Tyrosine inhibitors with immunosuppressive activity in human neonatal keratinocyte progenitors

Abstract

To provide tyrosine inhibitors having immunosuppressive activity in human neonatal keratinocyte progenitors.SOLUTION: Embodiments relate to tyrosine inhibitors that exhibit immunosuppressive activity in human neonatal keratinocyte progenitors. Particular embodiments feature the immunosuppressive effects of a decapeptide and / or oxyresveratrol, as measured by two different methods: blockade of stimulated cell growth, and inhibition of cytotoxic killing.SELECTED DRAWING: None

Description

[Technical Field]

[0001] explanation CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Patent Application No. 62 / 794,582, filed January 19, 2019, which is incorporated herein by reference along with all other references cited therein.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing entitled "20200120_ELIXP005_ST25.TXT" (3 kilobytes), which was created on January 20, 2020, and submitted electronically to this application. [Background technology]

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

[0004] Embodiments relate to tyrosine inhibitors that exhibit immunosuppressive activity in human neonatal keratinocyte progenitor cells. Certain embodiments feature the immunosuppressive effects of the decapeptide and / or oxyresveratrol as measured by two different methods: blockade of stimulated cell proliferation and inhibition of cytotoxic killing.

[0005] Some embodiments include a method of treating a subject for cellular immunosuppression, the method comprising administering to a subject in need of treatment a composition comprising an effective amount of one or more peptides, oxyresveratrol, or both, wherein the one or more peptides comprise SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12. The cells can be mammalian cells. The cells can be skin cells. The administration can include oral administration. Various embodiments are described in this patent.

[0006] In one embodiment, a method of treating a subject by immunosuppressing cells comprises administering to a subject in need of treatment a composition comprising an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0007] In various embodiments, the peptide consists of SEQ ID NO:9. The cell is a mammalian cell. The mammalian cell is a skin cell. The mammalian skin cell is a progenitor cell. The progenitor cell is an epidermal keratinocyte progenitor cell, a melanoblast, a fibroblast, a histoblast, or a dendritic cell. Administration is by oral administration. The cell is terminally differentiated. The cell is a keratinocyte, a melanocyte, a fibrocyte, a histiocyte, or a dendritic cell. The peptide is present at a concentration of about 1 millimolar or less. The composition further comprises oxyresveratrol.

[0008] In one embodiment, a method for treating a subject by cellular immunosuppression, comprising administering to a subject in need of treatment a composition comprising an effective amount of oxyresveratrol.

[0009] In various embodiments, the oxyresveratrol is present in a concentration of about 0.1 millimolar to about 1.0 millimolar. The composition further comprises an effective amount of one or more peptides, and one or more The peptide comprises SEQ ID NO: 9. The cell is a mammalian cell. The mammalian cell is a skin cell. The mammalian skin cell is a progenitor cell. The progenitor cell is an epidermal keratinocyte progenitor cell, a melanoblast, a fibroblast, a histoblast, or a dendritic cell. The administration is by oral administration. The cell is a terminally differentiated keratinocyte, a melanocyte, a fibrocyte, a histiocyte, or a dendritic cell.

[0010] Other objects, features, and advantages of the present invention will become apparent from a consideration of the following detailed description and the accompanying drawings, in which like reference designations represent like features throughout the drawings. [Brief explanation of the drawings]

[0011] [Figure 1A] (a) Dose-dependent increase in SIRT1 transcription. Data are expressed as fold increase compared to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1B] (b) Dose-dependent increase in SIRT3 transcription is shown. Data are expressed as fold increase compared to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1C] (c) Dose-dependent increase in SIRT6 transcription is shown. Data are expressed as fold increase compared to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 1D] (d) Dose-dependent increase in SIRT7 transcription is shown. Data are expressed as fold increase compared to the internal control gene 18S and represent the mean ± SEM of three independent experiments. [Figure 2A] Figure 1 shows the cytotoxic effects of decapeptide-12 and oxyresveratrol on epidermal keratinocytes. Data are expressed as percent 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 percent control and represent the mean ± SEM of three separate experiments. *P<0.05. [Figure 3] 1 shows the chemical structure of decapeptide P4 of SEQ ID NO:9. [Figure 4] 1 shows the chemical structure of oxyresveratrol. [Figure 5] 1 is a plot of the immunosuppressive effect of decapeptide P4 of SEQ ID NO:9. [Figure 6] 1 is a plot of the immunosuppressive effect of oxyresveratrol. [Figure 7] 1 is a plot of the immunosuppressive effect of decapeptide P4 of SEQ ID NO:9. [Figure 8] 1 is a plot of the immunosuppressive effect of oxyresveratrol. DETAILED DESCRIPTION OF THE INVENTION

[0012] The skin displays the consequences of chronological and photoaging, motivating us to constantly recognize the aging process and seek treatments to slow or reverse its effects. Skin aging has traditionally been classified as extrinsic or intrinsic. Recent evidence indicates that both types share key molecular features, including alterations in signaling pathways that promote matrix metalloproteinase expression, reduced procollagen synthesis, and connective tissue damage.

[0013] In human skin, aging is associated with an increase in the number of senescent cells and a decrease in the capacity for 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. have demonstrated in vivo evidence of a causal relationship between mitochondrial oxidative damage, cellular senescence, and the skin aging phenotype. We report in vivo evidence. Furthermore, ultraviolet (UV) radiation stimulates ROS synthesis, which is associated with mutagenesis and photoaging. Consistent with these findings, data suggest altered expression of sirtuin activity in UV-irradiated versus sun-protected skin, suggesting that these differences may be responsible for certain aspects of skin aging.

[0014] Cellular senescence is a process in which cells stop dividing and undergo significant chromatin and secretome changes. This refers to processes that result in specific phenotypic changes, such as the activation of tumor suppressors and tumor suppressors. Numerous reports have helped establish the concept of sirtuins as potent anti-aging proteins and detailed their multifaceted roles in delaying cellular senescence and 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.

[0015] Mammals have seven sirtuin genes (SIRT1-7) that are localized in different subcellular compartments and capable of diverse actions. Biochemically, sirtuins are a class of proteins that primarily possess NAD+-dependent lysine deacetylase activity. Sirtuins are widely recognized as key regulators of multiple metabolic pathways, sensors of intracellular energy and redox status, and modulators of oxidative stress.

[0016] These findings have sparked interest in developing small molecule activators or drugs that can help slow the progression of aging and its wide range of age-related disorders. Among the seven mammalian sirtuins, SIRT1 has been the most extensively studied in relation to aging and longevity. For example, the anti-aging effects of resveratrol are primarily due to activation of SIRT1. Indeed, Ido et al. reported that resveratrol improved cellular aging and proliferation dysfunction by enhancing the activity of AMP-activated protein kinase and sirtuins.

[0017] We previously reported the potent hypopigmentation effects of decapeptide-12 in human skin. Further clinical studies revealed overall improvement in facial skin appearance in patients with dyschromatosis treated twice daily for 8 weeks with a topical cream containing 0.01 percent decapeptide-12. These findings led us to hypothesize that decapeptide-12 could 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.

[0018] The report details the multifaceted roles that sirtuins play in inhibiting premature aging, delaying cellular senescence, extending lifespan, and ameliorating a wide range of aging disorders. Herein, we report our findings on a potent sirtuin activator, decapeptide-12, and compare its performance with the well-documented oxyresveratrol. Treatment of human epidermal keratinocyte progenitor cells with 100 micromolar 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 increased sirtuin transcription to levels similar to oxyresveratrol but with reduced cytotoxicity.

[0019] material and method reagent Decapeptide-12 (YRSRKYSSWY) SEQ ID NO: 9 was synthesized using solid-phase FMOC chemistry by Bio Basic, Inc. (Ontario, Canada). Oxyresveratrol was purchased from Sigma-Aldrich (St. Louis, MO).

[0020] cell culture Human neonatal epidermal progenitor cells (Thermo Fisher Scientific, NY) were added at 2 × 10 per well. 5 Cells were seeded into 6-well plates at a density of 1000 x g / ml. Each well received 2 ml of Epilife medium containing 60 micromolar calcium chloride (Thermo Fisher Scientific, NY). The plates were then incubated at 37°C for 1 hour. The cells were incubated in a humidified chamber with 5 percent CO2. After 24 hours, the 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.

[0021] Total RNA extraction, quantification, and cDNA synthesis After a 72-hour incubation period, cells were trypsinized and total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's protocol.

[0022] RNA concentration was determined using a nanodrop (Thermo Fisher Scientific, NY). 2 μg of total RNA was used to synthesize cDNA using oligo-dT primers and TaqMan reverse transcription reagents (Thermo Fisher Scientific, NY). The reaction was performed in a DNA Engine Peltier Thermal Cycler (Bio-Rad, Hercules, CA). The annealing temperature was 25°C for 10 minutes, followed by first-strand synthesis at 48°C for 1 hour and heat inactivation at 95°C for 5 minutes.

[0023] Semi-quantitative analysis SIRT1-7 primers (Table A) were designed using Primer3. Semi-quantitative PCR reactions were performed using a DNA Engine Peltier Thermo Cycler (Bio-Rad, Hercules, CA). PCR was performed under the following conditions: 34 cycles of denaturation at 94°C for 2 minutes and primer extension at 54°C for 30 seconds for SIRT1-7 and the housekeeping gene 18S. [Table 1]

[0024] Samples were separated on a 1.5 percent agarose gel containing 0.5 micrograms / milliliter ethidium bromide and imaged using a FluorChem HD2 Imaging System (Protein Simple, San Jose, CA). Densitometry analysis was performed using AlphaEase FC software (Protein Simple, San Jose, CA). Intensity ratios were calculated by dividing the intensity value of each gene by the intensity value of the internal control gene 18S.

[0025] 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 per well in 96-well plates in a humidified atmosphere containing 5 percent CO at 37°C. 4 After 24 hours, decapeptide-12 or oxyresveratrol was added to the corresponding wells at different concentrations (0, 3, 10, 30, 100, 300, and 1000 micromolar), and the cultures were incubated for 72 hours. The remainder of the procedure was performed according to the manufacturer's protocol.

[0026] Cytotoxicity was measured using a trypan blue dye exclusion assay. Cells were plated at 4 × 10 per well. 5 The cells were cultured in a 6-well plate at a density of 1000 x g. Each well contained a different concentration of dextran. Capeptide-12 or oxyresveratrol (0, 3, 10, 30, 100, 300, and 1000 micromolar) was added. Plates were incubated at 37 degrees Celsius in a 5 percent CO2 humidified chamber. After 72 hours, aliquots were taken and 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.

[0027] 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 ​​of P<0.05 were considered statistically significant.

[0028] result Effect of the decapeptide on proliferation rate and cytotoxicity: We first evaluated the cytotoxic effects of decapeptide-12 and oxyresveratrol on human epidermal progenitor cells. Figure 2A shows that treatment with 100 micromolar decapeptide-12 or oxyresveratrol resulted in 3±1 percent or 6±1 percent cell death, respectively. At 1 millimolar, decapeptide-12 or oxyresveratrol resulted in 7±2 percent or 16±2 percent cell death, respectively.

[0029] 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 micromolar decapeptide-12 or oxyresveratrol reduced cell proliferation by 2 ± 1 percent or 5 ± 1 percent, respectively. However, unlike 1 millimolar decapeptide-12, which reduced proliferation by 3 ± 2 percent, incubation with oxyresveratrol for 3 days reduced proliferation by 12 ± 2 percent.

[0030] Decapeptide-12 increased the transcription of SIRT1-7: Next, we evaluated the effects of oxyresveratrol and decapeptide-12 on sirtuin expression in human epidermal progenitor cells. Figures 1A-1D and Table B show that decapeptide-12 and oxyresveratrol dose-dependently regulated the transcription of SIRT1-7. At 30 micromolar oxyresveratrol, SIRT1 transcription levels increased by 125 ± 9 percent compared to control cells, whereas SIRT3, SIRT6, and SIRT7 increased by 133 ± 5 percent, 73 ± 8 percent, and 95 ± 7 percent, respectively.

[0031] Tables B and C. Gene expression profiles of SIRT1-7 in response to treatment with decapeptide-12 (Table B) and oxyresveratrol (Table C). Results are the average of three independent experiments. [Table 2] [Table 3]

[0032] The data show that 100 micromolar decapeptide-12 increased SIRT1 transcription by 141 ± 11 percent compared to untreated cells, whereas SIRT3, SIRT6, and SIRT7 increased by 121 ± 13 percent, 147 ± 8 percent, and 95 ± 14 percent, respectively ( Figures 1A–1D ).

[0033] Consideration The multifaceted roles that sirtuins play in delaying cellular senescence and preventing the progression of premature aging have helped validate sirtuins as potent anti-aging proteins. The therapeutic use of resveratrol as a SIRT1 activator and potential anti-aging agent has been extensively studied and demonstrated. Resveratrol protects human endothelium from H2O2-induced oxidative stress and aging through activation of SIRT1. Similarly, oxyresveratrol is also a potent antioxidant and free radical scavenger. However, unlike resveratrol, oxyresveratrol has low cytotoxicity and excellent water solubility. Therefore, we chose to use oxyresveratrol as a positive control against which we compared the performance of decapeptide-12 and its ability to modulate sirtuin transcription in human epidermal keratinocytes.

[0034] Even though all seven sirtuins were increased after treatment with decapeptide-12, our discussion focuses on the sirtuins directly related to skin aging.

[0035] At 100 micromolar and 1 millimolar concentrations, decapeptide-12 significantly increased SIRT1 transcription by 141 and 213 percent, respectively. SIRT1 is primarily a nuclear deacetylase. It regulates various cellular processes, including cell proliferation, differentiation, apoptosis, metabolism, stress response, genome stability, and cell survival. Cao et al. reported that SIRT1 protects against UVB- and H2O2-induced cell death in cultured skin keratinocytes through the regulation of p53 and c-Jun N-terminal kinase, suggesting that SIRT1 activators may serve as novel skin anti-aging agents. Other researchers have reported that SIRT1 suppresses NF-κB signaling, thereby delaying the aging process and extending lifespan. Activation of SIRT1 directly inhibits NF-κB signaling by deacetylating the p65 subunit of the NF-κB complex, promoting oxidative metabolism and inflammation resolution. SIRT1 can therefore be considered a key anti-aging protein that mediates a wide range of effects in preventing premature and accelerated aging by regulating multiple molecular pathways.

[0036] SIRT3 transcription increased by 121 percent after treatment with 100 micromolar decapeptide. SIRT3 primarily functions in various mitochondrial processes, including β-oxidation, ATP production, and ROS management. SIRT3 is associated with regulating endogenous processes. SIRT3 is also involved in maintaining the regenerative potential of hematopoietic stem cells. SIRT3 is suppressed with age, and increased expression of SIRT3 in senescent hematopoietic stem cells improves their regenerative potential. This finding establishes the critical role SIRT3 plays in maintaining regenerative potential and, more importantly, helps pave the way for future stem cell-based interventions for metabolic disorders that lead to premature aging.

[0037] SIRT6 can be considered an important anti-aging protein with multifaceted roles in DNA damage repair, metabolic regulation, inflammation, and tumor suppression. SIRT6 attracted attention when its knockout mouse model developed severe premature aging phenotypes and died within one month. Furthermore, SIRT6 is the only mammalian sirtuin that showed a clear increase in lifespan when overexpressed systemically in mice. Furthermore, 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, thereby strengthening the role of SIRT6 as an important anti-inflammatory protein.

[0038] Baohua et al. demonstrated that SIRT6 plays a key role in the skin aging process through the regulation of collagen metabolism and NF-κB signaling. They reported that blocking SIRT6 significantly reduced hydroxyproline content by inhibiting type 1 collagen transcription, promoting matrix metalloproteinase 1 secretion, and increasing NF-κB signaling. Collectively, SIRT6 stands out as a key modulator of the anti-aging process by regulating multiple pathways to delay cellular senescence and accelerated aging. Therefore, decapeptide-12, which enhanced SIRT6 transcription by 147 percent at 100 μM, may hold considerable promise as a therapeutic anti-aging candidate to address the phenotypes in which premature skin aging and photodamaged skin often coexist.

[0039] In summary, this report shows that decapeptide-12 significantly increases the transcription levels of SIRT1, SIRT3, and SIRT6, all three of which play important roles in combating skin aging and other age-related pathologies. Clinical trials using various topical formulations containing decapeptide-12 are currently being designed to help validate the in vitro findings and test the in vivo efficacy of this potent sirtuin activator. [Example]

[0040] In this example, specific modifications were made to the P4 decapeptide, as detailed in Table D below. [Table 4]

[0041] These modifications to decapeptide P4 may serve to improve stability to proteases and enhance transdermal or transcellular penetration, or both.

[0042] The peptides of the present invention can include residues from any naturally occurring amino acid or from non-naturally occurring amino acids. These naturally occurring and non-naturally occurring amino acids can be in the D or L configuration, or can include dextrorotatory forms of both. The terms D and L are used in this application as they are known in the art. The peptides of the present invention include single amino acids and short spans (e.g., 1-20) of amino acids. Furthermore, the modified peptides of the present invention can also include monomers or dimers.

[0043] In this application, the standard one-letter and three-letter amino acid codes are used and are shown in Table E below. [Table 5]

[0044] As noted above, the residues shown may be naturally occurring L-amino acids or modifications thereof, i.e., chemical modifications, optical isomers, or attachment to modifying groups. It is contemplated that certain modifications may be made within the peptides that maintain the ability of the peptides to specifically regulate expression of sirtuin genes.

[0045] The effects of decapeptides P4, P4A, P4B, and P4C on the transcription levels of sirtuins 1 to 7 were evaluated. Table F summarizes the transcription levels of all four decapeptides with their corresponding genes at tested concentrations of 10, 30, 50, 100, and 300 (all micromolar). [Table 6]

[0046] 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) increased the transcription levels of sirtuin genes compared to the control. At 100 micromolar concentrations, the effect on transcription levels was similar for all four decapeptides.

[0047] The proliferation rates of three human cell lines (epidermal progenitor cells, melanoblasts, and fibroblasts) were determined using the TACS® MTT Cell Proliferation Kit. Cells were seeded at 2.5 x 10 per well in a 96-well plate at 37°C in a humidified atmosphere containing 5 percent CO2. After 24 hours, the decapeptide was added at different concentrations to the corresponding wells and incubated for 72 hours. The remainder of the procedure was performed according to the manufacturer's protocol.

[0048] Table G shows the proliferation rate of epidermal progenitor cells after 72 hours. [Table 7]

[0049] Table H shows the melanoblast proliferation rate after 72 hours. [Table 8]

[0050] Table I shows the proliferation rate of fibroblasts after 72 hours. [Table 9]

[0051] Incubation of epidermal progenitor cells, melanoblasts, and fibroblasts with 100 micromolar decapeptide P4A for 72 hours resulted in a 3 percent reduction in the proliferation rate of all three cell lines.

[0052] At 1000 micromolar, the proliferation rate of epidermal progenitor cells was reduced by 6 percent, while the proliferation rates of melanoblasts and fibroblasts were reduced by 5 percent and 4 percent, respectively.

[0053] The effect of each decapeptide on cell viability was also tested. Specifically, cells were incubated with various concentrations of the decapeptide and then counted for viability using trypan blue compared to the control (untreated cells). 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.

[0054] Table J shows the survival rate of epidermal progenitor cells after 7 days. [Table 10]

[0055] Table K shows the survival rate of melanoblasts after 7 days. [Table 11]

[0056] Table L shows the viability of fibroblasts after 7 days. [Table 12]

[0057] At 100 micromolar concentrations, cell viability remained above 97 percent for all three cell lines. At 1,000 micromolar, cell viability decreased by 6 percent compared to the control.

[0058] In conclusion, recent reports detail the multifaceted roles that sirtuins play in inhibiting premature aging, delaying cellular senescence, extending lifespan, and ameliorating a wide range of aging disorders. Herein, we report our findings on decapeptide-12, a potent sirtuin activator, and compare it with oxyresveratrol, whose performance has been well documented. Treatment of human epidermal progenitor cells with 100 micromolar 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 increased sirtuin transcription to levels similar to oxyresveratrol but with reduced cytotoxicity. Therefore, decapeptide-12 may hold promise as a safer treatment for combating skin aging and other age-related pathologies.

[0059] While the above discussion refers to typical decapeptide concentrations of 100 micromolar or greater where an effect is evident, results also indicate that lower concentrations have a positive effect. Thus, in some embodiments, decapeptide concentrations of 1 micromolar or greater can be utilized, with particular embodiments using peptide concentration ranges of 100 micromolar or greater. Examples of peptide concentration ranges according to various embodiments include 1 micromolar or greater, 5 micromolar or greater, 10 micromolar or greater, 30 micromolar or greater, 50 micromolar or greater, 100 micromolar or greater, 300 micromolar or greater, 500 micromolar or greater, and 10 00 micromolar or more.

[0060] Furthermore, it should be noted that certain decapeptides can be used in combination with other ingredients to achieve the desired effect. For example, certain decapeptides can be used in combination with other peptides, such as decapeptides P4A, 4B, and / or 4C, and / or with other ingredients, such as oxyresveratrol. According to such embodiments, the synergistic effect achieved by including other ingredients may ultimately reduce the concentration of any individual ingredient (e.g., decapeptide, etc.) required to achieve the desired result.

[0061] Although decapeptide and oxyresveratrol are specifically listed above as possible additional ingredients, embodiments are not limited thereto.Other possible additives include, but are not limited to, α-lipoic acid, biotin, caffeine, ceramide, coenzyme Q10, glycolic acid, green tea, human stem cells, human stem cell extract, hyaluronic acid, hydroquinone, jojoba oil, kojic acid, lactic acid, malic acid, niacinamide, oligopeptides, peptides, plant stem cells, plant stem cell extract, resveratrol, retinol, vitamin C, vitamin E, and vitamin K.

[0062] It should be noted that embodiments may be utilized to treat a variety of skin cell types. Examples of terminally differentiated skin cells may include, but are not limited to, keratinocytes, fibrocytes, melanocytes, and immune cells such as Langerhans cells (e.g., histiocytes or dendritic cells), which also senesce over time.

[0063] Embodiments may also be utilized to treat skin progenitor cells to reduce skin aging and enable skin renewal throughout its lifespan, including, but not limited to, epidermal keratinocyte progenitor cells, fibroblasts, melanoblasts, histoblasts, or dendritic blasts, which are precursors to epidermis-resident Langerhans cells.

[0064] Finally, while the above describes the treatment of human skin cells, certain embodiments are not limited to such an approach. Alternative embodiments can utilize the treatment of skin cells from other organisms, including, but not limited to, mammals such as cattle (e.g., in leather manufacturing), pigs, and other animals (e.g., dogs, cats, and other animals that may be evaluated based on skin appearance for contest purposes).

[0065] Clause 1A. A peptide consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0066] Clause 2A. The peptide of Clause 1A, wherein the peptide consists of SEQ ID NO: 9 modified by a modifying group, wherein the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0067] Clause 3A. The peptide according to any of clauses 1A to 2A, consisting of SEQ ID NO: 11, having a tyrosine amino acid at position 6 as the D isoform, and all other amino acids as the L isoform.

[0068] Clause 4A. A composition comprising a first peptide consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0069] Clause 5A. The peptide consists of SEQ ID NO: 9 modified by a modifying group, wherein the modifying group is The composition of claim 4A, wherein the amino-terminal palmitoyl or acetyl group is either palmitoyl or acetyl, or the carboxy-terminal amidation is either palmitoyl or acetyl, or both.

[0070] Clause 6A. The composition according to any of clauses 4A to 5A, consisting of SEQ ID NO: 11, having a tyrosine amino acid at position 6 as the D isoform, and all other amino acids as the L isoform.

[0071] Clause 7A. The composition of any of clauses 4A-6A, wherein the peptide is present at a concentration of 1 μM or greater.

[0072] Clause 8A. A method for treating a subject by regulating the expression of a sirtuin gene in skin cells to reduce symptoms of skin aging, comprising administering to a subject in need of treatment 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.

[0073] Clause 9A. The method of Clause 8A, wherein the peptide consists of SEQ ID NO: 9 modified by a modifying group, wherein the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0074] Clause 10A. The method of any of clauses 8A-9A, wherein the peptide consists of SEQ ID NO: 11, having a tyrosine amino acid at position 6 as the D isoform, and all other amino acids as the L isoform.

[0075] Clause 11A. The method of any of clauses 8A-10A, wherein the skin cells are progenitor cells.

[0076] Clause 12A. The method of Clause 11A, wherein the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendritic blasts.

[0077] Clause 13A. The method of any of clauses 8A-10A, wherein the skin cells are terminally differentiated.

[0078] Clause 14A. The method of Clause 13A, wherein the skin cells are keratinocytes, melanocytes, fibrocytes, histiocytes, or dendritic cells.

[0079] Clause 15A. The method of any of clauses 8A-14A, wherein the peptide is present at a concentration of 1 μM or greater.

[0080] Clause 16A. The method of any of Clauses 8A-15A, wherein 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.

[0081] Clause 17A. The method of any of clauses 8A to 16A, wherein the composition further comprises oxyresveratrol.

[0082] Clause 18A. The method of any of clauses 8A to 17A, wherein the skin cells are mammalian cells.

[0083] Clause 19A. The method of Clause 18A, wherein the skin cells are human.

[0084] Clause 20. A method for regulating expression of a sirtuin gene in skin cells, comprising administering to a subject in need of treatment 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.

[0085] Further attention is focused on the immunosuppressive effects of peptide formulations and / or oxyresveratrol. Figure 3 shows the chemical structure of decapeptide P4 of SEQ ID NO: 9. Figure 4 shows the chemical structure of oxyresveratrol.

[0086] In particular, decapeptide-12 (P4) of SEQ ID NO: 9 and oxyresveratrol showed anti-inflammatory effects as measured by two different methods: 1) blocking of stimulated peripheral blood mononuclear cells (PBMCs), and 2) Inhibition of natural killer (NK)-mediated cytotoxic killing.

[0087] These results are detailed in the Examples below.

[0088] [Example] PBMC proliferation and NK cytotoxicity killing assays Cryopreserved human PBMCs were purchased from Astarte Biologics (Redmond, Washington, LA) and activated with phytohemagglutinin (PHA) to assess proliferation. Interleukin (IL)-2-activated human NK cells were assessed for cytotoxicity killing of human K562 cells using the CytoTox96 Nonradioactive Cytotoxicity Assay Kit (Promega). Protease inhibitors were added to the culture medium to prevent degradation of decapeptide-12.

[0089] Three independent experiments were performed for each experiment. Means and standard errors were calculated using Microsoft Excel (Seattle, Washington), and statistical significance was determined using independent analysis of variance or two-tailed Student's t-test. A p value of <0.05 was considered statistically significant.

[0090] The effects of decapeptide-12 and oxyresveratrol on PBMC proliferation rate after 72 h of exposure to PHA were investigated.

[0091] Figure 5 plots the immunosuppressive effect of decapeptide-12 (P4) on PHA-stimulated PBMC proliferation. Data are expressed as percent (%) control and represent the mean ± SEM of three separate experiments. *P<0.05. E:T indicates the ratio of effector cells to target cells.

[0092] Figure 5 shows that decapeptide-12 statistically significantly (p<0.02) reduced proliferation by 28.0±3.8 percent at 0.05 mM and 54.3±1.1 percent at 0.1 mM. No significant further reduction was achieved at 0.3 or 1 mM (p>0.05).

[0093] Figure 6 plots the immunosuppressive effect of oxyresveratrol on PHA-stimulated PBMC proliferation. Data are expressed as percent (%) control and represent the mean ± SEM of three separate experiments. *P<0.05. E:T indicates the ratio of effector cells to target cells.

[0094] Figure 6 shows that oxyresveratrol reduced proliferation by 35.3 ± 1.8 percent (p < 0.02) at 0.1 mM and 14.7 ± 3.4 percent (p < 0.02) at 3 mM. This indicates that the

[0095] The effect of decapeptide-12 on IL-2 primed NK-mediated cytotoxic killing of K562 cells was also evaluated.

[0096] Figure 7 plots the immunosuppressive effect of decapeptide-12 (P4) on NK92-mediated cytotoxic killing of K562 cells. Data are expressed as percent (%) control and represent the mean ± SEM of three separate experiments. *P<0.05. E:T indicates the ratio of effector cells to target cells.

[0097] Figure 7 shows that at an effector cell to target (E:T) cell ratio of 10:1, decapeptide-12 reduced NK killing by 81.4 ± 1.3 percent and 59.3 ± 3.6 percent (p>0.05) at 0.1 and 0.3 mmol, respectively. At a ratio of 30:1, decapeptide-12 reduced NK killing by 64.8 ± 5.3 percent and 44.8 ± 3.2 percent (p<0.04) at 0.1 and 0.3 mmol, respectively.

[0098] Figure 8 plots the immunosuppressive effect of oxyresveratrol on NK92-mediated cytotoxic killing of K562 cells. Data are expressed as percent (%) control and represent the mean ± SEM of three separate experiments. *P<0.05. E:T indicates the ratio of effector cells to target cells.

[0099] Figure 8 shows that oxyresveratrol reduced NK killing by 88.7 ± 1.8 percent and 86.1 ± 0.9 percent (p < 0.03) at 0.1 and 0.3 mmol / L at an E:T ratio of 10:1. At a 30:1 ratio, oxyresveratrol blocked NK killing by 72.8 ± 1.9 percent and 64.0 ± 3.4 percent (p < 0.03) at 0.1 and 0.3 mmol / L, respectively.

[0100] Thus, the study revealed that decapeptide-12 and oxyresveratrol exhibit anti-inflammatory effects as measured by two methods: 1) blockade of PHA-stimulated PBMC proliferation and 2) inhibition of NK-mediated cytotoxic killing.

[0101] For the blockade of proliferation assay, the effect of decapeptide-12 appeared to be dose-dependent. The effect of oxyresveratrol showed a narrow inhibitory concentration range.

[0102] Indeed, the general trend indicated that oxyresveratrol may have a biphasic effect, as concentrations of 0.3 and 1 mM were progressively less inhibitory than the 0.1 mM concentration.

[0103] Within the range of concentrations tested, decapeptide-12 showed a plateau or maximum inhibition at 0.1 mM or higher. This may be explained by dose-dependent differences in the activation of downstream signaling pathways or feedback loops. Indeed, careful examination of the dose-response curves of sirtuin expression patterns reveals a biphasic effect, with higher concentrations becoming inhibitory.

[0104] In contrast, abrogation of NK killing appeared to be dose-dependent for both decapeptide-12 and oxyresveratrol, with oxyresveratrol showing more pronounced inhibition at all concentrations tested.

[0105] The inhibitory effect was observed at an E:T ratio of 30:1 for both decapeptide-12 and oxyresveratrol. The NKG2D blockade and perforin-mediated cytotoxicity were 10:1 greater than those of the control group.

[0106] It should be noted that resveratrol (an analogue of oxyresveratrol) inhibits PHA-induced proliferation at 0.1 mM. This inhibitory effect may be due to the inhibition of NF-κB, which is also regulated by sirtuins and is associated with, among other effects, the regulation of immune and inflammatory responses, as well as cell proliferation and apoptosis.

[0107] Taken together, the immunosuppressive effects observed here suggest that unique and specific regulatory pathways are involved in different arms of the immune system. Further investigation may clarify these pleiotropic effects.

[0108] For example, evaluation of the effects of these two agents on proinflammatory mediators such as TNFα, IL-1β, IFNγ, and IL-6 may be useful. Additionally, determination of the translational and other transcriptional effects on activated versus resting PBMCs may be useful.

[0109] While the above description refers to a typical decapeptide concentration of about 0 to 1.0 millimolar, noting that a clear effect is observed, different concentrations may also provide positive effects. Thus, some embodiments may utilize decapeptide concentrations of 1.0 millimolar or greater. Exemplary peptide concentration ranges according to various embodiments include 0.025 millimolar, 0.05 millimolar, 0.1 millimolar, 0.2 millimolar, 0.3 millimolar, 0.4 millimolar, 0.5 millimolar, 0.6 millimolar, 0.7 millimolar, 0.8 millimolar, 0.9 millimolar, and 1.0 millimolar or greater.

[0110] Additionally, while the above description references a typical oxyresveratrol concentration of about 0.1 to 1.0 millimolar, focusing on when benefits are evident, different concentrations may also provide positive effects. Thus, some embodiments may utilize oxyresveratrol concentrations of 1.0 millimolar or greater. Exemplary oxyresveratrol concentration ranges according to various embodiments include 0.1 millimolar, 0.2 millimolar, 0.3 millimolar, 0.4 millimolar, 0.5 millimolar, 0.6 millimolar, 0.7 millimolar, 0.8 millimolar, 0.9 millimolar, and 1.0 millimolar or greater.

[0111] Furthermore, it should be noted that a particular component (e.g., a decapeptide, oxyresveratrol) can be used in combination with another component to achieve a desired effect. For example, a particular decapeptide can be used in combination with other peptides, such as decapeptides P4A, 4B, and / or 4C, and / or with other components, such as oxyresveratrol. According to such embodiments, the synergistic effect achieved by including other components may ultimately reduce the concentration of any individual component (e.g., a decapeptide, oxyresveratrol, etc.) required to achieve a desired result.

[0112] Although decapeptide and oxyresveratrol are specifically listed above as possible additional ingredients, embodiments are not limited thereto.Other possible additives include, but are not limited to, α-lipoic acid, biotin, caffeine, ceramide, coenzyme Q10, glycolic acid, green tea, human stem cells, human stem cell extract, hyaluronic acid, hydroquinone, jojoba oil, kojic acid, lactic acid, malic acid, niacinamide, oligopeptides, peptides, plant stem cells, plant stem cell extract, resveratrol, retinol, vitamin C, vitamin E, and vitamin K.

[0113] It should be noted that different embodiments may be utilized for immunosuppression of various skin cell types. Examples of terminally differentiated skin cells include keratinocytes, fibrocytes, melanocytes, and Similarly, examples include, but are not limited to, immune cells such as Langerhans cells (eg, histiocytes or dendritic cells) that senesce over time.

[0114] Certain embodiments may also be utilized to treat skin progenitor cells for immunosuppression and reduced skin aging, enabling lifelong skin renewal. Examples of such progenitor cells include, but are not limited to, epidermal keratinocyte progenitor cells, which are precursors to epidermis-resident Langerhans cells, fibroblasts, melanoblasts, histoblasts, or dendritic blasts.

[0115] While the above description focuses on 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 cattle (e.g., in the production of leather from skin), pigs, and other animals (e.g., dogs, cats, and other animals that may be evaluated based on skin appearance for contest purposes).

[0116] Additionally, while the above description focuses on treating skin cells, embodiments are not limited to this or any other cell type. Some embodiments can treat a variety of mammalian and even non-mammalian cell types.

[0117] According to some embodiments, treatment may occur via oral administration to a mammalian subject. Alternatively, treatment may involve other forms of delivery, such as direct application or targeted local application (e.g., injection).

[0118] Clause 1B. A method of treating a subject by immunosuppressing cells, comprising administering to a subject in need of treatment a composition comprising an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0119] Clause 2B. The method of Clause 1B, wherein the peptide consists of SEQ ID NO:9.

[0120] Clause 3B. The method of Clause 1B, wherein the peptide consists of SEQ ID NO: 9 modified by a modifying group, wherein the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxy terminus, or both.

[0121] Clause 4B. The method of Clause 1B, wherein the peptide consists of SEQ ID NO: 11, having a tyrosine amino acid at position 6 as the D isoform, and all other amino acids as the L isoform.

[0122] Clause 5B. The method of Clause 1B wherein the cell is a mammalian cell.

[0123] Clause 6B. The method of Clause 5B wherein the mammalian cell is a skin cell.

[0124] Clause 7B. The method of Clause 6B, wherein the mammalian skin cells are progenitor cells.

[0125] Clause 8B. The method of Clause 7B, wherein the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendritic blasts.

[0126] Clause 9B. The method of any of clauses 1B, 5B, 6B, 7B, and 8B, wherein administering is by oral administration.

[0127] Clause 10B. The method of Clause 1B, wherein the cells are terminally differentiated.

[0128] Clause 11B. The method of Clause 10B, wherein the cell is a keratinocyte, melanocyte, fibrocyte, histiocyte, or dendritic cell.

[0129] Clause 12B. The method of Clause 1B, wherein the peptide is present at a concentration of about 1 millimolar or less.

[0130] Clause 13B. The method of Clause 1B, wherein the composition further comprises oxyresveratrol.

[0131] Clause 14B. A method of treating a subject by cellular immunosuppression, comprising administering to a subject in need thereof a composition comprising an effective amount of oxyresveratrol.

[0132] Clause 15B. The method of Clause 14B, wherein the oxyresveratrol is present at a concentration of about 0.1 to 1.0 millimolar.

[0133] Clause 16B. The method of Clause 14B, wherein the composition further comprises an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0134] Clause 17B. The method of Clause 16B, wherein the peptide consists of SEQ ID NO:9.

[0135] Clause 18B. The method of Clause 16B, wherein the peptide is present at a concentration of about 1 millimolar or less.

[0136] Clause 19B. The method of Clause 14B, wherein the cell is a mammalian cell.

[0137] Clause 20B. The method of Clause 19B, wherein the mammalian cell is a skin cell.

[0138] Clause 21B. The method of Clause 20B, wherein the mammalian skin cells are progenitor cells.

[0139] Clause 22B. The method of Clause 21B, wherein the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendritic blasts.

[0140] Clause 23B. The method of any of clauses 14B, 19B, 20B, 21B, and 22B, wherein administering is by oral administration.

[0141] Clause 24B. The method of Clause 14B, wherein the cells are terminally differentiated.

[0142] Clause 25B. The method of Clause 24B, wherein the cell is a keratinocyte, melanocyte, fibrocyte, histiocyte, or dendritic cell.

[0143] 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 embodiment was chosen and described in order to best explain the principles of the invention and its practical application. This description will enable those skilled in the art to best utilize and practice the invention in various embodiments, with various modifications suited to particular applications. The scope of the invention is defined by the following claims. will be done.

Claims

1. A method for treating a subject by immunosuppressing cells, comprising administering to a subject in need of treatment a composition comprising an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

12.

2. 2. The method of claim 1, wherein the peptide consists of SEQ ID NO:

9.

3. The method of claim 1 , wherein the cell is a mammalian cell.

4. The method of claim 3 , wherein the mammalian cells are skin cells.

5. The method of claim 4 , wherein the mammalian skin cells are progenitor cells.

6. The method of claim 5 , wherein the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendritic cells.

7. 7. The method of claim 1, 2, 3, 4, 5, or 6, wherein the administration is by oral administration.

8. The method of claim 1 , wherein the cells are terminally differentiated.

9. 9. The method of claim 8, wherein the cells are keratinocytes, melanocytes, fibrocytes, histiocytes, or dendritic cells.

10. 10. The method of claim 1, wherein the peptide is present at a concentration of about 1 millimolar or less.

11. The method of claim 1 , wherein the composition further comprises oxyresveratrol.

12. A method for treating a subject by cellular immunosuppression, comprising administering to a subject in need of treatment a composition comprising an effective amount of oxyresveratrol.

13. 13. The method of claim 12, wherein the oxyresveratrol is present at a concentration of about 0.1 millimolar to about 1.0 millimolar.

14. 13. The method of claim 12, wherein the composition further comprises an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO:

9.

15. 16. The method of claim 15, wherein the cell is a mammalian cell.

16. 16. The method of claim 15, wherein the mammalian cell is a skin cell.

17. 17. The method of claim 16, wherein the mammalian skin cells are progenitor cells.

18. 18. The method of claim 17, wherein the progenitor cells are epidermal keratinocyte precursor cells, melanoblasts, fibroblasts, histoblasts, or dendritic blasts.

19. 19. The method of any of claims 12, 15, 16, 17, or 18, wherein the administration is by oral administration.

20. 16. The method of claim 15, wherein the cells are terminally differentiated keratinocytes, melanocytes, fibrocytes, histiocytes, or dendritic cells.

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