Method for the treatment and prophylaxis of hyperglycaemia and / or insulin resistance and drug for carrying out same

Abstract

The group of inventions relates to the field of medicine and pharmaceutics and can be used for producing therapeutic drugs based on human apolipoprotein A-I. For the purpose of permitting broader indications for use, a drug for the treatment or prophylaxis of hyperglycaemia and / or insulin resistance and / or related diseases and / or organ and system dysfunction is proposed which contains human apolipoprotein A-I (apoA-I) or a functionally significant fragment thereof. Said diseases related to hyperglycaemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus. Said organ and system dysfunction related to hyperglycaemia and / or insulin resistance is selected from the group consisting of dysfunction of the liver and kidneys, the bone marrow, the spleen, the myocardium or the reproductive system, locomotor activity disorders and cognitive function disorders. The functionally significant fragment of human apoA-I has a sequence consisting of the amino acid sequence of human apoA-I truncated at the N terminal. The drug is provided in the form of a pharmaceutical composition containing human apoA-I or the functionally significant fragment thereof, and a pharmaceutically acceptable carrier in the form of water for injection or saline solution. The use of said drug for the above-mentioned purpose is proposed. A method for the treatment and prophylaxis of hyperglycaemia and / or insulin resistance and / or related diseases and / or organ and system dysfunction is proposed which includes locally or parenterally administering said drug to a subject in need thereof in therapeutically effective doses.

Description

[0001] METHOD FOR THE TREATMENT AND PREVENTION OF HYPERGLYCEMIA AND / OR INSULIN RESISTANCE AND A MEANS FOR ITS IMPLEMENTATION

[0002] Field of technology to which the invention relates

[0003] The group of inventions relates to the field of medicine and pharmaceuticals and can be used to produce medicinal products based on human apolipoprotein AI, capable of reducing the manifestations of endocrine-metabolic disorders in type 2 diabetes mellitus, obesity and associated functional disorders of various organs and systems.

[0004] State of the art

[0005] The increasing incidence of type 2 diabetes mellitus (T2DM), manifested as hyperglycemia and insulin resistance, is a global problem of colossal proportions both in the world as a whole and in the Russian Federation, the main culprit of morbidity and mortality of patients and the main economic burden [Tinajero MG, Malik VS Endocrinol Metab Clin North Am. 2021. Vol. 50. N. 3. P. 337-355.; Dedov I.I. et al. Diabetes mellitus. 2021. Vol. 24. N. 3. P. 204-221.]. Although the treatment of T2DM has improved significantly in recent years, its results still do not achieve their goals or patients experience unwanted side effects.

[0006] Literature data indicate that lipid metabolism parameters in the classical course of type 2 diabetes change towards an increased risk of developing cardiovascular complications. It is known that patients with type 2 diabetes have an increase in total cholesterol. At the same time, low-density lipoprotein particles that carry cholesterol are smaller in size and have a higher density. A decrease in high-density lipoprotein cholesterol (HDL-C) and an increase in the concentration of triglycerides are noted [Vorobyov, S. V., Petrovskaya, E. Yu. Medical Bulletin of the South of Russia, 2012. N. 3. pp. 25-28.; Sabahelkhier M. K. et al. Nova Journal of Medical and Biological Sciences, 2016. Vol. 5. N. 2, pp. 1-9].

[0007] People with type 2 diabetes suffer from excess body weight, characterized by chronic hyperglycemia (elevated blood glucose levels), developing as a result of impaired mechanisms of interaction between insulin and tissue cells (insulin resistance) or its secretion. Type 2 diabetes is also associated with systemic dysfunction of various organs and systems that reduce the quality and duration of life, including dysfunction of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor and cognitive functions.

[0008] Diabetes mellitus is often accompanied by destructive changes in the musculoskeletal system. It is believed that excessive accumulation of advanced glycation end products provokes damage to periarticular tissues and joints (cheiroarthropathy), which in turn leads to limited joint mobility, usually in the absence of pain syndrome and, as a consequence, to limited physical (locomotor) activity [Panevin TS et al. Scientific and Practical Rheumatology. 2020. No. 3; Alabdali, Login Ahmed S., et al. / BMC Fam. Pract. 2019. Vol. 20. N. 1. P. 98.]. Joint mobility limitation syndrome is associated with other late complications of diabetes and can significantly impair functional activity, self-care and worsen quality of life [Panevin, TS, et al. Osteoporosis and Bone Diseases 2019.Vol. 22. N.3. P.19-26.].In this case, the impairment of locomotor function is aggravated by the state of obesity, in which physical activity is reduced due to an increase in physical and psychoemotional stress during any motor activity. Inadequate muscle glucose uptake in people with insulin resistance [Defronzo RA, Tripathy D. Diabetes Care. 2009. Vol. 32. Suppl 2. S157-S163.] leads to hyperglycemia and an increased risk of muscle fatigue.

[0009] To date, there is convincing evidence that type 2 diabetes is characterized by both a decrease in the overall assessment of cognitive functions and impairments in individual cognitive areas. Statistics indicate that deviations from the age norm are found in 20-40% of patients with type 2 diabetes. In the vast majority of cases, the cognitive defect remains insignificant, although it can negatively affect the quality of life of patients. A feature of the neuropsychological profile in patients with diabetes is the predominance of neurodynamic and regulatory disorders revealed in tests for attention, speed of psychomotor reactions, speech activity, ability to switch, conceptual thinking, etc. [Levin, O. S. Modern therapy in psychiatry and neurology 2015. No. 4. P. 18-25]. A study of the dependence of cognitive impairment on glycemia levels indicates that this main metabolic deviation in patients with diabetes may be the cause of cognitive impairment.It has been shown that a rapid increase in glucose levels, including after meals, is directly associated with a decrease in attention and other neurodynamic functions in patients with type 2 diabetes. Acute hypoglycemia can reduce regional brain perfusion and disrupt osmotic balance in cerebral neurons [ Ermatov U. European Multidisciplinary Journal of Modern Science 2022. N. 4. P. 698-703; MacKnight C. et al. Dement Geriatr Cogn Disord. 2002. Vol. 14. N. 2. P. 77-83.].

[0010] Other complications of diabetes, such as coronary heart disease, as well as associated risk factors such as arterial hypertension, insufficient physical activity, dyslipidemia, and obesity, can also contribute to cognitive impairment [Levin, O. S. Modern Therapy in Psychiatry and Neurology 2015. N.4. pp. 18-25; Xue M. et al. Ageing research reviews 2019. Vol. 55. p. 100944.].

[0011] The liver, as a key organ regulating carbohydrate metabolism, is a target of the pathological effects of diabetes mellitus. A diagnosis of non-alcoholic fatty liver disease (NAFLD) is made in patients with the most common metabolic disorders, such as obesity, metabolic syndrome and type 2 diabetes. This complication has several forms and includes fatty liver degeneration (steatosis), non-alcoholic steatohepatitis and fibrosis / cirrhosis, which can ultimately lead to end-stage liver failure or hepatocellular carcinoma. Recent studies show that patients with severe insulin resistance are associated with the state of NAFLD and its accelerated progression, which increases the risk of cardiovascular and renal diseases associated with type 2 diabetes mellitus and confirms the critical role of insulin resistance [Dewidar B. et al. Metabolism. 2020 Vol.11 IS. P.154299.; Shehri Z. Biomedical Research and Therapy. 2017. Vol. 4. N. 11. P. 1760-1774].

[0012] Bone marrow is one of the tissues affected by diabetes complications. Initial data from preclinical and clinical studies have shown that T2DM causes quantitative and qualitative disturbances in circulating stem cells, such as those in blood vessels. However, the initial damage occurs directly at the bone marrow level, before the cells are mobilized into the peripheral circulation, due to significant remodeling of all components of the bone marrow niche. In diabetes, the bone marrow itself is affected, as it can develop microangiopathy and neuropathy similar to other tissues of the body. Neuropathy leads to impaired mobilization of stem cells from the bone marrow, the so-called mobilopathy. These changes lead to impaired stem cell homeostasis, providing an additional mechanism to explain and interpret peripheral complications [Mangialardi G., Madeddu P. Curr Diab Rep. 2016. Vol. 16. N.5. P.43].The spleen plays an important role in the occurrence and development of diabetes, the development of splenic insufficiency in obesity, despite the fact that there is still little data on the associated mechanisms [Zhang J. et al. Stem Cells Dev. 2017. Vol. 26. No. 24. P. 1749- 1758.; Wang K. et al. Randomized Controlled Trial. Zhongguo Zhen Jiu. 2016. Vol. 36. No. 3. P.225-230; Tarantino G. et al. World J Gastroenterol 2013. Vol. 19. N. 23. P. 3534-3542.].

[0013] Androgens play a crucial role in the development of male reproductive organs such as the epididymis, vas deferens, seminal vesicles, prostate gland, and penis. High levels of intratesticular testosterone secreted by Leydig cells are essential for spermatogenesis [Dohle GR et al. World J. Urol. 2003. Vol. 21. N. 5. P. 341-345.]. Testosterone secretion has been shown to be significantly reduced in patients with type 2 diabetes mellitus [Spark RF Curr Urol Rep. 2007. Vol. 8. N. 6. P.467-471.].

[0014] It is known that patients with type 2 diabetes are characterized by specific pathological changes in the cardiovascular system, characterized by left ventricular hypertrophy and diastolic dysfunction at an early stage up to overt heart failure with a decrease in systolic function at later stages (diabetic cardiomyopathy) [Paolillo S. et alHeart Fail Clin. 2019. 15(3):341-347.] .

[0015] Antidiabetic agents developed in the previous era do not have sufficient therapeutic effects in relation to diseases and dysfunctions of organs and tissues associated with hyperglycemia and / or insulin resistance. However, in recent years, native and synthetic proteins, apolipoproteins, have attracted increasing attention for these purposes. Apolipoproteins are structural components of serum lipoproteins that perform important transport and regulatory functions in the body. The advantage of their use as drugs is their natural origin, since they are not foreign to the body's immune system. The possibility of using various types of apolipoproteins, their functional fragments and high-density lipoproteins (HDL) reconstructed on their basis is being studied. Currently, more than 10 apolipoproteins have been identified. According to the ABC nomenclature, individual classes of apolipoproteins (ApoA, ApoB, ApoC, etc.)) are families consisting of several individual proteins designated by Roman numerals - AnoAI, Apo AP, AnoAIV, etc. ApoA, ApoB are transport apoproteins. The main representatives of the group of regulatory apoproteins are apoproteins E (ApoE) and ApoC. However, the division into transport and regulatory apolipoproteins is conditional, since they have multifunctional properties. Lipoproteins (LP) containing ApoB are involved in the transport of endogenous cholesterol (CH) in LP particles from the liver to peripheral tissues, which is usually called direct or efferent, while LP, the structural protein of which is ApoA, are involved in the reverse or afferent transport of CH - the movement of CH from peripheral tissues through blood plasma components to the liver. ApoE is involved in the transfer of fatty acids into tissue cells and the transport of cholesterol into the liver [Boyko E.R., Kaneva A.M. ADVANCES IN PHYSIOLOGICAL SCIENCES, 2009, Vol. 40, No. 1, p.3-15; Bolanos-Garcia VM, Miguel RN Progress in Biophysics & Molecular Biology. 2003. N. 83. P. 47-68]. ApoM is a carrier of the lipid mediator sphingosine-1-phosphate in the blood, and its polymorphic substitutions T-855C and T-778C are associated with the development of obesity as a result of impaired HDL regulation [Kurano et al. Atheroscler Thromb. 2018. Vol. 25. N. 1. P.16-26.].

[0016] Publication WO2019238934 Al discloses the use of ApoM or a nucleic acid molecule encoding it for the treatment of insulin resistance by administering to a subject suffering from obesity or T2DM a therapeutically effective amount of the ApoM polypeptide. However, the publication does not concern the effect of ApoM on functional disorders of organs and systems in insulin resistance, T2DM, or obesity.

[0017] Publication W02012100010 A1 proposes apolipoprotein A-IV (AnoA-IV) as an antidiabetic peptide and a method for treating T2DM and lowering blood glucose levels in a subject, comprising administering to the subject an effective amount of anoA-IV or its biologically active, including a recombinant analogue. The effect of AnoA-IV was demonstrated in relation to glucose tolerance in AnoA-IV knockout mice and in T2DM obese mice, as well as in relation to an increase in insulin synthesis in a culture of human islet β-cells, i.e. the effect of AnoA-IV on carbohydrate metabolism. The publication does not cover the effect of AnoA-IV on the body weight of mice, as well as on functional disorders of organs and systems and the quality of life of patients with T2DM.

[0018] HDL and its major protein component, apolipoprotein AI (ApoA-1), play an important role in the transport and metabolism of cholesterol and other lipids in the bloodstream and are thought to prevent atherosclerosis and cardiovascular disease by promoting cholesterol efflux in patients with atherosclerosis [Gille A. et al., Arterioscler Thromb Vase Biol. 2018. Vol. 38. N. 4. P. 953-963; Rosenson RS et al., Nat Rev Cardiol. 2016. Vol. 13. N. 1. P. 48-60; Gibson CM et al., Am Heart J. 2021. N. 231. P. 121-127.

[0019] The article by Kalayci A. et al. Current Atherosclerosis Reports. 2022. Vol. 24. N. 7. P. 585-597 shows that therapy with reduced ApoA-I (CSL112) in patients who have suffered acute myocardial infarction reduces the recurrence of cardiovascular events. However, the study is limited to stating this connection, while this work did not study the effect of ApoA-I on dysfunctions of other organs and systems in patients with cardiovascular diseases, hyperglycemia and / or insulin resistance.

[0020] ApoA-I is also involved in the regulation of glucose control [Siebel AL et al. Front Pharmacol. 2015. N.6. P. 258; Liu J. et al. Diabetes. 2017. Vol. 66. Nl lP 2915-2926]. This suggests that ApoA-I may be an important link between diabetes and cardiovascular disease in the setting of carbohydrate metabolism disorders. However, the effect of ApoA-I on the impairment of other metabolic processes and organ functions in hyperglycemia remains unknown.

[0021] In the article Cochran BJ et al. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34:2261-2267 it was shown that HDL and ApoA-I of blood plasma enhance the synthesis and secretion of insulin in isolated pancreatic islets and clonal β-cell lines. However, in this work the influence of ApoA-I in the whole organism on metabolic processes and dysfunction of other organs (except the pancreas) in hyperglycemia and insulin resistance remained unstudied.

[0022] A similar effect of HDL and plasma ApoA-I was obtained in relation to the cells of the islets of Langerhans of the pancreas of Wistar rats in the article by Panin L.E. et al. Bulletin of the Siberian Branch of the Russian Academy of Medical Sciences. 2010. Vol. 30. No. 2. Pp. 28-32], as well as in the article by Usynina I.F. et al. Biomedical Chemistry. 2018. Vol. 64. No. 2. Pp. 195-200]. However, these studies did not study the effect of ApoA-I in vivo on the dysfunction of other organs and systems in type 2 diabetes, when hyperglycemia can be associated with insulin resistance with a normal level of insulin synthesis by the pancreas.

[0023] In the article by Stenkula KG et al. Diabetologia. 2014. Vol. 57. N. 4. P. 797-800, using a model of mice fed a high-fat diet, it was shown that native human ApoA-I and the Milan variant of ApoA-I with a single amino acid substitution at position 173 (Argl73Cys) equally improve insulin secretion, glucose tolerance, and reduce glucose levels by insulin-dependent and insulin-independent mechanisms. However, these studies are limited to studying carbohydrate metabolism, and the effect of ApoA-I on dysfunction of other organs and systems in hyperglycemia and insulin resistance remains unstudied.

[0024] In the article Domingo-Espin J. et al. Diabetes. 2016. Vol. 65. P. 1838-1848 on a mouse model of hyperglycemia created by acute and transient blockade of insulin secretion, it was shown that native ApoA-I equally enhances glucose-stimulated insulin secretion and acts as an activator of insulin-independent glucose uptake in peripheral tissue (cardiac and skeletal muscles). The analyses also identify the heart as an important target tissue for ApoA-1-stimulated glucose uptake with potential consequences in diabetic cardiomyopathy. However, the study of the effects of native ApoA-I in this work is limited to carbohydrate metabolism, including in muscle tissue, while the effect of ApoA-I on the dysfunction of other organs and systems in hyperglycemia is not studied.

[0025] In the article Drew BG et al. Circulation. 2009. Vol. 119. N. 15. P. 2103-2111 in patients with type 2 diabetes who were administered intravenously reduced HDL containing ApoA-I and in studying its effects on primary cultures of skeletal muscle cells obtained from these patients, a decrease in glucose levels was shown due to an increase in insulin levels under the influence of reduced HDL in vivo and activation of AMP-activated protein kinase in skeletal muscles. A conclusion was made about the role of therapy increasing HDL levels, in addition to atherosclerosis, for the treatment of type 2 diabetes mellitus. However, the study of the effects of HDL containing ApoA-I is limited to carbohydrate metabolism, while the effect of HDL on dysfunction of other organs and systems in patients with type 2 diabetes has not been studied.

[0026] In the article by Lehti M. et al. Circulation. 2013. Vol. 128. N. 22. P. 2364-2371, using genetic mouse models with elevated HDL levels (elevated ApoA-I content) and reduced HDL levels (ApoA-I deficiency), it was tested whether HDL modulates mitochondrial bioenergetics in skeletal muscles. It was found that increasing glucose utilization by skeletal muscles in mice with genetically elevated HDL and ApoA-I levels prevented the disruption of glucose homeostasis caused by a high-fat diet. Given that similar mitochondrial dysfunction and decreased HDL levels are observed in T2DM, it has been suggested that HDL-elevating therapy may preserve muscle mitochondrial function and address key aspects of type 2 diabetes associated with hyperglycemia and its associated muscle dysfunction, such as decreased exercise endurance.The effect of excess or deficiency of HDL and ApoA-I levels on dysfunction of other organs and systems associated with hyperglycemia remains unstudied. In the article by Tang S. et al. Scientific Reports. 2019. Vol. 9. N. 1. P. 1350, when primary human skeletal muscle cells are incubated with ApoA-1, the latter increases insulin-dependent and insulin-independent glucose uptake depending on time and concentration, while ApoA-1 increases glucose utilization in skeletal muscles by activating the IR / IRS-1 / PI3K / Akt / AS160 signaling pathway. Based on the results of the effect of ApoA-1 on hyperglycemia and insulin resistance in isolated skeletal muscles, the authors suggested the possibility of using therapeutic agents that increase ApoA-1 levels to improve glycemic control in people with type 2 diabetes, i.e. to correct carbohydrate metabolism.The use of ApoA-1 at the body level, including for the correction of other functions of organs and systems impaired by hyperglycemia and insulin resistance, remains unstudied.

[0027] In the article by Feng X. et al. Lipids in Health and Disease. 2017. Vol.16. N. lP 69. a negative association was found between blood ApoA-1 levels and insulin resistance in 108 Chinese patients with impaired glucose tolerance, but the use of ApoA-1 as a therapeutic agent to reduce insulin resistance in these patients with impaired glucose tolerance was not studied.

[0028] Since the content of ApoA-1 in human blood serum is very low, which limits the use of donor blood for its production and use for medical purposes, in the Russian Federation Patent No. 2573930, based on a recombinant strain of E. coli bacteria, recombinant mature human ApoA-1 (rApoA-I) was obtained, including a complete sequence of 243 amino acids, as well as its truncated version corresponding to amino acids 3-243. The similarity of the biological properties of rApoA-I with the natural human protein apoA-I in the ability to penetrate the nuclei of mammalian cells and stimulate the rate of DNA biosynthesis in them and the formation of specific antibodies during immunization of animals was shown. At the same time, the said patent did not study the possibility of using rApoA-I for the treatment of hyperglycemia and / or insulin resistance and associated diseases and functional disorders of organs and systems.

[0029] In the article by Dalla-Riva J. et al. J. Lipid Res. 2013. Vol. 54. N.5. P. 1275-1282 it was established that discoidal HDL and a fragment of apoA-I, including the sequence of amino acids No. 190-243, are potent agonists (inducers) of insulin-independent glucose uptake in skeletal muscles, and the C-terminal a-helical content of ApoA-I may be an important determinant of this process. However, the established effects are limited to demonstrating the influence of the claimed agents on carbohydrate metabolism in cultured mouse and human myotubes. Its effects on the whole organism in hyperglycemia and insulin resistance have not been studied, which does not allow us to assess the effect of these agents on other disorders of organ and system functions.

[0030] In the article Peterson SJ et al. J. Lipid Res. 2009. Vol. 50. N.7. P. 1293-1304 studied the possibilities of using the peptide L-4F, which has the amino acid sequence DWFKAFYDKVAEKFKEAF-NH2, synthesized from L-amino acids, and is a mimetic of ApoA-1, for the prevention of insulin resistance. In mice with obesity and diabetes, a decrease in visceral fat content with a reciprocal increase in adiponectin, a decrease in liver lipid content, and an improvement in insulin sensitivity, i.e. a decrease in insulin resistance, were shown. A disadvantage is the lack of studies of the effect of this mimetic of ApoA-1 on other functional disorders of organs and systems associated with insulin resistance, going beyond the disorders of carbohydrate and lipid metabolism.

[0031] The article by Edmunds SJ et al. Diabetologia. 2019. Vol. 62. N. 7. P. 1257-1267 studies the use of peptide RG54, a short peptide sequence derived from ApoA-1 and including 54 amino acids of its C-terminal region, as an antiatherosclerotic and antidiabetic agent. It is shown that the synthetic peptide RG54 induces glucose uptake in cultured rat muscle myotubes to the same extent as insulin, and also primes clonal pancreatic beta cells to improve glucose-stimulated insulin secretion. The results were tested in diet-induced insulin-resistant mice and Leprdb diabetic mice, which together confirmed the observed effect of the claimed functional fragment of ApoA-1 on carbohydrate metabolism parameters in cultured muscles and cultured pancreatic beta cells.A disadvantage is the lack of studies at the whole organism level in hyperglycemia and / or insulin resistance in terms of the influence of the effect of ApoA-1 on lipid metabolism, subject body weight, functional disorders of the liver, bone marrow, locomotor and cognitive activity associated with hyperglycemia and / or insulin resistance.

[0032] The closest analogue to the claimed invention is the technical solution from publication US2015353626 A1, which discloses a polypeptide containing a fragment of ApoA-1; a pharmaceutical composition including said polypeptide, as well as a method for treating diseases characterized by hyperglycemia and / or insulin resistance, including administering a therapeutically effective amount of said polypeptide to an individual in need thereof.The polypeptide comprises an amino acid sequence selected from the group consisting of: a) a fragment of the amino acid sequence of the C-terminal portion of ApoA-1 corresponding to amino acids 190-243; b) a biologically active variant of sequence a) containing at least 70% identity to the above-mentioned sequence; and c) a biologically active fragment of a) or b), wherein the fragment contains at least 10 consecutive amino acids of fragment a), wherein said polypeptide has a length of less than 100 amino acids, and wherein said biological activity is the induction of glucose uptake by cells. The pharmaceutical composition may additionally contain a second active ingredient for the treatment of a metabolic disease and / or cardiovascular disease, selected from the group of known drugs.In this publication, diseases and disorders associated with hyperglycemia and / or insulin resistance include, in particular, metabolic disorders and diseases selected from the group: type 2 diabetes mellitus, obesity, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, cardiovascular diseases (atherosclerosis, ischemic and other heart diseases).

[0033] The disadvantage of the indicated closest analogue is that its use is limited to the correction of endocrine-metabolic disorders in hyperglycemia and / or insulin resistance, including lipid and carbohydrate metabolism disorders in diabetes mellitus and obesity. The effects of the disclosed polypeptide on functional disorders of the liver, kidneys, bone marrow and spleen, locomotor and cognitive activity associated with hyperglycemia and / or insulin resistance remain unstudied.

[0034] Thus, the technical problem, the solution of which is provided by the implementation or use of the present invention, is the expansion of indications for the use of apolipoprotein AI or its functionally significant fragments for the correction of the functional state of a subject suffering from hyperglycemia and / or insulin resistance.

[0035] Disclosure of the essence of the invention

[0036] The general technical result is an expansion of the spectrum of functional disorders amenable to correction in a subject suffering from hyperglycemia and / or insulin resistance. The following are particular technical results: a decrease in functional disorders of the liver, kidneys, bone marrow and spleen, reproductive system organs, an improvement in the contractile function of the myocardium, an improvement in locomotor activity and cognitive function, and an associated improvement in the quality of life.

[0037] The invention relates to a means for treating or preventing hyperglycemia and / or insulin resistance and / or diseases and / or functional disorders of organs and systems associated therewith, comprising human ApoA-1 (ApoA-1) or a functionally significant fragment. Human ApoA-1 has the amino acid sequence SEQ ID NO 1 (amino acid residues 1-243).

[0038] In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group: obesity, type 2 diabetes mellitus.

[0039] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive function.

[0040] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine.

[0041] In one embodiment, the bone marrow dysfunction comprises suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0042] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total macrophage count and apoptosis, decreased immunoregulatory index or CD4 / CD8 ratio, increased cytotoxic lymphocyte and macrophage count.

[0043] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0044] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0045] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0046] In one embodiment, the functional disorders of organs and systems include disorders of cognitive functions of the subject. In one embodiment, said agent includes a functionally significant fragment of human ApoA-I.

[0047] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-I.

[0048] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0049] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

[0050] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0051] The above agent is also intended for use in the treatment and prevention of hyperglycemia and / or insulin resistance and related diseases and functional disorders of organs and systems. The invention also relates to the use of the above agent for the treatment and prevention of hyperglycemia and / or insulin resistance and related diseases and functional disorders of organs and systems.

[0052] In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

[0053] In one embodiment, the use is for the treatment of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

[0054] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive function.

[0055] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine. In one embodiment, the bone marrow dysfunctions include suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0056] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total number and apoptosis of macrophages, decreased immunoregulatory index or CD4 / CD8 ratio, and increased proportion of cytotoxic lymphocytes and macrophages.

[0057] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0058] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0059] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0060] In one embodiment, the functional impairments of organs and systems include impairments of the cognitive functions of the subject.

[0061] In one embodiment, the above agent comprises a functionally significant fragment of human ApoA-I.

[0062] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-I.

[0063] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0064] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

[0065] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0066] The invention also relates to a method for treating or preventing hyperglycemia and / or insulin resistance and / or diseases and functional disorders of organs and systems associated therewith, comprising administering the above agent to a subject in need thereof. In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

[0067] In one embodiment, the method is intended for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

[0068] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

[0069] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine.

[0070] In one embodiment, the bone marrow dysfunction comprises suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0071] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total macrophage count and apoptosis, decreased immunoregulatory index or CD4 / CD8 ratio, and increased cytotoxic lymphocyte and macrophage count.

[0072] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0073] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0074] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0075] In one embodiment, the functional impairments of organs and systems include impairments of the cognitive functions of the subject.

[0076] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an N-terminally truncated amino acid sequence of human ApoA-I.

[0077] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (amino acid residues 3-243).

[0078] In one embodiment, said agent is formulated as a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

[0079] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline. In one embodiment, the administration of the above agent is carried out locally or parenterally.

[0080] In one embodiment, parenteral administration is performed intravenously or intramuscularly.

[0081] In one embodiment, local administration is performed subcutaneously.

[0082] Brief description of the drawings

[0083] Fig. 1. Dynamics of body weight of mice in the control and experimental groups of mice from 1 to 16 weeks of the experiment.

[0084] K(-) - control group of intact healthy ICR mice, K(+) - control group of db / db mice (positive control), experimental group rApoA-1-3-243 - mice of the db / db mouse line that received rApoA-1-3-243. * - p<0.05 - significant difference in relation to the K(+) group.

[0085] Fig. 2. Comparison of body size of db / db J mice in the control and experimental groups.

[0086] A - db / db mice, positive control K(+), B - db / db mice, receiving injection of gApoA-1-3-243 (experimental group gApoA-1-3-243). 16th week of the experiment.

[0087] Fig. 3. Changes in glucose concentration in blood plasma of control and experimental groups of mice.

[0088] To - parameters before the start of the experiment, T1-T5 - control points of glucose measurement. K(-) - intact healthy ICR mice, K(+) - control group of db / db mice (positive control), experimental group hApoA-1-3-243 - db / db mice treated with hApoA-1-3-243. * - p<0.05 - reliable difference in relation to K(+).

[0089] Fig. 4. Dynamics of insulin levels in blood plasma of control and experimental groups of mice.

[0090] To - parameters before the experiment, Ti - 2 weeks after the experiment, Tg - 4 weeks after the experiment, T5 - 16 weeks after the experiment. K(-) - control group of intact healthy ICR mice, K(+) - control group of db / db J mice (positive control), experimental group gApoA-1-3-243 - db / db mice treated with gApoA-1-3-243. * - p<0.05 - significant difference compared to group K(+).

[0091] Fig. 5. Dynamics of cholesterol levels in blood plasma of control and experimental groups of mice.

[0092] To - parameters before the experiment, Ti - 2 weeks after the experiment, Tg - 4 weeks after the experiment, Ts - 16 weeks after the experiment. K(-) - control group of intact healthy ICR animals, K(+) - control group of db / db mice (positive control), experimental group rApo A-1-3-243 - db / db mice treated with rApoA-I. * - p<0.05 - significant difference in relation to group K(+).

[0093] Fig. 6. Dynamics of triglyceride levels in blood plasma of control and experimental groups of mice.

[0094] To - parameters before the start of the experiment, Ti - 2 weeks after the start of the experiment, Tg - 4 weeks after the start of the experiment, Ts - 16 weeks after the start of the experiment. K(-) - control group of intact healthy ICR mice, K(+) - control group of db / db mice (positive control), experimental group g Apo A- 1-3-243 - db / db mice receiving gApoA- 1-3-243.

[0095] Fig. 7. Dynamics of lactate levels in blood plasma of control and experimental groups of mice.

[0096] To - parameters before the experiment, Ti - 2 weeks after the experiment, Tg - 4 weeks after the experiment, Ts - 16 weeks after the experiment. K(-) - control group of intact healthy ICR mice, K(+) - control group of db / db mice (positive control), experimental group rApo A-1-3-243 - db / db mice treated with rApoA-1-3-243. * - p<0.05 - significant difference in relation to group K(+).

[0097] Fig. 8. Photo of mouse spleen, fifth control point (Ts)

[0098] A - control group of intact healthy ICR mice (K-), B - control group of db / db mice (positive control), K(+), C - experimental group of gApoA-1-3-243 - db / db mice treated with gApoA-1-3-243. In Fig. B, the necrotic zone is shown by an oval.

[0099] Fig. 9. Testosterone level in blood plasma of control and experimental groups of db / db mice 2 weeks after the start of the experiment (Me±w).

[0100] Ti - 2 weeks after the start of the experiment. * - p<0.05 - reliable difference in relation to the K(+) group. Implementation of the invention

[0101] Apolipoprotein AI (ApoA-1) is a native (natural) apolipoprotein of blood plasma, which is part of high-density lipoproteins (HDL).

[0102] Hyperglycemia is a clinical symptom indicating an increase in the glucose content in the blood serum compared to the norm. Chronic hyperglycemia in diabetes mellitus is accompanied by damage, dysfunction and failure of various organs and systems.

[0103] Hyperinsulinemia is a clinical syndrome characterized by high levels of insulin in the blood.

[0104] Diabetic cardiomyopathy is a specific pathological change in the cardiovascular system in patients with diabetes, characterized by left ventricular hypertrophy and diastolic dysfunction at an early stage up to overt heart failure with decreased systolic function at later stages [Paolillo S. et al. Heart Fail Clin. 2019. 15(3):341-347].

[0105] Dyslipidemia (DLP) is a condition in which the concentration of lipids and lipoproteins in the blood goes beyond the normal range and can be caused by both acquired (secondary) and hereditary (primary) reasons.

[0106] The immunoregulatory index or CD4 / CD8 ratio is a characteristic of the general dysfunction of the immune system of cytotoxic T lymphocytes CD8 + and T helpers CD 4 + , where cytotoxic T lymphocytes CD8 + - These are T-lymphocytes (a type of white blood cell) that kill damaged cells; T helper CD 4 +- is a type of T-cell that performs the functions of regulating the processes of other cells of the immune system (T-killers, B-lymphocytes, macrophages, NK-cells), recognizes antigens and "makes decisions" about starting or stopping the processes of functioning of the mechanisms of the acquired cellular immune response. The main phenotypic feature of T-helpers is the presence of the CD4 molecule on the cell surface, cytotoxic T lymphocytes - the presence of the CD8 molecule on the cell surface. CD4 is a monomeric transmembrane glycoprotein of the Ig superfamily, CD8 is a transmembrane glycoprotein that serves as a coreceptor for T-cell receptors.

[0107] Insulin resistance is a decrease in the sensitivity of cellular structures to insulin with its sufficient concentration in the blood, leading to chronic compensatory hyperinsulinemia. Quality of life is a reflection of the impact of the disease and the results of treatment on locomotor and cognitive functions, body weight, biochemical, immunological and other indicators reflecting the functional state of various organs and systems. A positive effect of drug therapy on any of the above criteria is considered an improvement in the quality of life.

[0108] Cognitive functions are complex functions of the brain, with the help of which, within the framework of the claimed invention, the process of rational cognition of the structured space in which experimental animals are placed is carried out, and their purposeful interaction with it is ensured.

[0109] Locomotor activity is a type of motor activity associated with the active movement of a subject in space, carried out by the muscular and skeletal systems of the body, which requires energy expenditure.

[0110] Obesity is a chronic disease characterized by excess accumulation of body fat, which poses a health risk and is a major risk factor for a number of other chronic diseases, including type 2 diabetes and cardiovascular disease (CVD).

[0111] Diabetes mellitus (DM) is a group of metabolic diseases characterized by chronic hyperglycemia, which results from impaired insulin secretion, insulin action, or both.

[0112] Type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus, T2DM) is a disorder of carbohydrate metabolism caused by predominant insulin resistance or relative insulin deficiency or predominant insulin secretion disorder with or without insulin resistance. Type 2 diabetes mellitus is expressed in a chronic increase in blood glucose concentration (hyperglycemia).

[0113] Recombinant ApoA-1 is a protein characterized by the amino acid sequence of natural ApoA-1, obtained by genetic engineering by placing the gene of the mature ApoA-1 protein into the genetic material of bacterial, mammalian or yeast cells. These microorganisms can be used as producers of ApoA-1 for medical and research purposes.

[0114] A therapeutically effective amount is a dose of the claimed agent when administered to a subject, sufficient to obtain a result aimed at treating or preventing hyperglycemia and / or insulin resistance and / or related diseases and / or functional disorders of organs and systems. A pharmaceutically acceptable carrier means that the carrier must be compatible with other ingredients of the composition and not cause harm to its recipient, i.e. be non-toxic to cells or mammals in the doses and concentrations in which it is used.

[0115] A functionally significant fragment is a fragment of the complete amino acid sequence of human ApoA-1 (amino acid residues No. 1-243), which has functional autonomy and functional activity, ensuring, as part of the claimed agent, the achievement of the technical results claimed in the invention.

[0116] The invention relates to a means for treating or preventing hyperglycemia and / or insulin resistance and / or diseases and / or functional disorders of organs and systems associated therewith, and includes native or recombinant human ApoA-1 or a functionally significant fragment thereof.

[0117] Human apoA-1 has an amino acid sequence of 243 aa and a molecular weight of 28.5 kDa SEQ ID NO 1 (amino acid residues 1-243). Native human apoA-1 is a component of high-density lipoproteins (HDL) in blood plasma.

[0118] Native human ApoA-1 SEQ ID NO 1 (amino acid residues 1-243, ApoA-1-1-243) can be obtained according to Mills GL et al. [Guidebook to Lipoprotein Techniques. Edited by RH Burden and PH van Knippenberg. - Amsterdam: Elsevier, 2000. - 475 p.] by isolating HDL from human blood plasma by isodense ultracentrifugation in a KBr solution, followed by delipidation of HDL [Cham VE, Knowlee B. RJ Lipid. Res. - 1976. - Vol. 17, No. 2. P. 176-181] and purification [Pykhtina MB et al. Biopharmaceutical Journal. - 2012. - T. 4. - No. 6. - P. 37-45].

[0119] Recombinant human ApoA-1 SEQ ID NO 1 (amino acid residues 1-243, rApoA-1-1-243) can be obtained using genetic engineering methods by constructing recombinant DNA that provides, as part of a plasmid vector, the expression of a synthetic gene comprising a nucleotide sequence encoding chimeric human ApoA-1 in E. coli cells, for example, according to Russian Patent No. 2573930, or in methylotrophic yeast cells Pichia pasloris, for example, according to Russian Patent No. 2605326], followed by obtaining mature human rApoA-1-1-243 by enzymatic hydrolysis of the corresponding chimeric protein, but not limited to the methods indicated. In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

[0120] In one embodiment, the agent is intended for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

[0121] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

[0122] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine.

[0123] In one embodiment, the bone marrow dysfunction comprises suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0124] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total number and apoptosis of macrophages, decreased immunoregulatory index or CD4 / CD8 ratio, and increased proportion of cytotoxic lymphocytes and macrophages.

[0125] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0126] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0127] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0128] In one embodiment, the functional impairments of organs and systems include impairments of the cognitive functions of the subject.

[0129] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-I.

[0130] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0131] In one embodiment, the functionally significant fragment of human ApoA-1 of 241 amino acids in size with a molecular weight of 28.3 kDa has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0132] A functionally significant fragment of human ApoA-I can be obtained using genetic engineering methods by constructing recombinant DNA that provides, as part of a plasmid vector, expression in a bacterial or yeast or other cell of a synthetic gene that has in its composition a nucleotide sequence encoding a functionally significant fragment of human ApoA-I, with subsequent isolation of the recombinant protein synthesized in the cell, encoded by the nucleotide sequence of the synthetic gene, and obtaining on its basis a functionally significant fragment of human ApoA-I using methods known to a specialist, but is not limited to the said genetic engineering method.A functionally significant fragment of human ApoA-I, having the amino acid sequence of human ApoA-1-1-243, shortened from the N-terminus by 2 amino acid residues (gApoA-1-3-243) was obtained by genetic engineering by cloning a synthetic gene, having in its composition the nucleotide sequence of the human ApoA-1-1-243 gene, in E. coli cells, with subsequent isolation of the target protein and its acid hydrolysis to obtain gApoA-1-3-243, as described in the patent according to Russian Federation Patent No. 2573930.

[0133] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0134] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0135] In one embodiment, the pharmaceutical composition comprises human hApoA-1-3-243 and water for injection.

[0136] The invention also relates to the use of human ApoA-1-1-243 or human hApoA-1-1-243 or a functionally significant fragment thereof for the treatment and prevention of hyperglycemia and / or insulin resistance and / or diseases and functional disorders of organs and systems associated therewith. In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

[0137] In one embodiment, the use of said agent for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance is proposed.

[0138] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

[0139] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine.

[0140] In one embodiment, the bone marrow dysfunction comprises suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0141] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total macrophage count and apoptosis, decreased immunoregulatory index or CD4 / CD8 ratio, and increased cytotoxic lymphocyte and macrophage count.

[0142] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0143] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0144] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0145] In one embodiment, the functional impairments of organs and systems include impairments of the cognitive functions of the subject.

[0146] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-I.

[0147] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0148] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0149] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0150] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline solution.

[0151] In one embodiment, the pharmaceutical composition comprises human hApoA-1-3-243 and water for injection.

[0152] The invention also relates to a method for treating or preventing hyperglycemia and / or insulin resistance and / or diseases and / or functional disorders of organs and systems associated therewith, comprising administering said agent to a subject in need thereof in a therapeutically effective amount.

[0153] In one embodiment, the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus. The main features of said diseases are lipid and carbohydrate metabolism disorders. In this case, lipid metabolism disorders include excess body weight, increased triglyceride and low-density lipoprotein cholesterol levels in blood plasma. Carbohydrate metabolism disorders include increased insulin and / or glucose levels in blood plasma.

[0154] In one embodiment, the method is intended for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

[0155] In one embodiment, the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

[0156] In one embodiment, the liver and kidney dysfunctions are selected from the group consisting of abnormalities in blood plasma levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine.

[0157] In one embodiment, the bone marrow dysfunctions include suppression of bone marrow cell proliferation and bone marrow cell apoptosis.

[0158] In one embodiment, the spleen dysfunction is selected from the group consisting of impaired organ integrity of the spleen, decreased total macrophage count and apoptosis, decreased immunoregulatory index or CD4 / CD8 ratio, and increased cytotoxic lymphocyte and macrophage count.

[0159] In one embodiment, the myocardial dysfunction comprises a myocardial contractile dysfunction.

[0160] In one embodiment, the reproductive dysfunction includes decreased serum testosterone levels.

[0161] In one embodiment, the functional impairments of organs and systems include impairments of the subject's locomotor activity.

[0162] In one embodiment, the functional impairments of organs and systems include impairments of the cognitive functions of the subject.

[0163] In one embodiment, said agent comprises a functionally significant fragment of human ApoA-I.

[0164] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus (SEQ ID NO: 1, amino acid residues 1-243, ApoA-1-1-243), which has functional autonomy and functional activity that ensures, as part of the claimed agent, the technical results claimed in the invention.

[0165] In one embodiment, the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-I truncated at the N-terminus by 2 amino acid residues (SEQ ID NO: 2, amino acid residues 3-243, ApoA-1-3-243).

[0166] In one embodiment, the above agent is formulated as a pharmaceutical composition comprising human ApoA-1-1-243 or hApoA-1-1-243 or a functionally significant fragment thereof, and a pharmaceutically acceptable carrier.

[0167] In one embodiment, the pharmaceutically acceptable carrier is water for injection or saline. In one embodiment, the pharmaceutical composition comprises human rApoA-1-3-243 and water for injection.

[0168] In one embodiment, the administration of the claimed agent is carried out locally or parenterally.

[0169] In one embodiment, parenteral administration is performed intravenously or intramuscularly.

[0170] In one embodiment, local administration is performed subcutaneously.

[0171] The duration of administration and the therapeutically effective amount of the administered claimed agent may vary widely depending on a variety of factors, including the severity of hyperglycemia and, therefore, the severity of insulin resistance, obesity, functional disorders of organs and systems, subject parameters, including size, weight, age, and other factors well known to a person skilled in the art.

[0172] A subject with signs of hyperinsulinemia and / or insulin resistance and / or associated diseases and functional disorders of organs and systems is a mammal.

[0173] EXAMPLES

[0174] All examples of the invention are carried out using a functionally significant fragment of ApoA-I, having the amino acid sequence of human ApoA-1-1-243, shortened from the N-terminus by 2 amino acid residues (rApoA-1-3-243).

[0175] Example 1. Obtaining a functionally significant fragment of human ApoA-I

[0176] A functionally significant fragment of human ApoA-I corresponding to amino acids 3-243 of human ApoA-I (SEQ ID NO 2) (hereinafter referred to as "gApoA-1-3-243") was obtained by genetic engineering as described in Russian Patent No. 2573930. In particular, recombinant DNA including the nucleotide sequence encoding chimeric ApoA-I and providing its synthesis in E. coli cells was cloned as part of a plasmid vector. Chimeric ApoA-I was isolated and purified. To obtain gApoA-1-3-243, the chimeric ApoA-I protein was subjected to acid hydrolysis, [deleted because I assume this is taken from the patent]

[0177] Example 2. Description of experiments.

[0178] Description of the mouse strain used for the experimental implementation of the claimed invention The implementation of the invention is shown in Examples 3-11 on an experimental model of hyperglycemia and insulin resistance, which used mice of the BKS.Cg-Dock7 strain. m Leprosy db / + / + / J (hereinafter referred to as db / db mice). To implement the invention, db / db mice with hyperinsulinemia, insulin resistance, type 2 diabetes, obesity and intact healthy ICR mice were used as a negative control.

[0179] Outbred (non-linear) ICR laboratory mice, which are the most frequently used research objects in the fields of oncology, toxicology, teratology, vaccine development, aging studies and for general purposes, were used as one of the controls.

[0180] The db / db mouse is a widely used model for studying obesity and T2DM. They carry a genetic mutation in the leptin receptor. Homozygous mice of this line used in the study are characterized by progressive obesity at the age of three to four weeks and the development of T2DM, pronounced hyperglycemia from the 4th to 8th week of life and the development of organ damage after the 8th to 10th week. An increase in the insulin level in the blood plasma begins at the age of 10 to 14 days. Exogenous insulin does not control the blood glucose level, and the activity of gluconeogenesis enzymes increases. Wound healing is slowed down, and the efficiency of metabolism increases. Homozygous females are characterized by a decrease in the weight of the uterus and ovaries, a decrease in the secretion of hormones by the ovaries and pronounced lipid inclusions in the follicles and endometrium. Impairments in cellular immunity in db / db mice and ICR controls were investigated both in vivo and in vitro.There is a significant suppression of delayed footpad edema and rejection time of the first and second installed skin allografts. In addition, DNA synthesis in spleen cells after nonspecific mitogen stimulation is markedly suppressed. Animals with T2DM with an average blood glucose level of 512 ± 101 mg / 100 ml do not respond to exogenous insulin therapy by lowering the blood glucose level, i.e., they are characterized by insulin resistance. In db / db mice, cell-mediated immune mechanisms are markedly suppressed [Yoon JW et al. Diabetes. 1988. 37 (9): 1287-93 .; Mandel MA et al. J Immunol. 1978. 120 (4): 1375-1377 .; Do, OH et al. Diabetologia. 2016. 59, 1222-1230. Conn PM Academic Press, Elsevier Inc. 2017:1175].

[0181] Thus, according to the above research data, db / db mice develop hyperinsulinemia, insulin resistance caused by a genetic mutation, and obesity and type 2 diabetes associated with it at the above-mentioned time points from birth. To study the effectiveness of the claimed agent, intact healthy ICR mice and db / db mice aged 6 weeks at the start of the experiment were used.

[0182] Data on experimental mice are presented in Table 1.

[0183] Table 1. Data on experimental animals

[0184] Means

[0185] The agent used was a functionally significant fragment of human ApoA-1-3-243 obtained in Example 1, formulated in the form of a pharmaceutical composition including a functionally significant fragment of human ApoA-I, hApoA-1-3-243, and a pharmaceutically acceptable solvent, in which water for injection was used as the pharmaceutically acceptable solvent.

[0186] Experimental methodology

[0187] db / db mice, like intact healthy ICR mice, were fed a normal diet with free access to water.

[0188] Three groups of experimental animals were formed:

[0189] K(-) - control group of intact healthy ICR mice, 10 individuals;

[0190] K(+) - control group of db / db mice with hyperglycemia, insulin resistance, and development of obesity and T2DM within a few weeks after birth.

[0191] The hApoA-1-3-243 group is an experimental group of db / db mice with hyperglycemia, insulin resistance, and the development of obesity and T2DM within a few weeks after birth.

[0192] Control mice were injected subcutaneously (into the withers area) with saline solution in an equivalent volume relative to the experimental group of gApoA-1.

[0193] The experimental group was administered hApoA-1-3-243 subcutaneously (into the withers area) at a dose of 7 mg / kg body weight, while hApoA-I was administered as part of a pharmaceutical composition in which water for injection was used as a pharmaceutically acceptable carrier, at a concentration of hApoA-1-3-243 in solution of 1.2 mg / ml.

[0194] T1 The experimental and control groups of db / db mice included 23 individuals in each group. Animals were distributed randomly among the groups.

[0195] Table 2 shows the schedule of manipulations and injections of hApoA-1-3-243 into experimental db / db mice or saline into control mice of this line.

[0196] Table 2 shows the sampling points for biomaterials to assess the indices of organ and system functions. In addition to the initial (To), the sampling points for biochemical blood indices were Ti - after 2 weeks, Tg - after 4 weeks, T3 - after 8 weeks, T4 - after 12 weeks, and T5 - after 16 weeks after the start of the experiment.

[0197] Based on the results of weekly weighing, the dynamics of body weight of experimental and control mice was determined during weeks 1–16 of the experiment (example 3).

[0198] Blood samples were collected from the tail vein to determine plasma glucose, insulin (sample 4), high-density lipoprotein cholesterol (HDL-C), triglycerides (sample 5), alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, creatinine (sample 7), lactate (sample 6), and testosterone (sample 10). The measurement points for blood biochemical parameters are listed in Table 2.

[0199] Bone marrow samples were used to assess proliferation and apoptosis of bone marrow cells (Example 8).

[0200] The spleen and its samples were used to assess splenic organ integrity, total macrophage count, macrophage apoptosis, immunoregulatory index, or CD4 / CD8 ratio (Example 9).

[0201] The methods and results of assessing locomotor and cognitive activity are described in Example 6.

[0202] The contractile function of the myocardium was studied using an isolated heart model (example 11).

[0203] Table 2. Schedule of manipulations and injections

[0204] Example 3. Effect of hApoA-1-3-243 on body weight of db / db mice

[0205] Body weight was assessed weekly on a Navigator Ohaus scale, USA, throughout the experiment. Data are presented as M ± w. Since db / db mice carry a spontaneous genetic mutation in the leptin receptor, body weight gain occurs mainly due to the growth of adipose tissue [Do O. H. et al. Diabetologia. 2016. Vol. 59, P. 1222-1230; Conn PM Animal Models for the Study of Human Disease (Second Edition), Academic Press, Elsevier Inc. 2017: 1175]. In this regard, the state of obesity was recorded by the increase in body weight relative to intact healthy ICR animals (Fig. 1).

[0206] The dynamics of body weight relative to the starting point of the experiment was assessed. As a result, it was shown that the dynamics of body weight throughout the experiment had a linear ascending trend in both experimental groups of db / db mice: the control group K(+) and the experimental group g Apo A-1-3-243. Moreover, after the first week of the experiment, the weight of animals in the experimental group gApoA-1-3-243 was reliably lower by 7% relative to the control group of intact healthy ICR animals. This trend persisted throughout the experiment and by week 16 the decrease in the experimental group gApoA-1-3-243 was 15.2% relative to the K(+) group.

[0207] Figure 2 shows the appearance of db / db mice:

[0208] A - K(+), B - group gApoA-1-3-243. 16th week of the experiment. Visually it is seen that the mouse from the gApoA-I group is smaller in size.

[0209] Thus, it was shown that the administration of hApoA-1-3-243 at a dosage of 7 mg / kg throughout the experiment led to a decrease in body weight in db / db mice.

[0210] Example 4. The effect of hApoA-1-3-243 on the state of hyperglycemia and hyperinsulinemia

[0211] Glucose control was carried out by spectrophotometric method using kits from VectorBest, Russia.

[0212] Insulin level monitoring was performed using the ELISA method, VectorBest, Russia. The experiment was started with animals at the age of 8 weeks after confirmation of hyperglycemia and hyperinsulinemia.

[0213] In addition to the initial (To) point, the points of biomaterial collection Tc Tg and Ts, as well as two intermediate points Tz, T4 (Fig. 3) were taken as the points of measurement of glucose concentration in the blood plasma of db / db mice. The assessment of the glucose concentration in the blood plasma of db / db mice showed a reliable decrease in the experimental group of gApoA-1-3-243 at points Ti and T2 by 64% and 37%, respectively, in relation to the control group of mice (positive control K(+)). However, these indicators were higher than the corresponding indicators of intact healthy ICR animals (K(-)). In the time interval, points Ti and T2 correspond to 2 and 4 weeks. It is worth noting that in the experimental group of gApoA-1-3-243, animals with glucose concentration within 6, 7-8, 2 were identified, whereas such effects were not observed in the K(+) group. The state of hyperinsulinemia was recorded in relation to intact healthy ICR mice (Fig. 4). At baseline, insulin levels in db / db mice were 6.7 times higher than those in the intact ICR mice group.Over time, the insulin level in db / db mice in both groups showed a gradual decrease and by week 16 was 10% lower than the initial level, but the state of hyperinsulinemia persisted.

[0214] Thus, it can be concluded that g Apo A-1-3-243 contributes to the reduction of hyperglycemia by increasing glucose uptake in db / db mice, bypassing the insulin resistance mechanism.

[0215] Example 5. The effect of gApoA-1-3-243 on lipid metabolism parameters

[0216] Evaluation of the cholesterol level in the blood plasma showed a reliable decrease in the gApoA-I group by 8.6% relative to K(+) at the first control point (Ti) (Fig. 5). However, the indicator then gradually increased and at week 16 in the gApoA-1-3-243 group it was higher by 11.3% relative to K(+).

[0217] Moreover, compared to the control group of intact healthy ICR K(-) mice, in both groups of db / db mice (control group K(+) and experimental group gApoA-1-3-243), cholesterol was almost 2 times higher throughout the entire experiment.

[0218] The cholesterol level in the control group of intact healthy animals ICR K(-) was 1.02±0.063 mmol / l. The data of the experimental group of gApoA-1-3-243 showed 1.11±0.098 mmol / l, which was 8.2% higher relative to K(-). At the same time, in the control group of db / db mice K(+), the cholesterol level in the control group was 1.395±0.079 mmol / l, which was 26.9% higher relative to K(-) and 20.5% higher relative to the experimental group of gApoA-1-3-243. Thus, gApoA-1-3-243 promotes a decrease in the cholesterol level in the blood plasma of db / db mice.

[0219] The change in the triglyceride (TG) level throughout the experiment is shown in Fig. 6. It is shown that in the control and experimental groups of db / db mice the indicator significantly exceeds the data in the control group of intact healthy ICR K(-) animals at all control points. Comparison of the experimental group gApoA-1-3-243 and the control group K(+) showed that at the first control point (Ti) the TG level in the gApoA-1-3-243 group is reliably higher by 16.9% relative to K(+). Then there is a decrease in both groups, and at T3 the TG level in the gApoA-1-3-243 group is 23.8% lower than the value in K(+).

[0220] Thus, administration of 7 mg / kg of gApoA-1-3-243 to db / db mice has a positive effect on triglyceride and LDL cholesterol levels during long-term use. Based on the data on the decrease in triglyceride and LDL cholesterol levels and the increase in total cholesterol in the gApoA-1-3-243 group of mice, it can be argued that the increase in total cholesterol occurs due to an increase in the volume of the HDL fraction. This effect of gApoA-1-3-243 can be considered as antiatherogenic and cardioprotective.

[0221] The results of the study of lipid metabolism allow us to conclude that lipid metabolism disorders in db / db mice are reduced under the influence of gApoA-1-3-243.

[0222] Example 6. Effect of hApoA-1-3-243 on locomotor and cognitive functions

[0223] Table 3 shows the effect of gApoA-1-3-243 on the locomotor function indices of db / db mice. It is shown that the experimental gApoA-1-3-243 group and the control K(+) group of db / db mice have impaired locomotor function in all indices relative to the control group of healthy K(-) mice. In the K(+) group there are 4 positions with a complete absence of the sign, while in the gApoA-1-3-243 group there are only two. Further comparison of the db / db mouse groups revealed the following differences. Mice in the gApoA-1-3-243 group started the first movement three times faster. They visited 2.17 times more squares, and in this group there were single wall rearings, while in the K(+) group they were completely absent. These data indicate that administration of hApoA-1-3-243 improves locomotor function in db / db mice. HApoA-1-3-243 mice had more frequent exits back to the center and 1.73 times more visits to the dummy burrows, indicating improved cognitive performance.

[0224] Table 3. Indicators of locomotor and cognitive functions in the control and experimental groups of mice after 16 weeks of the experiment

[0225] Note: K(-) - intact healthy ICR mice, K(+) - control group of db / db mice (positive control), experimental group gApoA-1-3-243 - db / db mice treated with gApoA-1

[0226] * - p<0.05 - significant difference in relation to group K(+)

[0227] # - p<0.05 - reliable difference in relation to group K(-)

[0228] Indirect confirmation of the data on the improvement of locomotor function is the lactate level in the blood plasma. Figure 7 shows that the lactate level in the db / db mouse groups is almost 1.5 times lower relative to K(-). At the same time, a comparison between the experimental group gApoA-1-3-243 and the control group K(+) shows a splitting of the two curves starting from T in favor of the experimental group gApoA-1-3-243. By the 16th week of the experiment, there is a reliable increase in this indicator by 14.4% in the experimental group gApoA-1-3-243 relative to the control group K(+), which confirms the improvement of locomotor function. Example 7. The effect of gApoA-3-243 on biochemical indicators of the functional state of the liver and kidneys

[0229] Analysis of biochemical indices of liver function (Table 4) indicates that both groups of db / db mice have liver dysfunction in all indices. Comparison of the experimental group of gApoA-1-3-243 and the control group of K(+) db / db mice revealed some peculiarities. With the same level of aspartate aminotransferase (AST), the alanine aminotransferase (ALT) index in the experimental group of gApoA-1-3-243 was 25.6% lower compared to the control group K(+). The gamma-glutamyl transferase (GGT) index was also reduced by 33.4%. At the same time, the alkaline phosphatase activity in the experimental group of gApoA-1-3-243 was 37.8% higher compared to the control group K(+). These data indicate that liver function in db / db mice in the experimental group gApoA-1-3-243 is less impaired compared to the control group K(+). The alkaline phosphatase index requires more detailed study, since this enzyme is related not only to liver function but also to damage or functional reorganization of bone tissue.

[0230] The blood creatinine level (Table 4) indicates renal impairment in both db / db mice. In the experimental group gApoA-1-3-243, this level was 26.18% lower than in the control group K(+). This indicates a less pronounced renal impairment in the experimental group gApoA-1-3-243 compared to the control group K(+).

[0231] Table 4. Effect of hApoA-1-3-243 on biochemical parameters of blood plasma of mice 16 weeks after the start of the experiment - Ts

[0232] Note: K(-) - intact healthy ICR mice, K(+) - control group of db / db mice (positive control K(+)), experimental group gApoA-1-3-243 - db / db mice treated with gApoA-1-3-243. * - p<0.05 - significant difference compared to group K(+), # - p<0.05 - significant difference compared to group K(-).

[0233] Thus, hApoA-1-3-243 has a hepatoprotective and nephroprotective effect, thereby reducing functional disorders of the liver and kidneys.

[0234] Example 8. Analysis of the functional state and qualitative composition of bone marrow.

[0235] Pathological events affecting the bone marrow contribute to cardiovascular complications in people with diabetes. Fadini and colleagues demonstrated that levels of circulating proangiogenic bone marrow-derived cells inversely correlate with coronary, peripheral, and cerebrovascular complications in patients with diabetes [Fadini GP et al. Stem Cells 2017. Vol. 35. NIP 106-116.].

[0236] It is known that cardiovascular risk factors, including T2DM, can have a negative impact on both the number and recruitment of circulating progenitor cells (PCs). Recent data suggest that less differentiated CD 133(+) PCs promote myocardial healing and are promising candidates for therapy. It has been shown that T2DM not only limits the number of CD133(+) after acute myocardial infarction, but also limits their activation. This can be explained by lower resistance of CD133(+) to oxidative stress [Voo S. et al. J Intern Med. 2009. Vol. 265. N.2. P.238-249.].

[0237] CD133 is a marker that is often found on multipotent progenitor cells, including immature hematopoietic stem and progenitor cells. CD 133 has been selected as one of the markers to detect bone marrow abnormalities in diabetes mellitus and to assess the efficacy of the claimed agent in reducing these abnormalities.

[0238] To analyze the effect of the claimed agent, hApoA-1-3-243, on the bone marrow of db / db mice, hApoA-I was administered subcutaneously to the mice according to the scheme presented in Table 2. At the indicated time points, the animals were taken out of the experiment by the method of cervical dislocation of the spinal cord in the cervical region. Bone marrow was isolated from the femur by the standard method [Goldberg E.D. et al., Methods of tissue culture in hematology. Tomsk. - 1992]. The bones were washed in cold phosphate buffer (PBS), the epiphyses were cut off and the bone marrow was carefully washed out using a syringe into test tubes containing PBS with 2% fetal calf serum (FCS). The cells were carefully suspended in 1 ml of this buffer and sedimented by centrifugation (300 g, 7 min). The pellet was resuspended in PBS buffer and used for staining with monoclonal antibodies to detect bone marrow stem cells and macrophages.To determine the proliferative activity of the BMC, the obtained cells were fixed with cold 70% ethanol until staining for at least 4 hours. After fixation, the cells were pelleted by centrifugation (400 g, 7 min), washed with PBS, and incubated for 5 minutes in a hypotonic low molecular weight DNA extraction buffer to determine the peak of apoptotic cells. After washing, the cells were stained in the dark for 30 minutes in a staining buffer of -50 pg / mL propidium iodide (PI, Sigma, USA) and 200 pg / mLPHKa3bi-A (Invitrogen, USA). The number of cells with different DNA content in the SubGl, G0 / G1, S, G2 / M cell cycle phases was estimated by flow cytometry, measuring the fluorescence of propidium iodide at LEt = 670 nm.

[0239] The bone marrow cell suspension was analyzed by flow cytometry (CYTOFLEX S100, BeckmanCoulter, USA) using monoclonal antibodies PE / Cy7 anti CD117 (ab95576 Abeam, United Kingdom); APC anti-mouse CD133 (397906, BioLegend, USA) and Per CP5.5 anti - mouse Sca-1 (108124, BioLegend, USA), FITC anti - mouse F4 / 80 (123108, BioLegend, USA). Cell samples (2xl0 6 ) in 10 μl were stained with fluorescently labeled antibodies for 30 min at room temperature in the dark. They were washed twice with PBS and analyzed on a flow cytometer. At least 10,000 events were collected per sample. Dead cells (apoptosis, debris) were determined by analyzing flow cytometry cytograms in FSC / SSC coordinates by selecting gates (cell populations) based on the size / granularity parameters; cells located near the zero point and forming a finely dispersed pool were considered dead. Also, the percentage of apoptotic cells was determined by the peak of hypodiploids during cell cycle analysis.

[0240] It was shown that the number of bone marrow cells with the CD 133 marker in the experimental group gApoA-1-3-243 was 5.7% higher than in the control group K(+) (Table 5). At the same time, apoptosis of CD 133 cells in the experimental group gApoA-1-3-243 was 11.1% lower than in the control group K(+), but these data did not differ significantly. Data for T3 and T3 were missing because the values ​​at these points were at zero in both groups. These data indicate that gApoA-1-3-243 exhibits a stimulating effect on stem cell proliferation at early stages of therapy. Table 5. Change in data on the total number of cells and cells in the apoptosis of the CD 133 population in the bone marrow of mice, in % of the total pool of studied cells, (M±w).

[0241] Note: K(+) - control group of db / db mice, hApoA-1-3-243 - experimental group of db / db mice that were administered hApoA-1-3-243.

[0242] * - p<0.05 - significant difference in relation to group K(+).

[0243] Sca-1 was chosen as the second marker for identifying bone marrow abnormalities in T2DM. Sca-1 is known to be a stem cell marker. Sea-1-containing resident vascular stem cells have been shown to be a regenerative pool for cardiomyocytes and also participate as regenerative cells of vascular smooth muscles [Shizuka U., et al. Stem Cell Reports. 2013. Vol. 1. N.5. P. 397-410.; Tang J. et al. Cell Stem Cell. 2020. Vol. 26. NIP 81-96.]. High glucose levels are known to trigger apoptosis of Scal+ progenitor cells in a dose- and time-dependent manner [Yang P. et al Biochem Biophys Res Commun. 2017. Vol. 482. N. 4. P. 575-581.].

[0244] In the study conducted at the first point, the number of Sca-1 containing bone marrow cells in the HApoA-1-3-243 group was 87.5% higher relative to the K(+) group (Table 6).

[0245] Table 6. Changes in the total number of cells and cells in the apoptotic population, Sca-1 in the bone marrow of mice, data are presented as % of the total pool of cells studied, (M±w).

[0246] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db mice, rAnoA-I-3-243 - experimental group of db / db mice that were administered rAnoA-1-3-243.

[0247] * - p<0.05 - significant difference in relation to group K(+).

[0248] At point Tg, a decrease in the growth of Sca-1-containing bone marrow cells to 20.4% is observed. At the fifth control point - Ts, the difference in the number of Sca-1-containing bone marrow cells was also in favor of the gApoA-1-3-243 group and amounted to 52.6%. Thus, throughout the experiment, gApoA-1-3-243 maintained the pool of Sca-1-containing bone marrow cells at a higher level. This fact suggests that gApoA-1-3-243, through stimulation of Sca-1-positive cells, can have a protective effect on the cardiovascular system in type 2 diabetes.

[0249] CD117 has been selected as a third marker to detect bone marrow abnormalities in type 2 diabetes + (with -kit). Impaired angiogenesis is a common feature of diabetes mellitus, while damage to CD 117+ bone marrow cells may be the cause of this complication. CD117 +(c-kit) is a receptor for stem cell factor (SCF) and plays an important role in early hematopoiesis. The CD117 cell population + considered suitable for organ regeneration, including the heart and pancreas [Liu Yunying et al. Cell Regeneration 2013. Vol. 2. NlP 1-12.; Marino F. et al. Frontiers in endocrinology / 2019. N.10. P.371.]. CD117 + (c-Kit) is a receptor for colony-stimulating growth factor (CSF).

[0250] The study showed that gApoA-1-3-243 has a clearly expressed stimulating effect on the number of cells expressing the SCF receptor, which in turn can have a positive effect on regenerative processes in the myocardium and pancreas. The reliable difference in Ti was 1.6 times, in Tg - 1.3, and in Ts - 1.36 times in favor of gApoA-1-3-243. Thus, this effect was maintained throughout the experiment.

[0251] The obtained data on CD117 apoptosis + In the first two control points, it was shown that the number of cells in a state of apoptosis in the gApoA-1-3-243 group was 1.53 times significantly lower relative to K(+). But in Tz, this indicator in the gApoA-1-3-243 group was 1.23 times significantly higher relative to K(+).

[0252] Reduction in the number of apoptotic cells CD117 + can be assessed as a cytoprotective effect, which has a positive effect on regenerative processes in the myocardium, as well as in the pancreas in mice with obesity and type 2 diabetes (Table 7). Table 7. Changes in the data of the total number of cells and cells in apoptosis of the CD117 population + , in the bone marrow of db / db mice, as a % of the total pool of cells studied, (M±w).

[0253] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db mice, hApoA-1-3-243 - experimental group of db / db mice that were administered hApoA-1-3-243.

[0254] * - p<0.05 - significant difference in relation to group K(+).

[0255] As a fourth marker for detecting bone marrow abnormalities in T2DM, F4 / 80+ macrophages were selected. It is known that the total cell count and immune function of F4 / 80+ macrophages are significantly impaired in mice with long-term exposure to a diabetic environment, which may be a mechanism responsible for the increase in macrophage-related complications in diabetic patients, such as high prevalence of infections and cardiovascular mortality [Ma H. J Mol Med (Berl). 2008. Vol. 86. N.4. P.391-400.].

[0256] When studying the effect of the declared hApoA-1-3-243 on F4 / 80+ macrophages from the bone marrow of db / db mice, the following results were obtained.

[0257] The number of macrophages in the experimental group gApoA-1-3-243 was significantly higher relative to the control group K(+) (Table 8). The increase was in Ti-308%, T2-180.3% and Ts-164.8% (Table 7). At the same time, the values ​​of the number of macrophages in the state of apoptosis at the first points Ti and T2 in the gApoA-1-3-243 group were 1.53 times significantly lower relative to K(+). Whereas in Ts the values ​​in gApoA-1-3-243 and K(+) were equal.

[0258] It is known that a decrease in the number of macrophages in the bone marrow negatively affects the hematopoietic system [Klei Th.RL et al. Frontiers in Immunology. 2017. N.8. P. 73.]. The obtained data indicate that hApoA-1-3-243 can have a protective effect on the hematopoietic system in db / db mice with obesity and T2DM. Table 8. Changes in the data of the total number of cells and cells in the apoptosis population of F4 / 80+ in the bone marrow of mice, data are presented as a percentage of the total pool of studied cells, (M±w)

[0259] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db mice, hApoA-1-3-243 - experimental group of db / db mice that were administered hApoA-1-3-243.

[0260] * - p<0.05 - significant difference in relation to group K(+).

[0261] Evaluation of the cell cycle phases of the total bone marrow cell pool using propidium iodide showed that in mice of the experimental group gApoA-1-3-243, the cell cycle phases were higher compared to the control group K(+). The increase was in G1 by 24.6%, in S by 59.6%, in G2 / MT by 42.2% (Table 9). Whereas apoptosis (SubGl) in the gApoA-1-3-243 group was significantly lower by 1.42 times compared to K(+).

[0262] This fact also explains the higher number of cells carrying stem markers in the gApoA-1-3-243 group. The obtained data indicate that gApoA-1-3-243 has a stimulating effect on proliferative processes in bone marrow cells of db / db mice with obesity and T2DM, thereby reducing functional disorders of the bone marrow.

[0263] Table 9. Phases of the cell cycle of bone marrow cells, % of the total pool of studied cells at week 16 of the experiment (Ts), (M±w) Note: K(+) - control group of db / db mice, hApoA-1 - experimental group of db / db mice that were administered hApoA-1-3-243.

[0264] * - p<0.05 - significant difference in relation to group K(+).

[0265] Example 9. Analysis of the functional state and qualitative composition of the spleen

[0266] To analyze the effect of the declared hApoA-1-3-243 on the spleen of db / db mice with type 2 diabetes, the animals were removed from the experiment by craniocervical dislocation, and the spleens of the animals were removed whole, photographed and weighed. A suspension of splenocytes was obtained by homogenizing the spleen by gentle crushing in a glass homogenizer in cold PBS buffer containing 2% FBS. Red blood cells were removed by osmotic shock to prepare samples for antibody staining. A suspension of spleen cells (2x10 6The cell count (cell count) was analyzed by flow cytometry (CYTOFLEX S100, BeckmanCoulter, USA) using monoclonal antibodies FITC anti-mouse F4 / 80 (123108, BioEegend, USA), PE anti-mouse CD8 (ab95772, Abeam, United Kingdom), APC anti-mouse CD4 (ab 18280, Abeam, United Kingdom), VP450 anti active cas3 (BD ​​biosciences, USA), CellEvent™ Caspase-3 / 7 Green Detection Reagent manufactured by Molecular Probes (USA). The assessment of dead cells, including apoptotic cells and debris, was performed similarly to the analysis of bone marrow cells (Example 7). Additionally, cells expressing caspases 3.7 were determined at point T2. At point T3, cells with active caspase 3 were determined on all splenocytes, T lymphocytes and macrophages.

[0267] Fig. 8. Shows photographs of the spleen of animals at the fifth control point (week 16 of the experiment). Upon external examination, it was noted that the organs in both groups of db / db mice were significantly smaller in relation to the spleen of the control group of intact healthy ICR mice.

[0268] Comparison of db / db mice, the hApoA-1-3-243 and K(+) groups indicates that in the hApoA-1-3-243 group the spleen has a fairly uniform, smooth structure, normal (scarlet) color, whereas in the K(+) group the spleen is drier, pale with black areas (presumably necrosis).

[0269] Comparison by weight showed that the spleen in the gApoA-1-3-243 group was 35.85% larger than in the K(+) group (Table 10). These data indicate that gApoA-1-3-243 in the group helps maintain organ integrity and spleen function in db / db mice with obesity and T2DM. Table 10. Dynamics of spleen weight in the experimental gApoA-1-3-243 group and control groups of mice, (M±m)

[0270] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment; K(-) - control group of intact healthy ICR mice, K(+) - control group of db / db mice (positive control K(+)), experimental group hApoA-1-3-243 - db / db mice treated with hApoA-1-3-243.

[0271] * - p<0.05 - significant difference in relation to group K(+);

[0272] # - p<0.05 - significant difference in relation to group K(-).

[0273] Analysis of the macrophage pool in the spleen showed that in all three points the number of macrophages in the gApoA-1-3-243 group was significantly higher in the first two control points relative to the K(+) group (Table 11). The increase was -220.3% in Ti and 148.6% in T2. ​​At point Ts the increase was 122.8%, but due to the large spread of values ​​it was not reliable. At the same time, the values ​​of the number of macrophages in the apoptotic state at points Ti and T2T3 in the gApoA-1-3-243 group, compared to the K(+) control group, were lower by 2.1, 1.63 and 1.69 times, respectively.

[0274] Thus, gApoA-1-3-243 stimulates the macrophage system of the spleen throughout the experiment, which in turn can have a regenerative, anti-inflammatory and immunomodulatory effect on the body of animals with obesity and type 2 diabetes. Table 11. Changes in the data of the total number of cells and cells in apoptosis of the population

[0275] F4 / 80+ macrophages in the spleen of mice, in % of the total pool of cells studied, (M±w)

[0276] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db mice, rApoA-1-3-243 - experimental group of db / db mice that were administered rApoA-1-3-243.

[0277] * - p<0.05 - significant difference in relation to group K(+).

[0278] Table 12 presents the results of determination of active caspase 3 in lymphocytes and macrophages of the spleen, which plays a central role in cellular apoptosis, in control and experimental db / db mice 16 weeks after the start of the experiment.

[0279] The data reflect a significant difference in the number of cytotoxic lymphocytes and macrophages with active caspase 3 in the control and experimental groups of db / db mice. The number of cytotoxic cells positive for caspase 3 in the gApoA-1-3-243 group was significantly lower relative to the K(+) group for both lymphocytes and macrophages.

[0280] Table 12. Percentage of cytotoxic T lymphocytes CD8 + , T helpers CD 4 + and macrophages F4 / 80 + , carrying active caspase 3 (cas3), in control and experimental groups of db / db mice 16 weeks after the start of the experiment

[0281] Note: K(+) - control group of db / db mice, rAnoA-1 - experimental group of db / db mice injected with rAnoA-I-3-243. CD4 + - T-lymphocyte marker, CD8 +- T-helper marker (a type of T-lymphocyte); F4 / 80+ - macrophage marker. The introduction of rAnoA-I-3-243 throughout the experiment not only reduces the number of cytotoxic cells with active caspase 3 and spleen cell apoptosis, but also maintains a certain average level of expression of the sign for both T lymphocytes and macrophages. In the control group, at week 16 of the experiment, animals with total cell death appeared, determined by a high percentage of cytotoxic T lymphocytes and macrophages carrying active caspase 3. In the group of mice with the introduction of the protein gApoA-1-3-243, at this time, there is an insignificant percentage of cells with active caspase 3. Thus, gApoA-1-3-243 maintains the viability of splenocytes, reducing the percentage of cells with pronounced active caspase 3, and therefore contributing to the preservation of the spleen as an important immune and lymphatic drainage organ.

[0282] The immunoregulatory index or CD4 / CD8 ratio largely characterizes the general dysfunction of the immune system. This indicator can be used as a biomarker of disease progression or response to treatment. A decrease in CD4 / CD8 indicates the development of inflammatory processes in the body. It was shown that at point Ti, CD4 / CD8 was 5% higher in the gApoA-1-3-243 group relative to the K(+) group. At point Tg, the indicators of both groups were almost equal, and at point Ts in the K(+) group, the indicator was below one, indicating a shift in the immune response towards cytotoxic (inflammatory) processes. The CD4 / CD8 indicator in the gApoA-1-3-243 group had a vector towards inflammation, but at Ts it was greater than one and 1.33 times higher relative to the K(+) group (Table 13).

[0283] Table 13. Changes in the total number of CD4 / CD8 cells in the spleen of mice, data are presented as % of the total pool of cells studied, (M±w)

[0284] Note: Ti - 2 weeks after the start of the experiment; T2 - 4 weeks after the start of the experiment; Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db mice, rAnoA-I-3-243 - experimental group of db / db mice administered rAnoA-1-3-243.

[0285] * - p<0.05 - significant difference in relation to group K(+).

[0286] The obtained data indicate that r Apo A-1-3-243 can exert immunomodulatory and anti-inflammatory effects in db / db mice with obesity and type 2 diabetes, thereby reducing functional impairment of the spleen.

[0287] Example 10. Evaluation of the effect of hApoA-1-3-243 on the level of testosterone in blood plasma as an indicator of reproductive function

[0288] The study conducted on db / db mice showed that plasma testosterone levels in the HAPOA-1-3-243 group were 5-fold higher than in the K(+) group in Ti (Fig. 9). Data for T3 and T3 were not fully available.

[0289] The results of the Ti-point indicate that early administration of hApoA-1-3-243 helps maintain testosterone levels in obese db / db mice with T2DM, thereby reducing reproductive dysfunction.

[0290] Example 11. Study of myocardial contractile function as an additional factor in the cardioprotective function of gApoA-1-3-243

[0291] The contractile function of the myocardium and the possibility of its correction using the declared gApoA-1-3-243 were studied in db / db mice.

[0292] The experiments were carried out on an isolated heart model.

[0293] Description of the method for assessing the reserve contractile function of the myocardium

[0294] After decapitation of the head, the skin was removed from the chest of the animals and access to the heart was performed by means of bilateral transabdominal thoracotomy. The heart was then quickly removed and placed in a Petri dish with Krebs-Henseleit buffer containing (in mM): NaCl - 118, KCl - 4.7, CaCl2 - 3, MgSO4 - 1.2, KH2PO4 - 1.2, NaHCO3 - 25, EDTA-Na2 - 0.5, glucose - 5, pH (7.4), solution temperature - 25°C. The following parameters were then assessed: time to first beat (seconds), duration of contraction (min), heart rate (beats / min). The data were expressed in M±m.

[0295] Table 14 presents the results of the evaluation of the myocardial contractile function in the control and experimental db / db mice. The parameter of the time of the first heart contraction was introduced because in the control K(+) carcass, 30% of the isolated hearts stopped before being placed in the Krebs Henseleit buffer, and after being placed in the buffer, contraction began. It was shown that in the gApoA-1-3-243 group, the time to the first contraction was 2.8 times shorter relative to the K(+) group. The contraction duration index varied significantly in both groups. The difference in this index was 2.2 times in favor of the animals of the gApo A-1-3-243 group. The HR parameter differed insignificantly.

[0296] Table 14. Myocardial contractile function indices in db / db mice in the K(+) and hApoA- 1-3-243 groups 16 weeks after the start of the experiment Note: Ts - 16 weeks after the start of the experiment. K(+) - control group of db / db' mice, gApoA- 1-3-243 - experimental group of db / db mice, which were administered g Apo A- 1-3-243.

[0297] * - p<0.05 - significant difference in relation to group K(+).

[0298] The obtained data indicate that hApoA-1-3-243 increases the contractile function of the heart muscle and exhibits a cardioprotective effect in db / db mice with T2DM and obesity.

[0299] Thus, the claimed agent allows a subject with hyperglycemia and / or insulin resistance to reduce not only lipid and carbohydrate metabolism disorders, body weight, but also functional disorders of the liver, kidneys, bone marrow and spleen, reproductive system organs, improve myocardial contractility, locomotor activity and cognitive function and the associated quality of life of the subject.

[0300] The work was carried out using the equipment of the FRC FTM Center for Collective Use “Proteomic Analysis”, supported by funding from the Ministry of Education and Science of Russia (agreement No. 075-15-2021-691) and the FRC FTM Center for Collective Use “Spectrometric Measurements”.

Claims

CLAUSE OF INVENTION 1. An agent for the treatment or prevention of hyperglycemia and / or insulin resistance and / or associated diseases and / or functional disorders of organs and systems, comprising human apolipoprotein AI (ApoA-1) or a functionally significant fragment thereof.

2. The agent according to claim 1, wherein the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

3. The agent according to item 1 for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

4. The agent according to any of paragraphs 1-3, wherein the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

5. The agent according to i.4, wherein the liver and kidney function disorders are selected from the group consisting of deviations from the normal levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine in the blood plasma.

6. The agent according to claim 4, wherein the disturbances of bone marrow function include suppression of bone marrow cell proliferation and apoptosis of bone marrow cells.

7. The agent according to claim 4, wherein the dysfunction of the spleen is selected from the group consisting of a disorder of the organ integrity of the spleen, a decrease in the total number and apoptosis of macrophages, a decrease in the immunoregulatory index or the CD4 / CD8 ratio, and an increase in the proportion of cytotoxic lymphocytes and macrophages.

8. The agent according to item 4, where the myocardial dysfunction includes a disorder of the contractile function of the myocardium.

9. The agent according to I.4, wherein the disorders of the reproductive system include a decrease in the level of testosterone in the blood serum.

10. The remedy according to i.4, where the functional disorders of organs and systems include disorders of the subject’s locomotor activity.

11. The remedy according to i.4, where the functional disorders of organs and systems include disorders of the cognitive functions of the subject. 47 12. The agent according to any one of claims 1-11, comprising a functionally significant fragment of human ApoA-I.

13. The agent according to claim 12, wherein the functionally significant fragment of human ApoA-I has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus.

14. The agent according to claim 13, wherein the functionally significant fragment of human ApoA-I has a sequence that is the amino acid sequence of human ApoA-I, shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243).

15. The agent according to any one of claims 1-14, formulated in the form of a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

16. The agent according to claim 15, wherein the pharmaceutically acceptable carrier is water for injection or a physiological solution.

17. Use of the agent according to any of paragraphs 1-16 for the treatment or prevention of hyperglycemia and / or insulin resistance and / or associated diseases and / or functional disorders of organs and systems.

18. Use according to i.17, wherein the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus.

19. Use according to I.17 for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

20. Use according to any one of claims 17-19, wherein the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

21. Use according to I.20, where the liver and kidney dysfunctions are selected from the group consisting of deviations from the normal levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine in the blood plasma.

22. Use according to claim 20, wherein the bone marrow dysfunction includes suppression of bone marrow cell proliferation and bone marrow cell apoptosis. 48 23. Use according to claim 20, wherein the disorders of spleen function are selected from the group consisting of a disorder of the organ integrity of the spleen, a decrease in the total number and apoptosis of macrophages, a decrease in the immunoregulatory index or CD4 / CD8 ratio, and an increase in the proportion of cytotoxic lymphocytes and macrophages.

24. Use according to item 20, where the myocardial dysfunction includes a disorder of the contractile function of the myocardium.

25. Use according to I.20, where the disorders of the reproductive system include a decrease in the level of testosterone in the blood serum.

26. Use according to I.20, where functional disorders of organs and systems include disorders of the subject’s locomotor activity.

27. Use according to I.20, where the functional disorders of organs and systems include disorders of the cognitive functions of the subject.

28. The use according to any one of claims 17-27, wherein said agent comprises a functionally significant fragment of human ApoA-I.

29. The use according to claim 28, wherein the functionally significant fragment of human ApoA-I has a sequence that is an amino acid sequence of human ApoA-I shortened at the N-terminus.

30. The use according to claim 29, wherein the functionally significant fragment of human ApoA-I has a sequence that is the amino acid sequence of human ApoA-I, shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243).

31. Use according to any one of claims 17-30, wherein said agent is formulated as a pharmaceutical composition comprising human ApoA-I or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

32. Use according to claim 31, wherein the pharmaceutically acceptable carrier is water for injection or a physiological solution.

33. A method for the treatment and prevention of hyperglycemia and / or insulin resistance and / or diseases and / or functional disorders of organs and systems associated therewith, comprising administering to a subject in need thereof a means according to any one of paragraphs 1-16.

34. The method according to i.33, wherein the diseases associated with hyperglycemia and / or insulin resistance are selected from the group consisting of obesity and type 2 diabetes mellitus. 49 35. The method according to paragraph 33 for the treatment or prevention of diseases and / or functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance.

36. The method according to any of paragraphs 33-35, wherein the functional disorders of organs and systems associated with hyperglycemia and / or insulin resistance are selected from the group consisting of disorders of the liver and kidneys, bone marrow, spleen, myocardium, reproductive system, locomotor activity and cognitive functions.

37. The method according to item 36, wherein the liver and kidney function disorders are selected from the group consisting of deviations from the normal levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and creatinine in the blood plasma.

38. The method according to claim 36, wherein the disturbances in bone marrow function include suppression of bone marrow cell proliferation and apoptosis of bone marrow cells.

39. The method according to claim 36, wherein the disorders of spleen function are selected from the group consisting of a disorder of the organ integrity of the spleen, a decrease in the total number and apoptosis of macrophages, a decrease in the immunoregulatory index or CD4 / CD8 ratio, and an increase in the proportion of cytotoxic lymphocytes and macrophages.

40. The method according to item 36, wherein the myocardial dysfunction includes a disorder of the contractile function of the myocardium.

41. The method according to item 36, wherein the disorders of the reproductive system include a decrease in the level of testosterone in the blood serum.

42. The method according to article 36, where the functional disorders of organs and systems include disorders of the subject’s locomotor activity.

43. The method according to item 36, where the functional disorders of organs and systems include disorders of the cognitive functions of the subject.

44. The method according to any of paragraphs 33-43, wherein said agent comprises a functionally significant fragment of human ApoA-1.

45. The method according to claim 44, wherein the functionally significant fragment of human ApoA-1 has a sequence that is an amino acid sequence of human ApoA-1 shortened at the N-terminus.

46. The method according to claim 45, wherein the functionally significant fragment of human ApoA-1 has a sequence that is the amino acid sequence of human ApoA-1, shortened from the N-terminus by 2 amino acid residues (amino acid residues 3-243). 50 47. The method according to any one of claims 33-46, wherein said agent is formulated as a pharmaceutical composition comprising human ApoA-1 or a functionally significant fragment thereof and a pharmaceutically acceptable carrier.

48. The method according to claim 47, wherein the pharmaceutically acceptable carrier is water for injection or a saline solution.

49. The method according to any of paragraphs 33-48, wherein the administration is carried out locally or parenterally.

50. The method according to Art. 49, wherein parenteral administration is carried out intravenously or intramuscularly.

51. The method according to Art. 49, where local administration is carried out subcutaneously.

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