Methods to counteract skin aging

Abstract

It is an object of the present invention to provide a non-therapeutic method for treating skin aging and hyperpigmentation, wherein the method comprises the administration of a cosmetic composition comprising one or more peptides.

Description

[0001] METHODS TO COUNTERACT SKIN AGING

[0002] STATE OF THE ART

[0003] Skin aging is a process influenced by intrinsic and extrinsic factors that negatively affect the proliferative capacity of cells, leading to cellular senescence that alters biosynthetic activity (Limbert G. et al., Biotribology of the ageing skin — Why we should care, Biotribology 2019, 17: 75-90). The main phenotypic consequences are the loss of skin elasticity and the development of wrinkles and a yellowish complexion with mottled pigmentation. Moreover, these factors contribute to the rapid deterioration of the dermal extracellular matrix (e.g. collagen, elastin, hyaluronic acid), loss of skin barrier function, oxidative stress and inflammatory processes.

[0004] In recent years, the use of cosmetics and personal care products has increased worldwide due to their corrective and preventive functions. The development of new cosmetic formulations based on bioactive compounds has rapidly expanded and consumer demand for natural products with protective and therapeutic functions has increased.

[0005] Cellular protection from oxidative damage is one of the targets for skin care products.

[0006] For this purpose, minerals and vitamins, phytochemicals (plant extracts, polyphenols) and microbial metabolites have been proposed. Some of these compounds have shown negative side effects such as skin irritation, an inflammatory response and allergenicity, as well as high costs and poor availability. Peptides derived from protein hydrolysates, bioactive peptides, have been widely used in the food industry as functional ingredients for dietary supplements and functional food production. Given their broad spectrum of activity, these peptides have been proposed as promising functional ingredients, as they show multiple advantages such as higher safety profiles, hypo-allergenicity and relatively cheap production.

[0007] Peptides derived from collagen have been used. Collagen is mainly obtained from bovine waste such as cartilage, hooves, skin and tendons. Specific processing conditions, including the pre-treatment used, the storage conditions of the hides and their characteristics (type of animal, age and sex) influence the quality of the extracted collagen. Other factors, such as the structural characteristics of the collagen, e.g. hydroxyproline content, influence the extraction process. The stability of collagen peptides during production and storage is crucial for their application as functional ingredients, as peptides are subject to chemical changes that include disulphide bond formation, dehydration, glycation and oxidation of the aromatic ring with consequent changes in structure and bioactivity. The factors listed here make peptides derived from collagen not ideal for cosmetic applications.

[0008] Industrial hemp seeds, which are non-pharmaceutical varieties of the plant Cannabis sativa L., are known for their high protein content of around 25%. Abraham T. et al., in Kinetics and Molecular Docking Studies of the Inhibitions of Angiotensin Converting Enzyme and Renin Activities by Hemp Seed (Cannabis sativa L.) Peptides, JAFC, 62: 4135-4144 describe hemp seed peptides, obtained by hydrolysing proteins with a combination of pepsin and pancreatin, having antioxidant and hypotensive activity both in vitro and in vivo.

[0009] Rodriguez-Martin NM et al. in Neuroprotective protein hydrolysates from hemp (Cannabis sativa L.) seeds, Food Funct. 2019, 10: 6732-6739, describe the antioxidant and neuroprotective activity of hydrolysed hemp seed fractions.

[0010] The authors of the present invention themselves, in Zanoni C. et al. Hempseed Peptides Exert Hypocholesterolemic Effects with a Statin-Eike Mechanism, JAFC, 2017, 65: 8829-8838, demonstrated that a hydrolysate obtained by digesting a total protein extract of hemp seeds with pepsin (HP) shows a cholesterol-lowering capacity through the inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase leading to a positive modulation of the low-density lipoprotein (LDL) receptor pathway in human hepatic HepG2 cells. Also, in Lammi C. et al. Enhancement of the Stability and Anti- DPPIV Activity of Hempseed Hydrolysates Through Self-Assembling Peptide-Based Hydrogels, Front. Chem. 2019, 6 it is shown how HP reduces dipeptidyl peptidase-IV (DPP-IV) activity in vitro and in human intestinal Caco-2 cells, suggesting a potential antidiabetic effect.

[0011] Among the peptides present in the starting HP, five peptides were able to cross mature Caco-2 cells. These are synthesised and evaluated for their antioxidant activity in vitro using DPPH, FRAP, ABTS, OPAC assays. H2 peptides (WVSPEAGRT, SEQ ID NO: 1) and H3 (IGFLIIWV, SEQ ID NO: 2) are shown to be the most effective antioxidants (Bollati C. et al. Investigation of the intestinal trans -epithelial transport and antioxidant activity of two hempseed peptides WVSPEAGRT (H2) and IGFLIIWV (H3), Food Res hat. 2021, 152:110720).

[0012] WO2021 / 081619 describes a mixture of peptides obtained from hemp seeds and their use in cosmetic preparations.

[0013] It is therefore an object of the present invention to identify peptides with a modulating effect on collagen and elastin homeostasis and modulation of the extracellular matrix, as well as tyrosinase inhibition, useful for the treatment of skin aging and hyperpigmentation, which meet the quality, safety and stability requirements, excluding problems related to animal origin.

[0014] DESCRIPTION

[0015] It is an object of the present invention to provide a non-therapeutic method for treating skin aging and hyperpigmentation, wherein the method comprises administering a cosmetic composition comprising a cosmeceutically effective amount of at least one peptide according to the present invention.

[0016] DESCRIPTION OF THE FIGURES

[0017] Figure 1: Cell viability in BJ-5ta cells after exposure to the indicated concentrations of Pepl (A) and Pep2 (B). C = Untreated control.

[0018] Figure 2: Cell viability in BJ-5ta cells after exposure to the indicated concentrations of H2O2 (B). C = Untreated control.

[0019] Figure 3: Collagen levels in cell lysate (A, D) and culture medium (B, C, E) from BJ-5ta cells exposed to the indicated treatments.

[0020] Figure 4: Phosphorylated Akt levels in cell lysate from BJ-5ta cells exposed to the indicated treatments.

[0021] Figure 5: Elastin levels in cell lysate (A, C, E) and culture medium (B, D, F) from BJ-5ta cells exposed to the indicated treatments.

[0022] Figure 6: Production of ROS (A) and lipid peroxidation (B) in BJ-5ta cells exposed to the indicated treatments.

[0023] Figure 7: Secretion levels of MMP-2 (A) and MMP-9 (B) measured in BJ-5ta cells exposed to the indicated treatments.

[0024] Figure 8: % inhibition of tyrosinase enzyme in BJ-5ta cells exposed to the indicated treatments (A, B, C).

[0025] Figure 9: Cell viability in BJ-5ta cells after exposure to Pepe at the indicated concentrations. C = Untreated control.

[0026] Figure 10: Secretion levels of MMP-2 (A) and MMP-9 (B) measured in BJ-5ta cells exposed to the indicated treatments.

[0027] Figure 11: ROS production in BJ-5ta cells exposed to the indicated treatments.

[0028] Figure 12: ROS production in BJ-5ta cells exposed to the indicated treatments under antiaging conditions.

[0029] Figure 13: Effect of the peptides Pepl, Pep2 in vivo on the indicated parameters.

[0030] DETAILED DESCRIPTION

[0031] It is an object of the present invention to use peptides obtained from hemp and lupin seeds for cosmetic applications.

[0032] The peptides used in this application are:

[0033] H2 (Pepl, WVSPLAGRT SEQ ID NO: 1), H3 (Pep2, IGFLIIWV SEQ ID NO: 2), H9 (GNASQVHQLVGMR SEQ ID NO: 3), WVSPL (Pepe, SEQ ID NO: 4), HHRE (SEQ ID NO: 5) and / or HGLP (SEQ ID NO: 6).

[0034] In an embodiment, said peptides are isolated from hemp and / or lupin seeds (HHRE and HGLP).

[0035] In an embodiment, said peptides are obtained synthetically, using methods known to a person skilled in the art. Advantageously, the synthetic route guarantees high product quality standards, reproducibility and purity. In an embodiment, said peptide is obtained by chemical or enzymatic synthesis from constituent amino acids or derivatives thereof; or it is obtained by controlled hydrolysis of natural proteins; or it is obtained by biotechnological processes.

[0036] In an embodiment, said peptides have the general formula

[0037] Ri - AA - R2 or stereoisomers, mixtures thereof and / or cosmeceutically acceptable salts, where the sequence AA is a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, where

[0038] Ri and R2 are independently selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl group, Rs-C(O)-, -NR3, R4, - OR3 and -SR3, where

[0039] R3 and R4 are selected independently from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl and unsubstituted aralkyl group.

[0040] In an embodiment, said composition comprises only one of said peptides or derivatives thereof.

[0041] In an embodiment, said composition comprises two of said peptides or derivatives thereof.

[0042] In an embodiment, said composition comprises three of said peptides or derivatives thereof.

[0043] In an embodiment, said peptides were modified, using methods known to a person skilled in the art. In a preferred embodiment, Pep2 was acetylated and methylated.

[0044] In an embodiment, said peptides or derivatives thereof are formulated in a cosmetic composition which comprises at least one cosmeceutically acceptable excipient or adjuvant.

[0045] Fibroblasts produce extracellular matrix (ECM) for the modulation of cell structure. Collagen is the main protein of the ECM and is responsible for the support of the cell structure and the stability and elasticity of the skin. However, its synthesis and degradation are finely regulated at the cellular level, with mechanisms that are still only partially understood. Most studies in the literature evaluate the ability of bioactive compounds to increase the production of type I collagen by comparing the effect with that of healthy cells. This approach and these experimental conditions indirectly correlate the effect of bioactives to a condition closer to the physiological condition of fibrosis.

[0046] The model used here, based on a human fibroblast cell line in which oxidative stress was induced, advantageously shows a condition of aging due to oxidative stress as happens in physiological skin aging conditions.

[0047] Surprisingly, it was shown here that the peptides Pepl, Pep2 and Pepe are effective in restoring oxidative stress-induced tissue damage and collagen I production under aging conditions.

[0048] Surprisingly, Pep 1, Pep2 and Pepe are more effective in modulating intracellular elastin levels and elastin secretion than observed with collagen, the reference compound.

[0049] Pepl and Pepe show in vitro inhibition of the enzyme tyrosinase (better than ascorbic acid and kojic acid), which is involved in melanin production, and for both Pepl and Pep2 positive results were observed in reducing ROS production and lipid peroxidation, high levels of which are typical in cases of oxidative stress, inflammation and aging. Pepe has no effect on ROS production.

[0050] The peptides according to the present invention were tested by means of a cosmetic study on volunteers, where there are no experimental models capable of showing antiaging efficacy in the skin. On the volunteer cohort, a surprising anti-aging effect was shown, evaluated in terms of improved hydration, improved skin firmness and decreased average wrinkling.

[0051] Thus, a cosmetic composition comprising said peptides, or derivatives thereof, stereoisomers, mixtures thereof and / or cosmeceutically acceptable salts, including modifications with fatty acids, acetylated and / or methylated derivatives, is an object of the present invention.

[0052] In an embodiment, said composition is in the form of a gel, or a cream, or a serum, or an emulsion.

[0053] It is a further object of the present invention to use said peptides in the treatment of skin aging and hyperpigmentation, to reduce or eliminate wrinkles on the skin of the face and / or neck.

[0054] In an embodiment, said cosmetic composition is administered topically, transdermally, enterally or parenterally, by adhesive or non-adhesive patches, orally, nasally or by inhalation, or by intradermal, intramuscular, intravenous, intraperitoneal or subcutaneous injection, iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive treatment, microinjections, needle-free pressure injections or microelectrical patches.

[0055] In an embodiment, said peptides are incorporated into a release or sustained release system selected from the group consisting of liposomes, mixed liposomes, oleosomes, niosomes, miniparticles, milliparticles, microparticles, nanoparticles and solid lipid nanoparticles, nanostructured lipid transporters, sponges, cyclo dextrins, vesicles, micelles, mixed surfactant micelles, mixed surfactant-phospholipid micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules, nanocapsules, microemulsions and nanoemulsions. In an embodiment, said cosmetic composition has a formulation selected from the group comprising creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, conditioners, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydrogel solutions, liniments, serums, soaps, shampoos, conditioners, serums, ointments, mousses, salves, powders, bars, pencils, sprays and aerosols.

[0056] In an embodiment, said cosmetic composition is contained in capsules, ampoules, syringes, pre-filled syringes, eye contour concealers, foundations, make-up remover lotions, make-up remover milks, eye shadows, lipsticks, lip glosses, lip protectants and powders, and mascaras.

[0057] In an embodiment, said cosmetic composition is incorporated into a fabric, a nonwoven fabric, a medical device.

[0058] In an embodiment, said cosmetic composition further comprises a cosmetically effective amount of at least one active agent selected from the group of cyclic adenosine monophosphate synthesis-stimulating agents, elastase-inhibiting agents, matrix metalloproteinase-inhibiting agents, melanin synthesis-stimulating agents or -inhibiting agents, whitening or depigmenting agents, propigmenting agents, self-tanning agents, anti-aging agents, NO-synthase-inhibiting agents, 5a-reductase-inhibiting agents, lysyl and / or prolyl hydroxylase-inhibiting agents, antioxidants, free radical scavengers and / or air pollution control agents, scavengers of reactive carbonyl species, antiglycation agents, antihistamine agents, antiviral agents, anti-parasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin and / or hair conditioners, humectants, moisture-retaining substances, alpha-hydroxy acids, beta-hydroxy acids, moisturisers, epidermal hydrolytic enzymes, vitamins, pigments or dyes, colouring agents, gelling polymers, thickeners, surfactants, softening agents, anti-wrinkle agents, agents that reduce or treat bags under the eyes, exfoliating agents, antimicrobial agents, antifungal agents, fungistatic agents, bactericidal agents, bacteriostatic agents, agents stimulating the synthesis of dermal or epidermal macromolecules and / or inhibiting or preventing their degradation, agents stimulating collagen synthesis, elastin synthesisstimulating agents, decorin synthesis-stimulating agents, laminin synthesis-stimulating agents, defensin synthesis-stimulating agents, chaperone synthesis-stimulating agents, aquaporin synthesis-stimulating agents, hyaluronic acid synthesis-stimulating agents, fibronectin synthesis-stimulating agents, sirtuin synthesis-stimulating agents, agents simulating the synthesis of lipids and components of the corneal layer, ceramide synthesis-stimulating agents, collagen degradation-inhibiting agents, elastin degradation-inhibiting agents, agents inhibiting serine proteases such as cathepsin G, fibroblast proliferation-stimulating agents, keratinocyte proliferation-stimulating agents, adipocyte proliferation-stimulating agents, melanocyte proliferation-stimulating agents, keratinocyte differentiation-stimulating agents, adipocyte differentiation-stimulating agents, acetylcholinesterase inhibiting agents, skin relaxing agents, acetylcholine receptor aggregation-inhibiting agents, muscle contraction-inhibiting agents, glycosaminoglycan synthesis-stimulating agents, anti-hyperkeratosis agents, comedolytic agents, antipsoriasis agents, DNA repair agents, DNA protection agents, stabilising agents, anti-itch agents, agents for the treatment and / or care of sensitive skin, firming agents, anti-stretch mark agents, binding agents, agents that regulate sebum production, lipolytic agents or agents that stimulate lipolysis, anti-cellulite agents, antiperspirant agents, agents that stimulate healing adjuvant healing agents, agents stimulating re-epithelialisation, agents adjuvant to re-epithelialisation, cytokine growth factors, calming agents, antiinflammatory agents, anaesthetic agents, agents acting on capillary circulation and / or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, venotonic agents, agents acting on cell metabolism, agents to improve the dermo-epidermal junction.

[0059] The following examples are intended to support and in no way limit the invention, the scope of which is defined by the claims.

[0060] EXAMPLES

[0061] Example 1: Peptides Pepl, Pep2 and Pepe are not toxic on human dermal fibroblasts

[0062] BJ-5ta cells, fibroblasts isolated from human dermis, were plated and exposed to increasing concentrations of Pepl and Pep2 for 48 hours.

[0063] Cell viability was assessed by MTT assay. The results are shown in the graphs of Figure 1A for Pepl and Figure IB for Pep2. Neither peptide tested affects cell viability.

[0064] The experiment was repeated with Pepe. The data are shown in the graph in Figure 9. Exposure to Pepe does not affect cell viability either.

[0065] Example 2: Development of the experimental skin aging model

[0066] BJ-5ta cells were exposed to increasing concentrations of hydrogen peroxide and cell viability was monitored. The aim of the experiment is to identify the dose of hydrogen peroxide that kills cells, and to select a dose that, without impacting vitality, mimics aging by stimulating oxidative stress.

[0067] The results are reported in Figure 2. The experiment made it possible to select a model of human BJ-5ta fibroblasts exposed to H2O2200 pM as an experimental model for the following examples.

[0068] Example 3: Modulation of intracellular collagen levels and its secretion

[0069] BJ-5ta cells were treated for 24 h with Pepl 100 pM, Pep250 pM or H9 100 pM. As a reference, the same cells were exposed to a collagen hydrolysate (5 mg / ml). At the end of the treatment, H2O2200 gM was added to the cell culture. After 18h, collagen levels in the cell lysate (Figure 3A) and collagen levels in the culture medium (Figure 3B) were measured. More precisely, after treatment, the supernatants were collected, and fibroblasts were lysed with lysis buffer (RIPA buffer + P-mercaptoethanol + protease and phosphatase inhibitors). The molecular levels of collagen were measured in the lysate by immunoblotting using the primary antibody Collagen Type I Monoclonal Ab (Proteintech), and secreted collagen was measured in the supernatants by specific ELISA test.

[0070] The data show that Pepl and Pep2 lead to a significant increase in intracellular collagen (Figure 3A), as well as in secreted collagen (Figure 3B). The effect obtained is greater than that observed during treatment with collagen hydrolysate. H9 does not modulate collagen production.

[0071] The same experiment was repeated, also testing Pepe 100 pM and a composition of Pep2 and Pepe. The data shown in Figure 3C, confirming the activity of Pep2 and Pepl, do not show a significant effect of Pepe on collagen secretion, nor do they show synergy by combining Pep2 with Pepe.

[0072] The experiment was repeated with lower doses of Pepe (50 pM). In the same experiment, the effects of Pepe 50 pM and Pep 1 100 pM on collagen production and release were evaluated. The results, shown in Figure 3D (production) and Figure 3E (secretion), show an unexpectedly improved efficacy of Pepe as the dose decreases, Pepe being even more active than Pepl. Surprisingly, the authors of the present invention have thus shown that Pepe, which is a portion of Pepl, is more active than the peptide from which it originates.

[0073] Example 4: Modulation of intracellular levels of active Akt (protein kinase B).

[0074] BJ-5ta cells were treated for 24 h with Pepl 100 pM or Pep250 pM. As a reference, the same cells were exposed to collagen hydrolysate (5 mg / mL). At the end of the treatment, H2O2 200 gM was added to the cell culture. After 18h, the levels of phosphorylated Akt on Ser 473 in the cell lysate were measured, following the same lysis method as in Example 3, where the antibody is Phospho- Akt (Ser473)(D9E) XP (Cell- Signaling).

[0075] The data shown in Figure 4 show that both peptides tested have comparable activity to that observed with collagen.

[0076] Example 5: Modulation of intracellular elastin levels and its secretion

[0077] Following a similar approach as described in Example 3, using the Elastin Polyclonal Ab antibody (Proteintech), the cellular elastin levels were measured.

[0078] The data show a strong increase in elastin production induced by Pepl and Pep2, to a greater extent by Pepl (Figure 5A). Release is also increased in the presence of Pepl and, to a greater extent, Pep2 (Figure 5B). H9, on the other hand, does not lead to a significant increase (Figure 5C).

[0079] The same experiment was repeated, also testing Pepe and a composition of Pep2 and Pepe. The data, shown in Figure 5D, confirming the activity of Pep2 and Pepl on elastin secretion, also show efficacy of Pepx. There is no evidence of a synergistic effect when combining Pep2 with Pepe (Figure 5D).

[0080] Again, the experiment was repeated decreasing the concentrations of Pepe, down to 50 pM. The data, shown in Figure 5E (production) and Figure 5F (secretion) show that Pepe is also effective on elastin production and secretion and is more effective than Pepl. This finding surprisingly confirms that the portion of Pepl relevant for anti-aging effects is the portion retained in Pepe.

[0081] Example 6: ROS production and modulation of lipid peroxidation

[0082] Following a similar approach as described in Example 3, the production of intracellular free radicals (ROS) was measured here through the formation of a fluorimetric product directly proportional to the levels of ROS generated. Lipid peroxidation was assessed by the formation of MDA (malonic aldehyde) detected by fluorimetric analysis.

[0083] The graph in Figure 6A shows a clear decrease in ROS production in the presence of Pepl and Pep2, where ROS levels are present at high levels in the experimental H2O2- induced oxidative stress model. It is shown that exposure to collagen hydrolysate has no effect in limiting ROS production.

[0084] The graph in Figure 6B shows the effectiveness of Pepl and Pep2 in decreasing lipid peroxidation. The experiment was repeated, including Pepe. The data, shown in Figure 11, unexpectedly show that Pepe does not impact ROS production. This finding indicates that the portion of Pepl retained in Pepe does not comprise the portion responsible for the antioxidant activity displayed by Pepl. These data, read in combination with those obtained in the preceding experiments (effect on collagen and effect on elastin), surprisingly conclude that Pepe retains anti-aging activity while losing the antioxidant activity demonstrated for Pepl.

[0085] Finally, the experiment was repeated including collagen among the treatment agents. The results are reported in Figure 12. Collagen leads to a marked increase in ROS production, up to 140, 48% higher than the values measured in the aging condition. Conversely, ROS decrease on cells exposed to Pepl or Pep2. Collagen, which is a known anti-aging agent, is not an anti-oxidant. The figure shows that anti-oxidant activity is not necessarily correlated with anti-aging activity.

[0086] Example 7: Modulation of the secretion of metalloproteases (MMP-2 and MMP-9)

[0087] Following a similar approach as described in example 3, the secretion of MMP-2 and MMP-9 was measured here using dedicated ELISA kits.

[0088] The effect of Pepl and Pep2, although present, is lower than the effect induced by collagen hydrolysate on MMP-2 (Figure 7A) while it is higher than the effect induced by collagen hydrolysate on MMP-9 (Figure 7B).

[0089] The experiment was repeated, also using Pepe at 50 pM. The results show that the effect of Pepe is comparable to the effect of Pep2 on MMP-2 secretion (Figure 10A). Pepe turns out to be slightly more effective than Pep2 on MMP-9 (Figure 10B). Here, too, Pepe was used at a lower concentration than the concentrations of Pep2 used.

[0090] Example 8: Evaluation of pigmentation control activity

[0091] The reference compound for anti-skin-staining activity is ascorbic acid, which was therefore taken as a positive control. The conducted assay measured the in vitro inhibition of the tyrosinase enzyme by optimising a specifically developed assay in which the purified recombinant form of the tyrosinase enzyme (5000 U) was initially activated at 37 °C and subsequently incubated with the inhibitory peptides at different concentrations for a further 15-20 minutes. Thus, the added substrate gives rise to a colorimetric reaction with the development of a campari red colour when the activity is 100%. Non-colour formation is associated with enzyme inhibition. The % inhibition is calculated: ODsampie X100 / OD100% Activity- Peptides Pepl 100 pM and Pep2 50 pM and ascorbic acid 100 pM were tested. A significant effect is observed for Pepl, not for Pep2 (Figure 8A).

[0092] The experiment was repeated by testing additional peptides, and kojic acid as a reference compound, resulting in a dose / response curve, shown in Figure 8B. The IC50s derived from these curves for the different peptides are 318.1 for Pepl, 208.8 for the peptide WVSPL (Pepe, SEQ ID NO: 4), 311.3 for the peptide HHRE (SEQ ID NO: 5), 730.4 for HGLP (SEQ ID NO: 6), 450.0 for kojic acid.

[0093] These data show the beneficial effect shown by Pepl, Pepe and HHRE, suggesting that they are more active than kojic acid.

[0094] Finally, Figure 8C shows that using the same test as described above, the ILSF and LLSFE peptides are not effective in inhibiting the enzyme tyrosinase.

[0095] Example 9: In vivo evaluation of Pepl and Pepe

[0096] A cohort of 20 volunteers, women aged between 41 and 63, was selected.

[0097] After obtaining the dermatologically tested certification for the Peptides according to the present invention, they were formulated and tested on volunteers as described below.

[0098] The volunteers used Pepl or Pep2, formulated in a cream base, Pep2 in a 0.2% concentration, Pepl in a 0.3% concentration for a period of 15 days.

[0099] After 15 days of daily treatment, the results obtained are depicted in Figure 13 and commented on here.

[0100] The formulation including Pepl did not induce any skin irritation, leading to a 5.68% improvement in skin hydration, a 7.87% improvement in skin firmness and an antiwrinkle effect measured in a 1.95% decrease.

[0101] The formulation including Pep2 did not induce any skin irritation, leading to a 6.89% improvement in skin hydration, a 12.46% improvement in skin firmness and an anti- wrinkle effect measured in a 2.25% decrease.

[0102] Data measured on the skin of subjects confirm a powerful anti-aging effect of the peptides according to the present invention.

Claims

CLAIMS1. A non-therapeutic method for treating skin aging, where the method comprises: administration of a cosmetic composition comprising a cosmeceutically effective amount of at least one peptide selected from the group consisting of Pepl of SEQ ID NO: 1, Pep2 of SEQ ID NO: 2, H9 of SEQ ID NO: 3, Pepe of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or general formulaRi - AA - R2 or stereoisomers, mixtures thereof and / or cosmeceutically acceptable salts, where the sequence AA is a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, whereRi and R2 are independently selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl alkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl group, Rs-C(O)-, -NR3, R4, -OR3 and -SR3, where R3 and R4 are selected independently from the group consisting of H, substituted or unsubstituted non- cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted hetero arylalkyl, substituted or unsubstituted aryl and unsubstituted aralkyl group; and at least one cosmeceutically acceptable adjuvant or excipient.

2. The method according to claim 1, where said peptide is selected from the group consisting of Pepl, Pep2 or Pepe.

3. The method according to claim 1, where said peptide is Pepe.

4. The method according to any one of claims 1 to 3, wherein said treatment is a treatment to reduce or eliminate wrinkles on the skin of the face and / or neck.

5. The method according to any one of claims 1 to 3, wherein said cosmetic composition is administered topically, transdermally, enterally orparenterally, by adhesive or non-adhesive patches, orally, nasally or by inhalation, or by intradermal, intramuscular, intravenous, intraperitoneal or subcutaneous injection, iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive treatment, microinjections, needle-free pressure injections or microelectrical patches.

6. The method according to any one of claims 1 to 3, wherein said peptides are incorporated into a release or sustained release system selected from the group consisting of liposomes, mixed liposomes, oleosomes, niosomes, miniparticles, milliparticles, microparticles, nanoparticles and solid lipid nanoparticles, nanostructured lipid transporters, sponges, cyclodextrins, vesicles, micelles, mixed surfactant micelles, mixed surfactant-phospholipid micelles, millispheres, microspheres, nanospheres, lipospheres, millicapsules, microcapsules, nanocapsules, microemulsions and nanoemulsions.

7. The method according to any one of claims 1 to 3, wherein the cosmetic composition has a formulation selected from the group comprising creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, conditioners, foams, lotions, gels, cream gels, hydroalcoholic solutions, hydrogel solutions, liniments, serums, soaps, shampoos, conditioners, serums, ointments, mousses, salves, powders, bars, pencils, sprays and aerosols.

8. The method according to any one of claims 1 to 3, wherein said cosmetic composition is contained in capsules, ampoules, syringes, pre-filled syringes, eye contour concealers, foundations, make-up remover lotions, make-up remover milks, eye shadows, lipsticks, lip glosses, lip protectants and powders.

9. The method according to any one of claims 1 to 3, wherein said cosmetic composition is incorporated into a textile, a non-woven fabric, a medical device.

10. The method according to any one of claims 1 to 3, wherein said cosmetic composition further comprises a cosmetically effective amount of at least one active agent selected from the group of cyclic adenosine monophosphate synthesis-stimulating agents, elastase-inhibiting agents, matrix metalloproteinaseinhibiting agents, melanin synthesis-stimulating agents or -inhibiting agents, whitening or depigmenting agents, propigmenting agents, self-tanning agents, anti-aging agents, NO-synthase-inhibiting agents, 5a-reductase-inhibiting agents, lysyl and / or prolyl hydroxylase-inhibiting agents, antioxidants, free radical scavengers and / or air pollution control agents, scavengers of reactive carbonylspecies, anti-glycation agents, antihistamine agents, antiviral agents, anti-parasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin and / or hair conditioners, humectants, moisture-retaining substances, alpha-hydroxy acids, beta-hydroxy acids, moisturisers, epidermal hydrolytic enzymes, vitamins, pigments or dyes, colouring agents, gelling polymers, thickeners, surfactants, softening agents, anti-wrinkle agents, agents that reduce or treat bags under the eyes, exfoliating agents, antimicrobial agents, antifungal agents, fungistatic agents, bactericidal agents, bacteriostatic agents, agents stimulating the synthesis of dermal or epidermal macromolecules and / or inhibiting or preventing their degradation, agents stimulating collagen synthesis, elastin synthesis-stimulating agents, decorin synthesis-stimulating agents, laminin synthesis-stimulating agents, defensin synthesis-stimulating agents, chaperone synthesis-stimulating agents, aquaporin synthesis-stimulating agents, hyaluronic acid synthesisstimulating agents, fibronectin synthesis-stimulating agents, sirtuin synthesisstimulating agents, agents simulating the synthesis of lipids and components of the corneal layer, ceramide synthesis-stimulating agents, collagen degradationinhibiting agents, elastin degradation-inhibiting agents, agents inhibiting serine proteases such as cathepsin G, fibroblast proliferation-stimulating agents, keratinocyte proliferation-stimulating agents, adipocyte proliferation-stimulating agents, melanocyte proliferation-stimulating agents, keratinocyte differentiationstimulating agents, adipocyte differentiation-stimulating agents, acetylcholinesterase inhibiting agents, skin relaxing agents, acetylcholine receptor aggregation-inhibiting agents, muscle contraction-inhibiting agents, glycosaminoglycan synthesis-stimulating agents, anti-hyperkeratosis agents, comedolytic agents, anti-psoriasis agents, DNA repair agents, DNA protection agents, stabilising agents, anti-itch agents, agents for the treatment and / or care of sensitive skin, firming agents, anti-stretch mark agents, binding agents, agents that regulate sebum production, lipolytic agents or agents that stimulate lipolysis, anticellulite agents, antiperspirant agents, agents that stimulate healing adjuvant healing agents, agents stimulating re-epithelialisation, agents adjuvant to re- epithelialisation, cytokine growth factors, calming agents, anti-inflammatory agents, anaesthetic agents, agents acting on capillary circulation and / or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, venotonic agents, agents acting on cell metabolism, agents to improve the dermo-epidermal junction.

11. The method according to any one of the preceding claims, characterised in that the peptide is obtained by chemical or enzymatic synthesisfrom constituent amino acids or derivatives thereof; or is obtained by controlled hydrolysis of natural proteins; or is obtained by biotechnological processes.

12. A composition that comprises a peptide that is Pepe, SEQ ID NO: 4 and pharmaceutically acceptable excipients.

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