RU2475531C2 - ANTIVIRAL MEDICATION BASED ON NEMATOPHAGOUS FUNGUS Duddingtonia flagrans - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: anti-viral preparation represents water extract from strain of namatophagous fungus Duddingtonia flagrans F-882, obtained by extraction of fungus biomass with ratio biomass:water 1:5, with further removal of insoluble deposit. Preparation possesses high activity against human immunodeficiency virus of the first type, viruses of human and avian influenza of type A and pox vaccine virus. Index of neutralisation of human influenza A virus in culture of MDCK cells constitutes 3.5 lg, of avian influenza A - 2.5-3.0 lg. Virus-neutralising preparation action with respect to pox vaccine virus no Vero cells constitutes 25-50% of cells. If preparation in concentration 25 mcg/ml is introduced into MT-4 cells simultaneously with virus adsorption inhibition of HIV-1reproduction, or in concentration 12.5 mcg/ml after adsorption in cells constitutes 100%.

EFFECT: increase of activity.

2 cl, 6 tbl, 5 ex

Description

The invention relates to antiviral agents, in particular to inhibitors of reproduction of human viruses and animals, and can be used in medicine, veterinary medicine, virology and pharmacology.

Viruses that infect humans and animals cause serious damage to public health, birds and other animals. Human immunodeficiency viruses and influenza are socially significant and dangerous to human health.

Known strain Duddingtonia flagrans T-89, registered in the Culture Collection of microorganisms of the SSC VB "Vector" under the number F-882 (RF patent 2253671, IPC C12N 1/14, publ. 10.06.2005), exhibiting properties against gall nematodes of plants and parasitic nematodes of animals and stimulating the growth and development of plants, as well as formulations based on its chlamydospores (RF patent No. 2366178, IPC AN 63/04, publ. 09/10/2009). The strain has high rates of growth stimulation, plant development and the manifestation of a nematophage effect. Studies with the predatory fungus strain Duddingtonia flagrans F-882 showed that it is capable of producing nematicides soluble in methyl tert-butyl ether, immobilizing nematodes of the species Steinernema carpocarsae and Panagrellus redivivus.

However, the antiviral activity of the predatory fungus Duddingtonia flagrans, including strain F-882, is not known from known sources of scientific, technical and patent information.

Anderson et al. (Anderson MG, Rickards RW, Lacey E. 1999. Structures of flagranones A, B and C, cyclohexenoxide antibiotics from the nematode-trapping fungus Duddingtonia flagrans. J Antibiot (Tokyo). 52 (11): 1023-1028 ) identified new metabolites in the surface culture of Duddingtonia flagrans - flagronons A, B and C, structurally related to the farnesylated cyclohexenoxides of the oligosporon group (famesylated cyclohexenoxides of the oligosporon group). Flagronons A and B had antibacterial and fungicidal activity similar to the oligosporon group, but did not show significant nematicidal activity. Studies of the antibacterial activity of the deep culture of D.flagrans F-882 strain revealed a pronounced antibiotic activity against Bacillus subtilis and less pronounced inhibition of the growth of Pseudomonas putida, Staphylococcus aureus and Salmonella thyphimurium.

As mentioned above, the authors did not find data on the antiviral activity of nematophage fungi. However, there is evidence of other non-micromycetic fungi, such as the nematophage fungus Duddingtonia flagrans.

When analyzing the antiviral activity of the extract of basidial Ganoderma pfeifferi against type A influenza virus and herpes simplex virus type 1, it was found that the main antiviral component of the extract were triterpenoids: ganodermadiol, lucidodiol, aplanoxic acid G (Mothana RAA, Awadh Ali NA, Jansen R., Wegner U., Mentel R., Lindequist U. Antiviral lanostanoid triterpenes from the fungus Ganoderma preifferi // Fitoterapia. - 74 (2003). - P.177-180).

Substances hispidin and chispolon having an isoprenoid nature and found in the ethanolic extract of the fungus Inonotus hispidus showed antiviral activity against type A and type B influenza viruses. Both fruit extracts and mycelial extracts showed antiviral activity [Ali NAA, Jansen R., Pilgrim H., Liberra K., Lindequist U. Hispolon, a yellow pigment from Inonotus hispidus // Phytochemistry. 1996.- V.41, I.3, P.927-929; Awadh Ali A.A., Mothana R.A.A., Lesnau A., Pilgrim H., Lindequist. Antiviral activity of Inonotus hispidus // Fitoterapia. - 74 (2003). - P.483-485].

American applications for inventions (No.20050238655, IPC A61K 35/84, publ. 10/27/2005 and No. 50,027,615 IPC A61K 35/84, publ. 12/15/2005) describe the activity of a composition of three species of Fomitopsis officinalis fungi, Piptoporus betulinus, Ganoderma resinaceum against smallpox viruses and human immunodeficiency.

It is known to use the extract of the black outer part of the fungus Fuscoporia oblique (Fr.) Aoshima as an active ingredient that inhibits the human immunodeficiency virus (Japanese Application No. 9191891, IPC A61K 35/84, C12P 1/02, published on July 29, 1997). The pointed mushroom grows on Japanese birch wood.

The closest analogue (prototype) is the use of the extract of the mycelial culture of the cells of the fungus Fuscoporia oblique (Fr.) Aoshima grown on an artificial liquid nutrient medium as an active ingredient that inhibits the human immunodeficiency virus (Application US No. 20040105869, IPC A61 35/78, publ. June 3, 2004).

However, this extract has only a prophylactic effect (the extract is added to MT-4 cells and incubated until the virus is adsorbed on the cells) and has a low therapeutic effect (weakly affects the virus when it penetrates into MT-4 cells).

The technical result of the present invention is to obtain an agent having an inhibitory effect against human immunodeficiency virus of the first type, human and avian influenza viruses of type A and vaccinia virus.

The specified technical result is achieved by obtaining an antiviral agent having activity against human immunodeficiency virus of the first type, human and avian influenza viruses of type A and vaccinia virus and representing an aqueous extract from a strain of nematophage fungus Duddingtonia flagrans strain F-882, obtained by extraction of the biomass of the fungus with water in the ratio biomass: water 1: 5, followed by removal of insoluble sediment. The biomass of the nematophagous fungus Duddingtonia flagrans of strain F-882 was obtained by culturing it in a liquid nutrient medium based on molasses and corn extract.

Below are examples 1-2 of obtaining an antiviral agent from the biomass of the nematophage fungus Duddingtonia flagrans.

Example 1. Obtaining sample 10-25 from a nematophage fungus Duddingtonia flagrans

Biomass was obtained by cultivating the fungus in a liquid nutrient medium containing molasses and corn extract. The composition of the medium,%:

Molasses 3.0 Corn extract 0.75 KN ₂ RO ₄ 0.5 NaNO ₃ 0.5 MgSO ₄ × 7H ₂ O 0.2 Tap water up to 1 l

Inoculation of the nutrient medium was carried out with an aqueous suspension of the fungus Duddingtonia flagrans, strain F-882 with a spore titer of 3 × 10 ⁴ in 1 ml, based on 1 ml of suspension per 75 ml of liquid nutrient medium. The fungus was cultured under stationary conditions for 20 days. The biomass had a jelly-like consistency, with microscopy on the mycelium numerous chlamydospores were revealed. Next, the biomass was mixed with distilled water in a ratio of 1: 5 by weight and ground in a homogenizer for 2 minutes at 600 rpm, then the biomass was subjected to sonication using an ultrasonic disintegrator (MSE, England) at an amplitude of 24 μm for 10 minutes. Centrifugation (10,000 rpm, 20 min) was used to precipitate the destroyed cell walls of the hyphae. The concentration of the obtained biologically active substances was 0.5 mg / ml.

The resulting product was dried to a residual moisture content of not more than 5 wt.% And ground to a powder state.

Example 2. Obtaining sample 10-25 / 1 from the nematophagous fungus Duddingtonia flagrans Biomass was obtained by submerged cultivation of the fungus in a nutrient medium containing molasses and corn extract. The composition of the medium,%:

Molasses 3.0 Corn extract 0.75 KN ₂ RO ₄ 0.5 NaNO ₃ 0.5 MgSO ₄ × 7H ₂ O 0.2 Tap water up to 1 l

Inoculation of the nutrient medium was carried out with an aqueous suspension of the fungus Duddingtonia flagrans, strain F-882 with a spore titer of 3 × 10 ⁴ in 1 ml, based on 1 ml of suspension per 75 ml of liquid nutrient medium. The fungus was cultivated for 6 days at 27 ° C in a thermostatically controlled shaker at 180 rpm. The biomass had a thick consistency and contained solids of at least 12-15 mg / ml. Chlamydospores on the mycelium were absent. Next, the biomass was mixed with distilled water in a ratio of 1: 5 by weight and processed in a homogenizer for 2 minutes at 600 rpm, then the biomass was subjected to sonication at an amplitude of 24 μm for 10 minutes. Centrifugation (10,000 rpm, 20 min) was used to precipitate the destroyed cell walls of the hyphae. The concentration of the obtained biologically active substances was 0.5 mg / ml.

Example 3. Determination of antiviral activity against influenza viruses

Cell culture. To test the antiviral activity of the preparations, a transplantable culture of MDCK cells obtained from the collection of cell cultures of the Federal State Pedagogical Research Center of the World Bank “Vector” was used. In a sterile place, the suspension of the MDCK cell culture was diluted with RPMI-1640 medium pre-heated to a temperature of + 37 ° C, containing 5% blood serum of cow fruits (Gibco, USA), to a concentration of 1.0-1.5 × 10 ⁵ cells / ml. 100 μl / well of the MDCK cell suspension was added to 96-well plates with an 8-channel automatic pipette. Tablets with cells were placed in a thermostat at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity for 1-2 days before the formation of a continuous cell monolayer.

The support medium for MDCK cells was identical in composition to the growth medium except that the medium contained 2 μg / ml TRPC trypsin (Sigma, USA) and did not contain serum.

Viruses. We used strains of avian influenza virus (A / chicken / Kurgan / 05/2005 (H5N1)) and human (A / Aichi / 2/68 (H3N2)) obtained from the Department of Microorganisms Collection of the Federal State Institution of Science and Science of the World Bank "Vector". The accumulation was made on 10-day-old developing chicken embryos (RCE). The concentration of the A / chicken / Kurgan / 05/2005 (H5N1) virus in the virus-allantoic fluid (IMPORTANT) when titrated on an MDCK cell culture was 8.5 lg TCD ₅₀ / ml (50% tissue cytopathic doses in ml), virus A / Aichi / 2/68 (H3N2) - 7.5 lg TCD ₅₀ / ml.

Determination of cytotoxicity of drugs on an MDCK cell culture. Investigated preparations based on nematophagous fungi: 10-25, 10-25 / 1 for cytotoxicity in MDCK cell culture (table 1). To determine toxic doses, the preparations were diluted several times and the presence of toxic effects in monolayers of MDCK cell culture was assessed using an inverted microscope. For this, dilutions of the starting preparations were done 2.5 times and then from 10 ^-1 to 10 ^-6 on RPMI-1640 medium, then 100 μl / well of dilutions were added to the wells of a 96-well plate and placed in a thermostat at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity for 1-2 days. After 1-2 days using an inverted microscope, the presence of toxic effects in monolayers of MDCK cells incubated with different drug concentrations was evaluated.

As a control, intact MDCK cells were used (without drug administration).

Table 1 The results of the determination of toxic concentrations of drugs in the monolayer of MDCK cells No. of drug and its initial concentration Dilutions of drugs that cause (+) and do not cause (-) toxic effects on cells 1: 2 1: 5 1:10 ^-1 1:10 ^-2 1:10 ^-3 1:10 ^-4 1:10 ^-5 10-25
0.5 mg / ml + - - - - - - 10-25 / 1
0.5 mg / ml + - - - - - -

To determine the antiviral activity (table 1) used non-toxic drug concentrations (0.1 mg / ml and below).

Determination of the antiviral effect of drugs against influenza viruses A / chicken / Kurgan / 05/2005 (H5N1) and A / Aichi / 2/68 (H3N2) on MDCK cells

Dilutions of virus-containing liquid (IMPORTANT) were prepared from 1 to 8 in ten-step increments using RPMI-1640 medium from Biolot LLC, St. Petersburg, containing 2 μg / ml TRPC trypsin (Sigma, USA).

The study of the antiviral activity of the drugs was carried out according to two schemes: prophylactic (first the drug is introduced into the cell culture and then the virus) and therapeutic (first the virus is introduced into the cell culture and then the drug).

Scheme 1 (preventive). To determine the antiviral activity of the preparations, 100 μl / well of the selected dilution of the preparation was added to the monolayer of the MDCK cell culture (Table 2), after 1 h of incubation at + 37 ° C, 5% CO ₂ and 100% humidity were added to 100 μl / well of the diluted from 1 to 8 dilutions IMPORTANT. Cells were incubated for 2-3 days at a temperature of + 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2-3 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% rooster erythrocytes. As a control, we used:

1. Control of MDCK cells cultured in RPMI-1640 nutrient medium, manufactured by Biolot LLC, St. Petersburg, containing 2 μg / ml TRPC trypsin (Sigma, USA).

2. Control of the reproduction of influenza viruses A / chicken / Kurgan / 05/2005 (H5N1) and A / Aichi / 2/68 (H3N2) in dilutions from 1 to 8 with a ten-fold step without application of drugs, but with preliminary introduction of RPMI- nutrient medium 1640 containing 2 μg / ml trypsin TRSC in a volume of 100 μl / well.

3. Repeated monitoring of the cytotoxicity of drugs in selected doses.

Scheme 2 (medical). To determine the antiviral activity of the preparations (Table 3), 100 μl / well diluted in RPMI-1640 medium containing 2 μg / ml TRPS trypsin from 1 to 8 dilutions of IMPORTANT was added to the monolayer of MDCK cell culture. After 1 h of incubation at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity, 100 μl / well of the selected dilution of the preparation was added. Cells were incubated for 2-3 days at a temperature of + 37 ° C in an atmosphere of 5% CO ₂ in a TS-1/80 SPU thermostat (Russia). After 2-3 days in each well, an inverted microscope was used to record the CPD in the cell monolayer and the presence of the virus in the culture medium was determined by the hemagglutination reaction (RGA) with 1% rooster erythrocytes.

As a control, we used:

1. Control of MDCK cells cultured in RPMI-1640 nutrient medium, manufactured by Biolot LLC, St. Petersburg, containing 2 μg / ml TRPC trypsin (Sigma, USA).

2. Control of the reproduction of influenza viruses A / chicken / Kurgan / 05/2005 (H5N1) and A / Aichi / 2/68 (H3N2) in dilutions from 1 to 8 with a ten-fold step without application of drugs, but with subsequent introduction of RPMI- nutrient medium 1640 containing 2 μg / ml trypsin TRSC in a volume of 100 μl / well.

3. Repeated monitoring of the cytotoxicity of drugs in selected doses.

Determination of the antiviral activity of the studied drugs showed that samples of preparations based on the nematophage fungus 10-25, 10-25 / 1 with respect to the influenza virus on MDCK cells have antiviral activity. At the same time, preparations 10-25 and 10-25 / 1 showed antiviral activity against both bird flu virus A / chicken / Kurgan / 05/2005 and human influenza virus A / Aichi / 2/68: with a prophylactic regimen for dilution of drugs 10 times the neutralization indices for avian influenza virus were 3.0 and 3.5 lg, respectively, while more concentrated preparations 10-25 and 10-25 / 1 neutralized 6.0 lg of this virus; drug 10-25 / 1 when diluted 10 times neutralized 5.0 lg of human influenza virus.

In the treatment regimen, the antiviral activity of all studied drug samples at the same dilutions was lower compared to the prophylactic regimen. Inhibitory activity against avian influenza virus showed drugs 10-25 and 10-25 / 1 at a dilution of 5 times (neutralization indices were 3.0 and 2.5 lg, respectively), and against human influenza, the drug 10-25 / 1 at dilution 10 times (neutralization index was 3.5 lg).

Example 4. Determination of antiviral activity against vaccinia virus

Cell culture. To test the antiviral activity of the drug based on the fungus Duddingtonia flagrans, we used a transplantable culture of Vero cells obtained from the collection of cell cultures of the Federal State Pedagogical Research Center of the World Bank “Vector”. To obtain a monolayer, Vero cells were grown in 96-well plastic plates for 2.0-3.0 days in RPMI-1640 growth medium containing 0.125 units / ml kanamycin, 150 μg / ml L-glutamine and 5% fetal fetal serum cows (Fetal Bovine Serum, HyClone, USA, Lot. No. CSC0518), inactivated at 56 ° C for 30 minutes).

The support medium for Vero cells was identical in composition to the growth medium except that the concentration of fetal serum was 1%.

Viruses. We used the vaccinia virus L-IVP strain obtained from the Collection of Microorganisms Department of the Federal State Institution of Science and Science of the World Bank Vektor with a titer of 4.6 lg TCD ₅₀ / ml.

Determination of cytotoxicity of the drug on a Vero cell culture.

To study the toxic effect of the drug on a monolayer of Vero cells grown in 96-well plates, the drug was added in dilutions with a double step from 1: 2 to 1: 512 in a support medium in a volume of 100 μl / well. Tablets with cells were incubated at a temperature of + 37 ° C, 5% CO ₂ and 100% humidity. The toxic effect of the drug was evaluated visually daily for 5 days using an inverted microscope (Lomo Biolam P2-1) and expressed as% damage to the cell monolayer. On the 6th day after the introduction of the preparation, the cells were stained with a solution of gentian violet (1 g of gentian violet, 50 ml of 96% ethanol, 700 ml of distilled water, 300 ml of 40% formalin solution), the results were taken into account visually and expressed as% damage to the cell monolayer.

Vero intact cells were used as control (without drug administration).

In experiments to determine the antiviral activity of the drug against the vaccinia virus, the most tolerated concentrations of the drug on Vero cells were used (Table 4).

Table 4 The toxicity of the drug based on the fungus Duddingtonia flagrans on Vero cells Sample concentration Dilution of the drug Cell damage (% CPD) after 6 days undiluted one hundred 1: 2 one hundred 1: 4 one hundred 10-25 / 1 1: 8 one hundred 0.5 mg / ml 1:10 fifty 1:16 0 1:32 0 1:64 0 1: 128 0 1: 512 0

Virus neutralizing effect of the drug against vaccinia virus on Vero cells

To determine the neutralizing effect, the virus was diluted in a supporting medium (volume 100 μl / well) at a dose of 100 TCD ₅₀ / well to double dilutions of the drug in a support medium (volume 100 μl / well). Tablets with infected cell cultures and the preparation added to them, as well as tablets with control cells, were incubated at + 37 ° C, 5% CO ₂ and 100% humidity for 5 days, while the cell monolayer was examined daily using an inverted microscope. On the 6th day after infection, the cells were stained with a solution of gentian violet (1 g of gentian violet, 50 ml of 96% ethanol, 700 ml of distilled water, 300 ml of 40% formalin solution) and the results were taken into account visually, assessing the damage to the cell monolayer in%.

For a 50% virus-inhibiting concentration of the drug (IC ₅₀ ), its maximum dilution was taken, at which 50% of the cells were protected from the action of the virus at a dose of 100 TCD ₅₀ / well.

As a negative control (control of the preservation of cell viability), intact cells were used in a support medium without adding a drug in at least 16 wells of each plate, which were incubated for the same time as in the experiment.

As a positive control used cells infected with the virus at a dose of 100 TCD ₅₀ / well without adding the drug (table 5).

Table 5 Virus-neutralizing effect of the drug based on the fungus Duddingtonia flagrans against the vaccinia virus on Vero cells Sample Name Initial drug concentration Dilution of the drug The defeat of cells (%) after 6 days 1:16 fifty 10-25 / 1 0.5 mg / ml 1:32 75 1:64 75 1: 128 one hundred 1: 512 one hundred Virus control (no drug) - - one hundred Note: The number of repetitions for all measurements is 4.

Determination of the toxicity of the preparation based on the fungus Duddingtonia flagrans on Vero cells showed that the preparation based on the nematophage fungus 10-25 / 1 on Vero cells was toxic at a dilution of 1: 8 at 100%, at a dilution of 1:10 - at 50%. In this regard, in experiments on the determination of antiviral activity, we used the drug in the most non-toxic dilutions, namely 10-25 / 1 - in dilutions from 1:16 to 1: 512. The determination of the neutralizing effect showed that the drug based on the nematophage fungus 10-25 / 1 at a dilution of 1:16 protected 50% of Vero cells from this virus and at a dilution of 1:32, 25% of these cells.

Based on the data obtained, it can be concluded that samples based on the fungus Duddingtonia flagrans are promising for further study in terms of the development of drugs against influenza viruses and vaccinia.

Example 5. Determination of antiviral activity against human immunodeficiency virus type 1

A study of the antiviral activity of compounds against HIV-1 was carried out on the transplanted line of human lymphocytes MT-4. Cells were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum pre-inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L-glutamine and 80 μg / ml gentamicin. Cells were used after passage 3, on day 3 of cultivation, in the logarithmic growth phase. After microscopy and morphological evaluation, the concentration and viability of the cells were calculated by the trypan blue exclusion method. For the experiment, cells with a concentration of at least 2.0 × 10 ⁶ cell particles per milliliter and a viability of at least 90% were used.

For infection, the supernatant of MT-4 cells infected with HIV-1 strain GKV 4046, stored in liquid nitrogen, was used. The multiplicity of infection of 0.2-0.5 infectious units per cell.

The toxicity of the compounds was investigated. For this, a suspension of MT-4 cells diluted to a seed concentration of 0.5 × 10 ⁶ cells per milliliter with RPMI-1640 nutrient medium supplemented with 10% fetal serum, previously inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L β-glutamine, 80 μg / ml gentamicin and 30 mg / ml lincomycin, were placed in the wells of 96-well plates, 180 μl per well. Solutions of test compounds were added to the cells (20 μl per well). Assessed toxicity in two stages: with final dilutions of the drug 1:10, 1:50, 1: 100, 1: 1000. When detecting low toxicity of the drug, an additional dilution of the drug to a final dilution of 1: 2.5 was used; 1: 5; 1:10; 1:20. Used 3 wells per dose. The control is cells to which instead of the test compound the same amount of solvent is added (RPMI-1640 medium without serum and antibiotics). The plates were incubated in a thermostat at 37 ° C. On the 3rd day of cultivation, the concentration and viability of the cells are calculated by intravital staining of MTT cells (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide). Dilution that does not have a toxic effect on MT-4 cells (in which the cells retained 50% and 100% viability) was determined according to the graph of the optical density versus cell concentration and viability.

The prophylactic effect of the preparations was evaluated by suppressing the virus production in MT-4 cells with the addition of the drug during virus adsorption.

Virus-containing material was added to the MT-4 cell suspension and immediately spread into the wells of 96-well plates. Immediately, preparations diluted in RPMI-1640 medium were added to final dilutions of 1:10, 1:20, 1:40, 1: 80 ... (three wells for each dose). The plates were incubated for one hour at 37 ° C to adsorb the virus. Then the cells were diluted to a seed concentration with RPMI-1640 nutrient medium supplemented with 10% fetal serum, pre-inactivated by heating at 56 ° C for 30 minutes, 300 mg / ml L-glutamine, 80 μg / ml gentamicin and 30 mg / ml lincomycin, containing the appropriate dose of drugs.

The therapeutic effect of the drugs was evaluated by suppressing the production of HIV-1 in MT-4 cells with the addition of the fungus preparation after the adsorption of the virus on the cell surface.

Virus-containing material was added to the suspension of sensitive cells and incubated for 37 hours at 37 ° C in an atmosphere of 5% CO ₂ with constant stirring of the contents to adsorb the virus.

After this, the cells were diluted to inoculum concentration with RPMI-1640 nutrient medium with serum and antibiotics and spread into 180-well plates of 96-well plates. Aliquots of the test compounds diluted in RPMI-1640 medium without additives were added to each well to final dilutions of 1:10, 1:20, 1:40, ... 1: 64000.

The controls were HIV-1 infected MT-4 cells without the addition of drugs. The plates were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 3 days. Anti-HIV drug activity was evaluated by suppressing the accumulation of p24 viral protein in infected cells using a quantitative determination of p24 by direct enzyme immunoassay. The inhibition of the growth of the virus-specific protein p24 by various dilutions of the studied drugs with the addition of the virus and the drug (50% and 100% virus inactivation) was determined from the graphs of the optical density versus p24 concentration, and the p24 concentration was calculated according to the schedule for standard antigen.

The total data on the assessment of toxicity and effectiveness against HIV-1 of the mushroom preparation 10-25 are presented in table 6.

Table 6 Suppression of HIV-1 reproduction in MT-4 cells upon application of a drug based on the fungus Duddingtonia flagrans before and after HIV-1 adsorption on cell surfaces and toxicity assessment Sample Name The concentration of the drug, inhibiting the reproduction of HIV-1 with simultaneous application with the virus (μg / ml) The concentration of the drug, inhibiting the reproduction of HIV-1 when making the drug after adsorption of the virus (μg / ml) Non-toxic drug concentration (mcg / ml) 50% off 100% 50% off 100% For 50% of the cells For 100% cells 10-25 / 1 12.5 25 6.25 12.5 more than 500 200

When the preparation of the fungus 10-25 / 1 was added at the same time as the virus was adsorbed, complete suppression of HIV-1 reproduction in MT-4 cells at a concentration of 25 μg / ml was shown. When the preparation of the fungus 10-25 / 1 was added after HIV adsorption, complete suppression of HIV-1 reproduction in MT-4 cells at a drug concentration of 12.5 μg / ml was shown.

It should be noted that this drug at a working concentration of more than 500 μg / ml does not show toxic properties for 50% of the cells.

At the same time, the chemotherapeutic index (calculated as the ratio of the maximum tolerated dose to the minimum effective dose) exceeds 80 when evaluating the therapeutic effect of the mushroom preparation. Example 5 shows the promise of using an anti-HIV drug based on the extract of the mushroom Duddingtonia flagrans (sample 10-25 /one).

Claims (2)

1. An antiviral agent that has activity against human immunodeficiency virus of the first type, influenza viruses of humans and birds of type A and vaccinia virus and is an aqueous extract from a strain of nematophage fungus Duddingtonia flagrans F-882, obtained by extraction of the biomass of the fungus with water at a ratio of biomass: water 1 : 5, followed by removal of the insoluble precipitate. 2. The tool according to claim 1, characterized in that the biomass of the nematophagous fungus Duddingtonia flagrans of strain F-882 is obtained by culturing it on a liquid nutrient medium based on molasses and corn extract.