RU2803286C1 - Composition for neuroprotection and stimulation of brain neuroregeneration after injury, agent based on it, a method of its production and use - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: group of inventions can be used to obtain and use medicines and biomaterials for neuroprotection and stimulation of brain tissue regeneration after injuries of various origins. A composition for neuroprotection and stimulation of brain neuroregeneration is proposed, the said composition is based on the components of the secretome of human mesenchymal stromal cells (MSCs) characterized by the presence of growth factors and cytokines in the culture medium including BDNF in an amount of at least 3 ng/ml, GDNF in an amount of at least 0.03 ng/l, VEGF in an amount of not less than 0.2 ng/ml, HGF in an amount of not less than 0.15 ng/ml, uPA in an amount of not less than 0.34 ng/ml and TGFb in an amount of not less than 0.055 ng/ml, besides the use of the indicated compositions as an agent for preventing glutamate-mediated death of neural cells, stimulating the polarization of cells of the monocyte-macrophage link in the anti-inflammatory direction (M2-phenotype), preventing the activation of microglial cells is proposed. The method of preparing the composition includes cultivating and conditioning primary human allogeneic MSCs isolated from adipose tissue for 2–5 passages in Dulbecco’s Modified Eagle Growth Medium (DMEM) with a glucose content of not more than 1 g/l without phenol red with the addition of penicillin/streptomycin, solutions of essential amino acids and vitamins, for 72–144 h, selection and purification of the culture medium from cell residues, concentration and purification of this medium to obtain a sterile concentrate. Also the following is proposed: an agent for neuroprotection and stimulation of neuroregeneration, including the specified composition in a therapeutically effective amount and auxiliary components, and a method of stimulating neuroprotection and regeneration of the brain after injury, including the introduction of the above composition or agent into the human body in a therapeutically effective amount.

EFFECT: reduction of neurological disorders severity within 14 days after modeling a stroke in experimental animals, and a decrease in the volume of brain damage by on average of 2 times according to MRI and histological examination compared with the control group (without composition), as well as a decrease in cell activation microglia to the basal level in the model of acute cerebrovascular accident by hemorrhagic type in the in vivo experiment.

24 cl, 17 dwg, 9 ex

Description

Field of technology to which the invention relates

The invention relates to the field of medicine and pharmacology and can be used for the production and use of drugs and biomaterials for neuroprotection and stimulation of regeneration of brain tissue after damage of various origins: mechanical, thermal, chemical (toxic, metabolic), radiation, combined, caused by acute brain damage blood circulation of the hemorrhagic or ischemic type, as well as infectious, including those caused by microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS-Cov2 virus.

State of the art

Brain damage (acute cerebrovascular accidents, trauma, toxic damage, etc.) is one of the main causes of death and disability in people in the world, and the number of such pathologies, according to expert estimates, will only increase due to increasing life expectancy and general aging of the Earth's population [Suslina Z.A. Clinical guidelines for early diagnosis, treatment and prevention of cerebrovascular diseases / Z.A. Suslina, Yu.Ya. Varakin. - M.: MEDpress-inform, 2017. - 352 p.; Strong, K. Preventing stroke: saving lives around the world / K. Strong, C. Mathers, R. Bonita // Lancet Neurol. - 2007. - Vol. 6. - P. 182-187]. At the same time, there are no available and effective therapeutic approaches that can stop the progression of the pathological process and stimulate the restoration of brain tissue.

In the pathogenesis of acute brain injuries of various origins, common key pathogenetic mechanisms are identified: ischemic damage to neurons and glial cells, toxic effects of blood and metabolites, glutamate-mediated excitotoxicity, inflammation and secondary damage to brain tissue. The main disadvantage of existing drugs and therapeutic approaches is their focus on relieving symptoms (manifestations of the disease) or influencing only part of the pathogenetic mechanisms of the disease: most often, this is the suppression of neuronal death due to ischemic, toxic or excitotoxic effects. Existing drugs (due to the lack of appropriate components or their origin from animal cells and tissues) do not have the ability to suppress the activation of microglial cells, inflammation at the site of injury and secondary damage to brain tissue. It is the complex counteraction to the pathogenetic mechanisms mentioned above that will increase the resistance of brain tissue to the negative effects of a damaging factor, stop the progression of the disease and accelerate the process of regeneration of brain tissue, and this approach can be considered effective for a wide range of pathologies due to the commonality of their pathogenetic mechanisms. In addition, the vast majority of existing patents and publications do not describe the key parameters (composition or agent, ratio of its components, method and frequency of administration, dosage and time therapeutic window) necessary for effective treatment of brain damage.

Compositions are known from the prior art for stimulating the restoration of nervous tissue after damage (including cerebral infarction, cerebral stroke, encephalorrhagia, subarachnoid hemorrhage, brain tumor, traumatic, ischemic, degenerative diseases of the cranial nerves) by administration (including . locally and intravenously) mesenchymal cells (including autologous) obtained from bone marrow, umbilical cord blood or peripheral blood [US20070178591]. A modification of this composition is the use of mesenchymal cells into which the BDNF gene, the PLGF gene, the GDNF gene or the IL-2 gene are introduced; or an immortalized mesenchymal cell introduced with the hTERT gene. However, the source does not disclose information about the range of therapeutic concentrations and the ratio of growth factors, does not contain data on the possibility of their use for neuroprotection of brain tissue and stimulation of brain recovery in thermal, chemical (toxic, metabolic), radiation and combined damage, as well as damage caused by infectious agents (including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS-Cov2 virus) and acute cerebrovascular accident of the hemorrhagic type, and the modes of administration of the composition are not presented, namely, the time therapeutic window, dose and the frequency of administration; in addition, the introduction of cells into the area of brain damage is possible only during a complex surgical intervention within a hospital; intravenous administration of cells is associated with a high risk of thrombosis and embolism; and the use of autologous cells makes it impossible to use this composition in the acute period, which greatly reduces the effectiveness of treating conditions after acute brain damage.

A composition is known from the prior art for stimulating the restoration of nervous tissue after hemorrhagic stroke, including components of a culture medium (including exosomes or other microvesicles (for example, apoptotic bodies)), in which fibroblasts isolated from bone marrow, adipose tissue, and placenta grew , umbilical cord with their introduction into the area of damage [WO2021097423]. The composition includes: leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF ), hepatocyte growth factor (HGF), IFN-g, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophic factors (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-b), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor -BB (PDGF-BB), transforming growth factors beta (TGF-b). The composition may also include a culture medium with one or more hematopoietic cells (can be obtained from one or more sources, including adipose tissue, bone marrow, peripheral blood, mobilized peripheral blood and umbilical cord blood) or mesenchymal stem cells (can be obtained from adipose tissue , bone marrow, peripheral blood, etc.), incl. genetically modified (do not express HLA, CD34 or CD14). However, the source does not disclose information about the range of therapeutic concentrations of growth factors, and also does not indicate the time therapeutic window and frequency of administration; moreover, the cells used are fibroblasts, which limits the regenerative potential of their conditioned environment, since fibroblasts synthesize profibrotic growth factors in large quantities (TGF-b) and components of the extracellular matrix, including collagens, which inhibit the regeneration of nervous tissue and stimulate gliosis.

The prior art knows a means for stimulating tissue regeneration based on components of a serum-free culture medium (secretion products) in which human MSCs isolated from adipose tissue grew and a method for its preparation [RU2620167]. The product contains secretion products of human MSCs, including growth factors: VEGF at a concentration of at least 200 pkg/ml, HGF at a concentration of at least 150 pkg/ml, FGF basic at a concentration of at least 0.29 pkg/ml, angiopoietin-1 at a concentration of less than 145 pg/ml, PDGF in a concentration of at least 500 pg/ml. However, this product does not contain growth factors with neuroprotective activity (for example, brain-derived neurotrophic factor BDNF) or molecules that regulate neuritogenesis processes (for example, urokinase plasminogen activator), as well as molecules with anti-inflammatory activity (interleukins, IDO, TNF), which does not imply the possibility of the components of this drug influencing key pathogenetic mechanisms in brain damage does not imply the possibility of its use for the treatment of brain damage. To obtain the product, cultivation of human MSCs isolated from adipose tissue is used in AdvanceSTEM Cell Culture Media, cultivation in which gives lower cell culture productivity than when cultivated in DMEM with a low glucose content without phenol red. In addition, the method of use of this drug does not describe its use for neuroprotection, as well as the critical parameters for the treatment of brain damage: the temporary therapeutic window, dose and frequency of administration.

In the prior art, a composition for treating, inhibiting, reducing, alleviating and/or preventing an inflammatory condition, an autoimmune disease, an autoinflammatory disease, a metabolic disorder or inflammation is known, which is a culture medium in which multipotential stromal cells isolated from adipose tissue have grown [WO2021011935] . However, the use of this composition is not intended for the purpose of neuroprotection and stimulation of brain tissue regeneration after mechanical, thermal, chemical (toxic, metabolic), radiation and combined damage, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents , including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS-Cov2 virus, information is not provided on the range of therapeutic concentrations of growth factors, the time therapeutic window, dose and frequency of administration are not indicated.

The prior art knows a therapeutic agent for neuroprotection after lesions of the central and peripheral nervous system of various origins by introducing into the area of damage components of the conditioned medium of mesenchymal stromal cells (MSCs) isolated from the human umbilical cord [RU2742034]. The conditioned medium contains G-CSF (granulocyte colony-stimulating factor), HGF (hepatocyte growth factor), IFN-a2 (interferon alpha-2), IL-6 (interleukin-6), IL-8 (interleukin-8), SCF (stem cell factor), SCGF (stem cell growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblast growth factor). However, the proposed remedy does not take into account the fact that the main neuroprotective function in the composition of the conditioned environment is performed by neurotrophic factors, does not take into account their contribution and does not evaluate their therapeutic concentrations. The description of this composition does not contain information about the time therapeutic window, dose and frequency of administration for the purpose of neuroprotection and stimulation of brain tissue regeneration after damage; moreover, the cells used are obtained from umbilical cord tissue, which imposes a number of ethical restrictions on their use.

The closest to the claimed composition is a composition of cells obtained from amniotic fluid or factors produced by these stem cells, as well as a method for inducing and accelerating neurological recovery after ischemic and hemorrhagic strokes, brain injury, etc. by introducing the specified composition [US2018071342]. These factors may include proteins, peptides, conditioned media, exosomes or microvesicles. Components of the medium may include activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, bFGF, B61, cadherins, CAM-RF, cGMP analogues, ChDI, CLAF, claudins, collagen, collagen receptors α1β1 and α2β1, connexins, Cox-2, ECDGF (endothelial cell growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growth inhibitor, maintenance factor endothelial cell viability, sphingolipid G protein-coupled receptor-1 (EDG1), ephrin, Epo, HGF, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor α5.beta.1, factor X, HB-EGF, HBNF, HGF, HUAF, cardiac inhibitor of vascular cell proliferation, IL1, IGF-2 IFN-gamma, integrin receptors, K-FGF, LIF, growth factor derived from leiomyomas, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, ME CIF, MMP 2, MMP3, MMP9, urocase plasminogen activator, neuropilin (NRP1, NRP2), neurothelin, nitric oxide donors , nitric oxide synthase (NOS), Notch, occludins, zonal occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2 , PPAR-gamma, PPAR-gamma ligands, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-smooth muscle cell-derived growth factor, sphingosine-1-phosphate-1 (SIP1), Syk, SLP76, tachykinins, TGF- beta, Tie 1, Tie2, TGF-(and TGF-(receptors, TIMP, TNF-alphatransferrin, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF164, VEGI, EG-VEGF. However, the description of the composition lists a wide range of growth factors and low molecular weight compounds without indicating the range of their concentrations or their ratios, and also simultaneously indicates factors that neutralize each other’s activity (MMP 2, MMP3, MMP9 and TIMP; TGF-(and TGF-(receptors; PDGF-B and PDGF receptors; urokinase, VEGF-A/B/C/D/E and cardiac inhibitor of vascular cell proliferation, etc.), which does not allow one to expect from the proposed composition (especially in the absence of information on the ratio of factor concentrations ) real therapeutic activity (the ability to provide a non-protective and pro-regenerative effect) in relation to damaged brain tissue. The physiological effects of growth factors and cytokines and the therapeutic activity of the drug based on them are largely determined by their concentrations and ratio in comparison with their antagonists, since in order to realize their effect they must penetrate the area of damage, overcome the inhibitory effect of the antagonist and saturate the corresponding receptor, providing the necessary duration and strength of the effect. Also, the source does not disclose details of the method for obtaining this composition, in particular, the medium for culturing MSCs is not indicated, although it is known that the composition of the culture growth medium for MSCs affects the qualitative and quantitative composition of the resulting secretome. In addition, the source does not disclose the route of administration, the temporary therapeutic window, the dose and frequency of administration for the prevention and treatment of acute damage to brain tissue. This information is integral in developing approaches for the treatment of brain damage and the prevention of complications, especially acute cerebrovascular accidents, since timely provision of medical care, an adequate delivery route and dose of a therapeutic drug are the basis for reducing mortality and the severity of neurological disorders in such conditions. In addition, to develop the composition, cells isolated from amniotic fluid were used, the obtaining of which requires the use of invasive and painful methods and is less promising in terms of its availability and safety for the donor, and is also associated with a number of ethical restrictions.

The technical problem to be solved by the claimed invention is the development of a composition for neuroprotection and stimulation of neuroregeneration of the brain after injury, a product based on it, as well as methods for its preparation and use, with the aim of reducing mortality and disability among such patients, which has a complex effect. Brain damage includes mechanical, thermal, chemical (toxic, metabolic), radiation and combined damage, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents, including microorganisms of the genera Neisseria, Borrelia and coronaviruses infections, including the SARS-Cov2 virus.

Disclosure of the Invention

The technical result of the claimed invention is the survival rate of experimental animals up to 100%, a reduction in the severity of neurological disorders - up to 100% of animals are visually healthy 14 days after a simulated stroke, and a reduction in the volume of brain damage by an average of 2 times according to MRI and histological examination compared with control group (without composition), as well as a decrease in the activation of microglial cells to the basal level in a model of acute hemorrhagic cerebrovascular accident in an in vivo experiment. In addition, the claimed composition stimulates the polarization of cells of the monocyte-macrophage series in the anti-inflammatory direction (M2 phenotype), including in pro-inflammatory conditions, 2-6 times (depending on the measured marker) more effectively compared to the control group (without compositions); and also stimulates the survival of SH-SY5Y neuroblastoma cells under conditions of glutamate-mediated toxicity 4-40 times (depending on the composition of the composition and the time of the study) more effectively compared to the control group (without composition) - in in vitro experiments.

A full comparison of the therapeutic activity of the claimed invention with the prototype is impossible due to the lack of publicly available experimentally confirmed information about the presence of such activity in text or graphic form.

The effectiveness of the claimed composition is achieved due to the balanced action of the components of the conditioned medium (CM) of human mesenchymal stromal cells (MSCs) or its individual fractions on the key participants in damage to nervous tissue during brain damage: on neurons, micro- and macroglial cells, as well as on monocytic cells. macrophage link of hematogenous origin.

The technical result is achieved by a composition for neuroprotection and stimulation of brain neuroregeneration based on components of the human MSC secretome, characterized by the presence of growth factors and cytokines in the culture medium, including BDNF in an amount of at least 3 ng/ml, GDNF - at least 0.03 ng/ml, VEGF - not less than 0.2 ng/ml, HGF - not less than 0.15 ng/ml, uPA - not less than 0.34 ng/ml and TGFb (total for TGFb1, TGFb2 and TGFb3 isoforms) - not less than 0.055 ng/ml and containing not less than 5 μg/ ml for total protein. The composition may additionally contain anti-inflammatory cytokines, including the IL-1 receptor antagonist IL-1Ra in an amount of at least 0.4 ng/ml, IL-4 - at least 0.08 ng/ml, IL-6 - at least 1 ng/ml, IL-10 - not less than 0.01 ng/ml, IL-11 - not less than 0.4 ng/ml, IL-13 - not less than 0.7 pg/ml, and molecules with anti-inflammatory effect, including IDO - not less than 0.08 ng/ml, soluble forms of receptors IL-1 - no less than 0.01 ng/ml and TNF - no less than 0.01 ng/ml. The composition may also additionally contain microRNA hsa-miR-23a-3p (SEQ ID NO: 1), hsa-miR-101 (SEQ ID NO: 2 and/or SEQ ID NO: 3), hsa-miR-146a (SEQ ID NO: 4 and/or SEQ ID NO: 5), hsa-miR-491-5p (SEQ ID NO: 6), hsa-miR-19b (SEQ ID NO: 7 and/or SEQ ID NO: 8), hsa -miR-21 (SEQ ID NO: 9 and/or SEQ ID NO: 10), hsa-miR-25 (SEQ ID NO: 11 and/or SEQ ID NO: 12), hsa-miR-125b (SEQ ID NO : 13 and/or SEQ ID NO: 14), hsa-miR-126a-3p (SEQ ID NO: 15). In this case, the composition is a concentrated solution of cytokines and growth factors in a culture medium, a frozen or freeze-dried solution, characterized by the presence of a set of properties, including the ability to prevent glutamate-mediated death of neural cells, the ability to stimulate the polarization of cells of the monocyte-macrophage link in the anti-inflammatory direction (M2- phenotype), preventing microglial cell activation.

The technical result is also achieved by using the claimed composition as a means of preventing glutamate-mediated death of neural cells, stimulating the polarization of cells of the monocyte-macrophage unit in the anti-inflammatory direction (M2 phenotype), preventing the activation of microglial cells.

Also, the technical result is achieved by a method for obtaining the claimed composition, including cultivation and conditioning of primary isolated allogeneic human mesenchymal stromal cells (MSCs) 2 - 5 passages in a growth medium for 72-144 hours, selection and purification of the culture medium from cell debris, concentration of the culture medium to reducing the initial volume by at least 10 times, purifying the culture medium to obtain a sterile concentrate. In this case, Dulbecco's modified Eagle's medium (DMEM) with a low glucose content (no more than 1 g/l) without phenol red with the addition of penicillin/streptomycin, as well as solutions of non-essential amino acids and vitamins, is used as a growth medium. The resulting sterile concentrate is frozen at a temperature from minus 80°C to minus 18°C or freeze-dried. Freeze drying is carried out in 6-10 stages at a pressure of 0.013-0.027 kPa, temperature from minus 45°C to 25°C, lyophilization time is no more than 24 hours at maximum freeze drying load. Adipose tissue is used as the primary isolated allogeneic human MSC, and MSC cultivation and medium conditioning are carried out at an oxygen content of 1-21 vol.%.

The technical result is also achieved by a means for neuroprotection and stimulation of neuroregeneration, including the claimed composition in a therapeutically effective amount and auxiliary components, which include any therapeutically acceptable additives, fillers, isotonic agents, stabilizers and/or preservatives. Additionally, the product may contain human albumin in an amount of at least 5 μg/ml. The inventive product is a concentrated solution of cytokines and growth factors in a culture medium, a frozen or freeze-dried solution.

Also, the technical result is achieved by a method of stimulating neuroprotection and brain regeneration after damage, including the introduction of the claimed composition or agent into the human body in a therapeutically effective amount. In this case, the composition or agent is administered locally into the area of brain damage at a dose of 5-30 mcg/ml (protein) or intranasally at a dose of 25-250 mcg of protein per 1 kg of body weight, or intravenously in a stream or drip at a dose of 30-300 mcg of protein per 1 kg of body weight, or into the spinal canal at a dose of 30-300 mcg of protein per 1 kg of body weight. The composition or agent is administered once or repeatedly over one or several days at equal intervals during the first three weeks after brain damage, while in the acute phase of brain damage, the composition or agent is administered no later than 6 hours from the moment of damage, in the acute phase of damage brain, the composition or agent is administered in the period from 6 to 120 hours from the moment of injury; in the subacute phase of brain damage, the composition or agent is administered in the period from 5 to 21 days from the moment of injury. Brain damage can be caused by acute cerebrovascular accident (ACVA) of the hemorrhagic type, regardless of its etiology, or of the ischemic type, regardless of the etiology of the disorder, or damage is caused by exposure to a mechanical factor, regardless of its nature, or exposure to a thermal factor, regardless from its nature, or a chemical factor, regardless of its nature (toxic effects, metabolic disorders), or penetrating radiation (radiation), regardless of its nature, or a combination of factors in any combination, regardless of their nature, as well as brain damage caused exposure to an infectious agent. At the same time, strokes of the hemorrhagic type are a consequence of congenital or acquired defects of the vascular bed of the brain. The infectious agent can be a bacterial or viral infection, namely a bacterial infection can be caused by microorganisms of the genera Neisseria and Borrelia, a viral infection - by the SARS-Cov2 virus.

Brief description of drawings

The invention is illustrated by the following drawings.

In fig. Figure 1 presents the results of in vivo studies of the neuroprotective activity of the unconcentrated secretome of human MSCs (hMSC1x) when injected directly into the area of damage 5 minutes after modeling a hemorrhagic stroke: A - survival of experimental animals; B - neurological status of experimental animals (LO - sham-operated); cognitive status of experimental animals: B - short-term memory (24 hours after intracerebral hemorrhage); D - long-term memory (10 days after intracerebral hemorrhage). The dotted line corresponds to the memory performance of LO animals. Data are presented as mean (25%; 75%). n=10; There are no significant differences between the groups.

In fig. Figure 2 presents the results of in vivo studies of the neuroprotective activity of a 10-fold concentrated secretome of human MSCs (hMSC10x) when injected directly into the area of damage 5 minutes after modeling a hemorrhagic stroke: A - survival of experimental animals; B - neurological status of experimental animals 3 and 10 days after intracerebral hemorrhage (LO - sham operation). Data are presented as mean (25%; 75%). [* - p<0.01; n ≥ 10; two-tailed Fisher's exact test].

In fig. Figure 3 shows the results of brain MRI on the 11th day after intracerebral hemorrhage: unconcentrated human MSC secretome (hMSC1x), 10-fold concentrated human MSC secretome (hMSC10x), recombinant BDNF (3.5 ng/μl) and unconcentrated human MSC secretome containing recombinant BDNF at a concentration of 3.5 ng/μl (hMSC-B) was injected directly into the damaged area 5 minutes after simulating a hemorrhagic stroke. In the diagram: the blue dotted line corresponds to the volume of the brain lesion in sham-operated animals. Data are presented as mean (25%; 75%). [* - p<0.05; n ≥ 8; ANOVA on ranks].

In fig. Figure 4 shows the results of a histochemical study of the brain on day 14 after intracerebral hemorrhage: unconcentrated human MSC secretome (hMSC1x), 10-fold concentrated human MSC secretome (hMSC10x), recombinant BDNF (3.5 ng/μl) and unconcentrated human MSC secretome containing recombinant BDNF at a concentration of 3.5 ng/μl (hMSC-B) was injected directly into the area of damage 5 minutes after simulating a hemorrhagic stroke. In the photographs: the red dotted line marks the lesion. In the diagram: the blue dotted line corresponds to the area of brain damage in sham-operated animals. Data are presented as mean (25%; 75%). n=5; There are no significant differences between the groups.

In fig. Figure 5 shows histological signs of damage to brain tissue (hematoxylin-eosin): A and B - focus of necrosis, restrained by neutrophil infiltration (IN) and glial scar (GR); B - signs of blood stagnation and migration of leukocytes through the capillary wall.

In fig. Figure 6 presents the results of a histological study of brain sections: unconcentrated secretome of human MSCs (hMSC1x), 10-fold concentrated secretome of human MSCs (hMSC10x), 10-fold concentrated secretome of rat MSCs (cMSC10x), recombinant BDNF (3.5 ng/μl) and unconcentrated secretome Human MSCs containing recombinant BDNF at a concentration of 3.5 ng/μl (hMSC-B) were injected directly into the area of damage 5 minutes after simulating a hemorrhagic stroke. Perls stain shows hemosiderin deposits (blue grains). Nissl staining reflects the functional state of neurons in the penumbra: red arrows - living but hypoxic neurons, black arrows - dead neurons (neuron shadows).

In fig. Figure 7 shows the results of immunohistochemical staining of brain sections 14 days after intracerebral hemorrhage: a - immunohistochemical staining for CD68; (b) - immunohistochemical staining for CD163. All drugs were injected directly into the injury area 5 minutes after simulating a hemorrhagic stroke. In the photo: green color corresponds to CD68 or CD163 staining; blue coloring corresponds to cell nuclei. In the diagram: the blue dotted line corresponds to the area of the brain lesion in CO animals. Data are presented as median (25%; 75%). [* - p<0.05, n ≥ 9, ANOVA on ranks; # - p<0.005, n ≥ 9, ANOVA on ranks].

In fig. Figure 8 presents the results of a study of the neuroprotective activity of a 10-fold concentrated rat (allogeneic) secretome of rat MSCs (cMSC10x) in vivo, introduced into the area of damage 5 minutes after modeling a hemorrhagic stroke: (a) - dynamics of survival of experimental animals; (b) - neurological status of experimental animals on days 3 and 10 after intracerebral hemorrhage (LO - sham-operated) [* - p<0.01, n=10, two-sided Fisher's exact test; ** - p<0.0005, n=18, two-sided Fisher’s exact test]; (c) - samples of MR images and histological sections of the brain obtained from experimental animals; (d) - results of MRI of the brain on the 11th day after intracerebral hemorrhage [# - p<0.005; n ≥ 8; ANOVA on ranks]; (e) - results of histochemical examination of the brain on the 14th day after intracerebral hemorrhage [## - p<0.05; n=5; ANOVA on ranks]. Data are presented as median (25%; 75%).

In fig. Figure 9 presents the results of an in vitro study of the neuroprotective activity of MSC secretomes (hMSC1x, hMSC10x, hMSC-B and cMSC10x): (a) - dynamics of survival of SH-SY5Y neuroblastoma cells under conditions of glutamate-induced neurotoxicity [* - p<0.001 compared with control ( K+); n=12; ANOVA on ranks]; (b) - neurite growth in neuroblastoma cell culture SH-SY5Y [** - p<0.004 compared to control; n=5; ANOVA on ranks]. Data are presented as median (25%; 75%).

In fig. Figure 10 shows sample images (raw data) obtained when studying the neuroprotective activity of MSC secretomes in a model of glutamate-induced neurotoxicity in vitro. Green color corresponds to dead cells (apoptosis).

In fig. Figure 11 shows sample images (raw data) obtained while studying the ability of MSC secretomes to stimulate neuritogenesis.

In fig. Figure 12 presents the results of a study of the ability of the human MSC secretome to influence the polarization of cells of the monocyte/macrophage unit of human peripheral blood. The human MSC secretome reduces the expression of markers of the pro-inflammatory phenotype (CD80, CD86) and increases the expression of markers of the anti-inflammatory phenotype (CD206, CD209) in human monocyte-macrophage cells (n≥3).

In fig. Figure 13 presents the results of assessing neurological deficits in rats with various methods of administration (n=5). The most pronounced neuroprotective effect of the secretome is observed with local injection of the MSC secretome into the area of damage, as well as with intravenous and intranasal administration of the MSC secretome.

In fig. Figure 14 presents the results of a comparison of the therapeutic activity of the rat MSC secretome upon intravenous administration and injection into the area of stroke (* - p<0.05, n ≥ 5) - MRI study.

In fig. Figure 15 presents the results of assessing neurological deficits in rats depending on the time of administration of the secretome (n=5). The most pronounced neuroprotective effect of the MSC secretome is observed when it is administered 1 and 3 hours after simulating a hemorrhagic stroke.

In fig. Figure 16 presents the results of assessing the volume of brain damage in rats with a hemorrhagic stroke model (according to an MRI study) depending on the time of secretome administration (n=3). The smallest amount of brain damage is observed with intravenous administration of the MSC secretome 1-3 hours after simulating a hemorrhagic stroke.

In fig. Figure 17 presents the results of assessing neurological deficits (A) and the volume of brain damage (according to an MRI study) (B) in rats with a model of hemorrhagic stroke, depending on the dose of the secretome of allogeneic MSCs administered intravenously 1 hour after modeling hemorrhagic stroke (n =4). The best neurological status (100% of animals are visually healthy) is observed in groups 5x-25x. The best therapeutic effect is observed with intravenous administration of rat MSC secretome at a dose of 5x-25x - 15-75 mcg/kg (by protein) 1 hour after modeling a hemorrhagic stroke.

Carrying out the invention

Below is a more detailed description of the claimed invention. The present invention is subject to various changes and modifications that will become apparent to those skilled in the art based on reading this specification. Such changes do not limit the scope of the claims.

To obtain the product, the claimed invention uses MSCs from adipose tissue, the production of which does not require the use of invasive and painful methods. It should also be taken into account that adipose tissue is considered one of the main sources of extracellular vesicles circulating in the human body. The claimed composition shows pronounced neuroprotective and anti-inflammatory properties, both in in vitro models of glutamate-mediated excitotoxicity, monocyte/macrophage polarization, and in vivo models of hemorrhagic stroke in rats. Mechanisms of damage to brain tissue, such as ischemic damage to neurons and glial cells, toxic effects of blood and metabolites, glutamate-mediated excitotoxicity, inflammation and secondary damage to brain tissue, are universal mechanisms of damage to brain tissue and are characteristic in certain combinations of mechanical, thermal, chemical (toxic, metabolic), radiation and combined damage to the brain, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents, including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS virus -Cov2. Compositions and agents based on them, capable of mitigating and inhibiting the action of the main pathogenetic components of brain damage, such as ischemic damage to neurons and glial cells, toxic effects of blood and metabolites, glutamate-mediated excitotoxicity, inflammation and secondary damage to brain tissue, in acute, acute and subacute stage of damage can be successfully used to treat a wide range of pathologies associated with acute brain damage, regardless of its genesis.

The technical problem of obtaining such a composition and the product obtained from it is solved by cultivating and conditioning primary human adipose tissue MSCs for 2 - 5 passages in serum-free growth medium DMEM with a low glucose content without phenol red, containing 1% antibiotic/antimycotic, 1% glutamine, including including, with additives supporting cell viability of at least 90% and suitable for therapeutic use. Unlike the prototype, the present invention uses mesenchymal stromal cells obtained from adipose tissue to obtain the secretome, which is a less invasive and painful, as well as safer and ethically sound approach than obtaining amniotic fluid cells. To obtain a conditioned environment, the claimed invention uses multipotent human mesenchymal stem/stromal cells (MSCs), which are responsible for the repair and regeneration of tissues and organs, both normally and when damaged. MSCs can be easily isolated by enzymatic treatment of adipose tissue samples obtained as a result of cosmetic liposuction or surgical removal of fat deposits. Cultivation of isolated AT MSCs results in a relatively homogeneous population of multipotent stromal fibroblast-like cells. It should also be taken into account that adipose tissue is considered one of the main sources of extracellular vesicles circulating in the human body.

The claimed invention can use MSCs obtained independently using any method known from the prior art (for example, from surgical biopsies, lipoaspirate, etc.), or in the form of commercial MSC preparations that have a quality certificate and are intended, including for clinical use. applications, such as the ATCC product (ATCC® PCS-500-011), which is human MSC isolated from lipoaspirate, cultured to passage 2 and cryopreserved.

Most protocols for the isolation and culture of adipose tissue MSCs use media supplemented with fetal bovine serum (FBS) or fetal bovine serum (FBS). These serums contain the necessary cocktail of growth factors, hormones and vitamins that stimulate cell adhesion and proliferation under in vitro culture conditions. The default culture medium also includes phenol red, which is a pH indicator. However, attempts to translate in vitro data into in vivo models and the clinic have shown that phenol red reduces the productivity of cell cultures and has a local irritant effect when concentrated cell-free products are administered at the transplant site.

A distinctive feature of the claimed invention is the use of a medium that does not contain phenol red for the cultivation of MSCs: DMEM with low glucose content without phenol red (Gibco, USA) with the addition of 10% fetal bovine serum FBS (HyClone, USA) with the addition of a solution of antibiotics Penicillin-Streptomycin ( HyClone, USA), L-Glutamine solution 200 mM (Gibco, USA), vol.%:

DMEM low glucose without phenol red (Gibco, USA) - 87.4-88.6;

Fetal bovine serum FBS (HyClone, USA) - 9.5-10.5;

Antibiotic solution Penicillin-Streptomycin (HyClone, USA) - 0.95 - 1.05;

L-Glutamine solution 200 mM (Gibco, USA) - 0.95 - 1.05;

MSCs are isolated from human subcutaneous adipose tissue and cultured in a growth medium prepared according to the above scheme for up to 2-5 passages. Then the cells are thoroughly washed from the components of the growth medium with Hanks' solution (from PanEco or similar) and subjected to conditioning. In a number of protocols, conditioning of producer cells occurs under the same conditions as cultivation - this leads to the fact that the main component of the resulting “secretome” is xenogeneic serum, which can activate a pronounced immune response on the part of the recipient’s body or even cause a graft-versus-host reaction " Other protocols use serum-free media as the conditioning medium, but there is no indication as to whether or not they contain phenol red.

An additional distinctive feature of the claimed invention is the use of a serum-free medium that does not contain phenol red for conditioning producer cells: DMEM with low glucose content without phenol red (Gibco, USA) with the addition of a solution of antibiotics Penicillin-Streptomycin (HyClone, USA), a solution of L-Glutamine 200 mM (Gibco, USA), solution of essential amino acids MEM NEAA (100x) (Gibco, USA), solution of vitamins MEM Vitamin Solution (100X) (Gibco, USA) in the following ratios by volume, vol.%:

DMEM with low glucose content (no more than 1 g/l) without phenol red (Gibco, USA) - 96.2 - 95.8;

Antibiotic solution Penicillin-Streptomycin (HyClone, USA) - 0.95 - 1.05;

L-Glutamine solution 200 mM (Gibco, USA) - 0.95 - 1.05;

Essential amino acid solution MEM NEAA (100x) (Gibco, USA) - 0.95 - 1.05;

Vitamin solution MEM Vitamin Solution (100X) (Gibco, USA) - 0.95 - 1.05.

The use of supplements of non-essential amino acids and vitamins is intended to compensate for the lack of fetal bovine serum in the conditioning medium and to increase the productivity of conditioned cells.

The specified conditioning medium is added to the cells in a volume of 0.1-0.2 ml/cm ² and cultured in a CO2 incubator at 37±1°C, 5% CO ₂ content, O2 content in the atmosphere not lower than 1% and relative humidity ≥ 95% within 72-144 hours. The thus obtained conditioned MSC medium containing all secretion products of human MSCs is collected in sterile containers for centrifugation, cell debris is removed by centrifugation, and the resulting supernatant is concentrated by centrifugation or tangential filtration using membranes and filters that allow molecules of no more than 10 kDa to pass through. with a decrease in the initial volume of the purified medium and obtaining a composition concentrated 1-50 times of the original volume and containing at least 1 μg/ml of total protein (https://www.thermofisher.com/order/catalog/product/23225), BDNF (not less than 3 ng/ml), GDNF (not less than 0.03 ng/ml), VEGF (not less than 0.2 ng/ml), HGF (not less than 0.15 ng/ml), uPA (not less than 0.34 ng/ml), TGFb (total for isoforms TGFb1, TGFb2 and TGFb3 not less than 0.055 ng/ml), IL-1 receptor antagonist IL-1Ra (not less than 0.4 ng/ml), IL-4 (not less than 0.08 ng/ml), IL-6 ( not less than 1 ng/ml), IL-10 (not less than 0.01 ng/ml), IL-11 (not less than 0.4 ng/ml), IL-13 (not less than 0.7 pg/ml), IDO (not less than 0.08 ng /ml), soluble forms of IL-1 receptors (not less than 0.01 ng/ml) and TNF (not less than 0.01 ng/ml), as well as microRNAs with neuroprotective and anti-inflammatory activity: hsa-miR-23a-3p (SEQ ID NO: 1), hsa-miR-101 (SEQ ID NO: 2 and/or SEQ ID NO: 3), hsa-miR-146a (SEQ ID NO: 4 and/or SEQ ID NO: 5), hsa-miR-491 -5p (SEQ ID NO: 6), which has an anti-inflammatory effect, and hsa-miR-19b (SEQ ID NO: 7 and/or SEQ ID NO: 8), hsa-miR-21 (SEQ ID NO: 9 and/or SEQ ID NO: 10), hsa-miR-25 (SEQ ID NO: 11 and/or SEQ ID NO: 12), hsa-miR-125b (SEQ ID NO: 13 and/or SEQ ID NO: 14), hsa -miR-126a-3p (SEQ ID NO: 15), which is determined by methods of analyzing the content of total protein or individual proteins/microRNAs in the mixture. When characterizing and assessing the quality of a composition, the qualitative composition of the specified microRNAs is assessed, since their quantity in mass units (ng) is not possible to measure. Their level is measured and indicated relative to the level of representation of another transcript (mRNA), which, in turn, can also vary greatly depending on the culture conditions, the type and source of cell production, etc.

Unlike the prototype, the resulting composition is characterized by the fact that it has the ability to prevent glutamate-mediated death of neural cells, the ability to stimulate the polarization of cells of the monocyte-macrophage link in the anti-inflammatory direction (M2 phenotype), and also prevent the activation of microglial cells, which was demonstrated in in vitro and in vivo models.

The resulting composition serves as the basis for obtaining a drug for neuroprotection and stimulation of brain tissue regeneration after injury. The product contains a composition in a therapeutically effective amount and auxiliary components that stabilize, preserve or improve the rheological properties of the composition as part of the medicinal product. To maintain the stability of the components included in the product, the latter can be frozen (-80...-18°C), followed by defrosting before use, or lyophilized in 6-10 stages at a pressure of 0.013-0.027 kPa, temperature from minus 45°C to 25°C for no more than 24 hours at maximum freeze-drying load, followed by reconstitution of the lyophilisate in water for injection or equivalent before use.

The resulting drug has a complex effect on the main components of the pathogenesis (ischemic damage to neurons and glial cells, toxic effects of blood and metabolites, glutamate-mediated excitotoxicity, inflammation and secondary damage to brain tissue) of a wide range of acute brain injuries, regardless of the mechanism and etiology of this damage, including mechanical, thermal, chemical (toxic, metabolic), radiation and combined brain damage, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents, including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS-Cov2 virus, which allows its use to reduce mortality and disability among such patients. In the claimed invention, the therapeutic potential of the resulting drug is demonstrated in a model of acute cerebrovascular accident of the hemorrhagic type (hemorrhagic stroke) in rats, since hemorrhagic stroke combines all the above-mentioned components of pathogenesis characteristic of damage to brain tissue.

Hemorrhagic stroke in rats is modeled by mechanical damage to the internal capsule of the brain (posterior to bregma - 2.0 mm, to the right of sigma - 3.5 mm) [Paxinos G. and Watson C. The rat brain in stereotaxic coordinates. 2007] followed by the introduction of 20-40 μl of autologous blood from the hypoglossal vein into the area of damage. Then, after 5 minutes, experimental animals are injected into the area of hemorrhage with MSC secretion, concentrated 10 times, containing growth factors: BDNF (at least 3 ng/ml), GDNF (at least 0.03 ng/ml), VEGF (at least 0.2 ng/ml ), HGF (not less than 0.15 ng/ml), uPA (not less than 0.34 ng/ml), TGFb (total for TGFb1, TGFb2 and TGFb3 isoforms not less than 0.055 ng/ml), cytokines: IL-4 (not less than 0.08 ng /ml), IL-6 (not less than 1 ng/ml), IL-10 (not less than 0.01 ng/ml), IL-11 (not less than 0.4 ng/ml), IL-13 (not less than 0.7 pg/ml ), anti-inflammatory molecules: IL-1 receptor antagonist IL-1Ra (at least 0.4 ng/ml), IDO (at least 0.08 ng/ml), soluble forms of IL-1 receptors (at least 0.01 ng/ml) and TNF (not less than 0.01 ng/ml), as well as microRNAs with neuroprotective and anti-inflammatory activity: hsa-miR-23a-3p (SEQ ID NO: 1), hsa-miR-101 (SEQ ID NO: 2 and SEQ ID NO: 3), hsa-miR-146a (SEQ ID NO: 4 and SEQ ID NO: 5), hsa-miR-491-5p (SEQ ID NO: 6), which have anti-inflammatory effects, and hsa-miR-19b (SEQ ID NO: 7 and SEQ ID NO: 8), hsa-miR-21 (SEQ ID NO: 9 and SEQ ID NO: 10), hsa-miR-25 (SEQ ID NO: 11 and SEQ ID NO: 12), hsa-miR- 125b (SEQ ID NO: 13 and SEQ ID NO: 14), hsa-miR-126a-3p (SEQ ID NO: 15) at a dose of at least 1 μg/kg (total protein). A modification of this method is the administration of the MSC secretome described above at a dose of at least 1 μg/kg (based on total protein) once intranasally, intravenously or intrathecally 6 hours or less after a simulated stroke. A modification of this method is the repeated administration of the MSC secretome described above at a dose of at least 1 μg/kg (based on total protein) using one of the methods described above after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days after stroke. The range of therapeutic doses for clinical use of the claimed composition and agent based on it must be established additionally, since it strongly depends on the volume of damage, localization and severity of the stroke, as well as the route of administration and time of administration after the stroke.

Recovery of animals after hemorrhagic stroke is assessed by the parameters of survival, preservation of neurological parameters, and the volume of the area of brain damage according to magnetic resonance imaging (MRI) and histological examination. The survival of the animals is assessed and recorded daily throughout the experiment; neurological parameters are assessed 3 and 10, 21 and 45 days after modeling a hemorrhagic stroke using the McGraw Stroke-index scale, modified for rodents by I.V. Ganushkina [McGraw C.P. et al., Stroke, 1976; Gannushkina I.V., Vestn Ross Akad Med Nauk, 2000]. “Visually healthy animals” show no signs of neurological deficits. Animals with “mild neurological impairment” are considered to be animals with signs of lethargy, limb weakness, tremors, ptosis and/or semiptosis. Animals with signs of paresis and/or paralysis of limbs, incoordination, or in a comatose state are counted as animals with “severe neurological impairment.”

The volume of brain damage by MRI is assessed 11 and 45 days after a simulated hemorrhagic stroke using a Clinscan 7T system (Bruker Biospin, USA) equipped with a rat brain analysis coil with TurboS Spin Echo and signal suppression from adipose tissue. Coronal views are obtained at the following parameters: TR (repetition time)=5220 ms; TE (time to echo)=53 ms; signal echo duration=9; base resolution 230x320; FoV (field of view) = 32 (40 mm; slice thickness = 0.5 mm; distance between slices = 0.75 mm. Transverse projections are obtained with the following parameters: TR = 4000 ms; TE = 40 ms; signal echo duration = 9; base resolution 288x320 ; FoV=40 (40 mm; slice thickness=0.5 mm; distance between slices=0.6 mm.

The volume of brain damage is assessed using histological examination 14 and 45 days after simulating a hemorrhagic stroke. To do this, rats are killed in a CO2 environment. The brain is removed, fixed in a 4% formaldehyde solution and embedded in paraffin. Brain sections containing the lesion are deparaffinized and the antigens are unmasked. Some sections are stained with hematoxylin-eosin, cresyl violet (Nissl stain) or Perls Prussian blue (Perls stain). The combination of these stains allows assessment of various aspects of morphological changes in the injured brain: lesion size, neuronal status, rate of leukocyte infiltration, and size of hemosiderin deposits.

For immunohistochemical staining, sections were treated with 50 mM ammonium acetate and blocked with 10% goat serum to reduce background fluorescence. After blocking, they are incubated with antibodies to CD68 (Abcam, #ab125212, USA) and CD163 (Abcam, #ab182422, USA), followed by incubation with fluorescently labeled secondary goat anti-rabbit antibodies (Invitrogen, #A11034, USA). Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma, #MBD0015-10ML, USA). Samples are examined using a Leica DM600 microscope equipped with a DFC360 FX and DFC420C camera (Leica Microsystems GmbH, Germany) or equivalent, using representative fields of view to obtain photographs. Image processing and analysis is carried out using LasX (Leica Microsystems GmbH, Germany) and FiJi software.”

In the present invention, based on data from a study of the effectiveness of the proposed composition in an in vivo model of hemorrhagic stroke, data is provided on the qualitative and quantitative composition of the composition used, on the optimal route of administration, time therapeutic window and dose, which is integral information in the development of approaches for the treatment of brain damage and prevention complications, especially acute cerebrovascular accidents.

The technical problem of stimulating neuroprotection and regeneration of brain tissue after damage is solved by using the drug using the method specified in the invention, which involves the introduction of a complex drug developed on the basis of the MSC secretome, concentrated 1-50 times, containing growth factors: BDNF (at least 3 ng/ml ), GDNF (not less than 0.03 ng/ml), VEGF (not less than 0.2 ng/ml), HGF (not less than 0.15 ng/ml), uPA (not less than 0.34 ng/ml), TGFb (total for TGFb1, TGFb2 isoforms and TGFb3 not less than 0.055 ng/ml), cytokines: IL-4 (not less than 0.08 ng/ml), IL-6 (not less than 1 ng/ml), IL-10 (not less than 0.01 ng/ml), IL- 11 (no less than 0.4 ng/ml), IL-13 (no less than 0.7 pg/ml), anti-inflammatory molecules: IL-1 receptor antagonist IL-1Ra (no less than 0.4 ng/ml), IDO (no less than 0.08 ng/ml ), soluble forms of IL-1 receptors (not less than 0.01 ng/ml) and TNF (not less than 0.01 ng/ml), as well as microRNAs with neuroprotective and anti-inflammatory activity: hsa-miR-23a-3p (SEQ ID NO: 1) , hsa-miR-101 (SEQ ID NO: 2 and SEQ ID NO: 3), hsa-miR-146a (SEQ ID NO: 4 and SEQ ID NO: 5), hsa-miR-491-5p (SEQ ID NO : 6), which have an anti-inflammatory effect, and hsa-miR-19b (SEQ ID NO: 7 and SEQ ID NO: 8), hsa-miR-21 (SEQ ID NO: 9 and SEQ ID NO: 10), hsa-miR -25 (SEQ ID NO: 11 and SEQ ID NO: 12), hsa-miR-125b (SEQ ID NO: 13 and SEQ ID NO: 14), hsa-miR-126a-3p (SEQ ID NO: 15), measured by enzyme immunoassay (for proteins), real-time PCR (for microRNAs) or similar. The composition used contains a combination of growth factors, extracellular vesicles carrying certain microRNAs, or other biologically active components secreted by human MSCs in an optimal combination of active components of various natures, does not contain proteins of animal origin and bacterial fragments, and also provides a balanced effect due to the presence of various the nature and mechanism of action of factors.

The MSC secretome is obtained by cultivating human MSCs in serum-free Dulbecco's modified Eagle's medium (DMEM), devoid of animal components, with a glucose content of no more than 1 g/l without phenol red with the addition of Penicillin-Streptomycin solution (HyClone, #SV30010) 0.95- 1.05%, L-Glutamine (200 mM) (Gibco, #25030081) 0.95-1.05%, MEM Non-Essential Amino Acids Solution (100X) (Gibco, #11140076) 0.95-1.05%, MEM Vitamin Solution (100X) (Gibco , #11120052) 0.95-1.05%), with a potentially safe composition for clinical use that maintains cell viability during a selected conditioning period by collecting the conditioned medium containing biologically active factors and extracellular vesicles secreted by cells into the culture medium, purifying the resulting medium and its fractionation using centrifugation and ultrafiltration. A product based on the claimed composition includes the composition in a therapeutically effective amount, as well as therapeutically acceptable additives, fillers (glucose, lactose, sodium citrate, human albumin, etc.), stabilizers, isotonic agents (sodium chloride) and/or preservatives (parabens, chlorobutanol, sorbic acid, etc.). Stabilization of the resulting compositions or agents is carried out by lyophilization. The resulting lyophilisate, diluted in sterile water for injection or an analogue, can be used as a medicine for the treatment or prevention of complications of acute hemorrhagic cerebrovascular accidents.

Thus, the technical result is achieved due to the fact that the proposed use of the composition, including local, intranasal, intravenous or intrathecal administration at a dose of at least 1 mcg/kg (based on total protein) within 6 hours or less after damage to brain tissue (in acute period), in the range of 6-120 hours for damage to brain tissue (acute period), in the range from 5 to 21 days for damage to brain tissue (subacute period) after mechanical, thermal, chemical (toxic, metabolic), radiation and combined damage, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents, including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS-Cov2 virus, has a complex effect, at least on neurons, macro- and microglial cells, as well as monocyte/macrophage cells of hematogenous origin. The effectiveness of the proposed drug presupposes the presence of a whole complex of effects aimed at treating or preventing complications in acute damage to brain tissue, regardless of the mechanism and etiology of the damage. When administered in the acute period after brain injury, the proposed composition prevents neuronal death caused by hypoxia, glutamate release and direct toxic effects of the blood, by reducing the activity of pro-apoptotic proteins Bax and increasing the activity of anti-apoptotic proteins Bcl-2. When administered in the acute and acute period after brain damage, the proposed composition suppresses the hyperactivation of microglial cells, which prevents the migration of an excess number of monocyte/macrophage cells from the blood to the site of injury and stimulates the polarization of the latter in an anti-inflammatory direction. When administered in the acute and subacute period after brain injury, the proposed composition prevents neuronal death caused by inflammation and secondary brain damage by reducing the activity of pro-apoptotic proteins Bax and increasing the activity of anti-apoptotic proteins Bcl-2 in neural cells and the polarization of microglial cells and monocytic cells /macrophage link in the anti-inflammatory direction. Secondary damage to brain tissue develops as a result of primary massive death of nervous tissue, which leads to disruption of the blood-brain barrier, inflammation and swelling, which leads to compression and disruption of the blood supply to the adjacent brain tissue, its ischemia and death with expansion of the primary source of damage. Timely neurotrophic support and suppression of inflammatory processes can break the vicious circle of secondary damage to nervous tissue.

Example 1. Method for culturing adipose tissue MSCs (AT MSCs). Method for obtaining secretome.

Human MSC cell cultures obtained from adipose tissue of healthy donors (n=3) were obtained from the biobank of the Institute of Regenerative Medicine, Moscow State University. M.V. Lomonosov, collection identifier: MSU_MSK_AD (https://human.depo.msu.ru). They were cultured in low glucose DMEM without phenol red (Gibco, USA) containing 10% fetal bovine serum (HyClone, USA) and 1% antibiotic/antimycotic (Gibco, USA), 1% glutamine (Gibco, USA). All procedures performed with patient tissue samples complied with the Declaration of Helsinki and were approved by the Ethics Committee of Moscow State University. M.V. Lomonosov (IRB00010587), protocol No. 4 (2018).

Rat MSCs were obtained from the adipose tissue of mature male Wistar rats in accordance with a previously described protocol [Aleksandrushkina NA, Danilova NV, Grigorieva OA, Mal'kov PG, Popov VS, Efimenko AY, et al. Bulletin of Experimental Biology and Medicine. 2019;167(1):159-63] and cultured in low glucose DMEM without phenol red (Gibco, USA) containing 10% fetal bovine serum (HyClone, USA) and 1% antibiotic/antimycotic (HyClone, USA), 1% glutamine (Gibco, USA). The resulting MSCs were characterized in accordance with the criteria of the International Society of Cell Therapy (ISCT) for the ability to adhere to plastic, express CD73, CD90 and CD105, the absence of hematopoietic and endothelial markers CD14, CD19, CD34, CD45 and HLA-DR and the ability to differentiate in vitro in adipogenic , chondrogenic and osteogenic direction [Viswanathan S, Shi Y, Galipeau J, Krampera M, Leblanc K, Martin I, Nolta J, Phinney DG, Sensebe L. Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT®) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy. 2019 Oct;21(10):1019-1024. doi: 10.1016/j.jcyt.2019.08.002; Sagaradze G, Grigorieva O, Nimiritsky P, Basalova N, Kalinina N, Akopyan Z, Efimenko A. Conditioned Medium from Human Mesenchymal Stromal Cells: Towards the Clinical Translation. Int J Mol Sci. 2019 Apr 3;20(7):1656. doi: 10.3390/ijms20071656]. The medium was changed every 3-4 days. All experiments were performed with cells within 5 passages.

Conditioned medium containing components of the human or rat MSC secretome was prepared according to a previously published protocol [Karagyaur M, Dzhauari S, Basalova N, et al. MSC Secretome as a Promising Tool for Neuroprotection and Neuroregeneration in a Model of Intracerebral Hemorrhage. Pharmaceutics. 2021; 13(12):2031, doi:10.3390/pharmaceutics13122031; RU2620167; RU2766707]. Briefly, subconfluent human or rat MSCs at passages 2-5 were thoroughly washed with Hanks' solution (PanEco, Russia) and then cultured for 144 hours in low-glucose DMEM without phenol red (Gibco, USA) supplemented with 1% antibiotic solution Penicillin-Streptomycin (HyClone, USA), 1% solution of L-Glutamine 200 mM (Gibco, USA), 1% solution of essential amino acids MEM NEAA (100x) (Gibco, USA), 1% solution of vitamins MEM Vitamin Solution (100X) ( Gibco, USA). At the end of conditioning, the medium was collected and centrifuged for 10 minutes at 300 g to remove cell debris (hMSC1x group), and some samples were further concentrated 10 times (hMSC10x group) using ultrafiltration cartridges with a molecular weight cutoff of less than 10 kDa (Merck, Germany).

The secretome of rat MSCs was concentrated 10 times in the same way (cMSC10x group). As a negative control (control group), we use low glucose DMEM without phenol red (Gibco, USA) with the addition of 1% Penicillin-Streptomycin antibiotic solution (HyClone, USA), 1% L-Glutamine 200 mM solution (Gibco, USA) , 1% solution of essential amino acids MEM NEAA (100x) (Gibco, USA), 1% solution of vitamins MEM Vitamin Solution (100X) (Gibco, USA). As a positive control, the same control medium was used with the addition of 3.5 ng/μl of recombinant human brain-derived neurotrophic factor (BDNF group) (#ab206642-2, Abcam, USA) [Karagyaur M, Dzhauari S, Basalova N, et al. MSC Secretome as a Promising Tool for Neuroprotection and Neuroregeneration in a Model of Intracerebral Hemorrhage. Pharmaceutics. 2021;13(12):2031, doi:10.3390/pharmaceutics13122031]. To enhance neuroprotective activity, Secretome 1x was supplemented with human BDNF to a final concentration of 3.5 ng/μL (hMSC-B group).

In the obtained secretomes of human and rat MSCs, the concentrations of key factors with neuroprotective and anti-inflammatory activity were assessed using enzyme immunoassay: BDNF, GDNF, VEGF, HGF, uPA, TGFb, IL-1Ra, IL-4, IL-6, IL-10, IL-11, IL-13, IDO, soluble forms of IL-1 and TNF receptors, as well as microRNAs: miR-23a-3p, miR-101, miR-146a, miR-491, miR-19b, miR-21, miR -25, miR-125b, 126a-3p. Their concentrations for the non-concentrated secretome of human/rat MSCs were in the amount of BDNF (no less than 3 ng/ml), GDNF (no less than 0.03 ng/ml), VEGF (no less than 0.2 ng/ml ml), HGF (not less than 0.15 ng/ml), uPA (not less than 0.34 ng/ml), TGFb (total for isoforms TGFb1, TGFb2 and TGFb3 not less than 0.055 ng/ml), IL-1 receptor antagonist IL-1Ra ( not less than 0.4 ng/ml), IL-4 (not less than 0.08 ng/ml), IL-6 (not less than 1 ng/ml), IL-10 (not less than 0.01 ng/ml), IL-11 (not less than 0.4 ng/ml), IL-13 (not less than 0.7 pg/ml), IDO (not less than 0.08 ng/ml), soluble forms of IL-1 receptors (not less than 0.01 ng/ml) and TNF (not less than 0.01 ng/ml ml).

During this study, several compositions were studied, differing in composition and concentration of active ingredients (based on 3 measurements):

Option 1 - (BDNF). Recombinant human BDNF at a concentration of 3.2 ng/μl in DMEM with low glucose and without phenol red.

Option 2 - (hMSC1x). BDNF (3.2 (1.2 ng/ml), GDNF (0.03 (0.018 ng/ml), VEGF (0.27 (0.13 ng/ml), HGF (0.15 (0.07 ng/ml), uPA (0.34 (0.16 ng/ml), TGF-b (0.055 (0.027 ng/ml) on DMEM with low glucose and without phenol red.

Option 3 - (hMSC-B). BDNF (3.2 (1.2 ng/ml), GDNF (0.03 (0.018 ng/ml), VEGF (0.27 (0.13 ng/ml), HGF (0.15 (0.07 ng/ml), uPA (0.34 (0.16 ng/ml), TGF-b (0.055 (0.027 ng/ml) on DMEM with low glucose and without phenol red.

Option 4 - (hMSC10x). Components of the human MSC secretome (xenogeneic for the rat model): BDNF (28.9 (14.3 ng/ml), GDNF (0.27 (0.11 ng/ml), VEGF (2.3 (0.7 ng/ml), HGF (1.4 (0.3 ng/ml)) , uPA (2.7 (0.74 ng/ml), TGF-b (0.45 (0.17 ng/ml), IL-1 receptor antagonist IL-1Ra (3.3 (0.7 ng/ml), IL-4 (0.77 (0.14 ng/ml) ), IL-6 (6 (0.7 ng/ml), IL-10 (0.13 (0.07 ng/ml), IL-11 (2.4 (0.88 ng/ml), IL-13 (6.5 (0.7 pg/ml), IDO (0.8 (0.27 ng/ml), soluble forms of IL-1 receptors (0.08 (0.05 ng/ml) and TNF (0.12 (0.067 ng/ml), as well as microRNA: hsa-miR-23a-3p (SEQ ID NO : 1), hsa-miR-101 (SEQ ID NO: 2 and SEQ ID NO: 3), hsa-miR-146a (SEQ ID NO: 4 and SEQ ID NO: 5), hsa-miR-491-5p ( SEQ ID NO: 6), hsa-miR-19b (SEQ ID NO: 7 and SEQ ID NO: 8), hsa-miR-21 (SEQ ID NO: 9 and SEQ ID NO: 10), hsa-miR-25 (SEQ ID NO: 11 and SEQ ID NO: 12), hsa-miR-125b (SEQ ID NO: 13 and SEQ ID NO: 14), hsa-miR-126a-3p (SEQ ID NO: 15) - on DMEM with low glucose content and without phenol red. Option 5 - (cMSC10x). Components of the rat MSC secretome (allogeneic for the rat model): BDNF (21.6 (6.8 ng/ml), GDNF (0.51 (0.19 ng/ml), VEGF (2.8 (1.2 ng/ml), HGF (0.94 (0.36 ng/ml), uPA (4.9 (1.8 ng/ml), TGF-b (0.73 (0.24 ng/ml), IL-1 receptor antagonist IL-1Ra (4.6 ( 1.4 ng/ml), IL-4 (0.86 (0.21 ng/ml), IL-6 (4.8 (0.9 ng/ml), IL-10 (0.24 (0.15 ng/ml), IL-11 (1.9 (0.69 ng) /ml), IL-13 (16.9 (4.9 pg/ml), IDO (1.4 (0.39 ng/ml), soluble forms of IL-1 receptors (0.12 (0.07 ng/ml) and TNF (0.19 (0.081 ng/ml) , as well as microRNAs: rno-miR-23a-3p (SEQ ID NO: 16), rno-miR-101 (SEQ ID NO: 17 and SEQ ID NO: 18), rno-miR-146a (SEQ ID NO: 19 and SEQ ID NO: 20), rno-miR-19b (SEQ ID NO: 21 and SEQ ID NO: 22), rno-miR-21 (SEQ ID NO: 23 and SEQ ID NO: 24), rno-miR- 25 (SEQ ID NO: 25 and SEQ ID NO: 26), rno-miR-125b (SEQ ID NO: 27 and SEQ ID NO: 28), rno-miR-126a-3p (SEQ ID NO: 29) - on DMEM low glucose and no phenol red.

Option 6 - (kMSK50x). Rat MSC secretome components (allogeneic for the rat model): BDNF (86.4 (14.5 ng/ml), GDNF (2.24 (0.87 ng/ml), VEGF (12.3 (4.9 ng/ml), HGF (4.57 (1.39 ng/ml)) , uPA (20.6 (4.6 ng/ml), TGF-b (3.3 (1.13 ng/ml), IL-1 receptor antagonist IL-1Ra (15.7 (3.9 ng/ml), IL-4 (3.8 (0.95 ng/ml) ), IL-6 (21.4 (4.3 ng/ml), IL-10 (1.22 (0.48 ng/ml), IL-11 (7.6 (1.94 ng/ml), IL-13 (0.06 (0.012 ng/ml), IDO (6.8 (1.6 ng/ml), soluble forms of IL-1 receptors (0.48 (0.19 ng/ml) and TNF (1.4 (0.37 ng/ml), as well as microRNA: rno-miR-23a-3p (SEQ ID NO : 16), rno-miR-101 (SEQ ID NO: 17 and SEQ ID NO: 18), rno-miR-146a (SEQ ID NO: 19 and SEQ ID NO: 20), rno-miR-19b (SEQ ID NO: 21 and SEQ ID NO: 22), rno-miR-21 (SEQ ID NO: 23 and SEQ ID NO: 24), rno-miR-25 (SEQ ID NO: 25 and SEQ ID NO: 26), rno -miR-125b (SEQ ID NO: 27 and SEQ ID NO: 28), rno-miR-126a-3p (SEQ ID NO: 29) - on DMEM with low glucose and without phenol red.

Concentration by more than 50 times (composition option 6), according to our data, is ineffective, which is due to the high viscosity of the resulting concentrate and the practical impossibility of further concentration. Thus, the upper limit of possible concentrations of growth factors in the compositions obtained by concentrating the secretome of adipose tissue MSCs is natural and corresponds to the concentrations obtained for composition option 6.

Example 2. Method for modeling intracerebral hemorrhage (hemorrhagic stroke) and introducing a secretome into the area of damage.

Mechanisms of damage to brain tissue, such as ischemic damage to neurons and glial cells, toxic effects of blood and metabolites, glutamate-mediated excitotoxicity, inflammation and secondary damage to brain tissue, are universal mechanisms of damage to brain tissue and are characteristic in certain combinations of mechanical, thermal, chemical (toxic, metabolic), radiation and combined damage to the brain, as well as damage caused by acute cerebrovascular accident of the hemorrhagic or ischemic type, and infectious agents, including microorganisms of the genera Neisseria, Borrelia and coronavirus infections, including the SARS virus -Cov2 [Ng SY, Lee AYW. Traumatic Brain Injuries: Pathophysiology and Potential Therapeutic Targets. Front Cell Neurosci. 2019;13:528. doi: 10.3389/fncel.2019.00528; Walter EJ, Carraretto M. The neurological and cognitive consequences of hyperthermia. Crit Care. 2016;20(1):199. doi:10.1186/s13054-016-1376-4; Dang J, Chen J, Bi F, Tian F. The clinical and pathological features of toxic encephalopathy caused by occupational 1,2-dichloroethane exposure. Medicine (Baltimore). 2019 Apr;98(17):e15273. doi: 10.1097/MD.0000000000015273; Kuriakose D, Xiao Z. Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. Int J Mol Sci. 2020 Oct 15;21(20):7609. doi: 10.3390/ijms21207609; Shao Z, Tu S, Shao A. Pathophysiological Mechanisms and Potential Therapeutic Targets in Intracerebral Hemorrhage. Front Pharmacol. 2019 Sep 19;10:1079. doi: 10.3389/fphar.2019.01079; Hoffman O, Weber RJ. Pathophysiology and treatment of bacterial meningitis. Ther Adv Neurol Disord. 2009;2(6):1-7. doi:10.1177/1756285609337975; Wijeratne T, Gillard Crewther S, Sales C, Karimi L. COVID-19 Pathophysiology Predicts That Ischemic Stroke Occurrence Is an Expectation, Not an Exception-A Systematic Review. Front Neurol. 2021 Jan 28;11:607221. doi: 10.3389/fneur.2020.607221].

Secondary damage to brain tissue develops as a result of primary massive death of nervous tissue, which leads to disruption of the blood-brain barrier, inflammation and edema, which leads to compression and disruption of the blood supply to adjacent brain tissue, its ischemia and death with expansion of the primary lesion [Ng SY, Lee AYW. Traumatic Brain Injuries: Pathophysiology and Potential Therapeutic Targets. Front Cell Neurosci. 2019;13:528. doi: 10.3389/fncel.2019.00528; Walter EJ, Carraretto M. The neurological and cognitive consequences of hyperthermia. Crit Care. 2016;20(1):199. doi:10.1186/s13054-016-1376-4]. Timely neurotrophic support and suppression of inflammatory processes can break the vicious circle of secondary damage to nervous tissue.

Compositions and agents based on them that can mitigate and inhibit the action of these main pathogenetic links of brain damage in the acute, acute and subacute stages of damage can be successfully used to treat a wide range of pathologies associated with acute brain damage, regardless of its genesis.

In the claimed invention, the therapeutic potential of the resulting drug is demonstrated in a model of acute cerebrovascular accident of the hemorrhagic type (hemorrhagic stroke) in rats, since hemorrhagic stroke combines all the above-mentioned components of pathogenesis, characteristic of damage to brain tissue, and compositions or agents that have demonstrated high therapeutic efficacy on models of hemorrhagic stroke are highly likely to be effective in the treatment of other acute brain injuries, regardless of their genesis.

Modeling of hemorrhagic stroke was carried out according to the previously described model of intracerebral hemorrhage in the internal capsule of the right hemisphere according to A.N. Makarenko and others [Makarenko, A.N.; Kositsyn, N.S.; Pasikova, N.V.; Svinov, M.M. Metod modelirovaniia lokal'nogo krovoizliianiia v razlich-nykh strukturakh golovnogo mozga u éksperimental'nykh zhivotnykh [Simulation of local cerebral hemorrhage in different brain structures of experimental animals]. Zh Vyssh Nerv Deiat Im I P Pavlova, 2002, 52(6), 765-768]. To simulate intracerebral hemorrhage, rats were anesthetized with a solution containing 2% Zoletil(50 (VirBac, Spain) and 1.5% xylazine (InterChemie, the Netherlands) in saline at a dose of 1 ml/kg body weight and placed in a stereotaxic apparatus.

The skin and tendon aponeurosis were incised, the skull was perforated (posterior to bregma -2.0 mm, to the right of sigma - 3.5 mm) [Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. 6th Edition. Cambridge: Academic Press, 2007]. The brain tissue in the perforation area was destroyed, and the superficial brain veins on the corresponding side were damaged to simulate bleeding. To standardize the volume of spilled blood, 20 μl of autologous blood taken from the hypoglossal vein was slowly injected into the lesion. After 5 min, 20 μl of the corresponding concentrated sample of the medium was injected, and the wound was sutured. In the course of studying the therapeutic activity of the composition for neuroprotection and stimulation of nerve tissue regeneration in an in vivo model, variants of compositions 1, 2, 3, 4, 5 and 6 were studied (see Example 1).

Animals in the LO (sham-operated) group underwent cranial perforation without any subsequent brain damage or injection of a blood/secretome sample.

Example 3. Method for assessing the effectiveness of restoration of brain morphology and function after modeling a hemorrhagic stroke.

Assessment of neurological status

The animals were observed for 14 days after modeling intracerebral hemorrhage with daily registration of rat death and assessment of neurological status 3 and 10 days after hemorrhage (according to the McGraw Stroke-index scale, modified for rodents by I.V. Ganushkina) [McGraw, C.P.; Pashayan, A. G.; Wendel, O.T.; Cerebral infarction in the Mongolian gerbil exacerbated by phenoxyben-zamine treatment. Stroke, 1976, 7(5), 485-488. doi: 10.1161/01.str.7.5.485.; Gannushkina, I.V.; Mozgovoe krovoobrashchenie pri razlichnykh vidakh tsirkuliatornoĭ gipoksii mozga [Cerebral circula-tion in different types of brain hypoxia]. Vestn Ross Akad Med Nauk, 2000, 9, 22-27. Russian.]. Briefly, “visually healthy animals” had no signs of neurological deficits. Animals exhibiting signs of lethargy, limb weakness, tremors, ptosis, and/or hemiptosis were considered to have “mild neurological impairment.” Animals that showed signs of paresis and/or paralysis of limbs, incoordination, or were in a coma were considered animals with “severe neurological impairment.” Cognitive impairment was assessed using the passive avoidance test [Fabbri, R., Furini, C. R., Passani, M. B., Provensi, G., Baldi, E., Bucherelli, C., et al. (2016). Memory retrieval of inhibitory avoidance requires histamine H1 receptor activation in the hippocampus. Proc. Natl. Acad. Sci. U.S.A. 113, E2714-E2720]. The animals developed the reflex 24 hours before intracerebral hemorrhage. To do this, each animal was placed in a passive avoidance apparatus (San Diego Instruments, USA) and the time until it entered the dark chamber of the apparatus was recorded. In the chamber, the rat received 8 painful electrical stimuli (0.4 mA, 1 sec each). 24 hours and 10 days after intracerebral hemorrhage (short-term and long-term memory, respectively), the animals were re-examined; the time they spent avoiding a dark chamber with an electric floor was recorded. The maximum observation time for each animal was 180 seconds (100%). The results obtained were normalized to 180 seconds, and the “efficiency” of short-term and long-term memory was calculated in %.

Assessing the extent of brain damage using MRI

MR images were obtained 11 days after simulated intracerebral hemorrhage using a Clinscan 7T system (Bruker Biospin, USA) equipped with a rat brain coil with TurboS Spin Echo and fat suppression. Coronal views were obtained using the following parameters: TR (repetition time)=5220 ms; TE (time to echo)=53 ms; signal echo duration=9; base resolution 230x320; FoV = 32 (40 mm; slice thickness = 0.5 mm; interslice distance = 0.75 mm. Transverse views were obtained using the following parameters: TR = 4000 ms; TE = 40 ms; echo duration = 9; baseline resolution 288x320; FoV=40 (40 mm; slice thickness=0.5 mm; distance between slices=0.6 mm.

Assessment of the volume of brain damage using histochemical and immunohistochemical methods

For histological studies, rats were sacrificed in a CO ₂ environment on day 14 after modeling intracerebral hemorrhage. The brain was removed, fixed in 4% formaldehyde solution and embedded in paraffin. Brain sections containing the lesion were deparaffinized and antigens were unmasked. Some sections were stained with hematoxylin-eosin, cresyl violet (Nissl stain) or Perls Prussian blue (Perls stain). The combination of these stains allows assessment of various aspects of morphological changes in the injured brain: lesion size, neuronal status, rate of leukocyte infiltration, and size of hemosiderin deposits.

For immunohistochemical staining, sections were treated with 50 mM ammonium acetate and blocked with 10% goat serum to reduce background fluorescence. After blocking, they were incubated with antibodies to CD68 (Abcam, #ab125212, USA) and CD163 (Abcam, #ab182422, USA), followed by incubation with fluorescently labeled secondary goat anti-rabbit antibodies (Invitrogen, #A11034, USA). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) solution (Sigma, #MBD0015-10ML, USA). Samples were examined using a Leica DM600 microscope equipped with a DFC360 FX and DFC420C camera (Leica Microsystems GmbH, Germany), using representative fields of view to take photographs. Image processing and analysis were performed using LasX (Leica Microsystems GmbH, Germany) and FiJi software.

Example 4. Method for assessing the neuroprotective and neurotrophic activity of allogeneic and xenogeneic secretomes of MSCs when they are introduced into the area of hemorrhagic stroke.

To assess the neuroprotective and neurotrophic activity of allogeneic and xenogeneic secretomes of MSCs when they were introduced into the area of hemorrhagic stroke, the hemorrhagic stroke model described above was used. The study included 98 animals: 40 rats for the pilot study (sham-operated (SO), control, BDNF and hMSC1x groups, 10 rats each), 30 rats to study the neuroprotective activity of the human MSC secretome (control, hMSC10x and hMSC-B groups , 10 rats each) and 28 rats to study species specificity (10 rats for the control group and 18 for the cMSC10x group). No rats were excluded from the study.

The results of the pilot study showed that hMSC1x secretome has potential therapeutic activity, but is less effective than the positive control BDNF drug (Figure 1B). Although the hMSC1x secretome did not have a significant effect on survival and alleviation of neurological deficits in experimental animals, it contributed to the preservation of long-term memory in rats [100% (94.4%; 100%) compared with 79.2% (12.2%; 100%) in the control group, n≥8] (Fig. 1D). While BDNF provided better neurological outcome (Figure 1B), it was less effective than hMSC1x in the cognitive test: 80.6% (42.8%; 100%) (Figure 1D). Presumably, this is due to the difference in the action of the secretome of BDNF and hMSC1x: BDNF is a protein with direct (immediate) neurotrophic activity, while the secretome of hMSC1x consists of a large number of molecular complexes (especially microvesicles, which have a delayed effect on the cell transcription program - targets) with a lower content of neurotrophic factors (for example, BDNF 3 concentration (1.2 pg/μl, n=9).

It has been suggested that the weak effect of the hMSC1x secretome is due to the low concentration of biologically active components within the secretome, so an expanded study was conducted using a combination of hMSC1x supplemented with human BDNF 3.5 ng/μl (hMSC-B) and a 10-fold concentrated human MSC secretome ( hMSC10x). The addition of the hMSC-B group to the experiment is due to the expectation of an additive therapeutic effect of BDNF protein (immediate effect) and MSC microvesicles (delayed effect) during the long-term process of brain recovery after intracerebral hemorrhage.

The secretome of hMSC10x contributed to the preservation of neurological parameters (10 days after intracerebral hemorrhage) and ensured the survival of 100% of experimental animals (up to 14 days after intracerebral hemorrhage), while in the negative control group (control) 30% of animals died, and 20% of animals had severe neurological deficit (10 days after intracerebral hemorrhage) [p<0.01; n (10; two-sided Fisher's exact test) (Fig. 2). Reduced neurological deficits without a significant increase in survival (80%) were observed in the hMSC-B experimental group (as well as in the BDNF and hMSC1x groups).

According to the MRI study, no significant differences in the volume of brain damage were detected in the experimental groups compared to the control. In the hMSC1x group, there was a tendency towards a decrease, and in the hMSC10x group, a tendency towards an increase in the volume of brain damage 11 days after intracerebral hemorrhage: 70.1 (53.5; 155.8) mm ³ and 261.4 (232.0; 337.4) mm ³ , respectively, against 176.6 ( 138.5; 201.5) mm ³ in the control group (Fig. 3). However, a significant difference in the size of the lesion volume was observed between the hMSC1x and hMSC10x groups [p<0.05; n (8; ANOVA on ranks].

Hematoxylin-eosin staining of brain sections in the area of damage showed a distribution of brain lesion sizes across groups similar to MRI results (Fig. 4). There was no significant difference between groups, although, as in the MRI study, lesion site tended to decrease in the hMSC1x and hMSC-B groups: 4.9% (3.9%; 6.2%) and 6.6% (2.8%; 7.7%), respectively. , versus 9.8% (6.6%; 13.4%) in the control group [p=0.220; n=5; ANOVA on ranks].

Hematoxylin-eosin and Nissl staining of brain sections also confirmed the presence of microscopic signs of brain tissue damage: necrosis, ischemia, edema, glial scar, neutrophil infiltration and blood stasis in the capillary bed (Figs. 5 and 6). Signs of damage were more pronounced in the control group, but no quantitative analysis was performed. Prussian blue staining (Perls) revealed hemosiderin deposits (blue) in the brain tissue adjacent to the lesion in all groups, but they were less pronounced in the control group and the hMSC10x group (Fig. 6). It was suggested that this was due to expansion of the lesion in the control and hMSC10x groups, which led to the absorption of hemosiderin deposits into necrotic masses.

Immunohistochemical staining of brain sections for CD68, a marker of pro-inflammatory M1-microglia cells and a marker of lysosomal activity, revealed its significantly higher representation in brain sections of the hMSC10x group: in this group, the area specifically stained with CD68 occupies 18.0% of brain sections (6.5%; 25.6%) versus 5.9%. % (5.0%; 7.4%) in the control group [p<0.05; n (9; ANOVA on ranks] (Fig. 7A). There was no significant difference in the area stained with CD68 antibodies in the BDNF, hMSC1x and hMSC-B groups compared to Control, although there was a trend towards an increase in the number of CD68+ cells these groups.

Staining for CD163, a marker of M2 microglial cells, showed a noticeable (but not statistically significant) decrease in the level of CD163+ cells in the hMSC10x group - 1.7% (0.6%; 5.3%) compared to 9.7% (4.6%; 15.0%) in the control group [p=0.052; n(8; ANOVA on ranks] (Fig. 7B). Similar reductions were observed in the BDNF, hMSC1x and hMSC-B groups, but they were not significantly different from the control group.

Since the area of sections specifically stained with antibodies to CD68 in the hMSC10x group was significantly higher than in the other groups and correlated with the volume of brain lesions according to MRI and histochemical studies, it was suggested that this negative effect is associated with the action of individual components of the xenogeneic secretome ( human) MSCs. To test this hypothesis, the potential therapeutic effect of the allogeneic rat MSC secretome hMSC10x was studied and its efficacy was compared with that of the xenogeneic human MSC secretome hMSC10x. It was revealed that the secretome of hMSC10x provides 100% survival and excellent neurological recovery of animals after intracerebral hemorrhage, as well as the secretome of hMSC10x [p<0.01 (n=10) for hMSC10x compared to the control; p<0.0005 (n=18) for cMSC10x compared to control; two-sided Fisher's exact test] (Fig. 8). According to an MRI study, the secretome of cMSC10x effectively reduced the volume of brain damage, in contrast to hMSC10x: 90.2 (48.3; 144.3) mm ³ for cMSC10x versus 261.4 (232.0; 337.4) mm ³ for hMSC10x [p <0.005; n (8; ANOVA on ranks]. Similar results were obtained from a histological study of the rat brain: the area of brain damage in the cMSC10x group was significantly less (3.9% (2.6%; 4.2%)) than in the control group (9.8% ( 6.6%; 13.4%) [p<0.05;n=5; ANOVA on ranks] (Fig. 8).

This reduction in lesion size also correlated with a decrease in CD68 immunostaining in brain sections from cMSC10x rats: 5.6% (1.1%; 7.7%) [p < 0.005 for cMSC10x compared with hMSC10x; n (9; ANOVA on ranks] (Fig. 7A). CD163 staining in sections of cMSC10x rats was not significantly different from other groups: 5.3% (2.2%; 5.5%) [p = 0.06; n (8; ANOVA on ranks] ( Fig. 7B).

Based on the results of this study, it was found that human and rat MSC secretomes, concentrated 10 times, have pronounced neuroprotective and neurotrophic activity (in terms of survival, neurological status), however, the xenogeneic components of the MSC secretome (in this case, the human MSC secretome) are capable of cause hyperactivation of immune cells, which leads to the growth of the lesion and in the future can lead to decreased survival and progression of neurological deficits. At the same time, the use of an allogeneic secretome (in this case, the secretome of rat MSCs) leads to a decrease in the volume of the lesion according to MRI and histological examination and does not lead to hyperactivation of immune cells surrounding the damage area, which, coupled with excellent survival and prevention of the development of neurological deficits corresponds to a positive prognosis for the treatment of hemorrhagic stroke by introducing the allogeneic secretome of MSCs, concentrated 10 times (at a dose of 15 μg/kg of protein) - composition option 5, into the area of hemorrhage. The remaining composition options do not allow achieving the desired therapeutic effect, which is manifested in lower animal survival (compared to the effect of composition option 5), worse recovery of neurological parameters, and a larger lesion according to MRI and histological examination. It is worth paying special attention to the effect of composition options 4 (xenogeneic secretome of human MSC cells) and 5 (allogeneic secretome of human MSC cells): the therapeutic effectiveness of these options in terms of survival rates of experimental animals and restoration of neurological status is almost identical, but the size of the lesion in the group with xenogeneic secretome higher, which, according to our data (increased expression of CD68 and decreased expression of CD163), as well as literature data, is due to the activation of immune-competent cells in the recipient’s body. Composition 4 itself is capable of polarizing allogeneic immune cells (monocytes and macrophages of human peripheral blood) into the anti-inflammatory M2 phenotype (see Example 6), and the observed effect of microglia activation and enlargement of the lesion in the in vivo experiment is due to the immunogenic effect of xenogeneic molecules/proteins . The totality of the results of in vivo and in vitro studies suggests high therapeutic activity of allogeneic composition 4 in relation to humans in case of brain damage.

Example 5. Method for assessing the neuroprotective and neurotrophic activity of the MSC secretome in vitro.

To assess the neuroprotective and neurotrophic activity of the cellular secretome in vitro, SH-SY5Y neuroblastoma cells (ATCC (CRL-2266™)) were seeded in 48-well plates in complete nutrient medium (DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin ( Gibco, USA)) in an amount of 40,000 cells/well in four replicates. The next day, the medium was removed and samples of serum-free medium (K+), serum-free medium with 3.5 ng/μl of human BDNF (BDNF) or MSC secretome (hMSC1x, hMSC10x, hMSC-B or hMSC10x) (composition options 1, 2, 3, 4 , 5 and 6 - see Example 1), 100 mM L-glutamate was added to each [Nampoothiri, M.; Reddy, N.D.; John, J.; Kumar, N.; Kutty Nampurath, G.; Rao Chamallamudi M. Insulin blocks glutamate-induced neurotoxicity in differentiated SH-SY5Y neuronal cells. Behav Neurol, 2014, 2014, 674164. doi:10.1155/2014/674164] and 5 µM apoptosis reagent IncuCyte (Caspase-3/7 (Essen Bioscience, #4440, USA). In the control group (K-) no L was added -glutamate, BDNF or MSC secretome to monitor spontaneous cell death in serum-free medium. Glutamate causes a rapid increase in cytosolic Ca2+ concentration in SH-SY5Y cells (similar to what occurs in neurons) with subsequent activation of caspase. IncuCyte reagent (Caspase-3 /7 for apoptosis, cleaved by activated Caspase-3/7, stains nuclear DNA (green fluorescence).To monitor SH-SY5Y cell death, the plate was placed in an Incucyte(ZOOM) live cell assay system (Essen Bioscience) located inside a CO ₂ incubator Time-lapse imaging of nine fields of view (phase and green channel) of each well was performed 1, 3, 6, 12, 24, 48, and 72 hours after media replacement.

To assess the ability of MSC secretomes to stimulate neurite growth, SH-SY5Y cells were seeded in 48-well plates in complete nutrient medium at 40,000 cells/well in four replicates. The next day, the medium was removed and serum-free medium (control), serum-free medium with 3.5 ng/μl human BDNF (BDNF) or MSC secretome (hMSC1x, hMSC10x, hMSC-B, or cMSC10x) were added. To monitor neurite outgrowth from SH-SY5Y cells, the plate was placed in an Incucyte(ZOOM) live cell assay system (Essen Bioscience) located inside a CO ₂ incubator. Time-lapse imaging of nine fields of view of each well was performed at 0, 6, and 24 hours after media exchange .

Micrographs to calculate the percentage of living cells (compared to the initial number of cells) and estimate the rate of neurite growth were analyzed using ImageJ software (NIH, USA). Cellular processes longer than twice the cell diameter were considered neurites.

When examining the neuroprotective and neurotrophic activities of the MSC secretome in vitro, the secretomes of hMSC1x, hMSC10x, cMSC10x and hMSC-B were found to support the survival of the human neuroblastoma cell line SH-SY5Y under conditions of glutamate-induced neurotoxicity (Figs. 9A and 10). Thus, the number of living cells in the hMSC1x, hMSC10x, hMSC10x and hMSC-B groups was significantly higher 3-24 hours after the addition of glutamate than in the positive control group “K+” without a secretome [p<0.001 compared to the control (K+ ); n=12; ANOVA on ranks]. SH-SY5Y survival in these groups was comparable to that observed in the BDNF positive control group. There were no significant differences in SH-SY5Y survival between MSC groups, with the exception of cMSC10x. Its neuroprotective activity was significantly higher compared to the K+ group and even compared to hMSC1x, hMSC-B and hMSC10x at certain time points. Secretomes of hMSC1x, hMSC10x and cMSC10x supported SH-SY5Y survival for up to 72 hours in neurotoxic conditions: 7% (0%; 9%), 8% (4%; 10%) and 19% (11%; 27%) of living cells, respectively. Whereas in the “K+” group no living cells were observed after 24 hours of the experiment. In addition to glutamate-induced death of SH-SY5Y cells, spontaneous cell death was observed. It increased significantly only 24 hours after the start of the in vitro experiment - this could be observed in the negative control group “K-” (serum-free medium without glutamate).

A study of the dynamics of SH-SY5Y neurite growth during 24 hours of the experiment showed spontaneous neurite growth in all experimental groups. The secretomes of hMSC1x, hMSC10x, hMSC-B and cMSC10x increased neurite growth compared to the control group 6 hours from the start of the experiment, but the difference was not statistically significant. Only in the BDNF group was neurite outgrowth significantly higher compared to the control group [p < 0.005 vs. control; n=5; ANOVA on ranks] (Figs. 9B and 11).

Thus, as a result of studying the neuroprotective and neurotrophic activity of the MSC secretome in vitro, it was found that the secretomes of hMSC1x, hMSC10x (composition option 4), cMSC10x (composition option 5) and hMSC-B (composition option 3) support the survival of the human neuroblastoma cell line SH-SY5Y under conditions of glutamate-induced neurotoxicity but did not significantly affect neurite outgrowth of the human neuroblastoma cell line SH-SY5Y over 24 hours.

Example 6. Method for assessing the ability of the MSC secretome to influence the activity of monocyte-macrophage cells of hematogenous origin in vitro.

In an in vitro model of glutamate-induced neurotoxicity, the secretomes of rat MSCs cMSC10x and human hMSC10x support the survival of the human neuroblastoma cell line SH-SY5Y, however, the introduction of these secretomes has the opposite effect on the volume of damage in the brain: the secretome of allogeneic rat MSCs reduces the volume of damage, and the secretome xenogeneic human MSCs leads to an increase in the volume of damage.

It was assumed that the difference in the observed effect of allogeneic and xenogeneic secretomes is due to the participation of a certain immune component in the body of rats, and for this purpose, brain sections were stained after exposure of rat and human MSC secretomes with antibodies to the phagocytosis marker CD68 (a marker of activated microglia). In sections of the rat brain after administration of the human MSC secretome, a pronounced expression of CD68, a marker of phagocytosis and microglial activation, was observed, while the introduction of the rat MSC secretome did not cause such an effect and the level of CD68 expression in the brain of such rats did not differ from control samples (Fig. 7A ). Thus, according to our data, the introduction of the secretome of xenogeneic cells, in particular MSCs, into the brain can activate microglial cells and phagocytosis processes, which can lead to the growth of the lesion, while no such negative effect was found for the allogeneic secretome.

To assess the ability of the MSC secretome to influence the activity of cells of the monocyte-macrophage series of hematogenous origin, a model of polarization of monocytes/macrophages into the M1- and M2-phenotype in vitro was used.

To do this, donor blood (20 ml) was collected in 50 ml tubes, thoroughly and carefully mixed with 200 µl of a 0.5 M EDTA*K2 solution (anticoagulant) and centrifuged at 400 g for 10 minutes in order to separate blood cells from plasma. Plasma was collected and an equivalent volume of sterile phosphate-buffered saline (PBS) was added to the cell pellet. A suspension of blood cells in PBS was layered onto a Ficoll solution with a specific gravity of 1.077 g/ml and then centrifuged at 400 g for 30 minutes without active braking. A layer of mononuclear cells was collected at the phase separation interface between Ficol and PBS, washed twice with PBS, pelleted by centrifugation, and resuspended in RPMI growth medium containing 10% FBS fetal bovine serum and 1% antibiotic/antimycotic solution. Cells were planted on culture plates with a highly adhesive surface BD Primaria at a concentration of 1.5 * 10 ⁶ cells/ml, and the plates were placed in a CO2 incubator for 35-40 minutes. After the end of the incubation time, unattached cells were washed, and serum-free culture medium X was added to the remaining ones -VIVO(15 (Lonza, #BE02-060F) with macrophage colony-stimulating factor (GM-CSF) (SciStore, #PSG030-10) at a concentration of 50 ng/ml. Cells were cultured for 8 days. On the 6th day after isolation populations of monocytes/macrophages were polarized into the M1 phenotype by adding recombinant human IFN ((R&D Systems, #285-IF-100/CF) (50 ng/ml) and lipopolysaccharide (10 ng/ml) (SigmaAldrich, #297-473-0) or in the M2 phenotype by adding recombinant IL-4 (R&D Systems, #204-IL-010/CF) (20 ng/ml) and TGF ((Peprotech, #100-21) to the growth medium ) (20 ng/ml) human. To some of the samples, the human MSC secretome hMSC10x (composition option 4) was added to achieve a final concentration of 1 μg/ml (by protein) - 1x in the growth medium.

The cells were incubated in polarizing media for 48 hours, after which they were removed from the plastic with culture scrapers, stained for markers of M1 and M2 phenotypes, and the composition of the populations was analyzed using flow cytometry FACSCantoTM II (BD Biosciences, USA). Forward and side light scattering of cells was also assessed to visualize the degree of purity of the resulting cell populations. To assess the efficiency of macrophage polarization in the M1 phenotype in the presence or absence of CD-008-0045, antibodies to the markers CD80 (BD, #566992) and CD86 (BD, #555660) were used. To assess the efficiency of macrophage polarization into the M2 phenotype in the presence or absence of the human MSC secretome, antibodies to the markers CD206 (BioLegend, #141720) and CD209 (BioLegend, #343006) were used.

The study showed that the addition of human MSC secretome to the culture medium significantly inhibits the polarization of monocytes/macrophages into the pro-inflammatory M1 phenotype compared to the control group (n=4; p<0.05) (Fig. 12). Thus, in the group without a secretome of human MSCs (control), the average color intensity (arithmetic mean signal intensity) of cells polarized to the M1 phenotype and stained with antibodies to CD86 (M1) was 60.3 (53.5;72.1)%, while the addition of a secretome Human MSCs reduced the average staining intensity of such cells to 50.5 (43.4;55.9)%.

The addition of the human MSC secretome to the culture medium stimulated the polarization of monocytes/macrophages into the anti-inflammatory M2 phenotype: the average staining intensity (arithmetic mean signal intensity) of cells polarized to the M2 phenotype and stained with antibodies to CD209 (M2) in the control group was 51.8 (18.7; 54.1)%, while in the human MSC secretome group - 68.8 (61.2;72.5)% (n (4) (Fig. 12).

Thus, the human MSC secretome reduces the expression of markers of the pro-inflammatory phenotype (CD80, CD86) and increases the expression of markers of the anti-inflammatory phenotype (CD206, CD209) on human monocyte-macrophage cells in vitro, which indicates the immunosuppressive activity of the MSC secretome (Fig. 12) in against allogeneic immune cells.

Example 7. Method for assessing the neuroprotective and neurotrophic activity of the MSC secretome using various methods of its administration.

The introduction of the allogeneic MSC secretome directly into the site of hemorrhage after injury stimulates survival, reduces the severity of neurological disorders and leads to a reduction in the site of injury, however, this method of delivering a therapeutic drug is practically impossible to implement in the clinic, so we studied the possibility of using clinically relevant routes of drug administration: intranasal, intravenous , as well as intrathecal (introduction into the spinal canal).

To assess the neuroprotective and neurotrophic activity of the MSC secretome with various methods of its administration, the model of hemorrhagic stroke described above was used; however, for this purpose we used the rat MSC secretome, concentrated 50 times (composition option 6), which was administered to rats intranasally, intravenously or intrathecally (in the spinal cord). channel) 5 minutes after simulating a hemorrhagic stroke. The study included 20 animals: 5 animals each in the groups of intravenous administration of the MSC secretome, intranasal administration of the MSC secretome, intrathecal administration of the MSC secretome, and the control group (intravenous administration of the corresponding volume of empty growth medium). No rats were excluded from the study.

For intranasal administration, an anesthetized rat after modeling a hemorrhagic stroke was placed belly up and 40 μl of rat MSC secretome, concentrated 50 times (at a dose of 20 μg/nostril (by protein)), was injected into each nostril. Intravenous administration of the cMSC50x secretome was carried out in a volume of 100 μl into the tail vein at a dose of 150 μg/kg (by protein) - according to the standard method. For injection into the spinal canal, an anesthetized rat after simulating a hemorrhagic stroke was placed on a high bolster with its back up, the skin in the lumbar spine was cut, and the cMSC50x secretion in a volume of 100 μl and a dose of 150 μg/kg (protein) was slowly injected into the spinal canal between vertebrae L5-S1.

To assess the effectiveness of restoration of brain structure and function depending on the method of drug administration, the methods described above were used: survival, neurological status, volume of brain damage according to MRI.

Due to the small sample (5 animals per group), 100% survival rate was observed in all groups, however, based on the results of neurological examination and MRI data, it was possible to establish that the most pronounced therapeutic effect of the secretome was observed when it was administered intravenously (Fig. 13 and 14). The neuroprotective effect of intravenous injection of the MSC secretome turned out to be almost as pronounced as with local injection of the MSC secretome of rats into the area of stroke - both according to the results of a neurological study (Fig. 13) and according to the results of MRI (n≥5) (Fig. 14 ). Intranasal administration of the drug had a less pronounced effect on the restoration of brain structure and function after a stroke and was accompanied by significant variations in parameters among animals, which, apparently, is associated with difficulties in dosing and maintaining the drug in the nasal cavity. Intrathecal administration of the drug had no effect on the restoration of brain structure and function, which is apparently due to the flow of cerebrospinal fluid in the caudal direction.

Thus, as a result of assessing the neuroprotective and neurotrophic activity of the MSC secretome with various methods of its administration, it was found that the neuroprotective, anti-inflammatory and pro-regenerative activity of the MSC secretome is most pronounced when administered intravenously or intranasally.

Example 8. Method for assessing the neuroprotective and neurotrophic activity of the MSC secretome depending on the timing of its administration after a stroke.

A fundamentally important step for the transition to the practical use of secretome in the treatment of strokes is to establish the therapeutic effectiveness of secretome depending on the time of its use after a hemorrhagic stroke (the so-called “therapeutic window”). To do this, we studied the effectiveness of intravenous and intranasal administration of the MSC secretome 1, 3 and 6 hours after hemorrhagic stroke using the hemorrhagic stroke model described above. The rat MSC secretome, concentrated 50 times (composition option 6), was administered intranasally (at a dose of 20 μg/nostril (by protein)) or intravenously (at a dose of 150 μg/kg (by protein)) 1, 3 or 6 hours after modeling hemorrhagic stroke. The study included 21 animals (7 groups of 3 animals each: intranasal administration 1 or 3 or 6 hours after modeling a hemorrhagic stroke, intravenous administration 1 or 3 or 6 hours after modeling a hemorrhagic stroke, control group (intravenous administration of the appropriate volume empty growth medium 1 hour after simulated hemorrhagic stroke) None of the rats were excluded from the study.

To assess the effectiveness of restoration of brain structure and function depending on the timing of drug administration, the methods described above were used: survival, neurological status, volume of brain damage according to MRI.

According to the results of a neurological study, the MSC secretome had a neuroprotective effect even 6 hours after a stroke (with intravenous administration), which was reflected in a decrease in the severity of neurological disorders in experimental animals (Fig. 15). However, according to MRI data, a decrease in the neuroprotective activity of the MSC secretome is observed with an increase in the period of administration from the moment of stroke. The smallest volume of the stroke focus is observed with intravenous administration of the MSC secretome 1 hour after the stroke (Fig. 16).

Thus, as a result of assessing the neuroprotective and neurotrophic activity of the MSC secretome depending on the timing of its administration after a stroke, it was found that the best therapeutic effect is achieved when the first dose of the drug is administered within 6 hours or less from the moment of acute hemorrhagic cerebrovascular accident.

Example 9. Method for assessing the neuroprotective and neurotrophic activity of the MSC secretome depending on the dose when administered intravenously

To assess the neuroprotective and neurotrophic activity of the MSC secretome depending on the dose upon intravenous administration, the hemorrhagic stroke model described above was used, and the drug was administered intravenously (in doses of 5x, 10x, 25x, 50x - 15-150 μg/kg (by protein)) after 1 an hour after simulating a hemorrhagic stroke in a volume of 100 μl. Doses of 5x, 10x and 25x were obtained by diluting the MSC secretome 50x (formulation option 6) in DMEM with low glucose content and without phenol red and without fetal bovine serum (FBS). The study included 20 animals: (5 groups of 4 rats each: dosages of 15, 30, 75, 150 mcg/kg (for protein) and a control group (intravenous administration of an appropriate volume of empty growth medium 1 hour after modeling a hemorrhagic stroke) ). No rats were excluded from the study.

To assess the effectiveness of restoration of brain structure and function depending on the dose during intravenous administration, the methods described above were used: survival, neurological status, volume of brain damage according to MRI.

In all experimental groups, survival rate was 100% (4 animals per group). According to the results of a neurological study, severe neurological disorders were observed in 25% of animals (1 rat out of 4) from the control group and 25% (1 rat out of 4) from the 50x group. Mild neurological disorders were observed in 25% of animals (1 rat out of 4) from the control group, the remaining animals were visually healthy. According to MRI data, the best therapeutic effect is observed when using the secretome of rat MSCs in doses of 5x-10x - 15-30 mcg/kg (by protein), and according to the results of reducing the severity of neurological disorders in doses of 5x-25x - 15-75 mcg/kg (by squirrel) (Fig. 17).

Thus, as a result of assessing the neuroprotective and neurotrophic activity of the MSC secretome depending on the dose when administered intravenously in a model of hemorrhagic stroke in rats, it was found that the best therapeutic effect is achieved with intravenous administration of the MSC secretome in the dose range 5x-10x - 15-75 μg/ kg (for protein)). The range of therapeutic doses for clinical use of the claimed composition and agent based on it must be additionally established, since it strongly depends on the volume of damage, localization and severity of the stroke, as well as the route of administration and time of administration after the stroke.

The claimed drug has a complex effect, at least on neurons, micro- and macroglial cells, monocyte/macrophage cells of hematogenous origin, as key participants in damage to nervous tissue.

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LIST OF SEQUENCES

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"Moscow State University named after M.V. Lomonosov" (MSU)

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<---

Claims (24)

1. Composition for neuroprotection and stimulation of neuroregeneration of the brain based on components of the secretome of human mesenchymal stromal cells (MSCs), characterized by the presence of growth factors and cytokines in the culture medium, including BDNF in an amount of at least 3 ng/ml, GDNF - at least 0.03 ng/ ml, VEGF - no less than 0.2 ng/ml, HGF - no less than 0.15 ng/ml, uPA - no less than 0.34 ng/ml and TGFb - no less than 0.055 ng/ml. 2. The composition according to claim 1, characterized in that the TGFb content is indicated in total for the TGFb1, TGFb2 and TGFb3 isoforms. 3. The composition according to claim 1, characterized in that it additionally contains anti-inflammatory cytokines, including the IL-1 receptor antagonist IL-1Ra in an amount of at least 0.4 ng/ml, IL-4 - at least 0.08 ng/ml, IL-6 - not less than 1 ng/ml, IL-10 - not less than 0.01 ng/ml, IL-11 - not less than 0.4 ng/ml, IL-13 - not less than 0.7 pg/ml, and molecules with anti-inflammatory effect, including IDO - not less than 0.08 ng/ml, soluble forms of IL-1 receptors - not less than 0.01 ng/ml and TNF - not less than 0.01 ng/ml. 4. The composition according to claim 1, characterized in that it additionally contains microRNA hsa-miR-23a-3p, hsa-miR-101, hsa-miR-146a, hsa-miR-491-5p, hsa-miR-19b, hsa-miR-21, hsa-miR-25, hsa-miR-125b, hsa-miR-126a-3p. 5. The composition according to claim 4, characterized in that they use microRNA hsa-miR-23a-3p with the sequence SEQ ID NO: 1, hsa-miR-101 - SEQ ID NO: 2 and/or SEQ ID NO: 3, hsa -miR-146a - SEQ ID NO: 4 and/or SEQ ID NO: 5, hsa-miR-491-5p - SEQ ID NO: 6, hsa-miR-19b - SEQ ID NO: 7 and/or SEQ ID NO : 8, hsa-miR-21 - SEQ ID NO: 9 and/or SEQ ID NO: 10, hsa-miR-25 - SEQ ID NO: 11 and/or SEQ ID NO: 12, hsa-miR-125b - SEQ ID NO: 13 and/or SEQ ID NO: 14, hsa-miR-126a-3p - SEQ ID NO: 15. 6. The composition according to claim 1, characterized in that it is a concentrated solution of cytokines and growth factors in a culture medium, a frozen or freeze-dried solution. 7. Use of the composition according to claim 1 as a means of preventing glutamate-mediated death of neural cells, stimulating the polarization of cells of the monocyte-macrophage unit in the anti-inflammatory direction (M2 phenotype), preventing the activation of microglial cells. 8. A method for producing a composition according to claim 1, characterized in that it includes culturing and conditioning primary isolated allogeneic human mesenchymal stromal cells (MSCs) isolated from adipose tissue for 2–5 passages in Dulbecco's modified Eagle growth medium (DMEM) with a glucose content of no more than 1 g/l without phenol red with the addition of penicillin/streptomycin, solutions of essential amino acids and vitamins, for 72-144 hours, selection and purification of the culture medium from cell debris, concentration of the culture medium to at least 10 -fold reduction of the initial volume, purification of the cultivation medium to obtain a sterile concentrate. 9. The method according to claim 8, characterized in that the sterile concentrate is frozen at a temperature from minus 80°C to minus 18°C or freeze-dried. 10. The method according to claim 8, characterized in that freeze-drying is carried out in 6-10 stages at a pressure of 0.013-0.027 kPa, temperature from minus 45°C to 25°C, lyophilization time is no more than 24 hours at maximum freeze-drying load . 11. The method according to claim 8, characterized in that the cultivation of MSCs and conditioning of the medium is carried out at an oxygen content of 1-21 vol.%. 12. A means for neuroprotection and stimulation of neuroregeneration, including the composition according to claim 1 in a therapeutically effective amount and auxiliary components. 13. The product according to claim 12, characterized in that therapeutically acceptable additives, fillers, isotonic agents, stabilizers and/or preservatives are used as auxiliary components. 14. The product according to claim 12, characterized in that it additionally contains human albumin in an amount of at least 5 μg/ml. 15. The product according to claim 12, characterized in that it is a concentrated solution of cytokines and growth factors in a culture medium, a frozen or freeze-dried solution. 16. A method for stimulating neuroprotection and brain regeneration after injury, including introducing the composition according to claim 1 or the agent according to claim 12 into the human body in a therapeutically effective amount. 17. The method according to claim 16, characterized in that the composition or agent is administered locally into the area of brain damage, intranasally, intravenously by stream or drip, intrathecally (into the spinal canal). 18. The method of claim 16, characterized in that the composition or agent is administered once or repeatedly over one or more days at regular intervals during the first three weeks after brain injury. 19. The method according to claim 16, characterized in that in the acute phase of brain damage, the composition or agent is administered no later than 6 hours from the moment of injury; in the acute phase of brain damage, the composition or agent is administered in the period from 6 to 120 hours from the moment of injury , in the subacute phase of brain damage, the composition or agent is administered in the period from 5 to 21 days from the moment of injury. 20. The method according to claim 16, characterized in that the brain damage is caused by an acute cerebrovascular accident (ACVA) of the hemorrhagic type, regardless of its etiology, or of the ischemic type, regardless of the etiology of this disorder, or the damage is caused by exposure to a mechanical factor, regardless from its nature, or by exposure to a thermal factor, regardless of its nature, or a chemical factor, regardless of its nature (toxic effects, metabolic disorders), or penetrating radiation (radiation), regardless of its nature, or a combination of factors in any combination, regardless from their nature, as well as brain damage caused by exposure to an infectious agent. 21. The method according to claim 20, characterized in that strokes of the hemorrhagic type are the result of congenital or acquired defects of the vascular bed of the brain. 22. The method according to claim 20, characterized in that the infectious agent can be a bacterial or viral infection. 23. The method according to claim 22, characterized in that the bacterial infection can be caused by microorganisms of the genera Neisseria and Borrelia. 24. The method according to claim 22, characterized in that the viral infection can be caused by the SARS-Cov2 virus.