WO2018085565A2 - Peptides bioactifs courts bloquant l'activité des produits finaux de glycation avancée, compositions et procédés d'utilisation - Google Patents

Peptides bioactifs courts bloquant l'activité des produits finaux de glycation avancée, compositions et procédés d'utilisation Download PDF

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WO2018085565A2
WO2018085565A2 PCT/US2017/059756 US2017059756W WO2018085565A2 WO 2018085565 A2 WO2018085565 A2 WO 2018085565A2 US 2017059756 W US2017059756 W US 2017059756W WO 2018085565 A2 WO2018085565 A2 WO 2018085565A2
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peptide
ages
skin
peptides
disease
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WO2018085565A3 (fr
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Lijuan Zhang
Robin Carmichael
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Helix Biomedix Inc
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Helix Biomedix Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to peptides for blocking activity of advanced glycation end products.
  • AGEs Advanced glycation end-products
  • the initial reaction is a non-enzymatic glycosylation also known as Maillard reaction, at the ⁇ -amino group of lysine or at its free amino group, although side chains of the amino acids cysteine, arginine or tryptophan can also be potential sites for glycation.
  • the intermediate products are known, variously, as Amadori, Schiff base, and Maillard products, named after the researchers who first described them. After further oxidation, dehydration, and condensation, the Amadori products undergo further structural changes to finally yield highly stable AGE compounds. Once AGEs are formed, they are nearly irreversible. The reaction causes the formation of protein adducts
  • AGEs exert their deleterious actions not only by deactivating proteins but also through their interaction with specific receptors.
  • the binding of ligands to certain AGE receptors stimulates various signaling pathways resulting in activation of the transcription factor nuclear factor kappa-B (NFkB) and subsequent transcription of many pro-inflammatory genes.
  • NFkB transcription factor nuclear factor kappa-B
  • AGE receptors and their ligands may be involved in the pathobiology of a wide range of diseases that share common features, such as enhanced oxidative stress, immune/inflammatory responses, and altered cell functions.
  • AGEs are formed both endogenously via glucose and fructose metabolism and exogenously via diet and tobacco.
  • the receptor of AGEs (RAGE) is a multi-ligand member of the immunoglobulin super-family of cell surface receptors and is highly expressed in many cell types including fibroblasts, keratinocytes, endothelial cells, immune cells, neurons, blood vessel walls, bone, and tumor cells, etc. RAGE activation induces multiple intracellular signaling pathways that have been implicated in the pathogenesis of serious systemic complications and aging.
  • a series of short peptides that effectively inhibit the progress of a Maillard reaction, block the AGEs and/or inhibit dicarbonyl induced cell damage, inflammation, apoptosis and death.
  • These manufactured peptides are promising therapeutics as antiglycation agents, skin anti-aging agents, and anti- diabetic complication agents.
  • a peptide for blocking the activity of advanced glycation end products (AGEs), the peptide being a 2-9 amino acid subsequence of SEQ ID NO:61.
  • composition including at least one peptide according to the disclosure and a pharmaceutically acceptable carrier.
  • provided herein is a method for treating a skin condition in a subject comprising administering to the subject a medicament comprising a pharmaceutically acceptable carrier and at least one peptide according to the disclosure.
  • the present disclosure provides a method for treating a condition, disease, or disorder in a subject comprising administering to the subject an effective amount of at least one peptide disclosed herein.
  • the at least one peptide is comprised in a medicament comprising a pharmaceutically acceptable carrier.
  • the condition, disease or disorder is diabetes, metabolic syndrome, diabetic skin chronic wounds, Alzheimer's disease, atherosclerosis, osteoporosis, osteoarthritis, cancer, cancer therapy associated condition, pollution associated assaults, body and skin detoxification, renal failure, diabetic retinopathy and glaucoma, periodontal disease, cystic fibrosis related diabetes, polycystic ovary syndrome, psoriasis associated condition, oxidative stress, complications associated with maternal chorioamnionitis or funisitis, or sarcopenia.
  • provided herein is a method of inhibiting formation of AGEs in a food or beverage comprising contacting the food or beverage with at least one peptide according to the disclosure.
  • a method of manufacturing a skin care product for blocking activity of AGEs including: synthesizing a first peptide, wherein the first peptide is between 2 and 9 amino acids in length and is a
  • Figure 1 shows the amino acid sequence of the AGE receptor isoform 1 of human (NCBI Reference Sequence: NP_001 127.1), where the underlined region indicates the active binding region of RAGE to AGEs from which the peptides disclosed herein have been generated.
  • Figure 2 shows the amino acid sequence of the V-region of the AGE receptor sequence of Figure 1.
  • peptides disclosed herein are advantageous as therapeutics, anti-glycation agents, skin anti-aging agents, and anti-diabetic complication agents. Accordingly, the peptides of the disclosure may be formulated as a composition or medicament (e.g., lotion, cream, etc.) that can be administered to a subject for the treatment or amelioration of a condition, disease or disorder.
  • a composition or medicament e.g., lotion, cream, etc.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term "about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.
  • a combination thereof refers to one of the all possible combinations of the listed items preceding the term.
  • A, B, C, or a combination thereof is intended to refer to any one of: A, B, C, AB, AC, BC, or ABC.
  • the term “combinations thereof” as used herein refers to all possible combinations of the listed items preceding the term. For instance, “A, B, C, and combinations thereof is intended to refer to all of: A, B, C, AB, AC, BC, and ABC.
  • AGEs Advanced glycation end-products
  • AGEs are a heterogeneous group of molecules but share some common characteristics including the formation of covalent cross-links between proteins, the effect of turning some foodstuffs a yellow-brown color, and the ability to generate fluorescence.
  • AGEs can be categorized as: 1) fluorescent cross-linking AGEs such as pentosidine, 2-(2-furoyl)- 4(5)-2(2-fluranyl)-1 H-imidazole, glyxal-lysine dimer, and methylglyoxal-lysine dimer (MOLD); and 2) non-fluorescent and non-cross-linking agents such as N- carboxymethyllysine (CML), l ⁇ l£-(carboxyethyl)-lysine (CEL), and pyrraline.
  • CML was the first described and represents the most prevalent AGE in vivo.
  • CML can form in vitro from low density lipids (LDL) incubated with copper ions and glucose, it has been postulated that it can form both lipid and protein adducts.
  • LDL low density lipids
  • AGEs are formed and accumulated both endogenously and exogenously. Endogenous AGE formation occurs slowly in normal aging. Tissue levels of AGEs increase with age. In diabetes and metabolic syndrome, hyperglycemia can accelerate the formation of AGEs. Endogenously, glucose auto-oxidation or glycolysis can generate highly reactive dicarbonyl compounds such as glyoxal, methylglyoxal (MG), and 3-deoxyglucosone as metabolic by-products, all of which can interact with proteins to form AGEs.
  • MG methylglyoxal
  • Methylglyoxal a potent glycating agent, is produced mainly during glucose and fructose metabolism. In comparison to the parent sugar, MG is more likely to bind to arginine, lysine, and cysteine residues of proteins, causing the formation of a number of AGEs and subsequently more cross-linking to various proteins. In addition to formation of AGEs, MG reacts rapidly with RNA and DNA and has both mutagenic and clastogenic activities. It can induce numerous adverse reactions if not efficiently detoxified. MG is not only found at high levels in the blood of diabetic patients but also in hypertension, where it covalently depletes glutathione, leading to serious
  • MG also contributes to dysfunction of adipose tissue during type 2 diabetes progressions.
  • glyoxal is also a toxic dicarbonyl compound capable of damaging cells via AGE formation.
  • CML One of the AGEs formed in the presence of glyoxal is CML, which has been used extensively as a biomarker for aging.
  • CML can be formed by different pathways: glucose can be oxidized to glyoxal, which can react with protein to form CML; glucose can also react with protein to form fructose-lysine (an Amadori product), which then undergoes oxidative cleavage to form CML.
  • AGEs may also be introduced into organisms by means of exogenous sources such as tobacco and diet.
  • the western style diet contains a substantial proportion of industrially processed foods that have been shown to contain high levels of AGEs, which is the main exogenous source and is associated with the development of several pathological conditions such as diabetes.
  • Food preparation methods using high temperatures enhance the production of AGEs.
  • MG can be generated by degradation of carbohydrates in food and beverages during food processing.
  • AGEs exert their deleterious actions not only by deactivating proteins but also through their interaction with specific receptors.
  • Receptor for AGEs (RAGE) is a multi-ligand member of the immunoglobulin superfamily of cell surface receptors. It is a pattern recognition receptor binding to various other molecules in addition to AGEs. Six receptors that recognize and bind AGEs have been identified, among which RAGE is the best characterized and most extensively studied.
  • RAGE is a type I transmembrane protein receptor, expressed mainly on the surface in many cell types. The binding of ligands to RAGE stimulates various signaling pathways resulting in activation of the transcription factor nuclear factor kappa-B (NFkB) and subsequent transcription of many pro-inflammatory genes.
  • NFkB transcription factor nuclear factor kappa-B
  • RAGE and its ligands are intimately involved in the pathobiology of a wide range of diseases that share common features, such as enhanced oxidative stress, immune/inflammatory responses, and altered cell functions.
  • RAGE is highly expressed in fibroblasts, keratinocytes, immune cells, neurons, blood vessel walls, bone, and tumor cells.
  • RAGE activation by high serum and tissue levels of AGEs induces multiple intracellular signaling pathways that have been implicated in the pathogenesis of serious complications including diabetes and diabetes associated atherosclerosis, cardiovascular disease, nephropathy and chronic inflammatory conditions.
  • RAGE is composed of an N-terminal extracellular domain with a ligand-engaging V-region and two cytosolic-regions; a single transmembrane domain and a C-terminal highly charged, short cytoplasmic domain essential for signal transduction.
  • a soluble form in contrast to the membrane bound form, has been characterized and is called sRAGE.
  • Figure 1 shows the amino acid sequence of isoform 1 of human RAGE.
  • the inventors focused on the V-region located in the N-terminal portion of the human isoform 1 of RAGE, which is underlined in Figure 1 and shown separately in Figure 2. Prior to this study, it was unknown whether the entire N-terminal 77-amino acid region shown in Figure 2 was required to bind AGE.
  • the inventors first utilized a "shotgun" approach to randomly generate overlapping sequences from 5 to 9 amino acids in length along the 77 amino acid region. These sequences are represented by SEQ ID NOs 1-17 (Table 1).
  • sequences are solely in s/7/co-designed sequences which do not exist or occur naturally, e.g. in microbes, animals to plants.
  • the binding activity was found encouraging as 10 out of the 17 sequences showed positive AGE binding (Table 2) suggesting there are multiple binding sites present in the 77-amino acid region shown in Figure 2, and the binding sites are linear, meaning a continuous linear sequence (from N-C) is required for binding.
  • the next step was to find out the minimal number of amino acids required for the binding, therefore shorter fragments of 2-4 amino acids in length were generated, also using in silico design.
  • SEQ ID NOs 18 to 59 shown in Table 1 , along with an indication of whether the peptides are in an amidated (NH2) or free acid (OH) form.
  • the peptides were tested for blocking AGEs as described in Example 1 , and the results are shown in Table 2.
  • Carnosine was used as antiglycation positive control in all of the experiments conducted in this study.
  • the peptides which effectively bind to a mixture of AGEs include SEQ ID NOs 1 , 4, 5, 8, 9, 12, 13, 14, 15, 16, 18, 19, 20, 21 , 22, 23, 24, 25, 27, 28, 29, 32, 33, 34, 35, 36, 38, 39, 41 , 42, 44, 45, 46, 47, 48, 49, 52, 54, 55, 56, 57, 58, and 59.
  • the specificity of each peptide was also tested for binding to CEL and CML.
  • the peptides that significantly reduced keratinocyte apoptosis include SEQ ID NOs 1 , 5, 8, 12, 13, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 29, 30, 31 , 32, 33, 35, 36, 37, 38, 39, 40, 41 , 43, 44, 45, 47, 48, 49, 52, 54, 55, and 58.
  • Such activity may be used in treating conditions associated with AGEs from aging to other chronic inflammatory conditions including, but not limited to, diabetes, metabolic syndrome, diabetic skin chronic wounds, Alzheimer's disease, atherosclerosis, osteoporosis, osteoarthritis, cancer therapy, pollution associated assaults, body and skin detoxification, renal failure, diabetic retinopathy and glaucoma, periodontal disease, cystic fibrosis related diabetes, polycystic ovary syndrome, psoriasis associated condition, and oxidative stress, among others.
  • These peptides include SEQ ID NOs 3, 8, 9, 12, 13, 14, 23, 24, 25, 29, 34, 35, 38, 39, 40, 41 , 42, 46, 48, 49, 52, 53, 55 and 58. These peptides can be useful to inhibit the Maillard reaction in a subject's body or elsewhere (e.g. added to a food or beverage product) so as to prevent this and similar reactions in food or beverages containing sugar and collagen.
  • the peptides disclosed herein were synthesized using standard Fmoc (9- fluorenylmethoxycarbonyl) solid-phase chemistry, although those skilled in the art will recognize that other synthesis technologies may be used. As the peptides are prepared synthetically, the disclosed peptides are essentially free of post-translational modifications, although various modifications may be added in a controlled manner to confer certain desired properties on the peptides. For example, the peptides may be prepared as either amidated (NH2) or free acid (OH) sequences using standard amino acids; Table 1 indicates the form of each peptide disclosed herein.
  • the peptides may include L- or D-amino acid enantiomers, either containing residues of one enantiomeric form or a
  • the peptides may be modified on one or both of the N- terminus and the C-terminus.
  • N-terminus lipidation or acylation may improve peptide penetration across skin without altering the bioactive function of the peptide, which may provide for enhanced skin penetration.
  • saturated or unsaturated fatty acids that can be used to provide a C12-18 lipid-component to the compounds disclosed herein include lauric acid, myristic acid, palmitic acid, stearic acid, myristoleic acid, palmitoleic acid, oleic acid, and linoleic acid, although other lipid sources may also be used.
  • the carboxy-terminus of the peptides can be modified to be acidic (-COOH) or amidated (e.g., -CONH2, -CONHR, or -CONR2). Amidation of the carboxy-terminus may confer advantageous properties on the peptides, for example rendering the inventive peptides less susceptible to protease degradation and/or increasing their polarity compared to the free acid forms, therefore providing heightened therapeutic potency.
  • the peptide functional groups that may typically be modified include hydroxyl, amino, guanidinium, carboxyl, amide, phenol, imidazol rings or sulfhydryl.
  • Peptides may also be conjugated to soluble or insoluble carrier molecules to modify their solubility properties as needed and to control (e.g. increase) the local concentrations of peptides in targeted tissues.
  • soluble carrier molecules include, but are not limited to, polymers of polyethyleneglycol (PEG) and
  • peptides may be micro-encapsulated using liposome technology or via nano- technology in the form of nano-emulsions, nanoliposomes, or other types of nanoparticles or nanomaterials including those disclosed in Sharma et al. (2012 Int J Pharm Pharm Sci, Vol 4, Issue 3, 57-66, incorporated herein by reference in its entirety), using methods known to those skilled in the art.
  • the peptides may be produced using any method known to those skilled in the art such as those disclosed in Merrifield (J Am Chem Soc.
  • compositions for using the above-described peptides such as in formulations or as therapeutic agents.
  • methods of using the above described peptides such as in formulations or as therapeutic agents. These methods may involve the use of a single peptide, or multiple peptides in combination or combined with other peptides having collagen stimulating activity or anti-inflammatory activity, or combined with materials such as antioxidants.
  • the inventive composition can be disposed within devices placed upon, in, or under the skin. Such devices include transdermal patches, implants, and injections which release the substances in such a manner as to contact the skin or hair follicle either by passive and/or active release mechanisms.
  • compositions used to deliver the peptides in the methods described herein can be in the form of an aerosol, emulsion, liquid, lotion, solution, gel, micro-encapsulation, cream, paste, ointment, powder, foam, or other pharmaceutically-acceptable formulation.
  • the delivery methods may also include nanotechnology or other advanced technologies to facilitate the penetration and delivery of peptides to desired areas or organs.
  • the peptides can be delivered using less involved formulations such as deionized/distilled water, PBS, or standard medical saline solutions.
  • the formulation may optionally have cosmetic appeal, and/or contain other agents such as retinoids, vitamin C, vitamin E or other peptides that can act as adjuvants for the therapeutic action of the inventive peptides.
  • Antibiotics can also be added to the formulation in order to ward off infection, thereby permitting maximal healing processes to occur.
  • the formulation may contain protease inhibitors.
  • a protease inhibitor can be selected to specifically target proteases that would be expected to degrade the selected bioactive peptide; such a selection would be determined based on the length and/or sequence of the bioactive peptide.
  • protease inhibitors need not necessarily be selected in any specific manner; for example, in some embodiments a protease inhibitor cocktail, which contains two or more inhibitors, can be employed in the formulation.
  • protease inhibitors can be incorporated in the formulation: serine protease inhibitors, cysteine protease inhibitors, aspartate protease inhibitors, metalloproteinase inhibitors, thiol protease inhibitors, and/or threonine protease inhibitors.
  • the protease inhibitor used in formulation may be a peptide, protein, or chemical.
  • Non-limiting examples of such inhibitors are the serpins, which include alpha-1-antitrypsin, complement 1 -inhibitor, antithrombin, alpha-1 - antichymotrypsin, plasminogen activator inhibitor 1 , and neuroserpin, or chemicals including, but not limited to, ursolic acid and tranexamic acid that can act as adjuvant for the therapeutic action of the inventive peptides.
  • the peptides disclosed herein are incorporated into an aqueous solution.
  • the peptides are incorporated into a cosmetically or pharmaceutically acceptable solvent.
  • the solubility of a peptide can be readily determined based on the charged or hydrophobic nature of the amino acid sequence and any modification at the N-terminal or C-terminal ends.
  • a pharmaceutically acceptable formulation includes any carrier suitable for use in or on humans.
  • Such pharmaceutically or cosmetically acceptable carriers include, but are not limited to, ethanol, propanol, isopropanol, propylene glycol, butylene glycol, polyethylene glycol, dimethyl sulfoxide, glycerol, silica, alumina, starch, or any combination thereof.
  • the pharmaceutically acceptable carrier or cosmetically acceptable carrier does not consist of water or does not consist essentially of water.
  • a condition, disease or disorder in a subject comprising administering to the subject an effective amount of at least one of the peptides of this disclosure.
  • Treatment refers to medical management of a condition, disease, or disorder of a subject (e.g., patient), which may be therapeutic,
  • an "effective amount” or “therapeutically effective amount” of a peptide or composition described herein refers to that amount of peptide or composition sufficient to result in amelioration of one or more symptoms of the condition, disease, or disorder being treated in a statistically significant manner.
  • the condition, disease, or disorder is associated with skin aging.
  • the method comprises administering at least one peptide disclosed herein to reduce or reverse the effects or appearance of aging with regard to skin (e.g. , anti-aging).
  • the condition, disease, or disorder is a chronic inflammatory conditions including, but not limited to, diabetes, metabolic syndrome, diabetic skin chronic wounds, Alzheimer's disease, atherosclerosis, osteoporosis, osteoarthritis, cancer therapy, pollution associated assaults, body and skin detoxification, renal failure, diabetic retinopathy and glaucoma, periodontal disease, cystic fibrosis related diabetes, polycystic ovary syndrome, psoriasis associated condition, and oxidative stress, complications associated with maternal chorioamnionitis or funisitis, sarcopenia, among others.
  • the peptides disclosed herein are useful for the treatment or amelioration of aging and symptoms associated with aging.
  • symptoms of aging include muscle weakness (e.g., sarcopenia), atherosclerosis, walking disability, Alzheimer's disease, and skin aging, such as drying, thinning, spots, decreased elasticity, increased stiffening, and wrinkles.
  • the peptides disclosed herein are useful for body and skin detoxification.
  • a sufficient amount of time of direct contact with the skin includes, for example, 1-24 hours, 1-2 days, 3-4 days, 5-6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, 12 months, or more.
  • taking peptides orally or systemically inhibits the formation of AGEs or the interaction of AGEs with its receptors therefore reducing toxic and inflammatory effects of AGEs and one or more associated symptoms, e.g., dry skin, rash, dull looking skin, sagging skin, wrinkles, fine lines, dark spots, uneven tone, or other inflammatory effects.
  • the peptides disclosed herein are useful for the treatment or amelioration of oxidative stress and symptoms associated with oxidative stress.
  • oxidative stress and symptoms of oxidative stress include inflammation (e.g., osteoporosis, osteoarthritis, arthritis, psoriasis), cancer (e.g., prostate cancer, breast cancer, gastric cancer, lung cancer), cancer therapy associated condition, renal failure.
  • cancer e.g., prostate cancer, breast cancer, gastric cancer, lung cancer
  • cancer therapy associated condition e.g., renal failure.
  • the peptides disclosed herein are useful for the treatment or amelioration of complications associated with or caused by diabetes.
  • diabetic complications include metabolic syndrome, hyper-coagulation of the blood, diabetic skin chronic wounds, skin ulcer, osteoporosis, periodontitis, cystic fibrosis related diabetes, diabetic retinopathy, diabetic nephropathy and glaucoma.
  • the peptides disclosed herein are useful for the treatment or amelioration of symptoms associated with exposure to pollution, such as indoor and outdoor air pollution.
  • pollution include carbonyl compounds (e.g., formaldehyde, acetaldehyde, hexanal, glyoxal, and methylglyoxal), oxygenated organic compounds (e.g, glyoxal, methylglyoxal, glycolaldehyde, and diacetyl).
  • pollution include carbonyl compounds (e.g., formaldehyde, acetaldehyde, hexanal, glyoxal, and methylglyoxal), oxygenated organic compounds (e.g, glyoxal, methylglyoxal, glycolaldehyde, and diacetyl).
  • exemplary symptoms of exposure to pollution include headache; eye, nose, or throat irritation; dry cough; dry, irritated, or itchy skin; dizziness and nausea; difficulty in
  • peptides disclosed herein may be used in a cosmetic or
  • the concentration of the peptide in the composition can be between 0.1 parts per million (ppm) and 500 ppm or between 0.1 ppm and
  • the concentration of the peptide in the composition is between 0.1 ppm and 100,000ppm; between 1 ppm and 50,000ppm; between 1 ppm and 10,000ppm; between 10 ppm and 5000 ppm; between 10ppm and 100,000 ppm.
  • the concentration of the peptide in the composition is at least about 0.1 ppm, at least about 0.5 ppm, at least about 1 ppm, at least about 5 ppm, at least about 10 ppm, at least about 50 ppm, at least about 100 ppm, at least about 500 ppm, at least about 1 ,000 ppm, at least about 5,000 ppm, at least about 10,000 ppm, or at least about 50,000 ppm.
  • the concentration of the peptide in the composition is no more than about 0.5 ppm, no more than about 1 ppm, no more than about 5 ppm, no more than about 10 ppm, no more than about 50 ppm, no more than about 100 ppm, no more than about 500 ppm, no more than about 1 ,000 ppm, no more than about 5,000 ppm, no more than about 10,000 ppm, no more than about 50,000 ppm, or no more than about 100,000ppm.
  • the peptides disclosed herein may be prepared as a premix peptide formulation.
  • a premix peptide formulation refers to a mixture containing at least one active peptide of the current disclosure plus at least one other ingredient disclosed herein, such as water, glycerin, a stabilizer, an oil, an emollient, an emulsifier, a humectant, a thickener, a neutralizer, a surfactant, a preservative, or any combination thereof.
  • a premix may exist in the form of solid (powder), semi-solid (emulsion), or solution. It may be made by agitation, stirring, or mixing under high shear force.
  • a premix is not a final formulation or product for use with a subject.
  • the peptides disclosed herein may be stabilized or are stable in a premix.
  • stabilized refers to the absence of or reduction of loss of activity over a period of time in a statistically significant manner compared to non-stabilized control peptide.
  • a peptide is "stable" in a premix (or another formulation) if no more than 50% of the peptide is degraded for at least 1 month.
  • any of the peptides disclosed herein in a premix may be stabilized or stable for at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, at least 54 months, or at least 60 months.
  • a premix formulation may comprise any of the peptide disclose herein at a concentration between 1 ppm and 100,000 ppm, such as between 1 ppm to 100 ppm, 100 ppm to 500 ppm, 500 ppm to 1 ,000 ppm, 1 ,000 ppm to 5,000 ppm, 5,000 ppm to 10,000 ppm, 10,000 ppm to 50,000 ppm, and 50,000 ppm to 100,000 ppm.
  • abrasives e.g. clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
  • anti-acne agents e.g. clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
  • anti-acne agents e.g. clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
  • anti-acne agents e.g. clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
  • anti-acne agents e.g. clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel
  • antioxidants binders, biological additives, buffering agents, bulking agents, chelating agents, chemical additives, cosmetic biocides, denaturants, drug astringents, external analgesics, film formers or materials, opacifying agents, pH adjusters, propellants, reducing agents, sequestrants, skin bleaching and lightening agents (e.g. hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucosamine), skin-conditioning agents (e.g. humectants), skin soothing and/or healing agents (e.g.
  • panthenol and its derivatives aloe vera, pantothenic acid and its derivatives, allantoin, bisabolol, and dipotassium glycyrrhizinate), skin treating agents, thickeners, and vitamins, and derivatives thereof.
  • the peptides are formulated in a lotion or cream that includes both water soluble ingredients and oil soluble ingredients.
  • These water soluble and oil soluble ingredients may comprise (but not be limited to) one or more moisturizing components, occlusive agents, humectants, emollients, emulsifiers, thickeners, diluents, fragrances, preservatives, and neutralizers.
  • the peptides may be formulated in a lotion or cream that may include one or more occlusive agents which creates a barrier that blocks water from escaping the skin.
  • occlusive agents may be present at a percentage of final volume of about 0.1 %-10%, about 0.5%-5%, about 0.001 %-1 %, about 1 %-10%, about 10%-50%, or about 50%-99%and may include but are not limited to petrolatum, mineral oil, dimethicone, isopropyl myristate, or any combination thereof.
  • One or more humectants may also be included in order to attract water to the skin.
  • Such humectants may be present at a percentage of final volume of 0.1 %-10%, about 0.5%-5%, about 1 %-5%, about 0.001 about 1 %-10%, about 10%-50%, or about 50%-99% and include but are not limited to glycerin, propylene glycol, PEG, silicones, urea, pyrrolidone carboxylic acid and salts thereof, aloe, honey, hyaluronic acid, or any combination thereof.
  • One or more emollients may also be added to improve the feel of the lotion on the skin and/or reduce the tackiness and greasiness caused by the other moisturizing ingredients.
  • Such emollients may be present at a percentage of final volume of about 0.1 %-10%, about 0.5%-5%, about 1 %-5%, about 0.001 %-1 %, about 1 %-10%, about 10%-50%, or about 50%-99%, and may include, but are not limited to, plant oils such as coconut oil, natural butters, cetyl esters, Argan oil, certain silicones, mineral oil, petrolatum, fatty acids, squalane , polymers, lenitin or any combination thereof.
  • the lotion also contains emulsifiers to help combine the oil soluble ingredient with the water soluble ingredients.
  • Such emulsifiers may be present at a percentage of final volume of about 0.1 %-10%, about 0.5%-5%, or about 1 %-5%, about 0.001-1 %, about 1 %-10%, about 10%-50%, or about 50%-99% and include but are not limited to lecithin, polyglyceryl oleate, glyceryl stearate, glyceryl oleate, sorbitan stearate, sorbitan oleate, laureth-3, PEG-8 beeswax, glycol distearate, stearic acid, polysorbates, creammaker ® ceteareth-20, gelmaker ® , gum Arabic, prehydrated, PEG, polysorbate stearic acid, cetearyl alcohol, cetyl phosphate, ceteareth, glyceryl stearate citrate; shea butter glycerides, creammaker®-wax, myristic acid, brassica glycerides and brassica alcohol
  • thickeners may also be provided to help keep the formula stable and make it more appealing to use.
  • Such thickeners may be present at a percentage of final volume of about 0.1 %-20%, about 1 %-10%, or about 3%-7%, about 0.001-1 %, about 1 %-10%, about 10%-50%, or about 50%-99% and include but are not limited to carbomer, natural wax, stearyl palmitate, or any combination thereof.
  • Other components such as one or more diluents at a percentage of final volume of about 50%-95%, about 60%-90%, about 70%-85%, 0.001 %-1 %, 1 %-10%, 10%-50%, 50%- 99% and include but not be limited to water, botanical extracts, or any combination thereof.
  • Fragrances or preservatives may be present at a percentage of final volume of about 0.01 %-3%, about 0.05%-2%, or about 0.1 %-1.5%, 0.001 %-1 %, 1 %-10%, 10%- 50%, 50-99% and include but not limited to methylparaben, propylparaben, benzylalcohol-DHA, phenoxyethanol disodium EDTA, or any combination thereof.
  • Neutralizers may be present at a percentage of final volume of about 0.05%-5%, about 0.1 %-3%, or about 0.2%-1.5%, 0.001 %-1 %, 1 %-10%, 10%-50%, 50%-99% and include but not limited to triethanolamine. Colorants may also be included to make a well-rounded formulation.
  • An example of a cream or lotion comprises the following phase ingredient quantity (%), A: glyceryl stearate 6, cetearyl alcohol 2.0, stearic acid 2.0, ethylhexyl stearate 3.0, caprylic/capric triglyceride 3.0, cetyl dimethicone 2.5, dimethicone 1.5, B: glycerin 5.5, Deionized Water 57.70, C: active peptide premix 2.5, urea 2.0, sodium bisulfate 39% 0.3, sodium ascorbyl phosphate 1.5, Z: preservative/perfume 0.5.
  • Heat phase A and B separately to approx 70-75°C. Add phase A to phase B with stirring. Homogenize, then cool with gentle stirring below 40°C and add phase C, then Z.
  • the peptides disclosed herein are combined or formulated with, for example, Palmitoyl hexapeptide-14, Heptapeptide-7, Hexapeptide- 21 , Oligopeptide-10, SD alcohol 40-B, dimethicone, cyclopentasiloxane, glycerin, neopentyl glycol diheptanoate, hydroxyethyl acrylate/sodium acryloyldimethyltaurate copolymer, polysilicone-1 1 , isohexadecane, pheyl trimethicone, sodium hydroxide, caprylyl glycol, hexylene glycol, polysorbate-20, polysorbate-60, ethylhexyglycerine, phenoxyehtanol, polyethylene, cocamidopropyl betaine, sorbitol, Peg-100 sterate, glyceryle sterate, glycol
  • the peptides disclosed herein are incorporated into cosmetic delivery systems, pharmaceutical delivery systems, or sustained release systems.
  • Such systems are related to a delivery system of a compound that provides either a targeted release of this compound e.g., delivery to intestine avoiding stomach enzymes or gradual release of this compound during a period of time and preferably, although not necessarily, with relatively constant compound release levels over a period of time.
  • Examples include, but not limited to, liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticules, nanoparticles and solid nanoparticules, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed micelles of surfactants, surfactant-phospholipiod mixed micelles, millispheres, microspheres and nanosperes, lipospheres, millicapsules, microcapsules and nanocapsules, or with the help of cell penetrating peptides to achieve a greater penetration of the active ingredient and/or improve its pharcokinetic and
  • inventive peptides and associated compositions may be administered to subjects such as humans and animals, including mammals. Application may also be made in combination with conventional and/or experimental materials such as tissue grafts, skin substitutes, tissue culture products and dressings.
  • tissue grafts tissue substitutes
  • tissue culture products and dressings tissue culture products and dressings.
  • the peptides disclosed herein can also be adsorbed on solid organic polymers or solid mineral supports.
  • the compositions which contain the peptides can also be
  • gauzes woven and non- woven, impregnated, nonadherent, packing, debriding
  • compression bandages and system wound fillers and cleansers
  • contact layers collagens; amniotic membranes; acellular human dermis; acellular matrices and combination products; and various commonly used dressings, including those listed below.
  • the cosmetic or pharmaceutical compositions can be applied to local areas to be treated by means of iontophoresis, sonophoresis, electroporation, microelectric pateches, mechanical pressure, osmotic gradient, occlusive cure, microinjections or needle-free injections by means of pressure, such as injection by oxygen pressure, or any combination thereof.
  • the cosmetic or pharmaceutical compositions containing the peptides disclosed herein, their stereoisomers or their cosmetically or pharmaceutically acceptable salts can be used in different types of formulations for oral administration, preferably in the form of oral cosmetics and pharmaceutical drugs, such as, but not limited to, capsules, tablets, powders, granules, chewing gum, solutions, suspensions, syrups, polysaccharide films, jellies or gelatins, and any other form known by the person skilled in the art.
  • the peptides disclosed herein can be incorporated into any forms of functional food, fortified food or nutritional supplements e.g., dietary bars or powders.
  • the powders can be dissolved in water, juices, soda, dairy products, or soya derivatives.
  • methods of treatment which include application of one or more of the disclosed peptides in a pharmaceutically- acceptable carrier composition and may be applied to treat one or more condition, including those disclosed herein.
  • compositions can be administered topically, orally, or parenterally including nasal, rectal, vaginal, ocular or subcutaneous implantation or injection directly into a specific body part, transdermally, systemically, or by any other method known to those of skill in the art to be useful to deliver the inventive peptides to the target tissue.
  • Compositions may also be applied in an in vitro or ex vivo manner, either to cells or patient grafts growing in culture, for example.
  • compositions disclosed herein may contain one or more additional agents that exert skin care activity.
  • the compositions disclosed herein can contain other active agents such as hyaluronic acid, niacinamide, phytantriol, farnesol, bisabolol, salicylic acid, retinol, retinoic acid, alphahydroxy acids, ascorbic acid and alguronic acid. It is expected that certain additional active agents will act synergistically with the bioactive peptide component, and/or will enhance the shelf-life of the formulation.
  • the peptides disclosed herein can be formulated in a number of carrier vehicles, for example, in a spray; an aerosol; a water and an oil-type emulsion; an oil and water-type emulsion; a face cream or body cream; a sun lotion or after-sun lotion; or other topical administration vehicle. Additionally, the peptides, and compositions containing them, may provide useful features for inclusion in general skin care and cosmetic formulations, such as various skin cosmetics, skin creams, lotions, sunscreens, and therapeutic lotions or creams. [0065] Areas for application
  • AGEs are toxic by-products of metabolism and are also acquired from diet, especially from high-temperature processed food. They promote oxidative damage to proteins, lipids and nucleotides. AGEs are strongly associated with chronic illnesses which account for about two-thirds of all premature deaths and 75% of all medical costs in the United States. AGE modified proteins are signaling molecules associated with several vascular and neurological complications. AGEs proved to be a marker of negative outcome that contribute to the pathophysiology of aging and long term complications of diabetes, atherosclerosis, osteoporosis, Alzheimer's disease, renal failure, and other chronic inflammatory conditions.
  • Diabetes is characterized by a high blood glucose level.
  • the chronic hyperglycemia increases basal rates of nonenzymatic glycation and higher serum AGE levels were detected in type 2 diabetic patients which are about 50% greater than that of healthy age-matched controls.
  • the subsequent loss of function of plasma proteins due to AGEs directly contributes to endothelial injury through irreversible glycation of collagen and other subendothelial structural proteins of the blood vessel.
  • AGE formation in ECM also interferes with matrix-cell interactions, with alterations in signaling and adhesion, which may be an important initial event in diabetic
  • AGE deposits have been found in atherosclerotic plaques and within myocardium fibers and associated with aortic stiffness and correlated with disease severity.
  • the cardiovascular complications are the leading cause of mortality in patients with diabetes mellitus.
  • Plasma AGEs such as CML and pentosidine levels has been shown to be an independent predictor of both re-hospitalization and mortality in heart failure patients.
  • Hyperglycemia induces the formation of AGE-modified low- density lipoprotein (LDL), resulting in a reduction in its plasma clearance therefore contributing to atherosclerosis.
  • LDL low- density lipoprotein
  • hypercoagulable phenotype also called hypercoagulable state.
  • the blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications.
  • hyperglycemia leads to reduced activity of the anticoagulant plasma protein antithrombin III, and methylglyoxal has been directly linked to such activity inhibition.
  • Other sources of dicarbonyls are in sugar-containing foods and beverages such as bread, coffee, honey, wine, and beer etc. These dicarbonyls including methylglyoxal are major precursors in the formation of intracellular AGEs in endothelial cells, triggers carbonyl stress and activates a series of inflammatory responses leading to
  • Diabetic skin ulcer is a major and increasing public health concern worldwide. It usually causes substantial morbidity, impairs quality of life, and results in high treatment costs for patients.
  • AGEs and dicarbonyl agents e.g., methylglyoxal
  • Both AGEs and free methylglyoxal contribute to delayed wound healing, which is associated with oxidative stress, keratinocyte injury, dysfunction and apoptosis, and defects in cell adhesion and endothelial dysfunction.
  • Hyperglycemia causes nephropathy through the formation of AGEs.
  • AGEs damage the kidneys through AGE- RAGE interaction, deposition of AGEs, or by in situ glycation. AGEs directly affect the structural integrity of the renal tissue through the cross-linking of matrix proteins (collagen). An inverse correlation has been evident between renal function and serum levels of AGEs. Chronic kidney disease and AGEs participate in what has been termed a "vicious cycle", as this condition is associated with increased oxidative stress and the production of reactive carbonyl compounds and AGEs. The ability of AGE to cross-link with ECM exacerbates glomerulosclerosis.
  • Activation of RAGE by circulating AGEs on mesangial cells, tubules, and podocytes all increase the production of intracellular reactive oxygen species and upregulation of transcription factor of nuclear factor kappa B, which further increase the production of some growth factors and inflammatory cytokines that contribute to the further decline of renal function.
  • AD Alzheimer's disease
  • AGEs have long been considered as potent molecules promoting neuronal cell death and contributing to neurodegenerative disorders.
  • AGEs co-localize with ⁇ in microglial cells in the brains of Alzheimer's individuals and a marked increase in AGE accumulation is observed at later stages of the disease, which is associated with the formation of insoluble deposits such as amyloid plaques and neurofibrillary tangles.
  • AGEs are also known to activate glia, resulting in inflammation and neuronal dysfunction. The interaction between AGEs and RAGE elicits intracellular oxidative stress, proinflammatory response and apoptosis all of which contribute to nerve cell damage.
  • the brain has high energy requirements and glucose is the main energy substrate for the brain.
  • Glucose utilization and glycolysis generate dicarbonyl by-products e.g., glyoxal, methyloglyxoal etc.
  • methyloglyoxal is one of the most potent glycating agents present in cells, making its accumulation highly deleterious.
  • the cerebrospinal fluid of AD patients shows high levels of methylogyoxal. Direct comparison of toxicity in primary cultures of mouse cortical neurons and astrocytes demonstrated that neurons are 6- fold more susceptible toward methylglyoxal toxicity than astrocytes.
  • Both type I and type II diabetes are known to compromise bone microstructure. Hyperglycemia and insulin resistance in type II diabetes induce osteoblast apoptosis and uncoupling of bone turnover. Advanced alterations of bone proteins by glycation are clearly detectable in osteoporotic bone.
  • AGE adducts form predominantly on the long-living and abundant matrix protein collagen type I (COL I).
  • the intermolecular cross-linking and side-chain modifications in COL I reduce the solubility and flexibility and increase the stiffness of the protein, thereby contributing to skeletal fragility. Elevated serum AGEs have been connected to poor grip strength in older community-dwelling women. AGEs also impair collagen's ability to dissipate energy.
  • AGEs have been implicated in vision loss associated with macular degeneration, cataract formation, diabetic retinopathy, and glaucoma.
  • AGEs have been detected at the site of the corneal epithelium and epithelial basement membrane in diabetic rats, monkeys, and humans. It was shown that AGEs were elevated in tears of diabetic patients. It was also evident that AGEs in ocular tissues mediate aberrant crosslinking of extracellular matrix proteins and disruption of endothelial junctional complexes that affect cell permeability and mediate angiogenesis and breakdown of the inner blood-retinal barrier. AGEs also severely affect cellular metabolism by disrupting ATP production, enhancing oxidative stress, and modulating gene expression of anti-angiogenic and anti-inflammatory genes. Treatment with aminoguanidine, an inhibitor of AGEs, prevented corneal structural abnormalities in diabetic rats.
  • apoptosis is a potential mechanism by which AGEs may exert effects.
  • AGEs induce apoptosis in retinal pericytes and corneal epithelium, and increases in corneal epithelial cell apoptosis contributes to delayed epithelial wound healing in diabetic cornea.
  • Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. Both glucose and ascorbic acid metabolism generate high levels of dicarbonyl compounds that participate in the glycation process of lens proteins.
  • Periodontitis is a globally prevalent inflammatory disease which causes the destruction of the periodontal structures (i.e. alveolar bone, periodontal ligament and root cementum) and potentially leads to tooth loss.
  • Type II diabetes patients have a 2- to 5-fold higher risk for periodontal diseases.
  • the chronic accumulation of AGEs in diabetes provides a potent and sustained stimulus to activation of the receptor RAGE and of tissue-destructive inflammatory cascades in diabetic periodontium. AGEs/RAGE interaction and activation are the primary concern in the development and progression of periodontitis.
  • CF related diabetes CFRD
  • CFRD patients have a higher mortality than CF alone and there is a strong association between CFRD and deterioration in lung function and clinical status.
  • the peptides disclosed herein can be formulated and delivered topically to the lung with or without the combination of other existing CF medications and provide an alternative to reduce the inflammatory status of the CFRD lung.
  • AGEs and receptor activation have been implicated to be responsible for the ovulation failure that characterizes polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the ovary is the main regulator of female fertility. Changes in maternal health and physiology can disrupt intraovarian homoeostasis thereby compromising oocyte competence and fertility. Both clinical and experimental research support the role of dicarbonyl overload and AGEs as key contributors to perturbations of the ovarian microenvironment leading to lower fertility.
  • women with PCOS have increased AGEs and RAGE expression in theca and granulosa cell layers compared with normal women.
  • the accumulation of AGE, pentosidine, and CML in the follicular fluid and AGE in serum negatively correlated with follicular growth, fertilization and embryonic development.
  • Maternal infection and inflammation are known risk factors for premature birth. For example, many premature infants are exposed to inflammatory stimuli even prior to birth, with additional inflammatory insults often occurring as a result of the subsequent resuscitation. Accordingly, the peptides disclosed herein can be administered to pregnant women to reduce inflammation associated with AGEs.
  • Psoriasis is a chronic inflammatory skin disease with a genetic
  • AGEs and anti-CML and anti-CEL antibodies were significantly higher in the active phase of psoriasis patients than those in healthy individuals.
  • patients with psoriasis vulgaris (PV) had significantly increased blood levels of total peroxide concentration (TPX) and methylglyoxal compared to healthy controls.
  • TPX total peroxide concentration
  • Methylgloxal serum level reflects increased oxidative stress as well as carbonyl stress and is positively correlated with psoriasis area and severity index.
  • Psoriasin was originally identified in psoriasis and its production is induced by reactive oxygen species. Psoriasin increases the expression of ROS and VEGF and acts through RAGE to promote endothelial cell proliferation.
  • the peptides disclosed herein may be used to treat or ameliorate osteoarthritis.
  • Aging and diabetes are known to be major causes which affect the progression of osteoarthritis (OA).
  • OA osteoarthritis
  • OA is a progressive degenerative joint disease with signs and symptoms of inflammation, including joint pain, swelling, and stiffness leading to significant functional impairment and disability in older adults.
  • Accumulation of AGEs in cartilage chondrocytes shows the decreased proteoglycan and collagen synthesis, which leads to stiffness and brittleness of the articular cartilage.
  • AGEs can also up-regulate the production of MMPs and IL-6 and IL-8 which mediate cartilage degradation, leading to joint destruction.
  • Rheumatoid arthritis is a chronic inflammatory disease of the synovium, a membrane lining the non-weight- bearing surfaces of the joint.
  • the peptides disclose herein may be used to treat or ameliorate loss of muscle mass and strength (sarcopenia).
  • Sarcopenia is a serious problem among older populations.
  • One third of women and half of men older than 60 suffer from sarcopenia in the U.S.
  • the pathogenesis of sarcopenia is multifactorial and may involve hormonal changes, oxidative stress and inflammation, changes in vasculature, and activity levels. It was shown that pentosidine concentrations were 200% higher in a group of older individuals with a mean age of 78 compared with their younger counterparts with a mean age of 25, suggesting therefore that AGEs may contribute to the decline of muscular function observed in aging.
  • the peptides disclose herein may be used to treat or ameliorate the risk of cancer.
  • a molecular link between glycotoxins and cancer biology involves the activation of an inflammatory cycle of persistent oxidative stress that favors cancer development.
  • In vitro studies of breast, prostate, and lung cancer cell lines imply a possible contribution of AGEs to cancer proliferation, migration, invasion, and survival.
  • Detection of AGEs e.g., CML and argpyrimidine, has been reported in various human tumors.
  • a higher accumulation of AGEs has been observed in malignant tissues (such as prostate cancer) compared to benign ones.
  • the peptides disclose herein may be used to treat or ameliorate skin aging.
  • skin aging One of the hallmarks of aging skin is the accumulation of AGEs resulted from the overlapping of an intrinsic chronological aging (individual, genetic) and of an extrinsic aging (external factors like UV, pollution, and lifestyle).
  • an intrinsic chronological aging individual, genetic
  • extrinsic aging external factors like UV, pollution, and lifestyle
  • the human skin becomes drier, thinner, spots appear, elasticity decreases and stiffening increases, together with the appearance of wrinkles over time.
  • glycation is involved in a very complex aging process and simultaneously affects, directly and indirectly, certain cells, their synthesis, and the organization of the matrix.
  • glycation affects cells including endothelial cells, fibroblasts, keratinocytes, and structural proteins such as collagen, elastin, glycoproteins, and glycosaminoglycans.
  • Glycation of collagens has been linked to the development of skin dullness and the decrease in skin elasticity and evidence supports that methylglyoxal induced AGEs deform the elasticity of the collagen molecules and reduce inherent collagen fibril viscoelasticity.
  • AGEs were reported to accumulate in dermal elastin and collagens and such glycation modified dermal ECM further affects growth,
  • AGEs Collagen cross-linking by AGEs has been increasingly implicated as a central factor in the onset and progression of connective tissue disease.
  • RAGE is highly expressed in the skin and upregulated by AGEs and TNF-a. In elderly subjects, the accumulation of AGEs causes decreased elasticity and increased stiffening.
  • Histological data indicate that changes in dermis including structural, biochemical, molecular, architectural, and functional changes start before the age of 35 and increase rapidly due to intrinsic processes and are amplified by UV exposure.
  • 35 is the age threshold that represents the best time for the initiation of anti- aging therapy, which may include a combination of products that both stimulate protein synthesis and inhibit the glycation process.
  • acetaldehyde, hexanal, glyoxal, and methylglyoxal were the dominant compounds in rainwater of an urban atmosphere. These carbonyl compounds are toxic and also formed in mainstream cigarette smoke. Both glyoxal and methylglyoxal are important elements in the cooking emissions.
  • a pilot hazardous airborne carbonyls study was carried out in Hong Kong and venue China. Workplace air samples in 14 factories of various types of manufacturing and industrial operations were collected and analyzed for a panel of 21 carbonyl compounds. The factories can be classified into five general categories, including food processing, electroplating, textile dyeing, chemical manufacturer, and petroleum refinery. The study found that glyoxal and methylglyoxal existed at variable levels in the selected workplaces, ranging from 0.2% to 5.5%.
  • these peptides not only effectively bind to AGEs and prevent AGE-RAGE interactions, but also effectively block dicarbonyl stress such as methylglyoxal-induced cell apoptosis and cell death, thereby blocking the signal transduction pathways that ultimately lead to cellular damage and cell death.
  • dicarbonyl stress such as methylglyoxal-induced cell apoptosis and cell death.
  • Such activity has not previously been reported or disclosed.
  • These peptides are novel as they are generated using an in silico design method which do not exist or occur in nature and the peptides produced by these methods cannot be isolated from microbes, animals, or plants.
  • the peptides disclosed herein significantly protect human skin keratinocytes against AGEs and precursors such as methyloglyoxal- induced apoptosis and improve fibroblast survival.
  • These peptides are promising therapeutic agents for neutralizing AGEs, alleviating AGE deposition, and blocking AGE-RAGE interactions and thus can be used for improving medical conditions associated with AGEs.
  • these peptides can be delivered systemically, topically, or via other routes, for the prevention of AGE formation as well as for the treatment, with and/or without the help of current available medical regimes, of AGE-associated complications and diseases.
  • the disclosed peptides effectively inhibit the Maillard reaction, as shown in Table 6, to block sugar and protein from forming AGEs.
  • These peptides can be used as Maillard reaction inhibitors, for example if mixed into foods and beverages containing proteins such as collagen, and therefore it may be possible to suppress the Maillard reaction to prevent the deterioration of the foods and beverages.
  • AGE-binding peptide binding to a mixture of various AGEs was conducted using an OxiSelect Advanced Glycation End Product (AGE) ELISA kit (Cat# STA-317) purchased from Cell BioLabs Inc (Atlanta GA).
  • This kit uses a standard AGE-BSA that was prepared by reacting BSA with glycolaldehyde followed by extensive dialysis and column purification. According to the product manual the AGE-BSA contains CML, pentosidine, and other AGE structures. The AGEs are probed with an anti-AGE polyclonal antibody, followed by an HRP- conjugated secondary antibody.
  • the current study design was that first AGE-BSA and a proper amount of peptide were mixed and the mixture was incubated at room temperature from 4-20 hours. Next the mixture was transferred to a 96-well protein binding plate supplied in the kit and incubated at +4°C for overnight. The ELISA assay was processed according to the manufacturer's instructions.
  • the principle of the experimental design is such that if a peptide binds to AGE it will compete against anti-AGE antibody for one or more binding sites.
  • AGE-BSA in the absence of a peptide competing for the AGE binding site, on the other hand, is expected to be fully accessible by the anti-AGE antibody, resulting in maximal reading which is taken as 100% binding/0% reduction.
  • the binding activity of a peptide to AGEs is calculated as percentage reduction, as indicated in the formula below.
  • the cutoff value was chosen to be equal or more than approximately 70-80% of the standard control of carnosine-induced activity and a value of ⁇ 30% is considered significant. The results are shown in Table 2. In most cases the peptides outperform the reference ("gold") standard carnosine.
  • N £ -(carboxyethyl) lysine (CEL) was evaluated using OxiSelectTM N £ -(carboxyethyl) lysine (CEL) ELISA kit (Cell BioLabs Inc., Atlanta, GA). Briefly, standard N £ -(carboxyethyl) lysine-BSA was diluted to 10 ⁇ g/ml and mixed with equal volume of 10 ⁇ g/ml reduced BSA to make a stock solution. Next, peptide (100 ⁇ g/ml) was added to an equal volume of the stock solution containing methylglyoxal-BSA and incubated at room temperature for 5hr.
  • Example 2 Activity of peptide to block methylglyoxal induced keratinocyte apoptosis
  • Human skin keratinocytes (ATCC CRL-2404, American Type Culture Collection, Manassas, VA, USA) were grown in serum-free keratinocyte growth media supplemented with 5ng/ml human recombinant epithelial growth factor (EGF) (Life TechnologiesTM, Grand Island, N.Y.). The cells were then trypsinized and seeded into a 96-well plate. The plate was incubated in 37°C, 5% C02 for 8hr to allow attachment of cells to the plate. Methylgloxal was purchased from Sigma-Aldrich (St. Louis, MO).
  • peptide 100 ⁇ g was pre-incubated with methylglyoxal (50 ⁇ g) for 6hr then transferred to a 96-well plate containing human keratinocytes. Carnosine was used as protective positive control. The cells were further incubated overnight prior to the apoptosis assay. Apoptotic cells release histone-associated DNA-fragments that were quantified using the Cell Death Detection ELISA P
  • the cell lysate was centrifuged then 20 ⁇ of the supernatant (cytoplasmic fraction) was carefully transferred into the streptavidin-coated plate; ELISA analysis then proceeded according to the manufacturer's instructions. A 20% protection/reduction in apoptosis was considered significant.
  • the cutoff value was chosen to be equal to or more than approximately 70-80% of the standard control carnosine-induced activity. Table 4 shows some peptides outperform the reference ("gold") standard carnosine.
  • Example 3 Protection of human skin fibroblast from methylglyoxal induced cell death
  • Human skin fibroblasts (ATCC CRL-7481 , American Type Culture Collection, Manassas, VA, USA) were grown in 96-well plates in Dulbecco's modified Eagle's medium (DMEM; 4 mM L-glutamine, 4.5 g/L glucose) adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum (FBS). After the cells reached >95% confluence the culture medium was replaced with fresh medium containing methylglyoxal (40ug/ml) and each peptide at 100ug/ml. The cells were incubated in a 37°C, 5% C02 incubator overnight and cell survival was measured using the XTT assay (from ATCC) according to the manufacturer's instructions.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Results are shown in Table 5. A protection of 10% and above is considered significant.
  • the cut off value was chosen to be equal to or more than approximately 70-80% of the standard control carnosine-induced activity and, as Table 5 shows, some peptides outperform carnosine.
  • Example 4 Activity of peptides to block AGE formation between sugar and collagen
  • Calf skin collagen (0.1 % solution in 0.1 M acetic acid) was purchased from Sigma-Aldrich. A 2xglycation stock solution was prepared which contained 3% glucose and 0.22% NaN3. Briefly, 300 ⁇ of collagen was mixed in a sterile 1.5ml Eppendorf tube with an equal volume of 2xglycation stock solution. Peptide was then added at final concentration of 2mg/ml. The mixture was incubated at 37°C for 3 month. Next, the mixture was diluted in PBS and the formation of AGE was quantified using the OxiSelect Advanced Glycation End Product (AGE) ELISA kit (Cat# STA-317) purchased from Cell BioLabs (Atlanta GA), and the results are shown in Table 6.
  • AGE OxiSelect Advanced Glycation End Product
  • Carnosine was used as a positive control which blocked AGE formation between collagen and sugar. Collagen alone in PBS was used as negative control. A value ⁇ 25 is considered significant. The cut off value was chosen to be equal to or more than approximately 70-80% of the standard control carnosine-induced activity. Some of the peptides outperform carnosine, as shown in Table 6.
  • Example 5 Formulation of the peptides in a lotion
  • a skin lotion is prepared from the following components according to the methods below:
  • the diluent (Item #1) is heated to 75°C. Items #2 through #5 (water soluble ingredients) are then added to the heated diluent. Items #6 through #1 1 (oil soluble ingredients) are separately mixed together. The mixture of oil soluble ingredients are slowly mixed with the mixture of water soluble ingredients and further mixed for thirty (30) minutes. The resulting mixture is then cooled to 25°C and fragrance (Item #12) is added after the temperature drops below 40°C. The pH and viscosity are checked and adjusted to a pH of 5.0-5.5 and a viscosity of 15,000-20,000. The peptides-containing suspension are then added to the resulting lotion.
  • Example 6 Formulation of the peptides in a cream
  • a skin cream is prepared according to the methods below:
  • 0.1-2% thickener such as natural wax or stearyl palmitate
  • 55-75% diluent such as water or water mixed with botanical extracts
  • An oil soluble phase is separately made by mixing 2-6% melted emulsifier (such as CreamMaker blend or a polysorbate) and 10-35% melted emollient (such as one or more plant oil or natural butter). The oil soluble mixture is then combined with the homogenous gel and stirred intensively until an emulsion is formed and then gently mixed while the emulsion is cooling.
  • Sensitive components like 0.1 %-10% active ingredients (peptide, anti-aging ingredients, skin whitening ingredients, antioxidants, etc.), 0.1-1.0% fragrances, and 0.1-1.0% preservatives (such as benzylalcohol-DHA) are added after the mixture is cooled (40-30°C) to keep their properties intact.
  • active ingredients peptide, anti-aging ingredients, skin whitening ingredients, antioxidants, etc.
  • fragrances 0.1-1.0%
  • preservatives such as benzylalcohol-DHA

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Abstract

Les produits finaux de glycation avancée (AGE) représentent une modification protéique post-traductionnelle de type non enzymatique. Les AGE sont très stables et leur accumulation dans le temps peut être utilisée comme marqueurs de stress carbonyle responsables de la malfonction de macromolécules bioactives dans le corps. La présente invention concerne une série de peptides courts capables de bloquer l'activité des produits finaux de glycation avancée (AGE), les peptides étant dérivés d'une région V d'une protéine sRAGE, ladite région V étant donnée par SEQ ID No: 61. Ces peptides inhibent efficacement l'avancement d'une réaction de Maillard, bloquant les AGE et inhibant l'apoptose et la mort cellulaires induites par les dicarbonyles. Les peptides peuvent être utilisés, par exemple, comme agent anti-glycation, agent anti-vieillissement de la peau, ou agent anti-complication diabétique.
PCT/US2017/059756 2016-11-03 2017-11-02 Peptides bioactifs courts bloquant l'activité des produits finaux de glycation avancée, compositions et procédés d'utilisation Ceased WO2018085565A2 (fr)

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JP7166039B1 (ja) * 2020-12-22 2022-11-07 株式会社マルハチ村松 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法
CN120554487A (zh) * 2025-05-12 2025-08-29 广东医科大学 一种糖化改性i型胶原蛋白及其制备方法和应用

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FR2815541B1 (fr) * 2000-10-24 2008-02-29 Lipha Utilisation d'inhibiteurs de l'apoptose des pericytes pour le traitement et/ou la prevention de la retinopathie diabetique
US20050118688A1 (en) * 2001-12-28 2005-06-02 Hudson Freeze Novel ligand involved in the transmigration of leukocytes across the endothelium and uses therefor
US8088734B2 (en) * 2003-01-21 2012-01-03 Unigene Laboratories Inc. Oral delivery of peptides
US20080207499A1 (en) * 2005-06-29 2008-08-28 Gaetano Barile Rage-related methods for treating and preventing diabetic retinopathy
US20100040623A1 (en) * 2005-12-20 2010-02-18 Elisabeth Bock Neuritogenic and neuronal survival promoting peptides derived from the family of s-100 proteins
US20100254983A1 (en) * 2007-06-07 2010-10-07 Ann Marie Schmidt Uses of rage antagonists for treating obesity and related diseases
MX2010012142A (es) * 2008-05-09 2011-04-05 Abbott Gmbh & Co Kg Anticuerpos para receptor de productos finales de glucacion avanzada (rage) y usos de los mismos.

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7166039B1 (ja) * 2020-12-22 2022-11-07 株式会社マルハチ村松 ペプチド、細胞増殖促進剤、タンパク質産生促進剤、培地、該ペプチドを用いた細胞増殖方法、及び、該ペプチドを用いたタンパク質産生方法
CN120554487A (zh) * 2025-05-12 2025-08-29 广东医科大学 一种糖化改性i型胶原蛋白及其制备方法和应用

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