Peptides and compositions for use in cosmetics
阅读说明：本技术 用于化妆品的肽和组合物 (Peptides and compositions for use in cosmetics ) 是由 A·格鲁-卡普斯坦尼 S·巴斯特 P·卡鲁亚 J·C·埃斯古德罗 J·A·波拉斯 M·J· 于 2020-03-26 设计创作，主要内容包括：本发明涉及能够增加同源域蛋白Mohawk的合成并因此可用作抗老化剂和嫩化剂的肽家族。(The present invention relates to a family of peptides capable of increasing the synthesis of the homeodomain protein Mohawk and therefore useful as anti-aging and tenderizing agents.)
1. A peptide capable of increasing Mohawk, an acceptable isomer, salt, solvate and/or derivative thereof and/or a mixture thereof.
2. The peptide according to claim 1, characterized in that it has a sequence according to formula (I):
R1-AA1-AA2-AA3-AA4-R2
(I)
their cosmetically acceptable isomers, salts, solvates and/or derivatives and mixtures thereof, wherein:
AA1is His;
AA2selected from the group of amino acids having aromatic side chains;
AA3selected from Lys or Arg;
AA4selected from the group of amino acids having aliphatic nonpolar side chains,
R1selected from the group consisting of H, substituted or unsubstituted acyclic aliphatic group, substituted or unsubstituted alicyclic group, substituted or unsubstituted heterocyclic group, substituted or unsubstituted heteroarylalkyl group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group and R5-CO-, wherein R5Selected from the group formed by: substituted or unsubstituted C1-C24Alkyl, substituted or unsubstituted C2-C24Alkenyl, substituted or unsubstituted C2-C24Alkynyl, substituted or unsubstituted C3-C24Cycloalkyl, substituted or unsubstituted C5-C24Cycloalkenyl, substituted or unsubstituted C8-C24Cycloalkynyl, substituted or unsubstituted C6-C30Aryl, substituted or unsubstituted C7-C24Aralkyl, substituted or unsubstituted 3 to 10 membered heterocyclyl ring, and substituted or unsubstituted heteroarylalkyl having 2 to 24 carbon atoms and an alkyl chain of atoms other than 1-3 carbons and 1 to 6 carbon atoms; and
R2Selected from H, -NR3R4-、-OR3and-SR3Wherein R is3And R4Independently selected from the group consisting of H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroaralkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted aralkyl group.
3. The peptide according to claim 2, characterized in that:
AA1is His;
AA2selected from Phe,Tyr and Trp;
AA3selected from Lys or Arg;
AA4selected from Ala, Val, Leu and Ile.
4. A peptide according to any one of claims 2 or 3, characterised in that the peptide of formula (I) is:
R1-His-Tyr-Arg-Ala-R2(R1-SEQ ID NO：1-R2)。
5. a peptide according to any one of claims 2 to 4, characterised in that R1Selected from Pal or Ac.
6. A peptide according to any one of claims 2 to 5, characterised in that R2Selected from H or NH2。
7. A peptide according to any one of claims 2 to 6, characterized in that R1Is Pal and R2Is NH2。
8. A peptide according to claim 4, characterized in that the peptide of formula (I) is:
Pal-His-Tyr-Arg-Ala-NH2(Pal-SEQ ID NO：1-NH2)。
9. a composition comprising a peptide according to any one of claims 1 to 8.
10. Use of a peptide according to any one of claims 1 to 8 or a composition according to claim 9 as a cosmetic product in a subject in need thereof.
11. Use as a cosmetic product according to claim 10, characterized in that the use as a cosmetic product is for reducing, preventing and/or eliminating the signs of skin aging, for skin rejuvenation, and/or for reducing, preventing and/or eliminating skin defects.
12. Use as a cosmetic product according to claim 11, characterized in that the signs of skin aging are wrinkles, roughness and/or laxity.
13. Use as a cosmetic product according to claim 11, characterized in that the use as a cosmetic product for skin rejuvenation and/or for reducing, preventing and/or eliminating skin imperfections is for skin tightening, body shaping, facial reduction, skin tightening and/or pore refining.
14. Cosmetic use of a peptide according to any one of claims 1 to 8 or a composition according to claim 9 in a subject in need thereof.
15. Cosmetic use according to claim 14, characterized in that it is for reducing, preventing and/or eliminating the signs of skin ageing, for skin rejuvenation, and/or for reducing, preventing and/or eliminating skin defects.
Examples
Abbreviations:
abbreviations for amino acids follow the recommendations of the 1983IUPAC-IUB Joint Commission on Biochemical nomenclature, listed in Eur.J.biochem. (1984)138: 937.
Ac, acetyl; ala, alanine; arg, arginine; c-terminal, carboxy-terminal; DCM, dichloromethane; DIEA, N' -diisopropylethylamine; DIPCDI, N' -diisopropylcarbodiimide; DMF, N-dimethylformamide; EDGS, growth supplement as defined by EpiLife; equiv, eq; ESI-MS, electrospray ionization mass spectrometry; fmoc, 9-fluorenylmethoxycarbonyl; his, histidine; HOBt, 1-hydroxybenzotriazole; hOSEC, human organ-typical 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-terminus, amino terminus; 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.
With respect to the chemical synthesis procedures included in the examples, it should be noted that all of the synthesis was carried out in polypropylene syringes equipped with porous polyethylene discs or in polypropylene syringes equipped with porous platesIs carried out in a reactor. All reagents and solvents were of synthetic quality and used without any further treatment. The solvent and soluble reagents were removed by aspiration. The Fmoc group was removed with piperidine-DMF (2:8, v/v) (at least 1X 1 min, 2X 10 min, 5mL/g resin) (Lloyd Williams P. et al, (1997) Chemical applications to the Synthesis of Peptides and Proteins, CRC, Boca Raton (Fla., USA)). Washing between deprotection, coupling and deprotection stages was performed with DMF (3X 1 min) and DCM (3X 1 min) each time using 10ml of solvent per g of resin. The coupling reaction was carried out with 3ml of solvent per g of resin. Control of coupling was performed by performing the ninhydrin test (Kaiser E. et al, anal. biochem.,1970,34: 595598). All synthesis reactions and washings were performed at room temperature.
Example 1. Synthesis and preparation of peptides.
1 2 3 4 1 2 3-obtaining Fmoc-AA-AA-AA-AA-Rink-MBHA-resin, wherein AA is L-His; AA is L-Tyr; AA 4Is L-Arg; AA is L-Ala.
The weight was normalized. 4.8g (2.5mmol) of Fmoc-Rink-MBHA resin with a functionality of 0.52mmol/g was treated with piperidine-DMF to remove the Fmoc group according to the general protocol described as known in the prior art. 2.33g of Fmoc-L-Ala-OH (7.5 mmol; 3 equivalents) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equivalents) and HOBt (1.01 g; 7.5 mmol; and 3 equivalents) using DMF as solvent for 1 h.
The resin was then washed as described in general methods known in the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocol, 4.87g of Fmoc-Arg (Pbf) -OH (7.5 mmol; 3 equivalents); followed by 3.45g of Fmoc-L-Tyr (tBu) -OH (7.5 mmol; 3 equivalents); and subsequently 4.65g of Fmoc-L-His (Trt) -OH (7.5 mmol; 3 equiv.) were coupled sequentially, each coupling being carried out in the presence of 1.01g of HOBt (7.5 mmol; 3 equiv.) and 1.17mL of DIPCDI (7.5 mmol; 3 equiv.). Deprotection of the Fmoc group was performed between each amino acid addition step as described above.
After synthesis, the peptide resin was washed with DCM (5 times for 3 min each) and dried under vacuum.
Using the above synthesis procedure, the following sequences were synthesized:
His-Tyr-Arg-Ala(SEQ ID NO：1)。
example 2. removal of the Fmoc N-terminal protecting group of the peptide synthesized according to example 1.
The N-terminal Fmoc group of the peptidyl resin was deprotected with 20% (v/v, hereinafter referred to as v/v) piperidine in DMF (1X 1 min + 2X 10 min) (Lloyd Williams P. et al (1997) Chemical applications to the Synthesis of Peptides and Proteins CRC, Boca Raton (Fla., USA)). Peptidyl resin was washed with DMF (5X 1 min), DCM (4X 1 min) and dried under vacuum.
Example 3 reaction of R1Procedure for introduction of palmitoyl group onto peptidyl resin obtained according to example 2.
1mmol (1 equivalent) of the peptidyl resin obtained according to 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 5mL of DMF as solvent. They were left to react for 30 min, then the peptidyl resin was washed with DMF (5 × 1 min), DCM (4 × 1 min) and dried under vacuum.
Example 4. cleavage procedure of polymeric support from peptidyl resin obtained according to examples 2 and 3.
The weight was normalized. 200mg of the dried peptidyl resin obtained from any of examples 2 or 3 was treated with 5mL of TFA/TIS/H at room temperature2O (90: 5) was stirred for 2 hours. CollectingThe filtrate was filtered and precipitated with 50mL (8 to 10 fold) of cold diethyl ether. The ethereal solution was evaporated to dryness under reduced pressure at room temperature, and the precipitate was redissolved in 50% (v/v) aqueous MeCN and lyophilized.
Example 5 characterization of the peptides synthesized and prepared according to example 4.
Using a Shimadzu apparatus (Kyoto, Japan), a reverse phase column (150X4.6mm, Xbridge Peptide BEH C18, 3.5 μm, Waters, USA), H was used2HPLC analysis of the peptide obtained according to example 4 was performed at a flow rate of 1.25mL/min in a gradient of MeCN (+ 0.045% (v/v) TFA (+ 0.036% (v/v) TFA) in O and detected at 220 nm. All peptides showed more than 80% purity. The identity of the peptide obtained was confirmed by ESI-MS on a Water ZQ 4000 detector using MeOH as the mobile phase at a flow rate of 0.2 mL/min. The results obtained demonstrate that the peptide Pal-His-Tyr-Arg-Ala-NH 2(Pal-SEQ ID NO：1-NH2) Is correctly and efficiently synthesized.
Example 6 peptide Pal-SEQ ID NO: 1-NH2Cytotoxicity assays in HEKa and HDFa cells.
Peptide Pal-SEQ ID NO: 1-NH2Was analyzed by viability assays in HEKa (human epidermal keratinocytes, adult) and HDFa (human dermal fibroblasts, adult) cells.
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
Cytotoxicity was assessed by MTT assay. Briefly, HEKa and HDFa cells were plated at 1 × 105The individual cells/mL were seeded in 96-well plates and cultured in Epilife medium supplemented with 1% (v/v) EDGS and 1% (v/v) penicillin/streptomycin (for HEKa cells) and medium supplemented with 2% (v/v) Low Serum Growth Supplement (LSGS) 106 (for HDFa cells) at 37 ℃ with 5% CO2And incubation at saturated humidity for 24 hours. The peptides were then expressed with varying concentrations of Pal-SEQ ID NO: 1-NH2(0.001 mg/mL, 0.005mg/mL, 0.01mg/mL, 0.05mg/mL, and 0.1mg/mL for HEKa cells; and 0.001mg/mL, 0.005mg/mL, and 0.01mg/mL for HDFa cells) cells were treated in triplicate for MTT assessment. After 24 hours of treatment with the corresponding concentration of peptide, 10. mu.L of yellow tetrazole (MTT) was addedIn each well, and the cells were incubated at 37 ℃ for an additional 4 hours. After incubation with MTT, the medium was aspirated from each well and 150 μ L of dimethyl sulfoxide (hereinafter DMSO) (100%) was added to dissolve the formazan crystals formed. The plate was placed on a shaker for 5 minutes to completely dissolve the crystals, and then absorbance at 450nm was measured for each well using a microplate reader Multiskan FC, which is directly proportional to the number of living cells in the culture. Absorbance values were normalized using data obtained for untreated cells (basal state).
The results are summarized in fig. 1 and 2.
As can be derived directly from the figure, the peptides of the invention (e.g.via Pal-SEQ ID NO: 1-NH)2Exemplified) did not alter the viability of HEKa and HDFa cells at the tested concentrations and thus were not cytotoxic.
Example 7 analysis of gene expression regulation in primary human dermal fibroblasts.
Analysis of the peptide Pal-SEQ ID NO: 1-NH2The ability to modulate the expression of genes associated with collagen and extracellular matrix production (genes analyzed see table 1).
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
A stock solution of 12.5mg/mL peptide in DMSO was prepared. The working solutions were freshly prepared at the indicated concentrations from the stock solutions in the respective supplemented media.
Untreated cells were used as negative control samples.
RNA (ribonucleic acid) extraction and RT-qPCR (reverse transcription quantitative polymerase chain reaction) were performed. Briefly, HDFa cells were plated in duplicate (n-2) at 4x105The density of individual cells/well was seeded in 6-well plates and maintained at standard culture conditions (106 medium supplemented with 1% (v/v) LSGS; 37 ℃, 95% room humidity, 5% CO)2) And then 24 hours. Then, the cells were treated with the peptide Pal-SEQ ID NO: 1-NH 2Treatment was for another 6 hours (or 24 hours for COL13a 1). Untreated cells were used as basal controls.
Finally cells were lysed and replicate samples were pooled together for RNA extraction using Qiagen RNeasy Mini kit according to the manufacturer's instructions. Purified RNA was used to generate corresponding cDNA (complementary deoxyribonucleic acid) as a template for amplification by reverse transcription using a commercial kit high-capacity cDNA reverse transcription kit (Applied Biosystems). RT-qPCR was performed using a StepOne plus Real-Time PCR instrument using appropriate TaqMan assay probe sets for the genes shown in Table 1 (plus GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the housekeeping gene) and 2x gene expression Master Mix. Amplification included the following 40 cycles: 15 seconds (denaturation) at 95 ℃ and 1 minute (annealing and extension) at 60 ℃ (Arya, M., Shergill, I.S., Williamson, M., Gommerall, L., Arya, N., Patel, H.R, (2005) Basic principles of real-time qualitative 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 1 genes analyzed in example 7.
The data obtained was analyzed using the Δ Δ Ct method, which provides target gene expression values as fold changes in the treated samples compared to the untreated base samples. Both samples were normalized with the relative expression of the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase).
The analysis step comprises:
1. calculate the average Ct for each condition
2. Calculating Δ CT test samples and Δ CT untreated samples
3. Calculating Δ Δ CT ═ Δ CT test sample- Δ CT untreated sample
4. Through 2-ΔΔCTObtaining the ratio
The results of this assay are summarized in figures 3 to 5.
From the figure, the peptide Pal-SEQ ID NO: 1-NH2：
-after 6 hours of treatment: down-regulating genes MMP1 and MMP3, both of which are known to degrade extracellular matrix proteins.
-after 6 hours of treatment: up-regulating several key genes in the collagen structural system and extracellular structure: COL3a1, COL4a5, COL5a1, COL6a1, COL7a1, COL12a1, COL14a1, COL13a1 (after 24 hours of treatment), MKX y ZEB 2.
Up-regulation of several collagen cross-linking related genes after 6 hours of treatment: TGFB1, FN1, LOXL3, LOXL2 and HSP 47.
Thus, the peptides of the invention (e.g., via Pal-SEQ ID NO: 1-NH)2Exemplified) to avoid or prevent degradation of the extracellular matrix, while helping to increase collagen synthesis and crosslinking, thus exhibiting anti-aging and rejuvenating activities.
Example 8 analysis of collagen VI synthesis in human dermal fibroblasts.
Analysis of the peptide Pal-SEQ ID NO: 1-NH 2The ability to induce collagen VI synthesis in human dermal fibroblasts.
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
In this assay, the ability of the tested peptides to increase collagen VI synthesis in human dermal fibroblasts was evaluated in vitro. This assessment was performed by using the Pal-SEQ ID NO: 1-NH2After treating the cells, the amount of collagen VI was determined.
Experimental protocols are provided for the following experimental groups:
negative control: cell cultures treated with solubilization vehicle (DMSO) only;
-with the peptide Pal-SEQ ID NO: 1-NH2Cell cultures treated at three concentrations (0.01mg/mL, 0.005mg/mL and 0.001 mg/mL).
Stock solutions of the peptide in DMSO were prepared, followed by three serial dilutions of 0.01mg/mL, 0.005mg/mL, and 0.001mg/mL in cell culture medium.
The biological model used in this case is composed ofNormal human dermal fibroblasts. Cells were plated at 1 × 104Individual cells/well were seeded in 96-well plates and cultured under standard culture conditions (37 ℃, 95% room humidity, 5% CO)2) And keeping for 24 hours.
After 24 hours of incubation, the medium was removed and the medium containing the treatment (DMSO or Pal-SEQ ID NO: 1-NH)2) The new medium of (2) is added to the wells. Sample treatment lasted 48 hours and cell culture medium was collected at the end of the treatment. Cells treated with DMSO were used as negative controls.
For test performance, the three concentrations of Pal-SEQ ID NO: 1-NH2Cell cultures of human dermal fibroblasts were treated.
After 48 hours of treatment, the amount of collagen VI produced and released by the cells (de novo collagen VI synthesis) was measured in cell culture medium by ELISA assay. The results were compared to those of the negative control (cells treated with DMSO). Treatments were performed in triplicate and at three different experimental stages.
The determination of collagen VI synthesis was performed by ELISA method. Commercial kits are used for this purpose. The test kit adopts a sandwich ELISA method to detect the content of human collagen VI. The micro ELISA plates provided in the kit were pre-coated with collagen VI specific antibodies. Standards or samples are added to appropriate microtiter ELISA plate wells and bound with specific antibodies. Then, a collagen VI specific biotinylated detection antibody and an avidin-horseradish peroxidase (HRP) conjugate were added to each microplate in sequence and incubated. The free components were washed off. Substrate solution (TMB) was added to each well. Only those wells containing collagen VI were blue. The enzyme-substrate reaction was stopped by adding a sulfuric acid solution and the color turned yellow. The Optical Density (OD) was measured by a microplate reader at a wavelength of 450 nm. The OD value is proportional to the concentration of collagen VI.
The quantitative determination used a calibration curve consisting of a known and increasing concentration of standard collagen VI. The percent change in collagen VI content between the negative control (DMSO) and the sample was calculated as the percent change in Pal-SEQ ID NO: 1-NH2A direct indicator of the efficacy of increasing collagen VI synthesis.
The results obtained in this experiment are summarized in table 2 and fig. 6.
Table 2 average results of collagen VI content of different groups in example 8 and percentage change of the content with respect to the negative control group.
As can be directly deduced from table 2 and fig. 6, in all cases (i.e. for all concentrations tested), a statistically significant increase in collagen VI synthesis was observed, which can furthermore be described as dose-dependent: an increase of 11% at 0.001mg/mL, 16% at 0.005mg/mL and 40% at 0.01 mg/mL. As shown in fig. 6, the observed difference in collagen VI content in the treated groups relative to the negative control was statistically significant.
The above results demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) increases the synthesis of the protein collagen VI and thus contributes to improved extracellular matrix assembly. Thus, the peptides may prevent or treat skin aging and/or skin signs of said aging, such as wrinkles, roughness and/or sagging.
In addition, the results of this example also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2Illustrated) can provide facial repositioning and/or facial skin tightening.
Example 9 analysis of Mohawk synthesis in human dermal fibroblasts.
Analysis of the peptide Pal-SEQ ID NO: 1-NH2The ability to induce Mohawk synthesis in human dermal fibroblasts.
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
The assay involves the peptide Pal-SEQ ID NO: 1-NH2In vitro capacity to increase synthesis of homeodomain protein Mohawk in human dermal fibroblastsAnd (6) evaluating. This evaluation was performed by determining the Mohawk quantity after treating the cells with the peptide.
Experimental protocols are provided for the following experimental groups:
-an untreated cell culture;
cell cultures treated with solubilizing vehicle (DMSO) only (negative control);
-the amino acid sequence of Pal-SEQ ID NO: 1-NH2Treated cell culture.
Stock solutions of the peptide in DMSO were prepared, followed by three serial dilutions of 0.01mg/mL, 0.005mg/mL, and 0.001mg/mL in cell culture medium.
As described above, the biological model used consisted of normal human dermal fibroblasts.
Cells were plated at 1 × 104Individual cells/well were seeded in 96-well plates and cultured under standard culture conditions (37 ℃, 95% room humidity, 5% CO)2) And keeping for 24 hours. After 24 hours of incubation, the medium was removed and fresh medium containing the test product was added to the wells.
Sample treatment lasted 24h and 48h, then cells were lysed to determine the intracellular Mohawk concentration by ELISA assay. The results were compared to a negative control (cells treated with DMSO).
Treatments were performed in triplicate at three different experimental stages.
As described above, Mohawk synthesis was determined by ELISA. Commercial kits are used for this purpose. The test kit detects the content of the human homeodomain protein Mohawk by a double-site sandwich ELISA method. Antibodies specific to Mohawk have been pre-coated onto microplates. Standards and samples were pipetted into the wells and any Mohawk present was bound by the immobilized antibody. After removing any unbound material, HRP-conjugated human Mohawk detection antibody was added to the wells. After washing to remove any unbound HRP reagent, a chromogen solution is added to the wells (substrate is TMB) and color development is proportional to the amount of Mohawk bound in the initial step. The color development was stopped and the concentration was determined colorimetrically at 450 nm.
The quantitative determination used a calibration curve consisting of a standard Mohawk of known and increasing concentration. Negative controls (DMSO) were calculated and compared with Pal-SEQ ID NO: 1-NH2Percent change in Mohawk content between treated groups, which is the peptide Pal-SEQ ID NO: 1-NH2Increasing a direct indicator of the efficacy of Mohawk synthesis.
The results obtained are summarized in table 3 and fig. 7.
TABLE 3 average results of the Mohawk content of the different groups in example 9 and the percentage change of said content with respect to the negative control group.
From table 3 and fig. 7 it can be directly deduced that in all cases (i.e. for all concentrations and durations tested) a statistically significant increase in the synthesis of the homeodomain protein Mohawk was observed, which can furthermore be described as dose and dependent: at 24 hours, the concentration increased by 9% at 0.001mg/mL, 21% at 0.005mg/mL and 43% at 0.01 mg/mL; further, at 48 hours, the concentration was increased by 31% at 0.001mg/mL, 51% at 0.005mg/mL and 81% at 0.01 mg/mL. As shown in fig. 7, the difference seen in Mohawk content in the treated groups relative to the negative control was statistically significant over each test time range.
The above results indicate that the peptide of the present invention (e.g., Pal-SEQ ID NO: 1-NH) 2Exemplified) increases Mohawk synthesis, thus demonstrating its potential to prevent or treat skin aging and/or skin signs associated with aging such as wrinkles, roughness, and/or sagging.
In addition, the results of this example also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2By way of example) canProviding facial repositioning and/or facial skin tightening.
Example 10 analysis of collagen crosslinking.
Analysis of the peptide Pal-SEQ ID NO: 1-NH2The ability to induce or improve collagen cross-linking.
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
The assay involves the peptide Pal-SEQ ID NO: 1-NH2In vitro (in tubo) assessment of the ability to increase and accelerate collagen fibrillation/collagen cross-linking. The assessment was performed by measuring collagen fibril formation after treatment of collagen with the peptides in solution (0.02M acetic acid, 0.125M NaCl, and 1/10 phosphate buffered saline (pH 7.4)) at room temperature.
Experimental protocols are provided for the following experimental groups:
-the expression of Pal-SEQ ID NO: 1-NH 2Treated collagen solution.
Collagen solution treated only with the same solution as the above-mentioned treatment group but without peptides (negative control), thus having the same volume of DMSO as the samples containing peptides;
stock solutions of the peptides in DMSO were prepared, then two serial dilutions were made so that the final concentration of the peptides in the collagen solution was 0.02mg/mL or 0.04 mg/mL.
In the control (solution identical to the treated group but without peptide and therefore having the same volume of DMSO as the sample containing peptide) and the treated group, the peptide Pal-SEQ ID NO: 1-NH2Immediately after addition of collagen to the solution of (a), collagen crosslinking and fibril formation are followed.
The results were compared to a negative control (containing DMSO but not containing Pal-SEQ ID NO: 1-NH)2Solution of (d) was compared.
The experiment was carried out in four different experimental stages.
The determination of collagen cross-linking is performed by measuring the absorbance at 450 nm.
The samples were pipetted into the wells and the absorbance at 450nm per well was measured every 2 minutes over a period of 100 minutes using a microplate reader Multiskan FC.
Negative controls (DMSO-only treated groups) and negative controls (positive controls) were calculated with Pal-SEQ ID NO: 1-NH2The percent change in collagen fibril formation between the treated groups and it is the peptide Pal-SEQ ID NO: 1-NH 2A direct indicator of increased efficacy of collagen fibrillation, furthermore, this can be described as dose-dependent: an increase of 59% at 0.02mg/mL and a 111% at 0.04 mg/mL.
The results obtained are summarized in fig. 8.
As can be seen from fig. 8, the peptide Pal-SEQ ID NO: 1-NH2Collagen crosslinking was increased and accelerated at two concentrations tested: 0.02(b) and 0.04(a) mg/mL.
The above supports the peptides of the invention (e.g., via Pal-SEQ ID NO: 1-NH)2Exemplified), and thus their utility in preventing or treating skin signs associated with aging, such as wrinkles, roughness, and/or sagging.
In addition, the results of this example also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2Illustrated) can provide facial repositioning and/or facial skin tightening.
Example 11. culture of human organ representative skin explants peptide Pal-SEQ ID NO: 1-NH2Analysis of cytotoxicity and collagen production.
By LDH cytotoxicity assay and resazurin assay for cytotoxicity; and analysis of collagen content and histological analysis for collagen production to analyze the peptide Pal-SEQ ID NO: 1-NH 2Cytotoxicity and collagen production.
The peptides Pal-SEQ ID NO were prepared according to examples 1 to 5: 1-NH2。
In this case, healthy and aged human organ representative skin explant culture (hOSEC) was used as experimental system.
Aged hOSEC were obtained by exposing healthy hOSEC to hydrocortisone (5 μ g/mL) for the first five days of the study.
The total number of treatment groups was as follows:
1. control group (negative control): untreated hOSEC.
2. Aged hOSEC: hOSEC treated with hydrocortisone at 5. mu.g/mL (Abraham A, Roga G. (2014) Topical stereo-damaged skin. Indian JDermatol.59(5),456-9.)
3. Aged hOSEC + product a (cream, this is placebo): hOSEC treated with hydrocortisone at 5. mu.g/mL and incubated with product A (10. mu.L).
4. Aged hOSEC + product B (containing Pal-SEQ ID NO: 1-NH at a concentration of 0.005 mg/mL)2Cream of (a): hOSEC treated with hydrocortisone at 5. mu.g/mL and incubated with product B (10. mu.L).
Each experimental set was performed in quadruplicate and one independent experiment was performed.
The total duration of the assay was 10 days (day 1 to day 10). Hydrocortisone was applied in the appropriate or corresponding groups daily on days 1 to 5. On the other hand, product B or product a was applied in the corresponding or appropriate group every day from day 2 to day 9.
Cytotoxicity assays were performed on day 0 (before study start), on days 1, 3, 5, 8 and 10.
Collagen production was measured on day 10.
LDH cytotoxicity assay
The LDH cytotoxicity test is a colorimetric assay that quantitatively measures Lactate Dehydrogenase (LDH), a stable cytosolic enzyme that is released into the culture supernatant when the plasma membrane of the cell is damaged. Released LDH in the culture supernatant was measured for a 30 min coupled enzymatic reaction: LDH oxidizes lactic acid to pyruvic acid, which then reacts with the tetrazolium salt WST-1 to form formazan. The increase in the amount of formazan measured in culture supernatant was directly correlated with the increase in the number of lysed cells (lesions) in the skin explants.
100 μ L of supernatant from each sample was removed and transferred to a 96-well microplate. LDH released in the culture supernatant was measured by a coupled enzymatic reaction: LDH oxidizes lactic acid to pyruvic acid, which then reacts with the tetrazolium salt WST-1 to form formazan. The increase in the amount of formazan is directly related to the increase in the number of lysed cells (damage) because LDH enzyme is released into the culture supernatant when the plasma membrane is damaged. Formazan dyes are water soluble and can be measured at 500nm using a standard ELISA plate reader.
The results of this assay were calculated by considering the negative control, i.e. untreated hOSEC, as 100% of the normal concentration of LDH. The results obtained are summarized in fig. 9.
As can be deduced from fig. 9, LDH values found in all groups were similar to the control group (negative control). This fact indicates that hydrocortisone does not have any adverse effect on hOSEC (does not cause plasma membrane damage) under the assay conditions. Likewise, product B (i.e., containing Pal-SEQ ID NO: 1-NH) was used2Cream) or product a and treatment with hydrocortisone at 5 μ g/mL showed no difference in LDH values compared to the control. These data imply that hydrocortisone incubation and both compounds did not adversely affect hOSEC under the assay conditions (plasma membrane damage).
Determination of Resazurin
Resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) has been widely used as an indicator of cell viability in proliferation and cytotoxicity assays. The assay is based on the ability of metabolically active living cells to reduce resazurin to resorufin and dihydroresorufin. This conversion is intracellular and is facilitated by mitochondrial, microsomal and cytosolic redox enzymes. Resazurin is non-toxic to cells and stable in the culture medium. Thus, it allows continuous measurement of cell proliferation in vitro as a kinetic or endpoint assay.
Therefore, resazurin dye has been widely used as an indicator of cell viability in several cytotoxicity assays. It is also an indicator of metabolic activity, 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.
Toxic damage that impairs cell viability and proliferation also affects the ability of the culture to reduce resazurin, and the rate of dye reduction is directly proportional to the number of viable cells present. Thus, since the reduction of resazurin is a direct measure of the metabolic capacity of the cell culture, it provides a convenient indicator of cell viability. The reduction in the amount of hOSEC-reduced resazurin was also directly correlated with the increase in the number of dead cells.
For resazurin assay, skin explants were treated with 6 μ M resazurin in NaCl for 1 hour. Subsequently, a volume of 100. mu.L of each sample was removed and transferred to a 96-well microplate. The resorufin formed was quantified in a fluorometer plate reader.
The fluorescence signal was monitored using an excitation wavelength of 530 and 560nm and an emission wavelength of 590 nm.
The results of the resazurin assay were calculated by considering the negative control (untreated healthy hOSEC) as 100% viability. The obtained results are shown in FIG. 10.
When hOSEC was treated with 5 μ g/mL hydrocortisone, a 10% reduction in the percentage of resorufin was observed compared to the control (negative control). With product B (containing Pal-SEQ ID NO: 1-NH)2Cream) or product a and hOSEC treated with hydrocortisone at 5 μ g/mL showed no reduction in resorufin values relative to the control (negative control).
These data support the idea that product B or product a produces metabolic activation in hOSEC under assay conditions. Since resorufin is a direct measure of the metabolic capacity of cells, an increase in resorufin might indicate metabolic activation of the oxidoreductase after exposure of hOSEC to both compounds.
Thus, from the results obtained in the LDH cytotoxicity assay and the resazurin assay, it can be concluded that Pal-SEQ ID NO: 1-NH2There is no toxicity and indeed the negative effects of hydrocortisone production are reversed.
Collagen production
-collagen content analysis:
the collagen assay is a dye binding method for the analysis of acid and pepsin soluble collagen (mainly type I, but also types II, III, IV and V). This assay can assess the rate at which newly synthesized collagen is produced during rapid growth and development.
Collagen dye reagent (1mL) was added to each sample (tube) and shaken for 30 minutes. The tube was centrifuged at 12,000rpm for 10 minutes. Subsequently, 750 μ L of ice-cold acid salt wash reagent was added to the collagen-dye precipitate to remove unbound dye from the precipitate surface and from the inner surface of the microcentrifuge tube. The tube was centrifuged again at 12,000rpm for 10 minutes. Finally 250. mu.L of alkaline reagent was added. When all bound dye dissolved (5 minutes), the sample was ready for measurement. The dissolved dye was measured at 550nm using a standard ELISA reader (200. mu.L per sample in 96 microwell plates).
As described above, collagen content analysis was performed on day 10 of the study. The results of the collagen content (μ g) assay per mg of fresh dermal tissue were calculated. The results obtained are summarized in fig. 11 and table 4.
Table 4 amount of collagen in different groups analyzed in example 11.
As shown in FIG. 11 and Table 4, product B (containing 0.005mg/mL Pal-SEQ ID NO: 1-NH) was observed2Cream of (b) increased collagen production in aged hOSEC compared to the remaining aged hOSEC group (treated with hydrocortisone at 5 μ g/mL only or with hydrocortisone and product a).
The aged hOSEc + product B group exhibited similar amounts of collagen as the untreated hOSEc.
As shown in fig. 11, the observed differences in collagen amounts between the untreated and aged hOSEC + product B and aged hOSEC + product a groups were statistically significant. There were no statistically significant differences between untreated hOSEC and aged hOSEC + product B.
These results demonstrate that Pal-SEQ ID NO: 1-NH2Topical application of (A) reverses the effects of hydrocortisone ageing and thus demonstrates that the peptides of the invention (e.g.via Pal-SEQ ID NO: 1-NH)2Examples) as an anti-aging product and preventing, reducing and/or eliminating signs of aging such as wrinkles, roughness and/or sagging.
In addition, these results also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2Illustrated) can provide facial repositioning and/or facial skin tightening.
Tissue collection and processing for TEM analysis:
skin samples were treated 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 overnight in 0.1M sucrose and in 0.1M 1% (v/v) osmium tetroxide. Finally, the sample was dehydrated with serialized ethanol solution and embedded in a light beam capsule.
Ultrathin sections (60-90nm thick) were stained with uranyl acetate for 15 minutes, followed by lead citrate for 5 minutes. The sample is then ready for analysis under an electron microscope.
Transmission electron microscopy studies were performed to observe the status of collagen fibers in treated skin explants.
The results are summarized in fig. 12A to 12D. Fig. 12A corresponds to untreated hOSEC, which shows the correct dermal structure (intact and structured dermal fibers in the dermis). Similarly, figure 12B shows aged hOSEC with unstructured collagen fibers (diffuse, non-dense fibers). This image demonstrates the adverse effect of hydrocortisone on skin, consistent with collagen quantification (included above). A similar image of non-dense collagen fibers was obtained for aged skin treated with product a (fig. 12C). However, as shown in FIG. 12D, when aged, product B for hOSEC (containing 0.005mg/mL peptide Pal-SEQ ID NO: 1-NH) 2The cream of (a) in the culture medium),good regeneration of collagen fibers (intact and non-dispersed fibers) is achieved. This image confirms the collagen values obtained by the ELISA method and mentioned above.
Also, the collagen fiber thickness and collagen density were analyzed by image analysis (see tables 5 and 6 below), and the obtained values confirmed the visual observation results.
The collagen density represents the degree of compaction of each collagen fiber observed on the TEM image. For this purpose, the optical image was analyzed by GIMP 2. Counterfactual analysis confirmed that healthy and aged hOSEC + product B groups exhibited higher density values than those observed in aged hOSEC and aged hOSEC + product a groups (see table 5 below and fig. 13).
Aged hOSEC + product B (containing 0.005mg/mL peptide Pal-SEQ ID NO: 1-NH)2Cream) wherein an increase in collagen production was observed as described above, exhibited a similar collagen density as untreated hOSEC (see table 5 below and figure 13).
Table 5 collagen density in different groups analyzed in example 11.
As shown in fig. 13, the observed differences in collagen density between the untreated hOSEC and aged hOSEC + product B groups and the aged hOSEC and aged hOSEC + product a groups were statistically significant. Untreated hOSEC and aged hOSEC + product B (with 0.005mg/mL peptide Pal-SEQ ID NO: 1-NH) 2Cream) was not statistically significantly different.
These results demonstrate that Pal-SEQ ID NO: 1-NH2Topical application of (A) reverses the effects of hydrocortisone ageing and thus demonstrates that the peptides of the invention (e.g.via Pal-SEQ ID NO: 1-NH)2Examples) as an anti-aging product and prevent, reduce and/or/andthe potential to counteract signs of aging such as wrinkles, roughness and/or sagging.
In addition, these results also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2Illustrated) can provide facial repositioning and/or facial skin tightening.
Collagen fiber thickness was also analyzed by GIMP 2. Fiber thickness analysis confirmed healthy and aged hOSEC + product B (containing 0.005mg/mL peptide Pal-SEQ ID NO: 1-NH)2Cream) group exhibited lower fiber thickness values than those observed in aged hOSEC and aged hOSEC + product group a due to better densification and structuring (intact and undispersed fibers). Pal-SEQ ID NO: 1-NH2The peptides exhibited similar collagen fiber thickness as untreated hOSEC (see table 6 and figure 14).
The higher fiber thickness in the aged hOSEC and aged hOSEC + product group a indicates an abnormal accumulation of collagen in the fibers, a phenomenon described as occurring in vivo in aged skin.
Untreated hOSEC and aged hOSEC + product B showed similar collagen fiber thickness.
Table 6 collagen fiber thickness in different groups analyzed in example 11.
As shown in fig. 14, the observed differences in collagen fiber thickness between the untreated and aged hOSEC + product B groups and the aged hOSEC group were statistically significant. The observed differences in collagen fiber thickness between the untreated and aged hOSEC + product B and the aged hOSEC + product a groups were approximately statistically significant. Untreated hOSEC and aged hOSEC + product B (with 0.005mg/mL peptide Pal-SEQ ID NO: 1-NH)2Cream) was not statistically significantly different.
These results demonstrate that Pal-SEQ ID NO: 1-NH2Topical application of (A) reverses the effects of hydrocortisone ageing and thus demonstrates that the peptides of the invention (e.g.via Pal-SEQ ID NO: 1-NH)2Examples) as an anti-aging product and preventing, reducing and/or eliminating signs of aging such as wrinkles, roughness and/or sagging.
In addition, these results also demonstrate that the peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH)2Exemplified) is capable of providing skin rejuvenation and/or reducing, preventing and/or eliminating skin defects, more specifically, peptides of the invention (e.g., Pal-SEQ ID NO: 1-NH2Illustrated) can provide facial repositioning and/or facial skin tightening.
Example 12 clinical evaluation of skin and wrinkle smoothing and anti-aging efficacy in female volunteers with different gloss types
44 female volunteers (50% light skin color (phototype II-III) and 50% dark skin color (phototype V-VI)) were evaluated for Pal-SEQ ID NO: 1-NH2Effect of peptides (synthesized according to examples 1 to 5) on anti-aging of facial skin.
Briefly, volunteers applied a vaccine containing 0.05% (m/v) Pal-SEQ ID NO: 1-NH2(from a 2% (m/v) stock solution) or a cosmetic preparation that does not contain Pal-SEQ ID NO: 1-NH2The cosmetic preparation of (1) (placebo). The application regimen was twice daily in the morning and before sleep over 56 days. Cosmetic formulations were applied to the whole face to compare the effect of placebo and active agents between volunteers and between volunteers of different light types.
On days 28 and 56, each volunteer evaluated wrinkle depth and cheek roughness using the AEVA system. A decrease in the parameters during the treatment/treatment days indicates a smoothing of the skin and thus anti-wrinkle and anti-aging benefits.
The obtained results are shown in FIG. 15.
As can be easily seen from fig. 15(a) to (C), the nucleic acid sequence comprising Pal-SEQ ID NO: 1-NH2The cosmetic formulations of (a) show a coarse thickness on the facial skin area studied at all study times and for all skin tone types studiedRoughness (Ra) and relief (Rz) as well as reduction in wrinkle depth (reduction of some or all of these parameters shows smoothing effect as well as anti-wrinkle and anti-aging effect). After 56 days of treatment, a reduction in cheek roughness of up to 4% (averaging 5% compared to placebo) and a reduction in cheek remission of 7% (averaging 10% compared to placebo) was observed. For wrinkle depth, a more pronounced effect was observed, reaching an average of 9% reduction, in particular 12% reduction in wrinkle depth (20% compared to placebo) for volunteers with skin of phototype V-VI, whereas volunteers with skin of phototype II-III experienced 6-7% reduction in wrinkle depth over time.
Sequence listing
<110> Lipotrue S.L.
<120> peptides and compositions for cosmetics
<130> 2020/10156
<150> EP19382222.8
<151> 2019-03-28
<160> 1
<170> BiSSAP 1.3.6
<210> 1
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> artificially synthesized sequence
<400> 1
His Tyr Arg Ala
1
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