Tyrosine inhibitors having immunosuppressive activity in human neonatal keratinocyte progenitor cells

Abstract

Tyrosine inhibitors with immunosuppressive activity in human neonatal keratinocyte precursor cells. 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.

Description

Technical Field

[0001] Description Cross - Reference to Related Applications This patent application claims the benefit of U.S. Patent Application No. 62 / 794,582, filed on January 19, 2019, and this patent application is incorporated herein by reference in its entirety together with all other references cited in this application.

[0002] Sequence Listing This application incorporates by reference a sequence listing entitled "20200120_ELIXP005_ST25.TXT" (3 kilobytes), created on January 20, 2020, and electronically submitted to this application.

Background Art

[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 are characterized by the immunosuppressive effects of decapeptides and / or oxyresveratrol, 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 that undergoes immunosuppression of cells, 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, the method 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.

[0007] In various embodiments, the peptide consists of SEQ ID NO: 9. The cells are mammalian cells. The mammalian cells are skin cells. The mammalian skin cells are progenitor cells. The progenitor cells are epidermal keratinocyte progenitor cells, melanoblasts, fibroblasts, histioblasts, or dendritic blast cells. The administration is by oral administration. The cells ultimately differentiate. The cells are keratinocytes, melanocytes, fibroblasts, histiocytes, or dendritic cells. The peptide is present at a concentration of about 1 millimolar or less. The composition further comprises oxyresveratrol.

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

[0009] In various embodiments, oxyresveratrol is present at a concentration of about 0.1 millimolar to about 1.0 millimolar. The composition further comprises an effective amount of one or more peptides, wherein the one or more peptides comprise SEQ ID NO: 9. The cells are mammalian cells. The mammalian cells are skin cells. The mammalian skin cells are progenitor cells. The progenitor cells are epidermal keratinocyte progenitor cells, melanoblasts, fibroblasts, histioblasts, or dendritic blast cells. The administration is by oral administration. The cells are terminally differentiated keratinocytes, melanocytes, fibroblasts, histiocytes, or dendritic cells.

[0010] Other objects, features, and advantages of the present invention will become apparent upon consideration of the following detailed description and the accompanying drawings. In the drawings, like reference numerals represent like features throughout the drawings.

Brief Description of the Drawings

[0011]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2A

Figure 2B

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Figure 8

Modes for Carrying Out the Invention

[0012] In the skin, the results of chronological and photoaging become apparent, so we will always recognize the aging process and seek treatments to delay or reverse its effects. Skin aging has traditionally been classified as either exogenous or endogenous. Recent evidence indicates that both types share important molecular features, including changes in signaling pathways that promote matrix metalloproteinase expression, a decrease in procollagen synthesis, and damage to connective tissue.

[0013] In human skin, aging is associated with an increase in the number of senescent cells and a decline in the ability of cells to proliferate and differentiate. Substantial evidence supports the theory that aging is mainly the result of free radical damage by various endogenous reactive oxygen species (ROS). Velarde et al. reported in vivo evidence of a causal relationship between mitochondrial oxidative damage, cellular senescence, and the aging phenotype of the skin. Furthermore, ultraviolet (UV) radiation stimulates ROS synthesis, which is associated with mutagenesis and photoaging. Consistent with these findings, data suggest changes in the expression of sirtuin activity in UV-irradiated skin versus skin protected by sunscreen, suggesting that these differences may contribute to specific aspects of skin aging.

[0014] Cellular senescence refers to the process by which cells stop dividing and undergo characteristic phenotypic changes, such as significant chromatin and secretome changes, as well as the activation of tumor suppressors. Numerous reports have helped establish the concept of sirtuins as powerful anti-aging proteins and have detailed their multifaceted roles in delaying cellular senescence and premature aging. Sirtuins are important effectors in pathways such as DNA damage repair, telomere shortening, cellular responses to oxidative stress, and the amelioration of ROS-induced pathologies.

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

[0016] These findings have attracted interest in the development of small molecule activators or pharmaceuticals that can help slow down aging and the progression of its wide range of age-related disorders. Among the seven mammalian sirtuins, SIRT1 has been the most widely studied with respect to aging and lifespan. For example, the anti-aging effect of resveratrol is mainly due to the activation of SIRT1. Indeed, Ido et al. reported that resveratrol improved cellular aging and proliferative dysfunction by enhancing the activities of AMP-activated protein kinase and sirtuin.

[0017] We previously reported the potent hypopigmenting effect of decapeptide-12 in human skin. Further clinical studies revealed an overall improvement in the facial skin appearance of patients with dyschromia 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 can regulate sirtuin activity and improve the 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 role of sirtuins in suppressing premature aging, delaying cellular senescence, extending lifespan, and improving a wide range of age-related impairments. Herein, we report our findings on a potent sirtuin activator, decapeptide-12, and compare its performance to the well-established resveratrol. Treatment of human epidermal keratinocyte progenitor cells with 100 micromolar decapeptide-12 increased SIRT1 transcription by 141 ± 11 percent compared to control cells, while the levels of SIRT3, SIRT6, and SIRT7 increased by 121 ± 13 percent, 147 ± 8 percent, and 95 ± 14 percent, respectively. Decapeptide-12 increased sirtuin transcription to levels similar to resveratrol but with reduced cytotoxicity.

[0019] Materials and Methods Reagents The decapeptide-12 (YRSRKYSSWY) of SEQ ID NO: 9 was synthesized by Bio Basic, Inc. (Ontario, Canada) using solid-phase Fmoc chemistry. Resveratrol was purchased from Sigma-Aldrich (St. Louis, MO).

[0020] Cell Culture Human neonatal epidermal progenitor cells (Thermo Fisher Scientific, NY) were seeded into 6-well plates at a density of 2 × 10 5 cells per well. To each well, 2 milliliters of Epilife medium containing 60 micromolar calcium chloride (Thermo Fisher Scientific, NY) was added. The plates were incubated in a humidified chamber at 37 degrees Celsius with 5 percent CO 2 2. After 24 hours, the cells were treated with various concentrations of resveratrol or decapeptide-12 dissolved in PBS containing 5 percent DMSO. Only vehicle (5 percent DMSO and PBS) was added to the control wells. The final concentration of DMSO in each well was 0.05 percent.

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

[0022] RNA concentration was determined using a nanodrop (Thermo fisher Scientific, NY). cDNA was synthesized using 2 μg of total RNA, oligo dT primers, and TaqMan reverse transcription reagents (Thermo fisher Scientific, NY). The reaction was carried out 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 carried out in a DNA Engine Peltier Thermo Cycler (Bio-Rad, Hercules, CA). PCR was performed under the following conditions: for SIRT1-7 and the housekeeping gene 18S, denaturation at 94°C for 2 minutes and primer extension at 54°C for 30 seconds were carried out for 34 cycles. [Table 1]

[0024] The samples were separated by running them on a 1.5% agarose gel containing 0.5 microgram / milliliter of 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). The intensity ratio was calculated by dividing the intensity value of each gene by the intensity value of the internal standard gene 18S.

[0025] Survival / growth and cytotoxicity assays The growth rate was determined using the TACS® MTT Cell Proliferation Kit (R&D Systems, Minneapolis, MN). Cells were seeded at 2.5×10 2 per well in a 96-well plate in a humidified atmosphere containing 5% CO 4 at 37 °C. After 24 hours, decapeptide-12 or oxyresveratrol was added to the corresponding wells at different concentrations (0, 3, 10, 30, 100, 300, and 1000 micromoles), and the cultures were incubated for 72 hours. The remaining part of the procedure was carried out according to the manufacturer's protocol.

[0026] Cytotoxicity was measured using the trypan blue dye exclusion assay. Cells were cultured in a 6-well plate at a density of 4×10 5 cells per well. Different concentrations of decapeptide-12 or oxyresveratrol (0, 3, 10, 30, 100, 300, and 1000 micromoles) were added to each well. The plates were incubated at 37 °C in a 5% CO 2 humidified chamber. After 72 hours, aliquots were 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 live cells in test sample) / number of cells in control] x 100%.

[0027] Statistical analysis The mean value and its standard error were calculated from three independent runs using Microsoft Excel, and statistical significance was determined using a paired analysis of variance. A P-value of P < 0.05 was considered statistically significant.

[0028] Results Effect of the decapeptide on growth rate and cytotoxicity: First, the cytotoxic effects of decapeptide-12 and oxyresveratrol on human epidermal progenitor cells were evaluated. Figure 2A shows that treatment with 100 micromolar of 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] Also, the effects of decapeptide-12 and oxyresveratrol on the viability and growth of human epidermal progenitor cells were evaluated. Figure 2B shows that treatment with 300 micromolar of decapeptide-12 or oxyresveratrol decreased cell growth by 2 ± 1 percent or 5 ± 1 percent, respectively. However, unlike 1 millimolar of decapeptide-12, which decreased growth by 3 ± 2 percent, a 3-day incubation with oxyresveratrol decreased growth by 12 ± 2 percent.

[0030] Decapeptide-12 increased the transcription of SIRT1-7: Next, the effects of oxyresveratrol and decapeptide-12 on sirtuin expression in human epidermal progenitor cells were evaluated. Figures 1A-1D and Table B show that decapeptide-12 and oxyresveratrol regulated the transcription of SIRT1-7 in a dose-dependent manner. At 30 micromolar of oxyresveratrol, the transcription level of SIRT1 increased by 125 ± 9 percent compared to control cells, while 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] Data showed that 100 μmol of decapeptide-12 increased the transcription of SIRT1 by 141 ± 11 percent compared to untreated cells, while SIRT3, SIRT6, and SIRT7 increased by 121 ± 13 percent, 147 ± 8 percent, and 95 ± 14 percent, respectively (Figures 1A-1D).

[0033] Discussion The multifaceted role of sirtuins in delaying cellular aging and preventing the progression of premature aging has helped to establish sirtuins as powerful anti-aging proteins. The therapeutic use of SIRT1 activators and resveratrol as a potential anti-aging agent has been widely studied and demonstrated. Resveratrol protects human endothelium from H2O2-induced oxidative stress and aging via 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 and compared its performance with that of decapeptide-12 and the ability to regulate sirtuin transcription in human epidermal keratinocytes.

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

[0035] At 100 micromoles or 1 millimole, the decapeptide-12 impressively increased SIRT1 transcription by 141 or 213 percent, respectively. SIRT1 is mainly 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 through the regulation of p53 and c-Jun N-terminal kinase in cultured skin keratinocytes, suggesting that SIRT1 activators could serve as new anti-skin-aging agents. Other researchers reported that SIRT1 can suppress NF-κB signaling, thus delaying the aging process and extending lifespan. The activation of SIRT1 directly suppresses NF-κB signaling by deacetylating the p65 subunit of the NF-κB complex, promoting oxidative metabolism and the resolution of inflammation. Therefore, SIRT1 can be regarded as an important anti-aging protein that mediates a broad effect of preventing premature aging and accelerating aging by regulating multiple molecular pathways.

[0036] SIRT3 transcription increased by 121 percent after treatment with 100 micromoles of the decapeptide. SIRT3 is mainly associated with the regulation of various mitochondrial processes such as β-oxidation, ATP production, and ROS management. SIRT3 is also involved in maintaining the regenerative capacity of hematopoietic stem cells. SIRT3 is suppressed with aging, and the increased expression of SIRT3 in aged hematopoietic stem cells improves their regenerative capacity. This discovery establishes the important role that SIRT3 plays in maintaining robustness and, more importantly, helps to open the way for future stem cell-based interventions against the metabolic disorders that lead to premature aging.

[0037] SIRT6 can be regarded as 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 early aging phenotypes and died within one month. Furthermore, SIRT6 is the only mammalian sirtuin that showed a clear increase in lifespan when overexpressed throughout the body of mice. Additionally, Kawahara et al. reported that SIRT6 reduces hyperactive NF-κB signaling by deacetylating histone H3 at K9 on the promoter of NF-κB target genes, thereby strengthening the role of SIRT6 as an important anti-inflammatory protein.

[0038] Baohua et al. showed that SIRT6 plays an important role in the skin aging process through the regulation of collagen metabolism and NF-κB signaling. According to their report, blocking SIRT6 inhibits the transcription of type I collagen, promotes the secretion of matrix metalloproteinase 1, and increases NF-κB signaling, resulting in a significant decrease in hydroxyproline content. In summary, SIRT6 stands out as an important modulator of the anti-aging process by regulating multiple pathways to delay cellular aging and the acceleration of aging. Therefore, decapeptide-12, which enhanced SIRT6 transcription by 147 percent at 100 μM, could be quite promising as a therapeutic anti-aging candidate to address phenotypes where early skin aging and photo-damaged skin often occur simultaneously.

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

Example

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

[0041] These modifications to the decapeptide P4 can help improve stability against proteases and enhance either transdermal or transcellular penetration, or both.

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

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

[0044] As noted above, the residues shown can be naturally occurring L-amino acids or their modifications, i.e., chemical modifications, optical isomers, or bonds to modifying groups. It is contemplated that specific modifications can be made within the peptide that maintain the ability of the peptide to specifically regulate the expression of the sirtuin gene.

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

Table 6

[0046] At low concentrations, the native decapeptide P4 showed an improvement in transcription level compared to the modified decapeptides. However, each of the three modified decapeptides (P4A, P4B, and P4C) increased the transcription level of the searchuin gene compared to the control. At a concentration of 100 micromolar, the effect on the transcription level was equivalent 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 2.5x104 per well in a 96-well plate in a humidified atmosphere containing 5 percent CO2 at 37 degrees Celsius. After 24 hours, decapeptides were added to the corresponding wells at different concentrations and incubated for 72 hours. The remaining part of the procedure was carried out 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 fibroblast proliferation rate after 72 hours.

Table 9

[0051] As a result of incubating epidermal progenitor cells, melanoblasts, and fibroblasts with 100 micromolar of decapeptide P4A for 72 hours, the growth rate of all three cell lines decreased by 3 percent.

[0052] At 1000 micromolar, the proliferation rate of epidermal progenitor cells decreased by 6 percent, while the proliferation rates of melanoblasts and fibroblasts decreased 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 the viability was counted using trypan blue and compared to a control (untreated cells). Cytotoxicity was measured according to the following formula: [1 - (number of cells in control - number of live cells in test sample) / number of cells in control] x 100 percent.

[0054] Table J shows the viability of epidermal progenitor cells after 7 days.

Table 10

[0055] Table K shows the viability of melanoblasts after 7 days.

Table 11

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

Table 12

[0057] At a concentration of 100 micromoles, the cell viability remained above 97 percent for all three cell lines. At 1000 micromoles, the cell viability decreased by 6 percent compared to the control.

[0058] In conclusion, recent reports have detailed the multifaceted role of sirtuins in the suppression of premature aging, the delay of cellular senescence, the extension of lifespan, and the improvement of a wide range of age-related disorders. Here, we report our findings regarding the potent sirtuin activator, decapeptide-12, and compare its performance to that of the well-established resveratrol. Treatment of human epidermal progenitor cells with 100 micromoles of decapeptide-12 increased the transcription of SIRT1 by 141 ± 11 percent compared to control cells, while the levels of SIRT3, SIRT6, and SIRT7 increased by 121 ± 13 percent, 147 ± 8 percent, and 95.4 ± 14 percent, respectively. Decapeptide-12 increased sirtuin transcription to levels similar to those of resveratrol but with reduced cytotoxicity. Therefore, decapeptide-12 may hold promise as a safer therapeutic for combating skin aging and other age-related pathologies.

[0059] The above description refers to typical decapeptide concentrations of 100 micromoles and above where the effect is clear, but the results also indicate that lower concentrations have a positive effect. Thus, in some embodiments, decapeptide concentrations of 1 micromole and above can be utilized, and certain embodiments use a peptide concentration range of 100 micromoles and above. Examples of peptide concentration ranges according to various embodiments are 1 micromole and above, 5 micromoles and above, 10 micromoles and above, 30 micromoles and above, 50 micromoles and above, 100 micromoles and above, 300 micromoles and above, 500 micromoles and above, and 1000 micromoles and above.

[0060] Furthermore, it should be noted that, to achieve the desired effect, a specific decapeptide can be used in combination with other components. For example, a specific 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 can ultimately reduce the concentration of any individual component (e.g., decapeptide, others) necessary to achieve the desired result.

[0061] In the above, decapeptides and oxyresveratrol were specifically mentioned as possible additional components, but the embodiments are not limited thereto. Examples of other 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 extract, hyaluronic acid, hydroquinone, jojoba oil, kojic acid, lactic acid, malic acid, niacinamide, oligopeptide, peptide, plant stem cells, plant stem cell extract, resveratrol, retinol, vitamin C, vitamin E, and vitamin K.

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

[0063] The embodiments can also be utilized to treat skin progenitor cells to reduce skin aging and enable skin regeneration over its lifetime. Examples of such progenitor cells include, but are not limited to, epidermal keratinocyte progenitor cells, fibroblasts, melanoblasts, histioblasts, or dendritic blast cells, which are the progenitor cells of Langerhans cells remaining in the epidermis.

[0064] Finally, although the above described the treatment of human skin cells, certain embodiments are not limited to such an approach. In alternative embodiments, skin cell treatments from other organisms can be utilized, including but not limited to mammals such as cows (e.g., in leather production), pigs, and other animals (e.g., dogs, cats, and other animals that can 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 according to Clause 1A, wherein the peptide consists of SEQ ID NO: 9 modified by a modifying group, and the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxyl terminus, or both.

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

[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 composition according to Clause 4A, wherein the peptide consists of SEQ ID NO: 9 modified by a modifying group, and the modifying group is either a palmitoyl group or an acetyl group at the amino terminus, or amidation at the carboxyl terminus, or both.

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

[0071] Clause 7A. The composition according to any one of Clauses 4A - 6A, wherein the peptide is present at a concentration of 1 μm or more.

[0072] Method for treating a subject by modulating the expression of sirtuin genes 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] The method according to clause 8A, wherein the peptide consists of SEQ ID NO: 9 modified by 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.

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

[0075] The method according to any one of clauses 8A to 10A, wherein the skin cells are progenitor cells.

[0076] The method according to clause 11A, wherein the progenitor cells are epidermal keratinocyte progenitor cells, melanoblasts, fibroblasts, histioblasts, or dendritic cells.

[0077] The method according to any one of clauses 8A to 10A, wherein the skin cells are terminally differentiated.

[0078] The method according to clause 13A, wherein the skin cells are keratinocytes, melanocytes, fibroblasts, histiocytes, or dendritic cells.

[0079] The method according to any one of clauses 8A to 14A, wherein the peptide is present at a concentration of 1 μm or more.

[0080] Article 16A. The method according to any one of Articles 8A to 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] Article 17A. The method according to any one of Articles 8A to 16A, wherein the composition further comprises oxyresveratrol.

[0082] Article 18A. The method according to any one of Articles 8A to 17A, wherein the skin cell is a mammalian cell.

[0083] Article 19A. The method according to Article 18A, wherein the skin cell is human.

[0084] Article 20. A method for regulating the expression of the sirtuin gene in a skin cell, 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] The immunosuppressive effects of peptide formulations and / or oxyresveratrol are further noted. The chemical structure of the decapeptide P4 of SEQ ID NO: 9 is shown in Figure 3. The chemical structure of oxyresveratrol is shown in Figure 4.

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

[0087] These results are detailed in the following examples.

[0088] [Examples] PBMC Proliferation and NK Cell Cytotoxic Killing Assay Cryopreserved human PBMCs were purchased from Astarte Biologies (Redmond, Washington, USA), activated with phytohemagglutinin (PHA), and proliferation was evaluated. The cytotoxic killing of human K562 cells by human NK cells activated with interleukin (IL)-2 was evaluated using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega). Protease inhibitors were added to the medium to prevent the degradation of decapeptide-12.

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

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

[0091] Figure 5 plots the immunosuppressive effect of decapeptide-12 (P4) on PHA-stimulated PBMC proliferation. Data are represented as percent (%) of control and show 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 significantly (p < 0.02) decreased proliferation by 28.0 ± 3.8 percent at 0.05 millimolar and 54.3 ± 1.1 percent at 0.1 millimolar. No further significant decrease was achieved at 0.3 or 1 millimolar (p > 0.05).

[0093] Figure 6 plots the immunosuppressive effect of oxyresveratrol on PHA-stimulated PBMC proliferation. Data are represented as percent (%) of control and show 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 decreased proliferation by 35.3 ± 1.8 percent (p < 0.02) at 0.1 millimole and by 14.7 ± 3.4 percent (p < 0.02) at 3 millimoles.

[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 represented as percent (%) of 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 to target (E:T) cell ratio of 10:1, decapeptide-12 decreased NK killing by 81.4 ± 1.3 percent and 59.3 ± 3.6 percent (p > 0.05) at 0.1 millimole and 0.3 millimoles, respectively. At a ratio of 30:1, decapeptide-12 decreased NK killing by 64.8 ± 5.3 percent and 44.8 ± 3.2 percent (p < 0.04) at 0.1 millimole and 0.3 millimoles, respectively.

[0098] Figure 8 plots the immunosuppressive effect of oxyresveratrol on NK92-mediated cytotoxic killing of K562 cells. Data are represented as percent (%) of 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 decreased NK killing by 88.7 ± 1.8 percent and 86.1 ± 0.9 percent (p < 0.03) at 0.1 millimolar and 0.3 millimolar, respectively, with an E:T ratio of 10:1. At a ratio of 30:1, oxyresveratrol blocked NK killing by 72.8 ± 1.9 percent and 64.0 ± 3.4 percent at 0.1 millimolar and 0.3 millimolar, respectively (p < 0.03).

[0100] Therefore, the investigation revealed that both decapeptide-12 and oxyresveratrol exhibited anti-inflammatory effects as measured by two methods: 1) blocking of PHA-stimulated PBMC proliferation and 2) inhibition of NK-mediated cytotoxic killing.

[0101] Regarding the blocking of the proliferation assay, the effect of decapeptide-12 appeared to be dose-dependent. The effect of oxyresveratrol showed a limited range of inhibitory concentrations.

[0102] Indeed, the general trend indicated that oxyresveratrol could have a biphasic effect. This is because the concentrations of 0.3 and 1 millimolar showed less inhibition gradually than the concentration of 0.1 millimolar.

[0103] Decapeptide-12 showed a plateau or maximum inhibition at 0.1 millimolar and above within the tested concentration range. This could be explained by dose-dependent differences in the activation of downstream signaling pathways or feedback loops. Indeed, a careful examination of the dose-dependent curve of the sirtuin expression pattern reveals a biphasic effect where high concentrations become inhibitory.

[0104] In contrast, when suppressing NK killing, both decapeptide-12 and oxyresveratrol appeared to be dose-dependent. Oxyresveratrol showed more significant inhibition at all concentrations tested.

[0105] The inhibitory effect was greater than 10:1 compared to an E:T ratio of 30:1 with both decapeptide-12 and oxyresveratrol. This could be due to the blockade of NKG2D and perforin-mediated cytotoxicity.

[0106] Note that resveratrol (a analogue of oxyresveratrol) inhibits PHA-induced proliferation at 0.1 millimolar. This inhibitory effect is also regulated by sirtuins and may be due to the inhibition of NF-κB, among other effects, related to the regulation of immune and inflammatory responses, as well as cell proliferation and apoptosis.

[0107] In summary, the immunosuppressive effects observed here suggest that distinct and specific regulatory pathways are involved in different arms of the immune system. Further investigation may clarify these multifaceted effects.

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

[0109] In the above description, attention has been paid to the case where the effect is clear, and a typical decapeptide concentration of about 0 to 1.0 millimolar has been mentioned, but different concentrations may also bring about a positive effect. Therefore, some embodiments may utilize a decapeptide concentration of 1.0 millimolar or more. Examples of peptide concentration ranges according to various embodiments are 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 more.

[0110] Also, in the above description, attention has been paid to cases where the effect is obvious, and a typical oxyresveratrol concentration of about 0.1 to 1.0 millimolar has been mentioned. However, different concentrations may also bring positive effects. Therefore, some embodiments may utilize an oxyresveratrol concentration of 1.0 millimolar or more. Examples of oxyresveratrol concentration ranges according to various embodiments are 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 more.

[0111] Furthermore, it should be noted that specific components (e.g., decapeptides, oxyresveratrol) can be used in combination with other components to achieve the desired effect. For example, a specific decapeptide can 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 embodiments, the synergistic effect realized by including other components can ultimately reduce the concentration of any individual component (e.g., decapeptide, oxyresveratrol, others) necessary to achieve the desired result.

[0112] In the above, decapeptides and oxyresveratrol have been specifically mentioned as possible additional components, but the embodiments are not limited thereto. Examples of other 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 extract, hyaluronic acid, hydroquinone, jojoba oil, kojic acid, lactic acid, malic acid, niacinamide, oligopeptide, peptide, plant stem cells, plant stem cell extract, resveratrol, retinol, vitamin C, vitamin E, and vitamin K.

[0113] Note that different embodiments can be utilized for the immunosuppression of various skin cell types. Examples of terminally differentiated skin cells can include, but are not limited to, keratinocytes, fibroblasts, melanocytes, and immune cells such as Langerhans cells (e.g., histiocytes or dendritic cells) that also age over time.

[0114] Certain embodiments can also be utilized to treat skin progenitor cells for immunosuppression and reduction of skin aging, enabling lifelong skin regeneration. Examples of such progenitor cells can include, but are not limited to, epidermal keratinocyte progenitor cells, which are the progenitors of Langerhans cells that remain in the epidermis, fibroblasts, melanoblasts, histioblasts, or dendritic blasts.

[0115] The above description focuses on the treatment of human skin cells, but certain embodiments are not limited to such an approach. In alternative embodiments, the treatment of skin cells from other organisms can be utilized, including but not limited to mammals such as cows (e.g., in the production of leather from skin), pigs, and other animals (e.g., dogs, cats, and other animals that can be evaluated based on skin appearance for contest purposes).

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

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

[0118] 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] The method according to clause 1B, wherein the peptide consists of SEQ ID NO: 9.

[0120] The method according to clause 1B, wherein the peptide consists of SEQ ID NO: 9 modified by 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.

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

[0122] The method according to clause 1B, wherein the cells are mammalian cells.

[0123] The method according to clause 5B, wherein the mammalian cells are skin cells.

[0124] The method according to clause 6B, wherein the mammalian skin cells are progenitor cells.

[0125] The method according to clause 7B, wherein the progenitor cells are epidermal keratinocyte progenitor cells, melanoblasts, fibroblasts, histiocytes, or dendritic cells.

[0126] The method according to any one of clauses 1B, 5B, 6B, 7B, and 8B, wherein the administration is by oral administration.

[0127] The method according to clause 1B, wherein the cells are finally differentiated.

[0128] The method according to clause 10B, wherein the cell is a keratinocyte, melanocyte, fibroblast, histiocyte, or dendritic cell.

[0129] The method according to clause 1B, wherein the peptide is present at a concentration of about 1 millimole or less.

[0130] The method according to clause 1B, wherein the composition further comprises oxyresveratrol.

[0131] A method of treating a subject by immunosuppressing a cell, the method comprising administering to a subject in need of treatment a composition comprising an effective amount of oxyresveratrol.

[0132] The method according to clause 14B, wherein oxyresveratrol is present at a concentration of about 0.1 to 1.0 millimole.

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

[0134] The method according to clause 16B, wherein the peptide consists of SEQ ID NO: 9.

[0135] The method according to clause 16B, wherein the peptide is present at a concentration of about 1 millimole or less.

[0136] The method according to clause 14B, wherein the cell is a mammalian cell.

[0137] The method according to clause 19B, wherein the mammalian cell is a skin cell.

[0138] The method according to clause 20B, wherein the mammalian skin cell is a progenitor cell.

[0139] The method according to clause 21B, wherein the progenitor cell is an epidermal keratinocyte progenitor cell, a melanoblast, a fibroblast, a histiocyte, or a dendritic cell.

[0140] The method according to any one of clauses 14B, 19B, 20B, 21B, and 22B, wherein the administration is by oral administration.

[0141] The method according to clause 14B, wherein the cell finally differentiates.

[0142] The method according to clause 24B, wherein the cell is a keratinocyte, a melanocyte, a fibrocyte, a histiocyte, or a 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 embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize and practice the invention in various embodiments with various modifications as are suited to the particular use contemplated. The scope of the invention is defined by the following claims.

Claims

1. A composition for treating a subject by immunosuppressing an immune cell, the composition comprising an effective amount of one or more peptides, the one or more peptides comprising 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 peptide consists of SEQ ID NO:

9.

3. The composition of claim 1 , wherein the immune cells are natural killer cells, peripheral blood mononuclear cells, Langerhans cells, histiocytes, or dendritic cells.

4. The composition of claim 1 , wherein the peptide is present at a concentration of 1 millimolar or less.

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

6. A composition for treating a subject by immunosuppressing immune cells, the composition comprising an effective amount of oxyresveratrol and an effective amount of one or more peptides, the one or more peptides comprising SEQ ID NO:

9.

7. 7. The composition of claim 6, wherein the oxyresveratrol is present in a concentration of 0.1 millimolar to 1.0 millimolar.

8. The composition of claim 6 or 7, wherein the immune cells are natural killer cells, peripheral blood mononuclear cells, Langerhans cells, histiocytes, or dendritic cells.

9. The composition of claim 7 or 8, wherein the composition is for administration by oral administration.

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