RU2747018C1 - Sars-cov-2 coronavirus replication inhibitor based on melanin from inonotus obliquus fungus - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: present invention relates to the pharmaceutical industry, namely, to a replication inhibitor of the SARS-CoV-2 coronavirus that causes severe acute respiratory syndrome COVID-19. The natural melanin from the basidiomycetes Inonotus obliquus is used, obtained by alkaline hydrolysis of Inonotus obliquus, acid precipitation, dissolution of the precipitate in ammonia water and drying of the final product at a temperature not exceeding 30-40ºC, as a replication inhibitor of SARS coronavirus. An aqueous solution of this inhibitor in the range of 6,218-8,488 mcg/ml exhibits 50% antiviral dose-dependent activity against SARS-CoV-2 in tests on Vero cell culture.EFFECT: above-described natural melanin exhibits 50% antiviral dose-dependent activity against SARS-CoV-2 in Vero cell culture tests.1 cl, 1 dwg, 1 tbl, 5 ex

Description

The present invention relates to a SARS-CoV-2 coronavirus replication inhibitor based on melanin from the fungus Inonotus obliquus and can be used in biotechnology, pharmaceutical industry and medicine.

The SARS-CoV-2 outbreak around the world and its associated consequences are a threat to public health and the economy of many countries. The lack of special therapy against the new virus and its high variability requires the creation of new drugs.

Natural substances of chaga (basidiomycete Inonotus obliquus) have a very wide range of different compounds that are formed by the close interaction of birch and fungus [Chaga and its medicinal use / Ed. prof. PC. Bulatov, prof. M.P. Berezina, prof. PA Yakimova / Medgiz, Leningrad branch, 1959]. In Siberia and in the Russian Federation in general, this fungus develops on warty birch and white birch (Betula pendula Roth and Betula alba L.), has a wide distribution area.

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

- extractives - not less than 20%;

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

- moisture - no more than 14%;

- ash - no more than 14%.

The fungus Inonotus obliquus contains a wide range of different biologically active substances, among which the dark pigment melanin occupies one of the important places.

All types of melanin pigments are long-chain polymers with gigantic molecular weights and complex liquid crystal structures.

There are reports of the practical use of synthetic, semi-synthetic and isolated from biological sources of melanins in medicine, cosmetology, and, thanks to their semiconductor properties, in technology and electronics.

Melanins isolated from microorganisms show similar properties to melanins of animal origin. These are pharmacologically active substances with antioxidant, antitoxic, anti-inflammatory, immunostimulating properties, protecting against photodynamic damage, etc.

Melanins are amorphous high-molecular substances insoluble in water, mineral acids, organic solvents; well soluble in alkalis, and then precipitate when acidifying solutions, which is used to isolate them.

According to their predecessors, melanins are divided into eumelanins, pheomelanins and allomelanins. Eumelanins (black) and pheomelanins (yellow, red and brown) are common in animals, allomelanins (black) - in plants, fungi, bacteria. The precursor of eumelanins is tyrosine, from which pigments containing C, H, N and O are obtained in the body. The precursors of allomelanins are diphenols (pyrocatechin, etc.), from which melanins that do not contain nitrogen are formed.

The melanin pigments contained in mushrooms are the most powerful bioprotectors that protect a living cell from adverse external and internal influences. They are also the most powerful natural antioxidants. Melanins are able to neutralize various free radicals that arise in a living cell under the influence of penetrating radiation, ultraviolet radiation, various toxins and enzymes of pathogenic microorganisms. Many medicinal fungi, especially tinder fungi, have a high melanin content and can reach 30% of dry weight and even more. Some of the fungal melanins are able to pass into a solution and can be absorbed in the gastrointestinal tract, spread by the blood throughout the body and protect living cells from the destructive action of free radicals (http://www.amrita.net.ua/pi/products_id/329).

It has been established that melanin from the chaga Inonotus obliquus and some other tinder fungi has photo- and radioprotective, antioxidant and gene-protective properties [VV Shcherba, VG Babitskaya, VP Kurchenko, NV Ikonnikova. Dukulyanskaya T.A. Antioxidant properties of melanin pigments of fungal origin // Applied Biochemistry and Microbiology. 2000. - T. 36. - No. 5. - S. 569-574; Sushinskaya N.V., Kurchenko V.P., Gorovoy L.F., Senyuk O.F. Obtaining and using melanins from tinder fungi in medicine // Advances in medical mycology. - 2005. - T. 6. - S. 255-259].

Known antiviral agent based on water-soluble melanins obtained by chemical synthesis from the following components: L-deoxidopamine, L deoxidopamine, cysteine, L-deoxidopamine / glutathione, L-tyrosine, serotonin, dopamine, adrenaline and norepinephrine [US Patent No. 5057325, IPC A61K 31 / 195, publ. 1991 g].

The specified antiviral agent can fully or partially protect in vitro human lymphocytes from the immunodeficiency virus (HIV-1 and HIV-2).

Known antiviral agent based on melanin obtained from the natural chaga Inonotus obliquus, which is effective against influenza viruses, herpes simplex virus type 2, vaccinia virus, human immunodeficiency virus (HIV-1) [Russian Patent 2480227, publ. 04/27/2013]. Isolation of melanin from natural chaga was carried out by the method of silk hydrolysis [Sushinskaya NV, Kurchenko VP, Gorovoy LF, Senyuk OF. Obtaining and using melanins from tinder fungi in medicine // Advances in medical mycology. - 2005. - T. 6. - S. 255-259]. The resulting pigments were identified using qualitative reactions [Kukulyanskaya TA, Kurchenko NV, Kurchenko VP, Babitskaya V.G. Physicochemical properties of melanins formed by chaga under natural conditions and during cultivation. // Applied Biochemistry and Microbiology. 2002. - T. 38. - No. 1. - S. 68-72]. The absorption of UV and visible light by aqueous and alkaline pigment solutions was recorded on a spectrophotometer (Genesys 5 Spectrophotometers, United States). The absorption spectrum of the melanin solution had the form of an inclined straight line, characteristic of melanins of fungal origin [Babitskaya VG, Shcherba VV, Ikonnikova NV. Melanin complex of the fungus Inonotus obliquus. II Applied Biochemistry and Microbiology. 2000. - T. 36. - No. 4. - C. 439-444].

However, in the above analogs, there is no information on the antiviral activity of melanin from fungi against the SARS-CoV-2 coronavirus.

Natural compounds such as flavonoids are known (https: /bioflavit.ru/wp-content/uploads/Taxifolin-basel-rus2.pdf) that inhibit the main protease SARS-CoV-1 and their effectiveness has been demonstrated using resonant energy transfer fluorescence (FRET) [Pillaiyar, T., Manickam, M., Namasivayam, V., Hayashi, Y., Jung, SH An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors: Peptidomimetics and small molecule chemotherapy. Journal of Medicinal Chemistry 2016, 59, 6595-6628]. In addition, in in vivo experiments, (-) - taxifolin is active against other viruses such as Coxsackie B4 virus. It is known that Siberian larch (Larix sibirica) produces (-) - taxifolin, which is a natural resource for its extraction. In addition, food preparations containing (-) - taxifolin are readily available from pharmacies and other companies, providing direct and quick access to a potential antiviral drug. It is noteworthy that (-) -taxifolin carries seven hydrogen bonds, which is the highest in the choice of antiviral compounds. Since hydrogen bonds are a key factor determining the specificity of the drug (-) - taxifolin can be a natural alternative to the proposed CP-1-CP-11 inhibitors [Wade, R.C. Goodford, P.J. The role of hydrogen-bonds in drug binding. Progress in clinical and biological research 1989, 289, 433-444].

It is also known that the heparin contained in algae is able to block the SARS-CoV-2 coronavirus better than, for example, the drug Remdesivir, which is actively used in the United States to treat COVID-19 [Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro / Paul S. Kwon, Hanseul Oh, Seok-Joon Kwon, Weihua Jin, Fuming Zhang, Keith Fraser, Jung Joo Hong, Robert J. Linhardt & Jonathan S. Dordick - Cell Discovery, volume 6, Article number: 50 (2020) ]. Scientists have purposefully investigated the properties of heparin, an anticoagulant known to all. This substance has been proven to have exceptional binding properties to the spike protein (S-protein) of SARS-CoV-2 and reduces the activity of this virus.

Known inhibitor of coronavirus SARS-CoV based on an aqueous plant extract, exhibiting dose-dependent activity against SARS-CoV in tests on cell cultures [US application No. 20080038382, IPC A61P 31/12, publ. February 14, 2008]. As an aqueous plant extract, it contains an aqueous extract of the perennial herb Scutellaria spp. (A species of the Shlemnik genus) effective in treating a patient with SARS-CoV infection. This agent inhibits the infectivity of SARS-CoV by about 50% at the highest concentration used (200 μg / ml). The effect is dose dependent, given that less inhibition is observed at the lower dose tested. The significantly higher inhibition was greater than ribavirin (100 μg / ml).

However, this analogue, the SARS-CoV coronavirus inhibitor, has not been tested for activity against the new SARS-CoV-2 coronavirus.

The closest analogue (prototype) is a method for producing melanin [US application No. 20110230562, IPC A61K 31/185, C12P 7/40, publ. 09/22/2011], including: contacting at least one precursor in an aqueous solution with an alkaline pH (sodium carbonate buffer with a pH of about 10.6), containing at least two hydroxyl groups, with a metal-containing catalyst in the presence of oxygen for time sufficient for the formation of melanin. The catalyst is selected from the group consisting of an iron complex, a cobalt complex, a nickel complex and salcomine. The precursor is selected from the group consisting of cinnamic acid, coumarin, phenol, hydroxyflavanone, hydroxynaphthalene, hydroxybenzoic acid, flavonoid, pyrocatechol, hydroxybenzaldehyde, and combinations thereof. The method involves additional purification of water-soluble melanin. Melanin is contemplated for use in a method for treating or preventing a disease in a human or animal, comprising: administering a therapeutically effective amount of melanin against SARS virus (Severe Acute Respiratory Syndrome), in particular SARS-CoV). Synthetic melanins can be used as components of multi-agent prophylaxis or treatment regimens that simultaneously target multiple mechanisms of microbial infection.

Thus, synthetic melanins obtained using the prototype method are active against several viruses, including SARS, and also have low toxicity towards animal cells. Synthetic melanins have shown moderate to high anti-SARS activity in the screening assay.

However, this closest analogue, a melanin-based SARS coronavirus inhibitor, has not been tested for activity against the novel SARS-CoV-2 coronavirus.

The technical result of the present invention is to obtain an inhibitor based on natural melanin from the fungus Inonotus obliquus to prevent replication of the SARS-CoV-2 coronavirus.

The specified technical result is achieved by the fact that in the melanin-based SARS coronavirus replication inhibitor, according to the invention, as melanin, the inhibitor contains natural melanin from the basidal fungus Inonotus obliquus, obtained by alkaline hydrolysis (according to patent RU 2480227 C2, publ. 27.04.2013), precipitation with acid, dissolving the precipitate in ammonia water and drying the final product at a temperature not higher than + 30-40 ° C, the inhibitory aqueous solution of which in the range of 6.218-8.488 μg / ml exhibits 50% antiviral dose-dependent activity against SARS-CoV-2 in cell culture tests Vero.

Changes in the preparation of samples for testing on the SARS-CoV-2 coronavirus are mainly related to the milder drying regime. Drying of the resulting melanin is carried out at a temperature not higher than + 30-40 ° C, which provides higher and more stable indicators of the antiviral activity of melanin in the Vero cell culture.

The invention is illustrated by the drawings shown in FIG. 1 - the results of OD measurement (for samples 20-24 and 20-30 of the inhibitor), depending on the concentration of the drug, are presented in a semi-logarithmic coordinate system. The abscissa (X) shows the concentration of drugs in a logarithmic measurement scale, and the ordinate (Y) shows the OD in a linear measurement scale.

Below are the options for obtaining the claimed inhibitor of the replication of the SARS-CoV-2 coronavirus based on melanin obtained from the pharmacopoeial crushed basidal fungus Inonotus obliquus.

Example 1. Obtaining water-soluble melanin from pharmacy chaga, sample 20-24.

To 5 g of dry crushed to 1 mm (universal mill MF-10 basik IKA) natural raw chaga was added 100 ml of 2% NaOH and kept in an autoclave for 30 minutes at an overpressure of 0.7 atm. Filtered through nylon and paper filters. Melanin was precipitated with concentrated hydrochloric acid and centrifuged in a Centra CL3 centrifuge for 20 min at 4000 rpm. Re-precipitation was carried out 3 times with 1N hydrochloric acid, the precipitate was washed three times with distilled water (1:10). The resulting preparation does not dissolve in water, but it dissolves well in alkalis. The water-soluble form of melanin is obtained by thoroughly dissolving the resulting pigment in a 10% aqueous solution of ammonia, bringing the pH to 7-8, followed by gentle evaporation of the residual ammonia and water to dryness at an air flow temperature of + 30 ° C. The resulting preparation is completely soluble in water.

The test piece was prepared by dissolving dry melanin in distilled water to a concentration of 2 mg in 1 ml.

An inhibitor was obtained containing an aqueous solution of melanin, obtained from the crushed basidal fungus Inonotus obliquus by the method of alkaline hydrolysis and dried at a temperature of + 30 ° C, the inhibitory aqueous solution of which at a concentration of 6.218 μg / ml exhibits 50% antiviral dose-dependent activity against SARS-CoV- 2 in tests on cell culture Vero.

Example 2. Obtaining water-soluble melanin from chaga sclerotia from birch, sample 20-29.

To 5 g of dry crushed to 1 mm (universal mill MF-10 basik IKA) natural raw chaga add 100 ml of 2% NaOH and incubate 3 times in a thermostat at 50 ° C for 24 hours. After each exposure, the supernatant was decanted and the process repeated. Melanin is precipitated from the collected supernatant with concentrated hydrochloric acid and centrifuged in a Centra CL3 centrifuge for 20 min at 4000 rpm for separation with the supernatant. Then purification is carried out by 3-fold reprecipitation with concentrated HCl (1: 1) to pH from 1.5-2.0, washed with distilled water in a ratio of 1:20, pH is adjusted to 7-8 with 10% aqueous ammonia solution. Melanin is dried at a temperature of + 35 ° C. The test piece is prepared by dissolving dry melanin in distilled water at a concentration of 2 mg in 1 ml.

An inhibitor containing an aqueous solution of melanin was obtained, obtained from the crushed basidal fungus Inonotus obliquus by the method of alkaline hydrolysis and dried at + 40 ° С, the inhibitory aqueous solution of which at a concentration of 8.488 μg / ml exhibits 50% antiviral dose-dependent activity against SARS-CoV-2 in tests on cell culture Vero.

Example 3. Obtaining water-soluble melanin from pharmacy chaga, sample 20-30.

To 5 g of dry crushed to 1 mm (universal mill MF-10 basik IKA) natural raw chaga add 100 ml of 2% NaOH and incubate 3 times in a thermostat at + 50 ° C for 24 hours. After each exposure, discard the supernatant and repeat the process. Melanin is precipitated from the collected supernatant with concentrated hydrochloric acid and centrifuged in a Centra CL3 centrifuge for 20 min at 4000 rpm for separation with the supernatant. Then purification is carried out by 3-fold reprecipitation with concentrated HCl (1: 1) to pH 1.5-2.0, washed with distilled water in a ratio of 1:20, adjusted to pH 7-8 with 10% aqueous ammonia solution and dried at + 30 ° C. The test piece was prepared by dissolving dry melanin in distilled water at a concentration of 2 mg in 1 ml.

An inhibitor containing an aqueous solution of melanin was obtained, obtained from the crushed basidal fungus Inonotus obliquus by the method of alkaline hydrolysis and dried at + 30 ° C, the inhibitory aqueous solution of which at a concentration of 8.188 μg / ml exhibits 50% antiviral dose-dependent activity against SARS-CoV-2 in tests on cell culture Vero.

Example 4. Determination of the antiviral activity of samples against SARS-Cov-2 coronavirus in Vero cell cultures.

Cell cultures. We used transplantable cell cultures of the kidney of the African green monkey Vero, obtained from the collection of cell cultures of the FBSI GNTs VB Vector. A monolayer of cells was grown in 96-well plates (0.1-0.15 ml / well of cell suspension with a concentration of 1.0-1.5 × 105 cells / ml) in DMEM medium (OOO Biolot, Russia) in presence of 10% fetal bovine serum (Gibco, USA) with the addition of penicillin (100 U / ml), streptomycin (100 μg / ml) and amphotericin B (0.25 μg / ml) (Antibiotic-Antimycotic (100X) (Gibco, USA) When cells with a virus were cultured, DMEM culture medium with antibiotics, without serum, was used as a maintenance medium.

The SARS-CoV-2 virus strain nCov / Victoria / 1/2020 with an infectious titer of 5.0 ± 0.29 (± 0.57) lg TCTs50 / ml was obtained from the State collection of viral pathogens and rickettsioses of the FBSI SSC VB "Vector".

The virus concentration was determined by titration in a Vero cell culture during cultivation for 3 days at + 37 ° C, 5% CO ₂ and humidity 85-90%. Virus titers were calculated by the Spearman-Kerber method, expressed in decimal logarithms of 50% tissue cytopathic doses in ml (lg TCID50 / ml) and presented as M ± m (± 195) for a 95% confidence level (195) [Zax L. Statistical estimation. M .: Statistics. 1976; 598 s .; Virology Methods Manual. Edited by: Brian WJ Mahy and Hillar O. Kangro. AcademicPress. 1996; 374 p.].

Colorimetric method for in vitro determination of cytotoxicity and antiviral activity of drugs.

The study of cytotoxicity and antiviral efficacy of drugs was carried out by the colorimetric method - by changing the optical density (OD) of a dye solution absorbed by living cells in a monolayer [Oestereich L., Ltidtke A., Wurr S., Rieger T., Munoz-Fontela C, Gtinther S. Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model // Antiviral Research, 2014., 105: 17-21; Baker R.O., Bray M., Huggins J.W. Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections // Antiviral Research. 2003, 57: 13-23; Paragas J., Whitehouse C. A., Endy T.P., Bray M. A simple assay for determining antiviral activity against Crimean-Congo hemorrhagic fever virus // Antiviral Research. 2004, 62: 21-25].

To evaluate the effectiveness of each, 8 consecutive 3-fold dilutions of the preparations were used. The initial concentration of extracts on the plates when assessing the cytotoxicity and antiviral activity of the drugs was 300 μg / ml.

When evaluating the antiviral activity of the preparations to the wells of 96-well plates with a monolayer of Vero cells was added 0.1 ml of a dilution of extracts in DMEM medium (OOO "Biolot", Russia), and after 2 h incubation at + 37 ° C, 5% CO ₂ added 0.1 ml of virus dilution in serum-free DMEM medium with a multiplicity of infection (MOI) of 0.1 TCID50 / cell. With such a multiplicity of infection, the cytopathic effect of the virus on the cell monolayer after 3 days of incubation reaches at least 90% (control of the virus without adding the drug). In addition, an intact monolayer (without virus and drugs) was used as a "cell control".

After 3 days of incubation in the wells of the plate with a monolayer of Vero cells, a vital (intravital) neutral red dye 0.05 ml was added to the culture medium for 1.5 hours at + 37 ° C. After that, the cell monolayer was washed twice with saline, lysis buffer was added, and after 30 min the optical density (OD) was determined.

Optical density was measured using a Multiskan FC plate reader (ThermoScientific, USA). The results of measuring OD depending on the concentration of the drug are presented in a semilogarithmic coordinate system (Fig. 1). In this case, the abscissa axis (X) shows the concentration of drugs in a logarithmic measurement scale, and the ordinate axis (Y) - OD in a linear measurement scale. The OD values were used to calculate the 50% toxic concentration (TC ₅₀ in μg / ml) and 50% inhibitory (effective) concentration (IC ₅₀ in μg / ml) of the preparation using the SoftMaxPro-4.0 software. TC ₅₀ is the concentration of the drug in the well of the plate that destroys 50% of the cells in the monolayer. IC ₅₀ is the concentration of drug that inhibits viral replication and keeps 50% of cells viable.

Based on these indicators, the selectivity index (SI) of the drug was calculated: SI = TC ₅₀ : .IC ₅₀ [Guidelines for experimental (preclinical) study of new pharmacological substances. Ed. Khabrieva R.U. M .: JSC "Medicine Publishing House". 2005; 832 s .; Methodical recommendations for studying the specific activity of interferon inducers. Guidelines for conducting clinical trials of medicinal products. Part One / Ed. A.N. Mironov. - M .: Grif and K, 2012. - 944 p.].

Example 5. Results of determining the antiviral activity of water-soluble melanin from chaga against SARS-Cov-2 coronavirus on Vero cells

Table 1 shows the results of evaluating the antiviral activity of melanin from natural chaga against coronavirus, strain nCov / Victoria / 1/2020.

The best melanin sample for inhibition of coronavirus replication, strain nCov / Victoria / 1/2020, in Vero cell culture is a 20-24 sample obtained by alkaline hydrolysis in an autoclave with an exposure of 30 minutes and an overpressure of 0.7 atm, followed by removal of an aqueous solution of ammonia from the product at a temperature of + 30 ° C and bringing it to a dry state.

Two samples of melanin 20-29 and 20-30 were obtained from chaga by alkaline hydrolysis in a thermostat at + 50 ° C (3 times for 24 hours). Chaga samples differed in origin. Melanin samples 20-24 and 20-30 were obtained from pharmacy chaga. Melanin sample 20-29 was obtained from chaga sclerotia collected from birch.

From the data obtained, the following conclusions can be drawn: alkaline hydrolysis using an autoclave makes it possible to obtain melanin samples from the natural fungus Inonotus obliquus, the inhibitory concentration of which in suppressing the replication of coronavirus, the nCov / Victoria / 1/2020 strain in the Vero cell culture, is higher than in the samples obtained in the thermostat (table 1).

To remove ammonia water, melanin samples from chaga are proposed to be dried at a temperature not higher than + 30-40 ° C. Melanin samples dried at temperatures above + 40-50 ° C are characterized by lower activity against coronavirus, as exemplified by the nCov / Victoria / 1/2020 strain. ... The inhibitory concentration IC ₅₀ μg / ml for two samples dried at + 50 ° C was 19.405; 22.992 μg / ml with the same toxicity of 666.7, while in the samples dried at a temperature not higher than + 30-40 ° С, presented in Table 1, IC50 μg / ml was in the range of 6.2-8.5 μg / ml.

Claims (1)

The use of natural melanin from the basidal fungus Inonotus obliquus, obtained by alkaline hydrolysis of Inonotus obliquus, acid precipitation, dissolution of the precipitate in ammonia water and drying the final product at a temperature not higher than 30-40 ° C, as an inhibitor of SARS coronavirus replication, the aqueous solution of which is in the range of 6.218 -8.488 μg / ml shows 50% antiviral dose-dependent activity against SARS-CoV-2 in Vero cell culture tests.

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