KR20210156389A - Pharmaceutical composition for the prevention or treatment of inflammatory disease comprising regorafenib an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising regorafenib as an active ingredient. Regorafenib of the present invention has an excellent inhibitory activity to inflammatory cytokines COX-2, IL-1β, IL-6, TNF-a or iNOS, and has an activity to inhibit TLR4 or AKT signaling related to inflammatory response, and to inhibit the level of p-STAT3, and thus can be used as a new therapeutic agent for the prevention or treatment of inflammatory diseases. Furthermore, Regorafenib of the present invention inhibits the activity of microglia or astrocytes by LPS, inhibits amyloid plaque, has an excellent inhibitory activity to dendrite increase and tau protein phosphorylation, and thus can be used as a new therapeutic agent for the prevention or treatment of neurodegenerative diseases caused by excessive inflammatory response.

Description

A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising regorafenib as an active ingredient {Pharmaceutical composition for the prevention or treatment of inflammatory disease comprising regorafenib an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising regorafenib as an active ingredient.

Inflammatory reaction is caused by tissue (cell) damage or infection with external infectious agents (bacteria, fungi, viruses, and various types of allergens). It exhibits a series of complex physiological reactions such as substance secretion, body fluid infiltration, cell migration, and tissue destruction and external symptoms such as erythema, edema, fever, and pain. In a normal case, the inflammatory response removes external infectious agents and regenerates damaged tissues, thereby restoring the function of the living organism. In some cases, it leads to diseases such as cancer.

Among the various factors that induce such an inflammatory response, lipopolysaccharide (LPS), a lipid-polysaccharide complex compound, is an endotoxin O-antigen found in the cell wall of Gram-negative bacteria, and lipid A (endotoxin) , a non-repetitive core oligosaccharide, and a polysaccharide (O-antigen). The lipid moiety (lipid A) forms a complex with the core polysaccharide moiety via glycosidic bonds. The core portion is linked to a highly immunogenic and variable O-chain polysaccharide composed of repeating oligosaccharide units, i.e. the third external site, an O-antigen site. The last region of the LPS molecule serves to activate a number of transcription factors, which performs a bacterial serospecies-specific function. The most important finding is that the plasma membrane protein TLR4 acts as a lipid A signaling receptor in animal cells. TLR4 belongs to the innate immune receptor family, and when LPS is recognized by TLR (Toll-like receptor)4, pro-inflammatory transcription factor initiators NF-κB (nuclear factor-kappa B) and MAPK (mitogen activated protein kinase) MyD88- By inducing dependent activation, various inflammatory cytokines are expressed. Therefore, inhibitors of these mechanisms can be used as therapeutic agents for inflammatory diseases.

Recently, it has been revealed that the inflammatory response is one of the main mechanisms that induce neurodegeneration. Microglial cells, which are immune cells in the central nervous system, can be activated by various exogenous and endogenous substances, and activated microglial cells Produces and releases substances such as inflammatory cytokines TNF-α and IL-1β, nitric oxide, prostaglandins, and superoxide. The production of these substances induces an immune response in the short term, but its excessive or continuous production induces the death of adjacent neurons, which ultimately leads to neurodegeneration. Also, since the substances released by dying nerve cells trigger the activity of microglia again, neurodegeneration falls into a continuous vicious cycle, and the activity of microglia actually decreases with Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, and Creutzfeldt's disease. It has been reported that it is related to various neurodegenerative diseases such as Creutzfelt-Jakob Disease (CJD) and multiple sclerosis.

On the other hand, regorafenib is known as a biomolecule with potential anti-angiogenic and anti-tumor activities (REF), and has an effect on various types of cancer such as colon cancer and liver cancer through inhibition of growth factors and angiogenesis. I know it affects

On the other hand, there is still no case that regorafenib can be used as a therapeutic agent for inflammatory diseases.

Republic of Korea Patent Publication 10-2015-0060869 Republic of Korea Patent Publication 10-2018-0118745

Accordingly, the present inventors completed the present invention by identifying a novel use as a novel therapeutic agent capable of preventing or treating inflammatory diseases by confirming that regorafenib, known as a conventional anticancer agent, has excellent anti-inflammatory activity.

Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, comprising regorafenib or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving inflammatory diseases containing regorafenib as an active ingredient.

Another object of the present invention is to provide a method for inhibiting the inflammatory activity of cells in vitro, comprising the step of treating the inflammatory activity-induced cells with regorafenib or a salt thereof.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, comprising regorafenib or a pharmaceutically acceptable salt thereof as an active ingredient.

In one embodiment of the present invention, the composition may be to inhibit the expression and production of inflammatory cytokines COX-2, IL-1β, IL-6, TNF-a or iNOS.

In one embodiment of the present invention, the composition may inhibit TLR4 or AKT signaling and inhibit the level of p-STAT3.

In one embodiment of the present invention, the composition inhibits the activity of microglia or astrocytes; amyloid plaque inhibition; increased dendrites; Alternatively, it may be to inhibit phosphorylation of the tau protein.

In one embodiment of the present invention, the inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis , hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, cancer and neurodegenerative diseases may be selected from the group consisting of.

In one embodiment of the present invention, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine nucleus-pony-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum-nigra degeneration , Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction it could be

In one embodiment of the present invention, the neurodegenerative disease may be caused by an excessive inflammatory response of nerve cells.

In addition, the present invention provides a health functional food for the prevention or improvement of inflammatory diseases containing regorafenib (regorafenib) as an active ingredient.

In one embodiment of the present invention, the inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory Bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, cancer and neurodegenerative disease may be selected from the group consisting of have.

In one embodiment of the present invention, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine nucleus-pony-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum-nigra degeneration , Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction it could be

In addition, the present invention provides a method for inhibiting inflammatory activity of cells in vitro, comprising the step of treating the inflammatory activity-induced cells with regorafenib or a salt thereof.

In one embodiment of the present invention, the inflammatory activity may be induced by LPS (Lipopolysaccharide) treatment.

Regorafenib of the present invention has excellent inhibitory activity of inflammatory cytokines COX-2, IL-1β, IL-6, TNF-a or iNOS, and inhibition of TLR4 or AKT signaling related to inflammatory response, p-STAT3 Since it has an activity to inhibit the level of Inhibits activity, suppresses amyloid plaques, increases dendrites, and has excellent phosphorylation inhibitory activity of tau protein. It is possible.

1 is a result of analyzing the levels of pro-inflammatory and anti-inflammatory cytokines induced by LPS according to regorafenib treatment in microglia, (A) is MTT assay after treatment with regorafenib by concentration The results are the results, (B) to (G) are the results of analyzing the level of inflammatory cytokines after pre-treatment with LPS and regorafenib treatment, (H)-(M) are the results of pre-treatment with regorafenib and after LPS treatment Results of analyzing the level of inflammatory cytokines, (N) to (P) are Western blot results using anti-p-AKT or anti-AKT antibody after LPS pretreatment and regorafenib treatment, (Q ) to (S) are LPS treatment and MK2206 (AKT inhibitor) treatment, then regorafenib, and then measure the level of pro-inflammatory cytokines, (T) to (W) are LPS pretreatment and Lego After lafenib treatment, the p-STAT3 levels in the cytoplasmic and nuclear fractions were measured, and (X) to (Y) are the results confirmed by a fluorescence microscope using an anti-CD11b antibody and an antibody to p-STAT3. it has been shown
2 is a result of measuring the level of anti-inflammatory cytokines in regorafenib-treated microglia, (A) to (B) are LPS pre-treatment and analysis of the level of anti-inflammatory cytokines after regorafenib treatment One result, (C) to (D) are the results of analyzing the level of anti-inflammatory cytokines after pre-treatment with regorafenib and LPS treatment.
3 is a result confirming that the effect of regorafenib on the level change of LPS-induced inflammatory cytokines is partially dependent on TLR4. The level of COX-2 and IL-1β in the cells was measured by treatment, (A) is a photograph of the RT-PCR result, and (B) to (C) are quantified and graphed.
4 is an analysis of the level of inflammatory cytokines according to regorafenib treatment in microglia and astrocytes, (A) to (E) are microglia pre-treated with LPS and inflammatory cytokines after treatment with regorafenib (F) to (K) are microglia pre-treated with LPS and regorafenib treatment followed by anti-p-AKT, anti-AKT, anti-p-P38 or anti-P38 antibody Western blot was performed, (L) to (P) are pre-treatment with LPS in astrocytes and analysis of the level of inflammatory cytokines after treatment with regorafenib, (Q) to (V) are LPS in astrocytes The results of Western blotting using anti-p-AKT, anti-AKT, anti-p-P38 or anti-P38 antibody after pretreatment and regorafenib treatment are shown.
Figure 5 is an analysis of the effect of regorafenib on the level of inflammatory cytokines caused by LPS in wild-type mice. After daily injection of regorafenib into wild-type mice for 3 days, LPS was administered for 8 hours, and then brain Shows the results of immunohistochemistry on cortex and hippocampal tissue. (A) to (E) were anti-Iba-1 antibodies, (F) to (J) were anti-GFAP antibodies, (K) to (O) are the results using the COX-2 antibody.
Figure 6 confirms the decrease in the activity of astrocytes in the cortex of 5xFAD mice, which is an animal model of Alzheimer's dementia administered with regorafenib, Iba-I and GFAP according to regorafenib treatment in the cerebral cortex and hippocampus tissue; It shows the result of confirming the level by immunohistochemical analysis and fluorescence microscopy.
Figure 7 confirms the increase in the number of dendritic spines by regorafenib in primary hippocampal neurons and brain tissue of 5xFAD mice, (A) to (B) are the density of dendrites for primary hippocampal neurons is the measurement result, and (C)-(H) shows the Golgi staining results for the hippocampus and cortical tissue.
Figure 8 confirms the reduction effect of amyloid plaques in 5xFAD mice injected with regorafenib, (A) to (E) are the results of measuring the level of Aβ in the cerebral cortex and hippocampal tissue, (F) ~ (I) is the result of measuring the levels of sAPPβ, full-length APP and APP CTF after treatment with regorafenib in APP-H4 cells, (J) to (S) are cortical and hippocampal tissues treated with regorafenib, respectively. This is the result of immunohistochemical analysis using anti-BACE1 or anti-ATG12 antibody.
9 is a result confirming that there is no change in the level of Aβ degrading enzyme NEP in 5xFAD mice injected with regorafenib. Immunohistochemical analysis of cortex and hippocampus tissues of 5xFAD mice injected with regorafenib with anti-NEP antibody. The results are shown. (A) to (B) are the analysis results for the cortex, and (C) to (E) are the analysis results for the hippocampal tissue.
10 is a result of confirming that there is no change in the level of α-secretase ADAM17 in 5xFAD mice injected with regorafenib. Immunohistochemical analysis of the cortex and hippocampus of 5xFAD mice injected with regorafenib was performed with an anti-ADAM17 antibody. One result is shown, (A) to (B) are the analysis results for the cortex, and (C) to (E) are the analysis results for the hippocampus tissue.
11 is a result confirming that regorafenib did not affect the level of autophagy ATG5 in 5xFAD mice injected with regorafenib. After injection of regorafenib into 5xFAD mice, immunohistochemical analysis was performed with anti-ATG5 antibody. As the results are shown, (A) to (B) are the analysis results for the cortex, and (C) to (E) are the analysis results for the hippocampus tissue.
Figure 12 confirms the decrease in tau phosphorylation for AT100 in 5xFAD mice injected with regorafenib, (A) to (T) are cortical and hippocampal tissues of 5xFAD mice injected with regorafenib, respectively, anti-AT100, anti The results of immunohistochemical analysis using an antibody against -p-GSK3β, anti-p-CDK5 or anti-DYRK1A are shown.
13 is a result of confirming that phosphorylation of tau AT8 and AT180 by regorafenib is reduced, (A) to (E) are cortical and hippocampal tissues, respectively, using anti-AT8 antibodies, (F) to (J) are Shows the results of immunohistochemical analysis using the anti-AT180 antibody.
14 is a result confirming that the level of tau-5 is reduced in 5xFAD mice injected with regorafenib, (A) to (B) for the cortex, (C) to (E) for the hippocampal tissue anti- Shows the results of immunohistochemical analysis using the Tau5 antibody.

The present invention has identified a novel use of regorafenib as a therapeutic agent for inflammatory diseases. Specifically, the present invention relates to a treatment for inflammatory diseases comprising regorafenib or a pharmaceutically acceptable salt thereof as an active ingredient. It is characterized by providing a pharmaceutical composition for prevention or treatment.

As mentioned in the prior art, regorafenib is known as an anticancer agent, but has not yet been studied for its therapeutic potential for inflammatory diseases.

Regarding the potential of regorafenib as a therapeutic agent for inflammatory diseases, according to an embodiment of the present invention, there is an activity of effectively inhibiting the expression and production of inflammatory and pro-inflammatory cytokines induced by LPS, known as an inflammatory factor. was confirmed, and specifically, it was confirmed that there was an activity of inhibiting the production of COX-2, IL-1β, IL-6, TNF-a or iNOS by LPS. Among various inflammation-inducing factors, LPS (lipopolysaccharide), one of the endotoxins, promotes the secretion of pro-inflammatory cytokines, which secretes inflammatory mediators such as nitric oxide and prostaglandins. become ill

Therefore, the present inventors found that regorafenib can inhibit the proinflammatory response caused by LPS, thereby preventing, improving or treating inflammatory diseases that may be caused by excessive inflammatory response.

Also, in another embodiment of the present invention, an experiment was performed to determine whether the inhibition of inflammatory cytokines by regorafenib is related to the TLR4/AKT/STAT3 signaling system.

Here, the TLR4 is known to induce the pathology of inflammatory diseases by generating various inflammatory factors through activation of NF-kB when cells are stimulated by LPS stimulation, and the AKT is known to activate NF-kB, AKT signaling is known to be related to the immune response induced by LPS through the TLR4 signaling pathway, and it is known that the level of pro-inflammatory cytokines is reduced in human liver cancer cell lines when AKT is knocked out. In addition, activated TLR4 is known to affect STAT3 and other transcription factors, and it is known that continuously activated STAT3 is closely related to the onset of inflammatory diseases.

In this regard, according to an embodiment of the present invention, it was confirmed that regorafenib inhibits the signaling activation of TLR4/AKT/STAT3, and LPS-induced inflammatory cytokines COX-2, IL-1β, IL- 6, it was found that the levels of TNF-a and iNOS were reduced in both microglia and astrocytes by regorafenib treatment, and the phosphorylation level of p-AKT was also reduced.

Therefore, it was found that regorafenib can inhibit the activation of intracellular signaling involved in the pathogenesis of inflammatory diseases.

In addition, regorafenib of the present invention has the effect of inhibiting the activity of microglia or astrocytes by LPS to inhibit the damage of activated microglia or activated astrocytes to nerve cells. confirmed that there is.

Microglial cells, which act as macrophages in the brain, are important effector cells that regulate immune responses in the central nervous system (CNS). Their activation plays an important role in maintaining CNS homeostasis by removing foreign substances caused by drugs or toxins and secreting nerve growth factors. However, when exposed to harmful stresses such as signals from damaged neurons, accumulation of abnormal proteins modified by external stimuli, and penetration of pathogens, the activity of microglia is excessively increased and causes nerve cell damage, leading to Alzheimer's disease, It can cause neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and cerebral infarction. Also, recently, research results have been revealed that an excessive inflammatory reaction in microglia or astrocytes is the cause of neurodegenerative diseases.

Specifically, excessively activated microglia, unlike microglia in a normal state, actively phagocytose, proliferate, and cytokines such as TNF-α, IL-1β and IL-6, chemokines, iNOS Genes such as (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) are expressed to produce inflammatory mediators. Activation of microglia removes damaged cells and protects nerve cells from invading bacteria or viruses, but nitric oxide (NO) produced by iNOS and prostaglandins produced by COX-2, TNF- Since α and the like are also toxic to nerve cells, as a result, activation of microglia aggravates nerve cell damage, and acts as a cause of degenerative brain disease or neurodegenerative disease.

In addition, astrocytes are also known to play an important role in maintaining normal brain activity. In particular, it is known that they play a role in synapse formation of neurons, synapse number regulation, synaptic function, and differentiation of neural stem cells into nerves. . However, if these astrocytes have excessive reactivity, that is, if they maintain an excessive activation state, it causes the death of nerve cells and also induces the death of neighboring nerve cells, and acts as a cause of degenerative brain disease. Therefore, suppression of excessive activation of astrocytes can also be a new treatment method for degenerative brain diseases.

Examples of substances that cause excessive activation of microglia and astrocytes include lipopolysaccharide (LPS), interferon-γ, beta-amyloid and ganglioside, which are bacterial endotoxins that can induce an inflammatory response.

In addition, in another embodiment of the present invention, it was confirmed that regorafenib can increase dendritic spines in primary hippocampal neurons, and furthermore, as a result of administering regorafenib to a mouse in which Alzheimer's disease was invented, Amyloid plaque inhibition, beta-amyloid inhibition, and phosphorylation inhibition of Tau protein were confirmed.

Beta amyloid (Aβ: amyloid beta) is derived from amyloid precursor protein (APP), which is broken down by beta secretase and gamma secretase to produce Aβ, mainly from amyloid plaques found in the brains of patients with brain diseases such as Alzheimer's disease. It is the main component and when it accumulates abnormally, it causes the onset of various brain diseases.

In addition, the excessive accumulation of beta-amyloid causes an inflammatory response in the hippocampus, cerebral cortex, and cells of the main cells, and thus nerve cells may be damaged and even the neural network that maintains the normal function of the brain may be damaged.

In addition, the tau protein is involved in the entanglement of nerve fibers with a schedule of microtubule binding proteins having a molecular weight of 50000 to 70000 Da. Tau protein is one of the microtubule-associated proteins present in the axon of normal neurons. When such tau protein is excessively accumulated, microtubules are disrupted and the network of normal neurons is disrupted. It is known that the cause of the accumulation of tau protein is that phosphorus is excessively attached to the tau protein to form a heavy body due to phosphorylation. .

In this regard, it is known that regorafenib of the present invention has beta-amyloid inhibition and phosphorylation inhibitory activity of Tau protein, so it can be used as a therapeutic agent for degenerative neurological diseases caused by inflammation in nerve cells or brain cells. could

Therefore, regorafenib or a pharmaceutically acceptable salt thereof of the present invention can be used as a therapeutic agent for inflammatory diseases.

The inflammatory disease according to the present invention is not limited thereto, but dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, pharyngitis, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, It may be selected from the group consisting of colitis, hemorrhoids, gout, ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, cancer and neurodegenerative diseases.

In addition, the neurodegenerative disease here is not limited thereto, but Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine nucleus-pondylo-cerebellar atrophy (OPCA), Shay-Drager syndrome, striatum-nigra degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Nieman-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction have.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablet pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose. ), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have one formulation.

In addition, these pharmaceutical compositions, as described above, the pharmaceutical composition of the present invention can be administered to treat various diseases including symptoms associated with an inflammatory response.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the subject type and severity, age, sex, activity of the drug, and the drug. It can be determined according to factors including sensitivity, administration time, administration route and excretion rate, duration of treatment, concomitant drugs, and other factors well known in the medical field.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects. A typical dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, oral administration, intranasal administration, intrapulmonary administration, or rectal administration as desired, but is not limited thereto. In addition, the composition may be administered by any device capable of transporting the active substance to a target cell.

The composition of the present invention can be used alone or in combination with surgery, hormone therapy, drug therapy, and methods using biological response modifiers for the prevention and treatment of inflammatory diseases.

Furthermore, the present invention can provide a health functional food for preventing or improving inflammatory diseases containing regorafenib as an active ingredient.

The health functional food of the present invention may further include a food-logically acceptable carrier.

The health functional food may have a function to help improve inflammatory diseases, and the food includes the form of pills, powders, granules, needles, tablets, capsules or liquids, and the active ingredient of the present invention may be added thereto. There is no particular limitation on the type of food that can be used, and for example, there are various beverages, gum, tea, vitamin complexes, health supplements, and the like.

In addition to the active ingredient of the present invention, other ingredients that do not interfere with the inhibitory activity of inflammatory diseases may be added to the health functional food, and the type thereof is not particularly limited. For example, it may contain various herbal extracts, food-logically acceptable food supplements or natural carbohydrates as additional ingredients, such as conventional food.

The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and pills using raw materials or ingredients useful for the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long period of time by using food as a raw material.

There is no particular limitation on the type of health functional food of the present invention, and specific examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, There are beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and may include all health functional foods in a conventional sense.

Furthermore, the present invention may provide a method for inhibiting inflammatory activity of cells in vitro, comprising the step of treating cells induced with inflammatory activity with regorafenib or a salt thereof, wherein the inflammatory activity is It may be induced by LPS (Lipopolysaccharide) treatment.

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Preparation example and experimental method>

All experiments performed in the examples of the present invention were performed with the approval of the Institutional Biosafety Committee (IBC) of the Korea Brain Research Institute (KBRI, approval number 2014-479).

Cell lines and culture conditions

BV2 microglial cells (obtained from Dr. Kyung-Ho Suk) were harvested from DMEM/high glucose (Invitrogen, Carlsbad, USA) with 5% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). CA, USA) was cultured in a 5% CO2 incubator using medium. APP-H4 cells (obtained from Dr. Young Young Koh, H4 cells capable of overexpressing human amyloid precursor protein and producing high levels of Aβ) were cultured using MEM/high glucose medium supplemented with 10% FBS and gentamicin. Cultured in a 5% CO2 incubator. In addition, the results obtained in all in vitro experiments were analyzed semi-automatically using Image J software, and the results were confirmed by independent researchers not currently participating in the experiment.

MTT assay

For cell viability, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay was used. BV2 microglia were aliquoted in 96-well plates and treated with various concentrations of regorafenib (0.1, 1, 5, 10, 20 μM) in the absence of FBS for 24 hours. Thereafter, the cells were treated with 0.5 mg/ml MTT and cultured in a 5% CO2 incubator at 37° C. for 3 hours, and then absorbance was measured at 580 nm.

RT-PCR (Reverse transcription-polymerase chain reaction)

Total RNA was extracted from the cells using TriZol (Invitrogene). Then, total RNA was reverse transcribed into cDNA using Superscript cDNA Premix Kit II containing oligoDT (GeNetBio, Korea). RT-PCR was performed with Prime Taq Premix (GeNetBio, Korea), and RT-PCR was performed on BV microglia using the following primers.

IL-1β Forward (F): AGC TGG AGA GTG TGG ATC CC

IL-1β Reverse (R): CCT GTC TTG GCC GAG GAC TA

IL-6 Forward (F): CCA CTT CAC AAG TCG GAG GC

IL-6 Rorward (R): GGA GAG CAT TGG AAA TTG GGG T

IL-18 Forward (F): TTT CTG GAC TCC TGC CTG CT

IL-18 Rorward (R): ATC GCA GCC ATT GTT CCT GG

COX-2 Forward (F): GCC AGC AAA GCC TAG AGC AA

COX-2 Rorward (R): GCC TTC TGC AGT CCA GGT TC

iNOS Forward (F): CCG GCA AAC CCA AGG TCT AC

iNOS Rorward (R): GCA TTT CGC TGT CTC CCC AA

TNF-α Forward (F): CTA TGG CCC AGA CCC TCA CA

TNF-α Rorward (R): TCT TGA CGG CAG AGA GGA GG

GAPDH Forward (F): CAG GAG CGA GAC CCC ACT AA

GAPDH Rorward (R): ATC ACG CCA CAG CTT TCC AG

Thereafter, the RT-PCR product was separated by electrophoresis on a 1.5% agarose gel containing Eco Dye (1: 5000, Korea). Electrophoretic RT-PCR image analysis was performed using Image J (NIH) and Fusion (Korea).

Real-time PCR (Q-PCR)

To determine the effect of regorafenib on LPS-induced levels of pro-inflammatory and anti-inflammatory cytokines, BV2 microglia and primary microglia from mice were treated with LPS (1 μg/ml) or PBS for 30 min. After treatment with regorafenib (5 μM) or vehicle (1% DMSO) for 5.5 hours. Thereafter, total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol, and reverse transcribed into cDNA using Superscript cDNA Premix Kit II (GeNetBio, Korea) with oligo (dT) primers. Then, real-time PCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific, CA, USA) and the QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific, San Jose, CA, USA). Cycle threshold (Ct) values for mRNA of inflammatory mediators were normalized to Ct values for Gapdh, and data were quantified as fold change compared to control.

Antibodies and inhibitors

The primary antibodies used for Western blot, immunocytochemistry (ICC) and immunohistochemistry (IHC) were rat anti-mouse CD11b (1:400 for ICC, Abcam), rabbit anti-COX-2 (1:200 for ICC, Abcam), rabbit anti-GFAP (1:500 for ICC, Wako, Japan), rabbit anti-Iba-1 (1:500 for ICC, Wako), rabbit anti-AKT (1:1000 for WB, Santa Cruz Biotechnology), rabbit anti-p-AKT (Ser473) (1:1000 for WB, Cell Signaling Technology), rabbit anti-STAT3 (1:1000 for WB, Cell Signaling Technology), rabbit anti-p- STAT3 (Ser727, 1:1000 for WB, 1:200 for ICC, Abcam), rabbit anti-P38 (1:1000 for WB, Cell Signaling Technology) and rabbit anti-p-P38 (1:1000 for WB, Cell Signaling) Technology) was used. In addition, TLR4 inhibitor (TAK-242, 500 nM; Calbiochem) and AKT inhibitor (MK2206, 10 mM; Selleckchem) were used as inhibitors. In addition, LPS was derived from E. coli O111:B4 and was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Western blotting

To determine whether regorafenib affects P38 and AKT signaling, BV2 microglia were treated with LPS (1 μg/ml) or PBS for 45 min, followed by regorafenib (5 μM) or vehicle (1% DMSO). ) was treated for 45 min. Cells were then lysed using RIPA buffer containing protease and phosphatase inhibitors (Roche, USA). Western blotting was performed by a conventional Western blot method, and images were analyzed using Fusion software or Image J software.

Immunocytochemistry

To determine the effect of regorafenib on dendritic spine formation, primary hippocampal culture was performed using embryonic day 18 Sprague-Dawley rat embryos. Primary hippocampal neurons were transfected with GFP plasmid DNA, treated with regorafenib (5 μM) or vehicle (1% DMSO) for 24 hours, followed by immunocytochemistry.

To determine whether regorafenib could alter LPS-mediated levels of nuclear p-STAT3, BV2 microglia were fixed with 4% paraformaldehyde for 10 min, washed 3 times with PBS, and then with GDB Using a buffer (0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate, pH 7.4, and 450 mM NaCl), it was reacted with anti-CD11b and anti-p-STAT3 antibodies overnight at 4°C.

In addition, the next day, cells were washed 3 times with PBS and reacted with the following secondary antibodies for 1 h at room temperature: Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit (1:200), molecules Probe, USA). Cells were then fixed in DAPI-containing solution (Vector Laboratories, CA, USA) and images were taken in a single plane using a confocal microscope (Nikon, Japan) and analyzed using ImageJ software. Samples were analyzed in a blinded manner with 6 to 10 individual images.

Cytosol and nuclear fractionation

To determine whether regorafenib affects the LPS-induced cytoplasmic and nuclear levels of p-STAT3, BV2 microglia were treated with LPS (1 μg/ml) or PBS for 30 min, followed by regorafenib. (5 μM) or vehicle (1% DMSO) for 5.5 hours. Cells were then lysed using buffer for cytoplasmic fraction (10 mM HEPES pH 8.0, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.1 % NP-40 and 0.5 mM PMSF). 5 minutes after the solution was added, the cell lysate was centrifuged at 10,000 rpm, 4° C. for 1 minute, and the supernatant was separated into a cytoplasmic fraction and stored. The pellet was dissolved in buffer for nuclear fractionation (10 mM HEPES pH 8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT and 0.5 mM PMSF) on ice for 15 min. Thereafter, centrifugation was performed at 10,000 rpm at 4 °C for 15 minutes, and Western blot analysis was performed using anti-STAT3 (Ser 727), PCNA and β-actin antibodies, and was analyzed using Fusion software.

Culture of mouse primary microglia and astrocytes

To determine whether regorafenib modulates the level of LPS-induced inflammatory cytokines in primary glia, a mouse mixed culture (a mixture of primary microglia and primary astrocytes) was prepared. Specifically, whole brains of postnatal 1-day-old C57BL/6 mice were minced and mechanically disrupted using a 70 μm nylon mesh. The mixed glial cells were aliquoted into 75 T culture flasks and grown in low-glucose DMEM medium supplemented with 10% FBS (including 100 unit/mL penicillin and 100 μg/mL streptomycin). Then, to obtain primary astrocytes from mice, the mixed glia were shaken at 250 rpm overnight. The next day, the conditioned medium was discarded, and the remaining cells were separated using trypsin-EDTA, followed by centrifugation (1200 rpm, 30 minutes). After centrifugation, the conditioned medium was removed, and cell pellets (primary astrocytes) were collected and analyzed for the effect of regorafenib on neuroinflammation in primary astrocytes.

To obtain mouse primary microglia, the mixed glia were subjected to mild trypsinization (5-fold dilution of 0.25% trypsin in serum-free DMEM) for 40 min in a 5% CO2 incubator at 37°C. After discarding all of the upper astrocyte layer, low-glucose DMEM containing 10% FBS was added, and the remaining primary microglia were used for the experiment.

wild type mice

All experiments were performed according to animal experiments and guidelines approved by the Korea Brain Research Institute (IACUC-2016-0013). C57BL6 / N mice were purchased from OrientBio Company, and male C57BL6 / N (8 weeks, 25-30 g) mice were bred in a pathogen-free facility at an ambient temperature of 22 °C under a light-dark cycle of 12 hours. Additionally, the data were analyzed in a semi-automated manner using Image J software, and the results were confirmed by an independent researcher not currently involved in the experiment.

Alzheimer's-induced mouse animal model production (5x FAD mice)

Male C57BL6/N (wild-type) mice were purchased from Orient-Bio Company (Gyeonggi Province, Korea), and F1 generation 5x FAD mice (stock # 34848-JAX, B6Cg-Tg APPSwFlLon, PSEN1 * M146L * L286V6799Vas/Mmjax) were obtained from Jackson Laboratory. purchased and used. 5x FAD mice (3 months old) were bred in a sterile facility under control of light and dark conditions for 12 hours per day at an ambient temperature of 22°C.

Immunohistochemistry

To determine the effect of regorafenib on LPS-induced neuroinflammation in vivo, wild-type mice were treated with regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween 80). ) was intraperitoneally administered every day for 3 days, and then LPS (Sigma, Escherichia coli, 10 mg/kg, ip) or PBS was continuously administered for 8 hours. After injection of LPS or PBS, mice were perfused and fixed with 4% paraformaldehyde (PFA) solution, then brain tissue was flash frozen and sectioned using a cryostat (35 μm thick). Each brain section was processed for immunohistochemical staining, and brain sections were rinsed with PBS and permeabilized for 1 hour at room temperature using PBS containing 0.2% Triton X-100 and 1% BSA. The next day, the tissue sections were washed three times with 0.5% BSA and reacted with biotin-conjugated anti-rabbit secondary antibody (1:400, Vector Laboratories) at room temperature for 1 hour. Then, the tissue sections were washed with 0.5% BSA, and reacted in an avidin-biotin complex solution (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature. After washing the sections with 0.1 M phosphate buffer (PB) 3 times, react the sections with 0.5 mg/ml 3,3'-diaminobenzidine (DAB, Sigma-Aldrich) in 0.1M PB containing 0.003% H2O2. signal was detected. Sections were also rinsed with 0.1 M PB and mounted on gelatin-coated slides, and then images were taken with a bright-field microscope (Leica).

Golgi staining

To determine the effect of regorafenib on dendritic spinogesis in vivo, Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neurotechnologies, Ellicott City, MD, USA). Specifically, animals injected with vehicle or regorafenib were immersed in solutions A and B, respectively, in the dark for 2 weeks, and then transferred to solution C and placed for 24 hours. Then, after 24 h, solution C was replaced, and the brains of each mouse were sliced at 150-μm thickness using a VT1000S Vibratome (Leica, Bannockburn, IL, USA). Dendritic images were taken with a bright field microscope using an Axioplan 2 (Zeiss, Oberkochen, Germany). Measurement of dendritic spine density was measured using 8-10 slices of each mouse brain of -1.70 mm to -2.30 mm for bregma. All data were analyzed using Image J software, and all experimental results were verified by independent researchers not currently participating in the experiment.

Statistical processing

All data were analyzed using two-tailed T-test or ANOVA using Graphpad Prism 6 software. Post-hoc analysis was performed by Tukey's Multiple Comparison test, and significance was set at p <0.05. Data are presented as mean ± S.E.M (* p <0.05, ** p <0.01, *** p <0.001).

<Example 1>

Analysis of whether regorafenib was cytotoxic to BV2 microglia and decreased levels of proinflammatory cytokines

To determine whether regorafenib is cytotoxic to BV2 microglia, BV2 microglia were treated with regorafenib by concentration (0.1, 1, 5, 10, 20uM), and then MTT analysis was performed. .

As a result of the analysis, as shown in FIG. 1A , it was found that regorafenib did not induce cytotoxicity against BV2 microglia.

In addition, in order to determine whether regorafenib affects the expression level of LPS-induced pro-inflammatory cytokines, the present inventors pretreated BV2 microglia with LPS (1 μg/ml) or PBS for 30 minutes, and After treatment with lafenib (5 μM) or vehicle (1% DMSO) for 5.5 hours, mRNA levels of inflammatory cytokines were analyzed.

As a result, as shown in FIGS. 1B to 1G, COX-2 and IL-1β were significantly inhibited by regorafenib treatment.

Also, unlike the above method, the present inventors pre-treated BV2 microglia with regorafenib (5 μM) or vehicle (1% DMSO) for 30 minutes first, and then LPS (1 μg / mL) for 5.5 hours after treatment , the levels of inflammatory cytokines were analyzed.

As a result, as shown in FIGS. 1H to 1M, when pretreatment with regorafenib, the mRNA levels of COX-2, IL-1β and IL-6 were significantly reduced.

Therefore, these results mean that regorafenib can inhibit the production of inflammatory cytokines by acting both before and after the inflammatory response is triggered.

To determine whether regorafenib also affects the anti-inflammatory response, the present inventors pre-treated BV2 microglia with LPS (1 μg/mL) or PBS for 30 min, and measured the mRNA levels of IL-4 and IL-10. measured.

As a result, as shown in Figures 2A to 2B, regorafenib was found to significantly increase IL-4 and IL-10 mRNA levels in LPS-treated BV2 microglia.

In addition, it was shown that IL-4 and IL-10 mRNA levels were significantly increased even when regorafenib was first pre-treated for 30 minutes and then LPS was treated for 5.5 hours ( FIGS. 2C-2D ).

Therefore, the present inventors found that regorafenib can effectively inhibit the expression and production of LPS-induced pro-inflammatory cytokines (COX-2, IL-1β and IL-6), as well as simultaneously anti-inflammatory cytokines (IL-4 and IL-10) has an activity to promote expression and production, and it was found that inflammatory diseases that may be caused by an excessive inflammatory reaction can be prevented, improved, or treated.

<Example 2>

Analysis of the effect of regorafenib on TLR4/AKT/STAT3 signaling

To determine whether regorafenib can modulate the LPS-induced inflammatory response, the effect of regorafenib on TLR4 signaling was analyzed. For this, BV2 microglia were pretreated with LPS (1 μg/ml) or PBS for 30 min, treated with TAK-242 (TLR4 inhibitor, 500 nM) or vehicle (1% DMSO) for 30 min, followed by regorafenib (5 μM) or vehicle (1% DMSO) for 5 hours. Thereafter, the level of inflammatory cytokines for each experimental group was analyzed.

As a result, as shown in FIG. 3 , treatment with regorafenib significantly reduced the mRNA levels of COX-2 and IL-1β, which are inflammatory cytokines induced by LPS, and combined treatment with TAK-242 and regorafenib It was also shown that LPS-induced COX-2 and IL-1β mRNA levels were significantly reduced.

On the other hand, when comparing the group treated with TAK-242, LPS and regorafenib with the group treated with both LPS and regorafenib, the difference in the mRNA level of LPS-induced IL-1β or COX-2 was not very large. appeared to be

Therefore, it was found that regorafenib inhibits the inflammatory response caused by LPS by participating in the TLR4 signaling pathway (FIG. 3).

In addition, the present inventors pre-treated BV2 microglia with LPS (1 μg/ml) or PBS for 45 min. Treatment with lafenib (5 μM) or vehicle (1% DMSO) for 45 min.

Subsequently, as a result of analyzing the phosphorylation level of AKT, it was found that the level of p-AKT induced by LPS was significantly reduced in BV2 microglia treated with regorafenib, whereas the total expression level of AKT was not affected. was not found (FIGS. 1N~1P).

Next, to determine whether regorafenib inhibits the level of LPS-mediated proinflammatory cytokines in an AKT-dependent manner, BV2 microglia were pretreated with LPS (1 μg/ml) or PBS for 30 min, and MK2206 (AKT Inhibitor, 500 nM) or vehicle (1% DMSO) for 30 minutes, followed by treatment with regorafenib (5 μM) or vehicle (1% DMSO) for 5 hours.

As a result of analyzing the level of pro-inflammatory cytokines thereafter, the LPS, MK2206, and regorafenib-treated group all treated with LPS and regorafenib compared to the MK2206 and LPS-treated group, IL-1β induced by LPS or to inhibit COX-2 mRNA levels (FIGS. 1Q-1S). These results suggest that regorafenib can suppress the level of LPS-induced pro-inflammatory cytokines by inhibiting AKT signaling.

In addition, to determine whether regorafenib can modulate p-STAT3 levels in the cytoplasm and nucleus, BV2 microglia were pretreated with LPS (1 μg/ml) or PBS for 30 min and regorafenib (5 μM) or vehicle (1% DMSO) for 5.5 hours. Thereafter, the cell lysate was fractionated into cytoplasmic and nuclear fractions, and the level of p-STAT3 was analyzed.

As a result, as shown in FIGS. 1T to 1W, it was found that p-STAT3 levels in the nucleus were significantly reduced by regorafenib, whereas there was little change in p-STAT3 levels in the cytosol. appear. In addition, as shown in Fig. 1X-1Y, as a result of immunocytochemical analysis with anti-p-STAT3 and anti-CD11b antibodies, regorafenib significantly reduced the p-STAT3 level in the nucleus in BV2 microglia. And it was found. In particular, activation of STAT3, that is, phosphorylation of STAT3 (p-STAT3), is known to play a role in inducing the expression of factors related to inflammatory responses in the nucleus. Ultimately, excessive inflammatory response can be suppressed.

<Example 3>

Analysis of the inhibitory effect of pro-inflammatory cytokines by LPS following regorafenib treatment in microglia and astrocytes

To analyze whether regorafenib is associated with LPS-induced neuroinflammation in primary microglia, primary microglia were cultured under low glucose conditions, and then primary microglia were incubated with LPS (1 μg/ml) or PBS, and then treated with regorafenib (5 μM) or vehicle (1% DMSO) for 5.5 hours, followed by real-time PCR to measure the level of proinflammatory cytokines.

As a result, as shown in FIGS. 4A to 4E , it was found that the level of LPS-induced pro-inflammatory cytokines was significantly reduced in the primary microblasts treated with regorafenib, and the level of p-AKT by LPS was found to be significantly reduced. It was also found that the level was significantly suppressed. On the other hand, it was found that there was no effect on the level of p-P38 (FIGS. 4F-4K).

In addition, the present inventors confirmed whether regorafenib can regulate the neuroinflammatory response caused by LPS in primary astrocytes. As a result, COX, an LPS-mediated inflammatory cytokine, also significantly in primary astrocytes treated with regorafenib. -2, IL-1β, IL-6, TNF-a and iNOS mRNA levels were found to be reduced ( FIGS. 4L-4P ). Furthermore, LPS-mediated phosphorylation levels of p-AKT and p-P38 were down-regulated in regorafenib-treated primary astrocytes, whereas the expression levels of total AKT and P38 showed little change (Fig. 4Q~4V).

These results mean that regorafenib can effectively inhibit the neuroinflammatory response caused by LPS in both microglia and astrocytes.

<Example 4>

Analysis of the effect of regorafenib on LPS-induced microglia/astrocyte activation and proinflammatory cytokine levels in wild-type mice

We analyzed whether regorafenib can regulate LPS-induced neuroinflammation in vivo. For this, wild-type mice were injected daily with regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween 80) for 3 days, followed by LPS (10 mg/kg, ip). Alternatively, PBS was treated for 8 hours. Thereafter, immunohistochemical staining was performed using anti-Iba-1 and anti-GFAP antibodies.

As a result, as shown in FIGS. 5A-5E , in the regorafenib-treated group, the activity of LPS-induced microglia in the hippocampus tissue was significantly reduced, whereas in the cortex, the microglia It was found that the effect of reducing the activity of glial cells was insignificant. In addition, in CA1 of hippocampus administered with regorafenib, LPS-mediated astrocyte activity was also decreased ( FIGS. 5F-5J ).

Furthermore, in order to determine whether regorafenib affects the level of pro-inflammatory cytokines by LPS, the present inventors tested regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween 80) was injected daily for 3 days, and then LPS (10 mg/kg, ip) or PBS was administered for 8 hours, followed by immunohistochemistry using an anti-COX-2 antibody.

As a result, as shown in Figures 5K-5O, it was found that the level of COX-2, a pro-inflammatory cytokine, by LPS was significantly reduced in both cortex and hippocampal tissues of wild-type mice to which regorafenib was administered.

In addition, we analyzed the effect of regorafenib on Aβ-mediated neuroinflammation. For this purpose, regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18) in 3 month old 5x FAD mice. % PEG + 5% Tween 80) were injected daily for 2 weeks, and immunohistochemistry was performed using anti-Iba-1 and anti-GFAP antibodies.

As a result, it was found that 5x FAD mice administered with regorafenib did not affect Aβ-mediated activation of microglia ( FIGS. 6A-6E ), and astrocytes in the cortex of 5x FAD mice administered with regorafenib It was found that the activation of cells was significantly reduced ( FIGS. 6F-6J ).

Therefore, through these results, the present inventors found that regorafenib can inhibit the activation of microglia and astrocytes by LPS even in neurons, and can inhibit the generation and expression of pro-inflammatory cytokines, It was confirmed that the inflammatory reaction can also be effectively suppressed.

<Example 5>

Analysis of increase in dendritic spine density by regorafenib in primary hippocampal neurons and 5x FAD mice

To determine whether regorafenib affects the formation of dendritic spines (associated with learning and memory), primary hippocampal neurons (neurons) were transfected with GFP (visualizing dendrite segments and spines). and treated with vehicle (1% DMSO) or regorafenib (5 μM) for 24 hours, and then the number of dendrites was measured.

As a result, as shown in FIGS. 7A-7B , it was confirmed that the number of dendrites was increased in the primary hippocampal neurons treated with regorafenib compared to the control group (vehicle treatment).

In addition, in order to determine whether regorafenib affects dendrite formation even in an Alzheimer's disease-induced mouse model, the present inventors studied regorafenib (30 mg/kg, ip) in 3-month-old 5x FAD mice (Alzheimer's induced mice). ) or vehicle (2% DMSO + 18% PEG + 5% Tween80, ip) was injected daily for 2 weeks, followed by Golgi staining.

As a result, interestingly, 5x FAD mice injected with regorafenib showed a significant increase in the number of dendrites in the hippocampus. On the other hand, there was no significant change in the cortex (FIGS. 7C-7H).

Therefore, it was found that regorafenib of the present invention can be used as a therapeutic agent for neurodegenerative diseases because it has an activity to increase the number of dendrites in normal brain tissue and Alzheimer's disease brain tissue.

<Example 6>

Analysis of Aβ plaque reduction effect by regorafenib in Alzheimer's disease mouse model

To analyze whether regorafenib influences the pathogenesis of amyloid plaques (Aβ), regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween 80, ip) was injected daily for 2 weeks, and then immunohistochemistry was performed using anti-4G8 antibody.

As a result, the group treated with regorafenib significantly reduced the level of amyloid plaques (Aβ) in the cortex and hippocampus compared to the control group ( FIGS. 8A-8E ).

In addition, to determine whether regorafenib affects the APP process to change the level of Aβ plaques, APP-H4 cells were treated with vehicle or regorafenib for 24 h, and sAPPβ, full-length APP (FL-APP) and APP CTF were measured.

As a result, regorafenib-treated APP-H4 cells showed decreased sAPPβ compared to the control group, and both FL-APP and APP CTF were also decreased ( FIGS. 8F-8I ).

Furthermore, the present inventors analyzed whether regorafenib affects the level of Aβ degrading enzyme in order to confirm the signaling mechanism regorafenib regulates the Aβ plaque level. For this, 3-month-old 5x FAD mice were injected daily with regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween80, ip) for 2 weeks, followed by the use of anti-NEP antibody. Thus, immunohistochemistry was performed.

As a result, it was found that regorafenib had no effect on NEP levels in Alzheimer's disease-induced 5x FAD mice (FIGS. 9a-9e).

In addition, the present inventors analyzed the effect of regorafenib on the levels of α-secretase ADAM17 and β-secretase BACE1. As a result, in the hippocampus of 5x FAD mice to which regorafenib was administered, BACE1 levels were decreased, but there was no change in the cortex. appeared (Figs. 8j-8n). In addition, ADAM17 levels in 5x FAD mice administered with regorafenib did not appear to change ( FIGS. 10A-10E ). In addition, as a result of investigating whether regorafenib regulates autophagy protein in the effect of Aβ pathogenesis, it was found that ATG12 levels were significantly decreased in the hippocampus of 5x FAD mice administered with regorafenib, whereas There was no significant decrease in ATG5 (FIGS. 8o-8s, 11a-11e).

<Example 7>

Analysis of the Reduction Effect of Regorafenib on AT100 on Tau Phosphorylation in Alzheimer's Disease Mouse Model

To determine the effect of regorafenib on tau phosphorylation, regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween) in Alzheimer's disease-induced 3-month-old 5x FAD mice. 80) was injected daily for 2 weeks, and immunohistochemistry was performed using anti-AT8, anti-AT100 or anti-AT180 antibody.

As a result, it was shown that total tau levels and phosphorylation of tau in AT100 were inhibited in 5x FAD mice injected with regorafenib, whereas inhibition of tau phosphorylation did not occur in AT8 and AT180 (FIGS. 12a-12e) , FIGS. 13 and 14).

Therefore, through these results, the present inventors found that regorafenib can prevent or treat neurodegenerative diseases such as Alzheimer's disease, which can be caused by neuroinflammation through inhibition of phosphorylation of tau.

<Example 8>

Analysis of the inhibitory effect of regorafenib on the tau kinase p-GSK3β

The present inventors investigated whether regorafenib could increase or decrease tau kinase in changes in tau phosphorylation. For this purpose, 3-month-old 5x FAD mice were injected daily with regorafenib (30 mg/kg, ip) or vehicle (2% DMSO + 18% PEG + 5% Tween 80) for 2 weeks, anti-p-GSK3β, Immunohistochemical analysis was performed using anti-p-CDK5 or anti-DYRK1A antibodies.

As a result, the level of p-GSK3β was decreased in the cortex and hippocampus of 5x FAD mice administered with regorafenib ( FIGS. 12f-12j ). In addition, in 5x FAD mice administered with regorafenib, p-CDK5 and DYRK1A levels were decreased in the cortex but not the hippocampus ( FIGS. 12k-12t ).

Therefore, through these results, the present inventors found that regorafenib can inhibit tau phosphorylation by regulating p-GSK3β kinase, a protein that phosphorylates tau.

Through the above results, the present inventors found that regorafenib can effectively inhibit the expression and generation of LPS-induced proinflammatory cytokines, so that regorafenib can be used as a new therapeutic agent for inflammatory diseases. Since lafenib can inhibit LPS-induced activation of microglia and astrocytes for nerve cells, and at the same time has all of amyloid plaque suppression, dendrite increase, and phosphorylation inhibitory activity of tau protein, regorafenib can be used for degenerative nerve cells. It was found that it can be used as a treatment for diseases.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising regorafenib or a pharmaceutically acceptable salt thereof as an active ingredient. According to claim 1,
The composition is a pharmaceutical composition for the prevention or treatment of inflammatory diseases, characterized in that it inhibits the expression and production of inflammatory cytokines COX-2, IL-1β, IL-6, TNF-a or iNOS. According to claim 1,
The composition inhibits the activity of TLR4 or AKT, characterized in that it suppresses the level of p-STAT3, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. According to claim 1,
The composition is
inhibiting the activity of microglia or astrocytes; amyloid plaque inhibition; increased dendrites; Or a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it inhibits phosphorylation of tau protein. According to claim 1,
The inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, ankylosing spondylitis, lupus , fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, cancer and neurodegenerative diseases, characterized in that selected from the group consisting of, the prevention or treatment of inflammatory diseases pharmaceutical composition . 6. The method of claim 5,
The degenerative neurological disease is Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine nucleus-pondy-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatal-nigular degeneration, Huntington's disease, amyotrophic lateral sclerosis ( ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction, characterized in that selected from the group consisting of, prevention of inflammatory diseases or a therapeutic pharmaceutical composition. 7. The method of claim 6,
The neurodegenerative disease is a pharmaceutical composition for preventing or treating an inflammatory disease, characterized in that it is caused by an excessive inflammatory reaction in nerve cells. A health functional food for the prevention or improvement of inflammatory diseases containing regorafenib as an active ingredient. 9. The method of claim 8,
The inflammatory disease is dermatitis, atopic dermatitis, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, iritis, sore throat, tonsillitis, pneumonia, gastric ulcer, pancreatitis, gastritis, Crohn's disease, inflammatory bowel disease, colitis, hemorrhoids, gout, Prevention or improvement of inflammatory diseases, characterized in that selected from the group consisting of ankylosing spondylitis, lupus, fibromyalgia, psoriasis, rheumatoid arthritis, osteoarthritis, osteoporosis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, cancer and neurodegenerative diseases Health functional food for use. 10. The method of claim 9,
The degenerative neurological disease is Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivine nucleus-pondy-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatal-nigular degeneration, Huntington's disease, amyotrophic lateral sclerosis ( ALS), essential tremor, cortical-basal nucleus degeneration, diffuse Lewy body disease, Parkinson's-ALS-dementia complex, Niemann-Pick's disease, Pick's disease, cerebral ischemia and cerebral infarction, characterized in that selected from the group consisting of, prevention of inflammatory diseases Or health functional food for improvement. A method of inhibiting inflammatory activity of cells in vitro, comprising the step of treating inflammatory activity-induced cells with regorafenib or a salt thereof. 12. The method of claim 11,
The inflammatory activity is characterized in that induced by LPS (Lipopolysaccharide) treatment, in vitro (in vitro) cell inflammatory activity inhibition method.

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