Methods for treating Alzheimer's disease

Abstract

at a therapeutically or prophylactically effective dose to treat or prevent a disease, 64 Zn e 10. A method for treating or preventing Alzheimer's disease, comprising administering a pharmaceutical composition comprising:

Description

[Technical Field]

[0001] The present disclosure relates to the treatment of Alzheimer's disease. [Background technology]

[0002] Alzheimer's disease ("AD") is the most common form of dementia, affecting over 37 million people worldwide (Mount C, Downton C., Nature Medicine 2006;12(7):780-784; Wimo A et al., Alzheimer's and Dementia 2010;6(2):98-103). In modern society, AD is of great medical and societal concern, given the increasing incidence of AD and the increasing social and economic demands caused by the prospect of an aging population.

[0003] AD is characterized by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles in affected brains, which cause neuronal loss in the neocortex, hippocampus, and basal forebrain, leading to progressive cognitive and behavioral decline (Watt NT et al., Int J Alzheimers Dis. 2010;2011:971021. Published 2010 Dec 20. doi:10.4061 / 2011 / 971021).

[0004] There is no cure for AD. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method of treating or preventing AD in a patient, comprising administering to the patient a pharmaceutical composition comprising zinc at a therapeutically or prophylactically effective dose to treat or prevent AD. In some embodiments, the composition comprises: 64 Zn-enriched zinc (the term 64 Zn e " is used herein to mean 64 (Used to refer to Zn-enriched zinc).

[0006] In some embodiments, 64 Zn-enriched zinc is 64 Zn e Compound 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 Zinc, e.g., 80% 64 Zn e , 85% 64 Zn e , 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e or 99.9% 64 Zn e It contains zinc.

[0007] In accordance with these and other aspects of the present invention, many other aspects are provided. Other features and aspects of the present invention will become more fully apparent from the following detailed description and the appended claims. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram of the experimental design for testing the therapeutic effects of 64Zn-asp on behavioral function in an experimental model of Alzheimer's disease.

[0009] [Figure 2] Figure 2 shows the Barnes maze.

[0010] [Figure 3]Figure 3 shows the effect of 64Zn-asp on body weight in a rat model of Aβ1-40-induced Alzheimer's disease. Data are presented as a percentage of the animal's final body weight (on the day of necropsy) relative to the animal's body weight before Alzheimer's disease mimicry, taken as 100%.

[0011] [Figure 4] Figures 4A and 4B show the effects of 64Zn-asp on feeding (Figure 4B) and drinking (Figure 4A) behavior in a rat model of Aβ1-40-induced Alzheimer's disease. Animals were housed in individual cages, and the amount of food and water consumed by each rat was measured daily, starting on day 18 (8 days after surgery) and continuing until the end of the experiment (day 37). Data were first averaged to one rat per day within groups, and then averaged to one rat per day across the entire observation period.

[0012] [Figure 5] Figures 5A-5D show immunohistochemical characterization of tyrosine hydroxylase (TH) activity in hippocampal neurons from untreated rats (Figure 5A), sham-operated rats (placebo) (Figure 5B), rat models of Aβ1-40-induced Alzheimer's disease injected with HO (Figure 5C), and rat models of Aβ1-40-induced Alzheimer's disease treated with 64Zn-asp (Figure 5D). TH-positive staining (dark). Oc. 40, ob. 10.

[0013] [Figure 6] Figure 6A (pre-surgery) and Figure 6B (post-surgery) are graphs showing the time required for spatial learning in the Barnes maze in a rat model of Aβ1-40-induced Alzheimer's disease. Data are expressed as the mean of four trials, 15 min / rat / day, and the mean / day within each group. M±SD

[0014] [Figure 7]Figure 7A (pre-surgery) and Figure 7B (post-surgery) show short-term and long-term spatial memory efficiency (time taken to find the entrance to the "escape box") and cognitive flexibility (time spent near the entrance to the "escape box") 24 hours (day 5 after 4 days of training) and 5 days (day 9 after 4 days of training) after training in a rat model of Aβ1-40-induced Alzheimer's disease. M±SD

[0015] [Figure 8] Figure 8 shows the therapeutic effect of 64Zn-asp on Aβ levels in the hippocampus of a rat model of AD induced by injection of Aβ1-40 (n=5 in all groups). A is the relative number of phagocytic cells; B is the phagocytic activity. *p≦0.05 vs. untreated animals

[0016] [Figure 9] Figure 9 shows the therapeutic effect of 64Zn-asp on tau protein levels in the hippocampus of a rat model of AD induced by Aβ1-40 injection (n=5 for all groups). A is the relative number of phagocytic cells; B is the phagocytic activity. *p≦0.05 vs. untreated animals

[0017] [Figure 10] Figures 10A and 10B show the therapeutic effect of 64Zn-asp on microglial phagocytosis in a rat model of AD induced by Aβ1-40 injection (n=5 for all groups). Figure 10A shows the relative number of phagocytic cells; Figure 10B shows phagocytic activity. *p≦0.05 vs. untreated animals

[0018] [Figure 11] Figure 11 shows the therapeutic effect of 64Zn-asp on oxidative metabolism in microglia in a rat model of AD induced by injection of Aβ1-40 (n=5 in all groups). *p≦0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0019] [Figure 12]Figures 12A and 12B show CD86 expression in microglia in a 64Zn-asp-treated rat model of Alzheimer's disease induced by Aβ1-40 injection (n=5 for all groups). Figure 12A shows the number of expressing cells in the analyzed population; Figure 12B shows the expression level. *p≦0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0020] [Figure 13] Figures 13A and 13B show CD206 expression in microglia in a 64Zn-asp-treated rat model of Alzheimer's disease induced by Aβ1-40 injection (n=5 for all groups). Figure 13A shows the number of expressing cells in the analyzed population; Figure 13B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0021] [Figure 14] Figure 14 shows the effect of 64Zn-asp on the level of circulating leukocytes in a rat model of Aβ1-40-induced AD. M±SD. Note: *p<0.05 vs. untreated animals, #p<0.05 vs. untreated animal model of AD.

[0022] [Figure 15] Figure 15A (number of phagocytic cells, %) and Figure 15B (phagocytic index, GMean) show the effect of 64Zn-asp on the phagocytic activity of circulating polymorphonuclear granulocytes in a rat model of Aβ1-40-induced AD. M±SD. Note: *p<0.05 vs. untreated animals. #p<0.05 vs. untreated animal model of AD.

[0023] [Figure 16] Figure 16A (number of phagocytic cells, %) and Figure 16B (phagocytic index, GMean) show the effect of 64Zn-asp on the phagocytic activity of circulating monocytes in a rat model of Aβ1-40-induced AD. M±SD. Note: *p<0.05 vs. untreated animals, #p<0.05 vs. untreated animal model of AD.

[0024] [Figure 17] Figures 17A and 17B show the effect of 64Zn-asp on oxidative metabolism in circulating granulocytes (Figure 17A) and monocytes (Figure 17B) in a rat model of Aβ1-40-induced AD. Note: *p<0.05 vs. untreated animals; #<0.05 vs. untreated AD animal model.

[0025] [Figure 18] Figures 18A and 18B show CD86 expression in circulating phagocyte populations in a 64Zn-asp-treated rat model of Alzheimer's disease induced by Aβ1-40 injection (n=5 for all groups). Figure 18A shows the number of expressing cells in the analyzed population; Figure 18B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0026] [Figure 19] Figures 19A and 19B show CD206 expression in circulating phagocyte populations in a 64Zn-asp-treated rat model of Alzheimer's disease induced by Aβ1-40 injection (n=5 for all groups). Figure 19A shows the number of expressing cells in the analyzed population; Figure 19B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0027] [Figure 20] Figure 20 shows the effect of 64Zn-asp on body weight in a rat model of Aβ25-35-induced Alzheimer's disease. Data are presented as a percentage of the animal's final body weight (on the day of necropsy), with the body weight of the animal before Alzheimer's disease mimicry set at 100%.

[0028] [Figure 21]Figures 21A, 21B, 21C, and 21D show immunohistochemical characterization of tyrosine hydroxylase activity in hippocampal neurons from untreated rats (Figure 21A), sham-operated rats (placebo) (Figure 21B), rat models of Aβ25-35-induced Alzheimer's disease injected with HO (Figure 21C), and rat models of Aβ25-35-induced Alzheimer's disease treated with 64Zn-asp (Figure 21D). TH-positive staining (dark). Oc. 40, ob. 10.

[0029] [Figure 22] Figure 22A (pre-surgery) and Figure 22B (post-surgery) are graphs showing the time required for spatial learning in the Barnes maze in rat models of Aβ25-35-induced Alzheimer's disease. Data are expressed as the mean of four trials, 15 min / rat / day, and the mean / day within each group. M±SD.

[0030] [Figure 23] Figures 23A and 23B show the therapeutic effect of 64Zn-asp on Aβ (A) and tau protein (B) levels in the hippocampus of a rat model of AD induced by injection of Aβ25-35 (n=5 for all groups). Figure 23A shows the relative number of phagocytic cells; Figure 23B shows the phagocytic activity. *p<0.05 vs. untreated animals

[0031] [Figure 24] Figures 24A and 24B show the therapeutic effect of 64Zn-asp on microglial phagocytosis in a rat model of AD induced by injection of Aβ25-35 (n=5 in all groups). Figure 24A shows the relative number of phagocytic cells; Figure 24B shows the phagocytic activity. *p<0.05 vs. untreated animals; #p≦0.05 vs. control rat model of AD.

[0032] [Figure 25]Figure 25 shows the therapeutic effect of 64Zn-asp on oxidative metabolism in microglia in a rat model of AD induced by injection of Aβ25-35 (n=5 in all groups). *p≦0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0033] [Figure 26] Figures 26A and 26B show CD86 expression in microglia in a 64Zn-asp-treated rat model of Alzheimer's disease induced by injection of Aβ25-35 (n=5 for all groups). Figure 26A shows the number of expressing cells in the analyzed population; Figure 26B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0034] [Figure 27] Figures 27A and 27B show CD206 expression in microglia in a 64Zn-asp-treated rat model of Alzheimer's disease induced by injection of Aβ25-35 (n=5 for all groups). Figure 27A shows the number of expressing cells in the analyzed population; Figure 27B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0035] [Figure 28] Figure 28 shows the effect of 64Zn-asp on the level of circulating leukocytes in a rat model of Aβ25-35-induced AD. M±SD. Note: *p<0.05 vs. untreated animals, #p<0.05 vs. untreated animal model of AD.

[0036] [Figure 29] Figures 29A and 29B show the therapeutic effect of 64Zn-asp on the phagocytic activity of circulating granulocytes in a rat model of Aβ25-35-induced AD (n=5 in all groups). Figure 29A shows the number of phagocytic cells; Figure 29B shows the phagocytic activity. Note: *p<0.05 vs. untreated animals, #p≦0.05 vs. control animal model of AD.

[0037] [Figure 30]Figures 30A and 30B show the therapeutic effect of 64Zn-asp on the phagocytic activity of circulating monocytes in a rat model of Aβ25-35-induced AD (n=5 in all groups). Figure 30A shows the relative number of phagocytic cells; Figure 30B shows the phagocytic activity. Note: *p<0.05 vs. untreated animals, #p≦0.05 vs. control animal model of AD.

[0038] [Figure 31] Figures 31A and 31B show the effect of 64Zn-asp on oxidative metabolism in circulating granulocytes (Figure 31A) and monocytes (Figure 31B) in a rat model of Aβ25-35-induced AD. Note: *p<0.05 vs. untreated animals; #<0.05 vs. untreated AD animal model.

[0039] [Figure 32] Figures 32A and 32B show CD86 expression in circulating phagocyte populations in a 64Zn-asp-treated rat model of Alzheimer's disease induced by injection of Aβ25-35 (n=5 for all groups). Figure 32A shows the number of expressing cells in the analyzed population; Figure 32B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model.

[0040] [Figure 33] Figures 33A and 33B show CD206 expression in circulating phagocyte populations in a 64Zn-asp-treated rat model of Alzheimer's disease induced by injection of Aβ25-35 (n=5 for all groups). Figure 33A shows the number of expressing cells in the analyzed population; Figure 33B shows the expression level. *p<0.05 vs. untreated animals; #≦0.05 vs. control AD ​​animal model. DETAILED DESCRIPTION OF THE INVENTION

[0041] As used herein, the word "a" or "plurality" before a noun refers to one or more of that particular noun.

[0042] The terms "for example" and "such as," and their grammatical equivalents, are understood to be followed by the phrase "and without limitation," unless clearly stated otherwise. As used herein, the term "about" is meant to account for variations due to experimental error. All measurements given herein are understood to be modified by the term "about," whether or not the term is expressly used, unless clearly stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0043] An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" refers to the amount of a drug or composition that provides a beneficial effect or favorable outcome in a subject, or the amount of a drug or composition that exhibits a desired in vivo or in vitro activity. An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" refers to the amount of a drug or composition that provides a desired biological, therapeutic, and / or preventative result. That result can be a reduction, amelioration, mitigation, reduction, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease, disorder, or condition in a patient / subject, or any other desired alteration of a biological system. An effective amount can be administered in one or more administrations.

[0044] An "effective amount," "prophylactically effective amount," or "therapeutically effective amount" can be initially estimated according to cell culture assays or using animal models, typically mice, rats, guinea pigs, rabbits, dogs, or pigs. Animal models can be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine appropriate doses 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" (US 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 will recognize additional guidelines that can also be used to determine human therapeutic dosages based on non-human data. Effective doses are generally between 0.01 mg / kg and 2000 mg / kg of active substance, preferably between 0.05 mg / kg and 500 mg / kg of active substance. The exact effective amount depends on the severity of disease, patient's general health condition, age, weight and sex, nutritional status, administration time and frequency, drug combination, response sensitivity, tolerance / response to administration and other factors that those skilled in the art will consider when determining the dosage and administration route for specific patient based on their knowledge in the art.This dosage can be determined by the judgment of a doctor through routine experimentation.Effective dosage also varies depending on the possibility of combination with other treatment procedures, such as the use of other drugs.

[0045] As used herein, "patient" and "subject" are interchangeable terms and may refer to human patients / subjects, dogs, cats, non-human primates, and the like.

[0046] All ranges disclosed herein should be understood to encompass all subranges subsumed therein. For example, a specified range of "1.0 to 10.0" should be deemed to include all subranges beginning with a minimum value greater than or equal to 1.0 and ending with a maximum value less than or equal to 10.0, such as 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

[0047] All ranges disclosed herein are also deemed to include the endpoints of the range unless expressly stated otherwise. For example, the ranges "between 5 and 10," "from 5 to 10," or "5-10" should be deemed to include the endpoints 5 and 10.

[0048] Furthermore, unless expressly prohibited by the nature of this disclosure or related embodiments, it should be understood that one or more features of one embodiment may generally be applied to other embodiments even if not specifically described or exemplified in other 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. Many modifications and / or adaptations of the compositions and methods described herein will be readily apparent to those skilled in the art without departing from the present subject matter.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein in their entirety by reference. In case of conflict, the present specification, including definitions, will control.

[0050] Alzheimer's Disease ("AD")

[0051] Amyloid or "senile" plaques are a major factor in the pathophysiology of AD. They are composed primarily of Aβ peptides derived from proteolytic processing of the amyloid precursor protein (APP). APP is a glycosylated transmembrane protein with a large N-terminal extracellular domain, a single hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain.

[0052] APP can be processed via one of two pathways: an amyloidogenic pathway leading to the production of Aβ, and a nonamyloidogenic pathway (Chow VW et al., Neuromolecular Medicine 2010;12(1):1-12). The primary APP processing pathway in the healthy brain is the nonamyloidogenic pathway, in which APP is cleaved by α-secretase within the Aβ region to form the secreted APPα (sAPPα) fragment and an 83-amino acid membrane-bound C-terminal fragment (C83). α-Secretase activity has been attributed to the disintegrin and metalloprotease (ADAM) family of zinc metalloproteases due to their long zinc-binding consensus sequence (Bode W, et al., Adv Exp Med Biol 1996;389:1-11. doi:10.1007 / 978-1-4613-0335-0_1). Subsequently, C83 is cleaved by the γ-secretase complex to form the APP intracellular domain (AICD) and p3. In the amyloidogenic pathway, APP is sequentially cleaved by aspartyl proteases to form the secreted APPβ (sAPPβ) fragment and a 99-amino acid membrane-bound C-terminal fragment (C99). The C99 fragment is then further processed by the γ-secretase complex to form AICD and Aβ peptides, primarily 40 and 42 amino acids in length. These aggregation-prone Aβ peptides are deposited in the brain and, over time, form oligomeric and fibrillar structures that cause AD (Zhang YW et al., Mol Brain 2011;4:3. Published 2011 Jan 7. doi:10.1186 / 1756-6606-4-3).

[0053] In healthy brains, the relatively small amounts of Aβ constitutively produced are mitigated by Aβ-degrading enzymes, many of which have been identified as candidates, most of which are zinc metalloproteases (Bateman RJ et al., Nature Medicine 2006;12(7):856-861).

[0054] Zinc is the most abundant trace metal in the brain and has multiple functions in Alzheimer's disease (AD). Zinc is important in the enzymatic nonamyloidogenic processing of amyloid precursor protein (APP) and the enzymatic degradation of amyloid beta (Aβ) peptides. Zinc binds to Aβ and promotes its aggregation into neurotoxic species; disruption of zinc homeostasis in the brain leads to synaptic and memory deficits. The specific binding site for zinc is located in a cysteine-rich region (within the extracellular domain) on the extracellular domain of APP (Bush AI et al., The Journal of Biological Chemistry. 1993;268(22):16109-16112; Bush AI, et al., The Journal of Biological Chemistry 1994;269(43):26618-26621). Clinical observations have shown that serum zinc levels in AD patients are significantly lower than those in healthy controls.

[0055] High free copper has shown promising evidence supporting its association with the pathogenesis of AD. Free copper generates reactive oxygen species, which activate neuroinflammation and neurodegeneration. Neuroinflammation plays an important role in the pathophysiology of AD and other neurodegenerative diseases in the synucleinopathy and tauopathy groups (Zhang F, Jiang L. Neuropsychiatr Dis Treat 2015;11:243-256). Zinc therapy is considered a promising approach for the treatment of free copper toxicity. Zinc induces the production of intestinal metallothionein and increases the excretion of free copper via the feces (Avan A, Hoogenraad TU. Journal of Alzheimer's Disease 46 (2015) 89-92 DOI 10.3233 / JAD-150186). Zinc may play a role in maintaining the adhesive properties of APP during cell-cell and cell-matrix interactions (Multhaup G et al., FEBS Letters 1994;355(2):151-154; Multhaup G, et al., Biochemistry 1998;37(20):7224-7230).

[0056] Methods and Compositions

[0057] In one aspect, the present disclosure provides a method of treating or delaying the onset (i.e., preventing) AD in a patient in need thereof, comprising administering to the patient a pharmaceutical composition comprising zinc in a therapeutically or prophylactically effective dose to treat or prevent AD. In some embodiments, the composition comprises: 64 Zn-enriched zinc (the term 64 Zn e " is used herein to mean 64 (Used to refer to Zn-enriched zinc).

[0058] In some embodiments, the solution is a natural 64 Zn e In some embodiments, 64 Zn eThe salt is an enoate or an aspartate. 64 Zn e The salt has two molecules of aspartic acid 64 Zn e It is an aspartate salt.

[0059] In some embodiments, 64 Zn-enriched zinc is 64 Zn e Compound 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 Zinc, e.g., 80% 64 Zn e , 85% 64 Zn e , 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e or 99.9% 64 Zn e It contains zinc.

[0060] In some embodiments, 64 Zn e is an aspartate (chemical formula C4H5O4N 64 Zn e ), sulfate, and citrate. 64 Zn e has two aspartic acid molecules 64 Zn e Aspartate (chemical formula C4H5O4N 64 Zn e ) form.

[0061] term" 64 Zn e " is used herein to mean 64 Used to refer to Zn-enriched zinc. 64 As Zn is enriched above its normal proportion in natural zinc, 64 Zn is enriched zinc.

[0062] Light isotopes 64 Zn e This form of zinc is much better absorbed by the body than naturally occurring zinc. 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 Zinc, e.g., 80% 64 Zn e , 85% 64 Zn e , 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e or 99.9% 64 Zn e It contains zinc.

[0063] In some embodiments, the composition contains between 0.05 mg and 110 mg of 64 Zn e In some embodiments, the composition comprises 1 to 10 mg of 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 It is an aqueous solution in which the concentration is 0.1 mg / ml to 10 mg / ml.

[0064] In some embodiments, the therapeutic dose for a human subject is 0.2 to 0.8 mg of Zn-64 per kg of body weight of the human subject.

[0065] In some embodiments, the composition or solution is administered by injection, hi other embodiments, the composition or solution is administered orally.

[0066] Formulation and Administration Compositions

[0067] The disclosed compositions can be administered to a subject in need thereof in any suitable mode of administration, at any suitable frequency, and in any suitable effective dose.

[0068] In some embodiments, the total amount of zinc administered is the same as the United States' recommended daily allowance or intake of zinc. In some embodiments, the total amount of zinc administered is 1 / 2, 2 times, 3 times, 5 times, or 10 times the United States' recommended daily allowance or intake of zinc. In some embodiments, the total amount of Zn is 1 / 2 to 10 times the United States' recommended daily allowance or intake of zinc. The composition for use in the disclosed methods can contain a predetermined daily amount administered once a day, or a portion thereof administered at a corresponding number of times per day. The composition for use in the disclosed methods can also contain an amount of Zn administered every two days, every three days, once a week, or any other suitable frequency.

[0069] The composition for use in the disclosed method can be in any suitable form and can be formulated for any suitable delivery means.In some embodiments, the composition for use in the disclosed method is 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 can provide immediate release, delayed release, sustained release, or enteric release, and can include one or more coatings, if appropriate.In some embodiments, the disclosed composition is provided in a form suitable for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or any other injection route.In some embodiments, the injectable composition is provided in a sterile and / or non-pyrogenic form, and can include preservatives and / or other suitable additives, such as sucrose, dibasic sodium phosphate heptahydrate, or other suitable buffers, pH adjusters, such as hydrochloric acid or sodium hydroxide, and polysorbate 80, or other suitable surfactants.

[0070] When provided in solution form, in some embodiments, the composition for use in the disclosed method is provided in glass or plastic bottle, vial or ampoule, any of which can be suitable for single or multiple use.The bottle, vial or ampoule that contains the disclosed composition can be provided in the form of a kit together with one or more needles and / or one or more syringes of suitable gauge, and all of them are preferably sterilized.Therefore, in certain embodiments, a kit is provided that includes the above-mentioned liquid solution, which is packaged in suitable glass or plastic bottle, vial or ampoule, and can further comprise one or more needles and / or one or more syringes.Kit can further comprise instructions for use.

[0071] In certain embodiments, the zinc dosage is proportional to various authoritative daily intake guidelines for the corresponding element (e.g., Recommended Dietary Allowance (USRDA), Adequate Intake (AI), Recommended Dietary Intake (RDI)).

[0072] In some embodiments, the Zn dosage is about 1 / 2 to about 20 times the recommended amount, more preferably about 1 to about 10 times the recommended amount, and even more preferably about 1 to about 3 times the recommended amount. Thus, in certain embodiments, a single dose of a composition for use in the disclosed methods for daily administration is formulated to contain an amount within these ranges, for example, about 1 / 2, about 1, about 3, about 5, about 10, and about 20 times the recommended amount. These amounts are generally for oral ingestion or topical application. In some embodiments, intravenous dosages are lower, such as about 1 / 10 to about 1 / 2 the recommended amount. Doses at the lower end of these ranges are appropriate for individuals with increased sensitivity to certain elements or types of elements (e.g., individuals with kidney problems). For zinc, the recommended daily amount ranges from 2 mg for infants to 8 to 11 mg (depending on gender) for individuals 9 years of age or older. The daily doses discussed throughout this application may be subdivided into fractional doses, with the fractional doses being administered an appropriate number of times per day to provide the total daily dose (e.g., 1 / 2 of the daily dose administered twice a day, 1 / 3 of the daily dose administered three times a day, etc.).

[0073] Compositions for use in the disclosed methods may be manufactured by methods adopted in accordance with 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").

[0074] In some embodiments, the composition for use in the disclosed method comprises at least one pharmaceutically acceptable vehicle or additive.These may include, for example, diluents, carriers, additives, excipients, disintegrants, solubilizers, dispersants, preservatives, wetting agents, preservatives, stabilizers, buffers (e.g., phosphates, citrates, acetates, tartrates), suspending agents, emulsifiers, penetration enhancers (e.g., DMSO).The composition may also include suitable auxiliary substances, such as solubilizers, dispersing agents, suspending agents and emulsifiers.

[0075] In certain embodiments, the composition may further comprise suitable diluents, glidants, lubricants, acidulants, stabilizers, excipients, binders, plasticizers or release aids, and other pharmaceutically acceptable additives.

[0076] A comprehensive 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 texts, such as Handbook of Pharmaceutical Excipients (Shesky et al. eds., 8th ed. 2017).

[0077] In some embodiments, compositions for use in the disclosed methods can be administered intragastrically, orally, intravenously, intraperitoneally, or intramuscularly, although other routes of administration are also possible.

[0078] Water can be used as carrier and diluent in the composition.In addition to or instead of water, other pharmaceutically acceptable solvents and diluents can also be used.In certain embodiments, deuterium-depleted water is used as diluent.

[0079] Large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers, can also be used as carrier compounds for the composition. Pharmaceutically acceptable carriers in therapeutic compositions can further contain liquids such as water, saline, glycerol, or ethanol. In addition, the composition can further contain additives such as wetting or emulsifying agents, buffer substances, etc. Such additives include, among others, conventional diluents and carriers in the art, and / or substances that promote the penetration of active compounds into cells, such as DMSO, as well as preservatives and stabilizers.

[0080] Compositions for use in the disclosed methods may be provided in a variety of dosage forms depending on the purpose of application; in particular, they may be formulated as solutions for injection.

[0081] The compositions for use in the disclosed methods can be administered systemically. Suitable administration routes include, for example, oral administration, or parenteral administration such as intravenous, intraperitoneal, or intragastric administration, as well as administration via drinking water. However, depending on the dosage form, the disclosed compositions can be administered via other routes.

[0082] In certain embodiments, a composition comprising Zn for use in the disclosed methods is administered intragastrically at a concentration of 2.25 mg / ml.

[0083] In some embodiments, the composition for use in the disclosed methods is about 2 ml.

[0084] In some embodiments, 64 Zn e In another further embodiment, the enrichment level of 2 ml of the composition is about 99% or greater. 64 Zn e is a zinc aspartate (chemical formula C4H5O4N 64 Zn e Doses of compositions for use 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 would be considered by a skilled artisan in determining the amount and route of administration for a particular patient based on knowledge in the art.

[0085] Light isotopes can be purchased commercially. Zn-64 oxide with the required enrichment can be purchased, for example, from Oak Ridge National Laboratory (Oak Ridge, Tenn., USA).

[0086] In some embodiments, zinc aspartate has the chemical formula C4H5O4N 64 Zn e In some embodiments, the structure of zinc aspartate is as follows: [ka]

[0087] In certain embodiments, the compositions for use in the disclosed methods comprise from about 20% to about 100% of the composition. 64 Zn e Includes.

[0088] The disclosed compositions can be administered in conjunction with other appropriate drugs or treatments.

[0089] As shown in the following examples, cognitive symptoms in a rat model of AD 64 Zn-aspartic acid ( 64 To test Zn-asp, two models induced by injection of a mixture of different β-amyloid peptides (1-40 and 25-35) were used. 64 Zn-asp is referred to as the "test substance" in the examples.

[0090] Animal models of AD are fundamental to the drug development process and must reproduce the phenotype with high certainty and be as relevant to the disease as possible. Over the past 20 years, transgenic AD models have made significant contributions to our understanding of the molecular mechanisms involved in disease onset and progression. However, extensive literature data indicates that genetic models of AD cannot reproduce the complete clinical picture of the disease, and that a rat model in which the AD phenotype is induced by injection of amyloid-β peptide provides a more reproducible outcome (Lecanu L, Papadopoulos V. Alzheimers Res Ther. 2013;5(3):17. doi:10.1186 / alzrt171). In these models, the AD phenotype is most commonly induced by intracerebroventricular administration of a solution containing the human 42-residue amyloid peptide (Aβ1-42) (Mudo G et al., J Neuroinflammation 2019;16(1):44. doi:10.1186 / s12974-019-1417-4). Aβ is a soluble amyloid peptide, which is thought to be the core of all amyloid plaque formation due to its excellent aggregation properties.1-42 was selected.

[0091] A newer AD model is based on the infusion of the β-amyloid peptide 25-35. Among the Aβ fragments studied to date, the Aβ(25-35) peptide is the shortest Aβ fragment formed in vivo as a result of the action of brain proteases (Kubo, T. et al., J. Neurosci. Res. 2002; 70, 474-483). This peptide lacks the metal-binding site but exhibits significant levels of molecular aggregation and retains the toxicity of the full-size peptide. Consistent with this finding, it has been proposed that the Aβ(25-35) peptide represents the biologically active region of Aβ.

[0092] Although Aβ deposition in the central nervous system is a hallmark of AD and may be the cause of neurodegeneration, several reports suggest that some non-aggregated amyloid molecules and their peptide fragments can penetrate neuronal membranes and directly alter membrane activity (Pike, CJ et al., J. Neurochem. 1995; 64, 253-265; Dahlgren, KN et al., J. Biol. Chem. 2002; 277, 32046-32053). Recent studies have shown that in the early stages of AD, non-aggregated forms of Aβ fragments, i.e., mono- / oligomeric Aβ(25-35) forms, are also able to penetrate cell membranes and trigger intracellular toxic mechanisms (Clementi ME et al., FEBS Lett. 2005;579(13):2913-2918 doi:10.1016 / j.febslet.2005.04.041).

[0093] Furthermore, the lag period between β-amyloid (Aβ) deposition and neurodegeneration in Alzheimer's disease (AD) suggests that age-dependent factors are involved in the pathogenesis. Racemization of Ser and Asp in Aβ is a typical age-dependent modification in AD. Recently, Ser 26 Racemized Aβ1-40 ([D-Ser 26]Aβ1-40) is soluble and non-toxic to neurons, but is cleaved by brain proteases to produce toxic fragments ([D-Ser 26 ]Aβ25-35 / 40). 26 Immunohistochemical analysis using Aβ25-35 / 40-specific antibodies revealed that [D-Ser 26 ]Aβ25-35 / 40 antigen was present in the brains of rats with aging, but not in those of age-matched controls. These results suggest that soluble [D-Ser] is likely produced with aging. 26 ]Aβ1-40 is released from plaques and proteolytically converted into toxic [D-Ser 26 ] supports the hypothesis that Aβ25-35 / 40 damages hippocampal CA1 neurons by enhancing excitotoxicity in AD (Kubo T. et al., J Neurosci Res. 2002;70(3):474-483 doi:10.1002 / jnr.10391).

[0094] Furthermore, studies have shown that a significant number of proteins and peptides can assemble into amyloid structures under experimental conditions. Although these polypeptides show no conformational or structural homology, their amyloid fibrils clearly share a common structural feature: a central beta-fold, indicating that amyloid formation is a common property of polypeptide backbones. Such a process can proceed in vivo if the cellular machinery is unable to remove protein aggregates.

[0095] Another common feature of amyloid aggregates is that they arise by a nucleation-dependent mechanism, and that the initial oligomeric and prefibrillar structures of various proteins are cytotoxic.

[0096] Mature fibrils are considered inert substances and can cause physical damage to organs and tissues (Moulias R et al., Ann Med Interne (Paris) 2002;153:441-445; Bucciantini M et al., Nature 2002;416: 507-511).

[0097] Recently, autoimmune components have been reported in the pathophysiology of dementia, including AD. Clinical trials have convincingly demonstrated that autoantibodies against various molecules are associated with the onset and progression of AD. Thus, antibodies against Aβ and tau proteins, multiple transmitter and receptor molecules (e.g., glutamate, dopamine), glial markers such as GFAP, lipids (ceramide, oxidized low-density lipoprotein), vascular markers such as RAGE (receptor for advanced glycation end products, a receptor involved in the pathogenesis of nearly all neurodegenerative diseases), cellular enzymes such as aldolase, and many other autoantigens have been found in the serum of AD patients. (Wu J, Li L. J Biomed Res. 2016;30(5):361-372. doi:10.7555 / JBR.30.20150131; MacLean M. et al., Neurochem Int. 2019;126:154-164. doi:10.1016 / j.neuint.2019.03.012). Although the role of these autoantibodies in AD remains unclear, their association with disease onset and progression convincingly demonstrates the primary role of the immune system in its pathophysiology. According to published clinical observations, Aβ25-35 is one of the autoantigens for which high titer antibodies are found in the serum of 90% of AD patients (Gruden MA et al., Dement Geriatr Cogn Disord. 2004;18(2):165-171 doi:10.1159 / 000079197). Therefore, it was reasonable to use an AD model induced by injection of Aβ25-35 here. [Example]

[0098] Example

[0099] In order that the present invention may be better understood, the following examples are set forth, which are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

[0100] Example 1 In a rat model of AD 64 Therapeutic effect of Zn-asp

[0101] (material and method)

[0102] experimental animals

[0103] Male Wistar rats (300-500 g) were used. Animals were maintained under standard conditions in the animal room of the ESC "Institute of Biology and Medicine" of Taras Shevchenko National University of Kyiv. Food and water were available ad libitum.

[0104] Modeling Alzheimer's disease in rats

[0105] Alzheimer's disease was induced in aged male rats (14 months old) by intrahippocampal injection of aggregated human amyloid beta (Aβ1-40) peptide (Cayman Chemical Company) and human amyloid beta (Aβ25-35) peptide (Tocris Bioscience, Brostol). Aβ1-40 and Aβ25-35 were dissolved in double-distilled water to a concentration of 15 μmol / L and incubated at 37°C for 24 hours for aggregation. Aβ aggregates were disrupted by ultrasound and sterilized immediately before injection.

[0106] Rats were anesthetized with a mixture of ketamine (75 mg / kg, diluted with sterile water for injection, Sigma, USA) and 2% xylazine (100 μl / rat, Alfasan International BV, Netherlands) administered intraperitoneally in a total volume of 1 ml. The rats were placed in a modified stereotaxic apparatus (SEZH-4) for rats. Then, a scalp incision was made 2 mm distal and 2 mm lateral to the intersection of the sagittal suture and bregma (zero point), with a depth of 3.5 mm, and a burr hole was created in the hippocampus with an injection needle. Dissolved Aβ1-40 or Aβ25-35 was taken into a homemade microinjector and its tip was dropped into the burr hole.

[0107] A volume of 10 μl of the suspension per animal was infused at a rate of 0.5 μl / min (every 15 seconds) for 5 minutes. After Aβ administration, the tip of the microinjector was left in the brain tissue for 4 minutes. The microinjector was then removed, and the soft tissue of the animal's scalp was sutured. Control animals received 10 μl of sterile deuterated water as a placebo instead of Aβ1-40 or Aβ25-35 (sham-operated animals).

[0108] Experimental design

[0109] The animals were divided into six groups:

[0110] I (n=7) - naive animals maintained under standard housing conditions and not subjected to any manipulation;

[0111] II (n = 7) - sham-operated rats that received 0.1 ml of deuterium-depleted water intravenously (iv) daily for 10 days after surgery (including days 18–27 of the experiment);

[0112] III (n=7) - rats that received 0.1 ml of deuterium-depleted water (iv) for 10 days (including days 18–27 of the experiment) after injecting Aβ1-40 to induce AD;

[0113] IV (n=7) - AD was induced by injection of Aβ1-40, followed by daily injection of 1.5 mg / kg for 10 days (including days 18-27 of the experiment). 64 Rats administered Zn-asp solution i.v.;

[0114] V (n = 7) - rats in which AD was induced by Aβ25-35 and then administered 0.1 ml of deuterium-depleted water (iv) daily for 10 days (including days 18–27 of the experiment);

[0115] VI (n=7) - AD to Aβ25-35 injection 64 Rats were induced with Zn-asp and then administered the solution intravenously at a dose of 1.5 mg / kg daily for 10 days (including days 18 to 27 of the experiment) (Figure 1).

[0116] Assessment of feeding and drinking behavior in rats

[0117] The animals were housed in individual cages, and the amount of food and water consumed by the rats was measured for each rat daily starting on day 18 (8 days after surgery) until the end of the experiment (day 37). Data were first averaged to one rat per day within groups, and then averaged to one rat per day over the entire observation period.

[0118] Immunohistochemical characterization of dopaminergic neurons

[0119] Hippocampal neuron degeneration was assessed using immunohistochemical analysis of tyrosine hydroxylase (TH) expression levels. Immunohistochemical staining was performed using a primary anti-TH antibody (Millipore, AB152) at a dilution of 1:200. Endogenous peroxidase activity was blocked with a blocking reagent (Dako, EnVision Flex, DM821). Nonspecific antibody binding was blocked with 4% dry milk dissolved in Tris-buffered saline (TBS) containing 0.2% Triton X-100.

[0120] The primary antibody was diluted in TBS containing 0.2% Triton X-100 and applied to the tissue sections. The sections were then incubated overnight (+4°C). The secondary antibody (anti-rabbit biotinylated antibody, 1:200) was incubated for 60 minutes. The immunoreaction was developed by applying diaminobenzidine (Dako, EnVision) for 5 minutes. The immunohistochemical staining results were evaluated at the light microscopic level using a Zeiss Primo Star microscope. The intensity of TH-positive staining was evaluated using a semiquantitative scoring system described in the quantitative scoring method (http: / / www.ihcworld.com / ihc_scoring.htm), which takes into account the number of positive (stained) cells and the intensity of staining (Table 1). The results were expressed as a quick score (Q), calculated according to the following formula: Q = P × I, where P is the percentage of positive cells and I is the staining intensity.

[0121] [Table 1]

[0122] Short-term and long-term memory change testing using the Barnes maze

[0123] The Barnes maze (Figure 2) is a tool used to measure spatial learning and memory in rodents and is useful for identifying cognitive impairments in rodents that model diseases such as Alzheimer's disease (Kinga Gawel et al., Assessment of spatial learning and memory in the Barnes maze task in rodents—methodological considerations, Naunyn Schmiedebergs Arch Pharmacol. 2019; 392(1): 1–18. doi: 10.1007 / s00210-018-1589-y). This test, first developed by Dr. Carol Barnes in 1979, is based on the subject's innate tendency to escape from an aversive environment—a brightly lit, open surface—and seek refuge in a small, dark "escape box." Animals learn to memorize the location of a target zone (the "escape box") using peripheral visual cues within the testing area as reference points. In this test, animals locate the "escape box" under stress-free conditions, in contrast to other tests that use strong aversive stimuli (stress induced by swimming) or deprivation (lack of food or water) as reinforcement to increase behavioral variability based on the animals' individual characteristics.

[0124] The Barnes maze used consisted of a circular table with 16 circular holes around its perimeter. To better orient the animals, peripheral visual cues such as black marks (a triangle on one wall and two parallel strips on the opposite wall) were placed. An "escape box" containing standard animal filler was fixed under one of the holes. Each animal was assigned the number of the hole where the "escape box" was fixed. The other holes remained open. Before the exploration phase (post-surgery), the hole numbers were changed.

[0125] On the first day of the experiment, during the rats' initial training, a habituation session was conducted but not repeated during the exploration phase. The rat was placed in the center of a circular table and left under an opaque hood for 10 seconds. After that, the light above the table was turned on and the hood was lifted. The rat was allowed to move freely around the table for 2–3 minutes. If the rat did not find the "escape box" during this time, it was helped to find the correct path.

[0126] After the 15-minute habituation session, the task training was repeated four times every 15 minutes (3 minutes on the table surface + 1 minute in the unlit "escape box") on days 2, 3, and 4. The latency for the animals to reach the "escape box" was recorded for each trial.

[0127] The animals' short-term memory was tested on day 5, and their long-term memory on day 9 (5 days after the last training). All holes in the maze were closed, and rats were allowed to freely explore the open area for 90 seconds, searching for their corresponding hole on the table top (under which the "escape box" had previously been placed). The time it took the animals to find the correct hole and the time they spent near the hole were recorded. The table surface was disinfected after each trial.

[0128] Eighteen days after surgery, animals underwent an additional four days of training sessions (exploratory phase) with the escape box relocated. As before surgery, short-term memory was tested on day 5, and long-term memory on day 9 (5 days after the last training session).

[0129] The following times were measured in seconds: 1) the time it took the animals to find the entrance to the "escape box" (assessing spatial learning and memory, which is related to hippocampal function); 2) the time the animals spent near the entrance (assessing cognitive flexibility, which is related to functioning of the brain's frontal cortex) - the less time the animals spent near the closed entrance, the faster they understood they needed to look elsewhere for escape.

[0130] Assessment of soluble amyloid-β and tau-pau protein levels in hippocampal homogenates

[0131] Soluble amyloid-β and tau protein levels were measured in hippocampal homogenates from a rat model of AD using ELISA kits according to the manufacturer's recommendations. Hippocampal homogenates were also prepared according to the manufacturer's recommendations. Amyloid-β proteolysis in the homogenates was prevented using a protease and phosphatase inhibitor complex.

[0132] Hematological tests

[0133] Blood counts were analyzed at the completion of the experiment (day 37). Absolute white blood cell counts, as well as absolute and relative numbers of lymphocytes, monocytes and neutrophil granulocytes were calculated.

[0134] Evaluation of endocytic activity of phagocytes at various locations

[0135] The phagocytic activity of microglia and peripheral blood phagocytes was analyzed by flow cytometry using FITC-labeled Staphylococcus aureus Wood 46 cells as phagocytic targets. S. aureus cells were obtained from the collection of the Department of Microbiology and Immunology, ERC Institute of Biology and Medicine, National Taras Shevchenko University. Differential assessment of phagocytic activity values ​​of circulating mononuclear and polymorphonuclear phagocytes was performed using a gating method.

[0136] Evaluation of oxidative metabolism in phagocytes at various locations

[0137] The oxidative metabolism of phagocytes at various localizations was analyzed by flow cytometry using cell-permeable 2'7'-dichlorodihydrofluorescein diacetate (DHP) (carboxy-H2DCFDA, Invitrogen, USA), which is converted by intracellular esterases to the non-fluorescent, membrane-impermeable carboxy-H2DCF form. Differential evaluation of oxidative metabolism values ​​of circulating mononuclear and polymorphonuclear phagocytes was performed using a gating method. To assess metabolic reserve, cells were treated with LPS (Sigma, USA).

[0138] Evaluation of the phenotypic profiles of phagocytes at different locations

[0139] The phenotypic profile of phagocytes of different localizations was characterized by the expression of markers of functional maturation and metabolic polarity (CD206 and CD86), which was determined by flow cytometry and the use of monoclonal antibodies of appropriate specificity labeled with fluorescent dyes (Abcam, Becton Dickinson).

[0140] Statistical data analysis methods

[0141] Numerical results were processed using statistical data analysis methods using the Statistica 12.0 software package. Student's t-test was used to determine the statistical significance of reliable differences between the results shown by each group. Significance was set at p<0.05.

[0142] (Test results)

[0143] Effects on cognitive activity and local and systemic immunoreactivity in a rat model of Alzheimer's disease induced by Aβ1-40 injection 64 Therapeutic effect of Zn-asp

[0144] On cognitive symptoms in a rat model of Aβ1-40-induced Alzheimer's disease 64 Effect of Zn-asp

[0145] Effect of Aβ1-40 on body weight changes in a rat model of Alzheimer's disease 64 Effect of Zn-asp

[0146] Animal weight is a typical clinical sign characterizing the animal's overall condition, and weight loss during the experiment indicates a deterioration in the animal's condition. Because this experiment involved aged animals with initial weights of 350–500 g, weight changes during the experiment were minimal compared to younger animals (120–200 g) over the same period. Despite this, a significant decrease in weight was observed in the Aβ1-40-induced AD animal model within one month of the experiment. The initial animal weight (before surgery) of the sham-operated animal group was 445.0 ± 41.1 g, and at the end of the experiment on the day of necropsy, it was 449.5 ± 37.4 g, i.e., a weight gain of 1.3 ± 4.0%. The weight of the Aβ1-40-induced AD rat model before surgery was 361.1 ± 25.3 g, and on the day of necropsy, it was 340.3 ± 33.5 g, indicating a weight loss of 4.3 ± 3.7% (P < 0.01, compared to sham-operated animals) (Figure 3).

[0147] 64 Administration of Zn-asp significantly improved this parameter. Therefore, it is useful in the preoperative treatment of Aβ1-40-induced Alzheimer's disease rat model. 64 The body weight of Zn-asp-treated rats was 432.1 ± 29.7 g and 425.4 ± 40.8 g at the end of the experiment on the day of necropsy, indicating a weight loss in the experimental group of 0.6 ± 2.3% ( P < 0.05, compared with sham-operated animals).

[0148] Effect on feeding and drinking behavior in a rat model of Aβ1-40-induced Alzheimer's disease 64 Effect of Zn-asp

[0149] The aortic changes in the rat model of Aβ1-40-induced AD were associated with reduced food and water intake compared with sham-operated animals (Figures 4A and 4B).

[0150] Aβ1-40-induced Alzheimer's disease 64 It was observed that the Zn-asp treated rat model recovered normal feeding and drinking behavior.

[0151] On tyrosine hydroxylase expression in the hippocampus of a rat model of Aβ1-40-induced Alzheimer's disease 64Effect of Zn-asp

[0152] Immunohistochemical analysis of hippocampal slice preparations from untreated animals showed that their tyrosine hydroxylase expression level was 6.0±0.0 score. In sham-operated animals, the staining intensity of TH-positive cells was 7.0±1.7 score, which was not significantly different from that of untreated rats (Table 2). The expression of tyrosine hydroxylase in the rat model of Aβ1-40-induced AD was 2.3±1.5 score, which was significantly lower than the values ​​obtained from untreated and sham-operated animals, indicating the destruction of hippocampal dopaminergic neurons during AD. 64 Administration of Zn-asp increased Q to a score of 4.0 ± 2.0, primarily due to an increase in the intensity of immunopositively stained cells, but not their number, compared to untreated AD rats and sham-operated rats, returning this parameter to nearly control values ​​(Figures 5A-D).

[0153] [Table 2]

[0154] The results obtained indicate a protective role of the test substances in relation to the function of dopaminergic neurons in the hippocampus.

[0155] On spatial memory in a rat model of Aβ1-40-induced Alzheimer's disease 64 Effect of Zn-asp

[0156] Alzheimer's disease, which is most common in elderly people, is associated with impairments in declarative memory, the memory of events. Human declarative memory shares similarities with spatial memory in rodents (which is why rodents provide an excellent model of declarative memory). Neurons responsible for declarative memory are expressed in the hippocampus and are associated with a specific neuronal process known as long-term potentiation. In rodents, the hippocampus is involved in coding spatial information and is studied in various mazes. The Barnes maze is used.

[0157] To evaluate the time required for spatial learning in Alzheimer's disease, we compared the time required to find the "escape box" during the 4-day pre-surgery training period and the 4-day post-surgery training period (starting on the 18th day after surgery). Different locations of the "escape box" were used during the training and exploration periods before and after surgery. As can be seen in Figure 6, all rat groups reduced the time required to find the escape hole entrance during the 4-day training period, both before and after surgery. No differences in time were observed between the control groups (untreated and placebo-treated animals) and the rat model of Aβ1-40-induced Alzheimer's disease.

[0158] To assess short-term memory, rats were placed in the Barnes maze 24 hours after the final 4-day training period (day 5 after the start of training), but the access to the "escape box" was closed. To assess long-term memory, the same test was repeated on day 5 after the 4-day training period.

[0159] The time it took each animal to find the "escape box" was measured. The faster an animal found the correct hole, the higher its level of spatial memory (hippocampal function). The level of cognitive flexibility was also assessed by measuring the amount of time the animal spent near the escape hole entrance. The shorter the time the animal spent there, the higher its level of cognitive flexibility (frontal cortex function) - i.e., the faster the animal realized it should look for the escape hole elsewhere.

[0160] As can be seen from Table 3 and Figures 7A and 7B, the values ​​exhibited by animals in all groups before surgery were highly variable, so it was logical to compare patterns of change rather than absolute values.

[0161] As shown in Table 3 and Figures 7A and 7B, the time taken by rats in all groups to search for the “escape box” before surgery increased spontaneously between testing this parameter 24 h and 5 days after the training trial.

[0162] [Table 3]

[0163] When the level of cognitive flexibility in rats was assessed by the time the animals spent near the escape hole, a natural decrease in this parameter was observed 24 hours and 5 days after the 4-day training phase, a pattern typical for all animal groups before surgery (Table 4, Figures 7A and 7B).

[0164] When the animals' short-term and long-term memory was tested during the exploration phase (18 days after surgery), the time taken by rats in all groups to find the "escape box" was reduced by an average of 40% and 33%, respectively, compared to within-group values. The rat model of Aβ1-40-induced AD showed the same pattern of changes in these parameters as untreated animals, and similar changes were observed in the control group. 64 It should be noted that this was observed in the rat model of Aβ1-40-induced AD treated with Zn-asp. Therefore, it can be concluded that there is no statistically significant impairment in either short-term or long-term memory in the rat model of Aβ1-40-induced Alzheimer's disease.

[0165] During the exploration phase, untreated and sham-operated animals showed an average 23% decrease in the time spent near the escape box 24 hours after the last training trial (short-term effect) and an average 12% decrease 5 days after the last training trial (long-term effect). This indicates a normal level of cognitive flexibility in rats (Table 4). It should be noted that the opposite results were observed in the rat model of Aβ1-40-induced Alzheimer's disease. The time spent near the escape box increased twofold (P = 0.02) (short-term memory), and this parameter was even stronger when testing long-term memory, with a fourfold increase in the time spent near the escape hole (P = 0.04). This fact indicates dysfunction of the frontal cortex, which is responsible for cognitive functions, in Aβ1-40-induced Alzheimer's disease.

[0166] [Table 4]

[0167] 64Administration of Zn-asp significantly improved cognitive function in a rat model of AD, virtually returning it to the values ​​obtained in untreated and sham-operated animals (Voikar V. Evaluation of methods and applications for behavioral profiling of transgenic mice. Academic dissertation. Faculty of Biosciences, University of Helsinki. 2006. 73 p.).

[0168] On Aβ and tau protein levels in hippocampal homogenates from a rat model of Alzheimer's disease induced by injection of Aβ1-40 64 Therapeutic effect of Zn-asp

[0169] The presence of soluble Aβ and tau proteins in the hippocampus is a clear sign of AD development in animal models and a marker for assessing disease severity and the effectiveness of etiologic treatments. Results showed that soluble Aβ levels in hippocampal homogenates in rat models of AD induced by Aβ1-40 injection were nearly four times higher than those in untreated animals (Figure 8). In placebo-treated animals (sham-operated animals), soluble Aβ levels were also slightly higher than in untreated rats. Aβ levels in the hippocampus of AD rat models treated with the test substance were 1.5-fold lower than those in control AD ​​rat models, but did not reach the levels of untreated / sham-operated animals. It should be noted that this parameter showed exceptional variability, which made it impossible to properly evaluate the evidence of the obtained data. This high degree of variability may be due to the low statistical sampling of animals, the difference in sample preparation method of the test system used in the study from similar procedures described in the literature for testing this parameter using ELISA kits from other manufacturers (Xuan A et al., J Neuroinflammation 2012;9:202. doi:10.1186 / 1742-2094-9-202; Wang L et al., Iran J Basic Med Sci. 2017;20(5):474-480. doi:10.22038 / IJBMS.2017.8669), as well as the natural variability of this parameter in AD models. The concentration of insoluble Aβ, as recently described in a paper (Zhao HF et al., Neuroscience. 2015;310:641-649 doi:10.1016 / j.neuroscience.2015.10.006), may be a more appropriate criterion for the formation of senile plaques in AD.

[0170] Levels of phosphorylated tau protein in hippocampal homogenates from a rat model of AD also exceed by more than four-fold the values ​​obtained from untreated animals, a measure of disease progression and validity of the model (FIG. 9). 64In animals that received a series of therapeutic treatments with Zn-asp, the levels of this protein in the hippocampus were lower than in control AD ​​models, but this difference is not considered significant, probably due to the large individual variability of this parameter in all animal groups, for the reasons mentioned above.

[0171] In general, analysis of the levels of proteins involved in the pathogenesis of AD indicates that the test substance has a pathologic therapeutic effect that results in a reduction in the concentration of plaque-forming components in an AD animal model.

[0172] Functional and phenotypic characteristics of microglia in a rat model of Alzheimer's disease induced by Aβ1-40 injection 64 Therapeutic effect of Zn-asp

[0173] Microglial phagocytic activity is an indicator of its activation state, and its changes should be viewed in the context of changes in other functional and phenotypic characteristics. Increased phagocytic activity of microglial cells can accompany both pro- and anti-inflammatory microglial activation. Furthermore, increased permeability of the blood-brain barrier (BBB) ​​leads to recruitment of resident microglial cell populations by peripheral blood phagocytes, but this differential assessment was not possible under these test conditions. Our results show that the relative number of phagocytic microglia (phagocytic index, (PI)) in AD animal models was two-fold higher than in untreated animals. It should be noted that this value was significantly lower in sham-operated (SO) animals. Endocytic activity (PI) in AD rats was also significantly higher (nearly five-fold) than in untreated animals and two-fold higher than in sham-operated rats (Figures 10A and 10B). Administration of the test substance completely normalized both the relative number of phagocytic microglia and their endocytic (phagocytic) activity levels, indicating the anti-inflammatory effects of the drug candidate.

[0174] Oxidative metabolism is another metabolic indicator of microglia. In this study, microglia from untreated animals were characterized by an unresponsiveness to in vitro stimulation with bacterial LPS, indicating their involvement in aging-associated inflammation (Figure 11) (Norden DM, Godbout JP. Review: microglia of the aged brain: primed to be activated and resistant to regulation. Neuropathol Appl Neurobiol. 2013;39(1):19-34. doi:10.1111 / j.1365-2990.2012.01306.x).

[0175] The oxidative metabolism of sham-operated animals was somewhat enhanced compared to untreated animals at the time of the experiment, supporting the hypothesis of a sustained reparative inflammatory process exacerbated by inflammation. A further criterion for this condition is the lack of functional reserve of microglial oxidative metabolism in this group of animals in response to in vitro LPS treatment.

[0176] The progression of AD was accompanied by a significant (5-fold) increase in the production of reactive oxygen species by microglial cells. Increased oxidative metabolism in microglia is an essential component of AD-associated neuroinflammation, and therefore, these data support the validity of the selected model. The response of microglial cells in this group of animals to in vitro LPS treatment was extremely negative, indicating extreme pro-inflammatory activation. Administration of the test substance completely normalized microglial oxidative metabolism in the AD rat model, i.e., both the basal level of ROS generation and the metabolic reserve capacity of this function. Thus, analysis of metabolic values ​​of microglial functional polarization demonstrated the presence of a pro-inflammatory metabolic shift in the AD rat model and its disappearance after a course of therapeutic treatment with the test substance.

[0177] To characterize the phenotypic profile of microglia, we used the markers CD206 (a scavenger receptor, a marker of alternative polarity of extracerebral phagocytes and a marker of activated resident microglia) and CD86 (a costimulatory molecule involved in the process of antigen presentation, a marker of pro-inflammatory activation of extracerebral phagocytes, also overexpressed by myeloid-derived suppressor cells, a negative regulator of pro-inflammatory responses of innate and adaptive immunity). There was considerable variability in the quantitative analysis of phenotypic microglial markers, likely due to the heterogeneity of the aging process and the low statistical sampling of animals. In general, the results of the evaluation of the phenotypic profile of microglial cells are shown in Figure 12A, Figure 12B, Figure 13A, and Figure 13B.

[0178] The number of CD86+ cells in the microglial population of AD animal models was 1.6-fold higher than in untreated animals (Figures 12A and 12B). The expression level of this marker in microglial cells was more than 2.5-fold higher than in untreated animals, demonstrating a proinflammatory shift in microglial function characteristic of AD and supporting the results of evaluating metabolic parameters of these cells. Administration of the test substance normalized both the number of CD86+ cells and the expression level of this marker in positive cells, indicating the potent anti-inflammatory effect of the drug candidate.

[0179] The data on CD86 expression are supported by data on the expression of another phenotypic marker, CD206 (FIGS. 13A and 13B).

[0180] The number of microglial cells expressing CD206 in the AD rat model was 3.5 times higher than in untreated animals, indicating the activation of phagocytes in the brains of AD rats. The expression level of this marker in positive cells of AD rats was more than 5 times higher than that of untreated rats. Treatment with zinc-based test substances caused a decrease in the above values ​​compared to the levels in untreated animals, which is further evidence of the anti-inflammatory effect of the test substances.

[0181] Thus, the progression of Aβ1-40-induced AD was accompanied by a marked pro-inflammatory functional transformation of microglial cells. 64 The use of Zn-asp normalizes the phenotypic and functional parameters of microglia: all analyzed characteristics of this phagocyte population at the time of the experiment were unchanged from those in healthy animals of the corresponding age group.

[0182] On blood counts in a rat model of Alzheimer's disease induced by Aβ1-40 injection 64 Therapeutic effect of Zn-asp

[0183] Literature data provide strong evidence that chronic inflammation is one of the most important pathophysiological components of synucleinopathies and tauopathies, including Alzheimer's disease. Leukograms in patients with AD reveal elevated monocyte and neutrophil counts and low lymphocyte counts. Elevated monocyte and neutrophil levels are hallmarks of chronic inflammation and may be both a precursor and a consequence of AD. Low lymphocyte counts indicate a significantly reduced body resistance to infection (Shad KF et al., Synapse. 2013;67(8):541-543. doi:10.1002 / syn.21651; Stock AJ, Kasus-Jacobi A, Pereira HA. ​​J Neuroinflammation. 2018;15(1):240 doi:10.1186 / s12974-018-1284-4). Increased permeability of the blood-brain barrier (BBB) ​​in AD promotes the translocation of neuroinflammatory mediators to the periphery and the recruitment of circulating leukocytes to the brain, which creates the prerequisites for a sustained meta-inflammatory process (Yamazaki Y, Kanekiyo T. Int J Mol Sci. 2017;18(9):1965 doi:10.3390 / ijms1809196). Blood counts of experimental animals were measured at the end of the experiment.

[0184] Analysis of blood samples from rat models of Aβ1-40-induced AD showed extremely high white blood cell (WBC) counts, with the number of circulating leukocytes in the blood being 2.5-fold higher compared to untreated animals (Figure 14). It should be noted that leukocytosis was also observed in placebo-treated rats, apparently due to the reduced ability of the immune system to repair itself in older animals. Treatment with the test substance completely normalized the absolute number of circulating leukocytes in this animal model of AD.

[0185] Analysis of the population composition of circulating leukocytes showed a slight decrease in lymphocyte counts and a significant decrease in monocyte counts (moderate monocytopenia). Induction of AD was also accompanied by a significant (>4-fold) increase in the neutrophil-lymphocyte ratio (the ratio of absolute neutrophil counts to absolute lymphocyte counts in peripheral blood, or NLR). The NLR is one of the early markers of AD progression (Kuyumcu ME et al., Dement Geriatr Cogn Disord. 2012;34(2):69-74. doi:10.1159 / 000341583) and an important biomarker for identifying patients with cognitive impairment (Dong X et al., Front Aging Neurosci. 2019;11:332 Published 2019 Dec 5. doi:10.3389 / fnagi.2019.00332). Administration of a zinc-based preparation completely normalized the NLR, with both an increase in lymphocyte count (a criterion for resolution of inflammation by activating the suppressive function of regulatory cells) and a significant decrease in the number of dividing neutrophils.

[0186] Functional and phenotypic characteristics of circulating phagocytes in a rat model of Alzheimer's disease induced by Aβ1-40 injection 64 Therapeutic effect of Zn-asp

[0187] As mentioned above, the development of AD is accompanied by the formation of systemic inflammation, which increases and maintains the persistence of the neuroinflammatory process. This situation makes effector cells of the systemic inflammatory process no less attractive than resident leukocytes as targets for anti-inflammatory treatment in AD. This was one of the reasons for analyzing the functional and phenotypic characteristics of circulating phagocytes in a rat model of Aβ1-40-induced AD. Furthermore, test substances were administered intravenously, which makes circulating phagocytes the first-line responder cells. As mentioned above, blood count results indicated the presence of a systemic inflammatory process in this rat model of Aβ1-40-induced AD, with significant leukocytosis, neutrophilia, and an increased neutrophil-to-lymphocyte ratio (a validated biomarker of the systemic inflammatory process in advanced AD). Analysis of the functional and phenotypic characteristics of circulating phagocytes confirmed these observations.

[0188] The neutrophilia detected in blood samples from AD rats was accompanied by a significant increase in phagocytic activity, which is a marker of cellular activation and, on the other hand, a sign of an anti-immune metabolic shift (Figure 15B). Furthermore, the relative number of phagocytic neutrophils in this group of animals did not differ significantly from those in untreated and SO rats (Figure 15A). It should be noted that the absorptive activity of polymorphonuclear phagocytes in SO rats was significantly higher than in untreated controls, which may be the result of surgery and the activation of the accompanying repair process, characterized by an anti-inflammatory shift in phagocyte metabolism.

[0189] Treatment with the test substance was accompanied by a decrease in the phagocytic activity of these cells substantially to the values ​​exhibited by untreated animals, indicating its homeostatic systemic effect.

[0190] The relative number of phagocytic monocytes in the rat model of AD was nearly four-fold higher compared to untreated and SO rats (Fig. 16A). Moreover, the phagocytic activity of these cells was not different from that of both control groups of animals (Fig. 16B).

[0191] Analysis of indicators of oxidative metabolism in both populations of circulating phagocytes showed no statistically significant differences between the AD animal model and untreated animals (Figures 17A and 17B). It should be noted that administration of zinc-based formulations to AD rats slightly created a functional reserve of oxidative metabolism in the analyzed circulating phagocyte populations, which may be a sign of the presence of "young" cells in the peripheral blood and therefore indicates the ability of the drug candidate to moderately stimulate myelopoiesis.

[0192] In sham-operated animals, a rapid increase in oxidative metabolic indicators was recorded in both peripheral blood granulocytes and mononuclear phagocytes. Concomitantly, there was a functional reserve of oxidative metabolism. This result likely reflects sustained reparative inflammation accompanied by activation of medullary myelopoiesis.

[0193] Analysis of phenotypic markers of circulating phagocytes in animal models of AD also supports the spontaneous resolution of systemic inflammation: the relative number of CD86+ circulating phagocytes in AD rats is significantly higher than in control animals (Figures 18A and 18B).

[0194] As mentioned above, this marker is characteristic of both phagocytes and myeloid suppressor cells with a proinflammatory metabolic transition. Considering the increased phagocytic activity of peripheral blood phagocytes, it can be speculated that the increased proportion of CD86+ cells is due to the presence of myeloid suppressor cells.

[0195] An increased proportion of CD86+ cells with increased expression levels of this marker was observed in sham-operated (SO) animals, which complements the results of the repair process induced by the surgical procedure.

[0196] Analysis of CD206 expression also supports the resolution of inflammation. In AD rats, the percentage of CD206 marker-positive cells was similar to that in untreated animals. However, its expression level by circulating phagocytes was higher than in untreated controls (Figures 19A and 19B).

[0197] Administration of the test substance as monotherapy normalized the number of cells expressing these markers and their expression levels, supporting a homeostatic effect on systemic immune reactivity in the progression of AD induced by Aβ1-40 infusion.

[0198] (Insights)

[0199] In a rat model of Aβ1-40-induced AD, a decrease in body weight and water and food intake was observed after 3 weeks of disease mimicry. These parameters were 64 It was recovered in AD rats treated with Zn-asp for 10 days.

[0200] In a rat model of Aβ1-40-induced AD, a decrease in the number of hippocampal dopaminergic neurons and a decrease in tyrosine hydroxylase (TH) expression in hippocampal dopaminergic neurons were observed. 64 Administration of Zn-asp increased the staining intensity rather than the number of TH-immunopositive cells.

[0201] The progression of Aβ1-40-induced AD is associated with impaired cognitive flexibility in AD rats, indicating frontal cortex dysfunction. The Aβ1-40-induced Alzheimer's disease rat model did not show alterations in spatial learning or short-term / long-term memory abilities (hippocampal function). 64 Administration of Zn-asp significantly improved cognitive function in the AD model, essentially returning it to the levels of untreated and sham-operated animals.

[0202] The progression of Aβ1-40-induced AD was characterized by a prolonged acute local (in microglia) inflammatory process and a moderately expressed systemic inflammation with signs of spontaneous resolution.

[0203] Treatment with zinc-based test substances resulted in almost complete resolution of neuroinflammation and homeostatic regulation of systemic immune reactivity, indicating the pathogenetic nature of its therapeutic effect.

[0204] (Results of Test II)

[0205] Effects on cognitive activity and local and systemic immune reactivity in a rat model of Alzheimer's disease induced by Aβ25-35 injection 64 Therapeutic effect of Zn-asp

[0206] On cognitive symptoms in a rat model of Aβ25-35-induced Alzheimer's disease 64 Effect of Zn-asp

[0207] Effect of Aβ25-35 on body weight changes in a rat model of Alzheimer's disease 64 Effect of Zn-asp

[0208] Animal weight is a typical clinical sign that characterizes the animal's overall condition, and weight loss during the experiment indicates a deterioration in the animal's condition. No significant changes in body weight were observed in the Aβ25-35-induced AD rat model during the 1-month experiment (Figure 20).

[0209] 64 Administration of Zn-asp had no effect on this parameter.

[0210] On tyrosine hydroxylase expression in the hippocampus of a rat model of Aβ25-35-induced Alzheimer's disease 64 Effect of Zn-asp

[0211] The results of immunohistochemical analysis of tyrosine hydroxylase expression showed that the quick score (Q) in the AD rat model was 6.0 ± 0.0. The staining intensity of TH-positive cells in sham-operated animals was 7.0 ± 1.7, which was not significantly different from that in untreated rats (Table 5).

[0212] [Table 5]

[0213] In the Aβ25-35-induced AD animal model, there were no statistically significant changes in either the number of TH-positive neurons or the intensity of staining compared with untreated and placebo-treated animals (Q = 5.3 ± 1.5). 64 Administration of Zn-asp did not affect the staining intensity of TH-positive cells; the Q value of this group was 5.0±1.4 (FIGS. 21A, 21B, 21C, and 21D).

[0214] This analysis showed no significant changes in the function or number of TH-positive hippocampal neurons.

[0215] On spatial memory in a rat model of Aβ25-35-induced Alzheimer's disease 64 Effect of Zn-asp

[0216] To evaluate the time required for spatial learning in Alzheimer's disease, we compared the time required to find the "escape box" during the 4-day pre-surgery training period and the 4-day post-surgery training period (starting on day 18 after surgery). Different locations of the "escape box" were used during the training and exploration periods before and after surgery. As can be seen in Figures 22A and 22B, all rat groups reduced the time required to find the escape hole entrance during the 4-day training period, both before and after surgery. No differences in time were observed between the control groups (untreated and placebo-treated animals) and the rat model of Aβ25-35-induced Alzheimer's disease.

[0217] To assess short-term memory, rats were placed in the Barnes maze 24 hours after the final 4-day training period (day 5 after training began), but the access to the "escape box" was closed. To assess long-term memory, the same test was repeated on day 5 after the 4-day training period.

[0218] The time it took each animal to find the "escape box" was measured. The faster an animal found the correct hole, the higher its level of spatial memory (hippocampal function). The level of cognitive flexibility was also assessed by measuring the amount of time the animal spent near the escape hole entrance. The shorter the time the animal spent there, the higher its level of cognitive flexibility (frontal cortex function) - i.e., the faster the animal realized it should look for the escape hole elsewhere.

[0219] As shown in Tables 6 and 7, the values ​​exhibited by animals in all groups before surgery varied considerably from individual to individual, so it was logical to compare patterns of change rather than absolute values.

[0220] As shown in Table 6, the time taken by rats in all groups to find the "escape box" before surgery spontaneously increased between the 24-h and 5-day post-exploratory testing of this parameter. Only rats that were subsequently injected with Aβ25-35 to mimic AD were observed to have a decreased time taken to find the "escape box," which may be related to the individual characteristics of these rats.

[0221] [Table 6]

[0222] During the exploration phase (18 days after surgery), the time taken by rats in all experimental groups to find the "escape box" in the short-term memory test either decreased slightly or remained the same. When animals were tested for long-term memory, the results were similar in untreated and sham-operated rats, but an increase in the time taken to find the correct hole was observed in the Aβ25-35-induced Alzheimer's disease rat model. The same pattern was observed in the 64 This was observed in the Zn-asp-treated rat model of AD. However, the changes observed in both groups were not statistically significant. Therefore, although there is no statistically significant impairment in short-term memory in the rat model of Aβ25-35-induced Alzheimer's disease, 64It can be concluded that there is a tendency for impairment in long-term memory that was not improved by Zn-asp.

[0223] When the level of cognitive flexibility of the rats was assessed by measuring the time the animals spent near the entrance to the "escape box" 24 hours and 5 days after the 4-day exploration phase, no spontaneous decrease or change in this parameter was observed (Table 7).

[0224] [Table 7]

[0225] This pattern was characteristic of all animal groups before surgery, with the exception of rats later used as a model for Alzheimer's disease, which showed an increased time spent near the escape hole, which may be related to the individual characteristics of these rats. During the exploration phase, untreated and sham-operated animals, as well as Aβ25-35 AD rats, were observed to spend slightly less time near the entrance to the "escape box." This indicates that AD rats have normal levels of cognitive flexibility and that Aβ25-35 has no effect on this parameter. No significant changes in this parameter were observed after treatment with the test substances.

[0226] On Aβ and tau protein levels in hippocampal homogenates from a rat model of Alzheimer's disease induced by injection of Aβ25-35 64 Therapeutic effect of Zn-asp

[0227] Analysis of the levels of proteins involved in the pathophysiological process of AD (Aβ and tau proteins) in hippocampal homogenates from the Aβ25-35 AD rat model showed results similar to those obtained from an animal model of AD induced by Aβ1-40 injection. The levels of both Aβ and tau proteins in AD controls significantly exceeded those in untreated and SO rats (Figures 23A and 23B). Therapeutic treatment with the test substance resulted in a significant decrease in the levels of these proteins, but they did not completely return to normal. As with the AD model induced by Aβ1-40 injection, the values ​​of both proteins in hippocampal homogenates were characterized by extremely high variability, and it should be noted that the results are inconclusive. This variability may be caused by the following factors: the sample preparation method recommended by the manufacturer of the test system used in the study differed from the method described in the protocols and presented in the literature for almost all test systems used for the same purpose; and the small number of animals in all experimental groups used to analyze this parameter (considering that the study was declared a pilot study).

[0228] However, the results of the analysis of these parameters suggest that the test substance has a pathologic therapeutic effect accompanied by a reduction in the quantitative characteristics of the pathologic markers of Alzheimer's disease.

[0229] Functional and phenotypic characteristics of microglia in a rat model of Alzheimer's disease induced by Aβ25-35 injection 64 Therapeutic effect of Zn-asp

[0230] As mentioned above, the AD model induced by Aβ25-35 injection was chosen due to the exceptional role of Aβ fragments in the formation of senile plaques in AD, their ability to exert direct toxicity on neurons leading to their death regardless of the formation of Aβ deposits, and the development of an autoimmune response to this peptide and the progression of AD. Analysis of the functional and phenotypic characteristics of microglia in a rat model of AD induced by Aβ25-35 injection showed the following:

[0231] The number of phagocytic microglial cells in control AD ​​rats was more than two-fold higher than in untreated and SO animals, indicating the activation state of a complex population of phagocytes in the brain (Figures 24A and 24B). However, the intensity of microglial resorption activity in the AD animal model was not different from that in untreated animals. Therefore, the increased resorption activity can be attributed to the anti-inflammatory activation of microglia caused by the natural resolution of neuroinflammation (as the analysis was performed at the end of the experiment).

[0232] It should be noted that a high level of microglial resorption activity was observed in SO animals, which is likely related to the post-surgery repair process. Treatment with the test substance caused a sharp decrease in the number of phagocytic microglial cells in the AD rat model, 10-fold compared to AD controls and 5-fold compared to untreated animals. At the same time, the rate of phagocytosis significantly increased. Changes in phagocyte function, regardless of their location, should be analyzed in conjunction with changes in other metabolic responses. In this case, it should be considered that AD modeling (including sham surgery) likely causes changes in BBB permeability and migration of circulating phagocytes toward microglia. As a result of these processes, the microglial population includes recruited mononuclear and polymorphonuclear phagocytes in addition to resident macrophages, although the differential assessment of this effect is unclear depending on the duration and conditions of the study. Considering the above, since the increased resorption activity of anti-inflammatory phenotype microglia is characteristic of extracerebral phagocytes (whose proportion in the complex microglial population may be crucial), data on phagocytic activity can be interpreted as an indication of stimulation of the repair process by the test substance.

[0233] The evaluation of microglial phagocytic activity in an animal model of AD induced by Aβ25-35 injection was supported by the evaluation of oxidative metabolism in these cells (FIG. 25).

[0234] The level of ROS generation in the Aβ25-35 AD rat model was unchanged from that in untreated animals and significantly lower than that in sham-operated rats. Evaluation of the microglial activation state in sham-operated rats as a marker of the sustained repair process induced by surgical intervention was verified by analysis of the physiological status of this group of animals, which was completely satisfactory, with no deviations in cognitive activity or behavioral responses. Therefore, the increased level of ROS generation in SO animals may be considered an indicator of the repair inflammatory process. The lack of differences in microglial oxidative metabolism between the Aβ25-35 AD rat model and untreated animals may indicate the spontaneous resolution of neuroinflammation and the imperfection of the AD model used in the study. Administration of the test substance to the Aβ25-35 AD rat model increased microglial oxidative metabolism, which may be evidence of stimulation of the repair process by the drug candidate.

[0235] The expression levels of phenotypic markers of microglial cells are generally consistent with their metabolic profile (Figures 26A and 26B), but contain factors that require more detailed examination of microglia in this AD model.

[0236] The number of CD86+ cells in the microglial population of AD animals was significantly higher than that of untreated animals. If CD86+ cells are considered a marker of myeloid suppressor cells, the obtained data are consistent with the concept of spontaneous regression of neuroinflammation in the AD rat model, indicating the imperfection of the model. However, if the increase in the percentage of CD86+ cells is attributed to an increase in the number of effector phagocytes activated by antigen presentation, the analysis of phenotypic markers indicates activation of the autoimmune response in the brain initiated by Aβ25-35 infusion, which is consistent with literature data on the involvement of Aβ25-35 in the autoimmune component of AD. In this case, the rapid decrease in the percentage of CD86+ cells in AD animals after a course of treatment with a zinc-based preparation can be considered evidence of the ability of the test substance to inhibit the development of the autoimmune response associated with AD.

[0237] This assumption is consistent with the evaluation of the expression of another microglial phenotype marker, CD206 (FIGS. 27A and 27B).

[0238] The expression level of this marker in microglia and the size of the percentage of positive cells in AD animals were not significantly different from the comparison values ​​in untreated animals. If this marker is considered to indicate the activation state of microglia, a decrease in the percentage of positive cells due to the action of a test substance can be considered evidence of its homeostatic therapeutic effect.

[0239] Functional and phenotypic characteristics of circulating phagocytes in a rat model of Alzheimer's disease induced by Aβ25-35 injection 64 Therapeutic effect of Zn-asp

[0240] Differential blood counts in the rat model of Aβ25-35-induced AD showed more pronounced inflammation than the Aβ1-40 AD model (FIG. 28), but with somewhat different characteristics.

[0241] The number of circulating leukocytes in the AD rat model was twice as high as in untreated animals, with lymphocyte and neutrophil granulocyte counts also doubled, and the number of monocytes increased nearly fourfold compared to untreated animals. At the same time, the neutrophil-lymphocyte ratio (NLR) in AD rats was significantly lower than in untreated animals. This increase in lymphocyte counts may be evidence of the activation of an autoreactive T cell immune response (autoimmunity), which is consistent with the proposed interpretation of the results of the evaluation of microglial functional and phenotypic profiles. A course of treatment with the test substance resulted in a slight decrease in leukocyte counts, but the leukocyte count did not return to normal. This decrease was primarily due to a normalization of monocyte counts. However, neutrophil and lymphocyte levels remained unchanged after treatment, and the NLR remained as high as in untreated AD rats.

[0242] The relative number of phagocytic neutrophils in the peripheral blood of a rat model of AD was higher than that of untreated animals, although the difference was small (Figures 29A and 29B).

[0243] The rate of phagocytosis by circulating polymorphonuclear phagocytes in a rat model of AD was significantly higher than in control animals, which is an indication of the activated state of these cells. Treatment with the test substance caused a small but statistically significant decrease in the phagocytic index of circulating granulocytes without a specific effect on their numbers, indicating the homeostasis of the immunomodulatory effect of the drug candidate on this circulating phagocyte population in AD progression.

[0244] The number of phagocytic monocytes and their phagocytic activity in the peripheral blood of a rat model of AD were significantly higher than those of controls (FIGS. 30A and 30B).

[0245] Treatment with the zinc-based preparation normalized the analyzed parameters, confirming our assumption of the homeostatic nature of the immunomodulatory effect of the test substances.

[0246] Indicators of oxidative metabolism in circulating mononuclear and polymorphonuclear phagocytes in these rat models of AD were higher than those in untreated animals and only slightly higher than those in SO animals (Figures 31A and 31B).

[0247] Treatment with the test substances did not cause significant changes in the oxidative metabolism of peripheral blood phagocytes.

[0248] The results of assessing the expression of phenotypic markers by peripheral blood phagocytes are difficult to interpret. The percentage of CD86+ cells and the expression level of this marker by circulating phagocytes in a rat model of Aβ25-35-induced AD were similar to those in untreated animals and lower than those in sham-operated rats (Figures 32A and 32B). This is most easily explained by the spontaneous regression of disease and the incompleteness of the AD model.

[0249] 64Zn-asp treatment resulted in an increase in the proportion of CD86+ cells and an increase in the expression of this marker, which may be evidence of accelerated resolution of inflammation under the action of the test substance. This assumption is also supported by the results of evaluation of the expression of another phenotypic marker, CD206 (Figures 33A and 33B).

[0250] The proportion of positive cells (cells with an anti-inflammatory phenotype) increased in rats injected with Aβ25-35. The expression level of this marker by blood phagocytes in AD animal models was not different from that of untreated animals. Treatment with the test substance rapidly decreased the proportion of cells positive for this marker, but significantly increased its expression.

[0251] (Insights)

[0252] The rat model of Aβ25-35-induced AD did not show significant changes in any of the selected and analyzed disease progression markers (animal weight, number and expression level of TH-positive neurons in the hippocampus, spatial learning, short- and long-term memory, cognitive flexibility). There was only a trend toward impairment of long-term spatial memory. 64 Zn-asp had no statistically significant effect on cognitive symptoms in animals injected with Aβ25-35.

[0253] The model of AD induced by Aβ25-35 injection does not feature the classical picture of neuroinflammation, and therefore the protocol used in this study does not reflect the clinical situation of Alzheimer's disease. Only the fact that there may be a local autoimmune process associated with this model is noteworthy.

[0254] The therapeutic effect of the test substances on this AD model is of an anti-inflammatory and homeostatic nature.

[0255] The present invention includes the following aspects and embodiments. [1] A method for treating Alzheimer's disease, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising Zn, wherein the composition comprises: 64 Zn e a compound or a salt thereof,64 Zn e At least 80% of the compound or its salt 64 Zn e That's the method. [2] 64 Zn e The compound or its salt is at least 95% 64 Zn e The method according to [1], [3] 64 Zn e The compound or its salt is at least 99% 64 Zn e The method according to [1] or [2], wherein [4] 64 Zn e The method according to any one of [1] to [3], wherein the compound is in the form of a salt selected from the group consisting of aspartate, sulfate, and citrate. [5] The method according to any one of [1] to [4], wherein the composition is administered by injection. [6] The method according to any one of [1] to [4], wherein the composition is administered orally. [7] A method for delaying the onset of Alzheimer's disease, comprising administering to a subject in need thereof a prophylactically effective amount of a composition comprising Zn, wherein the composition: 64 Zn e a compound or a salt thereof, 64 Zn e At least 80% of the compound or its salt 64 Zn e That's the method. [8] 64 Zn e The compound or its salt is at least 95% 64 Zn e The method according to [7]. [9] 64 Zn e The compound or its salt is at least 99% 64 Zn e The method according to [7] or [8], wherein

[10] 64 Zn e The method according to any one of [7] to [9], wherein the compound is in the form of a salt selected from the group consisting of aspartate, sulfate, and citrate.

[11] The method according to any one of [7] to

[10] , wherein the composition is administered by injection.

[12] The method according to any one of [7] to

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

Claims

1. 1. A pharmaceutical for treating Alzheimer's disease, comprising a therapeutically effective amount of a composition comprising Zn, the composition comprising: 64 Contains Zn-enriched salts, 64 At least 80% Zn-rich salt 64 A pharmaceutical agent which is Zn-enriched, and the 64 Zn-enriched salt is 64 Zn aspartate.

2. 64 Zn-enriched salt is at least 95% 64 The pharmaceutical composition of claim 1, which is enriched with Zn.

3. 64 Zn-enriched salt is at least 99% 64 The pharmaceutical composition according to claim 1 or 2, which is enriched with Zn.

4. The method of any one of claims 1 to 3, wherein the composition is administered by injection.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition is administered orally.

6. 1. A pharmaceutical for delaying the onset of Alzheimer's disease, comprising a prophylactically effective amount of a composition comprising Zn, the composition comprising: 64 Contains Zn-enriched salts, 64 At least 80% Zn-rich salt 64 A pharmaceutical agent which is Zn-enriched, and the 64 Zn-enriched salt is 64 Zn aspartate.

7. 64 Zn-enriched salt is at least 95% 64 The pharmaceutical composition of claim 6, which is enriched with Zn.

8. 64 Zn-enriched salt is at least 99% 64 The pharmaceutical composition according to claim 6 or 7, which is enriched with Zn.

9. The method of any one of claims 6 to 8, wherein the composition is administered by injection.

10. The pharmaceutical composition according to any one of claims 6 to 8, wherein the composition is administered orally.

Images