KR102005030B1 - Pharmaceutical Composition for preventing or treating neurodegenerative diseases comprising proteasome inhibitor as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a neurodegenerative disease of a tetrapeptide epoxyketone compound which is a proteasome inhibitor. The tetrapeptide epoxyketone compound (YU102) as a proteasome inhibitor according to the present invention has an effect of improving memory damage and an effect of reducing brain tissue nerve inflammation in the brain of an animal model of Alzheimer's dementia, and a composition comprising the compound as an active ingredient And can be usefully used as a composition capable of preventing / improving or treating neurodegenerative diseases including Alzheimer's disease.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating a neurodegenerative disease comprising a proteasome inhibitor as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating a neurodegenerative disease of a tetrapeptide epoxyketone compound which is a proteasome inhibitor.

Degenerative neurological disorders are degenerative changes in the nervous system of the central nervous system that cause various symptoms such as impaired motor and sensory function, memory, learning, and inhibition of high-order causative functions such as computational reasoning. Representative diseases include Parkinson ' s disease, Alzheimer ' s disease, and memory disorders. Degenerative neurological disease is characterized by necrosis or apoptosis, which causes rapid or slow progression of nerve cell death. Therefore, the understanding of the mechanism of death of neurons must be made in order to develop the prevention, control and treatment of central nervous system diseases.

The central nervous system consists of neurons and glial cells. The glial cells are the most distributed cells in the brain, accounting for 90% of total brain cells and 50% of the total brain volume. These cells, by themselves, do not produce nerve impulses, but they are known to play a very important role in helping neurons perform their functions and to restore them when brain tissue is damaged. The glial cells are again composed of three kinds of astrocytes, microglia and oligodendrocytes.

Among these, microglia are also referred to as microglial cells or microglia, and as immune cells which are supposed to be in the central nervous system (CNS) (Stoll and Jander, Prog Neurobiol 58: 233-247, 1999; Kim and de Vellis, J Neurosci Res 81: 302-313, 2005), which accounts for 5-10% of total brain cells.

The microglia function as a primary defense line in the CNS. Microglial cells are a major intracellular source of inflammatory mediators in the CNS. Microglial cells are involved in nerve inflammation by producing nitric oxide (NO), reactive oxygen species (ROS), proinflammatory cytokines and prostaglandins. Activated microglial cells migrate to the damaged neural tissue area and predispose to and destroy microorganisms and cell debris (Gehrmann et al., Brain Res Rev 20: 269-287, 1995).

However, the role of microglial cells as inflammatory cells is not always beneficial. Currently, the activation of uncontrolled microglia and persistent excessive neuroinflammation are thought to be the cause of various CNS pathologies, including neurodegenerative diseases (Gonzalez-Scarano and Baltuch, Annu Rev Neurosci 22: 219-240, 1999 ). In other words, it is known that functionally activated microglial cells produce and secrete inflammatory mediators, resulting in neuronal cell death. Thus, activation of microglial cells is associated with various types of degenerative neurological diseases. For example, it is well known that microglia play an important role in the formation of senile plaques in Alzheimer's disease (Wegiel, J. et al., Acta Neuropathol (Berl), 100 (4): 356-64,2000). (Weggen, S. et al., Nature, 414: 212-216, 2001; Wyss-Coray, T. and Lim, GP et al., J. Neurosci 20: 5709-5714, 2000).

On the other hand, it is known, or is believed, that various diseases or diseases are mediated by the catalytic function of proteasome. Proteasome inhibition has been proposed as a preventive and / or therapeutic treatment of a number of diseases. For example, proteasome inhibition has been clinically proven as an antitumor therapy and is known to be useful in reducing the degradation of muscle proteins and therefore in inhibiting muscle loss and fibrosis.

Accordingly, the present inventor sought to find a substance having an excellent effect for preventing or treating neurodegenerative diseases such as Alzheimer's disease, and to confirm the possibility of a proteasome inhibitor as an Alzheimer's disease drug through inhibition of neuroinflammation. In other words, we focused on the function and role of glial cells, which occupy the highest percentage of human brain, rather than developing therapeutic agents focused on the existing beta amyloid hypothesis for the treatment of Alzheimer's disease. The aim of this study was to investigate the effect of immunoproteasome inhibitor on the immune response by microglia. As a result, the inventors of the present invention completed the present invention by confirming the effect of reducing the neuronal inflammation in the brain tissue, as well as the effect of improving the memory damage in the experimental group in which the tetrapeptide epoxyketone compound YU102, which is a proteasome inhibitor,

Korean Patent Publication No. 2002-0084311 Korean Patent No. 10-0635451

Therefore, an object of the present invention is to provide a pharmaceutical composition useful for prevention or treatment of neurodegenerative diseases including Alzheimer's disease, which is stable to human body and has excellent neuroinflammatory activity even after long-term use.

In order to achieve the above-mentioned object of the present invention,

The present invention provides a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising, as an active ingredient, a compound of the following general formula (1) or a pharmaceutically acceptable salt thereof.

≪ Formula 1 >

The compound of formula (I) of the present invention is a compound of molecular formula C ₂₇ H ₃₈ N ₄ O ₆ , molecular weight of 514.6230.

In one embodiment of the present invention, the neurodegenerative disease is selected from the group consisting of alcoholism, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, HIV-associated dementia, Lewy body dementia, multiple sclerosis, Prion disease, Niemann-Pick Type C (NPC), and epilepsia ) ≪ / RTI > disease.

In one embodiment of the present invention, the compound may have the effect of preventing or treating neurodegenerative diseases through inhibition of neuroinflammation of brain tissue.

The tetrapeptide epoxyketone compound (YU102) as a proteasome inhibitor according to the present invention has an effect of improving memory damage and an effect of reducing brain tissue nerve inflammation in the brain of an animal model of Alzheimer's dementia, and a composition comprising the compound as an active ingredient And can be usefully used as a composition capable of preventing / improving or treating neurodegenerative diseases including Alzheimer's disease.

FIG. 1A is a simplified schematic diagram illustrating the process of evaluating the memory improvement effect after administration of the proteasome inhibitor YU102 peritoneal to an animal model of memory impairment by APPsw gene transplantation.
FIG. 1B is a graph showing the escape distance measured by the Morris water maze test for the memory-enhancing test according to the administration of the proteasome inhibitor YU102 in an animal model of memory impairment by APPsw gene transplantation.
FIG. 1C is a graph showing the escape delay time measured by Morris water maze test for the memory-enhancing test according to the administration of the proteasome inhibitor YU102 in an animal model of memory impairment by APPsw gene transplantation.
FIG. 1D is a graph showing the average time taken for a target quadrant by a probe test for memory-enhancing test according to the administration of a proteasome inhibitor, YU102, in an animal model of memory impairment by APPsw gene transplantation.
FIG. 1E is a graph showing the results of a manual-avoidance experiment for the memory-enhancing test according to the administration of the proteasome inhibitor YU102 in an animal model of memory impairment by APPsw gene transplantation.
FIG. 2A is a schematic diagram illustrating the process of evaluating the memory-enhancing effect after administration of YU102, a proteasome inhibitor, and LPS injection into an experimental animal mouse.
FIG. 2B is a graph showing the escape distance measured by the Morris water maze test for the memory-enhancing test according to the administration of the proteasome inhibitor YU102 in an animal model of memory impairment by LPS.
FIG. 2c is a graph showing the escape delay time measured by Morris water maze test for the memory-enhancing test according to the administration of YU102, a proteasome inhibitor, to the memory impaired animal model by LPS.
FIG. 2d is a graph showing the average time taken for the target quadrant by a probe test for the memory enhancement test according to the administration of the proteasome inhibitor YU102 in an animal model of memory impairment by LPS.
FIG. 2E is a graph showing the results of the manual-avoidance experiment for the memory-enhancing test according to the administration of the proteasome inhibitor YU102 in the animal model of memory impairment by LPS.
FIG. 3A is a photograph showing the number of GFAP and Iba-1 positive cells according to administration of YU102, which is a proteasome inhibitor, in an animal model of memory damage caused by APPsw gene transplantation through immunohistochemical staining.
FIG. 3B is a Western blot analysis showing the expression levels of iNOS, COX-2, GFAP and Iba-1 protein by administration of a proteasome inhibitor YU102 in an animal model of memory impairment by APPsw gene transplantation.
FIG. 4A is a photograph showing the number of GFAP and Iba-1 positive cells according to administration of YU102, which is a proteasome inhibitor, in an animal model of memory impairment by LPS through immunohistochemical staining.
FIG. 4B is a Western blot analysis showing the expression levels of iNOS, COX-2, GFAP and Iba-1 protein according to administration of YU102, a proteasome inhibitor, in an animal model of memory impairment caused by LPS.
FIG. 5A shows the amount of Aβ _{1 -42} protein accumulated in the brain tissue by administration of YU102, which is a proteasome inhibitor, by thioflavin T staining in an animal model of memory impairment by APPsw gene transplantation.
FIG. 5B shows the amount of A? _{1 -42} protein accumulated in brain tissue by ELISA after administration of YU102, a proteasome inhibitor, to an animal model of memory impairment caused by APPsw gene transplantation.
FIGS. 6A and 6B are Western blot analysis results of LPS-stimulated astrocytes, iNOS, COX-2, GFAP and Iba-1 protein expression in microcapsules treated with YU102, which is a proteasome inhibitor.

The present invention relates to a novel use of a proteasome inhibitor, YU102, which provides a composition for preventing, ameliorating or treating a neurodegenerative disease which comprises, as an active ingredient, a YU102 compound which is a proteasome inhibitor or a pharmaceutically acceptable salt thereof. .

The YU102 of the present invention is a tetrapeptide epoxyketone compound of the following formula (I) as a proteasome inhibitor.

≪ Formula 1 >

The YU102 compound according to the present invention can be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The YU102 compound according to the present invention may be a commercially available product or may be prepared by a chemical synthesis method known in the art.

The composition of the present invention is a pharmaceutical composition comprising the above YU102 compound or a pharmaceutically acceptable salt thereof as an active ingredient and can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, As the auxiliary agent, an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the active ingredient (YU102 compound or a pharmaceutically acceptable salt thereof) of the present invention may be contained in the composition at a concentration of 1 to 1000 μM, 0.1 to 95% by weight based on the total weight of the composition.

Neurodegenerative diseases in which the pharmaceutical composition of the present invention may exhibit therapeutic effects include Alcoholism, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, HIV-associated dementia, Lewy body dementia, multiple sclerosis, Prion disease, Niemann-Pick Type C, NPC ) And epilepsia diseases, but the present invention is not limited thereto.

In addition, the present invention provides the use of a composition comprising, as an active ingredient, a YU102 compound or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of neurodegenerative diseases. The composition of the present invention comprising the YU102 compound as an active ingredient can be used for the preparation of medicines for the prevention or treatment of neurodegenerative diseases.

The present invention also provides a method for the prevention or treatment of neurodegenerative diseases comprising administering to a mammal a compound of YU102 or a pharmaceutically acceptable salt thereof.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like. In the treatment method of the present invention, in the case of an adult, administering the compound of the present invention or a pharmaceutically acceptable salt thereof at a dose of 0.01 mg / kg to 250 mg / kg once to several times a day desirable.

In the therapeutic method of the present invention, the composition comprising the YU102 compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally, rectally, intravenously, intraarterally, intraperitoneally, intramuscularly, Can be administered in a conventional manner via intraocular or intradermal routes.

The present invention also provides a health functional food for preventing or ameliorating a neurodegenerative disease, which comprises a YU102 compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and improving degenerative neurodegenerative diseases.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant and other additives, granulating the mixture in a conventional manner, The mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of an active ingredient of the present invention and an additive such as an excipient and the soft capsule is prepared by mixing the active ingredient with an additive such as an excipient And filling the mixture into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The health functional food of the ring form can be prepared by molding a mixture of the active ingredient of the present invention and an excipient, a binder, a disintegrant, etc. by a conventionally known method, and if necessary, Alternatively, the surface may be coated with a material such as starch or talc.

The granular health functional food may be prepared by granulating a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and may contain a flavoring agent, have.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

Preparation of experimental animals

In this experiment, NSE-hAPPsw transgenic mice (9 months) in which hAPPsw gene was inserted into C57BL / 6 as an NSE-promoter were purchased from the Ministry of Animal Resources and Biotechnology (Osong, Chungcheongbuk-do, Korea, FDA). An 8-week-old ICR mouse was purchased from Samtako (Osan, Korea). Mice were placed under specific pathogen-free conditions at 40-60% relative humidity and 21-24 ° C for 12 hours / arm cycles. All animals were adapted at least one week before the experiment. The experimental method used in this experiment was approved by Chungbuk National University Animal Experimental Ethics Committee.

Preparation of other experimental materials

YU102 and YU102 isomers were synthesized in a laboratory of Professor Kim Kyungbo of the College of Pharmacy, University of Kentucky. Antibodies to iNOS, COX-2, and GFAP were purchased from Santa Cruz Biotechnology Inc (CA, USA). Antibodies against Iba-1 were purchased from Wako (Japan).

< Example  1>

APPsw  Effect of YU102 on memory impairment by gene transplantation

In this experiment, we investigated the memory effect of YU102, a proteasome inhibitor, in an animal model of memory impairment by APPsw gene transplantation.

<1-1> Morris water maze test

Memory impairment by APPsw transplantation An animal model was administered with YU102 at a dose of 10 mg / kg / day for 3 weeks (6 times in total) intraperitoneally at intervals of 3 days. In order to see the memory improvement effect, the escape delay time and the distance were measured through the Morris water maze test (see FIG. 1A). All mice were tested three times a day for seven days.

As a result, as shown in FIGS. 1 b and 1 c, the control mice were trained as the test proceeded, indicating short travel distance and time. As time passed, the time and distance for escape of drug - treated group decreased more slowly than those of drug - treated group. As time passed, the time and distance of escape of YU102 isomer treated with negative control group was slower than that of the drug treated group. YU102 treated mice showed improved time and distance. In the last test, the mean swimming distance and escape time after the test in the untreated group were 530.5 ± 88.7 cm and 35.3 ± 3.8 sec, respectively. The mean swimming distance and escape time of the YU102 isomer injected mice were 518.3 ± 72.8 ㎝ and 36.3 ± 5.8 sec, respectively. However, YU102-treated mice were 255.8 ± 21.54 cm and 20.3 ± 2.3 seconds, respectively.

<1-2> Probe (probe) test

Morris water maze After a day of testing, a probe test was performed to measure the maintenance of memory function. The platform used for Morris water maze is removed from the pool and the mouse is allowed to swim freely. The swimming pattern of each mouse was monitored and recorded for 60 seconds using the SMART-LD program (Panlab). The average time required for the target quadrant is measured and the required time is calculated from the graph.

As a result, as shown in Fig. 1d, during this test, the mean time required for the target quadrant was 21.25? In the YU102 group mice compared to the untreated group mice (14.50? 1.03%) and YU102 isomer injected mice (16.13? 1.31% 2.71%), indicating a significant difference.

<1-3> Passive-avoidance test

In addition, a manual-avoidance test was conducted to measure the learning ability one day after the probe test. The passive-evasion test used a "step-through" device (Med Associates Inc., Vermont, USA) separated by illuminated and dark compartments (20.3 x 15.9 x 21.3 cm each) And the bottom of 3.175mm stainless steel rod is installed at intervals of 8mm. On the first day, the mice were placed in an illuminated compartment facing away from the darkroom for training purposes. If the mouse moves completely into the dark compartment, give an electric shock (0.45 mA, lasting 3 seconds). After a day of manual-avoidance test training, the mice were again placed in the illuminated compartment and the waiting time was measured until the mouse entered the dark room. The test was set at a cutoff time limit of 3 minutes.

As a result, as shown in FIG. 1e, the latency of the untreated group was 43.59 ± 13.43 sec, while that of the YU102 group was significantly restored to 128.10 ± 20.72 sec.

These results show that administration of YU102, a proteosome inhibitor, can alleviate memory impairment and improve memory in an animal model of memory impairment caused by APPsw gene transplantation.

< Example  2>

LPS  Effect of YU102 on memory impairment by memory

In this study, we investigated the memory effect of YU102, a proteosome inhibitor, on LPS-induced memory damage.

<2-1> Morris water maze test

YU102 was intraperitoneally administered at a dose of 10 mg / kg / day for 3 weeks (6 times) for 3 weeks, and intraperitoneally for 7 days at a dose of 0.25 mg / kg / day for 3 weeks. In order to see the memory improvement effect, the escape delay time and distance were measured by Morris water maze test (see FIG. 2a). All mice were tested three times a day for seven days.

The results, as shown in Figures 2b and 2c, indicate that the control mice are trained as the test progresses, indicating a short travel distance and time. The mice treated with LPS slowed down the time and distance required to escape with time as compared to the control group, and thus the memory was damaged compared with the control group. The mice treated with YU102 showed improved time and distance to escape compared to the LPS group. In the final test, the mean swimming distance and escape time after the control test were 193.0 ± 58.8 cm and 16.5 ± 5.5 sec, respectively. In comparison, the average swimming distance and escape time of mice injected with LPS were 676.7.8 ± 211.0 ㎝ and 40.2 ± 10.0 sec, respectively (Figs. 2B and 2C). However, the number of mice treated with YU102 and LPS was 388.8 ± 181.8 cm and 22.5 ± 8.1 seconds, respectively.

<2-2> Probe (probe) test

Morris water maze After a day of testing, a probe test was performed to measure the maintenance of memory function. The probe test was carried out in the same manner as in Example <1-2> above.

As a result, during this test, the mean time required for the target quadrant was decreased in the LPS-injected mice (10.58 +/- 0.25%) compared to the control mice (16.92 +/- 4.75%) and the proteasome inhibitor YU102 And 14.08 ± 3.28% after LPS treatment, respectively, and showed significant difference from LPS alone group.

<2-3> Passive-avoidance test

In addition, a manual-avoidance test was conducted to measure the learning ability one day after the probe test. The passive-avoidance test was conducted in the same manner as in the above-mentioned embodiment <1-2>.

As shown in FIG. 1e, the latency of the control group was 141.0 ± 19.50 seconds, while that of the LPS group was significantly reduced to 78.0 ± 8.50 seconds, and that of the group administered with the proteasome inhibitor YU102 was 100.00 ± 14.16 seconds, And recovered from the group treated with LPS.

These results suggest that administration of YU102, a proteosome inhibitor, can alleviate memory impairment and improve memory in an animal model of memory impairment caused by LPS.

< Example  3>

APPsw  Effect of YU102 administration on neuroinflammation in memory impairment model by gene transplantation

Activation of glial cells increases with amyloid biosynthesis and nerve inflammation during the course of Alzheimer's disease. In order to evaluate the effects of the proteosome inhibitor YU102 on the neuronal inflammation of the brain in the memory impaired animal model of APPsw gene transplantation, immunohistochemical staining was performed using GFAP (a marker of astrocytic activation) and Iba -1 (markers of microglia activation) were examined; In addition, the expression of inflammatory proteins (iNOS, COX-2), GFAP, and Iba-1 was examined by Western blot analysis.

<3-1> Immunohistochemistry staining method

Brain tissues fixed in 4% formalin were transferred to a 30% sucrose solution, and the brain was cut into 30 μm sections using a low temperature maintenance cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea). The brain slices were washed several times with PBS (pH 7.4) and then reacted in PBS containing 3% hydrogen peroxide for 30 minutes to inhibit the activity of endogenous peroxidase in the tissues, followed by washing in PBS for 10 minutes. Subsequently, the brain slice was incubated with mouse polyclonal antibody (1: 300, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) For 2 hours at room temperature, and then the brain slices were washed three times with PBS for 10 minutes each. After washing, brain slices were reacted with biotinylated goat anti-rabbit or goat anti-mouse IgG secondary antibody (1: 1000; Gene Tex Inc., USA) for 1 hour at room temperature. Brain sections were washed 3 times with PBS for 10 min and visualized with a chromogenic DAB reaction for up to 10 min. Finally, brain slices were dehydrated in ethanol, washed with xylenes equipped with Permount, and photographed with an optical microscope.

As a result, as shown in FIG. 3A, the number of GFAP and Iba-1 positive cells in the APPsw gene-transfected mice increased in the non-treated group, whereas in the mice treated with the proteasome inhibitor YU102, GFAP and Iba-1 positive cells And the number of cells was decreased.

<3-2> Western Blat  analysis

In order to investigate whether protease inhibitor YU102 could affect neuroinflammation in the brain, we performed Western blot analysis of iNOS, COX-2, GFAP and Iba-1 protein expression in relation to neuroinflammation Respectively.

A total of 40 g of protein was isolated from mouse brain tissue, and the same amount of protein was dissolved in 8-15% SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The membrane was reacted with a primary antibody (1: 1000; Santa Cruz Biotechnologies Inc., CA, USA) for iNOS, COX-2, GFAP, Iba-1 and β-actin at room temperature for 2 hours, (Anti-mouse or anti-goat antibodies (1: 5000; Santa Cruz Biotechnology, Inc., CA, USA) for 2 hours at room temperature . Immunoreactive proteins were detected by chemiluminescent Western blotting detection system.

As a result, it was confirmed that iNOS, COX-2, GFAP and Iba-1 levels were decreased in the brain of the rats injected with YU102, as shown in Fig.

< Example  4>

LPS  Effects of YU102 on neuroinflammation in memory impairment model

In order to evaluate the effects of the proteosome inhibitor YU102 on neuronal inflammation in the brain of LPS-induced memory damage, immunohistochemical staining was performed using GFAP (a marker of astrocytic activation) and Iba-1 The markers of microglia activation); In addition, the expression of inflammatory proteins (iNOS, COX-2), GFAP, and Iba-1 was examined by Western blot analysis.

Immunohistochemical staining was performed in the same manner as in Example <3-1>, and Western blot analysis was performed in the same manner as in Example <3-2>.

As a result, as shown in FIG. 4A, the number of GFAP and Iba-1 positive cells in the LPS-injected mouse was increased compared to that of the control group, whereas the number of GFAP and Iba-1 positive cells was decreased in the YU102-treated mouse .

In addition, levels of iNOS, COX-2, GFAP, and Iba-1 in western blot using mouse hippocampal tissues were increased in the brain of mice injected with LPS, and this level was decreased by YU102.

< Example  5>

APPsw  YU102 administration to memory impairment model by gene transplantation Aβ _{One -42}  Impact on Accumulation

This experiment was to evaluate the bit of proteosome inhibitors YU102 how affected the brain Aβ accumulation _{1 -42.} Because accumulation of A? _{1 -42} was implicated in memory dysfunction, we examined the effect of YU102 on the level of A? _{1 -42} using A? _{1 -42} specific ELISA analysis and thioflavin T staining in the brain of APPsw mice.

For thioflavin T staining, the brain was first cut into 30 μm sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea). Brain sections were washed with distilled water for 5 minutes. Then transferred to a gelatin-coated slice and immersed in 1% Tioflavin T for 5 minutes. Then, 50%, 70%, 90%, and 100% ethanol were dewatered by soaking for 2 minutes continuously. After dyeing, the sample was covered with a sealing agent (Fluoromount ™, Sigma, USA). The thioflavin T staining was performed using a fluorescence microscope.

For ELISA analysis, an ELISA kit was purchased from Invitrogen (CA, USA). Lysis of brain tissue was obtained through protein extraction buffer containing protease inhibitors. A [beta] 1-42 levels were measured after addition of stop solution as described elsewhere using each specific ELISA kit (CUSABIO) using a microplate absorbance reader (SunriseTM, TECAN, Switzerland). The degree of color reaction was measured by spectrophotometer at 450nm wavelength. The absolute value of Aβ contained in the sample was calculated by comparing the value of each sample with the standard value.

As a result, as shown in FIGS. 5A and 5B, it was confirmed that the number of Aβ _{1 -42} cells was not significantly different between the untreated group, the YU102 treated group and the YU102 isomer treated group in the APPsw mouse model.

Consequently YU102 is focused on is judged to be not related to the generation of amyloid, nerve inflammation and inhibit Aβ _{1 -42-independent} immune response.

< Example  6>

Astrocytes  And Microglia  Effect of YU102 on activation

The effects of YU102 on LPS (1 μg / ml) in astrocytes and microglial cells were confirmed by western blot, with emphasis on the function and role of several glial cells occupying the highest proportion in the brain.

The astrocytes and microcyst cells were prematurely cultured from 1 day old rats born in the SD rats immediately before birth, purchased from Samtako (Osan, Korea). One day old rats were anesthetized with ice, the neck separated and the separated brain was washed with PBS and the cerebral cortex was dissociated by mechanical digestion. The resulting cells were resuspended in serum-supplemented culture medium [F12, FBS (5%), DMEM supplemented with antibiotic-antibiotic (Gibco, Rockville, MD 100 μg / ml)] by centrifugation And cultured in T75flasks. The cells were cultured in a 5% CO2 medium at 37 DEG C for 9 days. After confluence (9 days), the flask was shaken at 37 DEG C for 16 hours. The cultures were treated with cytosine arabinoside for 48 hours and the medium was replaced with DMEM / F12HAM containing 10% FBS.

The cultured cells were treated with LPS (1 μg / ml) and YU102 at various concentrations (0.5, 1.5, and 4.5 μM) in DMSO and treated at the same time. Cells were harvested after 24 h and analyzed by Bradford assay The amount of protein in each group was determined to be 40 mg. Western blotting was performed on 12% and 15% SDS gels. Transfer the specific antibodies (GFAP, COX-2, Iba-1) to the nitrocellulose membrane at 4 ° C for 15 hours after transfer. After 15 hours, the mice are incubated at room temperature for 2 hours with peroxidase-conjugated anti-mouse, anti-rabbit, anti-chlorine antibody. Immunoreactive proteins were detected using a chemiluminescence Western blotting detection system.

As a result, as shown in FIG. 6, the degree of protein expression of iNOS, COX-2 and GFAP in astrocytes was increased in the LPS-treated group compared to the control group, and the effect of the YU102 treatment was observed to fall to the control level (See FIG. 6A). In addition, the degree of protein expression of iNOS, COX-2 and Iba-1 in the microglial cells was increased in the LPS-treated group compared to the control group, and the effect of the YU102 treatment was reduced to the control level (FIG.

These results show that when the proteosome inhibitor YU102 is administered to a memory impairment model (Alzheimer's dementia animal model), the memory improvement can be improved by reducing the amount of amyloid beta protein accumulated in the brain But it is confirmed that it is accomplished by inhibiting neuroinflammation of brain tissue.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (3)

A pharmaceutical composition for preventing or treating Alzheimer's disease comprising, as an active ingredient, a compound of the following formula (1) or a pharmaceutically acceptable salt thereof:
&Lt; Formula 1 >

delete The method according to claim 1,
Wherein said compound has the effect of preventing or treating Alzheimer ' s disease through inhibition of nerve inflammation of brain tissue.

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