Compositions for the prevention or treatment of encephalitis and encephalodegenerative diseases, containing entrectinib

Abstract

The present invention provides compositions and methods for preventing, improving, or treating inflammatory brain diseases, degenerative brain diseases, memory impairments, and the like. [Solution] The present invention relates to a composition for the prevention or treatment of encephalitis and cerebral degenerative diseases comprising entrectinib, wherein the composition exhibits the effect of suppressing encephalitis and improving memory impairment.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising entrectinib for the prevention or treatment of encephalitis and cerebral degenerative diseases. [Background technology]

[0002] Inflammatory brain response is one of the representative pathological phenomena observed in most neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. In this response, inflammatory mediators such as inflammatory cytokines (cytoksine) and reactive oxygen species (ROS) produced by immune cells promote neuronal cell death.

[0003] Alzheimer's disease is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment, primarily affecting elderly individuals aged 65 and older. Pathologically, the main etiologies are β-amyloid protein deposits and tau protein entanglement in nerve fibers. As Alzheimer's disease progresses, it is often accompanied not only by cognitive decline but also by mental and behavioral symptoms such as personality changes, anxiety, depression, delusions, hallucinations, increased aggression, and sleep disturbances. In the terminal stages, neurological disorders such as muscle rigidity and gait abnormalities, as well as physical complications such as incontinence, infections, and pressure ulcers, are observed. Microscopic examination of brain tissue from Alzheimer's patients reveals characteristic lesions such as neuritic plaques and neurofibrillary tangles, and macroscopically, general brain atrophy due to nerve cell loss is observed. These neuropathological findings initially appear mainly in the hippocampus and entorhinal cortex, the major brain regions responsible for memory, but gradually spread to the parietal lobe, frontal lobe, and other areas, eventually reaching the entire brain. As the areas of neuropathological invasion progress, memory impairment is the primary symptom initially, but as the disease progresses, clinical symptoms become more diverse and progressively more severe. However, current dementia treatments are limited to symptom relief rather than addressing the underlying cause, and there is a need for the development of substances to suppress, delay, or treat the onset of Alzheimer's disease.

[0004] Microglia are a type of immune cell that, as macrophages in the brain, play a crucial role in host defense and tissue repair in the central nervous system (CNS). Under normal conditions, glial cells (i.e., astrocytes and microglia cells) support and protect neurons from pathogens and maintain synaptic homeostasis. Microglia are primarily found in the hippocampus and cortex, which are responsible for learning and memory. Abnormal activation of microglia due to external stimuli triggers a wide range of responses, including the initiation of inflammation, secretion of inflammatory mediators, neurotoxic factors, and various cytokines, and is involved in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and stroke. Therefore, suppressing abnormal microglial activity may have useful therapeutic potential in the treatment of inflammation-related diseases. BV2 microglia have been widely used as a cellular model of neuroinflammation to evaluate responses to pro-inflammatory cytokines and oxidative stress in the search for new drug candidates.

[0005] On the other hand, entrectinib is a targeted anticancer drug used to treat solid tumors expressed by NTRK gene fusion. A targeted anticancer drug is an anticancer drug that recognizes specific sites on the surface of cancer cells or signaling pathways inside cancer cells that differ from those of normal cells, and selectively suppresses the proliferation or survival of cancer cells. Generally, anticancer treatment is determined based on the site of cancer development, but entrectinib is a representative cross-cancer drug that targets NTRK gene mutations regardless of the site of cancer development. It is mainly used to treat certain solid tumors and non-small cell lung cancer.

[0006] Therefore, the inventors confirmed that entrectinib, an anticancer agent for the treatment of solid tumors or non-small cell lung cancer, has an effect of suppressing brain inflammation, and have found its potential use as a therapeutic agent for diseases other than cancer, thus completing the present invention. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Korean Registered Patent No. 10-2718538 [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a pharmaceutical composition comprising entrectinib for the prevention or treatment of inflammatory brain diseases or neurodegenerative diseases.

[0009] Furthermore, the present invention aims to provide a food composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

[0010] Furthermore, the present invention aims to provide an animal feed composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

[0011] Furthermore, the present invention aims to provide a pharmaceutical composition comprising entrectinib for the prevention or treatment of memory impairment.

[0012] Furthermore, the present invention aims to provide a food composition containing entrectinib for the prevention or improvement of memory impairment.

[0013] Furthermore, the present invention aims to provide an animal feed composition containing entrectinib for the prevention or improvement of memory impairment.

[0014] Furthermore, the present invention aims to provide a method for reducing encephalitis, which includes the step of administering a composition containing entrectinib to an individual.

[0015] Furthermore, the present invention aims to provide a method for preventing or improving memory impairment, which includes the step of administering a composition containing entrectinib to an individual.

[0016] Furthermore, an object of the present invention is to provide a method for enhancing the phagocytosis of microglia, which includes a step of treating a composition containing entrectinib in vitro or in an individual. **Means for Solving the Problems**

[0017] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a brain inflammatory disease or a brain degenerative disease, which contains entrectinib.

[0018] In addition, the present invention provides a food composition for preventing or improving a brain inflammatory disease or a brain degenerative disease, which contains entrectinib.

[0019] In addition, the present invention provides an animal feed composition for preventing or improving a brain inflammatory disease or a brain degenerative disease, which contains entrectinib.

[0020] The composition may be for patients in a group with increased tropomyosin receptor kinase (TRK) activity in a brain inflammatory disease or a brain degenerative disease.

[0021] The composition may reduce inflammatory cytokines and increase anti-inflammatory cytokines.

[0022] The composition may suppress the phosphorylation of JNK, p38, AKT, NF-κB or STAT3.

[0023] The composition may reduce the expression of CD16 / 32 and increase the expression of CD206.

[0024] The composition may reduce amyloid β (Aβ) aggregates.

[0025] The composition may enhance the phagocytosis of microglia.

[0026] The aforementioned composition may prevent or improve memory impairment induced in inflammatory brain diseases or neurodegenerative diseases.

[0027] The aforementioned neurodegenerative diseases may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, dementia, mild cognitive impairment, stroke, and cerebral infarction.

[0028] Furthermore, the present invention provides a pharmaceutical composition comprising entrectinib for the prevention or treatment of memory impairment.

[0029] Furthermore, the present invention provides a food composition for preventing or improving memory impairment, comprising entrectinib.

[0030] Furthermore, the present invention provides an animal feed composition containing entrectinib for the prevention or improvement of memory impairment.

[0031] The aforementioned memory impairment may be induced by neuroinflammation.

[0032] Furthermore, the present invention provides a method for reducing encephalitis, which includes the step of administering a composition containing entrectinib to a non-human organism.

[0033] Furthermore, the present invention provides a method for preventing or improving memory impairment, which includes the step of administering a composition containing entrectinib to a non-human organism.

[0034] Furthermore, the present invention provides a method for enhancing the phagocytic activity of microglia, comprising the step of treating an organism (either in vitro or in a non-human organism) with a composition containing entrectinib. [Effects of the Invention]

[0035] The composition containing entrectinib of the present invention exhibits the effect of suppressing brain inflammation and improving memory impairment, and can therefore be usefully used for the prevention, improvement, or treatment of inflammatory brain diseases, neurodegenerative diseases, memory impairment, etc. [Brief explanation of the drawing]

[0036] [Figure 1] This figure shows the results of Western blot analysis of TRK phosphorylation after treatment of LPS-treated microglia with entrectinib (n=4 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 2] This figure shows the results of immunofluorescence analysis of p-TRK expression after treatment of LPS-treated microglia with entrectinib (n=8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 3] This figure shows the results of real-time PCR analysis of pro-inflammatory and anti-inflammatory factors after treating LPS-treated microglia with entrectinib (n=4 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 4] This figure shows the results of Western blot analysis of JNK, P38, and AKT phosphorylation levels after treatment of LPS-treated microglia with entrectinib (JNK, n=4-7 / group; P38 and AKT, n=4 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 5] This figure shows the results of Western blot analysis of cytoplasmic and nuclear p-NF-κB and p-STAT3 levels after treatment of LPS-treated microglia with entrectinib (n=3 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 6]This figure shows the results of immunofluorescence analysis for the toxic and protective microglial markers CD16 / 32 and CD206 after LPS-treated microglia were treated with entrectinib (n=8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 7] This figure shows the results of live image analysis of phagocytosis in BV2 microglia treated with LPS, entrectinib, and Aβ oligomer (n=8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 8] This figure shows the results of real-time PCR analysis of phagocytic factor and cytoskeletal gene expression in microglia treated with LPS and entrectinib (n=3 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 9] This figure shows the results of Western blot analysis (n=4 / group) and immunofluorescence staining of TRK phosphorylation in mouse hippocampi after administration of entrectinib and LPS (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 10] This figure shows the results of Western blot analysis of JNK, P38, and AKT phosphorylation in mouse hippocampi after administration of entrectinib and LPS (n=4 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 11] This figure shows the results of immunofluorescence analysis for p-NF-κB and p-STAT3 in microglial nuclei in mouse hippocampi after administration of entrectinib and LPS (n=3 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 12] This figure shows the results of the Y-maze and NOR tests in mice administered entrectinib and LPS for 8 days (n=6-8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 13]This figure shows the results of the Y-maze and NOR tests in mice administered entrectinib and LPS for 16 days (n=6-8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 14] This figure shows the change in body weight of mice administered entrectinib and LPS for 8 or 16 days (n=6-8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 15] This figure shows the results of synaptic marker changes in mice administered entrectinib and LPS for 8 days (n=8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 16] This figure shows the results of synaptic marker changes in mice administered entrectinib and LPS for 16 days (n=8 / group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Modes for carrying out the invention]

[0037] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings, so that those with ordinary skill in the art to which the present invention pertains can easily implement it. However, the present invention can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0038] Where the specification as a whole states that a part "includes" a certain component, unless otherwise stated, this does not exclude other components, but rather means that it may include other components.

[0039] The present invention provides a pharmaceutical composition comprising entrectinib for the prevention or treatment of inflammatory brain diseases or neurodegenerative diseases.

[0040] In this invention, "entrectinib" is a targeted anticancer agent used to treat solid tumors expressed by NTRK gene fusion. It is a representative cross-cancer agent that targets NTRK gene mutations regardless of the site of cancer development. It is primarily used to treat certain solid tumors and non-small cell lung cancer.

[0041] The aforementioned inflammatory brain diseases refer to inflammatory diseases of the brain parenchyma, and are broadly classified into infectious, vasculitic, neoplastic, chemical, and idiopathic types depending on their cause, and include, but are not limited to, Alzheimer's disease or Parkinson's disease.

[0042] The aforementioned neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, dementia, mild cognitive impairment, stroke, and cerebral infarction, and may preferably be neurodegenerative diseases caused by encephalitis.

[0043] The composition may prevent or improve memory impairment, preferably preventing or improving memory impairment induced in inflammatory brain diseases or degenerative brain diseases, but is not limited thereto.

[0044] The aforementioned inflammatory brain disease or neurodegenerative brain disease may be, but is not limited to, a group of patients with increased TRK activity.

[0045] The composition may inhibit TRK (tropomyosin receptor kinase) activity, preferably inhibits TRK activity increased by neuroinflammation, and more preferably inhibits TRK activity in microglia, but is not limited thereto.

[0046] The composition may reduce inflammatory cytokines and increase anti-inflammatory cytokines, preferably reducing or increasing inflammatory cytokines and anti-inflammatory cytokines that have increased or decreased due to neuroinflammation, and more preferably reducing or increasing inflammatory cytokines and anti-inflammatory cytokines in microglia, but is not limited thereto.

[0047] The aforementioned inflammatory cytokines include, but are not limited to, IL-1β, IL-6, and TNF-α, while anti-inflammatory cytokines include, but are not limited to, IL-4, IL-10, and TGF-β.

[0048] The composition may suppress the phosphorylation of JNK, p38, AKT, NF-κB, or STAT3, preferably suppressing the phosphorylation of JNK, p38, AKT, NF-κB, or STAT3 that is increased by neuroinflammation, and more preferably suppressing the phosphorylation of JNK, p38, AKT, NF-κB, or STAT3 in microglia, but is not limited thereto.

[0049] The composition may decrease the expression of CD16 / 32 and increase the expression of CD206, preferably decreasing or increasing the expression of CD16 / 32 and CD206 that have increased or decreased due to neuroinflammation, and more preferably decreasing or increasing the expression of CD16 / 32 and CD206 in microglia, but is not limited thereto.

[0050] The composition may enhance the phagocytosis of microglia, and preferably enhances the phagocytosis of microglia that has been reduced by neuroinflammation, but is not limited thereto.

[0051] The composition may reduce amyloid-beta (Aβ) aggregates, and this reduction may be due to the activation of phagocytosis against amyloid-beta aggregates, but is not limited to this.

[0052] The pharmaceutical compositions of the present invention can be formulated as powders, granules, tablets, film-coated tablets, pills, sugar-coated tablets, capsules, liquids, suspensions, gels, syrups, slurries, suppositories, emulsions, pastes, ointments, creams, lotions, powders, sprays, or suspensions. For the formulation, the compositions may further contain excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and diluents that are commonly used, and are not limited to simple excipients but may also be used as lubricants such as magnesium stearate and talc.

[0053] The aforementioned pharmaceutical composition can be administered parenterally or orally depending on the purpose, and the dosage ranges widely depending on the patient's weight, age, sex, health condition, diet, administration time, method of administration, excretion rate, and severity of the disease. Furthermore, the therapeutically effective amount of the composition varies depending on the method of administration, target site, and patient condition, and the dosage when used in the human body should be appropriately determined considering safety and efficacy.

[0054] The pharmaceutical composition may further contain an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation.

[0055] Furthermore, the present invention provides a food composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

[0056] The food composition of the present invention may be a health functional food, a dairy product, a fermented product, or a food additive. The term "health functional food" refers to a food manufactured or processed using raw materials or components that have beneficial functions for the human body, and "functionality" means that it is consumed for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body.

[0057] The aforementioned food composition may take the form of pills, powders, granules, infusions, tablets, capsules, liquids, pastes, gels, jellies, etc. In addition to the active ingredient, each formulation of the food composition may be appropriately selected and blended by a person skilled in the art, depending on the formulation or intended use, with other ingredients commonly used in the field.

[0058] Furthermore, the present invention provides an animal feed composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

[0059] The animal feed composition of the present invention can be manufactured separately in the form of a feed additive and mixed with feed, or it can be manufactured by adding it directly during feed production, and it may further contain conventional additives to improve the shelf life of the feed.

[0060] The aforementioned animal feed composition includes, but is not limited to, plant-based materials such as grains, root vegetables, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meal products, and grain by-products, and animal-based materials such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, and leftover food.

[0061] The aforementioned animals refer to organisms that correspond to plants, primarily ingesting organic matter as nutrients, and possessing differentiated digestive, excretory, and respiratory organs. Specifically, these may include echinoderms, crustaceans, mollusks, fish, amphibians, reptiles, birds, and mammals.

[0062] The animal feed composition may further contain grains, plant-based protein feed, animal-based protein feed, sugars, or dairy products, and may also contain in combination ingredients such as nutritional supplements, digestive and absorption enhancers, growth promoters, or disease preventive agents.

[0063] The feed composition can be manufactured as a slightly viscous coarse or granular substance depending on the degree of grinding of the components, the composition can be supplied in a mesh or molded into a desired separated shape for further processing or packaging, and can undergo pelletizing, expansion or extrusion processes for storage, and preferably excess moisture can be dried out for easy storage.

[0064] Furthermore, the present invention provides a pharmaceutical composition comprising entrectinib for the prevention or treatment of memory impairment.

[0065] The aforementioned memory impairment may be induced by neuroinflammation, and preferably by LPS, but is not limited thereto.

[0066] The composition may increase synaptic proteins, and preferably increases synaptic proteins that have decreased due to neuroinflammation, but is not limited thereto.

[0067] The aforementioned synaptic proteins include, but are not limited to, SYP (synaptophysin) and PSD95 (postsynaptic density protein 95).

[0068] Furthermore, the present invention provides a food composition for preventing or improving memory impairment, comprising entrectinib.

[0069] Furthermore, the present invention provides an animal feed composition containing entrectinib for the prevention or improvement of memory impairment.

[0070] Furthermore, the present invention provides a method for reducing encephalitis, comprising the step of administering a composition containing entrectinib to an individual.

[0071] The aforementioned individuals include, but are not limited to, individuals whose brain inflammation needs to be controlled or reduced, and may preferably be non-human individuals.

[0072] Furthermore, the present invention provides a method for preventing or improving memory impairment, comprising the step of administering a composition containing entrectinib to an individual.

[0073] The aforementioned individuals include, but are not limited to, individuals that require prevention or improvement of memory impairment, and may preferably be non-human individuals.

[0074] Furthermore, the present invention provides a method for enhancing the phagocytic activity of microglia, comprising the step of treating an organism or an organism in vitro with a composition containing entrectinib.

[0075] The aforementioned individuals include, but are not limited to, individuals for which control or enhancement of microglial phagocytosis is necessary, and are preferably non-human individuals.

[0076] As used in this invention, "prevention" means all actions that suppress or delay a disease or the like by administering a composition according to this invention; "treatment" means all actions that improve or favorably alter the symptoms of an individual suspected of having or who has developed a disease by administering the composition; and "improvement" means all actions that at least reduce parameters related to the condition being treated by administering the composition of this invention, such as the severity of symptoms.

[0077] In this invention, unless otherwise specified, the "%" used to indicate the concentration of a specific substance shall be (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid. [Examples]

[0078] The present invention will be described in more detail through the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0079] [Preparation Example] Materials and Method 1. Entrectinib and LPS treatment In all in vitro experiments, Escherichia coli (Sigma, Cat No. L2630, St. Louis, MO, USA) lipopolysaccharide (LPS) dissolved in PBS was used. Primary cultured microglia and BV2 microglia were treated with LPS at a concentration of 200 ng / mL. In in vivo studies, mice were administered 250 μg / kg of LPS intraperitoneally (ip) once daily for 8 days to induce an inflammatory response.

[0080] Entrectinib (Selleckchem, Cat No. S7998, Houston, TX, USA) was prepared with dimethyl sulfoxide (DMSO) at a concentration of 1 μM for in vitro treatment of primary cultured microglia and BV2 microglia, unless otherwise specified. In in vivo experiments, the control group solution consisted of deionized water with 5% DMSO, 10% PEG, and 20% Tween 80. Mice were administered entrectinib at a dose of 10 mg / kg.

[0081] 2. BV2 microglia The BV2 microglia cell line was purchased from Koram Bio Tech (Seoul, South Korea). Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Cytiva, Cat No. SH30243.01, Marlborough, MA, USA) and supplemented with 5% fetal bovine serum (FBS, Gibco, Cat No. 16000-044, Waltham, MA, USA), 100 U / mL penicillin G, and 100 μg / mL streptomycin. The cultures were maintained in a 5% CO2 incubator at 37°C.

[0082] 3. Primary culture of microglia Primary cultured glial cells were isolated from the brains of postnatal C57BL / 6J mice on day 1. The cerebrum was placed in a cooled HBSS and washed twice with low-concentration glucose-DMEM. The brain was then filtered through a 70 μm nylon mesh and cultured in a 5% CO2 incubator with low-concentration glucose-DMEM containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. On day 14 of culture, astrocytes were removed using 0.25% trypsin-EDTA in low-concentration glucose-DMEM supplemented with EDTA and CaCl2. After washing to remove the astrocyte layer, primary cultured microglia were isolated with 0.25% trypsin-EDTA and centrifuged twice at 2,000 rpm for 10 minutes before use.

[0083] 4.CCK-8 analysis Place the cells in a 96-well plate, 1 x 10⁶ cells per well. 4 Cells were dispensed at a cell density and cultured for 1 hour in FBS-free medium. Subsequently, cells were treated with entrectinib at concentrations of 0.1, 1, 5, and 10 μM, or with 1% DMSO as a control group, for 12, 24, and 48 hours. At each time point, 10% CCK-8 solution (Dojindo, Cat No. CK04, Kumamoto, Japan) was added to the FBS-free medium, and the cells were cultured for a further 2 hours. Cell viability was confirmed by measuring the absorbance at 450 nm using a Multiskan Skyhigh microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA).

[0084] 5. Real-time PCR mRNA levels of genes related to inflammatory responses (Il6, Il1β, Tnfα, Inos, Ccl2, Il-23α, Il10, Il13, Il4), phagocytosis (Trem2, Sorl1, Cd33, Cr2), and the cytoskeleton (Vav1, Cdc42) were evaluated by real-time PCR in primary cultured microglia and hippocampal tissue. RNA was extracted from primary cultured microglia or hippocampal tissue using Nucleozol (Macherey-Nagel, Cat No. 740404.200, Dueren, Germany) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using Superscript cDNA Premix Kit II (GeNetBio, Cat No. SR-5000, Daejeon, South Korea) and analyzed using SensiFAST. TM Real-time PCR for 40 cycles was performed using the SYBR No-ROX Kit (Bioline, Cat No. BIO-98050, Memphis, TN, USA) on a CFX Duet Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The Gapdh cycle threshold (Ct) was used for normalization. The multiplicity changes of cells or tissues treated with LPS or LPS + entrectinib were calculated relative to the control group.

[0085] 6. Cytoplasmic and nuclear fractions Nuclear levels of phosphorylated NF-κB (p-NF-κB) and phosphorylated STAT3 (p-STAT3) were assessed in primary cultured microglia treated with 200 ng / mL LPS or phosphate PBS for 30 minutes, followed by exposure to 1 μM entrectinib or 1% DMSO (control group) for 5.5 hours. For cell lysis, cells were suspended in cytoplasmic fraction buffer consisting of 10 mM HEPES (pH 7.4), 10 mM KCl, and 0.05% NP-40, and allowed to stand on ice for 20 minutes. The cells were then centrifuged at 14,000 rpm at 4°C for 10 minutes, and the supernatant showing the cytoplasmic fraction was transferred to a new tube. Subsequently, RIPA lysis buffer (Thermo Scientific, Cat No. 89901) was added to the remaining pellet, and the nuclear components were lysed by sonication. After standing on ice for another 10 minutes, the nuclear lysate was again centrifuged at 14,000 rpm at 4°C for 10 minutes. The obtained nuclear fraction was used for Western blot analysis to detect p-NF-κB and p-STAT3 levels in the nuclei.

[0086] 7. Western blot Western blotting was performed on primary cultured microglia and hippocampal tissue treated with LPS or PBS, followed by treatment with entrectinib or DMSO (control group). Samples were lysed in RIPA lysis buffer (Thermo Scientific, Cat No. 89901), sonicated, and centrifuged at 12,000 rpm for 10 minutes. Protein concentration in the supernatant was quantified by comparison with standard BSA solution, and 20 μg of protein was loaded onto 8% or 10% SDS-PAGE gels depending on the experimental conditions. The protein was then loaded onto a nitrocellulose membrane (Amersham TM Protran TM0.2µm NC、Cytiva、Cat No.10600001, Wilmington, DE, USA) with 5% BSA and 5% BSA with a 1 hour freeze The solvent was prepared with the following dilutions and dilutions: anti-p-TRK(pan)(1:1000) Cell Signaling Technology, Cat No. 4621, Danvers, MA, USA, anti-TRK(pan) (1:1000), Cell Signaling Technology, Cat No. 92991, anti-p-JNK (1:1000), Cell Signaling Technology, Cat No.9251), anti-JNK(1:1000), MyBioSource, Cat No.MBS8509129, San Diego, CA, USA), anti-p-P38(1:1000), Cell Signaling Technology, Cat No.4511), anti-P38(1:1000), Cell Signaling Technology, Cat No.8690, anti-p-AKT(1:1000), Cell Signaling Technology, Cat No.9271, anti-AKT(1:1000), Cell Signaling Technology, Cat No.9272), anti-p-STAT3(1:1000, Abcam, Cat No.ab32143, Cambridge, MA, USA), anti-STAT3(1:1000, Abcam, Cat No.ab68153), anti-p-NF-κB (1:1000, Cell Signaling Technology, Cat No. 3033), anti-NF-κB (1:1000, Cell Signaling Technology, Cat No. 8242), anti-histone H3 (1:10,000), Cell Signaling Technology, Cat No. 9715, anti-GAPDH (1:10,000), Cell Signaling Technology, Cat No. 2118 (4°C).

[0087] Subsequently, the membranes were incubated for 1 hour with HRP-conjugated anti-mouse IgG (1:10,000, Invitrogen, Cat No. 31430, CA, USA) or anti-rabbit IgG (1:10,000, Promega, Cat No. W4011, WI, USA). ECL was used for detection. TM The procedure was performed using a Prime Western Blotting System (Cytiva, Cat No. RPN2232). For additional protein detection within the same membrane, the Restore system was used. TM Western Blot Stripping Buffer (Thermo Scientific, Cat No. 21059) was used. Images were processed using ChemiDoc MP. TM The data was acquired and analyzed using an imaging system (Bio-Rad, Hercules, CA, USA).

[0088] 8. Immunocytochemistry For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 10 minutes and washed three times with PBS. Subsequently, the following primary antibodies were incubated for one day in PBS containing FBS: anti-p-TRK(pan) (1:500, Cell Signaling Technology, Cat No. 4621), anti-p-JNK (1:100, Cell Signaling Technology, Cat No. 9251), anti-p-P38 (1:100, Cell Signaling Technology, Cat No. 4511), anti-p-AKT (1:1000, Cell Signaling Technology, Cat No. 9271), anti-p-STAT3 (1:100, Abcam, Cat No. ab32143), anti-p-NF-κB (1:100, Cell Signaling Technology, Cat No. 3033), anti-CD11b (1:100, Cell Signaling Technology, Cat No. 46512), anti-CD16 / 32 (1:100, Cell Cell Signaling Technology (Cat No. 80366), anti-CD206 (1:100, Cell Signaling Technology, Cat No. 24595). After culturing, cells were washed with PBS and treated with Alexa Fluor 488 or 594 conjugated secondary antibody at room temperature for 2 hours. Subsequently, cells were washed three times with PBS and incubated in DAPI solution (Thermo Scientific, Cat No. 62248) for 10 minutes. After DAPI staining, cells were washed, mounted on slides using mounting medium (Dako, Cat No. S3023, Santa Clara, CA, USA), and imaged using an A1 / Ni-E microscope (Nikon, Tokyo, Japan). Image analysis was performed using ImageJ software.

[0089] 9. Animal and immunofluorescence staining Male C57BL6 / J mice (8 weeks old; Orient-Bio Company, Gyeonggi-do, South Korea) were kept in a pathogen-free environment at 22±2°C, 50±5% humidity, and a 12-hour light-dark cycle, with free access to feed and water. The mice were randomly divided into three groups: a control group, an LPS group, and an LPS + entrectinib group. Depending on the group, mice received 10 mg / kg of entrectinib or a vehicle solution (5% DMSO, 10% PEG, 20% Tween 80 in deionized water) intraperitoneally daily for 8 days. 30 minutes after the final dose on day 8, 250 μg / kg of LPS or PBS was administered intraperitoneally. Eight hours after administration, the mice were perfused and fixed with PBS and 4% paraformaldehyde. Brain tissue was stored at 4°C in 4% paraformaldehyde for 24 hours, then immersed in PBS containing 30% sucrose for 72 hours, and 30 μm sections were prepared using a cryostat microtome (Leica CM1850, Wetzlar, Germany). Sections were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 2 hours, and then immunostained at 4°C for 1 day using the following primary antibodies: anti-p-TRK (1:150, Cell Signaling Technology, Cat No. 4621), anti-CD11b (1:500, Cell Signaling Technology, Cat No. 46512), anti-p-NF-κB (1:500, Cell Signaling Technology, Cat No. 3033), anti-p-STAT3 (1:200, Abcam, Cat No. ab32143), anti-CD16 / 32 (1:200, Cell Signaling Technology, Cat No. 80366), and anti-CD206 (1:50, Cell Signaling Technology, Cat No. 24595). Sections were washed with PBST buffer and incubated with Alexa 555 or Alexa 488 conjugated secondary antibody at room temperature for 2 hours. Subsequently, sections were washed three times with PBST buffer and incubated in DAPI solution (Thermo Scientific, Cat No. 62248) for 10 minutes.After DAPI staining, the samples were treated with VectaShield solution for fluorescence (Vector Labs, Cat No. H-1000, Burlingame, CA, USA), and fluorescence microscopy images were taken using a DMi8 microscope (Leica Microsystems, Wetzlar, Germany) and analyzed with ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

[0090] 10. Phagocytic activity evaluation and live cell imaging Phagocytosis of BV2 microglia was observed by Aβ clearance. Before exposure to Aβ, BV2 microglia were treated with 200 ng / mL LPS or PBS for 30 minutes and then with 1 μM entrectinib or 1% DMSO (control group) for 23.5 hours. During the treatment, 250 nM Alexa Fluor 488-conjugated Aβ 1-42 (Anaspec, Cat No. AS-60479-01, Fremont, CA, USA) was incubated in serum-free medium at 37 °C for 24 hours. Then, BV2 microglia were exposed to pre-prepared Aβ 1-42 for 1 hour. Subsequently, the cells were fixed with 4% paraformaldehyde for 20 minutes, washed three times with PBS, and then incubated with DAPI solution for 10 minutes. After DAPI staining, the cells were washed and mounted on glass slides using a fluorescence solution (Dako, Cat No. S302380-2, Santa Clara, CA, USA). All steps of this experiment were performed with minimal light exposure. Images were taken using an A1 / Ni-E microscope (Nikon, Tokyo, Japan), and image analysis was performed using ImageJ software.

[0091] Live imaging of Aβ clearance by BV2 microglia was also observed under similar conditions. BV2 microglia were cultured in a confocal dish and treated with 200 ng / mL LPS or PBS for 30 minutes, followed by treatment with 1 μM entrectinib or 1% DMSO (vehicle) for 23.5 hours before Aβ treatment. Then, immediately before observing the BV2 microglia with a confocal microscope, the prepared Aβ was used. 1-42 The subjects were exposed to the CO2 for 1 hour. Live images were acquired using a Dragonfly 502w high-speed confocal microscope system (Andor Technology, Belfast, UK) equipped with a CO2 and temperature-controlled chamber (5% CO2, 37°C). Video was recorded and snapshots were taken using Imaris analysis software.

[0092] 11.Y-shaped maze The effect of entrectinib on improving LPS-induced short-term and spatial memory impairment in wild-type mice was evaluated using a Y-maze test. The Y-maze consisted of three arms, each measuring 35cm × 7cm × 15cm, arranged at a 120° angle. In each test session, each mouse was allowed to freely explore the maze for 5 minutes. Mouse movements were recorded with an Exovision XT (Noldus, Leesburg, VA, USA) video camera and then manually calculated. The alternation rate was calculated using the following formula.

[0093] Substitution rate (%) = (Number of substitutions / Number of substitution attempts) × 100%

[0094] 12. Novel Object Recognition (NOR) Test A novel object recognition (NOR) test was used to evaluate the effects of entrectinib on long-term and cognitive memory. The NOR apparatus consisted of an open field box measuring 40 cm × 40 cm × 25 cm. The test was conducted in two phases: a training phase and a test phase, with a 24-hour interval between the two phases. In the training phase, two identical objects were placed in the box, and one mouse was allowed to explore freely for 5 minutes. Then, in the test phase, the mouse was returned to the box, and one familiar object and one novel object were placed inside, and the mouse was allowed to explore freely for 5 minutes. In the test phase, the objects were placed in the same positions to control for positional bias. To remove olfactory cues, the box and objects were cleaned with 70% ethanol between tests. Exploration time was automatically recorded by reviewing video footage of each session. Exploration behavior was defined as the state in which the mouse pointed its nose towards the object. The degree of preference for the novel object was calculated using the following formula. Object preference (%)={T Novel / (T Familiar +T Novel )} × 100% Here, T Novel This indicates the time taken to search for a new object, T Familiar This indicates the time taken to search for familiar objects.

[0095] 13.Statistical analysis Data analysis was performed using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). For comparisons between two groups, an unpaired two-tailed t-test (T-test) with Welch's correction was used. For multiple comparisons, one-way ANOVA and Tukey's test were used. A statistically significant difference was considered to be p<0.05. Results are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0096] [Example 1] Confirmation of the effect of suppressing encephalitis 1-1. TRK inhibitory activity Entrectinib is known to antagonize TRK (tropomyosin receptor kinase), and this study was conducted to confirm whether entrectinib inhibits TRK in microglia. Primary cultured microglia were treated with 200 ng / mL LPS or PBS for 15 minutes, and then treated with 1 μM entrectinib or 1% DMSO for 30 minutes.

[0097] Western blotting was performed to confirm protein levels, and it was confirmed that entrectinib significantly inhibited TRK by reducing phosphorylation in primary culture microglia treated with LPS (Figure 1). Furthermore, it was confirmed that phosphorylation on the surface of TRK was significantly reduced after exposure to entrectinib in primary culture microglia treated with LPS (Figure 2).

[0098] Therefore, we confirmed that entrectinib affects microglial function by antagonizing both TRK and its target receptor.

[0099] 1-2. Inflammatory response regulatory activity To confirm the effect of entrectinib on the inflammatory response of microglia, primary mouse cultured microglia were treated with 200 ng / mL LPS or PBS for 30 minutes, followed by treatment with 1 μM entrectinib or 1% DMSO for 5.5 hours.

[0100] Real-time PCR was performed to confirm the gene expression levels of inflammatory cytokines, and it was shown that entrectinib significantly suppressed the increase of LPS-induced inflammatory cytokines Il1β, Il6, Tnfα, Il23α, chemokine Ccl2, and Inos molecules, while significantly increasing the anti-inflammatory cytokine il13 (Figure 3).

[0101] Therefore, we confirmed that entrectinib can control neuroinflammatory factors that regulate TRK activity in microglia.

[0102] 1-3. Phosphorylation-modulating activity of JNK, P38, and AKT Multiple neuroinflammation-related signaling pathways have been identified in microglia, and the major inflammatory pathways in microglia are known to be the MyD88-MAPK (JNK, P38, ERK, etc.) or PI3K-AKT pathways, which activate inflammatory or anti-inflammatory cytokines and chemokines. To identify the signaling molecules involved in the effects of entrectinib on microglial regulation, we examined the phosphorylation levels of LPS-induced inflammatory molecules in microglia after entrectinib treatment.

[0103] To confirm this, primary cultured mouse microglia were treated with 200 ng / mL LPS or PBS for 15 minutes, followed by treatment with 1 μM entrectinib or 1% DMSO for 30 minutes.

[0104] After lysing the cells and performing Western blotting, we confirmed that LPS treatment significantly increased p-JNK, p-P38, and p-AKT, while entrectinib significantly decreased their phosphorylation levels (Figure 4).

[0105] Therefore, we confirmed that entrectinib modulates neuroinflammatory factors by reducing the protein levels of p-JNK, p-P38, and p-AKT in primary cultured microglia.

[0106] 1-4. Confirmation of NF-κB and STAT3 signaling pathway regulation Since entrectinib reduces LPS-stimulated inflammation-related signaling molecules, including mitogen-activated protein kinase (MAPK) and AKT, we investigated the involvement of transcription factors associated with neuroinflammation.

[0107] To confirm this, primary cultured microglia were treated with 200 ng / mL LPS or PBS for 30 minutes, and then cultured in 1 μM entrectinib or 1% DMSO for 5.5 hours. Nuclear and cytoplasmic fractionation was performed, and phosphorylation levels were detected.

[0108] Western blotting results showed that entrectinib significantly reduced the elevation of p-NF-κB and p-STAT3 levels in LPS-stimulated nuclei, but cytoplasmic phosphorylation levels increased after entrectinib treatment compared to LPS treatment (Figure 5). This suggests that LPS translocates p-NF-κB and NF-κB from the cytoplasm to the nucleus in microglia, stimulating the NF-κB pathway that transcribes neuroinflammation-related genes.

[0109] Therefore, we confirmed that entrectinib modulates LPS-induced microglial inflammation and anti-inflammatory factors by downregulating the Trk-bound NF-κB / STAT3 pathway in primary cultured microglia.

[0110] 1-5. Confirmation of CD16 / 32 and CD206 expression regulation Entrectinib downregulates the neuroinflammatory response of microglia by suppressing MAPK, AKT-related p-NF-κB, and p-STAT3 levels. Previous studies have elucidated the diverse stages of microglia, each playing a role in regulating inflammation-related homeostasis in the brain. Among the diverse phenotypes, the systems for pro-inflammatory activity and anti-inflammatory response states, and the restoration of their effects, have been well studied. Therefore, whether entrectinib modulates microglial characteristics under LPS conditions could be an important clue in elucidating its effects.

[0111] To confirm this, primary cultured microglia were treated with 200 ng / mL LPS or PBS for 30 minutes, followed by exposure to 1 μM entrectinib or 1% DMSO for 23.5 hours. Immunofluorescence analysis was performed using the toxic and protective microglia markers CD16 / 32 and CD206.

[0112] The results showed that exposure to entrectinib significantly reduced the fluorescence intensity of LPS-stimulated CD16 / 32, while the fluorescence intensity of CD206 significantly increased after entrectinib treatment compared to LPS treatment (Figure 6).

[0113] Therefore, we confirmed that entrectinib reduces CD16 / 32 while increasing CD206 in primary cultured microglia, thereby regulating microgliosis.

[0114] [Example 2] Confirmation of phagocytic enhancement effect Because entrectinib has the potential to control microglial phenotypes by modulating the TRK-related inflammatory pathway, we investigated the effects of entrectinib on microglial phagocytosis.

[0115] 2-1. Amyloid-beta (Aβ) scavenging activity Using BV2 microglia, we investigated the effect of entrectinib on microglial phagocytosis under LPS-induced neuroinflammatory conditions.

[0116] BV2 microglia were treated with 200 ng / mL LPS or PBS for 30 minutes, then treated with 1 μM entrectinib or 1% DMSO for 23.5 hours, and further treated with Aβ 1-42 The oligomers were treated for 1 hour. Subsequently, fluorescence of Alexa 488 was detected in the intracellular membrane of microglia.

[0117] Aβ 1-42 Data taken 1 hour after oligomer treatment showed that entrectinib was effective against LPS-induced Aβ 1-42 It significantly restored the decrease in fluorescence intensity of Aβ 1-42 We confirmed that the oligomer is present in the cytoplasmic region of BV2 microglia (Figure 7).

[0118] Therefore, we confirmed that entrectinib enhances the phagocytic activity of microglia against amyloid-beta aggregates.

[0119] 2-2. Phagocytic activity Given that entrectinib restores LPS-induced phagocytic dysfunction in BV2 microglia, we investigated the relationship between factors related to phagocytosis.

[0120] To confirm this, primary microglia were treated with 200 ng / mL LPS or PBS for 30 minutes, then exposed to 1 μM entrectinib or 1% DMSO for 23.5 hours, and real-time PCR was performed.

[0121] The results showed that gene expression of phagocytic receptors such as Trem2, Sorl1, Cd33, and Cr2, as well as cytoskeletal factors Vav1 and Cdc42, was significantly increased after entrectinib exposure compared to the LPS-treated group (Figure 8).

[0122] Therefore, we confirmed that entrectinib enhances the phagocytic capacity of microglia by suppressing neuroinflammation-related signaling pathways and upregulating the M2 phenotype of microglia.

[0123] [Example 3] Confirmation of the effect of suppressing encephalitis in animal experiments Since entrectinib downregulates LPS-induced neuroinflammatory responses in vitro, we confirmed the effect of entrectinib on neuroinflammation in vivo.

[0124] Eight-week-old male mice were intraperitoneally administered (ip) 10 mg / kg of entrectinib or vehicle (5% DMSO, 10% PEG, 20% Tween 80 in deionized water), followed by daily administration of LPS (250 ng / kg, ip) or PBS for 8 days 30 minutes later. On the final day, the brains were removed from the mice, the hippocampus was isolated, and then Western blotting and immunofluorescence staining were performed.

[0125] The results showed that entrectinib significantly reduced LPS-induced TRK phosphorylation in the hippocampus (Figure 9). Consistent with in vitro results, LPS-induced JNK, P38, and AKT phosphorylation were significantly downregulated by entrectinib (Figure 10).

[0126] Furthermore, immunofluorescence staining revealed that LPS significantly upregulated p-NF-κB and p-STAT3 levels in the hippocampus of mouse brains, but these increases were significantly downregulated by entrectinib treatment (Figure 11).

[0127] Therefore, we confirmed that entrectinib contributes to neuroinflammatory responses by inactivating the phosphorylation of MAPK / AKT and NF-κB / STAT3 in vivo.

[0128] [Example 4] Confirmation of the effect on improving memory impairment through animal experiments Since entrectinib alters LPS-induced neuroinflammatory responses in the hippocampus, this study was conducted to confirm the effect of entrectinib on memory impairment induced by neuroinflammation in mice administered LPS.

[0129] 4-1. Effect of improving memory impairment To confirm whether neuroinflammation-induced memory impairment improved after entrectinib administration, 8-week-old male mice were intraperitoneally administered LPS (1 mg / kg) or PBS, followed by daily administration of entrectinib 10 mg / kg or PBS for 8 or 16 days 30 minutes after administration. Behavioral assessments were performed on days 7-8 and 15-16 using the Y-maze test and the novel object recognition (NOR) test.

[0130] As a result, the spontaneous switching rate (%) and object preference were significantly increased in the Y-maze test and NOR test on days 8 and 16 after entrectinib administration (Figures 12 and 13). Furthermore, it was confirmed that all mice had recovered their body weight at the time of the behavioral tests (Figure 14).

[0131] Therefore, we confirmed that entrectinib improves memory impairment induced by neuroinflammation in mice.

[0132] 4-2. Confirmation of synaptic changes Memory impairment is known to be associated with changes in dendritic spine formation and synaptic plasticity of hippocampal neurons in the mouse brain. Therefore, to investigate synaptic changes associated with entrectinib, Western blot analysis was performed on mouse hippocampal tissue after behavioral testing. Representative presynaptic markers, such as SYP (synaptophysin), and postsynaptic markers, such as PSD95 (postsynaptic density protein 95), were used.

[0133] As a result, the expression levels of PSD95 and SYP, which were reduced by LPS exposure, were significantly restored on days 8 and 16 of daily entrectinib administration (Figures 15 and 16).

[0134] Therefore, we confirmed that entrectinib contributes to improving memory impairment induced by neuroinflammation by upregulating synaptic proteins in the mouse hippocampus.

Claims

1. A pharmaceutical composition comprising entrectinib for the prevention or treatment of inflammatory brain diseases or neurodegenerative diseases.

2. The composition according to claim 1, wherein the inflammatory brain disease or neurodegenerative brain disease is a group of patients in which TRK (tropomyosin receptor kinase) activity is increased.

3. The composition according to claim 1, wherein the composition reduces inflammatory cytokines and increases anti-inflammatory cytokines.

4. The composition according to claim 1, wherein the composition inhibits the phosphorylation of JNK, p38, AKT, NF-κB, or STAT3.

5. The composition according to claim 1, wherein the composition reduces the expression of CD16 / 32 and increases the expression of CD206.

6. The composition according to claim 1, wherein the composition reduces amyloid-beta (Aβ) aggregates.

7. The composition according to claim 1, wherein the composition enhances the phagocytosis of microglia.

8. The composition according to claim 1, wherein the composition prevents or improves memory impairment induced in inflammatory brain diseases or neurodegenerative diseases.

9. The composition according to claim 1, wherein the cerebral degenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Pick's disease, Huntington's disease, dementia, mild cognitive impairment, stroke, and cerebral infarction.

10. A food composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

11. A veterinary feed composition containing entrectinib for the prevention or improvement of inflammatory brain diseases or neurodegenerative diseases.

12. A pharmaceutical composition comprising entrectinib for the prevention or treatment of memory impairment.

13. The composition according to claim 12, wherein the memory impairment is induced by neuroinflammation.

14. The composition according to claim 12, wherein the composition increases synaptic proteins.

15. A food composition containing entrectinib for the prevention or improvement of memory impairment.

16. A veterinary feed composition containing entrectinib for the prevention or improvement of memory impairment.

17. A method for reducing encephalitis, comprising the step of administering a composition containing entrectinib to a non-human organism.

18. A method for preventing or improving memory impairment, comprising the step of administering a composition containing entrectinib to a non-human organism.

19. A method for enhancing the phagocytic activity of microglia, comprising the step of treating an organism, either in vitro or in a non-human body, with a composition containing entrectinib.

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