KR20210114790A - Composition for preventing, improving or treating Alzheimer's disease comprising N,N′-Diacetyl-p-phenylenediamine as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating Alzheimer's disease, including N,N'-diacetyl-p-phenylenediamine (DAPPD) compound as an active ingredient. According to the present invention, it is shown that DAPPD restores the damaged phagocytosis function of microglia in a test using Alzheimer's disease transformed mice to accelerate removal of amyloid-beta species, and thus effectively improves cognitive deficits. It is also shown that DAPPD having a significantly small and simple structure passes through the blood-brain barrier (BBB) and is absorbed to the brain, and has little toxicity characteristically. Therefore, DAPPD according to the present invention controls neurodegenerative inflammation as a neuroprotective formulation and is expected to be used as a hopeful medicine for treating neurodegenerative disease, such as Alzheimer's disease.

Description

A composition for preventing, improving or treating Alzheimer's disease comprising a N,N'-diacetyl-p-phenylenediamine compound as an active ingredient as an active ingredient}

The present invention relates to a composition for preventing, improving or treating Alzheimer's disease, comprising a N,N'-diacetyl-p-phenylenediamine (N,N'-Diacetyl-p-phenylenediamine; DAPPD) compound as an active ingredient.

Neurodegeneration is defined as the gradual loss of nerve structure and function, and various evidences have been reported to suggest that neuroinflammation, an innate immune mechanism of the central nervous system, is a major pathological contributor to neurodegeneration. Microglia are phagocytes residing in the central nervous system and are known to play an important role in the process by identifying and removing pathogens. Under normal conditions, the immune response of microglia is bidirectional, capable of secreting pro-inflammatory mediators involved in cell recruitment and removal of damaged neurons, or producing anti-inflammatory mediators that can promote neuronal proliferation and synaptic plasticity. adjust the balance In contrast, the persistent presence of pathological triggers such as nerve damage and protein aggregates results in chronic activation and damage of microglia, and dysfunction of microglia often results in 1) increased expression of neurotoxic pro-inflammatory mediators. ; 2) decreased production of neurotrophic anti-inflammatory mediators; and 3) impaired ability to remove pathogens due to loss of phagocytosis. In addition, these microglia changes have negative consequences for neurons through self-proliferation and positive-feedback loops. Therefore, dysfunction of microglia would be a potential therapeutic target for drug development for neurodegenerative diseases including Alzheimer's disease (AD), Parkinson disease and amyotrophic lateral sclerosis. can

Alzheimer's disease is the most common form of dementia, with approximately 47 million cases reported in 2016 and is expected to reach approximately 131 million patients by 2050. The multiple etiology of AD involves various pathological factors such as neuroinflammation and amyloid proteins including amyloid-β (Aβ). In addition, a close relationship between neuroinflammation and Aβ has been reported, and this correlation is considered to have a decisive influence on the development of Alzheimer's disease. Loss of phagocytosis ability due to dysfunction of microglia significantly reduces the Aβ clearance, thereby increasing Aβ may cause damage to microglia through chronic activation (Lancet Neurol. 14, 388–405). (2015)). Since this malignant cycle strongly induces neurodegeneration, functional restoration of microglia can restore neuronal homeostasis in Alzheimer's disease. Therefore, it is necessary to develop an agent that protects nerve cells by restoring the function of microglia.

As a result of research efforts to discover a low-molecular compound having an excellent therapeutic effect on Alzheimer's disease, the present inventors have made N,N'-diacetyl-p-phenylenediamine (N,N'-Diacetyl-p) having a structure similar to acetaminophen. -phenylenediamine; hereinafter, DAPPD) compound was confirmed to have excellent cognitive deficit improvement effect and amyloid-beta level reduction, and the mechanism of action through restoration of dysfunction of microglia was clarified, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing, improving or treating Alzheimer's disease, comprising the DAPPD compound as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention includes a compound represented by the following formula (1), a stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient, Alzheimer's disease prevention or treatment A pharmaceutical composition for use is provided.

[Formula 1]

In addition, the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition can repair damage to phagocytosis of microglia.

In another embodiment of the present invention, the composition may reduce neuroinflammation by mediating microglia.

In another embodiment of the present invention, the composition can reduce amyloid-beta levels in the brain.

In another embodiment of the present invention, the composition can restore impaired cognitive function.

In addition, the present invention provides a method for preventing or treating Alzheimer's disease, comprising administering to an individual a pharmaceutical composition comprising the compound represented by Formula 1, a stereoisomer, or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

In addition, the present invention provides the use of the pharmaceutical composition for preventing or treating Alzheimer's disease.

According to the present invention, it was confirmed that the DAPPD compound effectively ameliorated cognitive deficits by promoting the removal of amyloid-beta species by restoring the impaired phagocytic function of microglia in an experiment using Alzheimer's disease transgenic mice. has a very small and simple structure, passes through the blood-brain barrier (BBB) and is absorbed into the brain and has little toxicity. DAPPD according to the present invention is a neuroprotective agent, It is expected to be used as a promising therapeutic agent for neurodegenerative diseases, specifically Alzheimer's disease.

Figure 1 is the result of analyzing the characteristics of DAPPD, Figure 1a shows the chemical structure and mechanism of action of DAPPD, Figure 1b is the plasma (plasma) of C57BL/6 mice administered with DAPPD and acetaminophen (AAP), respectively , are chromatograms and quantitative numerical results analyzing the brain-absorption rate of each drug in whole brain and cerebrospinal fluid (CSF), and FIG. is the result of the analysis.
2 is an in vivo result of analyzing the effect of DAPPD in APP/PS1 mice, a mouse model of Alzheimer's disease. While the survival rate ( FIG. 2a ) and weight change ( FIG. 2b ) are measured, FIG. 2c is an escape-latency time and distance traveled during the MWM test for each mouse group. is the result of measuring , and the result showing the swimming path on the 10th day of training, FIGS. 2D and 2E are the time taken to reach the quadrant with the target platform in the probe test ( FIG. 2D ) and the swimming path ( FIG. 2E ) ) is a result showing, and FIG. 2f is a result of measuring the number of times the mouse in each group entered the target position during the probe test of 60 seconds (Crossing platform), distance traveled and swimming speed. and Figure 2g shows that Aβ plaques were produced through thioflavin-S (ThS) and 6E10 (anti-Aβ antibody) in cortex (Cortex) and hippocampus (Hippocampus) tissues of APP/PS1 mice administered vehicle or DAPPD, respectively. It is the result of observing and quantifying the degree of deposition.
3 is a result of administering vehicle or DAPPD to wild-type and APP/PS1 mice, respectively, and analyzing changes in neuroinflammation, FIGS. 3a and 3b are cortex and hippocampus tissues of each group mouse brain. It is the result of observing and quantifying activated microglia and astrocytes through immunostaining using Iba1 (Fig. 3b) and GFAP (Fig. 3b) antibodies, respectively, and Figs. 3c and 3d are the cerebral cortex of each group of mouse brains ( Figure 3c) and in the microglia isolated from the cortex (Figure 3d), pro-inflammatory cytokines ( Tnf-α , Il-1β , Il-6 , and iNos ) and anti-inflammatory cytokines (Anti- inflammatory Cytokines; Il-4, Tgf -β, and Arg 1) the results of measuring the level of mRNA, Fig. 3e are each mouse group MGnD microglia from microglia cells isolated from the cerebral cortex of the brain gene and M0- It is the result of measuring the mRNA level of the homeostatic microglia gene, and FIGS. 3f and 3g show the expression of CLEC7A ( FIG. 3f ) and P2RY12 ( FIG. 3g ) proteins respectively through immunostaining in the cerebral cortex of the mouse brain of each group. and quantified it.
Figure 4 is the result of analyzing the effect of DAPPD on the microglia phagocytosis of Aβ and the expression of NLRP3 inflammasome-related protein. Figure 4a is a microglia (Iba1, Immunostaining images for the green) and Aβ aggregates (6E10, red) and their quantification results, FIG. 4b shows the presence in the osteoclast (Lamp1, blue) of microglia (Iba1, red) in the mouse brain of each group. Immunostaining images of Aβ aggregates (6E10, green), and quantification results for the volume of osteolysosomes, Aβ-loaded osteolysosomes and microglia occupied by Aβ in osteolysosomes, Figure 4c is the cerebrum of mice in each group. 3D reconstructed quantitative results from immunostaining images and confocal image results showing the morphology of microglia surrounded by Aβ in the cortex, and FIG. 4D is an analysis result according to the size of Aβ plaques in each group of mice.
Figure 5 is the result of analyzing the effect of DAPPD on the NLRP3 inflammasome-related protein and the upstream pathways related to DAPPD-mediated regulation on the NLRP3 inflammasome. Figures 5a and 5b are wild-type and APP administered with vehicle or DAPPD In the cerebral cortex of /PS1 mice, the effect of DAPPD on the expression of proteins related to NLRP3 inflammasome (FIG. 5a), NF-κB signaling and cathepsin B (FIG. 5B) was analyzed by Western blot analysis.
6 is a result of analyzing the effects of DAPPD, PBMA, and AAP on microglia function in an in vitro Alzheimer's disease environment. PBMA, or AAP treatment, and NLRP3 inflammasome (FIG. 6a), NF-κB signaling and analysis of the expression level of proteins related to cathepsin B (FIG. 6b) through Western blot, Figure 6c is the above figure 6a and 6b are the results of measuring the mRNA levels of pro-inflammatory cytokines and anti-inflammatory cytokines in the same group, and FIG. 6d is the mRNA levels of MGnD microglia gene and M0-homeostasis gene in the same group as FIGS. 6a and 6b. is a result of measuring , and FIG. 6E is a result of analyzing the bead intake over time in the same group as in FIGS. 6A and 6B.
7 is a result of comparative analysis of the efficacy of a Tylenol drug containing DAPPD and acetaminophen having a similar structure according to the present invention as a main component. The vehicle or each drug was administered intraperitoneally and the Aβ ₄₂ and Aβ _17-24 levels were measured and compared, and FIG. 7b is the result of performing the MWM test to compare and analyze the cognitive deficit improvement effect for the mice in each group. am.

The present inventors have discovered N,N'-diacetyl-p-phenylenediamine (N,N'-Diacetyl-p-phenylenediamine; hereinafter, DAPPD) as a low-molecular compound having an excellent therapeutic effect on Alzheimer's disease, and its excellent Cognitive defect improvement effect and amyloid-beta level reduction were confirmed, and the mechanism of action through restoration of dysfunction of microglia was investigated, thereby completing the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease, comprising a compound represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In the present invention, the compound represented by Formula 1 is named N,N′-diacetyl-p-phenylenediamine (N,N′-Diacetyl-p-phenylenediamine; DAPPD), and two acetates at the para position It has a structure consisting of a benzene ring having only an acetamide group.

As used herein, the term “stereoisomer” refers to a molecule having the same compound structure and the same order of bonding between constituent atoms, but having a different three-dimensional structure. Stereoisomers of the compound represented by Formula 1 according to the present invention may exist as optical isomers, racemates, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers. These isomers can be separated by resolution such as column chromatography or HPLC.

In the present invention, as the pharmaceutically acceptable salt, an acid addition salt formed by a free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, 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, dinitrobenzoate, hydroxybenzoate, Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol late, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

It is also possible to make pharmaceutically acceptable metal salts using bases. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

"Alzheimer's disease", a disease to be prevented, improved, or treated in the present invention, is a degenerative neurological disease in which abnormal proteins such as amyloid-beta and tau proteins accumulate in the brain and brain neurons gradually die, and all of the definite causes are Although not identified, several genetic risk factors have been identified. The most common symptoms include memory impairment, language impairment, apraxia showing impairment in actions such as dressing in daily life, agnosia in which vision is normal but inability to distinguish objects, spatiotemporal ability impairment, impaired judgment, depressive symptoms, and emotional changes. appear.

As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of Alzheimer's disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms for Alzheimer's disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

In the present invention, the effect of the compound represented by Formula 1 on Alzheimer's disease and its mechanism of action were investigated through specific examples.

In one embodiment of the present invention, it is assumed that the DAPPD small molecule compound will exhibit an effective effect as an anti-neuroinflammatory compound based on the results of previous studies, and metabolic stability, BBB permeability and cytotoxicity were evaluated before evaluating the efficacy in vivo. As a result of the analysis, it was confirmed that the DAPPD compound is not chemically modified in the body, so it has high metabolic stability, penetrates the BBB and is absorbed into the brain, and does not exhibit significant cytotoxicity in neuroblastoma cells at a concentration of 500 μM or less (see Example 2). ).

In another embodiment of the present invention, as a result of verifying the effect of DAPPD using a mouse model of Alzheimer's disease, it was confirmed that DAPPD has an excellent effect in improving cognitive impairment through behavioral studies, and brain tissue analysis of the mouse model It was confirmed that the level of amyloid-beta aggregates was significantly reduced (see Example 3).

In another embodiment of the present invention, it was confirmed that neuroinflammation was reduced in a mouse model administered with DAPPD (see Example 4), and DAPPD modulates a functional phenotype in microglia to induce amyloid-beta. It was confirmed that alleviating neuroinflammation (see Example 5).

In another embodiment of the present invention, it was confirmed that DAPPD promotes the degradation of amyloid-beta by restoring the phagocytosis defect of microglia under neuroinflammatory conditions using a mouse model of Alzheimer's disease (see Example 6).

In another embodiment of the present invention, based on the results of previous studies that there is a correlation between Aβ-induced neuroinflammation and the NLRP3 inflammasome, whether the anti-inflammatory effect by DAPPD is related to the regulation of NLRP3 inflammasome formation. As a result of the analysis, it was confirmed that the expression of NLRP3 and ASC was inhibited by DAPPD and, as a result, the activity of caspase-1 and IL-1β was decreased. It was confirmed that it was caused (see Example 7).

In another embodiment of the invention, in using a human microglia vitro Analysis of the microglial cell function improved over the effect caused by DAPPD in Alzheimer's environment, and the NF-κB path inhibited by DAPPD Accordingly NLRP3 It was confirmed that inflammasome formation was down-regulated, and it was confirmed that the expression change of inflammatory cytokines changed by amyloid-beta in microglia was restored by DAPPD. Furthermore, it was confirmed that DAPPD restores amyloid-beta-induced dysfunction of microglia through a phagocytosis assay using FITC beads (see Example 8).

In another embodiment of the present invention, as a result of comparative analysis of the toxicity and efficacy of a Tylenol drug containing acetaminophen having a structure similar to DAPPD as a main component and DAPPD, the LD ₅₀ concentration of DAPPD is higher than that of Tylenol, so that even at a higher concentration It was confirmed that it was safe, and it was confirmed that DAPPD was more effective in reducing amyloid-beta levels and improving cognitive function than Tylenol (see Example 10).

The pharmaceutical composition according to the present invention includes the compound represented by Formula 1 as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as an injection, an inhalant, or an external preparation for skin.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or locally applied) according to a desired method, but preferably may be administered orally, and the dosage Although it varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the patient's disease type, severity, and drug activity, sensitivity to drugs, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, in general 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight, may be administered daily or every other day, or divided into 1 to 3 times a day. However, the dosage may be increased or decreased according to the route of administration, the severity of obesity, sex, weight, age, etc., and thus the dosage is not intended to limit the scope of the present invention in any way.

In addition, the present invention provides a food composition for preventing or improving Alzheimer's disease, comprising the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, before or after the onset of the disease, and simultaneously with the drug for treatment. or may be used separately.

As used herein, the term "food composition" is one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations, including one or more of carriers, diluents, excipients and additives. characterized. Foods that can be added to the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, tea, vitamin complexes, health functional foods, and the like. Additives that may be further included in the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers, lactic acid and salts thereof, At least one component selected from the group consisting of alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and pulp may be used. Examples of the above-mentioned 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 dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The composition according to the invention comprises various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and thickening agents, facic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners ; These components can be used independently or in combination.Specific examples of the carrier, excipient, diluent and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, Calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylkirolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylhydroxybenzoate, propylhydroxybenzoate At least one selected from the group consisting of , talc, magnesium stearate and mineral oil is preferably used.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental materials and methods

1-1. Cell viability assay

The mouse neuroblastoma Neuro-2a (N2a) cell line (ATCC) was prepared in a medium mixed with DMEM (GIBCO, NY, Grand Island, NY) and opti-MEM (GIBCO) in a 1:1 ratio with 5% (v/v) bovine Fetal serum (FBS; SigmaAldrich, MO, St. Louis, MO) and 100 U/mL penicillin and 100 mg/mL streptomycin (GIBCO) were added _{and cultured at 5% CO 2} and 37° C. conditions.

Meanwhile, cell viability according to DAPPD treatment was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. More specifically, cells were aliquoted into 96-well plates (10,000 cells in 100 μl medium per well) and then DAPPD was added at various concentrations (10, 50, 100, 200 and 500 μM; 1% v/v final DMSO concentration). ) and incubated for 24 hours. Then, MTT [25 μl; 5 mg/mL] solution in PBS (pH 7.4) was added to each well in which cells were seeded, and incubated at 37° C. for 4 hours. Then, the formazan produced by the cells was prepared using an acidic solution of N,N-dimethylformamide (DMF; 50% v/v, aq) and sodium dodecyl sulfate (SDS; 20% w/v). Reacted overnight at room temperature in the dark to allow solubilization. Then, absorbance was measured at 600 nm using a microplate reader, and cell viability was measured for cells containing an equivalent amount of DMSO.

1-2. in vivo Experimental Design

The purpose of this study was to investigate the neuroprotective function of DAPPD through anti-inflammatory effects using transgenic Alzheimer's disease (AD) mice.

1-2-1. Drug Treatment and Results Analysis in the APP/PS1 Mouse Model

Transgenic APP/PS1 mice overexpressing human amyloid precursor protein (APP) 695 [K670N/M671L (Sweden)] and presenilin-1 (PS1) (M146V) mutations were obtained from GlaxoSmithKline. (GlaxoSmithKline, Harlow, UK). Since APP/PS1 mice showed gender-dependent differences in disease progression, only male mice were used. Data analysis was performed at the age of 9.5 months, and age and sex-matched C57BL/6 mice were used as wild-type (WT) mice. A block randomization method was used to distribute the mice to the experimental group, and in order to eliminate bias, experimental procedures such as data collection and data analysis were performed as a blind test. Water and feed were freely supplied on a 12-hour day/night cycle, and all protocols were carried out after obtaining approval from the Kyungpook National University Experimental Animal Steering Committee (IACUC). DAPPD was freshly prepared immediately before administration by dissolving in a vehicle [85% DMSO in saline], and administered intraperitoneally every morning for 2 months at a dose of 2 mg/kg starting from the age of 7.5 months.

Thereafter, a behavioral study was performed to evaluate spatial learning and memory abilities through the Morris water maze (MWM) test for mice administered with the DAPPD. In order for the mice to learn in advance, a test was performed 4 times a day for 10 days, and a probe test with the platform removed on the 11th day was performed.

Meanwhile, for histological analysis, the anesthetized mice were perfused by cardiovascular perfusion with PBS to wash residual blood through cerebral circulation, and then additionally perfused with 4% paraformaldehyde (PFA). Then, the brain was excised and fixed with 4% PFA overnight at 4°C, and then the brain tissue was cut in a coronal plane to a thickness of 30 μm using a vibratome to obtain a section. Then, the tissue sections were subjected to Thioflavin-S (ThS) staining, and 6E10 (mouse, 1:100, SIG39300; Biolegend, CA, USA), Iba1 (rabbit, 1:500, 019) -19941; Wako Pure Chemical Industries, Ltd., Osaka, Japan), GFAP (rabbit, 1:500, N1506; Dako, CA, USA), Clec7a (rat, 1:100, MABG-MDECT; Invivogen, CA, USA) ), P2ry12 (rabbit, 1:100, ab188968; Abcam, Cambridge, UK), Lamp1 (rat, 1:100, ab25245; Abcam, Cambridge, UK) and Lamp1 (mouse, 1:200, ab24170; Abcam, Cambridge, UK) antibody was used for immunostaining for histological analysis. Results were analyzed with a laser scanning confocal microscope (FV1000; Olympus, Tokyo, Japan) or a BX51 microscope (Olympus), and values were quantified using MetaMorph software (Molecular Devices). Three-dimensional reconstructions of microglia were also recorded and analyzed using IMARIS software (Bitplane, Belfast, UK).

1-2-2. Drug treatment and outcome analysis in 5×FAD mouse model

We used transgenic 5×FAD mice overexpressing mutant human APP695 [K670N/M671L (Sweden), I716V (Florida), and V717I (London)] and PS1 (M146L and L286V) as another Alzheimer's disease animal model. did. Water and feed were freely supplied on a 12-hour day/night cycle, and the experiment was conducted after obtaining approval from the Asan Life Science Research Institute's Experimental Animal Steering Committee. DAPPD was mixed with vehicle [1% DMSO v/v; 20 mM HEPES, pH 7.4, 150 mM NaCl] was freshly prepared immediately before administration, and was administered intraperitoneally every morning at a dose of 2 mg/kg starting at 3 months of age for 30 days.

From the 27th day of administration in the mouse model administered with DAPPD, spatial learning and memory abilities were evaluated through the Morris water maze (MWM) test. For this, the test was conducted 3 times a day for 5 days, and 3 hours after the last test, the probe test was performed in the MWM without a platform for 60 seconds. Test parameters were collected and analyzed on a SMART video tracking system (Harvard Apparatus, Holliston, MA, USA).

On the other hand, the deposition of amyloid-beta (hereafter, Aβ) plaques was _{performed by immunohistochemistry using human Aβ 17-24} specific antibody 4G8 (Covance, Princeton, NJ, USA) and Accustain® Congo Red amyloid staining solution, respectively. After chemical staining, evaluation was performed in the sagittal plane (12 μm thickness) of the left hemisphere. Thereafter, the stained area was observed and photographed with an optical microscope (Eclipse 80i; Nikon, Tokyo, Japan). Amyloid deposits were expressed as the percentage area of 4G8-immunoreactive deposits or the number of congophilic plaques per ^{mm 2 of brain area.}

_{In addition, for quantification of Aβ, Aβ 40} , Aβ ₄₂ (Invitrogen, Carlsbad, CA, USA) and oligomeric Aβ in PBS-, SDS-, or formic acid-soluble fractions after successive centrifugation fractions of the right brain hemisphere were analyzed by sandwich ELISA. measured.

1-3. cell culture

Cortexes of E18 C57BL/6 mice were excised and dissociated by incubation in papain at 37° C. for 15 minutes. Neurons were cultured with neuronal culture medium, which is 2% v/v B27 supplement (GIBCO), 1 mM Glutamax supplement (GIBCO), and serum-free Neurobasal medium (GIBCO) containing 100 U/mL penicillin and 100 mg/mL streptomycin. -L-Lysine was plated on the coated coverslip and incubated at _{37° C., 5% CO 2 conditions.}

Primary astrocyte cultures were prepared from C57BL/6 mice. Briefly, after removing the meninges from 7-day-old (P7) mice, brain tissues were minced and incubated in a stirred water bath at 37° C. for 30 min in the presence of 0.25% v/v trypsin-EDTA (Sigma-Aldrich). Cells dissociated via enzymatic digestion were triturated under astrocyte-specific medium (DMEM/F12 with 10% v/v FBS, 0.2 and 1% v/v penicillin-streptomycin) and centrifuged at 1,300 rpm for 8 min. separated. The pellet was resuspended in DMEM/F12 and filtered using a 40-μm cell strainer, then allowed to pre-attach for 30 min to remove any contaminants from the fibroblasts before dispensing the cell filtrate into a culture dish and astrocyte- Specific medium was added. For the division of astrocytes, Ara-C was added to the culture dish and placed on a heat shaker for 6-7 hours. Thereafter, the medium was removed from the culture dish, trypsin-EDTA was added, and incubated at 37° C. for 5-10 minutes, followed by centrifugation at 1,300 rpm for 8 minutes. The supernatant was removed, and the astrocytes were maintained in culture by supplying an astrocyte-specific medium every 1-2 weeks.

In addition, primary microglia were isolated from the mouse brain according to the following procedure. Cortex of wild-type (1 month old) mice was minced in Hibernate A (GIBCO)/B27 (Invitrogen) medium and then dissociated using a papain (Worthington Biochemical Corporation, NJ, USA) solution. Thereafter, the tissue was crushed and the cells were isolated through Optiprep (Sigma-Aldrich) density gradient centrifugation. The fractionated microglia were cultured in DMEM/F12 containing 10% v/v FBS, 0.2 and 1% v/v penicillin-streptomycin, or used for expression level analysis of inflammatory cytokines or microglia genes. We have successfully isolated primary cells from the brain of mice according to each disclosed method. Immortalized human microglia (Applied Biologics Materials, T0251) were also cultured in PriGrow III (Applied Biologics Materials, TM003) (supplemented with 10% v/v FBS and 1% v/v penicillin/streptomycin) at 37° C., 5% CO. _It was cultured under 2 conditions.

1-4. Western blotting

After dissolving brain samples or human microglia in RIPA buffer (Cell Signaling Technology, MA, USA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, followed by nitro The separated proteins were transferred to the cellulose membrane. Next, 5% milk was added to the membrane to block a non-specific reaction, and after reaction with the primary antibody, it was incubated with the secondary antibody conjugated with horseradish peroxidase. The primary antibodies used in this experiment were: NLRP3 (rabbit, 1:1,000; 15101, Cell Signaling Technology), ASC (rabbit, 1:1,000; 67824, Cell Signaling Technology), Caspase-1 (mouse, 1) :1,000; AG-20B-0042, Adipogen Life Sciences, CA, USA), IL-1β (mouse, 1:1000; 12242, Cell Signaling Technology), pNF-κB p50 (rabbit, 1:1,000, 13586; Cell Signaling) Technology), pNF-κB p65 (rabbit, 1:1,000, 3033; Cell Signaling Technology), pIκB (rabbit, 1:1,000, 9246; Cell Signaling Technology), Cathepsin B (rabbit, 1:1,000, 31718; Cell Signaling Technology) ), COX-1 (mouse, 1:500, ab695; Abcam, Cambridge, UK), COX-2 (rabbit, 1:500, ab15191; Abcam, Cambridge, UK), and β-actin (1:1,000; SC) -47778, Santa Cruz Biotechnology, TX, USA). Rabbit-HRP (1:1,000; 7074s, Cell Signaling Technology) and mouse-HRP (1:1,000; sc2005, Santa Cruz Biotechnology) were used as secondary antibodies. Thereafter, the protein expression level was quantified using ImageJ software.

1-5. RNA Isolation and Real-time PCR Analysis

RNA was extracted from brain tissue homogenates and cell lysates using RNeasy Lipid Tissue Mini kit and RNeasy Plus Mini kit (QIAGEN, Hilden, Germany). Thereafter, cDNA was synthesized using a commercially available kit (Takara Bio Inc., Shiga, Japan) for 5 μg of the RNA extracted above. Quantitative real-time PCR was performed using a Corbett research RG-6000 real-time PCR instrument.

Example 2. Characterization of DAPPD

Based on the structural similarity of the DAPPD compound selected as a small molecule compound for the treatment of Alzheimer's disease to acetaminophen (AAP), an antipyretic and analgesic agent that exhibits mild anti-neuroinflammatory activity, the present inventors wanted to investigate the anti-neuroinflammatory effect of DAPPD. did. The small, symmetrical structure of DAPPD exhibits ideal properties and ease of synthesis, including lipophilicity, which gives the molecule the ability to penetrate the blood-brain barrier (BBB). The chemical structure of DAPPD is as shown in FIG. 1 , and is composed of phenylacetamide and an additional acetamide moiety. The phenylacetamide structure of DAPPD can be found in AAP, and additional acetamide functional groups present in DAPPD may exhibit improved enzymatic and metabolic stability under physiologically relevant conditions. Overall, it was determined that DAPPD could be an ideal candidate as an anti-neuroinflammatory compound in vivo.

Before investigating the efficacy of the DAPPD compound in vivo, the present inventors first tried to analyze metabolic stability, BBB permeability, and cytotoxicity to evaluate the biological applicability of DAPPD. First, in order to evaluate the 1) stability of the compound in plasma and 2) the ability to bind to plasma proteins, it was confirmed that 97% of the compound remained as a result of culturing DAPPD in human plasma for 120 minutes, through which it was confirmed that DAPPD was found to have significantly high plasma stability. Second, DAPPD showed significantly lower binding to plasma proteins (0% binding, 69% and 99% binding to dexamethasone and warfarin, respectively). These results suggest that DAPPD is stable in plasma and is relatively well distributed upon administration.

Next, in order to verify the possibility that DAPPD is changed to AAP by chemical modification, the metabolic stability of DAPPD in vivo was evaluated. More specifically, to verify brain accessibility and metabolism of DAPPD, the compound was intraperitoneally administered (10 mg/kg) to C57BL/6 mice, followed by liquid chromatography-mass spectrometry. As a result, it was confirmed that DAPPD was detected in plasma, brain and cerebrospinal fluid (CSF) after compound treatment as shown in FIG. 1b , whereas AAP was not detected. These results suggest that DAPPD can cross the BBB without being metabolized to AAP in vivo. In addition, even when DAPPD (10mg/kg) was orally administered in vivo, it was confirmed that it was absorbed into the brain.

Meanwhile, an in vitro metabolism study of DAPPD using human liver microsomes was conducted. As a result, as shown in Table 1 below, it was found that DAPPD had a moderate metabolic stability (half-life [t _1/2 ] > 60 min; intrinsic clearance [Cl _int ] = 3.9).

t _1/2 ^e Cl _int ^f DAPPD > 60 3.9 Verapamil
(Reference) 9.7 155

In addition, selectivity profiling of DAPPD against traditional pharmacological targets was performed through the CEREP Safety-screen44 panel. Screening showed that DAPPD had no appreciable activity (>50% inhibition) against the 44 targets tested. Finally, DAPPD was treated with various concentrations (10, 50, 100, 200, 500 uM) in Neuro-2a (N2a), a mouse neuroblastoma cell, and then cell viability was analyzed. As a result, as shown in FIG. 1c , it was confirmed that no significant cytotoxicity was exhibited at a concentration of 500 μM or less.

Overall, based on the above results, it was determined that DAPPD was suitable for evaluation of neuroprotective activity in vivo.

Example 3. Confirmation of improvement in cognitive function impairment and reduction of Aβ accumulation by DAPPD treatment in Alzheimer's disease mouse model

3-1. Confirmation of improvement effect on cognitive function impairment

First, wild-type (WT) and APP/PS1 mice (7.5 months old) were intraperitoneally (i.p.) administered with DAPPD or vehicle at 2 mg/kg/day for 2 months. At 30 days post-dose, a Morris Water Maze (MWM) test was performed to evaluate spatial learning and memory abilities of each mouse administered vehicle or DAPPD. As a result, as shown in FIGS. 2A and 2B , it was confirmed that DAPPD administration did not affect the survival and body weight of mice through the fact that neither wild-type nor APP/PS1 mice had any change in survival rate and body weight during the administration period of DAPPD. Furthermore, as a result of the MWM test, as shown in FIG. 2c , in the case of APP/PS1 mice treated with vehicle (Vehicle (APP/PS1)), escape latency was significantly longer than that of wild-type mice treated with vehicle and DAPPD. and it was confirmed that the platform was moved to a greater distance (Distance Traveled). In contrast, in the case of DAPPD-administered APP/PS1 mice (DAPPD (APP/PS1)), an escape platform was found at an early time point and appeared to move a shorter distance to the platform in a manner similar to that of wild-type mice. The difference in escape delay and movement distance as described above was particularly noticeable on the 5th and 7th days of training, where the escape delay time of APP/PS1 mice administered with vehicle and DAPPD, respectively, differed by approximately 2 times. confirmed that. In addition, as can be seen in FIGS. 2D to 2F , in the probe test performed after training for 10 days, APP/PS1 mice administered with DAPPD stayed in the target quadrant for a longer time than the non-target quadrant and were treated with vehicle. It appeared to traverse target sites more frequently compared to APP/PS1 mice. Interestingly, DAPPD-administered APP/PS1 mice showed similar results to wild-type mice, with no significant differences in the movement distance and swimming speed of mice between APP/PS1 mice administered vehicle or DAPPD in the probe test. It was found that APP/PS1 did not affect the motor performance of mice. Furthermore, in addition to the above results, the present inventors additionally confirmed that DAPPD has the effect of restoring cognitive function in AD transgenic mice using a 5xFAD mouse model.

Through the above results, it can be seen that DAPPD has the effect of effectively improving the impaired cognitive function in the Alzheimer's disease mouse model.

3-2. Confirmation of Aβ accumulation reduction effect

Furthermore, to verify whether DAPPD affects the accumulation of Aβ aggregates associated with cognitive impairment in AD, the present inventors investigated Aβ aggregates in the hippocampus and cortex of APP/PS1 mice for 2 months after administration of DAPPD. was monitored for the accumulation of As a result of observing the deposition of Aβ plaques through thioflavin-S (ThS) and 6E10 (anti-Aβ antibody), respectively, as shown in FIG. 2g, DAPPD was higher than that of APP/PS1 mice administered with the vehicle. It was confirmed that the accumulation of Aβ aggregates was reduced by 6-fold and 2-fold, respectively, in the administered mice. In addition, as a result of analysis using a 5xFAD AD transgenic mouse model, it was confirmed that the percentage area of 4G8-immunoreactive amyloid plaques in the brain of 5xFAD mice administered with DAPPD was reduced by approximately 40% compared to mice administered with vehicle. Moreover, _{total, soluble and insoluble levels of Aβ 40} were shown to be reduced by about 40, 20 and 50%, respectively (Approximately 30, 35 and 20% reductions were observed for _{Aβ 42, respectively), which are recognized as toxic Aβ species.} The level of soluble oligomeric Aβ was reduced by 30%, and the amount of congophilic amyloid plaques was also relieved by 10%. Collectively, the above experimental results using two AD transgenic mouse models demonstrate that DAPPD significantly restores cognitive deficits and reduces the accumulation of Aβ species.

Example 4. Confirmation of neuroinflammation reduction by DAPPD administration in Alzheimer's disease mouse model

In order to investigate whether the decrease in the accumulation of Aβ species by DAPPD is due to the anti-inflammatory effect, the present inventors investigated the inflammatory response in the cerebral cortex (Cortex) and hippocampus of wild-type and APP/PS1 mice administered with vehicle or DAPPD. Microglia and astrocytes, the major resident immune cells involved in , were histologically monitored using anti-Iba1 and anti-GFAP antibodies, respectively (2 mg/kg/day, ip; 2 months; 7.5 months of age). As a result, as shown in FIGS. 3A and 3B , it was found that the activity of microglia increased by chronic neuroinflammation in APP/PS1 mice was decreased by DAPPD administration. Specifically, in both cortical and hippocampal regions, compared to APP/PS1 mice administered with vehicle, in APP/PS1 mice administered with DAPPD, the activity of microglia was reduced by approximately 50%, and the activity of astrocytes was approximately 30%. was confirmed to be reduced. In addition, as can be seen in Figure 3c, expression of proinflammatory cytokines (Tnf-α , Il-1β , Il-6 and iNos ) and immunomodulatory cytokines ( Il-10 ) in the cerebral cortex of mice administered with vehicle Although this increased, it was confirmed that it was reduced to a level similar to that of wild-type mice by administration of DAPPD. On the other hand, the levels of down-regulated anti-inflammatory markers ( Il-4 , Tgf-β and Arg1 ) in vehicle-administered APP/PS1 mice were increased in the DAPPD-administered group. Therefore, it can be seen from the above results that DAPPD can inhibit the chronic activation of microglia and astrocytes under neuroinflammatory conditions.

Example 5. Confirmation of anti-neuroinflammatory effect by DAPPD in microglia

In the brain, various types of cells are involved in the inflammatory process. In an immune response, signaling molecules (eg, cytokines) released from cells play a role in regulating intercellular communication and inflammatory processes. Accordingly, the present inventors cultured three types of primary mouse brain cells, i.e., microglia, astrocytes, and neurons, in order to examine the cells that induce the anti-inflammatory activity of DAPPD in the brain. Then, for the cultured cells, first 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium (2-(4-iodophenyl)- The toxicity of DAPPD to these cells was investigated by 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; WST-1) and TUNEL assays. As a result, it was confirmed that DAPPD did not induce specific cytotoxicity or apoptosis with respect to the three types of cells.

Next, it was attempted to examine the expression change of inflammatory markers induced by Aβ in the cells treated with or without DAPPD at the in vitro level. To this end, a conditioned medium (CM) containing a mixture in which various signaling mediators are secreted from the three types of cells is collected and either astrocytes (microglia-derived CM) or microglia (astrocyte- or neuron-derived CM). ) was treated. As a result, in the presence of _{Aβ 42} , it was found that the expression levels of proinflammatory markers (Tnf-α , Il-1β , Il-6 and iNos ) and immune regulatory cytokines ( Il-10 ) were increased in microglia, as well as However, it was confirmed that the level of anti-inflammatory markers (Il-4 , Tgf-β and Arg1 ) was reduced compared to the control group of microglia not cultured with _{Aβ 42 .} Through the above results, it was confirmed that the expression of inflammatory markers was restored to a level similar to that of the control group by concurrently treating the _{microglia with DAPPD and Aβ 42, indicating that DAPPD can alleviate the Aβ-mediated inflammatory response in microglia.} will indicate

In the case of astrocytes, it was confirmed that the inflammatory response was induced in astrocytes by confirming the change in the expression of inflammatory markers in the Aβ-treated microglia-derived conditioned medium, and cultured in the DAPPD-treated microglia-derived conditioned medium. It was confirmed that the astrocytes expressed inflammatory markers at a level similar to that of astrocytes cultured in the conditioned medium obtained from the control group. In contrast, in astrocytes, direct treatment with DAPPD did not appear to alleviate the inflammatory response induced by Aβ. Conditioned medium obtained from astrocytes cultured by co-treatment with Aβ ₄₂ and DAPPD induced an inflammatory response in microglia, suggesting that DAPPD cannot convert astrocyte-mediated regulation of inflammatory signaling in microglia. do. In addition, DAPPD inhibits neuronal "off" signals that maintain the resting state of microglia (i.e., Bdnf, Cd47 and Cd200) and most "on" signals that induce activation of microglia (i.e., Cxcl10 and Ccl21). It was found that the expression induced by Aβ could not be inhibited. Moreover, similar to the neuron-derived conditioned medium treated with Aβ, the DAPPD-treated neuron-derived conditioned medium induced an inflammatory response in microglia. Collectively, these results indicate that among the three types of cells tested in this study, DAPPD can directly reduce the inflammatory response induced by Aβ in microglia.

Furthermore, in order to confirm the change of microglia-specific inflammatory cytokines, the present inventors investigated the expression of pro-inflammatory and anti-inflammatory cytokines in microglia isolated from the cortex of wild-type and APP/PS1 mice administered with vehicle or DAPPD. analyzed. As a result, as shown in FIG. 3D, it was confirmed that it was consistent with the histological analysis result of the cerebral cortex of FIG. 3C. Therefore, it can be seen from the above results that the anti-inflammatory effect of DAPPD is due to the direct effect of microglia responsible for the propagation of inflammatory signaling.

On the other hand, microglia homeostasis suggests a functional signatures expressing microglia homeostasis M0- genes such as P2ry12, Tmem119, Tgfßr1 and Sall1 in a healthy brain. It is reported that during neurodegenerative processes, microglia lose homeostatic molecular signatures and upregulate inflammatory molecules, including Clec7a , Trem2 , Apoe and Gpnmb (MGnD microglia gene), resulting in a disease-associated phenotype. Based on these findings, the present inventors investigated the mRNA levels of the M0-homeostasis gene and the MGnD microglia gene in wild-type or APP/PS1 mice administered with vehicle or DAPPD. As a result, as shown in FIG. 3E , it was found that the levels of Clec7a , Trem2 , Apoe and Gpnmb were upregulated in cortical-derived microglia of APP/PS1 mice administered with vehicle compared to wild-type mice administered with vehicle. However, it was confirmed that the expression of these genes was significantly reduced in APP/PS1 mice administered with DAPPD. In addition, it was confirmed that the expression of the M0-homeostasis microglia gene was decreased in APP/PS1 mice compared to wild-type mice, but was significantly increased by treatment with DAPPD.

In addition, as a result of analyzing the protein levels of CLEC7A and P2RY12A up-regulated and down-regulated in APP/PS1 mice through immunofluorescence experiments, as shown in FIGS. 3f and 3g, APP/PS1 mice administered with vehicle It was confirmed that the expression of the CLEC7A and P2RY12A protein was significantly decreased and increased, respectively, in DAPPD-administered APP/PS1 mice compared to . These results show that DAPPD attenuates neuroinflammation by regulating the functional phenotype of microglia in APP/PS1 mice.

Example 6. Confirmation of promotion of phagocytosis of microglia by DAPPD in chronic neuroinflammation

The phagocytosis capacity of microglia presents an important aspect of the inflammatory response to external stimuli such as Aβ, and the persistent presence of external stimuli can induce chronic activation of microglia. The present inventors confirmed that the anti-inflammatory activity of DAPPD is derived from microglia through the results of the above examples. In vivo analysis was performed using confocal microscopy, and volume quantification of overlapping fluorescent regions through three-dimensional reconstruction gave a numerical representation of the phagocytic process of microglia.

Specifically, brain tissue sections from APP/PS1 mice (2 mg/kg/day, ip; 2 months; 7.5 months old) administered vehicle or DAPPD were co-administered with anti-Iba1 (microglia) and 6E10 (Aβ aggregates) antibodies. As a result of observation by immunostaining, it was found that the migration of Aβ-associated microglia was increased as shown in FIG. 4a . In addition, in order to more closely investigate the function of phagocytosis of microglia, Lamp1, a membrane protein expressed in lysosomes of microglia, was monitored together with Iba1+/6E10+ amyloid plaques. As a result, as can be seen in Figure 4b, the volume of osteoclasts of microglia in APP/PS1 mice administered with DAPPD compared to APP/PS1 mice administered with vehicle through the overlap region of the three fluorescence images, Aβ - It was found that the microglia occupied by Aβ in rod osteolysosome and osteolysosome increased approximately 2.8-, 1.3- and 2.3-fold, respectively.

In addition, the effect of DAPPD on the phagocytosis function of microglia was analyzed by analyzing the morphology of microglia, which is closely related to the function of microglia. As a result, as shown in FIG. 4c , it was observed that Aβ-associated microglia were in an amoeba-shaped form in APP/PS1 mice administered with DAPPD, indicating that DAPPD could enhance the phagocytosis of microglia on Aβ. indicating that there is As a result of quantifying the morphological parameters, it was confirmed that the morphology of microglia cultured with DAPPD was changed for the phagocytosis state. Specifically, the number of segments, terminal points and branch points as well as the volume and dendrite length of microglia by DAPPD treatment was reduced and the cell body size ( Cell body size) was confirmed to increase.

Finally, the size of Aβ aggregates was compared in DAPPD-administered APP/PS1 mice and vehicle-administered APP/PS1 mice. As a result, as shown in Figure 4d, the amount of small Aβ species (<25 μm) was increased when DAPPD was administered compared to when vehicle was administered, whereas the amount of larger Aβ aggregates (25-50 μm, >50 μm) was decreased. confirmed that. Therefore, from the above results, it can be inferred that the phagocytosis of microglia enhanced by DAPPD treatment increases the degradation of larger Aβ species.

In conclusion, the results of the coexistence of Aβ plaques and lysosomes in microglia, changes in microglia morphology, and changes in the size of Aβ aggregates in microglia by DAPPD treatment as described above show that DAPPD is phagocytic of microglia in APP/PS1 mice. To demonstrate that functional defects can be repaired.

Example 7. Confirmation of down-regulation of NLRP3 inflammasome protein through inhibition of DAPPD-mediated NF-κB pathway

NLRP3 inflammasome is a signaling mediator composed of NLRP3, ASC (apoptosis-associated speck-like protein containing CARD) and procaspase-1 . Activated NLRP3 inflammasome plays a role in cleaving procaspase-1 to produce caspase-1, and then promotes the maturation of IL-1β, a representative proinflammatory cytokine. A recent study reported a correlation between the NLRP3 inflammasome and inflammation induced by Aβ. In addition, the expression of NLRP3 in microglia affects brain Aβ deposition and cognitive function in AD transgenic mice. is known Therefore, regulating NLRP3 inflammasome formation in microglia is attracting attention as a promising strategy to reduce neuroinflammation in AD.

In order to understand the anti-inflammatory activity of DAPPD, the present inventors found that DAPPD is an NLRP3 inflammasome-related protein, i.e., NLRP3, ASC and IL, in wild-type and APP/PS1 mice administered with vehicle or DAPPD, respectively, based on the results of previous studies. The effect on the expression of -1β was investigated. As a result, as shown in Figure 5a, DAPPD was found to downregulate the expression of NLRP3, ASC and IL-1β, it was confirmed that the ratio of cleaved caspase-1 to procaspase-1 was significantly reduced. These results suggest that inhibition of the expression of NLRP3 and ASC by DAPPD may consequently decrease the activity of caspase-1 and IL-1β. In addition, the direct interaction between DAPPD and NLRP3 was analyzed through nuclear magnetic resonance spectroscopy, but no direct interaction was observed.

On the other hand, it is known that NLRP3 inflammasome formation is regulated mainly through the NF-κB pathway in the upper level, and specifically, when NF-κB signaling is activated, it can induce upregulation of NLRP3 and increase the level of NLRP3 inflammasome. has been reported NF-κB is a protein complex composed of P50 and P65 dimers and is involved in cell survival, inflammation and immune responses. Under normal conditions, NF-κB dimers are present in the cytoplasm in an inactive state by binding to a specific inhibitor protein called IκB. However, when NF-κB is activated by extracellular or intracellular pathogens, the NF-κB dimer is dissociated from the IκB protein, migrates from the cytoplasm to the nucleus, and then mediates the transcription of subgenes such as NLRP3 and inflammatory cytokines. In addition, endosomal rupture and release of cathepsin B are known as other upstream processes regulating the NLRP3 inflammasome. In AD, it is known that intracellular Aβ fibrils can induce the destruction of the osteoclast and activation of the NLRP3 inflammasome upon release of cathepsin B.

Based on the above study results, the present inventors wanted to investigate whether the NF-κB and cathepsin B pathways are upstream pathways related to DAPPD-mediated regulation of the NLRP3 inflammasome. To this end, first, vehicle or DAPPD was intraperitoneally administered to wild-type and APP/PS1 mice (7 to 7.5 months old) at 2 mg/kg/day for 2 months, and then cerebral cortex was obtained from the mice and Western blot was performed. . As a result, as shown in FIG. 5B , the expression levels of pNF-κB p50, pNF-κB p65 and pIκB were increased in the cortex of APP/PS1 mice administered with vehicle, whereas when DAPPD was administered, the NF-κB It was confirmed that the expression of the related protein was significantly reduced. In contrast, there was no significant difference in the expression of cathepsin B in APP/PS1 mice administered with DAPPD.

Collectively, through the above results, it was found that DAPPD inhibits the expression of NLRP3 by affecting the NF-κB pathway.

Example 8. in vitro Confirmation of the improvement effect of DAPPD on microglia function in the Alzheimer's disease environment

To evaluate the effect of DAPPD on microglia, we analyzed the function of DAPPD-mediated microglia by using human microglia in vitro. In addition, in addition to DAPPD, PBMA and AAP were treated on microglia to analyze their effects, and the correlation between structure-activity was investigated. To this end, human microglia were cultured with Aβ for 24 hours, then treated with DAPPD, PBMA or AAP, and further cultured for 24 hours. As a result of recovering the cells and performing western blotting, as shown in FIG. 6a , NLRP3 and ASC were upregulated in human microglia cultured with Aβ, and caspase-1 cleaved in the supernatant and It was confirmed that the secretion of IL-1β was increased. However, as a result of subsequent DAPPD treatment, the expression of NLRP3 and ASC was significantly reduced, and the secretion of cleaved caspase-1 and IL-1β was also significantly reduced. In contrast, cleaved caspase-1 and IL-1β were down-regulated in human microglia when treated with PBMA or AAP, but at a much weaker level than that of DAPPD. Moreover, as can be seen in FIG. 6b , when DAPPD was treated, the activation of NF-κB signaling induced by Aβ was inhibited and the release of cathepsin B was not inhibited, and PBMA or It was confirmed that this effect did not appear when AAP was treated. These results indicate that DAPPD can downregulate NLRP3 by effectively inhibiting the NF-κB pathway in microglia. In addition, the results of inducing a weak effect on microglia when treated with PBMA and AAP suggest that the acetamide group of DAPPD may be important for the molecular efficacy of restoring the function of microglia.

Next, human microglia treated with or without Aβ were treated with DAPPD, PBMA or AAP, and expression of M0-homeostasis and MGnD microglia genes as well as pro-inflammatory and anti-inflammatory cytokines were analyzed. As a result, as shown in FIG. 6c , the human microglia treated with Aβ showed that the expression of pro-inflammatory cytokines was up-regulated and the anti-inflammatory cytokines were down-regulated, and when DAPPD was treated, the inflammatory It was confirmed that the change in cytokine expression was restored. In addition, as shown in FIG. 6d , similar results by DAPPD were confirmed in the results of analyzing the M0-homeostasis and the expression levels of MGnD microglia genes. On the other hand, PBMA and AAP had little effect on restoring the abnormal expression of inflammatory cytokines and microglia genes by Aβ. These results suggest that DAPPD can restore neuroinflammation by regulating the functional phenotype of NLRP3 inflammasome and microglia in the in vitro AD environment.

Finally, the present inventors verified the effect of DAPPD on the phagocytosis of microglia. As a result of the experiment, as shown in Fig. 6e, human microglia treated with Aβ took significantly longer time to phagocytose on FITC beads, and the total number of phagocytosed FITC beads was significantly reduced. showed a decrease. However, when DAPPD was treated, it was found that the deficient phagocytosis ability of Aβ-treated human microglia was restored, whereas PBMA and AAP-treated human microglia did not show the above effect. Confirmed. Collectively, these results suggest that DAPPD has the ability to alleviate Aβ-induced dysfunction of microglia.

On the other hand, AAP is known to inhibit inflammation by inhibiting the activity of cyclooxygenase 1 (COX-1) or 2 (COX-2) in microglia. Therefore, in order to further confirm whether the anti-neuroinflammatory effect of DAPPD is a result of the effect on COX, the present inventors analyzed the expression changes of COX-1 and COX-2 according to DAPPD treatment in microglia. As a result, it was found that Aβ upregulated the expression of COX-1 and COX-2 in microglia, and when AAP was treated, the expression of COX-1 and COX-2 upregulated by Aβ was effectively inhibited. . However, it was confirmed that the expression level of COX did not change when DAPPD and PBMA were treated. In addition, DAPPD showed very weak inhibitory activity against COX-2 in vitro _{, and the IC 50} (half maximal inhibitory concentration) value was 22 ± 1 mM. These results suggest that the DAPPD-mediated functional restoration effect of microglia occurs through the regulation of the NLRP3 inflammasome and functional phenotype of microglia, not by the expression or activity of COX.

Example 9. Investigation of the effect of DAPPD on Aβ aggregation and expression of Aβ degrading enzymes

To demonstrate that the decrease in Aβ accumulation observed in the brains of AD transgenic mice administered with DAPPD is indeed a product of the anti-inflammatory activity of DAPPD, we present aggregation of the two major subtypes of Aβ, Aβ ₄₀ and Aβ ₄₂ , and Aβ -The regulatory effect on the expression of degrading enzymes was investigated at the level in vitro and in vivo. First, to investigate the effect of DAPPD on the inhibition of Aβ aggregate formation and the effect of DAPPD on the degradation of preformed Aβ aggregates, we analyzed through gel/western blot and transmission electron microscopy. It was confirmed that there was no significant change in size distribution and shape. That is, DAPPD _{did not change the aggregation of both Aβ 40} and Aβ ₄₂ , indicating that there was no direct interaction between the compound and Aβ.

In addition, enzymes responsible for Aβ degradation in brain samples of APP/PS1 administered vehicle or DAPPD (i.e., neprilysin [Nep], matrix metallopeptidase 9 [Mmp9] and insulin-degrading enzyme [ The expression of insulin-degrading enzyme; Ide]) was analyzed. The results showed that Nep, Mmp9 and Ide were reduced in vehicle-administered APP/PS1 mice compared to vehicle-administered wild-type mice. However, it was confirmed that the expression of the enzymes was also reduced in the mice administered DAPPD (2mg / kg / day, i.p.; 2 months; 7.5 months of age). These results suggest that DAPPD does not affect the production of Aβ-degrading enzymes.

Therefore, the present invention suggests that the reduction in the accumulation of Aβ species in the brain of AD transgenic mice is due to the restored phagocytosis of microglia by DAPPD treatment.

Example 10. Toxicity and efficacy comparison of Tylenol drug and DAPPD

The present inventors tried to verify the excellent efficacy of the DAPPD compound according to the present invention by conducting a direct comparative experiment in terms of toxicity and efficacy with a drug Tyrenol, which is a main component of acetaminophen (AAP) similar to the structure of DAPPD.

10-1. Toxicity comparative analysis

In order to compare and analyze the single-dose toxicity of DAPPD and Tylenol drugs in vivo, the present inventors administered each drug once a day to rats by oral administration once a day before 11:53 according to a clinically scheduled route. The dose was calculated based on the fasting body weight measured on the day of the calculated administration, and the rats were fasted for 3-4 hours before administration to empty all gastric contents, and then administered directly into the stomach using a syringe tube equipped with a sonde for oral administration. Feed was re-feed approximately 1-2 hours after the end of administration.

_{As a result of analyzing the concentration (LD 50} ) causing 50% mortality in the test animal group for DAPPD and Tylenol, as shown in Table 2 below, Tylenol was 1,944 mg/kg, whereas DAPPD was >5,000 mg/kg. It was found that the concentration causing toxicity was significantly higher than that of Tylenol, indicating higher safety.

Compound Test Type Organism Route LD50 DAPPD LD ₅₀ rat oral > 5,000 mg/kg Tylenol LD ₅₀ rat oral 1,944 mg/kg

10-2. Comparative analysis of amyloid-beta reduction effect

In order to compare the effects of the two drugs on amyloid-beta deposition in a mouse model of Alzheimer's disease, the present inventors applied vehicle or each drug at 2 mg/kg/day for 30 days in 5xFAD mice (3.5 months old), an animal model of Alzheimer's disease. was intraperitoneally administered and the levels of _{Aβ 42} and Aβ _17-24 were measured in the brain tissues of the mice.

As a result, as shown in FIG. 7a , when Tylenol was administered compared to the control mice administered with the vehicle, the _{amounts of Aβ 42} and Aβ _17-24 were reduced, and when DAPPD was administered, compared to the case of administration of Tylenol. It was confirmed that it was reduced to a more significant level. From the above results, it can be seen that DAPPD according to the present invention is more effective in reducing the level of Aβ species than Tylenol.

10-3. Comparative analysis of the effect of improving cognitive function impairment

Finally, in order to compare and analyze the cognitive impairment improvement effect of DAPPD and Tylenol in the Alzheimer's disease mouse model, wild-type mice and 5xFAD mice (3.5 months old) administered with vehicle, Tylenol or DAPPD were subjected to the MWM test for 5 days. During the training period, the escape delay time was measured daily, and the time spent in each quadrant was measured and compared.

As a result, as shown in FIG. 7b , in mice administered with Tylenol, the escape delay time increased and the time remaining at the target site decreased, unlike wild-type mice. confirmed that. Through these behavioral study results, it was found that DAPPD had a significantly superior effect in improving the impaired cognitive function of Alzheimer's disease compared to Tylenol.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (10)

A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising a compound represented by Formula 1 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

According to claim 1,
The composition is characterized in that it restores the phagocytosis damage of microglia (Microglia), a pharmaceutical composition.
According to claim 1,
The composition is characterized in that for reducing neuroinflammation (Neuroinflammation) in the brain, a pharmaceutical composition.
According to claim 1,
The composition is characterized in that reducing the level of amyloid-beta in the brain, a pharmaceutical composition.
According to claim 1,
The composition is characterized in that it restores impaired cognitive function, a pharmaceutical composition.
A food composition for preventing or improving Alzheimer's disease, comprising a compound represented by Formula 1 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

7. The method of claim 6,
The composition is characterized in that to restore the phagocytosis damage of microglia, a food composition.
7. The method of claim 6,
The composition is characterized in that it reduces neuroinflammation in the brain, a food composition.
7. The method of claim 6,
The composition is characterized in that reducing amyloid-beta (Amyloid-beta) levels in the brain, a food composition.
7. The method of claim 6,
The composition is characterized in that it restores impaired cognitive function, a food composition.

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