RU2450812C1 - Drug for respiratory infectious diseases glucaferon® - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: drug for respiratory diseasescontains a basidiomycetes extract, herbal proteolytic enzyme Bromeline, succinic acid, rutin and calcium stearate in the following ratio (wt %): proteolytic enzyme Bromeline - 3-10; edible basidiomycetes extract - 20-25; rutin (vitamin) - 20-25; succinic acid - 40-50; calcium stearate - the rest. As the basidiomycetes extract, it contains hiratake extract, shiitake mushroom extract or reishi extract. The drug Glucaferon® has an integrated effect on a body, allows essentially increasing anti-infective properties. It can be used both independently, and with underlying conventional therapy with using antiviral preparations or antibiotics.

EFFECT: invention refers to medicine, namely to pathogenetic preparations for infectious respiratory diseases produced of natural herbal raw material.

4 cl, 5 tbl, 5 ex, 6 dwg

Description

The invention relates to medicine, namely to pathogenetic agents for the treatment of infectious diseases of the respiratory system, obtained on the basis of natural plant materials.

The existing therapy of acute respiratory diseases of the respiratory system of viral etiology is mainly based on the use of etiotropic, symptomatic and pathogenetic drugs [Kiselev OI, Marinich I.G., Sominina A.A. Influenza and other respiratory viral infections: epidemiology, prevention, diagnosis and therapy. SPb., RAMS, SZO, Research Institute of Influenza. 2003, 244 p .; Isakov V.A. Modern means of symptomatic therapy of influenza and acute respiratory infections. Terra Medica. 1999, No. 1 (14), p.3-5].

However, the well-known etiotropic antiviral chemotherapy drugs (remantadine, amantadine, ribavirin, acyclovir, etc.) are characterized by a narrow spectrum of action in relation to viruses that have an etiological relationship with acute respiratory viral infections (ARVI) and influenza; the presence of adverse adverse reactions; the absence or significant decrease in the therapeutic effect when using this group of drugs in the late stages of the development of the disease (2-3 days or more); rapid formation of virus resistance to chemotherapy drugs.

These shortcomings indicate the presence of unresolved problems of modern etiotropic chemotherapy for respiratory viral infections, which increases the relevance of the development and / or improvement of pathogenetic and symptomatic agents for the treatment of respiratory infections.

Symptomatic and pathogenetic drugs for the treatment of acute respiratory viral infections and influenza based on non-steroidal anti-inflammatory substances (NSAIDs) - paracetamol, sodium metamizole, acetylsalicylic acid, ibuprofen, etc. have gained widespread practice. [Vidal Handbook. Medicines in Russia: A Handbook. M .: AstraFarmService, 2007; Fisenko V.P. Safety and toxicity of multisymptomatic colds and flu agents in anticipation of the cold season, Pharmaceutical Bulletin, No. 28, Sept., 2002]. Against the background of such drugs as Fervex, Coldrex, Teraflu, Rinzasip, etc., containing NSAIDs, the severity of local inflammatory reactions (runny nose, cough, nasal congestion, pain or soreness in the nasopharynx) and general toxic effects (headache) pain, weakness, lethargy, fever, muscle pain) [McQuay HJ, Edvards JE, Moore RA Mechanism of action of anti-inflammatory drugs, Amer. J. Ther., 2000, v.9, p. 179-187].

However, with prolonged use of NSAIDs, the production of cytoprotective PGs in the mucous membranes of the gastrointestinal tract (GIT) is inhibited, which causes intestinal bleeding. The risk of developing these complications does not decrease with the use of instant forms of such drugs, as well as with the parenteral or rectal route of administration. In addition, NSAIDs provoke the development of asthma attacks, allergic reactions, reduce the rate of renal filtration, change the picture of peripheral blood, etc. [Rainsford K.D. Influenza ("Bird Flu"), inflammation and anti-inflammatory / analgesic drugs. Inflammopharmacology, 2006, vol. 14, No. 1-2, p. 2-9; Miller S.B. Prostaglandins in Health and Disease: An Overview. Seminars in Arthritis and Rheumatism. 2006, vol. 36, No. 1, p. 37-49; Rainsford K.D. Antiinflammatory drugs in the 21st century. Subcell Biochem., 2007, vol. 42, p.3-27; Karpov O.I., Zaitsev A.A. Nonsteroidal anti-inflammatory drugs safety aspects of long-term use. New St. Petersburg medical news. 2001, No. 4, pp. 79-82].

Short courses of use of NSAIDs (1-2 days) do not reduce the duration of the inflammatory process [Musil Y. Fundamentals of biochemistry of pathological processes. Per. from Czech., M .: Medicine, 1985, 432 p .; Marshall W.J. Clinical biochemistry. Translated from English, Moscow: SPb: Publishing House BINOM - Nevsky Dialect, 1999, 368 pp.], Lead to bacterial complications, contribute to the development of viral asthenia of the body. In addition, symptomatic and pathogenetic ARVI and influenza therapies containing NSAIDs have a number of pharmacodynamic limitations, the totality of which reduces the effectiveness of therapeutic procedures.

It is known to use for the treatment of influenza and acute respiratory viral infections drugs derived from plant-based medicinal products, such as extracts or tinctures of blackcurrant, onion, raspberry, sickle-shaped scapula, bitter wormwood, leaf roots, calamus, marsh cinquefoil, etc. [Complete Encyclopedia of Traditional Medicine, vol. 1, ANS, 1996, p.178-183; Sokolova I.S. Wild and cultivated plants. - M., Medicine, 1990].

Herbal preparations, as a rule, have a positive effect on the symptomatic and pathogenetic mechanisms of the development of infectious and inflammatory processes of the respiratory tract, mainly due to the activation of anti-inflammatory or immune defense of the body. However, compared with drugs of other classes, they usually have lower activity and a significant number of contraindications.

Extracts of basidiomycetes (Basidiomycetes spp.) Are one of the promising and currently insufficiently studied group of drugs of this type that can increase the effectiveness of the treatment of respiratory infections.

Mushroom extracts contain polysaccharides, one of the active components of which are beta-D-glucans. Basically, these biopolymers provide a more pronounced activation of factors of nonspecific defense of the body. In particular, the phagocytosis, chemotaxis and adhesive properties of neutrophils are enhanced, the functional activity of the complement system, natural killers, macrophages, dendritic cells is increased, hematopoiesis is stimulated, the production of immune system mediators (cytokines, interferons) that are responsible for the development of the inflammatory response, the immune response, and the antivirus are stimulated. body protection.

Extracts of basidiomycetes are non-toxic (according to the FDA classification, beta-D-glucans are classified as GRAS - “generally recognized as safe,” that is, they are safe), do not have antigenic and pyrogenic properties, which also indicate the advisability of their inclusion in multifunctional drugs intended for pathogenetic and / or symptomatic treatment of respiratory infections [Shamtsyan MM, Konusova VG, Goloshev AM et al. Immunomodulating and antitumour effect of basidiomycetes Pleorotus ostreatus. J. Biological Physics and Chemistry. 2004, No. 4, p. 157-161; Shamtsyan Mark, Konusova Valentina, Maksimova Yulia et al. Immunomodulating and antitumour action of extracts of several mushrooms. J. Biotechnology, No. 11, p. 77-83; Rop O., Micek J., Yurikava T. Beta-glucans in higher fungi and their health effects. Nutr. Rev., 2009, v. 11, p. 624-631; Besednova N.N., Ivanushko L.A., Zvyagintseva T.N., Elyakova L.A. Immunotropic properties 1-> 3; 1-> 6 β-D-glucans. Antibiotics and chemotherapy, 2000, No. 2, p. 37-44; Akramiene D., Kondrotos A., Didziapetriene J., Kevelaitis E. Effects of β-glucans on the immune system. Medicina (Kaunas), 2007, v. 43 (8), p. 597-606; Rondanelli M., Opizzi A., Montoferrario F. The biological activity of beta-glucans. Minerva Med., 2009, v. 100 (3), p. 237-245].

Currently, extracts from stroma and / or fruit bodies of such types of fungi as Oyster mushroom ordinary or oyster (Pleurotus ostreatus); Shiitake (Lentinus edodes); Reishi (Ganoderma lusidum), etc., is proposed to be used primarily for the treatment of cancer, prevention and treatment of diabetes and cardiovascular disease, as well as a means for antiviral therapy [http://www.rutravi.ru / e / status-1-364276-shiitake-ekstrakt; http://www.bezbolezni.ru/ishop/product/23; http://www.bezbolezni.ru/site/3; http://e-grib.ru/lentinus_edodes.php; http://nuskine.ru/zdorove/reishiax.html; http://www.bezbolezni.ru/ishop/product/24; Belova N.V. Prospects for the use of biologically active compounds of higher basidiomycetes in Russia. Mycology and phytopathology, 2004, v. 38, issue 2, p.1-7].

The main disadvantage of these drugs is the relatively low efficiency in the treatment of infectious diseases of the upper respiratory tract.

The closest in technical essence to the claimed invention is an extract of Shiitake mushroom, manufactured by Fungi Pharmaceutical Co., Ltd (China), 1 kg of which is obtained from 10 kg of dried mushrooms [http://e-grib.ru/lentinus_edodes.php ], proposed for the treatment of cancer, cardiovascular and infectious diseases, as well as diabetes. The drug is prescribed in a daily dose of 2-6 g.

The disadvantage of the extract is its relatively low pathogenetic effectiveness in the treatment of viral and bacterial diseases of the respiratory tract.

The problem solved by the authors of the invention is the creation on the basis of extracts of food basidiomycetes more effective multifunctional funds for the treatment of infectious diseases of the respiratory tract.

The problem was solved by creating a means for the pathogenetic treatment of infectious and inflammatory diseases of the respiratory tract, which received the provisional name "Glucaferon (Glucaferon)" (a positive decision on the application for trademark No. 20100721404 from 06/29/2010), which allows for oral administration complex effect, providing the optimal combination of immunostimulating, antioxidant and other anti-infectious properties, in particular by ensuring greater bioavailability of BA polysaccharides zidial fungi in the gastrointestinal tract (GIT).

The technical result of the invention was that in the creation of funds, which additionally includes the proteolytic enzyme of plant origin "Bromelain", succinic acid and rutin, as well as an auxiliary substance - calcium stearate in the following ratios of the ingredients of the final agent (% wt.):

Proteolytic enzyme Bromelain - 3-10;

Extract of food basidiomycetes - 20-25;

Rutin (vitamin P) - 20-25;

Succinic acid - 40-50;

Calcium stearate - the rest.

As a Bromelain, an industrially produced preparation Bromelain SGMV was used according to the certificate of state registration of SGR 77.99.11.3. U.1977.3.10 of 03.19.2010. The following food mushroom extracts were used as a source of polysaccharides:

- extract of edible fungus Oyster mushroom (Pleurotus ostreatus), obtained by technology protected by the authors of patent RU 2189825, 2001;

- Shiitake food mushroom extract (Lentinus edodes), manufactured by "Fungi Pharmaceutical Co., Ltd" (China);

- extract of crushed spores of Reishi edible fungus (Ganoderma lusidum) ReishiMax Gip® manufactured by Pharmanex as an immunostimulating agent.

When using the inventive composition of ingredients, a proteolytic enzyme in low concentrations together with succinic acid promotes the release of polysaccharides and, in particular, beta-D-glucans from the particles of the fungus extract and accelerates their interaction with the receptors of cells responsible for innate immunity, which increases and stabilizes the activity of all body defense systems against pathogens. A macrophage stimulated by beta-D-glucans triggers a spectrum of protective reactions, including the production of: cytokines, interferons, inflammatory enzymes, free radical forms of oxygen, nitric oxide, etc. The totality of the development of these reactions ensures the rapid formation of non-specific anti-infectious immunity of a wide spectrum that enhances the non-specific immune, anti-inflammatory and antioxidant defense of the body.

The introduction of rutin into the drug reduces negative reactions, protects blood vessels and capillaries from damage, reduces their fragility and permeability, and enhances the repair processes of body tissues. The introduction of calcium stearate improves the physico-chemical characteristics of the claimed funds (improves flowability, prevents clumping).

The claimed ratio of ingredients is optimal. A decrease in the concentration of the extract leads to a sharp decrease in efficiency, an increase in its concentration unreasonably increases the cost of the product. The decrease in the concentration of "bromelain" below the claimed concentration does not provide a complete yield of beta-D-glucans and also reduces the effectiveness of the drug, increasing its concentration is ineffective. A decrease in the concentration of succinic acid less than the declared one, apparently, reduces the effectiveness of the interaction of beta-D-glucans with cells, an increase in its concentration is economically unjustified. In addition, lowering the concentration of succinic acid less than the declared one reduces the efficiency of correction of cellular metabolism, increasing its concentration increases the permissible level of daily requirement of this component. A concentration of rutin of less than 20% does not guarantee the formation of effective therapeutic and prophylactic protection of blood vessels and capillaries against the background of the development of respiratory infectious diseases of a viral nature. An increase in rutin concentration of more than 25% exceeds the daily requirement of this component.

All used components of the drug are compatible with each other, as well as with antiviral agents and antibiotics used to treat acute respiratory viral infections and influenza.

The figures illustrate the effectiveness of the proposed tool.

Figure 1 shows the results of a study of the effect of the claimed funds based on the extract of Pleurotus ostreatus and its individual components on the intensity of luminol-dependent chemiluminescence (LHCL) of human blood neutrophils induced by PMA. At the same time, the effectiveness of the following compounds on the intensity of LCHL of neutrophils (the sum of pulses per 1 h) was shown: 1 - background (cells without drugs); 2 - cells treated with PMA (10 ng / ml); 3 - cells treated with PMA + Bromelain (0.5 mg / ml); 4 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Pleurotus ostreatus, 1.0 mg / ml); 5 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Pleurotus ostreatus, 1.0 mg / ml) + Rutin (1.0 mg / ml); 6 - cells treated with PMA + 3C ₁ - (Bromelain (0.5 mg / ml + Rutin (1.0 mg / ml) + Extract (Pleurotus ostreatus, 1.0 mg / ml) + Succinic acid (2.0 mg / ml)).

Figure 2 shows the results of a study of the effect of the claimed funds based on the extract of Lentinus edodes and its individual components on the intensity of luminol-dependent chemiluminescence (LHCL) of human blood neutrophils induced by PMA. At the same time, the effectiveness of the following compounds on the intensity of LCH of neutrophils (the sum of pulses per 1 h) was shown: 1 - background (cells without drugs); 2 - cells treated with PMA (10 ng / ml); 3 - cells treated with PMA + Bromelain (0.5 mg / ml); 4 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Lentinus edodes, 1.0 mg / ml); 5 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Lentinus edodes, 1.0 mg / ml) + Rutin (1.0 mg / ml); 6 - cells treated with PMA + AP ₂ - (Bromelain (0.5 mg / ml) + Rutin (1.0 mg / ml) + Extract (Lentinus edodes, 1.0 mg / ml) + Succinic acid (2.0 mg / ml)).

Figure 3 shows the results of a study of the effect of the claimed funds based on the extract of Ganoderma lusidum and its individual components on the intensity of luminol-dependent chemiluminescence (LHCL) of human blood neutrophils induced by PMA. At the same time, the effectiveness of the following compounds on the intensity of LCH of neutrophils (the sum of pulses per 1 h) was shown: 1 - background (cells without drugs); 2 - cells treated with PMA (10 ng / ml); 3 - cells treated with PMA + Bromelain (0.5 mg / ml); 4 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Ganoderma lusidum, 1.0 mg / ml); 5 - cells treated with PMA + Bromelain (0.5 mg / ml) + Extract (Ganoderma lusidum, 1.0 mg / ml) + Rutin (1.0 mg / ml); 6 - cells treated with PMA + AP ₃ [Bromelain (0.5 mg / ml) + Rutin (1.0 mg / ml) + Extract (Ganoderma lusidum, 1.0 mg / ml) + Succinic acid (2.0 mg / ml)].

Figure 4 presents data on the survival of mice (S _(t) ,%) with experimental intranasal influenza infection [A / Victoria / 72 (H ₃ N ₂ )] and oral administration of various drugs depending on the time elapsed after infection of animals in days . The time for starting the administration of drugs is 24 hours after infection of the animals. The following notation is used in the graphs: 1 - survival of the control group of infected animals; 2 - survival of the control group of infected animals when using "Glucaferon"; 3 - survival of the control group of infected animals when using remantadine; 4 - the survival of the control group of infected animals using a combination of remantadine and the claimed drug.

Figure 5 shows the risk assessment of the adverse outcome (R) of treatment of intranasal lethal influenza infection of mice on the 15th day of the experiment against the background of oral administration of remantadine and a combination of the claimed drug and remantadine. The following symbols are used in the graphs: 1 - parameter (R) values in the control group of infected animals that were not injected with drugs; 2 - parameter values (R) in the group of animals that received remantadine; 3 - values of the parameter (R) in the group of animals that were injected with the claimed drug and remantadine. The duration of oral administration of remantadine was 5 days, the claimed drug was 10 days.

Figure 6 shows the risk assessment of the adverse outcome (R) of treatment of intranasal pneumococcal infection of mice on the 15th day of the experiment on the background of the course oral administration of an antibiotic, as well as a combination of the claimed drug and antibiotic. The following notations are used in the graphs: 1 - parameter (R) values in the control (infected) group of animals (No. 1), which were not given drugs; 2 - parameter values (R) in the group of animals (No. 5), which were given an antibiotic; 3 - values of the parameter (R) in the combined group of animals that were administered the inventive tool and antibiotic. The duration of oral administration of drugs was 10 days.

The inventive tool is obtained by mixing these ingredients and subsequent packaging of the final mixture of substances in gelatin capsules. This approach is economical because during the production process, the primary synthesis of the components of the claimed drug is not carried out.

In the examples shown, the effectiveness of a number of use cases of the inventive means "Glucaferon".

Example 1. Evaluation of the immupotropic properties of the claimed drug in vitro

It is known that the system of innate immunity is an important component of the body’s anti-infectious defense [Galaktionov V.G. Immunology. M. 1998, p. 5-13; A.Royt, J. Brostoff, D. Mail. Immunology. - World, 2001, p. 38-90; Mejidov R., Janavei C. Innate immunity. Kazan honey. Journal 2004, 85 (3), pp. 161-163; Pirofski L.A., Casadevall A. Immunomodulators as an antimicrobial tool. Curr. Opin. Microbiol. 2006, v. 9, p. 489-495; Cristofaro P., Opal S.M. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs, 2006, v.66, p.15-29]. The functions of innate immunity are non-specific and are realized due to: mechanical protection (skin, mucous membranes); phagocytosis; destruction of infected cells (complement, natural killer cells); secretion of cytokines (interferon, interleukins); synthesis of antibacterial peptides, chemokines, etc.

Evaluation of the immunostimulating effect of the drug "Glucaferon" was carried out based on its effect on the phagocytic process. Studies were carried out on human blood cells using the method of luminol-dependent chemiluminescence (LCH) [Zemskov V.M. Chemiluminescence of blood. Guidelines. M. 1988; Vladimirov Yu.A., Sherstnev M.P. Chemiluminescence of animal cells. Results of science and technology, biophysics, 1989, v.24, p.3-176], which allows to determine the activity of the phagocytic reaction of neutrophils by the intensity of production of free radical forms of oxygen.

Zimosan, a drug that is widely used for experimental purposes to study the activity of phagocytic reactions [Ushijima Y., Tosune H., Nakano M. Chemiluminescence from human PMN leukocytes activated with opsonized zymosan, was used as a reference drug. Free radical Biology and Medicine. 1997, 22 (3), p. 401-409; Xia Y., Borland G., Huang J. Function of the lectin domain of Mac-1 / complement receptor type 3 (CD 11b / CD 18) in regulation neutrophil adgesion. J. Immmunol., 2002, 169, p. 6417-6426].

To exclude nonspecific activation of cells associated with the process of isolation of neutrophilic cells, all studies were performed on whole donated blood. Chemiluminescence was recorded using a Victor-2 chemiluminometer (Pribori Oy, Finland).

Donor blood was obtained by venipuncture of the ulnar vein. Heparin (20 IU / ml) was used as a stabilizer. The following reagents were used to carry out the LHLC method:

1. Luminol (Serva company, cat. No. 28085) - 5 amino 1,2,3,4 tetrahydro 1,4 phthalazinedione, aminophthalic acid hydrazide 3. To prepare the stock solution, a 1.77 mg sample of luminol was dissolved in 10 ml of dimethyl sulfoxide (DMSO). The stock solution was stored at + 4.0 ° C. Before the study, the basic solution of luminol was diluted 10 times in a 0.9% sodium chloride solution;

2. Hanks sterile solution (Biolot company, catalog No. 2-513);

3. The drug zymosan manufactured by Sigma, cat. No. 4250. Preparation of zymosan solution: 1 mg of zymosan was dissolved in 1 ml of 0.9% NaCl. To obtain a homogeneous suspension, the zymosan solution was boiled for 30 minutes.

As the studied preparations, we used the above extracts of food basidiomycetes, the proteolytic enzyme Bromelain, as well as the glucaferon agent of the following composition: mushroom extract 1.0 mg / ml, bromelain 0.05-5.0 mg / ml, succinic acid 1 , 0 mg / ml, rutin 1.0 mg / ml, calcium stearate - up to 100%.

The LZHL reaction was carried out in white opaque 96 well plates specially manufactured for this purpose (Costar company, cat. No. 3912). The following were added to the plate wells: 20 μl of whole donor blood, 20 μl of solutions of the studied preparations, 40 μl of a solution of luminol at a concentration of 10 ^-4 M. Hanks solution brought the volume of the reaction suspension to 200 μl. All studies were performed in three parallels.

The boards were placed in a thermostatic cuvette chemiluminometer at a temperature of + 37.0 ° C. The study was carried out for 1 hour, recording pulses every 3 minutes. During the experiment, 20 cycles were recorded. The results of the reaction were expressed by an integration indicator — the light sum or the number of pulses per second accumulated during the first hour of the experiment.

Table 1 presents the results of studies of both the intrinsic immunostimulating activity of these types of fungal polysaccharides and the immunostimulating activity of the drug (Glucaferon) against the background of various concentrations of the bromelain enzyme.

The results of the study show that, compared with the baseline values of LCH of neutrophils, the polysaccharides of basidiomycetes cause a significant increase in the level of LHCL (p <0.05), which indicates the formation of reactive oxygen species that accompany the phagocytic process in peripheral blood neutrophils [Segal A. How neutrophils kill microbes. Ann. Rev. Immunology, 2005, 23, p .98-223]. The biological activity of extracts of edible mushrooms is comparable to the stimulating effect of zymosan, used as a reference preparation. The introduction of Bromelain into the composition somewhat reduces the immunostimulating effect of polysaccharides, however, the use of Bromelain in the composition of Glucaferon at predetermined intervals increases the immunostimulating effect, reaching a maximum in the concentration range from 0.5 to 5.0 mg / ml.

This effect is practically independent of the source of mushroom polysaccharides, which allows you to use any source of polysaccharides of these types of basidiomycetes in the composition of the claimed funds as a natural immunotropic component.

Example 2. Evaluation of the antioxidant activity of the drug in vitro

One of the leading pathogenetic mechanisms for the development of acute respiratory viral infections (ARVI) and influenza is the formation of free radical oxygen forms in the cells of an infected organism [Karpukhin GI, Gripp. L. Medicine, 1996; Khaitov P.M., Pinegin B.B. Modern ideas about protecting the body from infection. Immunology, 2000, v. 1, pp. 61-64]. Oxygen radicals turn into highly toxic substances that damage cell membranes. This contributes to the destruction of the capillary network and capillary walls, impaired microcirculation, the rapid spread of the virus, generalization of the infectious process, the development of severe complications, such as pneumonia [Shepelev AP, Kornienko IV, Shestopalov AV, Antipov A.Yu. . The role of free radical oxidation processes in the pathogenesis of infectious diseases. Questions of medical chemistry, 2000, v.2, p.12-20].

The processes taking place in activated neutrophils are called “respiratory burst” [Mayansky AN, Mayansky DN Essays on neutrophil and macrophage. Novosibirsk The science. 1989; Vladimirov A.Yu., Azizova O.A., Deev A.I. et al. Free radicals in living systems. Results of science and technology. Biophysics, 1992, v.29, s.3-250]. Peroxide compounds formed as a result of a “respiratory explosion” interact with proteins, amino acids, and lipids, which leads to damage to cell membranes, impaired cell functions and their death [Kulinsky V.I. Active forms of oxygen and oxidative modification of macromolecules. Soros educational journal. 1999, No. 1, pp. 2-7]. This allows us to consider the effect on the intensity of the “respiratory explosion” as a model of the effect on the antioxidant processes that occur during acute respiratory viral infections and influenza.

For the occurrence of a “respiratory explosion” in neutrophils, it is possible to use, in particular, such preparations as calcium ionophore A23187, phorbol esters [Guide to Immunopharmacology, ed. Dale M.M., J.K. Formen. - M .: Medicine, 1998, p. 33-48]. To assess the effectiveness of Glucaferon for stopping the aforementioned processes, we studied the effect of its components and their combination on a “respiratory explosion” induced in the peripheral blood neutrophils phorbol-12-myristate-13-acetate (FMA).

The experiments were performed on neutrophils isolated from human whole donated blood. Whole donor blood in a ratio of 1:10 was mixed with 10% sterile gelatin solution. After 40 minutes, plasma was collected in a test tube in which white blood cells were concentrated using gelatin. Next, the plasma was layered on ficollpac ("Serva") in a ratio of 2: 1.

After centrifugation for 40 minutes at 400 g, neutrophils along with an admixture of red blood cells were at the bottom of the tube. By double centrifugation at 1200 rpm for 10 minutes, the ficollpac impurity was removed, after which red blood cells were removed from the cell suspension by hypotonic shock. Hypotonic shock was performed with a 10-fold volume of distilled water for 40 seconds. Then, an equivalent amount of a 1.8% sodium chloride solution was added, which made it possible to bring its concentration to 0.9%.

After a single wash at 1200 rpm for 10 minutes, a neutrophilic suspension of cells was ready for investigation. Cell viability was 96% (trypan blue score). The cell concentration used to study LHCL was 2.0 × 10 ⁶ .

The study of free radical processes accompanying the "respiratory explosion" was carried out using the method of luminol-dependent chemiluminescence (LCHL). The LZHL reaction was carried out as follows. At the same time, 20 μl of neutrophilic suspension, 20 μl of PMA at a concentration of 10 ng / ml, 20 μl of the solutions of the studied preparations, and 40 μl of luminol were simultaneously added to the wells of the chamber. Hanks solution was adjusted to 200 μl. All studies were conducted in three parallels.

After making the test material, the boards were placed in a thermostatic cuvette of a chemiluminometer (+ 37.0 ° С). The study was carried out for 1 hour, recording readings every 3 minutes. During the experiment, 20 cycles were recorded. The reaction results were expressed by an integrated indicator - light sum, i.e. the number of pulses per second during the 1st hour of the experiment.

The experiment was carried out as follows. To the neutrophils of the donor stimulated with PMA (10 ng / ml) was added sequentially:

- Enzyme bromelain (0.5 mg / ml);

- Enzyme bromelain (0.5 mg / ml) + extract of food mushrooms (1.0 mg / ml);

- Enzyme bromelain (0.5 mg / ml) + extract of food mushrooms (1.0 mg / ml) + rutin (1.0 mg / ml);

- Enzyme bromelain (0.5 mg / ml) + extract of food mushrooms (1.0 mg / ml) + rutin (1.0 mg / ml) + succinic acid (2.0 mg / ml) (Glucaferon).

The results of the experiment, presented in figures 1-3, showed that the PMA at a concentration of 10.0 ng / ml induces a powerful "respiratory burst" of neutrophilic cells. In this case, the LHLC cell intensity is more than 1200 pulses / s. The addition of the bromelinein proteolytic enzyme to the reactive mixture reduces the intensity of neutrophils LHCL by 6 times and indicates a decrease in the production of reactive oxygen species (ROS). The introduction of food mushroom extracts into the analyzed system, regardless of their nature (Oyster mushrooms, Shiitake or Reishi), as well as routine, does not significantly affect the intensity of free radical processes induced by PMA. However, the use of Glucoferon for this purpose leads to an almost complete suppression of the "respiratory explosion."

Thus, in vitro conditions, the possibility of suppressing the free radical processes, which are the effectors of the inflammatory response of the body during the development of infectious diseases of viral and / or bacterial etiology, has been demonstrated.

Example 3. The effect of the composition of "Glucoferon" on its antioxidant activity in vitro

In the conditions of example 2, the effect of the composition of the drug “Glucoferon” on the intensity of LHCL of human blood neutrophils stimulated by PMA was studied. The experimental results are shown in table 2.

The presented results of the study show that the maximum effect of inhibiting the intensity of stimulated LHCL of neutrophils is observed with the following ratios of the ingredients of GL (% wt.): Bromelain enzyme - 3-10; mushroom extract - 20-25; succinic acid - 40-50; rutin - 20-25, calcium stearate - the rest. With a decrease in the concentration of these components, there is a decrease in the efficiency of inhibition of the magnitude of neutrophil-stimulated LHLC. With an increase in the concentration of these ingredients, significant inhibition of neutrophil-stimulated LHCL does not occur.

Thus, in vitro conditions, the obtained ratios (% wt.) Of the main components of the proposed drug are optimal to achieve the maximum antioxidant and anti-inflammatory activity of the claimed drug.

Example 4. Evaluation of the effectiveness of the proposed drug on a model of experimental influenza infection

Assessment of the therapeutic efficacy of the claimed drug was carried out according to its effect on the course of experimental influenza infection of animals.

All studies were conducted on white mongrel mice weighing 16-18 g. During two-week quarantine and during the experiment, the animals were kept in standard conditions on a full diet [Guidance on experimental and clinical study of new drugs. M., 1984, 192 p.].

For infection of animals used the influenza virus A / Victoria / 72 (H ₃ N ₂ ), which was previously passivated in the allantoic cavity of 10-day-old chicken embryos. To reproduce the influenza infection, mice under mild ether anesthesia were intranasally injected with a virus-containing allantoic fluid with the following characteristics: titer EID ₅₀ (embryonic infectious dose) from 1.0 × 10 ^-6 to 1.0 × 10 ^-8 ; infectious dose from 3 to 5 LD ₅₀ .

Remantadine was used as an etiotropic antiviral drug, which was previously dissolved in a 0.9% sodium chloride solution and was orally administered to animals 24 hours after infection. An antiviral chemotherapy was used at a dose of 0.1 mg / mouse once a day. The duration of administration was 5 days.

As an immunotropic drug used various options of the proposed drug "Glucaferon (Glucaferon) ® of the following composition (wt.%): Bromelain enzyme - 5.0; extract of basidiomycetes - 25.0; succinic acid - 40; routine - 25; calcium stearate - 5.0.

The first option contained an extract of oyster mushrooms ordinary (ZC ₁ ), the second option - an extract of Shiitake mushrooms (ЗС ₂ ), the third option - an extract of Reishi mushrooms (ЗС ₃ ).

The indicated variants of Glucaferon were orally administered to animals 24 hours after their infection at a dose of 0.15 mg / mouse 1 time per day. The duration of administration was 10 days.

Observation of the animals was carried out for 15 days. Daily recorded mortality of animals in the control and experimental groups. On the 15th day of the experiment, survival rates (S _(t) ) and average life expectancy (S ₅₀ ) were determined in all groups of animals. For this, the Kaplan-Meier method was used [Glanz S. Biomedical statistics. Per. with the English., M., Practice, 1998, 459 S.]. To assess the risk (R) of the development of an unfavorable outcome of an experimental influenza infection in this animal species against the background of oral administration of drugs, the expression was used: R = A / A + B, where: A is the number of dead animals on the 15th day of the experiment, pcs .; In - the number of surviving animals on the 15th day of the experiment.

At the first stage of the study, the effectiveness of the oral administration of various variants of the claimed agent (ZC _1,2,3 ) in mice was assessed against the background of the development of experimental influenza infection. Table 3.1 presents the results of the study. Thus, the survival rate of mice that were injected with various variants of AP was at the level of 20-30% or 26.7 ± 8.1%. The absence of significant differences in the survival of animals in groups No. 3, 4 and 5 allows us to conclude that various extracts of basidiomycetes (Pleurotus ostreatus, Lentinus edodes or Ganoderma lusidum) can be used as part of the product.

Compared with control groups of animals (No. 1-2), the average life expectancy of mice (S ₅₀ ), in experimental groups No. 4, 5 and 6 increases by 2 times. This means that against the background of the development of experimental influenza infection, the oral administration of the drug provides a pronounced activation of non-specific factors of the immune, antioxidant and anti-inflammatory protection of the body of infected animals. Therefore, its use alone or in combination with antiviral chemotherapy drugs is advisable.

At the second stage of the study, the combination therapy of experimental influenza infection was evaluated based on the oral administration of remantadine and Glucaferon. In table 3 and figure 4. The results of this stage of work are presented.

The results of the studies demonstrate that, compared with the control group of mice, the survival rate of infected animals with the use of remantadine increases 7 times (58.3 ± 10.1%), and the survival rate of animals with the introduction of remantadine and Glucaferon increases by 9.2 times (76.4 ± 5.0%). This means that the combined administration of rimantadine and AP to animals against the background of influenza infection increases the effectiveness of etiotropic antiviral therapy 2.2 times (18.0%).

Figure 5 presents the risk values (R) of the development of an unfavorable outcome of an experimental influenza infection in mice following oral administration: remantadine, a combination of Glucaferon and remantadine, as well as a control group of infected animals that were not given these drugs.

A graphical interpretation of the results of the study shows that the risk of developing an unfavorable outcome of animal therapy with the administration of Glucaferon and remantadine is 3.8 times lower than in the control group of infected animals, and 1.75 times lower in the group of animals that received one etiotropic antiviral drug. Reducing the risk of developing an adverse outcome in the treatment of infected animals based on Glucaferon and remantadine indicates the effectiveness and feasibility of using combination therapy, which has an etiotropic and pathogenetic focus.

Thus, the above results allow us to draw the following conclusion:

1. Oral administration of Glucaferon and remantadine increases the effectiveness and efficiency of treatment of experimental intranasal influenza infection. The survival rate of animals against the background of the introduction of AP and remantadine reaches 76.4 ± 5.0%. The risk of an unfavorable outcome of therapy against the background of the drug and remantadine is significantly reduced and is 0.24. This indicates the need for the simultaneous use of etiotropic chemotherapy and AC, which enhances the pathogenetic effectiveness of antiviral therapy;

2. In the component composition of the claimed funds with equal opportunity to be used various extracts of basidiomycetes.

3. Against the background of the development of experimental influenza infection, Glucaferon "provides a sufficiently pronounced activation of nonspecific factors of the immune, anti-inflammatory and antioxidant defense of the body of infected animals. The combination of activation of these effects increases the average life expectancy of infected animals by 2 times;

4. The inventive tool is advisable to use in treatment regimens for infectious and inflammatory processes of the respiratory tract of viral etiology, including against the background of etiotropic antiviral chemotherapy drugs.

Example 5. Evaluation of the effectiveness of the proposed drug and antibiotics in a model of experimental pneumococcal infection.

Pneumonia of bacterial etiology is a common complication of acute respiratory viral infections (ARVI) and influenza [Kiselev OI, Marinich IG, Sominina AA Influenza and other respiratory viral infections: epidemiology, prevention, diagnosis and therapy. SPb, RAMS, SZO, Research Institute of Influenza. 2003, 244 p .; Rakhmanova A.G., Neverov V.A., Prigozhina V.K. Infectious diseases: a guide for general practitioners (2nd edition), St. Petersburg: Peter, 2001, 576 p .; Kiselev O.I., Isakov V.A., Sharonov B.P., Sukhinin V.P. The pathogenesis of severe forms of influenza. Vestnik RAMS, 1994, No. 9, p. 32-36.]. The development of pneumonia is facilitated by a decrease in the body's immune defense, which usually forms against the background of acute respiratory viral infections and influenza, which creates favorable conditions for the activation of pathogenic and conditionally pathogenic microorganisms.

For the treatment of bacterial pneumonia, antibiotics are used, which are usually prescribed on an empirical basis, taking into account the clinical condition of the patient.

However, the effectiveness of the release of an infected organism from a pathogen depends on both the activity of the immune system and the action of the antimicrobial drug. This is confirmed by a decrease in the effectiveness of antibiotic therapy in patients with acquired or congenital immune dysfunctions, as well as in complicated and chronic forms of infectious diseases. Consequently, the introduction of immunotropic drugs into treatment and prophylactic regimens seems to be the most direct and logical way, increasing the effectiveness of antibiotic therapy.

The effectiveness of the combined oral administration of various variants of the claimed drug and antibiotic was evaluated in a model of experimental pneumococcal infection.

The studies were conducted on white mongrel mice weighing 16-18 g, obtained from the Rappolovo nursery, quarantined for two weeks. During quarantine and during the experiment, the animals were kept under standard conditions on a full-fledged diet [Guidelines for experimental and clinical study of new drugs. M., 1984, 192 p.].

For infection of animals, bacteria of S. pneumoniae II serotype were used, which were previously cultured on blood agar at + 37.0 ° C for 24 hours. To simulate bacterial pneumonia, a suspension of S. pneumoniae cells under mild ether anesthesia was intranasally administered to animals in an amount of 0.05 ml (in each nasal passage) and a dose of 1.0 × 10 ⁷ CFU / ml. For this type of animal, the indicated value of the infectious dose and the method of its administration is optimal, because the death of animals in the control group (No. 1) was not observed during the first 24 hours after infection. However, by the end of the first day after infection in the animals of the control and experimental groups, the following clinical signs of the infectious process were noted: adynamia, shortness of breath, lack of appetite, swelling and hyperemia of the upper respiratory tract. The development of these signs of the infectious process served as an indication for the administration of drugs in all experimental groups of animals, i.e. 24 hours after infection. The control group of animals (No. 1) drugs were not administered.

As an antibacterial agent, the antibiotic ampicillin / sulbactam (ampicillin / sulbactam) was used. The use of this antibiotic is allowed for empirical therapy of community-acquired pneumonia [Rafailidis P.I., Ioannidou E.N., Falagas M.E. Ampicillin / sulbactam: current status in severe bacterial infections. Drugs., 2007, v. 67 (13), p. 1829-1849; Betrosian A.P., Douzinas E.E. Ampicillin-sulbactam: an update on the use of parenteral and oral forms in bacterial infections. Expert Opin Drug Metab Toxicol., 2009, v. 5 (9), p. 1099-1112; Sinopalnikov A.I., Sidorenko S.V. Community-acquired pneumonia: standards for empirical antibiotic therapy. Antibiotics and chemotherapy. 1999, No. 5, pp. 22-28].

Ampicillin / sulbactam when administered orally has a moderate bioavailability (40%), which minimizes its effect on the effectiveness of combination therapy associated with the simultaneous administration of an antibacterial drug and the claimed drug. The antibiotic was dissolved in a 0.9% sodium chloride solution and orally administered to animals at a dose of 0.5 mg / mouse once a day. The duration of antibiotic administration in the experimental groups (No. 5, 6, 7, 8) of the animals was 10 days.

As an immunotropic drug used the claimed tool "Glucaferon (Glucaferon) ®" of the following composition (wt.%): Bromelain enzyme - 5.0; extract of basidiomycetes - 25.0; succinic acid - 40; routine - 25; calcium stearate - 5.0.

Various varieties of mushroom extracts were used. "Glucaferon" was administered to animals of all experimental groups for 10 days orally at a dose of 0.12 mg / mouse 1 time per day. Animals of group No. 2 received a composition containing extract of oyster mushrooms ordinary (ZC ₁ ). Animals of group No. 3 received orally for 10 days, the composition, which contained the extract of Shiitake mushrooms (ZC ₂ ). Animals of group No. 4 received a composition containing an extract of Reishi mushrooms (ZS ₃ ). Animals in groups No. 6, 7 and 8 received the indicated options for AP against the background of the antibiotic, which made it possible to determine the effectiveness of the combined use of the antibiotic and the variants of the claimed drug (AP ₁ ), (AP ₂ ), (AP ₃ ).

To assess the effectiveness of combination therapy in each group, we determined: animal survival (S _(t) ,%), median survival or average life expectancy (S ₅₀ , day), as well as the risk of an adverse outcome (R, 0-1). Animal survival and life expectancy were determined by the Kaplan-Meier method [Glanz S. Medical and biological statistics. Per. from English., M.: Practice, 1998, 459 p.]. To estimate the value (R), the expression was used:

R = A / A + B, where: A is the number of dead animals on the 15th day of the experiment, pcs .; In - the number of surviving animals on the 15th day of the experiment.

The design and results of the experiment are presented in table 5.

An analysis of the results of the study shows that oral administration to animals of various variants of the claimed drug (ZC ₁ , ZC ₂ , ZC ₃ ) and an antibiotic (groups 6, 7, 8) increases, compared with groups 1 and 5, the survival of animals (S _{( t)} ). So, the value of the parameter S _(t) on the 15th day of the experiment was: in groups 6 and 7, 83.3 ± 10.8%, respectively; in group No. 8 - 75.3 ± 12.5%. No significant differences in the values of the parameter S _(t) between groups 6, 7 and 8 were found (p> 0.05). Therefore, in the component composition of the claimed funds can be used various extracts of basidiomycetes (Pleurotus ostreatus, Lentinus edodes, Ganoderma lusidum).

The absence of significant differences in the value of S _(t) between groups 6, 7 and 8 allows the results of the study in these groups to be combined, which increases the number of animals of the general experimental group and increases the information content of the parameter S _(t) . As a result of the calculations, the S _(t) value of animals that were given antibiotic and ZS was 80.6 ± 6.6%, the survival rate of animals of the control group No. 1 and comparison group No. 5 was 16.7 ± 10.8% and 41.7 ± 14.2%, respectively. Compared with the control group No. 1, the oral administration of an antibiotic and AP to animals increases their survival by 4.8 times, and the use of one antibiotic increases their survival by only 2.5 times. Therefore, the combined use of AP and an antibacterial drug increases the effectiveness of antibiotic therapy of infected animals by 2.3 times.

In Fig.6. the risk assessment (R) of the development of an adverse treatment outcome for an experimental pneumococcal infection of animals against the background of one antibiotic, AP, and antibiotic, as well as in the control group of animals that were not given drugs, is presented. A graphical interpretation of the results of the study shows that the risk of developing an unfavorable outcome of animal therapy with the administration of Glucaferon and an antibiotic is 4.0 times lower than in the control group of infected animals, and 3.0 times lower in the group of animals that received the antibiotic. Reducing the risk of an adverse outcome in the treatment of animals based on AP and antibiotic indicates an increase in the effectiveness of combined antibacterial therapy and the advisability of using AP against antibiotics with moderate bioavailability.

Significant differences in the survival of infected animals between groups No. 2, 3 and 4 were not found (P> 0.05). Therefore, these extracts of basidiomycetes do not significantly affect the final values of S _(t) , which confirms the possibility of their use as one of the components of Glucaferon, and also allows combining the results of the study in these groups. Thus, the survival rate of animals of the combined group was 33.3 ± 7.9%. The values of S _(t) in the combined group of animals and in control group No. 1 did not differ significantly.

The average life expectancy (S ₅₀ ) of the animals of the combined group was 10 days. Compared with the control group No. 1, the introduction of animals only one "Glucaferon" increases their average life expectancy by 3.3 times. This means that, against the background of its oral administration, there is a rather pronounced activation of nonspecific factors of the immune, anti-inflammatory and antioxidant defense of the body, the combination of which increases the average life expectancy of infected animals.

The value of S ₅₀ in the group of animals treated with one antibiotic was 13 days. Compared with control group No. 1, oral administration of an antibiotic increases the average life expectancy of infected animals by 4.3 times. However, an increase in the average life span of animals on the background of an antibiotic with moderate bioavailability does not significantly increase the final survival of animals of this group. In this case, the use of the drug against the background of an antibiotic is a logical and justified way that increases the effectiveness of antibiotic therapy.

The results obtained allow us to draw the following conclusion:

Table 3 Survival and average life expectancy of mice with experimental intranasal pneumococcal infection against the background of the antibiotic ampicillin / sulbactam and various variants of the claimed drug (ZC ₁ , ZS ₂ , ZS ₃ ) Study groups of animals, control / experience The number of animals in the group, pcs. The duration of the experiment, (t), day, number of dead animals, units / day Survival rate S _(t) ,%; Median survival, S ₅₀ , day one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen fifteen No. 1. Infected animals (without treatment), control 12 0 3 four 2 one 0 0 0 0 0 0 0 0 0 0 16.7 ± 10.8 3.00 No. 2. Infected animals (treatment), CC ₁ , experience 12 0 one 2 one 0 0 one one 0 0 one 0 one 0 0 33.3 ± 13.6 11.0 Number 3. Infected animals (treatment), AP ₂ , experience 12 0 one one 0 one one 0 one one one one 0 0 0 0 33.3 ± 13.6 10.00 Number 4. Infected animals (treatment), AP ₃ , experience 12 0 2 0 one one one 0 one 0 one 0 one 0 0 0 33.3 ± 13.6 8.00 No. 5. Infected animals (treatment). Antibiotic experience 12 0 0 0 one 2 0 one one one 0 0 0 one 0 0 41.7 ± 14.2 13.00 No. 6. Infected animals (treatment). Antibiotic + ZC ₁ , experience 12 0 0 0 0 0 0 one 0 one 0 0 0 0 0 0 83.3 ± 10.8, (*) Number 7. Infected animals (treatment), Antibiotic + AP ₂ , experience 12 0 0 0 0 0 0 0 one 0 one 0 0 0 0 0 83.3 ± 10.8 (*) No. 8. Infected animals (treatment). Antibiotic + AP ₃ , experience 12 0 0 0 0 0 0 one 0 one 0 0 one 0 0 0 75.3 ± 12.5 (*) No. 9. Control ZC ₁ (without infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 No. 10. ZS ₂ control (without infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 No. 11. ZS ₃ control (without infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 No. 12. Antibiotic control (no infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 U 100.0 Note: (*) - median survival was not determined, because the number of dead animals is less than the number observed (<12 pcs.).

Table 4 Survival and average life expectancy of mice on the background of experimental influenza infection and oral administration of various variants of the proposed drug (AP ₁ , AP ₂ , AP ₃ ) Study groups of animals The number of animals in the group, pcs. The duration of the experiment, (t), day, the number of dead animals, units / day Survival rate S _(t) ,%; Median survival, S ₅₀ , day one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen fifteen No. 1. Infection control 10 0 0 0 3 2 2 one 0 0 0 0 0 0 0 0 20.0 ± 12.6 the dose of the virus is 3.0 LD ₅₀ , (without treatment) 5,0 No. 2. Infection control 10 0 0 one four 3 one one 0 0 0 0 0 0 0 0 0.00 the dose of the virus is 5.0 LD ₅₀ , (without treatment) 4.0 Controlling the amount of infectious doses twenty 0 0 one 7 5 3 2 0 0 0 0 0 0 0 0 10.0 ± 6.7 virus, (without treatment) 5,0 Number 3. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (ZC ₁ ) 10 0 0 0 0 2 0 one one one 0 2 0 one 0 0 20.0 ± 12.6 9.0 Number 4. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (AP ₂ ) 10 0 0 0 0 0 2 0 one one one one one 0 0 0 30.0 ± 14.5 10.0 No. 5. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (AP ₃ ) 10 0 0 0 0 0 0 one 2 one one one one 0 0 0 30.0 ± 14.5 9.0 Experience, treatment of ZC _1-3 , thirty 0 0 0 0 2 2 2 four 3 2 four 2 one 0 0 26.7 ± 8.1 joint group, No. 3-5, 10.0 No. 6. Control ZC ₁ (without infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 Number 7. ZS ₂ control (without infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 No. 8. ZS ₃ control (without infection) 6 0 0 0 0 0 0 0 0 0 0 U 0 0 U U 100.0

Table 5 The survival and average life expectancy of mice on the background of experimental influenza infection and oral administration of remantadine and the claimed drug. Study groups of animals The number of animals in the group, pcs. The duration of the experiment, (t), day, number of dead animals, units / day Survival rate S _(t) ,%; Median survival, S ₅₀ , day one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen fifteen No. 1. Contamination control, virus dose 3.0 LD ₅₀ , (without treatment) 12 0 0 0 2 3 four one 0 0 0 0 0 0 0 0 16.7 ± 10.8 6.0 No. 2. Contamination control, virus dose 5.0 LD ₅₀ , (without treatment) 12 0 0 one four 5 one 0 one 0 0 0 0 0 0 0 0.00 5,0 Control of the amount of infecting doses of the virus, (without treatment) 24 0 0 one 6 8 5 one one 0 0 0 0 0 0 0 8.3 ± 5.6 5,0 Number 3. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (Remantadine) 24 0 0 0 0 one one 2 2 one one one one 0 0 0 58.3 ± 10.1 (*) Number 4. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (Remantadine + ZC ₁ ) 24 0 0 0 0 0 0 one 0 2 one one 0 one 0 0 75.0 ± 8.8 (*) No. 5. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (Remantadine + AP ₂ ) 24 0 0 0 0 0 2 0 0 one one 0 one 0 0 0 79.2 ± 8.3 0 No. 6. Experience, the dose of the virus is 5.0 LD ₅₀ + treatment (Remantadine + AP ₃ ) 24 0 0 0 0 0 0 one 0 2 0 one one one 0 0 75.0 ± 8.8 (*) Experience, combined treatment,
joint group No. 4, 5, 6 72 0 0 0 0 0 2 2 0 5 2 2 2 2 0 0 76.4 ± 5.0 (*) Number 7. Remantadine control (no infection) 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 Note: (*) - median survival was not determined, because the number of dead animals is less than the number observed.

1. Oral administration of the claimed agent and antibiotic increases the effectiveness of the treatment of experimental intranasal pneumococcal infection. Survival of animals against the background of the introduction of Glucaferon and the antibiotic ampicillin / sulbactam reaches 80.6 ± 6.6%. The risk of developing an adverse outcome of therapy against the background of the claimed drug and antibiotic is 0.2 and indicates the need for the use of the claimed drug in the etiotropic therapy;

2. In the component composition of the claimed funds can be used various extracts of basidiomycetes (Pleurotus ostreatus, Lentinus edodes or Ganoderma lusidum). The change in the nature of polysaccharides in the claimed tool does not have a significant effect on the survival of animals and, accordingly, on the effectiveness of antibiotic therapy;

3. The component composition of the claimed agent provides a sufficiently pronounced activation of non-specific factors of the immune, anti-inflammatory and antioxidant defense of the body of infected animals, which increases their average life expectancy 3.3 times;

4. The inventive tool is advisable to use in the schemes of combined etiotropic therapy of infectious and inflammatory processes of the respiratory tract against antibiotics with moderate bioavailability.

Claims (4)

1. An agent for the treatment of diseases of the respiratory tract containing an extract of basidiomycetes and excipients, characterized in that it additionally contains a proteolytic enzyme of plant origin "Bromelain", succinic acid and rutin, and calcium stearate as an adjuvant in the following ratios of ingredients, wt.%:
Proteolytic enzyme Bromelain 3-10 Extract of food basidiomycetes 20-25 Rutin (Vitamin P) 20-25 succinic acid 40-50 Calcium stearate Rest 2. The tool according to claim 1, characterized in that as an extract of basidiomycetes it contains an extract of Oyster mushroom ordinary. 3. The tool according to claim 1, characterized in that as an extract of basidiomycetes it contains Shiitake mushroom extract. 4. The tool according to claim 1, characterized in that as an extract of basidiomycetes it contains an extract of Reishi mushroom.

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