Peptides and compositions for use in cosmetics and pharmaceuticals

Abstract

The present invention relates to peptides and compositions thereof that are capable of modulating the expression of IL-33 and thus are useful in reducing, preventing and / or eliminating the signs of skin aging and / or skin defects.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, more precisely to molecular biology applied to cosmetics and / or pharmaceuticals, and even more precisely to peptides and compositions containing said peptides that can reduce, prevent and / or eliminate skin aging signs for skin rejuvenation and / or reduce, prevent and / or eliminate skin defects, more precisely skin defects in the eye. More preferably, the present invention relates to peptides effective for eye care and / or treatment of eye-related conditions and compositions containing them.

Background Art

[0002] Over the past few decades, people's interest in personal aesthetics has increased, and attempts have been made to delay or minimize the appearance of skin aging signs.

[0003] The skin is the largest organ in humans and, due to its position at the body interface, is subject to endogenous (chronic) aging and exogenous aging, the latter being caused by environmental factors (such as ultraviolet rays, smoking, pollution or sleep disruption, etc.).

[0004] In this sense, some of the most common skin aging signs are located on the face (facial skin aging signs), more precisely around the eyes, for example, eye bags, eyelid defects, wrinkles around the eyes, and dark circles (periorbital hyperpigmentation). These are also among the skin aging signs that cause more concern in society because their presence and / or increase provide a more aged appearance. Therefore, there is interest in the prevention and / or treatment of all these signs.

[0005] The most important eyelid defects are those related to eyelid ptosis and / or eyelid laxity. This is usually due to the weakening of the skin and muscles around the eyes with aging, causing the eyelids to sag. Exposure to the sun and the natural effects of aging can make the skin around the eyes sag more. Regarding aging, the skin loses elasticity with aging, and the face becomes less puffy and harder, so as a result, often the skin around the eyes becomes loose or excessive. Eyelid laxity occurs for various reasons, including medical conditions and cosmetic abnormalities. Usually, eyelid laxity is due to dermatochalasis, ptosis (e.g., aponeurotic ptosis and aponeurotic blepharoptosis), and eversion and inversion (Wendell Damasceno, R. et al. Eyelid aging: pathophysiology and clinical magement, Arq Bras Oftalmol. 2015; 78(5):328-31). When eyelid defects (preferably eyelid laxity) are usually called non-inflammatory based eyelid laxity, they are considered a cosmetic condition, but in the case of inflammatory based eyelid laxity defects (e.g., extensive eyelid laxity), they can result in visual field loss, eye or eyelid irritation, and headache, and thus are a medical condition.

[0006] As is well known in the state of the art, most of the signs of skin aging are produced or derived by oxidative stress and / or inflammation, which are caused by chronological aging or environmental factors (e.g., ultraviolet light or sleep disruption).

[0007] Interleukin-33 (hereinafter, IL-33) is a member of the IL-1 family. It is an inflammatory inducer that has a dual function, acting as both an extracellular alarm mediator and an intracellular regulator of gene expression. It is a ligand for ST2, a heterodimeric membrane-bound receptor of the orphan IL-1 family receptor.

[0008] IL-33 is expressed by a diverse range of cell types, including fibroblasts, mast cells, dendritic cells, macrophages, osteoblasts, endothelial cells, and epithelial cells (such as keratinocytes) (Martin, N.T. and Martin, M.U. Interleukin 33 is a guardian of barriers and a local alarmin, Nature Immunology (2016) 17, 122-131).

[0009] During homeostasis, nuclear IL-33 is constitutively expressed at high levels in epithelial barrier tissues such as the lung, skin, and stomach. Full-length bioactive IL-33 is released extracellularly from the nucleus upon tissue injury and cell death (or cell stress) following exposure to an allergen or infection with a virus or parasite. After release, IL-33 raises the "alarm" in the immune system by activating various types of immune cells, including mast cells, and most importantly, ILC2s that secrete large amounts of IL-5 and IL-13. After programmed cell death (apoptosis), IL-33 is inactivated by caspases to avoid unnecessarily warning the immune system. Although full-length IL-33 is active, it can be processed by inflammatory proteases (cathepsin G, elastase) into shorter "hyperactive" mature forms, which may be important bioactive forms in vivo.

[0010] The scope and duration of the action of IL-33 are regulated by its rapid oxidation, which inhibits its binding to ST2.

[0011] In recent years, IL-33 has attracted attention due to the numerous signaling processes it has been shown to participate in and thus the important role it appears to have in a number of cosmetic and medical features, such as the following: Skin inflammation: Exposure to UV radiation induces IL-33 expression in keratinocytes and dermal fibroblasts (Napier Byrne, S., et al. The immune-modulating cytokine and endogenous alarmin Interleukin-33 is upregulated in skin exposed to inflammatory UVB Radiation. The American Journal of Pathology, 179 (2011), 211-222). Skin barrier: IL-33 has an effect on skin barrier regulation. An increase in IL-33 results in a decrease in filaggrin expression, interfering with the skin barrier that promotes the entry of allergens, bacteria, and viruses (Seltmann, J. et.al. IL33 impacts on the skin barrier by downregulating the expression of filaggrin. J Allergy Clin Immunol, Volume 135, Number 6, pages 1659-1661). Vascular distribution: IL-33 promotes angiogenesis and vascular permeability (Choi, Y. et.al. Interleukin-33 induces angiogenesis and vascular permeability through ST2 / TRAF6-mediated endothelial nitric oxide production. Blood, 1 October 2009, volume 114, number 14, pages 3117-3126). Pigmentation: IL-33 has promelanogenic activity by improving melanin biosynthesis in melanocytes (Zhou, J. et.al. Enhancement of the p38 MAPK and PKA signaling pathways is associated with the pro-melanogenic activity of Interleukin 33 in primary melanocytes. Journal of Dermatological Science, 73 (2014), 110-116).

[0012] Therefore, IL-33 causes a decrease in keratinocyte cohesion, an increase in pigmentation and an increase in inflammation and vascular permeability, and is thus associated with all of the above skin symptoms.

[0013] Sleep disruption (which is a complete absence of the sleep period shorter than the optimal sleep time or a change in the circadian pattern) also has important effects on the skin and is a promoter of skin aging and / or skin defects. Some of the skin attributes associated with sleep disruption include rough, dull, and dry skin, as well as drooping eyelids and dark eye circles. This effect is not limited to visual characteristics, but sleep disruption can affect skin health and physiology such as exacerbation of skin disorders, impairment of skin barrier integrity, increased production of inflammatory cytokines, glucocorticoids, as well as increased glucose and cortisol levels and decreased melatonin levels (Kim, M.A. et al. The effect of sleep deprivation on the biophysical properties of facial skin, Journal of cosmetics, dermatological sciences and application (2017) 7, 34-47; and Guan L., Mehra R., Baron E. (2017) Sleep and Aging Skin. In: Farage M., Miller K., Maibach H. (eds) Textbook of Aging Skin. Springer, Berlin, Heidelberg).

[0014] An increase in glucose concentration negatively affects the proliferation of keratinocytes, similar to that of skin fibroblasts (Kruse, C.R. et al. The effect of local hyperglucemia on skin cells in vitro and on wound healing in euglycemic rats, Journal of Surgical Research (2016) 206, 418-426). Impaired proliferation of skin fibroblasts leads to delayed wound healing, similar to the delayed wound healing observed in the skin of the elderly (Buranasin, P., High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts, PLoS ONE (2018) 13).

[0015] There is also evidence that an increase in glucose concentration results in the terminal differentiation and morphological changes of these keratinocytes (Spravchikov, N. et al. Glucose effects on skin keratinocytes, Diabetes (2001) 50, 1627-1635). These terminally differentiated cells lose the ability to produce new cells and arrest in a terminal state through upregulation of cyclin-dependent kinase inhibitors and downregulation of positive mediators of the cell cycle.

[0016] Hyperglycemia is often accompanied by insulin deficiency or insulin resistance and also causes autoregulatory disorders and enhanced microvascular permeability.

[0017] Regarding cortisol, in the skin, an increase in glucocorticoid concentration is converted into a decrease in permeability barrier homeostasis, a decrease in stratum corneum cohesion, a decrease in wound healing, and a decrease in epidermal innate immunity (Altemus, M., Stress-induced changes in skin barrier function in healthy women, Journal of Investigative dermatology (2001) 117, 309-317), all of which may ultimately lead to intrinsic skin aging. Furthermore, sleep disruption may induce excessive glucocorticoid secretion and thus cause physiological stress that enhances skin aging. Excessive glucocorticoids also inhibit lipid synthesis and cause lower production and secretion of lamellar bodies, which can lead to further damage to the epidermal barrier.

[0018] Skin cells express both membrane-bound and nuclear melatonin receptors that enable melatonin to play roles in multiple life functions such as the hair growth cycle, hair pigmentation, melanoma control, antioxidant activity, and suppression of UV-induced damage to skin cells (Slominski, A., On the role of melatonin in skin physiology and pathology, Endocrine (2005) 27, 137-148). Melatonin has been shown to reduce aging-related skin changes from oxidative stress and prevent extrinsic skin aging through protection against UV-induced skin aging. Dysregulation of melatonin ultimately has an adverse effect on skin integrity and interferes with the natural anti-aging effect of melatonin on the skin. This leads to both an increase in extrinsic skin aging due to UV damage and an increase in intrinsic skin aging due to oxidative stress.

[0019] Furthermore, melatonin has antioxidant and scavenger properties (Reiter, R.J., Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans, Acta Biochim Pol (2003) 50, 1129-1146).

[0020] Melatonin also reduces hyperglycemic damage on keratinocytes and decreases inflammatory cytokine levels (Song, R., Melatonin promotes diabetic wound healing in vitro by regulating keratinocyte activity, Am J Transl Res (2016) 8, 4682-4693).

[0021] In recent years, the number of active ingredients for improving skin aging symptoms and / or skin defects has increased significantly. Examples of such active ingredients are retinoids, vitamins or plant extracts (Bradley E.J., Griffiths C.E.M., Sherratt M.J., Bell M. and Watson R.E.B. (2015), Over-the-counter anti-ageing topical agents and their ability to protect and repair photoaged skin. Maturitas, 80, 265-272).

[0022] In the case of retinoids, the retinoid acid is of note, and this retinoid acid is currently the "gold standard" for inducing the synthesis of several molecules of ECM (e.g., collagen, fibronectin, or laminin) (Varani J., Mitra R.S., Gibbs D., Phan S.H., Dixit V.M., Mitra R., Wang T., Siebert K.J., Nickoloff B.J., Voorhees J.J. (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). Therefore, retinoid acid can significantly improve the clinical appearance of facial wrinkles by upregulating the transcription and synthesis of ECM proteins such as collagen and fibronectin and the inhibition of matrix metalloproteinases (hereinafter, MMP), and is thus considered one of the most powerful compounds for treating the signs of aging (Varani J., Mitra R.S., Gibbs D., Phan S.H., Dixit V.M., Mitra R., Wang T., Siebert K.J., Nickoloff B.J., Voorhees J.J. (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 several drawbacks to the use of retinoid acid, for example, retinoid acid can easily cause skin irritation and must be used carefully, and the combination of retinoid acid and sun exposure is not recommended.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 J-H. (2006), Retinoids in cosmeceuticals. Dermatologic Therapy, 19, 289-296).

[0023] Antioxidants are also used to reduce the concentration of free radicals in the skin and thus prevent collagen degradation. An example of such an antioxidant is ascorbic acid (also known as vitamin C). In addition to its antioxidant action, ascorbic acid promotes epidermal differentiation, inhibits matrix metalloproteinase-1 (MMP1), and induces the synthesis of proteins from the ECM (collagen I and III and elastin). Unfortunately, ascorbic acid is extremely unstable and undergoes oxidation, especially at high temperatures, under aerobic conditions, at high pH, and / or when exposed to light (Manela-Azulay M., Azulay V., Aguinaga F., Issa M.C. (2017), Vitamins and other Antioxidants. Daily Routine in Cosmetic Dermatology, 1-13).

[0024] In addition to chemically synthesized compounds, a wide range of plant extracts and plant-derived compounds, such as grape extracts containing resveratrol (an antioxidant), green tea containing polyphenols, or soybeans containing isoflavones, are commercially available for numerous applications. However, the in vivo efficacy and composition of these compositions have not been sufficiently scientifically validated.

[0025] Due to its viscoelastic properties and its ability to retain water, hyaluronic acid is also used in the cosmetics industry to treat wrinkles by keeping the skin hydrated, maintaining elasticity, and improving roughness, or it is also used as a skin filler. However, currently, hyaluronic acid is obtained from several sources (e.g., rooster combs or bacterial extracts), and as a result, these products may contain impurities and need to be thoroughly 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).

[0026] On the other hand, peptides can also be incorporated into cosmetic formulations to improve signs of skin aging. Bioactive peptides mimic the body's own molecules and can affect processes such as collagen synthesis, and have the advantage of much better tolerance and stability. Furthermore, a wide range of activities, ingredients, and indications can be developed for them (Zhang L. and Falla T.J. (2009), Cosmeceuticals and peptides. Clinics in dermatology, 27, 485-494).

[0027] Despite a wide variety of compounds and / or extracts at the state of the art, there is still a need for improved active agents and compositions having a novel mechanism of action that enables reducing, preventing, and / or eliminating signs of skin aging and / or skin defects, and more precisely, there is a need to find novel molecules (preferably biomolecules) that target IL-33 and provide the desired and required cosmetic and medical effects. SUMMARY OF THE INVENTION

[0028] After extensive and thorough investigations, the inventors of the present invention surprisingly found that they could regulate (down-regulate) the expression of IL-33 and thus discovered peptides that are beneficial in cosmetics and pharmaceuticals. The peptides of the present invention can reduce, prevent and / or eliminate signs of skin aging and / or skin defects, more preferably signs of aging of facial skin, more preferably signs of aging and / or skin defects of the skin around the eyes or in the vicinity thereof, even more preferably eye bags, eyelid defects, wrinkles around the eyes and dark circles (periorbital hyperpigmentation). Furthermore, the peptides of the present invention can also surprisingly reduce, prevent and / or eliminate signs of skin aging and / or skin defects caused by sleep disruption. As described above, the peptides of the present invention are also effective in medicine due to their activities.

[0029] In a first aspect, the present invention refers to a peptide that can regulate (down-regulate) the expression of IL-33. As described above, the peptides of the present invention are effective in cosmetics and pharmaceuticals for the prevention, reduction and / or treatment of signs of skin aging and / or skin defects.

[0030] Furthermore, in a second aspect, the present invention refers to a composition comprising at least one peptide of the present invention.

[0031] In a third aspect, the present invention refers to the use of the peptide or cosmetic composition of the present invention as a cosmetic.

[0032] In a fourth aspect, the present invention refers to the cosmetic use of the peptide or cosmetic composition of the present invention.

[0033] In a fifth aspect, the present invention refers to a method for cosmetic treatment of a subject in need thereof, comprising the use of the peptide or cosmetic composition of the present invention.

[0034] In a sixth aspect, the present invention refers to the peptide or pharmaceutical composition of the present invention for use as a medicament.

[0035] In a seventh aspect, the invention refers to the use of the peptide or pharmaceutical composition of the invention for the manufacture of a medicament.

[0036] In a last aspect, the invention refers to a method for the medical treatment of a subject in need thereof, comprising the use of the peptide or pharmaceutical composition of the invention.

[0037] The terms "acyclic aliphatic group" and its plural forms, as used herein, have the common meaning given to such terms in the art. Thus, these terms refer to, for example, straight-chain or branched alkyl, alkenyl, and alkynyl groups, but are not limited thereto.

[0038] The term "alkyl group" and its plural forms, as used herein, have 1 to 24, preferably 1 to 16, more preferably 1 to 14, even more preferably 1 to 12, even more preferably 1, 2, 3, 4, 5, or 6 carbon atoms and are saturated, straight-chain or branched-chain groups attached to the rest of the molecule by a single bond and include, for example, 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, etc., but are not limited thereto. The alkyl group may be optionally substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio.

[0039] As used herein, the term "alkenyl group" and its plural forms refer to a straight-chain or branched-chain group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, even more preferably 2, 3, 4, 5 or 6 carbon atoms, preferably having 1, 2 or 3 carbon-carbon double bonds (conjugated or non-conjugated), and being bonded to the rest of the molecule via a single bond, including, but not limited to, vinyl, oleyl, linoleyl and similar groups. The alkenyl group may be optionally substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio.

[0040] As used herein, the term "alkynyl group" and its plural forms refer to a straight-chain or branched-chain group having 2 to 24, preferably 2 to 16, more preferably 2 to 14, even more preferably 2 to 12, even more preferably 2, 3, 4, 5 or 6 carbon atoms, preferably having 1, 2 or 3 carbon-carbon triple bonds (conjugated or non-conjugated), and being bonded to the rest of the molecule via a single bond, including, but not limited to, ethynyl group, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl and similar groups such as pentynyl. The alkynyl group may be optionally substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio.

[0041] As used herein, the term "alicyclic group" and its plural forms have the common meaning given to such terms in the art. Thus, these terms are used, for example, to refer to cycloalkyl or cycloalkenyl or cycloalkynyl groups, but are not limited thereto.

[0042] The term "cycloalkyl" and its plural forms, as used herein, 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, and being bonded to the remainder of the molecule via a single bond. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrophenalene, adamantyl, etc. Optionally, it can be substituted by one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio.

[0043] The term "cycloalkenyl" and its plural forms, as used herein, 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, preferably having 1, 2, or 3 carbon-carbon double bonds (conjugated or non-conjugated), and being bonded to the remainder of the molecule via a single bond. Examples include, but are not limited to, the cyclopent-1-en-1-yl group and similar groups. Optionally, it can be substituted by one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio.

[0044] The term "cycloalkynyl" and its plural forms, as used herein, 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, even more preferably 8 to 9 carbon atoms, preferably having 1, 2 or 3 carbon-carbon triple bonds (conjugated or non-conjugated), and being bonded to the rest of the molecule via a single bond, including, but not limited to, for example, the cyclooct-2-yn-1-yl group, and may in some cases be substituted by one or more groups such as alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio.

[0045] The term "aryl group" and its plural forms, as used herein, refer to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, even more preferably 6 or 10 carbon atoms, containing 1, 2, 3 or 4 aromatic rings bonded by carbon-carbon bonds or condensations, and being bonded to the remaining molecule via a single bond, including, but not limited to, for example, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthranyl. The aryl group may optionally be substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio.

[0046] The term "aralkyl group" and its plural forms, as used herein, refer to an alkyl group substituted by an aromatic group having 7 to 24 carbon atoms, including, but 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, etc. The aralkyl group may optionally be substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkoxythio.

[0047] As used herein, the term "heterocyclic group" and its plural forms refer to a 3- to 10-membered heterocycyl or hydrocarbon ring, where one or more of the ring atoms, preferably 1, 2, or 3 of the ring atoms, are elements different from carbon (e.g., nitrogen, oxygen, or sulfur), and which can be saturated or unsaturated. For the purposes of the present invention, the heterocyclic ring can be a cyclic, monocyclic, bicyclic, or tricyclic system that can include a fused ring system, and the nitrogen, carbon, or sulfur atoms can optionally be oxidized in the heterocyclic radical; the nitrogen atoms can optionally be quaternized; the heterocyclic radical can be partially or fully saturated or can be aromatic. Preferably, the term heterocyclic ring relates to a 5- or 6-membered ring. The heterocyclic group can optionally be substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio.

[0048] As used herein, the term "heteroarylalkyl group" and its plural forms refer to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group, the alkyl group having 1 to 6 carbon atoms and the aromatic heterocyclyl group having 2 to 24 carbon atoms and 1 to 3 atoms other than carbon, including, for example, -(CH2)1-6-imidazolyl, -(CH2)1-6-triazolyl, -(CH2)1-6-thienyl, -(CH2)1-6-furyl, -(CH2)1-6-pyrrolidinyl, etc., but not limited thereto. The heteroarylalkyl group can optionally be substituted by one or more substituents such as halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto, and alkoxythio.

[0049] As used herein, the term "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine, and its anions are called halides.

[0050] As used herein, the term "derivative" and its plural forms refer to both cosmetically and / or pharmaceutically acceptable compounds derived from a compound of interest for use in the preparation of a cosmetic or a drug, and cosmetically and / or pharmaceutically unacceptable compounds, because the latter may be useful in the preparation of cosmetically and / or pharmaceutically acceptable derivatives.

[0051] As used herein, the term "salt" and its plural forms refer to any type of salt from among those known in the art, for example, halide salts, hydroxyacid salts (such as oxyacid salts, acid salts, basic salts, and double salts), hydroxo salts, mixed salts, oxy salts, or other hydrated salts. This term includes both cosmetically and / or pharmaceutically acceptable salts; as well as cosmetically and / or pharmaceutically unacceptable salts, because the latter may be useful in the preparation of cosmetically and / or pharmaceutically acceptable salts.

[0052] As used herein, the term "isomer" and its plural forms refer to optical isomers, enantiomers, stereoisomers or diastereomers. Individual enantiomers or diastereomers, as well as mixtures thereof, can be separated by conventional techniques known in the art.

[0053] As used herein, the term "solvate" and its plural forms refer to any solvate known in the art, such as polar, nonpolar or amphiphilic solvates, and includes any cosmetically acceptable solvate that (directly or indirectly) provides the compound of interest (the peptide of the present invention) when administered or applied to the subject of interest. Preferably the solvate is a hydrate, a solvate with an alcohol such as methanol, ethanol, propanol or isopropanol, a solvate with an ester such as ethyl acetate, a solvate with an ether such as methyl ether, ethyl ether or THF (tetrahydrofuran), or a solvate with DMF (dimethylformamide), more preferably a hydrate or solvate with an alcohol such as ethanol.

[0054] Furthermore, as used herein, the terms “amino acid” and its plural forms include amino acids encoded by codons as well as non-encoded amino acids, whether they are natural or not and whether they are D- and L-amino acids. Examples of non-encoded amino acids are not particularly limited, but include 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-aminophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, cycloserine, carnitine, cysteine, penicillamine, pyroglutamic acid, thienylalanine, hydroxyproline, allo-isoleucine, allo-threonine, isonipecotic acid, isoserine, phenylglycine, statine, β-alanine, norleucine, N-methyl amino acids, α-amino acids and β-amino acids and their derivatives. Nevertheless, additional non-natural amino acids are known in the art (see, for example, “Unusual amino acids in peptide synthesis” by D. C. Roberts and F. Vellaccio, The Peptides, Vol. 5 (1983), Chapter VI, Gross E. and Meienhofer J., Eds., Academic Press, New York, USA).

[0055] As used herein, the "percent identity" with respect to peptides, polypeptides, and proteins has the meaning generally ascribed in the art and thus relates to the percentage of amino acids that are identical between two amino acid sequences being compared after optimal alignment of these sequences, where said percentage is merely statistical and the differences between the two amino acid sequences are randomly distributed throughout the sequences. "Optimal alignment" is understood as an alignment of the amino acid sequences that results in a greater percentage of identity. The percentage of identity is determined by counting the number of positions at which the amino acids are identical in the two sequences being compared, dividing the number of identical positions by the number of positions being compared, and multiplying the result obtained 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.

[0056] As used herein, "dark circles" and "periorbital hyperpigmentation" are used interchangeably and acquire the meaning they generally have in the art. Briefly, dark circles or periorbital hyperpigmentation typically feature melanin deposition around the eyes, a bluish color and visibility of the dermal capillary network around the eyes, and loss of subcutaneous fat providing thinning of the skin and cheeks.

[0057] As used herein, IL33 refers to the gene encoding the protein interleukin-33.

[0058] As used herein, IL-33 refers to the protein interleukin-33.

[0059] As described above, in a first aspect, the invention refers to a peptide comprising 6 amino acids, characterized by comprising at least 3 arginines.

[0060] As described above, the peptide of the present invention regulates the expression of the gene IL33 and / or the protein interleukin-33, and more preferably down-regulates the expression of the gene IL33 and / or the protein interleukin-33.

[0061] Isomers, salts, solvates and / or derivatives and mixtures of the peptides of the present invention; more preferably, cosmetic and / or pharmaceutically acceptable isomers, salts, solvates and / or derivatives and mixtures of the peptides of the present invention are also included in the present invention.

[0062] The amino acids used or present in the peptides of the present invention are intended to be 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.

[0063] Preferably, the above isomers are stereoisomers. The stereoisomers are intended to be enantiomers or diastereomers. Thus, in a preferred embodiment of the present invention, the peptide is a racemic mixture, a diastereomeric mixture, a pure enantiomer or a pure diastereomer.

[0064] In a preferred embodiment of the peptide of the present invention, the six amino acids are three arginines, one glutamine, one methionine and one glutamic acid.

[0065] Also preferably, the peptide of the present invention has the formula (I): R1-AA1-AA2-AA3-AA4-AA5-AA6-R2 having a sequence according to R1 is selected from H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R5-CO-, where R5 is substituted or unsubstituted C1-C 24Alkyl radical, 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 Aralkyl, a substituted or unsubstituted heterocyclyl ring consisting of 3 to 10 members, and a substituted or unsubstituted heteroarylalkyl consisting of an alkyl chain having 2 to 24 carbon atoms, 1 to 3 atoms other than carbon, and 1 to 6 carbon atoms, selected from R2 is independently selected from H, -NR3R4-, -OR3 and -SR3, and R3 and R4 are independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl.

[0066] R1 is preferably selected from H or R5-CO-, where R5 is a substituted or unsubstituted C1-C 24 Alkyl radical, 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 24It is selected from aralkyl, a substituted or unsubstituted heterocyclic ring consisting of 3 to 10 members, and a substituted or unsubstituted heteroarylalkyl having 2 to 24 carbon atoms, 1 to 3 atoms other than carbon atoms, and an alkyl chain having 1 to 6 carbon atoms. More preferably, R1 is selected from H, acetyl (hereinafter referred to as Ac), tert-butanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl, octanoyl, decanoyl, lauroyl myristoyl, palmitoyl (hereinafter referred to as Pal), stearoyl, oleoyl and linoleoyl. Even more preferably, R1 is Ac.

[0067] Preferably, R2 is -NH2.

[0068] Therefore, more preferably, R1 is Ac and R2 is -NH2.

[0069] Preferably, the peptide of the present invention contains Arg-Arg, Arg-Glu, Met-Arg or a combination thereof in its sequence. In the most preferred embodiment, the peptide of the present invention contains Arg-Arg, Arg-Glu and Met-Arg in its sequence.

[0070] In one of the most preferred embodiments, the sequence of the peptide of the present invention is: R1-Arg-Arg-Gln-Met-Arg-Glu-R2 (R1-SEQ ID NO:1-R2); R1-Met-Arg-Arg-Glu-Gln-Arg -R2 (R1-SEQ ID NO:2-R2); or R1-Arg-Glu-Gln-Met-Arg-Arg-R2 (R1-SEQ ID NO:3-R2) is.

[0071] As described above, R1 is preferably selected from H or R5-CO-, where R5 is a substituted or unsubstituted C1-C 24 alkyl radical, a substituted or unsubstituted C2-C 24 alkenyl, a substituted or unsubstituted C2-C 24Alkynyl, 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 Aralkyl, a substituted or unsubstituted heterocyclic ring consisting of 3 to 10 members and a substituted or unsubstituted heteroarylalkyl of an alkyl chain having 2 to 24 carbon atoms, 1 to 3 atoms other than carbon, and 1 to 6 carbon atoms. Even more preferably, R1 is selected from H, acetyl (hereinafter, Ac), tert-butanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl, octanoyl, decanoyl, lauroyl myristoyl, palmitoyl (hereinafter, Pal), stearoyl, oleoyl and linoleoyl, and even more preferably R1 is Ac.

[0072] Also, as described above, R2 is preferably -NH2.

[0073] Therefore, in the most preferred embodiment, the sequence of the peptide of the present invention is: Ac-Arg-Arg-Gln-Met-Arg-Glu-NH2 (Ac-SEQ ID NO:1-NH2); Ac-Met-Arg-Arg-Glu-Gln-Arg-NH2 (Ac-SEQ ID NO:2-NH2); or Ac-Arg-Glu-Gln-Met-Arg-Arg-NH2 (Ac-SEQ ID NO:3-NH2) is.

[0074] As can be directly derived from the examples included below, the peptides of the present invention can down-regulate the expression of the gene IL33 and the protein IL-33. Further, the peptides of the present invention provide for a preferred regulation of extracellular matrix-related genes, suppression of tyrosinase activity, and protection against protein glycation and lipid peroxidation. Accordingly, the peptides of the present invention are effective in cosmetics and pharmaceuticals for the treatment of skin aging signs and / or skin defects, more precisely as follows: In cosmetics, considering the above activities and the activities shown in the examples included below, the peptides of the present invention are effective at least for the treatment of eye bags, eyelid defects, wrinkles (preferably around the eyes) and hyperchromia (preferably periorbital hyperchromia). Further, the peptides of the present invention can prevent, reduce and / or reverse the effects on the skin of sleep disruption, which can treat skin aging signs and / or skin defects associated with sleep disruption. In pharmaceuticals, considering the above activities and the activities demonstrated in the examples included below, the peptides of the present invention are effective for the treatment of medical conditions related to inflammation, angiogenesis and / or hyperpigmentation, diseases related to lipid peroxidation, diseases related to protein glycation, or combinations thereof. Most preferably, the peptides of the present invention are useful for the treatment of at least eyelid defects, more preferably ichthyosis, ptosis (e.g., for aponeurotic ptosis and aponeurotic blepharoptosis), ectropion and entropion.

[0075] Accordingly, the peptides of the present invention exist in the state of the art and solve the above-mentioned needs and technical problems.

[0076] Furthermore, peptides having 70% identity, preferably 80%, more preferably 90%, more preferably 95%, and even more preferably 99% identity to any of the above specific sequences are included in the scope of the present invention, (which are R1-Arg-Arg-Gln-Met-Arg-Glu-R2; R1-Met-Arg-Arg-Glu-Gln-Arg-R2; or R1-Arg-Glu-Gln-Met-Arg-Arg-R2, and preferably Ac-Arg-Arg-Gln-Met-Arg-Glu-NH2; Ac-Met-Arg-Arg-Glu-Gln-Arg-NH2; or Ac-Arg-Glu-Gln-Met-Arg-Arg-NH2), which still exhibit the activities described herein against the peptides of the present invention.

[0077] The peptides of the present invention can be synthesized and produced by any method known in the art. For example, they can be synthesized and produced by chemical synthesis (preferably by solid-phase peptide synthesis), by expressing the peptides in cell culture, or by transgenic production of the peptides in plants or animals. Furthermore, the peptides of the present invention can be purified by any method known in the art.

[0078] In a preferred embodiment, the peptides of the present invention are cyclic peptides.

[0079] In a preferred embodiment, the peptides of the present invention are suitable or adapted to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), more preferably into and / or around the eyes.

[0080] In a further preferred embodiment, the peptides of the present invention are suitable or adapted to be applied subcutaneously, more preferably to the face of a subject (preferably a human), more preferably into and / or around the eyes.

[0081] In another preferred embodiment, the peptides of the present invention are suitable or adapted to be applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), more preferably in and / or around the eyes.

[0082] In a second aspect, the present invention refers to a composition comprising at least one peptide according to the present invention.

[0083] The composition of the present invention is intended to contain one type of peptide of the present invention, or a combination or mixture of different peptides of the present invention, preferably one type of peptide of the present invention.

[0084] In a preferred embodiment, the composition of the present invention is a cosmetic composition. Thus, in this embodiment, the cosmetic composition of the present invention contains an effective amount for cosmetic use of at least one peptide of the present invention. More preferably, the cosmetic composition of the present invention contains from 0.0001% (weight / volume g / 100 mL, hereinafter w / v) to 0.05% (w / v) of at least one peptide of the present invention. In the most preferred embodiment, the cosmetic composition of the present invention contains from 0.0001% (w / v) to 0.001% (w / v) of at least one peptide of the present invention, more preferably 0.0005% (w / v). In another most preferred embodiment, the cosmetic composition of the present invention contains from 0.05% (w / v) to 0.001% (w / v) of at least one peptide of the present invention.

[0085] The cosmetic composition of the present invention is also intended to contain at least one additional cosmetic component. The additional cosmetic component can be at least one excipient and / or at least one additional cosmetic active ingredient.

[0086] Additional cosmetic components include, for example, auxiliaries such as stabilizers, solubilizers, vitamins, colorants and fragrances; carriers; and / or other cosmetic active ingredients commonly used in the art.

[0087] The additional cosmetic ingredient(s) must be physically and chemically compatible with the other ingredients of the composition, and in particular with the peptide(s) of the invention included in the composition of the invention. Similarly, the nature of the additional cosmetic ingredient(s) must not be changed to such an extent that the advantages of the peptide(s) and composition of the invention are not tolerated. The additional cosmetic ingredient(s) may be, for example, of synthetic or natural origin, such as plant extracts, or they may be derived from biotechnological processes (see, for example, CTFA Cosmetic Ingredient Handbook, 11th Edition (2006)).

[0088] The above additional cosmetic ingredients are, inter alia, ingredients generally used in compositions for caring for and cleaning the skin and / or hair, and / or deodorants and / or creams for preventing excessive sweating, such as melanin synthesis inhibitors, whitening or depigmenting agents, anti-aging agents, NO synthase inhibitors, antioxidants, anti-air pollution and / or free radical scavengers, anti-glycation agents, emulsifiers, skin softeners, organic solvents, liquid propellants, skin conditioners such as humectants, moisture-retaining substances, alpha-hydroxy acids, moisturizers, vitamins, pigments or colorants, dyes, gelling polymers, thickeners, surfactants, softeners, other anti-wrinkle agents, agents capable of reducing or eliminating under-eye bags, exfoliants, antibacterial agents, antifungal agents, bactericides, agents that stimulate the synthesis of macromolecules in the dermis or epidermis and / or agents that can prevent or inhibit their degradation, such as collagen synthesis stimulants, elastin synthesis stimulants, laminin synthesis stimulants, collagen degradation inhibitors, elastin degradation inhibitors, fibroblast growth stimulants, keratinocyte growth stimulants, keratinocyte differentiation stimulants, lipid synthesis stimulants and stimulants for the synthesis of components of the stratum corneum (ceramides, fatty acids, etc.), skin relaxants, glycosaminoglycan synthesis stimulants, DNA repair agents, DNA protectants, proteasome activity stimulants, anti-itch agents, agents for treating sensitive skin, reaffirming agents, astringents, sebum production regulators, lipolysis stimulants, anti-cellulite agents, sedatives, anti-inflammatory agents, agents acting on capillary and / or microcirculation, agents acting on cell mitochondria, agents aimed at improving the dermo-epidermal junction, preservatives, perfumes, chelating agents, plant extracts, marine extracts, agents derived from biotechnological processes, mineral salts, cell extracts and / or are intended to contain solar filters (organic or inorganic photoprotective agents active against ultraviolet A and B).

[0089] In one embodiment, at least one of the additional cosmetic ingredients is a cosmetic active ingredient or substance that can exhibit the same, similar, complementary or different cosmetic activities as those disclosed above for the peptides of the present invention. The compositions of the present invention include other anti-wrinkle or anti-aging agents, such as collagen, elastin, growth factors, hyaluronic acid boosters, barrier function promoters, inducers, agents that stimulate the expression and / or synthesis of collagen I, III, IV and / or VI and / or laminin; agents that stimulate the synthesis of glycosaminoglycans or hyaluronic acid, agents that stimulate the expression and / or synthesis of elastin and / or other elastic fiber-related proteins; agents that inhibit the degradation of collagen and / or elastic fibers; agents that stimulate the expression and / or synthesis of mitochondrial-related proteins (e.g., sirtuin and aconitase); agents that stimulate the expression and / or synthesis of focal adhesion proteins; agents that stimulate the proliferation and / or differentiation of keratinocytes and / or fibroblasts; antioxidants, anti-air pollution agents and / or free radical scavengers; anti-glycation agents, detoxifying agents; agents that reduce chronological aging, environmental aging and inflammatory aging; and agents that reduce melanogenesis and / or inhibit tyrosinase and / or agents that stimulate the synthesis of lipid synthesis and epidermal components (keratin), more specifically the stratum corneum (keratin, ceramide, 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.

[0090] Furthermore, the cosmetic composition of the present invention (or the peptide of the present invention) can be formulated in any form commonly used in the art, such as, for example, solutions, suspensions, emulsions, pastes, gels, creams, powders, sprays, lotions, oils, liniments, serums, mousses, ointments, sticks or pencils, including "leave-on" and "rinse-off" formulations. The cosmetic composition of the present invention can also be incorporated into various types of solid accessories such as wet napkins, hydrogels, adhesive (or non-adhesive) patches or face masks by techniques known in the art, or, in particular, into various makeup line products such as concealers, makeup foundation, lotions or makeup removing lotions.

[0091] Also, both the cosmetic composition of the present invention and the peptide of the present invention disclosed herein can be incorporated into cosmetic sustained release systems and / or carriers such as liposomes, milliparticles, microparticles and nanoparticles, as well as sponges, vesicles, micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, and microemulsions and nanoemulsions in order to obtain greater penetration of the active ingredient.

[0092] In a preferred embodiment, the cosmetic composition of the present invention is suitable or adapted to be applied by iontophoresis, more preferably to the face of a subject (preferably human), more preferably in and / or around the eyes.

[0093] In a further preferred embodiment, the cosmetic composition of the present invention is suitable or adapted to be applied subcutaneously, more preferably to the face of a subject (preferably human), more preferably in and / or around the eyes.

[0094] In another preferred embodiment, the cosmetic composition of the present invention is suitable for, or adapted to be, applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), more preferably in and / or around the eyes.

[0095] In another preferred embodiment, the composition of the present invention is a pharmaceutical composition. Thus, in this embodiment, the pharmaceutical composition of the present invention contains a pharmaceutically effective amount of at least one peptide of the present invention, and more preferably, the pharmaceutical composition of the present invention contains from 0.0001% (w / v) to 0.05% (w / v) of at least one peptide of the present invention. In the most preferred embodiment, the pharmaceutical composition of the present invention contains from 0.0001% (w / v) to 0.001% (w / v) of at least one peptide of the present invention, more preferably 0.0005% (w / v). In another most preferred embodiment, the pharmaceutical composition of the present invention contains from 0.05% (w / v) to 0.001% (w / v) of at least one peptide of the present invention.

[0096] The pharmaceutical composition can be in any form known in the art suitable for the selected route. The pharmaceutical composition of the present invention is suitable for, or intended to be adapted for, administration by any method and any route known in the art (e.g., subcutaneous, intramuscular, intravenous, topical or oral; in the latter case, for example, in the form of physiological saline and / or biodegradable substances such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid copolymer, polycaprolactone and / or polymers of cholesterol). More preferably, the pharmaceutical composition of the present invention is suitable for, or adapted to be, administered topically, even more preferably in the form of a cream.

[0097] The pharmaceutical composition of the present invention is also intended to contain at least one additional pharmaceutical ingredient. The additional pharmaceutical ingredient can be at least one excipient and / or at least one additional pharmaceutically active ingredient.

[0098] As can be directly derived from the examples included below, the peptides of the present invention can down-regulate the expression of the gene IL33 and the protein IL-33. Further, the peptides of the present invention provide for a preferred regulation of extracellular matrix-related genes, suppression of tyrosinase activity, and protection against protein glycation and lipid peroxidation. Accordingly, the compositions of the present invention are effective in cosmetics and pharmaceuticals for the treatment of skin aging symptoms and / or skin defects, and more precisely as follows: In cosmetics, considering the above activities and the activities shown in the examples included below, the peptides of the present invention are effective at least for the treatment of eye bags, eyelid defects, wrinkles (preferably around the eyes) and hyperchromia (preferably periorbital hyperchromia). Further, the peptides of the present invention can prevent, reduce and / or reverse the effects on the skin of sleep disruption, which can treat skin aging symptoms and / or skin defects associated with sleep disruption. In pharmaceuticals, considering the above activities and the activities demonstrated in the examples included below, the peptides of the present invention are effective for the treatment of medical conditions related to inflammation, angiogenesis and / or hyperpigmentation, diseases related to lipid peroxidation, diseases related to protein glycation, or combinations thereof. Most preferably, the peptides of the present invention are useful for the treatment of at least eyelid defects, more preferably ichthyosis, ptosis (e.g., for aponeurotic ptosis and aponeurotic blepharoptosis), ectropion and entropion.

[0099] Accordingly, the compositions of the present invention address the above-described needs and technical problems existing in the state of the art.

[0100] As already described above, in a third aspect, the present invention refers to the use as a cosmetic of a peptide or a cosmetic composition according to the present invention.

[0101] The peptides and cosmetic compositions according to the present invention are as described above.

[0102] Preferably, the use as a cosmetic is for the treatment of inflammatory conditions, angiogenesis, extracellular matrix disorders, oxidative stress, alterations at the dermal cell junctions, apoptosis and / or hyperpigmentation, preferably cosmetic conditions related to cosmetic inflammation, cosmetic angiogenesis, cosmetic extracellular matrix disorders, cosmetic oxidative stress, cosmetic alterations at the dermal cell junctions, cosmetic apoptosis and / or cosmetic hyperpigmentation.

[0103] More preferably, the use as a cosmetic is for the treatment of signs of skin aging and / or skin defects, more preferably for the reduction, prevention and / or elimination of signs of skin aging and / or skin defects.

[0104] As is apparent, the above-mentioned signs of skin aging are cosmetic signs of skin aging and / or cosmetic skin defects.

[0105] Skin aging is due to chronological and / or environmental aging.

[0106] More preferably, the above-mentioned signs of skin aging and / or skin defects are generated by dysregulation of IL-33. As described above, the peptides and cosmetic compositions of the present invention down-regulate IL-33 (they down-regulate the expression of the IL33 gene and / or the expression of interleukin 33).

[0107] In the most preferred embodiment, the signs of skin aging and / or skin defects are eye bags, eyelid defects, wrinkles (preferably periorbital wrinkles), hyperchromasia (preferably periorbital hyperchromasia), or a combination thereof.

[0108] Eyelid defects are preferably eyelid ptosis, more preferably cosmetic dermatochalasis, cosmetic ptosis, cosmetic ectropion and cosmetic entropion, more preferably cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic degenerative ectropion and cosmetic degenerative entropion.

[0109] Therefore, preferably, the skin aging symptoms and / or skin defects are eye bags, wrinkles around the eyes, periorbital pigmentation, blepharoptosis, or a combination thereof, more preferably eye bags, wrinkles around the eyes, periorbital pigmentation, cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic ectropion, and cosmetic entropion, or a combination thereof.

[0110] In the most preferred embodiment, the skin aging symptoms and / or skin defects are eye bags, wrinkles around the eyes, periorbital pigmentation, or a combination thereof.

[0111] In another most preferred embodiment, the use as a cosmetic is for the treatment of the effects on the skin of sleep disruption, more preferably for reducing, preventing and / or eliminating skin aging symptoms and / or skin defects associated with sleep disruption.

[0112] The skin aging symptoms and / or skin defects associated with sleep disruption are preferably (as described above) blepharoptosis, periorbital pigmentation, symptoms associated with barrier dysfunction, or a combination thereof.

[0113] In a preferred embodiment, the skin aging symptoms and / or skin defects associated with sleep disruption are periorbital pigmentation.

[0114] In another preferred embodiment, the skin aging symptoms and / or skin defects associated with sleep disruption are impaired barrier function, more preferably altered skin permeability, skin inflammation, lack of cell aggregation in the skin, or a combination thereof, even more preferably symptoms associated with eye bags, wrinkles (preferably wrinkles around the eyes), or a combination thereof.

[0115] Also, in a preferred embodiment, the use as a cosmetic is for a subject. Preferably, the subject is a mammal, even more preferably a human.

[0116] The peptide or cosmetic composition of the present invention is intended to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0117] The peptide or cosmetic composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0118] In the most preferred embodiment, the peptide or cosmetic composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0119] Also, in the use as a cosmetic of the present invention, the peptide or cosmetic 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% (w / v) to 0.05% (w / v). In the most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (w / v) to 0.001% (w / v), more preferably 0.0005% (w / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (w / v) to 0.001% (w / v).

[0120] In a fourth aspect, the present invention refers to the cosmetic use of the peptide or cosmetic composition according to the present invention.

[0121] The peptide and cosmetic composition according to the present invention are as described above.

[0122] Preferably, the cosmetic use is for the treatment of cosmetic conditions related to inflammation, angiogenesis, extracellular matrix disorders, oxidative stress, modifications at the dermal cell junctions, apoptosis and / or hyperpigmentation, preferably cosmetic inflammation, cosmetic angiogenesis, cosmetic extracellular matrix disorders, cosmetic oxidative stress, cosmetic modifications at the dermal cell junctions, cosmetic apoptosis and / or cosmetic hyperpigmentation.

[0123] More preferably, the cosmetic use is for the treatment of skin aging signs and / or skin defects, more preferably for the reduction, prevention and / or elimination of skin aging signs and / or skin defects.

[0124] As is clear, the above skin aging signs are cosmetic skin aging signs and / or cosmetic skin defects.

[0125] Skin aging is due to chronological and / or environmental aging.

[0126] More preferably, the skin aging signs and / or skin defects are generated by dysregulation of IL-33. As described above, the peptides and cosmetic compositions of the present invention down-regulate IL-33 (they down-regulate the expression of the IL33 gene and / or the expression of interleukin 33).

[0127] In the most preferred embodiment, the skin aging signs and / or skin defects are eye bags, eyelid defects, wrinkles (preferably periorbital wrinkles), hyperpigmentation (preferably periorbital hyperpigmentation), or combinations thereof.

[0128] Eyelid defects are preferably eyelid ptosis, more preferably cosmetic dermatochalasis, cosmetic ptosis, cosmetic ectropion and cosmetic entropion, more preferably cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic degenerative ectropion and cosmetic degenerative entropion.

[0129] Therefore, preferably the skin aging signs and / or skin defects are eye bags, periorbital wrinkles, periorbital hyperpigmentation, eyelid ptosis, or combinations thereof, more preferably eye bags, periorbital wrinkles, periorbital hyperpigmentation, cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic degenerative ectropion, and cosmetic degenerative entropion, or combinations thereof.

[0130] In the most preferred embodiment, the skin aging signs and / or skin defects are eye bags, wrinkles around the eyes, periorbital hemosiderosis, or a combination thereof.

[0131] In another most preferred embodiment, the cosmetic use is for the treatment of the effects on the skin of sleep disruption, and more preferably for reducing, preventing and / or eliminating skin aging signs and / or skin defects associated with sleep disruption.

[0132] Skin aging signs and / or skin defects associated with sleep disruption are preferably (as described above) eyelid ptosis, periorbital hemosiderosis, signs associated with barrier dysfunction, or a combination thereof.

[0133] In a preferred embodiment, the skin aging signs and / or skin defects associated with sleep disruption are periorbital hemosiderosis.

[0134] In another preferred embodiment, the skin aging signs and / or skin defects associated with sleep disruption are impaired barrier function, more preferably altered skin permeability, skin inflammation, lack of cell aggregation in the skin, or a combination thereof, even more preferably signs associated with eye bags, wrinkles (preferably wrinkles around the eyes), or a combination thereof.

[0135] Also, in a preferred embodiment, the cosmetic use of the present invention is for a subject. Preferably, the subject is a mammal, even more preferably a human.

[0136] The peptide or cosmetic composition of the present invention is intended to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), even more preferably in and / or around the eyes.

[0137] The peptide or cosmetic composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), even more preferably in and / or around the eyes.

[0138] In the most preferred embodiment, the peptide or cosmetic composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), and even more preferably in and / or around the eyes.

[0139] Also, in the cosmetic use of the present invention, the peptide or cosmetic 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% (w / v) to 0.05% (w / v). In the most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (w / v) to 0.001% (w / v), more preferably 0.0005% (w / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (w / v) to 0.001% (w / v).

[0140] In a fifth aspect, the present invention refers to a method for the cosmetic treatment of a subject in need thereof, comprising the use of the peptide or cosmetic composition of the present invention in a subject in need thereof.

[0141] The peptides and cosmetic compositions according to the present invention are as described above.

[0142] Preferably, this method is for the cosmetic treatment of conditions related to inflammation, angiogenesis, extracellular matrix disorders, oxidative stress, modifications at the dermal cell junctions, apoptosis and / or hyperpigmentation, preferably cosmetic conditions related to cosmetic inflammation, cosmetic angiogenesis, cosmetic extracellular matrix disorders, cosmetic oxidative stress, cosmetic modifications at the dermal cell junctions, cosmetic apoptosis and / or cosmetic hyperpigmentation.

[0143] More preferably, this method is for the cosmetic treatment of signs of skin aging and / or skin defects, more preferably for the reduction, prevention and / or elimination of signs of skin aging and / or skin defects.

[0144] As is clear, the above skin aging symptoms are cosmetic skin aging symptoms and / or cosmetic skin defects.

[0145] Skin aging is due to chronological and / or environmental aging.

[0146] More preferably, the skin aging symptoms and / or skin defects are generated by dysregulation of IL-33. As described above, the peptides and cosmetic compositions of the present invention down-regulate IL-33 (they down-regulate the expression of the IL33 gene and / or the expression of interleukin 33).

[0147] In the most preferred embodiment, the skin aging symptoms and / or skin defects are eye bags, eyelid defects, wrinkles (preferably periorbital wrinkles), hyperpigmentation (preferably periorbital hyperpigmentation), or combinations thereof.

[0148] Eyelid defects are preferably eyelid ptosis, more preferably cosmetic dermatochalasis, cosmetic ptosis, cosmetic ectropion and cosmetic entropion, more preferably cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic degenerative ectropion and cosmetic degenerative entropion.

[0149] Therefore, preferably the skin aging symptoms and / or skin defects are eye bags, periorbital wrinkles, periorbital hyperpigmentation, eyelid ptosis, or combinations thereof, more preferably eye bags, periorbital wrinkles, periorbital hyperpigmentation, cosmetic dermatochalasis, cosmetic aponeurotic ptosis, cosmetic aponeurotic blepharoptosis, cosmetic degenerative ectropion, and cosmetic degenerative entropion, or combinations thereof.

[0150] In the most preferred embodiment, the skin aging symptoms and / or skin defects are eye bags, periorbital wrinkles, periorbital hyperpigmentation, or combinations thereof.

[0151] In another most preferred embodiment, the method is for the cosmetic treatment of the effects of sleep deprivation on the skin, more preferably for the cosmetic treatment of skin aging signs and / or skin defects associated with sleep deprivation, and even more preferably for cosmetically reducing, preventing and / or eliminating skin aging signs and / or skin defects associated with sleep deprivation.

[0152] Skin aging signs and / or skin defects associated with sleep deprivation are preferably (as described above) eyelid ptosis, periorbital hemosiderosis, signs associated with barrier dysfunction, or combinations thereof.

[0153] In a preferred embodiment, the skin aging signs and / or skin defects associated with sleep deprivation are periorbital hemosiderosis.

[0154] In another preferred embodiment, the skin aging signs and / or skin defects associated with sleep deprivation are impaired barrier function, more preferably altered skin permeability, skin inflammation, lack of cell aggregation in the skin, or combinations thereof, and even more preferably signs associated with eye bags, wrinkles (preferably periorbital wrinkles), or combinations thereof.

[0155] Also, in a preferred aspect, the subject in need thereof is a mammal, and even more preferably a human.

[0156] Preferably, in the method for cosmetic treatment of the present invention, the use of the peptide or cosmetic composition of the present invention in a subject in need thereof is by application of the peptide or cosmetic composition of the present invention in a subject in need thereof.

[0157] The peptide or cosmetic composition of the present invention is intended to be applied by iontophoresis, more preferably to the face of the subject (preferably human), and even more preferably in and / or around the eyes.

[0158] The peptide or cosmetic composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0159] In the most preferred embodiment, the peptide or cosmetic composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0160] Also, in the beauty 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% (w / v) to 0.05% (w / v). In the most preferred embodiment, the peptide of the present invention is used at a concentration of 0.0001% (w / v) to 0.001% (w / v), more preferably 0.0005% (w / v). In another most preferred embodiment, the peptide of the present invention is used at a concentration of 0.05% (w / v) to 0.001% (w / v).

[0161] In a sixth aspect, the present invention refers to the peptide or pharmaceutical composition of the present invention for use as a medicament.

[0162] The peptide and pharmaceutical composition according to the present invention are as described above.

[0163] Preferably, the peptide or composition of the present invention is used in a therapeutically effective amount.

[0164] In a preferred embodiment, the peptide and composition of the present invention are for use in mammals, more preferably in humans.

[0165] Preferably, the peptide or pharmaceutical composition of the present invention is for use in the treatment of diseases associated with inflammation, angiogenesis, extracellular matrix damage, oxidative stress, modification of dermal cell junctions, apoptosis and / or hyperpigmentation, lipid peroxidation, diseases associated with protein glycation, or a medical condition associated with a combination thereof.

[0166] Preferably, the diseases associated with lipid peroxidation are atherosclerosis, inflammatory bowel disease, retinopathy of prematurity, borderline personality disorder, asthma, Parkinson's disease, and kidney disorders.

[0167] Preferably, the diseases associated with protein glycation are diabetes (more preferably diabetic angiopathy), renal failure, and age-related degenerative diseases.

[0168] In the most preferred embodiment, the peptide or pharmaceutical composition of the present invention is for use in the treatment of eyelid defects, preferably blepharoptosis, more preferably dermatochalasis, ptosis, ectropion and entropion, even more preferably dermatochalasis, aponeurotic ptosis, aponeurotic blepharoptosis, degenerative ectropion and degenerative entropion.

[0169] The peptide or pharmaceutical composition of the present invention is intended to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0170] The peptide or pharmaceutical composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0171] In the most preferred embodiment, the peptide or pharmaceutical composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0172] In a seventh aspect, the invention refers to the use of the peptide or pharmaceutical composition of the invention for the manufacture of a medicament.

[0173] The peptides and pharmaceutical compositions according to the invention are as described above.

[0174] Preferably, the peptide or composition of the invention is used in a therapeutically effective amount.

[0175] In a preferred embodiment, the peptides and compositions of the invention are for use in mammals, more preferably in humans.

[0176] Preferably, this use is for the manufacture of a medicament for the treatment of a medical condition associated with inflammation, angiogenesis, extracellular matrix damage, oxidative stress, modification of dermal cell junctions, apoptosis and / or hyperpigmentation, lipid peroxidation, diseases associated with protein glycation, or combinations thereof.

[0177] Preferably, the diseases associated with lipid peroxidation are atherosclerosis, inflammatory bowel disease, retinopathy of prematurity, borderline personality disorder, asthma, Parkinson's disease and kidney disorders.

[0178] Preferably, the diseases associated with protein glycation are diabetes (more preferably diabetic angiopathy), renal insufficiency and age-related degenerative diseases.

[0179] In the most preferred embodiment, the use is for the manufacture of a medicament for the treatment of eyelid defects, preferably blepharoptosis, more preferably dermatochalasis, ptosis, ectropion and entropion, even more preferably dermatochalasis, aponeurotic ptosis, aponeurotic blepharoptosis, degenerative ectropion and degenerative entropion.

[0180] The peptide or pharmaceutical composition of the invention is intended to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), even more preferably into and / or around the eyes.

[0181] The peptide or pharmaceutical composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), and even more preferably into and / or around the eyes.

[0182] In the most preferred embodiment, the peptide or pharmaceutical composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), and even more preferably into and / or around the eyes.

[0183] In a final aspect, the present invention refers to a method for the medical treatment of a subject in need thereof, comprising the use of the peptide or pharmaceutical composition of the present invention.

[0184] The peptide and pharmaceutical composition according to the present invention are as described above.

[0185] Preferably, the peptide or composition of the present invention is used in a therapeutically effective amount.

[0186] In a preferred embodiment, the subject in need of medical treatment is a mammal, more preferably a human.

[0187] Preferably, in the method for cosmetic treatment of the present invention, the use of the peptide or cosmetic composition of the present invention in a subject in need thereof is by application of the said peptide or cosmetic composition of the present invention in a subject in need thereof.

[0188] Preferably, the method for medical treatment of the present invention is for the treatment of a medical condition associated with inflammation, angiogenesis, extracellular matrix disorders, oxidative stress, modification of dermal cell junctions, apoptosis and / or hyperpigmentation, lipid peroxidation, diseases associated with protein glycation, or combinations thereof.

[0189] Preferably, the diseases associated with lipid peroxidation are atherosclerosis, inflammatory bowel disease, retinopathy of prematurity, borderline personality disorder, asthma, Parkinson's disease, and kidney disorders.

[0190] Preferably, the diseases associated with protein glycation are diabetes (more preferably diabetic angiopathy), renal insufficiency, and age-related degenerative diseases.

[0191] In the most preferred embodiment, the method for medical treatment of the present invention is for the treatment of eyelid defects, preferably blepharoptosis, more preferably dermatochalasis, ptosis, ectropion and entropion, and even more preferably dermatochalasis, aponeurotic ptosis, aponeurotic blepharoptosis, degenerative ectropion and degenerative entropion.

[0192] The peptide or pharmaceutical composition of the present invention is intended to be applied by iontophoresis, more preferably to the face of a subject (preferably a human), and even more preferably into and / or around the eyes.

[0193] The peptide or pharmaceutical composition of the present invention is also intended to be applied subcutaneously, more preferably to the face of a subject (preferably a human), and even more preferably into and / or around the eyes.

[0194] In the most preferred embodiment, the peptide or pharmaceutical composition of the present invention is applied topically (preferably in the form of a cream), more preferably to the face of a subject (preferably a human), and even more preferably into and / or around the eyes.

[0195] For better understanding, the present invention will be described in more detail below with reference to the accompanying drawings presented as examples and with reference to exemplary and non-limiting examples.

Brief Description of the Drawings

[0196]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Example

[0197] 〔Abbreviations:〕 The abbreviations used for amino acids follow the 1983 IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in Eur. J. Biochem. (1984) 138:937.

[0198] Ac, acetyl; Arg, arginine; Boc, tert-butyloxycarbonyl; C-terminus, carboxyl terminus; DCM, dichloromethane; DIEA, N,N’-diisopropylethylamine; DIPCDI, N,N’-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; Glu, glutamic acid; Gln, glutamine; hiPSC, human induced pluripotent stem cell; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; INCI, International Nomenclature of Cosmetic Ingredients; MBHA, p-methylbenzhydrylamine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; Met, methionine; N-terminus, amino terminus; Palm, palmitoyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PFA, paraformaldehyde; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethanesulfonyl; RT, room temperature; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, tetramethylbenzidine; Trt, triphenylmethyl or trityl.

[0199] Regarding the chemical synthesis procedures included in this example, it should be noted that all synthesis processes were carried out in a polypropylene syringe equipped with a porous polyethylene disk or a Pyrex (registered trademark) reactor equipped with a porous plate. All reagents and solvents were of synthetic quality and were used without any additional treatment. The solvents and soluble reagents were removed by suction. The Fmoc group was 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., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC, 1997, Boca Raton (Fla., USA)). The washes between the deprotection, coupling, and re-deprotection steps were carried out with DMF (3×1 min) and DCM (3×1 min) each time using 10 ml of solvent / g resin. The coupling reaction was carried out with 3 ml of solvent / g resin. The control of the coupling was carried out by performing the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34: 595-598). All synthesis reactions and washes were carried out at room temperature.

[0200] Example 1. Synthesis and Preparation of Peptide Obtaining Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-Rink-MBHA-resin. AA1 is L-Arg, AA2 is L-Arg, AA3 is L-Gln, AA4 is L-Met, AA5 is L-Arg, AA6 is L-Glu. The weight was normalized. 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin having a functionalization of 0.52 mmol / g was treated with piperidine-DMF according to the general protocol described in the art for removing the Fmoc group. 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equivalents) was incorporated onto the deprotected resin using DMF as a solvent for 1 hour in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; 3 equivalents).

[0201] Subsequently, the resin was washed as described in common methods known in the art, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. According to the previously described protocol, 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents); subsequently 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equivalents); subsequently 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equivalents); subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) and subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) were successively coupled in the presence of 1.01 g of HOBt (7.5 mmol; 3 equivalents) and 1.17 mL of DIPCDI (7.5 mmol; 3 equivalents). As already described above, the deprotection treatment of the Fmoc group was performed during each amino acid addition step.

[0202] After synthesis, the peptide resin was washed with DCM (5 times for 3 minutes each) and dried under vacuum.

[0203] 〔Obtaining Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-Rink-MBHA-resin. AA1 is L-Met, AA2 is L-Arg, AA3 is L-Arg, AA4 is L-Glu, AA5 is L-Gln, AA6 is L-Arg.〕 The weight was normalized. 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin having a functionalization of 0.52 mmol / g was treated with piperidine-DMF according to a common protocol described and known in the art to remove the Fmoc group. 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) was incorporated onto the deprotected resin using DMF as a solvent for 1 hour in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; 3 equivalents).

[0204] Next, the resin was washed as described in common methods known in the art, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. According to the previously described protocol, 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equivalents); then 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equivalents); then 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents); then 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) and then 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equivalents) were successively coupled in the presence of 1.01 g of HOBt (7.5 mmol; 3 equivalents) and 1.17 mL of DIPCDI (7.5 mmol; 3 equivalents). As already described above, the deprotection treatment of the Fmoc group was carried out during each amino acid addition step.

[0205] After synthesis, the peptide resin was washed with DCM (5 times for 3 minutes each) and dried under vacuum.

[0206] Obtaining Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-Rink-MBHA-resin. AA1 is L-Arg, AA2 is L-Glu, AA3 is L-Gln, AA4 is L-Met, AA5 is L-Arg, AA6 is L-Arg. The weight was normalized. 4.8 g (2.5 mmol) of Fmoc-Rink-MBHA resin having a functionalization of 0.52 mmol / g was treated with piperidine-DMF according to a common protocol described in the art to remove the Fmoc group. 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) was incorporated onto the deprotected resin using DMF as a solvent for 1 hour in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; 3 equivalents).

[0207] Next, the resin was washed as described in common methods known in the art, and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. According to the previously described protocol, 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents); then 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equivalents); then 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equivalents); then 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equivalents) and then 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equivalents) were successively coupled in the presence of 1.01 g of HOBt (7.5 mmol; 3 equivalents) and 1.17 mL of DIPCDI (7.5 mmol; 3 equivalents). As already described above, the deprotection treatment of the Fmoc group was carried out during each amino acid addition step.

[0208] After synthesis, the peptide resin was washed with DCM (5 times for 3 minutes each) and dried under vacuum.

[0209] 〔Example 2. Removal of the Fmoc N-terminal protecting group of the peptide synthesized according to Example 1〕 The N-terminal Fmoc group of the peptidyl resin obtained in Example 1 was deprotected with 20% (v / v) piperidine in DMF (1×1 minute + 2×10 minutes) (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 minute), DCM (4×1 minute) and dried under vacuum.

[0210] 〔Example 3. Method for introducing an R1 acetyl group onto the peptidyl resin obtained in Example 2〕 1 mmol (1 equivalent) of the peptidyl resin obtained according to Example 2 was treated with 25 equivalents of acetic anhydride in the presence of 25 equivalents of DIEA using 5 mL of DMF as a solvent. After reacting them for 30 minutes, the peptidyl resin was washed with DMF (5 × 1 minute) and DCM (4 × 1 minute), and dried under vacuum.

[0211] 〔Example 4. Method for cleaving the peptidyl resin obtained according to Examples 2 and 3 from the polymer support〕 The weight was normalized. 200 mg of the dry peptidyl resin obtained in either Example 2 or 3 was treated with 5 ml of TFA / TIS / H2O (90:5:5) at room temperature for 2 hours with stirring. The filtrate was collected and precipitated using 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.

[0212] 〔Example 5. Characterization of the peptide synthesized and prepared according to Example 4〕 High-performance liquid chromatography analysis of the peptide obtained in Example 4 was performed using a reverse-phase column (150 × 4.6 mm, XBridge Peptide BEC18, 3.5 μm, Waters, USA) with a gradient of MeCN (+0.036% (v / v) TFA) in H2O (+0.045% (v / v) TFA) at a flow rate of 1.25 mL / min using a Shimadzu apparatus (Kyoto, Japan) and detection was carried out at 220 nm. All peptides showed a purity of over 80%. The identity of the obtained peptide was confirmed by ESI-MS in a Waters ZQ 4000 detector with a flow rate of 0.2 mL / min using MeOH as the mobile phase. The results obtained demonstrated that the peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2, and Ac-SEQ ID NO:3-NH2 were synthesized accurately and effectively.

[0213] 〔Example 6. Preparation of a facial cosmetic composition containing Ac-SEQ ID NO:1-NH2〕 A facial cosmetic composition was prepared according to Table 1 below. For this purpose, the components from Phase A were dissolved in a suitable container and the mixture was heated to 70 - 75°C. In another container, the components of Phase B were mixed together and the mixture was heated to 70 - 75°C. Next, Phase C (TEGOLON 12 - 10 (INCI: Nylon - 12)) was slowly added to Phase B with stirring until it was completely dissolved. The mixture was heated to 70 - 75°C. Next, the solution of Phase A was added to the mixture of Phases B and C under turbine stirring to form an emulsion. Next, Phase D was slowly added to the mixture and stirring was maintained until a homogeneous emulsion was obtained. Finally, at about 30°C, a commercially available formulation containing the compound Ac - SEQ ID NO:1 - NH2 (INCI: Water (Aqua), Glycerin, Caprylyl Glycol, Ac - SEQ ID NO:1 - NH2) was slowly added to the mixture with stirring.

[0214]

Table 1

[0215] 〔Example 7. Analysis of the Regulation of IL33 Expression in HEKa (Human Dermal Fibroblasts, Adult) Cells and HDFa (Human Dermal Fibroblasts, Adult) Cells〕 The peptides Ac - SEQ ID NO:1 - NH2, Ac - SEQ ID NO:2 - NH2 and Ac - SEQ ID NO:3 - NH2 were analyzed for their ability to regulate the expression of IL33 in HEKa cells (all three peptides) and HDFa (only analyzed for the peptide Ac - SEQ ID NO:1 - NH2).

[0216] The peptides used in this example were prepared according to Examples 1 - 5.

[0217] Stock solutions of the peptides were prepared in water at 1 mg / mL. Working solutions were freshly prepared at specific concentrations from the stock solutions in the corresponding supplemented medium.

[0218] Untreated cells were used as the basal or negative control sample.

[0219] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HDFa and HEKa cells were seeded in 6-well plates at a density of 4×10 5 cells / well in duplicates (n = 2) and maintained for 24 hours under standard culture conditions (106 medium supplemented with 1% (v / v) LSGS for HDFa cells; Epilife medium supplemented with 1% (v / v) EDGS and 1% (v / v) penicillin / streptomycin for HEKa cells; 37 °C, 95% indoor humidity, 5% CO2). The cells were then treated with the peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2 or Ac-SEQ ID NO:3-NH2 at a concentration of 0.01 mg / mL for 24 hours. Untreated cells were used as the basal or negative control.

[0220] The cells were finally lysed and the replicates were pooled together according to the manufacturer's instructions for RNA extraction using the Qiagen RNeasy Mini kit. Using the purified RNA, complementary DNA (cDNA), which serves as a template for amplification, was generated by reverse transcription using a commercially available high-capacity cDNA reverse transcription kit (Applied Biosystems). RT-qPCR was performed using a StepOne plus Real-Time PCR instrument with the appropriate TaqMan assay probe for IL33 (adding GAPDH - glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene) and 2x gene expression master mix. Amplification included 40 cycles of 15 seconds at 95 °C (denaturation) and 1 minute at 60 °C (annealing and extension) (Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., Patel, H.R. (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).

[0221] The data obtained were analyzed using the ΔΔCt method, which provided the target gene expression values as fold changes in the treated samples compared to the untreated basal samples. Both samples were normalized by the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0222] The analysis procedure is as follows: 1. Calculate the average Ct for each condition 2. Calculate ΔCT for the test sample and ΔCT for the untreated sample 3. Calculate ΔΔCT: ΔΔCT = ΔCT test sample - ΔCT untreated sample 4. 2 -ΔΔCT to obtain the ratio.

[0223] In this assay, a clear downregulation of the expression of the IL33 gene was observed in HEKa cells for all peptides tested: Ac-SEQ ID NO:1-NH2: -4.58-fold change in treated cells relative to the basal control. Ac-SEQ ID NO:2-NH2: -3.77-fold change in treated cells relative to the basal control. Ac-SEQ ID NO::3-NH2: -3.93-fold change in treated cells relative to the basal control.

[0224] Furthermore, for the peptide Ac-SEQ ID NO:1-NH2, a downregulation of the expression of IL33 was also observed in HDFa cells (-1.22-fold change in treated cells relative to the basal control).

[0225] The results of this example demonstrate the usefulness of the peptides of the present invention for downregulating the expression of IL33 and thus, as described above, mainly provide improvement of inflammation, further improvement of the skin barrier, reduction of angiogenesis, and reduction of pigmentation. Accordingly, the results of this example demonstrate the usefulness of the peptides of the present invention for reducing, preventing and / or eliminating skin aging signs and / or skin defects, more preferably eye bags, eyelid defects, wrinkles around the eyes, and dark circles (periorbital hyperpigmentation).

[0226] [Example 8. Analysis of the production of the protein IL-33 in HDFa cells] The peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to regulate IL-33 production in adult human dermal fibroblasts.

[0227] The peptide Ac-SEQ ID NO:1-NH2 was prepared according to Examples 1 to 5.

[0228] Adult human dermal fibroblasts (HDFa) were seeded onto 24-well plates at a concentration of 1×10 5 cells / mL in their corresponding growth media and incubated for 24 hours under normal culture conditions; at that point, three different concentrations (0.005, 0.01, and 0.05 mg / mL) of Ac-SEQ ID NO:1-NH2 were added in the presence of an IL-33-inducing stimulus (IFNγ, 300 U / mL). Cells without stimulation were also included as a basal group. The positive control group was cells treated with 300 U / mL of IFNγ but not with the peptide of the present invention. After 24 hours, the conditioned media was collected and the cells were processed for protein extraction.

[0229] The level of IL-33 in the cell lysates was analyzed using a commercially available ELISA kit (DuoSet ELISAR&D). Absorbance was measured using an automated spectrophotometer plate reader.

[0230] The results obtained in this example are summarized in Figure 1, and it can be seen that IFNγ (300 U / mL) induces an increase in the production of the protein IL-33 in HDFa cells, and that the peptide Ac-SEQ ID NO:1-NH2 dose-dependently decreases the level of IL-33 protein produced by HDFa cells exposed to IFNγ (300 U / mL) (this is that a greater decrease in the IL-33 protein level is observed as the concentration of the peptide Ac-SEQ ID NO:1-NH2 increases).

[0231] The results of this example demonstrate the usefulness of the peptide of the present invention for reducing inflammation through downregulation of the expression of IL-33, and thus, as described above, provide improvement of the skin barrier, reduction of angiogenesis, and reduction of pigmentation. Therefore, the results of this example demonstrate the usefulness of the peptide of the present invention for reducing, preventing, and / or eliminating skin aging symptoms and / or skin defects, more preferably eye bags, eyelid defects, wrinkles around the eyes, and dark circles (periorbital hyperpigmentation).

[0232] [Example 9. Analysis of Gene Expression Regulation in Human Epidermal Keratinocytes, Adult (hereinafter, "HEKa")] The peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2 and Ac-SEQ ID NO:3-NH2 were analyzed for their ability to regulate the expression of genes related to epidermal cell aggregation (see Table 2 for the genes analyzed).

[0233] The peptides used in this example were prepared according to Examples 1 to 5.

[0234] Stock solutions of the peptides were prepared in water at 1 mg / mL. Working solutions were freshly prepared at specific concentrations from the stock solutions in the corresponding supplemented medium.

[0235] Untreated cells were used as a basal or negative control sample.

[0236] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa cells were seeded in 6-well plates at a density of 4 × 10 5 cells / well in duplicate (n = 2) and maintained for 24 hours under standard culture conditions (medium Epilife supplemented with 1% (v / v) EDGS and 1% (v / v) penicillin / streptomycin; 37 °C, 95% indoor humidity, 5% CO2).

[0237] The cells were then treated with the peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2 or Ac-SEQ ID NO:3-NH2 at a concentration of 0.01 mg / mL for 24 hours. Untreated cells were used as a basal or negative control.

[0238] The cells were finally lysed and the replicates were pooled together according to the manufacturer's instructions for RNA extraction using the Qiagen RNeasy Mini kit. Using the purified RNA, complementary DNA (cDNA), which serves as a template for amplification, was generated by reverse transcription using a commercially available high-capacity cDNA reverse transcription kit (Applied Biosystems). RT-qPCR was performed using a StepOne plus Real-Time PCR instrument with a panel of appropriate TaqMan assay probes for the genes shown in Table 1 (including GAPDH - glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene) and 2x gene expression master mix. Amplification included 40 cycles of 15 seconds at 95°C (denaturation) and 1 minute at 60°C (annealing and extension) (Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., Patel, H.R. (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).

[0239]

Table 2

[0240] The data obtained were analyzed using the ΔΔCt method, which provided the target gene expression values as fold changes in the treated samples compared to the untreated basal samples. Both samples were normalized to the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0241] The procedure for the analysis was as follows: 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 to obtain the ratio.

[0242] The results of this assay are also summarized in Tables 3 to 5.

[0243]

Table 3

[0244]

Table 4

[0245]

Table 5

[0246] From Tables 3 to 5, the following can be easily derived: Peptide Ac-SEQ ID NO:1-NH2 significantly upregulated important genes related to adherens junctions and barrier function: JUP, OCLN, DSP, CLDN1, CLDN4, DSC2, CDSN, PCDH1, TJP1, PKP2, and PPL. Peptide Ac-SEQ ID NO:2-NH2 significantly upregulated important genes related to adherens junctions and barrier function: OCLN, CLDN4, PCDH1, TJP1, and PKP2. Peptide Ac-SEQ ID NO:3-NH2 significantly upregulated important genes related to adherens junctions and barrier function: JUP, OCLN, DSP, CLDN1, DSC1, CLDN4, DSC2, PCDH1, TJP1, PKP2, and PPL.

[0247] Therefore, the peptides of the present invention reduce, prevent and / or eliminate skin aging symptoms and / or skin defects (as exemplified by Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2 and Ac-SEQ ID NO:3-NH2), and more precisely, the results of this example demonstrate the usefulness of the peptides of the present invention for improving the tight junctions between keratinocytes, improving the aggregation between them, thus improving the firmness of the skin and reducing wrinkles.

[0248] 〔Example 10. Analysis of Gene Expression Regulation in Primary Human Dermal Fibroblasts, Adult〕 Peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2 and Ac-SEQ ID NO:3-NH2 were analyzed for their ability to regulate the expression of genes related to the production of collagen and extracellular matrix (see Table 6 for the genes analyzed).

[0249] The peptides used in this example were prepared according to Examples 1-5.

[0250] Stock solutions of the peptides were prepared in water at 1 mg / mL. Working solutions were freshly prepared at specific concentrations from the stock solutions in the corresponding supplemented medium.

[0251] Untreated cells were used as the basal or negative control samples.

[0252] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HDFa cells were seeded in 6-well plates at a density of 4×10 5 cells / well in duplicate (n = 2) and maintained for 24 hours under standard culture conditions (106 medium supplemented with 1% (v / v) LSGS; 37 °C, 95% indoor humidity, 5% CO2). The cells were then treated with peptide Ac-SEQ ID NO:1-NH2 at a concentration of 0.01 mg / mL for 24 hours and 48 hours. Untreated cells were used as the basal or negative control.

[0253] The cells were finally lysed and the replicates were pooled together according to the manufacturer's instructions for RNA extraction using the Qiagen RNeasy Mini kit. Using the purified RNA, complementary DNA (cDNA), which serves as a template for amplification, was generated by reverse transcription using a high-capacity cDNA reverse transcription kit (Applied Biosystems) of a commercially available kit. RT-qPCR was performed using a StepOne plus Real-Time PCR instrument with a panel of appropriate TaqMan assay probes for the genes shown in Table 5 (adding GAPDH - glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene) and 2x gene expression master mix. Amplification included 40 cycles of 15 seconds at 95 °C (denaturation) and 1 minute at 60 °C (annealing and extension) (Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., Patel, H.R. (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).

[0254]

Table 6

[0255] The data obtained were analyzed using the ΔΔCt method, which provided target gene expression values as fold changes in treated samples compared to untreated basal samples. Both samples were normalized to the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0256] The procedure for the analysis is as follows: 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 to obtain the ratio.

[0257] The results of this assay are summarized in Tables 7 to 10.

[0258]

Table 7

[0259]

Table 8

[0260]

Table 9

[0261]

Table 10

[0262] From Tables 7 and 8, the following can be readily derived for the peptide Ac-SEQ ID NO:1-NH2: At 24 hours, upregulation of LAMA1, COL3A1, and TIMP2 was observed. At 48 hours, downregulation of MMP1 and MMP3 was observed.

[0263] From Table 9, the following can be readily derived for the peptide Ac-SEQ ID NO:2-NH2: At 24 hours, upregulation of FBN1 and downregulation of MMP1 were observed.

[0264] From Table 10, the following can be readily derived for the peptide Ac-SEQ ID NO:3-NH2: At 24 hours, upregulation of FBN1 and downregulation of MMP1 and MMP3 were observed.

[0265] Therefore, the peptides of the present invention showed a favorable regulation of genes with respect to ECM synthesis and maintenance (as exemplified by Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2, and Ac-SEQ ID NO:3-NH2). Therefore, the peptides of the present invention can reduce, prevent and / or eliminate skin aging symptoms and / or skin defects. More precisely, the results of this example demonstrate the usefulness of the peptides of the present invention in improving skin firmness and reducing wrinkles.

[0266] 〔Example 11. Analysis of Tyrosinase Activity〕 The peptides Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2, Ac-SEQ ID NO:3-NH2, and Ac-Arg-Arg-Gln-Met-Glu-Glu--NH2 (Ac-SEQ ID NO:4-NH2) were analyzed for their ability to regulate tyrosinase activity.

[0267] The peptides used in this example were prepared according to Examples 1 to 5.

[0268] Briefly stated, Ac-SEQ ID NO:1-NH2 (0.05, 0.25, 0.5, and 2.5 mg / mL), Ac-SEQ ID NO:2-NH2 (0.05, 0.25, 0.5, and 2.5 mg / mL), Ac-SEQ ID NO:3-NH2 (0.05, 0.25, 0.5, and 2.5 mg / mL), and Ac-SEQ ID NO:4-NH2 (0.005, 0.025, 0.05, 0.25, 0.5, and 2.5 mg / mL), and the positive control kojic acid (400 and 800 μM) were incubated with recombinant mushroom tyrosinase for 30 minutes before the addition of L-DOPA (L-3,4-dihydroxyphenylalanine, 2.5 mg / mL in PBS). After reacting for 2 hours at room temperature, the absorbance at 450 nm of each well, which is directly proportional to the amount of dopachrome in the reaction mixture, was measured using a microplate reader Multiskan FC (Thermo Fisher Scientific, MA, USA).

[0269] The absorbance values were normalized with respect to non-treated wells (controls) established as 100%, thereby obtaining the percentage of activity for each state tested.

[0270] The results obtained in this example are summarized in Figure 2 (2A - 2D).

[0271] As can be directly derived from this figure, the peptide Ac-SEQ ID NO:1-NH2 (Figure 2A) inhibited tyrosinase at all concentrations tested, and this effect increased dose-dependently after 0.25 mg / mL.

[0272] On the other hand, the peptide Ac-SEQ ID NO:2-NH2 (Figure 2B) showed dose-dependent inhibition of tyrosinase activity from 0.05 mg / mL onwards.

[0273] The peptide Ac-SEQ ID NO:3-NH2 (Figure 2C) showed dose-dependent inhibition of tyrosinase activity at concentrations of 0.25 mg / mL and above.

[0274] On the other hand, the peptide Ac-SEQ ID NO:4-NH2 (Figure 2D) (which is not a peptide of the present invention) did not inhibit tyrosinase activity at any of the tested concentrations. On the contrary, it increased tyrosinase activity in a dose-dependent manner at concentrations of 0.05 mg / mL and higher.

[0275] In the state of the art, it is known that, among other things, dark circles are caused by the deposition of melanin, and it has also been established that the suppression of tyrosinase activity reduces melanin deposition (MI Ryung Roh, Kee Yang Chung: Infraorbital Dark Circles: Definition, Causes, and Treatment Options, Dermatological Surgery, 35:8:August 2009).

[0276] These results demonstrate the usefulness of the peptides of the present invention (such as those exemplified by Ac-SEQ ID NO:1-NH2, Ac-SEQ ID NO:2-NH2, and Ac-SEQ ID NO:3-NH2) for the treatment and / or prevention of hyperpigmentation in the eye, preferably periorbital hemosiderosis, rather than similar peptides (Ac-SEQ ID NO:4-NH2) that maintain or increase said activity instead of suppressing tyrosinase activity.

[0277] 〔Example 12. Effect of Peptide Ac-SEQ ID NO:1-NH2 on Angiogenesis〕 Peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to regulate angiogenesis in vitro.

[0278] Peptide Ac-SEQ ID NO:1-NH2 was prepared according to Examples 1 to 5.

[0279] In this case, a tube formation assay was performed using human umbilical vein endothelial cells (HUVEC). Briefly, 40 μL of geltrex was added to the wells of a 96-well plate and allowed to solidify for 30 minutes. Then, 8000 cells were applied to each well together: Complete medium (basal control). Complete medium + 20 μM suramin (an inhibitor of tube formation). Complete medium + 10 ng / mL IL-33. Complete medium + 10 ng / mL IL-33 + various concentrations of peptide Ac-SEQ ID NO:1-NH2 (more precisely, 0.01 mg / mL, 0.05 mg / mL, or 0.1 mg / mL).

[0280] After 16 hours of incubation, the cells were visualized by microscopy and photographed at 10× for each well. For each well, the number of meshes and the total area of the meshes were analyzed as indicators of angiogenesis (which is an indicator of neovascularization).

[0281] The results are summarized in Figure 3.

[0282] As can be readily derived from the figure, peptide Ac-SEQ ID NO:1-NH2 was able to cover angiogenesis or neovascularization induced by IL-33 at all concentrations tested, with respect to both the number of meshes and the total area of the meshes.

[0283] Thus, as exemplified by Ac-SEQ ID NO:1-NH2, the peptides of the present invention can inhibit and / or prevent neovascularization, and thus they are effective in the treatment of neovascularization or excessive angiogenesis, preferably cosmetic properties associated with eye bags and suborbital periorbital hemosiderosis.

[0284] [Example 13. Analysis of the protective ability of the peptides of the present invention against lipid peroxidation] Peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to protect against lipid peroxidation by the thiobarbituric acid reactive substance (hereinafter, TBAR) assay.

[0285] Peptide Ac-SEQ ID NO:1-NH2 was prepared according to Examples 1 to 5.

[0286] Briefly stated, 0.01, 0.05, 0.1 and 0.5 mg / mL of the peptide Ac-SEQ ID NO:1-NH2 in the presence of Trolox (positive control), small unilamellar vesicles of egg yolk phosphatidylcholine (EYPC), and a substrate for lipid peroxidation were incubated at 37 °C for 2 hours in the presence of thiobarbituric acid (TBA). Using fluorescence spectrometry, the % of lipid peroxidation was evaluated compared to the negative control (sample having only small unilamellar vesicles of egg yolk phosphatidylcholine).

[0287] The results obtained in this example are summarized and shown in Figure 4.

[0288] As can be directly derived from this figure, the peptide of the present invention exhibits antioxidant activity at all concentrations tested.

[0289] Therefore, as exemplified by Ac-SEQ ID NO:1-NH2, the peptide of the present invention was able to protect from lipid peroxidation.

[0290] Since melatonin is also a potent antioxidant, the ability of the peptide to protect from peroxidation was also compared to melatonin.

[0291] The tests, controls, procedures, materials and data analysis were the same as those described above.

[0292] Figure 8 shows a graphical representation of the data obtained for anti-peroxidation activity compared to 0.1 mg / mL and 0.5 mg / mL of melatonin.

[0293] This data indicates that the peptide of the present invention has a melatonin-like protective effect against lipid peroxidation.

[0294] As is known in the art, lipid peroxidation is associated with cell damage and death due to changes in cell membranes.

[0295] Therefore, the peptides of the present invention as exemplified by Ac-SEQ ID NO:1-NH2 are effective in cosmetics for the prevention and / or treatment of aging, and in pharmaceuticals for the prevention and / or treatment of diseases associated with lipid peroxidation, preferably atherosclerosis, inflammatory bowel disease, retinopathy of prematurity, borderline personality disorder, asthma, Parkinson's disease and kidney disorders.

[0296] [Example 14. Analysis of the ability of the peptides of the present invention to protect against glycation] The peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to reduce the production of advanced glycation end products (AGE).

[0297] The peptide Ac-SEQ ID NO:1-NH2 was prepared according to Examples 1-5.

[0298] Briefly, peptides Ac-SEQ ID NO:1-NH2 at 0.01, 0.05, 0.1 and 0.5 mg / mL were mixed with a glycating agent and its corresponding substrate. The samples were incubated at 37 °C for 72 hours. The % protein glycation was evaluated using fluorescence spectrometry, compared to a negative control (sample with only the sugar substrate).

[0299] The results obtained in this example are summarized in Figure 5.

[0300] As can be directly derived from this figure, the peptides of the present invention were able to almost completely reverse the glycation produced by the glycating agent at all concentrations tested.

[0301] Therefore, as exemplified by Ac-SEQ ID NO:1-NH2, the peptides of the present invention were able to protect against protein glycation.

[0302] Furthermore, the performance of the peptides of the present invention to protect against protein glycation was also tested ex vivo.

[0303] Briefly, human skin explants from 40-year-old women were kept alive in humidified 5% CO2 atmosphere, in BEM culture medium at 37 °C. A cosmetic composition according to Example 6 containing 1% (w / v) of Ac-SEQ ID NO:1-NH2 was prepared, and also a peptide-free one (placebo) was prepared. These creams were applied topically at 2 μL per explant (2 mg / cm 2 ) based on and spread using a small spatula on day 0 (D0), day 1, day 4, day 6 and day 8. Control explants received no treatment, except for the renewal of the culture medium. To induce glycation, a solution of methylglyoxal (MG) was incorporated into the BEM culture medium at a final concentration of 500 μM on day 4, day 6 and day 8 of the relevant batch “MG” (all samples except control untreated explants). The grafts were harvested at each time point and cut into two parts. Half were fixed in buffered formalin solution and half were frozen at -80 °C.

[0304] After fixation in buffered formalin for 24 h, the samples were dehydrated and impregnated with paraffin. Sections 5 μm thick were made using a Leica RM 2125 Minot microtome and the sections were placed on Superfrost® histological glass slides. Carboxymethyllysine (CML) immunostaining (CML is an advanced glycation end product (AGE) found on proteins and lipids as a result of oxidative stress and chemical glycation and is used as a marker of advanced glycation.) was performed overnight at room temperature using a monoclonal anti-CML antibody diluted 1:25 in PBS-BSA 0.3% (w / v) and revealed by VECTOR® VIP (Vector Laboratories), a substrate for peroxidase.

[0305] As seen in Figure 6, the peptide Ac-SEQ ID NO:1-NH2 was protected from MG-induced glycation after 9 days of culture and showed a significant decrease (-73%) in the CML signal when compared to untreated explants exposed to MG. Placebo-treated explants did not show a significant decrease in CML staining.

[0306] Melatonin is also a powerful antioxidant that can protect against glycation, so the analysis of the peptide's ability to reduce the formation of advanced glycation end products (AGE) (protection against glycation) was also compared with melatonin.

[0307] The tests, controls, procedures, materials, and data analysis were the same as those described above.

[0308] Figure 9 shows a graphical representation of the data obtained for antiglycation activity, compared with melatonin at 0.1 mg / mL and 0.5 mg / mL.

[0309] This data indicates that the peptide of the present invention has a melatonin-like protective effect against glycation.

[0310] As is known in the art, glycation occurs naturally over time but is also one of the endogenous aging mechanisms that occur pathologically during diabetes, renal failure, and inflammation. AGE accumulates highly in tissues and organs in many age-related degenerative diseases. These toxic adducts (glycotoxins) are associated with cell dysfunction, particularly in diabetic patients and older organisms. AGE formation and accumulation in diabetic patients lead to vascular changes that result in diabetic angiopathy.

[0311] Glycation is also associated with extracellular matrix damage (damage to collagen and elastic fibers), inflammation, oxidative stress, and apoptosis, and thus it is associated with aging.

[0312] Therefore, the peptide of the present invention, as exemplified by Ac-SEQ ID NO:1-NH2, can protect against protein glycation and is thus effective in cosmetics for the prevention and / or treatment of aging, as well as in pharmaceuticals for the prevention and / or treatment of glycation-related diseases, preferably diabetes (more preferably diabetic angiopathy), renal failure, inflammation, and degenerative diseases associated with aging.

[0313] 〔Example 15. Analysis of Fibrillin-1 in Ex Vivo Samples〕 The peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to protect the dermal extracellular matrix from glycation.

[0314] The peptide Ac-SEQ ID NO:1-NH2 was prepared according to Examples 1 to 5.

[0315] Briefly, human skin explants from 40-year-old women were kept viable in BEM culture medium at 37 °C in a humidified 5% CO2 atmosphere. Placebo cream and cream containing 1% Ac-SEQ ID NO:1-NH2 prepared according to Example 6 were topically applied at 2 μL per explant (2 mg / cm 2 ) and spread using a small spatula on days 0 (D0), 1, 4, 6, and 8. Control explants received no treatment except for renewal of the culture medium. To induce glycation, a solution of methylglyoxal (MG) was incorporated into the BEM culture medium at a final concentration of 500 μM on days 4, 6, and 8 for the relevant batches "MG" (all samples except control untreated samples). The grafts were harvested at each time point and cut into two parts. Half was fixed in buffered formalin solution and half was frozen at -80 °C.

[0316] Fibrillin 1 immunostaining was performed on frozen sections at room temperature for 1 hour using a monoclonal antifibrillin 1 antibody diluted 1:500 in PBS, 0.3% (w / v) BSA and 0.05% (v / v) Tween 20 and visualized using a biotin / streptavidin amplification system. Nuclei were counterstained with propidium iodide.

[0317] Data from Figure 7 show how exposure to MG induces a significant decrease in fibrillin 1 staining in the dermis compared to non-exposed controls on day 9. The peptide Ac-SEQ ID NO:1-NH2 was able to protect from this impairment and showed a significant increase (86%) in fibrillin 1 staining on day 9 compared to untreated controls exposed to MG.

[0318] As is known in the art, fibrillin-1 is a major component of oxytalan fibers found along the dermal-epidermal junction that exhibits essential functions in maintaining skin integrity and mechanical properties (Oxlund et al., 1980. J Anat. 131: 611-620). In skin cosmetic research, fibrillin-1 is commonly used as a biomarker for the structural state of the extracellular matrix of the dermis.

[0319] Therefore, the peptides of the present invention reduce, prevent and / or eliminate skin aging symptoms and / or skin defects (as exemplified by Ac-SEQ ID NO:1-NH2), and more precisely, the results of this example demonstrate the usefulness of the peptides of the present invention in maintaining skin integrity and mechanical properties, and thus improving skin firmness and reducing wrinkles.

[0320] [Example 16. Analysis of Gene Expression Related to Sleep Deprivation] The peptide Ac-SEQ ID NO:1-NH2 was analyzed for its ability to regulate the expression of genes related to the results of sleep deprivation in HDFa cells, HEKa cells and HUVECs (see Table 11 for the genes analyzed).

[0321] The peptides used in this example were prepared according to Examples 1-5.

[0322] A stock solution of the peptide was prepared in water at 1 mg / mL. The working solution was freshly prepared at a specific concentration from the stock solution in the corresponding supplemented medium.

[0323] Untreated cells were used as basal or negative control samples.

[0324] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa, HDFa or HUVEC cells were plated in duplicates (n = 2) at 4×10 5 cells / well (2×10 5Seeded in a 6-well plate at a density of (cells / well), and maintained for 24 hours under standard culture conditions (for HDFa cells, medium 106 supplemented with 1% (v / v) LSGS; for HEKa cells, Epilife medium supplemented with 1% (v / v) EDGS and 1% (v / v) penicillin / streptomycin; for HUVEC cells, endothelial cell growth medium; 37 °C, 95% indoor humidity, 5% CO2).

[0325] The cells were then treated with the peptide Ac-SEQ ID NO:1-NH2 at a concentration of 0.01 mg / mL for 24 hours. Untreated cells were used as the basal or negative control sample.

[0326] The cells were finally lysed and the replicates were pooled together according to the manufacturer's instructions for RNA extraction using the Qiagen RNeasy Mini kit. Using the purified RNA, complementary DNA (cDNA), which serves as a template for amplification, was generated by reverse transcription using a high-capacity cDNA reverse transcription kit (Applied Biosystems) of a commercially available kit. RT-qPCR was performed using a StepOne plus Real-Time PCR instrument with a panel of appropriate TaqMan assay probes for the genes shown in Table 10 (adding GAPDH - glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene) and 2x gene expression master mix. Amplification included 40 cycles of 15 seconds at 95°C (denaturation) and 1 minute at 60°C (annealing and extension) (Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., Patel, H.R. (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).

[0327]

Table 11

[0328] The data obtained were analyzed using the ΔΔCt method, which provided target gene expression values as fold changes in treated samples compared to untreated basal samples. Both samples were normalized by the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0329] The analysis procedure is as follows: 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 to obtain the ratio.

[0330] The results of this assay are also summarized in Tables 12 to 14.

[0331]

Table 12

[0332]

Table 13

[0333]

Table 14

[0334] Therefore, the peptide of the present invention reduces, prevents and / or eliminates skin aging symptoms and / or skin defects related to sleep disruption (as exemplified by Ac-SEQ ID NO:1-NH2). This is because the peptide Ac-SEQ ID NO:1-NH2 can regulate its signal by inducing the down-regulation of the IL33 receptor (IL1RL1) and the IL33-related inhibitor (SIGIRR) in addition to its effect on the IL-33 level (ELISA). The regulation of other inflammation-related genes (CXCL8 and NFKB1) has also been observed.

[0335] Apart from this effect on inflammation, the peptide Ac-SEQ ID NO:1-NH2 can also down-regulate the expression of the glucose transporter SLC2A1 and up-regulate the expression of the melatonin receptor MNTR1, both of which are involved in the physiological response to sleep disruption.

[0336] 〔Example 17. Analysis of Gene Expression Induced by Treatment with Ac-SEQ ID NO:4-NH2〕 The gene regulation of the peptide Ac-SEQ ID NO:4-NH2 was analyzed in this example in HDFa cells and HEKa cells (see Table 15 for the genes analyzed).

[0337] The peptides used in this example were prepared according to Examples 1 to 5 and subjected to the necessary adaptations.

[0338] A stock solution of the peptide was prepared in water at 1 mg / mL. The working solution was freshly prepared at a specific concentration from the stock solution in the corresponding supplemented medium.

[0339] Untreated cells were used as the basal or negative control sample.

[0340] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa cells or HDFa cells were seeded in 6-well plates in duplicates (n = 2) at a density of 4 × 10 5 cells / well and maintained for 24 hours under standard culture conditions (for HDFa cells, medium 106 supplemented with 1% (v / v) LSGS; for HEKa cells, medium supplemented with 1% (v / v) EDGS and 1% (v / v) penicillin / streptomycin; 37 °C, 95% indoor humidity, 5% CO2).

[0341] The cells were then treated with the peptide Ac-SEQ ID NO:4-NH2 at a concentration of 0.01 mg / mL for 24 hours. Untreated cells were used as the basal or negative control sample.

[0342] The cells were finally lysed, and replicates were pooled together according to the manufacturer's instructions for RNA extraction using the Qiagen RNeasy Mini kit. Using the purified RNA, corresponding cDNA (complementary deoxyribonucleic acid) that serves as a template for amplification was generated by reverse transcription using a high-capacity cDNA reverse transcription kit (Applied Biosystems) of a commercially available kit. RT-qPCR was performed using a StepOne plus Real-Time PCR instrument with a panel of appropriate TaqMan assay probes for the genes shown in Table 10 (adding GAPDH - glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene) and 2x gene expression master mix. Amplification included 40 cycles of 15 seconds at 95°C (denaturation) and 1 minute at 60°C (annealing and extension) (Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., Patel, H.R. (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).

[0343]

Table 15

[0344] The data obtained were analyzed using the ΔΔCt method, which provided target gene expression values as fold changes in treated samples compared to untreated basal samples. Both samples were normalized by the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

[0345] The analysis procedure is as follows: 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 to obtain the ratio.

[0346] The results of this assay are also summarized in Tables 16 and 17.

[0347]

Table 16

[0348]

Table 17

[0349] Thus, as can be directly obtained from the results included in Tables 16 and 17, the peptide Ac-SEQ ID NO:4-NH2 (which, as noted above, is not a peptide of the present invention but is very similar) does not exhibit the favorable gene regulation seen for the peptides of the present invention. On the contrary, this shows inverse gene regulation in a certain group of genes and a lack of effect on expression in another group of genes.

[0350] Thus, Ac-SEQ ID NO:4-NH2 is in contrast to what was demonstrated above for the peptides of the present invention: Ac-SEQ ID NO:4-NH2 showed down-regulation of COL3A1 and TIMP2. Therefore, Ac-SEQ ID NO:4-NH2 showed an unfavorable regulation of genes with respect to ECM synthesis and maintenance. Therefore, in contrast to the peptides of the present invention, Ac-SEQ ID NO:4-NH2 cannot reduce, prevent and / or eliminate skin aging signs and / or skin defects. More precisely, the results of this example demonstrate that the said peptides cannot improve skin firmness and reduce wrinkles. Ac-SEQ ID NO:4-NH2 shows a slight down-regulation or lack of regulation of the genes OCLN, CDSN and TJP1, i.e., important genes related to tight junctions and barrier functions. Therefore, Ac-SEQ ID NO:4-NH2 cannot improve the tight junctions between keratinocytes, and thus cannot improve the aggregation between them, and thus cannot improve skin firmness and reduce wrinkles, in contrast to what has been demonstrated for the peptides for the present invention. In addition, the peptide Ac-SEQ ID NO:4-NH2 showed down-regulation of SIGIRR and a slight down-regulation or lack of regulation of SLC2A1. Therefore, the peptide was unable to restore the response to sleep disruption. Finally, the peptide Ac-SEQ ID NO:4-NH2 was unable to regulate or change the expression of the genes IL33 and IL1RL1, and thus was unable to regulate all the reactions associated with IL33 and described above.

[0351] [Example 18. Analysis of the effect of Ac-SEQ ID NO:1-NH2 on the expression of emilin-1 protein in human lymphatic dermal microvascular endothelial cells (HDLMVEC)] Since the expression of emilin-1 protein may be associated with better lymphatic vessel structure and fluid clearance from tissues, an immunofluorescence assay was performed on emilin-1 protein in this example.

[0352] HDLMVEC was cultured on a coverslip in microvascular endothelial cell growth (MECG) medium at a density of 16,000 cells / cm 2 in humidified atmosphere containing 5% CO2 and at 37 °C. When the cell confluence was about 70 - 80%, the cells were treated with 0.05 mg / mL of Ac-SEQ ID NO:1-NH2 in MECG medium for 24 hours. Next, the cells were washed twice with PBS, fixed with 4% formaldehyde, and permeabilized with 0.2% Triton. Then, the samples were blocked with 5% PBS / BSA for 1 hour to avoid non-specific antibody binding.

[0353] Emilin-1 was stained with a 1:50 Emilin-1 antibody in 1% PBS / BSA at room temperature for 1.5 hours. After proper washing with PBS, actin was stained with 50 μg / mL phalloidin at room temperature for 1 hour. Then, the cells were washed with PBS and incubated with 5 μg / mL of secondary antibody goat anti-rabbit IgG at room temperature for 1 hour. Finally, the coverslip was mounted with Vectashield with DAPI to stain the nuclei, and the samples were protected from light at 4 °C until microscopic images were acquired.

[0354] Microscopic images were acquired using a 10× objective lens. Two replicates were stained for each condition, and images of 10 different fields of each coverslip were acquired using the same settings.

[0355] Figure 10 shows images obtained by fluorescence microscopy for basal cells (A) and cells treated with 0.05 mg / mL of Ac-SEQ ID NO:1-NH2 (B) (green: Emilin-1, red: phalloidin, blue: nuclei).

[0356] The images were analyzed using Image J software. Briefly, the threshold was adjusted to select Emilin-1 staining and the mean fluorescence was measured. The cell number was counted using DAPI staining. The mean fluorescence intensity of Emilin-1 was divided by the cell number. Finally, all data were normalized as follows to obtain the % of Emilin-1 compared to untreated cells: % against the basal = (fluorescence per cell in the treated well) / (fluorescence per cell in the untreated well) × 100.

[0357] Statistical data analysis was performed using Student's t-test by comparing the fluorescence of treated and untreated cells, *, p < 0.05; **, p < 0.01 and ***, p < 0.001.

[0358]

Table 18

[0359] The results show that, when compared to untreated cells, Ac-SEQ ID NO:1-NH2 significantly increased the expression of emilin-1 by more than 70% after 24 hours of treatment. Considering the important role of emilin-1 in lymphatic function, these results suggest a potential role of Ac-SEQ ID NO:1-NH2 in the clearance of fluid from tissues, which could be considered for cosmetic applications.

[0360] 〔Example 19. Analysis of the effect of peptide Ac-SEQ ID NO:1-NH2 on the expression of integrin α9β1 protein in human lymphatic dermal microvascular endothelial cells (HDLMVEC)〕 Since the expression of integrin α9β1 (Itg9) protein is associated with better lymphatic vessel structure and fluid clearance from tissues, an immunofluorescence assay was performed for integrin α9β1 (Itg9) protein in this example.

[0361] HDLMVECs were cultured on coverslips in microvascular endothelial cell growth (MECG) medium at a density of 16,000 cells / cm 2 and at 37 °C in a humidified atmosphere containing 5% CO2.

[0362] When the cell confluence was about 70 - 80%, the cells were treated with 0.05 mg / mL of Ac-SEQ ID NO:1-NH2 in MECG medium for 24 hours. Next, the cells were washed twice with PBS, fixed with 4% formaldehyde, and permeabilized with 0.2% Triton. Then, to avoid non-specific antibody binding, the samples were blocked with 5% PBS / BSA for 1 hour.

[0363] Itg9 was stained with a 1:50 anti-Itg9 antibody in 1% PBS / BSA at room temperature for 1.5 hours. After proper washing with PBS, actin was stained with 50 μg / mL phalloidin at room temperature for 1 hour. Then, the cells were washed with PBS and incubated with 5 μg / mL of secondary antibody goat anti-rabbit IgG at room temperature for 1 hour. Finally, the coverslip was mounted with Vectashield with DAPI to stain the nuclei, and the samples were protected from light at 4°C until microscopic images were acquired.

[0364] Microscopic images were acquired using a 10× objective lens. Two independent experiments were performed for each condition, and images of at least 13 different fields of view of each coverslip were acquired using the same settings.

[0365] Figure 11 shows fluorescence microscope images of integrin α9β1 expression on HDLMVECs after treatment with 0.05 mg / mL of Ac-SEQ ID NO:1-NH2 for 24 hours (green: integrin α9β1, red: phalloidin, blue: nuclei).

[0366] The images were analyzed using Image J software. Briefly, the threshold was adjusted to select the Itg9 staining, and the mean fluorescence was measured. The cell number was counted using DAPI staining. The Itg9 mean fluorescence intensity was divided by the cell number.

[0367] Normalization and statistics of the data were performed as described previously.

[0368]

Table 19

[0369] The results show that, when compared to untreated cells, the peptide Ac-SEQ ID NO:1-NH2 significantly increases the expression of Itg9 on primary human lymphatic dermal microvascular endothelial cells by more than 50% 24 hours after treatment. Considering the crucial role of Itg9 in lymphatic function, these results suggest a potential role of Ac-SEQ ID NO:1-NH2 in tissue fluid clearance, which is considered for cosmetic applications.

[0370] [Example 20. Analysis of the effects of peptide Ac-SEQ ID NO::1-NH2 and melatonin on the expression of genes related to innate antibacterial immunity in primary human epidermal keratinocytes (HEKa) isolated from human skin] The tests, controls, procedures, materials and data analysis were the same as those described in Examples 16 - 17.

[0371] The possibility that peptide Ac-SEQ ID NO:1-NH2 and melatonin regulate the expression of genes involved in the antiviral immune response in human epidermal keratinocytes was evaluated by RT-qPCR. The results show that for HEKa cells, a 6-hour treatment with 0.01 mg / mL of the Ac-SEQ ID NO:1-NH2 peptide can induce a strong upregulation of the important genes IRF3, DEFB4A, S100A7 and MX1 / MXA in HEKa. Other genes such as IRF1, DEFB1, OAS2, ISG15 were also upregulated by treatment with D18ChAH. Regarding the effect of melatonin in HEKa cells, after a 6-hour treatment at 0.01 mg / mL, the genes DEFB1, DEFB4A, S100A7, OAS2, MX1 were upregulated, but less so compared to the effect of Ac-SEQ ID NO:1-NH2 (Figure 12, Table 20).

[0372] Figure 12 shows the regulation of genes related to the antibacterial innate immune response by 0.01 mg / mL of peptide Ac-SEQ ID NO:1-NH2 or 0.01 mg / mL of melatonin in HEKa cells after 6 hours of treatment.

[0373]

Table 20

[0374]

Table 21

[0375] After treatment with peptide Ac-SEQ ID NO:1-NH2 at 0.01 mg / mL for 6 hours, gene expression in HEKa was regulated, leading to the regulation of genes involved in the antibacterial innate immune response in epidermal keratinocytes. Specifically, Ac-SEQ ID NO:1-NH2 induced strong upregulation of IRF3, DEFB4A, S100A7, and MX1. Furthermore, treatment with Ac-SEQ ID NO:1-NH2 also upregulated important genes IRF1, DEFB1, OAS2, and ISG16. Treatment of HEKa with melatonin upregulated genes DEFB1, DEFB4A, S100A7, OAS2, and MX1, but to a lesser extent compared to the action of peptide Ac-SEQ ID NO:1-NH2.

[0376] All these results strongly suggest that peptide Ac-SEQ ID NO:1-NH2 may be a modulator of genes involved in the host's antibacterial and antiviral innate immune responses, and thus can increase the immune defense of the epidermal barrier against invasion and infection. Furthermore, peptide Ac-SEQ ID NO:1-NH2 appears to exhibit a melatonin-like effect with respect to gene expression regulation. As a result, all of this makes Ac-SEQ ID NO:1-NH2 a compound of interest in the cosmetics industry.

[0377] 〔Example 21. In Vivo Results of Eye Bag Volume〕 A facial cosmetic composition containing a peptide was compared with a placebo composition in terms of in vivo reduction of eye bag volume. The same composition as in Example 6 was used in this example, but the weight of the commercial preparation of the peptide Ac-SEQ ID NO:1-NH2 was 3% (g / 100g). The placebo composition did not contain the peptide.

[0378] Figure 13 shows a graphical representation of the reduction in eye bag volume by the composition containing the peptide and the placebo composition.

[0379] The results show a 5% reduction in eye bag volume after 7 days and a 7% reduction after 14 days with the composition containing the peptide, compared to the volume reduction observed with the placebo composition.

Claims

1. A peptide, wherein the peptide has the following formula (I): R １ -AA １ -AA ２ -AA ３ -AA ４ -AA ５ -AA ６ -R ２ and has an array according to The array of the peptide is R １ -Arg-Arg-Gln-Met-Arg-Glu-R ２ , R １ -Met-Arg-Arg-Glu-Gln-Arg-R ２ , and R １ -Arg-Glu-Gln-Met-Arg-Arg-R ２ selected from the group consisting of R １ is selected from H, substituted or unsubstituted acyclic aliphatic, substituted or unsubstituted alicyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R ５ -CO-, where R ５ is substituted or unsubstituted C １ -C ２４ alkyl radical, substituted or unsubstituted C ２ -C ２４ alkenyl, substituted or unsubstituted C ２ -C ２４ alkynyl, substituted or unsubstituted C ３ -C ２４ cycloalkyl, substituted or unsubstituted C ５ -C ２４ cycloalkenyl, substituted or unsubstituted C ８ -C ２４ cycloalkynyl, substituted or unsubstituted C ６ -C ３０ aryl, substituted or unsubstituted C ７ -C ２４Selected from aralkyl, a substituted or unsubstituted heterocyclic ring consisting of 3 to 10 members, and a substituted or unsubstituted heteroarylalkyl consisting of an alkyl chain having 2 to 24 carbon atoms, 1 to 3 atoms other than carbon, and 1 to 6 carbon atoms. R ２ is H, -NR ３ R ４ -, -OR ３ and -SR ３ selected from, and R ３ and R ４ are independently selected from H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic, a substituted or unsubstituted heterocyclic, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl, a peptide characterized by that.

2. The sequence of the peptide is Ac - Arg - Arg - Gln - Met - Arg - Glu - NH ２ , Ac - Met - Arg - Arg - Glu - Gln - Arg - NH ２ , and Ac - Arg - Glu - Gln - Met - Arg - Arg - NH ２ selected from the group consisting of, the peptide according to claim 1.

3. A composition comprising the peptide according to claim 1 or 2.

4. Use of the peptide according to claim 1 or 2 or the composition according to claim 3 in the manufacture of a cosmetic composition for the treatment of skin aging signs and / or skin defects.

5. The skin aging signs and / or skin defects are selected from the group consisting of eye bags, eyelid defects, wrinkles around the eyes and periorbital periosseous hemochromatosis, or combinations thereof, the use according to claim 4.

6. The skin aging signs and / or skin defects are related to sleep disruption, the use according to claim 5.

7. The peptide according to claim 1 or 2 or the composition according to claim 3 for use as a medicament.

8. The peptide according to claim 1 or 2 or the composition according to claim 3 for use in the treatment of eyelid ptosis based on inflammation.

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