Skin care composition containing vitamin B3 combined with two peptides

Abstract

A skin care composition comprising a combination of a vitamin B3 compound, palmitoyl pentapeptide-4, acetyl tetrapeptide-11, and a dermatologically acceptable carrier, the combination of vitamin B3 compound, palmitoyl pentapeptide-4, and acetyl tetrapeptide-11 increasing (e.g., synergistically) activation of cellular antioxidant response elements (AREs) to improve visible signs of skin aging.

Description

[Technical Field]

[0001] The present disclosure is generally directed to improving skin health using synergistic combinations of skin care actives. More specifically, the present disclosure is directed to combinations of vitamin B3 with two or more peptides that synergistically stimulate cellular antioxidant and repair processes. [Background technology]

[0002] Skin plays an important role in a person's physical appearance, being the first line of defense against environmental insults that can damage otherwise sensitive underlying tissues and organs. Visible signs of aging, such as wrinkles and age spots on the skin, are undesirable reminders of the loss of youth. As a result, treating the signs of skin aging has become a booming business in a youth-conscious society.

[0003] Skin is made up of a variety of different cells that work together in a dynamic and complex relationship to maintain skin health. However, as skin cells age or become damaged, they generally lose their ability to function at the level required to maintain youthful, healthy-looking skin. Skin cells can be damaged by a variety of endogenous and exogenous stressors (e.g., UV radiation, pollution, and smoking). In some instances, these stressors trigger the production of reactive oxygen species (ROS), which disrupt normal cellular processes. In response, human cells have evolved defenses to combat ROS; however, cellular defenses can be overwhelmed by stressor-induced ROS spikes, leading to acute as well as chronic changes in cellular homeostasis. As ROS accumulate over time, they cause oxidative stress at the cellular level, which can ultimately manifest as visible signs of aging (e.g., fine lines, wrinkles, pigmented spots, and thinning skin).

[0004] The antioxidant defense system of most cells is typically controlled by a master switch called the antioxidant response element (ARE). AREs are cis-acting enhancer elements located in the regulatory regions of antioxidant and detoxification genes. AREs are activated by redox cycling phenolics and electrophiles. Several studies have shown that, although many other transcription factors also bind to ARE sequences, a protein called nuclear factor erythroid 2-related factor 2 (Nrf2) may be the primary transcription factor required for ARE activation. When AREs are activated in response to damage from ROS, the corresponding genes signal the cell to initiate the production of redox regulators and / or ROS-quenching proteins and enzymes. In addition to regulating the production of redox regulators and ROS-quenching compounds, AREs can also signal the cell to initiate repair processes. Therefore, it is desirable to enhance the activation of ARE in aging skin cells to combat the signs of skin aging caused by oxidative stress.

[0005] Several compounds have been discovered that enhance ARE activation. For example, U.S. Patent Application Publication No. 20160317419 provides data showing that a combination of niacinamide and nicotinamide riboside enhances ARE activation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 20160317419 Summary of the Invention [Problem to be solved by the invention]

[0007] However, there remains a need to identify compounds and methods that improve cellular antioxidant activity by enhancing ARE activation, and there is also a need to identify compounds and methods that beneficially regulate genes involved in cellular antioxidant and repair. [Means for solving the problem]

[0008] The present disclosure relates to a skin care composition comprising a combination of a vitamin B3 compound, palmitoyl pentapeptide-4 (pal-KTTKS) [SEQ ID NO: 1], acetyl tetrapeptide-11 (ac-PPYL) [SEQ ID NO: 2], and a dermatologically acceptable carrier. The combination of vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] has been found to activate cellular antioxidant response elements (AREs) and / or beneficially regulate, and in some cases synergistically regulate, genes involved in cellular antioxidant and repair. The present disclosure also relates to methods of using the aforementioned compositions. DETAILED DESCRIPTION OF THE INVENTION

[0009] Improving cellular antioxidant and repair is important for improving skin health and / or appearance.Therefore, there is a long-standing need to identify new ingredients for use in topical skin care compositions that provide these benefits.In particular, there is a need to identify compounds that enhance ARE activation in skin cells, which tend to be at higher risk from oxidative stress than some other types of cells due to high exposure to ultraviolet light and other exogenous stressors.Surprisingly, it has been discovered that the combination of vitamin B3 compound, palmitoyl pentapeptide-4 [SEQ ID NO: 1], and acetyl tetrapeptide-11 [SEQ ID NO: 2] can stimulate antioxidant and cellular repair processes.In particular, it has been found that this combination of ingredients synergistically enhances ARE activation and / or regulates certain genes that are thought to be involved in cellular antioxidant and repair.

[0010] References herein to "an embodiment" or the like mean that a particular material, feature, structure, and / or characteristic described in connection with that embodiment is included in at least one embodiment, and optionally in multiple embodiments, but do not mean that all embodiments incorporate the described material, feature, structure, and / or characteristic. Furthermore, materials, features, structures, and / or characteristics may be combined in any suitable manner across different embodiments, and materials, features, structures, and / or characteristics may be excluded or substituted from those described. Accordingly, the embodiments and aspects described herein can include or be combined with elements or components of other embodiments and / or aspects, even if not explicitly illustrated in combination, unless otherwise stated or unless incompatibility is explicitly stated.

[0011] In all embodiments, unless otherwise specified, all ingredient percentages are by weight of the cosmetic composition. Unless otherwise specified, all ratios are by weight. The number of significant digits does not represent a limitation on the stated amount or on the precision of the measurements. Unless otherwise specified, all quantities are understood to be modified by the word "about." Unless otherwise specified, all measurements are understood to be made at ambient conditions of approximately 25°C, where "ambient conditions" means conditions of about 1 atmosphere and about 50% relative humidity. All numerical ranges are inclusive and can be combined to form smaller ranges not expressly disclosed. For example, delimited upper and lower range limits are interchangeable to create further ranges.

[0012] The compositions of the present invention can comprise, consist essentially of, or consist of the essential and optional ingredients described herein. As used herein, "consisting essentially of" means that the composition or component may include only additional ingredients that do not materially alter the basic and novel characteristics of the claimed composition or method. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0013] Sequence Listing A Sequence Listing showing the amino acid sequences of SEQ ID NOS: 1 and 2, and the nucleotide sequences of SEQ ID NOS: 3-7, including their primary sequences and conservatively modified variants, is filed contemporaneously with this application as an ASCII text file entitled "15964_seq_list_ST25." This ASCII text file was created on January 19, 2021, and is 201 KB in size. Pursuant to MPEP 605.08 and 37 CFR 1.52(e), the subject matter of this ASCII text file is incorporated herein by reference.

[0014] definition "About" modifies a particular value by referring to a range equal to plus or minus 20 percent (±20%) or less (e.g., less than 15%, 10%, or even less than 5%) of the stated value.

[0015] "Applying" or "application" as used herein in reference to a composition or material means placing or spreading the composition onto a human skin surface, such as the epidermis.

[0016] "Cosmetic composition" means a composition that contains a cosmetic agent and is intended for non-therapeutic (i.e., non-medical) use. Examples of cosmetic compositions include color cosmetics (e.g., foundation, lipstick, concealer, and mascara), skin care compositions (e.g., moisturizers and sunscreens), personal care compositions (e.g., rinse-off and leave-in body washes and soaps), and hair care compositions (e.g., shampoos and conditioners).

[0017] "Derivative" means an amide, ether, ester, amino, carboxyl, acetyl, or alcohol derivative of a given compound.

[0018] "Effective amount" means an amount of compounds and compositions sufficient to significantly induce a positive effect on keratinous tissue over the course of a treatment period. The positive effect may be a health, appearance, and / or feel effect, including the effects disclosed herein, individually or in combination.

[0019] "Modulate" and variations thereof means to upregulate or downregulate gene expression.

[0020] "Skin care" means regulating and / or improving skin condition (e.g., skin health, appearance, or texture / feel). Some non-limiting examples of improving skin condition include improving the appearance and / or feel of skin by providing a smoother, more uniform appearance and / or feel, increasing the thickness of one or more layers of skin, improving skin elasticity or resilience, improving skin firmness, as well as reducing the oily, shiny, and / or dull appearance of skin, improving skin hydration or moisturization, improving the appearance of fine lines and / or wrinkles, improving skin exfoliation or scaling, plumping skin, improving skin barrier properties, improving skin tone, reducing the appearance of redness or skin blemishes, and / or improving skin brightness, radiance, or clarity.

[0021] "Skin care active" means a compound or combination of compounds that, when applied to the skin, produces an acute and / or chronic effect on the skin or a type of cell normally found in the skin. Skin care actives can regulate and / or improve the skin or cells associated therewith (e.g., improve skin elasticity, skin hydration, skin barrier function, and / or cellular metabolism).

[0022] "Skin care composition" means a composition that contains skin care actives and that regulates and / or improves the condition of the skin.

[0023] "Skin cells" refer to cell types commonly found in human skin. Non-limiting examples of skin cells are keratinocytes, fibroblasts, melanocytes, Langerhans cells, and Merkel cells.

[0024] "Synergy" and variations thereof mean that the cellular antioxidant and repair effect provided by the combination of niacinamide, palmitoyl pentapeptide-4 [SEQ ID NO: 1], and acetyl tetrapeptide-11 [SEQ ID NO: 2] is greater than the predicted additive effect of these components alone. For example, synergy is demonstrated when the combination of niacinamide, palmitoyl pentapeptide-4 [SEQ ID NO: 1], and acetyl tetrapeptide-11 [SEQ ID NO: 2] increases ARE activation beyond the calculated additive effect of these three components individually. ARE activation levels can be quantified using the ARE assay, described in more detail below.

[0025] As used herein, "treatment period" refers to the length of time and / or frequency that a material or composition is applied to the target skin surface.

[0026] "Up-regulation" and its variations refer to increasing gene expression. Conversely, "down-regulation" and its variations refer to decreasing gene expression. Gene expression can be quantified using conventional methods (e.g., microarray analysis, rt-PCR, Western blot).

[0027] Skin Care Composition The novel skin care composition described herein is intended for topical application to human skin to provide cellular antioxidant and / or repair effects. The skin care composition of the present invention contains safe and effective amounts of a vitamin B3 compound, palmitoyl pentapeptide-4 (pal-KTTKS) [SEQ ID NO: 1], and acetyl tetrapeptide-11 (ac-PPYL) [SEQ ID NO: 2]. The combination of effective amounts of these three ingredients can synergistically enhance the activation of antioxidant response elements, which is important for combating oxidative stress and reducing the visible signs of skin aging.

[0028] The combination of a vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] also inhibits the activity of Nuclear Factor Erythroid2-Related Factor 2 (NFE2L2) [SEQ ID NO: 3] (primary sequence) and conservatively modified variants thereof, Schlafen Family Member 5 (SLFN5) [SEQ ID NO: 4] (primary sequence) and conservatively modified variants thereof, Glycerophosphodiester Phosphodiesterase 1 (GDE1) [SEQ ID NO: 5] (primary sequence) and conservatively modified variants thereof, Multiple Inositol-Polyphosphate Phosphatase 1 (MIP1) [SEQ ID NO: 6] (primary sequence) and conservatively modified variants thereof, and The present invention may synergistically regulate one or more genes selected from the group consisting of 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) [SEQ ID NO: 7] (primary sequence) and its conservatively modified variants, and 3-hydroxy-3-methylglutaryl-CoA lyase (MINPP1) [SEQ ID NO: 6] (primary sequence) and its conservatively modified variants. Without being limited by theory, these genes are believed to play important roles in cellular antioxidant and / or repair, and these genes have been shown to be downregulated as a result of chronological and / or photoaging in the epidermis and / or dermis. For example, NRF2 is believed to play an important role in ARE activation (Annu Rev Pharmacol Toxicol. 2013; 53:401-426). In another example, SLFN5 is believed to play a role in regulating extracellular matrix (ECM) remodeling enzymes. Conditions of oxidative stress stimulate ECM degradation by upregulating metalloproteinases (MMPs). Upregulation of SLFN5 reduces the expression of these MMPs, thus enhancing ECM integrity.

[0029] With respect to enhancing ARE activation and / or modulating the expression of one or more of the above genes, the combination of a vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] may exhibit synergy of 1.2-fold or greater (e.g., 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or even greater than 2.0) relative to the sum of the responses from niacinamide, pal-KTTKS, and ac-PPYL treatments individually, such as a vehicle control or a predetermined threshold. In some cases, the composition may include a weight ratio of vitamin B3 compound to pal-KTTKS [SEQ ID NO: 1] to ac-PPYL [SEQ ID NO: 2] of 500:1:2 to 1:1:0.5 (e.g., 50:1:2 to 1:1:1). Methods for determining the synergy coefficient are described in more detail below.

[0030] The skin care compositions herein may be cosmetic compositions, pharmaceutical compositions, or cosmeceutical compositions and may be provided in a variety of product forms, including, but not limited to, solutions, suspensions, lotions, creams, gels, toners, sticks, sprays, aerosols, ointments, cleansing liquids and bars, pastes, foams, mousses, shaving creams, wipes, strips, patches, powered patches, hydrogels, film-forming products, facial and skin masks (with and without insoluble sheets); makeup such as foundations, eyeliners, and eye shadows; and the like. In some cases, the form of the composition may depend on the particular dermatologically acceptable carrier selected. For example, the composition (and carrier) may be provided in the form of an emulsion (e.g., water-in-oil, oil-in-water, or water-in-oil-in-water) or an aqueous dispersion.

[0031] The compositions herein can be prepared by conventional methods for making topical skin care compositions. Such methods typically involve mixing the ingredients in one or more steps to a relatively uniform state, with or without the use of heating, cooling, application of vacuum, etc. The compositions are preferably formulated to optimize stability (physical stability, chemical stability, photostability) and / or delivery of the active agent. This optimization can include an appropriate pH (e.g., below 7), excluding materials that may complex with the active agent and adversely affect stability or delivery (e.g., excluding tramp iron), using techniques to prevent complexation (e.g., appropriate dispersants or double-compartment packaging), and using appropriate photostability techniques (e.g., sunscreen / sunscreen formulations, use of opaque packaging), etc.

[0032] Vitamin B3 compounds The compositions herein contain a safe and effective amount of a vitamin B3 compound. In some examples, the compositions of the present invention may contain 0.01% to 10% by weight (e.g., 0.1% to 10%, 0.5% to 5%, or even 1% to 3%) of a vitamin B3 compound by weight or volume of the composition.

[0033] As used herein, a "vitamin B3 compound" refers to a compound having the formula:

[0034] [ka] wherein R is CONH2 (i.e., niacinamide), COOH (i.e., nicotinic acid), or CH2OH (i.e., nicotinyl alcohol), their derivatives, and salts of any of the foregoing.

[0035] Exemplary derivatives of vitamin B3 compounds include nicotinic acid esters, including non-vasodilatory esters of nicotinic acid (e.g., tocopheryl nicotinate, myristyl nicotinate), nicotinamide riboside, nicotinyl amino acids, nicotinyl alcohol esters of carboxylic acids, nicotinic acid N-oxide, and niacinamide N-oxide.

[0036] Pentapeptide The compositions herein comprise a safe and effective amount of palmitoylated pentapeptide, pal-KTTKS [SEQ ID NO: 1] (INCI: Palmitoyl Pentapeptide-4). In some examples, pal-KTTKS may be present in the compositions of the invention at 0.0001% to 3% (e.g., 0.001% to 2%, 0.01% to 1%, or 0.1% to 0.5%). Pal-KTTKS is available from Sederma (France) as PROMATRIXYL.

[0037] Tetrapeptides The compositions herein include a safe and effective amount of acetylated tetrapeptide, ac-PPYL [SEQ ID NO: 2] (INCI: Acetyl Tetrapeptide-11). In some cases, ac-PPYL may be present in the compositions at 0.0001% to 3% (e.g., 0.001% to 2%, 0.01% to 1%, or 0.1% to 0.5%). ac-PPYL is available as SYNIORAGE from BASF Care Creations (New Jersey).

[0038] Dermatologically acceptable carrier The compositions herein may include a dermatologically acceptable carrier (sometimes referred to as a "carrier"). The phrase "dermatologically acceptable carrier" means that the carrier is suitable for topical application to keratinous tissue, has good aesthetic properties, is compatible with the active agents in the composition, and does not raise any undue safety or toxicity concerns. In one embodiment, the carrier is present in a concentration of about 50% to about 99%, about 60% to about 98%, about 70% to about 98%, or alternatively, about 80% to about 95% by weight of the composition.

[0039] The carrier may be in a wide variety of forms. In some cases, the form and characteristics of the carrier may be determined by the solubility or dispersibility of the components (e.g., extracts, sunscreen actives, additional ingredients). Non-limiting examples include simple solutions (e.g., aqueous or anhydrous), dispersions, emulsions, and solid forms (e.g., gels, sticks, flowable solids, or amorphous materials). In some cases, the dermatologically acceptable carrier is in the form of an emulsion having a continuous aqueous phase (e.g., oil-in-water or water-in-oil-in-water emulsion) or a continuous oil phase (e.g., water-in-oil or oil-in-water-in-oil emulsion). The oil phase of the emulsion may include silicone oils, non-silicone oils (hydrocarbon oils, esters, ethers, etc.), and mixtures thereof. The aqueous phase may include water and water-soluble ingredients (e.g., water-soluble moisturizers, conditioning agents, antibacterial agents, humectants, and / or other skin care actives). In some cases, the aqueous phase may contain components other than water, including, but not limited to, water-soluble moisturizers, conditioning agents, antimicrobial agents, humectants, and / or other water-soluble skin care actives. In some cases, the non-water components of the composition include humectants, such as glycerin and / or other polyols.

[0040] In some cases, the compositions herein are in the form of oil-in-water ("O / W") emulsions, which provide a light, non-greasy sensory feel. Suitable O / W emulsions herein may comprise greater than 50% by weight of the composition as a continuous aqueous phase, with the remainder being a dispersed oil phase. The aqueous phase may comprise 1% to 99% water, based on the weight of the aqueous phase, along with any water-soluble and / or water-miscible ingredients. In these cases, the dispersed oil phase is typically present at less than 30% by weight of the composition (e.g., 1% to 20%, 2% to 15%, 3% to 12%, 4% to 10%, or even 5% to 8%) to help avoid some of the undesirable sensory effects of oily compositions. The oil phase may comprise one or more volatile and / or non-volatile oils (e.g., vegetable oils, silicone oils, and / or hydrocarbon oils). Some non-limiting examples of oils that may be suitable for use in the present compositions are disclosed in U.S. Pat. No. 9,446,265 and U.S. Patent Application Publication No. 2015 / 0196464.

[0041] The carrier may include one or more dermatologically acceptable diluents. As used herein, "diluent" refers to a substance capable of dispersing, dissolving, or otherwise incorporating the skin care actives herein. Some non-limiting examples of hydrophilic diluents include organic hydrophilic diluents such as water, lower monohydric alcohols (e.g., C1-C4), and low molecular weight glycols and polyols, including propylene glycol, polyethylene glycol (e.g., molecular weight 200-600 g / mol), polypropylene glycol (e.g., molecular weight 425-2025 g / mol), glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, sorbitol esters, butanediol, ether propanol, ethoxylated ethers, propoxylated ethers, and combinations thereof.

[0042] Conditioning Agent The compositions herein may comprise 0.1% to 50% by weight of a conditioning agent (e.g., 0.5% to 30%, 1% to 20%, or even 2% to 15%). The addition of a conditioning agent can help provide the composition with desirable feel characteristics (e.g., a silky, smooth feel upon application). Some non-limiting examples of conditioning agents include hydrocarbon oils and waxes, silicones, fatty acid derivatives, cholesterol, cholesterol derivatives, diglycerides, triglycerides, vegetable oils, vegetable oil derivatives, acetoglyceride esters, alkyl esters, alkenyl esters, lanolin, wax esters, beeswax derivatives, sterols and phospholipids, salts, isomers, and derivatives thereof, and combinations thereof. Particularly suitable examples of conditioning agents include volatile or non-volatile silicone fluids such as dimethicone copolyol, dimethylpolysiloxane, diethylpolysiloxane, mixed C1-30 alkylpolysiloxane, phenyldimethicone, dimethiconol, dimethicone, dimethiconol, silicone crosspolymers, and combinations thereof. Dimethicone may be particularly suitable because some consumers associate the feel characteristics provided by certain dimethicone fluids with good moisturizing. Other examples of silicone fluids that may be suitable for use as conditioning agents are described in U.S. Patent No. 5,011,681.

[0043] Rheology Modifiers The compositions herein may contain 0.1% to 5% of a rheology modifier (e.g., a thickener) to provide the composition with suitable rheological and skin-feel properties. Some non-limiting examples of thickeners include crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, gums, and mixtures thereof. In particularly suitable examples, the compositions may contain a superabsorbent polymer thickener, such as sodium polyacrylate, starch-grafted sodium polyacrylate, or a combination thereof. Some non-limiting examples of superabsorbent polymer thickeners are described, for example, in U.S. Pat. No. 9,795,552.

[0044] Some consumers find that compositions that use silicone fluid as conditioning agent have undesirable greasy or heavy feel.Therefore, it may be desirable to provide a composition that does not contain or is substantially free of silicone fluid.For example, it may be desirable to adjust the amount of water in the composition, the water:oil ratio (for example, 12:1 to 1:1), and / or the ratio of water to thickener or the ratio of oil to thickener, to provide a composition that has a light and airy feel.

[0045] emulsifier When the dermatologically acceptable carrier is in the form of an emulsion, it may be desirable to include an emulsifier that provides a stable composition (e.g., does not phase separate). If included, the emulsifier may be present in an amount of 0.1% to 10% (e.g., 1% to 5%, or 2% to 4%). The emulsifier may be nonionic, anionic, or cationic. Some non-limiting examples of emulsifiers that may be suitable for use herein are disclosed in U.S. Pat. Nos. 3,755,560 and 4,421,769, and McCutcheon's Detergents and Emulsifiers, North American Edition, pp. 317-324 (1986).

[0046] Other optional ingredients The present compositions may optionally contain one or more additional ingredients commonly used in cosmetic compositions (e.g., colorants, skin care actives, anti-inflammatory agents, sunscreens, emulsifiers, buffers, rheology modifiers, combinations thereof, etc.), provided that the additional ingredients do not undesirably alter the skin health or appearance benefits provided by the present compositions. If incorporated into the compositions, the additional ingredients should be suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic reaction, etc. Some non-limiting examples of additional actives include vitamins, minerals, peptides and peptide derivatives, sugar amines, sunscreens, oil control agents, microparticles, flavonoid compounds, hair growth regulators, antioxidants and / or antioxidant precursors, preservatives, protease inhibitors, tyrosinase inhibitors, anti-inflammatory agents, moisturizers, exfoliants, skin lightening agents, sunless tanning agents, lubricants, anti-acne actives, anti-cellulite actives, chelating agents, anti-wrinkle actives, anti-atrophy actives, phytosterols and / or plant hormones, N-acyl amino acid compounds, antibacterial agents, and antifungal agents. Other non-limiting examples of additional ingredients and / or skin care actives that may be suitable for use herein are described in U.S. Patent Application Publication Nos. 2002 / 0022040, 2003 / 0049212, 2004 / 0175347, 2006 / 0275237, 2007 / 0196344, 2008 / 0181956, 2008 / 0206373, 2010 / 00092408, 2008 / 0206373, 2010 / 0239510, 2010 / 0189669, 2010 / 019634 ... Nos. 010 / 0272667, 2011 / 0262025, 2011 / 0097286, 2012 / 0197016, 2012 / 0128683, 2012 / 0148515, 2012 / 0156146, and 2013 / 0022557, as well as U.S. Patent Nos. 5,939,082, 5,872,112, 6,492,326, 6,696,049, 6,524,598, 5,972,359, and 6,174,533.

[0047] When including optional ingredients in the compositions herein, it may be desirable to select ingredients that do not complex or otherwise interact undesirably with other ingredients in the composition, particularly pH-sensitive ingredients such as niacinamide, salicylates, and peptides. When present, optional ingredients may be included in an amount of 0.0001% to 50%, 0.001% to 20%, or even 0.01% to 10% (e.g., 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) by weight of the composition.

[0048] How to use The method of the present invention includes identifying a target area of ​​skin desired to be treated and applying a composition containing an effective amount of a vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2], and optionally one or more additional skin care actives, to the target area of ​​skin. The target area of ​​skin may be on the surface of the face, such as the forehead, perioral area, chin, periorbital area, nose, and / or cheeks, or on another part of the body (e.g., hands, arms, legs, back, chest). The person or target area of ​​skin in need of treatment may exhibit obvious signs of aging skin (e.g., fine lines, wrinkles, pigmented spots). In some cases, the target area of ​​skin may not exhibit signs of skin aging, but the user may still wish to treat the target area of ​​skin if the target area of ​​skin is an area commonly exposed to higher levels of exogenous stressors (e.g., sun-exposed skin such as facial skin and arm skin). In this manner, the methods and compositions of the present invention can be used prophylactically to help slow skin aging.

[0049] The composition can be applied daily to the target area of ​​skin, and, if desired, to surrounding skin, at least once daily, twice daily, or more frequently during the treatment period. If applied twice daily, the first and second applications are separated by at least 1-12 hours. The composition is typically applied in the morning and / or at night before bedtime. The treatment period herein is ideally long enough for the vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] to improve the appearance of the skin. The treatment period can last for at least one week (e.g., about two, four, eight, or even twelve weeks). In some cases, the treatment period will extend for several months (i.e., three to twelve months). In some cases, the composition can be applied at least once daily, or even twice daily, for the majority of days of the week (e.g., at least four, five, or six days per week) during a treatment period of at least two, four, eight, or twelve weeks.

[0050] The step of applying the composition can be performed by topical application. The terms "topical," "topical," or "topically," with respect to applying a composition, refer to delivering the composition to a target area (e.g., a pigmented spot or portion thereof) while minimizing delivery to skin surfaces where treatment is not desired. The composition can be applied to a skin area and gently massaged in. The form of the composition or dermatologically acceptable carrier should be selected to facilitate topical application. While certain embodiments herein contemplate applying the composition to a region topically, it will be understood that the compositions herein can also be applied systemically or broadly to one or more skin surfaces. In certain embodiments, the compositions herein can be used as part of a multi-step cosmetic regimen, in which the composition can be applied before and / or after one or more other compositions.

[0051] ARE assay This method provides a way to quantify ARE activation using the ARE-32 reporter cell line available from CXR Biosciences. The ARE-32 cell line is a stable MCF7 cell line containing pGL8x-ARE (eight copies of the rat GST ARE ligated to a luciferase gene) and pCDNA3.1, which contains a neomycin selectable marker. A detailed description of the ARE-32 cell line and its development and use can be found in "Generation of a Stable Antioxidant Response Element-Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents." Cancer Res 2006;66(22):10983-94. A general schematic of how the ARE reporter assay works to identify agents that promote transcription from the ARE is described in U.S. Patent Application Publication No. 2011 / 0262570.

[0052] Summary of the method: The ARE assay uses expanded and cryopreserved passage stocks. Cells are grown in culture flasks for 4-5 days and passaged every 3-4 days when the cells are approximately 80% confluent. When the cells are >70% confluent or ready to be seeded into 96-well plates, they are trypsinized, seeded into 96-well plates, and expanded. After 1 day of growth in the 96-well plates, the medium is replaced with fresh treatment medium (phenol red-free, FBS-free), and the cells are treated with compound and incubated overnight (24 hours). After treatment, the cells are rinsed and lysed with 1x PBS, luciferase kit reagent is added, and luminescence is measured.

[0053] Device: Biological safety cabinet Multichannel pipettes Inverted microscope ·Water bath Benchtop Centrifuge Incubator Plate reader (capable of reading luminescence) Corning 3275 cell culture flask (or equivalent) Pipettes and pipette controllers (e.g. Pipet Boy, Drummond Pipet) Aspirators that use a pipette tip and hook to apply a vacuum 96-well plate (Costar, catalog number 3903 or 3610)

[0054] Reagents and materials: Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, catalog number 11054-020) Fetal Bovine Serum One Shot, Heat-Inactivated (Gibco, Cat. No. A31604) Geneticin G418 sulfate (G418) (Gibco, catalog number 10131-027) Penicillin-streptomycin 100X (Gibco, catalog number 15140-148) GlutaMAX Supplement 100X (Gibco 35050-061) 0.25% Trypsin EDTA (Gibco 25200-056) If counting cells using a hemocytometer, use 0.4% trypan blue. 1×PBS Luciferase assay system with lysis buffer (Promega, catalog number E4530)

[0055] Plating medium: 500 ml of DMEM (Gibco, catalog number 11054-020) 0.8mg / ml G418 5ml of GlutaMAX 50ml FBS 5ml Pen / Strep

[0056] Processing medium: 500 ml of DMEM (Gibco, catalog number 11054-020) 5ml of GlutaMAX 5ml Pen / Strep

[0057] Starting a new culture: Quickly thaw frozen AREC32 cells (frozen in 90% FBS and 10% DMSO) and place them in a 50 ml conical tube with 25 ml of plating medium. Centrifuge at 1500-2000 RPM for 5 minutes. Remove the medium without disturbing the cell pellet. Resuspend the cells in 12 ml of medium and add to a T-75 tissue culture flask. Cells should be >80% confluent in 4-5 days. If more cells are needed, they can be split 1:3 into a T-150 flask for expansion to seed plates.

[0058] Cell maintenance and subculture: Maintain and plate cells in 96-well plates containing plating medium. Subculture (passage cells) every 3-4 days or when approximately 75-80% confluent. To passage cells, aspirate the medium from the flask and add 6 ml of 0.25% trypsin. Tilt the flask on all sides to distribute the trypsin to the bottom of the flask and place in the incubator. After 2-3 minutes, observe the cells under a microscope to see if they have detached. If the cells are not completely detached, place in the incubator for another 2-3 minutes. Once detached, neutralize the trypsin by adding 6 ml of maintenance medium and pipette into a centrifuge tube. Centrifuge at 1200 RPM for 5 minutes. Remove the medium without disturbing the cell pellet. Resuspend the pellet in 12 ml of maintenance medium and add to the flask. If splitting or separating cells from a T-150 flask, double all volumes and follow the same procedure.

[0059] Cell plating: Aspirate the medium from the flask and add the appropriate volume of 0.25% trypsin depending on the flask size (6 ml for a T-75 flask, 12 ml for a T-150 flask). Swirl the flask to distribute the trypsin to the bottom of the flask and return it to the incubator for 2-3 minutes. Observe the cells under a microscope. If the cells are not completely detached, return it to the incubator for an additional 2-3 minutes. Once the cells are detached, add DMEM equal to the volume of trypsin, mix gently, and place in a centrifuge tube. Centrifuge at 12,000 RPM for 5 minutes. Aspirate the medium without disturbing the cell pellet. Resuspend the pellet in 10 ml of DMEM. Dilute the cells 1:5 using 50 μl of cells + 200 μl of DMEM. Use 20 μl of this dilution and 20 μl of 0.4% trypan blue. Mix and add 10 μl per chamber to a disposable hemocytometer and insert into the Countess II Instrument. Nonviable cells will be blue, while viable cells will remain unstained. Cell seeding density can vary from approximately 10,000 cells / well or higher depending on cell passage and expansion. Cell density should be approximately 70-80% on the day of treatment / administration. On day 2, replace plating medium with treatment medium but do not add pen / strep. Medium must be phenol red-free.

[0060] Positive control: tert-Butylhydroquinone (tBHQ) (Aldrich, Catalog No. 11,294-1). Prepare a 100 mM stock (1M = 166.21 mg / ml, 100 mM = 16.62 mg / ml). Dilute to 750 μM (1:133) using 30 μl of 100 mM tBHQ and 3.62 ml of treatment medium. 2 μl of positive control should result in a final concentration of 7.5 μM in the test wells.

[0061] Test materials: Test materials can be prepared in DMSO or water, with the final concentration of DMSO not exceeding 1%.

[0062] procedure: Cell preparation and treatment: 1. In a 96-well plate, plate 1.5 x 10 cells in 100 µl of plating medium. 4 Seed cells / well. 2. After 15 minutes at room temperature, place the cells in an incubator. 3. Incubate the cells at 37°C, 5% CO2, and 95% humidity for 24 hours. 4. Replace the medium with 99 μl of treatment medium and treat with test compound (2 μl / well), vehicle control (2 μl / well), and positive control tBHQ (2 μl / well). 5. Add 99 μl of media after treatment (final assay volume 200 μl). Adding half of the media after dosing ensures better distribution of the material. 6. Incubate the cells for an additional 24 hours at 37°C, 5% CO2, and 95% humidity. 7. Remove the medium and wash the cells once with 100 μl of 1x PBS buffer. Remove the PBS and follow the instructions for the luciferase assay.

[0063] Luciferase assay: Prepare 1× lysis reagent by adding 1.4 volumes of water to 1 volume of 5× lysis reagent. 2. Add 20 μl of lysis buffer per well. Gently shake the plate to distribute the buffer among the wells. Place the plate in an 80°C freezer for 15 minutes to facilitate lysis. Allow the plate to thaw completely and check for lysis under a microscope. 3. Prepare the luciferase assay reagent by adding luciferase assay buffer to a vial of lyophilized luciferase assay substrate according to the manufacturer's instructions. Mix gently. Add 100 μl of luciferase assay reagent per well. Ensure there are no air bubbles. 4. Read the plate immediately in a plate reader (e.g., a Synergy™ Neo2 brand microplate reader available from BioTek).

[0064] Data calculation: ARE activation value = luminescence of test sample / (mean luminescence of vehicle control) Vehicle control = average cells + DMSO or water (n=8)

[0065] Gene regulation assays This method provides a way to measure the ability of a compound or agent to modulate the expression of a target gene. Cell: tert keratinocyte (tKC) BJ fibroblasts Plating: Cells are plated the day before treatment. For tert keratinocytes, 100,000 cells / well in 2 ml of medium / well for 12-well plates (e.g., collagen I coated plates, Corning, catalog no. 356500), or 50,000 cells / well in 1 ml of medium / well for 24-well plates. For BJ fibroblasts, 88,000 cells / well in 2 ml of medium / well for 12-well plates (e.g., Corning, Cat. No. 3512) or 44,000 cells / well in 1 ml of medium / well for 24-well plates. Culture medium: For tert keratinocytes, EpiLife (e.g., Thermo Fisher Scientific, catalog number MEPI500CA) + HKGS (e.g., Thermo Fisher Scientific, catalog number S-001-5). For BJ fibroblasts, EMEM (e.g., ATCC catalog number 30-2003) + 10% FBS (e.g., HyClone, catalog number SH30071.02).

[0066] Wafergen process: total RNA purification and qPCR The cell lysate is thawed at 4°C and then isolated using Biomek FxP and RNAdvance Tissue Isolation Kit (Beckman Coulter, p / n A32646). The resulting RNA is quantified using a Nandrop 8000 (Nanodrop, ND-8000). cDNA is generated using 500 ng of total RNA and Applied Biosystems High Capacity cDNA with a reverse transcription kit (Applied Biosystems p / n 4368814). Dilutions of cDNA, assay, and PrimeTime GeneExpression MasterMix (IDT, p / n 1055771) are plated onto a Wafergen MyDesign SmartChip (TakaraBio, p / n 640036) using a Wafergen Nanodispenser. The chip is then loaded into a SmartChip cycler, and qPCR is performed using the following PCR conditions:

[0067] Hold step: 50°C for 2 min (warm-up), then 95°C for 10 min; PCR step (40 cycles): 95°C for 15 seconds, then 60°C for 1 minute.

[0068] Export the data in .txt file format for analysis. [Example]

[0069] Example 1: Formulations. Table 1 below provides an example of a skin care composition of the present invention. This exemplary composition is prepared by blending the Phase A ingredients in a suitable mixer (e.g., a Tekmar RW20DZM or equivalent), heating to a temperature of 70-80°C, and maintaining that temperature while mixing. Separately, the Phase B ingredients are blended in a suitable mixer and heated to 70-75°C while maintaining the temperature while mixing. Phase B is added to Phase A and mixed thoroughly at the same time to form an oil-in-water (O / W) emulsion. The emulsion is then milled for 5 minutes using a suitable mill (e.g., a Tekmar T-25 or equivalent). When the emulsion is at 60°C, Phase C is added while continuing to mix. At 40°C, the ingredients of Phases D and E are added to the emulsion. The emulsion is then milled for 5 minutes to obtain a uniform composition.

[0070] [Table 1-1]

[0071] [Table 1-2]

[0072] Example 2: Vitamin B3 compounds, Pal-KTTKS [SEQ ID NO: 1], and Ac-PPYL [SEQ ID NO: 2] synergistically upregulate NRF2 [SEQ ID NO: 4]. This example demonstrates the ability of the combination of niacinamide, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] to synergistically activate the ARE. Test and control compositions were prepared as described above in the ARE assay and tested accordingly. The vitamin B3 compound used in this example is niacinamide [Sigma, Catalog No. N5535], pal-KTTKS is PROMATRIXYL from Sederma (France), and ac-PPYL is SYNIORAGE from BASF Care Creations (New Jersey). The results of the test are summarized in Table 2 below. N+P+A refers to the combination of niacinamide (N), pal-KTTKS (P) [SEQ ID NO: 1], and ac-PPYL (A) [SEQ ID NO: 2].

[0073] The synergy coefficient is calculated as follows:

[0074]

number

[0075] A synergy coefficient greater than 1.00 with a p-value < 0.05 indicates a statistically significant synergistic effect. A preferred synergy coefficient is greater than 1.3.

[0076] [Table 2]

[0077] As seen in Table 2, the data suggest that ratios of niacinamide to pal-KTTKS [SEQ ID NO:1] to ac-PPYL [SEQ ID NO:2] (N:P:A) of 500:1:2 to 1:1:0.5 synergistically activate the ARE. However, N:P:A ratios of 500:1:0.05, 50:1:2, 10:1:0.05, 1:1:2, and 1:1:0.05 do not appear to provide synergistic factors greater than 1.3. Therefore, selecting the correct combination of vitamin B3 compounds, pal-KTTKS [SEQ ID NO:1], and ac-PPYL, as shown in Table 2, to provide the desired synergistic effect may be important.

[0078] Example 3: Tetrapeptide specificity required for synergy. This example demonstrates the importance of selecting a specific tetrapeptide to provide the desired synergistic activation of the ARE. In this study, amino acids from ac-PPYL [SEQ ID NO: 2] were rearranged to form a new tetrapeptide, ac-YPLP [SEQ ID NO: 3]. Test and control compositions were prepared as described in the ARE assay above and tested accordingly. The test results are summarized in Table 3 below.

[0079] [Table 3]

[0080] Surprisingly, as can be seen in Table 3, a tetrapeptide with the same amino acids as ac-PPYL [SEQ ID NO: 2] but arranged in a different order does not provide the desired synergistic effect. These data suggest that the specific peptide sequence is important for providing the desired synergistic effect.

[0081] Example 4: Synergistic upregulation of genes involved in cellular antioxidant and repair. This example demonstrates the ability of the combination of niacinamide, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] to synergistically upregulate SLFN5 [SEQ ID NO: 4], GDE1 [SEQ ID NO: 5], MINPP1 [SEQ ID NO: 6], and HMGCL [SEQ ID NO: 7], which are involved in cellular antioxidant and repair processes. Test and control compositions were prepared as described in the gene regulation assay above and tested accordingly. The pal-KTTKS used in this example is PROMATRIXYL brand pal-KTTKS from Sederma (France), and ac-PPYL is SYNIORAGE brand tetrapeptide from BASF Care Creations (New Jersey). The results of the test are summarized in Table 4 below. The fold changes shown in Table 4 are based on the combination (N+P+A) versus the sum of the individual treatments (N, P, and A). A p-value of 0.05 or less is considered significant.

[0082] [Table 4]

[0083] Combination examples A. A skin care composition comprising: 1) a combination of a vitamin B3 compound, palmitoyl pentapeptide-4 (pal-KTTKS) [SEQ ID NO: 1], and acetyl tetrapeptide-11 (ac-PPYL) [SEQ ID NO: 2], wherein the combination of the vitamin B3 compound, pal-KTTKS, and ac-PPYL increases activation of the antioxidant response element (ARE) in cells by an ARE assay; 2) a dermatologically acceptable carrier. B. The skin care composition described in paragraph A, wherein the combination of a vitamin B3 compound, pal-KTTKS, and ac-PPYL synergistically enhances activation of the ARE. C. The composition described in paragraphs A or B, wherein the combination of the vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] exhibits a synergy coefficient of at least 1.3. D. The composition of any of the preceding paragraphs, wherein the combination of a vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] synergistically upregulates at least one gene selected from the group consisting of Nuclear Factor E2-Related Factor 2 (NRF2) [SEQ ID NO: 3], Schlafen family member 5 (SLFN5) [SEQ ID NO: 4], glycerophosphodiester phosphodiesterase 1 (GDE1) [SEQ ID NO: 5], multiple inositol-polyphosphate phosphatase 1 (MINPP1) [SEQ ID NO: 6], and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) [SEQ ID NO: 7]. E. The composition of any of the preceding paragraphs, wherein the vitamin B3 compound is present at 0.05% to 10% by weight of the composition. F. The composition of any of the preceding paragraphs, wherein the vitamin B3 compound is selected from the group consisting of niacinamide, nicotinic acid, nicotinyl alcohol, and combinations thereof. G. The composition of paragraph F, wherein the vitamin B3 compound is niacinamide. The composition of any of the preceding paragraphs, wherein H.pal-KTTKS [SEQ ID NO: 1] is present at 0.0001% to 2% by weight of the composition. I. A composition described in any of the preceding paragraphs, wherein ac-PPYL [SEQ ID NO: 2] is present at 0.0001% to 2% by weight of the composition. J. The composition of any of the preceding paragraphs, wherein the ratio of vitamin B3 compound to pal-KTTKS [SEQ ID NO: 1] to ac-PPYL [SEQ ID NO: 2] is 500:1:2 to 1:1:0.5. K. The composition of any of the preceding paragraphs, further comprising at least one additional ingredient selected from vitamins, minerals, peptides, sugar amines, sunscreens, oil control agents, flavonoid compounds, antioxidants, protease inhibitors, tyrosinase inhibitors, anti-inflammatory agents, moisturizers, exfoliants, skin lightening agents, anti-acne agents, anti-wrinkle agents, phytosterols, N-acyl amino acid compounds, antibacterial agents, antifungal agents, pH adjusters, thickeners, preservatives, and combinations thereof. L. A method for treating oxidative stress in the skin, comprising: Identifying a target area of ​​skin where treatment is desired; applying thereto the skin care composition of any of the preceding paragraphs. M. The method of paragraph L, wherein the composition improves the appearance of visible signs of skin aging. N. A method for upregulating NRF2 [SEQ ID NO: 4] in skin cells, comprising contacting the skin cells with effective amounts of a vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] in combination, wherein the effective amounts of the vitamin B3 compound, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] synergistically increase activation of antioxidant response elements by ARE assay.

[0084] Dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact numerical values ​​recited. Instead, unless otherwise indicated, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm."

[0085] All documents cited herein, including any cross-referenced or related patents or patent applications, and any patent applications or patents to which this application claims priority or benefit, are incorporated herein by reference in their entirety, unless expressly stated to the contrary. The citation of any document shall not be deemed to be prior art to any invention disclosed or claimed herein, or to teach, suggest, or disclose any such invention, either alone or in combination with any other reference(s). Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0086] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

1. 1. A skin care composition comprising: a) a combination of niacinamide, palmitoyl pentapeptide-4 (pal-KTTKS) [SEQ ID NO: 1], and acetyl tetrapeptide-11 (ac-PPYL) [SEQ ID NO: 2], wherein the combination of niacinamide, pal-KTTKS, and ac-PPYL synergistically increases the activity of a cellular antioxidant response element (ARE) according to an antioxidant response element (ARE) assay, exhibiting a synergy factor of at least 1.3; b) a dermatologically acceptable carrier; A skin care composition, wherein the ratio of the niacinamide to the pal-KTTKS [SEQ ID NO: 1] and to the ac-PPYL [SEQ ID NO: 2] is from 500:1:2 to 1:1:0.

5.

2. The composition of claim 1, wherein the combination of niacinamide, pal-KTTKS [SEQ ID NO: 1], and ac-PPYL [SEQ ID NO: 2] synergistically upregulates at least one gene selected from the group consisting of nuclear factor erythroid 2-related factor 2 (NRF2) [SEQ ID NO: 3], Schlafen family member 5 (SLFN5) [SEQ ID NO: 4], glycerophosphodiester phosphodiesterase 1 (GDE1) [SEQ ID NO: 5], multiple inositol-polyphosphate phosphatase 1 (MINPP1) [SEQ ID NO: 6], and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) [SEQ ID NO: 7].

3. 10. The composition of claim 1, wherein the niacinamide is present at 0.05% to 10% by weight of the composition.

4. 2. The composition of claim 1, wherein said pal-KTTKS [SEQ ID NO: 1] is present at 0.0001% to 2% by weight of said composition.

5. 2. The composition of claim 1, wherein the ac-PPYL [SEQ ID NO: 2] is present at 0.0001% to 2% by weight of the composition.

6. 10. The composition of claim 1, further comprising at least one additional ingredient selected from vitamins, minerals, peptides, sugar amines, sunscreens, oil control agents, flavonoid compounds, antioxidants, protease inhibitors, tyrosinase inhibitors, anti-inflammatory agents, moisturizers, exfoliants, skin lightening agents, anti-acne agents, anti-wrinkle agents, phytosterols, N-acyl amino acid compounds, antibacterial agents, antifungal agents, pH adjusters, thickeners, preservatives, or combinations thereof.

7. 1. A non-therapeutic method of treating oxidative stress in the skin, comprising: Identifying a target area of ​​skin where treatment is desired; applying thereto a skin care composition according to any one of claims 1 to 6; A method comprising:

8. 8. The method of claim 7, wherein the method improves the appearance of visible signs of skin aging over the course of a treatment period.

9. 9. The method of claim 8, wherein the treatment period is at least two weeks.

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