RU2635765C1 - Means with antiviral activity against influenza virus - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: means that is a bisulphite derivative of oxidized linear or cyclic non-reducing oligosaccharides containing up to 7 identical monosaccharide glucose residues containing a bisulfite moiety bound directly to a carbon atom of the general formula [(Glu_ox)_n-C_x]-(SO₃M)_x, where n=2-7; x=4-14, M = sodium or potassium metal) with a degree of substitution of 11.5-14.5% in terms of the quantitative content of sulfur.

EFFECT: increased antiviral activity.

4 dwg, 2 tbl, 7 ex

Description

The invention relates to the pharmaceutical industry, medicine, veterinary medicine and virology, and relates to agents having antiviral activity against influenza virus.

Flu is known to be a serious public health problem. Influenza viruses infect various organs and systems, cause enormous damage to the health of the population, in 5% of patients cause severe hypertoxic forms of the disease, mortality during the epidemic increases to 2.5%. The influenza virus is characterized by a high rate of emergence of new strains of the virus, as well as the emergence of strains resistant to existing drugs.

To prevent and treat the disease, a wide range of pathogenetic, immunomodulatory, general strengthening drugs is currently known, along with means of specific anti-influenza therapy. The latter drugs are represented by chemical compounds of two groups that differ in the mechanism of action and targets in the life cycle of the influenza virus. The drugs of the first group - rimantadine (α-methyl-1-adamantylmethylamine hydrochloride) and amantadine (1-aminoadamantane) block the influenza virus M2 protein, which plays the role of an ion channel in the viral membrane, thereby interfering with the process of fusion of the virus membranes and lysosomal vacuole (Scholtissek C ., Quack G., Klenk HD, Webster RG // Antiviral Res. 1998, v. 37, p. 83-95). The drugs of the second group are aimed at inhibiting viral neuraminidase, an enzyme necessary for the normal budding of viral particles and the manifestation of the infectious properties of the influenza virus. This group of compounds includes zanamivir [5- (acetylamino) -4 - [(aminoiminomethyl) amino] -2,6-anhydro-3,4,5-tridesoxy-D-glycero-D-galactonone-2-enonic acid] , oseltamivir [(3R, 4R, 5S) -ethyl-4-acetamido-5-amino-3- (pentan-3-yloxy) cyclohex-1-enecarboxylate] (Woodhead M., Lavanchy D., Johnston S., Colman P., Fleming D. // Int. J. Clin. Pract. 2000, v. 54 (9), p. 604-610) and peramivir [(1S, 2S, 3S, 4R) -3 - [(1S) -1-acetamido-2-ethyl-butyl] -4- (diaminomethylidenamino) -2-hydroxy-cyclopentane-1-carboxylic acid] (O'Malley P. // Clin. Nurse Spec. 2010, v. 24 (2) , p. 51-53).

Both groups of compounds have their drawbacks. In relation to the group of adamantane derivatives, one can note a relatively high toxicity, a narrow spectrum of action (the drugs are active against influenza A, but not against influenza B) and the rapid formation of virus resistance to drugs. Neuraminidase inhibitors are characterized by slightly lower clinical efficacy and high cost of synthesis, which makes these drugs less accessible for widespread use.

Known agent having interferon-inducing and immunomodulating activities (Arbidol), which is the hydrochloride of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole monohydrate (Patent RU 2033156 C1, op. 20.04.1995 )

Arbidol has a specific antiviral effect, inhibiting the fusion of the viral membrane with cell membranes, which prevents the virus from entering the cells and impairs its reproduction (Leneva I.A. et al. // Antiviral Res. 2009, 81 (2), 132-140). In addition, arbidol enhances the overall antiviral defense, stimulating the production of interferons and increasing the activity of cellular immunity (Glushkov R.G. et al. // Vestnik RAMS, 1999, 3, 36-40).

The disadvantages of arbidol are its low solubility and use in complex therapy as an immunostimulating, and not as an antiviral agent.

It should be noted that all of the listed drugs are synthetic drugs.

But natural and semi-synthetic compounds that act against the influenza virus are known, for example, sulfated carbohydrates have a high specific biological activity, including antiviral activity (Ikeda S, Neyts J, Verma S, Wickramasinghe A. "In Vitro and In Vivo Inhibition of Ortho- and Paramyxovirus Infections by a New Class of Sulfonic Acid. Polymers Interacting with Virus-Cell Binding and / or Fusion. "// Antimicrobial. Agents for Chemotherapy: 1994, v. 38, p. 256-259; Fitton JH" Therapies from fucoidan; multifunctional marine polymers ". Marine Drugs: 2011, v. 9, p. 1731-60).

М,

R, Kalthoff D, Pflugfelder В, Graf P, Frank-Gehrke B, Beer M, Fazekas T, Unger H, Prieschl-Grassauer E, Grassauer A. "Iota-carrageenan is a potent inhibitor of influenza A virus infection. // PLoS One: 2010, No.5, p. e14320; Grassauer A, Weinmuellner R, Meier C, Pretsch A, Prieschl-Grassauer E, Unger H. "Iota-Carrageenan is a potent inhibitor of rhinovirus infection." // Virol. J.: 2008, v. 5, p. 107).Sulfated derivatives of polysaccharides, mainly carrageenans, as well as dextran sulfate, chondroitin sulfate, heparin sulfate, sulfated derivatives of cellulose, Kurdlan, xylan, fucoidans - the so-called heparinoids, can directly interact with the influenza virus and other enveloped viruses, such as the herpes virus or the virus human immunodeficiency, preventing their adsorption and internalization into the cell. Unfortunately, their solubility is very limited, especially in aqueous solutions containing potassium or calcium ions. Their use is even more limited by the presence of anticoagulant activity, which causes significant side effects, the most dangerous of which are thrombocytopenia followed by heparin-induced thrombosis - the so-called "white clot syndrome", osteoporosis, skin necrosis, alopecia, hypoaldosteronism, blood aggregation and most commonly bleeding. The use of heparinoids is contraindicated in patients with active hemorrhage or hemophilia, purpura, thrombocytopenia, intracranial hemorrhage, bacterial endocarditis, active tuberculosis, increased capillary permeability, ulcerative lesions of the gastrointestinal tract, severe hypertension, and the threat of abortion or visceral carcinoma. However, for example, i-carrageenan, which is a sulfated polysaccharide, has been clinically tested and is currently licensed and sold in some countries as a flu medicine. (Leibbrandt A, Meier C,

M

R, Kalthoff D, Pflugfelder B, Graf P, Frank-Gehrke B, Beer M, Fazekas T, Unger H, Prieschl-Grassauer E, Grassauer A. "Iota-carrageenan is a potent inhibitor of influenza A virus infection. // PLoS One: 2010, No.5, p. E14320; Grassauer A, Weinmuellner R, Meier C, Pretsch A, Prieschl-Grassauer E, Unger H. "Iota-Carrageenan is a potent inhibitor of rhinovirus infection." // Virol. J .: 2008, v. 5, p. 107).

It is also known that c-carrageenan and some of its derivatives, with an average molecular weight of about 2 kDa, effectively inhibit the replication of influenza virus (Fengxiang Tang, Fang Chen, Feng Li "Preparation and potential in vivo anti-influenza virus activity of low molecular weight κ-carrageenans and their derivatives ", Journal of Applied Polymer Science, v. 127, No. 3, p. 2110-2115). The degree of polymerization of k-carrageenan oligosaccharide is from 3 to 13 units. The structural formula of k-carrageenan oligosaccharide mainly consists of (1,4) -β-d-galactose and (1,3) -α-d-galactose with a sulfation degree of 0.4 sulfate groups per sugar residue.

All of the above sulfated poly- and oligocharides are O-sulfates and / or O- and N-sulfates, such as:

K-carrageenan:

and / or O-, N-sulfates (chitosan sulfate):

in these polysaccharides is sulfur bound to the carbon atoms of the pyranose ring via oxygen or nitrogen, and for similar sulfo derivatives of carbohydrates in which sulfur is bound directly to the carbon atom (C-sulfates)? antiviral activity is not known.

At the same time, C-sulfates of other types of organic compounds are known to have various pharmacological properties: antiviral, anthelmintic, antiprotozoal, antitumor activities (Linhardt RJ et al. // Carbohydrate in Drug Designing. - New York, 1997), amyloid binding ability in in vivo experiments, which makes it possible to use them as potential agents for the treatment of Alzheimer's disease (Kisilevsky R. et al. // Nature Medicine. - 1995. - No. 1. - P. 143-148), but their widespread use limits high cytotoxicity and receptor non-select vnost, as well as a large number of side effects.

Synthetic polymers also belong to C-sulfates - N-sulfonate derivatives of poly (allylamine) hydrochloride with a molecular weight of 56 kDa and a degree of substitution of 98%, which showed strong antiviral activity against replication of influenza A virus in vitro and ex vivo, mainly in the late stages of infection . (J. Ciejka, A. Milewska, M. Wytrwal, J. Wojarski, A. Golda, M. Ochman, M. Nowakowska, K. Szczubialka, K. Pyrc, "Novel polyanions inhibiting replication of influenza". // Antimicrobal Agents for Chemotherapy: 2016, v. 60. No.4, p. 1955-1966). The mechanism of their antiviral action is similar to the mechanism of action of carrageenans and is to inhibit the release of the virus from the cell. Incubation of MDCK cells with N-sulfonated polyallylamine hydrochloride at a concentration of 5 mg / ml during infection and within 48 hours after infection resulted in a decrease in A / H3N2 / EVA virus replication. The virus titer in the supernatant of the treated cells decreased 10,000 times (from 2 × 10 ⁷ TCID ₅₀ to 7 × 10 ² TCID ₅₀ (tissue culture infectious doses). It was shown that N-sulfonated polyallylamine hydrochloride in concentrations lower than 5 mg / ml, not toxic to MDCK cells.

The closest in structure and molecular weight to the claimed agent - the prototype, are polyanionic low molecular weight (<10 kDa) sulfonate polymers such as, for example, a copolymer of urea and biphenyl disulfonic acid (C-sulfates): poly {imino [2,2'- disulfo (1,1'-biphenyl) -4,4'-diyl] iminocarbonyl-1,4-phenylenecarbonyl} synthetically prepared and having antiviral activity (US Pat. No. 5,728,731 (A), 1998; SP Ahmed, AD Cardin, CG Bridges, RL Jacson, MJ Mullins, AS Tyms. Potent inhibition of herpes simplex virus by MDL 101028, a novel biphenyl disulfonic acid urea co-polymer. // Antiviral Chemistry & Chemotherapy, 1995, v. 6. No.1, p . 3 4-42.), Possessing antiviral activity, namely against enveloped viruses, for example, herpes virus (ED ₅₀ = 0.29 mg / ml, or IC ₅₀ = 1.1 mg / ml).

The disadvantage of the prototype is the insufficient level of inactivation of the virus, since after the cells were incubated with the drug, part of the virus remained viable and, accordingly, could lead to the resumption of the infectious process.

The objective of the invention is to obtain a new effective, low toxic pharmacologically active agent with antiviral activity against influenza virus.

The technical result of the invention is to increase antiviral activity and the expansion of the range of products with antiviral activity against influenza virus.

This object is achieved by obtaining bisulfite derivatives of oxidized non-reducing homo-oligosaccharides having in their composition a bisulfite (or otherwise sulfonate) moiety directly bonded to a carbon atom (C-sulfates), the general formula [(Glu _ox ) _n -C _x ] - (SO ₃ R) _x , where

[(Glu _ox ) _n -C _x ] is an oxidized non-reducing homo-oligosaccharide, where n is the number of monosaccharide units equal to 2-7, and x is the number of sulfonated carbon atoms of a linear or cyclic structure equal to 4-14;

SO ₃ R - bisulfite (sulfonate) group,

R = H, M, where M are metals of groups I or II,

with the degree of substitution, in terms of quantitative sulfur content, 11.5-14.5%.

The proposed tool is obtained on the basis of non-reducing homo-oligosaccharides by periodically oxidizing them under standard conditions (General Organic Chemistry // Ed. By D. Barton and UD Ollis. T. 11. Lipids, carbohydrates, macromolecules, biosynthesis. // Ed. E. Khaslama - Translated from English / Edited by N.K. Kochetkov. - M .: Chemistry, 1986, p. 376), followed by isolation in the form of a bisulfite derivative.

To do this, the initial oligosaccharide is dissolved with stirring in water (1:50), then a 2-3% solution of potassium or sodium periodate (2: 5) is added, the mixture is kept at room temperature in the dark from several hours to several days, maintaining slightly acidic pH values using bicarbonate buffer. Then the reaction mixture is treated with a 40% aqueous solution of potassium or sodium pyrosulfite and the obtained bisulfite derivative of the oxidized oligosaccharide is purified from low molecular weight impurities by ultrafiltration and / or preparative gel permeation chromatography and dried by freeze-drying or spray drying.

As non-reducing homo-oligosaccharides, linear or cyclic carbohydrates containing up to 10 identical monosaccharide residues, most often glucose [(Glu) _n - (glucosyl) _n-1- glycosides, where n is the number of monosaccharide units] can be used. Moreover, as a result of oxidation, one to several oxidized monomers can be obtained, up to a completely oxidized oligosaccharide, while both primary and secondary hydroxyl groups of the monomer will undergo oxidation. Thus, in the macromolecule of an oxidized oligosaccharide, the same or different monomers oxidized by primary hydroxyl groups and / or secondary hydroxyl groups may be present.

The invention is illustrated by the following examples.

Example 1

1 g of trehalose was dissolved with stirring in 50 ml of purified water, then 20 ml of a 2% solution of potassium periodate (KJO ₄ ) was added. Oxidation was carried out with periodic stirring at room temperature in the dark for no more than 6 hours, maintaining slightly acidic pH values using a bicarbonate buffer. After that, the reaction mixture was treated with 18 ml of a 40% aqueous solution of potassium pyrosulfite (K ₂ S ₂ O ₅ ) and after 1 h, the low molecular weight impurities were purified by ultrafiltration through a polyethersulfone membrane module with a pore size of 1-5 kDa for 1 h. The obtained retant sprayed through a pneumatic nozzle (droplet size up to 150 microns) for 15 minutes at a system pressure of -65 mbar and a temperature at the inlet of the spray dryer + 120 ° C, and at the outlet + 50 ° C.

The yield of product [(Glu _ox ) ₂ -C ₄ ] - (SO ₃ K) ₄ was 56%, the quantitative sulfur content was 11.6%.

Example 2

Determination of the inhibitory effect of the bisulfite derivative of oxidized trehalose [(Glu _ox ) ₂ -C ₄ ] - (SO ₃ K) ₄ on the reproduction of the influenza virus subtype H1N1 in MDCK cell culture.

MDCK cells were added 5 × 10 ⁴ per well of a 24-well plate in DMEM medium containing 10% fetal calf serum and antibiotics and incubated for 48 hours at 37 ° C until a complete monolayer was formed. The cells were washed with PBS, then the compound [(Glu _ox ) ₂ —C ₄ ] - (SO ₃ K) _{4 was} added in various concentrations from 1.25 to 5 mg / ml and the virus was introduced simultaneously. Then, the cells were incubated with the compound [(Glu _ox ) ₂ -C ₄ ] - (SO ₃ K) ₄ and the virus without changing the medium for 48 h; MDCK cells incubated only in the presence of the influenza virus were used as a control. For infection of the cells used the virus A / WSN / 33 (H1N1) with a multiplicity of infection (MOI) of 0.01 IU / cell (focus-forming units). At the end of the incubation, the extracellular fluid was collected and the titer of the virus in it was determined according to the FOE method described earlier (Goncharova E.P., Kovpak M.P., Ryabchikova E.I., Konevets D.A., Silnikov V.N., Zenkova MA, Vlasov VV The cleavage of the viral genome using artificial ribonucleases is a new method of inactivation of RNA-containing viruses // Doklady AN AN 2009. T. 427. P. 840-843).

MDCK cells were grown in 96-well plates in DMEM until monolayer formation. Then the cells were infected with various dilutions of the obtained samples. After incubation at 37 ° С for 24 h, the cells were fixed for 10-15 min in chilled acetone and treated with a monoclonal antibody preparation for the NP protein of influenza A virus. After the incubation with antibodies, anti-species antibodies conjugated to horseradish peroxidase (Sigma) were added. The presence of peroxidase in the complexes on the surface of infected cells was detected in an enzymatic reaction with a chromogenic substrate 3-amino-9-ethylcarbazole (AEC) (Sigma). Virus-infected stained cells (foci) were counted and the titer of the virus was presented as a decimal logarithm (log10).

Table 1 shows virus titers (lg FOE / ml) in the culture medium 48 hours after infection of the cells with the influenza A / WSN / 33 virus, depending on the concentration of the compound [(Glu _ox ) ₂ -C ₄ ] - (SO ₃ K) ₄ .

Example 3

1 g of β-cyclodextrin was dissolved with stirring in 50 ml of purified water, then 50 ml of a 3% sodium periodate solution (NaJO ₄ ) was added. The oxidation was carried out with periodic stirring at room temperature in the dark for 5 days, maintaining slightly acidic pH values using a bicarbonate buffer. After that, the reaction mixture was treated with 50 ml of a 40% aqueous solution of sodium pyrosulfite (Na ₂ S ₂ O ₅ ) and after 15 minutes the obtained compound was purified from low molecular weight impurities by ultrafiltration through a polyethersulfone membrane module with a pore size of 1-5 kDa for 1 h, followed by purification by preparative gel permeation chromatography on a Bio Gel P4 column (BioRad) (d = 1.5 cm; l = 70 cm, eluent 0.2% solution (NH ₄ ) ₂ CO _{3. The} purified product was frozen at t = -40 ° C and subjected to sublimation at t≤-55 ° C for no more than 3 hours

The yield of product [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ , amounted to 60%, the quantitative sulfur content of 14.3%.

Example 4

Determination of the cytotoxicity of a bisulfite derivative of an oxidized β-cyclodextrin [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄

The cytotoxicity of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ was determined using the MTT test. 1.5 × 10 ⁴ MDCK cells were added to the wells of a 96-well plate in 100 μl of DMEM medium containing 10% fetal calf serum and antibiotics. Cells were incubated until a monolayer was formed at 37 ° C and 5% CO ₂ . Next, the medium was selected, the cells were washed with PBS (phosphate buffer, MP Biomedical), 100 μl of DMEM medium containing the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at various concentrations were added to the wells of the tablet, and incubated within 24 hours. At the end of the incubation, the medium was taken from the wells, 100 μl of MTT solution (0.5 mg / ml) in DMEM medium was added to the wells, and incubated for 3 hours at 37 ° C. Then the medium was taken, 100 μl of DMSO were added to the wells and incubated for 20 min. After incubation, the optical density was measured on a Multiscan RC multichannel spectrophotometer at a wavelength of 570 nm. The data were presented as the fraction of living cells, assuming the optical density of the solution in the control samples — cells incubated in the absence of compound as corresponding to 100% of the living cells in the sample. The concentration of the compound at which 50% of the cells survived after 24 hours of incubation was designated IC ₅₀ . The dependence of the number of living MDCK cells on the concentration of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ in the medium is shown in FIG. 1, while the number of cells in the wells incubated in the absence of compound was taken as 100%.

It was found that the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ in concentrations less than 5 mg / ml is not toxic to MDCK cells. The IC ₅₀ for the test compound is 15 ± 3.3 mg / ml.

Example 5

Determination of the inhibitory effect of the bisulfite derivative of the oxidized [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ on the reproduction of the influenza virus subtype H1N1 in MDCK cell culture.

MDCK cells were added 5 × 10 ⁴ per well of a 24-well plate in DMEM medium containing 10% fetal calf serum and antibiotics and incubated for 48 hours at 37 ° C until a complete monolayer was formed. Cells were washed with PBS, then the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) _{14 was} added in various concentrations from 0.6 to 5 mg / ml and the virus was introduced simultaneously. Then, the cells were incubated with the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ and the virus without changing the medium for 48 h; MDCK cells incubated only in the presence of the influenza virus were used as a control. When applying the treatment regimen, the cells were infected with the influenza virus, after 1 h the virus was removed, the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ was added at a concentration of 5 mg / ml and incubated for 48 h. Cells were used for infection virus A / WSN / 33 (H1N1) with a multiplicity of infection (MOI) of 0.01 IU / cell (focus-forming units). At the end of the incubation, the extracellular fluid was collected and the titer of the virus in it was determined by the FOE method described earlier [Goncharova EP, Kovpak MP, Ryabchikova E.I. et al. // Reports of the Academy of Sciences. - 2009. - T. 427. - No. 6. - S. 840-843].

MDCK cells were grown in 96-well plates in DMEM until monolayer formation. Then the cells were infected with various dilutions of the obtained samples. After incubation at 37 ° С for 24 h, the cells were fixed for 10-15 min in chilled acetone and treated with a monoclonal antibody preparation for the NP protein of influenza A virus. After the incubation with antibodies, anti-species antibodies conjugated to horseradish peroxidase (Sigma) were added. The presence of peroxidase in the complexes on the surface of infected cells was detected in an enzymatic reaction with a chromogenic substrate 3-amino-9-ethylcarbazole (AEC) (Sigma). Virus-infected stained cells (foci) were counted and the titer of the virus was presented as a decimal logarithm (log10).

Table 2 shows virus titers (lg FOE / ml) in the culture medium 48 hours after infection of the cells with the influenza A / WSN / 33 virus, depending on the concentration of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ and the time it was added to the cells.

It was found that as a result of treatment of cells with the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at a concentration of from 0.6 mg / ml to 5 mg / ml, virus reproduction in MDCK cells decreased simultaneously with the introduction of the virus 1,000,000 times (by 6 lg) (prophylactic regimen) compared with the control.

Incubation of cells at a compound concentration of 0.3 mg / ml led to a decrease in the titer of the virus by more than 10 times. When applying the treatment regimen for treating cells with the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at a concentration of 5 mg / ml, the titer of the virus decreased by 2.2 lg FOE / ml.

Example 6. Study of the toxicity of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ upon intranasal administration of the BALB / c line to mice.

The toxicity of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) _{14 was} studied in BALB / c mice (females weighing 14-16 g) with intranasal administration of the drug. The test compound at a dose of 312 mg / kg was once administered intranasally to mice under mild ether anesthesia in a volume of 30 μl. When assessing the toxicity of the compound, the general condition of the animals, the characteristics of their behavior, the intensity and nature of the motor activity, the condition of the hair and skin, consumption of feed and water, and change in body weight were regularly recorded. All mice were weighed daily, starting from the time of drug administration. The results of the effect of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at a dose of 312 mg / kg on the weight of mice after a single intranasal administration are plotted in FIG. 2. No significant differences were found in body weight, behavior changes, animal hair condition, feed and water consumption between the group of mice that received intranasally [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ and the control group that received PBS, indicating a low toxicity of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ .

Example 7. Determination of the antiviral activity of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ in vivo.

- контроль, животные получали среду разведения;

- животные получали соединение [(Glu_ox)₇-C₁₄]-(SO₃Na)₁₄ однократно,

- животные получали соединение [(Gfu_ox)₇-C₁₄]-(SO₃Na)₁₄ двукратно. Результаты данного эксперимента представлены на фигуре 3 (Б). Показано, что интраназальное применение соединения [(Glu_ox)₇-C₁₄]-(SO₃Na)₁₄ приводит к снижению специфической смертности животных. При однократном введении соединения [(Glu_ox)₇-C₁₄]-(SO₃Na)₁₄ выжило 66% животных, тогда как его двукратное введение обеспечивало полную защиту от заражения вирусом гриппа.To determine the antiviral activity of [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ in vivo, the test compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at a dose of 312 mg / kg was administered to mice line BALB / c (males weighing 14-16 g) intranasally under light ether anesthesia in a volume of 30 μl once a day and then infected with influenza virus A / WSN / 33 (H1N1) (dose 5 LD ₅₀ per mouse). The compound was administered once to the first group of mice (n = 6), the compound was administered twice to the second group of mice (n = 6): immediately after infection and one day after the first injection. The infected mice of the control group (n = 6) were injected with dilution medium. The experimental design is shown in FIG. 3 (A), where

- control, animals received a breeding medium;

- the animals received the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ once,

- the animals received the compound [(Gfu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ twice. The results of this experiment are presented in figure 3 (B). It was shown that the intranasal use of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ leads to a decrease in the specific mortality of animals. With a single injection of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ , 66% of the animals survived, while its double administration provided complete protection against infection with the influenza virus.

To determine the infectious titer of influenza virus in the lung tissue of animals, the lungs of mice recovered on the 4th day after infection were homogenized in a 10-fold volume of DMEM medium and serial dilutions were prepared to determine the titer of the virus by the FOE method. In FIG. 4. shows the values of the titer of influenza virus A / WSN / 33 (H1N1) in the lung tissue of mice, lg (FOE / ml) after intranasal administration of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ or medium, infected with a dose of 5 LD ₅₀ on the 4th day after infection (median values are indicated).

In FIG. Figure 4 shows that the titer of the virus in the lungs of mice receiving a single compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ decreased by 3 log CFU / ml (1000 times) compared with the titer of the virus in the lungs mice of the control group (virus + environment).

It was shown that the intranasal use of the compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ leads to a decrease in the specific mortality of animals at a dose of 312 mg / kg and a decrease in virus titers in lung tissue by 3 lg CFU / ml (in 1000 times).

Thus, it was found that:

one). Compounds [(Glu _ox ) _n -C _x ] - (SO ₃ M) _x (where n = 2-7; x = 4-14, linear or cyclic, M = metal sodium or potassium) in the tested doses effectively reduce reproduction influenza virus in MDCK cell cultures;

2). Maximum antiviral activity was observed for compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ under conditions when it was added before infection of MDCK cells, followed by incubation of infected cells with compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ . Under these conditions, a decrease in influenza virus titers under the action of the test compound at a concentration of from 0.6 to 5 mg / ml was 6.0 lg FOE / ml (1,000,000 times)

3). The compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) _{14 is} not toxic to model animals at a dose of 312 mg / kg with intranasal administration.

four). The compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ at a dose of 312 mg / kg with a single administration reduces the reproduction of influenza virus in lung tissue by 3 lg FOU / ml (1000 times).

5). The compound [(Glu _ox ) ₇ -C ₁₄ ] - (SO ₃ Na) ₁₄ provides complete protection of animals against infection with the influenza virus when administered intranasally twice at a dose of 312 mg / kg.

The proposed tool in the form of compounds [(Glu _ox ) _n -C _x ] - (SO ₃ M) _x (where n = 2-7; x = 4-14, linear or cyclic structure, M = metal sodium or potassium) can be used to prevent and treat influenza virus infection. The proposed tool has increased antiviral activity by reducing the reproduction of influenza virus in the lung tissue, which provides complete protection of animals from infection with the influenza virus.

Claims (1)

An agent with antiviral activity against influenza virus, which is a bisulfite derivative of an oxidized linear or cyclic non-reducing oligosaccharide containing up to 7 identical monosaccharide glucose residues, having a bisulfite group bonded directly to a carbon atom of the general formula [(Glu _ox ) _n - C _x ] - (SO ₃ M) _x , where n = 2-7; x = 4-14, M = metal sodium or potassium) with a degree of substitution, in terms of quantitative sulfur content, 11.5-14.5%.

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