Method of preventing or treating viral infections

Abstract

To provide a method of preventing or treating viral infections.SOLUTION: A method of treating or preventing a disease or condition caused (at least in part) by, or related to, a viral infection in a patient is provided, the method including a step of administering to the patient a pharmaceutical composition including, in certain embodiments, 64Zn-enriched zinc, at a therapeutically effective dose or a prophylactically effective dose for treating or preventing the disease or condition caused by the viral infection. In one aspect, the present disclosure provides a method of treating or preventing a disease or condition caused (at least in part) by, or related to, a viral infection in a patient, the method including a step of administering to the patient a pharmaceutical composition including zinc, at a therapeutically effective dose or a prophylactically effective dose for treating or preventing the disease or condition caused by the viral infection.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field The present disclosure relates to the prevention or treatment of viral infections (including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection) in patients.

Background Art

[0002] Background Viral infections (such as infections by coronaviruses) are becoming a problem from both the perspectives of treatment (or lack thereof) and the spread of infection and the exacerbation of diseases caused by the patient being infected.

Summary of the Invention

Means for Solving the Problems

[0003] Summary In one aspect, the present disclosure provides a method for treating or preventing a disease or condition caused by or associated with a viral infection (at least in part) in a patient, the method comprising administering to the patient a pharmaceutical composition comprising zinc in a therapeutically effective dose or a prophylactically effective dose for treating or preventing the disease or condition caused by the viral infection. In some embodiments, the composition comprises, in further embodiments, a complex of zinc and / or its isotope with an amino acid dissolved in either a culture medium (such as RPMI-1640, etc.) or deuterium-depleted water, or is the aforementioned complex. In some embodiments, the composition comprises 64 Zn-enriched zinc (the term " 64 Zn e " is used herein to refer to 64 Zn-enriched zinc). In some embodiments, the composition comprises natural Zn and / or Zn-64, or is a solution containing these.

[0004] In some embodiments, 64 Zn-enriched zinc is 64 a Zne compound or 64 Zne It is in the form of a salt. In certain embodiments, the disclosed compositions are at least 80% of 64 Zn e , at least 90% of 64 Zn e , at least 95% of 64 Zn e , or at least 99% of 64 Zn e zinc (e.g., 80% of 64 Zn e , 85% of 64 Zn e , 90% of 64 Zn e , 95% of 64 Zn e , 99% of 64 Zn e , or 99.9% of 64 Zn e zinc present).

[0005] The subject / patient can be a human or non - human mammal (such as a non - human primate or a pet dog or cat).

[0006] Numerous other aspects are provided in accordance with these and other aspects of the invention. Other features and aspects of the invention will become more fully apparent from the following detailed description and the appended claims. In embodiments of the invention, for example, the following items are provided. (Item 1) A method of treating or preventing a disease or condition caused by viral infection, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount or a prophylactically effective amount of Zn, wherein the composition 64 Zn e compound or a salt thereof, wherein the 64 Zn e compound or a salt thereof is at least 80% of 64 Zn e or the composition comprises a solution comprising natural Zn or Zn - 64. (Item 2) 2. The method of claim 1, wherein the composition comprises zinc dissolved in culture medium or deuterium-depleted water and / or zinc complexed with an amino acid of its isotope. (Item 3) 3. The method of claim 1, wherein the composition comprises a solution containing natural Zn or Zn-64, which is a citrate solution, a glutamic acid solution, a glycine-methionine solution, an EDDA solution, a sulfate solution, an aspartic acid solution, or a TBPDA solution. (Item 4) 10. The method of any of the preceding items, wherein the viral infection is an influenza virus, a herpes simplex virus, including herpes simplex virus type 2, a hepatitis virus, including hepatitis C virus, an Epstein-Barr virus, a coronavirus, including SARS-CoV-2, an Ebola virus, or an HIV infection. (Item 5) 5. The method of claim 4, wherein the viral infection is infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (Item 6) The method of any of the preceding items, further comprising a diluent or excipient. (Item 7) 7. The method of claim 6, wherein the diluent is deuterium-depleted water. (Item 8) The composition 64 Zn e The compound or a salt thereof 64 Zn e The compound is at least 95% 64 Zn e 3. The method of any of the preceding items, wherein (Item 9) The composition 64 Zn e The compound or a salt thereof 64 Zn e The compound is at least 99% 64 Zn e Item 9. The method according to Item 8, wherein (Item 10) 64 Zn eis in the form of a salt selected from the group consisting of aspartate (chemical formula -C4H5O4N 64 Zn e ) having two aspartic acid molecules, sulfate, and citrate, the method according to any of the preceding items. (Item 11) The method according to any of the preceding items, wherein the composition is administered by injection. (Item 12) The method according to any of Items 1 to 10, wherein the composition is administered orally.

Brief Description of the Drawings

[0007]

Figure 1

[0008]

Figure 2

[0009]

Figure 3

[0010]

Figure 4

Mode for Carrying Out the Invention

[0011] Detailed Description As used herein, the term "a" or "plural" before a noun represents one or more specific nouns.

[0012] Unless otherwise specified, for the terms "for example", "such as", and their grammatical equivalents, the clause "is not limited to" is understood to follow. As used herein, the term "about" is meant to account for variations due to experimental error. All measured values reported herein are understood to be modified by the term "about" whether or not this term is explicitly used, unless otherwise specified. Unless the context clearly dictates otherwise, as used herein, the singular forms "a", "an", and "the" include the plural forms.

[0013] Unless otherwise specified, for the terms "for example", "such as", and their grammatical equivalents, the clause "is not limited to" is understood to follow. As used herein, the term "about" is meant to account for variations due to experimental error. All measured values reported herein are understood to be modified by the term "about" whether or not this term is explicitly used, unless otherwise specified. Unless the context clearly dictates otherwise, as used herein, the singular forms "a", "an", and "the" include the plural forms.

[0014] All ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, the recited range "1.0 to 10.0" is to be considered to include any and all sub-ranges starting with a minimum value of 1.0 or greater and ending with a maximum value of 10.0 or less (e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9).

[0015] Also, all ranges disclosed herein are to be considered to include the endpoints thereof, unless explicitly stated otherwise. For example, a range of "between 5 and 10" or "from 5 to 10" or "5 - 10" is to be considered to include the endpoints 5 and 10.

[0016] Unless explicitly prohibited by the nature of the present disclosure or related embodiments, it should be further understood that the features of one embodiment can be applied generally to such other embodiments even though they are neither specifically described nor exemplified in the other embodiments. Similarly, the compositions and methods described herein can include any combination of features and / or steps described herein that are not inconsistent with the objectives of the present disclosure. Numerous modifications and / or adaptations of the compositions and methods described herein will be apparent to those skilled in the art without departing from the subject matter of the present invention.

[0017] "Effective amount", "preventive effective amount", or "therapeutically effective amount" refers to the amount of an agent or composition that produces an advantageous effect or a desirable result in a subject, or the amount of an agent or composition that exhibits a desired in vivo or in vitro activity. "Effective amount", "preventive effective amount", or "therapeutically effective amount" refers to the amount of an agent or composition that produces a desired biological, therapeutic, and / or preventive result. This result can be a reduction, remission, alleviation, delay, and / or mitigation of one or more symptoms, signs, or causes of a disease, disorder, or condition in a patient / subject, or any other desirable change in a biological system. The effective amount can be administered in one or more doses. [[ID=!]]

[0018] An "effective amount", "prophylactically effective amount", or "therapeutically effective amount" can first be estimated by following cell culture assays or using animal models, typically mice, rats, guinea pigs, rabbits, dogs, or pigs. Animal models can be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine appropriate dosages and routes of administration for humans. When calculating human equivalent doses, conversion tables (such as those provided in Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005)) can be used. Those skilled in the art are aware of additional guidance that can also be used to develop human therapeutic dosages based on non-human data. An effective dose is generally from 0.01 mg / kg to 2000 mg / kg of the active agent, preferably from 0.05 mg / kg to 500 mg / kg of the active agent. The exact effective dose will depend on the severity of the disease, the overall health of the patient, age, weight and gender, nutrition, time and frequency of administration, combination(s) of drugs, responsiveness and tolerance / response to the administration, and other factors that will be considered by those skilled in the art when determining dosages and routes of administration for a particular patient. Such dosages can be determined by routine experimentation and the discretion of the physician. Also, the effective dose will vary depending on the potential for combination use with other therapeutic procedures (such as the use of other drugs).

[0019] As used herein, "patient" and "subject" are interchangeable terms and can refer to human patients / subjects, dogs, cats, non-human primates, etc.

[0020] Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs. The methods and materials used in the present invention are described herein; other suitable methods and materials known in the art can 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 in this specification are hereby incorporated by reference in their entirety. In case of conflict, the specification including the definitions shall prevail.

[0021] Viral infection

[0022] Viral infection occurs when a pathogenic virus invades an animal's body, adheres to infectious virus particles (virions), and enters susceptible cells. These infections cause various diseases / conditions. Some infections are highly infectious (such as influenza virus infection). Other infections are highly lethal (such as Ebola virus infection). New viral diseases usually occur at a certain frequency when animal virus pathogens infect humans. Examples include HIV, Ebola virus, etc. Different from bacterial diseases, the treatment of diseases caused by viral infections is not readily available.

[0023] From 2019 to 2021, a pandemic caused by a virus struck humanity. The disease is called COVID-19 and was previously caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was formerly known as the novel coronavirus 2019 (2019-nCoV). This virus is thought to be of animal origin and first infected humans in Wuhan, China, in November or December 2019. The main source of infection soon became human-to-human transmission. It spread among humans mainly through respiratory droplets, primarily from coughing and sneezing.

[0024] Since the virus accesses host cells via ACE2, the most abundant enzyme in type II alveolar cells of the lung, the lung is the organ most affected by COVID-19. Zhang et al., Intensive Care Med (3 March 2020) https: / / doi.org / 10.1007 / s00134-020-05985-9. Angiotensin-converting enzyme 2 (ACE2), discovered as a homolog of ACE, acts as a substance maintaining physiological balance and controls the level of circulating angiotensin II (AngII) homeostasis. ACE2 is a zinc metalloenzyme and carboxypeptidase located as an extracellular enzyme on the surface of endothelial cells and other cells. The density of ACE2 in each tissue correlates with the severity of the disease in that tissue. When alveolar disease progresses, respiratory failure may develop and lead to death. Also, ACE2 can be a pathway for viruses that attack the heart and cause acute heart injury. Patients suffering from cardiovascular pathologies have a worse prognosis than those who do not.

[0025] Zinc

[0026] Zinc is due to a trace element essential for ensuring the proper metabolic state of the human body. More than 200 enzymes in the body depend on zinc. This element is either a component of enzymes targeting all of the following enzyme classes or a regulator of their activity: transferases (RNA and DNA polymerases, reverse transcriptase, thymidine kinase, nucleotidyl transferase, carboxypeptidase, and other peptidases), hydrolases (alkaline phosphatase, 5-nucleotidase, aminopeptidase, etc.), lyases (aldolase, carbonic anhydrase, etc.), oxidoreductases (alcohol dehydrogenase, superoxide dismutase, etc.), ligases, and isomerases. Without the presence of zinc, proteins, fats, or carbohydrates cannot be metabolized.

[0027] Also, zinc has been proven to mediate the effects of antioxidants. Zinc is an inhibitor of NADPH oxidase, an enzyme complex that catalyzes the production of highly aggressive superoxide anion radicals. Furthermore, zinc can directly affect the oxidation of free radicals at the initiation stage of the chain reaction; zinc is a component of some enzymes in the antioxidant defense system (such as Cu / Zn-containing superoxide dismutase). By linking to the thiol groups of proteins, zinc protects proteins from oxidation by reactive oxygen species. This trace element induces the synthesis of metallothionein, a cysteine-rich protein that acts as a free radical scavenger. Zinc suppresses the formation of reactive mixed-valence metal oxides and is involved in the stabilization of membrane structures.

[0028] The importance of zinc in metabolism and structure is clear from its wide range of biological activities. Thus, zinc is necessary for the normal operation of processes related to cell division and differentiation (such as 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 prostaglandin conversion reactions. Zinc exhibits significant lipophilic activity and has hepatoprotective properties. Haase H., Rink L. Zinc Signaling. Zinc in Human Health / / Amsterdam, Netherlands: IOS Press. 2011. 243.

[0029] Furthermore, zinc is a regulator of the activity of phagocytes and lymphocytes and affects neutrophil chemotaxis, thus playing a very important role in immunological reactions. 5'-Nucleotidase, a zinc-containing enzyme, is very important in the functional states of T lymphocytes and B lymphocytes. Deficiency of isolated zinc severely impairs various parameters of T cell function (including thymic involution, inhibition of cell-mediated cytotoxicity, and reduction in the total lymphocyte count). Zinc is involved in stimulating the metabolism and activity of pituitary hormones, the adrenal glands, the pancreas, the prostate, and the testes. It is clear that zinc plays a 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。

[0030] In addition, zinc acts as a co-drug / antagonist for the absorption of many trace elements and vitamins (such as iron, copper, magnesium, vitamin A, E, folic acid, etc.) and affects their metabolism.

[0031] In short, zinc is involved in various life processes and functions in the human body. Detailed studies on some of these functions are still incomplete, and many of the mechanisms of this trace element are still not fully understood or recognized. However, experimental and clinical studies shown in the literature indicate that zinc is one of the important elements, and a decrease in zinc levels in the body is associated with the onset and emergence of some of the most prevalent non-communicable diseases. Since the main metabolic processes in the body occur through the active participation of zinc-containing enzymes and zinc-dependent enzymes, its deficiency interferes with many life processes.

[0032] Even when using the classical pharmacological forms of zinc-zinc salts and their chelates, due to the low bioavailability of this element, it is not always possible to effectively compensate for zinc deficiency.

[0033] Treatment Methods and Compositions In one aspect, the present disclosure provides a method for treating or preventing a disease or condition caused by or associated with (at least in part) a viral infection in a patient, the method comprising administering a pharmaceutical composition comprising zinc to the aforementioned patient in a therapeutically effective dose or a prophylactically effective dose for treating or preventing the disease or condition caused by the viral infection. In some embodiments, the composition comprises a complex of zinc and / or its isotope with an amino acid dissolved in either a culture medium (such as RPMI-1640, etc.) or deuterium-depleted water, or is the aforementioned complex. In some embodiments, the composition is 64 Zn-enriched zinc (the term "64 Zn e " is used herein to mean 64 Zn (used to refer to enriched zinc). In some embodiments, the composition comprises or is a solution comprising natural Zn and / or Zn-64. In some embodiments, the composition comprises Zn in elemental form or in the form of a pharmaceutically acceptable salt, compound, or complex thereof. 64 Zn e Includes.

[0034] A subject may need to be prophylaxis, for example, but not limited to, if the subject is suspected of having been infected with the virus, is in a high-risk group for viral infection, or is in an area with a high incidence of viral infection.

[0035] In some embodiments, the solution containing native Zn or Zn-64 is a citrate solution, a glutamic acid solution, a glycine-methionine solution, an EDDA solution, a sulfate solution, an aspartic acid solution, or a TBPDA solution.

[0036] In some embodiments, 64 Zn concentrated zinc is 64 Zinc compounds or 64 Zn e In certain embodiments, the disclosed compositions 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 of zinc (e.g., 80% of 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 Contains zinc, which is present in

[0037] In some embodiments, 64 Zn e is in the form of a salt selected from the group consisting of aspartate, sulfate, and citrate. In further embodiments, 64 Zn e The chemical formula of aspartate is C4H5O4N 64 Zn e and has two aspartic acid molecules.

[0038] The SARS-CoV-2 virus accesses host cells via the enzyme ACE2, which has heavy isotopes of zinc. Therefore, COVID-19 patients should be treated by modulating ACE2 activity to homeostasis.

[0039] In some embodiments, the viral infection is an infection by SARS-CoV-2, and the patient is a human patient with SARS-CoV-2 infection or at risk of SARS-CoV-2 infection (e.g., having stayed in an area with a high infection rate).

[0040] In some embodiments, the viral infection is an infection by an influenza virus (such as influenza A virus), a herpes simplex virus (such as herpes simplex virus type 2), a hepatitis virus (such as hepatitis C virus), Epstein-Barr virus, a coronavirus (such as SARS-CoV-2), Ebola virus, or HIV.

[0041] The term " 64 Zn e " is used herein to refer to 64 Zn-enriched zinc. That is, zinc in which 64 Zn is enriched such that it is 64 more concentrated than the normal percentage of zinc in nature.

[0042] light isotopes 64 Zn eZinc in this form is absorbed into the body much more readily than naturally occurring zinc. In certain embodiments, the disclosed compositions comprise at least 80% of 64 Zn e 、at least 90% of 64 Zn e 、at least 95% of 64 Zn e 、or at least 99% of 64 Zn e zinc (e.g., 80% of 64 Zn e 、85% of 64 Zn e 、90% of 64 Zn e 、95% of 64 Zn e 、99% of 64 Zn e 、or 99.9% of 64 Zn e ).

[0043] In some embodiments, the composition or solution further comprises a diluent or excipient. In some embodiments, the diluent is water. In a further embodiment, the water diluent is deuterium-depleted water.

[0044] In some embodiments, 64 Zn e the compound or its salt is present between 20 - 100% of 64 Zn e . In a further embodiment, 64 Zn e the compound or its salt is present at least 80% of 64 Zn e . In a further embodiment, 64 Zn e the compound or its salt is present at least 95% of 64 Zn e . In some embodiments, the composition comprises between 0.05 mg and 110 mg of 64 Zn e . In some embodiments, the composition comprises between 1 mg and 10 mg of 64 Zn eIt includes. In some embodiments, 64 Zn e the compound or its salt has at least 90% of 64 Zn e present, and the composition is 64 Zn e an aqueous solution present at a concentration between 0.1 mg / ml and 10 mg / ml. In some embodiments, 64 Zn e is in the form of a salt selected from the group consisting of aspartate having two aspartic acid molecules (chemical formula -C4H5O4N 64 Zn e ), sulfate, and citrate.

[0045] In some embodiments, the composition or solution is administered by injection. In other embodiments, the composition or solution is administered orally.

[0046] Formulation and Administration of the Composition

[0047] The composition used in the disclosed method can be administered to a subject in need thereof in any suitable mode of administration, at any suitable frequency, and at any suitable effective dosage.

[0048] In some embodiments, the total dosage of zinc is the same as the recommended allowance or intake of zinc per day in the United States. In some embodiments, the total dosage of Zn is 1 / 2, 2 times, 3 times, 5 times, or 10 times the recommended allowance or intake of zinc per day in the United States. In some embodiments, the total amount of Zn is between 1 / 2 and 10 times the recommended allowance or intake of zinc per day in the United States. The composition used in the disclosed method can include a daily dose formulated to be administered once a day or several divided doses formulated to be administered at the corresponding number of times per day. Also, the composition used in the disclosed method can include a dosage of Zn once every two days, once every three days, once a week, or at any other suitable frequency.

[0049] The compositions used in the disclosed methods can be in any suitable form and can be formulated for any suitable delivery means. In some embodiments, the compositions used in the disclosed methods are provided in a form suitable for oral administration (such as tablets, pills, lozenges, capsules, liquid suspensions, solutions, or any other conventional oral dosage form). Oral dosage forms can be immediate release, delayed release, sustained release, or enteric release and can include one or more coatings where appropriate. 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 pyrogen-free form and can include preservatives and / or other suitable excipients (such as sucrose, disodium hydrogen phosphate heptahydrate or other suitable buffers), pH adjusters (such as hydrochloric acid or sodium hydroxide), and polysorbate 80 or other suitable surfactants.

[0050] When provided in the form of a solution, in some embodiments, the compositions used in the disclosed methods are provided in glass or plastic bottles, vials, or ampoules, any of which can be suitable for single or multiple uses. Bottles, vials, or ampoules containing the disclosed compositions can be provided in the form of a kit with one or more suitable gauge needles and / or one or more syringes, and it is preferred that all of them are sterile. Thus, in certain embodiments, a kit is provided that includes the above solution packaged in a suitable glass or plastic bottle, vial, or ampoule, and the kit can further include one or more needles and / or one or more syringes. The kit can further include instructions for use.

[0051] In certain embodiments, the dosage of Zn is proportional to the various reliable daily intake guidance for the corresponding element (such as the Recommended Dietary Allowance (USRDA), Adequate Intake (AI), Recommended Dietary Intake (RDI)).

[0052] In some embodiments, the composition used in the disclosed method comprises, or is, a complex of zinc and / or its isotope with an amino acid dissolved in either a culture medium (RPMI-1640) or deuterium-depleted water.

[0053] In some embodiments, the Zn dosage is between about 1 / 2 times and about 20 times the amount indicated by the guidance, more preferably between about 1 time and about 10 times the amount indicated by the guidance, and even more preferably between about 1 time and about 3 times the amount indicated by the guidance. Thus, in certain embodiments, the single dose of the composition used in the disclosed method for daily administration will be formulated to include amounts within these ranges (such as about 1 / 2 times, about 1 time, about 3 times, about 5 times, about 10 times, and about 20 times the amount indicated by the guidance). These amounts are generally for oral ingestion or topical application. In some embodiments, the intravenous dosage is less (such as about 1 / 10 to about 1 / 2 of the amount indicated by the guidance). For those who are sensitive to a particular element or class of elements (e.g., those with kidney problems), the lowest dosages within these ranges are appropriate. For zinc, the daily amount indicated by the guidance ranges from 2 mg for infants to 8 - 11 mg (depending on gender) for ages 9 years or older. The daily dosages considered throughout this application can be further divided into divided dosages, and the divided dosages can be administered at appropriate times per day such that the total daily dosage is administered (e.g., administering 1 / 2 of the daily dose 2 times a day, administering 1 / 3 of the daily dose 3 times a day, etc.). See Table 1.

[0054]

Table 1

[0055] The compositions used in the disclosed methods can be produced by methods used in common practice in the pharmaceutical industry (e.g., methods shown in Remington: The Science and Practice of Pharmacy (Pharmaceutical Press; 21st revised ed. (2011) (hereinafter, "Remington"))).

[0056] In some embodiments, the compositions used in the disclosed methods include at least one pharmaceutically acceptable vehicle or excipient. These include, for example, diluents, carriers, excipients, fillers, disintegrants, solubilizing agents, dispersants, preservatives, wetting agents, preservatives, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifying agents, and penetration enhancers (such as DMSO), as needed. The compositions can also include suitable adjuvants, such as solubilizing agents, dispersants, suspending agents, and emulsifying agents.

[0057] In certain embodiments, the composition further includes a suitable diluent, glidant, lubricant, acidulant, stabilizer, filler, binder, plasticizer, or release aid, and other pharmaceutically acceptable excipients.

[0058] A complete description of pharmaceutically acceptable excipients can be found, for example, in Remington’s Pharmaceutical Sciences (Mack Pub., Co., N.J. 1991) or other standard pharmaceutical science texts (such as Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017)).

[0059] In some embodiments, the compositions used in the disclosed methods can be administered intragastrically, orally, intravenously, intraperitoneally, or intramuscularly, although other routes of administration are possible.

[0060] Water can be used as a carrier and diluent in the composition. In addition to water, or instead of water, other pharmaceutically acceptable solvents and diluents may also be used. In certain embodiments, deuterium-depleted water is used as a diluent.

[0061] Furthermore, macromolecules with slow metabolism (such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, copolymers of amino acids, etc.) can be used as carrier compounds in the composition. Pharmaceutically acceptable carriers in the therapeutic composition may further contain a liquid (such as water, physiological saline, glycerol, or ethanol, etc.). Additionally, the aforementioned composition may further contain excipients (such as wetting agents or emulsifiers, buffering substances, etc.). Such excipients include, inter alia, diluents and carriers conventionally recognized in the art, and / or substances that promote the penetration of the active compound into cells (such as DMSO), as well as preservatives and stabilizers.

[0062] The composition used in the disclosed method can be provided in various dosage forms depending on the application; in particular, the aforementioned composition can be formulated as an injection solution.

[0063] The composition used in the disclosed method can be administered systemically. Suitable administration routes include, for example, oral or parenteral administration (intravenous, intraperitoneal, intragastric, etc.) and administration via drinking water. However, depending on the dosage form, the disclosed composition can be administered by other routes.

[0064] In certain embodiments, the composition used in the disclosed method containing Zn is administered at a concentration within 2.25 mg / ml.

[0065] In some embodiments, the composition used in the disclosed method is about 2 ml.

[0066] In some embodiments, 64 Zn e The enrichment level is about 99% or more. In other further embodiments, the 64 Zne comprises or consists of zinc aspartate having two aspartic acid molecules (chemical formula -C4H5O4N 64 Zn e ). The dosage of the compositions used in the disclosed methods can 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 one of ordinary skill in the art when determining the dosage and route of administration for a particular patient.

[0067] Light isotopes may be purchased. Zinc oxide - 64 of the required concentration may be purchased, for example, from Oak Ridge National Laboratory, Oak Ridge, TN, USA.

[0068] In some embodiments, the chemical formula of zinc aspartate is -C4H5O4N 64 Zn e and has two aspartic acid molecules. The structure of this zinc aspartate is as follows.

Chemical formula

[0069] In certain embodiments, about 20% to about 100% of the composition used in the disclosed method is 64 Zn e contained.

[0070] The compositions used in the disclosed methods can be co-administered with another suitable agent or therapeutic.

[0071] Viral infection can be any viral infection.

[0072] Examples

Examples

[0073] To better understand the present invention, the following examples are described. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

[0074] Example 1 Antiviral therapy using a zinc-64 isotope-based substance

[0075] Zinc is one of the most important micronutrients that play crucial roles in metabolism and is a component of numerous metalloenzymes and transcription factors. Barbosa, M.S., et al. 1989 J. Virol. 63:1404 - 1407. Zinc is part of 250 - 300 enzymes, and it is well-known that all of these belong to six enzyme classes. Barthel, A., E.A. Et al., 2007. Arch. Biochem. Biophys. 463:175 - 182. 10% of human proteins contain zinc. Beerheide, W. et al. 1999. J. Natl. Cancer Inst. 91:1211 - 1220. Bess, J.W. et al., 1992. J. Virol. 66:840 - 847. Zinc is essential for the normal functioning of the immune system as it increases thymocyte and peripheral T cell numbers. Boyle, W.J. et al., Cell 64:573 - 584.

[0076] Zinc is required for the growth and development of bone tissue. Briggs, M.W. et al. 2001. Virology 280:169 - 175. Zinc-containing enzymes involved in the synthesis and / or breakdown of carbohydrates, lipids, proteins, and nucleic acids target all known enzyme classes. Brottier, P. et al. 1992. J. Gen. Virol. 73:1931 - 1938. Zinc is a structural component of superoxide dismutase (SOD), an enzyme that is an important part of the antioxidant defense system. Culp, J.S. et al., 1988. Proc. Natl. Acad. Sci. USA 85:6450 - 6454; De Oliveira, W.R. et al., 2003. J. Eur. Acad. Dermatol. Venereol. 17:394 - 398.

[0077] Zn in the structure of zinc finger - proteins that bind to DNA +2Since ions are involved, this metal occupies a special position in cell biology. Zinc has long been known as a trace element involved in the most important processes, including the synthesis and function of many proteins in cells, signal transduction, proteins, and transcription factors.

[0078] Elucidating the details of these molecules in intracellular zinc homeostasis unexpectedly opened up new avenues in the field of virology and shed new light on host-virus interactions. Zn +2 has long been recognized as an important cofactor not only in cellular proteins but also in many viral proteins. Recent studies have demonstrated that the cellular environment itself, where the pool of free zinc is extremely small and tightly regulated, can be a limiting factor. Viruses are dependent on intracellularly stored zinc ions and use cellular Zn +2 for de novo protein synthesis. Therefore, cellular systems that control zinc balance can constitute a natural defense barrier that restricts zinc access and thereby interferes with virus replication.

[0079] In this regard, the aim of this study was to investigate the effect of a composition of natural zinc and its isotope (light Zn-64) on the replication of RNA and DNA viruses.

[0080] The aim of this study is to investigate the cytotoxicity and antiviral activity of the zinc isotope (Zn-64) against models of herpes simplex virus, Epstein-Barr virus, influenza virus, and a surrogate model of hepatitis virus (bovine viral diarrhea virus) in vitro and in vivo.

[0081] Materials and Methods

[0082] Test Substances Zinc and its light isotopes in various solvents such as citrate (citric acid), sulfate, aspartic acid and glutamic acid, glycine-methionine, TBPDA (n-n-toluenesulfonyl-n-benzoyl-o-phenylenediamine) and EDDA (ethylenediaminedisuccinic acid) were used in this study. 4 Zn-64 in citrate solution (0.9 mg / ml) 4k Natural Zn in citrate solution (0.9 mg / ml) 5 Zn-64 in EDDA (3 mg / ml) 5k Natural Zn in EDDA (3 mg / ml) 6 Zn-64 in sulfate solution (3 mg / ml) 6k Natural Zn in sulfate solution (3 mg / ml) 7 Zn-64 in aspartic acid solution (1.5 mg / ml) 7k Natural Zn in aspartic acid solution (1.5 mg / ml) 8 Zn-64 in glutamic acid solution (1.5 mg / ml) 8k Natural Zn in glutamic acid solution (1.5 mg / ml) 9-1 Zn-64 in glycine-methionine solution (2 mg / ml) 9-2 Natural Zn in glycine-methionine solution (1.5 mg / ml) 9-3 Glycine-methionine solution 10-1 Zn64 in TBPDA solution (3 mg / ml) 10-2 Natural Zn in TBPDA solution (0.9 mg / ml) 10-3 TBPDA solution (0.9 mg / ml) 11 Zn64 in TBPDA solution - for 14 days 9a Solution of complex 7 - aspartic acid - 3.41 mg / ml 10a Solution of complex 8 - glutamic acid - 4 mg / ml 8a Solution of complex 4 - citric acid - 2.3 mg / ml

[0083] Reference drug

[0084] Acyclovir (freeze preparation containing 250 mg sodium salt as the active ingredient), manufactured by KRKA, Slovenia from the active substance by The Welcome Foundation Limited; F. Hoffmann-La Roche Tamiflu manufactured by F. Hoffmann-La Roche Ltd, Switzerland

[0085] Cell cultures

[0086] Cell cultures were obtained from the Museum of Tissue Cultures of D.I. Ivanovsky Institute of Virology (RAMS, Moscow): - MDCK, a transplantable canine kidney cell culture - VNK, transplantable hamster embryo kidney epithelial cells - MDBK, a transplantable bovine kidney cell culture - B 95-8 (marmoset leukocytes) (transformed by Epstein - Barr virus (EBV), producing this virus chronically and serving as a source of EBV) - Raji, undifferentiated human B lymphoblastoid cells derived from Burkitt lymphoma

[0087] Cell cultures were grown in a growth medium consisting of 90% RPMI 1640 medium (Sigma, USA), 10% fetal bovine serum (Sigma, USA) and penicillin antibiotic (100 μg / ml), streptomycin (100 μg / ml) and L - glutamine (2 mM). A 0.25% Versene solution (Sigma, USA) was used to de - aggregate the monolayer of epithelial cells.

[0088] Cells were grown in plastic tissue culture flasks, 24 - well plates, and 96 - well plates in a thermostat at 37°C with 5% CO2. The growth activity of the cells was checked every two days using an optical inverted microscope.

[0089] Viruses

[0090] Influenza virus: infectious titer in allantoic cultures 5.0 - 9.0 lg EID 50 / 0.2 ml and hemagglutinin titer 1:512 GAO / 0.2 ml of influenza virus A / FM / 1 / 47 (H1N1) strain was obtained from the Museum of Viruses of D.I. Ivanovsky Institute of Virology (RAMS, Moscow) for use in this study.

[0091] Herpes simplex virus type 2 (HSV-2): BH strain was obtained from the Museum of Viruses of D.I. Ivanovsky Institute of Virology (RAMS, Moscow). The virus was maintained by serial passage in BNK cell cultures. The infectious titer for CPE in cell cultures was 6.0 - 9.0 lg TCD 50 / 0.1 ml.

[0092] Bovine viral diarrhea virus (BVDV): The virus material of the 4th passage was provided by Dr. A. Deryabin (a researcher at the Institute of Veterinary Medicine, UAAS). The infectious virus titer after 10 passages in MDBK cell cultures was 5 - 9 lg ID 50 .

[0093] Epstein - Barr virus (EBV) was recovered from lymphoblastoid cultures of B95 - 8 cells (B lymphocytes in marmoset), which are commonly used as a source of EBV, using the method described in Walt, Crawford. Finkel A., Czajke D. The effect of deuterium oxide on ascites tumor growth in mice / / Ed. F.N. Furness, New York: New York Acad. Sci, 1960. P.755 - 762.

[0094] Determination of the cytotoxic concentration (CC 50 ) of the drug

[0095] Using different cell cultures, the CC of each drug 50 was determined. At least 10 columns of wells in the cell culture plate were used for each dilution of the drug in the nutrient medium. The plates containing the cell cultures were incubated at 37 °C and 5% CO2 in air for 5 days. The test cultures and the control cultures were observed daily to determine the presence or absence of a cytopathic effect (CPE).

[0096] The degree of CPE was determined by changes in cell morphology (rounding, cell shrinkage, exclusion of cells undergoing degenerative changes from the well surface) using a 4-plus system from + to ++++:

[0097] "-" - No cytopathic effect is present

[0098] "+" - Up to 25% of the cell monolayer is affected (75% protection of the cell monolayer from the antiviral drug)

[0099] "++" - Up to 50% of the cell monolayer is affected

[0100] "+++" - Up to 75% of the cell monolayer is affected

[0101] "++++" - Complete degeneration of the cell monolayer

[0102] The CC of the drug 50 was the highest concentration at which no cytopathic effect was caused.

[0103] The colorimetric assay using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, USA) is based on the function of the mitochondrial dehydrogenase system of living cells, which reprocesses the artificial substrate MTT into formazan, which can be spectrophotometrically calculated, under normal conditions. The conversion of MTT to formazan decreases significantly in a dose-dependent manner when cells die under the action of a virus or substance toxic to the cells.

[0104] The MTT substrate (Sigma, USA) was dissolved in sterile phosphate-buffered saline (pH 7.2) at room temperature to a concentration of 5 mg / ml. 20 μl of the filtered MTT solution was added to the wells of a 96-well plate and incubated with the cells at 37 °C for 2 - 4 hours. After incubation, the medium was removed and 150 μl of 96-degree ethanol was added to the cells to dissolve the formazan crystals. The optical density of the solution was determined spectrophotometrically using a Multican FC reader (「Thermo Scientific」(USA)) at a wavelength of 540 nm. The drug concentration that inhibits cell viability by 50% (CC 50 ) was calculated using a linear regression program in Microsoft Excel created for Pentium (registered trademark) Pro.

[0105] Determination of the effective concentration (EC 50 )

[0106] EC 50 is the minimum drug concentration of the drug that inhibits the occurrence of virus-specific CPE by 50%. To determine EC 50 , cell cultures were infected with the test virus at a dose of 100 TCD 50 / 0.1 ml and then incubated at 37 °C for 60 minutes (min). After adsorption, unbound virus was removed, the cells were washed with nutrient medium, and drugs at different concentrations were added to the medium containing the cells (RPMI-1640 + 2% fetal serum). The absence of CPE in the experimental group (treated cultures) (which was present in the control group), as well as the decrease in the infectivity titer in the treated cultures compared to the virus control (the infectivity titer was present in the control group) and the difference in the infectivity titer of the experimental group, make it possible to determine the EC 50 of the drug.

[0107] Determination of the selectivity index (SI) of the drug

[0108] The selectivity index (SI) of the drug was determined as the ratio to the EC 50 of CC 50 .

[0109] Cytological analysis

[0110] Cytological analysis was performed after fixation of cells grown for 30 minutes in Shabadash's solution (9 parts copper nitrate + 1 part formalin in ethyl alcohol) on a coverslip. Samples for cytological analysis were stained using hematoxylin and eosin staining according to generally accepted procedures.

[0111] The mitotic index was calculated by analyzing 3000 - 10000 observed cells and expressed in ppm (‰) (number of mitoses per 1000 cells). At the same time, the percentage of pathological forms of mitosis was determined. The classification method for the analysis of pathological mitosis developed by V.N. Blyumkin was used.

[0112] Experiments on cytological preparations were carried out on a Zeiss Standard 20 microscope using 40x and 100x lenses and a 10x eyepiece.

[0113] In vitro amplification using polymerase chain reaction (PCR)

[0114] PCR was performed according to standard procedures using a reagent set for PCR (AmpliSens, Russia) and a DNA recombinant plasmid based on pUC vector 28 containing the coding sequence of the human leukemia inhibitory factor (LIF) gene as a template. The DNA concentration was 1 - 25 μg / 100 μl reaction mixture. DNA was amplified in a thermostat for PCR analysis (「Terzik」, DNA Technology, Moscow). The test substances were tested at concentrations of 5 - 40 μg / ml.

[0115] DNA viruses were isolated from samples using the innuPREP Virus DNA kit (Analityk Jena AC, Germany) or the DNA-sorb-B DNA kit (AmpliSens, Russia) according to the manufacturer's instructions. DNA concentration was measured using an Eppendorf BioPhotometer (Germany). Epstein-Barr virus DNA was analyzed using the AmpliSens® EBV-FL kit (FSIS CSRI, Russia) according to the manufacturer's instructions with real-time detection (qTOWER 2.2 amplifier, Germany).

[0116] Results

[0117] Determination of the cytotoxic concentration (CC 50 ) of the investigational drug

[0118] The CC 50 of each drug was determined using MDCK cells, VNK cells, and MDBK cells. At least 10 columns of wells in the cell culture plates were used for each dilution of the drug in nutrient medium. Plates containing cell cultures were incubated at 37 °C and 5% CO2 in air for 5 days. Test and control cultures were observed daily to determine the presence or absence of a cytopathic effect (CPE). The degree of CPE was determined by changes in cell morphology (rounding, cell shrinkage, elimination of cells undergoing degenerative changes from the well surface) using a 4-plus system from + to ++++.

[0119] The CC 50 of the drug was its highest concentration that did not cause cytopathic changes. The results are shown in Table 2.

[0120]

Table 2

[0121] In the toxicity analysis of substances in cultures of Raji lymphoblastoid cells, the substances with the lowest toxicity were glutamate- and glycine-methionine-based drugs, and the CC50 The values indicate that they are over 470 μg / ml and 200 μg / ml.

[0122] In the analysis of the results of the cytotoxic effect of the test substance, it is shown that the solvents of natural Zn and light isotopes of Zn are more toxic than the Zn complexes examined. Therefore, the CC of citric acid 50 is 1 / 160 or 0.014 mg / ml, and the CC of the complex in the citric acid solution 50 for Zn 64 is 0.45 mg / ml, and for natural Zn is 0.18 mg / ml, that is, its toxicity is 1 / 3 - 1 / 10. The CC of the glutamic acid solution 50 is 1:160 or 0.025 mg / ml, while in the Zn 64 complex it is 1 / 5 - 0.4 mg / ml, and for natural Zn it is 0.0046 mg / ml, that is, the light Zn-64 isotope reduces the toxicity of the solvent, and natural Zn increases it; the CC of aspartic acid 50 is 1 / 80 or 0.042 mg / ml, for the light Zn 64 complex it is 1 / 10 or 0.75 mg / ml, and for natural Zn it is 1 / 320 - 0.0046 mg / ml, that is, the same pattern is observed. The CC of the glycine and methionine solutions 50 is 1 / 160 or 0.018 mg / ml, and for the complexes of the light Zn 64 isotope and natural zinc they are 1 / 10 or 0.2 and 0.15 mg / ml respectively, that is, both the light Zn-64 isotope and natural zinc reduce the toxicity of the solvent to 1 / 10.

[0123] Regarding the TBPDA solvent, different regularities were observed: for MDCK cells, the light isotope increased the toxicity of the solvent by 30 times, while natural Zn had no effect on its toxicity.

[0124] In the cultures of BNK cells and MDCK cells, almost the same pattern as that of MDCK cells was observed.

[0125] Antiviral Activity of Natural Zinc and Zinc Isotopes

[0126] Assessment of Anti-Influenza Activity

[0127] To assess the antiviral activity of the drug solution in vitro, a transplantable MDCK cell culture was used daily. The cells were cultured in plates containing RPMI-1640 medium supplemented with 10% fetal serum (Nunclon, Surface, Denmark) in a thermostat with an atmosphere containing 37 °C and CO2. The cells were treated with trypsin from bovine pancreas type XIII (Sigma catalog number 8642, USA) to increase their sensitivity to influenza virus infection. The stock solution of trypsin was prepared by adding 2 mg of trypsin to 1 ml of DMEM culture medium.

[0128] The cells were washed three times with the solution by adding 50 μl to each well; the trypsin concentration was 2 μg / ml. Then, the cell growth medium was removed and replaced with 100 TCD 50 of the dose (50% tissue culture infective dose) of influenza virus, and then the test drug was added at different concentrations.

[0129] The cultures were incubated in a CO2 incubator for 3 days while being monitored daily using a microscope.

[0130] After 48 - 72 hours of incubation, the culture fluid was collected, and the infectivity titer of the influenza virus was determined by titration of the cell culture.

[0131] The influenza virus A / FM / 1 / 47 (H1N1) strain was used in the experiment. The infectivity titer in MDCK was 3.0 - 9.0 lg ID 50 .

[0132] The inhibitory effect of the substance was evaluated by the decrease in the infectivity titer of the virus under the action of the test substance compared with the control. 1.5 - 2.0 lg TCD 50The decrease indicates significant antiviral activity of the research compound, especially when the chemotherapy index is 8 or more.

[0133] The high influenza activity (EC 64 ) of solutions of natural Zn and its Zn 50 isotopes in MDCK cell cultures is shown in Table 3. See also FIGS. 1A and 1B.

[0134]

Table 3

[0135] The Zn-64 preparation and natural Zn in citrate solution, Zn-64 in glutamate solution and glycine-methionine solution were judged to exhibit anti-influenza activity.

[0136] Assay of anti-herpes activity

[0137] Transplantable VNK cell cultures were used to study the anti-herpes activity of zinc solutions, their isotopes and solvents. Cells were cultured in plates containing RPMI-1640 medium supplemented with 10% fetal serum (Nunclon, Surface, Denmark) in a thermostat with an atmosphere containing 37 °C and CO2.

[0138] Type 2 herpes simplex virus BH strain was used. Its infectious titer is 6.0 - 0.9 lg ID 50 .

[0139] A 1-day culture of VNK cells was selected to study the antiviral activity of the drug. The cell growth medium was removed and the test preparations were added to the cell monolayer at different concentrations. After 1 hour of contact, herpes virus was added to the cells at 100 TCD 50Inoculated at the dose of. Monitored daily using a microscope, and the cultures were incubated in a CO2 incubator for 5 days while recording virus replication by the cytopathic effect of HSV on VNK cells compared to control cultures where the cell monolayer was not exposed to any virus.

[0140] The cytopathic effect of HSV on cells, in combination with the proliferation and appearance of multinucleated giant cells, morphologically shows the formation of syncytia or rounded cells by the cells themselves (Figure 2).

[0141] After 3 days, the culture medium was recovered from the plate wells and the infectious titer in each sample was determined when each drug was added to each sample.

[0142] The anti-herpes activities (EC 50 ) of solutions of natural Zn and its Zn64 isotope in VNK cell cultures are shown in Table 4.

[0143]

Table 4

[0144] Natural Zn in EDTA and Zn-64 in glutamic acid solution and glycine-methionine solution showed anti-herpes activities.

[0145] Assessment of anti-HCV activity

[0146] Bovine viral diarrhea virus (BVDV), a surrogate HCV, was used in this study as it is a test model for hepatitis C virus.

[0147] The antiviral activity of the drug was studied in cultures of MDBK cells treated with different dilutions of the drug and then infected with BVDV at a dose of 100 TCD 50 . The cultures were incubated in a thermostat until specific cytopathic effects (Figure 3A and Figure 3B) in the virus control, and then the infectious virus titer was determined in the culture medium.

[0148] Antiviral activity (EC 50 ) of solutions of natural Zn and its Zn64 isotope in MDBK cell cultures against bovine viral diarrhea virus (BVDV) as a surrogate HCV virus is shown in Table 5.

[0149]

Table 5

[0150] Antiviral activity against surrogate hepatitis C virus (BVDV) was shown by natural Zn in citrate solution, EDDA, and sulfate solution and Zn-64 in glutamate solution and glycine-methionine solution.

[0151] In the analysis of the antiviral activity of solutions of natural zinc and light zinc isotopes in different solvents, the light isotopes of zinc in glutamate solution and glycine-methionine solution were the most active, indicating high values of the selectivity index for three virus infections. In citrate solution, its antiviral activity was significant against influenza virus and surrogate hepatitis C virus (BVDV), and the activities of both natural Zn and its light isotopes were equal here.

[0152] Salts of light zinc isotopes and natural zinc of EDDA, sulfate, and aspartic acid had antiviral activity against influenza virus and BVDV. Light Zn 64 isotopes in the TBFDA solvent showed no antiviral activity, and natural Zn showed activity only against influenza infection.

[0153] Assay of anti-EBV activity

[0154] The assay results of the antiviral activity of solutions of natural Zn and its Zn 64 isotope in cultures of Raji cells against Epstein-Barr virus infection model are shown in Table 6.

[0155]

Table 6

[0156] The drugs in glutamic acid solution and glycine-methionine solution were the most active against oncological Epstein-Barr virus, and the selectivity indices were 470 and 200, respectively.

[0157] Therefore, in the antiviral screening of natural zinc and its light isotopes in various solvents, it was shown that zinc compounds in glutamic acid, glycine-methionine, and citric acid were the most active against BVDV.

[0158] Study on the effectiveness of research compounds against the status of cell mitosis

[0159] Considering that the solvent showed high toxicity to cell cultures, the effects of zinc-based drugs in different solutions on the status of cell mitosis were investigated. Experiments were conducted on cultures of MDBK cells treated with various selected drugs. After 24 hours of contact, the cells were fixed and cytological preparations were made using standard techniques. The results obtained from this experiment are shown in Tables 7 and 8.

[0160]

Table 7

[0161] Table 7 shows that only three drugs - number 5 (EDDA solution of Zn64), number 6 (sulfate solution of Zn64), and number 7 (aspartic acid solution of Zn64) - significantly inhibited the mitotic activity of cells and increased the abnormal mitotic number.

[0162]

Table 8

[0163] Table 8 shows that the natural Zn in the glycine-methionine solution of drug-number 9-3 and the TBFDA solution of number 10-2 inhibited the mitotic activity of cells and increased the abnormal number of mitosis.

[0164] Further analysis of this research result on the mitotic situation of MDBK cells under the influence of drugs based on non-toxic concentrations of natural Zn and its light isotopes shows that Zn in the EDDA solution 64 Zn in the sulfate solution 64 as well as Zn in the aspartic acid solution 64 should be noted that natural Zn in the TBFDA solution and the lysine-methionine solvent significantly changed the mitotic situation of cells (that is, while the abnormal number of mitosis increased, the mitotic activity of cells significantly decreased). Therefore, the light isotopes and natural zinc of zinc in glutamic acid, glycine-methionine, and citric acid can be considered promising for further research.

[0165] Therefore, these studies on the cytotoxicity and antiviral activity of drugs show that Zn 64 citrate, Zn in EDDA 64 Zn 64 aspartate, Zn 64 glutamate, Zn 64 glycine-methionine are most promising as antiviral agents against EBV. Such a choice is explained by the fact that the solvent has no antiviral activity, the solvent as part of the zinc complex does not affect the mitotic situation of cells, and the Zn 64 complexes in glutamic acid and glycine-methionine effectively inhibit the replication of influenza virus and BVDV virus.

[0166] Evaluation of the antiviral activity of zinc glutamate and its isotope complexes in "light water"

[0167] Hydrogen isotopes are known to enter the body mainly with drinking water and food. When water enters the body, it becomes involved in various biochemical processes, and as a result, its atoms can become structural units of various compounds synthesized by the body. A clear example of how the isotope composition of water is reflected in the isotope composition of proteins synthesized by the body is described in Ehleringer J. et al. Proc. Nat. Acad. Sci USA, 2008, 105, pp. 2788 - 2793. The authors showed that there is a direct relationship between the isotope composition (H, O) of human hair (mainly consisting of α-keratin protein) and drinking water.

[0168] In cells, water has a special intermediate structure between liquid water and ice. Layers of oriented water molecules surround all hydrophilic macromolecules (including protein and nucleic acid molecules) in the protoplasm.

[0169] The strong antimitotic effect of D2O (heavy water) was detected in the first experiments. Thus, in 1938, H. Barbour and E. Allen described the growth retardation and regression of transplanted lymphosarcoma and breast cancer in mice given 40% D2O as drinking water in H., Allen E. Am. J. Cancer. 1938, 32. P. 440-446. However, the overall lifespan of tumor-bearing mice under the influence of D2O was shorter than that of the control group. Barbour H., Allen E. Am. J. Cancer. 1938, 32. P. 440-446. The same problem has been recognized in several other studies. Finkel A., Czajke D. The effect of deuterium oxide on ascites tumor growth in mice / / Ed. F. N. Furness, New York: New York Acad. Sci, 1960. P. 755-762. Hughes A. et al., Birch. Bimorph. Acta. 1958, 28. P. 58-61. Katz J. et al. J. Nat. Cancer Inst. 1957, 18. P. 641-659. Of particular note are recent studies showing that the activity of pancreatic carcinogenesis in cultures of AsPC-1 cells, BxPC-3 cells, and PANC-1 cells is significantly reduced by the continuous use of 10-30% D2O and gemcitabine (difluorodeoxycytigine). At the same time, the authors show that the consumption of water containing 10-30% D2O does not significantly affect the mononuclear cell levels in peripheral blood and that the harmful effects of D2O on bone marrow cells are limited. Hartmann J., et al. Anticancer Res., 2005, 25. P. 3407-3411. In contrast, other studies have described the positive (added to traditional treatment modalities) effects of light water (deuterium-depleted water) in the treatment of oncological diseases.

[0170] Therefore, the next stage of this study was the investigation of the antiviral activity of a complex of natural Zn and its Zn-64 isotope dissolved in "light water" (deuterium-depleted water).

[0171] Substance · Composition: 12-1-Zn-64 glutamate, concentration of Zn in deuterium-depleted water: 2.0 mg / ml · Composition: 12-2-Zn-64 glutamate, concentration of Zn in deuterium-depleted water: 1.5 mg / ml · Composition: 12-3 Zn glutamate, concentration of Zn in deuterium-depleted water: 2.0 mg / ml · 4-Distilled deuterium-depleted water ("light water")

[0172] Cytotoxicity and antiviral activity were analyzed according to procedures similar to those above in studies of natural zinc and zinc isotopes in normal distilled water.

[0173] The results are shown in Table 9.

[0174]

Table 9

[0175] It was determined that when the Zn-64 glutamate complex was dissolved in "light water", the isotopic toxicity of the cell culture increased significantly compared to natural zinc.

[0176] Table 9 shows the results of studies on the antiviral activity of the Zn-64 glutamate complex and the Zn glutamate complex dissolved in "light water" against experimental models of influenza, herpes, and surrogate hepatitis C virus. The results show that the zinc preparations in "light water" significantly inhibited the replication of all viruses. However, since the zinc preparations in "light water", especially the light isotopes of zinc, were toxic, the selectivity indices of these preparations were much lower than those of natural zinc.

[0177] Considering the high toxicity of the zinc complex to cell cultures, a study was conducted on the effect of the zinc preparation in "light water" on the mitotic status of cells. The MDBK cell culture was selected for the study. The aforementioned cells were treated with various selected drugs at concentrations non-toxic to the cells. After 24 hours of contact, the cells were fixed and cytological preparations were prepared according to the prior art. The results obtained from this experiment are shown in Table 10.

[0178]

Table 10

[0179] As can be seen from Table 10, the mitotic activities of preparations 12-1 and 12-3 were slightly different from those of the control group. The number of abnormal mitoses within these groups was also slightly different from that of intact cells.

[0180] Antiviral activity of the zinc preparation in vivo

[0181] The anti-herpes activity of the zinc preparation was studied in a herpes virus meningoencephalitis model of BALB / c mice (weighing 18-20 g) using intracerebral administration at a dose of 0.03 ml. In all experimental groups, the drug was administered intraperitoneally at a dose of 0.1 ml. The following zinc preparations were used in the experiment. 1. Zn-64 citrate - 3 mg / ml in 1.4B-deuterium-depleted water 2. Zn citrate - 3 mg / ml in 1.4B-2-deuterium-depleted water 3. Zn-64 glutamate - 3 mg / ml in 1.8B-deuterium-depleted water 4. Zn glutamate - 3 mg / ml in 1.8B-2-deuterium-depleted water 5. Zn-64 glycine-methionine - 1 mg / ml in 1.9B-deuterium-depleted water 6. Zn glycine-methionine - 1 mg / ml in 1.9B-2-deuterium-depleted water

[0182] The drug was injected 24 hours after infection with the herpes virus, and the treatment regimen was observed.

[0183] The drug activity was evaluated by comparing the lethality of the experimental group and the control group. The following factors were considered: - Mortality of animals - Multiplicity of protection (MP) - the multiplicity of the decrease in the number of mouse deaths in the experimental group compared to the control group - The effective index (EI) of the drug was determined using the following formula.

Number

[0184] In the observation of the treatment regimen, the following animal groups were used in the experiment. 1 - Mice injected with: Herpes virus + Drug 4B 2 - Herpes virus + Drug 4B - 2 3 - Herpes virus + Drug 8B 4 - Herpes virus + Drug 8B - 2 5 - Herpes virus + Drug 9B 6 - Herpes virus + Drug 9B - 2 7 - Virolex + Herpes virus 8 - Normal saline + Herpes virus

[0185] The results are shown in Table 11.

[0186]

Table 11

[0187] Based on the data shown in Table 11, it can be concluded that preparations based on light isotopes of zinc and natural zinc 9B, especially 9B - 2, have a significant therapeutic effect. However, with regard to survival time, the complex of natural zinc and glycine methionine dissolved in deuterium-depleted water was more effective as the lifespan here exceeded 30 days.

[0188] In vivo study on the anti-influenza activity of zinc preparations

[0189] The following zinc preparations were used in the experiments: 1. Zn-64 citrate - 3 mg / ml in 4B-deuterium-depleted water 2. Zn citrate - 3 mg / ml in 4B-2-deuterium-depleted water 3. Zn-64 aspartate - 3 mg / ml in 7B-deuterium-depleted water 4. Zn aspartate - 3 mg / ml in 7B-2-deuterium-depleted water 5. Zn-64 glutamate - 3 mg / ml in 8B-deuterium-depleted water 6. Zn glutamate - 3 mg / ml in 8B-2-deuterium-depleted water 7. Zn-64 glycine-methionine - 1 mg / ml in 9B-deuterium-depleted water 8. Zn glycine-methionine - 1 mg / ml in 9B-2-deuterium-depleted water

[0190] To determine the anti-influenza activity of the zinc preparations in vivo, a mouse model of influenza pneumonia was used.

[0191] For this purpose, the BALB / c mouse-adapted A / FM / 1 / 47 (H1N1) strain of influenza virus, which was induced at passage 15 with an infectious titer of 5.0 lg LD 50 was used, and the lethality rate of the mice for 5 days was 100%. In vivo studies on the anti-influenza activity of the drugs were conducted according to the treatment regimen. 24 hours after intranasal infection of the mice with the influenza virus, the mice were intraperitoneally injected with 0.1 ml of the zinc preparation solution. A control of the influenza virus and the reference drug Tamiflu were prepared. The effectiveness of the drugs was determined by the effectiveness index of animal lethality inhibition and the infectious titer of the influenza virus in the lung tissue of the mice. The results of the study are shown in Table 12.

[0192]

Table 12

[0193] Analyzing the data shown in Table 12, it should be noted that according to the results of IE and infectious titer, natural zinc and preparations of light zinc isotopes 4B, 4B-2, and 8B-2 completely protect mice from lethal influenza infection. Preparations 8B and 7B-2 protect mice from lethal influenza infection with effectiveness factors of 80.0 and 60.0. It should also be noted that the survival period of animals infected with influenza virus in the groups of mice treated with 4B, 4B-2, 8B, and 8B-2 was also significantly increased compared to the control.

[0194] Since the antiviral effect of deuterium-depleted water on the herpes simplex virus model was mentioned, the effects of drugs administered at various doses were studied. Therefore, it was administered to animals infected with 50, 100, and 200 μl per mouse.

[0195] Groups of 8 mice each were included in the experiment. Mice were injected intracerebrally at a dose of 10 LD50 (30 μl / mouse). The first drug administration was performed 24 hours later. A total of 4 injections were made every other day after infection. Non-infected mice injected intracerebrally with 30 μl of normal saline and mice infected with HSV served as controls.

[0196]

Table 13

[0197] Therefore, the protective effect of deuterium-depleted water against the herpes simplex virus model is shown, indicating that there is a prospect of investigating its properties as a pharmaceutical ingredient.

[0198] Discussion

[0199] This study is devoted to the effects of compositions of zinc and its isotopes with different amino acids and cell culture media or solutions further dissolved in deuterium-depleted water.

[0200] There is sufficient evidence that zinc is important in the process of virus infection of cells. Nevertheless, the molecular basis of this interaction between virus and cellular zinc remains poorly understood. There are two possible mechanisms for this phenomenon. First, zinc ions are known cofactors in the replication of some viruses and some cellular proteins, and zinc ions can change the activity of various transcriptional cofactors, and thus affect cellular and viral gene expression. The role of zinc as a protein cofactor is very common among viruses. Zinc-binding proteins of RNA viruses and DNA viruses (such as retroviruses, adenoviruses, herpesviruses, polyomaviruses, and papillomaviruses) have been described. Goswami R. et al. J. Virol., 1992, 66, p. 1746-1751. Turk B. et al. J. virol., 1993, 67. p-3671-3673. Erk I. et al. J. Virol., 2003, 77. P3595-3601. Fraefel et al. J. Virol., 1994, 68. p. 31-54-3162. Grossman S., Laimins L. Oncogene, 1989, 4. p. 1089-1093. These viral proteins containing zinc are similar to the zinc fingers of cellular proteins. Zinc fingers are one of the main groups of proteins that bind to DNA. They are transcriptional regulators and contain characteristic domains that contain two cysteine residues and two histidine residues. These amino acids interact with zinc ions, and the polypeptide chain arranged between them forms a finger-shaped loop. Zinc finger C2H2 forms an important family of DNA-binding protein domains that occur in eukaryotic C2H2 transcription factors.

[0201] Both viral and cellular zinc fingers that can be involved in protein - protein and protein - nucleus interactions are highly conserved and extremely important for protein function. Figure 4. The role of zinc fingers in HIV infection and the structural proteins of papillomavirus has been most studied. Mutations in zinc finger proteins, i.e., extraction of zinc from these compounds, disrupt the viral release function from cells, which can serve as one of the approaches in the treatment of viral infections. On the other hand, during viral replication within cells, zinc imbalance is observed within the cells, and exogenous introduction strategies of zinc into cell lines can be used to prevent this imbalance in order to normalize cell homeostasis.

[0202] Basically, the second concept, i.e., the use of complexes of zinc and its isotopes with amino acids and subsequent dissolution in culture medium (RPMI - 1640) or deuterium - depleted water, was used in this study.

[0203] The results obtained from a series of in vitro studies on the effects of complexes and solutions of zinc and its light isotopes with different amino acids on the replication of surrogate models of influenza, herpes, and HCV (BVDV) viruses indicate that solutions of zinc and its light isotopes effectively inhibit the replication of surrogate models of influenza, herpes, and HCV (BVDV) viruses.

[0204] Zn - 64 and Zn solutions in citric acid, Zn - 64 and Zn in glutamic acid, and Zn - 64 and Zn in glycine - methionine solution were the most promising, as confirmed by the following: - Inhibition of the replication of influenza and herpes viruses, Epstein - Barr virus, and surrogate hepatitis C virus (BVDV - bovine viral diarrhea virus); - Having no effect on the mitotic status of cells; - Inhibition of RNA and DNA synthesis by light isotopes of zinc and citrate and glutamate of Zn glycine - methionine

[0205] Zinc glutamate and zinc light isotope glutamate complexes in deuterium-depleted water effectively inhibited the replication of influenza, herpes, and surrogate hepatitis C viruses, but the CC of the light isotope of Zn in deuterium-depleted water 50 was much higher, so the effective coefficient of the light isotope in all virus replication systems was a fraction of that of natural zinc.

[0206] At non-toxic concentrations, neither the natural Zn complex dissolved in deuterium-depleted water nor the complex containing the light isotope of zinc had any effect on the mitotic status of cells.

[0207] The Zn-64 glutamate complex in deuterium-depleted water effectively inhibited RNA and DNA synthesis at concentrations of 50 μg / ml, 40 μg / ml, 10 μg / ml, and 1.1 μg / ml for preparations 12-1 (Zn-64 concentration is 1.5 mg / ml) and 12-2 (Zn-64 concentration is 1.5 mg / ml), respectively.

[0208] From the following examples of experimental models of herpes meningoencephalitis and influenza pneumonia, it was shown that the effectiveness of the preparations depends on the interaction of the solvent in the complexes of natural Zn and its isotopes: - The glycine-methionine complexes of natural Zn and its light isotopes produced a healing effect in the herpes meningoencephalitis model, as demonstrated by the fact that their selectivity index and lifespan of inhibition of infectious titer were more prominent than those of the reference drug Virolex; - The complexes of natural Zn and its light isotopes containing citric acid and glutamic acid had a significant therapeutic effect on the influenza pneumonia model, as indicated by the high effective coefficient, inhibition of infectious titer, and lifespan.

[0209] Shows the antiviral effect of deuterium-depleted water against the herpes simplex virus model.

[0210] Example 2 Zn against Epstein-Barr virus (EBV) 64 Assessment of the antiviral activity of the base preparation

[0211] This study was conducted using Epstein - Barr virus (EBV) - infected lymphoblastoid Raji cells. Raji cells are EBV - transformed human B - lymphocytes that contain 63 copies of the viral genome per cell in their cellular DNA but do not produce any virions. Cell cultures were grown by incubating them at 37 °C and 5% CO₂ in a growth medium consisting of 90% RPMI 1640, 10% fetal bovine serum, and antibiotics in 24 - well suspension culture plates. This cell line is a good model for studying the high antiviral activity of substances against Epstein - Barr virus.

[0212] When studying the in vitro antiviral activity of a novel substance, it is first necessary to determine its cytotoxicity level because drugs that show high toxicity to cell cultures are not desirable for subsequent studies. The value to be determined is defined as the cytotoxic concentration (CC 50 ) of the substance that reduces the viability of the cell population by 50%.

[0213] The cytotoxicity of the research preparation was assayed using the MTT (3 - (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyltetrazolium bromide) assay (one of the commonly used colorimetric assays) (Sigma USA). This assay determines cell viability by measuring the mitochondrial function of cells through the activity of mitochondrial enzymes such as succinate dehydrogenase. In this assay, MTT is reduced to purple formazan by NADH and can be quantified spectrophotometrically. The conversion of MTT to formazan decreases significantly in a dose - dependent manner when cells die under the action of a virus or substance toxic to the cells. The purple color was measured at an excitation wavelength of 540 nm using a Thermo Scientific (USA) reader.

[0214] The following table shows the detection results of the viability of cells treated with different doses of the research preparation.

[0215]

Table 14-1

Table 14-2

[0216] As can be seen from Table 14, the toxicity of glutamate-based preparations and glycine-methionine-based preparations is minimal. The CC 50 values of the test substances are above 470 and 200.

[0217]

Table 15

[0218] Therefore, the analysis of the cytotoxicity and antiviral activity assay results of the research preparations indicates that Zn 64 citrate, Zn 64 EDDA, Zn 64 aspartate, Zn 64 glutamate, and Zn in glycine-methionine 64 are the most promising substances against EBV infection.

[0219] Example 3 Use of KLS-1 in the treatment of COVID-19

[0220] The mechanism of action of KLS-1 ( 64 Zn e aspartate) is based on the prevention of virus entry by the receptor-zinc metalloenzyme ACE2 into new cells and the inhibition of coronavirus replication in already infected cells. The homeostatic repair effect is obtained by the modification of cellular protein production in ribosomes.

[0221] The synthesis of KLS and the Phase I-II study for COVID-19 patients are ready to start. A sufficient amount of KLS-1 can be produced in a short period for the treatment of a very large number of patients.

[0222] KLS-1 represents a novel platform that is crucial for combating not only the Covid-19 coronavirus but also any potential future mutagenic derivatives.

[0223] The results obtained from a series of in vitro studies on the effects of complexes and solutions of zinc and its light isotopes with different amino acids on the reproduction of surrogate models of influenza, herpes, and HCV (BVDV) viruses indicate that solutions of zinc and its light isotopes effectively inhibit the reproduction of surrogate models of influenza, herpes, and HCV (BVDV) viruses.

[0224] Solutions of Zn-64 and natural Zn in citric acid, Zn-64 and Zn in glutamic acid, and Zn-64 and Zn in glycine-methionine solution were the most promising, as confirmed below:

[0225] Inhibition of the reproduction of influenza and herpes viruses, Epstein-Barr virus, and surrogate hepatitis C virus (BVDV - bovine viral diarrhea virus).

[0226] No effect on the mitotic status of cells.

[0227] Inhibition of RNA and DNA synthesis by stable light isotopes of zinc-64 and Zn-64 glycine-methionine citrate and glutamate.

[0228] Zinc glutamate complexes and zinc-64 light isotope glutamate complexes in deuterium-depleted water effectively inhibited the reproduction of influenza, herpes, and surrogate hepatitis C virus, but the CC of the light isotopes of Zn in deuterium-depleted water 50 was much higher, so the effective coefficient of the light isotopes in all virus reproduction systems was a fraction of that of natural zinc.

[0229] The results of the assay of the cytotoxicity and antiviral activity of the research preparations showed that Zn 64 citrate, Zn 64 EDDA, Zn64 Aspartame, Zn 64 Glutamate, and Zn in glycine-methionine 64 are shown to be the most promising substances against EBV infection.

[0230] Anti-inflammatory effect and homeostasis effect

[0231] Another important feature of Zn-64-based KLS-1 is its potent systemic anti-inflammatory effect and homeostasis effect. Data were obtained during preclinical studies of the efficacy of KLS-1 in the treatment of obesity (which is also an important exacerbating factor in Covid-19), type 1 and type 2 diabetes, Parkinson's disease, and Alzheimer's disease.

[0232] Both the anti-inflammatory action and the homeostasis action of KLS-1 are extremely important for the treatment of Covid-19 patients to prevent or reduce the intensity of the cytokine storm and to reduce inflammation in a constitutive manner without sacrificing the efficiency of the immune system.

[0233] Adipose tissue is not only an energy storage site of the body but also an organ that actively participates in the regulation of metabolism via a complex of endocrine, paracrine, and autocrine signals that regulate the responses of numerous tissues and organs (including the hypothalamus, pituitary gland, pancreas, liver, skeletal muscle, kidney, endothelium, immune system, etc.). Thus, adipose tissue secretes more than 50 protein factors, hormones, and growth factors (including cytokines). There are pro-inflammatory cytokines (such as IL-1, IL-6, IL-8, IL-12, TNF-α, IFN-γ, etc.) and anti-inflammatory cytokines (such as IL-4, IL-10, IL-13, TGF, etc.).

[0234] One of the consequences of the overproduction of reactive oxygen species in adipocytes is the inhibition of signaling cascades, which in turn increases the production of pro-inflammatory cytokines by macrophages and the mass of adipose tissue due to macrophage infiltration. The result of such a disorder is the formation of systemic chronic inflammation in the body of patients suffering from obesity. According to a modern concept that is actively being considered, this is a preclinical chronic inflammation of adipose tissue and should be considered one of the important relevant matters in the pathogenesis of obesity and obesity-related diseases. Chronic inflammation of adipose tissue is characterized by cell infiltration, fibrosis, changes in microcirculation, adipokine secretion disorders, and adipose tissue metabolic disorders, as well as an increase in the blood levels of such non-specific inflammatory markers (such as C-reactive protein, fibrinogen, and white blood cells).

[0235] An increase in the levels of pro-inflammatory cytokines not only in adipose tissue but also in serum results in an inflammatory process in adipose tissue.

[0236] Cytokines, which control intercellular and intersystem interactions as endogenous biologically active mediators, affect cell survival by controlling cell growth, differentiation, functional activity, and apoptosis. Cytokines ensure the coordination of the actions of the immune, endocrine, and nervous systems in response to pathological effects under physiological conditions. Cytokines were previously thought to be produced by lymphocytes, monocytes, and tissue macrophages. However, recent research results show that in obesity, as seen in any inflammatory process, infiltration of neutrophils, T lymphocytes, and subsequent resident macrophages into adipose tissue occurs early, determining the initial inflammatory mechanism. Macrophages have been shown to contribute to adipocyte hypertrophy, which is accompanied by an increase in functional activity and cytokine synthesis, further enhancing the inflammatory response. Hypertrophied adipocytes strongly secrete chemokines and their receptors, thereby stimulating the influx of new neutrophils, macrophages, and lymphocytes, and thus contributing to further increases in hypertrophied adipocytes, preservation, and enhancement of the inflammatory response. Adipocytes increase the secretion of cytokines by macrophages, and macrophages act on adipocytes to hypertrophy and activate adipose tissue cells. Hypertrophied adipocytes have been found to produce cytokines, activate complement, and trigger a series of inflammatory processes, like lymphocytes and macrophages. As a result, inflammation becomes fixed and spreads throughout the body. Furthermore, lipid peroxidation products (such as trans-4-oxo-2-nonenal and malonic dialdehyde) are chemoattractants for monocytes and macrophages. Enhanced lipid peroxidation processes in accumulated adipose tissue contribute to the attraction and infiltration of macrophages into adipose tissue in obesity, and thus actively contribute to the initiation of the inflammatory reaction.

[0237] As a result, when adipose tissue mass increases, pro-inflammatory cytokines synthesized by both adipocytes and macrophages incorporated into adipose tissue are constantly supplied, thereby chronicling the inflammatory process and maintaining inflammation in the body. When the intensity of the inflammatory process is low, no direct clinical symptoms occur, but at the same time, this process becomes virtually systemic, meaning that it affects a wide range of organs and tissues, changing their metabolism, impairing their function and immune system response.

[0238] Taking the above into consideration, the next step is to elucidate whether the administration of the Zn-64 stable isotope in aspartate form affects the cytokine profile in obese animals. For this purpose, the concentrations of the main pro-inflammatory cytokines (IL-1, IL-6, IL-12, IFN-γ) and anti-inflammatory cytokines (IL-4, IL-10, TGF) in adipose tissue and serum in experimental animals were determined, whereby the inventors were able to draw conclusions about the intensity of the inflammatory process in adipose tissue and assess whether such an inflammatory process is systemic.

[0239] According to the results obtained, the onset of obesity was accompanied by an increase in the levels of all analyzed pro-inflammatory cytokines in the adipose tissue of animals fed a high-fat diet (Table 16), which indicates the activation of the inflammatory process.

[0240] Furthermore, when the inflammatory process is prolonged, various complications can develop. The intensification of the inflammatory process and the increased accumulation of inflammatory intermediates can lead to tissue damage and organ dysfunction.

[0241]

Table 16

[0242] It has been demonstrated that high levels of pro-inflammatory cytokines (including the above-mentioned cytokines) can cause apoptosis of β cells. High concentrations of IL-12, the expression of which is activated by IFN-γ, infiltrate CD8+ lymphocytes into the pancreas and cause acute pancreatitis. IL-1β activates NF-κB-mediated apoptosis through binding to specific receptors on the surface of these cells, whereby DNA is fragmented and the functional activity of the cells is lost. Furthermore, IL-1β can also be regarded as 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 phosphorylation of serine residues in insulin receptor substrate (IRS)-1. In addition, IL-1β can indirectly increase resistance to insulin action by activating lipogenesis in the liver and contributing to an increase in the levels of triglycerides and free fatty acids in adipocytes.

[0243] IL-6 has been shown to accumulate in direct proportion to the increase in adipose tissue mass in peripheral blood. Adipocytes are the second largest source of IL-6 after the immune system: 35% of circulating IL-6 is synthesized by fat cells. Its blood concentration is directly proportional to the body mass index and increases in obesity. At the same time, the blood levels of IL-6 decrease with weight loss. Excess IL-6 worsens 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 atherosclerotic arteriosclerosis. Furthermore, IL-6 induces an increase in the production of C-reactive protein (CRP), another factor associated with obesity.

[0244] One of the mechanisms for controlling the levels of pro-inflammatory cytokines and, thus, biological effects is carried out by a group of anti-inflammatory cytokines. These cytokines can inhibit the synthesis of pro-inflammatory cytokines by affecting the transcription of specific genes, induce the synthesis of the interleukin receptor antagonist RAIL, enhance the production of soluble receptors, and reduce the density of pro-inflammatory receptors on cells. Therefore, in order to clarify the possible mechanism of the effect of the Zn-64 stable isotope in aspartate form on the profile of pro-inflammatory cytokines, the levels of IL-4, IL-10, and TGF were determined.

[0245] Despite the slight decrease in anti-inflammatory cytokine levels in obese animals, changes in pro-inflammatory cytokine levels were detected. At the same time, in animals treated with the Zn-64 stable isotope in aspartate form, the anti-inflammatory cytokine levels were not only higher in the untreated model of obesity but also higher in animals from the control group.

[0246] It should be emphasized that the lack of change in animals from the control group treated with the test substance suggests that the long-term use of the Zn-64 stable isotope in aspartate form is safe and can show a therapeutic effect only against the onset of pathological conditions.

[0247] As mentioned above, the pathogenesis of obesity is accompanied by a systemic chronic inflammatory process, the intensity of which can be assessed by the serum levels of pro-inflammatory and anti-inflammatory cytokines.

[0248] Analysis of the cytokine profile in the serum of obese animals (Table 17) showed an increase in pro-inflammatory cytokine levels, which was even more significant when compared with data obtained from adipose tissue. The level of the anti-inflammatory cytokine IL-4 did not change statistically significantly. The slight increase in the serum level of IL-10 in obese animals can be regarded as a certain compensatory reaction of the body against metabolic disorders.

[0249] In animals treated with Zn-64-based KLS-1, pro-inflammatory cytokine levels decreased despite increased anti-inflammatory cytokine levels, which were much higher than those in animals from the control group.

Table 17

[0250] One of the basic mechanisms of the effect of zinc on the cytokine profile may be the inhibition of transcription factors sensitive to oxidative stress. Also, zinc-64 may partially block genes encoding pro-inflammatory cytokines (such as IL-6 and IL-8).

[0251] The anti-inflammatory effect of Zn-64 aspartate (KLS-1) is independent of the pathogenesis of inflammation. This is the result of the restoration to healthy homeostasis.

[0252] Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is to be understood as being defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Accordingly, while only certain specific features of the invention have been illustrated and described, those skilled in the art will be able to make many modifications and changes. 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

【Claim 1】 The invention described in the specification.

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