Treatment for non-alcoholic steatohepatitis

Abstract

a prophylactically or therapeutically effective amount 64 A method for preventing or treating non-alcoholic steatohepatitis (NASH), comprising administering Zn-enriched zinc.

Description

[Technical Field]

[0001] The present disclosure relates to the prevention and treatment of non-alcoholic steatohepatitis (NASH). [Background technology]

[0002] Nonalcoholic steatohepatitis (NASH) is liver inflammation and damage caused by the accumulation of fat in the liver. It is part of a group of diseases called nonalcoholic fatty liver disease. NASH can cause scarring of the liver, leading to cirrhosis. NASH resembles liver disease caused by long-term heavy alcohol consumption. However, NASH can also occur in people who do not abuse alcohol. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, drug therapies for NASH are limited, ineffective, and / or have side effects. [Means for solving the problem]

[0004] In one aspect, the present disclosure provides zinc ( 64 A composition containing Zn (" 64 Zn e " is used herein to mean 64 The composition is provided in a therapeutically or prophylactically effective dose for preventing and treating NASH. In another aspect, methods of using the composition are provided. In some embodiments, 64 Zn-enriched zinc is 64 Zn e Compound or 64 Zn e The disclosed compositions comprise: 64 In certain embodiments, the disclosed compositions contain at least 80% Zn enriched in zinc. 64 Zn e , at least 90%64 Zn e , at least 95% 64 Zn e , or at least 99% 64 Zn e Zinc, e.g., 80% 64 Zn e , 85% 64 Zn e , 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e , or 99.9% of 64 Zn e It contains zinc.

[0005] In accordance with these and other aspects of the present invention, numerous other aspects are provided. Other features and aspects of the present invention will become more fully apparent from the following detailed description and the appended claims. [Brief explanation of the drawings]

[0006] [Figure 1] FIG. 1 shows the kinetics of weight gain in the animals of the experimental group (M±n, n=10). [Figure 2] Figure 2 shows the calorie content of the food consumed by animals in the experimental groups (M ± n, n = 10). Note: 1 - control; 2 - control + 64Zne stable isotope in aspartic acid form; 3 - diet-induced obesity + 64Zne stable isotope in aspartic acid form; 4 - diet-induced obesity. [Figure 3] FIG. 3 shows the insulin levels in the blood of the experimental animals (M±n, n=10). [Figure 4] FIG. 4 shows the pancreatic islet area of ​​the experimental animals (M±n, n=10). [Figure 5]Figure 5 shows the kinetics of weight gain in animals in the experimental groups (M ± n, n = 10). Note: C - control; C + control against the background of administration of Zn-64 stable isotope in zinc-aspartate form; DIO - diet-induced obesity; DIO + diet-induced obesity against the background of administration of Zn-64 stable isotope in zinc-aspartate form. [Figure 6] Figure 6 shows the calorie content of the food consumed by animals in the experimental groups (M ± n, n = 10). Note: 1 - control; 2 - control + Zn-64 stable isotope in aspartic acid form; 3 - obese + Zn-64 stable isotope in aspartic acid form; 4 - obese. [Figure 7] Figures 7A-7F show photomicrographs of pancreatic sections in animals from the control (Figures 7A-7C) and obese (Figures 7D-7F) groups, hematoxylin and eosin, and arrows indicate exocrine cells with prominent fatty degeneration (10x objective 10, 10x objective 40). [Figure 8] Figures 8A-8F show photomicrographs (hematoxylin and eosin, 10x objective 10, 10x objective 40) of pancreatic sections from animals in the control group (Figures 8A-8C) treated with Zn-64 stable isotope in aspartic acid form and from animals in the obese group (Figures 8D-8F) treated with Zn-64 stable isotope in aspartic acid form. [Figure 9] Figure 9 shows the cross-sectional area of ​​the islets of Langerhans. * - The difference between the control and experimental groups is significant when p < 0.05; # - The difference between the obese group and the obese group treated with Zn-64 stable isotope aspartate is significant when p < 0.05. [Figure 10] Figures 10A-10D show photomicrographs of liver sections from animals in the control (Figures 10A and 10B) and obese (Figures 10C and 10D) groups (hematoxylin and eosin, 10x eyepiece objective 10, 10x eyepiece objective 40). [Figure 11]Figures 11A-11D show photomicrographs (hematoxylin and eosin, 10x objective 10, 10x objective 40) of liver sections from control (Figures 11A and 11B) and obese (Figures 11C and 11D) animals, all treated with Zn-64 stable isotope in aspartate form. [Figure 12] Figure 12A (hepatocyte nuclear area), Figure 12B (hepatocyte area), and Figure 12C (hepatocyte nuclear-to-cytoplasmic ratio) show the morphological analysis of the liver. * - The difference between the control group and the experimental group is significant when p < 0.05. # - The difference between the obese group treated with Zn-64 stable isotope aspartate and the obese group is significant when p < 0.05. [Figure 13] Figures 13A-13D show photomicrographs of liver sections from animals from the control group (Figures 13A and 13B) and the obese group (Figures 13C and 13D) (Van Gieson staining for collagen fibers (fibrosis), 10x eyepiece 10 objective, 10x eyepiece 40 objective). [Figure 14] Figures 14A-14D show photomicrographs of liver sections (Van Gieson staining for collagen fibers (fibrosis), 10x objective 10, 10x objective 40) from animals from the control group (Figures 14A and 14B) and the obese group (Figures 14C and 14D), all of which were treated with Zn-64 stable isotope in aspartic acid form. [Figure 15] Figure 15 shows the morphometric analysis of liver fibrosis. * - The difference between the control and experimental groups is significant when p < 0.05. # - The difference between the obese group and the obese group treated with Zn-64 stable isotope aspartate is significant when p < 0.05. [Figure 16] Figures 16A-16E show an analysis of body weight, food, and water consumption by animals in the experimental groups. Figure 16A shows food consumption rate in grams per animal; Figure 16B shows water consumption rate in ml per animal; Figure 16C shows average food consumption per animal per day (over the course of the experiment); Figure 16D shows average water consumption per animal per day (over the course of the experiment); and Figure 16E shows weight gain in rats after two weeks of drug administration. [Figure 17]FIG. 17 is a graph showing serum insulin levels (CU / mg total protein). [Figure 18] Figure 18A (serum) and Figure 18B (liver) are graphs showing superoxide dismutase activity (antioxidant Zn-dependent enzyme) CU / mg*min. [Figure 19] FIG. 19 is a graph showing measurements of the area of ​​the islets of Langerhans in the pancreas of experimental animals (microscopically, 7 days after the last drug administration). [Figure 20] Figures 20A-20F are photomicrographs of islets of Langerhans: Figures 20A and 20B - top panels; Figures 20C and 20D - middle panels; Figures 20E and 20F - bottom panels. [Figure 21] 21A-21C show the accumulation of metals in liver tissue of experimental animals. [Figure 22] 22A-22C show the accumulation of metals in kidney tissue of experimental animals. [Figure 23] Figures 23A-23F are photomicrographs of islets of Langerhans: Figures 23A and 23B - control group, magnification x10 and x40, respectively; Figures 23C and 23D - zinc acetate, comparison group, magnification x10 and x40, respectively; Figures 23E and 23F - zinc isotope, treatment group, magnification x10 and x40, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0007] As used herein, the word "a" or "plurality" before a noun refers to one or more of the particular noun.

[0008] The terms "for example" and "such as," and their grammatical equivalents, are understood to be followed by the phrase "and without limitation," unless otherwise specified. As used herein, the term "about" is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless otherwise specified, regardless of whether the term is explicitly used. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0009] An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" refers to the amount of an agent or composition that provides a beneficial effect or favorable outcome in a subject, or the amount of an agent or composition that exhibits the desired in vivo or in vitro activity. An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" refers to the amount of an agent or composition that provides the desired biological, therapeutic, and / or preventative result. That result can be reduction, amelioration, amelioration, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, disorder, or condition in a patient / subject, or any other desired alteration of a biological system. An effective amount may be administered in one or more administrations.

[0010] An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" can be initially estimated according to cell culture assays or using animal models, typically mice, rats, guinea pigs, rabbits, dogs, or pigs. Animal models may also be used to determine appropriate concentration ranges and routes of administration. Such information may then be used to determine appropriate doses and routes of administration for humans. Conversion tables, such as those found in "Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" [US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005], may be used to calculate human equivalent doses. Those skilled in the art will recognize additional guidance that can also be used to develop human therapeutic dosages based on non-human data. Effective doses generally range from 0.01 mg / kg to 2000 mg / kg of active agent, preferably from 0.05 mg / kg to 500 mg / kg of active agent. The exact effective dose depends on the severity of disease, the patient's general health condition, age, weight and sex, nutrition, administration time and frequency, drug combination, reaction sensitivity, and tolerance / response to administration, and other factors that will be considered by those skilled in the art when determining the dosage and administration route for a specific patient based on the knowledge of the art.This dosage can be determined at the discretion of a physician through routine experimentation.Effective dose also varies depending on the possibility of combination with other treatment procedures, such as the use of other drugs.

[0011] As used herein, "patient" and "subject" are interchangeable terms and may refer to human patients / subjects, dogs, cats, non-human primates, and the like.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein.Other suitable methods and materials known in the art may also be used.The materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0013] NASH Nonalcoholic steatohepatitis (NASH) is liver inflammation and damage caused by the accumulation of fat in the liver. It is part of a group of diseases called nonalcoholic fatty liver disease (NAFLD). NASH is the most severe form of NAFLD. NASH is closely associated with the triple epidemic of obesity, prediabetes, and diabetes. NASH can cause liver scarring, which can lead to cirrhosis. NASH resembles liver disease caused by long-term heavy alcohol consumption. However, NASH can also occur in people who do not abuse alcohol.

[0014] NASH worsens patients' cardiometabolic status and is associated with an increased risk of death from cardiovascular events. Up to 38% of deaths in NASH patients are directly related to cardiovascular events. Although patients are unaware of their liver condition, NASH can progress to more serious stages such as advanced fibrosis, cirrhosis, liver failure, or liver cancer due to hepatocyte ballooning and inflammation.

[0015] Currently, drug therapies for NASH are limited. Weight loss is commonly recommended for patients because it can reduce liver fat. At advanced stages of the disease, liver transplantation may be the only option for patients. However, this is a risky surgical procedure with a long waiting list.

[0016] zinc Zinc is an essential trace element for ensuring proper metabolic function in the human body. More than 200 enzymes throughout the body depend on zinc. This element is either a component of enzymes or a regulator of their activity, covering all classes of enzymes: transferases (RNA and DNA polymerases, reverse transcriptases, thymidine kinase, nucleotidyl transferases, carboxypeptidases and other peptidases), hydrolases (alkaline phosphatase, 5-nucleotidase), aminopeptidases, etc.), lyases (aldolases, carbonic anhydrases, etc.), oxidoreductases (alcohol dehydrogenase, superoxide dismutase, etc.), ligases, and isomerases. Without zinc, protein, fat, and carbohydrate metabolism would be impossible.

[0017] Zinc has also been shown to exhibit mediated antioxidant effects. Zinc is an inhibitor of NADPH oxidase, an enzyme complex that catalyzes the production of the highly aggressive superoxide anion radical. Furthermore, it may directly affect the oxidation of free radicals at the initiation stage of chain reactions; it is a structural component of several enzymes in the antioxidant defense system, including Cu / Zn-containing superoxide dismutase. Zinc protects proteins from oxidation by reactive oxygen species by binding their thiol groups. This trace element induces the synthesis of metallothionein, a cysteine-rich protein that acts as a free radical scavenger. Zinc inhibits the formation of reactive mixed-valence metal oxides and participates in stabilizing membrane structure.

[0018] The metabolic and structural importance of zinc is determined by its wide spectrum of biological activities. Thus, zinc is necessary for the normal execution of processes related to cell division and differentiation (growth, tissue regeneration, spermatogenesis, etc.) and is actively involved in nucleic acid metabolism and protein synthesis. This trace element is important for the metabolism of polyunsaturated fatty acids and reactions of prostaglandin conversion. It exhibits significant lipophilic activity and has hepatoprotective properties. Haase H., Rink L. Zinc Signaling. Zinc in Human Health / / Amsterdam, Netherlands: IOS Press. 2011. 243.

[0019] Furthermore, zinc plays a crucial role in the immune response, as it is a regulator of phagocyte and lymphocyte activity and influences neutrophil chemotaxis. The zinc-containing enzyme 5-nucleotidase is crucial for the functional status of T and B lymphocytes. Isolated zinc deficiency causes severe impairment of various parameters of T cell function, including thymic atrophy, inhibition of cell-mediated cytotoxicity, and a decrease in the total lymphocyte population. Zinc is involved in stimulating the metabolism and activity of pituitary hormones, adrenal glands, pancreas, prostate, and testes. Zinc plays a distinct role in the synthesis, storage, and secretion of insulin. Haase H., Rink L. Zinc Signaling. Zinc in Human Health / / Amsterdam, Netherlands: IOS Press. 2011. 243.

[0020] Zinc also acts as a synergist / antagonist for the absorption of many trace elements and vitamins (iron, copper, magnesium, vitamins A, E, folic acid, etc.) and affects their metabolism.

[0021] In summary, zinc is involved in a variety of important processes and functions in the human body. Detailed studies of some of these functions have not yet been fully completed, and many of the mechanisms of action of this trace element are not yet fully understood or recognized. However, experimental and clinical studies presented in the literature indicate that zinc is one of the key elements, and its reduced levels in the body are associated with the development and progression of many of the most prevalent non-epidemic diseases. Because the main metabolic processes in the body occur with the active participation of zinc-containing and zinc-dependent enzymes, its deficiency causes disruptions in many important processes.

[0022] The use of classical pharmacological forms of zinc (zinc salts and its chelates) does not always make it possible to achieve an adequate effect of compensating for zinc deficiency due to the low bioavailability of this element.

[0023] Treatment methods and compositions In one aspect, the present disclosure provides a method for the prevention and / or treatment of NASH in a therapeutically or prophylactically effective amount (dosage). 64 In some embodiments, a composition comprising zinc enriched in Zn is provided. 64 Zn-enriched zinc is 64 Zn e Compound or 64 Zn e In some embodiments, the disclosed compositions comprise: 64 Zn e However, aspartate (chemical formula - C4H5O4N 64 Zn e ), sulfate, and citrate.

[0024] " 64 Zn e " is used herein to mean 64 Zn is used to refer to concentrated zinc, i.e. 64 So that Zn is more concentrated than the normal proportion of zinc found in nature. 64 Zn is concentrated zinc.

[0025] The disclosed composition comprises: 64 Zn e Contains zinc enriched in light isotopes 64 Zn e In certain embodiments, the compositions of the present invention comprise at least 80% 64 Zn e , at least 90% 64 Zn e , at least 95% 64 Zn e , or at least 99% 64 Zn e Zinc, e.g., 80% 64 Zn e , 85% 64 Zinc, 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e , or 99.9% of 64 Zn e It contains zinc.

[0026] In another aspect, the present disclosure provides methods of treating and / or preventing NASH by administering a therapeutically or prophylactically effective amount of the disclosed compositions to a subject in need thereof.

[0027] 64 Zn e A method for preventing or treating NASH is provided, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising a compound or a salt thereof. In some embodiments, the composition further comprises a diluent or excipient. In some embodiments, the diluent is water. In further embodiments, the water diluent is deuterium-depleted water. In some embodiments, 64 Zn e The compound or its salt is 20 to 100% 64 Zn eIn a further embodiment, 64 Zn e The compound or its salt is at least 80% 64 Zn e In a further embodiment, 64 Zn e The compound or its salt is at least 95% 64 Zn e In some embodiments, the composition contains between 0.05 mg and 110 mg. 64 Zn e In some embodiments, the composition contains between 1 and 10 mg. 64 Zn e In some embodiments, 64 Zn e The compound or its salt is at least 90% 64 Zn e and the composition is 64 Zn e is present at a concentration between 0.1 mg / ml and 10 mg / ml. 64 Zn e is an aspartate salt (chemical formula - C4H5O4N) with two aspartic acid molecules. 64 Zn e In some embodiments, the composition is administered by injection. In other embodiments, the composition is administered orally. In certain embodiments, the proinflammatory cytokine is one or more of IL-1, IL-6, IL-12, and IFN-γ.

[0028] Formulation and Administration of Compositions The disclosed compositions can be administered to a subject in need thereof in any suitable mode of administration, at any suitable frequency, and in any suitable effective dosage.

[0029] In some embodiments, administered 64 Zn eThe total amount of zinc administered is the same as the recommended daily allowance or intake of zinc in the United States. 64 Zn e In some embodiments, the total amount of zinc is 1 / 2, 2, 3, 5, or 10 times the recommended daily allowance or intake of zinc in the United States. 64 Zn e The total amount of zinc is between 1 / 2 and 10 times the recommended daily allowance or intake of zinc in the United States. The disclosed compositions may comprise a prescribed daily dose administered once daily, or some fraction thereof administered a corresponding number of times per day. The disclosed compositions may also be administered once every two days, once every three days, once a week, or any other suitable frequency. 64 Zn e may contain an amount of

[0030] The disclosed compositions may be in any suitable form and formulated for any suitable delivery means. In some embodiments, the disclosed compositions are provided in a form suitable for oral administration, such as tablets, pills, lozenges, capsules, liquid suspensions, liquid solutions, or any other conventional oral dosage form. The oral dosage form may provide immediate release, delayed release, sustained release, or enteric release, and, where appropriate, may include one or more coatings. In some embodiments, the disclosed compositions are provided in a form suitable for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or any other injection route. In some embodiments, the injectable compositions are provided in a sterile and / or non-pyrogenic form and may contain preservatives and / or other suitable excipients, such as sucrose, sodium phosphate dibasic heptahydrate or other suitable buffers, pH adjusters, such as hydrochloric acid or sodium hydroxide, and polysorbate 80 or other suitable surfactants.

[0031] When provided in solution form, in some embodiments, the disclosed compositions are provided in glass or plastic bottles, vials or ampoules, any of which can be suitable for single or multiple use.The bottles, vials or ampoules containing the disclosed compositions can be provided in the form of a kit together with one or more needles and / or one or more syringes of appropriate gauge, all of which are preferably sterile.Therefore, in certain embodiments, a kit is provided that includes the liquid solution described above, packaged in a suitable glass or plastic bottle, vial or ampoules, and can further include one or more needles and / or one or more syringes.This kit can also include instructions for use.

[0032] In certain embodiments, 64 Zn eThe dosage is proportional to various authoritative daily intake guidelines for the corresponding element (e.g., the USRDA, the Adequate Intake (AI), or the Recommended Dietary Intake (RDI)). In some embodiments, the light isotope dosage is between about 1 / 2 and about 20 times the guidance amount, more preferably between about 1 and about 10 times the guidance amount, and even more preferably between about 1 and about 3 times the guidance amount. Thus, in certain embodiments, a single dose of the disclosed compositions for daily administration is formulated to contain amounts within these ranges, e.g., about 1 / 2, about 1, about 3, about 5, about 10, and about 20 times the guidance amount. These amounts are generally for oral ingestion or topical application. In some embodiments, intravenous dosages are lower, e.g., about 1 / 10 to about 1 / 2 of the guidance amount. Doses at the lower end of these ranges are appropriate for individuals with increased sensitivity to a particular element or class of elements (e.g., individuals with kidney problems). For zinc, daily guidance amounts range from 2 mg for infants to 8-11 mg (depending on gender) for those 9 years of age and older. The daily dosages discussed throughout this application may be subdivided into fractional doses and fractional doses administered an appropriate number of times per day (e.g., 1 / 2 of the daily dose administered twice daily, 1 / 3 of the daily dose administered three times daily, etc.) to provide the total daily dosage. See Table 1.

[0033] [Table 1]

[0034] The disclosed compositions can be produced by methods used in accordance with common practice in the pharmaceutical industry, such as those illustrated in Remington: The Science and Practice of Pharmacy [Pharmaceutical Press; 21st revised ed. (2011)] (hereinafter, "Remington").

[0035] In some embodiments, the disclosed compositions comprise at least one pharmaceutically acceptable vehicle or excipient. These include, for example, diluents, carriers, excipients, fillers, disintegrants, solubilizers, dispersants, preservatives, wetting agents, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifiers, and, if necessary, penetration enhancers such as DMSO. The compositions may also contain suitable auxiliary substances, such as solubilizers, dispersing agents, suspending agents, and emulsifying agents.

[0036] In certain embodiments, the composition further comprises suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids, and other pharmaceutically acceptable excipients.

[0037] A complete description of pharmaceutically acceptable excipients can be found, for example, in Remington's Pharmaceutical Sciences (Mack Pub., Co., NJ 1991) or other standard pharmaceutical texts, such as Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017).

[0038] In some embodiments, the disclosed compositions may be administered intragastrically, orally, intravenously, intraperitoneally, or intramuscularly, although other routes of administration are possible.

[0039] Water can be used as carrier and diluent in the composition.In addition to water or instead of water, other pharmaceutically acceptable solvents and diluents can also be used.In certain embodiments, deuterium-depleted water is used as diluent.

[0040] Large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers, may also be used as carrier compounds for the compositions. Pharmaceutically acceptable carriers in therapeutic compositions may further contain liquids such as water, saline, glycerol, or ethanol. In addition, the compositions may further contain excipients such as wetting or emulsifying agents, buffer substances, and the like. Such excipients include, among others, conventional diluents and carriers in the art, and / or substances that promote the penetration of active compounds into cells, such as DMSO, as well as preservatives and stabilizers.

[0041] The disclosed compositions may be presented in a variety of dosage forms depending on the purpose of application; in particular, they may be formulated as solutions for injection.

[0042] The disclosed compositions can be administered systemically.Suitable administration routes include, for example, oral administration or parenteral administration, such as intravenous administration, intraperitoneal administration, intragastric administration, and via drinking water.However, depending on the dosage form, the disclosed compositions can also be administered via other routes.

[0043] In certain embodiments, 64 Zn e is administered intragastrically at a concentration of 2.25 mg / ml to prevent and / or treat overweight or obesity in an animal subject. In a further embodiment, the disclosed composition is about 2 ml. 64 Zn e In other further embodiments, the level of concentration of 2 ml of the composition is about 99% or greater. 64 Zn e is a zinc aspartate (chemical formula - C4H5O4N) with two aspartic acid molecules. 64 Zn eThe dosage of the disclosed compositions may vary depending on the subject being treated, the severity of the disease, the condition of the patient, and other factors that would be considered by a skilled artisan in determining the dosage and route of administration for a particular patient based on the knowledge of one of ordinary skill in the art.

[0044] Lighter isotopes may also be purchased: Zn-64 oxide of the required enrichment can be purchased, for example, from Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.

[0045] Zinc aspartate has the chemical formula - C4H5O4N 64 Zn e and has two aspartic acid molecules. The structure of zinc aspartate is:

[0046] [ka]

[0047] In certain embodiments, the disclosed compositions comprise from about 20% to about 100% of the composition. 64 Zn e Includes:

[0048] 64 Zn e The disclosed compositions comprising the salts or chelates ( 64 The composition is metabolized in the body much better than compositions containing natural zinc in the form of zinc (not enriched in Zn). Furthermore, the composition helps reduce the toxic effects inherent in conventional pharmaceuticals with comparable levels of efficacy in preventing and treating NASH.

[0049] The disclosed compositions may be administered concurrently with another agent or therapy. [Example]

[0050] In order that the present invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way. [Example 1]

[0051] 64 Zn e Comparison between and naturally occurring zinc Effect on the absorption and utilization of glucose by the body 64 Zn e A comparative study of the potential effects of Zn and Zn acetate—Zn(CH3COO)2—showed that the zinc isotope has a more favorable effect on the absorption and utilization of glucose by the body on a number of parameters.

[0052] A positive kinetics of weight gain was recorded in control animals [vs. the group of animals injected with Zn acetate (Zn(CH3COO)2)] (Figure 1).

[0053] 64 Zn e Seven days after discontinuation, insulin levels in the animals' blood increased [relative to the group of animals injected with Zn acetate (Zn(CH3COO)2)] (Figure 3).

[0054] A significant increase in the area of ​​the pancreatic islets of the experimental animals was observed (microscopic examination 7 days after the last administration of the substance) [compared to the group of animals injected with Zn acetate (Zn(CHCOO))] (Figure 4). This is a positive kinetics, and similar to the development of type 1 diabetes, there is a significant lack of insulin due to problems with its synthesis by these pancreatic islets. The obtained results correlate with the results of determining insulin levels in the bloodstream (Figure 3).

[0055] Glucose tolerance tests showed a decrease in glucose levels in animals after administration of zinc isotopes compared to the control group and the group of animals injected with zinc acetate [Zn(CH3COO)2] (whose glucose levels also decreased, although to a lesser extent). This may indicate that zinc isotopes affect blood insulin levels, which leads to the initiation of mechanisms related to the utilization of glucose from the bloodstream. Given that insulin is a zinc-dependent protein, it can be assumed that the administration of zinc leads to an increase in the activity of this protein in association with receptors in tissues or an increase in the amount of this hormone in the bloodstream.

[0056] [Table 2]

[0057] The results obtained in a type 1 diabetes model indicate that this substance has a more positive effect on the pathogenesis of type 1 diabetes [vs. Zn acetate (Zn(CH3COO)2)] and could potentially be used to reduce the toxic effects of increased glucose levels in the bloodstream during the development of this condition.

[0058] Analysis of metal accumulation in kidney and liver tissues (zinc, manganese, and copper) revealed that zinc [Zn acetate (Zn(CH3COO)2) or 64 Zn e ] showed that only zinc was significantly increased in both groups of animals injected with . This indicates that the zinc injected into the animals accumulated and did not increase its utilization in the body. All other analyzed metals were within the same concentrations as in the control group of animals. The data obtained provide insight into the accumulation and utilization of zinc and related metals by the body. 64 Zn e It is shown that there are no adverse effects of

[0059] All these data suggest that Zn acetate [Zn(CH3COO)2] has a beneficial effect on the absorption and utilization of glucose by the body compared to Zn acetate [Zn(CH3COO)2]. 64 Zn eThis suggests a more pronounced and higher quality effect of

[0060] In this example, zinc acetate (natural zinc) was administered to the experimental group of animals at a dose of 3750 mcg of zinc (metallic) per kg of animal (rats) body weight. Zinc-64 in the form of zinc aspartate was also administered to the experimental group of animals at a dose of 3750 mcg of zinc (metallic) per kg of animal (rats) body weight. Administration of these compositions was via the intraperitoneal route. [Example 2]

[0061] in animal models of obesity 64 Zn e Anthropometric Effects of the Base Composition against the development of high-fat diet-induced obesity 64 Zn e To evaluate the efficacy of the base composition, untreated animal models of obesity and 64 Zn e Several anthropometric measurements were evaluated in an animal model of obesity treated with the solution.

[0062] White non-bred rats with an initial weight of 195–205 ± 10 g were used in the experiments. Animals were maintained in an accredited vivarium at the Academic and Research Center "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv, in accordance with standard rules on the arrangement, equipment, and maintenance of experimental biological clinics (vivariums). This study was conducted in accordance with the international standards and recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986) and was approved by the Bioethics Commission of the Academic and Research Center "Institute of Biology and Medicine."

[0063] Statistical analysis of the results was performed using the methods of variance statistics and correlation analysis using OriginLab Origin® Pro 9.1 and StatSoft STaStica® 10 software (Brandt, Z. Statistical methods for analysis of observation. M.:Mir, 1975-312p.). The assumption of normal distribution of the samples was tested using the Shapiro-Wilk test. If the samples met the criteria for normal distribution, the significance of differences between samples was determined using the Student's t-test. If the samples did not meet the criteria for normal distribution, the significance of differences between samples was determined using the Mann-Whitney U test. Differences were considered statistically significant if p<0.05.

[0064] Before the start of the experiment, the animals were fed a standard diet in the animal farm. To induce obesity in the experimental animals, they were fed a high-fat diet consisting of standard chow (60%), lard (10%), eggs (10%), sucrose (9%), peanuts (5%), milk powder (5%), and sunflower oil (1%) (see XH Shen et al., Exp. Biol. and Med. 235: 47-51 (2010)). The diet was prepared by the inventors. All animals were fed the high-fat diet for the first 4 weeks of the experiment, after which they were randomly divided into two experimental groups. - Animals in the first group (obese) continued to eat the high-fat diet and had free access to water for the next 6 weeks of the experiment. - Second group (obese + 64 Zn e The animals in the control group were also on a high-fat diet and had free access to water for the next 6 weeks of the experiment, but were given intragastrically 2 ml of 2.25 mg / ml of 2-mg ethanol every 3 days until the end of the experiment. 64 Zn e The solution contained 2.25 mg / ml of a pharmaceutically acceptable zinc salt, specifically zinc aspartate, 64 The Zn enrichment level was greater than 80 percent. Standard Dulbecco's Phosphate Buffered Saline (from the designated manufacturer) (based on deuterium-depleted water Langway) was used as the diluent (liquid medium) for the preparation of the claimed compositions.

[0065] There was also a group of animals (controls) that were fed a standard, field-prepared diet and had free access to water throughout the experiment.

[0066] After an overnight fast, animals in all groups were weighed weekly. The amount of food consumed by the animals was determined daily. At the end of the 10-week period, 24 hours after the last dose of zinc isotope solution, the animals were removed from their cages and decapitated.

[0067] Body Mass Index (BMI) (body length squared (cm) 2The ratio of body weight (g) to total body mass (g) was calculated at the end of the experiment. An increase in BMI is a characteristic morphological sign of obesity, which occurs as a result of the accumulation and redistribution of adipose tissue in the body. BMI allows for the evaluation of the weight-to-height (body length) ratio, thereby indirectly assessing whether a person is underweight, normal, or excessive. Furthermore, BMI is used as an integral value to characterize the body's composition and the degree of fat deposition. This is because the distribution of adipose tissue in the body determines the risk of obesity-related metabolic complications, which must be taken into account when examining patients for the development of obesity. BMI is not only a diagnostic criterion for obesity, but also an excellent measure of a patient's risk for diseases that can occur in overweight and obesity.

[0068] Data obtained during the experiment (Table 3) showed that at week 10 of the experiment, the mean body mass index of the control animals was 0.60 g / cm 2 The BMI of animals on a high-fat diet was 0.71 g / cm (0.71 g / cm ), which is within the reference range for animals in this age group. 2 ) was 1.14 times higher than that during the experiment. 64 Zn e The body mass index of the rats administered the solution was lower than that of the untreated obese animal model, but was higher than the control value (0.65 g / cm 2 ), which is slightly higher than 64 Zn e It has been shown that the solution has a positive effect on the general metabolic state of the animals.

[0069] [Table 3]

[0070] Since BMI is calculated based on weight, a decrease in BMI can 64 Zn e This may be directly related to the lower body weight of the animals given the solution. 64 Zn eWe further investigated whether administration of the solution affected body weight and weight gain in an animal model of obesity. The data obtained (Figure 1) show that the kinetics of weight gain by animals in the experimental groups were significantly different. Therefore, maintained on a high-fat diet, 64 Zn e The animals receiving the solution lost more weight than the animals receiving only the high-fat diet. Starting from the fourth week of the experiment, a particularly significant difference was observed in the weight gain of the animals in both groups. The weight gain of the animals receiving the high-fat diet reached 103% by the end of the experiment, while 64 Zn e Animals that received intragastric injections of the solution gained less weight than animals in the control group (62% vs. 59%).

[0071] The development of obesity, which is caused by the disruption of the coordinated actions of many neurotransmitters and hormone systems in the body, is known to lead to disorders at the level of appetite control and satiety regulation. These disorders promote excessive food intake and are often accompanied by the development of hyperphagia, a state of abnormally high appetite for food energy equivalent to that which exceeds the body's energy needs [L. Zhou et al., Cell Metabolism 6: 398(2007)].

[0072] 64 Zn e To define the possible mechanism of the reduction in weight gain in the animals treated with the solution, the amount of food consumed by the animals was analyzed. The data obtained are shown in Table 3.

[0073] When comparing the calculated data for all experimental groups, there was no significant difference in the average amount of food consumed by the animals per day. Therefore, animals in the control and obesity model groups consumed approximately 35 g of food per day. However, it should be noted that the control group animals were maintained on a standard diet, while the diet-induced obesity model group animals consumed a specially prepared high-fat diet with significantly higher calorie content.

[0074] Analysis of the results obtained, taking into account the calorie content of the food consumed by the animals, shows a significant difference in values. Although the animals ate the same amount of food, the calorie content of the food consumed by the group of diet-induced obesity models administered with the claimed compositions was lower than the calorie content of the control group of diet-induced obese animals (without administration of the disclosed compositions). Furthermore, at 10 weeks, the calorie content of the group of diet-induced obesity models administered with the disclosed compositions was approximately the same as that of the control group consuming standard food and the group consuming standard food simultaneously administered with the disclosed compositions.

[0075] The dynamics of the caloric content of the food consumed by the animals during the 10 weeks of the experiment are shown in Figure 2.

[0076] The data obtained is 64 Zn e This suggests that the base composition affects satiety, since with free access to food, animals treated with the disclosed composition of the disclosed method consumed significantly less food than untreated animals maintained on a high-fat diet. 64 Zn e The animals that received the solution 64 Zn e They ate less and gained less weight than animals that did not receive the solution, a difference that may be explained by both direct and indirect effects of zinc on energy homeostasis.

[0077] In this way, 64 Zn e Administration of the base composition has been shown to cause a reduction in the amount of food consumed per day, thus resulting in less pronounced weight gain in the animals and a normalization of their body mass index compared to similar values ​​in untreated animal models of obesity. [Example 3]

[0078] in animal models of obesity 64 Zn e Biochemical effects of the base composition Light zinc isotopes on blood biochemical variables affected by pathological changes in obesity, especially lipid profile 64 Zn e An experiment was conducted to study the effects of high-fat diet on the development of obesity in experimental animals. For this purpose, high-fat diet obesity was induced in experimental animals as described in Example 2. For the experiment, the following animals were used: control animals that consumed a standard diet; animals that were fed a high-fat diet for the next 6 weeks; and animals that were fed a high-fat diet but for the entire 6 weeks of the experiment. 64 Zn e The base composition (2.25 mg / ml concentration of 80% or more) 64 Zn e Animals were also given zinc aspartate containing α-aspartic acid (administered intragastrically in a volume of 2 ml). The experimental results are shown in Table 4.

[0079] [Table 4]

[0080] 64 Zn e The composition based on this invention has been found to have a positive effect on lipid metabolism in the body. 64 Zn e The reduction in serum triglyceride, cholesterol and free fatty acid levels in animals treated with was approximately the same as in the control group of animals. [Example 4]

[0081] Effect of redox state on experimental animals 64 Zn e Effect Numerous studies have shown that obesity is closely associated with altered redox status and increased metabolic risk. Oxidative stress is one of the factors that cause adipocyte dysfunction. Oxidative stress and the resulting tissue damage and cell death are the basis for the development of many chronic pathological conditions. Excessive production of free radicals and / or depletion of their detoxification systems leads to an imbalance between prooxidants and antioxidants, which in turn affects the structure of cell membrane lipids and proteins and nucleic acids. Lipid peroxidation (LPO) mediated by free radicals is one of the important causes of cell membrane destruction and further cell damage. Degradation of membrane lipids induces increased membrane fluidity and permeability to ions, which overall disrupts cellular homeostasis. Products of free radical oxidation (e.g., 4-hydroxyalkenes, malondialdehyde) are highly mutagenic and cytotoxic.

[0082] Furthermore, oxidative stress activates preadipocyte differentiation and stimulates the hypertrophy of mature adipocytes.Excessive production of ROS in accumulated adipose tissue further leads to the induction of oxidative stress in the bloodstream, which contributes to the propagation of oxidative stress to organs distant from fat depots.

[0083] The pro-oxidant-antioxidant balance in animals was evaluated using an obesity model as described in Example 2. A control group, an untreated animal model of obesity, and a group fed a high-fat diet were included. 64 Zn e A group of animals treated with was used in this experiment.

[0084] The concentration of lipid peroxidation products serves as a valuable criterion, allowing conclusions to be drawn about the intensity of the oxidation process. These include primary lipid peroxidation products (e.g., conjugated dienes) and secondary lipid peroxidation products (e.g., aldehydes, especially malonaldehyde), which are formed as a result of the cleavage of carbon-carbon double bonds in the carbon skeleton of oxidized molecules. Subsequently, the initiation of LPO leads to the formation of conjugated Schiff bases of phospholipids and malonaldehyde-like products, which disrupt the ordered orientation of phospholipid molecules and affect the intermolecular interactions of lipoproteins and the organization of basement membranes.

[0085] Considering the above, 64 Zn e The concentrations of primary LPO products [conjugated dienes (CD)], secondary LPO products [TBA-reactive substances (TBARS)] and final LPO products [Schiff bases (SB)] were determined in animals treated with . Considering that obesity is accompanied by the development of systemic oxidative stress that covers most tissues to varying degrees and leads to the disruption of cell membrane integrity and the influx of lipid peroxidation products into the bloodstream, values ​​characterizing the state of the pro-oxidant-antioxidant system were determined in the serum of the animals.

[0086] The obese model was found to have elevated serum levels of the primary products of free radical lipid oxidation (1.86-fold higher than in controls) (Table 5). These results can be explained in terms of disturbances in lipid metabolism, particularly in the transport processes of fatty acids, and therefore elevated plasma levels of free and esterified fatty acids, which are direct substrates for the action of reactive oxygen species.

[0087] On the other hand, the accumulation of lipid peroxidation products in serum may be a direct consequence of the oxidative destruction of their lipid components and the breach of cell membrane integrity due to the influx of lipid peroxidation products into the bloodstream.

[0088] [Table 5]

[0089] Therefore, the increase in the level of lipid peroxidation products at 10 weeks of obesity development clearly indicates that oxidative stress is systemic and that this process is chronic. These metabolites are highly toxic compounds, and their negative effects are manifested at various levels, leading to damage to DNA molecules, destruction of protein molecules and glycosaminoglycans, changes in the lipid composition of cell membranes, and disruption of membrane-related processes, making them an unfavorable prognostic marker.

[0090] To animals 64 Zn e Administration of the base composition helps normalize the levels of primary, secondary, and end-stage LPO products, which impacts the body's overall pro-oxidant-antioxidant status. 64 Zn e serves as additional evidence of the ability of

[0091] According to modern concepts, reactive oxygen species not only activate lipid peroxidation processes, but also cause the oxidative destruction of protein molecules, leading to the destruction of the higher-order structure of both soluble and membrane-bound enzymes, receptors, and ion channels, which ultimately leads to the loss of their biological activity (e.g., enzymes, receptors, transporters). Protein oxidation results in the formation of aldehyde and ketone (carbonyl) groups in amino acid residues in proteins.

[0092] Therefore, an increased number of oxidatively modified proteins may be considered an early criterion of free radical tissue damage and a marker of depletion of the body's antioxidant defense system. This study revealed increased serum levels of oxidatively modified proteins in animal models of obesity (Table 6).

[0093] [Table 6]

[0094] This experimental data shows that the mice were fed a high-fat diet throughout the experiment, 64 Zn eIn animals injected with the solution, aldehyde-dinitrophenyl-hydrazone levels exceeded the benchmark but were lower than those in obese, untreated animals. For ketone-dinitrophenyl-hydrazones, their concentrations remained within the control range. These results correlate with data showing reduced levels of LPO products, suggesting a reduced intensity of free radical oxidation reactions. [Example 5]

[0095] for cytokine profiles in animal models of obesity 64 Zn e Effect of the base composition The cytokine profile in animals was evaluated using an obesity model as described in Example 2. A control group, an untreated animal model of obesity, and a group fed a high-fat diet were included. 64 Zn e A group of animals treated with was used in the experiment.

[0096] The pathogenesis of obesity involves a systemic chronic inflammatory process, the degree of intensity of which can be assessed by serum levels of pro- and anti-inflammatory cytokines.

[0097] Analysis of serum cytokine profiles in animal models of obesity showed increased levels of pro-inflammatory cytokines (Table 7). 64 Zn e In animals that received the solution, serum levels of pro-inflammatory cytokines were reduced against a background of increased levels of anti-inflammatory cytokines, which were even higher than in animals from the control group.

[0098] [Table 7]

[0099] Isotopes 64 Zn eOne of the underlying mechanisms of the effect of enriched zinc on cytokine profiles may be its inhibition of transcription factors sensitive to oxidative stress. 64 Zn e The specific normalizing effect of the base composition may serve as evidence of the possible anti-inflammatory potential of the claimed composition in obesity.

[0100] Thus, experimental data support the role of obesity in a number of pathological variables in animal models of obesity. 64 Zn e The positive effects of the base composition were confirmed, especially in experimental animals. 64 Zn e Administration of the base composition has been demonstrated to reduce body mass index, weight gain and food consumption; 64 Zn e It was found to have a positive effect on lipid metabolism in the animals; demonstrated a normalization of pro-oxidant-antioxidant homeostasis due to a reduction in the intensity of free radical processes; and affected the serum cytokine profile of the animals. 64 Zn e The effects observed in this study are believed to be beneficial for the claimed prevention and treatment of obesity. 64 Zn e This confirms the effectiveness of the base composition.

[0101] For Examples 2-5, the disclosed compositions enriched with zinc-64 have zinc salts / compounds with the following structural formula:

[0102] [ka]

[0103] This compound is a crystalline hydrate containing two water molecules. The molar mass is 364 g / mol. 2,2H2O must be considered as ·2H2O. This is because 0,2H2O is unbound water and can evaporate when the powder is dried. ·2H2O is a crystalline hydrate and is part of the molecule. 4.5 mg of zinc aspartate was used (which was a 2 ml solution volume), which contained 17.8% pure zinc-64 (as metal). Therefore, each dose of 4.5 mg of zinc aspartate contained 800 μg of zinc-64 (as metal). [Example 6]

[0104] Zn64 stable isotope in aspartic acid form in relation to obesity and type 2 prediabetes in experimental animals (rats) fed a high-fat diet for a specific period of time List of abbreviations ROS - reactive oxygen species AOD-Antioxidant Defense FFA - Free Fatty Acids GI tract - gastrointestinal tract BMI - Body Mass Index IDO-indoleamine-2,3-dioxygenase IR - insulin resistance MAO - Monoamine oxidase Oxidative modification of OMP-proteins OS-oxidative stress SOD - superoxide dismutase IL-Interleukin

[0105] This study evaluated the effect of Zn-64 stable isotope in aspartic acid form on the development of high-fat diet-induced obesity in experimental animals.

[0106] To investigate the effects of Zn-64 stable isotope in aspartate form on a number of anthropometric (body mass index, body weight, weight gain) and biochemical (glucose concentration, insulin level, alkaline phosphatase activity, albumin content) values ​​in an animal model of obesity.

[0107] To investigate the effect of Zn-64 stable isotope in aspartate form on the morphofunctional characteristics of the pancreas and liver in animals fed a high-fat diet.

[0108] To evaluate the effects of Zn-64 stable isotope in aspartate form on the function of the central and peripheral serotonergic system (tryptophan and serotonin levels, tryptophan hydroxylase, tryptophan decarboxylase, monoamine oxidase, and indoleamine 2,3-dioxygenase activity) in an animal model of obesity.

[0109] To evaluate the effect of Zn-64 stable isotope in aspartic acid form on free radical processes (levels of primary, secondary and end products of lipid peroxidation, levels of products of oxidative modification of proteins) and on the activity of key antioxidant enzymes (superoxide dismutase, catalase) in serum and adipose tissue of animal models of obesity.

[0110] To evaluate the effect of Zn-64 stable isotope in aspartate form on cytokine profile (levels of pro- and anti-inflammatory cytokines) in serum and adipose tissue, as well as resistin and ghrelin levels in an animal model of obesity.

[0111] To investigate the effect of Zn-64 stable isotope in aspartic acid form on the distribution of divalent metals (e.g., zinc, copper, manganese) among different organs in an animal model of obesity.

[0112] material and method Development of obesity models This study used white non-bred rats. Animals were maintained in an accredited breeding facility at the Institute of Biological Medicine, Academic Research Center, Taras Shevchenko National University, Kyiv, in accordance with standard regulations for the arrangement, equipment, and maintenance of experimental biological clinics (breeding facilities). This study was conducted in accordance with the international standards and recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986) and was approved by the Bioethics Committee of the Institute of Biological Medicine, Academic Research Center. Murzin, OB, European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes / OB Murzin, / / Practical workbook on human physiology. - Dnipropetrovsk): Publishing House of Dnipropetrovsk University, 2004. - pp. 135-148.

[0113] Before the obesity model was implemented, animals were fed a standard diet in the animal farm, initially weighing 195–205 ± 10 g. To induce obesity, the animals were fed a high-fat diet consisting of standard chow (60%), lard (10%), eggs (10%), sucrose (9%), peanuts (5%), milk powder (5%), and sunflower oil (1%) (Shen X. et al., Experimental Biology and Medicine. -2010.-No.235.-pp.47-51). All animals were fed the high-fat diet for the first four weeks of the experiment, after which they were randomly divided into two experimental groups.

[0114] - The animals of the first group (obese) continued to eat the high-fat diet and had free access to water for the next 6 weeks of the experiment.

[0115] The animals in the second group (obese + solution of Zn-64 stable isotope in aspartic acid form) also followed their high-fat diet and had free access to water for the next six weeks of the experiment. However, every three days, and until the end of the experiment, the animals received intragastric administration of a solution of Zn-64 stable isotope in aspartic acid form. The dose of zinc aspartate administered to each animal was 4.5 mg (substance per animal), which was administered orally by gavage in a volume of 2 ml of solution.

[0116] There was also a group of animals (controls) that were fed a standard diet prepared by the vivarium and had free access to water throughout the experiment.

[0117] To determine whether or not Zn-64 stable isotope aspartate had any effect on the studied anthropometric and biochemical parameters, a group of animals was formed (control + solution of Zn-64 stable isotope aspartate), which ate a standard vivarium diet and had free access to water throughout the experiment, but every three days the animals were intragastrically administered Zn-64 stable isotope aspartate until the end of the experiment. The dose of zinc aspartate administered to each animal was 4.5 mg (substance per animal), which was administered by oral gavage in a volume of 2 ml of solution.

[0118] After an overnight fast, animals in all groups were weighed weekly. The amount of food consumed by the animals was determined daily. At the end of the 10-week period, 24 hours after the last dose of Zn-64 stable isotope aspartate, the animals were removed from their cages and decapitated.

[0119] Body mass index (BMI) (ratio of body weight (g) to body length squared (cm2)) was calculated at the end of the experiment.

[0120] Preparation of serum Animal serum was prepared from whole blood. To remove fibrinogen-related proteins, the blood was incubated at 37°C for 30 minutes, after which the clot was carefully separated from the tube wall with a clean, dry glass rod to facilitate serum production. The sample was centrifuged at 2500 g for 15 minutes. The resulting supernatant (serum) was immediately separated from the blood cells, frozen, and stored at -20°C until further investigation.

[0121] Preparation of adipose tissue homogenate At the end of the experiment, the animals were euthanized by decapitation. All procedures during tissue removal were performed at a temperature of 1-4°C.

[0122] The adipose tissue was pulverized using cold scissors and then transferred to a homogenizer equipped with a loosely fitting Teflon pestle. The tissue was homogenized in cold homogenization buffer (50 mM Tris-HCl (pH 7.4) containing 130 mM NaCl) using approximately 30 strokes of the pestle. The resulting primary homogenate was centrifuged at 600 g for 15 min. The supernatant was carefully collected and recentrifuged at 15,000 g for 15 min. The supernatant was then frozen and stored at -80°C until further investigation.

[0123] Preparation of brain tissue homogenate At the end of the experiment, the animals were euthanized by decapitation. All operations during organ removal were performed at a temperature of 1-4°C.

[0124] The animal's head was separated from the body and the skull was carefully cut. The brain was carefully lifted from the bony vault with a scalpel, all cranial nerves were severed, and the brain was removed from the skull. The brain was then divided into two halves, with a longitudinal incision made between the hemispheres.

[0125] Brain tissue was pulverized using cold scissors and then transferred to a homogenizer equipped with a loosely fitting Teflon pestle. The tissue was homogenized in cold homogenization buffer (50 mM Tris-acetate, pH 7.4, containing 5 mM EDTA and 10% sucrose) using approximately 30 strokes of the pestle. The tissue:buffer ratio was 1:10. The resulting homogenate was centrifuged at 1500 g for 20 minutes. The supernatant was then carefully collected, frozen, and stored at -80°C until further investigation.

[0126] Preparation of duodenal tissue homogenate At the end of the experiment, the animals were euthanized by decapitation. All operations during organ removal were performed at a temperature of 1-4°C.

[0127] After opening the abdomen, the duodenum was removed from the animal's body and washed in 0.9% sodium chloride solution in a Petri dish. The duodenal mucosa was mechanically separated using a scalpel and then homogenized in 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and 0.25 M sucrose. The tissue:buffer ratio was 1:10. The homogenate thus obtained was centrifuged at 1500 g for 10 minutes. The supernatant was then carefully collected, frozen, and stored at -80°C until further investigation.

[0128] Determination of serum glucose concentration Glucose concentrations were measured in the blood of animals that had fasted for at least two hours. Blood was collected from the tail vein using a catheter. Glucose concentrations were determined using a GLUTOFOT-II glucose meter (Ukraine) according to the manufacturer's instructions. Medical test "Glyukofot-II": [user's manual for "Glyukofot-II-Hemoglan]. - Kyiv: Norm, 2008. -12p. The test strip contained all the necessary reagent components for determining glucose concentrations by the glucose oxidase method, including absorption of glucose oxidase and peroxidase enzymes into a porous hydrophilic membrane. The formation of a colored complex was the result of the reaction. A drop of whole blood was applied to the strip and allowed to stand at room temperature for 30 seconds. The strip was then washed with distilled water and placed in a blood glucose meter. Glucose concentrations were expressed in mmol / L.

[0129] Determination of insulin, interleukin and adipokines levels in serum and adipose tissue homogenates Insulin, interleukin, and adipokines levels were determined using an immunoenzymatic method (Halenova TI et al. RSC Adv. 2016;6:100046-55). This method was performed in an adsorption-capable microplate according to the soluble protein test procedure. Antigen solutions, previously diluted to a concentration of 10 μg / ml in 0.1 M NaHCO3 buffer (pH 9.6), were incubated in the plate wells at 4°C for 12 hours. Unbound substances were removed by washing the wells three times with TBS buffer, first with 0.05% Tween-20 and then without Tween-20. Nonspecific binding sites were blocked by adding a solution of 5% skim milk or 1% bovine serum albumin to the plate wells and incubating them at 37°C for 60 minutes. After incubation, the wells were washed three times with TBS, first with 0.05% Tween-20 and then without Tween-20. The primary antibody was diluted in TBS according to the manufacturer's instructions and incubated with the antigen for 60 minutes at 37°C. After washing with TBS working buffer, the secondary antibody conjugate was added to the wells, first with 0.05% Tween-20 and then without Tween-20, and incubated for 60 minutes at 37°C. Similar to the primary antibody, the secondary antibody was diluted in TBS according to the manufacturer's instructions. After the washing procedure, phenyldiamine dihydrochloride as a substrate in 0.05 M phosphate-citrate buffer was added to each well, followed by 0.3% hydrogen peroxide. After 10 minutes (the reaction time required for color development), 2.5 nM H2SO4 was added.

[0130] The absorbance at a wavelength of 492 nm was measured using a μQuant microplate spectrophotometer (BioTek Instruments).

[0131] Determination of serum alkaline phosphatase activity Serum alkaline phosphatase activity in animals was measured spectrophotometrically using a Microlab 300 biochemical analyzer and a standard PLIVA-Lachema Diagnostika test kit (Czech Republic). Test kit / / Pliva-Lachema Diagnostika.-2008.

[0132] As a result of the hydrolytic cleavage of p-nitrophenyl phosphate catalyzed by alkaline phosphatase, p-nitrophenol is formed, which produces an intense yellow color in alkaline medium. The optical density of the samples was measured at a wavelength of 405 nm. Alkaline phosphatase activity was expressed in relative units.

[0133] Serum albumin determination Serum albumin levels in animals were determined spectrophotometrically using a Microlab 300 biochemistry analyzer and standard PLIVA-Lachema Diagnostica test kit (Czech Republic). Test kit / / Pliva-Lachema Diagnostika.-2008.

[0134] Determination of serum superoxide dismutase activity To measure superoxide dismutase activity, a method based on the ability of this enzyme to inhibit the autoxidation of adrenaline was used. (Syrota TV Questions of med. clin.-1999.-V.5, No.3.-P.263-272)

[0135] Serum aliquots were added to microplate wells containing 0.2 M bicarbonate buffer, pH 10. The reaction was initiated by adding 0.1% adrenaline solution to each well. The relevant volume of buffer was added to "blank" wells, to which no test sample was added. Optical density was measured at a wavelength of 347 nm using a μQuant microplate spectrophotometer (BioTek Instruments) 4 and 8 minutes after adding adrenaline to the wells. Enzyme activity was expressed as relative units / min / mg.

[0136] Determination of catalase activity To determine catalase activity, a spectrophotometric method was used, which relies on the ability of hydrogen peroxide to form a stable colored complex with molybdenum salts. Korolyuk MA et al., Lab. Business. -1988. -No.1.-P.44-67. During incubation, the concentration of hydrogen peroxide decreased due to enzymatic activity mediated by catalase in the test sample. A 4% ammonium molybdate solution and 0.03% hydrogen peroxide were used. The reaction was initiated by adding the test sample to the 0.03% hydrogen peroxide. An appropriate volume of distilled water was added to the blank sample instead of protein. The reaction was stopped after 10 minutes by adding 4% ammonium molybdate solution to the incubation medium. Optical density was measured at a wavelength of 410 nm using a μQuant microplate spectrophotometer (BioTek Instruments). Catalase activity was calculated using a calibration curve and estimated as μmol HO / mg protein x min.

[0137] Determination of conjugated diene and Schiff base levels in serum and adipose tissue homogenates Aliquots of test samples containing 0.1–0.5 mg of protein were placed in tight-fitting glass homogenizers, to which a 1:1 heptane / isopropyl alcohol mixture was added, and the mixture was homogenized for 10 minutes. The samples were then centrifuged at 1000 g for 15 minutes in test tubes closed with tight-fitting stoppers. The supernatant fraction was carefully collected, and distilled water was added to separate the heptane and isopropyl alcohol phases. The level of Schiff base was determined in the upper heptane phase by measuring the optical density of the sample using a spectrophotometer at an excitation wavelength of 360 nm and an emission wavelength of 420 nm. The level of Schiff base was expressed as units per mg of protein.

[0138] To determine the level of conjugated dienes, an aliquot of the heptane phase to which 96% ethanol had been added was taken and the sample was thoroughly mixed. The optical density of the sample was measured at a wavelength of 233 nm using a spectrophotometer. The level of conjugated dienes was determined based on the molar extinction coefficient (2.2 × 10) of conjugated dienes generated when polyunsaturated higher fatty acids are oxidized. 5 cm -1 ×M -1 ) and estimated in nmol per mg of protein. Nedzvetsky V et al., J Diabetes Metab. 2012;3(8): 1-9.

[0139] Determination of serum levels of TBA activity products and adipose tissue homogenates An aliquot of the test sample was added to the sample, and an equal volume of 17% trichloroacetic acid (TCA) was added. The sample was centrifuged at 1000 g for 15 minutes. Nedzvetsky V et al., J Diabetes Metab. 2012;3(8): 1-9. A solution of 0.8% thiobarbituric acid was added to the supernatant, and color was developed after incubation in a boiling water bath for 10 minutes. The optical density of the sample was measured at a wavelength of 532 nm using a spectrophotometer. The concentration of the TBA activity product was expressed in nmol per mg of protein and calculated using the molar extinction coefficient (1.56 × 10 5 cм -1 ×M -1 ) was calculated using

[0140] Determination of the levels of products of oxidative modification of proteins The estimation of the intensity of oxidative modification of proteins is based on the reaction between protein carbonyls and Schiff bases and 2,4-dinitrophenylhydrazine (DNPH) with the formation of 2,4-dinitrophenylhydrazones, which are neutral and basic in nature. Vartanyan LS, Gurevich SM Biochemistry.-1989.-Vol.54, No.6.-P.1020-1025.

[0141] An aliquot of the test sample (0.2 mg of protein) was added to a test tube containing 0.15 M potassium phosphate buffer, pH 7.4. Protein was precipitated by adding 20% ​​TCA solution. After centrifuging the sample at 1000 g for 15 minutes, a 0.1 M solution of 2,4-DNPH in 2 M HCl was added to the denatured protein precipitate. After incubation at room temperature for 1 hour, the precipitate was washed three times with an ethanol:ethyl acetate (1:1) mixture to remove lipids and 2,4-DNPH (not bound to carbonyl). The washed precipitate was dried and dissolved in 8 M urea in a boiling water bath for 10 minutes.

[0142] To determine the aldehyde and ketone products of oxidative modification of proteins, the optical density was measured at wavelengths of 356 nm and 370 nm, respectively, and the obtained values ​​were recalculated using the appropriate molar extinction coefficients.

[0143] Determination of serotonin and tryptophan levels in brain and duodenal homogenates and serum Aliquots of serum and tissue homogenates were mixed with 0.4 M perchloric acid in a 1:5 ratio to precipitate proteins. The samples were incubated at 4°C for 60 minutes and then centrifuged at 800 g for 5 minutes in a refrigerated centrifuge at 4°C. After phase separation, the supernatant was collected and the pH was adjusted to 5-6 with 2 M KOH. The sample was reprecipitated by centrifugation. The supernatant was applied to a KM-Sepharose column pre-equilibrated with 0.01 M sodium phosphate buffer, pH 6.2. Bound material was eluted at room temperature using Buffer 1 (0.01 M sodium phosphate buffer, pH 6.2) and Buffer 2 (0.03 M sodium phosphate buffer, pH 6.2). Tryptophan was eluted using Buffer 1, and serotonin was eluted using Buffer 2.

[0144] Tryptophan levels were measured using a spectrofluorometer at an excitation wavelength of 295 nm and an absorption wavelength of 550 nm against blank samples containing the corresponding volume of distilled water instead of the test sample.

[0145] Serotonin levels were measured using a spectrofluorometer at an excitation wavelength of 359 nm and an absorption wavelength of 485 nm in blank samples containing the corresponding amount of distilled water instead of the test sample. Gaitonde MK / / Biochem. S.-1974.-Vol.139.-P.625-631. Maksymenko EG, Savchenko VN / / Visnyk of VN Karazin Kharkiv National University. Medicine.-2000.-1, No.494.-P.40-43. H. Weissbach et al., / J Biol Chem / / -1957.-Vol.230, No.2.-P.865-71.

[0146] Determination of tryptophan hydroxylase activity in brain and duodenal homogenates Tryptophan hydroxylase activity was determined as described by Donald M. Kuhn et al., Biochemistry.-1980.-Vol.77.-P.4688-4691. Tissue homogenates were thawed at room temperature and centrifuged at 12,000 g for 30 minutes. The supernatant was used in further studies.

[0147] An aliquot of the supernatant was added to an incubation medium prepared in an Eppendorf microcentrifuge tube containing 500 mM Tris-HCl, pH 7.4, 20 mM dithiotrietol, 1 mM CaCl2, 4 mM L-tryptophan, and 50 μg catalase. The sample was incubated at 37°C for 15 minutes in a thermostat. The reaction was stopped by precipitating the proteins with 6 M HClO4. To separate the precipitated proteins, the sample was centrifuged at 600 g for 5 minutes.

[0148] The optical density of the samples was measured at 295 / 540 nm using a spectrofluorometer. Blank samples containing incubation medium and distilled water were used as controls.

[0149] Determination of indoleamine-2,3-dioxygenase activity in brain and duodenal homogenates Indoleamine-2,3-dioxygenase activity was determined as described in Y. Kudo, CAR Boyd, IL Sargent et al. / / Mol. Human reproduction.-2000.-Vol.6, N4.-P.369-374. Tissue homogenates were thawed at room temperature and centrifuged at 12,000 g for 30 minutes. The supernatant was used in further studies.

[0150] An aliquot of the supernatant was added to an incubation medium prepared in an Eppendorf microcentrifuge tube containing 100 mM potassium phosphate buffer, pH 7.5, 5 mM L-tryptophan, 10 mM ascorbate, 0.2 mM methylene blue, and 50 μg catalase. The sample was incubated at 37°C for 30 minutes in a thermostat. The reaction was terminated by precipitating the proteins with 10% trichloroacetic acid. To separate the precipitated proteins, the sample was centrifuged at 600 g for 5 minutes. 1 M Tris-HCl, pH 7.0, was then added to the supernatant aliquot.

[0151] The optical density of the samples was measured at 360 nm using a spectrofluorometer against blank samples containing incubation medium and distilled water.

[0152] Determination of serum monoamine oxidase activity Serum monoamine oxidase activity was determined using the method described by Balakleevsky AI / / Lab. business.-1976.-3.-P.151-152. This method involves the formation of benzaldehyde from benzylamine hydrochloride under the action of MAO. Benzaldehyde interacts with 2,3-dinitrophenylhydrazine to form an insoluble hydrazone, which can be precipitated by centrifugation. The hydrazone precipitate then forms a stable raspberry-colored compound in alkaline medium, the content of which can be determined spectrophotometrically.

[0153] Incubation medium was prepared in an Eppendorf microcentrifuge tube containing 0.2 M phosphate buffer, pH 7.4, distilled water, and a 1% solution of benzylamine hydrochloride. Blank samples did not contain benzylamine hydrochloride. The reaction was initiated by adding an aliquot of serum. The samples were incubated at 37°C for 3 hours in a thermostat. The reaction was terminated by precipitating the protein with 10% trichloroacetic acid. To separate the precipitated protein, the samples were centrifuged at 600g for 5 minutes. A 0.1% solution of 2,3-dinitrophenylhydrazine prepared in 2 M HCl was added to the resulting supernatant. The samples were vortexed and incubated at room temperature for 25 minutes. The samples were then centrifuged at 600g for 25 minutes to precipitate the hydrazone. 3 M NaOH and 96% ethanol were added sequentially to the hydrazone precipitate, resulting in the development of a raspberry color.

[0154] The optical density of the samples was measured using a spectrofluorometer with excitation at 460 nm for ethanol.

[0155] Morphofunctional analysis of pancreatic and liver tissues At the end of the experiment, the animals were euthanized by decapitation. The liver and pancreas, cut to 0.5-0.5 cm in size, were immediately placed in fixative. The organs were fixed in a 4% solution of paraformaldehyde at 25°C for 72 hours. After fixation, the pieces were rinsed with tap water. The material was then dehydrated by passing the organ pieces through increasing concentrations of alcohol (70%, 80%, 90%, and 96%), leaving them in each concentration for one day. Finally, after replacing the water with 96% alcohol, the material was placed in dioxane for 15 minutes, followed by xylene for 15 minutes. After complete clearing, the material was placed in a thermostatically controlled paraffin bath (a 1:1 mixture of paraffin and xylene) at 37°C for 30 minutes. The material was then immersed in two changes of paraffin (30-35 minutes) at 56°C to prepare paraffin blocks.

[0156] Serial 5 μm thick tissue sections of the tissue were cut using an MS-2 sliding microtome and placed on glass slides treated with a 1:1 mixture of protein and glycerol.

[0157] Dried preparations were stained with hematoxylin and eosin. Prior to staining, the sections were dewaxed in two changes of xylene for 5 minutes, then passed through decreasing strength alcohols (96%, 90%, 80%, and 70%, each for 3 minutes), and finally distilled water for 5 minutes. The sections were stained with Boehmer's hematoxylin for 1.5 minutes, then washed in running water for 15–20 minutes, and stained with eosin for 1 minute. Once stained, the sections were again dehydrated in 70% and 96% alcohol (30 seconds each) and cleared in dioxane and xylene for 2.5 minutes. The stained sections were surrounded with Canada balsam and covered with a coverslip. The cell nuclei were blue-purple, and the cytoplasm was pink.

[0158] To perform histochemical reactions to determine the level of liver fibrosis, we used Van Gierson's picrofuchsin staining. To do this, sections were first immersed in water and then restained with Boehmer's hematoxylin for 3–4 minutes. Next, sections were rinsed in distilled water and stained with Van Gierson's picrofuchsin for 3 minutes. Once stained, the sections were rinsed in distilled water, dehydrated in 96% alcohol, cleared with dioxane and xylene, and placed in balsam under a coverslip. The hepatocyte nuclei were dark brown, collagen fibers were red, and cytoplasm was yellow. All parameters were measured using ImageJ software.

[0159] Protein concentration determination Protein concentration was measured using the Bradford protein assay. Bradford MM. Anal Biochem. 1976;86:193-200. To measure protein concentration, 10% NaOH, distilled water, and Bradford reagent were added to the sample. Bradford reagent was prepared by mixing the initial solution (95% ethanol, 85% H3PO4, and Coomassie Brilliant Blue dye) with 95% ethanol and 85% H3PO4 and adjusting the resulting mixture to the desired volume with distilled water.

[0160] Absorbance was measured spectrophotometrically at 595 nm for control samples containing distilled water instead of the test samples. Protein concentrations were determined using a calibration curve and expressed in mg / ml.

[0161] Statistical treatment of the results Statistical analysis of the results was performed using the methods of variance statistics and correlation analysis using OriginLab Origin® Pro 9.1 and StatSoft STaStica® 10 software. Brandt Z. Statistical methods for observations. -M.:Mir, 1975.-312p. The hypothesis of normal distribution of the samples was tested using the Shapiro-Wilk test. If the samples met the criteria for normal distribution, the significance of differences between samples was determined using Student's t-test. If the samples did not meet the criteria for normal distribution, the significance of differences between samples was determined using the Mann-Whitney U test. Differences were considered statistically significant if p<0.05.

[0162] Results and Discussion Biochemical and anthropometric effects of Zn-64 stable isotope in aspartic acid form in animal models of obesity According to modern concepts, adipose tissue, which produces a wide range of biologically active substances, is actively involved in the pathogenesis of obesity. Therefore, overweight resulting from increased fat deposition is not only seen as a consequence of metabolic disorders during the development of obesity, but also as an important factor that causes and greatly complicates the course of this disease and contributes to the development of many obesity-related disorders.

[0163] To evaluate the effect of Zn-64 stable isotope aspartate on the development of obesity induced by the intake of a high-fat diet, several anthropometric measurements were evaluated in animal models of obesity and in animals treated with Zn-64 stable isotope aspartate.

[0164] A characteristic morphological sign of the onset of obesity is a significant increase in body weight due to the accumulation and redistribution of adipose tissue. To confirm the onset of obesity, the body mass index (BMI) or Quetelet index, calculated by dividing body weight (kilograms) by the square of height (meters), was first determined. Novelli E., Diniz Y., Galhardi C. Anthropometric parameters and markers of obesity in rats / / Laboratory Animals. -2007. -No. 41. -P. 111-119. BMI allows for the evaluation of the weight-height relationship and, thereby, indirectly assessing whether a person is underweight, normal, or overweight. Furthermore, BMI is used as an integral value, which allows for characterization of body composition and the degree of adipose tissue deposition. This is because the characteristics of adipose tissue distribution within the body determine the risk of developing obesity-related metabolic complications, which must be taken into consideration when examining obese patients. BMI is used not only to classify obesity but also to assess the risk of developing obesity-related diseases.

[0165] Data obtained during the study (Table 8) showed that at week 10 of the study, the mean body mass index of the control animals was 0.60 g / cm 2This value was shown to be within the reference range for animals of this age group. Novelli E., Diniz Y., Galhardi C. Anthropometrical parameters and markers of obesity in rats / / Laboratory Animals. -2007. -No.41. -P.111-119. The BMI of animals on a high-fat diet was 0.71 g / cm 2 compared with the BMI of animals in the control group. 2 The body mass index of rats administered Zn-64 stable isotope in aspartic acid form during the experiment was lower than that of obese animals, but was higher than the control value (0.65 g / cm). 2 It should be noted that the levels were slightly higher than those in the control group. The results obtained indicate that Zn-64 stable isotope aspartate has a positive effect on the general metabolic state of animals and lay the foundation for further studies aimed at elucidating the mechanism of action of Zn-64 stable isotope aspartate on obesity.

[0166] Because BMI is calculated based on body weight, a decrease in BMI may be directly related to the lower body weight of animals administered Zn-64 stable isotope in aspartate form. Therefore, we further investigated whether administration of Zn-64 stable isotope in aspartate form affects body weight and weight gain in an animal model of obesity. The data obtained in the experiment (Figure 5) show that the kinetics of weight gain by animals in the experimental groups were significantly different. Thus, animals maintained on a high-fat diet and administered Zn-64 stable isotope in aspartate form gained less weight than animals administered only a high-fat diet. Starting from the fourth week of the experiment, a particularly striking difference was observed in the weight gain of animals in both groups. The weight gain of animals on a high-fat diet reached 103% by the end of the experiment, while animals receiving intragastric injections of Zn-64 stable isotope in aspartate form weighed approximately the same as animals in the control group (62%).

[0167] The development of obesity, resulting from disruptions in the coordinated functioning of the body's many neurotransmitters and hormonal systems, is known to lead to disorders in appetite control and satiety regulation, promoting excessive food intake and often accompanied by the development of binge eating (a state of abnormally high appetite corresponding to food energy exceeding the body's energy needs). L. Zhou, G. Sutton, J. Rochford. / / Cell Metabolism. -2007. -Vol. 6, No. 5. -P.398-405.

[0168] To elucidate the possible mechanism of the weight loss effect in animals administered Zn-64 stable isotope in aspartic acid form, the amount of food consumed by the animals was analyzed. Table 8.

[0169] Comparing the calculated data for all experimental groups, we found no significant difference in the average amount of food consumed by the animals per day. Thus, the control and obese animals consumed approximately 35g of food per day. However, it should be noted that while the control animals were maintained on a standard diet, the animals in the diet-induced obesity model group were fed a specially formulated high-fat diet with significantly higher calorie content.

[0170] [Table 8]

[0171] Analysis of the results, taking into account the calorie content of the food consumed by the animals, shows significant differences. Although the control and diet-induced obesity model animals ate the same amount of food, the calorie content of the food differed by almost two-fold. The results obtained from the group administered Zn-64 stable isotope in aspartic acid form appear to be particularly interesting. Thus, the control and diet-induced obesity model animals ate smaller amounts of standard and high-fat diets, respectively.

[0172] The dynamics of the caloric content of the food consumed by the animals during the 10 weeks of the experiment are shown in Figure 6.

[0173] The data suggest that Zn-64 stable isotope aspartate affects satiety, as animals with free access to food and injected with Zn-64 stable isotope aspartate consumed significantly less food than animals fed only a high-fat diet. The reduced amount of food consumed, and consequently the slight weight gain in animals receiving Zn-64 stable isotope aspartate compared with animals in the diet-induced model, may be explained by both direct and indirect effects of zinc on energy homeostasis.

[0174] Thus, to summarize the results of this phase of the study, administration of Zn-64 stable isotope in aspartate form caused a decrease in the amount of food consumed per day, and therefore a less pronounced weight gain in the animals, and a normalization of their body mass index, compared to similar values ​​in obese animals not treated with Zn-64 stable isotope in aspartate form.

[0175] Early detection of zinc deficiency is of utmost importance to prevent the onset and development of metabolic disorders. The experimental sign of zinc deficiency is a decrease in its level in plasma (serum), but plasma zinc levels are unstable and are affected by many factors.

[0176] Other approaches to determining zinc status are based on measuring plasma (serum) concentrations of zinc-dependent proteins, primarily enzymes such as carbonic anhydrase, superoxide dismutase, lactate dehydrogenase, and alkaline phosphatase, as well as the serum retinol-binding protein, metallothionein. One of the earliest markers of zinc deficiency is a decrease in serum alkaline phosphatase and carbonic anhydrase activity. As a result, stress ulcers, caused by a high content of zinc-containing enzymes such as carbonic anhydrase in the mucosa, develop in the gastrointestinal tract. Therefore, to indirectly determine whether the development of obesity is accompanied by changes in zinc status, we studied alkaline phosphatase activity in the serum of obese animals and animals treated with the stable isotope Zn-64 in the aspartate form.

[0177] A significant decrease in the activity of this enzyme was observed in animals maintained on a high-fat diet (Table 9). Thus, the enzyme activity in these animals was 1.5-fold lower than in the control group. In animals treated with Zn-64 stable isotope in aspartic acid form, alkaline phosphatase activity was higher than in both the diet-induced obesity model and the control group.

[0178] The results obtained therefore indirectly confirm zinc deficiency in animal models of obesity and normal serum zinc levels in animals administered Zn-64 stable isotope in aspartate form.

[0179] The gastrointestinal tract maintains whole-body zinc homeostasis. The human body has no true reservoir of this trace element. Zinc absorbed from the intestine enters the bloodstream. Whole blood contains approximately 7–8 mg / L of zinc, and approximately two-thirds of this amount is transported by red blood cells. In plasma, approximately 80% of zinc is bound to albumin, and the remaining 20% ​​is bound to β2-macroglobulin and transferrin. Published data confirm that levels of this trace element depend on the plasma albumin concentration. Brown, KH International Zinc Nutrition Consultative Group (IZiNCG) technical document #1. Assessment of risk of zinc deficiency in populations and options for its control / KH Brown, JA Rivera, Z. Bhutta [et al.] / / Food Nutr. Bull. -2004. -Vol.25. -P.99-203.

[0180] Therefore, we further investigated albumin levels in untreated animal models of obesity and in obese animals treated with Zn-64 stable isotope aspartate. The available data from the experiments indicate that the pathogenesis of obesity is accompanied by a decrease in serum albumin levels in animals. At the same time, administration of Zn-64 stable isotope aspartate did not affect albumin levels, which remained similar to those in untreated obese animals (Table 9).

[0181] [Table 9]

[0182] Considering that albumin acts as the main transport protein for zinc, a decrease in its concentration would cause disruption of the timely delivery of zinc to organs, including the liver, where the synthesis of major zinc-containing proteins occurs.

[0183] In general, this result is in full agreement with the reduction in alkaline phosphatase activity established above.

[0184] Zn-64 stable isotope aspartate was also found to have a positive effect on lipid metabolism in the body. Animals fed a high-fat diet and treated with Zn-64 stable isotope aspartate had serum triglyceride, cholesterol, and free fatty acid levels reduced to levels similar to those of control animals.

[0185] The pathogenesis of obesity, which results from metabolic disorders, primarily those of carbohydrate metabolism, is usually accompanied by increased glucose levels, which, if maintained for a long period of time, initiate many pathological processes and become a key factor in inducing insulin resistance and the development of diabetes. It is now well established that there is a relationship between changes in trace element levels, particularly zinc, and the development of prediabetes and, in the absence of appropriate pharmacological correction, diabetes. Research has shown that, while the concentrations of most trace elements in the body remain constant, zinc levels are reduced in the serum of women with prediabetes. This element is also known to play a key role in insulin synthesis in pancreatic beta cells and to increase tissue sensitivity to this hormone. Chausmer, A.B. Zinc, insulin and diabetes. J. Am. Coll. Nutr. 1998, 17, 109-115.

[0186] Considering the above, the effect of Zn-64 stable isotope in aspartic acid form on serum glucose and insulin levels in animals on a high-fat diet was investigated.

[0187] Based on the literature, a fasting blood glucose level within the range of 3.5–5.5 mmol / L is considered normal. An increase in this value to 7.0 mmol / L or greater over a period of time is considered a state of hyperglycemia and may be a predictor of the development of diabetes mellitus.

[0188] The serum glucose levels of animals from the control group and those from the control group administered Zn-64 stable isotope aspartate were within the reference range (Table 10). The development of obesity was accompanied by a slight increase in glucose levels that was normalized by administration of Zn-64 stable isotope aspartate.

[0189] The effect of Zn-64 stable isotope aspartate on glucose levels may be directly related to its ability to stimulate the translocation of glucose transporters from internal cellular compartments to the adipocyte membrane, contributing to enhanced intracellular glucose transport. Zn-64 stable isotope aspartate has also been shown to increase tyrosine phosphorylation of the insulin receptor β subunit, thereby improving glucose transport in the absence of insulin. This data indicates that Zn-64 stable isotope aspartate can act as an inhibitor of tyrosine-1B-phosphatase, an enzyme involved in the suppression of insulin signaling.

[0190] [Table 10]

[0191] Increased glucose levels in obesity may be the result of decreased insulin secretion in pancreatic beta cells or insufficient insulin utilization by the body's tissues. High glucose levels in blood and other body fluids cause increased osmotic pressure, resulting in osmotic diuresis (increased water and salt loss through the kidneys), which leads to dehydration and a deficiency of sodium, potassium, calcium, and magnesium cations, chloride anions, phosphates, and hydrocarbonates. Furthermore, elevated glucose levels cause nonenzymatic glycosylation of proteins and lipids, the intensity of which is directly proportional to glucose concentration. This results in impaired function of many important proteins and various pathological changes in the body. (Skybchyk V. / / Ukrainian Medical Newspaper. - 2006. - No. 6. - pp. 61-68. Campos. / / Postgraduate Medicine. - 2012. - No. 126. - pp. 90-97.)

[0192] Given the changes in the animals' serum glucose levels, the next step in this study was to determine insulin levels. Furthermore, serum insulin levels are an important parameter in diagnosing the development of insulin resistance and prediabetes. In obesity and metabolic syndrome, hyperinsulinemia is often caused by excessive insulin production and secretion in pancreatic beta cells, which is a compensatory response to the decreased sensitivity of peripheral tissues to insulin action. However, in the later stages of the development of type 2 diabetes mellitus, serum insulin levels decrease significantly, which is directly related to a decrease in the ability of beta cells to produce insulin, impaired proinsulin processing and mature insulin secretion, and a decrease in the number of secretory cells and amyloid deposition in the islets of Langerhans. At the same time, beta cell dysfunction during development leads to further progression of diabetes mellitus. Boden. / / Diabetes.-1997.-Vol.46, No.3.-P.3-10. Robertson RP et al. / / Diabetes Mellitus.-2000.-P.125-132.

[0193] Increased serum insulin levels were observed in obese animals, and a normalizing effect of Zn-64 stable isotope aspartate on the studied parameters was observed in the group of animals maintained on a high-fat diet and administered Zn-64 stable isotope aspartate. It should be emphasized that administration of Zn-64 stable isotope aspartate to control animals resulted in a slight increase in insulin levels.

[0194] The effect of the aspartate form of Zn-64 stable isotope on insulin levels is associated with the direct involvement of this trace element in the synthesis, deposition, and release of insulin from the beta cells of the islets of Langerhans, and may also be related to its ability to inhibit insulin action. Zinc is known to be involved in the formation of hexameric proinsulin and contribute to insulin crystallization. Zinc ions have been shown to contribute to insulin uptake into transport complexes, ensuring delivery to target cells. Another possible mechanism was established in 1980 by Caulston and Dandona, who demonstrated that zinc exerts a potent, stimulatory effect on lipogenesis in rat adipocytes that is independent of and complementary to insulin action. This finding confirmed the involvement of zinc in regulating insulin action, as this cation is secreted along with insulin in response to high glucose levels.

[0195] Furthermore, zinc plays an important role in protecting insulin and pancreatic beta cells from free radicals because it is a structural component of antioxidant enzymes such as superoxide dismutase and a competitor for redox metals such as iron. Zinc stimulates the expression of metallothionein in pancreatic cells, which is known to be involved in the neutralization of many reactive oxygen metabolites and may prevent beta cell destruction.

[0196] Given the importance of maintaining physiological levels of zinc in the body to ensure insulin synthesis and secretion, as well as its crucial role in pancreatic function, we further investigated the effect of Zn-64 stable isotope in aspartate form on the overall morphofunctional properties of the pancreas.

[0197] Effects of Zn-64 stable isotope in aspartic acid form on morphofunctional properties of the pancreas and liver in an animal model of obesity The pancreas is a mixed gland with both exocrine and endocrine functions. Most of the pancreas is composed of exocrine cells arranged in acini. Secretions from the acini exit the pancreas through intercalated intralobular and interlobular ducts and the main pancreatic duct. The exocrine cell clusters of control animals, the acini (Figures 7A-7F), have a typical structure: the apical pole contains granular, brightly eosinophilic cytoplasm, while the basal pole contains strongly basophilic nuclei.

[0198] In the DIO (obese) animal model (Figures 7A-7F), acini with less prominent eosinophilic apical cytoplasm were present (Figures 7A-7F, arrows), which may be due to the accumulation of lipid inclusions and pancreatic steatosis.

[0199] Administration of Zn-64 stable isotope in aspartate form to rats fed a standard diet did not alter the morphology of exocrine cells (Figures 8A-8F). After administration of Zn-64 stable isotope in aspartate form to obese rats (Figures 8A-8F), no fatty degeneration was observed.

[0200] The endocrine portion of the pancreas consists of diffusely located islets of Langerhans. Morphometric studies of the functional state of the endocrine pancreas during the development of induced obesity showed clear differences between the values ​​obtained from all groups (Figure 9). The cross-sectional area of ​​the islets of Langerhans was significantly reduced (60%) in animals from the obese group, indicating a significant decrease in the functional activity of the endocrine pancreas. After administration of Zn-64 stable isotope in aspartate form to obese rats, the cross-sectional area of ​​the islets of Langerhans increased by 43% compared to the obese group, but still did not reach control levels (29% lower than the control value). Administration of Zn-64 stable isotope in aspartate form to rats fed a standard diet significantly reduced this value by 39% compared to the control group.

[0201] Since there is a direct correlation between morphological and functional indicators of pancreatic condition, the obtained data show that in a diet-induced obese rat model, the pancreatic hormone synthesis activity is significantly reduced, whereas administration of Zn-64 stable isotope in aspartic acid form significantly increases it, although it does not completely restore it to the level observed in the control group. Furthermore, against the background of the development of obesity, an improvement in the condition of the exocrine part of the pancreas after administration of the test substance was recorded, as evidenced by the disappearance of fatty degeneration, without any significant effect on the exocrine cells of rats fed a standard diet.

[0202] The liver of a control rat (Figures 10A-10D) has a classic lobular organization with a central vein running along the axis of each lobule. Polygonal hepatocytes, each with well-defined nuclei containing several nucleoli, are arranged in orderly hepatocyte cords extending from the central vein. Binucleate hepatocytes are also observed.

[0203] In the obese animals (Figures 10A-10D), the shape of hepatocytes changed from polygonal to rounded due to the deposition of lipid inclusions, a sign of hepatic steatosis. The structure of hepatic cords became disorganized, and the number of binucleated cells per field decreased.

[0204] Administration of Zn-64 stable isotope in the aspartate form to obese rats (Figures 11A-11D) restored the structure of hepatic cords, and most hepatocytes, with polygonal morphology, showed no signs of fatty degeneration. Binucleated cells were frequently observed. Administration of Zn-64 stable isotope in the aspartate form to rats on a standard diet (Figures 11A-11D) did not result in any changes in hepatocyte morphology or hepatic lobule structure.

[0205] With the onset of diet-induced obesity, significant morphometric changes occurred in hepatocytes (Figures 12A-C). Thus, in obese animals, nuclear area decreased by 25% (evidence of decreased nuclear transcriptional activity, also confirmed by their dark color and homogeneous structure without nuclei within the field of view), whereas hepatocyte area increased by 48%, due to the deposition of numerous lipid inclusions. At the same time, the nuclear-to-cytoplasmic ratio decreased significantly (45%), and its low level indicates a decrease in the functional activity of the cells.

[0206] Administration of the test substance to obese rats improved their morphometric parameters: thus, the area of ​​hepatocytes in animals treated with Zn-64 stable isotope in aspartic acid form was reduced by 41% compared to untreated obese animal models (only a 13% increase compared to control values, which is a sign of reduced deposition of lipid inclusions by hepatocytes), and their nucleus-to-cytoplasm ratio was increased by 31% compared to the obese group (a 30% decrease compared to the control group).

[0207] However, nuclear area was reduced by 35% relative to control values ​​(i.e., 14% relative to values ​​in the obese group, which may be the result of the combined effect of two factors (high-fat diet and Zn-64 stable isotope in aspartate form) on nuclear activity.

[0208] The effect of Zn-64 stable isotope in the aspartate form on morphometric parameters in rats maintained on a standard diet consisted of a 26% decrease in nuclear area, a 12% decrease in hepatocyte area, and a 17% decrease in the nucleus-to-cytoplasm ratio.

[0209] Liver fibrosis is characterized by excessive proliferation of connective tissue and increased collagen synthesis and deposition in extracellular material. In samples from control animals (Figures 13A-13D), most collagen fibers were located in the triangular regions formed by small interlobular vessels.

[0210] Samples taken from animals in the obese group (Figures 13A-13D), similar to the control group, showed a significant increase in the number of collagen fibers in the tritubular regions formed by the perilobular capillary plexus and larger interlobular vessels.

[0211] Samples taken from obese rats injected with Zn-64 stable isotope in the aspartate form (Figure 14) show similar levels of collagen fiber deposition in the indicated locations when compared to samples from untreated obese groups. Administration of the test substance to rats fed a standard diet (Figures 14A-14D) did not cause significant changes in the amount of collagen fibers in the perilobular and interlobular capillary plexuses.

[0212] Analysis of the area occupied by collagen fibers (Figure 15) shows significant changes with the onset of induced obesity. The area of ​​collagen fiber deposition in the obese group and the group treated with Zn-64 stable isotope aspartate increased 6.25-fold and 6-fold, respectively, compared to the control group. No significant difference was observed between the untreated obese group and the obese group treated with Zn-64 stable isotope aspartate. Administration of the test substance to rats eating a standard diet significantly increased the area of ​​collagen fiber deposition (2-fold).

[0213] Thus, summarizing the results obtained, it can be stated that Zn-64 stable isotope in aspartic acid form has a positive effect on the morphofunctional properties of the pancreas and liver in animal models of obesity.

[0214] Effects of Zn-64 stable isotope in aspartic acid form on the serotonergic system in animal models of obesity Despite the proven fact that the development of obesity is primarily a result of increased caloric intake and inadequate energy expenditure, the search for new pathogenetic mechanisms of weight gain remains relevant today.

[0215] According to current thinking, obesity, regardless of its etiology, is linked to a disruption of central regulatory mechanisms that influence behavioral responses, particularly feeding behavior. The hypothalamus, primarily in its paraventricular and lateral periphery, integrates numerous impulses from the sympathetic and parasympathetic nervous systems, from the cerebral cortex and subcortical structures. Disruption of this complex regulatory cascade can lead to alterations in food intake, fat deposition, and mobilization, ultimately leading to the development of obesity.

[0216] Important neurotransmitters involved in regulating feeding behavior, particularly appetite, and influencing satiety include many biogenic amines, of which serotonin plays a crucial role. (C. Portas et al., Progress in Neurobiology. -2000. -Vol.60, No.1. -P.13-35)

[0217] According to the theory of neurochemical imbalances in the central and peripheral nervous systems, overeating is a compensatory mechanism for obtaining pleasure due to insufficient production or sensitivity of neurotransmitters.

[0218] The central serotonergic system is fundamental in regulating hunger and satiety. Experiments have shown that increasing serotonergic transmission in the brain causes a decrease in food intake. Injection of 5-HT into the periventricular nucleus of the hypothalamus in rats resulted in a feeling of satiety in the animals, whereas food intake was accompanied by an increase in serotonin production in the lateral thalamus. These two sections of the hypothalamus are thought to play opposing functions in regulating appetite: insufficient inhibition of serotonergic transmission in the lateral hypothalamus may be responsible for excessive food intake during obesity, and enhanced release of serotonin in the periventricular nucleus of the hypothalamus may contribute to stress-induced appetite loss.

[0219] The brain and intestine are the main organs that produce serotonin in animals. Because serotonin does not cross the blood-brain barrier, the serotonin synthesis system is divided into central and peripheral systems that function independently. Small amounts of the hormone are present in plasma.

[0220] Several enzyme systems are involved in the metabolic conversion of serotonin. These systems include enzymes for the synthesis and degradation of serotonin, as well as enzymes that determine the amount of tryptophan entering the kynurenine pathway. A. Meneses, G. Liy-Salmeron. / / Annual Review of Neuroscience. -2012. -No.23. -P.543-553. M. Donovan, L. Tecott. / / Frontiers in Neuroscience. -2013. -No.7. -doi: 10.3389 / fnins.2013.00036.

[0221] Tryptophan, an essential amino acid naturally produced by the body, is the direct precursor of serotonin. Tryptophan is transported from the extracellular fluid into serotonergic neurons by a nonspecific membrane transporter that is thought to be involved in the transport of other neutral amino acids (valine, leucine, and isoleucine). Therefore, the level of tryptophan within neurons and the strength of its transport depend not only on the concentration of tryptophan but also on the ratio of the concentrations of competing neutral amino acids to the concentration of tryptophan.

[0222] Serotonin is synthesized via a two-step process catalyzed by two enzyme systems. First, tryptophan is converted to 5-hydroxytryptophan, the direct precursor of serotonin synthesis, as a result of hydroxylation at the fifth position of the indole ring. The tryptophan hydroxylation reaction is catalyzed by the rate-limiting enzyme tryptophan hydroxylase (tryptophan-5-monooxygenase, EC 1.14.16.4) in the presence of both molecular oxygen and the coenzyme pterin (tetrahydrobiopterin). The rate of tryptophan hydroxylation directly depends on the availability of substrate. The second step in serotonin synthesis is decarboxylation, catalyzed by DOPA decarboxylase (also known as aromatic L-amino acid decarboxylase, EC 4.1.1.28).

[0223] In addition to the involvement of these enzymes in the synthesis of serotonin and other biologically active amines, both enzymes are actively involved in the regulation of circadian rhythms, bone remodeling, cell differentiation processes, immune response mechanisms and inflammation.

[0224] Inactivation of serotonin occurs through enzymatic degradation, primarily provided by monoamine oxidase (EC 1.4.3.4). Under the action of MAO, serotonin is converted to 5-hydroxyindaldehyde, which can then be reversibly converted to 5-hydroxytryptophol under the action of alcohol dehydrogenase. Under the action of acetaldehyde dehydrogenase, 5-hydroxyindaldehyde is irreversibly converted to 5-hydroxyindoleacetic acid, which is then excreted in urine and feces. Sandler M. et al., Clinical Pathology. -1981. -No. 34. -pp. 292-302. S. Nilsson, N. et al., Acta medica Scandinavica. -1968. -No. 184. -pp. 105-108.

[0225] Once released, serotonin influences various biological processes by binding to serotonin receptors (HTRs), whose actions are then terminated by uptake into cells via the serotonin transporter (SERT, Slc6a4).

[0226] In addition to oxidative deamination of serotonin, other pathways of serotonin metabolism are possible, such as acetylation and glucuronide and sulfate ester conjugation. There is a pathway of serotonin metabolism that involves the formation of melatonin.

[0227] Medical research has shown that impaired metabolic conversion of serotonin is often not only a consequence but also one of the main factors that cause the development of overweight and obesity. Molecular and biochemical disorders at the level of various stages of serotonin metabolism, its transport through cell membranes, and deposition mechanisms that lead to altered serotonin concentrations in both the central nervous system and the periphery may provide the pathological basis for the formation of immune-neuroendocrine imbalances and the maintenance of proinflammatory responses. Imbalances in the serotonergic system are the underlying cause of many pathological conditions and psychiatric disorders, including schizophrenia, various psychoses, depression, and anxiety. Depressive states are often accompanied by increased appetite, which leads to excessive food intake, especially of carbohydrate-rich foods.

[0228] Recent research data indicate that zinc can regulate serotonergic function via the 5-HT1A receptor (5-HT1AR); however, the exact mechanism of action remains unclear. Considering the above, we further analyzed the importance of zinc for the proper functioning of the brain's neurohormonal system, its involvement in the metabolism of tryptophan, a key molecule in serotonin synthesis, and zinc's regulatory effect on the serotonergic system. We further analyzed key indicators that allow for a general assessment of serotonin metabolism in experimental animals. To this end, we determined the levels of serotonin and tryptophan, as well as the activities of key enzymes involved in serotonin metabolism (tryptophan hydroxylase, tryptophan decarboxylase, indoleamide dehydrogenase, and monoamine oxidase) in the serum, brain, and duodenum of animals fed a high-fat diet and injected with Zn-64 stable isotope in the aspartate form.

[0229] In general, the results of this study showed a significant imbalance of the serotonergic system in obese animals occurring in both the peripheral and central serotonergic systems, which may be one of the triggering mechanisms for the development and progression of obesity.

[0230] In the peripheral system, serotonin is localized in the enterochromaffin cells of the gastrointestinal mucosa. Approximately 80-95% of the total amount of this hormone in the body is synthesized there. Serotonin is also synthesized in the pineal gland. Cote F. et al, PNAS USA. -2003. -Vol.100. -P.13525-13530. Eddahibi S. The serotonin pathway in pulmonary hypertension. / Еddahibi S., Adnot S. / / Arch. Mai. Coeur. Vaiss. -2006. -Vol.99 -P.621-625.

[0231] Serotonin synthesized in the intestine is stored in platelets; it is also present in other peripheral tissues, such as the mammary gland, liver, and bone, as well as in the beta cells of the pancreas. Peripheral serotonin is involved in the regulation of intestinal motility, vasoconstriction, and blood pressure. Serotonin also regulates plasma glucose levels, thrombus formation, cardiac rhythm, and the strength of cardiac contractions.

[0232] A significant inhibition of the peripheral serotonergic system has been established, evident in the reduced activity of all key enzymes against the background of tryptophan pool depletion. Despite the identified changes, duodenal serotonin levels in animal models of obesity significantly exceeded those in controls (Table 11).

[0233] These results are generally consistent with the current concept that intestinal serotonin correlates with the development of obesity and that intestinal serotonin levels are significantly increased in obese people. Serotonin accumulation leads to elevated serum glucose levels, thus contributing to the development of diabetes and obesity.

[0234] [Table 11]

[0235] Analysis of serum serotonin and tryptophan levels in obese animals showed a significant decrease in the concentrations of both substances, which may be a direct result of an inhibition of the response of serotonin synthesis in the enterochromaffin cells of the gastrointestinal mucosa (Table 12).

[0236] An additional factor that contributes to low serotonin levels in the blood is the activation of monoamine oxidase.

[0237] Modulation of the peripheral serotonergic system may reduce obesity and increase insulin sensitivity, and therefore may be an excellent anti-obesity therapeutic strategy.

[0238] Despite the importance of maintaining physiological levels of peripheral serotonin, it is central serotonin that plays a key role in regulating energy homeostasis. An inverse relationship between central serotonin levels and food intake has been established. In the CNS, serotonin is synthesized in the hypothalamus and brainstem. It helps regulate mood, sleep-wake cycles, and diet.

[0239] Inhibition of brain serotonin synthesis via intracerebroventricular injection of p-chlorophenylalanine, an irreversible inhibitor of tryptophan hydroxylase, induces hyperphagia and weight gain in rats. Serotonin reuptake inhibitors and monoamine oxidase inhibitors reduce food intake. Thus, serotonin in the central nervous system functions as a neurotransmitter for anorexia.

[0240] [Table 12]

[0241] The pathogenesis of obesity in rats involves a significant decrease in tryptophan levels in the animals' brains, which may be caused by impaired transport of this amino acid across the blood-brain barrier (Table 13).

[0242] It is known that the transport of aromatic and branched-chain amino acids across the blood-brain barrier occurs through the participation of specific carriers and is competitive; therefore, the increase in serum concentrations of branched-chain amino acids, which is typical of obesity, affects the transport of tryptophan across the blood-brain barrier. C. Newgard, J. An, J. Bain. / / Cell Metabolism. -2009. -Vol.9, No.4. -pp.311-326. E. del Amo et al., / / European Journal of Pharmaceutical Sciences. -2008. -No.35. -pp.161-174.

[0243] [Table 13]

[0244] Given the close metabolic relationship between serotonin and tryptophan, a natural consequence of a lack of the latter is a decrease in serotonin levels.

[0245] Impairment of serotonergic transmission is one of the factors contributing to the development of depression and can be considered one of the major causes of obesity. It is known that people with congenital or acquired defects in the central serotonergic system develop subjective negative reactions to hunger, which is accompanied by a decrease in serotonin production. In such cases, even slight hunger can trigger the onset of depression. Therefore, such people consume food in amounts that exceed their physiological needs.

[0246] Thus, low serotonin levels in the ventromedial and paraventricular nuclei of the hypothalamus induce excessive food intake and cause insulin hypersecretion, which leads to a decrease in the sensitivity of peripheral tissues to the action of this hormone and the development of insulin resistance.

[0247] The changes in the activity of enzymes involved in the serotonin metabolic pathway identified during this study contribute to further depletion of serotonin stores in the brain. Thus, a decrease in the activity of tryptophan hydroxylase, the enzyme that limits the process of serotonin synthesis, was observed, along with an increase in the activity of monoamine oxidase, the enzyme responsible for serotonin degradation.

[0248] Increased activity of indoleamine-2,3-dioxygenase indicates activation of alternative tryptophan metabolic pathways, which not only contribute to further depletion of the pool of this amino acid but also serve as a source for the formation of numerous neurotoxic compounds.

[0249] Administration of Zn-64 stable isotope in the aspartate form to animals fed a high-fat diet resulted in normalization of most experimental parameters. Thus, increased serotonin levels in the brain were observed due to increased tryptophan levels, and thus activation of serotonin synthesis against the background of inhibition of alternative methods of its conversion and a decrease in the rate of serotonin degradation by monoamine oxidase. Similar effects were observed in the duodenum and serum.

[0250] Thus, the effects of zinc are complex and occur at the level of both central and peripheral serotonergic system function. These results support the therapeutic use of zinc preparations, either as monotherapy or in combination with other drugs, to improve overall metabolic status in the prevention of obesity and obesity-related disorders.

[0251] Effect of Zn-64 stable isotope in aspartic acid form on the pro-oxidant-antioxidant balance in animal models of obesity Free radical reactions, necessary for the formation of enzymes, activation of transcription factors, and oxidative and bactericidal protection of xenobiotics, are fundamental for the normal functioning of cells. Furthermore, they are involved in gene expression, transmit hormonal and cellular signals, and regulate the process of cellular regeneration. Thus, reactive oxygen and nitrogen species are naturally produced in the human body and are important by-products of metabolic processes. Antioxidants maintain the level of free radicals within physiological limits. A balance between antioxidant defenses and free radical oxidation is necessary for proper cellular functioning. When the amount of free radicals exceeds the activity of antioxidant defenses, a phenomenon known as oxidative stress occurs.

[0252] Oxidative stress and the resulting tissue damage and cell death contribute to many pathological conditions. Excessive production of free radicals and / or depletion of defense systems leads to an imbalance between prooxidants and antioxidants, which in turn causes damage to intracellular protein structures, the lipid bilayer of the cell membrane, and nucleic acids. Because the lipid bilayer is a component of all cell membranes, free radical-mediated lipid peroxidation is one of the important reasons for cell membrane damage and subsequent cell death. Degradation of membrane lipids causes increased membrane fluidity and permeability to ions, disrupting cellular homeostasis as a whole. Products of free radical oxidation (e.g., 4-hydroxyalkenes, malondialdehyde) are highly mutagenic and cytotoxic.

[0253] Epidemiological, clinical, and animal studies have shown that obesity is associated with altered redox status and increased metabolic risk. In this case, oxidative stress is not only a consequence but can also be a trigger for the development of disorders in obese people. One of the factors that causes adipocyte dysfunction is oxidative stress. Excess oxidants, initially produced by the increasing mass of adipose tissue, sensitively stimulate the oxidative stress signaling pathway mediated by the transcription factors NF-kB and JNK and p38-MAPK kinases, activating many protein kinases (e.g., PKB, PKC).

[0254] Furthermore, oxidative stress activates differentiation of preadipocytes and stimulates hypertrophy of mature adipocytes. Increased production of ROS in accumulated adipose tissue further leads to the induction of oxidative stress in the bloodstream, contributing to the propagation of oxidative stress to organs distant from fat depots.

[0255] Elevated glucose levels, together with impaired lipid metabolism and elevated free fatty acid concentrations, determine the mechanisms underlying the formation and progression of obesity-specific oxidative stress. Hyperglycemia-induced oxidative stress is thought to occur as a result of direct activation of ROS-generating reactions and as a result of disruption of cellular redox homeostasis.

[0256] The next phase of this study aimed to evaluate the pro-oxidant-antioxidant balance in an animal model of obesity treated with Zn-64 stable isotope in aspartate form.

[0257] The concentration of lipid peroxidation products (LOPs) serves as a valuable criterion, allowing conclusions to be drawn about the intensity of the oxidation process. These include primary lipid peroxidation products (e.g., conjugated dienes) and secondary lipid peroxidation products (e.g., aldehydes, especially malonaldehyde), which are formed as a result of the cleavage of carbon-carbon double bonds in the carbon skeleton of oxidized molecules. Subsequently, the initiation of lipid peroxidation leads to the formation of conjugated Schiff bases of phospholipids and malonaldehyde-like products, which disrupt the ordered orientation of phospholipid molecules and affect lipoprotein interactions and the organization of basement membranes.

[0258] Taking the above into account, the concentrations of primary LOP-conjugated dienes (CD), secondary products-TBA reactive substances (TBARS) and end products-Schiff bases (SB) were determined in animals treated with Zn-64 stable isotope aspartate. Considering that obesity is accompanied by the development of systemic oxidative stress that covers most tissues to varying degrees and leads to the disruption of cell membrane integrity and the influx of lipid peroxidation products into the bloodstream, values ​​characterizing the state of the pro-oxidant-antioxidant system were determined and analyzed in the serum of the animals.

[0259] The increase in serum levels of primary products of free radical lipid oxidation (1.86-fold) suggests that the initial stage of lipid peroxidation is still occurring actively after 10 weeks of experimental obesity (Table 14). This result can be explained in terms of disturbances in lipid metabolism, i.e., impaired fatty acid transport processes, and therefore elevated plasma levels of free and esterified fatty acids, which are direct substrates for the action of reactive oxygen metabolic species. On the other hand, the accumulation of lipid peroxidation products in serum could be a direct consequence of a breach in the integrity of cell membranes, resulting from the oxidative destruction of their lipid components and the permeability of lipid oxidation products to the bloodstream.

[0260] An additional factor that may contribute to increased oxidative stress in the development of obesity is the significant activation of MAO (demonstrated in the previous phase of this study), an enzyme involved in ROS production.

[0261] [Table 14]

[0262] The increased levels of CD were accompanied by the accumulation of secondary POLs, i.e., TBA-reactive substances. Thus, serum levels of TBA-reactive substances in obese animals were 4.8-fold higher compared to control values. In the case of Fe+2 ascorbic acid-dependent accumulation of TBA-reactive substances, this value exceeded the results obtained in the control group by 20-fold, suggesting a significant contribution of non-enzymatic reactions in the initiation of lipid peroxidation processes to the pro-oxidant / antioxidant imbalance in obese animals.

[0263] Such a significant increase in the concentration of aldehyde POL is considered an undesirable marker because these substances can bind to proteins and form stable adducts, which can affect protein function. Furthermore, such modified proteins may have immunological properties and may result in autoantibody production.

[0264] The changes in the levels of primary and secondary products were accompanied by the accumulation of Schiff bases, end products of lipid peroxidation, which are formed as a result of the condensation of aldehydes, such as malondialdehyde, or ketones with amino groups of proteins, leading to impairment of the structural and functional characteristics of the latter. According to the obtained data (Table), a significant increase in the level of Schiff bases was observed in the serum of obese animals. Thus, this value was four times higher than in the control group. The level of end products of lipid peroxidation characterizes periods of impaired oxidative homeostasis, and therefore, given that this value was significantly increased in this study, a prolonged activation of free radical reactions may be considered.

[0265] Therefore, the elevated levels of lipid peroxidation products at 10 weeks of obesity development clearly indicate that oxidative stress is systemic and that this process is chronic, and is an unfavorable prognostic marker since these metabolites are highly toxic compounds and their negative effects are manifested at various levels, leading to damage to DNA molecules, destruction of protein molecules and glycosaminoglycans, changes in the lipid composition of cell membranes, and disruption of membrane-related processes.

[0266] Activation of lipid peroxidation processes can indirectly indicate an increase in the concentration of ROS. Excess ROS can directly activate many serine-threonine kinases, such as PKC, AKT / PKB, mTOR, GSK-3, and p38MAPK. These synergistic protein kinases reduce cellular insulin sensitivity by selective phosphorylation of serine and threonine residues in IRS molecules, contributing to the development of insulin-dependent cell resistance to this hormone.

[0267] Administration of Zn-64 stable isotope in aspartate form to animals helped normalize the levels of primary, secondary, and terminal LOP, which serves as further evidence of the ability of Zn-64 stable isotope in aspartate form to affect the overall pro-oxidant-antioxidant status of the body.

[0268] According to modern concepts, reactive oxygen species not only activate lipid peroxidation processes, but also cause the oxidative destruction of protein molecules, leading to the disruption of the higher-order structure of soluble and membrane-bound enzymes, receptors, and ion channels, ultimately leading to the loss of their biological activity (enzyme, receptor, transport, etc.). Oxidative modification of proteins and the accumulation of structurally modified molecules are important factors that can potentially contribute to the production of new antibodies and thus to the development of autoimmune reactions. Berlett BS, Stadtman ERJ Biol. Chem. -1997. -V.272, No.33.-P.20313-20316.

[0269] Under the action of ROS, the native conformation of proteins is disrupted, resulting in the formation of large protein aggregates or, conversely, fragmentation of protein molecules. Hydroxyl radicals most often cause protein aggregation, and in combination with superoxide anions, cause fragmentation with the formation of low-molecular-weight fragments. Lipid radicals can also cause fragmentation of protein molecules. The formation of carbonyl groups (aldehyde or ketone groups of amino acid residues) can serve as a marker of oxidative protein damage.

[0270] According to modern concepts, all amino acid residues in proteins can be modified, but tryptophan, tyrosine, histidine, and cysteine ​​residues are the most susceptible. ROS attack the functional groups of the amino acids that make up proteins, leading to the formation of primary amino acid radicals that can interact with neighboring amino acid residues. In general, we are faced with a complex picture of the harmful effects of ROS on protein macromolecules. The radicals formed as a result of tyrosine oxidation can interact with each other to form bityrosine crosslinks in proteins. Bityrosine crosslinks increase the protein's resistance to the action of proteases and create the conditions for the accumulation of functionally inactive proteins in the body. Tryptophan oxidation is also associated with the formation of covalent crosslinks, an additional factor that leads to the aggregation of protein molecules. Rojas VC et al., Arch. Med. Res. -1996. -V.27, No1. -P.1-6. Archakov AI, Mokhosoev IM Modification of proteins with active oxygen and their decomposition / / Biochemistry. -1989. -Vol.54, No2. -P.179-185.

[0271] Oxidative modification of proteins plays an important role in protein metabolism in the body. The accumulation of oxidized proteins is considered one of the factors regulating protein synthesis and degradation, as well as the activation of multicatalytic proteases that selectively destroy oxidized proteins. The extent of oxidative damage to protein molecules can be assessed by the accumulation of carbonyl derivatives, particularly neutral aldehyde and ketone dinitrophenyl hydrazones. Aldehyde dinitrophenyl hydrazones, detected at a wavelength of 356 nm, are early markers of protein oxidative degradation and indicate the early stage of damage to protein molecules under the action of free radicals, while ketone dinitrophenyl hydrazones, detected at 370 nm, are considered late markers of oxidative damage to proteins.

[0272] Unlike lipid peroxidation products, carbonyl derivatives are much more stable, which allows the products of oxidative modification of proteins to be considered as markers of oxidative damage in tissues.

[0273] Therefore, an increase in the number of modified proteins can be considered an early criterion of tissue damage by free radicals and a marker of depletion of the body's antioxidant defense system.

[0274] These studies revealed increased serum levels of oxidatively modified proteins in obese animal models (Table 15), accompanied by more pronounced changes in the levels of aldehyde-dinitrophenyl-hydrazones, which indicate the active stage of oxidative stress development and metabolic disorders accompanied by enhanced formation of free radicals. In general, the increased concentration of carbonyl derivatives in oxidatively modified proteins in the serum of obese animals, against the background of enhanced lipid peroxidation processes, can be considered indisputable evidence of prolonged oxidative stress. Therefore, considering the obtained data, it can be said that the development of obesity is accompanied by the activation of free radical oxidation of proteins, which is manifested by an increased amount of carbonyl derivatives formed by oxidative modification of proteins, which have absorption peaks at 356 and 370 nm.

[0275] [Table 15]

[0276] The process of protein molecule modification by reactive oxygen species occurs not only in pathological conditions. Therefore, under physiological conditions, there is a certain level of oxidatively modified proteins in cells, reflecting the balance between the rate of proteolysis of these damaged "used" molecules and the rate of their synthesis. In some cases, proteolytic enzymes degrade modified protein molecules much faster than native ones, so oxidative inactivation is a marker step that increases the protein's susceptibility to the action of proteases. Therefore, increased levels of carbonyl derivatives in obese animals not only indicate the occurrence of oxidative stress, but may also provide evidence of significant impairments in the mechanisms of control and regulation of the degradation of structurally modified proteins, particularly the proteolytic enzymes that ensure this process.

[0277] In animals fed a high-fat diet throughout the experiment and injected with Zn-64 stable isotope in the aspartic acid form, the levels of aldehyde-dinitrophenyl-hydrazones exceeded the benchmark but were lower than those in untreated obese animals. As with ketone-dinitrophenyl-hydrazones, their concentrations remained within the control range. These results correlate with the data showing a decrease in LOP levels, which may suggest a decrease in the intensity of free radical oxidation reactions.

[0278] According to modern concepts, there exists a so-called nonoxidative pathway for the formation of carbonyl derivatives in cells, which consists of the modification of protein molecules with aldehydes in addition to the modification of proteins with ROS. Thus, experiments by Burcham PC et al. showed that incubation of proteins with various aldehydes, including MDA, induces a concentration-dependent increase in the number of oxidatively modified proteins. Since administration of Zn-64 stable isotope in the aspartate form to obese animals reduced the level of LOP, the observed decrease in the degree of oxidative modification of proteins appears to be partly related to the inhibition of the nonoxidative formation of carbonyl derivatives.

[0279] The identified positive effect of zinc on the overall oxidation state may be due both to its direct influence on the process of free radical oxidation at the initiation stage of the chain reaction and its inclusion in the active centers of antioxidant enzymes. It is also important to mention the membrane-stabilizing effect of this trace element, which may also be one of the mechanisms of its antioxidant action. The role of zinc as an antioxidant is confirmed by its ability to act as an intramolecular stabilizer, preventing the formation of disulfide structures. Furthermore, zinc competitively replaces copper and iron ions, which induce the formation of free radicals.

[0280] Free radical processes are controlled and regulated by a complex multi-component and multi-level system, the antioxidant defense (AOD). Under normal physiological conditions, a balance is maintained between the level of free radical oxidation reactions and the activity of this system, which ensures that lipid peroxidation processes are maintained at a steady and fairly low level.

[0281] The antioxidant defense system consists of non-enzymatic and enzymatic units. Non-enzymatic antioxidants provide primarily for the rapid inactivation of oxygen and nitrogen free radicals, while enzymatic antioxidants are referred to as the body's final system of long-term defense.

[0282] Superoxide dismutase (EC 1.15.1.1) is one of the key enzymes in SOD. This enzyme catalyzes the neutralization of superoxide anion radicals by dismutation of hydrogen peroxide and triplet oxygen into less reactive molecules. SOD is the only one of the most active antioxidant enzymes that interrupts the chain of oxygen-dependent free radical reactions in aerobic cells. Poberezkina NB, Osinskaya LF Biological role of superoxide dismutase / / Ukr. biohim journal. -1989. -Vol.61, No2. -P.14-27. Dudochnik LB, Tikhaze AK, Alesenko AV et al. Change in the activity of superoxide dismutase and glutahione peroxidase in the process of lipid peroxidation intensification in liver ischemia / / Bul. exp. biol. Med. -1981. -Vol.XCI, No4. -P.451-453.

[0283] Considering the major role of SOD in the metabolism of reactive oxygen species and the significant contribution of superoxide anion radicals to the induction and development of oxidative stress, we investigated the activity of Cu-Zn-dependent SOD in the serum of animal models of obesity treated and untreated with stable isotopes of Zn-64 in the aspartate form.

[0284] In this study, a statistically significant decrease in SOD activity was found in animals fed a high-fat diet. Administration of Zn-64 stable isotope in aspartic acid form to experimental animals increased SOD activity compared to controls as well as to values ​​in untreated obese animal models (Table 16).

[0285] [Table 16]

[0286] Taking into account the specific zinc deficiency characteristic of the pathogenesis of obesity, the restoration of the activity of this enzyme after administration of a solution of Zn-64 stable isotope in aspartic acid form may be the result of neutralizing its enzyme levels in the body and its active participation in the regulation and synthesis of zinc-dependent enzymes, in particular SOD.

[0287] The decrease in SOD activity can be seen as the result of a specific depletion of the antioxidant defense system due to the gradual damage of its components by free radicals and LOP. Therefore, according to modern concepts, the activity of this enzyme is closely related to the intensity of the LOP process, since the excessive accumulation of toxic secondary products of lipid oxidation causes the inhibition of the activity of SOD and other enzymes of the antioxidant system. A literature review and data analysis on the involvement of ROS in the oxidative degradation of proteins suggests that the decrease in SOD enzymatic activity may be due to oxidative modification of the enzyme molecule. Since SOD is a metal-containing enzyme, oxygen radicals that damage proteins can be formed directly in the active center of the enzyme. In this case, the hydroxyl radical OH (OH), formed in the Fenton and Haber-Weiss reactions from hydrogen and superoxide, is responsible for the formation of the hydroxyl radical OH (OH), which is formed in the Fenton and Haber-Weiss reactions from hydrogen and superoxide. - acts as a direct agent that inactivates the enzyme. Thus, experiments by Salo DC et al. show that incubation of superoxide dismutase in a medium containing oxygen radicals leads to the cleavage of the enzyme molecule and the formation of additional protein fractions. The authors explain their results by the oxidative inactivation of superoxide dismutase upon exposure to H2O2. This view is consistent with the ability of the copper atom in the active center of the enzyme to accelerate the formation of free radicals. Another argument supporting the involvement of metals with several common oxidation states in SOD inactivation is the fact that Mn2+-containing superoxide dismutase is less susceptible to oxidative degradation when incubated with H2O2.

[0288] A key antioxidant enzyme in this system is catalase, which neutralizes hydrogen peroxide formed as a result of the rearrangement of the superoxide anion radical by superoxide dismutase. Therefore, we evaluated the activity of this enzyme in animal models of obesity treated and untreated with the stable isotope Zn-64 aspartate. We observed an inhibition of catalase activity in untreated obese animals, as well as a reduction in SOD activity.

[0289] A decrease in the activity of both enzymes against the background of activation of lipid peroxidation processes is considered a negative prognostic event that contributes to further escalation of free radical processes.

[0290] Administration of Zn-64 stable isotope in aspartate form caused a slight increase in catalase activity when compared with the results obtained from a group of untreated animal models of obesity, which generally correlated with the previously established normalization of the pro-oxidant-antioxidant balance.

[0291] The normalization of the activity of the investigated antioxidant enzymes after administration of the test substance is consistent with literature data on other forms of this ion. Thus, pretreatment with the stable isotope of Zn-64 in the aspartate form was shown to increase the activity of SOD and catalase in rat hepatocytes during ethanol intoxication.

[0292] The revealed positive effect of the aspartic acid form of Zn-64 stable isotope on the activity of the main antioxidant enzymes can be explained, firstly, by an increase in their synthesis due to an increase in the concentration of zinc, a necessary structural element that ensures the proper functional activity of these enzymes.

[0293] The results obtained regarding the normalizing effect of the aspartic acid form of the Zn-64 stable isotope on the pro-oxidant-antioxidant balance confirm the significant antioxidant potential of this isotope. Zinc does not belong to the classic antioxidants that can directly interfere with free radical reactions, but has an indirect effect on the overall pro-oxidant-antioxidant balance. Thus, zinc is an inhibitor of NADPH oxidases, a group of enzymes involved in the formation of aggressive superoxide anion radicals.

[0294] Zinc interacts directly with sulfhydryl groups in proteins, protecting them from oxidation by reactive oxygen species, inducing the synthesis of metallothionein, a cysteine-rich metal-binding protein that acts as a trap for radicals, and inhibiting the formation of reactive mixed-valence metal oxides, exhibiting a membrane-stabilizing effect. Dietary zinc deficiency has been found to reduce vitamin E concentrations in plasma and several other organs, which in turn affects the body's general antioxidant reserves.

[0295] Another mechanism for the antioxidant properties of the aspartate form of Zn-64 stable isotope is related to its ability to stabilize cell membranes, which is particularly important in the context of ongoing oxidative stress. The membrane-protective effect of zinc can be explained by its mediating role in detoxifying heavy metals and in inducing the synthesis of metallothionein, which is directly involved in membrane stabilization. Increased levels of metallothionein can maintain membrane integrity and protect cells from the effects of alkylating agents.

[0296] It was found that zinc can stabilize cell membranes by affecting the synthesis of phospholipids (activation of PS synthase, PS decarboxylase, PEA methyltransferase and phospholipid methyltransferase with a concomitant decrease in the activity of PHI synthetase) and their asymmetric distribution.

[0297] Therefore, given the importance of controlling the intensity of free radical reactions and maintaining an adequate antioxidant status in the body, especially for patients with the pathogenesis of systemic chronic diseases closely linked to the development of oxidative stress, the use of stable isotopes of Zn-64 in aspartic acid form as an additional tool in the basic treatment of these diseases may contribute to improving the overall metabolic status.

[0298] Effects of Zn-64 stable isotope aspartate on cytokine profiles, resistin and ghrelin levels in animal models of obesity There is no doubt that adipose tissue is not only the body's energy reservoir but also an organ actively involved in regulating metabolism through a complex of endocrine, paracrine, and autocrine signals that regulate responses in many tissues and organs, including the hypothalamus, pituitary gland, pancreas, liver, skeletal muscle, kidneys, endothelium, and immune system. Accordingly, adipose tissue secretes more than 50 protein factors, hormones, and growth factors, including cytokines. These include proinflammatory cytokines such as IL-1, IL-6, IL-8, IL-12, TNF-α, and IFN-γ, as well as anti-inflammatory cytokines such as IL-4, IL-10, IL-13, and TGF-γ. Mohamed-Ali V., Pinkney J., Coppacf S. Adipose tissue as an endocrine and paracrine organ / / Int J Obes Relat Meabol Disord 1998;22:1145-1158.

[0299] One consequence of excessive production of reactive oxygen species in adipocytes is the initiation of a signaling cascade, leading to increased production of proinflammatory cytokines by macrophages infiltrating adipose tissue as its mass increases. The consequence of such a disturbance is the formation of systemic chronic inflammation in the body of obese individuals. Current and actively discussed concepts suggest that subclinical chronic inflammation in adipose tissue is a key link in the pathogenesis of obesity and obesity-related diseases. Chronic inflammation in adipose tissue is characterized by cellular infiltration, fibrosis, altered microcirculation, impaired adipokine secretion and adipose tissue metabolism, and elevated blood levels of nonspecific inflammatory markers such as C-reactive protein, fibrinogen, and leukocyte counts. Rajala M., Scherer E. / / Endocrinology 2003;144:3765-3773.

[0300] Increased levels of proinflammatory cytokines in serum as well as in adipose tissue occur as a result of inflammatory processes in adipose tissue.

[0301] Cytokines, endogenous biologically active mediators of cell-cell and system-wide interactions, affect cell survival by regulating cell growth, differentiation, functional activity, and apoptosis. They ensure the coordination of immune, endocrine, and nervous system functions under physiological conditions and in response to pathological conditions. Cytokines were previously believed to be produced by lymphocytes, monocytes, and tissue macrophages. However, recent studies have shown that in obesity, as with any inflammatory process, infiltration of neutrophils, T lymphocytes, and then resident macrophages into adipose tissue occurs early and determines the initial mechanism of inflammation. Macrophages have been shown to contribute to adipocyte hypertrophy, which is accompanied by increased functional activity and cytokine synthesis, further enhancing the inflammatory response. Enlarged adipocytes potently secrete chemokines and their receptors, stimulating the influx of new neutrophils, macrophages, and lymphocytes, contributing to further adipocyte hypertrophy, preservation, and enhanced inflammatory responses. Adipocytes increase cytokine secretion by macrophages, which then act on adipocytes, causing adipose tissue cell hypertrophy and activation. Enlarged adipocytes, like lymphocytes and macrophages, produce cytokines and activate complement, triggering a series of inflammatory processes. As a result, inflammation becomes stable and systemic. Furthermore, lipid peroxidation products, such as trans-4-hydroxy-2-nonenal and malondialdehyde, are chemoattractants for monocytes and macrophages. Enhanced lipid peroxidation in accumulated adipose tissue contributes to the attraction and infiltration of macrophages into obese adipose tissue, thus actively contributing to the initiation of inflammatory responses.

[0302] As a result, increasing adipose tissue mass is a constant source of proinflammatory cytokines synthesized by both adipocytes and macrophages embedded in adipose tissue, leading to the formation of a chronic inflammatory process and the maintenance of inflammation in the body. Although its low intensity does not cause direct clinical symptoms, this process is systemic in nature, affecting a wide range of organs and tissues, causing changes in their metabolism and impairing their function and immune system response.

[0303] Given the above, the next stage of the study was to investigate whether administration of Zn-64 stable isotope in aspartic acid form affects the cytokine profile in obese animals. To this end, the concentrations of the main pro-inflammatory (IL-1, IL-6, IL-12, IFN-γ) and anti-inflammatory (IL-4, IL-10, TGF-β) cytokines in the adipose tissue and serum of experimental animals were determined, which allowed us to draw conclusions about the intensity of the inflammatory process in adipose tissue and assess whether such an inflammatory process is systemic.

[0304] The results obtained show that the development of obesity is accompanied by an increase in the levels of all analyzed pro-inflammatory cytokines (Table 17) in the adipose tissue of animals fed a high-fat diet, indicating the activation of the inflammatory process.

[0305] Long-term inflammatory processes, in turn, can lead to the development of various complications and may be a risk factor for insulin resistance and diabetes. Cytokines not only reduce cellular sensitivity to insulin action, but also intensify the inflammatory process and increase the accumulation of inflammatory intermediates, causing tissue damage and organ dysfunction. J. Hirosumi et al. / / Nature. -2002. -Vol.420, No.6913. -P.333-336. C. Jiang, W. Wang, J. Tang. / / Journal of Endocrinological Investigation. -2013. -Vol.36, No.11. -P.986-992.

[0306] [Table 17]

[0307] High levels of proinflammatory cytokines, including those mentioned above, have been shown to induce β-cell apoptosis. High concentrations of IL-12, whose expression is activated by IFN-γ, induce CD8+ lymphocyte infiltration into the pancreas and the development of acute pancreatitis. IL-1β binds to specific receptors on the surface of these cells, triggering the activation of NF-κB-mediated apoptosis, which leads to DNA fragmentation and loss of functional activity of the cells. IL-1β may also be considered one of the factors contributing to the development of insulin resistance in peripheral tissues. IL-1β has been shown to activate IκB kinase-β, which affects insulin signaling by phosphorylating serine residues in insulin receptor substrate (IRS)-1. Furthermore, IL-1β may indirectly increase resistance to insulin action by activating hepatic lipogenesis and contributing to increased levels of triglycerides and free fatty acids in adipocytes.

[0308] IL-6 accumulation in peripheral blood has been shown to be directly proportional to the amount of adipose tissue. Adipocytes are the second largest source of IL-6 after the immune system; 35% of circulating IL-6 is synthesized by adipocytes. Circulating levels are directly proportional to body mass index and increase with obesity. At the same time, weight loss is accompanied by a decrease in IL-6 blood levels. In excess, IL-6 exacerbates insulin resistance by suppressing the synthesis of one of the insulin receptor subunits. By activating lipolysis in visceral adipose tissue, IL-6 contributes to the progressive development of fatty liver disease and systemic atherosclerosis. Furthermore, IL-6 induces increased production of C-reactive protein (CRP), another factor associated with obesity. V. Rotter et al. / / the Journal of Biological Chemistry. -2003. -No.278. -P.45777-45784. Fantuzzi G. / Journal of Allergy and Clinical Immunology. -2005. -Vol.115, No.5. -P.911-919.

[0309] One of the mechanisms controlling the levels, and therefore the biological effects, of proinflammatory cytokines is mediated by a group of anti-inflammatory cytokines. These cytokines can inhibit the synthesis of proinflammatory cytokines by affecting the transcription of specific genes, induce the synthesis of interleukin RAIL receptor antagonists, enhance the production of soluble receptors, and reduce the density of proinflammatory receptors on cells. Therefore, to clarify the possible mechanism of the effect of Zn-64 aspartate stable isotope on the proinflammatory cytokine profile, we measured the levels of IL-4, IL-10, and TGF-β.

[0310] The detected changes in pro-inflammatory cytokine levels occurred against a background of a slight decrease in the levels of anti-inflammatory cytokines in obese animals. At the same time, in animals treated with Zn-64 aspartate, the levels of anti-inflammatory cytokines were not only higher than in untreated obese animal models, but also higher than in control animals.

[0311] It should be emphasized that the absence of changes in the control group of animals treated with the test substance suggests that the long-term use of Zn-64 stable isotope in aspartic acid form is safe and may only show therapeutic effects upon the development of pathological conditions.

[0312] As mentioned above, the pathogenesis of obesity involves a systemic chronic inflammatory process, the intensity of which can be assessed by serum levels of pro- and anti-inflammatory cytokines.

[0313] Analysis of the cytokine profile in the serum of obese animals (Table 18) showed an increase in the levels of pro-inflammatory cytokines, which was more pronounced compared to the data obtained from adipose tissue. No statistically significant changes were observed in the levels of the anti-inflammatory cytokine IL-4. The slight increase in serum levels of IL-10 in obese animals may be considered a specific compensatory response of the body to metabolic disorders.

[0314] In animals treated with Zn-64 stable isotope in aspartic acid form, there was a decrease in the levels of pro-inflammatory cytokines against the background of an increase in the levels of anti-inflammatory cytokines, which was even higher in the control animals.

[0315] [Table 18]

[0316] One of the underlying mechanisms of zinc's effect on cytokine profiles may be its inhibition of transcription factors sensitive to oxidative stress. Zinc may also partially block genes encoding proinflammatory cytokines such as IL-6 and IL-8.

[0317] The specific normalizing effect of the aspartate form of Zn-64 stable isotope on cytokine profiles in animal models of obesity may serve as evidence of the possible anti-inflammatory potential of the studied test substances in obesity.

[0318] Therefore, the decreased levels of pro-inflammatory cytokines in the serum and adipose tissue of animals treated with Zn-64 stable isotope aspartate may be due, in part, to an increased level of anti-inflammatory cytokines. Considering the close relationship between the amount of adipose tissue and the levels of pro-inflammatory cytokines it produces, the revealed positive effect of Zn-64 stable isotope aspartate on cytokine profiles appears to primarily affect body weight and, therefore, adipose tissue mass.

[0319] In addition to cytokines, adipocytes secrete many biologically active substances involved in the regulation of energy metabolism. One such substance is resistin, or adipocyte-specific secretory factor (ADSF / FIZZ3). Resistin's involvement in stimulating inflammatory mechanisms, endothelial activation, and vascular smooth muscle cell proliferation allows it to be viewed as a marker or even a causative factor in the development of disease. This adipose-derived hormone exerts a feedback effect on fat metabolism: on the one hand, its concentration increases with adipocyte differentiation, and on the other hand, resistin suppresses adipogenesis. Resistin, as one of the causes of insulin resistance, may be a link between obesity and the development of diabetes. Literature data suggest that resistin levels can be used as a predictor of susceptibility to type II diabetes and obesity. It has been shown that resistin can reduce the sensitivity of peripheral tissues to the action of insulin and thus stimulate the development of insulin resistance. Resistin activates NF-κB-dependent expression and release of proinflammatory cytokines and adhesion molecules, including TNF-α and IL-6. Faantuzzi G. / Journal of Allergy and Clinical Immunology. -2005. -Vol.115, No5. -P.911-919. C. Jiang et al., / / Journal of Endocrinological Investigation. -2013. -Vol.36, No11. -P.986-992.

[0320] Therefore, we investigated the levels of resistin in adipose tissue and serum of obese animal models from all experimental groups. The results obtained from the experiment showed that the levels of this adipokine tended to increase, more significantly in adipose tissue (Table 19).

[0321] [Table 19]

[0322] Considering that resistin is a promoter of adipocyte maturation and acts as an autocrine regulator of the formation of insulating factors in adipose tissue, even a small increase in the levels of this adipokine would contribute to adipose tissue growth and the progression of obesity-associated metabolic disorders.

[0323] Resistin levels in animal models of obesity injected with the aspartate form of Zn-64 stable isotope were within the range of control values.

[0324] Since resistin is secreted primarily by preadipocytes and, to a lesser extent, by mature adipocytes of visceral adipose tissue, the positive effect of the aspartate form of Zn-64 stable isotope may be explained by its ability to influence animal body weight and therefore fat mass accumulation in animals maintained on a high-fat diet.

[0325] Another factor directly involved in appetite regulation is ghrelin, a lipophilic hormone secreted primarily by P / D1 cells lining the gastric fundus and, to a lesser extent, by other organs, such as the hypothalamus, pituitary gland, gonads, and ε-cells of the islets of Langerhans. This peptide plays a key role in regulating hunger and energy metabolism, stimulating food intake and contributing to the development of obesity. Ghrelin receptors are localized in the same hypothalamic structures as the leptin receptor, Ob-Rb, as well as in the arcuate and ventromedial nuclei. High levels may contribute to long-term weight gain. Lowering the ghrelin threshold in the body reduces appetite. F. Ferrini et al., / / Current Neuropharmacology. -2009. -Vol.7, No.1. -pp.37-49. J. Camina et al., / / Endocrine. -2003. -Vol.22, No.1. -pp.5-12.

[0326] Research carried out over the past few years has revealed the importance of ghrelin in regulating energy balance in the body through the effects of this hormone on the hypothalamus with the involvement of neuropeptide Y and the endocannabinoid system.

[0327] Ghrelin expression is increased in response to hypoglycemia and inhibited during hyperglycemia. This may indicate that ghrelin coordinates the body's metabolic and hormonal response to fasting with the involvement of insulin and mechanisms for maintaining adequate serum glucose levels. Ghrelin possesses anorexic, adipogenic, and somatotropic properties and acts as a leptin antagonist, increasing the need for food. Active immunization against ghrelin has been shown to induce weight loss. The appetite-stimulating effects of ghrelin are due to its ability to increase neuropeptide Y neuron activity and inhibit proopiomelanocortin neurons.

[0328] Ghrelin receptors are located both in the central nervous system (pituitary gland, hypothalamus) and other organs (pancreas, intestine, stomach). This peptide plays an important role in regulating hunger and energy metabolism, stimulating food intake and contributing to the development of obesity. Its levels increase during fasting, weight loss, high-calorie food intake, and hypoglycemia. Increased plasma levels of ghrelin after diet-induced weight loss are consistent with the hypothesis that ghrelin plays a role in the long-term regulation of body weight. Ghrelin levels are decreased in individuals with obesity, NASH, and hypertension.

[0329] Given the close relationship between impaired energy homeostasis and the development of obesity, we analyzed serum levels of ghrelin in treated and untreated animal models of obesity using the aspartate form of Zn-64 stable isotope.

[0330] These data indicate that the development of obesity is accompanied by a decrease in the levels of this hormone, and that administration of Zn-64 stable isotope in the aspartate form to control animals also caused a decrease in its serum levels (Figure 16). In general, circulating ghrelin levels are known to be inversely correlated with positive energy balance, total body weight and adipose tissue mass, adipocyte size, and leptin levels, so these data are consistent with information provided in the literature. For example, ghrelin levels in anorexic patients are higher than in patients who develop obesity. In an obese animal model injected with the test substance, ghrelin levels were similar to those determined in control animals. M. Kojima, K. Kangawa. / / Physiological Reviews -2005-Vol.85, No.2.-P.495-522.

[0331] Therefore, the data suggest that the aspartate form of Zn-64 stable isotope has a normalizing effect on the functional status of adipocytes, which may result in the restoration of cytokine balance to a physiologically normal state.

[0332] Divalent metal ion levels in organs of animal models of obesity The involvement of zinc in physiological and pathophysiological processes largely depends on the zinc level in the body. Zinc is found in all cells and organs, but its concentration in one organ or another varies considerably and depends on the specific activity of the organ. The human body's zinc reserves are very small, approximately 1.5–3 g. This figure depends on many factors (the person's age and gender, the condition of the gastrointestinal mucosa, associated diseases, pregnancy, etc.). Zinc is found in almost all tissues. Approximately 62–63% of zinc reserves are in skeletal muscle. According to data provided by many researchers, zinc is distributed in the human body as follows (μg / g): skin, adrenal glands - 6, ovaries - 12, brain - 13, lymph nodes - 14, digestive tract - 21, heart - 27, kidneys - 37, liver - 38, muscle - 48, bone - 66, prostate - 87, and sperm - 125. Whole blood contains approximately 2.5–5.3 μg / ml of zinc. Zinc in plasma is low (0.7-1.2 μg / ml, which is about 0.2-1% of the total amount of zinc in the body). Due to the breakdown of red blood cells, zinc levels in serum are slightly higher than in plasma (1.1-1.3 μg / ml).

[0333] The levels of zinc and several divalent metals (copper and manganese) were determined in the skeletal muscle, kidney and liver of animals with obesity, as well as the effect of Zn-64 stable isotope in aspartic acid form on the levels of these metals.

[0334] The selection of copper and manganese for analysis is explained by their exceptional importance and involvement in key metabolic processes. Furthermore, the studied metals affect each other's digestibility and bioavailability, so abnormal levels of one of them are often the result or cause of changes in the levels of the other metals mentioned above. In most cases, they compete with each other. Thus, for example, the high presence of iron in food reduces zinc absorption by approximately two-fold. The presence of copper also reduces zinc absorption in the gastrointestinal tract due to competitive association with transport metalloenzymes.

[0335] [Table 20]

[0336] Copper is an essential trace element. An adult's body contains approximately 110-150 mg of copper. Half of this amount is found in muscles. Smaller copper reserves are concentrated in the liver, the gray matter of the cerebral hemispheres, and the bone marrow. This important trace element is found in enzymes and proteins (mainly ceruloplasmin). The latter is a metalloenzyme that catalyzes the oxidation of many biologically active substances and ensures copper delivery to various tissues and organs. Copper is known to be a specific activator of cytochrome oxidase, tyrosinase, and copper oxidase. Copper activates the enzymes of protein metabolism, arginase and aminopeptidase, and promotes the synthesis of nucleic acids necessary for the healing process. This biosubstance is involved in the processes of blood formation, hemoglobin synthesis, and the functioning of the cytochrome system, and is part of the interstitium of blood cells. Copper used in microdoses increases glycogen levels in skeletal muscle and liver. It exhibits insulin-like activity, promotes glucose oxidation, and inhibits glycogen breakdown. Copper strengthens the liver's neutralizing function and normalizes mineral metabolism. Copper is part of many enzymes, determining their function and regulating their action. It is part of all oxidases and is itself a key component of redox reactions in the body. These enzymes are necessary for the processes of cellular respiration and protection of cells from the effects of free radicals, and are involved in the synthesis of myelin, connective tissue biosynthesis, and glandular metabolism. The antioxidant activity of copper is associated with its involvement in the structural formation of superoxide dismutase.

[0337] Manganese is an essential trace element necessary to ensure the body's proper metabolic state. Its highest levels are found in the bones, liver, and gray matter of the cerebral hemispheres. This biosubstance has an insulin-like effect, lowering blood glucose levels and increasing glycogen synthesis. Manganese has been shown to stimulate blood formation. It has high oxidative activity and a pronounced lipotropic (cholinomimetic) effect. This trace element influences the metabolism of fats and proteins, as well as the synthesis of many vitamins. Furthermore, it is part of the most important enzyme systems. Manganese salts attenuate the antihypertensive effect of adrenaline and induce a decrease in adrenaline hyperglycemia. Manganese has hypocholesterolemic and anti-sclerotic properties.

[0338] According to the results obtained (Table 20), there was a tendency for zinc to be redistributed between organs during the development of obesity. Thus, zinc levels were slightly increased in the kidney and decreased in the liver and muscle. The increase in muscle zinc levels in animals treated with Zn-64 stable isotope aspartate is quite natural, given that most of the zinc stores in normal physiological conditions are in skeletal muscle, and on the other hand, this can be considered as indirect confirmation that there is no visible dysfunction in the process of zinc transport and deposition in obese animals administered the test substance.

[0339] More importantly, zinc levels in the liver of obese animal models are reduced. After all, the liver is the primary site for the synthesis of zinc-containing proteins. Therefore, a reduction in zinc stores in this organ may contribute to the development and progression of disorders associated with zinc-dependent and zinc-containing enzyme deficiencies. One consequence of reducing hepatic zinc levels may be a reduction in serum superoxide dismutase activity in obese animals, as identified in the previous phase of this study.

[0340] Liver zinc levels in animals fed a high-fat diet and injected with Zn-64 stable isotope aspartate were still slightly higher than in control animals, indicating restoration of zinc homeostasis.

[0341] Regarding copper, there was a trend toward increased levels in muscle and decreased levels in kidney. The most significant changes in copper concentration were observed in the liver, the organ that plays a major role in the metabolism of this trace element.

[0342] It should be emphasized that zinc competes with copper in the intestinal absorption process and, at high concentrations, can cause copper deficiency in the body. Therefore, the lack of significant changes in copper levels in animals treated with Zn-64 stable isotope aspartate indicates that the dose level of this element was appropriately selected.

[0343] These results suggest a positive effect of the aspartate form of Zn-64 stable isotope on key systems directly involved in the development and progression of obesity.

[0344] In general, the results can be summarized as follows: Zn-64 stable isotope in aspartic acid form administered to animal models of obesity shows complex effects on many systems, the dysfunction of which can have the most serious consequences for the body.

[0345] Considering the importance of preserving zinc homeostasis to ensure the proper physiological state of all body tissues, especially adipose tissue, the obtained data on the positive effect of Zn-64 stable isotope in aspartic acid form in obesity seem to be partly related to the restoration of zinc levels reduced during the pathogenesis of obesity.

[0346] Accumulating data on the physiological role of zinc indicates that increasing its levels can trigger a cascade of biochemical shifts that ultimately determine its overall positive effects.

[0347] There are several potential mechanisms for the regulatory effect of the aspartate form of Zn-64 stable isotope. First, it is the enhancement of the synthesis of zinc-dependent enzymes and transcription factors. Against the background of enhanced free radical processes and the progression of oxidative stress, increasing the number of key antioxidant enzymes due to the availability of zinc (a necessary structural component of these enzymes) helps maintain an adequate antioxidant reserve. An important fact is the ability of zinc to induce the synthesis of many antioxidants, anti-inflammatory cytokines, and factors that are actively involved in the regulation of cell signaling cascades and are involved in the regulation of fundamental processes. Thus, according to literature data, the level of zinc-alpha-glycoprotein (ZAG), which contributes to the reduction of fat deposition by stimulating lipolysis in adipocytes, is significantly reduced in obesity. Furthermore, the process of adipocyte differentiation, especially of brown adipose tissue, is strictly determined and controlled by a group of transcription factors, many of which contain zinc.

[0348] Therefore, the results of a comprehensive analysis of the effect of the aspartate form of Zn-64 stable isotope on the pathogenesis of obesity indicate this trace element as a promising additive for use in the development of biologically active compounds not only for the treatment of pathologies accompanied by chronic inflammatory processes, systemic depletion of antioxidant reserves, and impaired functioning of the serotonergic system, but also for the prevention of diseases associated with systemic metabolic disorders.

[0349] conclusion For the first time, we conducted a comprehensive study on the effects of Zn-64 stable isotope aspartate on key pathogenic correlates of obesity development and progression in a diet-induced obesity model. The results provide a de facto basis for the use of Zn-64 stable isotope aspartate as an adjunctive therapeutic agent in the treatment of overweight and obese patients.

[0350] Administration of Zn-64 stable isotope in aspartate form to animals fed a high-fat diet has been shown to help reduce body weight and the amount of food consumed, accompanied by a reduction in body mass index, compared to untreated animal models of obesity.

[0351] Zn-64 stable isotope in the aspartate form was found to have a positive effect on lipid metabolism in animals fed a high-fat diet.

[0352] Zn-64 stable isotope in aspartate form was found to have a positive effect on the morphofunctional properties of the pancreas and liver in high-fat fed animals compared to untreated animal models of obesity.

[0353] It was found that Zn-64 stable isotope in aspartate form administered to animals fed a high-fat diet exerts a modulating effect on the activity of key enzymes involved in serotonin metabolism, which helps to restore central and peripheral serotonin levels compared to those of untreated obese animal models.

[0354] Administration of Zn-64 stable isotope in aspartate form has been shown to contribute to the normalization of pro-oxidant-antioxidant homeostasis (reduced levels of lipid peroxidation products and protein oxidative modifications) in animals fed a high-fat diet due to a reduction in the intensity of free radical processes against the background of activation of antioxidant defenses via an increase in the activity of antioxidant enzymes (superoxide dismutase and catalase).

[0355] The ability of the aspartate form of Zn-64 stable isotope to affect the cytokine profile in serum and adipose tissue of animals maintained on a high-fat diet, i.e., to reduce the levels of pro-inflammatory cytokines (IL-1, IL-6, IL-12, IFN-γ) against a background of a slight increase in the levels of anti-inflammatory cytokines (IL-4, IL-10, TGF), has been shown, which generally indicates a reduction in the intensity of systemic inflammation.

[0356] The aspartate form of Zn-64 stable isotope was found to have no appreciable effect on the levels of resistin in adipose tissue and serum of obese animals, as well as on serum levels of ghrelin.

[0357] Administration of Zn-64 stable isotope in aspartate form was found to cause a redistribution of divalent metal ions (zinc, copper, manganese) between muscle, kidney, and liver in an animal model of obesity, contributing to the restoration of their physiological levels. [Example 7]

[0358] Effects of experimental drugs on changes in blood insulin levels in experimental animals (rats) after intraperitoneal administration The first group consisted of animals (weighing approximately 180-220 grams) that were injected with saline.

[0359] Number of animals: 8 (4 animals per group); 2 sampling points - 2 days after drug administration and 7 days after the last injection of saline (4 x 2 = 8).

[0360] The second group consisted of animals (weighing approximately 180-220 grams) injected with zinc acetate, a naturally occurring isotope. The current dose was calculated based on the zinc content. Each animal was injected with 3750 μg of zinc per kg of animal weight. This was a comparison group to evaluate the effects of zinc on experimental animals. Zinc acetate is the standard zinc compound used in most animal and diabetes experiments.

[0361] Number of animals: 8 (4 animals per group); 2 sampling points - 2 days after drug administration and 7 days after the last injection of saline (4 x 2 = 8).

[0362] The third group is aspartic acid (Zn 64 The study consisted of animals (weighing approximately 180-220 grams) injected with zinc isotopes of the 2-form. The current dose was calculated based on the zinc. Each animal was injected with 3750 μg of zinc per kg of animal body weight.

[0363] Number of animals: 8 (4 animals per group); 2 sampling points - 2 days after drug administration and 7 days after the last injection of saline (4 x 2 = 8).

[0364] Experimental scheme: The animals were housed in cages (four per cage) with free access to food and water. Three days after the animals were housed, they were injected with the experimental drug (natural zinc or isotopic zinc). Administration was performed every other day, with seven injections per animal (intraperitoneal administration). The dose of the injected drug for each animal was calculated based on a ratio of 3750 μg of zinc per kg of animal body weight. After the last injection, half of the animals were kept fasting (12 hours) with free access to water. At the end of this period, the animals were removed from the experiment. Half of the animals continued to have free access to food and water for another seven days. Six days after the last injection, the animals were kept fasting (12 hours) with free access to water. At the end of this period, the animals were removed from the experiment.

[0365] research materials Compliance with animal welfare regulations governing animal research activities The International Recommendations for Conducting Biomedical Research Using Animals in Accordance with the General Principles of Working with Animals, approved by the First National Congress on Bioethics (Kiev, Ukraine, 2001) and in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, France, 1986), were followed during work with laboratory animals. Experimental work with rats was carried out in the animal farm of Taras Shevchenko National University in Kyiv. Research on animals was approved by the Scientific Council of this institution and, in turn, regulated by the Rules for Experimental Work with Laboratory Animals, adopted at the time and harmonized with the current legislation of Ukraine.

[0366] Conditions for research results in rats The study was conducted on albino rats aged 2-3 months and weighing 120-300 g. The experimental animals were maintained on a standard vivarium diet with free access to water. During the experiment, the animals were kept in plastic cages under natural day / night light conditions at room temperature of 19-24°C and humidity below 50%. Before the experiment, the animals were allowed to acclimate in the laboratory for 7 days.

[0367] Preparation of rat serum Rat serum was prepared from whole blood. To remove associated proteins and fibrinogen, the whole blood was incubated at 37°C for 30 minutes, after which the sample was centrifuged at 2500 g for 15 minutes. The resulting supernatant (serum) was immediately separated from blood cells and frozen at -20°C for further analysis.

[0368] Preparation of kidney and liver homogenates Whole kidney, liver, and muscle homogenates were prepared as follows. Organ removal and homogenization were performed at a temperature of 1–4°C. Tissue homogenization was performed in 50 mM Tris-HCl buffer (pH 7.4) containing 140 mM NaCl and 1 mM EDTA. The volume (ml) of buffer used was five times the mass (grams) of the isolated organ. The isolated liver was perfused with chilled saline (0.9% NaCl) via the portal vein using a syringe. The minced liver was transferred to a homogenizer equipped with a finely ground Teflon pestle and homogenized in chilled buffer. A pair of isolated kidneys was perfused with chilled saline to release the adipose tissue and then minced with scissors. The minced tissue was transferred to a homogenizer equipped with a finely ground Teflon pestle and homogenized in chilled buffer. Whole kidney and liver homogenates were centrifuged at 600 g for 15 min. After settling, the liquid was decanted and centrifuged again at 15,000 g for 15 minutes. These two steps allowed the removal of nuclear and mitochondrial debris. Aliquots of the homogenate were frozen in nitrogen (Rybalchenko VK, Koganov MM Structure and function of membranes 1988. -312 pp).

[0369] 1. Method for determining insulin levels in animal serum: Enzyme-linked immunosorbent assay (Insulin levels in rat serum were determined using an enzyme-linked immunosorbent assay based on a standard method used for soluble proteins. This was performed in a 96-well microplate with adsorption capacity for soluble proteins [Crowther JR ELISA Guidebook / JR Crowther. - Totowa, New Jersey: Humana Press Inc., 2001. - p. 436].)

[0370] Serum was prepared as a 1–10 dilution in 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl. 100 μl volumes of samples were incubated overnight at 4°C in microplate wells. After incubation, the wells were washed with a buffer containing 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween 20 to remove unbound material. Nonspecific binding sites were blocked with 5% nonfat milk blocking solution and incubated for 1 h at 37°C. After washing, primary rabbit anti-insulin antibodies were loaded into separate wells of the microplate and incubated for 1 h at 37°C. After incubation, the microplate wells were washed and loaded with the appropriate secondary antibody conjugated to horseradish peroxidase and incubated for an additional 1 h at 37°C. Secondary antibody binding was visualized by adding 100 μl of OPD solution to each well at a concentration of 0.4 mg / ml prepared in citrate buffer (pH 5.0) containing 0.013% HO. Optical density was measured at 492 nm. Insulin concentrations were calculated using a calibration curve generated under the given conditions and known concentrations of human insulin.

[0371] Determination of superoxide dismutase activity in rat liver and serum. To measure superoxide dismutase (SOD, EC 1.15.1.1) activity, a method based on the ability of the enzyme to inhibit the process of adrenaline autooxidation is used.

[0372] A 10 μl aliquot of the test sample (liver and kidney homogenate) was placed in a microplate well. 200 μl of 0.2 M bicarbonate buffer (pH 10.65) was added to each well. The reaction was initiated by adding 10 μl of a 0.1% solution of adrenaline to each well. No enzyme source was added to the blank sample. The optical density was measured at a wavelength of 347 nm using a microplate reader 4 and 8 minutes after the addition of adrenaline.

[0373] The classical method of measuring enzyme activity based on product accumulation or substrate reduction is not applicable to SOD because it is not possible to construct a calibration curve for the reaction product, which is the oxidation product of adrenaline. The activity of this enzyme was expressed in conventional units / min * mg protein, calculated using the following formula:

[0374]

number

[0375] where X is the optical density of the blank sample without the test sample, which is equal to the difference between the optical density of the blank sample at 8 minutes and the optical density of the same blank sample at 4 minutes; Y is the optical density of the blank sample with the test sample, which is equal to the difference between the optical density of the blank sample at 8 minutes and the optical density of the same blank sample at 4 minutes; a is the amount of protein (mg) in the blank sample; 4 is the incubation period during the determination of quenching, 4 minutes; and 50 / 100 is the conversion to conventional units [Sirota TV Novel approach to the study of adrenaline auto-oxidation and its use for the measurements of superoxide dismutase activity / / Vopr Med Khim -1999. -45(3). -P.263-272].

[0376] Determining glucose tolerance Animals that had access only to water 16 hours before the start of the experiment were used for the glucose tolerance test. Rats were anesthetized with an intraperitoneal injection of thiopental sodium at a dose of 40 mg / kg. Five animals were used in each experimental group. The basal blood glucose level was determined in the rats, and then the animals were given 2 ml of aqueous glucose solution at a dose of 3 g / kg. Glucose concentrations were determined 60 minutes after the start of the experiment. Blood samples were collected via the tail vein [OV Gorbulinska, Sugar-lowering effects of water extracts of yakon (Smallanthus sonchifolius poepp.&endl.) / OV Gorbulinska, MR Khokhla, LT Mishenko et al. / / Biological Studios -2014.-8(2).-P.57-64.]. Results are shown in Figures 16A-16E.

[0377] According to all the data analyzed, there is no reliable difference between the control group (rats injected with saline) and the group of rats given zinc. This can be attributed to two factors: either the zinc solution was administered incorrectly (intraperitoneally instead of orally), or intact rats were used, which may have compensatory mechanisms that prevent them from shifting the balance of the islet system towards overproduction of insulin when the amount of zinc in their body increases, since this would cause all the hypoglycemia that would accompany it.

[0378] At the same time, a significant increase in body weight was observed in the rats receiving zinc isotopes. This is a positive result, as people with type 1 diabetes often lose weight. The data obtained may indicate that zinc isotopes can have a positive effect on the dynamics of weight gain.

[0379] Analysis of insulin levels in the serum of the experimental group of animals showed the following results: no significant changes in insulin levels were observed in the control group of animals throughout the experiment. In the group of animals injected with zinc acetate, a significant increase in insulin levels was observed on the first day after drug cessation, followed by a decrease in insulin levels to the values ​​of intact / control animals. The opposite situation was observed in the group injected with zinc isotopes: on the first day after drug cessation, insulin levels were slightly higher than in the group of intact animals, and on the seventh day after cessation, a significant increase in insulin levels was observed in this group of animals. Figure 17.

[0380] The results indicate that compared to zinc acetate, zinc isotopes have a longer-lasting effect on serum insulin levels or are released more slowly from the deposition site, which also causes a slower effect of increasing insulin levels.

[0381] Analysis of superoxide dismutase activity showed no significant changes in its activity in serum in all experimental groups of animals. This may be due to the lack of effect of the infused zinc preparation on the activity of this enzyme or, most likely, to incorrect administration of the zinc preparation (Figures 18A-B).

[0382] Microscopic studies of the islets of Langerhans showed positive kinetics after administration of zinc isotopes—a significant increase in islet area compared to the control group and the group injected with zinc acetate.

[0383] The results obtained correlate with those from the analysis of insulin levels in the serum of experimental animals. Microscopic results indicate that administration of zinc preparations, especially zinc isotopes, leads to an increase in the area of ​​pancreatic islets, which in turn may indicate a potential increase in insulin production by these islets. This is a move in the right direction, since the development of type 1 diabetes is associated with a significant insulin deficiency due to problems with its synthesis by these islets. (Figures 20A-20F)

[0384] [Table 21]

[0385] A glucose tolerance test was performed to determine the potential effect of zinc isotopes on the dynamics of blood glucose levels. Experimental procedure: Experimental animals were intragastrically injected with either saline solution or zinc in saline solution (5 mg / kg) in a volume of 2 ml. Sixty minutes after the aforementioned injection, basal glucose levels were measured, and then a glucose solution was injected at a volume of 3 g / kg in a volume of 2 ml using the same administration route. Blood glucose levels were measured again 60 minutes after the injection of the glucose solution. A difference in the decrease in glucose levels indicates a positive effect of the injected effector on the dynamics of blood glucose levels.

[0386] Glucose tolerance tests showed a decrease in glucose levels after zinc isotope administration compared to the control group. This may indicate the effect of zinc isotope administration on insulin levels in the bloodstream, thereby triggering mechanisms related to clearing blood from glucose. Given that insulin is a zinc-dependent protein, it can be assumed that zinc administration either increases the activity of this protein relative to its receptor in tissues or increases the amount of this hormone in the bloodstream.

[0387] Analysis of metal (zinc, manganese, and copper) accumulation in liver tissue showed that only zinc levels were significantly increased in both groups of animals administered the zinc preparation, both 1 day after drug cessation and 7 days after cessation. This indicates that the zinc administered to the animals accumulates and its elimination does not increase. All other analyzed metals were within the range of their concentrations observed in the control group of animals. (Figures 21A-C).

[0388] Analysis of metal accumulation in kidney tissue (zinc, manganese, and copper) showed that only zinc levels were significantly increased in both groups of animals administered the zinc preparation, both 1 day after drug cessation and 7 days after cessation. This indicates that the zinc administered to the animals accumulates and its elimination is not increased. All other analyzed metals are within the range of their concentrations observed in the control group of animals. (Figures 22A-22C. Figures 23A-23F.)

[0389] Test substance (Zn) on type 1 diabetes in experimental animals (rats) 64 Research report on the potential effects of aspartic acid Oral administration First group - control. Animals weighed approximately 140-150 grams (initial weight of the animals).

[0390] Number of animals: 5 (male). All 5 animals were group-housed in one cage with free access to food and water. At the beginning of the experiment, each animal received a single intraperitoneal injection of 10 mM citrate buffer (pH 4.5). 24 hours later, seven doses of the test substance were orally administered to the animals. Frequency of administration: every other day. Dose: 800 μg of zinc per animal.

[0391] After the last dose, the animals were fasted for 12 hours with free access to water, at the end of which period they were removed from the study.

[0392] Group 2 - diabetes. The animals weighed approximately 140-150 grams (initial weight of the animals).

[0393] Number of animals: 10 (male). Animals were housed four per cage and had free access to food and water. To induce diabetes in the animals, each animal received a single intraperitoneal injection of 6 mg of streptozotocin solution per 100 g of animal weight dissolved in 10 mM citrate buffer (pH 4.5). Dose: 800 μg of zinc per animal. Fasted animals were tested for adequate blood glucose levels two days after induction of diabetes. Rats with blood glucose levels >20 mmol / L were used for the experiment.

[0394] Group 3 - diabetes + zinc: animals weighing approximately 140-150 grams (initial weight of the animals).

[0395] Number of animals: 10 (male). Animals were housed four per cage and had free access to food and water. To induce diabetes in the animals, each animal received a single intraperitoneal injection of streptozotocin solution dissolved in 10 mM citrate buffer (pH 4.5) at a dose of 6 mg per 100 g of animal body weight. Fasted animals were tested for adequate blood glucose levels two days after induction of diabetes. Rats with blood glucose levels >20 mmol / L were used for this experiment.

[0396] Twenty-four hours later, animals were orally administered seven doses of the test substance, every other day.

[0397] Dosage - 800 μg of zinc per animal.

[0398] research materials Compliance with animal welfare regulations governing animal research activities The experimental animals used in this study were maintained in accordance with international standards and recommendations for clinical and biological research involving animals and in accordance with the Basic Principles of Humane Animal Handling approved by the First National Bioethics Congress (Kyiv, Ukraine, 2001), which are in line with the standards of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, March 18, 1986). All experiments involving animals were conducted in the animal laboratory of Taras Shevchenko National University in Kyiv, in accordance with the Animal Experiments Regulations developed in accordance with applicable Ukrainian legislation and approved by the institution's Scientific Committee.

[0399] Conditions for maintaining laboratory animals Two- to three-month-old albino rats weighing 120-130 g were used in this experiment. The animals were fed a standard vivarium diet and had free access to water. During this experiment, the animals were housed in plastic cages, and the environmental conditions were controlled to maintain 19-24°C and 50% relative humidity with a 12-hour light-dark cycle. All animals were allowed to acclimate to the environment for 7 days before the start of the experiment.

[0400] Determination of glucose concentration in serum of rats Glucose concentrations in whole blood were measured by the glucose oxidase method using a GlucoDr Ato AGM-4000 blood glucose meter (Allmedicus Co., Ltd., Korea). All procedures were performed according to the manufacturer's instructions.

[0401] Induction of type 1 experimental diabetes in rats Experimental type 1 diabetes was induced by a single intraperitoneal injection of streptozotocin solution at a dose of 6 mg per 100 g of animal body weight dissolved in 10 mM citrate buffer (pH 4.5). Control rats were administered 10 mM citrate buffer (pH 4.5) as previously described. Whole blood glucose levels were measured 2 days after induction of diabetes in rats. Animals were considered diabetic when blood glucose levels reached 22–32 mmol / L (M. Zafar, S. Naqvi / / Int. J. Morphol. -2010. -Vol.28, No.1. -P.135–142.).

[0402] Preparation of rat serum Rat serum was prepared from whole blood. To remove associated proteins and fibrinogen, the whole blood was incubated at 37°C for 30 minutes, after which the sample was centrifuged at 2500 g for 15 minutes. The resulting supernatant (serum) was immediately separated from blood cells and frozen at -20°C for further analysis.

[0403] Determination of glycated hemoglobin in rat blood The levels of glycated hemoglobin in whole blood of rats were measured spectrophotometrically according to established procedures. The assay was performed using a standard assay kit manufactured by Lachema (Czech Republic).

[0404] The glycated hemoglobin assay is based on the fact that the stable form of glycated hemoglobin (HbA1c) contains 1-deoxy-1-(N-valyl) fructose, which is dehydrated with phosphoric acid to form a colored complex with an absorbance spectrum at 433 nm. Neither the unstable form of glycated hemoglobin nor fetal hemoglobin interferes with the determination of glycated hemoglobin.

[0405] Total hemoglobin was measured spectrophotometrically. 20 μl of whole blood was mixed with 5 ml of transformation solution. Absorbance readings were taken at a wavelength of 540 nm for the transformation solution. The total hemoglobin fraction was calculated according to the manufacturer's recommendations using the following formula:

[0406]

number

[0407] where Hb is total hemoglobin and A is the optical density of the test sample. The amount of total hemoglobin is expressed in g / L.

[0408] Hemolysates were prepared by adding a 1:10 diluted 3.8% solution of sodium citrate, an anticoagulant, to freshly collected blood. One milliliter of stabilized blood was withdrawn and centrifuged at 1000 g for 10 minutes to remove the plasma. Three milliliters of saline was added to the resulting red blood cell pellet, and the mixture was gently stirred and centrifuged again as described above. Three milliliters of distilled water was added to the pellet, and the well-mixed mixture was allowed to stand at room temperature for 10 minutes. After further centrifugation, 1.5 milliliters of the supernatant (hemolysate) was separated and mixed with 0.25 milliliters of 85% phosphoric acid solution. The test tubes were closed with rubber stoppers and heated in a boiling water bath for 30 minutes. Upon dehydration, the tubes were cooled under running water for 10 minutes. 0.5 milliliters of a 2.45 M solution of trichloroacetic acid was added to each tube. The contents of the tubes were shaken and centrifuged at 1000 g for 20 minutes. A 1 ml aliquot of the supernatant was pipetted into another set of dry tubes, to which a 2.5 μM thiobarbituric acid solution was added. The contents of the tubes were thoroughly mixed and incubated at 37°C for 40 min. The same procedure was performed for control samples, except that water was added to C1 instead of acid, and a mixture of acid and hemolysates from different samples was added to C2. The optical density of the samples was measured spectrophotometrically at a wavelength of 443 nm against distilled water.

[0409] The concentration of glycated hemoglobin was calculated using the following formula:

[0410]

number

[0411] where A1 is the optical density of the test sample, A2 is the optical density of the reagent control sample, A3 is the optical density of the positive control sample, K is the tangent angle calculated according to the fructose calibration curve, and Hb is the total hemoglobin content.

[0412] The concentration of glycated hemoglobin was expressed as μmoles of fructose per g of hemoglobin (Glycated Hemoglobin / Assay Kit / / Pliva-lachema diganostica.-2008.-10003258.).

[0413] Determination of insulin levels in rat serum Insulin levels in rat serum were determined using an enzyme-linked immunosorbent assay based on a common method used for soluble proteins, which was performed in 96-well microplates equipped with adsorption capacity for soluble proteins.

[0414] Serum was prepared as a 1–10 dilution in 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl. Samples in a volume of 100 μl were incubated overnight at 4°C in microplate wells. After incubation, the wells were washed with a buffer containing 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween 20 to remove unbound material. Nonspecific binding sites were blocked with 5% nonfat milk blocking solution and incubated at 37°C for 1 hour. After washing, primary rabbit anti-insulin antibodies were loaded into separate wells of the microplate and incubated at 37°C for 1 hour. After incubation, the microplate wells were washed and loaded with the appropriate secondary antibody conjugated to horseradish peroxidase and incubated at 37°C for an additional 1 hour. Secondary antibody binding was visualized by adding 100 μl of OPD solution to each well at a concentration of 0.4 mg / ml prepared in citrate buffer (pH 5.0) containing 0.013% HO. The peroxidase reaction was stopped after 10 min by adding 100 μl of 1 M HSO. Optical density was measured at 492 nm. Insulin, cytokine, and IgG concentrations were expressed in relative units related to the total protein concentration in serum, determined using the Bradford protein assay.

[0415] Protein concentration determination Protein concentration was measured using the Bradford protein assay, which is based on the absorbance shift of the dye Coomassie Brilliant Blue G-250.

[0416] To measure protein concentration, 10 μl of 30% NaOH, 70 μl of distilled water, and 2 ml of Bradford reagent were added to each sample. To prepare 100 ml of Bradford reagent, 6 ml of stock solution, 3 ml of 95% ethanol, 6 ml of 88% H3PO4, and 35 mg of Coomassie Brilliant Blue dye were mixed, and the resulting mixture was adjusted to a volume of 100 ml with distilled water. The stock solution contained 10 ml of 95% ethanol, 20 ml of 88% H3PO4, and 35 mg of Coomassie Brilliant Blue.

[0417] The absorbance, which was visible for 2–5 min, was then measured spectrophotometrically at 595 nm against a control sample containing 20 μl of distilled water instead of biological material. The protein concentration of each test sample was determined using a standard curve and expressed in mg / ml.

[0418] Statistical treatment of the results Statistical processing of the obtained results was performed using the method of variance statistics, and correlation analysis was performed using Origno Pro 7.0 and SPSS 16 software. Primary statistical values ​​were obtained by calculating the mean (M) and standard error of the mean (m). Differences between variables were evaluated using parametric statistical methods (ANOVA). Student's t-test was used to evaluate the statistical significance of differences between two samples. Differences were considered statistically significant if p<0.05.

[0419] result The effect of Zn on the development of type 1 diabetes in experimental groups of rats 64A study investigating the potential effects of aspartic acid showed the following results (Table 22). It was demonstrated that the main parameters characterizing the development of type 1 diabetes, namely the concentrations of glucose, glycated hemoglobin, and insulin, improved after administration of the test substance. A significant decrease in glucose levels (26%) and glycated hemoglobin (30%) was recorded. A 13% increase in serum insulin levels was also noted. The obtained results indicate that the test substance has a positive effect on the course of type 1 diabetes and can be used as a prophylactic agent to reduce the toxic effects of elevated glucose levels in the bloodstream during the development of this condition.

[0420] [Table 22]

[0421] Cytokines are endogenous biologically active polypeptide mediators represented by a large, heterogeneous group of small, nonspecific antigens and glycoproteins that are produced in response to external extracellular stimuli and are involved in the formation and regulation of specific immune responses in the body through interactions between nonspecific protective responses and specific immune responses. Proinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-12, TNF-α, and IFN-γ, are involved in the initiation of specific immune responses, while anti-inflammatory cytokines (IL-4, IL-10, IL-13, and TGF-β) are involved in the generation of anti-inflammatory responses and inhibit the synthesis of proinflammatory interleukins.

[0422] [Table 23]

[0423] One of the mechanisms controlling the levels, and therefore the biological effects, of proinflammatory cytokines is the anti-inflammatory cytokine group, which includes IL-4, IL-10, IL-13, and TGF-β. Cytokine imbalance not only determines the development of inflammatory processes, but also determines the further form of the immune response, particularly whether it is primarily cellular or humoral. These cytokines can inhibit the synthesis of proinflammatory cytokines by affecting the transcription of specific genes in producer cells, induce the synthesis of interleukin-1 receptor antagonists, enhance the production of soluble receptors, and reduce the density of proinflammatory receptors on cells. Thus, IL-4 and IL-10 inhibit the production of PGE2, superoxide and nitroxide radicals, block the formation of IL-1, IL-6, IL-8, and TNF, and inhibit the synthesis of IL-2 and IFN-γ in lymphocytes.

[0424] [Table 24]

[0425] The results of this study suggest that Zn has a beneficial effect on cytokine profiles. 64 It shows a specific positive effect of aspartic acid, which can be considered a factor that normalizes the inflammatory processes that occur in the body during the development of type 1 diabetes.

[0426] Summary of study findings This study on the potential effects of the test substance on glucose metabolism and related processes showed promise for its use in normalizing glucose metabolism in patients with type 1 diabetes, possibly as an independent agent and in combination with other drugs.

[0427] The intraperitoneal route of administration of the test substance is not suitable as a potential regulator of glucose metabolism. Oral administration was much more effective, resulting in a reduction in glucose levels in control animals.

[0428] The results indicate that compared to zinc acetate (natural zinc), zinc isotopes have a longer-lasting effect on serum insulin levels or are released more slowly from their deposition sites, which also delays the effect of increasing insulin levels.

[0429] Microscopic results indicate that administration of zinc preparations, especially zinc isotopes, results in an increase in the area of ​​pancreatic islets, which in turn may indicate the potential for increased insulin production by these islets. This is a move in the right direction, as the development of type 1 diabetes is associated with a significant insulin deficiency due to problems with insulin synthesis by these islets. The results correlate with those from analyses of insulin levels in the serum of experimental animals.

[0430] Glucose tolerance tests showed that zinc isotopes were significantly higher in the control group than in the control group. 64 The study showed a significant decrease in glucose levels after Zn administration. This may indicate that the administered test substance is affecting insulin levels in the bloodstream, resulting in a triggering mechanism related to the removal of glucose from the blood. Given that insulin is a zinc-dependent protein, zinc administration may be expected to either increase the activity of this protein relative to its receptor in tissues or increase the amount of this hormone in the bloodstream.

[0431] The results obtained using a type 1 diabetes model may show that the test substance has a positive effect on the course of type 1 diabetes and may be used as a preventive agent to reduce the toxic effects of elevated blood glucose levels in the bloodstream during the development of this pathology.

[0432] The results of this study suggest that Zn has a beneficial effect on cytokine profiles. 64 A specific positive effect of aspartic acid has been demonstrated, which can be considered a factor that normalizes the inflammatory processes that occur in the body during the development of type 1 diabetes.

[0433] Based on mathematical models of stable systems, mass spectrometry experimental data, and analyses of literature sources, we demonstrate that changes in amino acid chirality in proteins and subsequent violations of protein conformation and DNA defects are the result of helical distortion due to stable isotope substitution. Strong evidence suggests that the isotopic composition of the same chemical element influences chemical bond formation in the solenoidal / helical structure of biomolecules. Isotope-induced changes in the conformation of biomolecules appear to signal the onset of pathology. However, the most important feature of such modifications is their reversibility. The conformation of biomolecules can be modified by modulating the isotope ratios of the elements from which they are made. A perfect conformation of a protein implies complete, healthy / youthful homeostasis. It allows for complex effects on cell, tissue, organ, and organism pathological changes, mobilizing the immune, endocrine, and nervous systems against degenerative diseases. Isotope-selective therapy could be the next step after molecular signature-based therapeutic strategies.

[0434] While the present invention has been described in conjunction with the detailed description thereof, it should be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Thus, while only certain features of the invention have been illustrated and described, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. ６４ A pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis (NASH) comprising Zn-enriched zinc aspartate, wherein at least 80% of the total Zn content in the composition is ６４ Zn.

2. 10. The pharmaceutical composition of claim 1, wherein the composition further comprises a diluent or excipient.

3. 3. The pharmaceutical composition of claim 2, wherein the diluent is deuterium-depleted water.

4. At least 95% of the total Zn content in the composition ６４ The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound is Zn.

5. At least 99% of the total Zn content in the composition ６４ The pharmaceutical composition according to any one of claims 1 to 4, wherein the compound is Zn.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is administered by injection.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is administered orally.

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