NLRP3 inflammasome activation inhibitor, and asthma therapeutic agent comprising same

Abstract

The present application relates to an NLRP3 inflammasome activation inhibitor comprising trichospirolide A or a pharmaceutically acceptable salt thereof.

Description

NLRP3 inflammasome activation inhibitor and asthma treatment agent comprising the same

[0001] The present invention relates to an NLRP3 inflammasome activation inhibitor, an expression inhibitor of a cytokine comprising the same, and an asthma treatment comprising the same.

[0002] The inflammasome is a large multiprotein complex that plays a crucial role in innate immunity, producing cytokines. Cytokines produced by the inflammasome trigger various biological effects, including infection and autoimmune processes.

[0003] Among the above inflammasomes, NLRP3 (NOD-like receptor pyrin domain-containing protein 3, or NALP3), also called cryopyrin, secretes various cytokines such as IL-1β and IL-6 and induces pyroptosis, etc. However, if the NLRP3 inflammasome is abnormally or chronically activated, various diseases such as type 2 diabetes, gouty arthritis, cardiovascular disease, Alzheimer's disease, and CAPS (cryopyrin-associated-periodic-syndrome) may occur.

[0004] The technology underlying this application is disclosed in Korean Patent No. 10-2410055. The registered patent relates to a composition for treating, alleviating, or preventing respiratory inflammatory diseases.

[0005] The present invention aims to solve the problems of the above-mentioned prior art, and to provide an inhibitor capable of suppressing the activation of NLRP3 inflammasome and a cytokine expression inhibitor including the same.

[0006] In addition, it aims to provide an asthma treatment agent including an NLRP3 inflammasome activation inhibitor.

[0007] However, the technical tasks that the embodiments of the present invention seek to achieve are not limited to the technical tasks described above, and other technical tasks may exist.

[0008] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention relates to an NLRP3 inflammasome activation inhibitor comprising Trichospirolide A or a pharmaceutically acceptable salt thereof.

[0009] According to one embodiment of the present invention, the tricospiride A can be expressed as the following chemical formula 1, but is not limited thereto.

[0010] [Chemical Formula 1]

[0011]

[0012] According to one embodiment of the present invention, the tricospiride A can suppress the expression of the NLRP3 without affecting the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signal, but is not limited thereto.

[0013] According to one embodiment of the present invention, the tricospiride A can inhibit the binding of NLRP3 and NEK7 (NIMA-related kinase 7), but is not limited thereto.

[0014] According to one embodiment of the present invention, the tricospiride A can inhibit ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) oligomerization, but is not limited thereto.

[0015] According to one embodiment of the present invention, the tricospiride A can inhibit the activation of NLRP3 inflammasome regardless of the loss of intracellular potassium, but is not limited thereto.

[0016] According to one embodiment of the present invention, the tricospiride A can inhibit the activation of NLRP3 inflammasome regardless of the intracellular reactive oxygen species (ROS) level, but is not limited thereto.

[0017] It relates to an inhibitor of cytokine expression including an NLRP3 inflammasome activation inhibitor according to the first aspect above.

[0018] According to one embodiment of the present invention, the cytokine may include, but is not limited to, one selected from the group consisting of IL-1β, IL-6, TNF-α, and combinations thereof.

[0019] According to one embodiment of the present invention, the cytokine may include, but is not limited to, IL-1β.

[0020] It relates to an asthma treatment agent comprising a cytokine expression inhibitor according to the second aspect.

[0021] According to one embodiment of the present invention, the asthma may include, but is not limited to, neutrophilic asthma.

[0022] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0023] According to the above-described means for solving the problem of the present invention, the NLRP3 inflammasome activation inhibitor according to the present invention can suppress the expression of cytokines that aggravate diseases through tricospiride A.

[0024] Additionally, the NLRP3 inflammasome activation inhibitor can alleviate symptoms of neutrophilic asthma or treat neutrophilic asthma.

[0025] In addition, the above NLRP3 inflammasome activation inhibitor can be applied to all diseases associated with NLRP3, such as Alzheimer's, arteriosclerosis, and diabetes, to alleviate or treat symptoms.

[0026] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0027] Figure 1 shows data for tricospiride A according to one implementation example of the present invention.

[0028] Figures 2a and 2b are data for tricospiride A according to one implementation example of the present invention.

[0029] Figure 3 is a schematic diagram of an experiment to activate NLRP3 inflammasome in vitro according to one embodiment of the present invention.

[0030] Figures 4a and 4b show the cell viability of THP-1 and J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0031] FIG. 5a and FIG. 5b show THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, treated with nigericin and ATP, and FIG. 5c and FIG. 5d show J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, treated with nigericin and ATP.

[0032] Figure 6a shows the analysis of the protein expression levels of IL-1β, supernatant, and soluble Lys in J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention, and Figures 6b and 6c show the results after stimulation with dsDNA (2 μg / ml) and flagellin 1.25 μg / ml.

[0033] FIGS. 7A and 7B show J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, and then treated with nigericin, and then analyzed by immunoblot, and FIGS. 7C and 7D show THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, and then treated with nigericin, and then analyzed by immunoblot.

[0034] FIG. 8a shows nuclear fractionation in J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 8b shows measurement of NF-κB activity in 293T cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, and FIG. 8c and FIG. 8d show immunoblot analysis of J774A.1 cells and THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure.

[0035] Figures 9a and 9b are graphs analyzing J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0036] Figure 10 shows the relative activity of ATP converted by an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0037] Figures 11a and 11b show the results of immunoprecipitation and immunoblot analysis of HEK 293FT cells treated or not with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0038] Figure 12 is an image of J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0039] Figure 13a shows ASC specks of cells treated with an NLRP3 inflammasome activation inhibitor according to an example of the present invention, and Figure 13b shows THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an example of the present invention, analyzed by immunoblot.

[0040] Figure 14 is a ROS image of a cell treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0041] Figures 15a and 15b are graphs analyzing A549 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0042] Figures 16a and 16b show THP-1 cells and A549 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention, followed by treatment with nigericin and ATP, and then analyzed by immunoblot.

[0043] Figure 17 is a graph analyzing A549 cells treated with an NLRP3 inflammasome activation inhibitor according to one embodiment of the present invention.

[0044] Figure 18 is a schematic diagram of a neutrophilic asthma and eosinophilic asthma mouse model.

[0045] Figure 19 is an image of lung tissue stained with hematoxylin and eosin (H&E) and PAS.

[0046] Figures 20a to 20d show the measurement of the number of total cells, macrophages, neutrophils, and eosinophils in bronchoalveolar fluid (BALF).

[0047] Figures 21a to 21c show analysis of the levels of IL-1β, TNF-α, and IL-6 in bronchoalveolar fluid (BALF).

[0048] Figure 22 illustrates the extraction process of tricospiride A according to one embodiment of the present invention.

[0049] Below, with reference to the attached drawings, an embodiment of the present invention is described in detail so that a person having ordinary knowledge in the technical field to which the present invention pertains can easily carry out the present invention.

[0050] However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present invention in the drawings, parts irrelevant to the description have been omitted, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0051] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0052] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0053] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0054] Hereinafter, an NLRP3 inflammasome activation inhibitor, a cytokine expression inhibitor including the same, and an asthma treatment agent including the same according to one embodiment and example of the present invention are described.

[0055] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention relates to an NLRP3 inflammasome activation inhibitor comprising Trichospirolide A or a pharmaceutically acceptable salt thereof.

[0056] According to one embodiment of the present invention, the tricospiride A can be represented by the following chemical formula 1, but is not limited thereto:

[0057] [Chemical Formula 1]

[0058]

[0059] The above Trichospiride A may be derived from, but is not limited to, an extract of the plant Trichospira verticillata belonging to the Asteraceae family. The above Trichospiride A is known to have antimalarial activity.

[0060] Specifically, Trichospira verticillata was provided by the Korea Research Institute of Bioscience and Biotechnology and corresponds to specimen FBM303-049.

[0061] Figures 1 to 2b are data for Tricospiride A according to one embodiment of the present invention. Specifically, Figure 1 shows HPLC analysis data for Tricospiride A, using a SHIMADZU LC-20A instrument, and using 0.05% TFA in water and MeOH as solvents, and the ratio of solvents used by time period is as shown in Table 1 below:

[0062] Time A (0.05% TFA in water) B (MeOH) 0 1 0 0 1 0 1 0 0 6 0 1 0 7 0 1 0 7 8 1 0 0 8 5 1 0 0

[0063] In the above Table 1, the unit of time may be any one of seconds, minutes, and hours, but is not limited thereto. Preferably, the unit of time is minutes. In addition, FIG. 2a shows the unit of tricospiride A. 1 H-NMR data, and Figure 2b is of tricospiride A. 13 It refers to C-NMR data.

[0064] Figure 2a is 1 As H-NMR (700 MHz, CD3OD) data, δ: 6.82 (1H, dd, J = 1.0 & 1.0 Hz, H-5), 6.68 (1H, s, H-3a), 6.18 (1H, s, H-3'a), 6.05 (1H, s, H-1), 5.90 (1H, d, J = 8.0 Hz, H-8), 5.74 (1H, s, H-3'b), 4.93 (1H, d, J = 12.5 Hz, H-13a), 4.91 (1H, d, J = 12.5 Hz, H-13b), 3.70 (1H, d, J = 16.0 Hz, H-9a), 2.92 (1H, d, J = It contains data of 16.0 Hz, H-9b), 2.12 (3H, s, H-14), 2.02 (3H, s, H-15), 1.90 (3H, s, H-2''), and 1.95 (3H, s, H-4').

[0065] Also, Fig. 2b 13As C-NMR (175 MHz, CD3OD) data, δ: 194.5 (C-2), 191.8 (C-1''), 168.9 (C-12), 167.3 (C-1'), 159.5 (C-10), 155.0 (C-7), 149.4 (C-6), 138.5 (C-4), 136.7 (C-2'), 136.6 (C-3), 131.8 (C-1), 127.8 (C-3'), 127.3 (C-11), 120.1 (C-5), 74.4 (C-8), 56.5 (C-13), 48.8 (C-9), 30.0 (C-14), 20.8 (C-4'), 18.3 (C-2''), and 17.1 (C-15).

[0066] NLRP3 according to the present invention refers to a type of NOD-like receptor, specifically protein 3 including NLR, LRR, and PYD including NACHT protein. The NLRP3 refers to a component of the innate immune system that acts as a pattern recognition receptor (PRR). In addition, the NLRP3 inflammasome according to the present invention refers to a protein complex in which several molecules are combined, such as NLRP3 acting as a sensor, ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) acting as an adaptor, and caspase-1 acting as an effector.

[0067] Since the NLRP3 inflammasome can detect danger signals (e.g., ATP) or pathogen-associated molecular patterns (PAMPs or DAMPs) released from uric acid or damaged cells, when the danger signal occurs or the molecular pattern is detected, NLRP3 recruits the adaptor ASC (apoptosis-associated speck-like protein) to activate pro-caspase-1 and cause protein degradation, thereby activating the NLRP3 inflammasome.

[0068] Specifically, the NLRP3 inflammasome can be activated through a step including a priming step in which the transcription of cytokines or pattern recognition receptors is increased by NF-κB, and an activation step in which the NLRP3 inflammasome is formed by various mechanisms, such as the release of intracellular potassium ions by PAMPs and DAMPs or the increase in intracellular ROS levels, and caspase-1 included as a reactor in the NLRP3 inflammasome can activate the cytokine IL-1β. At this time, the process in which caspase-1 activates the cytokine includes a process in which the NLRP3 protein recruits ASC and caspase-1, which causes the cleavage and activation of caspase-1, resulting in the secretion of IL-1β from the NLRP3 protein.

[0069] In order to inhibit the activation of NLRP3 inflammasome, it is necessary to inhibit intracellular potassium movement or NF-κB activation. However, since unintended side effects may occur when intracellular potassium movement or NF-κB activation is inhibited, it is necessary to selectively inhibit only the activation of NLRP3 inflammasome and not inhibit intracellular potassium movement or NF-κB activation, and the NLRP3 inflammasome activation inhibitor according to the present invention, which includes tricospiride A, can selectively inhibit only the activation of NLRP3 inflammasome.

[0070] According to one embodiment of the present invention, the tricospiride A can suppress the expression of the NLRP3 without affecting the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signal, but is not limited thereto.

[0071] The above NF-κB is a protein complex involved in DNA transcription, cytokine production, and cell survival, and can be activated by signals such as IL-1β.

[0072] According to one embodiment of the present invention, the tricospiride A can inhibit the binding of NLRP3 and NEK7 (NIMA-related kinase 7), but is not limited thereto.

[0073] The above NEK7 is known to be a protein required for the activation of the NLRP3 inflammasome, and can specifically promote the oligomerization of NLRP3. That is, by inhibiting the binding of NLRP3 and NEK7, the activation of the NLRP3 inflammasome can be inhibited.

[0074] According to one embodiment of the present invention, the tricospiride A can inhibit ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) oligomerization, but is not limited thereto.

[0075] The above NLRP3 inflammasome may be activated by changes in intracellular potassium, but is not limited thereto.

[0076] According to one embodiment of the present invention, the tricospiride A can inhibit the activation of NLRP3 inflammasome regardless of the loss of intracellular potassium, but is not limited thereto.

[0077] When NLRP3 comes into contact with bacteria, viruses, fungal infections, endogenous DAMPs, environmental irritants, etc. that cause cell stress, the NLRP3 inflammasome can be activated by various signals such as potassium or chloride ion outflow, calcium ion influx, lysosomal rupture, mitochondrial dysfunction, generation of reactive oxygen species, and trans-Golgi degradation. In this regard, potassium ion outflow is known to be a common cause of NLRP3 inflammasome activation stimuli such as activation of P2X7 purinergic receptors by extracellular ATP, potassium ion ionophore, bacterial toxin nigericin, and particulate matter, and NLRP3 inflammasome activation can proceed when the concentration of intracellular potassium ions decreases. However, it was confirmed that the NLRP3 inflammasome activation inhibitor according to the present invention can inhibit NLRP3 inflammasome activation regardless of the concentration of intracellular potassium ions.

[0078] According to one embodiment of the present invention, the tricospiride A can inhibit the activation of NLRP3 inflammasome regardless of the intracellular reactive oxygen species (ROS) level, but is not limited thereto.

[0079] Specifically, the release of reactive oxygen species from damaged mitochondria can activate the NLRP3 inflammasome. However, the NLRP3 inflammasome activation inhibitor according to the present invention can suppress NLRP3 inflammasome activation without affecting intracellular ROS levels.

[0080] The NLRP3 inflammasome activation inhibitor according to the present invention can inhibit the activation of the NLRP3 inflammasome without affecting NF-κB signaling, independently of intracellular potassium movement, and independently of intracellular reactive oxygen levels.

[0081] In summary of the above, the NLRP3 inflammasome activation inhibitor according to the present invention includes tricospiride A, and tricospiride A can inhibit the binding of NLRP3 and NEK7, ASC oligomerization, and the expression of cytokines, which will be described later, without affecting NF-κB signaling, intracellular potassium efflux, or the level of intracellular reactive oxygen species.

[0082] The above Trichospiride A is obtained by sequentially separating an extract obtained from the seeds of Trichospira vercillata with water, ethyl acetate, butyl alcohol, dichloromethane, etc., eluting the material separated with ethyl acetate with a step gradient of acetonitrile and water, and purifying one of the separated products.

[0083] In addition, the second aspect of the present invention relates to an inhibitor of cytokine expression comprising an NLRP3 inflammasome activation inhibitor according to the first aspect.

[0084] When the activation of NLRP3 inflammasome is inhibited by the above NLRP3 inflammasome activation inhibitor, the activation of caspase-1 is inhibited, which may result in the inhibition of secretion and expression of cytokines.

[0085] According to one embodiment of the present invention, the cytokine may include, but is not limited to, one selected from the group consisting of IL-1β, IL-6, TNF-α, and combinations thereof.

[0086] According to one embodiment of the present invention, the cytokine may include, but is not limited to, IL-1β.

[0087] According to this invention, cytokines are a type of low-molecular-weight immune protein secreted by cells, playing a crucial role in cell signaling. It is known that the types of cytokines secreted and their roles vary from cell to cell.

[0088] An increase in IL-1β is known to be a major cause of steroid-resistant neutrophilic asthma, and patients with neutrophilic asthma are known to have higher expression levels of IL-1β-related proteins and genes than patients with eosinophilic asthma. In this regard, in a mouse model of neutrophilic asthma, asthma is known to be alleviated by IL-1β blockers instead of steroids such as DEX. The IL-1β can be secreted by NLRP3 inflammasomes, and thus, secretion of IL-1β can be suppressed by inhibiting the activation of NLRP3 inflammasomes.

[0089] In addition, the third aspect of the present invention relates to an asthma treatment agent comprising a cytokine expression inhibitor according to the second aspect.

[0090] According to one embodiment of the present invention, the asthma may include, but is not limited to, neutrophilic asthma.

[0091] Neutrophils are a type of white blood cell that responds to bacterial and fungal infections, and neutrophilic asthma refers to a type of bronchitis that occurs when the number of neutrophils in the lungs is high.

[0092] According to one embodiment of the present invention, at least one of the NLRP3 inflammasome activation inhibitor, the cytokine expression inhibitor, and the asthma treatment agent may act on immune cells, but is not limited thereto.

[0093] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0094] [Example 1]

[0095] An extract of Trichospira verticillata, belonging to the yellow Asteraceae family, was prepared (TV extract; TVE). Since the TVE contains trichospiride A, all TVEs used in Examples 1 to 11 contain trichospiride A.

[0096] The above Trichospira vercilata was provided by the Korea Research Institute of Bioscience and Biotechnology, is native to Costa Rica, and has the distribution number FBM303-049.

[0097] The dried seeds (3 kg) of the above Trichospira vercillata were extracted with 36 L of a mixed solvent (70% ethanol) and evaporated to dryness to obtain 313.2 g of a dark-colored syrupy extract. The syrupy extract was then dispersed in water (5 L) and sequentially separated with equal volumes of ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH), and dichloromethane (CH2Cl2), to obtain EtOAc product (8.67 g), n-BuOH product (10.5 g), CH2Cl2 product (76.5 g), and residual water-soluble product (217.5 g). More specifically, the extract extracted with a mixed solvent of Trichospira vercilata for 24 hours was first separated with dichloromethane to obtain a CH2Cl2 product, and then the extract that was not separated was separated with ethyl acetate to obtain an EtOAc product, and then the extract that was not separated with dichloromethane and ethyl acetate was extracted with butyl alcohol to obtain a BuOH extract and a residual water-soluble product.

[0098] Next, the EtOAc product (8.67 g) was applied to a C18 gel (120 g) column (Biotage sfar C18D) for chromatography, and the components in the EtOAc product were analyzed using acetonitrile and water. Fifteen isolates from F1 to F15 were obtained by stepwise gradient extraction using acetonitrile and water (from 1% to 100%), and the compound (5.5 mg) was purified from repeated JAI-GS310 chromatography of F9 through isocratic elution with 80% MeOH. The compound was identified as Trichospirolides A by comparison with NMR spectral data and literature. At this time, F1 is a mixture of 1 part by weight of acetonitrile and 99 parts by weight of water, F15 is a mixture of 99 parts by weight of acetonitrile and 1 part by weight of water, and F9 is a mixture of 65 parts by weight of acetonitrile and 35 parts by weight of water.

[0099] FIGS. 1 to 2b are data of tricospiride A according to one embodiment of the present invention, and FIG. 22 represents an extraction process of tricospiride A according to one embodiment of the present invention. Referring to FIGS. 1 to 2b and FIG. 22, data obtained by a method such as NMR spectroscopy of a material obtained through the extraction process of FIG. 22 may be the same as FIGS. 1 to 2b.

[0100] THP-1 cells, an immune cell line, were collected after differentiation with PMA (500 nM) for 3 hours and cultured for 2 days, and J774A.1 cells were cultured separately for 1 day from the THP-1 cells. Subsequently, A549 cells were placed in a transwell plate, and THP-1 cells were added to A549 cells at a ratio of 1:10, followed by priming with LPS (100 ng / mL) for 5 hours. Subsequently, cells were treated with LPS for 3 h, further treated with TVE (10 μg / mL, 50 μg / mL, or 100 μg / mL) for 2 h, treated with ATP (5 mM) for 30 min or 1 h, stimulated with nigericin (10 μM) and MCC950 (100 nM) for 1 h, and imiquimod (200 μM) and monosodium urate crystals (100 μg / ml) for 1 h, or transfected with dsDNA (2 μg / mL) for 2 h or flagellin (1.25 μg / mL) for 3 h using Lipofectamine 3000.

[0101] [Example 2]

[0102] Differentiated THP-1 cells were seeded in 96-well cell culture plates 2 days prior to the experiment, and J774A.1 cells were seeded 1 day prior to the experiment. The THP-1 and J774A.1 cells were primed with LPS for 1 h, treated with TVE for 2 h, or left untreated, and then incubated with EZ-CYTOX for 30 min.

[0103] Meanwhile, A549 cells were seeded in 96-well cell culture plates one day before the experiment and treated with TVE for 2 hours to overnight or incubated with EZ-CYTOX for 1 hour in a non-treated state. The absorbance of the 96-well cell culture plates was measured at 450 nm.

[0104] [Example 3]

[0105] For ASC oligomerization, THP-1 cell pellets were washed with phosphate-buffered saline (PBS) and cross-linked with DSS (2.5 mM). The mixture was then incubated at 25°C for 30 min, and the pellets were analyzed by immunoblotting.

[0106] For ASC speck staining, J774A.1 cells with activated NLRP3 inflammasome were fixed with 4% paraformaldehyde in PBS at 25°C for 10 min and washed three times with PBS at 0°C. The J774A.1 cells were then permeabilized with PBS containing 0.1% Triton X-100 at 25°C for 10 min and washed three times with PBS at 0°C for 5 min. The cells were then blocked with 1% bovine serum albumin (BSA) and 22.52 mg / mL glycine in PBS-T for 30 min and incubated overnight with ASC antibody at 4°C. Subsequently, the cells were washed three times with PBS for 5 minutes each, incubated for 1 hour with anti-mouse IgG antibody dissolved in 1% BSA and conjugated to AF488 in the dark at 25°C, and then washed three times with PBS for 5 minutes each.

[0107] [Example 4]

[0108] LPS-primed J774A.1 cells were treated with or without TVE (50 μg / ml or 100 μg / ml) for 2 h. The cell pellet was then washed with PBS, centrifuged at 500 ×g for 3 min, and the supernatant was discarded. The cell pellet was then homogenized on a cold CER I (CER 1) followed by the addition of a protease / phosphatase inhibitor mixture. The microtube containing the collected cells was then shaken for 20 s and incubated on ice for 10 min. After the addition of chilled CER II (CER 2), the tube was shaken for 5 s and incubated on ice for 1 min. The cell pellet was then centrifuged at 16,000 ×g for 5 min, and the supernatant containing cytoplasmic proteins was transferred to a cryotube. The insoluble pellet containing the nuclei was dispersed on a cold NER. The tube was shaken for 15 seconds, shaken for 15 seconds every 10 minutes, and placed on ice for 40 minutes. The tube was then centrifuged at 16,000 × g for 10 minutes, and the supernatant containing nuclear proteins was transferred to a cryotube on ice.

[0109] [Example 5]

[0110] To measure intracellular ROS levels, LPS-primed J774A.1 cells were treated with TVE (10 μg / mL, 50 μg / mL, and 100 μg / mL) or NAC (3 mM) for 2 h and activated with ATP (5 mM) for 25 min. The medium was then removed, and the cells were washed with 100 μL / well of 1x buffer. The cells were then treated with DCFDA solution (20 μM) for 45 min at 37°C in the dark, and after removal of the DCFDA solution, 100 μL / well of 1x supplement buffer was added. Intracellular ROS levels were then measured.

[0111] [Example 6]

[0112] Murine NLRP3 protein was treated with TVE (50 μg / ml) or DMSO in reaction buffer (100 mM Tris, pH 7.8, 2.8 mM EDTA, 100 mM MgCl2, 15 mM KCl, and 655 mM NaCl) at 37°C for 30 min. Ultrapure ATP (0.25 mM) was then added and incubated for an additional 40 min at 37°C. ATP hydrolysis by NLRP3 was measured by luminescent ADP detection, and ATP activity was measured using a BMG LabTech FLUOstar OPTIMA microplate reader.

[0113] [Example 7]

[0114] For NF-κB luciferase activity reporter gene analysis, 293T cells were first seeded in 96-well plates overnight. Subsequently, the 293T cells were transfected with pGL4.32 Luciferase Reporter Vector (0.1 μg / well) using Lipofectamine 3000 for 24 h. NF-κB activity was then induced for 5 h by TNF-α (20 ng / ml) in the presence or absence of TVE (10 μg / mL, 50 μg / mL, and 100 μg / mL).

[0115] [Example 8]

[0116] For immunoblotting and immunoprecipitation, whole cells were lysed using a cold lysis buffer containing 30 mM Tris-HCl pH 7.4, 2 mM EDTA, 120 mM NaCl, 2 mM KCl, 0.2% NP-40, 10% glycerol, and protease inhibitors. Proteins from the lysed cells and supernatants were then separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide (SDS) gel and transferred to a polyvinylidene difluoride membrane. The blots were then visualized using enhanced immunoluminescence (ENI) and detected with an Invitrogen iBright CL1500 imaging system.

[0117] [Example 9]

[0118] The asthma mouse model was a 6-week-old female mouse. Specifically, 6-week-old BALB / c mice (Orient BIO, Seongnam, Korea) were prepared in a specific pathogen-free environment. Then, to induce neutrophilic asthma, the mice were injected intraperitoneally (ip) with 10 μg ovalbumin (OVA, A5503, Sigma-Aldrich) emulsified in aluminum hydroxide gel on days 0 and 7. From days 14 to 17, the mice were injected with 6% OVA for 30 minutes using an ultrasonic nebulizer (n = 20) (NE-SM1; Ktmed Inc., Seoul, South Korea).

[0119] Additionally, mice in the neutrophilic asthma (NA) group were intranasally administered 10 μg of LPS (Sigma-Aldrich, L2880) on days 15 and 17. Subsequently, to determine the effects of TVE and MCC950 on the neutrophilic asthma mice and the neutrophilic asthma mice, mice in the TVE group were orally administered 30 mg / kg of TVE, the MCC950 group was intraperitoneally injected with 50 mg / kg of MCC950, and the dexamethasone (Dex) group was intraperitoneally injected with 1 mg / kg of Dex from day 14 to day 17 before OVA injection.

[0120] Additionally, to induce eosinophilic asthma (EA) in WT BALB / c mice, 10 μg OVA emulsified in aluminum hydroxide gel was injected intraperitoneally (ip) on days 0 and 7, and 6% OVA was injected intraperitoneally for 30 min using an ultrasonic nebulizer (n = 10) from days 14 to 17. To determine the effect of TVE on eosinophilic asthma, the TVE group was administered 30 mg / kg TVE orally from days 14 to 17 before OVA injection.

[0121] [Example 10]

[0122] Human IL-1β (DY201), rat IL-1β (DY401), rat TNF-α (DY410), and rat IL-6 (DY406) were measured and analyzed from supernatants collected from human A549 cells and rat bronchoalveolar lavage fluid (BALF). Fluorescence intensities were measured at 450 nm on a BioTek Epoch 2 Microplate Spectrophotometer.

[0123] [Example 11]

[0124] Right lower lung tissue sections from rats were fixed on slides in 4% paraformaldehyde. Subsequently, the lung tissues were stained with hematoxylin for 3 to 5 minutes, and then eosin Y solution was applied for 2 to 3 minutes to perform hematoxylin-eosin staining (H&E staining). Meanwhile, the tissues were stained with periodic acid solution for 5 minutes, and then Schiff's solution for 15 minutes to perform PAS staining.

[0125] Experimental examples 1 to 6 described below may refer to the results of the above examples 1 to 11.

[0126] [Experimental Example 1]

[0127] FIG. 3 is a schematic diagram of an in vitro NLRP3 inflammasome activation experiment according to an embodiment of the present disclosure, FIG. 4a and FIG. 4b show cell viability of THP-1 and J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 5a and FIG. 5b show THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, treated with nigericin and ATP, FIG. 5c and FIG. 5d show J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, treated with nigericin and ATP, and FIG. 6a shows analysis of protein expression levels of IL-1β, supernatant, and soluble Lys in J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, Figures 6b and 6c show the results after stimulation with dsDNA (2 μg / ml) and flagellin 1.25 μg / ml, Figures 7a and 7b show the results of immunoblotting analysis of J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an example of the present disclosure after treatment with nigericin, and Figures 7c and 7d show the results of immunoblotting analysis of THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an example of the present disclosure after treatment with nigericin.

[0128] Specifically, Fig. 3 is a schematic diagram showing the experimental protocol for TVE and NLRP3 activator treatment, in which THP-1 cells or J774A.1 cells were treated with LPS, treated with TVE, and then activated with NLRP3. In addition, Fig. 4a shows THP-1 cells differentiated with PMA (500 nM) treated with LPS (100 ng / ml) for 3 hours and then treated with TVE for 2 hours, and Fig. 4b shows J774A.1 cells differentiated with PMA (500 nM) treated with LPS (100 ng / ml) for 3 hours and then treated with TVE for 2 hours. In addition, Fig. 5a shows THP-1 cells treated with LPS, treated with TVE for 2 hours, and then stimulated with 10 μM nigericin for 1 hour, and Fig. 5b shows THP-1 cells stimulated with 5 mM ATP for 1 hour instead of 10 μM nigericin. In addition, Fig. 5c shows J774A.1 cells treated with LPS, treated with TVE for 2 hours, and then stimulated with 10 μM nigericin for 30 minutes, and Fig. 5d shows THP-1 cells stimulated with 5 mM ATP instead of nigericin. Also, Fig. 6a shows J774A.1 cells treated with LPS, treated with TVE for 2 hours, and then stimulated with 100 μg / ml of MSU (Monosodium urate crystals) for 3 hours, Fig. 6b shows J774A.1 cells pre-treated with LPS, treated with TVE for 2 hours, and then stimulated with dsDNA (2 μg / ml) using Lipofectamine 3000 for 3 hours, and Fig. 6c shows J774A.1 cells stimulated with flagellin (1.25 μg / mL) instead of dsDNA for 3 hours.

[0129] In addition, referring to FIGS. 4a and 4b, it can be confirmed that TVE has no cytotoxicity toward THP-1 cells and J774A.1 cells, and in particular, it does not significantly affect the cell viability of THP-1 cells and J774A.1 cells even at a concentration of 100 μg / ml.

[0130] Meanwhile, to analyze the effect of TVE on IL-1β, LPS-primed J774A.1 and THP-1 cells were treated with ATP, nigericin, and MSU, which activate NLRP3 inflammasomes. As a result, the amount of IL-1β secretion in the upper layer of cells was increased by NLRP3 activators, but the amount of IL-1β secretion was decreased in a dose-dependent manner depending on the treated TVE, which was similar to the effect of MCC950, an NLRP3 inhibitor, and the pro-IL-1β level of Lys (Lysates) was confirmed to be constant regardless of the presence or absence of TVE (see Figs. 5a to 5d and Figs. 7a to 7d).

[0131] In addition, since TVE treatment does not affect the secretion of IL-1β by activation of AIM2 and NLRC4 inflammasomes, it can be confirmed that TVE inhibits NLRP3 inflammasome activation.

[0132] [Experimental Example 2]

[0133] FIG. 8a shows nuclear fractionation in J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 8b shows measurement of NF-κB activity in 293T cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, and FIG. 8c and FIG. 8d show immunoblot analysis of J774A.1 cells and THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure.

[0134] Specifically, Fig. 8a shows the analysis of the nucleus and cytoplasm in lysed cells after 2 hours of TVE treatment of J774A.1 cells pretreated with LPS, Fig. 8b shows 293T cells transfected with pGL4.32 Luciferase Reporter vector simultaneously treated with TNF-α (20 ng / mL) and TVE for 5 hours, Fig. 8c shows J774A.1 cells pretreated with LPS treated with TVE for 2 hours and then stimulated with 10 μM nigiresin for 30 minutes, and Fig. 8d shows THP-1 cells pretreated with LPS treated with TVE for 2 hours and then stimulated with 10 μM nigiresin for 30 minutes.

[0135] After NF-κB activation, the transcription amount of pro-IL-1β increases, and the activating substance activates the inflammasome, thereby forming IL-1β. In this regard, to analyze the effect of TVE on NF-κB at the priming stage, the nuclear translocation of p65, a subunit of NF-κB, was analyzed.

[0136] Referring to Figures 8a to 8d, LPS treatment increased the nuclear translocation of p65 through TLR4 (toll-like receptor 4), but TVE treatment did not affect the nuclear translocation of p65, did not affect the NF-κB signaling induced by TNF-α, and did not affect the level of phospho p65 (an indicator of NF-κB activity) even when NLRP3 was activated by LPS and nigericin. In addition, since the level of p-ERK, a downstream target of NF-κB, did not change in J774A.1 cells and THP-1 cells treated with TVE, it was confirmed that TVE treatment inhibits the NLRP3 inflammasome without affecting the NF-κB signaling.

[0137] [Experimental Example 3]

[0138] FIG. 9a and FIG. 9b are graphs analyzing J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 10 shows the relative activity of ATP converted by an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, and FIG. 11a and FIG. 11b are results of immunoprecipitation and immunoblot analysis of HEK 293FT cells treated or not with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure.

[0139] Specifically, Fig. 9a shows LPS-primed J774A.1 cells treated with TVE for 2 hours and then stimulated with 200 μM imiquimod (IMQ) for 1 hour, Fig. 9b shows LPS-primed J774A.1 cells treated with TVE or NAC for 2 hours or not, and then stimulated with 5 mM ATP for 25 minutes, Fig. 10 shows the amount of ATP converted to ADP by NLRP3 by TVE, Fig. 11a shows the analysis of NLRP3-Myc transfected-HEK 293FT cells treated with TVE for 2 hours, and Fig. 11b shows the analysis of NLRP3-Myc transfected-HEK 293FT cells not treated with TVE.

[0140] It is known that the level of intracellular potassium efflux and intracellular ROS (reactive oxygen species) levels play an important role in the activation of NLRP3 inflammasome, which means that NLRP3 inflammasome activation can be inhibited by inhibiting intracellular potassium efflux or reducing intracellular ROS levels.

[0141] LPS-primed J774A.1 cells were treated with imiquimod (IMQ), which activates the NLRP3 inflammasome independent of potassium withdrawal, and then with TVE. Referring to Fig. 9a, TVE inhibited NLRP3 activation by IMQ in a concentration-dependent manner, indicating that TVE does not inhibit NLRP3 inflammasome activation by inhibiting potassium withdrawal. In addition, referring to Fig. 9b, intracellular ROS levels increased after ATP treatment, but the change in intracellular ROS levels was minimal after TVE treatment, and it was confirmed that the ROS levels decreased to the level before ATP treatment after NAC treatment.

[0142] In addition, NLRP3 inhibitors are known to bind to the NACHT domain to inhibit the activity of the ATPase of NLRP3, and when an in vitro ATPase assay was performed to determine whether TVE inhibits the activity of the ATPase, it was confirmed that TVE does not affect the activity of the ATPase (see Figure 10).

[0143] Additionally, the interaction between NEK7 and NLRP3 is known to influence the activation of the NLRP3 inflammasome. Referring to Figures 11a and 11b, the binding between NEK7 and NLRP3 was reduced after TVE treatment, indicating that TVE inhibits the binding between NLRP3 and NEK7, thereby inhibiting the activation of the NLRP3 inflammasome.

[0144] [Experimental Example 4]

[0145] FIG. 12 is an image of J774A.1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 13a shows ASC specks of cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 13b shows THP-1 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, analyzed by immunoblot, and FIG. 14 is an ROS image of cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure.

[0146] Specifically, the arrows in Fig. 12 represent ASC specks, Fig. 13a shows J774A.1 cells primed with LPS treated with TVE for 2 hours and then stimulated with 5 mM ATP for 30 minutes, and Fig. 13b shows THP-1 cells primed with LPS treated with TVE for 2 hours and then stimulated with 10 μM nigericin for 30 minutes.

[0147] During the activation of NLRP3 inflammasome, ASC, a component of NLRP3 inflammasome, moves to the surfactant-insoluble part, and ASC oligomers assemble to form ASC specks.

[0148] Referring to Figures 12 to 13b, ATP-treated J774A.1 cells showed a significant increase in the formation of ASC spectra, but J774A.1 cells treated with TVE or MCC950 showed an inhibition of ASC spectra formation. In addition, when ASC oligomerization was measured in THP-1 cells, ASC oligomerization in THP-1 cells was inhibited after treatment with TVE or MCC950. This indicates that TVE inhibits NLRP3 inflammasome activation by inhibiting ASC oligomerization.

[0149] [Experimental Example 5]

[0150] FIG. 15a and FIG. 15b are graphs analyzing A549 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure, FIG. 16a and FIG. 16b are graphs analyzing THP-1 cells and A549 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure by immunoblot after treatment with nigericin and ATP, and FIG. 17 is a graph analyzing A549 cells treated with an NLRP3 inflammasome activation inhibitor according to an embodiment of the present disclosure.

[0151] Specifically, Fig. 15a shows THP-1 cells treated with TVE for 2 hours, Fig. 15b shows THP-1 cells treated with nigericin, Fig. 16a shows THP-1 cells treated with ATP, and Fig. 17 shows A549 cells or THP-1 cells primed with LPS, and A549 cells or THP-1 cells primed with LPS stimulated with 5 mM ATP for 30 minutes, after which IL-1β was measured.

[0152] As a result of treating A549 lung epithelial cells with TVE for 2 hours to overnight, it was confirmed that TVE had no cytotoxicity toward A549 cells, as shown in Figures 15a and 15b.

[0153] A549 cells were co-cultured with PMA-differentiated THP-1 cells, and treated with LPS for 5 h and TVE for 2 h, followed by NLRP3 inflammasome activation. As a result, IL-1β secretion was not increased in A549 cells, whereas IL-1β secretion was slightly increased in the same number of THP-1 cells (see Fig. 17).

[0154] Referring to FIGS. 16A and 16B, when A549 cells and THP-1 cells were cultured together, a larger amount of IL-1β was secreted compared to when either A549 or THP-1 cells were cultured alone, and it was confirmed that TVE inhibited the secretion of IL-1β dependent on NLRP3 and / or NLRP3 inflammasome even in an environment where A549 and THP-1 cells were cultured together.

[0155] [Experimental Example 6]

[0156] Figure 18 is a schematic diagram of a neutrophilic asthma and eosinophilic asthma mouse model, Figure 19 is an image of lung tissue stained with hematoxylin and eosin (H&E) and PAS, Figures 20a to 20d show measurements of the number of total cells, macrophages, neutrophils, and eosinophils in bronchoalveolar fluid (BALF), and Figures 21a to 21c show analysis of the levels of IL-1β, TNF-α, and IL-6 in bronchoalveolar fluid (BALF). Specifically, the NA model in Figure 18 represents neutrophilic asthma, and the EA model represents eosinophilic asthma. In addition, NC in Figures 20a to 20d represents a control, meaning lung tissue of a normal mouse that does not have asthma.

[0157] Figure 18 illustrates the process for creating mouse models of eosinophilic asthma and neutrophilic asthma. Subsequently, H&E and PAS staining were performed to confirm pathological changes and mucus production in the lung tissues of mice with eosinophilic asthma and mice with neutrophilic asthma after TVE treatment.

[0158] Referring to Figure 19, accumulation of cells infiltrated around the lung tissue of a neutrophilic asthma mouse model was observed, and the number of cells infiltrated around the lung tissue significantly decreased when treated with TVE or MCC950. In addition, the analysis of mucus produced by airway epithelial cells in a PAS staining experiment showed that the amount of mucus significantly increased in the NA model, but mucus production was suppressed by treatment with TVE or MCC950.

[0159] Meanwhile, referring to FIGS. 20A to 20D, in lung tissues collected from a neutrophilic asthma mouse model, the number of total cells, macrophages, neutrophils, and eosinophils in BALF increased overall due to an increase in inflowed cells, indicating increased resistance to steroids. However, when the lung tissues were treated with TVE or MCC950, the number of immune cells such as macrophages and neutrophils decreased.

[0160] Meanwhile, lung tissues collected from a mouse model of eosinophilic asthma were found to have fewer neutrophils and more macrophages and eosinophils compared to neutrophilic asthma lung tissues. If TVE functions in a neutrophilic asthma model involving the NLRP3 inflammasome, it should have no effect in an eosinophilic asthma (EA) model. When TVE was treated in the lung tissue of the EA model, the number of macrophages and eosinophils remained constant, indicating that TVE functions only in the neutrophilic asthma model by inhibiting NLRP3 inflammasome activation.

[0161] In addition, cytokines IL-1β, IL-6, and TNF-α were significantly increased in the lung tissue of the neutrophilic asthma mouse model compared to the control group, and when treated with TVE, the amount of IL-1β secretion was significantly reduced, but it was confirmed that there was no effect on the secretion of IL-6 and TNF-α. In the group treated with MCC950, only the secretion of IL-1β was reduced, and IL-6 and TNF-α were confirmed to have no effect. At this time, in Fig. 21c, IL-6 was reduced when treated with TVE, but the decrease in IL-6 secretion is due to a downstream signal resulting from the decrease in IL-1β secretion, and TVE does not directly reduce the secretion of IL-6.

[0162] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0163] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. An NLRP3 inflammasome activation inhibitor comprising trichospirolide A or a pharmaceutically acceptable salt thereof.

2. In paragraph 1, The above tricospiride A is an NLRP3 inflammasome activation inhibitor represented by the following chemical formula 1: [Chemical Formula 1] .

3. In paragraph 1, The above tricospiride A is an NLRP3 inflammasome activation inhibitor that inhibits the expression of NLRP3 without affecting NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling.

4. In paragraph 1, The above-mentioned tricospiride A is an inflammasome activation inhibitor that inhibits the binding of NLRP3 and NEK7 (NIMA-related kinase 7).

5. In paragraph 1, The above tricospiride A is an NLRP3 inflammasome activation inhibitor that inhibits ASC (apoptosis-associated speck-like protein with a caspase-recruitment domain) oligomerization.

6. In paragraph 1, The above-mentioned tricospiride A is an NLRP3 inflammasome activation inhibitor that inhibits the activation of NLRP3 inflammasome regardless of the loss of intracellular potassium.

7. In paragraph 1, The above tricospiride A is an NLRP3 inflammasome activation inhibitor that inhibits the activation of NLRP3 inflammasome regardless of the level of intracellular reactive oxygen species (ROS).

8. A cytokine expression inhibitor comprising an NLRP3 inflammasome activation inhibitor according to paragraph 1.

9. In paragraph 8, A cytokine expression inhibitor, wherein the cytokine comprises a cytokine selected from the group consisting of IL-1β, IL-6, TNF-α, and combinations thereof.

10. In paragraph 9, A cytokine expression inhibitor, wherein the cytokine comprises IL-1β.

11. An asthma treatment agent comprising a cytokine expression inhibitor according to Article 8.

12. In paragraph 11, An asthma treatment agent, wherein the asthma includes neutrophilic asthma.

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