Peptides and compositions for use in cosmetics - Patents.com

Abstract

The present invention relates to a family of peptides that are capable of increasing the synthesis of the homeodomain protein Mohawk and are therefore effective as anti-aging and rejuvenating agents.

Description

Detailed Description of the Invention

[0001] The present invention relates to the field of molecular biology, more precisely to molecular biology applied to cosmetics, and even more precisely to a peptide capable of increasing the expression and synthesis of Mohawk, and to a composition comprising said peptide.

[0002] During the past few decades, people's life expectancy has increased significantly. Furthermore, people are increasingly concerned about their physical appearance and are trying to delay or minimize the appearance of signs related to aging.

[0003] Skin is the largest organ in humans and, due to its location as a body interface, is subject to intrinsic (chronological) and extrinsic aging. Skin aging is a complex biological process influenced by a combination of intrinsic and extrinsic factors (intrinsic: genetics, cellular metabolism, hormones and metabolic processes; extrinsic: chronic light exposure, pollution, ionizing radiation, chemicals, toxins), thus leading to changes in the skin and the appearance of defects therein (e.g., loss of firmness, wrinkles, roughness, and / or sagging).

[0004] These factors induce cumulative structural and physiological changes in the different layers of the skin, as well as changes in the overall skin appearance (Ganceviciene, R., Liakou, AI, Theodoridis, A., et al. (2012) Skin Anti-Aging Strategies. Dermato-Endocrinology, 4, 308-319).

[0005] The three main structural components of the dermis are collagen, elastin and glycosaminoglycans (GAGs), which are the subject of most of the anti-aging research and efforts for skin-related aesthetic anti-aging strategies, ranging from "anti-wrinkle creams" to various fillers (Baumann, L. (2007), Skin ageing and its treatment. J. Pathol., 211, 241-251.).

[0006] Collagens constitute a large family of extracellular matrix (ECM) proteins and play an essential role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent crosslinks between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Collagen biosynthesis is a highly complex process that encompasses numerous processes, including chain association and folding, secretion, procollagen processing, and crosslinking. As exemplified by human type I collagen, a heterotrimeric molecule consisting of two α1 and one α2 chains is synthesized on ribosomes and imported into the rough endoplasmic reticulum, after which the collagen chains undergo a series of post-translational modifications leading to the assembly of procollagen chains (Rodriguez-Pascual, F., Slatter, DA (2016) Collagen cross-linking: insights on the evolution of metazoan extracellular matrix. Scientific Reports, 6, 37374.).

[0007] Moreover, collagen is the most abundant protein in the body (70% by dry weight) and consists mainly of fibrillar collagen types I and III, which together provide most of the strength and stiffness of tissues. Although minor collagens constitute less than 10% of the total collagen content, they play a key role in ECM organization (Lovell, C., Smolenski, K., Duance, V., Light, N., Young, S., Dyson, M. (1987) Type I and III collagen content and fibre distribution in normal human skin during ageing. British Journal of Dermatology, 117, 419-428; and Theocharidis, G., Connelly, JT (2017) Minor collagens of the skin with not so minor functions. J. Anat.). Indeed, it has been demonstrated that collagen VI contributes to the proper binding and structure of collagen I (Bonaldo, P et.al. (1990) Structural and Functional Features of the α3 Chain Indicate a Bridging Role for Chicken Collagen VI in Connective Tissues. Biochemistry, 29, 1245-1254). In addition to their essential structural functions, collagens are also involved in cell adhesion, chemotaxis and migration.They dynamically interact with cells, growth factors and cytokines to regulate cell proliferation, differentiation, morphogenesis and tissue remodeling during wound repair (van der Rest, M., Garrone, R. (1991) Collagen family of proteins. FASEB J., 13, 2814-23.; Singer, AJ, Clark, RA (1999) Cutaneous wound healing. N Engl J Med., 341(10), 738-46; Gelse, K., Poschl, E.,Aigner, T. (2003) Collagens-structure, function, and biosynthesis. Advanced Drug Delivery Reviews, 55(12), 1531-1546; and Ricard-Blum S. (2011). The collagen family. Cold Spring Harbor perspectives in biology, 3(1), a004978).

[0008] Collagen destruction, together with damage to other structural components of the skin (i.e., elastic and reticular fibers), is thought to underlie the characteristic changes in the appearance of aged skin (Bailey, AJ (2001). Molecular mechanisms of ageing in connective tissue. Mech Ageing Dev, 122 pp. 735-755; Schwartz, E., Cruickshank, FA, Perlish, JS, Fleischmajer, R. (1989) Alterations in dermal collagen in ultraviolet irradiated hairless mice. J Invest Dermatol, 93, pp. 142-146; Schwartz, E., Cruickshank, FA, Christensen, CC, Perlish, JS, Lebwohl, M. (1993) Collagen alterations in chronically sun-damaged human skin. Photochem Photobiol, 58, pp. 841-844; Smith, JG, Davidson, EA, Sams, WM, Clark, RD (1962) Alterations in human dermal connective tissue with age and chronic sun damage. J Invest Dermatol, 39, pp. 347-350; Maloney, SJ, Edmonds, SH, Giddens, LD, Learn, DB (1992) The hairless mouse model of photoaging: Evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles. Photochem Photobiol, 56, pp. 505-511; Frigiel, SEG, Varani, J., Datta, S.C., Kang, S., Fisher, GJ, Voorhees, JJ (2003) Collagen Degradation in Aged / Photodamaged Skin in Vivo and after Exposure to Matrix Metalloproteinase-1 in Vitro. J. Invest. Dermatol., 120, pp. 842-848; and Marks, R. (Ed.) (1992) Sun-Damaged Skin. Martin Dunitz, London). .

[0009] Furthermore, it has been described that the homeodomain protein Mohawk (Mkx) is abundantly expressed in the skin (Uhlen, M. et al. (2015) Proteomics. Tissue-based map of the human proteome. Science 347, 1260419), and said protein has been associated with increased collagen synthesis and increased collagen fibrillogenesis, as well as the proper regulation of other genes related to the production of the extracellular matrix (Nakamichi, R. et.al. (2016) Mohawk promotes the maintenance and regeneration of the outer annulus fibrosus of intervertebral discs. nature communications, 7:12503; Nakahara, H. (2013) Transcription Factor Mohawk and the Pthogenesis of Human Anterior Criciate Ligament Degradation. arthritis & rheumatism, vol. 65, 8, 2081-2089).

[0010] In recent years, the number of active ingredients for improving the signs of skin aging, such as loss of firmness, skin texture and wrinkles, has increased considerably. Examples of such active ingredients are retinoids, vitamins or plant extracts (Bradley EJ, Griffiths CEM, Sherratt MJ, Bell M. and Watson REB (2015), Over-the-counter anti-ageing topical agents and their ability to protect and repair photoaged skin. Maturitas, 80, 265-272).

[0011] In the case of retinoids, noteworthy is retinoic acid, which is currently the "gold standard" for the induction of the synthesis of several molecules of the ECM (e.g. collagen, fibronectin or laminin; Varani J., Mitra RS, Gibbs D., Phan SH, Dixit VM, Mitra R., Wang T., Siebert KJ, Nickoloff BJ, Voorhees JJ (1990), All-Trans Retinoic Acid Stimulates Growth and Extracellular Matrix Production in Growth-Inhibited Cultured Human Skin Fibroblasts. The Journal of Investigative Dermatology, 94, 717-723). Thus, retinoic acid is considered one of the most powerful compounds for treating the signs of aging, since it can significantly improve the clinical appearance of facial wrinkles by upregulating the transcription and synthesis of proteins of the ECM, such as collagen and fibronectin, as well as inhibiting matrix metalloproteinases (hereinafter MMPs) (Varani J., Mitra RS, Gibbs D., Phan SH, Dixit VM, Mitra R., Wang T., Siebert KJ, Nickoloff BJ, Voorhees JJ (1990), All-Trans Retinoic Acid Stimulates Growth and Extracellular Matrix Production in Growth-Inhibited Cultured Human Skin Fibroblasts. The Journal of Investigative Dermatology, 94, 717-723). However, there are some drawbacks to the use of retinoic acid, for example, it must be used with caution as it can easily cause skin irritation, and it is not recommended to combine retinoic acid with sun exposure.Another major problem when using retinoids is their instability, especially in the presence of oxygen and light (Sorg O., Antille C., Kaya G. and Saurat JH. (2006), Retinoids in cosmeceuticals. Dermatologic Therapy, 19, 289-296).

[0012] Antioxidants are also used to reduce the concentration of free radicals in the skin, thus preventing collagen degradation. One example of said antioxidant is ascorbic acid (also known as vitamin C). In addition to its antioxidant effect, ascorbic acid, among other things, promotes epidermal differentiation and induces the synthesis of proteins from ECM (collagen I and III and elastin), while inhibiting matrix metalloproteinase-1 (MMP1). Unfortunately, ascorbic acid is highly unstable and undergoes oxidation, especially at high temperature, aerobic conditions, high pH, ​​and / or upon light exposure (Manela-Azulay M., Azulay V., Aguinaga F., Issa MC (2017), Vitamins and other Antioxidants. Daily Routine in Cosmetic Dermatology, 1-13).

[0013] In addition to chemically synthesized compounds, a wide range of plant extracts and plant-derived compounds are commercially available for various uses, such as grape extract containing resveratrol (antioxidant); green tea containing polyphenols; or soybean containing isoflavones.However, the in vivo efficacy and composition of these ingredients have not been fully scientifically verified.

[0014] Due to its viscoelastic properties and its ability to retain water, hyaluronic acid is also used in the cosmetic industry to treat wrinkles by keeping the skin hydrated, maintaining elasticity, and improving roughness, or as a dermal filler.However, currently, hyaluronic acid is obtained from multiple sources, such as rooster comb or bacterial extracts, and as a result, these products may contain impurities and need to be fully characterized (Kogan G., Soltes L., Stern R. and Gemeiner P. (2007), Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnological Letters 29, 17-25).

[0015] On the other hand, peptides can also be incorporated into cosmetic formulations to improve the signs of skin aging. Bioactive peptides have the advantage of mimicking the body's own molecules, being able to affect processes such as collagen synthesis, and having much better resistance and stability. Moreover, a wide range of activities, chemistries and applications can be developed for them (Zhang L. and Falla TJ (2009), Cosmeceuticals and peptides. Clinics in dermatology, 27, 485-494).

[0016] Despite the wide variety of compounds and / or extracts in the art, there remains a need for alternative compositions with novel mechanisms that allow for the prevention, reduction and / or elimination of the signs of skin aging (chronological and / or environmental aging) and / or provide skin rejuvenation.

[0017] The inventors of the present invention, after extensive and thorough research, have surprisingly found peptides that provide increased expression and synthesis of the homeodomain protein Mohawk, and as a result, increased expression and synthesis of collagen. Moreover, the peptides found by the inventors of the present invention regulate the expression of genes that favor or upregulate genes that govern the synthesis and structuring of extracellular matrix, while inhibiting or downregulating genes that govern the degradation of said extracellular matrix. The peptides of the present invention also increase collagen synthesis and improve collagen density and thickness in skin explants. Furthermore, their skin smoothing properties, as well as anti-wrinkle and anti-aging effects, have been demonstrated in vivo. Thus, the peptides of the present invention exhibit anti-aging and rejuvenation activity, thus solving the above-mentioned problems existing in the art.

[0018] The inventors are not aware of any prior art which provides peptides with the above-mentioned mechanism of action and therefore the above-mentioned activity.

[0019] In a first aspect, the present invention refers to a peptide capable of increasing the homeodomain protein Mohawk.

[0020] In a second aspect, the present invention refers to a composition comprising a peptide of the invention.

[0021] Moreover, in a third aspect, the present invention refers to the cosmetic use of a peptide or composition of the invention in a subject in need thereof.

[0022] In a fourth aspect, the present invention refers to the cosmetic use of a peptide or composition of the invention in a subject in need thereof.

[0023] In a fifth aspect, the present invention refers to a cosmetic method, characterized in that it comprises the use of a peptide or a composition of the invention in a subject in need thereof.

[0024] As used herein, the term "non-cyclic aliphatic group" and its plural forms have the ordinary meaning given to said term in the art, and thus refer to, for example, but not limited to, linear or branched alkyl, alkenyl, and alkynyl groups.

[0025] As used herein, the term "alkyl group" and its plurals refer to a saturated, straight or branched chain group having 1 to 24, preferably 1 to 16, more preferably 1 to 14, even more preferably 1 to 12, and even more preferably 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is attached to the remainder of the molecule by a simple bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, i-propyl, isobutyl, tert-butyl, n-butyl, sec-butyl, n-pentyl, n-hexyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, and the like. Alkyl groups may be optionally substituted with one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0026] As used herein, the term "alkenyl group" and its plurals refer to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, and even more preferably 2, 3, 4, 5, or 6 carbon atoms and one or more carbon-carbon double bonds, preferably 1, 2, or 3 carbon-carbon double bonds (conjugated or non-conjugated). The alkenyl group is attached to the remainder of the molecule through a single bond. Alkenyl groups include, but are not limited to, for example, vinyl, oleyl, linoleyl, and similar groups. The alkenyl group may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0027] As used herein, the term "alkynyl group" and its plurals refer to a linear or branched group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, and even more preferably 2, 3, 4, 5, or 6 carbon atoms and one or more carbon-carbon triple bonds, preferably 1, 2, or 3 carbon-carbon triple bonds (conjugated or non-conjugated). Alkynyl groups are attached to the remainder of the molecule through a single bond. Alkynyl groups include, but are not limited to, for example, ethynyl groups, pentynyl such as 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, and similar groups. Alkynyl groups may be optionally substituted with one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0028] As used herein, the term "alicyclyl" and its plural forms have the ordinary meaning given to said term in the art, and thus are used to refer to, for example, but not limited to, cycloalkyl or cycloalkenyl or cycloalkynyl groups.

[0029] As used herein, the term "cycloalkyl" and its plural forms refer to a saturated monocyclic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, and even more preferably 3, 4, 5, or 6 carbon atoms. The cycloalkyl is attached to the remainder of the molecule via a single bond. Cycloalkyls include, but are not limited to, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrophenalene, adamantyl, and the like. Cycloalkyls may be optionally substituted with one or more groups, such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0030] As used herein, the term "cycloalkenyl" and its plural forms refer to a non-aromatic monocyclic or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, and even more preferably 5 or 6 carbon atoms and having one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds (conjugated or non-conjugated). The cycloalkenyl is attached to the remainder of the molecule through a single bond. Cycloalkenyls include, but are not limited to, for example, cyclopent-1-en-1-yl groups, and similar groups. Cycloalkenyls may be optionally substituted with one or more groups, such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0031] As used herein, the term "cycloalkynyl" and its plural forms refer to a non-aromatic monocyclic or polycyclic aliphatic group having 8 to 24, preferably 8 to 16, more preferably 8 to 14, even more preferably 8 to 12, and even more preferably 8 or 9 carbon atoms and having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds (conjugated or non-conjugated). The cycloalkynyl is attached to the remainder of the molecule through a single bond. Cycloalkynyls include, but are not limited to, for example, cyclooct-2-yn-1-yl groups, and the like. Cycloalkynyls may be optionally substituted with one or more groups, such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0032] As used herein, the term "aryl group" and its plurals refer to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, and even more preferably 6 or 10 carbon atoms and containing 1, 2, 3 or 4 aromatic rings joined or fused by carbon-carbon bonds. The aryl group is attached to the remainder of the molecule through a single bond. Aryl groups include, but are not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthranyl, among others. Aryl groups may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0033] As used herein, the term "aralkyl group" and its plurals refer to an alkyl group substituted with an aromatic group having 7 to 24 carbon atoms. Aralkyl groups include, but are not limited to, for example, -(CH2)1-6-phenyl, -(CH2)1-6-(1-naphthyl), -(CH2)1-6-(2-naphthyl), -(CH2)1-6-CH(phenyl)2, and the like. Aralkyl groups may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0034] As used herein, the term "heterocyclyl group" and its plurals refer to a 3-10 membered heterocyclyl or hydrocarbon ring, in which one or more of the ring atoms, preferably one, two or three of the ring atoms, are elements different from carbon, such as nitrogen, oxygen or sulfur. A heterocyclyl group can be saturated or unsaturated. For the purposes of this invention, a heterocyclyl can be a ring system, monocyclic, bicyclic or tricyclic system, which can include fused ring systems; the nitrogen, carbon or sulfur atoms can be optionally oxidized in the heterocyclyl radical; the nitrogen atom can be optionally quaternized; the heterocyclyl radical can be partially or fully saturated or aromatic. Preferably, the term heterocyclyl refers to a 5- or 6-membered ring. A heterocyclyl group can be optionally substituted by one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.

[0035] As used herein, the term "heteroarylalkyl group" and its plurals refer to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group, the alkyl group having 1-6 carbon atoms and the aromatic heterocyclyl group having 2-24 carbon atoms and 1-3 non-carbon atoms. Heteroarylalkyl groups include, but are not limited to, for example, -(CH2)1-6-imidazolyl, -(CH2)1-6-triazolyl, -(CH2)1-6-thienyl, -(CH2)1-6-furyl, -(CH2)1-6-pyrrolidinyl, and the like. Heteroarylalkyl groups may be optionally substituted with one or more substituents, such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto, and alkoxythio.

[0036] As used herein, the term "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine, the anions of which are referred to as halides.

[0037] As used herein, the term "derivatives" and its plural forms refer to both cosmetically acceptable compounds, i.e., compounds derived from the compound of interest that can be used in the preparation of cosmetic products, and to derivatives that are not cosmetically acceptable, since derivatives that are not cosmetically acceptable may be useful in the preparation of cosmetically acceptable derivatives.

[0038] As used herein, the term "salt" and its plural forms refer to any type of salt from those known in the art, such as halide salts, hydroxy acid salts (oxy acid salts, acid salts, base salts, double salts, etc.), hydroxo salts, mixed salts, oxy salts, or other hydrated salts. The term includes both cosmetically acceptable and non-cosmetically acceptable salts, since the latter may be useful in the preparation of cosmetically acceptable salts.

[0039] As used herein, the term "isomer" and its plural forms refer to optical isomers, enantiomers, stereoisomers or diastereoisomers. The individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques known in the art.

[0040] As used herein, the term "solvate" and its plural forms refer to any solvate known in the art, such as polar, non-polar or amphiphilic solvate.Solvate includes any cosmetically acceptable solvate that provides the compound of interest (the peptide of the present invention) when administered or applied (directly or indirectly) to the subject of interest.Preferably, the solvate is: hydrate; solvate with alcohol such as methanol, ethanol, propanol or isopropanol; solvate with ester such as ethyl acetate; solvate with ether such as methyl ether, ethyl ether or THF (tetrahydrofuran); or solvate with DMF (dimethylformamide), more preferably: hydrate; or solvate with alcohol such as ethanol.

[0041] Furthermore, as used herein, the term "amino acid" and its plural forms include the amino acids encoded by the genetic code, as well as the non-encoded amino acids, whether or not they are naturally occurring, and whether or not they are D-amino acids and L-amino acids. Examples of non-encoded amino acids are, but are not limited to, citrulline, ornithine, sarcosine, desmosine, norvaline, 4-aminobutyric acid, 2-aminobutyric acid, 2-aminoisobutyric acid, 6-aminohexanoic acid, 1-naphthylalanine, 2-naphthylalanine, 2-aminobenzoic acid, 4-aminobenzoic acid, 4-chlorophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, cycloserine, carnitine, cysteine, penicillamine, pyroglutamic acid, thienylalanine, hydroxyproline, allo-isoleucine, allo-threonine, isonipecotic acid, isoserine, phenylglycine, statins, β-alanine, norleucine, N-methyl amino acids, α-amino acids and β-amino acids, among others, and derivatives thereof. Nonetheless, additional unnatural amino acids are known in the art (see, e.g., "Unusual amino acids in peptide synthesis" by DC Roberts and F. Vellaccio, The Peptides, Vol. 5 (1983), Chapter VI, Gross E. and Meienhofer J., Eds., Academic Press, New York, USA).

[0042] As used herein, "percentage of identity" with respect to peptides, polypeptides and proteins has the meaning generally ascribed to them in the art, and thus refers to the percentage of amino acids that are identical between two amino acid sequences compared after optimal alignment of the two amino acid sequences. Here, said percentage is merely statistical, and the differences between the two amino acid sequences are randomly distributed throughout the sequence. "Optimal alignment" is understood as the alignment of amino acid sequences that results in a larger percentage of identity. The percentage of identity is calculated by determining the number of identical positions in the two compared sequences where the amino acids are identical, dividing the number of identical positions by the number of positions compared, and multiplying the result by 100 to obtain the percentage of identity between the two sequences. The sequence comparison between two amino acid sequences can be performed manually or by computer programs known in the art, such as the BLAST (Basic Local Alignment Search Tool) algorithm.

[0043] As used herein, "homeodomain protein Mohawk," "Mohawk" and "Mkx" are synonymous and are used interchangeably, and unless otherwise specified, refer to the protein known as the homeodomain protein Mohawk.

[0044] As mentioned above, in a first aspect, the present invention refers to peptides capable of increasing Mohawk (hereinafter Mkx), their acceptable isomers, salts, solvates and / or derivatives, and / or mixtures thereof.

[0045] Mohawk is increased in the subject to which the peptide is applied, more preferably in a mammal, and even more preferably in a human.

[0046] Preferably, the increase in Mohawk is an increase in Mohawk gene expression and protein synthesis.

[0047] It is intended that the amino acids used or present in the peptides of the present invention are L-amino acids, D-amino acids, or combinations thereof. In a preferred embodiment, the amino acids used or present in the peptides of the present invention are L-amino acids.

[0048] Preferably, said isomer is a stereoisomer. Said stereoisomer is intended to be an enantiomer or a diastereoisomer. Thus, in a preferred embodiment of the present invention, the peptide is a racemic mixture, a diastereomeric mixture, a pure enantiomer, or a pure diastereoisomer.

[0049] Preferably, the peptide of the present invention comprises 2 to 50 amino acids, more preferably 4 to 10 amino acids, more preferably 4 to 6 amino acids, and even more preferably 4 amino acids.

[0050] It is intended that the peptide of the present invention comprises at least one moiety attached to its N-terminus and / or its C-terminus. The at least one moiety may be attached to the peptide by any means known in the art, preferably covalently. In a preferred embodiment, the peptide comprises one moiety covalently attached to its N-terminus and one moiety covalently attached to its C-terminus.

[0051] In one embodiment, at least one N-terminal moiety (preferably one N-terminal moiety) is selected from H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R5-CO-, where R5 is a substituted or unsubstituted C1-C6 alkyl group. 24 Alkyl radicals, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkynyl, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloalkynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 aralkyl, substituted or unsubstituted 3-10 membered heterocyclyl ring, and substituted or unsubstituted heteroarylalkyl with 2-24 carbon atoms and 1-3 non-carbon atoms and an alkyl chain of 1-6 carbon atoms. Even more preferably, at least one N-terminal moiety (preferably one N-terminal moiety) is selected from acetyl (hereinafter Ac) or palmitoyl (hereinafter Pal). In a most preferred embodiment, at least one N-terminal moiety (preferably one N-terminal moiety) is Pal.

[0052] In one embodiment, at least one C-terminal moiety (preferably one C-terminal moiety) is selected from H, -NR3R4-, -OR3 and -SR3, where R3 and R4 are independently selected from H, substituted or unsubstituted acyclic aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl. Even more preferably, at least one C-terminal moiety (preferably one C-terminal moiety) is selected from H or NH2. In the most preferred embodiment, at least one C-terminal moiety (preferably one C-terminal moiety) is NH2.

[0053] Thus, more preferably, the peptide of the present invention comprises one moiety attached to the N-terminus and one moiety attached to the C-terminus, wherein the moiety attached to the N-terminus is Pal and the moiety attached to the C-terminus is NH2.

[0054] In a preferred embodiment, the sequence of the peptide of the invention is according to formula (I): R1-AA1-AA2-AA3-AA4-R2(I) Cosmetically acceptable isomers, salts, solvates and / or derivatives thereof, and mixtures thereof: During the ceremony, AA1 is His; AA2 is selected from the group of amino acids having an aromatic side chain; AA3 is selected from Lys or Arg; AA4 is selected from the group of amino acids having an aliphatic, non-polar side chain.

[0055] R1 is selected from H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, and R5-CO-, where R5 is a substituted or unsubstituted C1-C 24 Alkyl radicals, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkynyl, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloalkynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 selected from the group formed by aralkyl, substituted or unsubstituted 3-10 membered heterocyclyl rings, and substituted or unsubstituted heteroarylalkyl having 2-24 carbon atoms and 1-3 non-carbon atoms and an alkyl chain of 1-6 carbon atoms; R2 is selected from H, -NR3R4-, -OR3 and -SR3, where R3 and R4 are independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl.

[0056] As mentioned above, it is intended that the amino acids used or present in the peptides of the invention are L-amino acids, D-amino acids, or combinations thereof. In a preferred embodiment, the amino acids used or present in the peptides of the invention are L-amino acids.

[0057] Also, as mentioned above, preferably, the above isomers are stereoisomers. Said stereoisomers are intended to be enantiomers or diastereoisomers. Thus, in a preferred embodiment of the present invention, the peptide is a racemic mixture, a diastereomeric mixture, a pure enantiomer, or a pure diastereoisomer.

[0058] Preferably, R1 is selected from Pal or Ac, and even more preferably, R1 is Pal.

[0059] Preferably, R2 is selected from H or NH2, and even more preferably, R2 is NH2.

[0060] Thus, more preferably, R1 is Pal and R2 is NH2.

[0061] Preferably, in formula (I), AA2 is selected from the group of Phe, Tyr and Trp, more preferably, AA2 is Tyr.

[0062] Preferably, in formula (I), AA3 is Arg.

[0063] Also preferably, in formula (I), AA4 is selected from the group of Ala, Val, Leu and Ile, more preferably, AA4 is Ala.

[0064] Thus, in a preferred embodiment, in formula (I): AA1 is His; AA2 is selected from the group of Phe, Tyr and Trp; AA3 is selected from Lys or Arg; AA4 is selected from the group of Ala, Val, Leu, and Ile; More preferably, in formula (I): AA1 is His; AA2 is selected from the group of Phe, Tyr and Trp; AA3 is Arg; AA4 is selected from the group of Ala, Val, Leu and Ile.

[0065] Even more preferably, the sequence of the peptide according to the invention (i.e. the peptide of formula (I)) is R1-His-Tyr-Arg-Ala-R2 (R1-SEQ ID NO:1-R2) It is.

[0066] In a most preferred embodiment, the sequence of the peptide of the invention (i.e. as set forth in formula (I)) is Pal-His-Tyr-Arg-Ala-NH2 (Pal-SEQ ID NO:1-NH2) It is.

[0067] The peptides of the present invention can be synthesized and produced by any means known in the art. For example, they can be synthesized and produced by chemical synthesis (preferably by solid-phase peptide synthesis), by expressing said peptides in cell culture, or by transgenic production of peptides in plants or animals. Furthermore, the peptides of the present invention can be purified by any means known in the art.

[0068] As will be apparent from the examples below, the peptides of the present invention provide increased expression and synthesis of Mohawk and collagen VI. Furthermore, the peptides of the present invention regulate the expression of genes that favor or upregulate genes that control the synthesis and structuring of extracellular matrix, while inhibiting or downregulating genes that control the degradation of said extracellular matrix. The peptides of the present invention also increase collagen synthesis and improve collagen density and thickness in skin explants. Furthermore, they have skin smoothing, anti-wrinkle, and anti-aging effects when applied topically to the face of female subjects.

[0069] Thus, the peptides of the present invention solve the above-mentioned problems and provide additional or alternative peptides with cosmetic activity (e.g., anti-aging and rejuvenating activity). The peptides of the present invention can improve signs of skin associated with aging, such as wrinkles, roughness and / or sagging, or can provide skin rejuvenation and / or improvement of skin imperfections, such as, for example, body tightening, body sculpting, facial reshaping, skin tightening, and / or pore refinement.

[0070] Also within the scope of the present invention are peptides that incorporate conservative amino acid substitutions with respect to any of the above-described peptides of the present invention and that still exhibit the activity described herein for the peptides of the present invention.

[0071] Furthermore, peptides having 70% percent identity with the peptide R1-His-Tyr-Arg-Ala-R2 (preferably Pal-His-Tyr-Arg-Ala-NH2), preferably 80%, more preferably 90%, more preferably 95%, even more preferably 99% percent identity therewith, which still exhibit the activity described herein for the peptides of the invention, are also included within the scope of the present invention.

[0072] In a preferred embodiment, the peptides of the present invention are suitable or adapted to be applied by iontophoresis, more preferably to the face and / or body of a subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of a subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0073] In a most preferred embodiment, the compositions of the present invention are suitable or adapted to be applied topically, more preferably to the face and / or body of a subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of a subject, even more preferably to the face and / or neck of a subject, preferably a human.

[0074] In a second aspect, the present invention refers to a composition comprising at least one peptide according to the invention.

[0075] A composition of the invention is intended to contain one peptide of the invention or a combination or mixture of various peptides of the invention, preferably one peptide of the invention.

[0076] In a preferred embodiment, the composition of the present invention is a cosmetic composition.

[0077] The composition of the present invention comprises a cosmetically effective amount of at least one peptide of the present invention. More preferably, the composition of the present invention comprises 0.0001% (mass / volume in g / 100 mL, hereinafter m / v) to 0.05% (m / v) of at least one peptide of the present invention. In a most preferred embodiment, the composition of the present invention comprises 0.0001% (m / v) to 0.001% (m / v), more preferably 0.0005% (m / v), of at least one peptide of the present invention. In another most preferred embodiment, the composition of the present invention comprises 0.05% (m / v) to 0.001% (m / v) of at least one peptide of the present invention.

[0078] The compositions of the present invention, as a result of the activity of the peptides of the present invention, provide improvement of signs of skin associated with aging, such as, for example, wrinkles, roughness and / or sagging; or provide skin rejuvenation and / or improvement of skin imperfections, such as, for example, body firming, body sculpting, facial reshaping, skin tightening, and / or pore refinement.

[0079] The composition of the present invention is also intended to include at least one additional cosmetic ingredient, which may be at least one excipient and / or at least one additional cosmetic active ingredient.

[0080] Additional cosmetic ingredients include those commonly used in the art as, for example, adjuvants such as stabilizers, solubilizers, vitamins, colorants and fragrances; carriers; and / or other cosmetic active ingredients.

[0081] Said additional cosmetic ingredient must be physically and chemically compatible with other components of the composition, especially with the peptide of the present invention contained in the composition of the present invention.Similarly, the nature of said additional cosmetic ingredient must not unacceptably alter the benefits of the peptide and composition of the present invention.Said additional cosmetic ingredient may be synthetic or of natural origin, such as, for example, plant extracts, or said additional cosmetic ingredient may be derived from a biological fermentation process (see, for example, CTFA Cosmetic Ingredient Handbook, Eleventh Edition (2006)).

[0082] The abovementioned additional cosmetic ingredients are intended to include those ingredients commonly used in care compositions for the care of the skin and / or hair, for cleaning, and / or in deodorants and / or creams for preventing excessive sweating. These are, among others, for example: melanin synthesis inhibitors, whitening or depigmenting agents, anti-aging agents, NO synthase inhibitors, antioxidants, air pollution inhibitors and / or free radical scavengers, antiglycationic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioners such as humectants, moisturizing substances, alphahydroxy acids, moisturizing agents, vitamins, pigments or colorants, dyes, gelling polymers, thickeners, surfactants, softeners, other anti-wrinkle agents, agents capable of reducing or eliminating dark circles under the eyes, exfoliants, antibacterial agents, antifungal agents, bactericides, agents that promote the synthesis of macromolecules in the dermis or epidermis and / or agents capable of preventing or inhibiting their degradation, such as collagen synthesis promoters, elastin synthesis promoters, laminin synthesis promoters, collagen degradation inhibitors. anti-inflammatory agents, agents acting on capillary circulation and / or microcirculation, agents acting on cellular mitochondria, agents aimed at improving the dermal-epidermal junction, preservatives, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from biological fermentation processes, mineral salts, cell extracts and / or solar filters (organic or inorganic photoprotective agents active against ultraviolet A and B).

[0083] In one embodiment, at least one of the additional cosmetic ingredients is a cosmetic active ingredient or substance that can exert the same, similar, complementary or different cosmetic activity as disclosed above for the peptide of the present invention.The composition of the present invention is intended to include the following: other anti-wrinkle or anti-aging agents, such as collagen, elastin, growth factors, hyaluronic acid boosters, barrier function agents, illuminating agents, promoters of collagen I, III, IV and / or VI and laminin expression and / or synthesis; promoters of glycosaminoglycan or hyaluronic acid synthesis; promoters of elastin and other elastic fiber-related protein expression and / or synthesis; inhibitors of collagen and / or elastic fiber degradation; promoters of mitochondria-related protein (e.g., sirtuins and aconitase) expression and / or synthesis. and / or promoters of synthesis; promoters of expression and / or synthesis of focal adhesion proteins; promoters of proliferation and / or differentiation of keratinocytes and / or fibroblasts; antioxidants; antipollutants and / or free radical scavengers; antiglycation agents; detoxifying agents; agents that reduce chronological, environmental and inflammatory aging; and agents that reduce melanin production and / or inhibit tyrosinase, and / or promoters of lipid synthesis and synthesis of epidermal components (keratin), more specifically the stratum corneum (keratin, ceramides, filaggrin, loricrin and SPRR1B). More preferably, at least one of the additional cosmetic ingredients is Argireline® (acetyl hexapeptide-8), Leuphasyl® (pentapeptide-3), Inyline® (acetyl hexapeptide-30), Syn-Ake® (tripeptide-3), or a combination thereof.

[0084] Furthermore, the compositions of the present invention (or the peptides of the present invention) can be formulated in any form commonly used in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, powders, sprays, lotions, oils, liniments, serums, mousses, ointments, sticks or brushes, including "leave-on" and "rinse-off" formulations. The compositions of the present invention can also be incorporated into various types of solid accessories, such as napkins, hydrogels, adhesive (or non-adhesive) patches or face masks, by techniques known in the art, or the compositions of the present invention can be incorporated into various makeup-based products, such as concealers, makeup foundations, lotions or makeup removal lotions, among others.

[0085] It is also contemplated that the composition of the present invention or the peptide of the present invention, both of which are disclosed herein, can be incorporated into a cosmetic sustained release system and / or carrier, such as liposomes, milliparticles, microparticles and nanoparticles, as well as sponges, vesicles, micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, to obtain greater penetration of the active ingredient.

[0086] In a preferred embodiment, the compositions of the present invention are suitable or adapted to be applied by iontophoresis, more preferably to the face and / or body of a subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of a subject, even more preferably to the face and / or neck of a subject, preferably a human.

[0087] In a most preferred embodiment, the compositions of the present invention are suitable or adapted to be applied topically (more preferably in the form of a cream), more preferably to the face and / or body of a subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of a subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0088] As already mentioned above, in a third aspect, the present invention refers to the cosmetic use of a peptide or a composition of the invention in a subject in need thereof.

[0089] As mentioned above, preferably the composition of the present invention is a cosmetic composition.

[0090] In a preferred embodiment, the cosmetic use is to reduce, prevent and / or eliminate the signs of aging of the skin.

[0091] As is evident, the above-mentioned signs of skin aging are cosmetic signs of skin aging.

[0092] Skin aging is due to chronological and / or environmental aging.

[0093] The cosmetic signs of skin aging are preferably wrinkles, roughness and / or sagging, more preferably facial wrinkles, facial sagging and / or roughness, even more preferably facial wrinkles.

[0094] In another preferred embodiment, the cosmetic use is for skin rejuvenation and / or reducing, preventing and / or eliminating skin imperfections, more preferably for skin tightening, body sculpting, facial reshaping, skin tightening and / or pore refinement, more preferably for body tightening, body sculpting, facial reshaping, facial skin tightening and / or facial pore refinement, even more preferably for facial reshaping and / or facial skin tightening.

[0095] Body toning and / or body sculpting preferably refers to toning and / or sculpting the buttocks, chest, arms and / or legs.

[0096] Thus, the peptides and compositions of the present invention may also be used to treat loss of firmness or tightness of the skin, preferably loss of tightness of the skin, even more preferably loss of tightness of the facial skin.

[0097] As mentioned above, the peptides of the invention can also be used for the treatment of loss of organized collagen fibers, and therefore preferably for the treatment of facial remodeling. As can be directly derived from the examples below, this treatment of loss of organized collagen fibers is carried out by improving the production of at least the homeodomain protein Mohawk and collagen VI.

[0098] Also in preferred embodiments, the subject is a mammal, and even more preferably a human.

[0099] In a preferred embodiment, the peptide or composition of the invention is applied by iontophoresis, more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of the subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0100] In a most preferred embodiment, the peptide or composition of the invention is applied topically (more preferably in the form of a cream), more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of the subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0101] In addition, in the use as a cosmetic product of the present invention, the peptide or composition of the present invention is used in an amount effective as a cosmetic product. More preferably, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.05% (m / v). In a most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.001% (m / v), more preferably 0.0005% (m / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (m / v) to 0.001% (m / v).

[0102] As already mentioned above, the composition of the present invention is also intended to include at least one additional cosmetic ingredient. Said additional cosmetic ingredient may be at least one excipient and / or at least one additional cosmetic active ingredient, which may be as described above. It is also intended that the peptide of the present invention is used in combination with at least one additional cosmetic ingredient as described above.

[0103] Furthermore, the peptides of the present invention and compositions of the present invention can be formulated in any form commonly used in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, powders, sprays, lotions, oils, liniments, serums, mousses, ointments, sticks or brushes, including "leave-on" and "rinse-off" formulations. The peptides and compositions of the present invention can also be incorporated into various types of solid accessories, such as napkins, hydrogels, adhesive (or non-adhesive) patches or face masks, by techniques known in the art, or the peptides and compositions of the present invention can be incorporated into various makeup-based products, such as concealers, makeup foundations, lotions or makeup removal lotions, among others.

[0104] It is also contemplated that the composition of the present invention or the peptide of the present invention, both of which are disclosed herein, can be incorporated into cosmetic sustained release systems and / or carriers, such as liposomes, milliparticles, microparticles and nanoparticles, as well as into sponges, vesicles, micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as into microemulsions and nanoemulsions, in order to obtain greater penetration of the active ingredients.

[0105] In a fourth aspect, the present invention refers to the cosmetic use of a peptide or composition of the invention (i.e. as described above) in a subject in need thereof.

[0106] As mentioned above, preferably the composition of the present invention is a cosmetic composition.

[0107] In a preferred embodiment, the cosmetic application is to reduce, prevent and / or eliminate the signs of aging of the skin.

[0108] As is evident, the above-mentioned signs of skin aging are cosmetic signs of skin aging.

[0109] Skin aging is due to chronological and / or environmental aging.

[0110] The cosmetic signs of skin aging are preferably wrinkles, roughness and / or sagging, more preferably facial wrinkles, facial sagging and / or roughness, even more preferably facial wrinkles.

[0111] In another preferred embodiment, the cosmetic application is for skin rejuvenation and / or reducing, preventing and / or eliminating skin imperfections, more preferably for skin tightening, body sculpting, facial reshaping, skin tightening and / or pore refinement, more preferably for body tightening, body sculpting, facial reshaping, facial skin tightening and / or facial pore refinement, even more preferably for facial reshaping and / or facial skin tightening.

[0112] Body toning and / or body sculpting preferably refers to toning and / or sculpting the buttocks, chest, arms and / or legs.

[0113] Thus, the peptides and compositions of the present invention may also be used to treat loss of firmness or tightness of the skin, preferably loss of tightness of the skin, even more preferably loss of tightness of the facial skin.

[0114] As mentioned above, the peptides of the invention can also be used for the treatment of loss of organized collagen fibers, and therefore preferably for the treatment of facial remodeling. As can be directly derived from the examples below, this treatment of loss of organized collagen fibers is carried out by improving the production of at least the homeodomain protein Mohawk and collagen VI.

[0115] Also in preferred embodiments, the subject is a mammal, and even more preferably a human.

[0116] In a preferred embodiment, the peptide or composition of the present invention is applied by iontophoresis, more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, legs, arms, chest and / or buttocks of the subject, even more preferably to the face and / or neck of the subject.

[0117] In most embodiments, the peptides or compositions of the invention are applied topically (more preferably in the form of a cream), more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of the subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0118] In addition, in the cosmetic use of the present invention, the peptide or composition of the present invention is used in a cosmetically effective amount. More preferably, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.05% (m / v). In a most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.001% (m / v), more preferably 0.0005% (m / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (m / v) to 0.001% (m / v).

[0119] As already mentioned above, the composition of the present invention is also intended to include at least one additional cosmetic ingredient. Said additional cosmetic ingredient may be at least one excipient and / or at least one additional cosmetic active ingredient, which may be as described above. It is also intended that the peptide of the present invention is used in combination with at least one additional cosmetic ingredient as described above.

[0120] Furthermore, the peptides of the present invention and compositions of the present invention can be formulated in any form commonly used in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, powders, sprays, lotions, oils, liniments, serums, mousses, ointments, sticks or brushes, including "leave-on" and "rinse-off" formulations. The peptides and compositions of the present invention can also be incorporated into various types of solid accessories, such as napkins, hydrogels, adhesive (or non-adhesive) patches or face masks, by techniques known in the art, or the peptides and compositions of the present invention can be incorporated into various makeup-based products, such as concealers, makeup foundations, lotions or makeup removal lotions, among others.

[0121] It is also contemplated that the composition of the present invention or the peptide of the present invention, both of which are disclosed herein, can be incorporated into cosmetic sustained release systems and / or carriers, such as liposomes, milliparticles, microparticles and nanoparticles, as well as into sponges, vesicles, micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as into microemulsions and nanoemulsions, in order to obtain greater penetration of the active ingredients.

[0122] As mentioned above, in a fifth aspect, the present invention refers to a cosmetic method, characterized in that it comprises the use of a peptide or a composition according to the invention in a subject in need thereof.

[0123] As mentioned above, preferably the composition of the present invention is a cosmetic composition.

[0124] As can be derived from the above, the use of the peptide or composition according to the present invention in the cosmetic method of the present invention is a use as a cosmetic product.

[0125] In a preferred embodiment, the cosmetic method is to reduce, prevent and / or eliminate the signs of skin aging in a subject in need thereof.

[0126] As is evident, the above-mentioned signs of skin aging are cosmetic signs of skin aging.

[0127] Skin aging is due to chronological and / or environmental aging.

[0128] The cosmetic signs of skin aging are preferably wrinkles, roughness and / or sagging, more preferably facial wrinkles, facial sagging and / or roughness, even more preferably facial wrinkles.

[0129] In another preferred embodiment, the cosmetic method is for skin rejuvenation and / or reducing, preventing and / or eliminating skin imperfections in a subject in need thereof, more preferably for skin tightening, body sculpting, facial reshaping, skin tightening and / or pore refinement, more preferably for body tightening, body sculpting, facial reshaping, facial skin tightening and / or facial pore refinement, even more preferably for facial reshaping and / or facial skin tightening.

[0130] Body toning and / or body sculpting preferably refers to toning and / or sculpting the buttocks, chest, arms and / or legs.

[0131] Thus, the peptides and compositions of the present invention may also be used to treat loss of firmness or tightness of the skin, preferably loss of tightness of the skin, even more preferably loss of tightness of the facial skin.

[0132] As mentioned above, the peptides of the invention can also be used for the treatment of loss of organized collagen fibers, and therefore preferably for the treatment of facial remodeling. As can be directly derived from the examples below, this treatment of loss of organized collagen fibers is carried out by improving the production of at least the homeodomain protein Mohawk and collagen VI.

[0133] Also in preferred embodiments, the subject is a mammal, and even more preferably a human.

[0134] In a preferred embodiment, the peptide or composition of the invention is applied by iontophoresis, more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of the subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0135] In a most preferred embodiment, the peptide or composition of the invention is applied topically (more preferably in the form of a cream), more preferably to the face and / or body of the subject, more preferably to the face, neck, hands, arms, legs, chest and / or buttocks of the subject, even more preferably to the face and / or neck of a subject (preferably a human).

[0136] Preferably, the subject is a mammal, and even more preferably a human.

[0137] In addition, in the cosmetic method of the present invention, the peptide or composition of the present invention is used in a cosmetically effective amount. More preferably, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.05% (m / v). In a most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (m / v) to 0.001% (m / v), more preferably 0.0005% (m / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (m / v) to 0.001% (m / v).

[0138] As already mentioned above, the composition of the present invention is also intended to include at least one additional cosmetic ingredient. Said additional cosmetic ingredient may be at least one excipient and / or at least one additional cosmetic active ingredient, which may be as described above. It is also intended that the peptide of the present invention is used in combination with at least one additional cosmetic ingredient as described above.

[0139] Furthermore, the peptides of the present invention and compositions of the present invention can be formulated in any form commonly used in the art, such as solutions, suspensions, emulsions, pastes, gels, creams, powders, sprays, lotions, oils, liniments, serums, mousses, ointments, sticks or brushes, including "leave-on" and "rinse-off" formulations. The peptides and compositions of the present invention can also be incorporated into various types of solid accessories, such as napkins, hydrogels, adhesive (or non-adhesive) patches or face masks, by techniques known in the art, or the peptides and compositions of the present invention can be incorporated into various makeup-based products, such as concealers, makeup foundations, lotions or makeup removal lotions, among others.

[0140] It is also contemplated that the composition of the present invention or the peptide of the present invention, both of which are disclosed herein, can be incorporated into cosmetic sustained release systems and / or carriers, such as liposomes, milliparticles, microparticles and nanoparticles, as well as into sponges, vesicles, micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as into microemulsions and nanoemulsions, in order to obtain greater penetration of the active ingredients.

[0141] To enable a better understanding, the invention will now be explained in more detail with reference to the accompanying drawings, which are presented by way of example and with reference to illustrative and non-limiting examples.

[0142] Figure 1 shows the percentage of HEKa cell viability after treatment with increasing concentrations of peptide Pal-SEQ ID NO:1-NH2 compared to the baseline condition (i.e., the cell viability of the baseline condition is set as 100% and then compared to the remaining samples tested). For Figure 1, the columns correspond from left to right on the x-axis to the following: baseline condition (cells without treatment) and cells treated with 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL of peptide Pal-SEQ ID NO:1-NH2, respectively. The y-axis shows the percentage of cell viability (relative to the baseline condition).

[0143] Figure 2 shows the percentage of HDFa cell viability after treatment with peptide Pal-SEQ ID NO:1-NH2 compared to the baseline condition (i.e., the cell viability of the baseline condition is set as 100% and then compared to the remaining samples tested). For Figure 2, the columns correspond from left to right on the x-axis to the following: baseline condition (cells without treatment) and cells treated with peptide Pal-SEQ ID NO:1-NH2 at 0.001 mg / mL, 0.005 mg / mL, and 0.01 mg / mL, respectively. The y-axis shows the percentage of cell viability (relative to the baseline condition). ** indicates p<0.01 and *** indicates p<0.001.

[0144] Figure 3 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 relative to untreated primary human dermal fibroblasts. The gene expression profile was normalized by the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Figure 3 shows the results obtained for treatment with the peptide (at a concentration of 0.005 mg / mL for 6 hours for all genes). Here, the bars refer to the following genes from top to bottom, respectively: MMP3 (matrix metalloproteinase-3), MMP1 (matrix metalloproteinase-1), COL14A1 (collagen type XIV alpha 1 chain), COL12A1 (collagen type XII alpha 1 chain), COL7A1 (collagen type VII alpha 1 chain), COL6A1 (collagen type VI alpha 1 chain), COL5A1 (collagen type V alpha 1 chain), COL4A5 (collagen type IV alpha 5 chain), COL3A1 (collagen type III alpha 1 chain), MKX (Mohawk homeobox), and ZEB2 (zinc finger E-Box binding homeobox 2). The x-axis refers to the fold change relative to the baseline state. A negative fold change refers to downregulation of gene expression, whereas a positive fold change refers to upregulation.

[0145] Figure 4 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 versus untreated primary human dermal fibroblasts. The gene expression profile was normalized by the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Figure 4 shows the results obtained for treatment with the above peptide (at a concentration of 0.005 mg / mL for 24 hours), where the bars correspond to the following genes: COL13A1 (collagen type XIII alpha 1 chain). The x-axis refers to the fold change relative to the baseline state. A negative fold change refers to a downregulation of gene expression, whereas a positive fold change refers to an upregulation.

[0146] FIG. 5 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 versus untreated primary human dermal fibroblasts. Gene expression profile was normalized by the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase). FIG. 5 shows the results obtained for treatment with the above peptides (6 hours for all genes at a concentration of 0.005 mg / mL). Here, the bars refer to the following genes, respectively, from top to bottom: TGFB1 (transforming growth factor beta-1), FN1 (fibronectin 1), LOXL3 (lysyl oxidase 3-like), LOXL2 (lysyl oxidase 2-like), and HSP47 (serpin H1). The x-axis refers to the fold change relative to the baseline state. A negative fold change refers to a downregulation of gene expression, whereas a positive fold change refers to an upregulation.

[0147] Figure 6 shows the percentage increase in collagen VI synthesis by human dermal fibroblasts treated with increasing concentrations of peptide Pal-SEQ ID NO:1-NH2 compared to the negative control (treated with dimethylsulfoxide) (i.e., collagen VI synthesis of the negative control is set as 100% and then compared to collagen VI synthesis of the remaining samples tested). For Figure 6, the columns correspond from left to right on the x-axis to the following: negative control (cells treated with dimethylsulfoxide) and cells treated with 0.001 mg / mL, 0.005 mg / mL, and 0.01 mg / mL of peptide Pal-SEQ ID NO:1-NH2, respectively. The y-axis shows the percentage of collagen VI synthesis (relative to the negative control). ** indicates p<0.01 and *** indicates p<0.001.

[0148] Figure 7 shows the percentage increase in Mohawk synthesis by human dermal fibroblasts treated with increasing concentrations of peptide Pal-SEQ ID NO:1-NH2 compared to the negative control (treated with dimethylsulfoxide) (i.e., the Mohawk synthesis of the negative control is set as 100% and then compared to the Mohawk synthesis of the remaining samples tested). For Figure 7, the black columns refer to 24 hours of treatment and the white columns refer to 48 hours of treatment. Furthermore, the groups of each of the two columns (one black and one white) correspond from left to right on the x-axis of this figure to the following: negative control (cells treated with dimethylsulfoxide), untreated cells, and cells treated with 0.001 mg / mL, 0.005 mg / mL, and 0.01 mg / mL of peptide Pal-SEQ ID NO:1-NH2, respectively. The y-axis shows the percentage of Mohawk synthesis (relative to the negative control). ** indicates p<0.01, and *** indicates p<0.001.

[0149] Figure 8 shows collagen cross-linking. The line marked "a" refers to cells treated with peptide Pal-SEQ ID NO:1-NH2 at a concentration of 0.04 mg / mL; the line marked "b" refers to cells treated with peptide Pal-SEQ ID NO:1-NH2 at a concentration of 0.02 mg / mL; the line marked "c" refers to cells treated with dimethylsulfoxide (negative control). The x-axis shows time in seconds and the y-axis shows optical density in absorbance units at 450 nm.

[0150] Figure 9 shows tissue viability by percentage of released LDH (lactate dehydrogenase) compared to negative control (untreated hOSEC) (i.e., the released LDH of the negative control is set as 100% and then compared to the released LDH in the remaining samples tested in Example 11). The line with diamonds refers to untreated hOSEC (negative control); the line with squares refers to senescent hOSEC; the line with triangles refers to senescent hOSEC + product A (cream); the line with crosses refers to senescent hOSEC + product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2). The x-axis represents time in days and the y-axis represents % of released LDH relative to the negative control (untreated hOSEC).

[0151] Figure 10 shows metabolic activity by percentage of resorufin compared to negative control (untreated hOSEC) (i.e., the resorufin of the negative control is set as 100% and then compared to the resorufin in the remaining samples tested in Example 11). The line with diamonds refers to untreated hOSEC (negative control); the line with squares refers to aged hOSEC; the line with triangles refers to aged hOSEC + Product A (cream); the line with crosses refers to aged hOSEC + Product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2). The x-axis represents time in days and the y-axis represents % of resorufin relative to the negative control (untreated hOSEC).

[0152] Figure 11 shows the quantification of collagen in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the amount of collagen in the negative control is set as 100% and then compared to the amount of collagen in the remaining samples tested). The columns correspond from left to right on the x-axis to the following: untreated hOSEC (negative control); aged hOSEC; aged hOSEC + product A (cream); and aged hOSEC + product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2). The y-axis refers to the percentage of collagen amount relative to the negative control. * indicates p<0.05; ** indicates p<0.01.

[0153] Figure 12 shows transmission electron microscopy images of the different experimental groups of Example 11. More precisely, Figure 12A corresponds to untreated hOSEC; Figure 12B corresponds to senescent hOSEC; Figure 12C corresponds to senescent hOSEC treated with product A (cream); Figure 12D corresponds to senescent hOSEC treated with product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2).

[0154] Figure 13 shows the collagen density in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the collagen density of the negative control is set as 100% and then compared to the collagen density of the remaining samples tested). The columns correspond from left to right on the x-axis to the following: untreated hOSEC (negative control); aged hOSEC; aged hOSEC + product A (cream); and aged hOSEC + product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2). The y-axis refers to the percentage of collagen density relative to the negative control. * indicates p<0.05.

[0155] Figure 14 shows the collagen fiber thickness in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the collagen fiber thickness of the negative control is set as 100% and then compared to the collagen fiber thickness of the remaining samples tested). The columns correspond from left to right on the x-axis to the following: untreated hOSEC (negative control); aged hOSEC; aged hOSEC + Product A (cream); and aged hOSEC + Product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2). The y-axis refers to the percentage of collagen fiber thickness relative to the negative control. * indicates p<0.05.

[0156] FIG. 15 shows the efficacy after topical application of Pal-SEQ ID NO:1-NH2 on 44 female volunteers (50% light pigmented (phototypes II-III) and 50% dark pigmented (phototypes V-VI)). A cosmetic formulation containing 2% (m / v) of Pal-SEQ ID NO:1-NH2 peptide from a 0.05% (m / v) stock or a placebo was applied to the entire face of the volunteers for 56 days. FIG. 15(A) shows the improvement of roughness on the volunteers' cheeks by AEVA (Eotech, France). The AEVA assessment is based on 3D images of the skin topography obtained by a stereo camera combined with an interference fringe projection system. On the x-axis, from left to right, the six column groups correspond respectively to: average of all volunteers on day 28, average of phototype V-VI volunteers on day 28, average of phototype II-III volunteers on day 28, average of all volunteers on day 56, average of phototype V-VI volunteers on day 58, average of phototype II-III volunteers on day 56, all counting from the start of treatment. Also on the x-axis, from left to right, in each column group, the columns correspond respectively to: volunteers treated with a cosmetic formulation (placebo), volunteers treated with the same formulation but also containing Pal-SEQ ID NO:1-NH2 peptide. The y-axis shows the percentage change in roughness parameter (Ra) relative to the start. FIG. 15(B) shows the improvement of the chiseled cheeks of volunteers by AEVA. On the x-axis, from left to right, the six column groups correspond respectively to: average of all volunteers on day 28, average of phototypes V-VI volunteers on day 28, average of phototypes II-III volunteers on day 28, average of all volunteers on day 56, average of phototypes V-VI volunteers on day 58, average of phototypes II-III volunteers on day 56, all counting from the start of treatment. Also on the x-axis, in each column group, from left to right, the columns correspond respectively to: volunteers treated with a cosmetic preparation (placebo), volunteers treated with the same preparation but also containing Pal-SEQ ID NO:1-NH2 peptide. The y-axis shows the percentage change in the engraving parameter (Rz) relative to the start.FIG. 15(C) shows the change in wrinkle depth (%) in the crow's feet wrinkle area. On the x-axis, from left to right, the six column groups correspond respectively to: average of all volunteers on day 28, average of all volunteers on day 56, average of phototypes V-VI volunteers on day 28, average of phototypes V-VI volunteers on day 56, average of phototypes II-III volunteers on day 28, average of phototypes II-III volunteers on day 56, all counting from the start of treatment. Also on the x-axis, in each column group, from left to right, the columns correspond respectively to: volunteers treated with a cosmetic formulation (placebo), volunteers treated with the same formulation but also containing Pal-SEQ ID NO:1-NH2 peptide. The y-axis shows the percentage change in wrinkle depth in the crow's feet wrinkle area relative to the start.

[0157] [Example] [Abbreviation] The abbreviations used for amino acids follow the 1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, as outlined in Eur. J. Biochem. (1984).

[0158] Ac, acetyl; Ala, alanine; Arg, arginine; C-terminus, carboxy-terminus; DCM, dichloromethane; DIEA, N,N'-diisopropylethylamine; DIPCDI, N,N'-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; EDGS, EpiLife Defined Growth Supplement; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; His, histidine; HOBt, 1-hydroxybenzotriazole; hOSEC, human organotypic skin explant culture; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; Ile, isoleucine; LSGS, Low Serum Growth Supplement;MBHA, p-methylbenzhydrylamine;Leu, leucine;Lys, lysine;Me, methyl;MeCN, acetonitrile;MeOH, methanol;Met, methionine;N-terminus, amino-terminus;Pal, palmitoyl;Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;Phe, phenylalanine;rpm, revolutions per minute;RT, room temperature;tBu, tert-butyl;TFA, trifluoroacetic acid;TIS, triisopropylsilane;TMB, 3,3',5,5'-tetramethylbenzidine;Trt, triphenylmethyl or trityl;Trp, tryptophan;Tyr, tyrosine;Val, valine.

[0159] Regarding the chemical synthesis procedures included in this example, it should be noted that all synthesis processes were carried out in polypropylene syringes fitted with porous polyethylene disks or in Pyrex® reactors fitted with porous plates. All reagents and solvents were of synthesis quality and were used without any additional treatment. Solvents and soluble reagents were removed by aspiration. Fmoc groups were removed with piperidine-DMF (2:8, v / v) (at least 1×1 min, 2×10 min, 5 mL / g resin) (Lloyd Williams P. et al, (1997), Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton (Fla., USA)). Washing between deprotection, coupling and deprotection again steps was performed with DMF (3×1 min) and DCM (3×1 min) each time using 10 ml solvent / g resin. Coupling reactions were carried out with 3 ml solvent / g resin. Coupling was controlled by carrying out the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34: 595-598). All synthesis reactions and washings were carried out at room temperature.

[0160] Example 1. Synthesis and preparation of peptides Obtain Fmoc-AA1-AA2-AA3-AA4-Rink-MBHA-resin (wherein AA1 is L-His; AA2 is L-Tyr; AA3 is L-Arg; AA4 is L-Ala). Weight was standardized. To remove Fmoc group, 4.8g (2.5mmol) of Fmoc-Rink-MBHA resin with 0.52mmol / g functionalization was treated with piperidine-DMF as described in the general protocol known in the art. In the presence of DIPCDI (1.17mL; 7.5mmol; 3eq.) and HOBt (1.01g; 7.5mmol; 3eq.), 2.33g of Fmoc-L-Ala-OH (7.5mmol; 3eq.) was loaded onto the deprotected resin using DMF as solvent for 1 hour.

[0161] The resin was then washed as described in the general methods known in the art, the deprotection of the Fmoc group was repeated and the next amino acid was coupled. Following the protocol described above, 4.87 g of Fmoc-Arg(Pbf)-OH (7.5 mmol; 3 eq.) were coupled in sequence, followed by 3.45 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 eq.) and then 4.65 g of Fmoc-L-His(Trt)-OH (7.5 mmol; 3 eq.), in the presence of 1.01 g of HOBt (7.5 mmol; 3 eq.) and 1.17 mL of DIPCDI (7.5 mmol; 3 eq.), respectively. Between each amino acid addition step, the deprotection of the Fmoc group was carried out as already described above.

[0162] After synthesis, the peptide resin was washed with DCM (5 times for 3 min each) and dried under vacuum.

[0163] Using the synthesis procedure described above, the following sequences were synthesized: His-Tyr-Arg-Ala (SEQ ID NO: 1).

[0164] Example 2. Removal of the Fmoc N-terminal protecting group of the peptide synthesized in Example 1 The N-terminal Fmoc group of the peptidyl resin was deprotected with 20% (volume / volume, hereafter, v / v) piperidine in DMF (1×1 min+2×10 min) (Lloyd Williams P. et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton (Fla., USA)). The peptidyl resin was washed with DMF (5×1 min), DCM (4×1 min) and dried under vacuum.

[0165] Example 3. Process for introducing R1 palmitoyl group onto the peptidyl resin obtained by Example 2 1 mmol (1 eq.) of the peptidyl resin obtained according to Example 2 was treated with 10 eq. of hexadecenoic acid (palmitic acid) in the presence of 10 eq. of DIEA and 10 eq. of HOBt, using 5 mL of DMF as solvent. After reacting them for 30 min, the peptidyl resin was washed with DMF (5×1 min), DCM (4×1 min) and dried under vacuum.

[0166] Example 4. Cleavage process of peptidyl resin obtained by Examples 2 and 3 from the polymer support Weights were standardized. 200 mg of dried peptidyl resin obtained in either Example 2 or 3 was treated with 5 mL of TFA / TIS / H2O (90:5:5) for 2 h at room temperature with stirring. The filtrate was collected and precipitated with 50 mL (8-10 times) of cold diethyl ether. The ether solution was evaporated to dryness under reduced pressure and at room temperature, and the precipitate was redissolved in 50% (v / v) MeCN in H2O and lyophilized.

[0167] Example 5. Characterization of peptides synthesized and prepared according to Example 4. HPLC analysis of the peptides obtained according to Example 4 was carried out using a Shimadzu instrument (Kyoto, Japan). Here, a reversed-phase column (150×4.6 mm, XBridge Peptide BEH C18, 3.5 μm, Waters, USA) was used in a gradient of MeCN (+0.036% (v / v) TFA) in HO (+0.045% (v / v) TFA) at a flow rate of 1.25 mL / min, with detection at 220 nm. All peptides showed a purity of more than 80%. The identity of the obtained peptides was confirmed by ESI-MS in a Water ZQ 4000 detector at a flow rate of 0.2 mL / min using MeOH as the mobile phase. The obtained results demonstrated that the peptide Pal-His-Tyr-Arg-Ala-NH2 (Pal-SEQ ID NO:1-NH2) was synthesized accurately and effectively.

[0168] Example 6. Analysis of the cytotoxicity of peptide Pal-SEQ ID NO:1-NH2 in HEKa and HDFa cells. The cytotoxicity of peptide Pal-SEQ ID NO:1-NH2 was analyzed by viability assay in HEKa (human epidermal keratinocytes, adult) and HDFa (human dermal fibroblasts, adult) cells.

[0169] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0170] Cytotoxicity was assessed by MTT assay. Briefly, HEKa and HDFa cells were cultured at 1 × 10 5 The cells were seeded in 96-well plates at a concentration of 10 ... After 24 h of treatment with the corresponding concentrations of peptides, 10 μL of yellow tetrazolium tetrazolium (MTT) was added to each well and the cells were incubated for another 4 h at 37 °C. After incubation with MTT, the medium in each well was aspirated and 150 μL of dimethyl sulfoxide (DMSO) (100%) was added to solubilize the formazan crystals formed. To complete the solubilization of the crystals, the plate was placed on a shaker for 5 min and then the absorbance at 450 nm of each well was determined using the microplate reader Multiskan FC. The absorbance is directly proportional to the number of living cells in the culture. The absorbance values ​​were normalized using the data obtained for untreated cells (reference condition).

[0171] The results are summarized in Figures 1 and 2.

[0172] As can be directly derived from the figure, the peptide of the invention (exemplified by Pal-SEQ ID NO:1-NH2) did not alter the viability of HEKa and HDFa cells at the concentrations tested, and is therefore not cytotoxic.

[0173] Example 7. Analysis of gene expression regulation in primary human dermal fibroblasts. The peptide Pal-SEQ ID NO:1-NH2 was analyzed for its ability to modulate the expression of genes related to collagen and extracellular matrix production (see Table 1 for genes analyzed).

[0174] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0175] Stock solutions of peptides were prepared at 12.5 mg / mL in DMSO. Standard curve solutions were freshly prepared from the stock solutions at the specified concentrations in the corresponding supplemented medium.

[0176] Untreated cells were used as a negative control sample.

[0177] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, 4 × 10 5 HDFa cells were seeded in duplicate (n=2) at a density of cells / well and maintained under standard culture conditions (106 medium supplemented with 1% (v / v) LSGS; 37°C, 95% room humidity, 5% CO2) for 24 hours. Cells were then treated with peptide Pal-SEQ ID NO:1-NH2 at a concentration of 0.005 mg / mL for an additional 6 hours (or 24 hours for COL13A1). Untreated cells were used as reference control.

[0178] Finally, cells were lysed and replicates were pooled together for RNA extraction using the Qiagen RNeasy Mini kit according to the manufacturer's instructions. The purified RNA was used to generate the corresponding cDNA (complementary deoxyribonucleic acid) by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), a commercially available kit that serves as a template for amplification. RT-qPCR was performed using a StepOne+ Real-Time PCR machine with a panel of appropriate TaqMan assay probes against the genes shown in Table 1 (plus GAPDH-glyceraldehyde 3-phosphate dehydrogenase, used as a housekeeping gene), and 2x Gene Expression Master Mix. Amplification involved 40 cycles of: 95°C for 15 s (denaturation) and 60°C for 1 min (annealing and extension); (Arya, M., Shergill, IS, Williamson, M., Gommersall, L., Arya, N., Patel, HR (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

[0179] [Table 1]

[0180] The data obtained were analyzed using the ΔΔCt method, which provides expression values ​​of the target genes as fold changes in treated samples compared to untreated reference samples. Both samples were normalized using the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0181] The steps for analysis include: 1. Calculate the average Ct for each condition. 2. Calculate the ΔCT test sample and the ΔCT untreated sample; 3. Calculate ΔΔCT (ΔΔCT = ΔCT test sample - ΔCT untreated sample); 4.2 ΔΔCT The ratio is obtained by

[0182] The results of this assay are summarized in Figures 3-5.

[0183] As can be easily derived from the figure, the peptide Pal-SEQ ID NO:1-NH2 is: After 6 hours of treatment: Downregulates the genes MMP1 and MMP3, which, as is widely known, degrade extracellular matrix proteins. - After 6 hours of treatment: upregulates several key genes in collagen architecture and extracellular architecture: COL3A1, COL4A5, COL5A1, COL6A1, COL7A1, COL12A1, COL14A1, COL13A1 (after 24 hours of treatment), MKX y ZEB2; After -6 hours of treatment, it upregulates several collagen cross-linking related genes: TGFB1, FN1, LOXL3, LOXL2 and HSP47;

[0184] Thus, the peptides of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) avoid or prevent extracellular matrix degradation while contributing to increased collagen synthesis and cross-linking, and therefore exhibit anti-aging and rejuvenating activity.

[0185] Example 8. Analysis of collagen VI in human dermal fibroblasts The peptide Pal-SEQ ID NO:1-NH2 was analyzed for its ability to induce the synthesis of collagen VI in human dermal fibroblasts.

[0186] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0187] In this assay, the ability of test peptides to increase collagen VI synthesis in human dermal fibroblasts was evaluated in vitro by measuring the amount of collagen VI after treatment of the cells with Pal-SEQ ID NO:1-NH2.

[0188] The experimental protocol defined the following experimental groups: - Negative control: cell cultures treated with the solubilization vehicle (DMSO) only; - Cell cultures treated with peptide Pal-SEQ ID NO:1-NH2 in three concentrations (0.01 mg / mL, 0.005 mg / mL and 0.001 mg / mL).

[0189] Stock solutions of the peptides were prepared in DMSO and then three serial dilutions of 0.01 mg / mL, 0.005 mg / mL and 0.001 mg / mL were prepared in cell culture medium.

[0190] The biological model used in this case consisted of normal human dermal fibroblasts. The cells were plated in 96-well plates at 1 × 10 4 Cells / well were seeded and maintained under standard culture conditions (37°C, 95% room humidity, 5% CO2) for 24 hours.

[0191] After 24 hours of incubation, the medium was removed and new medium containing treatment (DMSO or Pal-SEQ ID NO:1-NH2) was added to the wells. Sample treatment was continued for 48 hours, and at the end of treatment, cell culture medium was collected. DMSO-treated cells were used as negative control.

[0192] To carry out the test, cell cultures of human dermal fibroblasts were treated with Pal-SEQ ID NO:1-NH2 at the three concentrations mentioned above.

[0193] After 48 hours of treatment, the amount of collagen VI produced and released by the cells (exonov collagen VI synthesis) was measured in the cell culture medium by ELISA assay. The results were compared with those of the negative control (cells treated with DMSO). Treatments were performed in triplicate and in three different experimental sessions.

[0194] Quantification of collagen VI was performed by ELISA method. For this purpose, a commercial kit was used. The test kit detected the content of human collagen VI using a sandwich ELISA method. The micro ELISA plate provided with the kit was precoated with an antibody specific for collagen VI. Standards or samples were added to the appropriate micro ELISA plate wells and allowed to bind with the specific antibody. Then, a biotinylated detection antibody specific for collagen VI and an avidin-horseradish peroxidase (HRP) complex were added successively to the microplate wells, respectively, and incubated. The free components were washed away. Substrate solution (TMB) was added to each well. Only wells containing collagen VI developed a blue color. The enzyme-substrate reaction was stopped by the addition of sulfuric acid solution, and the color changed to yellow. The optical density (OD) was measured by a microplate reader at a wavelength of 450 nm. The OD value was proportional to the concentration of collagen VI.

[0195] Quantitative measurements were made using a standard curve consisting of increasing concentrations of known collagen VI standards. The % variation in collagen VI content between the negative control (DMSO) and the samples was calculated, which is a direct indicator of the effect of Pal-SEQ ID NO:1-NH2 in increasing collagen VI synthesis.

[0196] The results obtained in this experiment are summarized in Table 2 and FIG.

[0197] [Table 2]

[0198] As can be directly derived from Table 2 and Figure 6, in all cases (i.e. for all concentrations tested), a statistically significant increase in collagen VI synthesis was observed. This could further be described as dose-dependent: 11% increase at 0.001 mg / mL, 16% increase at 0.005 mg / mL, and 40% increase at 0.01 mg / mL. As shown in Figure 6, the difference in collagen VI content observed in the treated groups with respect to the negative control is statistically significant.

[0199] The above results demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) increases the synthesis of the protein collagen VI and thus contributes to improving extracellular matrix assembly. Thus, said peptide can prevent or treat skin aging and / or skin manifestations of said aging, such as wrinkles, roughness and / or sagging.

[0200] Furthermore, the results of this example also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0201] Example 9. Analysis of Mohawk synthesis in human dermal fibroblasts. The peptide Pal-SEQ ID NO:1-NH2 was analyzed for its ability to induce Mohawk synthesis in human dermal fibroblasts.

[0202] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0203] This assay involves the in vitro evaluation of the ability of the peptide Pal-SEQ ID NO:1-NH2 to increase the synthesis of the homeodomain protein Mohawk in human dermal fibroblasts by measuring the amount of Mohawk after treatment of the cells with said peptide.

[0204] The experimental protocol defined the following experimental groups: - untreated cell cultures; - cell cultures treated with solubilization vehicle (DMSO) alone (negative control); - Cell cultures treated with Pal-SEQ ID NO:1-NH2 at three concentrations (0.01 mg / mL, 0.005 mg / mL and 0.001 mg / mL).

[0205] Stock solutions of the peptides were prepared in DMSO and then three serial dilutions of 0.01 mg / mL, 0.005 mg / mL and 0.001 mg / mL were prepared in cell culture medium.

[0206] The biological model used consisted of normal human dermal fibroblasts, as described above.

[0207] Cells were plated in a 96-well plate at 1 x 10 4 Cells / well were seeded and maintained under standard culture conditions (37° C., 95% room humidity, 5% CO2) for 24 hours. After 24 hours of incubation, the medium was removed and fresh medium containing the test product was added to the wells.

[0208] Sample treatment was continued for 24 and 48 hours, then cells were lysed and intracellular Mohawk concentrations were determined by ELISA. Results were compared with negative controls (DMSO-treated cells).

[0209] Treatments were performed in triplicate and in three different experimental sessions.

[0210] Measurement of Mohawk synthesis was performed by ELISA method as described above. For this purpose, a commercial kit was used. The test kit applied a two-site sandwich ELISA method to detect the content of human homeodomain protein Mohawk. Mohawk-specific antibody was precoated on a microplate. Standards and samples were pipetted into the wells, and any Mohawk present was allowed to bind to the immobilized antibody. After removing unbound material, HRP-conjugated human Mohawk detection antibody was added to the wells. After washing and removing any unbound HRP reagent, a chromogen solution was added to the wells (substrate was TMB) and color was developed in proportion to the amount of Mohawk bound in the first step. Color development was stopped and the concentration was quantified colorimetrically at 450 nm.

[0211] Quantitative measurements were performed using a standard curve consisting of increasing concentrations of known Mohawk standards. The % variation in Mohawk content between the negative control (DMSO) and Pal-SEQ ID NO:1-NH2 treated groups was calculated. This was a direct indicator of the effect of peptide Pal-SEQ ID NO:1-NH2 in increasing Mohawk synthesis.

[0212] The results obtained are summarized in Table 3 and FIG.

[0213] [Table 3] JPEG0007652423000004.jpg161169

[0214] As can be directly derived from Table 3 and Figure 7, in all cases (i.e., for all concentrations and durations tested), a statistically significant increase in the synthesis of the homeodomain protein Mohawk was observed. This can be further described as dose-dependent: at 24 hours, 0.001 mg / mL increased by 9%, at 0.005 mg / mL increased by 21%, and at 0.01 mg / mL increased by 43%; and at 48 hours, 0.001 mg / mL increased by 31%, at 0.005 mg / mL increased by 51%, and at 0.01 mg / mL increased by 81%. As shown in Figure 7, the difference in Mohawk content observed in the treatment groups relative to the negative control at each time frame tested is statistically significant.

[0215] The results described above demonstrate that the peptides of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) increase the synthesis of Mohawk and therefore demonstrate their potential for preventing or treating skin aging and / or aging-related skin symptoms such as wrinkles, roughness and / or sagging.

[0216] Furthermore, the results of this example also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0217] Example 10. Analysis of collagen cross-links The peptide Pal-SEQ ID NO:1-NH2 was analyzed for its ability to induce or improve collagen cross-linking.

[0218] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0219] This assay involves the in-tube evaluation of the ability of peptide Pal-SEQ ID NO:1-NH2 to increase and promote collagen fibrillogenesis / collagen cross-linking, by measuring collagen fibrillogenesis after collagen treatment with a solution of said peptide (0.02 M acetic acid, 0.125 M NaCl and 1 / 10 phosphate buffered saline (pH=7.4)) at room temperature.

[0220] The experimental protocol defined the following experimental groups: - collagen solution treated with Pal-SEQ ID NO:1-NH2 at two concentrations (0.02 or 0.04 mg / mL); - A collagen solution (negative control) identical to the treatment groups described above, but without peptide, treated with a solution containing DMSO in the same volume as the samples containing peptide.

[0221] Stock solutions of peptides were prepared in DMSO, and then two serial dilutions were prepared so that the final concentrations of peptide in the collagen solution were 0.02 mg / mL or 0.04 mg / mL.

[0222] Collagen cross-linking and fibril formation were followed immediately after collagen addition to the control (a solution similar to the treatment group but without peptide and containing the same volume of DMSO as the peptide-containing sample) and to the solution containing Pal-SEQ ID NO:1-NH2 of the treatment group.

[0223] The results were compared to a negative control (a solution containing DMSO and no Pal-SEQ ID NO:1-NH2).

[0224] The experiment was conducted in four different experimental sessions.

[0225] Collagen cross-linking was determined by measuring absorbance at 450 nm.

[0226] Samples were pipetted into the wells and the absorbance of each well was measured at 450 nm using a microplate reader Multiskan FC every 2 minutes for 100 minutes.

[0227] The % variation in collagen fibril formation between the negative control (group treated with DMSO only) and the group treated with Pal-SEQ ID NO:1-NH2 was calculated, which is a direct indicator of the effect of peptide Pal-SEQ ID NO:1-NH2 in increasing collagen fibril formation, and could be described as dose-dependent: 59% increase at 0.02 mg / mL and 111% increase at 0.04 mg / mL.

[0228] The results obtained are summarized in FIG.

[0229] As can be derived from FIG. 8, the peptide Pal-SEQ ID NO:1-NH2 increases and promotes collagen cross-linking at the two concentrations tested: 0.02(b) and 0.04(a) mg / mL.

[0230] The above supports the anti-aging activity of the peptides of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) and therefore the usefulness of said peptides in preventing or treating signs of aging-related skin, such as wrinkles, roughness and / or sagging.

[0231] Furthermore, the results of this example also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0232] Example 11. Analysis of cytotoxicity and collagen production of peptide Pal-SEQ ID NO:1-NH2 in human organotypic skin explant cultures. The peptide Pal-SEQ ID NO:1-NH2 was analyzed for cytotoxicity and collagen production by LDH cytotoxicity assay and resazurin assay; and collagen production by collagen content analysis and histological analysis.

[0233] Peptide Pal-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0234] In this case, healthy and aged human organotypic skin explant cultures (hOSECs) were used as the experimental system.

[0235] Senescent hOSECs were obtained by exposing healthy hOSECs to hydrocortisone (5 μg / mL) for the first 5 days of the study.

[0236] The total numbers in the treatment groups were as follows: 1. Control group (negative control): Untreated hOSEC.

[0237] 2. Senescent hOSECs: hOSECs treated with 5 μg / mL hydrocortisone (Abraham A, Roga G. (2014) Topical steroid-damaged skin. Indian J Dermatol. 59(5),456-9.).

[0238] 3. Aged hOSECs + Product A (cream, i.e., placebo): hOSECs treated with 5 μg / mL hydrocortisone and incubated with Product A (10 μL).

[0239] 4. Aged hOSECs + product B (cream containing Pal-SEQ ID NO:1-NH2 at a concentration of 0.005 mg / mL): hOSECs treated with 5 μg / mL hydrocortisone and incubated with product B (10 μL).

[0240] Four replicates of each experimental group were performed in one independent experiment.

[0241] The total duration of the assay was 10 days (days 1-10). Hydrocortisone was applied to the appropriate or corresponding group daily on days 1-5. On the other side, product B or product A was applied to the corresponding or corresponding group daily on days 2-9.

[0242] Cytotoxicity assays were performed on days 0 (before the start of the study), 1, 3, 5, 8 and 10.

[0243] Collagen production was measured on the 10th day.

[0244] [LDH cytotoxicity assay] The LDH cytotoxicity test is a colorimetric assay that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released into the culture medium supernatant upon damage to the cytoplasmic membrane. The LDH released into the culture medium supernatant is measured for 30 min by coupling an enzymatic reaction: LDH oxidizes lactate to pyruvate, which then reacts with the tetrazolium salt WST-1 to form formazan. An increase in the amount of formazan measured in the culture supernatant directly correlates with an increase in the number of lysed cells (damage) in the skin explants.

[0245] 100 μL of the supernatant from each sample was removed and transferred to a 96-well microplate. LDH released into the culture medium supernatant was measured by a coupled enzymatic reaction: LDH oxidizes lactate to pyruvate, which then reacts with the tetrazolium salt WST-1 to form formazan. Since the LDH enzyme is released into the culture medium supernatant when the cytoplasmic membrane is damaged, an increase in the amount of formazan directly correlates with an increase in the number of lysed cells (damage). The formazan dye is water-soluble and could be measured at 500 nm using a standard ELISA plate reader.

[0246] The results of this assay were calculated taking into account the negative control (i.e., untreated hOSECs as 100% of the LDH standard concentration). The results obtained are summarized in FIG.

[0247] As can be derived from Figure 9, the LDH values ​​found in all groups were similar to the control group (negative control). This fact indicates that hydrocortisone did not cause any adverse effects (no damage to the cell membrane) in hOSECs under the conditions of the assay. Similarly, treatment with product B (i.e., cream containing Pal-SEQ ID NO:1-NH2) or product A and 5 μg / mL hydrocortisone did not show any difference in LDH values ​​compared to the control group. These data suggest that incubation with hydrocortisone and both compounds did not cause any adverse effects, no damage to the cell membrane, in hOSECs under the conditions of the assay.

[0248] [Resazurin Assay] Resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is widely used as an indicator of cell viability in proliferation and cytotoxicity assays. The assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion takes place intracellularly and is facilitated by mitochondrial, microsomal and cytosolic oxidoreductases. Resazurin is non-toxic to cells and stable in culture medium. Thus, resazurin allows for the continuous measurement of cell proliferation in vitro, either as a kinetic or end-point assay.

[0249] Therefore, resazurin dye has been widely used as an indicator of cell viability in several cytotoxicity assays and is also an indicator of metabolic activity since the assay is based on the ability of metabolically active cells to reduce resazurin to resorufin and dihydroresorufin by mitochondrial, microsomal and cytosolic oxidoreductases.

[0250] Toxic insults that impair cell viability and proliferation also affect the culture's ability to reduce resazurin. The rate of dye reduction is directly proportional to the number of viable cells present. Thus, reduction of resazurin provides a convenient indicator of cell viability, as it is a direct measure of the metabolic capacity of the cell culture. A decrease in the amount of resazurin reduced by hOSEC also directly correlates to an increase in the number of dead cells.

[0251] For the resazurin assay, skin explants were treated with 6 μM resazurin in NaCl solution for 1 h. Then, a volume of 100 μL of each sample was removed and transferred to a 96-well microplate. The resorufin formed was quantified using a fluorometer plate reader.

[0252] The fluorescent signal was monitored using an excitation wavelength of 530-560 nm and an emission wavelength of 590 nm.

[0253] The results of the resazurin assay were calculated considering the negative control, untreated healthy hOSECs, as 100% viability. The results obtained are shown in Figure 10.

[0254] When hOSECs were treated with 5 μg / mL hydrocortisone, a 10% decrease in resorufin was observed compared to the control group (negative control). hOSECs treated with Product B (cream containing Pal-SEQ ID NO:1-NH2) or Product A and 5 μg / mL hydrocortisone did not show a decrease in resorufin levels compared to the control group (negative control).

[0255] These data support the idea that both product B or product A caused metabolic activation in hOSECs under the conditions of the assay. Since resorufin is a direct measure of the metabolic capacity of cells, an increase in resorufin may suggest metabolic activation of oxidoreductase enzymes following exposure of hOSECs to both compounds.

[0256] Therefore, from the results obtained in the LDH cytotoxicity assay and in the resazurin assay it can be deduced that Pal-SEQ ID NO:1-NH2 is not toxic and actually reverses the negative effects caused by hydrocortisone.

[0257] [Collagen production] Collagen content analysis: The collagen assay is a dye-binding method for the analysis of acid- and pepsin-soluble collagen, primarily type I, but also types II, III, IV, and V. This assay can assess the proportion of newly synthesized collagen produced during periods of rapid growth and development.

[0258] Collagen dye reagent (1 mL) was added to each sample (tube) and shaken for 30 minutes. The tubes were centrifuged at 12,000 rpm for 10 minutes. Then, 750 μL of ice-cold acid-salt wash reagent was added to the collagen-dye pellet to remove unbound dye from the surface of the pellet and the inner surface of the microcentrifuge tube. The tubes were centrifuged again at 12,000 rpm for 10 minutes. Finally, 250 μL of alkaline reagent was added. When all bound dye was dissolved (5 minutes), the samples were ready for measurement. Dissolved dye (200 μL of each sample in a 96-microwell plate) was measured at 550 nm using a standard ELISA plate reader.

[0259] As mentioned above, the analysis of collagen content was carried out on the 10th day of the study. The results of the collagen content (micrograms) assay were calculated per milligram of fresh dermal tissue. The results obtained are summarized in FIG. 11 and Table 4.

[0260] [Table 4]

[0261] As shown in FIG. 11 and Table 4, increased collagen production was observed in aged hOSECs treated with Product B (cream containing 0.005 mg / mL Pal-SEQ ID NO:1-NH2) compared to the remaining aged hOSEC groups (treated with 5 μg / mL hydrocortisone alone or with hydrocortisone and Product A).

[0262] The aged hOSEc + Product B group showed collagen levels equivalent to untreated hOSEC.

[0263] As shown in Figure 11, the difference in collagen content between the untreated hOSEC and senescent hOSEC + Product B groups and the senescent hOSEC and senescent hOSEC + Product A groups is statistically significant. There was no statistically significant difference between untreated hOSEC and senescent hOSEC + Product B.

[0264] These results demonstrate that topical application of Pal-SEQ ID NO:1-NH2 reverses the anti-aging effects of hydrocortisone, thus demonstrating the potential of the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) as an anti-aging product to prevent, reduce and / or eliminate signs of aging, such as wrinkles, roughness and / or sagging skin.

[0265] Furthermore, these results also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0266] Tissue collection and processing for TEM analysis: Skin samples were processed for transmission electron microscopy analysis. Briefly, skin samples were fixed in 4% (v / v) formaldehyde and 1% (v / v) glutaraldehyde. Subsequently, skin samples were incubated overnight in 0.1 M sucrose and 0.1 M 1% (v / v) osmium tetroxide. Finally, samples were dehydrated in serialized ethanol solutions and embedded in a beam capsule.

[0267] Ultrathin sections (60-90 nm thick) were stained with uranyl acetate for 15 min and lead citrate for 5 min, after which the samples were ready for analysis under an electron microscope.

[0268] Transmission electron microscopy was performed to observe the state of collagen fibers in the treated skin explants.

[0269] The results are summarized in Figures 12A-12D. Figure 12A corresponds to untreated hOSECs and showed the correct dermal structure (intact and structured dermal fibers in the dermis). Similarly, Figure 12B represents aged hOSECs with unstructured collagen fibers (diffuse and uncompacted fibers). This image confirms the adverse effect of hydrocortisone in the skin, which is in agreement with the collagen quantification (included above). For aged skin treated with product A, a similar image of uncompacted collagen fibers was obtained (Figure 12C). However, as can be seen in Figure 12D, when aged hOSECs were incubated with product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2), a good regeneration of collagen fibers (intact and undiffused fibers) was achieved. This image confirms the collagen values ​​obtained by the ELISA method, as described above.

[0270] Similarly, collagen fiber thickness and collagen density were analyzed by image analysis (see Tables 5 and 6 below). The values ​​obtained confirm the visual assessment.

[0271] Collagen density indicates the compactness of each collagen fiber observed on the TEM image. For this purpose, optical images were analyzed by GIMP2. Counterfactual analysis confirmed that the group of healthy aged hOSEC + Product B showed higher density values ​​than those observed in the groups of aged hOSEC and aged hOSEC + Product A (see Table 5 below and Figure 13).

[0272] The aged hOSEC + Product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2) group showed increased collagen production and, as described above, showed collagen density equivalent to untreated hOSEC (see Table 5 below and Figure 13).

[0273] [Table 5]

[0274] As shown in Figure 13, the difference in collagen density between the groups of untreated hOSEC and aged hOSEC + Product B and the groups of aged hOSEC and aged hOSEC + Product A was statistically significant. There was no statistically significant difference between untreated hOSEC and aged hOSEC + Product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2).

[0275] These results demonstrate that topical application of Pal-SEQ ID NO:1-NH2 reverses the anti-aging effects of hydrocortisone, thus demonstrating the potential of the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) as an anti-aging product to prevent, reduce and / or eliminate signs of aging, such as wrinkles, roughness and / or sagging skin.

[0276] Furthermore, these results also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0277] Collagen fiber thickness was also analyzed by GIMP2. Fiber thickness analysis confirmed that the group of healthy aged hOSECs + product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2) showed lower fiber thickness values ​​than those observed in the groups of aged hOSECs and aged hOSECs + product A, due to better compaction and structuring (complete and not diffuse fibers). Pal-SEQ ID NO:1-NH2 peptide applied to aged hOSECs showed collagen fiber thickness comparable to untreated hOSECs (see Table 6 and Figure 14).

[0278] The higher fiber thickness in the aged hOSEC and aged hOSEC + Product A groups indicated an abnormal accumulation of collagen in the fibers, which has been described to occur in aged skin in vivo.

[0279] Untreated hOSEC and senescent hOSEC+Product B showed comparable collagen fiber thickness.

[0280] [Table 6]

[0281] As shown in Figure 14, the difference in collagen fiber thickness between the untreated hOSEC and aged hOSEC + Product B groups and the aged hOSEC group was statistically significant. The difference in collagen fiber thickness between the untreated hOSEC and aged hOSEC + Product B groups and the aged hOSEC + Product A group was approximately statistically significant. There was no statistically significant difference between untreated hOSEC and aged hOSEC + Product B (cream containing 0.005 mg / mL of peptide Pal-SEQ ID NO:1-NH2).

[0282] These results demonstrate that topical application of Pal-SEQ ID NO:1-NH2 reverses the anti-aging effects of hydrocortisone, thus demonstrating the potential of the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) as an anti-aging product to prevent, reduce and / or eliminate signs of aging, such as wrinkles, roughness and / or sagging skin.

[0283] Furthermore, these results also demonstrate that the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely, the peptide of the present invention (exemplified by Pal-SEQ ID NO:1-NH2) can provide facial reshaping and / or facial skin tightening.

[0284] Example 12. Clinical evaluation of skin and wrinkle smoothing and anti-aging effects on female volunteers with different phototypes The effect of Pal-SEQ ID NO:1-NH2 peptide (synthesized according to Examples 1-5) on anti-aging of facial skin was evaluated in 44 female volunteers (50% light pigmented (phototypes II-III) and 50% dark pigmented (phototypes V-VI)).

[0285] Briefly, volunteers applied a 2% (m / v) cosmetic formulation from a stock of Pal-SEQ ID NO:1-NH2 at 0.05% (m / v) or a cosmetic formulation without Pal-SEQ ID NO:1-NH2 (placebo). The application regime was twice daily, early in the morning and before going to bed, for 56 days. The cosmetic formulation was applied to the entire face, and the effects of placebo and active were compared between volunteers and also between volunteers with different phototypes.

[0286] Wrinkle depth and cheek roughness were assessed using the AEVA system on each volunteer on days 28 and 56. A decrease in the parameters within the treatment period suggested skin smoothing and therefore anti-wrinkle and anti-aging benefits.

[0287] The results obtained are shown in FIG.

[0288] As can be easily seen from Figures 15(A)-(C), the cosmetic formulation containing Pal-SEQ ID NO:1-NH2 showed a reduction in either roughness (Ra) and engraving (Rz) and wrinkle depth in the tested facial skin areas at all tested time points for all tested skin phototypes when compared to the start (a reduction in some or all of these parameters indicates a smoothing effect as well as an anti-wrinkle and anti-aging effect). After 56 days of treatment, a 4% reduction in cheek roughness (5% on average vs. placebo) and a 7% reduction in cheek engraving (10% on average vs. placebo) were observed. A more pronounced effect was observed for wrinkle depth, reaching an average reduction of 9%. In particular, for volunteers with phototypes V-VI skin, a 12% reduction in wrinkle depth (20% on average vs. placebo) was observed, while for volunteers with phototypes II-III skin, a 6-7% reduction in wrinkle depth was observed over time. [Brief description of the drawings]

[0289] [Figure 1]FIG. 1 shows the percentage of HEKa cell viability after treatment with increasing amounts of peptide Pal-SEQ ID NO:1-NH2 compared to baseline (i.e., cell viability in baseline is set at 100% and then compared to the remaining samples tested). [Diagram 2] FIG. 2 shows the percentage of HDFa cell viability after treatment with peptide Pal-SEQ ID NO:1-NH2 compared to baseline (i.e., cell viability in baseline is set at 100% and then compared to the remaining samples tested). [Diagram 3] FIG. 3 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 versus untreated primary human dermal fibroblasts. [Figure 4] FIG. 4 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 versus untreated primary human dermal fibroblasts. [Diagram 5] FIG. 5 shows the modulation of gene expression profile in primary human dermal fibroblasts induced by treatment with peptide Pal-SEQ ID NO:1-NH2 versus untreated primary human dermal fibroblasts. [Figure 6] FIG. 6 shows the increase in collagen VI synthesis by human dermal fibroblasts treated with increasing amounts of the peptide Pal-SEQ ID NO:1-NH2 compared to the negative control (treated with dimethylsulfoxide) (i.e., the collagen VI synthesis of the negative control is set at 100% and then compared to the collagen VI synthesis of the remaining samples tested). [Figure 7] FIG. 7 shows the percentage increase in Mohawk synthesis by human dermal fibroblasts treated with increasing amounts of the peptide Pal-SEQ ID NO:1-NH2 compared to the negative control (treated with dimethylsulfoxide) (i.e., the Mohawk synthesis of the negative control is set at 100% and then compared to the Mohawk synthesis of the remaining samples tested). [Figure 8] FIG. 8 shows collagen cross-linking. [Figure 9] FIG. 9 shows tissue viability by percentage of released LDH (lactate dehydrogenase) compared to the negative control (untreated hOSEC) (i.e., the released LDH of the negative control is set at 100% and then compared to the released LDH in the remaining samples tested in Example 11). [Figure 10] FIG. 10 shows metabolic activity as a percentage of resorufin compared to the negative control (untreated hOSEC) (i.e., the negative control resorufin is set at 100% and then compared to the resorufin in the remaining samples tested in Example 11). [Figure 11] FIG. 11 shows collagen quantification in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the collagen amount of the negative control is set at 100% and then compared to the collagen amount of the remaining samples tested). [Figure 12] FIG. 12 shows transmission electron microscope images of the various experimental groups of Example 11. [Figure 13] FIG. 13 shows the collagen density in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the collagen density of the negative control is set at 100% and then compared to the collagen density of the remaining samples tested). [Figure 14] FIG. 14 shows the collagen fiber thickness in the various experimental groups of Example 11 compared to the negative control (untreated hOSEC) (i.e., the collagen fiber thickness of the negative control is set at 100% and then compared to the collagen fiber thickness of the remaining samples tested). [Figure 15] FIG. 15 shows the efficacy of Pal-SEQ ID NO:1-NH2 after its topical application on 44 female volunteers (50% light-pigmented (phototypes II-III) and 50% dark-pigmented (phototypes V-VI)).

Claims

1. A peptide capable of increasing Mohawk, characterized in that it has a sequence according to formula (I): R １ -AA １ -AA ２ -AA ３ -AA ４ -R ２ (I) wherein said peptide of formula (I) is: The peptide is Pal-His-Tyr-Arg-Ala-NH 2 (Pal-SEQ ID NO:1-NH 2 ), its cosmetically acceptable salts and / or solvates, and mixtures thereof.

2. A composition comprising the peptide of claim 1.

3. 10. Cosmetic use of a peptide according to claim 1 or of a composition according to claim 2 in a subject in need thereof.

4. said cosmetic use being characterized in that it is for reducing, preventing and / or eliminating the signs of skin aging, for skin rejuvenation and / or for reducing, preventing and / or eliminating skin defects, Use as a cosmetic product according to claim 3.

5. The signs of skin aging are characterized by wrinkles, roughness and / or sagging. Use as a cosmetic product according to claim 4.

6. The cosmetic use for skin rejuvenation and / or for reducing, preventing and / or eliminating skin imperfections is characterized in that it is for skin tightening, body sculpting, facial reshaping, skin tightening and / or pore refinement, Use as a cosmetic product according to claim 4.

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