EP4313080A1 - Method of treating alzheimer's disease - Google Patents
Method of treating alzheimer's diseaseInfo
- Publication number
- EP4313080A1 EP4313080A1 EP21719803.5A EP21719803A EP4313080A1 EP 4313080 A1 EP4313080 A1 EP 4313080A1 EP 21719803 A EP21719803 A EP 21719803A EP 4313080 A1 EP4313080 A1 EP 4313080A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- induced
- disease
- animals
- asp
- rat models
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/30—Zinc; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
Definitions
- This disclosure relates to therapy for Alzheimer's disease.
- AD Alzheimer's disease
- Mount C Downton C., Nature Medicine 2006;12(7):780- 784; Wimo A et al., Alzheimer ’s and Dementia 2010;6(2):98-103.
- AD is of great medical and social concern, considering that the incidence of the illness grows and the prospect of an aging population will result in rising social and economic demands.
- AD is characterized by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles within the afflicted brain, which cause neuronal loss in the neocortex, hippocampus, and basal forebrain, leading to progressive cognitive and behavioral decline.
- Watt NT et al Ini J Alzheimer s Dis. 2010:2011:971021. Published 2010 Dec 20. doi: 10.4061 /2011/971021.
- this disclosure provides a method of treating or preventing AD in a patient, comprising administering to said patient a pharmaceutical composition comprising zinc, at a therapeutically effective or a prophylactically effective dose for treating or prevent the disease.
- the composition comprises 64 Zn-enriched zinc (the term “ 64 Zn e ” is used herein to refer to 64 Zn-enriched zinc).
- the 64 Zn-enriched zinc is in the form of a 64 Zn e compound or a 64 Zn e salt.
- the disclosed compositions contain zinc that is 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 , for example, zinc that is 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 .
- FIG. 1 is a graphical presentation of the experimental design in the study of therapeutic effects of 64 Zn-asp on behavioral functions of experimental models of Alzheimer's Disease.
- FIG. 2 shows the Barnes maze.
- FIG. 3 shows the effects of 64 Zn-asp on the body weight in rat models of Ab1-40- induced Alzheimer's disease.
- the data are presented as a percentage ratio of the final body weight of an animal (on the day of autopsy) to its body weight before mimicking Alzheimer's disease, which was taken as 100%.
- FIG. 4A and FIG. 4B show the effects of 64 Zn-asp on the eating (food intake) (FIG. 4B) and drinking (water intake) (FIG. 4A) behavior in rat models of AbI-40-induced Alzheimer's disease.
- the animals were placed in individual cages and the amounts of food and water they consumed were measured daily for each rat, starting from the 18th day (8 days after surgery) and until the end of the experiment (37th day). The data were first averaged into 1 rat per day within a group, and then into 1 rat per day for the entire period of observation.
- FIG. 5A - FIG. 5D Immunohistochemical identification of tyrosine hydroxylase (TH) activity in the neurons in the hippocampus of intact rats (FIG. 5A), sham-operated rats (placebo) (FIG. 5B), rat models of AbI-40-induced Alzheimer's disease injected with H2O (FIG. 5C) and rat models of AbI-40-induced Alzheimer's disease treated with 64Zn-asp (FIG. 5D). TH-positive staining (dark). Oc. 40, ob. 10.
- FIG. 6A (before the operation) and FIG. 6B (after the operation) are graphs showing time spent by rat models of AbI-40-induced Alzheimer's disease for spatial learning in the Barnes maze. Data are presented as an average of 4 trials every 15 min/rat/day and an average within each group/day. M ⁇ SD
- FIG. 7A shows the effects of 64 Zn-asp on the spatial short-term and long-term memory efficiency (time spent to find an entrance to the “escape box”) and cognitive flexibility (time spent near the entrance to the “escape box”) 24 hours (5 th day after the 4-day training) and 5 days after training (9 th day after the 4-day training) in rat models of AbI-40-induced Alzheimer's, disease, M ⁇ SD
- A is a relevant number of cells performing phagocytosis; B is phagocytic activity. * p ⁇ 0.05 versus intact animals
- FIG. 10A shows relevant number of cells performing phagocytosis;
- FIG. 10B shows phagocytic activity. *p ⁇ 0.05 versus intact animals
- FIG. 12A is the number of expressing cells in the population analyzed;
- FIG. 12B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- FIG. 13A is the number of expressed cells in the population analyzed;
- FIG. 13B is the level of expression. * - p ⁇ 0.05 versus intact animals; # - ⁇ 0.05 versus control AD animal models
- FIG. 14 shows the effects of 64 Zn-asp on the levels of circulating leukocytes in rat models of AbI-40-induced AD, M ⁇ SD. Note: *p ⁇ 0.05 vs. intact animals, # p ⁇ 0.05 vs. untreated animal models of AD
- FIG. 15A number of cells performing phagocytosis, %) and FIG. 15B (phagocytosis index, GMean) show effects of 64 Zn-asp on phagocytic activity of circulating polymorphonuclear granulocytes in rat models of AbI-40-induced AD, M ⁇ SD Note:
- FIG. 16A (number of cells performing phagocytosis, %) and FIG. 16B (phagocytosis index, GMean) show effects of 64 Zn-asp on phagocytic activity of circulating monocytes in rat models of AbI-40-induced AD, M ⁇ SD Note: *p ⁇ 0.05 vs. intact animals, # p ⁇ 0.05 vs. untreated animal models of AD [0024]
- FIG. 17A and FIG. 17B show Effect of 64 Zn-asp on the oxidative metabolism in circulating granulocytes (FIG. 17A) and monocytes (FIG.
- FIG. 18A is a number of expressing cells in the population analyzed;
- FIG. 18B is the level of expression.
- FIG. 19A is the number of expressing cells in the population analyzed;
- FIG. 19B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- FIG. 20 shows effects of 64 Zn-asp on the body weight in rat models of A ⁇ 25-35- induced Alzheimer's disease.
- the data are presented as a percentage ratio of the final body weight of an animal (on the day of autopsy) to its body weight before mimicking Alzheimer's disease, which was taken as 100%.
- FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show immunohistochemical identification of tyrosine hydroxylase activity in the neurons in the hippocampus of intact rats (FIG. 21A), sham-operated rats (placebo) (FIG. 21B), rat models of A ⁇ 25-35-induced Alzheimer's disease injected with H2O (FIG. 21C) and rat models of A ⁇ 25-35-induced Alzheimer's disease treated with 64 Zn-asp (FIG. 21D). TH-positive staining (dark). Oc. 40, ob. 10.
- FIG. 22A before the operation
- FIG. 22B after the operation
- M show time spent by rat models of A ⁇ 25-35-induced Alzheimer's disease for spatial learning in the Barnes maze. Data are presented as an average of 4 trials every 15 min/rat/day and an average within each group/day.
- FIG. 23A is the relevant number of cells performing phagocytosis;
- FIG. 23B is phagocytic activity. *p ⁇ 0.05 versus intact animals
- FIG. 24A is the relevant number of cells performing phagocytosis;
- FIG. 24A is the relevant number of cells performing phagocytosis;
- FIG. 24A is the relevant number of cells performing phagocytosis;
- FIG. 24A is the relevant number of cells performing phagocytosis;
- FIG. 24B is phagocytic activity. *p ⁇ 0.05 versus intact animals; # p ⁇ 0.05 versus control rat models of AD [0032]
- FIG. 26A is the number of expressing cells in the population analyzed;
- FIG. 26B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- FIG. 27A is the number of expressing cells in the population analyzed;
- FIG. 27B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- FIG. 28 shows the effects of 64 Zn-asp on the levels of circulating leukocytes in rat models of A ⁇ 25-35-induced AD, M ⁇ SD. Note: *p ⁇ 0.05 vs. intact animals, # p ⁇ 0.05 vs. untreated animal models of AD
- FIG. 29A is the number of cells performing phagocytosis;
- FIG. 29B is phagocytic activity. Note: *p ⁇ 0.05 vs. intact animals, # p ⁇ 0.05 vs. control animal models of AD
- FIG. 29A is the number of cells performing phagocytosis
- FIG. 29B is phagocytic activity. Note: *p ⁇ 0.05 vs. intact animals, # p ⁇ 0.05 vs. control animal models of AD
- FIG. 30A and FIG. 30B
- FIG. 30A is a relative number of cells performing phagocytosis; FIG. 30B is phagocytic activity.
- FIG. 31A and FIG. 31B show the effects of 64 Zn-asp on the oxidative metabolism in circulating granulocytes (FIG. 31A) and monocytes (FIG. 31B) in rat models of A ⁇ 25-35- induced AD.
- FIG. 32A and FIG. 32B show the effects of 64 Zn-asp on the oxidative metabolism in circulating granulocytes (FIG. 31A) and monocytes (FIG. 31B) in rat models of A ⁇ 25-35- induced AD. Note: *p ⁇ 0.05 vs. intact animals; # ⁇ 0.05 vs. untreated AD animal models [0039]
- FIG. 32A is the number of expressing cells in the population analyzed;
- FIG. 32B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- FIG. 33A is the number of expressing cells in the population analyzed;
- FIG. 33B is the level of expression. * p ⁇ 0.05 versus intact animals; # ⁇ 0.05 versus control AD animal models
- the word “a” or “plurality” before a noun represents one or more of the particular noun.
- Effective amount refers to an amount of an agent or composition that provides a beneficial effect or favorable result to a subject, or alternatively, an amount of an agent or composition that exhibits the desired in vivo or in vitro activity.
- Effective amount refers to an amount of an agent or composition that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the 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.
- an “effective amount,” “prophylactically effective amount,” or “therapeutically effective amount” may be first estimated either in accordance with cell culture assays or using animal models, typically mice, rats, guinea pigs, rabbits, dogs or pigs. An animal model may be used to determine an appropriate concentration range and route of administration. Such information can then be used to determine appropriate doses and routes of administration for humans.
- a conversion table such as that provided in Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005) may be used.
- An effective dose is generally 0.01 mg/kg to 2000 mg/kg of an active agent, preferably 0.05 mg/kg to 500 mg/kg of an active agent.
- An exact effective dose will depend on the severity of the disease, patient's general state of health, age, body weight and sex, nutrition, time and frequency of administration, combination(s) of medicines, response sensitivity and tolerance/response to administration and other factors that will be taken into account by a person skilled in the art when determining the dosage and route of administration for a particular patient based on his/her knowledge of the art. Such dose may be determined by conducting routine experiments and at the physician's discretion. Effective doses will also vary depending on the possibility of their combined use with other therapeutic procedures, such as the use of other agents.
- a “patient” and a “subject” are interchangeable terms and may refer to a human patient/subject, a dog, a cat, a non-human primate, etc.
- AD ALZHEIMER'S DISEASE
- amyloid or “senile” plaques are the main factors in the pathophysiology of AD.
- the amyloid or “senile” plaques predominantly consist of Ab peptides derived from the 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.
- APP can be processed by one of two pathways: the amyloidogenic pathway, leading to the production of Ab, and the non-amyloidogenic pathway. Chow VW et ak, Neuromolecular Medicine 2010;12(1): 1—12. The predominant APP-processing pathway in healthy brain is the nonamyloidogenic pathway, where APP is cleaved by the a-secretase within the Ab region, forming the secreted APPa (sAPPa) fragment and the membrane- bound C-terminal fragment of 83 amino acids (C83). a-secretase activity is attributed to the disintegrin and metalloprotease (ADAM) family of zinc metalloproteases, as they have a long zinc-binding consensus sequence.
- ADAM disintegrin and metalloprotease
- Zinc is the most abundant trace metal in the brain and it has multifactorial functions in Alzheimer's disease (AD). Zinc is critical in the enzymatic nonamyloidogenic processing of the amyloid precursor protein (APP) and in the enzymatic degradation of the amyloid-/? (A/?) peptide. Zinc binds to A/?, promoting its aggregation into neurotoxic species; disruption of zinc homeostasis in the brain results in synaptic and memory deficits. A specific binding site for zinc has been localized in the cysteine-rich region (within the extracellular domain) on the ectodomain of APP. Bush AI et al., The Journal of Biological Chemistry.
- Zinc may have a role in sustaining the adhesiveness of APP during cell-cell and cell-matrix interactions.
- this 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, at a therapeutically effective or a prophylactically effective dose for treating or preventing the disease.
- the composition comprises 64 Zn-enriched zinc (the term “ 64 Zn e ” is used herein to refer to 64 Zn-enriched zinc).
- the solution comprising natural 64 Zn e salt.
- the 64 Zn e salt is a citrate or aspartate.
- the 64 Zn e salt is 64 Zn e aspartate with 2 molecules of aspartic acid.
- the 64 Zn-enriched zinc is in the form of a 64 Zn e compound or a 64 Zn e salt.
- the disclosed compositions contain zinc that is 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 , for example, zinc that is 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 .
- the 64 Zn e is in a form of salt selected from the group consisting of aspartate (chemical formula - C 4 H 5 O 4 N 64 Zn e ) with 2 aspartic acid molecules, sulfate, and citrate. In some embodiments, the 64 Zn e is in a form of 64 Zn e aspartate (chemical formula - C 4 H 5 O 4 N 64 Zn e ) with 2 aspartic acid molecules.
- 64 Zn e is used herein to refer to 64 Zn-enriched zinc. That is, zinc that is enriched for 64 Zn such that 64 Zn is enriched greater than its usual percentage in zinc in nature.
- Zinc in the form of the light isotope 64 Zn e is absorbed in the body much better than naturally-occurring zinc.
- the disclosed compositions contain zinc that is 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 , for example, zinc that is 80% 64 Zn e , 85% 64 Zn e , 90% 64 Zn e , 95% 64 Zn e , 99% 64 Zn e , or 99.9%
- the composition contains between 0.05 mg and 110 mg of 64 Zn e . In some embodiments, the composition contains between 1 and 10 mg of 64 Zn e . In some embodiments, the 64 Zn e compound or a salt thereof is at least 90% 64 Zn e and the composition is an aqueous solution in which 64 Zn e is present at a concentration of between 0.1 mg/ml and 10 mg/ml.
- therapeutic doses for a human subject are between 0.2 and 0.8 mg of Zn-64 per kg of body weight of the human subject.
- the composition or solution is administered by injection. In other embodiments, the composition or solution is administered orally.
- the disclosed composition may be administered to a subject in need thereof by any suitable mode of administration, any suitable frequency, and at any suitable, effective dosage.
- the total amount of zinc administered is the same as the U.S. recommended daily allowance or intake of zinc.
- the total amount of Zn administered is 1/2, twice, three times, five times, or ten times the U.S. recommended daily allowance or intake of zinc.
- the total amount of Zn is between 1/2 and 10 times the U.S. recommended daily allowance or intake of zinc.
- a composition for use in a disclosed method may comprise the prescribed daily amount to be administered once a day or some fraction thereof to be administered a corresponding number of times per day.
- a composition for use in a disclosed method may also comprise an amount of Zn to be administered once every two days, once every three days, once a week, or at any other suitable frequency.
- the composition for use in a disclosed method may be in any suitable form and may be formulated for any suitable means of delivery.
- the composition for use in a disclosed method is provided in a form suitable for oral administration, such as a tablet, pill, lozenge, capsule, liquid suspension, liquid solution, or any other conventional oral dosage form.
- the oral dosage forms may provide immediate release, delayed release, sustained release, or enteric release, and, if appropriate, comprise one or more coating.
- the disclosed composition is provided in a form suitable for injection, such as subcutaneous, intramuscular, intravenous, intraperitoneal, or any other route of injection.
- compositions for injection are provided in sterile and/or non-pyrogenic form and may contain preservatives and/or other suitable excipients, such as sucrose, sodium phosphate dibasic heptahydrate or other suitable buffer, a pH-adjusting agent such as hydrochloric acid or sodium hydroxide, and polysorbate 80 or other suitable detergent.
- suitable excipients such as sucrose, sodium phosphate dibasic heptahydrate or other suitable buffer, a pH-adjusting agent such as hydrochloric acid or sodium hydroxide, and polysorbate 80 or other suitable detergent.
- the composition for use in a disclosed method is provided in a glass or plastic bottle, vial or ampoule, any of which may be suitable for either single or multiple use.
- the bottle, vial or ampoule containing the disclosed composition may be provided in kit form together with one or more needles of suitable gauge and/or one or more syringes, all of which preferably are sterile.
- a kit is provided comprising a liquid solution as described above, which is packaged in a suitable glass or plastic bottle, vial or ampoule and may further comprising one or more needles and/or one or more syringes.
- the kit may further comprise instruction for use.
- the dosage of Zn is proportional to various authoritative daily ingestion guidance (e.g., recommended dietary allowance (USRDA), adequate intake (AI), recommended dietary intake (RDI)) of the corresponding element.
- the Zn dosage is between about 1/2 and about 20 times the guidance amount, more preferably between about 1 and about 10 times the guidance amount, even more preferably between about 1 and about 3 times the guidance amount.
- a single dose of a composition for use in a disclosed method for daily administration would be formulated to comprise a quantity within these ranges, such as about 1/2, about 1, about 3, about 5, about 10, and about 20 times the guidance amount. These amounts generally are for oral intake or topical application.
- the intravenous dosage is lower, such as from about 1/10 to about 1/2 the guidance amount.
- Doses at the low end of these ranges are appropriate for anyone with a heightened sensitivity to a specific element or class of elements (e.g., those with kidney problems).
- the daily guidance amount ranges from 2 mg in infants to 8-11 mg (depending on sex) for ages 9 and up.
- Daily dosages discussed throughout this application may be subdivided into fractional dosages and the fractional dosages administered the appropriate number of times per day to provide the total daily dosage amount (e.g. 1/2 the daily dose administered twice daily, 1/3 the daily dose administered three times daily, etc.).
- composition for use in a disclosed method can be produced by methods employed in accordance with general practice in the pharmaceutical industry, such as, for example, the methods illustrated in Remington: The Science and Practice of Pharmacy (Pharmaceutical Press; 21st revised ed. (2011) (hereinafter “Remington”).
- the composition for use in a disclosed method comprise at least one pharmaceutically acceptable vehicle or excipient.
- vehicle or excipient include, for example, diluents, carriers, excipients, fillers, disintegrants, solubilizing agents, dispersing agents, preservatives, wetting agents, preservatives, stabilizers, buffering agents (e.g. phosphate, citrate, acetate, tartrate), suspending agents, emulsifiers, and penetration enhancing agents such as DMSO, as appropriate.
- the composition can also comprise suitable auxiliary substances, for example, solubilizing agents, dispersing agents, suspending agents and emulsifiers.
- the composition further comprises suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other pharmaceutically acceptable excipients.
- the composition for use in a disclosed method can be administered intragastrically, orally, intravenously, intraperitoneally or intramuscularly, but other routes of administration are also possible.
- Water may be used as a carrier and diluent in the composition.
- the use of other pharmaceutically acceptable solvents and diluents in addition to or instead of water is also acceptable.
- deuterium-depleted water is used as a diluent.
- compositions Large macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polygly colic acids, polymeric amino acids, copolymers of amino acids, can also be used as carrier compounds for the composition.
- Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids, such as water, saline, glycerol or ethanol.
- the said compositions may further comprise excipients, such as wetting agents or emulsifiers, buffering substances, and the like.
- excipients include, among others, diluents and carriers conventional in the art, and/or substances that promote penetration of the active compound into the cell, for example, DMSO, as well as preservatives and stabilizers.
- composition for use in a disclosed method may be presented in various dosage forms depending on the object of application; in particular, it may be formulated as a solution for injections.
- composition for use in a disclosed method may be administered systemically. Suitable routes of administration include, for example, oral or parenteral administration, such as intravenous, intraperitoneal, intragastric as well as via drinking water. However, depending on a dosage form, the disclosed composition may be administered by other routes. [0082] In certain embodiments, the composition for use in a disclosed method comprising Zn is administered intragastrically at a concentration of 2.25 mg/ml.
- the composition for use in a disclosed method is about 2 ml.
- the level of enrichment of 64 Zn e is about 99% or more.
- the 64 Zn e of the 2 ml composition comprises or consists of zinc aspartate (chemical formula - C 4 H 5 O 4 N 64 Zn e ) with 2 aspartic acid molecules.
- the dose of the composition for use in a disclosed method may vary depending on the subject being treated, severity of the disease, the patient's condition and other factors that will be taken into account by a person skilled in the art when determining the dosage and route of administration for a particular patient based on his/her knowledge in the art.
- Light isotopes may be purchased.
- Zn-64 oxide with the necessary degree of enrichment may be purchased from, for example, Oak Ridge National laboratory, Oak Ridge, TN, USA.
- zinc aspartate has a chemical formula - C 4 H 5 O 4 N 64 Zn e , with 2 aspartic acid molecules.
- the structure of zinc aspartate is:
- the composition for use in a disclosed method comprises 6 4 Zn e at about 20% to about 100% of the composition.
- composition can be co-administered with another appropriate agent or therapy.
- 64 Zn-aspartate 64 Zn-asp
- the test substance 64 Zn-aspartate
- the AD phenotype is most often induced by administering a solution containing the human form of the 42 residue amyloid peptide (Ab ⁇ - 42) via the intracerebroventricular route.
- Abi-42 was chosen because of its excellent aggregating properties and because it was thought to constitute the nucleus of any amyloid plaque formation.
- the AD model based on the infusion of beta-amyloid peptide 25-35 is a newer model.
- the Ab (25-35) peptide is the shortest Ab 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 exhibits significant levels of molecular aggregation, retaining the toxicity of the full-sized peptide, although it is lacking in metal binding sites. In line with this finding, it has been proposed that the Ab (25-35) peptide represents a biologically active region of Ab.
- amyloid fibrils apparently have a common structural feature - the presence of a b-folded structure in the center, indicating that amyloid formation is a common property of the polypeptide backbone. Such a process may progress in the body if cellular mechanisms are not able to eliminate protein aggregates.
- Another common feature of amyloid aggregates is that they develop by a nucleation- dependent mechanism and that the initial oligomeric and prefibrillar structures of various proteins are cytotoxic.
- Mature fibrils are considered as an inert material, which can cause physical damage to organs and tissues. Moulias R et al Ann M ⁇ Interne (Paris) 2002;153:441-445; Bucciantini M et al., Nature 2002;416: 507-511.
- autoimmune component is recently described of the pathophysiology of dementia, including AD.
- Clinical studies have convincingly demonstrated that autoantibodies to various molecules are associated with the development and progression of AD.
- antibodies to Ab and Tau protein, multiple transmitter and receptor molecules such as GFAP, lipids (ceramides, oxidized low-density lipoproteins), vascular markers, such as RAGE (Receptor for Advanced Gly cation End Products, a receptor involved in the pathogenesis of almost all neurodegenerative diseases), cellular enzymes such as aldolase and many other autoantigens are found in the serum of AD patients.
- mice Male Wistar rats (300-500 g) were used. The animals were maintained under standard conditions in the vivarium of the ESC “Institute of Biology and Medicine” of Taras Shevchenko National University of Kyiv. They were given ad libitum access to food and water.
- Alzheimer's Disease was induced in old male rats (14 months old) by giving them intrahippocampal injections of aggregated Amyloid Beta (Ab 1-40) Peptide (human) (Cayman Chemical Company) and Amyloid Beta (Ab 25-35) Peptide (human) (Tocris Bioscience, Brostol).
- Ab1-40 and A ⁇ 25-35 were dissolved in bidistilled water to the concentration of 15 pmol/L and incubated at 37°C for 24 hours for aggregation.
- Ab conglomerates were broken up by ultrasound and sterilized immediately prior to injection.
- Rats were anesthetized using a mixture of ketamine (75 mg/kg, diluted in sterile water for injection, Sigma, USA) and 2% xylazine (100 m ⁇ /rat, Alfasan International B.V., Netherlands) administered intraperitoneally in a total volume of 1 ml.
- a rat was placed in a stereotaxic apparatus (SEZH-4) modified for rats. The animal was then scalped from the point of intersection of the sagittal suture with bregma (zero point): 2 mm distally, 2 mm laterally and 3.5 mm in depth and a trephine opening was made with an injection needle, directly into the hippocampus.
- Dissolved Ab1-40 or A ⁇ 25-35 was collected into a homemade microinjector and its tip was dropped into the trephine opening.
- IV (n 7) - rats in which AD was induced by the infusion of Ab1-40 and which were then administered daily a solution of 64 Zn-asp in a volume of 1.5 mg/kg, i.v., for 10 days (from the 18 th to 27 th day of the experiment inclusively);
- V (n 7) - rats in which AD was induced by the of A ⁇ 25-35 and which were then daily administered 0.1 ml of deuterium-depleted water (i.v.,) for 10 days (from the 18 th to 27 th day of the experiment inclusively);
- TH tyrosine hydroxylase
- Immunohistochemical staining was performed using primary anti-TH antibodies at a 1:200 dilution (Millipore, AB152). Endogenous peroxidase activity was blocked with a blocking reagent (Dako, EnVision Flex, DM821). Nonspecific antibody binding was blocked using 4% dry milk dissolved in Tris-buffered saline (TBS) containing 0.2% Triton X-100.
- TBS Tris-buffered saline
- the primary antibody was diluted in TBS containing 0.2% Triton X-100 and applied to tissue sections. The sections were then incubated overnight (+4°C). Secondary antibodies (anti-rabbit biotinylated antibodies, 1:200) were incubated for 60 minutes. The immunoreaction was developed using diaminobenzidine (Dako, EnVision) applied for 5 minutes. The results of immunohistochemical staining were evaluated at the light-optical level using a Zeiss Primo Star microscope.
- the Bames maze (FIG. 2) is a tool used to measure spatial learning and memory of a rodent and helps to identify cognitive deficits in rodents that model for disease such as Alzheimer's disease. Kinga Gawel et al., Assessment of spatial learning and memory in the Bames maze task in rodents — methodological consideration, Naunyn Schmiedebergs Arch Pharmacol. 2019; 392(1): 1-18. doi: 10.1007/s00210-018-1589-y. The test was first developed by Dr.
- Bames maze used consisted of a circular table with 16 circular holes around its circumference. Peripheral visual cues, such as black marks (a triangle on one wall and two parallel strips on the opposite wall), were placed for beher orientation of the animal. Under one of the holes an "escape box” containing standard filler for animals was fixed. Each animal was assigned its number of the hole to which the “escape box” was fixed. The other holes remained open. Before performing the probe phase (after the operation), the number of the hole was changed.
- Hematologic study [00132] The blood count values were analyzed at the completion of the experiment (day 37). The absolute number of leukocytes, as well as the absolute and relative numbers of lymphocytes, monocytes and neutrophilic granulocytes were calculated.
- Oxidative metabolism of phagocytes of various localization was analyzed by flow cytometry using the cell-permeant 2'7'-dichlorodihydrofluorescein-diacetate (DHP) (carboxy-IBDCFDA, Invitrogen, USA) which is converted by intracellular esterases to the nonfluorescent membrane-impermeable carboxy-tUDCF form.
- DHP cell-permeant 2'7'-dichlorodihydrofluorescein-diacetate
- Differential assessment of oxidative metabolism values of circulating mono- and polymorphonuclear phagocytes was carried out using a gating method. To assess a metabolic reserve the cells were treated with LPS (Sigma, USA).
- phenotypic profile of phagocytes of various localization was characterized by the expression of markers of functional maturity and metabolic polarization (CD206 and CD86), which was determined by flow cytometry and the use of monoclonal antibodies of appropriate specificity marked with fluorescent dyes (Abeam, Becton Dickinson).
- Animal body weight is a classic clinical sign that characterizes the general condition of an animal, and its loss during the experiment indicates that the condition of the animal is deteriorating. Since this experiment involved old animals with an initial body weight of 350-500 g, changes in their body weights during the experiment were insignificant in comparison with young animals (120-200 g) for the same period of time. Despite this fact, a significant loss in the body weight of animal models of AbI-40-induced AD within 1 month of the experiment was observed. The initial animal body weight in the group of sham- operated animals (prior to the operation) was 445.0 ⁇ 41.1 g, and at the end of the experiment, on the day of autopsy, it was 449.5 ⁇ 37.4 g, i.e.
- the weight gain was 1.3 ⁇ 4.0%.
- the body weight of rat models of AbI-40-induced AD before the operation was 361.1 ⁇ 25.3 g, and on the day of autopsy it was 340.3 ⁇ 33.5 g, which indicates a weight loss of 4.3 ⁇ 3.7% (P ⁇ 0.01 compared with sham-operated animals) (FIG. 3).
- Table 2 Staining intensity and percentage of TH-positive hippocampal cells in rat models of AbI-40-induced Alzheimer's disease
- results obtained indicate a protective role of the test substance in relation to the functions of dopaminergic neurons in the hippocampus.
- Alzheimer's disease which is most common in people of old age, is associated with impairment of declarative memory, memory of events.
- Declarative memory in humans has parallels with the spatial memory in rodents (and this is why rodents provide a good model of declarative memory).
- Neurons responsible for declarative memory have representation in the hippocampus and are associated with a specific neuronal process known as long-term potentiation. In rodents, the hippocampus is involved in coding of spatial information which is studied in various mazes. The Barnes maze is used.
- Presence of soluble Ab and tau protein in the hippocampus is an unmistakable sign of AD development in animal models of AD and a marker for assessing the severity of disease and the effectiveness of pathogenetic methods of treatment.
- the level of soluble Ab in the hippocampal homogenates in rat models of AD induced by Ab1-40 infusion was almost 4 times higher than that in intact animals (FIG. 8).
- the level of soluble Ab was also slightly higher than in intact rats.
- the level of Ab in the hippocampus of AD rat models treated with the test substance was 1.5 times lower than in control AD rat models but did not reach the level in intact/sham-operated animals.
- this parameter showed exceptional variability, which made it impossible to adequately assess the evidence of the obtained data.
- Such high degree of variability could be due to a small statistical sampling of animals, a method of sample preparations for the test systems used in the study that differed from a similar procedure described in the literature for testing this parameter using the ELISA kits of 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.
- AD shows that the test substance has a pathogenetic therapeutic effect resulting in a decrease in the concentration of plaque-forming components in AD animal models.
- Phagocytic activity of microglia is an indicant of their activated state, any changes in which should be viewed in the context of changes in other functional and phenotypic characteristics. Increased phagocytic activity of microglial cells may accompany both pro- and anti-inflammatory microglial activation. In addition, with increased permeability of the blood brain barrier (BBB), the population of resident microglial cells is replenished by peripheral blood phagocytes, the differential assessment of which was not possible under the conditions of this study. According to the results, the relative number of phagocytic microglia (phagocytic index, (PI)) in animal models of AD was 2 times higher than in intact animals.
- phagocytic index, (PI) phagocytic index
- Oxidative metabolism is another metabolic indicator of microglia.
- microglia in intact animals were characterized by the absence of reaction to in vitro stimulation by bacterial LPS, which indicates their involvement in the inflammaging associated with aging (FIG. 11).
- Norden DM Godbout JP. Review: microglia of the aged brain: primed to be activated and resistant to regulation.
- Oxidative metabolism in sham-operated animals was somewhat enhanced compared with intact animals at the time of the experiment, which supports the assumption about a persistent reparative inflammatory process aggravated by inflammaging.
- An additional criterion for this condition is the lack of a functional reserve of oxidative metabolism in microglia in animals of this group in response to in vitro LPS treatment.
- AD progression was accompanied by a significant (5-fold) increase in the generation of reactive oxygen species by microglial cells.
- Enhanced oxidative metabolism in microglia is an essential component of neuroinflammation associated with AD, therefore, the data support the validity of the selected model.
- CD206 scavenger receptor, a marker of alternative polarization of phagocytes of extra-cerebral localization and also a marker of activated resident microglia
- CD86 a costimulatory molecule involved in the process of antigen presentation, a marker of pro- inflammatory activation of phagocytes of extra-cerebral localization and which is also overexpressed by myeloid-derived suppressor cells, negative regulators of proinflammatory reactions of innate and adaptive immunity.
- the number of CD86+ cells in the microglia population of animal models of AD was 1.6 times higher compared with intact animals (FIG. 12A and FIG. 12B).
- the expression level of this marker in microglial cells was more than 2.5 times higher than in intact animals, which evidences a pro-inflammatory shift in the microglial functions characteristic of AD and confirms the results of assessment of the 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, which indicates a powerful antiinflammatory effect of the drug candidate.
- CD86 expression are also supported by the data on expression of another phenotypic marker, CD206 (FIG. 13A and FIG. 13B).
- the number of microglial cells expressing CD206 in AD rat models was 3.5 times higher than that of intact animals, which indicates phagocytic activation in the brains of AD rats.
- the expression level of this marker in positive cells in AD rats was more than 5 times higher than that of intact rats.
- Treatment with the zinc-based test substance caused a decrease in the above values to the levels of intact animals, which is another evidence of antiinflammatory effect of the test substance.
- Literature data provide strong evidence that chronic inflammation is one of the most important pathophysiological components of synucleinopathies and taupathias, including AD.
- Leukograms of patients with AD reveal an increased number of monocytes and neutrophils and a low lymphocyte count. Escalated levels of monocytes and neutrophils are hallmarks of chronic inflammation and may be both precursor to AD and its consequence.
- a low number of lymphocytes specifies that the body's resistance to the fight infection is significantly reduced.
- BBB blood brain barrier
- Neutrophilia detected in the blood samples from AD rats was accompanied by a significant increase in the phagocytic activity, which is, on the one hand, a marker of an active state of the cells, and on the other hand, a sign of the anti-inflammatory shift in their metabolism (FIG. 15B).
- the relative number of phagocytic neutrophils in animals of this group did not differ significantly from that in intact and SO rats (FIG. 15A).
- the absorption activity of polymorphonuclear phagocytes in SO rats was significantly higher than in the intact controls, which may be a consequence of surgery and the associated activation of repair processes, characterized by an anti-inflammatory shift in phagocytic cell metabolism.
- FIG. 18A and FIG. 18B are identical to FIG. 18A and FIG. 18B.
- this marker is characteristic of both phagocytes with a pro-inflammatory metabolic shift and myeloid suppressor cells. Taking into account the increased phagocytic activity of peripheral blood phagocytes, it can be assumed that an increase in the fraction of CD86+ cells was due to the presence of myeloid suppressor cells. [00195] An increased fraction of CD86+ cells with an increased level of expression of this marker was found in sham-operated (SO) animals, which complements the picture of a reparative process induced by the surgical procedure.
- SO sham-operated
- test substance as monotherapy brought the number of cells expressing these markers and their expression levels to normal, which confirms its homeostatic effect on systemic immune reactivity in the progression of AD induced by Ab ⁇ - 40 infusion.
- a decrease in the body weight and a decrease in water and feed intake in rat models of AbI-40-induced AD was observed 3 weeks after mimicking the disease. These parameters were restored in AD rats treated with 64 Zn-asp for 10 days.
- a decreased number of hippocampal dopaminergic neurons and decreased expression of tyrosine hydroxylase (TH) in hippocampal dopaminergic neurons were recorded in rat models of AbI-40-induced AD.
- Administration of 64 Zn-asp to Ab1-40 AD rats increased the staining intensity of TH-immuno-positive cells rather than their number.
- Progression of AbI-40-induced AD was associated with impairment of cognitive flexibility in AD rats, which indicates impaired function of the frontal cortex. Rat models of AbI-40-induced Alzheimer's disease did not exhibit any changes in their ability to spatial learning or short-term/long-term memories (hippocampal function).
- Administration of 64 Zn-asp significantly improved the cognitive function in AD models and virtually returned it to the values in intact and sham-operated animals.
- Table 5 Staining intensity and percentage of TH-positive hippocampal cells in rat models of A ⁇ 25-35-induced Alzheimer's disease
- the level of cognitive flexibility was also assessed by measuring time the animal spent near the entrance to the escape hole - the less time the animal was there, the higher level of cognitive flexibility it possessed (the frontal cortex function), i.e., the animal realized faster that the escape hole should be sought elsewhere.
- test substance has a pathogenetic therapeutic effect accompanied by a decrease in the quantitative characteristics of the pathogenetic markers of Alzheimer's disease.
- the model of AD induced by A ⁇ 25-35 infusion was chosen because of an exceptional role of Ab fragment in the formation of senile plaques in AD, its ability to have a direct toxic effect on neurons leading to their death regardless of the formation of Ab deposits, as well as the fact of development of autoimmune reactions directed against this peptide and accompanying the progression of AD.
- Analysis of the functional and phenotypic characteristics of microglia in rat models of AD induced by A ⁇ 25- 35 infusion showed the following.
- the number of microglial cells performing phagocytosis in control AD rats was more than 2 times as high as in the intact and SO animals, which indicates an activated state of a complex population of phagocytes in the brain (FIG. 24A, and FIG. 24B).
- the microglia population includes, in addition to resident macrophages, recruited mononuclear and polymorphononuclear phagocytes, differential assessment of which was not provided by the terms and conditions of the study.
- the data on phagocytic activity can be interpreted as signs of stimulation of reparative processes by the test substance, since an increase in absorption activity of microglia is characteristic of extracerebral phagocytes (the proportion of which in the complex microglia population may be quite significant) of the anti-inflammatory phenotype.
- the levels of ROS generation in rat models of A ⁇ 25-35 AD did not differ from those in intact animals and were significantly lower than in sham-operated rats.
- Assessment of the activated state of microglia in sham-operated rats as a marker of a persistent reparative process caused by surgical intervention is validated by the analysis of physiological state of animals of this group, which was absolutely satisfactory, without deviations in their cognitive activity or behavioral reactions. Consequently, increased levels of ROS generation in SO animals may be considered as an indicant of the reparative inflammatory process.
- the absence of differences between the oxidative metabolism in microglia in the rat models of A ⁇ 25-35-induced AD and intact animals may indicate a spontaneous resolution of neuroinflammation and the imperfection of the AD model used in the study.
- Administration of the test substance to rat models of A ⁇ 25-35-induced AD increased oxidative metabolism in their microglia, which may be evidence of stimulation of the repair processes by the drug candidate.
- microglial cells The expression levels of phenotypic markers of microglial cells are generally consistent with their metabolic profile (FIG. 26A and FIG. 26B), however, they contain an element requiring a more detailed study of microglia in this AD model.
- CD86+ cells in the microglia population in AD animals was significantly higher than in intact animals. If we consider CD86+ cells as a marker of myeloid suppressor cells, then the data obtained are consistent with the concept of spontaneous regression of neuroinflammation in AD rat models, which indicates the imperfection of the model. However, if one attributes an increase in the fraction of CD86+ cells to the elevated number of effector phagocytes activated in antigen presentation, then the results of the analysis of phenotypic markers indicate activation of intracerebral autoimmune reactions initiated by the infusion of A ⁇ 25-35, which is consistent with literature data on the participation of A ⁇ 25-35 in the autoimmune component of AD. In this case, sharp reduction in the fraction of CD86+ cells in AD animals after a course of therapy with the zinc-based preparation can be considered as evidence of the ability of the test substance to inhibit the development of autoimmune reactions associated with AD.
- the number of circulating leukocytes in AD rat models was twice as high as in intact animals, lymphocytes and neutrophilic granulocytes also doubled in number, and the number of monocytes increased almost 4-fold compared with intact animals.
- the neutrophil-lymphocyte ratio (NLR) in AD rats was significantly lower than in intact animals.
- Such an increase in the number of lymphocytes can be evidence of the activation of self-reactive T-cell immune responses (autoimmunity), which is consistent with the proposed interpretation of the results of assessment of the functional and phenotypic profile of microglia.
- a therapeutic course with the test substance caused a slight decrease in leukocytosis but did not bring the leukocyte counts back to normal. This decrease was mainly due to normalization of the number of monocytes.
- the levels of neutrophils and lymphocytes after the treatment were unchanged and the NLR remained as high as in untreated AD rats.
- FIG. 30A and FIG. 30B are identical to FIG. 30A and FIG. 30B.
- the fraction of positive cells was increased in A ⁇ 25-35 injected rats.
- the levels of expression of this marker by blood phagocytes in AD animal models did not differ from those in the intact animals. Treatment with the test substance resulted in a sharp decrease in the fraction of cells positive for this marker but significantly increased its expression.
- Rat models of A ⁇ 25-35-induced AD showed no significant changes in any of the selected and analyzed markers of disease progression (animal body weight, number of TH-positive neurons in the hippocampus and level of TH expression, spatial learning, shortterm and long-term memories, cognitive flexibility). There was only a tendency for impairment of long-term spatial memory. 64 Zn-asp administered as monotherapy did not have any statistically significant effects on the cognitive symptoms in A ⁇ 25-35 injected animals.
- the model of AD induced by the infusion of A ⁇ 25-35 was not characterized by a classical picture of neuroinflammation and, therefore, the protocol used in the current study does not reflect the clinical picture of Alzheimer's disease. Only the fact of possible presence of local autoimmune processes accompanying this model is noteworthy.
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