RU2302236C2 - Preparation being of antihypoxic and cytoprotector activities and applied for decreasing remantadin toxicity at treating grippe infection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the suggested compound being 3,6-dioxocyclohexa-1,4-dien-1,2,4,5-sodium tetra-sulfonate is of antihypoxic and cytoprotector action. Moreover, the composition has been suggested being of anti-grippe action. The composition declared includes remantadin and 3,6-dioxocyclohexa-1,4-dien-1,2,4,5- sodium tetrasulfonate.

EFFECT: the compound mentioned enables to decrease remantadin toxicity during its usage at high dosage during treating the grippe infection.

4 cl, 6 dwg

Description

The invention relates to medicine, namely to pharmaceutical preparations. The invention revealed previously unknown properties of the compound 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium, namely: its cytoprotective effect, the ability to exhibit antihypoxic activity. The invention relates to the combined use of remantadine and the compound 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium. Such a composition is useful in the treatment of influenza infection. The combination of these two drugs can significantly reduce the toxic side effects of remantadine and ensure the use of the latter in a wide range of therapeutic dosages.

The literature indicates that, at standard dosages, remantadine is effective only against influenza A viruses, while increased doses expand its spectrum of action against other viruses, for example, influenza B viruses, which remantadine does not work with when used alone [Vidal Handbook. Medicines in Russia, 1998, AstraFarmService, M., E-197; Kiselev O.I., Deeva E.G., Slita A.V., Platonov V.G. Antiviral drugs for the treatment of influenza and acute respiratory infections. Design of drugs based on polymer carriers. "Time", St. Petersburg, 2000].

Influenza is a massive viral disease that is accompanied by complications and even those that are fatal. Annual influenza epidemics have a significant impact on the overall health of a nation and have serious economic and social consequences. Therefore, a constant search for new means for its treatment is ongoing.

Etiotropic anti-influenza drugs are arbidol, zanivir and oseltamivir. The mechanism of the antiviral action of arbidol has not yet been fully studied; moreover, it is effective only in the very early stages of influenza infection. Zanimivir and oseltamivir are neuraminidase inhibitors. These are imported very expensive drugs, due to which their mass use during the period of influenza epidemics is unlikely [Kiselev OI, Deeva E.G., Slita A.V., Platonov V.G. Antiviral drugs for the treatment of influenza and acute respiratory infections. Design of drugs based on polymer carriers. "Time", St. Petersburg, 2000].

Remantadine, one of the leading means of the etiotropic treatment of influenza caused by the A-type influenza virus, is a well-proven drug in the fight against influenza infection [Mashkovsky MD Medicines, part 2, M., "Medicine", 1993, S. 295-306]. Remantadine (rimantadine) - α-methyl-1-adamantylmethylamine hydrochloride - is used in medical practice in the form of white tablets, a bitter taste. Remantadine has antiviral activity against a wide range of strains of type A influenza virus, especially the H3N2 serotype.

The mechanism of its action has been sufficiently studied [Efimov E.I., Razgulin S.A., Military. Honey. Journal 1995, vol. 80 (2), 48-49; Kiselev O.I., Blinov V.M., Kozeletskaya K.I. and other Bulletin of RAMS, 1993, No. 3, 10-15]. It effectively interacts with the membranes of infected cells, in particular with negatively charged phospholipids. The hydrophobic core of adamantane intercalates into the membrane between fatty acid residues, where the compound almost completely blocks the function of the M2 viral protein, which acts as a proton pump. Thus, the suppression of the infectious activity of the virus at the stages of its receptor-dependent endocytosis, decapsidation in the phagolysosome, as well as self-assembly of viral particles and budding [Kiselev OI, Blinov V.M., Kozeletskaya K.I. and other Bulletin of RAMS, 1993, No. 3, 10-15].

Remantadine is prescribed to patients in a dose of 100-300 mg per daily intake, fractionally or simultaneously.

At the same time, remantadine in the conventional dosage form has a number of significant disadvantages, the main of which are: 1) the drug is active only against type A influenza virus; 2) relatively high toxicity - when taken in high doses, side effects are recorded in relation to the central nervous system, liver and kidneys, and the gastrointestinal tract; 3) the rapid formation of resistant strains of the virus. Due to the side effect of the drug, there are limitations in its administration to patients, for example, it is contraindicated in epilepsy [Vidal, 1998, AstraFarmServis, M., E-197; Mashkovsky M.D. Medicines M., "New Wave", 2000, V.2, s.325].

The aim of this invention was to reduce the toxicity of remantadine and the creation of a new composition based on remantadine for the treatment of influenza infection. Reducing the toxicity of remantadine would allow its use in high doses, without fear of complications due to the side effects of remantadine. The latter has a fairly high toxicity: when it is already 1-5 mg / l in the blood plasma, it causes toxic effects in the central nervous system, increases the risk of epileptic seizures, and increases the risk of hemorrhagic stroke [Vidal, 1998, AstraFarmServis, M., E-197; Mashkovsky M.D. Medicines M., "New Wave", 2000, V.2, s.325].

The task was accomplished by adding to the active principle - remantadine - the chemical compound 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium, described and characterized in RF patent No. 2244546: it has antioxidant activity, which is necessary to eliminate the effects of oxidative stress and antiviral activity against influenza infection. In the claimed invention it is established that this compound also has antihypoxic and cytoprotective effects.

The toxic effect of remantadine is due to its effect at the cellular level, therefore, testing was carried out in the laboratory using tests on cell cultures sensitive to influenza viruses.

Reducing the toxicity of drugs can be achieved through the joint use of drugs of the class of antioxidants and / or antihypoxants, since the oxidative stress accompanying the toxic effect of most xenobiotics provokes a serious increase in the level of peroxidation of lipids, proteins and nucleic acids, violates the physiologically adequate state of cell membranes and inhibits cellular respiration and, as a consequence, the production of macroergs. Therefore, a protective drug must be able to either quench free radical oxidation or maintain the integrity of the mitochondrial respiratory chain. Obviously, drugs that, by virtue of their structure, are able to take part in both processes simultaneously will be even more effective.

General prerequisites for reducing the toxicity of drugs were laid by us in the search for cytoprotective agents using remantadine. Previously, no drugs have been used to reduce the toxic effects of remantadine.

The results of our earlier work showed that not all protective pharmacological preparations approved for use show a protective effect when used with specific xenobiotics, and the most effective combinations should be selected and evaluated during an independent study [Eropkina EM, Mamaev E. G., Eropkin M.Yu. Exp. the clinic. pharmacol., 2003, No. 5, 48-52].

Pharmacological preparations with antioxidant activity can be classified depending on their effect on a specific link of the antioxidant system of the body [Mashkovsky M.D. Medicines M., "New Wave", 2000, T. 2, p. 188]. Derivatives of ubiquinone (coenzyme Q) are one of the classes of the most effective antioxidants that combine not only antioxidant but also antihypoxic activity. The following drugs of this class are known: ubiquinone itself (ubinon drug) is used in the treatment of coronary heart disease. A similar structure has idebenone [2- (10-Hydroxidecyl) -5,6-dimethoxy-3-methyl-2,5-cyclohexadiene-1,4-dione], related to nootropics, which also noted antihypoxic and antioxidant activity [Mashkovsky M.D. Medicines M., "New Wave", 2000, Vol. 2, p. 187; J. High-molecular compounds, 1975, v.17 (A), No. 3, p. 557-563; Ikejiri Y., Ishii K., Yasuda M. Neurology, 1996, v. 47, p. 583-585].

Based on the fact that sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate is similar in structure to natural quinones, in particular to ubiquinone, and has antioxidant properties, it was decided to study its protective effect on cells, check antihypoxic activity and cytoprotective effect, and also test it in combination with remantadine. Since sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate has its own antiviral activity against influenza viruses, which is associated with the initial stages of their replicative cycle [RF patent No. 2244546], and its combined the effect with remantadine was unknown; the effect on the influenza viruses of the composition of these 2 drugs was studied.

The essence of this invention is that previously unknown properties of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium are established - its antihypoxic effect, as well as cytoprotective effect, which allows to reduce toxicity caused by high doses of remantadine; a high biological effect was found from the combined use of remantadine and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium when acting on influenza infection.

The experimental part of the work

The in vitro in vitro evaluation complex of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate included:

1) a study of the drug’s own antiradical activity in the cell-free system (the antiradical activity test is the restoration of the stable free radical of diphenylpicrylhydrazyl (DPPH) by the drug [Pochinok T.V., Tarokhovsky M.L., Portnyagina V.A. et al. Chem.-farm Zh, 1985, No. 5, pp. 555-569] and inhibition of lipid peroxidation caused by Fe ^{2+ ions} in the system of vitelline liposomes - antioxidant activity [Klebanov GI, Babenkova IV, Teselkin Yu.A. and other Lab.delo, 1988, No. 5, pp. 59-62];

2) assessment of the antihypoxic effect of the drug on cells in culture by their ability to restore MTT reagent by mitochondrial respiratory enzymes [Mosmann T., J Immunol Meth, 1983, v. 65 (1), p. 55-63];

3) assessment of the range of safe concentrations of the drug and the establishment of its CTD50 (average cytotoxic concentration that reduces cell viability in culture by 50% of the control);

4) a study of the effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on the toxic effect of remantadine in cell cultures, with remantadine used in a concentration range including its toxic doses;

5) in vitro study of antiviral activity on cultures of influenza virus-sensitive MDCK cells of remantadine and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate both with isolated and combined use of drugs.

In all tests, sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was used in clinically adequate concentrations. The recommended single doses of the drug are from 0.5 to 0.75-1.25 g, which corresponds to an average body weight of 70 kg of 11-19 mg / kg (excluding kinetics of absorption, distribution and clearance), and the maximum therapeutic dose of the drug is determined as 50 mg / kg.

Highly effective in reducing the toxicity of remantadine and completely safe concentrations of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium, selected by the authors and then used in serial experiments, amounted to 20-40 mg / l ( on average about 55 micromoles per liter).

Own antiradical activity of the drug was determined using two methods:

1. Determination of antiradical activity with DPPH.

The determination was carried out according to a method based on the following principle: 2,2'-diphenylpicrylhydrazyl (DPPH) - a stable free radical with a maximum absorption at 517 nm during the reaction with an antioxidant turns into a nonradical form - 2,2'-diphenylpicrylhydrazine with a loss of characteristic absorption maximum [Pochinok T.V., Tarokhovsky M.L., Portnyagina V.A. and other Chem.-farm. F, 1985, No. 5, p. 565-569].

Determination process: A DPPH working solution (ICN, USA) was prepared on 95 ° ethanol and the concentration was selected so that the optical density at 517 nm was in the range 0.4-0.5 (the zone of maximum sensitivity of the test). To a sample of the test drug in a volume of 0.1 ml was added 2 ml of a working solution of DPPH and incubated for 15 minutes. The optical density was recorded on an SF-46 spectrophotometer (LOMO) against optical control. Samples were obtained by several serial binary dilutions of the starting preparation.

2. Determination of the total antioxidant activity of the drug (AOA) with a suspension of chicken egg yolk lipoproteins.

AOA of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was evaluated by the modified method of Klebanov et al. [Klebanov GI, Babenkova IV, Teselkin Yu.A. et al. Lab.delo, 1988, No. 5, pp. 59-62].

Principle of the method: When using a suspension of multilayer liposomes obtained from the yolk of chicken eggs as a peroxidation substrate, AOA manifests itself as a suppression of lipid peroxidation (LPO) reactions induced by ferrous ions, which are detected by the interaction of peroxidation products with thiobarbituric acid.

Definition: Egg yolk was mixed with 0.1 M Na-phosphate buffer, pH 7.4, in a 1: 1 ratio, suspended, diluted with the same buffer in a 1:25 ratio. Used only freshly prepared suspension. 1.6 ml of Na-phosphate buffer, 0.1 ml of working liposome suspension and 20 μl of FeSO ₄ solution (final concentration 1 mM) were added to 0.1 ml of the test sample, incubated for 15 min at 37 ° C, after which the reaction was stopped the introduction of 0.2 ml of 50% trichloroacetic acid. Next, the samples were centrifuged at 3000 g for 10 min and TBA-positive products were determined in reaction with 0.8% thiobarbituric acid by the photometric method at a wavelength of 532 nm. The degree of AOA was expressed as a percentage of control samples.

Dose-dependent reduction of free radicals in the test with DPPH and products of induced lipid peroxidation in the system with vitelline liposomes is presented in figure 1. (Figure 1. Antioxidant activity of 3,6-dioxocyclaghex-1,4-diene-1,2,4,5-tetrasulfonate Na in vitro). The abscissa represents the concentration of the drug in mg / l, the ordinate represents the optical density in% of the control. Curve 1 is the antiradical activity of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in the test with DPPH, curve 2 is the antioxidant activity in the test with liposomes. The vertical bars here and in the following drawings are standard deviation.

Figure 1 shows that the test with liposomes was more sensitive and, if we evaluate the concentration that inhibits 50% of free radical processes (IC50) in this method, this figure will be less than 10 mg / L. For comparison: in vivo data from mice showed an effective therapeutic concentration of about 50 mg / kg.

Cell Culture Experiments

The cell cultures used in the work - MDCK (epithelioid kidney line of a dog-spaniel) and human lung embryo fibroblasts are lines deposited in the collection of cell cultures of the Research Institute of Influenza RAMS, St. Petersburg, and are characterized in accordance with the requirements for the certification of deposited cell lines [Sominina A.A. (Ed.) Guidelines for working with cell cultures and media. L., All-Russian Research Institute of Influenza, Ministry of Health of the USSR, 1975, 43 pp.

Cells were grown on culture media recommended for the respective culture — Igla or DMEM (Biolot, St. Petersburg) supplemented with 10% fetal calf serum (Sigma, USA). During the experiments, the cells were scattered at a seed concentration of 200,000-250,000 cells / ml in plastic 24-hole or 96-well culture plates from Sarysedt (Germany) or Orange (Belgium) and incubated at 37 ° C in СО ₂ - incubator ("Sanyo", Japan) in an atmosphere of 5% CO ₂ . Remantadine and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate were dissolved in buffered saline (PBS) or in Eagle's growth medium, depending on the duration of exposure of the cultures with the preparations. Cells were incubated with preparations for 2 hours (PBS), 24 hours or 72 hours (growth medium).

Assessment of cell viability and cell metabolic rate

Method principle: Cell restoration of MTT water-soluble tetrazolium dye: 3- (4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (ICN Pharmaceuticals, USA) reflects the integral activity of mitochondrial dehydrogenases and serves as an adequate indicator of cell viability in culture and the intensity of oxidative processes . This method is one of the most commonly used indicators of cytotoxicity [Clemedson C. et al., ATLA, 1996, v.24, Suppl.1, p.251-311].

Definition: The microtetrazolium test was carried out according to Mosmann [Mosmann T. J Immunol Meth, 1983, v. 65 (1), p. 55-63]. Cell cultures grown in 24 or 96 well plates to a state of confluent monolayer were incubated for 3 hours in MTT solution (0.5 mg / ml) in buffered saline at 37 ° C in a CO ₂ incubator. The formed water-insoluble crystals of formazan were extracted with 95 ° ethanol for 30 min and the optical density of the samples was measured at a wavelength of 550 nm. Average cytotoxic doses - drug concentrations that suppress cell viability by 50%, were calculated from linear regression equations constructed from dose-response relationships. Mathematical and statistical processing of the results was performed using the Statistica 6.0 software package ("Statsoft Inc.", USA).

The antihypoxic effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate, expressed in an increase in the activity of mitochondrial dehydrogenases, especially succinate dehydrogenase, and recorded in the MTT test, is shown in FIG. 2. (Figure 2. Antihypoxic effect of 3,6-dioxocyclaghex-1,4-diene-1,2,4,5-tetrasulfonate Na in MDCK cultures under serum-free culture medium. The concentration of the drug is 31.3 μg / ml (≈55 μM ). The indicator of oxidative metabolism is MTT. 1 - control; 2 - incubation 2 hours; 3 - incubation 48 hours; 4 - incubation 48 hours under hypoxia).

The abscissa axis in this drawing is the composition of the experimental samples; along the ordinate axis is the optical density in the MTT test, expressed as a percentage of that of the intact control (cell culture without exposure). MDCK cell cultures were incubated with the drug for different periods in serum-free medium under normal conditions (leaky closed plastic plates in a CO ₂ incubator) and under hypoxia conditions (penicillin vials with hermetically sealed rubber stoppers). The antihypoxic effect of the drug was especially pronounced during a long period of incubation of cells and under conditions of hypoxia (figure 2): so, during incubation for 2 hours, the activity of respiratory enzymes of cells did not differ significantly from the control, during incubation for 48 hours it significantly exceeded control values by 22-24% and during 48 hours incubation under hypoxia, their activity under the action of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate exceeded the control values by more than two times.

The effect of a wide range of concentrations of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium on human fibroblasts in culture was also tested using the Mosmann method described above [Mosmann T. J Immunol Meth, 1983, v. 65 (1), p. 55-63]. The obtained results confirming that the drug has low toxicity - figure 3 (Figure 3. The effect of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate Na on human embryonic fibroblasts in culture. Test -method - MTT. * - Area of significant excess of control).

In this drawing, the abscissa axis represents the decimal logarithm of the drug concentration in mg / l, and the ordinate axis represents the opt. density at 550 nm in% of control. In the range of concentrations from 2 to 1000 mg / l, CTD50 is not achieved.

Moreover, at low concentrations of the drug (7.9-63.1 mg / l), an action stimulating the respiratory activity of cells was noted. When approximating the obtained data by linear regression analysis, the calculated value of CTD50 was 1700 mg / l. Evaluation of the pharmacological index, which shows in this case how many times the CTD50 is greater than the effective therapeutic concentration, gave a very good indicator - more than 50, whereas even with an indicator of ten, the drug is considered promising for further preclinical testing. Conclusion: the antihypoxant and antioxidant effects of the compound are responsible for its cytoprotective effect.

Next, the cytoprotective effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was determined in the presence of remantadine, i.e., the effect on the cells of two drugs when used together was studied to detect a decrease in the toxicity of remantadine in the presence of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

The cytoprotective effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was determined by the above MTT reduction procedure [Mosmann T. J Immunol Meth, 1983, v. 65 (1), p. 55 -63]. The evidence of the effective cytoprotective effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on cells in the presence of different concentrations of remantadine is illustrated by the examples in Figs. 4 and 5. In these figures, the abscissa shows the concentration remantadine, along the ordinate axis - wholesale. density at 550 nm in% of control. Reducing the toxicity of remantadine in cultures of human embryonic fibroblasts is shown in Fig.4. (Figure 4. The effect of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate Na on remantadine-induced cytotoxic response of human embryonic fibroblasts in culture. The toxicity index is MTT. * - difference from samples with the corresponding concentration of remantadine was significant, p <0.01).

Curve 1 corresponds to a dose-dependent decrease in the total activity of respiratory fibroblast enzymes in the presence of remantadine. At all concentrations of remantadine (125-500 mg / L) toxic to normal fibroblasts, a significant increase in cell viability is observed under the influence of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate. CTD50 in this case is defined as 378 mg / L. Curve 2 is a decrease in the respiratory activity of cells protected with 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium at a concentration of 31.2 μg / ml. The CTD50 in this case is significantly higher (862 mg / l), that is, a 2.3-fold decrease in the toxicity of remantadine is noted. The protective effect on cultures of MDCK cell lines (dog kidney epithelioid cells) was slightly lower — a 1.8-fold decrease in toxicity (Fig. 5), but in both cases this effect is significant over a wide range of remantadine concentrations: from 7.8 to 500 mg / l Similar data were obtained for concentrations of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate 20-80 μg / ml.

Thus, the high efficiency of in vitro 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium is shown not only as an antioxidant / antihypoxant, but also as an agent capable of significantly reducing the toxicity of remantadine when combined application.

Virological studies

The antiviral activity of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium was previously noted [RF patent No. 2244546]. It was interesting to determine how sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate and remantadine act on viral infection when used together.

Principle of the method: in order to assess whether sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate affects the antiviral effect of remantadine, the cytopathogenic effect of influenza A H3N2 virus on MDCK cell cultures was evaluated the presence of these drugs separately and their combination. The cytopathic response of cells when exposed to the virus was evaluated by the above MTT method. The degree of inhibition of cell viability in culture correlates with the development of viral infection in vitro.

The course of determination: as a reference, a highly infectious strain of influenza A virus Novosibirsk / 4/04 H3N2 was selected. Freeze-dried virus was restored on an MDCK cell culture: 1 ml of Eagle medium was added to the lyophilisate obtained from 1 ml of virus-containing medium and 0.2 ml of the resulting suspension was added to cell culture vials; after 1 hour, 2 ml of maintenance medium was added. Needle per bottle after which the cultures were incubated for 48-72 hours at 34 ° C. The presence of the virus in the incubation medium was determined by the micromethod of the hemagglutination reaction with a 0.5% suspension of human erythrocytes of I (0) blood group. As a result, the infectious dose of the working virus was determined and dilutions were selected for subsequent infection of the experimental cultures.

The test results are shown in Fig.6. (Fig. 6. Effect of remantadine and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate Na on the cytopathic response in MDCK cell cultures caused by the influenza A virus Novosibirsk / 4/04 H3N2. with the virus and drugs 24 hours 1 - control; 2 - virus; 3 - virus, remantadin-1; 4 - virus, remantadin-2; 5 - virus, drug; 6 - virus, remantadin-1 + drug; 7 - virus , Remantadin-2 + drug. * - the difference from the control is significant, p <0.001).

Here, on the abscissa axis, is the composition of the incubation medium, and on the ordinate axis, the optical density at 550 nm in% of the control samples (intact cells not infected with the virus). The concentrations of remantadine were - remantadine: 1-30 mg / l (which is the average effective antiviral dose in vitro against subtype A flu), remantadine: 2-10 mg / l. The concentration of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in this case: 31.3 mg / L. Shows the data obtained by infection with the virus in the titer of 0.5 × 10 ^-1 . As can be seen from Fig.6, the influenza virus caused a pronounced cytopathic reaction of cells in culture (inhibition of viability up to 25.2 ± 5.4% of control), while remantadine had a significant protective effect: at a dose of remantadine 10 mg / l inhibition of cell viability was 40.6 ± 1.88% of control; at a dose of 30 mg / l, the corresponding indicator was 54.4 ± 5.9 from the control.

Sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate, both by itself and in combination with remantadine, completely restored the cell viability: the integral indicator of their viability in the MTT test was 110, 5-112.4% in relation to intact cells. Therefore, in addition to the cytoprotective effect against toxicity caused by high concentrations of remantadine, sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate not only did not reduce the antiviral effect of the latter in effective antiviral doses, but on the contrary, significantly increased cell viability when exposed to a viral infection.

Conclusion: the toxicity of remantadine, which we established in vitro, was 200-350 mg / L (IC50) at 2 hours of exposure, which is in good agreement with the data established in vivo (LD50 = 160-210 mg / kg for mice with parenteral administration ) At final concentrations of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium 20-40 mg / l, the toxicity of remantadine when combined with the test drug (increase in IC50) was 1.8- 2.3 times (figures 4 and 5), therefore, therapeutic doses of remantadine can be doubled without any side effects. The chemical compound used does not worsen the effect of the main drug - remantadine, but gives it the opportunity to fully display its biological (antiviral) activity.

Based on the tests of the drug to reduce the toxicity of remantadine, the authors conducted studies on the optimal ratio of drugs when the antiviral effect of the action of remantadine is fully preserved and at the same time, the drug remantadine has no toxic effect (Figs. 4-6). Thus, it was found that the optimal ratio by weight of rimantadine: 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium in the composition is from 1: 1 to 1: 4. In this combination, patients can be prescribed treatment with remantadine, without fear of complications due to the presence of concomitant diseases.

Claims (4)

1. An antihypoxic agent, characterized in that it is sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate. 2. The use of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate as a cytoprotective agent. 3. The use of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate as a means to reduce the toxicity of remantadine at the cellular level. 4. A composition having an anti-influenza action, comprising remantadine, characterized in that it additionally contains 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in a ratio of ingredients, mg: remantadine one 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetra- sodium sulfonate 1-4

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