TWI498114B - Use of creosol for inhibiting expression and activation of nlrp3 inflammasome in cell - Google Patents

Description

Use of wood taritol for inhibiting activation and expression of NLRP3 complex inflammatory cells in cells

The present invention relates to the use of wood tar alcohol for the treatment of inflammatory diseases.

Wood tar alcohol, also known as 2-methoxy-4-methylphenol, 2-methoxy-4-cresol or 4-methyl-o-methoxyphenol, is a phenolic component of wood tar, generally used It is a disinfectant, a wood preservative, an antidiarrheal drug or an expectorant (Matsushima N et al., Eur J Pharmacol. 2007; 567(1-2): 59-66). Wood tar alcohol found in red wine cooked in oak barrels can also be prepared by hydrogenating vanillin (Pérez-Prieto LJ et al, J Agric Food Chem. 2003, 51(18): 5444-9). In addition, vanillin can be isolated from lignin on a large scale by commercial processes (Meylemans HA et al., ChemSus Chem. 2012, 5(1): 206-10). Wood taroleol, C ₈ H ₁₀ O ₂ is a colorless liquid having the following structure:

It has been reported that wood taritol can prevent cultured rats exposed to N-methyl-D-aspartate or H ₂ O ₂ by inhibiting Ca ²⁺ influx and generating intracellular reactive oxygen species. Cell death in hippocampal gyrus neurons (Nakamichi N et al , J Neurosci Res. 2010, 88(11): 2683-93). Wood taritol has also been reported to prevent bone loss caused by ovariectomy by inhibiting the anti-oxidative properties of osteoclasts in combination with osteoblasts (Moriguchi N et al , Biochem Pharmacol. 2007, 73(3): 385- 93).

There are many conditions in which an inflammatory response can occur, including physical injury, irritation, and tissue Tumor growth and infection with bacteria, parasites, fungi or viruses. The inflammatory response causes both local and systemic effects. Representative effects that can occur at the site of injury, irritation, or disease are increased vascular permeability, release of degrading enzymes including metalloproteinases, migration of white blood cells to affected sites, neutrophil bursts to destroy invading cells, and Interleukin secretion. Important systemic effects include pain, fever, and acute reactions in the liver.

Macrophages are a form of inflammatory cells that play an important role in the inflammatory response. Once activated, macrophages counteract the infection or foreign particles by triggering a series of inflammatory responses by releasing the inflammatory mediator. In addition, nitric oxide (NO), interleukin-6 (IL-6) and TNF-α are important early inflammatory mediators, mainly produced by lipopolysaccharide (LPS)-activated macrophages and regulate various biological effects. Including the activation of this immune response.

In addition, the inflammasome is a polyprotein message complex for the activation of caspase-1. Among the composite inflammatory bodies, the NLRP3 complex inflammatory body is one of the most well-studied. The NLRP3 complex inflammatory body is activated by adenosine triphosphate in LPS-activated macrophages, resulting in activation of apoptotic protease-1 and IL-1β secretion (Hu Y et al , J Immunol. 2010 Dec 15;185 ( 12): 7699-705).

Wood taritol has attracted the attention of medical researchers for its ability to prevent cell death in hippocampal gyrus neurons and bone loss due to oophorectomy. However, wood tar Alcohol has not been used to treat inflammation.

The present invention is based on the unpredictable discovery that 2-methoxy-4-methylphenol (wood tar alcohol) can be used to treat inflammation. Accordingly, the present invention provides a novel method for the treatment of inflammatory diseases.

In one aspect, the invention provides the use of a compound of formula (I) for the manufacture of a medicament for the treatment of an inflammatory disease, Wherein each of R ₁ and R ₂ is independently H or an alkyl group.

In a particular embodiment of the invention, the compound is 2-methoxy-4-methylphenol.

In another embodiment of the invention, the compound is effective to inhibit the expression of nitric oxide or interleukin-6 (IL-6) in an individual's inflammatory cells at a dose.

In a further embodiment of the invention, the compound is effective to inhibit apoptotic protease-1 activation or IL-1β secretion in an individual's inflammatory cells at a dose.

In another embodiment of the invention, the compound is effective to inhibit the expression of NLRP3 and IL-1β precursors in an individual's inflammatory cells at a dose.

In another embodiment of the invention, the inflammatory cell is a macrophage.

Details of one or more specific embodiments of the invention are set forth below. Other features of the present invention will become apparent from the Detailed Description of the Detailed Description of the Drawing. Or advantage.

To illustrate the invention, the presently preferred embodiments are shown in the drawings. However, it is to be understood that the invention is not to be limited to the preferred embodiments shown. In the drawings: Figures 1A to 1C show the effect of wood taritol on the performance of the following inflammatory mediators: NO (A), IL-6 (B) and TNF-α (C) (where *p < 0.05; **p <0.01).

Figure 2A shows the effect of wood taroleol on IL-1β after treatment with wood taritol for 30 minutes followed by LPS treatment (where *p < 0.05); Figure 2B shows when cells were treated with wood taritol for 30 minutes, Subsequent treatment with LPS for 6 hours followed by wood tar alcohol for caspase-1 (p45) and cleavage of caspase-1 (p10); Figure 2C shows that cells were incubated with wood tar alcohol before LPS-priming Effect of wood taroleol on IL-1β after minute (*p<0.05); Figure 2D shows that wood glycerol against apoptosis protease-1 after incubation with wood tar alcohol for 30 minutes before LPS-priming (p45) and the effect of cleavage of apoptotic protease-1 (p10); and Figure 2E shows that the tartar alcohol NLRP3 complex inflammatory body and IL-1β precursor were treated after treatment with wood taritol for 30 minutes followed by LPS treatment for 6 hours. The influence of things.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise stated, the following vocabulary used in the present invention has the meaning given.

The article "a" as used herein means one or more (i.e., at least one) grammatical object having the article. For example, "an element" means one element or a plurality of elements.

The table below shows abbreviations for some terms.

In one aspect, the invention relates to a method of treating an inflammatory disease comprising administering to a subject in need thereof an effective amount of a compound of formula (I):

Wherein each of R ₁ and R ₂ is independently H or an alkyl group.

Unless otherwise indicated, "alkyl" B, mean a straight or branched chain monovalent hydrocarbon (e.g. C ₁ -C _{8) 1} to 20 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. Unless otherwise indicated, the alkyl groups referred to in the present invention include both substituted and unsubstituted moieties. The term "substituted" means that one or more substituents each replace a hydrogen atom.

The term "treatment" as used in the present invention encompasses the prevention of a particular disease or condition or the alleviation of symptoms associated with a particular disease or condition and/or the elimination of such symptoms. For example, the term "treating an inflammatory disease" as used in the present invention means to inhibit oxygen in an individual's inflammatory cells. Nitrogen or IL-6 expression, inhibition of apoptosis proteinase-1 activation or IL-1β secretion and inhibition of NLRP3 and IL-1β precursor expression reduces local or systemic inflammatory overreaction.

The term "individual" as used in the present invention includes humans and animals, such as companion animals (such as dogs, cats, and the like), farm animals (such as cattle, sheep, pigs, horses, and the like) or laboratory animals (for example, Rat, mouse, guinea pig and similar animals).

The term "inflammatory disease" as used in the present invention includes rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic activity. Anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullous blister Sore, cardiomyopathy, celiac dermatitis, chronic fatigue syndrome immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, scar pemphigoid, cold agglutinin syndrome, Crest syndrome, Crohn's disease (Crohn's disease), Dego's disease, dermatomyositis, juvenile dermatomyositis, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Grave's disease, Guillain- Barre), Hashimoto's thyroiditis; idiopathic pulmonary interstitial fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (I ), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, nodular polyarteritis, polychondritis, Polygland syndrome, rheumatic polymyalgia, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's Syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, gradual freezing syndrome, Takayasu arteritis, radial artery Inflammatory/macrocellular arteritis, ulcerative colitis, uveitis, vasculitis, leukoplakia and Wegener's granulomatosis.

As used herein, the term "therapeutically effective amount" means the amount of each active agent required to administer a therapeutic effect to an individual, either alone or in combination with one or more active agents. As will be appreciated by those skilled in the art, an effective amount can vary depending on the route of administration, the excipients employed, and the co-use with other active agents.

In the present invention, it is unpredictable to find that the compound of formula (I) can stimulate apoptosis in LPS-activated macrophages by reducing the expression of NO and IL-6 in LPS-activated macrophages by reducing ATP. Protein-1 activation and inhibition of NLRP3 complex inflammatory body mediates IL-1β expression and secretion for the treatment of inflammatory response.

Accordingly, the present invention provides the use of a compound of formula (I) for the manufacture of a medicament for the treatment of an inflammatory disease, wherein each of R ₁ and R ₂ is independently H or alkyl. The invention also provides a method of treating an inflammatory disease comprising administering a therapeutically effective amount of a compound of formula (I) to an individual in need thereof.

In a specific embodiment, the compound of the present invention inhibits NO or IL-6 secretion, apoptosis protease-1 activation, IL-1β secretion and/or in a inflammatory cell, particularly a macrophage, in a therapeutically effective amount. Or NLRP3 performance and performance of IL-1β precursors.

Without further elaboration, it is believed that the foregoing description may be Therefore, the following examples are to be construed as illustrative only and not in any way limiting. All publications, including patents, cited herein are hereby incorporated by reference in their entirety.

Example

Material

LPS (from Escherichia coli 0111:B4) and anti-actin antibodies were purchased from Sigma (St. Louis, MO). Anti-IL-1β and anti-apoptosis protease-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The TNF-[alpha], IL-6 and IL- l[beta] ELISA kits were purchased from R&D Systems (Minneapolis, MN). The anti-NLRP3 antibody was obtained from Enzo Life Science Inc. (Exeter, UK).

2. Cell culture

Mouse macrophage RAW 264.7 and J774A.1 cells were obtained from the American Type Culture Collection (Rockville, MD). Stably transfected with NF-κB gene reported RAW (RAW-Blue ^TM cells) 264.7 macrophages were purchased from InvivoGen (San Diego, CA). All cells were propagated in RPMI-1640 medium supplemented with 10% heat-reduced fetal bovine serum and 2 mM L-glutamic acid, and cultured in a 5% CO ₂ incubator at 37 ° C (RAW-Blue ^TM cells cultured in in the presence of zeocin ^TM).

3. Enzyme-linked immunosorbent assay (ELISA)

The cells were seeded in a 6-well plate at a density of 5 x 10 ⁵ cells/ml, followed by incubation with or without LPS (1 μg/ml) for 24 hours without or with the test sample. The effect of test samples on TNF-[alpha], IL-6 and IL- l[beta] production was measured by ELISA according to manufacturer's guidelines. Briefly, 50 μl of biotinylated antibody reagent and 50 μl of supernatant were added to anti-mouse TNF-α, IL-6 and IL-1β precoated wells and incubated for 2 hours at room temperature. After the dish was washed three times with washing buffer, 100 μl of a diluted horseradish peroxidase-labeled streptavidin-HRP concentrate was added to each well, and the dish was incubated at room temperature for 30 minutes. The washing process was repeated; after that, 100 μl of a premixed tetramethylbenzidine base solution was added to each well and developed in the dark at room temperature for 30 minutes. After 100 μl of the stop solution was added to each well to terminate the reaction, the absorbance of the culture dish was measured by a microplate reader at a wavelength of 450 nm.

4.NO inhibition test

RAW 264.7 cells were seeded in a 24-well plate at a density of 5 x 10 ⁵ cells/ml, followed by incubation with a medium containing or not containing LPS (1 μg/ml) for 24 hours without or with the test sample. The effect of wood taritol on NO production was measured indirectly by analyzing the nitrite concentration using the Griess reaction.

5. Western Blot Assay

Whole cell lysates were separated by SDS-PAGE and electrotransferred to PVDF (polyvinylidene fluoride) membranes. The membrane was incubated for 1 hour at room temperature in a sealing solution (5% dehumidified bovine milk phosphate buffered saline containing 0.1% Tween 20). Each membrane was incubated with a specific primary antibody for 2 hours at room temperature. After washing the membrane three times with PBS containing 0.1% Tween 20, the primary antibody was incubated with HRP-conjugated secondary antibody. The film is developed by an enhanced chemiluminescence western blot detection system.

6. Statistical analysis

All values are listed as mean ± SE. Data analysis included a one-way ANOVA for subsequent Scheffé tests.

result

1. Wood taroleol reduces NO and IL-6 in LPS-activated macrophages

The effect of wood tar alcohol on the performance of inflammatory mediators was analyzed. In the present invention, RAW 264.7 macrophages (5 × 10 ⁵ /ml) were pretreated with wood tar alcohol for 30 minutes, followed by stimulation with LPS (1 μg/ml) for 24 hours. The concentration of NO in the medium was measured by the Griess reaction. Data are expressed as a percentage (%) of individual LPS ± SE from three separate experiments (where * p <0.05; ** p < 0.01), as shown in Figure 1A.

As shown in Fig. 1B and Fig. 1C, RAW 264.7 macrophages (5 × 10 ⁵ /ml) were pretreated with wood tar alcohol for 30 minutes, followed by stimulation with LPS (1 μg/ml) for 24 hours. The concentration of IL-6 and TNF-α in the medium was measured by ELISA. Data are expressed as a percentage (%) of individual LPS ± SE from three separate experiments (where * p <0.05; ** p < 0.01).

As mentioned previously, NO, IL-6 and TNF-[alpha] are important early inflammatory mediators produced primarily by activated macrophages and mediate a variety of biological effects, including activation of this immune response. In the present invention, the Griess reaction is used for determining the dose-response characteristics of NO in cells pretreated with wood tar alcohol, and it is found that cells pretreated with wood tar alcohol inhibit the expression of NO in a dose-dependent manner, such as Figure 1A shows. To assess the effect of wood taritol on the expression of interleukins in LPS-activated macrophages, ELISA was used to determine the characteristics of the interleukin-expressing dose response in cells pretreated with wood tar alcohol. As shown in Figure 1B, wood tar alcohol was found to inhibit IL-6 expression in a dose-dependent manner; however, wood tar alcohol did not inhibit TNF-α expression in LPS-activated macrophages, as shown in Figure 1C.

2. Wood tar alcohol reduces IL-1β secretion by inhibiting NLRP3 complex inflammatory body

J774A.1 cells (1 x 10 ⁶ /ml) were challenged with LPS for 5.5 hours, followed by treatment with wood taritol for 30 minutes, followed by stimulation with ATP (5 mM) for an additional 30 minutes. As shown in Figure 2A, the IL-1β concentration in the medium was measured by ELISA. Data are expressed as mean ± SE from three respective experiments (where * p < 0.05). In Figure 2B, the expression of caspase-1 (p45) and lytic protease-1 (p10) was analyzed by Western blotting. The results of one of the three sets of respective experiments are shown below. In the present invention, J774A.1 cells (1 × 10 ⁶ /ml) were treated with wood tar alcohol for 30 minutes, followed by treatment with LPS for 6 hours. After washing, the cells were stimulated with ATP (5 mM) for an additional 30 minutes. As shown in Figure 2C, the IL-1β concentration in the medium was measured by ELISA. Data are expressed as mean ± SE from three respective experiments (where * p < 0.05). The expression of the caspase-1 (p45) and lytic protease-1 (p10) was analyzed by Western blotting. The results of one of the three sets of experiments are shown in Fig. 2D. In addition, J774A.1 cells (1 x 106/ml) were treated with wood tar alcohol for 30 minutes followed by LPS for an additional 6 hours. The expression of the NLRP3 complex inflammatory body and the IL-1β precursor was analyzed by Western blotting. The results of one of the three sets of respective experiments are shown in Figure 2E.

ATP-activated LPS promotes NLRP3 complex inflammatory bodies in macrophages, which cause activation of apoptotic protease-1 and secretion of IL-1β. To test whether wood taritol can modulate NLRP3 complex inflammatory body activation, the mouse macrophage cell line J774A.1 was selected and the effect of wood taritol on the activation of NLRP3 complex inflammatory bodies was tested.

Figure 2A shows the effect of wood taritol on IL-1 β , and Figure 2B shows that xylool against caspase-1 (p45) and cleavage after cells were treated with wood taritol for 30 minutes followed by LPS for 6 hours. Effect of Apoptosis Protease-1 (p10). Initially, activation of NLRP3 complex inflammatory body in J774A.1 cells after LPS-induced treatment and ATP stimulation was detected by measuring IL-1β secretion and activation of apoptotic protease-1. As shown in Figure 2A, ATP induced IL-1β secretion in LPS-promoted J774A.1 cells. To determine whether IL-1β secretion was reduced by wood tar alcohol, LPS-promoted J774A.1 cells were incubated with wood tar alcohol for 30 minutes, followed by addition of ATP and incubation for an additional 30 minutes. Wood tar alcohol slightly inhibited IL-1β secretion relative to the vehicle control group. The effect of wood taritol on the activation of apoptotic protease-1 was also tested by immunopolating the p10 subunit of mature apoptotic protease-1 in J774A.1 cells induced by ATP-stimulated LPS. ATP induces activation of apoptotic protease-1 in J774A.1 cells induced by LPS. To determine whether activation of apoptotic protease-1 was reduced by wood taroleol, LPS-promoted J774A.1 cells were incubated with wood tar alcohol for 30 minutes, followed by addition of ATP and incubation for an additional 30 minutes. Wood taritol slightly inhibits activation of apoptotic protease-1 as shown in Figure 2B.

Figure 2C shows the effect of wood taritol on IL-1 β , and Figure 2D shows that when the cells were incubated with wood tar oleol for 30 minutes, wood taritol apoptotic protease-1 (p45) and lytic apoptosis before LPS initiation. The effect of protease-1 (p10). In addition, J774A.1 cells were incubated with wood tar alcohol for 30 minutes prior to LPS initiation in order to determine whether xylanol inhibited NLRP3 complex inflammatory activation via LPS-mediated signaling. After LPS induction, wood tar alcohol and LPS were washed out before measuring IL-1β secretion and activation of caspase-1, and ATP was added for another 30 minutes. As shown in Figure 2C, wood tar alcohol inhibited IL-1β secretion in a dose-dependent manner. The activation of caspase-1 was also inhibited in a dose-dependent manner by wood tar alcohol as shown in Figure 2D.

Figure 2E shows the effect of wood taritol on NLRP3 complex inflammatory bodies and IL-1β precursors. In addition, it was tested whether wood taritol can inhibit NLRP3 expression, which is an essential component of a composite inflammatory body, and whether it can inhibit IL-1β precursors in LPS-activated cells. Cells were incubated with wood tar alcohol for 30 minutes followed by stimulation with LPS for an additional 6 hours. It was found that wood taritol slightly inhibited the expression of NLRP3, but significantly inhibited the expression of IL-1β precursor in LPS-activated macrophages. These results indicate that wood taritol inhibits NLRP3 complex inflammatory body activation by affecting LPS mediation signaling.

A person skilled in the art can make changes to the foregoing specific embodiments without departing from the broad inventive concept. Therefore, the invention is not limited to the specific embodiments disclosed, but is intended to be included within the spirit and scope of the invention.

A person skilled in the art can readily ascertain the essential features of the present invention from the foregoing description, and various modifications and changes can be made to the various uses and conditions without departing from the spirit and scope of the invention. Therefore, other specific embodiments are also within the scope of the patent application.

Claims (3)

Use of a 2-methoxy-4-methylphenol compound for the preparation of a medicament for inhibiting the activation and expression of NLRP3 complex inflammatory cells of cells. The use of claim 1, wherein the cell is a macrophage. The use of claim 1, wherein the compound is effective for inhibiting apoptosis protease-1 activation, IL-1β secretion or IL-1β precursor expression in the cell.