LT4102B - Biologically active immunomodulating material, process for preparing thereof, pharmaceutical preparation for the human and animals physiological state conditioning - Google Patents

Abstract

Bio-active substance, possessing immune-modulating properties, incorporating in its contents water soluble organic compound hydrolysis products of animal finely ground tissue ingredients; in hydrolysis the product composition incorporates thermo-stable, low-molecular components of morpho-plasma and glucocalix cells. These components are modified during the processes of embrio-genesis and/or proliferation, and/or cell differentiation, and/or pathology, followed by and/or after the already accomplished tissue regeneration and/or repair processes.The method of production of the bio-active substance with immune-modulating properties incorporates the operations of clean raw-material grinding, hydrolysis of ground product, filtering of the hydrolysis product, and supernatant evolution. The raw material consists of morpho-plasma and glucocalix cell components, and, before hydrolysis it is homogenized and cleaned of non-dissociated tissue elements.The human and animal physiological state normalization preparation consists of a bio-active substance and a filling agent. The bio-active substance in its hydrolysis product composition incorporates thermo-stable low-molecular modified morpho-plasma and glucocalix cell components (from 0.001 to 85%) and the remaining part is filling agent.The preparation is used to normalize human physiologic state using pharmacologically efficient quantity.

Description

The invention relates to the pharmaceutical chemical industry, namely, to the development of active substances having immunomodulatory properties and their use in medicine and veterinary medicine to harmonize the physiological state of humans and animals, and to promote animal productivity.

The biologically active substance according to the present invention is classified as animal substance containing organic compounds. Pathological disorders of various origins in humans and animals cause abnormal physiological parameters. In this case, the treatment, including / and prophylaxis, consists in maintaining the physiological parameters (if abnormal), by exerting an effect on the body to normalize them.

The invention includes a biologically active substance, a process for its preparation and a preparation thereof for use in the normalization of the physiological state of the body.

Normalizing the physiological state of humans and animals can be done in two directions, first by direct action on pathologies and second, by direct action on the systems, organs, and tissues responsible for maintaining the homeostatic parameters in the norm.

The introduction of hormonal agents, mediators, growth promoters, chemotherapy, and the like directly affect the cells - targets by prescription.

For example, to treat diabetes, insulin is used to restore pancreatic function; in the case of functional damage of the nervous system, neurotransmitters are prescribed, which normalize neuronal marginal sensitivity and improve nerve impulse transmission accordingly; in the case of dysfunction of the body's immune system, immune mediators are used to correlate immune responses.

Intermediate treatment of diseases through the mobilization of internal resources of the body.

The processes involved in the regeneration of organs and tissues stimulate the body's immune system through inflammatory processes.

The body's immune system is stimulated by processes involved in tissue and organ regeneration, rehabilitation, and inflammatory and infectious processes. This improves the immune and general condition of the body.

Known biologically active substance derived from the germ material, which has immunomodulatory action, restores the physiological state of the body and regulates cell turnover (FR - A - 2413912). The biologically active substance (hereinafter BAM) is derived from crushed and homogenized animal tissue derived from such components, germinated and mixed with physiological serum.

Because of its relatively high immunomodulatory activity in mice, its use normalizes physiological conditions, heals and enhances animal productivity compared to a control group of 5-15%.

The efficacy of known BAMs is inadequate due to the presence of immunodepressants in combination with immunomodulatory factors, which prevent full immunomodulatory effects. The immunomodulatory activity of a given substance is essentially the result of co-acting immunomodulatory factors and depressants. Thus, the specific activity of the known BAM, which is dependent on the ratio of immunomodulatory factors to depressant concentrations, is relatively small due to the significant concentration of depressant in it.

Another disadvantage of the known BAM is its increased content of large molecule compounds, which are immunogens that trigger immune reactions (allergies, delayed-acting hypersensitivity, and 1.1).

The disadvantages of the known BAM described above are to a large extent dependent on the manner in which the material is obtained, whereby the process ends with the crushing and homogenization of the raw material of animal tissue, which necessarily entails a high content of depressants and high molecular weight compounds.

BAM having immunomodulatory properties, which normalize the physiological state of the organism, is also known to be obtained from crushed and homogenized tissue isolated from animal germ tissue extract (EP-A-0249563).

BAM is obtained by the following steps: comminution and homogenization of animal tissue, removal of insoluble compounds and use of the extracted dissolved material as the final product (EP-A-0249563).

The specific activity of the substance obtained as described is the result of those immunostimulatory and immuno-inhibitory effects and is also deficient.

The method of preparation is characterized in that the extract contains only soluble proteins and the structural proteins are removed, which significantly reduces the amount of immunostimulatory factors in the material.

Also known is BAM from crushed and homogenized animal tissue, obtained in the form of an extract from the tissue organs constituting the immune system (thymus, spleen and lymph nodes), thermo-stable components, or hormone-producing tissues (placenta), including for normalization. to increase animal resistance and productivity (SU-A-1442064).

The known BAM is intended to regulate the immune T-system and, in practice, does not induce any immune response (allergies, slow-acting hypersensitivity, etc.). Its specific activity is higher than that of the already mentioned BAMs due to the presence of a number of immunostimulatory factors.

The raw material used, as well as the absence of hydrolysis operations to homogenize and purify whole tissue elements and the resulting supematant containing ingredients with morphoplasmic and glycocalyx cellular components, do not allow the final product to be present in high concentrations (> 30%). as well as reduce immunodepressant, neutral and ballast compounds.

v

A known drug for the normalization of the physiological state of the human and animal body from the constituents of hydrolysis products of animal tissue, expressed as organic compounds, which is applied to the affected areas of the body (US-A-4455302). In addition to the above-mentioned disadvantages of the drug from the known BAM, the stimulation of the body's immune system can only be applied locally to the injured areas of the body. Therefore, the activity of such a preparation is relatively low. These deficiencies have been partially overcome by a known preparation having immunomodulatory activity for the normalization of the physiological state of the human and animal body, containing an active substance which is a biologically active substance consisting of hydrolysis products of animal tissue components and a filler which is injected intramuscularly 0 , 75-1.0 m / g body weight every 3 days, and thus its use is not localized (SUA-904707).

The disadvantage of the known therapeutic application is that it is administered only by injection, and also because of the immunogens it contains, it causes side effects (allergy, delayed hypersensitivity, etc.).

The use of animal tissue containing relatively high amounts of immunodepressants as a feedstock and the inability to improve the formulation of the BAM on which the therapeutic preparation is based significantly reduces the efficacy of the known BAM and its formulation.

v

The major disadvantage of the known preparation is that it contains a large amount of various biologically active substances to achieve the desired effect, which, on the one hand, leads to the risk of overdose, side effects and, on the other hand, increases the cost of the preparation.

v

There is a known method of normalizing the physiological state of humans and animals in the case of pathologically altered and / or foreign cells, microorganisms and products of their viability caused by diseases, which involves the hydrolysis of animal tissue components. (SU-A-904707).

The known method is ineffective due to the drawbacks described above, including the need to use it in high doses (up to 10 mg / kg), which can lead to overdose and cause side effects.

The foregoing drawbacks are overcome by the claimed biologically active substance having an immunomodulatory property, the claimed method for the preparation of the substance and the preparation thereof, as well as its use in the normalization of the physiological state of the body.

It is an object of the present invention to provide a biologically active substance having immunomodulatory properties, a method of preparation thereof and a medicament based thereon which is highly biologically active, without toxic effects and other side effects, as well as providing a final product with predetermined properties.

It is an object of the present invention to provide a biologically active substance having immunomodulatory properties consisting of water-soluble hydrolysis products of crushed animal tissue constituents expressed as organic compounds containing thermostable low molecular weight morphoplasmic and glycocalyx cellular components and modified embryogens and processes. and / or cell differentiation and / or pathology prior to and / or accompanied by regeneration and tissue repair.

It should be borne in mind that the term "morphoplasma" is used herein in its broadest sense and includes the structural part of the cytoplasmic cells presented by cellular organelles, membranes, cytoskeletons, and similar cellular structures (Yasuo Karaba. Biomembranes. M. Vyssaya Schola - 1995 13-15) and glycocalyx. used to label a carbohydrate-containing layer attached to an outer plasma membrane cell (B. Alberts, D. Brei, J. Liuis. Molecular Biology kletki. Mir - 1968, p. 97).

This is also achieved because the claimed BAM-modified morphoplasmic and glycocalyx cellular components contain thermostable components (at a temperature T ^ <= 120 ° C). In addition, the ratio of the amount of thermostable components to the modified elements is 30-100% by weight.

In addition, known BAM-modified morphoplasmic and glycocalyx cellular components contain low molecular weight M <= 10.0 kDa.

In addition, the ratio of low molecular weight components to modified morphoplasmic and glycocalyx cellular components is 30-100% by weight.

The object of the invention is also achieved by the fact that the claimed material contains hydrolysis products with modified morphoplasmic and glycocalyx cellular components containing by weight (%) organic compounds.

Polypeptides up to 26.0

Peptides 10.0-22.5

Amino acids 32.0-60.1

Carbohydrates- 10.5-28.7

Lipids, nucleotides and other organic compounds - 2.0-11.0

In addition, the claimed material contains carbohydrate organic compounds in the following weight ratio (%)

- 0 kDa for large molecular weight compounds -

to 2.0

- low molecular weight compounds of molecular weight KL, _n = 0,8-10,0kDa 2,5-11,5

The object is also achieved by the fact that the claimed substance contains hydrolysis products with modified morphoplasmic and glycocalyx cellular components containing by weight (%) organic compounds.

Polypeptides Peptides Amino Acids Carbohydrates 16.3-24.7

11.9-17.1

32.0-48.2

14.6-20.8

Lipids, nucleotides and other organic compounds 5.1-9.3.

In addition, the claimed substance contains hydrolysis products with modified morphoplasmic and glycocalyx cellular components containing% by weight of organic compounds

Polypeptides Peptides Amino Acids Carbohydrates 5.5 - 12.7

13.6-21.0

37.1-52.9

16.1-25.1

Lipids, nucleotides and other organic compounds - 5.3-11.0

In addition, the claimed material contains hydrolysis products with modified morphoplasmic and glycocalyx cellular components containing% by weight of organic compounds

Polypeptides - less 0.3

Peptides 14.9-22.5

Amino acids 43.5-60.1

Carbohydrates- 15.5-28.7

Lipids, nucleotides and other organic compounds - 5.0-9.2

The object is also achieved by the use of a modified process for the production of B AM having immunomodulatory properties, which involves the digestion of the washed raw material from the animal tissue, followed by hydrolysis of the hydrolysis products, their filtration and isolation of the supematant as the final product. performs homogenization and purification of non-disrupted tissue elements during the process of embryogenesis and / or poliferation, and / or cell differentiation and / or pathology, before and / or accompanying regeneration and / or tissue repair, and isolation of the disintegrated tissue elements by isolating the supernatant with morphoplasma and glycocalyx cellular components.

The raw materials used are embryonic tissue, thymocyte differentiated thymus gland tissue, testicular spermatogonia and spermatocytes, placenta, intestinal epithelial tissue, bone marrow cells, regenerating skin tissue, regenerating liver tissue, regenerating stage of amphibian limb, regenerating tissue.

In addition, after homogenization in saline and / or buffer, the undissolved tissue cells are cleaned, filtered and centrifuged at 150-250 g for 10-15 min.

In addition, crude animal tissue is crushed prior to homogenization prior to cell disruption, followed by extraction by rinsing and filtering and / or centrifugation at 30015000 g for 10-20 min. and isolating the crushed material already extracted.

This is achieved because the hydrolysed ingredients contain a ratio of morphoplasmic to glycocalyx cellular components of 3060% of the total weight of the organic compounds. Hydrolysis is performed at Tp at 445 ° C. In addition, hydrolysis may be carried out in the presence of a preservative.

The objective is further achieved by subjecting the hydrolysis products of the ingredients to thermal treatment at T _n = 60-120 ° C until complete denaturation of the thermolabile compounds and filtering and / or centrifugation at 1500 g in 20-40 min to isolate a supernatant having a thermostable final product. components.

In addition, the hydrolysis products of the ingredients are fractionated by dialysis through a semipermeable film and / or ultrafiltrated and / or gel-filtered to a final fraction having low molecular weight M <10kDa of low molecular weight morphoplasmic and glycocalyx cellular components.

The object is achieved by the fact that a known preparation for normalization of the human and animal body containing a biologically active substance based on hydrolysis products of animal tissue ingredients and a filler, the hydrolysis product contains thermostable, low molecular weight morphoplasmic and glucocalyx cell components, modified embryogens and or in the process of poliferation, and / or cell differentiation and / or pathology, before and / or accompanied by regeneration and / or tissue repair, and are present in the preparation in an amount of 0.001 to 85.0% (w / w) and the rest is a filler.

The object is also achieved by the fact that the claimed preparation of a biologically active substance contains hydrolysis products with modified morphoplasmic and glucocalix cellular components containing by weight (%) organic compounds.

Polypeptides up to 26.0

Peptides 10.0-22.5

Amino acids 32.0-60.1

Carbohydrates- 10.5-28.7

Lipids, nucleotides and other organic compounds - 2.0-11.0

In addition, the objective may be achieved by the fact that the biologically active substance (drug) contains organic compounds in the following ratio, weight (%)

Polypeptides 16.3-24.7

Peptides 11.9-17.1

Amino acids 32.0-48.2

Carbohydrates- 14.6-20.8

Lipids, nucleotides and other organic compounds 5.1-9.3

The objective is achieved by the fact that the biologically active substance (drug) contains organic compounds in the following weight ratio:

Polypeptides 5.5 - 12.7

Peptides 13.6-21.0

Amino Acids- 37.1-52.9

Carbohydrates- 16.1-25.1

Lipids, nucleotides and other organic compounds - 5.3-11.0

The claimed preparation may contain a biologically active substance containing by weight%

Polypeptides - less 0.3

Peptides 14.9-22.5

Amino acids 43.5-60.1

Carbohydrates- 15.5-28.7

Lipids, nucleotides and other organic compounds - 5.0-9.2

The formulation excipient may be saline and / or saline buffer, and / or carbohydrate compounds and / or fats of mineral, animal, vegetable and synthetic origin, and mixtures thereof.

Additionally, the product may contain a preservative.

The object is also achieved by the use of a preparation of hydrolysis products of animal constituents for the normalization of physiological conditions in human and animal diseases related to pathological alterations and / or alien cellular microorganisms and / or viability products and is a biologically active substance containing hydrolysis products. products containing modified tissues affected by embryogenesis and / or poliferation processes and / or cell differentiation and / or pathology affected before and / or accompanied by regeneration and / or tissue repair, morphoplasma and glycocalyx cell components, composition, 0.001-85.0 by weight (%) and filler, 15.0-99.999 mass (%). The preparation is used for intramuscular injection at 0.005-0.5 mg / kg body weight, at intervals of 1-7 days, and / or for inhalation 0.012-0.12 mg / kg every 4-48 hours, and / or 0.012-0.12 mg sublingually. / kg every 4-48 hours, and / or oral 0.02-1.0 mg / kg every 1-10 days, and / or 0.02-1.0 mg / kg intermittently every 1-10 days, and / or intranasally - daily dose 0.001-0.05 mg / kg every 2-24 hours, and / or compresses, suppositories, ointments applied to the abscess or wound every 1-10 days - until normalization of physiological parameters.

In addition, the physiological state of humans and animals is normalized by stimulation of the immune system, in particular activation of the reticuloendothelial system, T-, Bsystems, neutrophils and / or the natural killer population.

The claimed efficacy of the claimed BAM obtained in the manner indicated is characterized by high biological activity, and in particular an enhanced immunomodulatory effect.

Upon activation of the immune system, and in particular macrophage granulocyte cells, BAM and its preparation are expressed:

1) It does not act on a specific pathology, but by mobilizing internal reserves that normalize all parameters of homeostasis, including pathology, improve the overall physiological state of the body. In addition, corrects pathological abnormalities without side effects. Unlike other drugs, expressed BAM does not compromise other parameters of the body's homeostasis by normalizing altered parameters.

2) has a complex activity that allows the use of BAM to be extended to a range of immune-mediated disorders

3) have a degree of activity directed toward normalization of parameters of homeostasis that correlate with abnormalities.

The stated results are achieved due to the particular properties of the claimed BAM and its formulation.

It has been found that the amount of modified morphoplasmic and glycocalyx cellular components is increased by utilizing animal tissue affected by embryogenesis and / or proliferation processes and / or differentiation and / or pathology before and / or accompanying regeneration and / or tissue repair.

Such fabrics include:

- Differentiated thymocytes of the thymus

- testicular spermatogonia and spermatocytes, bone marrow cells, intestinal epithelial tissue

- Embryonic tissue, placenta

- Reconstructive tissue of amphibian limbs, regenerated liver tissue, regenerated skin tissue following partial hepatectomy or poisoning such as CCI4, as well as tissue affected by pathology such as gangrene, chemically or heat treated.

The use of the aforementioned fabrics as a raw material gives B AM the expected properties.

This property of the modified morphoplasmic and glycocalyx cell components enables them to be used in the production of expressed BAM and its preparation.

Administration to the human or animal body stimulates the body's response to pathology.

The immune system plays a key role in this process.

The tangible carriers of the above processes and / or abnormalities of the indicated tissues contain specific components that are part of the morphoplasmic and glycocalyx cell structure and undergo modification upon the physiological status of the cells. The claimed BAM and its preparation have modified morphoplasmic and glycocalyx cellular components, which in turn stimulate the body's response.

The hydrolysis is preceded by flushing of the crushed tissue from the tissue extract components and blood, followed by homogenization followed by removal of unbroken tissue, high levels (30-60%) of expressed BAM and its preparation morphoplasmic and glycocalyx cells, and reduction of immunodepressants, the amount of neutral and ballast compounds. This increases the body's immune response.

The body's response is enhanced by the introduction of a substance that contains thermostable low molecular weight M <= 10 kDa components of the morphoplasma and glycocalyx cells (stable at a heating temperature of <120 ° C).

The stated BAM is used as an active base in the formulation, and the fillers are used depending on the application.

For internal use, the product is administered by injection and the filler is saline or buffered saline.

For topical use, in addition to the solutions listed above, gels for filling are vegetable or animal carbohydrate compounds such as honey, juice, vegetable pulp and the like, and / or fats of animal, vegetable or synthetic origin, or mixtures thereof.

The efficacy of the preparation is highly dependent on the composition of BAM. As a result, the required efficacy can be achieved by significantly reducing the amount of active ingredient in the preparation, which avoids overdose and side effects and reduces the cost of the preparation.

In addition, it has been found that the above-described properties of the preparation allow it to be used to normalize the physiological state of the body by administering any dosage, in particular from 0.001 to several g / pg.

The following features illustrate specific embodiments of the invention; 1,2,4,8,9,12,16,18-25 irkt.

Illnesses caused by pathological lesions and / or foreign cells, microorganisms and / or products of their activity treated with a novel preparation are illustrated in specific doses and in specific ways in specific examples of the use of the invention: 3,4,7,8,11-13,15-17 et al. .

The biologically active substance having immunomodulatory properties is obtained as follows.

The raw material is animal modified tissue during embryogenesis and / or proliferation processes and / or during cell differentiation and / or pathology before and / or in combination with tissue regeneration and / or repair.

The animal tissue is washed, crushed, then homogenized in saline and / or buffer until complete cell disruption, and the homogenized mixture is purified from non-disintegrating material by filtration and / or centrifugation to isolate morphoplasmic and glycocalyx cell-containing ingredients and then hydrolyzed. Hydrolysis products after centrifugation and / or filtration are used as end products to normalize the physiological state of the organism.

The efficacy of the final product depends on the amount of modified morphoplasmic and glycocalyx cell components it contains. The amount is increased by the optimum selection of the raw material with intensive tissue modification processes and the optimum way to obtain it.

The raw material is selected on the basis that the cellular components of the morphoplasma and glycocalyx alter their structure in response to changes in the physiological state of the cells, and this alteration results in an immune alteration of the tissue and consequently in the body's immune responses. It was found that the immunogenicity of the modified structure is maintained in all stages of the process (embryogenesis, etc.).

Animal tissue used

- Thyroid cells differentiated by the parathyroid gland (cell differentiation process)

- testicular spermatogonia and spermatocytes, bone marrow cells, epithelial intestinal tissue (cell proliferation and differentiation processes) embryonic tissue, placenta (proliferation, differentiation, embryogenesis processes)

- Tissue repair of amphibian limbs, regenerated rat liver after partial hepatectomy or poisoning, such as CCl4, regenerated skin tissue, and tissue affected by pathology such as gangrene, chemically or heat treated (combination of various processes).

The use of the described animal tissues as a raw material in the claimed process enables to obtain a biologically active substance with expected properties.

It has been found that the most characteristic feature of the various tissues described above is the alteration of the structural components of their morphoplasmic and glycocalyx cells, allowing their differentiation from intact or dormant tissue cells.

v

The properties of these modified morphoplasmic and glycocalyx cellular components in the indicated tissues allow their utilization in the expressed BAM, which, when introduced into the body, provides an opportunity to stimulate the body's response to pathology. The body's immune system plays a key role.

It is best to use animal tissue taken from the modification process v

in the incremental stage. This stage is determined empirically by the nature of the immune disorder. In the case of embryonic tissue use, this period corresponds to the first half of embryogenesis. The regenerated rat liver is used after partial hepatectomy after 4 to 12 hours, and in the case of regenerated limbs it is used at the stage of blastema formation.

The relative increase in the amount of morphoplasmic and glycocalyx cellular components is achieved by the basic condition, i.e. removal of unbreakable tissue elements.

Uncleaned items are cleaned by centrifugation at 150-250 g for 10-15 minutes. At higher centrifugation speeds and longer time, morphoplasmic and glycocalyx cell elements are precipitated, and at slower than 150 g in 10-15 min. - cleaning is ineffective.

The cleaning of unbreakable elements is carried out in the filtering process by a coarse-grained filter, for example, through several layers of gauze, through a cotton cloth or a capron net.

The immunomodulatory property in the final product is enhanced by the fact that, prior to homogenization, the tissue is pulverized to cellular injury, followed by extraction with saline and / or filtration with organic acid solution and / or centrifugation at 300-15000 g for 10-20 min. and homogenizing the resulting extracted extruded tissue.

In addition, on the one hand, more immunodepressants in blood serum and tissue extract are removed and, on the other hand, there is a relative increase in morphoplasmic and glycocalyx cells.

The additional flushing of the crushed animal tissue prior to honogenization by centrifugation at 300-15000 g for 10-20 minutes enables a higher quality separation of the extracted tissue from the extract under industrial conditions.

A similar effect is achieved by coarse filtering, for example, through several layers of gauze, through cotton cloth, or through a capron filter.

The relative proportions of morphoplasmic and glycocalyx cellular components in tissue components secreted for hydrolysis are determined by the amount of protein and carbohydrate in the structures liberated from intact tissue.

The efficiency of the end product is significantly enhanced by the use of low molecular weight morphoplasmic and glycocalyx cellular components.

On the one hand, this helps to prevent adverse events and, on the other hand, to obtain a broad-spectrum preparation. To this end, tissue components containing modified morphoplasmic and glycocalyx cells are hydrolyzed by disrupting the macromolecular structures of the morphoplasmic and glycocalyx cells into lower molecular weight components. The efficiency of hydrolysis products depends on the specificity of hydrolysis, which depends on temperature-time characteristics.

The immunomodulatory property, namely, the partial (in vitro) activity of the rat peritoneal exudate macrophages was found to be maximized at the hydrolysis passage temperature T = 4-45 ° C.

Temperature deviations are undesirable because

- At Tp <4 ° C, the hydrolysis process is significantly prolonged, which reduces the economic efficiency

- Tp> 45 ° C reduces the activity of enzymes catalyzing the hydrolysis of the raw material and accelerates the degradation of the active components.

This leads to a deterioration in the quality of the final product.

The duration of hydrolysis is determined by the expected maximal immunomodulatory effect, in part by the activity of the peritoneal squadrate macrophages in accordance with the NST assay.

Prolonged hydrolysis is carried out with a preservative.

The relative increase in the amount of low-molecular-weight components in the hydrolysis products corresponding to the corresponding high molecular weight thermolabile compounds is achieved by heat treatment of the hydrolyzate to complete denaturation of the thermolabile compounds, followed by removal by centrifugation

1500 g in 20-40 min. and / or filtration. Use of a supernatant containing thermostable components of morphoplasmic and glycocalyx cells as a final product.

The hydrolysis products are heat treated in the temperature range of Tpj> 60-120 ° C.

At Tpj <60 ° C, not all proteins are denatured, which results in an increase in thermolabile compounds in the final product. This reduces the specific activity of the substance. At T ^> 120 ° C, the peptide and carbohydrate components of the final product exhibit a dramatic increase in the rate of structural alteration, which results in degradation of the product.

After heat treatment, the thermostable components are removed by centrifugation at a rate of at least 1500 g for 20-40 min. Lower centrifugation times reduce the rate of removal of denatured thermolabile compounds. Centrifugation time 20-40 min. is optimal.

Coagulated denatured thermostable compounds can be separated from the supematant by filtration through a paper filter.

The final product, which contains thermostable components of marfoplasma and glycocalyx cells, exhibits high specific activity. It can also be used as an injection device in both veterinary and medical applications.

Further enhancement of BAM-specific activity is achieved by the low molecular weight M <10.0 kDa components. Dialysis of the hydrolysis products by semi-permeable film and / or ultrafiltration and / or gel filtration completely removes the large molecule compounds which may cause side effects.

The efficiency achieved in this way allows the end product to be used in medicine.

The claimed BAM is used as an active ingredient in the manufacture of a medicament, the concentration of which depends on the intended use. Preparations for watering and feeding animals have low levels of BAM and preparations for injections, as well as ointments, gels and powders have a higher concentration.

Fillers are used depending on the destination. Sodium chloride and saline buffer solutions are used in preparations for injection, inhalation, application and intranasal administration. Carbohydrate compounds as fillers are used in preparations for feeding bees (e.g. 10% sugar solution) or gel preparation (e.g. honey, juice and plant pulp). Fats used as fillers are mineral, animal, vegetable and synthetic fats or mixtures thereof.

Specific examples of the preparation and use of the claimed invention are described below, as well as methods for determining the amount and properties of biologically active substances.

example

Embryonic tissue taken from a cow in the first half of pregnancy was used to obtain the claimed BAM.

Following disinfection of the embryo, muscle and skin tissue, liver, spleen, pancreas, kidney, lungs and heart are dissected in a weak solution of potassium permanganate (KMnO ^) and coarsely minced.

Mix 1 kg of the resulting mince with 30 kg of saline and homogenise with a knife blender until smooth. Decantation is used to clean the mixture from the disintegrated tissue elements and the resulting supernatant fluid with tissue components containing morphoplasmic and glycocalyx cellular components is autolysed (Th ⁼ 45 ° C for 48 hours). The resulting hydrolyzate was filtered through a paper filter and the filtrate containing the morphoplasmic and glycocalyx cellular components was used as the final product having the organic compound composition and specific activity shown in Tables 1 and 2.

The final product, in which BAM is 1.0% of the total weight and the other part is a filler, is mixed with 9 parts of cucumber-pear pulp and used as a therapeutic product - a gel mask on the face for cosmetic purposes.

For prolonged storage, preservatives (nipagin and nipazole in a weight ratio of 5: 1) are added to the gel at a final concentration of 0.1% by weight. %. The gel is applied to the face for 2-3 hours. The procedure is repeated 1-2 times a week. The healing effect is achieved after 4-10 sessions and is monitored according to the skin condition.

example

The embryogenic tissue obtained in the same manner as in Example 1 is shredded. 10 kg of the resulting mince are mixed with 30 kg of physiological fluid and homogenized to a uniform weight.

The homogenized material is centrifuged at 250 g for 10 min to remove unbroken tissue. After centrifugation, the residue is removed and the floated fat is removed by filtration of the supernatant through a cotton cloth. To the filtered homogenate containing ingredients containing 30% by weight of morphoplasmic and glycocalyx cellular components, a preservative (quinosol) is added to a final concentration of 0.08% by weight. After mixing, the homogenate is hydrolyzed at Tp = 4 ° C for 6 weeks.

The resulting hydrolyzate was centrifuged at 15000 g for 20 min. and isolating the supematant with the components of the morphoplasmic and glycocalyx cells, already as a final product, whose organic compounds and their specific activity are shown in Tables 1 and 2.

The final product, containing 1.0% by weight of B AM and the remainder of the filler, is used as a medicament for the preservation of depleted fur animals as well as for inhalation of chickens in a special chamber for 15-20 minutes. In the chamber, the preparation is sprayed with 0.5 ml per 1 ml for 1 min. The procedure is repeated for 7 weeks. The effect is controlled by a decrease in the number of fall chicks. The fall of pups decreases by 30-40%.

example

The embryonic tissue obtained as in Example 1 is pulverized. The resulting mince is poured into two volumes of physiological fluid and mixed. The resulting mass was centrifuged at 1500 g for 20 min. The precipitate is removed and re-mixed with twice the volume of physiological fluid and centrifuged again. The mince wash operation is performed 3 times. Thereafter, 10 kg of the washed mince is mixed with 30 kg of centimolar phosphate saline buffer (0.01 M PBS) pH 7.2 containing centimolar sodium phosphate substitution and fifteen centimolar sodium chloride (0.01 M Na2HPC> 4 and 0, 15 M NaCl) and homogenized in a colloidal mill to a uniform weight. Undissociated tissue is removed by sedimentation by centrifugation at 150 g for 15 min.

The supernatant, after flotation fat removal by filtration, is treated with a preservative to a final concentration of 0.08% by weight.

The mixed homogenate containing 60% by weight of morphoplasmic and glycocalyx cellular components was hydrolyzed for 6 weeks at Tp = 4 ° C. The resulting hydrolyzate was centrifuged at 1500 g for 40 min. until the release of the supematant containing the components of the morphoplasmic and glycocalyx cells as a final product having the composition of the organic compounds and their specific activity are shown in Tables 1 and 2.

The final product, containing 1.0% w / w BAM and the remainder of the filler, is used as a therapeutic agent for parainfluenza calves, administered by intramuscular injection at 0.05 ml / kg every third day. The treatment effect occurs after 1-5 injections. It is controlled by temperature and the amount of virus in the blood.

example

Coarsely minced minced meat obtained in Example 1 is poured

2 times more 3% acetic acid and mix. The mixture is vacuum filtered through a cotton cloth suction filter. The precipitate is removed, re-mixed with two volumes of saline and centrifuged at 300 g for 20 min. Mix 10 kg of wash mince with 30 kg of centimolar phosphate saline buffer (0.01 M PBS) pH 7.2. containing a centimolar amount of double-substituted sodium phosphate and 15 centimolar sodium chloride (0.01 M Na2HPO2 _Z1 and 0.15 M NaCl), and the homogenized mass is filtered through a cotton pad (canvas). The resulting filtrate is supplemented with 0.08% w / w preservative. The mixed homogenate containing 50.0% by weight of morphoplasmic and glycocalyx cellular components was hydrolyzed at Tp = 20 ° C for 8 days. Further processed as in Example 3.

The organic compound composition of the final product and its specific activity indices are shown in Tables 1 and 2. The final product, containing 1.0% by weight of BAM, and the rest is a filler used to treat dermatoses.

Oily preparation, prepared by mixing lanolin petroleum jelly and end product in a BAM ratio of 1: 3: 1 parts by weight. The skin is greased daily. The healing effect occurred after 4-12 days and was controlled by skin condition.

example

The relative percentage (w / w) of morphoplasmic and glycocalyx cellular components with connective tissue components and tissue constituents extract for hydrolysis is determined by the amount of protein and carbohydrate in the cellular structures liberated from unmodified tissue particles.

For this purpose, the homogenate was ultracentrifuged at 100,000 g for 60 min after being cleaned of unbroken tissue elements as in Examples 1-4.

The ratio of total protein content to total amount of supernatant indicates the ratio of protein morphoplasmic and glycocalyx cell components to extract proteins.

The protein content of the residue and the supernatant is determined after ultracentrifugation of the homogenate. After removal of supernatant, 4.9 ml of saline is added to 0.1 ml of the residue and, after suspension, 0.1 ml of protein is determined by SDS-Louri photometric determination of the protein intensity. (U.K. Laemmli. - Nature, - 1970, - V 227, - 5259 - pp. 680-685).

To determine the amount of protein in the supernatant, add 0.9 ml of saline to 0.1 ml of supernatant and, after mixing, take 0.1 ml of the protein for determination according to SDS-Louri.

The amount of connective tissue components is determined by the amino acid analysis of oxylysine and oxypyroline.

Unlike the known BAM, no amino acids were found.

The amount of glycocalyx cell components is determined by the amount of carbohydrates in the compound of greater than 0.8 kDa homogenate by reaction with the anthron after removal of the glycogen granules from the homogenate (S. Seifter, S. Dayton, C. Honiv - Arh. Biochemistiy and Biophysical, 1950 , -25, - 1, pp. 191-200).

For this purpose, 20 ml of tissue homogenate is layered in centrifuge tubes at 30 ml on 5 ml of sucrose (d = 1, 40 density) and ultracentrifuged at 100,000 g for 60 min. Glycogen deposited at the bottom of the tubes is removed and morphoplasmic and glycocalyx cellular elements are selected in interphase and, after washing with sucrose, centrifuged in saline and used for assays.

The carbohydrate content of the test samples is determined by the total carbohydrate content after hydrolysis and the free carbohydrate monomer content. The difference between these data determines the amount of free carbohydrates in the various compounds.

For total hydrolysis, take 1,0 ml of the test sample and mix with 1 ml of 4,0 N saline. After hydrolysis at 105 ° C for 2 hours, the hydrolyzate is evaporated under vacuum in a desiccator containing NaOH. The evaporated sample is exposed to 1.0 ml distilled water and used for analysis.

The free carbohydrate content of BAM or after acid hydrolysis is determined as follows:

Mix 1 ml of the sample with 1 ml of distilled water, then add 4 ml of 0,2% anthronov reagent, prepared with 95% sulfuric acid (H2SO4), with constant stirring at -5 ° C. The resulting mixture was heated at ° C for 10 min and, after cooling, the optical density at 620 nm was determined. The standard is 20 pg / ml of glucose concentrate. The BAM protein obtained in Examples 1 and 2 was 37.6 + -7.4% for morphoplasmic and glycocalyx cell components, and the BAM protein obtained in Examples 3 and 4 was 51.8 + -8.2%.

Here and again in the text, as well as in the tables, the experimentally obtained values are expressed as the mean with the mean deviation calculated according to the Student's criterion.

example

The hydrolysis supernatant obtained in Example 3 was heated to Tp || = 55 ° C, ΤΗ2 ⁼ 6θ Th3 ⁼ 95 ° C, 1 ^ = 120 ° C, and Τ ^ = 135 ° C until complete denaturation of the thermolabile compounds of the hydrolysis products, followed by heating. centrifuge at 1500 g for 30 minutes. until the supernatant is released as a final product containing thermolabile marfoplasma and glycocalyx cells.

The final product obtained has in each case an immunomodulatory effect, the values of which are shown in Table 3.

v

The following table shows the distribution of BAM specific activity values depending on the selected hydrolyzate heating temperature range and beyond. The optimum heating temperature is Tpj = 95 ° C, when the final product obtained has maximum enzymatic and phagocytic activity, 72.8% and 64.3%.

The mean specific activity at Th ⁼ 55 ° C compared to control is 61.1% by NST and 52.3% by phagocyte. At > 120 &lt; 0 &gt; C, there is a sharp structural change in the peptides and carbohydrate compounds which results in a structural change in the components of the final product resulting in a decrease in the specific activity of BAM. At 135 = 135 ° C, the specific BAM activity was 57.3% on average for the NST assay and 43.0% for the phagocytes compared to the control. The maximal specific activity of BAM as compared to the control is reached at Tj = 60-120 ° C, in which rat peritoneal squamate macrophage activity (in vitro) is 72.8% by enzymatic activity (NST assay) and by phagocytic activity (E. Coli). - 64.3%.

example

The hydrolysis supernatant obtained in Example 3 was heated at T = 95 ° C until complete denaturation of the thermolabile elements and after centrifugation at 1500 g for 40 min after cooling.

The residue of the denatured compounds is removed and the final product used is a supernatant containing 60% by weight of the thermostable components by peptides for the hydrolysis products of the water-soluble compounds prior to heat treatment.

The content of organic compounds in the hydrolysis products and the activity indexes of the resulting material obtained experimentally are shown in Tables 1 and 2.

The final product, containing 0.5% by weight of BAM and the remainder of the filler, is used to treat mastitis in cows by intramuscular injection of 0.05 ml / kg every third day. The treatment effect is observed after 1-4 injections and is controlled by the udder and purulent secretions.

example

The hydrolyzate supematant obtained in Example 2 is heat treated to complete denaturation of the thermolabile compounds and centrifuged at 50,000 g for 20 min after cooling. The precipitate is removed and the final product used is a supernatant containing 30% by weight of thermally stable components by peptides of the hydrolysis products, respectively, to water-soluble compounds prior to heat treatment. The content of organic compounds in the hydrolysis products and the specific activity rates of the substance obtained experimentally are shown in Tables 1 and 2.

The final product, containing 0.5% BAM by weight, and the remainder of the excipient, is used as a therapeutic preparation for the treatment of parainfluenza calves with intramuscular injection of 0.05 ml / kg twice every 14 days. The efficacy of the preparation was recorded on the basis of the number of sick cattle per month and did not exceed 10%, while the control rate reached 22-28%.

example

The hydrolyzate obtained in Example 4 is heat treated at Tpj = 95 ° C until complete denaturation of the thermolabile compounds and, after cooling, filtered by suction through a filter paper. The filtrate containing 45% of the peptide thermostable elements for the hydrolysis products of the water soluble compounds, respectively, is used as the final product.

The content of organic compounds in the hydrolysis products and the values of the substance specific activity obtained experimentally are shown in Tables 1 and 2.

The final product, lyophilized and sublingually used as a therapeutic parparate for the treatment of mastitis in women. The procedure is performed daily. The treatment effect is observed after 1-4 days and is controlled by breast condition (hardening, purulent discharge) and blood tests.

example

The supernatant hydrolyzate obtained in Example 3 is fractionated in an Anicon ultrafiltration unit. Ultrafiltration-carrying compounds with a molecular weight of less than or equal to 50.0 kDa, 10.0 kDa, 5.0 kDa, 1.0 kDa, 0.8 kDa, 0.5 kDa are used.

The resulting fractions containing compounds with molecular weight were: 1) Mj> 50.0 kDa, 2) 10.0 kDa <M ₂ <50.0 kDa, 3) M ₃ <10.0 kDa, 4) M ₄ <5.0 The biological activity of kDa, 5) M ₅ <1.0 kDa, 6) M ^ <0.8 kDa, 7) Μγ <0.5 kDa was analyzed by the ability to activate rat peritoneal exudate macrophages by NST assay. The results of the studies are shown in Table 4.

The conclusions are based on the analysis of Table 4:

1) the fractional distribution of specific activity has a pronounced peak, in the mass range of 0.8 to 10.0 kDa

2) the decrease in specific activity at molecular weight M> 10.0 kDa is related to the action of inhibitors in the final product and the decrease of active substance concentration;

3) The decrease in specific activity at molecular weight M <0.8 kDa is associated with a decrease in the concentration of active substance in the form of oligomeric compounds, M> 0.8 kDa.

example

The supernatant hydrolyzate obtained in Example 3 was filtered on a millipore filter BPY-15-PA (TY6-12-151-87) with AP-2.0.

The resulting ultrafiltrate having low molecular weight morphoplasmic and glycocalyx cellular components M <= 10.0 kDa is used as the final product, wherein the low molecular weight components with M <= 10.0 kDa represent 60% by weight of peptides for the hydrolysis products of the water soluble compounds after ultrafiltration.

The amounts of organic compounds in the hydrolysis products and the specific activity rates of the resulting material are shown in Tables 1 and 2.

The final product containing 0.1% w / w BAM and the remainder of the excipient is used as a drug for experimental treatment of rat hepatitis by intramuscular injection of 0.05 ml / kg every other day (treatment and results are described in Examples 34 and 35).

example

The hydrolyzate supematant obtained in Example 2 is dialyzed for 16-24 hours. through a cellophane film, in saline containing 0.8 g of preservative per kg. The resulting dialysate is used as the final product, wherein the low molecular weight components, where M <10 kDa, represent 30% by weight of the peptides of the corresponding products of water soluble compounds prior to dialysis.

The amount of organic compounds in the hydrolysis products and the specific activity rates of the resulting material are shown in Tables 1 and 2.

The final product, containing 0.1% by weight of BAM, and the remainder of the filler is used as a medicament for injection to normalize calf homeostasis parameters. For calves, abnormal intramuscular injection of 0.05 ml / kg for 1-2 months. The effect is controlled by weighing calves monthly. The daily gain is 4770% higher than the control.

example

The hydrolyzate supematant obtained in Example 7 was gel-filtered on a Sephadex G-25 d = 26 mm diameter and h = 600 mm high column with decimolar (0.01 M) phosphate buffer pH 7.2 containing fifteen centimeters of sodium chloride (0.15). M NaCl).

Molecular weight fractionation of the hydrolyzate supematant is performed by column elution with the same buffer, and the chromatographic profile of the eluate is controlled spectrometrically at 280 nm.

The fraction of the low molecular weight components of the hydrolyzate containing the morphoplasmic and glycocalyx cellular components is eluted as the final product.

The amount of organic compounds in the hydrolysis products and the BAM specific activity score obtained experimentally are shown in Tables 1 and 2.

The final product containing 0.1% by weight of BAM and the remainder of the filler is used as a medicinal product in calves with bronchopneumonia or acute respiratory disease. The preparation is injected intramuscularly at 0.05 ml / kg every three days.

The effect of treatment is monitored after 1-7 injections and is controlled by body temperature.

example

The dependence of the claimed BAM efficiency on the amount of modified morphoplasmic and glycocalyx cellular components is determined from the raw material of animal tissue undergoing embryogenesis and / or proliferation and / or cell differentiation and / or pathology prior to and / or accompanying tissue regeneration and / or repair. recovery.

The resulting materials are processed in the same manner as in Example 7. The final product obtained using tissue from each of the aforementioned modified cellular structures, namely morphoplasma and glycocalyx cells, is shown in Table 5.

The table analysis shows that the presence and potency of an immunomodulatory effect on BAM depends on the raw material used to derive hydrolysis products containing modified morphoplasmic and glycocalyx cellular components.

v

Raw material derived from animal tissue at the stage of cellular proliferation and differentiation used in rat testicular spermatogonia and spermatocytes, bone marrow cells, rat intestinal epithelial tissue. Raw embryonic tissue from animal tissue harvested by embryogenesis is used.

v

The raw material with pathology and pre-regeneration processes is the rat liver after partial hepatectomy, and the raw material with pathological processes is regenerated amphibian limb tissue, the rat liver after subcutaneous administration of carbon tetrachloride (CCl ^).

Together with the data obtained, Table 5 shows the immunomodulatory effect results from intact tissue of rat female muscle and liver.

the table analysis shows that the immunomodulatory effect is expressed in B AM obtained from tissues affected by embryogenesis, proliferation, and pathology prior to and / or accompanied by regeneration and / or tissue repair, higher than the tissues unaffected by said processes.

In addition, it can be concluded that the greatest immunomodulatory effect is achieved by the use of reconstituted liver tissue, embryonic tissue and reconstructed tissue of amphibian limbs.

example

The raw material used is the regenerated liver of rats 4 to 12 hours after hepatectomy by a known method (G.M. Higgins, R.M. Anderson Experimental pathology if the liver. - Areh. Pathology, - 1931- N 12p.186).

The liver is finely trimmed and mixed with three volumes of saline, followed by centrifugation at 1500 g / min, and the resulting residue is mixed with three volumes of saline and centrifuged again under the same conditions. The pulverized tissue is washed 3 times and further steps in the final product preparation are performed as in Examples 3, 7 and 11.

The specific activity of the resulting final products is determined experimentally by the activity of the hepatocytes repairing rat liver exposed to CCl4, where Na, K - AT Phase activity is restored, respectively, 32.7 + -3.1%; 43.6 + -3.9% and 85.4 + -7.8% compared to the animal group for which the claimed material was not used.

Blood aminotransferase activity is restored by 37.4 + -2.8%; 47.2 + 3.4% and 98.3 + -1.7%, - and hepatic hepatocyte potassium concentration 22.6 + 1.8%; 28.9 + -2.5% and 58.1 + -4.7%.

The final product containing BAM 0.1% by weight and the remainder of the excipient used as a therapeutic agent for the treatment of calves with hepatitis by intramuscular injection of 0.05 ml / kg every 3 days. The treatment effect is monitored after 7-14 injections and is controlled by aminotransferase activity, bilirubin and blood nitrogen levels.

example

The raw material used is the bull testis cake obtained after extraction of the lidases by extraction of tissue homogenate with 3% acetic acid.

The resulting testicular cake, the residue from the lidase production, is re-homogenized in three parts of saline in knife homogenizers and centrifuged at 250 g for 15 minutes to remove undissolved tissue elements.

Alkaline decinormal aqueous solution (0.1 N NaOH) raises the acidity of the medium to pH 7.4 and adds to the homogenate containing the morphoplasmic and glycocalyx cellular components a modified preservative of 0.8 g / l, followed by mixing and is hydrolyzed with further processing as in Examples 3.7 and 11.

Specific activity of the resulting end products is determined experimentally by the activity of rat peritoneal exudate macrophages and blood neutrophils. Using the final product obtained as in Example 11, the enzymatic activity of macrophages increased by 108.3 ± 13.2% and the phagocytic activity according to E. Coli by 92.2 ± 10.2%. For blood neutrophils, these rates increased by 99.2 ± 14.1% and 72.4 ± 8.3%, respectively.

The final product, containing 0.1% by weight of BAM and the remainder of the filler, is mixed with 50 volumes of water and used as a nutritional preparation to normalize chicken homeostasis parameters. Chickens are watered at 5 ml / kg for 2-6 weeks.

Effect control was observed with a 15-30% increase in egg strength compared to the control group.

example

The raw material is used in the cow placenta, which is treated in the same manner as in Examples 3, 7 and 11, and the final products are obtained with specific activity determined experimentally by the activity of rat peritoneal exudate macrophages and blood neutrophils.

The end product obtained in Example 11 shows an increase in enzymatic macrophage activity of 150.6 ± 17.4% and a phagocytic macrophage activity of 109.3 ± 12.7%.

Corresponding indices of blood neutrophils are 137.6 ± 16.8% and 95.7 ± 11.3% respectively.

The final product containing 0.5% by weight of BAM and the remainder of the filling is used to treat cows suffering from endometritis. Intramuscular injection is given at 0.05 ml / kg every 3 days. The effect of treatment is monitored after 6-10 injections and is controlled by inflammatory reactions, histological examination and purulent discharge rates.

example

The raw material used is the thymus gland obtained after isolation of insoluble tissue components that are waste from the production of thymosin from the homogenate.

Thyroid gland pulp is processed in the same manner as in Examples 3, 7, and 11 to give final products experimentally determined by stimulation of the T-lymphocyte blast transformation reaction and increased by 165.6 ± 13.7% compared to the control used. the final product was obtained according to Example 11.

The final product, containing 0.5% by weight of BAM and the rest of the excipient, is used intranasally as a drug for the prophylaxis of human infectious diseases. Apply 2 drops to each nasopharynx and massage to drain them into the mucosa. This procedure is performed twice a day. The effect is controlled by body condition and body temperature.

example

A therapeutic preparation containing B AM was obtained in the same manner as in Examples 1-4, 7-9, 1113 and 15-17 with 0.001% B AM by weight. Filler - 10% sucrose solution.

The preparation is used to feed bees.

In one bee, 11 formulations per meal improved compared with the control:

- survival 85-140%

- departures 100-160%

- 27-64% sample of honey collection

A therapeutic preparation containing B AM prepared in the same manner as in Examples 1-4, 7-9, 11-13 and 15-17 is prepared with 85% by weight of lyophilized BAM. Fillers use antiseptic. The preparation in the form of sprays of lyophilized dried powder is used to treat open wounds, burns, injuries, mucous membrane erosion.

Application of the preparation accelerates the regeneration processes 2-7 times compared to the control.

example

A therapeutic preparation containing BAM obtained in the same manner as in Examples 1-6, 9-14, 16-21 and 23-25 is prepared with 1.0% by weight of BAM.

Filling gel and plant pulp: cucumber, apple, peach purée. The preparation is used in cosmetics. The therapeutic effect is observed after

4-8 sessions and controlled by skin elasticity.

example

A therapeutic preparation containing B AM prepared in the same manner as in Examples 1-6, 9-14, 16-21 and 23-26 is prepared with 1.0% by weight of BAM.

The filling gel is 15 parts honey, 3 parts turnip juice, 1 part garlic juice and 1 part burdock oil.

This product is used to strengthen the roots of the hair. The gel is applied on the head for 2-8 hours. The procedure is performed once a week. The treatment effect is monitored after 5-12 sessions and controlled by the hair condition.

example

A therapeutic preparation containing BAM obtained in the same manner as in Examples 1-6, 9-14, 16-21 and 23-2 is prepared with 1.0% by weight of BAM.

The filler used is a 1: 3 mixture of lanolin and spermaceti. The product is used to restore the skin layer after dermatosis. Application of the preparation accelerated the recovery process 2-5 times compared to the control.

example

A therapeutic preparation containing BAM obtained in the same manner as in Examples 1-6, 9-14, 16-21 and 23-26 is prepared with 1.0% by weight of BAM.

Cocoa is used as filler. The preparation is used to treat endometritis with suppositories. The effect of treatment is observed after 14-21 days and is controlled by the indicators of inflammatory response, histological examination, blood analysis.

example

A therapeutic preparation containing B AM prepared in the same manner as in Examples 1-6, 9-14, 16-21 and 23-26 is prepared with 1.0% by weight B AM.

The filler used is a 1: 3 mixture of lanolin and petroleum jelly. It is used to treat mucous membrane erosion. The restorative processes are 3-5 times faster than those of the control.

example

The B AM obtained in the same manner as in Examples 1-18 is subjected to amino acid analysis in three directions which determine:

- total amino acid composition;

- amino acid composition of peptides and free amino acids, free of acid-insoluble proteins;

- composition of free amino acids.

1) The total amino acid analysis of B AM obtained in the same manner as in Examples 1-18 is carried out by hydrolysis under vacuum in 6N hydrochloric acid (6N HCl) at 110 ° C for 24 hours.

After hydrolysis, the sample is evaporated in vacuo over dry sodium hydroxide (NaOH). The dry residue is diluted with 0.2 N sodium citrate buffer pH 2.2 and analyzed on an amino acid analyzer ion exchange column.

2) To remove acid-insoluble proteins, add 0.5 ml of 3% sulfosalicylic acid to the 0.5 ml sample for 1 hour, stirring periodically. Denatured proteins are removed by centrifugation at 8000 g for 15-20 min. The resulting supematant is subjected to a general amino acid analysis.

3) Determine the free amino acid content of the B AM obtained in the same manner as in Examples 1-18 after equilibration of the samples with 0.2 N sodium citrate buffer pH 2.2 and filtering through an ion exchange column on an amino acid analyzer. .

Elution of the amino acid column was performed with sodium citrate buffer pH 3.25; pH 4.25 and pH 5.28. Fractionated amino acids are treated with a ninhydride reagent and the optical density is determined at 440 and 570 nm with a passing photometer.

example

The BAM obtained in the same manner as in Examples 1-18 is determined as follows in the total lipid content. 0.5 ml of BAM is mixed with 1.5 ml of concentrated sulfuric acid (H2SO4) and heated at 95 ° C for 15 min. To the cooled 0.1 ml of hydrolyzate is added 1.5 ml of phosphovanil reagent (Chempal.) According to a known general lipid determination method by specific staining. (J.M. Knight, S. Anderson, J.M. Rawle. - Clinical Chemistry - 18, p. 199). The optical density of the solution is determined at 530 nm.

example

Nucleotide analysis of BAM obtained in the same manner as in Examples 1-8 is performed as follows. The purine and pyrimidine acid-soluble non-nucleotides are extracted from 1.5 to 5.5 ml of a 5% sample of chilled chloric acid (HCIO4). Samples after 20 min. extraction, centrifugation in the cold, 2500 g in 10 min, the residue was washed three times with 5% chloric acid, the supernatants combined and used to isolate the acid soluble nucleotides. The acid soluble fraction is hydrolyzed with one molar chloric acid (1 M HClO4) at 100 ° C for 1 h. to pyrimidine monophosphates and purines. The solutions are neutralized with potassium alkali (KOH) and the hydrolysis products distributed on a cation exchanger DAUEKS 50x4 by step elution:

distillate (Hio) - uric acid and uridine monophosphate (UMF); 0.2 M chloric acid - xanthine and cytosine monophosphate; 0.4 M Chloric Acid - Hypoxanthine;

1.0 M chlorinic acid - guanine; 2 M chlorinic acid - for adenine.

Nucleotides are identified chromotographically by movement in the cation exchanger relative to the standard as well as by absorption spectra at 200-300 nm. The amount of nucleotides is determined using the magnitude of the molar extinction coefficient.

example

Immunomodulatory activity of BAM obtained in the same manner as in Examples 1-18 was determined by the activity of peristereal macrophages in the wister line rat in vitro, the potency of the tetrazole enzyme in nitromel reduction (NST assay) and the phagocytic activity E. With respect to Coli.

20 ml of Chenx's solution containing 20 units / ml heparin is injected into the abdominal cavity of the decapitated animals. After 2-3 minutes abdominal massage, incision is made and at the same time the injected fluid is withdrawn from the cavity, which is filtered through a nylon filter and centrifuged at 150-200 g for 15 minutes. The resulting residue is resuspended in 0.5 volumes of fresh Chenx solution and centrifuged again under the same conditions. The leached cells are washed 3 to 4 times, after which the cell concentration in the suspension is 80x10 5 cells / ml. The macrophage content of whole cell tissue escudate is 25-35%.

v

The enzymatic activity of rat peritineal exudate macrophages is determined spectrometrically by the reduction of nitro tetrazolium blue to v

diformazan. The rat peritoneal exudate cell residue was diluted with colorless Chenx solution to a concentration of 80 x 10 4 cells / ml. A sample containing pH 7.4 and 1.0 mg / ml peptide was added to 5 μΐ cell suspension. The mixture was incubated in a water bath at 37 ° C for 30 min, then 5 μΐ of saturated nitro tetrazolium blue Reanal at 20 ° C was added and incubated for another 30 min. 3 ml of acetone are then added and centrifuged at 2000 g for 20 min. The precipitated acetone extract is photometric at 515 nm.

A sample with a preservative in saline is used to control enzyme activity comparison.

The phagocytic activity of rat peritoneal exudate cells is determined by their ability to occupy E. coli.

To 50 μΐ of cell suspension is added 50 μΐ of sample with pH 7.4 and peptide concentration of 0.05 mg / ml. The mixture was incubated at 37 ° C in a water bath for 30 min. shaking constantly. After incubation, the mixture is suspended in 10 ml of colorless Chenx solution at 4 ° C and centrifuged at 150-200 g for 15 min. The cellular residue of rat peritoneal exudate was diluted with Chenx's solution to a concentration of 2.5 x 10 4 cells / ml. 0.2 ml of the resulting suspension is placed on an 18x18 mm glass and incubated at 37 ° C for 30 min. 5% carbon dioxide (CO2) in the atmosphere to attach the macrophages to the glass. After incubation, non-adherent cells are removed by rinsing 3 times in a beaker with Chenx solution. To the remaining cells on the glass is added 0.2 ml of E. coli suspension at 20 x 10 4 cells / ml and incubated under the same conditions for 45 min. The glass is then rinsed again three times in the glass, fixed with methanol and stained by the Romanovsky-Grimza method. The phagocytosis index (FI) is determined by the formula:

FI = (O + 2.5n + 6t + 9p + 10R) / cell number, (1) where O is the number of cells not phagocytosed according to E. Coli, n is the number of cells losing 1-4 cells, t is the number of cells , losing 5-7 cells, p = number of cells losing 8-9 cells,

R is the number of cells losing 10 or more E. coli cells.

Macrophage identification is performed by the color of non-specific esterase.

example

Immunomodulatory activity of BAM obtained as in Examples 1-18 is determined by the activity of wistar rat rat blood neutrophils in in vitro assays, by enzymatic activity by nitro tetrazolium blue reduction to diformazan (NST assay) and by phagocytic activity against E. coli.

Rat tail artery blood is collected in tubes with colorless Chenx-heparin solution and mixed. To the resulting mixture was added 6% dextran T-500 at a ratio of 3: 1 and tubes for 1 hour. placed in the refrigerator.

After sedimentation of the erythrocytes, the supernatant leukocyte layer is pipetted and centrifuged at 250 g for 15 min. Erythrocytes are removed from the leukocyte mass by hemolysis - the leukocyte residue is suspended in distilled water, Chenx's solution is added and centrifuged. The leukocyte residue is further suspended in fresh Chenx's solution and centrifuged under the same conditions. The leukocytes are washed 3 times.

The enzymatic activity of blood neutrophils is determined spectrophotometrically by the reduction of nitro tetrazole blue to dipomarzan.

The rat erythrocyte residue is diluted in colorless Chenx solution to a concentration of 80 x 10 4 cells / ml. Add 100 ml to 100 ml of cell suspension. BAM obtained according to Examples 1-2 having a pH of 7.4 and a peptide concentration of up to 1.0 mg / ml. The mixture was incubated for 30 min. Under constant shaking in a water bath at 37 ° C, add 400 μalo of Reanal nitro tetrazolium blue solution and proceed as in Example 29.

The phagocytic activity of the rat blood is determined by their ability to occupy E. coli.

To 5 μΐ leukocyte suspension was added 50 μΐ BAM, pH 7.4, peptide concentration 0.05 mg / ml. The mixture was incubated, followed by addition of 10 mL of colorless Chenx solution at 4 ° C and centrifugation at 150-200 g for 15 min. The leukocyte residue is diluted in 0.3 ml of Chenx's solution and 0.05 ml of E. coli suspension is added at a concentration of 500 x 10 4 cells / ml, followed by incubation for 30 min. At 37 ° C. After incubation, smears are prepared in which the number of phagocytic cells and the phagocytosis index after staining are determined according to Example 29.

example

Immunomodulatory activity of BAM obtained in the same manner as in Examples 1-18 was determined by in vivo assays in BALB / C mice.

Animals are given two subcutaneous injections of perperate at 0.5 ml / kg at a peptide concentration of 1.0 mg / ml every two days. The immunomodulatory properties are analyzed two days after the last injection. At least 10 animals are used per test. Animals used for control were treated with 0.08% preservative dissolved in saline according to the given scheme.

Phytohemagglutinin, a E. coli lipopolysaccharide of B cell mitogen, is used to determine the lymphocyte blast transformation reaction. Lymphocytes isolated from mouse germ are incubated with the given mitogens for 72 hours. At 37 ° C in an atmosphere of 5% carbonic acid. Cell proliferation levels are determined radiometrically by the amount of ^ Htimidine on. Therefore, H-thymidine (3.7x10 Bk / sample) is added to the cultures and incubated for 3 hours. The samples are then placed on millipore filters, washed three times with 199 medium, followed by 5% trichloroacetic acid solution and ethanol. The filters are dried, placed in vials with scintillation solution and the Mark 111 "counter for radioactivity.

example

Immunomodulatory efficacy of BAM obtained in the same manner as in Examples 1-18 was determined in mouse assays and in vivo by stimulation of the natural killer activity of splenocytes. Animals are dosed with two subcutaneous injections of 0.05 ml / kg and peptide concentrations of 1.0 mg / ml for two days.

Immunomodulatory properties are analyzed one day after injection. At least 10 animals are used per test. Animals used for control are treated with 0.08% preservative diluted in saline according to the above scheme. The natural killer activity of splenocytes is determined in murine lymphoma cells labeled with the chromium isotope ^ Cr. Incubation is performed for 18 hours at a cell target to cell effector ratio of 1:25 (1x10 ^: 2.5x10 cells).

The specific chromium isotope release corresponding to the cytotoxic activity of effector cells is calculated using the formula:

Cr = [(experimentally released) - (sustained release) / (maximum release) - (steady release)] x 100 (2) where maximum release is the radioactivity at which all cells are lysed. A preservative dissolved in saline is used to determine the natural activity of the killer.

example

Immunomodulatory activity of BAM obtained in the same manner as in Examples 1-18 is determined by the mouse thymus functional activity in in vivo assays similar to those in Example 31.

The effect is determined by the Thyroid Serum Factor (LSF) titer.

The test mice serum is ultrafiltered with CENTRIFLOCF - 50A. The reaction uses 0.1 ml of condensed and diluted serum in 199 media. Controlled in 199 media.

The germ of mice is harvested 10-14 days after thymectotirovation. Serum-harvested cells are prepared by needling the tissue in Medium 199. Filtration through a capron mesh is followed by 2 washes in Medium 199, centrifugation in the medium to a concentration of 4 x 10 ^ cells / ml, and the resulting 0.1 ml suspension is added to serum and 199 media. The contents of the tubes were mixed and incubated at 37 ° C for 60 min.

After incubation, 0.1 ml of anti-thymocytic serum and 1:10 dilution of guinea-pig complement are added to each tube. The suspension was further incubated at 37 ° C for 30 min. 0.1 ml of ram's erythrocyte suspension (12 x 10 4 cells / ml) is then added to the mixture, centrifuged at 250 g for 5 min. and incubated for 60 min. At 4-8 ° C. Residue in tubes for 5 min. Slots are counted in the Goriajav cell. A nest is a lymphocyte with 4 or more erythrocytes attached.

USF titer is considered the last dilution of serum that induces 50% cell lysis reduction (LSR) as compared to control. The results are reflected in the logarithm of the titre with 2 basis, i.e. log A, where A is the titre value.

example

BAM received as well as 1-18 samples of rat liver hepatocytes reparative strengthening efficiency of subcutaneous introduction of CC1 ₄ is determined by the Na, K - -ATPase activity of ATP-restoration in this manner.

Male rats weighing 230-250 g daily are injected subcutaneously with CCl in a mixture of 1: 1, 4.0 ml / kg vegetable oil. 4 hours later after the first injection of CCI4, test animals are given the first of 4 Subcutaneous injections of 0.05 ml / kg / day. The efficacy of the substance is recorded the day after the last injection.

Na K-ATF -ase activity is determined using hepatocyte cell membrane fractions. 15 g of chilled liver of 3 rats is homogenized in 150 g of 1 m M pH 7.5 with 0.5 mM CaCl 2 using a glass homogenizer.

The homogenized mixture was centrifuged at 150 g at 4 ° C for 10-12 min. The supernatant is centrifuged again at 100,000 g for 20 minutes, and the resulting residue is washed 3 times in 130 ml buffer under the same centrifugation conditions.

After washing, the residue is resuspended in buffer solution to a protein concentration of 3.0 mg / ml by SDS - Louri and used for assays. All operations to obtain the membrane fraction are performed in the cold.

Well, membrane plasma activity of K-ATF-assay hepatocytes is determined in standard medium from 66 mM NaCl, 34 mM KCl, 5 mM MgCl 2, to which 5 mM ATF is added prior to assay.

To 1.8 ml of the indicated medium, add 0.2 ml of membrane fraction suspension with a protein concentration of 3.0 mg / ml and incubate at 37 ° C for 15 minutes, then stop the reaction by adding 0.6 ml of 5% trichloroacetic acid solution. . The mixture was centrifuged at 300-500 g for 15 min. To 1.0 ml of the resulting supernatant was added 4 ml of 0.2 M acetate buffer pH = 4.0 0.5 ml of 1% ammonium T molybdate solution in 1% H2SO4, 0.5 ml of ascorbic acid dissolved in 16% CuSO4. in solution and after mixing, incubate for 10 min. At 25 ° C. Optical density is determined spectrometrically at 700 nm.

Well, K-ATFase activity is calculated from the difference between total ATFase activity and Mg-ATFase activity. The activity of Mg-ATFase is determined analogously by adding 0.3 ml of 0.05% strophanthine solution instead of 0.6 ml of trichloroacetic acid.

It was found that the liver exposed to CCl3 Na had a 3.5-fold decrease in K-AF -ase activity and a 50% increase in BAM activity as exemplified in Example 11.

example

BAM obtained in the same manner as in Examples 1-18, the efficacy of hepatic hepatocyte reparation enhancement in rats following subcutaneous administration of CCI was determined by the recovery of blood aminotransferase activity and rat hepatic potassium (K ⁺ ) concentration in the following manner.

Animals treated as in Example 34 are tested for blood aminotransferase activity in a test system by specific staining photometrically (S.Reitman, S.Franklin. - American Journal of Clinical Pathology, 1957-18-56).

To a 0.25 mL solution of 0.1 M L-aspartate, 0.002 M 2-oxaglutarate and 0.1 M phosphate buffer, pH 7.4, 0.05 mL of rat serum is added and incubated at 37 ° C for 60 min. .

After incubation, 0.001 M 2-4-dinitrophenylhydrazine dissolved in 1 M HCl was added to the solution and the sample was added for 20 min. left at room temperature, followed by addition of 2.5 mL of 0.4 M NaOH, stirred and after 10 min. Measure the optical density at 520 nm against the control solution, replacing 0.05 ml of blood with 0.05 ml of saline.

Hepatic toxicity of CCI4 is accompanied by hepatocyte degradation and aminotransferase depletion, resulting in a 2-fold increase in serum leg activity.

Potassium (K ⁺ ) concentration in hepatitis hepatitis test rats is determined by a standard flame photometric method according to Brikker using a flame photometer.

The K-ATFase activity of the liver exposed to CCl ^, Na, was found to be 3.5-fold reduced, resulting in a 40% decrease in K ⁺ concentration in hepatic hepatitis.

And using the BAM obtained in Example 11, the K ⁺ concentration is restored by at least 43%.

The invention is not limited to these examples. Various embodiments may be used within the spirit and scope of the invention.

ANALYSIS OF THE COMPOSITION AND PROPERTIES OF THE BIOLOGICAL ACTIVE SUBSTANCE

Identification of the claimed BAM among other biologically active substances is possible directly through biochemical composition analysis and indirectly by assessing the immunomodulatory effect of BAM.

Significant enhancement of the body's immune system, compared to control, was found to be due to modified components of the morphoplasma and glycocalyx cells, which are modified in BAM. This allows BAM to be identified by the following indicators:

(a) Increased macrophage activity in rat peritoneal exudate, ie at least 54% increase in macrophage enzymatic activity as determined by NST assay (using nitrotetrazole blue in the experiment - Hungarian Reanal NST assay) and E. coli phagocytic activity not less than 39%;

b) Based on an increase in rat neutrophil activation of blood, ie at least 67% increase in enzymatic activity as determined by NST assay (using nitrotetrazol blue - NST assay by Hungarian firm Reanal) and at least 44% increase in E. coli phagocytic activity %;

(c) Not less than 42% for T-lymphocyte blast transformation and not less than 50% for Lymphocytes;

d) an increase in thymic gland functional activity, i.e., an increase in serum thymine serum factor of at least 167%;

(e) Not less than 66% based on an increase in the natural killer activity of rat splenocytes;

(f) In terms of capacity to stabilize plasma membranes of rat hepatocytes following administration of carbon tetrachloride (CCI4):

- at least 24% according to recovery of Na, K-ATF -ase activity;

- at least 22% based on the recovery of transferase activity in rat blood;

- Not less than 13.8% based on the ratio of potassium (K) to sodium (Na) in rat hepatocytes.

Based on comparisons, a control preservative dissolved in saline was used in controls.

The present system of BAM immunomodulatory and regenerative effects (properties) allows BAM to be identified by its biological activity, i.e., as an immunomodulator. BAM and its apparatus have a tendency to act as an enhancer of the body's immune function, directed at both elimination of internal pathology (stimulation of natural killer activity) and enhancement of the body's overall resistance to infections, well above the known levels of BAM described above.

Immunomodulatory properties of various biologically active substances have been experimentally determined by a set of indices that identify the test substances. The results of the experiments are shown in Table 6, which gives quantitative effects of known BAM N 1, N 2, N 3 obtained by the technology described in EP-A-02499563, SU-A-139404 and US-A-4455302, respectively, and claimed by BAM. represented by WS, TS and LM groups summarizing the results:

WS - Examples 1-4 (morphoplasmic and glycocalyx cell components);

TS - Examples 7-9 (thermostable components of morphoplasma and glycocalyx cells);

LM - 11-13 (low molecular weight components of morphoplasma and glycocalyx cells with M <10.0 kDa).

A saline solution with a preservative was used as a control subject. The results obtained are given in Table 6 and characterize the expressed and known BAM by comparison. The presented results describe the characteristics of the claimed BAM, which is obtained as a mixture of an unidentified structure, and the data necessary for the identification of the BAM, which determines its purpose.

In addition to the ability to identify the candidate material, the analysis of the data in Table 6 also allows the evaluation of the specific result obtained with BAM, which is a significant increase in biological activity due to BAM hydrolysis products containing modified morphoplasmic and glycocalyx cells with modified structure unspecified.

The claimed properties of BAM and its formulation are closely related to its composition.

According to the results of biochemical analysis (Table 7), BAM contains organic compounds such as polypeptides, peptides, amino acids, bound and free carbohydrates, lipids, nucleotides and other compounds. The data in Table 7 reveal the weight distribution of organic compounds in BAM using raw horn embryo tissue as the starting material. In addition, Table 7 reflects the results of obtaining the claimed BAM for the different groups (WS, TS, and LM), for all claimed pathways. In addition to the results of the method for producing BAM-WS, which utilizes animal tissues in the morphoplasmic and glycocalyx cell modification process as a starting material, Table 7 also states BAM-ST and BAM-LM thermostable components of BAM production. results of the implementation of the method

the data in the table allow for a comparative analysis of the methods of obtaining BAM expressed and known, namely, comparing the known and claimed BAM, bearing in mind that BAM N 3 (US-A-4455302), BAM N 2 ( SU-A139404) and BAM N 1 (EP-A-0249563).

Comparative analysis of the specific and known BAM specific activity shows (Tables 6 and 7) that their specific activity depends on the composition of the ingredients, the ratio of which significantly influences the amount of specific activity.

A characteristic feature of the claimed biochemical composition of BAM is the presence of carbohydrate-containing components, the modification of which significantly determines the properties of BAM.

The carbohydrate content of the most active fractions of the low molecular weight components with molecular weight M = 0.8-10.0 kDa (oligonucleotides, glycopeptides, glycolipids, nucleotides) in Table 3 of Table 3) is crucial for the identification and characterization of the expressed BAM.

Comparative analysis of the specific activity and composition of the ingredients (Tables 6 and 7) shows that the higher BAM activity compared to its counterparts correlates with the higher carbohydrate content of components with M = 0.8-10.0 kDa. For the material declared, these values are: 6.8 ± 3.5%; 7.5 ± 3.5% and 8.2 ± 3.3%, while the BAM N 1, N 2 and N 3 analogues are not more than 0.2%, 2.7 ± 1.1% and 3 ± 0.5%, respectively. Even better, this dependence correlates with the proportion of carbohydrates relative to the amount of other organic matter in the fraction.

In this respect, the ratio of carbohydrate to peptide content of components with molecular weight M = 0.8-10.0 kDa (Tables 2 and 3), expressed as formula (3), is an essential indicator:

C _y

G = C _P (3) where C _y and C _p are carbohydrate and peptide concentrations, respectively, in the components of molecular weight M = 0.8-10.0 kDa.

Reportedly, the BAM has a value of G3 of 0.25-0.60, whereas for known BAMs its value does not exceed G _ž <0.25.

Thus, the value of G is a generalized characteristic of the hydrolysis products and depends on the raw material used in the claimed process, on the specified set of feedstock processing operations and on the mode of implementation of the hydrolysis processes and is one of the characteristic parameters of BAM biochemical identification.

The totality of the properties of the claimed biologically active substance and the general idea of the claimed route of preparation and the medicinal product based on this substance are related to the specific activity of BAM.

INDUSTRIAL APPLICABILITY

Inventions relating to a biologically active substance, its method of preparation and its pharmaceutical formulation are based on the selection of the starting material, the process conditions for the preparation of the BAM and the preparation with a guaranteed high specific activity. The selected BAM production technology ensures repeatability of results, i.e., allows the standardization of the expressed BAM with the desired properties, namely, the desired fixed immunomodulatory activity that determines the pharmaceutical, therapeutic and general biological effects of the invention.

The resulting end products, in general and in individual embodiments of the claimed route of production of BAM, are equivalent in their biological activity profile but differ in their specific activity. Therefore, the suitability of one or another particular BAM is determined by the requirements of the object for which it is intended, i.e. veterinary and / or medical requirements. In this regard, the choice between one or the other BAM variant for each specific end use of the product is determined by the relationship between the requirements for its immunomodulatory activity and overall efficacy and the cost (cost effectiveness). The high efficiency of the product containing the expressed low molecular weight BAM-LM component with molecular weight M <10 kDa is fully manifested in the enhancement of macrophage, neutrophil, lymphocyte, natural killer activity, which enhances the regenerative processes (Table 6). In domestic animals, these effects are accompanied by an increase in extra weight during intensive fattening (Table 8). In the treatment of respiratory and infectious diseases (para-influenza), such an advantage is less important and in this case it is economically feasible to use an agent containing water-soluble morphoplasmic and glycocalyx cellular components BAM50

WS. For the treatment of inflammatory processes (mastitis), it is preferable to use an agent containing BAM-TS thermostable components.

In the manufacture of a preparation for external use in the form of an ointment, suppositories, flowers, compresses, intranasal or animal watering solution, it is economically feasible to use BAM containing BAM-ST thermostable components instead of BAM-LM.

For maximum effect, it is more appropriate to use a candidate material to stimulate the immune function of the body, both to eliminate internal pathology (stimulation of natural killer activity) and to enhance overall body resistance to infectious diseases, far beyond the analogous levels of known BAMs discussed above.

The use of the claimed BAM for the treatment of infectious diseases of the large horned adult (para-influenza, acute respiratory distress of the calves) provides animal recovery in 82-93% of cases. Meanwhile, with known BAM, recovery under the same conditions is observed in up to 75% of cases, and spontaneous recovery in the control group is within 55-67% of cases.

The claimed preparation gave positive results when administered to the body for the treatment of inflammatory processes.

Prophylactic use of BAM reduced the incidence of para-influenza in calves by up to 10%, compared with 12-17% in animals using known means and 2228% in the control group.

The use of the claimed preparation increased the resistance of the animals to infectious diseases while reducing the drop in the herd.

The normalization of the general biological condition of livestock by means of the preparation has improved the production indices, including the increase of the live weight without compromising the quality of the meat, bringing these indices closer to the genetically programmed level possible with intensive fattening. This property advantageously distinguishes the pronounced BAM and preparations based thereon from hormonal and other growth stimulants which, in quantitative terms, adversely affect homeostasis and impair the quality of production.

Intravenous administration of the expressed BAM and preparations based thereon on an animal with abnormal homeostatic parameters resulted in additional weight gain of approximately 37-100% on average in the untreated BAM control group. Meanwhile, using the known BAM analogues (N 1, N 2 and N 3) under the same conditions, the additional weight gain compared to the weight in the control group was on average 12-24%.

Normalization of homeostatic parameters also increases:

- milk yield of coarse and fine horns;

- poultry production;

- the quality of the fur of fur animals;

- survival of the adolescent;

- amount of honey harvested, etc.

The stated BAM is non-toxic and does not cause side effects (allergies, etc.).

Comparative data on the pharmaceutical, therapeutic and general biological effects of the claimed BAM and its formulation are presented in Table 8. Analysis of the data in this table shows that the biological activity of the claimed BAM is significantly higher than that of the analogs.

Thus, BAM is expressed and has a broad range of veterinary and medical uses and can be used to normalize the physiological state of the body in the treatment of disorders controlled by the body's immune system.

According to the invention, the claimed material and the preparation thereof are used in the following manner. Adjusts homeostatic parameters, then, if abnormal, stimulates the immune system by injecting BAM until the indicated homeostatic parameters return to normal.

The above immune system stimulation is accomplished by activation of the reticuloendothelial system, T- and B-systems, neutrophils and / or the natural killer population, which results in increased peritoneal exudate and blood neutrophil macrophage activity in vitro, increased lymphocyte blast-transformation response in vivo, thymus. an increase in the stimulation of functional activity, an increase in the stimulation of the natural killer activity of splenocytes in vivo, as well as an increase in hepatic hepatocyte repair following the administration of toxic substances or after partial hepatectomy and the like.

For this purpose, the route of administration of the claimed BAM and the formulation based on it shall be selected in the order of:

- intramuscular injection at a dose of 0.005-0.5 ml / kg body weight at intervals of 1-7 days;

- inhalation doses of 0.012-0.12 ml / kg body weight at intervals of 4-48 hours between inhalations;

- sublingually, at doses of 0.012-0.12 ml / kg body weight at intervals of 4-48 hours;

- watering at doses of 0.02-1.0 ml / kg body weight at intervals of 1 to 10 days;

- orally at a dose of 0.02-1.0 ml / kg body weight at intervals of 1-10 days;

- intranasally at daily doses of 0.001-0.05 ml / kg body weight at intervals of 2-24 hours;

- applications on the mucous membrane or wound surface in the form of compresses, suppositories, lighteners, ointments, as well as flowers, or combinations thereof until normalization of homeostatic parameters.

Data demonstrating the efficacy of the claimed BAM thermostable components in the form of BAM-ST and in the formulation thereof in which we used NaCl saline as filler are presented in Tables 9-12.

The tables show the effect of the normalization effect of homeostatic parameters on the above doses and the route of administration, which was controlled by the size of the calves that initially had abnormalities in the homeostatic parameters. Tables 9-12 present the efficacy results of the claimed preparation,

- Intramuscular injection every 3 days (Table 9);

- by inhalation every 24 hours (Table 10);

- watered every 7 days (Table 11);

- intranasally every 12 hours. (Table 12).

Administration of the claimed BAM in the form of a claimed preparation stimulates:

- treatment of infectious diseases (para-influenza, acute respiratory disease);

- treatment of inflammatory conditions (mastitis, pneumonia, endometritis);

- resistance of animals to infectious and inflammatory processes;

- improvement of the general biological state of the body;

- weight gain in livestock;

- hepatic function in hepatitis;

- liver regeneration after partial hepatectomy;

- recovery of blood formula after bleeding.

The absence of side effects from the use of BAM and its preparation and data on the pharmaceutical, therapeutic and general effects on the body of the animal made possible the clinical approval of BAM and its preparation. Positive effects on the physiological state of the body were found in a group of patients, as well as a practically complete analogy with the results of experimental animal preclinical studies.

The method of obtaining the claimed biologically active substance can be implemented under both industrial and laboratory conditions.

-55LT4102 B

Table

Immunomodulatory and regenerative effects of biologically active substance (BAM) Quantification of BAM effects (by examples) N 1 N2 N3 N4 1. Peritoneal activity of rat exudate macrophages enzymatic activity (NST test) 52.3 54.2 71.0 58.3 % increase compared to control phagocytic activity on productivity in E.Coli 37.3 39.1 54.5 43.8 2. Increase in blood neutrophil activity in%, enzymatic activity (NST test) 66.7 71.2 85.0 75.4 compared to the control phagocytic activity by E.Coli 44.5 49.8 59.8 52.2 3. Blast transformations T - lymphocytes 40.2 44.8 51.3 46.2 response stimulation% compared to control B - lymphocytes 48.7 50.2 61.0 55.2 4. Increase in germline thymic factor titer in% compared to control 156.3 167.4 249.8 210.4 5. Stimulation of natural killer activity of rat splenocytes in% relative to control. 65.7 66.1 78.5 72.3 6. Reproductive potential of rat hepatocytes Well, K - ATF-assays 20.1 24.3 29.1 26.7 % increase in activity recovery after CCl administration, of blood aminotransferase 18.7 22.1 32.3 27.4 compared to the control. K ⁺ concentration in hepatocytes 10.3 13.8 19.8 16.8

-56LT 4102 B

Continuation of the table

Quantification of BAM Effects (by Examples)

Immunomodulatory and regenerative effects of biologically active substance (BAM)

N7 N8 N9 N 11 N 12 N 13 1. Peritoneal activity of rat exudate macrophages enzymatic activity (NST test) 102.6 73.0 87.6 158.2 127.0 151.1 % increase compared to control phagocytic activity according to E.Colli 73.9 54.7 64.1 124.7 116.1 122.4 2. Increase in blood neutrophil activity in%, enzymatic activity (NST test) 109.2 81.6 95.4 179.5 138.3 167.6 compared to the control phagocytic activity according to E.Colli 78.9 61.9 60.2 134.3 112.9 125.4 3. Blast transformations T - lymphocytes 79.7 59.3 69.2 149.3 120.3 141.3 response stimulation% compared to control B - lymphocytes 93.3 71.9 83.4 189.6 146.8 176.2 4. Increase in germline thymic factor titer in% compared to control 364.3 273.1 307.4 706.0 543.8 650.8 5. Stimulation of natural killer activity of rat splenocytes in% relative to control. 120.6 90.0 105.0 211.0 178.4 194.4 6. Reproductive potential of rat hepatocytes Well, K - ATF-assays 34.6 28.2 30.8 64.8 50.4 60.2 % increase in activity recovery after CCl administration, of blood aminotransferase 53.6 38.8 45.2 82.0 59.2 75.6 compared to the control. K ⁺ concentration in hepatocytes 27.0 22.2 24.6 52.5 43.9 50.4

-572 Table

Organic compounds % By weight of organic compounds obtained as examples and present in BAM N 1 N2 N3 N4 Polypeptides 26.0 16.3 21.8 20.5 Peptides 10.0 11.6 20.0 11.9 Amino acids 51.5 48.2 32.0 44.3 Carbohydrates containing: - high molecular weight compounds with molecular weight M> 10,0kDa 2.0 0.3 1.1 0.6 - low molecular weight compounds with molecular weight M = 0.8h-10.0 kDa 2.5 3.3 10.3 5.0 - free monomers or compounds with a molecular weight M <0.8 kDa 6.0 11.0 9.4 12.0 ALTOGETHER: 10.0 14.6 20.8 18.2 Lipids, nucleotides and other organic compounds 2.0 9.3 5.4 5.1 TOTAL: 100.0 100.0 100.0 100.0 Carbohydrate to peptide ratio G 0.25 0.28 0.52 0.42

-582 Continuation of Table

G is a coefficient characterizing the ratio of carbohydrates to molecular weight M = 0.8-? 10.0 in relation to the amount of peptides.

Organic compounds % By weight of organic compounds obtained as examples and present in BAM N7 N8 N9 N 11 N 12 N 13 less Polypeptides 11.0 5.5 12.7 0.2 0.3 0.3 Peptides 21.0 13.6 18.6 19.1 14.9 22.5 Amino acids 37.1 52.9 47.3 43.5 60.1 49.8 Carbohydrates containing: - high molecular weight compounds with molecular weight M> 10.0 kDa 0.6 0.2 0.4 0.2 0.2 0.2 - Low molecular weight compounds with M = 0.84-10.0 kDa 11.0 4.0 7.4 11.5 4.9 11.0 - free monomers or compounds with a molecular weight M <0.8 kDa 13.5 12.8 8.3 17.0 10.4 11.2 ALTOGETHER: 25.1 17.0 16.1 28.7 15.5 22.4 Lipids, nucleotides and other organic compounds 5.8 11.0 5.3 8.5 9.2 5.0 TOTAL: 100.0 100.0 100.0 100.0 100.0 100.0 Carbohydrate to peptide ratio G 0.52 0.30 0.40 0.60 0.33 0.50

-593 Table

Heating temperature (heat treatment) ° C Rat peritoneal exudate macrophage activity (in vitro) obtained with BAM expressed at different temperatures (%) relative to control. Increase in enzymatic activity (NST test) Increase in phagocytic activity by E.Coli 55 61.1 ± 2.2 52.3 ± 2.8 60 65.7 ± 3.8 55.1 ± 2.1 95 72.8 ± 4.8 64.3 ± 5.6 120 64.3 ± 5.1 58.3 ± 4.2 135 57.3 ± 5.3 43.0 ± 3.4

Differences with control were correct at P <0.05

Table

Order no. Molecular weight of fraction, M, kDa Macrophage activity of rat peritoneal exudate, expressed by action of agent BAM in low molecular weight fractions (%) versus control 1 Me> 50.0 52.1 ± 4.8 2 10.0 <M ₂ <50.0 93.8 ± 7.6 3 M3 <10.0 148.4 ± 17.4 4 M4 <5.0 160.7 ± 15.1 5 M ₅ <1.0 120.5 ± 11.7 6th Mg <0.8 87.3 ± 8.7 7th M ₇ <0.5 53.4 ± 7.2

Differences with control were correct at P <0.05.

-605 Table

Animal tissues are used Rat peritoneal exudate macrophage activity (in vitro) against BAM-TS obtained from various substances (%) compared to control Increase in enzymatic activity (NST test) Increase in phagocytic activity by E.Colli Hepatocytes of intact liver 32.2 ± 5.4 30.8 ± 2.7 Intact muscular tissue of adult animals 43.6 ± 6.8 35.2 ± 2.6 Testicular spermatogonia and spermatocytes Intestinal epithelial tissue 63.8 ± 3.5 55.4 ± 3.2 Liver hepatocytes after CClas input 68.2 ± 6.3 61.3 ± 5.2 Bone marrow cells Reconstructive tissue of amphibian limbs * 72.1 ± 4.2 64.4 ± 4.3 Embryonic muscle 74.2 ± 6.1 67.5 ± 4.1 cloth 82.5 70.2 Rat hepatocytes regenerated in rats after 87.3 77.4 hepatectomies 83.7 ± 5.2 73.6 ± 4.8 94.8 ± 5.7 78.8 ± 5.6

Differences from control were correct at P <0.05.

* Tissue with characteristics obtained from a specific experiment.

-61 Table 6

Immunomodulatory and regenerative effects of biologically active substance (BAM) Quantity of effects (by examples) Known BAM BAM would claim N 1 N2 N3 WS TS LM 1. Peritoneal activity of rat exudate macrophages increase in% relative to control enzymatic activity (NST test) 27.1 ± 2.6 42.2 ± 2.4 46.8 ± 4.3 62.6 ± 8.4 87.8 ± 4.8 142.6 ± 15.6 phagocytic activity according to E.Colli 15.6 ± 2.1 21.4 ± 4.2 33.4 ± 4.3 46.8 ± 7.7 64.3 ± 9.6 120.4 ± 4.3 2. Increase in blood neutrophil activity in%, compared to controls enzymatic activity (NST test) 33.8 ± 5.9 60.6 ± 3.7 67.2 ± 3.8 76.2 ± 8.8 95.4 ± 13.8 158.9 ± 20.6 phagocytic activity according to E.Colli 25.4 ± 1.6 34.6 ± 3.7 44.2 ± 5.7 52.3 ± 5.7 70.4 ± 8.5 123.6 ± 10.7 3.Blast transformations response stimulation% versus control T - lymphocytes 24.6 ± 2.8 25.1 ± 3.7 38.4 ± 4.6 46.6 ± 4.7 69.5 ± 10.2 134.8 ± 14.5 B - lymphocytes 22.1 ± 3.2 28.8 ± 2.4 46.3 ± 4.7 55.6 ± 5.4 82.6 ± 10.7 168.2 ± 21.4 4.% increase in germline thymic factor titer compared to control 52.4 ± 4.2 56.7 ± 6.2 117.8 ± 9.1 208.6 ± 41.2 318.7 ± 45.6 624.7 ± 81.3 5. Stimulation of natural killer activity of rat splenocytes in% relative to control. 65.6 ± 4.8 17.6 ± 4.2 53.8 ± 5.4 72.3 ± 6.2 105.3 ± 15.3 194.7 ± 16.3 6. Reproductive potential of rat hepatocides % increase in activity recovery after CCl administration, compared to controls. Well, K - ATF-assays 0 13.2 ± 3.1 22.6 ± 3.4 26.7 ± 2.4 31.4 ± 3.2 57.6 ± 7.2 of blood aminotransferase 0 0 23.4 ± 4.8 27.2 ± 5.1 46.2 ± 7.4 70.6 ± 11.4 K ⁺ concentrations in hepatocytes 0 5.2 ± 1.2 14.2 3.1 16.8 ± 3.0 24.6 ± 2.4 48.2 ± 4.3

The difference with control is correct at P> 0.05.

-627 Table

Organic compounds Content of BAM in organic compounds In the known In the pleading N 1 N2 N3 WS TS LM Polypeptides 72.7 47.7 32.1 20.0 9.1 less ± 9.4 ± 8.7 ± 6.1 ± 3.5 ± 3.6 0.3 Peptides 13.5 35.8 16.3 15.0 17.3 18.7 ± 2.1 ± 7.4 ± 3.2 ± 5.0 ± 3.7 ± 3.8 Amino acids 4.7 3.7 31.7 40.1 45.0 51.8 ± 1.7 ± 0.8 ± 6.2 ± 8.1 ± 7.9 ± 8.3 ALTOGETHER: 90.9 86.9 80.1 75.1 71.4 70.5 ± 6.0 ± 5.1 ± 5.1 ± 5.1 ± 4.8 ± 5.3 Carbohydrates containing: - high molecular weight 0.5 ± 0.2 0.9 ± 0.2 1.9 ± 0.8 0.7 ± 0.4 0.4 ± 0.2 compounds with molecular weight M> 10.0 kDa  i u * 0.2 - low molecular weight compounds with molecular weight less 2.7 3.0 6.8 7.5 8.2 M = 0.8-H0.0 kDa 0.2 ± 1.1 ± 0.5 ± 3.5 ± 3.5 ± 3.3 - of free monomers or of compounds with molecular weight M <0.8 kDa 7.3 ± 2.4 2.1 ± 0.6 10.2 '± 2.7 10.0 ± 2.4 12.7 ± 2.9 13.7 ± 3.5 ALTOGETHER: 8.0 5.7 14.9 17.7 20.6 22.2 ± 1.8 ± 1.6 ± 2.7 ± 3.1 ± 4.5 ± 6.6 Lipids, nucleotides, and others 1.1 7.4 5.0 7.2 8.0 7.1 organic compounds ± 0.3 ± 2.1 ± 1.6 ± 2.1 ± 2.7 ± 2.1 TOTAL: 100.0 100.0 100.0 100.0 100.0 100.0 Carbohydrate to Peptide Ratio G * <0.01 0.08 0.18 0.45 0.43 0.44 BAM weight of dry residue 20.0 11.4 32.6 12.8 8.4 6.1 (mg / ml) ± 4.2 ± 3.7 ± 9.6 ± 3.6 ± 2.2 ± 1.8 Antiseptic weight (mg / ml) 0.7 0.7 0.7 BAM concentration in powder - - - 94.8 92.3 89.7 in composition, by weight% ± 1.1 ± 1.5 ± 2.2

The difference with the control is correct at P <0.05 * Values were calculated from statistical values from the table.

Table -638

Pharmaceutical, therapeutic and total biological effect- using a biologically active substance (BAM) Quantitative effect characteristic Of course BAM Pronounced BAM N1 N2 N3 WS ST LM 1. Inflammatory processes Cows mast this, good luck Kim % Ban- diction Con- troll 56 ± 8 59 ± 7 66 + 6 * 57 + 6 70 ± 5 61 ± 5 76 ± 3 58 ± 8 83 ± 4 63 + 7 88 ± 5 53 ± 8 Lung chicks closed birth, good luck Kim % Ban- diction Con- troll 55 ± 8 58 ± 7 64 + 7 * 60 + 7 69 + 6 55 + 7 78 + 5 51 ± 8 89 ± 6 61 ± 6 91 ± 5 48 ± 6 2. Infectious diseases Para influenza and severe respiratory disease, recovery% Ban- diction Con- troll 55 ± 5 * 60 ± 5 65 ± 7 62 ± 6 70 ± 6 64 ± 7 82 ± 6 58 ± 6 90 ± 7 61 ± 7 93 ± 5 67 + 5 3.% increase in overweight of calves compared to control -12 ± * 10th 15 ± 6 24 ± 7 37 + 7 58 ± 8 102 ± 15th 4. Acute toxicity LU5q, ml / mg (mg / kg) body weight more on 25 (260) more on 25 (260)

The difference with control is correct at P <0.05 * P> 0.05

Table -649

Effectiveness of intramuscular injection Amount ml / kg body weight less 0.005 (0.001) 0.005 0.05 0.5 more 0.5 (1.0) Growth,% as compared to control 32 ± 2 47 ± 2 55 ± 5 61 ± 5 42 ± 3

The difference with control is correct at P <0.05

Effectiveness of inhalations Amount ml / kg body weight 0.001 0.012 0.05 0.12 0.6 Growth,% as compared to control 32 ± 2 47 ± 2 55 ± 5 61 ± 5 42 ± 3

The difference with control is correct at P <0.05

Efficiency of watering Amount ml / kg body weight 0.001 0.01 0.05 0.5 2.0 Growth,% as compared to control 10 ± 4 32 ± 3 35 ± 3 42 ± 3 30 ± 4

The difference with control is correct at P <0.05

Efficacy when administered intranasally Amount ml / kg body weight 0.0005 0.001 0.005 0.05 5.0 Growth,% as compared to control 3 ± 1 12 ± 2 17 ± 3 20 ± 4 8 ± 2

The difference with control is correct at P <0.05

Claims (20)

DEFINITION OF INVENTION 1. A biologically active substance having an immunomodulatory property of water-soluble organic compound hydrolysis products of crushed animal tissue ingredients, characterized in that the hydrolysis products contain thermostable low molecular weight morphoplasmic and glycocalyx cellular components, modified by embryogenesis and / or proliferation, and / or and / or pathogenesis prior to and / or accompanied by regeneration and / or tissue repair. 2. The material of claim 1, wherein the modified morphoplasmic and glycocalyx cellular components contain thermostable Tjj <120 ° C. 3. Material according to claim 2, characterized in that it contains 30-100% by weight of thermostable components. 4. A material according to claim 1, wherein the weight of the low molecular weight components of the modified morphoplasma and glycocalyx cells is M <10.0 kDa. 5. The material of claim 4, wherein the low molecular weight component is present in an amount of 30-100% by weight. Substance according to Claim 1 or 2, or 3, or 4, or 5, characterized in that its hydrolysis products with modified morphoplasmic and glycocalyx cellular components contain% by weight of organic compounds. Polypeptides up to 26.0 Peptides 10.0-22.5 Amino acids 32.0-60.1 Carbohydrates - 10.5-28.7 Lipids, nucleotides and other organic compounds - 2.0-11.0 7. A substance according to claim 6, wherein the organic compounds contain carbohydrate by weight (%) - for high molecular weight compounds having a molecular weight My ^> 10.0 kDa to 2.0 - for low molecular weight compounds having a molecular weight My _n = 0,8-10,0 kDa-2,5,5,5,5 - 6.0-17.0 for free monomers Μ ^ Ο, δ kDa 8. A process for the preparation of a biologically active substance having an immunomodulatory property, comprising digesting the washed raw material from animal tissue, hydrolysis of the resulting ingredients, filtration of the hydrolysis products and isolation of the supernatant as the final product using animal tissue, modified embryogenesis and / or during cell proliferation processes and / or cell differentiation and / or pathology, before and / or accompanying regeneration and / or tissue repair, and prior to hydrolysis homogenize and purify non-disintegrating tissue elements, secrete a supematant containing morphoplasmic and glycocalyx cells. 9. The method of claim 8, wherein the raw material utilizes tissue selected from the group consisting of embryonic tissue, differentiated thymic thymocyte tissue, testicular spermatogonia and spermatocytes, bone marrow cells, epithelial intestinal tissue, placenta, reconstituted liver tissue. tissue, amphibian recovery tissue, pathologically abnormal tissue taken prior to regeneration, or any combination thereof. 10. The method of claim 8, wherein the intact tissue elements are homogenized in saline and / or buffer after purification by filtration and / or centrifugation at a rate of 150-250 g per 10 min. 11. The method of claim 8, wherein, prior to homogenization, the raw material is shredded from animal tissue to complete cell lysis, followed by extraction, additional washing, filtration and / or centrifugation at a rate of 300-15000 g for 10-20 min. to secrete extruded crushed tissue. 12. A process according to claim 8 or 9, or 10 or 11, wherein the hydrolysable ingredients contain 30-60% by weight of the organic compounds of the morphoplasma and glycocalyx cellular components. 13. A process according to claim 8, wherein it hydrolyses at a temperature of Tp = 4-45 ° C. 14. A process according to claim 14, wherein the hydrolysis is carried out with a preservative. Process according to claim 8 or 9, or 10, or 11, or 13, or 14, characterized in that the ingredients of the hydrolysis products are thermally treated at Th ⁼ 60-120 ° C until complete denaturation of the thermolabile components and filtered and / or centrifuged. , at a rate of 1500 g in 20-40 min. and isolates the supematant as the final product containing the thermostable component. The process according to claim 8 or 9, or 10, or 11, or 13, or 14, wherein the hydrolysis products of the ingredients are fractionated through a semipermeable film by dialysis and / or ultrafiltration and / or gel filtration to low molecular weight components. M <10.0 kDa fraction as final product isolation. 17. A process according to claim 15, wherein the hydrolysis products of the ingredients are fractionated by dialysis through semipermeable film and / or ultrafiltration and / or gel filtration to isolate the low molecular weight M <10.0 kDa fraction as the final product. 18. A preparation comprising a biologically active substance and filler in hydrolysis products of animal tissue ingredients, characterized in that the biologically active substance contains thermostable, low molecular weight morphoplasmic and glycocalyx cell components modified by embryogenesis and / or proliferation and / or and / or pathology, before and / or accompanying regeneration and / or tissue repair, 0.001% to 85% by weight in the formulation. 19. A formulation according to claim 18, wherein the biologically active agent contains organic compounds in a weight ratio of up to 26.0. Peptides 10.0-22.5 Amino acids 32.0-60.1 Carbohydrates- 10.5-28.7 Lipids, nucleotides and other organic compounds - 2.0-11.0 A preparation according to claims 18 and 19 comprising from 0.001% to 85% of the biologically active substance and 15.0% to 99.999% of the excipient, for use in the normalization of the physiological state of humans and animals by a pharmaceutically effective amount by intramuscular and / or inhalation and / or either sublingually and / or orally and / or via pills and / or intramuscularly and / or by application.