KR20110090058A - Pharmaceutical composition for treating inflammatory disease - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing 2-[2-chloro-6-(3-iodobenzyl)-purine-9-yl]-tetrahydro-thiophene-3,4-diol(LJ-1888) is provided to prevent and treat inflammatory diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammatory diseases contains 2-[2-chloro-6-(3-iodobenzyl)-purine-9-yl]-tetrahydro-thiophene-3,4-diol(LJ-1888) or pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition additionally contains pharmaceutically acceptable carrier, excipient, or mixture thereof.

Description

Pharmaceutical Composition for Treating Inflammatory Disease

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases, more specifically 2- [2-chloro-6- (3-iodobenzyl) -purin-9-yl] -tetrahydro-thiophen-3,4 It relates to a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases comprising diol (hereinafter abbreviated as 'LJ-1888') or a pharmaceutically acceptable salt thereof as an active ingredient.

Inflammation is a defensive reaction in the body when biological tissues are damaged by physicochemical factors such as trauma or burns, or biological factors such as bacteria or viruses. Symptoms such as hyperemia, swelling, fever, and pain This is characteristic. Biochemically, inflammation refers to the local exudation of plasma components or tissue fluids containing antibodies, histamine, serotonin, or other chemicals from inflammatory cells when the pro-inflammatory agents act on the body, infiltration of white blood cells, and fibrosis for recovery. Refers to a reaction phenomenon occurring on the living body. Depending on the type, amount, and immune status of the body, the inflammatory response may vary.

Factors that control the inflammatory response can be divided into substances that greatly increase the permeability of blood vessels and chemical delivery agents that promote leukocyte migration (Rubin. Lippincott Williams and wilkins, 24-46, 2001). Inflammation can also occur in various organs in the body, and in chronic inflammatory diseases, carcinogenesis is closely related to cancer (Shacter et al ., Oncology, 16, 217-26, 229, 2002; Coussens et al ., Nature, 420, 860-7, 2002).

Adenosine (adenosine) is produced by the breakdown of ATP in cells and is released into cells when adenosine accumulates in the cell.In the presence of pathophysiological processes such as inflammatory diseases, ischemic heart disease and tissue damage, metabolism of ATP in cells It is known to become more active and result in increased release of adenosine (Linden et. al ., Annu Rev Pharmacol Toxicol, 41, 775-787, 2001; Stiles, Clin Res, 38, 10-18, 1990). Adenosine receptors are G-protein-coupled receptors, and there are four subtypes of A ₁ , A _2A , A _2B and A _3. A _2A and A _2B increase the cyclic adenosine monophosphate (cAMP). On the other hand, since A ₁ and A ₃ reduce cAMP, intracellular signaling is affected by which receptors are expressed (Fredholm BB et. al ., Pharmacol Rev , 53, 527-552, 2001; Jacobson KA et al ., Trends pharmacol Sci, 19, 184-191, 1998).

Adenosine receptors are also expressed in macrophages, and substances that affect the interaction between adenosine and its selective receptors can affect the phagocytosis or NO production of these macrophages. TNF-α, IL-1ß, It also reduces the expression of inflammatory cytokines such as IL-6 (Hasko Get et. al ., J Immunol, 157, 4634-40, 1996; Sajjadi FGet et al ., J Immunol, 156, 3435-42, 1996). A ₃ AR selective antagonists are known to activate A ₃ AR, resulting in cerebroprotective, anti-asthma and anti-inflammatory activity (Fredholm et al. al ., Pharmacol Rev, 53, 527-552, 2001).

However, all known A3 adenosine receptor antagonists are compounds of a heterocyclic compound, and thus have low affinity for animal receptors, and thus are not suitable for animal testing for drug development. The development of a new class of antagonists that act on both animal and human receptors is urgent worldwide. In addition, according to the conventional A ₃ AR antagonist, such as A ₃ AR agonists such as Cl-IB-MECA should be used in high concentrations in the treatment of inflammatory diseases, such as side effects such as cytotoxicity was used according to restrictions.

As a result of continuous research by the present inventors to solve the above-mentioned problems of the prior art, it has a new structure that acts on both animal and human receptors away from the existing antagonist compound structure, has excellent anti-inflammatory effect even at low concentration, and side effects such as toxicity. A free A ₃ AR antagonist was discovered and the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases, which has a novel structure that acts on both animal and human receptors, has excellent anti-inflammatory effect even at low concentration, and does not have side effects such as toxicity.

In order to achieve the object of the present invention described above, the present invention

2- [2-chloro-6- (3-iodobenzyl) -purin-9-yl] -tetrahydro-thiophen-3,4-diol (LJ-1888) or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising as an active ingredient.

The active ingredient LJ-1888 in the composition of the present invention is represented by the formula (I):

[Formula I]

Unlike the A ₃ AR selective antagonists such as MRS 1191 and MRS 1220, which are compounds of a heterocyclic compound, the active ingredient LJ-1888 of the present invention has a nucleoside skeleton as an adenosine derivative and their pharmacology There is a difference in the positive response and the binding force. In particular, the A ₃ AR antagonist, a bicyclic compound, is inadequate for animal testing for the development of new drugs because its affinity for A ₃ AR is very poor. However, LJ-1888 has a nucleoside backbone that acts on both animal and human receptors. Therefore, it is excellent in the new drug development using it.

The active ingredient LJ-1888 of the present invention may be prepared by synthesizing according to the following reaction scheme, but is not limited thereto, and may be synthesized by modified synthesis methods by those skilled in the art. The synthesis according to the scheme is described in detail in the Applicant's International Application Publication No. WO08 / 108508, all of which are incorporated herein by reference:

Examples of pharmaceutically acceptable salts herein are the organic addition salts of LJ-1888 formed with acids that form physiologically acceptable anions such as: tosylate, methanesulfonate, malate, acetate, citrate, Malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate. In addition, inorganic salts including hydrochloride, sulfate, nitrate, bicarbonate and carbonate salts can also be used. Pharmaceutically acceptable salts can be obtained by standard reactions well known in the art, for example, by sufficiently reacting a basic compound such as an amine with a suitable acid that supplies a physiologically acceptable anion. Alkali metal (such as sodium, potassium or lithium) or alkaline earth metal (such as calcium) salts of carboxylic acids may also be made.

Inflammatory diseases that can be treated with the pharmaceutical compositions according to the present invention are concepts that are irrelevant to their cause and include diseases mediated by an inflammatory response. Specifically sepsis, septic shock; Rheumatoid arthritis, osteoarthritis, ankylosing spondylitis; Vasculitis, pleurisy, pericarditis, associated inflammation, inflammatory aneurysms; Nephritis; hepatitis; Chronic pulmonary inflammatory disease; Bronchial inflammation, rhinitis; dermatitis; gastritis; Colitis, irritable bowel syndrome; Fever and muscle pain caused by infection.

In inflammatory diseases, several factors are involved in the inflammatory response. Gene expression of enzymes and cytokines involved in the inflammatory response is mainly regulated by NF-κB. NF-κB is composed of RelA (p65), RelB, c-Rel, NF-κB 1 (p50), and NF-κB2 (p52), which are members of the Rel family, and are homodimers or heterodimers. It is a form of transcription factor. Most known as NF-κB are present in the cytoplasm in the form of heterodimers formed by the binding of RelA / NF-κB 1 (p65 / p50).

NF-κB is inactivated by binding to a κB (IκBs) inhibitor in the cytoplasm. When stimulated by LPS or an inflammatory substance, NF-κB is activated. The process is caused by three substances that regulate IκB: IκB kinase (IKK), ubiquitin ligase and 26s proteosomes. After IκB is phosphorylated by IKK, NF-κB is ubiquitinated by uniquitin ligase and finally separated from the 26s proteosome and IκB to separate NF-κB. When free NF-κB moves into the nucleus and binds to the κB-binding site, gene transcription occurs to regulate the expression of genes such as iNOS and COX-2, which are inflammation-related enzymes, and TNF-α and inflammatory cytokines TNF-α and IL-1β. Done.

Therefore, the expression / inhibition test of NF-κB, inflammation-related enzymes iNOS, COX-2 and inflammatory cytokines TNF-α, IL-1β can be used to analyze the effects of inflammatory disease therapeutic substances.

The pharmaceutical composition according to the present invention may contain one or more selected from pharmaceutically acceptable carriers or excipients, for example diluents, glidants, binders, disintegrants, sweeteners, stabilizers, and preservatives commonly used in the art. It may include, and may be formulated into a liquid, tablets, granules, capsules, powders and the like.

The pharmaceutical compositions according to the invention can be administered by route of intravenous administration, intraperitoneal administration or oral administration.

The pharmaceutical composition according to the present invention may be administered with other known anti-inflammatory agents in addition to the active ingredient according to the present invention to enhance the therapeutic effect.

The dosage and frequency of administration of the pharmaceutical composition according to the present invention can be appropriately adjusted by those skilled in the art according to the age, condition, weight, severity of the disease, drug form, route of administration, and duration of the patient. Preferably, the active ingredient of the pharmaceutical composition may be administered 1 to several times per day at 1 to 50 mg / kg (subject) per body weight of the patient.

The pharmaceutical composition according to the present invention exhibits excellent anti-inflammatory efficacy even at low concentrations (about 1/6) without side effects such as toxicity compared to the conventional A ₃ adenosine receptor agonists and antagonists.

Figure 1a and 1b is a graph showing the NO production inhibitory effect (Fig. 1a) and cytotoxic effect (Fig. 1b) of the active ingredient of the present invention composition LJ-1888.
2A and 2B are photographs showing the iNOS protein and COX-2 protein expression inhibitory effect (FIG. 2A) and the iNOS gene and COX-2 gene expression inhibitory effect (FIG. 2B) of LJ-1888 as an active ingredient of the present invention composition.
Figures 3a and 3b shows the inhibitory effect of the IL-1b protein and TNF-α protein expression (Fig. 3a) and IL-1b gene and TNF-α gene expression inhibitory effect of the composition of the present invention LJ-1888 (Fig. 3b) The picture shown.
Figure 4 is a photograph showing the expression inhibitory effect on IκBa protein degradation of the active ingredient of the composition of the present invention LJ-1888.
5 is a photograph showing the inhibitory effects on the DNA binding of NF-κB and the protein binding of p65, a subunit of NF-κB, of LJ-1888 as an active ingredient of the present invention.
6A and 6B are graphs showing the increase rate of foot edema (FIG. 6A) and the rate of foot edema (FIG. 6B) in an animal model inducing foot edema with carrageenan of LJ-1888, an active ingredient of the present invention composition.
FIG. 7 is a graph showing ear edema inhibition rate in an animal model in which ear edema was induced by TPA of LJ-1888 of the composition of the present invention.

The invention is explained in more detail by the following examples. These examples are intended to illustrate the invention and the scope of the invention should not be limited by them.

Example

The pharmaceutical composition according to the present invention was tested for anti-inflammatory activity in mouse-derived macrophage RAW 264.7 cells and inflammatory animal models.

Example  One: NO  Generation inhibitory activity detection ( iNOS assay )

The inhibitory activity of the pharmaceutical compositions according to the invention against the enzyme iNOS (inducible nitric oxide synthase), which increases expression in the presence of inflammatory reactions and trauma, was tested.

Specifically, RAW 264.7 (5 × 10 ⁵ cells / ml) cells were put in 1 ml of each well of a 24-well plate in DMEM medium containing 10% FBS and incubated for 24 hours. After 24 hours, the attached cells were washed with PBS (phosphate-buffered saline) and treated with 10% FBS-DMEM (without phenol red) medium and the composition of the present invention (LJ-1888, 5, 10, 15, 20 μM, respectively). After 30 minutes, 1 μg / ml LPS was added and incubated at 37 ° C. and 5% CO ₂ for 20 hours (experimental group). RAW cells cultured without addition of LPS as a blank and the composition according to the present invention and RAW cells cultured with only LPS added as a control were used.

100 μl of the supernatant of each well was reacted with 90 μl of sulfanilamide solution and N- (1-naphtyl) -ethylenediamine solution, respectively, at 540 nm. Absorbance was measured to confirm the amount of nitrate or nitrite produced in the culture, and the results are shown in FIG. 1.

A standard calibration curve was prepared using NaNO ₂ solution, and based on the measured absorbance, Equation 1 was used as a method to compare with the LPS-only group in terms of NaNO ₂ concentration according to the treatment of each experimental substance in the experimental group. The NO production inhibitory activity of the sample was evaluated. After the inhibition rate (%) was calculated from the nitrate concentration using the following equation, the IC ₅₀ value was calculated.

&Quot; (1) "

As shown in FIG. 1, treatment of the composition (LJ-1888) of the present invention with concentrations of 5, 10, 15, and 20 μM resulted in concentration-dependent inhibition of NO production (see FIG. 1A). IC ₅₀ was 10.16 μΜ. The IC ₅₀ value of the composition of the present invention is about 1/6 of the IC ₅₀ value (65.7 μM: measured value) of IB-MECA, which is a conventional A ₃ AR agonist. I could see this excellent.

MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide) analysis to determine whether the iNOS production inhibitory effect is due to toxicity of the sample itself (composition of the present invention, LJ-1888) As a result, cell viability (43%) was shown at 20 μM, the highest concentration of the composition of the present invention, but RAW 264.7 cells were at least 80% alive at 15 μM (FIG. 1B). It was confirmed that the effect was not due to toxicity, the experiment was based on 5, 10, 15 μM without cytotoxicity.

Example  2: iNOS  And COX -2 Protein and Gene Expression Inhibition Efficacy Evaluation

Expression of mRNA of iNOS protein and COX-2 protein induced by iNOS of the composition of the present invention was confirmed by Western blot analysis and RT-PCR.

Specifically, washed twice with PBS and incubated for 24 hours at 37 ℃, 5% CO ₂ condition and diluted such that 1 100mm culture ⅹ 10 ⁶ per dish in a medium containing 10% FBS to RAW 264.7 cells. The medium containing 10% FBS was pretreated with LJ-1888 15, 10, and 5 μM of the composition of the present invention, the highest concentrations showing no cytotoxicity, and incubated for 18 hours by inducing an inflammatory response with LPS (1 μg / ml). . The cells that were not attached to the cell medium and attached cells were collected and washed twice with PBS, and then, the cells were suspended in boiling cell lysis buffer and heated for 5 minutes at 100 ° C. After cooling, the solution was stored at 20 ° C and immediately melted at 37 ° C to be used for protein quantification and electrophoresis.

Protein quantitation was performed using the BCA method and 30-50 mg of protein was electrophoresed at 150 V for 110 minutes using an 8-12% SDS-polyacrylamide gel. The gel of the desired site was cut and transferred to a polyvinylidene fluoride (PVDF) membrane for 1 hour, washed twice with PBST, and stirred in a blocking buffer for 1 hour at room temperature. After washing 3 times with PBST for 5 minutes, the following primary antibody was diluted with 3% skim milk / PBST at a ratio of 1: 1,000 to 1: 2,000, sealed with a membrane, and incubated for 12 hours while stirring at 4 ° C. . After washing the membrane 2-3 times with PBST for 5 minutes, HRP-bound secondary antibody was diluted 1: 1,500 ~ 1: 2,000 and incubated with the membrane for 2-3 hours at room temperature. After rinsing three times with PBST for 5 minutes, the luminescence generated by treatment with western blotting substrate (WESTSAVE Up ^TM ) was confirmed using LAS-3000. As a control, protein expression was compared using the group treated with only LPS (+ control) and the group not treated with both LPS and the composition of the present invention, and the results are shown in FIG. 2A.

For 24 hours at 37 ℃, 5% CO ₂ condition and diluted such that 1 100mm culture ⅹ 10 ⁶ per plate for RAW 264.7 cells in a culture medium containing 10% FBS were washed twice with PBS and incubated. After diluting the composition of the present invention to 15, 10, 5 μM concentration of LJ-1888 in a medium containing 10% FBS, 10 ml of the prepared medium was added to a 100 mm culture dish and cultured for a predetermined time. Collect the cells that are not attached and the cells attached in the cell medium and wash them twice with PBS. Then, break down the cells with TRI reagent (TRIzol), collect the cells, and add RNA (CHCl ₃₎ to extract RNA, and then use isopropyl alcohol. Precipitated. RNA precipitate was washed with 70% ethanol, dried in air and then suspended in nuclease-free water. Heating at 55 ° C. for 10 minutes and heating at 70 ° C. for 5 minutes allowed RNA to be in single chain state. Total RNA was quantified by nano drop, and diluted to 1 μg / μl concentration to make cDNA using avian myeloblastosis virus (AMV) reverse transcriptase and oligo (dT) ₁₅ primers. After amplification of 0.2 mM dNTP mixture, 10 pmol target gene-specific primers (Table 1), and 0.25 unit Taq DNA polymerase in GeneAmp PCR system 2400, the resulting PCR product was electrophoresed at 100 V for 2 min agarose gel for 40 min. After staining with SYBR Safe and confirmed the stained RNA using Alpha Imager and the results are shown in Figure 2b.

<Primer> gene order iNOS sense 5'-ATGTCCGAAGCAAACATCAC-3 ' Antisense 5'-TAATGTCCAGGAAGTAGGTG-3 ' COX-2 sense 5'-CCC CCA CAG TCA AAG ACA CT-3 ' Antisense 5'-CCC CAA AGA TAG CAT CTG GA-3 ' IL-1β sense 5'-TGCAGAGTTCCCCAACTGGTACATC-3 ' Antisense 5'-GTGCTGCCTAATGTCCCCTTGAATC-3 ' TNF-α sense 5'-ATGAGCACAGAAAGCATGATC-3 ' Antisense 5'-TACAGGCTTGTCACTCGAATT-3 ' β-actin sense 5'-TGTGATGGTGGGAATGGGTCAG-3 ' Antisense 5'-TTTGATGTCACGCACGATTTCC-3 '

As can be seen in Figure 2a, the composition of the present invention (LJ-1888) inhibited iNOS protein expression in a concentration-dependent manner. However, the effect of inhibiting COX-2 protein expression was insufficient.

However, as shown in Figure 2b, it LPS only (compositions of the present ^invention) as compared to (2 references the second from the left in Fig. 2b) a control treatment, a composition (LJ-1888 5, 10, 15 μM) of the present invention It can be seen that the concentration-dependent inhibition of iNOS and COX-2 gene expression.

From these results, it can be seen that the anti-inflammatory activity of the composition (LJ-1888) of the present invention inhibits the expression of the enzyme through the regulation of gene levels of inflammatory reaction-related enzymes iNOS and COX-2.

Example  3: IL -1ß and TNF Effect on -α Expression

IL-1ß and TNF-α are cytokines with increased expression in LPS-stimulated cells, which affect the expression of IL-1β and the progression of the inflammatory response, and thus the composition of the present invention determines whether IL-1ß and TNF-α are expressed. This effect was tested. Specifically, the composition of the present invention was pre-treated in RAW 264.7 cells at concentrations of 15, 10, and 5 μM of LJ-1888, followed by incubation for 4 hours and 8 hours, respectively, by inducing an inflammatory response with LPS (1 μg / ml). The protein fractions of the cells were collected, and the degree of expression of IL-1ß and TNF-α proteins was measured by Western blotting. The results are shown in FIG. 3A.

In addition, to determine whether expression is inhibited at the gene level, pretreatment with LJ-1888 at a concentration of 15, 10, or 5 μM and inducing an inflammatory response with LPS (1 μg / ml) to collect RNA fractions after 4 hours of incubation, The mRNA expression level was measured using the RT-PCR method and the results are shown in Figure 3b.

As shown in Figure 3a, the experimental group treated with the composition (LJ-1888) of the present invention showed a protein-inhibitory effect of IL-1ß and TNF-α concentration-dependently compared to the control group. It was confirmed that IL-1ß and TNF-α mRNA expression was also inhibited in a concentration dependent manner in the composition (LJ-1888) of the present invention (FIG. 3B).

Example  4: NF - kB  Influence on signal transmission

NF-κB is a transcription factor consisting of p65 / p50, which is inactivated by binding to IκB in the cytoplasm, but undergoes a series of processes in which IκB is phosphorylated and degraded by IκB kinase (IKK) upon stimulation such as LPS. Is activated. When the free NF-κB state moves into the nucleus, it binds to the κB-binding site and regulates the expression of genes such as iNOS, TNF-α, IL-1β which are involved in the inflammatory response. Therefore, the effect on the NF-κB signaling system of the composition of the present invention was tested using Western blot and electrophoresis mobility shift assay (EMSA).

As a result, it was found that IκB protein degradation was significantly inhibited in the experimental group treated with the composition (LJ-1888) of the present invention compared to the control group treated with only LPS (see FIG. 4).

EMSA confirmed that NF-κB migrates into the nucleus and binds to DNA. As a result, the composition (LJ-1888) of the present invention inhibits DNA binding of NF-κB and protein binding of p65, a subunit of NF-κB. It was confirmed that (see Figure 5).

Example  5: Carrageenan ( carrageenan By induction Foot edema  Animal model

The anti-inflammatory efficacy of the composition of the present invention was tested through an animal model inducing an inflammatory response using carrageenan as the base material.

Measure the volume of the ankle joint of the right hind paw of the SD rat (150 ~ 200g, male, n = 7) using a plethysmometer to determine the initial volume of the foot (basal line), carboxymethyl cellulose The composition LJ-1888 (experimental group) and the positive control indomethacin (Indomethacin) dissolved in the rats were orally administered to rats, respectively. After 30 minutes, 1% carrageenan was dissolved in sterile distilled water, and 0.1 ml was injected subcutaneously into the sole of the foot. In the preliminary experiments (after the administration of the base material, the volume of the foot is determined by measuring the volume of the foot every hour to determine the time of the experiment), the volume of the base due to the base material rises highest between 2 to 4 hours, and then the volume subsides over time. In this experiment, the observation was performed for 6 hours after the administration of the chlorine substance, and the swelling rate was measured at one hour intervals. The anti-inflammatory activity of the composition of the present invention was evaluated, and the results are shown in FIGS. 6A and 6B.

<Equation 2>

&Quot; (3) &quot;

As shown in Figure 6a, looking at the 6 hours after the start of the experiment, compared to the increase rate of edema of the control group administered only carrageenin edema of the group administered the present invention composition (LJ-1888) and the indomethacin positive control group The rate of increase was significantly reduced.

As a result of measuring the edema inhibition rate, the inhibition rate was the highest between 2 and 4 hours and the drug effect was decreased after 6 hours (see FIG. 6B). In addition, the inhibition rate of the composition (LJ-1888) of the present invention was higher than that of indomethacin, and the composition of the present invention (LJ-1888) showed an inhibitory effect in a concentration-dependent manner.

Example  6: TPA  Judo Edema  Animal model

Phorbol 12-myristate 13-acetate (tetradecanoyl phorbol acetate; TPA), a substance derived from croton oil, is known as a toxic substance that activates protein kinase C and causes carcinogenesis. Was applied locally to the ear surface of the mouse (ICR mouse (20-25g), male, n = 7) to induce redness and inflammation.

Specifically, the composition of the present invention (LJ-1888) dissolved in carboxymethyl cellulose (CMC) and indomethacin, a positive control, were respectively orally administered to mice, and after 1 hour, TPA (2.5 μg / 20 μl acetone) was applied to the outside of the right ear. Each applied to the inner surface caused edema. After 4 hours, the left (control) and the outside of the right ear were punched to the same position in the same size and placed in a pre-weighed eppentrope tube to measure the weight difference from the left ear with a microbalance. Indicated.

As shown in FIG. 7, the edema growth rate of the group administered with the active ingredient LJ-1888 and the positive control soldier indomethacin of the present invention was reduced by 50% or more at a concentration of 20 mg / kg, and the composition of the present invention (LJ-1888). ) Showed inhibitory effect on ear edema in a concentration-dependent manner by oral administration.

Claims (8)

2- [2-chloro-6- (3-iodobenzyl) -purin-9-yl] -tetrahydro-thiophen-3,4-diol (LJ-1888) or a pharmaceutically acceptable salt thereof Pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising as an active ingredient. According to claim 1, The inflammatory disease is sepsis, septic shock; Rheumatoid arthritis, osteoarthritis, ankylosing spondylitis; Vasculitis, pleurisy, pericarditis, ischemic related inflammation, inflammatory aneurysms; Nephritis; hepatitis; Chronic pulmonary inflammatory disease; Bronchial inflammation, rhinitis; dermatitis; gastritis; Colitis, irritable bowel syndrome; Pharmaceutical composition is selected from the group consisting of fever and muscle pain caused by infection. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or mixture thereof. The pharmaceutical composition of claim 1, wherein the inflammatory disease is pulmonary pulmonary or pulmonary shock. The pharmaceutical composition of claim 1, wherein the inflammatory disease is rheumatoid arthritis, osteoarthritis, or ankylosing spondylitis. The pharmaceutical composition of claim 1, wherein the inflammatory disease is chronic pulmonary inflammatory disease, bronchial inflammation, or rhinitis. The pharmaceutical composition of claim 1, wherein the inflammatory disease is vasculitis, pleurisy, pericarditis, ischemia related inflammation, or inflammatory aneurysm. The pharmaceutical composition of claim 1, wherein the inflammatory disease is gastritis, colitis, or irritable bowel syndrome.

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