RU43767U1 - DRY FORM OF MEDICINAL PREVENTIVE DRUG - PROBIOTICS OF VETOCILES - Google Patents

Abstract

The utility model relates to biotechnology, namely to the production of biological products, probiotics. The utility model allows to obtain a simpler and cheaper drug in tablet, or encapsulated, or powder packaged form while preserving all its therapeutic and prophylactic properties. The probiotic preparation consists of a dose of biomass spores of bacteria of the genus Bacillus and a sorbent. The sorbent contains natural zeolite. The biomass of bacterial spores of the genus Bacillus has a titer in the final preparation of at least 10 ⁶ cells / g, and the dry form of the treatment and prophylactic drug is a tablet made in the form of a monolithic disk-shaped solid, consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus, or cylindrical a hollow, detachable capsule or packet filled with a powder consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus. The bacterial spore biomass of the genus Bacillus contains a bacterial strain Bacillus subtilis VKPM B 7048, and / or a bacterial strain Bacillus subtilis VKPM B 7092, and / or a bacterial strain Bacillus I icheniformis VKPM B 7038. Glauconite mineral powder with a particle size of 0.01 is used as zeolite -0.1 mm. The detachable hollow capsule is made of material melting under the influence of body temperature, for example, gelatin. In a tablet, or capsule, or in powder form, a mixture of Bacillus subtilis bacterial strains VKPM B 7048, VKPM B 7092 and Bacillus licheniformis VKPM B 7038, or a mixture of Bacillus subtilis bacterial strains VKPM B 7048 and VKPM B 7092, or a mixture of Bacillus subtilis bacterial strains VKPM 7048 and Bacillus licheniformis VKPM B 7038, or a mixture of strains of bacteria Bacillus subtilis VKPM B 7092 and Bacillus licheniformis VKPM B 7038 are contained in equal proportions.

Description

The utility model relates to biotechnology, namely to the production of biological products, probiotics.

Currently, there are a number of biological products from bacteria of the genus Bacillus that inhibit the growth of pathogenic and conditionally pathogenic bacteria: biosporin, bactisporin, bactisubtil, etc. Their therapeutic effectiveness is determined by the complex effect of a wide range of metabolites that can produce live microbial cells of bacterial strains included in bacteria composition of drugs.

The study of the mechanism of the therapeutic and prophylactic effect of probiotic preparations on the human and animal body shows that bacilli have the ability to penetrate from the gastrointestinal tract (GIT) into the bloodstream, and from there into the lesion, remain viable, produce antibiotics, proteolytic enzymes and immunomodulators. It has been established that after oral administration, already in the first minutes, approximately 0.1% of the total number of bacteria penetrate the parenchymal organs through the mucous membranes of the oropharynx, esophagus and stomach - asymptomatic translocation of microorganisms; it lasts 6-8 hours after a single dose of the drug until the elimination of microbial cells from organs, during which the effect of the drug on the body is carried out (Smirnov V.V., Reznik S.R., Sorokulova I.B., Vyunitskaya V.A. Discussion questions of the creation and use of bacterial preparations for micro correction warm-blooded flora // Mikrobiol. Zh. 1992. - T.54, N6. - S.82-92.).

The antagonistic effect of bacilli is due to the production of biologically active substances of a different nature: polypeptide antibiotics, lysozyme, lytic enzymes (Sorokulova I. B. Prospects for the use of bacteria of the genus Bacillus for the construction of new biological products // Antibiotics and chemotherapy. - 1996. -T.41 N10. - S.13-15).

High enzymatic activity of bacilli has a positive value with

points of view of the enrichment of gastric secretion with additional, including paradigmatic enzymes (Slabospitskaya A.T., Pirog T.P., Grinberg T.A. et al. Influence of some components of the environment of bacteria of the genus Bacillus on the synthesis of exopolysaccharides // Control and management of biotechnological processes: Abstract of the report of the All-Union Conference - Gorky, 1985. - P. 33 .; Slabospitskaya A.T., Krymovskaya S.S., Reznik S.R. Enzymatic activity of bacilli, promising for inclusion in biological products // Microbiol. J. -1990. -N2. - S. 9-14). Bacilli do not form biofilms, since their adhesive properties are poorly expressed. Their activity is carried out in the lumen of the gastrointestinal tract and is primarily associated not with competitive relationships for attachment sites to the mucosa, but with high antagonistic activity against pathogenic microorganisms, without antagonistic effects on representatives of normal microflora, which creates the conditions for uncompetitive restoration of autoflora.

A known method of obtaining the drug "Baktisporin" based on a strain of Bacillus subtilis 3 N (GISK N 248), including cultivating the strain in a liquid nutrient medium, separating the biomass from the nutrient medium by ultrafiltration, concentrated and freeze drying (RF Patent No. 2067616, IPC C 12 N 1 / 20, publ. 10.10.96).

Known therapeutic and biological product "Baktisporin" based on an antagonistically active strain of B. subtilis 3H, which provides for obtaining the biomass of the strain, mixing it with a protective medium (sugar gelatin mixture or lactose), ampouling and freeze drying (RF Patent No. 2130316, IPC A 61 K 35 / 74, publ. 05.20.99). The drug is used to treat a wide range of diseases and before use, the ampoule is opened, dry biomass is dissolved in a small amount of water and used orally in the form of a solution. Since the strain, which is part of Baktisporin, is a strict aerobic, it can carry out its vital activity only in the upper parts of the gastrointestinal tract, where there are aerobic conditions. However, the ampoule form of the drug "Baktisporin" requires dilution with water before use, which reduces usability and limits consumer demand. In addition, when

opening the ampoule requires caution to prevent ampoule glass from entering the preparation.

The closest analogue (prototype) is a tablet probiotic preparation made in the form of a disk-shaped monolithic body containing biomass based on Bacillus subtilis, stabilizers based on sugar gelatin medium or lactose, characterized in that it additionally contains calcium gluconate in an amount of 1-10 wt. %, or sorbent and excipients. Activated carbon, carbolong, lactulose, acetylphthalyl cellulose or aluminum phosphate gel are used as a sorbent. The biomass of bacteria and excipients in the preparation are in the ratio (2: 1) - (3: 1). As auxiliary substances, calcium stearate, talc, and (or) starch, and (or) aerosil, glucose, milk powder and (or) methyl cellulose are used (RF patent No. 2182009, IPC A 61 K 35/74, A 61 K 9 / 20, publ. 2002).

The disadvantage of the prototype drug is the complex multicomponent composition of the tablet form of the drug and the expensive and energy-intensive technology for its preparation, associated with suspension cultivation of bacteria, their concentrated and freeze drying.

The technical result of the proposed utility model is to obtain a simpler and cheaper drug in tablet, or capsule, or powder packaged form, while maintaining all its therapeutic and prophylactic properties.

The specified technical result is achieved by the fact that in the dry form of a therapeutic and prophylactic drug-probiotic, consisting of a dose of biomass spores of bacteria of the genus Bacillus and a sorbent, according to a utility model, the sorbent consists of natural zeolite, the biomass of spores of bacteria of the genus Bacillus has a titer of at least 10 ⁶ cells / g, and the dry form of the therapeutic drug is a tablet made in the form of a monolithic disk-shaped solid, consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus, or a cylinder An organic hollow, detachable capsule or packet filled with a powder consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus.

Bacillus bacteria spore biomass contains Bacillus bacteria strain

subtilis VKPM B 7048, and / or a bacterial strain Bacillus subtilis VKPM B 7092, and / or a bacterial strain Bacillus licheniformis VKPM B 7038.

As a zeolite, glauconite mineral powder with a particle size of 0.01-0.1 mm is used.

The detachable hollow capsule is made of material melting under the influence of body temperature, for example, gelatin.

In a tablet, or capsule, or in powder form, a mixture of Bacillus subtilis bacterial strains VKPM B 7048. VKPM B 7092 and Bacillus licheniformis VKPM B 7038, or a mixture of Bacillus subtilis bacterial strains VKPM B 7048 and VKPM B 7092, or a mixture of Bacillus subtilis bacterial strains VKPM 7048 and Bacillus licheniformis VKPM B 7038, or a mixture of bacterial strains of Bacillus subtilis VKPM B 7092 and Bacillus licheniformis VKPM B 7038 are contained in equal proportions.

Zeolite-based enterosorbent is used in medicine in many pathological processes. In particular, the drug normalizes the functional state of the liver, taking on a significant part of its antitoxic function. Entering direct contact with the intestinal wall and potentiating regenerative processes, the zeolite has a pronounced positive effect in diseases such as colitis, enteritis, enterocolitis. At the same time, the functions of the neuromuscular apparatus of the intestine are improved and diarrhea and constipation are eliminated. Zeolites are able to selectively remove pathogenic microflora from the intestine without affecting the normal microflora, which eliminates dysbiosis.

Natural zeolites specifically affect the microorganisms of the rumen, stomach and intestines. They reduce the processes of fermentation and decay in the intestines. The bactericidal effects of natural zeolites in the digestive tract are associated with the release of oxygen free radicals.

As the zeolite, the mineral glauconite obtained from the Kari deposit in the Chelyabinsk region by grinding it to a fraction of 0.01-0.1 mm can be used. The name glauconite comes from the Greek "glaucos" - blue-green. By decision of the International Nomenclature Committee, glandular dioctahedral mica should be called glauconite. The content of the main components is usually in the range,%: Si ₂ -47-50,5; A; l ₂ O ₃ -5-10;

Fe ₂ O ₃ -15-22; FeO-2-4; MgO-3-4; CaO-0-0.8; Na ₂ O-0-0.5; K ₂ O-6-8; H ₂ O-7-9. Fluctuations in composition are associated with isomorphic substitutions.

The bacterial strain Bacillus subtilis VKPM B 7048 belongs to aerobes. This is a gram-positive spore forming bacterium. The strain multiplies at 15-50 ° C. Optimum growth is observed at a temperature of 36-37 ° C. The bacterial strain Bacillus subtilis VKPM B 7048 produces a broad-spectrum antibiotic that inhibits the growth and development of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

The bacterial strain Bacillus lichenifbrmis VKPM B 7038 belongs to aerobes. This is a gram-positive stick that can form spores. The bacterial strain Bacillus lichenifbrmis VKPM B 7038 produces a broad-spectrum antibiotic that inhibits the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

The recombinant bacterial strain Bacillus subilis VKPM B 7092 was obtained by transforming the Bacillus subtilis strain VKPM B 7048 with the recombinant plasmid pBMB DNA 5 containing the alpha-2 interferon gene. The resulting strain IC-16 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) GNI Genetics under the number VKPM B 7092. The plasmid rVMV 5 is able to replicate in bacilli and contains the human alpha-2 interferon gene, a promoter for the expression of this gene, a landing site ribosomes (SD site) and B. amyloliquefaciens alpha-amylase signal peptide for secretion of interferon into the culture medium, as well as carrying resistance to kanamycin.

The antibiotic activity of production strains of Bacillus subtilis VKPM B 7048, VKPM B 7092 and Bacillus lichenifbrmis VKPM B 7038 with respect to each other as components of complex preparations was studied. In this case, we can conclude that the antagonism of Bacillus subtilis and Bacillus lichenifbrmis cells is not a significant obstacle to their joint use in drugs.

Figure 1 shows a tablet form of the drug in the context. Figure 2 presents the encapsulated form of the drug. Figure 3 shows a diagram of a package with a powder form of the drug.

The tablet form of Vetacil is made in the form of a disk-shaped monolithic body 1 (Fig. 1), which disintegrates under the influence of moisture, organic acids and body temperature in the gastrointestinal tract. The body weight of 1 tablet contains the following components:

Example 1

A mixture of natural zeolite with biomass spores of bacteria Bacillus subtilis VKPM 7048 with a titer of at least 10 ⁶ cells / g

Example 2

A mixture of natural zeolite with biomass spores of bacteria Bacillus subtilis VKPM 7048 and VKPM B 7092 in a 1: 1 ratio with a titer of at least 10 ⁶ cells / g.

Example 3

A mixture of natural zeolite with biomass spores of bacteria Bacillus subtilis VKPM 7048, VKPM B 7092 and Bacillus licheniformis VKPM B 7038 in a 1: 1: 1 ratio with a titer of at least 10 ⁶ cells / g.

In another embodiment (FIG. 2), the embodiment of the preparation is a powdered mass 2, which is placed in a gelatin capsule 3, which disintegrates under the influence of temperature. The powdered mass of 2 preparations contains the following components:

Example 4

A mixture of natural zeolite (glauconite) with the biomass of bacterial spores Bacillus subtilis VKPM 7048 and Bacillus licheniformis VKPM B 7038 in a 1: 1 ratio with a titer of at least 10 ⁶ cells / g.

Example 5

A mixture of natural zeolite (glauconite) with the biomass of bacterial spores of Bacillus subtilis VKPM B 7092 with a titer of at least 10 ⁶ cells / g.

Example 6

A mixture of natural zeolite (glauconite) with the biomass of bacterial spores Bacillus subtilis VKPM 7048, VKPM B 7092 and Bacillus licheniformis VKPM B 7038 in a 1: 1: 1 ratio with a titer of at least 10 ⁶ cells / g /

In the third embodiment (figure 3), the form of the preparation is a powdered mass 4, which is placed in a plastic or paper hermetically sealed bag 5. Powdered mass 4 of the drug

contains the following components (wt.%):

Example 7

A mixture of natural zeolite with biomass spores of bacteria Bacillus subtilis VKPM B 7048 with a titer of at least 10 ⁶ cells / g

Example 8

A mixture of natural zeolite with biomass spores of bacteria Bacillus subtilis VKPM 7048, VKPM B 7092 and Bacillus licheniformis VKPM B 7038 in a 1: 1: 1 ratio with a titer of at least 10 ⁶ cells / g /

For medical purposes, it is advisable to release the VETOCIL drug in the form of tablets or capsules. Use the drug inside 2 tablets or capsules (1 g) in the morning and evening until recovery.

For veterinary purposes, the drug is produced packaged in 2, 5, 10, 50, 100, 200, 500 and 1000 grams in plastic film bags. By agreement with the consumer, other types of packaging are possible.

Example 9. Pharmacological properties of the drug

The drug VETOCIL has the following properties:

1. High antagonistic activity against a wide range of pathogenic and opportunistic microorganisms, including with respect to pathogens - salmonellosis, coccidiosis, colibacteriosis, dysentery.

2. It has adsorption, ion exchange, detoxification and anti-stress action.

3. Promotes the adsorption of exo - and endotoxins and their removal from the body.

4. Prevents the development of dysbiosis.

5. Stimulates the growth and development of young farm animals and poultry.

6. Normalizes and activates oxidative processes, cellular respiration and the metabolism of minerals in the body.

7. Increases the buffer activity of blood.

8 Increases nonspecific resistance of the body.

9. Reduces the processes of fermentation and decay in the intestine and increases

digestibility of feed nutrients.

10. Regulates the content of free fluid in the intestine and promotes the formation of dense feces in diarrheal conditions.

11. When using a recombinant strain VKPM 7092, the drug has an antiviral effect.

Example 10. Methods of using the drug in veterinary medicine.

1. VETOCIL is used for the prevention and treatment of gastrointestinal diseases with diarrhea syndrome, as well as for the prevention of immunodeficiency conditions in calves, piglets and dogs, stimulating the growth and development of young farm animals. As a detoxifying agent, it is prescribed for toxicosis of exo - and endogenous origin.

2. The drug is prescribed to animals inside individually or in a group method with food (milk, colostrum, boiled cooled water, etc.). Solutions are prepared before use.

3. For therapeutic purposes, VETOCIL is used 4 times a day, 75-100 mg / kg of body weight daily until recovery.

4. For the prevention of diseases of the digestive system, VETOCIL is prescribed to animals immediately after birth once a day 100 mg / kg body weight for 15-30 days.

5. To stimulate growth and development, animals are given a dose of 75 mg / kg weight once a day or two days for 15-30 days.

6. At the recommended doses, VETOCIL has no side effects and does not cause complications.

Example 11. Data on the study of the therapeutic and prophylactic properties of the drug VETOCIL in veterinary medicine

For this, 2 experimental and control groups of 10 calves in each at the age of 1-10 days were formed. Animals of the experimental group received vetotsil 25 mg / kg of live weight 1 time per day for a month. The calves of the 2 experimental group of vetotsil were assigned 100 mg / kg of live weight. The calves of the control group did not receive the drug.

Research results

Under the influence of vetotsil-2 the intensity of growth and development of calves increases. A month after the start of the drug in calves 1

in the experimental group, the average daily gain was 1060 g., in calves of the 2 experimental group - 939 grams, in animals of the control group - 856 g. Thus, experimental calves of group 1 in terms of growth intensity exceeded the control group of animals by 23.8%, the second experimental group - by 9.7%. Diseases in animals of the 1st experimental group were not recorded. In 5 calves of the 2nd experimental group, dyspepsia was noted, which proceeded in a mild form and recovery of the animals occurred mainly on the 4th - 5th day. The average daily gain in ill calves was 232 g., And therefore the average daily gain in experimental calves of group 2 was lower than analogues from the 1st experimental group. When using vetotsil-2 at the indicated dosages, no side effects were noted.

Hematological studies of calf blood prior to drug administration did not record characteristic differences in animals from the experimental and control groups (Table 1).

Table 1

Calves leukogram indicators before drug administration

Group Control 1st experienced 2nd experienced Indicator M ± m C _v M ± m C _v M ± m C _v Er 10 ¹² / l 5.05 ± 0.222 8.7 5.05 ± 0.24 9,4 4.96 ± 0.28 12.6 Lake 10 ⁹ / l 6.93 ± 0.21 6 7.35 ± 0.646 17.5 6.92 ± 0.424 13.6 Нb, g% 6.7 ± 0.342 10.1 6.6 ± 0.374 11.3 7.04 ± 0.331 10.5 Neutrophils: Yu.N.,% 0.3 ± 0.25 200 1.3 ± 0.25 40 0.6 ± 0.24 91.2 P.N.,% 5.5 ± 1.32 48.1 6.3 ± 1.49 47.7 3.4 ± 0.75 49.2 C.N.,% 61 ± 1.96 6.4 56 ± 2,83 10.1 58.4 ± 2.82 10.8 Eosin,% 2.3 ± 1.11 98.5 2 ± 0.71 70.7 2.6 ± 0.98 84.2 Basophil,% 0.8 ± 0.25 66.6 0.8 ± 0.25 66.6 0.6 ± 0.24 91.2 Monocyte,% 1.3 ± 0.75 120 1.3 ± 0.63 100.6 0.8 ± 0.37 104.5 Lymphocyte% 30.5 ± 2.22 14.5 32.5 ± 2.53 15,5 33.4 ± 2.73 18.2

By the 10th day of studies in animals of the control group, compared with the initial period of the study, the concentration of hemoglobin increased from (6.7 ± 0.342) g /% to (7.5 ± 0.265) g /%, the content of red blood cells from (5.05 ± 0.222) 10 ¹² / l to (5.45 ± 0.132) 10 ¹² / l, white blood cells from (6.93 ± 0.21) 10 ⁹ / l to (10.73 ± 0.65) 10%, lymphocytes from (30, 5 ± 2.22)% to (44.5 ± 2.1)% (Table 2).

Table 2

Calcium leukogram indices on the 30th day after drug administration

Group Control 1st experienced 2nd experienced Indicator M ± m C _v M ± m C _v M ± m C _v Er 10 ¹² / l 5.45 ± 0.132 4.8 5.47 ± 0.111 4 5.32 ± 0.185 7.7 Lake 10 ⁹ / l 10.73 ± 0.65 12.1 8.45 ± 0.189 4.4 11.4 ± 0.51 10 Нb, g% 7.5 ± 0.265 7 7.1 ± 0.367 10.3 7.32 ± 0.287 8.7 Neutrophils: Yu.N.,% 0.5 ± 0.29 115.4 0.3 ± 0.25 200 0.2 ± 0.2 223.6 P.N.,% 5.8 ± 0.85 29.7 2.8 ± 0.25 18.1 3.2 ± 0.37 26.1 S. n.,% 48 ± 1,58 6.5 36, ± 2.84 15,5 45 ± 2.45 12.1 Eosin,% 3.3 ± 1.25 76.9 2.5 ± 0.65 51.6 2.6 ± 0.93 79.7 Basophil,% 0.5 ± 0.29 115.4 0.5 ± 0.29 115.4 0.2 ± 0.2 223.6 Monocyte,% 1.5 ± 0.65 86 1.5 ± 0.29 38,4 1.4 ± 0.24 39.1 Lymphocyte% 44.5 ± 2.1 9,4 51 ± 1.96 7.6 47.8 ± 1.24 5.8

Thus, in both calves of the experimental and control groups, quantitative changes in the hematological parameters and all populations of T-lymphocytes occur in the direction of their increase, which is due to age specificity. However, in animals of the experimental groups, their changes were more pronounced compared with the analogues from the control. The nature of the changes in the studied blood hematological parameters depended both on the drug used and on the duration of its administration.

In the calves of the first experimental group, when the blood test was performed 10 days after the start of the administration of Vetocil-2, the leukocyte count increased by 6.2%, compared with the analogues from the control.

In animals of the second experimental blood, the hemoglobin content increased by 4.7%, the number of erythrocytes by 2.35%, leukocytes by 4.6% and lymphocytes by 22.5%, compared with analogues in the control group.

On day 30 of the study, the calves of the first experimental group showed an increase in lymphocytes in blood by 14.6%, in the second experimental group, the number of leukocytes increased by 6.3% and lymphocytes by 7.4%.

Conclusion Thus, Vetocil has a growth-promoting effect, stimulates cellular factors of immunity and increases the body's resistance to the action of adverse environmental factors.

Calves tolerate the drug well, they did not have any side effects during the experiment.

Claims (5)

1. The dry form of the therapeutic and prophylactic drug probiotic, consisting of a dose of biomass spores of bacteria of the genus Bacillus and sorbent, characterized in that the sorbent consists of natural zeolite, the biomass of spores of bacteria of the genus Bacillus has a titer in the final preparation of at least 10 ⁶ cells / g, and the dry form of the therapeutic drug is a tablet made in the form of a monolithic disk-shaped solid, consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus, or a cylindrical hollow split capsule or a bag filled Powders consisting of a mixture of zeolite and biomass spores of bacteria of the genus Bacillus. 2. The dry form of the therapeutic and probiotic preparation according to claim 1, characterized in that the biomass of the bacterial spores of the genus Bacillus contains a bacterial strain Bacillus subtilis VKPM B 7048, and / or a bacterial strain Bacillus subtilis VKPM B 7092, and / or a bacterial strain Bacillus licheniformis VKPM B 7038. 3. The dry form of the therapeutic and probiotic preparation according to claim 1, characterized in that glauconite mineral powder with a particle size of 0.01-0.1 mm is used as zeolite. 4. The dry form of the therapeutic and probiotic preparation according to claim 1, characterized in that the detachable hollow capsule is made of material melting under the influence of body temperature, for example, gelatin. 5. The dry form of the treatment-prophylactic probiotic preparation according to claim 2, characterized in that in a tablet, or capsule, or powder packaged form, a mixture of the bacterial strains Bacillus subtilis VKPM B 7048, VKPM B 7092 and Bacillus licheniformis VKPM B 7038, or a mixture of Bacillus subtilis bacterial strains VKPM B 7048 and VKPM B 7092, or a mixture of Bacillus subtilis bacterial strains VKPM 7048 and Bacillus licheniformis VKPM B 7038, or a mixture of Bacillus subtilis bacterial strains VKPM B 7092 and Bacillus licheniformis VKPM B 7038 are contained in equal proportions.