KR20200109537A - trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea as an active ingredient for preventing and treating Parkinson's disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Parkinson′s diseases which contains trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)urea (UC2288) as an active component. The UC2299 of the present invention blocks the decrease in protein which affects the development of neurons through inhibition of p21 activity, thereby being able to be usefully used for preventing and treating Parkinson′s disease.

Description

A pharmaceutical composition for the prevention and treatment of Parkinson's disease using trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea as an active ingredient {trans- 1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea as an active ingredient for preventing and treating Parkinson's disease}

The present invention is trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl) )-3-(4-hydroxy-cyclohexyl)-urea, UC2288) as an active ingredient for the prevention and treatment of Parkinson's disease relates to a pharmaceutical composition.

Parkinson's disease (PD) is a common chronic neurodegenerative disease with reduced muscle vibration and ability to control mobility. Parkinson's etiology is associated with degeneration of dopaminergic neurons. The degeneration of these dopaminergic neurons decreases the dopamine content in the cerebral cortex. L-dopa therapy has been widely used in the treatment of Parkinson's disease since 1969, but the limitations of its efficacy and side effects are required to develop new target-oriented drugs. In addition, since dopaminergic drugs cannot easily pass through the cerebrovascular barrier, they must be administered systemically at high concentrations.Therefore, gene therapy for AADC (aromatic amino acid decarboxylase), GAD (glutamic acid decarboxylase) and GDNF (glial cell line-derived neurotrophic factor) is used. Have been used.

Reactive Oxygen Species (ROS) can cause serious damage to cellular structures, and are a major cause of neuronal cell death in Parkinson's disease. In an animal model of symptoms similar to Parkinson's disease, free radicals trigger activation of microglial cells that attack adjacent dopaminergic neurons.

Dj-1 (ananti-oxidative stress reaction and shaperone) knockout mice showed rapid loss of dopaminergic neurons in the black matter, and vitamin E, which removes several free radicals, is known to reduce the severity of Parkinson's disease by inhibiting lipid peroxidation. have.

The combination therapy of ascorbate and α-tocopherol is also used in patients with early Parkinson's disease. The neuroprotective effect of CoQ10 was confirmed in MPTP-induced mouse and monkey models. Several studies have shown an association of the mitogen activated protein kinase (MAPK) pathway in the development of Parkinson's disease.

The p38 MAPK activity was increased in a 6-OHD (A6-hydroxydopamine)-induced Parkinson's disease mouse model, which was also related to the mechanism of neuronal cell death.

It is known that α-Synuclein, a marker of Parkinson's disease, activates p38, ERK, and JNK pathways to produce IL-1β and TNF-α, thus promoting neuroinflammation in human microglial cells. Rotenone, which destroys dopamine-producing neurons, causing nervous system toxicity similar to Parkinson's disease, such as tremors, seizures, and confusion, induces the activities of p38 MAPK and JNK, which induce apoptosis in dopaminergic SH-SY5Y cell death. However, inhibition of JNK attenuated neuronal cell death and increased dopamine levels in both in vitro and in vivo models of Parkinson's disease.

In addition, JNK3 deficient mice were more resistant to the survival of dopaminergic neurons by MPTP than wild-type mice. In addition to pharmacological inhibition of the ERK pathway, treatment of dopaminergic cells with the 6-OHDA-activated ERK1/2 pathway also improved neuronal survival. As such, various studies have confirmed that ROS can induce activation of the MAPK pathway in neuronal cell death.

Parkin ubiquitinizes proteins to regulate various cellular processes with ubiquitin E3 ligase. Mutations generated during this process are known to be another cause of Parkinson's disease. Parkin knockout mice show normal function in several behavioral tests, but experience a lack of dopamine supply from the nerves and memory confusion. In addition, overexpression of Parkin protected acute α-synuclein-induced dopaminergic neurons in primary culture.

In Parkinson's disease, p21 has recently been shown to play an important role in the activation of inflammatory factors and MPP+-induced NF-κB in mouse microglial cells. Moreover, it was confirmed that the expression of p21ras was increased in the brain of MPTP-induced mice. In addition, the degradation of p21, which was increased in lymphocytes by Parkin's disease, was performed. It was recently shown that simvastatin inhibits p21 activation and prevents the loss of dopaminergic neurons in a mouse Parkinson animal model.

(001) Korean Patent Registration KR 10-1220182

Accordingly, the inventors of the present invention have shown that Parkin knockout in mice inhibits neurodevelopment through the decomposition of p21. Through the previous study, the accumulation of knockout Parkin failed to decompose p21 inhibits neurogenesis, resulting in neurodegenerative diseases such as Parkin's disease. Since it can cause the progression of P21, the present invention has been completed because an optimal material for inhibiting P21 was discovered.

The present invention is trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-) An object of the present invention is to provide a pharmaceutical composition for preventing and treating Parkinson's disease or a health food for improving Parkinson's disease using phenyl)-3-(4-hydroxy-cyclohexyl)-urea) as an active ingredient.

In order to achieve the above object, the present invention provides trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea represented by the following formula (1). It provides a pharmaceutical composition for preventing and treating Parkinson's disease as an active ingredient.

[Formula 1]

In addition, the present invention is a Parkinson containing trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea represented by Chemical Formula 1 as an active ingredient. Provide health food for disease improvement.

Trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl) of the present invention )-3-(4-hydroxy-cyclohexyl)-urea, UC2288) can be usefully used in the prevention and treatment of Parkinson's disease by blocking protein reduction that affects neuron development through p21 activity inhibition. .

1 is a graph showing the effect of MPTP on inhibiting behavioral disorders and cell death of UC2288.
Figure 2 is a graph showing the effect of UC2288 on ROS production, lipid peroxidation and inflammatory cytokine levels.
3 is a photograph and graph showing the effect of UC2288 on dopaminergic neurodegeneration.
4 is a photograph and graph showing the MAPK pathway in the reduced ROS generation in the rat brain.
5 is a photograph and graph showing UC2288 suppression of cell death and ROS production in vitro.
6 is a photograph and graph showing the results of inhibiting the translocation of p-p21 to UC2288 nuclear and MAPK activation.
7 is a graph showing the results of inhibition of MAPK inhibiting cell death and U0126 treatment reducing ROS damage caused by MPP+.

Hereinafter, the present invention will be described in detail.

The present inventors noted that the development of the nervous system was slow in the Parkin knockout model, and predicted that the accumulation of P21 reduces the protein that affects the development of neurons, resulting in disorders in the development of the nervous system, thereby causing Parkinson's disease, so that p21 is the target (Target). As a result of discovering a pharmaceutical composition for the treatment and prevention of Parkinson's disease, trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans Since it was confirmed that -1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy cyclohexyl)-urea) is an optimal material for inhibiting P21, the present invention was completed.

The present invention is trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-) It provides a pharmaceutical composition for the prevention and treatment of Parkinson's disease using phenyl)-3-(4-hydroxy-cyclohexyl)-urea) as an active ingredient.

The trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea is represented by the following formula (1).

Trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl) -3-(4-hydroxy-cyclohexyl)-urea) may be referred to as "UC2288" in the present invention.

Trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea inhibits the activity of p21, and when p21 activity is inhibited, neurons (Neuron) Blocks the production of inhibitory proteins, so it can prevent and treat Parkinson's disease.

Trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl) -3-(4-hydroxy-cyclohexyl)-urea) may further include a pharmaceutically acceptable salt thereof.

Preferably, the pharmaceutically acceptable salt is composed of sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt. It is specified that it may be one or more basic salts selected from the group, but is not limited thereto.

Preferably, the pharmaceutically acceptable salts are hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, citric acid, acetic acid, maleic acid, fumaric acid, glucoic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, Malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and aspartic acid may be one or more acidic salts selected from the group consisting of, However, it is stated that this is not limited thereto.

When the composition of the present invention is a pharmaceutical composition, it may be formulated as a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta, and cataplasma. On the other hand, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the active ingredient, and such a pharmaceutically acceptable carrier is commonly used in pharmaceutical formulations, lactose, dextrose, sucrose, sorbitol, Mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, Talc, magnesium stearate, mineral oil, and the like may be included, but are not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like as an additive.

The method of administration of the pharmaceutical composition is determined according to the degree of symptoms, and a topical administration method is usually preferred. In addition, the dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times a day.

Trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3- It provides health food for improving Parkinson's disease using trifluoromethyl-phenyl)-3-(4-hydroxy cyclohexyl)-urea) as an active ingredient.

The health food composition may be provided in the form of powder, granule, tablet, capsule, syrup, or beverage, and the health food composition is used with other foods or food additives in addition to the active ingredient, and appropriately used according to a conventional method. I can. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the active ingredient contained in the health food composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it is within the above range. It is clear that the active ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Example > Experimental materials and methods

<1-1> Manufacturing method of animal model

A Parkinson's disease mouse (C57BL6) model was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Each mouse in the MPTP group was administered MPTP 20mg/kg intraperitoneally 4 times for 7 days, and the CON concentration was administered with the same amount of physiological saline as in the normal group. In the UC2288 group, UC2288 10mg/kg, MPTP + UC2288 group, UC2288 10mg/kg and MPTP 20mg/kg were injected intraperitoneally 4 times for 7 days. The animal model was National approved by IACUC of CBNUR-1117-18, Chungbuk National University. It was carried out according to the guidelines of the Institute of Toxicological Research Korea and Drug Administration. 1-2 hours after the last injection, behavioral change experiments and biochemical experiments were performed.

<1-2> Behavioral change analysis

Behavioral changes were analyzed by Rota rod, pole test and gait test. Motor performance and tuning used a Rota rod treadmill (MED Associates Inc., St. Albans, VT) consisting of a 3.6 cm diameter cylindrical treadmill connected to a computer controlled stepper motor as previously described. The pole test was performed using a wooden stick with three rough surfaces on the animals, and the average of three experiments per animal was measured. The gait test measured the length of footsteps and the distance between successive footsteps on a bright runway (4.5cm wide) and a dark target box 20 × 17 × 10cm), and the data was repeated 5 times for each animal to derive an average value.

<1-3> Crecy Violet dyeing

Brains were fixed in 4% paraformaldehyde at 4° C. for 24 hours and then transferred to a 30% sucrose solution. A 25μm-sized brain section was washed with phosphate-buffered saline (PBS), removed the excess fixative, transferred to a gelatin-coated slide glass, stained with 0.1% cresyl violet (2-5 minutes), and the dorsal layer The cytostructural characteristics of the cortical layer of the isocortical region were confirmed. The brain sections were then washed with distilled water and then dehydrated for 2 minutes at each grade using ascending grade ethanol, 50, 70, 90 and 100% ethanol, followed by a 1:1 mixture of alcohol and xylene. Soak for minutes. Removed from xylene for 5-10 minutes and fixed in Cytoseal™ XYL (Thermo Scientific, Pittsburgh, CA).

<1-4> Oxidative stress analysis

The concentration of malondialdehyde (MDA) in brain tissue and nerve cells was analyzed based on TBA (thiobarbituric acid) reactivity (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer's guidance.

Specifically, after centrifuging the TBA reacted with the mixture of HaCaT keratinocytes, the supernatant was mixed with TBA. The reactor color was measured at 532 nm with a spectrophotometer.

The binding of H ₂ O ₂ to the molybdic acid complex was evaluated using a commercially available kit (DCFH-DA, Sigma Aldrich) with H ₂ O ₂ content at 405 nm, and then the H ₂ O ₂ content was calculated. GSH-Px activity was measured by a rate method using a GSH-Px kit (DCFH-DA, Sigma Aldrich). The change in absorbance during the conversion of GSH to GSSG was recorded with a spectrophotometer at 412 nm as GSH-Px activity. ARBEC's SOD production was evaluated with a superoxide anion assay kit (catalog number CS1000, Sigma, USA). Cells were cultured in serum-free medium and treated with 100 μM MPP ⁺ for 24 hours or with 10, 20, 50 nM UC2288. Cell suspensions (90 μl each) were added to a 96-well plate containing 5 μl of luminol solution (catalog number L5043) and 5 μl enhancer solution (catalog number E4281), and the samples were mixed with a micropipette. After a 15 minute incubation period, the luminescence intensity was measured with an Orion II Microplate Luminometer (Berthold, Germany).

<1-5> Western Blot analysis

Brain tissue was homogenized with a lysis buffer solution (PROPREP; iNtRON, Snamnam, Korea, n = 8 mice/group) and centrifuged at 2,500 x g for 15 minutes at 4°C. The same amount of total protein (40 μg) isolated from brain tissue was separated on an 8 or 10% sodium dodecyl sulfate polyacrylamide gel and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ). Membrane with anti-parkin, anti-ubiquitin, anti-ERK, anti-p-ERK, anti-p-P21, anti-p-P38, anti-P38, anti-p40 antibody and anti-p40 antibody at room temperature for 2 hours. Incubated at. Anti-JNK (anti-signaling Mol Neurobiol Technology) including anti-cleaved caspase-3 (1:1,000, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) and anti-cleaved caspase-3 (anti-cleaved caspase-3) , Inc., Beverly, anti-β-actin (1:2,500; Sigma, St Louis, MO).The blot was then blotted at room temperature for 2 hours with the corresponding peroxidase-binding anti-mouse or anti-rabbit antibody (1:2,000 ;Santa Cruz Biotechnology, Inc., Santa Cruz, CA) Immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) Western blotting detection system The relative density of the protein bands was My Image (SLB, Seoul) , Korea) using a densitometer and quantified with Lab Works 4.0 (UVP, Upland, CA).

<1-6> Immunohistochemistry and Immunity Fluorescence method

After incubation with p-p21 anti-mouse IgG antibody (1:1000 dilution; Vector Laboratories, Burlingame, CA, USA) and washing with paraffin-embedded tissue sections, avidin-conjugated peroxidase complex (ABC kit , 1:200 Vector Laboratories) tissue sections were stained with 3,3'-diaminobenzidine tetrahydrochloride (DAB, 0.02%) with a chromogen. And it is evaluated through an optical microscope (Olympus, Tokyo, Japan). For immunofluorescence, the sections were incubated with an anti-mouse secondary antibody labeled with Alexa-Fluor 568 (1:400 dilution, Invitrogen). Final images were acquired using a confocal laser scanning microscope (TCS SP2, Leica Microsystems AG, Werzlar, Germany).

<1-7> IL-6, IL- of proteins 1β And TNF -α level analysis

According to the manufacturing instructions, the levels of IL-6, IL-1β and TNF were measured by sandwich enzyme-linked immunosorbent assay (ELISA) (Li et al., 2018). The levels of IL-6, IL-1β and TNF in the blood were expressed as picograms of cytokines per milligram of protein.

<1-8> Cell culture and treatment

Pregnant SD rats are purchased from DBL Company 28 hours prior to use in experiments and placed in a quiet room. Embryos were removed from SD rats (DBL, Korea) who were pregnant (17 days) under intraperitoneal pentobarbitol (Sigma-Aldrich, UK) anesthesia. Cortices were dissected and exposed to a phosphate buffer (PBS) solution containing DNAase I (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and B27 (Invitrogen) for 5 minutes, followed by papain (10 U mL, Sigma-Aldrich, UK) ( Gibco, Thermo Fisher Scientific, Waltham, USA) and D-glucose (33 mmol/L, Sigma-Aldrich, UK). Subsequently, the cells were crushed and dissociated and filtered through a membrane (70 μm, BD Falcon). Cells were purified with BSA solution (8%, Sigma-Aldrich, UK) diluted with Neurobasal A-25 (Invitrogen, Thermo Fisher Scientific, Waltham, USA) (0.1 mg/mL, Sigma-Aldrich, UK). For each experiment, 8-12 embryos per mouse were mixed. The experiment was repeated 3 to 8 times. Cells were harvested with 2 mmol/L glutamine, 0.1% penicillin and streptomycin (Gibco, Thermo Fisher Scientific, USA), 250 U/mL amphotericin, Invitrogen, Thermo Fisher Scientific, Waltham, USA) and 1 mmol/L of lactic acid (Sigma-Aldrich, USA). UK) containing B27 medium (Invitrogen, Thermo Fisher Scientific, Waltham, USA) was added in poly-D-lysine coated dishes Neurobasal (Eurobio) at a density of 6× ^{10 5} cells/cm ² and cultured. The proportion of connective cells in the culture system of the present invention is determined to be 90% or more by cell morphology and immunostaining. Cells were cultured at 37° C. and 95% humidity with 5% CO _2, and when cells reached ~80% confluence, cells were treated with newly prepared MPP ⁺ in DMSO (final concentration 100 nM).

<1-9> TUNEL analysis

Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for TUNEL analysis according to the manufacturer's instructions.

After fixing a 25 mm sheet with ₄ % paraformaldehyde, 0.1% NaBH ₄ and 0.1 Triton X-100, the slides were incubated with a mixture reaction buffer containing deoxynucleotide transferase and FITC-dUDP for at least 1 hour. (Roche, Reinach, Switzerland).

The slides were incubated for 15 minutes in the dark at room temperature with a mounting medium for fluorescence containing DAPI (Vector Laboratories, Cambridgeshire, UK) for 40, 60-diamidino-2-phenylindole dihydrochloride (DAPI) staining. The tissue was examined through a fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany), and the nuclei were visualized through DAPI staining.

<1-10> Statistical analysis

GraphPad Prism 4 software (GraphPad Software, La Jolla, CA) was used for statistical ananlysin. One-way analysis of variance (ANOVA) was applied to evaluate the differences between the graphs. When significance was found, the differences were further analyzed with Dunnett's test. Data are expressed as mean ± standard deviation (SD), and values of P <0.05 are considered statistically significant.

< Example 2> Experiment result

<2-1> MPTP Analysis of the Effect of UC2288 on Behavioral Disorder

The effect of UC2288 on MPTP-induced behavioral disorder was investigated. Rota rod analysis was performed to analyze the coordination ability. The MPTP treatment group significantly reduced the waiting time of the Rota rod. However, mice treated with MPTP and UC2288 (10mg/kg) at the same time (44.7±49.5s) had a reduced delay time compared to MPTP-treated mice (94.8±32.5s) (Fig. 1A).

In addition, a pole inspection was performed to measure the time from the top of the pole to the bottom. Time reflected bradykinesia. There was no significant difference between the control group and the UC2288 administration group in terms of behavioral disorders. However, UC2288 (10mg/kg) and MPTP-treated mice (22.8±6.49s) significantly reduced the descent time compared to MPTP-treated mice (41.0±1.0s) (Fig. 1A). In the mice treated with UC2288 and MPTP at the same time (4.9±0.6cm), the hind limb stride was longer than that of the MPTP-treated mice (4.5±0.3cm) (Fig. 1A). However, no significant difference was found in terms of behavioral disorder in MPTP-treated mice and UC2288 and MPTP-treated mice.

<2-2> in the mouse brain MPTP Analysis of the effect of UC2288 on induced cell death

Cresyl violet staining was used to evaluate cell death of striatum. As a result of comparing with the number of stained cells indicated by Cresile purple, the MPTP-treated mice were 691.658/cm ² in the UC2288 and MPTP-treated mice compared to 390.29/cm ^2. UC2288 inhibited the death of nerve cells by MPTP (Fig. 1B).

In addition, Western blot data on the expression of BAX and cleave-caspase3 (a pro-apoptotic protein) showed that the expression of BAX and cleave-caspase3 was reduced in the mouse brain treated with UC2288 and MPTP at the same time compared to the mouse brain treated with only MPTP. (Figure 1C).

<2-3> ROS Analysis of the effect of UC2288 on production and lipid peroxidation

MPTP is well known to induce oxidized lipids by forming high ROS levels, which is an important factor in neuronal cell death. Therefore, ROS production and lipid oxidation were examined in MPTP-treated mouse brains.

As a result, ROS levels were elevated by MPTP, but ROS elevated with treatment of UC2288 (10 mg/kg) decreased. There was no significant difference in ROS levels between the control group and the UC2288 (10 mg/kg)-treated mouse group (Fig. 2A).

In addition, MDA levels are the determining factor in lipid peroxidation. MDA levels were also significantly higher in the MPTP treatment group, but decreased with UC2288 treatment. There was no significant difference in MDA levels between the control group and the UC2288 (10 mg/kg)-treated mouse group (Fig. 2A).

In addition, the normal cell state maintains the antioxidant state by effectively removing oxidative stress. When the oxidative stress is excessively produced, the intracellular antioxidant glutathione (GSH) is converted into oxidized glutathione (GSSG) and accumulated in the cells, so the higher the GSH/GSSG ratio, the more the antioxidant state is maintained. Compared with MPTP-treated mice, UC2288 treatment increased the GSH/GSSG ratio (FIG. 2A).

<2-4> Analysis of the effect of UC2288 on cytokine levels

TNF-α, IL-6 and IL-1β all play an important role in neurodegenerative diseases, and cytokines can be released by oxidative stress of nerve cells, and thus cytokine levels were investigated.

As a result, MPTP treatment increased TNTP-α, IL-6 and IL-1β levels in mouse brains. However, TNF-α and IL-6 secretion was significantly reduced by UC2288 treatment in MPTP-treated mice, but IL-1β was not (Fig. 2B). There was no significant difference in cytokine levels between the control and UC2288 (10 mg/kg) treated mice.

<2-5> Dopaminergic neurodegeneration ( dopaminergic neurodegeneration Analysis of the impact of UC2288 on)

In striatum and substantia nigra, TH-positive fibers were high in the control group and UC2288-treated group, but the number of TH-positive neurons was lower in the striatum and black matter of MPTP-treated mice. However, the number of TH-positive neurons was recovered in MPTP-treated mice further treated with UC2288 (Fig. 3A).

In addition, GFAP reactivity decreased after UC2288 treatment in MPTP-treated mice (68.79 vs 53.43 cells/mm ² ) (Fig. 3A).

Further treatment with UC2288 increased dopamine levels, which are associated with the recovery of TH positive cells, from 1.40 ng/ml to 2.55 ng/ml (Fig. 3B).

<2-6> MAPK Analysis of the impact of UC2288 on pathway activation

It was speculated that MPTP-induced ROS production might be related to the activation of the MAPK pathway. The activation of the MAPK pathway was increased in MPTP-treated mice (Fig. 4A). UC2288 and p21 inhibitors inhibited the phosphorylation of p38 and ERK in the brain in MPTP-treated mice (Fig. 4A).

In addition, the expression of MPTP-elevated iNOS and COX-2 was decreased by the treatment of UC2288 (Fig. 4B).

<2-7> Analysis of the effect of UC2288 concentration on neuronal cell death in vitro

Treatment of MPTP causes dopaminergic neurodegeneration. Exposure to MPTP for up to 24 hours causes cell death in cultured neurons. As a result of the MTT analysis, when UC2288 (10, 20 and 50 nM) was treated on the neurons exposed to MPTP, the neuronal cell viability was increased in a concentration-dependent manner (FIG. 5A).

In addition, in order to investigate apoptosis of neurons, TUNEL analysis in primary cultured neurons showed that TUNEL-positive apoptotic cells in the MPTP group were significantly higher in the MPTP-treated group, but the number of cells killed by the treatment of UC2288 decreased. And, as the treatment concentration of UC2288 increased, the number of dead cells significantly decreased (FIG. 5B).

In addition, ROS production in whole cells was also quantified as an indicator of MPTP-induced oxidative stress. MPTP exposure of 100 μM for 24 hours increased H ₂ O ₂ and decreased GSH levels, but the group treated with UC2288 recovered to the previous level, and recovered significantly as the treatment concentration of UC2288 increased (FIG. 5C ). .

<2-8> p21 expression in vitro and MAPK Analysis of the effect of UC2288 concentration on the pathway

In the first cultured neuron, UC2288 and MPP+ were treated together. There was no significant difference with UC2288 (50 nM) treatment alone. However, MPP+ treatment increased the expression of p-p21, and UC2288 decreased its expression (Fig. 6A).

In addition, the expression of MAPK group, p-ERK, p-JNK, p-P38, iNOS, and COX-2 by MPK+ increased in early cultured neurons, but decreased with UC2288 treatment, and was significant as the treatment concentration of UC2288 increased. Decreased to (Fig. 6B).

In the MAPK group, phosphorylation of ERK induced by MPP+ was decreased by UC2288, and significantly decreased as the concentration of UC2288 increased (FIG. 6B ).

<2-9> ERK Inhibition of the pathway is MPP+ induced ROS And analysis of the recovery effect of p21 inhibitors on IL-6

Treatment with MPP+ (100 μM) for 24 hours causes cell death in cultured neurons. However, inhibition of ERK kinase (U0126) increased cell viability. Inhibition of ERK reduced cell death when U0126 was treated with 5 and 10 μM (Fig. 7A).

SB203580 is a p39 inhibitor, and SP600125 is a JNK inhibitor, and because UC2288 exhibits efficacy through MAPK, it was confirmed that the ERK inhibitors were the most affected by treatment with ERK, p38, and JNK inhibitors, which are MAPK components (Fig. 7A).

In addition, the abolition of the ERK pathway by U0126 treatment further increased the expression of H ₂ O ₂ and decreased cell survival, but did not change the level of IL-6, whereas UC2288 inhibited the expression of IL-6. It was confirmed that the regression was suppressed (Fig. 7B).

Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (6)

Trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl)- Parkinson's disease prevention and treatment pharmaceutical composition containing 3-(4-hydroxy-cyclohexyl)-urea) as an active ingredient. The Parkinson according to claim 1, wherein the trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea is represented by Formula 1 below. Pharmaceutical composition for disease prevention and treatment:
[Formula 1]

. The method of claim 1, wherein the trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea inhibits the activity of p21. Pharmaceutical composition for disease prevention and treatment. The pharmaceutical composition for preventing and treating Parkinson's disease according to claim 3, wherein when the activity of p21 is inhibited, the production of neurons inhibiting protein is blocked. Trans-1-(4-Chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea (trans-1-(4-Chloro-3-trifluoromethyl-phenyl)- A health food for improving Parkinson's disease using 3-(4-hydroxy cyclohexyl)-urea) as an active ingredient. The Parkinson according to claim 5, wherein the trans-1-(4-chloro-3-trifluoromethyl-phenyl)-3-(4-hydroxy-cyclohexyl)-urea is represented by the following formula (1). Health food for disease improvement:
[Formula 1]

.

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