RU2720800C1 - Agent for cattle, having anti-inflammatory, regenerative, immunomodulating, detoxification, adaptogenic activity and able to regulate metabolism - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a cattle remedy having regenerative, immunomodulating, detoxification and adaptogenic activity. Agent for cattle contains protein-peptide complex with weight less than 10 kDa, produced by ultrafiltration from conditioned medium from cultivation of mesenchymal stem cells from cattle adipose tissue after at least 3–4 passages, glycine in a concentration of ±2.5 to ±22.5 mg/cm³ as a stabilizer, a standardized method of biological control of regenerative activity on dermal fibroblasts, has regenerative, immunomodulatory, detoxification and adaptogenic activity.

EFFECT: agent described above has a complex action through regulation of the bivalent tissue elements, provides recovery of the injured structure and disturbed functions of the organs and systems.

4 cl, 6 tbl, 8 ex

Description

Technical field

The invention relates to veterinary medicine, in particular to veterinary therapy, and can be used to stimulate regenerative processes in the body of cattle, for immunocorrection, correction of metabolic disorders, for detoxification of the body in infectious diseases, food or chemical substances, inflammatory processes in the animal’s body , as well as to adapt to changing conditions.

State of the art

In the prevention and treatment of cattle diseases associated with inflammation and tissue damage, impaired immune system functions, metabolic disorders, as well as for adaptation of animals to transport stress and stress when changing the conditions of detention, various approaches are used, including highly targeted means and methods.

A known method of treating inflammatory diseases of the musculoskeletal system in cattle, including local exposure to acupuncture points, which is performed by an external therapeutic massage electrode with alternating frequencies of 77 and 10 Hz on biologically active points located in the area of the metacarpal and carpal joints, with the subsequent transition to biologically active points of the forearm, shoulder, shoulder blades and withers of the chest of the limbs, while therapy sessions are carried out daily for 11-15 minutes for 7-10 days (patent RU 2626599). This method has a very narrow focus and carries risks associated with the impact of currents on the animal.

In the prevention and treatment of postpartum complications in cows, such as endometritis, various antibiotic methods are used. However, a known method for the prevention of postpartum endometritis in cows (patent application RU 2016122507), comprising administering a bacteriophage preparation once in a dose of 30 ml intrauterine through a plastic disposable catheter after separation of the placenta. The advantage of this method is the non-use of antibiotics, however, the safety of the bacteriophage preparation for the animal and its products is not well understood.

Antibiotic drugs are also often used to prevent and treat mastitis. However, other synthetic agents are known. So, for the treatment of subclinical mastitis, as well as for the prevention of mastitis of other forms, a drug, dioxomast is used (the incidence of cows with mastitis and the use of a new effective drug for the treatment of its subclinical form // V.Yu. Komarov, B.L. Belkin), which includes dioxidine, xanthan gum, lactam tetramethylenediethylenetetramine, prednisolone and distilled water. The drug has a high antimicrobial effect, a wide range of effects.

To reduce the effects of transport stress in cattle, a method is used (patent RU 2271211), including the introduction of a mineral additive into the feed - a neutralized white mud of alumina production of known composition, which is introduced into the feed once a day for 5-7 days before transportation in a dose 200-300 g per head per day. The use of mineral additives obtained as waste from the mining industry carries great risks, both for animals and for products derived from them.

However, to adapt cattle to new conditions of detention, the method described in patent RU 2490011 is used. For this, a mixture of formaldehyde medical solution is administered intramuscularly at a dose of 5.0-6.0 ml per head with an interval between injections of 7-10 days with a solution of sodium chloride 0.85-0.95% concentration at a ratio of weight parts 2-6: 994-998, while the composition is administered twice. The formaldehyde solution carries risks not only for the animal and the products received from it, but for personnel who work with the delivered product.

As can be seen from the above analogues, various scattered narrowly targeted methods are used to solve the described health problems in cattle, which do not allow to fully restore the structure and function of damaged organs, as well as restore the violation in the regulatory systems of the body (immune, nervous) and eliminate systemic metabolic disorders.

For a comprehensive solution to these problems, it is possible to use the achievements of regenerative medicine.

In the field of regenerative medicine, a number of inventions are known. In particular, peptides of the general formula obtained by chemical synthesis (relatively short (11, 12, 13, and 14 amino acid residues) fragments of human interleukin-2) are inductors and / or potentiators of growth-promoting macrophage activity in vitro and have regenerative and reparative effects in vivo ( patent application RU 94023808/04). These peptides can be used in medicine for the treatment of a number of diseases accompanied by generalized activation of macrophages (liver damage, skin wounds, burns, polytrauma, diabetes, etc.). Similar peptides obtained synthetically have a lower affinity, and as a result, activity, compared with compounds extracted from biological systems using biotechnology.

Currently, a number of tools and methods are being developed in the field of regenerative medicine based on the use of mesenchymal stem cells.

Known invention, which sets forth methods and compositions for use in cell therapy (patent RU 2610427). In the described invention, the use of mesenchymal stem cells alone or in combination with various antigens by simultaneous or sequential introduction into the lymphatic system in case of immune-mediated inflammatory diseases.

Also known is a conditioned medium (patent RU 2292212), which has a therapeutic effect in thermal burns based on mesenchymal stem cells of human bone marrow and a medium for their cultivation. The specified environment is obtained by collecting at the stage of stationary growth of a culture of a stable cell line of mesenchymal stem cells located in the G ₀ period of the cell cycle.

Based on various cells, a tool has been developed to change the growth rate or reproduction of cells, which stimulates the healing of wounds and burns, allows you to correct a cosmetic defect, as well as inhibition of skin aging and a method of stimulating hair growth (patent RU 2280459). The conditioned cell medium compositions of the present invention can be composed of any known defined or uncertain medium and can be conditioned using any eukaryotic cells (stromal cells, parenchymal cells, mesenchymal stem cells, reserve liver cells, stem nerve cells, pancreatic stem cells and / or embryonic stem cells). The conditioned medium may also be processed to isolate and purify the product, for example, to remove unwanted proteases (gel chromatography (using matrices such as Sephadex), ion exchange chromatography, affinity chromatography on a metal chelate with an insoluble matrix, such as cross-linked crosslinked agarose, HPLC purification and hydrophobic chromatography of conditioned medium). Alternatively, sterilization may be necessary, and it can be carried out by methods to ensure the preservation of the desired biological activity.

It should be noted that the invention described above is characterized by an extremely narrow focus of action, while mesenchymal stem cells have a complex effect. A tool based on them is able to solve the whole complex of problems associated with inflammation and tissue damage, impaired immune system functions, metabolic disorders, as well as for adaptation of animals to transport stress and stress when changing the place of detention.

The isolation of animal mesenchymal stem cells is also known. This also applies to cattle (patent RU 2482182).

As a prototype of the claimed invention can be considered a comprehensive tool immunomodulator-metabolic-detoxicant-adaptogen-radioprotector (patent RU 2194502). This product contains at least one placenta extract, one nucleic acid derivative, and additionally at least one vitamin. The main advantage of the described invention is the complexity of the action. However, the use of placenta cells carries some risks associated with the possible malignancy of cells. Supplementing the composition with derivatives of nucleic acids and vitamins does not contribute to the stabilization of the pharmaceutical composition, can antagonistically affect the main active substance. At the same time, the possibility of using the specified means to solve the health problems of cattle, as well as the side effects, are unknown.

SUMMARY OF THE INVENTION

The present invention sets itself the task of creating a comprehensive tool for cattle (hereinafter - cattle), which, through exposure to the stomatologic tissue elements, provides replacement of damaged cellular elements, which leads to the restoration of the damaged structure and impaired functions of organs and systems. Such an approach will allow constructing a safe agent that practically does not have side effects, but at the same time exhibits a number of pharmacological activities: regenerative (restoration of the liver, organs of the musculoskeletal system, skin and its derivatives), immunomodulating (prevention and treatment of infectious diseases, prevention of immunodeficiencies in animals and their offspring), detoxification (eliminating the effects of intoxication of various etiologies), adaptogenic (reducing stress and strengthening immunity when changing the place of detention).

The technical result is achieved by developing a veterinary agent for cattle based on a conditioned medium obtained by cultivating species-specific mesenchymal stem cells containing a complex of biologically active substances of a protein-peptide nature that provide the listed types of pharmacological activity. In the development of the described tools applied biological standardization.

Mesenchymal stem cells (MSCs) have the ability to secrete a wide range of chemokines and growth factors, to respond quickly to changes in tissue homeostasis by modifying their secretion profile, which provides a natural mechanism for tissue regeneration. It was shown that the main contribution to tissue regeneration is made not by replacing damaged cells with introduced cells, but by stimulating the body's own stologic potential. The specified fact was used to create the described tool.

The described invention is a tool consisting of a conditioned environment of cattle mesenchymal stem cells, may contain mesenchymal stem cell extract, a stabilizer (glycine, sucrose or other stabilizer), a preservative (chlorobutanol hydrate, phenol, chloroform, nipagin or another preservative) that has regenerative, immunomodulating detoxification, adaptogenic, metabolic activity. The use of species-specific mesenchymal stem cells can minimize the risks from the use of the claimed invention associated with the occurrence of allergic reactions, intolerance of the drug to target animals, and also oncogenicity.

The basis of the therapeutic activity of the claimed drug provide low molecular weight peptides weighing up to 10 kDa. This allows, if necessary, to apply product purification by ultrafiltration, cutting off high molecular weight protein fractions.

The manufacture of the claimed funds is carried out in the following steps:

The accumulation of cell mass and the formation of a conditioned environment. At this stage, both freshly isolated cattle mesenchymal stem cells and cells after cryogenic storage can be used. Adipose tissue or bone marrow are used as a source of cattle mesenchymal stem cells. Cultivation is carried out on a liquid nutrient medium (DMEM, Igla medium, 199 or other medium suitable for these purposes) with the addition of fetal cattle serum at a concentration of 10 ± 5% at a temperature of 37 ± 2 ° C, CO ₂ content 5 ± 0.2% . The accumulation of cells is carried out in order to collect a conditioned medium, the suitability of which is evaluated by the biological method to ensure regenerative activity on the model of mouse dermal fibroblasts. The resulting conditioned medium is an active pharmaceutical substance.

Preparation of products based on a conditioned environment. The resulting conditioned medium is sterilized (sterilized filtration using a filter with a pore diameter of not more than 0.45 μm or another acceptable method), after which it is aseptically poured and sealed. Additionally, information steps may be provided with cattle mesenchymal stem cell extract (the extract is obtained by destroying the cells by physical means — freezing-thawing, ultrasonic waves — or by other methods), adding a stabilizer (glycine, sucrose or other acceptable stabilizer), adding a preservative (chlorobutanol hydrate, phenol chloroform, nipagin or other acceptable preservative), as well as freeze-drying.

Brief description of illustrative material

The invention is illustrated in tables.

Table 1. Regenerative activity of fractions of a conditioned medium.

Table 2. Data on biochemical parameters of rat blood.

Table 3. Data on the concentration of immunoglobulins in the blood of calves.

Table 4. Data on the content of somatic cells in milk.

Table 5. Data on biochemical blood parameters of cattle.

The possibility of carrying out the invention and the effectiveness of its application is illustrated by examples.

Example 1. Description of the receipt of funds.

Mesenchymal stem cells were obtained from subcutaneous adipose tissue of a cow: a 2-4 cm long skin incision was made under local anesthesia, a piece of subcutaneous fat 0.5-1.0 cm ^{3 was} taken from the incision site, and placed in a test tube with buffer solution (Biowest, USA ) containing gentamicin (80 μg / cm ³ , Biolot, Russia). Adipose tissue was fragmented to a homogeneous mass and enzymatic treatment was carried out with the addition of an antibiotic and antimycotics, as well as trypsin enzyme (Biowest, USA), incubated for 20 ± 10 minutes at a temperature of 37 ± 2 ° С and constant stirring on a magnetic stirrer. The described operation was repeated twice with a decrease in incubation time to 20 ± 5 minutes. The cell suspension was purified from tissue residues by centrifugation, cells were sown in culture bottles in a growth medium containing 90% DMEM growth medium (PanEco, Russia) and 10% fetal cattle serum, cultured at a temperature of 37 ± 2 ° С and carbon dioxide concentration 5, 0 ± 0.2%.

After 24 hours of cultivation, the medium was replaced. Subsequently, the medium was replaced every 3-4 days. Upon the confluence of the monolayer (visually checked using an inverted microscope), at least 85% of the monolayer was disaggregated with a dispersing solution (0.05-0.1% trypsin (Biowest, USA) in Versen solution (PanEco, Russia) and inoculated into new culture bottles with reseeding coefficient from 1: 3 to 1: 6.

To obtain a conditioned medium, the culture was transplanted for at least 3-4 passages, after which they were inoculated into culture bottles at the rate of 5-20 × 10 ³ cells per 1 cm ^{2 of} the growth surface area and cultivated under the conditions described above. The quality of the conditioned medium was evaluated by the biological method to ensure regenerative activity on the model of mouse dermal fibroblasts.

The resulting conditioned medium can be used directly for its intended purpose to obtain funds or stored for a long time under certain conditions (up to seven days at a temperature of 5 ± 3 ° C, longer storage requires a temperature of no higher than minus 18 ° C).

After collecting the conditioned medium, an MSC extract was obtained: the cell monolayer was disaggregated with the above dispersing solution, a 0.9% sodium chloride solution (5-10 cm ³ per 25 cm ² of growth medium area) was added, it was frozen at a temperature of minus 20 ± 2 ° C, after which thawing was performed at room temperature. The extract obtained was purified from cell debris by centrifugation for 10 minutes and added to the conditioned medium.

Upon receipt of the product, glycine was added in a concentration of 22.5 ± 2.5 mg / cm ³ , sterilizing filtration through a filter with a pore diameter of 0.22 μm, aseptic filling and capping.

The remaining conditioning medium was divided into three parts: the first part was subjected to sterilizing filtration without preliminary influences, for the second part, ultrafiltration was performed on a membrane with a pore rating of 50 kDa before sterilizing filtration, a third part was filtered on a membrane with pores that cut off substances weighing more than 10 kDa. Then, glycine was added to all three fractions obtained at a concentration of 22.5 ± 2.5 mg / cm ³ , after which they were subjected to individually sterilizing filtration through a filter with a pore diameter of 0.22 μm, after which they were aseptically poured and freeze-dried.

Thus, the product was obtained in the form of two dosage forms - a solution for injection and a lyophilisate for the preparation of a solution for injection.

Example 2. Determination of the fraction containing the active substance.

To study the fraction of the conditioned medium containing the active substance, ultrafiltration was carried out through membranes with a pore rating of 1, 10 and 50 kDa. The obtained fractions were studied by a biological method on a model of mouse dermal fibroblasts in order to establish biological activity. The control was a growth medium; at least 10% of overgrowth of the damage applied to the cell monolayer was considered the norm of activity.

It was found (table 1) that in the control and ultrafiltrate containing substances less than 1 kDa activity is below normal. Fractions of the whole conditioned medium, ultrafiltrates containing fractions of less than 10 and less than 50 kDa have activity. No statistically significant differences were found between them. From the above it can be concluded that the active components of the conditioned medium have a mass of from 1 to 10 kDa.

Example 3. Correction of liver pathology.

The use of an agent for the correction of liver pathology was investigated on a model of acute liver damage (MOPP) in rats. The model of acute liver damage was performed by a single injection of rats into the stomach through a probe of paracetamol (N-4-hydroxyphenyl) acetamide) in the form of a suspension at a dose of 700 mg per 1 kg of body weight. The study had three groups of animals:

- intact animals (did not simulate pathology);

- experimental animals (simulated pathology, carried out therapy with the claimed agent; the course of intramuscular injection on 0, 3 and 5 days at 0.5 cm ³ per 100 g of body weight);

- control animals (simulated pathology, did not carry out therapy, 1.0 cm ^{3 of} physiological saline was administered).

The animals were monitored for 10 days, after which the animals were taken out of the experiment by decapitation under mild ether anesthesia. Blood was collected from animals in which a biochemical study was performed. Before slaughter, a detoxification function of the liver was studied using a hexenal test.

The concentration of total protein (biuret method), enzymes such as alkaline phosphatase, aspartate (AsAT) and alanine aminotransferase (AlAT), as well as total and conjugated bilirubin, were determined in blood serum. The studied biochemical parameters reflect the functional state of the liver. The enzymes AsAT and AlAT are present in significant quantities in the liver, kidneys and heart, therefore, normally, the concentrations of AsAT and AlAT in the blood are low. When hepatocytes are damaged, the enzymes AsAT and AlAT are lost, and the concentration of these biochemical markers in the blood increases.

The results of the study are shown in table 2. The results obtained indicate the following. The pathology model worked, because in the control group all the studied parameters differ from those in the group of intact animals (the time of hexanal sleep and the concentration of enzymes exceed, and the protein concentration is greatly reduced). Since the studied parameters in the group of experimental animals do not differ from the group of intact animals, which indicates the correction of the simulated pathology, we can conclude that the tool can be used to correct liver pathology.

Example 4. The use of funds in the treatment of diseases of the skin and its derivatives.

The effectiveness of the drug in skin diseases (wounds, ulcers, necrosis, abscesses, dermatitis and other diseases), as well as its derivatives - hooves (corolla phlegmon, dermatitis) in cattle was investigated.

Animals with the indicated diseases were selected in the control and experimental groups. In the control group, treatment was applied with traditional means of external use, usual for this livestock farm, in the experimental group, the claimed drug in the form of a course of intramuscular injections. It was shown a reduction in recovery time (on average by 31.4%), a more complete cure of the disease, restoration of the skin and structure of the hooves. In the experimental group, in contrast to the control group, no relapses were observed.

Example 5. The use of funds in the treatment of diseases of the musculoskeletal system.

The efficacy of using the drug for diseases of the musculoskeletal system (joint diseases - arthritis, arthrosis, bursitis; bone diseases - fracture, osteomalacia in calves) was investigated.

Animals with the indicated diseases were selected in the control and experimental groups. In the control group, conservative treatment was used, the usual means for this animal husbandry, in the experimental group - the claimed tool in the form of a course of intramuscular injection. It was shown a reduction in recovery time (on average by 42.3%), a more complete cure of the disease, restoration of the structure and functions of the bone and joint. In the experimental group, in contrast to the control group, no relapses were observed.

Example 6. The use of funds for the prevention of immunodeficiencies in the offspring of cows.

The effect of the drug on the formation of immunity in calves in early life was investigated. Three groups of calves were formed: from cows to which the claimed agent was not administered, from cows to which the claimed agent was administered intramuscularly on the day of calving, and also from cows who were administered the claimed agent twice — three days before and on the day of calving.

It is known that the concentration of immunoglobulin G (Ig G) in the blood of calves indicates the state of the calf's immune system in early life and allows its survival to be predicted. So, with an Ig G concentration of less than 5 mg / cm ^3, survival is extremely low, with a concentration of 5 to 10 mg / cm ³ - average, if higher than 10 mg / cm ³ , then high.

In the study (the results are shown in table 3), it was shown that in the control group, calves Ig G were on average below 10 mg / cm ³ , while in the experimental groups - above 15 mg / cm ³ . The differences between the control and experimental groups are statistically significant. When applied to the cows of the claimed agent twice, there is a tendency to increase this indicator in calves, but it is not statistically significant.

Example 7. The use of funds for subclinical mastitis.

The use of the claimed drug in the treatment of latent (subclinical) mastitis was investigated. This diagnosis was established based on the content of somatic cells in milk (with a content of over 1 million cells in cm ³ ). Three groups of animals were formed - the control (an intracisternal agent was used once), two experimental animals - they once administered the drug without ultrafiltration intramuscularly (whole conditioned medium) and the agent after ultrafiltration on a 50 kDa membrane.

The results are shown in table 4. In the control group, the concentration of somatic cells in milk did not change during the entire observation period. At the same time, in the experimental groups, the content of somatic cells increased in milk by the third day, which indicates an accelerated yield of damaged tissue elements, and already from the fifth day of observation it returned to normal and subsequently decreased to a value of about 300 thousand cells / cm ³ , which is typical for high-grade milk.

Example 8. The use of funds for postpartum complications in cows.

The effectiveness of the drug in postpartum complications in cows was studied (retention of the placenta, endometritis, and others).

Animals with the indicated diseases were selected in the control and experimental groups. In the control group, conservative treatment was used, the usual means for this animal husbandry, in the experimental group - the claimed tool in the form of a course of intramuscular injection. It was shown a reduction in recovery time (on average by 23.4%), a more complete cure of the disease, restoration of the shape of the uterus (shown by rectal examination), and also at an accelerated pace, an improvement in the general well-being of the animal (motor activity, appetite, intake of food and water) began.

Example 9. The use of funds as an adaptogen.

The effectiveness of the claimed agent as an adaptogen for cattle was investigated when changing the place of keeping. For this, animals intended to be sent to another farm (distance more than 200 km) were divided into two groups: control (did not use any manipulations) and experimental (introduced once declared means). After transportation, animals were observed for one month.

It was shown that the incidence of animals in the control group of infectious diseases is statistically significantly higher (47.5% of the livestock group) than in the experimental (12.5% of the livestock group).

Example 10. The use of funds for detoxification of the body of cattle.

The detoxification activity of the claimed drug was investigated. For this, animals were selected with atony of the pancreas of various etiologies, in which a pronounced picture of intoxication was observed.

Animals with this disease were divided into control and experimental groups. For animals of the control group, conservative treatment was used; for animals of the experimental group, it was supplemented with a course of intramuscular administration of the claimed drug.

After 5 days of observation, the animals of the control group began to show interest in food and water, weak rumination was observed. At the same time, the animals of the experimental group showed interest in food and water, there was rumination and increased activity by the end of the third day, and on the fifth day they were completely healthy.

Biochemical blood tests conducted to study the detoxification function of the liver showed that in the animals of the control group, the clinical picture of intoxication is confirmed by biochemical markers (table 5). In animals of the experimental group, the studied biochemical parameters were normal already on the fifth day of observation.

Note * - hereinafter, statistically significant differences at the level of p <0.05 compared with the control group

Claims (4)

1. Means for cattle (cattle) containing a protein-peptide complex weighing less than 10 kDa, obtained by ultrafiltration from a conditioned medium from culturing mesenchymal stem cells from cattle adipose tissue after at least 3-4 passages, glycine at a concentration of 22.5 ± 2.5 mg / cm ³ as a stabilizer, standardized by the method of biological control of regenerative activity on dermal fibroblasts, has regenerative, immunomodulating, detoxifying and adaptogenic activity. 2. The tool according to p. 1, characterized in that it is made in liquid form. 3. The tool according to p. 1, characterized in that it is made in lyophilized form. 4. The tool according to p. 1, characterized in that it also contains an extract of mesenchymal stem cells from cattle adipose tissue obtained by freezing and thawing cells, purified by centrifugation.