BACKGROUND OF THE INVENTION

Field of the Invention

The present disclosure relates to an acetylcholine receptor-binding peptide and, more particularly, to peptides which exhibit a wrinkle amelioration effect by binding the peptides to an acetylcholine receptor on which acetylcholine acts, thereby blocking secretion of acetylcholine.

Related Art

Recently, consumers' needs for cosmetics have been gradually changed from requesting uses for decorating themselves beautifully to requesting functional uses of the cosmetics due to an increase in interest for healthy life, improvement of the standard of living, an increase in the entry of women in public affairs, the acceleration of aging, etc.

Research activities intended for developing bioactive substances having a wrinkle amelioration effect has consistently progressed to prevent skin aging phenomenon and maintain more healthy and elastic skin. Typically, tretinoin (trans-retinoic acid) as a therapeutic agent for improving photoaged skin received United States FDA permission in 1995, and wrinkle ameliorating cosmetics have been started to be marketed in earnest while retinol having less side effects than tretinoin has been used in raw materials for cosmetic products from the middle and late 1990s. Thereafter, female hormone-like substances, antioxidants extracted from various plants, etc. as wrinkle ameliorating raw materials have been introduced into cosmetics.

However, most of such raw materials for cosmetic products have been had various problems including inept efficacy, causing of skin side effects, etc. Further, the present raw materials for cosmetic products have not been able to sufficiently satisfy needs of consumers wanting newer, stronger and more fundamental amelioration of wrinkles since the present raw materials for cosmetic products have limited application ranges to the skin, and most of the present raw materials for cosmetic products are similar in efficacies on the skin such as promotion of collagen synthesis, inhibition of collagen decomposition, and removal of active oxygen although present raw materials for cosmetic products are raw materials with good efficacies. Accordingly, researches on raw materials and technologies which are capable of establishing new skin aging mechanisms, and blocking or delaying skin aging based on recent dermatological theories have been actively progressed in cosmetic industries.

Recently, a study of ameliorating skin wrinkles by using a peptide component in cosmetics has been actively proceeded. Peptides, as material formed by coupling two or more amino acids, are produced by chemical synthesis, enzyme reaction, or hydrolysis from protein.

On the other hand, acetylcholine is involved in movements of skeletal muscles and visceral muscle in the peripheral nervous system, and has an effect on learning and memory in the brain. When secretion of acetylcholine, i.e., neurotransmitter is hindered at places where a motor nerve and muscles meet, acetylcholine inhibits contraction of the muscles such that wrinkles are spread while the muscles are being paralyzed. Botox corresponds to an example using this. Botox blocks a process of secreting acetylcholine, i.e., material that is essential in contraction of the muscles at a motor nerve terminal. As a result, the muscles cannot be moved, and wrinkles caused by the muscles are removed.

Therefore, if peptides of inhibiting secretion of acetylcholine are developed by the same principle, it is predicted that amelioration and prevention of skin wrinkles can be expected.

PRIOR ART DOCUMENT

Patent Document

(Patent document 1) Korea Patent Registration Publication No. 10-0553174

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor.

To achieve the purpose, an acetylcholine receptor-binding peptide comprising an amino acid sequence represented by any one of the following general formula 1 according to an aspect of the present disclosure is provided:
WTWKG(SEQ ID NO:32)-Xn[General Formula 1]
TWKG(SEQ ID NO:33)-Xn[General Formula 2]
WKG-Xn[General Formula 3]
KG-Xn[General Formula 4]
G-Xn[General Formula 5]
Xn[General Formula 6]

wherein the Xnindicates a sequence comprised of 1 to 6 any amino acids.

The term in the present disclosure, “peptides or fragments thereof”, means a polymer comprised of two or more amino acids connected by an amide bond (or peptide bond). For the purpose of the present disclosure, the peptides or the fragments thereof means peptides which exhibit a wrinkle amelioration effect or fragments thereof.

Peptides or fragments thereof of the present disclosure may include an additional amino acid sequence devised as a specific purpose for increasing stability of targeting sequence, tag, labelled residues, half-life or peptides.

Peptides or fragments thereof of the present disclosure may be obtained by various methods well known in the art. Specifically, peptides or fragments thereof of the present disclosure may be produced by using genetic recombination or protein expression system, or may be produced by a method of synthesizing the peptides or the fragment thereof in vitro through chemical synthesis such as peptide synthesis, and a cell-free protein synthesis method.

More specifically, although the peptides or the fragment thereof not only may be synthesized by a method well known in the art, e.g., an automatic peptide synthesizer, but also may be produced by genetic engineering technology, the present disclosure is not limited thereto. For example, desired peptides can be produced by cutting and separating peptides according to the present disclosure from the fusion protein by using protease or a compound after preparing a fusion gene which encodes a fusion protein formed of a fusion partner and peptides according to the present disclosure through gene manipulation, transforming the fusion gene into a host microbe, and expressing the host microbe in the form of a fusion protein. To this end, for instance, a DNA sequence encoding amino acid residues which can be cut by protease such as Factor Xa or enterokinase and a compound such as CNBr or hydroxylamine may be inserted between the fusion partner and a peptide gene of the present disclosure.

In peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor of the present disclosure, the Xnmay have any one amino acid sequence among the following SEQ ID NO: 1 to SEQ ID NO: 11:

[Sequence number 1]KGTLNR,[Sequence number 2]RKSLLR,[Sequence number 3]EDKGKN,[Sequence number 4]RDKLQM,[Sequence number 5]QLGQLS,[Sequence number 6]GRLSAS,[Sequence number 7]RQLNNQ,[Sequence number 8]DNLQNN,[Sequence number 9]LYQRLG,[Sequence number 10]NKQVKF,and[Sequence number 11]ETYDSK

In peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor of the present disclosure, the peptides may comprise any one amino acid sequence among the following SEQ ID NO: 12 to SEQ ID NO: 22:

Sequence number 12:WTWKGKGTLNR,Sequence number 13:WTWKGRKSLLR,Sequence number 14:WTWKGEDKGKN,Sequence number 15:WTWKGRDKLQM,Sequence number 16:WTWKGQLGQLS,Sequence number 17:WTWKGGRLSAS,Sequence number 18:WTWKGRQLNNQ,Sequence number 19:WTWKGDNLQNN,Sequence number 20:WTWKGLYQRLG,Sequence number 21:WTWKGNKQVKF,andSequence number 22:WTWKGETYDSK

In peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor of the present disclosure, the acetylcholine receptor-binding peptide comprises any one sequence among amino acid sequences represented by the following SEQ ID NO: 23 to SEQ ID NO: 26:

Sequence number 23:WTWKGKGTLNR,Sequence number 24:WTWKGRKSLLR,Sequence number 25:WTWKGEDKGKN,andSequence number 26:WTWKGRDKLQM

In peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor of the present disclosure, the acetylcholine receptor-binding peptide comprises any one sequence among amino acid sequences represented by the following SEQ ID NO: 27 to SEQ ID NO: 31. The amino acid sequences represented by the following SEQ ID NO: 27 to SEQ ID NO: 31 are structures in which some of amino acid sequences represented by the SEQ ID NO: 23 to SEQ ID NO: 26 bear fruit for optimization:

Sequence number 27:TWKGKGTLNR,Sequence number 28:WKGKGTLNR,Sequence number 29:WTWKGKGTLN,Sequence number 30:WTWKGKGTL,andSequence number 31:KGTLNR

A method of screening an acetylcholine receptor-binding peptide according to another aspect of the present disclosure is provided. The method comprises the steps of:

(1) preparing a recombinant phage by inserting the peptide library into a vector after preparing a peptide library;

(2) mixing the recombinant phage with an acetylcholine receptor, and biopanning a mixture of the recombinant phage and the acetylcholine receptor to select a phage which is bound to the acetylcholine receptor;

(3) performing an enzyme-linked immunosorbent assay (ELISA) of the acetylcholine receptor and a control group with respect to the phage selected in the step (2); and

(4) analyzing performance results of the ELISA to select a phage having an acetylcholine receptor-binding signal intensity of 1.5 time or more compared to the control group.

In a method of selecting peptides which suppress secretion of acetylcholine by binding the peptides to an acetylcholine receptor of the present disclosure, the peptide library in the step (1) of preparing a recombinant phage by inserting the peptide library into a vector after preparing a peptide library may be prepared by using a DNA library comprised of any one base sequence among SEQ ID NO: 1 to SEQ ID NO: 11.

Furthermore, the present disclosure provides a polynucleotide encoding peptides according to the present disclosure. Further, as long as a polynucleotide comprising base sequences showing homology with the base sequence can encode peptides which are capable of showing a bonding activity with respect to the biostructure, the polynucleotide can be also included in a category of the polynucleotide provided in the present disclosure, wherein the polynucleotide may become a polynucleotide comprising base sequences showing preferably 80% or more of homology, more preferably 90% or more of homology, or most preferably 95% or more of homology.

Furthermore, a cosmetic composition for wrinkle amelioration comprising peptides according to the present disclosure as an active ingredient according to another aspect of the present disclosure is provided.

Furthermore, a pharmaceutical composition for wrinkle amelioration comprising the above-mentioned peptides as an active ingredient according to another aspect of the present disclosure is provided.

A pharmaceutical composition for wrinkle amelioration of the present disclosure may comprise peptides according to the present disclosure or pharmaceutically acceptable salts thereof alone, or may further comprise one or more pharmaceutically acceptable carriers, excipients or diluents thereof.

In the present disclosure, the term “pharmaceutically acceptable” means that the salts, carriers, excipients or diluents of the peptides are contained in the pharmaceutical composition in such sufficient amount extents that can exhibit a treatment effect, do not cause side effects, and may be easily determined by a person of ordinary skill in the art according to elements well-known to the medical field including types of diseases, age, weight, health and gender of a patient, sensitivity of the patient to drug, administration route, administration method, administration frequency, treatment period, mixing, a drug simultaneously used, etc.

For example, the pharmaceutically acceptable carriers may further comprise carriers for oral administration or carriers for non-oral administration.

The carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium, stearate, stearic acid, etc. Further, the carriers for non-oral administration may include water, suitable oil, a saline solution, water-based glucose, glycol, etc., and may additionally include a stabilizer and a preservative. A suitable stabilizer may include an antioxidant such as sodium bisulfate, sodium sulfite or ascorbic acid. A suitable preservative may include benzalkonium chloride, methyl- or propyl-paraben, or chlorobutanol. Carriers described in the following document may be referred to as other pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

A pharmaceutical composition for wrinkle amelioration of the present disclosure can be administered to mammals including human by any method. For example, a pharmaceutical composition for wrinkle amelioration of the present disclosure can be administered by oral administration or non-oral administration. Although the present disclosure is not limited thereto, the non-oral administration may be intravenous administration, intramuscular medication, intraarterial administration, intramedullary administration, intrathecal administration, intraperitoneal administration, dermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, intestinal administration, topical administration, sublingual administration, or intrarectal administration. Preferably, a pharmaceutical composition according to the present disclosure can be dermally administered. The ‘dermal administration’ in the above description indicates that an active ingredient contained in the composition according to the present disclosure is enabled to be transferred into the skin by administering a pharmaceutical composition according to the present disclosure into cells or skin. For example, a pharmaceutical composition according to the present disclosure is prepared into an injection type formulation such that a pharmaceutical composition according to the present disclosure may be administered by a method of lightly pricking the injection type formulation into the skin with a 30-gauge thin injection needle or a method of directly applying the injection type formulation to the skin.

A pharmaceutical composition according to the present disclosure may be formulated into a preparation for oral administration or a preparation for non-oral administration along the above-described administration routes.

In case of the preparation for oral administration, the composition according to the present disclosure can be formulated into powder, granule, tablet, pill, sugar-coated table, capsule, liquid, gel, syrup, slurry, suspension, etc. by methods known in the art. For example, the preparation for oral administration may include tablet or sugar-coated tablet which are obtained by mixing an active ingredient with a solid excipient, pulverizing a mixture of the active ingredient and the solid excipient, adding a suitable supplemental agent to a pulverized material, and processing a supplemental agent-added pulverized material into a granule mixture. Examples of a suitable excipient may comprise saccharides including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, etc., starches including corn starch, wheat starch, rice starch, potato starch, etc., celluloses including cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, etc., and fillers including gelatin, polyvinylpyrrolidone, etc. Further, in some cases, crosslinked polyvinylpyrrolidone, agar, alginic acid, sodium alginate, or the like may be added as a disintegrating agent.

The preparation for non-oral administration can be formulated in the form of injection, cream, lotion, ointment for external application, oil, moisturizer, gel, aerosol, and nasal inhaler by methods known in the art. All of these formulations are described in a document, i.e., a prescription generally known in the pharmaceutical field (Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pa. 18042, Chapter 87: Blaug, Seymour).

Total therapeutically effective amount of peptides according to the present disclosure may be administered to patients with single dose or multiple dose of fractionated treatment protocol. An active ingredient included in a pharmaceutical composition according to the present disclosure may vary according to the severity of a disease.

