RU2476205C1 - Composition with anti-infectious activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry and represents a composition with anti-infectious activity presented in the solid dosage form for oral administration in the form of a powder, representing a balanced complex containing a probiotic agent, an adsorbent and an excipient; the probiotic agent is sterilised cultural fluid containing metabolites of the strain Bacillus subtilis VKPM (Russian National Collection of Industrial Microorganisms) No. B-2335; the adsorbent is zeolite; the excipient is calcium stearate or aerosil; the composition is characterised by the fact that it additionally contains a high-active immunomodulatory agent representing a complex of polysaccharides - products of polymer enzymatic hydrolysis of internal and external layers of brewers' yeast cell coating in the form of β-glucane and mannan with the ingredients of the composition taken in specific proportions, wt %.

EFFECT: invention provides high effectiveness and versatility with respect to dangerous bacterial and viral infections; it is storage safe and stable.

11 ex, 10 tbl, 10 dwg

Description

The invention relates to the field of medicine and the pharmaceutical industry, in particular to means for the prevention and treatment of dangerous infectious pathologies of bacterial and viral nature.

Infectious diseases of various nature continue to be one of the urgent problems of modern medicine [1-3]. At the same time, so-called dangerous infectious diseases (OIDs) are becoming increasingly important, outbreaks of which are recorded primarily in developing countries with unstable socio-economic conditions [4-7]. To date, OIZs have not lost their relevance for developed countries, including Russia, as evidenced by the periodic activation of natural foci of infection in the country (anthrax, hemorrhagic fever with renal syndrome, Congo-Crimean hemorrhagic fever, tick-borne encephalitis), as well as periodic outbreaks of West Nile fever, bird flu, swine flu. California encephalitis. Japanese encephalitis, Sakhalin fever, Issyk-Kul fever and other infections [8].

According to experts of the World Health Organization (WHO) [8] in the near future we should expect epidemics of yellow fever on the Asian continent, a new pandemic of smallpox caused by the vector-borne variant of Monkeypox, the spread of the Ebola virus transmitted by an aerogenic route. The potential danger of bioterrorism with the use of pathogens as biological agents should not be ruled out. According to domestic and foreign experts, priority among these agents will be given to genetically non-traditional pathogens of infections, against which known means of prevention and treatment may not be effective enough [9, 10].

Infectious diseases are of great relevance for healthcare, as they occupy leading positions in terms of the overall incidence and average prevalence of the population [11], causing significant damage to its working capacity, as well as to the quality of life in general. Analysis of the sanitary-epidemiological situation in various regions of Russia showed that despite its general deterioration after the collapse of the USSR, stabilization of the situation has recently been noted [12, 13]. Organizational and practical measures carried out by the sanitary-epidemiological service of the country led to the creation of a powerful system of sanitary-epidemiological surveillance, contributing to the improvement of the sanitary-epidemiological situation in the regions [12-14].

The above indicates that the problem of creating and improving effective means and methods for the prevention and treatment of infections, including acute respiratory infections, remains extremely urgent, and at present, it should be given the closest attention.

Intensification of studies on the pathogenesis of OIDs has established that in the development of this pathology there is the formation of functional cellular anergy and secondary immune deficiency, the main reasons for which are the imbalance between various components of the immune system, primarily the imbalance between cyclic nucleotides, inflammatory mediators, cytokines and cellular suppressor factors [14].

Induction of cellular immunosuppression occurs, as a rule, with systemic hyperproduction of these molecules or with an increase in their local concentration above the physiological norm. Soluble molecules secreted by infected cells can also contribute to cellular immunosuppression. The latter is most characteristic of viral infections. In particular, to date, proteins secreted by cells infected with variola, cowpox, Ebola, Las, Marburg and others hemorrhagic fevers have been described, which have a powerful suppressive effect on the production of interferon in the body. In relation to the causative agents of bacterial infections, a similar suppressing effect is exerted by products secreted by cells that are infected with the causative agents of plague, anthrax, tularemia, etc.

In this case, the main forms of manifestation of an imbalance in the immune system are:

- a change in the ratio of cells with CD4 + and CD8 + phenotypes (a change in the immunoregulatory index) downward due to a decrease in the number of cells with the CD4 + phenotype and an increase in the number of cells with the CD8 + phenotype under the influence of the pathogen;

- violation of the ratio of effector and suppressor cells resulting from failures in the process of their differentiation;

- a change in the subpopulation composition of T-helper lymphocytes, leading to a modification of the set of cytokines produced by these cells and, as a result, to an imbalance between the cellular and humoral components of the immune system;

- imbalance between proliferative and apoptotic regulatory cell programs, manifested in a violation of the ratio between the number of cells with phenotypes CD25 +, CD30 + and CD95 +;

- imbalance due to a violation of the relationship between the immune, nervous and endocrine systems;

- violation of the principle of clonal activation, manifested by the formation of a polyclonal type of immunoreactivity and leading to autosensitization - aggression of the immune system against its own cells and tissues [15-17].

So, in the course of the infectious process, first of all, there are dysfunctions of the immune system. In this case, the response of the immune system to infection of the body is characterized by the development of a systemic acute phase response. Pathogens, launching special mechanisms to counteract the formation of anti-infectious immunity, have a fairly powerful suppressive effect on the cellular and humoral components of the immune system, thereby creating a condition for their own reproduction.

In addition to cellular, the humoral mechanisms of the immune system are also disturbed, manifested:

- violation of the synthesis of immunoglobulins (Ig) due to damage and deformation by the antigen of immunocompetent cells and suppression of the production of specific antibodies B-l with the participation of T-helpers, as well as direct contact of the pathogen with Ig and damage to their molecular structure, leading to their loss of ability inactivate antigen;

- damage to the synthesis and secretion of immunocompetent cells of mediators, energy and metabolite-protective substances involved in immune processes (prostaglandins, adenylate cyclase, guanylate cyclase, phosphodiesterase, cyclic adenosine monophosphate, adenosine triphosphate, etc.);

- violation of the absorbing and digesting functions of phagocytes circulating in the blood.

The combination of these negative shifts adversely affects the formation of an adequate immune response. Initially, pathogen recognition processes are disrupted by immunocompetent cells, and later on, intercellular relationships within the immune system. These changes lead to suppression of specific antibody production, lymphokine production by sensitized lymphocytes and the formation of immune deficiency in compensated or subcompensated forms [18].

The depletion of the adaptive capabilities of the immune system is accompanied by severe metabolic disorders and indicates the transition of functional and structural immune deficiency to a decompensated form. Further depletion of the reserve capabilities of the body leads to total immune deficiency, which is characterized by a complete disorganization of the entire system of immunoreactivity and the simultaneous loss of the functional capabilities of the blood forming organs. This indicates the depletion of the last "strategic" reserves.

Protective reactions against a number of viral and bacterial pathogens can be enhanced by activation of the immune system [19], when the stimulating signal is nonspecific in nature, directed not selectively against the pathogen that caused the infection, but increases overall immunological resistance.

Currently described various ways and means of correcting immune deficiency. With a certain degree of conditionality, the means of immuno-oriented therapy can be divided into etiotropic (extra-immune) and pathogenetic or immunotropic proper, which realize their effect through the immune system. The action of extraimmune drugs is aimed at improving the general condition of the body and its metabolism, eliminating the causes of the dysfunction of the immune system. These drugs are also called mediated immunotherapy agents. They allow you to increase non-specific immunological resistance to various environmental influences, including infectious agents. Pathogenetic drugs normalize the functioning of the immune system and are intended to stimulate or suppress immunoreactivity, correct imbalances in various parts of the immune system, compensate for existing immunoreactivity disorders and prevent the development of immune deficiency.

The use of immunotropic drugs in the treatment of acute respiratory infections or for prophylactic purposes seems to be very relevant and important. In the infectious process, immune dysfunctions arise and amplify, while the possibilities of etiotropic therapy are limited by the emergence of new and transformation of known etiopathogens, the formation of pathogenic virus-bacterial associations and resistant strains, a change in the ratio between symbiotic and pathogenic flora, an increase in the number and severity of complications arising from this, an increase the duration of treatment, and therefore its cost [20]. The incidence of immunologically compromised individuals is also increasing.

The spectrum of immunotropic drugs today is quite extensive and includes both natural and synthetic products [15, 20, 21]. Among these drugs are probiotics - drugs that have a positive effect on the homeostasis of the system "microorganism and its normal microflora", as well as having a fairly pronounced immunomodulating effect, manifested in the stimulation of the lymphoid apparatus, the synthesis of immunoglobulins, interferons, cytokines (primarily IL-1 and IL-2), processes of synthesis and secretion of humoral factors of non-specific resistance. In addition, probiotic preparations inhibit colonization of the intestine by pathogenic bacteria and protect the macroorganism from potentially harmful factors that enter the gastrointestinal tract from the outside or are synthesized in the intestine during a number of processes [22]. The antagonistic effect of probiotics against pathogens is based on binding to mucosal receptors and dense colonization of the intestinal mucin layer, activation of the synthesis and release of substances with a bactericidal or bacteriostatic effect, such as short-chain fatty chains, hydrogen sulfide, hydrogen peroxide and antibiotics. Bacteriostatic effect is exerted by low molecular weight metabolites of saccharolytic microflora, primarily short-chain fatty acids (propionic, butyric, acetic, formic, lactic), as well as lactate.

Considering the mechanisms of the immunotropic action of probiotic agents, as well as the immunopathogenesis of OIZ, the following strategic directions for their application seem appropriate:

- for the purpose of immunoprotection - to prevent the development of immune deficiency. An indication for the appointment of immunotherapy is the fact of exposure to an extraordinary factor or situation;

- for the purpose of immunocorrection - to compensate for manifestations of immune deficiency associated with the cellular component of immunoreactivity and eliminate the regulatory imbalance of the immune system with the subsequent restoration of normal immunoreactivity algorithms, as well as microflora imbalance;

- for the purpose of immuno-restoration - to recreate elements of immunoreactivity and restore the morphological and functional integrity of the immune system.

As for medicines that have antiviral activity and are used in the prevention and treatment of dangerous infectious pathologies of viral etiology, it is known that at present they can be divided into 3 groups:

1. Chemotherapy drugs that are widely used in medical practice for the prevention and treatment of dangerous viral infections: acyclovir (zovirax), foscarnet, ganciclovir, vidarabine for herpetic and cytomegalovirus infections; remantadine and its analogues for influenza; ribamidil, virazole, ribavirin for respiratory syncytial infection; azidothymidine against retroviruses and others [22-24].

2. Inductors of interferon (ridostin, kamedon, cycloferon, neovir, etc.), which have already found their place in the prevention and treatment of herpes, hepatitis, multiple sclerosis [25-27].

3. Drugs previously used in other fields of medicine and which have revealed biological activity against viral pathogens: dibazole, nicotinamide [28-30], etc.

Until now, interest remains in nucleotide and nucleoside analogues, which are inhibitors of various stages of the biosynthesis of nucleic acids. The antiviral effect of these substances is apparently associated with the presence in their structure of hydroxyl groups capable of undergoing phosphorylation in the body. It is important that phosphorylation proceeds to a greater extent in a virus-infected cell than in an uninfected one. The resulting triphosphates selectively inhibit virus-coding DNA polymerases, which is due to the antiviral effect of these drugs. Typical representatives of this class of compounds are acyclovir, widely used to treat herpetic diseases, and ribavirin, which is becoming more widespread in MS infections.

One way to increase the body's resistance to viral infections is to stimulate all parts of the immunity chain using interferon or its inducers. Recent studies on experimental animals and patients have confirmed the fact that interferon inducers are effective in the prevention and early treatment of viral infections. For this purpose, domestic preparations of ridostin, cycloferon, neovir, anadine, etc. are allowed for use in medical practice.

At the same time, the well-known basic schemes for emergency prevention and treatment of dangerous infectious diseases of a bacterial nature are based on the use of broad-spectrum antibiotics [31-34], the use of which to provide antibacterial protection is associated with a number of difficulties. So, there is a pronounced dynamics of a decrease in the number of antibiotics and chemotherapeutic agents registered in Russia over the past eight years. If in 2002 the total number of the main practically significant antibacterial compounds allowed for use was 65, in 2009 only 43 (66%). This negative process is especially evident in relation to the nomenclature of betalactams (28 - in 2002, 19 - in 2008), aminoglycosides (11 and 7, respectively), tetracyclines (5 and 1, respectively) and sulfonamides (8 and 4, respectively). The most stable in this regard is the class of fluoroquinolones, since the elimination of one drug (enoxacin) from its composition is compensated by the appearance on the Russian market in 2008 of two new representatives of this class - gemifloxacin and gatifloxacin [35-38].

A constant decrease in the nomenclature of antibiotics and chemotherapeutic agents registered in Russia fully correlates with the general state of production of these drugs in the country. In Russia, a powerful antibiotic industry was created in the 50s. Production volumes amounted to over 3,000 tons per year, and this was enough to provide antibiotics for all the republics of the former USSR and the countries of the socialist community. At the same time, production was based on domestic selection strains. In connection with the crisis of the Russian economy, by the mid-1990s, the production of domestic antibiotic substances based on microbial synthesis had become unprofitable. Currently, a significant part of the fermentation capacity for the production of antibiotic substances is idle. So, in 2005, capacities for the production of antibiotic substances were used only 17%, for the production of finished dosage forms for injections - 29%. Recently, the production of antibiotic substances has decreased by almost 4 times (2363 tons in 1985, 891 tons in 1995, 624 tons in 2004) [10]. The main reasons for the decline in the production of antibiotic substances in Russia are the state-of-the-art biosynthesis and chemical transformation technologies for antibiotics, the high cost of substances, the lack of state support for substance producers (loans, tax incentives, etc.) and the dumping policy of global manufacturers of substances. Due to a sharp reduction in the production of domestic antibiotic substances, Russian manufacturers are forced to purchase a significant part of the substances abroad, moreover, their price is 2-3 times lower than the cost of domestic products.

Thus, the disappearance of a significant number of antimicrobial compounds from the market of antibacterial chemotherapeutic drugs in Russia could not but affect the general state of the problem of prevention and treatment of infections, since many of the drugs that have lost registration in our country are still included in the current regulatory documents regulating the procedure for prevention and treating this kind of dangerous pathology.

Currently, to protect against infectious diseases of a bacterial nature, the use of a sufficiently large list of antibacterial agents is recommended. The basic antimicrobial agents include doxycycline, rifampicin, rifamethoprim, ampicillin, sulfatone, sisomycin, gentamicin, etc.

As a result of recent studies, promising drugs have been identified, which include a number of modern developments, namely:

- from the group of penicillins - azlocillin, amoxicillin, piperacillin and others;

- from the group of cephalosporins - cefotaxime, ceftazidime, ceftriaxone, cefpirome, cefepime, etc .;

- from the group of carbapenems - imipenem, meropenem;

- from the group of aminoglycosides - isenamycin;

- from the macrolide group - azithromycin, clarithromycin, spiramycin, roxithromycin, etc .;

- from the group of fluoroquinolones - pefloxacin, ciprofloxacin, ofloxacin, norfloxacin, etc.

However, it should be noted that in order to achieve the required level of antibacterial effect, these anti-infective drugs, as a rule, must be taken often, for a long time and in large dosages. Such antibacterial drugs, on the one hand, make it possible to effectively combat infectious diseases, but on the other hand, with the most commonly used dosing regimens, they often cause the development of a number of side effects from various organs and systems of the body, such as a negative effect on the state of human intestinal microbiocenosis due to the lack of selectivity of the action of antibiotics on microorganisms, allergic reactions, etc. In addition, basic antibacterial drugs are toxic, have nonspecific bactericidal action and cause the appearance of pathogenic microorganisms resistant to the action of antibacterial agents, as a result of which antibiotic therapy requires at least subsequent restoration of intestinal microbiocenosis (imbalance in the system "macroorganism and normal microflora"). We should not forget about the significant dependence of the Russian pharmaceutical market on imported drugs, which could negatively affect the level of anti-epidemic care.

One of the promising ways to overcome these shortcomings, manifested in the case of using antibacterial drugs, is not their independent use, but in combination with means for correcting intestinal microbiocenosis - probiotics. It has been established that in the case of combined use with antibiotics, priority should be given to such probiotic agents that contain non-integral living microorganisms (for example, bifidobacterin, lactobacterin, sporobacterin, laminolact), foreign to the human gastrointestinal tract, which will be destroyed by antibacterial agents, creating an additional antigenic load on the body, and metabolites of probiotic strains of microorganisms [39-41].

Among these agents is the probiotic complex Bactistatin [42]. It is based on (wt.%): Sterilized culture fluid (SCF) obtained by culturing bacteria of the species Bacillus subtilis and containing their metabolites - 1.0, soy flour hydrolyzate (GSM) - 20.0, a natural mineral zeolite with sorption and ion exchange properties - 78.0 and calcium stearate (SC) or aerosil - 1.0. The specified probiotic complex in composition, mechanism of action on the body and the achieved effect is closest to the claimed composition and adopted as a prototype.

The basis of the prototype is constituted by exogenous and endogenous biologically active substances (BAS) immobilized on zeolite, synthesized by Bacillus subtilis bacteria during deep growth, which largely determine the therapeutic and prophylactic effect of this probiotic complex. Their list is quite extensive and includes substances that are heterogeneous in chemical composition and biological properties: proteolytic and amylolytic enzymes, amino acids, polysaccharides (hexosamine, glucosamine), B vitamins (pyridoxine hydrochloride, riboflavin), nitrogenous bases and their derivatives (adenine, guanine, tim , uracil, cytosine) [43-45]. Among them, various natural antibacterial substances (bacteriocins, lysozyme, catalases) are widely represented, which selectively inhibit the growth and reproduction of pathogenic and conditionally pathogenic microorganisms in the intestine, without affecting the symbiotic microflora. All of these biologically active substances have a positive effect on the human body and animals in general.

The zeolite, which is part of the prototype agent, has a pronounced sorption effect (mainly with respect to compounds with a low molecular weight, such as methane, ammonia hydrogen sulfide and other toxic substances), without directly interacting with vitamins, amino acids, proteins, due to which these substances remain in the gastrointestinal tract. Ions contained in the body can be included in the crystalline structure of the mineral, and vice versa, the body receives those inorganic elements that it needs from the mineral. Due to an increase in the height of intestinal villi, improved digestion, and an increase in the absorption area of nutrients in the small intestine, the so-called selective ion exchange occurs [46].

An important component of the prototype tool is the hydrolyzate of soy flour, which is, on the one hand, part of the protective environment of metabolites, which is largely responsible for the strength of their sorption on the surface of the zeolite, and on the other hand, is a source of amino acids that provide the nutritional needs of normal intestinal microflora and macroorganism cells. The main component of the soy flour hydrolyzate, soy oligosaccharide (SOE), has bifidogenic properties [47].

Calcium stearate (SC) or Aerosil are included in the composition of the prototype as a processing aid.

The main purpose of the prototype tool is to restore and maintain optimal microecological parameters in the intestine, treat gastrointestinal diseases by introducing substances into the body that inhibit the development of pathogenic microflora and stimulate the development of beneficial microorganisms. The action of the drug is based on the fact that during its transit through the gastrointestinal tract in a given area, the protective capsule is destroyed and the probiotic components immobilized on zeolite particles are released into the intestinal cavity. At the same time, micellar structures are formed around zeolite particles, which are gradually released from the porous surface of the zeolite during movement along the gastrointestinal tract. On the one hand, this allows maintaining the activity of the biological components of the probiotic in the gastrointestinal tract during the day, which is sufficient to restore and stimulate the functional activity of the normal intestinal microflora. On the other hand, the effect of the gradual release of biologically active substances (BAS) immobilized on it from the zeolite surface leads to the appearance of open surfaces of its porous structure, which ensures the inclusion of ion exchange mechanisms and selective sorption of toxic compounds that help to clean the gastrointestinal tract from toxins and decomposition products. This is especially important for general detoxification of the body.

Thus, due to the complex composition, the prototype tool contributes to the general normalization of microecological conditions in the intestine through various mechanisms, such as suppression of opportunistic flora, sorption and elimination of toxins, improvement of digestion processes, and improvement of the trophic base for normal microflora and gastrointestinal epithelium.

Clinical trials of the prototype agent showed its high efficiency in stopping the phenomena of gastric and intestinal dyspepsia in patients with acid-dependent diseases of the upper gastrointestinal tract. The use of this drug in patients with erosive antral gastritis led to a clear positive clinical effect in most patients, manifested in the form of a decrease in symptoms of intestinal dyspepsia, developing against the background of eradication therapy. The use of this drug contributed to the improvement of lipid metabolism, an increase in high density lipoproteins and a decrease in total blood cholesterol. In addition to improving somatic symptoms in patients, the level of depression and neurotization decreased during therapy [48].

The clinical efficacy of the prototype agent was also proved when prescribing it to patients with chronic pancreatitis with exocrine pancreatic insufficiency combined with enzyme replacement therapy (mesim-forte, penzital), which is manifested in the elimination of intestinal microbiocenosis disorders, the correction of secondary exocrine pancreatic insufficiency, caused by excessive pancreatic microflora, caused by developing against the background of primary exocrine insufficiency and significantly exacerbating it section [49].

The expediency of prescribing a prototype agent in the implementation of fundamentally new treatment regimens for coronary heart disease (CHD), a disease that currently has extremely high clinical and social significance due to the high incidence rate among people of working age, as well as the severity of complications [50], has also been established. Along with the main reasons for the development of this pathology, there are also a number of additional factors that indirectly contribute to its occurrence and development. First of all, they include intestinal dysbiosis, since so far an opinion has been formed about the relationship of IHD with quantitative and qualitative violations of the composition of the microbiota, as well as the key role of the intestinal microbiota with the participation of enterohepatic circulation mechanisms in the regulation of lipid metabolism. The development of intestinal dysbiosis mediates the formation and progression of negative changes in lipid metabolism as one of the triggers of cholesterol aggression. Hypolipidemic therapy is currently considered as one of the main tasks in the treatment of coronary heart disease and involves the use of statins as the main and most effective cholesterol lowering drugs. However, statins of the latest generation, which radically changed the approach to the prevention of coronary heart disease and its complications, have side effects that significantly limit their widespread use: hepatotoxicity, as well as a decrease in the effectiveness of treatment with prolonged use. The additional use of the prototype against the background of standard basic therapy, which prescribes the use of nitrates, antiplatelet agents, β-blockers or calcium channel blockers, angiotensin converting enzyme inhibitors, diuretics, is accompanied by a lipid-lowering effect that exceeds the effect under simvastatin under similar conditions. This is due to the positive effects of the prototype due to the correction of intestinal microbiocenosis.

The absence of living microorganisms in the prototype means allows it to be used in conjunction with antibiotics. Their combined introduction into the gastrointestinal tract makes it possible to improve microecological conditions, which helps to restore the qualitative and quantitative characteristics of symbiotic microorganisms. However, when assessing the prospects of the combined use of an antibacterial agent (doxycycline) and bactistatin in cases of emergency prophylaxis and treatment of OID of a bacterial nature (anthrax infection), an insufficiently high level of effect from the action of the prototype agent was established [51]. The effect of the prototype agent in relation to other RPIs of a bacterial nature was not evaluated.

At the same time, along with the observed effects, the prototype agent is characterized by such a significant drawback as the inability to concentrate the culture fluid obtained on soybean medium using fuels and lubricants, due to its high viscosity. In addition, the use in the technological process of obtaining the prototype as a nutrient medium of native soy flour does not allow the full use of the nutritional properties of the feedstock. In this case, additional chemical or enzymatic hydrolysis of soy flour is required, which significantly complicates the technology for obtaining the prototype.

It should also be noted that the feasibility is theoretically substantiated and the possibility of using the prototype is practically proved only when correcting microbiota disorders and treating the corresponding gastrointestinal diseases by the general normalization of microecological conditions in the intestine. The prospects of using probiotics for the prevention and treatment of infectious pathologies were reported in the works of Sadovy N.V. et al. (1998) [52], I. Ryzhko et al. (2000) [53], Vorobeichikova E.V. et al. (2005) [39]. However, the data of systematic studies evaluating the immunotropic effects of the drug Bactistatin (prototype agent), indicating the fundamental possibility and prospects of its use in the prevention and treatment of a wide range of infectious diseases of various etiologies, including dangerous infectious diseases, both in terms of monotherapy and no combination of course prescription with basic anti-infective agents. At the same time, the available theoretical information allows us to rely on the promise of using probiotic-based agents for highly effective prophylaxis and treatment of such a pathology.

The aim of the invention was to increase the effectiveness of the prevention and treatment of dangerous infectious diseases by creating a composition for pathogenetic therapy based on a complex of biologically active substances with probiotic and immunomodulating activity, which is highly effective and versatile against infections of a bacterial and viral nature when administered orally as a means of monotherapy, and when combined with etiotropic agents of basic therapy, safety and stability spine during storage.

Achieving this goal is possible by creating a composition for pathogenetic therapy, which is a balanced complex of biologically active substances with probiotic and immunomodulating activity, the qualitative and quantitative composition of which will ensure the normalization of the functioning of the body's immune system by eliminating the dysfunctional changes in the immune system and, above all, overcoming the powerful suppressive action of pathogens on its cellular and humoral components , which impedes the formation of anti-infective immunity, the compensation of existing violations of immunoreactivity and the prevention of the development of immune deficiency.

When forming the qualitative composition of the claimed composition, intended for the prevention and treatment of acute respiratory infections of various etiologies, it was assumed that its main components should:

- provide an immunomodulatory effect and help eliminate dysfunctional changes in the immune system that develop when exposed to pathogens of OIZ;

- combined with the basic means of etiotropic therapy of acute respiratory infections of bacterial and viral etiology;

- reduce the negative impact of antibacterial agents on the qualitative and quantitative characteristics of the normal microflora of the gastrointestinal tract;

- increase the body's resistance to viral infections by stimulating all parts of the immune chain by inducing the synthesis of endogenous interferon;

- activate the mechanisms of elimination of pathogens from the body;

- exert an antitoxic effect by binding and eliminating toxic components that accumulate in the body both due to the development of the infectious process and the negative effects of antibacterial drugs during their course use.

An important distinguishing feature of the claimed composition is the ability to effectively increase the nonspecific and specific immune resistance of the body, which is realized through a complex effect on the immune system of all components of the claimed composition exhibiting immunomodulatory activity.

To this end, in the composition of the claimed composition, which is a balanced complex, in addition to the probiotic agent, adsorbent and auxiliary additives, an immunomodulating agent is additionally included.

A probiotic agent is a sterilized culture fluid (SCF) containing metabolites obtained by culturing and sterilizing bacteria of the species Bacillus subtilis. In this case, VKPM strain No. B-2335 was used, the metabolites of which are characterized by high antagonistic activity against a wide range of pathogenic (Salmonella, Staphylococcus, Shigella, Pseudomonas, etc.) and opportunistic (Proteus, Klebsiella, Candida, etc.) microorganisms and are capable of inhibit the growth of pathogenic microflora, without exerting an inhibitory effect on lactobacilli and bifidobacteria. In addition, metabolites of Bacillus subtilis bacilli, increasing the phagocytic activity of macrophages, themselves are able to exhibit immunomodulating activity.

Biologically active substances produced by microorganisms are concentrated during their cultivation in the culture fluid. The list of biologically active substances synthesized by Bacillus subtilis during deep growth is quite representative, and the manifestation of their biological activity is an integral component of the therapeutic properties of the claimed composition. Among them, various natural antibacterial substances (bacteriocins, lysozyme, catalases) are widely represented, which selectively inhibit the growth and reproduction of pathogenic and conditionally pathogenic microorganisms in the intestine, without affecting the symbiotic microflora. It is especially valuable that during growth and spore formation, the microbial producers of Bacillus subtilis synthesize nitrogenous bases (purine and pyrimidine derivatives). These nitrogenous bases and their derivatives, which are a separate group of immunomodulatory substances, are contained in significant quantities in the SCR (table 1). This circumstance is of decisive importance for the claimed composition, since nitrogenous bases and their derivatives restore the functional activity of a single macrophage system of the body, inhibited due to exposure to pathogens, and significantly increase the effectiveness of nonspecific and specific protection at various stages of the infectious process.

Moreover, the pharmacological activity of the probiotic agent is manifested through the indirect interaction of biologically active substances produced with immunocompetent organs after overcoming physiological protective barriers.

The adsorbent in the composition of the claimed composition is intended primarily for the immobilization of metabolites produced by bacilli during their cultivation and concentrated in the SCR, their transportation and targeted delivery throughout the intestine. The gradual release of biologically active substances immobilized on it allows maintaining a high level of activity of the claimed composition.

The possibility and expediency of using zeolite as a component of the claimed composition (adsorbent) are determined by reliable representations of the nature and mechanism of its influence on the body. Thus, due to the unique chemical structure of molecules, zeolite provides selective binding and excretion of low molecular weight toxins from the body: methane, hydrogen sulfide, ammonia, heavy metals, radionuclides, etc. There is no direct interaction with useful nutrients: vitamins, amino acids, proteins, etc. Zeolite contains minerals and has ion-exchange properties, since the main skeleton of its crystal lattice consists primarily of a tetrahedron and has cavities in which ions are located (on dence, potassium, calcium), easily exchangeable with each other and with the surrounding substrate. Zeolite, not absorbed in the digestive tract and passing in transit, is involved in selective ion exchange and selective sorption, being an additional source of a wide range of essential trace elements. It is also important that this natural mineral improves digestion as a whole by activating biochemical reactions in the intestine, sorption of low molecular weight metabolites and normalizing the state of intestinal microflora. The particles of the zeolite used are no more than 500 microns in size and have an oval crystal shape, which, in turn, eliminates the formation of microtraumas in the intestine and is completely safe [54-56].

A significant difference of the claimed composition from the prototype means is that it additionally contains a highly active immunomodulating agent, which is a complex of polysaccharides - products of enzymatic hydrolysis of polymers of the inner and outer layers of the cell wall of brewer's yeast, namely β-glucan and mannan. The action of these easily digestible BAS in the body is complex and consists, first of all, in the fact that β-glucan and mannan, through immunomodulating effects on immunocompetent cells, effectively inhibit the growth and reproduction of microbes and viruses that enter the body.

The immunomodulatory effect of the used β-glucan of brewer's yeast is due to the fact that it is transported through the walls of the intestinal tract cells to the lymph, in which it interacts with the key cells of the immune system - macrophages - by attaching to specific receptors located on their cell membranes and activates macrophages. By acting on macrophages, β-glucan enhances their phagocytic activity - motility, as well as the ability to find and eliminate alien, including pathogenic microorganisms. In addition, brewing yeast β-glucan stimulates cells such as neutrophils NK (natural killer cells) and LAK (lymphokine activated killer cells), significantly increasing the antibacterial activity of the claimed composition. It also allows you to activate the production in the cells of the macrophage system of such a cytokine as interferon, thereby increasing the antiviral activity of the claimed composition.

At the same time, the mannan polysaccharide, possessing the ability of antigenic stimulation, increases the production of various classes of immunoglobulins in lymphocytes, which determine the specific protection of the body from the effects of pathogens.

To improve the manufacturability of the process of obtaining the inventive composition in solid dosage form for oral administration in the form of a powder, an auxiliary additive is introduced into its composition. As an auxiliary additive to prevent sticking of the claimed composition to the equipment used in the technological process, calcium stearate or aerosil is used.

As you can see, the qualitative composition is selected in such a way that the claimed composition is a balanced complex with pronounced immunomodulating activity in the following ratio of ingredients, wt.%:

Sterilized culture fluid containing metabolites of the strain VKPM№B-2335 Bacillus subtilis bacteria 1.6-2.3 Zeolite 43.0-46.0 The complex of polysaccharides (β-glucan and mannan) 51.0-54.0 Calcium Stearate or Aerosil 0.5-1.5

The ability to achieve the objectives of the invention is confirmed by the results of the studies presented in the following examples.

Example 1. Obtaining the inventive composition in the form of a solid dosage form for oral administration (powder).

To obtain the claimed composition in powder form, the following algorithm of actions is performed. Bacillus subtilis microorganisms are grown by the method of deep cultivation in a biological reactor (fermenter). After the completion of the cultivation process, the culture fluid (QL) with microorganisms is first subjected to centrifugation to separate and subsequently remove living cells of microorganisms, and then sterilize. Before sterilization, QOL is mixed with an adsorbent - zeolite, previously crushed to particles no larger than 500 microns in size. Then, a complex of polysaccharides, as well as calcium stearate or aerosil, are introduced into the obtained sterilized culture fluid (SCF) containing producer metabolites. The resulting mixture is subjected to lyophilization, in which the biologically active metabolites of the producer are immobilized on zeolite particles. Thus prepared mass is placed in the tank of the granulation plant for the purpose of mixing and drying.

The used complex of polysaccharides (BITRA LLC, Moscow) includes biologically active products of fermentolysis of polymers of the inner and outer layers of the cell wall of brewing yeast: β-glucan (30.7%) and mannan (10.3%).

As an adsorbent, natural zeolite is used — natural microporous silicate mineral of the Kholinsky deposit (Buryatia) (CJSC NPF NOV, Novosibirsk) [57, 58].

As an auxiliary additive, calcium stearate [59] or aerosil [60] are used.

The inventive composition can also be made in solid dosage form for oral administration in the form of capsules or tablets, which will not only greatly simplify its use, but also provide reliable protection of its constituent components from the effects of factors causing their degradation.

As you can see, the process of obtaining the inventive composition in the form of a solid dosage form for oral administration (powder) is simple, and the ingredients used are inexpensive, are produced by the domestic industry and are available.

Example 2. Safety assessment of the claimed composition.

The safety of the claimed composition was evaluated by conducting a sanitary-chemical analysis, sanitary-microbiological studies, as well as determining the important toxicological characteristics - acute toxicity.

Sanitary chemical analysis.

Sample preparation was carried out according to GOST 26929-94 “Raw materials and food products. Sample preparation. Mineralization to identify toxic elements. Interstate standard. " Lead and cadmium were determined according to GOST 30178-96 “Raw materials and food products. Atomic absorption method for the determination of toxic elements. Interstate standard. " Arsenic was determined according to GOST 26930-86 “Raw materials and food products. Method for the determination of arsenic. " Mercury was determined according to the "Guidelines for the detection and determination of total mercury in food products by flameless atomic absorption" (No. 5178-90).

The content of organochlorine pesticides (toxicants) was determined according to the “Methodological guidelines for the determination of residues of organochlorine pesticides” (No. 1766-77) and the official methods of analysis of AOAC (“Official methods of analysis of the AOAC”, 1984, 14th ed., Chapter 29, pp .537-538).

The studies were carried out using laboratory weights VL-210, a complex of a voltammetric CTA, a Kristallux 4000 chromatograph with PID, ECD detectors, a Kvant-2A atomic absorption spectrometer, a KFK-2 photoelectric colorimeter, an SF-26 spectrophotometer, and a gamma-ray spectrometer. and beta radiation MKGB-01 "Radek".

The results of the measurements are presented in tables 2 and 3 and showed that in the claimed composition the content of toxicants (organochlorine pesticides HCTs and DDT), hazardous substances (lead, cadmium, mercury, arsenic), radionuclides ( ⁹⁰ Sr, ¹³⁷ Cs) does not exceed permissible levels established by the “Unified Sanitary and Epidemiological and Hygienic Requirements for Goods Subject to Sanitary and Epidemiological Surveillance (Control)” of the EurAsEC Customs Union (chapter II, section I, index 10.6) (ET standard (chapter II, section I, index 10.6) ) The same pesticides as aldrin and heptachlor in the claimed composition were not found.

Sanitary and microbiological studies.

Microbiological studies were carried out in accordance with the requirements of MUK 4.2.577-96 “Methods of microbiological control of baby food, therapeutic nutrition and their components”, GOST R 52814-2007 “Food products. Method for the detection of bacteria of the genus Salmonella ", GOST R 52815-2007" Food products. Methods for the identification and determination of the number of coagulase-positive staphylococci and Staphylococcus aurous ”, GOST R 52816-2007“ Food products. A method for identifying and determining the number of bacteria of the group of Escherichia coli (coliform bacteria) ", GOST 10444.8-88" Food. Method for determination of Bacillus cereus ”, GOST 10444.12-88“ Food products. Method for determination of yeast and mold fungi ”, GOST 30726-2001“ Food products. Methods for the identification and quantification of bacteria of the species Escherichia coli. "

The research results on the assessment of sanitary-microbiological safety indicators are presented in table 4. As can be seen, according to the sanitary-microbiological safety indicators, the claimed composition meets the "Unified sanitary-epidemiological and hygienic requirements for goods subject to sanitary-epidemiological surveillance (control)" of the EurAsEC Customs Union ( chapter II, section I, index 10.6.10.10) (ET standard (chapter II, section I, index 10.6.10.10)).

Acute toxicity study.

To determine acute toxicity indicators, the claimed composition was administered to healthy outbred white male mice weighing 22-24 g and healthy outbred female rats weighing 130-150 g orally in the form of an aqueous suspension stabilized with tween-80, in a volume of not more than 0.5 ml per animal to mice and not more than 5.0 ml to rats. The studies were carried out using Litchfield-Wilcoxon probit analysis modified by Z. Roth [61]. From samples of each animal species of 50 animals, 2 groups of 25 animals were formed each: control (introduced the same volume of distilled water) and experimental (introduced the inventive composition twice a day at a dose of 0.33 mg for mice and 2.0 mg for rats).

Observation of experimental animals for 8 days made it possible to establish that not one animal died, there were no visible changes in their behavior. The duration of observation was 1 month from the date of administration of the claimed composition. The results of studying the body weight dynamics of experimental animals during 21 days of observation showed that in none of the experimental groups were there any statistically significant differences by t-student criterion from the corresponding control group (table 5).

Since the toxic effect was not detected at the maximum used dose of the claimed composition, in accordance with the requirements of the Pharmaceutical Committee of the Ministry of Health and Social Development of Russia [62], the administered dose was increased 10 times. The effect of the claimed composition was evaluated on young sexually mature white male mice weighing 24-27 g using an acute toxicity test. The experimental animals were kept in groups of 10 animals per cage under standard conditions: room temperature 20 ± 2 ° C, PK-121-2 feed (000 INFORMKORM), drink at libitum. An hour before the start of the experiment, the animals stopped feeding with free access to water.

In an experiment, the inventive composition was administered orally in the form of an aqueous suspension by means of a metal probe. Immediately prior to each injection, the sample was mixed until a uniform turbidity. The control group of animals was injected with saline - 0.9% NaCl (placebo).

The volume of injected samples was calculated for each animal, taking into account its weight according to the formula:

where V is the sample volume, µl; m is the mass of the animal, g

During the experiment, the inventive composition was administered at 6.6 mg per animal, which corresponds to the maximum value allowed for the test [63]. The experimental and control groups contained 20 animals. The criterion was the survival of animals from the experimental and control groups, which was evaluated after 1 day, 2 days, 4 days, 8 days, 16 days from the moment of administration of the claimed composition. The number of animals in the indicated observation periods is shown in Table 6. During the experiment, no deaths were observed in the control and experimental groups. Thus, it was experimentally established that the claimed composition does not differ in acute toxicity, since it does not cause any visible changes in the general condition of the experimental animals and does not cause their death.

Example 3. Evaluation of the stability of the claimed composition during storage.

We investigated the possibility of maintaining the activity and safety of the developed solid dosage form of the claimed composition in the form of a powder and established a guaranteed shelf life in case of storage at various temperatures and without the use of special equipment.

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№V-2335 of bacteria Bacillus subtilis (1.6 wt.%), Zeolite (43.4 wt.%), A complex of polysaccharides (53.6 wt.%) Calcium stearate (1.4 wt.%).

During the experiment, the powder was placed in 80 consumer packages in the form of double plastic bags and stored for 2 years at temperatures 5 ± 2 ° C, 10 ± 3 ° C, 25 ± 5 ° C, 35 ± 6 ° C, 20 packages for each temperature storage mode.

After every 6 months of storage, 5 packs were taken from the corresponding temperature regime and the characteristics of the powder were determined: moisture, amylolytic and proteolytic activities, antagonistic activity, and microbiological purity. To assess changes in these characteristics during storage, their values were determined at the beginning of the study.

The data presented in table 7 indicate that at the selected storage temperature conditions (from 5 to 40 ° C), the estimated parameters of the claimed composition practically do not change for 2 years. The average values of the indicators were: humidity - 6.8 ± 2.5%, amylolytic activity - 4.8 ± 3.5 units. AA / g, proteolytic activity - 0.66 ± 3.0 units. PA / g The microbiological purity of the claimed composition, assessed by the absence of pathogenic microorganisms (Pseudomonas aeruginosa, Enterobacteriaceae, Staphylococcus aureus and Bacillus cereus) in it, was maintained for 2 years, that is, the entire study period. The antagonistic activity of the claimed composition against Staphylococcus aureus, Candida albicans, Shigella sonnei and Salmonella typhimurium also persisted throughout the storage period.

Thus, the guaranteed shelf life of the claimed composition, made in solid dosage form (powder), is at least 2 years at a temperature not exceeding 40 ° C, subject to the safety of packaging. Special temperature storage conditions or the use of special equipment is not required.

Example 4. The study of the immunotropic effects of the prototype and the claimed composition.

The purpose of the research was to assess the ability of the prototype and the claimed composition to activate the mechanisms of anti-infective immunity and, above all, to influence the factors of nonspecific resistance of the body. And also in determining the role in this of the active components (β-glucan and mannan), additionally introduced into the composition of the claimed composition, since it is known that the vital products of Bacillus subtilis bacilli themselves are quite powerful stimuli for immunocompetent cells, leading to their activation. Studies were conducted in comparison with the antibiotic doxycycline, an antibacterial drug widely used in clinical practice.

The studies were performed on white outbred laboratory mice (Rappolovo nursery, Russian Academy of Medical Sciences) weighing 18-23 g. In total, 200 animals were used, divided into four groups of 50 animals each: control (received isotonic sodium chloride solution) and three experimental animals in which animals were injected with the antibiotic doxycycline, a prototype agent or the claimed composition.

Doxycycline was administered intragastrically 1 time per day at a dose of 0.05 mg / mouse.

The prototype was administered orally 1 time per day at a dose of 150 μg / mouse.

The inventive composition was administered orally 1 time per day at a dose of 150 μg / mouse.

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of a strain VKPM No. B-2335 of bacteria Bacillus subtilis (2.3 wt.%), Zeolite (45.3 wt.%), A complex of polysaccharides (51.7 wt.%) Calcium stearate (0.7 wt.%).

The duration of the introduction of the antibiotic, prototype and the claimed composition was 7 days. Evaluation of the impact of the used funds was carried out after 1, 5 and 7 days. For this, before the administration of the preparations (control), as well as 1, 3, and 7 days after the administration, animals of each group were sampled for the study. At each time period, material was collected from 10 animals from the group, individually from each mouse by decapitation.

The dynamics of the development of changes in non-specific resistance factors was assessed by determining the functional state of the cells of the phagocytic system (the digesting activity of granular leukocytes), as well as the concentration in the blood serum of lysozyme and myeloperoxidase, which not only have a direct bactericidal effect, but also mediate intercellular interaction during the development of the immune response to any antigenic effect.

The digesting activity of granular leukocytes of peripheral blood of experimental animals was evaluated by the method of Zelenova E.G. and Nikiforova V.A. (1972). Serum was obtained by standard methods, separating the blood clot from the liquid phase. To assess the functional state of phagocytic cells, blood was collected in tubes pretreated with silicone (Serva, Germany) and containing a heparin solution at the rate of 20 PIECES per 1 ml of blood.

The phagocytic index (Phi), which is the ratio of the severity of the absorbing and digesting functions of phagocytic cells, was determined after 15 minutes and 30 minutes of incubation with the test object of phagocytosis - a test microbe (M. lysodeicticus), which was grown on meat and peptone agar in a thermostat at a temperature of 37 ° C for 24 hours. The index of digesting activity of granular leukocytes (PI), which is the ratio of the number of phagocytic cells in a smear after 30 minutes of blood incubation with the test object gotsitoza to the amount of phagocytic cells in the smear after 15 min incubation with the blood of the test subject phagocytosis.

The IP values were determined by the formula:

where A is the number of phagocytic cells per 100, calculated in a smear of granular leukocytes, which absorbed the test microbe during 15 minutes of incubation;

B is the number of phagocytic cells per 100, counted in a smear of granular leukocytes, which absorbed the test microbe within 30 minutes of incubation.

With the satisfactory functioning of the granular leukocytes of peripheral blood, the IP values are always greater than 1. The IP value was expressed in arbitrary units (Fig. 1).

The concentration of lysozyme in serum was determined by the method of Bukharin O.V. et al. (1974). The blood of the experimental animal was collected in a centrifuge tube and received serum. Then 0.1 ml of blood serum was transferred to a bacteriological tube and 0.9 ml of physiological saline was added. The resulting mixture was mixed and 1.5 ml of a suspension of the test microbe was added to it, which was previously standardized on an FEK-56M photoelectrocolorimeter to an extinction of 0.66.

The prepared suspension containing the test microbe and the test serum was mixed and placed in a thermostat for 30 min at a temperature of 37 ° C. After the incubation time, the tubes were removed from the thermostat and the extinction of the material was determined on FEK-56M. The obtained extinction values using special tables were translated into values characterizing the concentration of lysozyme in the blood serum, and expressed in μg / ml.

The level of myeloperoxidase (MPO) in blood serum was determined by the method developed on the basis of generally accepted approaches to detect this substrate in blood serum and immunocompetent cells (Gell P.G.H. et al. (1968); Page R.C. et al. (1978)). To determine the concentration of MPO in serum in 96-well plates for immunological reactions with a flat bottom, starting from the second well of row A, 0.1 ml of sera from experimental animals were poured. Then, 0.1 ml of the reagent, which is a mixture of benzidine and 3% hydrogen peroxide, was added to them. Instead of serum, 0.1 ml of physiological saline was poured into the first well of row A of the panel (control). After adding the reagent, the panels were shaken, placed in a thermostat and kept at a temperature of 37 ° C for 60 minutes. After the incubation time, the panels were removed from the thermostat and viewed on a vertical Dynatech spectrophotometer (Germany) at a wavelength of 495 nm. The concentration of the enzyme in blood serum was obtained in units of extinction and expressed in arbitrary units.

As shown in figures 2-3, the immunotropic effects of the prototype agent and the claimed composition are manifested at the level of the blood phagocytic system cells and processes associated with the synthesis and secretion of lysozyme and myeloperoxidase enzymes into the blood. The dynamics of changes in the values of factors of nonspecific resistance of experimental animals of the experimental groups, which were introduced the prototype tool or the claimed composition, is almost the same, as evidenced by the absence of statistically significant differences (p> 0.05). The revealed effects, which consisted in a significant increase in the functional activity of factors of nonspecific immunity, were recorded 1 day after the administration of these drugs, and they lasted at least 7 days from the time of administration. That is, the stimulating effect of both the prototype agent and the claimed composition on the studied mechanisms of immunological resistance took place after their single administration.

The values of non-specific resistance factors of animals treated with doxycycline for 1, 3, and 7 days remain practically unchanged with respect to the control, which indicates the absence of the ability to stimulate the functional activity of immunocompetent cells in this basic antimicrobial agent.

The introduction of the prototype for 1, 3 and 7 days leads to a significant increase in the activity of factors of nonspecific resistance compared with the control. This is manifested in an increase in the index of digestive activity of granular leukocytes, as well as in the concentration of lysozyme and myeloperoxidase. A sufficiently significant increase in the functional activity of these factors is observed after a single injection of the prototype means during the first day. On the one hand, this indicates the activation of factors of nonspecific immunity and the possibility of providing quick nonspecific protection of the body from pathogens. On the other hand, this means that the primary mechanisms of action of the prototype agent are associated not only with its direct effect on the microflora of the gastrointestinal tract, but also with a direct effect on the components of local immunity, which, inter alia, are associated with the regulation of the balance of endogenous cytokines that enhance protective reactions.

In the case of using the claimed composition, the composition of which additionally included a complex of polysaccharides in the form of biologically active products of hydrolysis of polymers of the cell wall of brewer's yeast (β-glucan and mannan), its greater effectiveness in the established effects was compared with the prototype agent. So, the claimed composition had a significantly more pronounced effect on the cells of the phagocytic system of blood and their oxygen-dependent bactericidal mechanism (increased concentration of myeloperoxidase in blood serum). However, it is known that an increase in the concentration of serum myeloperoxidase indicates not only the activation of nonspecific immunological resistance, but also an increase in the likelihood of its activating effect on immunocompetent cells, since their membrane has a corresponding specific receptor.

The claimed composition was superior in activity to the prototype agent in relation to the actually bactericidal mechanisms of action of blood serum, which include the activity of serum lysozyme. The observed more pronounced effects of the claimed composition on the specified component of the immune defense in relation to the investigated infectious pathology of a bacterial nature allow it to be preferred as an anti-infectious protection.

Thus, the studies showed that the claimed composition significantly activates the mechanisms of bacterial defense. In the course of further studies, the appropriateness and prospects of using the claimed composition as a means of preventing and treating dangerous infections of bacterial etiology, including generalized salmonella and anthrax infections, were assessed.

Example 5. The study of interferonogenic properties of the prototype and the claimed composition.

Since non-specific immunological resistance is designed to provide protection not only for bacterial, but also for viral infections, we investigated the extent to which the prototype agent and the claimed composition are able to influence interferonogenesis.

The studies were performed on white outbred laboratory mice (Rappolovo nursery RAMS) weighing 18-23 g. Experimental groups of animals were formed as described in example 4.

In a particular case, the composition of the claimed composition: CCM containing metabolites of the strain VKPM№B-2335 of bacteria Bacillus subtilis (2.1 wt.%), Zeolite (44.6 wt.%), A complex of polysaccharides (52.4 wt.%) Calcium stearate (0.9 wt.%).

To determine the interferonogenic properties of the prototype and the claimed composition in dynamics, blood was collected from experimental animals from the retroorbital sinus, combining material from three animals in one sample. The serum obtained after centrifugation at 1000 rpm for 10 minutes was aspirated and stored at a temperature of minus 20 ° С. The internal organs of mice were triturated with physiological saline, the homogenate was centrifuged (4000 rpm) for 20 minutes, and a 10% suspension in physiological saline was prepared from the supernatant. The samples thus obtained were stored at a temperature of minus 20 ° C. Interferon titration (IFN) in the obtained samples was carried out by micromethod in cell culture. The L-929 cell culture, diluted with growth medium to the required concentration (3-4 × 10 ⁵ cells / ml), was added to the wells of 96-well plastic panels and incubated in an incubator for 24 hours. Throughout the entire titration process, the cell cultures were incubated under identical conditions: temperature 37 ° C in an atmosphere with 5% CO ₂ and 98% humidity. After the formation of a cell monolayer at the bottom of the wells, the growth medium was removed from the panel and a support medium was introduced, in which successive twofold dilutions of the tested samples were made (in paired wells). At the end of the daily incubation, the test samples were removed and the test virus encephalomyocarditis (EMC) was introduced in a working dilution of 100 CPD ₅₀ (cytopathogenic dose, at which 50% of the cells died). After a day, the results were recorded using an inverted microscope. IFN titer is the reciprocal of the last dilution of serum or organ homogenate sample, at which 50% protection against the test virus is observed.

It was found that the prototype tool practically does not have interferonogenic properties (figure 4). In experimental animals that were injected with the prototype agent, the level of serum interferon practically did not change with respect to the control values, although it increased slightly (up to 20 PIECES / ml), however, these differences with the control level (5 PIECES / ml) were statistically unreliable. At the same time, a single injection of the claimed composition in the same dose as the prototype agent (150 μg / mouse) had a rather pronounced interferonogenic effect. Already 1 day after the introduction of the claimed composition, the level of serum interferon increased to 160 IU / ml (the differences were significant (p <0.05) compared with the control and the level of serum interferon in animals treated with the prototype). In the future, a decrease in the level of serum interferon was observed, however, in the subsequent periods of the study, the estimated indicator significantly exceeded the data of the control group and the group of animals receiving the prototype agent (p <0.05).

Thus, the studies showed that the claimed composition in addition to activating the mechanisms of bacterial defense, significantly activated and interferonogenesis - a key mechanism of antiviral protection. Sufficiently fast production of endogenous interferon allows already at an early stage to provide reliable protection of the body against infectious diseases of viral etiology.

In the course of further studies, the appropriateness and prospects of using the claimed composition as a means of preventing and treating dangerous viral infections, including tick-borne encephalitis and West Nile fever, having general epidemiological significance, the causative agents of which are among potential biological damaging agents and bioterrorism agents, were assessed.

Example 6. The study of the specific anti-infective activity of the prototype and the claimed composition on a model of experimental generalized salmonella infection.

The experiments were performed on white outbred laboratory mice (Rappolovo cattery RAMS) at the age of 2-3 months and weighing 18-23 g. Before the experiment, the experimental animals were compulsorily quarantined for 1 week.

The most appropriate experimental model for studying the effectiveness of the prototype and the claimed composition is the model of salmonellosis of white outbred mice, induced by the oral administration of the pathogen. To simulate an experimental generalized salmonella infection, a collection strain of the Enterobacteriaceae family microorganism (Salmonella typhimurium breslau, strain No. 20) was used. As a result of determining the dose of the pathogen that causes the death of 50% of infected animals (LD ₅₀ / ml), it was found that LD ₅₀ / ml S. typhimurium for outbred mice is 10 ⁷ MK / ml. In further studies, a pathogen dose of 5 LD ₅₀ / ml was used to infect the animals.

In the experiment, 3 groups of animals (control and 2 experimental) of 10 animals each were formed.

The animals of the first group were not prescribed any drugs, an isotonic sodium chloride solution (placebo) was administered and used as a control.

The animals of the second group were injected with a prototype agent in the form of a suspension of powder (orally at a dose of 150 μg / mouse).

The animals of the third group were injected with the claimed composition in the form of a suspension of powder (orally at a dose of 150 μg / mouse).

For oral administration of suspensions of the prototype agent and the claimed composition, syringes with a special probe were used.

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of a strain VKPM№B-2335 of bacteria Bacillus subtilis (1.8 wt.%), Zeolite (46.0 wt.%), A complex of polysaccharides (51.4 wt.%) Calcium stearate (0.8 wt.%).

The introduction of the prototype and the claimed composition regarding infection by the pathogen: once 24 hours before infection (prophylactic); once 24 hours after infection (therapeutic); three times: 24 hours before, simultaneously and 24 hours after infection (combined).

The criterion for anti-infective activity of the drugs used was the survival of animals in the experimental groups. The percentage of surviving animals was determined according to the tables of Genes V.S. Infected animals were monitored for 21 days from the time of infection, daily recording the number of live and dead animals in the experimental groups.

As follows from the data presented in figa, a single prophylactic administration of the prototype before infection of experimental animals with the causative agent of salmonella infection at a dose of 5 LD ₅₀ exerted a certain protective effect in relation to this type of infection, expressed in ensuring the survival rate of 20% of infected animals compared to 100% mortality in control. At the same time, the prophylactic effectiveness under the similar conditions of the claimed composition was higher - against the background of 100% mortality in the control, the animal survival rate was 40%, that is, 2.0 times higher than the prophylactic effectiveness of the prototype.

With therapeutic use of the prototype and the claimed composition (Fig.5b) it was found that the effectiveness of the use of both drugs was 10% less pronounced than with their single prophylactic use. However, in this case, the effectiveness of the claimed composition compared to the prototype was 3.0 times higher.

The most pronounced protective effect of both the prototype tool and the claimed composition was recorded when they were used according to the combined scheme (Fig. 5c). Moreover, in the case of using the prototype means, 60% survived, and the claimed composition - 100% of infected animals on the background of 100% mortality in the control. The protective properties of the claimed composition not only significantly exceeded the control values (p <0.05), but also 1.7 times higher than the specified indicator of the prototype.

Thus, the protective properties of the claimed composition in relation to experimental generalized salmonella infection are more pronounced than that of the prototype. Under the condition of a single prophylactic use, the excess was 2.0 times, a single treatment - 3.0 times, and with combined use - 1.7 times.

Example 7. The study of the specific anti-infective activity of the prototype and the claimed composition on a model of experimental anthrax infection.

The studies were performed on white outbred laboratory mice (Rappolovo cattery of the Russian Academy of Medical Sciences) at the age of 2-3 months and weighing 18-23 g. Before the experiment, the experimental animals were mandatory quarantined for 1 week.

To simulate an experimental anthrax infection, the collection strain of Bacillus anthracis 71/12 (the second Zenkovsky vaccine) was used. The spore form of the pathogen was prepared as follows: in the meat and peptone broth (pH = 7.2-7.4) at a temperature of 37 ° C, uterine material was grown for 24 hours. The daily broth culture was evenly distributed over the surface of wheat agar spilled into mattresses, and cultivated in a thermostat at a temperature of 32 ± 0.5 ° C for 72 hours. After cultivation in a thermostat, the mattresses were wrapped in dark paper and kept in a dark place at a temperature of 10 ° C for 5-7 days. The grown culture was washed from the surface of the agar with sterile distilled water. The resulting spore suspension was transferred to a sterile flask with a ground stopper, which was shaken in a joker apparatus for 60 minutes. After the time of shaking, the contents of the flask were filtered through a sterile cotton-gauze filter, placed in a sterile bottle with beads and stored at 4 ± 0.5 ° C.

As a result of determining the dose of the pathogen causing the death of 50% of infected animals (LD ₅₀ / ml), it was found that the LD ₅₀ / ml of Bacillus anthracis for outbred mice was 20 spores / ml. In further studies, a pathogen dose of 10 LD ₅₀ / ml was used to infect the animals.

Experimental groups of animals were formed as described in example 6. The prototype and the inventive composition were administered at a dose of 150 μg / mouse each.

Studies have been conducted with the aim of evaluating the protective properties of the prototype and the claimed composition with a single administration of them at different times relative to infection with the causative agent of anthrax infection. The criterion for the anti-infective effectiveness of the drugs used was the survival of the animals in the experimental groups.

In a particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№B-2335 of bacteria Bacillus subtilis (1.7 wt.%), Zeolite (45.7 wt.%), A complex of polysaccharides (51.1 wt.%) Calcium stearate (1.5 wt.%).

As follows from the data presented (figa), a single prophylactic administration of the prototype or the claimed composition is most effective if then the infection occurred 1 day after the introduction of funds. In this case, against the background of 100% mortality in the control, 40% survived in the groups of experimental animals (p <0.05). The effectiveness of the prototype and the claimed composition in these conditions was the same. With an increase in the time interval between a single administration of each agent and subsequent infection of animals with the causative agent of anthrax infection, their protective effectiveness decreased. Moreover, the dynamics of the decrease in the protective properties of both agents was the same: with the introduction of both the prototype agent and the claimed composition 2 days before infection, 10% of the experimental animals survived, and subsequently, these agents did not show anti-infection effectiveness with increasing time.

Slightly different results were obtained with a single therapeutic use of the prototype means or the claimed composition (Fig.6b). So, the prototype tool was practically not effective, providing only 20% protection of infected animals against 100% mortality in the control when it was introduced 1 day after infection. In similar conditions, the claimed composition provided a more pronounced level of protection. When it was introduced 1 day after infection, the survival rate of infected animals was 50%, after 2 days - 30%, after 3 days - 10%. The inventive composition was practically ineffective when administered 4 days after infection.

The most effective was the use of both drugs in a single administration at the same time as infection (Fig.6c), which manifested itself in an increase in the survival rate of infected mice by 40% (prototype agent) and 60% (inventive composition) against 100% mortality in the control. As can be seen, the effectiveness of the claimed composition is 1.5 times higher in comparison with the prototype tool.

The totality of the presented results indicates that a single use of the claimed composition for protection against experimental anthrax infection is more effective than a single use of the prototype.

Example 8. The study of the protective properties of the claimed composition on a model of experimental tick-borne encephalitis.

The experiments were performed on white non-inbred male mice weighing 16-18 g. Before the experiment, the experimental animals were mandatory quarantined for 1 week.

Tick-borne encephalitis was simulated using tick-borne encephalitis virus (TBE) (pathogenic Absettarov strain). The accumulation of virus-containing material for infection of laboratory animals was carried out on sucker mice. Initially, five consecutive ten-fold dilutions of virus-containing material were prepared, which were a centrifugate of a 10% suspension of the brain of previously infected mice or virus-containing material rehydrated from the lyophilic state. 0.02 ml of each dilution was injected into the brain of sucker mice, which was monitored for 24-48 hours, after which the animals were killed with ether, the brain was removed and three samples were deposited in penicillin vials, which were stored in a freezer at a temperature of minus 20 ± 0.5 ° С. Subsequently, a 10% suspension of the brain was used as a virus-containing material. The initial titer of the virus was 10 ² -10 ³ LD ₅₀ / ml

In the experiment, 3 groups of animals were formed (control and 2 experimental) of 14 animals each. Experimental animals of all groups were infected with a virus at a dose of 8 LD ₅₀ .

The animals of the control group were not prescribed any drugs and used them as a control.

The animals of the first and second experimental groups were administered the inventive composition orally in a volume of 0.5 ml and at a dose of 150 μg / mouse. In this case, the animals of the first experimental group of the claimed composition was administered 5 days, 4 days, 3 days, 2 days, 1 day before infection (preventive regimen), and the animals of the second experimental group - simultaneously with infection and then 1 time per day for 5 days in a row (treatment regimen).

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№V-2335 of bacteria Bacillus subtilis (2.2 wt.%), Zeolite (43.8 wt.%), A complex of polysaccharides (53.6 wt.%) Calcium stearate (1.4 wt.%).

The criterion of anti-infective effectiveness of the claimed composition was the survival of animals in the experimental groups. During the experiment it was established (table 8) that when the inventive composition was introduced for prophylactic or therapeutic purposes, the resistance of experimental animals to tick-borne encephalitis virus infection was 64.28 ± 10.52% or 57.14 ± 12.34%, respectively, with 100% mortality in control (p <0.05). This circumstance indicates that the claimed composition exhibits antiviral activity due to its interferonogenic properties.

Example 9. The study of the specific anti-infective activity of the claimed composition on a model of experimental West Nile fever.

The studies were performed on white outbred male mice weighing 18-23 g, obtained from the Rappolovo nursery of the Russian Academy of Medical Sciences. Before the experiment, experimental animals quarantined without fail for 1 week.

West Nile Experimental Fever was modeled using West Nile Fever Virus (LUS) (pathogenic strain HP91). The accumulation of virus-containing material for infection of experimental animals was carried out on sucker mice. Initially, five consecutive ten-fold dilutions of virus-containing material were prepared, which were a centrifugate of a 10% suspension of the brain of previously infected mice or virus-containing material rehydrated from the lyophilic state. 0.02 ml of each dilution was introduced into the brain of sucker mice and they were monitored for 24-48 hours, after which the animals were killed with ether, the brain was removed and three samples were deposited in penicillin vials, which were stored in a freezer at a temperature of minus 20 ± 0.5 ° С. Subsequently, a 10% suspension of the brain was used as a virus-containing material. The initial titer of the virus was 10 ³ -10 ⁴ LD ₅₀ / ml

In the experiment, 3 groups of animals (control and 2 experimental) of 10 animals each were formed. The virulent pathogen strain was used at an infectious dose of 10 LD ₅₀ and was injected subcutaneously in a volume of 0.3 ml.

The animals of the control group were not prescribed any drugs.

The animals of the first and second experimental groups were administered the inventive composition orally in a volume of 0.5 ml and at a dose of 150 μg / mouse. In this case, the animals of the first experimental group of the claimed composition was administered 5 days, 4 days, 3 days, 2 days, 1 day before infection (preventive regimen), and the animals of the second experimental group - simultaneously with infection and then 1 time per day for 5 days in a row (treatment regimen).

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№V-2335 of bacteria Bacillus subtilis (1.9 wt.%), Zeolite (43.2 wt.%), A complex of polysaccharides (53.4 wt.%) Calcium stearate (1.5 wt.%).

The criterion of anti-infective effectiveness of the claimed composition was the survival of animals in the experimental groups. Infected animals were monitored for 21 days, taking into account the number of live and dead animals daily.

It was found that all experimental animals that received the claimed composition orally repeatedly, under conditions of infection with their West Nile fever virus at a dose of 10 LD _50, survived at 100% mortality in the control group (table 9). Moreover, in the control group, the death of two animals (20%) was observed already 5 days after infection, after 7 days - 8 animals (80%), and after 10 days - all 10 animals (100%) (table 10).

Thus, the feasibility of using the inventive composition as a means for the prevention and treatment of viral infections, including a dangerous viral infection - West Nile fever, has been proved once again.

Example 10. The study of the effectiveness of the combined use of the claimed composition and the antibiotic doxycycline on a model of experimental anthrax infection.

The studies were performed on white outbred laboratory mice (Rappolovo nursery, Russian Academy of Medical Sciences) weighing 18-23 g. Before the experiment, the experimental animals were compulsorily quarantined for 1 week.

To simulate an experimental anthrax infection, a collection strain of Bacillus anthracis 71/12 (the second Zenkovsky vaccine) was used. The spore form of the pathogen was prepared as described in example 7. To infect animals, the pathogen was administered at doses of 10, 100 and 1000 LD ₅₀ / ml.

In the experiment, 4 groups of animals were formed, 10 animals each.

The animals of the first group were not prescribed any drugs, an isotonic sodium chloride solution (placebo) was administered and used as a control.

The animals of the second group were prescribed antibiotic doxycycline in the form of 0.1 g capsules manufactured by Fereyn, the contents of which were dissolved in a 0.9% sodium chloride solution and administered orally 1 time per day. The dose of the antibacterial agent was equivalent to one human dose and was 1 mg / mouse.

For the animals of the third and fourth groups, the combination was prescribed the antibiotic doxycycline and the claimed composition and administered orally. Doxycycline was prescribed according to the generally accepted scheme, that is, exclusively after infection. Doxycycline was administered orally 1 time per day and at a dose of 1 mg / mouse. The inventive composition was administered in a single dose of 150 μg / mouse and in a volume of 0.5 ml. The animals of the third group of the claimed composition was administered according to the prophylactic regimen (before infection), and the animals of the fourth group - according to the treatment regimen (after infection).

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№V-2335 of bacteria Bacillus subtilis (2.3 wt.%), Zeolite (45.4 wt.%), A complex of polysaccharides (51.2 wt.%) Calcium stearate (1.1 wt.%).

As shown in Fig.7, the average life expectancy of animals in the control group infected with the causative agent of anthrax infection in doses of 10, 100 and 1000 LD ₅₀ is 4.1 days, 3.5 days and 2.5 days, respectively.

It was found that, in contrast to antibiotic monotherapy, the combined therapeutic prescription of the antibiotic and the claimed composition contributes to a significant (p <0.05) increase in the average life expectancy of experimental animals with infectious doses of 10 and 100 LD ₅₀ . So, the combined oral administration of an antibiotic and the claimed composition 1 day or 2 days after infection contributes to an increase in the average life expectancy of experimental animals by 56.6% or 35.6%, respectively, with a pathogen dose of 10 LD ₅₀ and 26.6% or 25, 0% - at a dose of the pathogen 100 LD ₅₀ . With a high pathogen dose of 1000 LD ₅₀ , the increase in the average life expectancy of animals was only 14.3% or 13.6% and did not have statistical significance (p> 0.05).

Thus, it has been experimentally established that the therapeutic appointment of a combination of doxycycline and the claimed composition is advisable 1 day or 2 days after infection with the infectious agent in low doses. To significantly increase the average life expectancy of experimental animals infected with high doses of the pathogen (1000 LD ₅₀ ), the combined administration of an antibiotic and the claimed composition must be started no later than 0.5 days after infection (Fig. 8). This will increase the average life expectancy of experimental animals by 25.0%.

In addition, it was found that the combined use of doxycycline and the claimed composition, regardless of the route of administration of the claimed composition, was more effective in terms of forming protection against infection by the causative agent of anthrax infection than using only the claimed composition or only doxycycline. So, when prescribing the inventive composition in combination with doxycycline before infection, a high level of resistance of experimental animals to infection was ensured not only with an infectious dose of the pathogen 10 LD ₅₀ , but also 100 LD ₅₀ , that is, in the case when the single exposure of the inventive composition was ineffective, and doxycycline provided protection at the level of 70% (figa).

A similar pattern was observed with the introduction of the inventive composition in combination with doxycycline after infection (figb). However, in this case, a high protective effect from the combined use of these drugs was noted not only at low infectious doses of the causative agent of anthrax infection (10 LD ₅₀ and 100 LD ₅₀ ), but also 1000 LD5o. In the latter case, doxycycline monotherapy provided protection at the level of 40%, and the claimed composition was not effective. The effect of the combined administration of drugs under similar conditions reached 90% against the background of 100% mortality in the control (p <0.001).

Thus, the totality of the experimental data obtained indicates the promise of the combined use of antibacterial and probiotic agents in cases of emergency prevention and etiotropic treatment of dangerous infectious diseases of a bacterial nature, in particular anthrax infection. The use of the claimed composition in a similar situation is highly effective, which is associated with the development between the claimed composition and the antimicrobial agent used of a certain mutually potentiating effect. This effect is due to the presence in the composition of the claimed composition of polysaccharides in the form of a complex of biologically active products of enzymatic hydrolysis of cell wall polymers of brewer's yeast (β-glucan and mannan), which have an additional significant activating effect on immunocompetent cells, which accounts for the observed potentiating effect.

Example 11. The study of the effectiveness of the combined appointment of the claimed composition and the antibiotic doxycycline on a model of generalized salmonella infection.

The studies were performed on white outbred laboratory mice (Rappolovo nursery of the Russian Academy of Medical Sciences) weighing 18-23 g. Before the experiment, the experimental animals were mandatory quarantined for 1 week.

Used a collection strain of microorganisms of the Enterobacteriaceae family (S. typhimurium breslau, strain No. 20). The infectious dose of the pathogen was 5 LD ₅₀ / ml. Formed 4 groups of animals, as described in example 10.

In a particular particular case, the composition of the claimed composition: SKZh containing metabolites of the strain VKPM№V-2335 of bacteria Bacillus subtilis (1.6 wt.%), Zeolite (43.7 wt.%), A complex of polysaccharides (53.2 wt.%) Calcium stearate (1.5 wt.%).

During the experiment, it was found that in order to increase the effectiveness of protection during infection with salmonella infection, it is advisable to prescribe the inventive composition in conjunction with doxycycline, and enter it before infection (Fig. 10a). In this case, the protective effect is provided even in cases of infection with the pathogen at a dose of 1000 LD ₅₀ , that is, when monotherapy using the inventive composition is not effective, and the effect of the antibacterial drug is reduced by 60%. With the prophylactic purpose of the claimed composition, both a direct effect on pathogenic microorganisms of its probiotic component and a significant activation of immunocompetent cells are provided. As a result of such exposure, pathogenic organisms lose their stability, and the antibacterial agent exhibits a more pronounced anti-infectious effect.

The appointment of both drugs after infection according to the treatment regimen is also more effective than atibiotic monotherapy (Fig. 10b).

Confirmed are the results obtained under conditions of infection with anthrax infection, which showed the advisability of the combined administration of the claimed composition and antibiotic also when infected with a generalized salmonella infection.

Thus, the totality of the obtained experimental data indicates the achievement of the goal of the invention - the creation of a composition for effective pathogenetic prophylaxis and treatment of dangerous infectious diseases, which significantly activates the mechanisms of antibacterial protection and interferonogenesis (a key mechanism of antiviral protection) and is universal for bacterial and viral etiology when administered orally both with monotherapy, and with a combination of prescribing with basic etiotropic drugs. The inventive composition is safe, because the content of toxicants (organochlorine pesticides HCTs and DCTs), as well as hazardous substances (lead, cadmium, mercury, arsenic) and radionuclides ( ⁹⁰ Sr and ¹³⁷ Cs) does not exceed acceptable levels, and it does not has acute toxicity. The guaranteed shelf life of the claimed composition is at least 2 years at a temperature not exceeding 40 ° C and the use of special equipment is not required during storage.

The claimed invention meets the criterion of "novelty", since for the first time for the prevention and treatment of dangerous infectious diseases, it is proposed to use a composition that provides for the combined action of metabolites of probiotic microorganisms and products of enzymatic hydrolysis of polymers of the inner and outer layers of the cell wall of brewer's yeast with immunomodulating activity, characterized by high specific efficiency and universality in relation to infections of a bacterial and viral nature.

The claimed invention satisfies the criterion of "inventive step", since it is not obvious from the available information sources that the complex use of metabolites of probiotic microorganisms and fermentolysis products of the cell wall of brewer's yeast (β-glucan and mannan) as part of the claimed composition, as well as their optimal content to ensure highly effective anti-infection protection.

The compliance of the claimed invention with the criterion of "suitability for use" is confirmed by the above examples, which showed a fairly simple technology for producing a solid dosage form of the claimed composition in the form of a powder, the ingredients of which are inexpensive, are available from the domestic industry and are available, and the results of numerous studies confirming its safety and stability when stored in 2 years without the use of special equipment, as well as the presence of immunotropic effects, interferonog nnyh anti-infectious properties and high efficiency, indicating that the inventive composition will be widely used in clinical practice for preventing and treating hazardous infections like bacterial and viral etiology in case administration of the monotherapy or combination scheme assigning a conventionally used basic drugs.

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Claims (1)

A composition with anti-infective activity, made in solid dosage form for oral administration in powder form, which is a balanced complex comprising a probiotic agent, an adsorbent and an auxiliary additive, in which a sterilized culture fluid containing metabolites of VKPM strain No. B-2335 is used as a probiotic agent bacteria Bacillus subtilis, as an adsorbent - zeolite, as an auxiliary additive - calcium stearate or aerosil, characterized in that it additionally includes a highly active immunomodulating agent, which is a complex of polysaccharides - products of enzymatic hydrolysis of polymers of the inner and outer layers of the cell wall of brewing yeast - in the form of β-glucan and mannan, in the following ratio of ingredients, wt.%:
Sterilized culture fluid containing metabolites of VKPM strain No. B-2335 of Bacillus bacteria subtilis 1.6-2.3 Zeolite 43.0-46.0 Polysaccharide Complex: β-Glucan and Mannan 51.0-54.0 Calcium Stearate or Aerosil 0.5-1.5

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