KR102499338B1 - Pharmaceutical composition for preventing or treating Alzheimer's disease - Google Patents

Abstract

It relates to a pharmaceutical composition for preventing or treating Alzheimer's disease containing gadolinium, and more particularly, to prevent or treat Alzheimer's disease by reducing the secretion of beta-amyloid protein, which is the main cause of Alzheimer's disease. It relates to pharmaceutical compositions.

Description

Pharmaceutical composition for preventing or treating Alzheimer's disease {Pharmaceutical composition for preventing or treating Alzheimer's disease}

The present invention relates to a pharmaceutical composition for preventing or treating Alzheimer's disease containing gadolinium, and more particularly, to prevent or treat Alzheimer's disease by reducing the secretion of beta-amyloid protein, which is a major cause of Alzheimer's disease. It relates to a pharmaceutical composition that can be treated.

Alzheimer's disease is the most prevalent form of dementia among the 65-year-old elderly population in Korea as well as in Western societies. According to the dementia prevalence survey conducted by the Ministry of Health and Welfare in Korea, as of 2012, dementia patients aged 65 years or older appear to be about 9.18%, of which Alzheimer's disease is estimated to be about 71.3%. Dementia treatment costs increased from 56.1 billion won [48,000 people] in 2002 to 621.1 billion won [215,000 people] in 2009, and management is in full swing at the national level, such as the implementation of the Dementia Management Act from 2011.

The main causative agents of Alzheimer's dementia identified so far are beta amyloid and tau protein from the viewpoint of molecular biology and pathology. Compared to tau protein, beta amyloid is considered to be important in the early and middle stages of dementia in Alzheimer's disease, and compared to tau protein, which is also related to frontotemporal dementia and dementia with Lewy bodies, beta amyloid is It is known to be highly specific for Alzheimer's disease.

Beta amyloid is produced from beta amyloid precursor protein ("APP"). The production mechanism is that APP is produced as beta amyloid by activation of APP-specific cleavage enzymes, beta-secretase and gamma-secretase. On the other hand, when alpha-secretase is activated, the production of beta-amyloid is suppressed because the middle of beta-amyloid is cut. Based on this mechanism, alpha secretase activators or beta and gamma secretase inhibitors have been developed, but there are no officially approved drugs due to various side effects. Currently, cholinesterase inhibitors or glutamate inhibitors are used clinically for the treatment of Alzheimer's disease, but it is judged that only the symptoms are treated and not the fundamental treatment.

On the other hand, gadolinium is a rare earth metal (Gd) and corresponds to atomic number 64. This mainly produces a trivalent cation (Gd ³⁺ or Gd[III]), and this ion is used for various purposes. Gadolium-157 radioactive isotope is used in surgery to remove neutron tumors and is also used as an MRI contrast agent due to its magnetic field properties.

In addition, although it has no use for diseases, gadolinium ion has a use as an inhibitor of intracellular TRP (Transient Receptor Potenial) channel, and is known to play an important role in regulating intracellular calcium ion. As a related physiological activity, it has been reported to regulate the secretion of hormones or neurotransmitters such as vasopressin or catecholamine. Human-derived diseases have not yet been reviewed in the literature, and gadolinium ions have not been explored for use in prevention or treatment in Alzheimer's disease either.

Korea Patent Registration 10-1856838 Korea Patent Registration 10-2187922

Therefore, the present invention is intended to solve various drawbacks and problems caused by the prior art in consideration of the above situation, and is an Alzheimer's disease containing gadolinium ion having an effect of reducing the concentration of beta amyloid, a causative agent of Alzheimer's disease. It is an object of the present invention to provide a pharmaceutical composition for preventing or treating disease.

In addition, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above object, according to an embodiment of the present invention, a pharmaceutical composition for preventing or treating Alzheimer's disease containing gadolinium ions as an active ingredient may be provided.

According to one embodiment of the present invention, the gadolinium ion may be derived from a pharmaceutically acceptable salt of gadolinium ion.

According to one embodiment of the present invention, the pharmaceutically acceptable salt of the gadolinium ion may be at least one of gadolinium halide, gadolinium oxide, and gadolinium hydroxide.

According to one embodiment of the present invention, the gadolinium ion may decrease secretion of beta amyloid protein.

According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating Alzheimer's disease further comprising a pharmaceutically acceptable carrier may be provided.

According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating neurodegenerative diseases containing gadolinium ions as an active ingredient may be provided.

According to one embodiment of the present invention, the neurodegenerative disease is Parkinson's disease, Pick's disease, Huntington's disease, multiple sclerosis, epilepsy, stroke, stroke, ischemic encephalopathy, memory loss, traumatic central nervous system disease, spinal cord injury disease, peripheral nerve It may be selected from the group consisting of injury, amyotrophic axonal sclerosis, peripheral nerve disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome, or schizophrenia.

According to one embodiment of the present invention, a health functional food composition for preventing or improving neurodegenerative diseases containing gadolinium ions as an active ingredient is provided.

The pharmaceutical composition for preventing or treating Alzheimer's disease of the present invention contains gadolinium ions as an active ingredient, so it is not toxic to cells and has an effect of reducing the concentration of beta amyloid, a causative agent of Alzheimer's disease. There is an advantage of providing a pharmaceutical composition useful for the treatment of degenerative diseases.

1 relates to APP cleavage (cleavage) by processing APP (amyloid precursor protein) and a method for confirming the same. The upper sAPP (soluble APP) is secreted into the extracellular space. This means that total sAPPs (sAPPα + sAPPβ) can be detected in the medium by the 22C11 antibody [red], and only sAPPα can be detected by the 2B3 antibody [orange]. In cells, CTF (C-terminal fragments) and APP-FL (full length of APP) can be detected using A8717 [green] or Y188 [blue] antibodies according to their size. Among CTFs, C83 is produced by alpha secretase, C89 and C99 are produced by beta secretase, and APP intracellular domain (AICD) is produced by gamma secretase. The activity of beta and gamma secretase causes the production of beta amyloid, and the activity of gamma secretase causes the decrease of beta amyloid.
FIG. 2 confirms that no significant cytotoxicity was observed through LDH release assay when the HEK293-APPsw cell line, which is an Alzheimer's disease cell model capable of secreting beta amyloid into the culture medium, was treated with gadolinium ions at different concentrations.
FIG. 3 confirms that no significant cytotoxicity was observed through MTT reduction assay when the HEK293-APPsw cell line, which is an Alzheimer's disease cell model capable of secreting beta amyloid into the culture medium, was treated with gadolinium ions at different concentrations.
FIG. 4 is confirmed by ELISA (enzyme-linked immunosorbent assay) to show that beta amyloid 1-40 and 1-42 secreted into the culture medium are reduced by concentration when the cell model is treated with gadolinium ions by concentration. .
FIG. 5 confirms, by western blot analysis, that the beta amyloid-reducing effect is due to increased activity of alpha secretase among beta-amyloid-generating enzymes and inhibition of secretion of soluble APPs (sAPPs).

Hereinafter, the present invention will be described in detail. Prior to this, terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor appropriately uses the concept of terms in order to describe his/her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical spirit of the present invention based on the principle that it can be defined in the following way. Therefore, the configuration described in the embodiments described in this specification is only one of the most preferred embodiments of the present invention and does not represent all of the technical ideas of the present invention, so various equivalents and equivalents that can replace them at the time of this application It should be understood that variations may exist.

According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating Alzheimer's disease containing gadolinium ions as an active ingredient is provided.

As used throughout this specification, the term "prevention" refers to any action that inhibits or delays the onset of Alzheimer's disease.

In addition, the term "treatment" used throughout the present specification refers to any activity in which symptoms caused by Alzheimer's disease are improved or beneficially changed by administration of the composition.

The term "Gadolinium" used throughout this specification is a rare earth metal (Gd), an element with atomic number 64, which reacts with acids, alkalis, and metals to mainly trivalent cations (Gd ³⁺ or Gd [ III]), and this ion is used for various purposes.

In this case, according to an embodiment of the present invention, the gadolinium ion may be derived from a pharmaceutically acceptable salt of gadolinium ion, and the term "pharmaceutically acceptable salt" used throughout the present specification is non-toxic in an administered dose. It means a salt, and includes commonly used salts such as inorganic acids, organic acids, or non-isotropic salts.

Examples of the inorganic acid herein include, but are not limited to, hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, and phosphorous acid, and examples of the organic acid include acetic acid, lactic acid, citric acid, malic acid, succinic acid, fumaric acid, and maleic acid. acids, tartaric acid, benzoic acid, phthalic acid, gluconic acid, saccharic acid, methane sulphonic acid, ethane disulphonic acid, aliphatic mono and dicarboxylates, phenyl-substituted al phenylsubstituted alkanoates, hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, but not limited thereto. .

The non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride , acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, Fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, Phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, beta-hydroxybutyrates, glycolates, malates , tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate, but is not limited thereto.

In this case, according to a preferred embodiment, the pharmaceutically acceptable salt of the gadolinium ion may be at least one of gadolinium halide, gadolinium oxide, and gadolinium hydroxide, and more preferably may be gadolinium halide.

Meanwhile, the pharmaceutical composition for preventing or treating Alzheimer's disease of the present invention may further include a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" used throughout the present specification may refer to a carrier or diluent that does not inhibit biological activities and properties of a compound to be injected without irritating living organisms. The type of the carrier that can be used herein is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

On the other hand, in one embodiment of the present invention, the pharmaceutical composition for preventing or treating Alzheimer's disease does not increase the drug efficacy, but is commonly used in pharmaceutical compositions to improve odor, taste, vision, etc. can do.

In addition, the pharmaceutical composition of the present application may additionally include pharmaceutically acceptable additives. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, and talc, but are not limited thereto.

In one embodiment of the present invention, the pharmaceutical composition including a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It may be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions for internal use, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.

Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

Meanwhile, according to another embodiment of the present invention, a pharmaceutical composition for preventing or treating neurodegenerative diseases containing gadolinium ions as an active ingredient is provided.

The term "neurodegenerative disease" used throughout the present specification refers to a disease that occurs in the nerves among degenerative diseases that occur with aging, and for reasons unknown to date, certain brain cell groups in the brain and spinal cord gradually lose their function. It is known to be caused by the death of cranial nerve cells, which are most important for the transmission of information in the cranial nervous system, the formation or functional problems of synapses that transmit information between cranial nerve cells, and the abnormal increase or decrease in electrical activity of cranial nerves. . Nerve cells in the brain and spinal cord have very diverse functions depending on their location, so they show very different clinical features depending on which area of the nerve cells are damaged and lose their function first, and how these dysfunctions progress. .

In one embodiment of the present invention, the neurodegenerative disease is dementia, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, multiple sclerosis, epilepsy, stroke, stroke, ischemic encephalopathy, memory loss, traumatic central nervous system disease , It may be selected from the group consisting of spinal cord injury disease, peripheral nerve injury, amyotrophic axonal sclerosis, peripheral nerve disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome, or schizophrenia, but is not limited thereto.

The pharmaceutical composition for preventing or treating neurodegenerative diseases of the present invention can be administered in an effective amount.

The term "pharmaceutically effective amount" used throughout the present specification means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, It may be determined according to factors including the type of infected virus, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. For example, the composition or a pharmaceutically acceptable salt thereof may be administered at 00001 to 1000 mg/kg per day, preferably 0001 to 100 mg/kg per day.

The administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Specifically, it can be administered orally or parenterally depending on the desired method, and in the case of parenteral administration, external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method can be selected. It may be, but is not limited thereto.

The pharmaceutical composition for preventing or treating neurodegenerative diseases of the present application may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into 2 to 3 times. The number of administrations when the two active ingredients are each a single agent may be the same or may be different. In addition, the pharmaceutical composition of the present application may be used alone or in combination with other drug treatments for the prevention or treatment of neurological diseases. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

According to another embodiment of the present invention, a health functional food composition for preventing or improving neurodegenerative diseases containing gadolinium ions as an active ingredient is provided.

The term "functional food" used throughout the present specification is the same term as food for special health use (FoSHU), and is processed to efficiently display bioregulatory functions in addition to nutrient supply. It means food with high medicinal and medical effects. Here, function (sex) refers to obtaining useful effects for health purposes such as regulating nutrients or physiological functions with respect to the structure and function of the human body. The health functional food composition of the present application can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The health functional food composition can be prepared in various types of formulations, and unlike general drugs, it has the advantage of using food as a raw material and has no side effects that may occur when taking drugs for a long time, and is excellent in portability, so the food of the present invention It can be taken as an adjuvant to enhance the immune enhancing effect. Health food (health food) means a food that has an active health maintenance or promotion effect compared to general food, health supplement food (health supplement food) means a food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, There are beverages, alcoholic beverages, vitamin complexes, dairy products and milk-processed products, and includes all health functional foods in a conventional sense.

According to one embodiment of the present application, the health functional food composition may include additional components that are commonly used in food compositions to improve odor, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr) may be included. In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine, but is not limited thereto.

According to one embodiment of the present application, the health functional food composition contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericide (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxy Anisole (BHA), butylhydroxytoluene (BHT), etc.), coloring agent (tar colorant, etc.), coloring agent (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (dulcin, cyclamate, saccharin, sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogentartrate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base, foam It may include food additives such as inhibitors, solvents, and improvers, but is not limited thereto.

The additive may be selected according to the type of food and used in an appropriate amount.

According to one embodiment of the present application, the health functional food composition for preventing or improving mild degenerative diseases may be added to food as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, the amount of the composition in the health functional food may be added in an amount of 01 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for the purpose of health maintenance or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

The health functional food composition according to an embodiment of the present application is not particularly limited in other ingredients except for containing the active substance as an essential ingredient in the indicated ratio, and various flavoring agents or natural carbohydrates are added as additional ingredients like conventional beverages. may contain Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the health functional food composition of the present application, but is not limited thereto.

The health functional food composition according to an embodiment of the present application contains various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the health functional food composition of the present application may contain natural fruit juice, fruit juice beverages, and fruit flesh for the preparation of vegetable beverages.

These components may be used independently or in combination. The ratio of these additives is not so critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health functional food composition of the present application.

Hereinafter, the present invention is further detailed through examples, but it is obvious that the present invention is not limited by the following examples.

Establishment of the HEK293-APPsw cell line

Plasmids expressing human APPsw (amyloid precursor protein swedish mutants) K670N and M671L were introduced into HEK293 cells. After about 12 hours, the culture medium was replaced with DMEM (Dulbecco's Modified Eagle Medium) containing 800 μg/mL of G418. Only the cells forming the colony were transferred to each well of a 24-well plate and cultured, and the grown cells were transferred to a 12-well plate and selectively cultured in DMEM medium supplemented with G418 at a concentration of 600 μg/mL. The culture was selectively cultured by lowering the concentration of G418 in the medium to 200 μg/mL while subculturing again through the 60 mm dish to the 100 mm dish. The stable colony formed through this was confirmed by Western blot analysis and APPsw protein expression and beta-amyloid 1-40/1-42 ELISA (enzyme-linked immunosorbent assay; Biosource, USA) in the culture medium.

Cell culture and gadolinium ion treatment

The HEK293-APPsw cell line was placed in DMEM medium supplemented with 10% Fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) and cultured in an incubator (37°C, 5% CO ₂ ). After culturing the cells in a 12-well plate to 70-80% confluency, they were washed once with DPBS the next day. 1 ml of DMEM medium containing 1% FBS was added to each well. The gadalinium ion was diluted in DMEM medium supplemented with 1% FBS to a concentration of 25, 50, and 100 μM and treated in each well. After 48 hours, the medium and cells were harvested and subjected to ELISA and Western blot analysis of beta-amyloid 1-40/1-42.

Cytotoxicity test of gadolinium ion

Cytotoxicity was measured through MTT reduction assay and LDH release assay. The MTT reduction assay measures the redox capacity of mitochondria in living cells, and the LDH rerelase assay measures the amount of cell damage by using the LDH enzyme in cells to leak into the medium when the cell membrane is damaged in dead cells. Cytotoxicity was measured through MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide analysis, and after treatment with gadolinium ions of each concentration for 8 hours, 5mg/mL MTT solution was treated with 1/5 of the culture medium and allowed to react for 1 hour, then the culture medium was removed and dissolved by adding DMSO. The absorbance of the dissolved sample was measured at 570 nm using a 96-well spectrophotometer (molecular probe, USA). LDH release assay was measured using Takara's LDH cytotoxicity detection kit. After treatment with gadolinium ions of each concentration for 8 hours, the cultured cell medium and LDH enzyme substrate were reacted 1:1 at room temperature for 30 minutes to 1 hour, Absorbance was measured at 492 nm using a 96-well spectrophotometer (molecular probe, USA).

ELISA analysis of beta-amyloid in media

The amount of beta-amyloid 1-40 and 1-42 in medium samples treated with gadolinium ions of each concentration for 8 hours was performed using an ELISA (Invitrogen) kit. 50 μl each of the medium diluted (2 to 20 times) with the primary antibody was mixed with the primary antibody and dispensed onto a 96-well ELISA plate coated with other antibodies, and incubated at room temperature for about 2 hours. After washing about 5 times with the washing buffer, the secondary antibody labeled with HRP (horse radish peroxidase) was again treated. After final washing, color was developed with a tetrametyhlybenzidine (TMB) solution, fixed with a 0.01N HCl solution, and the degree of color development was measured at absorbance at 450 nm using a 96-well ELISA plate (Thermo). The measured value was substituted into a standard curve of beta-amyloid dissolved in advance to calculate the amount of beta-amyloid in the medium.

Confirmation of APP processing through western blot to measure changes by gadolinium ion

In order to confirm APP processing after treatment with gadolinium ions, western blot was performed using cells and media. In the case of the medium, in order to measure the amount of sAPP, 8 μl of the medium was mixed with 2 μl of 5x sample buffer, boiled, and loaded on an 8% acrylamide gel. Cells were dissolved in a buffer containing 10mM Tris (pH 7.4), 150mM NaCl, 1% SDS and protease inhibitors and phosphatase inhibitors for APP and APP-CTF (C-terminal fragment) measurement, and samples were then disrupted by ultrasonication. got The obtained sample was mixed with 5x sample buffer, loaded, and electrophoresed through 15 and 8% acrylamide gels. After electrophoresis of the two types of samples at 100V for about 2 hours, the gel was transferred to a PVDF membrane at 100V for 1 hour and then blocked with 5% skim milk at room temperature for 1 hour. After washing three times with TBS-T (Tris buffered saline with 0.1% Tween20), the membrane was reacted with primary antibody diluted in TBS-T overnight. After washing three times with TBST, the secondary antibody (horseradish peroxidase-conjugated Ig anti-mouse antibody) was reacted at room temperature for 1 hour, and then washed three times with TBST. To detect sAPP in the culture medium, anti-APP N-terminal antibody (22C11; sAPPs or total sAPP) and anti-soluble APPα (2B33; sAPPα or sAPPalpha) were used. To detect CTF in cells, anti-APP C -terminal antibody (Y188; detects C83, C89, C99, and AICD), anti-APP C-terminal antibody (A8717; detects all APP-FL or APP precursors), and anti-soluble APPα antibody were used. Detection was confirmed using ECL (Enhanced chemiluminescence) and LAS4000 chemiluminescence detector (GE healthcare). The obtained images were quantified using Image J software (NIH, USA). The detection principle of the antibody used in Western blot analysis is shown in FIG. 1. In the above results, in the detection of CTF, C83 represents the activity of alpha secretase, C89 and C99 represent the activity of beta secretase, and AICD represents the activity of gamma secretase.

Result 1: Confirmation of gadolinium ion toxicity by LDH release assay

When the HEK293-APPsw cell line was treated with gadolinium chloride (GdCl3) at concentrations of 25, 50, and 100 μM and LDH release was analyzed, no change in cell death was observed. From this, it can be seen that gadolinium ions are not toxic to human-derived cells (FIG. 2).

Result 2: Confirmation of toxicity of gadolinium ion by MTT reduction assay

When the HEK293-APPsw cell line was treated with gadolinium chloride (GdCl3) at concentrations of 25, 50, and 100 μM and analyzed for MTT, no change in cell viability was observed. From this, it can be seen that gadolinium ions are not toxic to human-derived cells (FIG. 3).

Result 3: Beta amyloid secretion concentration change according to gadolinium ion treatment

After treating the HEK293-APPsw cell line with gadolinium chloride (GdCl3) at concentrations of 25, 50, and 100 μM, the concentration of beta-amyloid in the cell culture medium was confirmed by ELISA. As a result of examining 1-40 and 1-42, the most abundant types of beta amyloid, it was found that both types decreased with increasing gadolinium ion concentration. Considering that toxicity was not shown as in Results 1 and 2 at the corresponding concentration, it was confirmed that the beta amyloid concentration was reduced by about 80% and 65% in beta amyloid 1-40 and 1-42, respectively (FIG. 4).

Result 4: Investigation of the activity of APP processing

In order to analyze the cause of the beta amyloid reduction effect shown in the beta amyloid concentration test, the cleavage pattern of APP processing was investigated through western blot. As shown in FIG. 1, the activity of beta-secretase produces water-soluble APPβ secreted into the culture medium in addition to beta-amyloid and CTFβ in the cells, and the activation of alpha-secretase produces water-soluble APPα and intracellular CTFα in the culture medium. , the activity of gamma secretase results in the production of AICD within the cell. After treating the cells with 100 μM gadolinium ions, first, when the increase or decrease of CTF was analyzed using the Y188 antibody in the cells, it was observed that C83, C89, and AICD were increased. Since the activities of alpha, beta, and gamma secretases all increased, and beta amyloid decreased in result 3, it can be judged that this decrease is due to increased activity of alpha secretase. In addition, when total water-soluble APP (sAPPs: water-soluble APPα + water-soluble APPβ) and water-soluble APPα were measured using the 22C11 antibody in the culture medium and water-soluble APPα using the 2B3 antibody, it was found that both water-soluble APPα and APPβ were not secreted by 100 μM gadolinium ion treatment. could Therefore, it can be determined that the decrease in beta amyloid concentration in Result 3 is due to the increase in activity of alpha secretase and the decrease in extracellular secretion of water-soluble APP (FIG. 5).

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

In a pharmaceutical composition for preventing or treating Alzheimer's disease containing gadolinium ions as an active ingredient,
The gadolinium ion is derived from a pharmaceutically acceptable salt of gadolinium ion,
The pharmaceutical composition for preventing or treating Alzheimer's disease, wherein the pharmaceutically acceptable salt of the gadolinium ion is gadolinium chloride. delete delete According to claim 1,
The gadolinium ion is to reduce the secretion of beta amyloid protein, a pharmaceutical composition for preventing or treating Alzheimer's disease. According to claim 1,
A pharmaceutical composition for preventing or treating Alzheimer's disease, further comprising a pharmaceutically acceptable carrier. delete delete In the health functional food composition for preventing or improving Alzheimer's disease containing gadolinium ions as an active ingredient,
The gadolinium ion is derived from a pharmaceutically acceptable salt of gadolinium ion,
The pharmaceutically acceptable salt of the gadolinium ion is gadolinium chloride, a health functional food composition for preventing or improving Alzheimer's disease.

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