WO2024014620A1 - Composition for inflammation inhibition and immunoregulation of degenerative brain diseases, comprising syk inhibitor as active ingredient - Google Patents
Composition for inflammation inhibition and immunoregulation of degenerative brain diseases, comprising syk inhibitor as active ingredient Download PDFInfo
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- WO2024014620A1 WO2024014620A1 PCT/KR2022/014986 KR2022014986W WO2024014620A1 WO 2024014620 A1 WO2024014620 A1 WO 2024014620A1 KR 2022014986 W KR2022014986 W KR 2022014986W WO 2024014620 A1 WO2024014620 A1 WO 2024014620A1
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- syk
- degenerative brain
- lps
- inhibitor
- active ingredient
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Definitions
- the present invention relates to a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- Common pathological features found in the brains of patients with degenerative brain diseases include death of nerve cells and abnormal aggregation of specific proteins.
- Abnormal aggregation of proteins such as amyloid ⁇ , senile plaques, neurofibrillary tangles, ⁇ -synuclein, or Lewy bodies is itself a phenomenon in nerve cells.
- By inducing death or activating microglial cells to cause an inflammatory response it can cause Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, and olivopontocerebellar atrophy.
- OPCA Shy-Drager syndrome
- striatonigral degeneration Huntington's disease
- amyotrophic lateral sclerosis ALS
- essential tremor Essential tremor, cortico-basal ganlionic degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, Pick's disease It is known to play an important role in the development of degenerative brain diseases such as
- Syk spleen tyrosine kinase
- BAY61-3606 a selective Syk inhibitor, is effective in suppressing various inflammation-mediated pathological models, including sepsis and acute kidney injury, through regulating macrophage activation.
- Korean Patent No. 1754451 discloses a composition for preventing and treating degenerative brain diseases containing a PCAF inhibitor (P300/CBP-associated factor's inhibitor) as an active ingredient
- Korean Patent No. 1723514 discloses a pharmaceutical composition for preventing or treating Alzheimer's disease and degenerative brain diseases containing a SNO-OGT inhibitor, but it is also disclosed in a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing the Syk inhibitor of the present invention as an active ingredient.
- a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing the Syk inhibitor of the present invention as an active ingredient.
- the present invention was developed in response to the above-mentioned needs, and provides a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient, and comprising the Syk inhibitor, specifically BAY61-3606 ( Hereinafter referred to in parallel with BAY61), Syk, Iba-1, inflammatory cytokines (TNF- ⁇ , IL-1 ⁇ , IL-6) and inflammation-related factors (NF- ⁇ B, Cox-2, iNOS) upregulated by LPS.
- Syk spleen tyrosine kinase
- the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the present invention provides a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the present invention provides a method for preventing or treating degenerative brain diseases, comprising administering to an animal other than a human a composition containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the present invention provides a veterinary composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the present invention relates to a composition for suppressing inflammation and regulating immunity in degenerative brain diseases comprising a Syk inhibitor as an active ingredient, wherein the Syk inhibitor, specifically BAY61-3606, inhibits Syk, Iba-1, and inflammatory cytokines upregulated by LPS.
- a Syk inhibitor specifically BAY61-3606, inhibits Syk, Iba-1, and inflammatory cytokines upregulated by LPS.
- Figure 1 is a schematic diagram of the animal testing schedule of the present invention.
- Figure 2 shows the results of confirming the effect of Syk on the expression of microglial activation factor (Iba-1) and inflammation mediating factor (TNF ⁇ ) in an animal model in which neuroinflammation was induced by LPS.
- Iba-1 microglial activation factor
- TNF ⁇ inflammation mediating factor
- BAY61 (BAY61-3606) is a Syk inhibitor. *, **, *** mean there is a statistically significant difference from each other, * is p ⁇ 0.05, ** is p ⁇ 0.005, *** is p ⁇ 0.0001.
- Figure 3 shows the results confirming the effect of alleviating inflammation by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS.
- A) and (C) are the results of confirming and quantifying the degree of phosphorylation of NF- ⁇ B and changes in protein expression of iNOS and COX-2 in the cortex and hippocampus by Western blot
- B) and (D) is an immunofluorescence image confirming changes in protein expression of iNOS and COX-2 in the cortex and hippocampus and the results of their quantification (scale bars, 100 ⁇ m).
- Figure 4 shows the level of microglial activation factor (Iba-1) and inflammation mediating factors (TNF ⁇ , IL-1 ⁇ , IL-6) by administration of Syk inhibitor (BAY61) in BV2 microglial cells inducing inflammation with LPS. This is the result of confirming the expression change.
- A is the result of confirming and quantifying the protein expression levels of Syk, Iba-1, TNF ⁇ , and IL-1 ⁇ by Western blot
- (B) is the result of confirming the mRNA expression of TNF ⁇ , IL-1 ⁇ , and IL-6.
- Figure 5 shows the results of confirming changes in iNOS and COX-2 protein expression by administration of a Syk inhibitor (BAY61) in BV2 microglial cells inducing inflammation with LPS (A), the results of confirming changes in mRNA expression (B), and nitrite production. This is the result of confirming the degree (C).
- Figure 6 shows the results confirming the effect of alleviating neuronal cell death by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS.
- A is a Nissl staining image (scale bars, 50 ⁇ m) confirming neuron density in the cortex and hippocampus areas [Cornu ammonis (CA)1, CA3, and dentate gyrus (DG)], and
- B is a Nissl staining image (scale bars, 50 ⁇ m).
- (Nissl) This is the result of quantifying positive neurons.
- C is an image confirming the protein expression levels of cytochrome C and Bim in the cortex and hippocampus by Western blot, and
- D is the result of quantification.
- Figure 7 shows the results confirming the effect of Syk inhibitor (BAY61) on alleviating neurotoxin-mediated neurodegeneration.
- A is a diagram showing the experimental plan for microglial conditioned medium and the results of confirming the cytotoxicity of microglial conditioned medium in the hippocampal neuron cell line HT22
- B is the conditioned medium for LPS-treated microglial cells ( LCM);
- LCM LPS-treated microglial cells
- FIG. C shows the experimental plan of the transwell-based co-culture system of microglial cells (BV2) and hippocampal neuron cell line (HT22) and the results of measuring nitrite in the transwell-permeable support containing BV2 and HT22 medium.
- D shows the results of confirming and quantifying the protein expression levels of cytochrome C and Bim in HT22 cultured by a transwell-based co-culture system by Western blot.
- *, **, ***, **** mean there is a statistically significant difference from each other, * is p ⁇ 0.05, ** is p ⁇ 0.005, *** is p ⁇ 0.0001, **** is p ⁇ 0.00001. ## means there is a statistically significant difference, p ⁇ 0.005.
- Figure 8 shows SNAP25 (synaptosome-associated protein, 25kDa), Syp (synaptophysin), and PSD95 (postsynaptic) by administration of Syk inhibitor (BAY61) in the cortex (A) and hippocampus (B) of an animal model in which neuroinflammation was induced by LPS. This is the result of confirming and quantifying changes in protein expression of density protein-95) by Western blot.
- Figure 9 is an immunofluorescence image (scale bars, 25 ⁇ m) confirming changes in PSD95 protein expression by administration of a Syk inhibitor (BAY61) in the cortex (A) and hippocampus (B) of an animal model in which neuroinflammation was induced by LPS. This is a quantitative result. **, *** mean that there is a statistically significant difference from each other, ** is p ⁇ 0.005, *** is p ⁇ 0.0001.
- Figure 10 shows the results of the Y-maze test confirming the effect of short-term spatial working memory recovery by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS.
- A is the total number of entries into the Y-maze passage
- B is the result of spontaneous alteration measured using an automatic video tracking system.
- *, ** mean that there is a statistically significant difference from each other, * is p ⁇ 0.05, ** is p ⁇ 0.005.
- Figure 11 shows the results of confirming the learning memory recovery effect by administration of Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS using a passive avoidance test.
- the test results were expressed as entry latency when passing from a bright room to a dark room. **** means there is a statistically significant difference from each other, and **** is p ⁇ 0.00001.
- the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the Syk inhibitor may be any substance capable of inhibiting the expression or action of Syk, and is preferably a compound of the following formula (BAY61-3606), but is not limited thereto.
- the degenerative brain disease may be any one selected from Parkinson's disease, Huntington disease, and amyotrophic lateral sclerosis, and is preferably Parkinson's disease, but is not limited thereto.
- the degenerative brain disease is characterized by neuroinflammation or apoptosis.
- the pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.
- pharmaceutically acceptable saline solution sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia Rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals.
- It may further contain one or more carriers selected from oils, and in addition to the above active ingredients, pharmaceutically acceptable antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, and suspending agents. And it may further contain one or more auxiliaries selected from among preservatives.
- the pharmaceutical composition can be administered in an oral or parenteral dosage form according to a conventional method, and when formulated, 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 tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
- Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups.
- suppositories Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
- non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
- injectable ester such as ethyl oleate.
- suppositories witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.
- the present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the health functional food composition may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
- the health functional food composition of the present invention When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods.
- the amount of active ingredient can be appropriately used depending on the purpose of use (prevention or improvement).
- the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total raw materials.
- the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
- Health functional foods There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.
- the health functional food composition of the present invention can be manufactured into food, especially functional food.
- the functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings.
- ingredients commonly added during food production includes, for example, proteins, carbohydrates, fats, nutrients and seasonings.
- natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient.
- the natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., For example, xylitol, sorbitol, erythritol, etc.) is preferable.
- the flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
- the ratio of the ingredients added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
- the present invention provides a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the feed additive of the present invention corresponds to supplementary feed under the Feed Management Act.
- the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals.
- the type of feed is not particularly limited, and feed commonly used in the art can be used.
- Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products;
- Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or food. These may be used alone or in combination of two or more types.
- the present invention provides a method for preventing or treating degenerative brain diseases, comprising administering to an animal other than a human a composition containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the Syk inhibitor may be any substance capable of inhibiting the expression or action of Syk, and is preferably a compound of the following formula (1), but is not limited thereto.
- the present invention provides a veterinary composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
- the veterinary composition containing the active ingredient of the present invention may further include appropriate excipients and diluents according to conventional methods.
- Excipients and diluents that may be included in the veterinary composition containing the active ingredient of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium.
- Phosphate calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, Examples include sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.
- the veterinary composition containing the active ingredient according to the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc.
- the veterinary composition according to the present invention is active after being administered to animals.
- the veterinary composition according to the present invention may contain the Syk (spleen tyrosine kinase) inhibitor in an effective amount or as an active ingredient, depending on its intended use.
- the effective amount can be appropriately selected depending on the individual animal. Factors including the severity of the disease or condition, the individual's age, weight, health, gender, the individual's sensitivity to the active ingredient, administration time, administration route and excretion rate, administration period, other compositions combined or used simultaneously with the composition, and It may be determined according to factors well known in the fields of other physiology or veterinary medicine.
- mice 7-week-old male C57BL/6 mice were purchased from Samtaco Bio (Osan, Korea). Mice were acclimatized for one week at room temperature (23 ⁇ 2°C) with a 12-hour night/day cycle and with free access to food and water.
- the experimental groups were divided as described in Table 1 below, and LPS (L2630, Sigma Aldrich, USA) and BAY61-3606 (11423, Cayman, USA) were treated by intraperitoneal injection.
- the overall schematic diagram of the experimental schedule is as disclosed in Figure 1.
- the Y-maze test measurement equipment consists of three branches. The experimental animal was placed at the end of one arm of the equipment and allowed to move freely around the maze for 8 minutes. Afterwards, the maze was disinfected with 70% (v/v) ethanol to avoid orientation bias. The total number of entries into the Y-maze passage, spontaneous alteration, and average zone speed were recorded using SMART V3.0 (Harvard apparatus, USA).
- the manual avoidance test was conducted using a manual avoidance box.
- the passive avoidance box is divided into a dark room and a bright room, and the habit of the experimental animal moving to the dark room when placed in the bright room was taken advantage of.
- subjects were allowed to explore freely in a bright room while the dark room was closed for 3 minutes.
- the mouse was placed in a bright room, and after 30 seconds, the door to the dark room was opened and the mouse moved to the dark room.
- the door was closed and an electric shock derived from the grid floor was delivered. (0.2mA, 2 seconds).
- the mouse was placed back in the bright room under the same conditions as day 2 except that an electric shock was applied, and the entry latency when passed through the dark room with a cutoff time of 5 minutes. was measured.
- mice were perfused transcardially with cold PBS and 4% (v/v) formaldehyde, and sequentially post-fixed with 4% (v/v) formaldehyde and 20% (w/v) sucrose for 24 hours. .
- the brains were then fixed in optimal cutting temperature compound (OCT) composite (Sakura, Torrance, CA, USA) and frozen in liquid nitrogen.
- OCT optimal cutting temperature compound
- frozen brain pieces were cut into 20 ⁇ m sections, mounted on slides, stored at -70°C, and incubated overnight at room temperature before each use before staining and immunofluorescence.
- Citrate buffer-based heat-induced antigen retrieval was performed by incubating brain hippocampal slices at 90-100°C for 20 minutes. After drying for 10 minutes, the slides were washed with PBS (PBST) containing 0.1% (v/v) Tween 20, then incubated with 5% (v/v) normal goat serum and 0.1% (v/v) TritonX- It was stored in PBST containing 100% for 1 hour. After 1 hour, the slides were reacted with primary antibodies at 4°C overnight, and the next day, exposed to room temperature for 1 hour, washed with PBST, and then incubated with goat-anti-mouse conjugated with Alexa fluor 488 or conjugated with Alexa fluor 594.
- BV2 a microglial cell line, was cultured in DMEM medium containing 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin.
- HT22 a mouse hippocampal neuronal cell line, was cultured in DMEM medium containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.
- BV2 cells were inoculated into a culture dish at 2.0 ⁇ 10 5 cells per 1 mL, and when the cells were 70% full, 100 ng/mL of LPS was treated. RNA was extracted 6 hours after LPS treatment, and protein was extracted 24 hours after LPS treatment.
- LPS was treated at 100ng/mL
- BAY61-3606 was treated at 10nM or 100nM.
- Grease reagent was used according to the manufacturer's protocol.
- the conditioned medium of microglia with or without BAY61-3606 and LPS treatment was treated with HT22 for 24 hours and then subjected to MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] analysis. Cell viability was then measured.
- BV2 cells were inoculated into transwells (0.4 ⁇ m pores), HT22 was inoculated into 6-well plates, and BV2 was treated with LPS (100ng/ml) and treated with or without BAY61-3606 (10nM or 100nM). was cultured for 24 hours. Afterwards, the medium from the transwell and 6-well plate was collected to measure nitrite, and HT22 was collected to perform Western blotting to evaluate neuronal death.
- Bim (#2933, 1:3,000) and Syk (#13198, 1:3,000) were purchased from Cell Signaling (Cell Signaling Technology, USA).
- iba-1 (016-20001, 1:1,000) was purchased from Wako (WAKO, Japan).
- ⁇ -actin (sc-47778, 1:10,000), cytochrome C (sc-13156, 1:3,000), SNAP25 (sc-20038, 1:3,000), SYP (sc-17750, 1:3,000), PSD95 (sc-17750, 1:3,000) -71933, 1:3,000), p-NF-kB (sc-136548, 1:1,000), and TNF ⁇ (sc-52746, 1:1,000) were purchased from Santa Cruz (Santa Cruz, USA).
- iNOS (610432, 1:3,000) was purchased from BD Lifescience (BD lifescience, USA).
- COX-2 160126, 1:3,000 was purchased from Simon (Cayman, USA).
- qPCR quantitative polymerase chain reaction
- Example 1 In an animal model in which neuroinflammation was induced by LPS, changes in microglial activity and expression of inflammatory mediators were confirmed by Syk inhibitor administration.
- BAY61 a Syk inhibitor, reduced the degree of phosphorylation of NF- ⁇ B and protein expression of iNOS and CPX-2, which were increased by LPS in the cortex and hippocampus.
- BAY61 a Syk inhibitor
- LPS lipoprotein
- Example 2 Confirmation of changes in microglial activity and expression of inflammatory mediators by administration of Syk inhibitor in BV2 microglial cells induced inflammation with LPS
- BAY61 effectively reduced the iNOS and COX-2 protein and mRNA expressions increased by LPS, and also significantly suppressed the production of nitrite, as shown in Figure 5C.
- Syk is an inflammation regulator of microglial cells.
- Example 3 Confirmation of the effect of alleviating neuronal cell death by administration of a Syk inhibitor in an animal model in which neuroinflammation was induced by LPS
- conditioned medium (LCM) of LPS-treated microglial cells induced HT22 cell death, and when treated with conditioned medium treated with BAY61 [(L+BAY61)CM], apoptosis of HT22 occurred.
- the severity has eased.
- cytochrome C and Bim which are cell death-related factors
- BV2 cells were inoculated onto the transwell-permeable support and stimulated with LPS and BAY61, and then the transplanted support was placed on a plate containing HT22.
- the nitrite concentration of BV2 cells decreased upon LPS stimulation. It increased and then decreased upon BAY61 treatment, but no effect of reducing nitrite concentration was observed in wells containing HT22, and cytochrome C and Bim increased in LPS-treated BV2 cells and transwell-cultured HT22 cells, as shown in Figure 7D. It was confirmed that the expression of was decreased in BV2 cells treated with LPS and BAY61 and in HT22 cells cultured in transwells.
- Example 5 In an animal model in which neuroinflammation was induced by LPS, the effect of Syk inhibitor administration on recovering synaptic dysfunction, cognitive and spatial working memory impairment was confirmed.
- the Y-maze test is an experiment that measures short-term memory and can evaluate the ability to act sequentially.
- the total number of entries into each arm of the Y-maze was increased in mice with neuroinflammation induced by LPS, but recovered to the normal group level when administered with BAY61, and the automatic video tracking system
- the reduced spontaneous alteration behavior ratio of alternation and exploration
- BAY61 can restore spatial working memory damage that occurred in mice with neuroinflammation induced by LPS.
- mice in which neuroinflammation was induced by LPS had a decreased time spent in the bright room compared to the normal group, even though they had been exposed to an electric shock the day before, and mice administered together with BAY61 showed similar activity to the normal group. I had time to stay in a bright room.
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Abstract
The present invention relates to a composition for inflammation inhibition and immunoregulation of degenerative brain diseases, comprising an Syk inhibitor as an active ingredient. The Syk inhibitor, specifically, BAY61-3606, has the effects of: significantly reducing the expression of Syk, Iba-1, inflammatory cytokines (TNF-α, IL-1α and IL-6) and inflammation-related factors (NF-κB, Cox-2 and iNOS), all of which are up-regulated by LPS; significantly inhibiting microglia-mediated neuronal cell death by regulating the expression of cytochrome C and BCL-2 interacting mediator of cell death (Bim) in LPS-treated mouse brains and HT22 cells; restoring the expression of synaptic markers of synaptosomal-associated protein, 25 kDa (SNAP25), synaptophysin (Syp), and postsynaptic density protein-95 (PSD95) in LPS-treated mouse brains and HT22 cells; and inhibiting cognitive and memory dysfunctions caused by LPS in Y-maze tests and passive avoidance tests, which are related to memory and cognitive dysfunctions, and thus the composition of the present invention, comprising an Syk inhibitor as an active ingredient, can be effectively used for the prevention, alleviation or treatment of degenerative brain diseases.
Description
본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 염증 억제 및 면역 조절용 조성물에 관한 것이다. The present invention relates to a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
퇴행성 뇌질환 환자의 뇌에서 공통으로 발견되는 병리학적 특징으로는 신경세포의 사멸과 특정단백질의 비정상적 응집이 있다. 아밀로이드 베타(amyloid β), 노인반(senile plaque), 신경섬유농축제(neurofibrillary tangle), 알파 시뉴클린(α-synuclein) 또는 루이소체(lewy body) 등의 단백질의 비정상적 응집이 그 자체로 신경세포의 사멸을 유도하거나, 미세아교세포를 활성화해 염증반응을 일으킴으로써 파킨슨병(Parkinson's disease), 진행성 핵상마비(Progressive supranuclear palsy), 다계통 위축증(Multiple system strophy), 감람핵-뇌교-소뇌 위축증(Olivopontocerebellar atrophy; OPCA), 샤이-드래거 증후군(Shy-Drager syndrome), 선조체-흑질 퇴행증 (Striatonigral degeneration), 헌팅톤병(Huntington's disease), 근위축성 측색 경화증(Amyotrophic lateral sclerosis; ALS), 본태성 진전증(Essential tremor), 피질-기저핵 퇴행증 (Cortico-basal ganlionic degeneration), 미만성 루이 소체 질환(Diffuse Lewy body disease), 파킨스-ALS-치매 복합증(Parkinson-ALS-dementia complex of Guam), 픽병(Pick's disease) 등의 퇴행성 뇌질환의 발병에 중요한 역할을 하는 것으로 알려져 있다.Common pathological features found in the brains of patients with degenerative brain diseases include death of nerve cells and abnormal aggregation of specific proteins. Abnormal aggregation of proteins such as amyloid β, senile plaques, neurofibrillary tangles, α-synuclein, or Lewy bodies is itself a phenomenon in nerve cells. By inducing death or activating microglial cells to cause an inflammatory response, it can cause Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, and olivopontocerebellar atrophy. atrophy; OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor ( Essential tremor, cortico-basal ganlionic degeneration, diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, Pick's disease It is known to play an important role in the development of degenerative brain diseases such as
한편, Syk(spleen tyrosine kinase)는 다양한 신호 전달의 중요한 조절자이며, 대식세포의 적응 면역 및 식세포 활동에 관여하는 것으로 알려져 있다. 그 중 선택적 Syk 억제제인 BAY61-3606은 대식세포 활성화 조절을 통해 패혈증 및 급성 신장 손상을 포함한 다양한 염증 매개 병리학적 모델을 억제하는 효과가 있다. Meanwhile, Syk (spleen tyrosine kinase) is an important regulator of various signal transduction and is known to be involved in adaptive immunity and phagocytic activity of macrophages. Among them, BAY61-3606, a selective Syk inhibitor, is effective in suppressing various inflammation-mediated pathological models, including sepsis and acute kidney injury, through regulating macrophage activation.
퇴행성 뇌질환 관련 선행기술로는 한국등록특허 제1754451호에 PCAF 저해제(P300/CBP-associated factor's inhibitor)를 유효성분으로 함유하는 퇴행성 뇌질환의 예방 및 치료용 조성물이 개시되어 있고, 한국등록특허 제1723514호에는 SNO-OGT 억제제를 포함하는 알츠하이머병, 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물이 개시되어 있지만, 본 발명의 Syk 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 염증 억제 및 면역 조절용 조성물에 관해 전혀 개시된 바 없다. As prior art related to degenerative brain diseases, Korean Patent No. 1754451 discloses a composition for preventing and treating degenerative brain diseases containing a PCAF inhibitor (P300/CBP-associated factor's inhibitor) as an active ingredient, and Korean Patent No. No. 1723514 discloses a pharmaceutical composition for preventing or treating Alzheimer's disease and degenerative brain diseases containing a SNO-OGT inhibitor, but it is also disclosed in a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing the Syk inhibitor of the present invention as an active ingredient. Nothing has been disclosed about it at all.
본 발명은 상기와 같은 요구에 의해 도출된 것으로, Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 염증 억제 및 면역 조절용 조성물을 제공하고, 상기 Syk 저해제, 구체적으로 BAY61-3606(이하, BAY61 병행 표기)은 LPS에 의해 상향 조절된 Syk, Iba-1, 염증성 사이토카인(TNF-α, IL-1α, IL-6) 및 염증관련 인자(NF-κB, Cox-2, iNOS)의 발현을 유의미하게 감소시키고, LPS 처리된 마우스 뇌와 HT22 세포에서 시토크롬 C와 Bim(BCL-2 상호작용 세포사 매개체)의 발현을 조절함으로써 미세아교-매개 신경세포 사멸을 유의미하게 억제하였으며, LPS 처리한 마우스의 뇌 및 HT22 세포에서 시냅스 마커인 SNAP25(synaptosomal-associated protein, 25kDa), Syp(synaptophysin) 및 PSD95(postsynaptic density protein-95)의 발현을 회복하고, 기억 및 인지기능 장애 관련 Y-미로 테스트와 수동 회피 테스트에서 LPS로 인한 인지 및 기억 장애를 억제할 수 있다는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and provides a composition for suppressing inflammation and regulating immunity in degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient, and comprising the Syk inhibitor, specifically BAY61-3606 ( Hereinafter referred to in parallel with BAY61), Syk, Iba-1, inflammatory cytokines (TNF-α, IL-1α, IL-6) and inflammation-related factors (NF-κB, Cox-2, iNOS) upregulated by LPS. significantly reduced the expression of and significantly inhibited microglial-mediated neuronal death by regulating the expression of cytochrome C and Bim (BCL-2 interacting cell death mediator) in LPS-treated mouse brain and HT22 cells. Restoring the expression of synaptic markers SNAP25 (synaptosomal-associated protein, 25 kDa), Syp (synaptophysin), and PSD95 (postsynaptic density protein-95) in the brain and HT22 cells of one mouse, Y-maze test related to memory and cognitive dysfunction The present invention was completed by confirming that cognitive and memory impairment caused by LPS can be suppressed in the and passive avoidance tests.
상기 과제를 해결하기 위하여, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다. In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 사료 첨가제를 제공한다. In addition, the present invention provides a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 조성물을 인간을 제외한 동물에게 투여하는 단계;를 포함하는 퇴행성 뇌질환의 예방 또는 치료방법을 제공한다. Additionally, the present invention provides a method for preventing or treating degenerative brain diseases, comprising administering to an animal other than a human a composition containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 수의학적 조성물을 제공한다. Additionally, the present invention provides a veterinary composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
본 발명은 Syk 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 염증 억제 및 면역 조절용 조성물에 관한 것으로, 상기 Syk 저해제, 구체적으로 BAY61-3606은 LPS에 의해 상향 조절된 Syk, Iba-1, 염증성 사이토카인(TNF-α, IL-1α, IL-6) 및 염증관련 인자(NF-κB, Cox-2, iNOS)의 발현을 유의미하게 감소시키고, LPS 처리된 마우스 뇌와 HT22 세포에서 시토크롬 C와 Bim(BCL-2 상호작용 세포사 매개체)의 발현을 조절함으로써 미세아교-매개 신경세포 사멸을 유의미하게 억제하였으며, LPS 처리한 마우스의 뇌 및 HT22 세포에서 시냅스 마커인 SNAP25(synaptosomal-associated protein, 25kDa), Syp(synaptophysin) 및 PSD95(postsynaptic density protein-95)의 발현을 회복하고, 기억 및 인지기능 장애 관련 Y-미로 테스트와 수동 회피 테스트에서 LPS로 인한 인지 및 기억 장애를 억제하는 효과가 있다.The present invention relates to a composition for suppressing inflammation and regulating immunity in degenerative brain diseases comprising a Syk inhibitor as an active ingredient, wherein the Syk inhibitor, specifically BAY61-3606, inhibits Syk, Iba-1, and inflammatory cytokines upregulated by LPS. significantly reduced the expression of (TNF-α, IL-1α, IL-6) and inflammation-related factors (NF-κB, Cox-2, iNOS), and increased cytochrome C and Bim ( By regulating the expression of BCL-2 interacting cell death mediator), microglial-mediated neuronal death was significantly inhibited, and the synaptic markers SNAP25 (synaptosomal-associated protein, 25kDa) and Syp were expressed in LPS-treated mouse brains and HT22 cells. It has the effect of restoring the expression of (synaptophysin) and PSD95 (postsynaptic density protein-95) and suppressing cognitive and memory impairment caused by LPS in the Y-maze test and passive avoidance test related to memory and cognitive dysfunction.
도 1은 본 발명의 동물실험 일정 개략도이다. Figure 1 is a schematic diagram of the animal testing schedule of the present invention.
도 2는 LPS로 신경염증이 유발된 동물모델에서, Syk가 미세아교세포의 활성화 인자(Iba-1) 및 염증 매개 인자(TNFα)의 발현에 미치는 효과를 확인한 결과이다. (A) 및 (C)는 피질(Cortex) 및 해마(Hippocampus)에서 Syk, Iba-1 및 TNFα의 단백질 발현 정도를 웨스턴 블롯으로 확인하고 이를 정량한 결과이고, (B) 및 (D)는 피질(Cortex) 및 해마(Hippocampus)에서 Iba-1 발현을 확인한 면역형광 이미지 및 이를 정량한 결과이다(Scale bars, 100㎛). BAY61(BAY61-3606)은 Syk 억제제이다. *, **, ***은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001이다.Figure 2 shows the results of confirming the effect of Syk on the expression of microglial activation factor (Iba-1) and inflammation mediating factor (TNFα) in an animal model in which neuroinflammation was induced by LPS. (A) and (C) are the results of confirming and quantifying the protein expression levels of Syk, Iba-1, and TNFα in the cortex and hippocampus by Western blot, and (B) and (D) are the results of quantifying the protein expression levels of Syk, Iba-1, and TNFα in the cortex and hippocampus. Immunofluorescence images confirming Iba-1 expression in (Cortex) and hippocampus and the results of quantification (Scale bars, 100㎛). BAY61 (BAY61-3606) is a Syk inhibitor. *, **, *** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001.
도 3은 LPS로 신경염증이 유발된 동물모델에서, Syk 억제제(BAY61) 투여에 의한 염증완화 효과를 확인한 결과이다. (A) 및 (C)는 피질(Cortex) 및 해마(Hippocampus)에서 NF-κB의 인산화 정도, iNOS 및 COX-2의 단백질 발현 변화를 웨스턴 블롯으로 확인하고 이를 정량한 결과이고, (B) 및 (D)는 피질(Cortex) 및 해마(Hippocampus)에서 iNOS 및 COX-2의 단백질 발현 변화를 확인한 면역형광 이미지 및 이를 정량한 결과이다(Scale bars, 100㎛). *, **, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001, ****은 p<0.00001이다.Figure 3 shows the results confirming the effect of alleviating inflammation by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS. (A) and (C) are the results of confirming and quantifying the degree of phosphorylation of NF-κB and changes in protein expression of iNOS and COX-2 in the cortex and hippocampus by Western blot, (B) and (D) is an immunofluorescence image confirming changes in protein expression of iNOS and COX-2 in the cortex and hippocampus and the results of their quantification (scale bars, 100㎛). *, **, ***, **** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001, **** is p < 0.00001.
도 4는 LPS로 염증을 유발시킨 BV2 미세아교세포에서, Syk 억제제(BAY61) 투여에 의한 미세아교세포의 활성화 인자(Iba-1) 및 염증 매개 인자(TNFα, IL-1β, IL-6)의 발현 변화를 확인한 결과이다. (A)는 Syk, Iba-1, TNFα 및 IL-1β의 단백질 발현 정도를 웨스턴 블롯으로 확인하고 이를 정량한 결과이고, (B)는 TNFα, IL-1β 및 IL-6의 mRNA 발현을 확인한 결과이다. *, **, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001, ****은 p<0.00001이다.Figure 4 shows the level of microglial activation factor (Iba-1) and inflammation mediating factors (TNFα, IL-1β, IL-6) by administration of Syk inhibitor (BAY61) in BV2 microglial cells inducing inflammation with LPS. This is the result of confirming the expression change. (A) is the result of confirming and quantifying the protein expression levels of Syk, Iba-1, TNFα, and IL-1β by Western blot, and (B) is the result of confirming the mRNA expression of TNFα, IL-1β, and IL-6. am. *, **, ***, **** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001, **** is p < 0.00001.
도 5는 LPS로 염증을 유발시킨 BV2 미세아교세포에서, Syk 억제제(BAY61) 투여에 의한 iNOS 및 COX-2 단백질 발현 변화를 확인한 결과(A), mRNA 발현 변화를 확인한 결과(B) 및 아질산염 생성 정도를 확인한 결과(C)이다. *, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, ***은 p<0.0001, ****은 p<0.00001이다. ##은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.005이다.Figure 5 shows the results of confirming changes in iNOS and COX-2 protein expression by administration of a Syk inhibitor (BAY61) in BV2 microglial cells inducing inflammation with LPS (A), the results of confirming changes in mRNA expression (B), and nitrite production. This is the result of confirming the degree (C). *, ***, **** mean that there is a statistically significant difference from each other, * is p < 0.05, *** is p < 0.0001, and **** is p < 0.00001. ## means there is a statistically significant difference, p <0.005.
도 6은 LPS로 신경염증이 유발된 동물모델에서, Syk 억제제(BAY61) 투여에 의한 신경세포 사멸 완화 효과를 확인한 결과이다. (A)는 피질 및 해마 영역[Cornu ammonis(CA)1, CA3 및 Dentate gyrus(DG)]의 신경세포 밀도를 확인한 니슬(Nissl) 염색 이미지(Scale bars, 50㎛)이고, (B)는 니슬(Nissl) 양성 신경세포를 정량한 결과이다. (C)는 피질과 해마에서 시토크롬 C 및 Bim의 단백질 발현 정도를 웨스턴 블롯으로 확인한 이미지이고, (D)는 이를 정량한 결과이다. *, **, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001, ****은 p<0.00001이다.Figure 6 shows the results confirming the effect of alleviating neuronal cell death by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS. (A) is a Nissl staining image (scale bars, 50㎛) confirming neuron density in the cortex and hippocampus areas [Cornu ammonis (CA)1, CA3, and dentate gyrus (DG)], and (B) is a Nissl staining image (scale bars, 50㎛). (Nissl) This is the result of quantifying positive neurons. (C) is an image confirming the protein expression levels of cytochrome C and Bim in the cortex and hippocampus by Western blot, and (D) is the result of quantification. *, **, ***, **** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001, **** is p < 0.00001.
도 7은 Syk 억제제(BAY61)의 신경독 매개 신경변성 완화 효과를 확인한 결과이다. (A)는 미세아교세포 조절 배지에 대한 실험 계획을 나타낸 그림 및 해마 신경세포주인 HT22에서 미세아교세포 조절 배지의 세포 독성을 확인한 결과이고, (B)는 LPS 처리된 미세아교세포의 조절 배지(LCM); 또는 BAY61와 LPS가 함께 처리된 미세아교세포의 조절 배지[(L+BAY61)CM];를 처리한 HT22의 시토크롬 C 및 Bim의 단백질 발현 정도를 웨스턴 블롯으로 확인하고, 이를 정량한 결과이다. (C)는 미세아교세포(BV2) 및 해마 신경세포주(HT22)의 트랜스웰 기반 공동배양 시스템의 실험 계획을 나타낸 그림 및 BV2를 포함하는 트랜스웰 투과성 지지체 및 HT22 배지에서 아질산염을 측정한 결과이다. (D)는 트랜스웰 기반 공동배양 시스템에 의해 배양된 HT22의 시토크롬 C 및 Bim의 단백질 발현 정도를 웨스턴 블롯으로 확인하고, 이를 정량한 결과이다. *, **, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001, ****은 p<0.00001이다. ##은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, p<0.005이다.Figure 7 shows the results confirming the effect of Syk inhibitor (BAY61) on alleviating neurotoxin-mediated neurodegeneration. (A) is a diagram showing the experimental plan for microglial conditioned medium and the results of confirming the cytotoxicity of microglial conditioned medium in the hippocampal neuron cell line HT22, and (B) is the conditioned medium for LPS-treated microglial cells ( LCM); Alternatively, the protein expression levels of cytochrome C and Bim in HT22 treated with conditioned medium [(L+BAY61)CM] of microglial cells treated with BAY61 and LPS were confirmed by Western blot and quantified. (C) shows the experimental plan of the transwell-based co-culture system of microglial cells (BV2) and hippocampal neuron cell line (HT22) and the results of measuring nitrite in the transwell-permeable support containing BV2 and HT22 medium. (D) shows the results of confirming and quantifying the protein expression levels of cytochrome C and Bim in HT22 cultured by a transwell-based co-culture system by Western blot. *, **, ***, **** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001, **** is p < 0.00001. ## means there is a statistically significant difference, p <0.005.
도 8은 LPS로 신경염증이 유발된 동물모델의 피질(A) 및 해마(B)에서, Syk 억제제(BAY61) 투여에 의한 SNAP25(synaptosome-associated protein, 25kDa), Syp(synaptophysin) 및 PSD95(postsynaptic density protein-95)의 단백질 발현 변화를 웨스턴 블롯으로 확인하고, 이를 정량한 결과이다. *, **, ***, ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005, ***은 p<0.0001, ****은 p<0.00001이다.Figure 8 shows SNAP25 (synaptosome-associated protein, 25kDa), Syp (synaptophysin), and PSD95 (postsynaptic) by administration of Syk inhibitor (BAY61) in the cortex (A) and hippocampus (B) of an animal model in which neuroinflammation was induced by LPS. This is the result of confirming and quantifying changes in protein expression of density protein-95) by Western blot. *, **, ***, **** mean there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005, *** is p < 0.0001, **** is p < 0.00001.
도 9는 LPS로 신경염증이 유발된 동물모델의 피질(A) 및 해마(B)에서, Syk 억제제(BAY61) 투여에 의한 PSD95 단백질 발현 변화를 확인한 면역형광 이미지(Scale bars, 25㎛) 및 이를 정량한 결과이다. **, ***은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, **은 p<0.005, ***은 p<0.0001이다.Figure 9 is an immunofluorescence image (scale bars, 25㎛) confirming changes in PSD95 protein expression by administration of a Syk inhibitor (BAY61) in the cortex (A) and hippocampus (B) of an animal model in which neuroinflammation was induced by LPS. This is a quantitative result. **, *** mean that there is a statistically significant difference from each other, ** is p < 0.005, *** is p < 0.0001.
도 10은 LPS로 신경염증이 유발된 동물모델에서, Syk 억제제(BAY61) 투여에 의한 단기 공간 작업 기억 회복 효과를 Y-미로 테스트로 확인한 결과이다. (A)는 Y-미로 통로 총 입장 횟수(total entry)이고, (B)는 자발적 변경 행동력(Spontaneous alteration)을 자동 비디오 추적 시스템으로 측정한 결과이다. *, **은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, *은 p<0.05, **은 p<0.005이다.Figure 10 shows the results of the Y-maze test confirming the effect of short-term spatial working memory recovery by administration of a Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS. (A) is the total number of entries into the Y-maze passage, and (B) is the result of spontaneous alteration measured using an automatic video tracking system. *, ** mean that there is a statistically significant difference from each other, * is p < 0.05, ** is p < 0.005.
도 11은 LPS로 신경염증이 유발된 동물모델에서, Syk 억제제(BAY61) 투여에 의한 학습 기억 회복 효과를 수동 회피 테스트로 확인한 결과이다. 시험 결과는 밝은 방에서 어두운 방을 통과할 때 진입대기 시간(Entry latency)으로 나타냈다. ****은 서로 통계적으로 유의미한 차이가 있다는 것을 의미하며, ****은 p<0.00001이다.Figure 11 shows the results of confirming the learning memory recovery effect by administration of Syk inhibitor (BAY61) in an animal model in which neuroinflammation was induced by LPS using a passive avoidance test. The test results were expressed as entry latency when passing from a bright room to a dark room. **** means there is a statistically significant difference from each other, and **** is p < 0.00001.
본 발명의 목적을 달성하기 위하여, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물을 제공한다. In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
상기 Syk 저해제는 Syk의 발현 또는 작용을 저해할 수 있는 물질은 어느 것이라도 사용할 수 있고, 바람직하게는 하기 화학식 1의 화합물(BAY61-3606)인 것이지만, 이에 제한되지 않는다. The Syk inhibitor may be any substance capable of inhibiting the expression or action of Syk, and is preferably a compound of the following formula (BAY61-3606), but is not limited thereto.
상기 퇴행성 뇌질환은 파킨슨병(Parkinson's disease), 헌팅턴병(Huntington disease) 및 근위축성 축삭경화증(amyotrophic lateral sclerosis) 중에서 선택된 어느 하나인 것일 수 있고, 바람직하게는 파킨슨병인 것이지만, 이에 제한되지 않는다. The degenerative brain disease may be any one selected from Parkinson's disease, Huntington disease, and amyotrophic lateral sclerosis, and is preferably Parkinson's disease, but is not limited thereto.
상기 퇴행성 뇌질환은 신경염증(Neuroinflammation) 또는 세포사멸(Apoptotic cell death)에 의한 것이 특징이다. The degenerative brain disease is characterized by neuroinflammation or apoptosis.
상기 약학 조성물은 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함할 수 있다. The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.
상기 약학 조성물의 유효성분 이외에 약학적으로 허용 가능한 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 중에서 선택된 1종 이상의 담체를 더 함유할 수 있으며, 상기 유효성분 이외에 약학적으로 허용 가능한 항산화제, 완충액, 정균제, 희석제, 계면활성제, 결합제, 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제 및 보존제 중에서 선택된 1종 이상의 보조제를 더 함유할 수 있다.In addition to the active ingredients of the pharmaceutical composition, pharmaceutically acceptable saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia Rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals. It may further contain one or more carriers selected from oils, and in addition to the above active ingredients, pharmaceutically acceptable antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, and suspending agents. And it may further contain one or more auxiliaries selected from among preservatives.
상기 약학 조성물은 통상의 방법에 따라 경구 또는 비경구의 제형으로 투여될 수 있으며, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로오스, 락토오스, 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 또한, 비수성용제, 현탁제로는 프로필렌 글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition can be administered in an oral or parenteral dosage form according to a conventional method, and when formulated, 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 tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. In addition, non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
상기 건강기능식품 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조될 수 있지만, 이에 한정하는 것은 아니다. The health functional food composition may be manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup and beverage, but is not limited thereto.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 양은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 총 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 하는 장기간의 섭취인 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수 있다.When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of active ingredient can be appropriately used depending on the purpose of use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total raw materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 건강기능식품의 종류에 특별한 제한은 없다. 상기 건강기능식품 조성물을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.
또한, 본 발명의 건강기능식품 조성물은 식품, 특히 기능성 식품으로 제조될 수 있다. 본 발명의 기능성 식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., For example, xylitol, sorbitol, erythritol, etc.) is preferable. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages. The ratio of the ingredients added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 사료 첨가제를 제공한다. In addition, the present invention provides a feed additive for preventing or improving degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
본 발명의 사료 첨가제는 사료관리법상의 보조사료에 해당한다. 본 발명에서 용어 '사료'는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미할 수 있다. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. In the present invention, the term 'feed' may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals.
상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or food. These may be used alone or in combination of two or more types.
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 조성물을 인간을 제외한 동물에게 투여하는 단계;를 포함하는 퇴행성 뇌질환의 예방 또는 치료방법을 제공한다. Additionally, the present invention provides a method for preventing or treating degenerative brain diseases, comprising administering to an animal other than a human a composition containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
상기 Syk 저해제는 Syk의 발현 또는 작용을 저해할 수 있는 물질은 어느 것이라도 사용할 수 있고, 바람직하게는 하기 화학식 1의 화합물인 것이지만, 이에 제한되지 않는다.The Syk inhibitor may be any substance capable of inhibiting the expression or action of Syk, and is preferably a compound of the following formula (1), but is not limited thereto.
[화학식 1][Formula 1]
또한, 본 발명은 Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 수의학적 조성물을 제공한다.Additionally, the present invention provides a veterinary composition for preventing or treating degenerative brain diseases containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient.
본 발명의 유효성분을 포함하는 수의학적 조성물은 통상의 방법에 따른 적절한 부형제 및 희석제를 더 포함할 수 있다. 본 발명의 유효성분을 포함하는 수의학적 조성물에 포함될 수 있는 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 세탄올, 스테아릴알콜, 유동파라핀, 솔비탄모노스테아레이트, 폴리소르베이트 60, 메칠파라벤, 프로필파라벤 및 광물유를 들 수 있다.The veterinary composition containing the active ingredient of the present invention may further include appropriate excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition containing the active ingredient of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, Examples include sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.
본 발명에 따른 유효성분을 포함하는 수의학적 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향신료, 유화제, 방부제 등을 추가로 포함할 수 있는데, 본 발명에 의한 수의학적 조성물은 동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 잘 알려진 방법을 사용하여 제형화될 수 있고, 제형은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 용액, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 좌제, 멸균 주사용액, 멸균 외용제 등의 형태일 수 있다.The veterinary composition containing the active ingredient according to the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc. The veterinary composition according to the present invention is active after being administered to animals. Can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the ingredients, and the dosage forms can be powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, softgels. Alternatively, it may be in the form of hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, etc.
본 발명에 의한 수의학적 조성물은 그 용도에 따라 상기 Syk(spleen tyrosine kinase) 저해제를 유효한 양 또는 유효성분으로서 포함할 수 있다. 상기 유효한 양은 동물의 개체에 따라 적절하게 선택할 수 있다. 질환 내지 상태의 중증도, 개체의 연령, 체중, 건강, 성별, 개체의 유효성분에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 투여 기간, 상기 조성물과 배합 또는 동시 사용되는 다른 조성물을 포함한 요소 및 기타 생리 내지 수의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The veterinary composition according to the present invention may contain the Syk (spleen tyrosine kinase) inhibitor in an effective amount or as an active ingredient, depending on its intended use. The effective amount can be appropriately selected depending on the individual animal. Factors including the severity of the disease or condition, the individual's age, weight, health, gender, the individual's sensitivity to the active ingredient, administration time, administration route and excretion rate, administration period, other compositions combined or used simultaneously with the composition, and It may be determined according to factors well known in the fields of other physiology or veterinary medicine.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.
[재료 및 방법][Materials and Methods]
1. 실험용 마우스1. Laboratory mice
7주령 수컷 C57BL/6 마우스는 샘타코 바이오(Osan, Korea)에서 구입하였다. 마우스는 실온(23±2℃)에서 12시간 밤/낮의 주기로, 사료와 물을 자유롭게 섭취할 수 있는 환경에서 일주일간 순응시켰다. 실험 그룹은 하기 표 1에 개시된 바와 같이 구분하였으며, LPS(L2630, Sigma Aldrich, USA) 및 BAY61-3606(11423, Cayman, USA)은 복강 내 주사로 처리하였다. 실험 일정의 전체 개략도는 도 1에 개시된 바와 같다.7-week-old male C57BL/6 mice were purchased from Samtaco Bio (Osan, Korea). Mice were acclimatized for one week at room temperature (23 ± 2°C) with a 12-hour night/day cycle and with free access to food and water. The experimental groups were divided as described in Table 1 below, and LPS (L2630, Sigma Aldrich, USA) and BAY61-3606 (11423, Cayman, USA) were treated by intraperitoneal injection. The overall schematic diagram of the experimental schedule is as disclosed in Figure 1.
구분division | 처리 기간processing period | 처리 시료processed sample | 그룹 당 마리 수Number of animals per group | |
Control 그룹Control group |
9일 | 식염수saline solution | 44 | |
LPS 그룹LPS Group | 9일9th |
LPS: 0.25mg/kgLPS: 0.25mg/ |
44 | |
LPS+BAY61 그룹LPS+BAY61 Group | 9일9th |
LPS: 0.25mg/kg BAY61-3606: 10mg/kgLPS: 0.25mg/kg BAY61-3606: 10mg/ |
44 | |
BAY61 그룹BAY61 Group | 9일9th |
BAY61-3606: 10mg/kgBAY61-3606: 10mg/ |
44 |
2. Y-미로 및 수동 회피 테스트2. Y-maze and passive avoidance test
Y-미로 테스트 측정 장비는 세 개의 가지로 구성되어 있다. 실험동물을 장비의 한쪽 팔(arm) 끝에 넣고 8분 동안 미로 주변을 자유롭게 움직일 수 있게 하였다. 그 후, 미로는 방향 편향을 피하기 위해 70%(v/v) 에탄올로 소독하였다. Y-미로 통로 총 입장 횟수(total entry), 자발적 변경 행동력(Spontaneous alteration) 및 평균 구역 속도는 SMART V3.0 (Harvard apparatus, USA)으로 기록하였다.The Y-maze test measurement equipment consists of three branches. The experimental animal was placed at the end of one arm of the equipment and allowed to move freely around the maze for 8 minutes. Afterwards, the maze was disinfected with 70% (v/v) ethanol to avoid orientation bias. The total number of entries into the Y-maze passage, spontaneous alteration, and average zone speed were recorded using SMART V3.0 (Harvard apparatus, USA).
수동 회피 테스트는 수동 회피 상자를 이용하여 진행하였다. 수동 회피 상자는 어두운 방과 밝은 방으로 나누어져 있으며, 밝은 방에 실험동물을 넣으면 어두운 방으로 실험동물이 이동하는 습성을 이용하였다. 실험 첫날 3분 동안 어두운 방이 닫힌 조건에서 밝은 방에서 자유롭게 탐색하도록 하였다. 2일 차에 마우스를 밝은 방에 넣고, 30초 후 어두운 방의 문이 열리면 마우스가 어두운 방으로 이동하는데, 마우스가 어두운 방을 통과할 때 문을 닫고 그리드 바닥(grid floor)에서 파생된 전기 충격을 주었다(0.2mA, 2초). 24시간 동안 마우스를 안정화시킨 후, 전기 충격을 가한 것을 제외하고는 2일 차와 동일한 조건에서, 밝은 방에 마우스를 다시 넣고 5분의 컷오프 시간으로 암실을 통과할 때 진입대기 시간(Entry latency)을 측정하였다. The manual avoidance test was conducted using a manual avoidance box. The passive avoidance box is divided into a dark room and a bright room, and the habit of the experimental animal moving to the dark room when placed in the bright room was taken advantage of. On the first day of the experiment, subjects were allowed to explore freely in a bright room while the dark room was closed for 3 minutes. On day 2, the mouse was placed in a bright room, and after 30 seconds, the door to the dark room was opened and the mouse moved to the dark room. When the mouse passed through the dark room, the door was closed and an electric shock derived from the grid floor was delivered. (0.2mA, 2 seconds). After stabilizing the mouse for 24 hours, the mouse was placed back in the bright room under the same conditions as day 2 except that an electric shock was applied, and the entry latency when passed through the dark room with a cutoff time of 5 minutes. was measured.
3. 조직 처리3. Tissue processing
실험 종료 후, 차가운 PBS 및 4%(v/v) 포름알데히드로 마우스를 경심관류하고, 4%(v/v) 포름알데히드 및 20%(w/v) 자당으로 24시간 동안 순차적으로 후고정시켰다. 그 후 뇌를 OCT(optimal cutting temperature compound) 복합체(Sakura, Torrance, CA, USA)에 고정한 다음 액체 질소에서 동결시켰다. After completion of the experiment, mice were perfused transcardially with cold PBS and 4% (v/v) formaldehyde, and sequentially post-fixed with 4% (v/v) formaldehyde and 20% (w/v) sucrose for 24 hours. . The brains were then fixed in optimal cutting temperature compound (OCT) composite (Sakura, Torrance, CA, USA) and frozen in liquid nitrogen.
염색 및 면역형광 실험을 위해, 동결시킨 뇌 조각을 20㎛로 절단한 다음 슬라이드에 고정하고, 이를 영하 70℃에서 보관하였고, 염색 및 면역형광 전에 사용할 때마다 실온에서 밤새 배양하였다.For staining and immunofluorescence experiments, frozen brain pieces were cut into 20 μm sections, mounted on slides, stored at -70°C, and incubated overnight at room temperature before each use before staining and immunofluorescence.
4. 니슬 염색(Nissl staining)4. Nissl staining
니슬 염색을 위해, 뇌 절편을 자일렌으로 탈수하고, 100% 에탄올에서 재수화한 후, 슬라이드를 0.1%(w/v) 크레실 바이올렛과 함께 15분 동안 배양하였고, 이후 70% 에탄올(v/v)로 세척한 다음 D.P.X 장착제(D.P.X mounting medium)로 장착한 후, 슬라이드를 커버 슬립으로 덮고 니슬 양성 세포를 광학 현미경(Axioskpo2 plus, Zen, Zeiss, Germany)으로 관찰하였다. 관찰 결과는 이미지 J 소프트웨어로 분석하였다. For Nissl staining, brain sections were dehydrated in xylene, rehydrated in 100% ethanol, and slides were incubated with 0.1% (w/v) cresyl violet for 15 min, followed by 70% ethanol (v/v). After washing with v) and mounting with D.P. The observation results were analyzed with Image J software.
5. 면역형광5. Immunofluorescence
구연산염 완충액 기반 열 유도 항원 회복은 뇌 해마 슬라이스를 90~100℃에서 20분 동안 반응시켜 수행하였다. 그 후 10분 동안 건조하고, 슬라이드를 0.1%(v/v) 트윈 20을 포함한 PBS(PBST)로 세척한 다음, 5%(v/v) 정상 염소 혈청 및 0.1%(v/v) TritonX-100을 포함한 PBST에 1시간 동안 보관하였다. 1시간 후, 슬라이드를 밤새 4℃에서 1차 항체들과 반응시키고, 다음날, 실온에 1시간 동안 노출시킨 뒤, PBST로 세척한 다음 Alexa fluor 488이 결합된 염소-항 마우스 또는 Alexa fluor 594가 결합된 2차 항체(Thermo Fisher Scientific, USA)로 인큐베이션하였다. 인큐베이션 후 슬라이드를 실온에서 90분 동안 세척한 다음 핵 염색을 위하여, DAPI(4',6-diamidino-2-phenylindole)를 10분 동안 처리하였다. 관심 표적 단백질은 공초점 레이저 스캐닝 현미경(FV 1000MPE, Fluoview, Olympus, Japan)에 의해 검출되었다. Citrate buffer-based heat-induced antigen retrieval was performed by incubating brain hippocampal slices at 90-100°C for 20 minutes. After drying for 10 minutes, the slides were washed with PBS (PBST) containing 0.1% (v/v) Tween 20, then incubated with 5% (v/v) normal goat serum and 0.1% (v/v) TritonX- It was stored in PBST containing 100% for 1 hour. After 1 hour, the slides were reacted with primary antibodies at 4°C overnight, and the next day, exposed to room temperature for 1 hour, washed with PBST, and then incubated with goat-anti-mouse conjugated with Alexa fluor 488 or conjugated with Alexa fluor 594. was incubated with a secondary antibody (Thermo Fisher Scientific, USA). After incubation, the slides were washed at room temperature for 90 minutes and then treated with DAPI (4',6-diamidino-2-phenylindole) for 10 minutes for nuclear staining. Target proteins of interest were detected by confocal laser scanning microscopy (FV 1000MPE, Fluoview, Olympus, Japan).
6. 세포 배양6. Cell culture
미세아교세포주인 BV2는 5%(v/v) FBS 및 1%(v/v) 페니실린/스트렙토마이신이 포함된 DMEM 배지에서 배양하였다. 마우스 해마 신경세포주인 HT22는 10%(v/v) FBS 및 1%(v/v) 페니실린/스트렙토마이신이 포함된 DMEM 배지에서 배양하였다. BV2, a microglial cell line, was cultured in DMEM medium containing 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin. HT22, a mouse hippocampal neuronal cell line, was cultured in DMEM medium containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.
BV2 세포는 1mL당 2.0×105개의 세포 수로 배양 접시에 접종하였고, 세포가 70% 찼을 때 100ng/mL의 LPS를 처리하였다. LPS 처리 6시간 후, RNA를 추출하였고, LPS 처리 24시간 후 단백질을 추출하였다. BV2 cells were inoculated into a culture dish at 2.0 × 10 5 cells per 1 mL, and when the cells were 70% full, 100 ng/mL of LPS was treated. RNA was extracted 6 hours after LPS treatment, and protein was extracted 24 hours after LPS treatment.
세포 실험에서 LPS는 100ng/mL 처리하였고, BAY61-3606은 10nM 또는 100nM 처리하였다. In cell experiments, LPS was treated at 100ng/mL, and BAY61-3606 was treated at 10nM or 100nM.
BAY61-3606 및 LPS 처리 유무에 의해 유도된 아질산염을 검출하기 위해 제조사의 프로토콜에 따라 그리스 시약을 사용하였다. To detect nitrite induced by BAY61-3606 and LPS treatment with or without treatment, Grease reagent was used according to the manufacturer's protocol.
BAY61-3606 및 LPS 처리 유무에 따른 미세아교세포의 조절 배지는 HT22에 24시간 동안 처리한 후, MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] 분석을 진행하여 세포 생존율을 측정하였다.The conditioned medium of microglia with or without BAY61-3606 and LPS treatment was treated with HT22 for 24 hours and then subjected to MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] analysis. Cell viability was then measured.
7. 트랜스웰 공동 배양 분석(Transwell Co-culture Assay)7. Transwell Co-culture Assay
BV2 세포를 트랜스웰(0.4㎛ 기공)에 접종하고, HT22를 6웰 플레이트에 접종한 후, BV2를 LPS(100ng/ml)로 처리하고 BAY61-3606(10nM 또는 100nM)을 처리하거나 처리하지 않은 조건에서 24시간 동안 배양하였다. 그 후, 트랜스웰 및 6-웰 플레이트의 배지를 수집하여 아질산염을 측정하고 HT22를 수집하여 신경세포 사멸을 평가하기 위한 웨스턴 블롯팅을 수행하였다.BV2 cells were inoculated into transwells (0.4㎛ pores), HT22 was inoculated into 6-well plates, and BV2 was treated with LPS (100ng/ml) and treated with or without BAY61-3606 (10nM or 100nM). was cultured for 24 hours. Afterwards, the medium from the transwell and 6-well plate was collected to measure nitrite, and HT22 was collected to perform Western blotting to evaluate neuronal death.
8. 웨스턴 블롯 분석8. Western blot analysis
마우스 조직 또는 세포를 인산가수분해효소 및 단백질분해효소 억제제가 포함된 RIPA 버퍼에 용해한 후, 용해물을 30분 동안 4℃에서 13000rpm으로 원심분리하였다. 단백질은 브래드 포드 분석으로 정량하였다. Mouse tissues or cells were dissolved in RIPA buffer containing phosphatase and protease inhibitors, and the lysate was centrifuged at 13000 rpm at 4°C for 30 minutes. Proteins were quantified by Bradford analysis.
SDS PAGE Gel(sodium dodecyl polyacrylamide gel)에 미리 염색해 놓은 단백질 마커(GangNam-STAIN, iNTRON Biotechonology, 서울, 한국)와 함께 30㎍의 단백질을 로딩하였고, 전기영동 후에 크기별로 분리된 단백질을 PVDF(polyvinylidene fluoride membrane)로 옮긴 후, 5%(w/v) 탈지유로 블로킹하였다.30 μg of protein was loaded onto SDS PAGE Gel (sodium dodecyl polyacrylamide gel) along with a pre-stained protein marker (GangNam-STAIN, iNTRON Biotechonology, Seoul, Korea), and after electrophoresis, the proteins separated by size were transferred to PVDF (polyvinylidene). After transferring to a fluoride membrane, it was blocked with 5% (w/v) skim milk.
분석하고자 하는 각각의 단백질에 대응하는 1차 및 2차 항체를 처리하고, ECL(enhanced chemiluminescent) 시약을 이용하여 시각화하였다. β-액틴은 로딩 컨트롤로 사용되었고, β-액틴으로 광학 밀도를 정규화하기 위해 이미지 J 소프트웨어(National Institutes of Health, Bethesda, MD, USA)를 사용하였다. Primary and secondary antibodies corresponding to each protein to be analyzed were treated and visualized using ECL (enhanced chemiluminescent) reagent. β-Actin was used as a loading control, and Image J software (National Institutes of Health, Bethesda, MD, USA) was used to normalize optical density to β-actin.
9. 항체9. Antibodies
Bim(#2933, 1:3,000), Syk(#13198, 1:3,000)은 셀 시그날링사로부터 구매하였다(Cell Signaling Technology, USA). iba-1(016-20001, 1:1,000)은 와코사로부터 구매하였다(WAKO, Japan). β-액틴(sc-47778, 1:10,000), 시토크롬 C(sc-13156, 1:3,000), SNAP25(sc-20038, 1:3,000), SYP(sc-17750, 1:3,000), PSD95(sc-71933, 1:3,000), p-NF-kB(sc-136548, 1:1,000), TNFα(sc-52746, 1:1,000)는 산타크루즈사로부터 구매하였다(Santa cruz, USA). iNOS(610432, 1:3,000)는 BD 라이프사이언스사로부터 구매하였다(BD lifescience, USA). COX-2(160126, 1:3,000)는 사이만사로부터 구매하였다(Cayman, USA).Bim (#2933, 1:3,000) and Syk (#13198, 1:3,000) were purchased from Cell Signaling (Cell Signaling Technology, USA). iba-1 (016-20001, 1:1,000) was purchased from Wako (WAKO, Japan). β-actin (sc-47778, 1:10,000), cytochrome C (sc-13156, 1:3,000), SNAP25 (sc-20038, 1:3,000), SYP (sc-17750, 1:3,000), PSD95 (sc-17750, 1:3,000) -71933, 1:3,000), p-NF-kB (sc-136548, 1:1,000), and TNFα (sc-52746, 1:1,000) were purchased from Santa Cruz (Santa Cruz, USA). iNOS (610432, 1:3,000) was purchased from BD Lifescience (BD lifescience, USA). COX-2 (160126, 1:3,000) was purchased from Simon (Cayman, USA).
10. RNA 분리 및 정량적 PCR10. RNA isolation and quantitative PCR
TRIzol 시약(Invitro-gen, USA)을 이용하여 조직 또는 세포에서 총 RNA를 추출한 후, 2㎍의 총 RNA로부터 상보적인 DNA를 합성하였다. 정량적 중합효소 연쇄 반응(qPCR)은 LightCycler 480 시스템(Roche, Rotkreuz, Switzerland)에서 SYBR Green I(GeNet Bio, Korea)을 사용하여 수행하였고, mRNA의 상대적 발현은 비교 CT(ΔΔCT)를 사용하여 측정하였으며, GAPDH로 정규화하고, 대조군에 대한 배수 변화로 실험값을 나타내었다. Total RNA was extracted from tissues or cells using TRIzol reagent (Invitro-gen, USA), and then complementary DNA was synthesized from 2 μg of total RNA. Quantitative polymerase chain reaction (qPCR) was performed using SYBR Green I (GeNet Bio, Korea) on a LightCycler 480 system (Roche, Rotkreuz, Switzerland), and relative expression of mRNA was measured using comparative CT ( ΔΔ CT). It was normalized to GAPDH, and the experimental values were expressed as fold change relative to the control group.
11. 데이터 분석 및 통계11. Data analysis and statistics
모든 데이터는 4개의 독립적인 실험의 평균±표준편차(S.E.M)로 표시하였다. 각 그룹들 간의 비교를 위하여, Tukey의 사후검정을 사용한 one-way ANOVA가 사용되었다. 계산은 Prism 8.0 소프트웨어(GraphPad Software, San Diego, CA, USA)를 이용하여 수행하였으며, p<0.05를 기준으로 유의한 차이(significant difference) 여부를 결정하였다.All data were expressed as mean ± standard deviation (SEM) of four independent experiments. For comparison between groups, one-way ANOVA with Tukey's post hoc test was used. Calculations were performed using Prism 8.0 software (GraphPad Software, San Diego, CA, USA), and significant differences were determined based on p <0.05.
실시예 1. LPS로 신경염증이 유발된 동물모델에서, Syk 억제제 투여에 의한 미세아교세포 활성 및 염증 매개 인자 발현 변화 확인Example 1. In an animal model in which neuroinflammation was induced by LPS, changes in microglial activity and expression of inflammatory mediators were confirmed by Syk inhibitor administration.
LPS로 신경염증이 유발된 동물모델에서, Syk 억제제 투여에 의한 미세아교세포 활성 및 염증 매개 인자 발현 변화를 확인한 결과, 도 2에 개시된 바와 같이 피질 및 해마 모두에서 LPS 처리에 의해 증가된 Syk의 발현이 Syk 억제제인 BAY61 처리에 의해 감소하였고, LPS 처리에 의해 증가된 미세아교 활성 인자인 Iba-1 및 염증 매개 인자인 TNFα의 발현도 감소하였다. In an animal model in which neuroinflammation was induced by LPS, changes in microglial activity and expression of inflammatory mediators due to Syk inhibitor administration were confirmed. As shown in Figure 2, the expression of Syk increased by LPS treatment in both the cortex and hippocampus. This was decreased by treatment with BAY61, a Syk inhibitor, and the expression of Iba-1, a microglial activator, and TNFα, an inflammatory mediator, which were increased by LPS treatment, also decreased.
또한, 도 3에 개시된 바와 같이 Syk 억제제인 BAY61은 피질 및 해마에서 LPS에 의해 증가된 NF-κB의 인산화 정도, iNOS 및 CPX-2의 단백질 발현을 감소시켰다. In addition, as shown in Figure 3, BAY61, a Syk inhibitor, reduced the degree of phosphorylation of NF-κB and protein expression of iNOS and CPX-2, which were increased by LPS in the cortex and hippocampus.
이를 통해 Syk 억제제인 BAY61은 LPS에 의해 유도된 미세아교세포의 활성을 감소시키고, 염증반응을 억제할 수 있다는 것을 확인하였다. Through this, it was confirmed that BAY61, a Syk inhibitor, can reduce the activity of microglial cells induced by LPS and suppress the inflammatory response.
실시예 2. LPS로 염증을 유발시킨 BV2 미세아교세포에서, Syk 억제제 투여에 의한 미세아교세포의 활성 및 염증 매개 인자 발현 변화 확인Example 2. Confirmation of changes in microglial activity and expression of inflammatory mediators by administration of Syk inhibitor in BV2 microglial cells induced inflammation with LPS
Syk 억제제인 BAY61이 미세아교세포인 BV2에서도 LPS에 의해 유도된 염증을 억제하는지 확인하였다. 그 결과, 도 4A에 개시된 바와 같이 LPS에 의해 유도된 Syk, Iba-1, TNFα 및 IL-1β의 단백질 발현은 BAY61 처리 용량 의존적으로 하향 조절되었다. 또한, 도 4B에 개시된 바와 같이 LPS 처리시 증가된 TNFα, IL-1β 및 IL-6의 mRNA 발현도 BAY61 처리에 의해 감소되었다. It was confirmed whether BAY61, a Syk inhibitor, suppresses LPS-induced inflammation in BV2 microglial cells. As a result, as shown in Figure 4A, the protein expression of Syk, Iba-1, TNFα, and IL-1β induced by LPS was down-regulated in a BAY61 treatment dose-dependent manner. In addition, as shown in Figure 4B, the mRNA expression of TNFα, IL-1β, and IL-6, which was increased upon LPS treatment, was also reduced by BAY61 treatment.
또한, 도 5A 및 5B에 개시된 바와 같이 BAY61은 LPS에 의해 증가된 iNOS와 COX-2 단백질 및 mRNA 발현을 효과적으로 감소시켰으며, 도 5C에 개시된 바와 같이 아질산염의 생성량도 유의미하게 억제하였다. In addition, as shown in Figures 5A and 5B, BAY61 effectively reduced the iNOS and COX-2 protein and mRNA expressions increased by LPS, and also significantly suppressed the production of nitrite, as shown in Figure 5C.
이를 통해 Syk는 미세아교세포의 염증 조절제임을 확인하였다. Through this, it was confirmed that Syk is an inflammation regulator of microglial cells.
실시예 3. LPS로 신경염증이 유발된 동물모델에서, Syk 억제제 투여에 의한 신경세포 사멸 완화 효과 확인Example 3. Confirmation of the effect of alleviating neuronal cell death by administration of a Syk inhibitor in an animal model in which neuroinflammation was induced by LPS
Syk의 억제가 LPS에 의해 유도된 신경세포 사멸을 개선하는지 확인하였다. 니슬(Nissl) 염색 결과, 도 6A 및 6B에 개시된 바와 같이 LPS에 의해 유도된 신경세포 밀도 감소는 BAY61 처리에 의해 회복되었다. It was confirmed whether inhibition of Syk improved neuronal cell death induced by LPS. As a result of Nissl staining, as shown in Figures 6A and 6B, the decrease in neuronal density induced by LPS was recovered by BAY61 treatment.
또한, 도 6C 및 6D에 개시된 바와 같이 피질 및 해마에서 LPS에 의해 유도된 세포사멸 관련 단백질 시토크롬 C(Cyto C) 및 Bcl-2-유사 단백질 11(Bim)의 발현이 BAY61 처리에 의해 하향 조절되었고, 이는 상기 조직학적 분석과 일치하였다.Additionally, as shown in Figures 6C and 6D, the expression of LPS-induced apoptosis-related proteins cytochrome C (Cyto C) and Bcl-2-like protein 11 (Bim) in the cortex and hippocampus was downregulated by BAY61 treatment. , which was consistent with the above histological analysis.
실시예 4. Syk 억제제(BAY61)의 신경독 매개 신경변성 완화 효과 확인Example 4. Confirmation of the effect of Syk inhibitor (BAY61) on alleviating neurotoxin-mediated neurodegeneration
Syk 억제제(BAY61) 처리에 의한 신경세포 사멸 억제 효과가 측분비 방식에 의한 미세아교세포 활성화 정도에 의존하는지 확인하기 위해, 조건 배지 및 트랜스웰 배양의 두 가지 실험을 진행하였다. To confirm whether the effect of suppressing neuronal cell death by treatment with Syk inhibitor (BAY61) depends on the degree of microglial activation in a paracrine manner, two experiments were conducted: conditioned medium and transwell culture.
도 7A에 개시된 바와 같이 LPS 처리된 미세아교세포의 조절 배지(LCM)는 HT22 세포 사멸을 유발하였고, BAY61와 함께 처리된 조절배지[(L+BAY61)CM]를 처리한 경우, HT22의 세포 사멸 정도가 완화되었다. 또한, 세포 사멸 관련 인자인 시토크롬 C 및 Bim의 발현 변화를 확인한 결과, 도 7B에 개시된 바와 같이 LCM 처리에 의해 증가된 시토크롬 C 및 Bim의 발현이 (L+BAY61)CM 처리에 의해 감소하는 것을 확인하였다. As shown in Figure 7A, conditioned medium (LCM) of LPS-treated microglial cells induced HT22 cell death, and when treated with conditioned medium treated with BAY61 [(L+BAY61)CM], apoptosis of HT22 occurred. The severity has eased. In addition, as a result of confirming changes in the expression of cytochrome C and Bim, which are cell death-related factors, it was confirmed that the expression of cytochrome C and Bim, which was increased by LCM treatment, was decreased by (L+BAY61)CM treatment, as shown in Figure 7B. did.
다음으로, BV2 세포를 트랜스웰 투과성 지지체에 접종하고 LPS 및 BAY61로 자극한 후, 이식된 지지체를 HT22가 포함된 플레이트에 배치한 결과, 도 7C에 개시된 바와 같이 BV2 세포의 아질산염 농도는 LPS 자극에 의해 증가하였다가 BAY61 처리시 감소하였으나, HT22가 포함된 웰에서는 아질산염 농도의 감소 효과가 나타나지 않았고, 도 7D에 개시된 바와 같이 LPS 처리된 BV2 세포와 트랜스웰 배양된 HT22 세포에서 증가된 시토크롬 C 및 Bim의 발현이 LPS 및 BAY61 처리된 BV2 세포와 트랜스웰 배양된 HT22 세포에서 감소하는 것을 확인하였다. Next, BV2 cells were inoculated onto the transwell-permeable support and stimulated with LPS and BAY61, and then the transplanted support was placed on a plate containing HT22. As a result, as shown in Figure 7C, the nitrite concentration of BV2 cells decreased upon LPS stimulation. It increased and then decreased upon BAY61 treatment, but no effect of reducing nitrite concentration was observed in wells containing HT22, and cytochrome C and Bim increased in LPS-treated BV2 cells and transwell-cultured HT22 cells, as shown in Figure 7D. It was confirmed that the expression of was decreased in BV2 cells treated with LPS and BAY61 and in HT22 cells cultured in transwells.
실시예 5. LPS로 신경염증이 유발된 동물모델에서, Syk 억제제 투여에 의한 시냅스 기능 장애, 인지 및 공간 작업 기억 장애 회복 효과 확인Example 5. In an animal model in which neuroinflammation was induced by LPS, the effect of Syk inhibitor administration on recovering synaptic dysfunction, cognitive and spatial working memory impairment was confirmed.
기억 장애는 시냅스 기능 장애에 이어 신경세포 사멸과 밀접한 관련이 있으므로, Syk의 억제가 LPS로 신경염증이 유발된 마우스에서 시냅스 및 인지 기능 장애에 영향을 미치는지 여부를 확인하였다. Since memory impairment is closely related to synaptic dysfunction followed by neuronal death, we examined whether inhibition of Syk affects synaptic and cognitive dysfunction in mice with neuroinflammation induced by LPS.
그 결과, 도 8에 개시된 바와 같이 LPS로 신경염증이 유발된 마우스 피질 및 해마에서 SNAP25(synaptosome-associated protein, 25kDa), Syp(synaptophysin) 및 PSD95(postsynaptic density protein-95)의 감소된 발현이 BAY61 투여 후 정상군과 유사한 수준으로 회복되었고, 면역형광분석으로 PSD95 단백질 발현을 확인한 결과, 도 9에 개시된 바와 같이 LPS로 신경염증이 유발된 마우스 피질 및 해마에서 감소된 PSD95의 발현이 BAY61 투여 후 정상군과 유사한 수준으로 회복되었다. As a result, as shown in Figure 8, decreased expression of SNAP25 (synaptosome-associated protein, 25 kDa), Syp (synaptophysin), and PSD95 (postsynaptic density protein-95) in the mouse cortex and hippocampus in which neuroinflammation was induced by LPS was observed in BAY61. After administration, it recovered to a level similar to that of the normal group, and as a result of confirming PSD95 protein expression by immunofluorescence analysis, as shown in Figure 9, the expression of PSD95 was reduced in the mouse cortex and hippocampus in which neuroinflammation was induced by LPS, which was normal after BAY61 administration. It recovered to a level similar to that of the military.
또한, LPS로 신경염증이 유발된 마우스에서 기억 관련 기능에 대한 Syk 억제 효과를 확인하기 위해 Y-미로 테스트 및 수동 회피 테스트를 수행하였다. Additionally, the Y-maze test and passive avoidance test were performed to confirm the effect of Syk inhibition on memory-related functions in mice in which neuroinflammation was induced by LPS.
Y-미로 테스트는 단기 기억력을 측정하는 실험으로, 순차적으로 행동하는 능력을 평가할 수 있다. Y-미로 테스트 결과, 도 10A에 개시된 바와 같이 Y-미로 각 팔(arm) 총 입장 횟수가 LPS로 신경염증이 유발된 마우스에서 증가되었으나, BAY61 투여시 정상군 수준으로 회복하였고, 자동 비디오 추적 시스템으로 자발적 변경 행동력(Spontaneous alteration)을 측정한 결과, 도 10B에 개시된 바와 같이 LPS로 신경염증이 유발된 마우스에서 감소된 자발적 변경 행동력(교대 및 탐색의 비율)이 BAY61 투여시 정상군 수준으로 회복되었다. 이는 BAY61이 LPS로 신경염증이 유발된 마우스에서 발생된 공간 작업 기억 손상을 회복시킬 수 있다는 것을 의미한다. The Y-maze test is an experiment that measures short-term memory and can evaluate the ability to act sequentially. As a result of the Y-maze test, as shown in Figure 10A, the total number of entries into each arm of the Y-maze was increased in mice with neuroinflammation induced by LPS, but recovered to the normal group level when administered with BAY61, and the automatic video tracking system As a result of measuring spontaneous alteration, as shown in Figure 10B, the reduced spontaneous alteration behavior (ratio of alternation and exploration) in mice induced by neuroinflammation with LPS was restored to the normal group level when BAY61 was administered. . This means that BAY61 can restore spatial working memory damage that occurred in mice with neuroinflammation induced by LPS.
또한, 도 11에 개시된 바와 같이 수동 회피 테스트에서 LPS로 신경염증이 유도된 마우스는 전날 전기 충격에 노출되었음에도 밝은 방에 머무는 시간이 정상군 대비 감소하였고, BAY61이 함께 투여된 마우스는 정상군과 유사한 밝은 방에 머무는 시간을 가졌다.In addition, as shown in Figure 11, in the passive avoidance test, mice in which neuroinflammation was induced by LPS had a decreased time spent in the bright room compared to the normal group, even though they had been exposed to an electric shock the day before, and mice administered together with BAY61 showed similar activity to the normal group. I had time to stay in a bright room.
Claims (10)
- Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating degenerative brain diseases containing a Syk (spleen tyrosine kinase) inhibitor as an active ingredient.
- 제1항에 있어서, 상기 퇴행성 뇌질환은 파킨슨병(Parkinson's disease), 헌팅턴병(Huntington disease) 및 근위축성 축삭경화증(amyotrophic lateral sclerosis) 중에서 선택된 어느 하나인 것을 특징으로 하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물.The method of claim 1, wherein the degenerative brain disease is any one selected from Parkinson's disease, Huntington disease, and amyotrophic lateral sclerosis. Pharmaceutical composition.
- 제1항에 있어서, 상기 퇴행성 뇌질환은 신경염증(Neuroinflammation) 또는 세포사멸(Apoptotic cell death)에 의한 것임을 특징으로 하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating degenerative brain diseases according to claim 1, wherein the degenerative brain diseases are caused by neuroinflammation or apoptosis.
- 제1항에 있어서, 상기 Syk 저해제 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 더 포함하는 것을 특징으로 하는 퇴행성 뇌질환의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating degenerative brain diseases according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent in addition to the Syk inhibitor.
- Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving degenerative brain diseases containing a Syk (spleen tyrosine kinase) inhibitor as an active ingredient.
- 제6항에 있어서, 상기 조성물은 분말, 과립, 환, 정제, 캡슐, 캔디, 시럽 및 음료 중에서 선택된 어느 하나의 제형으로 제조되는 것을 특징으로 하는 퇴행성 뇌질환의 예방 또는 개선용 건강기능식품 조성물.The health functional food composition for preventing or improving degenerative brain disease according to claim 6, wherein the composition is manufactured in any one formulation selected from powder, granule, pill, tablet, capsule, candy, syrup, and beverage.
- Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 개선용 사료 첨가제.A feed additive for preventing or improving degenerative brain diseases containing a Syk (spleen tyrosine kinase) inhibitor as an active ingredient.
- Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 조성물을 인간을 제외한 동물에게 투여하는 단계;를 포함하는 퇴행성 뇌질환의 예방 또는 치료방법.A method for preventing or treating a degenerative brain disease comprising: administering a composition containing a spleen tyrosine kinase (Syk) inhibitor as an active ingredient to an animal other than a human.
- Syk(spleen tyrosine kinase) 저해제를 유효성분으로 포함하는 퇴행성 뇌질환의 예방 또는 치료용 수의학적 조성물.A veterinary composition for preventing or treating degenerative brain diseases containing a Syk (spleen tyrosine kinase) inhibitor as an active ingredient.
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Non-Patent Citations (6)
Title |
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HE XUEJUN, HUANG YIMIN, LIU YANCHAO, ZHANG XINCHENG, YUE PENGJIE, MA XIAOPENG, MIAO ZHUANGZHUANG, LONG XIAOBING, YANG YIPING, WAN : "BAY61‑3606 attenuates neuroinflammation and neurofunctional damage by inhibiting microglial Mincle/Syk signaling response after traumatic brain injury", INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 49, no. 1, 9 November 2021 (2021-11-09), GR , pages 1 - 13, XP093128446, ISSN: 1107-3756, DOI: 10.3892/ijmm.2021.5060 * |
KANURI SRI HARSHA: "Role of spleen tyrosine kinase in tumor necrosis factor like weak inhibitor of apoptosis induced dopaminergic neuronal cell death and microglial activation in invitro and in vivo models of Parkinson disease", DISSERTATION PHD, IOWA STATE UNIVERSITY, 10 August 2018 (2018-08-10), XP093128452, Retrieved from the Internet <URL:https://www.proquest.com/docview/2059154957?pq-origsite=gscholar&fromopenview=true&sourcetype=Dissertations%20&%20Theses> [retrieved on 20240207], DOI: 10.31274/etd-180810-6019 * |
KIM MIN WOO, CHOE KYONGHWAN, PARK JUN SUNG, LEE HYEON JIN, KANG MIN HWA, AHMAD RIAZ, KIM MYEONG OK: "Pharmacological Inhibition of Spleen Tyrosine Kinase Suppressed Neuroinflammation and Cognitive Dysfunction in LPS-Induced Neurodegeneration Model", CELLS, vol. 11, no. 11, 28 May 2022 (2022-05-28), pages 1777, XP093128454, ISSN: 2073-4409, DOI: 10.3390/cells11111777 * |
RICCABONI, M. ; BIANCHI, I. ; PETRILLO, P.: "Spleen tyrosine kinases: biology, therapeutic targets and drugs", DRUG DISCOVERY TODAY, ELSEVIER, AMSTERDAM, NL, vol. 15, no. 13-14, 1 July 2010 (2010-07-01), AMSTERDAM, NL , pages 517 - 530, XP027121509, ISSN: 1359-6446 * |
SCHWEIG JONAS ELIAS, YAO HAILAN, COPPOLA KYLE, JIN CHAO, CRAWFORD FIONA, MULLAN MICHAEL, PARIS DANIEL: "Spleen tyrosine kinase (SYK) blocks autophagic Tau degradation in vitro and in vivo", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 294, no. 36, 1 September 2019 (2019-09-01), US , pages 13378 - 13395, XP093128444, ISSN: 0021-9258, DOI: 10.1074/jbc.RA119.008033 * |
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