RU2375073C1 - Inhibitor of reproduction of human immunodeficiency virus type 1 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to inhibitor of reproduction of human immunodeficiency virus type 1. Inhibitor of reproduction of human immunodeficiency virus type 1, containing aqueous or water-alcohol extract of natural biologically active substances of basidium fungus Inonotus obliquus in specified concentration.

EFFECT: effective inhibitor of reproduction of human immunodeficiency virus type 1.

4 tbl, 4 ex

Description

The invention relates to inhibitors of reproduction of the human immunodeficiency virus of the first type of plant origin and can be used in medicine, virology and pharmacology.

Today, the only way to prevent the development of HIV infection and the progression of the disease is antiretroviral combination therapy (ART).

ARVT is designed to suppress the replication of the human immunodeficiency virus (HIV), i.e. is an etiotropic therapy for HIV infection.

To date, HIV infection remains an incurable disease and it is not possible to completely eliminate the virus from the body using modern methods of treatment. However, with the help of ARV drugs, it is possible to block the multiplication of HIV and reduce the virus content in the blood (up to an undetectable level). This leads to the restoration of a subpopulation of CD4 lymphocytes (full or partial), which helps prevent development, facilitates the course or contributes to the disappearance of opportunistic diseases. As a result, quality improves and life expectancy of patients with HIV infection increases. In addition, the use of ARV drugs for HIV-positive pregnant women can significantly reduce the likelihood of vertical transmission.

Despite the large number of currently developed anti-HIV drugs, there is a problem with the effectiveness of the antiviral therapy used.

The main obstacles to solving this problem are the toxicity of effective drugs, due to the fact that the virus uses intracellular systems during its development (Inouye M. et al. Annu. Rev. Micribiol. 1991. V.45. P.163-186) , the high cost of the drugs used and the ability of HIV-1 to develop resistance to antiviral drugs.

Therefore, the problem of developing effective and inexpensive antiviral agents that do not have a toxic effect on the human body remains extremely urgent.

Known natural compounds that have an inhibitory effect on the human immunodeficiency virus.

There is evidence of HIV-1 suppression by Trametes versicolor fungus extract (Collins RA and Ng TB (1997). Polysaccaropeptide from Trametes versicolor has potential for use against human immunodeficiency virus type 1 infection. Life Scince, 60 (25), P. 383-387 )

Some triterpenes of the fungus Ganoderma lucidum are active against immunodeficiency virus in cell culture MT-4 (EI-Mekkawy S., Meselhy MR, Nakamura N., Tezuki Y., Hatori M., Kakiuchi et al. (1998) Anti-HIV-1 and anti-HIV protease substances from Ganoderma lucidum. Phytochemistry, 49, P.1651-1657).

Isolation of a novel ubiquitin-like protein from Pleurotus ostrestus mushroom with anti-human immunodeficiency virus and translation-inhibitor is isolated from oyster mushroom Pleurotus ostreatus Biochem Biopnys Res Commun, 256, 587-593).

Similar results were obtained with extracts of the fungus Grifola frondosa (Nanba H., Kodama N., Schar D. and Turner D. 2000. Effect of Maitake (Grifola frondosa) glucan in HIV-infection patients. Mycoscience, 41, 293-295).

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 officinales fungi , Piptoporus betulinus, Ganoderma resinaceum against smallpox viruses and human immunodeficiency.

In the patent of the Russian Federation No. 2246500, IPC C07J 63/00, publ. 02/20/2005 describes a method for producing betulin from betulin, which can be used to obtain anti-tumor and anti-HIV drugs. The description of the invention does not provide data on the antiviral activity of the drug.

It is known to use the extract of birch fungus Inonotus obliquus, called chaga, as an anticancer agent and capable of enhancing immunity in immunodeficient conditions of a non-viral origin (Application for Korean Invention No. 20040042119, IPC A 61 K 35/78, publ. 05.20.2004 ) However, studies on the anti-HIV activity of this extract have not been conducted.

It is known to use the extract of the mycelial culture of the cells of the fungus Fuscoporia oblique (Fr.) Aoshima (Japanese chaga) grown on an artificial liquid nutrient medium as an active ingredient that inhibits the human immunodeficiency virus (Application US No. 20040105859, IPC A61 K 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).

One of the closest analogues (prototype) is the use of the extract of the black outer part of the growth of the fungus Fuscoporia oblique (Fr.) Aoshima as an active ingredient that inhibits the human immunodeficiency virus (Japanese Application No. 9191891, IPC АКК 35/84, С12Р 1/02, publ. July 29, 1997). The indicated fungus grows on Japanese birch wood of two species Betula platyphyla Sukatchev var. japonica and Betula ermanii.

However, the reserves of Fuscoporia oblique in Japan are very limited and do not have the prospect of being used as a raw material for the treatment and prophylactic against HIV infection even in this country. This fungus is not found on the territory of the Russian Federation, which complicates its use as a therapeutic antiviral agent in the Russian Federation and other countries.

The technical result of the invention is the expansion of the range of sources of raw materials based on fungi of basidiomycetes, which have an inhibitory effect against human immunodeficiency virus of the first type.

The indicated technical result is achieved through the discovery of new properties of the fungus Inonotus obliquus, which consists in inhibiting the reproduction of the human immunodeficiency virus of the first type, i.e. The object of the invention is the use of the basidiomycete Inonotus obliquus (Pers.) Pil. as an inhibitor of reproduction of the human immunodeficiency virus of the first type.

The first type of human immunodeficiency virus reproduction inhibitor contains natural biologically active substances from the basidiomycete Inonotus obliquus, soluble in water or alcohol (alcohol content of 70% or more), or aqueous-alcoholic solutions.

Moreover, the content in the inhibitor of reproduction of the human immunodeficiency virus of natural biologically active substances from the basidiomycete Inonotus obliquus is at a concentration of not less than 156 ng / ml.

In the present invention, the aqueous, alcoholic and aqueous-alcoholic extracts of the fungus Inonotus obliquus were studied and selected using a viral model - a cell culture of the transplantable human lymphocyte line MT-4 infected with various strains of HIV-1, in particular strains reproducing as acute and chronic infections . This approach allows conducting studies close to the real processes of HIV infection in the body. The use of laboratory strains of HIV-1 makes it possible to evaluate the effect of drugs on the natural pool of the viral population, which obviously has genetic diversity. It also allows for a more objective assessment of the antiviral efficacy of drugs.

The therapeutic result was the complete suppression of type 1 human immunodeficiency virus in the transplanted MT-4 human lymphocyte line, both when it was introduced into the cell culture with the virus and after the virus was adsorbed (preventive and therapeutic effect of the drug).

This suggests the future use of drugs based on the natural compounds of the fungus Inonotus obliguus for the treatment of HIV-infected patients after appropriate clinical trials.

Prior art analysis

In the description of US application No. 20040105869, it was noted (in the application text paragraph [0012]) that the anti-HIV activity of extracts from the cells of the fungus Fuscoporia oblique (Fr.) Aoshima obtained both from natural raw materials (Japanese application No. 9191891) and grown on artificial nutrient media (US application No. 20040105869) is almost the same. However, it is preferable to use artificially grown cells of the fungus Fuscoporia oblique (Fr.) Aoshima, because natural raw materials in Japan are not enough.

Compared with the analogue (US Application No. 20040105869) and the prototype (Japanese Application No. 9191891) for a preparation obtained from the fungus Inonotus obliquus, it was shown that 50% and 100% of the effect of suppressing the reproduction of HIV-1 on human cell culture is achieved at lower concentrations the drug. So, 50% suppression of HIV-1 when using the most effective drugs obtained in the specified analogue is achieved when using more than 35,000 ng / ml of the drug. For the drug from the fungus Inonotus obliquus, claimed by the authors, 50% suppression of HIV-1 is achieved at a drug concentration of less than 156 ng / ml. 100% suppression of HIV-1 is achieved by adding 312 ng / ml of the preparation of the fungus Inonotus obliquus, while a concentration of 218750-875000 ng / ml is required to achieve a similar inhibitory effect of the preparation based on the cell cultures of the fungus Fuscoporia oblique.

Unlike the analogue (US Application No. 20040105869) and the prototype (Japanese Application No. 9191891), the present invention shows not only prophylactic (adding the drug to cells and incubating until the virus is adsorbed on the cells), but also a significant therapeutic effect of the chaga drug. The authors of the present application for the invention demonstrated that even with the introduction of 312 ng / ml of the preparation of the fungus Inonotus obliquus into the cell culture after adsorption of the virus, 100% inhibition of HIV-1 is achieved.

The fungus Inonotus obliquus develops in the form of dark growths on the trunks of living birch trees and some other tree species; it has long been used in folk medicine (chaga with birch trees) for the treatment of gastrointestinal diseases and for cancer of various localization.

In general, the natural substances of chaga (fungus Inonotus obliquus} have a very wide range of different compounds, which are formed by the close interaction of birch and fungus (Chaga and its therapeutic use / Edited by Prof. P.K. Bulatov, Prof. M.P. Berezina, Prof. P.A. Yakimova / Medgiz, Leningrad Branch, 1959.) In Siberia and in the whole of the Russian Federation, this fungus develops on the warty birch and the white birch (Betula pendula Roth and Betula alba L.), and has a wide distribution range.

After extensive clinical and chemical studies, the fungus Inonotus obliquus was approved by the Pharmacological Committee of the USSR Ministry of Health for use in 1955. Based on the pharmacopoeial article FS 42-53-72, the finished raw material of the fungus Inonotus obliquus has the following indicators:

- extractive substances - not less than 20%;

- chromogenic complex - not less than 50% of the mass of the total dry residue of extractive substances;

- moisture - not more than 14%;

- ash - not more than 14%.

The fungus Inonotus obliquus contains a wide range of various biologically active substances: water-soluble pigments in large quantities (20%), which form a chromogenic polyphenol-carboxylic complex, which exhibits antitumor activity, due to the fact that phenolic compounds regulate the activity of cytoplasmic and mitochondrial ATPases and reduce the formation of ADP, and since Magnetized cells are more than normal, dependent on glycolysis, then a violation of this process negatively affects their development; pterins (derivatives of pteridine), the presence of which determines the cytostatic effect of chaga; polysaccharides (6-8%); agaricin and humic chagic acids (up to 60%); organic acids, the total content of which is 0.5-1.3% (oxalic, acetic, formic, vanillic, lilac, p-hydroxybenzoic, as well as 2 triterpenic acids from the group of tetracyclic triterpenes - inotonic and obliquin); lipids (di- and triglycerides); steroidal substances (sterols - ergosterol, as well as tetracyclic triterpenes - lanosterol iinotodiol, showing antiblastic activity); lignin; cellulose; free phenols; flavonoids; coumarin peucedanin; cellulose; resins; traces of alkaloids of unknown structure; ash (12.3%), rich in manganese, which, perhaps, is important in the therapeutic effect of chaga as an activator of enzymes; other trace elements in the form of oxides: copper, barium, zinc, iron, silicon, aluminum, calcium, magnesium.

Inonotus obliquus mushroom preparations are widely used in medicine, in particular:

- in diseases of the gastrointestinal tract: gastrointestinal dyskinesia with a predominance of atony, chronic gastritis with decreased secretory function and anacid gastritis, peptic ulcer of the stomach and duodenum, polyposis of the stomach and intestines, gastralgia, enterralgia, liver and spleen diseases;

- with low bowel tone;

- in case of malignant neoplasms of different localization in inoperable cases and the impossibility of radiation therapy: cancer of the stomach, intestines, pancreas, liver, esophagus, lungs and other well-vascularized organs, less effective is the chaga in the localization of tumors in the bones, brain and skin;

- to prevent the occurrence of malignant tumors (with the constant use of Chaga infusion, the percentage of cancer diseases is much lower);

- with radiation leukopenia and to prevent its development during radiation therapy, the renewal of the blood formula and to improve blood circulation;

- in otorhinolaryngological practice as an adjunct in the treatment of laryngeal tumors in the form of inhalations. At the same time, the general condition of patients improves, the process of swallowing normalizes, voice hoarseness decreases, breathing improves, and the accompanying inflammatory process decreases;

- with insomnia, to calm the nervous system;

- in violation of catalase and protease metabolism;

- after suffering serious illnesses and operations as a restorative agent;

- to increase the body's resistance to infectious diseases;

- in small concentrations as a substitute for tea (renews strength, gives vitality, increases appetite, relieves headaches);

- in dentistry for the treatment of periodontal disease (injected into the gum pockets and taken orally);

- with psoriasis, eczema and other skin diseases.

To assess the antiviral activity against HIV-1, we used the entire spectrum of water and alcohol-soluble natural compounds contained in dry growths of birch fungus - Chaga Inonotus obliguus.

In the case of using dried growths of the fungus Inonotus obliguus, they were crushed to a powder state. A portion of the crushed mushroom of pharmaceutical production or the powder obtained by us was suspended in sterile distilled water.

To extract water-soluble substances, in some cases, the mixture was heated in a water bath for 20 min or shaken on a rocking chair at a temperature of 28-30 ° C for 18 hours with a stirring speed of 120 rpm.

For tests on viruses used as just prepared samples, and stored for a long time at temperatures of 6-8 ° C or at minus 20 ° C.

Befungin and chaga tincture were also used.

Example 1. Evaluation of the therapeutic and prophylactic effect of preparations of the fungus Inonotus obliquus, obtained as a result of aqueous extraction, on the reproduction of the human immunodeficiency virus in cell culture MT-4.

A portion of the crushed mushroom Inonotus obliquus from a pharmacy (manufactured in Moscow) 5 g is suspended in 100 ml of sterile distilled water and incubated for 7-14 days at a temperature of 6-8 ° C, released from sediment (samples 08-11, 07-05).

The study of the antiviral activity of the compounds against HIV-1 is 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 100 μg / ml gentamicin. Use cells after 3 passages, on the 3rd day of cultivation, in the logarithmic growth phase. After microscopy and morphological evaluation, the concentration and viability of the cells are 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% are used.

For infection, the supernatant of MT-4 cells infected with HIV-1 strain GKV 4046 and strain GKV 4005, stored in liquid nitrogen, is used. The multiplicity of infection of 0.2-0.5 infectious units per cell.

The toxicity of the compounds is 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 are placed in wells of 96-well plates, 180 μl per well. To the cells add solutions of the studied compounds (20 μl per well) to a final dilution of 1:10, 1:20, 1:40, 1: 80 ... 1: 1280. Use 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 are 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) is determined by the graph of the dependence of optical density (filter 450 nm) on the concentration and viability of cells.

The prophylactic effect of the drugs is evaluated by the suppression of 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 add the preparations diluted in RPMI-1640 medium to the final dilutions of 1:10, 1:20, 1:40, 1: 80 ... (three wells for each dose). The plates are incubated for one hour at 37 ° C to adsorb the virus. Then the cells are diluted to a seed concentration with RPMI-1640 nutrient medium with the addition of 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 is evaluated by suppressing the production of HIV-1 in MT-4 cells with the addition of the chaga mushroom preparation after the adsorption of the virus on the cell surface.

Virus-containing material is introduced into 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 are diluted to a seed concentration with RPMI-1640 nutrient medium with serum and antibiotics and distributed into wells of 96-well plates, 180 μl per well. Aliquots of the test compounds diluted in RPMI-1640 medium without additives are added to each well to final dilutions of 1:10, 1: 20.1: 40, ... 1: 64000.

The controls are HIV-1 infected MT-4 cells without the addition of drugs. The plates are incubated at 37 ° C in an atmosphere of 5% CO ₂ for 3 days.

Anti-HIV drug activity is 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 when the virus and the drug (50% and 100% virus inactivation) are added is determined from the graphs of the dependence of the optical density (filter 490 nm) on the concentration of p24, recalculating the concentration of p24 according to the schedule for standard antigen. Summary data on the assessment of toxicity and efficacy against HIV-1 preparations of the fungus Inonotus obliquus are presented in table 1.

TABLE 1
Suppression of HIV-1 reproduction in MT-4 cells when introducing drugs of the fungus inonotus obliquus before and after adsorption of HIV-1 on the cell surface and evaluation of the toxicity of drugs A drug Dilution of the drug, inhibiting the reproduction of HIV-1 (with simultaneous application with the virus) Dilution of the drug, inhibiting the reproduction of HIV-1 (with the introduction of the drug after adsorption of the virus) Nontoxic drug dilutions 50% off 100% 50% off 100% For 50% of the cells For 100% cells 07-05 > 1: 25600 > 1: 12800 > 1: 12800 > 1: 8000 1:40 1: 320 08-11 > 1: 32000 > 1: 25600 > 1: 32000 1: 25600 1:80 1: 640

When the preparations of the fungus Inonotus obliquus (07-05 and 08-11) were added simultaneously with the adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells was shown when the preparations were diluted 1: 12800 and 1: 25600. When drugs of the fungus Inonotus obliquus (07-05 and 08-11) were added after the adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells was shown upon dilution of the preparations 1: 8000 and 1: 25600. Moreover, in dilutions of more than 1: 640, the preparations do not exhibit properties toxic to human cells.

Example 2. Evaluation of the therapeutic and prophylactic effect of preparations of the fungus Inonotus obliquus, obtained as a result of water extraction in a water bath at a temperature of 98 ° C, on the reproduction of human immunodeficiency virus in cell culture MT-4.

A portion of the crushed growth of the fungus Inonotus obliquus (Siberia) and the crushed mushroom from the pharmacy (manufactured in Moscow) 3 g and 5 g, respectively, are suspended in 100 ml of sterile distilled water and heated in a boiling water bath for 20 minutes, free from sediment (sample 07 -45 and 08-10).

The study of the antiviral activity of the compounds against HIV-1 is 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 100 μg / ml gentamicin. Use cells after 3 passages, on the 3rd day of cultivation, in the logarithmic growth phase. After microscopy and morphological evaluation, the concentration and viability of the cells are 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% are used.

For infection, the supernatant of MT-4 cells infected with HIV-1 (strain GKV 4046 and strain GKV 4005), stored in liquid nitrogen, is used. The multiplicity of infection of 0.2-0.5 infectious units per cell.

The toxicity of the compounds is 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, are placed in wells of 96-well plates, 180 μl per well. To the cells add solutions of the studied compounds (20 μl per well) to a final dilution of 1:10, 1:20, 1:40, 1: 80 ... 1: 1280. Use 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 are 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) is determined by the graph of the dependence of optical density (filter 450 nm) on the concentration and viability of cells.

The prophylactic effect of the drugs is 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 add the preparations diluted in RPMI-1640 medium to the final dilutions of 1:10, 1:20, 1:40, 1: 80 ... (three wells for each dose). The plates are incubated for one hour at 37 ° C to adsorb the virus. Then the cells are diluted to a seed concentration with RPMI-1640 nutrient medium with the addition of 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 is 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 is introduced into 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 are diluted to a seed concentration with RPMI-1640 nutrient medium with serum and antibiotics and distributed into wells of 96-well plates, 180 μl per well. Aliquots of the test compounds diluted in RPMI-1640 medium without additives are added to each well to a final dilution of 1:10, 1: 20.1: 40 ... 1: 64000.

The controls are HIV-1 infected MT-4 cells without the addition of drugs. The plates are incubated at 37 ° C in an atmosphere of 5% CO ₂ for 3 days.

Anti-HIV drug activity is 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 when the virus and the drug (50% and 100% virus inactivation) are added is determined by the graphs of the dependence of optical density (filter 490 nm) on the concentration of p24, recalculating the concentration of p24 according to the schedule for standard antigen. Summary data on the assessment of toxicity and effectiveness against HIV-1 preparations of the fungus fungus Inonotus obliquus are presented in table 2.

TABLE 2
Suppression of HIV-1 reproduction in MT-4 cells upon application of an aqueous extract of the fungus Inonotus obliquus before and after the adsorption of HIV-1 on the surface of cells and evaluation of the toxicity of drugs A drug Dilution of the drug, inhibiting the reproduction of HIV-1 (with simultaneous application with the virus) Dilution of the drug, inhibiting the reproduction of HIV-1 (with the introduction of the drug after adsorption of the virus) Non-toxic drug dilutions 50% off 100% 50% off 100% For 50% of the cells For 100% cells 07-45 > 1: 64000 1: 16000 1: 32000 1: 16000 1:40 1: 320 08-10 > 1: 64000 > 1: 32000 > 1: 64000 1: 32000 1:80 1: 320

When drugs of the fungus Inonotus obliquus (07-45 and 08-10) were added simultaneously with the adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells was shown when the preparations were diluted 1: 16000 and 1: 32000, respectively. When drugs are added after the adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells is shown when the drugs are diluted 1: 16000 (for 07-45) and 1: 32000 (for 08-10). Moreover, in a dilution of more than 1: 320, the preparations do not exhibit properties toxic to human cells.

Example 3. Evaluation of the prophylactic and therapeutic effect of the preparation of the fungus Inonotus obliquus (Befungin) on the reproduction of human immunodeficiency in MT-4 cell culture.

Befungin, which is a semi-thick extract from a mushroom growth with the addition of cobalt salts (cobalt chloride 0.175% or cobalt sulfate 0.2%) per 100 ml, is diluted in distilled water 1:10 (sample 08-08).

The study of the antiviral activity of the compounds against HIV-1 is 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 100 μg / ml gentamicin. Use cells after 3 passages, on the 3rd day of cultivation, in the logarithmic growth phase. After microscopy and morphological evaluation, the concentration and viability of the cells are 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% are used.

For infection, the supernatant of MT-4 cells infected with HIV-1 strain GKV 4046 and strain GKV 4005, stored in liquid nitrogen, is used. The multiplicity of infection of 0.2-0.5 infectious units per cell.

The toxicity of the compounds is 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, are placed in wells of 96-well plates, 180 μl per well. To the cells add solutions of the studied compounds (20 μl per well) to a final dilution of 1:10, 1:20, 1:40, 1: 80 ... 1: 1280. Use 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 are 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) is determined by the graph of the dependence of optical density (filter 450 nm) on the concentration and viability of cells.

The prophylactic effect of the drugs is 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 add the preparations diluted in RPMI-1640 medium to the final dilutions of 1:10, 1:20, 1:40, 1: 80 ... (three wells for each dose). The plates are incubated for one hour at 37 ° C to adsorb the virus. Then the cells are diluted to a seed concentration with RPMI-1640 nutrient medium with the addition of 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 is 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 is introduced into 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 are diluted to a seed concentration with RPMI-1640 nutrient medium with serum and antibiotics and distributed into wells of 96-well plates, 180 μl per well. Aliquots of the test compounds diluted in RPMI-1640 medium without additives are added to each well to final dilutions of 1:10, 1:20, 1: 40 ... (1: 64000 (three wells per dose).

The controls are HIV-1 infected MT-4 cells without the addition of drugs. The plates are incubated at 37 ° C in an atmosphere of 5% CO ₂ for 3 days.

Anti-HIV drug activity is 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 (50% and 100% inactivation of the virus) is determined by the graphs of the dependence of optical density (filter 490 nm) on the concentration of p24, recalculating the concentration of p24 according to the schedule for standard antigen. Summary data on the assessment of toxicity and efficacy against HIV-1 preparation of the fungus Inonotus obliquus Befungin are presented in table 3.

TABLE 3
Suppression of HIV-1 reproduction in MT-4 cells upon application of the inonotus obliquus fungus (Befungin) before and after HIV-1 adsorption on the surface of cells and assessment of the toxicity of the drug The drug Befungin Dilution of the drug inhibiting the reproduction of HIV-1 Non-toxic drug dilutions 50% off 100% For 50% of the cells For 100% cells When introducing the drug to the adsorption of the virus > 1: 64000 > 1: 32000 1:32 1: 100 When making the drug after virus adsorption > 1: 64000 1: 32000 1:32 1: 100

With the addition of the preparation of the fungus Inonotus obliquus Befungin, before and after adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells is shown with a dilution of 1: 32000. Moreover, with a dilution of more than 1: 100, the drug does not exhibit properties toxic to human cells.

Example 4. Evaluation of the preventive and therapeutic effect of the preparation of the fungus Inonotus obliquus (tincture of chaga) on the reproduction of the human immunodeficiency virus in cell culture MT-4.

Tincture of chaga, which is an alcoholic (ethanol) 70% fungus extract, is diluted 1:10 in distilled water (sample 08-15).

The study of the antiviral activity of the compounds against HIV-1 is 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 100 μg / ml gentamicin. Use cells after 3 passages, on the 3rd day of cultivation, in the logarithmic growth phase. After microscopy and morphological evaluation, the concentration and viability of the cells are 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% are used.

For infection, the supernatant of MT-4 cells infected with HIV-1 strain GKV 4046 and strain GKV 4005, stored in liquid nitrogen, is used. The multiplicity of infection of 0.2-0.5 infectious units per cell.

The toxicity of the compounds is 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, are placed in wells of 96-well plates, 180 μl per well. To the cells add solutions of the studied compounds (20 μl per well) to a final dilution of 1:10, 1:20, 1:40, 1: 80 ... 1: 1280. Use 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 are 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) is determined by the graph of the dependence of optical density (filter 450 nm) on the concentration and viability of cells.

The prophylactic effect of the drugs is 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 add the preparations diluted in RPMI-1640 medium to the final dilutions of 1:10, 1:20, 1:40, 1: 80 ... (three wells for each dose). The plates are incubated for one hour at 37 ° C to adsorb the virus. Then the cells are diluted to a seed concentration with RPMI-1640 nutrient medium with the addition of 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 is 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 is introduced into 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 are diluted to a seed concentration with RPMI-1640 nutrient medium with serum and antibiotics and distributed into wells of 96-well plates, 180 μl per well. Aliquots of the test compounds diluted in RPMI-1640 medium without additives are added to each well to final dilutions of 1:10, 1:20, 1: 40 ... (1: 64000 (three wells per dose).

The controls are HIV-1 infected MT-4 cells without the addition of drugs. The plates are incubated at 3 ° C in an atmosphere of 5% CO ₂ for 3 days.

Anti-HIV drug activity is 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 (50% and 100% inactivation of the virus) is determined by the graphs of the dependence of optical density (filter 490 nm) on the concentration of p24, recalculating the concentration of p24 according to the schedule for standard antigen. The total data on the assessment of toxicity and effectiveness against HIV-1 of the preparation of the fungus Inonotus obliquus (tincture of chaga) are presented in table 4.

TABLE 4
Suppression of HIV-1 reproduction in MT-4 cells upon application of the inonotus obliquus fungus The drug tincture of chaga Dilution of the drug inhibiting the reproduction of HIV-1 Non-toxic drug dilutions 50% off 100% For 50% of the cells For 100% cells When introducing the drug to the adsorption of the virus > 1: 16000 1: 12800 1:40 1: 320 When making the drug after virus adsorption > 1: 12800 1: 8000 1:40 1: 320

When the preparation of the fungus Inonotus obliquus Tincture of chaga was added before and after the adsorption of the virus, complete suppression of HIV-1 reproduction in MT-4 cells was shown when the preparations were diluted 1: 12800 and 1: 8000, respectively. Moreover, with a dilution of more than 1: 320, the drug does not exhibit properties toxic to human cells.

Evaluation of the effectiveness of the antiviral effect of various extracts of the fungus Inonotus obliquus on the human cell culture of MT-4 infected with HIV-1 showed that, with low toxicity to cells, chaga preparations have a pronounced inhibitory effect on human immunodeficiency virus type I as if simultaneously with the virus cell culture, and in post-adsorption conditions.

Claims (1)

The first type of human immunodeficiency virus reproduction inhibitor containing an aqueous or hydroalcoholic extract of natural biologically active substances from the basal fungus Inonotus obliquus at a concentration of at least 156 ng / ml, providing 100% antiviral protection of the cells.