KR20150098983A - Pharmaceutical composition for prevention and treatment of inflammatory disease comprising compound having anti-inflammatory activity as an effective ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating inflammatory disease. According to the present invention, a pharmaceutical composition for preventing and treating inflammatory disease has effects in preventing and treating inflammatory disease, by inhibiting generation of NO which is a main inducing substance of inflammation, PGE_2, and tumor necrosis factor-alpha (TNF-α), and activation of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS).

Description

[0001] PHARMACEUTICAL COMPOSITION FOR PREVENTION AND TREATMENT OF INFLAMMATORY DISEASE COMPRISING COMPOUND HAVING ANTI-INFLAMMATORY ACTIVITY AS AN EFFECTIVE INGREDIENT [0002]

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases.

In general, the inflammatory reaction is associated with various inflammatory mediators and immune cells present in the local blood vessels and body fluids when the tissue is damaged, exposed to bacteria or viruses, and the enzyme reaction, inflammatory mediator secretion, cell migration, tissue destruction, Swelling, fever and pain.

In vivo inflammation has been implicated in a variety of biochemical phenomena and is associated with nitric oxide synthase (NOS), an enzyme that generates nitric oxide, and prostaglandin Enzymes are known to play an important role in mediating inflammatory responses.

Thus, NOS, an enzyme that produces NO from L-arginine, and COX-2, a cyclooxygenase, an enzyme involved in the synthesis of prostaglandins from arachidonic acid, It is becoming the main target.

Meanwhile, it has been known that NO, which is rapidly over-induced by various cytokines or external stimulants induced by iNOS (inducible NOS), causes cytotoxicity and various inflammatory responses. Chronic inflammation is also associated with increased iNOS activity (Miller MJ et al., Mediators of inflammation, 4, pp 387-396, 1995: Appleton L. et al., Adv. Pharmacol., 35, pp 27-28, 1996).

When LPS (lipopolysaccharide, a kind of toxin produced by microorganisms) is administered to experimental animals, a large amount of nitrate (NO ₃ ^- ) is produced, which is proven to be derived from oxidized nitrogen (NO) generated from macrophages. In addition, it has been found that such nitric oxide is an important mediator of antimicrobial and anticancer effects associated with macrophages. In antibiotic therapy, LPS released from the extracellular membrane as the bacteria die may also cause an endotoxin shock (Dunn, DL, Surg., 2000, Am 1994, Giroir, BP, Crit. Med., 21, 1993: Yamaguchi, Y. et al., J. Reticuloendothel. Soc., 409, 1982). Thus, drug treatment that reduces the action of LPS may also have an effective anti-inflammatory effect.

On the other hand, macrophages are derived from blood mononuclear cells (white blood cells), which remove cellular debris, kill pathogenic microorganisms, collect into lymphocytes, act as antigens and play an important role in an effective immune system. If the body is exposed to pathogenic stimuli or damage, macrophages can be treated with a variety of proinflammatory cytokines such as tumor necrosis factor-α [TNF-α], interleukin-1 [IL-1], interleukin-2 [IL-2] of which is responsible for signaling chemokine (chemokines), inflammatory molecules, nitric oxide (NO), reactive oxygen species (ROS) and PGE glycoprotein emit ₂ (Prostaglandin E2), and (co-stimulation molecules CD80 and CD86, adhesion molecules (Bresnihan, 1999; Burmester et al, 1997; Gracie et al, 1999).

In addition, depending on the magnitude of the host immune response, it is possible to prevent damage associated with inflammatory diseases such as rheumatoid arthritis, arteriosclerosis (Gracie et al, 1999; Michaelsson et al, 1995; Stuhlmuller et al, 2000). Therefore, recovery from severe disease depends on the regulation of macrophage function and the regulation of LPS.

However, the anti-inflammatory drugs known so far reduce inflammation and relieve pain, but due to side effects such as gastrointestinal disorders, it is difficult to use them for a long period of time. Therefore, Korean Patent Publication Nos. 10-1058805 and 10-0597211 disclose that inflammation A new pharmaceutical composition for suppressing or alleviating the cytotoxic effect of the present invention has been proposed. In addition, there is an urgent need to develop a novel anti-inflammatory drug having low cytotoxicity.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a pharmaceutical composition containing a compound having antiinflammatory activity as an active ingredient.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide an apparatus and method for controlling the same.

In order to achieve the above object, a pharmaceutical composition for preventing and treating inflammatory diseases according to one aspect of the present invention may comprise, as an active ingredient, a compound represented by the following formula (1).

[Chemical Formula 1]

In addition, the pharmaceutical composition for the prevention and treatment of inflammatory diseases can inhibit the production of NO (nitric oxide).

In addition, pharmaceutical compositions for the prophylaxis and treatment of inflammatory diseases can inhibit the production of PGE ₂ (Prostaglandin E2).

Furthermore, pharmaceutical compositions for the prophylaxis and treatment of inflammatory diseases can inhibit the production of TNF-alpha (Tumor Necrosis Factor-alpha).

The pharmaceutical composition for the prevention and treatment of inflammatory diseases can inhibit the activity of COX-2 (Cyclooxygenase-2), IL-6 (Interleukin-6) and iNOS (inducible nitric oxide synthase).

As described above, according to the present invention, the production of NO, PGE ₂ and TNF-α, which are the main inducers of inflammation, and the production of COX-2 (Cyclooxygenase-2), IL- ) And iNOS (inducible nitric oxide synthase), thereby preventing and treating inflammatory diseases.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the description of the claims.

FIG. 1 is a graph (Griess assay) showing the results of inhibiting the production of NO, PGE ₂ , and TNF-? By treating LPS with a compound according to one embodiment of the present invention in RAW 264.7 cells for 24 hours.
FIG. 2 is a graph showing the results of inhibiting the production of NO, PGE ₂ , and TNF-? By reacting a compound according to one embodiment of the present invention with peritoneal macrophages and then reacting LPS for 24 hours assay.
FIG. 3 is a graph (MTT assay) showing the results of treating the compounds according to one embodiment of the present invention with RAW 264.7 cells for 24 hours in order to confirm toxicity of the compounds according to one embodiment of the present invention.
FIG. 4 is a graph (ELISA) showing the results of inhibiting the production of PGE ₂ by treating PAM3 or Poly (I: C) for 24 hours after treating a compound according to one embodiment of the present invention in RAW 264.7 cells.
FIG. 5 is a graph showing the mRNA levels of TNF-a, iNOS, COX-2, and IL-6 after RAW 264.7 cells were treated with LPS and treated with LPS for 6 hours -time PCR).
FIG. 6 is a graph showing the mRNA levels of TNF-a and COX-2 after treatment with PMA (Phorbol Myristate Acetate) -treated U937 cells treated with a compound according to one embodiment of the present invention and LPS for 6 hours (real-time PCR).
FIG. 7 is a graph showing ear edema suppression effect (ear thickness comparison ratio) by oral administration of a compound according to one embodiment of the present invention to a mouse for induction of ear edema.
FIG. 8 is a graph showing the effect of the compound or the resveratrol extract of the present invention on the experimental mice treated with 3% DSS (Dextran Sodium Sulfate) or 1% CMC (CarboxyMethyl Cellulose) for 1 week, Fig.

The preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings, in which the technical parts already known will be omitted or compressed for simplicity of explanation.

The pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases according to one embodiment of the present invention may contain, as an active ingredient, a compound represented by the following general formula (1), which will be described with reference to FIG. 1 to FIG.

[Chemical Formula 1]

Herein, the compound of formula 1 (hereinafter abbreviated as 'DHB') may be 2,4-dihydroxy-N- (4-hydroxyphenyl) benzamide, hydroxyphenyl) benzamide).

The above-mentioned method for producing DHB of formula (1) includes the following three steps.

Step 1: introducing a protecting group into the hydroxyl group of 2,4-dihydroxy benzoic acid

Step 2: Chloride is prepared by reacting benzoic acid with thionyl chloride introduced with the protecting group prepared in Step 1, and 4-hydroxyl phenylamine (4-hydroxyphenylamine) To prepare a DHB to which a protecting group is introduced

Step 3: Preparation of the DHB of Formula 1 by removing the protecting group from the DHB to which the protecting group prepared in Step 2 is introduced in an aqueous base solution

The above-described three-step method for producing DHB is schematized in the following reaction formula (1).

[Reaction Scheme 1]

- Step 1

- Step 2

- Step 3

≪ Example 1 > Synthesis of DHB

(Synthesis of 2,4-diacetyloxybenzoic acid: II)

2,4-Dihydroxybenzoic acid (10.0 g, 0.065 mol) and triethylamine (32.8 g, 0.32 mol) were added to tetrahydrofuran (200 ml) and stirred for 10 minutes. Acetic anhydride (19.9 g, 0.19 mol) was added dropwise to the reaction mixture and refluxed for 3 hours. The reaction mixture is cooled to room temperature and evaporated under reduced pressure. Dichloromethane and water are added. The organic layer is washed several times with water and 1N hydrochloric acid aqueous solution, and then evaporated under reduced pressure. Hexane was added to the oil to form a precipitate, which was filtered under reduced pressure to give the product (11.6 g, 75%).

(Synthesis of 2,4-diacetyloxy-N- (4-hydroxyphenyl) benzamide: III)

2,4-Diacetyloxybenzoic acid (5.0 g, 0.021 mol) was added to tetrahydrofuran (200 ml) and cooled to 0 占 폚. Thionyl chloride (2.9 g, 0.025 mol) was added dropwise to the reaction mixture, stirred for 20 minutes, and then tetrahydrofuran was distilled off under reduced pressure to obtain a chloride compound. 4-Aminophenol (3.4 g, 0.031 mol) is dissolved in tetrahydrofuran (200 ml) and the temperature is cooled to 0 占 폚. The chloride compound prepared above was dissolved in tetrahydrofuran (200 ml) and slowly added dropwise. The reaction was stirred at 0 ° C for 4 hours and then evaporated under reduced pressure. The residue was chromatographed using ethyl acetate / hexane as the solvent to give the product (3.4 g, 50%).

(Synthesis of 2,4-dihydroxy-N- (4-hydroxyphenyl) benzamide:

2,4-Diacetyloxy-N- (4-hydroxyphenyl) benzamide (3 g, 0.009 mol) was added to an aqueous solution of 0.5 M potassium hydroxide (200 ml) and refluxed for 40 minutes. The solution is cooled to room temperature and then acidified to a pH of 4 to 3 with 1N aqueous hydrochloric acid solution. The neutralized reaction product was extracted with ethyl acetate (300 ml) and distilled under reduced pressure. The residue was purified by column chromatography using ethyl acetate / hexane as the solvent to give the product (1.4 g, 65%). ¹ H-NMR was used for analysis of the obtained product.

^{1 H-NMR (300MHz, DMSO-} d 6): δ 12.45 (s, 1H), 10.19 (s, 1H), 9.89 (s, 1H), 9.26 (s, 1H), 7.82 (d, 1H, J = 9.0 Hz), 7.35 (d, 2H, J = 9.0 Hz), 6.70 (d, 2H, J = 9.0 Hz), 6.32 (d, 1H, J = 9.0 Hz), 6.23 (s, 1H).

FIG. 1 is a graph showing the results of inhibiting the production of NO, PGE ₂ , and TNF-? By reacting RAW 264.7 cells with a compound according to one embodiment of the present invention followed by LPS for 24 hours.

That is, DHB was pretreated with RAW 264.7 cells (1 × 10 ⁶ cells / ml) supplemented with 100 u / ml of penicillin, 100 μg / ml of streptomycin and 10% (1 [mu] g / ml) for 24 hours. Measurement of NO, PGE ₂ , and TNF-α was then carried out through the Griess assay (Int J Biochem Cell Biol, 41 (4), pp. 811-821, 2009).

As shown in FIG. 1, the pharmaceutical composition for the prevention and treatment of inflammatory diseases according to an embodiment of the present invention showed significant differences (*: P <0.05, **: P <0.01) Inhibiting the production of inflammation inducers NO, PGE ₂ , and TNF- ?, thereby confirming the prevention and treatment of inflammation.

FIG. 2 is a graph showing the results of inhibiting the production of NO, PGE ₂ , and TNF-α by treating LPS with a compound according to one embodiment of the present invention in peritoneal macrophages for 24 hours.

That is, DHB was pretreated for 30 minutes in the abdominal macrophages (2 × 10 ⁶ cells / ml) extracted from the experimental mice and treated with LPS (1 μg / ml) for 24 hours. ₂ , the pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases according to one embodiment of the present invention is administered by administering (P <0.05, **: P <0.01), and inhibited the production of NO, PGE ₂ , and TNF-α, which are inflammation inducers, have.

FIG. 3 is a graph (MTT assay) showing the results of treating the compounds according to one embodiment of the present invention with RAW 264.7 cells for 24 hours in order to confirm toxicity of the compounds according to one embodiment of the present invention.

That is, the present inventors conducted a cytotoxicity test to see whether a compound according to one embodiment of the present invention can be used as a safe anti-inflammatory agent. For this purpose, RAW 264.7 cells (1 × 10 ⁶ cells / ml) were pretreated with DHB compounds at different concentrations for 18 hours and cultured for 24 hours to perform MTT assays (J Virol Methods 1988; 20: 309-21) .

As a result, the pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases according to one embodiment of the present invention was found to be free from cytotoxicity as shown in Fig.

FIG. 4 is a graph (ELISA) showing the results of inhibiting the production of PGE ₂ by treating PAM3 or Poly (I: C) for 24 hours after treating a compound according to one embodiment of the present invention in RAW 264.7 cells.

Namely, Pam3 (10 μg / ml) or Poly (I: C) (200 μg / ml) was treated for 24 hours after RAW 264.7 cells (1 × 10 ⁶ cells / ml) . Subsequently, the measurement of PGE ₂ was performed by ELISA (Endogen, Cambridge, Mass.).

As shown in FIG. 4, the pharmaceutical compositions for the prevention and treatment of inflammatory diseases according to one embodiment of the present invention showed significant differences (*: P <0.05, **: P <0.01) Inhibiting the production of PGE ₂ , which is an inflammation inducing substance, is effective in preventing and treating inflammation.

FIG. 5 is a graph showing the mRNA levels of TNF-a, iNOS, COX-2, and IL-6 after RAW 264.7 cells were treated with LPS and treated with LPS for 6 hours -time PCR).

That is, RAW 264.7 cells (1 × 10 ⁶ cells / ml) were treated with DHB and LPS at different concentrations for 6 hours, and quantitative real-time PCR was performed. First, all the RNAs were isolated from the cells using a triazole reagent (Trizol, Invitrogen, USA). CDNA was synthesized using a reverse transcriptase (superscript II reverse transcriptase) and oligo (dT) primer as a template, Real-time PCR was performed.

The results of the quantitative real-time PCR are shown in FIG. 5, and the pharmaceutical compositions for the prophylaxis and treatment of inflammatory diseases according to one embodiment of the present invention showed significant differences (*: P <0.05, **: P <0.01). The inhibition of mRNA expression of TNF-α, iNOS, COX-2 and IL-6, which are inflammation inducers,

FIG. 6 is a graph showing the mRNA levels of TNF-a and COX-2 after treatment with PMA (Phorbol Myristate Acetate) -treated U937 cells treated with a compound according to one embodiment of the present invention and LPS for 6 hours (real-time PCR).

That is, U937 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and treated with PMA (20 ng / ml) for 24 hours in cultured U937 cells (2 × 10 ⁶ cells / ml). After that, the PMA was washed away, treated with LPS (1 μg / ml) and DHB by concentration for 6 hours after standing for 40 minutes.

Quantitative real-time PCR of U937 cells was performed in the same manner as in Fig. 5, and the results are shown in Fig.

As a result, the pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases according to an embodiment of the present invention showed a significant difference (*: P <0.05, **: P <0.01) alpha] and COX-2 mRNA expression, thereby confirming the prevention and treatment of inflammation.

FIG. 7 is a graph showing ear edema suppression effect (ear thickness comparison ratio) by oral administration of a compound according to one embodiment of the present invention to a mouse for induction of ear edema.

After pre-treatment with 0.5% CMC (CarboxyMethyl Cellulose) or DHB (100 mg / kg) for 3 days in ICR mice (7 rats per group), arachidonic acid (2% w / ) Was used to induce ear edema (Biochem Pharmacol, 37, pp 2161-2165, 1988). After 1 hour of treatment with arachidonic acid, the thickness of the edema was measured with a constant-pressure thickness gauge. The effect of suppressing ear edema upon administration of DHB was numerically calculated by the following equation (1).

[Equation 1]

(Where _ET is the ear thickness of the group treated with DHB, N _ET is the ear thickness of the group not treated with DHB, and C _ET is the ear thickness of the control group)

As shown in FIG. 7, the thickness of the ear edema of the rats pretreated with 0.5% CMC was hardly changed, whereas the experimental composition for the prevention and treatment of inflammatory diseases according to one embodiment of the present invention was pretreated Showed a significant difference (**: P <0.01) between ear edema thickness and decreased inflammation.

FIG. 8 is a graph showing the effect of the compound or Resveratrol extract according to an embodiment of the present invention on experimental mice to which 3% DSS (Dextran Sodium Sulfate) or 1% CMC was administered for 1 week, &Lt; / RTI &gt;

Seven rats were orally administered with 3% DSS (w / v) for 7 days to induce enteritis, and DHB (50 mg / kg) or resveratrol (50 mg / kg) was orally administered every morning for the same 7 days After 7 days, the length of the small intestine was measured.

As a result, the experimental group pretreated with the pharmaceutical composition for the prophylactic and therapeutic treatment of the inflammatory disease according to the present invention had longer intestinal length than the test group without the anti-inflammatory drug or the test group administered with the resveratrol extract, (*: P < 0.05). Therefore, it can be confirmed that the pharmaceutical composition for the prevention and treatment of inflammatory diseases according to one embodiment of the present invention is effective for prevention and treatment of inflammation.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. And the scope of the present invention should be understood as the following claims and their equivalents.

Claims (5)

A pharmaceutical composition comprising a compound represented by the following formula (1) as an active ingredient:
A pharmaceutical composition for the prevention and treatment of inflammatory diseases.
[Chemical Formula 1]

The method according to claim 1,
Characterized in that said composition inhibits the production of NO (Nitric Oxide)
A pharmaceutical composition for the prevention and treatment of inflammatory diseases. The method according to claim 1,
Wherein said composition inhibits the production of PGE ₂ (Prostaglandin E2)
A pharmaceutical composition for the prevention and treatment of inflammatory diseases. The method according to claim 1,
Wherein said composition inhibits the production of TNF-alpha (Tumor Necrosis Factor-alpha)
A pharmaceutical composition for the prevention and treatment of inflammatory diseases. The method according to claim 1,
The composition is characterized by inhibiting the activity of COX-2 (cyclooxygenase-2), IL-6 (Interleukin-6) and iNOS (inducible nitric oxide synthase)
A pharmaceutical composition for the prevention and treatment of inflammatory diseases.

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