Polypeptides having Anti-senescent effects and uses thereof

Abstract

SOLUTION: Polypeptides which can provide a senotherapeutic effect are provided herein. The polypeptides can be formulated for topical application and can be applied topically to a subject to provide a senotherapeutic effect in the subject or in the cells of the subject.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] cross reference This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 877,164, filed July 22, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The accumulation of senescent cells is not simply a product of organismal aging; rather, it may actively contribute to further senescence through a positive feedback cycle. Among the hallmarks of aging, cellular senescence occupies a central position and may integrate key, antagonistic, and integrative aspects of aging. First, senescence may reduce the proliferative capacity of progenitor cells, thereby impairing the tissue repair and regeneration capacity of affected tissues. Second, senescent cells may alter the paracrine signaling environment and become characterized by a senescence-associated secretory phenotype (SASP), which may induce inflammation and further cellular senescence, potentially exacerbating harmful inflammatory responses and promoting tissue injury. Third, the accumulation of senescent cells with age has been documented in several tissues, including, but not limited to, the skin. Fourth, cellular senescence may play an active role in diseases such as macular degeneration, dementia, atherosclerosis, and cancer.

[0003] Senescent cells can be identified by increased senescence-associated beta-galactosidase (SA-BGal) production, p16 expression, and accumulation of alpha-thalassemia / mental retardation X-linked chromatin remodeling protein (ATRX) foci in the nucleus. Altered functionality also distinguishes senescent cells, including reduced proliferative capacity and resistance to mitogenic stimuli. Summary of the Invention

[0004] Compositions comprising isolated, synthetic, and / or recombinant polypeptides are described herein, wherein the polypeptide comprises the amino acid sequence of LKGI (SEQ ID NO:5) or an analog thereof, the amino acid sequence of LKGIL (SEQ ID NO:6) or an analog thereof, or the amino acid sequence of WLKGI (SEQ ID NO:7) or an analog thereof. In some embodiments, such polypeptides can comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 amino acids. Alternatively, or additionally, such polypeptides can comprise up to 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, or 20 amino acids. In some examples, the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs:1-4.

[0005] Also described herein is an isolated, synthetic, and / or recombinant polypeptide comprising the amino acid sequence of SEQ ID NO:8, which is 10 or its analogs, 1. In the formula, X1 is E, X2 is T, X4 is K, X6 is W, X7 is L, X9 is G, and X 10 is I; and (i) X3 is not S; or 2. (ii) if X5 is any amino acid, then X8 is not G; or 3. (iii) If X8 is any amino acid, then X5 is not N; 4. Or, (iv) any one of (i), (ii), or (iii), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or 5. (b) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a second sequence, SEQ ID NO:2, wherein X1 is A, X2 is T, X3 is A, X4 is K, X5 is A, X6 is W, X7 is L, X8 is K, X9 is G, and X 10 is I, optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (c) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a third sequence, SEQ ID NO:3, wherein X1 is K, X2 is L, X5 is I, X6 is L, X8 is G, and X 10 is A; and (i) when X9 is any amino acid, X3 is not N; or (ii) when X3 is any amino acid, X9 is not S; or (iii) when X4 is any amino acid, X7 is not L; or (iv) when X7 is any amino acid, X4 is not S; and, or (v) any one of (i), (ii), (iii), or (iv), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (d) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a fourth sequence, SEQ ID NO:4, wherein X1 is W, X2 is L, X3 is K, X4 is G, X5 is I, X6 is L, X7 is R, X8 is E, X9 is A, and X 10 is A, optionally with 1, 2, 3, or 4 conservative amino acid substitutions.

[0006] In some embodiments, the amino acid sequence comprises LKGI (SEQ ID NO:5). In some embodiments, the amino acid sequence comprises WLKGI (SEQ ID NO:7). In some embodiments, the amino acid sequence comprises LKGIL (SEQ ID NO:6). In some embodiments, the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO:1. In some embodiments, the amino acid sequence is SEQ ID NO:1. In some embodiments, the amino acid sequence is SEQ ID NO:2. In some embodiments, the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO:3. In some embodiments, the amino acid sequence is SEQ ID NO:3. In some embodiments, the amino acid sequence is SEQ ID NO:4. In some embodiments, the recombinant polypeptide comprises at least 10 amino acids, 15 amino acids, or 20 amino acids.

[0007] The compositions provided herein can be formulated for therapeutic, nutraceutical, or cosmetic use.

[0008] In some embodiments, the formulation further comprises a therapeutic, nutraceutical, or cosmetic excipient. In some embodiments, the excipient is configured for topical application. In some embodiments, the excipient is configured as an external supplement. In further embodiments, the formulation is configured for application to human skin. In some embodiments, the formulation is a cream, transdermal patch, topical patch, ointment, oil, gel, liquid, powder, lotion, serum, emulsion, moisturizer, toner, foam, face mask, mousse, aerosol, spray, cleanser, hydrogel patch, powder, or shampoo. In some embodiments, the formulation may be used in conjunction with sonic treatment, ultrasound treatment, LED treatment, light treatment, electrical treatment, or radiofrequency treatment. In some embodiments, the transdermal patch delivers the formulation to the epidermal layer of the skin. In some embodiments, the transdermal patch delivers the formulation to both the epidermal and dermal layers of the skin. In some embodiments, the formulation is intended for systemic delivery in a subject with minimal or low amounts or is not intended for direct delivery to the subject's bloodstream. In some embodiments, the formulation acts locally, at and near the site of delivery, hi some embodiments, the formulation has minimal or no systemic effects.

[0009] In some embodiments, the formulation is configured as a dietary supplement. In some cases, the formulation is configured as a beverage.

[0010] Described herein are therapeutic, nutraceutical, or cosmetic formulations comprising at least one recombinant or synthetic polypeptide and a therapeutic, nutraceutical, or cosmetic excipient.

[0011] In some embodiments, the excipient is configured for topical application. In some embodiments, the excipient is configured as an external supplement. In further embodiments, the formulation is formulated for application to human skin. More specifically, the formulation can be configured to penetrate locally from the epidermis to the dermis. In some embodiments, the formulation can be configured to penetrate locally through the epidermal and dermal layers. In some embodiments, the formulation can be configured to penetrate locally through the epidermal layer and have low penetration into the dermal layer. Often, the penetration of ingredients in the formulation can be evaluated using various penetration tests, including, but not limited to, those using Franz diffusion cells. In some embodiments, the formulation includes a carrier, microsphere, liposome, or micelle to carry the polypeptide and control the release time and / or penetration depth of the polypeptide through the skin. In some cases, the formulations herein are creams, ointments, gels, liquids, oils, powders, lotions, serums, emulsions, moisturizers, foams, face masks, mousses, aerosols, sprays, cleansers, toners, topical patches, hydrogel patches, or shampoos.

[0012] In some embodiments, the formulation is configured as a dietary supplement, in some embodiments, the formulation is configured as a drink, in some embodiments, the formulation is configured as a tablet, capsule, gel, gummy, or powder.

[0013] Described herein are methods for treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutic, nutraceutical, or cosmetic formulation comprising at least one amino acid sequence of SEQ ID NO:5-7.

[0014] In some embodiments, the administering step comprises topically applying the formulation to the subject. In further embodiments, the subject is a human or other animal. In some embodiments, the method comprises administering an effective amount of the formulation to the subject.

[0015] In some embodiments, the disease is a disorder associated with the accumulation of senescent cells in a subject. In some embodiments, the disorder associated with the accumulation of senescent cells comprises skin aging. In some embodiments, the disease is progeria and / or a disorder associated with the effects of progeria. In some embodiments, progeria comprises a disease having signs of premature aging in the epidermal and dermal layers of the skin.

[0016] Described herein are methods for reducing cellular senescence in a subject in need thereof, the methods comprising administering to the subject a therapeutic, nutraceutical, or cosmetic formulation comprising a polypeptide comprising the amino acid sequence of LKGI (SEQ ID NO: 5), optionally with one conservative amino acid substitution.

[0017] In some embodiments, the polypeptide comprises at least 4 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids. In some embodiments, the formulation comprises the amino acid sequence of WLKGI (SEQ ID NO: 7), optionally with one conservative amino acid substitution. In some embodiments, the formulation comprises the amino acid sequence of LKGIL (SEQ ID NO: 6), optionally with one conservative amino acid substitution. In some embodiments, the polypeptide comprises at least 5 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids. In some embodiments, the polypeptide comprises at most 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, or 40 amino acids.

[0018] In some embodiments, the formulation further comprises a therapeutic, nutraceutical, or cosmetic excipient.

[0019] Described herein are methods for reducing cellular senescence in a subject in need thereof, the methods comprising administering to the subject a therapeutic, nutraceutical, or cosmetic formulation comprising at least one polypeptide described herein.

[0020] In some embodiments, the formulation further comprises a therapeutic, nutraceutical, or cosmetic excipient. In some embodiments, the administering step comprises applying the formulation to a portion of the subject's skin. In some embodiments, the formulation extends the lifespan of a plurality of cells of the subject, induces SIRT6 expression in a plurality of cells of the subject, increases the cell renewal rate in a plurality of cells of the subject, promotes apoptosis in a plurality of cells of the subject, promotes DNA repair in a plurality of cells of the subject, increases collagen production in a plurality of cells of the subject, increases hyaluronic acid synthase production in a plurality of cells of the subject, reduces ATRX nuclear foci accumulation in a plurality of cells of the subject, reduces p16 expression in a plurality of cells of the subject, reduces senescence-associated beta-galactosidase production in a plurality of cells of the subject, reduces IL8 expression in a plurality of cells of the subject, reduces MMP1 expression in a plurality of cells of the subject, increases BLM expression in a plurality of cells of the subject, and / or prevents UV-induced DNA damage in a plurality of cells of the subject.

[0021] Described herein are methods for treating a disease in a subject in need thereof, the methods comprising administering to the subject a therapeutic, nutraceutical, or cosmetic formulation comprising the amino acid sequence of SEQ ID NO:5, optionally with one conservative amino acid substitution.

[0022] In some embodiments, the therapeutic, nutraceutical, or cosmetic formulation comprises the amino acid sequence of SEQ ID NO: 6, optionally with one conservative amino acid substitution. In some embodiments, the therapeutic, nutraceutical, or cosmetic formulation comprises the amino acid sequence of SEQ ID NO: 7, optionally with one conservative amino acid substitution.

[0023] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0024] The novel features of the invention are set forth with particularity in the appended claims. For a better understanding of the features and advantages of the present invention, reference should be made to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings.

[0025] [Figure 1] The effect of individual polypeptides on progeria fibroblast number and level of senescence is shown. [Figure 2A] The effects of senotherapeutic treatment with four polypeptides, peptide 14 (Panel A), peptide 13 (Panel B), peptide 15 (Panel C), and peptide 16 (Panel D), on senescent fibroblasts are shown. *P<0.05; **P<0.01; ***P<0.001. [Figure 2B] The effects of senotherapy with four polypeptides, peptide 14 (Panel A), peptide 13 (Panel B), peptide 15 (Panel C), and peptide 16 (Panel D), on senescent fibroblasts are shown. *P<0.05; **P<0.01; ***P<0.001. [Figure 2C] The effects of senotherapy with four polypeptides, peptide 14 (Panel A), peptide 13 (Panel B), peptide 15 (Panel C), and peptide 16 (Panel D), on senescent fibroblasts are shown. *P<0.05; **P<0.01; ***P<0.001. [Figure 2D] The effects of senotherapy with four polypeptides, peptide 14 (Panel A), peptide 13 (Panel B), peptide 15 (Panel C), and peptide 16 (Panel D), on senescent fibroblasts are shown. *P<0.05; **P<0.01; ***P<0.001. [Figure 3A] Shown are the effects of polypeptide senotherapy in promoting an increase in the number of cells with fewer ATRX foci / nucleus (Panel A), a decrease in the mean ATRX foci / nucleus (Panel B), and an increase in the number of cells with less than 10 ATRX foci / nucleus (Panel C). *P<0.05; **<0.01. [Figure 3B]Shown are the effects of polypeptide senotherapy in promoting an increase in the number of cells with fewer ATRX foci / nucleus (Panel A), a decrease in the mean ATRX foci / nucleus (Panel B), and an increase in the number of cells with less than 10 ATRX foci / nucleus (Panel C). *P<0.05; **<0.01. [Figure 3C] Shown are the effects of polypeptide senotherapy in promoting an increase in the number of cells with fewer ATRX foci / nucleus (Panel A), a decrease in the mean ATRX foci / nucleus (Panel B), and an increase in the number of cells with less than 10 ATRX foci / nucleus (Panel C). *P<0.05; **<0.01. [Figure 4A] The effect of senotherapeutic polypeptides is shown to be able to reduce the number of senescent fibroblasts in the cell population during a 3-week long-term exposure, maintaining the effect of senotherapy for at least 1 week after treatment (Panel A), without inducing cytotoxicity or significantly affecting cell proliferation during this period (Panel B). ***P<0.001; ***, <0.0001. [Figure 4B] The effect of senotherapeutic polypeptides is shown to be able to reduce the number of senescent fibroblasts in the cell population during a 3-week long-term exposure, maintaining the effect of senotherapy for at least 1 week after treatment (Panel A), without inducing cytotoxicity or significantly affecting cell proliferation during this period (Panel B). ***P<0.001; ***, <0.0001. [Figure 5] 1 shows that treatment with the polypeptides can promote a dose-responsive decrease in cellular senescence as measured by the average ATRX foci accumulation per cell in cells derived from multiple donors. [Figure 6A] These results show that cellular senescence can be induced in fibroblasts by exposure to etoposide (Panel A), that treatment of etoposide-induced senescent cells with polypeptides can result in reduced senescence (Panel B), that cellular senescence can be induced by UVB exposure (Panel C), and that treatment of UVB-treated samples with polypeptides can result in reduced senescence (Panel D). *P<0.05; **P<0.01; ***P<0.001. [Figure 6B]These results show that cellular senescence can be induced in fibroblasts by exposure to etoposide (Panel A), that treatment of etoposide-induced senescent cells with polypeptides can result in reduced senescence (Panel B), that cellular senescence can be induced by UVB exposure (Panel C), and that treatment of UVB-treated samples with polypeptides can result in reduced senescence (Panel D). *P<0.05; **P<0.01; ***P<0.001. [Figure 6C] These results show that cellular senescence can be induced in fibroblasts by exposure to etoposide (Panel A), that treatment of etoposide-induced senescent cells with polypeptides can result in reduced senescence (Panel B), that cellular senescence can be induced by UVB exposure (Panel C), and that treatment of UVB-treated samples with polypeptides can result in reduced senescence (Panel D). *P<0.05; **P<0.01; ***P<0.001. [Figure 6D] These results show that cellular senescence can be induced in fibroblasts by exposure to etoposide (Panel A), that treatment of etoposide-induced senescent cells with polypeptides can result in reduced senescence (Panel B), that cellular senescence can be induced by UVB exposure (Panel C), and that treatment of UVB-treated samples with polypeptides can result in reduced senescence (Panel D). *P<0.05; **P<0.01; ***P<0.001. [Figure 7A] The results show that polypeptide-treated human skin equivalents can exhibit higher quality by overall structural analysis score (Panel A), contain significantly fewer senescent cells than untreated human skin equivalents (Panels A and B), and contain gene expression changes (Panel C) in which p16 can have significantly lower expression in the polypeptide-treated epidermis and dermis compared to untreated controls, and IL-8 and MMP-1 were significantly reduced in the polypeptide-treated dermis compared to untreated equivalents. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7B] The results show that polypeptide-treated human skin equivalents can exhibit higher quality by overall structural analysis score (Panel A), contain significantly fewer senescent cells than untreated human skin equivalents (Panels A and B), and contain gene expression changes (Panel C) in which p16 can have significantly lower expression in the polypeptide-treated epidermis and dermis compared to untreated controls, and IL-8 and MMP-1 were significantly reduced in the polypeptide-treated dermis compared to untreated equivalents. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 7C] The results show that polypeptide-treated human skin equivalents can exhibit higher quality by overall structural analysis score (Panel A), contain significantly fewer senescent cells than untreated human skin equivalents (Panels A and B), and contain gene expression changes (Panel C) in which p16 can have significantly lower expression in the polypeptide-treated epidermis and dermis compared to untreated controls, and IL-8 and MMP-1 were significantly reduced in the polypeptide-treated dermis compared to untreated equivalents. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 8A] These results show that pAkt S473 can be significantly reduced in both epidermal and dermal samples treated with the polypeptide (Panel A), that polypeptide treatment can reduce SA-Bgal staining in UVB-exposed samples, indicating protection from UVB-induced cellular senescence (Panel B), and that the polypeptide can increase the expression of SIRT6 and BLM (Panel C). *P<0.05; **P<0.01; ***P<0.001. [Figure 8B]These results show that pAkt S473 can be significantly reduced in both epidermal and dermal samples treated with the polypeptide (Panel A), that polypeptide treatment can reduce SA-Bgal staining in UVB-exposed samples, indicating protection from UVB-induced cellular senescence (Panel B), and that the polypeptide can increase the expression of SIRT6 and BLM (Panel C). *P<0.05; **P<0.01; ***P<0.001. [Figure 8C] These results show that pAkt S473 can be significantly reduced in both epidermal and dermal samples treated with the polypeptide (Panel A), that polypeptide treatment can reduce SA-Bgal staining in UVB-exposed samples, indicating protection from UVB-induced cellular senescence (Panel B), and that the polypeptide can increase the expression of SIRT6 and BLM (Panel C). *P<0.05; **P<0.01; ***P<0.001. [Figure 9] Increased epidermal layer thickness in human skin equivalents is shown as histological images (Panel A) and statistical analysis of the acquired data (Panel B). **p<0.01. [Figure 10A] Shown are the predicted three-dimensional structures of two polypeptides, peptide 14 (panel A) and peptide 13 (panel B), and a superposition of the two polypeptides (panel C). [Figure 10B] Shown are the predicted three-dimensional structures of two polypeptides, peptide 14 (panel A) and peptide 13 (panel B), and a superposition of the two polypeptides (panel C). [Figure 10C] Shown are the predicted three-dimensional structures of two polypeptides, peptide 14 (panel A) and peptide 13 (panel B), and a superposition of the two polypeptides (panel C). [Figure 11A] Hematoxylin-eosin (H&E) stained histological images of 3D skin equivalents (top row) and ex vivo skin biopsy samples (bottom row) cultured without peptide 14 (control) and with 12.5 μM of peptide 14 (12.5 μM Pep14) over a 5-day period are shown. [Figure 11B] The predicted age, also known as molecular DNA age, for 3D skin model (top graph) and ex vivo skin biopsy (bottom graph) samples treated with 12.5 μM peptide 14 was lower than the predicted age for untreated control (ctrl) samples. **p<0.01. [Figure 11C] 1 shows that the predicted age of ex vivo skin biopsies treated with 12.5 μM peptide 14 (12.5 μM Pep14) was lower than the predicted age of untreated control (ctrl) samples. [Figure 12A] Figure 1 shows the relative melanin content of cell pellets of MeWo cells treated with the positive control of isobutylmethylxanthine (IBMX), the negative control, peptide 14 with IBMX, and retinoic acid with IBMX for 7 days after 14 days of pretreatment culture with IBMX. Data are shown as the mean and standard deviation of melanin content normalized to the negative control (100%). **P<0.01; ***P<0.001; ****P<0.0001. [Figure 12B] Figure 1 shows the relative melanin content of cell culture supernatants from MeWo cells treated with a positive control, a negative control, peptide 14, and retinoic acid with IBMX for 7 days after 14 days of isobutylmethylxanthine (IBMX) pretreatment. Data are shown as the mean and standard deviation of melanin content normalized to the negative control (100%). *P<0.05; **P<0.01. [Figure 13A] Figure 1 shows the relative melanin content of cell culture pellets of MeWo cells treated with a positive control of IBMX, a negative control, peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days, peptide 14 with IBXM for 7 days, and retinoic acid with IBMX for 7 days after pretreatment with isobutylmethylxanthine (IBMX) for 14 days. Data are shown as the mean and standard deviation of melanin content normalized to the negative control (100%). *P<0.05; **P<0.01; ****P<0.0001. [Figure 13B]Figure 1 shows the relative melanin content of cell culture supernatants of MeWo cells pretreated with isobutylmethylxanthine (IBMX) for 14 days, followed by treatment with the positive control (IBMX), the negative control (negative control), peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days, peptide 14 with IBXM for 7 days, and retinoic acid with IBMX for 7 days. Data are shown as the mean and standard deviation of melanin content normalized to the negative control (100%). *P<0.05; **P<0.01; ****P<0.0001. [Figure 14A] Figure 1 shows the relative mRNA expression levels of tyrosinase in MeWo cells treated with the positive control IBMX, the negative control, peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days, peptide with IBXM for 7 days, and retinoic acid with IBMX for 7 days. Data are shown as the mean and standard deviation of 2-ddCt normalized to GAPDH and negative control expression (100%). *P<0.05; **P<0.01; ****P<0.0001. [Figure 14B] Figure 1 shows the relative mRNA expression levels of melanocyte-induced transcription factor (MITF) in MeWo cells treated with the positive control IBMX, the negative control, peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days, peptide with IBXM for 7 days, and retinoic acid with IBMX for 7 days. Data are shown as the mean and standard deviation of 2-ddCt normalized to GAPDH and negative control expression (100%). *P<0.05; **P<0.01; ***P<0.001. [Figure 14C] Figure 1 shows the relative mRNA expression levels of dopachrome tautomerase (DCT) in MeWo cells treated with the positive control IBMX, the negative control, peptide 14 with IBXM for 14 days, retinoic acid with IBMX for 14 days, peptide with IBXM for 7 days, and retinoic acid with IBMX for 7 days. Data are shown as the mean and standard deviation of 2-ddCT normalized to GAPDH and negative control expression (100%). **P<0.01; ***P<0.001; ****P<0.0001. [Figure 15A]H&E stained histological images of an in vitro human skin model treated with vehicle only (control), peptide 13, or peptide 14 are shown. [Figure 15B] The mean histological scores of human skin models treated with vehicle only (control), peptide 14, or peptide 13 were 21.00, 23.83, and 23.44, respectively. **p<0.01. [Figure 16] An example of the left side of the face treated with peptide 14 is shown at baseline before treatment (left, baseline) and after 12 weeks of treatment (right, 12 weeks). [Figure 17] Figure 1 shows the relative mRNA expression levels of p16, BLIMP1, ZYG11B, IL-8, Ki-67, ZIC1, MMP1, and HAS2 in the epidermal and dermal layers of 3D skin equivalents treated with control, peptide 14, peptide 13, or retinoic acid. Data are presented as 2-ddCt normalized to GAPDH and untreated control. *p<0.05. [Figure 18A] Data obtained show the extension of lifespan and healthspan of Caenorhabditis elegans worms (C. elegans), demonstrating that treatment with either 1 μM or 2 μM of the polypeptide improved worm thrashing (Panel A), pumping (Panel B), and median lifespan (Panel C). *P<0.05; **p<0.01. [Figure 18B] Data obtained show that treatment with either 1 μM or 2 μM of the polypeptide improved worm thrashing (Panel A), pumping (Panel B), and median lifespan (Panel C). *P<0.05; **p<0.01. [Figure 18C]Data obtained show that treatment with either 1 μM or 2 μM of the polypeptide improved worm thrashing (Panel A), pumping (Panel B), and median lifespan (Panel C). *P<0.05; **p<0.01. [Figure 19A] Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. [Figure 19B] Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. [Figure 19C] Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. [Figure 19D]Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. [Figure 19E] Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. [Figure 19F] Figures 1A-1F show the effects of the polypeptide sequence LKGIL (SEQ ID NO: 6) (A, B, C) and the polypeptide sequence WLKGI (SEQ ID NO: 7) (D, E, F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis indicates the relative cell number normalized to the untreated control. In panels C and F, the Y-axis indicates the average ATRX foci accumulation per cell. *P<0.05; **p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0026] Aging may be primarily due to a functional decline in the capacity to maintain tissue homeostasis and integrity, possibly coupled with a reduced response to physiological demands under conditions of stress.

[0027] In the mosaic model of skin aging, senescent cells can be induced by endogenous and exogenous stimuli, such as time / age, UV exposure, and smoking, among others. According to this model, senescent cells accumulate in the skin and actively promote tissue aging by altering the local microenvironment through, among other things, the senescence-associated secretory phenotype (SASP), which is composed of inflammatory cytokines. In some cases, senescent cells further accelerate skin aging by impairing epidermal stem cell renewal and promoting the senescence of otherwise normal cells. Thus, senescent cells are not simply a product of skin aging but also play an active role in the aging process.

[0028] Skin dysfunction can affect organismal aging and the progression of aging-related diseases and disorders. For example, healthy skin and normal barrier function may be associated with lower levels of the inflammatory and age-related cytokines IL-1β and IL-6 compared to counterparts with impaired skin barriers. Increased levels of IL-1β and IL-6 have been observed in the serum of patients with several age-related disorders, including cardiovascular disease (CVD), Alzheimer's disease, and type II diabetes. In some cases, IL-6 in the serum of elderly individuals may be associated with total mortality, CVD, cancer, and liver-related mortality. In some cases, restoring epidermal function can effectively reduce circulating cytokine levels of TNFα, IL-1β, and IL-6.

[0029] Provided herein are polypeptides, compositions comprising the polypeptides and other components, and methods of use thereof. The polypeptides and compositions comprising the polypeptides can provide anti-aging effects (e.g., in a subject's cells). The polypeptides can promote a reduction in the level of aging in cells and tissues by promoting apoptosis, promoting DNA repair, and / or inhibiting DNA damage-induced aging. Examples of anti-aging effects include, but are not limited to, increased cell renewal rates, increased collagen production, increased hyaluronic acid synthase production, reduced ATRX nuclear foci accumulation, reduced p16 expression, reduced SASP production, reduced aging-associated beta-galactosidase production, reduced uneven pigmentation, maintenance or improvement of the epidermal barrier, and reduced transepidermal water loss (TEWL). The polypeptides provided herein and compositions comprising the polypeptides may inhibit, prevent, or slow age-related and / or aging-associated diseases or disorders. Furthermore, the polypeptides and compositions comprising the polypeptides may promote or improve healthspan and / or promote longevity.

[0030] It is understood that the methods and compositions described herein are not limited to the particular methodology, protocols, and reagents described herein, and as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims.

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention described herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention described herein, the preferred methods, devices, and materials are described herein.

[0033] Suitable conservative substitutions of amino acids in peptides or polypeptides are well known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. Watson et al. (1987, Molecular Biology of the Gene, 4th Edition, The Benjamin Cummings Pub. Co., p. 224) is incorporated herein by reference. Amino acids may be either L- or D-isomers. When an amino acid residue is part of a polypeptide chain, the D-isomer of the amino acid can be substituted for an L-amino acid residue as long as the desired functional properties are retained. Amino acids herein can be represented by their standard IUPAC one-letter or three-letter codes. Amino acid residues represented by "X" or "Xxx" refer to any one of the naturally occurring or non-naturally occurring amino acid residues, or modifications of adjacent residues, as known in the art. Amino acid substitutions are generally single residues, and such substitutions are preferably made with those set forth in Table 1, but may also be clustered or dispersed among multiple residues. Amino acids can be replaced with various naturally occurring or non-conventional amino acid residues. Such substitutions can be classified as "conservative", in which the amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid that has similar properties in terms of polarity, side chain functionality, or size. Additions include the addition of one or more naturally occurring or non-conventional amino acid residues. Deletions include the deletion of one or more amino acid residues.

[0034] [Table 1]

[0035] Substitutions encompassed by the present disclosure may be "non-conservative," in which an amino acid residue present in the polypeptide is substituted with an amino acid having different properties, such as a naturally occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino acid with alanine), or alternatively, a naturally occurring amino acid is substituted with a non-conventional amino acid.

[0036] As used herein, the term "analog(s)" refers to a composition that retains the same structure or function (e.g., binding to a receptor) as a polypeptide, such as the same protein from a different organism. Examples of analogs herein include mimetics or peptidomimetics, peptides, small organic and inorganic compounds, and large organic and inorganic compounds, as well as derivatives and variants of polypeptides. Such derivatives and variants refer to polypeptides that differ from naturally occurring polypeptides by deletion, addition, substitution, or side chain modification of one or more amino acids. In some embodiments, a peptide analog is a peptide in which one or more of the amino acids has undergone a side chain modification. Examples of side chain modifications contemplated by the present disclosure include modifications of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of the amino group with cyanate; trinitrobenzylation of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group with succinic anhydride and tetrahydrophthalic anhydride; and lysination with pyridoxal-5-phosphate followed by pyridoxylation by reduction with NaBH4. In some embodiments, peptide analogs are those in which the guanidine groups of arginine residues are modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal; the carboxyl groups are modified by carbodiimides via O-acylisourea formation followed by subsequent derivatization, for example, to the corresponding amide; and the sulfhydryl groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; mixed disulfide formation with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromercuribenzoic acid and 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuri-4-nitrophenol, and other mercurial agents; carbamoylation with cyanate at alkaline pH, and the like.In any of the analogs herein, any modification of the cysteine ​​residue preferably does not affect the ability of the peptide to form the necessary disulfide bond. In some embodiments, peptide analogs include tyrosine residues modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halide compounds; tyrosine residues modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives; imidazole ring modifications of histidine residues by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate; proline residues modified by, for example, hydroxylation at the 4th position; glycosylation variants from completely unglycosylated molecules to modified glycosylation; and tryptophan residues modified by altered glycosylation patterns resulting from the expression of recombinant molecules in different host cells.

[0037] The term "isolated" means altered from its natural state; that is, changed or removed from its original environment if it occurs in nature, or both. As the term is used herein, a naturally occurring polynucleotide or polypeptide that is naturally present, for example, in an animal living in the wild, is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is.

[0038] As used herein, the terms "protein," "peptide," "oligopeptide," or "polypeptide" refer to any composition comprising two or more amino acids joined together by peptide bonds. Polypeptides often contain amino acids other than the 20 commonly referred to as the 20 naturally occurring amino acids, and it will be understood that many amino acids, including the terminal amino acids, may be modified in a given polypeptide by either natural processes, such as glycosylation and other post-translational modifications, or by chemical modification techniques that are known in the art. Known modifications that may be present in the polypeptides of the present disclosure include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavonoid or heme moiety, covalent attachment of a polynucleotide or polynucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, glycosylphosphatidylinositol GPI membrane anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to polypeptides such as arginylation, and ubiquitination. The term "protein" also includes "artificial proteins," which refer to linear or non-linear polypeptides consisting of alternating repeats of a polypeptide (e.g., SEQ ID NOs: 1-7) and a spacer. DNA constructs encoding alternating repeats of a polypeptide and a spacer can be synthesized using methods well known in the art (see Rotzschke et al., 1997, Proc. Natl. Acad. Sci. USA 94:14642-14647).

[0039] The term "purified," when used herein to describe a polypeptide, polynucleotide, or other composition, refers to a polypeptide, polynucleotide, or other composition that has been separated from one or more compounds with which it is normally associated in nature. Such other compositions may be, for example, other polypeptides or polynucleotides, carbohydrates, lipids, etc. The term "purified" may also be used to specify separation of a monomeric polypeptide of the disclosure from oligomeric forms, examples of which include homodimers, heterodimers, trimers, etc. A substantially pure polypeptide generally comprises at least about 50%, 60%, 70%, 80%, or 90% weight / weight of the polypeptide sample, or more preferably at least about 95%, 96%, 97%, 98%, 99%, or 99.5% weight / weight of the polypeptide sample. In preferred embodiments, the polypeptides of the present disclosure are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% pure relative to heterologous polypeptides.

[0040] As used herein, the term "subject" or "patient" includes animals. In some embodiments, the subject may be a mammal. Examples of mammals include, but are not limited to, any member of the following mammalian classes: humans, non-human primates such as chimpanzees, and other apes and monkey species; livestock such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. The mammal may be a human.

[0041] As used herein, the terms "treat," "treating," or "treatment" include delaying the onset, reducing the occurrence, or ameliorating at least one symptom of a disease or disorder, preventing additional symptoms, inhibiting the disease or disorder, e.g., arresting the progression of the disease or disorder, relieving the disease or disorder, causing regression of the disease or disorder, alleviating the conditions caused by the disease or disorder, or arresting the symptoms of the disease or disorder prophylactically and / or therapeutically.

[0042] As used herein, the term "pharmaceutically acceptable" refers to a substance, including but not limited to salts, carriers, or diluents, that do not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the substance may be administered to a subject without causing undesired biological effects or interacting adversely with any of the components of the composition in which it is included.

[0043] As used herein, the term "carrier" refers to relatively nontoxic chemical compounds or agents that facilitate the incorporation of a compound into cells or tissues.

[0044] As used herein, the term "diluent" refers to a compound used to dilute a desired compound prior to delivery. Diluents may also be used to stabilize compounds, as they can provide a more stable environment. Salts dissolved in buffers (which can control or maintain pH) are used as diluents in the art, including, but not limited to, phosphate buffered saline solutions.

[0045] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or disorder being treated. This may result in a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound as disclosed herein that is required to produce a clinically significant reduction in a disease symptom. An appropriate "effective" amount in any individual may be determined using techniques such as a dose escalation study.

[0046] Senescent cells can be identified by senescence-associated beta-galactosidase (SA-BGal) production, p16 expression, SASP presentation, and / or alpha thalassemia / mental retardation X-linked chromatin remodeling protein (ATRX) foci accumulation in the nucleus. Functional alterations that can also identify senescent cells include, but are not limited to, reduced proliferative capacity and resistance to mitogenic stimuli.

[0047] Aging is usually attributed to a decline in the ability to maintain tissue homeostasis and integrity and is associated with a reduced response to physiological demands under conditions of stress.

[0048] A mosaic model of skin aging has been proposed, in which senescent cells are induced by endogenous and exogenous stimuli, such as time / age, UV exposure, and smoking, among other stimuli. According to this mosaic model, senescent cells accumulate in the skin and actively promote tissue aging by altering the local microenvironment through, among other things, the senescence-associated secretory phenotype (SASP), which is composed of inflammatory cytokines. Senescent cells have been shown to promote skin aging by impairing epidermal stem cell renewal and by promoting the senescence of otherwise normal cells. Thus, senescent cells may not simply be a product of skin aging but may also play an active role in the aging process.

[0049] Polypeptides possess properties such as multifunctional behavior that make them useful for cosmetic or therapeutic applications, including skin treatment. Skin dysfunction observed with aging can influence the progression of age-related diseases and disorders.

[0050] Polypeptides The polypeptides and compositions comprising the polypeptides provided herein can provide senotherapy effects, for example, the polypeptides can reduce senescence by stopping senescence, preventing senescence, inhibiting senescence, reversing senescence, destroying senescent cells, killing senescent cells, removing senescent cells, or by any suitable mechanism that reduces the burden or effects of senescent cell accumulation. Such polypeptides can, in some cases, comprise the amino acid sequence LKGI (SEQ ID NO: 5). Compositions comprising such polypeptides can be used to provide senotherapy effects.

[0051] A polypeptide (e.g., a senotherapeutic polypeptide) can contain at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more amino acids. In some cases, a polypeptide can be up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. In some cases, a polypeptide can be 4 to 25, 4 to 15, or 4 to 10 amino acids in length. In some embodiments, a polypeptide can contain at least 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids. In certain embodiments, a polypeptide may contain less than 100, 90, 80, 70, 60, 50, 40, 30, or fewer amino acids.

[0052] Examples of polypeptides that can provide a therapeutic effect are provided in Table 2 below.

[0053] [Table 2]

[0054] Polypeptides are X1X2X3X4X5X6X7X8X9X 10 The polypeptide may be an isolated or recombinant polypeptide that may comprise the amino acid sequence of the formula (I). The amino acids comprised in the polypeptide may include naturally occurring amino acids, which may include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Glu, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0055] In some cases, X1X2X3X4X5X6X7X8X9X 10 The isolated or recombinant polypeptide comprising the amino acid sequence of can comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to a first sequence, ETAKHWLKGI (SEQ ID NO: 1), wherein X1 is E, X2 is T, X4 is K, X6 is W, X7 is L, X9 is G, and X 10is I, and (i) X3 is not S, or (ii) if X5 is any amino acid, then X8 is not G, or (iii) if X8 is any amino acid, then X5 is not N, or (iv) in any one of (i), (ii), or (iii), the sequence may optionally include 1, 2, 3, or 4 conservative amino acid substitutions. In some cases, the isolated or recombinant polypeptide may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to the sequence of ETAKHWLKGI (SEQ ID NO: 1). The present disclosure further contemplates analogs, such as peptidomimetics, of the above.

[0056] In some cases, X1X2X3X4X5X6X7X8X9X 10 The isolated or recombinant polypeptide comprising the amino acid sequence of can comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to a second sequence, ATAKAWLKGI (SEQ ID NO:2), wherein X1 is A, X2 is T, X3 is A, X4 is K, X5 is A, X6 is W, X7 is L, X8 is K, X9 is G, and X 10 is I. Such recombinant polypeptides may optionally contain one, two, three, or four conservative amino acid substitutions. In some cases, the isolated or recombinant polypeptide may contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to the sequence of ATAKAWLKGI (SEQ ID NO: 2). The present disclosure further contemplates analogs such as peptidomimetics of the above.

[0057] In some cases, X1X2X3X4X5X6X7X8X9X 10may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% identity to a third sequence, KLKGILRGAA (SEQ ID NO:3), wherein (i) X3 is not N when X9 is any amino acid, or (ii) X9 is not S when X3 is any amino acid, or (iii) X7 is not L when X4 is any amino acid, or (iv) X4 is not S when X7 is any amino acid, or (v) in any one of (i), (ii), (iii), or (iv), the sequence may optionally include 1, 2, 3, or 4 conservative amino acid substitutions. In some cases, the isolated or recombinant polypeptide may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity to the sequence of KLKGILRGAA (SEQ ID NO: 3). The present disclosure further contemplates analogs such as peptidomimetics of the above.

[0058] In some cases, X1X2X3X4X5X6X7X8X9X 10 The isolated or recombinant polypeptide comprising the amino acid sequence of X3 can comprise an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to a fourth sequence, WLKGILREAA (SEQ ID NO: 4), wherein X1 is W, X2 is L, X3 is K, X4 is G, X5 is I, X6 is L, X7 is R, X8 is E, X9 is A, and X 10 is A. Such recombinant polypeptides may optionally contain one, two, three, or four conservative amino acid substitutions. In some cases, the isolated or recombinant polypeptide may contain an amino acid sequence having at least 80%, 85%, 90%, 95%, or 100% identity to the sequence of WLKGILREAA (SEQ ID NO:4). The present disclosure further contemplates analogs such as peptidomimetics of the above.

[0059] In some cases, the polypeptide may comprise the amino acid sequence LKGI (SEQ ID NO:5), LKGIL (SEQ ID NO:6), or WLKGI (SEQ ID NO:7) (see Table 3 below).

[0060] [Table 3]

[0061] A polypeptide comprising one of SEQ ID NOs:5-7 may contain at least 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 amino acids. In some embodiments, a polypeptide comprising one of SEQ ID NOs:5-7 may contain up to 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. SEQ ID NOs:5-7 may be at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or between the N-terminus and C-terminus of the polypeptide. In some cases, a polypeptide may contain more than SEQ ID NOs:5-7.

[0062] A polypeptide can be isolated, substantially pure, or purified. Optionally, an isolated polypeptide can be (i) chemically synthesized, or (ii) expressed in a host cell and purified away from associated and contaminating proteins. Optionally, a polypeptide can be present in a host cell as an expression product as part of an expression vector, can be linked to a protein moiety, or can be linked to a chemical moiety.

[0063] Analogs of the disclosed polypeptides, including peptidomimetics, can provide therapeutic benefits. The peptides and polypeptides disclosed herein may include peptidomimetic equivalents.

[0064] In some cases, as discussed above, a polypeptide may have sequence identity to a polypeptide described herein. The sequence identity of a polypeptide may refer to the exact amino acid-to-amino acid correspondence of two polypeptide sequences. In some cases, techniques for determining sequence identity may include determining an amino acid sequence and comparing it to a second amino acid sequence. Two or more sequences may be compared by their percent identity, or by dividing the number of exact matches between two aligned sequences by the length of the longer sequence and multiplying by 100. Percent identity may also be determined by comparing sequence information using an advanced BLAST computer program, including, for example, version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and is as discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). The program may be used to determine percent identity over the entire length of the polypeptides being compared. Default parameters are provided to optimize searches using, for example, short query sequences in the blastp program. The program further allows the use of the SEG filter to mask off segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993).

[0065] composition Disclosed herein are compositions comprising one or more of the polypeptides described herein. In some embodiments, the compositions may be used for senotherapy. In some cases, the compositions may be used to treat age-related diseases or conditions or age-related disorders, for example, to delay the onset, reduce the occurrence, or ameliorate the onset of age-related diseases or conditions or age-related disorders. In some cases, the compositions may be used to treat tissue lesions, for example, to delay the onset, reduce the occurrence, or ameliorate tissue lesions, such as UV damage caused by sun exposure.

[0066] In some cases, the composition may include, for example, an effective amount of the polypeptide, alone or in combination with one or more vehicles (e.g., a therapeutically acceptable composition or a therapeutically acceptable carrier) and other therapeutically effective compounds. In some embodiments, an effective amount of the polypeptide refers to having a desired effect on a subject, including, but not limited to, a cell, tissue, or organism treated with the composition. In some embodiments, an effective amount of the polypeptide has minimal or low systemic effect on the treated subject. In some embodiments, an effective amount of the polypeptide exerts maximal effect locally at or near the treatment area. In some embodiments, the formulation can be configured to penetrate locally through the epidermis to the dermis. In some embodiments, the formulation is configured to penetrate locally through the epidermis layer. In some embodiments, an effective amount of the polypeptide is at least 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, at least 10 μM, at least 25 μM, at least 50 μM, at least 75 μM, at least 100 μM, at least 150 μM, at least 200 μM, at least 250 μM, at least 300 μM, at least 350 μM, at least 400 μM, at least 450 μM, or at least 500 μM. In some examples, an effective amount of the polypeptide is from about 1 nM to about 1000 nM, from about 5 nM to about 750 nM, from about 25 nM to about 750 nM, or from about 50 nM to about 500 nM. In some examples, an effective amount of the polypeptide is about 1 μM to about 500 μM, about 25 μM to about 250 μM, about 50 μM to about 200 μM, or about 75 μM to about 125 nM. In some examples, an effective amount of the polypeptide is at least 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, at least 0.005%, at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, or at least 5% (w / w) of the final composition.In some examples, an effective amount of polypeptide is from about 0.00001% to about 5%, 0.00001% to about 1%, 0.00001% to about 0.1%, about 0.001% to about 5%, about 0.005% to about 4%, about 0.005% to about 3%, about 0.005% to about 2%, about 0.005% to about 1%, or about 0.005% to about 0.5% of the final composition. In some embodiments, an effective amount of polypeptide for in vivo applications may be at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 times the amount used for in vitro applications. An effective amount of polypeptide causes the polypeptide to penetrate into the dermis, and in some cases, causes about 1% penetration, about 2% penetration, about 4% penetration, about 5% penetration, or about 10% penetration.In some cases, up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the polypeptide in the composition applied to the skin penetrates into the dermis.In some cases, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the polypeptide in the composition applied to the skin penetrates into the dermis.In some cases, the amount used for in vivo application is multiple of the amount of skin penetration in in vitro penetration test. In some examples, the amount used in in vivo applications is at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 1000 times the amount of skin penetration in vitro.

[0067] In some cases, the compositions described herein can be administered with (e.g., simultaneously, sequentially, or formulated together with) one or more additional nutraceutical, cosmetic, therapeutic, or pharmaceutical treatments.

[0068] In some embodiments, the formulation may be used in conjunction with one or more treatments. In some embodiments, the formulation may be used in conjunction with sonic treatment, ultrasound treatment, LED treatment, light treatment, electrical treatment, radiofrequency treatment, or other dermatological treatment. In some embodiments, the composition is applied to the skin before, after, or during the treatment.

[0069] The composition can be formulated for topical application. For example, the composition may be formulated for application on the skin. In some embodiments, the composition is configured as a topical supplement. Formulations for topical application, etc., can be creams, ointments, gels, liquids, powders, lotions, serums, emulsions, moisturizers, foams, face masks, mousses, aerosols, sprays, cleansers, toners, topical patches, hydrogel patches, or shampoos. Topically applied polypeptides can be applied to the affected area, areas that may be affected in the future, a portion of the subject, or substantially the entire subject. In some cases, topical treatments can be applied with a buffer, another topical treatment, a cream, or a moisturizer.

[0070] Compositions for external application can be formulated as cosmetic compositions. Examples of cosmetic compositions include makeup, foundation, sunscreen, after-sun lotion, and skin care products, including anti-aging skin care products. In some cases, the makeup composition can leave color on the face and can include foundation, bronzer, mascara, concealer, eyeliner, eyebrow color, eyeshadow, blusher, lip color, powder, solid emulsion compact, or other makeup items. In some cases, the skin care product can be a product used to treat or care for the skin, or in some way moisturize, improve, accelerate regeneration, protect, prevent damage, or cleanse the skin. The skin care product can be applied as a cream, topical patch, hydrogel patch, transdermal patch, ointment, gel, liquid, powder, lotion, serum, emulsion, oil, clay, moisturizer, foam, face mask, mousse, aerosol, spray, cleanser, toner, or shampoo. In some cases, the skin care product may be in the form of an adhesive, a bandage, an exfoliant, a toothpaste, a moisturizer, a lotion, a primer, a lipstick, a lip balm, an anhydrous occlusive moisturizer, an antiperspirant, a deodorant, a personal cleansing product, an occlusive drug delivery patch, nail polish, a powder, a tissue, a wipe, a hair conditioner, or a shaving cream.

[0071] The compositions contemplated herein can also be edible, i.e., can be formulated as a dietary supplement or beverage, so that the composition is formulated to be safe for human consumption.In some cases, the dietary composition can be therapeutically effective in treating age-related diseases or illnesses or age-related disorders.In some cases, the dietary supplement can be configured as a tablet, capsule, chew, gummy, powder, food bar, meal replacement bar, or food additive.In some cases, the beverage can be formulated to contain water, soda, tea, coffee, milk, juice, shake, drink, or other edible liquid.

[0072] In some cases, the composition may contain a skin conditioning agent (e.g., a moisturizer, an exfoliant, an emollient, or a hydrator). The moisturizer may be for moisturizing, reducing scaling, or stimulating the removal of accumulated scale from the skin. The exfoliant may be for removing dead skin cells from the surface and may be a physical exfoliant or a chemical exfoliant. The emollient may be a formulation or ingredient that can soften dry, rough, or flaky skin. The hydrator may be for moisturizing, reducing scaling, or stimulating the removal of accumulated scale from the skin. In some cases, the emollient is an agent that prevents moisture loss and has a softening and soothing effect on the skin. In some embodiments, the emollient may comprise at least one of vegetable oil, mineral oil, shea butter, cocoa butter, petrolatum, fatty acids (including emu, mink, and lanolin), triglycerides, benzoic acid, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, Calycophyllum spruceanum oil, almond oil, argan oil, caprylic / capric triglyceride, jojoba butter, jojoba oil, Spectrastat G2, ceramides, and algae extract. In some embodiments, the composition comprises a skin moisturizer, also known as a skin hydrator. In some cases, the skin moisturizer includes, but is not limited to, glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract. In some embodiments, the composition comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a skin conditioning agent.In some embodiments, the composition comprises from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 50%, from about 5% to about 50%, from about 5% to 45%, or from about 5% to 40% of a skin conditioning agent.

[0073] The composition may contain a sheen control agent that can improve or adjust the glossy appearance of skin. Sheen control agents may be porous in nature. Such agents may provide a reservoir for absorbing excess moisture and reducing the appearance of shine. Sheen control agents may be silica, magnesium aluminum silicate, talc, sericite, and various organic copolymers. Particularly effective sheen control agents may include silicates or carbonates formed by the reaction of alkali (IA) metals, alkaline earth (IA) metals, or transition metals with silica (silicon dioxide). Preferred sheen control agents are selected from the group consisting of calcium silicate, amorphous silica, calcium carbonate, magnesium carbonate, zinc carbonate, bentonite clay, and combinations thereof.

[0074] The composition may contain a film-forming agent that can help the film last and adhere to the skin. The film-forming agent can improve the long-term wear and non-transfer performance of the composition. The film-forming agent may be water-soluble, water-insoluble, or water-dispersible. Film formers include: 1) organic silicone resins, fluorinated silicone resins, copolymers of organic silicone resins, trimethylsiloxysilicate, GE's silicone resin copolymers, SF1318 (organic ester of silicone resin and isostearic acid copolymer) and CF1301 (silicone resin and alpha-methylstyrene copolymer), Dow Corning's silicone resin pressure-sensitive adhesive copolymer and various PDMS (BIO-PSA series); 2) acrylic and methacrylic acid polymers and resins, silicone-acrylate type copolymers, and fluorinated versions including 3M's silicones and polymers, Shin-Etsu Chemical's KP545, alkyl-acrylate copolymers, and Shin-Etsu Chemical's KP561 and 562; 3) Collaborative Labs' decene / butene copolymers; 4) polyvinyl-based materials, PVP, PVP / VA (including ISP's Antaron / Ganex (PVP / triacontene copolymer), BASF's Luviskol materials), polyurethanes, Polyderm PE / PA, Polyderm series from Alzo, including but not limited to Polyderm PPI-SI-WS, Polyderm PPI-GH, Luviset P.UR from BASF; 6) polyquaternium materials, Luviquat series from BASF; 7) acrylate copolymers and acrylate / acrylamide copolymers, Luvimer and Ultrahold series, both available from BASF; 8) styrene-based materials; and 9) chitosan and chitosan-based materials, including cellulose and cellulose-based materials.

[0075] The composition may include a thickener or emulsifier. Thickeners may be used to increase the viscosity of liquid base materials used in cosmetic compositions. The selection of a particular thickener may depend on the type of composition desired (e.g., gel, cream, lotion, or wax-based), the desired rheology, the liquid base material used, and other materials used in the composition. Examples of thickeners or emulsifiers include candelilla, carnauba wax, beeswax, spermaceti, carnauba, bayberry, montan, ozokerite, ceresin, paraffin, synthetic waxes such as Fischer-Tropsch wax, silicone wax (DC 2503 from Dow Corning), waxy materials such as microcrystalline wax, soaps, such as high fatty acids, sodium and potassium salts of acids having 12 to 22 carbon atoms, amides of high fatty acids, high fatty acid amides of alkylolamines, dibenzaldehyde-monosorbitol acetal, alkali metal and alkaline earth metal salts of acetates, propionates, and lactates, and mixtures thereof. Also useful are polymeric materials such as locust bean gum, sodium alginate, sodium caseinate, egg albumin, gelatin agar, carrageenan gum, sodium alginate, xanthan gum, quince seed extract, tragacanth gum, starch, modified starch, and the like; semi-synthetic polymeric materials such as cellulose, cellulose derivatives, cellulose ethers, hydroxyethyl cellulose, methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, guar gum, hydroxypropyl guar gum, soluble starch, cationic cellulose, cationic guar; and synthetic polymeric materials such as carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcohol-polyacrylic acid polymers, poly(acrylic acid), carbomer, polymethacrylic acid polymers, polyvinyl acetate polymers, polyvinyl chloride polymers, polyvinylidene chloride polymers, and the like. Inorganic thickeners may also be used, for example aluminum silicates such as bentonite, or mixtures of polyethylene glycol and polyethylene glycol stearate or distearate.Emulsifiers may be used to help keep the hydrophilic and hydrophobic components from separating within the emulsion. In some cases, emulsifiers include, but are not limited to, Olivem, Oliwax LC, polysorbate, laureth-4, and potassium cetyl sulfate.

[0076] Cosmetic compositions can provide a temporary change in appearance, or they can provide a long-term change in appearance. In some cases, cosmetic compositions can be formulated to provide a short-term change in appearance (e.g., skin pigmentation or plumping), as well as a long-term change in appearance (e.g., reduction in age spots, the appearance of fine lines, the appearance of wrinkles, or other characteristics that may affect appearance).

[0077] The compositions may contain additives that have additive or synergistic effects when applied together with the polypeptides disclosed herein. For example, a composition containing a polypeptide and an additive may have an effect on aging and age-related diseases or disorders (e.g., delaying the onset, reducing the occurrence, or ameliorating one or more symptoms) that is greater than the individual effects of the additive, the polypeptide, or the sum of the individual effects of the additive and the polypeptide. The additive may be an additional polypeptide, glycosaminoglycan, carbohydrate, polyphenol, protein, lipid, aqueous or oily plant extract, nucleic acid, antibody, small molecule, vitamin, moisturizer, emollient, or another suitable additive. In some embodiments, the composition includes a UV blocker. In some embodiments, UV blockers may include, but are not limited to, aminobenzoic acid, avobenzone, cinoxate, dioxybenzone, homosalate, meladimate, octocrylene, octinoxate, octisalate, oxybenzone, padimate O, ensulizole, sulisobenzone, titanium dioxide, trolamine salicylate, and zinc oxide.

[0078] Frequently, the methods, systems, and compositions provided herein include a vitamin. In some examples, the vitamin provides a skin-soothing effect, a skin-restoring effect, a skin-replenishing effect, and / or a moisturizing effect. In some examples, the vitamin provides an antioxidant effect. In some examples, the vitamin acts as an emollient. In some examples, the vitamin improves the appearance of enlarged pores, uneven skin tone, fine lines, dullness, and / or superficial weakness of the skin. In some examples, the vitamin is vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, and mixtures thereof. In some examples, the composition includes a derivative of a vitamin. In some examples, a derivative of a vitamin is used to improve the stability of the vitamin in the composition and / or the composition compatibility of the vitamin derivative with other ingredients in the composition. In some examples, the composition includes vitamin B3 or a derivative thereof and vitamin E or a derivative thereof. In some examples, the composition includes niacinamide and vitamin E or a derivative thereof. In some examples, the composition includes vitamin C or a derivative thereof, vitamin B3 or a derivative thereof, and vitamin E or a derivative thereof. In some embodiments, the composition includes at least 0.01%, 0.05%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a vitamin. In some embodiments, the composition includes about 0.1% to about 10%, about 0.1% to about 5%, about 0.5% to about 10%, about 0.5% to about 5%, about 1% to 10%, or about 1% to 5% of a vitamin.

[0079] Compositions for topical administration may further comprise a carrier. The carrier may be a solution, emulsion, ointment, oil, or gel base. The gel base may, for example, comprise one or more of the following: petrolatum, lanolin, PEG(s), beeswax, mineral oil, diluents such as water and alcohol, emulsifiers, and / or stabilizers. A thickener may be present in a therapeutic composition for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device. In some cases, biodegradable microspheres (e.g., polylactic acid) may also be used as a carrier for the composition. In some cases, transdermal patches are configured to deliver the formulation to the epidermal layer of the skin. In some cases, transdermal patches are configured to deliver the formulation to both the epidermal and dermal layers of the skin. In some cases, the formulation is configured for minimal systemic delivery in a subject or is not intended to be delivered directly to the subject's bloodstream.

[0080] The composition may further comprise one or more diluents, such as a buffer solution, or one or more antioxidants, such as ascorbic acid, low molecular weight polypeptides, polypeptides, amino acids, carbohydrates, including glucose, sucrose, or dextrin, chelating agents, such as EDTA, glutathione, and other stabilizers. Neutral buffered saline, or saline mixed with nonspecific serum albumin, are exemplary diluents. The product can be formulated as a lyophilizate using a suitable excipient solution (e.g., sucrose) as a diluent.

[0081] The composition may include one or more excipients, such as therapeutic, nutraceutical, or cosmetic excipients. Examples of excipients may include antiadherents, binders, coatings, colorants, disintegrants, flavors, glidants, lubricants, preservatives, adsorbents, sweeteners, or vehicles.

[0082] Suitable excipients or stabilizers may be nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citric acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid, vitamin E, and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride, benzalkonium chloride, benzethonium chloride, gluconolactone and sodium benzoate; phenol, butyl, or benzyl alcohol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; EDTA or EDTA substitutes (e.g., Biopure GLDA, Spectrastat G2), sugars such as sucrose, mannitol, trehalose, or sorbitol, counterions that form salts such as sodium, metal complexes (e.g., Zn-protein complexes), and / or surfactants.In some examples, the surfactant is polysorbate 20, polysorbate 80, alginate, poloxamer, TRITON® (t-octylphenoxypolyethoxyethanol), non-ionic surfactant, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, lauryl-sulfohetam, myristyl-sulfohetam, linoleyl-sulfohetam, or stearyl-sulfohetam; lauryl-sarcosine, iristylsarcosine, linoleyl-sarcosine, or stearyl-sarcosine; linoleyl-betaine, myristyl Lauraamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidropropyl betaine, or isostearamidopropyl betaine (e.g., lauraarnidopropyl); myristamidopropyl taurate, palmidopropyl taurate, or isostearamidopropyl dimethylamine methyl cocoyl taurate, or disodium oleylmethyl taurate; sorbitan monopalmitate, and the MONAQUAT series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol (PEG), polypropylene glycol (PPG), copolymers of polyoxyethylene and polyoxypropylene glycol (e.g., Pluronies / Poloxamer, PLURONIC® F68, etc.), or another suitable surfactant. In some cases, the composition may contain squalene, natural oil, plant extract, hyaluronic acid, or clay.In some cases, the composition may contain a skin penetration enhancer to enhance the penetration of active ingredients into the skin.In some cases, the skin penetration enhancer includes, but is not limited to, fatty acid, aromatic oil, urea, liposome, microsphere, DMSO, azone, sodium PCA, and squalene.

[0083] In some embodiments, the formulation includes a carrier, microsphere, liposome, or micelle, to deliver the polypeptide and control the release frequency and / or penetration depth of the polypeptide in the skin. In some embodiments, the polypeptide is functionalized. In some embodiments, the polypeptide is functionalized with a chemical group. In some embodiments, the polypeptide is functionalized with a functional group containing 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or fewer carbons, or 20 or more carbons. In some embodiments, the polypeptide is functionalized with acetyl or palmitoyl.

[0084] A polypeptide or composition applied to a subject can be sterilized, which can be accomplished, for example, by filtration through sterile filtration membranes or other art-recognized sterilization methods.

[0085] The composition may contain a therapeutically effective amount of a polypeptide or peptidomimetic that can delay the onset of an age-related disease, or the onset of a disease or age-related disorder, reduce its occurrence, or improve one or more symptoms, such as skin conditions. In some cases, a therapeutically effective amount can be an amount of a therapeutic agent (e.g., a polypeptide) that can induce a therapeutic (e.g., senotherapy) or desired response in a subject. A therapeutically effective amount can be sufficient to cause a therapeutic effect in a subject. The therapeutically effective amount can vary depending on various factors, including the active agent selected for use and the age, weight, height, and / or general health of the subject being treated.

[0086] As understood in clinical contexts, an effective therapeutic amount of an active agent may or may not be achieved in combination with another drug, compound, therapeutic, or pharmaceutical composition. Thus, an effective therapeutic amount may be considered in the context of administering one or more active agents, and a single active agent may be considered to be given in an effective amount when, in combination with one or more other active agents, a desired result is achieved or is likely to be achieved. Accordingly, in some instances, one or more active agents may be administered to a subject. In other instances, treatment with an active agent described herein is performed before or after one or more treatment modalities described herein.

[0087] Polypeptide Synthesis Furthermore, the present disclosure provides an isolated polynucleotide encoding one or more of the polypeptides of the present disclosure.The isolated polynucleotide can be present in an expression vector comprising the isolated polynucleotide operably linked to a promoter.The expression vector can be present in an isolated cell (i.e., a recombinant cell transfected or transformed with the expression vector).

[0088] Suitable expression vectors can include those that replicate in bacterial, plant, fungal, insect, or animal host cells and / or express the disclosed peptides, polypeptides, and variants thereof. Expression vectors can be used to transform suitable host cells (e.g., E. coli). Transformed host cells can be cultured or fermented so that the peptide or polypeptide is constitutively expressed or expressed after addition of an agent that induces expression (e.g., via an inducible promoter). Expression vectors as contemplated herein can include control sequences that regulate expression of the encoded polypeptide. Expression control sequences can include constitutive or inducible promoters (e.g., T3, T7, Lac, trp, or phoA), ribosome binding sites, or transcription terminators.

[0089] The expression vector can be used to transform a host cell. Suitable host cells include bacterial, plant, fungal, insect, or animal host cells. Suitable bacteria include, but are not limited to, Gram-negative bacteria such as Escherichia species (e.g., Escherichia coli), other Gram-negative bacteria (e.g., Pseudomonas species such as Pseudomonas aeruginosa, or Caulobacter species such as Caulobacter crescentus), or Gram-positive bacteria (e.g., Bacillus species such as Bacillus subtilis). Suitable bacterial cells may include yeast (e.g., Saccharomyces cerevisiae).

[0090] An expression vector can provide a mechanism for the synthesis of, for example, a polypeptide. Synthesis can be carried out in cells (e.g., animal cells, plant cells, bacterial cells, or yeast cells). An expression vector can contain nucleic acid, such as DNA derived from a plasmid, cosmid, phasmid, or bacteriophage, or synthesized by chemical or enzymatic means, into which one or more fragments of nucleic acid capable of encoding one or more of the polypeptides described herein can be inserted or cloned. An expression vector can be autonomously replicable in a defined host or organism, such that the cloned sequences are replicated. An expression vector can have a linear, circular, or supercoiled configuration and can be complexed with other vectors or other materials for specific purposes. Components of an expression vector can include, but are not limited to, (1) DNA, (2) a sequence encoding a therapeutic or desired product, or (3) a DNA molecule incorporating regulatory elements for transcription, translation, RNA stability, and replication.

[0091] Polypeptides can be produced using expression vectors. In some cases, such production can involve culturing or fermenting transformed host cells (e.g., bacterial host cells as contemplated herein) containing an expression vector (as contemplated herein) that includes a nucleic acid molecule encoding the disclosed peptides, polypeptides, or variants thereof (as contemplated herein), where the culturing occurs under conditions that result in expression of the peptide, polypeptide, or variant, and isolating, separating, or purifying the peptide, polypeptide, or variant. Transformed bacteria can be cultured or fermented using methods known in the art to express the peptide, polypeptide, or variant. Exemplary isolation, separation, or purification methods can include one or more of the following steps: cell disruption, clarification (e.g., by centrifugation or filtration), chromatographic separation, dialysis, and precipitation.

[0092] In some other embodiments, the polypeptide may be chemically synthesized. Synthesis of the polypeptide may be carried out using liquid phase techniques, solid phase methods, or other suitable methods of polypeptide synthesis.

[0093] method Methods for using the polypeptides and compositions disclosed herein are provided. Such methods can include administering one or more polypeptides described herein to a subject. The methods described herein can delay, reduce, or ameliorate the onset of age-related diseases or disorders or age-related disorders.

[0094] The methods described herein can delay the onset, reduce the occurrence, reduce the occurrence, or ameliorate a disease, disorder, or condition associated with the accumulation of senescent cells. The disease or disorder associated with the accumulation of senescent cells can be age-related. In some cases, the disease or disorder can worsen over time if left untreated.

[0095] The polypeptide or composition may be applied or administered to a subject to treat a condition that is directly or indirectly affected by skin health. Such methods may include administering to a subject a compound that promotes skin health or applying a topical treatment to the skin.

[0096] Age-related disorders can involve a biological progression of events that occurs during a disease process that can affect the body, which can mimic or substantially mimic all or some of the aging events that occur in a normal subject. In some cases, this biological progression of events can occur in an accelerated time frame.

[0097] Age-related diseases or disorders or age-associated disorders may be related to normal processes in the body, such as the ability to move or eat.

[0098] The age-related disease or condition or age-related disorder can be a disease, illness, or disorder affecting the skin, such as a skin disorder or dermatosis, which can include wrinkles, lines, dryness, itchiness, blemishes, age spots, bedsores, ulcers, cancer, hypopigmentation, infection (e.g., fungal infection), or a decrease in skin properties such as clarity, texture, elasticity, color, tone, flexibility, firmness, firmness, smoothness, thickness, radiance, luminosity, hydration, moisture retention, skin barrier, uniformity, laxity, or oiliness, or other skin conditions. In some examples, the age-related disease or condition or age-related disorder is hyperpigmentation of the skin. In some examples, the hyperpigmentation disorder is melasma, age spots, lentigines, and / or progressive pigmentary purpura. In some examples, the hyperpigmentation is the result of sun damage, inflammation, hormonal changes, or skin injury. In some instances, hyperpigmentation occurs after cosmetic procedures, including but not limited to laser treatment, phototherapy, or chemical peels, administration of antibiotics, oral contraceptives, or photosensitizing drugs, or application of topical medications. In some instances, hyperpigmentation is the result of overproduction of melanin.

[0099] In some examples, treatment of an age-related disease or condition or age-related disorder with the methods, systems, and compositions disclosed herein results in a brighter, more luminous, radiant, smoother, smoother, and / or firmer appearance of skin. In some examples, treatment with the methods, systems, and compositions disclosed herein improves the epidermal barrier, skin hydration levels, skin water retention, the appearance of wrinkles, smoothness, firmness, elasticity, radiance, and luminosity, and / or improves or maintains ceramide levels in the skin. In some examples, the effects of treatment with the methods, systems, and compositions disclosed herein are assessed by measuring skin moisture content, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis, skin viscoelastic properties, or skin surface profile. In some examples, the effects of treatment with the methods, systems, and compositions disclosed herein are assessed by assessing the reduction in the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, roughness, skin hyperpigmentation, or overall appearance. In some examples, the effects of treatment with the methods, systems, and compositions disclosed herein are measured using a keratometer to measure skin water content / hydration, a VapoMeter to measure transepidermal water loss (TEWL), ultrasound to measure skin thickness (density) and echogenicity, a non-invasive optical skin imaging device to measure skin evenness and chromophore mapping, a suction-based cutometer to measure skin's viscoelastic properties (firmness and elasticity), skin profilometry, multispectral analysis, and colorimetry to measure the skin's surface profile, lines, and wrinkles.

[0100] In some examples, treating an age-related disease or condition or age-related disorder reduces the appearance of wrinkles or skin pigmentation by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some examples, instrumental measurements show at least a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% improvement in at least one of the following: appearance of lines / wrinkles, appearance of skin tone (evenness), appearance of pores, appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, skin roughness, skin radiance, or overall appearance after use of the composition compared to before use of the composition. In some examples, the improvement is expressed as a mean percent improvement (MPI) compared to a baseline before use of the composition. In some examples, the MPI is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after use of the composition compared to before use of the composition. In some examples, measurements are taken 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 5 months, 6 months, or 1 year after use of the composition. In some examples, the effectiveness of the treatment is assessed by a skin disease, disorder, or condition specialist who analyzes one or more of the skin measurements. In some examples, the effectiveness of the treatment is assessed by the user themselves. In some examples, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of users may report an improvement in skin barrier, skin roughness, skin radiance, appearance of lines / wrinkles, appearance of skin tone (evenness), appearance of pores, appearance of texture / smoothness, firmness (visual), elasticity (tactile), or overall appearance after using the methods, systems, and compositions disclosed herein. In some examples, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of users may report an improvement in skin hydration.In some instances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of users may report an improvement in skin barrier function.

[0101] In some examples, the methods, systems, and compositions provided herein can reduce hyperpigmentation of skin. In some examples, hyperpigmentation is associated with the overproduction of melanin. In some examples, the methods, systems, and compositions provided herein reduce the overproduction of melanin. In some examples, the methods, systems, and compositions provided herein reduce the presence of melanin pigment in skin. In some examples, the methods, systems, and compositions provided herein reduce the expression levels of proteins involved in melanogenesis, including tyrosinase, melanocyte-induced transcription factor (MITF), and dopachrome tautomerase (DCT), by treated skin cells. In some examples, the methods, systems, and compositions provided herein reduce tyrosinase activity, reduce tyrosinase expression or activation, remove intermediate products of melanin synthesis, reduce the transfer of melanosomes to keratinocytes, reduce the amount of melanin present, or reduce the activity or viability of melanocytes.

[0102] In some cases, the methods, systems, and compositions provided herein may reduce skin inflammation. In some cases, the methods, systems, and compositions provided herein may reduce the expression levels of proteins involved in inflammation, interferon gamma (IFN-γ) and interleukin 10 (IL-10), by treated skin cells.

[0103] In some embodiments, the compositions described herein are administered once daily, twice daily, three times daily, or more. In some embodiments, the compositions described herein are administered twice daily, for example, in the morning and at night. In some embodiments, the compositions described herein are administered daily, daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. In some embodiments, the compositions described herein are administered for at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, 4 years, 5 years, or more. In some embodiments, the compositions are instructed to be applied as a smooth layer to clean, dry skin on the face and / or neck in the morning and at night. In some embodiments, the formulation is a daily essential topical supplement scientifically formulated to improve skin elasticity and strengthen the epidermal barrier for long-term skin health. In some embodiments, the user applies a composition described herein comprising at least one polypeptide to the face and / or neck. In some embodiments, the composition is directed to be applied to the skin of the body. In some embodiments, the composition described herein is used in combination with other topical compositions, such as UV blockers. In some embodiments, the composition described herein is applied before, together with, or after the application of other topical compositions. In some embodiments, the composition comprises a UV blocker.

[0104] The polypeptide or composition may be applied topically, ie, to the skin, to delay the onset of, reduce the occurrence of, or ameliorate a disease, illness, or disorder affecting the skin.

[0105] Age-related diseases or disorders, or age-associated disorders, can be caused by UV damage, DNA damage, accumulation of ATRX foci in cell nuclei, increased p16 expression, increased senescence-associated β-galactosidase activity, accumulation of senescent cells in tissues, increased SASP production, chemically induced aging, chronological aging, decreased hyaluronic acid production, decreased expression of sirtuin 6, altered insulin-like growth factor-1 (IGF-I) pathway signaling, increased production of matrix metallopeptidase 1 (MMP1), a thin epidermal layer of skin, or genetic mutations. In some cases, age-related diseases or disorders, or age-associated disorders, can be initiated or exacerbated by side effects of treatment regimens, such as therapeutic drugs. Age-related diseases or disorders, or age-associated disorders, can directly or indirectly affect the health or appearance of the skin. Topical application of the polypeptides or compositions herein can improve the health or appearance of the skin in some such cases.

[0106] Age-related diseases or illnesses or age-related disorders may include cell proliferative disorders. Cell proliferative disorders may affect the health or appearance of the skin. In some cases, treatments for cell proliferative disorders, such as chemotherapy or radiation therapy, may affect the health or appearance of the skin. Topical application of the polypeptides or compositions herein may improve the health or appearance of the skin in some such cases.

[0107] Further provided herein is a method for treating the skin of a subject, comprising administering to the subject a composition that can promote a reduction in the number of senescent cells in a tissue or organism, induce a pro-apoptotic state in the treated cells, induce SIRT6 expression, prevent DNA-induced aging, and / or enhance DNA repair ability. In some cases, skin disorders, such as skin diseases or skin disorders, can include sagging or wrinkling of the skin, accumulation of senescent cells in tissue, reduced epidermal thickness, reduced collagen production, increased MMP-1 production, reduced DNA repair ability, reduced SIRT6 expression, skin breakdown, a thin epidermal layer of the skin, inflammation, a secretory phenotype associated with aging, or depletion of stem cells in the skin.

[0108] The method may include administering to a subject a composition comprising a polypeptide that can promote a significant reduction in the number of senescent cells in a tissue or organism.The reduction in the number of senescent cells may include promoting the pro-apoptotic state of the treated cells, inducing SIRT6 expression, preventing DNA-induced senescence, or enhancing DNA repair ability.In some cases, the number of senescent cells in a sample, a part of a subject (for example, the facial skin of a subject), and / or in a subject can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0109] The polypeptide or composition can be applied or administered to a cell, tissue, or subject. In some cases, the application or administration of the polypeptide can cause a senotherapy effect in a cell, tissue, or subject. In some cases, the polypeptide can be administered to a subject, applied topically to a subject, or incubated with cultured cells to provide a senotherapy effect.

[0110] The cells may be cultured cells or cells isolated from a subject or cell line. Some examples of cultured cells may include keratinocytes, fibroblasts, or melanocytes. The cells may be wild-type or genetically modified. Some genetic modifications, such as genetic modifications of the p53 / p21 pathway, p16 / RB pathway, mRNA, or miR genes, among other RNA classes, may promote senescence. In some cases, applying a polypeptide or composition to the cells may reduce cellular senescence. In some cases, the cells may include cells in vivo or in situ in organisms, including, but not limited to, animals, nematodes, and humans.

[0111] The tissue can be a tissue of a subject, or tissue that is isolated from a subject, i.e., ex vivo.In some cases, the tissue is artificially grown.Examples of tissue can include healthy skin, diseased skin, aging skin, or scalp.In some cases, applying polypeptide or composition to tissue can reduce the aging of one or more cells of the tissue or the entire tissue.In some cases, the tissue can include tissue in vivo or in situ in organisms, including but not limited to animals, nematodes, and humans.

[0112] If the subject is a human, the subject may be of any age. In some cases, the subject has an age-related disease or condition or an age-related disorder, is at risk of an age-related disease or condition or an age-related disorder, or is healthy. The subject may be male or female.

[0113] The method may involve topical application of the polypeptide or composition, which may involve rubbing, spraying, dipping, dabbing, or otherwise applying the polypeptide or composition to the skin or mucous membrane. [Example]

[0114] Example 1 Primary fibroblasts isolated from progeria patients may constitute a genetic model of early aging and cellular senescence in humans. Primary fibroblasts from progeria patients were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). Cells were maintained at 37°C, 5% CO2, and 95% humidity. After expansion, these cells were seeded into 96-well plates (1,000 cells per well) and incubated for 48 hours with individual polypeptides from a proprietary library at 50 μM, 6 hours after plating. Negative controls included untreated cells receiving only vehicle; positive controls were incubated for the same period with 10 μM ABT-263, a senolytic compound. After incubation, relative cellular senescence (assessed by the activity of senescence-associated B-galactosidase staining compared to untreated controls) was analyzed as shown in Figure 1, where the Y-axis indicates the total number of cells in the well (normalized to the untreated control) and the X-axis represents the senescence-associated B-galactosidase staining intensity / nucleus (i.e., senescence level), also normalized to the negative control. Three individual experiments with three technical replicates were performed. Polypeptides that promoted a significant reduction in cellular senescence, below 75% of that of untreated control samples, were considered positive hits.

[0115] A total of 764 polypeptides were tested, of which 56 promoted a reduction in cellular senescence below 75% of that of untreated control samples. They were therefore considered positive hits and putative senotherapy compounds. ABT-263 was considered a positive control in the experiment and further promoted a significant reduction in cellular senescence and also in cellular toxicity. This observation confirmed the senolytic properties of ABT-263 and the senotherapy potential of several of the tested polypeptides (Figure 1).

[0116] Example 2 Primary fibroblasts isolated from three healthy, chronologically aged patients were used. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). Cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After growth, these cells were seeded into 96-well plates (4,000 cells per well) and treated with one of four senotherapeutic polypeptides (peptide 14, peptide 13, peptide 15, and peptide 16) 6 hours after plating and incubated for 48 hours. Each polypeptide was tested at six different concentrations: 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, and 1.56 μM, except for peptide 16, which was tested at five different concentrations: 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, and 1.56 μM. Negative controls included untreated cells receiving vehicle only. After incubation, relative cellular senescence (as assessed by the activity of senescence-associated B-galactosidase staining compared to untreated controls) was analyzed (Figures 2A-D, panels A-D). In Figures 2A-D, the Y-axis indicates the relative senescence level normalized to the untreated control. Each column corresponds to a different concentration of polypeptide. Three individual experiments (biological replicas) with three technical replicates were performed. Data were analyzed using analysis of variance (ANOVA) with Bonferroni post-hoc test. Statistical significance was determined when p-value was less than 0.05.

[0117] All polypeptides showed senotherapeutic potential at at least one of the concentrations tested, as evidenced by a significant reduction in cellular senescence compared to untreated controls: *p<0.05; **p<0.01; ***p<0.001 compared to untreated controls (ctrl) (Figures 2A-D, panels A-D).

[0118] Example 3ATRX is a chromatin-remodeling enzyme that contributes to the formation of senescence-associated heterochromatin foci. It accumulates increasingly in nuclear foci with aging. Therefore, it constitutes a marker of cellular senescence. To investigate whether peptide 14 reduced the level of cellular senescence, ATRX foci were analyzed in treated (1 μM, 500 nM, 100 nM, and 10 nM) and untreated cells. To do so, primary fibroblasts isolated from three healthy, chronologically aged (aged) donors were used. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). Cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After proliferation, these cells were seeded into 96-well plates (4,000 cells per well) and incubated for 48 hours with peptide 14 polypeptide at the aforementioned concentrations, 6 hours after plating. Negative controls included untreated cells receiving only vehicle; relative cell senescence was assessed after incubation. Briefly, cells were fixed, permeabilized, and immunostained by incubating with an anti-ATRX antibody followed by a secondary antibody. The number of nuclei and stained ATRX foci was counted. Figure 3A (Panel A) shows a representative graph showing the number of cells (Y axis) exhibiting a specific amount of ATRX foci / cell, represented as columns (X axis). The top graph shows untreated cells, while the bottom graph shows cells treated with 500 nM peptide 14. Figure 3B (Panel B) shows the average number of ATRX foci / nucleus in fibroblasts treated with different peptide 14 conditions (columns). Figure 3C (Panel C) shows the percentage of cells exhibiting fewer than 10 ATRX foci / nucleus in fibroblasts treated with different conditions (columns) of peptide 14. Three individual experiments with three technical replicates (biological replicates) were performed. Data from Figure 3B and Figure 3C (Panels B and C) were analyzed using ANOVA and Bonferroni post-hoc comparison tests.Statistical significance was determined when the p-value was less than or equal to 0.05.

[0119] Peptide 14 treatment significantly reduced ATRX foci / nucleus when used at 500 nM and 50 nM compared to untreated cells. At the same concentrations, peptide 14 also increased the number of cells exhibiting fewer than 10 foci / nucleus. *p<0.05; **p<0.01 compared to untreated control (ctrl) (Figures 3A-3C).

[0120] Example 4 Human primary fibroblasts isolated from three healthy, chronologically aged (elderly) donors were used. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). Cells were maintained at 37°C in a 5% CO2, 95% humidified atmosphere. After expansion, the cells were seeded into T-75 flasks (250,000 cells per flask) and incubated with peptide 14 at 3.12 μM for 6 hours after plating for 3 weeks (21 days). Negative controls included untreated cells that received only vehicle; day 0 was defined as the day cells began to be treated with peptide 14. Treatment with peptide 14 was not performed between days 21 and 28. Cellular senescence was assessed weekly according to the level of senescence-associated β-galactosidase staining. Data were normalized to the untreated group and plotted (Figure 4A, panel A). Cell proliferation was also determined weekly. On days 7, 14, 21, and 28, cells were trypsinized and counted (Figure 4B, panel B). After counting, 250,000 cells were plated into new T-75 flasks. Three individual experiments (biological replicas) with three technical replicas were performed. Data were analyzed using a T-test. Statistical significance was determined when p-value was ≤ 0.05.

[0121] Peptide 14 promoted a significant reduction in cellular senescence (***p<0.001; ****p<0.0001), beginning at week 2. After 21 days of treatment, the effects of peptide 14 senescence were maintained for at least 7 days (between days 21 and 28 of the experiment) after removal of the polypeptide. No significant differences in cell proliferation were observed between the peptide 14-treated group and untreated controls (Figures 4A-B).

[0122] Example 5 Primary fibroblasts isolated from seven healthy, sequentially aged patients were used (patients incidentally identified as patients 2, 3, 4, 5, 6, 7, or 8). These cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U mL-1). Cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After proliferation, these cells were seeded into 96-well plates (4,000 cells per well) and incubated for 48 hours with peptide 14 at five different concentrations: 25 μM (concentration 5), 12.5 μM (concentration 4), 6.25 μM (concentration 3), 3.12 μM (concentration 2), and 1.56 μM (concentration 1). Negative controls included untreated cells (concentration 0) receiving only the vehicle. After incubation, relative cellular senescence was determined according to the average number of ATRX foci / nuclei quantified after ATRX immunostaining. Seven individual experiments (biological replicas), including three technical replicas, were performed. Data were analyzed using a covariance test. Statistical significance was determined when p-values ​​were less than or equal to 0.05 (Figure 5).

[0123] Analysis of covariance showed that the number of ATRX foci / nuclei was significantly reduced after treatment with peptide 14. The efficacy of peptide 14 followed a dose-response pattern, with a significant correlation between the concentration and the number of ATRX foci / nuclei (p<0.0004) (Figure 5).

[0124] Example 6 Cellular senescence can be triggered by several different stimuli. To evaluate whether peptide 14 is effective against UVB-induced and chemically induced senescence, we used human primary fibroblasts isolated from three healthy donors. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U mL-1). Cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After growth, these cells were seeded into 96-well plates (4,000 cells per well) and treated with etoposide (20 μM) or 0.05 J / cm for 6 h after plating and 24 h after treatment with 0.05 J / cm. 2 Cells were submitted for either two exposures to UVB radiation (1000 kJ / s). Each UVB exposure corresponds to approximately 1-3 hours of daily sunlight exposure in April in major cities around the world (e.g., Auckland, New Zealand; Los Angeles, USA; and Brasilia, Brazil). After different senescence induction protocols, etoposide-treated cells were incubated with peptide 14 at 5 μM, 2.5 μM, or 1 μM for 48 hours. UVB-exposed cells were treated with peptide 14 at 5 μM for 48 hours. Negative controls included untreated cells exposed to stress but administered only the vehicle. After incubation, relative cellular senescence (assessed by the activity of senescence-associated β-galactosidase staining compared to untreated controls) was analyzed and plotted on a bar graph. ATRX foci were further assessed after ATRX immunofluorescence staining. Graphs were constructed using the average ATRX foci detected per nucleus. Three individual experiments (biological replicas) with three technical replicates were performed. Data were analyzed using either t-tests or ANOVA followed by Bonferroni post-hoc comparison tests. Statistical significance was determined when p-values ​​were 0.05 or less (Figures 6A-D).

[0125] Etoposide treatment promoted a significant increase in the level of cellular senescence (p<0.001) and also a significant increase in the nuclear accumulation of ATRX foci (expressed as the mean number of ATRX foci / cell; p<0.05) (Figure 6A, (Panel A)). Treatment of etoposide-stressed cells with 2.5 μM or 5 μM peptide 14 significantly reduced senescence-associated β-galactosidase staining (*p<0.05) (Figure 6B, (Panel B), left graph). As shown in the right graph of Figure 6B (Panel B), treatment of etoposide-exposed cells with 2.5 μM peptide 14 further reduced the mean ATRX foci / cell. UVB exposure also promoted a significant increase in cellular senescence, as assessed by senescence-associated β-galactosidase staining, as shown in the left graph of Figure 6C (Panel C), and treatment with 5 μM peptide 14 significantly prevented cellular senescence (*p<0.05). As shown in the right graph of Figure 6C (Panel C), treatment with peptide 14 did not significantly alter cell number. UVB exposure promoted a significant increase in the average number of ATRX foci per nucleus, and treatment with peptide 14 at 5 μM significantly prevented cellular senescence, resulting in a significant decrease in ATRX foci / nucleus compared to UVB-treated samples that did not receive peptide 14 (*p<0.01) (Figure 6D, Panel D).

[0126] Example 7 Human skin equivalents were constructed using primary human fibroblasts and keratinocytes isolated from healthy elderly donors. The skin equivalents were treated with 0.01% wv. Peptide 14 for 5 days was characterized according to the level of aging using senescence-associated β-galactosidase staining and overall structure as indicated by epidermal thickness. Quality assessment based on several parameters was performed by blinded analysts. Observed parameters included the general organization of the cell layers and, in particular, the thickness of the stratum corneum, which were shown to decrease with aging and aging levels. The assessment was conducted with a maximum score of 28, with higher scores correlating with decreased age and aging. A minimum score of 19 was required for batch use. This score was internally validated and shown to decrease with age / aging of the skin equivalents or cultured cells. Furthermore, the skin equivalents were characterized according to the expression of specific genes by reverse transcription quantitative polymerase chain reaction (RT-qPCR). After treatment, the epidermis and dermis were separately processed for RT-qPCR. Epidermal samples were analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ubiquitously expressed); p16 (associated with aging), IL-8 (associated with skin irritation), and Ki-67 (associated with cell proliferation). Dermal samples were analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ubiquitously expressed); p16 (associated with aging), IL-8 (associated with skin irritation), and Ki-67 (associated with cell proliferation); hyaluronan synthase 2 (HAS-2; associated with hyaluronan production), and matrix metalloproteinase 1 (MMP1; associated with extracellular matrix protein degradation). CT values ​​were calculated as 2. -ΔΔCt The data were analyzed using the **p < 0.05 method. Average mRNA expression was normalized to GAPDH (ΔCt) and the negative control group (ΔΔCt). The negative control group received the formulation only. Three separate experiments were performed with three technical replicates. Data were analyzed using a t-test. Statistical significance was determined when p-values ​​were ≤ 0.05 (Figures 7A-C).

[0127] As shown in Figure 7A (Panel A), peptide 14 treatment of human skin equivalents promoted an increase in skin equivalent score (peptide 14 group 24 ± 1 vs. 19.0 ± 2), suggesting the safety, tolerability, and beneficial effects of treatment. This contrasts with other senolytic agents, which may have safety and tolerability issues. See, e.g., Tse et al., Cancer Res. 68: 3421 (2008); Wilson et al., Lancet Oncol. 11:1149 (2010). In addition, treatment promoted a significant decrease in senescence-associated β-galactosidase staining (***p<0.001), as shown in Figure 7A (Panel A) and Figure 7B (Panel B), demonstrating the efficacy of polypeptide senotherapy. Furthermore, peptide 14 treatment resulted in a significant decrease in p16 in the epidermis (****p<0.0001) and dermis (**p<0.01); a decrease in IL-8 expression in the dermis (*p<0.5); and a decrease in MMP-1 expression in the dermis (**p<0.01). The data, shown in Figure 7C (Panel C), demonstrate the therapeutic potential, safety, and tolerability of peptide 14, as well as the beneficial effects of the polypeptide on skin gene expression.

[0128] Example 8 To shed light on the mechanism of action of peptide 14 and the similar peptide 13, we investigated Akt S473 phosphorylation (Figure 8A, panel A), senescence-associated β-galactosidase staining (Figure 8B, panel B), and mRNA expression (Figure 8C, panel C). For Akt S473 phosphorylation analysis using Western blotting, human primary fibroblasts and keratinocytes were used. These cells were used to construct human skin equivalents, which were maintained at an air-liquid interface in a 5% CO2, 37°C, and 95% humidity atmosphere. The skin equivalents were then treated with either 1 μM peptide 14 or peptide 13 for 5 days, and protein analysis was performed on the skin equivalents. Proteins were isolated and quantified. Equal amounts of protein were loaded onto polyacrylamide gels and transferred to nitrocellulose membranes. GAPDH (loading control) and pAkt S473 antibodies were incubated with the plate, and staining was revealed by chemiluminescence. Relative pAkt S473 / GAPDH signals were compared between treated and untreated samples. For senescence-associated B-galactosidase experiments, fibroblasts were used. These cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U mL-1). Cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After proliferation, these cells were seeded into 96-well plates (4,000 cells per well) and incubated in basal medium for 6 hours to allow cell attachment. Cells were then exposed to 0.05 J / cm2 of irradiance. 2The cells were exposed twice to 1000 μg / ml of senescence-associated B-galactosidase (SAG). This was followed immediately by a second incubation in which peptide 14 or peptide 13 was added to the medium and left for 48 hours, at which point the medium was replaced and the cells were stained for senescence-associated B-galactosidase. Untreated cells were incubated with vehicle alone as a negative control (-). Relative staining was obtained after normalizing the senescence-associated B-galactosidase levels of the untreated control to 100%. For mRNA analysis, fibroblasts were used. These cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). The cells were maintained in a 5% CO2, 37°C, 95% humidified atmosphere. After proliferation, these cells were seeded into 6-well plates (50,000 cells per well) and incubated in basal medium for 6 hours to allow cell attachment. Cells were then incubated with either peptide 14 or peptide 13 for 48 hours. Negative controls included untreated cells and those administered with vehicle only; total RNA was isolated, samples were reverse transcribed, and mRNA expression of GAPDH, sirtuin 6 (SIRT6), BLM, and exonuclease 1 (EXO1) genes was determined using qPCR. The negative control group received only vehicle. CT values ​​were calculated as 2 -ΔΔCt Analysis was performed using the **p < 0.05 method. Average mRNA expression was normalized to GAPDH (ΔCt) and the negative control (ΔΔCt). For all analyses, three individual experiments were performed with three technical replicates. Data were analyzed using a T-test. Statistical significance was determined when p-values ​​were ≤ 0.05 (Figures 8A-8C).

[0129] pAkt S473 was significantly reduced in both epidermal and dermal samples treated with peptide 14 (*p<0.05 and **p<0.01, respectively). Peptide 13 reduced pAkt S473 only in dermal samples (***p<0.001) (Figure 8A, panel A). For UVB and aging-associated B-galactosidase staining, staining was observed to always increase after UVB exposure. Furthermore, both peptide 14 and peptide 13 reduced staining in UVB-exposed samples (***p<0.001). Peptide 14, in particular, led to an increase in SIRT6 and BLM expression in treated samples (*p<0.05) (Figures 8A-C).

[0130] Example 9 Human skin equivalents were constructed using human primary fibroblasts and keratinocytes isolated from healthy elderly donors. The skin equivalents were treated with 0.01% wv of peptide 14 for 5 days and characterized according to epidermal thickness, which was quantified according to total epidermal area. The negative control was treated with the formulation alone. An exemplary histological image is shown in Panel A of Figure 9. Three individual experiments were performed using three technical replicates. Data were analyzed using a t-test. Statistical significance was determined when p-values ​​were 0.05 or less (Panel B of Figure 9).

[0131] Peptide 14 treatment of human skin equivalents promoted an increase in epidermal thickness compared to untreated controls (**p<0.01), suggesting a beneficial effect of the polypeptide on the skin epidermis (Figure 9).

[0132] Example 10 The predicted three-dimensional structures of polypeptides having the amino acid sequences ETAKHWLKGI (SEQ ID NO: 1) and ATAKAWLKGI (SEQ ID NO: 2) were determined in water. The structural predictions are shown in Figure 10A, (Panel A) (SEQ ID NO: 1) and Figure 10B, (Panel B) (SEQ ID NO: 2). The structures were overlaid (Figure 10C, (Panel C)) to show the similarity of the structures. Example 11 A topical formulation of peptide 14 is created containing niacin, vitamin E, at least one preservative, at least one emulsifier, and 50-150 μM of peptide 14. The topical formulation is applied to human skin, resulting in a reduction in the appearance of wrinkles.

[0133] Example 12 A topical formulation of peptide 14 was created containing niacin, vitamin E, at least one preservative, at least one emulsifier, and 75-100 μM of peptide 14. The topical formulation was applied to human skin, resulting in a reduction in the appearance of wrinkles.

[0134] Example 13 Exemplary topical formulations are shown below in Table 4. The topical formulations were applied to human skin and resulted in a reduction in at least one of the appearance of wrinkles, skin tone (evenness), the appearance of pores, the appearance of texture and smoothness, firmness, elasticity, and overall appearance. The polypeptide in the formulation comprises at least one of the polypeptides disclosed herein.

[0135] [Table 4]

[0136] Example 14 Exemplary topical formulations containing peptide 14 are shown below in Table 5. The topical formulations were applied to human skin and resulted in a reduction in at least one of the appearance of wrinkles, skin tone (evenness), the appearance of pores, the appearance of texture and smoothness, firmness, elasticity, and overall appearance. The polypeptide in the formulation comprises peptide 14.

[0137] [Table 5]

[0138] Example 15 A three-dimensional in vitro skin model and ex vivo human skin samples were evaluated for skin aging after treatment with peptide 14.

[0139] In vitro 3D skin model: A 3D skin model was prepared using a modified method based on that described in Pennacchi, PC et al., "Glycated Reconstructed Human Skin as a Platform to Study the Pathogenesis of Skin Aging." Tissue Eng. Part A, 21, 2417-2425, 2015. Briefly, normal human epidermal keratinocytes (NHEKs) were seeded on top of fibroblast-embedded type I collagen gels and cultured for 24 hours to allow the NHEKs to reach a monolayer. The top gel containing the NHEKs was then elevated to the air-liquid interface and cultured for an additional 10 days to allow the epidermis to keratinize. The gels were treated with 12.5 μM peptide 14 by adding peptide 14 to the culture medium.

[0140] Ex vivo human skin model: Skin samples from healthy human donors were obtained from ZenBio (Research Triangle, NC) and maintained in air-liquid interface culture using Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% (v / v) FBS. Skin samples were treated with either control vehicle or 12.5 μM peptide 14 in medium on days 1 and 3. After 5 days, samples were harvested and fixed in formalin for histology or used for DNA isolation.

[0141] DNA methylation analysis: Predicted biological age, also known as molecular DNA age, was determined from DNA methylation levels in 3D skin model samples and ex vivo human skin biopsy samples. Intact DNA samples were obtained from the samples using a QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. DNA methylation assessment, as a marker of skin aging, was performed using a human Illumina Infinium EPIC 850K chip. DNA samples included: i) four biopsy samples (from the same donor) considered as untreated controls; ii) four skin biopsy samples (from the same donor) treated with 12 μM peptide 14; iii) three 3D skin samples considered as untreated controls (each sample was a pool of three skin samples from one donor, for a total of three donors); and iv) three 3D skin samples treated with 12 μM peptide 14 (each sample was a pool of three skin samples from one donor, for a total of three donors). Raw image data were processed using the preprocessRaw() and then preprocessSWAN() commands. Methylation signals (M-values) were then converted to ratios using ratioConvert() and then to beta values ​​using getBeta(), all functions implemented in the "minfi" (registered trademark) package. Beta values ​​were normalized using the betaqn() method, a quantile normalizing beta implemented by the "watermelon" package, and then normalized using the "preprocessQuantile" normalization method implemented in the "minfi" (registered trademark) package. Normalized beta values ​​were used for age estimation.

[0142] Statistical analysis: Data were tested for normal distribution by the Shapiro-Wilk test. When more than two groups were compared, one-way analysis of variance was performed followed by Bonferroni's multiple comparison test. When paired samples were compared, a paired t-test was performed. p=<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism (GraphPad software) or R software.

[0143] Results: In vitro 3D skin models and ex vivo human skin samples showed reduced skin aging after 5 days of peptide 14 treatment, as shown in Figures 11A, 11B, and 11C. Figure 11A shows hematoxylin-eosin (H&E)-stained histological images of 3D skin equivalents (top row) and ex vivo skin biopsy samples (bottom row) cultured without peptide 14 (control) and with 12.5 μM peptide 14 for 5 days. The peptide 14-treated 3D skin equivalents and ex vivo skin samples generally exhibited similar epidermal thickness or greater epidermal thickness than the untreated control samples. Figure 11B shows that the predicted age, also referred to as molecular DNA age, for 3D skin model samples treated with 12.5 μM peptide 14 (treated) was lower than that of the untreated control (control) samples. The mean predicted age for untreated 3D skin models was approximately 80, while the mean predicted age for peptide 14-treated 3D skin models was approximately 66 (p=0.25 by t-test). Figure 11C shows that the predicted age for ex vivo skin biopsies treated with 12.5 μM peptide 14 was lower than the predicted age for untreated control (ctrl) samples. The mean predicted age for ex vivo skin biopsy samples was approximately 71, while the mean predicted age for peptide 14-treated ex vivo skin biopsy samples was approximately 68 (**p<0.01).

[0144] Example 16 In vertebrates, specialized cells called melanocytes normally produce melanin. MeWo cells are a human immortalized melanocyte cell line that represent late-differentiated melanocytes expressing retinoic acid receptors and can provide an experimental model for in vitro investigations of melanogenesis. Additional information regarding the use of MeWo cells as an in vitro model of melanogenesis can be found in Schadendorf et al., 1994. Retinoic Acid Receptor-gamma-selective Retinoids Exert Antiproliferative Effects on Human-melanoma Cell-growth In-vitro. International Journal of Oncology. doi:10.3892 / Ijo.5.6.1325 and Malaspina et al., Depigmenting Potential of Lichen Extracts Evaluated by In Vitro and In Vivo Tests. PeerJ 8:e9150 https: / / doi.org / 10.7717 / peerj.9150.

[0145] To test whether peptide 14 promotes melanocyte depigmentation, MeWo cells were cultured in DMEM supplemented with 10% v / v FBS and 1% v / v penicillin / streptomycin solution (1,000 U / mL) at 5% CO2, 37°C, and 95% humidity. Two weeks before treatment with one of the experimental conditions (pretreatment), MeWo cells were cultured with isobutylmethylxanthine (IBMX) to stimulate melanin synthesis. MeWo cells were seeded at 1,000,000 cells per well in six-well plates for 6 hours, and then cultured for 7 days under one of the experimental conditions and analyzed for intracellular and supernatant melanin content.

[0146] Experimental conditions included a positive control group, a negative control group, a peptide 14(IBMX)7d group, and a retinoic acid (IBMX)7d group. The negative control group was stimulated with 25 μM IBMX for a 2-week pretreatment period and then, after plating for the experiment, was left untreated for 7 days and administered vehicle alone. The positive control group included MeWo cells incubated with 25 μM IBMX for the entire experiment, i.e., a 2-week pretreatment period and a 7-day treatment period. The peptide 14(IBMX)7d group included MeWo cells incubated with 25 μM IBMX for a 2-week pretreatment period and then treated with 25 μM IBMX and 3.12 μM peptide 14 for 7 days. The retinoic acid (IBMX) 7d group contained MeWo cells incubated with 25 μM IBMX for 2 weeks during the pretreatment period and then treated with 25 μM IBMX and 2 μM retinoic acid for 7 days.

[0147] After treatment, melanin content in the cell pellet and cell culture supernatant was assessed by measuring the absorbance of the samples at 492 nm. Cell pellets were prepared by trypsinization, counted, and incubated in 200 μL of 1 M NaOH for 16 hours (Matsuda et al., 2004; Yoo et al., 2007). Cell culture supernatants were obtained after centrifugation at 1.2 × g for 10 minutes. Data are expressed as relative melanin content, the absorbance of a sample at a specific wavelength (492 nm) normalized to the absorbance of a negative control sample at the same wavelength. Three individual experiments with three technical replicates were performed. When statistical significance was detected, data were analyzed using ANOVA and Bonferroni post-hoc comparison tests. Statistically significant p-values ​​between groups are marked with * for p < 0.05; ** for p < 0.01; *** for p < 0.001; and **** for p < 0.0001.

[0148] Peptide 14 treatment promoted a significant decrease in relative melanin content compared to the positive control, negative control, and retinoic acid-treated groups in the cell pellets shown in Figure 12A and in the cell supernatants shown in Figure 12B. In the cell pellets shown in Figure 12A, the positive control had a relative melanin content that was approximately 170% of the negative control group (****p<0.0001), and the retinoic acid-treated group had a relative melanin content that was approximately 125% of the negative control group (**p<0.01), while the peptide 14 treatment had a relative melanin content that was approximately 90% of the negative control group (***p<0.001), or lower than the negative control group. In the cell culture supernatants as shown in Figure 12B, the positive control and retinoic acid treatment groups had relative melanin content very similar to that of the negative control group, while the peptide 14 treatment had a relative melanin content that was approximately 70% of that of the negative control group (*p<0.05), or lower than that of the negative control group.

[0149] This indicates that peptide 14 may have applications for reducing various melanogenesis-related conditions, including, but not limited to, age spots, hyperpigmentation caused by skin inflammation, hormonal changes, aging, sun exposure, and chronic lesions. Furthermore, peptide 14 appeared to outperform retinoic acid, which is currently considered the gold standard molecule for anti-aging skin care.

[0150] Example 17 Skin often undergoes rebound hyperpigmentation, in which pigmentation is restored to the skin after cessation of lightening treatment. To test whether the depigmenting effect of peptide 14 persists after cessation of peptide 14 treatment, MeWo cells were cultured in DMEM supplemented with 10% v / v FBS and 1% v / v penicillin / streptomycin solution (1,000 U / mL) at 5% CO2, 37°C, and 95% humidity. Two weeks before treatment with one of the experimental conditions (pretreatment), MeWo cells were cultured with isobutylmethylxanthine (IBMX) to stimulate melanin synthesis. MeWo cells were seeded at 1,000,000 cells per well in 6-well plates for 6 hours, and then cultured for 14 days under one of the experimental conditions and analyzed for intracellular and supernatant melanin content.

[0151] Experimental conditions included a positive control group, a negative control group, a 14-day peptide 14 (IBMX) group, a 14-day retinoic acid (IBMX) group, a 7-day peptide 14 (IBMX) withdrawal group, and a 7-day retinoic acid (IBMX) withdrawal group. The negative control group was stimulated with 25 μM IBMX for a 2-week pretreatment period and then, after plating for the experiment, was left untreated for 14 days and administered vehicle alone. The positive control group included MeWo cells incubated with 25 μM IBMX for the entire experiment, including the 2-week pretreatment period and the 14-day treatment period. Some groups were treated with both 25 μM IBMX and 3.12 μM peptide 14 for 7 or 14 days, or both 25 μM IBMX and 2 μM retinoic acid (for 7 or 14 days). The peptide 14 (IBMX) 14-day group contained MeWo cells that had been pretreated with 25 μM IBMX for 2 weeks, followed by 14 days of treatment with 25 μM IBMX and 3.12 μM peptide 14. The retinoic acid (IBMX) 14-day group contained MeWo cells that had been pretreated with 25 μM IBMX for 2 weeks, followed by 14 days of treatment with 25 μM IBMX and 2 μM retinoic acid. The peptide 14 (IBMX) 7-day group contained MeWo cells that had been pretreated with 25 μM IBMX for 2 weeks, followed by 7 days of treatment with 25 μM IBMX and 3.12 μM peptide 14, followed by an additional 7 days of treatment with 25 μM IBMX alone. The retinoic acid (IBMX) 7-day group contained MeWo cells incubated with 25 μM IBMX for 2 weeks during the pretreatment period, then treated with 25 μM IBMX and 2 μM retinoic acid for only 7 days, and then cultured with 25 μM IBMX alone for an additional 7 days.

[0152] After treatment, melanin content in the cell pellet and cell culture supernatant was assessed by measuring the absorbance of the samples at 492 nm. Cell pellets were prepared by trypsinization, counted, and incubated in 200 μL of 1 M NaOH for 16 hours (Matsuda et al., 2004; Yoo et al., 2007). Cell culture supernatants were obtained after centrifugation at 1.2 × g for 10 minutes. Data are expressed as relative melanin content, which is the absorbance of a sample at a specific wavelength (492 nm) normalized to the cell number of a negative control sample at the same wavelength. Cell proliferation was significant over the 14-day period; in this experiment only, melanin content data were normalized to the cell number in the sample within each group. Three individual experiments with three technical replicates were performed. When statistical significance was detected, data were analyzed using ANOVA and Bonferroni post-hoc comparison tests. Statistically significant p-values ​​are marked as * for p<0.05; ** for P<0.01; *** for P<0.001; **** for p<0.0001 between groups.

[0153] In addition, the mRNA levels of key genes involved in melanogenesis, including tyrosinase, melanocyte-induced transcription factor (MITF), and dopachrome tautomerase (DCT), were analyzed.

[0154] In the cell pellets (shown in Figure 13A) and in the cell supernatants (shown in Figure 13B), peptide 14 treatment promoted a significant decrease in relative melanin content compared to the positive control and retinoic acid-treated groups. In the cells shown in Figure 13A, the positive control had a relative melanin content of approximately 130%, and the 14-day retinoic acid treatment group had a relative melanin content of approximately 130%, while the 14-day peptide 14 treatment group had a relative melanin content of approximately 90% of the negative control group, or lower than the negative control group. The 7-day peptide 14 treatment group had a relative melanin content of approximately 40%, the lowest relative melanin content among the experimental groups. The 7-day retinoic acid treatment group had a relative melanin content of approximately 90%. In the cell culture supernatants shown in Figure 13B, the positive control had a relative melanin content of approximately 110%, while the 14-day retinoic acid treatment group and the 14-day peptide 14 treatment group had a relative melanin content of approximately 50%. The 7-day retinoic acid treatment group and the 7-day peptide 14 treatment group had a relative melanin content of approximately 100%.

[0155] The mRNA levels of melanogenesis-related genes are shown in Figures 14A, 14B, and 14C. Peptide 14 treatment appears to result in a significant decrease in the expression of tyrosinase, MITF, and DCT genes, with the decrease being lower or at least similar to the mRNA levels in the retinoic acid-treated group. As shown in Figure 14A, the relative expression level of tyrosinase was lowest in the 14-day peptide 14 treatment group compared to the other experimental groups. As shown in Figure 14B, the relative expression level of MITF was lowest in the 14-day peptide 14 treatment group and the 14-day retinoic acid treatment group. As shown in Figure 14C, the relative expression level of DCT was lowest in the 14-day peptide 14 treatment group and the 7-day peptide 14 treatment group compared to the other experimental groups.

[0156] This indicates that peptide 14 can reduce melanin formation and continue to result in lower melanin formation even after treatment with peptide 14 is discontinued. Furthermore, peptide 14 appeared to outperform retinoic acid, which is currently considered the gold standard molecule for anti-aging skin care.

[0157] Example 18 To test the effects of peptide 13 and peptide 14 treatment on human skin models and skin morphology, human skin equivalents were constructed using primary human fibroblasts and keratinocytes isolated from healthy elderly donors (71, 84, and 90 years old). Skin equivalents were treated with 0.01% w / v (or 1 μM) peptide 13 or peptide 14 for 5 days. Negative controls were treated with formulation alone. After 5 days of incubation, the skin equivalents were analyzed for epidermal thickness, which was quantified according to total epidermal area. A quality assessment based on several parameters was performed by blinded analysts. Observed parameters included the general organization of cell layers, as well as the thickness of the stratum corneum in particular, which were shown to decrease with age and aging. The assessment was based on a maximum score of 28, with higher scores correlating with decreased age and aging. A minimum score of 19 was required for batch use. This score was internally validated and shown to decrease with age / aging of the skin equivalents or cultured cells. Three individual experiments were performed with three technical replicates. Data were analyzed using a t-test. Statistical significance was determined when p-values ​​were less than or equal to 0.05. Statistically significant p-values ​​between groups are marked with * for p<0.05; ** for p<0.01; *** for p<0.001; and **** for p<0.0001.

[0158] Figure 15A shows H&E-stained histological images of in vitro human skin models treated with vehicle only (control), peptide 13, or peptide 14. Human skin models treated with peptide 13 or peptide 14 generally exhibit similar or increased thickness of the stratum corneum (indicated by the dark gray layer at the top of the image, or stained bright pink by H&E) and epidermal layer (indicated by the medium gray layer in the center of the image, or stained purple by H&E) relative to untreated control samples. Figure 15B shows the average histological scores of human skin models treated with vehicle only (control), peptide 14, or peptide 13, which were 21.00, 23.83, and 23.44, respectively. Histological scores were higher for samples treated with peptide 13 or peptide 14 than for the negative control. Treatment of skin samples with peptide 13 or peptide 14 appeared to improve skin morphology as assessed by epidermal thickness and epidermal barrier.

[0159] Example 19 To test the level of skin depth penetration by peptide 14, a diffusion study was performed using Franz cells and on ex vivo skin culture samples. Fresh human skin from the abdomen of a female donor (79 years old) was cut into ~2.5 cm x 2.5 cm pieces.

[0160] In the Franz cell test, the skin was treated with 10 μL of a formulated cream containing 0.01% peptide 14, and a 5 mm diameter contact area (0.2 cm 2 The cells were placed in a Franz cell at 32°C. The receptor chamber contained 2 mL of PBS at pH 7.4. The Franz cell was kept under stirring at 32°C for 24 hours.

[0161] For ex vivo skin culture studies, skin was treated with 2 μL of formulated cream containing 0.01% peptide 14 (total of 200 ng of peptide 14). The skin samples were then placed at the air-liquid interface with the DMEM medium underneath and maintained at 37°C for 24 hours.

[0162] After 24 hours, excess formulation on the skin was removed with tissue paper, and the skin samples were washed four times in PBS. All surrounding skin was also removed. The skin was then incubated at 60°C for 1-2 minutes to separate the epidermis from the dermis. The PBS, epidermal layer, and dermal layer in the receptor chamber (2 mL) were collected and frozen at -80°C until further analysis. The dermal layer was analyzed by mass spectrometry for peptide 14 to determine the amount of peptide 14 that had penetrated the dermis.

[0163] In Franz cell studies, approximately 1.37% to 2.60% of applied peptide 14 was detected in the dermal layer. In ex vivo human skin culture studies, approximately 1.94% to 1.96% of applied peptide 14 was detected in the dermal layer. When peptide 14 was applied topically to the surface of the skin, little or no peptide 14 penetrated into the dermis. This demonstrates that the skin penetration of peptide 14 achieved by topical application is very low to minimal.

[0164] Example 20 A clinical trial was conducted on human subjects to evaluate the effects of topical application of peptide 14 to the face. The study was approved by an IRB committee. Twenty-two human subjects participated in the clinical trial and were asked to use two products, one on each side of their face. On the right side of their face, subjects used a negative control formulation containing only the formulation. On the left side of their face, subjects used a treatment formulation containing 0.01% peptide 14. Subjects were evaluated before starting product use (baseline) and after 6 and 12 weeks of daily topical application of the formulation to each side of their face. The effects of topical peptide 14 application on facial skin were evaluated using various assessment criteria detailed below: skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis, skin viscoelastic properties, and skin surface profile, including the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), and overall appearance.

[0165] Clinical Expert Ratings Rating for Efficacy: Visual and tactile assessments were performed on both sides of the face using 10 cm Visual Analog Scales (VAS) at baseline, week 6, and week 12. The following parameters: fine lines / wrinkles, skin tone (evenness of color), texture / smoothness (visual), firmness (visual), elasticity (tactile), skin pores, radiance / luminosity, and overall appearance were assessed using the instruments and methods detailed below.

[0166] Skin moisture / hydration was assessed by measuring skin capacitance using a Corneometer CM825 (Courage + Khazaka, Germany). Measurements were taken three times on both sides of the face at baseline, week 6, and week 12 and averaged. A test site map was used to ensure the same location was measured for each measurement.

[0167] VapoMeter: Transepidermal water loss (TEWL) of the skin was measured with a VapoMeter (Delfin Technologies Ltd., Finland) using a sealed cylindrical chamber equipped with sensors to measure relative humidity and temperature. Changes in TEWL rate provide a measure of barrier disruption or integrity, thereby providing an indication of the effect of peptide 14 on skin integrity. All subjects underwent two VapoMeter measurements on each side of the face at baseline, week 6, and week 12, which were averaged. The assessment locations were recorded on each subject's body map.

[0168] Ultrasound-DermaScan: The DermaScan C USB (Cortex Technology ApS, Hadsund, Denmark) is a compact, high-resolution ultrasound scanner. All subjects underwent ultrasound assessments on both sides of the face at baseline and week 12. The location of the assessments was the same for each assessment and recorded on a facial map. After ultrasound scans were obtained, they were analyzed for skin thickness (density) and echogenicity.

[0169] SIAScope: The COSMETRICS™ SIAScope (Astron Clinical, Toft, UK) is a non-invasive optical skin imaging device that uses Spectrophotometric Intracutaneous Analysis (SIA) or chromophore mapping. Skin collagen and hemoglobin were measured on both sides of the face at baseline, 6 weeks, and 12 weeks.

[0170] The Cutometer: Cutometer MPA 580 (Courage + Khazaka, Germany) measures the viscoelastic properties (firmness and elasticity) of the skin by applying suction to the skin surface, drawing the skin into the probe opening, and using an optical measurement system to determine penetration depth. Measurements were taken on both sides of the face at baseline, 6 weeks, and 12 weeks. The same locations were measured at each time point and recorded using a face map. Measurements included firmness, elasticity, and net elasticity.

[0171] VISIA-CR: Photographic documentation was performed using a VISIA-CR imaging system (Canfield Scientific, Paramus, NJ, USA), which captures high-resolution images in multiple illumination modes. Photographs were captured at baseline, week 6, and week 12 for central, right lateral, and left lateral views in standard and parallel polarized light.

[0172] ANTERA 3D®: The Antera 3D® (Miravex, Ireland) is a device that combines skin topography, multispectral analysis, and colorimetry to provide a three-dimensional reconstruction of the skin surface and subsequent image analysis. Images were captured at baseline, week 6, and week 12 in the left and right crow's feet areas of all subjects. The same locations were measured at each time point and recorded using a face map. Images were analyzed for texture, width, and line / wrinkle depth.

[0173] Expert Clinical Rating

[0174] Weeks 6 and 12 compared to baseline

[0175] Peptide 14 Formulation: Comparing the mean scores of the left side of the Peptide 14-treated face at baseline with subsequent time points revealed statistically significant improvements in clinical ratings at week 6 that continued through week 12 for the appearance of lines / wrinkles, appearance of skin tone (evenness), appearance of pores, texture / smoothness, firmness (visual), elasticity (tactile), and overall appearance. Additionally, there was a statistically significant improvement in the appearance of radiance / luminosity at week 12 compared to baseline on the left side of the Peptide 14-treated face.

[0176] Control Formulation: Comparing the mean scores for the right side of the face treated with the negative control formulation at baseline with subsequent time points revealed statistically significant improvements in clinical ratings at week 6 that continued through week 12 for the appearance of lines / wrinkles, appearance of skin tone (evenness), appearance of pores, texture / smoothness, firmness (visual), elasticity (tactile), radiance / luminosity, and overall appearance.

[0177] Comparison of peptide 14 treatment to negative control (left vs. right side of face)

[0178] Comparison of peptide 14 treatment to negative control for treatment time

[0179] When compared to baseline (before treatment), the left side of the face treated with Peptide 14 appeared better than the right side of the face treated with the negative control by several measures. The left side of the face treated with Peptide 14 had higher levels of skin hydration as measured by the corneometer, higher levels of collagen as measured by the SIAscope, and better TEWL (transepidermal water loss) improvement as measured by the vapometer at week 6 than the right side of the face treated with the negative control. No significant differences were found between the left side of the face treated with Peptide 14 and the right side of the face treated with the negative control formulation in Dermascan assessments of skin thickness (density) and echogenicity, and in Visia assessments of overall skin appearance. The left side of the face treated with Peptide 14 exhibited better Antera measurements of texture (roughness) at week 12 than the right side of the face treated with the negative control formulation.

[0180] The results of 22 patients using a topical cream containing 0.01% peptide 14 after 12 weeks were analyzed. Figure 16 shows an example of the left side of a face treated with peptide 14 at baseline (left, baseline) and 12 weeks after treatment (right, 12 weeks). Those treated with 0.01% peptide 14 for 12 weeks appeared to have a reduced appearance of lines and wrinkles, and improved smoothness, skin tone (evenness), texture / smoothness, and overall appearance compared to the baseline time point.

[0181] A blinded expert opinion analysis was performed to determine the percentage of patients among all subjects who showed improvement and the mean percent improvement (MPI), where MPI represents the average percent improvement compared to baseline (pre-treatment). The analysis determined that 87% of patients were rated as having a reduced appearance of skin wrinkles (MPI: 3.3%), 90% were rated as having improved skin elasticity (MPI: 4.75%), and 95.5% were rated as having improved skin evenness (MPI: 4.4%), radiance (MPI: 5.06%), pore appearance (MPI: 4.58%), and firmness (MPI: 5.43%). All subjects were rated as having better skin texture / smoothness (MPI: 7.46%) and a better overall appearance (MPI: 6.17%).

[0182] The subjects' faces were evaluated by various instruments to determine the percentage of patients who showed improvement among all subjects and the mean percent improvement (MPI), where MPI represents the mean percent improvement compared to baseline (before treatment). From the instrumental evaluation, it was confirmed that 81% of subjects had a better skin barrier (MPI: 14.19%) as assessed by Vapometer. 73% of subjects showed improvement in skin roughness (MPI: 5.05%) as assessed by Antera. 73% of subjects showed improvement in skin radiance (MPI: 16.63%) as assessed by VISIA.

[0183] Subject perception was also assessed. Analysis of subject perception showed that over 80% of subjects believed that formulations containing Peptide 14 promoted better skin appearance, better skin texture, better skin firmness, and better hydration. 78% of subjects noticed an improvement in skin radiance.

[0184] Example 21 The topical formulation comprising at least one of the polypeptides disclosed herein is used on human skin.The formulation is directed to be applied as a smooth layer to clean, dry skin of face and / or neck in the morning and evening.The formulation is a chemically formulated, essential daily external supplement, which improves skin resilience and strengthens the epidermal barrier for lasting skin health.

[0185] Example 22 The user applies a topical formulation described herein containing at least one of the polypeptides to the face and / or neck, and the user also applies another topical formulation containing a UV blocker after applying the topical formulation.

[0186] Example 23 The user applies a topical formulation described herein containing at least one of the polypeptides and a UV blocker to the face or neck. The topical formulation may also be applied to the skin of the user's body.

[0187] Example 24 Sample 3D skin equivalent sections constructed with cells from elderly donors (71, 84, or 90 years old) were treated with 1 μM peptide 14 or peptide 13, or 20 μM retinoic acid (RA), and the effects of the various treatments on various markers of senescence, aging, and health were assessed.

[0188] Figure 17 shows the relative mRNA expression levels of p16, BLIMP1, ZYG11B, IL-8, Ki-67, ZIC1, MMP1, and HAS2 in the epidermal (Epi) and dermal (Der) layers of 3D skin equivalents treated with control, peptide 14, peptide 13, or retinoic acid. Data are presented as 2-ddCt normalized to GAPDH and untreated controls. *p<0.05. Peptide 13- and peptide 14-treated samples generally had similar relative mRNA expression levels for p16, BLIMP1, ZYG11B, IL-8, and Ki-67 in the epidermal layer and for p16, MMP1, HAS2, IL-8, and Ki-67 in the dermal layer. Samples treated with peptide 13 and peptide 14 generally had lower relative mRNA expression levels for p16, BLIMP1, ZYG11B, and IL-8 in the epidermal layer, and for p16, MMP1, IL-8, and Ki-67 in the dermal layer than samples treated with RA. Samples treated with peptide 13 and peptide 14 generally had higher relative mRNA expression levels for Ki-67 in the epidermal layer and HAS2 for the dermal layer than samples treated with RA. Samples treated with peptide 13 and peptide 14 had similar relative mRNA expression levels for ZIC1 and Ki-67 for the dermal layer as samples treated with RA.

[0189] Example 25 Senotherapy strategies can be associated with healthspan and lifespan extension in vivo. Caenorhabditis elegans worms were used to evaluate whether peptide 14 promotes healthspan and lifespan extension. Peptide 14 was added to nematode culture medium (M9 buffer medium) at different concentrations of 1 μM or 2 μM. Negative control nematodes were administered vehicle only. Two healthspan parameters were assessed: i) pharyngeal pumping and ii) nematode movement. Lifespan was also determined. For pharyngeal pumping analysis, 15 nematodes were observed daily for pharyngeal movement (pumping) and counted for 20 seconds. This experiment was repeated three times by two different blinded observers using different groups of animals on different days. Statistical differences were detected by analyzing each individual time point using one-way ANOVA and Dunnett's post hoc test. All groups were compared with the HO group. For worm movement analysis, the basic movement of worms, also known as thrashing, was measured for 15 worms daily. The observation time was 30 seconds. The experiment was repeated three times by two different blinded observers using different populations of worms on different days. Statistical differences were detected by analyzing each individual time point using one-way ANOVA and Dunnett's post hoc test. All groups were compared with the HO group. For lifespan analysis, 15 worms were observed daily for lifespan until the last worm died. This experiment was repeated three times by two different blinded observers using different populations of worms on different days. The mean lifespan was measured. Statistical differences were detected by analyzing each individual time point by one-way ANOVA and Dunnett's post-hoc comparison test, and all groups were compared with the H2O group (Figs. 18A-18C).

[0190] Treatment with either 1 μM or 2 μM peptide 14 improved thrashing (Figure 18A, panel A), pumping (Figure 18B, panel B), and lifespan (Figure 18C, panel C). Thrashing was significantly improved on day 1 (*p<0.05), whereas pumping was significantly decreased on this day (*p<0.5). Taken together, the data suggest the safety and efficacy of the tested peptides in promoting healthspan and lifespan. For example, on day 8, both thrashing (*p<0.05) and pumping (*p<0.05) were increased in the peptide 14-treated group compared to the control. Pumping decreased with worm aging, primarily as a result of a loss of muscle integrity. Although not directly measured, gross anatomical defects in the worm pharynx (curvature or swelling of the pharynx, both common features of aging in these worms) were also reduced in the 1 μM peptide 14 group compared with the HO control group. No reduction in worm movement was detected in any sample at any time point, suggesting that the peptide was not toxic to worms at the concentrations tested. When used at 1 μM and 2 μM, peptide 14 promoted a statistically significant increase in worm mean lifespan (1 μM peptide 14 **p<0.01; 2 μM peptide 14 *p<0.05).

[0191] Example 26 Due to their high levels of cellular senescence, skin cells from progeria patients were used as a model of aging. Primary fibroblasts from progeria patients were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% vv fetal bovine serum (FBS) and 1% vv penicillin / streptomycin solution (1,000 U / mL). Cells were cultured at 37°C in a 5% CO2, 95% humidified atmosphere. After proliferation, cells were seeded into 96-well plates (4,000 cells per well) and incubated with 500 nM, 5 μM, or 50 μM of the polypeptide sequence LKGIL (SEQ ID NO: 6) or WLKGI (SEQ ID NO: 7) for 6 hours after plating. Negative controls included untreated cells receiving only the vehicle; after incubation, relative cellular senescence, assessed by the activity of senescence-associated β-galactosidase staining and by quantification of ATRX foci / nuclei relative to the untreated control, was analyzed. Three individual experiments (biological replicas) with three technical replicas were performed. Data were analyzed using ANOVA and Bonferroni post-hoc comparison tests. Statistical significance was determined when p-values ​​were ≤0.05, where *p<0.05 and **p<0.01. Figures 19A-19F show the effects of the polypeptide sequences LKGIL (SEQ ID NO: 6) (Figures 19A, 19B, 19C) and WLKGI (SEQ ID NO: 7) (Figures 19D, 19E, 19F) on reducing cellular senescence without promoting cell death. In panels A and D, the Y-axis indicates the relative senescence level normalized to the untreated control. In panels B and D, the Y-axis shows relative cell number normalized to untreated controls. In panels C and F, the Y-axis shows average ATRX foci accumulation per cell. Treatment of cells with LKGIL (SEQ ID NO: 6), when used at 5 μM and 50 μM, significantly reduced senescence-associated β-galactosidase staining and the average number of ATRX foci / nucleus compared to untreated cells (p<0.01 for β-galactosidase staining and p<0.05 for ATRX foci / nucleus).Treatment of cells with WLKGI (SEQ ID NO: 7) reduced senescence-associated β-galactosidase staining levels compared to untreated cells when used at 5 μM and 50 μM (p<0.05 for 5 μM and p<0.01 for 50 μM). No cytotoxicity was observed at the concentrations tested.

[0192] Example 27 Additional polypeptides were tested for their suitability as anti-aging agents. Cells were incubated with one of the polypeptides. Negative controls included untreated cells receiving only the vehicle; after incubation, the relative cellular senescence (assessed as the activity of senescence-associated β-galactosidase staining relative to the negative control) and relative cellular proliferation relative to the negative control were analyzed. Many polypeptides exhibited a relative cellular senescence of less than 1, reduced cellular senescence compared to the untreated negative control group, and maintained cellular proliferation equal to or greater than that of the untreated negative control. Examples of such polypeptides are shown in Table 6.

[0193] Furthermore, the dose-dependent effects of the polypeptides in Table 6 on cellular senescence were examined. Cells were incubated with various amounts of the polypeptides ranging from 1.26 μM to 50 μM (1.26 μM, 3.12 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM). Even lower doses of the polypeptides significantly reduced cellular senescence.

[0194] [Table 6]

[0195] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0196] Illustrative Embodiments Among the exemplary embodiments are the following:

[0197] Embodiment 1 includes a composition for treating skin, comprising an isolated, synthetic, or recombinant polypeptide comprising the amino acid sequence of LKGI (SEQ ID NO: 5) or an analog thereof, wherein the polypeptide comprises up to 100 amino acids. Embodiment 2. The composition of embodiment 1, wherein the isolated, synthetic, or recombinant polypeptide comprises the amino acid sequence of WLKGI (SEQ ID NO: 7) or an analog thereof. Embodiment 3. The composition of embodiment 1 or embodiment 2, wherein the isolated, synthetic, or recombinant polypeptide comprises the amino acid sequence of LKGIL (SEQ ID NO: 6) or an analog thereof. Embodiment 4. The composition of any one of embodiments 1-3, wherein the isolated, synthetic, or recombinant polypeptide comprises at least 4 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids. Embodiment 5. The composition of any one of embodiments 1-4, further comprising a therapeutic, nutraceutical, or cosmetic excipient. Embodiment 6. The composition of embodiment 5, wherein the excipient is configured for topical application. Embodiment 7. The composition of any one of embodiments 1-6, wherein the composition is configured for application to human skin.Embodiment 8. The composition of any one of embodiments 1-7, wherein the composition is a cream, transdermal patch, topical patch, ointment, oil, gel, liquid, powder, lotion, serum, emulsion, moisturizer, foam, face mask, mousse, aerosol, spray, cleanser, toner, or shampoo.Embodiment 9. The composition of any one of embodiments 1-5, wherein the composition is configured as a dietary supplement.Embodiment 10. The composition of embodiment 9, wherein the composition is configured as a beverage.Embodiment 11. The composition of any one of embodiments 1-9, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.Embodiment 12. The composition of any one of embodiments 1-9, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oils, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extracts.Embodiment 13. The composition of embodiment 12, wherein the vegetable oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, caliphyllum pulcanum oil, almond oil, and argan oil. Embodiment 14. The composition of any one of embodiments 1-13, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or a derivative thereof. Embodiment 15. The composition of any one of embodiments 1-14, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM. Embodiment 16. The composition of any one of embodiments 1-14, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 0.001% to about 5% (w / w). Embodiment 17. The composition of any one of embodiments 1-16, wherein the composition is formulated to achieve low skin penetration of the isolated, synthetic, or recombinant polypeptide into the dermis of up to 10%.Embodiment 18. The composition of any one of embodiments 1-17, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis of the user's skin, skin viscoelastic properties, or skin surface profile after using the composition on the skin compared to before using the composition.Embodiment 19. The composition of any one of embodiments 1-17, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or the overall appearance of the skin of a user's skin after using the composition on the skin compared to before using the composition.Embodiment 20. The composition of any one of embodiments 1-19, wherein treating the skin with the composition reduces or treats the effects of aging on the skin.Embodiment 21. The composition of any one of embodiments 1-20, wherein treating the skin with the composition reduces the effects of cellular aging on the skin. Embodiment 22. The composition of any one of embodiments 20-21, wherein the effects of aging are assessed by at least one of senescence-associated β-galactosidase activity levels, the percentage of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

[0198] Embodiment 23 is X1X2X3X4X5X6X7X8X9X 10 or an analog thereof, wherein (a) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a first sequence SEQ ID NO:1, wherein X1 is E, X2 is T, X4 is K, X6 is W, X7 is L, X9 is G, and X 10 is I; and (i) X3 is not S; or (ii) if X5 is any amino acid, then X8 is not G; or (iii) if X8 is any amino acid, then X5 is not N; or (iv) any one of (i), (ii), or (iii), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (b) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a second sequence, SEQ ID NO:2, wherein X1 is A, X2 is T, X3 is A, X4 is K, X5 is A, X6 is W, X7 is L, X8 is K, X9 is G, and X10 is I, optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (c) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a third sequence, SEQ ID NO:3, wherein X1 is K, X2 is L, and X5 is I , X6 is L, X8 is G, and X 10 is A; and (i) when X9 is any amino acid, X3 is not N; or (ii) when X3 is any amino acid, X9 is not S; or (iii) when X4 is any amino acid, X7 is not L; or (iv) when X7 is any amino acid, X4 is not S; and, or (v) any one of (i), (ii), (iii), or (iv), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (d) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a fourth sequence, SEQ ID NO:4, wherein X1 is W, X2 is L, X3 is K, X4 is G, X5 is I, X6 is L, X7 is R, X8 is E, X9 is A, and X 10is A, optionally with 1, 2, 3, or 4 conservative amino acid substitutions. Embodiment 24. The polypeptide of embodiment 23, wherein the amino acid sequence comprises LKGI (SEQ ID NO: 5). Embodiment 25. The polypeptide of embodiment 23 or embodiment 24, wherein the amino acid sequence comprises WLKGI (SEQ ID NO: 7). Embodiment 26. The polypeptide of any one of embodiments 23-25, wherein the amino acid sequence comprises LKGIL (SEQ ID NO: 6). Embodiment 27. The polypeptide of embodiment 23, wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO: 1. Embodiment 28. The polypeptide of embodiment 23, wherein the amino acid sequence is SEQ ID NO: 1. Embodiment 29. The polypeptide of embodiment 23, wherein the amino acid sequence is SEQ ID NO: 2. Embodiment 30. The polypeptide of embodiment 23, wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO: 3. Embodiment 31. The polypeptide of embodiment 23, wherein the amino acid sequence has at least 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO: 3. Embodiment 32. The polypeptide of embodiment 23, wherein the amino acid sequence is SEQ ID NO: 3. Embodiment 33. The polypeptide of embodiment 23, wherein the amino acid sequence is SEQ ID NO: 4. Embodiment 34. The polypeptide of any one of embodiments 23-33, wherein the recombinant polypeptide comprises at least 10 amino acids, 15 amino acids, or 20 amino acids, and up to 100 amino acids. Embodiment 35 comprises a composition for treating skin, comprising at least one synthetic or recombinant polypeptide of any one of embodiments 23-34 and a therapeutic, nutraceutical, or cosmetic excipient. Embodiment 36. The composition of embodiment 35, wherein the excipient is configured for topical application. Embodiment 37. The composition of embodiment 35 or embodiment 36, wherein the composition is formulated for application to human skin. Embodiment 38. The composition of any one of embodiments 35-37, wherein the composition is a cream, ointment, gel, liquid, oil, powder, lotion, serum, emulsion, moisturizer, foam, face mask, mousse, aerosol, spray, cleanser, toner, topical patch, or shampoo.Embodiment 39. The composition of embodiment 35, wherein the composition is configured as a dietary supplement.Embodiment 40. The composition of embodiment 39, wherein the composition is configured as a beverage.

[0199] Embodiment 41. The composition of any one of embodiments 35-40, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.Embodiment 42. The composition of any one of embodiments 35-40, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oils, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extract. Embodiment 43. The composition of embodiment 42, wherein the plant oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, calicophilum pulcanum oil, almond oil, and argan oil.Embodiment 44. The composition of any one of embodiments 35-43, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivatives thereof.Embodiment 45. The composition of any one of embodiments 35-44, wherein the composition comprises an effective amount of an isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM. Embodiment 46. The composition of any one of embodiments 35-44, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 0.001% to about 5% (w / w).Embodiment 47. The composition of any one of embodiments 35-44, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 0.001% to about 1%.Embodiment 48. The composition of any one of embodiments 35-47, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis, skin viscoelastic properties, or skin surface profile of the user's skin after using the composition on the skin compared to before using the composition.Embodiment 49. The composition of any one of embodiments 35-47, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or the overall appearance of the skin of the user's skin after using the composition on the skin compared to before using the composition.Embodiment 50. The composition of any one of embodiments 35-49, wherein treating the skin with the composition reduces or treats the effects of aging on the skin.Embodiment 51. The composition of any one of embodiments 35-50, wherein treating the skin with the composition reduces the effects of cellular aging on the skin. Embodiment 52. The composition of any one of embodiments 50-51, wherein the effects of aging are assessed by at least one of senescence-associated β-galactosidase activity levels, the percentage of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

[0200] Embodiment 53 includes a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a composition comprising the amino acid sequence of SEQ ID NO:5. Embodiment 54. The method of embodiment 53, wherein the formulation comprises the amino acid sequence of SEQ ID NO:6. Embodiment 55. The method of embodiment 53 or embodiment 54, wherein the composition comprises the amino acid sequence of SEQ ID NO:7. Embodiment 56. The method of any one of embodiments 53-55, wherein the administering step comprises topically applying the composition to the subject. Embodiment 57. The method of any one of embodiments 53-56, wherein the subject is a human or other animal. Embodiment 58. The method of any one of embodiments 53-57, wherein the method comprises administering to the subject an effective amount of the composition. Embodiment 59. The method of any one of embodiments 53-58, wherein the disease is a disorder associated with the accumulation of senescent cells in the subject. Embodiment 60. The method of embodiment 53, wherein the disorder associated with the accumulation of senescent cells comprises skin aging. Embodiment 61. The method of any one of embodiments 53-60, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.Embodiment 62. The method of any one of embodiments 53-61, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oils, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extract. Embodiment 63. The method of embodiment 62, wherein the plant oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, Calycophyllum spruceanum oil, almond oil, and argan oil.Embodiment 64. The method of any one of embodiments 53-63, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivatives thereof. Embodiment 65. The method of any one of embodiments 53-64, wherein the composition comprises an effective amount of an isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM. Embodiment 66. The method of any one of embodiments 53-64, wherein the composition comprises an effective amount of an isolated, synthetic, or recombinant polypeptide from about 0.001% to about 5% (w / w). Embodiment 67. The method of any one of embodiments 53-66, wherein the composition is formulated to achieve up to 10% reduced skin penetration of the isolated, synthetic, or recombinant polypeptide into the dermis.Embodiment 68. The method of any one of embodiments 53-67, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis of the user's skin, skin viscoelastic properties, or skin surface profile after using the composition on the skin compared to before using the composition.Embodiment 69. The method of any one of embodiments 53-68, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or the overall appearance of the skin of the user's skin after using the composition on the skin compared to before using the composition. Embodiment 70. The method of any one of embodiments 53-69, wherein treating the skin with the composition reduces or treats the effects of aging on the skin. Embodiment 71. The method of any one of embodiments 53-70, wherein treating the skin with the composition reduces the effects of cellular aging on the skin.Embodiment 72. The method of any one of embodiments 70-71, wherein the effects of aging are assessed by at least one of senescence-associated β-galactosidase activity levels, the percentage of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

[0201] Embodiment 73. A method of reducing cellular senescence in a subject in need thereof, the method comprising administering to the subject a polypeptide comprising the amino acid sequence of LKGI (SEQ ID NO: 5), optionally with one conservative amino acid substitution, wherein the polypeptide comprises up to 100 amino acids.Embodiment 74. The method of embodiment 73, wherein the polypeptide comprises at least 4 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids.Embodiment 75. The method of embodiment 73, wherein the composition comprises the amino acid sequence of WLKGI (SEQ ID NO: 7), optionally with one conservative amino acid substitution.Embodiment 76. The method of embodiment 73, wherein the composition comprises the amino acid sequence of LKGIL (SEQ ID NO: 6), optionally with one conservative amino acid substitution.Embodiment 77. The method of embodiment 75 or embodiment 76, wherein the polypeptide comprises at least 5 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids, and up to 100 amino acids. Embodiment 78. The method of any one of embodiments 73-77, wherein the composition further comprises a therapeutic, nutraceutical, or cosmetic excipient.Embodiment 79. A method of reducing cellular senescence in a subject in need thereof, the method comprising administering to the subject a therapeutic, nutraceutical, or cosmetic composition comprising at least one polypeptide of any one of embodiments 23-34.Embodiment 80. The method of embodiment 79, wherein the composition further comprises a therapeutic, nutraceutical, or cosmetic excipient.Embodiment 81. The method of embodiment 79 or embodiment 80, wherein the administering step comprises applying the composition to a portion of the subject's skin.Embodiment 82. The method of any one of embodiments 79-81, wherein the composition extends the lifespan of a plurality of cells of the subject, induces SIRT6 expression in a plurality of cells of the subject, increases the cell renewal rate in a plurality of cells of the subject, promotes apoptosis in a plurality of cells of the subject, promotes DNA repair in a plurality of cells of the subject, increases collagen production in a plurality of cells of the subject, increases hyaluronic acid synthase production in a plurality of cells of the subject, reduces ATRX nuclear foci accumulation in a plurality of cells of the subject, reduces p16 expression in a plurality of cells of the subject, reduces senescence-associated beta-galactosidase production in a plurality of cells of the subject, reduces IL8 expression in a plurality of cells of the subject, reduces MMP1 expression in a plurality of cells of the subject, increases BLM expression in a plurality of cells of the subject, and / or prevents UV-induced DNA damage in a plurality of cells of the subject.

[0202] Embodiment 83 includes a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutic, nutraceutical, or cosmetic composition comprising the amino acid sequence of SEQ ID NO:5, optionally with one conservative amino acid substitution.Embodiment 84. The method of embodiment 83, wherein the therapeutic, nutraceutical, or cosmetic composition comprises the amino acid sequence of SEQ ID NO:6, optionally with one conservative amino acid substitution.Embodiment 85. The method of embodiment 83 or embodiment 84, wherein the therapeutic, nutraceutical, or cosmetic composition comprises the amino acid sequence of SEQ ID NO:7, optionally with one conservative amino acid substitution.

Claims

1. A composition for treating skin, said composition comprising an isolated, synthetic, or recombinant polypeptide comprising the amino acid sequence of LKGI (SEQ ID NO: 5) or an analog thereof, wherein said polypeptide comprises up to 100 amino acids.

2. The composition of claim 1 , wherein the isolated, synthetic, or recombinant polypeptide comprises the amino acid sequence of WLKGI (SEQ ID NO: 7) or an analog thereof.

3. 3. The composition of claim 1 or claim 2, wherein the isolated, synthetic, or recombinant polypeptide comprises the amino acid sequence of LKGIL (SEQ ID NO: 6) or an analog thereof.

4. 4. The composition of any one of claims 1 to 3, wherein the isolated, synthetic, or recombinant polypeptide comprises at least 4 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids.

5. The composition of any one of claims 1 to 4, further comprising a therapeutic, nutraceutical, or cosmetic excipient.

6. The composition of claim 5 , wherein the vehicle is configured for topical application.

7. The composition of any one of claims 1 to 6, wherein the composition is adapted for application to human skin.

8. 8. The composition of any one of claims 1 to 7, wherein the composition is a cream, a transdermal patch, a topical patch, an ointment, an oil, a gel, a liquid, a powder, a lotion, a serum, an emulsion, a moisturizer, a foam, a face mask, a mousse, an aerosol, a spray, a facial cleanser, a toner, or a shampoo.

9. The composition according to any one of claims 1 to 5, wherein the composition is configured as a dietary supplement.

10. The composition of claim 9 , wherein the composition is configured as a beverage.

11. 10. The composition of any one of claims 1 to 9, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.

12. 10. The composition of any one of claims 1 to 9, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oil, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extracts.

13. 13. The composition of claim 12, wherein the plant oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, calicophilum pulcanum oil, almond oil, and argan oil.

14. 14. The composition of any one of claims 1 to 13, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivatives thereof.

15. 15. The composition of any one of claims 1 to 14, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM.

16. 15. The composition of any one of claims 1 to 14, wherein the composition comprises an effective amount of about 0.001% to about 5% (w / w) of the isolated, synthetic, or recombinant polypeptide.

17. 15. The composition of any one of claims 1 to 14, wherein the composition comprises an effective amount of about 0.01% to about 1% of the isolated, synthetic, or recombinant polypeptide.

18. 18. The composition of any one of claims 1 to 17, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis, skin viscoelastic properties, or skin surface profile of a user's skin after using the composition on the skin compared to before using the composition.

19. 18. The composition of any one of claims 1 to 17, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or overall appearance of the skin of a user after using the composition on the skin compared to before using the composition.

20. A composition according to any one of claims 1 to 19, wherein treatment of skin with said composition reduces or treats the effects of ageing on the skin.

21. The composition of any one of claims 1 to 20, wherein treatment of skin with the composition reduces the effects of cellular aging on the skin.

22. The composition of any one of claims 20 to 21, wherein the effects of aging are assessed by at least one of senescence-associated β-galactosidase activity level, the ratio of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

23. X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ or an analog thereof, wherein: (a) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a first sequence, SEQ ID NO:1, wherein: X １ is E, X ２ is T, X ４ is K, X ６ W, X7 は L, X ９ is G, and X １０ is I; and, (i) X ３ is not S; or (ii) X ５ is any amino acid, X ８ is not G; or (iii) X ８ is any amino acid, X ５ is not N; or (iv) any one of (i), (ii), or (iii), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (b) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a second sequence, SEQ ID NO:2, wherein: X １ A, X ２ is T, X ３ A, X ４ is K, X ５ A, X ６ is W, X ７ is L, X ８ is K, X ９ is G, and X １０ is I, optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (c) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a third sequence, SEQ ID NO:3, wherein: X １ is K, X ２ is L, X ５ is I, X ６ is L, X ８ is G, and X １０ is A; and, (i) X ９ is any amino acid, X ３ is not N; or (ii) X ３ is any amino acid, X ９ is not S; or (iii) X ４ is any amino acid, X ７ is not L; or (iv) X ７ is any amino acid, X ４ is not S; and, or (v) any one of (i), (ii), (iii), or (iv), optionally with 1, 2, 3, or 4 conservative amino acid substitutions; or (d) the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to a fourth sequence, SEQ ID NO:4, wherein: X １ is W, X ２ is L, X ３ is K, X ４ is G, X ５ is I, X ６ is L, X ７ is R, X ８ is E, X ９ is A, and X １０ is A, optionally with 1, 2, 3, or 4 conservative amino acid substitutions.

24. 24. The polypeptide of claim 23, wherein the amino acid sequence comprises LKGI (SEQ ID NO: 5).

25. 25. The polypeptide of claim 23 or claim 24, wherein the amino acid sequence comprises WLKGI (SEQ ID NO: 7).

26. The polypeptide of any one of claims 23-25, wherein the amino acid sequence comprises LKGIL (SEQ ID NO: 6).

27. 24. The polypeptide of claim 23, wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO:

1.

28. 24. The polypeptide of claim 23, wherein the amino acid sequence is SEQ ID NO:

1.

29. 24. The polypeptide of claim 23, wherein the amino acid sequence is SEQ ID NO:

2.

30. 24. The polypeptide of claim 23, wherein the amino acid sequence has at least 70%, 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO:

3.

31. 24. The polypeptide of claim 23, wherein the amino acid sequence has at least 80%, 85%, 90%, or 95% identity to the sequence of SEQ ID NO:

3.

32. 24. The polypeptide of claim 23, wherein the amino acid sequence is SEQ ID NO:

3.

33. 24. The polypeptide of claim 23, wherein the amino acid sequence is SEQ ID NO:

4.

34. The polypeptide of any one of claims 23-33, wherein the recombinant polypeptide comprises at least 10 amino acids, 15 amino acids, or 20 amino acids, and up to 100 amino acids.

35. A composition for treating skin comprising at least one isolated, synthetic or recombinant polypeptide according to any one of claims 23-34 and a therapeutic, nutraceutical or cosmetic excipient.

36. 36. The composition of claim 35, wherein the vehicle is configured for topical application.

37. 37. The composition of claim 35 or claim 36, wherein the composition is formulated for application to human skin.

38. 38. The composition of any one of claims 35-37, wherein the composition is a cream, ointment, gel, liquid, oil, powder, lotion, serum, emulsion, moisturizer, foam, face mask, mousse, aerosol, spray, cleanser, toner, topical patch, or shampoo.

39. 36. The composition of claim 35, wherein the composition is configured as a dietary supplement.

40. 40. The composition of claim 39, wherein the composition is configured as a beverage.

41. 41. The composition of any one of claims 35-40, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.

42. 41. The composition of any one of claims 35-40, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oil, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extracts.

43. 43. The composition of claim 42, wherein the plant oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, calicophilum pulcanum oil, almond oil, and argan oil.

44. 44. The composition of any one of claims 35-43, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivatives thereof.

45. 45. The composition of any one of claims 35-44, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM.

46. 45. The composition of any one of claims 35-44, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 0.001% to about 5% (w / w).

47. 45. The composition of any one of claims 35-44, wherein the composition comprises an effective amount of about 0.01% to about 1% of the isolated, synthetic, or recombinant polypeptide.

48. 48. The composition of any one of claims 35-47, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis of a user's skin, skin viscoelastic properties, or skin surface profile after using the composition on the skin compared to before using the composition.

49. 48. The composition of any one of claims 35-47, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or overall appearance of the skin of a user after using the composition on the skin compared to before using the composition.

50. 50. The composition of any one of claims 35-49, wherein treatment of skin with said composition reduces or treats the effects of aging on the skin.

51. 51. The composition of any one of claims 35-50, wherein treatment of the skin with the composition reduces the effects of cellular aging on the skin.

52. 52. The composition of any one of claims 50-51, wherein the effects of aging are assessed by at least one of senescence-associated B-galactosidase activity level, the percentage of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

53. A method of treating a disease in a subject in need thereof, said method comprising administering to the subject a composition comprising the amino acid sequence of SEQ ID NO:

5.

54. 54. The method of claim 53, wherein the composition comprises the amino acid sequence of SEQ ID NO:

6.

55. 55. The method of claim 53 or claim 54, wherein the composition comprises the amino acid sequence of SEQ ID NO:

7.

56. 56. The method of any one of claims 53-55, wherein the administering step comprises topically applying the composition to the subject.

57. 57. The method of any one of claims 53-56, wherein the subject is a human or other animal.

58. 58. The method of any one of claims 53-57, wherein the method comprises administering to the subject an effective amount of the composition.

59. 59. The method of any one of claims 53-58, wherein the disease is a disorder associated with the accumulation of senescent cells in a subject.

60. 54. The method of claim 53, wherein the disorder associated with accumulation of senescent cells comprises skin aging.

61. 61. The method of any one of claims 53-60, wherein the composition comprises at least one skin moisturizer selected from the group consisting of glycerin, squalene, sorbitol, hyaluronic acid, hyaluronic acid derivatives, sodium hyaluronate, sodium hyaluronate crosspolymer, niacinamide, glycoproteins, pyrrolidone carboxylic acid (PCA), lysine HCl, allantoin, and algae extract.

62. 62. The method of any one of claims 53-61, wherein the composition comprises at least one emollient selected from the group consisting of vegetable oils, mineral oil, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristic acid, palmitic acid, stearic acid, glycolipids, phospholipids, squalene, glycerin, ceramides, and algae extracts.

63. 63. The method of claim 62, wherein the plant oil is selected from the group consisting of rosehip oil, andiroba oil, grapeseed oil, avocado oil, plum seed oil, pratense oil, calicophilum pulcanum oil, almond oil, and argan oil.

64. 64. The method of any one of claims 53-63, wherein the composition comprises at least one vitamin selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin F, vitamin K, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B7 (biotin), vitamin B6, vitamin B12 (cyanocobalamin), vitamin B9, folic acid, niacinamide, or derivatives thereof.

65. 65. The method of any one of claims 53-64, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 500 nM to about 500 μM.

66. 65. The method of any one of claims 53-64, wherein the composition comprises an effective amount of the isolated, synthetic, or recombinant polypeptide from about 0.001% to about 5% (w / w).

67. 67. The method of any one of claims 53-66, wherein the composition is formulated to achieve low skin penetration of up to 10% of the isolated, synthetic, or recombinant polypeptide into the dermis.

68. 68. The method of any one of claims 53-67, wherein the composition improves at least one of skin hydration, transepidermal water loss (TEWL), skin thickness and echogenicity, intradermal analysis of the user's skin, skin viscoelastic properties, or skin surface profile after using the composition on the skin compared to before using the composition.

69. 69. The method of any one of claims 53-68, wherein the composition reduces the appearance of lines / wrinkles, the appearance of skin tone (evenness), the appearance of pores, the appearance of texture / smoothness, firmness (visual), elasticity (tactile), epidermal barrier, rough skin, skin hyperpigmentation, or overall appearance of the skin of a user after using the composition on the skin compared to before using the composition.

70. 70. The method of any one of claims 53-69, wherein treating skin with the composition reduces or treats the effects of aging on the skin.

71. 71. The method of any one of claims 53-70, wherein treating the skin with the composition reduces the effects of cellular aging on the skin.

72. 72. The method of any one of claims 70-71, wherein the effects of aging are assessed by at least one of senescence-associated B-galactosidase activity level, the percentage of ATRX foci / cells, p16 expression, IL-8 expression, Ki-67 expression, hyaluronan synthase 2 expression, matrix metalloproteinase 1 (MMP1) expression, sirtuin 6 (SIRT6) expression, BLM expression, or exonuclease 1 (EXO1) expression.

73. 1. A method of reducing cellular senescence in a subject in need thereof, the method comprising administering to the subject a polypeptide comprising the amino acid sequence of LKGI (SEQ ID NO: 5), optionally with one conservative amino acid substitution, wherein the polypeptide comprises up to 100 amino acids.

74. 74. The method of claim 73, wherein the polypeptide comprises at least 4 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids.

75. 74. The method of claim 73, wherein the composition comprises the amino acid sequence of WLKGI (SEQ ID NO: 7), optionally with one conservative amino acid substitution.

76. 74. The method of claim 73, wherein the composition comprises the amino acid sequence of LKGIL (SEQ ID NO: 6), optionally with one conservative amino acid substitution.

77. 77. The method of any one of claims 75 or 76, wherein the polypeptide comprises at least 5 amino acids, 10 amino acids, 15 amino acids, or 20 amino acids, and up to 100 amino acids.

78. 78. The method of any one of claims 73-77, wherein the composition further comprises a therapeutic, nutraceutical, or cosmetic excipient.

79. 35. A method of reducing cellular senescence in a subject in need thereof, said method comprising administering to said subject a therapeutic, nutraceutical or cosmetic composition comprising at least one polypeptide according to any one of claims 23-34.

80. 80. The method of claim 79, wherein the composition further comprises a therapeutic, nutraceutical, or cosmetic excipient.

81. the administering step comprises applying the composition to a portion of the subject's skin; 81. The method of claim 79 or claim 80.

82. 82. The method of any one of claims 79-81, wherein the composition extends the lifespan of a plurality of cells of the subject, induces SIRT6 expression in a plurality of cells of the subject, increases the rate of cell renewal in a plurality of cells of the subject, promotes apoptosis in a plurality of cells of the subject, promotes DNA repair in a plurality of cells of the subject, increases collagen production in a plurality of cells of the subject, increases hyaluronan synthase production in a plurality of cells of the subject, reduces ATRX nuclear foci accumulation in a plurality of cells of the subject, reduces p16 expression in a plurality of cells of the subject, reduces senescence-associated beta-galactosidase production in a plurality of cells of the subject, reduces IL8 expression in a plurality of cells of the subject, reduces MMP1 expression in a plurality of cells of the subject, increases BLM expression in a plurality of cells of the subject, and / or prevents UV-induced DNA damage in a plurality of cells of the subject.

83. A method for treating a disease in a subject in need thereof, said method comprising administering to the subject a therapeutic, nutraceutical, or cosmetic composition comprising the amino acid sequence of SEQ ID NO: 5, optionally with one conservative amino acid substitution.

84. 84. The method of claim 83, wherein the therapeutic, nutraceutical, or cosmetic composition comprises the amino acid sequence of SEQ ID NO: 6, optionally with one conservative amino acid substitution.

85. 85. The method of claim 83 or claim 84, wherein the therapeutic, nutraceutical, or cosmetic composition comprises the amino acid sequence of SEQ ID NO: 7, optionally with one conservative amino acid substitution.

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