Protein aggregation inhibitor

Abstract

To provide technology that can be used to inhibit aggregation of β-amyloid protein (Aβ) and microtubule-binding protein Tau, which bring about senile plaque (senile plaque: AP) and neurofibrillary tangle (Neurofibrillary Tangle: NFT).SOLUTION: The present invention provides a protein aggregation inhibitor containing mesostructured particles of calcium hydrogen carbonate as an active ingredient.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a protein aggregation inhibitor, and more particularly to an aggregation inhibitor for proteins causing neurodegenerative diseases, such as beta-amyloid protein and tau, which contains mesostructured particles of calcium bicarbonate as an active ingredient. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes dementia. Characteristic neuropathological changes called senile plaques (APs) and neurofibrillary tangles (NFTs) are observed in the brains of AD patients. AP is an extracellular deposition lesion of beta-amyloid protein (Aβ), which consists of approximately 40 amino acid residues in total. As the AD pathology progresses, the lesions spread from the frontal and temporal cortex to the entire cerebral neocortex. NFTs are intracellular lesions in which the microtubule-associated protein Tau aggregates and accumulates in the form of fibrils within nerve cells. In the cerebral cortex, lesion formation begins in the entorhinal cortex and hippocampus, and if the lesion spreads to the cerebral neocortex, it can lead to the onset of dementia.

[0003] Aβ is produced by two-step cleavage of the amyloid precursor protein (APP), which is primarily expressed in neurons, by β- and γ-secretases. There are multiple molecular species ranging in length from 38 to 43 amino acid residues depending on the cleavage site of γ-secretase. The most prevalent molecular species in the brain is Aβ40, which contains 40 amino acid residues. On the other hand, Aβ42, which contains two more amino acid residues, is highly aggregatable and highly neurotoxic. Three causative genes for familial AD have been identified to date: APP, presenilin-1 (PS1), and presenilin-2 (PS2). Because PS1 and PS2 are both active sites of the protein complex that constitutes γ-secretase, all causative genes for familial AD are involved in Aβ production. For this reason, the amyloid hypothesis, which holds that Aβ is the causative molecule for AD, has remained supported since the 1990s.

[0004] Tau is a microtubule-binding protein that is primarily localized in the axons of neurons and plays a role in stabilizing microtubule structures. Tau exists as either a 3-repeat (3R) Tau with three microtubule-binding domains or a 4-repeat (4R) Tau with four, due to differences in splicing of exon 10. Furthermore, splicing variants occur in exons 2 and 3, resulting in a total of six isoforms expressed in humans. NFTs are closely correlated with neuronal cell death and the onset of dementia, suggesting their potential as a fundamental pathology in neurodegeneration.

[0005] Currently, lecanemab, developed by Eisai and Biogen (USA), is approved as a treatment for dementia. Lecanemab is a humanized IgG1 monoclonal antibody that targets soluble and insoluble Aβ aggregates.

[0006] CAC-717 is a suspension of mesostructured calcium bicarbonate particles (50-500 nm) produced by applying ultrasonic vibrations to water containing plant-derived calcium bicarbonate (Ca(HCO3)2), applying a high DC voltage, and irradiating it with infrared light (see Patent Documents 1 and 2, and Non-Patent Document 1). Calcium particles with mesostructures are formed by splitting H from water molecules. + Attract Oh - It is strongly alkaline (pH 12.6) because it releases CAC-717 into solution, but becomes nearly neutral upon contact with human or animal tissue, and it has been confirmed that it does not irritate mucous membranes or skin. Non-Patent Document 1 reports that CAC-717 significantly reduces the infectivity of influenza viruses. Furthermore, Non-Patent Document 2 reports that CAC-717 inactivates abnormal prion protein (PrPSc), an infectious pathogen.

[0007] PrPSc is a pathogen that causes transmissible spongiform encephalopathy (TSE), which can be transmitted across species. PrPSc aggregation is induced by the structural transformation of normal prion protein with an α-helical structure into abnormal prion protein with a β-sheet structure. On the other hand, Aβ and tau are not infectious. Aβ aggregation is initiated by the cleavage of APP by secretases, which generates highly aggregative hydrophobic peptides. Tau aggregation is also associated with excessive phosphorylation of tau. Therefore, PrPSc, Aβ, and tau have different molecular structures and their production and aggregation processes are completely different. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5778328 [Patent Document 2] Patent No. 5864010 [Non-patent literature]

[0009] [Non-Patent Document 1] "Evaluation of calcium hydrogen carbonate mesoscopic crystals as a disinfectant for influenza A viruses", Nakashima et. al., J Vet Med Sci, 2017, 79, 939-942. [Non-patent document 2] "Inactivation of Scrapie Prions by the Electrically Charged Disinfectant CAC-717", Sakudo et. al., Pathogens, 2020, 9, 536 Summary of the Invention [Problem to be solved by the invention]

[0010] Suppression of the formation of senile plaques (APs) and neurofibrillary tangles (NFTs) may be a promising target for the treatment and / or prevention of Alzheimer's disease (AD). Therefore, the main objective of the present disclosure is to provide a technology that can be used to inhibit the aggregation of amyloid beta protein (Aβ) and the microtubule-associated protein Tau, which cause APs and NFTs. [Means for solving the problem]

[0011] To solve the above problems, the present disclosure provides the following [1]-

[27] . [1] A protein aggregation inhibitor containing mesostructured particles of calcium bicarbonate as the active ingredient. [2] The protein aggregation inhibitor according to [1], wherein the mesostructured particles of calcium bicarbonate have an activity of inhibiting aggregation of the protein and / or an activity of dissociating the aggregated protein. [3] The protein aggregation inhibitor according to [1] or [2], wherein the protein exhibits neurodegenerative disease-causing properties through aggregation. [4] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal locomotion dementia (FTD), frontotemporal locomotion dementia (FTD ... The protein aggregation inhibitor according to [3], wherein the protein aggregation inhibitor is one or more selected from the group consisting of familial amyloidosis-related dementia (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA). [5] The protein aggregation inhibitor according to any one of [1] to [4], wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[0012] [6] A pharmaceutical for treating and / or preventing neurodegenerative diseases caused by protein aggregation, comprising mesostructured particles of calcium bicarbonate as an active ingredient. [7] The pharmaceutical according to [6], wherein the mesostructured particles of calcium bicarbonate have an activity of inhibiting aggregation of the protein and / or an activity of dissociating the aggregated protein. [8] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD ... The pharmaceutical according to [6] or [7], wherein the disease is one or more selected from the group consisting of: Loeber's degeneration-related polyneuropathy (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, Senecan amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA). [9] The pharmaceutical according to any one of [6] to [8], wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[0013]

[10] Use of calcium bicarbonate mesostructured particles to inhibit protein aggregation.

[11] The use according to

[10] , wherein the mesostructured particles of calcium bicarbonate have the activity of inhibiting aggregation of the protein and / or the activity of dissociating the aggregated protein.

[12] The use according to

[10] or

[11] , wherein the protein expresses neurodegenerative disease-causing properties through aggregation.

[13] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD ... The use according to

[12] , wherein the condition is one or more selected from the group consisting of familial amyloidosis-related polyneuropathy (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, Seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA).

[14] The use according to any one of

[10] to

[13] , wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[0014]

[15] Use of mesostructured particles of calcium bicarbonate for the treatment and / or prevention of neurodegenerative diseases caused by protein aggregation.

[16] The use according to

[15] , wherein the mesostructured particles of calcium bicarbonate have an activity of inhibiting aggregation of the protein and / or an activity of dissociating the aggregated protein.

[17] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal locomotive syndrome (FSS), frontotemporal rheumatoid arthritis (FTD ... The use according to

[15] or

[16] , wherein the condition is one or more selected from the group consisting of familial amyloidosis (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA).

[18] The use according to any one of

[15] to

[17] , wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[0015]

[19] Use of mesostructured particles of calcium bicarbonate for the manufacture of a medicament for the treatment and / or prevention of a neurodegenerative disease caused by protein aggregation.

[20] The use according to

[19] , wherein the mesostructured particles of calcium bicarbonate have the activity of inhibiting aggregation of the protein and / or the activity of dissociating the aggregated protein. [twenty one] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal locomotive syndrome (FSS), frontotemporal rheumatoid arthritis (FTD ... The use according to

[19] or

[20] , wherein the condition is one or more selected from the group consisting of familial amyloidosis (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA). [twenty two] The use according to any one of

[19] to

[21] , wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[0016] [twenty three] A method for treating and / or preventing a neurodegenerative disease caused by protein aggregation in a subject, comprising the step of administering to the subject a therapeutically effective amount of mesostructured particles of calcium bicarbonate. [twenty four] The method according to

[23] , wherein the mesostructured particles of calcium bicarbonate have an activity of inhibiting aggregation of the protein and / or an activity of dissociating the aggregated protein. [twenty five] The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal locomotive syndrome (FSS), frontotemporal rheumatoid arthritis (FTD ... The method according to

[23] or

[24] , wherein the amyloidosis is one or more selected from the group consisting of familial amyloidosis-related degeneration (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA).

[26] The method according to any one of

[23] to

[25] , wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

[27] The method according to any one of

[23] to

[26] , wherein the subject is a human or non-human mammal.

[0017] [Definition] In the present disclosure, protein "aggregation" refers to the process by which two or more protein molecules come together to form larger molecular weight clusters. Aggregation can be a physical or chemical process. A protein "aggregate" refers to a cluster formed by the aggregation of two or more single protein molecules (monomers). In the present disclosure, aggregates of 2-30 protein molecules may be referred to as "oligomers," and aggregates of more than 30 protein molecules may be simply referred to as "aggregates." "Dissociation" of aggregated proteins refers to the process by which protein aggregates are separated into two or more smaller aggregates or monomers. Dissociated aggregates can be smaller aggregates (e.g., oligomers), monomers, or a mixture of these. [Effects of the Invention]

[0018] The present disclosure provides techniques that can be used to inhibit the aggregation of amyloid beta protein (Aβ) and the microtubule-associated protein Tau, which lead to AP and NFT. [Brief explanation of the drawings]

[0019] [Figure 1] The schedule for the cell culture experiment is shown below. [Figure 2] Western blot analysis was performed to examine the dissociation effect of CAC-717 on recombinant human Aβ and recombinant human tau aggregates. 5 μL of Aβ (50 μM) and tau (1 μg / μL) aggregates were treated with 5 μL, 2.5 μL (2-fold dilution), or 1.25 μL (4-fold dilution) of CAC-717. Three different CAC-717 lots, Lot #A, Lot #B, and Lot #C, were used. CAC-717 significantly dissociated Aβ and tau aggregates in a concentration-dependent manner. [Figure 3] N2a cells were seeded in a 6-well plate, and 2000 μL of medium was treated with CAC-717 at 50 μL (25 μL / mL), 100 μL (50 μL / mL), or 200 μL (100 μL / mL) per 2000 μL of medium. Twenty-four hours after the addition of CAC-717, Triton-soluble and Triton-insoluble fractions were extracted from the N2a cells and analyzed by Western blot (A). Soluble and insoluble tau were significantly reduced in cells treated with CAC-717 at 50 μL / mL and 100 μL / mL. (B) shows the relative amount of tau protein, with the amount in the control (CT) group set at 1 (*p<0.05). [Figure 4]Triton-soluble and -insoluble fractions were extracted from N2a cells treated with CAC-717 and lysosomal inhibitor (PEL) for 24 hours and analyzed by Western blot. CAC-717 significantly reduced soluble and insoluble tau, even in the presence of lysosomal inhibitor. (B) The amount of tau protein is shown relative to the amount in the control group (CT) (*p<0.05). [Figure 5] In N2a cells expressing P301L-Tau (CT), Tau accumulated widely throughout the cytoplasm. In N2a cells treated with CAC-717 (CAC), Tau staining was significantly reduced. In N2a cells treated with a lysosomal inhibitor simultaneously with CAC-717 (CAC+PEL), significant accumulation of enlarged autophagosomes due to impaired lysosomal protein metabolic function was observed, but Tau staining was reduced to the same extent as in CAC. [Figure 6] The Aβ fluorescence intensity of samples in which Aβ aggregates were treated with CAC-717 was measured. Aβ aggregates were dissociated by CAC-717. [Figure 7] Aβ aggregates were treated with CAC-717 for 5, 15, and 30 minutes, and the amounts of oligomers and monomers formed in the samples were measured. CAC-717 dissociated Aβ aggregates, resulting in an increase in monomers and oligomers. [Figure 8] Recombinant human Aβ1-42 was treated with CAC-717 and then aggregated, and the fluorescence intensity of the aggregated sample was measured. CAC-717 inhibited Aβ aggregation. [Figure 9] The amounts of oligomers and monomers formed in aggregated samples after treatment with CAC-717 were measured. CAC-717 inhibited Aβ aggregation and dissociated Aβ aggregates, resulting in an increase in oligomers and monomers. [Figure 10]The results of evaluating the neurodegeneration-suppressing effect of CAC-717 in Drosophila expressing mutant Tau are shown below. The decrease in R cell number and abnormalities in R cell arrangement were suppressed in individuals raised on a diet containing CAC-717 (DF) compared to individuals raised on a diet without CAC-717 (AC). [Figure 11] The results of evaluating the neurodegeneration-suppressing effect of CAC-717 in Drosophila expressing mutant Tau are shown below. The decrease in R cell number was suppressed in individuals raised on a diet containing CAC-717 compared to individuals raised on a diet without CAC-717. [Figure 12] The results of evaluating the inhibitory effect of CAC-717 on tau aggregation in Drosophila expressing atypical tau are shown below. Aggregated tau was reduced in individuals raised on a diet containing CAC-717 compared to individuals raised on a diet without CAC-717. DETAILED DESCRIPTION OF THE INVENTION

[0020] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the present disclosure.

[0021] 1. Protein aggregation inhibitors The protein aggregation inhibitor according to the present disclosure contains mesostructured particles of calcium bicarbonate as an active ingredient. It has been found that the mesostructured particles of calcium bicarbonate have the activity of inhibiting protein aggregation in vivo and in vitro and / or the activity of dissociating aggregated proteins.

[0022] Mesostructured particles of calcium bicarbonate can be produced by applying ultrasonic vibrations to water containing plant-derived calcium bicarbonate (Ca(HCO3)2), applying high DC voltage, and irradiating it with infrared light. Mesostructured particles of calcium hydrogen carbonate can be produced according to the methods described in Patent Documents 1 and 2. Specifically, for example, mesostructured particles (50-500 nm) can be obtained by applying ultrasonic vibrations (50 kHz, amplitude 1.5 / 1000 mm) to water containing plant-derived calcium bicarbonate while applying a high DC voltage (8300 V, 100 mA) and then irradiating it with far-infrared rays with a wavelength of 6-14 μm. Furthermore, mesostructured particles of calcium bicarbonate may be commercially available, for example, "Terra Protect CAC-717" (also simply referred to as "CAC-717") manufactured by Santa Minerals.

[0023] The target protein is not particularly limited as long as it has the property of aggregating in vivo or in vitro, but is preferably a protein that manifests pathogenesis upon aggregation, and more preferably a protein that manifests neurodegenerative disease pathogenicity upon aggregation in vivo.

[0024] Target proteins include, for example, amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid proteins, and transthyretin. The protein of interest is preferably Aβ or tau.

[0025] The protein aggregation inhibitor according to the present disclosure exhibits the activity of inhibiting protein aggregation in vitro (cell-free systems and cellular systems) and in vivo (in living organisms), and / or the activity of dissociating aggregated proteins. Therefore, the protein aggregation inhibitor according to the present disclosure can be an active ingredient of the pharmaceutical according to the present disclosure described below, and can also be incorporated into disinfectants for surgical instruments, for example. For example, it is expected to be used to dissociate Aβ, tau, and the like that adhere to instruments used in brain surgery.

[0026] 2. Drugs for the treatment and / or prevention of neurodegenerative diseases The pharmaceutical for treating and / or preventing diseases caused by protein aggregation according to the present disclosure contains the above-mentioned mesostructured calcium bicarbonate particles as an active ingredient. The mesostructured calcium bicarbonate particles have the activity of inhibiting protein aggregation in vivo and in vitro and / or the activity of dissociating aggregated proteins, and therefore may be effective in treating and / or preventing diseases caused by such protein aggregation. The therapeutic effect of the medicament according to the present disclosure includes alleviating or improving symptoms or delaying the progression of symptoms by suppressing further protein aggregation or dissociating aggregated proteins in patients who have developed a disease. The preventive effect of a pharmaceutical also includes preventing or delaying the onset of symptoms by inhibiting protein aggregation or dissociating aggregated proteins in patients who have not yet developed the disease.

[0027] The target diseases can broadly include diseases caused by the above-mentioned proteins that exhibit pathogenicity through aggregation. Examples of the target diseases include the following:

[0028] Diseases involving Aβ aggregation include Alzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy.

[0029] Diseases involving tau aggregation include Alzheimer's disease, Down's syndrome, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, and Creutzfeldt-Jakob disease.

[0030] Diseases involving alpha-synuclein aggregation include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy (MSA).

[0031] Diseases involving aggregation of the Huntington protein include Huntington's disease.

[0032] Diseases involving aggregation of SOD1, TDP-43, or FUS include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and frontotemporal Lober's degeneration (FTLD-TDP).

[0033] Diseases involving aggregation of amyloid proteins include Alzheimer's disease, light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, and Seneca amyloidosis.

[0034] Diseases involving transthyretin aggregation include transthyretin amyloidosis, FAP, familial amyloidotic cardiomyopathy (FAC), and senile systemic amyloidosis (SSA).

[0035] The target disease is preferably a neurogenic disease, and particularly preferably Alzheimer's disease.

[0036] The medicament according to the present disclosure may contain a therapeutically effective amount of mesostructured particles of calcium bicarbonate together with a pharmaceutically acceptable carrier. The carrier may be a solid such as an excipient or a liquid such as a diluent. Specific examples include magnesium stearate, lactose, starch, gelatin, agar, talc, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol, and distilled water.

[0037] The dosage of the pharmaceutical according to the present disclosure can be determined appropriately taking into consideration the severity of symptoms, sex, age, etc. of the subject to be administered. For example, the amount of mesostructured calcium bicarbonate particles can be 0.0001 to 1000 mg per adult per day. This daily dosage may be administered once a day, but is preferably administered in divided doses several times a day.

[0038] The animals to be administered may be humans or non-human mammals such as pigs, cows, horses, sheep, goats, dogs and cats, and are not particularly limited. The therapeutically effective amount for non-human animals may vary depending on the target animal, and can be adjusted and optimized as appropriate within the scope of conventional farming techniques for industrial animals.

[0039] The pharmaceutical composition according to the present disclosure can be prepared into a dosage form suitable for administration. Oral administration forms include solid and liquid forms such as granules, pills, tablets, capsules, powders, and liquids. Parenteral administration forms include injections such as intravenous and intramuscular injections, and transdermal absorption forms such as ointments, gels, patches, films, tapes, and sprays.

[0040] The present disclosure provides, in addition to the above-mentioned medicines, foods, beverages, supplements, and feeds as compositions containing mesostructured particles of calcium bicarbonate as an active ingredient. "Food" includes health foods, functional foods, health foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), health supplements, and nutritional supplements. The form of the food can be selected appropriately, such as solid, liquid, or paste. "Beverages" includes soft drinks, dairy drinks and alcoholic drinks. The "supplement" may be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, emulsions, etc. In addition, to protect the supplement from gastric acid and allow it to act in the intestines, it may be in the form of an enteric-coated agent with different solubility at different pH levels. "Feed" can be obtained by mixing the active ingredient with fresh grass, hay, green forage crops (green corn, etc.), grains (corn, milo, barley, oats, rice, foxtail millet, barnyard millet, millet, sorghum, etc.), grain by-products (rice bran, bran, etc.), root vegetables, straw, oilseed meal (peanut meal, cottonseed meal, sunflower meal, rapeseed meal, sesame meal, flaxseed meal, etc.), etc. [Example]

[0041] Test Example 1: Evaluation of the Tau aggregation inhibitory effect of CAC-717 1. Materials and Methods (1) Reagents CAC-717 (Santa Mineral Co., Ltd.) was stored in a dark place at room temperature, and the required amount was used at the time of experiment. Recombinant human Aβ1-42 (Peptide, 4349-v) and recombinant human mutant tau (P301S-tau) aggregates (StressMarq Biosciences Inc., SPR-329B) were used for in vitro experiments. To inhibit protein metabolism in lysosomes, a mixture of the cathepsin D inhibitor pepstatin A (Merck, P5318), the cathepsin B / H / L inhibitor E64d (Cayman, 13533), and the serine / cysteine ​​proteinase inhibitor leupeptin (Merck, L2884) was used. The primary antibodies used were mouse monoclonal anti-β-actin antibody (Mecrk, A5441), mouse monoclonal anti-Tau antibody (Tau12; Merck, MAB2241), and rabbit monoclonal anti-LC3A / B antibody (Cell Signaling Tech, 12741), which is related to autophagy. The secondary antibodies used were HRP-labeled anti-mouse IgG antibody (Bethyl, A90-116P), HRP-labeled anti-rabbit IgG antibody (Bethyl, A120-101P), Alexa488-labeled anti-mouse IgG antibody (Thermo, A-11001), and Alexa594-labeled anti-rabbit IgG antibody (Thermo, A-11012). In addition, DAPI (Cayman, 14285) was used to stain the cell nuclei.

[0042] (2) Expression plasmid We used a plasmid expressing human mutant tau (P301L-Tau) derived from FTDP17 (familial frontotemporal dementia on chromosome 17), which was provided by Dr. Akihiko Takashima of Gakushuin University. The plasmid was constructed by inserting P301L-Tau into pCI-neo (Promega, E1841) using EcoRI and SalI.

[0043] (3) In vitro experiments Human recombinant Aβ1-42 was solubilized with 0.02% aqueous ammonia to a final concentration of 250 μM and stored at -80°C until immediately before the experiment. 600 μL of Aβ1-42 solution was adjusted to a final concentration of 50 μM by adding 50 mM PBS (pH 7.5) and 100 mM NaCl, and incubated at 37°C for 4 hours to obtain aggregates. 5 μL of the resulting Aβ aggregates were treated with equal, half, or one-quarter volumes of CAC-717 at room temperature for 30 minutes, followed by addition of Laemmuli buffer and biochemical analysis by Western blotting. Human recombinant mutant tau aggregates (1 μg / μL) were thawed on ice, and then 5 μL of the aggregates were treated with equal, half, or one-quarter volume of CAC-717 at room temperature for 30 minutes. After that, Laemmuli buffer was added and biochemical analysis was performed by Western blotting.

[0044] (4) Cell culture experiments Mouse neuroblastoma Neuro2a cells (N2a cells) were used. N2a cells were maintained in 5% fetal bovine serum (FBS)-Dulbecco's modified Eagle's medium (DMEM), and during experiments, they were cultured in 1% FBS-DMEM to prevent cell overgrowth. The time schedule for the cell culture experiment is shown in Figure 1. N2a cells were cultured at 3 x 10 cells per well in a 6-well plate (for biochemical studies) coated with 0.1% polyethyleneimine (FujiFilm, 161-17831) or a 4-well plate with a 12 mm coverslip (for immunocytochemical staining) on ​​the bottom. 4 / cm 2The cells were seeded at a cell density of 1000 kJ / well. The next day (Day 2), P301L-Tau was transfected into N2a cells using polyethyleneimine-MAX (PEI-MAX; Polyscience, 24765-100). DNA was adjusted to 1 μg / well for 6-well plates and 0.25 μg / well for 12-mm coverslips. The following day (Day 3), cells were treated with CAC-717 and lysosomal inhibitors (pepstatin A, E64d, and leupeptin, each adjusted to a final concentration of 10 μM: PEL) by complete medium replacement. Sterile water was added to the control group for CAC-717, and DMSO (Nacalai Tesque, 13408-64), the solvent, was added to the medium for PEL. The following day (Day 4), cells were harvested and used for various experiments.

[0045] N2a cells seeded in a 6-well plate were washed twice with ice-cold phosphate-buffered saline (PBS) and then 300 μl of 1% Triton buffer (10 mM HEPES (pH 8.0), 1% Triton X-100, 150 mM NaCl, 2 mM EGTA, complete mini) was applied and shaken at 4°C for 30 minutes. The cell lysate was collected in a 1.5 ml Eppendorf tube and centrifuged at 20,000 xg for 20 minutes. The supernatant was collected as the Triton-soluble fraction. The pellet was sonicated in 200 μL of cell lysis buffer (62.5 mM Tris-HCl (pH 6.8), 0.5% Triton X-100, 2.3% SDS, 2 mM EDTA, 2.5% 2-mercaptoethanol), boiled for 5 minutes, and collected as the Triton-insoluble fraction. The cells seeded on a 12 mm coverslip were washed twice with ice-cold PBS, fixed in 3% paraformaldehyde buffer (PFA) for 20 minutes, and then used for immunocytochemical staining.

[0046] (5) Evaluation by Western blotting Western blotting was performed on in vitro samples and Triton-soluble and Triton-insoluble fractions recovered from N2a cells seeded in 6-well plates. Protein content of each sample was adjusted to 10 μg, and the resulting fractions were electrophoresed on a 10% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck, IPVH00010). The membrane was blocked with 5% skim milk and 0.1% Tween 20 in PBS (Phosphate Buffered Saline with Tween 20: PBST) and then placed in a hybridization bag with anti-tau antibody (1:5000) and anti-β-actin antibody (1:10,000) at 4°C overnight. The next day, the membrane was washed with PBST and treated with secondary antibody (1:20,000) for 1 hour at room temperature. Specific antibody bands were detected by chemiluminescence using HRP substrate (Merck, WBLUF0500). The bands were quantified using Image J (https: / / imagej.nih.gov / ij / download.html), and statistical analysis was performed using R (https: / / cran.r-project.org / ).

[0047] (6) Immune cell staining N2a cells fixed with 3% PFA were pretreated with 0.1% Triton X-100 in PBS and then blocked with 10% normal goat serum (NGS) in PBST for 30 minutes at room temperature. After blocking, anti-tau antibody (1:1000) and anti-LC3 antibody (1:2000) were applied to the cells in 10% NGS-PBST and incubated overnight at 4°C. The next day, the cells were washed with PBST and incubated with 10% NGS-PBST containing fluorescently labeled secondary antibody (1:2000) and DAPI (4',6-diamidino-2-phenylindole, 1:2000) for 1 hour at room temperature. The cells were then mounted and observed under a fluorescence microscope (KEYENCE, BZ-X800).

[0048] 2.Results (1) CAC-717 reduces aggregated Aβ and tau in vitro experiments To clarify whether CAC-717 has a dissociation effect on Aβ and tau aggregates, we performed in vitro assay experiments using recombinant human Aβ1-42 and recombinant human P301S-tau aggregates. CAC-717 significantly dissociated Aβ and tau aggregates in a concentration-dependent manner (see Figure 2). This indicates that CAC-717 has a potent dissociation effect on Aβ and tau, which are the main pathological proteins in AD.

[0049] (2) CAC-717 reduces soluble and insoluble tau levels in N2a cells Since tau aggregates and causes pathological changes within cells, we investigated whether CAC-717 can also dissociate intracellular protein aggregates. We evaluated the effect of adding CAC-717 to the culture medium of N2a cells overexpressing human mutant P301L-tau. As a result, we confirmed a significant decrease in aggregated tau levels in both the Triton-soluble and Triton-insoluble fractions (Figure 3).

[0050] (3) CAC-717 reduces aggregated tau even in the presence of lysosomal inhibitors It has been shown that insoluble tau aggregates in cells are degraded in lysosomes via autophagy. Therefore, to prove that the tau dissociation effect of CAC-717 is not due to the lysosomal proteolytic system, we examined the effect of CAC-717 on tau in the presence of lysosomal inhibitors. As a result, a significant tau dissociation effect by CAC-717 was confirmed even in the presence of lysosomal inhibitors (see Figure 4). It was revealed that the dissociation effect of CAC-717 on Aβ and tau is due to an action independent of the lysosomal metabolic system.

[0051] Furthermore, the dissociation effect of CAC-717 was histologically verified by immunocytochemistry. In N2a cells treated with CAC-717, the intracellular localization of tau was clearly reduced (see Figure 5). On the other hand, in N2a cells treated with lysosomal inhibitors, intracellular accumulation of enlarged LC3-positive autophagosomes was observed, but the intracellular localization of tau was significantly reduced.

[0052] Test Example 2: Evaluation of the Aβ aggregation inhibitory effect of CAC-717 1. Materials and Methods (1) Reagents Recombinant human Aβ1-42 was dissolved in phosphate-buffered saline (PBS) at 10 μM.

[0053] (2) Dissociation of Aβ aggregates by CAC-717 The Aβ1-42 solution was adjusted to 5 μM with PBS. The mixture was incubated at 37°C for 24 hours to allow Aβ aggregate formation. An equal volume of CAC-717 was added, either undiluted or diluted with distilled water (2x, 4x, or 8x dilutions). The mixture was left to stand at 25°C for 30 minutes, after which the CAC-717 reaction was stopped by adding 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES).

[0054] (3) Inhibition of Aβ aggregation by CAC-717 An equal volume of CAC-717 was added to an Aβ1-42 solution (10 μM), either undiluted or diluted with distilled water (2-, 4-, or 8-fold dilutions), and incubated at 37°C for 24 hours to allow Aβ aggregate formation. The CAC-717 reaction was stopped by adding HEPES.

[0055] (4) Measurement of aggregate amount using thioflavin T Thioflavin T reacts with Aβ aggregates and emits fluorescence, and the fluorescence is known to change depending on the amyloid fibril surface structure and the size of the amyloid core region. Thioflavin T was added to the Aβ sample placed in a well of a black plate to a final concentration of 0.2 mM. After 5 minutes of incubation at 25°C, the amount of Aβ aggregates was determined by measuring the fluorescence intensity (Excitation: 450 nm; Emission: 570 nm) using a fluorescent plate reader. Fluorescence intensity was high for Aβ aggregates (large polymer: LP), intermediate for Aβ oligomers (assemblies of 2-30 particles, Oligomer: O), and low for Aβ monomers (Monomer; M).

[0056] (5)ELISA (Enzyme-Linked Immuno Sorbent Assay) We used a kit (Wako Human and Rat β Amyloid (42) ELISA Kit) that can distinguish between Aβ aggregates (LP), Aβ oligomers (O), and Aβ monomers (M). The percentage of Aβ aggregates in the Aβ samples was determined from the calibration curve. The larger the aggregates, the more likely they are to mask the antigenic site where the detection antibody binds. Therefore, the fluorescence intensity is low for Aβ aggregates (LP), medium for Aβ oligomers (O), and high for Aβ monomers (M).

[0057] 2.Results (1) Dissociation of Aβ aggregates by CAC-717 The fluorescence intensity of Aβ in samples in which Aβ aggregates were treated with CAC-717 was measured using thioflavin T. When CAC-717 was added to the Aβ aggregates, the fluorescence intensity decreased significantly (p<0.05) compared to samples in which distilled water was added, confirming the dissociation of Aβ aggregates (see Figure 6). The time for dissociating Aβ aggregates with CAC-717 was varied to 5, 15, and 30 minutes, and the amounts of oligomers and monomers produced were measured by ELISA. Dissociation of Aβ aggregates was confirmed by reacting Aβ aggregates with CAC-717 for just 5 minutes (see Figure 7).

[0058] (2) Inhibition of Aβ aggregation by CAC-717 The fluorescence intensity of the sample in which Aβ1-42 was treated with CAC-717 and then aggregated was measured using thioflavin T. When CAC-717 was added to the Aβ1-42 solution, the fluorescence intensity was significantly reduced (p<0.01) compared to the sample in which distilled water was added, confirming that Aβ aggregation was inhibited (see Figure 8). The amounts of oligomers and monomers produced were measured by ELISA. Larger amounts of oligomers and monomers were detected in samples treated with CAC-717 than in samples treated with distilled water (see Figure 9(A)). The aggregation inhibitory effect of CAC-717 was significantly observed even when CAC-717 was diluted (see Figure 9(B)). It is thought that treatment with CAC-717 inhibited the formation of Aβ aggregates and simultaneously caused dissociation of Aβ aggregates, resulting in an increase in the amounts of oligomers and monomers.

[0059] [Test Example 3: Evaluation of the inhibitory effect of CAC-717 on tau aggregation in genetically modified Drosophila] 1. Materials and Methods (1) Feed, rearing conditions and drug administration methods The agar diet was prepared according to a standard recipe for Drosophila experiments. CAC-717 solution (Terra Protect CAC-717, CAC-717 concentration approximately 2.5 mg / ml) was stored in the dark at 4°C. The CAC-717 solution was added to the agar diet at a concentration of 5% (v / v). Rearing bottles (22 mm diameter x 96 mm height) were used for rearing. The rearing temperature was 18°C ​​before emergence and before the start of the adult test, and 29°C after the start of the adult test. The light period was 12 hours (9:00 AM - 9:00 PM).

[0060] (2) Drosophila strains used The following three strains of Drosophila were used. Emerged adults (F1 individuals) were anesthetized with CO2, and the required number of individuals were selected for the test. The F1 individuals were reared at 29°C to synchronously express the introduced gene.

[0061] Line 1: Oregon-R: wild-type Drosophila. Line 2: w; UAS-TauR406W: A Tau model in which a mutant Tau (TauR406W), which causes frontotemporal dementia, was transfected. Line 3: yw, GMR-Gal4, rh1-GFP; tub-gal80ts; tub-gal80ts: a line for evaluating neurons transfected with a conjugated protein of rod opsin (rh1) and a fluorescent protein (GFP) under the control of the photoreceptor neuron-specific promoter GMR and a temperature-sensitive promoter (tub-gal80ts).

[0062] (3) Neurodegenerative phenotype observation F1 individuals (males) obtained by crossing line 2 with line 3 were raised at 29°C to induce TauR406W expression specifically in the compound eye. As a control, an F1 antibody obtained by crossing line 2 with line 1 was also used. After 7 days of rearing on a diet containing or without CAC-717, photoreceptor neurons (R cells) were observed for fluorescence using the following method.

[0063] A 1% agar medium was melted at 100°C, and when the temperature dropped to 65°C, an anesthetized Drosophila was placed in it (it was confirmed under a stereomicroscope that half of the fly's body was submerged in the agar medium). After the agar had completely solidified, water was added so that the entire fly was submerged, and the survival of photoreceptor cells was observed under fluorescent light using a 60x depth-of-field lens. Fluorescent images were captured using a CCD camera (Olympus, DP-86). The number of surviving R cells (rh1-GFP positive cells) was counted, and the survival rate of R cells was calculated using the following formula. Viability rate (%) = (number of rh1-GFP positive cells / number of ommatidia x 6) x 100

[0064] (4) Quantification of Tau aggregation After induction of TauR406W expression, the flies were raised on a diet containing or not containing CAC-717 for 3 weeks. Then, the heads of approximately 30 flies were lysed in a buffer containing 1% TritonX100 to obtain a Triton-insoluble fraction. Western blotting was performed as described in Example 1, 1.(5).

[0065] 2.Results (1) Feeding CAC-717 suppressed neurodegeneration In individuals fed a diet containing no CAC-717 (control group), the number of R cells was reduced (see arrows in Figure 10(A)-(C)), and many ommatidia with abnormal cell arrangement were observed. On the other hand, in individuals fed a diet containing CAC-717 (CAC-717-treated group), many ommatidia with normal numbers and arrangements of R cells were observed (Figure 10(D)-(F)). The viability of R cells is shown in Figure 11. In the control group (80-406-standard), 30 individuals (707 ommatidia) were evaluated, and the average viability was 90%. On the other hand, in the CAC-717-treated group (80-406-CAC), 29 individuals (726 ommatidia) were evaluated, and the viability was 99%. R cell viability was significantly maintained in the CAC-717-treated group compared to the control group (P<0.01). These results indicate that the neuronal degeneration caused by the expression of mutant Tau was suppressed by the administration of CAC-717.

[0066] (2) Feeding CAC-717 reduced aggregated tau. The results of Western blotting are shown in Figure 12. TauR406W was reduced in the CAC-717-administered group (CAC) compared to the control group (CT). The reduction in aggregated tau in the CAC-717-administered group suggests that CAC-717 has the effect of inhibiting tau aggregation and / or dissociating aggregated tau in vivo.

Claims

1. A protein aggregation inhibitor containing mesostructured particles of calcium bicarbonate as the active ingredient.

2. The protein aggregation inhibitor according to claim 1 , wherein the calcium bicarbonate mesostructured particles have an activity of inhibiting aggregation of the protein and / or an activity of dissociating the aggregated protein.

3. The protein aggregation inhibitor according to claim 2, wherein the protein exhibits neurodegenerative disease-causing properties due to aggregation.

4. The neurodegenerative disease is Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, frontotemporal dementia, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia, neurofibrillary tangle senile dementia, Pick's disease, Creutzfeldt-Jakob disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy (MSA), Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), frontotemporal lobar The protein aggregation inhibitor of claim 3, wherein the protein aggregation inhibitor is one or more selected from the group consisting of familial amyloidosis (FTLD-TDP), light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloid polyneuropathy (FAP), diarylidosis, hemosiderin amyloidosis, seneca amyloidosis, transthyretin amyloidosis, familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA).

5. The protein aggregation inhibitor according to claim 4, wherein the protein is one or more selected from the group consisting of amyloid beta protein (Aβ), tau, alpha-synuclein, huntingtin protein, superoxide dismutase 1 (SOD1), TDP-43 (TAR DNA-binding protein 43), fused in sarcoma (FUS), amyloid protein, and transthyretin.

6. A pharmaceutical for treating and / or preventing neurodegenerative diseases caused by protein aggregation, comprising mesostructured particles of calcium bicarbonate as an active ingredient.

7. Use of calcium bicarbonate mesostructured particles to inhibit protein aggregation.

8. Use of mesostructured particles of calcium bicarbonate for the treatment and / or prevention of neurodegenerative diseases caused by protein aggregation.

9. Use of mesostructured particles of calcium bicarbonate for the manufacture of a medicament for the treatment and / or prevention of a neurodegenerative disease caused by protein aggregation.

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