Bioactive nut peptides and compositions for topical skin treatment

Abstract

Bioactive Walnut Peptides and Compositions for Topical Skin Treatment Bioactive walnut peptides and a use of the bioactive walnut peptides for topical application to the skin are described. The bioactive walnut peptides, which may be incorporated into a pharmaceutical or cosmetic composition, are useful for treating, preventing, and improving skin damage and combating or preventing physiological changes associated with skin aging. Figure for abstract: none

Description

Title of Invention: Bioactive Walnut Peptides and Compositions for Topical Skin Treatment FIELD OF DISCLOSURE

[0001] The present disclosure relates to bioactive walnut peptides and the use of the walnut peptides for topical application to the skin. The walnut peptides and compositions comprising them are useful for improving the appearance of the skin, preventing or reversing skin aging, and treating various skin conditions associated with aging. CONTEXT

[0002] The skin is a unique and complex organ that extends throughout the body. It is essential for protecting the body from external threats, controlling body temperature, and preventing moisture loss. It also serves as a sensory organ and produces vitamin D. There are different types of skin in different parts of the body. For example, the skin on the face is different from that of the scalp, and even the skin on the palm of the hand is different from the skin on the back of the hand.

[0003] The skin constitutes the integumentary system, which includes three layers: the epidermis, the dermis, and the hypodermis. The epidermis is the outermost layer of the skin and the first line of defense against external threats such as microbes. The human epidermis is primarily composed of keratinocytes containing other cell types, including melanocytes and Langerhans cells. Each of these cell types contributes, through its specific function, to the essential role played by the skin. Melanocytes impart a characteristic color and tone to the skin. The epidermis is essentially impermeable to water and therefore helps maintain hydration and prevents the body from absorbing water when bathing. Unlike other cells, epidermal cells have a unique ability to regenerate and can therefore heal after being injured.

[0004] The middle layer of the skin that lies just beneath the epidermis is the dermis, which includes connective tissue, collagen, elastin, hair follicles, lymph nodes, and sweat glands. The dermis provides a strong support for the epidermis. It is also its nourishing layer. The dermis primarily includes fibroblasts, but leukocytes, mast cells, or tissue macrophages are also present. The dermis also contains blood vessels and nerve fibers. The acellular portion (i.e., the area between the cells) of the dermis is called the extracellular matrix. The extracellular matrix of the skin is composed of various extracellular components, including proteins, particularly collagen fibers and elastin. Other components of the extracellular matrix of the skin include glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, etc.), proteoglycans (e.g., fibromodulin, decorin, biglycan, perlecan, heparan sulfate proteoglycan 2, agrin, versican, aggrecan, lumican, collagen type IX, collagen type XII, collagen type XIV, testican 1, testican 2, etc.), and various glycoproteins (e.g., fibrillin 1, thrombospondin-1 and -2, tenascin-C and -X, osteopontin, fibronectin, laminin-5 and -6, vitronectin, etc.). These extracellular components are synthesized by dermal fibroblasts, which make them the main constituent of the structural assembly of the dermis.

[0005] The extracellular matrix is ​​a highly heterogeneous amalgam of morphologically diverse architectural entities. It organizes and confers structural integrity to individual tissues, in addition to modulating cellular behavior by interacting with cell surface receptors and soluble growth factors. Dysfunctions and changes in extracellular matrix components can therefore interfere with both tissue integrity and cellular performance. Dysfunctions and changes in extracellular matrix components of skin and mucosa in humans can lead to skin aging, skin atrophy, skin damage, skin wounds, and other problems.

[0006] The hypodermis is the deepest layer of the skin and is composed primarily of fatty and connective tissue. It connects the skin to the underlying fascia (tissue) of bones and muscles. The hypodermis is not always classified as an official layer of the skin, but it nevertheless performs important functions such as storing energy, insulating the body, and preventing skin sagging. The hypodermis houses fat storage cells that function as energy reservoirs and act as a cushion against impacts.

[0007] Over time, skin ages, which can lead to wrinkles, brown spots, and a loss of elasticity and volume. Skin aging includes both chronological aging (endogenous aging), which is natural aging that occurs over time due to natural biological processes, and degenerative aging due to external influences such as exposure to harmful sunlight, pollution, lack of hydration, etc. Ultraviolet rays such as UVB have a wavelength in the range of 280 nm to 320 nm and are known to cause skin damage and accelerate aging. UVB rays stimulate reactive oxygen species (ROS) and free radicals in skin cells, which which accelerates the intracellular signaling system and induces oxidative stress on biomolecules such as DNA, proteins, and lipids. An increase in oxidative stress of skin cells can cause stimulation of epidermal keratinocytes or dermal fibroblasts, and through a series of intracellular signal transduction, the expression of genes such as matrix metalloproteinase (MMP), a collagen-degrading enzyme, can be increased.

[0008] There are many treatments available for treating the skin that promote anti-aging benefits. For example, the importance of collagen in the aging process has led to the development of many topical products containing collagen. Other components such as retinoic acid, vitamin C, and hyaluronic acid are used in topical formulations to stimulate collagen synthesis. Some cosmetic procedures, such as laser skin resurfacing, are used to reduce facial wrinkles and skin irregularities. Topical administration of proteins known to inhibit one or more signaling pathways that exhibit altered activity after UV exposure has been explored. However, there are difficulties associated with the use of whole proteins, which cannot be effectively delivered across the epidermis.Additionally, large proteins can be unstable, difficult to formulate, and not effective topically. Despite the number of products and treatments proposed, none of them satisfactorily address the intrinsic mechanisms of skin repair and renewal. SUMMARY OF DISCLOSURE

[0009] The present disclosure relates to bioactive walnut peptides and the use of the walnut peptides for topical application to the skin. The bioactive walnut peptides, which can be incorporated into a pharmaceutical or cosmetic composition, treat and prevent conditions that negatively impact skin aging. Furthermore, topical application of the bioactive walnut peptides to the skin surprisingly reduces age-related inflammation, increases epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor alpha (TGFa), promotes epidermal stem cell expression, induces autophagy and improves mitochondrial function. It has been surprisingly found that the bioactive walnut peptides influence multiple physiological pathways beneficial to the skin.Due to the plethora of benefits, bioactive walnut peptides and the pharmaceutical and cosmetic composition containing them are particularly suitable for the prevention and treatment of skin aging and for improving or reversing the effects of skin aging.

[0010] Walnuts are one of the most widespread and oldest nuts in the world. They have high nutritional value and are rich in oleic acid, linoleic acid, a-linolenic acid and other unsaturated fatty acids, vitamins and proteins. Walnuts are commonly used to make walnut oil because they contain a high lipid content. The residue remaining after lipid extraction is considered a by-product although it contains nut protein and other useful components. Walnut protein is mainly composed of albumin, globulin, gliadin and glutenin.

[0011] The bioactive nut peptides may be derived from nut proteins or may be synthesized. For example, the nut peptides are obtained by enzymatic hydrolysis, fermentation hydrolysis, or chemical hydrolysis of nut proteins or produced synthetically, for example, by solid-phase synthesis. The bioactive peptides that are particularly useful according to the present disclosure typically have a molecular weight of less than 6,000 Da and often less than 1,000 Da. These nut peptides may have from 2 to 50 amino acid residues, but typically have from 2 or 3 amino acid residues to about 20 amino acid residues.

[0012] Preferably, the bioactive nut peptides have a minimum of 2 or 3 amino acid residues up to about 20 amino acid residues and confer a positive physiological or dermatological effect on skin cells. Bioactive peptides include amino acids joined by covalent bonds, also referred to as amidic or peptide bonds, while proteins are higher molecular weight (MW) polypeptides, i.e., having more than 50 amino acid residues. Bioactive nut peptides generally exhibit hormonal or drug-like activities and are classified according to their mode of action. Many bioactive peptides share certain structural features, for example, an amino acid residue length for 2 to 20 amino acids.

[0013] The bioactive walnut peptides useful according to the present disclosure often include one or more amino acid residues selected from leucine, proline, or combinations thereof. In other embodiments, the walnut peptides include three or more amino acid residues selected from leucine, proline, or combinations thereof. Non-limiting examples of amino acid residues within the walnut peptides that include leucine and proline include Leu-Pro-Leu (LPP), Leu-Leu-Pro, Pro-Pro-Leu (PPL), and Pro-Leu-Pro (PLP). Other non-limiting examples of amino acid sequence that may be included in the bioactive nut peptides include Thr-Trp-Leu-Pro-Leu-Pro-Arg (TWLPLPR), Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys (YVLLPSPK), Lys-Val-Pro-Pro-Leu-Leu-Tyr (KVPPLLY), and combinations thereof.

[0014] In various embodiments, one or more of the following nut peptides are preferred:

[0015] Peptide I Thr-Trp-Leu-Pro-Leu-Pro-Arg (TWLPLPR),

[0016] Peptide II Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys (YVLLPSPK), and / or

[0017] Peptide III Lys-Val-Pro-Pro-Leu-Leu-Tyr (KVPPLLY).

[0018] As already mentioned, bioactive nut peptides are particularly useful for topical application to the skin. Accordingly, the present disclosure relates to the use of walnut peptides in methods of treating the skin. In various embodiments, the one or more walnut peptides are applied to the skin in a pharmaceutical or cosmetic composition, which typically includes a physiologically acceptable carrier, e.g., water and optionally water-soluble solvents. The pharmaceutical or cosmetic composition includes an amount of the one or more bioactive walnut peptides sufficient to ensure that a therapeutically effective amount of the one or more walnut peptides is delivered to the skin during use.

[0019] Bioactive walnut peptides are useful for treating age-related inflammation. For example, bioactive walnut peptides surprisingly and beneficially reduce, treat, or prevent pro-inflammatory cytokines in the skin. Pro-inflammatory cytokines are among the first factors to be produced in response to skin damage, and regulate the functions of immune cells in epithelialization. In various embodiments, the bioactive peptides of the present disclosure prevent or downregulate the production, release, or abundance of interleukin-6 (IL-6), interleukin-8 (IL-8), or a combination thereof.

[0020] Bioactive walnut peptides are useful in stimulating skin cells called fibroblasts, which produce collagen and elastin to clarify, thicken, and firm the skin. Growth factors are extremely important for regulating cellular processes in the human body, including the skin. The bioactive walnut peptides of the present disclosure are useful in stimulating the production, release, and abundance of epidermal growth factor (EGF). EGF is a restorative compound that signals cells to stimulate the production of collagen and elastin. This helps repair and renew cells, resulting in a brighter, more youthful complexion. In addition, bioactive walnut peptides are useful in stimulating the production, release, and abundance of hepatocyte growth factor (HGF).Increasing HGF improves tissue fibrosis and reverses the imbalance in collagen metabolism. Finally, bioactive walnut peptides are useful in potentiating the production, release, and abundance of transforming growth factor alpha (TGFa or TGF-a). TGFa is a factor of . transforming growth factor that functions as a ligand for the epidermal growth factor receptor, which activates a signaling pathway for cell proliferation, differentiation, and development. Therefore, an increase in TGFα may accelerate skin rejuvenation and repair.

[0021] In other embodiments, the bioactive walnut peptides of the present disclosure are useful for potentiating, inducing, and increasing autophagy in the skin, for example, in senescent fibroblasts. Senescent cells are characterized by their inability to proliferate. They tend to accumulate with age and contribute to age-related changes and pathologies of the skin. An induction of autophagy allows the body to destroy and metabolize damaged or redundant cellular components that occur in vacuoles within cells. A natural reduction in autophagy occurs with age. Thus, the restoration or induction of autophagy helps improve skin health and prevent premature aging of the skin, for example, aging of the skin due to sun damage.

[0022] Due to their bioactive properties, including those described above, bioactive walnut peptides are useful in methods of improving the appearance of the skin; reducing, treating or preventing age-related inflammation of the skin and skin conditions associated with age-related inflammation; promoting fibroblast migration; potentiating hyaluronic acid synthesis, increasing or promoting synthesis or abundance of collagen, elastin and / or fibronectin; improving skin elasticity; promoting skin autophagy; and combinations thereof.

[0023] In various embodiments, the use of two or more bioactive walnut peptides is preferred. The two or more bioactive walnut peptides may have similar activities or may provide different activities beneficial to the skin. In other embodiments, the use of three or more bioactive walnut peptides is preferred. The use of multiple bioactive peptides allows for the modification of more than one physiological mechanism in the treatment of the skin. For example, one or more bioactive walnut peptides may be useful for preventing and / or treating age-related inflammation while another bioactive walnut peptide may be useful for stimulating fibroblasts that produce collagen and elastin to clarify, thicken, and firm the skin.Furthermore, combinations of bioactive nut peptides may interact synergistically and provide benefits that go beyond the sum of the individual contributions of the peptides. For example, the synergistic activity of a combination may be at least 5%, at least 10%, or at least 25% greater than the sum of the individual activities of the corresponding amounts of the bioactive nut peptides.

[0024] The one or more bioactive nut peptides are often incorporated into a pharmaceutical or cosmetic composition for application to the skin. The pharmaceutical and cosmetic composition typically includes one or more bioactive nut peptides and one or more physiologically acceptable carriers, for example, water. Non-limiting examples of physiologically acceptable carriers include water, water-soluble solvents such as alcohols, polyols, and glycols, a fatty compound such as oils, triglycerides, fatty acids, fatty alcohols, and the like. The pharmaceutical and cosmetic compositions include lotions, creams, serums, sprays, emulsions, gels, powders, dispersions, ointments, sticks, pastes, and mousses. Brief Description of the Drawings

[0025] An implementation of the present technology is described, by way of example only, with reference to the accompanying figures, in which:

[0026] [Fig.lA] Figure 1(a) shows the concentration of interleukin-6 (IL-6) released by adult human skin fibroblasts in response to treatment with 100 uM and 250 uM of peptide I, peptide II and peptide III.

[0027] [Fig.lB] Figure 1(b) shows the concentration of interleukin-8 (IL-8) released by adult human skin fibroblasts in response to treatment with 100 uM and 250 uM of peptide I, peptide II and peptide III.

[0028] [Fig.2A] Figure 2(a) shows the amounts of epidermal growth factor (EGF) generated with reconstructed human epidermis in response to treatment with 250 µM of peptide I, peptide II and peptide III, and their combinations.

[0029] [Fig.2B] Figure 2(b) shows the amounts of hepatocyte growth factor (HGF) generated by reconstructed human epidermis in response to treatment with 250 µM of peptide I, peptide II and peptide III, and their combinations.

[0030] [Fig.2C] Figure 2(c) shows the amounts of transforming growth factor alpha (TGFa) generated by reconstructed human epidermis in response to treatment with 250 uM of peptide I, peptide II and peptide III, and their combinations.

[0031] [Fig.4] [Fig.4] shows the average intensity of active mitochondria of Adult human skin fibroblasts in response to treatment with 250 μM of peptide I, peptide II, peptide III, and their combinations.

[0032] The various aspects of the disclosure are not limited to the results, arrangements and representations shown in the drawings. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure relates to bioactive walnut peptides and their topical use for treating the skin. The bioactive walnut peptides include two to several dozen amino acids linked together by peptide bonds. Their weight molecular weight is generally less than 6,000 Da, preferably less than 3,000 Da, and more preferably less than 1,000 Da. The term "peptide" in accordance with the present disclosure is a compound that includes an uninterrupted sequence of at least two amino acids within its structure and has a maximum of about 50 amino acids. The terms "di-peptide" or "dipeptide" as used herein refer to a compound that includes an uninterrupted sequence of two amino acids within its structure. The terms "tri-peptide" or "tripeptide" as used herein refer to a compound that includes an uninterrupted sequence of three amino acids within its structure. As used herein, a "tetra-peptide" or "tetrapeptide" is a compound that includes an uninterrupted sequence of four amino acids within its structure.These amino acids are listed here using a traditional one-letter convention from left (N-terminus) to right (C-terminus). In this nomenclature, G is glycine, H is histidine, K is lysine, E is glutamic acid, and the like, according to nomenclature well known and accepted in the art.

[0034] A “bioactive” peptide as used in the present disclosure has a minimum of 2 or 3 amino acid residues up to about 20 amino acid residues in length and has a measurable physiological effect on skin cells. Bioactive peptides include amino acids linked by covalent bonds, also known as amidic or peptide bonds, while proteins are polypeptides having a higher molecular weight (MW) and generally more than 50 amino acid residues. Bioactive peptides generally exhibit hormonal or drug-like activities and are classified according to their mode of action. Many bioactive peptides share certain structural features which include, for example, a peptide residue length of 2 to 20 amino acids.

[0035] The term "amino acid" as used herein includes and encompasses all naturally occurring amino acids, in the D or L configuration if optically active, and known non-native, synthetic and modified amino acids, such as homocysteine, omithine, norleucine and p-valine. A list of non-naturally occurring amino acids can be found in The Peptides, Vol. 5 (1983), Academie Press, Chapter VI, by D.C. Roberts and F. Vellaccio, which is incorporated herein by reference in its entirety. The amino acids in the peptides of the present invention may be present in their naturally occurring L configuration, their non-naturally occurring D configuration or as a racemic mixture.

[0036] As used herein, the term "peptide" also refers to salts, unprotected forms, acylated forms of the peptide, deacylated forms of the peptide, enantiomers, diastereomers, racemates, prodrugs and hydrates of the aforementioned peptide. Diastereomers of the peptide are obtained when the stereochemical or chiral center of one or more amino acids is modified. The enantiomer exhibits the opposite stereochemistry in all chiral centers. In various embodiments, the C-terminus of a peptide is synthesized as an amide to neutralize the negative charge created by the C-terminal COOH. This modification may be added to help prevent enzymatic degradation.

[0037] The term "prodrug" refers to any precursor compound that can generate or release the aforementioned peptide under physiological conditions. Such prodrugs are, for example, larger peptides that are selectively cleaved to form the peptide of the invention. Other prodrugs are protected amino acids having protecting groups at the carboxylic acid and / or amino group. Suitable protecting groups for amino groups include, for example, benzyloxycarbonyl, t-butyloxycarbonyl (BOC), formyl, and acetyl or acyl group. Suitable protecting groups for the carboxylic acid group are esters such as benzyl esters or t-butyl esters.

[0038] The present disclosure includes methods of treating skin comprising topically applying a therapeutically effective amount of one or more walnut peptides to the skin. In various embodiments, the one or more bioactive walnut peptides have a molecular weight of less than 10,000 Da. In other embodiments, the one or more bioactive walnut peptides have a molecular weight of less than 8,000 Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Da, less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, and even less than 500 Da. Preferably, the one or more bioactive walnut peptides have a molecular weight of less than 5,000 Da, more preferably less than 2,000 Da, and even more preferably less than 1,000 Da.

[0039] The number of amino acid residues in the one or more bioactive nut peptides will vary and may be limited based on the molecular weights described above. However, in various embodiments, the one or more bioactive nut peptides comprise from 2 to about 50 amino acid residues.In other embodiments, the one or more bioactive peptides have from 2 to about 25 amino acid residues, from about 2 to about 20 amino acid residues, from about 2 to about 18 amino acid residues, from about 2 to about 15 amino acid residues, from about 2 to about 12 amino acid residues, from about 2 to about 10 amino acid residues, from about 3 to about 25 amino acid residues, from about 3 to about 20 amino acid residues, from about 3 to about 18 amino acid residues, from about 3 to about 18 amino acid residues, from about 3 to about 15 amino acid residues, from about 3 to about 12 amino acid residues, or from about 3 to about 10 amino acid residues. of amino acid. Preferably, the one or more bioactive peptides comprise 2 to 20 amino acid residues, more preferably 2 to about 15 amino acid residues, even more preferably 3 to about 12 amino acid residues.

[0040] In various embodiments, the one or more bioactive nut peptides include one or more amino acid residues selected from leucine, proline, and combinations thereof. In another embodiment, the one or more bioactive nut peptides include three or more amino acid residues selected from leucine, proline, or a combination thereof. Non-limiting examples of a bioactive peptide of three or more amino acid residues selected from leucine, proline, and combinations thereof include Leu-Pro-Leu, Leu-Leu-Pro, Pro-Pro-Leu, and Pro-Leu-Pro. For example, the one or more bioactive nut peptides may comprise an amino acid sequence selected from Thr-Trp-Leu-Pro-Leu-Pro-Arg, Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys, or Lys-Val-Pro-Pro-Leu-Leu-Tyr.As already mentioned, bioactive nut peptides include salts, deprotected forms, acylated forms, deacylated forms, enantiomers, diastereomers, racemates, prodrugs and hydrates of a particular amino acid sequence.

[0041] In a preferred embodiment, at least one, and preferably all, of the one or more bioactive walnut peptides are selected from peptide I, peptide II, and peptide III, including salts, deprotected forms, acylated forms, deacylated forms, enantiomers, diastereomers, racemates, prodrugs, and hydrates thereof.

[0042] Peptide I TWLPLPR (Thr-Trp-Leu-Pro-Leu-Pro-Arg),

[0043] Peptide II YVLLPSPK (Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys), and

[0044] Peptide III KVPPLLY (Lys-Val-Pro-Pro-Leu-Leu-Tyr).

[0045] In various embodiments, the use of two bioactive walnut peptidesor more is desirable. The two or more bioactive walnut peptides may share similar activities or may provide different activities beneficial to the skin. In other embodiments, the use of three or more bioactive walnut peptides is preferred. The use of multiple bioactive peptides allows for more than one physiological mechanism to be leveraged for skin treatment. For example, one or more bioactive walnut peptides may be useful for preventing and / or treating age-related inflammation while another bioactive walnut peptide may be useful for stimulating fibroblasts that produce collagen and elastin to clarify, thicken, and firm the skin. Furthermore, as shown later, combinations of bioactive walnut peptides interact synergistically to provide benefits that go beyond the sum of the individual contributions of the peptides.For example, the synergistic activity of a combination of bioactive nut peptides may be at least 5%, preferably at least 10%, more preferably at least 25% greater than the sum of the activity of the corresponding individual amounts of the bioactive nut peptides.

[0046] Combinations of peptide I, peptide II, and peptide III are particularly useful. For example, a combination of peptide I and peptide II, a combination of peptide I and peptide III, a combination of peptide II and peptide III, or a combination of peptide I, peptide II, and peptide III may be used. The peptides in the combinations may be included in various weight ratios relative to each other as described below.

[0047] Peptide I and peptide II may be used together in a weight ratio of about 1:10 to about 10:1. In other embodiments, peptide I and peptide II may be used together in a weight ratio of about 8:1 to about 1:8, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1.

[0048] Peptide I and peptide III may be used together in a weight ratio of about 1:10 to about 10:1. In other embodiments, peptide I and peptide III may be used together in a weight ratio of about 8:1 to about 1:8, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1.

[0049] Peptide II and peptide III may be used together in a weight ratio of about 1:10 to about 10:1. In other embodiments, peptide II and peptide III may be used together in a weight ratio of about 8:1 to about 1:8, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1.

[0050] Bioactive walnut peptides are useful for treating age-related inflammation and for preventing, reducing, and even reversing the effects of skin aging. Bioactive walnut peptides beneficially reduce, treat, or prevent pro-inflammatory cytokines in the skin. Pro-inflammatory cytokines are among the first factors to be produced in response to skin damage and regulate immune cell functions in epithelialization. In various embodiments, the bioactive peptides of the present disclosure prevent the production, release, or abundance of interleukin-6 (IL-6), interleukin-8 (IL-8), or a combination thereof.

[0051] Bioactive walnut peptides are useful for stimulating skin cells called fibroblasts, which produce collagen and elastin to clarify, thicken, and firm the skin. Growth factors are extremely important for regulating cellular processes in the human body, including the skin. In particular, the bioactive walnut peptides of the present disclosure are useful for potentiating the production, release, and abundance of epidermal growth factor (EGF). EGF is a restorative compound that signals cells to stimulate the production of collagen and elastin. This Helps repair and renew cells, resulting in a brighter, more youthful complexion. Additionally, bioactive walnut peptides are also helpful in potentiating the production, release, and abundance of hepatocyte growth factor (HGF). Increasing HGF improves tissue fibrosis and reverses imbalance in collagen metabolism. Finally, bioactive walnut peptides are helpful in potentiating the production, release, and abundance of transforming growth factor alpha (TGFa or TGF-a). TGF-a is a transforming growth factor that functions as a ligand for the epidermal growth factor receptor, which activates a signaling pathway for cell proliferation, differentiation, and development. Therefore, an increase in TGF-a can accelerate skin rejuvenation and repair.

[0052] In other embodiments, the bioactive walnut peptides of the present disclosure are useful for potentiating, inducing, or increasing autophagy, for example, in senescent fibroblasts. Senescent cells are characterized by their inability to proliferate. They tend to accumulate with age and contribute to age-related changes and pathologies of the skin. An induction of autophagy allows the body to destroy and metabolize damaged or redundant cellular components that occur in vacuoles within cells. A natural reduction in autophagy occurs with age. Thus, restoring autophagy using the bioactive peptides of the present disclosure helps improve or prevent premature aging of the skin, for example, aging of the skin due to sun damage.

[0053] Due to their bioactive properties, including those described above, bioactive walnut peptides are useful in methods of improving the appearance of skin; reducing, treating and / or preventing skin aging and / or the effects of skin aging; increasing fibroblast proliferation; stimulating epithelial cell proliferation, motility, morphogenesis and / or angiogenesis; promoting fibroblast migration; increasing the abundance or synthesis of hyaluronic acid, and / or increasing the synthesis or abundance of collagen, elastin and / or fibronectin; improving skin elasticity; and / or enhancing autophagy in skin fibroblasts.

[0054] The one or more bioactive walnut peptides are often incorporated into a pharmaceutical or cosmetic composition for application to the skin. Pharmaceutical and cosmetic compositions typically include one or more bioactive walnut peptides and a physiologically acceptable carrier. A "physiological carrier" as used herein is a carrier suitable and safe for application to the skin of a human. Water is a physiologically acceptable carrier. acceptable carrier particularly common. However, physiologically acceptable carriers may be oil, fats, organic solvents, and the like, provided they are suitable and safe for application to the skin. Non-limiting examples of physiologically acceptable carriers include water, water-soluble solvents such as alcohols, polyols, and glycols, fatty compounds such as oils, triglycerides, fatty acids, fatty alcohols, and the like. The pharmaceutical and cosmetic compositions of the present disclosure may be lotions, creams, serums, sprays, emulsions, gels, powders, dispersions, ointments, sticks, pastes, or mousses.

[0055] In a preferred embodiment, the one or more bioactive walnut peptides reduce, treat, or prevent the production, release, or abundance of pro-inflammatory cytokines in the skin, e.g., interleukin-6 (IL-6), interleukin-8 (IL-8), or a combination thereof.

[0056] In a preferred embodiment, the one or more bioactive walnut peptides potentiate the production, release or abundance of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and / or transforming growth factor alpha (TGFa) in the skin.

[0057] In a preferred embodiment, the one or more bioactive walnut peptides potentiate or upregulate skin autophagy, for example in aging or senescent fibroblasts.

[0058] In a preferred embodiment, the one or more bioactive walnut peptides potentiate or enhance mitochondrial function or activity of skin cells, for example, in fibroblasts or in senescent fibroblasts.

[0059] Bioactive nut peptides may be derived from the hydrolysis of nut proteins into small molecular peptides with molecular weights between the molecular weights of individual amino acids and the molecular weights of proteins. This can be done using biological or chemical processes. For example, bioactive peptides can be prepared by enzymatic procedures, fermentation, and chemical processes.

[0060] Bioactive walnut peptides can be synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. Due to the possibility of unforeseen reactions, protecting groups are sometimes required. The chemical synthesis of peptides begins at the C-terminus of the peptide and ends at the N-terminus. Peptides can be synthesized either by solid-phase peptide synthesis, liquid-phase peptide synthesis, or fragment condensation. In principle, the seemingly simple formation of a peptide bond can be achieved using of all available procedures in organic chemistry for the synthesis of carboxylic acid amides.

[0061] The general process of solid-phase peptide synthesis (e.g., of a resin) begins with the attachment of the first amino acid, the C-terminal residue, to the resin. To prevent polymerization of the amino acid, the alpha-amino group and reactive side chains are protected by a temporary protecting group. Once the amino acid is attached to the resin, the resin is filtered and washed to remove by-products and excess reactants. Next, the N-alpha protecting group is removed in a deprotection process, and the resin is washed again to remove by-products and excess reactants. Next, the next amino acid is coupled to the attached amino acid. This is followed by another washing procedure, which leaves the resin-peptide assembly ready for the next coupling cycle. The cycle is repeated until the peptide sequence is completed.Then, typically, all protecting groups are removed, the peptide resin is washed, and the peptide is cleaved from the resin.

[0062] Enzymatic hydrolysis involves the use of commercial enzymes to obtain bioactive peptides. Enzymes are responsible for cleaving the peptide bonds established in the protein, thereby releasing the encrypted peptide. For the enzyme to carry out its activities, it is important that the enzyme binds to the substrate and continues the enzymatic catalysis. The enzyme has specific active sites containing residues that form temporary bonds with the substrate and residues that catalyze the reaction with the substrate. In this way, binding sites and catalytic sites are formed respectively. The bonds forming the enzyme-substrate complex are usually hydrogen bonds, hydrophobic bonds or Van der Waals interactions.Enzymatic hydrolysis can generally be performed in three ways: (i) under traditional batch conditions; (ii) using immobilized enzymes; or (iii) using ultrafiltration membranes. Many proteolytic enzymes are known and include those described, for example, in Cruz-Casas et al Enzymatic Hydrolysis and Microbial Fermentation: The Most Favorable Biotechnological Methods for the Release of Bioactive Peptides (Food Chem (Oxf). 3:100047, Dec. 30, 2021), which is incorporated herein by reference in its entirety.

[0063] Microbial fermentation is a biotechnological process that allows obtaining bioactive peptides. This process involves the use of microorganisms capable of producing proteolytic enzymes with the objective that these enzymes hydrolyze proteins into shorter peptides. The microorganisms generally used are bacteria, fungi or yeasts, which can be present in the substrate indigenously or added as a starter culture. The microbial fermentation process can be divided into several systems. However, the Immersion fermentation and solid-state fermentation are the most widely used.

[0064] Immersion fermentation uses a culture of microorganisms in a liquid medium, which contains nutrients. This system is suitable for microorganisms with high activities with the water level, such as bacteria, and offers the advantage that the generated bioactive peptides are easy to purify. Solid-state fermentation uses microbial growth on nutrient-rich solid substrates. It has the advantage of releasing nutrients in a controlled manner and is suitable for fungi and microorganisms with lower moisture requirements.

[0065] The bioactive walnut peptides can be produced using any method known to those skilled in the art such as those disclosed in Merrifield, R.B., Solid Phase Peptide Synthesis I., J. AM. CHEM. SOC. 85:2149-2154 (1963); Carpino, L.A. et al., [(9-Fluorenylmethyl)Oxy] Carbonyl (Fmoc) Amino Acid Chlorides: Synthesis, Characterization, And Application To The Rapid Synthesis Of Short Peptides, J. ORG. CHEM. 37:51:3732-3734; Merrifield, R.B. et al., Instrument For or Automated Synthesis Of Peptides, ANAL. CHEM. 38:1905-1914 (1966); or Kent, SBH et al., High Yield Chemical Synthesis Of Biologically Active Peptides On An Automated Peptide Synthesizer OfNovel Design, IN: PEPTIDES 1984 (Ragnarsson U., ed.) Almqvist and Wiksell Int., Stockholm (Sweden), p. 185-188.

[0066] The therapeutically effective amount of the one or more bioactive walnut peptides will vary depending on the bioactive walnut peptide and the combination of bioactive walnut peptides. However, in various embodiments, the therapeutically effective amount may be from about 1 μg to about 50 mg (50,000 μg) per cm2 of skin. In other embodiments, the therapeutically effective amount of the one or more walnut peptides is from about 1 pg to about 40 mg, from about 1 pg to about 30 mg, from about 1 to about 20 mg, from about 1 pg to about 10 mg, from about 1 pg to about 8,000 pg, from about 1 pg to about 5,000 pg, from about 1 pg to about 2,000 pg, from about 1 pg to about 1,000 pg, from about 10 pg to about 40 mg, from about 10 pg to about 30 mg, from about 10 to about 20 mg, from about 10 pg to about 10 mg, from about 10 pg to about 8,000 pg, from about 10 pg to about 5,000 pg, from about 10 pg to about 2,000 pg, from about 10 pg to about 1,000 pg,from about 100 pg to about 50 mg, from about 100 pg to about 40 mg, from about 100 pg to about 30 mg, from about 100 pg to about 20 mg, from about 100 pg to about 10 mg, from about 100 pg to about 8,000 pg, from about 100 pg to about 5,000 pg, from about 100 pg to about 2,000 pg, from about 100 pg to about 1,000 pg, from about 500 pg to about 50 mg, from about 500 pg to about 40 mg, from about 500 pg to about 30 mg, from about 500 pg to about 30 mg, from about 500 pg to about 20 mg, from about 500 pg to about 10 mg, from about 500 to about 8,000 pg, from about 500 pg to about 5,000, pg, from about 500 pg to about 2,000 pg or from about 500 |ig to about 1,000 |ig per cm2 of skin.

[0067] The bioactive walnut peptides are typically formulated with a physiologically acceptable carrier and applied to the skin as a pharmaceutical or cosmetic composition. The amount of the one or more bioactive walnut peptides will vary depending on their activity, use, and interaction with additional bioactive walnut peptides that may optionally be included in the pharmaceutical or cosmetic composition. However, in various embodiments, the pharmaceutical or cosmetic composition includes from about 0.01 to about 10% by weight of the one or more bioactive walnut peptides, based on the total weight of the pharmaceutical or cosmetic composition.

[0068] In other embodiments, the pharmaceutical or cosmetic composition includes from about 0.01 to about 8% by weight, from about 0.01 to about 5% by weight, from about 0.01 to about 3% by weight, from about 0.01 to about 1% by weight, from about 0.01 to about 0.5% by weight, from about 0.1 to about 10% by weight, from about 0.1 to about 8% by weight, from about 0.1 to about 5% by weight, from about 0.1 to about 3% by weight, from about 0.1 to about 1% by weight, from about 0.5 to about 10% by weight, from about 0.5 to about 8% by weight, from about 0.5 to about 5% by weight, from about 0.5 to about 3% by weight, from about 0.5 to about 2% by weight, about 1 to about 10% by weight, about 1 to about 8% by weight, about 1 to about 5% by weight, about 1 to about 3% by weight, or about 0.1 to about 2% by weight of the one or more bioactive nut peptides, based on the total weight of the pharmaceutical or cosmetic composition.

[0069] In one embodiment, the one or more bioactive peptides and pharmaceutical or cosmetic compositions described throughout the present disclosure are useful for improving the tensile properties of the skin, potentiating collagen synthesis and / or activating the collagen biosynthetic pathway.

[0070] Collagen appears in many places in the body. To date, at least 28 types of collagen have been identified and described, which offer a variety of structural and functional properties that collagen exhibits throughout the body. The five most common types are collagen I, II, III, IV, and V. However, more than 90% of the collagen in the body is type I.

[0071] In human skin, collagen types I and III are the predominant collagen types. They are present in the form of fibrils and are responsible for the strength and resistance of the dermis. Since type I collagen is the predominant collagen in adult human skin, constituting approximately 80% of the total collagen mass, it plays a major role in providing resistance to traction to the skin. However, it is clear that type III collagen, which constitutes approximately 10% of total dermal collagen, also plays a vital role in providing additional tensile properties to the skin and other tissues.

[0072] Structurally, three collagen polypeptides wrap around each other in a helix to form a triple helix collagen I or III molecule. These molecules are organized into a five-stranded rope-like structure in which each collagen molecule is offset by one-quarter from the next to form a microfibril. The microfibrils are then wrapped around other microfibrils to form fibrils, which in turn wrap around other fibrils to produce even larger fibrils.

[0073] Using histological and ultrastructural approaches in the past, it has been well described that chronologically aged skin is manifested by a reduced synthesis of both types I and III collagen. Regarding photoaging, Schwarz et al. (Photochem Photobiol 1993, 58, 841-844) demonstrated that collagen loss in sun-damaged human skin is due to an increased degradation of both types I and III collagen. Furthermore, it has been shown that fibroblasts from sun-exposed skin synthesize a lower proportion of collagen III than cells from sun-protected skin. (J Photochem Photobiol B. 1995, 27: 33-38).

[0074] Other types of collagen are also present in the skin and may change with skin aging. For example, collagen VII, responsible for anchoring the basement membrane to the dermal matrix, decreases with aging. (See Eur J Dermatol 2008;18:297-302). One of the main additional morphological features of aged skin is an altered epidermal dermal junction that is structurally manifested by a flattening of the epidermal dermal junction contour with loss of insertion pedicels and re-duplication of the lamina densa.Since the epidermal-dermal junction is involved in the cohesion between the dermis and epidermis, age-related alterations of the epidermal-dermal junction resulting from the decrease in collagen VII lead to functional changes in skin resistance to mechanical stress and tissue homeostasis, which likely contributes to wrinkle formation.

[0075] Collagen V assembles into various molecular forms and is believed to be expressed in the skin as different subtypes with important but distinct roles in matrix organization and stability. (See J Invest Dermatol 2012, 132:1841-1849). While collagen V is the defective product in most cases of classic Ehlers-Danlos syndrome, which is an extracellular matrix disorder typically characterized by skin fragility and scarring abnormal wounds, it does not appear to change significantly with skin aging.

[0076] Other types of collagen such as collagens VI, XIV and XVI are also expressed in the collagen-rich dermis.

[0077] Collagen production in vivo requires activation of the collagen biosynthetic pathway whereby transcription in the cell nucleus promotes polypeptide synthesis via translation of mRNA, organization of polypeptides into a procollagen triple helix in the cytoplasm, secretion of procollagen from the cell, and subsequent cleavage reactions, fibrillar assembly, and crosslinking extracellularly. Unlike many proteins that are stored in secretory granules and then secreted from the cell on demand, collagen is secreted continuously.

[0078] In one embodiment of the present disclosure, the one or more bioactive walnut peptides and pharmaceutical or cosmetic compositions comprising them are useful for improving skin elasticity, potentiating elastin synthesis and preventing elastin deterioration.

[0079] Elastin is a vital component of the vertebrate extracellular matrix and provides exceptional properties, including elasticity and tensile strength, to many tissues and organs, including the skin. Mature elastin is an insoluble and extremely durable protein that undergoes little turnover, but prolonged exposure to proteases can lead to irreversible and severe damage, and thus functional loss of the elastic fiber network. In general, elastin content decreases with age in non-sun-exposed skin (i.e., buttocks) (i.e., elastin content decreases by approximately 44% between ages 50 and 70). A similar decrease has been observed in severely sun-exposed skin (i.e., face) (i.e., elastin content decreases by approximately 31% between ages 50 and 70).Interestingly, elastin content in moderately sun-exposed areas (i.e., the forearm) does not change significantly during aging. This phenomenon could be explained by a combination of age-induced reduction and sun-dependent increase in elastin, which appears to be at least partially regulated by UV-induced lysozyme deposition in elastin fibers. (See JEADV, 2006, 20, 980-987).

[0080] Fibrillins (e.g., fibrillin 1) are ubiquitous glycoproteins of the extracellular matrix that self-polymerize into filamentary microfibrils in which individual molecules are organized into head-to-tail longitudinal arrays and also associate laterally. (See Fibrogenesis & Tissue Repair, 2010, 3, 24). Fibrillin microfibrils may further serve as a structural template for tropoelastin deposition and / or crosslinking during formation of elastic fibers. Specific segments of fibrillins interact in vitro with numerous extracellular and cell-surface signaling molecules, including fibronectin, fibulins, syndecans, and integrins. The multiple molecular interactions of fibrillins are thought to result in the assembly of morphologically distinct macroaggregates, which help confer structural integrity to individual tissues and organs (structural role), and target TGF-beta and BMP complexes into the architectural matrix, which helps instruct cell behavior (instructive role). TGF-beta and BMP are potent modulators of extracellular matrix metabolism that are under the control of a complex network of relays and servomechanisms operating within and outside the cell, and at the cell surface.Thus, fibrillins are important components of the extracellular matrix that are required for the formation of other extracellular matrix components such as elastin and the formation of elastic fibers.

[0081] In one embodiment of the present disclosure, the one or more bioactive peptides and pharmaceutical or cosmetic compositions comprising them are useful for increasing hyaluronic acid levels in the extracellular matrix of the skin, for potentiating the production of glycosaminoglycans, or a combination thereof.

[0082] Hyaluronic acid (also called hyaluronan) is a non-sulfated, anionic glycosaminoglycan. It is unique among glycosaminoglycans in that it is non-sulfated and can be very large, with its molecular weight (g mol ') often reaching millions. As the predominantly large molecule, hyaluronic acid is a major component of the extracellular matrix of the skin. It provides structure, volume (associated with the excellent water-retaining properties of hyaluronic acid) and organization (e.g., facilitates the transport of ionic solutes and nutrients), but also contributes significantly to cell proliferation and migration in the dermis. In addition, due to the water-absorbing properties of hyaluronic acid, it contributes to skin hydration.

[0083] Glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, etc.) and in particular hyaluronic acid are major components of the cutaneous extracellular matrix involved in wound healing and tissue regeneration. Wound healing is a dynamic and interactive process involving many precisely interdependent phases, which overlap in time and lead to the restoration of tissue integrity. The healing process reflects the body's complex and coordinated response to tissue damage resulting from the interaction of different cell types and matrix components. Extracellular. Hyaluronan plays a key role in every phase of wound healing by stimulating cell migration, differentiation, and proliferation, as well as regulating the organization and metabolism of the extracellular matrix. Glycosaminoglycans, and hyaluronic acid in particular, are also involved in skin aging.

[0084] As summarized by R. Stem in 2010 (Textbook of Aging Skin, Springer), although dermal hyaluronic acid is responsible for most of the hyaluronic acid in the skin, epidermal cells (e.g., keratinocytes) are also capable of synthesizing hyaluronic acid. The most considerable histochemical change observed in aged skin is the marked decrease in epidermal hyaluronic acid. In the skin of an elderly person, hyaluronic acid is still present in the dermis, while hyaluronic acid in the epidermis has completely disappeared. The reasons for this precipitous decline with aging are unknown. It has been described that the synthesis of epidermal hyaluronic acid is influenced by the underlying dermis, thus indicating that epidermal hyaluronic acid is under separate control from dermal hyaluronic acid.

[0085] In one embodiment of the present disclosure, the one or more bioactive peptides and pharmaceutical or cosmetic compositions comprising them are useful for wound healing, minimizing scar formation, or a combination thereof.

[0086] Besides skin aging, collagens I and III are also major components of the extracellular matrix involved in scar formation. Scar formation occurs after trauma, injury, or surgery to a tissue or organ in the body. These scars are the consequence of a repair mechanism that replaces the missing normal tissue with an extracellular matrix composed predominantly of collagen types I and III as well as fibronectin and some other components of the extracellular matrix. Wound healing represents imperfect tissue regeneration. While skin wounds on early mammalian embryos (e.g., up to about 24 weeks of gestation in humans) heal perfectly without signs of scarring and with complete restoration of normal skin architecture, postnatal wounds heal with scars. (See Dang C et al., Clin Plast Surg 2003: 30, 13-23). .

[0087] There are phenotypic differences between collagen content and crosslinking patterns in fetal and postnatal wounds (see Clin Plast Surg 2003, 30, 13-23 and Curr Opin Pediatr 2012, 24, 371-378). In fetal wounds, type III collagen is rapidly deposited into a fine reticular network that is indistinguishable from uninjured skin. After birth, the ratio of type I collagen to Type III collagen in wounds increases. Of the many collagen types identified, fetal skin is known to contain a greater proportion of type III collagen, whereas adult skin is predominantly composed of type I collagen. The predominance of type I collagen in postnatal wounds provides regenerating tissues with greater strength and stiffness. Early scar formation in fetal wounds during late gestation shows larger collagen fibers with greater interfiber spacing.

[0088] In one embodiment of the present disclosure, the one or more bioactive peptides and pharmaceutical or cosmetic compositions comprising them are useful for preventing, treating or reversing skin aging and / or signs of skin aging.

[0089] All expressions such as "aging skin", "signs of aging skin", "topical application" and the like are used in the sense that they are generally and widely used in the art of developing, testing and marketing pharmaceutical, cosmetic and personal care products, as well as medicaments indicated for aging skin.

[0090] Skin aging is classified into intrinsic aging and extrinsic aging depending on its cause. Intrinsic aging is a process by which the skin structure and physiological functions of the skin deteriorate independently of environmental changes as a human ages. Extrinsic aging is caused by continuous exposure to the external environment such as sunlight and air pollutants. In particular, skin aging caused by sunlight is called photoaging. Ultraviolet (UV) light from the sun is the main cause of physiological and morphological changes in aging skin.

[0091] As the skin intrinsically ages, it dries out and wrinkles and fine lines form, which become more visible and deeper with age. In addition, due to structural and functional changes in the epidermis and dermis, the skin loses its elasticity and appears to sag. With age, the dermis becomes thinner and clearly visible skin folds (e.g., the nasolabial fold) form. It is estimated that the total amount of collagen lost each year in adults is approximately 1%. In addition, the remaining collagen fibers gradually become thicker, while the crosslinking of the collagen fibers increases, so that solubility, elasticity, and the like decrease. In addition, elastin fibers become thicker and their crosslinking also increases.In addition, the proliferative activity of fibroblasts in the dermis decreases over time, and the ability of aging fibroblasts to form (i.e., synthesize) new collagen, elastin, . hyaluronic acid and other components of the extracellular matrix also decrease.

[0092] Continuous exposure to sunlight is the primary cause of extrinsic skin aging. The UV component of sunlight, particularly UVA and UVB, is generally considered the primary causative agent in this process, termed photoaging. The extent of UV exposure required to cause “photoaging” is currently unknown, although the amount sufficient to cause erythema (redness, commonly described as sunburn) on human skin is quantified as the “minimum perceptible erythema” (MED) of a given UV light source. Repeated exposure to sunlight at levels causing erythema and tanning are nevertheless frequently associated with photoaging.

[0093] There is a difference between the physiology of intrinsically aged (i.e., chronologically aged) skin and that of photoaged skin. Chronologically aged skin generally retains a smooth and flawless appearance, compared to the tanned, mottled, and often deep wrinkles of photoaged skin. Photoaging is clinically characterized by roughness, wrinkles, mottled pigmentation, a yellowish complexion, laxity, telangiectasia, lentigines, purpura, and relative ease of bruising, atrophy, depigmented areas, and finally premalignant and ultimately malignant neoplasia (i.e., an abnormal tissue mass due to neoplasia, which is the abnormal proliferation of cells). Photoaging commonly occurs on skin typically exposed to sunlight, such as the face, ears, bald areas of the scalp, neck, décolletage, forearms, and hands.

[0094] Although the typical appearance of photoaged and chronologically aged human skin can be readily distinguished, recent evidence indicates that chronologically aged and UV-irradiated skin share important molecular features, including altered signal transduction pathways that promote matrix-metalloproteinase (e.g., collagenase, gelatinase) expression resulting in extracellular matrix degradation, decreased collagen formation, and alteration or damage to the skin's extracellular matrix, such as the accumulation of amorphous elastin-containing material residing beneath the epidermal-dermal junction. This concordance of molecular mechanisms suggests that UV irradiation accelerates many key aspects of the chronological aging process in human skin.

[0095] “Signs of skin aging” include, but are not limited to, all external manifestations that are visible and perceptible to the touch as well as any other macro or micro effect due to skin aging. These signs may be induced or caused by intrinsic factors (presenting as chronological aging of the skin) and extrinsic factors (presenting as environmental damage to the skin, including, but not limited to, photoaged skin). These signs may result from processes that include, but are not limited to, the development of textural discontinuities such as coarse deep wrinkles and lines, fine lines or skin lines, fissures, bumps, enlarged pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, etc.), or irregularity or roughness, loss of skin elasticity (loss and / or inactivation of functional skin elastin), sagging (including eye area puffiness and jowls), loss of skin firmness, loss of skin tightening, loss of skin recoil due to deformation, discoloration (including dark circles), blotching, yellowish complexion, hyperpigmented areas of the skin such as age spots and ephelides, keratoses, abnormal differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the stratum corneum, dermis, epidermis, cutaneous vasculature (e.g., telangiectasia or spider vessels), and underlying tissues, especially those close to the skin.

[0096] In various embodiments of the present disclosure, the one or more bioactive peptides and pharmaceutical or cosmetic compositions comprising them are useful for prophylactically preventing, treating, or regulating a skin condition. As used herein, preventing, treating, or prophylactically regulating a skin condition includes delaying, minimizing, and / or preventing visible and / or tactile discontinuities in the skin (e.g., textural irregularities in the skin that can be detected visually or by touch), including signs of skin aging. This includes improving, for example, decreasing, minimizing, and / or erasing skin discontinuities, including signs of skin aging.

[0097] The pharmaceutical and cosmetic compositions including the one or more bioactive nut peptides may be formulated in various forms, for example, lotions, creams, serums, sprays, emulsions, gels, powders, dispersions, ointments, sticks, pastes and mousses. In addition to a physiologically acceptable carrier, the pharmaceutical and cosmetic composition may optionally include one or more of the following: • ascorbates and other vitamins, • active sunscreen agents, • anti-wrinkle active agents and anti-atrophy active agents, • wetting agents, humectants and revitalizing agents, • emollients, • antioxidants and free radical scavengers, • active desquamating agents, keratolytic agents and exfoliating agents, • suspending agents, • emulsifying agents, • thickeners, and / or • antihistamines. Ascorbates and other vitamins

[0098] The pharmaceutical or cosmetic compositions of the present disclosure may contain one or more vitamins, such as ascorbates (e.g., vitamin C, vitamin C derivatives, ascorbic acid, ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, tetrahexadecyl scorbate, ascorbyl phosphate, aminopin-3-aminopinamine), B, vitamin B derivatives, vitamin B1 - vitamin B12 and their derivatives, vitamin K, vitamin K derivatives, vitamin H, vitamin D, vitamin D3, vitamin D derivatives, vitamin E, vitamin E derivatives and its provitamins, such as ntenol, and mixtures thereof. The vitamin compounds may be included as substantially pure material or as an extract obtained by suitable physical and / or chemical isolation from natural (e.g., plant) sources.

[0099] The amount of the one or more vitamin compounds in the pharmaceutical or cosmetic compositions will vary but is typically from about 0.0001% to about 25%, more preferably from about 0.001% to about 10%, more preferably from about 0.01% to about 5%, and more preferably from about 0.1% to about 1%, based on the total weight of the pharmaceutical or cosmetic compositions. Active sunscreen agents

[0100] The pharmaceutical or cosmetic composition of the present disclosure may optionally contain sunscreen active agents, which may also be referred to as UV filtering agents. As used herein, "sunscreen active agents" include both solar reflectors and physical sunscreen agents. Suitable sunscreen active agents may be organic or inorganic. A wide variety of standard organic or inorganic sunscreen active agents are suitable for use in accordance with the present invention.

[0101] The amount of the one or more sunscreen active agents in the pharmaceutical or cosmetic compositions will vary. However, in some embodiments, the pharmaceutical or cosmetic compositions include from about 0.1% to about 25%, more often from about 0.5% to about 10%, of active sunscreen agents, relative to the total weight of the pharmaceutical or cosmetic compositions.

[0102] Non-limiting examples of organic sunscreen active agents include ethylhexyl salicylate, butyl methoxybenzoylmethane, ethylhexyl methoxycinnamate, octocrylene, phenylbenzimidazole sulfonic acid, terephthalilide dicamphor sulfonic acid, benzophenone-4-benzophenone-3 methylbenzylidene camphor, benzimidazylate, anisotriazine, ethylhexyltriazone, diethylhexylbutamidotriazone, methylene-bis-benzotriazolyltetramethylbutylphenol, dromethrazole trisiloxane, and mixtures thereof.

[0103] Non-limiting examples of inorganic sunscreen agents include nanopigments (with an average primary particle size: generally between 5 nm and 100 nm, preferably between 10 nm and 50 nm), or aggregates, coated or not with metal oxides, such as, for example, titanium oxide nanopigments (amorphous or crystalline in the form of rutile and / or anatase), iron, zinc, zirconium or cerium oxides, and mixtures thereof. Coating agents are also alumina and / or aluminum stearate and silicones.

[0104] Anti-wrinkle active agents and anti-atrophy active agents

[0105] The pharmaceutical or cosmetic compositions of the present disclosure may optionally include one or more anti-wrinkle active agents or anti-atrophy active agents. Non-limiting examples of anti-wrinkle active agents and anti-atrophy active agents include proxylan, amino acids, N-acetyl derivatives of amino acids (e.g., N-acetylcysteine), hydroxyl acids (e.g., alpha hydroxy acids, such as lactic acid and glycolic acid, or beta hydroxy acids, such as salicylic acid and salicylic acid derivatives, such as octanylic derivative, lactobionic acid), keto acids (e.g., pyruvic acid), hydrochloric acid, ascorbic acid (vitamin C), retinoids (e.g., retinoic acid, tretinoin, isotretinoin, adapalene, retinol, retinylaldehyde, retinyl palmitate and other retinoid derivatives), kinetin (N6-furfuriladenenine), zeatin, and their derivatives, for example,furunyrenifenir ... ®, TNS® or CCM™ Complex; etc.); cell extracts, stem cell extracts, stem cell components; ingredients stimulating epidermis or other adult stem cells; skin conditioning agents, stilbenes, cinnamates, sirtuin 1 activating ingredients (e.g., resveratrol); ingredients enhancing mitochondrial function; dimethylaminoethanol, synthetic anti-aging peptides, peptides from natural sources (e.g., soy peptides) and sugar salts (e.g., Mn gluconate, Zn gluconate), lipoic acid; lysophosphatidylic acid, vitamin B3 compounds and other vitamin B compounds (e.g., thiamine (vitamin B1), pantothenic acid (vitamin B5), riboflavin (vitamin B2)) and their derivatives and salts (e.g., HCl or calcium salts).

[0106] The total amount of anti-wrinkle and / or anti-atrophy active agents in the pharmaceutical or cosmetic compositions will vary. However, in some embodiments, the pharmaceutical or cosmetic compositions include from about 0.0001% to about 10%, more preferably from about 0.001% to about 8%, more preferably from about 0.01% to about 5% and more preferably from about 0.1% to about 1% of the one or more anti-wrinkle and / or anti-atrophy active agents by weight, based on the total weight of the composition.

[0107] Wetting agents, humectants and conditioning agents

[0108] The pharmaceutical or cosmetic composition of the present invention may optionally include an effective amount of a wetting agent, moisturizer, and / or skin conditioning agent. The total amount of the one or more wetting agents, humectants, and conditioning agents, if any, will vary. However, in some embodiments, the pharmaceutical or cosmetic compositions include from about 0.01% to about 80%, more preferably from about 0.1% to about 25%, and most preferably from about 0.5% to about 10% by weight of the one or more wetting agents, humectants, and / or conditioning agents, based on the total weight of the pharmaceutical or cosmetic composition.

[0109] Wetting agents are ingredients that help maintain moisture levels in the skin. Non-limiting examples of wetting agents include polyhydric alcohols, water-soluble alkoxylated nonionic polymers, and mixtures thereof. Non-limiting examples of polyols include the polyhydroxy alcohols listed above and glycerol, hexylene glycol, ethoxylated glucose, 1,2-hexanediol, dipropylene glycol, trehalose, diglycerin, maltitol, maltose, glucose, fructose, sodium chondrophosphate gum, sodium nitrophosphate, sodium lactate, pyrrolidone carbonate, glucosamine, cyclodextrin, and mixtures thereof. Water-soluble alkoxylated nonionic polymers useful in this application include polyethylene glycols and polypropylene glycols, with molecular weights up to about 1,000, such as polyethylene glycols and polypropylene glycols, named according to the Association for Perfumes and Cosmetics and Fragrances (CTFA) PEG-200, PEG-400, PEG-600, PEG-1000, and mixtures thereof.Additional wetting agents include acetylarginine, seaweed extract, aloe barbadensis leaf extract, 2,3-butanediol, chitosan lauroylglycinate, diglyceret-7 malate, diglycerol, diglycol guanidine succinate, erythritol, fructose, glycosylated glucose hydrochloride, glycosylated hydrochloride, glycosylated hydrochloride, inositol, lactitol, maltitol, maltose, mannitol, mannose, methoxypolyethylene glycol, myristamidobutyl guanidine acetate, polyglyceryl sorbitol, potassium pyrrolidone carboxylic acid (PCA), propylene glycol, butylene glycol, atria pyrrolidone carboxylic acid (PCA), sorbitol, sucrose, dextran.

[0110] Non-limiting examples of conditioning agents include guanidine, urea, glycolic acid, glycolates (e.g., ammonium and quaternary alkylammonium), salicylic acid, lactic acid, lactates (e.g., ammonium and quaternary alkylammonium), aloe vera in one of its various forms (e.g., aloe vera gel), polyhydric alcohols such as sorbitol, mannitol, xylitol, erythritol, hexanetriol, butanetriol, propylene glycol, butylene glycol, hexylene glycol, etc., polyethylene glycols, propoxylated glycol alternatives, sugars (e.g., melibioses), starches, sugar and starch derivatives (e.g., alkoxylated glucose, fructose, glucosamine), C1-C30 monoesters and polyesters of sugars and related materials, hyaluronic acid, lactamide monoethanolamine, acetamide monoanthenol, acetamide monoanthenol allantoin, and mixtures thereof.Skin conditioning agents may also include fatty acids, fatty acid esters, lipids, ceramides, cholesterol, cholesterol esters, beeswax, petroleum jelly, and mineral oil. Emollients

[0111] One or more emollients may also optionally be included in the pharmaceutical or cosmetic composition described herein. An emollient generally refers to an ingredient that can help maintain a soft, smooth, and supple appearance of the skin. Emollients generally remain on the surface of the skin or in the stratum corneum and act as a moisturizer or lubricant and reduce delamination. Non-limiting examples of emollients include acetylarginine, acetylated lanolin, seaweed extract, polyethylene glycol-6 esters from apricot kernel oil, polyethylene glycol-11 esters from avocado oil, bis-polyethylene glycol-4 dimethicone, butoxyethyl stearate, esters glycol, alkyl glycol esters, cetyl laurate, polyethylene glycol-10 coco esters, alkyl tartrates, diethyl sebacate, dihydrocholesteryl butyrate, dimethiconol, dimyristyl tartarate, distearate-5 lauroylg utamate, etilavokadate, ethylhexyl myristate, glyceryl isostearate, glyceryl oleate, geksildetsilstearate, geksilizostearate, hydrogenated palm glycerides, hydrogenated soy glycerides, hydrogenated fat glycerides isostearilneopentanoate, isostearyl palmitate, isostearylsilizononanoate, laureth-2 acetate, lauryl polyglyceryl-6 cetearyl glycol ether, methylglyutset-20 benzoate, mineral oil, palm oil, coconut oil, miret-3 palmitate, octyldecanol, octyldodecanol, odontella aurita oil, 2-oleamido-l,3 octadecandiol, commercial glycerides pal, polyethylene glycols glycerides of avocado,Castor oil polyethylene glycol, polyethylene glycol-2 / dodecyl glycol copolymer, shea butter polyethylene glycol glycerides, phytol, raffinose, stearyl citrate, sunflower seed oil glycerides, non-ointment, petit tocopheryl glucoside. , Antioxidants and free radical scavengers

[0112] The pharmaceutical or cosmetic composition of the present invention may optionally include one or more antioxidants and / or free radical scavengers. For example, the compositions may include from about 0.001% to about 10%, more preferably from about 0.01% to about 8%, and more preferably from about 0.1% to about 5% of the one or more antioxidants and / or free radical scavengers, based on the total weight of the composition.

[0113] Non-limiting examples of antioxidants and free radical scavengers include falcata bark extract, ascorbic acid (vitamin C) and its salts, fatty acid ascorbyl esters and other ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl sorbate, ascorbyl tetrabeplite, tetra ascorbyl tetrabal and ascorbyl tetraxalde, etc.), tocopherol (vitamin E), tocopheryl sorbate, tocopheryl acetate, other tocopherol esters, beta-carotene, butylated hydroxybenzoic acids and their salts, ferulic acid, peroxides, including hydrogen peroxide, perborate, thioglycolates, persulfate salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (commercially available as Trolox™), gallic acid and its alkyl esters, in particular propyl gallate, uric acid and its salts and alkyl esters, amines (e.g., N,N-diethylhydroxylamine, aminoguanidine), nordihydroguayaretic acid, bioflavonoids, sulfhydryl compounds (e.g., glutathione), dihydroxyfumaric acid and its salts, silymarin lysine ligninphenolate, lysine, 1-methionine, proline, superoxide dismutase, sorbic acids and their salts, lipoic acid, olive extracts, tea extracts, respheratrol, polyphenols such as . such as Bunge proanthrocyanidin, carotenoids, curcumin compounds such as tetrahydrocurcumin, coenzyme Q10 (L-2-oxo-4-thiazolidinecarboxylic acid), selenium, creatine, glutathione, N-acetylcysteine, N-acetylcysteine, N-acetylcysteine ​​esters, dimethylmethoxychromanol, lipoic acid, melanin; plant extracts containing polyphenols, including but not limited to coffee bean extracts, green tea extracts, rosemary extracts, witch hazel extracts, and grape skin / seed extracts that may be used. Preferred antioxidants / free radical scavengers may be selected from ascorbic acid esters, tocopherol, ferulic acid, polyphenols, creatine, and derivatives thereof; as well as plant extracts containing polyphenols, such as green tea extract. Suspensing agents

[0114] The pharmaceutical or cosmetic composition of the present invention may optionally include one or more suspending agents, preferably in a concentration effective to suspend the water-insoluble material in a dispersed form in the compositions or to modify the viscosity of the composition. These concentrations will vary. However, in certain embodiments, the pharmaceutical and cosmetic composition includes from about 0.1% to about 10%, more preferably from about 0.25% to about 5.0% of the one or more suspending agents, based on the total weight of the composition. Non-limiting examples include vinyl polymers, such as crosslinked acrylic acid polymers, referred to as carbomer, cellulose derivatives and modified cellulose polymers such as methylcellulose, ethylcellulose, nitrocellulose, carboxymethylcellulose, crystalline cellulose, cellulose powder, polyvinylpyrrolidone,polyvinyl alcohol, guar gum, hydroxypropyl guar gum, gum arabic, galactan, locust bean gum, pectin, agar, starch (rice, corn, potato, wheat), seaweed colloids (seaweed extract), microbiological polymers such as dextran, succinoglycan, pullulan, starch-based polymers such as carboxymethyl starch, methyl starch, alginic acid polymers such as sodium alginate, propylene glycol esters of alginic acid, acrylate polymers such as sodium polyacrylate, polyacrylate, polyacrylamide, polyethyleneimine and water-soluble inorganic materials such as bentonite, magnesium aluminum silicate, laponite, hectonite and anhydrous silicic acid. ,

[0115] Other optional suspending agents include crystalline suspending agents that can be resolved into acyl derivatives, long chain amine oxides, long chain acyl derivatives, and mixtures thereof. Said preferred suspending agents include fatty acid ethylene glycol esters, fatty acid alkanolamides, fatty acid esters long-chain fatty acids (e.g., stearyl stearate, cetyl palmitate, etc.); long-chain esters of long-chain alkanolamides (e.g., stearamide distearate diethanolamide, stearamide stearate monoethanol amide); and glyceryl esters (e.g., glyceryl distearate, trihydroxystearin, tribhengene). Other suitable suspending agents include primary amines containing one fatty alkyl moiety containing at least about 16 carbon atoms, examples of which include palmitamine or stearamine, and secondary amines containing two fatty alkyl moieties, each of which contains at least about 12 carbon atoms, examples of which include dipalmitoylamine or di(hydrogenated fat) amine. Other suitable suspending agents include phthalic acid diamide (hydrogenated fat) and a crosslinked copolymer of maleic anhydride / methyl vinyl ether. Emulsifying agents

[0116] Non-limiting examples of emulsifying agents include condensation products of alkylene oxides with fatty acids (i.e., fatty acid alkylene oxide esters), condensation products of alkylene oxides with 2 moles of fatty acids (i.e., fatty acid alkylene oxide diesters), condensation products of alkylene oxides with fatty alcohols (i.e., fatty alcohol alkylene oxide esters), condensation products of alkylene oxides with both fatty acids and fatty alcohols [i.e., where a portion of the polyalkylene oxide is esterified at one end with a fatty acid and esterified (i.e., via an ether linkage) at the other end with a fatty alcohol].Non-limiting examples of nonionic surfactants derived from said alkylene oxide include cetet-6, cetet-10, cetet-12, cetet-6, cetet-10, cetet-12, stearet-6, stearet-10, stearet-12, stearet-21, PEG-6 stearate, PEG-10 stearate, PEG-100 stearate, PEG-12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryl tallowate, PEG-10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80 glyceryl cocoate, PEG-200 glyceryl diisostearate, PEG-8 dilaurate, PEG-10 distearate, and mixtures thereof. Other applicable nonionic surfactants include polyhydroxyamide fatty acid surfactants. A particularly preferred surfactant corresponding to the above structure is coco N-methylglucoside alkylamide.Preferred surfactants among the nonionic surfactants are surfactants selected from the group consisting of stearet-21, ceteareth-20, ceteareth-12, sucrose cocoate, stearet-100, PEG-100 stearate, and mixtures thereof. Other nonionic surfactants suitable for use in this application include sugar esters and polyesters, alkoxylated sugar esters and polyesters, C1-C30 fatty acid esters, C1-C30 fatty alcohols, alkoxylated C1-C30 ester derivatives, . C1-C30 fatty alcohol fatty acids, alkoxylated C1-C30 fatty alcohol esters, polyglyceryl C1-C30 fatty acid esters, C1-C30 polyol esters, C1-C30 polyol esters, alkyl phosphates, polyoxyalkylene fatty ether phosphates, fatty acid amides, acyl lactylates, and mixtures thereof. Non-limiting examples of such emulsifiers include: polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soy sterol, stearet-20, cetearet-20, PPG-2 methyl glucose ether distearate, cetet-10, polysorbate 80, cetyl phosphate, cetyl phosphate, cetyl phosphate, cetyl phosphate, cetyl phosphate, polysorbate 60, glyceryl stearate, polyoxyethylene 20 sorbitan triolcat (polysorbate 85), sorbitan monolaurate, polyoxyethylene 4 lauryl ether sodium stearate, polyglyceryl-4 isostearate, hexyl laurate, PPG-2 methyl glucose ether distearate, PEG-100 and their PEG-100.Another group of nonionic surfactants useful herein is a mixture of fatty acid esters based on a mixture of sorbitan or a sorbitol fatty acid ester and a sucrose fatty acid ester, wherein the fatty acid in each example is preferably C8-C24, more preferably C10-C20. Thickeners

[0117] Thickeners suitable for inclusion in the pharmaceutical or cosmetic composition are described herein.Non-limiting examples include acrylamide copolymer, agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethylcellulose, carbomer, carboxymethylchitin, cellulose gum, dextrin, gelatin, hydroxymethyl hydroxycellulose hydroxypropyl, hydroxyethyl hydroxypropyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxyethyl hydroxypropyl, hydroxypropyl, hydroxypropyl, hydroxypropyl, hydroxypropyl, hydroxypropyl, hydroxypropyl; magnesium alginate, methylcellulose, microcrystalline cellulose, pectin, various polyethylene glycols, polyacrylic acid, polymethacrylic acid, polyvinyl alcohol, various isopropylene glycols, sodium acrylate copolymers, sodium carrageenan, xanthan gum and / or yeast beta-glucan, or mixtures thereof.

[0118] More generally, carboxylic acid polymers are useful thickeners. Carboxylic acid polymers are crosslinked compounds containing one or more monomers derived from acrylic acid, substituted acrylic acids, and salts and esters of said acrylic acids and substituted acrylic acids, wherein the crosslinking agent contains two or more carbon-carbon double bonds and is derived from a polyhydric alcohol. Examples of commercially available carboxylic acid polymers useful herein include carbomers, which are homopolymers of acrylic acid crosslinked with allyl ethers of sucrose or pentaerythritol. Carbomers are available in the Carbopol® 900 range from BF Goodrich (e.g., Carbopol® 954). In addition, other suitable carboxylic acid-based polymeric agents include copolymers of C10-30 alkyl acrylates with one or more monomers of acrylic acid, methacrylic acid, or esters of one of its short chains (i.e., a C1-4 alcohol), the crosslinking agent being an allyl ether of sucrose or pentaerythritol. These copolymers are known as C10-C30 alkyl acrylate / acrylate crosslinked polymers and are commercially available as Carbopol® 1342, Carbopol® 1382, Pemulen TR-1 and Pemulen TR-2. Goodrich.Examples of preferred carboxylic acid polymer-based thickeners useful in this application include thickeners selected from carbomers, acrylate / C10-30 alkyl acrylate crosslinked polymers, and mixtures thereof.

[0119] Further, in some embodiments, the thickeners are selected from polysaccharides. Non-limiting examples of polysaccharide thickeners include cellulose, carboxymethyl hydroxyethyl cellulose, cellulose acetate propionate carboxylate, hydroxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl hydroxyethyl cellulose hydroxyethyl cellulose sodium and hydroxyethyl methyl hydroxyethyl hydroxyethyl cellulose hydroxyethyl methyl hydroxyethyl hydroxyethyl cellulose hydroxyethyl hydroxyethyl cellulose hydroxyethyl methyl hydroxyethyl hydroxyethyl. Alkyl-substituted celluloses are also useful.In these polymers, the hydroxy groups of the cellulose polymer are hydroxylated (preferably hydroxyethylated or hydroxypropylated) to form hydroxylated cellulose, which is then further modified with a C10-30 straight-chain or branched-chain alkyl group via an ether linkage. Typically, these polymers are straight-chain or branched-chain esters of C10-30 alcohols and hydroxy alkyl celluloses. Examples of alkyl groups applicable in this application include groups selected from stearyl, isostearyl, lauryl, myristyl, cetyl, isocetyl, cocoyl (e.g., an alkyl group derived from coconut oil alcohols), palmityl, oleyl, linoleyl, linoleyl, retinoyl, behenyl, and mixtures thereof.A preferred hydroxyalkyl cellulose ester is a material called cetyl hydroxyethyl cellulose, which is an ester of cetyl alcohol and hydroxyethyl cellulose, according to the Perfume and Cosmetics Association (CTFA). The material listed is sold under the trade name Natrosol® CS Plus by Aqualon Corporation (Wilmington, Delaware). Other examples can be found in the International Cosmetic Ingredient Dictionary and Handbook, the Cosmetic Bench Reference - . Directory of Cosmetic Ingredients, offered by the United States Pharmacopeia (USP) and the National Formulary (NF), and other references to cosmetic and pharmaceutical ingredients known in the art. Other useful polysaccharides include scleroglucans, which are a linear chain of (1 to 3) linked glucose units (1 to 6), where every third glucose unit is linked, a commercially available example of which is Clearogel™ CS 11 from Michel Mercier Products Inc. (Mountainside, New Jersey).

[0120] Other useful thickeners include those derived from natural sources. Non-limiting examples include gum arabic, agar, algin, alginic acid, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum hydrochloride, gidro hydrochloride, gidro hydrochloride, gidro hydrochloride, gidro hydrochloride, hyaluronic acid, hydrated silicon dioxide, hydroxypropyl chitosan, hydroxypropyl gum, karaya gum, kelp, fruit tree resin, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, sodium carboxymethyl dextran, dextran, sodium carrageenan, tragacanth gum, xanthan gum and / or mixtures thereof.

[0121] Additionally, the compositions may optionally contain polyacrylamide polymers, particularly nonionic polyacrylamide polymers, including substituted branched or unbranched polymers. Other polyacrylamide polymers useful herein include multiblock copolymers of acrylamides and acrylamides substituted with acrylic acids and substituted acrylic acids. Antihistamines

[0122] Antihistamines, also called histamine antagonists, are substances that inhibit the action of histamine by blocking its binding to histamine receptors or by inhibiting the enzymatic activity of histidine decarboxylase, which catalyzes the conversion of histidine to histamine and the like.Non-limiting examples of antihistamines are acrivastine, azelastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, chlordiphenhydramine, cimetidine, clemastine, cyproheptadine, desloratine, dimenden nizatidine, olopadadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, ranitidine, roxatidine, rupatadine, tripelennamine and triprolidine. EXAMPLES

[0123] Various changes may be made to the compositions and methods described above without departing from the scope of the invention. Accordingly, it is intended that any disclosure contained in the above description and in the examples given below, should be interpreted in an illustrative and not limiting sense. Example 1 Reduction of inflammatory cytokines

[0124] Adult human dermal fibroblasts (batch 2485, passage 5, cell applications) were expanded and seeded in a 48-well plate at a density of 5,000 cells / well. Cells were cultured in Eagle's minimal essential medium (EMEM) supplemented with fetal bovine serum (FBS) and penicillin-streptomycin. Cells were fixed overnight, then the culture medium was aspirated and replaced with medium containing either 100 µM or 250 µM of peptide I, peptide II, or peptide III. Cells were cultured for 4 days with treatment with renewal of the treatment medium every other day except for weekends. Media were collected at the end of the culture period. Inflammatory cytokines were quantified using a LEGENDplex Human Inflammation Panel 1 13-plex (catalog no. 740809, Biolegend.Protein in the medium supernatant was also quantified using the Pierce BCA Protein Assay Kit (catalog no. 23225, ThermoFisher). Microsoft Excel and GraphPad Prism were used for further data analysis and presentation. The amounts of interleukin 6 (IL-6) and interleukin 8 (IL-8) released from adult human skin fibroblasts are shown below in Table 1 and graphically diagrammed in Figure 1(a) (for IL-6) and Figure 1(b) (for IL-8).

[0125] [Tables 1] Table 1 Peptide Concentration Pg IL-6 / pg protein Pg IL-8 / pg protein Aged control 292.81 + 76.91 55.69 + 24.27 Peptide I 154.52 + 32.33 154.52 + 32.33 22.72 + 6.45 Peptide I 126.97 + 29.10 126.97 + 29.10 42.71 + 11.29 Peptide II 265.86 + 58.08 265.86 + 58.08 20.36 + 5.39 Peptide II 257.53 + 38.82 257.53 + 38.82 35.68 + 11.37 Peptide III 291.73 + 61.03 291.73 + 61.03 31.14 + 22.26 Peptide III 333.61 + 38.37 333.61 + 38.37 32.58 + 4.06 Example 2 Increased growth factor

[0126] Reconstructed human epidermis (RHE) (EPISKIN / S / 13, Episkin) was received and samples were prepared for use. Upon arrival, the inserts containing the RHE were removed from the multiplate and placed in a 12-well plate containing maintenance medium. Tissues serving as untreated control were placed in regular maintenance medium; treatment groups were placed in maintenance medium containing 250 µM of peptide I, II, or III alone or in combination. The medium was aspirated and replaced every other day except for weekends. The RHE was cultured at the air-liquid interface at 37 C, 5% CO2, and saturated humidity for 7 days. Media were collected at the end of the culture period. Growth factors were quantified using a LEGENDplex Human Growth Factor Panel (catalog no. 740809, Biolegend.Protein in the medium supernatant was also quantified using the Pierce BCA Protein Assay Kit (catalog no. 741061, ThermoFisher). Microsoft Excel and GraphPad Prism were used for further data analysis and presentation. The amounts of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor alpha (TGFa) generated by RHE are shown below in Table 2 and graphically in Figure 2(a) (for EGF), Figure 2(b) (for HGF), and Figure 2(c) (for TGFα).

[0127] [Tables2] Table 2 Peptide Concentration EGF / pg protein HGF / pg protein TGFα / pg protein Control 1.00 + 0.23 1.00 + 0.09 1.00 + 0.29 Peptide I 250 pM 1.58 + 0.17 0.98 + 0.20 1.37 + 0.23 Peptide II 250 pM 1.23 + 0.16 1.28 + 0.25 1.56 + 0.29 Peptide III 250 pM 1.79 + 0.28 1.01 + 0.17 1.93 + 0.60 Peptides I+II (1:1) 250 pM 1.49 + 0.31 1.40 + 0.25 1.62 + 0.47 Peptides I+III (1:1) 250 pM 1.59 + 0.21 1.16 + 0.18 1.04 + 0.39 Peptides II+III (1:1) 250 pM 1.36 + 0.16 1.26 + 0.09 1.92 + 0.94 Peptides I+II+III (1:1:1) 250 pM 1.92 + 0.16 1.21 + 0.33 2.21+0.88 Example 3 Increased epidermal stem cells

[0128] Keratinocyte stem cells (lot KSC870, passage 2, Bioalternatives) were seeded in 96-well plates and cultured in keratinocyte-SFM supplemented with epidermal growth factor and pituitary extract. After 4 hours, the medium was replaced with that containing peptide II or peptide III, alone or in combination. The cells were incubated for 96 hours with renewal of the treatment after 24 hours. After 96 hours, the medium was removed and the cells were fixed with methanol at -20C. The cells were then stained for 10 minutes with Papanicolaou solution and imaged. The number of holoclone clones was counted. The data were analyzed and plotted using GraphPad Prism.The holocolone count relative to the control (normalized) is shown below in Table 3 for different concentrations of peptide II, peptide III, and combinations of peptide II and peptide III. The results are shown graphically in [Fig.3] [Fig.3]. .

[0129] [Tables3] Table 3 Peptide Concentration Holoclone Count at Control (Normalized) Control 24.80 + 6.14 Peptide II 0.2 mM 27.60 + 7.59 Peptide II 0.4 mM 34.30 + 8.85 Peptide III 0.2 mM 27.40 + 6.13 Peptide III 0.4 mM 36.50 + 5.97 Peptides II+III (1:1) 0.2 mM 32.30 + 6.82 Peptides II+III (4:1) Peptide II at 0.4 mM Peptide III at 0.1 mM 27.40 + 5.85 Peptides II+III (2:1) Peptide II at 0.4 mM Peptide III at 0.2 mM 25.20 + 5.05 Example 4 Autophagy induced in aged fibroblasts

[0130] Adult human dermal fibroblasts (batch 2485, passage 5, cell applications) were expanded and seeded in a glass-bottomed plate at 12-well plates at a density of 10,000 cells / well. Cells were cultured in Eagle's Minimal Essential Medium (EMEM) supplemented with fetal bovine serum (FBS) and penicillin-streptomycin. Cells were fixed overnight, then the culture medium was aspirated and replaced with medium containing either 100 µM or 250 µM peptide I, peptide II, or peptide III. Cells were cultured for 7 days with treatment with renewal of treatment medium every other day except weekends. On day 7, cells were stained and imaged for the presence of autophagic vacuoles according to the protocol of the Autophagy Assay Kit (abl39484, Abcam). Images were compared to determine whether the number of autophagic vacuoles increased, decreased, or remained unchanged.The results are reported below in Table 4, where “î” indicates an increase, “J,” indicates a decrease, and “++” indicates no change.

[0131] [Tables4] Table 4 Peptide Concentration Presence of autophagic vacuoles (compared to the control) Control n / a Peptide I 100 pM î Peptide I 250 pM î Peptide II 100 pM î Peptide II 250 pM î Peptide III 100 pM î Peptide III 250 pM Example 5

[0132] Improved mitochondrial function in aged fibroblasts

[0133] Adult human dermal fibroblasts (batch 2485, passage 5, cell applications) were expanded and seeded in a 12-well glass-bottom plate at a density of 10,000 cells / well. Cells were cultured in Eagle's minimal essential medium (EMEM) supplemented with fetal bovine serum (FBS) and penicillin-streptomycin. Cells were fixed overnight, then the culture medium was aspirated and replaced with medium containing either 100 µM or 250 µM of peptide I, peptide II, or peptide III. Cells were cultured for 7 days with treatment with renewal of the treatment medium every other day except for weekends. On day 7, cells were stained with ester tetramethylrhodamine ethyl ester (TMRE) and imaged for mitochondrial activity according to the TMRE-Mitochondrial Membrane Potential Assay Kit protocol (abl 13852, Abcam). Active mitochondria (mean intensity normalized to control) were determined and are shown below in Table 5. Results are shown graphically in [Fig.4].

[0134] [Tables5] Table 5 Peptide Concentration Active mitochondria (average intensity normalized to control) Control 1.00 + 0.15 Peptide I 250 pM 1.158 + 0.17 Peptide II 250 pM 1.00 + 0.19 Peptide III 250 pM 0.97 + 0.15 Peptides I+II (1:1) 250 pM 1.45 + 0.26 Peptides I+III (1:1) 250 pM 1.26 + 0.21 Peptides II+III (1:1) 250 pM 1.21+0.23 Peptides 1+11+111(1:1:1) 250 pM 0.95 + 0.22 Example 6 Facial treatment

[0135] An exemplary facial treatment containing about 0.01 to about 1% by weight of peptide I, peptide II, peptide III, or combinations thereof, is shown below.

[0136] [Tableauxô] Table 6 INGREDIENTS % by weight PEPTIDE I, II, III OR THEIR COMBINATIONS 0.01 - 2 METHYLPARABEN 0.3 2-PHENOXYETHANOL 0.5 SODIUM POLYACRYLATE 0.8 XANTHAN GUM 0.2 WATER / AQUA 98.2

[0137] Salts referred to throughout the disclosure may include salts having a counterion such as an alkali metal, alkali metal, or alkyl metal counterion. earthy or ammonium. This list of counterions, however, is not exhaustive. Suitable counterions for the components described herein are known in the art.

Claims

Claims

1. One, two, three or more bioactive walnut peptides or a composition comprising the one or more bioactive walnut peptides, for use in the topical treatment of skin for: - improving skin barrier function or re-epithelization of the skin; and / or - treating damaged skin, preferably wherein the damaged skin has been damaged by physical damage, chemical damage, environmental damage, sun damage, damage caused by disease, or combinations thereof; and / or - improving wound healing of damaged skin, wherein the damage is caused by an ablative laser procedure, a non-ablative laser procedure, a microneedle procedure, a cryotherapy procedure, a radiofrequency microneedle procedure, dermabrasion, a chemical peel, an exfoliant or a mechanical device that damages the skin;and / or - improve skin barrier function in healthy, intact skin; and / or - treat dry and / or itchy skin; and / or - potentiate terminal differentiation and keratinization of skin cells; and / or - potentiate ceramide synthesis (ACER1); and / or - facilitate terminal differentiation (involucrin (IVL), Loricrin (Lor) or Transglutaminase 1 (TGM1)); and / or - increase keratinization (KRT); and / or - promote the formation of the late horny cell envelope (LCE); and / or - increase the abundance or synthesis of small proline-rich proteins (SPRR).;

2. A peptide or composition according to claim 1, used for the treatment of a skin condition, disease or disorder selected from psoriasis, dermatitis, atopic dermatitis, allergic dermatitis, eczema, spongiosis, edema, hereditary ichthyosis, senile xerosis, palmar hyperkeratosis, plantar hyperkeratosis, cuts, bruises, pore size, skin cancer, wound healing or re-epithelialization disorders, keloids, scars hypertrophic, cellulite, orange peel, elastosis, actinic elastosis, keratosis, rosacea, telangiectasia, couperose, or combinations thereof.

3. A peptide or composition according to claim 1, wherein the skin is inflamed or suffers from an inflammatory condition, preferably wherein the inflammatory condition is selected from atopic dermatitis, psoriasis or a combination thereof.

4. A composition according to any preceding claim, wherein the one or more walnut peptides are combined with one or more additional skin active agents, preferably wherein the one or more additional skin active agents are selected from anti-atrophic agents, antioxidants, depigmenting agents, or combinations thereof, and more preferably wherein the one or more additional skin active agents are selected from ceramides, alpha-hydroxy acids, beta-hydroxy acids, vitamin A, vitamin C, vitamin D, vitamin E, niacinamide, caffeine, ferulic acid, salicylic acid, madecassoside, retinoic acid, benzoyl peroxide, or a combination thereof;and more preferably still, wherein the one or more additional skin active agents include one or more ceramides, preferably wherein the one or more ceramides are selected from ceramide EOP, ceramide AS, ceramide AP, ceramide NS, ceramide NP, ceramide NH, ceramide AH, ceramide EOH, ceramide EOS, ceramide AdS, ceramide NdS, ceramide EOdS, or combinations thereof.;

5. A peptide or composition according to any one of the above claims, wherein the one, two, three or more bioactive walnut peptides have a molecular weight of less than 5,000 Da, preferably less than 2,000 Da, more preferably less than 1,000 Da and / or wherein the one or more bioactive walnut peptides have 2 to 20 amino acid residues, preferably 3 to 15 amino acid residues, more preferably 4 to about 10 amino acid residues; and / or wherein the one or more bioactive walnut peptides include one or more amino acids selected from leucine, proline and combinations thereof, preferably wherein the one or more bioactive walnut peptides include three or more amino acids selected from leucine, proline or combinations thereof.

6. A peptide or composition according to any one of the above claims, wherein the one, two, three or more bioactive walnut peptides comprise an amino acid sequence selected from Leu-Pro-Leu, Leu-Leu-Pro, Pro-Pro-Leu or Pro-Leu-Pro; and / or Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys or Lys-Val-Pro-Pro-Leu-Leu-Tyr; preferably wherein the one, two, three or more bioactive nut peptides are selected from: Peptide I TWLPLPR (Thr-Trp-Leu-Pro-Leu-Pro-Arg), Peptide II YVLLPSPK (Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys), or Peptide III KVPPLLY (Lys-Val-Pro-Pro-Leu-Leu-Tyr), or a combination thereof.

7. Peptides or composition according to any one of the preceding claims, wherein the topical application of the combination of the two, three or more walnut peptides provides a synergistic effect which is greater than the additive sum provided by the topical application of corresponding amounts of the individual bioactive walnut peptides, preferably wherein the synergistic effect is at least 5%, preferably at least 10%, more preferably at least 25% greater than the additive sum.

8. A pharmaceutical composition comprising one, two, three or more walnut peptides according to any one of the above claims, wherein the pharmaceutical composition comprises: (a) a therapeutically effective amount of one or more bioactive walnut peptides having a molecular weight of less than 5,000 Da, preferably less than 2,000 Da, more preferably less than 1,000 Da; wherein preferably the one or more bioactive walnut peptides have 2 to 20 amino acid residues, preferably 3 to 15 amino acid residues, more preferably 5 to about 10 amino acid residues;even more preferably wherein the one or more bioactive nut peptides include one or more amino acids selected from leucine, proline and combinations thereof, preferably wherein the one, two, three or more bioactive nut peptides comprise an amino acid sequence selected from Leu-Pro-Leu, Leu-Leu-Pro, Pro-Pro-Leu, or Pro-Leu-Pro and / or Tyr-Val-Leu-Leu-Pro-Ser-Lys or Lys-Val-Pro-Pro-Leu-Leu-Tyr; even more preferably wherein the one, two, three or more bioactive nut peptides are selected from:;

9. Peptide I TWLPLPR (Thr-Trp-Leu-Pro-Leu-Pro-Arg), Peptide II YVLLPSPK (Tyr-Val-Leu-Leu-Pro-Ser-Pro-Lys), or Peptide III KVPPLLY (Lys-Val-Pro-Pro-Leu-Leu-Tyr), or a combination thereof; And (b) a physiologically acceptable carrier, wherein preferably the physiologically acceptable carrier comprises, consists essentially of, or consists of water, oil or combinations thereof. The pharmaceutical composition of claim 8 comprising two or more walnut peptides, wherein the two or more walnut peptides include a first bioactive walnut peptide and a second bioactive walnut peptide, and the first bioactive walnut peptide and the second bioactive walnut peptide are in a weight ratio of about 1:10 to about 10:1, preferably about 1:5 to about 5:1, more preferably about 1:2 to about 2:1, and even more preferably about 1:1;and / or the pharmaceutical composition comprises three or more bioactive walnut peptides, preferably wherein the three or more bioactive walnut peptides include a first bioactive walnut peptide, a second bioactive walnut peptide and a third bioactive walnut peptide, wherein: (i) the first bioactive walnut peptide and the second bioactive walnut peptide are in a weight ratio of about 1:10 to about 10:1, preferably about 1:5 to about 5:1, more preferably about 1:2 to about 2:1, even more preferably about 1:1; (ii) the first bioactive walnut peptide and the third bioactive walnut peptide are in a weight ratio of about 1:10 to about 10:1, preferably about 1:5 to about 5:1, more preferably about 1:2 to about 2:1, even more preferably about 1:1; and (iii) the second bioactive walnut peptide and the third bioactive walnut peptide are in a weight ratio of about 1:10 to about 10:1, preferably about 1:5 to about 5:1, more preferably about 1:2 to about 2:1, and even more preferably about 1:1.

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