Decapeptide-12 modulation of sirtuin gene expression in epidermal keratinocyte precursor cells

Abstract

Recent reports have detailed the multifaceted roles played by sirtuins in suppressing premature aging, delaying cell senescence, extending lifespan, and ameliorating a wide range of aging disorders. Here, we report findings on decapeptide-12, a potent sirtuin activator, and compare its performance with the well-documented oxyresveratrol. Treatment of human epidermal keratinocyte progenitor cells with 100 μM decapeptide-12 increased SIRT1 transcription by 141 ± 11% compared to control cells, while SIRT3, SIRT6, and SIRT7 levels increased by 121 ± 13%, 147 ± 8%, and 95.4 ± 14%, respectively. Decapeptide-12 upregulated sirtuin transcription to levels similar to oxyresveratrol but with reduced cytotoxicity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 479,248, filed March 30, 2017, entitled "Decapeptide-12 Modulation of Sirtuin Gene Expression in Epidermal Keratinocytes," which is incorporated by reference along with all other references cited in this application.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing entitled “ELIXP004US_ST25.txt” (3 KB), created on March 21, 2018, and submitted electronically herewith.

[0003] 2. Background of the Invention The present invention relates to the field of novel biological agents. [Background technology]

[0004] overview Recent reports have detailed the multifaceted roles played by sirtuins in suppressing premature aging, delaying cellular senescence, extending life span, and ameliorating a wide range of aging disorders. Herein, we report findings on decapeptide-12, a potent sirtuin activator, and compare its performance to the well-documented oxyresveratrol. Treatment of human epidermal keratinocyte progenitor cells with 100 μM decapeptide-12 increased SIRT1 transcription by 141 ± 11 percent compared to control cells, while SIRT3, SIRT6, and SIRT7 levels increased by 121 ± 13 percent, 147 ± 8 percent, and 95 ± 14 percent, respectively. Decapeptide-12 upregulated sirtuin transcription to similar levels as oxyresveratrol, but with reduced cytotoxicity. Summary of the Invention [Means for solving the problem]

[0005] The peptide according to one embodiment consists of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0006] A peptide according to certain embodiments consists of SEQ ID NO:9 modified by a modifying group, either a palmitoyl or acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0007] A peptide according to various embodiments consists of SEQ ID NO:11, which has a tyrosine amino acid at position 6 as the D-isoform, with all other amino acids being the L-isoform.

[0008] A composition according to one embodiment comprises a first peptide consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0009] A composition according to certain embodiments consists of SEQ ID NO:9 modified by a modifying group, either a palmitoyl or acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0010] A composition according to some embodiments consists of SEQ ID NO:11, having a tyrosine amino acid at position 6 as the D-isoform, with all other amino acids in the L-isoform.

[0011] Compositions according to certain embodiments comprise peptides present at a concentration of 1 μM or greater. An embodiment of a method for treating a subject by modulating expression of a sirtuin gene in skin cells to reduce symptoms of skin aging comprises administering to a subject in need thereof a composition comprising an effective amount of one or more peptides, wherein the one or more peptides consist of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0012] In certain embodiment methods, the peptide consists of SEQ ID NO:9 modified by a modifying group, either a palmitoyl or acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0013] In methods according to some embodiments, the peptide consists of SEQ ID NO:11, having a tyrosine amino acid at position 6 as the D-isoform, with all other amino acids being the L-isoform.

[0014] In the methods according to various embodiments, the skin cells are progenitor cells. According to some embodiments, the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendroblasts.

[0015] In methods according to certain embodiments, the skin cells are terminally differentiated. According to various method embodiments, the skin cells are keratinocytes, melanocytes, fibrocytes, histiocytes, or dendrocytes.

[0016] In certain embodiments of the method, the peptide is present at a concentration of 1 μM or greater. In certain embodiments, the sirtuin gene comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0017] In some embodiments, the composition further comprises oxyresveratrol. In certain embodiments, the skin cells are mammalian cells.

[0018] In some embodiments, the skin cells are human. An embodiment of a method for modulating expression of a sirtuin gene in a skin cell includes administering to a subject in need thereof a composition comprising an effective amount of one or more peptides, wherein the one or more peptides consist of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0019] Other objects, features and advantages of the present invention will become apparent from consideration of the following detailed description and the accompanying drawings, in which like reference designations represent like features throughout the drawings. [Brief description of the drawings]

[0020] [Figure 1A] Figure 1 shows dose-dependent transcriptional upregulation of SIRT1 (a). Data are expressed as fold increase relative to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1B] (b) Dose-dependent transcriptional upregulation of SIRT3. Data are expressed as fold increase relative to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1C] (c) Dose-dependent transcriptional upregulation of SIRT6. Data are expressed as fold increase relative to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1D] (d) Dose-dependent transcriptional upregulation of SIRT7. Data are expressed as fold increase relative to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 2A] Figure 1 shows the cytotoxic effect of decapeptide-12 and oxyresveratrol in epidermal keratinocytes. Data are expressed as percentage control and represent the mean ± SEM of three separate experiments. *P<0.05. [Figure 2B] Figure 1 shows the effect of decapeptide-12 and oxyresveratrol on epidermal keratinocyte proliferation. Data are expressed as percentage control and represent the mean ± SEM of three separate experiments. *P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description Skin represents the outcome of chrono- and photoaging, forcing us to constantly be aware of the aging process and to seek treatments that could slow or reverse its effects. Skin aging has traditionally been classified as extrinsic or intrinsic. Recent evidence has shown that both types share key molecular features, including alterations in signaling pathways that promote expression of matrix metalloproteinases, reduced procollagen synthesis, and connective tissue damage.

[0022] In human skin, aging is associated with an increase in the number of senescent cells and a decrease in the capacity of cell proliferation and differentiation. Substantial evidence supports the theory that aging is primarily the result of free radical damage caused by various endogenous reactive oxygen species (ROS). Velarde et al. reported in vivo evidence of a causal relationship between mitochondrial oxidative damage, cell senescence, and the skin aging phenotype. Furthermore, ultraviolet (UV) radiation stimulates ROS synthesis, which is related to mutagenesis and photoaging. Consistent with these findings, data suggest altered expression of sirtuin activity in UV-irradiated versus sun-protected skin, and these differences may be responsible for certain aspects of skin aging.

[0023] Cell senescence is described as a process in which cells stop dividing and undergo characteristic phenotypic changes, including prominent chromatin and secretome alterations, as well as activation of tumor suppressors. Numerous reports have helped establish the concept of sirtuins as potent anti-aging proteins detailing their multifaceted roles in cell senescence and delaying premature aging. Sirtuins are key effectors in pathways such as DNA damage repair, telomere shortening, cellular response to oxidative stress, and amelioration of ROS-induced pathologies.

[0024] In mammals, there are seven sirtuin genes (SIRT1-7) that are localized in different cellular compartments and can act in a variety of ways. Biochemically, sirtuins mainly regulate NAD + Sirtuins are a class of proteins with kinase-dependent lysine deacetylase activity. They are widely recognized as key regulators of multiple metabolic pathways, sensors of cellular energy and redox status, and modulators of oxidative stress.

[0025] These findings have sparked interest in developing small molecule activators or drugs that can help slow the progression of aging and its broad range of age-related disorders. Among the seven mammalian sirtuins, SIRT1 has been the most widely studied in relation to aging and longevity. For example, the anti-aging effects of resveratrol are primarily attributed to the activation of SIRT1. Indeed, Ido et al. reported that resveratrol ameliorated cell senescence and proliferation dysfunction by increasing the activity of AMP-activated protein kinase and sirtuins.

[0026] We previously reported the potent hypopigmentation effects of decapeptide-12 in human skin. Further clinical studies revealed an overall improvement in facial skin appearance in pigmentation disorder patients treated for eight weeks with a topical cream containing 0.01 percent decapeptide-12 twice daily. These findings led us to hypothesize that decapeptide-12 may modulate sirtuin activity to improve overall skin appearance. To clarify this possibility, we evaluated the effect of decapeptide-12 on sirtuin transcription in human epidermal progenitor cells.

[0027] material and method reagent Decapeptide-12 (YRSRKYSSWY) SEQ ID NO:9 was synthesized by Bio Basic, Inc. (Ontario, Canada) using solid phase FMOC chemistry. Oxyresveratrol was purchased from Sigma-Aldrich (St. Louis, MO).

[0028] cell culture Human neonatal epidermal progenitor cells (Thermo Fisher Scientific, NY) were cultured at 2 × 10 5 Cells were seeded into 6-well plates at a density of 1000 cells / well. Two ml of Epilife medium containing 60 μM calcium chloride (Thermo Fisher Scientific, NY) was added to each well. Plates were incubated at 37 degrees Celsius and 5 percent CO2 in a humidified chamber. After 24 hours, cells were treated with various concentrations of oxyresveratrol or decapeptide-12 dissolved in PBS containing 5 percent DMSO. Control wells received vehicle only (5 percent DMSO and PBS). The final concentration of DMSO in each well was 0.05 percent.

[0029] Total RNA extraction, quantification, and cDNA synthesis After a 72-h incubation period, cells were trypsinized and total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's protocol.

[0030] RNA concentration was determined using Nanodrop (Thermo Fisher Scientific, NY). cDNA was synthesized using 2 μg of total RNA with oligo dT primer and TaqMan reverse transcription reagent (Thermo Fisher Scientific, NY). Reactions were performed in a DNA Engine Peltier Thermal Cycler (Bio-Rad, Hercules, CA). Annealing temperature was 25 degrees Celsius for 10 min, followed by first strand synthesis at 48 degrees Celsius for 1 h and heat inactivation at 95 degrees Celsius for 5 min.

[0031] Semi-quantitative analysis SIRT1-7 primers (Table 1) were designed using Primer3. Semi-quantitative PCR reactions were performed in a DNA Engine Peltier Thermo Cycler (Bio-Rad, Hercules, CA). PCR was performed under the following conditions: 34 cycles of denaturation at 94 degrees Celsius for 2 minutes and primer extension at 54 degrees Celsius for 30 seconds for SIRT1-7 and housekeeping gene 18S.

[0032] Table 1: Primer sequences for SIRT1-7 and 18S

[0033] [Table 1]

[0034] Samples were run and separated on a 1.5 percent agarose gel containing 0.5 μg / ml ethidium bromide and imaged using a FluorChem HD2 Imaging System (Protein simple, San Jose, CA). Densitometric analysis was performed using AlphaEase FC software (Protein simple, San Jose, CA). Intensity ratios were calculated by dividing the intensity value for each gene by the intensity value of the internal control gene 18S.

[0035] Viability / proliferation and cytotoxicity assays Proliferation rates were determined using the TACS® MTT Cell Proliferation Kit (R&D systems, Minneapolis, MN). Cells were plated at 2.5 × 10 cells / well in 96-well plates in a humidified atmosphere of 37 degrees Celsius and 5 percent CO2. 4 After 24 h, decapeptide-12 or oxyresveratrol was added to the corresponding wells at various concentrations (0, 3, 10, 30, 100, 300, and 1000 μM), and the cultures were then incubated for 72 h. The remaining procedures were carried out according to the manufacturer's protocol.

[0036] Cytotoxicity was measured using a trypan blue dye exclusion assay. 5 Cells were plated at a density of 1000 cells / well. Different concentrations of decapeptide-12 or oxyresveratrol (0, 3, 10, 30, 100, 300, and 1000 μM) were added to each well. The plates were incubated at 37 degrees Celsius in a humidified 5 percent CO2 chamber. After 72 hours, an aliquot was taken and the cells were counted using a hemocytometer. Cytotoxicity was measured according to the following formula: [1-(number of cells in control-number of viable cells in test sample) / number of cells in control] x 100 percent.

[0037] statistical analysis Mean values ​​and their standard errors were calculated from three independent runs using Microsoft Excel, and statistical significance was determined using paired analysis of variance. P values ​​were considered statistically significant at P < 0.05.

[0038] result Effect of the decapeptide on proliferation rate and cytotoxicity: We first evaluated the cytotoxic effects of decapeptide-12 and oxyresveratrol in human epidermal progenitor cells. Figure 2A shows that treatment with 100 μM decapeptide-12 or oxyresveratrol resulted in 3 ± 1 or 6 ± 1 percent cell death, respectively. At 1 mM, decapeptide-12 or oxyresveratrol resulted in 7 ± 2 or 16 ± 2 percent cell death, respectively.

[0039] We also evaluated the effects of decapeptide-12 and oxyresveratrol on the viability and proliferation of human epidermal progenitor cells. Figure 2B shows that treatment with 300 μM decapeptide-12 or oxyresveratrol reduced cell proliferation by 2 ± 1 percent or 5 ± 1 percent, respectively. However, unlike 1 mM decapeptide-12, which reduced proliferation by 3 ± 2 percent, incubation with oxyresveratrol for 3 days reduced proliferation by 12 ± 2 percent.

[0040] Decapeptide-12 upregulated the transcription of SIRT1-7: We next evaluated the effects of oxyresveratrol and decapeptide-12 on sirtuin expression in human epidermal progenitor cells. Figures 1A-1D and Table 2 show that decapeptide-12 and oxyresveratrol dose-dependently modulated the transcription of SIRT1-7. At 30 μM oxyresveratrol, SIRT1 transcript levels were upregulated by 125 ± 9 percent compared to control cells, whereas SIRT3, SIRT6, and SIRT7 were upregulated by 133 ± 5, 73 ± 8, and 95 ± 7 percent, respectively.

[0041] Table 2. Gene expression profiles of SIRT1–7 in response to treatment with decapeptide-12 (a) and oxyresveratrol (b). Results are the average of three independent trials.

[0042] [Table 2]

[0043] [Table 3]

[0044] The data show that 100 μM decapeptide-12 increased the transcription of SIRT1 by 141 ± 11 percent compared to untreated cells, whereas SIRT3, SIRT6, and SIRT7 were increased by 121 ± 13 percent, 147 ± 8 percent, and 95 ± 14 percent, respectively (Figure 1A-D).

[0045] discussion The multifaceted role of sirtuins in slowing cell senescence and blocking the progression of premature aging has helped to demonstrate that they are potent anti-aging proteins. The therapeutic use of resveratrol as a SIRT1 activator and potential anti-aging agent has been widely studied and documented. Resveratrol protects human endothelium from H2O2-induced oxidative stress and senescence via activation of SIRT1. Similarly, oxyresveratrol is also a potent antioxidant and free radical scavenger. However, unlike resveratrol, it is less cytotoxic and better water soluble. Therefore, it was chosen to use it as a positive control to compare the performance of decapeptide-12 with its ability to modulate sirtuin transcription in human epidermal keratinocytes.

[0046] Although all seven sirtuins were upregulated following treatment with decapeptide-12, our discussion focuses on those sirtuins directly related to skin aging.

[0047] At 100 μM or 1 mM, decapeptide-12 increased SIRT1 transcription by an impressive 141 or 213 percent, respectively. SIRT1 is primarily a nuclear deacetylase. It controls various cellular processes, such as cell proliferation, differentiation, apoptosis, metabolism, stress response, genomic stability, and cell survival. Cao et al. reported that SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and c-Jun N-terminal kinase in cultured skin keratinocytes, suggesting that SIRT1 activators may act as novel anti-skin aging agents. Other researchers reported that SIRT1 can suppress NF-κB signaling, thus delaying the aging process and extending lifespan. SIRT1 activation directly inhibits NF-κB signaling by deacetylating the p65 subunit of the NF-κB complex, promoting oxidative metabolism and inflammation resolution. Therefore, SIRT1 can be considered as an important anti-aging protein that mediates its broad effects in preventing premature senescence and accelerated aging by regulating multiple molecular pathways.

[0048] SIRT3 transcription was increased by 121 percent after treatment with 100 μM decapeptide. SIRT3 is primarily associated with regulating various mitochondrial processes, including β-oxidation, ATP generation, and ROS management. SIRT3 is also involved in maintaining the regenerative capacity of hematopoietic stem cells. SIRT3 is suppressed with aging, and SIRT3 upregulation in aged hematopoietic stem cells improves regenerative capacity. This finding establishes a critical role for SIRT3 in maintaining stemness and, more importantly, helps pave the way for future stem cell-based interventions against metabolic disorders that lead to premature aging.

[0049] SIRT6 can be regarded as an important anti-aging protein with multifaceted roles in DNA damage repair, metabolic regulation, inflammation, and tumor suppression. SIRT6 became famous for its knockout mouse model developing a severe premature aging phenotype that results in death within one month. Moreover, SIRT6 is the only mammalian sirtuin that shows a clear increase in lifespan when overexpressed whole-body in mice. Moreover, Kawahara et al. reported that SIRT6 attenuates hyperactive NF-κB signaling by deacetylating histone H3 at K9 on the promoters of NF-κB target genes, reinforcing the role of SIRT6 as a key anti-inflammatory protein.

[0050] Baohua et al. showed that SIRT6 plays a key role in the process of skin aging through the modulation of collagen metabolism and NF-κB signaling. They reported that blocking SIRT6 significantly reduced hydroxyproline content by inhibiting collagen type 1 transcription, promoting matrix metalloproteinase 1 secretion, and increasing NF-κB signaling. Taken together, SIRT6 stands out as a key modulator of the anti-aging process by regulating multiple pathways that delay cell senescence and accelerated aging. Therefore, decapeptide-12, which enhanced SIRT6 transcription by 147 percent at 100 μM, may hold great promise as a therapeutic anti-aging candidate to combat premature skin aging and photodamaged skin phenotypes that often occur simultaneously.

[0051] In summary, this report shows that decapeptide-12 significantly upregulates the transcriptional levels of SIRT1, SIRT3, and SIRT6, all three of which play important roles in combating skin aging and other aging-related pathologies. Clinical studies of different topical formulations containing decapeptide-12 are currently being designed to help validate the in vitro findings and test the efficacy of this potent sirtuin activator in vivo. EXAMPLES

[0052] In this example, specific modifications were made to the P4 decapeptide, as detailed in Table 3 below.

[0053] [Table 4]

[0054] These modifications to decapeptide P4 may serve to improve stability to proteases and facilitate transdermal or transcellular penetration, or both.

[0055] The peptides of the invention may include residues from any of the naturally occurring amino acids or non-naturally occurring amino acids. These naturally occurring and non-naturally occurring amino acids may be in the D or L configuration or may include dextrorotatory forms of both. The terms D and L are used in this application as they are known to be used in the art. The peptides of the invention include single amino acids and short spans (e.g., 1-20) of amino acids. Additionally, modified peptides of the invention may also include monomers or dimers.

[0056] Standard one-letter and three-letter amino acid codes are used in this application and are set forth below in Table A.

[0057] [Table 5]

[0058] As noted above, the indicated residues may be naturally occurring L-amino acids, or modifications thereof, i.e., chemical modifications, optical isomers, or links to modifying groups. It is contemplated that certain modifications may be made within the peptides to maintain the ability of the peptides to specifically modulate expression of sirtuin genes.

[0059] We evaluated the effects of decapeptides P4, P4A, P4B, and P4C on the transcript levels of sirtuins 1 to 7. Table 4 summarizes the transcript levels of all four decapeptides with their corresponding genes at the tested concentrations of 10, 30, 50, 100, and 300 (all in μM).

[0060] [Table 6] TIFF0007620429000007.tif103131

[0061] At low concentrations, the native decapeptide P4 showed enhanced transcription levels compared to the modified decapeptides. However, each of the three modified decapeptides (P4A, P4B, and P4C) upregulated the transcription levels of sirtuin genes compared to the control. At a concentration of 100 μM, the effect on transcription levels was comparable for all four decapeptides.

[0062] The proliferation rates of three human cell lines (epidermal progenitor cells, melanoblasts, and fibroblasts) were determined using the TACS® MTT Cell Proliferation Kit. 2.5 × 10 cells were cultured in 96-well plates at 37 degrees Celsius in a humidified atmosphere of 5 percent CO2. 4 Cells were seeded at 1000 x g / well. After 24 h, the decapeptide was added at various concentrations to the corresponding wells and incubated for 72 h. The remaining procedures were carried out according to the manufacturer's protocol.

[0063] The proliferation rate of epidermal progenitor cells after 72 hours is shown in Table 5.

[0064] [Table 7]

[0065] The melanoblast proliferation rate after 72 hours is shown in Table 6.

[0066] [Table 8]

[0067] The fibroblast proliferation rate after 72 hours is shown in Table 7.

[0068] [Table 9]

[0069] Incubation of epidermal progenitor cells, melanoblasts, and fibroblasts with 100 μM decapeptide P4A for 72 hours resulted in a 3 percent decrease in proliferation of all three cell lines.

[0070] At 1000 μM, the proliferation rate of epidermal progenitor cells was reduced by 6 percent, while the proliferation rates of melanoblasts and fibroblasts were reduced by 5 and 4 percent, respectively.

[0071] The effect of each decapeptide on cell viability was also tested. Specifically, cells were incubated with various concentrations of the decapeptide and viability counted compared to the control (untreated cells) using trypan blue. Cytotoxicity was measured according to the following formula:

[0072] [1-(number of cells in control-number of viable cells in test sample) / number of cells in control] x 100 percent.

[0073] Table 8 shows the survival rate of epidermal progenitor cells after 7 days.

[0074] [Table 10]

[0075] The survival rate of melanoblasts after 7 days is shown in Table 9.

[0076] [Table 11]

[0077] The viability of the fibroblasts after 7 days is shown in Table 10.

[0078] [Table 12]

[0079] At a concentration of 100 μM, cell viability remained above 97 percent in all three cell lines. At 1,000 μM, cell viability decreased by 6 percent compared to the control.

[0080] In conclusion, recent reports have detailed the multifaceted roles played by sirtuins in inhibiting premature aging, delaying cell senescence, extending life span, and ameliorating a wide range of aging disorders. Herein, we report findings on decapeptide-12, a potent sirtuin activator, and compare its performance with the well-documented oxyresveratrol. Treatment of human epidermal progenitor cells with 100 μM decapeptide-12 increased SIRT1 transcription by 141 ± 11 percent compared to control cells, while SIRT3, SIRT6, and SIRT7 levels increased by 121 ± 13 percent, 147 ± 8 percent, and 95.4 ± 14 percent, respectively. Decapeptide-12 upregulated sirtuin transcription to similar levels as oxyresveratrol, but with reduced cytotoxicity. Thus, decapeptide-12 may hold promise as a safer therapeutic agent to combat skin aging and other aging-associated pathologies.

[0081] Although the above description refers to typical decapeptide concentrations of 100 μM or more where an effect is evident, results have also demonstrated that lower concentrations have positive effects. Thus, in some embodiments, decapeptide concentrations of 1 μM or more are utilized, and in certain embodiments, peptide concentration ranges of 100 μM or more may be used. Examples of peptide concentration ranges according to various embodiments are 1 μM or more, 5 μM or more, 10 μM or more, 30 μM or more, 50 μM or more, 100 μM or more, 300 μM or more, 500 μM or more, and 1000 μM or more.

[0082] It should be further noted that a particular decapeptide may be used in combination with other components to achieve a desired effect.For example, a particular decapeptide may be used in combination with other peptides such as decapeptides P4A, 4B, and / or 4C, and / or other components such as oxyresveratrol.According to such an embodiment, the synergistic effect achieved by including other components may ultimately reduce the concentration of individual components (e.g., decapeptides, etc.) that are required to achieve a desired result.

[0083] The above specifically includes decapeptides and oxyresveratrol as possible additional ingredients, but the embodiments are not limited thereto.Other examples of possible additives include, but are not limited to, alpha-lipoic acid, biotin, caffeine, ceramide, coenzyme Q10, glycolic acid, green tea, human stem cells, human stem cell extracts, hyaluronic acid, hydroquinone, jojoba oil, kojic acid, lactic acid, malic acid, niacinamide, oligopeptides, peptides, plant stem cells, plant stem cell extracts, resveratrol, retinol, vitamin C, vitamin E, and vitamin K, among others.

[0084] It should be noted that embodiments may be utilized to treat various types of skin cells. Examples of terminally differentiated skin cells include, but are not limited to, keratinocytes, fibrocytes, melanocytes, and immune cells such as Langerhans cells (e.g., histiocytes or dendrocytes), which also age over time.

[0085] Embodiments may also be utilized to treat skin progenitor cells to reduce skin aging and allow for skin regeneration over its lifespan. Examples of such progenitor cells include, but are not limited to, epidermal keratinocyte progenitor cells, fibroblasts, melanoblasts, histoblasts, or dendroblasts, which are precursors to Langerhans cells that reside in the epidermis.

[0086] Finally, although the above describes the treatment of human skin cells, certain embodiments are not limited to such an approach. Alternative embodiments may utilize the treatment of skin cells from other organisms, including but not limited to mammals such as cows (e.g., for leather production), pigs, and other animals (e.g., dogs, cats, and other animals that may be evaluated based on the appearance of their skin for athletic purposes).

[0087] This description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the above teachings. The embodiments have been chosen and described in order to best explain the principles of the invention and its practical application. This description will enable others skilled in the art to best utilize and practice the invention in various embodiments and with various modifications suited to particular applications. The scope of the invention is defined by the following claims.

Claims

1. A composition for use in a method for treating a skin aging disorder associated with premature senescence in a subject's skin cells by modulating expression of a sirtuin gene in skin cells, the method comprising administering the composition to a subject, wherein the composition comprises an effective amount of one or more peptides consisting of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

12.

2. The composition of claim 1 , wherein the skin cells are progenitor cells, and the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendroblasts.

3. The composition of claim 1 or 2, wherein the skin cells are terminally differentiated, wherein the skin cells are keratinocytes, melanocytes, fibrocytes, histiocytes, or dendrocytes.

4. The composition of any one of claims 1 to 3, wherein the peptide is present at a concentration of 1 μM or greater.

5. The composition according to any one of claims 1 to 4, wherein the sirtuin gene comprises the base sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

6. The composition according to any one of claims 1 to 5, further comprising oxyresveratrol.

7. The composition of any one of claims 1 to 6, wherein the skin cells are mammalian cells, wherein the skin cells are human skin cells.

8. The composition according to any one of claims 1 to 7, wherein the peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 9 modified with a modifying group, and the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

9. The composition of any one of claims 1-8, wherein the peptide comprises residues independently selected from naturally occurring L-amino acids or non-naturally occurring D-amino acids and maintains the ability to specifically modulate expression of a sirtuin gene.

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