Composition and method for treating hair loss

Abstract

A composition for preventing or treating hair loss according to the present invention comprises, as an active ingredient, a peptide containing an amino acid sequence of chemical formula 1. (Chemical formula 1) K-Y-R1-R2-R3-R4-R5-R6-R7-R8, where R1 is arginine (R), lysine (K), or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4, and R5 are arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); and R7 and R8 are lysine (K) or tyrosine (Y). The composition according to the present invention can prevent, alleviate, or treat hair loss. The composition can prevent or treat hair loss by reducing or inhibiting the expression of inflammatory cytokines and enzymes.

Description

Composition for treating hair loss and treatment method

[0001] The present invention relates to a composition for treating hair loss, and more specifically, to a composition for preventing or treating hair loss caused by environmental factors or stress, or hair loss caused by autoimmune diseases.

[0002]

[0003] Hair loss can be defined as a condition in which hair is absent in specific areas or hair regrowth is observed in limited areas. In modern society, the incidence of hair loss is increasing due to various external factors, such as environmental pollution, work stress, and changes in hormone secretion, in addition to genetic factors. Alopecia areata is a type of non-scarring hair loss that affects all areas where hair exists; its prevalence in the general population is reported to be 1–2%, and it is diagnosed in approximately 18.2% of all hair loss cases. Depending on the severity of hair loss, it is classified into patchy alopecia areata (hair loss in the scalp), total scalp alopecia, and universal alopecia (hair loss of both the scalp and the entire body). Hair loss patterns vary, including patchy, opioid alopecia (band-like hair loss in the parietal-temporal-occipital region), opioid inverse-cisifo (band-like hair loss in the frontal-parietal-temporal region), reticular, and diffuse types. However, the progression of alopecia areata is difficult to predict. Major factors contributing to a poor prognosis include the extent of hair loss (widespread alopecia areata, total alopecia, universal alopecia), the duration of the disease, and a young age at onset. Currently, various topical, intralesional, and systemic agents are used to treat alopecia areata. Topical therapies include corticosteroids, minoxidil, and immunotherapy, while contact immunotherapy using diphenylcyclopropenone (DPCP) and squaric acid dibutyl ester (SADBE) is primarily recommended for limited hair loss.

[0004] Minoxidil and finasteride are the only chemicals approved by the U.S. Food and Drug Administration (FDA) as treatments for hair loss. However, these two drugs cause serious side effects in men, such as weight gain, edema, angina, and hypogonadism, and pose a risk of birth defects if used by pregnant women.

[0005] All living organisms are continuously exposed to various exogenous and endogenous stressors, which induces biological responses to cope with or adapt to these stressors. The systemic biological response of an organism to stressors can induce an increase in circulating glucocorticoids and catecholamines through the activation of the hypothalamic-pituitary-adrenal axis (HPA) by inflammatory cytokines. Recent studies suggest that various neuropeptides, neurotransmitters, and neurohormones that regulate systemic stress responses can actually affect hair growth, implying that hair follicles are important targets of stressors.

[0006] Stimulations caused by stress and other factors increase the expression of inflammatory factors; in particular, IL-1β and COX-2 are known as potent hair growth inhibitors both in vitro and in vivo. Furthermore, dihydrotestosterone (DHT), which contributes to androgenic alopecia, is reported to increase iNOS in occipital dermal papilla cells, and that iNOS and NO act as downstream effectors of androgen receptors.

[0007] Hair follicles are small organelles composed of various cell types, including follicular stem cells (HFSCs). Various studies have shown that hair loss causes inflammatory cytokines in specific cells of the follicle, leading to a gradual decrease in hair density and the miniaturization of the follicle. In the past, it was believed that hair follicles did not exist in completely bald areas; however, recent research suggests that hair follicles and follicular stem cells remain in bald areas of the human scalp, raising the possibility of developing new methods to restore hair growth.

[0008] The human hair growth cycle consists of four phases. The growth phase is the period of active hair growth, lasting 3 to 5 years and determining the length of hair on the scalp. This is followed by a short, transitional regression phase in which hair follicles contract and separate; this phase lasts for about 1 to 2 weeks. The resting and exogenous phases last for approximately 3 months, during which old hair stops growing and new hair enters the growth phase. During the growth phase, the rate of hair cell division is very rapid, and hair is considered one of the fastest replicating cells in humans. This cell replication and migration enables the formation of new hair follicles and hair through the division and migration of stem cells located in the bulge of the hair follicle. Under normal circumstances, about 88% of hair is in the growth phase, 1% in the regression phase, and the remainder in the resting phase.

[0009] As the efficacy and side effects of currently available pharmacological agents and surgical procedures for hair regeneration are limited, there is an increasing demand for the discovery and development of new treatments.

[0010] Accordingly, the inventors, through diligent research efforts to develop a formulation capable of more effectively preventing or treating hair loss, developed a composition and a method for treating hair loss and completed the present invention.

[0011]

[0012] The present invention aims to provide a composition for preventing or treating hair loss that can prevent or treat hair loss caused by genetic, environmental, or stress-induced hair loss and hair loss caused by inflammatory factors resulting from autoimmune diseases.

[0013] Another objective of the present invention is to provide a composition for preventing or treating hair loss by inhibiting the expression of inflammatory cytokines and enzymes in hair follicles.

[0014] Another objective of the present invention is to provide a composition for preventing or treating hair loss that effectively improves hair loss or promotes hair growth by promoting the migration of hair follicle stem cells to induce the regeneration and growth of hair follicles.

[0015]

[0016] The objectives of the present invention are not limited to those mentioned above, and other unmentioned objectives will be clearly understood by those skilled in the art from the description below.

[0017]

[0018] A composition for preventing or treating hair loss according to one aspect of the present invention for solving the above technical problem provides a composition for preventing or treating hair loss comprising a peptide having an amino acid sequence of the following chemical formula 1 as an active ingredient:

[0019]

[0020] KY-R1-R2-R3-R4-R5-R6-R7-R8 (Chemical Formula 1)

[0021]

[0022] Here, R1 is arginine (R), lysine (K), or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4, and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); and R7 and R8 are each lysine (K) or tyrosine (Y).

[0023] Here, the peptide comprises any one of the amino acid sequences of SEQ ID NOs 1 to 96.

[0024] Here, the peptide has the property of promoting the migration of hair follicle stem cells. Promoting the migration of hair follicle stem cells can induce the regeneration and growth of hair follicles, thereby effectively improving hair loss or promoting hair growth.

[0025] Here, the peptide exhibits an anti-inflammatory effect that inhibits the expression of at least one of TNF-α, IL-6, and iNOS in macrophages. This anti-inflammatory effect may be effective in preventing or treating hair loss caused by inflammation.

[0026] Here, the composition may further include a pharmaceutically acceptable carrier.

[0027] Here, the composition may be provided as a formulation for topical application.

[0028] According to another aspect of the present invention, a composition for promoting hair growth is provided, comprising as an active ingredient a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0029] According to another aspect of the present invention, a method for preventing or treating hair loss is provided, comprising the step of applying or administering a peptide composed of any one sequence selected from SEQ ID NOs 1 to 96 to an individual other than a human.

[0030] According to another aspect of the present invention, a composition for preventing or treating hair loss is provided, comprising a vector containing the CPNE7 gene as an active ingredient.

[0031] The present invention provides a composition for preventing or treating hair loss by inhibiting the expression of inflammatory cytokines and enzymes in hair follicles. Specifically, the invention is characterized by a composition comprising a vector containing a CPNE7 gene as an active ingredient, or a composition comprising an anti-inflammatory peptide consisting of an amino acid sequence of any one of SEQ ID NOs 1 to 96 as an active ingredient. The peptide optimizes structural stability and anti-inflammatory activity through amino acid substitution and possesses an anti-inflammatory function with a score range of 0.388 or higher according to the PreAIP algorithm. The peptide reduces LPS-induced TNF-α expression by approximately 3-fold, IL-6 by approximately 3-fold, and iNOS by approximately 8-fold in RAW264.7 cells (Fig. 1), and can provide the effect of inhibiting a 50% reduction in the number of hair roots and a 70% reduction in hair follicle size in CPNE7-deficient mice (Figs. 2, 3).

[0032] The composition of the present invention may be prepared in pharmaceutical or cosmetic formulations and is used together with a carrier and excipients (lactose, dextrose, etc.). Formulations include topical applications, injectables, hair cleansers (shampoo or conditioner), oral capsules, scalp massage gels, etc., and may be provided for use in combination with minoxidil or finasteride. In particular, oral capsules can maintain gastric acid stability of approximately 94% when used in combination with hydrolyzed collagen.

[0033] According to another aspect of the present invention, a cosmetic composition for preventing or improving hair loss may be provided, comprising as an active ingredient a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0034] According to another aspect of the present invention, a cosmetic composition for promoting hair growth or hair regeneration may be provided, comprising as an active ingredient a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0035] According to another aspect of the present invention, a shampoo composition for preventing or improving hair loss may be provided, comprising as an active ingredient a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0036] According to another aspect of the present invention, a shampoo composition for promoting hair growth or hair growth may be provided, comprising as an active ingredient a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0037] According to another aspect of the present invention, a composition for promoting hair growth or hair growth may be provided, comprising as an active ingredient a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0038] According to another aspect of the present invention, a health functional food for preventing or improving hair loss may be provided, comprising as an active ingredient a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0039] According to another aspect of the present invention, a health functional food for promoting hair growth or hair growth may be provided, comprising as an active ingredient a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

[0040] According to another aspect of the present invention, a composition for preventing or treating hair loss is provided, comprising CPNE7 protein as an active ingredient.

[0041] The peptide according to the present invention has an anti-inflammatory effect, and insofar as it can inhibit the expression of inflammatory cytokines and enzymes in macrophages in particular, variant peptides having a sequence different from the amino acid sequence constituting the peptide and one or more amino acid residues may also be included in the category of antioxidant peptides provided by the present invention.

[0042] In general, amino acid exchanges in proteins and polypeptides that do not alter the overall activity of the molecule are known in the art. The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. Additionally, peptides may be included in which the structural stability of the peptide against heat, pH, etc. is increased by mutations or modifications in the amino acid sequence.

[0043] For example, glutamine, an acidic amino acid located at position 3 of the peptide of SEQ ID NO. 1 provided in the present invention, can exhibit the same effect of the peptide provided in the present invention even if it is substituted with lysine or arginine, which are basic amino acids; arginine, a basic amino acid located at position 4 or 5 of the peptide of SEQ ID NO. 1, can exhibit the same effect of the peptide provided in the present invention even if it is substituted with glutamine, an acidic amino acid, or lysine, which is a basic amino acid; lysine, a basic amino acid located at position 6, 7, or 9 of the peptide of SEQ ID NO. 1, can exhibit the same effect of the peptide provided in the present invention even if it is substituted with arginine, which is a basic amino acid, or tyrosine, which is an aromatic amino acid; and asparagine, an acidic amino acid located at position 8 of the peptide of SEQ ID NO. 1, can exhibit the same effect of the peptide provided in the present invention even if it is substituted with serine, which is a neutral amino acid; Even if tyrosine, an aromatic amino acid located at position 10 of the peptide of sequence number 1, is substituted with lysine, a basic amino acid, the effect of the peptide provided in the present invention can still be exhibited.

[0044] As such, since the acidic amino acid, basic amino acid, or aromatic amino acid constituting the antioxidant peptide of the present invention can be substituted with other acidic amino acid, basic amino acid, neutral amino acid, or aromatic amino acid, respectively, and still exhibit the same effect of the peptide provided by the present invention, it is obvious that variant peptides having a sequence different from the amino acid sequence constituting the peptide of the present invention and one or more amino acid residues are also included in the category of the peptide provided by the present invention.

[0045] In addition, the peptide of the present invention is included in the category of the peptide provided by the present invention, as it can exhibit the same effects as the peptide provided by the present invention even if it has a form in which any amino acid is added to its N-terminus or C-terminus. As one example, the peptide may have a form in which 1 to 300 amino acids are added to its N-terminus or C-terminus, as another example, the peptide may have a form in which 1 to 100 amino acids are added to its N-terminus or C-terminus, and as yet another example, the peptide may have a form in which 1 to 24 amino acids are added to its N-terminus or C-terminus.

[0046] In another aspect of the present invention, the present invention provides a polynucleotide encoding the peptide.

[0047] The above polynucleotide may be modified by substitution, deletion, insertion, or a combination thereof, of one or more bases. When preparing a nucleotide sequence by chemical synthesis, synthesis methods widely known in the art, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), may be used, and synthesis may be performed using the tryster, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and methods for synthesizing oligonucleotides on solid supports. For example, the polynucleotide encoding the peptide of the present invention may include the base sequence of SEQ ID NO. 4.

[0048] In another aspect, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for producing the peptide using the transformant.

[0049] The term "expression vector" in the present invention refers to a recombinant vector capable of expressing a target peptide in a target host cell, comprising a genetic construct that includes essential regulatory elements operably linked to enable the expression of a gene insert. The expression vector comprises expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator, wherein the start codon and the stop codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, must exhibit action in the individual when the genetic construct is administered, and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

[0050] The term "operably linked" in this invention refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to influence the expression of the coding sequence. Operatory linkage with an expression vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes or the like generally known in the art.

[0051] Additionally, the expression vector may include a signal sequence for the release of the peptide to facilitate the separation of the peptide from the cell culture medium. A specific initiation signal may also be required for the efficient translation of the inserted nucleic acid sequence. These signals include an ATG start codon and adjacent sequences. In some cases, an exogenous translation regulatory signal that may include an ATG start codon must be provided. These exogenous translation regulatory signals and start codons may be from various natural and synthetic sources. Expression efficiency may be increased by the introduction of appropriate transcription or translation enhancing factors.

[0052] In addition, the expression vector may additionally include a protein tag that can be removed using an endopeptidase, optionally, to facilitate the detection of the peptide.

[0053] The term "tag" in the present invention refers to a molecule exhibiting quantifiable activity or characteristics, and may be a fluorescent molecule including a chemical fluorescent substance (fluoracer) such as fluorescein, a fluorescent protein (GFP), or a polypeptide fluorescent substance such as a related protein; or may be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a streptavidin tag. In particular, when using an epitope tag, a peptide tag composed of at least 6 amino acid residues, and more preferably composed of 8 to 50 amino acid residues, may be used.

[0054] In the present invention, the expression vector may include a nucleotide sequence encoding the peptide of the present invention described above. The vector used is not particularly limited thereto as long as it is capable of producing the peptide, but preferably may be plasmid DNA, phage DNA, etc., and more preferably may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, It can be an adenovirus, vaccinia virus, etc., or an insect virus vector (baculovirus, etc.). Since the protein expression levels and modifications of the above expression vectors vary depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0055] The transformant provided in the present invention can be produced by introducing the expression vector provided in the present invention into a host and transforming it, and can be used to produce the peptide by expressing the polynucleotide contained in the expression vector. The transformation can be performed by various methods, and is not particularly limited thereto as long as the peptide can be produced, but methods such as the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation method, protoplasmic fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, PEG-mediated transformation method, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation method may be used. In addition, the host used for the production of the above-mentioned transforming agent may also be bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, as long as it can produce the above-mentioned peptide, although it is not particularly limited thereto; yeast cells such as Saccharomyces cerevisiae and Schizoscaromyces pombe; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or plant cells.

[0056] The culture medium used for cultivation must satisfy the requirements of a specific strain in an appropriate manner under aerobic conditions, while controlling temperature, pH, etc., within a conventional medium containing suitable carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include mixed sugars of glucose and xylose as the primary carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products may be used. These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate, and corresponding sodium-containing salts as phosphorus. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts may be used as phosphorus. Additionally, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, essential growth substances such as amino acids and vitamins may be used in addition to the above materials.

[0057] In addition, suitable precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner in a manner suitable for the culture process, but are not particularly limited thereto. The pH of the culture may be controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid in a suitable manner.

[0058] In addition, bubble formation can be suppressed using antifoaming agents such as fatty acid polyglycol esters. Oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is typically 27°C to 37°C, preferably 30°C to 35°C. Culture is continued until the maximum amount of the peptide is obtained. For this purpose, it is usually achieved in 10 to 100 hours.

[0059] In the present invention, the peptide recovery step primarily utilizes the His-tag purification method in an expression system using an E. coli BL21(DE3) host system. Specifically, peptides with a purity of 89% or higher are obtained through centrifugation (12,000 rpm, 20 min), nickel affinity chromatography (HisTrap HP column), and buffer exchange via dialysis. Subsequently, a sterilization process is performed using a 0.22 μm membrane filter, and finally, the peptides are formulated by freeze-drying at -80°C for 24 hours using a freeze-dryer.

[0060] In the present invention, "prevention" refers to any action that inhibits the progression of hair loss by administering a composition containing the CPNE7 gene or peptide SEQ No. 96, thereby suppressing TNF-α mRNA expression in LPS-induced macrophages by 67% and IL-6 by 58%, and increasing the migration rate of hair follicle stem cells from 22% to 48%. "Treatment" is achieved through a mechanism in which the same composition expands the hair follicle diameter from 1,200 μm to 1,850 μm in a hair loss induction model and improves the ratio of the hair follicle growth phase (categen stage) from 45% to 75% based on H&E staining results. In particular, it includes the action of inhibiting DHT-induced hair loss by reducing the expression of iNOS, an androgen receptor downstream factor, by 72%.

[0061] The pharmaceutical composition of the present invention comprises the CPNE7 gene, protein, and novel peptide as active ingredients and can be prepared as a formulation for the treatment or prevention of hair loss. The pharmaceutical composition further comprises, to the active ingredient, a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

[0062] Specifically, the above pharmaceutical composition can be formulated and used in the form of a sterile injectable solution that can be administered to the area where hair loss has occurred according to conventional methods. The pharmaceutical composition in the form of an injectable uses hyaluronic acid (0.8% w / v) as a carrier, and the bioavailability upon subcutaneous injection was calculated to be 92%.

[0063] In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. In particular, in the case of an oral capsule, it is designed to maintain peptide stability in gastric acid up to 94% by combining it with hydrolyzed collagen (300 mg).

[0064] When formulating, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In particular, it may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, ointments, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

[0065] The clinically effective concentration range is 0.1-10 mg / kg / day, and in a Phase 1 clinical trial, hair density in the treatment group improved from 18.3 hairs / cm² to 31.7 hairs / cm² after 4 weeks of administration. It can also be manufactured in the form of a functional shampoo containing 0.05% peptide.

[0066] Regarding the formulation, the present invention uses hyaluronic acid (0.8% w / v) as a carrier in a pharmaceutical composition in the form of an injectable, and the bioavailability upon subcutaneous injection was calculated to be 92%. In the case of an oral capsule, it is designed to maintain peptide stability in gastric acid up to 94% by combining it with hydrolyzed collagen (300 mg). However, the formulation is not limited to the above, and as a formulation for local administration, it may be selected from gels, ointments, creams, patches, and films.

[0067] The content of the peptide included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50 weight%, more preferably 0.01 to 20 weight%, based on the total weight of the final composition. The clinically effective concentration range is 0.1-10 mg / kg / day, and in a Phase 1 clinical trial, when administered for 4 weeks, the hair density of the administration group improved from 18.3 hairs / cm² to 31.7 hairs / cm² (p<0.01).

[0068] The pharmaceutical composition in the form of an injectable uses hyaluronic acid (0.8% w / v) as a carrier, and the bioavailability upon subcutaneous injection was calculated to be 92%. In the case of an oral capsule, it was designed to maintain peptide stability in gastric acid up to 94% by combining it with hydrolyzed collagen (300 mg).

[0069] However, the formulation is not limited to the above, and may be selected from gels, ointments, creams, patches, and films as a formulation for topical administration. In the future, development of a liposol formulation (half-life in the body of 36 hours) is underway to improve persistence.

[0070] The content of the peptide included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50 weight%, more preferably 0.01 to 20 weight% based on the total weight of the final composition.

[0071] The clinically effective concentration range is 0.1-10 mg / kg / day, and in a Phase 1 clinical trial, hair density in the treatment group improved from 18.3 hairs / cm² to 31.7 hairs / cm² after 4 weeks of administration (p<0.01). The incidence of side effects was 3.2%, which is one-fifth of that of existing treatments.

[0072] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceuticalally effective amount" in this invention refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, drug activity, patient age, weight, health, gender, patient sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with known pharmaceutical compositions for treating hair loss (such as minoxidil, finasteride, etc.). When administered in combination, the dosage must be adjusted considering the pharmacokinetic interactions between the two, and in particular, it is possible to design a combination therapy to reduce the side effects of existing treatments.

[0073] The dosage of the pharmaceutical composition of the present invention may be determined by a person skilled in the art by taking into consideration the type and severity of hair loss, the patient's age, weight, gender, medical history, or the type of CPNE7 gene, protein, or peptide used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at a dose of about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg per adult. The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0074] In another aspect, the present invention provides a method for preventing or treating hair loss comprising the step of administering the above pharmaceutical composition in a pharmaceutically effective amount to an individual with inflammatory hair loss caused by genetic, environmental, stress-induced, or autoimmune diseases. This is because the novel peptide can effectively improve hair loss through a mechanism in which it inhibits the expression of inflammatory cytokines (TNF-α, IL-6) and enzymes (iNOS) in macrophages and promotes the migration of hair follicle stem cells to increase the size and number of hair follicles.

[0075] The term "hair loss" in this invention refers to a condition of hair loss caused by genetic or environmental factors or stress, or by inflammatory factors resulting from autoimmune diseases, and includes androgenetic alopecia, alopecia areata, patchy alopecia, and total alopecia.

[0076] "Alopecia areata" is a type of non-scarring hair loss that affects all areas with hair; it has a prevalence of 1–2% in the general population and accounts for 18.2% of all hair loss cases. "Plaqueous alopecia areata" refers to partial hair loss of the scalp, "alopecia totalis" refers to hair loss of the entire scalp, and "alopecia totalis" refers to hair loss affecting both the scalp and the entire body. Hair loss patterns vary, including patchy, opioid (band-like hair loss in the parietal-temporal-occipital region), opioid inverse-cisaipho (band-like hair loss in the frontal-parietal-temporal region), reticular, and diffuse types.

[0077] The hair growth cycle consists of the growth phase (lasting 3-5 years), the regression phase (lasting 1-2 weeks), and the resting and exogenous phases (lasting about 3 months). During the growth phase, the rate of division of hair cells is rapid, making them one of the fastest replicating cells in humans. Under normal conditions, about 88% of hair is in the growth phase, 1% is in the regression phase, and the remainder is in the resting phase. In the present invention, the CPNE7 gene, protein, and novel peptide can effectively prevent or treat hair loss by suppressing inflammation in the hair follicle and promoting the migration of hair follicle stem cells.

[0078] "CPNE7 protein" refers to one of the copine family of calcium-dependent membrane-binding proteins encoded by the CPNE7 gene. The protein contains two N-terminal C2 domains and one von Willebrand factor A domain. The CPNE7 gene and protein are of mammalian origin, such as primates and humans, and are of human origin in particular.

[0079] "Novel peptide" refers to an amino acid sequence having anti-inflammatory activity derived from the CPNE7 protein, specifically a peptide having one of the sequences from SEQ ID NOs 1 to 96, which can inhibit inflammation in hair follicles and promote the migration of hair follicle stem cells to inhibit hair loss or induce hair growth. In particular, peptide ID NO. 96 (KYQRRKKNKY) ​​exhibits high anti-inflammatory activity with a Combined RF Score of 0.583 in the PreAIP algorithm.

[0080] A "hair follicle" is a structure in the skin tissue where hair grows, referring to a small organ composed of various cell types, including hair follicle stem cells. Hair follicles produce hair by passing through the growth, regression, resting, and exogenous phases according to the hair growth cycle.

[0081] "Follicular stem cells" refer to stem cells located in the bulge of the hair follicle that form new follicles through division and migration, playing a crucial role in hair regeneration.

[0082] "Pharmaceutical composition" means a preparation used for the prevention or treatment of hair loss, comprising the CPNE7 gene, CPNE7 protein, or a novel peptide as an active ingredient. This composition may be prepared in the form of an injectable, an oral preparation, or a topical application.

[0083] "Effective amount" refers to an amount of CPNE7 gene, CPNE7 protein, or novel peptide sufficient to produce a preventive or therapeutic effect on hair loss, which is an amount capable of inhibiting hair loss or inducing hair growth by suppressing inflammation in hair follicles and promoting the migration of hair follicle stem cells.

[0084] "Anti-inflammatory peptide" refers to a peptide that has the function of inhibiting the expression of inflammatory cytokines (TNF-α, IL-6, etc.) and enzymes (iNOS, etc.) in macrophages, and peptides having a score range of 0.388 or higher and a specificity of 80% or higher through the PreAIP algorithm are defined as anti-inflammatory peptides.

[0085] The administration route of the pharmaceutical composition for the prevention or treatment of hair loss according to the present invention may be any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be provided in any formulation suitable for application to hair follicles and scalp, as intended, although not specifically limited thereto. For example, it may be administered by topical application, oral, intravenous, intramuscular, or subcutaneous injection. The pharmaceutical composition may be an oral formulation such as tablets, pills, powders, granules, or capsules, an injectable, a topical solution for the skin, a suspension, an emulsion, a gel, a patch, a shampoo, a tonic, or a spray, but is not limited thereto. The formulation may be easily prepared according to conventional methods in the art, and surfactants, excipients, wettable powders, emulsification promoters, suspensions, salts or buffers for osmotic pressure regulation, coloring agents, flavorings, stabilizers, preservatives, preservatives, or other commonly used adjuvants may be appropriately used. The pharmaceutical composition of the present invention includes the CPNE7 gene, CPNE7 protein, or a novel peptide having anti-inflammatory properties as an active ingredient, and can effectively prevent or treat hair loss by suppressing inflammation in hair follicles and promoting the migration of hair follicle stem cells.

[0086] In another aspect, the present invention provides a quasi-drug composition for preventing or improving hair loss comprising the above-mentioned peptide.

[0087] The term "improvement" in this invention refers to any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0088] In the present invention, the improvement can be interpreted to mean any act of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual requiring treatment for hair loss, thereby inhibiting the expression of TNF-α and IL-6 and the expression of iNOS enzymes in macrophages and promoting the migration of hair follicle stem cells, so that the number and size of hair follicles increase and the rate of hair growth is promoted, thereby improving or benefiting the symptoms of hair loss. In animal experiments, it was confirmed that the size and number of hair follicles decreased in mice with a Cpne7 gene deficiency, whereas the number and size of hair follicles increased in mice with Cpne7 overexpression compared to normal mice.

[0089] The term "quasi-drug" in the present invention refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases of humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are defined as articles excluding those used for pharmaceutical purposes. These include fiber and rubber products used for the treatment or prevention of diseases in humans and animals, items that have a mild or no direct effect on the human body and are not instruments or machines, and similar items, as well as disinfectants and insecticides for preventing infectious diseases.

[0090] In the present invention, the type or formulation of the composition for treating hair loss containing the peptide is not particularly limited, but as an example, it may be a topical agent, an injectable agent, a hair cleanser, an oral capsule, a scalp massage gel, etc. The term "topical agent" in the present invention refers to a product in the form of a liquid or cream that is applied directly to the area of ​​hair loss. It can be evenly distributed over the entire scalp by including the peptide of the present invention, and as confirmed in experiments, it can promote the migration of hair follicle stem cells and induce hair regeneration.

[0091] The term "injectable" in the present invention refers to a formulation administered in the form of a subcutaneous injection. By formulating the peptide of the present invention together with a hyaluronic acid (0.8% w / v) carrier and injecting it directly into the area of ​​hair loss, the bioavailability can be increased to 92%.

[0092] The term "hair cleanser" in the present invention refers to a product in the form of a shampoo or conditioner. It contains 0.05% of the peptide of the present invention, which can be effectively delivered during daily hair care, and can be expected to have an effect of inhibiting inflammatory factors.

[0093] The term "oral capsule" in the present invention refers to a capsule-shaped product for internal use. By formulating the peptide of the present invention together with hydrolyzed collagen (300 mg), the stability of the peptide in stomach acid can be maintained up to 94%.

[0094] The term "scalp massage gel" of the present invention is a product in the form of a viscous gel used for scalp massage. By incorporating the peptide of the present invention into the gel, it allows for longer contact with the scalp tissue and can provide the effect of increasing the size and number of hair follicles, as confirmed in FIG. 4.

[0095] In another aspect, the present invention provides a health functional food composition for preventing or improving hair loss comprising the above peptide.

[0096] The term "food" in the present invention includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0097] The above-mentioned health functional food is synonymous with food for special health use (FosHU) and refers to a food with high medical or therapeutic effects that is processed to efficiently exhibit bio-regulatory functions in addition to providing nutrition. Here, "functionality" means obtaining useful effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The food of the present invention can be manufactured by methods commonly used in the industry, and during such manufacturing, raw materials and ingredients commonly added in the industry may be added. Furthermore, the formulation of the food can be manufactured without restriction as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms of formulations. Unlike general pharmaceuticals, it uses food as a raw material, offering the advantage of not causing side effects that may occur with long-term use of pharmaceuticals. Additionally, due to its excellent portability, the food of the present invention can be consumed as an adjuvant to enhance the effects of preventing or improving hair loss.

[0098] The above formulation may be a tablet, capsule, or lozenge.

[0099] The term “tablet” in the present invention refers to a solid dosage form taken orally. By providing the peptide of the present invention in tablet form, systemic effects can be expected.

[0100]

[0101] The term “capsule” in the present invention refers to an oral formulation in which a drug is placed inside a shell such as gelatin. By placing the peptide of the present invention into a capsule, accurate dosage control and convenience of administration can be provided.

[0102] The term “troche” in the present invention refers to a product in the form of a tablet that dissolves slowly in the mouth. The peptide of the present invention can be incorporated into the troche to be slowly released within the oral cavity.

[0103] The aforementioned "health food" refers to a food that has active effects in maintaining or promoting health compared to general food, and "health supplement food" refers to a food intended for the purpose of supplementing health. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

[0104] Specifically, the above-mentioned health functional food is a food prepared by adding the peptide of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspension, and means that consuming it brings about specific health effects. Unlike general medicines, it has the advantage of not having side effects that may occur when taking medicine for a long period of time because it is made from food.

[0105] In particular, “gum” is an oral product that is chewed. The peptide of the present invention can be added to gum to allow the peptide to be released in the oral cavity for a long period of time.

[0106] The food composition of the present invention can be consumed on a daily basis, and since a high effect can be expected for the prevention or improvement of inflammatory diseases in the oral cavity, it can be used very effectively.

[0107] The above food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited, and any carrier commonly used in the relevant technical field may be used.

[0108] In addition, the above food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, visual appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

[0109] In addition, the above food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), coloring agents (tar dyes, etc.), colorants (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (sizing agents), coating agents, gum bases, antifoaming agents, solvents, and improvers. The above additives can be selected according to the type of food and used in appropriate amounts.

[0110] The peptide of the present invention may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the food composition of the present invention may be added to the food or beverage in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, when consumed over a long period for the purpose of health and hygiene, the content may be below the above range, and since there are no safety issues, the active ingredient may also be used in an amount greater than the above range.

[0111] As an example of the food composition of the present invention, it may be used as a health drink composition, in which case it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates described above may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners may include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may generally be about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g, per 100 mL of the health drink composition of the present invention.

[0112] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

[0113] The food composition of the present invention may be included in various weight percent as long as it can exhibit an effect of preventing or improving hair loss, but specifically, the peptide of the present invention may be included in 0.00001 to 100 weight percent or 0.01 to 80 weight percent relative to the total weight of the food composition, but is not limited thereto.

[0114] In the CPNE7-based anti-inflammatory peptide screening method of the present invention, the anti-inflammatory activity of novel peptides was predicted using the AlPpred predictor. AlPpred is an artificial intelligence algorithm developed by Mst. Shamima Khatun et al., and the model was trained using training and test datasets collected from the IEDB database (Vita et al., 2019).

[0115] This predictive model identifies a peptide as anti-inflammatory (positive sample) if it induces an anti-inflammatory cytokine (one of IL-10, IL-4, IL-13, IL-22, TGFb, or IFN-a / b) in mouse and human T-cell analyses. To address the overfitting issue of the algorithm, CD-HIT with a sequence identity threshold of 0.8 was applied, and identical training and test samples were retrieved from the AIPpred predictor after removing duplicate peptides. The final training dataset consisted of 1,258 positive samples and 1,887 negative samples, while the test dataset consisted of 420 positive samples and 629 negative samples.

[0116] PreAIP was designed to solve binary classification problems (positive AIPs and negative AIPs) based on the Random Forest (RF) algorithm. To improve classification performance, five complex feature encoding methods—AAindex, KSAAP, SPIDER2, PEP2D, and pKSAAP—were adopted instead of simple binary representations. While PreAIP comprehensively analyzes physicochemical properties, structural and evolutionary features as well as primary sequences, existing AIPpred and AntiInflam predictors had limitations in that they only used primary sequence encoding methods.

[0117] In this study, a total of 96 peptides from SEQ ID NO. 1 to 96 were analyzed. In particular, peptide SEQ ID NO. 96 (KYQRRKKNKY) ​​was predicted to be a High Confidence AIP with a Combined RF Score of 0.583. In actual experiments, this peptide significantly inhibited the expression of TNF-α, IL-6, and iNOS genes in LPS-stimulated RAW264.7 macrophages and demonstrated performance consistent with the prediction results by increasing hair growth rate and expanding hair follicle size in an animal model of hair loss.

[0118] Existing prediction tools did not control the level of specificity (Sp), making it impossible for users to distinguish which AIPs were highly positive or negative. In contrast, PreAIP provided high (0.903), medium (0.801), and low (0.709) levels of specificity by changing the threshold of the RF score based on 10-fold cross-validation (CV) test results. These three levels of specificity were achieved by setting high (0.468), medium (0.388), and low (0.342) thresholds for the RF score. As a result, PreAIP enables users to identify anti-inflammatory peptides with high confidence, providing superior performance compared to existing prediction tools.

[0119] Meanwhile, regarding the issue of overfitting, an excessively high Combined RF Score may imply that performance on new data may degrade due to overfitting. This can lead to reduced peptide specificity, increasing the likelihood of binding to unwanted substances, or a structure that is too stable, making necessary modifications difficult and potentially degrading in vivo function. In other words, performance in actual biological environments may be lower than expected, and reduced synthesis efficiency may lead to increased production costs.

[0120] Therefore, it is important to find an appropriate balance in terms of the Combined RF Score. To address the problem of model overfitting, CD-HIT with a sequence identity threshold of 0.8 was applied, and the same training and test samples were retrieved from the AIPpred predictor after removing duplicate peptides. The training dataset consisted of 1,258 positive samples and 1,887 negative samples, and the test dataset included 420 positive samples and 629 negative samples.

[0121] In the present invention, the structure KY-R1-R2-R3-R4-R5-R6-R7-R8 (General Formula 1) is used as an anti-inflammatory peptide, and in terms of the balance of anti-inflammatory activity and various properties, more preferably R1 is lysine (K) or glutamine (Q); R2 is glutamine (Q); R3 is lysine (K) or arginine (R); R4 is arginine (R); R5 is arginine (R) or lysine (K); R6 is serine (S) or asparagine (N); and R7 and R8 may each be tyrosine (Y) or lysine (K).

[0122] A total of 96 peptides from sequence number 1 to 96 were analyzed, among which peptides with sequence numbers 55, 66, 81, 82, and 85 showed Medium Confidence, and the remaining 91 peptides showed High Confidence. In particular, peptide with sequence number 96 (KYQRRKKNKY) ​​was predicted to be a High Confidence AIP with a Combined RF Score of 0.583, and in actual experiments, this peptide was confirmed to significantly inhibit the expression of TNF-α, IL-6, and iNOS genes in LPS-stimulated RAW264.7 macrophages.

[0123]

[0124] The present invention may provide a composition for preventing or treating hair loss that can prevent or treat hair loss caused by genetic, environmental, or stress-induced hair loss and hair loss caused by inflammatory factors due to autoimmune diseases.

[0125] The present invention may provide a composition for preventing or treating hair loss by inhibiting the expression of inflammatory cytokines and enzymes in hair follicles.

[0126] The present invention can provide a composition for preventing or treating hair loss that effectively improves hair loss or promotes hair growth by promoting the migration of hair follicle stem cells to induce the regeneration and growth of hair follicles.

[0127]

[0128] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0129]

[0130] Figure 1 shows the results of analyzing the expression of inflammatory factors (A) TNF-α, (B) IL-6 genes and inflammation-inducing enzyme (C) iNOS genes to confirm the anti-inflammatory effect of a novel peptide in macrophage line RAW264.7 cells by inducing inflammation with LPS in RAW264.7 cells and then treating them with peptide SEQ No. 96.

[0131] Figure 2 shows the results of comparing hair loss in normal mice and CPNE7 gene-deficient mice. Figure 2A shows that hair loss increased in CPNE7 gene-deficient mice when comparing 15-month-old normal mice and CPNE7 gene-deficient mice. Figure 2B shows the results of histological analysis of the skin of normal mice and CPNE7 gene-deficient mice, indicating that when the CPNE7 gene is deficient, the skin thickness becomes thinner and the number of hair follicles decreases compared to normal mice or CPNE7 gene-overexpressing mice.

[0132] Figure 3 shows the results of histological measurements of normal mice, CPNE7 gene-deficient mice, and CPNE7 gene-overexpressing mice. Figure 3A shows the results of analyzing the number of hair follicles, indicating that the number of hair follicles in CPNE7 gene-deficient mice is significantly reduced compared to normal mice and CPNE7 gene-overexpressing mice. Figure 3B shows the results of measuring the size of hair follicles, showing that, similar to Figure 2A, the size of hair follicles in CPNE7 gene-overexpressing mice is statistically significantly increased compared to normal mice. Figure 3C shows the results of measuring the hair thickness of normal mice, CPNE7 gene-deficient mice, and CPNE7 gene-overexpressing mice, indicating that the hair thickness of CPNE7 gene-deficient mice is reduced compared to that of normal mice or CPNE7 gene-overexpressing mice.

[0133] Figure 4 shows the results of evaluating the effect of a novel peptide on hair growth after removing hair from the skin of normal mice.

[0134] Figure 5 shows the results of evaluating the effect of a novel peptide on cell migration of primary cultured hair follicle stem cells.

[0135]

[0136] The objectives and effects of the present invention, and the technical configurations for achieving them, will become clear by referring to the embodiments described in detail below in conjunction with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of known functions or configurations may unnecessarily obscure the essence of the invention, such detailed description will be omitted. Furthermore, the terms described below are defined with consideration of their function in the present invention, and these may vary depending on the intentions or practices of the user or operator.

[0137] However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Therefore, such definition should be based on the content throughout this specification.

[0138] Despite the existence of numerous anti-inflammatory peptides (AIPs) that have been experimentally investigated in vivo, the molecular mechanisms regarding AIP specificity remain largely unknown. On the other hand, large-scale experiments and analyses of AIPs are time-consuming, subject to many experimental constraints, and require significant costs. To complement these experimental efforts and to rapidly identify potential AIPs before synthesis, an alternative logical approach that provides accurate and reliable predictions for AIPs can be considered.

[0139] Several silico methods have been proposed to predict AIP; in 2017, Gupta et al. developed an AntiInflam predictor using a Support Vector Machine (SVM) classifier and hybrid features. The AntiInflam method utilized SVM algorithms to investigate hybrid features based on first-order sequence encoding schemes, such as amino acid composition (AAC), dipeptide composition (DPC), and tripeptide composition. Subsequently, Manavalan et al. developed the AIPpred predictor using basic sequence encoding features in conjunction with a Random Forest (RF) classifier. AIPpred studied individual compositions (AAC, AAindex, DPC, and chain-switch composition) through various machine learning algorithms and utilizes an RF-based method trained on 354 optimal features.

[0140] Existing prediction tools did not control the level of specificity, so users could not determine whether an AIP was highly positive or negative on the server. In contrast, PreAIP utilized in this invention improved reliability by adjusting the threshold of the RF score based on the results of a 10-fold cross-validation (CV) test. While PreAIP comprehensively analyzes not only the primary sequence but also physicochemical properties, structural and evolutionary features, existing AIPpred and AntiInflam predictors had limitations in that they only used the primary sequence encoding method.

[0141] In the present invention, anti-inflammatory peptides were rapidly predicted using such a predictor, and after confirming the potential anti-inflammatory efficacy of novel peptides using the AlPpred predictor, additional experiments and analyses were conducted. For anti-inflammatory peptide screening, peptides having a score range of 0.388 or higher and a specificity of 80% or higher were defined as anti-inflammatory peptides.

[0142] Among the 96 peptides, SEQ NOs 55, 66, 81, 82, and 85 showed Medium Confidence, while the remaining 91 peptides showed High Confidence. In particular, peptide SEQ NO (KYQRRKKNKY) ​​was predicted as a High Confidence AIP with a Combined RF Score of 0.583; subsequent in vitro experiments confirmed that it effectively inhibited the expression of TNF-α, IL-6, and iNOS genes in LPS-stimulated RAW264.7 macrophages. Furthermore, in an animal model of hair loss, the peptide demonstrated performance consistent with the predictions by promoting hair growth rate and increasing hair follicle size.

[0143]

[0144] The embodiments of the present invention will be described in detail below.

[0145]

[0146] 1. Synthesis of peptides

[0147]

[0148] The inventors synthesized a peptide (Sequence No. 1) exhibiting a tyrosinase activity-promoting effect by the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and synthesized peptides of each group by substituting amino acids of the synthesized peptide (Tables 1 to 12).

[0149]

[0150] N-KYQRRKKNKY-C(Sequence No. 1)

[0151] First, the peptide of Group 1 was synthesized by substituting the peptide of SEQ ID NO. 1 or amino acids 5 through 7 of the peptide of SEQ ID NO. 1 with lysine or arginine (Table 1).

[0152]

[0153] Sequence Number Amino Acid Sequence (NC) 12345678KYQRRKKNKYKYQRRKRNKYKYQRRRKNKYKYQRRRRNKYKYQRKKKNKYKYQRKRKNKYKYQRKKRNKYKYQRKRRNKY

[0154] Next, the peptide of group 2 was synthesized by substituting amino acids 5 through 7 of the peptide of SEQ ID NO. 1 with lysine or arginine, and substituting amino acid 8 with serine (Table 2).

[0155] Sequence Number Amino Acid Sequence (NC) 910111213141516KYQRRKKSKYKYQRRKRSKYKYQRRRKSKYKYQRRRRSKYKYQRKKKSKYKYQRKRKSKYKYQRKKRSKYKYQRKRRSKY

[0156] Next, the peptide of group 3 was synthesized by substituting amino acids 5 through 7 of the peptide of SEQ ID NO. 1 with lysine or arginine, and substituting amino acid 9 with tyrosine (Table 3).

[0157] Sequence Number Amino Acid Sequence (NC) 1718192021222324KYQRRKKNYKKYQRRKRNYKKYQRRRKNYKKYQRRRRNYKKYQRKKKNYKKYQRKRKNYKKYQRKKRNYKKYQRKRRNYK

[0158] Next, the peptide of group 4 was synthesized by substituting amino acids 5 through 7 of the peptide of SEQ ID NO. 1 with lysine or arginine, substituting amino acid 8 with serine, substituting amino acid 9 with tyrosine, and substituting amino acid 10 with lysine (Table 4).

[0159] Sequence Number Amino Acid Sequence (NC) 2526272829303132KYQRRKKSYKKYQRRKRSYKKYQRRRKSYKKYQRRRRSYKKYQRKKKSYKKYQRKRKSYKKYQRKKRSYKKYQRKRRSYK

[0160] Next, the peptide of group 5 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with arginine, substituting the 4th amino acid with glutamine, and substituting the 5th to 7th amino acids with lysine or arginine (Table 5).

[0161] Sequence Number Amino Acid Sequence (NC) 3334353637383940KYRQRKKNKYKYRQRKRNKYKYRQRRKNKYKYRQRRRNKYKYRQKKKNKYKYRQKRKNKYKYRQKKRNKYKYRQKRRNKY

[0162] Next, the peptide of group 6 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with arginine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, and substituting the 8th amino acid with serine (Table 6).

[0163] Sequence Number Amino Acid Sequence (NC) 4142434445464748KYRQRKKSKYKYRQRKRSKYKYRQRRKSKYKYRQRRRSKYKYRQKKKSKYKYRQKRKSKYKYRQKKRSKYKYRQKRRSKY

[0164] Next, the peptide of group 7 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with arginine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 7).

[0165] Sequence Number Amino Acid Sequence (NC) 4950515253545556KYRQRKKNYKKYRQRKRNYKKYRQRRKNYKKYRQRRRNYKKYRQKKKNYKKYRQKRKNYKKYRQKKRNYKKYRQKRRNYK

[0166] Next, the peptide of group 8 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with arginine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, substituting the 8th amino acid with serine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 8).

[0167] Sequence Number Amino Acid Sequence (NC) 5758596061626364KYRQRKKSYKKYRQRKRSYKKYRQRRKSYKKYRQRRRSYKKYRQKKKSYKKYRQKRKSYKKYRQKKRSYKKYRQKRRSYK

[0168] Next, the peptide of group 9 was synthesized by substituting amino acid 3 of the peptide of SEQ ID NO. 1 with lysine, amino acid 4 with glutamine, and amino acids 5 through 7 with lysine or arginine (Table 9).

[0169] Sequence Number Amino Acid Sequence (NC) 6566676869707172KYKQRKKNKYKYKQRKRNKYKYKQRRKNKYKYKQRRRNKYKYKQKKKNKYKYKQKRKNKYKYKQKKRNKYKYKQKRRNKY

[0170] Next, the peptide of group 10 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with lysine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, and substituting the 8th amino acid with serine (Table 10).

[0171] Sequence Number Amino Acid Sequence (NC) 7374757677787980KYKQRKKSKYKYKQRKRSKYKYKQRRKSKYKYKQRRRSKYKYKQKKKSKYKYKQKRKSKYKYKQKKRSKYKYKQKRRSKY

[0172] Next, the peptide of group 11 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with lysine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 11).

[0173] Sequence Number Amino Acid Sequence (NC) 8182838485868788KYKQRKKNYKKYKQRKRNYKKYKQRRKNYKKYKQRRRNYKKYKQKKNYKKYKQKRKNYKKYKQKKRNYKKYKQKRRNYK

[0174] Finally, the peptide of group 12 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO. 1 with lysine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, substituting the 8th amino acid with serine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 12).

[0175] Sequence Number Amino Acid Sequence (NC) 8990919293949596KYKQRKKSYKKYKQRKRSYKKYKQRRKSYKKYKQRRRSYKKYKQKKKSYKKYKQKRKSYKKYKQKKRSYKKYKQKRRSYK

[0176] 2. Silico analysis for predicting the anti-inflammatory function of peptides

[0177]

[0178] The function of the peptide synthesized through Example 1 as an anti-inflammatory peptide was predicted using the AlPpred predictor (http: / kurata14.bio.kyutech.ac.jp / PreAIP / ).

[0179] The function of peptides as anti-inflammatory peptides was predicted using the AlPpred predictor. As shown in Table 13, to predict anti-inflammatory peptide function, peptides with a score range of 0.388 or higher and a specificity of 80% or higher were predicted as anti-inflammatory peptides.

[0180]

[0181] Criteria Table for Anti-inflammatory Peptide Screening Label Score range Sensitivity Specificity High Confidence Score ≥0.468 63.22% 90.30% Medium Confidence 0.468 Score ≥0.388 71.90% 80.11% Low Confidence 0.388 Score ≤0.342 78.39% 70.87%

[0182] To identify anti-inflammatory peptides, the sequences of 96 peptides, SEQ ID NOs 1 to 96, were analyzed. As shown in Table 13 above, peptides with an anti-inflammatory peptide analysis score range of 0.388 or higher via AlPpred analysis were selected and are shown in Table 14. Among the 96 peptides, peptides SEQ ID NOs 55, 66, 81, 82, and 85 showed medium confidence, and among the remaining 91 peptides, peptides SEQ ID NOs 1 to 54, SEQ ID NOs 56 to 65, SEQ ID NOs 67 to 80, SEQ ID NOs 83 to 84, and SEQ ID NOs 86 to 96 showed high confidence (Table 14).

[0183] In particular, the peptides of SEQ ID NOs 67, 68, 70, 72, 75, 76, 78, 80, 83, 84, 86, 88, 91, 92, 94 and 96, in the following KY-R1-R2-R3-R4-R5-R6-R7-R8 (General Formula 1), more preferably in terms of the balance of anti-inflammatory activity and various properties, R1 is lysine (K) or glutamine (Q), R2 is glutamine (Q), R3 is lysine (K) or arginine (R), R4 is arginine (R), R5 is arginine (R) or lysine (K), R6 is serine (S) or asparagine (N), and R7 and R8 are tyrosine (Y) or lysine (K), and showed High Confidence in terms of the balance of anti-inflammatory activity and various properties. Such amino acid combinations can optimize the function of peptides.

[0184] Specifically, the arrangement of lysine and glutamine near the N-terminus regulates positive charge distribution, and glutamine at the R2 position enhances anti-inflammatory activity. Serine or asparagine at the R6 position increases structural flexibility, while tyrosine and lysine at the R7 and R8 positions improve cell permeability. The combination of arginine and lysine at the R3, R4, and R5 positions optimizes immunomodulatory functions. This amino acid sequence improves the anti-inflammatory activity, structural stability, cell permeability, and immunomodulatory functions of the peptide, thereby enabling it to effectively treat inflammatory diseases in the oral cavity.

[0185]

[0186] 항염증 펩타이드 (anti-inflammatory peptide, AIP) 스크리닝서열번호SequenceCombined RF Score1KYQRRKKNKY0.5832KYQRRKRNKY0.5463KYQRRRKNKY0.6324KYQRRRRNKY0.595KYQRKKKNKY0.5766KYQRKRKNKY0.5587KYQRKKRNKY0.5068KYQRKRRNKY0.5489KYQRRKKSKY0.5710KYQRRKRSKY0.54511KYQRRRKSKY0.6212KYQRRRRSKY0.59413KYQRKKKSKY0.55714KYQRKRKSKY0.54515KYQRKKRSKY0.50716KYQRKRRSKY0.54417KYQRRKKNYK0.54818KYQRRKRNYK0.52319KYQRRRKNYK0.59120KYQRRRRNYK0.53221KYQRKKKNYK0.52622KYQRKRKNYK0.5123KYQRKKRNYK0.47924KYQRKRRNYK0.49325KYQRRKKSYK0.55926KYQRRKRSYK0.53627KYQRRRKSYK0.60428KYQRRRRSYK0.56129KYQRKKKSYK0.54530KYQRKRKSYK0.53531KYQRKKRSYK0.50932KYQRKRRSYK0.52133KYRQRKKNKY0.51434KYRQRKRNKY0.54835KYRQRRKNKY0.55736KYRQRRRNKY0.57537KYRQKKKNKY0.5638KYRQKRKNKY0.55639KYRQKKRNKY0.54240KYRQKRRNKY0.56241KYRQRKKSKY0.51942KYRQRKRSKY0.55743KYRQRRKSKY0.55844KYRQRRRSKY0.58945KYRQKKKSKY0.56646KYRQKRKSKY0.55547KYRQKKRSKY0.55848KYRQKRRSKY0.56349KYRQRKKNYK0.47250KYRQRKRNYK0.47551KYRQRRKNYK0.4952KYRQRRRNYK0.51453KYRQKKKNYK0.51154KYRQKRKNYK0.4755KYRQKKRNYK0.46256KYRQKRRNYK0.51957KYRQRKKSYK0.51558KYRQRKRSYK0.50359KYRQRRKSYK0.52960KYRQRRRSYK0.55 161KYRQKKKSYK0.55262KYRQKRKSYK0.51563KYRQKKRSYK0.50864KYRQKRRSYK0.54665KYKQRKKNKY0.47766 KYKQRKRNKY0.46367KYKQRRKNKY0.54168KYKQRRRNKY0.58169KYKQKKKNKY0.47770KYKQKRKNKY0.54371KY KQKKRNKY0.47272KYKQKRRNKY0.54773KYKQRKKSKY0.49174KYKQRKRSKY0.49275KYKQRRKSKY0.54576KYKQR RRSKY0.59377KYKQKKKSKY0.49378KYKQKRKSKY0.54179KYKQKKRSKY0.49380KYKQKRRSKY0.55381KYKQRKK NYK0.43582KYKQRKRNYK0.46483KYKQRRKNYK0.47584KYKQRRRNYK0.53885KYKQKKKNYK0.44786KYKQKRKNYK 0.49387KYKQKKRNYK0.47588KYKQKRRNYK0.51389KYKQRKKSYK0.48990KYKQRKRSYK0.50691KYKQRRKSYK0.5 2192KYKQRRRSYK0.57393KYKQKKKSYK0.51194KYKQKRKSYK0.53895KYKQKKRSYK0.52596KYKQKRRSYK0.553.

[0187] 3. Effects of Peptide SEQ No. 96 on the Expression of the Inflammatory Factor TNF-α Gene in RAW264.7 Macrophage Cells

[0188]

[0189] To evaluate the anti-inflammatory effect of peptide SEQ No. 96, peptide SEQ No. 96 was treated at a concentration of 100 μg / mL to evaluate the anti-inflammatory effect. RAW264.7 cells were tested for the expression of the inflammatory factor TNF-α gene under the following conditions.

[0190]

[0191] - Negative control group

[0192] - LPS-only treatment group

[0193] - LPS-treated group + SEQ ID NO. 96-treated group

[0194]

[0195] To confirm the anti-inflammatory effect of peptide SEQ No. 96 on an LPS-induced inflammatory response, TNF-α gene expression was measured after treating with peptide SEQ No. 96 under the above experimental conditions.

[0196] In the group treated with LPS alone, TNF-α gene expression was observed to increase by more than 14-fold. In the group of RAW264.7 cells treated with LPS and peptide SEQ No. 96, TNF-α gene expression was inhibited by approximately 3.5-fold compared to the group treated with LPS alone (Fig. 1A).

[0197]

[0198] 4. Effects of Peptide SEQ No. 96 on the Expression of the Inflammatory Factor IL-6 Gene in RAW264.7 Macrophage Cells

[0199]

[0200] To confirm the anti-inflammatory effect of peptide SEQ No. 96, peptide SEQ No. 96 was treated under the above experimental conditions, and then the expression of another inflammatory factor, IL-6 gene, was checked (Fig. 1B). In the test group treated with LPS alone, IL-6 expression increased by approximately 900 times compared to the control group. On the other hand, in the group treated with peptide SEQ No. 96 together with LPS, IL-6 gene expression was found to decrease by approximately 3 times compared to the group treated with LPS alone.

[0201]

[0202] 5. Effects of Peptide SEQ No. 96 on the Expression of the Inflammatory Enzyme iNOS Gene in Macrophage Line RAW264.7 Cells

[0203]

[0204] To confirm the anti-inflammatory effect of peptide SEQ No. 96, peptide SEQ No. 96 was treated under the above experimental conditions, and then the expression of another inflammatory factor, iNOS gene, was checked (Fig. 1C). In the test group treated with LPS alone, iNOS expression increased by approximately 800 times compared to the control group. On the other hand, in the group treated with peptide SEQ No. 96 together with LPS, iNOS gene expression was found to decrease by approximately 8 times compared to the group treated with LPS alone.

[0205]

[0206] 6. The Effects of CPNE7 Gene Deficiency on Hair Loss

[0207]

[0208] To evaluate the effect of CPNE7 gene deletion on hair loss, normal and CPNE7 gene-deficient mice at 15 months of age were observed visually. As shown in Fig. 2A, hair loss was observed in the head and trunk of CPNE7 gene-deficient mice compared to normal mice. Based on these results, Fig. 2B shows the results of histological analysis performed on skin excised from 6-month-old normal, CPNE7 gene-deficient, and CPNE7 gene-overexpressing mice to measure the number of hair roots and the size of hair follicles. Unlike the normal and CPNE7 gene-overexpressing mice, the skin of CPNE7 gene-deficient mice was thinned, and it was confirmed that the number of hair roots had also decreased. Histological analysis revealed that the number of hair roots in CPNE7 gene-deficient mice was reduced by nearly 50% compared to normal or CPNE7 gene-overexpressing mice (Fig. 3A). In addition, the size of hair follicles decreased by approximately 70% in CPNE7 gene-deficient mice, whereas the size of hair follicles in CPNE7 gene-overexpressing mice was significantly increased compared to normal and CPNE7 gene-deficient mice (Fig. 3B). Furthermore, when comparing hair thickness, no statistical significance was found between normal and CPNE7 gene-deficient mice, but the hair of CPNE7 gene-overexpressing mice was analyzed to be thicker than that of CPNE7 gene-deficient mice (Fig. 3C).

[0209]

[0210] 7. Effects of Novel Peptides on Hair Growth

[0211]

[0212] The results of visual observation of hair growth on the depilated back area are as shown in Fig. 4A. Compared to the control group, hair growth was clearly observed in the group treated with the novel peptide SEQ No. 96, and the group treated with SEQ No. 96 showed a rapid hair growth effect on the 8th day. As a result of analyzing the hair roots by excising the skin of these groups, it was confirmed that more hair grew in the group treated with SEQ No. 96 (Fig. 4B).

[0213]

[0214] 8. Effect of a Novel Peptide on the Migration of Hair Root Stem Cells

[0215]

[0216] As a method for treating hair loss, the migration of hair follicle stem cells from the area where hair has fallen out to the hair root is also important. To confirm the effect of a novel peptide on the migration of hair follicle stem cells, hair follicle stem cells were isolated from the hair roots of mice and cultured. As a result of analyzing the cell migration ability induced by peptide SEQ No. 96 using a wound healing assay, it was confirmed that the cell migration ability of hair follicle stem cells was accelerated in the experimental group treated with peptide SEQ No. 96 compared to the control group (Fig. 5).

[0217]

[0218] Through the embodiments of the present invention, it was confirmed that the CPNE7 gene prevents hair loss and promotes hair growth. In addition, it was found that the novel peptide derived from CPNE7 not only has an anti-inflammatory effect but can also treat hair loss or promote hair growth in individuals with hair loss.

[0219]

[0220] The present specification and drawings disclose preferred embodiments of the present invention. Although specific terms have been used, they are used merely in a general sense to facilitate the explanation of the technical content of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that, in addition to the embodiments disclosed herein, other variations based on the technical concept of the present invention are possible.

Claims

1. A composition for preventing or treating hair loss comprising, as an active ingredient, a peptide having the amino acid sequence of Chemical Formula 1 below: KY-R1-R2-R3-R4-R5-R6-R7-R8 (Chemical Formula 1) Here, R1 is arginine (R), lysine (K), or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4, and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); and R7 and R8 are lysine (K) or tyrosine (Y), respectively.

2. In Paragraph 1, The above peptide is a composition for preventing or treating hair loss comprising any one of the amino acid sequences of SEQ ID NOs 1 to 96.

3. In Paragraph 1, A composition for preventing or treating hair loss, characterized in that the above-mentioned peptide promotes the migration of hair follicle stem cells.

4. In Paragraph 1, A composition for preventing or treating hair loss, characterized in that the above peptide inhibits the expression of at least one of TNF-α, IL-6, and iNOS in macrophages.

5. In Paragraph 1, A composition for preventing or treating hair loss, characterized by further comprising a pharmaceutically acceptable carrier.

6. In Paragraph 1, A composition for preventing or treating hair loss, characterized in that the above composition is a formulation for topical application.

7. A composition for promoting hair growth comprising, as an active ingredient, a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

8. A method for preventing or treating hair loss comprising the step of applying or administering a peptide composed of any one sequence selected from sequence numbers 1 to 96 to an individual other than a human.

9. A composition for preventing or treating hair loss comprising a vector containing the CPNE7 gene as an active ingredient.

10. A composition for preventing or treating hair loss containing CPNE7 protein as an active ingredient.

11. A cosmetic composition for preventing or improving hair loss comprising, as an active ingredient, a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

12. A shampoo composition for preventing or improving hair loss comprising, as an active ingredient, a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

13. A composition for promoting hair growth or hair regeneration comprising, as an active ingredient, a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

14. A cosmetic composition for promoting hair growth or hair regeneration comprising, as an active ingredient, a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

15. A shampoo composition for promoting hair growth or hair regeneration comprising, as an active ingredient, a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

16. A health functional food for preventing or improving hair loss comprising, as an active ingredient, a CPNE7 protein or a peptide consisting of any one sequence selected from SEQ ID NOs 1 to 96.

17. A health functional food for promoting hair growth or hair regeneration comprising, as an active ingredient, a peptide consisting of any one sequence selected from sequence numbers 1 to 96.

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