Anti-aging compositions and methods of use thereof

Abstract

Compositions containing specific tetrapeptides are provided for use in treating, preventing, minimizing, reducing, or reversing various signs of skin aging. The compositions are useful for improving skin firmness or elasticity, smoothing fine lines or wrinkles, reducing skin pores and hyperpigmentation, and increasing skin thickness, radiance, and / or softness.

Description

[Technical field]

[0001] The present invention relates to compositions useful for providing improvements in the appearance and texture of skin, and methods of their use. In particular, the compositions are useful for treating, preventing, minimizing, reducing, or reversing various signs of skin aging. [Background technology]

[0002] Skin aging is characterized by features such as wrinkles, loss of volume and elasticity, fragility and increased pigmentation. Although it is a natural phenomenon that occurs with age, there are clinically proven environmental factors that contribute to skin aging, such as exposure to sunlight and various toxins. The most common treatments for skin aging include over-the-counter topical creams and moisturizers. These typically contain fatty acids and alcohols, various vitamins and vitamin derivatives, fillers such as collagen and hyaluronic acid, as well as botanical extracts.

[0003] US 4,619,916 describes 13 tripeptides made from L-amino acids corresponding to the formula p-GLU-X-TRP, where X is a specific amino acid different from p-GLU and Trp, as well as methods for their preparation, pharmaceutical formulations containing them, and their use as antihypertensive and analgesic agents. The tripeptides are designed for systemic administration.

[0004] US 7,220,725 and WO 2002 / 012269 describe novel peptides comprising pGlu-Asn-Trp-Lys(octanoyl)-OH (ZEP3) and pGlu-Asn-Trp-Thr-OH (ZEP4), as well as pharmaceutical compositions comprising an analgesically effective amount of the peptides for topical administration in the treatment of pain.

[0005] US9,012,397 and WO2012 / 131676 describe topical pharmaceutical compositions comprising peptides ZEP3 or ZEP4 and their use for treating skin disorders selected from the group consisting of herpes virus infections, chickenpox virus infections, rashes, insect bites, jellyfish stings, burns, psoriasis, itch, skin allergic reactions, skin lesions as a result of side effects or complications of drugs or medical treatments, and hypopigmentation.

[0006] Gaynes et al. (Invest. Ophthalmol. Vis. Sci. 54, E-Abstract 5416, 2013) describe the analgesic effect of the peptide ZEP4 in reducing ocular pain and altering nociceptive pathways in a rat model of experimentally induced chemical corneal injury.

[0007] WO2019 / 186561 describes pharmaceutical compositions comprising certain tetrapeptides for use in reducing the release of or inhibiting the activity of inflammatory cytokines and mediators, and the treatment of diseases and disorders related thereto.

[0008] There is an unmet need for compositions and methods useful for treating symptoms associated with natural or premature aging. Summary of the Invention

[0009] The present invention provides compositions useful for treating, preventing, minimizing, reducing, or reversing epidermal conditions associated with aging. Improvements in at least one of skin firmness or elasticity, smoothing fine lines or wrinkles, shrinking skin pores, reducing hyperpigmentation, and increasing skin thickness, radiance, and / or softness are within the scope of the present invention.

[0010] The present invention is based, in part, on the unexpected discovery that peptides designated ZEP3 and ZEP4, or salts thereof, attenuate the formation of reactive oxygen species (ROS) and enhance the survival of epidermal cells under stress conditions.

[0011] According to one aspect of the present invention, there is provided a composition for use in treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging, comprising as an active ingredient a compound of formula I: pGlu-X 1 -X 2 -X 3 -OH(wherein, X 1 is a polar amino acid residue, and X 2 is an aromatic or hydrophobic amino acid residue, and X 3 is a positively charged or polar amino acid residue), or a salt or derivative thereof, and a dermatologically acceptable carrier.

[0012] According to another aspect of the present invention, there is provided a method for treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging in a subject in need thereof, comprising administering to said subject a compound of formula I: pGlu-X as an active ingredient. 1 -X 2 -X 3 -OH (wherein X1 is a polar amino acid residue, and X 2 is an aromatic or hydrophobic amino acid residue, and X 3 is a positively charged or polar amino acid residue), or a salt or derivative thereof, and a dermatologically acceptable carrier.

[0013] According to some embodiments, X 1 is selected from the group consisting of Asn, Gln, His, Ser, Thr, Tyr and Cys; 2 is selected from the group consisting of Trp, Phe, Tyr, Ala, Ile, Leu, Met, Val, and Gly; 3 is selected from the group consisting of Lys, Lys derivatives, Arg, His, Asn, Gln, Ser, Thr, and Tyr. Each possibility represents a separate embodiment.

[0014] According to another embodiment, X 1 is selected from the group consisting of Asn and Thr, 2 is selected from the group consisting of Trp, Phe and Tyr, and X 3 is selected from the group consisting of Lys, a Lys derivative, and Thr. Each possibility represents a separate embodiment.

[0015] According to various embodiments, peptide derivatives include peptides of Formula I having alkyl groups attached to free functional groups of the peptide sequence.

[0016] According to yet other embodiments, the alkyl group is attached to a free amino group at the side chain or N-terminus of the peptide by an amide bond or linkage.

[0017] According to some embodiments, alkyl is C 4 ~C 30 It is an alkyl.

[0018] According to some particular embodiments, C 8 The alkyl group (herein octanoyl) is attached by an amide linkage to the terminal amino group of the peptide or to the side chain of a Lys residue in the peptide sequence. According to the latter embodiment, the Lys derivative is Lys(octanoyl).

[0019] According to some embodiments, the C-terminus of the peptide is modified to form an amide, alcohol, or ester terminus, each possibility representing a separate embodiment.

[0020] According to a particular embodiment, the peptide of formula I or a salt or derivative thereof is selected from the group consisting of pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1), pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2) and salts thereof.

[0021] According to some embodiments, the peptide of Formula I, or a salt or derivative thereof, has the amino acid sequence set forth in SEQ ID NO:1, or a salt thereof.

[0022] According to another embodiment, the peptide of formula I, or a salt or derivative thereof, has the amino acid sequence set forth in SEQ ID NO:2, or a salt thereof.

[0023] According to various embodiments, the composition comprises the sodium salt of a peptide of formula I, or a derivative thereof, having a sequence as set forth in any one of SEQ ID NO: 1 and SEQ ID NO: 2. Each possibility represents a separate embodiment.

[0024] According to certain embodiments, the composition comprises about 0.1% to about 5% w / w of a peptide of formula I or a salt or derivative thereof, with each value falling within the range specified. According to certain embodiments, the composition comprises about 0.5% to about 2% w / w of a peptide of formula I or a salt or derivative thereof, with each value falling within the range specified.

[0025] In some embodiments, the compositions are formulated for topical or intradermal administration.

[0026] In other embodiments, the composition is in a form selected from the group consisting of an oil, a gel, a stick, a lotion, a cream, a milk, an aerosol, a spray, a foam, a mousse, an ointment, a drop, an atomized liquid, a liquid wash, an emulsion, a suspension, a liposome, an adhesive patch, and a powder. Each possibility represents a separate embodiment. In exemplary embodiments, the composition is in the form of a gel, an ointment, a cream, or an emulsion. In one embodiment, the composition is in the form of an emulgel.

[0027] In certain embodiments, the dermatologically acceptable carrier comprises at least one of a thickening agent, a filler, a humectant, an emulsifier, a humectant, a surfactant, a buffering or pH adjusting agent, a film former, a foaming agent, an antifoaming agent, a preservative, an antioxidant, a fragrance, a solvent, a propellant, a colorant, and combinations or mixtures thereof. Each possibility represents a separate embodiment.

[0028] According to certain embodiments, the aging-associated epidermal condition is selected from the group consisting of elastosis, skin atrophy, fine lines, wrinkles, enlarged pores, hyperpigmentation, sagging skin, rough skin, and dry skin, with each possibility representing a separate embodiment.

[0029] In further embodiments, the aging-associated epidermal condition is selected from the group consisting of elastosis, skin atrophy, fine lines, wrinkles, enlarged pores, sagging skin, rough skin, and dry skin. Each possibility represents a separate embodiment.

[0030] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, shall prevail. Moreover, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention relates to compositions comprising certain peptides and their salts or derivatives, which are useful for treating, preventing, minimizing, reducing or reversing epidermal conditions associated with aging.

[0032] The principles and operation of the present invention may be better understood with reference to the accompanying description. Before describing at least one embodiment of the present invention in detail, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The present invention is capable of other embodiments or can be practiced or carried out in various ways. It should also be understood that the phraseology or terminology used herein is for the purpose of description and should not be construed as limiting.

[0033] The present invention is based in part on the surprising discovery that peptides ZEP3 and ZEP4, and their sodium salt forms, reduce the formation of reactive oxygen species (ROS) and increase the viability of epidermal cells under stress conditions (70 mM NaCl). Reactive oxygen species (ROS) can cause significant damage to cells by oxidizing lipids, proteins, carbohydrates, and DNA in cells and tissues. This unwanted oxidation results in membrane damage, protein modifications, and DNA damage that can cause cell and tissue death. The reduction in the formation of ROS and the increase in the viability of epidermal cells under stress conditions using the peptides of the present invention indicates that the peptides claimed herein provide beneficial cosmetic effects in providing at least one of the following properties: improved skin firmness or elasticity, smoothing fine lines and wrinkles, shrinking skin pores, reducing hyperpigmentation, increasing skin thickness, radiance, softness, and reducing dry skin. Each possibility represents a separate embodiment.

[0034] According to certain aspects and embodiments, a composition for use in treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging is provided, the composition comprising a peptide of formula I or a salt or derivative thereof as an active ingredient and a dermatologically acceptable carrier. According to other aspects and embodiments, a method for treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging in a subject in need of such treatment, comprises administering to the subject a composition comprising a peptide of formula I or a salt or derivative thereof as an active ingredient and a dermatologically acceptable carrier. According to further aspects and embodiments, a use of a peptide of formula I or a salt or derivative thereof and a dermatologically acceptable carrier is provided for the preparation of a composition for treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging in a subject in need of such treatment, prevention, minimizing, reducing or reversing an epidermal condition associated with aging.

[0035] According to various aspects and embodiments, the peptide of formula I has the following structure: pGlu-X 1 -X 2 -X 3 -OH (wherein pGlu is pyroglutamic acid; X 1 is a polar amino acid residue, and X 2 is an aromatic or hydrophobic amino acid residue, and X 3 is a positively charged or polar amino acid residue. It is understood that the -OH in formula I represents the natural carboxy terminus.

[0036] As used herein, the term "polar amino acid residue" refers to the following amino acids: asparagine (Asn; N), glutamine (Gln; Q), histidine (His; H), serine (Ser; S), threonine (Thr; T), tyrosine (Tyr; Y), and cysteine ​​(Cys; C). Each possibility represents a separate embodiment.

[0037] As used herein, the term "aromatic amino acid residue" refers to the following amino acids: tryptophan (Trp; W), and tyrosine (Tyr; Y). Each possibility represents a separate embodiment.

[0038] As used herein, the term "hydrophobic amino acid residue" refers to the following amino acids: Alanine (Ala; A), Isoleucine (Ile; I), Leucine (Leu; L), Methionine (Met; M), Phenylalanine (Phe; F), Valine (Val; V), and Glycine (Gly; G). Each possibility represents a separate embodiment.

[0039] As used herein, the term "positively charged amino acid residue" refers to the following amino acids: lysine (Lys; K) and its derivatives, arginine (Arg; R), and histidine (His; H). Each possibility represents a separate embodiment.

[0040] The amino acid residues of the present invention are intended to include both D- and L-amino acids, preferably L-amino acids.

[0041] In a further embodiment of the invention, X 1 is selected from the group consisting of Asn and Thr. Each possibility represents a separate embodiment.

[0042] In another embodiment of the present invention, X 2 is selected from the group consisting of Trp, Phe, and Tyr. Each possibility represents a separate embodiment.

[0043] In yet another embodiment of the present invention, X 3 is selected from the group consisting of Lys, a Lys derivative, and Thr. Each possibility represents a separate embodiment.

[0044] In a further embodiment of the invention, X 1 is selected from the group consisting of Asn and Thr, 2 is selected from the group consisting of Trp, Phe, and Tyr; 3 is selected from the group consisting of Lys, a Lys derivative, and Thr. Each possibility represents a separate embodiment.

[0045] Salts and derivatives of the peptides used in the disclosed compositions and methods are also included within the scope of the invention.

[0046] As used herein, the term "salt" refers to salts of carboxyl groups, also named base addition salts of peptide molecules, and acid addition salts of amino or guanidino groups. Suitable base addition salts include, but are not limited to, metal salts of sodium, calcium, lithium, magnesium, potassium, aluminum, ferric and zinc; ammonium salts derived from ammonia, primary amines, secondary amines, tertiary amines and quaternary amines (non-limiting examples of which are trimethylamine, cyclohexylamine, benzylamine, dibenzylamine, 2-hydroxyethylamine, bis(2-hydroxyethyl)amine, phenylethylbenzylamine, dibenzylethylenediamine, procaine, chloroprocaine, piperidine, monoethanolamine, triethanolamine, quinine, choline and N-methylglucosamine). Each possibility represents a separate embodiment. Salts with amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine are contemplated. Each possibility represents a separate embodiment. Additionally, any zwitterionic salts formed with carboxylic acids and amino or guanidino groups on the peptide molecule are also contemplated.

[0047] Suitable acid addition salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and salts derived from organic acids such as acetic acid or oxalic acid, aliphatic mono- and dicarboxylic acids, including phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic acids, and aromatic sulfonic acids, etc. Each possibility represents a separate embodiment. Thus, salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Each possibility represents a separate embodiment. Salts of amino acids such as arginate and gluconate or galacturonate are also contemplated. Each possibility represents a separate embodiment.

[0048] Acid addition salts can be prepared by methods known in the art in which the free base form is contacted with a sufficient amount of the desired acid to produce the salt, and base addition salts are prepared by methods known in the art in which the free acid form is contacted with a sufficient amount of the desired base to produce the salt.

[0049] As used herein, "derivatives" of the peptides of the invention encompass derivatives that may be prepared from functional groups occurring as side chains of the residues or N- or C-terminal groups by means known in the art, and are included in the present invention so long as they remain pharma- ceutically acceptable, i.e., they do not adversely affect the benefits of the peptide and do not impart toxicity to compositions containing it.

[0050] These derivatives can include, for example, aliphatic esters of the carboxyl group, amides of the carboxyl group produced by reaction with ammonia or a primary or secondary amine, N-acyl derivatives of the free amino group of an amino acid residue formed by reaction with an acyl moiety (e.g., an alkanoyl or aroyl group), or O-acyl derivatives of the free hydroxyl group (e.g., of a seryl or threonyl residue) formed by reaction with an acyl moiety.

[0051] According to a particular embodiment of the invention, the peptide derivative comprises an alkyl group attached to a free functional group of the peptide sequence.

[0052] According to certain embodiments, the alkyl group is attached to the side chain or to the free amino group at the N-terminus of the peptide by an amide bond or linkage. 4 -C 30 C, which may be alkyl and is attached by an amide linkage to the side chain of a Lys residue in the peptide sequence (Lys(octanoyl)) or to the terminal amino group of the peptide. 8 Alkyl groups (herein octanoyl) are preferred. One of skill in the art can appreciate that lysine has an amino-containing side chain. Thus, peptides that include lysine can be modified via the aforementioned lysine side chain amino functionality. Specifically, the lysine side chain amino group is a primary amine (-NH 2 ), which is converted to an amide by reaction with a carboxylic acid-containing moiety. The term "Lys(octanoyl)" refers to the reaction of the lysine amino side chain with octanoic acid, thereby forming an octanoyl group (C 7 H 15 C(O)) containing octanoyl amide (C 7 H 15 It should be understood that the reference to the product of such a reaction forms a carboxyl group, i.e., a carboxy group, such as carboxyl, methyl, ethyl ... 7 H15 It is.

[0053] According to some embodiments, the C-terminus of the peptide is modified to form an amide, alcohol, or ester terminus, each possibility representing a separate embodiment.

[0054] The peptides, derivatives and salts used in the compositions and methods of the invention may be synthesized using any method known in the art, including, but not limited to, solid phase and solution phase peptide synthesis. Some of the peptides used in the compositions of the invention may be produced using recombinant methods or a combination of recombinant and synthetic methods.

[0055] In an exemplary embodiment of the invention, the peptide derivative is pGlu-Asn-Trp-Lys(octanoyl)-OH (SEQ ID NO: 1; hereinafter referred to as "ZEP3"), where pGlu is pyroglutamic acid. ZEP3 can be produced, for example, by the procedure described in U.S. Patent No. 7,220,725.

[0056] In another exemplary embodiment of the present invention, the peptide derivative is pGlu-Asn-Trp-Thr-OH (SEQ ID NO: 2; hereinafter referred to as "ZEP4"). ZEP4 can be produced, for example, by the following procedure.

[0057] The synthesis of ZEP4 is carried out by sequential synthesis of 9-fluoromethoxycarbonyl (Fmoc) amino acids on the solid support of chlorotrityl chloride resin (CTC). CTC resin (125 gr) is loaded with Fmoc-threonine (t-butyl; 79 gr) and diisopropylethylamine (DIPEA; 160 gr) is used as coupling agent for the amino acid to the solid support. The Fmoc protecting group is removed with a mixture of 25% piperidine and dimethylformamide (DMF) and the resin-peptide is filtered and washed with DMF. The second amino acid, Fmoc-Trp (85 gr), is activated by a mixture of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) / hydroxybenzothiazole (OHBT) and coupled to the first amino acid by addition of DIPEA. The Fmoc group is removed as above, the resin-peptide is filtered and washed with DMF. The third amino acid, Fmoc-Asn(trt) (119 gr), is activated by HBTU / HOBT and coupled by addition of DIPEA. The Fmoc group is removed as above, the resin-peptide is filtered and washed with DMF. The fourth amino acid, pGlu (26 gr), is activated by HBTU / HOBT and coupled with DIPEA.

[0058] The peptide-resin is thoroughly washed with DMF, then with IPA and dried under reduced pressure. The peptide is cleaved from the resin and the Thr and Asn protecting groups with TFA (95%) and triisopropylsilane (TIS) (5%) at room temperature for 2 hours. The peptide is precipitated by adding methyl tert-butyl ether (MTBE), filtered and dried (yield 46 gr).

[0059] The crude product (46 gr) is dissolved in a mixture of acetonitrile (ACN) / water and loaded onto a preparative HPLC system (4", RP C-18 100-120A pore size) and purified using a gradient system containing phase A - 0.1% TFA in water and phase B - ACN. Elution is performed by gradually increasing phase B (from 3% to 33%) in 45 min. Fractions with a purity greater than 97% are collected. The combined fractions are eluted on the same HPLC system using a gradient containing phase A: 0.2% acetic acid and phase B: ACN. Elution is performed by gradually increasing phase B (from 10% to 40%) in 30 min. Fractions with a purity greater than 97% are collected, combined and lyophilized (yield 29 gr). The final product has a MW (MS) of 530.5 and a purity (HPLC) of 97.3%.

[0060] In one embodiment of the present invention, the peptide or peptide derivative is the sodium salt of the peptide derivative set forth in SEQ ID NO:1 (pGlu-Asn-Trp-Lys(octanoyl)-OH nNa, where n is 1 or 2, hereinafter referred to as "ZEP3 sodium salt" or "ZEP3Na"). In a particular embodiment, the sodium salt of the peptide derivative comprises the following formula: pGlu-Asn-Trp-Lys(octanoyl)-ONa. ZEP3 sodium salt can be produced, for example, by the following procedure. ZEP3 (3.1 g) was dissolved in NaHCO 3 (100 mM) (50 g / l). The solution is injected onto an HPLC ion exchange column (2.5 x 22 cm Luna C18, 100A, 15 microns) and the mobile phase A:H 2 NaHCO in O 3 2mM; Mobile phase B:CH 3 CN / H 2 NaHCO in O(8 / 2) 3 2 mM; and mobile phase C: NaHCO in water 3Elute with a gradient consisting of 100 mM. Load per run: maximum 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). Gradient sequence: 20 min phase C; 5 min phase A; 18 min phase B; and 7 min phase C. The fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile (110 g / l) and then lyophilized [yield 2.2 g (71%)]. The final product has a purity of 99.7% (HPLC), a sodium content of 3.1% and a solubility in water of 50 mg / ml.

[0061] In one embodiment of the invention, the peptide is the sodium salt of the peptide set forth in SEQ ID NO:2 (pGlu-Asn-Trp-Thr-OH·nNa, where n is 1 or 2, hereafter referred to as "ZEP4 sodium salt" or "ZEP4Na"). In a particular embodiment, the sodium salt of the peptide comprises the following formula: pGlu-Asn-Trp-Thr-ONa. ZEP4 sodium salt can be produced, for example, by the following procedure. ZEP4 (5 g) was dissolved in NaHCO 3 (100 mM) (50 g / l). The solution is injected onto an HPLC ion exchange column (2.5 x 22 cm Luna C18, 100A, 15 microns) and the mobile phase A:H 2 NaHCO in O 3 2mM; Mobile phase B:CH 3 CN / H 2 NaHCO in O(8 / 2) 3 2 mM; and mobile phase C: NaHCO in water 3 Elute with a gradient consisting of 100 mM. Load per run: maximum 5% (W / W% peptide / stationary phase). Flow rate: 4.8 cm / min (24 ml / min). Gradient procedure: 20 min phase C, then 5 min phase A, then 20 min phase B, and 10 min phase C. The fractions containing the product are collected and concentrated under reduced pressure to remove acetonitrile (110 g / l) and then lyophilized [yield 4 g (80%)]. The final product has a purity of 97.5% (HPLC), a sodium content of 2.5%, and a solubility in water of 50 mg / ml.

[0062] The peptides, peptide derivatives and / or salts of the present invention can be used as anti-aging agents themselves or as part of a composition (active ingredient) together with a dermatologically acceptable carrier. According to these embodiments, the composition comprises about 0.1% to about 5% w / w of the peptide, peptide derivative or salt thereof, each value within the specified range. According to other embodiments, the composition comprises about 0.5% to about 2% w / w of the peptide, peptide derivative or salt thereof, each value within the specified range. According to yet other embodiments, the composition comprises about 1% of the peptide, peptide derivative or salt thereof. In various embodiments, the amount of the peptide, peptide derivative or salt thereof ranges from about 200 μg to about 800 μg per gram of the composition, each value within the specified range. In further embodiments, the amount of the peptide, peptide derivative or salt thereof ranges from about 300 μg to about 700 μg per gram of the composition, each value within the specified range. In further embodiments, the amount of the peptide, peptide derivative or salt thereof ranges from about 400 μg to about 600 μg per gram of the composition, each value within the specified range. In certain embodiments, the amount of peptide, peptide derivative, or salt thereof is about 500 μg per gram of composition.

[0063] As used herein, a "composition" refers to a preparation of a peptide of formula I with one or more chemical components, such as a dermatologically acceptable carrier, designed to facilitate administration of the compound to a subject, preferably a human subject. As used herein, the term "dermatologically acceptable carrier" refers to an excipient that does not cause significant irritation to the skin and does not negate the beneficial activity and properties of the peptide of the present invention. Suitable dermatologically acceptable carriers within the scope of the present invention include, but are not limited to, thickeners, fillers, moisturizers, emulsifiers, humectants, surfactants, buffers or pH adjusters, film formers, foaming agents, antifoaming agents, preservatives, antioxidants, fragrances, solvents, propellants, colorants, and combinations or mixtures thereof. Each possibility represents a separate embodiment.

[0064] Suitable thickening agents include fatty acids and alcohols, such as stearic acid and stearyl alcohol; gums, such as xanthan, carrageenan, gelatin, cellulose gum, agarose, karaya, pectin, amylopectin, locust bean gum; and hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, calcium carboxymethyl cellulose, polyvinylpyrrolidone (povidone, PVP), polyvinyl alcohol, medium to high molecular weight polyethylene glycols (PEG-3350, PEG-6 Examples of suitable thickening agents include, but are not limited to, glyceryl ether ...

[0065] Suitable fillers include, but are not limited to, mica, talc, silicon dioxide (e.g., silica), nylon, polyethylene, polymethacrylate, kaolin, calcium carbonate, calcium phosphate, microcrystalline cellulose, various sugar and starch types, polysaccharides, dextrins, cyclodextrins (e.g., β-CD, hydroxypropyl-β-CD, sulfobutylether-CD), and Teflon. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0.5%-50% w / w filler, with each value falling within the specified range.

[0066] Suitable moisturizing agents include, but are not limited to, glycerin, hydrocarbon oils and waxes, such as mineral oil, petrolatum, paraffin, ceresin, ozokerite, microcrystalline wax, polyethylene, and perhydrosqualene; silicone oils; triglyceride oils, such as those derived from plant, animal, and marine sources, including jojoba oil and shea butter; acetoglyceride esters, such as acetylated monoglycerides; ethoxylated glycerides, such as ethoxylated glyceryl monostearate; fatty acids, fatty alcohols, and their derivatives.Other suitable emollients include, but are not limited to, caprylic or capric triglycerides; lanolin and lanolin derivatives; polyhydric alcohols and polyether derivatives; polyhydric alcohol esters; wax esters; vegetable waxes; phospholipids, such as lecithin and derivatives; sterols, including, but not limited to, cholesterol and cholesterol fatty acid esters; amides, such as fatty acid amides, ethoxylated fatty acid amides, and solid fatty acid alkanolamides.Each possibility represents a separate embodiment. In one embodiment, the composition comprises from about 0% to about 10% w / w humectant, with each value falling within the range specified.

[0067] Suitable emulsifiers include, but are not limited to, polyethylene glycol ethers of stearic acid, such as steareth-2, steareth-4, steareth-6, steareth-7, steareth-10, steareth-11, steareth-13, steareth-15, steareth-20, glyceryl stearate, stearyl alcohol, cetyl alcohol, cetearyl alcohol, behenyl alcohol, diethanolamine, lecithin, and polyethylene glycol. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 10% w / w of the emulsifier, with each value within the specified range. In another embodiment, the composition comprises about 0% to about 5% w / w of the emulsifier, with each value within the specified range.

[0068] Suitable humectants include, but are not limited to, glycols such as triethylene glycol, tripropylene glycol, propylene glycol, polypropylene glycol, butylene glycol, polyethylene glycol, sugar alcohols such as sorbitol, hexylene, urea, and collagen. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 5% w / w of humectant, with each value falling within the specified range.

[0069] Suitable surfactants are cationic, anionic or zwitterionic, including but not limited to polyoxyethylene glycol octylphenol ether, polyoxyethylene glycol alkylphenol ether, polyoxyethylene glycol sorbitan alkyl ester (such as polysorbate 60, polysorbate 80), sorbitan alkyl ester, block copolymers of polyethylene glycol and polyethylene glycol (poloxamer), polyethylene glycol tocopheryl succinate, DL alpha tocopheryl acetate, polyethoxylated castor oil derivatives (Cremophor EL, Cremophor RH40), dioctyl sodium sulfosuccinate, perfluorooctane sulfonate, alkylbenzene sulfonate, sodium lauryl ether sulfate, ammonium laureth sulfate, ammonium lauryl sulfate, disodium laureth sulfonate, lignosulfonate, sodium stearate, benzalkonium chloride, cetylpyridinium chloride, benzethonium chloride, cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, and betaine. Each possibility represents a separate embodiment. In one embodiment, the composition comprises from about 0% to about 30% w / w surfactant, with each value falling within the range specified. In another embodiment, the composition comprises from about 0% to about 20% w / w surfactant, with each value falling within the range specified. In yet another embodiment, the composition comprises from about 0% to about 5% w / w surfactant, with each value falling within the range specified.

[0070] Suitable buffering or pH adjusting agents include, but are not limited to, acidic buffering or pH adjusting agents, such as short chain fatty acids, citric acid, acetic acid, hydrochloric acid, sulfuric acid, and fumaric acid; basic buffering or pH adjusting agents, such as Tris, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, and magnesium hydroxide. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 10% w / w of the buffering or pH adjusting agent, with each value within the specified range. In another embodiment, the composition comprises about 0% to about 1% w / w of the buffering or pH adjusting agent, with each value within the specified range.

[0071] Suitable film formers include, but are not limited to, polyvinylpyrrolidone, acrylates, acrylamides, methacrylates, shellac, acacia, and hydroxyethylcellulose. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 20% w / w of a film former, with each value falling within the specified range.

[0072] Suitable foaming agents include, but are not limited to, isopropyl myristate, oxyalkylated sulfates, and ethoxylated alcohol sulfates. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 10% w / w of foaming agent, with each value falling within the specified range.

[0073] Suitable anti-foaming agents include, but are not limited to, simethicone, and sorbitan sesquioleate. Each possibility represents a separate embodiment. In one embodiment, the composition comprises from about 0% to about 10% w / w of anti-foaming agent, with each value falling within the specified range.

[0074] Suitable preservatives include, but are not limited to, methylparaben, propylparaben, butylparaben, ethylparaben, potassium sorbate, trisodium EDTA, tetrasodium EDT, disodium edetate, benzophenone, 2-bromo-2-nitropane-1,3-diol, butylhydroxytoluene, chlorhexidine digluconate, citric acid, DMDM ​​hydantoin, formaldehyde, methylchloroisothiazolinone, methylisothiazolinone, methyldibromoglutaronitrile, sodium benzoate, phenoxyethanol, ethyl alcohol, benzyl alcohol, diazolidinyl urea, imidazolidinyl urea, and quaternium-15. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 5% w / w of preservative, with each value within the specified range.

[0075] Suitable antioxidants include, but are not limited to, ascorbic acid, ubiquinone, tocopherol acetate, and sodium bisulfite. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 10% w / w antioxidant, with each value falling within the specified range.

[0076] Suitable fragrances include, but are not limited to, various plant extracts such as chamomile oil, lavender oil, and camellia sinensis. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 5% w / w fragrance, with each value within the specified range.

[0077] Suitable solvents include, but are not limited to, water, lower alcohols such as ethanol and isopropanol, propylene glycol, ammonium xylene sulfonate, and low molecular weight polyethylene glycols such as, for example, PEG-300, PEG-1450. Each possibility represents a separate embodiment. In one embodiment, the composition comprises from about 0% to about 90% w / w of the solvent, with each value falling within the range specified. In another embodiment, the composition comprises from about 0% to about 50% w / w of the solvent, with each value falling within the range specified. In yet another embodiment, the composition comprises from about 0% to about 10% w / w of the solvent, with each value falling within the range specified.

[0078] Suitable propellants include, but are not limited to, hydrocarbons having 4-7 carbon atoms, such as isopentane. Additional propellants include, but are not limited to, chlorinated, fluorinated, and chlorofluorinated low molecular weight hydrocarbons, as well as nitrous oxide, carbon dioxide, butane, and propane. Each possibility represents a separate embodiment. The propellant may be used in an amount and under a pressure suitable to adequately release the peptide from the container in the appropriate amount.

[0079] Suitable colorants include, but are not limited to, alumina (dried aluminum hydroxide), annatto extract, calcium carbonate, canthaxanthin, caramel, beta-carotene, cochineal extract, carmine, potassium sodium copper chlorophyllin (chlorophyllin copper complex), dihydroxyacetone, bismuth oxychloride, synthetic iron oxide, ferric ammonium ferrocyanide, ferric ferrocyanide, chromium hydroxide green, chromium oxide green, guanine, mica-based pearlescent pigments, pyrophyllite, disodium distyrylbiphenyl, mica, dentifric powder, talc, titanium dioxide, aluminum powder, bronze powder, copper powder, and zinc oxide, or mixtures or combinations thereof. Each possibility represents a separate embodiment. In one embodiment, the composition comprises about 0% to about 5% w / w colorant, with each value within the specified range.

[0080] Further components that may be included in the compositions of the present invention are sunscreens and tanning agents. Sunscreens include materials commonly used to block ultraviolet radiation. Exemplary compounds include derivatives of PABA, cinnamate, and salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone are also known as Parsol MCX and Benzophenone-3, respectively. When present in the composition, the amount of sunscreen used may vary depending on the degree of protection desired from ultraviolet radiation. The sunscreen should be compatible with the active compound, but generally, the composition may contain from about 0.5% to about 20% w / w of sunscreen, with each value within the specified range. The exact amount will vary depending on the sunscreen selected and the desired sun protection factor (SPF).

[0081] Additional ingredients that may be included in the compositions of the present invention are vitamins and vitamin derivatives, such as vitamin A, vitamin B, vitamin D, vitamin E, vitamin K, and derivatives thereof, including, for example, alpha tocopherol, as well as various plant extracts, such as, for example, aloe vera, aloe barbadensis, castor oil, lemon essential oil, grapefruit, sweet orange, oil palm, etc. Each possibility represents a separate embodiment.

[0082] The composition of the present invention can be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, suspending, solubilizing, granulating, wet grinding, emulsifying, encapsulating, encapsulating, spray drying or freeze drying processes.The composition can be formulated in a conventional manner using one or more dermatologically acceptable carriers as described above, which facilitates the processing of peptides and peptide derivatives and salts into dermatologically usable preparations.The appropriate formulation depends on the route of administration selected.In particular, the composition of the present invention is formulated for topical application or intradermal administration.Each possibility represents a separate embodiment.

[0083] For topical or intradermal administration, the compositions of the present invention may be formulated as oils, gels, sticks, lotions, creams, milks, aerosols, sprays, foams, mousses, ointments, drops, atomized liquids, liquid washes, emulsions, suspensions, liposomes, adhesive patches, and powders. Each possibility represents a separate embodiment. Currently preferred are compositions formulated as gels, ointments, creams, or emulsions. Each possibility represents a separate embodiment. Included within the present invention are emulsions (e.g., oil-in-water or water-in-oil) and gels (e.g., hydrogels or hydroalcoholic gels), and emulgel compositions that have the advantages of both emulsions and gels, e.g., easily spreadable and easily removable. Alternative forms of the compositions of the present invention may also be used, including forms that are designed to be reconstituted with a suitable vehicle before use. Intradermal delivery of peptides, peptide derivatives or salts thereof via nanoneedles or microneedles, such as patches as described in Larraneta et al. (Mater. Sci. Eng. R104:1-32, 2016), is also contemplated within the scope of the present invention. Optionally, combinations with other anti-aging treatments can be used.

[0084] The compositions of the present invention are useful for treating, preventing, minimizing, reducing, or reversing epidermal conditions associated with aging.As used herein, the term "epidermal conditions associated with aging" refers to aging-associated elastosis, skin atrophy, fine lines or wrinkles, skin imperfections, enlarged pores, blemishes including lentigines or solar lentigines, uneven skin tone or texture, UV-induced or photodamaged skin, hyperpigmented skin or melasma, dry skin, sagging skin, rough skin, and any combination thereof.Each possibility represents a separate embodiment.The compositions described herein can also be used to treat, prevent, minimize, reduce, or reverse the visible signs of scarring, including, but not limited to, acne scarring and chickenpox scarring.The treatment of skin aging, skin imperfections, and scarring is within the scope of the present invention. It is to be understood that the treatment, prevention, minimization, reduction, or reversal of epidermal conditions associated with aging in accordance with the present invention does not include treatment of skin disorders selected from the group consisting of herpes virus infections, chickenpox virus infections, rashes, insect bites, jellyfish stings, burns, psoriasis, itch, skin allergic reactions, skin lesions as a result of side effects or complications of drugs or medical treatments, and hypopigmentation.

[0085] The term "treat, prevent, minimize, reduce, or reverse" in the context of the present invention includes at least one of the following: improving skin firmness or elasticity, smoothing fine lines or wrinkles, shrinking skin pores, reducing hyperpigmentation, increasing skin thickness, radiance, and / or softness, and reducing dry skin. Each possibility represents a separate embodiment. Treating, preventing, minimizing, reducing, or reversing a condition can be evaluated as known in the art and includes beneficial results or improvements manifested by, for example, enhancing the appearance of the skin by reducing the appearance of wrinkles. This includes, for example, reducing oxidative damage to the skin.

[0086] Compositions suitable for use in connection with the present invention include compositions containing the peptides, derivatives, or salts of the present invention in an amount effective to achieve the intended purpose. More specifically, an effective amount refers to an amount of the peptide, derivative, or salt effective to treat, prevent, minimize, reduce, or reverse at least one symptom of an epidermal condition associated with aging. Determination of a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the method of the present invention, the effective amount can be initially estimated from in vitro and cell culture assays, for example, as described in Nakamura et al. (Exper.Derma.27:495-500,2018). For example, a particular dose can be formulated in an animal model to achieve a desired concentration or potency. Such information can be used to more accurately determine a useful dose in humans. Dosage can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected in consideration of the patient's condition. The compositions of the present invention can be administered in a single dose, although multiple doses at specific intervals are contemplated within the scope of the present invention. These can be adjusted according to individual characteristics and routes of administration, depending on the severity and reactivity of the epidermal condition. The duration of treatment may last from a few days to a few weeks, months, or years, as necessary. Typical dosages of peptides, derivatives, or salts encompassed by the present invention include, but are not limited to, values ​​within the specified ranges, such as about 0.01 to about 1,000 mg / kg body weight, about 0.1 mg / kg to about 100 mg / kg, about 1 mg / kg to about 100 mg / kg, about 10 mg / kg to about 75 mg / kg, and about 0.1 mg / kg to about 1 mg / kg. Exemplary non-limiting amounts include about 0.1 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 50 mg / kg, about 60 mg / kg, about 75 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, or about 1,000 mg / kg.Each possibility represents a separate embodiment. Alternatively, dosages may be measured and expressed as the molar concentration of the peptide, derivative, or salt administered. By way of example and not limitation, the peptide, derivative, or salt may be administered in the range of about 0.1 to about 10 mM, for example, about 0.1, about 0.25, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mM, inclusive of each value within the range specified. Each possibility represents a separate embodiment. Alternatively, dosages may be measured and expressed as mg / ml, μg / ml, or ng / ml.

[0087] As used herein, the term "administering" refers to contacting with the peptide, derivative or salt of the present invention, or a composition comprising the same. Typically, administration can be achieved by topically applying an effective amount of the composition to an area of ​​skin exhibiting an epidermal condition associated with aging. Administration can be achieved to a living organism, for example, a human.

[0088] As used herein, the term "about" refers to ±10%.

[0089] The words "comprise," "comprising," "include," "including," "having" and their conjugations mean "including but not limited to."

[0090] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "peptide" or "carrier" can include a plurality of peptides and carriers, including mixtures thereof.

[0091] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values ​​within that range. For example, C 4 -C 30 The description of the range of alkyl, etc. includes, but is not limited to, C 4 -C 25 Alkyl, C 4 -C 20 Alkyl, C 4 -C 15 Alkyl, C 4 -C 10 Alkyl, C 6 -C 10 Subranges such as alkyl, as well as individual numbers within the ranges, e.g., C 5 Alkyl, C 7 Alkyl, C 8 alkyl, etc. should be considered to be specifically disclosed. This applies regardless of the breadth of the range. Whenever a numerical range is given herein, it is meant to include any cited numbers (fractional or integer) within the given range. The expressions "ranging / ranges" between a first and a second denoted number and "ranging / ranges" "from" a first denoted number to a second denoted number are used interchangeably herein and are meant to include the first and second denoted numbers and all fractional and integer numbers therebetween.

[0092] As used herein, the term "method" refers to methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, methods, means, techniques, and procedures known by practitioners of the arts of chemistry, pharmacology, biology, biochemistry, and medicine, or methods, means, techniques, and procedures readily developed from known methods, means, techniques, and procedures.

[0093] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination or with any other described embodiment of the invention as appropriate. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment cannot function without those elements.

[0094] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below are found to be experimentally supported in the following examples. EXAMPLES

[0095] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting manner.

[0096] Example 1. Formulation: Exemplary formulations of the present invention are outlined in Tables 1-3 below. [Table 1] [Table 2] [Table 3]

[0097] ZEP-3Na chitosan gel: The gel was prepared by dissolving 2% (w / v) of high molecular weight (2,000,000) chitosan with a degree of deacetylation (DD) of 92% in 1% (v / v) aqueous acetic acid. Methylparaben sodium salt (0.1%, w / w) was then added as a preservative. The sample was stirred and the resulting gel was sonicated to remove air bubbles. The resulting gel composition and ZEP3Na are outlined in Table 4 below. [Table 4]

[0098] ZEP-3Na Chitin Ointment: Water-soluble chitin (WSC) (0.35 g) was dissolved in 0.01% acetic acid-containing water (15 ml) at room temperature. Zep-3Na (0.35 g) was dissolved in propylene glycol and added to the aqueous solution. White petrolatum (6 g) and stearyl alcohol (6 g) were mixed and slowly heated to 70°C, then stirred in the WSC solution at 70°C to obtain an emulsion. The emulsion was slowly cooled to room temperature to obtain a water-soluble ointment. The composition is outlined in Table 5 below. [Table 5]

[0099] ZEP-3Na Emulgel: An emulsion composed of DL-α tocopheryl acetate solubilized in a medium chain triglyceride (caprylic / capric oil, Mygliol) forms the oil phase of the emulsion. The aqueous phase composed of ZEP3Na, propylene glycol and glycerin as humectants, and Tween 80 and polyethylene glycol as surfactants was dispersed in water. Methylparaben and propylparaben as preservatives were dispersed in propylene glycol. Both the oil and aqueous phases were heated separately in a water bath at 70-75°C and then mixed with continuous stirring using a high shear homogenizer for 2 hours to obtain a stable emulsion. The composition is outlined in Table 6 below. [Table 6]

[0100] Cyclodextrin / Poloxamer ZEP3Na Topical Formulation: Cyclodextrin inclusion complexes with ZEP-3Na were prepared after stirring a solution of cyclodextrin in water at room temperature for several hours, followed by the addition of ZEP-3Na and further mixing for 2 hours until completely dissolved. DL-α tocopheryl acetate, methylparaben, and propylparaben were then added sequentially, followed by mixing until completely dissolved. Poloxamer as a thickening agent was then added to provide viscosity and a semi-solid consistency. The composition is outlined in Table 7 below. [Table 7]

[0101] ZEP-3Na cream: ZEP3Na was dissolved in propylene glycol and added to an aqueous phase containing glycerin, methylparaben, and deionized water. An oil phase was prepared containing stearic acid, cetyl alcohol, and liquid paraffin. Both the oil and aqueous phases were heated separately in a water bath at 70-75°C and then mixed with continuous stirring using a high shear homogenizer for 2 hours to obtain a homogenous cream, after which it was cooled for an additional 2 hours. The composition is outlined in Table 8 below. [Table 8]

[0102] Example 2. Absorption into the epidermis: A skin permeation study was performed to evaluate the amount of peptide remaining on the skin after application. This study compared the permeation profiles of ZEP3 and ZEP3Na applied as either a 1% w / w peptide-containing cream or ointment, as detailed in Example 1, Tables 2 and 3, respectively. Skin permeation studies were performed using vertical diffusion cells (Franz cells) by taking samples at specific time intervals. In vitro reconstructed human epidermal tissue from normal keratinocytes was used, which shows a structure and morphology very similar to that of human epidermal tissue in vivo.

[0103] skin tissue Mattek's EpiDerm™ X (EPI-606-X) tissue was used as a skin model for reconstruction. The integrity of the tissue cultures was visually inspected prior to testing. Transepidermal water loss (TEWL) was measured using a Cortex Technology DermaLab® instrument equipped with a DermaLab® Series TEWL probe. -2 h -1 Exclusion criteria for TEWL values ​​above 0.05 were set for possible impairment of skin barrier integrity (Pinnagoda et al. Contact Dermatitis, 22, 164-178, 1990; McPhail et al. Presentation at American Academy of Dermatology, New Orleans, LA, February 19, 2005). To ensure that the metabolic activity of Mattek's EpiDerm™ X (EPI-606-X) tissues was maintained throughout the in vitro permeation experiments without being affected by the experimental conditions and the activity of the peptide, a skin viability test was performed after the test. Tissue viability was determined by monitoring the conversion of MTT (a water-soluble tetrazolium dye) to formazan, an insoluble purple metabolite. The skin tissue viability test showed that the reconstructed human epidermal tissues maintained their viability and their metabolic activity throughout the in vitro permeation studies.

[0104] Vertical diffusion cell setup - in vitro permeation studies For this study, a SES Gmbh Analysesyteme vertical diffusion cell apparatus was used, consisting of six cells, each containing a donor and a receptor chamber. A Julabo GmbH model ED open bath circulator was used for temperature regulation of the receptor medium. Prior to the study, the tissue culture inserts were placed on several paper towels soaked in phosphate-buffered saline. Appropriate sections were then cut using a sterile scalpel and mounted between the donor and receptor chambers of each cell of the vertical diffusion cell. The tissue was allowed to equilibrate for 30 min with 5 mL of degassed receptor medium prefilled in the receptor compartment. The temperature was maintained at 32 ± 1 °C during the experiment and 30 min before the start of the experiment. The receptor medium was continuously stirred using a magnetic stirrer.

[0105] Mattek EPI-100-MM maintenance medium was selected as the receptor medium with DMSO as a diluent (Mattek EPI-100-MM:DMSO=9:1). In each diffusion cell, the available contact area of ​​the skin tissue with the receptor medium was 0.636 cm 2 Following skin integrity testing, 12.7 mg of cream or ointment formulation was applied uniformly to the skin tissue using an inoculation loop. Control solutions of ZEP3 and ZEP3Na reference standards were also tested by themselves. Approximately 127 μg amounts of ZEP3 or ZEP3Na were applied to two separate skin tissues using concentrated solutions diluted in receptor medium.

[0106] After the dose was applied, the vertical cell was properly assembled and visually inspected to ensure that there were no air bubbles in the receptor compartment. Samples were collected at 0, 4, 8, 20, 22, and 24 h. At each sampling time point, 1 mL of receptor fluid was collected in a glass vial and replaced with fresh receptor medium. A portion of the collected sample equivalent to 0.9 mL was transferred to a sample vial for analysis and 0.1 mL of DMSO was added. The solution was mixed on a vortex mixer for 10 s and placed under autosampler conditions for analysis. At the end of the permeation experiment, the remaining amount of ZEP3 or ZEP3Na in the applicator, donor compartment, and receptor compartment was determined.

[0107] Measurement of ZEP3 and ZEP3Na from in vitro permeation test samples Skin samples were tested using a Thermo Accela ultra-performance liquid chromatography system consisting of a pump, autosampler, PDA 80Hz detector and connected to a Thermo Scientific LTQ-Velos (Linear Trap Quadrupole-Orbitrap) mass spectrometer using HESI (heated electrospray source). The column used was a Fortis C18 1.7μm, 100×2.1mm, Fortis Technologies, part number: F18-020501A, with the following chromatographic parameters: Mobile phase A: 0.01% v / v trifluoroacetic acid in water, Mobile phase B: 0.01% v / v trifluoroacetic acid in 80 / 20 acetonitrile / water, Autosampler solution: 80 / 20 v / v% acetonitrile / water, Injection volume: 10μL, Column oven temperature: 40°C, Autosampler temperature: 25°C, Run time: 20min. The gradient elution program is outlined in Table 9 below. [Table 9]

[0108] The following MS parameters were used for the determination of ZEP3 and ZEP3Na: acquisition mode: positive ionization mode, full scan 110-1500 m / z, MS acquisition time: 20 min, sheath gas flow rate (arb): 30, auxiliary gas flow rate (arb): 10; spray voltage: 3.5 kV, capillary temperature: 250 °C, and heater temperature: 350 °C.

[0109] To generate calibration curves for the determination of ZEP3 and ZEP3Na concentrations, standard stock solutions of ZEP3 in DMSO were prepared at concentrations of 10 μg / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, and 5 ng / mL.

[0110] To calculate the cumulative percentage of ZEP3 permeated, the total applied dose was determined, as well as the amount of ZEP3 permeated at the first measurement time point, and the cumulative amount of drug permeated at subsequent measurement time points. The results show that both ZEP3 and ZEP3Na molecules show negligible skin permeation in both ointment and cream formulations at less than 0.02% of the applied dose. Furthermore, ZEP3 and ZEP3Na control solutions also show negligible skin permeation (<0.02%). All measurements performed on receptor fluid samples are below the limit of quantification, which corresponds to a concentration of 5ng / mL. In all cases, the % permeation was less than 0.02% of the total applied dose. Therefore, it can be concluded that substantially all peptides remain in the skin after application, thereby exerting their anti-aging effect according to embodiments of the present invention.

[0111] Skin metabolism of ZEP3 and ZEP3Na was also investigated. Full-scan HRMS chromatograms were acquired for samples in both positive and negative ESI modes to identify possible metabolites of the target analytes. Differential analysis between samples from the experiment (t = 4, 8, 20, 22, and 24 h) and the time zero sample was performed. Evaluation of fragmentation profiles from MS / MS experiments was also performed to provide further information. No metabolites of ZEP3 and ZEP3Na were identified.

[0112] Example 3. Effects of ZEP3 and ZEP3Na in human keratinocytes: To determine the anti-aging effects of ZEP3 and ZEP3Na on human keratinocytes, SCCE020 cells (EpiGRO™ human epidermal keratinocytes) were grown for four passages in complete growth medium (EpiGRO™ human keratinocyte complete medium; Millipore catalog number SCMK001). At passage 5, cells were seeded in triplicate (3×10 cells) in a volume of 1 ml of complete growth medium per well. 5 cells / well). An additional three control wells contained medium only without cells. Once attached, the cell medium was replaced with basal medium containing L-glutamine without supplements (starvation medium) and cells were starved overnight. The next day, 24 hours after seeding, cells were treated with ZEP3 or ZEP3Na or the appropriate diluent (PBS or DMSO). After 4 hours of incubation, LPS (from E. Coli 055:B5; Sigma catalog number L6529-1MG) was added to the wells at a final concentration of 20 or 30 μg / ml.

[0113] Cells were incubated for 24 hours in a tissue culture incubator. After collecting cell supernatants from all wells, cells from treatment groups (1, 2, 3, 8, 9, 10, 13, 14 (no DMSO group)) were pelleted and stored at -80°C for further analysis. Cells were collected and processed as follows: after collection of supernatants, wells were flushed with trypsin, then incubated with trypsin until cells detached from the plate surface, centrifuged, washed with PBS, and stored as cell pellets.

[0114] Example 4. In vitro assay: Two model systems are used to evaluate the in vitro efficacy of the peptides of the invention: the first system utilizes naturally immortalized human keratinocytes, referred to as the "HaCat" cell line (Ockenfels et al., Arch. Dermatol. Res., 287:304-309, 1995; Paramio et al., Brit. J. Dermatol., 137:44-50, 1997), and the second system utilizes rapidly proliferating human keratinocytes (Nickoloff et al., Am. J. Pathol., 131(1):12-8, 1988). Immortalized human keratinocyte HaCat cells were cultured in Eagle's minimum essential medium (MEM-EAGLE) supplemented with 5% fetal calf serum (FCS) and 1% antibiotics (penicillin 20 units / ml; streptomycin 20 μg / ml and nystatin 2.5 units / ml) at 37 °C in 95% air / 5% CO. 2 Bottom, 75cm 2 They are usually cultured in flasks, with the medium changed every 3-4 days.

[0115] Long-term cultures of HaCat cells grown with ZEP3, ZEP4, or their salts are obtained by culturing HaCat cells for 6 months in a commonly used medium. Human epidermal keratinocytes (passages 3-6) obtained from a routine facelift surgery are cultured in serum-free KGM®-2 BulletKit® (Clonetics, USA) medium with low calcium to promote keratinocyte proliferation.

[0116] The viability and / or proliferation of HaCat cells and cultured human epidermal keratinocytes after treatment with ZEP3, ZEP4, or their salts is determined by MTT assay in 96-well microtiter plates according to the manufacturer's instructions. Briefly, equal numbers of cells are seeded in each well and incubated for 24 hours. ZEP3, ZEP4, or their salts are added and the wells are incubated for another 72 hours. Then, 20 μl of 5 mg / ml MTT in phosphate-buffered saline (PBS) without calcium and magnesium ions is added to each well. The plate is placed in an incubator for about 3.5 hours to allow the MTT to be converted by mitochondrial dehydrogenase into insoluble MTT-formazan crystals. The medium is then removed and the resulting formazan crystals are dissolved in 0.2 ml of DMSO. The amount of formazan is quantified in an ELISA reader at 550 nm. The background value at 650 nm is subtracted. The experiment is performed in triplicate.

[0117] The late differentiation process in HaCat cells treated with ZEP3, ZEP4, or their salts is measured by determining keratinocyte envelope formation according to the procedure described by Sun et al. (Cell 9(4 Pt 1):511-2, 1976). Briefly, cells are seeded in 24-well tissue culture plates and exposed to ZEP3, ZEP4, or their salts after attachment (24 hours). The cells are then detached and resuspended in medium. Total and basal (small, rounded) cell counts are performed in quadruplicate aliquots using a hemocytometer. The remaining cells are spun down and treated with 10 mM Tris-HCl (pH 7.4) supplemented with 1% μ-mercaptoethanol and 1% SDS for 10 minutes, and keratinocyte envelope cells are counted in quadruplicate using a hemocytometer.

[0118] The effects of ZEP3, ZEP4, or their salts on early (keratin k10 expression) and late (involucrin expression) differentiation processes in HaCat cells are estimated by indirect immunofluorescence. 4Cells / ml are seeded on glass coverslips in Petri dishes containing ZEP3, ZEP4, or their salts. After 72 h of incubation, the cells on the glass coverslips are washed with PBS, fixed with an ice-cold mixture of methanol:acetone (1:1), and incubated at -20°C for 10 min. The fixed cells are then washed with PBS and incubated for 10 min with blocking buffer (1% BSA in PBS) to minimize nonspecific absorption of the primary antibody to the coverslip. The cells are then incubated for 1 h at 37°C in a humidified chamber with the primary monoclonal antibodies (Keratin 10 expression is detected by anti-human mouse monoclonal antibody at 1 / 50 final dilution, and Involucrin expression is detected by anti-human involucrin mouse monoclonal antibody at 1 / 100 final dilution). The extensive PBS washed cells are incubated with fluorophore-conjugated goat anti-mouse IgG at 1 / 50 final dilution for 30 min at room temperature. Observe the slides under a Zeiss microscope (Axioskop-2) equipped with epifluorescence optics and appropriate filters to avoid cross-channel contamination. Estimate the expression levels of keratin 10 and involucrin by counting positive cells relative to the total cell number. In each slide, at least 500-1000 cells are scored.

[0119] Example 5. Protection against ROS: To evaluate the effect of the protection of the peptides of the invention against ROS, the following experimental model is used: Primary normal human epidermal keratinocytes (NHEK) from a 20-year-old Caucasian male are used. Flow cytometry analysis with mean fluorescence intensity readings shows the percentage of cells with high ROS content. ZEP3, ZEP4, or their salts at concentrations of 15, 30, or 60 μg / mL are tested. Cells are incubated with ZEP3, ZEP4, or their salts for 24 hours before treatment and co-treated with ROS inducers selected from menadione, tert-butyl hydroperoxide (tBHT), and cumene hydroperoxide (2 concentrations) for 1 hour. Vehicle control is used as negative control and 10 mM N-acetylcysteine ​​(NAC) is used as positive control. Each experiment is performed in triplicate.

[0120] Example 6. Assessment of cell viability after induction of oxidative stress: To evaluate the viability of cells treated with the peptides of the invention after oxidative stress induction, the following experimental model is used: Primary normal human epidermal keratinocytes (NHEK) derived from a 20-year-old Caucasian male are used. Live cell imaging (one image every 2-4 hours) provides the percentage of cells lysed. ZEP3, ZEP4, or their salts at concentrations of 15, 30, or 60 μg / mL are tested. Cells are co-treated with ZEP3, ZEP4, or their salts for 24 hours before treatment and with ROS inducers selected from tert-butyl hydroperoxide (tBHT) and hydroperoxides (2 concentrations) for 24-28 hours. Vehicle control is used as negative control and 10 mM N-acetylcysteine ​​(NAC) as positive control. Each experiment is performed in triplicate.

[0121] Example 7. Protection against oxidative stress-induced aging: To evaluate the protective effect of the peptides of the invention against oxidative stress-induced aging, the following experimental model is used: Primary normal human epidermal keratinocytes (NHEK) from a 20-year-old Caucasian male are used. Senescence is evaluated by measuring beta-galactosidase levels using flow cytometry. ZEP3, ZEP4, or their salts are tested at concentrations of 15, 30, or 60 μg / mL. Cells are incubated with ZEP3, ZEP4, or their salts for 24 hours before treatment, 2 hours during co-treatment, and 48 hours after treatment with hydroperoxide (200 μM) as ROS inducer. Vehicle control is used as negative control and 10 mM N-acetylcysteine ​​(NAC) is used as positive control. Each experiment is performed in triplicate.

[0122] Example 8. Efficacy of compositions containing ZEP3, ZEP4 or salts thereof in human subjects: The effect of the composition containing the peptide of the present invention on human subjects was tested. In particular, the cream composition containing 1.0% ZEP3, ZEP4, or their salts as detailed in Example 1, Table 2, or 0.1-1.0% ZEP3, ZEP4, or their salts supplemented in a commercially available cream was applied to six individuals, and positive results were reported as follows:

[0123] An 80-year-old female subject who applied the cream every other day for four years reported an improvement in the appearance of wrinkles.

[0124] A 52-year-old female subject who applied the cream daily for 18 months reported that wrinkles had disappeared and the smoothness of her facial skin had improved.

[0125] A 70-year-old female subject who applied the cream daily to her face and both arms for four years reported an improvement in the appearance of her skin.

[0126] An 80-year-old female subject who applied the cream daily for one year reported that her skin had become smoother and softer.

[0127] A 70-year-old female subject with dark pigmentation marks over most of her body applied the cream 3-4 times a day and reported that her skin hyperpigmentation had disappeared.

[0128] A 42 year old female subject who suffered from sagging skin on her chest applied the cream daily for two years and reported that the skin in the treated areas was significantly smoother.

[0129] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0130] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

Claims

1. A composition for use in treating, preventing, minimizing, reducing or reversing an epidermal condition associated with aging selected from the group consisting of elastosis, skin atrophy, fine lines, wrinkles, enlarged pores, hyperpigmentation, sagging skin, rough skin and dry skin, comprising a compound of formula I: pGlu-X １ -X ２ -X ３ -OH (wherein, X １ is selected from the group consisting of Asn and Thr, X 2 is selected from the group consisting of Trp, Phe, and Tyr, and X 3 is selected from the group consisting of Lys, Lys attached via an amino side chain to a C 4 -C 30 alkyl group, and Thr, or a salt thereof, and a dermatologically acceptable carrier.

2. The composition for use according to claim 1, wherein the peptide of formula I or a salt thereof has the amino acid sequence pGlu-Asn-Trp-Lys(octanoyl)-OH as set forth in SEQ ID NO:1 or a salt thereof.

3. The composition for use according to claim 2 , wherein the peptide is the sodium salt of the peptide having the amino acid sequence set forth in SEQ ID NO:

1.

4. 2. The method of claim 1, wherein the peptide of formula I or a salt thereof has the amino acid sequence pGlu-Asn-Trp-Thr-OH as set forth in SEQ ID NO:2 or a salt thereof.

5. The composition for use according to claim 4, wherein the peptide is the sodium salt of the peptide having the amino acid sequence set forth in SEQ ID NO:

2.

6. The composition for use according to any one of claims 1 to 5, wherein said composition comprises from about 0.1% to about 5% w / w of said peptide of formula I or its salt.

7. The composition for use according to any one of claims 1 to 6, wherein the composition is formulated for topical or intradermal administration.

8. 8. The composition for use according to claim 7, wherein the composition is in a form selected from the group consisting of an oil, a gel, a stick, a lotion, a cream, a milk, an aerosol, a spray, a foam, a mousse, an ointment, a drop, an atomized liquid, a liquid wash, an emulsion, a suspension, a liposome, an adhesive patch, and a powder.

9. The composition for use according to claim 7, wherein the composition is in the form of a gel, an ointment, a cream, an emulsion, or an emulgel.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the dermatologically acceptable carrier comprises at least one of a thickening agent, a filler, a moisturizer, an emulsifier, a humectant, a surfactant, a buffer or pH adjusting agent, a film former, a foaming agent, an antifoaming agent, a preservative, an antioxidant, a fragrance, a solvent, a propellant, a colorant, and combinations or mixtures thereof.

11. 11. The composition for use according to any one of claims 1 to 10, wherein treating, preventing, minimizing, reducing or reversing said aging-related epidermal conditions comprises at least one of improving skin firmness or elasticity, smoothing fine lines or wrinkles, shrinking skin pores, reducing hyperpigmentation, increasing skin thickness, radiance and / or softness, and reducing dry skin.

12. A composition for use according to any one of claims 1 to 10, wherein treating, preventing, minimizing, reducing or reversing the aging-related epidermal condition includes at least one of smoothing fine lines or wrinkles and reducing hyperpigmentation.

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