PEPTIDES AND COMPOSITIONS FOR USE IN COSMETICS AND MEDICINE

Abstract

Peptides and compositions thereof which are able to modulate the expression of IL-33 and, hence useful for the prevention, reduction and/or elimination of signs of skin aging and/or skin imperfections.

Claims

1. A peptide comprising 6 amino acids, its isomers, salts, solvates and/or solvates of the salts and mixtures thereof, wherein the peptide comprises at least 3 arginines.

2. The peptide in accordance with claim 1, wherein said 6 amino acids are 3 arginines, 1 glutamine, 1 methionine and 1 glutamic acid.

3. The peptide in accordance with claim 1, comprising a sequence in accordance with formula (I):
R.sub.1-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-R.sub.2 wherein: 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 heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R.sub.5—CO—, wherein R.sub.5 is selected from the group formed by substituted or unsubstituted C.sub.1-C.sub.24 alkyl radical, substituted or unsubstituted C.sub.2-C.sub.24 alkenyl, substituted or unsubstituted C.sub.2-C.sub.24 alkynyl, substituted or unsubstituted C.sub.3-C.sub.24 cycloalkyl, substituted or unsubstituted C.sub.5-C.sub.24 cycloalkenyl, substituted or unsubstituted C.sub.8-C.sub.24 cycloalkynyl, substituted or unsubstituted C.sub.6-C.sub.30 aryl, substituted or unsubstituted C.sub.7-C.sub.24 aralkyl, substituted or unsubstituted heterocyclyl ring of 3 to 10 members, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon and an alkyl chain of 1 to 6 carbon atoms; 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 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.

4. The peptide in accordance with claim 1, wherein the peptide comprises Arg-Arg within its sequence.

5. The peptide in accordance with claim 1 wherein the peptide comprises Arg-Glu within its sequence.

6. The peptide in accordance with claim 1, wherein the peptide comprises Met-Arg within its sequence.

7. The peptide in accordance with claim 1, wherein the sequence of the peptide is: TABLE-US-00024 R.sub.1-Arg-Arg-Gln-Met-Arg-Glu-R.sub.2, R.sub.1-Met-Arg-Arg-Glu-Gln-Arg-R.sub.2, or R.sub.1-Arg-Glu-Gln-Met-Arg-Arg-R.sub.2.

8. The peptide in accordance with claim 1, wherein the sequence of the peptide is: TABLE-US-00025 Ac-Arg-Arg-Gln-Met-Arg-Glu-NH.sub.2); Ac-Met-Arg-Arg-Glu-Gln-Arg-NH.sub.2); or Ac-Arg-Glu-Gln-Met-Arg-Arg-NH.sub.2.

9. A composition comprising the peptide in accordance with claim 1.

10. A cosmetic comprising the peptide in accordance with claim 1.

11. A method for treating signs of skin aging and/or imperfections comprising applying the cosmetic in accordance with claim 10 to a subject in need thereof.

12. The method in accordance with claim 11, wherein the signs of skin aging and/or skin imperfections are eyebags, eyelid imperfections, wrinkles in the surrounding of the eyes, periorbital hyperchromia or combinations thereof.

13. The method in accordance with claim 12, wherein the signs of skin aging and/or skin imperfections are associated with sleep deprivation.

14. A medicament comprising the peptide in accordance with claim 1.

15. A method for treating inflammation-based sagging eyelid comprising applying the peptide in accordance with claim 1 to a subject in need thereof.

Description

[0206] 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.

[0207] FIG. 1 shows the IL-33 protein levels in human dermal fibroblasts adult cells treated with peptide Ac-SEQ ID NO:1-NH.sub.2. Results are shown as percentage in comparison with the positive control (treatment with 300 U/mL of Interferon-γ), which is stablished as 100%. The columns in the x-axis, from left to right refer to: basal state of the cells, positive control and cells treated with 0.005 mg/mL, 0.01 mg/mL or 0.05 mg/mL of Ac-SEQ ID NO:1-NH.sub.2, respectively. Samples treated with the peptide of the present invention were also treated with 300 U/mL of Interferon-γ. The y-axis refers to the % of IL-33 in pg/mL, with regard to the positive control that is stablished as 100%. **refers to a p<0.01; and ***refers to a p<0.001.

[0208] FIG. 2 shows the results for the inhibition of mushroom tyrosinase activity obtained in example 11. Tyrosinase activity with regard to the control (without peptide), which is stablished as 100%, is measured. FIG. 2A refers to the results of tyrosinase activity obtained with Ac-SEQ ID NO:1-NH.sub.2 and, hence, columns in the x-axis, from left to right, refer to: control wells and wells containing 400 μM and 800 μM of kojic acid and wells containing 0.05 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 2.5 mg/mL of Ac-SEQ ID NO:1-NH.sub.2, respectively. FIG. 2B refers to the results of tyrosinase activity obtained with Ac-SEQ ID NO:2-NH.sub.2 and, hence, columns in the x-axis, from left to right, refer to: control wells, wells containing 400 μM and 800 μM of kojic acid and wells containing 0.05 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 2.5 mg/mL of Ac-SEQ ID NO:2-NH.sub.2, respectively. FIG. 2C refers to the results of tyrosinase activity obtained with Ac-SEQ ID NO:3-NH.sub.2 and, hence, columns in the x-axis, from left to right, refer to: control wells, wells containing 400 μM and 800 μM of kojic acid and wells containing 0.05 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 2.5 mg/mL of Ac-SEQ ID NO:3-NH.sub.2, respectively. FIG. 2D refers to the results obtained with Ac-SEQ ID NO: 4-NH.sub.2 and, hence, columns in the x-axis, from left to right, refer to: control wells and wells treated with 0.005 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.25 mg/mL, 0.5 mg/mL and 2.5 mg/mL of Ac-SEQ ID NO:4-NH.sub.2, respectively. In FIGS. 2A, 2B, 2C and 2D, the y-axis refers to the percentage of tyrosinase activity, stablishing as 100% the tyrosinase activity of the control. *refers to a p<0.05; **refers to a p<0.01; and ***refers to a p 21 0.001.

[0209] FIG. 3 shows the tube formation (angiogenesis) in HUVEC treated with Ac-SEQ ID NO:1-NH.sub.2 in example 12. More precisely, columns in black refer to the number of meshes as percentage in comparison with the basal state (stablishing the basal state as 100%); and columns in white refer to the total area of meshes as percentage in comparison with the basal state (stablishing the basal state as 100%). In addition, each group of two columns (one black and one white) from left to right in the x-axis in this figure correspond to: basal; negative control (treatment with 20 μM of Suramin); positive control (treatment with 10 ng/mL IL-33); treatment with 0.01 mg/mL of Ac-SEQ ID NO:1-NH.sub.2 and 10 ng/mL IL-33; treatment with 0.05 mg/mL of Ac-SEQ ID NO:1-NH.sub.2 and 10 ng/mL IL-33; and treatment with 0.1 mg/mL of Ac-SEQ ID NO:1-NH.sub.2 and 10 ng/mL IL-33. The y-axis refers to the percentage of number of meshes (for black columns) or of total area of meshes (for white columns) (depending on the column considered) in comparison with the basal state (stablished as 100%). *refers to a p<0.05; **refers to a p<0.01; and ***refers to a p<0.001.

[0210] FIG. 4 shows the results of example 13, this is the peroxidation of lipids in comparison with the negative control, which is stablished as 100%. In the x-axis, columns from left to right correspond to: negative control (lipid substrate without antioxidant compound), positive control (lipid substrate treated with 250 μg/mL of Trolox (a stable vitamin E analogue) but not with a peptide of the present invention) and treatment with 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL or 0.5 mg/mL of Ac-SEQ ID NO:1-NH.sub.2, respectively. All samples had a lipid substrate and all samples except the negative control also comprised a lipid peroxidant (thiobarbituric acid). A 2-hour incubation at 37° C. was performed before reading the plate at Excitation at: 500 nm; Emission at: 530 nm. The y-axis refers to the percentage of lipid peroxidation in comparison with the negative control, which is stablished as 100%. *refers to a p<0.05; **refers to a p<0.01; and ***refers to a p<0.001.

[0211] FIG. 5 shows the results of example 14, this is the glycation of proteins in comparison with the negative control, which is stablished as 100%. In the x-axis, columns from left to right correspond to: negative control (sugar substrate), positive control (sugar substrate treated with 690 μg/mL of aminoguanidine (AG, a representative antiglycation compound)) and treatment with 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL or 0.5 mg/mL of Ac-SEQ ID NO:1-NH.sub.2, respectively. All samples had a sugar substrate and all samples except the negative control also comprised a glycation agent. A 72-hour incubation at 37° C. was performed before reading the plate at Excitation at: 370 nm; Emission at: 440 nm. The y-axis refers to the percentage of protein glycation in comparison with the negative control, which is stablished as 100%. *refers to a p<0.05; ** refers to a p<0.01; and ***refers to a p<0.001.

[0212] FIG. 6 shows the ex-vivo results of example 14, this is the protective effect of Ac-SEQ ID NO:1-NH.sub.2 in front methylglyoxal (MG)-induced glycation in comparison with the negative control (untreated explants), which is stablished as 100%. In the x-axis, columns from left to right correspond to: negative control (untreated explants), positive control (MG, not treated with any cream), and MG with placebo cream or cream containing 1% (w/v) of Ac-SEQ ID NO:1-NH.sub.2, respectively. The y-axis refers to the percentage of CML staining (N(ε)-carboxymethyl-Lysine) in comparison with the negative control, which is stablished as 100%.

[0213] FIG. 7 shows the ex-vivo results of example 15, this is the protective effect of Ac-SEQ ID NO:1-NH.sub.2 in front methylglyoxal (MG)-induced fibrillin-1 degradation in comparison with the negative control (untreated explants), which is stablished as 100%. In the x-axis, columns from left to right correspond to: negative control (untreated explants), positive control (MG, not treated with any cream), and MG with placebo cream or cream containing 1% (w/v) of Ac-SEQ ID NO:1-NH.sub.2, respectively. The y-axis refers to the percentage of fibrillin-1 staining in comparison with the negative control, which is stablished as 100%.

[0214] FIG. 8 shows a graphic representation of the data obtained for anti-peroxidation activity in comparison with 0.1 mg/mL and 0.5 mg/mL of melatonin. In the x-axis, columns from left to right correspond to: negative control (lipid substrate without antioxidant compound), positive control (lipid substrate treated with 250 μg/mL of Trolox (a stable vitamin E analogue) but not with a peptide of the present invention), 0.1 mg/mL melatonin, 0.5 mg/mL melatonin.

[0215] FIG. 9 shows a graphic representation of the data obtained for anti-glycation activity against 0.1 mg/mL and 0.5 mg/mL of melatonin. In the x-axis, columns from left to right correspond to: negative control (sugar substrate), positive control (sugar substrate treated with 690 μg/mL of aminoguanidine (AG, a representative antiglycation compound)), 0.1 mg/mL melatonin, 0.5 mg/mL melatonin.

[0216] FIG. 10 shows immunofluorescence images acquired with a fluorescence microscope for basal cells (A) and emilin-1 expression on HDLMVEC after 24 h treatment with D18ChAH (B) (green: emilin-1, red: phalloidin, blue: nuclei).

[0217] FIG. 11 shows immunofluorescence images acquired with a fluorescence microscope for basal cells (A) and integrin α9β1 expression on HDLMVEC after 24 h treatment with Ac-SEQ ID NO:1-NH.sub.2 (B) (green: integrin α9β1, red: phalloidin, blue: nuclei).

[0218] FIG. 12 shows the regulation of genes related with antimicrobial innate immune response by 0.01 mg/mL of Ac-SEQ ID NO:1-NH.sub.2 or 0.01 mg/mL of melatonin in HEKa cells after 6 h of treatment. The x-axis shows Fold change vs. basal.

[0219] FIG. 13 shows a graphic representation of the reduction of eyebags volume with a composition comprising the peptide and a placebo composition. In the x-axis, columns from left to right correspond to: T7 days and T14 days.

EXAMPLES

[0220] Abbreviations:

[0221] 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.

[0222] Ac, acetyl; Arg, arginine; Boc, tert-butyloxycarbonyl; C-terminal, carboxy-terminal; DCM, dichloromethane; DIEA, N,N′-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; Glu, Glutamic acid; Gln, Glutamine; hiPSC, human induced pluripotent stem cells; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; HRP, Horseradish peroxidase; INCI, International Nomenclature of Cosmetic Ingredients; MBHA, p-methylbenzhydrylamine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; Met, Methionine; N-terminal, amino-terminal; Palm, palmitoyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PFA, paraformaldehyde; PMA, phorbol 12-myristate 13-acetate; PMSF, Phenylmethanesulfonyl; RT, room temperature; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, Tetramethylbenzidine; Trt, triphenylmethyl or trityl.

[0223] 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 1×1 min, 2×10 min, 5 mL/g resin) (Lloyd Williams P. et al., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC, 1997, Boca Raton (Fla., USA)). Washes between stages of deprotection, coupling, and, again, deprotection, were carried out with DMF (3×1 min) and DCM (3×1 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

[0224] Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-Rink-MBHA-resin, wherein AA.sub.1 is L-Arg; AA.sub.2 is L-Arg; AA.sub.3 is L-Gln; AA.sub.4 is L-Met; AA.sub.5 is L-Arg; and AA.sub.6 is L-Glu.

[0225] 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 known in the state of the art in order to remove the Fmoc group. 3.19 g of Fmoc-L-Glu(OtBu)-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.

[0226] The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equiv); subsequently 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv) and subsequently 4.86 g of Fmoc-L-Arg(Pbf)-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.

[0227] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

[0228] Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-Rink-MBHA-resin, wherein AA.sub.1 is L-Met; AA.sub.2 is L-Arg; AA.sub.3 is L-Arg; AA.sub.4 is L-Glu; AA.sub.5 is L-Gln; and AA.sub.6 is L-Arg.

[0229] 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 known in the state of the art in order to remove the Fmoc group. 4.86 g of Fmoc-L-Arg(Pbf)-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.

[0230] The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equiv); subsequently 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv) and subsequently 2.78 g of Fmoc-L-Met-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.

[0231] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

[0232] Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-Rink-MBHA-resin, wherein AA.sub.1 is L-Arg; AA.sub.2 is L-Glu; AA.sub.3 is L-Gln; AA.sub.4 is L-Met; AA.sub.5 is L-Arg; and AA.sub.6 is L-Arg.

[0233] 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 known in the state of the art in order to remove the Fmoc group. 4.86 g of Fmoc-L-Arg(Pbf)-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.

[0234] The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equiv); subsequently 4.58 g of Fmoc-L-Gln(Trt)-OH (7.5 mmol; 3 equiv); subsequently 3.19 g Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv) and subsequently 4.86 g of Fmoc-L-Arg(Pbf)-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.

[0235] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

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

[0236] The N-terminal Fmoc group of the peptidyl resins obtained in Example 1 was deprotected with 20% (volume/volume, hereinafter v/v) piperidine in DMF (1×1 min+2×10 min) (Lloyd Williams P. et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton (Fla., USA)). The peptidyl resins were washed with DMF (5×1 min), DCM (4×1 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

[0237] 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 (5×1 min), DCM (4×1 min), and were dried under vacuum.

Example 4. Cleavage Process from the Polymeric Support of the Peptidyl Resins obtained in Accordance with Example 2 and 3

[0238] Weights were normalized. 200 mg of the dried peptidyl resin obtained in any of Examples 2 or 3 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 were redissolved in 50% (v/v) MeCN in H.sub.2O and lyophilized.

Example 5. Characterization of the Peptides Synthesized and Prepared in Accordance with Example 4

[0239] HPLC analysis of the peptides obtained in accordance with example 4 was carried out with a Shimadzu equipment (Kyoto, Japan) using a reverse-phase column (150×4.6 mm, XBridge Peptide BEH C18, 3.5 μm, Waters, USA) in gradients of MeCN (+0.036% (v/v) TFA) in H.sub.2O (+0.045% (v/v) 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. Results obtained demonstrated that peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2 were correctly and effectively synthesized.

Example 6. Preparation of a Cosmetic Facial Composition containing Ac-SEQ ID NO: 1-NH.SUB.2

[0240] 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-00003 TABLE 1 Cosmetic facial composition prepared in Example 6. % in weight Phase Ingredient (g/100 g) A Water q. s. (quantum satis) 100 A Disodium EDTA 0.15 A Magnesium sulfate 1.5 A Glycerin 2.5 B Caprylic/Capric triglyceride 8 B Isononyl/lsononanoate 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, 1.7 Dimethicone/Vinyl dimethicone crosspolymer): 0.3 Dimethicone Dimethicone/Vinyl dimethicone crosspolymer 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 (Aqua) 0.9445 Glycerin 0.05 Caprylyl glycol 0.005 Ac-SEQ ID NO: 1-NH.sub.2 0.0005

Example 7. Analysis of IL33 expression modulation in HEKa (Human Epidermal Keratinocytes, adult) and HDFa (Human Dermal Fibroblasts, adult) cells

[0241] Peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2 were analysed for its capacity to modulate expression of IL33 in HEKa cells (all three peptides) and in HDFa (only analysed for peptide Ac-SEQ ID NO: 1-NH.sub.2).

[0242] The peptides used in this example were prepared in accordance with examples 1 to 5.

[0243] A stock solution of the peptide was prepared in water at 1 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.

[0244] Untreated cells were used as basal or negative control samples.

[0245] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HDFa and HEKa cells were seeded in duplicate (n=2) in 6-well plates at a density of 4×10.sup.5 cells/well and maintained at standard culture conditions (106 Medium supplemented with 1% (v/v) LSGS for HDFa cells; Medium Epilife supplemented with 1% (v/v) EDGS and 1% (v/v) penicillin/streptomycin for HEKa cells; 37° C., 95% room humidity, 5% CO.sub.2) for 24 hours. Then, cells were treated with peptide Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 or Ac-SEQ ID NO: 3-NH.sub.2 at the concentration of 0.01 mg/mL for 24 hours. Untreated cells were used as basal or negative control.

[0246] Cells were finally lysed, and replicates were pooled together for RNA extraction using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs (complementary deoxyribonucleic acids) by reverse transcription using a commercial kit High capacity cDNA reverse transcription kit (Applied Biosystems) which served as templates for amplification. RT-qPCR was performed with the appropriate TaqMan assay probes for IL33 (plus GAPDH—Glyceraldehyde 3-phosphate dehydrogenase—that was used as housekeeping gene) and 2×gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension) (Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

[0247] The obtained data was analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. Both samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

[0248] The steps for analysis included:

[0249] 1. Calculate the average Ct for each condition

[0250] 2. Calculate the ΔCT test sample and the OCT untreated sample

[0251] 3. Calculate the ΔΔCT: ΔΔCT=ΔCT test sample−ΔCT untreated sample

[0252] 4. Obtain ratio by 2.sup.−ΔΔCT

[0253] In this assay, a clear downregulation of the expression of IL33 gene was observed in HEKa cells for all the peptides tested: [0254] Ac-SEQ ID NO: 1-NH.sub.2: −4.58 fold change in treated cells with regard to basal control. [0255] Ac-SEQ ID NO: 2-NH.sub.2: −3.77 fold change in treated cells with regard to basal control. [0256] Ac-SEQ ID NO: 3-NH.sub.2: −3.93 fold change in treated cells with regard to basal control.

[0257] In addition, for peptide Ac-SEQ ID NO: 1-NH.sub.2 a downregulation of the expression of IL33 was also observed in HDFa cells (−1.22 fold change treated cells with regard to basal control).

[0258] The results of the present example demonstrate the usefulness of the peptides of the present invention to downregulate the expression of IL33, thus, as explained above, providing for an improvement mainly in inflammation, and additionally in the skin barrier, a decrease in angiogenesis and a decrease in pigmentation. Therefore, the results of this example demonstrate the usefulness of the peptides of the present invention to prevent, reduce and or eliminate signs of skin aging and/or skin imperfections, more preferably, eyebags, eyelid imperfections, wrinkles in the surrounding of the eyes and dark circles (periorbital hyperchromia).

Example 8. Analysis of the Production of Protein IL-33 in HDFa Cells

[0259] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to modulate IL-33 production in adult human dermal fibroblasts.

[0260] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.

[0261] Adult human dermal fibroblasts (HDFa) were seeded on 24-well plate at concentration of 1×10.sup.5 cells/mL in their corresponding growth media and incubated for 24 hours in standard culture conditions; at that time, three different concentrations (0.005, 0.01 and 0.05 mg/mL) of the Ac-SEQ ID NO: 1-NH.sub.2 were added in presence of IL-33-induction stimulus (IFNγ, 300 U/mL). Cells without stimuli were also included as basal group. Positive control group was cells treated with 300 U/mL of IFNγ, but without being treated with a peptide of the present invention. 24 hours later, conditioned media was collected, and cells processed for protein extraction.

[0262] IL-33 levels in the cell lysates were analysed with a commercial ELISA kit (DuoSet ELISA. R&D). Absorbances were measured with an automated spectrophotometer plate-reader.

[0263] Results obtained in this example appear summarized in FIG. 1, wherein it can be seen that IFNy (300 U/mL) induces an increase in the production of protein IL-33 in HDFa cells and that peptide Ac-SEQ ID NO: 1-NH.sub.2 reduces the IL-33 protein levels produced by HDFa cells exposed to IFNγ (300 U/mL) in a dose-dependent manner (this is, a greater reduction of IL-33 protein levels is observed as concentration of peptide Ac-SEQ ID NO: 1-NH.sub.2 is increased).

[0264] The results of the present example demonstrate the usefulness of the peptides of the present invention to reduce inflammation through the downregulation of the expression of IL-33, thus, as explained above, providing for an improvement in the skin barrier, a decrease in angiogenesis and a decrease in pigmentation. Therefore, the results of this example demonstrate the usefulness of the peptides of the present invention to prevent, reduce and or eliminate signs of skin aging and/or skin imperfections, more preferably, eyebags, eyelid imperfections, wrinkles in the surrounding of the eyes and dark circles (periorbital hyperchromia).

Example 9. Analysis of the Gene Expression Modulation in Human Epidermal Keratinocytes, Adult (hereinafter, HEKa)

[0265] Peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2 were analysed for its capacity to modulate the expression of genes related epidermal cell cohesion (see table 2 for the analysed genes).

[0266] The peptides used in this example were prepared in accordance with examples 1 to 5.

[0267] A stock solution of the peptide was prepared in water at 1 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.

[0268] Untreated cells were used as basal or negative control samples.

[0269] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa cells were seeded in duplicate (n=2) in 6-well plates at a density of 4×10.sup.5 cells/well and maintained at standard culture conditions (Medium Epilife supplemented with 1% (v/v) EDGS and 1% (v/v) penicillin/streptomycin; 37° C., 95% room humidity, 5% CO.sub.2) for 24 hours.

[0270] Then, cells were treated with peptide Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 or Ac-SEQ ID NO: 3-NH.sub.2 at the concentration of 0.01 mg/mL for 24 hours. Untreated cells were used as basal or negative control.

[0271] Cells were finally lysed, and replicates were pooled together for RNA extraction using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs (complementary deoxyribonucleic acids) by reverse transcription using a commercial kit High capacity cDNA reverse transcription kit (Applied Biosystems) which served as templates for amplification. RT-qPCR was performed with the panel of appropriate TaqMan assay probes for genes shown in table 1 (plus GAPDH—Glyceraldehyde 3-phosphate dehydrogenase—that was used as housekeeping gene) and 2×gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension) (Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

TABLE-US-00004 TABLE 2 Genes analysed in example 9 (abbreviation and complete name) Abbreviation Gene JUP Junction Plakoglobin OCLN Occludin DSP Desmoplakin CLDN1 Claudin 1 CLDN4 Claudin 4 DSC2 Desmocollin 2 CDSN Corneodesmosin PCDH1 Protocadherin 1 TJP1 Tight Junction Protein 1 PKP2 Plakophilin 2 PPL Periplakin

[0272] The obtained data was analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. Both samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

[0273] The steps for analysis included:

[0274] 1. Calculate the average Ct for each condition

[0275] 2. Calculate the ΔCT test sample and the ΔCT untreated sample

[0276] 3. Calculate the ΔΔCT: ΔΔCT=OCT test sample−ΔCT untreated sample

[0277] 4. Obtain ratio by 2.sup.−ΔΔCT

[0278] The results of this assay appear also summarized in tables 3 to 5.

TABLE-US-00005 TABLE 3 Fold change between treated cells and basal control obtained in HEKa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 1-NH.sub.2 for 24 hours. Ac-SEQ ID NO: 1-NH.sub.2 Abbreviation (Fold change) JUP 1.42 OCLN 1.98 DSP 1.28 CLDN1 1.57 CLDN4 2.64 DSC2 1.23 CDSN 1.46 PCDH1 2.41 TJP1 1.25 PKP2 1.72 PPL 1.54

TABLE-US-00006 TABLE 4 Fold change between treated cells and basal control obtained in HEKa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 2-NH.sub.2 for 24 hours. Ac-SEQ ID NO: 2-NH.sub.2 Abbreviation (Fold change) OCLN 1.58 CLDN4 1.51 PCDH1 2.14 TJP1 1.34 PKP2 1.42

TABLE-US-00007 TABLE 5 Fold change between treated cells and basal control obtained in HEKa cells treated with 0.01 mg/mL . of Ac-SEQ ID NO: 3-NH.sub.2 for 24 hours Ac-SEQ ID NO: 3-NH.sub.2 Abbreviation (Fold change) JUP 1.23 OCLN 1.99 DSP 1.28 CLDN1 1.5 DSC1 1.41 CLDN4 1.90 DSC2 1.43 PCDH1 2.48 TJP1 1.43 PKP2 1.86 PPL 1.52

[0279] As can be readily derived from tables 3 to 5: [0280] Peptide Ac-SEQ ID NO: 1-NH.sub.2 clearly upregulated key genes related to tight junctions and barrier function: JUP, OCLN, DSP, CLDN1, CLDN4, DSC2, CDSN, PCDH1, TJP1, PKP2 and PPL. [0281] Peptide Ac-SEQ ID NO: 2-NH.sub.2 clearly upregulated key genes related to tight junctions and barrier function: OCLN, CLDN4, PCDH1, TJP1 and PKP2. [0282] Peptide Ac-SEQ ID NO: 3-NH.sub.2 clearly upregulated key genes related to tight junctions and barrier function: JUP, OCLN, DSP, CLDN1, DSC1, CLDN4, DSC2, PCDH1, TJP1, PKP2 and PPL.

[0283] Therefore, the peptides of the present invention (as exemplified by means of Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2) reduce, prevent and/or eliminate signs of skin aging and/or skin imperfections, more precisely, the results of this example demonstrate the usefulness of the peptides of the present invention to improve tight junctions between keratinocytes, improving cohesion between them and, hence, improving firmness of the skin and reducing wrinkles.

Example 10. Analysis of Gene Expression Modulation in Primary Human Dermal Fibroblasts, Adults

[0284] Peptides Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2 were analysed for its capacity to modulate expression of genes related with the production of collagen and the extracellular matrix (see table 6 for the analysed genes).

[0285] The peptides used in this example were prepared in accordance with examples 1 to 5.

[0286] A stock solution of the peptide was prepared in water at 1 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.

[0287] Untreated cells were used as basal or negative control samples.

[0288] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HDFa cells were seeded in duplicate (n=2) in 6-well plates at a density of 4×10.sup.5 cells/well and maintained at standard culture conditions (106 Medium supplemented with 1% (v/v) LSGS; 37° C., 95% room humidity, 5% CO.sub.2) for 24 hours. Then, cells were treated with peptide Ac-SEQ ID NO: 1-NH.sub.2 at the concentration of 0.01 mg/mL for 24 and 48 hours. Untreated cells were used as basal or negative control.

[0289] Cells were finally lysed, and replicates were pooled together for RNA extraction using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs (complementary deoxyribonucleic acids) by reverse transcription using a commercial kit High capacity cDNA reverse transcription kit (Applied Biosystems) which served as templates for amplification. RT-qPCR was performed with the panel of appropriate TaqMan assay probes for genes shown in table 5 (plus GAPDH—Glyceraldehyde 3-phosphate dehydrogenase—that was used as housekeeping gene) and 2×gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension) (Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

TABLE-US-00008 TABLE 6 Genes analysed in example 10 (abbreviation and complete name) Abbreviation Gene COL3A1 Collagen Type III Alpha 1 Chain MMP1 Matrix Metalloproteinase 1 MMP3 Matrix Metalloproteinase 3 FBN1 Fibrillin 1 TIMP2 Tissue Inhibitor of Metalloproteinases 2 LAMA1 Laminin Subunit Alpha 1

[0290] The obtained data was analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. Both samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

[0291] The steps for analysis included:

[0292] 1. Calculate the average Ct for each condition

[0293] 2. Calculate the ΔCT test sample and the ΔCT untreated sample

[0294] 3. Calculate the ΔΔCT: ΔΔCT=ΔCT test sample−ΔCT untreated sample

[0295] 4. Obtain ratio by 2.sup.−ΔΔCT

[0296] The results of this assay appear summarized in tables 7 to 10.

TABLE-US-00009 TABLE 7 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 1-NH.sub.2 for 24 hours. Abbreviation Fold change COL3A1 1.32 TIMP2 1.35 LAMA1 1.16

TABLE-US-00010 TABLE 8 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 1-NH.sub.2 for 48 hours. Abbreviation Fold change MMP1 −1.30 MMP3 −1.69

TABLE-US-00011 TABLE 9 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 2-NH.sub.2 for 24 hours. Abbreviation Fold change MMP1 −1.21 FBN1  1.20

TABLE-US-00012 TABLE 10 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 3-NH.sub.2 for 24 hours. Abbreviation Fold change MMP1 −1.46 MMP3 −1.30 FBN1  1.29

[0297] As can be readily derived from tables 7 and 8, for peptide Ac-SEQ ID NO: 1-NH.sub.2: [0298] At 24 hours: an upregulation of LAMA1, COL3A1 and TIM P2 was observed. [0299] At 48 hours: a downregulation of MMP1 and MMP3 was observed.

[0300] As can be readily derived from table 9, for peptide Ac-SEQ ID NO: 2-NH.sub.2: [0301] At 24 hours: an upregulation of FBN1 and a downregulation of MMP1 were observed.

[0302] As can be readily derived from table 10, for peptide Ac-SEQ ID NO: 3-NH.sub.2: [0303] At 24 hours: an upregulation of FBN1 and a downregulation of MMP1 and MMP3 were observed.

[0304] Therefore, the peptides of the present invention (as exemplified by means of Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2) showed a favorable regulation of genes with regard to the ECM synthesis and maintenance. Therefore, the peptides of the present invention are able to reduce, prevent and/or eliminate signs of skin aging and/or skin imperfections, more precisely, the results of this example demonstrate the usefulness of the peptides of the present invention to improve firmness of the skin and reduce wrinkles.

Example 11. Analysis of Tyrosinase Activity

[0305] 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 Ac-Arg-Arg-Gln-Met-Glu-Glu-NH.sub.2 (Ac-SEQ ID NO: 4-NH.sub.2) were analysed for their capacity to modulate tyrosinase activity.

[0306] The peptides used in this example were prepared in accordance with examples 1 to 5.

[0307] Briefly, different concentrations of Ac-SEQ ID NO: 1-NH.sub.2 (0.05, 0.25, 0.5 and 2.5 mg/mL), Ac-SEQ ID NO: 2-NH.sub.2 (0.05, 0.25, 0.5 and 2.5 mg/mL), Ac-SEQ ID NO: 3-NH.sub.2 (0.05, 0.25, 0.5 and 2.5 mg/mL) and Ac-SEQ ID NO: 4-NH.sub.2 (0.005, 0.025, 0.05, 0.25, 0.5 and 2.5 mg/mL) and the positive control kojic acid (400 and 800 μM) were incubated with recombinant mushroom tyrosinase for 30 minutes before the addition of L-DOPA (L-3,4 dihidroxiphenilalanine, 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, Mass., USA), which is directly proportional to the amount of dopachrome in the reaction mixture.

[0308] 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.

[0309] Results obtained in this example appear summarized in FIG. 2 (2A to 2D).

[0310] As it can be directly derivable from said figure, peptide Ac-SEQ ID NO: 1-NH.sub.2 (FIG. 2A) inhibited tyrosinase in all the concentrations tested, but this effect increased in a dose-dependent from 0.25 mg/mL and onwards.

[0311] On its side, peptide Ac-SEQ ID NO: 2-NH.sub.2 (FIG. 2B) showed inhibition of tyrosinase activity in a dose-dependent manner from 0.05 mg/mL and onwards.

[0312] Peptide Ac-SEQ ID NO: 3-NH.sub.2 (FIG. 2C) showed a dose-dependent inhibition of tyrosinase activity at concentrations of 0.25 mg/mL and higher.

[0313] On its side, peptide Ac-SEQ ID NO: 4-NH.sub.2 (FIG. 2D) (a peptide not of the present invention) did not inhibit tyrosinase activity in any of the concentrations tested. On the contrary, from concentration 0.05 mg/mL and onwards it increased tyrosinase activity in a dose-dependent manner.

[0314] In the state of the art it is known that dark circles are caused, among others, due to the deposit of melanin and it is also established that inhibition of tyrosinase activity reduces melanin deposits (MI Ryung Roh, Kee Yang Chung: Infraorbital Dark Circles: Definition, Causes, and Treatment Options, Dermatological Surgery, 35:8:August 2009).

[0315] These results demonstrate the usefulness of the peptides of the present invention (as exemplified by means of Ac-SEQ ID NO: 1-NH.sub.2, Ac-SEQ ID NO: 2-NH.sub.2 and Ac-SEQ ID NO: 3-NH.sub.2) for the treatment and/or prevention of hyperpigmentation, preferably periorbital hyperchromia in eyes, but not of similar peptides (Ac-SEQ ID NO: 4-NH.sub.2) which instead of inhibiting tyrosinase activity, it maintained or increased said activity.

Example 12. Effect of Peptide Ac-SEQ ID NO: 1-NH.SUB.2 .on Angiogenesis

[0316] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to modulate blood vessel formation in vitro.

[0317] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.

[0318] In this case, a tube formation assay was performed using Human umbilical vein endothelial cells (HUVECs). Briefly, 40 μL of geltrex were added to the wells of a 96-well plate and it was left solidifying for 30 minutes. Afterwards 8000 cells were applied to each well together with: [0319] Complete medium (basal control). [0320] Complete medium+20 μM Suramin (inhibitor of tube formation). [0321] Complete medium+10 ng/mL IL-33. [0322] Complete medium+10 ng/mL IL-33+different concentrations of peptide Ac-SEQ ID NO: 1-NH.sub.2 (more precisely, 0.01 mg/mL, 0.05 mg/mL or 0.1 mg/mL).

[0323] After 16 hours of incubation cells were visualized by means of microscopy and photos were taken at 10x of each of the wells. For each of the wells, the number of meshes and the total area of the meshes was analysed as an indication of vessel formation (this is, of angiogenesis).

[0324] Results appear summarized in FIG. 3.

[0325] As it is readily derivable from said figure, peptide Ac-SEQ ID NO: 1-NH.sub.2 was able to reverse the vessel formation or angiogenesis induced by IL-33, at all the concentrations tested, both, with regard to the number of meshes and with regard to the total area of meshes.

[0326] Therefore, the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, are able to inhibit and/or prevent angiogenesis and, hence, they are useful for the treatment of cosmetic traits related with angiogenesis or excessive blood vessel formation, preferably eyebags and periorbital hyperchromia.

Example 13. Analysis of the Ability of the Peptides of the Present Invention to Protect against Lipid Peroxidation

[0327] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to protect from lipid peroxidation by means of a Thiobarbituric Acid Reactive Substances (hereinafter, TBARS) assay.

[0328] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.

[0329] Briefly, Trolox (positive control), 0.01, 0.05, 0.1 and 0.5 mg/mL of peptide Ac-SEQ ID NO: 1-NH.sub.2 in presence of small unilamellar vesicles of egg yolk phosphatidylcholine (EYPC), a substrate for lipid peroxidation were incubated for 2 hours at 37° C. in the presence of thiobarbituric acid (TBA). A fluorimetric method was used to evaluate the % of lipid peroxidation compared to the negative control (sample only with small unilamellar vesicles of egg yolk phosphatidylcholine).

[0330] The results obtained in this example, appear summarized in FIG. 4.

[0331] As it can be directly derived from said figure, the peptide of the present invention shows anti-oxidant activity at all the concentrations tested.

[0332] Therefore, the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, were able to protect from lipid peroxidation.

[0333] Analysis of the peptide capacity to protect against peroxidation was also compared with melatonin, since melatonin is also a potent antioxidant.

[0334] Tests, controls, procedures, materials and data analysis were the same as explained above.

[0335] FIG. 8 shows a graphic representation of the data obtained for anti-peroxidation activity in comparison with 0.1 mg/mL and 0.5 mg/mL of melatonin.

[0336] The data shows that the peptide of the present invention has a melatonin-like protecting effect against lipid peroxidation.

[0337] As it is known in the state of the art, lipid peroxidation is related with cellular damage and death due to alterations in the plasmatic membrane.

[0338] Therefore, the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, are useful in cosmetics for the prevention and/or treatment of ageing and in medicine for the prevention and/or treatment of diseases related with lipid peroxidation, preferably, atherosclerosis, Inflammatory Bowel Disease, Retinopathy of Prematurity, Borderline Personality Disorder, Asthma, Parkinson's disease and kidney damage.

Example 14. Analysis of the Ability of the Peptides of the Present Invention to Protect against Glycation

[0339] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to reduce formation of Advanced glycation end products (AGEs).

[0340] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.

[0341] Briefly, 0.01, 0.05, 0.1 and 0.5 mg/mL of peptide Ac-SEQ ID NO: 1-NH.sub.2 were mixed together with a glycation agent and its corresponding substrate. Samples were incubated for 72 hours at 37° C. A fluorimetric method was used to evaluate the % of protein glycation compared to the negative control (sample only with sugar substrate).

[0342] The results obtained in this example, appear summarized in FIG. 5.

[0343] As it can be directly derived from said figure, the peptide of the present invention was able to reverse almost completely the glycation produced by the glycation agent, at all the concentrations tested.

[0344] Therefore, the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, were able to protect from protein glycation.

[0345] In addition, the ability of the peptides of the present invention to protect from protein glycation was also tested ex vivo.

[0346] Briefly, human skin explants from a 40-year-old woman were kept in survival in BEM culture medium at 37° C. in a humid, 5% CO.sub.2 atmosphere. A cosmetic composition in accordance with Example 6 was prepared containing 1% (w/v Ac-SEQ ID NO: 1-NH.sub.2and without the peptide (placebo). These creams were applied topically on the basis of 2 μL per explant (2 mg/cm.sup.2) and spread using a small spatula on day 0 (DO), day 1, day 4, day 6 and day 8. The control explants did not receive any treatment except the renewal of culture medium. To induce glycation, a solution of Methylglyoxal (MG) was incorporated in the BEM culture medium at a final concentration of 500 μM on day 4, day 6 and day 8, on the concerned batches “MG” (all samples, except control untreated explants). Explants were collected at each timepoint and cut in two parts. Half was fixed in buffered formalin solution and half was frozen at −80° C.

[0347] After fixation for 24 hours in buffered formalin, the samples were dehydrated and impregnated in paraffin. 5-μm-thick sections were made using a Leica RM 2125 Minot-type microtome, and the sections were mounted on Superfrost® histological glass slides. Carboxy.methyl lysine (CML) immunostaining (CML is an advanced glycation end product (AGE) found on proteins and lipids as a result of oxidative stress and chemical glycation and is used as a marker of advanced glycation) was performed with a monoclonal anti-CML antibody diluted at 1:25 in PBS-BSA 0.3% (w/v) for 1 night at room temperature and revealed by VECTOR® VIP (Vector Laboratories), a substrate of peroxidase.

[0348] As can be observed in FIG. 6, peptide Ac-SEQ ID NO: 1-NH.sub.2 protected from MG-induced glycation after 9 days of culture, showing a significant decrease in CML signal (−73%) when compared with MG-exposed untreated explants. Placebo-treated explants did not show a significant decrease in CML staining.

[0349] Analysis of the peptide capacity to reduce formation of Advanced glycation end products (AGEs) (protection against glycation) was also compared with melatonin, since melatonin is also a potent antioxidant able to protect from glycation.

[0350] Tests, controls, procedures, materials and data analysis were the same as explained above.

[0351] FIG. 9 shows a graphic representation of the data obtained for anti-glycation activity compared with 0.1 mg/mL and 0.5 mg/mL of melatonin.

[0352] The data shows that the peptide of the present invention has a melatonin-like protecting effect against glycation.

[0353] As it is known in the state of the art, glycation is one of the endogenous aging mechanisms that occurs spontaneously with time, but also in a pathological manner during diabetes, renal failure, and inflammation. AGEs are highly accumulated in tissues and organs in numerous age-related degenerative diseases. These toxic adducts (glycotoxins) are implicated in cell dysfunction, especially in diabetic patients and older organisms. AGE formation and accumulation in diabetic patients results in vascular alterations leading to diabetic vasculopathy.

[0354] Glycation is also related with extracellular matrix damage (damage to collagen and elastic fibers), inflammation, oxidative stress and apoptosis, and, hence, it is related with ageing.

[0355] Therefore, the peptides of the present invention, as exemplified by Ac-SEQ ID NO: 1-NH.sub.2, are able to protect from protein glycation and, hence, are useful in cosmetics for the prevention and/or treatment of ageing and in medicine for the prevention and/or treatment of diseases related with glycation, preferably, diabetes (more preferably diabetic vasculopathy), renal failure, inflammation and age-related degenerative diseases.

Example 15. Analysis of Fibrillin-1 in Ex Vivo Samples

[0356] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to protect the dermal extracellular matrix structure from glycation.

[0357] Peptide Ac-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.

[0358] Briefly, human skin explants from a 40-year-old woman were kept in survival in BEM culture medium at 37° C. in a humid, 5% CO.sub.2 atmosphere. A placebo cream and a cream containing 1% Ac-SEQ ID NO: 1-NH.sub.2, prepared according to Example 6, were applied topically on the basis of 2 μL per explant (2mg/cm.sup.2) and spread using a small spatula on day 0 (DO), day 1, day 4, day 6 and day 8. The control explants did not receive any treatment except the renewal of culture medium. To induce glycation, a solution of Methylglyoxal (MG) was incorporated in the BEM culture medium at a final concentration of 500 μM on day 4, day 6 and day 8, on the concerned batches “MG” (all samples except control untreated samples). Explants were collected at each timepoint and cut in two parts. Half was fixed in buffered formalin solution and half was frozen at −80° C.

[0359] Fibrillin 1 immunostaining was performed on frozen sections with a monoclonal anti-fibrillin 1 antibody diluted at 1:500 in PBS, BSA 0.3% (w/v) and Tween 20 at 0.05% (v/v) for 1 h at room temperature, with a biotin/streptavidin amplifying system and revealed with FITC. The nuclei were counterstained with propidium iodide.

[0360] Data from FIG. 7 shows how exposure to MG induces a significant reduction of fibrillin 1 staining in the dermis when compared to the unexposed control at day 9. Peptide Ac-SEQ ID NO: 1-NH.sub.2 was able to protect from this damage, showing a significant increase in fibrillin 1 staining (86%) at day 9 when compared with the MG-exposed untreated control.

[0361] As it is known in the state of the art, fibrillin-1 is the major component of oxytalan fibers, that are found along the dermal-epidermal junctions revealing essential functions in maintaining the integrity and the mechanical proprieties of the skin (Oxlund et al., 1980. J Anat. 131: 611-620). In dermo-cosmetic research, fibrillin-1 is usually used as a bio-marker of the structural state of the extracellular matrix of the dermis.

[0362] Therefore, the peptides of the present invention (as exemplified by means of Ac-SEQ ID NO: 1-NH.sub.2) reduce, prevent and/or eliminate signs of skin aging and/or skin imperfections, more precisely, the results of this example demonstrate the usefulness of the peptides of the present invention maintain the integrity and the mechanical proprieties of the skin and, hence, improve firmness of the skin and reduce wrinkles.

Example 16. Analysis of the Expressions of Genes related with the Response to Sleep Deprivation

[0363] Peptide Ac-SEQ ID NO: 1-NH.sub.2was analysed for its capacity to modulate expression of genes related with the consequences of sleep deprivation (see table 11 for the analysed genes) in HDFa cells, HEKa cells and HUVECs.

[0364] The peptide used in this example was prepared in accordance with examples 1 to 5.

[0365] A stock solution of the peptide was prepared in water at 1 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.

[0366] Untreated cells were used as basal or negative control samples.

[0367] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa, HDFa or HUVEC cells were seeded in duplicate (n=2) in 6-well plates at a density of 4×10.sup.5 cells/well (2×10.sup.5 cells/well for HUVEC cells) and maintained at standard culture conditions (106 Medium supplemented with 1% (v/v) LSGS for HDFa cells; Medium Epilife supplemented with 1% (v/v) EDGS and 1% (v/v) penicillin/streptomycin for HEKa cells; Endothelial Cell Growth Medium for HUVEC cells; 37° C., 95% room humidity, 5% CO.sub.2) for 24 hours.

[0368] Then, cells were treated with peptide Ac-SEQ ID NO: 1-NH.sub.2 at the concentration of 0.01 mg/mL for 24 hours. Untreated cells were used as basal or negative control.

[0369] Cells were finally lysed, and replicates were pooled together for RNA extraction using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs (complementary deoxyribonucleic acids) by reverse transcription using a commercial kit High capacity cDNA reverse transcription kit (Applied Biosystems) which served as templates for amplification. RT-qPCR was performed with the panel of appropriate TaqMan assay probes for genes shown in table 10 (plus GAPDH—Glyceraldehyde 3-phosphate dehydrogenase—that was used as housekeeping gene) and 2×gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension) (Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

TABLE-US-00013 TABLE 11 Genes analysed in example 16 (abbreviation and complete name) Abbreviation Gene IL1RL1 Interleukin 1 Receptor Like 1 (IL33 receptor) MTNR1A Melatonin Receptor 1A SIGIRR Single Ig IL-1 related receptor (IL33 binding inhibitor) SLC2A1 Glucose Transporter Type 1 (GLUT1) CXCL8 Interleukin-8 NFKB1 Nuclear Factor Kappa B Subunit 1

[0370] The obtained data was analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. Both samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

[0371] The steps for analysis included:

[0372] 1. Calculate the average Ct for each condition

[0373] 2. Calculate the ΔCT test sample and the ΔCT untreated sample

[0374] 3. Calculate the ΔΔCT: ΔΔCT=ΔCT test sample−ΔCT untreated sample

[0375] 4. Obtain ratio by 2.sup.−ΔΔCT

[0376] The results of this assay appear also summarized in tables 12 to 14.

TABLE-US-00014 TABLE 12 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/ml of Ac-SEQ ID NO: 1-NH.sub.2 for 24 hours. Abbreviation Fold change IL1RL1 −1.26

TABLE-US-00015 TABLE 13 Fold change between treated cells and basal control obtained in HEKa cells treated with 0.01 mg/ml of Ac-SEQ ID NO: 1-NH.sub.2 for 24 hours. Abbreviation Fold change MTNR1A 2.11 SIGIRR 1.27 SLC2A1 −1.21 CXCL8 −1.33

TABLE-US-00016 TABLE 14 Fold change between treated cells and basal control obtained in HUVECs treated with 0.01 mg/ml of Ac-SEQ ID NO: 1-NH.sub.2 for 24 hours. Abbreviation Fold change NFKB1 −1.24

[0377] Therefore, the peptides of the present invention (as exemplified by means of Ac-SEQ ID NO: 1-NH.sub.2) reduce, prevent and/or eliminate signs of skin aging, and/or eliminate skin imperfections related with sleep deprivation. This is so because peptide Ac-SEQ ID NO: 1-NH.sub.2, besides its effects on IL-33 levels (ELISA) is able to modulate its signalling by inducing a downregulation of IL33 receptor (IL1RL1) and the IL33-related inhibitor (SIGIRR). Regulation of other inflammatory-related genes (CXCL8 and NFKB1) has also been observed.

[0378] Apart from this effect on inflammation, peptide Ac-SEQ ID NO: 1-NH.sub.2 is also able to downregulate the expression of glucose transporter SLC2A1 and upregulate the expression of melatonin receptor MNTR.sub.1, both involved in the physiological response to sleep deprivation.

Example 17. Analysis of Gene Expression Induced by the Treatment with Ac-SEQ ID NO: 4-NH.SUB.2.)

[0379] Gene modulation of peptide Ac-SEQ ID NO: 4-NH.sub.2 was analysed in this example (see table 15 for the analysed genes) in HDFa cells and HEKa cells.

[0380] The peptide used in this example was prepared in accordance with examples 1 to 5, making the required adaptations.

[0381] A stock solution of the peptide was prepared in water at 1 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.

[0382] Untreated cells were used as basal or negative control samples.

[0383] RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HEKa or HDFa cells were seeded in duplicate (n=2) in 6-well plates at a density of 4×10.sup.5 cells/well and maintained at standard culture conditions (106 Medium supplemented with 1% (v/v) LSGS for HDFa cells; Medium Epilife supplemented with 1% (v/v) EDGS and 1% (v/v) penicillin/streptomycin for HEKa cells; 37° C., 95% room humidity, 5% CO.sub.2) for 24 hours.

[0384] Then, cells were treated with peptide Ac-SEQ ID NO: 4-NH.sub.2 at the concentration of 0.01 mg/mL for 24 hours. Untreated cells were used as basal or negative control.

[0385] Cells were finally lysed, and replicates were pooled together for RNA extraction using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs (complementary deoxyribonucleic acids) by reverse transcription using a commercial kit High capacity cDNA reverse transcription kit (Applied Biosystems) which served as templates for amplification. RT-qPCR was performed with the panel of appropriate TaqMan assay probes for genes shown in table 10 (plus GAPDH—Glyceraldehyde 3-phosphate dehydrogenase—that was used as housekeeping gene) and 2×gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension) (Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., Patel, H. R. (2005) Basic principles of real-time quantitative PCR. Expert Rev. Mol. Diagn.; 5(2):209-19; and Jozefczuk, J. and Adjaye, J. (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol. 500; 99-109).

TABLE-US-00017 TABLE 15 Genes analysed in example 17 (abbreviation and complete name) Abbreviation Gene COL3A1 Collagen Type III Alpha 1 Chain TIMP2 Tissue Inhibitor of Metalloproteinases 2 IL1RL1 Interleukin 1 Receptor Like 1 (IL33 receptor) OCLN Occludin SIGIRR Single Ig IL-1 related receptor (IL33 binding inhibitor) CDSN Corneodesmosin SLC2A1 Glucose Transporter Type 1 (GLUT1) TJP1 Tight Junction Protein 1 IL33 Interleukin-33

[0386] The obtained data was analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. Both samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

[0387] The steps for analysis included:

[0388] 1. Calculate the average Ct for each condition

[0389] 2. Calculate the ΔCT test sample and the ΔCT untreated sample

[0390] 3. Calculate the ΔΔCT: ΔΔCT=ΔCT test sample−ΔCT untreated sample

[0391] 4. Obtain ratio by 2.sup.−ΔΔCT

[0392] The results of this assay appear also summarized in tables 16 and 17.

TABLE-US-00018 TABLE 16 Fold change between treated cells and basal control obtained in HDFa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 4-NH.sub.2 for 24 hours. Abbreviation Fold change COL3A1 −1.28 TIMP2 −1.26 IL1RL1 −1.12

TABLE-US-00019 TABLE 17 Fold change between treated cells and basal control obtained in HEKa cells treated with 0.01 mg/mL of Ac-SEQ ID NO: 4-NH.sub.2 for 24 hours. Abbreviation Gene OCLN −1.08 SIGIRR −1.49 CDSN −1.04 SLC2A1 −1.07 TJP1 −1.04 IL33 −1.01

[0393] Therefore, as can be directly derived from the results included in tables 16 and 17, peptide Ac-SEQ ID NO: 4-NH.sub.2, which as noted above is not a peptide of the present invention, but is very similar, does not show the favorable gene regulation seen for the peptides of the present invention, on the contrary, it shows an opposite gene regulation in a group of genes and a lack of effect in the expression in another group of genes.

[0394] Hence, Ac-SEQ ID NO:4-NH.sub.2, contrary to what has been demonstrated above for the peptides of the present invention: [0395] Showed a downregulation of COL3A1 and TIMP2. Therefore, Ac-SEQ ID NO: 4-NH.sub.2 showed an unfavourable regulation of genes with regard to the ECM synthesis and maintenance. Therefore, contrary to the peptides of the present invention, Ac-SEQ ID NO: 4-NH.sub.2 was not able to reduce, prevent and/or eliminate signs of skin aging and/or skin imperfections, more precisely, the results of this example demonstrate that said peptide is not able to improve firmness of the skin and reduce wrinkles. [0396] Showed a slightly downregulation or a lack of regulation of genes OCLN, CDSN and TJP1, this is, key genes related to tight junctions and barrier function. Therefore, Ac-SEQ ID NO: 4-NH.sub.2, contrary to what has been demonstrated for the peptides for the present invention, is not able to improve tight junctions between keratinocytes, and, hence, cannot improve cohesion between them and, hence, it cannot improve firmness of the skin and reduce wrinkles. [0397] Also, peptide Ac-SEQ ID NO: 4-NH.sub.2 showed a downregulation of SIGIRR and a slight downregulation or lack of regulation of SLC2A1. Therefore, the peptide was unable to revert the response to sleep deprivation. [0398] Finally, peptide Ac-SEQ ID NO: 4-NH.sub.2 was not able to regulate or alter the expression of genes IL33 and IL1RL1, therefore, being unable to modulate all the response associated with IL33 and explained above.

Example 18. Analysis of the Effects of Ac-SEQ ID NO:1-NH.SUB.2 .over the Expression of Emilin-1 Protein on Human Lymphatic Dermal Microvascular Endothelial Cells (HDLMVEC)

[0399] Immunofluorescence assays were performed in this example for emilin-1 protein, since its expression could be related with a better lymphatic vasculature and fluid clearance from tissues.

[0400] HDLMVEC were cultured on coverslips in Microvascular Endothelial Cell Growth (MECG) Medium at a density of 16.000 cell/cm.sup.2 and 37° C. in a humidified atmosphere with 5% CO.sub.2. When cell confluence was around 70-80%, cells were treated with 0.05 mg/mL Ac-SEQ ID NO:1-NH.sub.2 in MECG medium for 24 hours. Then, cells were washed twice with PBS, fixed with 4% formaldehyde and permeabilized with Triton 0.2%. Next, samples were blocked for 1 hour with PBS/BSA 5%, in order to avoid nonspecific antibody binding. Emilin-1 was stained with 1:50 Emilin-1 antibody in PBS/BSA 1%, during 1.5 hours at room temperature. After proper washing with PBS, actin was stained with 50 μg/mL Phalloidin during 1 hour at room temperature. Then, cells were washed with PBS and incubated with 5 μg/mL of the secondary antibody Goat anti-Rabbit IgG for 1 hour at room temperature. Finally, coverslips were mounted using Vectashield with DAPI, that stains nuclei, and samples were kept protected from light at 4° C. until microscopic images were acquired.

[0401] Microscopic images were acquired using 10×objective. Two replicates were stained for each condition and images of ten different fields of each coverslip were acquired using the same settings.

[0402] FIG. 10 shows images acquired with a fluorescence microscope for basal cells (A) and cells treated with 0.05 mg/mL Ac-SEQ ID NO:1-NH.sub.2 (B) (green: emilin-1, red: phalloidin, blue: nuclei).

[0403] Images were analyzed using Image J software. Shortly, threshold was adjusted to select emilin-1 staining and mean fluorescent was measured. The number of cells was counted using the DAPI staining. Emilin-1 mean fluorescent intensity was divided by the number of cells. Finally, all data was normalized as follows to obtain the % of emilin-1 compared to untreated cells:

[00001]$\%\mathrm{vs}\mathrm{Basal}=\frac{\mathrm{fluo}res\mathrm{cence}\mathrm{per}\mathrm{cell}\mathrm{in}\mathrm{treated}\mathrm{wells}}{fluoresc\mathrm{ence}\mathrm{per}\mathrm{cell}\mathrm{in}\mathrm{untreated}\mathrm{wells}}\times 100$

[0404] Statistical data analysis was performed using Student t-test by comparison of fluorescence from treated cells vs non-treated cells, *, p<0.05; **, p<0.01 and ***, p<0.001.

TABLE-US-00020 TABLE 18 Data of the analysis of results for HDLMVEC % p-value (two- p-value Mean ±SEM tailed) summary Basal 100.00 25.25 — — 0.05 mg/mL Ac-SEQ 170.88 21.23 0.0395 * ID NO: 1-NH.sub.2

[0405] The results show that Ac-SEQ ID NO:1-NH.sub.2 significantly increased the expression of emilin-1 more than 70% after 24 h of treatment, when compared with untreated cells. Given that emilin-1 plays an important role in lymphatic function, these results suggest a potential role of Ac-SEQ ID NO:1-NH.sub.2 in fluid clearance from tissues, with possible applications in cosmetics.

Example 19. Analysis of the Effects of Peptide Ac-SEQ ID NO:1-NH.SUB.2 .over the Expression of Integrin α9β1 Protein on Human Lymphatic Dermal Microvascular Endothelial Cells (HDLMVEC)

[0406] Immunofluorescence assays were performed in this example for integrin α9β1 (Itg9) protein since its expression is related with a better lymphatic vasculature and fluid clearance from tissues.

[0407] HDLMVEC were cultured on coverslips in Microvascular Endothelial Cell Growth (MECG) Medium at a density of 16.000 cell/cm.sup.2 and 37° C. in a humidified atmosphere with 5% CO.sub.2.

[0408] When cell confluence was around 70-80%, cells were treated with 0.05 mg/mL Ac-SEQ ID NO:1-NH.sub.2 in MECG medium for 24 hours. Then, cells were washed twice with PBS, fixed with 4% formaldehyde and permeabilized with Triton 0.2%. Next, samples were blocked for 1 hour with PBS/BSA 5%, in order to avoid nonspecific antibody binding.

[0409] Itg9 was stained with 1:50 anti-Itg9 antibody in PBS/BSA 1%, during 1.5 hours at room temperature. After proper washing with PBS, actin was stained with 50 μg/mL Phalloidin during 1 hour at room temperature. Then, cells were washed with PBS and incubated with 5 μg/mL of the secondary antibody Goat anti-Rabbit IgG for 1 hour at room temperature. Finally, coverslips were mounted using Vectashield with DAPI, that stains nuclei, and samples were kept protected from light at 4° C. until microscopic images were acquired.

[0410] Microscopic images were acquired using 10×objective. Two independent experiments were performed for each condition and images of at least thirteen different fields of each coverslip were acquired using the same settings.

[0411] FIG. 11 shows images acquired with a fluorescence microscope for basal cells (A) and integrin α9β1 expression on HDLMVEC after 24 h treatment with 0.05 mg/mL Ac-SEQ ID NO:1-NH.sub.2 (B) (green: Integrin α9β1, red: phalloidin, blue: nuclei).

[0412] Images were analyzed using Image J software. Shortly, threshold was adjusted to select Itg9 staining and mean fluorescent was measured. The number of cells was counted using the DAPI staining. Itg9 mean fluorescent intensity was divided by the number of cells.

[0413] Data normalization and statistic was performed as previously explained.

TABLE-US-00021 TABLE 18 Data of the analysis of results shown in FIG. 11 for HDLMVEC % p-value p-value Mean ±SEM (two-tailed) summary Basal 100.00 13.92 — — 0.05 mg/mL Ac-SEQ 153.56 22.10 0.0463 * ID NO: 1-NH.sub.2

[0414] The results show that peptide Ac-SEQ ID NO:1-NH.sub.2 significantly increases the expression of Itg9 on primary human lymphatic dermal microvascular endothelial cells after 24 hours of treatment, more than a 50% when compared with the untreated cells. Given that Itg9 plays an important role in lymphatic function, these results suggest a potential role of Ac-SEQ ID NO:1-NH.sub.2 in fluid clearance from tissues, with possible applications in cosmetics.

Example 20. Analysis of the Effect of Peptide Ac-SEQ ID NO:1-NH.SUB.2 .and Melatonin on the Expression of Innate Antimicrobial Immunity related Genes in Primary Human Epidermal Keratinocytes (HEKa) Isolated from Human Skin

[0415] Tests, controls, procedures, materials and data analysis were the same as explained in examples 16-17.

[0416] The potential of peptide Ac-SEQ ID NO:1-NH.sub.2 and melatonin to modulate the expression of genes involved in virus immune response in Human Epidermal Keratinocytes was evaluated by RT-qPCR. Results show that for HEKa cells, a 6 h-treatment with 0.01 mg/mL of Ac-SEQ ID NO:1-NH.sub.2 peptide is able to induce a strong upregulation of the key genes IRF3, DEFB4A, S100A7 and MX1/MXA in HEKa. Other genes as IRF1, DEFB1, OAS2, ISG15 were also upregulated upon treatment with D18ChAH. Regarding the effect of melatonin in HEKa cells, after 6 h-treatment at 0.01 mg/mL, genes DEFB1, DEFB4A, S100A7, OAS2, MX1 were upregulated, but less compared to the effect of Ac-SEQ ID NO:1-NH.sub.2 (FIG. 12, Table 20).

[0417] FIG. 12 shows the regulation of genes related with antimicrobial innate immune response by 0.01 mg/mL of peptide Ac-SEQ ID NO:1-NH.sub.2 or 0.01 mg/mL of melatonin in HEKa cells after 6 h of treatment.

TABLE-US-00022 TABLE 20 Selection of genes involved in host viral response in epidermal keratinocytes ID Gene IRF1 Interferon regulatory factor 1 IRF3 Interferon regulatory factor 3 DEFB1 Defensin beta 1 DEFB4A Defensin beta 4A S100A7 S100 calcium binding protein A 7 OAS2 2′-5′-oligoadenylate synthetase 2 MX1 MX dynamin like GTPase 1 ISG15 ISG15 ubiquitin like modifier

TABLE-US-00023 TABLE 21 Normalized expression. Mean normalized expression, % SEM and fold change vs. basal for each gene analyzed in Human Epidermal Keratinocytes treated for 6 h with 0.01 mg/mL of Ac-SEQ ID NO: 1-NH.sub.2 or melatonin. Fold change vs. Basal IRF1 0.01 mg/mL Melatonin 1.05 IRF1 0.01 mg/mL Ac-SEQ 1.25 ID NO: 1-NH.sub.2 IRF3 0.01 mg/mL Melatonin 1.13 IRF3 0.01 mg/mL Ac-SEQ 1.54 ID NO: 1-NH.sub.2 DEFB1 0.01 mg/mL Melatonin 1.24 DEFB1 0.01 mg/mL Ac-SEQ 1.31 ID NO: 1-NH.sub.2 DEFB4A 0.01 mg/mL 1.35 Melatonin DEFB4A 0.01 mg/mL Ac-SEQ 1.58 ID NO: 1-NH.sub.2 S100A7 0.01 mg/mL 1.31 Melatonin S100A7 0.01 mg/mL Ac-SEQ 2.04 ID NO: 1-NH.sub.2 OAS2 0.01 mg/mL Melatonin 1.34 OAS2 0.01 mg/mL Ac-SEQ ID 1.39 NO: 1-NH.sub.2 MX1 0.01 mg/mL Melatonin 1.20 MX1 0.01 mg/mL Ac-SEQ ID 1.93 NO: 1-NH.sub.2 ISG15 0.01 mg/mL Melatonin 0.98 ISG15 0.01 mg/mL Ac-SEQ ID 1.38 NO: 1-NH.sub.2

[0418] Peptide Ac-SEQ ID NO:1-NH.sub.2 modulates gene expression in HEKa after 6 h-treatment at 0.01 mg/mL, resulting into a modulation of genes involved in the innate antimicrobial immune response in epidermal keratinocytes. Concretely, Ac-SEQ ID NO:1-NH.sub.2 induced a strong upregulation of IRF3, DEFB4A, S100A7 and MX1. Moreover, treatment with Ac-SEQ ID NO:1-NH.sub.2 upregulated key genes IRF1, DEFB1, OAS2 and ISG16 as well. Treatment of HEKa with melatonin upregulated genes DEFB1, DEFB4A, S100A7, OAS2 and MX1, but to a lesser extent compared to the effect of peptide Ac-SEQ ID NO:1-NH.sub.2.

[0419] All these results strongly suggest that peptide Ac-SEQ ID NO:1-NH.sub.2 could be a modulator of genes involved in host antimicrobial and antiviral innate immune response, therefore being capable of increasing immune protection of the epidermal barrier against invasion and infection. Moreover, peptide Ac-SEQ ID NO:1-NH.sub.2 seems to display a melatonin-like effect when it comes to gene expression modulation. Consequently, all of this makes Ac-SEQ ID NO:1-NH.sub.2 a compound of interest in the cosmetics industry.

Example 21. In Vivo Results for Eyebags Volume

[0420] A cosmetic facial composition comprising the peptide was compared against a placebo composition in in vivo reduction of eyebags volume.

[0421] The same composition of Example 6 was used for this example, but with 3% in weight (g/100 g) of Commercial formulation of peptide Ac-SEQ ID NO: 1-NH.sub.2. The placebo composition does not comprise the peptide.

[0422] FIG. 13 shows a graphic representation of the reduction of eyebags volume with a composition comprising the peptide and a placebo composition.

[0423] The results show a reduction of the eyebags volume of 5% after 7 days and a reduction of 7% after 14 days with the composition comprising the peptide in comparison with the reduction of volume observed with the placebo composition.