RU2396968C2 - Agent for treating gastrointestinal disturbances and method for making thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to agents for treating gastrointestinal disorders (GID) based on probiotics, and their metabolites. There is offered a preparation containing (wt %): zeolite - 70÷80; soya flour hydrolysate - 15÷25; calcium stearate - 0.5÷1.5; lytic enzymes - 0.05÷3.5; microorganism cell lysate - the rest. The method involves microorganism cultivation, mixing of a metabolite mixture with zeolite and adjuvants, sterilisation and packing of an end product; and the prepared producing microorganism cells are treated with lytic enzymes for 2-10 hours at temperature +20-50°C. If necessary, the composition additionally contains enzymatic preparations to the preset parametres. An agent represents a preparation of broad clinical effectiveness; it is recommended in treatment regimens for the patients suffering from intestinal dysbacteriosis of various geneses.

EFFECT: when implementing the technology, constant biochemical characteristics of the composition of the declared agent are provided; living producing cells are eliminated from the end product; storage-stability of the agent is improved; preparation of the end agent is simplified due to eliminated drying stage.

8 tbl, 16 ex

Description

The invention relates to medicine, namely to drugs for the treatment of diseases of the gastrointestinal tract (GIT), obtained on the basis of probiotic microorganisms and / or their metabolites. When introduced into the body, drugs of this group have a positive effect on the physiological, biochemical and immune responses of the host through stabilization and optimization of the functional activity of the normal gastrointestinal microflora and can be used in applied biotechnology, medicine, food industry, veterinary medicine and related industries.

Currently, to normalize the activity of the gastrointestinal tract, preparations containing live microorganisms are orally administered into the macroorganism, in particular biological preparations based on lactic acid microorganisms [EP 0097484, 1984 cl. A23C 1/05: EP 0043962, 1980 cl. A23C 19/086: US 4289888, 1979, cl. A23C 9/123]. As a rule, the composition of such drugs includes: bacterial cells, residues of the culture fluid (QOL), fats, proteins, lactose, gum and other additives. The specific composition of such compositions is determined by the characteristics of the microorganisms used and the nature of the task.

The disadvantage of these drugs is their relatively low biological activity associated with the process of inactivation of live microorganisms during their passage through the upper gastrointestinal tract (stomach, duodenum), as well as the lack of stability of their biological properties both upon receipt and during long-term storage, which leads to reduce the effectiveness of treatment procedures.

More stable during storage are probiotic preparations containing spore-forming bacteria as an active principle. The probiotic Bactisubtil (firm Marion Merrell, France) and its analogue Flonivin BS (firm Galenika, Serbia) containing a culture of the strain Bacillus cereus IP 5832 (collection of the Pasteur Institute, Paris), as well as Enterohermine (firm Sanofi) were widely used. Winthrop ", Italy) and Cereobiogen (" Xing Jian ", China), the active principle of which are various strains of B. cereus [Discussion questions of the creation and use of bacterial preparations for the correction of warm-blooded microflora. // Microbiol. Zh., 1992, t. 54, No. 6, p. 82-93]. The disadvantage of these probiotics is the limited scope of their action, as they are directed against a relatively narrow group of pathogens.

Known therapeutic and biological product "Baktisporin" used to treat a wide range of diseases. It contains freeze-dried biomass of bacteria of the Bacillus subtilis 3H strain and a protective medium based on a sugar gelatin mixture or lactose [RU 2130316, 1999]. However, the biological product also has low effectiveness against pathogens of acute intestinal infections such as Pseudomonas spp., Streptococcus spp., Enterococcus spp.

Known drugs "Biosporin" and "Irilis", which are characterized by high antagonistic activity to a wide range of pathogenic and conditionally pathogenic microorganisms. The preparations contain living cells of strains of Bacillus subtilis and Bacillus licheniformis, as well as an excipient [RU 1722502, 1992; RU 2264454, 2005]. The disadvantage of these drugs is the sensitivity of their microorganisms to antibiotics, which excludes the possibility of their combined use with antibacterial agents. Therapeutic doses of antibiotics a priori cause the death of pathogenic forms of bacteria and the inevitable suppression of the functional activity of living strains of probiotic microorganisms. This leads to a significant reduction and / or lack of the expected clinical effect, which limits the practical application of such treatment regimens.

Of the analogues in technical essence and the claimed effect, the closest to the claimed invention is the drug "Bactistatin" [RU 2287335, 2006], taken orally in the form of capsules. The composition of the drug includes (wt.%): Sterilized culture fluid (SCF) containing metabolites of the Bacillus subtilis strain - 0.1 ÷ 2.0, zeolite - 68.0 ÷ 85.0, soy flour hydrolyzate (GSM) - 15, 0 ÷ 30.0 and calcium stearate (SC) - 0.5 ÷ 5.0.

The action of the Bactistatin preparation is based on the fact that during its transit through the gastrointestinal tract in a given zone, the protective gelatin capsule is destroyed and probiotic components immobilized on zeolite particles are released into the intestinal cavity. In the process of movement of zeolite particles along the gastrointestinal tract from the porous surface of the adsorbent, biologically active compounds are gradually released, which allows maintaining the activity of the biological components of the probiotic in the gastrointestinal tract for at least a day, which is necessary to restore and stimulate the functional activity of the normal intestinal microflora. At the same time, the effect of the gradual release of the drug components from the zeolite surface leads to the appearance of open surfaces of its structure, which ensures the inclusion of ion exchange mechanisms and selective adsorption of toxic compounds.

The drug is prepared as follows. Microorganisms of B. subtilis are grown by the method of deep cultivation. Then the culture fluid (QL) with microorganisms is subjected to centrifugation and sterilization, and the obtained sterilized QL containing producer metabolites is mixed with fuel, lubricant, zeolite and SC (as an option, a mixture of QL with zeolite is sterilized). The resulting mixture is subjected to lyophilization, in which the biologically active components of the preparation are immobilized on zeolite particles, and Packed in capsules.

The disadvantages of the drug Bactistatin and its production method are:

- the instability of the composition due to the use of deep cultivation of B.subtilis bacteria, in which the accumulation of producer metabolites in the QOL depends on a large number of factors, as well as the presence of high molecular weight proteins and peptides in the preparation, which increase the likelihood of allergic reactions;

- a relatively rapid decrease in the activity of the drug during prolonged storage without special equipment;

- the possibility of the presence in the QOL of spore forms of the producer, which makes it necessary to apply harsh sterilization methods, leading simultaneously to the destruction of biologically active substances that make up the active principle of the drug.

The problem solved by the inventors is the creation of a standard compositionally complex probiotic agent with higher activity and storage stability, as well as a more economical and technologically advanced method for its preparation.

The technical result of the invention in relation to the claimed "means" is that as a source of metabolites and / or other biologically active substances (BAS), it is not a culture fluid containing producer metabolites that is used, but the products of microorganism cell breakdown by lytic enzymes ("lysate") in the following ratios of the ingredients of the final product (wt.%): zeolite - 70.0 ÷ 80.0; hydrolyzate of soy flour (fuels and lubricants) - 15.0 ÷ 25.0; calcium stearate (SC) - 0.5 ÷ 1.5; lytic enzymes - 0.05 ÷ 3.5; producer cell lysate - the rest.

It was shown that a significant suppression of the growth of test cultures of the microorganisms Sh.sonnei and S.aureus (table 1) compared with the analogue drug can be achieved regardless of the nature of the microorganisms. Lithium enzymes either remain in the composition after the completion of lysis as part of the lysate, or, in particular, when low concentrations of the lysate (less than 0.05 ÷ 0.1%) are introduced into the composition of the claimed agent, they are introduced into the composition in the form of enzyme preparations introduced from the outside.

The best results on the impact on these test cultures of microorganisms were achieved using the product, received the code name "SL08", containing (wt.%): Cell lysate of Bacillus subtilis - 0.10; lytic enzymes - 0.5; zeolite - 76.0; soy flour hydrolyzate - 22.4; calcium stearate - 1.0. The SL08 agent was provided with an enteric-soluble capsule coated with an acid-protective layer.

The technical result of the invention in relation to the "method" is that after cultivation, cells of microorganisms are isolated and subjected to lytic enzymes for 2 ÷ 10 hours at a temperature

+ 20.0 ÷ 50.0 ° C, after which it is mixed with excipients, and then packaged in gelatin capsules (0.5 or 1.0 g each).

As a result of using the proposed method at the stage of separation of microorganism cells from the nutrient base from further processing, the ingress of undesirable components of the culture medium into the final substance “means” is excluded. Under the influence of lytic enzymes, an increased controlled release of useful cell metabolites occurs, at the same time destruction of potential allergens (proteins, polysaccharides, etc.) is provided, and the possibility of live microorganisms and spores entering the final preparation is excluded. In this case, there is no need to use strict sterilization procedures for the biologically active substances, and the zeolite and / or fuel and lubricants can be sterilized using conventional methods, for example, autoclaving or dry heat.

In addition, the process of sequential mixing of biologically active components of the claimed means eliminates the use of the subsequent drying procedure of the resulting final substance by lyophilic or any other method.

Thus, the implementation of the above technology for producing a preparation based on an enzymatic lysate of microorganism cells ensures the constancy of biochemical characteristics of the composition of the claimed “agent”; excludes the subsequent appearance in the final product of living cells; increases the stability of the composition during storage; simplifies the process of obtaining the final substance of the composition by eliminating the drying stage.

Example 1. Microorganisms B.subtilis VKPM No. B-2335 (3) were grown by surface cultivation. After the cultivation process, mechanical separation of cells from the culture medium was performed. Then the cells were subjected to destruction using the lytic enzyme subtilisin (0.8 wt.%) For 2 hours at a temperature of + 50 ° C. The obtained enzymatic cell lysate was mixed with GSM, zeolite and SC. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. The result was a preparation of the following composition (wt.%): B.subtilis cell lysate — 0.25; lytic enzymes - 0.75; zeolite - 78.0; hydrolyzate of soy flour - 20.0; calcium stearate - 1.0. The yield of the final product is 200 ± 5 kg.

Example 2. Microorganisms B.subtilis strain VKPM No. 8-2335 (3) was grown by the method of deep cultivation. After the cultivation process, cells were separated from the culture medium by centrifugation. Then the cells were subjected to destruction using the lytic enzyme papain (0.1 wt.%) For 3 hours at a temperature of + 42 ° C. The obtained enzymatic lysate of B.subtilis cells was mixed with GSM, zeolite and SC. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 0.5; Fuels and lubricants - 20.2; zeolite - 78.2; SC - 1.0; lytic enzymes - 0.1. The yield of the final product is 180 ± 5 kg.

Example 3. Microorganisms B.subtilis strain TPI 13-07.06.21-DEPT / VGNKI were grown by surface cultivation. After the cultivation process, mechanical separation of cells from the culture medium was performed. Then the cells were subjected to destruction using the lytic enzyme pepsin (0.2 wt.%) For 6 hours at a temperature of + 35 ° C. The obtained enzymatic lysate of B.subtilis cells was mixed with GSM, zeolite and SC. Before mixing, fuels and lubricants and zeolite were subjected to autoclave sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 1.65; Fuels and lubricants - 16.8; zeolite - 80.0; SC - 1.5; lytic enzymes - 0.05. The yield of the final product is 215 ± 5 kg.

Example 4. Microorganisms B.subtilis strain VKPM No. B4759 (3) were grown by surface cultivation. After the cultivation process, cells were separated from the culture medium by separation. Then the cells were destroyed using a neutral protease lytic enzyme (2.08 wt.%) For 4 hours at a temperature of + 37 ° C. The obtained enzymatic lysate of B.subtilis cells was mixed with GSM, zeolite and SC. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. The result was a preparation of the following composition (wt.%): Cell lysate - 1.3; Fuels and lubricants - 25.0; zeolite - 72.4; SC - 0.5; lytic enzymes - 0.8. The yield of the final product is 140 ± 5 kg.

Example 5. Microorganisms of the mixed culture of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus (Yo-Flex® culture for yogurt production, Christian Hansen / Denmark) were grown by the method of deep cultivation. After the cultivation process, cells were separated from the culture medium by separation. Then the cells were destroyed using the lytic enzyme trypsin (2.1 wt.%) For 4 hours at a temperature of + 37 ° C. The obtained enzymatic cell lysate of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus was mixed with GSM, zeolite and SK. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 2.5; Fuels and lubricants - 20.5; zeolite - 74.6; SC - 1.2; enzymes - 1.2. The yield of the final product is 170 ± 5 kg.

Example 6. Microorganisms B.subtilis strain VKPM No. B4759 (3) was grown by surface cultivation. After the cultivation process, cells were separated from the culture medium by separation. Then the cells were destroyed using the lytic enzyme Biopron (Nagase / Japan - 1.1 wt.%) For 10 hours at a temperature of + 37 ° C. The obtained enzymatic lysate of producer B.subtilis was mixed with fuel and lubricants, zeolite and SK. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 0.1; Fuels and lubricants - 22.4; zeolite - 76.0; SC - 1.0; lytic enzymes - 0.50. The yield of the final product is 140 ± 5 kg.

Example 7. Microorganisms of the mixed culture Lactococcus lactis subspecies lactis and cremoris, Leuconostoc mesenteroides subspecies cremoris and Leuconostoc pseudomesenteroides (culture eXact® for the production of sour cream, cottage cheese and cheese, the company Christian Hansen / Denmark) were grown by deep cultivation. After the cultivation process, cells were separated from the culture medium by microfiltration. Then the cells were destroyed using the lytic enzyme chymotrypsin (0.28 wt.%) For 2 hours at a temperature of + 32 ° C. The obtained enzymatic cell lysate was mixed with GSM, zeolite and SC. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 0.08; Fuels and lubricants - 24.0; zeolite - 73.9; SC - 1.5; lytic enzymes - 0.52. The yield of the final product is 185 ± 5 kg.

Example 8. Microorganisms B.subtilis strain TPI 9 were grown by the method of deep cultivation. After the cultivation process, cells were separated from the culture medium by microfiltration. Then the cells were subjected to destruction using the proteolytic enzyme acid protease (1.2 wt.%) For 2 hours at a temperature of + 32.0 ° C. The resulting product was introduced with the proteolytic bacterial preparation Protozyme MN (Danisco, Netherlands) - 1.5 wt.% And the amylolytic bacterial preparation Zimajunt NT340 (Enmex, Mexico) - 2.0 wt.%. The obtained enzyme-containing lysate of B.subtilis cells was mixed with GSM, zeolite and SK. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. As a result, a preparation of the following composition was obtained (wt.%): Cell lysate - 0.02; Fuels and lubricants - 25.0; zeolite - 70.0; SK - 1.48; enzymes - 3.5. The yield of the final product is 180 ± 5 kg.

Example 9. Microorganisms of a mixed culture Lactobacillus acidophilus, Bifldobacterium lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subspecies bulgaricus, Bifidobacterium longum, Lactobacillus casei . After the cultivation process, mechanical separation of cells from the culture medium was performed. Then the cells were subjected to destruction using pancreatin (0.5 wt.%) For 2 hours at a temperature of + 20 ° C. The resulting product was introduced with the proteolytic bacterial preparation Neutrase (Novozymes, Denmark) - 1.5 wt.% And the amylolytic bacterial preparation Likvamil (Genencor, USA) - 1.1 wt.%. The obtained enzymatic cell lysate was mixed with fuel, lubricant, zeolite and SK in the following proportions (wt.%): Cell lysate - 0.95; Fuels and lubricants - 15.0; zeolite - 80.0; SK - 1.45. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. The yield of the final product is 160 ± 5 kg.

Example 10. Microorganisms Saccharomyces cerevisiae, race 9 were grown by deep cultivation. After the cultivation process, mechanical separation of cells from the culture medium was performed. Then the cells were destroyed using the proteolytic composition of the enzymes of yeast cathepsins (0.23 wt.%) For 10 hours at a temperature of 50 ° C. The resulting product was introduced proteolytic bacterial preparation Protosubtilin G3x - 1 wt.% And amylolytic bacterial preparation Amilosubtilin G3x - 0.45 wt.%. The obtained enzymatic cell lysate of S. cerevisiae was mixed with GSM, zeolite and SK in the following proportions (wt.%): Cell lysate - 0.05; Fuel and lubricants - 21.3; zeolite - 76.0; SC - 1.2. Before mixing, fuels and lubricants and zeolite were subjected to dry heat sterilization. The yield of the final product obtained is 195 ± 5 kg.

Example 11. The study of the effects of the component composition of the claimed funds on the inhibition of the growth of pathogenic and conditionally pathogenic microflora.

Table 1 shows the results of the effect of the same doses of drugs (500 mg), obtained on the basis of the claimed funds and the drug of the analogue, introduced into the meat-peptone agar (MPA), on the inhibition of growth of Sh.sonnei and S.aureus 1479. The presented results show that, practically , in all cases there is a more effective inhibition of the growth of these microorganisms in comparison with the drug analogue. However, with the introduction in MPA of the drug obtained according to example No. 6 in the following ratios of components (wt.%): Lysate - 0.10; zeolite - 76.0; Fuels and lubricants - 22.4; lytic enzymes - 0.50 and SK - 1.0, maximum inhibition of growth of Sh.sonnei (20.9 mm) and S.aureus 1479 (26.6 mm) is ensured. Further studies were conducted on the preparation of the above formulation, which received the code name "SL08".

It should be noted the presence of close in magnitude of the effects of inhibition of the growth of bacteria Sh.sonnei and S.aureus 1479 on MPA, which were obtained with the introduction of the composition of the proposed drug lysates of other microorganisms. Therefore, the implementation of the technology for producing the claimed agent on the basis of an enzymatic lysate of cells of various types of microorganisms allows the creation of metabolite-like preparations with probiotic and prebiotic properties similar in composition.

Table 1 The influence of the component compositions of the claimed funds and drug-analogue on the growth of microorganisms in the environment MPA Example No. The composition of the claimed funds, wt.% Sh.sonnei growth inhibition zone after 24 h, mm Zone of growth inhibition of S.aureus 1479 after 24 hours, mm Lysate Zeolite Fuels and lubricants Lytic enzymes SC one 0.25 78.0 20,0 0.75 1,0 18.2 25.7 2 0.5 78,2 20,2 0.10 1,0 19.2 18.2 3 1.65 80.0 16.8 0.05 1,5 18.5 19.3 four 1.3 72,4 25.0 0.80 0.5 17.4 22.2 5 2.5 74.6 20.5 1,2 1,2 19.8 25.6 6 0.10 76.0 22.4 0.50 1,0 20.9 26.6 7 0.08 73.9 24.0 0.52 1,5 20,2 24.1 8 0.02 70.0 25.0 3,5 1.48 16,4 24.7 9 0.95 80.0 15.0 2.6 1.45 16.7 24.9 10 0.05 76.0 21.3 1.45 1,2 19,4 25.6 Analog drug (optimal composition for comparison) - SCW 1.0 78 twenty - 1,0 16.7 24.2

Example 12. Comparison of the component composition of the claimed funds and drug-analogue "Bactistatin"

Analyzed the composition of the samples of the claimed funds obtained according to example No. 1, and the drug-analogue (PA) with the same content in it (wt.%): Zeolite - 78, fuel and lubricants - 20.0 and SK - 1.0. Table 2 presents the results of high performance liquid chromatography analysis of substances in samples containing the enzymatic lysate of the producer cells (FLKP, the claimed tool) and SCR (Bactistatin). Table 3 shows the content of biologically active substances in the final products (finished substances) of the claimed funds and drug-analogue "Bactistatin".

table 2 The content of biologically active substances in sterilized culture fluid (SCF) and in the enzymatic lysate of producer cells (FLCP) Name of the analyzed indicators SCG FLKP Protein Content *, μg / cm ^-3 541.5 496.0 Proteolytic activity (PA), units PA, dm ^-3 8.7 2.2 Amylolytic activity (AA), units AA, dm ^-3 42.7 19.2 The total concentration of total amino acids, μg / cm ^-3 568.8 2540 The total concentration of free amino acids, μg / cm ^-3 60.8 357.0 The concentration of hexosamines, mcg / cm ^-3 104.9 92.1 Glucosamine concentration, mcg / cm ^-3 95.6 87.1 The concentration of diaminopimelic acid, μg / cm ^-3 19.6 7.6 Adenine / Xa ^** concentration, μg / cm ^-3 12.2 / 3.2 20.8 / 0.2 Guanine / Xg ^** concentration, μg / cm ^-3 0 / 16.3 0.6 / 5.7 Thymine concentration / Xt ^** , μg / cm ^-3 8.2 / 0 6.2 / 1.0 The concentration of uracil / Xy ^** , mcg / cm ^-3 11.4 / 8.6 4.7 / 1.6 Cytosine / Chz ^** concentration, μg / cm ^-3 0,0 / 12,1 0,0 / 3,9 Notes: * protein content was determined by the Lowry method; ** Xa, Xg, Xm, Xy, Xc - unidentified derivatives of adenine, guanine, thymine, uracil and cytosine in terms of the basic substance.

Table 3 The content of biologically active substances in the final substances of the drug-analogue "Bactistatin" and the claimed funds Name of the analyzed indicators The drug "Bactistatin" The inventive tool Proteolytic activity (PA), units PA / g 2.00 1.8 Amylolytic activity (AA), units AA / g 30,20 21,2 The total concentration of total amino acids, mg / g 109.00 610.00 The total concentration of free amino acids, mg / g 2.06 12.5 The concentration of total hexosamines, mg / g 50,2 62.3 The concentration of total glucosamine, mg / g 45.6 41.2 The concentration of diaminopimelic acid, μg / g 2.90 2.01 The concentration of free / bound adenine, mg / g 0.02 / 0.26 0.26 / 0.20 The concentration of free / bound guanine, mg / g 0.00 / 1.03 0.10 / 2.02 The concentration of free / bound thymine, mg / g 0.47 / 0.00 3.01 / 0.09 The concentration of free / bound uracil, mg / g 0.08 / 1.06 0.35 / 0.11 The concentration of free / bound cytosine, mg / g 0.00 / 1.82 0.00 / 1.15

Analysis of the results of the chromatographic analysis of substances contained both in the samples of FLKP and SKZh (table 2), and in the final finished substances of the preparations (table 3) show that in the composition of the active principle of the claimed drug are all the main groups of biochemical compounds that were found in the current the beginning of the drug-analogue "Bactistatin." Moreover, the content of nucleic bases (products of the cleavage of nucleic acids), diaminopimelic acid and hexosamines (products of the cleavage of the cell wall of the producer), as well as amino acids (products of the cleavage of the cell protein) in the composition of the claimed funds are significantly higher, which indicates the greater biological value of the latter. In addition, upon receipt of the claimed agent based on the enzymatic lysate of B.subtilis cells, the component composition of the product is constant due to the controlled concentration of the vegetative forms of the cells of this microorganism during their treatment with lytic enzymes, which subsequently also excludes the possibility of the appearance of living cells in the final substance.

Example 13. Evaluation of the immunotropic effects of the claimed drug and drug-analogue

The functional activity of innate immunity is non-specific and is realized due to: mechanical protection (skin, mucous membranes); phagocytosis; destruction of infected cells (complement, natural killer cells); secretion of cytokines (interferon, interleukins); synthesis of antibacterial peptides, chemokines, etc. [Finger M.A., Onishchenko G.G., Zverev V.V., Ivanov A.A. and other Biological safety. / M. Medicine .: 2006; 220-238; Pirofski L.A., Casadevall A. Immunomodulators as an antimicrobial tool. Curr. Opin. Microbiol. 2006; 9: 489-495; Cristofaro P., Opal S.M. Role of Toll-like receptors in infection and immunity: clinical implications. Drugs 2006; 66: 15-29.]. To activate this system, immunotropic preparations of microbial origin containing microbial lysates can be used, as well as partially purified cellular elements (lipopolysaccharides, peptidoglycans) or biologically active fragments obtained by synthesis (muramyl dipeptide, glucosamine muramyl dipeptide). The use of such drugs allows you to activate non-specific factors of immunity, which also take part in the formation of anti-infection protection of the body.

Changes in nonspecific immunity factors were evaluated against the background of the enteral administration of the inventive SL08 drug obtained in Example No. 6 and the Bactistatin drug analogue. To solve this problem, the functional activity of factors of nonspecific resistance of the organism was determined for various durations of administration of the claimed drug and the analogue drug.

The studies were conducted on white outbred male mice weighing 16-18 g, obtained from the Rappolovo nursery. During the experiment, animals were divided into two groups and kept under standard conditions on a complete diet. The first group of animals was enterally administered with "SL08" according to the following scheme: 1 time per day, dose - 1.5 mg / mouse, duration of administration - 1, 5, 10 and 20 days. Bactistatin was administered enterally to the second group of animals according to a similar pattern. Before the administration of the preparations, as well as on the day after the end of the corresponding duration of their course administration, blood was taken from animals of both groups to assess the dynamics of changes in non-specific resistance factors. The following indicators were determined: the digesting activity of granular leukocytes, the concentration of lysozyme and myeloperoxidase.

The digesting activity of granular leukocytes of peripheral blood of animals was evaluated as follows. For this, blood was taken from each animal of the corresponding group into a centrifuge tube containing 0.1 ml of a 1:10 dilution of heparin solution. To avoid blood coagulation, the resulting suspension was thoroughly mixed. The total volume of the suspension was 1.0 ml. Then 0.1 ml of the phagocytosis test object, a one-day culture of M.lysodeicticus at a concentration of 10 ⁹ CFU / ml, grown on meat and peptone agar at a temperature of 37.0 ° C, was added to the test tube. The resulting mixture of blood with a test object was placed in a thermostat for 15 minutes. After this time, a blood smear was made on a glass slide. After this, the blood tube was again placed in a thermostat for 15 minutes. After this time, another blood smear was made on a glass slide. The smears were dried at room temperature, fixed in 96 ° ethanol and stained according to Romanovsky-Giemsa. Smears were examined under a light microscope under immersion (objective × 40).

In smears obtained, respectively, after 15 and 30 minutes, the number of phagocytic cells containing 1 or more absorbed phagocytosis objects was counted per 100 granulocytes. As an estimated indicator, the digestion index (PI) was determined, which is the ratio of the number of phagocytic cells in a smear after 30 min of incubation of blood with a phagocytosis test object to the number of phagocytic cells in a smear after 15 min of incubation of blood with a phagocytosis test object. The IP values were determined by the formula:

where A is the number of phagocytic cells per 100 granular leukocytes counted in a smear, which absorbed the test object within 15 minutes of incubation; B is the number of phagocytic cells per 100 granular leukocytes counted in a smear, which absorbed the test object within 30 minutes of incubation. With satisfactory functioning of the granular leukocytes of peripheral blood, the IP values are always> 1. This value was expressed in relative units (pu).

The concentration of lysozyme in serum was determined as follows. Blood from the animal was collected in a centrifuge tube and serum was obtained. Next, 0.1 ml of blood serum was transferred to a bacteriological tube, to which 0.9 ml of physiological saline was added. The resulting mixture was stirred and 1.5 ml of a suspension of the test object (M.lysodeicticus culture) was added to it, which was previously standardized on FEK-56M before extinction 0.66 (against green filter No. 6). The test object was grown on meat and peptone agar for 24 hours in a thermostat at a temperature of + 37.0 ° C. The prepared suspension containing the test object and the test serum was mixed and placed in a thermostat for 30 min at a temperature of + 37.0 ° C. At the end of the incubation time, the tubes with the material were removed from the thermostat and its extinction was determined on FEK-56M. Obtained during the determination of the magnitude of extinction using spice tables was translated into values characterizing the concentration of lysozyme in the blood serum, and expressed in μg / ml.

The level of myeloperoxidase in blood serum was determined spectrophotometrically. Blood from the animal was collected in a centrifuge tube and serum was obtained. Then 0.1 ml of blood serum was transferred to a well with a flat bottom of the ELISA panel and 0.1 ml of a 5% benzidine solution was added to the same well. Instead of the test serum, 0.1 ml of physiological saline was added to the control well. The test samples were gently mixed in the tablets and placed in a thermostat for 60 min at a temperature of 37.0 ° C. At the end of the incubation time, the plates were removed from the thermostat and viewed on a Dynatech vertical spectrophotometer at a wavelength of 492 nm. The serum myeloperoxidase concentration was obtained in units of extinction and expressed in units of activity (EA / ml).

Statistical processing of the experimental results was carried out using a data analysis package included in Excel spreadsheets. In this case, one-way analysis of variance with fixed effects was used [Yunkerov V.I., Grigoriev S.G. Mathematical and statistical processing of medical research data. SPb .; WMA: 2005; 292 .; Medic V.A., Tokmachev M.S., Fishman B.B. Statistics in medicine and biology. / Ed. Yu.M. Komarova. T.1. Theoretical statistics. M .: 2000; 412].

Table 4 presents the results of a comparative assessment of the dynamics of changes in factors of nonspecific resistance in animals of the first and second groups with different durations of enteral administration of the claimed drug and the analogue drug. An analysis of the results shows that the dynamics of changes in the values of factors of nonspecific resistance of animals of the first and second groups against the background of the introduction of these drugs is almost identical. This is evidenced by the absence of statistically significant differences (P> 0.05) between the values of the studied factors recorded in animals of the two groups 1, 5, 10 and 20 days after the administration of the claimed drug and the analogue preparation.

At the same time, the dynamics of the development of changes in factors of nonspecific immunity in animals of the two groups with respect to the background (control values of the factors before drug administration) has statistically significant differences (P <0.05). This indicates a significant increase in the functional activity of factors of nonspecific immunity in animals against the background of enteral administration of the claimed drug and drug-analogue.

The maximum activity of factors of nonspecific resistance in two groups of animals is observed on the 5-10th day after drug administration. Then, on the 20th day of the use of drugs, the functional activity of these indicators decreases. It is possible that with prolonged administration of drugs (more than 20 days), immunosuppressive effects begin to develop, which are explained by the impossibility of endless stimulation of the entire immune system and its individual components. The same dynamics of the development of the severity and direction of changes in factors of nonspecific immunity in animals that were administered enterically the claimed agent and the analogue drug, as well as the similarity of the component composition of these drugs prove the possibility of using the claimed agent according to the indications that were given for the analogue drug.

Thus, the practical use of the claimed agent based on the enzymatic lysate of the producer Bacillus subtilis in the form of a drug and / or food additive obtained by the claimed technology is, in comparison with the analogue preparation, more appropriate.

A further assessment of the possibility of increasing the functional activity of these factors of nonspecific immunity was carried out against the background of enteral administration of the inventive agent obtained in Example No. 10 and the Bactistatin analogue to the animal organism. For this, we used a similar experimental design (doses of drugs, groups of animals, etc.), which was presented above. However, in this case, the duration of enteral administration to animals of the claimed drug and the analogue drug was: 1, 5, and 10 days. In addition, the samples of the injected drugs had the main differences in the sources of microbial metabolites, as well as in the content (wt.%) In their composition of the cell lysate and lytic enzymes (table 1).

Table 4 Factors of nonspecific resistance of animals with varying durations of enteral administration of the claimed drug and drug-analogue Names of non-specific resistance factors Duration of drug administration to animals, day / days The values of the factors with the introduction of "SL08" The values of the factors with the introduction of Bactistatin Index of digesting activity of granular leukocytes, relative units, p.u. 0 background, control 1.21 ± 0.015
δ = 0.05 (N = 12) 1.21 ± 0.015
δ = 0.05 (N = 12) one

5

10

twenty

The concentration of lysozyme in serum, mcg / ml 0 background, control 6.95 ± 0.025
δ = 0.50 (N = 12) 6.95 ± 0.025
δ = 0.50 (N = 12) one

5

10

twenty

The concentration of myeloperoxidase in serum, EA / ml 0 background, control 1.57 ± 0.10
δ = 0.063 (N = 12) 1.57 ± 0.10
δ = 0.063 (N = 12) one

5

10

twenty 1.48 ± 0.52
δ = 0.35 (N = 10 1.61 ± 0.44
δ = 0.34 (N = 10) Note: M ± m is the average value and its error; δ is the standard deviation; N is the number of animals; the underlined values of factors have statistically significant differences with respect to the background (control, before drug administration) at P <0.05

Table 5 presents the results of a comparative assessment of the dynamics of changes in factors of nonspecific resistance in animals of the two groups with different durations of enteral administration of the inventive preparation obtained on the basis of a cell lysate of Saccharomyces cerevisiae, race 9, and an analogue preparation based on metabolites of the producer B.subtilis strain VKPM No. B-2335 (3).

The experimental results presented in table 5 show that the dynamics of changes in the values of factors of nonspecific resistance in animals of the two groups also has the same orientation and, practically, does not depend on the species source of microbial metabolites, on the basis of which samples of the claimed drug and the analogue preparation were prepared. This is evidenced by the absence of statistically significant differences (P> 0.05) between the values of the studied factors recorded in animals after 1, 5 and 10 days of administration of samples of these drugs.

Therefore, the component compositions of the claimed drug, including that prepared on the basis of a cell lysate of Saccharomyces cerevisiae, and an analogue preparation prepared on the basis of metabolites of B. subtilis cells, provide comparable biological effects associated with the stimulation of non-specific immunity factors. This means that for the production of the claimed funds can be used enzymatic cell lysates of various types of probiotic strains of microorganisms.

Table 5 Factors of nonspecific resistance of animals with varying durations of enteral administration of the claimed drug obtained according to example No. 10, and the drug-analogue "Bactistatin" Names of non-specific resistance factors Duration of drug administration to animals, day / days The values of the factors with the introduction of the proposed drug, example No. 10 The values of the factors with the introduction of the drug-analogue "Bactistatin" Index of digesting activity of granular leukocytes, relative units, p.u. 0 background, control 1.23 ± 0.014
δ = 0.05 (N = 10) 1.23 ± 0.014
δ = 0.05 (N = 10) one

5

10

The concentration of lysozyme in serum, mcg / ml 0 background, control 6.97 ± 0.024
δ = 0.52 (N = 10) 6.97 ± 0.024
δ = 0.52 (N = 10) one

5

10

The concentration of myeloperoxidase in serum, EA / ml 0 background, control 1.62 ± 0.10
δ = 0.063 (N = 10) 1.62 ± 0.10
δ = 0.063 (N = 10) one

5

10 2.95 ± 0.26
δ = 0.50 (N = 10) 2.69 ± 0.33
δ = 0.46 (N = 10) Note: M ± m is the average value and its error; δ is the standard deviation; N is the number of animals; the underlined values of factors have statistically significant differences with respect to the background (control, before drug administration) at P <0.05

It should be noted that the claimed agent contains substantially less (wt.%) Enzymatic lysate of Saccharomyces cerevisiae cells (0.05%) and, accordingly, metabolites than they contain (wt.%) In the composition of the analogue (1, 0%). Therefore, to obtain a comparable increase in the functional activity of factors of nonspecific immunity in the composition of the claimed drug, lower concentrations of metabolites than that of the analogue drug can be used, which reduces the risk of potential development of adverse events that are observed in humans when taking immunobiological preparations of microbial origin.

Example 14. Evaluation of the effectiveness of "SL08" in patients with erosive antral gastritis after a course of eradication therapy

The study of the effectiveness of the use of the drug "SL08" included 20 patients with H. pylori-associated acid-dependent diseases of the stomach and duodenum in the acute stage. The age of patients ranged from 20 to 60 years. Patients were divided into two groups of 10 people - the test group (patients received the drug in a dose of 2 capsules 2 times a day with food “SL08” in the treatment regimen) and 10 - the control group (patients received only basic therapy).

The research results are presented in table 6. As can be seen from the table, the means "SL08" is effective for 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. During treatment with SL08, all patients noted good tolerability of the drug, and no side effects were detected.

Table 6 The dynamics of the severity of clinical manifestations in patients with erosive antral gastritis in the main (test) and control groups before and during treatment Symptom Names Patients of the test group Patients in the control group before treatment (%) 10 day of treatment (%) 30 day of treatment (%) before treatment (%) 10 day of treatment (%) 30 day of treatment (%) Stomach ache 90 40 0 90 60 0 Heaviness and discomfort in the abdomen. 90 thirty 10** 90 fifty 40 Nausea 40 10 0 70 twenty 10 Heartburn 60 ** 10** 0 90 fifty twenty Vomiting twenty 0 0 10 0 0 Bitterness in the mouth 60 ** twenty** 0 twenty twenty 0 Rumbling 90 40 ** twenty** 90 80 70 Bloating 90 ** 10** 0 ** twenty 80 fifty Frequency of stool (once a day) 0.86 * 1,0 1,2 ** 0.86 2.06 1.7 Bristol chair character 2.12 * 3 * 3.2 * 3,5 5.2 4.01 Notes: ** p <0.05 (differences are statistically significant)

Example 15. Evaluation of the effectiveness of the use of "SL08" for the treatment of patients with celiac disease with dysbiotic disorders of the colon of varying severity

Under observation were 10 people with celiac disease. All patients had a clinical diagnosis of celiac enteropathy based on the clinical course of the disease, morphometric examination of the duodenal mucosa, and immunological blood tests. The drug was prescribed for six weeks, 2 capsules 2 times a day. Analysis of feces for dysbiosis was performed before treatment and on 25-31 days after treatment. The results of the study of changes in microbiocenosis in patients with celiac disease before and after treatment are presented in table 7.

Table 7 Changes in the composition of the colon microflora after treatment with the drug "SL08" in patients with celiac disease The name of the group of microorganisms The number of patients with a modified indicator before treatment Change in indicator after treatment (number of patients,%) - Percentage is indicated on the number of changed indicators Increase in quantity Quantity reduction Did not change Bifidumbacteria 8 (80%) 6 (60%) 1 (10%) 1 (10%) Lactobacilli 10 (100%) 7 (70%) 0 3 (30%) Bacteroids 5 (50%) 3 (30%) 2 (20%) 0 E. coli with normal enzymatic activity 9 (90%) 9 (90%) 0 0 E. coli with reduced enzymatic activity 5 (50%) 0 5 (50%) 0 Enterococci 5 (50%) 0 4 (40%) 1 (10%) Hemolytic microorganisms 0 0 0 0 Opportunistic bacteria 4 (40%) 0 4 (40%) 0 Staph. aureus 0 0 0 0 Staph. saprophyticus, epidermidis 0 0 0 0 Mushrooms of the genus Candida 0 0 0 0 Clostridia 0 0 0 0

A study of microbiocenosis in patients with celiac disease revealed significant changes in the composition of the colon microflora, characterized by a significant decrease in the content of harmful bioflora. While taking the drug, there was a significant improvement in indicators of both the anaerobic flora and the aerobic component. An increase in bifidobacteria and lactobacilli, bacteroids was noted, an increase in the number of Escherichia with normal enzymatic activity was observed with a decrease in the percentage of E. coli with altered properties. After the therapy, the number of hemolytic organisms, conditionally pathogenic bacteria decreased.

Example 16. The study of the effects of drugs "SL08" and a drug analogue "Bactistatin" on the inhibition of growth of pathogenic and conditionally pathogenic microflora during storage at + 4.0 ° C

The inhibitory activity of SL08 and Bactistatin preparations of a similar composition was studied under conditions of different storage periods for the growth of Sh.sonnei and S.aureus 1479 with the introduction of 500 mg of the drug into meat-peptone agar (MPA). The inhibitory activity of the preparations was evaluated in mm.

The results are shown in table 8. The above data indicate that the claimed tool is comparable in activity and effectiveness to the body with the drug "Bactistatin", but significantly exceeds it in terms of stability and manufacturability of the production processes.

Table 8 The effect of the shelf life of the product on the growth of microorganisms on MPA Shelf life, days, at + 18 ° С Name of drugs Sh.sonnei growth inhibition zone after 24 h, mm Zone of growth inhibition of S.aureus 1479 after 24 hours, mm one Bactistatin 16.7 24.2 one SL08 20.9 26.6 fourteen Bactistatin 16.6 24.7 fourteen SL08 20.9 26.8 thirty Bactistatin 12.0 18.2 thirty SL08 19.6 ** 25,4 60 Bactistatin 10.6 15.8 60 SL08 17.4 ** 23.0 ** 90 Bactistatin 5,6 9,4 90 SL08 15.8 ** 21.3 ** 180 Bactistatin 0.5 1.4 180 SL08 16.2 ** 20.7 ** 360 Bactistatin 0.2 0.2 360 SL08 15.4 ** 17.7 ** Note: ** p <0.05 (differences are statistically significant).

Claims (8)

1. The tool for the treatment of diseases of the gastrointestinal tract, containing a source of microbial metabolites, zeolite, soy flour hydrolyzate, calcium stearate, characterized in that the source of metabolites of the claimed agent contains an enzymatic lysate of microorganisms, as well as additional lytic enzymes in the following ratio of ingredients, wt.%:
Zeolite 70 ÷ 80 Hydrolyzed Soybean Meal 15 ÷ 25 Calcium stearate 0.5 ÷ 1.5 Lytic enzymes 0.05 ÷ 3.5 Enzymatic cell lysate of microorganisms rest 2. The tool according to claim 1, characterized in that as a lysate of microorganisms, it contains a lysate of Bacillus subtilis cells. 3. The tool according to claim 1, characterized in that as a lysate of microorganisms, it contains a lysate of lactic acid bacteria cells. 4. The tool according to claim 1, characterized in that as a lysate of microorganisms it contains a lysate of yeast cells. 5. The tool according to claim 1, characterized in that it contains, wt.%: Cell lysate of Bacillus subtilis 0.10; lytic enzymes 0.5; zeolite 76.0; soy flour hydrolyzate 22.4; calcium stearate 1.0. 6. The tool according to claim 1, characterized in that it is provided with an enteric-soluble capsule coated with an acid-protective layer. 7. The method of obtaining funds for the treatment of diseases of the gastrointestinal tract according to claim 1 on the basis of microbial metabolites, including the cultivation of microorganisms, the obtained cells of the producer microorganism are exposed to lytic enzymes for 2-10 hours at a temperature of + 20-50 ° C then the enzymatic lysate of microorganism cells is mixed with zeolite, soy flour hydrolyzate and calcium stearate, sterilized and packaged in the final product. 8. The method according to claim 7, characterized in that the enzyme preparations are additionally introduced into the composition to predetermined parameters.