Methods for preventing or treating viral infections

Abstract

A method of treating or preventing a disease or condition caused by or associated (at least in part) with a viral infection in a patient, comprising, in certain embodiments, 64 A method comprising administering to said patient a pharmaceutical composition comprising Zn-enriched zinc at a therapeutically or prophylactically effective dose for treating or preventing a disease or condition caused by a viral infection. In one aspect, the present disclosure provides a method of treating or preventing a disease or condition in a patient caused by or associated (at least in part) with a viral infection, comprising administering to said patient a pharmaceutical composition comprising zinc at a therapeutically or prophylactically effective dose for treating or preventing a disease or condition caused by a viral infection.

Description

[Technical Field]

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

[0002] background Viral infections (such as those caused by coronaviruses) are becoming problematic both in terms of treatment (or lack thereof) and the spread of infection and the severity of the disease caused by infected patients. Summary of the Invention [Means for solving the problem]

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

[0004] In some embodiments, 64 Zn concentrated zinc is 64 Zinc compounds or 64 Zne 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

[0005] The subject / patient can be a human or a non-human mammal (such as a non-human primate or a domestic 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 present invention will become more fully apparent from the following detailed description and the appended claims. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for treating or preventing a disease or condition caused by a viral infection, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition comprising Zn, wherein the composition 64 Zn e or a salt thereof, 64 Zn e The compound or its salt is at least 80% 64 Zn e or wherein the composition comprises a solution containing 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 e However, asparaginate (chemical formula -C) has two aspartic acid molecules. 4 H 5 O 4 N 64 Zn e ), sulfate, and citrate. (Item 11) 10. The method of any of the preceding items, wherein the composition is administered by injection. (Item 12) 11. The method according to any one of items 1 to 10, wherein the composition is administered orally. [Brief explanation of the drawings]

[0007] [Figure 1]1A and 1B show the cytopathic effect of influenza virus on MDCK cells as revealed by cell disruption of the cell monolayer. Magnification: 10x40.

[0008] [Figure 2] Figure 2 shows a culture of VNK cells (symplasts) infected with herpesvirus. Magnification: 10x40.

[0009] [Figure 3] Figures 3A and 3B show the cytopathic effect of BVDV, as evidenced by minor cytopathies in monolayers of MDBK cells. Magnification: 10x40.

[0010] [Figure 4] FIG. 4 shows a computer model of a zinc finger protein. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description As used herein, the word "a" or "plurality" before a noun refers to one or more of that particular noun.

[0012] The terms "for example," "such as," and their grammatical equivalents are understood to be followed by the phrase "without limitation," unless otherwise specified. As used herein, the term "about" is meant to account for variation due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless otherwise specified, regardless of whether this term is expressly used. As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0013] The terms "for example," "such as," and their grammatical equivalents are understood to be followed by the phrase "without limitation," unless otherwise specified. As used herein, the term "about" is meant to account for variation due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless otherwise specified, regardless of whether this term is expressly used. As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0014] All ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1.0 to 10.0" shall be deemed to include any and all subranges beginning 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] Additionally, all ranges disclosed herein are to be construed as including the endpoints of the range unless expressly stated otherwise. For example, a range "between 5 and 10" or "from 5 to 10" or "5-10" is to be construed as including the endpoints 5 and 10.

[0016] It should further be understood that features of one embodiment may be generally applied to other embodiments, even though they are not specifically described or exemplified therein, unless expressly prohibited by the nature of the present disclosure or related embodiments. Similarly, the compositions and methods described herein may include any combination of the features and / or steps described herein that is 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 scope of the present invention.

[0017] An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" refers to the amount of an agent or composition that produces a beneficial effect or favorable outcome in a subject, or that exhibits 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 produces a desired biological, therapeutic, and / or prophylactic result. This result can be reduction, remission, amelioration, relief, 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 can be administered one or more times.

[0018] 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. Using animal models, appropriate concentration ranges and routes of administration can be determined. Such information can then be used to determine appropriate doses and routes of administration for humans. 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 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. An effective dose is generally between 0.01 mg / kg and 2000 mg / kg of active agent, preferably between 0.05 mg / kg and 500 mg / kg of active agent. The exact effective dose will depend on the severity of the disease, the patient's overall health, age, weight, and sex, nutrition, time and frequency of administration, drug combination(s), response 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 their knowledge. Such doses can be determined by routine experimentation and the discretion of the physician. In addition, the effective dose will vary depending on the possibility of co-administration with other treatment 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, and the like.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. Methods and materials used in the present invention are described herein; other suitable methods and materials known in the art can also be used. 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, shall control.

[0021] Viral infection

[0022] Viral infections occur when a pathogenic virus invades an animal's body and infectious virus particles (virions) attach and enter susceptible cells. These infections cause a variety of diseases / conditions. Some infections are highly contagious (such as influenza virus infections). Other infections are highly lethal (such as Ebola virus infections). Novel viral diseases usually occur with some frequency when animal viral pathogens infect humans. Examples include HIV, Ebola virus, etc. Unlike bacterial diseases, treatments for diseases caused by viral infections are not as readily available.

[0023] A viral pandemic ravaged humanity from 2019 to 2021. The disease, known as COVID-19, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019 novel coronavirus (2019-nCoV). The virus is thought to have originated in animals and appears to have first been transmitted to humans in Wuhan, China, in November or December 2019. The primary source of infection soon shifted to person-to-person transmission. It appears to spread between humans primarily via respiratory droplets from coughing and sneezing.

[0024] The lungs are the organ most affected by COVID-19 because the virus gains access to host cells via the enzyme ACE2, which is most abundant in type II pneumocytes in the lungs. 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 physiological counterbalance, homeostatically controlling circulating angiotensin II (Ang II) levels. 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 disease in that tissue. Progression of alveolar disease can lead to respiratory failure and death. ACE2 may also be a pathway for the virus to attack the heart, causing acute cardiac injury. Patients with cardiovascular conditions have a worse prognosis than those without.

[0025] zinc

[0026] Zinc is 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 or a regulator of the activity of enzymes in all the following enzyme classes: 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, proteins, fats, or carbohydrates cannot be metabolized.

[0027] Zinc has also been shown to mediate antioxidant effects. It is an inhibitor of NADPH oxidase, an enzyme complex that catalyzes the production of the highly aggressive superoxide anion radical. Furthermore, zinc can directly influence the oxidation of free radicals at the initiation stage of chain reactions; it is a component of several enzymes of the antioxidant defense system (including Cu / Zn-containing superoxide dismutase). By ligating thiol groups in proteins, zinc protects them 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 inhibits the formation of reactive mixed-valence metal oxides and participates in stabilizing membrane structure.

[0028] The importance of zinc in metabolism and structure is evident from the wide range of its biological activities. Thus, zinc is necessary for the normal functioning 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 prostaglandin conversion reactions. Zinc exhibits significant lipotropic activity and is hepatoprotective. Haase H., Rink L. Zinc Signaling. Zinc in Human Health / / Amsterdam, Netherlands: IOS Press. 2011. 243.

[0029] Furthermore, zinc plays a crucial role in immunological responses, as it is a regulator of phagocyte and lymphocyte activity and influences neutrophil chemotaxis. 5-Nucleotidase, a zinc-containing enzyme, is crucial for the functional status of T and B lymphocytes. Isolated zinc deficiency severely disrupts various parameters of T cell function, including thymic involution, inhibition of cell-mediated cytotoxicity, and a decrease in total lymphocyte count. Zinc is involved in stimulating the metabolism and activity of pituitary hormones, adrenal glands, pancreas, prostate, and testes. Zinc appears to play 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] 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.

[0031] In short, zinc is involved in various vital processes and functions in the human body. Detailed research into some of these functions has yet to be completed, and many of the mechanisms of this trace element are still not fully understood or recognized. However, published experimental and clinical studies indicate that zinc is one of the key elements, and that reduced zinc levels in the body are associated with the onset and emergence of several of the most prevalent and non-prevalent diseases. Because major metabolic processes in the body occur through the active involvement of zinc-containing and zinc-dependent enzymes, zinc deficiency disrupts numerous vital processes.

[0032] Zinc - The use of classical pharmacological forms of zinc salts and their chelates does not always allow for an adequate compensation of zinc deficiency due to the low bioavailability of this element.

[0033] Treatment methods and compositions In one aspect, the present disclosure provides a method of treating or preventing a disease or condition caused by or associated (at least in part) with a viral infection in a patient, comprising administering to said patient a pharmaceutical composition comprising zinc at a therapeutically or prophylactically effective dose for treating or preventing the disease or condition caused by the viral infection. In some embodiments, the composition comprises or is a complex of zinc and / or its isotopes with an amino acid dissolved in either culture medium (e.g., RPMI-1640, etc.) or deuterium-depleted water. In some embodiments, the composition comprises: 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 asparaginate, sulfate, and citrate. 64 Zn e The chemical formula for asparaginate is C4H5O4N 64 Zn e and has two aspartic acid molecules.

[0038] The SARS-CoV-2 virus gains access to host cells via the enzyme ACE2, which contains a heavy isotope of zinc. Therefore, COVID-19 patients should be treated by homeostatically correcting ACE2 activity.

[0039] In some embodiments, the viral infection is infection with SARS-CoV-2 and the patient is a human SARS-CoV-2 infected patient or is at risk for SARS-CoV-2 infection (e.g., has been in an area with high infection rates).

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

[0041] term" 64 Zn e "teeth, 64 Zn is used herein to refer to enriched zinc, i.e., zinc that is present in a higher percentage than the normal percentage of zinc found in nature. 64 As Zn becomes concentrated 64 Zn is concentrated zinc.

[0042] Light isotopes 64 Zn e In certain embodiments, the disclosed compositions contain 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

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

[0044] In some embodiments, 64 Zn e The compound or its salt is present in an amount between 20 and 100% 64 Zn e In 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 mg 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 asparaginate (chemical formula - C4H5O4N 64 Zn e ), sulfate, and citrate.

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

[0046] Formulation and Administration of Compositions

[0047] The compositions used in the disclosed methods 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.

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

[0049] The compositions used in the disclosed methods may be in any suitable form and 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). The oral dosage form may be immediate-release, delayed-release, sustained-release, or enteric-release, and may contain one or more coatings, if 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 may contain 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 composition used in the disclosed method is provided in a glass or plastic bottle, vial, or ampoule, any of which can be suitable for single or multiple use.The bottle, vial, or ampoule containing the disclosed composition can be provided in the form of a kit together with one or more needles of suitable gauge and / or one or more syringes, all of which are preferably sterile.Therefore, in certain embodiments, a kit is provided that includes the above-mentioned 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 Zn dosage is proportional to various reliable daily intake guidance for the corresponding element (e.g., United States Recommended Dietary Allowance (USRDA), Adequate Intake (AI), Recommended Dietary Intake (RDI)).

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

[0053] In some embodiments, the Zn 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 composition used in the disclosed methods for daily administration will be formulated to contain an amount 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 the guidance amount). For individuals with increased sensitivity to a particular element or class of elements (e.g., those with kidney problems), doses at the lower end of these ranges are appropriate. For zinc, guidance daily amounts range from 2 mg for infants to 8-11 mg (depending on gender) for individuals 9 years of age or older. The daily dosages discussed throughout this application may be further divided into sub-doses, which may be administered at appropriate times per day such that the total daily dosage is administered (e.g., 1 / 2 of the daily dose administered twice daily, 1 / 3 of the daily dose administered three times daily, etc.). See Table 1.

[0054] [Table 1]

[0055] Compositions used in the disclosed methods can be produced by methods used according to common practice in the pharmaceutical industry, such as those set forth in Remington: The Science and Practice of Pharmacy (Pharmaceutical Press; 21st revised ed. (2011) (hereinafter "Remington").

[0056] In some embodiments, the composition used in the disclosed method comprises at least one pharmaceutically acceptable vehicle or excipient.These include, for example, diluents, carriers, excipients, fillers, disintegrants, solubilizers, dispersants, preservatives, wetting agents, preservatives, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifiers, and penetration enhancers (DMSO, etc.), as needed.The composition can also comprise suitable auxiliary agents, such as solubilizers, dispersing agents, suspending agents, and emulsifiers.

[0057] In certain embodiments, the composition further comprises suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers, or release aids, 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., NJ 1991) or other standard pharmaceutical science textbooks, such as Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017).

[0059] In some embodiments, 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 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.

[0061] Also, slowly metabolized polymers (proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, etc.) can 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). Furthermore, the compositions may further contain excipients (wetting or emulsifying agents, buffer substances, etc.). Such excipients include, inter alia, diluents and carriers conventionally recognized in the art, and / or substances that promote the penetration of active compounds into cells (e.g., DMSO), as well as preservatives and stabilizers.

[0062] The compositions used in the disclosed methods may be provided in a variety of dosage forms depending on the application; in particular, the compositions may be formulated as injectable solutions.

[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 dosage form, the disclosed composition can be administered by other routes.

[0064] In certain embodiments, compositions used in the disclosed methods that include Zn are administered at a concentration of 2.25 mg / ml or less.

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

[0066] In some embodiments, 64 Zn e In another further embodiment, the concentration level of 2 ml of the composition is about 99% or greater. 64 Zn e contains or consists of zinc asparaginate with two aspartic acid molecules (chemical formula -C4H5O4N 64 Zn eThe dosage of the compositions used in the disclosed methods may vary depending on the subject being treated, the severity of the disease, the condition of the patient, and other factors that one of ordinary skill in the art would consider when determining the dosage and route of administration for a particular patient based on the knowledge of one of ordinary skill in the art.

[0067] Light isotopes may be purchased commercially. Zn-64 oxide of the required enrichment may be purchased, for example, from Oak Ridge National Laboratory, Oak Ridge, Tenn., USA.

[0068] In some embodiments, the chemical formula of zinc asparaginate is —C4H5O4N 64 Zn e and has two aspartic acid molecules. The structure of this zinc asparaginate is: [ka]

[0069] In certain embodiments, the compositions used in the disclosed methods comprise from about 20% to about 100% of the composition. 64 Zn e Includes.

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

[0071] The viral infection can be any viral infection.

[0072] Example [Example]

[0073] In order that this 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 this invention in any way.

[0074] Example 1 Antiviral Treatment Using Zinc-64 Isotope-Based Substances

[0075] Zinc is one of the most important micronutrients, playing a key role in metabolism and is a component of numerous metalloenzymes and transcription factors. (Barbosa, MS, et al. 1989 J. Virol. 63:1404-1407) Zinc is known to be part of 250-300 enzymes, all of which belong to six enzyme classes. (Barthel, A., EA et al. 2007 Arch. Biochem. Biophys. 463:175-182) Ten percent of human proteins contain zinc. (Beerheide, W. et al. 1999 J. Natl. Cancer Inst. 91:1211-1220) Bess, J. Wet al. 1992 J. Virol. 66:840-847) Zinc is essential for the normal functioning of the immune system by increasing the number of thymocytes and peripheral T cells. Boyle, WJet al., Cell 64:573-584.

[0076] Zinc is required for the growth and development of bone tissue. Briggs, MW et al. 2001. Virology 280:169-175. Zinc-containing enzymes involved in the synthesis and / or degradation of carbohydrates, lipids, proteins, and nucleic acids cover 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 a key part of the antioxidant defense system. Culp, J. Set al., 1988. Proc. Natl. Acad. Sci. USA 85:6450-6454; De Oliveira, WR et al., 2003. J. Eur. Acad. Dermatol. Venereol. 17:394-398.

[0077] Zinc finger binding to DNA - Zn in protein structure +2This metal has a special place in cell biology because of the ions involved. Zinc has long been known as a trace element involved in essential processes in cells, including the synthesis and function of many proteins, signal transduction proteins, and transcription factors.

[0078] Uncovering these molecular details of intracellular zinc homeostasis has unexpectedly opened new avenues in virology and shed new light on host-virus interactions. +2 It has long been recognized that zinc is an important cofactor not only for cellular proteins but also for many viral proteins. Recent studies have demonstrated that the cellular environment itself, where the pool of free zinc is extremely small and tightly regulated, may be the limiting factor. Viruses rely on intracellular stores of zinc ions and require cellular Zn for de novo protein synthesis. +2 Thus, cellular systems that control zinc balance may constitute a natural protective barrier that limits zinc access and thereby interferes with viral replication.

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

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

[0081] Materials and Methods

[0082] Substance under consideration Zinc and its light isotopes in various solvents such as citrate (citric acid), sulfate, aspartic and glutamic acid, glycine-methionine, TBPDA (nn-toluenesulfonyl-n-benzoyl-o-phenylenediamine) and EDDA (ethylenediaminedisuccinic acid) were used in this study. 4 Zn-64 in citrate solution (0.9 mg / ml) Natural Zn in 4k Citrate Solution (0.9mg / ml) Zn-64 in 5 EDDA (3 mg / ml) Natural Zn in 5k EDDA (3mg / ml) 6 Zn-64 in sulfate solution (3 mg / ml) Natural Zn in 6k sulfate solution (3 mg / ml) 7. Zn-64 in aspartic acid solution (1.5 mg / ml) Natural Zn in 7k aspartic acid solution (1.5mg / ml) 8. Zn-64 in glutamic acid solution (1.5 mg / ml) Natural Zn in 8k Glutamic Acid Solution (1.5mg / 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 (0.9mg / ml) Zn in 11-TBPDA solution for 14 days 9a Solution of Compound 7 - Aspartic acid - 3.41 mg / ml 10a Solution of Compound 8 - Glutamic Acid - 4 mg / ml 8a Solution of Compound 4 - Citric Acid - 2.3 mg / ml

[0083] Reference drug

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

[0085] cell culture

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

[0087] Cell cultures were grown in growth medium consisting of 90% RPMI 1640 medium (Sigma, USA), 10% fetal bovine serum (Sigma, USA), and the antibiotics penicillin (100 μg / ml), streptomycin (100 μg / ml), and L-glutamine (2 mM). Epithelial cell monolayers were disaggregated using 0.25% Versene solution (Sigma, USA).

[0088] Cells were grown in plastic tissue culture flasks, 24-well plates, and 96-well plates in a thermostat at 37° C. and 5% CO. Cell proliferation activity was checked every 2 days using an inverted optical microscope.

[0089] virus

[0090] Influenza virus: Infectious titer in allantoic culture: 5.0-9.0 lg EID 50 Influenza virus A / FM / 1 / 47 (H1N1) strain with a GAO / 0.2 ml and hemagglutinin titer of 1:512 GAO / 0.2 ml was obtained from the Museum of Viruses of DI 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 DIIvanovsky Institute of Virology (RAMS, Moscow). The virus was maintained by serial passage in BNK cell cultures. The infectious titer for CPE in cell culture was 6.0-9.0 lg TCD. 50 / 0.1ml.

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

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

[0094] Cytotoxic concentration of drug (CC 50 ) determination

[0095] CC of each drug using different cell cultures 50 was determined. At least 10 rows of wells in the cell culture plate were used for each dilution of drug in nutrient medium. The plates containing the cell cultures were incubated at 37°C and 5% CO2 in air for 5 days. The test and control cultures were observed daily to determine the presence or absence of 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 ranging from + to ++++:

[0097] "-" - no cell degeneration at all

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

[0099] "++" - 50% or less of the cell monolayer is affected

[0100] "+++" - 75% or less of the cell monolayer is affected

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

[0102] Drug CC 50 was the highest concentration that did not cause cell pathology.

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

[0104] MTT substrate (Sigma, USA) was dissolved at a concentration of 5 mg / ml in sterile phosphate-buffered saline (pH 7.2) at room temperature. A volume of 20 μl of filtered MTT solution was added to 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°C 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. Cell viability was defined as 50% (CC) compared to the control. 50 ) Inhibitory drug concentrations were calculated using a linear regression program in Microsoft Excel written for a Pentium® Pro.

[0105] Effective concentration (EC 50 ) determination

[0106] EC 50 EC is the minimum drug concentration that inhibits the development of virus-specific CPE by 50%. 50 To determine the TCD, the test virus was injected into the cell culture. 50 The cells were infected with a dose of 0.1 ml / 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 different concentrations of drugs were added to the medium containing the cells (RPMI-1640 + 2% fetal serum). The EC of the drug was determined by the lack of CPE in the experimental group (treated cultures) (which was present in the control group), as well as the reduced infectious titer in the treated cultures compared to the virus control (which was present in the control group) and the difference in infectious titer between the experimental groups. 50 It becomes possible to determine.

[0107] Determination of drug selectivity index (IS)

[0108] The selectivity index (IS) of a drug is calculated by CC 50 EC 50 was determined as a ratio to

[0109] Cytological analysis

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

[0111] The mitotic index was calculated by analyzing 3,000–10,000 observed cells and expressed as ppm (‰) (mitotic divisions per 1,000 cells). The percentage of pathological forms of mitosis was also determined. The classification system for analyzing pathological mitosis developed by V.N.Blyumkin was used.

[0112] Examination of cytological preparations was performed 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 PCR reagent set (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 ranged from 1 to 25 μg / 100 μl of reaction mixture. DNA was amplified in a PCR analysis thermostat ("Terzik", DNA Technology, Moscow). The substances tested were tested at concentrations ranging from 5 to 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 concentrations were 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] result

[0117] Cytotoxic concentration (CC) of the study drug 50 ) determination

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

[0119] Drug CC 50 was the highest concentration that did not cause cell degeneration. The results are shown in Table 2.

[0120] [Table 2]

[0121] In toxicity assays of substances in cultures of Raji lymphoblastoid cells, the least toxic substances were glutamate and glycine-methionine-based drugs, whereas CC50 Values ​​of 470 μg / ml and above 200 μg / ml are shown.

[0122] The analysis of the results of the cytotoxic effects of the test substances shows that the solvents of natural Zn and light Zn isotopes are more toxic than the Zn compounds studied. Therefore, the CC of citric acid 50 is 1 / 160 or 0.014 mg / ml, and the CC of the compound in citric acid solution 50 Zn 64 The concentration of glutamic acid in the CC solution is 0.45mg / ml, while that of natural Zn is 0.18mg / ml, meaning that its toxicity is 1 / 3 to 1 / 10. 50 is 1:160 or 0.025 mg / ml, while Zn 64 The compound had a 1 / 5-0.4 mg / ml concentration, while the natural Zn had a 0.0046 mg / ml concentration. This means that the light Zn-64 isotope reduces the toxicity of the solvent, while the natural Zn increases it. 50 is 1 / 80 or 0.042 mg / ml, and light Zn 64 The CC of the glycine and methionine solutions was 1 / 10 or 0.75 mg / ml for the complex and 1 / 320-0.0046 mg / ml for the native Zn. 50 is 1 / 160 or 0.018 mg / ml, and light Zn 64 The isotope and natural zinc complexes are 1 / 10 or 0.2 and 0.15 mg / ml, respectively, i.e., both the light Zn-64 isotope and natural zinc reduce the toxicity of the solvent by 10 times.

[0123] A different pattern was observed for the TBPDA solvent: for MDCK cells, the light isotope increased the toxicity of the solvent by 30-fold, whereas natural Zn had no effect on its toxicity.

[0124] In cultures of BNK and MDCK cells, a pattern almost identical to that of MDCK cells was observed.

[0125] Antiviral activity of natural zinc and zinc isotopes

[0126] Assessment of anti-influenza activity

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

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

[0129] Cultures were incubated in a CO2 incubator for 3 days with daily monitoring using a microscope.

[0130] After 48 to 72 hours of incubation, the culture medium was harvested, and the infectious titer of influenza virus was determined by cell culture titration.

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

[0132] The inhibitory effect of the substances was evaluated by the reduction in the viral infectivity titer under the action of the test substance compared to the control. 50A decrease in β indicates significant antiviral activity of the studied compound, especially when the chemotherapeutic index is 8 or greater.

[0133] Natural Zn and its Zn in MDCK cell cultures 64 High influenza activity (EC 50 ) assessment results are shown in Table 3. See also Figure 1A and Figure 1B.

[0134] [Table 3]

[0135] Zn-64 preparations in citrate solution and natural Zn, Zn-64 in glutamic acid solution and glycine-methionine solution were determined to exhibit anti-influenza activity.

[0136] Assessment of anti-herpes activity

[0137] To study the antiherpetic activity of zinc solutions, their isotopes, and solvents, transplantable VNK cell cultures were used. Cells were grown in plates containing RPMI-1640 medium supplemented with 10% fetal serum (Nunclon, Surface, Denmark) at 37°C in a thermostat containing CO2.

[0138] The herpes simplex virus type 2 BH strain was used. The infectivity titer was 6.0 to 0.9 lg ID 50 is.

[0139] One-day cultures of VNK cells were selected to study the antiviral activity of drugs. The cell growth medium was removed, and the test preparations were added to the cell monolayer at different concentrations. After 1 hour of contact, the cells were infected with 100 TCD herpesvirus. 50Cultures were incubated for 5 days in a CO2 incubator with daily monitoring using a microscope to document virus recovery by the cytopathic effect of HSV on VNK cells compared to control cultures in which the cell monolayer was not exposed to any virus.

[0140] The cytopathic effect of HSV on cells is morphologically manifested by the formation of symplasts or rounded cells, combined with the proliferation and appearance of multinucleated giant cells (Fig. 2).

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

[0142] Antiherpetic activity (EC) of solutions of natural Zn and its Zn64 isotope in cultures of VNK cells 50 The assessment results are shown in Table 4.

[0143] [Table 4]

[0144] Neutral Zn in EDDA and Zn-64 in glutamic acid and glycine-methionine solutions showed antiherpes activity.

[0145] Assessment of anti-HCV activity

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

[0147] The antiviral activity of the drugs was evaluated by treating with different dilutions of the drugs and then infecting BVDV at 100 TCD 50 The virus was studied in cultures of MDBK cells infected with a dose of 1000 μg / ml ...

[0148] Antiviral activity (EC) of solutions of natural Zn and its Zn64 isotope in MDBK cell culture against the bovine viral diarrhea virus (BVDV) model as a surrogate for HCV. 50 ) assessment results are shown in Table 5.

[0149] [Table 5]

[0150] Antiviral activity against a surrogate hepatitis C virus (BVDV) was demonstrated by native Zn in citrate, EDDA, ​​and sulfate solutions, and Zn-64 in glutamic acid and glycine-methionine solutions.

[0151] In analyzing the antiviral activity of solutions of natural zinc and light zinc isotopes in different solvents, the light zinc isotopes in glutamic acid and glycine-methionine solutions were found to be the most active, demonstrating high selectivity indices for three viral infections. In citrate solutions, their antiviral activity was most pronounced against influenza virus and a surrogate hepatitis C virus (BVDV), with the activity of both natural zinc and its light isotopes being equivalent.

[0152] Light zinc isotope and natural zinc salts of EDDA, ​​sulfate, and aspartic acid had antiviral activity against influenza virus and BVDV. 64 The isotope did not show any antiviral activity, and natural Zn was only active against influenza infection.

[0153] Assessment of anti-EBV activity

[0154] Natural Zn and its Zn in Raji cell cultures in response to Epstein-Barr virus infection model 64 The results of the assessment of the antiviral activity of the isotope solutions are shown in Table 6.

[0155] [Table 6]

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

[0157] Thus, in an antiviral screening of natural zinc and its light isotopes in various solvents, zinc compounds in glutamic acid and glycine-methionine, as well as in citrate, showed the most activity against BVDV.

[0158] Study of the effectiveness of the investigated compounds on the mitotic status of cells

[0159] Given the high toxicity of the solvents used in cell cultures, the effects of zinc-based drugs in different solutions on the mitotic status of the cells were investigated. Experiments were performed on cultures of MDBK cells treated with various selective drugs. After 24 hours of exposure, 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 - No. 5 (Zn64 in EDDA solution), No. 6 (Zn64 in sulfate solution), and No. 7 (Zn64 in aspartate solution) - significantly inhibited the mitotic activity of the cells and increased the number of mitotic abnormalities.

[0162] [Table 8]

[0163] Table 8 shows that three drugs—number 9-3, a glycine-methionine solution, and number 10-2, a natural Zn in TBFDA solution—inhibited the mitotic activity of cells, resulting in an increased number of mitotic abnormalities.

[0164] Further analysis of the results of this study on the mitotic status of MDBK cells under the influence of drugs based on natural Zn and its light isotopes at non-toxic concentrations showed that Zn in EDDA solution 64 , Zn in sulfate solution 64 , and Zn in aspartic acid solution 64 It should be noted that natural zinc in TBFDA solution and lysine-methionine solvent significantly altered the mitotic status of cells (i.e., the number of mitotic abnormalities increased, while the mitotic activity of cells significantly decreased). Therefore, light isotopes of zinc and natural zinc in glutamate, glycine-methionine, and citrate can be considered promising for further research.

[0165] Therefore, these studies on the cytotoxic and antiviral activity of the drug Zn 64 Citrate, Zn in EDDA 64 , Zn 64 Aspartate, Zn 64 Glutamate, Zn 64 Glycine-methionine is shown to be the most promising antiviral agent against EBV. This selection is based on the fact that the solvent has no antiviral activity, that the solvent does not affect the mitotic status of cells as part of the zinc complex, and that the Zn in glutamic acid and glycine-methionine is not a factor. 64 This is explained by the fact that the compound effectively inhibits the reproduction of influenza and BVDV viruses.

[0166] Assessment of the antiviral activity of zinc glutamate and its isotopic conjugates in "light water"

[0167] It is known that hydrogen isotopes enter the body primarily through drinking water and food. Once inside the body, water 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 isotopic composition of water is reflected in the isotopic 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 demonstrate a direct correlation between the isotopic composition (H, O) of human hair (mainly composed of α-keratin protein) and drinking water.

[0168] In cells, water is structured in a special state intermediate between liquid water and ice: a layer of oriented water molecules surrounds all hydrophilic macromolecules in the cytoplasm, including proteins and nucleic acid molecules.

[0169] The strong antimitotic effect of DO (heavy water) was first detected in experiments. Thus, in 1938, H. Barbour and E. Allen (Barbour H., Allen E., Am. J. Cancer. 1938, 32, pp. 440-446) described the retardation of growth and reversal of development of transplanted lymphosarcoma and breast cancer in mice receiving 40% DO in their drinking water. However, the overall lifespan of tumor-bearing mice exposed to DO was shorter than that of the control group. (Barbour H., Allen E., Am. J. Cancer. 1938, 32, pp. 440-446) Several other studies have noted the same problem. (Finkel A., Czajke D., The effect of deuterium oxide on ascites tumor growth in mice / / Ed. FN Furness, New York: New York Acad. Sci., 1960, pp. 755-762) Hughes A. et al., Birch. Bimorph. Acta. 1958, 28, pp. 58-61; Katz J. et al., J. Nat. Cancer Inst. 1957, 18, pp. 641-659. A recent study was particularly noteworthy, showing that the activity of pancreatic carcinogenesis in cultures of AsPC-1, BxPC-3, and PANC-1 cells was significantly reduced by the continuous use of 10-30% D2O and gemcitabine (difluorodeoxycytidine). At the same time, the authors demonstrated that the consumption of water containing 10-30% D2O did not significantly affect mononuclear cell levels in peripheral blood, suggesting that the adverse effects of D2O on bone marrow cells were limited. Hartmann J. et al., Anticancer Res. 2005, 25, pp. 3407-3411. Other studies, in contrast, have described the positive (additional to traditional treatment forms) effects of light water (deuterium-depleted water) in the treatment of oncological diseases.

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

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

[0172] Cytotoxicity and antiviral activity were analyzed following procedures similar to those described above in the study of natural zinc and zinc isotopes in ordinary distilled water.

[0173] The results are shown in Table 9.

[0174] [Table 9]

[0175] They determined that dissolving Zn-64 glutamate complexes in "light water" significantly increased isotope toxicity in cell cultures compared to natural zinc.

[0176] Table 9 shows the results of a study of the antiviral activity of Zn-64 glutamate and Zn glutamate complexes dissolved in "light water" against experimental models of influenza, herpes, and surrogate hepatitis C viruses. The results show that the zinc preparations in "light water" significantly inhibited the reproduction of all viruses. However, because the zinc preparations in "light water" are toxic, especially the light isotope of zinc, the selectivity index of these preparations was much lower than that of natural zinc.

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

[0178] [Table 10]

[0179] As can be seen from Table 10, the mitotic activity of preparations 12-1 and 12-3 was slightly different from the control group. The number of aberrant mitoses in these groups was also slightly different from intact cells.

[0180] Antiviral activity of zinc preparations in vivo.

[0181] The antiherpes activity of zinc preparations was studied in a herpesvirus meningoencephalitis model in BALB / c mice (18-20 g body weight) using intracerebral administration at a volume of 0.03 ml. In all experimental groups, the drug was administered intraperitoneally at a volume of 0.1 ml. The following zinc preparations were used in the experiments: 1.4B-Zn-64 citrate in deuterium-depleted water - 3 mg / ml 2.4B-2-Zn citrate in deuterium-depleted water - 3 mg / ml 3.8B-Zn-64 glutamate in deuterium-depleted water - 3 mg / ml 4.8B-2-Zn glutamate in deuterium-depleted water - 3 mg / ml 5.9B-Zn-64 Glycine-Methionine in Deuterium-Depleted Water - 1 mg / ml 6.9B-2-Zn Glycine-Methionine in Deuterium-Depleted Water - 1 mg / ml

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

[0183] Drug activity was assessed by comparing the lethality of experimental and control groups. The following factors were taken into consideration: - Animal mortality - Multiplicity of Protection (MP) - the multiplicity of reduction in the number of mouse deaths in the experimental group compared to the control group -The effectiveness index (EI) of the drug was determined using the following formula:

number

[0184] In observing the treatment regimen, the following animal groups were used in the experiment: 1- Mice injected with: herpesvirus + 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 solution + herpes virus

[0185] The results are shown in Table 11.

[0186] [Table 11]

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

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

[0189] The following zinc preparations were used in the experiments: 1.4B-Zn-64 citrate in deuterium-depleted water - 3 mg / ml 2.4B-2-Zn citrate in deuterium-depleted water - 3 mg / ml 3.7B-Zn-64 asparaginate in deuterium-depleted water - 3mg / ml 4.7B-2-Zn Asparaginate in Deuterium-Depleted Water - 3mg / ml 5.8B-Zn-64 glutamate in deuterium-depleted water - 3 mg / ml 6.8B-2-Zn glutamate in deuterium-depleted water - 3 mg / ml 7.9B-Zn-64 glycine-methionine in deuterium-depleted water - 1 mg / ml 8.9B-2-Zn glycine-methionine in deuterium-depleted water - 1 mg / ml

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

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

[0192] [Table 12]

[0193] Analyzing the data presented in Table 12, it should be noted that, according to the IE and infectivity results, preparations of natural zinc and 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 efficacy factors of 80.0 and 60.0. It should also be noted that the survival time of animals infected with influenza virus in the groups of mice treated with 4B, 4B-2, 8B, and 8B-2 was significantly increased compared to the control.

[0194] Having mentioned the antiviral effect of deuterium-depleted water on the herpes simplex virus model, we investigated the effect of the drug administered at various doses: 50, 100, and 200 μl per mouse were administered to infected animals.

[0195] Groups of eight mice each were included in the experiment. Mice were injected intracerebrally with a dose of 10LD50 (30 μl / mouse). The first drug administration was administered 24 hours later. After infection, injections were administered every other day for a total of four times. Non-infected mice and HSV-infected mice were injected intracerebrally with 30 μl of normal saline and served as controls.

[0196] [Table 13]

[0197] Thus, the protective effect of deuterium-depleted water against a herpes simplex virus model is demonstrated, indicating promise for exploring its properties as a pharmaceutical ingredient.

[0198] Consideration

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

[0200] There is ample evidence that zinc is important for the viral infection process. Nevertheless, the molecular basis of this interaction between virus and cellular zinc remains largely unknown. Two possible mechanisms for this phenomenon exist. First, zinc ions are known cofactors in the viral regeneration of some viral and cellular proteins. Zinc ions can alter the activity of various transcription cofactors, thus affecting cellular and viral gene expression. The role of zinc as a protein cofactor is highly common among viruses. Zinc-binding proteins have been described in RNA and DNA viruses (e.g., retroviruses, adenoviruses, herpesviruses, polyomaviruses, and papillomaviruses). Goswami R. et al., J. Virol., 1992, 66, pp. 1746-1751; Turk B. et al., J. Virol., 1993, 67, pp. 3671-3673; Erk I. et al., J. Virol., 2003, 77, pp. 3595-3601. Fraefel et al. J. Virol., 1994, 68, pp. 31-54-3162. Grossman S., Laimins L. Oncogene, 1989, 4, pp. 1089-1093. These zinc-containing viral proteins resemble zinc finger proteins of cellular proteins. Zinc fingers are one of the major groups of proteins that bind to DNA. They are transcriptional regulators and contain a characteristic domain containing two cysteine ​​and two histidine residues. These amino acids interact with the zinc ion, and the polypeptide chain located 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, which may be involved in protein-protein and protein-nuclear interactions, are highly conserved and crucial for protein function. (Figure 4) The role of zinc fingers in HIV infection and papillomavirus structural proteins has been most studied. Mutation of zinc finger proteins, i.e., extraction of zinc from these compounds, disrupts the virus's release function from cells, which may serve as one approach for treating viral infections. On the other hand, during intracellular viral replication, an imbalance of zinc is observed within the cell, and exogenous introduction of zinc into cell systems can be used to prevent this imbalance and normalize cellular homeostasis.

[0202] Essentially, the second concept, namely 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 in a series of in vitro studies on the effect of complexes and solutions of zinc and its light isotopes with different amino acids on the regeneration of surrogate models of influenza, herpes, and HCV (BVDV) viruses show that solutions of zinc and its light isotopes effectively inhibit the regeneration of surrogate models of influenza, herpes, and HCV (BVDV) viruses.

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

[0205] Zinc glutamate and light zinc isotope glutamate conjugates in deuterium-depleted water effectively inhibited the reproduction of influenza, herpes, and surrogate hepatitis C viruses, whereas CC of light Zn isotopes in deuterium-depleted water did not. 50 is much higher, the effectiveness factors of the light isotopes in all virus reproduction systems were several times lower than that of natural zinc.

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

[0207] Zn-64 glutamate conjugate 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] The following examples of experimental models of herpes meningoencephalitis and influenza pneumonia show that the effectiveness of the preparation depends on the interaction of the solvent in the complex with natural Zn and its isotopes: - Natural Zn and its light isotope glycine-methionine complex produced a curative effect in a herpes meningoencephalitis model, as evidenced by its selectivity index of inhibition of infectious titers, longevity being more pronounced than the reference drug Virolex; -Natural Zn and its light isotope conjugates with citrate and glutamate were significantly therapeutically effective against influenza pneumonia models, as indicated by high efficacy coefficients, inhibition of infectivity titers, and life span.

[0209] 1 shows the antiviral effect of deuterium-depleted water on a herpes simplex virus model.

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

[0211] This study was performed using lymphoblastoid Raji cells infected with EBV. 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 in 24-well suspension culture plates in a growth medium consisting of 90% RPMI 1640, 10% fetal bovine serum, and antibiotics, incubated at 37°C and 5% CO2. This cell line is a good model for studying the high viroactivity of substances against Epstein-Barr virus.

[0212] When studying the in vitro antiviral activity of a new substance, it is first necessary to determine its cytotoxicity level, since drugs that are highly toxic to cell cultures are undesirable for further studies. The value to be determined is the cytotoxic concentration (CC) of a substance that reduces the viability of a cell population by 50%. 50 ) is defined as

[0213] The cytotoxicity of the study preparations was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay (Sigma, USA), a commonly used colorimetric assay. This assay determines cell viability by determining mitochondrial function of cells through the measurement of the activity of mitochondrial enzymes such as succinate dehydrogenase. In this assay, MTT is reduced by NADH to purple formazan, which can be quantified spectrophotometrically. The conversion of MTT to formazan significantly decreases in a dose-dependent manner when cells are killed 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 table below shows the results of the detection of the viability of cells treated with different doses of the preparation studied.

[0215] [Table 14-1] [Table 14-2]

[0216] As can be seen from Table 14, the glutamate-based and glycine-methionine-based formulations are least toxic. 50 The values ​​are 470 and over 200.

[0217] [Table 15]

[0218] Therefore, analysis of the results of the assessment of the cytotoxic and antiviral activity of the studied preparations revealed that Zn 64 Citrate, Zn 64 EDDA, ​​Zn 64 Aspartate, Zn 64 Zn in glutamate and glycine-methionine 64 are the most promising substances for anti-EBV infection.

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

[0220] KLS-1( 64 Zn e The mechanism of action of ACE2 (aspartate) is based on preventing viral entry into new cells via the receptor-zinc metalloenzyme ACE2 and inhibiting coronavirus reproduction in already infected cells. The homeostatic restoration effect is achieved by correcting cellular protein production in ribosomes.

[0221] The synthesis of KLS and Phase I-II studies in Covid-19 patients are ready to begin. Sufficient amounts of KLS-1 can be produced in a short period of time to treat a very large number of patients.

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

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

[0225] Inhibition of 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 conjugates and zinc-64 light isotope glutamate conjugates in deuterium-depleted water effectively inhibited the reproduction of influenza, herpes, and surrogate hepatitis C viruses, whereas CC of light isotopes of Zn in deuterium-depleted water did not. 50 is much higher, the effectiveness factors of the light isotopes in all virus reproduction systems were several times lower than that of natural zinc.

[0229] The results of the assessment of the cytotoxic and antiviral activity of the study preparations showed that Zn 64 Citrate, Zn 64 EDDA, ​​Zn64 Aspartate, Zn 64 Zn in glutamate and glycine-methionine 64 are the most promising substances for anti-EBV infection.

[0230] Anti-inflammatory and homeostatic effects

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

[0232] Both the anti-inflammatory and homeostatic effects of KLS-1 are crucial for the treatment of Covid-19 patients to prevent or reduce the intensity of the cytokine storm and to reduce inflammation in a homeostatic manner without compromising the efficiency of the immune system.

[0233] 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 coordinate responses of numerous tissues and organs (including the hypothalamus, pituitary gland, pancreas, liver, skeletal muscle, kidneys, endothelium, and immune system). Accordingly, adipose tissue secretes over 50 protein factors, hormones, and growth factors (including cytokines). There are pro-inflammatory cytokines (e.g., IL-1, IL-6, IL-8, IL-12, TNF-α, IFN-γ) and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13, TGF-β).

[0234] One consequence of excessive production of reactive oxygen species in adipocytes is the inhibition of signaling cascades, which increases the production of pro-inflammatory cytokines by macrophages and their infiltration into adipose tissue, resulting in an increase in its mass. The consequence of such disturbances is the formation of systemic chronic inflammation in obese patients. According to current and actively discussed concepts, this is subclinical chronic inflammation of adipose tissue and should be considered one of the key correlates of the pathogenesis of obesity and obesity-related diseases. Chronic inflammation of adipose tissue is characterized by cellular infiltration, fibrosis, altered microcirculation, impaired adipokines secretion, and impaired adipose tissue metabolism, as well as increased blood levels of nonspecific inflammatory markers (e.g., C-reactive protein, fibrinogen, and leukocytes).

[0235] Increased levels of pro-inflammatory cytokines in serum as well as in adipose tissue result in inflammatory processes in adipose tissue.

[0236] Cytokines, endogenous biologically active mediators that regulate cell-cell and intersystem interactions, affect cell survival by regulating cell growth, differentiation, functional activity, and apoptosis. Cytokines ensure the coordination of immune, endocrine, and nervous system functions under physiological conditions and in response to pathological conditions. Cytokines were previously thought to be produced by lymphocytes, monocytes, and tissue macrophages. However, recent findings indicate that in obesity, as in any inflammatory process, the infiltration of neutrophils, T lymphocytes, and subsequently resident macrophages into adipose tissue occurs early and determines the initial inflammatory mechanism. 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, thereby stimulating the influx of new neutrophils, macrophages, and lymphocytes, thereby contributing to the further expansion of hypertrophic adipocytes and the preservation and enhancement of the inflammatory response. Adipocytes increase cytokine secretion by macrophages, which then act on adipocytes to hypertrophy and activate adipose tissue cells. Enlarged adipocytes, like lymphocytes and macrophages, produce cytokines, activate complement, and trigger a series of inflammatory processes. As a result, inflammation becomes fixed and spreads throughout the body. Furthermore, lipid peroxidation products (e.g., trans-4-hydroxy-2-nonenal and malonic dialdehyde) are chemoattractants for monocytes and macrophages. Enhanced lipid peroxidation in accumulated adipose tissue contributes to the attraction and infiltration of macrophages into adipose tissue in obesity, thus actively contributing to the initiation of inflammatory responses.

[0237] As a result, increased adipose tissue mass provides a constant supply of pro-inflammatory cytokines synthesized by both adipocytes and macrophages incorporated into the adipose tissue, thereby chronicating the inflammatory process and maintaining inflammation in the body. At low levels of intensity, the inflammatory process does not produce direct clinical symptoms, but at the same time, the process is systemic in nature, meaning that it affects a wide range of organs and tissues, altering their metabolism and impairing their function and immune system response.

[0238] Considering the above, the next step is to determine whether administration of Zn-64 stable isotope in aspartate form affects the cytokine profile in obese animals. To this end, 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 of experimental animals are determined, which allows us to draw conclusions about the intensity of the inflammatory process in adipose tissue and to assess whether such an inflammatory process is systemic.

[0239] The results obtained show that the development of obesity is 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), indicating an activation of the inflammatory process.

[0240] Furthermore, as the inflammatory process prolongs, various complications can develop: Intensification of the inflammatory process and increased accumulation of inflammatory intermediates can result in tissue damage and organ dysfunction.

[0241] [Table 16] * - difference is significant relative to the control group of animals; # - difference is significant relative to the obese animal model group NOTE: C - control; C + control administered with Zn-64 stable isotope in the form of zinc-aspartate; DIO - diet-induced obesity; DIO + diet-induced obesity administered with Zn-64 stable isotope in the form of zinc-64-aspartate.

[0242] It has been shown that high levels of pro-inflammatory cytokines (including those listed above) can induce β-cell apoptosis. High concentrations of IL-12 (whose expression is activated by IFN-γ) induce CD8+ lymphocyte infiltration into the pancreas, leading to acute pancreatitis. IL-1β activates NF-κB-mediated apoptosis through binding to specific receptors on the surface of these cells, resulting in DNA fragmentation and loss of functional activity. 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. IL-1β can also activate lipogenesis in the liver, contributing to increased levels of triglycerides and free fatty acids in adipocytes, thereby indirectly increasing resistance to insulin action.

[0243] IL-6 has been shown to accumulate in peripheral blood in direct proportion 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 adipose cells. Its blood concentration is directly proportional to body mass index and increases with obesity. Concurrently, blood levels of IL-6 decrease with weight loss. 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 hepatic steatosis and systemic atherosclerosis. Furthermore, IL-6 induces increased production of C-reactive protein (CRP), another factor associated with obesity.

[0244] One mechanism for controlling proinflammatory cytokine levels, and therefore their biological effects, is through the anti-inflammatory cytokines. These cytokines inhibit the synthesis of proinflammatory 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 proinflammatory receptors on cells. Therefore, to clarify the possible mechanism of the effect of Zn-64 stable isotope aspartate on the proinflammatory cytokine profile, we measured the levels of IL-4, IL-10, and TGF-β.

[0245] Despite only a 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 Zn-64 stable isotope aspartate, anti-inflammatory cytokine levels were elevated not only in untreated models of obesity but also in animals from the control group.

[0246] It should be emphasized that the absence of changes in animals from the control group treated with the test substance suggests that the long-term use of Zn-64 stable isotope in the aspartate form is safe and may only show therapeutic effects against the development 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 serum levels of pro- and anti-inflammatory cytokines.

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

[0249] In animals treated with Zn-64-based KLS-1, pro-inflammatory cytokine levels were reduced, despite the fact that anti-inflammatory cytokine levels were increased and much higher than in animals from the control group. [Table 17] * - difference is significant relative to the control group of animals; # - difference is significant relative to the obese animal model group NOTE: C - control; C + control administered with Zn-64 stable isotope in the form of zinc-aspartate; DIO - diet-induced obesity; DIO + diet-induced obesity administered with Zn-64 stable isotope in the form of zinc-aspartate.

[0250] One of the mechanisms underlying zinc's effect on cytokine profiles may be the inhibition of transcription factors sensitive to oxidative stress, and 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 and is a result of the restoration of healthy homeostasis.

[0252] While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of 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. 1. A composition comprising Zn for treating or preventing a disease or condition caused by a viral infection, the composition comprising: ６４ Zn-enriched compounds or salts thereof, wherein at least 80% of the total Zn content in said composition is ６４ Zn, and the viral infection is caused by influenza virus, herpes simplex virus, hepatitis C virus, or Epstein-Barr virus; wherein, when the viral infection is caused by influenza virus, the composition is a 64 Zn citrate solution, a 64 Zn glutamic acid solution, or a 64 Zn glycine-methionine solution comprising a 64 Zn enriched compound or a salt thereof; wherein, when the viral infection is caused by herpes simplex virus, hepatitis C virus, or Epstein-Barr virus, the composition is a 64 Zn glutamate solution or a 64 Zn glycine-methionine solution comprising a 64 Zn enriched compound or a salt thereof.

2. The aforementioned ６４ 10. The composition of claim 1, wherein the Zn-enriched compound or salt thereof is dissolved in culture medium or deuterium-depleted water.

3. The composition of any one of claims 1 to 2, further comprising a diluent or excipient.

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

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

6. The composition according to any one of claims 1 to 5, characterized in that the composition is administered by injection.

7. The composition according to any one of claims 1 to 5, characterized in that the composition is administered orally.

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