KR101499007B1 - A composition for prevention and treatment of inflammatory diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing a compound having a structure of chemical formula 1 for preventing or treating inflammatory diseases. In chemical formal 1, R1 to R5 each are independently hydrogen, C1-C5 alkyl, or halogen.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases,

And a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising a compound having the structure of the following formula (1).

Leukotriene B4 (LTB ₄ ) is a group of inflammatory lipid mediators synthesized from arachidonic acid (AA) by the 5-lipoxygenase pathway mediating acute and chronic inflammation. LTB ₄ is known to have biological effects by binding to two types of receptors, BLT1 and BLT2. BLT2 is one of the GPCR (G protein-coupled receptor) family of receptors with low affinity for LTB ₄ and is a lipid mediator of arachidonic acid (AA) derived via a 5-lipoxygenase dependent pathway.

On the other hand, it has been reported that BLT2 is associated with a number of inflammatory diseases including asthma and cancer (Yoo, MH, Song, HW, Woo, CH, Kim, HG, Yoo, YJ, _{of the BLT2, a leukotriene B 4} receptor, in Ras -transformation (2004) Oncogene, 23, 9259-9268;.. Cho KJ, Shin YH, Kim HG Seo JM, Yoo MH, Chang YS, Cho SH and Kim JH Blockade of airway inflammation and hyperesponsiveness by inhibition of BLT2, a low-affinity leukotriene B4 receptor (2010) Am . J. Res Cell Mol Biol . , 42, 293-303, etc.). For example, overexpression of BLT2 in Rat-2 cells (Rat2-BLT2 cells stably transfected with human BLT2) is explained by increased cell growth and chemotactic responses.

Thus, the present inventors have repeatedly conducted studies for screening a compound exhibiting a BLT2-inhibiting activity. As a result, it has been experimentally confirmed that a compound having the structure of the formula (1) has an excellent BLT2 inhibitory activity, The present invention has been completed on a composition for preventing and treating various BLT2-mediated inflammatory diseases such as cancer and asthma.

Yoo, M. H., Song, H. W., Woo, C. H., Kim, H. G., Yoo, Y. J., and Kim JH. Role of the BLT2, a leukotriene B4 receptor, in Ras-transformation (2004) Oncogene, 23, 9259-9268; Cho KJ., Shin YH, Kim H.G. Seo JM, Yoo MH, Chang YS, Cho SH and Kim JH. Blockade of airway inflammation and hyperesponsiveness by inhibition of BLT2, a low-affinity leukotriene B4 receptor (2010) Am. J. Res Cell Mol Biol., 42, 293-303

The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising a compound having the structure of Chemical Formula (1).

The compound having the structure of formula (I) of the present invention has an effect of inhibiting cell growth by BLT2 with a very high efficiency and thus has an effect of preventing or treating inflammatory diseases caused by overexpression of BLT2.

FIG. 1 is a graph showing cell growth of cells in which BLT2 was not expressed (control group; Rat2-pcDNA3.1 cells) and cells in which BLT2 was expressed (experimental group: Rat2-BLT2) (Control: Rat2-pcDNA3.1 cells), and the right picture shows cell growth of BLT2-expressing cells (experimental group: Rat2-BLT2 stable cells).
2A-2E are graphs showing the effect of the compounds of the present invention (represented by Formulas 1-1 to 1-5) in BLT2-expressing cells (control group; Rat2, right panel) and BLT2-expressing cells (Rat2- Compound), LY255283 and DMSO, respectively.

The present invention relates to a novel use for the prophylaxis or treatment of inflammatory diseases of compounds having the structure of formula

(Formula 1)

The present inventors have found that a compound having the structure of the above formula (1) is a conventional BLT2 inhibitor such as LY255283 (1- [5-ethyl-2-hydroxy-4- [[6-methyl- 6- (1H-tetrazol- ) heptyl] oxy] phenyl] -ethanone) exhibited remarkably excellent BLT2 inhibition or cell growth inhibition effect by BLT2, the present invention has been completed.

Specifically, the present invention relates to a composition for preventing or treating an inflammatory disease comprising a compound having the structure of Chemical Formula 1, and a method for preventing or treating an inflammatory disease using the compound having the structure of Chemical Formula 1 or the composition .

Formula 1 has the following structure:

(Formula 1)

In the general formula (1), R1 to R5 each independently represent hydrogen, C1-C5 alkyl or halogen. Preferably, each of R1 to R5 independently represents hydrogen, a methyl group or -Cl.

Specifically, the compound having the structure of Formula 1 may be a compound having any one of the following Formulas 1-1 to 1-5:

(1-1)

N-butyl-6- (2,4-dimethylphenoxy) -1-hexanamine)

(1-2)

N-butyl-6- (4-chlorophenoxy) -1-hexanamine)

(1-3)

N-butyl-6- (2,3-dichlorophenoxy) -1-hexanamine was used instead of N-butyl-6- (2,3-dichlorophenoxy)

(Formula 1-4)

N-butyl-6- (2,6-dichlorophenoxy) -1-hexanamine)

(Formula 1-5)

N-butyl-6- (3,5-dimethylphenoxy) -1-hexanamine) .

In the present invention, the inflammatory disease is a disease caused by overexpression of BLT2, and may be at least one selected from asthma, atherosclerosis, cancer, skin itching, rheumatoid arthritis and inflammatory bowel disease, but is not limited thereto. In addition to the diseases exemplified herein, all BLT2-related inflammatory diseases known in the art are considered to be included in inflammatory diseases that can be prevented or treated with compounds having the structure of Formula 1 of the present invention.

In one embodiment, the cancer may be any cancer caused by overexpression of BLT2 or the tumor gene Ras. The cancer may be selected from the group consisting of bladder cancer, prostate cancer, pancreatic cancer, breast cancer, brain tumor, skin cancer, and liver cancer. We have shown that BLT2 is overexpressed in these cancers via PCT / KR2008 / 001649 and can be used to treat cancer using BLT2 inhibitors. When the BLT2 inhibitor is administered, cancer can be prevented or treated by inhibiting induction of cell death, inhibition of cancer cell metastasis or angiogenesis of tumor.

The compound having the structure of formula (I) of the present invention inhibits cell growth by BLT2, thereby exhibiting an effect of preventing or treating inflammatory diseases. The inhibitory effect of BLT2 on cell growth by the compound having the structure of formula (1) may be due to the action of inhibiting either BLT2 binding to LTB4, BLT2 activation, or its intracellular signal transduction pathway . More specifically, a compound having the structure of Formula 1 of the present invention inhibits Akt phosphorylation by binding to BLT2, inhibits LTB ₄ -induced ROS production, and consequently inhibits LTB ₄ -induced chemotaxis have. In the present invention, 'inhibition' is understood to include inhibition of any step during transcription, mRNA processing, translation, translocation and maturation of a gene, inhibition of protein-protein binding, protein activation or signal transduction through it do.

In one embodiment, the composition may be administered sequentially or concurrently with other active ingredients for the prevention or treatment of inflammatory diseases. Combination therapy with other active ingredients may inhibit inflammatory diseases through the same or different mechanisms, thus providing a synergistic therapeutic effect.

For example, the other active ingredient may be an anticancer agent or an asthma treatment agent. The inventors of the present invention have confirmed through PCT / KR2008 / 001649 that a synergistic effect on breast cancer is manifested by using LY255283, a BLT2 antagonist, and epirubicin, an anti-cancer chemotherapeutic agent.

The composition for preventing or treating inflammatory diseases of the present invention may be a pharmaceutical composition or a food composition.

The pharmaceutical composition for the prevention or treatment of inflammatory diseases of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, , A swelling agent, a lubricant, a lubricant or a flavoring agent may be used.

The pharmaceutical composition for the prevention or treatment of inflammatory diseases of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient for administration.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

Pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, suspending agents or injectable solutions or suspensions .

The pharmaceutical compositions of the present invention may be formulated and administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI >

The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating diseases or inducing bone growth. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, perpurin or chloranil may be administered at a dose of 0.1 μg / kg to 1 g / kg once or several times a day when administered once to several times a day .

In the present invention, the 'object' includes, but is not limited to, a human, an orangutan, a chimpanzee, a mouse, a rat, a dog, a cow, a chicken, a pig,

The food composition according to the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverage such as drink, teabe type granule, stick type diet bar, chewable tablet type vitamin, candy, chocolate, food, confectionery, Other noodles, gums, ice cream, alcoholic beverages, vitamin complexes, and health supplement foods.

When the composition of the present invention is used as a food composition, the composition of the present invention may further comprise at least one ingredient selected from the group consisting of amino acids, vitamins, minerals, sweeteners and dietary fibers. These ingredients can improve the taste and nutrition of the food composition.

The present invention will be described in more detail by the following examples. These examples are for illustrating the present invention, and the scope of the present invention should not be limited by them.

Reference Example  One: BLT2 Preparation of Expressed Cells

Cells without BLT2 expression (control; Rat2-pcDNA3.1 cells) and cells with BLT2 expression (experimental group: Rat2-BLT2) were prepared by the following method.

(DMEM) containing 0.1 mM MEM non-essential amino acids (Life technologies, Inc.), 10% fetal bovine serum (FBS), and antibiotic ntimycotic solution (Life technologies, Inc.) modified Eagle medium (Life Technologies, Inc.), Rat2-cells were added and maintained at 37 ° C and atmospheric conditions of 5% CO ₂ . The cells were maintained for 3 days in triplicate using Trypsin-EDTA. The cells were maintained in growth phase and washed twice with PBS (phosphate-buffered saline; 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ ) , And then added to a fresh medium to prepare cells in which BLT2 was not expressed (control; Rat2-pcDNA3.1 cells).

For the production of stable Rat2 / BLT2 clones, Rat-2 cells were transformed with pcDNA3-short form BLT2 encoding HA-tagged human BLT2 and stained with 0.4 mg / ml G418 (Invitrogen, Carlsbad , CA, USA). To screen for BLT2 expression, the selected clones were analyzed by RT-CR using human-specific BLT2 primers and representative clones were used for experiments with BLT2-expressing cells (Rat2-BLT2).

Reference Example  2: BLT2 0.0 > cells < / RTI >

To test the effect of on cell proliferation, BLT2, BLT2 are not expressing cells prepared in the above Reference Example 1; the (control-pcDNA3.1 Rat2 cells) and BLT2 the expressing cells (experimental group: Rat2-BLT2) to 1X10 ⁵ Each was dispensed on a 60 mm culture dish and cultured for 24 hours. The cell morphology was then observed under a phase-contrast microscope and counted by the trypan-blue exclusion method, as shown in Fig.

FIG. 1 is a graph showing cell growth of cells without BLT2 expression (control group; Rat2-pcDNA3.1 cells) and cells expressing BLT2 (experimental group: Rat2-BLT2) (Control: Rat2-pcDNA3.1 cells), and the right picture shows cell growth of BLT2-expressing cells (experimental group: Rat2-BLT2 stable cells).

The results showed that the cells expressing BLT2 (in the experimental group: Rat2-BLT2, right panel) showed increased cell growth as compared to the cells not expressing BLT2 (control: Rat2-pcDNA3.1 cells, left panel) Fold increase). That is, it was confirmed that Rat-2 cells stably transformed into human BLT2 exhibited increased growth.

Example  1: Preparation of the compounds of the formulas 1-1 to 1-5

Compounds of formulas 1-1 through 1-5 were prepared (ChemBridge, San Diego, USA).

Comparative Example  One: DMSO Preparation of

As a control, DMSO was prepared (Sigma, Co.).

Comparative Example  2: BLT2  Preparation of inhibitors

As a positive control, LY255283 (1- [5-ethyl-2-hydroxy-4 - [[6-methyl- 6- (lH-tetrazol- 5- yl) heptyl] oxy] phenyl] -ethanone, (BioMol, Plymouth Meeting, PA).

Experimental Example  One: BLT2 - Mediated  Determining the Effect of the Compounds of the Invention on Cell Growth

In order to confirm the effect of the compounds of the formulas 1-1 to 1-5 on the BLT2-mediated cell growth, the compounds of the above Examples and Comparative Examples were added to each of the cells not expressing BLT2 and the cells expressing RBLT2, The proliferation was analyzed.

Specifically, the reference example 1 cells BLT2 has not been prepared in the expression (control; Rat2-pcDNA3.1 cells) and BLT2 the expressing cells (experimental group: Rat2-BLT2) to 1 X10 ⁵ 60 mm culture dish (culture dish) , 7.5 ug / ml of the compound of Formulas 1-1 to 1-5 prepared in Example 1, 10 μM of DMSO of Comparative Example 1 and 3 g of the positive control LY255283 (an inhibitor of BLT2) of Comparative Example 2 / ml was added thereto, followed by culturing for 24 hours to obtain the cells, which were counted by trypan-blue exclusion method. The results of the experiment are shown in FIG.

2A-2E are graphs showing the effect of the compounds of the present invention (each represented by the formulas 1-1 to 1-7, respectively) in cells in which BLT2 was not expressed (control group; Rat2, right panel) and cells in which BLT2 was expressed (Rat2- 5), LY255283, and DMSO, respectively.

Referring to FIG. 2A, in the cells expressing BLT2 (Rat2-BLT2, left panel), the compound of formula 1-1 showed a proliferation rate of 39.3% as compared with that of Comparative Example 1 (DMSO treatment; Rat2-BLT cells without compound treatment) . However, the compound did not show an inhibitory effect on Rat-2 cells in which BLT2 was not expressed (control; Rat2, right picture).

Referring to FIG. 2B, the compound of the formula 1-2 was found to have a proliferation rate of 36.6% as compared with the compound of Example 1 (DMSO treatment; Rat2-BLT cells without compound treatment) in cells BLT2-expressing cells (Rat2-BLT2, . However, the compound did not show an inhibitory effect on Rat-2 cells in which BLT2 was not expressed (control; Rat2, right picture).

Referring to FIG. 2C, in the cells expressing BLT2 (Rat2-BLT2, left panel), the compound of Formula 1-3 had a proliferation rate of 37.7% as compared with Comparative Example 1 (DMSO treatment; Rat2-BLT cells without compound treatment) . However, the compound did not show an inhibitory effect on Rat-2 cells in which BLT2 was not expressed (control; Rat2, right picture).

Referring to FIG. 2d, in the cells expressing BLT2 (Rat2-BLT2, left panel), the compounds of Formulas 1-4 were found to have a proliferation rate of 37.2% as compared with Comparative Example 1 (DMSO treatment; Rat2-BLT cells without compound treatment) . However, the compound did not show an inhibitory effect on Rat-2 cells in which BLT2 was not expressed (control; Rat2, right picture).

Referring to FIG. 2E, the compound of the formula 1-5 was found to have a proliferation rate of 34.7% as compared with the compound 1 (DMSO treatment; Rat2-BLT cells without compound treatment) in cells BLT2-expressing cells (Rat2-BLT2, . However, the compound did not show an inhibitory effect on Rat-2 cells in which BLT2 was not expressed (control; Rat2, right picture).

On the other hand, when 3 ug / ml of LY255283 (an inhibitor of BLT2) of Comparative Example 2 used as a positive control for cells expressing BLT2 (Rat2-BLT2, left picture) was pretreated with Comparative Example 1 (DMSO treatment; Rat2- BLT cells without compound treatment) showed a proliferation rate of 79.45%. (Table 1)

That is, it has been experimentally confirmed that the compounds of formulas 1-1 to 1-5 exhibit remarkably enhanced BLT2 inhibitory effect as compared with LY255283, which is known as BLT2 inhibitor.

From the above experimental results, it was confirmed that the compounds of formulas 1-1 to 1-5 can inhibit BLT2-induced cell proliferation with excellent efficiency. Accordingly, the compounds are judged to be BLT2-blocking pharmacological molecules that can be used as therapeutic agents for the inhibition of anti-cancer, anti-asthma or other forms of BLT2-related inflammatory diseases.

Claims (6)

A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases comprising a compound having a structure of the following formula
(Formula 1)

In the above formula (1), X is oxygen, and R 1 to R 5 are each independently hydrogen, a methyl group or -Cl.
delete The pharmaceutical composition according to claim 1, wherein the compound having the structure of Formula 1 is a compound having any one of the following Formulas 1-1 to 1-5:
(1-1)

(1-2)

(1-3)

(Formula 1-4)

(Formula 1-5)

.
The method according to claim 1,
Wherein the inflammatory disease is at least one selected from asthma, atherosclerosis, cancer, skin itching, rheumatoid arthritis and inflammatory bowel disease.
The method according to claim 1,
Wherein said inflammatory disease is due to overexpression of BLT2.
The method according to claim 1,
Wherein the inflammatory disease is prevented or treated by inhibiting cell growth by BLT2.

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