Novel peptides and their applications

Abstract

This invention relates to a peptide that has preventive, ameliorative and / or therapeutic effects against 4R tauopathy, is safe for the body, and has few side effects including abnormal reactions, as well as pharmaceutical compositions and health functional foods containing the same.

Description

Technical Field

[0001] The present invention relates to novel peptides and their uses.

Background Art

[0002] Tau is abundantly present in nerve cells of the central nervous system (CNS) and mainly plays a role in stabilizing microtubules within axons.

[0003] When hyperphosphorylation occurs, where phosphate attaches excessively to the tau protein, tau separates from the microtubules and forms neurofibrillary tangles (NFTs), which are insoluble aggregates. Neurodegenerative diseases associated with such abnormal tau aggregation are collectively called tauopathy.

[0004] Tauopathy is roughly classified into 3R tauopathy and 4R tauopathy according to the isoform of the tau protein that causes the disease.

[0005] 4R tauopathy includes Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), Argyrophilic grain disease (AGD), Globular glial tauopathy (GGT), and Aging-related tau astrogliopathy (ARTAG), etc.

[0006] With aging, the number of patients with 4R tauopathy is increasing, but currently, there is no drug that can effectively treat or prevent this disease.

[0007] While Phase 3 clinical trials are underway for LMTM, a known tau aggregation inhibitor, the specific effects of LMTM on 4R tauopathy have not been reported. Furthermore, LMTM is found to be within the tau aggregation inhibition interval (EC). 50 Cytotoxicity (GI) in (=2.2±0.2μM) 50 There is a problem in that it shows (=6.4±0.3μM).

[0008] Therefore, the present inventors diligently researched the treatment of 4R tauopathy by suppressing tau hyperphosphorylation and aggregation, and as a result, completed the present invention. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a novel peptide that has a preventive or therapeutic effect on 4R tauopathy.

[0010] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of 4R tauopathy.

[0011] The present invention aims to provide a health functional food for the prevention or improvement of 4R tauopathy. [Means for solving the problem]

[0012] 1. A peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. [ka]

[0013] 2. A pharmaceutical composition for the prevention or treatment of 4R tauopathy, comprising the peptide of item 1 or a pharmaceutically acceptable salt thereof.

[0014] 3. A pharmaceutical composition for the prevention or treatment of 4R tauopathy, wherein, in item 2 above, 4R tauopathy is one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, argyrophilic granulopathy, spheroid glial tauopathy, and age-related tauastrogliopathies.

[0015] 4. In item 3 above, prevention or treatment of 4R tauopathy is a pharmaceutical composition for the prevention or treatment of 4R tauopathy by inhibiting the hyperphosphorylation and aggregation of tau protein.

[0016] 5. A health functional food for the prevention or improvement of 4R tauopathy, comprising the peptide of the following chemical formula 1 or a food-safe salt thereof. [ka]

[0017] 6. In item 5 above, 4R tauopathy is one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, argyrophilic granulopathy, spheroid glial tauopathy, and age-related tauastrogliopathies, and is a health functional food for the prevention or improvement of 4R tauopathy.

[0018] 7. In item 6 above, prevention or improvement of 4R tauopathy refers to a health functional food for the prevention or improvement of 4R tauopathy by inhibiting the hyperphosphorylation and aggregation of tau protein. [Effects of the Invention]

[0019] The peptide of chemical formula 1 of the present invention and its pharmaceutically acceptable salt have excellent inhibitory effects on tau protein hyperphosphorylation.

[0020] The peptide of chemical formula 1 of the present invention and its pharmaceutically acceptable salt have excellent tau protein aggregation inhibitory effects.

[0021] The pharmaceutical composition and the functional food for health containing the peptide of Chemical Formula 1 of the present invention or a salt thereof can exhibit an effect on the prevention, improvement and / or treatment of 4R tauopathy by suppressing the hyperphosphorylation and aggregation of tau protein.

[0022] The peptide, pharmaceutical composition and functional food for health of the present invention can stably exert a medicinal effect in vivo for a long time.

Brief Description of Drawings

[0023] [Figure 1] Figure 1 shows the experimental results of confirming the stability of GV2002 in human plasma. [Figure 2] Figure 2 shows the results of confirming the pharmacokinetic profile (PK profile) after intravenous administration of GV2002 to rats. [Figure 3] Figure 3 shows the results of confirming the pharmacokinetic profile after subcutaneous administration of GV2002 to rats. [Figure 4] Figure 4 shows the mouse groups used in the in vivo experiment to verify the effect of GV2002. [Figure 5] Figure 5 shows the schedule of the effect evaluation experiment of GV2002 on the 4R tauopathy mouse model. [Figure 6] Figure 6 shows the results of the evaluation experiment on the improvement of motor ability of the 4R tauopathy mouse model. [Figure 7a] Figure 7 shows the results of the evaluation experiment on the improvement of motor ability of the 4R tauopathy mouse model. [Figure 7b] Same as above [Figure 8a] Figure 8 shows the method and results of the evaluation experiment on the improvement of cognitive ability of the 4R tauopathy mouse model. [Figure 8b] Same as above [Figure 9a] Figure 9 shows the method and results of the evaluation experiment on the improvement of spatial memory ability of the 4R tauopathy mouse model. [Figure 9b] Same as above [Figure 10a] Figure 10 shows the results of an experiment evaluating the tau hyperphosphorylation inhibitory effect of GV2002 using immunofluorescence staining of brain tissue. [Figure 10b] Same as above [Figure 10c] Same as above [Figure 10d] Same as above [Figure 11a] Figure 11 shows the results of an experiment evaluating the inhibitory effect of GV2002 on tau oligomer formation in brain tissue using Tau-BiFC imaging. [Figure 11b] Same as above [Figure 11c] Same as above [Figure 11d] Same as above [Figure 12a] Figure 12 shows the results of an experiment evaluating the inhibitory effect of GV2002 on tau hyperphosphorylation and aggregation by analysis of brain lysates. [Figure 12b] Same as above [Figure 12c] Same as above [Modes for carrying out the invention]

[0024] The present invention provides a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0025] [ka]

[0026] In this invention, the peptide of chemical formula 1 includes its functional equivalent. "Functional equivalent" means a peptide that exhibits substantially the same physiological activity as the peptide of chemical formula 1.

[0027] The present invention provides a pharmaceutical composition for the prevention or treatment of 4R tauopathy, comprising a peptide of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0028] In this invention, "pharmaceutically acceptable" means that the composition does not exhibit toxicity to cells or individuals exposed to it.

[0029] In the present invention, 4R tauopathy is a general term for neurodegenerative diseases caused by aggregation of 4R tau protein, and includes progressive supranuclear palsy, corticobasal degeneration, argyrophilic granulopathy, spheroid glial tauopathy, and age-related tauastrogliopathies.

[0030] In this invention, "prevention" means all actions that suppress or delay 4R tauopathy.

[0031] In this invention, "improvement" and "treatment" mean all actions that improve or change to a favorable state the symptoms of an individual suspected of having or who has developed 4R tauopathy.

[0032] In one embodiment, prevention, improvement, and / or treatment may be achieved by inhibiting the hyperphosphorylation of tau protein.

[0033] In one embodiment, prevention, improvement, and / or treatment may be achieved by inhibiting the aggregation of tau protein.

[0034] In one embodiment, prevention, improvement, and / or treatment may involve improving the motor skills of an individual suspected of or having developed 4R tauopathy.

[0035] In one embodiment, prevention, improvement, and / or treatment may involve improving the cognitive abilities of individuals suspected of or who have developed 4R tauopathy.

[0036] In one embodiment, prevention, improvement, and / or treatment may involve improving the spatial memory ability of individuals suspected of or who have developed 4R tauopathy.

[0037] The pharmaceutical composition of the present invention may contain an active ingredient alone, or may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0038] Examples of carriers, excipients, or diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0039] The pharmaceutical composition of the present invention can be provided by administering it to an individual.

[0040] In this invention, "administration" means introducing a predetermined substance into an individual in an appropriate manner. Furthermore, "individual" means all animals such as livestock and mice that have developed or are likely to develop 4R tauopathy, and may include mammals, such as humans.

[0041] The administration route of the pharmaceutical composition of the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal.

[0042] In one embodiment, the composition of the present invention may be administered orally or parenterally.

[0043] When administering the composition of the present invention parenterally, it is preferable to select an injection method such as topical application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but is not limited to these.

[0044] The pharmaceutical composition of the present invention may be provided in the form of a solid dosage form for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0045] The pharmaceutical composition of the present invention may be provided as a liquid formulation for oral administration, such as a suspension, an oral solution, an emulsion, or a syrup.

[0046] The pharmaceutical composition of the present invention may be provided as a formulation for parenteral administration, such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, or a suppository.

[0047] The pharmaceutical composition of the present invention can be administered to an individual in a pharmaceutically effective amount. "Pharmacologically effective amount" means an amount sufficient to treat 4R tauopathy with a reasonable benefit / risk ratio applicable to medical treatment.

[0048] The pharmaceutically effective amount of the pharmaceutical composition of the present invention may be determined based on factors including the severity of 4R tauopathy, the activity of the pharmaceutical composition, the sensitivity of the individual or patient to the pharmaceutical composition, the time of administration, the route of administration, the elimination rate, the duration of treatment, and concomitant drugs, as well as other factors well known in the medical field, and may be appropriately selected by those skilled in the art.

[0049] The pharmaceutical compositions of the present invention may be administered as a single therapeutic agent or in combination with other conventional therapeutic agents. When administered in combination, they may be administered sequentially or simultaneously, and may be administered individually or in multiple doses, which can be easily determined by a person of ordinary skill.

[0050] The present invention provides a health functional food for the prevention or improvement of 4R tauopathy, comprising a peptide of chemical formula 1 or a food-safe salt thereof.

[0051] In this invention, "health functional foods" means foods manufactured and processed using raw materials and ingredients that have beneficial functional properties for the human body, in accordance with the Act on Health Functional Foods. Furthermore, "functionality" means being ingested for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients in relation to the structure and function of the human body, or physiological effects.

[0052] The food composition of the present invention may contain common food additives. Unless otherwise specified, the suitability of food additives shall be determined according to the standards and criteria for the item in question, based on the general provisions and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety.

[0053] Examples of items listed in the Food Additives Code include chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar dye preparations.

[0054] The functional health food of the present invention may contain 0.01 to 95% by weight, preferably 1 to 80% by weight, of the peptide of chemical formula 1 relative to the total weight of the functional health food, for the purpose of preventing and / or improving 4R tauopathy. Furthermore, for the purpose of preventing and / or improving 4R tauopathy, it can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

[0055] The present invention will be described in detail below with reference to examples. However, the following examples are provided to make the present invention easier to understand and do not limit the scope of the invention. [Examples]

[0056] [Example 1] Manufacturing of GV2002 A peptide having the structural formula of chemical formula 1 below (hereinafter referred to as "GV2002") was prepared. [ka]

[0057] GV2002 was synthesized by coupling amino acids one by one from the C-terminus according to a known Fmoc solid-phase peptide synthesis (SPPS) method.

[0058] All amino acid starting materials used in peptide synthesis were those protected with Fmoc at the N-terminus and all residues removed by acid, such as Boc, t-Bu (t-butylester), or Pbf (2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl). Examples of amino acid starting materials used in peptide synthesis include: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Leu- OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser-OH, Fmoc-Thr-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH.

[0059] The coupling reagents used were DIC (Diisopropylcarbodiimide), DMF (Dimethylformamide), Oxyma (Ethyl(hydroxyamino)Cyanoacetate), and IPA (Isopropyl alcohol). 20% piperidine in DMF was used to remove Fmoc. The synthesized peptide was separated from the resin, and a cleavage cocktail [95% TFA (trifluoroacetic acid) / 5% H2O / 50 mg / mL DTT (Dithiothreitol)] was used to remove the protecting groups from the residues.

[0060] For N-terminal capping of the peptide, the N-terminus was acetylated using Ac2O (Acetic anhydride) / DIEA (Diisopropylethylamine) / DMF (Dimethylformamide). The C-terminus of the peptide was aminated.

[0061] Peptides were synthesized by sequentially reacting each amino acid, washing with a solvent, and deprotecting them while the starting amino acids, to which amino acid protecting groups were attached, were bound to a solid support. After the synthesized peptides were cleaved from the resin, they were purified by HPLC, their synthesis was confirmed by MS, and they were freeze-dried.

[0062] As a counter ion exchange process, GV2002, dissolved in water in the form of HOAc salt, was converted to its chloride form using AmberChrom ion exchange resin. The converted GV2002 was filtered through a PVDF filter and freeze-dried.

[0063] The specific synthesis process for GV2002 is as follows: (1) Coupling The amino acids were coupled to the Fmoc-Rink Methylbenzhydrylamine resin by adding each amino acid from the C-terminus to the N-terminus and the coupling reagent DIC / Oxyma / IPA dissolved in DMF. The resin was then washed with DMF. (2) Fmoc deprotection and N-terminal capping After deprotecting Fmoc by treating it with 20% piperidine in DMF, the N-terminus was acetylated. (3) Cutting The resin to which the synthesized peptides were bound was treated with a cleavage cocktail, and the peptides were separated from the resin. (4) Filtration The resulting mixture was concentrated and precipitated with MTBE / hexane. The precipitate was then filtered and dried. (5) Purification The resulting dried filtrate was purified by Prep-HPLC, its molecular weight was determined by LC / MS, and it was then freeze-dried. (6) Production of powder The product was prepared by replacing the chloride with AmberChrom resin, filtering it through PVDF, and then freeze-drying it to obtain a powder.

[0064] [Example 2] Confirmation of the stability of GV2002 in human plasma The peptide was added to 100% human plasma, diluted to 5 μM, and reacted with stirring at 37°C for 10, 60, and 240 minutes. The mixture was collected and pretreated by plasma protein precipitation using methanol containing 0.1% formic acid. The supernatant was collected and analyzed by LC-MS under the conditions shown in Table 1 below, and the ratio (%) of residual peptide to the amount of peptide before reaction was calculated.

[0065] [Table 1]

[0066] As shown in Figure 1, the ratio of residual peptide to the amount of peptide before reaction in GV2002 was 57.23% at 60 minutes and 46.54% at 240 minutes. This confirmed the stability of GV2002 in plasma.

[0067] [Example 3] Confirmation of the pharmacokinetic profile of GV2002 To confirm the pharmacokinetic profile of GV2002 prepared in Example 1, plasma was collected from rats administered GV2002. This plasma was pretreated by protein precipitation with methanol containing 9 times the volume of 0.1% formic acid, centrifuged at 16,100 g for 10 minutes, and the supernatant was collected and analyzed by LC-MS under the conditions shown in Table 2 below.

[0068] [Table 2]

[0069] 3-1. Confirmation of the pharmacokinetic profile of GV2002 by intravenous administration. GV2002 was administered intravenously (IV) to three Sprague Dawley rats per group. Blood samples were collected at 0.033, 0.083, 0.17, 0.25, 0.5, 1, and 2 hours after administration. Blood samples were centrifuged to separate the plasma, pretreated, and analyzed using LC-MS. Pharmacokinetic parameters were calculated using the Non-compartmental analysis model of the Phoenix WinNonlin (Pharsight ver 6.4, USA) program. As a result, as shown in Figure 2, the AUC of GV2002 t The value was 0.438 μg·h / mL. This confirms that GV2002 remains stable in vivo without being degraded and persists for a long period of time.

[0070] 3-2. Confirmation of the pharmacokinetic profile of GV2002 by subcutaneous administration. GV2002 was administered subcutaneously (SC) to three Sprague Dawley rats per group. Blood samples were collected at 0.033, 0.083, 0.17, 0.25, 0.5, 1, and 2 hours after administration. Blood samples were centrifuged to separate the plasma, pretreated, and analyzed using LC-MS. Pharmacokinetic parameters were calculated using the Non-compartmental analysis model of the Phoenix WinNonlin (Pharsight ver 6.4, USA) program. As a result, as shown in Figure 3, it was confirmed that even when administered subcutaneously, GV2002 remained stable in the body for a long period of time without being degraded.

[0071] [Example 4] Evaluation of the effects of GV2002 based on an animal model of 4R tauopathy In vivo experiments were conducted using the 4R tauopathy mouse group shown in Figure 4, based on the schedule shown in Figure 5 and the summary shown in Table 3 below.

[0072] [Table 3]

[0073] 4-1. Evaluation of improvement in motor function using the Rota-rod test The rotarod test was performed on a 4R tauopathy mouse model administered the drug for 5 months, from 7.5 months to 12.5 months of age, to evaluate the effect of GV2002 on improving motor performance.

[0074] (1) Experimental method Habituation training was conducted on day 1, and behavioral experiments were performed on day 2. Before the start of habituation training and behavioral experiments, one hour was allowed for the mice to acclimate to the environment. In habituation training, mice were made to walk on a treadmill at a speed of 5 rpm to 25 rpm for 300 seconds, and mice that lost their balance and fell were made to walk again. In behavioral experiments, mice were made to walk on a treadmill that gradually increased in speed from 5 rpm to 40 rpm for 300 seconds, and the time (in seconds) until a mouse lost its balance and fell was measured. The measured values ​​were analyzed using two-way ANOVA with Dunnett's multiple comparison test.

[0075] (2) Experimental results As shown in Figure 6, the GV2002-treated group showed significantly improved exercise capacity compared to the vehicle-treated group, and relatively improved exercise capacity compared to the LMTM-treated group, which is the reference compound.

[0076] 4-2. Evaluation of improvement in motor skills using open field tests An open-field study was conducted on a 4R tauopathy mouse model that received drug administration for 5 months, from 7.5 months to 12.5 months of age, to evaluate the effect of GV2002 on improving motor performance.

[0077] (1) Experimental method To prevent changes in the mice's behavioral responses, handling was performed for five days prior to the experiment (days 1-5). Each handling session lasted two minutes. On day 6, the mice were placed in a 40cm x 40cm enclosure and subjected to a 10-minute open-field test. One hour was allowed before the start of the experiment to allow the mice to acclimate to the environment. During the open-field test, the mice's movement trajectory, distance traveled, and movement speed were recorded. A constant illumination level was maintained throughout the experiment. The recorded values ​​were analyzed using one-way ANOVA with Dunnett's multiple comparison test.

[0078] (2) Experimental results As shown in Figure 7, the total distance traveled and movement speed in the open field were significantly higher in the GV2002-administered group compared to the vehicle-administered group. This suggests that GV2002 administration improved motor skills.

[0079] 4-3. Evaluation of cognitive ability improvement using a novel object recognition test A novel object recognition test was conducted on a 4R tauopathy mouse model administered with the drug for five months, from 7.5 months to 12.5 months of age, to evaluate the cognitive ability-improving effect of GV2002.

[0080] (1) Experimental method Mice that had undergone experiments from day 1 to day 6 using the same method as in Example 4-2 were subjected to a novel object recognition test on days 7 and 8. On day 7, as shown in Figure 8a, the mice were placed in a 40cm x 40cm enclosure containing two similar objects and allowed to learn for 10 minutes. On day 8, one of the two objects was replaced with a new one, and the mice were placed back into the enclosure. Their behavior was observed for 10 minutes, and the time they spent near the new object was measured. Before the start of the experiments on days 7 and 8, the mice were given one hour to acclimate to the environment. A constant illumination level was maintained throughout the experiment. The recorded values ​​were analyzed using two-way ANOVA with Sidak's multiple comparison test.

[0081] (2) Experimental results As shown in Figure 8b, the vehicle administration group was unable to significantly distinguish between learned and novel objects, whereas the GV2002 administration group showed a significant increase in the time spent searching for novel objects compared to the time spent searching for learned objects, indicating an improvement in cognitive ability towards novel objects.

[0082] 4-4. Evaluation of spatial memory ability improvement using the Y-maze test The Y-maze test was performed on 4R tauopathy mouse models administered the drug for 5 months, from 7.5 months to 12.5 months of age, to evaluate the effect of GV2002 on improving spatial memory ability.

[0083] (1) Experimental method Before the experiment began, the mice were given one hour to acclimate to the environment. Then, the mice were placed at starting point A of a Y-shaped platform as shown in Figure 9a, and their behavior was recorded for eight minutes.

[0084] (2) Experimental results As shown in Figure 9b, the GV2002-administered group showed a significantly increased rate of spontaneous alternation—exploring new areas that were not previously explored—compared to the vehicle-administered group. This suggests an improvement in spatial memory ability.

[0085] 4-5. Evaluation of tau hyperphosphorylation suppression by immunofluorescence staining of brain tissue Brains from 4R tauopathy mouse models, administered drugs for five months from 7.5 months to 12.5 months of age, were extracted, and brain tissue was analyzed by immunofluorescence staining.

[0086] (1) Experimental method For brain tissue sampling, mouse models were anesthetized, saline solution was injected into the heart, and blood was removed from the body using a perfusion pump. The brain was then extracted and weighed. The brain tissue to be used for tissue staining experiments was fixed in a 4% PFA solution for 24 hours. The fixed brain tissue was then immersed in a 20% sucrose solution for 24 hours, followed by immersion in a 30% sucrose solution for 48 hours to remove moisture. After removing the dehydrated brain tissue and removing excess moisture, the brain tissue and a cryogenic tissue embedding compound (OCT compound) were placed together in a mold and frozen as quickly as possible with dry ice while protecting the tissue. The frozen brain tissue mold was stored in a -80°C freezer. Subsequently, the brain tissue was cut into 30 μm thick sections using a cryostat and stored in a 0.05% sodium azide solution.

[0087] For immunofluorescence staining of brain tissue, 30 μm thick frozen sections of brain tissue were sorted by brain region and immersed in PBS solution, washing three times for 10 minutes each. They were fixed with 3.7% formaldehyde for 5 minutes. They were then washed three times in PBS solution for 10 minutes each. Brain tissue samples were permeabilized in 0.3% PBS-T solution. Brain tissue samples were blocked for 1 hour in 5% BSA solution in PBS. As the primary antibody, AT8 (phospho-Tau S202 / T205), a tau hyperphosphorylated antibody, was diluted 1:200 in PBS with 3% BSA and 0.1% Tween-20 solution. Brain tissue was placed in this solution and allowed to bind overnight. Brain tissue was then washed three times in PBS solution for 10 minutes each. The secondary antibody was diluted 1:500 in PBS with 3% BSA and 0.1% Tween-20 solution. Brain tissue was placed in this solution and allowed to bind at room temperature for 1 hour. Brain tissue was immersed in PBS solution and washed once for 10 minutes. The brain tissue was immersed in PBS diluted to 1:2000 and stained the nuclei with Hoechst (stock conc. 1 mg / mL) solution. The brain tissue was immersed in PBS solution and washed three times for 10 minutes each. Fluorescence imaging of the brain tissue was performed using a slide scanner.

[0088] To obtain fluorescence images of stained brain tissue, brain tissue slides were scanned using a Zeiss Axio Scan (Zeiss, Oberkochen, Germany). AT8 fluorescence values ​​for different brain tissue regions obtained by fluorescence imaging—namely, the somatosensory cortex, hippocampus (CA1), motor cortex, and substantia nigra—were calculated using Image J software (NIH). The calculated values ​​were analyzed using one-way ANOVA with Dunnett's multiple comparison test.

[0089] (2) Experimental results As shown in Figure 10, tau hyperphosphorylation was significantly suppressed in all brain regions of the GV2002-treated group compared to the vehicle-treated group. As shown in Figure 10a, in the somatosensory cortical region, the AT8 fluorescence intensity in the GV2002-administered group was significantly reduced by 57% compared to the vehicle-administered group. As shown in Figure 10b, in the hippocampal region, the AT8 fluorescence intensity in the GV2002-treated group was significantly reduced by 62% compared to the vehicle-treated group. As shown in Figure 10c, in the motor cortex region, the AT8 fluorescence intensity in the GV2002-administered group was significantly reduced by 65% ​​compared to the vehicle-administered group. As shown in Figure 10d, in the substantia nigra region, the AT8 fluorescence intensity in the GV2002-administered group was significantly reduced by 69% compared to the vehicle-administered group. These findings confirm the inhibitory effect of GV2002 on 4R tau hyperphosphorylation.

[0090] 4-6. Evaluation of Tau-BiFC Imaging of Brain Tissue to Inhibit Tau Oligomer Formation Brains from 4R tauopathy mouse models, administered drugs for five months from 7.5 months to 12.5 months of age, were extracted, and brain tissue was analyzed by lipid staining.

[0091] (1) Experimental method Brain tissue samples were collected from a mouse model using the same method as in Examples 4-5. To remove autofluorescence from lipid staining of brain tissue, a 0.05% Sudan Black B solution in 70% ethanol was stirred overnight, and the particles were filtered through a 0.22 μm filter. Frozen sections of brain tissue, each 30 μm thick, were immersed in deionized water and washed three times for 5 minutes. Brain tissue was immersed in the prepared 0.05% Sudan Black B solution and stained for 10 minutes. Brain tissue stained with Sudan Black B was immersed in 0.1% PBS-T solution, and the degree of staining was adjusted by washing at 30-second intervals. It was washed three times for 5 minutes with deionized water. Nuclear staining was performed for 20 minutes by immersing brain tissue in 0.2 μg / mL Hoechst solution in deionized water. It was washed three times for 5 minutes with deionized water. Tau-BiFC imaging was performed using a slide scanner.

[0092] To obtain fluorescence images of stained brain tissue, brain tissue slides were scanned using a Zeiss Axio Scan. Tau-BiFC fluorescence values ​​for different brain tissue regions—namely the somatosensory cortex, hippocampus, motor cortex, and substantia nigra—obtained by fluorescence imaging were calculated using ImageJ software. The calculated values ​​were analyzed using one-way ANOVA with Dunnett's multiple comparison test.

[0093] (2) Experimental results As shown in Figure 11, tau oligomer formation was significantly suppressed in all brain regions of the GV2002-treated group compared to the vehicle-treated group. As shown in Figure 11a, in the somatosensory cortical region, the Tau-BiFC fluorescence intensity in the GV2002-administered group was significantly reduced by 58% compared to the vehicle-administered group. As shown in Figure 11b, in the hippocampal region, the Tau-BiFC fluorescence intensity in the GV2002-treated group was significantly reduced by 63% compared to the vehicle-treated group. As shown in Figure 11c, in the motor cortex region, the Tau-BiFC fluorescence intensity in the GV2002-administered group was significantly reduced by 56% compared to the vehicle-administered group. As shown in Figure 11d, in the substantia nigra region, the Tau-BiFC fluorescence intensity in the GV2002-administered group was significantly reduced by 56% compared to the vehicle-administered group. These findings confirm the inhibitory effect of GV2002 on tau oligomer formation.

[0094] 4-7. Evaluation of tau hyperphosphorylation and aggregation inhibition by analysis of brain lysates. Brain lysates were obtained from 4R tauopathy mouse models administered drugs for 5 months, from 7.5 months to 12.5 months of age. Soluble and insoluble fractions were subjected to immunoblotting using total tau (Tau5) and phosphorylated tau (pS199, pS396) antibodies to quantitatively analyze the levels of total tau and phosphorylated tau.

[0095] (1) Experimental method Mouse models were anesthetized, and saline solution was injected into their hearts. Blood was then removed from their bodies using a perfusion pump. The brains were then removed and weighed. One mL of RIPA buffer (containing a cocktail of protease and phosphatase inhibitors) was added to the removed brains and homogenized.

[0096] The homogenized lysate was transferred to an EP tube and incubated in an orbital shaker at 4°C for 2 hours. The mixture was then centrifuged at 4°C and 13,000 rpm for 20 minutes, and the supernatant (soluble fraction) was collected in a new EP tube and stored at -80°C.

[0097] To prepare the immunoblot samples, the concentrations of the fractional samples were quantified using the Bradford protein quantification method. RIPA buffer and 4X SDS-Laemmli sample buffer (containing 2.5% β-mercaptoethanol for reducing conditions) were added to each sample to a final concentration of 2 mg / mL. The samples were then boiled at 97°C for 5 minutes.

[0098] Meanwhile, to sample the insoluble fraction, the supernatant was transferred and the remaining pellet was dissolved in RIPA buffer containing 1 mL of 1 M sucrose and DNase I (1 mg / mL concentration). After centrifugation at 13,000 rpm at 4°C for 20 minutes, the supernatant was removed, and the remaining pellet was resuspended in 2% SDS solution (1 mL per gram of tissue) and incubated at room temperature for 1 hour. Centrifuged at 13,000 rpm at room temperature for 1 minute, the supernatant (insoluble fraction) was collected and transferred to a new ep tube, and an equal volume of 2 × SDS Laemmli sample buffer (2.5% β-mercaptoethanol for reducing conditions) was added. The sample was boiled at 97°C for 5 minutes.

[0099] 20 μg each of soluble and insoluble brain lysate samples were loaded onto 10% SDS-PAGE gels (SDS-PAGE running buffer: 1X Tris / Glycine / SDS buffer in 3'DW). Electrophoresis was performed at 80V for 30 minutes, then at 90V for 1 hour. PVDF membranes, cut to fit the size of the transfer tray, were immersed in 100% methanol for 1 minute, autoclaved in 3'DW for 3 minutes, and then immersed in chilled 1X transfer buffer (Transfer buffer: 1X Tris / Glycine buffer w / 20% Methanol in 3'DW) for 10 minutes. Wet transfer was performed at 100V for 1 hour and 20 minutes. The membranes were immersed in 5% BSA solution in TBS-T (containing 0.1% Tween-20) and blocked at room temperature for 1 hour. The membrane was immersed in a 2.5% BSA solution in TBS-T (containing 0.1% Tween-20) containing primary antibodies (Anti-Tau(Tau5)antibody(ab80579,1:5000), Anti-Tau(phospho S199)antibody(ab109390,1:5000), and Anti-Tau(phospho S396)antibody(ab81268,1:5000)) and conjugated overnight in an orbital shaker at 4°C. The membrane was washed three times for 10 minutes each time by immersing it in TBS-T (containing 0.1% Tween-20) solution. The membrane was immersed in a 2.5% BSA solution in TBS-T (containing 0.1% Tween-20) containing secondary antibodies (Goat Anti-Mouse IgG H&L (HRP) secondary antibody (ab6789, 1:10000) and Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab6721, 1:10000)) and allowed to bind at room temperature for 1 hour. The membrane was washed three times for 10 minutes each time by immersing it in TBS-T (containing 0.1% Tween-20) solution. Bands on the membrane were detected by ChemiDoc using ECL solution, which is an HRP substrate.The intensity of the detected bands was quantified using Image J software (NIH).

[0100] (2) Experimental results As shown in Figure 12, tau immunoblotting results using the soluble fraction showed that the levels of phosphorylated tau pS199 and pS396 were significantly reduced in the GV2002-treated group compared to the vehicle-treated group. Tau immunoblotting results using the insoluble fraction also showed a significant reduction in total tau in the GV2002-treated group compared to the vehicle-treated group. As shown in Figure 12a, phosphorylated tau pS199 in the GV2002 administration group was significantly reduced by 66% compared to the vehicle administration group. As shown in Figure 12b, phosphorylated tau pS396 in the GV2002 administration group was significantly reduced by 56% compared to the vehicle administration group. As shown in Figure 12c, the level of insoluble tau in the GV2002 administration group was significantly reduced by 57% compared to the vehicle administration group. These findings confirm that GV2002 has an inhibitory effect on phosphorylated tau formation and an inhibitory effect on insoluble tau aggregation.

Claims

1. A peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. A pharmaceutical composition for the prevention or treatment of 4R tauopathy, comprising the peptide described in claim 1 or a pharmaceutically acceptable salt thereof.

3. The 4R tauopathy is one selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrolytic granulopathy (AGD), globular glial tauopathy (GGT), and aging-related tau astrogliopathy (ARTAG), as described in claim 2, a pharmaceutical composition for the prevention or treatment of 4R tauopathy.

4. The prevention or treatment of 4R tauopathy is achieved by inhibiting the hyperphosphorylation and aggregation of tau protein, as described in claim 3, for the pharmaceutical composition for the prevention or treatment of 4R tauopathy.

5. A health functional food for the prevention or improvement of 4R tauopathy, comprising the peptide of the following chemical formula 1 or a food-safe salt thereof. 【Chemistry 2】

6. The 4R tauopathy is one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, argyrophilic granulopathy, spheroid glial tauopathy, and age-related tauastrogliopathies, as described in claim 5, a health functional food for the prevention or improvement of 4R tauopathy.

7. The prevention or improvement of 4R tauopathy is achieved by inhibiting the hyperphosphorylation and aggregation of tau protein, as described in claim 6, for the health functional food for the prevention or improvement of 4R tauopathy.

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