Uses of the peptide of sequence His-D-Trp-Ala-Trp-d-Phe-Lys-NH2 for reducing or delaying the appearance of cell senescence and signs of skin aging
11628133 · 2023-04-18
Assignee
Inventors
Cpc classification
A61K8/64
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to the cosmetic use of a composition including, as the active agent, 0.1 to 1 μM of a synthetic peptide of sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 or one of the salts thereof in a physiologically suitable medium, in order to reduce or delay the appearance of cell senescence and signs of skin aging. The present invention also relates to a method for cosmetic treatment which relates to these novel uses, including comprising the steps of applying the composition, topically, at least once per day, for a period of at least two days.
Claims
1. A method for increasing the activity of prolidase and the synthesis of collagen in skin, the method comprising topically applying a composition comprising, as the only active agent, between 0.1 and 1 μM of a synthetic peptide with sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (SEQ ID NO: 1) or one of its salts in a physiologically acceptable medium, wherein the composition is applied topically, at least once per day, for a period of at least 2 days.
2. The method of claim 1, wherein the composition comprises between 0.5 and 1 μM of said synthetic peptide.
3. The method of claim 1, wherein the composition comprises less than 1 μM of said synthetic peptide.
4. The method of claim 1, wherein the composition is applied 2 times per day.
5. The method of claim 1, wherein the composition is applied over a period of at least 6 weeks.
6. The method of claim 1, wherein the composition is applied over a period of at least 3 months.
7. A cosmetic treatment method for reducing or delaying the appearance of cellular senescence and the signs of skin aging, the method comprising the topical application, at least once per day over a period of at least 2 days, of a composition comprising, as the only active agent, between 0.1 and 1 μM of a synthetic peptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (SEQ ID NO: 1) in a physiologically acceptable medium, where the composition increases the activity of prolidase and the synthesis of collagen.
8. The method of claim 1, wherein the method reduces or delays the appearance of cellular senescence and signs of skin aging selected from thinning of the skin, sagging, toss of moisture, skin atony, deep wrinkles and fine lines, loss of firmness and tone, dermal atrophy with the exception of pigmentary anomalies of the skin.
9. The method of claim 8, wherein the signs of skin aging are wrinkles.
Description
(1) The invention and its resulting advantages will be better understood from the following non-limiting embodiment made with reference to the accompanying figures in which:
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(11) In the description and examples, unless specified otherwise, it should be understood that when a range it given it includes the upper and lower limits of said range.
EXAMPLE 1
Study of the Expression of Prolidase in Human Fibroblasts, in the Presence of Hexapeptide-2
(12) The aim of this study was to determine the influence of hexapeptide-2 on the expression of prolidase in human fibroblasts.
(13) Protocol: Cultured human fibroblasts were treated with hexapeptide-2 in a final concentration of 0.5 and 1 μM after dilution in a culture medium, for 24 hours (2 applications per day). The cells were then washed, fixed with cold methanol for 4 minutes at 4° C. The cells were incubated in the presence of a mouse monoclonal antibody specific for prolidase (LifeSpan BioSciences, Ref. LS-C115644), then of an anti-mouse secondary antibody coupled to a fluorochrome (Invitrogen, Ref. A21202). The cells were then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). Quantification of the fluorescence was carried out from the photographs obtained.
(14) Results: The microscope observations shown in Table 1 exhibited a significantly more intense cytoplasmic fluorescence in cells treated with hexapeptide-2 (using the Student t-test).
(15) TABLE-US-00001 TABLE 1 0.5 μM 1 μM Untreated hexapeptide-2 hexapeptide-2 Expression of prolidase 100 118 128 (intensity/area of cells (%)
(16) Conclusion: Hexapeptide-2 stimulates the expression of prolidase in human fibroblasts.
EXAMPLE 2
Study of the Activity of Prolidase in Human Fibroblasts, in the Presence of Hexapeptide-2
(17) The aim of this study was to determine the influence of hexapeptide-2 on the activity of prolidase in human fibroblasts.
(18) Protocol: Cultured human fibroblasts were treated with hexapeptide-2 in a concentration of 0.5 and 1 μM after dilution in a culture medium, for 24 hours (2 applications per day). At the same time, fibroblasts were treated with an activator and an inhibitor of prolidase: retinoic acid (Sigma, Ref. R2625) and Cpz-Pro (Bachem, Ref. 4001343). Next, the cells were detached from the dish with the aid of a 150 mM NaCl solution. After having recovered the cellular pellet and activating the prolidase with a solution containing 2 mM MnCl.sub.2 for 2 hours at 37° C., the prolidase substrate was added: 47 mM of Glycine-Proline (Sigma, Ref. G3002) for 1 hour at 37° C. Next, the amount of proline contained in each sample was determined at a wavelength of 515 nm using Chinard's reagent. At the same time, a proline concentration range was produced in order to establish a calibration curve. The amount of protein was determined using the BCA protein assay kit (Thermo Scientific, Ref. 23225), which enabled the ratio of proline to the number of proteins in each sample to be recorded.
(19) Result: The treatment with hexapeptide-2 enabled the activity of prolidase (using the Student t-test) to be significantly increased. The results are presented in Table 2 below.
(20) TABLE-US-00002 TABLE 2 1 μM 1 μM 0.5 μM hexapeptide- retinoic 10 mM Untreated hexapeptide-2 2 acid Cpz-Pro Activity of 100 122 114 105 90 prolidase (%)
(21) Conclusion: Hexapeptide-2 stimulates the activity of prolidase in human fibroblasts.
EXAMPLE 3
Study of the Expression of Intracellular Collagen I in Human Fibroblasts, in the Presence of Hexapeptide-2
(22) The aim of this study was to determine the influence of hexapeptide-2 on the expression of intracellular collagen I in human fibroblasts by flow cytometry.
(23) Protocol: Cultured human fibroblasts were treated with hexapeptide-2 in a concentration of 0.5 and 1 μM after dilution in a culture medium, for 48 hours (2 applications per day). At the same time, fibroblasts were treated with an activator for collagen I: TGF-β (R&D system, Ref. 240B). The fibroblasts were trypsinated and the cellular pellet was recovered. The cells were then fixed with cold methanol for 15 minutes at 4° C. Immunolabelling was carried out with the aid of a mouse monoclonal antibody specific for collagen I (Millipore, Ref: anti-Collagen Type I, Clone: 5D8-G9) coupled with a fluorochrome. Next, 10 000 cells were analysed by flow cytometry (Guava EasyCyte) in order to determine the amount of fluorescence in each of them.
(24) Results: Following analysis by flow cytometry, a more intense cytoplasmic fluorescence was detected in cells treated with hexapeptide-2. The results are presented in Table 3 below.
(25) TABLE-US-00003 TABLE 3 0.5 μM 1 μM 10 ng/ml Untreated hexapeptide-2 hexapeptide-2 TGF-β Expression of 100 120 145 182 collagen I (%)
(26) Conclusion: Hexapeptide-2 stimulates the expression of collagen I in human fibroblasts.
EXAMPLE 4
Study of the Expression of Collagens I and III in Human Skin, in Ex Vivo Culture in the Presence of Hexapeptide-2
(27) The aim of this study was to determine the influence of hexapeptide-2 on the expression of collagens I and III in human skin ex vivo.
(28) Protocol: Samples of human skin were cultured at the air/liquid interface. Hexapeptide-2 was applied topically to the samples in concentrations of 0.5 and 1 μM after dilution in a culture medium, in an amount of 2 applications per day, then the samples were incubated for 48 hours at 37° C.
(29) These skin samples were subsequently fixed with formaldehyde then included into paraffin. 4 μm sections were then produced.
(30) Labelling of the collagens I and III was then carried out after demasking specific sites by microwave incubation then by treatment with trypsin. The immunolabelling was carried out with the aid of a rabbit polyclonal antibody specific for collagen I (Rockland, Ref. 600-401-103-0.5), a rabbit polyclonal antibody specific for collagen III (Rockland, Ref 600-401-105-0.5), then an anti-rabbit secondary antibody coupled to a fluorochrome (Invitrogen, Ref. A21206). The cells were then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). A quantification of the fluorescence from the photographs obtained was carried out.
(31) Results: The microscopic observations exhibited a significantly more intense fluorescence in the dermis of biopsies treated with hexapeptide-2. The results are presented in Table 4 below.
(32) TABLE-US-00004 TABLE 4 0.5 μM 1 μM Untreated hexapeptide-2 hexapeptide-2 Expression of Collagen I 100 119 154 (%) Expression of Collagen III 100 155 155 (%)
(33) Conclusions: Hexapeptide-2 stimulates the expression of collagens I and III in human skin ex vivo.
EXAMPLE 5
Study of the Expression of miRNA-29a 3p in Human Fibroblasts Deriving from Donors of Different Ages
(34) The aim of this study was to determine the influence of hexapeptide-2 on the expression of miRNA-29a 3p in human fibroblasts from donors of different ages, by qPCR. The miRNAs play a fundamental role in the post-transcriptional regulation of their targets by inhibiting them. It has been demonstrated in the literature that the expression of miRNA-29a increases with senescence (Mancini M. et al., 2014). By using databases such as miRanda, the targets for miRNA-29a were identified as being collagen I and prolidase.
(35) Protocol: Human fibroblasts from 3 donors of 33, 45 and 66 years of age were treated with hexapeptide-2 in a concentration of 0.5 and 1 μM in culture medium overnight (1 application).
(36) The miRNAs were extracted with the aid of an extraction kit (Ambion, Ref. AM1561), then reverse transcribed with a specific kit (Applied Biosystem, Ref 4374966). A real time PCR was carried out in a thermocycler using a TaqMan Gene Expression Assay specific for miRNA-29a 3p (Applied Biosystems, Ref 002112) and a TaqMan Gene Expression Assays specific for RNU44 used as the control endogen (Applied Biosystems, Ref. 001094). The relative quantification if the expression of miRNA-29a 3p was carried out by the comparative Ct method.
(37) Result: Comparing the three ages, the expression of miRNA-29a 3p increases significantly with senescence by +32% (between 33 and 45 years old) and +48% (between 45 and 66 years old) (Student t test). At each age: 33, 45 and 66 years old, the cells treated with hexapeptide-2 showed, respectively, a significant reduction of −13%, −14% and −27% in the expression of miRNA-29a 3p (using the Student t-test). The results are presented in
(38) Conclusion: The expression of miRNA-29a 3p is reduced when the cells are treated with hexapeptide-2, which means that the negative impact of its expression on targets such as collagen and prolidase can be limited.
EXAMPLE 6
Study of the Activity of β-Galactosidase in Human Fibroblasts Made Senescent by Transfection with the siRNA FOXO3a, in the Presence of Hexapeptide-2
(39) The aim of this study was to determine the influence of hexapeptide-2 on senescent human fibroblasts. Senescence was induced by inhibition of the FOXO3a gene using interfering RNA (siRNA) from FOXO3a. FOXO3a is a Forkhead type transcription factor involved in cellular longevity. By inhibiting the expression of FOXO3a, the cells over-express β-galactosidase, which is characteristic of senescent cells.
(40) Protocol: Cultured human fibroblasts were treated or not treated with a specific siRNA of FOXO3a (Invitrogen, Ref. HSS177176) in a final concentration of 25 nM using the technique of transfection by Lipofectamine™ RNAiMAX (Invitrogen, Ref 56531) and treated or not treated with hexapeptide-2 in a final concentration of 1 μM in culture medium, for 48 hours (2 applications per day).
(41) The cells were rinsed and fixed in a fixing buffer (0.2% glutaraldehyde, 2% formaldehyde). The cells were then incubated at 37° C. without CO.sub.2 for 24 hours, with a solution of 1 mg/mL X-Gal in 40 mM of citric acid/phosphate (pH 6), 5 mM K.sub.3FeCN.sub.6, 5 mM K.sub.4FeCN.sub.6, 150 mM NaCl and 2 mM MgCl.sub.2. The cells were then examined under white light with a microscope (Nikon Eclipse E600 microscope).
(42) Results: The senescent cells having a specific β-galactosidase activity were stained blue. The cells treated with the specific siRNA of FOXO3a exhibited an increase in β-galactosidase linked to senescence, resulting in an increase in the number of blue-stained cells. The cells transfected with the specific siRNA of FOXO3a and treated with hexapeptide-2 exhibited a visible reduction in the activity of β-galactosidase linked to senescence, resulting in a reduction in the number of blue-stained cells (results not shown).
(43) Conclusion: Treatment with hexapeptide-2 allowed cells to be observed for which the level of senescence was lower than untreated transfected cells.
EXAMPLE 7
Study of the Expression of Collagen I in Human Fibroblasts Made Senescent by Transfection with the siRNA FOXO3a, in the Presence of Hexapeptide-2
(44) The aim of this study was to determine the influence of hexapeptide-2 on the expression of collagen I in senescent human fibroblasts by transfection with the siRNA FOXO3a.
(45) Protocol: Cultured human fibroblasts were treated or not treated with a specific siRNA of FOXO3a (Invitrogen, Ref. HSS177176) in a final concentration of 25 nM using the technique of transfection by Lipofectamine™ RNAiMAX (Invitrogen, Ref. 56531) and treated or not treated with hexapeptide-2 in a concentration of 0.5 or 1 μM in culture medium, for 48 hours (2 applications per day).
(46) The fibroblasts were trypsinated and the cellular pellet was recovered. The cells were then fixed with cold methanol for 15 minutes at 4° C. Immunolabelling was carried out with the aid of a mouse monoclonal antibody specific for collagen I (Millipore, Ref. anti-Collagen Type I, Clone: 5D8-G9) coupled with a fluorochrome. Next, 10 000 cells were analysed using flow cytometry (Guava EasyCyte) in order to determine the level of fluorescence in each of them.
(47) Results: Following the induction of senescence by transfection with the siRNA FOXO3a, a reduction in the expression of collagen I was observed in these cells. The cells which were senescent and treated with hexapeptide-2 per se exhibited a smaller reduction in the expression of collagen I. The results are presented in
(48) Conclusion: Treatment with hexapeptide-2 can be used to limit the effects of senescence such as the reduction in the expression of collagen I.
EXAMPLE 8
Study of the Activity of β-Galactosidase in Human Fibroblasts Made Senescent by Replicative Senescence, in the Presence of Hexapeptide-2 (Long-Term Treatment)
(49) The aim of this study was to determine the influence of hexapeptide-2 on senescent human fibroblasts. In contrast to the model of senescence obtained by transfection of the siRNA FOXO3a for which senescence was induced over 48 hours, replicative senescence is a long process which necessitates approximately 3 months. The fibroblasts were regularly sub-cultured by trypsinization in order to cause them to divide, and as a consequence to make them age.
(50) Protocol: Human fibroblasts were cultured in a specific medium and maintained under culture (for more than 27 passes) and treated or not treated with a daily application of hexapeptide-2 in a concentration of 1 μM in the culture medium, (i.e. 5 applications per week). A portion of the cells was frozen in each pass. Next, key passes were selected: passes 4, 12, 16, 20, 24 and 27. After freezing, the senescent phenotype of the fibroblasts treated or not treated with 1 μM hexapeptide-2 was evaluated by assaying the activity of β-galactosidase.
(51) To this end, the cells were rinsed and fixed in a fixing buffer (0.2% glutaraldehyde, 2% formaldehyde). The cells were then incubated, at 37° C. without CO.sub.2 for 24 hours, with a solution of 1 mg/mL X-Gal in 40 mM of citric acid/phosphate (pH 6), 5 mM K.sub.3FeCN.sub.6, 5 mM K.sub.4FeCN.sub.6, 150 mM NaCl and 2 mM MgCl.sub.2. The cells were then examined under white light with a microscope (Nikon Eclipse E600 microscope).
(52) Results: During the course of the cell passes, the activity of the β-galactosidase increased, indicating that the level of senescence increased with each pass. When these cells were treated with 1 μM hexapeptide-2, we observed a general, significant reduction of −22% in the activity of beta-galactosidase compared with untreated cells (using the Student t-test). The results are shown in
(53) Conclusion: Following a long-term treatment of approximately 3 months, we observed that the degree of senescence in cells treated with hexapeptide-2 was lower than in the untreated cells. Thus, the treatment with hexapeptide-2 could slow down cell aging.
EXAMPLE 9
Study of the Activity of β-Galactosidase in Senescent Human Fibroblasts by Replicative Senescence, in the Presence of Hexapeptide-2 (Short-Term Treatment)
(54) The aim of this study was to determine the influence of hexapeptide-2 on senescent human fibroblasts by replicative senescence.
(55) Protocol: Human fibroblasts were cultured in a specific medium and maintained under long-term culture treatment (for more than 28 passes, i.e. approximately 3 months). A portion of the cells was frozen in each pass. Next, key passes were selected: passes 4, 12, 16, 20 and 28. After freezing, the fibroblasts were treated with 1 μM hexapeptide-2 in culture medium, for 48 hours (2 applications per day). The senescent phenotype of the fibroblasts treated or not treated with 1 μM hexapeptide-2 was evaluated by assaying the activity of the β-galactosidase.
(56) To this end, the cells were rinsed and fixed in a fixing buffer (0.2% glutaraldehyde, 2% formaldehyde). The cells were then incubated, at 37° C. without CO.sub.2 for 24 hours, with a solution of 1 mg/mL X-Gal in 40 mM of citric acid/phosphate (pH 6), 5 mM K.sub.3FeCN.sub.6, 5 mM K.sub.4FeCN.sub.6, 150 mM NaCl and 2 mM MgCl.sub.2. The cells were then examined under white light with a microscope (Nikon Eclipse E600 microscope).
(57) Results: During the course of the cell passes, the activity of β-galactosidase increased, indicating the level of senescence of each pass. When these cells, which had been made senescent, were treated with 1 μM hexapeptide-2, we observed a general significant reduction of −31% in the activity of the β-galactosidase compared with untreated cells (using the Student t-test). The results are presented in
(58) Conclusion: Following a short-term treatment of 48 hours, we observed that the cells treated with hexapeptide-2 had a lower level of senescence than the untreated cells. Thus, treatment with hexapeptide-2 could be used to slow down cellular aging.
EXAMPLE 10
Study of the Activity of Prolidase in Human Fibroblasts Made Senescent by Replicative Senescence, in the Presence of Hexapeptide-2 (Long-Term Treatment)
(59) The aim of this study was to determine the influence of hexapeptide-2 on the activity of prolidase in senescent human fibroblasts. It has been shown in the literature that the activity of prolidase reduces with cellular aging, and this is correlated with the reduction in collagen I observed in senescent cells (Palka et al. Tokai J Exp Clin Med 1996).
(60) Protocol: Human fibroblasts were cultured in a specific medium and maintained under long-term culture treatment (for more than 20 passes, i.e. approximately 3 months), treated or not treated with the aid of a daily application of hexapeptide-2 in a concentration of 1 μM in culture medium, (5 applications per week). A portion of the cells was frozen for each pass. Next, two passes were selected: passes 12 and 20. After freezing, the activity of prolidase was observed for these conditions.
(61) To this end, the cells were detached from their support with a solution of 150 mM NaCl. After recovering the cellular pellet and activating the prolidase with a solution containing 2 mM of MnCl.sub.2 for 2 hours at 37° C., the substrate for the prolidase was added: 47 mM of Glycine-Proline (Sigma, Ref. G3002), for 1 hour at 37° C. Next, the proline content contained in each sample was determined at a wavelength of 515 nm using Chinard's reagent. At the same time, a concentration range for proline was produced in order to establish a calibration curve. The protein content was determined using the BCA protein assay kit (Thermo Scientific, Ref. 23225), in order to relate the proline content with the number of proteins in each sample.
(62) Result: The comparison of the passes P12 (cells considered to be young) and P20 (cells considered to be senescent) show that the activity of the prolidase reduces by 36% (significant value using the Student t-test) during replicative senescence. Interestingly, while hexapeptide-2 had little or no effect on young cells (P12), the application of hexapeptide-2 to senescent cells (P20) showed a restoration of 20% (significant value using the Student t-test) in the activity of prolidase compared with untreated control cells in the same pass.
(63) The results are shown in
(64) Conclusion: During senescence, the activity of prolidase reduces, along with the expression of collagen. Treatment with hexapeptide-2 could allow the activity of prolidase to be maintained, thereby resulting in collagen recycling being maintained.
EXAMPLE 11
Study of the Expression of miRNA-29a 3p in Human Fibroblasts Made Senescent by Replicative Senescence, in the Presence of Hexapeptide-2 (Long-Term Treatment)
(65) The aim of this study was to determine the influence of hexapeptide-2 on the expression of miRNA-29a 3p in senescent human fibroblasts, by replicative senescence. The miRNAs play a fundamental role in post-transcriptional regulation of their targets by inhibiting them. It has been shown in the literature that the expression of miRNA-29a increases with senescence (Mancini M. et al., 2014). By means of databases such as miRanda, the targets for miRNA-29a were identified, including collagen I and prolidase.
(66) Protocol: Human fibroblasts were cultured in a specific medium and maintained under a long-term culture treatment (for more than 24 passes, i.e. approximately 3 months), treated or not treated by means of a daily application of hexapeptide-2 in a concentration of 1 μM in culture medium, (5 applications per week). For each pass, a portion of the cells was frozen. Next, three passes were selected: passes 4, 12 and 24. After freezing, the expression of miRNA-29a 3p was observed for these conditions, by quantitative PCR.
(67) The miRNAs were extracted with the aid of an extraction kit (Ambion, Ref AMI1561), then reverse transcribed with a specific kit (Applied Biosystems, Ref 4374966). A real-time PCR was carried out in a thermocycler with the aid of a TaqMan Gene Expression Assay specific for miRNA-29a 3p (Applied Biosystems, 002112) and a TaqMan Gene Expression Assay specific for RNU44 used as an endogenic control (Applied Biosystems, 001094). The relative quantification of the expression of miRNA-29a 3p was carried out using the comparative Ct method.
(68) Result: Following a comparison of the three passes P4, P12 and P20, we could observe that the expression of miRNA-29a 3p increased significantly by +25% (between P4 and P12) and +52% (between P4 and P24) (Student t test) with senescence. At pass 4, no effect of hexapeptide-2 was observed on the expression of miRNA-29a 3p. In contrast, at passes 12 and 24, the cells treated with hexapeptide-2 respectively exhibited a significant reduction of −16% and of −7% in the expression of miRNA-29a 3p (using the Student t-test). The results are shown in
(69) Conclusion: The cells treated with 1 μM hexapeptide-2 appeared to maintain a certain level of expression of miRNA-29a 3p despite senescence, thus allowing the negative impact of its expression on targets such as collagen and prolidase to be limited.
EXAMPLE 12
Preparation of an Anti-Aging Cosmetic Composition for the Face
(70) TABLE-US-00005 % by Commercial names INCI names weight PHASE A Demineralized water Aqua 76.80 Na4 EDTA Tetrasodium EDTA 0.05 Lubrasil ™ II DM Glycerin & Glyceryl Acrylate/Acrylic 3.00 Acid Copolymer & Laureth-23 & Dimethicone Liquapar MEP Phenoxyethanol & Methylparaben & 1.00 Ethylparaben & Propylparaben PHASE B UltraThix ® P-100 Acrylic Acid/VP Crosspolymer 0.60 PHASE C NaOH Pearls Sodium Hydroxide 0.02 Demineralized water Aqua 0.50 PHASE D Belsil SDM 6022 Stearoxy Dimethicone & Dimethicone 2.00 Simulsol 165 PEG-100 Stearate & Glyceryl Stearate 2.00 Refined Shea Butter Butyrospermum Parkii (Shea) Butter 2.00 Ceraphyl ® 28 Cetyl Lactate 1.50 Ceraphyl ® 791 Isocetyl Stearoyl Stearate 2.00 Ceraphyl ® ODS Octyldodecyl Stearate 3.00 Ceraphyl ® 368 Ethylhexyl Palmitate 4.00 PHASE E NaOH Pearls Sodium Hydroxide 0.03 Demineralized water Aqua 0.50 PHASE F Hexapeptide-2 1.00 100.00
(71) Under the conditions for the preparation of this formula, the 1% hexapeptide-2 corresponds to a concentration of 1 μM.
(72) The cosmetic composition for the face termed the “placebo” was produced in exactly the same manner, with the exception that the hexapeptide-2 was replaced with purified water.
EXAMPLE 13
Study of the Effect of 1% Hexapeptide-2 on the Appearance of Wrinkles (Clinical Study)
(73) Aim: Clinical study regarding the appearance of wrinkles after topical treatment with a cosmetic composition comprising 1% hexapeptide-2, against placebo.
(74) Protocol: Number of participants: 35 (aged 35 to 65 years of age). Study: double blind study against placebo. Products tested: hexapeptide-2, 1% formulation (i.e. a final concentration of 1 μM) in a cosmetic composition as described in Example 12 versus the placebo composition. Under the conditions of: Number of applications: 2 applications per day, morning and evening, in a dose of 2 mg/cm.sup.2. Duration of test: 6 weeks. Control visits: Day 0 (D0), and Day 42 (D42). Measurements: analysis of silicone replicas made of the crow's feet using Visioline® VL650 software Statistical analyses: either the “t” Student test or Wilcoxon test was used, depending on the normality of the distribution of the data.
(75) Results: After application of hexapeptide-2 in a 1% formula in the composition in accordance with Example 12, the analysis of silicone replicas exhibited a statistically significant reduction on D42 in the number, total surface area, total length, total depth and maximum depth of the wrinkles compared with the placebo. The results are presented in
(76) TABLE-US-00006 TABLE 5 % % improvement Treatment Time Mean Sem p change in participants Number of Placebo D42-D0 −7.83 14.97 0.0206* −12.47% 57% wrinkles 1% D42-D0 −53.14 17.36 hexapeptide-2 Total surface Placebo D42-D0 −1.29 1.64 0.0093** −27.59% 63% area of 1% D42-D0 −6.85 1.33 wrinkles (mm.sup.2) hexapeptide-2 Total length Placebo D42-D0 −10.17 11.84 0.0281* −17.48% 66% (mm) 1% D42-D0 −43.99 10.62 hexapeptide-2 Total depth Placebo D42-D0 −364.44 716.25 0.0088** −15.75% 60% (μm) 1% D42-D0 −3030.34 850.01 hexapeptide-2 Maximum Placebo D42-D0 8.68 10.81 0.0021** −23.13% 63% depth (μm) 1% D42-D0 −43.06 10.29 hexapeptide-2 *significant; **very significant, with the Student test or the Wilcoxon test (test selected as a function of the normality of the distribution of the data); mean n = 35 +/− sem.
(77) Conclusions: The topical application of 1% hexapeptide-2 (i.e. in a concentration of 1 μM) over 42 days causes a statistically significant reduction in the appearance of wrinkles.
(78) Applicants incorporate by reference the material contained in the accompanying computer readable Sequence Listing entitled “BV15-176PCT Sequence Listing ST25”, which was created on Oct. 4, 2017, and is 731 bytes in size.