PEPTIDES AND COMPOSITIONS FOR USE IN COSMETICS
20220142899 · 2022-05-12
Inventors
- Ariadna GRAU-CAMPISTANY (Barcelona, ES)
- Silvia PASTOR (Alicante, ES)
- Patricia CARULLA (Barcelona, ES)
- Juan Carlos ESCUDERO (Barcelona, ES)
- Julia A BORAS (Begas (Barcelona), ES)
- Marco Jan KLEIN (L'Hospitalet de Llobregat (Barcelona), ES)
Cpc classification
C07K5/1024
CHEMISTRY; METALLURGY
International classification
Abstract
A family of peptides which are able to increase synthesis of homeodomain protein Mohawk and useful as anti-aging agents and as rejuvenating agents.
Claims
1. A peptide capable of increasing Mohawk, acceptable isomers, salts, solvates and/or derivatives and/or mixtures thereof.
2. 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-R.sub.2 (I) cosmetically acceptable isomers, salts, solvates and/or derivatives and mixtures thereof, wherein: AA.sub.1 is His; AA.sub.2 is selected from the group of amino acids with an aromatic side-chain; AA.sub.3 is selected from Lys or Arg; AA.sub.4 is selected from the group of amino acids with an aliphatic non-polar side-chain 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.
3. The peptide in accordance with claim 2, wherein AA.sub.1 is His; AA.sub.2 is selected from the group of Phe, Tyr and Trp; AA.sub.3 is selected from Lys or Arg; AA.sub.4 is selected from the group of Ala, Val, Leu and Ile.
4. The peptide in accordance with claim 2, wherein the peptide of formula (I) is:
R.sub.1-His-Tyr-Arg-Ala-R.sub.2(R.sub.1-SEQ ID NO:1-R.sub.2).
5. The peptide in accordance with claim 2, wherein R.sub.1 is Pal or Ac.
6. The peptide in accordance with claim 2, wherein R.sub.2 is H or NH.sub.2.
7. The peptide in accordance with claim 2, wherein R.sub.1 is Pal and R.sub.2 is NH.sub.2.
8. The peptide in accordance with claim 4, wherein the peptide of formula (1) is:
Pal-His-Tyr-Arg-Ala-NH.sub.2(Pal-SEQ ID NO:1-NH.sub.2).
9. A cosmetic composition comprising the peptide in accordance with claim 1.
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. A method for rejuvenating skin, reducing signs of skin aging and/or eliminating skin imperfections comprising applying the cosmetic according to claim 9 to a subject in need thereof.
16. The method of claim 15, wherein the signs of skin aging are wrinkles, roughness and/or sagginess.
17. The method of claim 15, wherein the method provide for skin firming, body sculpturing, facial repositioning, skin tightening and/or pore refining in the subject in need thereof.
Description
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EXAMPLES
[0171] Abbreviations:
[0172] 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.
[0173] Ac, acetyl; Ala, alanine; Arg, arginine; C-terminal, carboxy-terminal; DCM, dichloromethane; DIEA, N, N′-diisopropylethylamine; DIPCDI, N, N′-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; EDGS, EpiLife Defined Growth Supplement; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; His, histidine; HOBt, 1-hydroxybenzotriazole; hOSEC, human organotypical skin explant culture; HPLC, high performance liquid chromatography; HRP, Horseradish peroxidase; Ile, Isoleucine; LSGS, Low Serum Growth Supplement; MBHA, p-methylbenzhydrylamine; Leu, leucine; Lys, lysine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; Met, Methionine; N-terminal, amino-terminal; Pal, palmitoyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Phe, Phenylalanine; rpm, revolutions per minute; RT, room temperature; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, 3,3′,5,5′-Tetramethylbenzidine; Trt, triphenylmethyl or trityl; Trp, Tryptophan; Tyr, Tyrosine; Val, Valine.
[0174] 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, (1997), Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, 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
[0175] Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Rink-MBHA-resin, wherein AA.sub.1 is L-His; AA.sub.2 is L-Tyr; AA.sub.3 is L-Arg; and AA.sub.4 is L-Ala.
[0176] 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. 2.33 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.
[0177] 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 4.87 g of Fmoc-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 3.45 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv); and subsequently 4.65 g of Fmoc-L-His(Trt)-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.
[0178] After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.
[0179] Using the synthesis procedure mentioned above, the following sequence was synthesized:
His-Tyr-Arg-Ala(SEQ ID NO:1).
Example 2. Removal of Fmoc N-Terminal Protective Group of the Peptides Synthesized in Accordance with Example 1
[0180] The N-terminal Fmoc group of the peptidyl resins 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 .Palmitoyl Group onto the Peptidyl Resins Obtained in Accordance with Example 2
[0181] 1 mmol (1 equiv) of the peptidyl resins obtained in accordance with Example 2 was treated with 10 equivalents of hexadecenoic acid (palmitic acid) in the presence of 10 equivalents of DIEA and 10 equivalents of HOBt 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
[0182] 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
[0183] 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 peptide Pal-His-Tyr-Arg-Ala-NH.sub.2 (Pal-SEQ ID NO: 1-NH.sub.2) was correctly and effectively synthesized.
Example 6. Analysis of Cytotoxicity of the Peptide Pal-SEQ ID NO: 1-NH.SUB.2 .in HEKa and HDFa Cells
[0184] The cytotoxicity of peptide Pal-SEQ ID NO: 1-NH.sub.2 was analysed by means of viability assay in HEKa (Human Epidermal Keratinocytes, adult) and HDFa (Human Dermal Fibroblasts, adult) cells.
[0185] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0186] Cytotoxicity was assessed by means of the MTT assay. Briefly HEKa and HDFa cells were seeded in 96-well plates at a concentration of 1×10.sup.5 cells/mL and incubated in Epilife Medium supplemented with 1% (v/v) EDGS and 1% (v/v) of Penicillin/Streptomycin (for HEKa cells) and Medium 106 supplemented with 2% (v/v) of Low Serum Growth Supplement (LSGS) (for HDFa cells) for 24 h at 37° C., 5% CO.sub.2 and saturated humidity. Cells were then treated with different concentrations of peptide Pal-SEQ ID NO: 1-NH.sub.2 (0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, and 0.1 mg/mL for HEKa cells; and 0.001 mg/mL, 0.005 mg/mL and 0.01 mg/mL for HDFa cells) in triplicates, for MTT evaluation. After 24 h of treatment with the peptide at the corresponding concentration, 10 μL of the yellow tetrazolium (MTT) were added to each well and the cells were incubated at 37° C. for additional 4 h. After incubating with MTT, the medium of each well was aspirated and 150 μL of dimethyl sulfoxide (hereinafter DMSO) (100%) were added in order to solubilise the formazan crystals formed. The plates were placed on a shaker for 5 minutes for complete solubilisation of the crystals and then the absorbance at 450 nm of each well was determined using the microplate reader Multiskan FC, which is directly proportional to the number of living cells in culture. Absorbance values were normalized using the data obtained for non-treated cells (basal state).
[0187] Results appear summarized in
[0188] As can be directly derived from said figures, the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) did not alter viability of HEKa and HDFa cells at the tested concentrations and, hence, are not cytotoxic.
Example 7. Analysis Gene Expression Modulation in Primary Human Dermal Fibroblasts
[0189] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to modulate expression of genes related with the production of collagen and the extracellular matrix (see table 1 for the analysed genes).
[0190] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0191] A stock solution of the peptide was prepared in DMSO at 12.5 mg/mL. Working solution was freshly prepared at the specified concentration from stock solution in the corresponding supplemented medium.
[0192] Untreated cells were used as negative control samples.
[0193] 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 Pal-SEQ ID NO: 1-NH.sub.2 at the concentration of 0.005 mg/mL for additional 6 hours (or 24 hours, for COL13A1). Untreated cells were used as basal control.
[0194] 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-00001 TABLE 1 Genes analysed in example 7. Abbreviation Gene TGFB1 Transforming growth factor beta-1 FN1 Fibronectin 1 LOXL3 Lysyl oxidase like 3 LOXL2 Lysyl oxidase like 2 HSP47 Serpin H1 COL3A1 Collagen Type III Alpha 1 Chain COL4A5 Collagen Type IV Alpha 5 Chain COL5A1 Collagen Type V Alpha 1 Chain COL6A1 Collagen Type VI Alpha 1 Chain COL7A1 Collagen Type VII Alpha 1 Chain COL12A1 Collagen Type XII Alpha 1 Chain COL14A1 Collagen Type XIV Alpha 1 Chain COL13A1 Collagen Type XIII Alpha 1 Chain MKX Mohawk homeobox ZEB2 Zinc Finger E-Box Binding Homeobox 2 MMP1 Matrix metalloproteinase-1 MMP3 Matrix metalloproteinase-3
[0195] 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).
[0196] The steps for analysis included:
[0197] 1. Calculate the average Ct for each condition
[0198] 2. Calculate the ΔCT test sample and the ΔCT untreated sample
[0199] 3. Calculate the ΔΔCT: ΔΔCT=ΔCT test sample—ΔCT untreated sample
[0200] 4. Obtain ratio by 2.sup.−ΔΔCT
[0201] The results of this assay appear summarized in
[0202] As can be readily derived from said figures, peptide Pal-SEQ ID NO: 1-NH.sub.2: [0203] After 6 hours of treatment: downregulates genes MMP1 and MMP3, which, as it is widely known, degrade extracellular matrix proteins. [0204] After 6 hours of treatment: upregulates several key genes in collagen architecture and extracellular architecture: COL3A1, COL4A5, COL5A1, COL6A1, COL7A1, COL12A1, COL14A1, COL13A1 (after 24 hours of treatment), MKX y ZEB2. [0205] Upregulates several collagen crosslinking-related genes after 6 hours of treatment: TGFB1, FN1, LOXL3, LOXL2 and HSP47.
[0206] Therefore, the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) avoid or prevent extracellular matrix degradation while contributing to an increases synthesis and cross-linking of collagen, showing, hence, an antiaging activity and rejuvenating activity.
Example 8. Analysis of Collagen VI Synthesis in Human Dermal Fibroblasts
[0207] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to induce synthesis collagen VI in human dermal fibroblasts.
[0208] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0209] In this assay, it was evaluated in vitro the capability of the tested peptide to increase collagen VI synthesis in human dermal fibroblasts. This evaluation was carried out by determination of collagen VI amount after cell treatment with Pal-SEQ ID NO: 1-NH.sub.2.
[0210] Experimental protocol provided for the following experimental groups: [0211] negative control: cell culture treated only with solubilisation vehicle (DMSO); [0212] cell culture treated with peptide Pal-SEQ ID NO: 1-NH.sub.2 at three concentrations (0.01 mg/mL, 0.005 mg/mL and 0.001 mg/mL).
[0213] A stock solution of the peptide in DMSO was prepared and then three serial dilutions at 0.01 mg/mL, 0.005 mg/mL and 0.001 mg/mL in cell culture medium were prepared.
[0214] The biological model used in this case consisted of normal human dermal fibroblasts. Cells were seeded in 96-well plate at 1×10.sup.4 cells/well and maintained for 24 h at standard culture conditions (37° C., 95% room humidity, 5% CO.sub.2).
[0215] After 24 h incubation, medium was removed and new medium containing the treatment (DMSO or Pal-SEQ ID NO: 1-NH.sub.2) was added to the wells. Sample treatment lasted 48 hours and at the end of treatment cell culture media were collected. Cells treated with DMSO were used as negative control.
[0216] For the test execution, cell culture of human dermal fibroblasts were treated with Pal-SEQ ID NO: 1-NH.sub.2 at the three concentrations noted above.
[0217] After a 48-hour treatment the amount of collagen VI produced and released by the cells (ex-novo collagen VI synthesis) was measured in cell culture medium by means of ELISA assay. The results were compared to those of the negative control (cells treated with DMSO). The treatments were performed in triplicate and in three different experimental sessions.
[0218] The determination of collagen VI synthesis was carried out by means of ELISA method. Commercial kits were used for this purpose. The test kit used Sandwich-ELISA method to detect the content of human collagen VI. The micro ELISA plate provided in the kit was pre-coated with an antibody specific to collagen VI. Standards or samples were added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then, a biotinylated detection antibody specific for collagen VI and Avidin-Horseradish Peroxidase (HRP) conjugate were added to each micro plate well successively and incubated. Free components were washed away. The substrate solution (TMB) was added to each well. Only those wells that contained collagen VI developed a blue color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color turned yellow. The optical density (OD) was measured by microplate reader at a wavelength of 450 nm. The OD value was proportional to the concentration of collagen VI.
[0219] The quantitative determination used a calibration curve made-up of known and growing concentrations of standard collagen VI. The % variation in collagen VI content between negative control (DMSO) and sample was calculated, which is a direct index of the efficacy of Pal-SEQ ID NO: 1-NH.sub.2 to increase collagen VI synthesis.
[0220] The results obtained in this experiment appear summarized in table 2 and in
TABLE-US-00002 TABLE 2 Mean results for the collagen VI content in example 8 for the different groups as well as percentage variation of said content with regard to the negative control group. Collagen VI content % variation with regard to Group (ng/mL) the negative control Negative control 11.06 ± 0.75 — cell culture treated with 15.52 ± 0.80 40% peptide Pal-SEQ ID NO: 1- NH.sub.2 at 0.01 mg/mL cell culture treated with 12.87 ± 0.97 16% peptide Pal-SEQ ID NO: 1- NH.sub.2 at 0.005 mg/mL cell culture treated with 12.22 ± 0.78 11% peptide Pal-SEQ ID NO: 1- NH.sub.2 at 0.001 mg/mL
[0221] As can be directly derivable from table 2 and
[0222] The above results demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) increase the synthesis of the protein collagen VI and, hence, contribute to improve extracellular matrix assembly. Therefore, said peptides can prevent or treat aging of the skin and/or the skin signs of said aging as, for example, wrinkles, roughness and/or sagginess.
[0223] In addition, the results of this example also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening.
Example 9. Analysis of Mohawk Synthesis in Human Dermal Fibroblasts
[0224] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to induce synthesis of Mohawk in human dermal fibroblasts.
[0225] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0226] This assay concerns the in vitro evaluation of the capability of peptide Pal-SEQ ID NO: 1-NH.sub.2 to increase Homeodomain protein Mohawk synthesis in human dermal fibroblasts. This evaluation was carried out by determination of Mohawk amount after cell treatment with said peptide.
[0227] The experimental protocol provided for the following experimental groups: [0228] untreated cell culture; [0229] cell culture treated only with solubilisation vehicle (DMSO) (negative control); [0230] cell culture treated with Pal-SEQ ID NO: 1-NH.sub.2 at three concentrations (0.01 mg/mL, 0.005 mg/mL and 0.001 mg/mL).
[0231] A stock solution of the peptide in DMSO was prepared and then three serial dilutions at 0.01 mg/mL, 0.005 mg/mL and 0.001 mg/mL in cell culture medium were prepared.
[0232] The biological model used, as noted above, consists of normal human dermal fibroblasts.
[0233] Cells were seeded in 96-well plate at 1×10.sup.4 cells/well and maintained for 24 h at standard culture conditions (37° C., 95% room humidity, 5% CO.sub.2). After 24 h incubation, medium was removed and new medium containing tested product was added to the wells.
[0234] Sample treatment lasted 24 h and 48 h and then cells were lysed in order to determine the concentration of intracellular Mohawk by means of ELISA assay. The results were compared to negative control (cells treated with DMSO).
[0235] The treatments were performed in triplicate in three different experimental sessions.
[0236] The determination of Mohawk synthesis, as noted above, was carried out by means of ELISA method. Commercial kits were used for this purpose. The test kit applied a two-site sandwich ELISA method to detect the content of human Homeodomain protein Mohawk. An antibody specific for Mohawk had been pre-coated onto a microplate. Standards and samples were pipetted into the wells and any Mohawk present was bound by the immobilized antibody. After removing any unbound substances, HRP-Conjugated Human Mohawk detection antibody was added to the wells. Following a wash to remove any unbound HRP reagent, a chromogen solution was added to the wells (substrate was TMB) and color developed in proportion to the amount of Mohawk bound in the initial step. The color development was stopped and the concentration was determined colorimetrically at 450 nm.
[0237] The quantitative determination used a calibration curve made-up of known and growing concentrations of standard Mohawk. The % variation in Mohawk content between negative control (DMSO) and the group treated with Pal-SEQ ID NO: 1-NH.sub.2 was calculated and was a direct index of the efficacy of peptide Pal-SEQ ID NO: 1-NH.sub.2 to increase Mohawk synthesis.
[0238] The results obtained appear summarized in table 3 and
TABLE-US-00003 TABLE 3 Mean results for the Mohawk content in example 9 for the different groups as well as percentage variation of said content with regard to the negative control group. % variation with Mohawk content regard to the Group Time (h) (pg/mL) negative control Untreated cells 24 137.40 ± 6.87 — Negative control 24 136.14 ± 5.44 — cell culture treated 24 194.37 ± 7.30 43% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.01 mg/mL cell culture treated 24 165.31 ± 7.24 21% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.005 mg/mL cell culture treated 24 148.19 ± 8.44 9% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.001 mg/mL Untreated cells 48 154.98 ± 9.91 — Negative control 48 157.20 ± 7.57 — cell culture treated 48 284.72 ± 13.51 81% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.01 mg/mL cell culture treated 48 237.03 ± 10.44 51% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.005 mg/mL cell culture treated 48 205.30 ± 11.53 31% with peptide Pal- SEQ ID NO: 1- NH.sub.2 at 0.001 mg/mL
[0239] As can be directly derivable from table 3 and
[0240] The above results demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) increase the synthesis of Mohawk and, hence, its potential for the prevention or treatment of aging of the skin and/or the skin signs related with aging as, for example, wrinkles, roughness and/or sagginess.
[0241] In addition, the results of this example also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening.
Example 10. Analysis of Collagen Cross-Linking
[0242] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was analysed for its capacity to induce or improve collagen cross-linking.
[0243] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0244] This assay concerns the in tubo evaluation of the capability of peptide Pal-SEQ ID NO: 1-NH.sub.2 to increase and accelerate collagen fibril formation/collagen cross-linking. This evaluation was carried out by determination of collagen fibril formation after collagen treatment with said peptide in solution (0.02 M acetic acid, 0.125 M NaCl and 1/10 phosphate buffered saline (pH=7.4)) at RT.
[0245] The experimental protocol provided for the following experimental groups: [0246] collagen solution treated with Pal-SEQ ID NO: 1-NH.sub.2 at two concentrations (0.02 or 0.04 mg/mL). [0247] collagen solution only treated with the same solution as the treatment groups mentioned above but without the peptide, and hence, with the same volume of DMSO as the samples containing the peptide (negative control);
[0248] A stock solution of the peptide in DMSO was prepared and then two serial dilutions were prepared so that the final concentration of the peptide in the collagen solution was 0.02 mg/mL or 0.04 mg/m L.
[0249] The collagen cross-linking and fibril formation was followed immediately after addition of collagen to the control (same solution as the treatment groups but without the peptide, and hence, with the same volume of DMSO as the samples containing the peptide) and the solution containing Pal-SEQ ID NO: 1-NH.sub.2 in the treated groups.
[0250] The results were compared to negative control (solution containing DMSO and no Pal-SEQ ID NO: 1-NH.sub.2).
[0251] The experiments were performed in four different experimental sessions.
[0252] The determination of collagen cross-linking, was carried out by measurement of the absorbance at 450 nm.
[0253] Samples were pipetted into the wells and the absorbance at 450 nm of each well was determined every 2 min for a time period of 100 min using the microplate reader Multiskan FC.
[0254] The % variation in collagen fibril formation between negative control (group only treated with DMSO) and the groups treated with Pal-SEQ ID NO: 1-NH.sub.2 was calculated and was a direct index of the efficacy of peptide Pal-SEQ ID NO: 1-NH.sub.2 to increase collagen fibril formation, which, moreover, could be described as dose dependent: increase of 59% at 0.02 mg/mL and of 111% at 0.04 mg/mL.
[0255] The results obtained appear summarized in
[0256] As can be derived from
[0257] The above gives support to the anti-ageing activity of the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) and, hence, their utility in preventing or treating skin signs related with aging as, for example, wrinkles, roughness and/or sagginess.
[0258] In addition, the results of this example also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening.
Example 11. Analysis of Cytotoxicity and Collagen Production of the Peptide Pal-SEQ ID NO: 1-NH.SUB.2 .in Human Organotypical Skin Explant Cultures
[0259] The cytotoxicity and collagen production of peptide Pal-SEQ ID NO: 1-NH.sub.2 were analysed by means of the LDH cytotoxicity assay and the resazurin assay for cytotoxicity; and collagen content analysis and histological analysis with regard to collagen production.
[0260] Peptide Pal-SEQ ID NO: 1-NH.sub.2 was prepared in accordance with examples 1 to 5.
[0261] In this case healthy and aged human organotypical skin explant cultures (hOSECs) were used as experimental systems.
[0262] The aged hOSEC was obtained by exposition of the healthy hOSEC to hydrocortisone (5 μg/mL) for the first five days of the study.
[0263] The total number of treatment groups was as follows:
[0264] 1. Control group (negative control): Untreated hOSEC.
[0265] 2. Aged hOSEC: hOSECs treated with hydrocortisone at 5 μg/mL (Abraham A, Roga G. (2014) Topical steroid-damaged skin. Indian J Dermatol. 59(5),456-9.)
[0266] 3. Aged hOSEC+Product A (cream, this is, placebo): hOSECs treated with hydrocortisone at 5 μg/mL and incubated with product A (10 μL).
[0267] 4. Aged hOSEC+Product B (cream with Pal-SEQ ID NO: 1-NH.sub.2 at a concentration of 0.005 mg/mL): hOSECs treated with hydrocortisone at 5 μg/mL and incubated with Product B (10 μL).
[0268] Four replicates of each experimental group were carried out and one independent experiment was performed.
[0269] The total duration of the assay was of 10 days (days 1 to 10). Hydrocortisone was applied in the appropriate or corresponding groups each day on days 1 to 5. On its side, Product B or Product A were applied in the corresponding or appropriate groups each day on days 2 to 9.
[0270] The cytotoxicity assays were performed on days 0 (before beginning the study), 1, 3, 5, 8 and 10.
[0271] Collagen production was measured on day 10.
[0272] LDH Cytotoxicity Assay
[0273] The LDH Cytotoxicity test is a colorimetric assay that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released into the culture medium supernatant upon damage of the cytoplasmic membrane. The released LDH in culture medium supernatants is measured for 30 minutes coupled enzymatic reaction: LDH oxidizes lactate to pyruvate which then reacts with the tetrazolium salt WST-1 to form formazan. The increase in the amount of formazan measured in the culture supernatant directly correlates to the increase in the number of lysed cells (damage) in the skin explant.
[0274] 100 μL supernatant of each sample were removed and transferred into a 96-well microplate. The released LDH in the culture medium supernatants was measured by a coupled enzymatic reaction: LDH oxidizes lactate to pyruvate which then reacts with the tetrazolium salt WST-1 to form formazan. The increase in the amount of formazan correlates directly with the increase in the number of lysed cells (damage) as the LDH enzyme is released into the culture medium supernatant when the cytoplasmic membrane is damaged. The formazan dye is water-soluble and can was measured using a standard ELISA plate reader at 500 nm.
[0275] The results of this assay were calculated considering the negative control, this is, untreated hOSEC as 100% of the LDH normal concentration. The results obtained appear summarized in
[0276] As it is derivable from
[0277] Resazurin Assay
[0278] The resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) has been broadly used as an indicator of cell viability in proliferation and cytotoxicity assays. The assay is based on the ability of viable, metabolically active cells to reduce resazurin to resorufin and dihydroresorufin. This conversion is intracellular, facilitated by mitochondrial, microsomal and cytosolic oxidoreductases. Resazurin is non-toxic to cells and it is stable in culture medium. Therefore, it allows continuous measurement of cell proliferation in vitro as either a kinetic or an endpoint assay.
[0279] Resazurin dye, therefore, has been broadly used as an indicator of cell viability in several cytotoxicity assays. It is also a metabolic activity indicator as the assay is based on the ability of metabolically active cells to reduce resazurin to resorufin and dihydroresorufin by mitochondrial, microsomal and cytosolic oxidoreductases.
[0280] Toxic insult that impairs cell viability and proliferation also affects the capacity of cultures to reduce resazurin, and the rate of dye reduction is directly proportional to the number of viable cells present. Therefore, as the resazurin reduction is a direct measure of the metabolic competence of cell cultures, it provides a convenient index of cell viability. The decrease in the amount of resazurin reduced by hOSECs also directly correlates to the increase in the number of dead cells.
[0281] For the resazurin assay, the skin explants were treated with 6 μM of resazurin in NaCl solution for 1 hour. Subsequently, a volume of 100 μL of each sample was removed and transferred into a 96-well microplate. The resorufin formed was quantified in a fluorometer plate reader.
[0282] The fluorescent signal was monitored using 530-560 nm excitation wavelength and 590 nm emission wavelength.
[0283] The results of the resazurin assay were calculated considering the negative control, untreated healthy hOSECs, as 100% viability. The obtained results appear in
[0284] A 10% reduction of Resorufin percentage was observed when the hOSECs were treated with 5 μg/mL of hydrocortisone with respect to the Control group (negative control). hOSECs treated with Product B (cream with Pal-SEQ ID NO: 1-NH.sub.2) or Product A and with hydrocortisone at 5 μg/mL did not show a decrease of Resorufin values with respect to the Control group (negative control).
[0285] These data support the idea that both Product B or Product A produced a metabolic activation in hOSEC in the conditions of the assay. As the resorufin is a direct measure of metabolic competence of cells, resorufin increase may indicate a metabolic activation of the oxide-reductase enzymes following exposure of hOSECs to both compounds.
[0286] Therefore, from the results obtained in the LDH cytotoxicity assay and the resazurin assay it can be deduced that Pal-SEQ ID NO: 1-NH.sub.2 was not toxic and, in fact, reverses the negative effects produced by hydrocortisone.
[0287] Collagen production [0288] Collagen content analysis:
[0289] The Collagen Assay is a dye-binding method for the analysis of acid and pepsin-soluble collagens (mainly, Type I, but also types II, III, IV and V). The assay can assess the rate of newly synthesized collagen produced during periods of rapid growth and development.
[0290] Collagen Dye Reagent (1 mL) was added to each sample (tubes) and shaken for 30 minutes. The tubes were centrifuged at 12,000 rpm for 10 minutes. Subsequently, 750 μL ice-cold Acid-Salt Wash Reagent was added to the collagen-dye pellet to remove unbound dye from the surface of the pellet and from the inside surface of the microcentrifuge tube. The tubes were again centrifuged at 12,000 rpm for 10 minutes. Finally, 250 μL of alkali reagent was added. When all the bound dye was dissolved (5 minutes), the samples were ready for measurement. The dissolved dye (200 μL of each sample in 96 micro well plates) was measured using a standard ELISA plate reader at 550 nm.
[0291] As noted above, the collagen content analysis was performed on day number 10 of the study. The results of collagen content (micrograms) assay were calculated per milligram of fresh dermal tissue. The results obtained appear summarized in
TABLE-US-00004 TABLE 4 Collagen quantity in the different groups analysed in example 11. Mean collagen Standard quantity (in deviation (in collagen μg/ collagen μg/ Group skin mg) skin mg) Untreated hOSEC 0.863 0.09 (Control group) Aged hOSEC 0.617 0.07 Aged hOSEC + Product A 0.610 0.06 Aged hOSEC + Product B 0.858 0.03
[0292] As shown in the
[0293] Group aged hOSEc+Product B, presented a collagen quantity similar to untreated hOSEC.
[0294] As noted in
[0295] These results demonstrate that the topical application of Pal-SEQ ID NO: 1-NH.sub.2 reversed the hydrocortisone aging effects and, hence, demonstrate the potential of the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) as anti-ageing products and to prevent, reduce and/or eliminate the signs of aging as, for example, wrinkles, roughness and/or sagginess.
[0296] In addition, these results also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening. [0297] Tissue Collection and Processing for TEM Analysis:
[0298] Skin samples were processed for Transmission Electron Microscopy analysis. Briefly, skin samples were fixed in 4% (v/v) formaldehyde and 1% (v/v) glutaraldehyde. Subsequently, skin samples were incubated in sucrose 0.1 M overnight and in 1% (v/v) osmium tetroxide in 0.1 M. Finally, samples were dehydrated with serialized ethanol solutions and embedded in beam capsules.
[0299] Ultrathin sections (60-90 nm thick) were stained with uranyl acetate for 15 minutes and lead citrate for 5 minutes. Subsequently samples were ready to analyze under electron microscope.
[0300] Transmission electron microscopy study was performed to observe the state of collagen fibres in treated skin explants.
[0301] Results appear summarized in
[0302] Likewise, collagen fibres thickness and collagen density were analysed by imagen analysis (see tables 5 and 6 below), and the values obtained corroborate the visual appreciations.
[0303] Collagen density indicates the compactness of each collagen fibre observed on the TEM image. To this end, the optical images were analyzed by GIMP2. The counterfactual analysis corroborated that healthy and aged hOSECs+Product B group presented density values higher than the values observed in aged hOSECs and aged hOSECs+Product A groups (see table 5 below and
[0304] Group aged hOSEC+Product B (cream with 0.005 mg/mL of peptide Pal-SEQ ID NO: 1-NH.sub.2), where it was appreciated an increase in collagen production, as noted above, presented a collagen density similar to untreated hOSEC (see table 5 below and
TABLE-US-00005 TABLE 5 Collagen density in the different groups analysed in example 11. Collagen density (in % with regard to Standard deviation Group untreated hOSEC) (in %) Untreated hOSEC 100 11.62 (Control group) Aged hOSEC 82.22 6.54 Aged hOSEC + Product A 77.22 8.39 Aged hOSEC + Product B 95.56 3.63
[0305] As noted in
[0306] These results demonstrate that the topical application of Pal-SEQ ID NO: 1-NH.sub.2 reversed the hydrocortisone aging effects and, hence, demonstrate the potential of the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) as anti-ageing products and to prevent, reduce and/or eliminate the signs of aging as, for example, wrinkles, roughness and/or sagginess.
[0307] In addition, these results also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening.
[0308] The collagen fibres thickness was also analyzed by GIMP2. The fibre thickness analysis corroborated that healthy and aged hOSEC+Product B (cream with 0.005 mg/mL of peptide Pal-SEQ ID NO: 1-NH.sub.2) group presented fibre thickness values lower than the values observed in aged hOSEC and aged hOSEC+Product A groups due to a better compaction and structuration (complete and not diffused fibres). Pal-SEQ ID NO: 1-NH.sub.2 peptide applied on aged hOSEC presented a collagen fibre thickness similar to untreated hOSEC (see table 6 and
[0309] Higher fibres thickness in aged hOSEC and aged hOSEC+Product A groups indicated abnormal accumulation of collagen in the fibres which is described to occur in aged skin in vivo.
[0310] Untreated hOSEC and aged hOSEC+Product B showed similar thickness of collagen fibres.
TABLE-US-00006 TABLE 6 Collagen fibre thickness in the different groups analysed in example 11. Collagen fibre thickness Standard deviation Group (nm) (nm) Untreated hOSEC 88.26 7.82 (Control group) Aged Skin 98.77 8.44 Aged Skin + Product A 95.44 8.70 Aged Skin + Product B 84.87 6.85
[0311] As noted in
[0312] These results demonstrate that the topical application of Pal-SEQ ID NO: 1-NH.sub.2 reversed the hydrocortisone aging effects and, hence, demonstrate the potential of the peptides of the present invention (as exemplified by means of Pal-SEQ ID NO: 1-NH.sub.2) as anti-ageing products and to prevent, reduce and/or eliminate the signs of aging as, for example, wrinkles, roughness and/or sagginess.
[0313] In addition, these results also demonstrate that the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for skin rejuvenation and/or to reduce, prevent and/or eliminate skin imperfections, more precisely, the peptides of the present invention (as exemplified by Pal-SEQ ID NO: 1-NH.sub.2) are able to provide for facial repositioning and/or facial skin tightening.
Example 12. Clinical Evaluation of Skin and Wrinkle Smoothing and Antiaging Efficacy on Female Volunteers with Different Phototypes
[0314] The effect of Pal-SEQ ID NO: 1-NH.sub.2 peptide (synthesized in accordance with examples 1 to 5) on facial skin antiaging was evaluated on 44 female volunteers (50% light-pigmented (phototype and 50% dark-pigmented (phototype V-VI)).
[0315] Briefly, volunteers applied a cosmetic formulation with 2% (m/v) from a stock at 0.05% (m/v) of Pal-SEQ ID NO: 1-NH.sub.2 or the cosmetic formulation without Pal-SEQ ID NO: 1-NH.sub.2 (placebo). The application regime was of two times per day during 56 days, on early morning and before bedtime. Cosmetic formulation were applied on the whole face to compare the effect of placebo and active between volunteers and also between different phototype volunteers.
[0316] On days 28 and 56, an AEVA system was used on each volunteer in order to assess wrinkles depth and cheek roughness. A decrease of the parameters within the days of treatment indicated a smoothing of the skin, hence an antiwrinkle and antiaging benefit.
[0317] The results obtained are shown in
[0318] As can be readily seen in