Novel peptide and use thereof

Abstract

The present invention relates to: a peptide which has the effects of preventing, alleviating, and / or treating 4R tauopathy, and which is safe with respect to the human body, and thus has reduced side effects including an abnormal reaction; and a pharmaceutical composition and a health functional food, which comprise same.

Description

Novel peptides and their uses

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

[0002]

[0003] Tau is abundant in neurons of the central nervous system (CNS) and plays a role in maintaining the stability of microtubules in axons.

[0004] When hyperphosphorylation occurs, where excessive phosphate is attached to the tau protein, tau detaches from microtubules and forms insoluble aggregates called neurofibrillary tangles (NFTs). Neurodegenerative diseases associated with these abnormal tau aggregates are collectively called tauopathies.

[0005] Tauopathies are classified into 3R tauopathy and 4R tauopathy depending on the isoform of the tau protein that causes them.

[0006] 4R tauopathies include progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), globular glial tauopathy (GGT), and age-related tau astrogliopathy (ARTAG).

[0007] The number of patients with 4R tauopathy is increasing with the aging population, but there are currently no drugs that can effectively treat or prevent it.

[0008] Phase 3 clinical trials are underway for LMTM, known as a tau aggregation inhibitor, but there have been no reports on the specific efficacy of LMTM for 4R tauopathy. In addition, LMTM has a tau aggregation inhibition interval (EC).50 Cytotoxicity (GI) at 2.2±0.2 μM 50 There is a problem that (= 6.4±0.3 μM) appears.

[0009] Accordingly, the inventors of the present invention completed the present invention as a result of extensive research to treat 4R tauopathy by inhibiting tau hyperphosphorylation and aggregation.

[0010]

[0011] The present invention aims to provide a novel peptide having a preventive or therapeutic effect on 4R tauopathy.

[0012] The present invention aims to provide a pharmaceutical composition for preventing or treating 4R tauopathy.

[0013] The purpose of the present invention is to provide a health functional food for preventing or improving 4R tauopathy.

[0014]

[0015] 1. A peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0016] [Chemical Formula 1]

[0017]

[0018] 2. A pharmaceutical composition for preventing or treating 4R tauopathy, comprising the peptide of 1 above or a pharmaceutically acceptable salt thereof.

[0019] 3. A pharmaceutical composition for preventing or treating 4R tauopathy, wherein in the above 2, the 4R tauopathy is any one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, arginine affinity tauopathy, globular tauopathy, and age-related tauoastrocytopathy.

[0020] 4. A pharmaceutical composition for preventing or treating 4R tauopathy, wherein the prevention or treatment of 4R tauopathy in the above 3 is achieved by inhibiting hyperphosphorylation and aggregation of tau protein.

[0021] 5. A health functional food for preventing or improving 4R tauopathy, comprising a peptide of the following chemical formula 1 or a food-based salt thereof:

[0022] [Chemical Formula 1]

[0023]

[0024] 6. In the above 5, the 4R tauopathy is any one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, arginine affinity tauopathy, globular tauopathy, and age-related tauoastrocytopathy, a health functional food for preventing or improving 4R tauopathy.

[0025] 7. A health functional food for preventing or improving 4R tauopathy, wherein the prevention or improvement of 4R tauopathy in the above 6 is achieved by suppressing hyperphosphorylation and aggregation of tau protein.

[0026]

[0027] The peptide of chemical formula 1 of the present invention and its pharmaceutically acceptable salt have an excellent tau protein hyperphosphorylation inhibitory effect.

[0028] The peptide of chemical formula 1 of the present invention and its pharmaceutically acceptable salt have an excellent tau protein aggregation inhibitory effect.

[0029] The pharmaceutical composition and health functional food comprising the peptide of chemical formula 1 of the present invention or a salt thereof can exhibit a preventive, ameliorating and / or therapeutic effect on 4R tauopathy by inhibiting tau protein hyperphosphorylation and aggregation.

[0030] The peptide, pharmaceutical composition and health functional food of the present invention can stably exert a long-term medicinal effect in vivo.

[0031]

[0032] Figure 1 shows the results of an experiment confirming the stability of GV2002 in human plasma.

[0033] Figure 2 shows the results of confirming the pharmacokinetic profile (PK profile) of GV2002 after intravenous administration to rats.

[0034] Figure 3 shows the results of confirming the pharmacokinetic profile of GV2002 after subcutaneous administration to rats.

[0035] Figure 4 shows the mouse groups used in the in vivo experiment to verify the efficacy of GV2002.

[0036] Figure 5 shows the schedule of the efficacy evaluation experiment of GV2002 performed on a 4R tauopathy mouse model.

[0037] Figures 6 and 7 show the results of an experiment evaluating improvement in motor ability in a 4R tauopathy mouse model.

[0038] Figure 8 shows the experimental method and results for evaluating cognitive ability improvement in a 4R tauopathy mouse model.

[0039] Figure 9 shows the experimental method and results for evaluating improvement in spatial memory ability of a 4R tauopathy mouse model.

[0040] Figure 10 shows the results of an experiment evaluating the inhibitory efficacy of GV2002 on tau hyperphosphorylation through brain tissue immunofluorescence staining.

[0041] Figure 11 shows the results of an experiment evaluating the inhibitory efficacy of GV2002 on tau oligomer formation through brain tissue Tau-BiFC imaging.

[0042] Figure 12 shows the results of an experiment evaluating the inhibitory efficacy of GV2002 on tau hyperphosphorylation and aggregation through brain lysate analysis.

[0043]

[0044] The present invention provides a peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0045] [Chemical Formula 1]

[0046]

[0047] In the present 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.

[0048]

[0049] The present invention provides a pharmaceutical composition for preventing or treating 4R tauopathy, comprising a peptide of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0050] In the present invention, “pharmaceutically acceptable” means that the composition exhibits the property of being non-toxic to cells or organisms exposed to the composition.

[0051] In the present invention, 4R tauopathy is a general term for neurodegenerative diseases caused by the aggregation of 4R tau protein, and includes progressive supranuclear palsy, corticobasal degeneration, arginine affinity neuropathy, globular astrocytic tauopathy, and age-related tauoastrocytopathy.

[0052] In the present invention, “prevention” means any action that inhibits or delays 4R tauopathy.

[0053] In the present invention, “improvement” and “treatment” mean any action that improves or beneficially changes the symptoms of an individual suspected of having or having developed 4R tauopathy.

[0054] In one embodiment, prevention, improvement and / or treatment may be achieved by inhibiting hyperphosphorylation of tau protein.

[0055] In one embodiment, prevention, improvement and / or treatment may be achieved by inhibiting tau protein aggregation.

[0056] In one embodiment, the prevention, improvement and / or treatment may be to improve the motor skills of an individual suspected of having or having developed a 4R tauopathy.

[0057] In one embodiment, the prevention, improvement and / or treatment may be to improve cognitive abilities in an individual suspected of having or having developed a 4R tauopathy.

[0058] In one embodiment, the prevention, improvement and / or treatment may be to improve spatial memory abilities in an individual suspected of having or having developed a 4R tauopathy.

[0059] The pharmaceutical composition of the present invention may contain the active ingredient alone, or may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0060] The carrier, excipient or diluent that may be included in the pharmaceutical composition of the present invention may be, but is not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0061] The pharmaceutical composition of the present invention can be administered to a subject.

[0062] In the present invention, “administration” means introducing a predetermined substance to an individual by an appropriate method, and “individual” means any animal, such as livestock and mice, that has developed or may develop 4R tauopathy, and may be a mammal, including a human.

[0063] The route of administration of the pharmaceutical composition of the present invention may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal, but is not limited thereto.

[0064] In one embodiment, the composition of the present invention may be administered orally or parenterally.

[0065] When administering the composition of the present invention parenterally, it is preferable to select an injection method such as external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection, but is not limited thereto.

[0066] The pharmaceutical composition of the present invention may be provided in the form of a solid preparation for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0067] The pharmaceutical composition of the present invention may be provided in a liquid form for oral administration, such as a suspension, a solution, an emulsion, or a syrup.

[0068] The pharmaceutical composition of the present invention may be provided as a preparation for parenteral administration, such as a sterile aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized preparation or suppository.

[0069] The pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount, whereby a “pharmaceutically effective amount” means an amount sufficient to treat 4R tauopathy at a reasonable benefit / risk ratio applicable to medical treatment.

[0070] The pharmaceutically effective amount of the pharmaceutical composition of the present invention can be determined based on factors including the severity of 4R tauopathy, activity of the pharmaceutical composition, sensitivity of the subject or patient to the pharmaceutical composition, time of administration, route of administration, excretion rate, duration of treatment, and concomitant medications, and other factors well known in the medical field, and can be appropriately selected by a person skilled in the art.

[0071] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other conventional therapeutic agents, and when administered in combination, may be administered sequentially or simultaneously, and may be administered singly or in multiple doses, which can be readily determined by a person skilled in the art.

[0072]

[0073] The present invention provides a health functional food for preventing or improving 4R tauopathy, comprising a peptide of chemical formula 1 or a food-related salt thereof.

[0074] The health functional food of the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to the Health Functional Food Act, and functionality means consuming it for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0075] The food composition of the present invention may include conventional food additives, and the suitability of food additives is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0076] Items listed in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0077] The health functional food of the present invention may contain 0.01 to 95 wt%, preferably 1 to 80 wt%, of the peptide of chemical formula 1 based on the total weight of the health functional food for the purpose of preventing and / or improving 4R tauopathy. In addition, the health functional food may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing and / or improving 4R tauopathy.

[0078]

[0079] Hereinafter, the present invention will be described in detail with examples to specifically illustrate the invention. However, the following examples are provided merely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0080]

[0081] Example

[0082]

[0083] [Example 1] Preparation of GV2002

[0084] A peptide (hereinafter referred to as GV2002) having the structural formula of the following chemical formula 1 was prepared.

[0085]

[0086] [Chemical Formula 1]

[0087] GV2002 was synthesized by coupling amino acids one by one from the C-terminus according to the known Fmoc solid phase peptide synthesis (SPPS) method.

[0088] All amino acid starting materials used for peptide synthesis were N-terminally protected with Fmoc, and all residues were protected with Boc, t-Bu (t-butylester), or Pbf (2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl), which can be removed by acid. The amino acid starting materials used for peptide synthesis are as follows:

[0089] 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.

[0090] Diisopropylcarbodiimide (DIC), dimethylforamide (DMF), ethyl(hydroxyamino)cyanoacetate (Oxyma), and isopropyl alcohol (IPA) were used as coupling reagents. 20% piperidine in DMF was used for Fmoc removal. Cleavage cocktail [95% trifluoroacetic acid (TFA) / 5% H2O / 50 mg / mL dithiothreitol (DTT)] was used to detach the synthesized peptide from the resin and remove the protecting groups of the residues.

[0091] 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 aminolated.

[0092] Peptides were synthesized by sequentially reacting each amino acid, washing with a solvent, and deprotecting the starting amino acid, while the amino acid protecting group was bound to a solid support. The synthesized peptide was excised from the resin, purified by HPLC, confirmed to be synthesized by MS, and lyophilized.

[0093] In the counter ion exchange process, GV2002, dissolved in water in the form of HOAc salt, was replaced with a chloride form using AmberChrom ion exchange resin. The replaced GV2002 was filtered through a PVDF filter and lyophilized.

[0094] The specific synthesis process of GV2002 is as follows:

[0095] (1) Coupling

[0096] Amino acids were coupled to Fmoc-Rink Methylbenzhydrylamine resin by adding each amino acid from the C-terminus to the N-terminus and coupling reagents DIC / Oxyma / IPA dissolved in DMF. The resin was then washed with DMF.

[0097] (2) Fmoc deprotection and N-terminal capping

[0098] After Fmoc deprotection by treatment with 20% piperidine in DMF, the N-terminus was acetylated.

[0099] (3) Cutting

[0100] The peptide was separated from the resin by treating the resin with a cleavage cocktail to which the synthesized peptide was bound.

[0101] (4) Filtration

[0102] The resulting mixture was concentrated and precipitated with MTBE / hexane. The precipitate was then filtered and dried.

[0103] (5) Purification

[0104] The obtained dried filtrate was purified by Prep-HPLC, the molecular weight was confirmed by LC / MS, and then freeze-dried.

[0105] (6) Powder manufacturing

[0106] AmberChrom resin was used to replace chloride, filtered through PVDF, and then freeze-dried to produce a powder.

[0107]

[0108] [Example 2] Confirmation of the stability of GV2002 in human plasma

[0109] Peptides were added to 100% human plasma, diluted to 5 μM, and incubated with stirring at 37°C for 10, 60, and 240 minutes. The resulting solution 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 to determine the ratio (%) of residual peptide to the amount of peptide before the reaction.

[0110]

[0111] As shown in Figure 1, the ratio of residual peptide to the total peptide amount before the reaction of GV2002 was 57.23% at 60 minutes and 46.54% at 240 minutes. This confirmed the stability of GV2002 in plasma.

[0112]

[0113] [Example 3] Confirmation of the pharmacokinetic profile of GV2002

[0114] In order to confirm the pharmacokinetic profile of GV2002 manufactured in Example 1, plasma from rats administered GV2002 was collected, pretreated by protein precipitation using methanol containing 9 times the amount of 0.1% formic acid, centrifuged at 16,100 g for 10 minutes, and the supernatant was collected and subjected to LC-MS analysis under the conditions of Table 2 below.

[0115]

[0116]

[0117] 3-1. Confirmation of the pharmacokinetic profile of GV2002 through intravenous administration

[0118] 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 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.

[0119] As a result, as shown in Fig. 2, the AUC of GV2002 t The value was found to be 0.438 ug·h / mL. This confirmed that GV2002 remains stable in vivo without being decomposed for a long time.

[0120]

[0121] 3-2. Confirmation of the pharmacokinetic profile of GV2002 through subcutaneous administration

[0122] 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 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.

[0123] As a result, as shown in Fig. 3, it can be seen that even when subcutaneous administration was performed, GV2002 remained stable in the body for a long time without being decomposed.

[0124]

[0125] [Example 4] Evaluation of GV2002 Efficacy Based on 4R Tauopathy Animal Model

[0126] In vivo experiments were performed on the 4R tauopathy mouse group shown in Fig. 4 according to the schedule shown in Fig. 5 and the outline in Table 3 below.

[0127]

[0128]

[0129] 4-1. Evaluation of improved motor skills through the Rota-rod experiment

[0130] The efficacy of GV2002 in improving motor ability was evaluated by conducting a rotarod experiment on a 4R tauopathy mouse model administered with the drug for 5 months from 7.5 to 12.5 months of age.

[0131]

[0132] (1) Experimental method

[0133] On the first day, acclimation training was conducted, and on the second day, behavioral experiments were conducted. Before acclimation training and behavioral experiments, mice were allowed 1 hour to adapt to the space. During acclimation training, mice were made to walk on a treadmill at a speed of 5 to 25 rpm for 300 seconds. Mice that lost their balance and fell to the floor were made to walk again. In the behavioral experiment, mice were made to walk on a treadmill at a speed that gradually increased from 5 to 40 rpm for 300 seconds, and the time (in seconds) until the mouse lost its balance and fell to the floor was measured. The measured values ​​were analyzed by two-way ANOVA with Dunnett's multiple comparisons test.

[0134]

[0135] (2) Experimental results

[0136] As shown in Fig. 6, the GV2002 administration group showed significantly improved exercise ability compared to the vehicle administration group, and showed relatively improved exercise ability compared to the reference compound LMTM administration group.

[0137]

[0138] 4-2. Evaluation of motor ability improvement through open space experiments

[0139] The efficacy of GV2002 in improving motor ability was evaluated in an open field experiment using a 4R tauopathy mouse model administered the drug for 5 months from 7.5 to 12.5 months of age.

[0140]

[0141] (1) Experimental method

[0142] To prevent changes in the behavioral responses of mice, handling was performed for 5 days (days 1 to 5) prior to the experiment. Handling lasted 2 minutes per session. On day 6, mice were placed in a 40 cm x 40 cm enclosure and subjected to an open-space experiment for 10 minutes. The mice were allowed 1 hour to acclimate to the space before the experiment. In the open-space experiment, the movement trajectories, distance traveled, and movement speed of the mice were recorded. The lighting was maintained at a constant level throughout the experiment. The recorded values ​​were analyzed using a one-way ANOVA followed by Dunnett's multiple comparisons test.

[0143]

[0144] (2) Experimental results

[0145] As shown in Fig. 7, the total distance traveled and movement speed in the open space were significantly higher in the GV2002 administration group than in the vehicle administration group, indicating that the exercise ability of the GV2002 administration group was improved.

[0146]

[0147] 4-3. Evaluation of cognitive ability improvement through novel object recognition experiments.

[0148] A novel object recognition experiment was conducted on a 4R tauopathy mouse model administered the drug for 5 months from 7.5 to 12.5 months of age to evaluate the cognitive ability improvement efficacy of GV2002.

[0149]

[0150] (1) Experimental method

[0151] A novel object recognition experiment was conducted on mice that had been tested from day 1 to day 6 according to the experimental method of Example 4-2 on days 7 and 8. On day 7, the mice were placed in a 40 cm x 40 cm partition with two similar objects arranged as shown in Fig. 8a and allowed to learn for 10 minutes. On day 8, one of the two objects was replaced with a novel one, and the mice were placed in the partition and their behavior was observed for 10 minutes, measuring the time spent on the novel object. Before performing the experiment on days 7 and 8, the mice were allowed 1 hour to adapt to the space. A constant lighting level was maintained throughout the experiment. The recorded values ​​were analyzed by Two-way ANOVA with Sidak's multiple comparisons test.

[0152]

[0153] (2) Experimental results

[0154] As shown in Figure 8b, the vehicle-administered group failed to significantly distinguish between learned objects and novel objects, whereas the GV2002-administered group showed an improvement in cognitive ability for novel objects, with the time spent exploring novel objects significantly increasing compared to the time spent exploring learned objects.

[0155]

[0156] 4-4. Evaluation of spatial memory improvement through the Y-maze experiment

[0157] The efficacy of GV2002 in improving spatial memory was evaluated by conducting a Y-maze experiment on a 4R tauopathy mouse model that was administered the drug for 5 months from 7.5 to 12.5 months of age.

[0158]

[0159] (1) Experimental method

[0160] Before conducting the experiment, the mice were allowed one hour to acclimate to the space. Then, the mice were placed at starting point A of the Y-shaped platform, as shown in Figure 9a. The mice's behavior was recorded for eight minutes.

[0161]

[0162] (2) Experimental results

[0163] As shown in Figure 9b, the GV2002-administered group showed a significantly increased rate of spontaneous alternation behavior, which involves exploring a new area rather than a previously explored area, compared to the vehicle-administered group, indicating improved spatial memory ability.

[0164]

[0165] 4-5. Evaluation of inhibition of tau hyperphosphorylation through brain tissue immunofluorescence staining

[0166] The brains of a 4R tauopathy mouse model that was administered drugs for 5 months from 7.5 to 12.5 months of age were removed and brain tissues were analyzed using immunofluorescence staining.

[0167]

[0168] (1) Experimental method

[0169] For brain tissue sampling, mouse models were anesthetized, saline was perfused intracardiacly, and blood was removed using a perfusion pump. The brains were then removed and weighed. Brains to be used for tissue staining experiments were fixed in a 4% PFA solution for 24 hours. The fixed brain tissue was then immersed in a 20% sucrose solution for 24 hours and then in a 30% sucrose solution for 48 hours to remove moisture. The dehydrated brain tissue was removed, excess moisture was removed, and the brain tissue and a cryoembedding agent (OCT compound) were placed together in a mold to protect the tissue and rapidly freeze the brain tissue on dry ice. The frozen brain tissue molds were stored in a -80°C freezer. Afterwards, the brain tissue was sectioned into 30 μm-thick sections using a cryostat and stored in a 0.05% sodium azide solution.

[0170] For immunofluorescence staining of brain tissue, 30 μm-thick frozen brain sections were selected by brain region, washed three times in PBS for 10 minutes each, fixed in 3.7% formaldehyde for 5 minutes, and washed three times in PBS for 10 minutes each. The brain tissue samples were permeabilized in 0.3% PBS-T. The brain tissue samples were blocked in 5% BSA in PBS for 1 hour. The brain tissues were placed in a solution of 3% BSA and 0.1% Tween-20 in PBS containing a 1:200 dilution of AT8 (phospho-Tau S202 / T205), a tau hyperphosphorylation antibody, and bound overnight. The brain tissues were washed three times in PBS for 10 minutes each. Brain tissues were placed in a solution of 3% BSA and 0.1% Tween-20 in PBS, in which the secondary antibody was diluted 1:500, and bound for 1 hour at room temperature. The brain tissues were soaked in PBS and washed once for 10 minutes. Nuclei were stained by soaking the brain tissues in a solution of Hoechst (stock concentration 1 mg / mL) in PBS, diluted 1:2000. The brain tissues were soaked in PBS and washed three times for 10 minutes. Fluorescence imaging of the brain tissues was performed using a slide scanner.

[0171] To obtain fluorescent images of stained brain tissue, brain tissue slides were scanned using a Zeiss Axio Scan (Zeiss, Oberkochen, Germany). AT8 fluorescence levels in each brain region obtained through fluorescent imaging, i.e., the somatosensory cortex, hippocampus (CA1), motor cortex, and substantia nigra, were calculated using Image J software (NIH). The calculated values ​​were analyzed by one-way ANOVA with Dunnett's multiple-comparisons test.

[0172]

[0173] (2) Experimental results

[0174] As shown in Figure 10, tau hyperphosphorylation was significantly suppressed in all brain regions of the GV2002 administration group compared to the vehicle administration group.

[0175] As shown in Fig. 10a, the AT8 fluorescence intensity in the GV2002-administered group in the sensorimotor cortex area was significantly reduced by 57% compared to the vehicle-administered group.

[0176] As shown in Fig. 10b, the AT8 fluorescence intensity in the GV2002-administered group in the hippocampal region was significantly reduced by 62% compared to the vehicle-administered group.

[0177] As shown in Fig. 10c, the AT8 fluorescence intensity in the motor cortex area of ​​the GV2002-administered group was significantly reduced by 65% ​​compared to the vehicle-administered group.

[0178] As shown in Fig. 10d, the AT8 fluorescence intensity in the substantia nigra region of the GV2002-administered group was significantly reduced by 69% compared to the vehicle-administered group.

[0179] This confirmed the inhibitory effect of GV2002 on 4R tau hyperphosphorylation.

[0180]

[0181] 4-6. Evaluation of inhibition of tau oligomer formation through brain tissue Tau-BiFC imaging

[0182] The brains of a 4R tauopathy mouse model that was administered drugs for 5 months from 7.5 to 12.5 months of age were removed and brain tissue was analyzed using lipid staining.

[0183]

[0184] (1) Experimental method

[0185] Brain tissue sampling of the mouse model was performed using the same method as in Example 4-5.

[0186] To remove autofluorescence through lipid staining of brain tissue, 0.05% Sudan Black B solution in 70% ethanol was stirred overnight and then filtered through a 0.22 μm filter. 30 μm-thick frozen brain sections were washed three times for 5 minutes each in deionized water. The brain tissue was immersed in the prepared 0.05% Sudan Black B solution and stained for 10 minutes. The brain tissue stained with Sudan Black B was washed in 0.1% PBS-T solution at 30-second intervals to adjust the staining intensity. The tissue was washed three times for 5 minutes each in deionized water. The brain tissue was immersed in a 0.2 μg / mL Hoechst solution in deionized water for 20 minutes to stain the nuclei. The brain tissue was washed three times for 5 minutes each in deionized water. Tau-BiFC imaging was performed using a slide scanner.

[0187] To obtain fluorescent images of stained brain tissue, brain tissue slides were scanned using a Zeiss Axio Scan. Tau-BiFC fluorescence levels in each brain region obtained through fluorescent imaging, i.e., the sensorimotor cortex, hippocampus, motor cortex, and substantia nigra, were calculated using ImageJ software. The calculated values ​​were analyzed using one-way ANOVA with Dunnett's multiple-comparisons test.

[0188]

[0189] (2) Experimental results

[0190] As shown in Figure 11, tau oligomer formation was significantly suppressed in all brain regions of the GV2002 administration group compared to the vehicle administration group.

[0191] As shown in Figure 11a, the Tau-BiFC fluorescence intensity in the sensorimotor cortex area of ​​the GV2002-administered group was significantly reduced by 58% compared to the vehicle-administered group.

[0192] As shown in Figure 11b, the Tau-BiFC fluorescence intensity in the hippocampal region of the GV2002-administered group was significantly reduced by 63% compared to the vehicle-administered group.

[0193] As shown in Figure 11c, the Tau-BiFC fluorescence intensity in the motor cortex area of ​​the GV2002-administered group was significantly reduced by 56% compared to the vehicle-administered group.

[0194] As shown in Figure 11c, the Tau-BiFC fluorescence intensity in the substantia nigra region of the GV2002-administered group was significantly reduced by 56% compared to the vehicle-administered group.

[0195] This confirmed the inhibitory effect of GV2002 on tau oligomer formation.

[0196]

[0197] 4-7. Evaluation of inhibition of tau hyperphosphorylation and aggregation through brain lysate analysis

[0198] Brain lysates from a 4R tauopathy mouse model administered drugs for 5 months from 7.5 to 12.5 months of age were obtained, and immunoblots were performed using total tau (Tau5) and phosphorylated tau (pS199, pS396) antibodies on soluble and insoluble fraction samples, and the levels of total tau and phosphorylated tau were quantitatively analyzed.

[0199]

[0200] (1) Experimental method

[0201] Mouse models were anesthetized, saline was perfused into the heart, and blood was removed using a perfusion pump. The brains were then removed and weighed. The extracted brains were homogenized in 1 mL of RIPA buffer (containing a cocktail of protease and phosphatase inhibitors).

[0202] The homogenized brain lysate was transferred to an EP tube and incubated for 2 h on an orbital shaker at 4°C. After centrifugation at 13,000 rpm for 20 min at 4°C, the supernatant (soluble fraction) was collected in a new EP tube and stored at -80°C.

[0203] To prepare immunoblot samples, the concentration of fractionated samples was 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.

[0204] 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 for 20 min at 4°C, the supernatant was removed, and the remaining pellet was resuspended in 2% SDS solution (1 mL per 1 g of tissue) and incubated at room temperature for 1 h. Centrifugation was performed at 13,000 rpm for 1 min at room temperature, and the supernatant (insoluble fraction) was collected and transferred to a new ep-tube, followed by the addition of an equal volume of 2x SDS Laemmli sample buffer (2.5% β-mercaptoethanol for reducing conditions). The sample was boiled at 97°C for 5 min.

[0205] Twenty μg of brain lysate samples from the soluble and insoluble fractions were loaded onto a 10% SDS-PAGE gel (SDS-PAGE running buffer: 1X Tris / Glycine / SDS buffer in 3'DW). Electrophoresis was performed at 80 V for 30 min and then at 90 V for 1 h. PVDF membranes cut to the size of the transfer tray were soaked in 100% methanol for 1 min, autoclaved 3'DW for 3 min, and then in chilled 1X Transfer buffer (Transfer buffer: 1X Tris / Glycine buffer w / 20% methanol in 3'DW) for 10 min. Wet-transfer was performed at 100 V for 1 h 20 min. The membrane was blocked in 5% BSA solution in TBS-T (containing 0.1% Tween-20) for 1 hour at room temperature. The membrane was incubated with 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)) overnight at 4°C on an orbital shaker. The membrane was washed three times for 10 minutes in TBS-T (containing 0.1% Tween-20). The membrane was soaked in 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 bound for 1 hour at room temperature. The membrane was washed three times for 10 minutes in TBS-T (containing 0.1% Tween-20).Membrane bands were detected using ChemiDoc using ECL solution, an HRP substrate. The intensity of the detected bands was quantified using Image J software (NIH).

[0206]

[0207] (2) Experimental results

[0208] As shown in Figure 12, the results of tau immunoblotting using the available fraction showed that the levels of phosphorylated tau pS199 and pS396 in the GV2002 administration group were significantly reduced compared to the vehicle administration group, and the results of tau immunoblotting using the insoluble fraction also showed that the total tau in the GV2002 administration group was significantly reduced compared to the vehicle administration group.

[0209] As shown in Figure 12a, phosphorylated tau pS199 was significantly reduced by 66% in the GV2002 administration group compared to the vehicle administration group.

[0210] As shown in Figure 12b, phosphorylated tau pS396 was significantly reduced by 56% in the GV2002 administration group compared to the vehicle administration group.

[0211] 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.

[0212] This confirmed that GV2002 has the effect of inhibiting phosphorylated tau formation and insoluble tau aggregation.

Claims

1. A peptide of the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 2. A pharmaceutical composition for preventing or treating 4R tauopathy, comprising the peptide of claim 1 or a pharmaceutically acceptable salt thereof.

3. A pharmaceutical composition for preventing or treating 4R tauopathy according to claim 2, wherein the 4R tauopathy is any one selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), globular glial tauopathy (GGT), and age-related tau astrogliopathy (ARTAG).

4. A pharmaceutical composition for preventing or treating 4R tauopathy according to claim 3, wherein the prevention or treatment of 4R tauopathy is achieved by inhibiting hyperphosphorylation and aggregation of tau protein.

5. A health functional food for preventing or improving 4R tauopathy, comprising a peptide of the following chemical formula 1 or a food-based salt thereof: [Chemical Formula 1] 6. A health functional food for preventing or improving 4R tauopathy, wherein the 4R tauopathy according to claim 5 is any one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, arginine affinity neuropathy, globular tauopathy, and age-related tauoastrocytopathy.

7. A health functional food for preventing or improving 4R tauopathy according to claim 6, wherein the prevention or improvement of 4R tauopathy is achieved by suppressing hyperphosphorylation and aggregation of tau protein.

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