Protein aggregation inhibitor

Abstract

Provided is a protein aggregation inhibitor containing meso-structured particles of calcium hydrogencarbonate as an active ingredient, as a technology that can be used in order to inhibit the aggregation of β-amyloid protein (Aβ) or microtubule-associated protein Tau which causes a senile plaque (AP) and a neurofibrillary tangle (NFT).

Description

Protein aggregation inhibitor

[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.

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to dementia. Characteristic neuropathological changes known as senile plaques (APs) and neurofibrillary tangles (NFTs) are observed in the brains of AD patients. APs are extracellular deposits of the β-amyloid protein (Aβ), consisting of approximately 40 amino acid residues. As AD progresses, these lesions spread from the frontal and temporal cortices to the entire cerebral neocortex. NFTs are intracellular lesions in which the microtubule-associated protein Tau aggregates and accumulates in fibrils within neurons. In the cerebral cortex, NFTs begin to form in the entorhinal cortex and hippocampus, and their spread to the cerebral neocortex leads to the onset of dementia.

[0003] Aβ is produced by two-step cleavage of the amyloid precursor protein (APP), primarily expressed in neurons, by β-secretase and γ-secretase. There are several molecular species ranging in length from 38 to 43 amino acid residues depending on the cleavage site by 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 aggregative and has been shown to be highly neurotoxic. Three causative genes for familial AD have been identified to date: APP, presenilin-1 (PS1), and presenilin-2 (PS2). Since PS1 and PS2 are both active centers of the protein complex that constitutes γ-secretase, all causative genes for familial AD are involved in Aβ production. Therefore, the amyloid hypothesis, which proposes that Aβ is the causative molecule of AD, has been 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 a mesostructure are formed by splitting water molecules into H + 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 has been confirmed to cause no irritation to mucous membranes or skin. Non-Patent Document 1 reports that CAC-717 significantly reduces the infectivity of influenza viruses. Non-Patent Document 2 reports that CAC-717 inactivates the pathogenic prion protein (PrPSc).

[0007] PrPSc is a pathogen that causes transmissible spongiform encephalopathy and can be transmitted across species, as in bovine spongiform encephalopathy (BSE). 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, resulting in the generation of highly aggregating hydrophobic peptides. Furthermore, tau aggregation is associated with excessive tau phosphorylation. Therefore, PrPSc, Aβ, and tau have different molecular structures and are also completely different in their generation and aggregation processes.

[0008] Patent No. 5778328 Patent No. 5864010

[0009] "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. "Inactivation of Scrapie Prions by the Electrically Charged Disinfectant CAC-717", Sakudo et. al., Pathogens, 2020, 9, 536

[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.

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

[27] . [1] A protein aggregation inhibitor containing mesostructured particles of calcium bicarbonate as an active ingredient. [2] The protein aggregation inhibitor according to [1], wherein the mesostructured particles of calcium bicarbonate have activity to inhibit aggregation of the protein and / or activity to dissociate the aggregated protein. [3] The protein aggregation inhibitor according to [1] or [2], wherein the protein exhibits neurodegenerative disease pathogenicity 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 locomotive syndrome (FSS), frontotemporal lateral sclerosis (LS ... The protein aggregation inhibitor according to [3], wherein the protein is one or more selected from the group consisting of 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). [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 beta-amyloid protein (Aβ), tau, alpha-synuclein, huntingtin protein, SOD1 (superoxide dismutase 1), TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), amyloid proteins, and transthyretin.

[0012] [6] A pharmaceutical for treating and / or preventing a neurodegenerative disease 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 activity to inhibit protein aggregation and / or activity to dissociate the aggregated protein. [8] The neurodegenerative disease is selected from the group consisting of 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), and frontotemporal dementia. The pharmaceutical according to [6] or [7], wherein the protein is one or more selected from the group consisting of FTLD-TDP, light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloidotic 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 beta amyloid protein (Aβ), tau, alpha-synuclein, huntingtin protein, SOD1 (superoxide dismutase 1), TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), amyloid protein, and transthyretin.

[0013]

[10] Use of mesostructured calcium bicarbonate particles for inhibiting protein aggregation.

[11] The use according to

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

[12] The use according to

[10] or

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

[0014]

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

[16] The use according to

[15] , wherein the mesostructured calcium bicarbonate particles have activity to inhibit protein aggregation and / or activity to dissociate the aggregated protein.

[17] The neurodegenerative disease is selected from the group consisting of 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), and frontotemporal rheumatoid arthritis. The use according to

[15] or

[16] , wherein the protein is one or more selected from the group consisting of FTLD-TDP, light-chain (AL) amyloidosis, atrine (AA) amyloidosis, familial amyloidotic polyneuropathy (FAP), dialidosis, hemosiderin amyloidosis, Seneca amyloidosis, transthyretin amyloidosis, familial amyloidotic 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 beta amyloid protein (Aβ), tau, alpha-synuclein, huntingtin protein, SOD1 (superoxide dismutase 1), TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), amyloid proteins, and transthyretin.

[0015]

[19] Use of mesostructured calcium bicarbonate particles 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 calcium bicarbonate particles have activity to inhibit protein aggregation and / or activity to dissociate the aggregated protein.

[21] The neurodegenerative disease is selected from the group consisting of 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), and frontotemporal rheumatoid arthritis. The use according to

[19] or

[20] , wherein the protein is one or more selected from the group consisting of 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).

[22] The use according to any one of

[19] to

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

[0016]

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

[24] The method according to

[23] , wherein the mesostructured particles of calcium bicarbonate have activity to inhibit aggregation of the protein and / or activity to dissociate the aggregated protein.

[25] The neurodegenerative disease is selected from the group consisting of 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), and frontotemporal rheumatoid arthritis.

[26] The method according to any one of

[23] to

[25] , wherein the protein is one or more selected from the group consisting of beta-amyloid 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.

[27] The method according to any one of

[23] to

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

[0017] [Definitions] In the present disclosure, protein "aggregation" refers to the process in which two or more protein molecules come together to form a mass (cluster) with a larger molecular weight. Aggregation can be a physical or chemical process. Protein "aggregates" refer to clusters in which two or more single protein molecules (monomers) aggregate together. 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 in which protein aggregates are separated into two or more aggregates or monomers with smaller molecular weights. Dissociated aggregates can become aggregates with smaller molecular weights (e.g., oligomers), monomers, or a mixture of these.

[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.

[0019] The following shows the experimental schedule for cultured cells. 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. N2a cells were seeded in a 6-well plate, and 2000 μL of medium was added with 50 μL (25 μL / mL), 100 μL (50 μL / mL), or 200 μL (100 μL / mL) of CAC-717. Triton-soluble and Triton-insoluble fractions were extracted from N2a cells 24 hours after the addition of CAC-717 and analyzed by Western blot (A). Cells treated with 50 μL / mL or 100 μL / mL of CAC-717 showed a significant decrease in soluble and insoluble tau. (B) The amount of tau protein is shown relative to the amount in the control (CT) group (*p<0.05). Triton-soluble and insoluble fractions were extracted from N2a cells treated with CAC-717 and a lysosomal inhibitor (PEL) in the medium for 24 hours and analyzed by Western blot. CAC-717 significantly reduced soluble and insoluble tau, even in the presence of a lysosomal inhibitor. (B) The amount of tau protein is shown relative to the amount in the control (CT) group (*p<0.05). In N2a cells expressing P301L-Tau (CT), tau accumulated widely throughout the cytoplasm. Tau staining was significantly reduced in N2a cells treated with CAC-717 (CAC). 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. Aβ fluorescence intensity was measured in samples treated with CAC-717 to remove Aβ aggregates. CAC-717 dissociated Aβ aggregates.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. The fluorescence intensity of the aggregated samples was measured after treatment of recombinant human Aβ1-42 with CAC-717. CAC-717 inhibited Aβ aggregation. The amounts of oligomers and monomers formed in the 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. The neurodegenerative effects of CAC-717 were evaluated in Drosophila expressing mutant tau. The reduction in R cell number and abnormalities in cell alignment were suppressed in individuals fed a diet containing CAC-717 (D-F) compared to individuals fed a diet without CAC-717 (A-C). The results of evaluating the neurodegeneration inhibitory 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 lacking CAC-717. The results of evaluating the Tau aggregation inhibitory effect of CAC-717 in Drosophila expressing atypical Tau are shown below. The aggregated Tau was reduced in individuals raised on a diet containing CAC-717 compared to individuals raised on a diet lacking CAC-717.

[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 Inhibitor The protein aggregation inhibitor according to the present disclosure contains mesostructured calcium bicarbonate particles as an active ingredient. It has been found that 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.

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

[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] Examples of target proteins include β-amyloid protein (Aβ), tau, alpha-synuclein, huntingtin protein, SOD1 (superoxide dismutase 1), TDP-43 (TAR DNA-binding protein 43), FUS (fused in sarcoma), amyloid protein, transthyretin, etc. The target protein 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 vivo), and / or the activity of dissociating aggregated proteins. Therefore, the protein aggregation inhibitor according to the present disclosure can be used as an active ingredient in the pharmaceutical according to the present disclosure, as 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. Drug for the Treatment and / or Prevention of Neurodegenerative Diseases A drug for the treatment and / or prevention of a disease caused by protein aggregation according to the present disclosure contains the above-described mesostructured calcium bicarbonate particles as an active ingredient. Because the calcium bicarbonate mesostructured particles have the activity of inhibiting protein aggregation and / or dissociating aggregated proteins in vivo and in vitro, they may be effective for the treatment and / or prevention of such diseases caused by protein aggregation. The therapeutic effect of the drug according to the present disclosure includes alleviating or improving symptoms or delaying the progression of symptoms in patients who have developed the disease by suppressing further protein aggregation or dissociating aggregated proteins. The preventive effect of the drug also includes preventing or delaying the onset of symptoms in patients who have not yet developed the disease by suppressing protein aggregation or dissociating aggregated proteins.

[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 lobar 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 Senecan 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 animal to be administered may be, but is not limited to, a human or a non-human mammal such as a pig, cow, horse, sheep, goat, dog, or cat. The therapeutically effective amount for a non-human animal may vary depending on the target animal, and can be appropriately adjusted and optimized 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 pharmaceuticals, foods, beverages, supplements, and feeds as compositions containing mesostructured calcium bicarbonate particles as an active ingredient. "Foods" include health foods, functional foods, health claim foods (e.g., foods for specified health uses, foods with nutrient functions, foods with functional claims), dietary supplements, and nutritional supplements. The form of the food can be selected appropriately, such as solid, liquid, or paste. "Beverages" include soft drinks, dairy drinks, and alcoholic beverages. "Supplements" can be in any form, such as tablets, granules, powders, sugar-coated tablets, capsules, syrups, suspensions, liquids, and emulsions. Furthermore, enteric-coated formulations with different solubility at different pH levels can be used to protect the supplement from gastric acid and allow it to act in the intestines. "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 cakes (peanut meal, cottonseed meal, sunflower meal, rapeseed meal, sesame meal, flaxseed meal, etc.), etc.

[0041] Test Example 1: Evaluation of the Inhibitory Effect of CAC-717 on Tau Aggregation 1. Materials and Methods (1) Reagents CAC-717 (Santa Minerals) was stored in the dark at room temperature and used in the required amount 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-conjugated anti-mouse IgG antibody (Bethyl, A90-116P), HRP-conjugated anti-rabbit IgG antibody (Bethyl, A120-101P), Alexa488-conjugated anti-mouse IgG antibody (Thermo, A-11001), and Alexa594-conjugated anti-rabbit IgG antibody (Thermo, A-11012). DAPI (Cayman, 14285) was used to stain cell nuclei.

[0042] (2) Expression Plasmid: A plasmid expressing human mutant Tau (P301L-Tau) derived from FTDP17 (familial frontotemporal dementia on chromosome 17), provided by Dr. Akihiko Takashima of Gakushuin University, was used. 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 experiments. 600 μL of Aβ1-42 solution, adjusted to a final concentration of 50 μM with 50 mM PBS (pH 7.5) and 100 mM NaCl, was incubated at 37°C for 4 hours to obtain aggregates. 5 μL of the resulting Aβ aggregates were added with equal, half, or one-quarter volumes of CAC-717 and incubated at room temperature for 30 minutes. After incubation with Laemmuli buffer, biochemical analysis was performed by Western blotting. Human recombinant mutant tau aggregates (1 μg / μL) were thawed on ice, and then added with equal, half, or one-quarter volumes of CAC-717 to 5 μL of the aggregates. After incubation at room temperature for 30 minutes, 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 experiments is shown in Figure 1. N2a cells were cultured at a density of 3 × 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 immunocytostaining) 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). The 6-well plate was adjusted to 1 μg DNA / well, and the 12-mm coverslip was adjusted to 0.25 μg DNA / well. The following day (day 3), the 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) was added to the medium as a solvent for PEL. The following day (day 4), the 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 g 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 immunocytochemistry.

[0046] (5) Western Blotting Analysis Biochemical analysis was performed by Western blotting using in vitro experimental samples and Triton-soluble and Triton-insoluble fractions recovered from N2a cells seeded in 6-well plates. Each sample was adjusted to 10 μg of protein and electrophoresed on a 10% polyacrylamide gel. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane (Merck, IPVH00010). The transferred membrane was blocked with 5% skim milk-0.1% Tween 20 (Phosphate Buffered Saline with Tween 20: PBST) and then placed in a hybrid 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 a secondary antibody (1:20,000) for 1 hour at room temperature. Specific antibody-reactive 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) Immunocytochemistry. 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 at room temperature for 30 minutes. After blocking, 10% NGS-PBST containing anti-Tau antibody (1:1000) and anti-LC3 antibody (1:2000) was applied and incubated overnight at 4°C. The next day, after washing with PBST, the cells were incubated with 10% NGS-PBST containing fluorescently labeled secondary antibody (1:2000) and DAPI (4',6-diamidino-2-phenylindole, 1:2000) at room temperature for 1 hour, then mounted and observed under a fluorescence microscope (KEYENCE, BZ-X800).

[0048] 2. Results (1) CAC-717 reduces aggregated Aβ and tau in in vitro experiments. To determine whether CAC-717 has the ability to dissociate Aβ and tau aggregates, in vitro assays were performed 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). These results demonstrate that CAC-717 has a potent dissociation effect on Aβ and tau, the main pathological proteins of AD.

[0049] (2) CAC-717 reduces soluble and insoluble tau levels in N2a cells. Since tau aggregates intracellularly, we investigated whether CAC-717 can also dissociate intracellular protein aggregates. We added CAC-717 to the culture medium of N2a cells overexpressing human mutant P301L-tau and evaluated its effect. As a result, we confirmed a significant reduction in aggregated tau levels in both the Triton-soluble and insoluble fractions (Fig. 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 demonstrate that the tau dissociation effect of CAC-717 is not due to the lysosomal proteolysis system, we examined the effect of CAC-717 on tau in the presence of lysosomal inhibitors. As a result, we confirmed that CAC-717 significantly dissociated tau even in the presence of lysosomal inhibitors (see Figure 4). These results demonstrate that the dissociation effect of CAC-717 on Aβ and tau is independent of the lysosomal metabolic system.

[0051] Furthermore, we histologically verified the dissociation effect of CAC-717 using 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, the 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 with CAC-717 The Aβ1-42 solution was adjusted to 5 μM in PBS. It 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). 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) Aβ aggregation inhibition treatment with CAC-717. An equal volume of CAC-717, either undiluted or diluted with distilled water (2x, 4x, or 8x dilutions), was added to an Aβ1-42 solution (10 μM) and incubated at 37°C for 24 hours to form Aβ aggregates. The CAC-717 reaction was stopped by adding HEPES.

[0055] (4) Measurement of Aggregate Amount Using Thioflavin T Thioflavin T reacts with Aβ aggregates to emit fluorescence, and its fluorescence intensity is known to vary 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 black plate well to a final concentration of 0.2 mM. After 5 minutes of incubation at 25°C, the amount of Aβ aggregates was measured 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 Immunosorbent Assay) A kit (Human / Rat β-Amyloid (42) ELISA Kit Wako) capable of distinguishing and detecting Aβ aggregates (LP), Aβ oligomers (O), and Aβ monomers (Monomer; M) was used. The percentage of Aβ aggregates in the Aβ sample was determined from the calibration curve. The larger the aggregates, the more easily the antigen site to which the detection antibody binds becomes masked. Therefore, the fluorescence intensity is low for Aβ aggregates (LP), intermediate 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 Aβ aggregates, the fluorescence intensity significantly decreased (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, or 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 aggregated samples of Aβ1-42 treated with CAC-717 was measured using thioflavin T. Addition of CAC-717 to the Aβ1-42 solution significantly reduced the fluorescence intensity (p<0.01) compared with the sample treated with distilled water, confirming the inhibition of Aβ aggregation (see Figure 8). The amounts of oligomers and monomers formed were measured by ELISA. Higher amounts of oligomers and monomers were detected in the CAC-717-treated sample compared with the sample treated with distilled water (see Figure 9(A)). The aggregation inhibitory effect of CAC-717 remained significant even when CAC-717 was diluted (see Figure 9(B)). It is believed that treatment with CAC-717 inhibited Aβ aggregate formation and simultaneously dissociated Aβ aggregates, resulting in an increase in the amounts of oligomers and monomers.

[0059] [Test Example 3: Evaluation of the Tau Aggregation Inhibitory Effect of CAC-717 in Transgenic Drosophila] 1. Materials and Methods (1) Diet, Rearing Conditions, and Drug Administration Methods Agar diet prepared according to a standard recipe for Drosophila experiments was used. CAC-717 solution (Terra Protect CAC-717, CAC-717 concentration approximately 2.5 mg / ml) was stored in the dark at 4°C. 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. Rearing temperatures were 18°C ​​before emergence and before the start of the adult test, and 29°C after the start of the adult test. Light exposure 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 use in the experiment. The F1 individuals were reared at 29°C to synchronously express the introduced gene.

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

[0062] (3) Observation of neurodegenerative phenotypes. F1 individuals (males) derived from a cross between line 2 and line 3 were reared at 29°C to induce TauR406W expression specifically in the compound eye. As a control, an F1 antibody derived from a cross between line 2 and line 1 was also used. After rearing the mice for 7 days 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 had cooled to 65°C, anesthetized individual fruit flies were added (it was confirmed under a stereomicroscope that half of the fruit fly's body was buried in the agar medium). After the agar had completely solidified, water was added so that the entire fly was immersed, and the survival of photoreceptor cells was observed under fluorescent light using a 60x water depth lens. Fluorescent images were observed and captured with 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: Survival 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 in the same manner as described in 1.(5) of Example 1.

[0065] 2. Results (1) CAC-717 Feeding Suppressed Neurodegeneration. In mice fed a diet lacking 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. In contrast, in mice 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)). R cell viability is shown in Figure 11. In the control group (80-406-standard), 30 mice (707 ommatidia) were evaluated, with an average survival rate of 90%. In the CAC-717-treated group (80-406-CAC), 29 mice (726 ommatidia) were evaluated, with a survival rate of 99%. R cell survival 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 blot analysis are shown in Figure 12. TauR406W was reduced in the CAC-717-treated group (CAC) compared to the control group (CT). The reduction in aggregated tau in the CAC-717-treated 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 mesostructured particles of calcium bicarbonate have the activity of inhibiting the aggregation of the protein and / or the activity of dissociating the aggregated protein.

3. The protein aggregation inhibitor according to claim 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 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 calcium bicarbonate mesostructured particles 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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