RU2787479C1 - Kit of a pharmacological agent intended for improving the blood flow in the cerebral cortex and restoring motor and cognitive functions of the body - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular, to a kit for improving the blood flow of the cerebral cortex and restoring motor and cognitive functions of the body. Kit for improving the blood flow of the cerebral cortex and restoring motor and cognitive functions of the body includes two biologically active substances extracted from blood serum and bovine brain tissue, protein-peptide complexes containing peptides with molecular weights of 1,000 to 6,000 Da, and a protein being a member of the mammalian albumin family under No. gi|1351907 with the molecular weights of 66,267 Da and 66,667 Da, extracted from blood serum and bovine brain tissue, respectively, contained in separate vials and administered separately in a pharmaceutically acceptable carrier intranasally, orally, intramuscularly, or intravenously, exhibiting biological activity in the concentration range thereof from 10^-15 to 10^-8 mg/ml when diluted in a pharmaceutically acceptable carrier and/or diluent.

EFFECT: above kit provides a possibility of expanding the range of medical products for preventing and treating nervous diseases associated with brain pathology, improving the blood flow of the cerebral cortex and restoring motor and cognitive functions of the body.

4 cl, 5 dwg, 6 ex

Description

The invention relates to biotechnology and medicine, and concerns a pharmacological kit containing biologically active substances, which are bioregulators in the form of protein-peptide complexes isolated from blood serum and brain tissue of various animals, which can be used together as the main active substances in development of drugs to restore the functional state of the brain, its motor and cognitive functions of diseases of the nervous system, in neurodegenerative diseases associated with impaired cerebral circulation and / or damage to brain neurons, including hemorrhagic stroke and Parkinson's disease.

A stroke is an acute circulatory disorder resulting in damage to brain tissue due to blockage or rupture of a cerebral vessel. Hemorrhagic stroke is characterized by rupture of the vessel and hemorrhage in the brain; Ischemic stroke (cerebral infarction) is also caused by impaired blood circulation in a part of the brain due to blockage or narrowing of an artery in the brain. As a result, the nervous tissue suffers, in which the death of neurons occurs, the conduction of nerve impulses is disturbed, resulting in a significant change in the motor and cognitive functions of the nervous system. The main treatment is to take antihypertensives, diuretics, as well as cardiac drugs, which generally support the work of the myocardium and blood vessels and prevent the occurrence of re-ischemia of the cardiovascular system.

During the rehabilitation period, exercise therapy is also prescribed, physiotherapeutic methods designed to restore the conduction of nerve signals in the nervous system. Almost all of these drugs are synthetic and are characterized by the ability to initiate a large number of adverse reactions and processes in the body of patients.

However, taking these pharmacological agents does not guarantee the development of repeated relapses of the disease. Strokes are one of the most common diseases in the world. It is known that many patients after a stroke remain permanently disabled, experiencing significant consequences in the violation of the motor and cognitive functions of the brain for the rest of their lives.

Impaired blood circulation in the brain is one of the common causes of neurodegenerative changes in the brain tissue, in which there is a progressive death of nerve cells. The resulting neurodegeneration always leads to dementia and impaired motor function in some patients. Neurodegenerative diseases differ in localization, symptoms, duration of the development of the pathological process, but they always end in dementia and loss of personality. In addition to impaired blood flow and vascular function, neurodegeneration can also be caused by a genetic predisposition, which is based on a violation of the function of certain genes, oncological diseases of the brain, traumatic brain injuries, infectious diseases, and prolonged intoxication of the body. Neurodegeneration can occur at any age, this process is irreversible, and the main goal of therapy in this case is the ability to slow down the development of this pathological process.

Parkinson's disease (PD) is a neurodegenerative disease and is a fairly common pathology: about 6 million people are ill every year in the world. The pathogenesis of PD remains poorly understood.

However, it has recently been shown that PD is associated with the degeneration of nigrostriatal dopaminergic neurons and/or with a decrease in the content of dopamine in the striopallidar system.

The developing activity of cholinergic interneurons leads to the formation of an imbalance of neurotransmitter systems, which is expressed in the appearance of stiffness of movements, hypertonicity of skeletal muscles, rest tremor. In patients with PD, postural disorders also appear, salivation, sweating and secretion of the sebaceous glands increase, behavior changes - tearfulness, irritability, etc.

The basis of modern PD chemotherapy is the restoration of the disturbed balance between dopaminergic and cholinergic neurotransmission, namely: enhancement of dopaminergic functions or suppression of cholinergic hyperactivity.

One of the drugs widely used in PD is Levodopa, which is able to eliminate the deficiency of endogenous dopamine in the neurons of the striopallidar system. However, taking Levodopa is characterized by the development of such negative processes in the body as tachycardia, arrhythmia, hypotension, vomiting, etc.

It should also be noted that a significant increase in the level of dopamine in the brain can lead to involuntary movements and mental disorders in patients with PD. In order to avoid the development of negative side reactions in the body when taking levodopa, other drugs are prescribed that have their own specific side effects. Thus, patients with PD have to take a large number of potent drugs, characterized by the presence of a significant number of negative side effects for the body. Therefore, the search and study of endogenous biologically active substances that regulate regenerative processes in the nervous tissue and do not cause the development of negative side processes in the body continues to be an urgent problem of modern biotechnology in medicine.

These biologically active substances should be able to initiate regeneration processes in pathologically altered tissues, including the nervous and blood vessels, and control their course and direction in such a way that the most complete restoration and repair of the structure and function of damaged tissues occurs.

The invention allows to expand the scope of drugs recommended for nervous diseases, and of natural origin in the form of peptide-protein compositions, to enhance the biological effects of their action by developing a combined joint method for their use, to obtain substances that function in the body for a long time without losing the ability to activate regeneration processes in the tissues of blood vessels and the brain with various pathological disorders in the functioning of the nervous system, and, in particular, with hemorrhagic

From patent RU 2315606, 27.01.2008, a method for producing a drug for the treatment of myopia and diseases of the sclera of the eye is known, characterized in that the tissue of the central region of the sclera, including the cribriform plate, is isolated from freshly enucleated eyes of vertebrate animals, washed in water-salt physiological solution, keep the sclera tissue in a water-salt solution at 4-7°C for 2-4 hours, centrifuge the tissue extract, collect the supernatant, separate it by dissolving it in a 100% ammonium sulfate solution at 4-6°C for 5-7 days, the solution is filtered, it is dialyzed in dialysis bags at 4-7°C until ammonium sulfate ions are completely removed, it is separated by isoelectric focusing in a sucrose density gradient at pH 3.5-10.0 at a temperature of 4-6°C and voltage of 500-2000 V for 72-96 hours, after which acidic protein fractions are collected, combined, dialyzed until sucrose and ampholines are completely removed in dialysis bags at 4-7 ° C for 10-14 days, the resulting aqueous protein solution is dried until a dry substance is obtained, which is purified by electrophoresis in a 7.5-15% polyacrylamide gel, a low molecular weight fraction Rf = 0.9-0.95 is collected, containing regulatory peptide, by elution with deionized water from the gel for 2-5 days at 4-7°C, the resulting fraction is dialyzed in dialysis bags with a pore limit of 2-8 kDa at 4-7°C for 7-10 days with deionized water , the dialyzed regulatory peptide solution is then re-dried to obtain a white flaky fine powder, which is then dissolved in a physiological aqueous saline solution

From the monograph by A.A. Konstantinovsky, M.S. Krasnov et al. “Study of the wound-healing effect of bioregulators isolated from eye tissues and bull blood serum on the model of experimental corneal injury in rabbits “in vivo”, Biochemistry. 2009, volume 74, no. 9, p. 1195-1203, new bioregulators isolated from eye tissues and bull blood serum are known. Bioregulators belong to a new group of biologically active substances previously found in different tissues of animals. Bioregulators of this group are localized extracellularly, have a complex structure - they contain proteins, carbohydrates, lipids. Bioregulators of this group affect adhesion, migration, differentiation, and proliferation of cells in the tissues that were the source of their release. In low doses, bioregulators stimulate the processes of recovery and repair in tissues. It was shown that the bioregulator isolated from the cornea of the bull's eye had a pronounced protective effect on the state of the tissue during the cultivation of the cornea of the eye of vertebrates in vitro due to the ability to additionally activate cellular sources of regeneration. A bioregulator isolated from blood serum stimulated corneal wound healing in a rabbit in an in vivo experiment, as well as in a person with post-traumatic recurrent erosion and corneal burns. The bioregulator isolated from the pigment epithelium of the bull's eye contributed to the stabilization of the differentiation of pigment epithelium cells and the maintenance of the viability of bipolar and Muller cells under conditions of organotypic cultivation of eye tissues in vitro.

All bioregulators were obtained according to a previously developed scheme for isolation and purification, consisting of obtaining a tissue extract, salting out impurity proteins in a saturated solution of ammonium sulfate, obtaining a supernatant fraction and its subsequent separation by isoelectric focusing in a sucrose gradient in the pH range of 3.5–10.0. Fractions of acidic proteins of blood serum and cornea were studied, which were collected in the pH range of 1.5–3.0, as well as the main fraction of the pigment epithelium, collected in the pH range of 8.5–9.5. During the purification process, the presence of bioregulators in the fractions was determined by the adhesiometric method, which is based on the assessment of their membranotropic activity. Bioregulators were studied at a concentration corresponding to 10-12 mg protein/ml, which was obtained by successive 10-fold dilution of the stock solution. The protein concentration in the initial fractions of bioregulators was determined by the colorimetric method.

The bioregulators described above are mainly used in ophthalmology in the treatment of various eye diseases.

In particular, from RU 2701566, 09/30/2019, a pharmacological composition is known that has a protective effect on the state of the tissues of the posterior part of the eye - the sclera, retina, pigment epithelium, choroid. This composition is an aqueous solution of a protein-peptide complex at a concentration of 10 ^-7 -10 ^-17 mg / ml and contains peptides with molecular weights from 1000 to 6000 Da and a protein with a molecular weight of 66000-68000 Da, belonging to the blood serum albumin family, as well as lipids and carbohydrates. The complex was obtained by isolating IEF-fractions from the sclera of the eye of vertebrate animals, separated and collected by isoelectrofocusing (IEF) in the pH range <3.0, mixed in equimolecular ratios. The complex was obtained as a result of successive extraction of the sclera of the eye of vertebrate animals, fractionation and separation of the obtained fractions using the method of reversed-phase high-performance liquid chromatography HPLC. This invention ensures the preservation of adhesive intercellular interactions in the eye tissue, the integrity of collagen fibers and an increase in the viability of fibroblasts, as well as maintaining adhesion between the tissues of the posterior part of the eye, increasing cell viability, maintaining the status of cell adhesion, proliferation and differentiation. EFFECT: invention makes it possible to obtain a highly purified drug intended for the prevention and treatment of eye diseases associated with the claimed disorders. The disadvantage of this invention is the study of only one representative of the MHTB group isolated from scleral tissue. For a complete characterization of this group of bioregulators, it was necessary to study MHTB bioregulators isolated from other animal tissues, as well as to obtain the results of a study of their composition, structure, and biological (pharmacological) action.

There is also known a method for obtaining a biologically active complex and a biologically active protein-peptide complex (patent RU2428196, 07/02/2020), which has a neuroregenerative and neuroprotective effect and stimulates the physiological and reparative regeneration of nervous tissue, for use in the manufacture of drugs for diseases and the peripheral nervous system. This method makes it possible to obtain a mixture of proteins and polypeptides from the brain of embryos of ungulates. The disadvantages of this invention are the incomplete purification of the final substance, which is a protein-peptide complex, as well as the use of embryonic material, which may contain other biologically active substances that cause harmful side reactions in the tissues of an adult organism.

From RU 2725491, 07/02/2020, a substance is known for obtaining funds that increases the growth of spermatozoa, mammals and exhibits biological activity when diluted in an aqueous pharmaceutically acceptable carrier and / or diluent to a total protein concentration of 10 ^-8 to 10 ^-15 mg / ml, including peptides with molecular weights of 1000-10000 Da and a protein of the mammalian albumin family, bovine serum albumin, with a molecular weight of 65712 Da with an amino acid sequence that has 100% homology with the N-terminal sequence of a mature molecule of bovine serum albumin, when used in bulk peptides :protein 1:1-7, isolated from the testes of bulls IEF-fractions, separated and collected by isoelectric focusing in the range of pH<3.0 using the method of reversed-phase chromatography HPLC.

Closest to the claimed invention is the technical solution according to the patent RU2290936 C1, 01/10/2007, describing the agent for maintenance therapy and tissue-specific therapy, which is a low molecular weight complex purified from protein, nucleic and lipid impurities, the peptide component of which does not denature and retains its regulatory properties. The peptide complex contains peptide components with a molecular weight of 1000 to 12000 Da, amino acids, minerals, trace elements and vitamins in a biologically bound form, exhibiting pronounced tissue-specific activity. The method for obtaining this agent includes grinding raw materials of animal origin, extraction, which is carried out with a 3% solution of acetic acid with the addition of zinc chloride, cooling to a temperature of plus 7-16 ° C with constant stirring for 48 hours, separating the extract from the ballast substances by separation, precipitation, which is carried out with acetone in a volume ratio of 1:5 at a temperature of plus 3-5°C for 4 hours, settling until a formed precipitate is formed. The precipitate is then washed with two times the volume of acetone, rubbed through a metal sieve. The resulting target product is dried at a temperature of 18±2°C until complete removal of traces of acetone. In this case, the maximum absorption of the target product in the ultraviolet spectrum is (270±5) nm. The technology used makes it possible to obtain a peptide complex purified from nucleic, protein and lipid impurities with a molecular weight of its constituent peptide components from 1000 to 12000 Da, containing microelements, minerals and vitamins in a biologically bound form, which has a pronounced tissue-specific activity.

However, this tool has a number of disadvantages: firstly, it contains a mixture of peptides of an unclear nature, an insufficient degree of purification of their individual state, and secondly, the use of the target product in high doses can lead to adverse effects on the body, since peptides in high doses can significantly affect on metabolism.

The technical objective of the claimed invention is the creation of a pharmacological set consisting of two biologically active substances in the form of bioregulators belonging to the group of membranotropic homeostatic tissue-specific bioregulators MHTB, which are protein-peptide complexes isolated from blood serum and from the brain tissue of a bull, which provide regeneration pathologically. altered nervous tissue and restoration of impaired motor and cognitive functions of the brain in case of nervous pathologies, various neurodegenerative diseases.

In accordance with the set technical task, the technical result of the claimed invention is the expansion of the arsenal of agents related to endogenous biologically active substances in the form of protein-peptide complexes (bioregulator) obtained from blood serum and from bull brain tissue, contributing to the restoration of motor and cognitive functions of the body during the rehabilitation period after nervous diseases due to the restoration of cerebral blood flow and regeneration of the nervous tissue in the affected area.

The set technical task and the technical result are achieved by a set of pharmacological agents designed to improve the blood flow of the cerebral cortex and restore the motor and cognitive functions of the body, containing biologically active substances placed in different vials as bioregulators, which are isolated from blood serum and from brain tissue bovine protein-peptide complexes containing peptides with molecular weights of 1000-6000 Da and a protein that is a member of the mammalian albumin family under the number gi| ^1351907 in the SwissProt database, and exhibiting biological activity in the range of their concentrations from 10 ^-15 to 10 mg/ml when diluted in a pharmaceutically acceptable carrier and/or diluent.

At the same time, a set of a pharmacological agent according to the invention for improving blood flow in the cerebral cortex and restoring the motor and cognitive functions of the body is intended for the combined combined intake of two biologically active substances as bioregulators placed in different vials.

The protein of the albumino family, which is part of the protein-peptide complex of the kit, is numbered gi|1351907 in the SwissProt database and has a molecular weight of 66367 Da or 66667 Da. In addition, the protein-peptide complexes included in its composition, isolated from the blood serum and from the brain tissue of a bull, respectively, are administered separately intranasally, orally or intramuscularly, or intravenously.

When using a set of pharmacological agents intended to improve blood flow and restore the motor and cognitive functions of the body, the protein-peptide complexes included in its composition are administered in an aqueous saline physiological solution intranasally, intramuscularly, parenterally or administered in distilled water for oral administration. The set of pharmacological agents according to the invention is intended to improve blood flow in the cerebral cortex and restore the motor and cognitive functions of the body, moreover, the protein-peptide complex from bull blood serum included in the kit is administered intranasally, and the protein-peptide complex isolated from bull brain tissue administered intramuscularly.

As pharmaceutically acceptable carriers and/or diluents, physiological saline, distilled water (water for injection), and possibly auxiliary additives traditionally used in the preparation of pharmacological agents, such as ascorbic acid, oxidation inhibitors, are used.

The two substances included in the pharmacological kit in the form of protein-peptide complexes isolated from the blood serum and brain tissue of a bull are administered separately in a pharmaceutically acceptable carrier, in particular, they are administered in an aqueous saline physiological solution intranasally, intramuscularly, parenterally or administered in distilled water for oral administration.

Biologically active substances isolated from blood serum and brain tissue of a bull are highly purified protein-peptide complexes obtained using modern methods of biochemistry and bioorganic chemistry, isoelectrofocusing in a sucrose gradient, reverse-phase HPLC chromatography, the degree of homogeneity of which was assessed by electrophoresis methods ,

Below are figs. 1-5 and examples illustrating the invention but not limiting it.

Fig. 1. Changes in local cerebral blood flow (LCF) in the parietal region of the cerebral cortex and blood pressure (BP) in rat No. 1 under conditions of hemorrhagic stroke (control group 2). Designations from top to bottom: local cerebral blood flow (LCB) in c.u.; blood pressure (BP) in mmHg; timestamp in minutes.

Fig. Fig. 2. Effect of MHTB-C (0.6 ml, i.v.) on local cerebral blood flow (LCB) and blood pressure in rat No. 3 in a model of hemorrhagic stroke (HI).

Designations from top to bottom: local cerebral blood flow (LCB) in c.u., arterial pressure (BP) in mmHg, time stamp in min.

Fig 3. The effect of MHTB-M on local cerebral blood flow (LCB) in the parietal region of the cerebral cortex and blood pressure (BP) in rat No. 5 in conditions of hemorrhagic stroke. Designations from top to bottom: local cerebral blood flow (LCB) in c.u., arterial pressure (BP) in mmHg, time stamp in min.

Fig. Fig. 4. Effect of MHTB-C (0.6 ml, i.v.) and MHTB-M (40 μl intranasally) on local cerebral blood flow (LCB) in the parietal region of the cerebral cortex and blood pressure (BP) in rat No. 7 under conditions hemorrhagic stroke.

Designations from top to bottom: local cerebral blood flow (LCB) in c.u.; blood pressure (BP) in mmHg; timestamp in min.

Fig. 5 Effect of mexidol (0.6 ml, i.v.) on local cerebral blood flow (LCB) in the parietal region of the cerebral cortex and blood pressure (BP) in rat No. 10 under conditions of hemorrhagic stroke.

Designations from top to bottom: local cerebral blood flow (LCB) in c.u.; blood pressure (BP) in mmHg; timestamp in min.

Example. 1. Method for obtaining BOD from bull blood serum.

Ammonium sulphate is gradually added to the cooled solution of bull's blood serum with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml of serum). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The resulting supernatant fractions are concentrated using a rotary vacuum evaporator at a temperature below 40°C. The blood serum supernatant fraction is separated using the isoelectrofocusing (IEF) method in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range of 3.5 - 10.0 (Serva, Germany) (Osterman, 1983) . The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% H ₃ PO ₄ solution; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. IEF is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. Protein detection is carried out on any spectrophotometer at 280 nm, for each fraction, the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by time-of-flight mass spectrometry with an UltraFlex 2 mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; including peptides with molecular masses of 1151, 1338, 1448, 1666, 1812, 1915, 2016 Da. Determination of the primary sequence using C-carboxypeptidase shows that they are products of proteolysis, respectively, for the 1448 Da peptide -MGLRGGPLASLLLR - there is complete homology with the N-terminal sequence of P-cadherin identified in bovine aortic endothelium. For a peptide with a molecular weight of 1151Da -(dkhgc) _n-5 -PAYVSP-homology with the amino acid sequence of a fragment of the kinase domain of the regulatory protein tribble-2 (TRB-2), obtained from Bostaurus granulocytes (granulosacells). For a peptide with a molecular weight of 1338 Da, homology with adrenomodulins, proteins produced by the vascular endothelium of Bostaurus, was found.

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band is cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in gel followed by mass spectrometric study of the hydrolysis products shows that the isolated protein with a molecular weight of 66367 Da is a protein of the mammalian albumin family under the number gi|1351907 in the SwissProt database. The partial N-terminal amino acid sequence of this protein DTHKSEIAHRFKDLGE was determined by the Edman method, which has 100% homology with the N-terminal sequence of the mature bovine BSA molecule.

Thus, the IEF fraction collected in the pH range <3.0 contains the gi|1351907 protein, a representative of the bovine serum albumin multifamily, with a pI value in the pH range of 3.90–4.00, and a mixture of small peptides with a molecular weighing 1000-6000 Yes. Identification of three peptides that make up the VPC showed that they are products of proteolysis of the adhesive protein P-cadherin, the regulatory protein tribble-2 (TRB-2) isolated from granulocytes, and adrenomolulin.

Example 2. Method for obtaining BOD from bovine brain tissue

Obtaining a tissue extract of the bull's brain Freshly isolated bull's brain was washed several times in cold saline until the blood was completely removed, cut into small fragments weighing 1-2 g, washed again with saline and placed for 2-3 hours in saline at 0°C-+4 °C. Then the resulting extract was poured off, centrifuged at 2500g for 30 min. The obtained tissue extract was further fractionated to obtain BOD or frozen and stored at -90°C.

Preparation of BOD from bovine brain tissue extract

Ammonium sulphate is gradually added to the cooled solution of the lung tissue extract with constant stirring until a saturated salt solution is formed (780 g of salt was added per 1000 ml). The protein suspension is left for 7 days at a temperature of +4°C, after which it is centrifuged at 12000g for 30 minutes. Collect a fraction of the supernatant and sediment. The supernatant fraction is dialyzed against distilled water until ammonium sulfate is removed in a volume ratio of 1:100 with five water changes. The presence of ammonium ions in a solution is determined by reaction with Nessler's reagent. The supernatant fraction is concentrated in a rotary vacuum evaporator at a temperature below 40°C. The supernatant fraction was dialyzed against distilled water to remove ammonium sulfate and separated by isoelectric focusing (IEF) in a sucrose density gradient on an LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10.0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution - 60% sucrose in 1% H ₃ PO ₄ solution; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The solutions are layered into the column in the following order: heavy electrode solution, heavy and light gradient solutions, which are fed from the gradient mixer, and then light electrode solution. Isoelectric focusing (IEF) is carried out at +4°C for 96 hours at a voltage of 500-1500 V. Fractions of 10 ml are collected. The detection of proteins is carried out on a spectrophotometer at 280 nm, for each fraction the pH value is determined using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH values <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:100 with a fivefold change of water. This fraction is analyzed by time-of-flight mass spectrometry with an UltraFlex 2 mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1000 Da - 6000 Da are detected. This fraction is analyzed by time-of-flight mass spectrometry with an UltraFlex 2 mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; including peptides with molecular weights 967, 1241, 1437, 1665, 2694, 2874, 2932, 3306, 3695, 4283, 4747, 4963, 6023Da. Determination of the primary sequence of the 474Da peptide -N-myristoyl-G-palmitoyl-S-CTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLGAG - using tryptic hydrolysis of peptides and their identification in the Mascot software package shows that this peptide is an N-terminal fragment of the guanine-nucleotide-binding G _o protein, predominant in the mammalian brain.

Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH<3.0. A band was cut from the gel corresponding to a molecular weight of approximately 66,000 Da. The tryptic hydrolysis of the protein in a gel followed by mass spectrometric study of the hydrolysis products showed that the isolated protein with a molecular weight of 66667 Da is a protein of the mammalian albumin family under the number gi|1351907 in the SwissProt database. The partial N-terminal amino acid sequence of this protein, DTHKSEIAHRFKDLGE, was determined by the Edman method, which has 100% homology with the N-terminal sequence of the mature BSA Bostaurus molecule. Thus, the IEF fraction collected in the pH range <3.0 contained the gi|1351907 protein, a representative of the bovine serum albumin multifamily with a pI value in the pH range of 3.90–4.00, and a mixture of peptides with molecular weights from 1000 to 6000 Yes. Example 3. Obtaining drugs MHTB-S and MHTB-M based on protein-peptide complexes isolated from blood serum and brain tissue of a bull, respectively.

As BOD solutions, IEF fractions collected in the pH <3.0 region, isolated from blood serum and brain tissue of a bull, respectively, are used, as described in Examples 1 and 2. Into a vial containing 0.1 ml of an aqueous solution of the corresponding BOD with a protein concentration of 100±10 µg/ml, add 0.9 ml of saline or distilled water and shake vigorously 25 times. Take 0.1 ml of the resulting solution into the next vial and add 0.9 ml of saline or distilled water, shake 25 times. This procedure is repeated 17 times, thus obtaining solutions of BOD with 10-fold serial dilutions from 1 to 19 degrees. Thus, solutions of the corresponding BOD are obtained from 10 ^-1 to 10 ^-19 mg / ml, prepared in saline (for intranasal, intramuscular administration), as well as BOD solutions in the same concentration range, prepared in water (for oral administration). In experiments to study the safety of a BOD solution isolated from bull blood serum, solutions were studied at concentrations of 10 ^-5 , 10 ^-12 , 10 ^-19 mg/ml. All solutions are sterilized by passing through sterile GV membrane filters with a pore diameter of 0.22 μm (Millipore, USA). As an MHTB-C preparation, a BOD solution isolated from the blood serum of a bull is examined at a concentration of ^10–12 mg protein/ml; BOD solution isolated from bovine brain tissue at a concentration of ^10–12 mg protein/ml is used as MHTB-M.

Example 4. Study of the effect of MHTB isolated from the blood serum and brain of a bull on the restoration of motor functions, as well as the state of the brain, in experimental hemorrhagic stroke in Wistar rats in vivo.

4.1. An in vivo model of hemorrhagic stroke in rats

Description of experimental animals and investigational preparations.

The study of the action of drugs MHTB-S and MHTB-M with chronic administration was carried out on 60 animals (Wi rats, males, 2.5 months, about 190 g) on the model of experimental hemorrhagic stroke (HI). A total of 6 groups of 10 animals were formed.

1. control group No. 1 - sham-operated animals

2. control group No. 2 - with a model of experimental GI

3. experimental group No. 3 - with a model of experimental GI and intramuscular (im) administration of the drug MHTB-S

4. experimental group No. 4 - with a model of experimental GI and intranasal administration of the drug MGTB-M

5. experimental group No. 5 - with a model of experimental GI and combined joint administration of drugs MHTB-S (im) and MHTB-M (i/n), administered every other day (one day - one drug, the next day - another drug) .

6. experimental group No. 6 - with a model of experimental GI and the introduction of the reference drug mexidol.

To administer the drug MHTB-M, the intranasal route of administration was chosen as an effective way to deliver the drug to the brain. Drug delivery by this route is seen as a modern alternative to oral administration, since the nasal cavity is easily accessible, richly vascularized, well permeable and has a large surface area, which contributes to good absorption and increased bioavailability. In addition, drugs enter the bloodstream, avoiding the "first pass" effect through the liver.

For the drug MHTB-C, an intramuscular route of administration was chosen, corresponding to the route of administration of the reference drug Mexidol.

The domestic synthetic drug Mexidol was chosen as the reference drug, the drug for which its antioxidant and membrane-protective effects were previously presented. The effectiveness of mexidol was revealed at the neuronal and vascular levels. The effective dose is 100 mg/kg.

In this experiment, the animals were injected with the maximum possible volume of the MHTB-M preparation applicable for i / n administration (the volume of a separate single injection was up to 40.0 μl, 20 μl into the nasal passage, with each injection). For the MGTB-C preparation, the volume of daily administration was 50 μl, which corresponds to the volume of administration of the reference drug.

The studied MHTB preparations were obtained as indicated in examples 1 and 2, then sterile solutions were prepared with concentrations: MHTB-C - 10 ^-12 mg protein / ml and MHTB-M - 10 ^-12 mg protein / ml (example 3).

The reference drug mexidol was used ready-made (OOO NPK FARMASOFT, 50 mg/ml) at a dose of 50 µl per rat.

Control animals were injected with saline by intranasal administration.

Intranasal administration of drugs was carried out with an automatic pipette nasally, 40 μl at the same time of the day, intramuscular, in a volume of 0.5 ml, was carried out alternately into the right and left hind limbs.

Obtaining a model of hemorrhagic stroke in rats

Despite significant differences in the etiology and pathogenesis of ischemic and hemorrhagic strokes (HI), they are united by a deterioration in the blood supply to the brain. So, hemorrhagic stroke is accompanied by a generalized increase in the tone of the brain vessels and, as a result, a significant decrease in the level of blood supply to the brain. This determines the need for a pharmacological effect on the cerebral vessels under these conditions in order to restore the initial level of cerebral circulation.

Intracerebral hemorrhage was reproduced in anesthetized rats according to A.N. Makarenko et al. (2002). Urethane (1.3 g/kg, ip) was used as general anesthesia. Symmetrical holes were drilled with a dental burr on the left and right in the projection of the internal capsule: H=4 mm, L=3 mm, A=-1.5 mm from bregma). Then the dura mater was pierced using a sharpened cannula needle and immersed with a stereotaxic device to the required depth (4 mm). Then the mandrin-knife was pulled out to the stop, which is provided by the upper latch, and three turns were made to the left and right, thereby cutting the vessels. The next stage: the mandrin - the knife is removed, and autologous blood 0.15 ml is injected into the needle-cannula after 2-3 minutes with a Hamiltonian syringe into the area of mechanical destruction of the brain tissue. In most cases, blood is distributed in the form of a well-defined hematoma at the end of the injection needle with deformation and destruction of subcortical structures in the region of the thalamic nuclei, less often in the subcortical white matter. Blood flow was determined in the ipsilateral hemisphere 3 mm from the injury and in the contralateral hemisphere at a point symmetrical to the injury. After the operation, the wound was decontaminated and sutured, the rats were placed in cages and their condition and healing process were observed for 2 weeks [1].

4.2. Study of the effect of MHTB-S, MHTB-M and Mexidol on blood flow and blood pressure in rats in vivo

The experiments performed showed that hemorrhagic brain damage causes a significant decrease in the level of cerebral blood flow in the cerebral cortex of rats in the area of damage in the ipsilateral hemisphere and in the contralateral hemisphere in the area symmetrical to the damage. The initial level of local cerebral blood flow decreased immediately after hemorrhagic brain injury and after 30 minutes was lower than the initial level by 47% and 60%. It should be noted that with hemorrhagic brain damage, a decrease in local cerebral blood flow in most experiments was observed within 1.5 - 2 hours of observation and by the end of the experiment was -50% and -70%. Hemorrhagic damage to the brain was accompanied by a decrease in blood pressure by 16 and 22% by the end of the experiment (Fig. 1).

Changes in local cerebral blood flow in the cerebral cortex of rats in a model of hemorrhagic stroke with the introduction of MHTB-C.

Experiments have shown that MHTB-C, when administered intravenously in 0.6 ml to anesthetized rats under conditions of a hemorrhagic stroke model, increases the blood supply to the affected brain. The cerebrovascular effect of the drug begins to develop immediately after administration (7% and 17% for 10 minutes) and gradually increases until the end of the experiment (60% and 50% for 90 minutes). Under these conditions, the introduction of MHTB-C causes an increase in blood pressure in the first experiment within 90 minutes after administration, in the second - the increase began from the fortieth minute.

Changes in local cerebral blood flow in the cerebral cortex of rats in a model of hemorrhagic stroke with the introduction of MHTB-M.

The experiments showed that MHTB-M, when intranasally administered 40 μl to anesthetized rats under conditions of a hemorrhagic stroke model, caused a slight decrease in cerebral blood circulation (-7% and -10%) by 40 minutes after drug administration, on the 80th and 90th minutes this effect was not. In the experiments, MHTB-M had a multidirectional effect on blood pressure depending on the background, in the experiment with a higher value of blood pressure at the point "background 2" it decreased, and in the experiment with a low value it increased.

Changes in local cerebral blood flow in the cerebral cortex of rats in a model of hemorrhagic stroke with combined combined administration of MHTB-S and MHTB-M

Immediately after the joint administration of MHTB-C (0.6 ml IV) and MHTB-M (40 μl intranasally) to anesthetized rats in a model of hemorrhagic stroke, local cerebral blood flow (LCB) increased by 47% in the first experiment and by 14% during second. In the first animal, LMC increased to a maximum of 90% at 40 minutes and gradually decreased by the end of the experiment to the level of "background 2". In the second animal, the level of LMK gradually increased until the 60th minute of the experiment (an increase of 64%), and then decreased to 36% at the 90th minute.

Arterial pressure in the first case increased immediately by 11%, by the middle of the experiment (40 minutes) the increase was 21%, and at the end of the experiment 9%. In the second case, blood pressure initially decreased by 6%, by the 40th minute it was restored to the value of "background 2" and at the 70th and 80th minutes it exceeded it by 6% and 5%, respectively.

Changes in local cerebral blood flow in the cerebral cortex of rats in a model of hemorrhagic stroke with the introduction of Mexidol

As early as 10 minutes after administration of Mexidol (0.6 ml IV), the increase in LMC was 10% and 7% for the first and second experiments, respectively. The level of blood flow under the influence of Mexidol increases throughout the experiment to 70.0% and 29% by 90 minutes (Fig. 5).

In this series of experiments, blood pressure under the influence of mexidol decreased after the administration of the drug and remained below the values of "Background 2" until the 90th minute (up to -17% and -25% for the first and second experiments).

The results obtained allow us to draw the following conclusions:

1. MHTB-C at a dose of 0.6 ml IV increased blood flow in the cerebral cortex of anesthetized rats in a model of hemorrhagic stroke, while causing a decrease in blood pressure.

2. MHTB-M, when administered intranasally, lowered local cerebral blood flow in the parietal region of the brain of rats against the background of a multidirectional effect on blood pressure.

3. With the combined combined use of MHTB-S and MHTB-M, an increase in blood flow in the cerebral cortex of rats was observed under conditions

4. models of hemorrhagic stroke without significant changes in blood pressure.

5. The drug mexidol in rats under conditions of hemorrhagic brain damage increased blood flow in the cerebral cortex of rats against the background of developing hypotension.

4.3. The results of the study of the effectiveness of the action of MHTB isolated from the blood serum and brain of cattle in behavioral tests

To assess the therapeutic effects of membranotropic homeostatic tissue-specific bioregulators (MHTB) isolated from the blood serum and brain of mammals (bovine), which in solutions of low concentrations can influence the main biological processes and stimulate the recovery and reparative processes in damaged tissues, a study of the anti-stroke effect of MHTB was carried out. on the GI model in rats in comparison with Mexidol. The introduction of drugs was started on the day of surgery, after 1 hour.

Animal observations

Animals were observed for 28 days after surgery. The weight, behavior and condition of the animals were recorded on the 1st, 3rd, 7th, 14th, 28th days. Survival of animals: during the operation and immediately after it, 5 rats died - 2 from the control group of sham-operated animals, 3 from the GI control group. During the experiment, the death of a rat from the comparison drug group was recorded on the 3rd day after the operation.

Animal weight dynamics

The weight of the animals of the experimental groups practically did not differ from the weight of the animals of the control group during the entire experiment. The maximum weight loss during the entire experiment, observed in the group of animals receiving MHTB-M, did not exceed 7±% of the value of the control group of l/o animals. The data are presented in table 13.

Neurological assessment according to the Stroke-index McGrow scale

No severe neurological disorders (paresis, limb paralysis, etc.) were observed on the first postoperative day. Were noted such violations as weakness, lethargy and slowness of movement, as well as in the control group of GI in one rat, the first degree semiptosis parameter was noted - 1 point; in the group with mexidol, one rat had lethargy, tremor, ptosis grade 2, paresis of 2 limbs. Total - 0.5+1.0+1.5+4=7 points. In the rest of the animals, clearly expressed disorders were not found.

4.4. The results of weekly tests to identify the effect of study drugs on the course of HI disease in dynamics

Scheme of the frequency of test execution during the experiment

Operation day - 0 day

Assessment on a neurological scale - 1, 3 days

Test "Open field" -3, 2, 9, 16, 23 days

Test "Vertical grid" - 2, 3, 10, 17, 24 days.

Test "Horizontal crossbar" - 2, 4, 11, 18, 25 days.

Test "Extrapolation deliverance) - 1, 2, 7, 14, 21, 27 days.

Weighing - -2, 3, 10, 17, 24 days.

Sampling of material for histology on the 7th, 14th, 28th day.

The results of the test "Hold on the horizontal bar" to assess the severity of inhibition of motor functions, coordination of movement and therapeutic effects of drugs MHTB-S and MHTB-M.

The test was performed to register neurological deficit, expressed in impaired coordination of movements, before the administration of drugs (background value) and 1 time per week throughout the entire period of administration - 4 weeks. The inability of the animals to maintain balance on the rod for 2 minutes was considered as a manifestation of impaired coordination of movements.

The holding time on the bar was estimated in seconds. Animals were tested immediately prior to administration to rule out the effect of injection on test performance. Testing animals in this mode with chronic administration made it possible to reveal the therapeutic effects of MHTB-S and MHTB-M, as well as to study the change in the action of drugs over time.

It should be noted that in the first week after the operation, in the control groups of both sham-operated animals and those with the GI model, motor coordination was impaired significantly more compared to the background value for these animals - by 35-50%, respectively, which is probably possible. explained by the stressful state of animals as a result of operations and everyday manipulations. At the same time, two and three weeks after the operation, the test performance in group No. 2 - animals with the HI model (negative control) remained the most (significantly) low, which can serve as an indirect confirmation of the development of HI. By the end of the fourth week, the difference in the ability to perform the test in animals of the control groups (No. 1 and No. 2) disappeared, which may indicate the restoration of CNS functions after surgery.

The greatest efficacy in chronic administration of the study drugs was found in MHTB-S during the entire period of administration. Significant differences in the average results of the test for the group of animals treated with MHTB-C and the control group of animals with hemorrhagic stroke were detected in the first week of drug administration, while the average retention time on the crossbar was twice the value of the negative control and reached the background value. The effect persisted until the end of the administration period - for 4 weeks. It is important to note that the action of MHTB-C led to a greater or comparable effect in this test compared to mexidol during the entire study period, approximately 2-4 times for groups No. 3 and No. 6.

The effectiveness of the second study drug MHTB-M was also found, but to a much lesser extent. The most significant difference from the negative GI control (45%) was found by the end of the second week of administration, with a subsequent decrease in efficacy.

With the combined administration of the two study drugs, i.m. was detected after two weeks of drug use.

The results of the test "Hold on the vertical grid"

This test was used to record muscle tone and assess the effect on the physical performance of rats under static load.

Under the experimental conditions, a rigidly fixed vertically oriented grid 40 cm wide and 100 cm high, bounded on all sides by wooden boards, was used. Under experimental conditions, this procedure provides an even distribution of the load on all muscle groups, especially on the muscles of the limbs. The advantages of this method are that a sufficiently large installation height contributes to the fact that the animal independently controls the level of physical activity and ceases to hold the body only when fatigue appears (it moves to the lower horizontal surface).

One-time testing consists of 5 presentations (cycle): the experimenter returns the animal to the vertical grid immediately after it moves to the horizontal surface 4 times. The total time of "hanging" on the grid was recorded. The criterion for the effectiveness of drugs is a significant increase in the total time of the test under conditions of static physical activity after the administration of the drug in comparison with the control and the reference drug.

It should be noted that due to the fact that the results in the negative control group, obtained when performing the test both a week later and in the following periods of the study, practically did not differ from the values of the group of sham-operated animals, we can conclude that this test cannot fully reflect the effect of drugs on the GI model. Nevertheless, it is important to note that the intranasal administration of MHTB-M in the group of animals No. 4 led to a significant increase in both the average retention time over 5 sets and the maximum time per set by 53±23% and 100±36%, respectively, during the entire period of drug administration, which indicates an increase in endurance and improvement in muscle tone of animals with intranasal administration of MHTB-M.

The introduction of MHTB-C under the conditions of this test led to the opposite effect - a decrease in the total and maximum time of the test, but without reaching the level of significance. At the same time, the joint use of drugs for one week contributed to an increase in the values of the studied parameters (total and maximum test execution time by 40 and 60%, respectively) compared with the control group of animals with hemorrhagic stroke - group No. 2. The use of the reference drug Mexidol had practically no effect on the ability of the animals to perform the test.

The results of the test "Open field" (shown in Fig. 1-5)

Neurological assessment according to the Stroke-index McGrow scale

No severe neurological disorders (paresis, limb paralysis, etc.) were observed on the first postoperative day. Were noted such violations as weakness, lethargy and slowness of movement, as well as in the control group of GI in one rat, the first degree semiptosis parameter was noted - 1 point; in the group with mexidol, one rat had lethargy, tremor, ptosis grade 2, paresis of 2 limbs. Total - 0.5+1.0+1.5+4=7 points. In the rest of the animals, clearly expressed disorders were not found.

4.5. The results of weekly tests to identify the effect of study drugs on the course of HI disease in dynamics

Scheme of the frequency of test execution during the experiment

Operation day - 0 day

Assessment on a neurological scale - 1, 3 days

Test "Open field" - 3, 2, 9, 16, 23 days

Test "Vertical grid" - 2, 3, 10, 17, 24 days.

Test "Horizontal crossbar" - 2, 4, 11, 18, 25 days.

Test "Extrapolation deliverance) - 1, 2, 7, 14, 21, 27 days.

Weighing - -2, 3, 10, 17, 24 days.

Sampling of material for histology on the 7th, 14th, 28th day.

The results of the test "Hold on the horizontal bar" to assess the severity of inhibition of motor functions, coordination of movement and therapeutic effects of drugs MHTB-S and MHTB-M

The test was performed to register neurological deficit, expressed in impaired coordination of movements, before the administration of drugs (background value) and 1 time per week throughout the entire period of administration - 4 weeks. The inability of the animals to maintain balance on the rod for 2 minutes was considered as a manifestation of impaired coordination of movements. The holding time on the bar was estimated in seconds. Animals were tested immediately prior to administration to rule out the effect of injection on test performance. Testing animals in this mode with chronic administration made it possible to reveal the therapeutic effects of MHTB-S and MHTB-M, as well as to study the change in the action of drugs over time.

It should be noted that in the first week after the operation, in the control groups of both sham-operated animals and those with the GI model, motor coordination was impaired significantly more compared to the background value for these animals - by 35-50%, respectively, which is probably possible. explained by the stressful state of animals as a result of operations and everyday manipulations. At the same time, two and three weeks after the operation, the test performance in group No. 2 - animals with the HI model (negative control) remained the most (significantly) low, which can serve as an indirect confirmation of the development of HI. By the end of the fourth week, the difference in the ability to perform the test in animals of the control groups (No. 1 and No. 2) disappeared, which may indicate the restoration of CNS functions after surgery.

The greatest efficacy in chronic administration of the study drugs was found in MHTB-S during the entire period of administration. Significant differences in the average results of the test for the group of animals treated with MHTB-C and the control group of animals with hemorrhagic stroke were detected in the first week of drug administration, while the average retention time on the crossbar was twice the value of the negative control and reached the background value. The effect persisted until the end of the administration period - for 4 weeks. It is important to note that the action of MHTB-C led to a greater or comparable effect in this test compared to mexidol during the entire study period, approximately 2-4 times for groups No. 3 and No. 6.

The effectiveness of the second study drug MHTB-M was also found, but to a much lesser extent. The most significant difference from the negative GI control (45%) was found by the end of the second week of administration, with a subsequent decrease in efficacy.

The combined administration of the two study drugs, i.m. MHTB-S and i/n MHTB-M, also showed an improvement in the ability of animals to perform the test in the second and third weeks of the study, compared with the negative control, with a significant difference in the results of this group animals were identified after two weeks of drug use.

The results of the test "Hold on the vertical grid"

This test was used to record muscle tone and assess the effect on the physical performance of rats under static load.

Under the experimental conditions, a rigidly fixed vertically oriented grid 40 cm wide and 100 cm high, bounded on all sides by wooden boards, was used. Under experimental conditions, this procedure provides an even distribution of the load on all muscle groups, especially on the muscles of the limbs. The advantages of this method are that a sufficiently large installation height contributes to the fact that the animal independently controls the level of physical activity and ceases to hold the body only when fatigue appears (it moves to the lower horizontal surface).

One-time testing consists of 5 presentations (cycle):

the experimenter returns the animal to the vertical grid immediately after it has moved to the horizontal surface 4 times. The total time of "hanging" on the grid was recorded. The criterion for the effectiveness of drugs is a significant increase in the total time of the test under conditions of static physical activity after the administration of the drug in comparison with the control and the reference drug.

It should be noted that due to the fact that the results in the negative control group, obtained when performing the test both a week later and in the following periods of the study, practically did not differ from the values of the group of sham-operated animals, we can conclude that this test cannot fully reflect the effect of drugs on the GI model. Nevertheless, it is important to note that the intranasal administration of MHTB-M in the group of animals No. 4 led to a significant increase in both the average retention time over 5 sets and the maximum time per set by 53±23% and 100±36%, respectively, during the entire period of drug administration, which indicates an increase in endurance and improvement in muscle tone of animals with intranasal administration of MHTB-M.

The introduction of MHTB-C under the conditions of this test led to the opposite effect - a decrease in the total and maximum time of the test, but without reaching the level of significance. At the same time, the joint use of drugs for one week contributed to an increase in the values of the studied parameters (total and maximum test execution time by 40 and 60%, respectively) compared with the control group of animals with hemorrhagic stroke - group No. 2. The use of the reference drug Mexidol had practically no effect on the ability of the animals to perform the test.

The results of the test "Open field"

The Open Field test was also performed before the start of drug administration and weekly for 4 weeks of drug administration. We determined such indicators as the number of changes in the position of the animal - vertical stance, grooming, exit to the center, which allow us to evaluate the motor and exploratory functions of animals

Analysis of the data obtained during the "Open Field" test did not reveal a significant level of differences between groups of animals in any of the measured parameters on all days of the study, which is associated with a rather high individual scatter of results and, as a result, a large standard deviation.

At the same time, there is a steady trend towards an increase in horizontal activity in animals of group No. 5 (MHTB-S / MHTB-M) at the first, third and fourth weeks compared with the control group of GI No. 2, as well as in rats of group No. 3 and No. 4 - by the end of the introduction of drugs. An increase in vertical activity was noted in the group receiving a complex of MHTB preparations (MGTB-S/MGTB-M) during the entire study period.

In the group of animals receiving the reference drug Mexidol, starting from the second week, a decrease in horizontal and vertical activity can be observed, which may indicate a decrease in exploratory activity (and, possibly, an increase in fear).

Thus, the results obtained allow us to say that the effect of MHTB preparations on the exploratory and motor activity of animals was revealed, but without a significant level of significance, at the level of a trend.

The results of the test "Extrapolation deliverance"

The "Extrapolation Deliverance" test (ETI) was proposed in 1982 by N.A. Bondarenko (Laboratory of "Pharmacology of Emotional Stress" of the Research Institute of Pharmacology of the Academy of Medical Sciences, Moscow) - an employee of Academician A.V. Waldman. In this setup, a rat, placed in water inside a narrow transparent cylinder, either jumps up or tries to dive under the bottom edge of the cylinder. Previously, it was shown that the behavior of diving in EMT is purposeful (search for a place that allows you to escape from the water), and upon reinforcement, it is quickly learned by animals [7] (Bondarenko N.A. 2013). The jumping behavior of rats, on the contrary, increases with a decrease in the cognitive functions of animals, including the administration of high (attention-disturbing) doses of benzodiazepine tranquilizers.

The maladaptive jumping behavior of rats when placed in is a consequence of the increased anxiety of these animals.

Consequently, the fewer jumps and faster diving, the less anxiety and the higher the cognitive function of animals.

When performing the test, the freezing time, the number of jumps, and the time before the animals dived were recorded.

In the EEI in the control groups No. 1 and 2 - l / o and GI, there was a difference in values from the background level with a multidirectional character. In the first week, in the group of sham-operated animals, there was a significant increase in the number of jumps and the latent period of diving in relation to the background value, which may indicate an increase in the stress state of the animals. Against this background, the decrease in the values of such parameters as jumping and diving (by approximately 25-60)%) in the groups receiving Mexidol - No. 6, as well as with the combined use of MHTB-S and MHTB-M - No. 5 in the first week (approximately by 25-50%) may indicate a positive effect of these drugs on reducing anxiety in animals.

There was also a significant decrease in the latent period of diving in group No. 5 with the combined use of MHTB-S and MHTB-M during the entire period of the experiment, as well as in group No. 4 with the use of MHTB-M at the third or fourth week. It is possible that these data indicate not only a decrease in anxiety, but also an increase in an important cognitive ability - the ability to remember. However, the differences were not significant due to the scatter of individual data.

Conclusions on the action of a set of pharmacological agents when used in hemorrhagic stroke

The results obtained allow us to draw the following conclusions. The best effect on the restoration of blood flow has a combined use of drugs MHTB-S and MHTB-M, which leads to an increase in blood flow in the cerebral cortex while maintaining constant blood pressure parameters. The used comparator drug mexidol caused a decrease in blood pressure, also increasing local blood flow in the brain, complete overgrowth of the wound space with nerve cells that formed processes.

Based on the dynamics of overgrowth and recovery of the wound channel for 4 weeks after the formation of a hemorrhagic stroke, we can conclude that the most effective effect on the restoration of the nervous tissue in the group of animals No. 5 with the combined effect of MHTB-C and MHTB-M. It was in this case that a rapid recovery of both neuroepithelial cells and choroid plexus cells was observed. This ensured the most rapid and complete restoration of blood flow in the area of damage, as well as the transmission of intercellular signals of connections in the nervous tissue, which resulted in the restoration of motor and cognitive abilities in rats with experimental hemorrhagic stroke.

The results of the study of the therapeutic effects of membranotropic homeostatic tissue-specific bioregulators (MHTB) isolated from the blood serum and brain tissue of cattle (bovine) in comparison with the standard drug Mexidol allow us to draw the following conclusions:

- Preparations MHTB-S and MHTB-M affect all the studied parameters, including cerebral circulation, motor coordination, exploratory activity (cognitive functions of animals), but they have different significance and direction of the effect.

- MHTB-S (at a dose of 0.6 ml IV) increased blood flow in the cerebral cortex of anesthetized rats in a model of hemorrhagic stroke, causing a decrease in blood pressure, and MHTB-M, when administered intranasally, decreased local cerebral blood flow in the parietal areas of the brain of rats against the background of multidirectional effects on blood pressure.

- With the combined administration of MHTB-C and MHTB-M, an increase in blood flow in the cerebral cortex of rats was observed in a model of hemorrhagic stroke without significant changes in blood pressure.

- The drug mexidol in rats in conditions of hemorrhagic brain damage increased blood flow in the cerebral cortex of rats against the background of developing hypotension, possibly due to low production of cerebrospinal fluid.

- In the test "Retention on the horizontal bar" the highest efficiency in chronic administration of the study drugs was found in MHTB-S during the entire period of administration. The effect persisted until the end of the administration period - 4 weeks.

The improvement in motor activity can be explained by the restoration of blood flow in the area of damage and the maintenance of the work of motor neurons. The action led to a greater or comparable effect in this test compared to mexidol during the entire study period in comparison with the group of sham-operated animals. Combined administration of the two study drugs, IM MHTB-C and IM MHTB-M, also showed an improvement in the ability of animals to perform the test in the second and third weeks of the study, compared with the negative control, with a significant difference in the results obtained in the study of animals of this group and all others, except for group No. 1 (sham operated), was detected after two weeks of using the drugs.

In the vertical grid hold test, the use of MHTB-M resulted in a significant increase in both the average retention time over 5 approaches and the maximum time per approach by 53±23% and 100±36%, respectively, during the entire period of drug administration, which indicates an increase in endurance and improvement in muscle tone of animals. The introduction of MHTB-C under the conditions of this test led to the opposite effect - a decrease in the total and maximum time of the test. At the same time, the combined use of MHTB-S and MHTB-M preparations for one week contributed to an increase in the values of the studied parameters (total and maximum test execution time by 40% and 60%, respectively) compared with animals in the control group with hemorrhagic stroke. The use of the reference drug Mexidol had practically no effect on the ability of the animals to perform the test.

The open field test did not reveal significant differences between the groups. There is a steady trend towards an increase in horizontal and vertical activity (motor and exploratory activity) in animals of the MHTB-C/MHTB-M group during the entire study period, but without affecting their level of anxiety. In the group receiving mexidol, starting from the second week, there is a tendency to an increase in anxiety (or, possibly, an increase in lethargy), which is expressed in a decrease in grooming acts, horizontal and vertical activity.

In the "extrapolation escape" test, in the first week in the group of sham-operated animals, there is a significant increase in the number of jumps and the latent period of diving in relation to the group of experimental hemorrhagic stroke. In the group treated with Mexidol, in the first week there was a significant decrease in the latent period of diving, which indicates a decrease in anxiety.

The studied preparations prepared on the basis of protein-peptide complexes obtained from bovine serum (MHTB-S) and brain tissue (MHTB-M) are effective in the tests used in the model of experimental hemorrhagic stroke in rats. Particularly high efficiency drugs MHTB-S and MHTB-M show when used in combination: they improved the coordination of animal movements, motor and exploratory activity, restored blood flow. Thus, it can be concluded that the studied drugs MHTB-S and MHTB-M may have clinical potential and are promising for their use as agents for the treatment of various pathologies of the brain and nervous diseases, for example, hemorrhagic stroke, especially during the rehabilitation period after this diseases as drugs with potential neuroprotective and psychoactivating effects.

Example 5. Study of the effect of drugs MGTB-S and MTGB-M on the development of Parkinson's disease in an experiment in rats in vivo.

5.1. An experimental model of Parkinson's disease in rats in vivo

Description of experimental animals, investigational preparations and experimental conditions.

The study of the action of drugs MHTB-S and MHTB-M with chronic administration was carried out on 60 animals (Wi rats, males, 2.5 months, about 190 g) on an experimental and induced model of Parkinson's disease (PD). A total of 6 groups of 10 animals were formed.

- control group No. 1 - animals were not injected with anything - control group No. 2 - with a model of experimental BP

- control group No. 3 - with induced PD and administration of the reference drug L-DOPA/benserazide (1:4)

- experimental group No. 4 - with induced PD and intranasal administration of the drug MHTB-M (from the brain)

- experimental group with induced PD and intranasal administration of MHTB-C (from blood serum)

- experimental group with induced PD and combined intranasal administration of drugs MHTB-M and MHTB-S administered every other day (one day - one drug, the next day

- another drug).

For the administration of MHTB-M and MHTB-S preparations, the intranasal route of administration was chosen as an effective method of delivery to the brain. Drug delivery by this route is seen as a modern alternative to oral administration, since the nasal cavity is easily accessible, richly vascularized, well permeable and has a large surface area, which contributes to good absorption and increased bioavailability. In addition, drugs enter the bloodstream, avoiding the "first pass" effect through the liver.

It is known that the high efficiency of intranasal administration of dopamine was confirmed experimentally in Wistar rats with a model induced by 6-hydroxydopamine (6-OHDA). Found relief of symptoms of the disease, assessed in tests for rotation and staging of the forelimb [PumM. et al., 2009; OwenY.C. et2012].

In addition, intranasal administration is traditionally used to deliver peptide drugs to the brain [Kamensky A.A. et al., 1988; Veronica G.D. et al., 2009].

3,4-dihydroxyphenylalanine (L-DOPA, Sigma) was chosen as a reference drug in combination with a peripheral decarboxylation inhibitor that prevents the drug from being converted into dopamine in peripheral tissues (benserazide, Sigma) in a ratio of 1:4, which reproduces the standard drug for treatment Parkinson's disease, used in therapy to reduce severe symptoms of the disease. The drug eliminates hypokinesia, rigidity, tremor. The action of L-DOPA is due to the conversion to dopamine directly in the central nervous system, which leads to the replenishment of dopamine deficiency in

CNS. The therapeutic effect in humans is observed after 6-8 days, and the maximum - after 25-30 days.

In this experiment, the animals were injected with the maximum possible dose, taking into account the route of administration, the minimum amount of administration and the low solubility of the L-DOPA substance.

All preparations were administered to animals in the nasal passages. The volume of a single single injection was up to 40.0 μl (20 μl in the nasal passage, with each injection). The reference drug L-DOPA at a concentration of 3.0 mg/ml was prepared weekly, immediately before the start of the experiment. A saline solution with the addition of ascorbic acid (1%, pH 2.6) was used as a solvent to improve solubility and prevent oxidation of the drug upon dissolution. For optimal dissolution of L-DOPA, the pH of the solution was reduced to 1.5, and after dissolution of the preparations, the pH value was increased to a value no higher than 7.0 (strictly). The solution was stored at +4°C for a week, stopping use when a yellowish tint of the solution appeared, indicating oxidation of the active substance. The concentration of benserazide inhibitor was 0.75 mg/ml.

Control animals were injected with saline by intranasal administration.

The experiment was carried out on 60 Wistar rats, male, 4 months old, weighing about 400 g.

Animals were kept under controlled environmental conditions (18-26°C and 30-70% relative humidity). Temperature and humidity were constantly monitored in each experimental room and manually documented once a day. Animal housing rooms were maintained on a 12 hour lighting cycle and at least 10 times the room air volume change per hour.

Animals were adapted/acclimatized in the laboratory for 14 days prior to administration. During this period, daily examination of the external condition of the animals was carried out. Animals with abnormalities detected during the examination were not included in the experimental group.

Animals without signs of appearance deviations were selected in the experimental group, according to the results of preliminary tests, so that the individual weight value did not deviate from the average value within the sex by no more than 20%, and the average body weight values in the groups did not differ significantly.

Each animal was assigned an individual number, marked with color and fixed on the cage card.

Mortality, severe condition, general health, and signs of toxicity were assessed daily.

Clinical examination was carried out weekly.

Body weight was recorded before administration and weekly during the administration period.

Obtaining a pharmacological model of Parkinson's disease in rats in vivo

The experiment used a pharmacological model of Parkinson's disease (PD) using the neurotoxin 6-OHDA (6-hydroxydopamine, Sigma) to destroy the compact part of the substantia nigra. The toxin was injected unilaterally, into the right side of the brain, using a stereotaxic device for rats according to the standard method, along the coordinates АР-4.4; L-1.2; H 7.8, which corresponds to the right medial anterior bundle [CenciM. etal, 1998; Hanna S.L. et al., 2007]. The operation was performed under general anesthesia. Rats were intraperitoneally injected with a solution of chloral hydrate (Chloralhydrat, ALDRICH) at a dose of 400 mg/kg, 100 mg/ml in physiological saline (Nizhpharm). After fixing the animal, a hole was drilled in the skull at the selected coordinates, then the needle of a cannula filled with a solution of 6-OHDA in degassed saline (4 µg/µl) or, in the case of animals from the intact control group, with degassed saline was inserted. Using a 10 µl measuring syringe, 4.5 µl of the solution was injected at a rate of 1 µl/min. After insertion, the needle was left in the hole for another 5 minutes. After the operation, the wound was decontaminated and sutured, the rats were placed in cages and their condition and healing process were observed for 2 weeks.

Tests to confirm the development of the Parkinson's disease model

a) "Rotation caused by the administration of apomorphine" for evidence of the development of Parkinson's disease

The development of neurodegeneration was confirmed 3 weeks after surgery by registration of rotation in response to intraperitoneal administration of a solution of apomorphine (R-(-)-Apomorphine hydrochloride hemihydrate, Sigma) 1.6 mg/kg (0.8 mg/ml, in saline). 10 minutes after the injection, the rat was placed in a glass cylinder with a radius of 30 cm and a height of 40 cm. Over the next 10 minutes, the number of complete revolutions made by the rat in each direction was recorded. The degree of neurodegeneration in animals was judged by the number of revolutions per minute in the direction opposite to the side of brain damage. Animals that performed more than 2 revolutions/min were selected for participation in the further experiment.

b) "Open field" to obtain preliminary data on motor and exploratory activity.

The "Open field" test was used to assess the possible consequences of chronic administration of drugs on the motor and exploratory activity of experimental animals in conditions of free behavior. The test was carried out according to the standard method [Titov S.A. et al., 1980]. The following parameters were recorded for 2 minutes: horizontal activity, the number of exits to the center of the "open field", the number of racks, the number of grooming acts.

c) "Setting the limb" to assess the severity of inhibition of motor functions.

The limb positioning test was used to assess the coordination of movements of animals with unilaterally destroyed substantia nigra. The animal was held with one hand under the abdomen, parallel to the edge of the laboratory table, below its surface. Then slowly lifted so that the vibrissae touched the edge of the table, and at the end point of the lift, the front paw of the corresponding side of the body was higher than the edge of the tabletop. The criterion for performing an attempt was the placement of the forelimb of the side of the body closest to the table on the surface of the table. The test was repeated 10 times for each side, recording the number of successful attempts.

5.2. Tests to identify the effect of investigational drugs on the course of Parkinson's disease over time.

Weekly:

Test "Staging of the limb" to assess the severity of inhibition of motor functions and the effectiveness of treatment

The test was used to assess the coordination of movements of animals with unilaterally destroyed substantia nigra. The animal was held with one hand under the abdomen, parallel to the edge of the laboratory table, below its surface. Then slowly lifted so that the vibrissae touched the edge of the table, and at the end point of the lift, the front paw of the corresponding side of the body was higher than the edge of the table top. The criterion for performing an attempt was the placement of the forelimb of the side of the body closest to the table on the surface of the table. The test was repeated 10 times for each side, recording the number of successful attempts. The test is carried out twice, 30 minutes before and 30 minutes after the next dose of the drug to assess the duration of the effect.

One-time tests (at least a month interval between tests):

a) "Open field" for obtaining data on motor and exploratory activity, general condition of animals, depression.

The "Open field" test was used to assess the possible consequences of chronic administration of drugs on the motor and exploratory activity of experimental animals in conditions of free behavior. The test was carried out according to the standard method [Titov S.A. et al., 1980].

The behavior of the rats was studied in the "open field" in accordance with generally accepted standards. "Open field" - a square evenly illuminated area 80 × 80 cm in size, fenced with opaque walls 15 cm high. The floor of the "open field" is divided into squares with a side of 20 cm. Initially, the animal is placed in the center of the field, after which the experimenter fixes behavioral activity for 5 minutes.

The following parameters were recorded for 2 minutes: horizontal activity, the number of exits to the center of the "open field", the number of racks, the number of grooming acts.

b) Moving along a horizontal grid with video recording to assess the severity of the inhibition of motor functions and the effectiveness of treatment.

The "Movement on a horizontal grid" test was used to reveal asymmetry in the coordination of limb movements caused by unilateral degradation of the substantia nigra. The installation was a square frame 40 × 40 cm raised above the table surface by 40 cm with a metal mesh stretched over it. The side of the cell was 2.5 cm. The animal was placed on the device and its movements were recorded on a video camera for 2 minutes. The subsequent analysis of the video recording included counting the total number of steps of each of the forelimbs in 2 minutes, as well as the total number of “mistakes” - misses in the setting, when the forelimb does not find support and falls below the grid level [Chaoetal., 2012].

The percentage of errors for the limb was calculated using formula 1:

Asymmetry coefficient (%) was calculated by formula 2:

Data analysis

For all data, descriptive statistics were used: the mean value and standard deviation were calculated, which, together with the N value, are presented in the final tables.

Statistical analysis was carried out using the Statistica program. Differences were determined at 0.05% confidence level and indicated by *.

5.3. RESULTS AND DISCUSSION

Limb staging test, acute administration

The test was performed 30 minutes after the first administration of the drugs. The average result in the healthy control group was taken as 100% of the result. The results of the remaining groups were:

in group No. 2 (negative control) - 63±19%;

in group No. 3 (receiving the reference drug L-DOPA/benserazide) - 85*±15%;

in group No. 4 (receiving MHTB-M) - 68±19%;

in group No. 5 (receiving MHTB-S) - 59±27%;

in the group (treated with MHTB-S/MHTB-M) - 70±19%

There was a significant difference in the mean test results for the L-DOPA/benserazide group compared to the negative control group (p<0.05). Animals from the other treated groups did not show a difference in mean test results from the untreated group of animals with the PD model (negative control). The results of all groups of animals with an induced PD model were significantly different from the results of animals in the healthy control group.

Test "Staging of the limb", subchronic administration.

The test was carried out once a week, throughout the entire period of administration - 4 weeks. Animals were tested immediately before drug administration and 30 minutes after administration. Testing animals in this mode with chronic administration made it possible to identify the effectiveness of the drug immediately after administration and to study the effect of the drugs a day after administration.

The test results were obtained 30 minutes after the administration of drugs in dynamics. It is important to note that the comparator drug L-DOPA was effective 30 minutes after administration throughout the entire study period. At the same time, significantly significant differences from the negative control group were obtained only with acute use (85±15%).

With subchronic administration, the effectiveness of the study drug MHTB-M was revealed during the entire period of administration. Significant differences in the average results of the test for the group of animals No. 4, treated with MHTB-M, from the group No. 2 of the negative control of PD were revealed by the end of the third week of the drug administration, while the value of the average result reached 87±21%, while maintaining the effect until the end of the period introduction - within 2 weeks.

The effectiveness of the second study drug MHTB-C was also found, but to a lesser extent, without achieving significantly significant results.

With combined administration (combined use of two study drugs MHTB-S and MHTB-M in a set with a time interval of one day), an improvement in the ability of animals to perform the test during the entire study period was also found, compared with the negative control, while a significant difference in the results this group of animals was detected after a week of drug use (90±13%).

It is important to note that by the end of the drug administration period, all groups of animals treated with study drugs, when tested 30 minutes after administration, showed an improvement in the ability to perform the test to a greater extent compared to the group of animals receiving the reference drug. Mean values were 74±27%, *87±14%, 71±18%, and 65±24%, respectively, for groups treated with MHTB-S, MHTB-M, the combination of MHTB-S and MHTB-M (set of two drugs with a time interval of one day), as well as the comparison drug, while this ability to perform the test in animals of the negative control group decreased to 50%.

The results of testing animals before the next dose of the drug or, respectively, 24 hours after the previous dose showed that significantly significant results were obtained for the drug MHTB-M after three weeks of administration (82±21%), with the effect remaining by the end of the fourth week (82±14%). Thus, the ability of the drug to improve the motor functions of animals was revealed, which is reflected in the increase in the efficiency of the "Staging of the limb" test after 3 weeks of administration, 30 minutes after administration, with the preservation of the effect after 24 hours.

At the same time, for groups of animals treated with MHTB-C or a combination of MHTB preparations, by the end of the period of drug administration (4 weeks), it was also noted that the test was more effective until a new dose was administered compared with the group of animals that received the comparator drug. This, of course, indicates a positive effect of the studied drugs.

The study of this time point allows us to estimate the duration of the effect of the drug administration.

Thus, the ability of the drug MHTB-M to improve the motor functions of animals was revealed, which is reflected in an increase in the efficiency of the "Setting the limb" test after 3 weeks of administration, 30 minutes after administration, with the preservation of the effect after 24 hours.

The results of the study of the effectiveness of drugs in the test "Staging the limb" by weeks of administration also made it possible to evaluate the effectiveness of the studied MHTB drugs.

1 week of introduction. Reliable efficacy of using a combination of MHTB drugs (MHTB-S + MHTB-M) in the form of a pharmacological kit placed in separate vials was revealed. It should be noted that the result was detected only 30 minutes after the next injection of the drug and was absent 24 hours after the injection. The comparator effect shown with acute administration was not seen one week after the start of therapy, which can be explained by the "dose wasting effect" for L-DOPA.

2 weeks of introduction. When testing after two weeks of administration, no significant effect was found for any of the study drugs.

3 weeks of introduction. Testing of animals with a PD model after three weeks of drug administration revealed a significant improvement in the test results for the group that received the MHTB-M drug - the result of the group 30 after the next injection of the drug was 87 ± 21% of the healthy control result, while the result of the negative control group control was expressed as 52±36%.

It is important to note that significant differences with the results of the negative control were also recorded during the test immediately before the next administration of the drug, that is, the effect of therapy persists for at least 24 hours after administration. The result in testing before the administration of the drug was for the group treated with MHTB-M, 82±21% of the result of the healthy control group.

4 weeks of introduction. Testing after four weeks of drug administration confirmed a significant improvement in the results of the group that received the MHTB-M drug - the result of the group 30 minutes after the next injection of the drug was 87 ± 14% of the healthy control result and significantly differed from the PD control group.

Significant differences with the results of the negative control were also recorded immediately before the next administration of the drug, which confirms the observation of a prolongation of the effect of therapy.

It is important to note that significant differences were recorded not only in relation to the negative control group, but also in relation to the group receiving the reference drug.

The results of the test "Open field"

The "Open field" test was performed twice, before the start of drug administration and after 3 weeks of drug administration. There were no significant changes in any of the studied parameters. Such studied parameters as the total mileage and the number of racks did not differ between the groups, and the number of grooming acts and exits to the center had too high an individual scatter for a qualitative analysis.

Due to the fact that modeling of PD practically does not affect the test results (the negative control has no significant differences from the healthy control), the absence of changes and differences between the groups receiving drugs and the control indicates the absence of the effect of drugs on the exploratory and motor activity of animals. .

The results of the test "Movement on a horizontal grid"

The test was performed once, after three weeks of drug administration, 30 minutes after drug administration.

The number of steps with each forelimb and the number of errors during their setting were recorded. Based on these data, the asymmetry coefficient was calculated: K(asymmetry)=K(errors of the left) - K(errors of the right)

Animals with an induced unilateral PD model (damaged substantia nigra on the right side of the brain) were found to be less able to control movements of the left forelimb. In animals of the healthy control group, the asymmetry coefficient is almost zero, while in animals of the negative control it is on average 5%.

It is important to note that, according to the results of this test, all study drugs effectively managed the symptoms of PD. The results of the three groups, animals treated with the studied MHTB preparations individually and in combination, significantly differed from both the result of the negative control group and the result of the group that received the comparator drug. At the same time, the results obtained in the study of groups of animals treated with MHTB preparations did not have significant differences with the results of the healthy control group.

The obtained results of the "Movement on a horizontal grid" test are fully consistent with the results obtained in the "Limb staging" test and confirm both the presence of motor dysfunctions in animals with induced Parkinson's disease and the significantly higher efficacy of nasal therapy with MHTB drugs compared to standard drugs. in the treatment of Parkinson's disease.

findings

1. The development of a PD model in rats was confirmed three weeks after the administration of the neurotoxin 6-ONDA (standard method).

2. A study was made of the effect on the motor functions of animals with a model of PD of drugs-bioregulators of the MHTB group isolated from the serum of cattle (MHTB-S) and from the brain of cattle (MHTB-M), as well as their combined use by administration every other day. versus generic standard antiparkinsonian L-DOPA/benserazide (1:4)

3. The following effects were found: the reference drug significantly improved the motor functions of animals only after a single application. At the same time, the effect of daily nasal administration, detected in the “limb staging” test 30 minutes after administration, was noticeable throughout the entire administration period and differed by 10-20% from the corresponding values of the PD control group. It is important to note that the effect of the reference drug was not maintained when testing animals 24 hours after administration. The obtained results are consistent with the literature data, as well as the known characteristics of the drug L-DOPA/benserazide (1:4), as it is rapidly metabolized and has an effect in a single dose for a short time, which leads to the need for a constant increase in the dose and frequency of taking the drug in humans with PD .

4. The effectiveness of the study drug MHTB-M was revealed in the subchronic administration in the "Staging of the limb" test.

Significant differences in the average results of the test 30 minutes after administration for the group of animals treated with MHTB-M, from the group of negative PD control, were revealed by the end of the third week of drug administration, while the average result reached 87 ± 21% of the healthy control group, with the preservation of the effect until the end of the period of administration - within 2 weeks.

It is important to note the significant preservation of the drug effect even 24 hours after administration, which indicates a stable improvement in motor functions in animals for at least a week, from the end of the third to the end of the fourth week of administration.

In addition, the introduction of the drug MHTB-M led to a complete restoration of the coordination of animals in the test "Movement on a horizontal grid". The results obtained had a significant difference from the PD control group and the group that received the comparator drug (p<0.01), and did not have significant differences from the results of the healthy control group.

The efficacy of the second study drug, MHTB-S, was also found, but to a lesser extent, without achieving significantly significant results in the limb staging test. At the same time, by the end of the third week of daily administration of the drug, the results of the “Movement along the horizontal grid” test had a significant difference from the PD control group and the group that received the reference drug (p<0.01), and did not have significant differences from the results of the healthy group. control.

With the combined administration of the two studied drugs MHTB-S and MHTB-M (in the form of a kit according to the invention), an improvement was also found in the ability of animals to perform the limb staging test during the entire study period compared to the negative control, with a significant difference in the results of this group animals were detected 30 minutes after a week of drug use. The effect did not persist 24 hours after administration, but the results of the group were slightly higher relative to the reference drug. The test "Movement along the horizontal grid" also revealed a significant difference between this group and the PD control group and the reference drug (p<0.05) and the absence of significant differences from the results of the healthy control group.

The "Open Field" test did not reveal any differences between the groups and, accordingly, any effect of drugs on the research activity of animals and their level of anxiety.

Conclusion.

The studied preparations MHTB-S and MHTB-M, prepared on the basis of bioregulators of the MHTB group obtained from the serum and brain of cattle (bovine), when administered nasally in the form of a solution at a concentration of ^10–12 mg/ml, are highly effective in two of the used tests on a pharmacological model of Parkinson's disease in Wistar rats. Along with efficiency, 30 minutes after injection, an increase in the duration of the effect of the MHTB-M preparation was found, which lasted at least a day between injections already after three weeks of drug administration, with confirmation of the effect after a week. The drugs significantly improved the coordination of animal movements. Thus, it can be concluded that the studied drugs have a high clinical potential and are promising for use as antiparkinsonian drugs.

Example. 6. Effect of MHTB-S and MHTB-M preparations on the inhibition of DTT-induced BSA aggregation (chaperone activity)

We studied the effect of MHTB-C and MHTB-M preparations on dithiothreitol (DTT) induced aggregation of model proteins (bovine serum albumin-BSA) over a wide range of chaperone concentrations.

DTT-induced aggregation of BSA was studied in water at 50°C. The BSA solution was placed in a cylindrical vial with an inner diameter of 6.3 mm and preincubated for 5 min at 50°С. The vials were stoppered to prevent evaporation. The aggregation process was initiated by adding an aliquot of DTT to the protein solution to a final volume of 0.5 ml. The measurement was carried out on a PhotocorCompact-Z analyzer (Russia), obtaining the correlation function of the scattered light intensity fluctuations and the integrated scattering intensity. The correlation function was processed using the DynaLS software from Alango Ltd (Israel). The range of acceptable measured sizes of nanoparticles is in the range from fractions of a nm to 5-10 microns. The measurements were carried out at a scattering angle of 90°.

To study the effect of the studied MHTB preparations on DTT-induced BSA aggregation, a solution of the studied MHTB was added in a wide range of concentrations to a preheated solution of the model protein before adding DTT.

According to the results of the study, it was found that MHTB-C at a protein concentration corresponding to 18 μg/ml exhibits chaperone-like activity, which was expressed in blocking the intermolecular aggregation of BSA (75-200 nM) induced by dithiothreitol. The MHTB-M preparation at a protein concentration corresponding to 10 μg/ml also showed chaperone-like activity, which was expressed in the complete suppression of intermolecular aggregation of BSA (75-200 nM) induced by dithiothreitol.

The manifestation of MHTB-C and MHTB-M chaperone-like activity suggests that these drugs can be effective in neurodegenerative diseases, which are based on the formation of intermolecular protein associates in the brain tissue that disrupt the transmission of nerve signals (for example, Alzheimer's disease, in which amyloid-beta aggregation and plaque formation in the brain tissue).

Claims (4)

1. A kit for improving the blood flow of the cerebral cortex and restoring the motor and cognitive functions of the body, which includes two biologically active substances placed in different vials and administered separately in a pharmaceutically acceptable carrier intranasally, orally or intramuscularly or intravenously, isolated from blood serum and from brain tissue bovine brain, protein-peptide complexes containing peptides with molecular weights of 1000-6000 Da, and a protein that is a member of the mammalian albumin family under the number gi | 1351907 with molecular weights of 66267 Da and 66667 Da, isolated from blood serum and from brain tissue bull, respectively, and exhibiting biological activity in the range of their concentrations from 10 ^-15 to 10 ^-8 mg/ml when diluted in a pharmaceutically acceptable carrier and/or diluent. 2. A set for improving blood flow in the cerebral cortex and restoring the motor and cognitive functions of the body according to claim 1, characterized in that it is intended for the combined joint intake of two biologically active substances as bioregulators placed in different vials. 3. A kit for improving blood flow and restoring the motor and cognitive functions of the body according to claim 1 or 2, characterized in that the protein-peptide complexes included in it are administered in an aqueous saline physiological solution intranasally, intramuscularly, parenterally or administered in distilled water for oral intake. 4. A kit for improving blood flow in the cerebral cortex and restoring the motor and cognitive functions of the body according to claim 3, characterized in that the protein-peptide complex from blood serum is administered intranasally, and the protein-peptide complex isolated from bull brain tissue is administered intramuscularly.

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