Use of methylated peptide as therapeutic agent for degenerative brain diseases

Abstract

The present invention relates to a use of a methylated peptide as a therapeutic agent for degenerative brain diseases. Specifically, it has been confirmed that the methylated peptide according to the present invention exhibits improved solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to a conventional unmodified peptide, and exhibits excellent amyloid beta reduction efficacy compared to a conventional unmodified peptide in an animal model of degenerative brain diseases. Therefore, the methylated peptide according to the present invention can be usefully used as an active ingredient of a composition for preventing or treating degenerative brain diseases and cognitive impairment, learning disability, or memory impairment associated therewith.

Description

Uses of methylated peptides as therapeutic agents for neurodegenerative diseases

[0001] The present invention relates to the use of a methylated peptide as a therapeutic agent for degenerative brain diseases, and more specifically, to a peptide having an amino acid sequence represented by SEQ ID NO. 1, in which at least one proline residue is N-methylated, and the use of the same as a therapeutic agent for degenerative brain diseases.

[0002] Neurodegenerative diseases refer to diseases that occur in the brain among age-related degenerative conditions. They can be classified based on major symptoms and the affected brain regions, with representative examples including Alzheimer's disease and Parkinson's disease. Neurodegenerative diseases are known to be caused by the death of nerve cells resulting from the aggregation of proteins due to age-related neurodegeneration as well as genetic and environmental factors.

[0003] Furthermore, degenerative brain diseases involve the death or degeneration of specific brain cells progressing either temporarily or over a long period. Since brain cells do not regenerate once they die, it is known to eventually lead to a fatal loss of brain function. In particular, brain dysfunction accompanied by the progressive decline of cognitive, sensory, motor, and systemic functions ultimately leads to changes in personality and behavior, rendering patients unable to care for themselves.

[0004] It has been reported that the activity of microglia and astrocytes is involved in the onset and progression of neurodegenerative diseases. Microglia are the major glial cells of the brain and spinal cord, playing an important role in maintaining normal brain function and neuroprotection, and contributing to the removal of excessive synapses, amyloid beta (Aβ), and neurofibrillary tangles.

[0005] The role of microglia in the pathogenesis of Alzheimer's disease, the most common form of dementia, is being elucidated. For example, microglia or monocytes are observed surrounding amyloid beta in the brains of Alzheimer's patients and mouse models; while microglia perform the beneficial function of removing amyloid beta through phagocytosis, they can also induce damage to neurons by triggering excessive inflammatory responses.

[0006] Parkinson's disease is a common degenerative brain disease affecting approximately 1.2% of people over the age of 65. Neurodegeneration in Parkinson's disease occurs in the substantia nigra, accompanied by dopaminergic deneurization and the accumulation of Lewy bodies containing alpha-synuclein. Activated microglia are frequently found in the parenchymal tissues of these patients, and microglia are known to mediate the inflammatory response of the central nervous system in Parkinson's disease.

[0007] Huntington's disease, caused by mutations in the Huntington protein, is characterized by progressive atrophy of the striatum and cortex. Additionally, pro-inflammatory microglia are observed early in this disease. In Huntington's disease, pro-inflammatory microglia are closely associated with the severity of the disease.

[0008] As the importance of the role of microglia in neurodegenerative diseases has been revealed, microglia are being proposed as targets for therapies for these diseases.

[0009] The inventors have filed a patent application for a peptide that targets the microglia to increase phagocytosis as a treatment for degenerative brain diseases, promotes the reduction of amyloid beta, and induces improvement of cognitive function by penetrating the blood-brain barrier (Republic of Korea Published Patent No. 10-2025-0117287).

[0010] Meanwhile, the inventors prepared a peptide in which a proline residue is N-methylated in order to maximize the efficacy of the above peptide through post-translational modification. Furthermore, it was confirmed that the above peptide improves solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to the existing peptide that has not been modified. In addition, it was confirmed that the above peptide exhibits a higher amyloid beta reduction efficacy than the existing peptide that has not been modified in an animal model of a neurodegenerative disease. Therefore, the present application was made by revealing that the methylated peptide of the present invention can be usefully utilized as an active ingredient in a composition for the prevention or treatment of neurodegenerative diseases and the resulting cognitive impairment, learning impairment, or memory impairment.

[0011] [Prior Art Literature]

[0012] [Patent Literature]

[0013] Republic of Korea Published Patent No. 10-2021-0055626

[0014] [Non-patent literature]

[0015] Efthymiou, AG and AM Goate, Late onset Alzheimer's disease genetics implicates microglial pathways in disease risk. Mol Neurodegener, 2017. 12(1): p. 43.

[0016] Hong, S., et al., Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science, 2016. 352(6286): p. 712-716.

[0017] Crotti, A.et al., Mutant huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors. Nat. Neurosci. 17, 513-521(2014).

[0018] The object of the present invention is to provide a methylated peptide with improved solubility, bioavailability, and efficacy as a therapeutic agent for degenerative brain diseases, and the use thereof.

[0019] To achieve the objective of the present invention, the present invention provides a peptide having an amino acid sequence represented by SEQ ID NO. 1, in which at least one proline residue is N-methylated, and a pharmaceutically acceptable salt thereof.

[0020] In addition, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain disease comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide or a pharmaceutically acceptable salt thereof for use in the pharmaceutical composition for preventing or treating degenerative brain disease; the use of the peptide or a pharmaceutically acceptable salt thereof for preparing the pharmaceutical composition for preventing or treating degenerative brain disease; and a method for treating degenerative brain disease comprising administering the peptide or a pharmaceutically acceptable salt thereof to an individual.

[0021] In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain disease, comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide or a pharmaceutically acceptable salt thereof for use in the health functional food composition for preventing or improving degenerative brain disease; the use of the peptide or a pharmaceutically acceptable salt thereof for preparing the health functional food composition for preventing or improving degenerative brain disease; and a method for preventing or improving degenerative brain disease, comprising administering the peptide or a pharmaceutically acceptable salt thereof to an individual.

[0022] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide or a pharmaceutically acceptable salt thereof for use in the pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment; the use of the peptide or a pharmaceutically acceptable salt thereof for preparing the pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment; and a method for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising administering the peptide or a pharmaceutically acceptable salt thereof to an individual.

[0023] In addition, the present invention provides a health functional food composition for enhancing cognitive ability, learning ability, or memory, comprising the peptide or a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide or a pharmaceutically acceptable salt thereof for use in the health functional food composition for enhancing cognitive ability, learning ability, or memory; the use of the peptide or a pharmaceutically acceptable salt thereof for preparing the health functional food composition for enhancing cognitive ability, learning ability, or memory; and a method for enhancing cognitive ability, learning ability, or memory, comprising administering the peptide or a pharmaceutically acceptable salt thereof to an individual.

[0024] The methylated peptide according to the present invention has improved solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to an unmodified conventional peptide, and exhibits improved amyloid beta reduction efficacy compared to the conventional peptide in an animal model of neurodegenerative disease, so it can be usefully utilized as an active ingredient in a composition for the prevention or treatment of neurodegenerative disease, or in a composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment.

[0025] In addition, the methylated peptide according to the present invention, including existing peptides, consists of very small peptides, so side effects from the administration of external substances can be minimized.

[0026] Figure 1 is a figure showing the plasma exposure levels of KINE-101, a trimer-type peptide prepared according to one embodiment of the present invention, and KINE-101M1, a variant in which the N-terminal proline (P) residue of KINE-101 is N-methylated, in an animal model.

[0027] Figure 2 is a figure comparing the efficacy of KINE-101 and KINE-101M1 in promoting microglia phagocytosis.

[0028] Figure 3 is a figure comparing the efficacy of KINE-101 and KINE-101M1 in inducing a microglial anti-inflammatory state (M2 state).

[0029] Figure 4 is a schematic diagram illustrating the method of administering KINE-101M1 to an animal model to confirm whether KINE-101M1 penetrates the blood-brain barrier.

[0030] Figure 5 is a figure showing the concentration of KINE-101M1 in the brain tissue of an animal model administered KINE-101M1.

[0031] Figure 6 is a schematic diagram illustrating the method of administering KINE-101 or KINE-101M1 to an animal model of Alzheimer's disease.

[0032] Figure 7 is a figure comparing the amyloid beta plaque reduction efficacy of KINE-101 and KINE-101M1 in an animal model of Alzheimer's disease.

[0033] The present invention will be described in more detail below.

[0034] The present invention provides a peptide having an amino acid sequence represented by SEQ ID NO. 1, in which at least one proline residue is N-methylated, and a pharmaceutically acceptable salt thereof.

[0035] In the present invention, the peptide may have at least one proline residue N-methylated, and specifically, may have an N-terminal proline residue N-methylated.

[0036] In the present invention, the peptide may have at least one proline residue N-methylated, specifically the N-terminal proline residue N-methylated so as to improve solubility.

[0037] The above peptide may have at least one proline residue N-methylated, specifically the N-terminal proline residue N-methylated, so that its bioavailability can be improved.

[0038] More specifically, the peptide has N-methylated N-terminal proline residues, which improves solubility and bioavailability, thereby ensuring flexibility in dose adjustment during clinical trials and achieving improved efficacy in terms of in vivo exposure and duration of drug action.

[0039] In the present invention, the peptide may be administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically. Specifically, the peptide has at least one proline residue N-methylated, specifically the N-terminal proline residue N-methylated to improve solubility and bioavailability, thereby ensuring in vivo exposure and sustained efficacy even with oral administration, intramuscular administration, intravenous administration, intraperitoneally, subcutaneously, intradermally, or topically.

[0040] The peptide of the present invention can be obtained by various methods widely known in the field. For example, it can be prepared by using a polynucleotide recombination and protein expression system, by synthesizing in vitro through chemical synthesis such as peptide synthesis, and by cell-free protein synthesis methods.

[0041] In addition, the peptide according to the present invention may exist in the form of various pharmaceutically acceptable salts commonly used in the art. Here, "pharmaceutically acceptable salt" means a salt formed to improve the physicochemical properties (e.g., solubility, stability, absorption rate, etc.) of the peptide or to facilitate formulation within a range that is toxicologically safe and pharmaceutically acceptable.

[0042] The above salt may be an acid-added salt or a base-added salt. Examples of acid-added salts may include hydrochloride, sulfate, phosphate, acetate, formate, propionate, succinate, citrate, maleate, tartrate, lactate, methanesulfonate, p-toluenesulfonate, trifluoroacetate, etc.

[0043] Examples of base-added salts may include sodium, potassium, calcium, magnesium, ammonium, triethylamine, and tromethamine.

[0044] The present invention also includes hydrates, solvates, and mixtures thereof of the peptide. These various forms of pharmaceutically acceptable salts do not limit the scope of the present invention and can be easily prepared by a person skilled in the art using ordinary techniques.

[0045] In a specific embodiment of the present invention, the inventors prepared a trimer-type peptide KINE-101 using a PSP fragment (PSP monomer) and prepared a variant KINE-101M1 in which the N-terminal proline residue of KINE-101 was N-methylated.

[0046] Furthermore, the inventors confirmed that the solubility and bioavailability of the KINE-101M1 are improved compared to KINE-101, and confirmed that significant improvements in pharmacokinetic indicators can be achieved in terms of in vivo exposure by securing significantly improved pharmacokinetic indicators upon intravenous, subcutaneous, and oral administration.

[0047] Accordingly, the peptide according to the present invention and the pharmaceutically acceptable salt thereof can be usefully used through various routes of administration, such as oral, intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or topical.

[0048] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain disease comprising, as an active ingredient, a peptide according to the present invention and a pharmaceutically acceptable salt thereof; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for the prevention or treatment of degenerative brain disease; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for the prevention or treatment of degenerative brain disease; and a method for treating degenerative brain disease comprising administering the peptide according to the present invention and a pharmaceutically acceptable salt thereof to an individual.

[0049] The term "prevention" as used in this invention refers to any act of suppressing a disease or delaying its onset by administering a pharmaceutical composition according to this invention.

[0050] As used in the present invention, the term "treatment" refers to any act in which symptoms of a disease are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0051] In the present invention, "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, rodents (mouse, rat), dogs, cats, horses, and cows.

[0052] In the present invention, the degenerative brain disease may be dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Pick's disease, senile dementia, Lewy body dementia, progressive supranuclear palsy, or frontotemporal dementia, but is not limited thereto.

[0053] In the present invention, the composition targets microglia and can prevent or treat the degenerative brain disease through the following properties by targeting microglia:

[0054] i) reduction of neuroinflammation by inducing microglia from an inflammatory state to an anti-inflammatory state; and

[0055] ii) Removal of amyloid beta by activation of microglia phagocytosis.

[0056] Specifically, the above composition can prevent or treat the degenerative brain disease by passing through the blood-brain-barrier (BBB) ​​to target microglia in the brain, inducing the inflammatory microglia to become anti-inflammatory to reduce neuroinflammation, and also by increasing phagocytosis to remove the toxic protein amyloid beta.

[0057] In a specific embodiment of the present invention, the inventors confirmed that KINE-101M1 can cross the blood-brain barrier.

[0058] In addition, the inventors confirmed that KINE-101M1 has enhanced efficacy in inducing an anti-inflammatory state in microglia and promoting microglia phagocytosis compared to KINE-101.

[0059] In addition, the inventors confirmed that KINE-101M1 has an enhanced effect of reducing amyloid beta in the brain of an Alzheimer's disease mouse model compared to KINE-101.

[0060] Accordingly, the inventors have confirmed that the methylated peptide according to the present invention has improved solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to an unmodified conventional peptide, and that the peptide has improved amyloid beta reduction efficacy compared to an unmodified conventional peptide in an animal model of neurodegenerative disease. Therefore, the methylated peptide according to the present invention and its pharmaceutically acceptable salt can be usefully utilized as an active ingredient in a pharmaceutical composition for the prevention or treatment of neurodegenerative disease.

[0061] The peptide of the present invention or a pharmaceutically acceptable salt thereof may be carried on a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. These may form a complex with or be associated with a transport means and may be transported in vivo using a transport system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption-enhancing substances, or fatty acids.

[0062] In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. Furthermore, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be additionally included.

[0063] The pharmaceutical composition of the present invention may be administered orally or parenterally, specifically intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically, depending on the intended method, and the dosage may be appropriately selected by a person skilled in the art, although it varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0064] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art.

[0065] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, and may increase or decrease depending on the route of administration, severity of obesity, gender, body weight, age, etc.

[0066] In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain disease, comprising a peptide according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for use in the health functional food composition for preventing or improving degenerative brain disease; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for preparing the health functional food composition for preventing or improving degenerative brain disease; and a method for preventing or improving degenerative brain disease, comprising administering the peptide according to the present invention and a pharmaceutically acceptable salt thereof to an individual.

[0067] As used in the present invention, the term "improvement" refers to any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0068] In the present invention, the details regarding the peptide, the pharmaceutically acceptable salt thereof, and degenerative brain disease are the same as those described above, so the specific description is based on the above details, and below, only the specific composition of the health functional food composition will be described.

[0069] Meanwhile, the inventors have confirmed that the methylated peptide according to the present invention has improved solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to an unmodified conventional peptide, and that the peptide has improved amyloid beta reduction efficacy compared to an unmodified conventional peptide in an animal model of neurodegenerative disease. Therefore, the methylated peptide according to the present invention and its pharmaceutically acceptable salt can be usefully utilized as an active ingredient in a health functional food composition for the prevention or improvement of neurodegenerative disease.

[0070] The health functional food composition of the present invention may be used for the prevention or improvement of a disease, either simultaneously with or separately from a drug for treatment, before or after the onset of the disease.

[0071] In the composition of the health functional food of the present invention, the active ingredient may be added directly to the food or used together with other food or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverages, the composition of the present invention may be added to the raw material in an amount preferably 15% by weight or less, more preferably 10% by weight or less. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0072] The health functional food composition of the present invention may contain other ingredients as essential components without special limitations, in addition to containing the active ingredient described above. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, such as in conventional beverages. Examples of the natural carbohydrates described above may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0073] In addition to the above, the health functional food composition of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

[0074] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising a peptide according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment; the use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment; and a method for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising administering the peptide according to the present invention and a pharmaceutically acceptable salt thereof to an individual.

[0075] The term "cognitive impairment" as used in this invention refers to an abnormality in cognitive ability regarding the manner of acquiring, remembering, and using knowledge, and cognitive ability includes cognitive functions such as knowledge, memory, comprehension, thinking ability, problem-solving ability, critical thinking, and creativity.

[0076] The term "learning disability" as used in this invention refers to a disability that exhibits significant difficulty in learning functions such as listening, speaking, attention, perception, memory, and problem-solving, or in academic achievement areas such as reading, writing, and mathematics.

[0077] The term "memory impairment" as used in this invention refers to a state in which one is unable to remember newly learned facts, unable to remember the names of objects or people, or finds it difficult or impossible to recall past experiences.

[0078] Such cognitive impairment, learning disability, or memory(ability) impairment may be caused by various causes and environments, and the causes are not particularly limited in the present invention. For example, it may be due to degenerative brain diseases, such as Alzheimer's disease, dementia, mild cognitive impairment, cognitive deficits, and attention deficits.

[0079] In the present invention, the details regarding the peptide, the pharmaceutically acceptable salt thereof, the degenerative brain disease, and the pharmaceutical composition are the same as those described above, so the specific description is made by reference to the above.

[0080] Meanwhile, the inventors have confirmed that the methylated peptide according to the present invention exhibits enhanced amyloid beta reduction efficacy compared to an unmodified conventional peptide in an animal model of a degenerative brain disease, specifically an animal model of Alzheimer's disease. Therefore, the methylated peptide according to the present invention and its pharmaceutically acceptable salt can be usefully used as an active ingredient in a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment.

[0081] In addition, the present invention provides a health functional food composition for enhancing cognitive ability, learning ability, or memory, comprising a peptide according to the present invention and a pharmaceutically acceptable salt thereof as an active ingredient; use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for use in the health functional food composition for enhancing cognitive ability, learning ability, or memory; use of the peptide according to the present invention and a pharmaceutically acceptable salt thereof for preparing the health functional food composition for enhancing cognitive ability, learning ability, or memory; and a method for enhancing cognitive ability, learning ability, or memory, comprising administering the peptide according to the present invention and a pharmaceutically acceptable salt thereof to an individual.

[0082] In the present invention, the details regarding the peptide, the pharmaceutically acceptable salt thereof, degenerative brain disease, and the health functional food composition are the same as those described above, so the specific description is taken by reference from the above.

[0083] Meanwhile, the inventors have confirmed that the methylated peptide according to the present invention exhibits enhanced amyloid beta reduction efficacy compared to an unmodified conventional peptide in an animal model of a degenerative brain disease, specifically an animal model of Alzheimer's disease. Therefore, the methylated peptide according to the present invention and its pharmaceutically acceptable salt can be usefully used as active ingredients in a health functional food composition for enhancing cognitive ability, learning ability, or memory.

[0084] The present invention will be explained in detail below through examples.

[0085] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0086] <Example 1> Preparation of Methylated Peptides by Post-Translational Modification

[0087] In this example, the polymer KINE-101 in the form of a trimer was synthesized using a PSP fragment (PSP monomer) as shown in Table 1 below. Next, a variant KINE-101M1 (or named "KINE-501") in which the proline (P) of the N-terminal first residue of KINE-101 was N-methylated was synthesized by applying a conventional solid-phase peptide synthesis (SPPS) and purification method. Subsequently, the synthesized peptide was purified using high-performance liquid chromatography (SHIMADZU Prominence HPLC), and a Shiseido capcell pak C18 column (4.6 x 50 mm) was used. In addition, the mass of the synthesized peptide was confirmed using a mass spectrometer (AXIMA Assurance, MALDI-TOF, Shimadzu).

[0088] Peptide Name Amino Acid Sequence Molecular Weight (MW) KINE-101PSPPSPPSP(Sequence No. 1) 861.9 g / mol KINE-101M1P * SPPSPPSP875.9780 g / molP * N-methylated proline

[0089] <Example 2> Confirmation of Improved Solubility of Proline (P) Methylated Peptide KINE-101M1

[0090] In order to determine the effect of N-methylation of proline (P) residues on the effect of KINE-101, the solubility of KINE-101 and KINE-101M1 prepared in <Example 1> above was compared.

[0091] Specifically, KINE-101 and KINE-101M1 powders of various concentrations were placed in DPBS (Dulbecco's Phosphate-Buffered Saline), a commonly used peptide solvent, and mixed by vigorously shaking for 30 seconds at 5-minute intervals at room temperature. The dissolved samples were visually inspected for dissolution at 30 minutes and 2 hours, and the results are shown in Tables 2 and 3.

[0092] KINE-101 Solubility Test Results Concentration (Peptide / Solvent) Time Elapsed After Dissolution Term 30 min 2 hours 500 mg / 0.5 ml XX Dissolves very well 500 mg / 0.5 ml ~ 100 mg / ml XX Dissolves well 100 mg / ml ~ 33.3 mg / ml XX Dissolves 33.3 mg / ml ~ 10 mg / ml OO Dissolves slightly 10 mg / ml ~ 1 mg / ml OO Difficult to dissolve 1 mg / ml ~ 0.1 mg / ml OO Very difficult to dissolve 500 mg / 0.5 ml OO Almost no dissolution Result: The DPBS solubility of KINE-101 was confirmed as 'slightly soluble'.

[0093] KINE-101M1 Solubility Test Results Concentration (Peptide / Solvent) Time Elapsed After Dissolution Term 30 min 2 hours 500 mg / 0.5 ml XX Dissolves very well 500 mg / 0.5 ml ~ 100 mg / ml XX Dissolves well 100 mg / ml ~ 33.3 mg / ml OO Dissolves 33.3 mg / ml ~ 10 mg / ml OO Dissolves slightly 10 mg / ml ~ 1 mg / ml OO Difficult to dissolve 1 mg / ml ~ 0.1 mg / ml OO Very difficult to dissolve 500 mg / 0.5 ml OO Almost no dissolution Result: The DPBS solubility of KINE-101M1 was confirmed as 'dissolves well'.

[0094] As a result, as shown in Tables 2 and 3, KINE-101 showed solubility in DPBS used as a solvent up to a maximum concentration of 33.3 mg / ml (Table 3), whereas KINE-101M1 showed excellent solubility in DPBS up to a concentration of 100 mg / ml (Table 4). Through the above results, it can be seen that by N-methylating the proline residue of KINE-101, the solubility of KINE-101 is increased, thereby ensuring flexibility in dose adjustment in animal and clinical trials.

[0095] <Example 3> Confirmation of Improved Bioavailability of Proline (P) Methylated Peptide KINE-101M1

[0096] In order to determine the effect of N-methylation of proline residues on the efficacy of KINE-101, the bioavailability of KINE-101 and KINE-101M1 prepared in <Example 1> above was compared.

[0097] Specifically, the plasma exposure characteristics and absolute bioavailability of KINE-101 and KINE-101M1 were evaluated using 6-week-old male SD rats (Sprague-Dawley rats). Animals were acclimatized at least 7 days prior to the start of the experiment, and water and feed were provided ad libitum without fasting conditions.

[0098] In addition, each group consisted of three animals, and a total of six groups were established as shown in Table 4 below (combinations of administration routes IV, SC, PO and two types of peptides). Administration was performed as a single dose as follows: intravenous injection (IV) 25 mg / kg, subcutaneous injection (SC) 25 mg / kg, and oral administration (PO) 250 mg / kg. After administration, blood samples were collected at 1, 2, 5, 15, and 30 minutes, as well as at 1, 2, and 4 hours. The collected blood was retrieved into tubes pre-cooled using an anticoagulant, and plasma was separated by centrifugation at 2,000 ×g for 10 minutes at 4°C. The separated plasma was immediately stored at -70°C or below until analysis.

[0099] Group Administration Substance Administration Method and Dosage Feeding KINE-101 IV 25 mg / kg KINE-101 Intravenous Injection (IV) 25 mg / kg Administer without fasting KINE-101 SC 25 mg / kg KINE-101 Subcutaneous Injection (SC) 25 mg / kg Administer without fasting KINE-101 PO 250 mg / kg KINE-101 Oral Administration (PO) 250 mg / kg Fast for 16 hours prior to administration KINE-101 M1 IV 25 mg / kg KINE-101 M1 Intravenous Injection (IV) 25 mg / kg Administer without fasting KINE-101 M1 SC 25 mg / kg KINE-101 M1 Subcutaneous Injection (SC) 25 mg / kg Administer without fasting KINE-101 M1 PO 250 mg / kg KINE-101 M1 Oral Administration (PO) Fasting for 16 hours before administering 250 mg / kg

[0100] Peptide concentrations in plasma were quantified by LC-MS / MS following pretreatment using the conventional protein precipitation method. The accuracy and precision of the analysis were ensured by including standard curves and quality control samples. Pharmacokinetic parameters were calculated using non-compartmental analysis. Cmax and Tmax were determined from observed values, and AUC was calculated using the area under the curve up to the point of final quantification [AUC(last)]. The elimination half-life (t 1 / 2 ) was calculated from the terminal slope.

[0101] The above parameters (Cmax, Tmax, AUC(last), t 1 / 2 ) was calculated for each of the IV, SC, and PO administration groups. In addition, bioavailability was evaluated in two ways. Absolute bioavailability (%Fabs) was calculated by comparing the dose-adjusted AUC(last) of each administration route with the intravenous (IV) administration AUC(last), and relative bioavailability (%Frel) was calculated by comparing the AUC(last) between different formulations in the same route for comparison between formulations.

[0102] PK parametersIV 25 mg / kgSC 25 mg / kgPO 250 mg / kgKINE-101KINE-101M1KINE-101KINE-101M1KINE-101KINE-101M1AUC(last)(ng·h / ml)171.9643515.6583. 2044913.02ND3545.21Cmax8198.21175098.33414.0823978.97ND1360.80Tmax(h)0.0160.0160.0831.00ND0.50t 1 / 2 (h)0.010.340.120.61ND2.20Bioavailability,%Fabs--48.38103.21-0.81Bioavailability,%Frel-25305.98-53981.78--

[0103] As a result, as shown in Table 5 and Figure 1, KINE-101 exhibits pharmacokinetic characteristics in which the drug concentration is rapidly lost in the body, whereas KINE-101M1 exhibits a high AUC and an appropriate half-life when administered subcutaneously, which is advantageous in terms of in vivo exposure and duration of efficacy, and it was confirmed that reproducible exposure was observed even when administered orally. Through the above results, it can be seen that by N-methylating the proline residue of KINE-101, significantly improved pharmacokinetic indicators (AUC, half-life, bioavailability) can be obtained at the same dosage and route of administration as KINE-101, and distinct improvements can be achieved in terms of ensuring duration and in vivo exposure.

[0104] <Example 4> Confirmation of Enhanced Efficacy of Proline (P) Methylated Peptide KINE-101M1 Promoting Microglia Phagocytosis

[0105] In order to investigate the effect of N-methylation of proline residues on the effect of KINE-101, microglia that had been induced with environmental conditions of Alzheimer's disease, the biggest cause of dementia, were treated with KINE-101 and KINE-101M1 prepared in <Example 1> above, and the phagocytosis of the microglia was compared.

[0106] Specifically, an Alzheimer's disease environment was created by treating cultured human microglia (hMG) with oligomeric amyloid beta protein (oAβ), known as a cause of Alzheimer's disease, and interferon gamma (INF-γ), an inflammatory substance. Under these conditions, KINE-101 and KINE-101M1 were treated at concentrations of 0, 1, 5, and 10 ng / ml. After two days, the medium was changed and the same drugs were treated again. Then, after two days, the intracellular amyloid beta protein was measured to quantify the amyloid beta protein that had moved into the cell via phagocytosis. Amyloid beta protein is labeled with a pH-sensitive red fluorescent substance and has the characteristic of emitting fluorescence due to the low pH (pH 3-5) of the lysosome when it moves into the lysosome, an intracellular organelle that plays a role in protein degradation. Therefore, the fact that red fluorescence is emitted within the cell indicates that amyloid beta is being degraded in lysosomes after moving into microglia via phagocytosis. Red fluorescence images were obtained using a fluorescence microscope, and quantified and compared using software programs.

[0107] As a result, as shown in Figure 2, the intracellular migration of amyloid beta by phagocytosis of microglia increased in a concentration-dependent manner with KINE-101, and the intracellular migration was further increased by treatment with KINE-101M1.

[0108] From the above results, it can be seen that N-methylating the proline residue of KINE-101 enhances the efficacy of increasing the phagocytosis of microglia by KINE-101.

[0109] <Example 5> Confirmation of Enhanced Efficacy of Proline (P) Methylated Peptide KINE-101M1 in Inducing Anti-inflammatory State in Microglia

[0110] In order to investigate the effect of N-methylation of proline residues on the effect of KINE-101, microglia that had been induced with environmental conditions of Alzheimer's disease, the biggest cause of dementia, were treated with KINE-101 and KINE-101M1 prepared in <Example 1> above, and the change of microglia to an anti-inflammatory state (M2 state) was compared.

[0111] Specifically, an Alzheimer's disease environment was created in microglia using the same method as described in <Example 4> above, and KINE-101 and KINE-101M1 were treated at concentrations of 0, 1, 5, and 10 ng / ml. After two days, the culture medium was changed and the same drugs were treated again. Then, after two days, the cells were fixed using a fixative, and CD206, a marker of the anti-inflammatory state of microglia, was stained with an antibody, and the CD206 antibody was stained with a fluorescently labeled secondary antibody. Images were obtained using a fluorescence microscope, the fluorescence signal was quantified using a software program, and the degree of change from an inflammatory state to an anti-inflammatory state induced by KINE-101 and KINE-101M1 was compared.

[0112] As a result, as shown in Figure 3, CD206, an anti-inflammatory status marker of microglia, increased in a concentration-dependent manner with treatment with KINE-101, and it was confirmed that treatment with KINE-101M1 increased more strongly at all concentrations.

[0113] From the above results, it can be seen that by N-methylating the proline residue of KINE-101, the change of microglia to an anti-inflammatory state by KINE-101 is achieved more effectively.

[0114] <Example 6> Confirmation of Increased Blood-Brain-Barrier Permeation Efficiency of Proline (P) Methylated Peptide KINE-101M1

[0115] To investigate the effect of N-methylation of proline residues on the efficacy of KINE-101, KINE-101M1 prepared in <Example 1> was administered intravenously or subcutaneously to a mouse model, and the blood-brain barrier penetration and concentration in brain tissue were compared.

[0116] Specifically, as shown in the schematic diagram of Fig. 4, a guide cannula was implanted into the hippocampus of 9-week-old mice using a stereotaxic device, and a 2-week recovery period was granted. After the recovery period ended, at weeks 11, 12, and 13, a microdialysis probe was inserted into the guide while the animals were anesthetized, and baseline samples were collected prior to peptide administration. KINE-101M1 fluorescently labeled with FITC (KINE-101M-FITC) or Dextran (40 kDa) fluorescently labeled with FITC as a negative control (Dextran-FITC) were administered via intravenous (IV) or subcutaneous (SC) routes, with each concentration set to 1.44 mM. Cerebrospinal fluid was collected in serial fractions at 20-minute intervals starting from 0 minutes after the injection of the test substance for a total of 120 minutes, and the fluorescence intensity of each fraction was measured using Cytation5. The exposure to the test substance in the hippocampus (concentration-time profile) was estimated using the obtained time-dependent fluorescence signals.

[0117] As a result, as shown in Figure 5, Dextran in the negative control group (Dex-FITC-SC) showed only extremely low signals close to baseline levels in all fractions, confirming blood-brain barrier impermeability. On the other hand, the KINE-101M intravenous administration group (KINE-101M-FITC-IV) showed a pattern where fluorescence intensity increased sharply in the first fraction immediately after administration, then rapidly decreased within a short time to approach baseline. Meanwhile, in the KINE-101M subcutaneous administration group (KINE-101M-FITC-SC), fluorescence intensity continuously increased as time passed after the start of observation, and no decreasing trend was observed even at the 120-minute mark.

[0118] The above results indicate that KINE-101M1, in which the proline residues of KINE-101 are N-methylated, effectively crosses the blood-brain barrier and reaches brain tissue, particularly the hippocampus. Furthermore, intracerebral concentrations were confirmed with both intravenous and subcutaneous administration; in the case of subcutaneous administration, the concentration was maintained continuously, albeit relatively gradually. This demonstrates that KINE-101M1 reaches the brain and exerts pharmacological activity regardless of the route of administration.

[0119] <Example 7> Confirmation of Enhanced Efficacy by Proline (P) Methylated Peptide KINE-101M1 in an Alzheimer's Disease Mouse Model

[0120] To investigate the effect of N-methylation of proline residues on the effect of KINE-101, the amyloid beta reduction ability was compared after administering KINE-101 and KINE-101M1 prepared in <Example 1> to an Alzheimer's disease mouse model.

[0121] Specifically, 5xFAD mice, an amyloid beta-induced Alzheimer's disease mouse model, were provided by Jackson Laboratory. This Alzheimer's disease mouse model is known to cause dementia by overexpressing human amyloid beta protein, undergoing oligomerization outside the cell, eventually precipitating as insoluble protein clumps, and damaging neurons. Accordingly, as shown in the schematic diagram of Fig. 6, the effects of KINE-101 and KINE-101M1 were evaluated using 4-month-old 5xFAD mice. Each peptide was administered subcutaneously at a dose of 100 mg / kg twice a week for 4 weeks. Four male mice were used for each of the KINE-101 and KINE-101M1 administration groups, while five male mice administered PBS in the same manner were used as a negative control. After the administration was completed, the mouse brains were fixed in a fixative, and tissue sections of the cortex and hippocampus were prepared using a cryoslice. Subsequently, amyloid beta plaques were immunostained using a 6E10 antibody specific to amyloid beta protein and a fluorescently labeled secondary antibody. The number of plaques in the stained brain tissue was quantified using the Imaris program.

[0122] As a result, as shown in Figure 7, the number of amyloid beta plaques was significantly reduced by approximately 17% in the KINE-101 administration group and by approximately 32% in the KINE-101M1 administration group compared to the negative control group. In particular, a statistically significant reduction effect was observed in the KINE-101M1 administration group even when compared to the KINE-101 administration group.

[0123] From the above results, it can be seen that the efficacy of KINE-101 in reducing amyloid beta is enhanced by N-methylating the proline residue of KINE-101.

[0124] The methylated peptide according to the present invention has improved solubility, bioavailability, phagocytosis by microglia, and anti-inflammatory efficacy compared to an unmodified conventional peptide, and has superior amyloid beta reduction efficacy compared to the conventional peptide in an animal model of neurodegenerative disease, so it can be usefully utilized as an active ingredient in a composition for the prevention or treatment of neurodegenerative disease and the resulting cognitive impairment, learning impairment, or memory impairment.

Claims

1. A peptide having the amino acid sequence represented by SEQ ID NO. 1, wherein at least one proline residue is N-methylated, and a pharmaceutically acceptable salt thereof.

2. The peptide according to claim 1, wherein the N-terminal proline residue of the peptide is N-methylated.

3. The peptide according to claim 1, wherein the solubility of the peptide is enhanced by N-methylation.

4. The peptide of claim 1, wherein the peptide has improved bioavailability through N-methylation.

5. The peptide according to claim 1, wherein the peptide is administered orally, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically.

6. A pharmaceutical composition for the prevention or treatment of degenerative brain diseases, comprising as an active ingredient a peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition for the prevention or treatment of a degenerative brain disease according to claim 6, wherein the degenerative brain disease is selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch amyloidosis, Pick's disease, senile dementia, Lewy body dementia, progressive supranuclear palsy, and frontotemporal dementia.

8. A pharmaceutical composition for the prevention or treatment of degenerative brain disease, wherein the composition passes through the blood-brain barrier in claim 6.

9. A pharmaceutical composition for the prevention or treatment of degenerative brain diseases, wherein the composition targets microglia in claim 6.

10. A pharmaceutical composition for the prevention or treatment of degenerative brain diseases, wherein the composition targets microglia and has the following characteristics: i) reduction of neuroinflammation by inducing microglia from an inflammatory state to an anti-inflammatory state; and ii) Removal of amyloid beta by activation of microglia phagocytosis.

11. A pharmaceutical composition for the prevention or treatment of degenerative brain disease according to claim 6, wherein the composition is formulated for oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, or topical administration.

12. A health functional food composition for the prevention or improvement of degenerative brain diseases, comprising as an active ingredient a peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising as an active ingredient a peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

14. A health functional food composition for enhancing cognitive ability, learning ability, or memory, comprising as an active ingredient a peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

15. A method for treating a degenerative brain disease comprising administering to an individual the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

16. A method for preventing or improving degenerative brain disease, comprising administering to an individual the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

17. A method for the prevention or treatment of cognitive impairment, learning impairment, or memory impairment, comprising administering to an individual the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

18. A method for enhancing cognitive ability, learning ability, or memory, comprising administering to an individual the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof.

19. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for use as a pharmaceutical composition for the prevention or treatment of degenerative brain diseases.

20. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for use as a health functional food composition for the prevention or improvement of degenerative brain diseases.

21. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for use in a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning disability or memory impairment.

22. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for use as a health functional food composition for enhancing cognitive ability, learning ability or memory.

23. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for the prevention or treatment of degenerative brain diseases.

24. Use of the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof for manufacturing a health functional food composition for the prevention or improvement of degenerative brain diseases.

25. Use of the peptide of any one of claims 1 to 5 and the pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for the prevention or treatment of cognitive impairment, learning disability or memory impairment.

26. Use of the peptide of any one of claims 1 to 5 and a pharmaceutically acceptable salt thereof for preparing a health functional food composition for enhancing cognitive ability, learning ability or memory.

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