KR101937660B1 - Pharmaceutical composition for preventing or treating inflammasome mediated inflammatory disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of inflammus-mediated inflammatory diseases, which inhibits the formation of intracellular ASC polymer when 5-methylphenazin-1-one is used The present invention can promote the inhibition of the activity of inflammazol by the above-mentioned process, thereby exerting an effect for improving, preventing or treating the above-mentioned diseases.

Description

[0001] PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING INFLAMMASOME MEDIATED INFLAMMATORY DISEASE [0002]

The present invention relates to a composition for the prophylaxis or treatment of an inflammatory mediated inflammatory disease.

Pseudomonas aeruginosa is a relatively common bacterium that causes disease in mammals, including humans. The Pseudomonas aeruginosa is widely distributed in surrounding soil, water, skin, etc., and can be grown in a relatively low oxygen concentration, so that it is found in various environments. Animal infections can cause inflammation and sepsis, and can be fatal if infected with human organs such as the lungs and kidneys. It is rapidly recognized on wet surfaces and is frequently detected in medical facilities and devices, and thus is recognized as a major germ causing cross infection in hospitals. In the environmental field, it is also used as a microbial agent that removes tarballs when an oil spill accident occurs due to the ability to decompose hydrocarbons.

In addition, many other factors that are involved in virulence in P. aeruginosa have been reported to be regulated by an intercellular signaling mechanism called quorum sensing (QS), as in other Gram-negative bacteria (Fuqua et al., 2001; Welch et al., 2005; Coggan and Wolfgang, 2012). In general, the quorum sensing mechanism is controlled by a diffusible signaling substance, an enzyme that synthesizes it, and a receptor protein that accepts it (Henke and Bassler, 2004; Welch et al., 2005). The QS signaling substance is very different in structure and type depending on the bacteria. In the case of Gram-positive bacteria, a small peptide is used as a signal substance, and Gram-negative bacteria are mostly used as acyl homoserine lactone (acyl-HSL) , And some Vibrio and intestinal bacteria use furanone-based materials called AI-2 (Fuqua et al., 2001; Henke and Bassler, 2004; Reading and Sperandio, 2006).

In the meantime, inflammmasomes are substances that induce the maturation of inflammatory cytokines, such as IL-1, associated with innate immune defenses, such as cell infections or stress. And acquired inflammatory diseases have been emphasized in the immune response modulating mechanism, and there have been active developments of therapies targeted therewith.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of an inflammatory mediator inflammatory disease comprising 5-methylphenazin-1-one as an active ingredient.

It is another object of the present invention to provide a food composition for improving or preventing inflammatory disease mediated by inflammation which comprises 5-methylphenazin-1-one as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present inventors administered a pharmaceutical composition containing 5-methylphenazin-1-one to an inflammatory disease mediated by NLRP3 (NOD-like receptor family, pyrin domain-containing 3) , It was confirmed that NLRP3 inflammation activity can be effectively inhibited and the above diseases can be treated, thereby completing the present invention.

In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammatory-mediated inflammatory diseases comprising 5-methylphenazin-1-one represented by the following formula 1 as an active ingredient .

[Chemical Formula 1]

In the present invention, the 5-methylphenazin-1-one is a representative example of a gram-negative bacterium, Pseudomonas aeruginosa, produced through quorum sensing for communication between bacteria It is used as a cell signaling substance because it is composed of a structure capable of easily transmitting a cell membrane.

In one embodiment of the present invention, the pharmaceutical composition according to the present invention may inhibit the activity of inflammose.

However, in the present invention, the inhibition of the activation of the inflammase refers to a modification in vivo that causes a decrease in the activation of the inflammation of the target cell or the like. For the purpose of the present invention, Inhibition of cytokine production or inhibition of the formation of ASC (oligomer formation) of a caspase-recruitment domain (ASC) containing associated apoptosis-associated spec-like protein. Preferably, the pharmaceutical composition according to the present invention may inhibit, but is not limited to, inhibiting the formation of ASC polymer oligomer corresponding to the pre-inflammation stage.

In the present invention, the inflammasomes are caspase-1-activated complex protein complexes comprising 1) a sensory protein NLRP3 (NLRP3), 2) a linking protein (ASC), an adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain, and 3) an effector protein, caspase-1. The above inflammase components are assembled when a microorganism infection or tissue injury occurs. The inflammation activated by assembly in cytosol activates caspase-1 to produce interleukin (IL) -1β or interleukin- 18, and performs the innate immune defense function of the host (Schroder K, Tschopp J, Cell 140: 821-832 (2010); Franchi L, Munoz-Planillo R, Nunex, Nat Immunol 13: 325-332 ). Preferably, the inflammase may be, but is not limited to, NLRP3 inflammoma.

In the present invention, the NLRP3 inflammase is capable of inducing and / or promoting the onset of inflammatory mediated inflammatory disease when it is excessively active (Henao-Mejia J, Elinav Em Thaiss CA, Flavell RA (2014) Inflammasomes and Metabolic disease. Annu Rev Physiol 76: 57-78; Wen H, TingJP, eil LA 2012) A role for the NLRP3 inflammasome in metabolic diseases-did Warburg miss inflammation Nat Immunol 13: 352-357.

However, in the present invention, the inflammation-mediated inflammatory disease is an abnormal disease that occurs when the inflammoma is abnormally overactivated, preferably an autoinflammatory disease or neuroinflammatory disease, but the present invention is not limited thereto .

However, in the present invention, the autoinflammatory disease is classified as a disease group in which systemic inflammation is frequently repeated in a state where no autoantibody or antigen-specific T cell is found, unlike autoimmune disease, (21), which is characterized by an impaired regulation of innate immunity (Journal of Rheumatic Disease Vol. 21, No. 5, October, 2014). In addition, the autologous inflammatory disease plays an important role in the correlation of innate immunity and autoimmune disease with NLRP3 inflammatory activity through activation of caspase-1 and interleukin-1? (Am J Hum Genet. 2002 Jul; 71 (1): 198-203., Annu Rev Immunol. 2009; 27 (): 229-65, Cell, Mar. Mar., 140 (6): 784-790.).

Preferably the autoinflammatory disease is selected from the group consisting of Muckle-Wells syndrome (MWS), LADA (adult delayed autoimmune diabetes), familial cold autoimmune inflammatory syndrome (FCAS), krypyrin-related periodic syndrome Specific forms of pediatric arthritis, such as neuropathic pain syndrome (CAPS), neonatal-onset manifestation inflammatory syndrome (NOMID), chronic infantile nerve dermal joint (CINCA) syndrome, FMF, and / or systemic onset idiopathic arthritis (SJIA) And / or certain forms of adult rheumatoid arthritis, including, but not limited to, certain forms of pediatric rheumatoid arthritis, such as systemic onset idiopathic rheumatoid arthritis, and / or certain forms of adult rheumatoid arthritis.

In the present invention, the neuroinflammatory disease refers to a disease caused by damage to nerve tissue by an inflammatory reaction. Preferably, the neuroinflammatory disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt Jakob disease, multiple sclerosis, But is not limited to, any one or more diseases selected from the group consisting of lateral sclerosis, diffuse rheumatoid disease, white matter encephalitis, temporal lobe epilepsy and inflammatory spinal cord injury, more preferably Alzheimer's disease.

Alzheimer's disease has been reported to be an important mechanism of NLRP3 inflammation activation of microglia, and in animal models of Alzheimer's disease, caspase-1, which is a cytokine that is caused by inflammase in the microglial cells, The amount of IL-1 is increased (Immunol Rev. 2015 May; 265 (1): 63-74).

Recent studies have shown that compounds that inhibit NLRP3 inflammation formation, such as MCC950 or ketone metabolite beta-hydroxybutyrate, Glibenclamide, Alzheimer's disease and CNS damage such as neuroinflammatory diseases, etc., have been reported to exert a therapeutic effect through the process of alleviating the disease (Nature Medicine Volume: 21, Pages: 248-255 Year published: 2015) DOI: doi: 10.1038 / .3806, Nature Medicine Volume: 21, Pages: 263-269, published by (2015) DOI: doi: 10.1038 / nm.3804). Thus, a pharmaceutical composition comprising 5-Methylphenazin-1-one inhibiting NLRP3 inflammation according to the present invention significantly inhibits NLRP3 inflammation, thereby inhibiting NLRP3 Inflammatory diseases and neuroinflammatory diseases which are inflammatory-mediated inflammatory diseases (NLRP3).

In the present invention, the term "preventive " may include, without limitation, any action that inhibits the symptoms of obesity or lipid-related metabolic diseases using the pharmaceutical composition according to the present invention, or inhibits or delays the symptoms thereof.

In the present invention, "treatment" may include, without limitation, any action that alleviates or alleviates the symptoms of obesity or lipid-related metabolic diseases using the pharmaceutical composition according to the present invention.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be a human.

The pharmaceutical composition of the present invention may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterilized injection solutions according to a conventional method, have. The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. For topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. The formulation of the pharmaceutical composition according to the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other forms may be formulated as solutions, suspensions, tablets, capsules, sustained release formulations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The route of administration of the pharmaceutical compositions according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated. And the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of administration, the route of administration and the period of time, and may be 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

According to another embodiment of the present invention, there is provided a food for preventing or ameliorating an inflammatory disease mediated by inflammatory disease comprising 5-methylphenazin-1-one represented by the following formula (1) Lt; / RTI >

[Chemical Formula 1]

In the present invention, the 5-methylphenazin-1-one can prevent or ameliorate inflammatory mediated inflammatory diseases by inhibiting the activity of NLRP3 inflammas.

The contents of the 5-Methylphenazin-1-one, inflammus-mediated inflammatory diseases, prevention and inflammoses in the food composition of the present invention are the same as those described in the above pharmaceutical composition And the detailed description thereof will be omitted.

On the other hand, in the present invention, "improvement" may include, without limitation, any action that improves or alleviates symptoms of an autoinflammatory disease or neuroinflammatory disease using the food composition of the present invention.

The food composition according to the present invention may be prepared in the form of various foods such as beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes and breads. When the compound of the present invention is contained in the food composition, the amount thereof may be added in a proportion of 0.1 to 50% of the total weight.

Herein, when the food composition is prepared in a beverage form, there is no particular limitation other than that the food composition is contained at the specified ratio, and it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like and sugar sugars such as polysaccharide, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol can do. Examples of the flavors include natural flavors (tau martin, stevia extract (for example, rebaudioside A and glycyrrhizin), and synthetic flavors (for example, saccharine and aspartame).

In addition, the food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

These components may be used independently or in combination. The proportion of these additives is not a key element of the present invention, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.

In addition, the food composition of the present invention may be ingested as food or nutritional products such as milk or dairy fermented milk products, or as food supplements or health functional foods. Specifically, examples include foods such as dairy products, beverages, juices, soups or foods for children, but are not limited thereto. The dairy product means any liquid or semi-solid milk or liquid product having various fat contents. The milk product may be, for example, milk, goat's milk, positive milk, cream, full-fat milk, whole milk, low-fat milk or skim milk, Dyfiltered milk, microfiltered milk, recombinant milk obtained from milk powder or whey through any processing, processed products such as yoghurt, curd, milk oil, sour whole milk, , Buttermilk, other fermented milk products, e.g., viili, filling of snack bars, and the like. Another important group may include milk-based foods such as milk beverages, such as whey drinks, fermented milk, concentrated milk, infant and baby milk, ice cream, and sweets.

In the case of using 5-methylphenazin-1-one, a composition according to the present invention, the formation of ASC (adaptive protein apoptosis-associated spec-like protein containing a caspase-recruitment domain) Inhibiting the activity of inflammation, and thus can exert an effect on improvement, prevention or treatment of inflammatory disease mediated by inflammoma.

FIG. 1 shows the change in protein expression in Western blot analysis after administration of 5-methylphenazin-1-one to bone marrow-derived macrophages in Experimental Example 1. FIG.
FIG. 2 shows the results of ELISA for the release of interleukin (IL) -1β after administration of 5-methylphenazin-1-one to bone marrow-derived macrophages in Experimental Example 2 The results are shown.
FIG. 3 shows changes in protein polymerization by Western blot analysis after administration of 5-methylphenazin-1-one to bone marrow-derived macrophages in Experimental Example 3. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

[ Preparation Example ] mouse Bone marrow origin  Macrophage isolation and culture

(AIM2), which is the most potent inhibitor of innate immunity to Francisella turalrensis, was obtained from C57BL / 6 mice (Fernandes-Alnemri T, et al. 385-393.). All mice were grown under specific disease-free conditions. Protocols for animal experiments were approved by the Ethics Committee of Yonsei University. All BMDMs were cultured in L929-regulated DMEM supplemented with 10% FBS and 100 U / ml penicillin / streptomycin at 37 ° C, 5%, in a CO ₂ incubator.

[ Experimental Example  1] mouse Bone marrow origin  In macrophages Inflammase  Active inhibition

5-Methylphenazin-1-one (hereinafter referred to as " pyocyanin ") was confirmed to inhibit NLRP3 inflammation activity in the following manner.

The BMDMs of the above preparations were pretreated for 30 minutes with pyocyanin (20, 50, and 100 μM), treated with LPS (0.25 μg / ml for 3 hours), treated with ATP (2.5 mM, 40 minutes) I activated the moss. After the culture supernatant was obtained, western blot analysis was performed to confirm protein expression changes in cell lysate and supernatant after protein precipitation using methanol / chloroform method.

The preparation method of the cell lysate used in the Western blot analysis is as follows. The BMDMs were harvested by treatment with PBS, washed with PBS, and then dissolved in a RIPA buffer, which is a cell lysis buffer containing a protein degradation inhibitor. The dissolved solution was fractionated using a centrifuge, and only the protein-containing solution was extracted. The cell culture medium and cell lysate proteins were quantified by the Bradford method. Proteins at the same concentration as obtained by quantification were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membranes from which the proteins were transferred were blocked with TBS-T (Tris-buffered saline / 0.1% Tween-20) solution containing 5% non-fat milk for 1 hour at room temperature to reduce non-specific binding After the reaction, the primary antibody diluted in 5% BSA solution was reacted at 4 ° C for 12 hours or more, and the secondary antibody was reacted at room temperature for 1 hour. An enhanced chemiluminescence system was used for visualization.

Expression of p20, interleukin (IL) -1β and pro-interleukin (pro-IL) -1β, which are procaspase-1, activated caspase-1, And the results are shown in Fig.

As shown in FIG. 1, when LPS and ATP were treated together, secretion of activated form of interleukin-1? And activated caspase-1 p20 into cell culture was increased, and NLRP3 infectivity It was found that the llama activity was well induced.

As described above, secretion of interleukin-1 beta was remarkably inhibited at 50 [mu] M and 100 [mu] M in the case of treatment with pyocyanin when NLRP3 inflammation was induced. In addition, secretion was also remarkably reduced in the cell culture of p20.

From the above results, it can be seen that phycocyanin significantly inhibits NLRP3 inflammase activity.

[ Experimental Example  2] mouse Bone marrow origin  Interleukin- 1β  Confirmation of secretion inhibition

In order to examine the inhibition of NLRP3 inflammation activity of the phycocyanin identified above, LPS, quantified by ELISA (enzyme-linked immunosorbent assay), was assayed for interleukin-1 beta secreted by nigericin stimulation

The prepared BMDMs were treated with LPS (0.25 μg / ml, 3 hours), followed by treatment with Nigericin (5 μM, 40 minutes), followed by treatment with NLRP3 After activation of inflammase, each cell culture was obtained.

The ELISA was prepared by adding 100 쨉 l of the cell culture solution to each well of a 96-well plate coated with an interleukin-1β antibody, and reacted at room temperature for 1 hour. After washing with PBS for 4 times, the reaction mixture was incubated with a secondary antibody conjugated with an enzyme for coloring for 1 hour at room temperature. Subsequently, a substrate buffer solution (3,3 ', 5,5'-Tetramethylbenzidine (TMB ) And hydrogen peroxide) was added, coloring was stopped, color development was stopped with 2N sulfuric acid, and absorbance was measured at 450 nm. The results are shown in Fig.

As shown in FIG. 2, the values of interleukin-1 beta increased up to about 2000 pg / ml when LPS and nigericin were treated together, and the levels of 1000 pg / ml and 10 pg / Was remarkably decreased.

From the above results, it can be seen that phycocyanin effectively inhibits NLRP3 inflammase activity by reducing the secretion of interleukin-1 beta into cell culture medium by NLRP3 inflammas activation stimulation.

[ Experimental Example  3] mouse Bone marrow origin  In macrophages ASC  Confirm polymerization inhibition

The following Western blot analysis was carried out to confirm that the inhibition effect of NLRP3 inflammase activity of phycocyanin is through the process of inhibiting ASC polymerization.

The BMDMs of the above preparations were pretreated for 30 minutes with pyocyanin (100 μM), treated with LPS (0.25 μg / ml for 3 hours), treated with Nigericin (5 μM, 40 minutes), and treated with NLRP3 After activation of the moxa, the cell lysate then induced cross-linking of bound proteins present in the lysate using DSS (disuccinimidyl suberate), and then subjected to Western blotting as in Experimental Example 1 Respectively.

 Western blot analysis confirmed the polymerization of the ASC protein, and the results are shown in FIG.

As shown in FIG. 3, the production of ASC polymer (dimer, oligomer) was increased in cell lysate by LPS and nigerisin stimulation, and production of ASC polymer was remarkable in the case of treatment with pyocyanin , Respectively.

From the above results, it can be seen that phycocyanin effectively inhibits NLRP3 inflammase activity which induces autologous inflammatory diseases or neuroinflammatory diseases by inhibiting ASC polymer production.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the present invention is not limited to the disclosed exemplary embodiments, but various changes and modifications may be made without departing from the scope of the invention. It will be obvious to those who have knowledge of

Claims (12)

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

Wherein said inflammoma-mediated inflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injuries. The method according to claim 1,
Wherein said compound inhibits inflammose activity. 3. The method of claim 2,
Wherein the inflammase is NLRP3 (NOD-like receptor family, pyrin domain-containing 3). delete delete delete 1. A food composition for preventing or ameliorating inflammatory disease mediated inflammatory diseases comprising, as an active ingredient, a compound represented by the following formula (1)
[Chemical Formula 1]

Wherein said inflammus-mediated inflammatory disease is any one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, multiple sclerosis, amyotrophic lateral sclerosis, and inflammatory spinal injuries. 8. The method of claim 7,
Wherein said compound inhibits inflammose activity. 9. The method of claim 8,
Wherein the inflammase is NLRP3 (NOD-like receptor family, pyrin domain-containing 3). delete delete delete

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