KR102168228B1 - Novel 2-hydroxy-4-methylbenzoic anhydride and Composition for Preventing or Treating Neuroinflammation Disease Comprising the same - Google Patents

Abstract

The present invention relates to a novel 2-hydroxy-4-methylbenzoic anhydride (HMA) and a composition for preventing or treating neuroinflammatory diseases containing the same as an active ingredient. In the present invention, it has been confirmed that HMA not only inhibits the production of inflammatory factors including NO, iNOS and COX-2, but also effectively controls neuroinflammation through inhibition of expression of MAC-1, Iba-1, GFAP and COX-2 in experimental animal models of Parkinson′s disease, and thus HMA can be favorably used in a pharmaceutical composition for preventing or treating neuroinflammatory diseases.

Description

Novel 2-hydroxy-4-methylbenzoic anhydride and composition for preventing or treating neuroinflammation disease containing it as an active ingredient {Novel 2-hydroxy-4-methylbenzoic anhydride and Composition for Preventing or Treating Neuroinflammation Disease Comprising the same}

The present invention relates to a novel 2-hydroxy-4-methylbenzoic anhydride and a composition for preventing or treating neuroinflammatory diseases containing the same as an active ingredient, and more particularly, to NO generation of microglia and iNOS/COX- 2 It relates to a composition for preventing or treating neuroinflammatory diseases containing 2-hydroxy-4-methylbenzoic anhydride as an active ingredient, which has an effect of inhibiting neuroinflammation through inhibition of expression.

Neurodegenerative diseases are of major concern to researchers due to the lack of effective therapeutics, increased environmental exposure to chemicals, and predisposing genetic factors. The various types of neurodegenerative diseases have a specific set of neural circuits that undergo degeneration and cause specific disease symptoms (Teeling and Perry, Neuroscience, 158:1062-1073, 2009).

The body's immune system reacts delicately to the risk of internal or external stimuli. However, sustained or long-term immune responses in the central nervous system (CNS) eventually cause excessive nerve inflammation, damaging adjacent neurons. In the case of immune stimulation in the brain, microglia plays an important role and are macrophages that reside in the central nervous system. Microglia have sensitive reactions (infectious diseases, oxidative stress) and high mobility. When activated, these antigen-presenting cells produce various intermediate mediators (pro-inflammatory cytokines, chemokines, ROS) that can destroy pathogens, but can cause various inflammatory mediated neurodegenerative diseases.

Recent studies suggest that neuroinflammation is one of the key mechanisms involved in acute brain injury and chronic neurodegeneration. In particular, microglia-mediated neuroinflammation is associated with the pathogenesis of various neurodegenerative diseases including Parkinson's disease. Parkinson's disease is a progressive neurodegenerative motor disorder caused by the loss of dopaminergic neurons in the basal ganglia. According to previous reports, it has been reported that microglia activation induces dopaminergic neuron damage seen in Parkinson's disease (Alam, Q. et al. , Curr Pharm Des, 22:541-548, 2016). Therefore, the regulation of microglia activation can serve as an important therapeutic strategy to attenuate nerve inflammation.

Triflusal, derived from salicylic acid, has been used for centuries to treat cardiovascular and inflammatory diseases in Central and South America and several European countries. Triflusal and 2-hydroxy-4-trifluoro in previous studies Methyl benzoic acid (2-hydroxy-4-trifluoromethyl benzoic acid; HTB) is based on the inhibitory properties of cyclooxygenase-2 (COX-2) and nuclear factor kappa beta (NF-κB). It was confirmed to have neuroprotective properties (Fernandez De Arriba, A. et al. , Mol Pharmacol., 55:753-760, 1999). Metabolic deacetylation of triflusal produces HTB with antiplatelet drug efficacy (Anninos, H. et al. , Hellenic J Cardio., 50:199-207, 2009). HTB has also been reported to be effective in inhibiting inflammatory signaling molecules such as cAMP, NF-κB, VCAM-1 and E-selection (Hernandez, M. et al. , Br J Pharmacol, 132:547-555, 2001).

However, doses of 10 μM or more for both triflusal and HTB are limited in view of undesirable effects and safety profiles, and in particular, non-steroidal anti-inflammatory drugs (NSAIDs), warfarin And when a combination therapy with other therapeutic agents such as glisentide is required, use is limited (Mis, R., Ramis, J. et al. ,. Eur J Clin Pharmacol, 42:175- 179,1992). In addition, HTB derivatives OPTBA (HTB-pyruvate ester) and OPMBA (conversion of HTB-pyruvate ester) have been developed, but the derivatives have a lower anti-inflammatory effect than HTB and have a problem of showing cytotoxicity.

Accordingly, the present inventors have made diligent efforts to develop a new HTB anhydride derivative, and as a result, 2-hydroxy-4-methylbenzoic anhydride (HMA) was synthesized, and the 2-hydroxy-4 -It was confirmed that methylbenzoic acid anhydride showed a strong inhibitory effect on neuroinflammation control at the Parkinson's disease experimental animal model and cell level, and the present invention was completed.

An object of the present invention is to provide a novel compound, 2-hydroxy-4-methylbenzoic anhydride.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing or improving neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride as an active ingredient.

To achieve the above object,

The present invention provides a 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1).

[Formula 1]

According to a preferred embodiment of the present invention, the 2-hydroxy-4-methylbenzoic acid anhydride is 2-hydroxy-4-trifluoromethyl benzoic acid represented by the following formula (2). : HTB) may be derived.

[Formula 2]

In addition, the present invention is a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1) as an active ingredient Provides.

[Formula 1]

According to a preferred embodiment of the present invention, the 2-hydroxy-4-methylbenzoic acid anhydride is 2-hydroxy-4-trifluoromethyl benzoic acid represented by the following formula (2). : HTB) may be derived.

[Formula 2]

According to another preferred embodiment of the present invention, the 2-hydroxy-4-methylbenzoic acid anhydride can control neuroinflammation by inhibiting the production of inflammatory mediators including NO, iNOS and COX-2. have.

According to another preferred embodiment of the present invention, the 2-hydroxy-4-methylbenzoic acid anhydride may inhibit the expression of MAC-1, Iba-1 and GFAP proteins in substantia nigra pars compacta (SNpc).

According to another preferred embodiment of the present invention, the neuroinflammatory disease is multiple sclerosis, neuroblastoma, ischemic stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob's disease, post-traumatic stress disorder, depression, psychosis. It may be selected from the group consisting of schizophrenia or amyotrophic lateral sclerosis.

In addition, the present invention is a health functional food for preventing or improving neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1) as an active ingredient The composition is provided.

[Formula 1]

In the present invention, 2-hydroxy-4-methylbenzoic anhydride (HMA) not only inhibits the production of inflammatory factors including NO, iNOS and COX-2, but also in a Parkinson's disease experimental animal model. Since it was confirmed that it can effectively control neuroinflammation by inhibiting the expression of MAC-1, Iba-1, GFAP and COX-2, it can be usefully used in a pharmaceutical composition for preventing or treating neuroinflammatory diseases.

1 is data confirming the effect on HTB synthesis and BV-2 microglia viability and NO generation. A shows the synthesis process of the HTB derivative. B is data confirming the degree of NO generation when HMA (0.1, 1.0, and 10 μM) is treated, and C is data confirming the cytotoxicity of HMA on BV-2 cells. Data are expressed as mean ± standard measurement error value (n=3) ( ^### P <0.001, compared with control; ***P <0.001, compared with LPS alone treatment group).
FIG. 2 is data showing the effect of HMA on iNOS and COX-2 production in LPS-stimulated BV-2 microglia. A and B are data showing the level of iNOS and COX-2 mRNA expression, and C and D are data showing the level of iNOS and COX-2 protein expression. Data are expressed as mean ± standard measurement error value (n=3) ( ^### p <0.001, compared with control; *p <0.05, **p <0.01 and ***p <0.001, LPS Compared to treatment alone).
3 is data confirming the HMA effect on the expression of MAC-1, Iba-1, GFAP and COX-2 in MPTP poisoned mice. The protein levels of MAC-1 (A), Iba-1 (B), GFAP (C) and COX-2 (D) were confirmed by Western blot in SNpc. The results were expressed as the ratio of MAC-1/β-actin, Iba-1/β-actin GFAP/β-actin and COX-2/β-actin in the lower panel. Data are expressed as mean ± standard measurement error value (n=6) ( ^### p <0.001, compared with control; **p <0.01 and ***p <0.001, compared with MPTP alone treatment group ).

Hereinafter, the present invention will be described in detail.

The present invention relates to a 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1).

[Formula 1]

In the present invention, the 2-hydroxy-4-methylbenzoic anhydride is derived from 2-hydroxy-4-trifluoromethyl benzoic acid (HTB) represented by the following formula (2). It can be characterized.

[Formula 2]

Previous studies have shown that salicylic acid-related derivatives such as triflusal and HTB (2-hydroxy-4-trifluoromethyl benzoic acid) have anti-inflammatory effects, but doses of 10 μM or more have undesirable effects and safety profiles. , In particular, when combination therapy is required under certain disease conditions, there is a problem that its use is limited.

In the present invention, it was intended to synthesize a novel HTB derivative showing effective neuroinflammatory activity even at low concentrations. Figure 1A shows the synthesis process of the HTB synthetic derivative, in a specific embodiment of the present invention 2-hydroxy-4-methylbenzoic acid (2-hydroxy-4-methylbenzoic acid) and 2-hydroxy-4-methylbenzoic acid The active ester of 2-hydroxy-4-methylbenzoic acid is reacted with tetrahydrofuran (THF) containing dicyclocarbodiimide (DCC) to obtain 2-hydroxy-4-methyl Benzoic anhydride (2-hydroxy-4-methylbenzoic anhydride; hereinafter referred to as'HMA') was prepared.

In addition, the present invention is a pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1) as an active ingredient It is about.

[Formula 1]

The microglia stimulated by LPS in vitro and the MPTP-addicted mouse model of Parkinson's disease in vivo are considered as a standard effective model for investigating substances that inhibit neuroinflammation. In the present invention, the above two models were used to evaluate the effects and underlying mechanisms of HMA.

In the present invention, the 2-hydroxy-4-methylbenzoic anhydride may be characterized in that it regulates neuroinflammation by inhibiting the production of inflammatory mediators including NO, iNOS and COX-2. .

In a specific embodiment of the present invention, in order to confirm the efficacy of HMA to inhibit neuroinflammation, it was attempted to confirm whether HMA inhibits NO generation in neurons. As a result, it was confirmed that NO generation was inhibited in a concentration-dependent manner in LPS-stimulated microglia pretreated with 0.1, 1 and 10 μM of HMA as shown in FIG. 1B, and cytotoxicity appeared as shown in FIG. 1C. It was confirmed that it did not.

In another specific embodiment of the present invention, when HMA was treated by concentration in LPS-stimulated BV-2 microglia, the levels of NOS and COX-2 mRNA and protein expression were confirmed, and as shown in FIG. 2, HMA It was confirmed that the levels of iNOS and COX-2 mRNA and protein expression were suppressed in LPS-stimulated BV-2 microglia in a concentration-dependent manner, and as shown in FIG. 3, LPS-stimulated BV-2 microglia in a HMA concentration-dependent manner. It was confirmed that the production of pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α was inhibited in glial cells.

In the present invention, the 2-hydroxy-4-methylbenzoic acid anhydride inhibits the expression of MAC-1, Iba-1, and glial fibrillary acidic protein (GFAP) in substantia nigra pars compacta (SNpc). It can be characterized.

Microglia activity by MPTP-addiction is a well-known pathological characteristic of rodents. In another specific embodiment of the present invention, in order to confirm the HMA prophylactic effect in an MPTP-addicted mouse model of Parkinson's disease, MAC- 1, Iba-1 and GFAP protein expression levels were confirmed.

As shown in FIGS. 3A to 3C, as a result of MPTP-addiction, the expression of GFAP proteins increased in SNpc, whereas GFAP protein expression was increased in SNpc as a result of microglia activation-related markers. It was confirmed that the expression of MAC-1 and Iba-1 proteins in SNpc was reduced by preventing glial activity, and GFAP, whose expression was increased by MPTP-addiction, was also confirmed to be suppressed by HMA administration. In addition, as shown in Fig. 3D, it was confirmed that the expression of COX-2 protein was significantly reduced by the administration of HMA.

That is, in the present invention, since it was confirmed that HMA exhibits a strong inhibitory effect on the control of neuroinflammation at the Parkinson's disease experimental animal model and cell level, it can be usefully used in a pharmaceutical composition or health functional food for the prevention or treatment of neuroinflammatory disease.

In the present invention, the neuroinflammatory disease is multiple sclerosis, neuroblastoma, ischemic stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia or amyotrophic lateral sclerosis. It may be selected from the group consisting of.

Each of the pharmaceutical compositions of the present invention can be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and may be formulated in the form of external preparations, suppositories and sterile injectable solutions. It may further include a pharmaceutically acceptable carrier, excipient, and diluent according to each formulation. In addition, it may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to a conventional method.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the composition, such as starch, calcium carbonate, and sucrose. , Lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.

As used herein, the term "administering" means providing a pharmaceutical composition of the present invention to an individual by any suitable method. The pharmaceutical composition of the present invention is an amount of an active ingredient or a pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human considered by a researcher, veterinarian, doctor or other clinician, that is, relief of symptoms of the disease or disorder to be treated It can be administered in a therapeutically effective amount, which is an amount that induces. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, the patient's age, weight, and general health condition. , Sex and diet, administration time, administration route and composition ratio, treatment period, and various factors including drugs used simultaneously. The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual, or topical administration, but is not limited thereto. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or divided into several times.

In addition, the present invention is a health functional food for preventing or improving neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride (HMA) represented by the following formula (1) as an active ingredient It relates to the composition.

[Formula 1]

The characteristics of the 2-hydroxy-4-methylbenzoic anhydride contained in the health functional food composition for preventing or improving neuroinflammatory diseases are the same as described above.

The health functional food composition of the present invention can be used as a health functional food, food additive or dietary supplement. When the 2-hydroxy-4-methylbenzoic anhydride of the present invention is used as a food additive, it may be appropriately used according to a conventional method, such as being added as it is or used in combination with other foods or food ingredients.

In addition, the mixed amount of the health functional food composition may be appropriately changed according to the purpose of use (prevention, health or therapeutic treatment). As a specific example, when preparing food or beverage, the 2-hydroxy-4-methylbenzoic anhydride of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for health control purposes, it may be added in an amount below the above range, and since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. have.

There is no particular limitation on the type of food, but examples of foods to which the 2-hydroxy-4-methylbenzoic anhydride of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, Others include noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and all health foods in the usual sense are included.

When the health functional food composition of the present invention is prepared as a beverage, it may include additional ingredients such as various flavoring agents or natural carbohydrates, like a normal beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is included in 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

The health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , Carbonation agents used in carbonated beverages, and the like, and may include flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage, but are not limited thereto. These components may be used independently or in combination. The ratio of the additives is not largely limited, but is preferably included in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Material preparation

LPS (Escherichia coli 0111:B4, Sigma, St. Louis, USA), MPTP, N-1-naphthylethylenediaminedihydrochloride (N-(1-naphthyl) ethylenediamine dihydrochloride), MTT(3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), bovine serum albumin (BSA), Tween-20, dimethyl sulfoxide (DMSO); Compound Diluent/(1%) Vehicle control), sulfanilamide and sodium nitrite were purchased from Sigma (USA).

DMEM (Dulbecco's modified Eagle's medium), PBS (phosphate buffered saline) and other culture reagents were purchased from Gibco/Invitrogen (USA). Fetal bovine serum (FBS) was purchased from PAA Laboratories Inc. (Canada). Protease cocktail tablets and phosphatase inhibitors were supplied from Roche (USA).

Antibodies against iNOS and β-actin were purchased from Cell Signaling Technology (USA), and COX-2 was purchased from Santa Cruz Biotechnology (USA).

Statistical analysis

All data were analyzed using Graph Pad Prism version 5.01 (Graph Pad, Inc., USA). All data are expressed as mean ± standard error (as mean ± standard error) of three or more independent experiments performed in vitro or for histological and biochemical experiments.

Synthesis of 2-hydroxy-4-methylbenzoic anhydride (HMA)

HMA is the active ester of 2-hydroxy-4-methylbenzoic acid 1 (2.00 g, 9.70 mM) and 2-hydroxy-4-methylbenzoic acid, dicyclocarbodiimide (DCC, 2.71 g, 13.1 mM). It was prepared by reacting with included tetrahydrofuran (THF). The formed solid was filtered and the residue was evaporated under vacuum.

After the crude product was purified by silica gel chromatography, a white solid HMA compound (560 mg, 31%) was obtained.

Mp 171 °C; 1H NMR (CDCl3) δ 2.44 (s, 6H), 7.18-7.22 (d, 1H, J = 8.0 Hz), 7.31 (s, 2H), 7.82-7.86 (d, 2H, J = 8.0 Hz); 13C NMR (CDCl3) δ 21.81, 120.86, 124.17, 126.77, 131.50, 144.31, 148.40, 164.52.

As a result of measuring the molecular weight using the Chemdraw program, the molecular weight of the purified HMA was 286.28, and the synthesis process is shown in FIG. 1A.

HMA effect on NO generation and cell viability in LPS-stimulated BV-2 microglia

In order to confirm the efficacy of inhibiting neuroinflammation of HMA prepared in Example 1, the inhibitory effect of NO generation was confirmed.

BV-2 microglia were obtained by a known method (Kim, BW et al. , PLoS One, 8:e55792. 2013). Briefly, cells were DMEM (Dulbecco's modified Eagle's medium; Gibco/Invitrogen; USA) medium supplemented with 5% Fetal bovine serum (PAA Laboratories Inc.; Canada) and 50 μg/ml penicillin-streptomycin. At 37°C, 5% CO ₂ was cultured under conditions.

BV-2 cells were dispensed into a 96-well plate at a concentration of 2.5 × 10 ⁴ cells/ml per well, and then HMA was treated at concentrations of 0.1, 1 and 10 μM, followed by pretreatment for 1 hour. Then, LPS (200 ng/ml), which is a neuroinflammation inducing factor, was treated and BV-2 cells were stimulated for 24 hours, and the concentration of nitrite (NO) produced at this time was measured.

Nitrite (NO) was measured using a NO assay kit (Abcam) using a grease reagent, and the measurement method was carried out according to the instruction manual of the kit. 100 µl of the cell culture solution and grease reagent were mixed, reacted for 10 minutes, and observed at an absorbance of 540 nm, and the concentration was finally confirmed using a standard curve.

As a result, stimulation with LPS increased NO production compared to the control group, but as shown in FIG. 1B, NO generation in LPS-stimulated microglia pretreated with 0.1, 1 and 10 μM HMA was 94.5 ± 3.15, respectively. %, 79.9 ± 1.56% and 28.6 ± 1.32%, it was confirmed that NO generation was suppressed in a concentration-dependent manner.

In the present invention, in order to confirm whether HMA has cytotoxicity, the effect of HMA on the viability of BV-2 microglia at various concentrations (0.1, 1 and 10 μM) with or without LPS was evaluated. BV-2 cell viability was measured according to a known method (Kim, JJ, et al. , Journal of Ethnopharmacology , 140:213-221, 2012) to measure the degree of cytotoxicity according to HMA concentration.

First, BV-2 cells were dispensed into a 96-well plate so that each well was 2.5 × 10 ⁴ cells/ml, and then the synthesized HTB derivatives were treated at each concentration and pretreated for 1 hour. Then, it was treated with LPS (200 ng/ml), which is a neuroinflammation factor, and BV-2 cells were stimulated for 24 hours.

After 24 hours, the supernatant was removed, and then 100 µl DMEM medium containing MTT (500 µg/ml) was added to each well, followed by incubation at 37°C for 4 hours. After incubation, the supernatant was removed and dimethyl sulfoxide (DMSO); Compound Diluent/(1%) Vehicle control, Sigma, USA) was added to each well to dissolve formazan. Optical density was measured at 550 nm using a microplate reader (Tecan Trading AG, Switzerland), and the value was compared with the HTB derivative untreated group (control).

As a result of treatment with HMA alone or LPS at various concentrations, it was confirmed that no cytotoxicity appeared, as shown in FIG. 1C.

HMA effect on iNOS and COX-2 mRNA and protein expression levels in LPS-stimulated BV-2 microglia

3-1: iNOS and COX-2 mRNA expression level confirmation

BV-2 cells were dispensed into a 6-well plate so that each well was 5 × 10 ⁵ cells/well, and then, according to the presence or absence of the synthesized HTB derivative and LPS (100 ng/ml), the cells were subjected to the same method as above under each condition. Was cultured. After 6 hours, total RNA was isolated using TRIzol reagent. 2.5 μg of total RNA was reverse transcribed using a cDNA synthesis kit (First-Strand cDNA Synthesis kit; Invitrogen, USA), and then cDNA was amplified by PCR using a specific primer according to a known method (Park, SY et al. , Oxid Med Cell Longev, 2018: 3175214, 2018).

PCR was performed for each target using the synthesized cDNA as a template, and the PCR product was analyzed using a 1% agarose gel.

PCR primer sequence and reaction conditions Primer sequence (5'-3') order
number Product size (bp) Annealing
Temperature(℃) cycle iNOS F: 5'- GAG GTA CTC AGC GTG CTC CA-3' One 444 56 27 R: 5'-AGG GAG GAA AGG GAG AGA GG-3' 2 COX-2 F: 5'-TGA GTG GTA GCC AGC AAA GC-3' 3 319 56 27 R: 5'-CTG CAG TCC AGG TTC ATG G-3' 4 GAPDH F: 5'-ACC ACA GTC CAT GCC ATC AC-3' 5 472 57 25 R: 5'-CCA CCA CCC TGT TGC TGT AG-3' 6

3-2: Confirmation of iNOS and COX-2 protein expression levels

BV-2 cells were dispensed into a 6-well plate at 5 × 105 cells/well per well, and then the cells were harvested in the same manner as above under each condition depending on the presence or absence of the synthesized HTB derivative and LPS (100 ng/ml). Cultured. After 24 hours, the cells were washed twice with ice cold PBS, and then 50 ml of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, containing fresh protease inhibitor cocktail) or 100 ml was added to obtain total cell lysate. Bio-Rad DC Protein Assay was used to determine protein concentration.

40 µg of the total cell lysate was electrophoresed on a 10% SDS-PAGE gel, and then the separated protein was transferred to a PVDF membrane (Millipore, USA). In order to block non-specific binding, the PVDF membrane was reacted with PBS containing 5% skim milk for 1 hour. PVDF membrane was treated with anti-iNOS antibody (1:1000), anti-COX-2 antibody (1:1000), and anti-β-actin antibody (1:2000) and reacted overnight at 4°C, followed by secondary antibody (horseradish peroxidase-conjugated secondary antibody; 1:1000-2000) was treated and reacted for 1 hour.

Then, the optical density of the antibody-specific band was analyzed using an ECL detection kit and a luminescent image analyzer (LAS-3000, Fujifilm, Japan). Nuclear proteins were extracted using a nuclear and cytoplasmic extraction kit from Thermo Scientific (USA).

2A and 2B, as a result of pretreatment with 0.1, 1, and 10 μM HMA, iNOS mRNA expression was suppressed to 99.8 ± 10.5, 80.9 ± 3.39, and 56.4 ± 7.88%, respectively, and iNOS enzyme production was 103.1, respectively. It was confirmed that it was suppressed to ± 5.49, 74.2 ± 1.63 and 40.4 ± 10.7%. The results were consistent with the HMA inhibitory effect in the NO generation assay.

2C and 2D, it was confirmed that COX-2 mRNA and protein expression were increased in BV-2 cells by LPS stimulation. On the other hand, as a result of pretreatment with 0.1, 1 and 10 μM of HMA, COX-2 mRNA expression was suppressed to 88.0 ± 4.77, 67.3 ± 5.17 and 43.2 ± 6.20%, respectively, and COX-2 protein levels were 70.8 ± 8.20, 69.1, respectively. It was confirmed that it was suppressed to ± 6.01 and 47.3 ± 11.8%.

That is, it was confirmed that the expression levels of iNOS and COX-2 mRNA and protein were suppressed in LPS-stimulated BV-2 microglia in a HMA concentration-dependent manner.

HMA effect in MPTP-addicted mouse model of Parkinson's disease

4-1: animal preparation and processing

Six-week-old male C57BL/6 mice of 25 to 28 g were purchased from Samtako Bio Korea (Korea). All mice were acclimated in a laboratory environment for 2 weeks prior to the experiment. All experiments were performed according to the laboratory animal care principles (NIH publication no.85-23, revised 1985) and Konkuk University Laboratory Animal Committee guidelines (No. KU17024, 2017). Animals were freely ingested food and water in an environment controlled at 23 ± 1°C temperature and 50% ± 5% humidity, and the lighting was controlled at intervals of 12 hours.

All mice were divided into 3 groups with 12 mice per group (total 36).

Group 1: Vehicle Group

Group 2: MPTP 20 mg/kg induction group (4 times a day at 2 hour intervals, intraperitoneal injection)

Group 3: HMA 30 mg/kg (orally administered for 14 days) + MPTP 20 mg/kg (4 times a day at 2 hour intervals, intraperitoneal injection) group

HMA was administered orally for 14 days, and MPTP was administered intraperitoneally on the 14th day of HMA administration. Both HMA and MPTP solutions were prepared with saline immediately before administration. In particular, MPTP safety measures were performed according to Parkinson's MPTP mouse model protocol (Jackson-Lewis and Przedborski, NatProtoc, 2:141-151, 2007).

4-2: Confirmation of HMA effect on expression of MAC-1, Iba-1, GFAP and COX-2 in MPTP poisoned mice

To confirm the HMA effect on the expression of MAC-1, Iba-1, glial fibrillary acidic protein (GFAP) and COX-2, SNpc in the MPTP-addicted mouse model of Parkinson's disease of Example 4-1 (substantia nigra pars compacta) was isolated, and then a protein was obtained and Western blot was performed.

Specifically, mice administered with MPTP or vehicle/HMA were anesthetized by administration of sodium pentobarbital (sodium pentobarbital; 50 mg/kg, intraperitoneal injection), and then the brains of each mouse were obtained and SNpc was isolated. Thereafter, 40 μg of the protein obtained using a known method was subjected to electrophoresis on a 10% SDS-PAGE gel, and then the separated protein was transferred to a PVDF membrane (Millipore, USA). In order to block non-specific binding, the PVDF membrane was reacted with PBS containing 5% skim milk for 1 hour. PVDF membrane is treated with anti-MAC-1 antibody, anti-Iba-1 antibody, anti-GFAP antibody, and anti-β-actin antibody, reacted overnight at 4°C, and then secondary antibody (horseradish peroxidase-conjugated secondary antibody) And reacted for 1 hour.

Then, the optical density of the antibody-specific band was analyzed using an ECL detection kit and a luminescent image analyzer (LAS-3000, Fujifilm, Japan). Nuclear proteins were extracted using a nuclear and cytoplasmic extraction kit from Thermo Scientific (USA).

As shown in FIGS. 3A to 3C, as a result of MPTP-addiction, the expression of GFAP proteins increased in SNpc, whereas GFAP protein expression was increased in SNpc as a result of microglia activation-related markers. It was confirmed that the expression of MAC-1 and Iba-1 proteins in SNpc was reduced by preventing glial activity, and GFAP, whose expression was increased by MPTP-addiction, was also confirmed to be suppressed by HMA administration. In addition, as shown in Fig. 3D, it was confirmed that the expression of COX-2 protein was significantly reduced by the administration of HMA.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Novel 2-hydroxy-4-methylbenzoic anhydride and Composition for Preventing or Treating Neuroinflammation Disease Comprising the same <130> 1065253 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_F <400> 1 gaggtactca gcgtgctcca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_R <400> 2 agggaggaaa gggagagagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_F <400> 3 tgagtggtag ccagcaaagc 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX-2_R <400> 4 ctgcagtcca ggttcatgg 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 5 accacagtcc atgccatcac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 6 ccaccaccct gttgctgtag 20

Claims (7)

delete delete A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases comprising 2-hydroxy-4-methylbenzoic anhydride represented by the following formula (1) as an active ingredient:
[Formula 1]

.
The method of claim 3, wherein the 2-hydroxy-4-methylbenzoic anhydride is derived from 2-hydroxy-4-trifluoromethyl benzoic acid represented by the following formula (2). A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases characterized by:
[Formula 2]

.
The nerve according to claim 3, wherein the 2-hydroxy-4-methylbenzoic acid anhydride regulates nerve inflammation by inhibiting the production of inflammatory mediators including NO, iNOS and COX-2. Pharmaceutical composition for the prevention or treatment of inflammatory diseases.
The method of claim 3, wherein the 2-hydroxy-4-methylbenzoic anhydride inhibits the expression of MAC-1, Iba-1, and GFAP proteins in substantia nigra pars compacta (SNpc). The therapeutic pharmaceutical composition.
The method of claim 3, wherein the neuroinflammatory disease is multiple sclerosis, neuroblastoma, ischemic stroke, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Creutzfeldt-Jakob disease, post-traumatic stress disorder, depression, schizophrenia or amyotrophic lateral sclerosis. A pharmaceutical composition for the prevention or treatment of neuroinflammatory diseases, characterized in that selected from the group consisting of.

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