PEPTIDES AND COMPOSITIONS FOR USE IN COSMETICS

Abstract

A family of peptides with antioxidant and brightening activities is disclosed as well as cosmetic compositions having the peptides and cosmetic uses and methods of the peptides or cosmetic compositions.

Claims

1. Peptide A peptide of formula (I):
R.sub.1X.sub.m-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.nR.sub.2 (I) their cosmetically acceptable isomers, salts, solvates, derivatives and mixtures thereof, wherein: X is selected from the group of amino acids with an aliphatic non-polar side-chain; AA.sub.1 is selected from Asp, Glu, His or Thr; AA.sub.2 is selected from the group of aromatic amino acids; AA.sub.3 is selected from Lys, Arg, Phe, Trp or Tyr; AA.sub.4 is selected from Val, lie, Leu, Lys or Arg; Y is selected from the group of amino acids with an aliphatic non-polar side-chain; n and m are selected independently of each other from 0 and 1; R.sub.1 is selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyi, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R.sub.5CO wherein R.sub.5 is selected from H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyi, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl; and R.sub.2 is selected from the group consisting of H, NR.sub.3R.sub.4, OR.sub.3 and SR.sub.3, wherein R.sub.3 and R.sub.4 are independently selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl.

2. The peptide according to claim 1, wherein the amino acids are L-amino acids.

3. The peptide according to claim 1, wherein m is 1 and X is Leu.

4. The peptide according to claim 1, wherein n is 1 and Y is Ala.

5. The peptide according to claim 1, wherein R.sub.1 is ferulic acid.

6. The peptide according to claim 1, wherein R.sub.1 is acetyl.

7. The peptide according to claim 1, wherein R.sub.2 is H or NH.sub.2.

8. The peptide according to claim 1, wherein the peptide of formula (I) is: TABLE-US-00015 (R.sub.1-SEQ ID NO: 1-R.sub.2) R.sub.1-Asp-Tyr-Lys-Val-R.sub.2; (R.sub.1-SEQ ID NO: 2-R.sub.2) R.sub.1-His-Trp-Phe-Lys-R.sub.2; (R.sub.1-SEQ ID NO: 3-R.sub.2) R.sub.1-Leu-His-Trp-Phe-Arg-Ala-R.sub.2; or (R.sub.1-SEQ ID NO: 4-R.sub.2) R.sub.1-Thr-Phe-Phe-Lys-R.sub.2.

9. The peptide according to claim 1, wherein the peptide of formula (I) is: TABLE-US-00016 (Ac-SEQ ID NO: 1-NH.sub.2) Ac-Asp-Tyr-Lys-Val-NH.sub.2; (Ac-SEQ ID NO: 2-NH.sub.2) Ac-His-Trp-Phe-Lys-NH.sub.2; (Ferulic acid-SEQ ID NO: 2-NH.sub.2) Ferulic acid-His-Trp-Phe-Lys-NH.sub.2; or (Ac-SEQ ID NO: 3-NH.sub.2) Ac-Leu-His-Trp-Phe-Arg-Ala-NH.sub.2.

10. The peptide according to claim 1, wherein the peptide of formula (I) is: TABLE-US-00017 (Ac-SEQ ID NO: 1-NH.sub.2) Ac-Asp-Tyr-Lys-Val-NH.sub.2; or (Ac-SEQ ID NO: 2-NH.sub.2) Ac-His-Trp-Phe-Lys-NH.sub.2.

11. A cosmetic composition comprising the peptide according to claim 1.

12. The cosmetic composition according to claim 11, further comprising 0.1%-0.0001% (m/v) of the peptide.

13. Method for reducing or removing oxidative stress in the skin of a subject comprising applying the cosmetic composition according to claim 11 to the skin of the subject.

14. A method for brightening the skin of a subject comprising applying the cosmetic composition according to claim 11 to the subject.

15. Method for reducing or removing oxidative stress in the skin of a subject, comprising applying the peptide in accordance with claim 1.

16. Method for brightening the skin of a subject comprising applying the peptide in accordance with claim 1.

Description

[0117] To allow a better understanding, the present invention is described in more detail below with reference to the enclosed drawings, which are presented by way of example, and with reference to illustrative and non-limitative examples.

[0118] FIG. 1 shows the antioxidant power of Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 in the form of trolox equivalents (see example 9). FIG. 1(A) shows the trolox equivalents obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2 in the three concentrations tested, columns from left to right (x-axis): 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. FIG. 1(B) shows the trolox equivalents obtained for the treatment with Ac-SEQ ID NO: 2-NH.sub.2 in the three corresponding concentrations tested, columns from left to right (x-axis): 0.05 mg/ml, 0.1 mg/ml and 0.5 mg/ml. FIG. 1(C) shows the trolox equivalents obtained for the treatment with Ac-SEQ ID NO: 3-NH.sub.2 in the three corresponding concentrations tested, columns from left to right (x-axis): 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. FIG. 1(D) shows the trolox equivalents obtained for the treatment with Ferulic acid-SEQ ID NO: 2-NH.sub.2 in the three corresponding concentrations tested, columns from left to right (x-axis): 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. As positive controls of the assay, ascorbic acid 2-glucoside (hereinafter, AA-2G) and magnesium ascorbyl phosphate (hereinafter, MAP) were tested. FIG. 1(E) shows the trolox equivalents obtained for the treatment with AA-2G in the three corresponding concentrations tested, columns from left to right (x-axis): 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. FIG. 1(F) shows the trolox equivalents obtained for the treatment with MAP in the three corresponding concentrations tested, columns from left to right (x-axis): 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. For FIGS. 1(A) to 1(F), the y-axis shows the trolox equivalents in M.

[0119] FIG. 2 shows the antioxidant activity of Ferulic acid-SEQ ID NO: 2-NH.sub.2 at four concentrations and the reference compound ascorbic acid at two concentrations, assessed by means of the 2,2-diphenyl-1-picrylhydrazyl (hereinafter, DPPH) assay and normalized on the basis of the control sample (as explained in example 10). Columns from left to right in the x-axis correspond to: control (no peptide added); ascorbic acid at 30 M and 60 M, respectively; and concentrations 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL of Ferulic acid-SEQ ID NO: 2-NH.sub.2. The y-axis shows the antioxidant activity (% of DPPH reduction) versus the control condition (no peptide added).

[0120] FIG. 3 shows the percentage of lipid peroxidation normalized on the basis of the control sample (as explained in example 11), this is, setting the percentage of lipid peroxidation in the control sample as 100% and then performing the comparison with the rest of the samples. FIG. 3(A) shows the results of percentage of lipid peroxidation obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2 in the three concentrations tested. Columns from left to right in the x-axis in FIG. 3(A) correspond to: control and treatments with Ac-SEQ ID NO: 1-NH.sub.2 concentrations of 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml, respectively. FIG. 3(B) shows the results of percentage of lipid peroxidation obtained for the treatment with Ac-SEQ ID NO: 2-NH.sub.2 in the three corresponding concentrations tested. Columns from left to right in the x-axis in FIG. 3(B) correspond to: control and treatments with Ac-SEQ ID NO: 2-NH.sub.2 concentrations of 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml, respectively. For both FIGS. 3(A) and 3(B), the y-axis shows the percentage of lipid peroxidation versus the lipid peroxidation observed in the positive control sample.

[0121] FIG. 4 shows the percentage of reactive oxygen species (induced by hydrogen peroxide) in comparison with the positive control sample (sample treated only with 100 M of hydrogen peroxide, as explained in example 12), this is, setting the percentage of reactive oxygen species in the positive control sample as 100% and then performing the comparison with the rest of the samples. FIG. 4(A) shows the results of the percentage of reactive oxygen species obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2 in the three concentrations tested. Columns from left to right in the x-axis of FIG. 4(A) correspond to: basal state (cells to which no treatment is applied), positive control, negative control of oxidation (sample treated with 1 mM ascorbic acid) and treatments with Ac-SEQ ID NO: 1-NH.sub.2 concentrations of 0.001 mg/ml, 0.005 mg/ml and 0.01 mg/ml, respectively. FIG. 4(B) shows the results of the percentage of reactive oxygen species obtained for the treatment with Ac-SEQ ID NO: 2-NH.sub.2 in the four corresponding concentrations tested. Columns from left to right in the x-axis of FIG. 3(B) correspond to: basal state (cells to which no treatment is applied), positive control, negative control (sample treated with 1 mM ascorbic acid) and treatments with Ac-SEQ ID NO: 2-NH.sub.2 concentrations of 0.001 mg/ml, 0.005 mg/ml, 0.01 mg/ml and 0.05 mg/ml, respectively. For both FIGS. 4(A) and 4(B), the y-axis shows the percentage of reactive oxygen species versus the reactive oxygen species observed in the positive control sample.

[0122] FIG. 5 shows the percentage of advanced glycation end-products (induced by heavy metals) obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2, in comparison with the control sample (sample treated only with 300 M of heavy metals, as explained in example 13), this is, setting the percentage of advanced glycation end-products in the control sample as 100% and then performing the comparison with the rest of the samples. The x-axis shows the samples tested, this is, columns from left to right: basal state (cells to which no treatment is applied), positive control and the three Ac-SEQ ID NO: 1-NH.sub.2 concentrations tested, 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml, respectively. The y-axis shows the percentage of Advanced Glycation End Products versus the Advanced Glycation End products observed in the control sample.

[0123] FIG. 6 shows the protection of DNA oxidation (induced by synthetic smoke) in comparison with the control sample (sample treated only with synthetic smoke, as explained in example 14). To this end, FIG. 6 shows the percentage of 8-hydroxydeoxyguanosine (as measure of DNA oxidation) obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2 in comparison or normalized with that of the control sample (setting the percentage of the control sample as 100% and then performing the comparison with the rest of the samples). The x-axis shows, from left to right: basal state (cells to which no treatment is applied), positive control and the three Ac-SEQ ID NO: 1-NH.sub.2 concentrations tested, 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml, respectively. The y-axis shows the percentage of 8-hydroxydeoxyguanosine versus the 8-hydroxydeoxyguanosine observed in the control sample.

[0124] FIG. 7 shows the protection of lipoxidation (induced by synthetic smoke) in comparison with the control sample (sample treated only with synthetic smoke, as explained in example 14). To this end, FIG. 7 shows the percentage of MDA (as measure of lipoxidation) obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2 in comparison with that of the control sample (setting the percentage of the control sample as 100% and then performing the comparison with the rest of the samples). The x-axis shows, from left to right: basal state (cells to which no treatment has been applied), control and the three concentrations tested for Ac-SEQ ID NO: 1-NH.sub.2, this is, 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml, respectively. The y-axis shows the percentage MDA versus the MDA observed in the control sample.

[0125] FIG. 8 shows the effect of Ac-SEQ ID NO: 1-NH.sub.2 on the gene expression profile of HEKa cells after treatment with a peptide concentration of 0.05 mg/mL. Changes in gene expression levels are represented as a positive or negative fold-change with regard to the basal control (untreated cells). Bars from top to bottom in the y-axis refer to: CYP2R1 (Vitamin D 25-hydroxylase), NFE2L2 (Nuclear factor-like 2), HMOX1 (Heme oxygenase-1), GSTP1 (Glutathione S-transferase P), GSS (Glutathione synthetase), GPX1 (Glutathione peroxidase) and TRX (Thioredoxin). The x-axis refers to fold change versus or with regard to the basal control. A negative fold change means that the corresponding gene is downregulated; and a positive fold change means that the corresponding gene is upregulated.

[0126] FIG. 9 shows the antioxidant and anti-pollutant efficacy of peptide Ac-SEQ ID NO: 1-NH.sub.2 on human skin explants. FIGS. 9(A) and 9(B) show the percentage of epidermal surface occupied by AhR (FIG. 9(A)) and heme oxygenase-1 (hereinafter, HO-1) (FIG. 9(B)) on human skin explants, previously treated or not with peptide Ac-SEQ ID NO: 1-NH.sub.2, after exposure to a mixture of pollutants (heavy metals and hydrocarbons), while FIG. 9(C) shows the percentage of epidermal surface occupied by HO-1, of explants treated or not with Ac-SEQ ID NO: 1-NH.sub.2 with no exposure to pollutants. The x-axis shows, for FIGS. 9(A) and 9(B), from left to right: untreated explants exposed to the mixture of pollutants and explants exposed to the mixture of pollutants and treated with Ac-SEQ ID NO: 1-NH.sub.2. On its side, the x-axis for FIG. 9(C) shows, from left to right, untreated explants and explants treated with Ac-SEQ ID NO: 1-NH.sub.2. The y-axis shows the percentage of surface occupied by AhR (FIG. 9(A)) and HO-1 (FIGS. 9(B) and 9(C)), establishing as 100% the value obtained for the untreated but exposed to the mixture of pollutants explants for FIGS. 9(A) and 9(B), and untreated and unexposed explants for FIG. 9(C).

[0127] FIG. 10 shows the brightening efficacy of Ac-SEQ ID NO: 1-NH.sub.2. To this end, FIG. 10 shows the percentage of melanin per cell in comparison with the melanin per cell present in the basal state (cells to which no treatment has been applied) (setting the percentage of the basal state as 100% and then performing the comparison with the rest of the samples), obtained for the treatment with Ac-SEQ ID NO: 1-NH.sub.2. Two concentrations of kojic acid were used as positive control. The x-axis shows, from left to right: basal state, 10 M kojic acid, 50 M kojic acid and the three concentrations of Ac-SEQ ID NO: 1-NH.sub.2 tested, 0.01 mg/ml (17.7 M), 0.05 mg/ml (88.5 M) and 0.1 mg/ml (177.1 M), respectively. The y-axis shows the percentage of melanin per cell (stablishing as 100% the value obtained for the basal state).

[0128] FIG. 11 shows the brightening efficacy of Ac-SEQ ID NO: 2-NH.sub.2. To this end, FIG. 11 shows the percentage of melanin per cell in comparison with the melanin per cell present in the basal state (cells to which no treatment has been applied) (setting the percentage of the basal state as 100% and then performing the comparison with the rest of the samples), obtained for the treatment with Ac-SEQ ID NO: 2-NH.sub.2. Two concentrations of kojic acid were used as positive control. The x-axis shows, from left to right: basal state, 10 M kojic acid, 50 M kojic acid and the three concentrations of Ac-SEQ ID NO: 2-NH.sub.2 tested, 0.01 mg/ml (15.2 M), 0.05 mg/ml (76 M) and 0.1 mg/ml (152 M), respectively. The y-axis shows the percentage of melanin per cell (stablishing as 100% the value obtained for the basal state).

[0129] FIG. 12 shows the brightening efficacy of Ac-SEQ ID NO: 3-NH.sub.2. To this end, FIG. 12 shows the percentage of melanin per cell in comparison with the melanin per cell present in the basal state (cells to which no treatment has been applied) (setting the percentage of the basal state as 100% and then performing the comparison with the rest of the samples), obtained for the treatment with Ac-SEQ ID NO: 3-NH.sub.2. Kojic acid was used as positive control. The x-axis shows, from left to right: basal state, 70 M kojic acid and the three concentrations of Ac-SEQ ID NO: 3-NH.sub.2 tested, this is, 0.01 mg/ml (11.5 M), 0.05 mg/ml (57.5 M) and 0.1 mg/ml (115 M), respectively. The y-axis shows the percentage of melanin per cell (stablishing as 100% the value obtained for the basal state).

[0130] FIG. 13 shows the brightening efficacy of Ferulic acid-SEQ ID NO: 2-NH.sub.2. To this end, FIG. 13 shows the percentage of melanin per cell in comparison with the melanin per cell present in the basal state (cells to which no treatment has been applied) (setting the percentage of the basal state as 100% and then performing the comparison with the rest of the samples), obtained for the treatment with Ferulic acid-SEQ ID NO: 2-NH.sub.2. Kojic acid was used as positive control. The x-axis shows, from left to right: basal state, 70 M kojic acid and the three concentrations of Ferulic acid-SEQ ID NO: 2-NH.sub.2 tested, this is, 0.005 mg/mL (6.3 M), 0.01 mg/ml (12.6 M) and 0.05 mg/ml (63 M), respectively. The y-axis shows the percentage of melanin per cell (stablishing as 100% the value obtained for the basal state).

[0131] FIG. 14 shows the inhibitory effect of peptide Ac-SEQ ID NO: 3-NH.sub.2 on mushroom tyrosinase activity in tubo. Results are shown as percentage of mushroom tyrosinase inhibition compared to basal (non-treated cells). Kojic acid was used as positive control. The x-axis shows, from left to right: basal state, 400 M and 800 M of kojic acid and the three concentrations of Ac-SEQ ID NO: 3-NH.sub.2 tested, this is 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL. The y-axis shows mushroom tyrosinase percentage activity (stablishing as 100% the value obtained for the basal state).

[0132] FIG. 15 shows the inhibitory effect of peptide Ferulic acid-SEQ ID NO: 2-NH.sub.2 on mushroom tyrosinase activity in tubo. Results are shown as percentage of mushroom tyrosinase inhibition compared to basal (non-treated wells). Kojic acid was used as positive control. The x-axis shows, from left to right: basal state, 400 M and 800 M of kojic acid and the three concentrations of Ferulic acid-SEQ ID NO: 2-NH.sub.2 tested, this is, 0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL. The y-axis shows mushroom tyrosinase percentage activity (stablishing as 100% the value obtained for the basal state).

[0133] FIG. 16 shows the effect of Ac-SEQ ID NO: 3-NH.sub.2 on the gene expression profile of HEMn cells after 11-day (FIG. 16(A)) and 24-hour (FIG. 16(B)) treatment with a peptide concentration of 0.1 mg/mL. Changes in gene expression levels are represented as a positive or negative fold-change with regard to the basal control (untreated cells). For FIG. 16(A), bars from top to bottom in the y-axis refer to: COX-1 (Cyclooxigenase-1), MITF (Melanogenesis associated transcription factor), MC1R (Melanocortin-1 receptor), MLAN-A (Melanoma antigen recognized by T-cells), C-KIT (Protoongogen receptor tyrosine kinase KIT), PMEL17 (Pre-melanosome protein), DCT-TYRP2 (Dopachrome tautomerase), TYRP-1 (Tyrosinase related protein 1) and TYR (Tyrosinase). For FIG. 16(B), bars from top to bottom in the y-axis refer to: BLOC-1 (Biogenesis of lysosome-related organelles complex-1), MITF (Melanogenesis associated transcription factor), MC1R (Melanocortin-1 receptor), MLAN-A (Melanoma antigen recognized by T-cells), C-KIT (Protoongogen receptor tyrosine kinase KIT), PMEL17 (Pre-melanosome protein), DCT-TYRP2 (Dopachrome tautomerase), TYRP-1 (Tyrosinase related protein 1) and TYR (Tyrosinase). The x-axis refers to fold change versus basal control. A negative fold change means that the corresponding gene is downregulated; and a positive fold change means that the corresponding gene is upregulated.

[0134] FIG. 17 shows the effect of Ferulic acid-SEQ ID NO: 2-NH.sub.2 on the gene expression profile of HEMn cells after 11-day treatment with a peptide concentration of 0.05 mg/mL. Changes in gene expression levels are represented as a positive or negative fold-change with regard to the basal control (untreated cells). Bars from top to bottom in the y-axis refer to: VDR (Vitamin D receptor), PMEL17 (Pre-melanosome protein) and DCT-TYRP2 (Dopachrome tautomerase). The x-axis refers to fold change versus basal control. A negative fold change means that the corresponding gene is downregulated; and a positive fold change means that the corresponding gene is upregulated.

[0135] FIG. 18 shows the effect of Ac-SEQ ID NO: 3-NH.sub.2 on the gene expression profile of HEKa cells after 24-hour treatment with a peptide concentration of 0.1 mg/mL. Changes in gene expression levels are represented as a positive or negative fold-change with regard to the basal control (untreated cells). Bars from top to bottom in the y-axis refer to: KITLG (KIT ligand), TP53 (Tumor protein p53), DKK1 (Dickkopf-related protein 1) and NGF (Nerve growth factor). The x-axis refers to fold change verus basal control. A negative fold change means that the corresponding gene is downregulated; and a positive fold change means that the corresponding gene is upregulated.

[0136] FIG. 19 shows the effect of Ferulic acid-SEQ ID NO: 2-NH.sub.2 on the gene expression profile of HEKa cells after 24-hour treatment with a peptide concentration of 0.05 mg/mL. Changes in gene expression levels are represented as a positive or negative fold-change with regard to the basal control (untreated cells). Bars from top to bottom in the y-axis refer to: KITLG (KIT ligand), POMC (Pro-opiomelanocortin), EDN1 (Endothelin-1) and NGF (Nerve growth factor). The x-axis refers to fold change versus basal control. A negative fold change means that the corresponding gene is downregulated; and a positive fold change means that the corresponding gene is upregulated.

EXAMPLES

Abbreviations

[0137] 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.

[0138] 2-CITrt, 2-chlorotrityl; Ac, acetyl; Arg, arginine; Asp, Aspartic acid; Boc, tert-butyloxycarbonyl; cDNA, complementary DNA; C-terminal, carboxy-terminal; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DIPCDI, N,N-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; DNA, deoxyribonucleic acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; Glu, glutamic acid; HDFa, Human Dermal Fibroblasts, adult; HEKa, Human Epidermal Keratinocytes, adult; HEMn, Human Epidermal Melanocytes neonatal; His, histidine; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; Ile, isoleucine; INCI, International Nomenclature of Cosmetic Ingredients; MBHA, p-methylbenzhydrylamine; Leu, leucine; L Lys, lysine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; N-terminal, amino-terminal; Palm, palmitoyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Phe, phenylalanine; RNA, ribonucleic acid; RT, room temperature; Ser, serine; tBu, tert-butyl; TFA, trifluoroacetic acid; Thr, threonine; TIS, triisopropylsilane; Trp, tryptophan; Trt, triphenylmethyl or trityl; Tyr, tyrosine; Val, valine; Z, benzyloxycarbonyl.

[0139] Regarding the chemical synthesis procedures included in the examples, it is noted that all synthetic processes were carried out in polypropylene syringes fitted with porous polyethylene discs or Pyrex reactors fitted with porous plates. All the reagents and solvents were synthesis 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 11 min, 210 min, 5 mL/g resin) (Lloyd Williams P. et al., Chemical Approaches to the Synthesis of Peptides and Proteins, C R C, 1997, Boca Raton (Fla., USA)). Washes between stages of deprotection, coupling, and, again, deprotection, were carried out with DMF (31 min) and DCM (31 min) each time using 10 mL solvent/g resin. Coupling reactions were performed with 3 mL solvent/g resin. The control of the couplings was performed by carrying out the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34: 595598). All synthetic reactions and washes were carried out at RT.

Example 1. Synthesis and Preparation of the Peptides of the Present Invention

[0140] Obtaining Fmoc-X.sub.m-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.nR.sub.2-Rink-MBHA-resin, wherein AA.sub.1 is -L-Asp- or -L-Glu-; AA.sub.2 is -L-Tyr-, -L-Trp- or -L-Phe-; AA.sub.3 is -L-Lys- or -L-Arg-; AA.sub.4 is -L-Val-, -L-Ile- or -L-Leu-; and n and m, are 0.

[0141] Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol in order to remove the Fmoc group. 2.55 g of Fmoc-L-Val-OH, 2.65 g of Fmoc-L-Ile-OH or 2.65 g Fmoc-L-Leu-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.

[0142] The resin was then washed as described in the general methods and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 3.51 g of Fmoc-L-Lys(Boc)-OH or 4.87 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 3.45 g of Fmoc-L-Tyr(tBu)-OH, 3.95 g of Fmoc-L-Trp(Boc)-OH or 2.91 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equiv); and subsequently 3.09 g of Fmoc-L-Asp(tBu)-OH or 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.

[0143] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum. [0144] Obtaining Fmoc-X.sub.m-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.nR.sub.2-Rink-MBHA-resin, Wherein X is -L-Leu-; AA.sub.1 is -L-His - or -L-Thr-; AA.sub.2 is -L-Tyr-, -L-Trp- or -L-Phe-; AA.sub.3 is -L-Tyr-, -L-Trp- or -L-Phe-; AA.sub.4 is -L-Lys- or -L-Arg- and Y is -L-Ala-; and n and m, are 1.

[0145] Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol in order to remove the Fmoc group. 2.34 g of Fmoc-L-Ala-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.

[0146] The resin was then washed as described in the general methods and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 3.51 g of Fmoc-L-Lys(Boc)-OH or 4.87 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 3.45 g of Fmoc-L-Tyr(tBu)-OH, 3.95 g of Fmoc-L-Trp(Boc)-OH or 2.91 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equiv); subsequently 3.45 g of Fmoc-L-Tyr(tBu)-OH, 3.95 g of Fmoc-L-Trp(Boc)-OH or 2.91 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equiv); subsequently 4.65 g Fmoc-L-His(Trt)-OH or 2.98 g Fmoc-L-Thr(tBu)-OH (7.5 mmol; 3 equiv); and subsequently 2.65 g of Fmoc-L-Leu-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.

[0147] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum. [0148] Obtaining Fmoc-X.sub.m-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Y.sub.nR.sub.2O-2-CITrt-resin, Wherein AA.sub.1 is -L-Asp- or -L-Glu-; AA.sub.2 is -L-Tyr-, -L-Trp- or -L-Phe-; AA.sub.3 is -L-Lys- or -L-Arg-; AA.sub.4 is -L-Val-, -L-Ile- or -L-Leu-; and n and m, are 0.

[0149] Weights have been normalized. A mixture of 1.77 g of Fmoc-L-Ile-OH, 1.77 g of Fmoc-L-Leu-OH or 1.70 g of Fmoc-L-Val-OH (5 mmol; 1 equiv) with 0.85 mL of DIEA (5 mmol; 1 equiv) dissolved in approximately 30 mL of DCM were coupled to dry 2-chlorotrityl resin (3.8 g; 5 mmol). They were stirred for 5 min, after which 1.7 mL of DIEA were added (10 mmol; 2 equiv). The mixture was allowed to react for 40 min. The remaining chloride groups were blocked by treatment with MeOH.

[0150] The resin was then washed as described in the general methods and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 7.02 g of Fmoc-L-Lys(Boc)-OH or 9.74 g of Fmoc-L-Arg(Pbf)-OH (15 mmol; 3 equiv); subsequently 6.90 g of Fmoc-L-Tyr(tBu)-OH, 7.90 g of Fmoc-L-Trp(Boc)-OH or 5.82 g of Fmoc-L-Phe-OH (15 mmol; 3 equiv); and subsequently 6.18 g of Fmoc-L-Asp(tBu)-OH or 6.38 g of Fmoc-L-Glu(OtBu)-OH (15 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 2.03 g of HOBt (15 mmol; 3 equiv) and 2.3 mL of DIPCDI (15 mmol; 3 equiv).

[0151] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

[0152] Using the synthesis procedures mentioned above, with the required selection of amino acids, the following sequences were synthesized:

TABLE-US-00012 (SEQ ID NO: 1) Asp-Tyr-Lys-Val; and (SEQ ID NO: 3) Leu-His-Trp-Phe-Arg-Ala.

[0153] In addition, making the appropriate arrangement, also the following sequences were synthesized:

TABLE-US-00013 (SEQ ID NO: 2) His-Trp-Phe-Lys; and (SEQ ID NO: 4) Thr-Phe-Phe-Lys.

Example 2. Removal of Fmoc N-Terminal Protective Group of the Peptides Synthesized in Accordance with Example 1

[0154] The N-terminal Fmoc group of the peptidyl resins was deprotected with 20% piperidine in DMF (11 min+210 min) (Lloyd Williams P. et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins CRC, Boca Raton (Fla., USA)). The peptidyl resins were washed with DMF (51 min), DCM (41 min), and dried under vacuum.

Example 3. Process for Introducing the R.SUB.1 .Acetyl Group onto the Peptidyl Resins Obtained in Accordance with Example 2

[0155] 1 mmol (1 equiv) of the peptidyl resins obtained in accordance with 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. They were left to react for 30 minutes, after which the peptidyl resins were washed with DMF (51 min), DCM (41 min), and were dried under vacuum.

Example 4. Process for Introducing the R.SUB.1 .Palmitoyl Group onto the Peptidyl Resins Obtained in Example 2

[0156] 10 equivalents of pre-dissolved palmytic acid in DMF (1 mL) were incorporated onto 1 mmol (1 equiv) of the peptidyl resins obtained in Example 2, in the presence of 10 equivalents of HOBt and 10 equivalents of DIPCDI. They were allowed to react overnight (approximately 15 hours), after which the resins were washed with DMF (51 min), DCM (41 min), MeOH (51 min) and were dried under vacuum.

Example 5. Process for Introducing the R.SUB.1 .Ferulic Acid onto De Peptidyl Resins Obtained in Example 2

[0157] 10 equivalents of pre-dissolved ferulic acid in DMF (1 mL) were incorporated onto 1 mmol (1 equiv) of the peptidyl resins obtained in Example 2, in the presence of 10 equivalents of HOBt and 10 equivalents of DIPCDI. They were allowed to react for approximately 3-4 hours, after which the resins were washed with DMF (51 min), DCM (41 min), MeOH (51 min) and were dried under vacuum.

Example 6. Cleavage Process from the Polymeric Support of the Peptidyl Resins Obtained in Accordance with Example 2, 3, 4 and 5

[0158] Weights were normalized. 200 mg of the dried peptidyl resin obtained in any of Examples 2, 3, 4 or 5 were treated with 5 mL of TFA/TIS/H.sub.2O (90:5:5) for 2 hours at room temperature under stirring. The filtrates were collected and precipitated using 50 mL (8 to 10-fold) of cold diethyl ether. The ethereal solutions were evaporated to dryness at reduced pressure and room temperature, the precipitates are redissolved in 50% MeCN in H.sub.2O and lyophilized.

Example 7. Characterization of the Peptides Synthesized and Prepared in Accordance with Example 6

[0159] HPLC analysis of the peptides obtained in accordance with example 5 was carried out with a Shimadzu equipment (Kyoto, Japan) using a reverse-phase column (1504.6 mm, XBridge Peptide BEH C18, 3.5 m, Waters, USA) in gradients of MeCN (+0.036% TFA) in H.sub.2O (+0.045% TFA) at a flow rate of 1.25 mL/min and detection was carried out at 220 nm. All peptides showed a purity exceeding 80%. The identity of the peptides obtained was confirmed by ESI-MS in a Water ZQ 4000 detector using MeOH as the mobile phase and a flow rate of 0.2 mL/min.

Example 8. Preparation of a Cosmetic Facial Composition Containing Ac-SEQ ID NO: 1-NH.SUB.2

[0160] A cosmetic facial composition in accordance with table 1 below was prepared. To that end, components from phase A were dissolved in a suitable vessel and the mixture was heated to 70-75 C. In another vessel 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, under 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, maintaining the stirring until an homogeneous emulsion was obtained. Finally, with the mixture at about 30 C., the commercial formulation containing the compound Ac-SEQ ID NO: 1-NH.sub.2 (INCI: Water (Aqua), Glycerin, Caprylyl Glycol, Ac-SEQ ID NO: 1-NH.sub.2) was slowly added maintaining stirring.

TABLE-US-00014 TABLE 1 Details of the cosmetic facial composition of example 8. Phase Ingredient % in weight A Water Quantity required to arrive at 100 A Disodium EDTA 0.15 A Magnesium sulfate 1.5 A Glycerin 2.5 B Caprylic/Capric triglyceride 8 B Isononyl/Isononanoate 15 B Polyglyceryl-4 3 diisostearate/polyhydroxystearate/sebacate B Verstatil TBO (INCI: Triethyl citrate, Caprylyl glycol, Benzoic acid): Thriethyl citrate 0.455 Caprylyl glycol 0.37 Benzoic acid 0.175 B Synthetic beeswax 3 C Nylon-12 2 D BRB SG 516 (INCI: Dimethicone, Dimethicone/ Vinyl dimethicone crosspolymer): Dimethicone 1.7 Dimethicone/Vinyl dimethicone crosspolymer 0.3 E Commercial formulation of Ac-SEQ ID NO: 1- NH.sub.2 (INCI: Water (Aqua), Glycerin, Caprylyl glycol, Ac-SEQ ID NO: 1-NH.sub.2): Water (Aqual) 1.8384 Glycerin 0.101 Caprylyl glycol 0.0101 Ac-SEQ ID NO: 1-NH.sub.2 0.0505

Example 9. Trolox Equivalent Antioxidant Capacity (Hereinafter, TEAC)

[0161] Peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 synthesized in accordance with examples 1-6, were used in this example.

[0162] TEAC assay was used in this case. Said assay measures the antioxidant capacity of a given substance, as compared to the standard, Trolox, an analogue of Vitamin E.

[0163] Antioxidant capacity was measured using the ABTS Decolorization Assay. In this assay, the pre-formed radical monocation of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) was generated by oxidation of ABTS with potassium persulfate and was reduced in the presence of antioxidants (Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 or Ferulic acid-SEQ ID NO: 2-NH.sub.2). The ABTS.+ has a blue-green color, with maximum absorptions at 650, 734 and 820 nm. Antioxidants in the sample reduce ABTS.+ suppressing this color production to a degree that is proportional to their concentrations and antioxidant capacity. Quantification of absorbance was conducted at 734 nm using a Polarstar Omega equipment (BMG Labtech).

[0164] Results obtained for this experiment appear summarized in FIG. 1. Briefly, for Ac-SEQ ID NO: 1-NH.sub.2 the following values of trolox equivalents were obtained: 15 M at 0.01 mg/mL, 24 M at 0.05 mg/ml and 94 M at 0.1 mg/mL; for Ac-SEQ ID NO: 2-NH.sub.2 the following values of trolox equivalents were obtained: 22 M at 0.05 mg/mL, 49 M at 0.1 mg/mL and 130 M at 0.5 mg/mL; for Ac-SEQ ID NO: 3-NH.sub.2 the following values of trolox equivalents were obtained: 28 M at 0.01 mg/mL, 59 M at 0.05 mg/mL and 74 M at 0.1 mg/mL; and for Ferulic acid-SEQ ID NO: 2-NH.sub.2 the following values of trolox equivalents were obtained: 59 M at 0.01 mg/mL, 112 M at 0.05 mg/mLl and 113 M at 0.1 mg/mL. Results also show that the antioxidant power of Ferulic acid-SEQ ID NO: 2-NH.sub.2 is higher than the one of AA-2G at concentrations >0.05 mg/mL and equals MAP values of trolox equivalents at this same concentration. Also, the higher concentration tested of Ac-SEQ ID NO: 2-NH.sub.2 showed a higher antioxidant power than AA-2G and MAP.

[0165] As derived from the results shown in said figure, the four peptides tested show an important antioxidant power which reaches 94, 130, 74 and 113 M of Trolox in the higher concentration tested for Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2, respectively. Other antioxidants in the state of the art, like Resveratrol, show an antioxidant capacity of 29 M of Trolox at 0.02 mg/ml, this is, a markedly lower antioxidant capacity when compared with peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2.

Example 10. Evaluation of Antioxidant Activity

[0166] Antioxidant activity of peptide Ferulic acid-SEQ ID NO: 2-NH.sub.2 was evaluated in tubo by means of a scavenging assay.

[0167] The capacity to scavenge the stable DPPH free radical can be expressed as a measure of antioxidant activity.

[0168] During this assay, the purple chromogen (DPPH) radical was reduced by antioxidant/reducing compounds to the corresponding pale-yellow hydrazine. The reduction of the purple chromogen radical by hydrogen-donating antioxidants was monitored by the decrease of optical density at long wavelengths (515-520 nm).

[0169] Briefly, different concentrations of Ferulic acid-SEQ ID NO: 2-NH.sub.2 (mainly, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL) were incubated together with 300 M DPPH (in MeOH) in triplicate. Ascorbic acid was also included in the assay as positive control for scavenger activity evaluation at 30 M and 60 M. After 30 minutes of incubation at room temperature and gentle shaking, the absorbance at 520 nm of each well was determined using the microplate reader Multiskan FC (Thermo Fisher Scientific, MA, USA), being directly proportional to the amount of DPPH left (not-scavenged) in the reaction mixture. Absorbance values were normalized on the basis of the control (non-treated wells) which was stablished as 100%, obtaining the corresponding percentage of activity for each of the treatments.

[0170] As shown in FIG. 2, Ferulic acid-SEQ ID NO: 2-NH.sub.2 presents a significant antioxidant activity following a dose-response at the higher concentrations tested (0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL) with a reduction of DPPH of 48.3%, 72.9% and 90.6% respectively. The antioxidant activity of the compound at 0.05 mg/mL and 0.1 mg/mL is comparable to that of ascorbic acid at 30 M (31.3%) and 60 M (67.6%), respectively.

Example 11. Thiobarbituric Acid Reactive Substances (Hereinafter, TBARS) Assay

[0171] Peptides Ac-SEQ ID NO: 1-NH.sub.2 and Ac-SEQ ID NO: 2-NH.sub.2, synthesized in accordance with examples 1-3 and 6, were also used in this example.

[0172] TBARS are naturally present in biological samples and increase as a result of oxidative stress. This assay measures lipid peroxidation levels through the detection of MDA, a compound that results from the decomposition of polyunsaturated fatty acid lipid peroxides and it is an indicator of oxidative stress in cells and tissues.

[0173] Small Unilamellar Vesicles (hereinafter, SUVs) from egg yolk were prepared and incubated with the analysed compounds (peptides Ac-SEQ ID NO: 1-NH.sub.2 or Ac-SEQ ID NO: 2-NH.sub.2 and the corresponding controls). Then, oxidation of SUVs was induced by the addition of 2,2-Azobis(2-amidinopropane) dihydrochloride (hereinafter, AAPH), a free radical able to oxidize different molecules. Oxidative reaction was then stopped with Butylated hydroxytoluene (hereinafter, BHT), a lipophilic organic compound with antioxidant properties. Finally, Thiobarbituric Acid (hereinafter, TBA) was added for the detection and quantification of MDA by fluorescence at 530 nm. Fluorescence quantification was performed by Cytation 3 (Biotek).

[0174] Results obtained for this assay appear summarized in FIG. 3.

[0175] For the two peptides, a statistically significant inhibition of lipid peroxidation was observed in all the concentrations tested.

Example 12. In Vitro Evaluation of the Protective Efficacy Against Oxidative Stress Induced by Hydrogen Peroxide (H.SUB.2.O.SUB.2.)

[0176] Ac-SEQ ID NO: 1-NH.sub.2 and Ac-SEQ ID NO: 2-NH.sub.2, synthesized in accordance with examples 1-3 and 6, were then tested for their capacity to protect against oxidative stress induced by hydrogen peroxide.

[0177] Primary human epidermal keratinocytes were seeded in black 96 well-plates in the corresponding culture medium and, 48 hours later, treated with the different compounds analysed (Ac-SEQ ID NO: 1-NH.sub.2 or Ac-SEQ ID NO: 2-NH.sub.2) for 3 hours at 37 C. or with ascorbic acid as an antioxidant control. After incubation, cells were loaded with a fluorescence probe for 45 minutes and then H.sub.2O.sub.2 was added for 30 minutes.

[0178] Finally, fluorescence was determined at Excitation 485 nm/Emission 520 nm.

[0179] Results obtained appear summarized in FIG. 4.

[0180] As can be directly derived from this figure, both peptides show an inhibition of the oxidative activity exerted by hydrogen peroxide, materialized by a reduction in the reactive oxygen species detected in the sample when treated with any one of the above-mentioned peptides. Noteworthy is the fact that Ac-SEQ ID NO: 1-NH.sub.2 has shown increased activity in all concentrations tested when compared with Ac-SEQ ID NO: 2-NH.sub.2.

Example 13. In Vitro Evaluation of the Protective Efficacy Against Glycation Induced by Heavy Metals (Advanced Glycation End Products Modulation)

[0181] Peptide Ac-SEQ ID NO: 1-NH.sub.2, synthesized in accordance with examples 1-3 and 6, was also tested for its capacity to protect against glycation induced by heavy metals.

[0182] Primary human epidermal keratinocytes were seeded in 96-well plates in standard culture medium and, 24 hours later, subjected to a simultaneous treatment with heavy metals (containing a mixture of Fe, Pb and Cr) and peptide Ac-SEQ ID NO: 1-NH.sub.2 for 48 hours. Levels of Advance Glycation End (AGE) products were measured by means of a competitive ELISA assay with absorbance at 450 nm.

[0183] The results obtained in this assay are summarized in FIG. 5.

[0184] As can be seen in FIG. 5, all concentrations tested of the peptide Ac-SEQ ID NO: 1-NH.sub.2 produced a statistically significant decrease in the quantity of advanced glycation end-products, reducing it to levels near those observed in the basal state (quantity or level in control samples not treated with heavy metals or in the treated samples prior to the application of the treatment with the heavy metals).

Example 14. In Vitro Evaluation of the Protective Efficacy Against DNA Oxidation and Lipoxidation Induced by Synthetic Smoke (8-Hydroxy-2-Deoxyguanosine(8-OHdG) Modulation)

[0185] The antioxidant activity of peptide Ac-SEQ ID NO: 1-NH.sub.2, synthesized in accordance with examples 1-3 and 6, was also analysed by treating the samples with synthetic smoke.

[0186] Primary human epidermal keratinocytes were seeded in 96-well plates in standard culture medium and, 24 hours later, subjected to a simultaneous treatment with synthetic smoke containing selected compounds from the particle phase (for example, cadmium and nicotine) and vapour phase (for example, formaldehyde (hereinafter, FA) and ethyl carbamate) of tobacco smoke and the analysed compound (Ac-SEQ ID NO: 1-NH.sub.2) for 48 h.

[0187] After treatment, cells were lysed and DNA oxidation on cell homogenates was analysed measuring levels of 8-OHdG by means of a competitive ELISA assay with absorbance at 450 nm.

[0188] In addition, after treatment, lipoxidation was analysed by means of the levels of intracellular MDA, which were measured by the reaction between MDA and N-methyl-2-phenylindole (hereinafter, NMPI) rendering a stable blue chromophore with an absorption peak at 586 nm.

[0189] The outcome of this assays is shown in FIGS. 6 and 7.

[0190] From both figures, it can be derived that Ac-SEQ ID NO: 1-NH.sub.2 is able to protect cells against DNA oxidation and lipoxidation. In fact, for these two parameters, the behavior was similar: the lower tested concentration showed a slight decrease (non-statistically significant) for said parameters while the other two tested concentrations showed a higher, statistically significant, effect which, in the case of protection of DNA oxidation leads to an almost complete inhibition of the effects of synthetic smoke as the levels of DNA oxidation observed in the samples treated with 0.1 mg/ml approaches to that observed in the basal state (this is, in the non-treated control sample).

Example 15. Modulation of the Expression of Antioxidant Genes

[0191] Modulation of gene expression by Ac-SEQ ID NO: 1-NH.sub.2 was evaluated in vitro on HEKa cells.

[0192] Briefly, HEKa cells were seeded in duplicate in 6-well plates at a density of 410.sup.5 cells/well and maintained at standard culture conditions (37 C., 95% humidity, 5% CO.sub.2) for 24 hours. Then, cells were treated with non-cytotoxic concentrations of Ac-SEQ ID NO: 1-NH.sub.2 (0.05 mg/mL) for an additional 24 hours.

[0193] Untreated cells were used as basal control. Cells were then lysed for RNA extraction with a RNA purification commercial kit following manufacturer instructions (RNeasy mini kit; Qiagen; Netherlands). RNA was then quantified by nanodrop, adjusted in concentration and processed for retrotranscription to cDNA using a commercially available kit (High-Capacity cDNA Reverse Transcription kit; Thermofisher Scientific, USA). Resulting cDNA was used to perform a RTqPCR (Real Time Quantitative Polymerase Chain Reaction) using Taqman technology and a panel of probes designed to target specific genes related to melanogenesis and HEMn-HEKa and HEMn-HDFa communication.

[0194] The results of this example appear summarized in FIG. 8.

[0195] Said figure shows that treatment with 0.05 mg/mL of As-SEQ ID NO: 1-NH.sub.2 induces the upregulation of key antioxidant genes with the following fold change: CYP2R1 (1.44), NFE2L2 (1.23), HMOX1 (1.48), GSTP1 (1.13), GSS (1.10), GPX1 (1.22) and TRX (1.34). Therefore, peptide Ac-SEQ ID NO: 1-NH.sub.2 not only shows a high intrinsic antioxidant activity, as seen in Example 9, but, also, through the increase in the expression of key antioxidant genes such as NFE2L2 and HMOX-1, it is able to protect several biological structures, such as proteins, lipids and DNA against multiple stressors, as heavy metals and tobacco smoke.

Example 16. Antioxidant and Anti-Pollutant Capacity of Ac-SEQ ID NO: 1-NH.SUB.2 .on Human Skin Explants

[0196] The protection efficacy of peptide Ac-SEQ ID NO: 1-NH.sub.2 against pollution aggression on human living explants was assessed by observation of the general morphology, immunostaining of AhR and HO-1 (both are known markers of oxidative stress as they have been related to anti-oxidative response) (Esser C., Bargen I., Weighardt H., Haarmann-Stemmann T., Krutmann J., Functions of the aryl hydrocarbon receptor in the skin, Semin Immunopathol (2013) 35:677-691; Kohen R., Nyska A., Oxidation of Biological Systems: Oxidative Stress Phenomena, Antioxidants, Redox Reactions, and Methods for Their Quantification, Toxicologic Pathology (2002) vol 30, no 6, 620-650).

[0197] Briefly, human skin explants from a 49-year-old Caucasian woman were put in culture under controlled conditions. 21 skin explants of an average diameter of 11 mm (1 mm) were prepared and kept in survival in BEM culture medium at 37 C. in a humid, 5% CO.sub.2 atmosphere. The explants were distributed in 5 batches: explant control (Time=0), control batch, explants treated with the above-mentioned peptide, explants treated with pollutant and explants treated with pollutant and with the above-mentioned peptide. A composition containing Ac-SEQ ID NO: 1-NH.sub.2 in accordance with Example 8 was prepared and it was topically applied on days 0, 1, 3 and 4. Untreated explants were used as controls and did not received any treatment. For each condition, half of the explants were afterwards exposed to a pollutant mixture containing Benzene, Xylene, Toluene, heavy metals and hydrocarbons. Explant exposure was performed 3 hours after the treatment with Ac-SEQ ID NO: 1-NH.sub.2 using a Pollubox system (BIO-EC, Longjumeau, France) and lasted for 1.5 hours. Samples were processed for histology at day 0 and day 5. The observation of the general morphology was performed after staining of paraffinized sections according to Masson's trichrome, Goldner variant. AhR immunostaining was performed on paraffin sections with a monoclonal anti-AhR antibody (Thermoscientific, ref. MA1-514, clone RPT1, MA, USA) and HO-1 with a monoclonal anti-HO-1 antibody (Novus biologicals, ref. NBP1-97507, CO, USA). Finally, image analyses and quantification were performed using Cell{circumflex over ()}D software (Olympus, Tokyo, Japan).

[0198] Results of this experiment appear summarized in FIG. 9, where the antioxidant efficacy (amount of AhR and HO-1) of peptide Ac-SEQ ID NO: 1-NH.sub.2 on human skin explants, treated with said peptide in a formulation, can be observed.

[0199] As it can be seen in FIG. 9(C), it was confirmed the antioxidant effect of Ac-SEQ ID NO: 1-NH.sub.2 as it is directly derivable from its efficacy in increasing the expression on HO-1 for the prevention of oxidative damage and oxidative stress. After treatment of skin explants with the peptide in a final formulation (without exposure to pollutants), the levels of HO-1 significantly increased by 69% (FIG. 9C) when compared to untreated and unexposed explants.

[0200] As observed in FIGS. 9(A) and 9(B), Ac-SEQ ID NO: 1-NH.sub.2 showed an anti-pollution and anti-oxidant activity by reducing the anti-oxidant response required by the explants, which can be readily ascertained by the observed decrease in AhR (16%) and HO-1 (23%), respectively, compared to the levels expressed with pollutants and without treatment with the peptide. This is so because, the pretreatment with the peptide of the present invention (in this case Ac-SEQ ID NO: 1-NH.sub.2) would already induce an anti-oxidant response preparing the explants for any future or subsequent oxidative insult and, hence, when said oxidative insult arrives or is performed (in the present case, exposure of the cells to pollutants), the cells require less anti-oxidative response and the anti-oxidative machinery already prepared is used or consumed against said oxidative insult (see, for example, FIG. 9(C)).

[0201] Therefore, the peptides of the present invention, are effective in the prevention of oxidative stress and its consequences.

[0202] It is of special interest the enzyme HO-1 or HMOX-1, detected both at the genetic level and in the skin explants, which is known to be an enzyme activated via NFE2L2 (also detected in the smart data gene panel), known as the antioxidant switch, which is itself a transcription factor that binds to antioxidant-responsive elements (AREs), enhancer DNA sequences that initiate the transcription of a battery of genes encoding potent antioxidant enzymes (Nguyen, T., Nioi, P., and Pickett, C. B., The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress, J. Biol. Chem., 284 (2009) 13291-13295). Ac-SEQ ID NO: 1-NH.sub.2 also acts as a modulator of the increased expression of AhR induced after pollutants exposure, as observed on human skin explants, therefore, preventing the oxidative damage associated with AhR activation and the subsequent hyperpigmentation, also observed in vitro through inhibition of melanin synthesis.

[0203] The results shown in Examples 9 to 16 demonstrate that the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2, have not only a potent antioxidant activity but also that said activity is wide-spectrum. This is, in all the oxidative conditions tested and for all the parameters tested the peptides have shown a significant and important activity.

[0204] Hence, these experimental results demonstrate the feasibility of these peptides to be used in cosmetic compositions or methods to prevent, reduce and/or remove skin imperfections related with oxidation and oxidative stress as, for example, skin complexion, pigmentation alterations or other age related or environment related skin imperfections. This is so, because, the analysed activities are directly related with oxidative stress (Gkogkolou P, Bhm M, Advanced glycation end products Key players in skin aging?, Dermatoendocrinol., 2012 Jul. 1; 4(3): 259-270; ciskalska M, Zalewska M, Grzelak A, and Milnerowicz H, The Influence of the Occupational Exposure to Heavy Metals and Tobacco Smoke on the Selected Oxidative Stress Markers in Smelters, Biol Trace Elem Res., 2014; 159(1-3): 59-68; Tchounwou P B, Yedjou C G, Patlolla A K, and Sutton D J, Heavy Metals Toxicity and the Environment, EXS., 2012; 101: 133-164; Aflanie I, Effect of Heavy Metal on Malondialdehyde and Advanced Oxidation Protein Products Concentration: A Focus on Arsenic, Cadmium, and Mercury, Journal of Medical and Bioengineering Vol. 4, No. 4, August 2015; van der Vaart H, Postma D S, Timens W, ten Hacken N H, Acute effects of cigarette smoke on inflammation and oxidative stress: a review, Thorax., 2004 August; 59(8):713-21; and Danielsen PH1, Mller P, Jensen K A, Sharma A K, Wallin H, Bossi R, Autrup H, Mlhave L, Ravanat J L, Bried J J, de Kok T M, Loft S, Oxidative stress, DNA damage, and inflammation induced by ambient air and wood smoke particulate matter in human A549 and THP-1 cell lines, Chem Res Toxicol., 2011 Feb. 18; 24(2):168-84).

Example 17. Brightening Activity Studies

[0205] Peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 synthesized in accordance with examples 1-6, were used in this example.

[0206] Primary human epidermal melanocytes were incubated in 6-well plates for 4 days and then treated with the compounds to be analysed (Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 or Ferulic acid-SEQ ID NO: 2-NH.sub.2) for 7 days. After treatment, cells were homogenized an intracellular melanin content was quantified by absorbance at 450 nm by Multiskan FC (Thermo Fisher Scientific, MA, USA).

[0207] Results obtained in this example appear summarized in FIGS. 10 to 13.

[0208] As can be readily derivable from the results shown in FIGS. 10 and 11, both peptide Ac-SEQ ID NO: 1-NH.sub.2 and Ac-SEQ ID NO: 2-NH.sub.2 in all the concentrations tested have a significantly higher brightening or whitening activity when compared with kojic acid, a reference compound for this activity. This increased activity ranges from two to more than four times the activity seen for the two concentrations of kojic acid. In addition, it is also noteworthy that even at lower concentrations, the peptides of the present invention exert a markedly increased brightening activity with regards to kojic acid (see lower concentration used for Ac-SEQ ID NO: 1-NH.sub.2 17.7 Mand Ac-SEQ ID NO:2-NH.sub.2 15.2 Mwhen compared with 50 M of kojic acid).

[0209] As observed in FIGS. 12 and 13, peptides Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 also show a significant decrease in melanin content, which is higher than the positive reference control (treatment with kojic acid) in case of Ac-SEQ ID NO: 3-NH.sub.2 (up to 49.3% at 0.1 mg/mL) but comparable in the case of Ferulic acid-SEQ ID NO: 2-NH.sub.2 (36% decrease at 0.05 mg/mL).

Example 18. Evaluation of Tyrosinase Activity

[0210] The potential of peptides Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 to inhibit tyrosinase activity was evaluated in tubo.

[0211] Briefly, different concentrations of Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 (0.1, 0.5 and 1 mg/mL) and the positive control kojic acid (400 and 800 M) were incubated with recombinant mushroom tyrosinase for 30 minutes before the addition of L-DOPA (L-3,4 dihydroxyfenylalanine, 2.5 mg/mL in PBS). After 2 hours' reaction at room temperature, the absorbance at 450 nm of each well was determined using the microplate reader Multiskan FC (Thermo Fisher Scientific, MA, USA), which is directly proportional to the amount of dopachrome in the reaction mixture.

[0212] Absorbance values were normalized with regard to non-treated wells (control) which were stablished as 100%, obtaining, hence, the percentage of activity for each condition tested.

[0213] Results appear summarized in FIGS. 14 and 15.

[0214] Results in FIG. 14 show a significant decrease in tyrosinase activity after treatment with 0.5 mg/mL (30.6%) and 1 mg/mL (58.1%) of Ac-SEQ ID NO: 3-NH.sub.2, reaching percentages close to those of kojic acid at 800 M. Similarly, Ferulic acid-SEQ ID NO: 2-NH.sub.2 induced a dose-response reduction in tyrosinase activity, with a decrease of 12%, 46.5% and 71% at 0.05 mg/mL, 0.1 mg/mL and 0.5 mg/mL, respectively (see FIG. 15).

Example 19. Modulation in Gene Expression of Melanogenic Pathways

[0215] Modulation of gene expression by Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2 peptides was evaluated in vitro on two different cell types (HEMn and HEKa).

[0216] Briefly, HEKa cells were seeded in duplicate in 6-well plates at a density of 410.sup.5 cells/well and maintained at standard culture conditions (37 C., 95% humidity, 5% CO.sub.2) for 24 hours. Then, cells were treated with non-cytotoxic concentrations of Ac-SEQ ID NO: 3-NH.sub.2 (0.1 mg/mL) or Ferulic acid-SEQ ID NO: 2-NH.sub.2 (0.05 mg/mL) for an additional 24 hours.

[0217] In the case of HEMn cells, cells were left in culture for 12 days (from day 0 to day 11, 11-day treatment) at standard culture conditions (37 C., 95% humidity, 5% CO.sub.2) and treated with non-cytotoxic concentrations of Ac-SEQ ID NO: 3-NH.sub.2 (0.1 mg/mL) or Ferulic acid-SEQ ID NO: 2-NH.sub.2 (0.05 mg/mL) from day 4 to day 11.

[0218] In addition, HEMn cells were left in culture for 24 h at standard culture conditions (37 C., 95% humidity, 5% CO.sub.2) and treated with a non-cytotoxic concentration of Ac-SEQ ID NO: 3-NH.sub.2 (0.1 mg/mL) for additional 24 h.

[0219] Untreated cells were used as basal control. Cells were then lysed for RNA extraction with a RNA purification commercial kit following manufacturer instructions (RNeasy mini kit, Qiagen, Netherlands). RNA was then quantified by nanodrop, adjusted in concentration and processed for retrotranscription to cDNA using a commercially available kit (High-Capacity cDNA Reverse Transcription kitThermofisher Scientific, USA). Resulting cDNA was used to perform a RTqPCR (Real Time Quantitative Polymerase Chain Reaction) using Taqman technology and a panel of probes designed to target specific genes related to melanogenesis and HEMn-HEKa and HEMn-HDFa communication.

[0220] The results of this example appear summarized in FIGS. 16 to 19 (FIGS. 16 and 17 for modulation in HEMn and, FIGS. 18 and 19 for modulation in HEKa).

[0221] Said FIG. 16 shows that treatment with 0.1 mg/mL of Ac-SEQ ID NO: 3-NH.sub.2 (FIG. 16) induces a downregulation of several genes involved in the melanogenic pathway at 11-day treatment, with the following fold change: COX-1 (1.51), MITF (1.48), MC1R (1.28), MLAN-A (1.88), C-KIT (1.78), PMEL17 (2.30), DCT-TYRP2 (1.40), TYRP-1 (1.64) and TYR (1.66). When treatment was performed at 24 h, this tendency to downregulate gene expression was also observed.

[0222] In the case of Ferulic acid-SEQ ID NO: 2-NH.sub.2 (FIG. 17), an upregulation of VDR gene (fold change of 1.48) and a slight tendency to downregulate PMEL17 and DCT-TYRP2 expression was observed.

[0223] FIGS. 18 and 19 show the regulation of genes involved in HEMn-HEKa communication and release of melanogenic factors. A 24-hour treatment of HEKa cells with 0.1 mg/mL of Ac-SEQ ID NO: 3-NH.sub.2 induced the downregulation of KITLG, TP53 and NGF and the upregulation of DKK-1 (FIG. 18). A lower concentration of Ferulic acid-SEQ ID NO: 2-NH.sub.2 (0.05 mg/mL) induced de downregulation of KITLG, POMC, EDN1 and NGF (FIG. 19).

[0224] The above results demonstrate the feasibility of the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2, Ac-SEQ ID NO: 3-NH.sub.2 and Ferulic acid-SEQ ID NO: 2-NH.sub.2, to be used in cosmetic compositions or methods to lighten or brighten skin.