE.

The data obtained are presented in table 5. From the presented data it is seen that BAS has a virucidal effect against influenza viruses A / duck / Altai / 1285/91 (H5N3), A / VChP / Rostock / 34 (H7N1). As an additional control, the virucidal effect of the well-known antiviral drug remantadine was studied under similar conditions.

At the concentrations of BAS indicated in the table, in contrast to remantadine, a decrease in the infectious titer of the influenza A / duck / Altai / 1285/91 (H5N3) virus in CE was observed by 1.0-1.75 lg EID ₅₀ / 0.1 ml.

BES, unlike remantadine, exerted a virucidal effect against influenza A virus / HPV / Rostock / 34 (H7N1). The drug at concentrations of 20 and 50 μg / ml reduced the infectious titer of the virus in CE by 0.75-1.75 Ig EID ₅₀ / 0.1 ml in comparison with the viral control.

II. In vivo studies

2.1. Materials and methods

The study of the therapeutic and prophylactic activity of “Dry birch bark extract” (BES) was carried out in in vivo experiments on a model of influenza pneumonia in mice according to the “Methodological guidelines for the study of the antiviral activity of pharmacological substances” (Guskova TA, Nikolaeva IS, Peters V. B. Guidance on the experimental (preclinical) study of new pharmacological substances. Moscow. Remedium, 2000, pp. 274-278).

For research, the influenza virus A / duck / Altai / 1285/91 (H5N3) isolated from wild birds was used. The virus was cultured in the allantoic cavity of 10-day-old chicken embryos (CE). Female outbred mice weighing 16-18 g were used in the experiment. BES was administered per os once a day at doses of 100 and 200 μg / ml in 0.2 ml of 1% starch gel 72, 48, 24, 1 hour before and 24, 48, 72 hours after infection of mice with influenza virus. Mice in the control group (10 pieces) were given a placebo (0.2 ml of 1% starch gel) under the same conditions. A total of 30 mice were used in the experiment.

Infection of mice with the influenza A / duck / Altai / 1285/91 (H5N3) virus was performed intranasally one hour after the last prophylactic administration of BAS at a dose of 1 LD ₇₀ under mild ether anesthesia in a volume of 0.05 ml. The animals were monitored for 14 days after infection, taking into account the death of mice from influenza pneumonia in groups of treated animals and control. For a more detailed study of the antiviral activity of the drug, not a 100% lethal dose of the virus was used, but doses that caused 70% mortality in mice.

The specificity of the death of animals from influenza pneumonia was confirmed by the registration of pathological changes in the lungs of the fallen animals of the experimental and control groups.

The activity of the study drug was evaluated by comparing lethality in animals taking the drug and in the control group. The decrease in mortality of the treated animals in relation to the control was expressed as a percentage. In addition, differences in the average life expectancy of the experimental and control animals were taken into account.

Statistical processing of the results was carried out, determining the reliability of the difference in the average values of the life expectancy of animals according to the Student table.

2.2. results

The results of the study of the therapeutic and prophylactic effect of BES are presented in table 6.

In the control group, the death of mice from influenza pneumonia began on the 3rd day after infection. Killed 70% of the mice. The average life expectancy in the control group was 6.3 days. As a result of the studies, it was found that BAS at a concentration of 200 mg / kg after a single oral administration 72, 48, 24, 1 hour before and 24, 48, 72 after infection has a clear protective effect against experimental influenza infection caused by influenza A virus / H5. The survival rate of infected mice receiving BAS at a concentration of 200 mg / kg, compared with the control increased by 70%.

2.3. Pathological changes in the lungs of infected animals.

The lungs of animals that died from influenza infection in the control are increased in size, red, dense. The specific degree of damage is 3.5 points. In the lungs of mice treated with the drug at a dose of 100 mg / kg, basal lesions were noted and the degree of damage was estimated at 2 points. In the lungs of surviving animals, single small focal affected areas were noted. The use of the drug at a dose of 200 mg / kg according to the therapeutic and prophylactic dose was accompanied by infection of mice with subsequent recovery from the death of mice in the control group.

2.4. Detection of serum antibody titer in surviving mice in an enzyme-linked immunosorbent assay.

The presence of foci in the lungs and the subsequent recovery of mice suggested the development of specific protective antibodies. To clarify this assumption, we determined the titer of antibodies in the blood serum of surviving mice on day 14 after infection in an enzyme-linked immunosorbent assay. Influenza virus A / duck / Altai / 1285/91 (H5N3) was sorbed onto 96-well plates in bicarbonate buffer (KBB, pH 9.2) in a volume of 100 μl. After 24 hours, the plates were washed 3 times with phosphate-buffered saline (PBS, PBS with 0.05% TWEEN 20). These and all further washing procedures were performed with the indicated solution. Then, 100 μl of serum in PBS solution with 1% fetal serum and 0.05% TWEEN 20 in various dilutions were added to the wells. Also, negative and positive controls were added to the wells in a PBS solution with 1% fetal serum and 0.05% TWEEN 20. After incubation with antibodies for 1 hour at 37 ° C, the wells were washed and 100 μl of rabbit IgG against mouse IgG labeled horseradish peroxidase at a dilution of 1: 1000. After 4-fold washing, ligated peroxidase was detected by adding 100 μp TMB to the wells (33'55 'Tetramethylbenzidine - substrate buffer). The reaction was taken into account by optical density at 450 nm in a Biocom spectrophotometer. Each serum dilution was examined in 2 repetitions, for which the average OD-450 value was calculated.

Industrial applicability

Research results indicate that birch bark extract has a prophylactic effect in influenza pneumonia in mice caused by avian influenza A / H5N3 virus using an infectious dose of 1 LD ₇₀ . The protective effect is dependent on the dose of the drug and at a concentration of 200 mg / kg was 70% compared with the control.

Therapeutic and prophylactic administration of the drug contributed to an increase in the life span of mice compared with the control group:

when the concentration of the drug is 100 mg / kg - for 2.3 days;

at a concentration of 200 mg / kg was accompanied by the survival of 70% of the animals.

Treatment-and-prophylactic administration of the drug was accompanied by a significant decrease in the intensity of lung damage in the treated mice (single foci) compared with the control group.

The results obtained indicate the promising use of birch bark extract as an effective tool for the prevention and treatment of avian influenza.

Table 1.
Study of the cytotoxic effect of remantadine in MDCK cell culture Preparations CTD ₅₀ (μg / ml) Visual determination method Neutral Method BES
Remantadine 60
60 62.5
62.5

Table 2.
The influence of BAS on the reproduction of various strains of influenza A virus in birds with a multiplicity of infection of 0.01 EID _{50 /} cell in a cell culture of MDSK. Strain The percentage of inhibition of OP-450 BES (10 μg / ml) BES (20 mcg / ml) BES (50 mcg / ml) Remantadine (10 mcg / ml) A / duck / Altai / 1285/91 (H5N3) 5% 10% 12% 65% A / FPV / Rostok / (H7N1) 8% fifteen% 16% 72%

Table 3.
Influence of BAS on the reproduction of avian influenza viruses A / H5 and A / H7 in MDSC cells depending on the time of drug addition Time The percentage of inhibition of OP-450 A / duck / Altai / 1285/91 (H5N3) A / FPV / Rostok / (H7N1) The concentration of BAS, μg / ml The concentration of BAS, μg / ml fifty twenty 10 fifty twenty 10 24 hours twenty% fifteen% 13% 24% fifteen% 13% 6 o'clock 21% fourteen% 13% 19% 17% 8% 3 hours fifteen% 9% 3% 13% 12% 5% 1 hour 16% eleven% 3.5% fifteen% eleven% 8%

Table 4.
The influence of BAS on the reproduction of influenza A viruses in TBE. Viruses BEJ concentration (μg / embryo) 0 twenty 0 fifty 0 150 0 250 A / duck / Altai / 1285/91 (H5N3) 9.0 9.0 9.0 9.0 9.0 9.25 9.0 9.5 A / VChP / Rostock / (N7N1) - - 7.5 7.5 7.5 7.5 7.5 7.5 The table shows the average results of three experiments.

Table 5.
The virucidal effect of BAS on influenza A viruses in TBE. VIRUSES Virus titer, EID ₅₀ / 0.1 ml Original * After incubation for 17 hours at 37 ° C No drugs + BES (10 μg / ml) + BES (20 mcg / ml) + BES (50 mcg / ml) + rimantadine (20 mcg / ml) A / duck / Altai / 1285/91 (H5N3) 10 ⁴ 10 ^4.25 10 ^3.25 10 ^2.75 10 ^2.5 10 ^4.25 A / VChP / Rostock / 34 (N7N1) 10 ⁴ 10 ^5.25 10 ^4,5 10 ^3,5 10 ^3,5 10 ^5.25 The table shows the average results of three identical experiments.
* - working dilution of the virus.

Table 6
The therapeutic and prophylactic effect of “Birch bark extract dry” on a model of influenza pneumonia in mice caused by the bird flu virus A / H5. Group of animals Dose, mg / ml Number of animals Survival rate,% Life expectancy, days (M + m) BES one hundred 10 40 8.6 + 1.9 BES 200 10 70 11.1 + 2.5 Control (placebo) - 10 thirty 6.3 + 1.1

Table 7
The antibody titer in mice to influenza virus A / duck / Altai / 1285/91 (H5N3) in ELISA. Group of animals IgG BES 100 mg / kg 1: 1600 BES 200 mg / kg 1: 3200 Control (placebo) 1: 3200

Claims (1)

The use of birch bark extract as a means for the prevention and treatment of avian influenza.