KR20140062939A - Composition for preventing or treating of inflammation - Google Patents

Abstract

The present invention relates to fimasartan, a pharmaceutically acceptable salt thereof, a solvate of the same, or a pharmaceutical composition comprising a hydrate thereof as an active ingredient for preventing and treating inflammation and inflammatory diseases. The pharmaceutical composition can be used for preventing or treating Hyperlipidemia, arthritis, gastric ulcers, respiratory disease, spondylitis, urethritis, cystitis, nephritis, allergic disease, dermatomyositis, rhinitis, tonsillitis, acute pain, chronic pain, periodontal disease, gingivitis, inflammatory bowel disease, pancreatitis, respiratory syncitial virus (RSV) disease , autoimmune diseases, asthma, atopic dermatitis, Crohn′s disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and psoriasis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating inflammation,

The present invention relates to a composition for preventing or treating inflammation, which comprises as an active ingredient Pimersartan, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof.

The pharmacological induction of LPS-induced inflammation mediators (eg, nitrite oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin Downregulation is considered to be an essential requirement for the alleviation of many diseases due to macrophage activation. Thus, RAW 264.7 macrophages are good models for the screening of anti-inflammatory drugs and the evaluation of inhibitors of pathways leading to the induction of pro-inflammatory enzymes and induction of pro-inflammatory cytokines [Shin JS, Yun CH, Cho YW, Baek NI, Choi MS, Jeong TS, Chung HG, Lee KT., J Med Food., 14 (12), 1527-1537 (2011)]. Macrophages and other progenitor cells cause resolution of inflammation. One of the important steps during inflammation is leukocyte infiltration, which is mainly regulated by chemokines for neutrophils and monocytes. This chemokine production is regulated positively or negatively by inducible nitric oxide synthase (iNOS) -induced NO. The mechanism underlying these two effects of NO is not yet known, but the level of NO and the duration of NO exposure appear to be crucial factors [Kobayashi Y., JLeukoc Biol., 88 (6), 1157-1162 (2010)] . Anyway, NO production and weakening of iNOS expression are good targets for the development of anti-inflammatory drugs.

Nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) play an important role in the regulation of the genes involved in cell survival, It is known to regulate expression. The promoter sequence of the iNOS gene has a number of sites that bind to a variety of transcription factors including NF-κB and AP-1 [Ratajczak-Wrona W., Jablonska E., Garley M., Jablonski J., Radziwon P. , Iwaniuka .. JImmunotoxicol., Epub (2012)]. In particular, NF-κB is expressed as a central regulator of the immune response, regulating the expression of pro-inflammatory enzymes, cytokines, and chemokines, and thus is the development of the treatment of NF-κB signaling cascade inflammation and autoimmune diseases , 11 (5), 335-346 (2011). This study was conducted to investigate the effect of anti- )]. On the other hand, AP-1 can regulate the expression of many genes. Thus, AP-1 is involved in a wide range of cellular processes, including proliferation, apoptosis, survival and differentiation, and is involved in a variety of biological and pathological processes such as development of epidermis and neurons, Reactions, and tumor formation. With respect to inflammation, AP-1 is an important regulator of inflammatory diseases [Meng Q, Xia Y., Protein Cell., 2 (11), 889-898 (2011)].

In the meantime, Pimassartan has proposed a compound {2-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl-3 - [[2 ' Yl] methyl] pyridin-4 (3H) -one} (Korean Patent Registration No. 0354654). Pimassartan is a new angiotensin II receptor antagonist used in hypertension and has a safety profile even after a rapid oral dose of 20-480 mg and is effective [Chi YH, Lee H., Paik SH, Lee JH, Yoo BW, Kim JH , Tan HK, Kim SL., Am J Cardiovasc Drugs., 11 (5), 335-346 (2011)]. However, there is no known anti-inflammatory effect against Pimassaran.

Thus, the present inventors have conducted studies using the inflammation-related models, indicators, and modulators to complete the composition of the present invention, which can be used as a safe and effective anti-inflammatory drug.

Korean Patent Registration No. 0354654, October 7, 2005, all

14 (12), 1527-1537 (2011). This study was conducted to investigate the effects of dietary supplements on dietary fiber intake, Kobayashi Y., JLeukoc Biol., 88 (6), 1157-1162 (2010) Ratajczak-Wrona W., Jablonska E., Garley M., Jablonski J., Radziwon P., Iwaniuka .. JImmunotoxicol., Epub (2012) 11 (5), 335-346 (2011)). In this study, we have investigated the effect of anti- Meng Q, Xia Y., Protein Cell., 2 (11), 889-898 (2011)

It is therefore an object of the present invention to provide a safe and effective composition for preventing or treating inflammation.

In order to achieve the above object, the present invention provides a composition for preventing and treating inflammatory or inflammatory diseases, which comprises as an active ingredient, Pimacartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof.

In the present invention, Pimassartan is a compound having the chemical structure shown in the following FIG. 1A, {2-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl- Yl) biphenyl-4-yl] methyl] pyridin-4 (3H) -one}.

In the present invention, Pimassartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof may be crystalline or amorphous, and the present invention includes the crystalline form and / or the amorphous form thereof.

In the present invention, pharmaceutically acceptable salts generally refer to inorganic acid salts, organic acid salts and metal salts which are used by pharmaceutical manufacturers in the manufacture of medicines. As the "inorganic acid ", hydrochloric acid, bromic acid, sulfuric acid, Organic acids "include organic acids such as citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, maleic acid, benzoic acid, gluconic acid, glycolic acid, Succinic acid, glutamic acid, aspartic acid, adipate, camsylate or besylate salts, and the like can be used, for example, "Metal" includes sodium, potassium, calcium, magnesium, and the like.

In the present invention, the pharmaceutically acceptable salt of the palmatartan is preferably a potassium salt potassium salt, a hydrochloride salt, a calcium salt, a sulfate salt, an adipate salt, a camylate salt or a besylate salt, desirable.

In the present invention, as the hydrate of the pharmaceutically acceptable salt of the palmatartan, potassium palmitate trihydrate potassium salt is preferable.

In the present invention, the solvent in the "solvate" means a conventional organic solvent used in the production of an organic compound. Examples of the solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, There may be mentioned acetone, acetone, acetic acid, anisole, tetrahydrofuran, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, n-butyl acetate, dimethyl sulfoxide, pentane, heptane and the like The solvates of the present invention are not limited.

In the present invention, "hydrate" and "solvate" may be contained in an amount of 0.25 to 10 molar equivalents relative to 1 mol of the palmatheartan, for example, 0.5 mol, 1 mol, 1.5 mol, 2 mol, 2.5 mol, , 5 moles, and the like, but the present invention is not limited thereto.

In the present invention, Pimassartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof is commercially available and can be prepared by a known method (for example, Korean Patent Registration No. 0354654 And 0521980).

In the present invention, the inflammatory diseases are selected from the group consisting of sepsis, arthritis, gastric ulcer, respiratory disease, spondylitis, urethritis, cystitis, nephritis, allergic diseases, dermatomyositis, rhinitis, tonsillitis, acute pain, chronic pain, periodontitis, gingivitis, , Respiratory syncitial virus (RSV) disease, and autoimmune disease.

In the present invention, the autoimmune disease may be at least one selected from the group consisting of asthma, atopic dermatitis, Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and psoriasis.

The composition of the present invention may be prepared by incorporating at least one pharmaceutically acceptable carrier in addition to the above-mentioned components for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as 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 according to a suitable method in the art or using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the body weight, age, sex, The range varies depending on diet, administration time, method of administration, excretion rate, and severity of the disease. The composition of the present invention can be administered once or divided into several times a day. For example, the effective amount of the pigsartan of the composition of the present invention is from 150 mg to 900 mg based on the general adult weighing about 60 kg.

The composition of the present invention may be in the form of injectable solutions such as aqueous solutions, suspensions, emulsions and the like, pills, capsules, granules or tablets, or a form for oral administration in the form of a kit, more preferably for oral administration, desirable.

The present invention also provides a pharmaceutical composition comprising as an active ingredient a composition comprising Pimassartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof, to a mammal, including a human, in need thereof to produce an inflammatory or inflammatory disease Prevention or treatment of cancer. Humans may be excluded from the method.

Also provided is the use of a composition comprising phimacartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof, as an active ingredient, for pharmaceutical manufacture for the prevention or treatment of an inflammatory or inflammatory disease.

The composition according to the present invention is effective for the prevention or treatment of inflammation and is therefore useful as a medicament for the prevention and treatment of inflammation such as inflammation, arthritis, gastric ulcer, respiratory disease, respiratory syncitial virus (RSV), autoimmune diseases such asthma, atopic dermatitis, Crohn's disease, rheumatoid arthritis, Lupus erythematosus, multiple sclerosis, psoriasis, and the like.

1A is a diagram showing the chemical structure of Pimassartan.
Figure 1B is a graph showing the effect of Pimassarthan on cell viability in RAW 264.7 macrophages.
Figure 2a shows the effect of Pimassartan on LPS-induced NO production in RAW 264.7 macrophages.
Figure 2b shows the effect of Pimassarthan on LPS-induced iNOS expression in RAW 264.7 macrophages.
Figure 2c shows the effect of Pimassartan on LPS-induced iNOS expression in RAW 264.7 macrophages.
Figure 2d shows the effect of Pimassartan on LPS-induced iNOS promoter activity in RAW 264.7 macrophages.
Figure 3a shows the effect of Pimacarthan on LPS-induced NF-kB transcriptional activity in RAW 264.7 macrophages.
Figure 3b shows the effect of Pimassartan on the potential of LPS-induced NF-kB in RAW 264.7 macrophages.
Figure 3c shows the effect of Pimassartan on the DNA binding activity of NF-kB in RAW 264.7 macrophages.
Figure 3D shows the effect of Pimassarthan on the DNA binding activity of NF-kB in RAW 264.7 macrophages.
Figure 4a shows the effect of Pimassartan on LPS-induced AP-1 transcriptional activity in RAW 264.7 macrophages.
Figure 4b shows the effect of Pimassartan on the potential of LPS-induced AP-1 in RAW 264.7 macrophages.
Figure 4c shows the effect of Pimassartan on the DNA binding activity of AP-1 in RAW 264.7 macrophages.
Figure 4d shows the effect of Pimassartan on the DNA binding activity of AP-1 in RAW 264.7 macrophages.
Figure 5 is a graph showing the effect of Pimassartan in an LPS-induced septic shock model.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention. In the following examples, the term " phimassartan "

Example . Pima Saltan  Identification of anti-inflammatory effects

1. Materials and Methods

end. Experimental material

DMEM, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). The peroxidase-conjugated secondary antibody was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz Biotechnology, Inc., Santa Cruz Biotechnology, Inc., Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Optional oligonucleotide primers and M-MLV reverse transcriptase were purchased from Promega (Madison, Wis., USA). SYBR green ex Taq is TaKaRa (Cigar, Japan). iNOS, beta -actin oligonucleotide primers were purchased from Bioni (Seoul, Korea). Pimassartan triphosphate potassium salt was obtained from Boryung Pharmaceutical (Seoul, Korea). MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyl tertiazolium bromide), PMSF, DTT (DL-Dithiothreitol), NS-398, LPS ( Escherichia coli , serotype 0111: B4) All compounds were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

I. Cell culture and sample treatment

RAW 264.7 macrophage cell line was obtained from Korean Cell Line Bank (Seoul, Korea). These cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin 100 units / ml and streptomycin 100 ug / ml at a CO ₂ concentration of 5%, humid conditions, at 37 ° C. Cells were incubated with Fimasartan at concentrations of 62.5, 125, 250 μM, and then stimulated with 1 μg / ml LPS for the indicated time.

All. Cell viability measurement

RAW 264.7 macrophages were plated in 96 well-plates at 10 ⁵ cells / well and then cells were seeded at various concentrations of Pimassartan (3.9 [mu] M to 500 [mu] M) in order to determine the appropriate concentration of Pimacartan not affecting cell viability mu M) for 24 hours. After 24 hours, the cells were treated with 1 mg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) and then cultured for another 4 hours. Respectively. After incubation for 10 minutes, the absorbance was measured at 540 nm using a microplate reader (Perkin Elmer Cetus, Forster City, CA, USA). The cell survival rate was calculated as the cell viability of the sample-added group (pimeticl-treated group) and the cell survival rate of the sample-added group (pimeticl-untreated group)

la. Nitrite measurement ( Nitrite Determination )

RAW 264.7 for the macrophage cells 24-well - LPS (1 μg / ml) in the presence or absence of Pima saltan of 5 × 10 ⁵ cells / well plated, and then various concentrations (62.5, 125, 250 μM) on the plate and And cultured for 24 hours with or without. Nitrite accumulated in the culture medium was measured as an indicator of nitrite oxide (NO) production based on the Griess reaction. Briefly, 100 [mu] l of culture medium and 1 [mu] l of the same volume in 100 [mu] l of grease reaction [5% (v / v) phosphoric acid and 0.1% (w / v) naphthylethylenediamine- w / v) sulfanilamide], incubated at room temperature for 10 minutes, and then absorbance at 540 nm was measured in a microplate reader (Perkin Elmer Cetus, CA, USA). Fresh culture medium was used as a blank for all experiments. The amount of nitrite in the samples was measured using a sequential dilution standard curve of sodium nitrite and nitrite production was measured.

hemp. Western Blot  analysis

RAW 264.7 macrophages were harvested by centrifugation and washed once with PBS (phosphate-buffered saline). The washed cell pellet was resuspended in PRO-PREP ™ protein extraction solution (IntronBiotechnology, Seoul, Korea) and incubated at 4 ° C for 20 min. Cell debris is removed by microcentrifugation and then the supernatant is rapidly cooled. Protein concentration was determined using the Bio-Rad protein assay reagent according to the manufacturer's instructions. Cell proteins from treated and untreated cell extracts were separated on 10-12% SDS-PAGE and electroblotted on PVDF membranes. Immunoblot was incubated with blocking solution (5% skim milk) overnight at 4 ° C, followed by incubation with primary antibody for 4 hours. The blot was washed four times with Tween 20 / TBS (Tris-buffered saline) and incubated with horseradish peroxidase-conjugated secondary antibody diluted 1: 1000 for 2 hours at room temperature . The blot was washed again with Tween 20 / TBS three times and then developed with ECL (enhanced chemiluminescence) [Amersham Life Science].

bar. Quantitative real-time Reverse transcriptase  Polymerase chain reaction Quantitative Real - time  Reverse-transcriptase Polymerase Chain Reaction ; qRT - PCR )

Total cellular RNA was isolated by Easy Blue kits ^® (Intron Biotechnology, Seoul, Korea). 1 μg of RNA from each sample was reverse-transcribed (RT) using MuLV reverse transcriptase, 1 mM dNTP (deoxyribonucleotide triphosphate), and 0.5 μg / μl oligo (dT12-18). PCR amplification was performed using the incorporation of SYBR green. The oligonucleotide primer for iNOS was designed from mouse [National Center for Biotechnology Information (NCBI), NM_010927], 5'-CATGCTACTGGAGGTGGGTG-3 '(forward primer; SEQ ID NO: 1) -CATTGATCTCCGTGACAGCC-3 '(reverse primer; SEQ ID NO: 2), and the suitable size of the synthesized cDNA is 209 bp. The primers for β-actin used as a housekeeping gene were designed from mouse (NCBI, NM - 007393), 5'-ATCACTATTGGCAACGAGCG-3 '(forward primer; SEQ ID NO: 3) and 5'-TCAGCAATGCCTGGGTACAT-3 (Reverse primer; SEQ ID NO: 4), and the suitable size of the synthesized cDNA is 200 bp. The steady-state mRNA levels of iNOS and β-actin were measured by real-time quantitative PCR (qPCR) using Takara thermal cycler dice ㄾ (Takara Bio Inc., Japan). The results are expressed as a ratio of OD (optimal density) to? -Actin.

four. Nuclear extraction ( Nuclear Extraction ) And EMSA ( Electrophoretic Mobility Shift Assay )

RAW 264.7 macrophages were plated on 100-mm dish (1 × 10 ⁶ cells / ml), treated with Pimassaran (62.5, 125, or 250 μM), stimulated with LPS for 1 hour, washed once with PBS, Scraped in cold PBS, and pelleted by centrifugation. The nuclear extracts were prepared in the same manner as in the method disclosed in Kim JY, Park SJ, Yun KJ, Cho YW, Park HJ, Lee KT, Eur J Pharmacol., 584, 175-184 (2008). Nuclear extracts (5 μg) were mixed with double-stranded NF-κB and AP-1 oligonucleotides, 5'-AGTTGAGGGGACTTTCCCAGGC-3 ', and 5'-CGCTTGATGACTCAGCCGGAA-3' respectively and ligated with biotin by end- . DNA-binding activity was measured using a LightShft Chemiluminescent EMSA kit (Thermo Scientific, USA).

Ah. temporary Transfection ( Transient Transfection ) And Luciferase  analysis( Luciferase Assay )

The mouse iNOS promoter plasmid (pGL3-iNOS; -1592 / + 185) and the COX-2 promoter plasmid (pGL3-COX-2; -965 / + 39) were obtained from Lowenstein CJ, Alley EW, Raval P., Snowman AM, Snyder SH , Russell SW, Murphy WJ, Proc Natl Acad Sci US A., 90, 9730-9734 (1993). RAW 264.7 macrophages as provided by the manufacturer instructions Lipofectamine LTX ^TM (Invitrogen, CA, USA) and pGL3-iNOS, NF-κB-Luc, or AP-1-Luc reporter plasmid vector plus phRL-TK plasmid (promega use, Madison, < RTI ID = 0.0 > WI). ≪ / RTI > After transfection for 4 hours, the cells were pretreated with Pimassaran for 1 hour and then stimulated with LPS (1 μg / ml) for 18 hours. Each well was washed with cold PBS and cells were lysed and luciferase activity was measured using the Promega luciferase assay system (Promega, Madison, CA, USA).

character. animal

All animal experiments were conducted under the University Guidelines of the Animal Experimental Ethics Committee at Kyung Hee University according to the animal protocol (KHP-2012-05-2). 20-25 g C57BL / 6 male mice were purchased from Orient Bio Inc. (Seongnam city, Korea) and maintained under constant conditions (temperature: 20 ± 2 ℃, humidity: 40-60%, name / cancer cycle: 12 hours). Twelve hours before the experiment, only water was provided.

car. In mice, septic shock ( Septic shock )

Sepsis was induced in C57BL / 6 mice (male, 5-6 weeks old) by intraperitoneal (i.p.) injection of LPS (Salmonella enteric, 50 mg / kg). Mice were dosed with Pimassartan (10, 20, and 50 mg / kg, oral (p.o.)) 1 hour before LPS injection. Survival of the mice was monitored over the following 24 hours.

Car. Statistical analysis

The results are expressed as mean ± standard deviation (SD) of 3 experiments. Statistically significant values were compared using ANOVA and Dunnett's post-hoctest test, and p-values less than 0.05 were considered statistically significant.

2. Results

end. Pima Saltan LPS -Judo NO  production, iNOS  Expression and transcriptional activity ( transcriptional  activity.

The cytotoxic effect of Pimassarthan was confirmed using MTT assay. As a result, the cytotoxic effect of Pimassartan was 90% or more at a concentration of at least 250 μM, indicating almost no cytotoxic effect on RAW 264.7 macrophages (see FIG.

Fig. 1b shows the results of treatment of RAW 264.7 macrophages with Pima sacalin (3.9 [mu] M to 500 [mu] M) and cell viability by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) .

At the identified concentrations, the culture medium was harvested and the nitrite levels were measured to determine the effect of the pharmacata on LPS-induced NO production in RAW 264.7 macrophages. Pimassartan (62.5, 125, or 250 μM) inhibited LPS-induced NO production in a potent and concentration dependent manner (see FIG. 2a). In this experiment, L-NIL (20 μM) was used as a positive NO production inhibitor.

Since iNOS catalyzes the oxidative deamination of L-arginine to produce NO (Moncada S., Palmer RM, Higgs EA Pharmacol Rev., 43, 109-142 (1991)), iNOS protein and mRNA Expression was measured by Western blot and qRT-PCR, respectively. iNOS protein and mRNA levels were markedly expressed in LPS-induced RAW 264.7 macrophages and it was found that Pimassartan significantly inhibited this upregulation by LPS in a dose-dependent manner (Figures 2b and 2c Reference).

In addition, it can be confirmed that Pimassartan inhibits LPS-induced iNOS and promoter luciferase activity in a concentration-dependent manner (see Fig. 2d).

The experimental conditions in FIG. 2A are as follows. Pimersartan (62.5, 125, or 250 μM) was pretreated for 1 h before cells were treated with LPS (1 μg / ml) for 24 h. L-NIL (20 [mu] M) was used as a positive control.

The experimental conditions of FIG. 2B are as follows. Lysates were prepared from the control and treated with LPS (1 μg / ml) alone or LPS + Pimacartan (62.5, 125, or 250 μM) for 24 hours. Total cellular protein (30 μg) was separated by SDS-PAGE and transferred to PVDF membrane and detected as a specific antibody. The lower graph of FIG. 2B shows the density of each band of the target protein (iNOS) relative to that of the inner control β-actin. When the LPS treatment and the pigsartan treatment group were regarded as 1, the LPS treatment group, Fold increase in the amount of iNOS protein expression in the salting-treated group.

The experimental conditions of FIG. 2c are as follows. Total RNA was prepared for qRT-PCR analysis of iNOS from cells stimulated with LPS (1 μg / ml) with / without Pimasartan (62.5, 125, or 250 μM) for 4 hours. mRNA expression of iNOS was determined as described in Experimental Materials and Methods. FIG. 2C is a graph comparing the amount of the target mRNA (iNOS) with that of the housekeeping gene β-actin. When the LPS treatment and the pigsartan-free treatment group were taken as 1, the LPS treatment group and the LPS treatment group and the pigsartan treatment group and a fold increase in the amount of iNOS mRNA expression (Fold increase).

The experimental conditions of FIG. 2d are as follows. Figure 2d is an internal control, in which cells were transfected with pGL3-iNOS promoter (-1592 / + 185) vector and phRL-TK vector. The luciferase activity levels were determined as described in Experimental Materials and Methods. Figure 2d is a graph showing iNOS promoter activity.

In Figures 2a-2d the data are presented as the mean mean ± standard deviation (SD) of three independent experiments. # p < 0.05 versus control cells; * p < 0.05 ** p < 0.01, *** p < 0.001 vs. LPS-stimulated cells. ANOVA and Dunnett's post-hoctest test were used to determine the significance of the differences.

I. Pima Saltan LPS -Judo NF -KB transcriptional activity, nuclear potential ( Nuclear  Translocation) and DNA  Reducing binding activity.

Since NF-κB activation is required for the expression of iNOS by LPS (Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS, Mutat.Res., 1, 243-268 (2001) ; Lappas M., Permezel M., Georgiou HM, Rice GE, Biol Reprod., 67, 668-673 (2002)) to determine if Pimersartan affects the transcriptional activity of NF- Respectively. Pimacartan significantly reduced the transcriptional activity of NF-kB in a concentration dependent manner (see Figure 3a). Whether Pimacarthan inhibits the nuclear translocation of p65 and p50 subunits of NF-kB was tested by Western blot. LPS significantly induces the translocation of p65 and p50 to the nucleus, but pretreatment of the pimassartan significantly inhibits this process (see FIG. 3b). In addition, EMSA was performed to determine whether permsartan inhibited the DNA binding activity of NF-kB. For this analysis, nuclear extracts were obtained from RAW 264.7 macrophages stimulated with LPS (1 [mu] g / ml) for 30 minutes or 1 hour under the presence or absence of Pimacartan for 1 hour. LPS treatment at 1 hour increases the binding activity of NF-κB to their consensus DNA sequences. However, pre-treatment of the pigsartan reduces this LPS-induced NF-κB-DNA binding in a concentration-dependent manner. The specificity of binding was tested by competition with 100-fold unlabeled oligonucleotides (6 lanes) and competitive inhibition (7 lanes) with p65 antibodies (see Figures 3c and 3d).

The experimental conditions in FIG. 3A are as follows. Cells were transiently cotransfected with pNF-κB-luc reporter and then either not treated with Pimassartan (control; Con), or with Pimassartan at different concentrations (62.5, 125, or 250 μM) for 1 hour Lt; / RTI &gt; LPS (1 μg / ml) was then added and the cells were incubated for an additional 4 hours. Cells were then harvested and luciferase activity was determined using the Promega luciferase assay system and luminometer. Figure 3A is a graph showing NF-κB reporter activity.

The experimental conditions of FIG. 3B are as follows. Nuclear extracts were prepared for western blotting of p65 and p50 using specific anti-p65 or anti-p50 monoclonal antibodies. PARP was used as an internal control for nuclear extracts. The lower graph of FIG. 3B shows the density of each band of the target protein (p65, p50) in comparison with that of the internal control PARP. When the LPS treatment and the pigsartan treatment group were regarded as 1, the LPS treatment group, Fold increase in the amount of protein expression of p65 and p50 in the salting-treated group, respectively.

The experimental conditions in FIG. 3c are as follows. After treatment with Pimassaran (250 μM) for 1 hour, the cells were treated with LPS (1 μg / ml) for 30 minutes or 1 hour. Nuclear extracts were prepared and analyzed for NF-κB-DNA binding by EMSA. The arrow indicates the position of the NF-kB band.

The experimental conditions in Figure 3d are as follows. Pimersartan (62.5, 125, or 250 μM) was pretreated for 1 hour and cells were treated with LPS (1 μg / ml) for 1 hour. Binding specificity was tested by competitive inhibition of competition (cp) with a 100-fold excess of unlabeled NF-κB nucleotide and anti-p65 monoclonal antibody.

In Figures 3a-3d # p < 0.05 versus control cells; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-stimulated cells. ANOVA and Dunnett's post-hoctest test were used to determine the significance of the differences.

All. Pima Saltan LPS -Judo AP -1 transcriptional activity, nuclear potential and DNA  Reducing binding activity.

Since the transcription factor AP-1 plays an important role in the transcriptional regulation of iNOS expression (Cai TG., Cai Y., Chem Biodivers., 8, 2135-2143 (2011)), the AP-1 signaling pathway The inhibitory activity of Pimacartan was measured. Whether Pimersartan affects the transcriptional activity of AP-1 was determined by luciferase assay. Pimacartan significantly reduced the transcriptional activity of AP-1 (see Fig. 4A). Pimacartan also significantly inhibited the nuclear translocation of cFos and cJun subunits of AP-1 (see Fig. 4b). The DNA-binding activity of AP-1 was measured by EMSA. At 30 min, LPS treatment increases the binding activity of AP-1 to their consensus DNA sequence. Pre-treatment of the pigsartan, however, reduces this LPS-induced AP-1-DNA binding in a concentration-dependent manner. The specificity of binding was tested by competition with 100-fold unlabeled oligonucleotides (6 lanes) and competitive inhibition with p65 antibodies (7 lanes) (see Figures 3c and 3d).

The experimental conditions of FIG. 4A are as follows. Cells were transiently cotransfected with pNF-κB-luc reporter and then either not treated with Pimassartan (control; Con), or with Pimassartan at different concentrations (62.5, 125, or 250 μM) for 1 hour Lt; / RTI &gt; LPS (1 μg / ml) was then added and the cells were incubated for an additional 4 hours. Cells were then harvested and luciferase activity was determined using the Promega luciferase assay system and luminometer. Figure 4A is a graph showing AP-I reporter activity.

The experimental conditions of FIG. 4B are as follows. Nuclear extracts were prepared for western blotting of cFos and cJun using specific anti-cFos or anti-cJun monoclonal antibodies. PARP was used as an internal control for nuclear extracts. The lower graph of FIG. 4B shows the density of each band of the target protein (cFos, cJun) in comparison with that of the internal control PARP. When the LPS treatment and the pigsartan treatment group were regarded as 1, the LPS treatment group, Fold increase in protein expression of cFos and cJun in the salting-treated group, respectively.

The experimental conditions of FIG. 4c are as follows. After treatment with Pimassaran (250 μM) for 1 hour in FIG. 4 (C), the cells were treated with LPS (1 μg / ml) for 30 minutes or 1 hour. Nuclear extracts were prepared and analyzed for AP-1-DNA binding by EMSA. The arrow indicates the position of the AP-1 band.

The experimental conditions in FIG. 4D are as follows. Pimersartan (62.5, 125, or 250 μM) was pretreated for 1 hour and cells were treated with LPS (1 μg / ml) for 1 hour. Binding specificity was tested by competitive inhibition of competition (cp) with 100-fold unlabeled AP-1 nucleotides and anti-cJun monoclonal antibodies.

In Figures 3a-3d # p < 0.05 versus control cells; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-stimulated cells. ANOVA and Dunnett's post-hoctest test were used to determine the significance of the differences.

la. Pima Saltan LPS - Reduces the induced septic shock model.

Survival of LPS-induced endotoxin shock was measured in mice to determine the inhibitory effect of the pigsartan against LPS-induced endotoxin shock. Under the absence of Pimacarthan, injection of LPS (50 mg / kg, ip) only survived 20% of the mice. When Pima salatans (10, 20, and 50 mg / kg) were administered 1 hour before LPS injection, the survival rate was improved to 40% (see FIG. 5).

The experimental conditions of FIG. 5 are as follows. Six mice per group were treated with vehicle alone or with Pimalacan (10, 20, or 50 mg / kg, orally) and 1 hour later with LPS (50 mg / kg, intraperitoneally) . The survival rate of these mice was observed over the next 2 hours. * P <0.05, ** P <0.01, *** P <0.001 versus LPS-injected group. ANOVA and Dunnett's post-hoctest test were used to determine the significance of the differences.

Claims (3)

Or a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof, as an active ingredient, for the prevention and treatment of inflammatory or inflammatory diseases. The method of claim 1, wherein the inflammatory disease is selected from the group consisting of sepsis, arthritis, gastric ulcer, respiratory disease, spondylitis, urethritis, cystitis, nephritis, allergic disease, dermatomyositis, rhinitis, tonsillitis, acute pain, chronic pain, periodontitis, , Pancreatitis, respiratory syncitial virus (RSV) disease, and autoimmune disease. The composition according to claim 2, wherein the autoimmune disease is at least one selected from the group consisting of asthma, atopic dermatitis, Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and psoriasis.

Images