Acetylcholine receptor-binding optimized short peptide and its use

Abstract

Regarding the acetylcholine receptor binding optimized truncated peptide and its uses, the optimized truncated peptide of the present invention, which is truncated compared to conventional acetylcholine receptor inhibitory peptides but has high binding strength with acetylcholine receptors and acetylcholine receptor action inhibitory effect, can be used to provide a cosmetic composition for improving wrinkles, a pharmaceutical product for preventing or treating acetylcholine receptor-related diseases, and a functional health food for improving the same.

Description

Technical Field

[0001] The present invention relates to an acetylcholine receptor-binding optimized short peptide and its use, and more specifically, to an acetylcholine receptor-binding optimized short peptide in which specific amino acids important for binding to an acetylcholine receptor and suppressing its action are represented by a certain formula for peptide optimization, and its use.

Background Art

[0002] Acetylcholine is a chemical substance present in the nerve tissue of animals. It is secreted from the nerve endings and plays a role in transmitting nerve stimuli to muscles. As neurotransmitters secreted from nerve endings, acetylcholine is known in motor nerves and parasympathetic nerves, and epinephrine (adrenaline) is known in sympathetic nerves. When acetylcholine is secreted, it exhibits physiological effects such as blood pressure drop, heart rate suppression, intestinal contraction, and skeletal muscle contraction. When a muscle contracts, the nerve gives an order to the muscle to contract, and in response, the muscle contracts. By this order, at the part where the nerve and the muscle meet (neuromuscular junction), the nerve secretes acetylcholine, and this substance binds to the acetylcholine receptor of the muscle, causing the muscle to contract (Non-Patent Document 1: Vincent, A., 1985; Lindstrom, J.M., et al., 1976). Blocking the peripheral acetylcholine receptor that innervates the thigh skeletal muscle causes muscle movement paralysis, and blocking the acetylcholine receptor of smooth muscle and cardiac muscle responsible for respiration or heart movement causes respiration and heart movement paralysis.

[0003] Acetylcholine receptors are classified into muscarinic acetylcholine receptors (mAchR) and nicotinic acetylcholine receptors (nAchR). Muscarinic acetylcholine receptors are G protein-coupled receptors that can be activated by muscarine, and different subtypes activate different signaling mechanisms. Muscarinic acetylcholine receptors are distributed throughout the body, including the central nervous system and peripheral organs, and primarily mediate the physiological effects of acetylcholine secreted from postganglionic fibers of the parasympathetic nervous system.

[0004] The nicotinic acetylcholine receptor is a receptor that mimics the pharmacological action of nicotine and is an ion channel operated by neurotransmitters. It is a non-selective cation channel that non-selectively allows the passage of sodium, potassium, calcium ions, etc. through the opening and closing of the ion channel, and regulates the electrical signaling between nerve cells and muscle cells. The nicotinic acetylcholine receptor is classified into a muscle type and a neuronal type according to the expression site. The muscle-type nicotinic acetylcholine receptor is expressed at the neuromuscular junction where the motor neuron meets the skeletal muscle, and acetylcholine secreted by the motor neuron contributes to inducing the end plate potential (EPP) of the skeletal muscle cell membrane. On the other hand, since the neuronal-type nicotinic acetylcholine receptor is expressed in the peripheral ganglia of the autonomic nervous system (ANS), acetylcholine secreted from the preganglionic fiber contributes to exciting the postganglionic fiber.

[0005] Drugs that interfere with or inhibit the activity of acetylcholine or mimic its action are very usefully used. Acetylcholine receptor agonists are used to treat myasthenia gravis and Alzheimer's disease. Myasthenia gravis is an autoimmune disease caused by the body producing antibodies against nicotinic acetylcholine receptors and inhibiting normal acetylcholine signaling. It can be treated by using an acetylcholine esterase (AChE) inhibitor to increase the time that acetylcholine can interact with each receptor before being inactivated in the synaptic cleft between the nerve and the muscle.

[0006] In addition, when the secretion of acetylcholine is interfered with, muscle contraction is suppressed and wrinkles are stretched while the muscle becomes paralyzed. Botox uses this principle. Botox interferes with the process by which acetylcholine, a substance essential for muscle contraction, is secreted at the ends of motor nerves. As a result, the muscle becomes immobile and the wrinkles induced by the muscle disappear. Since the muscle relaxation effect of Botox gradually disappears after 3 to 6 weeks, repeated administration is necessary.

[0007] In addition, a cosmetic peptide was developed using the mechanism of inhibiting the binding of acetylcholine to the acetylcholine receptor to stretch wrinkles. This is DSM's Synake. It is a snake venom-derived peptide that has the best clinical effect (about 52%) among wrinkle-improving peptides and is widely used as a raw material for peptide cosmetics.

[0008] Therefore, while researching acetylcholine receptor-binding peptides, the present inventors screened and secured peptides with a high binding affinity to the acetylcholine receptor, and confirmed that such peptides bind to the acetylcholine receptor and prevent acetylcholine binding, thereby suppressing the action of the acetylcholine receptor. However, when the acetylcholine receptor-binding peptide is provided as a peptide fragment, there is a problem that the production cost per unit increases when the length of the peptide becomes longer, and the skin permeability decreases when it is manufactured as a cosmetic. Therefore, it was necessary to study shorter peptides while maintaining a high binding affinity to the acetylcholine receptor.

[0009] As prior art, Patent Document 1 (Korean Patent Publication No. 10-2020-0080179), the inventor's prior art, describes acetylcholine receptor inhibitory peptides and their uses, but only describes 8-mer, 11-mer, 14-mer, and 18-mer peptides as acetylcholine receptor inhibitory peptides, and does not describe the optimized shortened peptides of the present invention and their effects. Furthermore, Korean Patent Registration No. 1216008 describes peptides that bind to acetylcholine receptors, selected using biopanning, but does not describe peptides or libraries containing the amino acid sequences of the present invention.

[0010] Furthermore, Patent Document 2 (Korean Patent Publication No. 2018-0028748) describes a neurotransmitter release-regulating peptide containing acetylcholine and its wrinkle-improving effect, but does not describe the acetylcholine receptor binding strength of a peptide containing the amino acid sequence of the present invention and the resulting acetylcholine receptor activity inhibitory effect. Patent Document 3 (Korean Patent Publication No. 2014-0139010) describes a peptide for promoting transdermal penetration, but the composition and effect are different from the activity-inhibiting peptide that uses acetylcholine receptor binding of the present invention. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Korean Patent Publication No. 10-2020-0080179 [Patent Document 2] Korean Patent Publication No. 2018-0028748 [Patent Document 3] Korean Patent Publication No. 2014-0139010 [Non-patent literature]

[0012] [Non-Patent Document 1] Vincent, A., 1985; Lindstrom, J. M., et al., 1976 Summary of the Invention

Problems to be Solved by the Invention

[0013] An object of the present invention is to provide an acetylcholine receptor-binding optimized shortened peptide. Another object of the present invention is to provide the optimized shortened peptide of the acetylcholine receptor-binding optimized shortened peptide.

[0014] Another object of the present invention is to provide a wrinkle-improving cosmetic composition containing an acetylcholine receptor-binding optimized shortened peptide. Another object of the present invention is to provide a composition for preventing or treating an acetylcholine receptor overactivity-related disease containing an acetylcholine receptor-binding optimized shortened peptide.

[0015] An object of the present invention is to provide a health functional food composition and a composition for medical devices for improving an acetylcholine receptor overactivity-related disease containing an acetylcholine receptor-binding optimized shortened peptide.

Means for Solving the Problems

[0016] In order to achieve the above object, the present invention provides an acetylcholine receptor-binding optimized shortened peptide.

[0017] In the present invention, the "amino acid" or "any amino acid" includes natural amino acids and other amino acids used for producing synthetic peptides in the field of peptides, for example, natural amino acids, unnatural amino acids, amino acids not encrypted as base sequences, etc., including both L- and D-isomers.

[0018] The natural amino acids are alanine (Ala, A), cysteine (Cys, C), aspartic acid (Asp, D), and glutamic acid (Glutamic acid acid, Glu, E), phenylalanine (phenylalanine, Phe, F), glycine (glycine, Gly, G), histidine (histidine, His, H), isoleucine (isoleucine, Ile, I), lysine (lysine, Lys, K), leucine (leucine, Leu, L), methionine (methionine, Met, M), asparagine (asparagine, Asn, N), proline (proline, Pro, P), glutamine (glutamine, Gln, Q), arginine (arginine, Arg, R), serine (serine, Ser, S), threonine (threonine, Thr, T), valine (valine, Val, V), tryptophan (tryptophan, Trp, W), and tyrosine (tyrosine, Tyr, Y).

[0019] The other amino acids are 2-aminoadipic acid (2-aminohexanedioic acid), α-asparagine, 2-aminobutanoic acid, 2-aminocapric acid (2-aminodecanoic acid), α-glutamine, α-aminoisobutyric acid (α-methylalanine), 2-aminopimelic acid (2-aminohepanedioic acid), γ-amino-β-hydroxybenzenepentanoic acid, 2-aminosuberic acid (2-aminooctanedioic acid), 2-carboxyazetidine, β-alanine, β-aspartic acid, 3,6-diaminohexanoic acid (3,6-diaminohexanoic acid, β-lysine), butanoic acid, 4-amino-3-hydroxybutanoic acid, γ-amino-β-hydroxycyclohexanepentanoic acid, 3-cyclohexylalanine, N5-aminocarbonylornithine, 3-sulfoalanine, 2,4-diaminopropionic acid, 2,7-diaminosuberic acid, 2,7-diaminooctanedioic acid, S-ethylthiocysteine, γ-glutamic acid, γ-carboxylglutamic acid, hydroxyacetic acid (glycolic acid), pyroglutamic acid, homoarginine, homocysteine, homohistidine, 2-hydroxyisovaleric acid, homoserine, 2-hydroxypentanoic acid, 5-hydroxylysine, 4-hydroxyproline, isovaline, 2-hydroxypropanoic acid (lactic acid), mercaptoacetic acid acid, mercaptobutanoic acid, 3-hydroxy-4-methylproline, mercaptopropanoic acid, 3-naphthylalanine, norleucine, nortyrosine, norvaline, 2-carboxyoctahydroindole, ornithine, penicillamine, 3-mercaptovaline, 2-phenylglycine, 2-carboxypiperidine, sarcosine, N-methylglycine, 1-amino-1-carboxycyclopentane, statin,4-amino-3-hydroxy-6-methylheptanoic acid, 3-thienylalanine, 3-carboxyisoquinoline, 3-methylvaline, ε-N-trimethyllysine, 3-thiazolylalanine, α-amino-2,4-dioxopyrimidinepropanoic acid, and the like.

[0020] In the present invention, the term "peptide" refers to a polymer consisting of two or more amino acids linked by amide bonds or peptide bonds.

[0021] In the present invention, "acetylcholine receptor (AchR)" refers to a receptor to which acetylcholine secreted from nerve endings binds, and acts as a pathway for transmitting nerve stimulation by acetylcholine. For example, when muscle contraction is required, if a nerve issues a command to the muscle to contract, the nerve secretes acetylcholine at the interface between the nerve and muscle, and the secreted acetylcholine binds to the acetylcholine receptor in the muscle, causing the muscle to contract.

[0022] The acetylcholine receptor of the present invention is classified into a muscarinic acetylcholine receptor and a nicotinic acetylcholine receptor, and the acetylcholine receptor of the present invention is preferably a nicotinic acetylcholine receptor.

[0023] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by Chemical Formula 1 below.

[0024] [Chemical formula 1]

[0025] (R / K)XXX(R / K)

[0026] (However, the R / K is arginine or lysine, and X is any one amino acid.)

[0027] The peptide consists of a 5 - amino acid sequence, where the first and fifth amino acids are arginine or lysine respectively, and the second, third, and fourth sequences are peptides of any amino acid.

[0028] The acetylcholine receptor - binding peptide consists of a 5 - amino acid sequence represented by the above [Chemical Formula 1], and the K or R, which are the first and fifth amino acids, may be important sites in acetylcholine receptor binding. For the amino acid sequence represented by the above [Chemical Formula 1], the second, third, and fourth amino acids may be any amino acids, which means that as long as the first and fifth amino acids are K or R, even if the types of the second, third, and fourth amino acids change, it can show a certain level of acetylcholine receptor - binding ability.

[0029] The present invention provides an acetylcholine receptor - binding peptide consisting of the amino acid sequence represented by the following Chemical Formula 1 - 1.

[0030] [Chemical Formula 1 - 1]

[0031] (R / K)XYZ(R / K)

[0032] (However, the R / K is arginine or lysine, XYZ are any amino acids arranged sequentially, X is one amino acid selected from R, Q, G, V, L, S, W, Y is one amino acid selected from R, Q, L, I, F, V, Y, and Z is one amino acid selected from R, S, L, C, Y, Q, T.)

[0033] Also, the peptide of the amino acid sequence represented by the above Chemical Formula 1 - 1 may be a peptide of one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 600.

[0034] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following Chemical Formula 1-2.

[0035] [Chemical Formula 1-2]

[0036] XRRQRR

[0037] (However, R is arginine, Q is glutamine, and X is any one amino acid.)

[0038] The peptide of the amino acid sequence represented by the above Chemical Formula 1-2 may be a peptide of one amino acid sequence selected from the group consisting of SEQ ID NOs: 3601 to 3620.

[0039] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following Chemical Formula 1-3.

[0040] [Chemical Formula 1-3]

[0041] RRQRRX

[0042] (However, R is arginine, Q is glutamine, and X is any one amino acid.)

[0043] The peptide of the amino acid sequence represented by the above Chemical Formula 1-3 may be a peptide of one amino acid sequence selected from the group consisting of SEQ ID NOs: 3621 to 3640.

[0044] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following [Chemical Formula 2].

[0045] [Chemical Formula 2]

[0046] (R / K)(R / K)XXX(R / K)

[0047] (However, R / K is arginine or lysine, and X is any one amino acid.)

[0048] The acetylcholine receptor-binding peptide consists of a six-amino acid sequence represented by the above [Chemical Formula 2], and the first, second, and sixth amino acids, K or R, may be important sites for acetylcholine receptor binding. In the amino acid sequence represented by the above [Chemical Formula 2], the third, fourth, and fifth amino acids are any amino acids, which means that as long as the first, second, and sixth amino acids are K or R, the peptide can exhibit a certain level of acetylcholine receptor binding ability even if the types of the third, fourth, and fifth amino acids are different.

[0049] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following Chemical Formula 2-1.

[0050] [Chemical formula 2-1]

[0051] (R / K)(R / K)XYZ(R / K)

[0052] (wherein R / K is arginine or lysine, XYZ are any amino acids arranged in order, X is one amino acid selected from R, Q, G, V, L, S, and W, Y is one amino acid selected from R, Q, L, I, F, V, and Y, and Z is one amino acid selected from R, S, L, C, Y, Q, and T.)

[0053] The peptide having the amino acid sequence represented by Chemical Formula 2-1 above may be a peptide having one amino acid sequence selected from the group consisting of SEQ ID NOs: 1201 to 1800.

[0054] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following Chemical Formula 2-2.

[0055] [Chemical formula 2-2]

[0056] XRRGVRR

[0057] (However, R is arginine, G is glycine, V is valine, and X is any one amino acid.)

[0058] The peptide of the amino acid sequence represented by the above Chemical Formula 2-2 may be a peptide of one amino acid sequence selected from the group consisting of SEQ ID NOs: 3641 to 3660.

[0059] The present invention provides an acetylcholine receptor-binding peptide comprising an amino acid sequence represented by the following Chemical Formula 2-3.

[0060] [Chemical Formula 2-3]

[0061] RRGVRRX

[0062] (However, R is arginine, G is glycine, V is valine, and X is any one amino acid.)

[0063] The peptide of the amino acid sequence represented by the above Chemical Formula 2-3 may be a peptide of one amino acid sequence selected from the group consisting of SEQ ID NOs: 3661 to 3680.

[0064] The present invention provides an acetylcholine receptor-binding peptide comprising an amino acid sequence represented by the following [Chemical Formula 3].

[0065] [Chemical Formula 3]

[0066] (R / K)XXX(R / K)(R / K)

[0067] (However, R / K is arginine or lysine, and X is any one amino acid.)

[0068] The acetylcholine receptor-binding peptide consists of a six-amino acid sequence represented by the above [Chemical Formula 3], and the first, fifth, and sixth amino acids, K or R, may be important sites for acetylcholine receptor binding. In the amino acid sequence represented by the above [Chemical Formula 3], the second, third, and fourth amino acids may be any amino acids, which means that as long as the first, fifth, and sixth amino acids are K or R, the peptide can exhibit a certain level of acetylcholine receptor binding ability even if the types of the second, third, and fourth amino acids are different.

[0069] The present invention provides an acetylcholine receptor-binding peptide consisting of an amino acid sequence represented by the following Chemical Formula 3-1.

[0070] [Chemical formula 3-1]

[0071] (R / K)XYZ(R / K)(R / K)

[0072] (wherein R / K is arginine or lysine, XYZ are any amino acids arranged in order, X is one amino acid selected from R, Q, G, V, L, S, and W, Y is one amino acid selected from R, Q, L, I, F, V, and Y, and Z is one amino acid selected from R, S, L, C, Y, Q, and T.)

[0073] The peptide having the amino acid sequence represented by Chemical Formula 3-1 above may be a peptide having one amino acid sequence selected from the group consisting of SEQ ID NOs: 2401 to 3000.

[0074] The amino acid sequence of the acetylcholine receptor-binding peptide excludes the amino acid sequence disclosed in Korean Patent Publication No. 10-2020-0080179, which is a prior patent of the present invention. The amino acid sequence disclosed in Korean Patent Publication No. 10-2020-0080179 includes RKSLLR.

[0075] The present invention relates to an acetylcholine receptor-binding peptide, characterized in that the N-terminus or C-terminus of the peptide is modified.

[0076] The modified N-terminus or C-terminus is palmitoylated, acetylated, formylated or PEGylated, or is 2-mercaptoacetic acid, 3-mercaptopropionic acid, 6-mercaptohexanoic acid, pyroglutamic acid, succinimide acid, amidation, cystramine, methyl ester, ethyl ester, benzyl ester, etc., and may be an acetylcholine receptor-binding peptide characterized by one or more bonds selected from the group consisting of fatty acid bonds such as myristic acid, stearic acid, palmitic acid, cholesterol, 6-aminohexanoic acid and 8-aminooctanoic acid.

[0077] The peptides of the present invention can be obtained by methods widely known in the art. Specifically, they can be produced using genetic recombination and protein expression systems, or synthesized in vitro by chemical synthesis methods such as peptide synthesis and cell-free protein synthesis methods. More specifically, they can be synthesized by methods well known in the art, for example, using an automatic peptide synthesizer, and can be produced by genetic engineering techniques, but are not limited thereto. For example, a gene encoding a fusion protein consisting of a fusion partner and the peptide of the present invention is produced by genetic engineering, and after the produced gene is transformed into a host microorganism, it is expressed in the form of a fusion protein in the host microorganism, and the peptide of the present invention is cleaved and separated from the fusion protein using a proteolytic enzyme or a compound to produce the desired peptide.

[0078] The peptides of the present invention may exist in the form of salts. The salt forms available for use in the present invention may be those made during the final separation and purification of the compound or by reacting an amino group with a suitable acid. For example, as acid addition salts, they may be acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate, but are not limited thereto. Also, examples of acids that can be used to form acid addition salts may be inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid, but are not limited thereto.

[0079] The peptide can be modified by adding an amino acid sequence, targeting, enhancing efficacy, or increasing stability designed for the purpose of increasing the targeting sequence, tag, labeled residue, half-life, or stability of the peptide, binding with an antibody or antibody fragment, human serum albumin (HSA), etc., or modifying the N-terminus or C-terminus of the peptide.

[0080] The "antibody" means a specific protein molecule directed against an antigenic site. Preferably, the antibody means an antibody that specifically binds to a specific protein or its immunogenic fragment, and can include any of monoclonal antibody (mAb), polyclonal antibody (pAb), and recombinant antibody. The antibody can be easily produced using techniques widely known in the art.

[0081] In addition to the complete form having two full-length light chains and two full-length heavy chains, the antibody can also include functional fragments of the antibody molecule. The functional fragment of the antibody molecule means a fragment that possesses at least the antigen-binding function, such as Fab, F(ab’), F(ab’)2, F(ab)2, Fv, etc.

[0082] The peptide can be encapsulated or immobilized in nanoparticles, microparticles, metal particles, ceramic particles, hydrogels, etc., but is not limited thereto, for the purpose of transmission to a specific tissue or ensuring stability.

[0083] The nanoparticles, microparticles, metal particles, ceramic particles, hydrogels, etc. may be biocompatible and non-toxic.

[0084] The acetylcholine receptor-binding peptide can bind to the acetylcholine receptor, prevent acetylcholine from binding to the receptor, and inhibit the action of the acetylcholine receptor. Preferably, it can inhibit muscle contraction, exhibit the effect of improving wrinkles and suppressing abnormal muscle contraction, and promote muscle relaxation during surgery to ensure the convenience of surgery.

[0085] The present invention also provides a polynucleotide that encodes the acetylcholine receptor-binding peptide. As long as it can encode a peptide that can exhibit binding activity to the acetylcholine receptor, a polynucleotide containing a nucleotide sequence homologous to the nucleotide sequence constituting the polynucleotide may also be included in the scope of the polynucleotide provided by the present invention. Preferably, it is a polynucleotide containing a nucleotide sequence showing 80% or more homology, more preferably, a polynucleotide containing a nucleotide sequence showing 90% or more homology, and most preferably, a polynucleotide containing a nucleotide sequence showing 95% or more homology.

[0086] The present invention also provides a cosmetic composition for improving wrinkles, which contains the acetylcholine receptor-binding peptide.

[0087] The acetylcholine receptor-binding peptide can suppress the action of the acetylcholine receptor, prevent muscle contraction from occurring, and improve wrinkles.

[0088] The cosmetic composition may contain the acetylcholine receptor-binding peptide and auxiliary agents commonly used in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, deodorants or dyes.

[0089] The amount of the auxiliary agent is an amount commonly used in the art, and in any case, the auxiliary agent and its ratio can be selected so as not to adversely affect the preferred properties of the cosmetic composition according to the present invention.

[0090] The cosmetic composition for improving wrinkles can be produced by including additional additives.

[0091] The additives may be moisturizers, functional raw materials, thickeners, softeners, emulsifiers, surfactants, pH adjusters, etc.

[0092] The moisturizer may include, but is not limited to, glycerin, propylene glycol, butylene glycol, hyaluronic acid, ceramide components, etc.

[0093] The thickener may be, but is not limited to, polymers, xanthan gum, guar gum.

[0094] The softener may be, but is not limited to, mineral oil, shea butter, paraffin.

[0095] The emulsifier may be, for example, dimethicone, beeswax, etc.

[0096] The cosmetic composition for improving wrinkles can be used by mixing with raw materials having a wrinkle-improving effect.

[0097] The raw materials having a wrinkle-improving effect may be, but are not limited to, vitamin A, vitamin A derivatives (such as retinyl palmitate, retinyl acetate, etc.), adenosine, polyethoxylated retinamides.

[0098] The cosmetic composition may be one or more dosage forms selected from the group consisting of lotion, skin softener, skin toner, astringent, cream, foundation, essence, pack, mask pack, soap, body cleanser, cleansing foam, body oil, and body lotion, but is not limited thereto.

[0099] The cosmetic composition may be used daily or for an unspecified period, and preferably, the amount of use, the number of uses, and the period can be adjusted according to the age, skin condition, or skin type of the user.

[0100] In addition, the present invention provides a pharmaceutical composition for preventing or treating an acetylcholine receptor overactivity-related disease, which contains the acetylcholine receptor-binding peptide.

[0101] By binding to the acetylcholine receptor and suppressing the activation of the acetylcholine receptor, the pharmaceutical composition can prevent or treat the diseases associated with overactive acetylcholine receptor.

[0102] The diseases associated with overactive acetylcholine receptor are diseases with abnormal excessive contraction of muscles, such as cervical dystonia, limb dystonia, truncal dystonia, blepharospasm (facial tremor), spasticity, hemifacial spasm, strabismus, nystagmus, tics, chronic pain, chronic migraine, neurogenic bladder, detrusor-sphincter dyssynergia, achalasia cardia, hyperhidrosis, neuropathic pain, skin wrinkles, clenched jaw, sialorrhea, pediatric cerebral palsy, muscle rigidity after stroke, low back pain, benign prostatic hyperplasia, urinary incontinence, vocal cord nodules and correction, hemorrhoids, anal fissure, etc.

[0103] In addition, the pharmaceutical composition can be used to promote muscle relaxation during surgery to ensure the convenience of surgery, and can be used as a therapeutic agent or adjuvant for diseases caused by nicotine poisoning, for wrinkle removal, correction of clenched jaw or calf, but is not limited thereto.

[0104] The pharmaceutical composition can contain the acetylcholine receptor-binding peptide and a pharmaceutically acceptable excipient.

[0105] Each of the pharmaceutical compositions can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterilized injection solutions by ordinary methods. Examples of carriers, excipients, and diluents that can be contained in the pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is usually prepared using diluents or excipients such as ordinary fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, gelatin, etc., with the acetylcholine receptor-binding peptide. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As the base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerogelatin, etc. can be used.

[0106] Moreover, although there is no particular limitation on the dosage form, it may be used as a topical skin preparation having one dosage form selected from ointments, lotions, sprays, patches, creams, gels, and jellies. Formulations that increase transdermal absorption, for example, but not limited to, may contain dimethyl sulfoxide, dimethylacetamide, dimethylformamide, surfactants, alcohols, acetone, propylene glycol, or polyethylene glycol. The frequency of application may vary considerably depending on the age, sex, weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. The frequency of application may be once a month or 10 times a day, preferably once a week or 4 times a day, more preferably 3 times a week or 3 times a day, and even more preferably 1 to 2 times a day.

[0107] The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration may be considered, for example, it may be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, dermal, intrauterine, epidural, or intracerebrovascular injection. Preferably, it is dermal administration.

[0108] The present invention also relates to a health functional food composition for improving acetylcholine receptor overactivity-related diseases, which contains the acetylcholine receptor-binding peptide.

[0109] The health functional food composition may contain the acetylcholine receptor-binding peptide and a food pharmaceutically acceptable adjuvant additive.

[0110] The health functional food composition of the present invention includes forms such as tablets, capsules, pills, or liquids. Foods to which the acetylcholine receptor-binding peptide of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, and the like.

[0111] Still another aspect of the present invention provides a composition for medical devices containing the acetylcholine receptor-binding peptide.

[0112] The composition for medical devices may be a filler, but is not limited thereto.

[0113] In the present invention, "filler" is a substance capable of replenishing skin tissue and is intended to be injected and filled for restoring facial firmness, improving facial lines, and alleviating wrinkles.

[0114] The composition for medical devices can suppress muscle contraction by an acetylcholine receptor-binding peptide to alleviate wrinkles, and can also obtain an effect of improving facial lines. Biocompatible microparticles, nanoparticles, hydrogels, etc. to which the acetylcholine receptor-binding peptide is fixed can be injected to fill the tissue.

Effects of the Invention

[0115] The present invention relates to an acetylcholine receptor-binding optimized shortened peptide and its use. More specifically, a pentamer containing arginine or lysine at both ends and having a sequence of a certain amino acid XYZ in the center, and a hexamer further containing arginine or lysine at its N or C terminus are very excellent in acetylcholine receptor-binding ability compared with existing long peptides or existing pentamers and hexamers, and it is expected that they can be developed as a cosmetic composition for improving wrinkles, a pharmaceutical for preventing or treating acetylcholine receptor-related diseases, and a health functional food for improvement by confirming that the skin permeability is improved.

Brief Description of the Drawings

[0116]

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Figure 21

Mode for Carrying Out the Invention

[0117] The inventors of the present invention selected AchR-specific peptides with high binding affinity and specificity to acetylcholine receptors (AchRs) in a prior patent (Korean Patent Publication No. 10-2020-0080179). These peptides were created by identifying the sequence regions important for peptide AchR binding based on the ESPEP-2 peptide consisting of WTWKGRKSLLR, and then identifying optimized 8-mer, 11-mer, and 14-mer peptides with increased affinity to AchRs. These peptides demonstrated high selective binding affinity to AchRs through multiple biopanning rounds. However, the peptides, consisting of 8- to 14-mer peptides, had problems with high peptide production costs and poor skin penetration. In particular, increasing the peptide length not only increases production costs but also adversely affects skin penetration.

[0118] To solve these problems, the present inventors devised the present invention while searching for a shortened peptide that has excellent selective binding to AchR. In particular, we confirmed that the RKSLLR and KSLLR peptides, which are simply shortened versions of Espep2 to 6mers and 5mers, have superior binding ability compared to the syn-ake peptide. Based on the Espep2 structure, we searched for optimal 6mer and 5mer peptide structures. To determine the optimized shortened peptides, we created and screened random libraries of 6mers and 5mers and analyzed the peptide sequences. As a result, we confirmed that peptides with specific amino acid sequences at certain positions exhibit higher acetylcholine receptor binding ability than the existing Espep2 (11mer), 6mer, and 5mer.

[0119] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to preferred embodiments thereof. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, this disclosure is provided so that the present invention will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art.

[0120] Example 1. Measurement of affinity of peptides shortened in size to generate an optimized shortened library

[0121] To create an optimized shortened library of the prepro-eserpentide 2 peptide, the affinity of peptides with one amino acid removed step by step at the N-terminus and C-terminus was confirmed. First, a surface plasmon resonance experiment (SPR) was performed using a biosensor chip (Biacore 3000, Biacore AB, Uppsala, Sweden). The selected acetylcholine receptor protein was immobilized on a CM5 chip (Biacore) using EDC / NHS, and association and dissociation were observed up to a maximum of 500 seconds. The observation conditions were a running buffer of 20 mM Tris (pH 7.4), a flow rate of 30 μl / min, and a concentration of 20 μM (peptide), and the results are shown in Figure 1.

[0122] As shown in Figure 1, the affinities of the derivatives with amino acids removed at the N-terminus and C-terminus to shorten the size were 1.2 μM for 11mer (eserpentide 2-11mer, WTWKGRKSLLR), 3.1 μM for 8mer (eserpentide 2-8mer, KGRKSLLR), and 57 μM for 6mer (eserpentide 2-6mer, RKSLLR). Compared to Synake, the acetylcholine receptor affinities of 11mer were 1916 times higher, 8mer were 740 times higher, and 6mer were 40 times higher.

[0123] Synake is a snake venom-derived peptide that inhibits the binding of acetylcholine to the acetylcholine receptor and is used for wrinkle improvement. As seen in Figure 1, the high affinity of the eserpentide-2 shortened peptides (11mer, 8mer, 6mer) for the acetylcholine receptor protein is superior to that of Synake. Therefore, it was confirmed that it can be used not only for cosmetic compositions including wrinkle improvement but also for the prevention or treatment of diseases related to acetylcholine receptor overactivity by suppressing the acetylcholine receptor.

[0124] Conventionally, botulinum toxin, which has been used to treat various muscle spasms and diseases caused by muscle hyperactivity due to acetylcholine receptor hyperactivity, has extremely high toxicity such that just 1 g can cause approximately one million people to die. Despite its excellent medical uses, in light of it being a substance regulated by the Biological Weapons Convention, it has been confirmed that a high acetylcholine receptor inhibitor efficacy can be achieved using short peptides.

[0125] <Example 2. Alanine Scanning for Confirming the Core Sequence and Measurement of the Affinity of Short Peptides>

[0126] Alanine scanning was performed to confirm the important sequence of the short peptide. Espep 2-6mer (RKSLLR) and Espep 2-5mer (KSLLR) were used as wild type (WT), and peptides in which the amino acid sequences of each peptide were sequentially substituted with alanine (Ala, A) were synthesized. Then, the affinity was measured by the same method used in Example 1 and normalized based on WT, as shown in Figure 2.

[0127] As can be seen in Figure 2, it was confirmed that positive charge amino acids near the N- and C-termini are important for binding.

[0128] Also, among the six amino acids of the RKSLLR sequence, it was confirmed that the R sequences at the N- and C-termini are important. Based on such results, 5mer-ND (KSLLR) and 5mer-CD (RKSLL) peptides were prepared by removing one sequence from each of the two ends of the N- and C-termini based on the 6mer, and the affinity of each was measured and shown in Figure 3.

[0129] As can be seen in Figure 3, Espep 26mer had an RU value of 177 and a higher affinity compared to Syn-Ake. The 5mer-ND, which was shorter than this, showed a higher binding force compared to Syn-Ake. 5mer-CD showed a somewhat lower binding force compared to Syn-Ake. Thereby, it was confirmed that a structure in which the 5mer short peptide contains lysine (K) or arginine (R) at both ends is advantageous for acetylcholine receptor binding.

[0130] <Example 3. Preparation of shortened libraries of 6mer-1, 6mer-2, and 5mer-ND>

[0131] Based on the important sequences confirmed by the alanine scanning in Example 2 above, an optimized library was prepared for 6mer-1; (K / R)(K / R)XXX(K / R), 6mer-2; (K / R)XXX(K / R)(K / R), 5mer-ND; (K / R)XXX(K / R) [(K / R) is either K or R, and X is a random amino acid].

[0132] Example 3-1. Preparation of an optimized shortened library vector

[0133] To create a 6mer-optimized peptide library, 6mer-1-F (SfiI_ARAARANNKNNKNNKARA_NotI), 6mer-2-F (SfiI_ARANNKNNKNNKARAARA_NotI), 5mer-ND-F (SfiI_ARANNKNNKNNKARA_NotI), and Back (AACAGTTTCTGCGGCCGC) (N is A, T, G, or C; K is G or T; M is C or A) were synthesized (Bioneer, Daejeon, Korea). Using the synthesized DNA library in Table 1 as a template, 5mer and 6mer inserts were subjected to a PCR reaction (5 minutes at 94°C, 60 cycles: 30 seconds at 40°C, 30 seconds at 72°C, and 7 minutes at 72°C) to form double strands, and then purified (using a PCR purification kit, GeneAll, Seoul, Korea) to obtain library genes. To insert this into a phagemid vector (pIGT), the phagemid vector and insert DNA were treated with restriction enzymes. Approximately 10 μg of insert DNA was reacted with SfiI (New England Biolabs (NEB, Ipswich)) and NotI (NEB, Ipswich) for 8 hours, and then purified DNA was obtained using a PCR purification kit. Also, approximately 10 μg of the phagemid vector was treated with SfiI and NotI for 8 hours, CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich) was added, and the reaction was carried out for 1 hour, followed by purification using a PCR purification kit. After ligating the insert DNA with the phagemid vector at 18°C for 15 hours using T4 DNA ligase (Bioneer, Daejeon, Korea), it was precipitated with ethanol and the DNA was dissolved in 100 μl of TE buffer.

[0134] [Table 1] TIFF0007715435000001.tif77166

[0135] Example 3-2. Electroporation

[0136] 100 μl of the ligation reaction mixture containing 10 μg of phagemid vector and 3 μg of 6-mer or 5-mer random insert DNA was aliquoted into 10 portions and subjected to electroporation. The competent cells were thawed on ice, mixed with 4 μl of the ligation reaction solution, cooled, placed in a prepared 0.2 cm cuvette, and left on ice for 1 minute. The electroporator (BioRAD, Hercules, CA) was programmed under the conditions of 200 Ω, 25 μF, and 2.5 kV. After removing the moisture from the prepared cuvette and positioning it in the electroporator, a pulse was applied. At this time, the time constant was 4.5 - 5 msec. Immediately thereafter, the mixture was transferred into 1 ml of LB liquid medium containing 20 mM glucose pre-warmed to 37°C, and the resulting total 25 ml of cells was transferred to a 100 ml test tube. Then, the cells were cultured with mixing at 200 rpm for 1 hour at 37°C. Then, 10 μl was taken and diluted and spread on an ampicillin agar medium to measure the number of libraries. The remaining cells were cultured in 1 L of LB containing 20 mM glucose and 50 μg / ml of ampicillin at 30°C for 1 day. Then, the cells were centrifuged at 4,000 g for 20 minutes at 4°C, and all the supernatant except the precipitated cells was removed. After resuspending in 40 ml of LB, glycerol was added to a final concentration of 20% or more, and the cells were stored at -80°C.

[0137] Example 3-3. Production of Recombinant Phage with Optimized Peptide Library

[0138] Recombinant phage was produced using the 6-mer and 5-mer optimized peptide libraries stored at -80°C. 1 ml of the library stored at -80°C was added to 30 ml of SB liquid medium, and the mixture was cultured with mixing at 200 rpm for 20 minutes at 37°C. Here, helper phage (10 10Pfu) and ampicillin (final concentration 50 μg / ml) were added and cultured for an additional 1 hour under the same conditions. Then, it was transferred to 30 ml of SB liquid medium containing ampicillin (50 μg / ml) and kanamycin (10 μg / ml) and cultured for 16 hours or more under the same conditions to produce recombinant phage. PEG / NaCl was mixed with the supernatant obtained by centrifuging the culture solution at 5,000 rpm for 10 minutes at 4°C in a ratio of 5:1, left standing on ice for 1 hour, then centrifuged at 13,000 rpm for 20 minutes at 4°C, and the supernatant was carefully removed. The pellet was resuspended in 1 ml of PBS.

[0139] To probabilistically include all possible amino acid sequences in the library, for the 5-mer case, the number of completed libraries is, for the 5-mer optimized library, the number 2×2×20×20×20 = 3.2×10 4 or more. Therefore, in this study, the number of 5-mer libraries was set to 1.95×10 6 For the 6-mer optimized library, the number is 2×2×2×20×20×20 = 6.4×10 4 or more, and in this study, as a result of preparation, the library numbers of 6-mer-1 and 6-mer-2 were 1.23×10 6 , 1.53×10 6 were obtained, and it was confirmed that the library was successfully developed. This is shown in Table 2 below. Also, by partially sequencing each library, it was confirmed that there were no errors in the specified sequences. The results are shown in Table 3.

[0140] [Table 2] TIFF0007715435000002.tif32166

[0141] [Table 3] TIFF0007715435000003.tif166166

[0142] [Example 4. Biopanning and Screening of Short Peptide Library]

[0143] Example 4-1. Biopanning method

[0144] The process of treating a phage library expressing antibodies with an immobilized antigen and selecting antibody candidates that bind to the antigen is called biopanning, and it consists of three steps: bind / wash / elution. During the washing process, phages with weakly binding antibodies are removed, and as a result, only phages expressing highly binding antibodies remain. By repeating such a process, antibody candidates with excellent antigen-binding ability and specificity can be discovered. Therefore, the biopanning method was used to screen for an acetylcholine receptor-binding peptide with excellent binding ability and specificity to the acetylcholine receptor of the present invention.

[0145] 5 μg / ml of AchR α1 was placed in 8 wells of a 96-well plate at 50 μl per well and left overnight at 4°C. The next day, after washing once with 200 μl of Tris (20 mM pH 7), 200 μl of 2% BSA (bovine serum albumin) was added and blocked at room temperature for 2 hours, then all the solution was discarded and washed 3 times with 200 μl of Tris (20 mM pH 7). 400 μl of the random peptide recombinant phage (input phage) suspended in the PBS of Example 3-3 and 400 μl of 2% BSA were mixed and placed in each well at 100 μl per well and left at 30°C for 1 hour. All the solution in the well was removed, washed 3 times with Tris (20 mM pH 7), then 100 μl of 0.2 M glycine (pH 2.2) was added to each well to release the phage for 20 minutes, and then all were collected into one tube and 200 μl of 1 M Tris (pH 9.0) was added to obtain the output phage.

[0146] To repeat the biopanning, 500 μl of the released phage was mixed with 5 ml of Escherichia coli (E. coli) and cultured at 37°C at 200 rpm for 30 minutes, then 1×10 10The pfu helper phage and ampicillin were added to a final concentration of 50 μg / ml and further cultured for 30 minutes. Then, the culture solution was transferred to SB liquid medium containing 50 μg / ml ampicillin and 10 μg / ml kanamycin and cultured overnight under the same conditions to reproduce the random peptide recombinant phage. The reproduced random peptide recombinant phage was centrifuged at 5,000 rpm for 10 minutes at 4°C to obtain the supernatant. PEG / NaCl was mixed with the supernatant at 5:1 [v:v], left on ice for 1 hour, then centrifuged at 13,000 rpm for 20 minutes at 4°C to remove the supernatant. The precipitate was suspended in 1 ml of PBS (phosphate buffered saline) and then used for the second biopanning.

[0147] Random recombinant phages were reproduced and biopanned in the same manner as above, except that the washing process was increased stepwise by 3, 3, 4, 4, 5, and 6 times (0.05% PBST) for each biopanning step.

[0148] To measure the number of input phage and output phage for each biopanning, they were mixed with Escherichia coli (E. coli) with an absorbance at 600 nm of 0.7 (OD600 = 0.7) and spread on an agar plate containing ampicillin. The results are shown in Tables 4 to 6 below.

[0149] [Table 4] TIFF0007715435000004.tif51166

[0150] [Table 5] TIFF0007715435000005.tif51166

[0151] [Table 6] TIFF0007715435000006.tif51166

[0152] Example 4-2. ELISA (enzyme-linked immunosorbent assay) method using a recombinant phage input with a random peptide library

[0153] Using the phages input separately for each round of the biopanning in Example 4-1, ELISA was performed on BSA (Bovine serum albumin) and AchR.

[0154] In a 96-well ELISA plate, 50 μl of 10 μg / ml AchR or BSA was added to each well and left overnight at 4°C. The next day, it was washed three times with Tris (20 mM pH 7), then 2% BSA diluted with PBS was added and blocked at room temperature for 2 hours. After discarding all the solution, it was washed three times with Tris (20 mM pH 7). Then, 800 μl of the phages input for each round (1st, 2nd, 3rd, 4th, 5th, and 6th) in Table 4-6 of Example 4-1 was mixed with 200 μl of 10% BSA, and 100 μl of the mixture was added to each well and left at 30°C for 1 hour. After removing all the solution in the wells and washing three times with Tris (20 mM pH 7), 100 μl of HRP (horseradish peroxidase)-conjugated anti-M13 antibody (HRP-conjugated anti-M13Ab, GE Healthcare), diluted 1:1,000, was added to each well and reacted at 30°C for 1 hour. Then, after washing three times with Tris (20 mM pH 7), 100 μl of tetramethylbenzidine (TMB) solution, which is a substrate for HRP, was added to each well to induce a color reaction. Then, 100 μl of 1M HCl was added to stop the reaction, and the absorbance (OD450) was measured at 450 nm. The results are shown in Figure 4. As can be seen in Figure 4, as each round of biopanning progresses, the OD value of acetylcholine / BSA increases, which means that phages with high specificity for the acetylcholine receptor have been successfully screened.

[0155] <Example 5. X amino acid analysis of each library peptide>

[0156] After biopanning, it was confirmed whether amino acids with a particularly high binding affinity to the acetylcholine receptor could be identified in relation to the random sequence XXX in the middle part of the peptide sequences of each library. Among the libraries of Example 4 above, as a result of analyzing the sequences of phages after 6 rounds of highly specific biopanning, it was confirmed that the number of repetitions of specific amino acids was high for both 5-mers and 6-mers. The XXX part was designated as XYZ in order, and the number of occurrences of individual amino acids at the positions of X, Y, and Z of the peptide with a high number of repetitions was shown in Table 7.

[0157] [Table 7] TIFF0007715435000007.tif115166

[0158] Specifically, at the X position, 7 amino acids, namely R, Q, G, V, L, S, and W; at the Y position, 7 amino acids, namely R, Q, L, I, F, V, and Y; and at the Z position, 7 amino acids, namely R, S, L, C, Q, T, and Y, had a much higher number of occurrences compared to other amino acids.

[0159] <Analysis of the binding affinity of peptides combined with the top 7 amino acids in the repeated sequences of XYZ in Example 6>

[0160] Based on Table 7 of Example 5 above, peptides combined with the top 7 amino acids with a high number of occurrences (top combination), and peptides combined with the bottom 13 amino acids with a low number of occurrences (bottom combination) were synthesized 600 each as 5-mer, 6-mer-1, and 6-mer-2 series, and the acetylcholine receptor binding affinity of each sequence was measured. The binding affinity (Ru; Resonance Units) with AchR was measured using the surface plasmon resonance (SPR) analysis method, and the results are shown in Tables 8 to 13. At this time, the peptides were tested under a concentration condition of 10 μM. Also, the average binding affinities of the top combination and the bottom combination of each series are shown in Figures 5 to 7.

[0161] [Table 8] TIFF0007715435000008.tif242166TIFF0007715435000009.tif229166TIFF0007715435000010.tif229166TIFF0007715435000011.tif229166TIFF0007715435000012.tif229166TIFF0007715435000013.tif229166TIFF0007715435000014.tif229166TIFF0007715435000015.tif121166

[0162] [Table 9] TIFF0007715435000016.tif242166TIFF0007715435000017.tif229166TIFF0007715435000018.tif229166TIFF0007715435000019.tif229166TIFF0007715435000020.tif229166TIFF0007715435000021.tif229166TIFF0007715435000022.tif229166TIFF0007715435000023.tif121166

[0163] [Table 10] TIFF0007715435000024.tif242166TIFF0007715435000025.tif229166TIFF0007715435000026.tif229166TIFF0007715435000027.tif229166TIFF0007715435000028.tif229166TIFF0007715435000029.tif229166TIFF0007715435000030.tif229166TIFF0007715435000031.tif121166

[0164] [Table 11] TIFF0007715435000032.tif242166TIFF0007715435000033.tif229166TIFF0007715435000034.tif229166TIFF0007715435000035.tif229166TIFF0007715435000036.tif229166TIFF0007715435000037.tif229166TIFF0007715435000038.tif229166TIFF0007715435000039.tif121166

[0165] [Table 12] TIFF0007715435000040.tif242166TIFF0007715435000041.tif229166TIFF0007715435000042.tif229166TIFF0007715435000043.tif229166TIFF0007715435000044.tif229166TIFF0007715435000045.tif229166TIFF0007715435000046.tif229166TIFF0007715435000047.tif121166

[0166] [Table 13] TIFF0007715435000048.tif242166TIFF0007715435000049.tif229166TIFF0007715435000050.tif229166TIFF0007715435000051.tif229166TIFF0007715435000052.tif229166TIFF0007715435000053.tif229166TIFF0007715435000054.tif229166TIFF0007715435000055.tif121166

[0167] Among the 5-mers in Table 8 above, the acetylcholine receptor binding ability of the RRQRR sequence of SEQ ID NO: 73 5-mer-73 was found to be extremely high compared to other 5-mers (binding ability 179). Accordingly, extended 6-mer sequences XRRQRR and RRQRRX of SEQ ID NO: 73 were prepared and their acetylcholine receptor binding abilities were confirmed, and the results are shown in Table 14 as relative values of the RRQRR sequence.

[0168] [Table 14] TIFF0007715435000056.tif140166

[0169] As can be seen in Table 14 above, the extended sequences XRRQRR and RRQRRX of RRQRR showed extremely high acetylcholine receptor binding abilities in most sequences.

[0170] Among the 6-mers in Table 9 above, the acetylcholine receptor binding ability of the RRGVRR sequence of SEQ ID NO: 1410 6-mer-1-210 was found to be extremely high compared to other 6-mers (binding ability 184). Accordingly, extended 7-mer sequences XRRGVRR and RRGVRRX of SEQ ID NO: 1410 were prepared and their acetylcholine receptor binding abilities were confirmed, and the results are shown in Table 15 as relative values of RRGVRR.

[0171] [Table 15] TIFF0007715435000057.tif127166

[0172] As can be seen in Table 15 above, the extended sequences XRRGVRR and RRGVRRX of RRGVRR showed extremely high acetylcholine receptor binding abilities in most sequences.

[0173] [Example 7. Comparison of the binding abilities of two peptides with high binding abilities in each library with the positive control group>

[0174] Two representative peptides from each library confirmed in Example 6 above were selected, two each of 5mer (73,311), 6mer-1 (43,210), and 6mer-2 (136,233), and their binding affinities were compared with those of the pre-optimized 6mer, 5mer-ND, and Synake. The results are shown in Table 16 and Figure 8. As seen in Table 16 and Figure 8, in Example 6 above, the optimized truncated peptides of 5mer and 6mer limited by XYZ were superior in binding affinity to the acetylcholine receptor not only to Synake but also to the pre-optimized Espep-2 truncated peptide. The affinity for the 6mer-1-43 peptide among the optimized truncated peptides was measured, and the results are shown in Figure 9. The affinity of the 6mer-1-43 peptide according to the present invention for the acetylcholine receptor was 609 nM.

[0175] [Table 16] TIFF0007715435000058.tif64166

[0176] <Example 8. Confirmation of Inhibition of Acetylcholine Receptor Activity by 6 Peptides with High Binding Affinity>

[0177] The inhibitory effects of the 5mer (73,311), 6mer-1 (43,210), and 6mer-2 (136,233) peptides, which were confirmed to have excellent binding affinity for AchR in Example 5 above, on AchR activity were confirmed.

[0178] TE671 cells overexpressing the acetylcholine receptor were cultured in DMEM medium containing 10% FBS and 1% P / S at 37°C and 5% CO2. When the TE671 cells had grown sufficiently for about 4 days, an 18 mm cover-slip was placed in a 12-well cell culture plate, and the cultured cells were detached with trypsin. Then, 1 ml aliquots were dispensed to give 2×10 4 / cells per well and cultured for 4 days.

[0179] Transfer the coverslip on which the cells were cultured to a new 12-well cell culture plate. Pipette 997 μl of HBSS buffer and 3 μl of Fura-2-AM, mix them gradually, and then culture at 37 °C with 5% CO₂ for 15 minutes. After culturing, wash 3 - 4 times with 1 ml of HBSS buffer to remove the remaining Fura-2-AM, and pipette another 1 ml. After fitting the coverslip with the growing cells into the chamber, pipette 500 μl of HBSS buffer. Then, adjust the nicotine concentration to a final concentration of 400 μM and confirm the calcium imaging reaction for each. After that, keep the nicotine concentration fixed and search for the inhibitory concentration by adjusting the concentration of the sample. Treat with Synake as a control group and show the results in Figures 10 - 13.

[0180] As shown in Figures 10 - 13, at 500 μM, Synake inhibited the acetylcholine receptor, and at 5 μM, the 11-mer, which is the precursor peptide, inhibited the acetylcholine receptor. On the other hand, it was confirmed that the optimized shortened peptides 5-mer (73,311), 6-mer-1 (43,210), and 6-mer-2 (136,233) according to the present invention were inhibited at 10 μM. Thus, the newly discovered 6-mer peptide showed an inhibitory ability similar to that of the precursor peptide 11-mer, but was about 100 times superior to the average Synake, and it was confirmed that the 5-mer peptide showed an inhibitory ability about 33 times superior.

[0181] <Example 9. Evaluation of the inhibitory ability on the acetylcholine receptor using the modification of the peptide terminus>

[0182] The terminals of the optimized shortened peptides according to the present invention were modified with palmitate, and the inhibitory ability against acetylcholine receptors was evaluated. The inhibitory abilities of Palmitate-6mer-1-43 and Palmitate-5mer-73 peptides against acetylcholine receptors were confirmed in the same manner as in Example 6. As a result, it was confirmed that 100% inhibition occurred at 1 μM, and the results are shown in FIGS. 14 and 15. In addition, the inhibitory ability (IC 50 ) of the peptide obtained by binding and modifying the optimized shortened peptide according to the present invention with palmitate against acetylcholine receptors was compared with that of Cinepazide and shown in FIG. 16. As seen in FIGS. 14 to 16, when the terminals of the optimized shortened peptides according to the present invention were modified with palmitate, it was confirmed that the inhibitory ability against acetylcholine receptors was significantly increased compared to the unmodified shortened peptides.

[0183] <Example 10. Cytotoxicity Evaluation of Optimized Shortened Peptides>

[0184] The cytotoxicity of the optimized shortened peptides according to the present invention was evaluated. Using dermal fibroblast cells, which are dermal cells isolated from human skin, cytotoxicity was evaluated by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The pre-cultured cells were seeded at a concentration of 1×10 5 cells / ml in a 24-well plate and cultured for 18 hours. Then, the medium was removed, the diluted sample was added to the medium without added FBS, and the cells were cultured for 24 hours. After that, the medium was removed, the MTT solution was added at a concentration of 1 μg / ml, and the reaction was allowed to proceed for 3 hours. The unreacted MTT was removed, 100 μl of DMSO was added to dissolve the formazan formed, and the absorbance at 540 nm was measured with an ELISA reader for evaluation.

[0185] As shown in Fig. 17, the cytotoxicity of 6mer-1-43 (6mer peptide), 5mer-73 (5mer peptide), Pal-6mer-1-43, and Pal-5mer-73 peptides on dermal cells of the skin was measured by the MTT method. As a result, both peptides were evaluated to have no cytotoxicity at concentrations of 100 nM to 100 μM.

[0186] <Example 11. Analysis of Skin Permeation Rate by Peptide Length>

[0187] Using a Franz diffusion cell system, the skin permeation rates of the prior peptide 11mer and the optimized shortened peptide of the present invention were compared.

[0188] After adding 5 ml of PBS (containing 0.05% polysorbate 80, pH 7.4) to the receptor chamber of the Franz diffusion cell system, one or two cellulose acetate membranes (artificial skin) were placed and fixed between the receptor chamber and the donor chamber. Each existing 11mer and the optimized 5mer-73 (RRQRR) peptide were added to the donor chamber of the Franz cell. The conditions of the receptor chamber were adjusted to 37°C and 600 rpm. Samples were collected at 0.5, 1, 2, 4, 8, 12, 18, and 24 hours, 500 μl each time. The amount of drug that permeated through the skin over time was measured by HPLC, and the results are shown in Fig. 18. As shown in Fig. 18, it was confirmed that the skin permeation rate of the optimized shortened peptide 5mer according to the present invention was 66.87% after 8 hours, while the 11mer did not permeate at all.

[0189] As described above, the acetylcholine receptor shortened peptide of the optimized shortened peptide 5mer of the present invention exhibits similar acetylcholine receptor inhibitory activity to the 11mer peptide that showed excellent acetylcholine receptor inhibitory effects in previous studies. However, by shortening the peptide length by half, the skin permeation rate can be dramatically increased, and the production cost can be epochally reduced.

[0190] <Example 12. Clinical Efficacy Evaluation of Crow's Feet of Peptide Cosmetic Formulations>

[0191] The clinical efficacy of the optimized short peptide according to the present invention against crow's feet on the skin was evaluated. A test product containing the optimized short peptide according to the present invention and a control group were manufactured as shown in Table 17 below, and a human application test was conducted. Twenty-one subjects who met the selected conditions and did not fall under the selected exclusion conditions were recruited, and one dropped out due to withdrawal of consent to participate in the test, and finally 20 people were tested. All measurements were carried out under constant temperature and humidity conditions (22 ± 2 °C, 50 ± 10% RH) without air movement and direct sunlight after the subjects had taken at least 30 minutes of rest. The selected subjects visited a clinical institution before using the product (week 0), measured the texture (wrinkles) at the crow's feet, and conducted a demographic survey. The test product was applied twice a day (morning and evening) for 6 weeks, and the subjects visited the clinical institution again at the 3rd and 6th weeks after using the product and received the same evaluation as before using the product (week 0).

[0192] [Table 17] TIFF0007715435000059.tif96166

[0193] As evaluation items, the overall size of wrinkles, depth of wrinkles, and maximum depth of wrinkles were measured, and ANTERA 3D (R) CS (Miravex, Ireland) was used to photograph and analyze the crow's feet skin wrinkles, and the results were compared with those of week 0 and shown in Table 18 and Figure 19. In the case of the control group, no significant difference was found in any of the evaluation items.

[0194] [Table 18] TIFF0007715435000060.tif70166

[0195] As shown in Table 18 and Figure 19 above, the overall size of the crow's feet wrinkles decreased by 19.1% (p < 0.001) at the third week of use and 31.5% (p < 0.001) at the sixth week of use compared to before product use. The depth decreased by 21.0% (p < 0.001) at the third week of use and 30.1% (p < 0.001) at the sixth week of use compared to before product use. The maximum depth decreased by 14.2% (p < 0.001) at the third week of use and 27.9% (p < 0.001) at the sixth week of use compared to before product use.

[0196] <Example 13. Clinical Efficacy Evaluation of the Elasticity of Peptide Cosmetic Formulations>

[0197] The clinical efficacy on skin elasticity (Ur / Ue) was evaluated in the same way as in Example 12 above. The dermal density was measured using the DUB (R) Skinscanner (tpm, Germany). The dermal density of the subject's cheek area was measured before product use (0 week), at the third week after product use, and at the sixth week after product use, and the results were compared before and after and shown in Figure 20.

[0198] As can be seen in Figure 20, the skin elasticity (Ur / Ue) increased by 6.9% (p < 0.001) at the third week of use and 17.8% (p < 0.001) at the sixth week of use compared to before product use. The dermal density increased by 18.9% (p < 0.001) at the third week of product use and 28.5% (p < 0.001) at the sixth week of product use compared to before product use.

[0199] <Example 14. Clinical Efficacy Evaluation of Forehead Wrinkles of Peptide Cosmetic Formulations>

[0200] The clinical efficacy of the optimized shortened peptide cosmetic formulation according to the present invention was evaluated against forehead wrinkles. Before using the product, the forehead wrinkle area was photographed with Antera 3D and a DSLR. Then, after applying a gauze (5 cm × 5 cm) soaked with the test product and the control group in Table 17 above to the forehead wrinkle area for 8 hours, after removing the gauze, when 40 minutes and 2 hours and 40 minutes had elapsed, the forehead wrinkle area was photographed with Antera 3D and a DSLR, and the results are shown in Figure 21.

[0201] As can be seen in Figure 21, compared with before removing the product, the improvement effect of forehead wrinkles appeared significantly later, and it was confirmed that the wrinkle improvement effect was maintained even after more than 2 hours had elapsed.

Claims

1. The following chemical formula 2-1 or chemical formula 3-1: [Chemical formula 2-1] (R / K)(R / K)XYZ(R / K) [Chemical formula 3-1] (R / K)XYZ(R / K)(R / K) (However, R / K is arginine or lysine, XYZ are any amino acids arranged sequentially, X is one amino acid selected from R, Q, G, V, L, S, W, Y is one amino acid selected from R, Q, L, I, F, V, Y, and Z is one amino acid selected from R, S, L, C, Y, Q, T) An acetylcholine receptor-binding peptide consisting of the amino acid sequence represented by the peptide is a peptide of one amino acid sequence selected from the group consisting of SEQ ID NO: 1243 (6mer-1-43, RKRIRR), SEQ ID NO: 1410 (6mer-1-210, RRGVRR), SEQ ID NO: 2536 (6mer-2-136, RWRYKR), and SEQ ID NO: 2633 (6mer-2-233, KWRQKR) The acetylcholine receptor-binding peptide characterized by the above.

2. The N-terminus or C-terminus of the peptide is modified The acetylcholine receptor-binding peptide according to Claim 1.

3. The fact that the N-terminus or C-terminus of the peptide is modified means that the N-terminus or C-terminus of the peptide is palmitoylated, acetylated, formylated, amidated, or PEGylated, or A compound selected from 2-mercaptoacetic acid, 3-mercapto propionic acid, 6-mercaptohexanoic acid, pyroglutamic acid, succinimide acid, cysteamine, methyl ester, ethyl ester, and benzyl ester binds, or by binding of one or more fatty acids selected from the group consisting of myristic acid, stearic acid, palmitic acid, cholesterol, 6-aminohexanoic acid, and 8-aminooctanoic acid The acetylcholine receptor-binding peptide according to claim 2

4. Coding the peptide according to any one of claims 1 to 3 A polynucleotide characterized by this

5. Containing the peptide according to any one of claims 1 to 3 A cosmetic composition for improving wrinkles, characterized by this

6. Containing the peptide according to any one of claims 1 to 3 A composition for preventing or treating an acetylcholine receptor overactivity-related disease, characterized by this

7. The acetylcholine receptor overactivity-related disease is Cervical dystonia, limb dystonia, truncal dystonia, blepharospasm (eyelid spasm, facial tremor), spasticity, hemifacial spasm, strabismus, nystagmus, tics, chronic pain, chronic migraine, neurogenic bladder, detrusor-sphincter dyssynergia, achalasia cardia, hyperhidrosis, neuropathic pain, skin wrinkles, clenched jaw, and sialorrhea, which is any one or more selected from the group consisting of The composition for preventing or treating a disease associated with acetylcholine receptor overactivity according to claim 6.

8. Containing the peptide according to any one of claims 1 to 3 A health functional food composition for improving a disease associated with acetylcholine receptor overactivity, characterized by the above.

9. Containing the peptide according to any one of claims 1 to 3 A composition for a medical device containing an acetylcholine receptor-binding peptide, characterized by the above.

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