Method of preparation of biological products

Abstract

This invention relates to the field of pharmaceutics, namely, to a method of preparation of biological products employing successive repeated vibration treatment of a neutral carrier in the presence of biological objects, which play the role of the initial biological substance, as well as to the biological products themselves, which acquire biological properties similar to those of the initial biological substance in the course of preparation. Consequently, they can be used as biological (pharmaceutical) products, the action of which in the body is directed to the same target molecules, physiological or biological targets, to which the action of a biological object is directed.

Description

[0001] Method of preparation of biological products

[0002] This invention relates to the field of pharmaceutics, namely, to a method of preparation of biological products employing successive repeated vibration treatment of a neutral carrier in the presence of biological objects, which play the role of the initial biological substance, as well as to the biological products themselves, which acquire biological properties similar to those of the initial biological substance in the course of preparation. Consequently, they can be used as biological (pharmaceutical) products, the action of which in the body is directed to the same target molecules, physiological or biological targets, to which the action of a biological object is directed.

[0003] It is known from the prior art that certain long-lasting structural changes can appear in water after exposure to any vibration treatment - both mechanical [Gudkov S.V. et al., 2019; Astashev M.E. et al., 2023; Demangeat JL., 2022; Duval E. et al., 2012] and electromagnetic [D'Emilia E. et al., 2017; Sronsri C. et al., 2021; Wu T. et al., 2020],

[0004] It is also known from the prior art that when solutions of an initial substance are prepared by multiple dilutions using an external mechanical effect in the form of vertical shaking (vibration) at each dilution cycle up to the level of ultra-high dilutions, the latter acquire a number of new physical properties that determine their activity even at speculatively small amounts in dilutions of the initial substance.

[0005] Such properties include the ability of high dilutions obtained by applying multiply repeated vibration treatment to exert a non-contact (distant) effect on other substances or solvents [Jerman, I.; Ruzic, R.; Krasovec, R.; Skarja, M.; Mogilnicki, L. «Electrical Transfer of Molecule Information into Water, Its Storage, and Bioeffects on Plants and Bacteria», Electromagnetic Biology and Medicine 2005, 24 (3), 341-353. https: / / doi.org / 10. 1080 / 15368370500381620; Ruzic, R., Jerman, I., Skarja, M., Leskovar, R., Mogilnicki, L. «Electromagnetic Transference of Molecular Information in Garden Cress Germination», Int J High Dilution Res 2008; 7(24): 122-131 2008, 7, 122-131; Igor Jerman, Linda Ogrizek, Vesna Pericek Krapez and Luka Jan, Physicochemical study of the molecular signal transfer of ul-tra-high diluted antibodies to interferon gamma. Int. J. Mol. Sci. 2023, 24; N.Penkov, N.Penkova. Analysis of Emission Infrared Spectra of Protein Solutions in Low Concentrations. Front. Phys., 18 December 2020 Sec. Interdisciplinary Physics, Volume 8 - 2020 | http s : / / doi . org / 10.3389 / fph y .2020.624779; Penkov N., 2021; Novikov V.V. and Yablokova E.V., 2022)], as well as the ability of dilutions to exert a modifying effect on the initial substance [WO2012017324, US7,229,648, US4,311,897, RU2192888, 2577137, 2577136, 2536230, 2519695, 2778521, 2509572, 2531048, 2517085, 2505312, 2577299], Modifying effects are manifested in changes in the structure of the molecules of the initial substance, their hydration shell, which leads to modulation of biological activity of the initial substance [Tarasov SA, Gorbunov EA, Don ES, Emelyanova AG, Kovalchuk AL, Yanamala N, Schleker ASS, Klein- Seetharaman J, Groenestein R, Tafani JP, van der Meide P, Epstein OI. Insights into the Mechanism of Action of Highly Diluted Biologies. J Immunol. 2020;205(5): 1345-1354. doi: 10.4049 / jimmunol.2000098; Woods KN. Modeling of protein hydration dynamics is supported by THz spectroscopy of highly diluted solutions. Front Chem. 2023; 11 : 1131935. doi: 10.3389 / fchem.2023.1131935] and determines the presence of biological (pharmacological) activity in high dilutions.

[0006] The modifying effect underlying the specific pharmacological activity of multiply diluted drugs is quite pronounced, which allowed evaluation of modifying effects using generally accepted methods and standardization of high dilutions of biological substances (see State Pharmacopoeia of the Russian Federation XV, General Pharmacopoeia Monograph (GPM) 1.7.0001).

[0007] The technology of obtaining solutions in the form of serial dilution of biological substances and vibration treatment of dilutions after the release of GPM received the official name ‘gradual technology’. Currently, preparations based on gradual technology, gradualized drugs, are produced mainly from antibodies and therefore belong to the category of biological products [Mkrtumyan A, Ametov A, Demidova T, Volkova A, Dudinskaya E, Vertkin A, Vorobiev S. A New Approach to Overcome Insulin Resistance in Patients with Impaired Glucose Tolerance: The Results of a Multicenter, Double-Blind, Placebo-Controlled, Randomized Clinical Trial of Efficacy and Safety of Subetta. J Clin Med. 2022; 11(5): 1390. doi: 10.3390 / jcml 1051390; Geppe NA, Blokhin BM, Shamsheva OV, Abdrakhmanova ST, Alikhanova KA, Myrzabekova GT. Efficacy and Safety of Ergoferon in Children from 6 Months to 6 Years Old with Acute Respiratory Viral Infections in Contemporary Outpatient Practice: A Multicenter, Double-Blind, Placebo-Controlled Randomized Trial. Can Respir J. 2021; 2021 :5570178. doi: 10.1155 / 2021 / 5570178; Lashch NU, Kamchatnov PR, Fedorova TN, Muzychuk OA, Khacheva KK, Pizova NV, Malygin AU, Shavlovskaya OA, Fateeva VV, Nikulina KV, Abrosimov AV, Gerasimova YA, Glushkov KS, Lebedeva AV. Efficacy and Safety of Divaza for the Correction of Oxidative Disturbances in Patients with Cerebral Atherosclerosis: A Randomized Controlled Trial. Cerebrovasc Dis. 2021; 50(4):472-482. doi: 10.1159 / 000515233; Avdeev SN, Vizel AA, Abrosimov VN, Zaicev AA, Ignatova GL, Khamitov RF, Mikhaylusova MP, Shapovalova JS, Pavlysh EF, Trofimov BI, Emelyanov AV, Martynenko TI, Martynenko VA, Kostina NE, Chizhov DA, Chizhova OY, Kuzubova NA, Makova EV, Makarova EV. Management of Cough in Patients with Chronic Obstructive Pulmonary Disease: Results of the Multicenter Randomized Placebo-Controlled Clinical Trial. Int J Chron Obstruct Pulmon Dis. 2021; 16: 1243-1253. doi: 10.2147 / COPD.S292109; Parfenov VA, Zhivolupov SA, Poverennova IE, Nesterova MV, Ushakova SE, Zhukova NG, Glazunov AB, Nikulina KV, Alexandrov MV, Lapatukhin VG, Zhestikova MG. Treatment of Cognitive Impairment and the Role of Demographic Factors in Disease Progression: The Final Results of the Russian Observational Program "DIAMANT". Eur Neurol. 2020; 83(6):591-601. doi: 10.1159 / 000508184; Ivashkin VT, Poluektova EA, Glazunov AB, Putilovskiy MA, Epstein OI. Pathogenetic approach to the treatment of functional disorders of the gastrointestinal tract and their intersection: results of the Russian observation retrospective program COMFORT. BMC Gastroenterol. 2019; 20(l):2. doi: 10.1186 / sl2876-019-l 143-5; Parfenov VA, Ostroumova OD, Ostroumova TM, Kochetkov Al, Fateeva VV, Khacheva KK, Khakimova GR, Epstein OI. Vascular cognitive impairment: pathophysiological mechanisms, insights into structural basis, and perspectives in specific treatments. Neuropsychiatr Dis Treat. 2019; 15: 1381-1402. doi: 10.2147 / NDT.S197032; Pushkar D, Vinarov A, Spivak L, Kolontarev K, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Afalaza in men with symptomatic benign prostatic hyperplasia at risk of progression: a multicenter, double-blind, placebo-controlled, randomized clinical trial. Cent European J Urol. 2018; 71(4):427-435. doi:

[0008] 10.5173 / ceju.2018.1803; Mkrtumyan A, Romantsova T, Vorobiev S, Volkova A, Vorokhobina N, Tarasov S, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Subetta add-on therapy in type 1 diabetes mellitus: The results of a multicenter, double-blind, placebo- controlled, randomized clinical trial. Diabetes Res Clin Pract. 2018; 142: 1-9. doi: 10.1016 / j.diabres.2018.04.044; Rafalsky V, Averyanov A, Bart B, Minina E, Putilovskiy M, Andrianova E, Epstein O. Efficacy and safety of Ergoferon versus oseltamivir in adult outpatients with seasonal influenza virus infection: a multicenter, open-label, randomized trial. Int J Infect Dis. 2016; 51 :47-55. doi: 10.1016 / j .ijid.2016.09.002; Don E, van der Meide N, Egorov V, Putilovskiy M, Tarasov S. The level of natural autoantibodies to IFN-gamma in varicella infection treated with antiviral drug Anaferon for children: A pilot study. Immunol Lett. 2020; 222:90-94. doi: 10.1016 / j.imlet.2019.10.015; Tarasov SA, Gorbunov EA, Don ES, Emelyanova AG, Kovalchuk AL, Yanamala N, Schleker ASS, Klein-Seetharaman J, Groenestein R, Tafani JP, van der Meide P, Epstein 01. Insights into the Mechanism of Action of Highly Diluted Biologies. J Immunol. 2020; 205(5): 1345-1354. doi: 10.4049 / jimmunol.2000098; Woods KN. Modeling of protein hydration dynamics is supported by THz spectroscopy of highly diluted solutions. Front Chem. 2023; 11 : 1131935. doi: 10.3389 / fchem.2023.1131935; Alinkina E, Don E, Gizitdinova O, Samsonova L, Petrova A, Stepanov G, Tarasov S. A novel technique for studying the effects of technologically processed antibodies by evaluating the rate of oxidation of ascorbic acid during the reduction of the green-blue ABTS + radical. Spectrochim Acta A Mol Biomol Spectrosc. 2024; 304: 123323. doi: 10.1016 / j.saa.2023.123323; Emelianova AG, Petrova NV, Fremez C, Fontanie M, Tarasov SA, Epstein OI. Therapeutic potential of highly diluted antibodies in antibiotic-resistant infection. Eur J Pharm Sci. 2022; 173: 106161. doi: 10.1016 / j.ejps.2022.106161; Petrova NV, Tarasov SA, Epstein OI, Dubroca C, Sulpice T. Highly Diluted Antibodies to eNOS Restore Endothelium Function in Aortic Rings From Hypertensive Rats. Dose Response. 2022; 20(2): 15593258221099281. doi: 10.1177 / 15593258221099281; Don ES, Bobrovnik SA, Sherriff G, Myslivets AA, Tarasov SA, Epstein OI. Advanced approach to activity evaluation for released-active forms of antibodies to interferon-gamma by enzyme-linked immunoassay. J Immunoassay Immunochem. 2019;40(3):250-268. doi: 10.1080 / 15321819.2019.1567536; Kardash EV, Ertuzun IA, Khakimova GR, Kolyadin AN, Tarasov SA, Wagner S, Andriambeloson E, Ivashkin VT, Epstein OI. Dose-Response Effect of Antibodies to SI 00 Protein and Cannabinoid Receptor Type 1 in Released-Active Form in the Light-Dark Test in Mice. Dose Response. 2018; 16(2):1559325818779752. doi: 10.1177 / 1559325818779752; Don ES, Emelyanova AG, Yakovleva NN, Petrova NV, Nikiforova MV, Gorbunov EA, Tarasov SA, Morozov SG, Epstein OI. Dose-dependent antiviral activity of released-active form of antibodies to interferon-gamma against influenza A / California / 07 / 09(H1N1) in murine model. J Med Virol. 2017; 89(5):759- 766. doi: 10.1002 / jmv.24717; Don E, Farafonova O, Pokhil S, Barykina D, Nikiforova M, Shulga D, Borshcheva A, Tarasov S, Ermolaeva T, Epstein O. Use of Piezoelectric Immunosensors for Detection of Interferon-Gamma Interaction with Specific Antibodies in the Presence of Released-Active Forms of Antibodies to Interferon-Gamma. Sensors (Basel). 2016;16(l):96. doi: 10.3390 / sl6010096; Gorbunov EA, Nicoll J, Kachaeva EV, Tarasov SA, Epstein OI. Subetta increases phosphorylation of insulin receptor P-subunit alone and in the presence of insulin. Nutr Diabetes. 2015; 5(7):el69. doi: 10.1038 / nutd.2015.20; Gorbunov EA, Ertuzun IA, Kachaeva EV, Tarasov SA, Epstein 01. In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to SI 00 protein in released- active form. Neuropsychiatr Dis Treat. 2015; 11 :2837-46. doi: 10.2147 / NDT.S92456; Bailbe D, Philippe E, Gorbunov E, Tarasov S, Epstein O, Portha B. The novel oral drug Subetta exerts an antidiabetic effect in the diabetic Goto-Kakizaki rat: comparison with rosiglitazone. J Diabetes Res. 2013; 2013:763125. doi: 10.1155 / 2013 / 763125; Nicoll J, Gorbunov EA, Tarasov SA, Epstein OI. Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro. Int J Endocrinol. 2013; 2013:925874. doi: 10.1155 / 2013 / 925874; Andrianov VV, Epstein OI, Gainutdinova TKh, Shtark MB, Timoshenko AKh, Gainutdinov KL. Antibodies to calcium- binding SIOOB protein block the conditioning of long-term sensitization in the terrestrial snail. Pharmacol Biochem Behav. 2009; 94(l):37-42. doi: 10.1016 / j.pbb.2009.07.003; Castagne V, Lemaire M, Kheyfets I, Dugina JL, Sergeeva SA, Epstein OI. Antibodies to SI 00 proteins have anxiolytic-like activity at ultra-low doses in the adult rat. J Pharm Pharmacol. 2008; 60(3):309- 16. doi: 10.121 l / jpp.60.3.0005; Chu X, Zhavbert ES, Dugina JL, Kheyfets IA, Sergeeva SA, Epstein OI, Agmo A. Sildenafil and a compound stimulating endothelial NO synthase modify sexual incentive motivation and copulatory behavior in male Wistar and Fisher 344 rats. J Sex Med. 2008; 5(9):2085-99. doi: 10.1111 / j.1743-6109.2008.00937.x; Chu X, Agmo A. Sexual incentive motivation in old male rats: the effects of sildenafil and a compound (Impaza) stimulating endothelial NO synthase. Pharmacol Biochem Behav. 2008; 89(2):209-17. doi: 10.1016 / j.pbb.2007.12.012; Epstein OI, Zapara TA, Simonova OG, Ratushnyak AS, Shtark MB. Plasticity of neuronal responses induced by low concentrations of exogenous ligands affecting cellular calcium stores. Front Biosci. 2004; 9:809-15. doi: 10.2741 / 1244; Epstein OI, Beregovoy A, Sorokina NS, Starostina MV, Shtark MB, Gainutdinov KhL, Gainutdinova TKh, Muhamedshina DI. Membrane and synaptic effects of anti-S-100 are prevented by the same antibodies in low concentrations. Front Biosci. 2003; 8:a79-84. doi: 10.2741 / 1025],

[0009] Biological substances have a great potential for implementation, since unlike synthetic drugs, they have a wide spectrum of activity and a gentle character of pharmacological action. However, the use of biological molecules not exposed to gradual treatment has a number of limitations in the form of adverse events, such as toxicity of monoclonal antibodies.

[0010] Currently, individual therapy of various diseases using various biological objects, primarily fetal tissues containing stem cells and used in the form of cell transplants, has seen substantial progress. This therapy has both advantages, first of all, the ability to have a regenerative effect, and disadvantages. Thus, cell transplants are difficult to standardize and store for a long time, which hinders their widespread use in the form of medicines. The use of cell transplants can be accompanied by immunologic rejection reactions, viral or prion contamination. In this regard, the use of gradualized biological substances subjected to multiple dilution accompanied by vibration treatment is a priori a promising direction. However, obtaining high dilutions of a biological substance is a difficult task, since during dilution, with preliminary homogenization, the biological substance loses part of its initial activity.

[0011] The applicant found that even without dilution of the initial substance, successive vibration treatment of a test tube with a solvent in the presence of a nearby test tube containing various molecules, including biological ones, i.e., antibodies, results in the solvent changing its physical properties and turning into an artificial material object called ‘iteration’. It was found that iterations were distributed by physical-chemical properties into fractions, each of which reproduced some pharmacological properties of the initial molecule. In a number of cases, iterations exerted multidirectional effects, which allows them to be widely used as products, both in the cases when one or another physiological process needs to be suppressed and in the cases when it needs to be enhanced.

[0012] The technical objective of this invention was to develop a method of obtaining biological products from the initial biological substance (biological object), which have an action similar to that of the biological object, directed to the same endogenous molecules in the organism, targets or functional -metabolic processes, which are affected by the biological object.

[0013] The applicant has discovered a way of obtaining a product, reproducing the properties of a biological object, affecting molecular or cellular targets in the body, physiological and metabolic processes regulated by the biological object, which is a biological (pharmacological) product - iterations of a biological substance, prepared using external rhythmic effect (Fig. 1).

[0014] There are articles in the prior art relating to high dilutions. For example, there is the article 'Interaction between highly diluted samples, protein solutions and water in a controlled magnetic field' by V. V. Novikov, published in Applied Sciences in 2022. Another example is the article 'Antibodies processed using high dilution technology distantly change the structural properties of an IFN gamma aqueous solution' by N. Penkov, published in Pharmaceutics in 2021. These articles describe a stock solution of a substance that is diluted multiple times (HD), and an experiment on the remote action of HD IFNg on IFNg. However, this is not relevant to the present invention, as the application discloses iterations that are prepared from a neutral carrier in the presence of a biological product. Another known article is “The Supramolecular Matrix Concept” by O. Epstein, Symmetry, 2023, describes ultrahigh dilutions (i.e. preparations obtained by successive reduction of concentration and shaking of solutions of various substances) and the probable mechanism of action of such preparations. It describes the so-called distant effect, which implies that ultrahigh dilutions can affect their targets from a distance. However, the above article does not contain information on the preparation and use of iterations of biological objects.

[0015] Iteration preparation technology is fundamentally different from the technology used to prepare high dilutions. High dilutions involve a sequential reduction in the concentration of the starting substance by physical action on the solution. In contrast, iteration technology involves sequential vibratory action on a neutral carrier in the presence of a solution of the substance. These are two distinct technologies.

[0016] Therefore, the articles cited above are not relevant to the present application. Some articles provide information on the distance effects of products obtained using the high dilution preparation technique. However, this information relates to the properties of such preparations, whereas the presence of the substance in close proximity to a neutral carrier within the iterative preparation technology is a condition for its realization, which is in no way disclosed in the articles. Thus, the average person skilled in the art, having knowledge of the high dilution technology or the properties thereof, would not be able to implement the iteration technology.

[0017] A distinctive feature of the claimed method of obtaining biological products is the possibility of using integral biological object - cells and their structures (culture of cells, tissues, organs) as an initial biological substance without dilution of the biological substance in order to transfer the properties of the native biological object into iterations.

[0018] The iterations obtained in the course of successive vibration treatment of biological objects have a biological (pharmacological) effect, partially or fully reproducing the biological effects of a biological object (substance). The range, direction and extent of pharmacological effect of different fractions of iterations of biological objects differ and can be established experimentally.

[0019] Furthermore, an obligatory part of the solution is the separation of the obtained iterations into fractions by evaluating their physical-chemical properties using generally accepted analytical methods and further determination of their biological (pharmacological) properties using also generally accepted methods for experimental biology and pharmacology.

[0020] The method includes obtaining of ‘iteration zero’ at the initial stage by vibration treatment of tubes with the initial biological substance and solvent, which can be carried out both simultaneously and alternately, leading to the transformation of the solvent into a material object with new physical, chemical and biological (pharmacological) properties, different from the properties of both the initial substance and the solvent and further successive vibration treatment of the previous iteration and the solvent to obtain subsequent iterations 1, 2, 3, etc. from the solvent.

[0021] A biological object within the scope of this invention is understood to be an integral (intact), retaining original activity, cell or cell associations in the form of a group of cells, tissue or organ, or cells of microorganisms, mainly bacteria and viruses, or extracellular structures, prions. A micro- or macroorganism cell or relatively functionally and morphologically homogeneous macroorganism cells in the form of a cellular structure (tissues, body fluids, organs, as well as a cellular transplant from fetal, embryonic or other tissue may be used, representing both the recipient’s own previously obtained biomaterial (autotransplant) and biomaterial taken from other donors (allogeneic transplant), as well as biomaterial obtained from other biological species (xenotransplant), which are used as a biological substance immediately or after storage, and can also be grown outside the organism.

[0022] Since some of the iterations have a suppressive effect, it is possible to select iterations that reduce the activity of microorganisms experimentally. In the vast majority of cases, the biological object is cells or cellular ensembles of the macroorganism. Depending on the goals of therapy, in this case, iterations with the necessary directional effect are selected.

[0023] A biological object is selected depending on which altered or pathological functional and metabolic processes or which target molecules are supposed to be affected. This approach allows to use a wide range of experimental and clinical data on the activity of biological objects, which facilitates the development of new drugs.

[0024] Cells in the form of an isolated cell or group of cells may be used as biological objects, and the claimed product will affect the same functions that these cells perform in the body.

[0025]

[0026] As a biological object, both structurally normal cells and cell cultures can be used, and in some cases, if necessary to weaken the activity of the biological object - its homogenates or supernatants, as well as cells of microorganisms weakened by physical or chemical effects can be used. Both normal physiological cells and atypical cells can be used.

[0027] The biological object may be a cell, tissue or organ culture grown outside the body.

[0028] Organs or organ system isolated from an organism or grown outside the organism can be used as biological objects:

[0029] Both a homogeneous set of cells and a combination thereof may be used as an initial substance for obtaining the claimed product. For example, both individually derived stem cells (undifferentiated cells capable of transforming into any of the cell types in the body, derived from embryos, fetuses, umbilical cord blood, bone marrow, induced pluripotent stem cells) and stem cells contained directly in fetal tissues commonly used as cell transplants for personalized biological regenerative medicine can be used.

[0030] As a biological object, fetal or embryonic cells and tissues can be used — whether they are of autologous, allogeneic, or heterologous origin — containing, in addition to stem cells, regional blast cells along with differentiated cells, specialized cells, and biologically active substances. Due to this, iterations of such cells (or tissues), can be used to treat a wide range of diseases of various systems: nervous system, endocrine system, metabolic disorders, diseases of blood, hematopoietic organs, conditions associated with disorders of immune mechanisms, treatment of neoplasms, diseases of the eye and adnexa, ear, blood circulation, respiratory organs, digestion, diseases of skin and subcutaneous tissue, musculoskeletal system, connective tissue, conditions arising in the perinatal period, traumas and poisonings, since it is known from the prior art that fetal tissues and cells exert an effect on these organs (targets) and functions of the organism [RU2160112],

[0031] The biological (pharmacological) effect of a biological preparation obtained by the claimed method of successive vibration treatment is understood as activating or inhibiting effect on functional and metabolic processes involving or affected by the initial biological object, or the regulatory effect on targets affected by the initial biological object, or on the biological object itself, which determines the specific (directed) pharmacological (biological) effect of the biological preparation of this type, which is similar to the biological effect of the initial biological object.

[0032] The targets of action of the iteration, biological preparation obtained by the discussed method of successive vibration treatment, can be not only endogenous targets or processes, but also the initial biological objects themselves, for example, viruses, bacteria, prions or atypical cells of the organism, as well as targets are pathological morpho-functional or metabolic processes in the initial biological object itself, for example, a thyroid cyst or processes controlled by thyroid gland, for example, the content of hormones, calcium, phosphorus, vitamin D3 in the organism during treatment, for example, by iteration of the native thyroid gland isolated from the body.

[0033] The claimed invention provides a method for producing a biological preparation comprising the following steps: 1) preparation of ‘iteration zero’ - an artificial material object obtained by exposure of the solvent to external vibration treatment in the presence of the initial biological substance (with their further exposure - incubation);

[0034] 2) obtaining the first iteration by exposure of the intact solvent to vibration treatment in the presence of ‘iteration zero’;

[0035] 3) obtaining a series of iterations, where each successive iteration is obtained by exposure of the solvent to vibration treatment in the presence of the previous iteration.

[0036] In the first step, a ‘iteration zero’ is obtained, into which the solvent is transformed by joint vibration treatment of the solvent and the initial biological object (with their further incubation). Then, by vibration treatment of the preceding iteration and the solvent from the solvent, subsequent iterations, the first, second, third, etc., are obtained.

[0037] The process of obtaining iterations may differ in terms of the time during which the external vibration treatment is applied, or incubation (exposure) - post-vibration contact of the substance (or iterations) with the solvent; the type of external rhythmic physical effect - horizontal or vertical mechanical effect (shaking, rotation on a vortex), ultrasound, electromagnetic field, etc., as well as variants of external rhythmic effect in frequency and amplitude. Various solvents, such as water or a water-alcohol mixture, may also be used. The series of iterations may be derived from a single biological substance, or a combination thereof, such as an auto-, allo- or heterogeneous cell transplant containing different cell types

[0038] The time of external vibration treatment and incubation (co-incubation of test tubes with intact solvent and iteration or initial biological substance at room temperature) depends on the amount of the initial biological object and solvent and is selected experimentally for each specific case. Test tubes and vials are placed side by side - close to each other or at a distance, mostly 1-3 cm. Separate vibration treatment of substance (or iterations in the case of the preparing subsequent iterations) and solvent with their further incubation is also possible, when tubes with substance (or iterations in the case of the preparing subsequent iterations) and solvent are placed next to each other for a period of 1 sec or more.

[0039] Iterations obtained by successive external vibration treatment on test tubes with solvent and preceding iterations or initial substance (to obtain the ‘iteration zero’) represent a biological product because they are derived from biological raw materials and their mechanisms of action are biological because they are directed to biological targets in the body.

[0040] In contrast to individually applied biological products, such as cell transplants, biological products obtained by the claimed method have a number of advantages: they can be characterized by relatively simple physical-chemical criteria and validated using commonly available analytical methods, they can be stored for a long time, administered orally, do not cause rejection reactions and other side effects, for example, there is no risk of viral or prion contamination. In certain cases, some iteration fractions activate the targeted processes and others inhibit them, thus extending the range of their use.

[0041] It has been experimentally established that fractions of iterations with common physical, first of all spectral properties, have common biological properties; therefore, the separation of iterations into fractions is a necessary technological step in the creation of biological products of this type, with further experimental determination of their biological (pharmacological) activity.

[0042] The method of separation into fractions, according to this invention, comprises the following steps:

[0043] 1. Using known analytical methods, each iteration obtained is evaluated in terms of physical -chemi cal properties, differing from the properties of the intact solvent, using the following assays: determination of specific electrical conductivity (SEC), radiometry, terahertz spectroscopy and immunoassay (ELISA), pH measurement, detection of the amount of dissolved oxygen, dynamic light scattering, high-resolution thermography, etc.

[0044] 2. Depending on the evaluation, iterations are divided into fractions exhibiting their own physical -chemi cal properties different from those of the initial neutral solvent;

[0045] 3. The specific pharmacological (biological) activity, which is similar to the activity of the biological object used as an initial substance, is determined experimentally for each fraction.

[0046] Thus, knowing the properties of the biological object selected as an initial substance for obtaining iterations, it is possible to foresee what properties may be exhibited by the obtained iterations (technological product) and to study them using generally accepted methods.

[0047] The external vibration treatment used to obtain iterations means horizontal, vertical mechanical shaking or a combination thereof, as well as external effect exerted by acoustic and microfluidics methods [RU2724254], electromagnetic, ultrasonic influence or other external rhythmic influence. The test tubes with solvent and substance (or the preceding iteration) may be subjected to joint (simultaneous) vibration treatment or each of the test tubes is independently of the other exposed to vibration treatment. In this case, different types of external rhythmic effect may also be used. Intrinsic physical-chemical properties mean the appearance of long-lasting physicalchemical properties in iterations that are quantitatively or qualitatively different from the physical -chemi cal properties of the initial substance or neutral solvent.

[0048] The neutral carrier can be either a solvent whose transformation results in iterations: water, aqueous-alcoholic solution, or solids - lactose or any pharmaceutically acceptable solvent or excipient.

[0049] An intact carrier / solvent is understood to be a neutral carrier / solvent, until the moment of vibration treatment.

[0050] The initial biological object, biological substance, can be used in a conditionally solid, unaltered, form or in the form of homogenate, lyophilizate, supernatant, solution or high dilution of homogenate in a specially selected solvent, in fresh or frozen form. The concentration of the initial biological substance in the vial may be from 0.1 pg / mL to 10 mg / mL; the concentration is selected in experiments for each particular case.

[0051] The product obtained using the claimed method can be used in liquid or solid dosage form. For example, a product obtained using the claimed method can be used in solid dosage form and contain a technologically necessary (effective) amount of a neutral carrier saturated with iterations and pharmaceutically acceptable additives, which include, for example, lactose, microcrystalline cellulose, magnesium stearate and others. To obtain the solid oral form of the claimed product in a fluidized bed unit (for example, the ‘Huttlin Pilotlab’ type manufactured by Huttlin GmbH), irrigation is performed until the granules of a neutral substance, lactose (milk sugar), introduced into the fluidized bed are saturated with a previously obtained aqueous or aqueous alcohol solution of iterations with simultaneous drying in the stream under the heated air grate at a temperature not exceeding 40 °C. The resulting tablet mass is evenly mixed and tableted by direct dry pressing (for example, in a Korsch - XL 400 tablet press) [W02007105981(A1), 09 / 20 / 2007], After tableting, tablets weighing 300 mg and impregnated with aqueous or aqueous-alcoholic solution of iterations are obtained.

[0052] The figures below, together with the drawings attached, are offered to illustrate the present invention in more detail:

[0053] Fig. l. The scheme of the technology for obtaining iterations.

[0054] Fig.2. Results of assessing the edema of the affected auricle in animals on the 2nd and 4th days (5 animals per group); Note - * - statistically significant differences from “Control” (p <0.05), @ - statistically significant differences from the Placebo group (p <0.05),A- statistically significant differences from the “Induction of pathology ” group (p <0.05) . Fig.3. Effect of CHO-S iterations on glucose consumption by CHO-S cells normalized to cell number as a function of insulin concentration (- statistically significant differences (p<0.05) from intact water).

[0055] Fig.4. Comparison of groups 1-4: by tumor mass between each other and with positive control (A), by the number of metastases between each other and with Control 1 and 2 (B, C, respectively).* significant differences from the corresponding control (p < 0.005).Asignificant differences from group 2 (p < 0.05).

[0056] Fig 5. Effect (% of intact A549 cell survival ± SD) of Active fraction of vibrational iterations of K-A21 normal lung cells (I3-act), Native fraction of vibrational iterations of K-A21 normal lung cells (I6-nat), and neutral carrier (ultrapure water, MQ) on the survival of A549 lung carcinoma cells after exposure to UV for 2 min. * statistically significant difference from neutral carrier (purified water, MQ) (p < 0.01).

[0057] The invention is now described in more detail with reference to the enclosed Figure 1.

[0058] The production of an artificial product (iterations) consists of several stages:

[0059] 1. A test tube with the initial substance and a test tube with a neutral carrier (water) are placed close together and subjected to joint vibrational treatment using a vortex.

[0060] 2. The test tubes are then incubated at room temperature. Empirically, a minimum incubation time of 1 minute has been established as sufficient for obtaining iterations. In some cases, the incubation time can be reduced or skipped if the transformation of the neutral solvent into a material object (iteration) has been experimentally confirmed.

[0061] After stages 1 and 2, a change occurs in the physical properties of the solvent in the first test tube compared to the intact solvent.

[0062] The test tube with the neutral carrier (solvent), altered by the first vibrational treatment is called the ‘iteration zero’ of the substance (10).

[0063] 3. Then, 10 is placed close to a test tube with water (a new portion), and both test tubes are subjected to vibrational treatment and incubation at room temperature. As a result, the water in the second test tube also changes its properties and transforms into the first iteration of the substance (II).

[0064] 4. The described vibrational treatment procedure is repeated to obtain subsequent iterations from the previous ones — 12, 13,..., In iterations of the substance. 5. Afterward, the stage of selecting the obtained iterations, which have separated into fractions after vibrational treatment.

[0065] Fractions that are potential candidates for biological products after vibrational treatment should differ in physical properties from the solvent. For practical purposes, it is sufficient to select candidates after obtaining a series of six iterations and choose two main groups that differ the most in physical properties, especially in their influence on the spectral characteristics of the initial substance. It has been empirically established that these fractions also differ the most in biological and chemical properties, allowing for the selection of the most suitable iteration for a specific task. It is sufficient to use only one iteration from the entire series of obtained iterations or to mix several iterations of one fraction together.

[0066] Given the methods we use, the first six iterations (11-16) can only be divided into four fractions based on their physical characteristics, the ratio of which may vary for each initial substance, or one of the fractions may be absent. Technical terms proposed for the fractions are native, seminative, semi-active, and active.

[0067] 1. Native - Iterations that, according to conductometry (C) and radiometry (GHz) results, do not have significant changes in physicochemical properties compared to the intact neutral carrier (water) and, according to terahertz (Thz) spectroscopy (effect on initial substance) and immunoassay (ELISA), do not possess modifying activity towards the initial substance, i.e., C-, GHz-, THz-, ELISA.

[0068] 2. Semi-native - Iterations in which statistically significant changes in physicochemical properties were found compared to the intact neutral carrier (water), but like natives, do not possess modifying activity towards the initial substance, i.e., C+ and / or GHz+, THz-, ELISA-.

[0069] 3. Semi-active - Iterations that do not have significant changes in physicochemical properties compared to the intact neutral carrier (water), but unlike natives, possess modifying activity towards the initial substance, i.e., C-, GHz-, THz+ and / or ELISA+.

[0070] 4. Active - Iterations in which statistically significant differences in physicochemical properties were found compared to the intact neutral carrier (water) and which possess modifying activity towards the initial substance, i.e., C+ and / or GHz+, THz+ and / or ELISA+.

[0071] Usually, the choice is made between the semi-native and active fractions, which are tested on the same biological or pharmacological experimental models to determine their activity, which in varying degrees of different fractions of iterations reproduces the effects of the initial substance. Example 1. Iterations obtained using human adipocytes and water as a neutral carrier.

[0072] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0073] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0074] The process of obtaining iterations included several stages. A vial with a capacity of 60 ml with a freshly prepared suspension of differentiated human adipocytes as initial substance at a concentration of 35* 103cells / ml in a volume of 50 ml and a vial with a capacity of 250 ml with purified water (neutral carrier) in a volume of 180 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0075] Next, a 40 ml vial with a Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0076] Each of the obtained iterations also has its own physical and chemical properties, determined in the following ways: Conductometry. To evaluate the physicochemical properties of a series of iterations, the conductometry method was used. To measure specific electrical conductivity, 10 ml of sample was taken into 15 ml vials and an electrode (Mettler Toledo, USA) was positioned so that the electrode membrane was completely immersed in the solution, after which the SEC was measured. In total, 21 iterations were obtained. Using the conductometry method, it was revealed that iterations No. 1, 4, 11, 14, 17 have significant differences from the neutral carrier. Radiometry. The assessment of the iterations' intrinsic electromagnetic radiation was carried out using the radiometry method on a special installation, which consisted of a Faraday cage - an aluminum frame covered with a copper mesh. Inside the cage there was placed a tripod with a claw for attaching a detector, a thermal shaker (BIOSAN PST- 60HL-4) for heating the samples, and a TES-92 electromagnetic radiation (EMR) detector (TES Electrical Electronic Corp., Taiwan), which makes it possible to detect EMR flux density in the range from 1 pW / m2to 30.93 W / m2and frequency range from 50 MHz to 3.5 GHz. The measurement mode used was maximum average value (MAX AVG). The test sample (10 ml) was placed in a Petri dish, then the Petri dish with the sample was placed on a thermal shaker and the sample was heated to a temperature of 37±1°C. After this, the detector was placed above the sample at a distance of 0.5 cm, and the lid of the Petri dish was removed. The measurements were carried out with a closed Faraday cage. The measurement time was 10 minutes. After the time had elapsed, the maximum average value of the EMR flux density from the device display was recorded. Using the radiometric method, it was revealed that iterations No. 6, 13, 15, 16, 20 have significant differences from neutral carrier. 3. Terahertz spectroscopy. Analysis of changes in spectral characteristics in the terahertz region of the target molecule after adding iterations was carried out using a TeraView TeraPulse Lx terahertz spectrometer (England). To do this, 1 part (5 pL) of the iterations or control was added to 99 parts (495 pl) of a solution of the substance, which is a specific target for the iterations under study, and the refractive and transmission spectra were recorded. To analyze the data, the coefficients of the equation of the well- known Debye function that describes the resulting spectra (Penkov N., Fesenko E. Development of terahertz time-domain spectroscopy for properties analysis of highly diluted antibodies. Appl. Sci. 2020;10:7736. doi: 10.3390 / appl0217736.) and characterizes the dielectric constant in relaxation regions were used. Using terahertz spectroscopy, it was revealed that iterations No. 1, 4, 9, 12, 13, 18, 21 have significant differences from the neutral carrier in the value of the coefficients Asl (amplitude of the relaxation process Rl) or As2 (amplitude of the relaxation process R2).

[0077] 4. Enzyme-linked immunosorbent assay (ELISA). Also, the modifying properties of iterations were studied using ELISA. To do this, the studied iterations were added to the antigen - the target molecule, and the modifying activity was indirectly assessed by changes in the degree of its binding to monoclonal antibodies depending on their concentration. Using the ELISA method, it was revealed that iterations No. 6, 7, 10, 11, 14, 15, 16, 19 have significant differences from the neutral carrier in terms of the curve of dependence of optical density on the concentration of monoclonal antibodies to adiponectin after their interaction with adiponectin (1 pg / ml) in the presence of samples.

[0078] Thus, using the above methods, the previously obtained iterations with numbers from 1 to 21 (I1-I21) were divided into the following fractions: having altered physicochemical properties and exhibiting a modifying effect (Ii, I4, L, In, I13, I14, I15, IK); having altered physicochemical properties and not exhibiting a modifying effect (I17, I20); not having altered physicochemical properties, but exhibiting a modifying effect (I7, I9, ho, I12, Iis, I19, I21); not having altered physicochemical properties and not exhibiting a modifying effect (I2, I3, h, h).

[0079] It is known that one of the functions of white adipose tissue adipocytes is the secretion of a number of hormones, in particular, adiponectin, leptin, resistin. The obtained iterations may exhibit specific activity in relation to the secretory function of adipocytes.

[0080] The assessment of the specific activity was performed by an in vitro method on a culture of differentiated human adipocytes. Preadipocytes were obtained from the adipose tissue of healthy donors with a BMI of 27.1±1.1, pooled, and seeded into a tissue culture plate at an initial density of 40,625 cells / cm2in a culture medium for the growth of preadipocytes PM-1. The plate was incubated in a CO2 incubator overnight. Then, primary preadipocytes were differentiated in a CO2 incubator for a week in a culture medium for adipocyte differentiation DM-2, and for another week in a mixed medium DM-2 + AM-1 (1 :2).

[0081] Sample 1 (mixture of iterations I4, L, In, I13, I14, I15, 116 of human adipocytes) was added to mature adipocytes in AM-1 medium (50% v / v) or 0.1% dimethyl sulfoxide (negative control) or Sample 2 (control, purified water) in AM-1 medium and incubated in a CO2 incubator for 72 hours. Then, the level of adiponectin secretion in the culture medium was measured using quantitative solid-phase ELISA (catalog number ADIP-1, Zen-Bio, Inc., USA) in accordance with the manufacturer's instructions. To do this, 20 pl of the collected medium was added to 80 pl of the pretreatment solution from the ELISA kit. The resulting samples were heated to a temperature of 100°C for 5 minutes. After cooling to room temperature, 50 pl of each sample was added to 200 pl of dilution buffer from the ELISA kit, mixed, and then 100 pl of the mixture was added to the wells of primary antibody-coated ELISA plate. The plate was incubated at room temperature for 1 hour. After washing, secondary antibodies were added and further incubated at room temperature for 1 hour. After 1 hour, plate was washed again and detection antibodies were added and incubated at room temperature for another 1 hour. After 1 hour, plate was washed, detection reagents were added and absorbance was measured at 570 nm using a microplate reader (SpectraMax 250, Molecular Devices).

[0082] For statistical processing of the results, one-way analysis of variance (one-way ANOVA) with Tukey's post-hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0083] The results of the study are presented in Table 1.

[0084] Table 1. Effect of samples on adiponectin production by mature human adipocytes

[0085] (M±m)

[0086] Note: * - differences are significant compared to Sample 2 and 0.1% DMSO groups.

[0087] It was shown that the addition of Sample 1 to the culture of human mature adipocytes significantly increased the production of adiponectin compared to both controls: purified water - 2.9 times, and 0.1% dimethyl sulfoxide - 3.4 times.

[0088] Thus, it has been demonstrated that Sample 1 (mixture of fractions L, L, In, I13, 114, 115, 116 of human adipocytes) exhibits specific activity in relation to the secretory function of adipocytes, namely: it statistically significantly increases the production of adiponectin in a culture of differentiated human adipocytes. Example 2. Iterations obtained using murine adipocytes and water as a neutral carrier.

[0089] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared suspension of adipocytes derived from 3T3-L1 murine embryonic cell line (1.0 x 106cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0090] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0091] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0092] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0093] In this study E (iteration No. 3) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1. The study was conducted in a model of diet-induced metabolic syndrome in mature C57B1 / 6 mice (5 groups of 13 each). In the first group, rats received a standard diet (STD), in the second - STD and a placebo drug (purified water, Sample 2), in the third - a high-fat diet (HFD), in the fourth - HFD and Sample 2, in the fifth - HFD and Sample 1 (iteration No. 3 of adipocytes). Animals received HFD for 10 weeks. During the entire study, animals in HFD groups received standard synthetic complete food, manufactured at the research center according to the recipe AIN-93 G, with the addition of rendered pork fat (lard). The drinking water of the animals in HFD groups was replaced with an 8% fructose solution. Animals of the first and second groups received standard synthetic complete food and water throughout the study. Experimental samples were administered to animals orally in the form of an aqueous solution daily for 10 weeks immediately from the start of the experimental diet-induced metabolic syndrome modeling.

[0094] During the experiment, the body weight and body weight gain of laboratory animals in the control and experimental groups, as well as indicators of food and water consumption were monitored. At the end of the administration of experimental samples (10 weeks after the start of the experiment), mice of all groups were subjected to humane euthanasia by CO2 asphyxia with immediate collection of blood serum for biochemical analyzes and epidi dymal adipose tissue for histological examination. When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0095] The effectiveness of experimental samples against obesity was assessed by the body weight of the animals at the end of the experiment, as well as by the weight of epididymal adipose tissue.

[0096] For statistical processing of the results, the normality of distribution was assessed using the Shapiro-Wilk test, and the homogeneity of variance was assessed using the Bartlett test. Groups were compared using analysis of variance and Dunn's test. Holm's correction for multiple comparisons was used. Differences between groups were considered statistically significant at p<0.05.

[0097] The experiment showed that animals on a high-fat diet experienced a statistically significant increase in body weight and epididymal fat mass. Administration of Sample 1 resulted in a significant reduction in body weight (32% compared to placebo) as well as epididymal fat (41%). The results of the study are presented in Table 2. Table 2. Results of determining body weight at the end of the experiment and visceral fat weight

[0098] Note: * - differences are significant compared to groups maintained on standard diet, # - differences are significant compared to groups “high-fat diet” and “high-fat diet + Sample 2

[0099] Thus, the Sample 1 (iteration No. 3 of adipocytes) demonstrated the ability to counteract the development of metabolic syndrome and pathological fat formation in mice.

[0100] Example 3. Iterations obtained using murine melanoma cells or keratinocytes and water as a neutral carrier.

[0101] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared suspension of murine melanoma cell line B16 (l.OxlO6cells / ml) or suspension of primary normal keratinocytes (l.OxlO6cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0102] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0103] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0104] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0105] In this study I? (iteration No. 7) obtained using suspension of murine melanoma cell line B16 as initial substance (hereinafter referred to as Sample 1) and I23 (iteration No. 23) obtained using suspension of primary normal keratinocytes (hereinafter referred to as Sample 3) were used. The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0106] The study was conducted on a mouse melanoma cell line B16. The following groups were formed: the first group was intact (n=3), the second was with the addition of the reference drug Docetaxel at a dose of 40 pg / ml (n=3), the third was with the addition of Sample 1 (iteration No. 7, n=3), the fourth - with the addition of Sample 2 (placebo, which is purified water, n=3 repeats), the fifth - with the addition of Sample 3 (iteration No. 23, n =3). Cells were cultured in 96-well plates in a total volume of 200 pl / well at a concentration of 15,000 cells per well. Test samples were introduced into the medium in a volume of 50 pl per 150 pl ml of medium and incubated for 24 hours. Complete culture medium was used as a control. The cell culture was kept in a 5% CO2 incubator at a temperature of 37°C and a relative humidity of 45%. The nutrient medium used was DMEM / F12 with the addition of 10% bovine fetal serum, 0.3 mg / ml L-glutamine, 50 units / ml penicillin, and 50 pg / ml streptomycin.

[0107] The direct cytotoxic effect of test samples was assessed by MTT test. The cells were incubated with the test drugs at 37°C in 5% CO2 for 24±4 hours. Then the medium was removed, the MTT reagent dissolved in DMEM / F-12 was added to each well to a final concentration of 0.5 mg / ml, and incubated under the same conditions for 4 hours. Then 100 pl of DMSO (dimethyl sulfoxide) was added. The formazan was dissolved for 15 min at room temperature, and then the absorbance was measured at a wavelength of 540 nm on a Multiskan GO microplate reader.

[0108] Tumor cell apoptosis was analyzed using flow cytometry with annexin V staining. Cells were removed from the plate with a 0.05% trypsin solution, centrifuged in phosphate-buffered saline, and the pellet was resuspended in lx binding buffer at a concentration of 1 x 106 / ml. 1 * 105cells in 100 pl in lx binding buffer were placed in Eppendorf tubes. 5 pl of Annexin V-AF 488 dye was added, mixed, and incubated for 15 min at room temperature in the dark. 1 pl of a 100 pg / ml solution of propidium iodide (PI) was added to the samples, mixed, and incubated for 5 min at room temperature in the dark. 0.4 ml of lx binding buffer was added to the cells. The entire volume was transferred into cytometric tubes and analyzed on a NovoCyte Advanteon flow cytometer. Based on the results of the study, the % of apoptotic cells was determined.

[0109] For statistical processing of the results, the normality of distribution was assessed using the Shapiro-Wilk test, and the homogeneity of variance was assessed using the Bartlett test. Groups were compared using analysis of variance and Dunn's test. Holm's correction for multiple comparisons was used. Differences between groups were considered statistically significant at p<0.05.

[0110] The results of the study are presented in Tables 3 and 4. It was shown that both iterations of melanoma cells (Sample 1) and iterations of healthy keratinocytes (Sample 3) reduced the functional metabolic activity of the B 16 melanoma cell culture compared to placebo (Sample 2) and intact group. These results are consistent with the flow cytometry data where a statistically significant increase in the proportion of cells undergoing apoptosis was observed compared with placebo in the docetaxel groups (especially noticeable), as well as Samples 1 and 3.

[0111] Table 3. Results of MTT test (M±m) Table 4. Results of flow cytometry (M±m)

[0112] Note: * - differences are significant compared to Sample 2 (placebo),#- differences are significant compared to intact group

[0113] Thus, the ability of iterations of both tumor and healthy cells to suppress the metabolic activity of a tumor cell culture, as well as induce their apoptosis, has been demonstrated.

[0114] Example 4. Iterations obtained using thyroid gland and water as a neutral carrier.

[0115] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with the thyroid gland (14.6 mg) of an adult male Wistar rat in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0116] Next, a 40 ml vial with a Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0117] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0118] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0119] In this study I4 (iteration No. 4) was used (hereinafter referred to as Sample 2). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0120] One of the most physiological models of obesity and metabolic syndrome, in which thyroid dysfunction is a concomitant pathology, was used in this study. The study was conducted on male Wistar rats weighing 160-180 g and aged 1.5-2 months (n=60). The animals were randomly divided into 5 groups of 12 each, in the first group rats received a standard diet, in the second - a standard diet and Sample 1 (placebo, purified water) from week 9, in the third - a high-fat diet, in the fourth - a high-fat diet and Sample 1 (placebo, purified water) from week 9, in the fifth - a high-fat diet and Sample 2 (iteration No. 4) from week 9. The standard diet included granulated feed in accordance with the approved diet for laboratory animals in the vivarium. The high-calorie diet included granulated feed and additional lard (45% of the calorie content of the standard diet), drinking water was replaced with a 10% fructose solution. Such a diet causes the development of metabolic syndrome and obesity in experimental animals. Test samples and placebo were administered intragastrically once a day for 8 weeks in a volume of 4 ml / kg. At the end of the experiment, the animals were decapitated under anesthesia (sodium thiopental). When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0121] The body weight of the animals was recorded before the experiment, and then weekly for 16 weeks. Determination of visceral fat mass was carried out after the animals were removed from the experiment by isolating perinephric and epididymal fat deposits and weighing them.

[0122] Animals were deprived of food at the end of the study (for 12-16 hours), before blood sampling. Before necroscopy, mixed arterial and venous blood was collected using the decapitation method. After clotting, blood samples were centrifuged at 3000 rpm for 20 minutes to obtain serum. Determination of hormones in blood serum (T3 and T4) was carried out by enzyme immunoassay using commercial Fine test kits and a microplate reader.

[0123] Thyroid tissue was collected for analysis of TPO levels in accordance with the anatomical atlas of the rat after animals were removed from the experiment. Tissue homogenization was carried out in 0.05 mol / L phosphate buffer (pH 7.4) using an IKA T10 basic UltraTurrax homogenizer. Homogenates were prepared in a dilution of 1 :80, centrifuged at 4 °C at 10,667 g on an Allegra 64R Centrifuge (Beckman Coulter). The supernatant was collected and the protein content was determined by the biuret method on an automatic biochemical analyzer Mindrey BS 200 using Diassens reagent kits. Spectrophotometric studies were carried out on a SOLAR CM 2203 spectrofluorimeter. Buffer and other components were added to a thermostated cuvette (with an optical layer width of 1 cm), the final volume of the incubation mixture was about 3 ml. The contents of the cuvette were kept for 3 min at a temperature of 25°C. After adding hydrogen peroxide, the sample was stirred and the optical density was recorded at a wavelength of 353 nm for 1-3 min.

[0124] Statistical data processing was carried out using the Statistica 10 program (Tibco, USA). Normality of distribution was assessed using the Shapiro-Wilk test, homogeneity of variances - using the Bartlett test. Group comparisons were made using Student's t-test and Mann-Whitney test. Differences were considered statistically significant at p <0.05.

[0125] Maintaining animals on a high-fat diet led to a significant increase in body weight and visceral fat mass. It was shown that oral administration of placebo (Sample 1) had no significant effect on animals fed either a standard or high-fat diet. At the same time, an 8-week administration of Sample 2 (iteration of thyroid gland) to animals led to a slight decrease in the manifestations of obesity (see Table 5). Administration of the thyroid gland preparation to animals led to a decrease in total body weight, and a statistically significant reduction in visceral fat mass.

[0126] Table 5. Results of determining visceral fat weight (M±m)

[0127] Note: * - differences are significant compared to groups maintained on standard diet, #

[0128] - differences are significant compared to groups “high-fat diet” and “high-fat diet + Sample 1

[0129] A study of the effect of diet and Sample 2 (iterations of thyroid gland) on the functional activity of the thyroid gland showed that a high-fat diet causes symptoms of hypothyroidism - a decrease in thyroperoxidase (TPO) activity and an increase in thyroxine levels (see Table 6). Administration of Sample 2 (iterations of thyroid gland) to the animals was able to compensate for these changes, bringing these indicators closer to the levels of groups fed a standard diet.

[0130] Table 6. Biochemical indicators of the functional activity of the thyroid gland (M±m)

[0131] Note: * - differences are significant compared to groups maintained on standard diet, #

[0132] - differences are significant compared to groups “high-fat diet” and “high-fat diet + Sample 1

[0133] Thus, an 8-week administration of iteration of thyroid gland to rats maintained on a high-fat diet allowed the restoration of impaired thyroid function, as well as a reduction in visceral adipose tissue deposits. Example 5. Iterations obtained using influenza virus and water as a neutral carrier.

[0134] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (A / California / 04 / 2009 pndm (H1N1), 104TCID50 / 0.1ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0135] Next, a 40 ml vial with a Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 15 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0136] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0137] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0138] In this study Is (iteration No. 8) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1. The study assessed the cytotoxic and antiviral activity of iterations of influenza virus in vitro. The study was conducted on MDCK cell culture. In the experiments, the following experimental groups were formed: the first group (control) - cells infected with the virus, to which neither test samples nor reference drug were added (n=5), the second - to the cells infected with the virus, the reference drug oseltamivir carboxylate was added at a dose of 10 pM (n=5), the third-tenth group - test Sample 1 (iteration No. 8) was added to the cells infected with the virus in 4 different amounts (n=5 for each one), the eleventh-eighteenth - to the cells infected with the virus, test Sample 2 (placebo, purified water) was added in 4 different amounts (n=5 for each one). The samples were used in the form of aqueous solutions. The cell culture was kept in a 5% CO2 incubator at a temperature of 37°C and a relative humidity of 45%. The nutrient medium used was DMEM / F12 with the addition of 10% bovine serum, 0.3 mg / ml L-glutamine and an antibiotic mixture of 50 units / ml penicillin and 50 pg / ml streptomycin.

[0139] To assess the cytotoxic effect of the tested samples, MTT test was performed. Several final dilutions of the test samples were tested: 1 / 4, 1 / 8, 1 / 16, 1 / 32 (calculated in volume fractions of the total volume of the incubation medium). After removing the medium from the plates, an MTT solution was added to each well; after incubation for 4 hours in a CO2 incubator, the solution was removed and DMSO was added. After mixing and incubation, absorbance measurements were taken at 540 and 670 nm. The concentration of the drug that did not change the OD540 value compared to the control cells was taken as the tolerable concentration.

[0140] Testing of the antiviral activity of the drugs was carried out using MDCK cell culture. Cells were seeded into a 96-well plate and incubated for 24 hours at 37°C in 5% CO2 atmosphere until a monolayer was formed. The test drugs were added to the cell culture 24 hours before inoculation with influenza A virus (A / Califomia / 04 / 09 (H1N1)). The tested samples in a volume of 100 pl in the medium were added to the cell culture in 2 amounts, selected based on the results of assessing the cytotoxic effect (maximum non-toxic doses). Infection with the virus was carried out 24 hours after the addition of the test samples; the virus was introduced into the cell culture in the amount necessary to achieve a dose of 1 and 0.1 PFU / cell (volume 100 pl) in the culture. After incubating infected cells with the test samples approximately 24 hours after infection of the culture (before the first signs of cytotoxicity appeared in control cells), the cells were fixed and virus reproduction was determined by determining the expression level of viral antigens (NP and Ml) by ELISA.

[0141] Statistical analyses were performed using the statistical package R 3.2.1 (R Foundation for Statistical Computing, Vienna, Austria). Data were checked for outliers using the 1.5*IQR (inter quartile range) rule. The significance of differences in optical density values was assessed using the Kruskal-Wallis test with Dunn's post hoc test. Differences between groups were considered statistically significant at p<0.05.

[0142] Based on MTT test, the death of a significant number of cells was shown when the Sample 1 (iteration No. 8) and Sample 2 (placebo) (as well as purified water as a control) were administered only at a concentration of 14 of the well volume. Therefore, for further study of the antiviral activity of the test samples in MDCK cell culture, 1 / 8 and 1 / 16 doses were chosen as the maximum tolerated dose (MTD) of samples that do not have a cytotoxic effect on cells.

[0143] High inhibition of virus activity was observed in Sample 1 (see Table 7). This level was comparable to the level of inhibition by the reference drug oseltamivir carboxylate, which in turn is the gold standard in influenza therapy. Significant differences were demonstrated between the Sample 1 (iteration No. 8) (1 / 8 dose) and Sample 2 (placebo).

[0144] Table 7. Antiviral activity of test samples in vitro (average optical density values and the degree of inhibition of viral activity in % of the corresponding controls are indicated)

[0145] Note: * - p<0.05 for 0.1 PFU / cell; ** - p<0.001 for multiplicity of infection of 1 PFU / cell.

[0146] Thus, iterations of the influenza virus demonstrated antiviral activity that was not inferior to the effectiveness of the comparator drug Oseltamivir carboxylate.

[0147] Example 6. Iterations obtained using bacterium and water as a neutral carrier.

[0148] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (Salmonella enteritidis rif92 bacteria, 1,35 x 106CFU / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0149] Next, a 40 ml vial with a Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 15 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0150] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0151] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0152] In this study In (iteration No. 11) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0153] The study was conducted on female C57B1 mice weighing 21-24 g, aged 8 weeks. Animals were intragastrically infected with Salmonella enteritidis rif92 (1.35 x 106CFU / mouse). On days 3-7 after infection, mice in the first two groups (n=10 in each) were intragastrically administered with Sample 1 (iterations of Salmonella enteritidis rif92 bacteria; 0.2 ml / day) or Sample 2 (control, purified water; 0.2 ml / day). Animals of the third group (n=10) were a positive control; on the same days of the experiment, they were administered ciprofloxacin (twice a day intragastrically at 1 mg / kg (ED 100)).

[0154] At the end of the study, the presence or absence of Salmonella enteritidis rif92 in the liver and feces of mice was assessed. If present, the average value of CFU / g, frequency of occurrence of pathological signs of internal organs (enlarged mesenteric lymph nodes, enlarged liver, enlarged spleen, necrotic foci and hemorrhages in the liver, spleen and intestines) were also assessed.

[0155] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0156] Statistical analysis of the results was performed using Fisher's exact test; pairwise comparison was performed using the Benjamini -Hochberg post-hoc test. Differences between groups were considered statistically significant at p<0.05.

[0157] The results of the study are presented in Table 8.

[0158] Table 8. Presence and concentration of Salmonella enteritidis rif92 in the liver and feces of mice treated with Sample 1 or ciprofloxacin

[0159] It was shown that Sample 1 reduced both the percentage of sick animals and the concentration of the pathogen in the liver and feces compared to Sample 2 (control). Moreover, with regard to the study of the pathogen in the liver, the percentage of sick animals, as well as the average content of Salmonella enteritidis rif92. was minimal in this group. The number of Salmonella enteritidis rif92 in the feces was reduced by several orders of magnitude. The therapeutic activity of Sample 1 was comparable to the effect of the reference drug, the effective antibiotic ciprofloxacin.

[0160] Thus, Sample 1 (iterations of Salmonella enteritidis rif92 bacteria) showed a significant antibacterial effect against Salmonella enteritidis rif92. The revealed effectiveness was expressed by a decrease in bacterial contamination of organs and the number of sick animals.

[0161] Example 7. Iterations obtained using herpes simplex virus and water as a neutral carrier.

[0162] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (herpes simplex virus type 2, 5.3 1g PFU / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0163] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 20 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration. Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0164] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0165] In this study In (iteration No. 11) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0166] The study was conducted on female Agouti smooth-haired guinea pigs (400-420 g). Animals were infected with herpes simplex virus type 2 (strain EC) by applying a viruscontaining liquid to the scarified surface of the genitals. Sample 1 (iteration of HSV-2, n=15) or Sample 2 (control, purified water; n=15) was administered intragastrically at 5 ml / kg twice a day (daily dose 10 ml / kg) for 15 days after infection. The reference drug, Acyclovir (KRKA, n=15), was administered intragastrically at a dose of 50 mg / kg twice a day (daily dose 100 mg / kg) for 15 days after infection. Another group consisted of 15 animals that received Sample 1 for 20 days (5 ml / kg 2 times a day) starting 5 days before infection and for 15 days after infection. The following indicators were assessed:

[0167] • The severity of the infectious process within 2 months after infection, assessing the main signs of the disease on a conditional 4-point scale: hyperemia, swelling, specific elements (vesicles, pustules, ulcerations), and animal activity.

[0168] • Virus titer in vaginal swabs on days 2, 5, 8, 11, 15, and 60 after infection. The virus content in vaginal smears was assessed using VERO cells (4-5xl05cells / ml). Herpes simplex virus type 2, strain VN, with an activity of 5.0-5.5 Lg TCD50 / 0.1 ml was used as a test virus. The virus was prepared in Eagle medium with the addition of 100 pg / ml streptomycin and 100 units / ml penicillin, in equal volumes (0.4 ml of each component), and incubated in an incubator at +37°C for 60 minutes. Then the mixture was introduced into the VERO cell culture (in which the growth medium was discarded, and the cell monolayer was washed three times with Hanks’ solution) and incubated in a thermostat at +37°C for 45 minutes. Using an inverted microscope, the cytopathic effect of the virus was studied throughout the entire period of observation of the cell monolayer (after 24, 48, 72, and 96 hours), and the virus titer was calculated. The virus titer was determined using standard methods and expressed in 1g.

[0169] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0170] For statistical processing of the results, the Wilcoxon test, one-way ANOVA and oneway repeated measures ANOVA were used, followed by comparison of all groups with each other using Tukey's post-hoc test. Differences between groups were considered statistically significant at p<0.05.

[0171] The results of the study are presented in Table 9.

[0172] Table 9. The effect of the test samples on the total index of symptom severity and virus titer in vaginal smears of animals infected with herpes simplex virus type 2 (strain EC).

[0173] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to acyclovir group; °- there were no mucosal lesions It was shown that the therapeutic, and to an even greater extent, the therapeutic and prophylactic use of Sample 1, significantly reduced the severity and duration of general and local symptoms of herpes infection, and also significantly reduced viral shedding. It should be noted that Sample 1, when administered therapeutically and prophylactically, was more effective than the reference drug acyclovir, reducing the duration of clinical symptoms compared to the control as well as completely suppressing mucosal damage. The therapeutic effect index of Sample 1 during therapeutic and prophylactic administration was 4.6 times (p<0.05) higher than that of acyclovir.

[0174] Thus, Sample 1 (iteration of HSV-2) showed specific activity against genital herpes type 2 virus (HSV-2 DNA virus). The maximum therapeutic effect was observed when using Sample 1 (iteration of HSV-2) with a combined (preventive and therapeutic) administration regimen. At the same time, minimal clinical manifestations of herpesvirus infection and minimal virus titers in smears were observed. For all studied indicators, Sample 1 (iteration of HSV-2) with a combined (preventive and therapeutic) administration regimen was superior to the comparison drug acyclovir.

[0175] Example 8. Iterations obtained using liver cells and water as a neutral carrier.

[0176] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared suspension of pig liver cells (1.0 x 106cells / ml) in a volume of 5 ml (initial substance) and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the abovedescribed procedure, 35 ml of iteration 0 (Io) was obtained.

[0177] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0178] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0179] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0180] In this study E (iteration No. 5) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0181] In the experiment 35 female Balb / c mice aged 6-7 weeks were used. All animals were housed under standard conditions in a 12: 12 light-dark cycle. When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010). The animals received pelleted food and drinking water ad libitum. The animals were randomly assigned into 4 groups:

[0182] 1. Control group (administration of purified water at a dose corresponding to the dose of the Sample 1, n=5).

[0183] 2. Group of animals with experimental pathology - an acne model (n=10).

[0184] 3. Experimental group (acne modeling + administration of the Sample 1, n=10).

[0185] 4. Placebo (acne modeling + administration of purified water at a dose corresponding to the dose of the Sample 1, n=10).

[0186] Each animal was individually marked using a permanent marker. In the groups in which pathology was induced (acne modeling), animals were injected intradermally into the central part of the outer side of the ear (at the base of the scaphoconchal angle) with Staphylococcus aureus (pathogenic properties were confirmed by a coagulation reaction with rabbit plasma and the zone of active hemolysis on the blood agar) at a concentration of 1 x 109CFU / ml in a volume of 20 pl, grown on yolk-salt agar.

[0187] On the 2nd and 4th days after acne modeling, 5 animals from each group of animals were killed in a CO2 chamber. Next, the auricle was excised at the base of the ear and the “control” and “experimental” (affected) auricles were weighed to assess the relative amount of edema.

[0188] Student's t-test was used to compare ear inflammation index data between groups. Statistical analysis was performed using Statistica 10.0 software (StatSoft, USA). Differences between groups were considered statistically significant at p<0.05.

[0189] Local inflammation, characterized by redness and swelling, was observed in all animals at 6 hours after the induction of pathology (day 1). On the 2nd day, all animals developed a pustule filled with purulent contents. On the 3rd day after injection of the bacterial suspension, a decrease in swelling and a decrease in the size of the pustule was observed in the animals. On the 4th day, practically no signs of inflammation were visually observed in the mice. To quantitatively assess edema (%) in dynamics, the weight of the experimental (affected) and control ears was measured on the 2nd and 4th day after the induction of pathology. The results are presented in Fig. 2.

[0190] Thus, the data obtained indicate the anti-inflammatory activity of the Sample 1 (iteration of pig liver cells) in the acne model. The therapeutic effect of the drug was expressed by inhibition of the dynamics of the inflammatory reaction (reduction in the volume of edema during therapy).

[0191] Example 9. Iterations obtained using CHO-S cells and water as a neutral carrier.

[0192] The process of obtaining iterations included several stages. A vial with a capacity of 60 ml with a freshly prepared suspension of CHO-S cells (1.0* 105cells / ml) as initial substance in a volume of 50 ml and a vial with a capacity of 60 ml with purified water (neutral carrier) in a volume of 50 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the abovedescribed procedure, 50 ml of iteration 0 (Io) was obtained.

[0193] Next, a 60 ml vial Io (50 ml) was placed adjacent to a new 500 ml vial with 480 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named h. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0194] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0195] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0196] In this study, iteration No. 5 and iteration No. 7 were used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0197] The activity of the samples was tested on CHO-S cell line (Thermo Fisher Scientific, USA). To assess the effect of the test samples, a 7 w% solution was prepared in a volume of 10 ml. To do this, a 0.07 g of each sample (iteration No. 5 and iteration No. 7) was dissolved in Hybris medium without insulin by carefully pipetting the solution, then the solution was passed through syringe filters with a PVDF membrane with a pore size of 0.2 pm (Macherey-Nagel, Germany). Each time, freshly prepared sample solutions were used. To evaluate the effect of water iteration samples, they were added directly to the cell culture.

[0198] CHO-S cells were cultured in 6-well plates (Nest, China) in Hybris medium without insulin for 7 days (37°C, 5% CO2), and the proportion of the test samples solution samples was 1 / 6 of the total well volume, iteration samples on water - 1 / 10 of the well volume. Next, the cells were transferred into 48-well plates (Nest, China) with the addition of test samples and various concentrations (0.5 pg / ml, 5 pg / ml and 50 pg / ml) of human insulin. The final cell concentration was 0.15x l06cells / ml, the proportion of tested samples was 1 / 6 or 1 / 10 of the total well volume (500 pl). The plates were incubated for 72 hours at 37°C, 5% CO2. Glucose consumption analysis was then performed for each experimental well using the hexokinase method, as well as WST analysis to calculate normalized glucose consumption.

[0199] For the hexokinase method, 2 ml plastic tubes (JetBiofil, China) were used. Each tube contained 780 pl of Tris buffer (0.1 M, pH 7.8) and 20 pl of cell suspension from the corresponding sample well. The resulting solutions were mixed using a vortex (Biosan, Latvia), and 50 pl was added to the wells of a 96-well plate (Corning Costar, USA). Then, 50 pl of a mixture of enzymes and coenzymes (Tris buffer, hexokinase, NAD+and ATP) was added and incubated for 1 hour at 37°C. After incubation, 200 pl of Tris buffer (0.1 M, pH 7.8) was added to each well. Absorption spectra were measured on a Multiskan FC microplate photometer (Thermo Fisher Scientific, USA) at wavelengths from 300 nm to 450 nm. The optical density values in each well were obtained using the formula: ODgiu=OD340-OD450-ODtris(caiibration). Next, glucose consumption was calculated using the formula: Cgiu=30-((ODgiu-b) / a*40), using the calibration equation y=ax+b. For replicates within the same insulin concentration, the mean value, standard deviation and coefficient of variation were calculated using Excel software (Microsoft, USA).

[0200] To assess the metabolic activity of cells, WST-1 reagent (Biosynth, China) was used. The analysis was carried out in accordance with the manufacturer's instructions. CHO-S cells were added to 96-well plates (Coming Costar, USA) at 100 pl / well in duplicates, then WST-1 reagent was added in a volume of 8 pl / well and incubated for 3 hours at 37°C. Optical density was measured on a Multiskan FC microplate photometer (Thermo Fisher Scientific, USA) at wavelengths of 440 nm and 650 nm. The final optical density was calculated using the formula: ODWst = OD440-OD650.

[0201] Calculation of normalized glucose consumption by CHO-S cells was carried out using the formula: N= Cgiu / ODwst (where ODwst is the average value for each duplicate).

[0202] Outliers within groups were determined using interquartile range. Comparisons between groups at each concentration point were made using the Student / Welch t-test and the Kruskal- Wallis test followed by Dunn's test. P-values were adjusted using the Holm method. Differences between groups were considered statistically significant at p<0.05.

[0203] The results of the study are presented in Fig. 3.

[0204] Based on the data obtained, we can conclude that the use of iterations of CHO-S has a statistically significant effect on glucose consumption by CHO-S cells in the presence of various insulin concentrations. Depending on the physicochemical properties of the studied fractions, glucose consumption by cells can either decrease or increase.

[0205] Example 10. Iterations obtained using placenta cells and water as a neutral carrier.

[0206] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of porcine placenta cells, 5* 104cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0207] Next, a 40 ml vial with a Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 3 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named h. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0208] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0209] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0210] In this study, I34 (iteration No. 34) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0211] The study of the chondroprotective activity of the drug was carried out on 70 outbread white male rats (age 4-5 months, body weight 265-290 g). The animals were divided into four groups of 15 each. The first group was intact rats; the second group - animals with experimental osteoarthritis (OA), without treatment; the third group - rats with experimental OA, who received Sample 1 at a dose of 50 mg of ointment; fourth group - rats with experimental OA, who received placebo (purified water) in an amount equivalent to Sample 1.

[0212] To induce OA, rats were injected with dexamethasone three times at an interval of 7 days (intramuscularly in the thigh, single dose - 7 mg / kg). The OA modeling was monitored by biochemical studies of biological material obtained in the 4th week of the experiment from 10 animals from the additional control group sacrificed by decapitation under ether anesthesia. From the 28th day of the experiment and over the following 4 weeks, all animals from the third and fourth groups received the appropriate drugs daily, which were applied topically to the knee joints of both paws in equivalent quantities - 50 mg each. The drugs were carefully rubbed into the skin of the rats for faster absorption, while preventing them from being licked or wiped off by the animals themselves.

[0213] During the study, clinical examination of the animals was carried out: the functional state of their joints was monitored, including the degree of mobility, resistance to physical activity, swelling, and hyperemia. Upon completion of the application of the study drugs (on the 56th day of the experiment), all animals were decapitated under ether anesthesia, and the total content and fractions of glycosaminoglycans (GAGs) were determined in the blood serum.

[0214] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0215] For statistical analysis, two-way ANOVA with Tukey's post-hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0216] The fractional composition and total content of GAGs in the blood serum of rats with OA that were administered the Sample 1 (group 3) or placebo (group 4) are presented in Table 10. It was found that when the Sample 1 was applied to the joints of animals, there was a decrease in the total level of GAGs. This was caused not only by a decrease in the content of chondroitin-4-sulfates, but also by a decrease in the fraction of chondroitin-6-sulfates to the level of healthy animals (group 1). When using placebo (group 4), the levels of total content and content of GAG fractions remained virtually unchanged compared to untreated animals with OA (group 2).

[0217] Table 10. The fractional composition and total content of glycosaminoglycans (GAGs) in the blood of animals with experimental osteoarthritis

[0218] Note. * p <0.05 compared to group 1; @ -p<0.05 compared to group 2;A- p<0.05 compared to group 4.

[0219] Thus, we can conclude that the Sample 1 (iteration of porcine placenta cells) has chondroprotective properties, demonstrating effectiveness in the treatment of experimental arthrosis in rats.

[0220] Example 11. Iterations obtained using umbilical cord cells and water as a neutral carrier.

[0221] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of pig umbilical cord cells, I x lO6cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0222] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 30 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named h. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration. The procedure was repeated the required number of times to obtain the final iteration.

[0223] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0224] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0225] In this study mixture of E (iteration No. 6) and Is (iteration No. 8) (hereinafter referred to as Sample 1) was used. The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0226] The experiment was carried out on male Wistar rats weighing 200-250 g. The animals were divided into 4 groups of 15 each: groups I, III, and IV were on a high cholesterol diet for 180 days (6 months), which consisted of supplementing animal food with cholesterol powder, Mercazolil, and vitamin D. 15 days before putting rats on an atherogenic diet, a surgery was performed: ligation of the left renal pedicle with non-absorbable sutures and suturing the upper pole of the right kidney, leaving 2 / 3 of the organ. During the experiment, 15 healthy rats fed a normal diet served as controls (group II). Animals in group III were administered Sample 1. Animals in group IV were administered an equivalent amount of placebo (purified water). The experiment was carried out in strict compliance with the requirements of the European Convention on the maintenance, feeding, and care of experimental animals, as well as their removal from the experiment and subsequent disposal. The experiments were carried out in accordance with the requirements of the World Society for the Protection of Animals (WSPA) and the European Convention for the Protection of Experimental Animals. After 6 months of the study, the animals of each group were removed from the experiment by decapitation under ether anesthesia. Fragments of the aorta, femoral arteries, and microvessels of the anterior abdominal wall (AW) were collected.

[0227] The determination of total cholesterol (TC), triglycerides (TG), LDL, and HDL levels was carried out using standard colorimetric method reagents. The atherogenicity index (Al) was calculated using the formula: (total cholesterol - HDL) / HDL. Blood pressure was measured in the tail artery using an MLU / 4C 501 analyzer (MedLab, China). The magnetic resonance imaging was performed as follows: before scanning, the animals were euthanized with overdose of solutions of Rometar (Xylazine, SPORA, PRAHA) at a concentration of 1 mg / ml and Relanium at a concentration of 2 mg / ml, injected intraperitoneally. MRI diagnostics were performed on a tomograph for experimental research “PharmaScan US 70 / 16” (Bruker, Germany) with a magnetic field strength of 7.0 Tesla, a frequency of 300 MHz, and a BGA 09P coil. For angiography, the Head Angio protocol was used with the following parameters: TR / TE=50.0 / 5.6; tilt angle 25.0; image field 3.0 / 3.0 / 3.0; effective cutting thickness 30 mm; overlap 30.0 mm; matrix 256 / 256 / 64 elements; one signal averaging, scanning time 14 min. Histological preparations were fixed in 10% neutral formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and Sudan IV (Okamoto’s method). The description of micropreparations was carried out on an Olympus BX 41 microscope. Images were taken with an Olympus DR 12 camera at lOOx or 400x magnification. Morphometry was performed using an ocular micrometer.

[0228] To assess the NADPH-diaphorase activity, a histochemical method was used. Enzyme activity was measured in the endothelium and smooth myocytes of the aorta, femoral arteries, and AW. Enzyme activity values were determined using Imaged v. 1.37 software and expressed in optical density units. For statistical analysis, SPSS v. 16 software was used. Comparison of mean values between the groups was carried out using one-way ANOVA, followed by comparison of all groups with each other using Tukey’s post-hoc test. Differences between groups were considered statistically significant at p<0.05.

[0229] Results of the study are shown in Tables 12, 13 and 14.

[0230] Table 12. Indicators of blood pressure in rats with experimental atherosclerosis.

[0231] Note: SBP is systolic blood pressure, DBP is diastolic blood pressure. Data are presented as means ± standard error of the mean.A- differences are significant compared to group I (p<0.05); • - differences are significant compared to group II (p<0.05); * - differences are significant compared to group IV (p<0.05).

[0232] Table 13. The lipid spectrum of blood serum of rats with experimental atherosclerosis.

[0233] Note: Data are presented as average values.A- differences are significant compared to group I (differences from the value in the corresponding month, p<0.05); # - differences are significant compared to group II (differences from the value in the corresponding month, p<0.05); * - differences are significant compared to group IV (differences from the value in the corresponding month, p<0.05).

[0234] Table 14. NADPH-diaphorase activity in rat vessels.

[0235] Note: Data are presented as average values.A- differences are significant compared to group I (p<0.05); # - differences are significant compared to group II (p<0.05); * - differences are significant compared to group IV (p<0.05).

[0236] Thus, the data indicate the influence of the Sample 1 (mixture of U (iteration No. 6) and Is (iteration No. 8) of pig umbilical cord cells) on indicators of structural and functional damage to blood vessels in experimental atherosclerosis.

[0237] Example 12. Iterations obtained using embryonic cells and water as a neutral carrier.

[0238] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of porcine embryonic cells, 5* 105cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 25 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0239] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration. The procedure was repeated the required number of times to obtain the final iteration.

[0240] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0241] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0242] In this study E (iteration No. 4) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0243] Hypertensive encephalopathy was modeled using awake 12-month-old Wistar rats with a moderately active behavior. The animals were divided into 6 groups of 10 each. The animals were placed in plexiglass containers located along the edges of the horizontal centrifuge rod in a strictly caudal-cranial direction and were subjected to 9 g force for 5 minutes twice a day for 4 weeks. Animals of the 6th group were administered Sample 1 (iteration of porcine embryonic cells) daily for 5 days before modeling hypertensive encephalopathy. Animals in the 5th group were administered Sample 1 daily for 5 days, starting 1 (iteration of porcine embryonic cells) hour after modeling hypertensive encephalopathy. The 4th group consisted of rats that received placebo (purified water) daily for 5 days before modeling hypertensive encephalopathy. The 3rd group consisted of rats that received placebo (purified water) after modeling hypertensive encephalopathy, similar to group 5. The 2nd group consisted of control rats in which hypertensive encephalopathy was modeled but who were not subjected to any therapy. Group 1 consisted of control (intact) rats, which were not exposed to any effect. On the 40th day from the day of modeling the pathology, the animals were removed from the experiment: they were decapitated, and brain samples were taken. Brain samples were fixed in 10% neutral formalin and embedded in paraffin.

[0244] Sections (5-7 pm thick) were stained with hematoxylin and eosin, thionin using the Nissl method. Microphotography of histological preparations was carried out on a Micros microscope (Austria) with an Olympus digital camera (Japan). The main morphometric parameters of the pyramidal layer of the hippocampus of the right hemisphere were studied: the average area of neuronal perikarya, the average area of neuronal nuclei, the specific area of neuronal perikarya, as well as the proportion of hyperchromic neurons.

[0245] Statistical data processing was carried out using MS Office Excell 2007 (Microsoft Inc., USA), and Statistica 6.0 (StatSoft, USA) software. The summarized data are presented as median (IQR). The data were analyzed using one-way ANOVA, followed by comparison of all groups with each other by Tukey's post-hoc test and were considered statistically significant at p<0.05.

[0246] The results of the study are shown in Table 15.

[0247] Table 15. Changes in the morphometric parameters of the pyramidal layer of the hippocampus of the right hemisphere of rats with simulated hypertensive encephalopathy.

[0248]

[0249] Note: * - p <0.05 - differences are significant compared to group 1;A- p <0.05 - differences are significant compared to group 2; $ - p <0.05 - differences are significant compared to group 3; & -p <0.05 - differences are significant compared to group 4.

[0250] Thus, when modeling hypertensive encephalopathy by four-week exposure to high gravitation forces, in animals from group 2, a pronounced increase in the number of damaged cells in all zones was observed. This was accompanied by a significant decrease in the specific area of the perikaryon of neurons in the CAI, CA2, and CA3 zones, accompanied by signs of circulatory disorders in the microvasculature, which indicates the development of deep degenerative changes in the hippocampus and the presence of signs of damage contributing to the development of psychoneurological disorders. These pathological changes also occurred when placebo samples were used (groups 3 and 4). Moreover, in animals from groups 5 and 6, the observed differences from the indicators of intact rats were expressed to a significantly lesser extent compared to placebo. The differences between the performance of the Sample 1 groups (groups 5 and 6) and the performance of the placebo groups (groups 3 and 4, respectively) were statistically significant for most of the parameters studied.

[0251] Thus, we can conclude that the Sample 1 (iteration of porcine embryonic cells) demonstrated effectiveness in the treatment of experimental hypertensive encephalopathy in rats.

[0252] Example 13. Iterations obtained using cardiac progenitor cells and water as a neutral carrier.

[0253] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of cardiac progenitor cells, 2* 104cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0254] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 15 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0255] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0256] Type 1 (ultrapure) water with a resistivity of 18.2 mO- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0257] In this study E (iteration No. 3) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0258] The study was conducted on a model of myocardial infarction in male Wistar rats weighing 254-270 g aged 2.5 months, followed by therapy in the form of transplantation of cardiac progenitor cells (CPC).

[0259] Myocardial infarction was induced by ligation of the anterior descending coronary artery. Before transplantation, CPCs were labeled with the fluorescent dye CM-Dil in accordance with the recommendations of the manufacturer. The animals were divided into 3 groups of 24 each: control group 1 (4 injections of M199 medium without additives, 50 pl each, were performed intramyocardially); Sample 1 group (intramyocardial transplantation of CPCs - 4 intramyocardial injections of 25* 104cells in 50 pl of M199 medium, followed by intragastric administration of Sample 1 (iterations of cardiac progenitor cells; 5 ml / kg / day for 14 days, starting an hour after modeling the pathology); group Sample 2 (intramyocardial transplantation of CPCs - 4 intramyocardial injections of 25* 104cells in 50 pl of M199 medium, followed by intragastric administration of Sample 2 (control, purified water; 5 ml / kg / day for 14 days, starting an hour after modeling the pathology).

[0260] To obtain a culture of c-kit+ CPCs, rat hearts were cut using scissors to obtain pieces measuring 2-3 mm3, washed with phosphate-buffered saline (PBS), and treated with enzyme solution (0.1% collagenase A, and 0.2% trypsin) in for 15 minutes. After this, a triple volume of DMEM / F12 medium with 10% fetal bovine serum was added, and the mixture was centrifuged for 3 min at 50 g. The treated myocardium pieces were washed with PBS and placed on fibronectin-coated cell culture dishes to obtain an explant culture. After 2 weeks, the explant cells were used for immunomagnetic selection using primary antibodies to c-kit and CD45 and secondary antibodies conjugated to magnetic beads. Cultivation of c-kit+CD45-CPC was carried out in DMEM / F12 medium supplemented with 10% fetal calf serum, 100 units / ml penicillin / streptomycin, 2 mM L-glutamine, 2% B27, insulin-transferrin-selenium solution, and growth factors (20 ng / ml bFGF, 20 ng / ml EGF, and 10 ng / ml LIF).

[0261] On the 15th day of the experiment, the animals were sacrificed, and the migration of CPCs from the transplantation area was assessed. Before collecting hearts, 0.1 ml of a saturated KC1 solution was injected into the left ventricle, which led to a stop of contractions in diastole. The atria and large vessels were dissected, the hearts were washed with isotonic sodium chloride solution, placed in O.C.T. cryomedium and frozen in liquid nitrogen. Heart cryosections (7 pm thick, cut at 300 pm intervals between sections across the apex to the base of the left ventricle) were stored at -70°C. Next, we assessed the number of CPCs surviving after transplantation containing the Cell Tracker CM-Dil fluorescent label and the viability of transplanted CPCs stained with antibodies to the proliferation marker Ki67. A Zeiss Axiovert 200 M fluorescence microscope and Axiovision 3.1 software were used in this work.

[0262] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0263] For statistical processing of the results, one-way analysis of variance with Tukey's post- hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0264] The results of the study are presented in Table 16.

[0265] Table 16. Influence of the test samples on the viability of cardiac progenitor cells (CPCs) transplanted into the hearts of rats in a model of myocardial infarction (M±m).

[0266] Note: * - differences are significant compared to Sample 2 (control, purified water) and control group (without sample administration).

[0267] Analysis of the data showed that in the group without the introduction of samples (Control) and in Sample 2 group (purified water), the transplanted CPCs were localized exclusively in the form of extensive clusters and practically did not migrate from the area of primary injection. At the same time, the retention of the transplanted cells did not differ; there were no statistically significant differences between these groups. Sample 1 treatment resulted in pronounced integration of PSCs into the myocardium and retention of significantly more transplanted cells. This indicator for Sample 1 group exceeded the value of the Control group by 4 times (p<0.05) and the Sample 2 group by 3.5 times (p<0.05).

[0268] When assessing the viability of transplanted CPCs based on the level of proliferation, the number of labeled ACLs expressing the proliferation marker Ki67 in response to the administration of Sample 1 was significantly higher than in Control and Sample 2 groups. For this indicator, the average value for the Sample 1 group exceeded the value for Control group by 2.8 times (p<0.05) and Sample 2 group by 3.6 times (p<0.05).

[0269] Thus, it has been shown that Sample 1 (iteration suspension of cardiac progenitor cells) exhibits specific activity regarding the effectiveness of cell transplantation: it significantly increases the distribution of transplanted cells and ensures the preservation of a significantly larger number of proliferating cells in the damaged myocardium compared to Sample 2 group (purified water).

[0270] Example 14. Iterations obtained using neonatal testicular tissue and water as a neutral carrier.

[0271] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (neonatal testicular tissue of outbred male rat, 1 mg) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 30 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0272] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 30 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0273] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0274] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0275] In this study E (iteration No. 4) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0276] The study was conducted in a model of abdominal cryptorchidism using outbred male rats weighing 262-274 g and aged 3 months. In 16 animals, in order to disrupt spermatogenesis, the pathology was previously modeled by moving both testicles from the scrotum through the inguinal canal into the abdominal cavity and fixing them with a ligature to the side wall. After 2 weeks, the testicle was returned to the scrotum. At the same time, testicular tissue obtained from newborn rat pups (1-2 days after birth) was transplanted under the tunica albuginea. Then the animals were assigned into two groups of 8 each and intragastrically administered for 14 days either Sample 1 (iterations of neonatal testicular tissue; 5 ml / kg / day) or Sample 2 (control, purified water; 5 ml / kg / day). The transplantation technique consisted of incising the tunica albuginea in the avascular zone, forming a tunnel under the tunica albuginea using a microsurgical probe, and placing a fragment of neonatal testicular tissue into it, followed by suturing the incision in the tunica testis.

[0277] The animals were examined 1 month after testicular retraction. Blood samples were taken to determine the level of testosterone in the blood. The testicles were removed, their weight was determined by weighing, and tissue samples were taken from the transplant area for histological examination. The concentration of testosterone in the blood serum was determined by chemiluminescence immunoassay using an Access 2 immunochemical analyzer (Beckman Coulter, USA). Histological examination was carried out according to the standard procedure with staining of paraffin sections with hematoxylin and eosin.

[0278] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0279] For statistical processing of the results, one-way analysis of variance with Tukey's post- hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0280] Analysis of the data showed that all transplants got engrafted, and their sizes increased with increasing observation periods. At the same time, the mass of the organ in Sample 1 group increased to a greater extent than in Sample 2 group, approaching the physiological norm values of 1.61 ±0.03 g by the end of the experiment.

[0281] A quantitative analysis of indicators characterizing the state of cryptorchid testicles, namely, the number of empty tubules per unit area of the histological preparation, the number of Sertoli cells per 1 tubule, the number of Leydig cells per unit area of the preparation, as well as the presence of spermatogenic cells of varying degrees of differentiation, revealed the therapeutic effect of Sample 1. Thus, in control group (Sample 2), the proportion of completely empty tubules 1 month after testicular retraction was 71.4±7.3%, while in the Sample 1 group their proportion decreased significantly, amounting to only 8.0±0.7%. The number of Sertoli cells per tubule in control group averaged 3.3±0.7 cells, and in Sample 1 group this figure increased to 36.6±2.7 cells. The number of interstitial Leydig cells per unit of area in the control group was 3.7±0.6, while in Sample 1 group this value was 40.8±4.1 cells.

[0282] An important indicator of the state of spermatogenesis is its completeness, determined histologically by identifying cells. Analysis of the results showed that in control group (Sample 2), in the vast majority of tubules with preserved spermatogenic epithelium, a block of spermatogenesis was detected at the level of spermatogonia or first-order spermatocytes. At the same time, in Sample 1 group, spermatogenesis reached the stage of 2nd-order spermatocytes, and, in most cases, was fully completed (spermatozoa were detected in the lumen of the tubules) (see Table 17).

[0283] Table 17. Effect of the test samples on neonatal testicular tissue transplantation to restore impaired spermatogenesis in cryptorchid testes

[0284] Note: * - differences are significant compared to Sample 2 (control, purified water)

[0285] The determination of testosterone concentration in the blood (an indicator of the functional activity of testicular tissue) showed that in Sample 2 group, 1 month after testicular retraction into the scrotum, this indicator was 1.48±0.3 ng / ml. At the same time, in response to Sample 1 administration, the testosterone level increased to 3.17±0.7 ng / ml.

[0286] Thus, it has been demonstrated that Sample 1 (iterations of neonatal testicular tissue) has a positive effect on the effectiveness of neonatal tissue transplantation: the transplanted immature testicular tissue underwent normal development with the formation of typical structures of a mature organ, which was not observed in the Sample 2 (control, purified water).

[0287] Example 15. Iterations obtained using mast cells and water as a neutral carrier.

[0288] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (suspension of adult outbred male mice mast cells, 2xl04cells / ml) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0289] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 10 seconds at 3000 rpm in close contact. Then both vials were incubated for 1 minute at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration. Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0290] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0291] In this study U (iteration No. 6) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0292] The study was conducted on outbred male mice weighing 19-21 g and aged 2-2.5 months. The animals were divided into three groups 12 each. The mice of the intact group were not subjected to any manipulations for 14 days, while the animals of the remaining two groups were sensitized with an epidermal allergen from wool. The allergen at a dose of 500 PNU / ml was administered intraperitoneally in a volume of 0.1 ml twice with an interval of one day. One day after the last injection, mice were injected with the allergen at a dose of 1000 PNU / ml in a volume of 0.1 ml. Two injections were performed with an interval of one day. From the day of the last injection, seven days were counted, and animals were sacrificed by cervical dislocation. Then 5 ml of phosphate buffered saline with pH=7.4, heated to a temperature of 37°C, was injected into the abdominal cavity, and the abdominal wall of the mouse was massaged for 1-2 minutes. Peritoneal washout was collected with a syringe and transferred to a test tube with heparin (20 units / ml), centrifuged at 1000 rpm for 10 minutes. Cell pellet was dissolved in phosphate-buffered saline (PBS) with pH=7.4. The mast cell suspension obtained from each animal was divided into two subgroups so that the following substances could be added: PBS, PBS + allergen, Sample 1 + allergen or Sample 2 + allergen (see Table 18). PBS was added with pH=7.4 and the allergen was added at a dose of 100 PNU / ml in a volume of 0.1 ml. Sample 1 (iteration of mast cells, 10 pl / ml) or Sample 2 (control, purified water, 10 pl / ml) were added in a volume of 0.1 ml. The tubes were incubated at 37°C for 15 minutes. Then a 0.1% solution of toluidine blue was added and samples were additionally incubated at a temperature of 37°C for 20 minutes. In 20 pl of the resulting solution, 100 mast cells were counted, among which the number of degranulated mast cells was determined. The test was considered negative if the percentage of cells with such a reaction did not exceed 15%.

[0293] When working with animals, all generally accepted standards of experimental ethics were observed (Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010).

[0294] For statistical processing of the results, one-way analysis of variance with Tukey's post- hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0295] The results of the study are presented in Table 18.

[0296] Table 18. Effect of the test samples on the percentage of degranulation of mast cells obtained from naive mice and mice sensitized with epidermal wool allergen (M±m)

[0297] Note: * - differences are significant compared to the indicator of the combination “PBS + allergen ” in the corresponding group, # - differences are significant compared to intact group from the indicator “PBS” in the corresponding group

[0298] According to the data, in mast cells obtained from intact animals, when exposed to an allergen, there is an increase in the number of degranulated mast cells by 1.7 times ( s PBS). However, this value of the percentage of mast cell degranulation does not indicate the presence of sensitization in animals of intact group to this allergen. The number of degranulated mast cells when Sample 1 was added to the cells of animals from intact group decreased by 4.5 times compared to the effect of the combination “PBS + allergen” (p<0.05) and by 4 times compared to the combination “Sample 2 + allergen” (p<0.05), which indicates a stabilizing effect on mast cells of Sample 1.

[0299] In mast cells obtained from sensitized animals, the percentage of mast cells that degranulated when the allergen was added was 2.7 times higher than in the corresponding control (PBS). The indicated increase in the number of degranulated mast cells indicates a pronounced manifestation of an allergic reaction (p<0.05). When Sample 1 was added to the cells of the sensitized group in the presence of the allergen, there was a significant decrease in the number of degranulated mast cells (2.9 times compared to the effect of the combination “PBS + allergen” (p <0.05) and 2.5 times relative to the combination “Sample 2 + allergen” (p<0.05)). At the same time, the Sample effect in absolute value did not differ statistically significantly from the value of the physiological norm (i.e., the percentage of degranulation of mast cells obtained from intact animals under the influence of PBS).

[0300] Thus, it has been demonstrated that Sample 1 (iteration of mast cells) has a stabilizing effect on mast cells (anti -allergic effect): it suppresses the process of mast cell degranulation.

[0301] Example 16. Iterations obtained using myocardium and water as a neutral carrier.

[0302] The process of obtaining iterations included several stages. A vial with a capacity of 5 ml with a freshly prepared stock solution of the initial substance (myocardium of adult male Wistar rat, 15 mg) in a volume of 5 ml and a vial with a capacity of 40 ml with purified water (neutral carrier) in a volume of 35 ml were rotated on a vortex for 10 seconds at 3000 rpm in close contact. After this, both vials were incubated for 1 minute at room temperature in close contact. Using the above-described procedure, 35 ml of iteration 0 (Io) was obtained.

[0303] Next, a 40 ml vial with Io (35 ml) was placed adjacent to a new 40 ml vial with 35 ml of neutral carrier and vortexed for 15 seconds at 3000 rpm in close contact. Then both vials were incubated for 2 minutes at room temperature in close contact. As a result, the vial with the neutral carrier was named Ii. Then the procedure for obtaining the next iteration (No. 2, No. 3, etc.) was repeated several times by joint vortexing of a vial with a new portion of neutral carrier and the previous iteration.

[0304] Temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer; the temperature was 22±3°C, and the humidity was 20-70%. To prepare iterations, transparent borosilicate glass vials (40 and 60 ml, Glastechnik Grafenroda, Infochroma AG, Switzerland) or vials (100, 250, and 500 ml, Simax, Czech Republic) were used.

[0305] Type 1 (ultrapure) water with a resistivity of 18.2 mQ- cm (Milli-Q Integral 5, Millipore) was used as the neutral carrier for the preparation of iterations, as well as for all other reagents used in this work. The quality of purified water was monitored daily by measuring resistivity using a SevenCompact S230 conductivity meter (Mettler Toledo) and pH using a SevenCompact S220 pH meter (Mettler Toledo). For liquids, automatic pipettes of various volumes were used (Eppendorf, Germany; Socorex, Switzerland), as well as measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine). Dry reagents were weighed using an analytical balance of accuracy class I (Pioneer PA214C, Ohaus, USA). A calibrated laboratory timer (Traceable, VWR) was used to measure all incubation times. The vibration effect was carried out using an MS 3 basic shaker (vortex) (IKA-Werke, Germany) with a standard insert.

[0306] In this study I9 (iteration No. 9) was used (hereinafter referred to as Sample 1). The distribution of the obtained iterations into fractions was made as described in the Example 1.

[0307] The study was conducted on a model of pulmonary heart failure in male Wistar rats. Animals aged four weeks (weight 80-100 g) were randomly divided into three groups: intact control (n=15), group with experimental pathology and therapy with Sample 1 (iterations of myocardium, n=15), and group with experimental pathology and therapy with Sample 2 (control, purified water; n=15).

[0308] To simulate pulmonary heart failure, animals were subjected to a single intraperitoneal injection of 2 ml of saline with the addition of the alkaloid monocrotaline at 50 mg / kg of animal weight. Intact rats were injected with saline according to a similar scheme without the addition of monocrotaline. An hour after the administration of these substances and then daily throughout the experiment, animals of the second and third groups were administered, respectively, Sample 1 or Sample 2 (2 ml / kg / day, intragastrically).

[0309] At the seventh to eighth week of life of the animals (the time of appearance of clinical signs of heart failure: weight loss, shortness of breath at rest), animals of all groups were sacrificed. 15-20 minutes before euthanasia, the animals were intramuscularly administered a muscle relaxant (Rometar, 1 ml / kg), an anesthetic (Zoletil, 0.02 ml / kg) and heparin (1000 U / kg).

[0310] After euthanasia of the animal, the heart was removed and placed in a preparation bath with Krebs-Henseleit solution (in mM): NaCl - 118, KC1 - 4.7, MgSCU - 1.2, NaHCOs - 14.5, KH2PO4 - 1.2, CaCE - 2.5, glucose - 11.1, pH 7.35, with the addition of 30 mM 2,3- butanedione monoxime. A thin trabecula was excised from the right ventricle of the heart and placed in an experimental bath with a flowing Krebs-Henseleit solution without 2,3 -butanedione monoxime at constant saturation with a mixture of 95% O2 and 5% CO2. One end of the trabecula was fixed on the rod of the force sensor, the other end - on the rod of the length servomotor. Biomechanical measurements in the muscle preparation were performed using a system for studying muscle activity (Muscle Research System, Scientific Instruments GmbH, Heidelberg, Germany) in pseudo-real time (with an interval of 100 ps) using an ADC / DAC PCI- 17168 (Ad-L ink Technology Inc., Taiwan) and software operating in the real-time HyperKernel (Arc Systems Ltd., Japan). The measurements were performed at an electrical stimulation frequency of 1 Hz and a saline temperature of 25°C. Isometric muscle tension was determined as the ratio of the magnitude of muscle strength to its cross-sectional area, which was determined by the formula S = 7td2 / l 2, where d is the diameter of the muscle in an unstretched state.

[0311] When working with animals, all generally accepted standards of experimental ethics ((Directive 2010 / 63 / EU of the European Parliament and of the council on the protection of animals used for scientific purposes, September 22, 2010)) were observed.

[0312] For statistical processing of the results, one-way analysis of variance with Tukey's post- hoc test was used. Differences between groups were considered statistically significant at p<0.05.

[0313] The results of the study are presented in Tables 19 and 20.

[0314] Table 19. The effect of the test samples on the morphometric parameters of the cardiac muscle of the right ventricle of rats with experimental pulmonary heart failure (M±m)

[0315] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal

[0316] Table 20. The effect of the test samples on the contractility parameters of the unstretched cardiac muscle of the right ventricle of rats with experimental pulmonary heart failure (M±m)

[0317] Note: * - differences are significant compared to Sample 2 (control, purified water), # - differences are significant compared to intact animal

[0318] According to the obtained results, morphometric indicators of the whole heart (see Table 19) and indicators of contractility of isolated muscle preparations (see Table 20) confirm the development of severe right ventricular myocardial hypertrophy and subsequent pulmonary- cardiac failure in rats in the Sample 2 group (control, purified water). Thus, in Sample 2 group, three to four weeks, after a single use of monocrotaline an increase in the mass of the whole heart was observed (1.2 times vs. intact animals), as well as the mass of the right ventricle (1.9 times vs. intact animals). At the same time, the amplitude of isometric tension sharply decreased (by 2.6 times vs. intact animals), and the time to reach the peak of tension increased (by 1.3 times vs. intact animals) and the time of relaxation from the peak of tension to 50% of the amplitude (by 1.4 times vs. intact animals).

[0319] In the Sample 1 group there were no pathological changes in both the morphological parameters of the heart and the functional activity of the myocardium. All measured parameters corresponded to the physiological norm, as evidenced by the absence of statistically significant differences between Sample 1 group and intact animals.

[0320] Thus, it has been demonstrated that Sample 1 (iterations of myocardium) has a positive effect on the morphological parameters of the heart and the functional activity of the myocardium in a model of pulmonary heart failure.

[0321] Example 17. Study of antitumor activity of vibrational iterations using an in vivo Lewis lung carcinoma (LLC) model

[0322] Materials and methods

[0323] Mouse model 120 conventional C57B1 / 6 male mice (18-22 g, aged 2.5-3 months) were obtained from the department of experimental biological models of the E.D. Goldberg Research Institute of Fertilizers and Microorganisms (Russia).

[0324] Right before the experiment, mice were divided into 8 groups (n=10) as following (Table 21):

[0325] Table_21. Experimental groups

[0326] On the first day of the experiment, the samples were administered to animals of prophylactic groups. LLC cells grown in culture were injected into the paws of animals of all experimental groups on the 10th day of the experiment. The experiment ended on day 22. The animals were euthanized, after which the experimental parameters (tumor weight, number of metastases) were evaluated.

[0327] The criterion for assessing the efficacy of Endoxan 200 in the in vivo system was the presence of statistically significant differences in the parameters of tumor development compared to Control 2 (solvent). The efficacy of the samples was assessed based on statistically significant inhibition of tumor progression relative to the Control 1 group (no treatment).

[0328] Preparation of iterations

[0329] The vibrational iterations were prepared using technology described in Example 1, but the initial substance is selected from the normal lung cell line (FLECH-104 cell line from human embryo, lung fibroblasts), LLC cell line or ultrapure water and as a neutral carrier used ultrapure water at the first stage. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier (ultrapure water) in a volume of 200 mL in close contact with the bottle made of transparent borosilicate glass (250 mL) with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200mL of vibrational iteration zero was obtained from neutral carrier (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (type 1) with a resistivity of 18.2 MQ ' cm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0330] Samples

[0331] In this work, vibrational iterations were analyzed using intact water as a control, classified into four groups (fractions) by their unique physico-chemical properties and modifying effect (Table 22). These four types of fractions were named "Active", "Native", "Semi-Native" and "Semi -Active".

[0332] Table 22.

[0333] Note: the test result was considered positive (“+”) if it met the acceptance criteria. Otherwise, the result was taken as negative The acceptance criteria: the values obtained for vibrational iterations should statistically significantly (p < 0.05) differ from those of intact water by ±5% or more (by conductometry and THz spectroscopy) and by +10% or more (by radiometry). The values obtained for intact water were taken as 100%.

[0334] The test samples used in the study are shown in the table below.

[0335] Table 23. The test samples and the initial substances that were used in the study for vibrational iterations preparation.

[0336] Note: LLC - Lewis lung carcinoma; N / D - Not done; I - Vibrational iteration.

[0337] Statistical analysis Analysis and data visualization were performed using the R statistical computing environment (R Foundation for Statistical Computing, Vienna, Austria). Descriptive statistics included arithmetic means, medians, quartiles, and outliers.

[0338] Normality of distribution was assessed using the Shapiro-Wilk test, and homogeneity of variances using Bartlett’s test. Group comparisons were carried out using the Student’s t-test, Welch’s t-test, or the Kruskal-Wallis test followed by Dunn’s post hoc test, depending on the conformity of the raw data to the assumptions of each test.

[0339] Holm’s correction was used to adjust for multiple comparisons. Differences were considered statistically significant at p < 0.05.

[0340] Results

[0341] The results of the study (day 22) are presented below (Fig. 4)

[0342] Conclusions

[0343] Among the studied vibrational iterations, the most promising reduction in primary tumor mass and the number of metastasis was observed in the group 4. In group 4 a statistically significant reduction in the number of metastases compared to the Control 1 and 2 was observed. Meanwhile there was no significant difference between positive control group and group 4 in tumor weight. Thus, these findings might suggest that Sample 4 may have a potential in affecting tumor growth and metastatic progression.

[0344] Example 18. Study of antitumor activity of iterations using an in vivo lung carcinoma (A549) model.

[0345] Materials and methods

[0346] Mouse model

[0347] 120 specific pathogen-free inbred female BALB / c nude mice (18-22 g, aged 2.5-3 months) were obtained from the department of experimental biological models of the E.D. Goldberg Research Institute of Fertilizers and Microorganisms (Russia).

[0348] Right before the experiment, mice were divided into 12 groups (n=10) as following

[0349] (Table 24): Table 24. Experimental groups

[0350] On the first day of the experiment, the samples were administered to animals of the prophylactic group. A549 cell carcinoma cells grown in culture were injected into the paws of animals of all experimental groups on the 10th day of the experiment. The experiment ended on day 22. The animals were euthanized, after which the experimental parameters (body weight, tumor weight, number and area of metastases) were evaluated.

[0351] The criterion for assessing the efficacy of the drug Endoxan 200 in the in vivo system was the presence of statistically significant differences in the parameters of tumor development compared to Control 2 (solvent). The efficacy of the tested samples was assessed based on statistically significant inhibition of tumor progression relative to the Control 1 group (no treatment).

[0352] Preparation of iterations

[0353] The vibrational iterations were prepared using technology described in Example 1, but the initial substance is normal lung cell line and neutral carrier is intact water. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier (ultrapure water) in a volume of 200 mL in close contact with the bottle with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (type 1) with a resistivity of 18.2 MQxcm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0354] Samples

[0355] The samples used in the study are shown in the table below.

[0356] Table 25. The test samples and the initial substances that are used in the study for vibrational iterations preparation.

[0357]

[0358] Statistical analysis

[0359] Analysis and data visualization were performed using the R statistical computing environment (R Foundation for Statistical Computing, Vienna, Austria). Descriptive statistics included arithmetic means, medians, quantiles, standard deviation (SD), standard error (SE), and others.

[0360] Normality of distribution is assessed using the Shapiro-Wilk test, and homogeneity of variances using Bartlett’s test. Group comparisons were carried out using the Student’s t-test, Welch’s t-test, or the Kruskal-Wallis test followed by Dunn’s post hoc test, depending on the conformity of the raw data to the assumptions of each test.

[0361] Holm’s correction is used to adjust for multiple comparisons. Differences were considered statistically significant at p < 0.05.

[0362] Results

[0363] The results of the study (day 22) are presented in the table below.

[0364] Table 26. significant differences compared to the Control 2 group.

[0365] Conclusions Studied vibrational iterations have an antitumor effect. The effect of Sample 2, which is an Active fraction of vibration iterations of the normal lung cell line (15), was the most pronounced. The use of Sample 2 led to a statistically significant (compared to the control) reduction in tumor mass, number and area of metastases.

[0366] Example 19. Evaluation of A549 survival in culture after short-term exposure to UV in the presence of vibrational iterations

[0367] Materials and methods

[0368] Cell culture

[0369] The A549 lung cancer cell line was grown adherent using T-75 flasks in low-glucose (1 g / L) DMEM medium supplemented with 10% FBS, glutamine, and penicillin-streptomycin. The culture medium was changed every 2-3 days. Cells up to 25 passages and viability of at least 80% were used for experiments.

[0370] Preparation of vibrational iterations

[0371] K-A21 normal lung cell suspension (the initial substance) and intact water were used for preparing the iterations using technology described in Example 1. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier (ultrapure water) in a volume of 200 mL in close contact with the bottle with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 17) were obtained from each previous one. Samples from 10 to 17 comprised a row of vibrational iterations. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (Type 1) with a resistivity of 18.2 MQxcm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of the vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0372] In this study, the following iterations belonging to different fractions were selected as test samples (Table 27).

[0373] Table 27.

[0374] Scheme of the experiment

[0375] 1. After routine cultivation, A549 cells were washed with Versen solution once. Then the cells were removed from the bottom of the flask using trypsin and a scraper. The suspension was centrifuged for 5 min at 1500 rpm. The supernatant was removed, cells were resuspended in fresh culture medium, counted, and their viability was measured.

[0376] 2. Cells were added to 96-well plates at 15,000 cells per well in a volume of 50 pl. Two plates were used: one for further UV treatment and one control plate (used for evaluation of the control sample (ultrapure water without UV treatment)). Then 50 pl of a mixture of cell culture with experimental samples in a 3 / 2 ratio was added to the cells. To the intact cells, 50 pl of undiluted culture medium was added. The final sample-to-medium ratio in each well was 1:5 (v / v), the total volume per well was 100 pl, and 6 wells were used for each sample. The edge wells of the plate were filled with Versen solution (100 pl per well) to avoid the evaporation effect.

[0377] 3. The plates were placed in an incubator for 24 h at 37 °C, 5% CO2.

[0378] 4. One plate was placed in a UV cabinet (LEDVANCE TIBERA UVC 15W G13, 100-280 nm) with the lid open for 2 minutes. The control plate was placed in a laminar flow cabinet with the lid open for the same time.

[0379] 5. Incubation for 24 h at 37 °C, 5% CO2.

[0380] 6. MTT test was performed.

[0381] Conducting MTT test

[0382] MTT solution (10 pl) with a concentration of 5 mg / mL was added into each well (except edge wells), after which the plates were incubated for 2 h at 37 °C, 5% CO2. Next, the culture medium was carefully removed from the wells, and 100 pl of DMSO was added to dissolve the formed formazan crystals, after which the plates were incubated at room temperature for 10 min. After shaking, plate for 2 min, the absorbance values were measured using Multiscan GO at wavelengths of 554 nm and 700 nm.

[0383] Statistical analysis

[0384] For statistical processing of the data, the RStudio 2024.12.1+563 software was used. The Shapiro-Wilk test was used to assess the normality of the distribution of data obtained in repetitions. The Student t-test was used to compare data between groups. Differences between groups were considered statistically significant at p < 0.01.

[0385] Results

[0386] The results of the study are presented in Fig 5.

[0387] In the presence of sample 13 (Active fraction of vibrational iterations of K-A21 normal lung cells), the survival rate of A549 lung carcinoma cells after UV exposure was statistically significantly decreased by 27% relative to the control (ultrapure water).

[0388] Conclusion Vibrational iterations of K-A21 normal lung cells have the property of reducing the survival of A549 lung carcinoma cells when they are exposed to UV.

[0389] Example 20. Study of the action of the vibrational iterations on human prostate tumor cell lines in vitro.

[0390] Materials and methods

[0391] Cell lines

[0392] To evaluate the activity of test samples and Cisplatin on survival of human prostate tumor cell lines (22RV1, PC3, and LNCaP), cells were seeded into 96-well culture plates and incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO. Following incubation, the test samples or Cisplatin were added to the culture medium in a 1 :5 ratio by volume. The cells were incubated with the compounds for 48 hours. Untreated cells served as the negative control. Cell survival was assessed using microscopy and a colorimetric MTT assay.

[0393] Preparation of iterations

[0394] The vibrational iterations (Table 28) were prepared using the crossing technology. The initial substance (chosen from PC3 cell line, LNCap cell line, 22RV1 cell line, CHO cell line or Cisplatin) and intact water acted as crossing partners at the first stage. A bottle made of transparent borosilicate glass (250 mL) with neutral carrier (ultrapure water) in a volume of 200 mL in close contact with the bottle made of transparent borosilicate glass (250 mL) with the initial substance was vibrated on a vortex for 10 s at 3000 rpm (MS 3 basic with the MS 1.21 platform, IKA-Werke, Germany). Next, both bottles were incubated for 1 min at room temperature, keeping them in close contact. As a result of the above treatment, 200 mL of vibrational iteration zero was obtained from neutral carrier (hereinafter referred to as 10). To obtain the first vibrational iteration (II), the bottle with vibrational iteration 10 was placed next to the bottle with the neutral carrier in a volume of 200 mL and subjected to joint (in close contact) vibration treatment on the vortex for 10 s at 3000 rpm. Then both bottles were incubated for 1 min at room temperature, keeping them in close contact. After this procedure, the resulting neutral carrier was considered to be the first vibrational iteration of the initial substance (II). Then the subsequent vibrational iterations (up to 110) were obtained from each previous one. Samples from 10 to 110 comprised a row of vibrational iterations. A row of iterations II -Il 0 was prepared from each of the initial substances separately. All vibrational iterations were prepared on the same day. The temperature and humidity in the laboratory during sample preparation and subsequent measurements were monitored using a verified IVA-6N thermohygrometer (NPK MICROFOR LLC, Russia). During the experiment, the humidity in the room was 45-50%, and the temperature was 24.5 °C. Ultrapure water (type 1) with a resistivity of 18.2 MQxcm (Milli-Q Integral 5, Millipore, France) was used as a neutral carrier for the preparation of vibrational iterations and as a control. Automatic pipettes of various volumes (Eppendorf, Germany; Socorex, Switzerland) and measuring glassware of accuracy class A (Borosil, India; Steklopribor, Ukraine) were used for sampling liquids.

[0395] In this study, the following vibrational iterations of initial substances (PC3 cell line, LNCap cell line, 22RV1 cell line, CHO cell line or Cisplatin) and belonging to different fractions were selected as test samples (Table 28).

[0396] Table 28.

[0397]

[0398] Statistical analysis

[0399] Analysis was performed using the R statistical computing environment (R Foundation for Statistical Computing, Vienna, Austria). Descriptive statistics include arithmetic means. Group comparisons were carried out using the Student’s t-test. If necessary, other more appropriate tests were applied. Differences were considered statistically significant at p < 0.05.

[0400] Results The results of the study are presented below (Table 29).

[0401] *significant differences from ultrapure water (control) (p < 0.05)

[0402] Conclusions

[0403] Among the studied vibrational iterations, the promising reduction in cell survival was observed in groups where Active fractions of iterations (LNCap, PC3, 22RV1, CHO cell lines, and Cisplatin) were used. The introduction of these test samples led to a statistically significant decrease in cell survival. Thus, vibrational iterations of cell lines or antitumor substances may have a potential in antitumor therapy.

Claims

1. Claims1. A method for obtaining a biological product, which is an artificial material object - iteration, having a biological activity similar to that of a biological object, by successive vibration treatment of a neutral carrier in the presence of biological object acting as a biological substance.

2. A method according to claim 1, wherein the biological activity comprises influencing biological objects or their molecular or cellular targets in the organism, and functional and metabolic processes regulated by the biological objects used to produce the biological product.

3. A method according to claim 1, wherein the biological substance is a cellular object comprising a single cell, a group of cells, or a plurality of cells, or an extracellular object - prion.

4. A method according to claim 3, wherein the plurality of cells is a tissue, a body fluid medium - blood and lymph, or an organ.

5. A method according to claim 3, wherein the cells, their associations or cell structures are isolated from the organism.

6. A method according to claim 3, wherein the cells, their associations and cell structures are grown outside the organism.

7. A method according to claim 3, wherein the cell is a microorganism cell.

8. A method according to claim 7, wherein the microorganism cell is a bacteria, virus.

9. A method according to claim 3, wherein the cells are normally functioning cells.

10. A method according to claim 3, wherein the cells are atypical cells.

11. A method according to claim 1, wherein the biological object is a biological substance comprising an extracellular structure.

12. A method according to claim 1, wherein the biological object used as a biological substance in relation to the organism of a patient is auto-, allo- or heterologous.

13. A method according to claim 1, wherein the successive vibration treatment of the biological object is a process including:a) external vibration treatment of test tubes containing biological object and neutral carrier to obtain from neutral carrier the primary artificial object - ‘iteration zero’; b) external vibration treatment of the previous iteration and neutral carrier to obtain from neutral carrier the subsequent iteration;14. A method according to claims 1, 13, wherein neutral carrier is water or an aqueous alcoholic solution, lactose or a pharmaceutically acceptable carrier.

15. A method according to claim 13, wherein the vibration treatment may be horizontal or vertical mechanical shaking.

16. A method according to claim 13, wherein the vibration treatment is accomplished by electromagnetic, ultrasonic, acoustic or other rhythmic physical action.

17. An artificial object obtained using the method according to claim 1, comprising a neutral carrier subjected to vibration treatment in the presence of a biological object or a preceding iteration of a biological object having biological (pharmacological) properties similar to those of the biological object.

18. A method for obtaining a fraction of a biological product- iteration, obtained using the method according to claim 1 having specific activity, including:1) preliminary separation of the obtained iterations into fractions depending on the physical -chemi cal properties they acquire after vibration treatment, which are different from the properties of neutral carrier,2) determination of specific biological or pharmacological activity for each fraction using standard methods.

19. A method according to claim 18, wherein the physical-chemical properties are evaluated using generally accepted analytical methods.

20. A method according to claim 18, wherein the distribution into fractions is based on the presence of physical -chemi cal properties altered compared to the neutral carrier.

21. A method according to claim 18, wherein the determination of specific activity is accomplished by determining the biological and / or chemical activity of the fraction using methods generally accepted in experimental biology.

22. A method according to claim 19, wherein the analytical method is any generally known validated analytical method for determining physical-chemical properties of the substance.

23. A method according to claim 22, where the evaluation of specific electrical conductivity (SEC), radiometry, terahertz spectroscopy, immunoassay (ELISA), pH measurement, determination of the amount of dissolved oxygen, dynamic light scattering, high- resolution thermography are selected as the analytical method.

24. A method according to claim 18, wherein iterations for further experimental determination of the biological (pharmacological) action are selected from two fractions that markedly differ in their physico-chemical properties both from the neutral carrier and from each other - in the presence or absence of a modifying effect on the physical characteristics of the initial biological substance.

25. A drug product containing a biological product obtained using the method according to claim 1, exhibiting biological activity similar to biological activity of a biological object.

26. A drug product according to claim 25, wherein the biological object is selected from an extracellular structure, a microorganism, a cell, a cell line, a tissue, an organ, an organ system.

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