TW201924656A - Methods and compositions for modulating melanogenesis - Google Patents

Abstract

Disclosed herein is a composition including a peptide consisting of the sequence of SEQ ID NO:1 for increasing the production of melanin. The present disclosure provides methods and formulations for the treatment of pigmentation disorders or conditions. In some embodiments, provided herein are compositions and methods for sunless tanning. In other embodiments, the present disclosure is directed to such compositions that are formulated for use as pharmaceuticals and cosmetic products, and to medical devices comprising the disclosed compositions.

Description

Method and composition for regulating melanin production

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The skin is the largest organ of the human body, and its main structure is two layers of the epidermis layer and the dermis layer. The epidermis is in contact with the outside world, and its various biological and physical properties make the epidermis resistant to external environmental stimuli that occur repeatedly and avoid damage. The epidermis is composed of several cells, such as keratinocytes, melanocytes, Langerhans cells, Merkel cells, and inflammatory cells.

The keratinocytes in the upper layer of the basal layer differentiate into a chemically and physically resistant stratum corneum, which is coated with proteins and lipids (including neuropterin, cholesterol and fatty acids). The upper layer of keratinized epithelial tissue, after being naturally detached or removed by external force, induces a metabolic mechanism that causes the underlying cells to replace damaged or lost cells. The most important function of keratinocytes is to form a skin barrier, which protects the body from various chemical, physical and mechanical, microbial invasion, heat, ultraviolet rays and the like, and prevents water loss. Keratinocytes are also the major components of mucosal tissue that is connected to the epidermal layer.

The epidermal melanocytes of the human body are distributed in the skin, hair follicles, eyes, inner ear, bones, heart and brain, and their main function is to produce melanin. Melanocytes are derived from pluripotent neural ridge cells and are constantly differentiated by complex regulatory pathway network interactions. When melanocytes accumulate in the epidermal layer, it means that most melanocytes are completely differentiated and have long life cycle proliferative ability like neurons. The melanocytes are dendritic cells of the neuroectoderm, and the precursor cells are melanocytes which are non-pigmented and differentiated from embryonic neural crest cells. After the neural tube is closed, melanocytes migrate to multiple parts of the body and develop into melanocytes and peripheral nervous system cells, skull and head cartilage, and the choroid of the eye. Melanocytes that can develop into melanocytes are mostly distributed in the basal layer of the epidermis and hair follicles, and are characterized by melanocyte-specific markers. The melanocytes in human skin are covered with keratinocytes (about 36 keratinocytes around a melanocyte), and melanocytes transport melanin to keratinocytes. Melanin has a molecular structure suitable for absorbing ultraviolet light and visible light, thus helping the body to block ultraviolet rays in the sun.

Melanin is widely found in the animal kingdom and generally acts as a source of resistance to environmental stress. Although melanin is distributed in epidermal keratinocytes, it is actually synthesized by a large number of melanocytes present in the lowest layer of the epidermis and a small amount in the dermis, and is a pigment molecule synthesized intracellularly. It is well known that people who are not easy to tanned with lighter skin are more likely to suffer from skin cancer (such as melanoma) than those with darker skin. Animals will synthesize melanin in the body to deepen the skin color to prevent the sun from harming the skin.

Skin melanin plays a pivotal role in camouflage, mimicry, socializing, and resistance to sun radiation damage. As black particles continue to accumulate in keratinocytes, pigmentation occurs in the skin, and keratinocytes form a natural sunscreen structure that absorbs ultraviolet light to prevent UV rays from invading the deeper areas of the skin where the proliferating cells are located. Therefore, pigmentation in the skin protects the DNA from damage caused by sunlight, and effectively prevents cancer from forming.

Melanin is divided into three categories, namely pheomelanin, eumelanin and neuromelanin. The main factor regulating mammalian melanin synthesis in mammalian melanocytes is the melanocortin-1 receptor (MC1R). MC1R is a receptor coupled to the G protein, which has seven transmembrane domains. MC1R is expressed on the surface of melanocytes and signals are transmitted via activation of the cyclic adenosine monophosphate (cAMP) pathway. MC1R is a member of the melanocortin receptor family (MC1-5R) and is the only melanocortin receptor that is expressed on melanocytes. In vivo studies have shown that injection of purified melanocortin into human subjects induces an increase in pigmentation. In the early 1990s, the MC1R gene was cloned from human melanocytes and showed that the expressed receptor is functional. This result strongly suggests that MC1R directly regulates the melanin synthesis in melanocytes to regulate the importance of human skin pigmentation.

The MC1R gene line leads to a major cause of human skin color diversity. Epidemiological studies have found that each species will exhibit multiple MC1R polymorphisms (nearly 200 allelic variants). Most of the wild-type MC1R genes are found in Africa. The black skin of local people has high melanin content, which is a necessary condition for adapting to the equatorial sunshine. Certain variants associated with light-colored skin and red-haired phenotypes, including R151C, R160W and D294H, are abundantly found in Nordic and Australian Celts. The skin phenotype of these people increases the incidence of melanoma and also shows that the MC1R gene is closely related to the occurrence of melanoma. The MC1R gene has additional cytoprotective functions, including antioxidant defense machinery turnover, DNA repair, and regulation of inflammatory responses via the NF-κB signaling pathway. More than 100 MC1R gene variants form different amino acids on specific codons, and these variants are generally referred to as non-synonymous variants and are highly variable.

For decades, a significant number of people have been involved in melanocortin studies, hoping to develop melanocortin analogs that promote pigmentation (sunning) without exposure to sunlight. Many people have also attempted to calibrate melanocortin analogs on chemotherapeutic agents to selectively eradicate melanoma tumor cells. The most well-known and most well-known melanin analogue is NDP-MSH (Ac-[Nle ⁴ , D-Phe ⁷ ]-α-MSH), and studies have shown that NDP-MSH is more potent than amphibians, reptiles and mammals. The native alpha-MSH in the body is at least 100 times higher and is significantly more stable and long lasting. For the activity of tyrosinase in cultured human melanocytes, NDP-MSH can have a stronger and longer-lasting effect than α-MSH. The results of a systemic administration of NDP-MSH to human subjects showed that NDP-MSH was effective in promoting pigmentation in the absence of sun exposure. When the analog was administered to the subject, the degree of DNA damage caused by sunlight in the body decreased, and it was apparent that NDP-MSH had a photoprotective effect. However, administration of NDP-MSH also has side effects, including nausea and loss of appetite, which can be attributed to NDP-MSH binding and activation of melanocortin receptors expressed on other cells than melanocytes.

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Disclosed herein is a short peptide, referred to herein as P131 (SEQ ID NO: 1), which has the advantage of modulating melanin production. This article shows that P131 can upregulate the expression of MC1R and other genes involved in melanin production. Specifically, it is shown herein that P131 can increase the amount of melanin production. Another advantage of P131 is the ability to modulate the expression of genes associated with interstitial repair and remodeling that maintains skin barrier function. In addition, P131 significantly reduced the amount of gene involved in the pro-inflammatory pathway. In view of this, the P131 peptide in the present invention can be formulated into a composition or a drug (such as an emulsion, a cream, etc.) for administration to a subject for sunburning, color change, or treatment or improvement of condition, disease or abnormality. . P131 peptides have not been found to regulate melanin production in the past.

In the description herein, any concentration range, percentage range, ratio range or integer range is understood to include any integer within the range, and if necessary, a fractional part of the integer (eg an integer) The first or second decimal point). Unless otherwise stated, any numerical range associated with a physical feature, such as a polymer subunit, size or thickness, as used herein, is to be understood to include any integer within the range. The term "about" as used herein, unless otherwise indicated, means within the range of ±20% of the stated range, value or structure.

It is to be understood that the phrase "or" or "an" or "an"

In addition, the singular <RTI ID=0.0>" </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt;

Words "including", "having", "including" and the like are synonymous and should be construed as not limiting.

The term "a combination thereof" as used herein refers to one of all possible combinations of items listed in the preceding paragraph. For example, "A, B, C or a combination thereof" is intended to mean any combination of A, B, C, AB, AC, BC or ABC. Similarly, the term "combinations thereof" as used herein refers to all possible combinations of items listed in the preceding paragraph. For example, "A, B, C, and combinations" are intended to refer to all combinations of A, B, C, AB, AC, BC, and ABC.

The term "melanogenesis" as used herein refers to the process by which melanin is produced. Melanogenesis produces long-lasting sunburn on mammalian skin. Melanogenesis is divided into a ground state and an excited state. In general, the melanin of the light-colored skin species has a lower ground state. As described herein, various embodiments herein provide methods of promoting or stimulating melanin production in a subject by administering to the subject a composition comprising a peptide having the sequence of SEQ ID NO: 1. The melanin production promoted or stimulated can restore the skin of the pigmented condition to normal pigmentation, or the skin can maintain the sunburn without the risk of ultraviolet exposure.

SEQ ID NOs: 1, 2, and 3 herein are included in a study that proposes novel dermatological therapies that have not been disclosed.

The three peptides of SEQ ID NOs: 1, 2, and 3 were subjected to a melanin screening test using B16F10 melanocytes. After co-culture with B16F10 cells, SEQ ID NO: 1 significantly promoted melanin production with a dose-dependent increase (see Table 1). When the melanin production rate of the water-treated melanocytes was set to 100%, the melanin production rate of the melanocytes added with SEQ ID NO: 1 at 50 μg/ml was 153% at 200 μg/ml. The rate of increase in melanin production was 346% (see Table 1). It was found that the aforementioned induced yield conditions were all caused by SEQ ID NO: 1, while SEQ ID NO: 2 and SEQ ID NO: 3 did not have any ability to induce melanin production.

To confirm that the cause of melanin production is cytotoxic or cellular abnormality, cell viability was determined using the MTT assay. As shown in Table 2, all of the peptides did not significantly affect the activity of B16F10 melanocytes or neonatal dermal fibroblasts at the necessary concentrations that promote melanin production.

Further study of SEQ ID NO: 1, using MatTek's MelanoDerm model, consisting of common human epidermal keratinocytes (NHEK) and melanocytes (NHM), is a highly differentiated human epidermal multi-layered model formed by cell culture. NHM in co-cultures produces melanin, which causes pigmentation in tissues. The MelanoDerm tissue was treated with SEQ ID NO: 1 for 24 hours, and all 107 groups of genes were analyzed.

As shown in Table 3, SEQ ID NO: 1 significantly increased the amount of expression of the following genes: MC1R (4.02 fold), MITF (1.93 fold), BMP4 (1.92 fold). These three groups of genes are the key to melanin production. In addition, genes related to interstitial repair and remodeling, such as MMP9 and KLK8, have also been significantly increased in performance. The genes whose expression levels were also up-regulated included FLG (1.92 times) and AQP5 (1.82 times), which are important genes for maintaining the stability of the skin barrier function. SEQ ID NO: 1 significantly reduced the expression of most (or even all) genes involved in the pro-inflammatory pathway, including IL1-β, IL8, PTGS2, TNF-α, LIF, IL1-α, and IL6 (Table 3).

All peptides contained herein are synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid state chemistry. The peptides can be prepared as the amide or free acid sequence using standard amino acids. The amidation of the C-terminus makes the peptide of the present invention less susceptible to protease degradation and makes it more soluble than the free acid form, thus providing a higher therapeutic effect. The peptides may comprise L- and/or D-amino acid mirror image isomers. The peptides may include D- and/or L-amino acids. The peptides can include all D-amino acids. The N-terminus and C-terminus of the peptides can be modified. For example, lipidation or acetylation of the N-terminus increases the ability of the peptide to penetrate the skin without altering its biological activity (see Samah A and Haerd CM. Int J Cosmet Sci. 2011 Dec; 33 (6 ): 483-90, the entire disclosure of which is incorporated herein by reference. Thus, the peptides can be lipidated and increase skin penetration. Saturated or unsaturated fatty acids useful in providing the C12-18 lipid component of the compounds described herein include lauric acid, myristic acid, palmitic acid, stearic acid, myristic acid, palmitoleic acid, oleic acid, and linoleic acid. The C-terminus of the peptides may be modified by an acid group (-COOH) or a guanamine group (-CONH ₂ , -CONHR or -CONR ₂ ). The amidation of the C-terminus renders the peptides less susceptible to protease degradation and makes them more polar than the free acid form, thereby providing a higher therapeutic effect. Groups which may be modified in such peptides also include hydroxyl, amine, guanidinium, carboxyl, phenolic, imidazole or sulfhydryl.

The peptide can also be conjugated to a soluble or insoluble carrier molecule to adjust its solubility as needed and increase the local peptide concentration of the target tissue. Soluble carrier molecules include, but are not limited to, polymers of polyethylene glycol (PEG) and polyvinylpyrrolidone; insoluble polymers include, but are not limited to, silicate, polystyrene, and cellulose. . The liposome technology or nanotechnology can be utilized to place the peptide into the microcapsules to increase their stability and to easily control their release. The peptides can be transported by other peptides (such as cell penetrating peptides) to make it easier for the peptide to penetrate the cell membrane and reach the target between cells. In the general procedure described above, the peptides can be prepared by any method known to those of ordinary skill in the art to which the present invention pertains, such as the method disclosed in Merrifield ( J Am Chem Soc. 85: 2149, 1963). Carpino et al. ( J Org Chem. 51:3732, 1986); Merrifield et al. ( Anal Chem. 38:1905, 1966); or Kent et al. [ High Yield Chemical Synthesis Of Biologically Active Peptides On An Automated Peptide Synthesizer Of Novel Design , IN: PEPTIDES 1984 (Ragnarsson, ed.) Almqvist and Wiksell Int., Stockholm (Sweden), pp. 185-188]. The entire contents of the above documents are hereby incorporated by reference.

In certain embodiments, the present invention provides a cosmetic composition comprising a peptide having the amino acid sequence of SEQ ID NO: 1 or consisting of the amino acid sequence of SEQ ID NO: 1, pharmaceutically or cosmetically The accepted carrier, as well as the second active agent. In certain embodiments, the cosmetic composition is a tanning composition. In some embodiments, the cosmetic composition is a tanning composition and the second active agent is a tanning agent. Exemplary tanning agents include dihydroxyacetone, erythrulose, canthaxanthin, or any combination of the foregoing.

In certain embodiments, the cosmetic composition allows the hair to grow healthily to prevent white growth. In some embodiments, the cosmetic composition is used to restore white hair to natural pigmentation. In a particular embodiment, the cosmetic composition comprises a second formulation, such as methionine, cysteine, guanidine, coconut oil, jojoba oil, mustard seed oil (muster seed oil), castor oil, pantothenic acid, biotin, copper, vitamin B6, ammonia, henna or any combination of the foregoing. In a specific embodiment, the second active agent is a hair dye and comprises 1,4-diaminobenzene, 1,3-diaminobenzene, 2, 5-Diaminotoluene, phenol and naphthols, such as 3-aminophenol (3-aminophenol (CAS #591-27-5)), 5-amino-2- Methylphenol (5-amino-2-methylphenol (CAS #2835-95-2)) and 1-naphthol (1-naphthol (CAS #90-15-3)), resorcinol (resorcinol), 4 - 4-chlororesorcinol and benzodioxoles, couplants, oxidizing agents or any derivative or combination of the foregoing. The combination of 2,5-diaminotoluene and the couplant 3-aminophenol forms a magenta-brown dye, and the combination of 2,5-diaminotoluene and the couplant 1-naphthol forms a purple dye. Resorcinol, 4-chlororesorcinol and benzodioxol react with each other to form a dye, which results in broad-band absorption and makes hair color more natural. The combination of 2,5-diaminotoluene and the couplant resorcinol forms a greenish brown dye.

In certain embodiments, the present invention provides a pharmaceutical composition comprising a peptide having the amino acid sequence SEQ ID NO: 1 or consisting of the amino acid sequence SEQ ID NO: 1, a pharmaceutically acceptable carrier, And a penetration enhancer, a broad-acting sunscreen lotion or a second active agent for treating or ameliorating symptoms associated with abnormal pigmentation. Exemplary penetration enhancers include liposomes, lipid nanocarriers, polysorbates, N-methylpyrrolidone, laurocapram, transcutol P, terpineol, eucalyptus oil Cineole, steroids (such as dimethyl hydrazine (DMSO)), azones (such as azone), pyrrolidone (such as 2-pyrrolidone), alcohols and alkanols (such as ethanol or hydrazine) Alcohols), glycols (such as propylene glycol, excipients often included in external dosage forms), surfactants (common in dosage form), and terpenes. Exemplary broad-spectrum sunscreens include titanium dioxide, zinc oxide, avobenzone, botanical extracts, or any combination of the foregoing.

In certain embodiments, the pharmaceutical composition comprises the second active agent, and the second active agent is for treating or ameliorating symptoms associated with vitiligo and comprising a calcineurin inhibitor (calcineurin) Inhibitor), oxsoralen, methoxsalen, all-trans-retinoic acid, psoralens, or any combination of the foregoing.

In certain embodiments, the pharmaceutical composition comprises the second active agent, and the second active agent is for treating or ameliorating symptoms associated with hypopigmentation, and comprises all-trans retinoic acid, supplement A psoralen, an anti-inflammatory preparation, a corticosteroid, an antibacterial or antifungal formulation, an extracellular matrix enhancer, a lipid that aids in the skin barrier, a botanical component, or any combination of the foregoing.

In certain embodiments, the concentration of the peptide in the composition ranges from 0.01 μg/ml to about 50 mg/ml, by weight of the composition. The concentration range may include, but is not limited to, 0.1 μg/ml to 20 mg/ml; 0.5 μg/ml to 10 mg/ml; 1 μg/ml to 5 mg/ml; 2 μg/ml to 2 mg/ml; 3 μg /ml to 1.5 mg/ml; 4 μg/ml to 1 mg/ml; 5 μg/ml to 800 μg/ml; 6 μg/ml to 500 μg/ml; 10 μg/ml to 450 μg/ml; 15 μg /ml to 400 μg/ml; 20 μg/ml to 350 μg/ml; 25 μg/ml to 300 μg/ml; 30 μg/ml to 250 μg/ml; 40 μg/ml to 200 μg/ml; 45 μg /ml to 150 μg/ml; 50 μg/ml to 100 μg/ml; 10 μg/ml to 100 μg/ml; 100 μg/ml to 300 μg/ml; 300 μg/ml to 500 μg/ml; 500 μg /ml to 1 mg/ml; or 1 mg/ml to 10 mg/ml; 50 μg/ml to 200 μg/ml, 200 μg/ml to 500 μg/ml, 1 mg/ml to 10 mg/ml or 10 From mg/ml to 100 mg/ml, based on the weight of the composition. The final concentration to be formulated can vary outside of the foregoing ranges depending on the nature of the tissue conditions, the biological activity of the peptide, and the use of any adjuvant or technique used to enhance absorption of the composition.

In general, a pharmaceutically or cosmetically acceptable dosage form includes any suitable carrier (e.g., a pharmaceutically or cosmetically acceptable carrier) that can be applied to human skin. The aforementioned pharmaceutically or cosmetically acceptable carriers include ethanol, dimethyl hydrazine, glycerin, cerium oxide, aluminum oxide, starch, and the like carriers and diluents. In certain embodiments, the pharmaceutically or cosmetically acceptable carrier is not comprised of water or consists essentially of water.

In the Personal Care Products Council International Cosmetic Ingredient Dictionary and Handbook (Sixteenth Edition 2016), a number of non-limiting cosmetic and pharmaceutical ingredients are described which are often used on the skin. The maintenance industry also applies to the compositions herein. The aforementioned ingredients may include abrasives, absorbents, cosmetic ingredients such as aromas, pigments, pigments/colorants, essential oils, skin sensitizers, astringents, etc. (such as clove oil, menthol, camphor oil, eucalyptus oil, eugenol, Menthol lactate, witch hazel distillate), anti-acne agents, anti-caking agents, anti-foaming agents, antibacterial agents (such as iodopropyl butylcarbamate), antioxidants, Adhesives, biological agents, buffers, swelling agents, chelating agents, chemical additives, cosmetic biocides, denaturants, drug astringent, topical analgesics, film formers Or materials, opacifiers, pH adjusters, propellants, reducing agents, sequestrants, skin bleaching and skin lightening agents (eg hydroquinone, niacin, ascorbic acid, magnesium ascorbyl phosphate) , ascorbyl glucosamine, skin conditioners (such as humectants), skin soothing and/or therapeutic agents (such as panthenol and its derivatives, aloe vera gel, pantothenic acid and its Biology, allantoin (Allantoin), Bisabolol (Bisabolol) and dipotassium glycyrrhizinate (dipotassium glycyrrhizinate)), skin treating agents, thickeners, and vitamins and derivatives thereof.

Furthermore, any of the compositions disclosed herein can comprise a penetration enhancer. As used herein, the term "penetration enhancers" or "permeation enhancers" refers to substances that enable therapeutic peptides to penetrate the skin barrier more easily. It is well known that the skin (especially the stratum corneum) forms a physical barrier that blocks the harmful stimuli of the external environment. However, the skin can also interfere with the absorption and transdermal transport of topical drugs. In general, penetration enhancers reduce the level of interference or blockage of the skin, allowing therapeutic drugs to penetrate the skin more easily. In particular, such substances that interfere with the normal structure of the stratum corneum can interfere with intercellular lipid tissue and reduce its barrier effect. Such materials may include any lipid material that decomposes into small particles that penetrate the stratum corneum lipid and cause a direct effect, or any lipid material that affects the protein and indirectly interferes with the lipid structure. In addition, solvents such as ethanol can remove lipids in the stratum corneum, thereby destroying its lipid organization and barrier function.

Examples of penetration enhancers or barrier function interferers include, but are not limited to, alcohol enhancers such as alkanols with from 1 to 16 carbons, benzyl alcohol, butylene glycol, diethylene glycol, tetrahydrofuran polyglycol ethers (glycofurol), glycerides, glycerol, glycerol, phenylethyl alcohol, polypropylene glycol, polyvinyl alcohol and phenol; guanamine enhancers such as N-butyl-N-dodecylacetamide (N-butyl- N-dodecylacetamide, crotamiton, N,N-dimethylformamide, N,N-dimethylacetamide, N - N-methyl formamide and urea; amino acids such as L-α-amino acids and water-soluble proteins; azones and azone compounds such as azacycloalkanes; essential oils, Such as almond oil, amyl butyrate, apricot kernel oil, avocado oil, camphor oil, castor oil, 1-carvone, coconut oil, corn oil, cottonseed oil, eugenol, Menthol, fennel oil, clove oil, orange oil, peanut oil, peppermint oil, rose oil, safflower oil, sesame oil, shark liver oil (squalen) e)), soy oil, sunflower oil and walnut oil; vitamins and herbs such as aloe vera, allantoin, black walnut extract, chamomile extract, panthenol, papain, tocopherol and vitamin A palmitate Wax, such as candelilla wax, carnauba wax, ceresin wax, beeswax, lanolin wax, jojoba oil, petrolatum; mixed ester, For example, mixing a fraction of a vegetable oil fatty acid monoglyceride with glycerin or propylene glycol, and interesterified medium chain triglyceride oils; fatty acids and fatty acid esters such as amyl hexanoate, butyl acetate, octanoic acid, Cetyl ester, diethyl sebacate, dioctyl malate, elaidic acid ethyl caprylate, palmitic stearic acid Ethyl alcohol (ethyl glycol palmitostearate), glyceryl behenate, glucose glutamic acid, isobutyl acetate, laureth-4, lauric acid, malic acid, citrate , mineral oil, myristic acid, oleic acid, palmitic acid, polyethylene glycol fatty acid ester, polyoxylene sorbitan monooleate, polypropylene glycol, propylene glycol, sucrose stearate, water Salicylic acid, sodium citrate, stearic acid, soap and caproic acid/caprylic acid/capric acid/lauric acid triglyceride; macrocyclics such as butylated hydroxyanisole, cyclopentadecanolide ( Cyclopentadecanolide), cyclodextrins; phospholipids and phosphate enhancers, such as dialkylphosphates, ditetradecyl phosphate, lecithin, 2-pyrrolidone derivatives, such as Alkyl pyrrolidone-5-carboxylate esters, pyroglutamic acid esters, N-methyl pyrrolidone, biodegradable soft penetration enhancers (biodegradable soft penetration enhancer), such as dioxane derivatives and dioxolane derivatives; hydrazine enhancers such as dimethyl hydrazine Decylmethyl sulphoxide; acid enhancer such as alginic acid, sorbic acid, succinic acid; cyclic amine; imidazolinones, imidazoles Ketones such as acetone, dimethicone, methyl ethyl ketone and pentanedione; lanolin derivatives such as lanolin alcohol, polyethylene glycol 16 lanolin and acetylated wool Fat; oxazolines; oxazolindinones; proline esters; pyrroles, urethanes; and surfactants such as nonoxynol , polysorbates, polyoxylene alcohols, polyoxylene fatty acid esters, sodium lauryl sulfate, and sorbitan monostearate .

In certain embodiments, the compositions disclosed herein further comprise a compound that promotes microcirculation of the skin and exposes melanocytes to more oxygen; enhances photoprotection and protects melanocytes from natural and artificial UV exposure Threat; can promote melanin production and stability; can induce extracellular interstitial metabolism; or can be reacted with amino acids in the stratum corneum to produce pigments when applied to the skin, including but not limited to benzyl nicotinic acid (benzyl nicotinate), phenylalanine, methionine, acetyl cysteine, tyrosine; peptides that induce collagen and extracellular matrix, anti-inflammatory Peptide, dihydroxyacetone, erythrulose, canthaxanthin, vitamins, botanical ingredients, minerals such as copper, zinc and/or beta-carotene.

In certain embodiments, any of the compositions disclosed herein are in the form of a gel, a solution, a liquid, a spray, a spray (eg, a tanning spray), an emulsifier, an emulsion, a mousse, a wipe, a micro Capsules, serums, creams, ointments, powders, foams, ointments or other pharmaceutically or cosmetically acceptable dosage forms. In certain embodiments, the peptide is coated in a liposome or microsponge.

In certain embodiments, any of the compositions disclosed herein further comprise a sunscreen, a skin conditioning agent, a tanning agent, a skin lightening agent, a skin protectant, a moisturizer, a thickener, an excipient, Humectant or any combination of the foregoing.

In certain embodiments, any of the compositions disclosed herein further comprise a fatty alcohol, a fatty acid, an organic base, an inorganic base, a preservative, an ester wax, a steroid, a triglyceride, a phospholipid, a polyol ester, a fatty alcohol. Ether, hydrophilic lanolin derivative, hydrophilic beeswax derivative, cocoa butter wax, eucalyptus oil, pH balance agent, cellulose derivative, hydrocarbon oil or any combination of the foregoing.

In certain embodiments, the compositions may optionally have a cosmetic effect and/or comprise other agents such as retinol, citric acid, vitamin C, hyaluronic acid, stem cell extracts or other peptides (eg, The cell penetrates the peptide) and can be used as an adjuvant for the treatment of the peptide of the present invention. It is also possible to add antibiotics to the medicament to prevent the symptoms of infection, thereby maximizing the therapeutic effect.

In certain embodiments, the composition can comprise a protease inhibitor. Protease inhibitors can be selected to specifically inhibit proteases that may degrade the bioactive peptide. The basis for selecting a protease inhibitor is based on the length and/or sequence of the biologically active peptide. However, no particular manner is required to select a protease inhibitor; for example, a protease inhibitor cocktail comprising two or more inhibitors may be suitable for use herein. The protease inhibitors listed below can be incorporated herein: serine protease inhibitors, cysteine protease inhibitors, aspartate protease inhibitors, Metalloproteinase inhibitors, thiol protease inhibitors, and threonine protease inhibitors. The protease inhibitors used herein can be peptides or proteins or chemicals. Non-limiting such inhibitors are serine protease inhibitors, including alpha-1-antitrypsin, complement 1-inhibitor, antithrombin ( Antithrombin), alpha-1-antichymotrypsin, plasminogen activator inhibitor 1 and neuroserin inhibitor may be used as the case The chemical used for the adjuvant in the treatment of the peptide includes, but is not limited to, ursolic acid and tranexamic acid.

In a particular example, the composition can be placed in a device that is placed on the skin, intradermally or subcutaneously. The foregoing devices comprise a transdermal patch, an implant, and an injectable that release a substance in contact with the skin or hair follicle via a passive or active release mechanism. In certain embodiments, any of the compositions disclosed herein can be placed in a device that delivers the peptide via iontophoresis or ultrasound. In certain embodiments, any of the compositions disclosed herein are integrated with a device that can be used to administer the composition under skin tissue, mucous tissue, skin surface or scalp.

The peptides and related compositions herein can be administered to a subject. "Subject" means humans and animals, including all mammals. When the above substances are administered, they can also be handled with typical materials and/or experimental materials such as tissue grafts, skin substitutes, tissue cultures, and dressings. Examples include, but are not limited to, gauze (woven or non-woven, impregnated, non-stick, stuffing, debridement); compression bandages and systems; wound fillers and cleaners; contact layers; Collagen; amniotic membrane; acellular human dermis; decellularized interstitial and composition; and a variety of commonly used dressings.

The most commonly used dressings include, but are not limited to, absorbents; alginic acid; antibacterial agents; cell and/or tissue products; collagen; compression dressings; composites/laminates; contact layers; elastic bandages; foam dressings; gauze and non-woven fabrics; Gelatinized fiber dressings; hydrocolloids; hydrophilic fiber dressings; hydrogel dressings (unshaped, impregnated and lamellar); hyaluronic acid dressings; impregnated dressings; medical grade honey; Glycan dressings, protease-regulating dressings; skeleton and interstitial; enamel film; special absorbent dressings; topical growth factors; transparent film and wound filler.

In general, the compositions can be administered topically, orally, transdermally, systemically, or by any other method known to those of ordinary skill in the art to transport the peptides of the present invention to the target tissue. The compositions may also be administered in vitro or ex vivo, for example, for cells or patient grafts grown in the culture medium.

The compositions herein may comprise one or more additional preparations that aid in skin care. In addition to the components of the bioactive peptide, other active agents may be included herein, such as hyaluronic acid, ergothioneine, DNA repair enzymes, stem cell extracts, niacinamide, phytane Phytantriol, farnesol, bisabolol, salicylic acid, retinol, acid A, fruit acid, ascorbic acid, and alguronic acid. It is contemplated that these additional agents will work synergistically with the bioactive peptide component or will extend the shelf life of the formulation.

For the formulation and application techniques of drugs, refer to Remington Pharmacology 22nd Edition (Pharmaceutical Press, London UK). Although topical external administration is an ideal mode of administration, other methods such as oral, parenteral, spray, intramuscular, subcutaneous, transdermal, intramedullary, intrathecal, intraventricular, intravenous, and intraperitoneal may be used. Administered internally or intranasally. The invention can be formulated in a variety of carriers, such as sprays, sprays, water-in-oil emulsifiers, oil-in-water emulsifiers, creams or body creams, sunscreen or after-sun cream or other topical pharmaceutical carriers. In addition, the peptide of the present invention and the composition comprising the peptide can have practical properties, including general skin care and cosmetic preparations, such as various skin beauty products, moisturizers, lotions, sunscreens, and anti-acne preparations. A curative lotion or cream.

In certain embodiments, provided herein is a method of increasing melanin production, such as melanin production, comprising administering to a subject a composition comprising an effective amount of a peptide having the amine of SEQ ID NO: 1. The acid sequence consists of or consists of the amino acid sequence of SEQ ID NO: 1. The composition can be any of the compositions disclosed herein or variants thereof. The melanin may be brown melanin, true melanin, neuromelanin or any combination of the foregoing.

"Improving melanin production" refers to a statistically significant increase in the amount of melanin production at the target site of treatment compared to the same subject before or after treatment. The melanin production amount can be measured by a visual inspection or a non-invasive method, and the above measurement method is performed for the characteristic that melanin contributes to the reduction of the amount of light. Exemplary methods include single point measurement, ie, collecting light reflected from a defined skin area, and then calculating a pigment index, which refers to the average pigmentation amount of the measured area; or use for the scanned skin area An imaging technique that produces a distribution of melanin concentrations. There are many commercially available devices that measure the melanin index (or pigmentation index) and the erythema index. The principle is to measure the reflectance at a selected wavelength (see, for example, Statamas GN et al., Pigment Cell Res. 17:618- 626. 2004).

As used herein, an "effective amount" or "therapeutically effective amount" of a peptide or composition is indicative of the condition, disease, or abnormal condition being treated, and the amount of the peptide or composition has been improved to one or more symptoms. Degree, and statistically significant. The "therapeutically effective amount" of a compound herein will vary depending on the compound, the disease or condition and its severity, the mode of administration, and the age of the mammal being treated, but those of ordinary skill in the art to which the invention pertains may According to their own knowledge and the content of this article, the "therapeutically effective amount" is routinely judged. Preferably, for the purposes of this document, "therapeutically effective amount" means that the amount of the compound herein has reached the level at which melanin production can be induced to treat an abnormal state of a particular pigmentation.

In a particular embodiment, the method is for treating or ameliorating symptoms of an abnormal state of pigmentation. The words "treatment" or "improvement" as used herein may be used interchangeably and refer to the medical treatment of the condition, disease or abnormal state of a subject (such as a patient), which may be therapeutic, preventive. Or a combination of the foregoing. Further, "an abnormality or condition of pigmentation" includes an abnormal state in which the amount of pigmentation in the skin of the subject is low, insufficient or lacking. Examples of pigmentation abnormalities or conditions include, but are not limited to, leukoplakia, hypopigmentation, hypopigmentation, albinism, white pityriasis alba, tinea versicolor, post-inflammatory hypomelanosis, hypopigmentation Hypopigmented mycosis fungoides, tuberculoid leprosy, hypomelanosis of Ito, naevus anaemicus, ocular albino 1-4, chubby Wiley disease Prader-Will & Angelman syndrome, Hermansky-Pudlak syndrome, Tietz syndrome, oculocutaneous albinism, seborrheic dermatitis, and atopic dermatitis, dryness, drops Hypothermia caused by guttate parapsoriasis, rosacea, and erythema.

In some embodiments, the abnormality of the pigmentation after treatment results in a return to the original pigmentation. As used herein, the term "recovering the original pigmentation" refers to an increase in the amount of pigmentation in the skin of any of the aforementioned pigmentation abnormalities or conditions. For conditions such as leukoplakia, the amount of pigmentation in the skin area of the non-pigmented precipitate can be increased after the treatment is applied. However, pigmentation in this area does not necessarily return completely to the same pigmentation as normal skin around it.

In some embodiments, the method is used to perform a tanning. The term "fake" refers to the appearance of sunburn without subjecting the subject to UV exposure (whether UV is natural or derived from a tanning machine). The method can comprise topically applying a composition comprising a dose of SEQ ID NO: 1 to increase the amount of melanin produced in the topical site.

In certain embodiments, the method comprises contacting a composition having the effect and comprising SEQ ID NO: 1 with a hair follicle to modulate the color development of the subject, increasing the concentration of the pigment that is new from the hair follicle. In some embodiments, the hair follicle is part of a scalp, eyebrow or lash area.

In certain embodiments, the method comprises treating or ameliorating a condition of a condition of the skin comprising administering to the subject a composition comprising an effective amount of a peptide having the amino acid sequence of SEQ ID NO: 1, The skin condition is dermabraison, chemical peel, prostaglandin treatment for treating eyes or eyelashes, intralesional steroid therapy, dryness, acne, The result of rose spots, uneven skin tone, atopic dermatitis or skin expansion. In certain embodiments, the prostaglandin derivative therapy is administered bimatoprost, tafluprost, travoprost or latanoprost. In certain embodiments, the condition of the skin condition is inflammation.

In any of the methods disclosed herein, the composition can be applied to a target area. "Target area" means the area on the skin or hair follicle where the desired cosmetic or therapeutic effect occurs. For example, the target area can include a reduced or abnormal skin area of the same subject relative to other skin areas. In another example, the target area may comprise hair follicles with low or no pigmentation, i.e., where silver or white hair is grown. The target area can include sub-areas of the subject's skin, eye area, or other location.

The compositions disclosed herein can be administered by topical, transdermal, injection, microneedle injection, mesotherapy, nanotechnology, or any combination of the foregoing. Upon treatment or administration of the composition, the amount of pigmentation of the target area is significantly increased as compared to the skin of the subject prior to or after administration of the composition. In some embodiments, the composition is administered continuously for a sufficient period of time to allow the desired effect to be detected. Long enough to include, for example, 1-24 hours, 1-2 days, 3-4 days, 5-6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer. In certain embodiments, the composition is administered one, two, three or more times daily.

In certain embodiments, the methods and compositions disclosed herein can be practiced in conjunction with other therapies, including but not limited to calcineurin inhibitors, oxosarin, methoxalin, topical all-trans yellow Acids, topical steroids or immunomodulators and immunomodifiers, topical and oral psoralen and psoralen UVA range (PUVA) therapy, narrow-band UVB therapy, decolorization therapy and surgical transplantation techniques.

The highly polymorphic MC1R gene has a key influence on the amount of pigmentation. Variants of this gene have been shown to induce squamous cell carcinoma of the skin (SCC). Among the more than 100 genes that affect pigmentation, the key message-regulating gene MC1R , its antagonist ASIP, and the downstream melanin-regulated genes TYR and TYRP1 are the most thoroughly studied genes in skin cancer-related research. SEQ ID NO: 1 significantly induced the expression of the MC1R gene and increased the expression of MITF. One specific embodiment herein is directed to compositions using the above peptides. Uses of the peptide include, but are not limited to, inducing melanin production to prevent and treat skin cancer. The method is generally such that the peptide is contacted with human skin, and the contacting step can be by in vivo, in vitro, external, oral, transdermal, systemic, or any other manner known to those of ordinary skill in the art to which the present invention pertains. get on.

White spot is a skin abnormality characterized by fading plaques that are prone to progressive deterioration. The report pointed out that the disease has high family aggregation. 1% of the world's population suffer from leukoplakia, and it occurs in the ethnic group of colored people. At present, the causes of leukoplakia are different, including neurochemical theory, autoimmune theory, oxidative stress related theory and gene defect theory. White spot syndrome is a complex hereditary disease that is regulated by a set of recessive alleles located at several different chromosomal locations and may affect oxidative stress generation, melanin production, autoimmune function, and collectively cause leukoplakia phenotypes. . If it induces melanin production and produces melanin in the skin, it should be effective in treating leukoplakia and improving fading plaque so that the rickets can return to pigmentation. Accordingly, another specific embodiment herein is directed to a composition for preventing and treating skin hypopigmentation using the above peptides. The method generally involves contacting the peptide with human skin, and the contacting step can be by in vivo, in vitro, external, oral, transdermal, systemic, or any other manner known to those of ordinary skill in the art to which the present invention pertains. get on.

In terms of beauty, skin pigmentation can have a great impact. Some population groups prefer tanned skin. Since the 1960s, the activity of tanning skin has become popular, while the skin of bronze has been widely accepted by young women. According to the American Academy of Dermatology, 80% of tanning skin activity is carried out through sunbathing and 30% by sun dryer. Even if the positive publicity of various sun protection factors is on the rise, the market is also full of various sunscreen products, and the trend of tanning skin still leads to an increasing incidence of skin cancer. Inducing melanin production to improve skin pigmentation, in theory, should extend the tanning effect while avoiding exposure to sunlight for too long. Thus, another specific embodiment herein is directed to a composition for use in the above-described peptides for soaking. The method is generally such that the peptide is contacted with human skin, and the contacting step can be by in vivo, in vitro, external, oral, transdermal, systemic, or any other manner known to those of ordinary skill in the art to which the present invention pertains. get on.

Hypopigmentation is also a common skin condition that can result from skin diseases, illnesses, burns, trauma, and trauma. This condition is commonly referred to as skin loss, also known as skin hypopigmentation. Although everyone may suffer from this disease, the disease occurs in the dark-skinned ethnic group. The most common cause of hypopigmentation is skin trauma. Acne, blisters, chickenpox, scratches, and even inappropriate dermatological treatments (such as laser resurfacing, chemical peels, and other therapies) can cause hypopigmentation. Other causes of hypopigmentation include albino (hair, skin and eyes due to skin cells that produce little or no melanin and lack of color) and liposuction dermatitis (an inflammatory skin disease that occurs on the surface of the patient's skin) Itchy tingles, red plaques that produce scaly, and the skin of the affected area is also prone to oil.) Another specific embodiment herein is directed to a composition for treating skin tone unevenness caused by hypopigmentation using the above peptide. The method is generally such that the peptide is contacted with human skin, and the contacting step can be by in vivo, in vitro, external, oral, transdermal, systemic, or any other manner known to those of ordinary skill in the art to which the present invention pertains. get on.

In social activities, people immediately notice the color of other people, while white hair is a symbol of old age, poor health, and physical decline. Conditions that affect hair color include aging, achromotricha, stress, medical conditions, and non-natural factors. People are always eager to keep young and energetic, so researchers pay special attention to juvenile white phenomenon. At the same time, the pharmaceutical or health food industry is also developing means to prevent and treat white hair. Hair color is affected not only by genes, age, environment, etc., but also by many cytokines and proteins. Human hair color is also regulated by MC1R and melanin production. The study found that up-regulating MC1R performance was one of the main targets for the treatment of white occurrence with Polygonum Multiflori Radix. Thus, another specific embodiment herein is directed to a composition for preventing and treating whitening using the above peptides. The method generally involves contacting the peptide with a human scalp and a hair follicle. The contacting step can be by in vivo, in vitro, external, oral, transdermal, systemic circulation, or any of the ordinary knowledge of those skilled in the art to which the present invention pertains. Other ways to proceed.

SEQ ID NO: 1 significantly promotes wound healing as shown by the keratinocytes scratch wound test. The keratinocyte scratch test is a test that can be used to measure the ability of an active compound to promote wound healing. As shown in Table 4, SEQ ID NO: 1 promotes wound healing with a promotion rate of 226% of phosphate buffered saline (PBS), the best example being a diabetic wound or a wound associated with pressure sore. . These types of wounds are inflamed, prone to infection, and take a long time to recover, so they are usually quite tricky. Part of the healing step of a skin or mucosal wound involves activation of basal keratinocytes. Upon activation, the basal keratinocytes at the edge of the wound move and form a single layer covering the wound, the so-called epithelialization process. Studies have shown that keratinocytes at the edge of the unhealed wound of chronic wounds will proliferate a lot, but will not move to other places, so they will not be epithelialized and will become the main cause of chronic ulcers. The peptide disclosed herein can also be used to treat damage associated with keratinocytes in the skin and mucosa. The term "related mucosal tissue" is used to refer to any tissue that is structurally similar to the skin and that includes epithelial cells/keratinocytes, including but not limited to the inner skin surface associated with the mouth, nose, throat, ear, genitals, genitals, and palpebral conjunctiva. . Examples of wounds or lesions/injuries that may affect the aforementioned tissue and that can be treated with the peptide of the present invention are abrasion, blisters, burns, lacerations, puncture wounds, ulcers, blood stasis, rashes and scars. Post-operative wounds can also be treated with these peptides.

Skin aging is accumulated over a long period of time and eventually reduces skin function. There are two main ways of skin aging: endogenous aging (chronic aging) of skin that is not exposed to sunlight, and exogenous aging (photoaging) of sunlight exposed to the skin. Endogenous aging is genetically related and the extent varies over time. In any case, aged skin is associated with one or more of the following characteristics: wrinkles, fine lines, increased pigmentation, spots, erythema, tarnish, smoothness, toughness, skin tone brightness and flatness, and changes in pore appearance . In addition to the above obvious features, the less obvious features are due to genetic inheritance, or pathological and cellular changes caused by acute or chronic exposure to external environmental stimuli such as ultraviolet light or contaminants. Cosmetic problems such as wrinkles, dry skin, thinning, sagging, or prone to blood stasis are all obvious features of epidermal damage; the above characteristics may be caused by exposure to harmful stimuli (such as ultraviolet rays and pollutants). Long appeared early. Therefore, the peptide disclosed herein can be directed to skin aging due to endogenous and exogenous stimuli to prevent and repair skin damage, and to regenerate healthy skin tissue, thereby reversing the aging condition. In a similar situation, the peptides can be used on tissues that are damaged by exposure to various external stimuli such as sunlight. Under the above circumstances, the peptide disclosed in the present invention can also be used as a cosmetic product to rejuvenate the appearance and skin texture. Unmodified or chemically modified and/or specially transported short peptides can penetrate the epidermis after treatment to enhance the effect of resisting skin thinning, wrinkles, fragility and thickening/hardening. Since the most important component of the keratinocyte epidermis surface will disappear due to aging or damage of the skin, it is expected that the keratinocytes can be supplemented by peptide stimulation to counteract the above conditions.

SEQ ID NO: 1 significantly increased the expression levels of the filaggrin gene (1.92 fold), the aquaporin 5 gene (1.82 fold), and the KLK8 gene (1.71 fold), as shown in Table 3. The aforementioned genes have a critical impact on epidermal integrity and constancy.

The skin is a relatively elastic tissue, but its extent is still limited. Stretch mark/striae is a kind of discolored scar on the skin, usually associated with an inflammatory reaction caused by tearing of the skin, which fades over time but does not completely disappear. The initial appearance is characterized by reddish or purple lines, but gradually fades to a smaller extent. Skin stretch marks may occur in areas where the body does not exhibit significant or excessive pull or swelling. Repairing and restoring the function of keratinocytes in the skin/skin and reducing the inflammatory response is the key to eliminating skin expansion. The peptide disclosed herein promotes scratch healing and increases anti-inflammatory activity and should be an ideal treatment for improving skin expansion.

SEQ ID NO: 1 significantly reduces the amount of expression of genes associated with the pro-inflammatory pathway and is therefore suitable for treating skin inflammatory conditions. Inflammation is one of the characteristics of chronic wounds. Cognac is a chronic inflammatory skin disease that affects approximately 2% of the world's population. At the onset of dryness, an inflammatory cytokine that induces excessive proliferation of keratinocytes is apparent. The study found that SEQ ID NO: 1 significantly reduced the expression of TNF-α and IL8, as well as the performance of several other factors associated with inflammatory responses, suggesting that the peptide in this case can be used to treat inflammatory conditions. Inflammatory conditions include, but are not limited to, dryness, systemic lupus erythematosus, and rheumatoid arthritis.

Rose plaque is another inflammatory condition that occurs in adults. Current theoretical studies suggest that clinically induced factors of rose plaque include the modulation of Toll-like receptor signaling or the induction of active oxides and inflammatory cytokines by UV radiation, heat, cold, stress, and pungency. Food and microbes. The use of peptides in this case to reduce the amount of inflammatory cytokines can ensure significant and practical efficacy, improve the inflammatory condition of rose spot disease.

Instance

Example 1: Inducing melanin expression of B16F10 melanocytes

Determination of Effect of peptides on the cell growth and the amount of melanin production using Sunny Biodiscovery ^TM. Briefly, the assay was performed by culturing neonatal dermal fibroblasts (p. 8, Cell Applications, San Diego, CA) and B16F10 melanocytes (ATCC, Manassas, VA) in a 96-well culture dish. The wells were cultured for 5,000 cells in DMEM/5% FBS. After one day of culture, test substances at a concentration of 200 μg/mL and a concentration of 50 μg/mL were added to the exponentially growing cell population, three replicates in each group. After 8 days, cell growth was quantified by the sulforhodamine B method (Voigt, 2005) and the Molecular Devices MAX190 microplate spectrophotometer. In addition, the coloring of the medium in the B16 culture was quantified at 490 nm (proportional to the amount of melanin secreted), and the results are shown in Table 1. Results with a difference of ≥ 25% and p < 0.05 (using a two-tailed Student's test) were considered statistically significant.

Table 1: Melanin performance after 8 days of addition of various peptide reactions

^* : P < 0.005 means significant, in bold.

Example 2: Effect of peptide on cell growth

After each of the peptides was added for reaction, cell viability tests were performed on all kinds of peptides to confirm that the melanin production-inducing effect of melanin P131 on B16F10 melanocytes was not based on cell proliferation or inhibition. As shown in Table 2, all of the peptides did not significantly exhibit inhibition of B16F10 melanocytes and human neonatal dermal fibroblasts.

Table 2: Effect of various peptides on the growth of B16F10 melanocytes and neonatal skin fibroblasts (nHF)

^* : p<0.05 means significant

Example 3: Gene expression analysis of skin cultures reacted with P131

This experiment was performed by Genemarkers LLC (Kalamazoo, MI) using a full thickness in vitro skin culture model (ETF-400, MatTek). P131 was dissolved in sterile water and (sterile) water itself was used as a vehicle control group. One hundred microliters of test substance (equivalent to 500 μg per tissue) was placed in the center of each EFT-400 culture. The test substance was dispensed using a sterile glass applicator stick. Each culture was visually inspected to confirm that the amount of distribution was uniform. After the test substance was dispensed, the culture was placed in an incubator and incubated at 37 ° C for 24 hours in 5% CO ₂ . The test substance on the surface of each culture was washed away with sterile PBS. After removing the test substance, each tissue was cut into four portions to prepare for RNA isolation. RNA was isolated from tissues using the Maxwell 16 LEV simply RNA Tissue kit according to the manufacturer's instructions (Promega) and RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer. cDNA was generated using the High Capacity cDNA Synthesis kit according to the manufacturer's instructions (Life Technologies). Gene expression was analyzed using the qPCR-based panel, Genemarkers Standard Skin Panel, which measures 107 target genes and 5 endogenous control genes, each of which is assayed. Two repetitions. Unpaired t assays (P < 0.05, N = 4) were performed using StatMiner software to compare the differences between the P131 group and the vehicle control group.

The purpose of this experiment was to clarify how gene expression in the skin would be affected when externally applied with P131. The results are shown in Table 3. P131 affects several major pathways associated with skin health. The first path that will be affected is melanin generation. Studies have shown that this peptide significantly increases (more than 4 times) the amount of MC1R (melanocortin receptor) gene expression. At the same time, the gene expression of MITF (small eye deformity-related transcription factor) is nearly doubled. Both MC1R and MITF are the major regulators of melanin production. The data in Table 3 is complementary to the experimental results in Table 1; in other words, P131 upregulates the melanin content of B16F10 melanocytes, and the increase is dose dependent.

Table 3: Linear fold change values for genes with statistical significance

Example 4: Promoting keratinocyte movement and scratch healing

In serum-free keratinocyte growth medium cultured human skin keratinocytes (ATCC CRL-2404), and was added 5 ng / ml of human recombinant epithelial growth factor ^{(Life Technologies TM, Grand Island,} NY). The keratinocytes were seeded into a 12-well culture plate and allowed to reach 100% confluent. Monolayer cells were starved for 24 hours and scratched on a single layer using the tip of a P200 (200 μl) dropper. The scratch was cleaned and the wound was taken at time 0, and a peptide with a final concentration of 40 μg/ml was added. The cells were placed in an incubator at 37 ° C, 5% CO ₂ , and humidity greater than 90%, and were taken into the incubator only when the cell photographs were taken briefly at room temperature. After 7-8 hours of treatment, the wounds healed and the results are shown in Table 4. Compared with the control group treated with PBS, all three peptides can significantly promote keratinocyte movement and wound healing.

Table 4: Healing conditions after cultured keratinocytes were scratched. After 7 hours of treatment, the degree of wound healing treated with PBS was set to 100%, and the degree of wound healing treated with peptide was calculated in comparison with the former.

The various specific embodiments described above may be combined to each other to provide more specific embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent disclosures contained in the specification and/or application data sheets are hereby incorporated by reference in their entirety. In addition, the cited literature or one of the documents is excluded from the content disclosed in the present invention. The present application also claims priority to U.S. Provisional Patent Application Serial No. 62/592, 724, filed on Jan. Various aspects of the foregoing specific embodiments may be modified to implement the concepts in the various patents, applications, and disclosures described above, in order to provide more specific embodiments.

The above and other changes can be made to the specific embodiments of the present invention in light of the foregoing description. In general, the words used in the following claims are not to be interpreted as limiting the scope of the claims to the specific embodiments in the specification, but are to be construed as including all possible specific embodiments and The scope is equivalent to all ranges. Therefore, the following claims are not limited by the disclosure herein.

no

Claims (29)

A method for increasing melanin production, comprising A composition is administered to a subject comprising an effective amount of one peptide consisting of the amino acid sequence KWKLF (SEQ ID NO: 1). The method of claim 1, wherein the peptide is a monoamidated or free acid sequence; the N-terminus is lipidated or acetylated; the peptide is a soluble or insoluble carrier. Conjugation; or any combination of the foregoing. The method of claim 1 or 2, wherein the melanin is pheomelanin, eumelanin, neuromelanin or any combination of the foregoing. The method of any one of claims 1 to 3, wherein the method is for treating a pigmentation abnormality or condition. The method of claim 4, wherein the pigmentation abnormality or condition comprises reduced or inhibited melanin production. The method of claim 4, wherein the abnormal pigmentation or condition is vitiligo, albino, white pityriasis alba, tinea versicolor, post-inflammatory pigment Postinflammatory hypomelanosis, hypopigmented mycosis fungoides, tuberculoid leprosy, hypomelanosis of Ito, naevus anaemicus, ocular albino 1 4 (OCA 1-4), Prader-Will & Angelman syndrome, Hermansky-Pudlak syndrome, Tietz syndrome, oculocutaneous albinism, or by liposuction dermatitis ( Seborrheic dermatitis), atopic dermatitis, psoriasis, guttate parapsoriasis, rosacea, erythema-induced hypopigmentation or hypopigmentation ( Depigmentation), or any combination of the foregoing. The method of any one of claims 1 to 3, wherein the method is for tanning. The method of any one of claims 1 to 3, wherein the method comprises contacting the composition with a hair follicle to adjust color development, thereby increasing a new pigment concentration emanating from the hair follicle . The method of claim 8, wherein the hair follicle is part of a scalp, eyebrow or lash area. A method of treating or ameliorating one of the symptoms of a skin condition comprising administering to a subject a composition comprising an effective amount of a peptide having the amino acid sequence of SEQ ID NO: 1, wherein The skin condition is dermabraison, chemical peel, prostaglandin treatment for the treatment of the eye or eyelashes, intralesional steroid therapy, or by dry acne, acne, Caused by hypopigmentation or hypopigmentation caused by rose spots, uneven skin tone, atopic dermatitis or skin expansion marks. The method of claim 9, wherein the prostaglandin derivative therapy is administered bimatoprost, tafluprost, travoprost or latanoprost ( Latanoprost). A method according to any one of the preceding claims, wherein the composition is applied to a target by topical application, transdermal, injection, microneedle injection, mesotherapy, nanotechnology or any combination of the foregoing. region. The method of claim 12, wherein the target area is a sub-area of the subject's skin, eye area or other part. The method of claim 12, wherein the amount of pigmentation of the target area is significantly increased as compared to the skin of the subject prior to or after administration of the composition. A cosmetic composition comprising: a) a peptide consisting of the amino acid sequence SEQ ID NO: 1; b) a carrier; and c) A second active agent which is a tanning agent or hair dye. The cosmetic composition of claim 15, wherein the tanning agent comprises dihydroxyacetone, erythrulose, canthaxanthin, or any combination of the foregoing. The cosmetic composition of claim 15, wherein the composition prevents white growth or restores natural coloration, and the hair dye comprises methionine, cysteine,蓟, coconut oil, jojoba oil, muster seed oil, castor oil, pantothenic acid, biotin, vitamin B6, copper, zinc or the foregoing Any combination. A pharmaceutical composition comprising: a) a peptide consisting of the amino acid sequence SEQ ID NO: 1; b) a carrier; and c) a penetration enhancer, a broad-acting sunscreen lotion or a second active agent for treating or ameliorating symptoms associated with abnormalities or conditions of pigmentation. The composition of claim 18, wherein the pigmentation abnormality or condition is leukoplakia, albinism, white pityriasis, sweat stains, hypopigmentation after hypopigmentation, hypogranulomatous granuloma, tuberculous leprosy , Ito pigmentation, anemia, eye whitening 1-4, chubby Wiley / Angel syndrome, Hermansky-Pudlak syndrome, Tietz syndrome, ocular albino, or by liposuction dermatitis, atopic skin Hypopigmentation or hypopigmentation caused by inflammation, dryness, drip-like dryness, rose spots or erythema. The composition of claim 18, wherein the pharmaceutical composition comprises the second active agent, and the second active agent is for treating or ameliorating symptoms associated with leukoplakia, and comprises a Calcineurin inhibitor, oxsoralen, methoxsalen, all-trans-retinoic acid, psoralens or the foregoing Any combination. The composition of claim 18, wherein the pharmaceutical composition comprises the second active agent, and the second active agent is for treating or ameliorating symptoms associated with hypopigmentation, and includes Trans retinoic acid, psoralen or any combination of the foregoing. The composition of any one of clauses 15 to 21, wherein the concentration of the peptide ranges from 0.01 μg/ml to about 50 mg/ml, based on the weight of the composition. The composition of any one of claims 15 to 22, wherein the composition is a gel, a solution, a spray, an emulsifier, an emulsion, a mousse, a spray, a wet wipe, a microcapsule, Essence, cream, ointment, powder or foam. The composition according to any one of claims 15 to 23, further comprising a sunscreen agent, a skin conditioner, a sunscreen agent, a skin lightening agent, a skin protectant, a moisturizer, and a thickening Agent, excipient, humectant or any combination of the foregoing. The composition of any one of claims 15 to 24, wherein the peptide is coated in a liposome or microsponge. The composition according to any one of claims 15 to 25, wherein the peptide is formulated in a device, and the peptide is transported by iontophoresis or ultrasonic wave through the device. . The composition according to any one of claims 15 to 26, further comprising a fatty alcohol, a fatty acid, an organic base, an inorganic base, a preservative, an ester wax, a steroid, a triglyceride, a phospholipid, A polyol ester, a fatty alcohol ether, a hydrophilic lanolin derivative, a hydrophilic beeswax derivative, a cocoa butter wax, an emu oil, a pH balance agent, a cellulose derivative, a hydrocarbon oil or any mixture of the foregoing. The composition of any one of claims 15 to 27, wherein the composition is integrated with a device for applying the composition to the underlying skin tissue, mucous tissue, skin surface or scalp . The composition of any one of claims 15 to 28, wherein the peptide is a monoamidated or free acid sequence; the N-terminus is lipidated or acetylated; the peptide Conjugated with a soluble or insoluble carrier; or any combination of the foregoing.