Composition containing peptide WKDEAGKPLVK

Abstract

To provide an alternative that helps muscle recovery, maintenance and / or muscle growth.SOLUTION: A composition comprises a peptide of SEQ ID NO: 1, or a glucose transport-promoting variant of SEQ ID NO: 1 with 1 to 3 amino acid alterations relative to SEQ ID NO: 1. The amino acid alterations occur in one or more positions of positions 1, 2, 3, 9, 10 and 11 of SEQ ID NO: 1. The amino acid alterations in the variant include insertion, addition, deletion and substitution of amino acids.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This invention relates to cosmetic compositions and pharmaceutical compositions. [Background technology]

[0002] Many compositions are described. Improvements are always desired.

[0003] Currently, various methods exist to improve muscle health or muscle glucose absorption. However, it is desirable to discover alternatives that can aid in muscle recovery, maintenance, and / or muscle growth.

[0004] In particular, as people now live longer, healthier lives, the growing desire to maintain a youthful appearance is indeed leading to more and more research into new cosmetics and dermatological procedures to treat skin aging. In recent years, there has been growing interest in minimally invasive treatments and techniques designed to address issues such as wrinkles, weight loss, and other skin damage. The most common topical anti-aging products are creams and serums.

[0005] U.S. Patent Application Publication No. 2004 / 0132667 discloses a composition comprising a peptide, which can be optionally combined with one or more further components. The disclosed composition provides relief of one or more skin conditions, including skin conditions caused by a variety of factors such as stress, pollution, and general aging.

[0006] U.S. Patent Application Publication No. 2014 / 0120141 discloses cosmetic and pharmaceutical compositions containing peptides for use in the treatment and / or care of skin and / or mucous membrane conditions, disorders, and / or diseases. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2004 / 0132667 [Patent Document 2] U.S. Patent Application Publication No. 2014 / 0120141 [Patent Document 3] European Patent Application Publication No. 2050437 [Patent Document 4] International Publication No. 2005 / 023290 Pamphlet [Patent Document 5] U.S. Patent Application Publication No. 2010 / 098660 [Patent Document 6] U.S. Patent Application Publication No. 2007 / 0053845 [Patent Document 7] U.S. Patent Application Publication No. 2014 / 120131 [Patent Document 8] U.S. Patent Application Publication No. 2004 / 132667 [Patent Document 9] U.S. Patent No. 4,186,183 [Patent Document 10] U.S. Patent No. 4,217,344 [Patent Document 11] U.S. Patent No. 4,235,871 [Patent Document 12] U.S. Patent No. 4,261,975 [Patent Document 13] U.S. Patent No. 4,485,054 [Patent Document 14] U.S. Patent No. 4,501,728 [Patent Document 15] U.S. Patent No. 4,774,085 [Patent Document 16] U.S. Patent No. 4,837,028 [Patent Document 17] U.S. Patent No. 4,235,871 [Patent Document 18] U.S. Patent No. 4,261,975 [Patent Document 19] U.S. Patent No. 4,485,054 [Patent Document 20] U.S. Patent No. 4,501,728 [Patent Document 21] U.S. Patent No. 4,774,085 [Patent Document 22] U.S. Patent No. 4,837,028 [Patent Document 23] U.S. Patent No. 4,946,787 [Patent Document 24] PCT International Open Brochure No. 91 / 17424 [Patent Document 25] U.S. Patent Application Publication No. 2014120141 [Patent Document 26] U.S. Patent No. 4,766,106 [Patent Document 27] U.S. Patent No. 4,179,337 [Patent Document 28] U.S. Patent No. 4,495,285 [Patent Document 29] U.S. Patent No. 4,609,546 [Patent Document 30] U.S. Patent No. 3,654,090 [Patent Document 31] U.S. Patent No. 3,850,752 [Patent Document 32] U.S. Patent No. 4,016,043 [Patent Document 33] U.S. Patent No. 6,165,779 [Patent Document 34] U.S. Patent No. 6,225,289 [Patent Document 35] U.S. Patent No. 5,708,025 [Patent Document 36] U.S. Patent No. 5,994,106 [Patent Document 37] Brochure for International Patent Application Publication No. 98 / 32859 [Patent Document 38] International Publication No. 00 / 55119 Pamphlet [Patent Document 39] U.S. Patent No. 5,656,722 [Patent Document 40] U.S. Patent No. 5,750,497 [Patent Document 41] U.S. Patent No. 6,251,856 [Patent Document 42] U.S. Patent No. 6,268,335 [Patent Document 43] U.S. Patent No. 6,451,970 [Patent Document 44] International Publication No. 01 / 62218 Pamphlet [Patent Document 45] International Publication No. 98 / 07744 [Patent Document 46] International Publication No. 99 / 18927 Pamphlet [Non-patent literature]

[0008] [Non-Patent Document 1] Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis [Non-Patent Document 2] O'Riordan et al(Respir Care,2002,Nov.47) [Non-Patent Document 3] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller [Non-Patent Document 4] Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro edit. 1985) [Non-Patent Document 5] JMStewart and JDYoung, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984)

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[0009] An object of the present invention is to overcome at least one of the problems mentioned above and to provide a composition. [Means for solving the problem]

[0010] A first aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 (hereinafter referred to as "the peptide of the present invention").

[0011] A second aspect of the present invention provides a composition comprising an effective amount of the peptide of the present invention (hereinafter referred to as "the composition of the present invention"). Preferably, the composition is a topical composition. Typically, the composition may be a cosmetic composition containing a cosmetically effective amount of peptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof. Suitablely, the composition may be a pharmaceutical composition comprising a pharmaceutically effective amount of a peptide or a variant thereof containing the amino acid sequence of SEQ ID NO: 1. Preferably, the peptide is a bioactive peptide. Ideally, the composition comprises multiple peptides. Typically, the composition further comprises at least one cosmetically or pharmaceutically acceptable excipient or additive. Typically, the composition further comprises at least one cosmetically or pharmaceutically acceptable active ingredient. Ideally, the peptide contains the amino acid sequence consisting of SEQ ID NO: 1. Typically, the peptide contains about 3 to 50 amino acids in length. Preferably, it contains about 5 to about 20 amino acids in length, and preferably about 11 amino acids in length.

[0012] The present invention also relates to a fragment of SEQ ID NO: 1 having a length of at least three amino acids. Typically, such fragments are bioactive. Preferably, the fragment has a length of at least 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. Preferably, the peptide or fragment has one or more activities selected from anti-aging activity, glucose transport-promoting activity, cell proliferation-promoting activity, or anabolic activity. The activity may be cosmetic activity (i.e., non-therapeutic activity), therapeutic activity, or both. Ideally, the peptide or fragment possesses anti-aging activity. Ideally, the peptide or fragment possesses glucose transport-promoting activity. Appropriately, the peptide or fragment has cell proliferation-promoting activity. Appropriately, peptides or fragments possess anabolic activity. Typically, anabolic activity is muscle growth activity. Peptides or fragments stimulate anabolic metabolism in mammals.

[0013] Preferably, the peptide or fragment exhibits GLUT4 transport-promoting activity. Preferably, the peptide or fragment exhibits increased protein synthesis (stimulation of anabolic metabolic activity).

[0014] More preferably, the peptide comprises (or consists of) any one amino acid sequence from SEQ ID NOs: 1 to 85, in which case the peptide typically has anti-aging activity. More preferably, the peptide comprises (or consists of) any one amino acid sequence from SEQ ID NOs: 1 to 85, in which case the peptide typically has glucose transport-promoting activity. More preferably, the peptide comprises (or consists of) any one amino acid sequence from SEQ ID NOs: 1 to 85, in which case the peptide typically has cell proliferation-promoting activity. More preferably, the peptide comprises (or consists of) any one amino acid sequence from SEQ ID NOs: 1 to 85, in which case the peptide typically has anabolic activity. More preferably, the peptide comprises (or is essentially composed of) one of the amino acid sequences from SEQ ID NOs: 1 to 85, in which case the peptide typically exhibits GLUT4 translocation-promoting activity. More preferably, the peptide comprises (or is essentially composed of) one of the amino acid sequences from SEQ ID NOs: 1 to 85, in which case the peptide typically exhibits increased protein synthesis (stimulation of anabolic metabolic activity). More preferably, the peptide comprises (or is essentially composed of) one of the amino acid sequences from SEQ ID NOs: 1 to 85, in which case the peptide typically exhibits cell proliferation-promoting activity.

[0015] A further aspect of the present invention provides a non-therapeutic use of the composition of the present invention as a cosmetic.

[0016] A further aspect of the present invention provides a composition of the present invention as a pharmaceutical.

[0017] The following embodiments relate to cosmetics, and may be non-therapeutic or therapeutic.

[0018] Aspects of the present invention provide compositions for use in methods for anti-aging.

[0019] A further aspect of the present invention provides compositions of the present invention for use as pharmaceuticals.

[0020] The present invention also provides peptides or compositions for use in methods for slowing, inhibiting, or preventing the aging of human skin. Typically, administration may be by means of a plaster or patch or formulation suitable for topical application.

[0021] A further aspect of the present invention provides peptides or compositions of the present invention for use in methods for promoting tissue growth. The present invention also provides peptides or compositions for use in methods for promoting the proliferation of epithelial tissue. The present invention also provides peptides or compositions for use in methods for promoting skin proliferation. The present invention also provides peptides or compositions for use in methods for promoting organ growth. The present invention also provides peptides or compositions for use in methods for promoting the growth of organisms. Preferably, cells, tissues, or organisms have a normal pathological condition (e.g., aging skin). Typically, cells, tissues, or skin have an abnormal pathological condition (e.g., tissue damaged due to trauma, drug use, or gastrointestinal epithelial tissue damaged due to inflammatory disorders). Growth-promoting uses may be in vivo or in vitro. Growth-promoting uses may involve administration to the external (i.e., skin) or internal (i.e., gastrointestinal tract) of mammals.

[0022] Further aspects of the present invention provide peptides or compositions for use in methods for improving muscle condition in mammals.

[0023] Further aspects of the present invention provide peptides or compositions for use in methods for promoting muscle recovery, typically after physical exercise.

[0024] Further aspects of the present invention provide peptides or compositions for use in methods for maintaining or restoring muscle health (e.g., lean tissue mass) in mammals.

[0025] A further aspect of the present invention provides peptides or compositions for use in methods for enhancing physical performance by topical administration.

[0026] Further aspects of the present invention provide peptides or compositions for use in methods for treating or preventing diseases or conditions characterized by lethargy or low energy levels.

[0027] Further aspects of the present invention provide peptides or compositions for use in methods for muscle growth or muscle enhancement. Compositions of the present invention can be used in methods for increasing lean muscle mass in beef cattle and other farm animals.

[0028] A further aspect of the present invention provides peptides or compositions of the present invention for use in methods for increasing protein synthesis.

[0029] Further aspects of the present invention provide peptides or compositions for use in methods for treating muscle weakness.

[0030] Another aspect of the present invention provides peptides or compositions of the present invention for use in methods for treating wounds in mammals.

[0031] Further embodiments of the present invention provide peptides or compositions for use in methods for treating or preventing pain in mammals.

[0032] Another aspect of the present invention provides peptides or compositions for use in methods for treating or preventing diseases or conditions characterized by damage to epithelial cells or tissues, and / or damage to dermal or epithelial cells or tissues. Preferably, the disease or condition is characterized by damage to dermal or epithelial cells or tissues and is selected from cancer, trauma, etc.

[0033] Further aspects of the present invention relate to a method for treating, preventing, or caring for any one of the diseases, conditions, and / or disorders described herein, comprising the step of administering a peptide or composition of the present invention. The composition can be administered topically.

[0034] The use of the present invention may be therapeutic or cosmetic, i.e., non-therapeutic.

[0035] Further aspects of the present invention provide peptides or compositions of the present invention for use in methods of treating or preventing metabolic disorders in patients. In one embodiment, the metabolic disorder is a disease or syndrome characterized by a decrease in insulin response or production in a patient. In one embodiment, the metabolic disorder is selected from diabetes and prediabetes. The present invention also provides peptides or compositions of the present invention for use in treating or preventing diseases or conditions selected from hyperlipidemia, obesity, hypertension, appetite-related syndromes, diabetes-related syndromes or diabetes-related conditions (e.g., reduced rates of stroke, heart disease, kidney disease, blindness, and / or loss of sensation in the limbs, associated with glucose levels and / or diabetes), and syndrome X.

[0036] Further embodiments of the present invention provide peptides or compositions for use in methods of treating diseases or conditions characterized by muscle wasting, reduction of lean muscle mass, reduced anabolic metabolism, catabolic wasting, or reduced protein synthesis. In one embodiment, the disease or condition is selected from sarcopenia or cachexia. In one embodiment, the patient to be treated is elderly (i.e., over 60 years of age), and the disease is age-related sarcopenia or cachexia. In one embodiment, the patient has cancer. In one embodiment, the patient has lethargy.

[0037] Further aspects of the present invention provide peptides or compositions for use in methods of treating or preventing diseases or conditions characterized by damage to epithelial cells or tissues, and / or damage to dermal or epithelial cells or tissues. Preferably, the disease or condition is characterized by damage to epithelial cells or tissues and is selected from cancer, trauma, drug use-induced tissue damage, or gastrointestinal epithelial tissue damage-induced by inflammatory disorders.

[0038] Further aspects of the present invention provide peptides or compositions for use in methods for maintaining or restoring muscle health (e.g., lean tissue mass) in mammals.

[0039] Further aspects of the present invention provide peptides or compositions for use in methods of treating or preventing diseases or conditions characterized by lethargy or low energy levels.

[0040] Another aspect of the present invention provides peptides or compositions for use in methods of treating wounds in mammals.

[0041] Further embodiments of the present invention provide peptides or compositions for use in methods for treating or preventing pain in mammals.

[0042] Another aspect relates to topical compositions of the present invention for use in the treatment or prevention of metabolic disorders in mammals. Metabolic disorders may include diabetes mellitus. In one embodiment, the use of the present invention includes the step of administering a therapeutically effective amount of the peptide of the present invention. In one embodiment, the composition of the present invention is administered systemically. In one embodiment, the composition of the present invention (particularly the pharmaceutical composition) is formulated for oral or parenteral administration. Other methods of administration are also described herein.

[0043] A further aspect of the present invention relates to a composition comprising one or more peptides selected from SEQ ID NOs: 1 to 85. In one embodiment, the composition of the present invention comprises substantially all of the peptides of SEQ ID NOs: 1 to 85.

[0044] Further aspects of the present invention relate to artificial treatment compositions comprising the compositions of the present invention. Preferably, the treatment composition is an anti-aging composition. Preferably, the treatment is a muscle treatment composition. In embodiments, the composition is a topical composition. In one embodiment, the composition comprises a cream, gel, lotion, rub, or powder. Preferably, the treatment is a wound treatment composition.

[0045] The present invention also relates to plasters, bandages, or dressing materials that are suitable for application to keratinous tissue or wounds and contain the peptide or composition of the present invention. Preferably, the composition or product is artificial.

[0046] (definition) All publications, patents, patent applications, and other references referenced herein are incorporated herein by reference in whole, for all purposes, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and fully enumerated content.

[0047] As used herein, and unless otherwise specifically indicated, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings that the terms may have in the art:

[0048] Unless otherwise required by the context, the use of the singular form in this specification shall be interpreted as including the plural form and vice versa. The terms “indefinite article (a)” and “indefinite article (an)” used in relation to entities shall be interpreted as referring to one or more of these entities. Thus, in this specification, the terms “indefinite article (a)” (or “indefinite article (an)”), “one or more,” and “at least one” are used interchangeably.

[0049] Throughout this specification, the word “comprise,” or its variations such as “comprises,” or “comprises,” shall be read as referring to the inclusion of an enumerated integer (e.g., features, elements, characteristics, properties, methods / process steps, or limits) or group of integers (e.g., multiple features, elements, multiple characteristics, multiple methods / process steps, or multiple limits), and not to the exclusion of any other integer or group of integers. Therefore, as used herein, the term “comprises” is inclusive or open-ended and does not exclude further, unenumerated integers or methods / process steps.

[0050] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, disease, abnormality, pathology, illness, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology (or, in fact, whether the etiological basis of the disease is established). Thus, it includes conditions resulting from infection, trauma, injury, surgery, radiation ablation, poisoning, or malnutrition.

[0051] As used herein, the terms “treatment” or “treating” refer to an intervention (e.g., administration of a drug to a subject) that cures, improves, or alleviates the symptoms of a disease, or eliminates its cause (or reduces its effects) (e.g., reduction of the accumulation of lysosomal enzymes at pathological levels). In this context, the term is used synonymously with the term “therapy.”

[0052] In addition, the terms “treatment” or “treating” refer to an intervention (e.g., administering a drug to a subject) that prevents, delays, or reduces (or eradicates) the onset or progression of a disease within the treated population. In this case, the term “treatment” is used synonymously with the term “prevention.”

[0053] When used herein, the effective dose or therapeutic dose of a drug is defined as the amount that can be administered to a subject in proportion to a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic response, or other problems or complications, and is sufficient to produce the desired effect, such as a treatment or prevention manifested by a permanent or temporary improvement of the subject's condition. The dose will vary from subject to subject depending on the individual's age and overall condition, method of administration, and other factors. Therefore, it is impossible to specify an exact effective dose, but a person skilled in the art will be able to determine an appropriate “effective” dose in any individual case using prescribed experiments and general background knowledge. Therapeutic outcomes in this context include eradication or relief of symptoms, reduction of pain or discomfort, extension of survival, improvement of movement, and other markers of clinical improvement. Therapeutic outcomes do not necessarily have to be a complete cure.

[0054] In this specification, the term “composition” shall be understood to mean something that is made by human hands and does not contain any natural compositions.

[0055] As used herein, the term “bioactive” refers to a peptide or fragment that possesses biological activity. For example, biological activity may be one or more of the following: glucose transport-promoting activity, growth-promoting activity, anti-aging activity, anabolic activity, anabolic metabolism-promoting activity, GLUT4 transport-promoting activity, and protein synthesis-promoting activity. Activity may be anti-inflammatory activity or antibacterial activity. Activity may be antioxidant activity.

[0056] When applied to peptides or fragments, "glucose transport enhancement" or "glucose transport enhancement activity" means peptides, variants, or fragments capable of increasing glucose uptake into cells in the glucose uptake assays described below.

[0057] The terms "GLUT4 transport enhancement" or "GLUT4 transport enhancement activity" applied to peptides or fragments refer to peptide fragments capable of increasing GLUT4 transport to skeletal muscle compared to untreated controls in the in vitro assays described below.

[0058] "Anti-aging" means inhibiting or delaying the onset of aging in human skin, and / or reversing the onset of aging. "Delaying or inhibiting skin aging" means delaying or inhibiting the aging process in the skin, and / or reversing the onset of aging. In one embodiment, "anti-aging" or "anti-aging activity," applied to a peptide or fragment, means a peptide or fragment capable of increasing collagen production or elastin production in human dermal fibroblasts compared to an untreated control, as examined in the in vitro assays described below, and / or a peptide or fragment capable of increasing cell proliferation in the cell proliferation assays described below.

[0059] When applied to peptides or fragments, “promoting cell proliferation” means peptides, variants, or fragments capable of increasing elastin production, collagen production, or cell proliferation in the assays described below. Peptides that increase cell proliferation or possess “promoting cell proliferation activity” are anti-aging peptides or anti-aging variants.

[0060] The term "increased protein synthesis" as applied to the peptides of this invention means peptides that significantly increase the degree of MTOR phosphorylation in the phospho-MTOR assay described below, or peptides that significantly increase the degree of protein synthesis in the puromycin assay described below. Peptides and compositions that increase protein synthesis can be used to stimulate anabolic metabolism in mammals and / or treat diseases or conditions characterized by catabolic exhaustion.

[0061] The term "anti-inflammatory" applied to peptides or fragments means that when macrophages are treated with 100 μM of the peptide, variant, or fragment, the peptide or fragment is capable of significantly reducing TNFα secretion by LPS-stimulated J774.2 macrophages (compared to untreated LPS-stimulated J774.2 macrophages). J774.2 macrophages were treated with 100 μM of the synthetic peptide for 24 hours, then stimulated with either (A) LPS (10 ng / ml) for 5 hours, or (B) LPS (10 ng / ml) for 5 hours, followed by stimulation with ATP (5 mM) for 1 hour. The supernatant was collected, and TNFα levels were determined by ELISA.

[0062] The terms "antimicrobial" or "antimicrobial activity" applied to peptides or fragments mean peptides, variants, or fragments capable of visually inhibiting bacterial growth in the following agar plate-based growth inhibition assays: peptide stock solution = 5 mg / mL dissolved in DMSO. Bacterial inoculum was prepared to McFarland 0.5 standard and spread onto MHA plates. Blank disks were placed in the plates and 10 samples of each compound (at the maximum test concentration of 64 μg / mL) were added. The plates were incubated at 37°C for 16–18 hours. Appropriate controls (DMSO; Mueller-Hinton medium alone; and two antibiotic disks: ciprofloxacin and tetracycline) were also prepared.

[0063] The term "antioxidant activity" as applied to the peptides of this invention means a peptide exhibiting antioxidant activity determined by the DPPH radical capture assay described below. This invention relates to the use of the peptides or compositions of this invention as antioxidants and in the treatment or prevention, or slowing of the progression of, diseases or conditions characterized by oxidative stress (or an imbalance between prooxidative and antioxidant factors). Examples include cardiovascular diseases (particularly atherosclerosis), cancer, neurodegenerative diseases (i.e., Parkinson's disease, Alzheimer's disease, and ALS), cataract formation, diabetes, and rheumatoid arthritis. Oxidation, the same chemical reaction that causes iron to rust, similarly plays a role in corrosion in our bodies. The process is called oxidative stress. Numerous studies have demonstrated that because these diseases are mediated by oxidative stress and the loss of balance between prooxidative and antioxidant factors, antioxidants can play a crucial role in preventing or slowing the progression of these conditions. Several studies have shown a link between decreased dietary antioxidant intake and an increased incidence of heart disease. Conversely, individuals with higher blood levels of antioxidants have a lower risk of heart disease. For example, as seen in a study of nurses, those who regularly consumed large amounts of vitamin E had a 41% lower incidence of heart disease than those who consumed small amounts. Increasing dietary antioxidant vitamins may reduce the risk of heart disease by 20-30%.

[0064] Cancer kills millions of people worldwide. Diet can be a contributing factor in up to 35% of all human cancers. Low levels of antioxidants in the diet can also be a contributing factor.

[0065] Promoting oxidants, or substances that generate free radicals, stimulate cell division, initiating mutagenesis and tumorigenesis. When cells with damaged DNA strands divide, they result in disrupted and deformed clusters of cells, which then form cancer.

[0066] Antioxidants are • Reduces oxidative damage to DNA, • To reduce the abnormal increase in cell division. This provides them with protective effects.

[0067] In addition, smoking and chronic inflammation contribute to the generation of powerful free radicals, which is thought to be the cause of many cancers. Some studies indicate that smokers have lower antioxidant levels than non-smokers, which increases the risk of cancer in smokers.

[0068] The respiratory system is a well-known target of free radical attack. This stems from endogenous factors as well as exposure to air pollutants and toxins, and cigarette smoke. Recent studies suggest that free radicals may be involved in the development of lung disorders, such as asthma. Antioxidants are thought to reduce the incidence of asthmatic symptoms. Supplementation with vitamins C, E, and beta-carotene is associated with improved lung function.

[0069] Free radicals can also damage nerves and the brain. Nerve tissue is particularly susceptible to oxidative damage. This is because the brain receives overwhelmingly the majority of oxygen and contains large amounts of polyunsaturated fatty acids, which are highly prone to oxidation and oxidative damage.

[0070] Diseases in which oxidative stress is suggested to be involved include: Alzheimer's disease Parkinson's disease • Dementia, etc. Includes.

[0071] Cataract formation is thought to involve damage to lens proteins by free radicals, which leads to clouding of the lens. Cataract formation can be delayed by the regular intake of antioxidants in supplements, such as vitamin E, vitamin C, and carotenoids. Other diseases, such as diabetes and rheumatoid arthritis, are also associated with low levels of antioxidants in the blood.

[0072] The term “topical composition” refers to a composition that is a formulation for topical administration. “Topical administration” refers to application to keratinous tissue, such as skin, hair, and nails. Topical delivery generally means delivery to the skin, but may also mean delivery to body cavities lined with epithelial cells, such as the lungs or airways, the digestive tract, and the oral cavity. In particular, formulations for topical delivery are described in their complete form in *Topical drug delivery formulations* edited by David Osborne and Antonio Aman, Taylor & Francis, which is incorporated herein by reference. Compositions or formulations for delivery to the airways are described in O'Riordan et al (Respir Care, 2002, Nov. 47), European Patent Application Publication No. 2050437, International Publication No. 2005023290, U.S. Patent Application Publication No. 2010 / 098660, and U.S. Patent Application Publication No. 2007 / 0053845. Compositions and formulations for delivering active agents to the ileum, particularly the proximal ileum, include microparticles and microcapsules in which the active agent is encapsulated within a protective matrix formed from polymers or dairy proteins that are acid-resistant but tend to dissolve in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. An alternative means of transdermal administration is by the use of skin patches. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. Similarly, the active ingredient can be incorporated into an ointment consisting of a white wax or white soft paraffin base at a concentration between 1 and 10% by weight, together with any required stabilizers and preservatives. Injectable forms may contain between 10 and 1000 mg, preferably between 10 and 250 mg, of the active ingredient per dose.

[0073] The composition may be formulated in unit dosage forms, that is, in the form of individual parts containing a unit dose, or multiple unit doses or subunits of unit doses.

[0074] As used herein, the term “cosmetic composition” refers to a composition that may be used for cosmetic, personal care, and / or hygienic purposes. A composition may have two or more cosmetic purposes, and it will be understood that it may be used for two or more of these purposes simultaneously. As used herein, “cosmetics” may include, but are not limited to, lipsticks, mascaras, lip glosses, foundations, blushes, eyeliners, face powders and body powders, sunscreens, sunblocks, nail polishes, compacts, solids, and pencils.

[0075] "Pharmaceutical Compositions": Further aspects of the present invention relate to pharmaceutical compositions comprising the peptides of the present invention, or compositions of the peptides of the present invention mixed with one or more pharmaceutically acceptable diluents, excipients, or carriers. The peptides and compositions of the present invention may be administered alone, but will generally, and especially for human therapeutics, be administered in mixture with pharmaceutical carriers, excipients, or diluents. Pharmaceutical compositions may be compositions for human or animal use in human and veterinary medicine. Examples of such excipients suitable for a variety of different forms of the pharmaceutical compositions described herein can be found in Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ Weller. In particular, formulations for topical delivery are described in their complete form in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical technology field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be made in light of the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may include, or in addition to, any suitable binders, lubricants, suspending agents, coatings, or solubilizers as carriers, excipients, or diluents.Suitable binders include natural sugars such as starch, gelatin, glucose, anhydrous lactose, fluid lactose, and β-lactose, corn sweeteners, natural and synthetic gums such as acacia and tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include esters of sodium benzoate, sorbic acid, and hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0076] The term “mammal” is understood to mean higher mammals, in particular humans. However, the term also includes non-mammals, such as fish. Humans can be infants, toddlers, children, young adults, adults, or elderly humans. In one embodiment, human is, for example, an elderly person aged 55 years or older. In one embodiment, human is an elderly person experiencing a decrease in lean body mass. In one embodiment, human is an athlete. In one embodiment, human is a pregnant woman. In one embodiment, human suffers from lethargy or lack of energy.

[0077] As used herein, the term “dermatologically acceptable” means that a topical composition or component of a composition is suitable for use in contact with human skin or keratinous tissue without hazards such as toxicity, incompatibility, instability, and / or allergic reactions.

[0078] The term "sustained release" is used in the conventional sense in relation to the delivery system of a compound or active ingredient, resulting in the gradual release of the compound or active ingredient over a period of time. Preferably, though not necessarily, the gradual release of the compound or active ingredient occurs at a relatively constant compound release level over a period of time.

[0079] As used herein, the term “peptide” typically refers to a polymer composed of monomers of up to 50 amino acids, e.g., 5 to 50 amino acids, linked via peptide bond linkages. The peptides of the present invention and peptides for use in the present invention (including their fragments and variants) can be produced entirely or partially by chemical synthesis or by expression from nucleic acids. For example, the peptides of the present invention and peptides for use in the present invention can be readily prepared according to well-established, standard liquid-phase peptide synthesis methods known in the art, or preferably solid-phase peptide synthesis methods (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), and M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)). If necessary, any of the peptides used in the present invention can be chemically modified to increase their stability. Chemically modified peptides or peptide analogs include any functional chemical equivalent of a peptide, characterized in relation to the implementation of the invention by increased stability and / or efficacy in vivo or in vitro. The term peptide analog also refers to any amino acid derivative of the peptides described herein. Peptide analogs can be prepared by procedures including, but not limited to, modification of the side chain, incorporation of non-natural amino acids and / or their derivatives during peptide synthesis, and the use of crosslinking agents and other methods to impart conformational constraints to the peptide or its analog.Examples of side-chain modifications include reductive alkylation by reaction with aldehydes and subsequent reduction with NaBH4; amidation with methyl acetimidate; acetylation with acetic anhydride; carbamylation of the amino group with cyanates; trinitrobenzylation of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS); alkylation of the amino group with succinic anhydride and tetrahydrophthalic anhydride; and modification of the amino group, such as pyridoxylation of lysine by reduction with pyridoxa-5'-phosphate and subsequent reduction with NaBH4. The guanidino group of the arginine residue can be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. The carboxyl group can be modified by carbodiimide activation by o-acyl isourea formation and subsequent derivatization (e.g., derivatization to the corresponding amide). The sulfhydryl group can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performation of cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromerclybenzoate, 4-chloromerclyphenylsulfonic acid, phenylmercury chloride, 2-chloromerclyc-4-nitrophenol, and other mercury compounds; and carbamylation of cyanates at alkaline pH. The tryptophan residue can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or halogenated sulfonyl. The tyrosine residue can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives. Modification of the imidazole ring of a histidine residue can be achieved by alkylation with an iodoacetic acid derivative or by N-carboethoxylation with diethyl pyrocarbonate.Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or the use of D-isomers of amino acids. Peptide structure modification includes the creation of retroinversopeptides containing the reverse sequence encoded by the D-amino acid. Changes may be those that reduce susceptibility to proteolysis, reduce susceptibility to oxidation, alter the binding affinity of the mutant sequence (typically, increasing affinity is desired), and / or confer or modify other physicochemical or functional properties to the associated mutant / analog peptide.

[0080] As used herein, the term “peptide containing the amino acid sequence of SEQ ID NO: 1” typically refers to a polymer composed of monomers of up to 50 amino acids, e.g., 5 to 50 amino acids, linked via peptide bond linkages, and containing an amino acid sequence essentially identical to SEQ ID NO: 1, or a variant essentially derived from SEQ ID NO: 1 (hereinafter referred to herein as a “variant” or “peptide variant”) that is substantially identical to SEQ ID NO: 1 but modified by a change in one or more amino acid residues, e.g., one, two, or three residues. Preferably, such modifications involve insertions, additions, deletions, and / or substitutions of 11 or fewer amino acids, more preferably 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, preferably 5 or fewer, 4 or fewer, even more preferably 3 or fewer amino acids, most preferably just one or two amino acids. Insertions, additions, and substitutions by native and modified amino acids are assumed. The peptide may have a conserved amino acid change in which the introduced amino acid is structurally, chemically, or functionally similar to the substituted amino acid. Generally, peptides will have at least 70% amino acid sequence identity, preferably at least 80%, more preferably at least 90%, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity with the parent sequence.

[0081] The term "fragment" is understood to mean a segment of amino acid sequence number 1. Typically, fragments have a length between 3 and 10 consecutive amino acids. Generally, fragments have a charge between -5 and +3. The charge of a peptide, fragment, or region is determined using the method described in Cameselle, JC, Ribeiro, JM, and Sillero, A. (1986). Derivation and use of a formula to calculate the net charge of acid-base compounds. Its application to amino acids, proteins and nucleotides. Biochem. Educ. 14, 131-136.

[0082] In this specification, the term “sequence identity” includes both sequence identity and sequence similarity; that is, a variant (or homolog) that shares 70% sequence identity with a reference sequence is understood to mean that any 70% of the aligned residues of the variant (or homolog) are identical to, or are conserved substitutions of, the corresponding residues in the reference sequence over the entire length of the sequence. Sequence identity is the amount of letters that exactly match between two different sequences. In this specification, gaps are not counted, and the measurement relates to the shorter of the two sequences.

[0083] In relation to "sequence homology," the term shall be understood to mean that a specified percentage of aligned residues in a mutant (or homolog) is identical to or a conserved substitution of the corresponding residues in the reference sequence, and the mutant (or homolog) shares the same function as the reference sequence.

[0084] This alignment and homology percentage or sequence identity percentage can be determined using software programs known in the art, for example, one alignment program is BLAST using default parameters. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0085] "Inflammatory disorders" refer to immune-mediated inflammatory conditions affecting humans that are generally characterized by dysregulation of the expression of one or more cytokines. Examples of inflammatory disorders include inflammatory disorders of the skin and joints, inflammatory disorders of the cardiovascular system, certain autoimmune diseases, inflammatory disorders of the lungs and airways, and inflammatory disorders of the intestines. Examples of inflammatory disorders of the skin include dermatitis, e.g., atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis. Examples of inflammatory disorders of the joints include rheumatoid arthritis. Examples of inflammatory disorders of the cardiovascular system include cardiovascular diseases and atherosclerosis. Examples of autoimmune diseases include type 1 diabetes, Graves' disease, Guillain-Barré disease, lupus, psoriatic arthritis, and ulcerative colitis. Examples of inflammatory disorders of the lungs and airways include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome. Examples of inflammatory disorders of the intestines include colitis and inflammatory bowel disease. Other inflammatory disorders include cancer, hay fever, periodontal disease, allergies, hypersensitivity, ischemia, depression, systemic diseases, post-infection inflammation, and bronchitis. The peptides and compositions of the present invention can also be used in non-therapeutic treatments of inflammation. Examples of non-therapeutic treatments of inflammation include use to alleviate normal, non-pathological inflammation, such as inflammation of muscles and joints after physical exercise.

[0086] "A disease or condition characterized by damage to dermal or epithelial cells or tissues" means any condition or disease that results in damage to dermal or epithelial tissues, cells or organs. One example is trauma, which often results in skin damage. Another example is inflammatory skin conditions, such as psoriasis or eczema, which often result in skin damage. Another example is inflammatory disorders of the lower intestine, which result in damage to the epithelial cells / tissues that make up the inner wall of the lower intestine. Another example is damage to the epithelial cells / tissues that make up the inner wall of the lower intestine, caused by ingestion of toxic or damage-inducing substances (e.g., toxic chemicals or toxic drugs). Another example is cancer, such as colon cancer, which results in damage to the epithelial tissue in the intestine. Another condition is peripheral inflammatory disorders, such as atopic dermatitis, which can result in skin damage in humans.

[0087] In this specification, the term “metabolic disorder” shall be understood to include prediabetes, diabetes; type 1 diabetes; type 2 diabetes; metabolic syndrome; obesity; diabetic dyslipidemia; hyperlipidemia; hypertension; hypertriglyceridemia; hyperfatty acidemia; hypercholesterolemia; hyperinsulinemia; and MODY.

[0088] "Bacterial infection characterized by disease or condition" means any condition or disease characterized by a pathological state caused by bacterial growth or infection, including, for example, MRSA, Salmonella, Listerine pneumonia, Staphylococcal food poisoning, and bacterial meningitis. Specific examples are given at https: / / en.wikipedia.org / wiki / List_of_infectious_diseases.

[0089] When applied to edible foods, the term "artificial" is understood to mean something that has been created by humans and does not exist in nature.

[0090] "Improvement of muscle condition" means improvement in muscle health, such as skeletal muscle protein synthesis, enhanced glucose absorption by the skeleton, improvement in lean tissue mass in therapeutic or non-therapeutic situations, enhanced muscle recovery after generally active physical exercise, or improved muscle capacity. The methods or uses may be therapeutic or non-therapeutic. The term "improvement of lean tissue condition" shall be understood to mean an increase in lean tissue mass, or inhibition or prevention of the rate of breakdown of lean tissue mass.

[0091] "Promoting muscle recovery" means causing an increase in glucose absorption within skeletal muscle compared to untreated skeletal muscle.

[0092] "Disorders or conditions characterized by lethargy or low energy levels" means any condition or disorder characterized by fatigue or low energy. Examples include allergies, asthma, anemia, cancer and its treatment, chronic pain, heart disease, infection, depression, eating disorders, grief, sleep disorders, thyroid problems, medication side effects, alcohol use, or drug use.

[0093] The term "catabolic depletion" encompasses both sarcopenia and cachexia ("catabolism" refers to the breakdown of tissue, and is the opposite of "anabolism," which means the construction of tissue).

[0094] The loss of muscle and adipose tissue due to chronic disease is called cachexia. The common age-related decrease in body weight and muscle mass is called sarcopenia. In both cachexia and sarcopenia, muscle weakness leads to frailty and has detrimental effects on various clinical outcomes (Rolland 2011; Fearon 2013; Muscaritoli 2013). Individuals with cachexia and / or sarcopenia are at increased risk of death, infection, and falls; delayed wound healing; markedly lower physical and respiratory capacity; and an overall decline in quality of life.

[0095] Cachexia typically causes more rapid and significant weight loss than sarcopenia, generally characterized by a loss of more than 5% of body weight in total, of muscle and adipose tissue, although a loss of more than 20% of body weight is common. In many cases, people with cachexia continue to lose weight even with sufficient calorie intake. Severe chronic diseases such as cancer, AIDS, and chronic obstructive pulmonary disease (COPD) are known causes of cachexia. 50% to 80% of all cancer patients experience cachexia, and it is estimated that cachexia is the leading cause of more than 20% of all cancer-related deaths. Cachexia in HIV / AIDS patients is common and was already present before the advent of anti-HIV drugs. Sarcopenia (derived from the Greek word meaning "lack of meat") generally refers to age-related loss of muscle mass and muscle function. Approximately 50% of people over 80 years of age experience sarcopenia. Sarcopenia can also result from physical inactivity, malnutrition, or disease. Some researchers refer to age-related, unrelated muscle weakness as "primary sarcopenia," and muscle weakness resulting from one or more other causes as "secondary sarcopenia." Sarcopenia is associated with an increased risk of insulin resistance and type 2 diabetes in non-obese adults over 60 years of age. The conventional medical community has failed to provide early, aggressive intervention for cachexia, often resulting in poor clinical outcomes, including premature death and physical disability. Standard medical treatments for cachexia include promoting fluid and food intake, as well as the use of certain medications. However, many standard medical treatments for sarcopenia and cachexia carry the risk of adverse effects, such as nausea, edema, and fatigue, some of which have not been adequately investigated even in clinical trials. Numerous nutritional, lifestyle, and innovative pharmacological interventions may be useful in preventing and treating catabolic waste. Whey protein, creatine, and amino acids such as glutamine, arginine, leucine, and methyl hydroxybutyrate or HMB (leucine derivatives) are particularly important for building and maintaining lean muscle mass. Omega-3 fatty acids, conjugated linoleic acid, and vitamin D also counteract lean tissue loss.There is a real and urgent need for new and emerging strategies to prevent muscle wasting. (Ribeiro S, Keheyias J. Sarcopenia and the analysis of body composition. Adv Nutr 2014:5:260-267. Muscaritoli A, Lucia S, Molfino A, Cederholm MT, Rossi Fanelli F, Muscle atrophy in aging and chronic diseases: is it sarcopenia or cachexia? Intern Emerg Med 2013;8: 553-560. Rolland Y, VanKan GA, Gillette-Guyonnet S, Vellas B. Cachexia versus sarcopenia. Curr Opin Clin Nutr Metab Care. 2010;14(1):15-21. Evans WJ, Morley JE, Argiles JM, et al. Cachexia: a new definition. Clin Nutr 2008;27:793-799. Fearon K, Arends J, Baracos V. Understanding the mechanisms and treatment options in cancer cachexia. Nat Rev Clin Oncol 2013;10(2):90-99. )

[0096] "Maintaining or restoring muscle health" means helping to maintain muscle health in mammals or to recover from injuries sustained during physical exercise. By promoting glucose transport within skeletal muscle, peptides facilitate recovery from physical exercise and alleviate muscle pain and injury associated with physical exercise. They can also be used to reduce and prevent muscle cramps and enable faster recovery from muscle cramps. Cramps can result from physical stress, mental stress, and / or repetitive over-injury stress. By promoting glucose transport, peptides reduce muscle myopathy, prevent sarcopenia in mammals, facilitate recovery from injuries during physical exercise, and alleviate muscle pain and injury associated with physical exercise. The present invention also relates to peptides or compositions of the present invention for use in maintaining or restoring muscle health in mammals.

[0097] In this specification, the term “personal care products” shall be understood to mean compositions formulated for human use in the cleansing or treatment of the human body, in particular the skin, teeth, nails, feet, and hair. Examples include shampoos, conditioners, skin creams and lotions, powders, toothpastes, shower gels or shower creams, bath gels or shower gels, hair dyes, soaps, body scrubs, exfoliants, anti-dandruff solutions, body lotions, shaving solutions, moisturizers, cleansing solutions, masks, oils, serums, as well as rinses, deodorants, and antiperspirants.

[0098] The term "skin aging" is used in the sense commonly and broadly used in the field of cosmetics and personal care products. Signs of skin aging include wrinkles, creases, fissures, nodules, red spots, enlarged pores, roughness, dullness, loss of elasticity, sagging, loss of firmness, discoloration, blemishes, hyperpigmentation, spots, keratosis, inflammation, collagen breakdown, and other histological changes within the skin layers, including the underlying tissue.

[0099] The term “cosmetically or pharmaceutically acceptable salts” means salts whose use in animals, more specifically, in humans, is recognized, and includes salts used to form base-addition salts or acid-addition salts. Base-addition salts are inorganic salts (in particular, but not limited to lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum) or organic salts (in particular, but not limited to ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine). Acid-addition salts are organic salts (in particular, but not limited to acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid, or gluconic acid) or inorganic salts (in particular, but not limited to chloride, sulfuric acid, boric acid, or carbonic acid). The properties of the salt are not particularly important, provided that it is cosmetically or pharmaceutically acceptable. Cosmetic or pharmaceutically acceptable salts of the peptides of the present invention can be obtained by conventional methods, which are well known in the prior art [Berge SM et al., "Pharmaceutical Salts", J. Pharm. Sci., (1977), 66, 1-19].

[0100] The "C-terminal domain" applied to a fragment refers to the first three amino acids at the C-terminus of the fragment.

[0101] The "N-terminal domain" applied to a fragment refers to the last three amino acids at the N-terminus of the fragment.

[0102] The term "homologous" to a reference protein is understood to mean a protein derived from a different plant species that has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology to the reference protein.

[0103] In this specification, the terms “comprise, comprises, comprised, comprising,” or any variations thereof, and the terms “include, includes, included, including,” or any variations thereof, are considered to be fully interchangeable, all given the broadest possible interpretation, and vice versa.

[0104] The present invention will be more clearly understood from the following description of embodiments provided for illustrative purposes only, with reference to the attached drawings. [Brief explanation of the drawing]

[0105] [Figure 1] This figure illustrates the results of a cell proliferation assay using untreated and peptide-treated human dermal fibroblasts (HDFs). [Figure 2] This figure illustrates the results of a cell proliferation assay using untreated and peptide-treated HACAT cells. [Figure 3] This figure illustrates the results of a collagen expression assay using untreated and peptide-treated human dermal fibroblasts (HDFs). [Figure 4] This figure illustrates the results of an elastin expression assay using untreated and peptide-treated human dermal fibroblasts (HDFs). [Figure 5] This figure illustrates the results of a glucose uptake assay using untreated and peptide-treated human skeletal myoblasts. [Figure 6] This figure illustrates the results of a glucose uptake assay using untreated and peptide-treated human skeletal myoblasts. [Figure 7] This figure illustrates the results of a glucose uptake assay using untreated and peptide-treated human skeletal myoblasts. [Figure 8]This figure illustrates the results of a GLUT4 migration assay using untreated and peptide-treated human skeletal myoblasts. [Figure 9] This figure illustrates the results of a muscle protein synthesis (mTOR) assay. [Figure 10] This figure illustrates the results of a muscle protein synthesis (puromycin) assay. [Figure 11] This figure illustrates the results of a cytotoxicity study in HSKMC cells using the peptide of Sequence ID No. 1. [Figure 12] This figure illustrates the results of a phosphoacetyl-CoA carboxylase (Ser79) ELISA (peptide of SEQ ID NO: 1). [Figure 13] This figure illustrates the results of a phosphoAkt1 ELISA (peptide of SEQ ID NO: 1). [Figure 14] This figure illustrates the results of the phosphoAMPKα ELISA (peptide of SEQ ID NO: 1). [Figure 15] This figure illustrates the blood glucose levels of KKAY mice after a 5-day treatment with the peptide of Sequence ID No. 1. [Figure 16] This figure illustrates the blood glucose levels of KKAY mice after a 7-day treatment with the peptide of Sequence ID No. 1. [Figure 17] This figure illustrates the body weight of KKAY mice after a 13-day treatment with the peptide of Sequence ID No. 1. [Modes for carrying out the invention]

[0106] The present invention will now be described with reference to specific examples. These are illustrative for illustrative purposes only and are not intended to limit in any way to any claimed exclusive rights or the scope of the invention described herein. These examples constitute the best possible method currently available for carrying out the present invention.

[0107] In its broadest sense, the first aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 (hereinafter referred to as "the peptide of the present invention").

[0108] In a second aspect, the present invention provides a composition comprising an effective amount of the peptide of the present invention. The composition may be a topical composition. The composition may be a cosmetic composition containing a cosmetically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof. The composition may be a pharmaceutical composition comprising a pharmaceutically effective amount of a peptide or a variant thereof containing the amino acid sequence of SEQ ID NO: 1. It will be understood that the composition may have cosmetic effects, i.e., non-therapeutic effects, or therapeutic effects. The composition may have both cosmetic and therapeutic effects.

[0109] Examples of cosmetic applications include, but are not limited to, anti-aging, slowing, inhibiting or preventing the aging of human skin, promoting tissue proliferation, promoting epithelial tissue proliferation, promoting skin proliferation, promoting organ proliferation, promoting the proliferation of organisms, improving muscle condition in mammals, typically promoting muscle recovery after physical exercise, maintaining or restoring muscle health in mammals, enhancing physical ability, promoting muscle growth or muscle strengthening, and treating muscle weakness.

[0110] The present invention also provides therapeutic uses / applications of the compositions or peptides of the present invention, in particular non-topic therapeutic applications.

[0111] Examples of therapeutic applications include, but are not limited to, promoting tissue growth, promoting epithelial tissue growth, promoting skin growth, promoting organ growth, promoting organism growth, improving muscle condition in mammals, promoting muscle recovery, maintaining or restoring muscle health in mammals, muscle growth or muscle strengthening, treating muscle weakness, treating or preventing diseases or conditions characterized by muscle wasting or reduced anabolic activity or reduced protein synthesis (catabolic wasting), treating or preventing diseases or conditions characterized by or mediated by oxidative stress, treating or preventing metabolic diseases (particularly diabetes or prediabetes), treating or preventing diseases or conditions characterized by lethargy or low energy levels, treating wounds in mammals, treating or preventing pain in mammals, treating or preventing diseases or conditions characterized by damage to epithelial cells or tissues, and / or damage to dermal or epithelial cells or tissues, and treating or preventing metabolic disorders (such as diabetes) in mammals.

[0112] Sequence ID 1 is the following sequence: It has WKDEAGKPLVK.

[0113] In embodiments of the present invention, the peptide is bioactive.

[0114] In one embodiment, the peptide has glucose transport-promoting activity. In one embodiment, the peptide has cell proliferation-promoting activity. In one embodiment, the peptide has anti-aging activity. In one embodiment, the peptide has anabolic activity. It will be understood that the peptide may have two or three of the following: glucose transport-promoting activity, cell proliferation-promoting activity, anti-aging activity, and anabolic activity. The peptide may have glucose transport-promoting activity, cell proliferation-promoting activity, anti-aging activity, and anabolic activity.

[0115] In embodiments of the present invention, the peptides include lengths of about 3 to 50 amino acids, about 14 to about 50 amino acids, preferably about 15, 20, 25, 30, 35, 40, 45, or 49 amino acids, preferably about 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0116] In embodiments of the present invention, the peptide has a length of about 3 to about 11 amino acids, preferably about 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0117] In one embodiment, the peptide has 1 to 5 amino acid changes compared to SEQ ID NO: 1. In one embodiment, the peptide has 1 to 4 amino acid changes compared to SEQ ID NO: 1. In one embodiment, the peptide has 1 to 3 amino acid changes compared to SEQ ID NO: 1. In one embodiment, the peptide has 1 to 2 amino acid changes compared to SEQ ID NO: 1. In one embodiment, the amino acid changes are conservative amino acid changes. In one embodiment, the amino acid changes are amino acid substitutions. In one embodiment, the amino acid substitutions are conservative substitutions. In one embodiment, the amino acid changes are amino acid additions. In one embodiment, the amino acid changes are amino acid deletions.

[0118] The peptide of the present invention includes the following variant peptides.

[0119] Variant of sequence number 1 Below are variants of Sequence ID No. 1 (WKDEAGKPLVK) that include variants having one, two, or three conserved amino acid substitutions, one, two to three non-conserved amino acids, one, two, or three amino acid additions, or one, two, or three amino acid deletions: One-site conservative amino acid substitution: WKEEAGKPLVK(sequence code 2);FKDEAGKPLVK(sequence code 3); WKDEAGKPMVK(sequence code 4);WKDEAGRPLVK(sequence code 5); WRDEAGKPLVK(sequence number 6); WKDEAGKPLMK(sequence number 7); WKDQAGKPLVK(sequence ID 8); WKDEATKPLVK(sequence ID 9) Two conservative amino acid substitutions: YKNEAGKPLVK(sequence code 10);WKNESGKPLVK(sequence code 11);WKDEAGKTLVR(sequence code 12);FKDEATKPLVK(sequence code 13);FKDEAGKPLIK(sequence code 14);WKDEAGKTLLK(sequence code 15);WKNEAGKPVVK(sequence code 16);WKDEAGRTLVK(sequence code 17) Three conservative amino acid substitutions: WEDESGKPLLK(sequence code 18);WKEEAGKPIVQ(sequence code 19);YKNEAGKPLVR(sequence code 20);WKDQATRPLVK(sequence code 21);WKDESGKPVLK(sequence code 22);WQDDSGKPLVK(sequence code 23);WKNEAGKTLLK(sequence code 24);WKDKAGEPLVR(sequence code 25) One non-conservative amino acid substitution: WKDEAGNPLVK(sequence code 26);CKDEAGKPLVK(sequence code 27);WKDEAGKPLGK(sequence code 28);WKDENGKPLVK(sequence code 29);WKDEARKPLVK(sequence code 30);WKDEAGKPLVT(sequence code 31);WKDEAGKRLVK(sequence code 32);WKWEAGKPLVK(sequence code 33) Two non-conservative amino acid substitutions: WKDEAGFPTVK(sequence number 34);WYDMAGKPLVK(sequence number 35);WKDYEGKPLVK(sequence number 36);WKREAGKPGVK(sequence number 37);WKLEKGKPLVK(sequence number 38);WKDEAGKPCVK(sequence number 39);WKKEAPKPLVK(sequence number 40);SKDEAGPPLVK(sequence number 41) Three non-conservative amino acid substitutions: WKHEPGKPLAK(sequence code 42);WKDEREKPFVK(sequence code 43);WKQEAGKPWRK(sequence code 44);VKDEAKKPLVH(sequence code 45);NWDEAGKMLVK(sequence code 46);IKDEDGPPLVK(sequence code 47);LKDEYGKPLVN(sequence code 48);WKDRAGKELTK(sequence code 49) Amino acid addition WKDEAGKPLPVK(array_50); WKGDENYAGKPLVK(Sequence ID 51); LWKDEAGRKYPLVK(sequence code 52); WKDCEGAGKPLVK(Sequence ID 53); WKDEPAGKPLVVK(Sequence ID 54); WKDEAGPKPLVK(sequence number 55); WKDEAGWADKPLVK(array_56); WKNDEAGKPLVK(Sequence ID 57) amino acid deficiency WKDAKPLVK(sequence code 58); WKEAGKPVK(sequence code 59); WKDEAKPLVK(sequence code 60);WDEAGKPV(sequence code 61); WKDEAGKPVK(sequence number 62);WDAGKPLVK(sequence number 63); WKDEAGKPLV(sequence code 64);WEAGKPLV(sequence code 65)

[0120] Mutant peptides can be bioactive variants.

[0121] The present invention also provides the fragment of Sequence ID No. 1, and peptides comprising one or more of these fragments.

[0122] The fragment may be a bioactive fragment. In one embodiment, the fragment is an anti-aging fragment. In one embodiment, the fragment is a cell proliferation-promoting fragment. In one embodiment, the fragment is a glucose transport-promoting fragment. In one embodiment, the fragment is an anabolic fragment. In one embodiment, it will be understood that the bioactive fragment is two or more of the anti-aging fragment, glucose transport-promoting fragment, cell proliferation-promoting fragment, and anabolic fragment. In one embodiment, the bioactive fragment is an anti-aging fragment, a glucose transport-promoting fragment, a cell proliferation-promoting fragment, and an anabolic fragment.

[0123] The fragment may be a bioactive fragment. In one embodiment, the fragment is a cell proliferation-promoting fragment. In one embodiment, the fragment is a GLUT4 transport-promoting fragment. In one embodiment, the fragment exhibits anabolic metabolic stimulating activity. In one embodiment, the fragment exhibits antioxidant or anti-inflammatory activity.

[0124] An example of the fragment for sequence number 1 is shown below. WKDEAG(sequence number 66);WKDEA(sequence number 67);KDEAGKPL(sequence number 68);KDEAG(sequence number 69);DEAGKPL(sequence number 70);GKPLV(sequence number 71);DEAGK(sequence number 72);WKDEAGKPL(sequence number 73);WKD(sequence number 74);KDE(sequence number 75);KPLVK(sequence number 76);WKDE(sequence number 77);AGKPL(sequence number 78);EAG(sequence number 79);AGK(sequence number 80);KPL(sequence number 81);LVK(sequence number 82);GKP(sequence number 83);DEA(sequence number 84);PLV(sequence number 85)

[0125] It will be understood that the composition may contain multiple peptides or fragments. Preferably, the composition contains at least two of the peptides of the present invention.

[0126] Preferably, the composition contains at least three peptides of the present invention. Preferably, the composition contains at least four peptides of the present invention. Preferably, the composition contains at least five peptides of the present invention. Preferably, the composition contains at least six peptides of the present invention. Preferably, the composition contains at least seven peptides of the present invention. Preferably, the composition contains at least eight peptides of the present invention. Preferably, the composition contains at least nine peptides of the present invention. Preferably, the composition contains at least ten peptides of the present invention. In one embodiment, the composition contains substantially all peptides. In one embodiment, the composition contains substantially all variants. In one embodiment, the composition contains substantially no other peptides.

[0127] In embodiments, the peptides or compositions of the present invention have an activity selected from one or more of the following: antimicrobial activity, anti-inflammatory activity, and antioxidant activity. The activity may be cosmetic activity (i.e., non-therapeutic activity), therapeutic activity, or both. The present invention also provides compositions of the present invention for use in methods for maintaining or restoring gastrointestinal health in mammals. The present invention also provides compositions of the present invention for use in methods for treating bacterial infections. Further embodiments of the present invention provide compositions of the present invention for use in methods for treating or preventing inflammatory disorders and / or inflammation in mammals. Preferably, the inflammation is symptomatic inflammation. This use may be in addition to or alternative to the uses of the present invention discussed above.

[0128] In embodiments, the peptide, fragment, or composition of the present invention has one or more activities selected from antibacterial activity, anti-inflammatory activity, and antioxidant activity.

[0129] The composition may be a topical composition. Topical compositions can be provided as formulations selected from the group including creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, water-alcohol solutions, water-glycol solutions, cosmetics, personal care products, hydrogels, liniments, serums, soaps, dusting powders, pastes, semi-solid formulations, liniments, serums, shampoos, conditioners, ointments, any rinsing formulations, talc, mousse, powders, sprays, aerosols, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, patches, gel patches, bandages, adhesives, water-in-oil emulsions, oil-in-water emulsions, and silicone emulsions.

[0130] In embodiments of the present invention, the emulsion contains lipids or oils. The emulsion may, but is not limited to, oil-in-water, water-in-oil, water-in-water, and silicone-in-water emulsions. The emulsion may contain a humectant. The emulsion may contain an antifoaming agent such as a silicone. The emulsion may have any suitable viscosity. The emulsion may further contain an emulsifier and / or an antifoaming agent. Methods for preparing emulsions are known to those skilled in the art.

[0131] The compositions of the present invention may be provided, prepared, and / or administered in a variety of suitable forms. Such forms include, but are not limited to, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injectable and infusion solutions), dispersions or suspensions, emulsions, microemulsions, tablets, pills, powders, liposomes, dendrimers and other nanoparticles, microparticles, and suppositories. It will be understood that the form may depend on the intended method of administration, the nature of the composition or combination, and the therapeutic application or other intended use. Formulations may also include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, endoplasmic reticulum containing lipids (cationic or anionic), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsions, carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes.

[0132] It is important to understand that an ingredient considered "active" in one product may be a "functional" or "excipient" in another product, and vice versa. It should also be understood that some ingredients may play a dual role as both an active ingredient and a functional or excipient.

[0133] In particularly preferred embodiments, the methods and uses of the present invention involve the administration of the peptide or composition of the present invention in combination with one or more other active agents (e.g., commercially available, existing growth promoters or pharmacological enhancers). In such cases, the compounds of the present invention can be administered sequentially, concurrently, or sequentially with one or more other active agents.

[0134] In a preferred embodiment, repeated use of the composition is provided.

[0135] The compositions of the present invention can be incorporated into medical devices for administration. Such devices include, but are not limited to, woven fabrics, patches, bandages, gauze, socks, tights, underwear, bandages, gloves, masks, adhesive patches, non-adhesive patches, occlusive patches, and microcurrent patches, or suitable adhesive systems. In such embodiments, the device is in direct contact with a keratin layer, such as skin, thereby releasing the peptides of the present invention. It will be understood that the topical compositions can be incorporated in any suitable form, as detailed herein. For example, the topical compositions or topical peptides of the present invention may be incorporated into or present on the surface of the device, or present in a cream, gel, or wax formulation, or any suitable formulation as defined herein, and can be incorporated into or on the surface of the device.

[0136] The device can be adapted for adhesion or bonding to the skin. In one embodiment, the device is adapted for the release of a certain amount of the composition or peptide of the present invention. It will be understood that the amount of composition contained in the sustained-release system will depend, for example, where the composition is administered, the dynamics and duration of the release of the composition of the present invention, as well as the nature of the symptoms, disorders, and / or diseases being treated and / or cared for. The device may be such that the composition is released due to the biodegradation of the device or friction between the device and the body, for the sake of body moisture, skin pH, or body temperature.

[0137] In embodiments of the present invention, the composition may further comprise at least one cosmetically or pharmaceutically acceptable excipient. The excipient can be used interchangeably with functional components or additives. While the topical compositions of the present invention may be administered alone, it will generally be understood that they are administered in mixture with cosmetic or pharmaceutical excipients. Cosmetically or pharmaceutically acceptable excipients are well known in the art, and any known excipient can be used, provided that it is suitable for topical administration and is dermatologically acceptable without inappropriate toxicity, incompatibility, and / or allergic reactions.

[0138] Preferably, any excipients incorporated are present in trace amounts. The amount of excipients incorporated depends on a number of factors, including the type of excipient used, the properties of the excipient, the components of the topical composition, the amount of the active ingredient or peptide in the topical composition, and / or the intended use of the topical composition. The properties and amounts of any excipients shall not alter the beneficial properties of the peptide of the present invention in an unacceptable manner.

[0139] In embodiments of the present invention, the excipient may be a suitable diluent, carrier, binder, lubricant, suspending agent, coating agent, preservative, stabilizer, dye, medium, solubilizer, base, softener, emulsifier, fragrance, humectant, and / or surfactant.

[0140] Examples of suitable diluents include, but are not limited to, any diluents disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667. Examples include ethanol, glycerol, and water. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and any suitable carriers disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0141] Examples of suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, anhydrous lactose, fluid lactose, and β-lactose, corn sweeteners, natural and synthetic gums such as acacia and tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol, as well as any suitable binders disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0142] Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride, as well as any suitable lubricants disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0143] The carrier may be any suitable carrier known in the Art or disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667. In some embodiments, the carrier may include, but is not limited to, liquids such as water and oil, or surfactants including those of petroleum, animal, plant, or synthetic origin, polymers, oils such as peanut oil, mineral oil, castor oil, and soybean oil, alcohols, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, dextroses, glycerols, or digitonin. The carrier will be understood to be dermatologically acceptable. Preferred carriers include emulsions such as oil-in-water, water-in-oil, water-in-oil-in-water, and silicone-in-water emulsions. The emulsion may further contain emulsifiers and / or defoamers.

[0144] In embodiments of the present invention, the composition may further comprise one or more additional components. The composition of the present invention may be administered sequentially, simultaneously, or sequentially with one or more other additional agents. Such additional components may be beneficial to incorporate into the topical composition or may be beneficial components depending on the intended use of the topical composition. The additional components may be active ingredients or functional ingredients, or both.

[0145] Examples of such additional ingredients include, but are not limited to, one or more of the following: cleansing agents, conditioning agents, sunscreens, pigments, moisturizers, thickeners, gelling agents, essential oils, astringents, pigments, anticoagulants, defoamers, binders, additives, buffers, chelating agents, topical analgesics, film-forming agents or materials, fillers, polymers, opacifiers, pH adjusters, propellants, reducing agents, chelating agents, skin depigments and skin whitening agents, skin conditioning agents, aloe, healing agents, sedatives, smoothing agents, pantothenic acid, treatment agents, thickeners, vitamins, colorants, pharmaceuticals, preservatives, defoamers, buffers, astringents, polymers, pH adjusters, body odor inhibitors, or any other dermatologically acceptable carriers or surfactants.

[0146] It should be understood that the additional components listed may offer more than one benefit. The classifications made herein are for clarity and simplicity only and are not intended to limit further components to the listed, specific applications or types.

[0147] Any additional ingredients should be suitable for application to the skin without causing inappropriate toxicity, incompatibility, and / or allergic reactions.

[0148] In some embodiments, additional components have glucose transport activity or assist in glucose transport activity. In some embodiments, additional components have anabolic activity. In some embodiments, additional components have anti-inflammatory activity or assist in anti-inflammatory activity. In some embodiments, additional components have anti-aging activity or assist in anti-aging activity. In some embodiments, additional components are components for keratin layer health and / or development, skin health and / or development, and / or muscle health, recovery, and / or development. The active agent may be a pharmacological enhancer. Such active agents are known and commercially available. In such cases, the topical composition of the present invention may be administered sequentially, simultaneously, or sequentially with one or more other active agents.

[0149] In some embodiments, further components may be farnesol ([2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol), phytantriol (3,7,11,15-tetramethylhexadecane-1,2,3-triol), desquamating active ingredients, enzymes, enzyme inhibitors, enzyme activators, plant extracts, and marine extracts, anti-acne active ingredients, anti-wrinkle active ingredients or anti-atrophy active ingredients, antioxidants / radical scavengers, chelating agents, flavonoids, anti-inflammatory agents, anti-cellulite agents, local anesthetics, sunburn active ingredients, skin whitening agents, skin healing agents, bisabolol, antimicrobial or antifungal active ingredients, sunscreen active ingredients, particulate materials, conditioning agents, structuring agents, and thickeners. The desquamating active ingredient may be any suitable agent that enhances the appearance or texture of the skin, as disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0150] Examples of anti-acne active ingredients are disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667 and include resorcinol, salicylic acid, erythromycin, zinc, sulfur, and benzoyl peroxide.

[0151] Examples of thickeners are disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667 and include carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, and polysaccharides.

[0152] Examples of conditioning agents are disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667 and include humectants, moisturizers, or skin conditioners.

[0153] Examples of structuring agents are disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667 and include any agents that impart rheological characteristics to a composition and contribute to the stability of the composition.

[0154] Any suitable antimicrobial or antifungal active ingredient can be used, examples of which are disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667. Such active ingredients can destroy microorganisms or prevent their growth or action. Examples include, but are not limited to, beta-lactams, quinolones, tetracyclines, erythromycin, streptomycin sulfate, salicylic acid, and benzoyl peroxide.

[0155] Examples of particulate materials include metal oxides.

[0156] Examples of anti-cellulite agents include xanthine-based drugs.

[0157] Examples of sunscreen active ingredients include 1,3-dihydroxy-2-propanone and sunscreen active ingredients disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0158] Examples of local anesthetics include benzocaine, lidocaine, and bupivacaine, as well as local anesthetics disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0159] Examples of skin whitening agents include kojic acid, ascorbic acid, and any agents known in the art, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0160] Examples of sunscreen active ingredients include any suitable organic or inorganic sunscreen active ingredients. Examples include metal oxides, 2-ethylhexyl p-methoxycinnamate, and sunscreen active ingredients disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0161] Examples of skin healing agents include pantheonic acid disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0162] Examples of anti-inflammatory agents include, but are not limited to, any agents that enhance skin appearance, shade, or color, including non-steroidal drugs such as corticosteroids, hydrocortisone, ibuprofen, and aspirin, as well as anti-inflammatory agents disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0163] Examples of flavonoids include flavanones, methoxyflavonones, unsubstituted chalcones, and mixtures thereof, as well as flavonoids disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667.

[0164] Examples of enzymes include lipase, protease, catalase, superoxide dismutase, amylase, peroxidase, glucuronidase, ceramidase, and hyaluronidase. Examples of enzyme inhibitors include trypsin inhibitors, Bowman-Burk inhibitors, chymotrypsin inhibitors, plant extracts, flavonoids, quercetin chalcone, and enzymes disclosed in U.S. Patent Application Publication 2014 / 0120131 or U.S. Patent Application Publication 2004 / 0132667, as well as mixtures thereof. Examples of enzyme activators include coenzyme A, coenzyme Q10 (ubiquinone), glycyrrhizin, berberine, chrysin, and enzyme activators disclosed in U.S. Patent Application Publication 2014 / 0120131 or U.S. Patent Application Publication 2004 / 0132667, as well as mixtures thereof.

[0165] Examples of anti-wrinkle or anti-atrophy active ingredients include sulfur-containing D and L amino acids, in particular N-acyl derivatives such as N-acetyl-L-cysteine, hydroxy acids, phytic acids, lipoic acid, lysophosphatidic acid, exfoliants, vitamin B3, retinoids, and anti-wrinkle or anti-atrophy active ingredients disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667, as well as mixtures thereof.

[0166] Antioxidants / radical scavengers may be any agent useful for providing protection against UV radiation or other environmentally active substances that can cause skin damage, such as antioxidants / radical scavengers disclosed in U.S. Patent Application Publication No. 2014 / 0120131 or U.S. Patent Application Publication No. 2004 / 0132667. Examples of antioxidants / radical scavengers include ascorbic acid, its salts and derivatives (vitamin C), tocopherol, its salts and derivatives (vitamin E), butylated hydroxylbenzoic acid and its salts, peroxides, gallic acid and alkyl esters, sorbic acid, lipoic acid, amines, lysine pyrolates, arginine pyrolates, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silmarin, tea extracts, and mixtures thereof.

[0167] Examples of chelating agents include EDTA, NTA, hydroxyacid, phytic acid, lactoferrin, and chelating agents disclosed in U.S. Patent Application Publication 2014 / 0120131 or U.S. Patent Application Publication 2004 / 0132667, as well as mixtures thereof. A chelating agent means an agent capable of removing metal ions by forming a complex such that the metal ions cannot participate in or catalyze a chemical reaction. Chelating agents are useful for protection against UV radiation or other environmental agents that can cause skin damage.

[0168] It will be understood that multiple additional components may be added. The amount of additional components may be about 0.001% to about 50% by weight of the composition, preferably about 0.01% to about 20%, preferably about 0.1% to about 10%, about 0.5% to about 10%, about 1% to about 5%, preferably 2% by weight. The amount of additional components depends on a number of factors, including the type of additional component used, the nature of the additional component, the components of the topical composition, the amount of the active ingredient or peptide in the topical composition, and / or the intended use of the topical composition. The nature and amount of any additional component should not alter the beneficial properties of the peptide of the present invention in an unacceptable manner.

[0169] Topical compositions may be alcohol-free.

[0170] In some embodiments of the present invention, the composition further comprises one or more additional activating agents in addition to the peptide of the present invention (which is also known as an active ingredient of the composition). In addition, or alternatively, the composition may be administered with one or more other additional activating agents. Typically, additional activating agents are present only in trace amounts. In some embodiments, no additional activating agents may be present in the composition. The amount of additional activating agents incorporated depends on a number of factors, including the type of additional activating agent used, the nature of the additional activating agent, the components of the topical composition, the amount of the active ingredient or peptide in the topical composition, and / or the intended use of the topical composition. The nature and amount of any additional activating agent should not alter the beneficial effects of the peptide of the present invention in an unacceptable manner.

[0171] It is important to understand that an ingredient considered "active" in one product may be a "functional" or "excipient" in another product, and vice versa. It should also be understood that some ingredients may play a dual role as both an active ingredient and a functional or excipient.

[0172] Examples of additional active agents include glucose transport promoters, skin supplements, agents for skin treatment and / or care, anti-inflammatory agents, anti-aging agents, anabolic agents, cell proliferation promoters, and pharmacological enhancers. Such agents are well known in the art, and it will be understood that any appropriate additional active agents may be used. Additional active agents for skin treatment and / or care may include collagen synthesis agents, retinoids, exfoliants, anti-cellulite agents, elastase inhibitors, melanin synthesis stimulants or inhibitors, self-tanning agents, anti-aging agents, antimicrobial agents, antifungal agents, fungicidal agents, and healing agents. Active agents also include anti-inflammatory agents.

[0173] Any additional active agent should be suitable for topical application without undue toxicity, incompatibility, and / or allergic reaction. It will be understood that the classifications made herein are for the sake of clarity and convenience only and are not intended to limit additional ingredients, excipients, or active ingredients to those listed for this particular application or type.

[0174] In particularly preferred embodiments, the methods and uses of the invention involve administration of the peptides or compositions of the invention in combination with one or more other active agents (e.g., commercially available, existing growth promoters or pharmacological enhancers). In such cases, the compounds of the invention can be administered with one or more other active agents continuously, simultaneously, or sequentially.

[0175] In embodiments, the effects of the invention are achieved by topical application or administration of the topical compositions of the invention described herein to a human, animal, or patient in need of treatment or care. Topical delivery preferably means delivery to a keratin layer such as the skin, hair, and / or nails, but can also mean delivery to a body cavity lined with epithelial cells (e.g., the lung or airway, gastrointestinal tract, mouth). The effects can be limited to the surface of the skin, or can be within the skin, or can be a combination of both.

[0176] The topical composition of the present invention is administered in a cosmetically effective or pharmaceutically effective amount. In other words, it is administered in an amount that is non-toxic but sufficient to produce the desired effect. Those skilled in the art will understand that it is possible to determine an appropriate dose for administering the topical composition of the present invention without unnecessary experimentation. Alternatively, a physician will determine the actual dose that is most appropriate for the patient, depending on the specific symptoms, disease, or disorder being treated or cared for, as well as the person's age, weight, and / or health. This will depend on a variety of factors, including the activity, metabolic stability, and duration of action of the specific compound used, age, weight, overall health, sex, diet, method and frequency of administration, elimination rate, drug combination, severity of the specific symptoms, and the individual being treated. Naturally, there may be individual cases where a high-dose range or a low-dose range is appropriate, and such cases are also within the scope of the present invention. For example, the composition can be administered in doses of 0.01 to 50 mg per kg of body weight, more preferably 0.1 to 20 mg per kg of body weight, more preferably 0.1 to 10 mg per kg of body weight, and preferably 0.1 to 5 mg per kg of body weight, such as 0.1 to 30 mg / kg of body weight. In exemplary embodiments, the patient is administered one or more doses of 10 to 300 mg per day, or more preferably 10 to 150 mg per day. The amount and frequency should be as best suited to the purpose. The frequency of application or administration may vary considerably depending on the needs of each subject, and a range of application or administration is recommended from once a month to 10 times a day, preferably once a week to 4 times a day, more preferably three times a week to 3 times a day, and even more preferably once or twice a day.

[0177] In a preferred embodiment, the composition is subjected to repeated use.

[0178] Topical compositions may be applied to keratinous tissue, skin, or areas of the body being treated or cared for by rubbing or massaging, but are not limited to these means. In some embodiments, the composition is left on the area of ​​the body or not removed from the area of ​​the body. In other embodiments, the composition is removed after a time of about 2 to 60 minutes, about 5 to about 30 minutes, preferably about 10 to about 20 minutes, etc., but not limited to these times. The composition can be removed quickly after application. In some embodiments of the present invention, the compositions of the present invention may be applied to the area being treated by means of achieving greater penetration of the compositions and / or peptides of the present invention, such as iontophoresis, sonophoresis, electroporation, microcurrent patch, mechanical pressure, osmotic gradient, shielded healing method, microinjection, or pressure-assisted needle injection (such as oxygen pressure injection), or any combination thereof, but are not limited to these means.

[0179] The peptides or fragments of the present invention are used in the topical cosmetic or pharmaceutical composition of the present invention at concentrations effective for cosmetic, pharmaceutical, or therapeutic purposes to achieve the desired effect; in a preferred form, the concentration is between 0.00000001% (by weight) and 20% (by weight), based on the total weight of the composition; preferably between 0.000001% (by weight) and 15% (by weight), more preferably between 0.0001% (by weight) and 10% (by weight), and even more preferably between 0.0001% (by weight) and 5% (by weight). Ideally, the peptides of the present invention are used in the composition at a concentration of, preferably, about 0.00001% w / w to about 0.5% w / w, more preferably 0.00005% w / w to about 0.05% w / w, and most preferably about 0.0001% w / w to about 0.01% w / w. Ideally, the peptide of the present invention is used in the composition, preferably at a concentration of about 0.0001% w / w to about 0.004% w / w.

[0180] The compositions of the present invention can be administered individually or in combination with other pharmacologically active agents. Such combination therapies will be understood to encompass, without limitation, different treatment regimens, including the administration of multiple agents combined in a single dosage form or the administration of multiple agents in separate, independent dosage forms. Where the agents are present in different dosage forms, administration may be simultaneous or nearly simultaneous, or may follow any predetermined regimen encompassing the administration of different agents. Suitable active agents may be as described herein.

[0181] In some embodiments of the present invention, compositions can be delivered via any one of the following: liposomes, mixed liposomes, oleosomes, niosomes, etosomes, millicapsules, capsules, macrocapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, surfactant mixed micelles, surfactant-phospholipid mixed micelles, myrispheres, spheres, lipospheres, particles, nanospheres, nanoparticles, milliparticles, solid nanoparticles, as well as microemulsions containing water-in-oil microemulsions with an inverse micelle internal structure, nanoemulsion microspheres, and microparticles.

[0182] Various methods are available for preparing liposomes. For example, see Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT International Publication No. 91 / 17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976);Fraley, et al, PNAS 76:3348-3352 (1979);Hope et al, Biochim. Biophys. Acta 812:55-65 (1985);Mayer et al, Biochim. Biophys. Acta 858:161-168 (1986);Williams et al, PNAS 85:242-246 (1988);Liposomes (Ostro (ed.), 1983, Chapter 1);Hope et al, Chem. Phys. Lip. 40:89 (1986);Gregoriadis, Liposome Technology (1984);and Lasic, Liposomes: from Physics to Applications (1993). Suitable methods include, for example, sonication, extrusion, high-pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome media, and ether fusion, all of which are well known in the art.

[0183] These delivery systems can be adapted to achieve greater penetration of the compounds and / or peptides of the present invention. This can improve pharmacokinetic and pharmacodynamic properties. The delivery system may be a sustained-release system that gradually releases the compounds or peptides of the present invention over a set period of time, preferably over a set period of time, at a constant release rate. The delivery system is prepared by methods known in the art. The amount of peptide contained in the sustained-release system depends on the location to which the composition is delivered, the duration of release, and the type of symptom, disease, and / or disorder being treated or cared for.

[0184] The compounds of the present invention can be administered orally. The compounds (and, if desired, other components) can also be encapsulated in hard or soft gelatin capsules, compressed into tablets, or directly incorporated into the diet of the subject. For therapeutic oral administration, the compounds can be incorporated with excipients and used in the form of edible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. The compounds can be coated with a material that prevents their inactivation, or the compounds can be co-administered together with such a material.

[0185] The compositions of the present invention may be foods or beverages. In one embodiment, the artificial food is a sports nutrition product, such as a beverage, snack, or supplement. In one embodiment, the artificial food is a beverage. In one embodiment, the artificial food is a bread product. In one embodiment, the artificial food is a dairy product. In one embodiment, the artificial food is a snack product. In one embodiment, the artificial food is a baked extruded food. In one embodiment, the artificial food is powdered milk. In one embodiment, the artificial food is an artificial nutritional dairy product. In one embodiment, the artificial food is a confectionery product. In one embodiment, the artificial food is yogurt. In one embodiment, the artificial food is a yogurt drink. In one embodiment, the artificial food is an ice cream product. In one embodiment, the artificial food is a frozen food. In one embodiment, the artificial food is a breakfast cereal. In one embodiment, the artificial food is sliced ​​bread. In one embodiment, the artificial food is a flavored milk drink. In one embodiment, the artificial food is a candy bar. In one embodiment, the artificial food is tea or a tea product. In one embodiment, the artificial food is a base extruded snack product. In one embodiment, the artificial food is a fried snack product. In one embodiment, the artificial food is a nutritional supplement. In one embodiment, the artificial food is a sports nutritional supplement. In one embodiment, the artificial food is baby food. In one embodiment, the artificial food is a specialized food for immunocompromised individuals. In one embodiment, the artificial food is food for elderly patients.

[0186] In one embodiment, the composition is a plant-based food. In one embodiment, the composition is a cell culture medium. In one embodiment, the composition is animal feed. In one embodiment, the composition is an animal feed supplement. In one embodiment, the composition is a medical food.

[0187] In embodiments, the compositions of the present invention may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, and / or intramuscularly). For example, it may be administered by intravenous infusion or intravenous injection, or by intramuscular or subcutaneous injection.

[0188] The compositions of the present invention may be used in human or animal medicine in human and veterinary medicine.

[0189] The compositions of the present invention can be used for pharmaceutical, personal care, and / or cosmetic purposes.

[0190] The composition can be used to treat or care for any skin disease, disorder, or condition, including but not limited to psoriasis, dermatitis, allergic dermatitis, eczema, spongiform eczema, edema, skin cancer, ulcers, acne, scarring, cellulitis, elastic fibrosis, keratosis, rosacea, esophageal varices, and inflammatory disorders.

[0191] The composition may be used for non-topic treatment of any skin disease, disorder, or symptom, including but not limited to psoriasis, dermatitis, allergic dermatitis, eczema, spongiform eczema, edema, skin cancer, ulcers, acne, scarring, cellulitis, elastic fibrosis, keratosis, rosacea, esophageal varices, and inflammatory disorders.

[0192] The composition can be used for non-topic treatment of wounds in mammals. In another embodiment, the composition is for use in the non-topic treatment or prevention of diseases or conditions characterized by damage to epithelial cells or tissues, and / or damage to dermal or epithelial cells or tissues. Diseases may be, but are not limited to, cancer and trauma.

[0193] The composition can be used to treat or care for visible signs of aging, including but not limited to wrinkles, stretch marks and eye corners, dryness, fine lines, age spots, red patches, skin sagging, and conditions caused by sun exposure, stress, pollution, and diet such as sunburn. The topical composition can also be used to delay, retard, or inhibit the onset of skin aging. The composition can be administered by medical devices such as the plasters or patches described herein.

[0194] The composition can be used to treat or care for wounds in mammals. In another embodiment, the topical composition is a topical composition for use in the treatment or prevention of diseases or conditions characterized by damage to epithelial cells or tissues, and / or damage to dermal or epithelial cells or tissues. The diseases can include but are not limited to cancer and trauma.

[0195] The composition can be used to treat or care for any muscle condition to improve muscle condition in mammals, typically promote muscle recovery after physical exercise, maintain or restore muscle health (e.g., lean body mass) in mammals, and enhance physical ability in the treatment or prevention of diseases or conditions characterized by drowsiness or low energy levels.

[0196] The composition can be used to increase or stimulate muscle growth activity.

[0197] The composition can be used to promote tissue growth, promote epithelial tissue growth, promote skin growth, promote organ growth, and promote the growth of organisms. The skin can have normal and / or abnormal conditions.

[0198] The composition can also be used to treat or care for any inflammatory disorder. In one embodiment, the inflammatory disorder is an inflammatory disorder of the joints. In one embodiment, the inflammatory disorder is an inflammatory disorder of the cardiovascular system. In one embodiment, the inflammatory disorder is an autoimmune disease. In one embodiment, the inflammatory disorder is an inflammatory disorder of the lungs and airways. In one embodiment, the inflammatory disorder is an inflammatory disorder of the intestines. In one embodiment, the inflammatory disorder is dermatitis. In one embodiment, the inflammatory disorder is acne vulgaris. In one embodiment, the inflammatory disorder is psoriasis. In one embodiment, the inflammatory disorder is rheumatoid arthritis. In one embodiment, the inflammatory disorder is a cardiovascular disease. In one embodiment, the inflammatory disorder is atherosclerosis. In one embodiment, the inflammatory disorder is type 1 diabetes mellitus.

[0199] In one embodiment, the inflammatory disorder is Graves' disease. In one embodiment, the inflammatory disorder is Guillain-Barré disease. In one embodiment, the inflammatory disorder is lupus. In one embodiment, the inflammatory disorder is psoriatic arthritis. In one embodiment, the inflammatory disorder is ulcerative colitis. In one embodiment, the inflammatory disorder is asthma. In one embodiment, the inflammatory disorder is cystic fibrosis. In one embodiment, the inflammatory disorder is COPD. In one embodiment, the inflammatory disorder is emphysema. In one embodiment, the inflammatory disorder is acute respiratory distress syndrome. In one embodiment, the inflammatory disorder is colitis. In one embodiment, the inflammatory disorder is inflammatory bowel disease.

[0200] The composition can also be used to treat or care for metabolic disorders. In one embodiment, the metabolic disorder is prediabetes. In one embodiment, the metabolic disorder is diabetes. In one embodiment, the metabolic disorder is type 1 diabetes. In one embodiment, the metabolic disorder is type 2 diabetes. In one embodiment, the metabolic disorder is metabolic syndrome. In one embodiment, the metabolic disorder is obesity. In one embodiment, the metabolic disorder is diabetic dyslipidemia. In one embodiment, the metabolic disorder is hyperlipidemia. In one embodiment, the metabolic disorder is hypertension. In one embodiment, the metabolic disorder is hypertriglyceridemia. In one embodiment, the metabolic disorder is hyperfatty acidemia. In one embodiment, the metabolic disorder is hypercholesterolemia. In one embodiment, the metabolic disorder is hyperinsulinemia. In one embodiment, the metabolic disorder is MODY. The composition of the present invention can also be used in methods for treating patients suffering from disease symptoms associated with or caused by hyperinsulinemia, hypoglycemia, hypokalemia, and / or hypophosphatemia. The present invention relates to a method for treating a blood glucose-related disease or disorder, further comprising the administration of an insulin derivative or insulin conjugate. Blood glucose-related diseases or disorders include type 1 and type 2 diabetes mellitus, as well as gestational diabetes mellitus. Cystic fibrosis, polycystic ovary syndrome, pancreatitis, and other pancreatic-related diseases can also be treated.

[0201] In embodiments of the present invention, the composition is a composition for use in maintaining or restoring gastrointestinal health.

[0202] In embodiments of the present invention, the composition is a composition for use in a method for treating or preventing local pain.

[0203] The composition has uses as a personal care product, supplement, cosmetic, and pharmaceutical.

[0204] Also provided are methods for treating, preventing, or caring for any one of the diseases, disorders, or symptoms described herein, the methods comprising the step of administering a composition or topical composition of the present invention. The compositions can be administered to the skin, hair, or nails. The compositions can be administered in any dose or frequency, by any method of administration or any method of topical application, as disclosed herein.

[0205] In one embodiment, the composition is a cosmetic composition. In another embodiment, the composition is a pharmaceutical composition. It will be understood that the composition may have a dual function and may be both a cosmetic composition and a pharmaceutical composition.

[0206] It will be understood that a composition may be a therapeutic composition or a non-therapeutic composition. A composition may have a dual role.

[0207] The composition may be formulated in unit dosage forms, that is, in the form of individual parts containing a unit dose, or multiple unit doses or subunits of unit doses.

[0208] In particularly preferred embodiments, the methods and uses of the present invention involve the administration of the peptide or composition of the present invention in combination with one or more other commercially available active agents. In such cases, the compounds of the present invention can be administered sequentially, concurrently, or sequentially with one or more other active agents.

[0209] Modified peptides In one embodiment, the peptides of the present invention (including peptide fragments and peptide variants) may be modified peptides. The term “modified peptide” is used interchangeably with the term “derived peptide.” In one embodiment, the term “modified peptide” means a peptide modified to exhibit one or more of the following properties compared to an unmodified peptide: extended plasma half-life; increased lipophilicity of the peptide; increased renal clearance of the modified peptide; increased activity of the modified peptide; and increased resistance of the modified peptide to proteolysis (i.e., by mammalian, and especially human, gastrointestinal proteases). Various methods for modifying the peptides of the present invention to exhibit these properties are disclosed herein, and these methods include conjugating the peptide with a binding partner (e.g., albumin that binds to small molecules, large polymers, long-lived plasma proteins, or antibodies or antibody fragments), cyclization, addition of an N-terminal protecting group or a C-terminal protecting group or a side-chain protecting group, replacement of one or more L-amino acids with D-isomers, amino acid modification, increased plasma protein binding, and increased albumin binding. Modified peptides include, but are not limited to, peptides that are substituted with one or more groups, conjugated with a binding partner, or cyclized, as defined herein. Generally, peptides are modified to extend their half-life in vivo in animals. A variety of modification methods are presented below.

[0210] In one embodiment, the modification may be any modification that results in an increased ability of the peptide and / or composition of the present invention to permeate cell membranes. In one embodiment, the modification may be any modification that extends the half-life of the composition or peptide of the present invention. In one embodiment, the modification may be any modification that increases the activity of the composition or peptide of the present invention. In one embodiment, the modification may be any modification that increases the selectivity of the composition or peptide of the present invention.

[0211] In one embodiment, the group is a protecting group. The protecting group may be an N-terminal protecting group, a C-terminal protecting group, or a side-chain protecting group. The peptide may have one or more of these protecting groups.

[0212] Those skilled in the art are familiar with suitable techniques for reacting amino acids with these protecting groups. These groups can be added by preparation methods known in the art, for example, by the methods outlined in paragraphs

[0104] to

[0107] of U.S. Patent Application Publication No. 2014 / 0120141. The groups may remain on the peptide or be removed. Protecting groups can be added during synthesis.

[0213] In embodiments of the present invention, the peptide may be substituted with one or more groups selected from linear or branched, long or short, saturated or unsaturated, having 1 to 29 carbon atoms, and substituted with a hydroxyl, amino, aminoacyl, sulfuric acid, or sulfide group, or unsubstituted. The N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isostearic acid, elaidic acid, 2-ethylhexanoic acid, coconut oil fatty acid, tallow fatty acid, hydrogenated tallow fatty acid, palm kernel fatty acid, lanolin fatty acid, or similar acids. These may be substituted or unsubstituted. If substituted, they are preferably substituted with hydroxyl or sulfur-containing groups such as, but not limited to, SO3H, SH, or SS.

[0214] In embodiments of the present invention, the peptide is R1-X-R2.

[0215] The R1 group and / or R2 group are attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the peptide sequence, respectively.

[0216] In one embodiment, the peptide is R1-X, or the peptide is X-R2.

[0217] Preferably, R1 is H, C 1~4 The molecule is alkyl, acetyl, benzoyl, or trifluoroacetyl; X is the peptide of the present invention; and R2 is OH or NH2.

[0218] In embodiments, R1 is selected from a group formed by H, an acyclic, substituted or unsubstituted aliphatic group, a substituted or unsubstituted cyclic aliphatic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-[wherein R5 is selected from a group formed by H, an acyclic, substituted or unsubstituted aliphatic group, a substituted or unsubstituted cyclic aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted heteroarylalkyl group]; R2 is selected from groups formed by -NR3R4, -OR3, and -SR3 [wherein R3 and R4 are independently selected from groups formed by H, an acyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted cyclic aliphatic group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroarylalkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted aralkyl group], provided that R1 and R2 are not α-amino acids.

[0219] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3 [wherein R3 and R4 are independently H, substituted or unsubstituted C1-C]. 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), substituted or unsubstituted C2-C 24 Alkinyl, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C5-C 24Cycloalkynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 Aralkyl, a 3- to 10-membered, substituted or unsubstituted heterocycle, and a substituted or unsubstituted heteroarylalkyl composed of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, wherein the alkyl chain is selected from groups formed by heteroarylalkyl composed of 1 to 6 carbon atoms]. Optionally, R3 and R4 can be bonded by a saturated or unsaturated carbon-carbon bond to form a ring with a nitrogen atom. More preferably, R2 is -NR3R4 or -OR3 [wherein, R3 and R4 are independently H, substituted or unsubstituted C1-C 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkynyl, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 15 Aryl, a group formed by a 3- to 10-membered, substituted or unsubstituted heterocyclic group, and a substituted or unsubstituted heteroarylalkyl having a 3- to 10-membered ring and an alkyl chain composed of 1 to 6 carbon atoms]. More preferably, R3 and R4 are selected from groups formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. Even more preferably, R3 is H, and R4 is selected from groups formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. According to an even more preferred embodiment, R2 is selected from -OH and -NH2.

[0220] According to another embodiment of the present invention, R1 is selected from groups formed by H, acetyl, lauroyl, myristoyl, or palmitoyl, R2 is -NR3R4 or -OR3 [wherein, R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl, and hexadecyl], and preferably, R2 is -OH or -NH2. More preferably, R1 is acetyl or palmitoyl, and R2 is -NH2.

[0221] In a preferred embodiment, an acyl (or acetyl) group is attached to the N-terminus of at least one amino acid of the peptide.

[0222] In embodiments of the present invention, the peptide is modified to include a side-chain protecting group. The side-chain protecting group may be one or more groups including benzyl or benzyl-based groups, t-butyl-based groups, benzyloxycarbonyl (Z) groups, and allyloxycarbonyl (alloc) protecting groups. The side-chain protecting group may be derived from an achiral amino acid such as achiral glycine. The use of an achiral amino acid helps to stabilize the resulting peptide and also facilitates the simplified synthetic route of the present invention. Preferably, the peptide further includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue may be α-aminoisobutyric acid (methylalanine). It will be understood that the specific side-chain protecting group used will depend on the sequence of the peptide and the type of N-terminal protecting group used. In one embodiment of the present invention, the peptide is conjugated, linked, or fused with one or more polyethylene glycol polymers or other compounds such as compounds that increase molecular weight. The molecular weight increasing compounds are any compounds that increase the molecular weight of the resulting conjugate by typically 10% to 90% or 20% to 50%, and may have a molecular weight between 200 and 20,000, preferably between 500 and 10,000. The molecular weight increasing compounds may be PEG, any water-soluble (amphiphilic or hydrophilic) polymer moiety, homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG), and polyoxyethylene glycerol (POG), polylysine, polyglutamic acid, polyaspartic acid, and other polyamino acids (especially those having the L conformation), pharmacologically inactive proteins such as albumin, gelatin, fatty acids, polysaccharides, lipids, amino acids, and dextran. The polymer moiety can be linear or branched and may have molecular weights of 500 to 40,000 Da, 5,000 to 10,000 Da, or 10,000 to 5,000 Da. The compound can be any suitable cell membrane permeable compound, such as TAT ​​peptide, penetratin, or PEP-1. The compound can be an antibody molecule. The compound can be a lipophilic or polymeric moiety.

[0223] In the art, lipophilic substituents and polymeric substituents are known. Lipophilic substituents include acyl groups, sulfonyl groups, N atoms, O atoms, or S atoms, which form part of esters, sulfonyl esters, thioesters, amides, or sulfonamides. The lipophilic portion may include a hydrocarbon chain having 4 to 30 C atoms, preferably 8 to 12 C atoms. It may be linear or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted. It may be a cycloalkane or heterocycloalkane. The peptide may be modified at the N-terminus, C-terminus, or both. The polymer or compound may be conjugated to an amino, carboxyl, or thio group, which may be conjugated at the N-terminus or C-terminus of the side chain of any amino acid residue. The polymer or compound may be conjugated to the side chain of any suitable residue.

[0224] Polymers or compounds can be conjugated via spacers. These spacers may be natural or non-natural amino acids, succinic acid, lysyl, glutamyl, asparagyl, glycyl, β-alanyl, or γ-aminobutanoyl.

[0225] Polymers or compounds can be conjugated via esters, sulfonyl esters, thioesters, amides, carbamates, ureas, and sulfonamides.

[0226] Those skilled in the art are aware of suitable means for preparing the described conjugates.

[0227] Peptides can be chemically modified by covalent conjugation to polymers to extend their cyclic half-lives, for example. Exemplary polymers and methods for conjugating such polymers to peptides are exemplified, for example, in U.S. Patents 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Further exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) moieties.

[0228] The peptides of the present invention may undergo one or more site-level modifications to manipulate the storage stability, pharmacokinetics, and / or any aspect of the peptide's bioactivity (e.g., potency, selectivity, and drug interactions). Chemical modifications to the peptides include, but are not limited to, conjugations of the peptide to one or more of the following: polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colomic acid, or other carbohydrate-based polymers, amino acid polymers, and biotin derivatives. PEG conjugations of proteins at Cys residues are disclosed, for example, in Goodson, RJ & Katre, NV (1990) Bio / Technology 8, 343; and Kogan, TP (1992) Synthetic Comm. 22, 2417.

[0229] Modified peptides may include sequences in which one or more residues are modified (i.e., by phosphorylation, sulfation, acylation, amidation, PEGylation, etc.), and mutants containing one or more modified residues relative to the parent sequence. Furthermore, amino acid sequences can also be modified directly or indirectly by labels capable of producing a detectable signal, including, but not limited to, radioisotope labels, fluorescent labels, and enzymatic labels. Fluorescent labels include, for example, Cy3, Cy5, Alexa, BODIPY, fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC), auramine, Texas Red, AMCA blue, and Lucifer Yellow. Preferred isotope labels are: 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 286Contains Re. Preferred enzyme labels include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase + peroxidase, and alkaline phosphatase (see, for example, U.S. Patent No. 3,654,090; No. 3,850,752; and No. 4,016,043). The enzymes can be conjugated by reactions involving molecules such as carbodiimide, diisocyanate, and glutaraldehyde. The enzyme labels can be detected visually or measured by calorimetry, spectrophotometry, fluorescence spectroscopy, amperometry, or gas quantification. In this technical field, other labeling systems are also known and commercially available, such as avidin / biotin and Tyramide Signal Amplification (TSA®) (see, for example, ABC Kit (Vector Laboratories, Inc., Burlingame, Calif); NEN® (Life Science Products, Inc., Boston, Mass.)).

[0230] In embodiments, peptides, variants, and / or compositions are modified to enhance drug efficacy. In embodiments, peptides, variants, and / or compositions are modified to increase stability, permeability, maintain potency, avoid toxicity, and / or extend half-life. Modifications may be as described above. For example, modifications may involve protecting the N-terminus and C-terminus, which may be amino acid modification, cyclization, amino acid substitution, and / or conjugation with high molecular weight or large polymer or long-lived plasma proteins. Strategies for extending the half-life are described by Strohl, et al (BioDrugs, 2015), Schlapschy, et al (Protein Eng Des Sel. 2013), Podust, VN, et al (Protein Eng Des Sel. 2013), Zhang, L, et al (Curr Med Chem. 2012), and Gaberc-Porekar, V, et al (Curr Opin Drug Discov Devel. 2008). Examples include PEGylation, lipidation (covalent bonding of fatty acids to peptide side chains), fusion to Fc domains and human serum albumin, fusion with hydrophilic amino acid polymers (e.g., XTEN or PAS), and / or fusion with half-life-extending proteins.

[0231] Peptides or proteins may contain vulnerable sites within their sequence that are susceptible to proteolysis in environments with enhanced proteolytic activity, such as in the blood or gastrointestinal tract. In embodiments, peptides, variants, and / or compositions include modifications to one or more vulnerable sites such that the peptide, variant, and / or composition is not proteolytic / cleaved, or the amount of proteolytic / cleaved is reduced compared to unmodified peptides or proteins. Thus, peptides can be modified to increase their resistance to proteolysis by mammalian gastrointestinal proteases. Suitable modifications are described in Diao et al (Clinical pharmacokinetics 52. 10 (2013): 855-868).

[0232] Peptide modifications that extend the in vivo half-life of peptides are described in the literature, for example, Strategies to improve plasma half life time of peptide and protein drugs. Werle M, Bernkop-Schnurch A. Amino Acids. 2006 Jun; 30(4): 351-67.

[0233] Due to the clear advantages of long-acting peptide and protein drugs, there is a great need for strategies to extend the plasma half-life of such compounds. Short plasma half-lives are generally due to rapid renal clearance as well as enzymatic degradation that occurs during systemic circulation. Peptide / protein modifications can result in extended plasma half-lives. By shortening the total amino acid content of somatostatin and replacing L-analogous amino acids with D-amino acids, the plasma half-life of the derivative octreotide was 1.5 hours, compared to only a few minutes for somatostatin. The PEG(2.40K) conjugate of INF-α-2b exhibited a plasma half-life 330-fold longer than that of the native protein. In addition to possible strategies for extending plasma half-life, such as N-terminal and C-terminal modifications or PEGylation, the aim of this review was to provide an overview of methods for assessing the efficacy of drug modifications. Furthermore, to predict the enzymatic cleavage of peptides and protein drugs in systemic circulation, fundamental data on the most important proteolytic enzymes in human blood, liver, and kidneys, as well as their cleavage specificity and inhibitors, are presented. (Strategic Approaches to Optimizing Peptide ADME Properties. Li Di AAPS J. 2015 Jan; 17(1): 134-143)

[0234] Strategies for stabilizing peptides against proteolysis Many methods are available to enhance peptide stability through structural modification. Some methods not only improve stability but also enhance other ADME properties; for example, cyclization can increase stability and permeability, while conjugation with macromolecules can improve stability and reduce renal clearance. It is important to improve stability and ADME properties while maintaining peptide potency and avoiding toxicity.

[0235] • Protection of the N-terminus and C-terminus Numerous proteolytic enzymes in blood / plasma, liver, or kidneys are exopeptidases, aminopeptidases, and carboxypeptidases, which degrade peptide sequences from the N-terminus and C-terminus. Modifications to the N-terminus and / or C-terminus can often improve peptide stability. Many studies have reported that N-acetylation and C-amidation increase resistance to proteolysis.

[0236] • Substitution of L-amino acids with D-amino acids Substitution of natural L-amino acids with natural D-amino acids reduces the substrate recognition and binding affinity of proteolytic enzymes and increases their stability. One example is vasopressin, which contains L-Arg and has a half-life of 10–35 minutes in humans. Desmopressin, a D-Arg analog, has a half-life of 3.7 hours in healthy human volunteers. In studies of bicyclic peptide inhibitors of urokinase-type plasminogen activators (uPAs), a cancer-related protease, substitution of specific glycines with D-serine not only improved potency by 1.8 times but also increased stability in mouse plasma by 4 times.

[0237] • Amino acid modification Modification of natural amino acids can improve peptide stability by introducing steric hindrance or disrupting enzymatic recognition. For example, gonadotropin-releasing hormone has an extremely short half-life (a few minutes), while buserelin, in which one glycerin molecule is substituted with t-butyl-D-Ser and another with ethylamide, has a much longer half-life in humans.

[0238] ·Cyclization Cyclization introduces conformational constraints, reducing peptide flexibility and increasing stability and permeability. Depending on the functional group, peptides can be cyclized head-to-tail, head / tail-to-side-chain, or side-chain-to-side-chain. Cyclization is generally achieved by lactamization, lactonization, and sulfide-based crosslinking. Disulfide crosslinking creates folding and conformational constraints that can improve potency, selectivity, and stability. Numerous disulfide-rich peptides, such as linaclotide, repiridine, and diconotide, are commercially available or in preclinical or clinical development.

[0239] • Conjugation with polymers Conjugation with polymers (e.g., polyethylene glycol (PEG), albumin) is an effective strategy for improving peptide stability and reducing renal clearance.

[0240] Renal clearance Many peptides exhibit promising in vitro pharmacological activity but fail to demonstrate in vivo efficacy due to their extremely short in vivo half-lives (several minutes). Rapid clearance and short half-lives of peptides hinder their successful drug development. The primary causes of rapid peptide clearance by systemic circulation are enzymatic proteolysis and / or renal clearance. Glomeruli have a small pore size of approximately 8 nm, and hydrophilic peptides with MW < 2-25 kDa are readily filtered rapidly through the renal glomeruli. Since peptides are not readily reabsorbed by the renal tubules, peptides often have large renal clearances and short half-lives. Other secondary pathways of peptide clearance include endocytosis and degradation by the proteasome and liver. Comparison of systemic and renal clearance in animal models provides useful information on whether renal clearance is the primary efflux pathway.

[0241] Inappropriate administration in patients with renal failure can lead to toxicity or treatment failure; therefore, dose adjustments may be necessary in patients with renal impairment to avoid peptide drug accumulation and high drug exposure. Several strategies have been developed to reduce peptide renal clearance and extend half-life. These will be reviewed below.

[0242] • Increases plasma protein binding The renal clearance of peptides is reduced when they are bound to membrane proteins or serum proteins. An example is octreotide, a cyclic peptide drug used to treat endocrine tumors, which has a half-life of approximately 100 minutes (unbound fraction: 0.65 minutes) in humans due to its binding to lipoproteins.

[0243] • Covalent linkage with albumin-binding low-molecular-weight molecules The covalent binding of albumin-binding small molecules to peptides can reduce glomerular filtration, improve stability against proteolysis, and extend half-life by indirectly interacting with albumin through highly bound small molecules.

[0244] • Conjugation with large polymers Conjugation of peptides with large synthetic or natural polymers or carbohydrates can increase their molecular weight and hydrodynamic capacity, thereby reducing their renal clearance. Common polymers used for peptide conjugation include PEG, polysialic acid (PSA), and hydroxyethyl starch (HES).

[0245] • Fusion with long-lived plasma proteins Plasma proteins such as albumin and immunoglobulin (IgG) fragments have long half-lives of 19–21 days in humans. Due to their high MW (67–150 kDa), these proteins have low renal clearance, and their binding to neonatal Fc receptors (FcRn) reduces their efflux via phagocytosis by vascular epithelium. Covalent linkage of peptides to albumin or IgG fragments may reduce renal clearance and prolong their half-lives. Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters William R. Strohl BioDrugs. 2015; 29(4): 215-239 Schlapschy, M, Binder, U, Borger, C et al. PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel. 2013; 26(8): 489-501 Podust, VN, Sim, BC, Kothari, D et al. Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer. Protein Eng Des Sel. 2013; 26(11): 743-53 Zhang, L, Bulaj, G. Converting Peptides into Drug Leads by Lipidation. Curr Med Chem. 2012; 19(11): 1602-18 Gaberc-Porekar, V, Zore, I, Podobnik, B et al. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel. 2008; 11(2): 242-50 By Dr Ronald V. Swanson-Long live peptides evolution of peptide half-life extension technologies and emerging hybrid approaches. From Drug Discovery World on line. Spring 2014

[0246] PEGylation PEGylation (the attachment of long chains of polyethylene glycol, a hydrophilic polymer, to a target molecule) was originally thought to be a modification that prevents recognition of foreign proteins by the immune system, thereby enabling their usefulness as therapeutic agents. Once formed, antibodies against unmodified drugs can rapidly neutralize and remove protein drugs. Unexpectedly, PEGylation improved the pharmacokinetics of proteins even in the absence of anti-drug antibodies. Simply by making the drug molecule larger, PEGylation resulted in drugs that were filtered more slowly by the kidneys. Subsequently, the empirical observation that increasing size or hydrodynamic radius led to reduced renal clearance and extended half-life became the main rationale for PEGylation of protein and peptide drugs. PEGylation can have various effects on molecules, including increasing the water solubility of proteins or peptides and protecting them from degradation by proteolytic enzymes. PEGylation also affects the binding of therapeutic proteins to their cognitive cell receptors, typically reducing affinity. Changes in the size, structure, and bonding mechanism of PEG polymers can affect the biological activity of the drug to which they are bonded.

[0247] First-generation PEGylation methods were fraught with challenges. However, the chemical reaction of PEGylation is remarkably simple. The process involves the covalent bonding of polyethylene glycol chains to reactive side chains of a protein or peptide. For example, PEG readily bonds to lysine amino groups on the surface of a protein or peptide. The reaction is pH-dependent. At high pH (above 8.0), N-hydroxysuccinimide covalently bonds the lysine side chain amino groups to PEG. This method typically results in a family of products containing different numbers of PEG chains bonded to different sites on the protein, rather than a single, distinct product. The first approved PEGylated pharmaceuticals were bovine peg-ademase (PEGylated bovine siadenosine deamidase), an enzyme replacement therapy for severe combined immunodeficiency, and pegaspargase (PEGylated asperginase), for the treatment of acute lymphoblastic leukemia. These drugs were complex mixtures of diverse PEGylated species, but they offered improved therapeutic properties compared to natural enzymes, including prolonged serum half-life and reduced immunogenicity of the proteins. Due to the polydispersity inherent in PEG, quality and batch-to-batch reproducibility were challenging. Despite this limitation, two PEGylated interferons (Peg interferon alpha 2b and Peg interferon alpha 2a), a heterogeneous population of numerous monoPEGylated positional isomers, were approved by the FDA for the treatment of hepatitis C. These drugs were marketed in 2001 and 2002, respectively.

[0248] Various enhancements and modifications have been made to the basic PEGylation technology. Second-generation PEGylation processes have introduced alternative chemical reactions for PEG conjugation, in addition to the use of branched structures. In particular, PEGs with cysteine-reactive groups such as maleimide or iodoacetamide allow for PEGylation targeting of single residues within peptides or proteins, reducing heterogeneity in the final product without losing the heterogeneity caused by the polydispersity of PEG itself.

[0249] Although the original rationale for PEGylation was to reduce immunogenicity, a small number of immunogenic PEGylated proteins exist. One example is PEGylated urate oxidase, an enzyme that lowers plasma uric acid levels in gout patients. In clinical trials, a relatively high proportion of gout patients did not respond to treatment and to developed antibodies that were specific to PEG but not to the uricase protein. Some studies have also found that PEGylated liposomes, which are generally considered non-immunogenic, are immunogenic. PEGylated liposomes induce a potent anti-PEG immunoglobulin M (IgM) response. In addition, multiple injections of PEG-glucuronidase have been shown to induce the development of specific anti-PEG IgM antibodies, thus accelerating the clearance of PEG-modified proteins from the body.

[0250] A major potential drawback of using PEG as a modifier is its non-biodegradability. The U.S. Food and Drug Administration (FDA) has approved PEG for use as a medium in pharmaceutical formulations, including injectable, topical, rectal, and nasal preparations. PEG is largely non-toxic and is excreted unchanged from the body by the kidneys (for PEG <30 kDa) or feces (for PEG >20 kDa). Repeated administration of some PEGylated proteins to animals has resulted in the observation of vacuolation of cells in the renal tubules. More recently, vacuolation of choroid plexus epithelial cells has also been observed in toxicity studies of proteins conjugated with large (>40 kDa) PEG. Choroid plexus epithelial cells produce cerebrospinal fluid and form the blood-CSF barrier. The long-term negative consequences of cellular vacuolation are unclear, but represent an undesirable outcome for some potential therapeutic agents. One possible alternative would be the substitution of PEG with a biodegradable polymer. Polymers such as hydroxyethyl starch (HES) are possible alternatives. HES is non-toxic, biodegradable, and used as a blood volume expander. The HESation process functions similarly to PEGation in reducing renal clearance by increasing the hydrodynamic radius of the peptide, but with less conferring of accumulation tendency due to its biodegradability. However, HES, as a PEG alternative, and other proposed biodegradable polymers, like PEG, are polydisperse, making it difficult to characterize the final product and metabolites. One emerging solution that mitigates both concerns is to use polypeptides with a clearly defined chemical composition as polymer components, a method that will be discussed later in this paper.

[0251] Lipidization A second major chemical modification method for extending the peptide half-life is lipidization, which involves the covalent bonding of fatty acids to the peptide side chain. Originally conceived and developed as a method for extending the half-life of insulin, lipidization shares the same fundamental mechanism of half-life extension as PEGylation, namely, increasing the hydrodynamic radius and reducing renal filtration. However, the lipid portion itself is relatively small, and the effect is brought about indirectly, via the non-covalent bonding of the lipid portion to circulating albumin. Albumin, a large protein (67 kDa) with a high abundance in human serum (35-50 g / L), inherently has the function of transporting molecules (including lipids) throughout the body. As also seen with PEGylation, binding to plasma proteins protects the peptide from peptidase attack via steric hindrance. One consequence of lipidization is that it reduces the water solubility of the peptide, but this effect can be regulated by manipulating the linker between the peptide and the fatty acid, for example, by using glutamate or mini-PEG in the linker. Manipulation of the linker and alteration of the lipid portion can affect autoaggregation, which can contribute to extending the half-life by delaying in vivo distribution independently of albumin.

[0252] Following pioneering research on insulin, the lipidization of various peptides, particularly those in the field of diabetes, including human glucagon-like peptide 1 (GLP-1) analogs, glucose-dependent insulin-secreting polypeptides, and GLP-1R / glucagon receptor coagonists, has been explored. Currently, two lipidized peptide drugs are approved by the FDA for use in humans. These are both long-acting antidiabetic agents: the GLP-1 analog liraglutide and insulin detemir.

[0253] A potentially pharmacologically significant difference between PEGylation and lipidation is that therapeutically active peptides are covalently linked to much larger PEG molecules, while smaller fatty acid acyl-peptide conjugates acovalently associate with larger albumin molecules, resulting in equilibrium between bound and unbound forms. This can lead to differences in biodistribution, potentially resulting in different pharmacologies, because access to receptors localized in different tissues can induce differential effects. In some cases, more restricted biodistribution may be desired, while in others, greater tissue penetration may be important. An interesting modification of the PEG method addressing this problem, developed by Santi et al., uses PEG conjugates that can be released at predictable cleavage rates.

[0254] Both PEGylation and lipidation confer protection from proteases and peptidases, directly or indirectly, through steric hindrance-mediated shielding, and extend the circulating half-life by directly or indirectly increasing the hydrodynamic radius. Both methods are flexible in that they utilize chemical conjugation and are tolerant of the means used to generate the peptide to be modified (whether biologically or synthetically produced). The advantage of using synthetic peptides is that they can incorporate non-natural amino acids designed to address a number of unique problems (including known instability due to susceptibility to proteolytic cleavage). When activity or potency is highly dependent on modified residues such as free ends or C-terminal amides, these methods can also be more flexible in terms of the choice of the critically important junction site.

[0255] Classical gene fusions: Fc and HSA Classical gene fusions with long-lived serum proteins offer an alternative method for half-life extension, distinct from chemical conjugation with PEG or lipids. Two key proteins—antibody Fc domains and human serum albumin (HAS)—have been used as fusion partners (Figure 2). Fc fusions involve the fusion of a peptide, protein, or receptor extracellular domain to the Fc portion of an antibody. Both Fc and albumin fusions achieve half-life extension not only by increasing the size of peptide drugs but also by utilizing the body's natural circulatory mechanism: the neonatal Fc receptor, FcRn. pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in endosomes. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, much longer than typical PEGylated or lipid-conjugated peptides. Fusion with antibody Fc can improve the solubility and stability of peptide or protein drugs. An example of a peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein utilized by fatty acid acylated peptides, is another common fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is the dimeric nature of Fc compared to the monomeric structure of HAS, resulting in the expression of the fusion peptide as a dimer or monomer, depending on the choice of fusion partner. If the target receptors are sufficiently closely spaced or are dimers themselves, the dimeric nature of the peptide-Fc fusion can lead to an avidity effect. This may or may not be desirable, depending on the target.

[0256] Designed polypeptide fusion: XTEN and PAS An interesting variation of the recombinant fusion concept was the development of a low-complexity sequence designed as a fusion partner (Figure 1). This sequence is essentially an unstructured, hydrophilic amino acid polymer, a functional analogue of PEG. The inherent biodegradability of the polypeptide platform makes it a potentially better and more attractive alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule, in contrast to the polydispersity of PEG. Unlike HSA and Fc peptide fusions, which require maintaining the three-dimensional folding of the fusion partner, recombinant fusions with unstructured partners can often be subjected to harsh conditions such as high temperatures or HPLC purification.

[0257] The most advanced of this class of polypeptides is called XTEN(Amunix), which is 864 amino acids long and contains six amino acids (A, E, G, P, S, and T) (Figure 1). This is made possible by the polymer's biodegradable properties, as it is much larger than the typically used 40 kDa PEG, and consequently confers a significant extension of its half-life. XTEN fusions with peptides result in a 60 to 130-fold extension of the half-life of the native molecule. Two fully recombinant XTENylated products, namely VRS-859 (exenatide-XTEN) and VRS-317 (human growth hormone-XTEN), are undergoing clinical trials. In a Phase Ia study, VRS-859 was found to be highly tolerable and effective in patients with type 2 diabetes. VRS-317 has been reported to have superior pharmacokinetic and pharmacodynamic properties compared to previously studied rhGH products, suggesting the potential for monthly administration. A second polymer based on a similar concept is PAS (XL-Protein GmbH). The random coil polymer consists of an even more constrained set of only three small, uncharged amino acids: proline, alanine, and serine (Figure 1). It is still unknown whether the differences in biophysical properties between PAS and the highly negatively charged XTEN contribute to differences in in vivo distribution and / or in vivo activity. However, this will likely become clear as these polypeptides are incorporated into more therapeutic agents and the behavior of the fusions is characterized.

[0258] Regardless of whether the partner is Fc, HSA, XTEN, or PAS, all peptide-protein fusions are genetically encoded and consequently subject to similar limitations. One limitation is that only native amino acids are incorporated, unlike methods that utilize chemical conjugations, which allow the use of synthetic peptides incorporating non-native amino acids. Methods to overcome this by extending the genetic code are being developed by companies such as Ambrx and Sutro, but these are not widely used. A second limitation is that the N-terminus or C-terminus of the peptide must be fused to the partner. Often, the peptide ends are involved in receptor interactions, and fusion to one or both ends can significantly impair activity. The site of PEG or lipid conjugation can be anywhere on the peptide, so it can be optimized to maximize the biological activity of the resulting therapeutic agent.

[0259] A hybrid method for integrating synthetic peptides with half-life extension proteins. While gene fusions have historically offered potential for greater half-life extension, they lack the advantages offered by methods using chemical conjugation (PEGylation and Lipidization) in terms of the flexibility of the junction site and the incorporation of non-natural amino acids or modifications to the peptide backbone. One of the first attempts to integrate the advantages of gene fusions with chemical conjugation for half-life extension was carried out by researchers at the Scripps Research Institute in La Jolla, with technology that later formed the basis for the biotech company CovX. Using catalytic aldolase antibodies, these researchers developed a platform in which the lysine active site of the antibody forms a reversible covalent enamine bond with a β-diketone incorporated into a peptide or small molecule. The resulting complex is referred to as CovXBody®. This method combines the functional qualities of a peptide drug or small molecule with the long serum half-life of an antibody, not through gene fusion, but through chemical linkage. Following initial technical validation, researchers expanded to the use of CovX-Body® prototypes based on peptide-mimicking pharmacophores that target integrins. Based on this architecture, at least three molecules—CVX-096, a Glp-1R agonist; CVX-060, an angiopoietin II-binding peptide; and CVX-045, a thrombospongin mimetic—have entered clinical development.

[0260] Similarly, in recent years, XTEN polypeptides have also been used in chemical conjugation methods that make them even more directly analogous to PEG. The first example of an XTENized peptide created using this method is GLP2-2G-XTEN, in which the peptide was chemically conjugated to an XTEN protein polymer using a maleimide-thiol chemical reaction. The chemically conjugated GLP2-2GXTEN molecule exhibited in vitro activity, in vitro plasma stability, and pharmacokinetics in rats that were comparable to recombinant fusion GLP2-2G-XTEN.

[0261] The number and spacing of reactive groups, such as lysine or cysteine ​​side chains, within the fully designed sequences of XTEN or PAS polypeptides can be precisely controlled via site-directed changes, resulting from the constrained sets of amino acids that constitute them. This offers greater flexibility than methods that can use Fc or albumin, whose sequences inherently contain many reactive groups, and contrasts with CovX technology, which relies on reactive residues within highly specialized active sites. In addition, the lack of tertiary structure in XTEN or PAS should provide greater flexibility over the conditions and chemical reactions used in the coupling and purification of conjugates.

[0262] In summary, hybrid peptide half-life extension methods are emerging that combine the advantages of chemical conjugation and gene fusion, overcoming their respective limitations. These methods confer long half-lives and free the therapeutic peptide moiety from the limitations of being composed solely of native L-amino acids or linear, unidirectional polypeptides fused at the N-terminus or C-terminus, enabling the creation of molecules based on recombinant polypeptide partners. This opens the door to a wide range of long-acting peptide-based drugs.

[0263] Pharmaceutical composition In situations where a pharmaceutically acceptable composition containing one or more peptides of the present invention is being prepared, the peptides of the present invention (including variants and modified peptides) can be combined with one or more carriers (diluents, excipients, etc.) suitable for one or more intended routes of administration.

[0264] The peptides of the present invention can be mixed with, for example, lactose, sucrose, powder (e.g., starch powder), cellulose esters of alkanates, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, acacia, gelatin, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, the peptides of the present invention can be dissolved in physiological saline, water, polyethylene glycol, propylene glycol, carboxymethylcellulose colloidal solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and / or various buffers. Other carriers, adjuvants, and administration methods are also well known in the pharmaceutical art. The carrier or diluent may contain a time-delaying material, such as glyceryl monostearate or glyceryl distearate, alone or together with wax or other functionally similar materials.

[0265] Pharmacoherent carriers generally include all suitable solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, as well as any other suitable solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, etc., that are physiologically compatible with the insulin analogue. Examples of pharmaceutically acceptable carriers include water, physiological saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and any combination thereof. In many cases, it may be desirable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride. Small amounts of auxiliary substances (such as humectants or emulsifiers, preservatives or buffers) that may enhance the shelf life or efficacy of pharmaceutically acceptable substances, or insulin analogues, related compositions, or combinations thereof, such as wetting agents. The suitability of the carrier and other components of the pharmaceutical composition can be determined based on the absence of any significant negative impact on the desired biological properties of the insulin analog, associated composition, or combination (e.g., a reduction of about 20%, 15%, 10%, 5%, or less than 1% of insulin receptor (IR) binding and / or activation; or the ability to reduce blood glucose in the target host).

[0266] Compositions, related compositions, and combinations according to the present invention may be presented, prepared, and / or administered in a variety of suitable forms. Such forms include liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusion solutions), dispersions or suspensions, emulsions, microemulsions, tablets, pills, powders, liposomes, dendrimers and other nanoparticles (see, e.g., Baek et al, Methods Enzymol. 2003; 362: 240-9; Nigavekar et al, Pharm Res. 2004 March; 21(3): 476-83), microparticles, and suppositories. The optimal form for any peptide of the compositions related to the present invention depends on the intended method of administration, the nature of the composition or combination, and the therapeutic application or other intended use. The formulations may also include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) vesicles, DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil emulsions, emulsions, carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. Any of the aforementioned mixtures may be suitable for treatments and therapies according to the present invention, provided that the binding of the peptide to cognitive IR is not significantly inhibited by the formulation and the formulation is physiologically compatible and tolerable with respect to the route of administration. For further information related to excipients and carriers well known to medicinal chemists, see, for example, Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52: 238-311 (1998), and the references therein.

[0267] In certain embodiments, the peptide of the present invention is administered via liposomes. In other embodiments, the peptide of the present invention is administered via liposomes together with one or more adjunct agents, such as one or more antidiabetic drugs.

[0268] Furthermore, the compositions of the present invention also include compositions comprising any suitable combination of the peptide of the present invention and a suitable salt thereof. Any suitable salt, such as any suitable form of alkaline earth metal salt (e.g., buffer salt), can be used in stabilizing the peptide of the present invention (preferably, the amount of salt is such that oxidation and / or precipitation of the peptide is avoided). Suitable salts typically include sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride. Compositions comprising a base and one or more peptides of the present invention are also provided.

[0269] Typical methods for delivering the compositions of the present invention are parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, and / or intramuscular administration). In one embodiment, the composition or peptide of the present invention is administered to a human patient by intravenous infusion or intravenous injection. In another embodiment, the composition or peptide of the present invention is administered by intramuscular or subcutaneous injection. As indicated above, in certain therapeutic regimens, intratumoral administration may also be useful.

[0270] Accordingly, the peptides of the present invention can be formulated, for example, in solid form (including granules, powders, projectile particles, or suppositories), semi-solid form (such as gels or creams), or liquid form (such as solutions, suspensions, or emulsions). The compositions or peptides of the present invention can be applied by various solutions. Solutions suitable for use according to the present invention are typically sterile, capable of dissolving sufficient amounts of the peptides and other components of the composition, stable under manufacturing and storage conditions, and non-toxic to the target for the proposed application. The peptides of the present invention may also be subjected to conventional pharmaceutical processes, such as sterilization, and / or may contain conventional adjuvants (such as preservatives, stabilizers, humectants, emulsifiers, or buffers). The compositions can also be formulated as solutions, microemulsions, dispersions, powders, macroemulsions, liposomes, or other ordered structures suitable for high drug concentrations. For example, the desired particle size can be maintained in the case of dispersions by using coatings such as lecithin, and the desired fluidity of solutions can be maintained by using surfactants. Sustained absorption of an injectable composition can be achieved by incorporating absorption-delaying agents (e.g., monostearate and gelatin) into the composition. These and other components of the pharmaceutically acceptable compositions of the present invention can confer advantageous properties such as improved ingestion, delivery, and tolerance.

[0271] Compositions for pharmaceutical use may include a variety of diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for incorporation into compositions for pharmaceutical use. Examples of suitable components are also described, for example, in Berge et al, J. Pharm. Sci., 6661), 1-19 (1977); Wang and Hanson, J. Parenteral. Sci. Tech: 42, S4-S6 (1988); U.S. Patent No. 6,165,779 and No. 6,225,289; and other documents cited herein. Such pharmaceutical compositions may also include preservatives, antioxidants, or other additives known to those skilled in the art. In this technical field, further pharmaceutically acceptable carriers are known, for example, Urquhart et al, Lancet, 16, 367 (1980); Lieberman et al, Pharmaceutical Dosage Forms-Disperse Systems (2nd ed., vol.3, 1998); Ansel et al, Pharmaceutical Dosage Forms & Drug Delivery Systems (7th ed.2000); Martindale, The Extra Pharmacopeia (31st edition); Remington's Parmaceutical Sciences (16th-20th editions); The Pharmacological Basis Of Therapeutics, Goodman and Gilman, Eds. (9th ed.-1996); Wilson and Gisvolds' TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, Eds. (10th edition) This is described in (ed.-1998), as well as in U.S. Patent No. 5,708,025 and U.S. Patent No. 5,994,106.The principles for formulating pharmaceutically acceptable compositions are also described, for example, in Piatt, Clin. Lab Med., 7: 289-99 (1987); Aulton, Pharmaceutics: The Science Of Dosage Form Design, Churchill Livingstone (New York) (1988); EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP (1998); and “Drug Dosage,” J. Kans. Med. Soc, 70(I), 30-32 (1969). Further pharmaceutically acceptable carriers particularly suitable for the administration of the peptides or compositions of the present invention and related compositions (e.g., compositions comprising nucleic acids encoding the peptides of the present invention, or vectors containing nucleic acids encoding the peptides of the present invention) are described, for example, in International Patent Application Publication No. 98 / 32859.

[0272] The peptides or compositions of the present invention can be prepared with carriers that protect the compounds from rapid release, such as controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. To produce such compositions, biodegradable and biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, and any combination thereof. Methods for preparing such compositions are known. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0273] In another embodiment, the composition of the present invention is administered orally, for example, with an inert diluent or an assimilable edible carrier (specific oral formulations and methods of oral administration are also described separately elsewhere in this specification). The compound (and, if desired, other components) may also be encapsulated in hard or soft gelatin capsules, compressed into tablets, or directly incorporated into the diet of the subject. For therapeutic oral administration, the compound may be incorporated with excipients and used in the form of edible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For administration of the compound of the present invention by means other than parenteral administration, it may be necessary to coat the compound with a material that prevents its inactivation or to co-administer it together with such material.

[0274] The preparation of pharmaceutical compositions containing peptides as active ingredients is well understood in the art. Typically, such compositions are prepared as injectable solutions or suspensions, but they can also be prepared in solid form suitable for dissolution or suspension in liquid before injection. The preparations can also be emulsified. The therapeutic active ingredient is often mixed with excipients that are pharmaceutically (i.e., physiologically) acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, ethanol, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as humectants or emulsifiers, pH buffers, etc., that enhance the efficacy of the active ingredient.

[0275] The peptides of the present invention can be formulated into pharmaceutical compositions as physiologically acceptable neutralized salt forms. Suitable salts are acid addition salts (i.e., formed from the free amino groups of the peptide molecule), including, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or acid addition salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0276] In the case of combination compositions (further discussed herein), the peptides of the present invention can be co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., antidiabetic agents such as insulin, insulin analogs, metformin, or other antidiabetic biguanides, glucagon receptor antagonists, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, meglitinide, glucagon-like peptide-1 (GLP-1), GLP-1 analogs, etc.). Such combination therapies may require low doses of the peptides of the present invention and / or co-administered agents to avoid possible toxicity or complications associated with diverse monotherapies.

[0277] therapeutic application As indicated above, the peptides or compositions of the present invention can be administered individually or in combination with other pharmacologically active agents. It will be understood that such combination therapies encompass different treatment regimens. Treatment regimens include, but are not limited to, the administration of multiple agents combined in a single dosage form or the administration of multiple agents in separate, independent dosage forms. If the agents are present in different dosage forms, the administration may be simultaneous or nearly simultaneous, or may follow any predetermined regimen that encompasses the administration of different agents.

[0278] For example, when used to treat diabetes or other diseases or syndromes associated with a decreased insulin response or production, such as hyperlipidemia, obesity, or appetite-related syndromes, the peptides of the present invention may be advantageous when administered in combination with one or more agents, including but not limited to insulin, insulin analogues, insulin derivatives, glucagon-like peptide 1 or 2 (GLP-1, GLP-2), or derivatives or analogues of GLP-1 or GLP-2 (e.g., those disclosed in International Publication No. 00 / 55119). As used herein, “analogue” of insulin, GLP-1, or GLP-2 means, where applicable, a peptide containing one or more amino acid substitutions compared to the natural sequence of insulin, GLP-1, or GLP-2; as used herein, “derivative” of insulin, GLP-1, or GLP-2 means, in particular, a natural or analogue insulin, GLP-1, or GLP-2 peptide that has undergone one or more further chemical modifications of its amino acid sequence compared to the natural sequence. Insulin derivatives and insulin analogs are disclosed, for example, in U.S. Patent Nos. 5,656,722, 5,750,497, 6,251,856, and 6,268,335. In some embodiments, the combination agent is one of LysB29(ε-myristoyl)des(B30) human insulin, LysB29(ε-tetradecanoyl)des(B30) human insulin, and B29-Nε-(N-lithocoryl-γ-glutamyl)-des(B30) human insulin. Non-peptide hypoglycemic agents and anti-dyslipidemia agents, such as those well known in the art, are also suitable for combination therapy.

[0279] In one embodiment, the present invention provides a method for treating diabetes mellitus or related syndromes or conditions (e.g., reducing the rates of stroke, heart disease, kidney disease, blindness, and / or loss of sensation in the limbs that are related to glucose levels and / or associated with diabetes), comprising the step of delivering an effective amount of at least one peptide of the present invention (by gene expression, by delivery of homogeneous peptides, or by administration of a pharmaceutically acceptable composition typically comprising one or more peptides as described above and one or more pharmaceutically acceptable carriers). In another embodiment, the present invention provides the use of the peptides or compositions (such as combination compositions) of the present invention in the manufacture of pharmaceuticals used in the treatment of type 1 or type 2 diabetes mellitus.

[0280] The peptides of the present invention can generally be used in the treatment of type 1 diabetes or type 2 diabetes, i.e., both insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM).

[0281] In exemplary combination therapy embodiments, the present invention provides a method for treating diabetes (e.g., a method for reducing one or more associated symptoms in a host, and / or a method for administering to a host an amount of a composition proven to be therapeutically effective in at least a substantial proportion of similar host populations), comprising the steps of delivering a first amount of the peptide or composition of the present invention and a second amount of a long-acting insulin analog (e.g., LysB29(ε-myristoyl)des(B30) human insulin, LysB29(ε-tetradecanoyl)des(B30) human insulin, or B29-Nε-(N-litocoryl-γ-glutamyl)-des(B30) human insulin), wherein the first and second amounts together are effective for treating the syndrome. As used herein, a long-acting insulin analog is an insulin analog that exhibits a sustained action profile compared to natural human insulin, as disclosed, for example, in U.S. Patent No. 6,451,970. In another aspect, the present invention provides the use of combination compositions comprising a therapeutically effective combination of at least one peptide or composition of the present invention and at least one insulin or insulin analog in the manufacture of a pharmaceutical used in the treatment of a disease (such as the treatment of type 1 diabetes or type 2 diabetes). Similar compositions comprising a combination of one or more peptides or compositions of the present invention and one or more long-acting insulin analogs and / or short-acting insulin analogs may also be suitable for therapeutic methods such as the treatment of diabetes.

[0282] In one embodiment, the present invention provides a method for treating symptoms and / or underlying conditions associated with type 2 diabetes in a patient requiring treatment (due to a substantial risk of diagnosis and / or development of the disease), comprising the step of treating such symptoms and / or conditions by delivering to the patient an amount of the peptide or composition of the present invention effective for treatment or prevention. In a particular embodiment, the present invention provides a method for treating a patient having type 2 diabetes and high insulin blood levels (hyperinsulinemia). In such one embodiment, the patient is obese. In another embodiment, the patient also includes, simultaneously or alternatively, an insulin resistance genotype / mutation.

[0283] In another aspect, the present invention provides a method for lowering blood pressure in a patient having insulin / IR-related hypertension, comprising the step of administering or otherwise delivering a therapeutically effective amount of the peptide or composition of the present invention to lower the patient's blood pressure.

[0284] In another aspect, the present invention provides a method for treating the symptoms and / or underlying conditions of syndrome X, or manifestations thereof (e.g., hyperlipidemia, hypertension, and / or obesity), comprising the step of administering to the patient an amount of the peptide or composition of the present invention effective for treatment and / or prevention, or otherwise delivering it, thereby treating syndrome X or the syndrome X condition.

[0285] In yet another aspect, the present invention provides a method for treating non-diabetic IR-mediated symptoms, disorders, or diseases in a patient, such as IR-related neurodegenerative diseases; IR-related non-diabetic autoimmune diseases, comprising the step of administering to the patient an amount of the peptide or composition of the present invention effective for treatment or prevention to treat such condition / symptoms.

[0286] In another aspect, the present invention provides a method for preventing weight gain in a patient in need thereof, comprising the step of administering to the patient an effective amount of the peptide or composition of the present invention for treatment or prevention to prevent IR-related weight gain.

[0287] In a related aspect, the present invention provides a method for treating obesity, comprising the step of administering to a patient an amount of the peptide or composition of the present invention effective for treatment or prevention, thereby treating obesity or an associated condition (by stabilizing and / or reducing the patient's weight).

[0288] In another aspect, the present invention provides a method for treating a patient suffering from disease symptoms associated with or caused by hyperinsulinemia, hypoglycemia, hypokalemia, and / or hypophosphatemia, comprising the step of treating such condition / symptom by administering (or otherwise delivering, as in any case) an amount of the peptide or composition of the present invention effective for treatment or prevention to the patient. The present invention further relates to a method for treating a blood glucose-related disease or disorder, comprising the administration of an insulin derivative or insulin conjugate. Blood glucose-related diseases or disorders include type 1 and type 2 diabetes mellitus, as well as gestational diabetes mellitus. Cystic fibrosis, polycystic ovary syndrome, pancreatitis, and other pancreatic-related diseases can also be treated by the administration of the insulin derivative or insulin conjugate of the present invention. Insulin is also known as a growth factor, and therefore the insulin derivative or insulin conjugate of the present invention may also be useful in topical administration for wound healing and other related indications.

[0289] In another aspect, the present invention provides the use of the peptide or composition (such as a combination composition) of the present invention in the manufacture of a pharmaceutical used in any of the conditions described above.

[0290] In one general aspect, the present invention provides a method for adjusting glucose levels in an individual, comprising the step of administering a physiologically effective amount of the peptide or composition of the present invention to adjust the glucose level in the patient to a detectable level. In another aspect, the present invention provides the use of the peptide or composition (such as a combination composition) of the present invention in the manufacture of a pharmaceutical used in reducing blood glucose levels.

[0291] In another general embodiment, the present invention provides a method for mediating IR activity, comprising the step of administering a physiologically effective amount of the peptide or composition of the present invention such that a responsive IR on an IR-presenting cell binds in an amount and under conditions sufficient to induce, promote, enhance, and / or otherwise modulate IR-mediated activity or response. For example, the peptide of the present invention can be delivered to a host by binding to site 1 or site 2 to guide an insulin or insulin analog molecule to the other site and modify the profile of insulin or insulin analog treatment.

[0292] In more detail, the present invention provides methods for inducing, promoting, and / or enhancing such physiological responses by delivering or otherwise administering to a patient an amount of the peptide or composition of the present invention effective for treatment or prevention: methods for regulating nitric oxide production levels in a patient, such as in patient endothelial cells; methods for mediating RAS, RAF, MEK, and / or mitogen-activated protein (MAP) kinase pathways; methods for regulating vascular tissue proliferation and / or proliferation and / or migration of smooth muscle cells, monocytes, macrophages, and / or endothelial cells; methods for stimulating the production of type 1 plasminogen activator inhibitor (PAI-1); methods for regulating endothelin production; methods for regulating IR-related atherosclerotic pathway biological events; methods for regulating IR-related inflammation; methods for treating and / or reducing the risk of arterial injury; methods for treating and / or preventing atherosclerosis; and / or reducing IR-related inflammatory molecules, such as LDL cholesterol in the patient's vascular wall.

[0293] The peptides and compositions of the present invention can also be used to treat catabolic loss (e.g., cachexia and sarcopenia), and can also be used for non-therapeutic purposes, such as increasing muscle mass in athletes, sportsmen, or bodybuilders. The peptides are also used for their anabolic effects on muscle mass in athletes (GHRP refers to growth hormone-releasing hexapeptide, a type of growth hormone-releasing hormone).

[0294] This can be useful in several ways. Obviously, athletes need to heal quickly and be productive immediately after injury. Peptides can aid in muscle or soft tissue during this reconstructive healing process. Supplements that provide anabolic effects can also be used during the preseason and at other times when building muscle mass is important. Muscle mass can be built rapidly because athletes induce small tears in their muscles, heal them on a rapid schedule, and repeat the process: the end effect is increased muscle mass and reduced body fat within a short timeframe. Since new peptides do not have the side effects of anabolic steroids, the bodybuilding community uses the most effective peptides in this second method.

[0295] Exemplary Dosage and Dosage Strategy As described above, the compositions of the present invention may contain a "therapeutic effective dose" or a "preventive effective dose" of the peptide of the present invention (or, in the case of a combination composition comprising the peptide of the present invention and a second component, a first and second amount; or, in the case of a combination composition comprising two peptides of the present invention and a secondary agent, or a first, second, and third amount comprising the peptide of the present invention and two secondary agents). To better illustrate specific embodiments, a further detailed discussion of the dosage principles is presented here.

[0296] The amount or dosage range of the peptide of the present invention used in carrying out the present invention is typically a range that effectively induces, promotes, or enhances the physiological response associated with the binding of the peptide of the present invention to cognitive IRs. In one embodiment, the dosage range of the peptide of the present invention used is selected so that it induces, promotes, or enhances a medially significant effect in patients who have, or are at substantial risk of developing, a related condition (such as a form of diabetes) that is at least partially modulated by IR activity. The medially significant effect is associated with the activation, signaling, and / or biological modification (e.g., phosphorylation) of cognitive IRs.

[0297] In yet another embodiment, the patient is provided with an active ingredient (e.g., the peptide of the present invention) in doses of about 0.01 to 100 milligrams per kilogram of body weight daily. Typically, doses of about 1 to 5 or 1 to 10 milligrams per kilogram per day, administered in divided doses of about 1 to 6 times daily, or in a sustained-release form, may be effective in obtaining the desired results.

[0298] As a non-limiting example, treatment of IR-related conditions in humans or animals may be a single dose or divided doses administered approximately every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or also on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 This can be administered by administering a daily dose of the peptide of the present invention in an amount of approximately 0.1 to 100 mg / kg per day (such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg) during at least one of weeks 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof.

[0299] In one embodiment, the method of the present invention comprises the step of administering or otherwise delivering two different peptides of the present invention over a period of one month, at the initiation of treatment with the second peptide of the present invention, or at any point in time when a significant immune response to the first peptide of the present invention occurs in the host, and as a result, continued use of the first peptide of the present invention becomes harmful to the patient. [Examples]

[0300] The peptides mentioned in the examples are peptides having the sequence of SEQ ID NO: 1.

[0301] (Example 1) Cell proliferation assay Research Description BrDu is incorporated into the newly synthesized DNA strands of actively proliferating cells. After partial denaturation of double-stranded DNA, BrDu can be detected immunochemically, enabling evaluation of cell populations that are synthesizing DNA.

[0302] method Human dermal fibroblasts (HDF-Sigma 10605a) were seeded at a rate of 10,000 cells per well in DMEM containing 10% fetal bovine serum (FCS), 1% Pen / strep, and 1% L-glutamine in a 96-well plate, and allowed to adhere for 24 hours.

[0303] After an initial 24-hour incubation, cells were incubated for 24 hours with synthetic peptides at concentrations of 5 μg / ml, 0.5 μg / ml, or 0.05 μg / ml, respectively.

[0304] After 18 hours of incubation with the synthetic peptide, 20 μL of BrDu reagent was added to each well. After 24 hours of incubation, the cells were fixed, and the amount of 2-DG6P was measured using the BrdU cell proliferation assay. All steps were performed according to the manufacturer's instructions.

[0305] Next, this experiment was repeated using HACAT cells.

[0306] result The results were calculated as a percentage relative to the untreated control. As illustrated in Figure 1 (HDF cells) and Figure 2 (HACAT cells), an increase in optical density readings indicates increased BrDu incorporation and increased cell proliferation. As illustrated in Figures 1 and 2, samples stimulated with the peptide of the present invention showed increased cell proliferation compared to the untreated control.

[0307] conclusion These results support the effectiveness of peptides that facilitate cell proliferation.

[0308] (Example 2) Collagen production assay Research Description Hydroxyproline in tissue preparations is a direct measure of the amount of collagen present. A FIRELISA human hydroxyproline ELISA kit assay was designed to measure hydroxyproline in tissue or peptide composition. method

[0309] Human dermal fibroblasts (HDF Sigma 10605a) were seeded at a rate of 50,000 cells per well in DMEM containing 10% fetal bovine serum (FCS), 1% Pen / strep, and 1% L-glutamine in a 24-well plate, and allowed to adhere for 24 hours.

[0310] After an initial 24-hour incubation, cells were incubated for 96 hours with synthetic peptides at concentrations of 5 μg / ml, 1 μg / ml, or 0.1 μg / ml, respectively.

[0311] After the procedure, the cell supernatant was removed and centrifuged. Each supernatant was assayed using the FIRELISA Human Hydroxyproline ELISA Kit, 50 μL in each volume. All steps were performed according to the manufacturer's instructions.

[0312] result The results were calculated as a percentage relative to the untreated control. An increase in optical density readings indicates an increase in collagen content. As illustrated in Figure 3, an increase in collagen production was observed compared to the untreated control.

[0313] conclusion These results support the effectiveness of peptides that facilitate collagen production.

[0314] (Example 3) Elastin Production Assay Research Description Elastin is a highly elastic protein found in connective tissue, enabling many tissues in the body to regain their shape after stretching. Design a FIRELISA human elastin ELISA kit assay to measure elastin in tissue composition or protein / peptide composition. method

[0315] Human dermal fibroblasts (HDFs) were seeded at a rate of 50,000 cells per well in DMEM containing 10% fetal bovine serum (FCS), 1% Pen / strep, and 1% L-glutamine in 24-well plates, and allowed to adhere for 24 hours.

[0316] After an initial 24-hour incubation, cells were incubated for 96 hours with synthetic peptides at concentrations of 5 μg / ml, 1 μg / ml, or 0.1 μg / ml, respectively.

[0317] After treatment, cells were lysed using cell lysis buffer and sonicated for 10 seconds. The lysed cells were centrifuged, and the supernatant was collected from each sample. The total protein concentration was determined using a BCA assay. Each sample was diluted with assay buffer to contain 10 μg of total protein. Each 50 μL sample was then assayed using the FIRELISA Human Elastin ELISA Kit. All steps were performed according to the manufacturer's instructions.

[0318] result The results were calculated as a percentage relative to the untreated control. An increase in optical density reading indicates an increase in elastin content. As illustrated in Figure 4, samples stimulated with the peptide of the present invention showed increased elastin production compared to the untreated control.

[0319] conclusion These results support the effectiveness of peptides that facilitate elastin production.

[0320] (Example 4) glucose uptake Research Description We measured glucose uptake in skeletal muscle cells using 2-deoxyglucose (2-DG). 2-DG is taken up by glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P). The accumulation of unmetabolized 2-DG6P is proportional to glucose uptake by cells. method

[0321] Human skeletal myoblasts (Sigma 150-05a) were seeded in skeletal muscle differentiation medium in a 96-well plate at a rate of 10,000 cells per well, and differentiated for 72 hours prior to the experiment.

[0322] Differentiated cells were serum-starved for 24 hours before stimulation with insulin or synthetic peptides. After starvation, the serum-free medium was removed, the cells were rinsed with phosphate-buffered saline (PBS), and the medium was replaced with 100 μL of Krebs-Ringer-phosphate-HEPES (KRPH) and incubated for 1 hour.

[0323] The cells were then stimulated with 100 nM insulin for 30 minutes, or with synthetic peptides at concentrations of 5 μg / ml, 0.5 μg / ml, or 0.05 μg / ml, respectively, for 3 hours.

[0324] Following stimulation, cells were incubated with 10 μL of 2-DG solution per well for 40 minutes, and glucose uptake was measured using the "PrismColor Glucose Uptake Assay Kit" (Molecutools). All steps were performed according to the manufacturer's instructions. The experiment was repeated using HACAT cells.

[0325] result Results were calculated as a percentage relative to the untreated control. Increased optical density readings indicate increased 2-DG6P incorporation and increased glucose uptake. All experiments were performed on three plates, in a double-row configuration (six wells per state). Student's t-test ( * Compared to the control, p<0.05.** Compared to the control, p<0.01. *** Significance was determined using p<0.001) compared to the control. Figure 6 illustrates the results for 0.5 μg / ml of the synthetic peptide, and Figure 7 illustrates the results for 5 μg / ml of the synthetic peptide. Figure 5 shows the results for HACAT cells. As illustrated by these figures, samples stimulated with the peptide of the present invention showed increased glucose uptake compared to the untreated control.

[0326] conclusion These results support the effectiveness of peptides that facilitate glucose uptake within skeletal muscle.

[0327] (Example 5) GLUT-4 migration method HSKMC cells were cultured in a 6-well plate and then treated with peptide / hydrolyzate at concentrations of 0.5 μg / ml and 5 μg / ml for 2 hours. After treatment, cell membrane proteins were collected. Briefly, cells were collected from the wells using a cell scraper and centrifuged at 300 × g for 5 minutes. The cell pellet was washed with 300 μl of cell wash and then centrifuged again at 300 × g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1.5 ml of cell wash and transferred to a 2 ml centrifuge tube. The cells were then centrifuged at 300 × g for 5 minutes, and the supernatant was discarded. 150 μl of permeabilization buffer was added to the cell pellet and vortexed briefly to obtain a homogeneous cell suspension. The suspension was then incubated at 4°C for 10 minutes with constant mixing. Next, the permeabilized cells were centrifuged at 16000 × g for 15 minutes, and the supernatant containing cytosolic proteins was transferred to a new tube. 100 μl of solubilizing buffer was added to the pellet and resuspended by gentle pipetting. The sample was then incubated at 4°C for 10 minutes with continuous mixing. The sample was then centrifuged at 16000 × g for 15 minutes and at 4°C for 10 minutes. The supernatant containing the solubilized membrane and membrane-related protein fractions was transferred to a new tube and stored at -80°C until ready for evaluation.

[0328] GLUT-4 in the cell membrane fraction was evaluated using an LSBio sandwich ELISA. 10 μg of protein from each sample was loaded into individual wells on a 96-well plate to a final volume of 100 μl. GLUT-4 standards (0.3 ng / ml to 20 ng / ml) were also added to the plate in the same volume. The plate was then sealed and incubated at 37°C for 1 hour. The liquid was aspirated from each well, and 100 μl of detection reagent A was added to each well. At this point, the plate was sealed, gently agitated to ensure thorough mixing, and incubated at 37°C for 1 hour. The liquid was aspirated from each well, and the wells were washed three times with 350 μl of wash buffer. 100 μl of detection reagent B was added to each well. At this point, the plate was sealed and incubated at 37°C for 30 minutes. The wells were washed five times as previously outlined, and 90 μl of TMB substrate was added to each well. The plate was sealed, protected from light, and incubated at 37°C for 10 minutes to confirm optimal color development. 50 μl of stop solution was added to each well, and the optical density of the sample was determined using a microplate reader set to 450 nm. The GLUT-4 concentration in the sample was calculated from the calibration curve after generating a 4-parameter logistic (4-PL) curve fit. The sample concentration readings on the curve were then corrected by multiplying the results by the dilution factor.

[0329] result As illustrated in Figure 8, increased GLUT4 migration was observed in samples stimulated with both 0.5 μg / ml and 5 μg / ml peptides.

[0330] conclusion The peptide demonstrated a stimulating effect on skeletal muscle GLUT4 transport and its ability to facilitate glucose transport within skeletal muscle.

[0331] (Example 6) Muscle protein synthesis (phosphomTOR assay) Research Description In mammals, the target of rapamycin (mTOR) is a Ser / Thr protein kinase that functions as an ATP and amino acid sensor to balance nutrient availability and cell proliferation. mTOR activity is regulated by insulin, amino acids, physical exercise, oxidative stress, and growth factors, and its phosphorylation consistently promotes protein synthesis pathways. However, dysregulation of mTOR leads to accelerated aging, among many other pathological conditions. In muscle, activation (phosphorylation) of mTOR results in skeletal muscle hypertrophy. This fact is widely reported in the literature in both rodents and humans. Amino acids such as leucine enhance mTOR phosphorylation, and its activation upregulates protein translation via phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K), leading to cell proliferation (growth and proliferation).

[0332] Phospho-mTOR sandwich ELISA detects the endogenous level of phosphorylated mTOR at Ser2448.

[0333] method Day 1: Seed HSkMC cells in 500 μL of HSkMC growth medium per well and allow them to adhere to the wells for 16–24 hours. Day 2: Remove the growth medium and add 500 μL of HSKMC differentiation medium per well, allowing them to differentiate for 5-7 days. Day 7: Treat the cells for 2 hours. Collect the cell lysates in the lysis buffer. Day 8: Perform BCA (protein determination) and assay. In the first incubation step, mTOR (phosphorus and non-phosphorus) is captured from the cell lysate using mTOR antibody coated on ELISA microwells. Then, after thorough washing, a specific phospho-mTOR detection antibody is added to detect only the already captured phospho-mTOR protein. Finally, an HRP-conjugated antibody is used to recognize the detection antibody, and the addition of TMB (HRP substrate) causes color development in proportion to the amount of mTOR phosphorylated at Ser2448.

[0334] result The results are illustrated in Figure 9. Samples stimulated with 0.5 μg / ml and 5 μg / ml showed an increase in mTOR.

[0335] conclusion These results support the effectiveness of peptides that facilitate muscle protein synthesis and muscle proliferation.

[0336] (Examples 7-15) formulation In one embodiment, the composition is formulated as a topical composition. In one embodiment, it may be an emulsion, a cream, or a lab such as a muscle lab.

[0337] In the embodiment, the cream comprises an excipient or diluent, a suspending agent, a preservative, and an unspecified amount of at least one peptide of the present invention.

[0338] In embodiments, the cream comprises alcohol, carbomer, sorbate, water, and at least one peptide of the present invention.

[0339] Preferably, the alcohol is butylene glycol (1,3-butanediol). The alcohol may be present in an amount between 1 and 10% of the composition, preferably between 2 and 6%, preferably between 4%. The sorbate may be polysorbate 20. The sorbate may be present in an amount between 0.01 and 1% of the composition, preferably between 0.05 and 0.5%, preferably between 0.10%. Water may be present in an amount between 10% and 90%, preferably between 30% and 75%. The carbomer may be present in an amount between 0.05% and 1%, preferably between 0.1% and 0.5%, preferably between 0.15%. The carbomer may be Ultrez 10.

[0340] The peptide of the present invention may be present in an amount of 0.5% to 10%, preferably 1% to 5%, and preferably 3%.

[0341] The composition may further comprise one or more of the following: sugar alcohols such as glycerin, parabens, silicones such as cyclohexasiloxane, fatty alcohols or phosphoric acid, or mixtures of fatty alcohols and phosphoric acid (such as cetearyl alcohol, dicetyl phosphate, and Cereth 10 phosphate, or combinations thereof), polyoxyethylene stearyl ethers (such as Stereth 2 and 10), and fragrances.

[0342] (Example 7) An example of a rub or emulsion in Example 7 is as follows:

[0343] [Table 1]

[0344] The resulting emulsion is suitable as a lab coat. In addition, the lab coat is suitable for fragile, aging skin. The emulsion is suitable for improving fine lines, wrinkles, and dryness, and for reducing redness and irritation.

[0345] The percentages are merely examples, and it should be understood that any appropriate percentage can be used depending on the context.

[0346] The emulsion is prepared in the following manner: Phase A: Ultrez 10 (carbomer) is dispersed in water and allowed to swell for 20 minutes, then Phase B is added and heated to 75°C. Phase C is heated separately to 75°C. The two phases are mixed under stirring and homogenized, then Phase D is added, neutralized with Phase E, cooled to 30°C, then Phases F and G are added, and the pH is adjusted to 6 with NaOH. This is merely an example, and it will be understood that any suitable method known in the art may be used.

[0347] In further embodiments, the composition may comprise one or more of the following: water, carbomer, sorbates such as potassium sorbate, sugar alcohols such as glycerin, alcohols such as 2-(2-ethoxyethoxy)ethanol, polyoxyethylene stearyl ethers such as Steareth 2, fatty alcohols or phosphoric acid, or mixtures of fatty alcohols and phosphoric acid such as cetearyl alcohol, dicetyl phosphate, and Cereth 10 phosphate, or combinations thereof, siloxanes such as cyclomethicone, caprylic / capric triglyceride, sorbitan stearate, parabens, sodium hydroxide, glycerin (and) butylene glycol (and) a mixture of Arcostaphylus Uva Ursi leaf extract (and) Mitracarpus Scaber extract (ETIOLINE®), or muscle health agents, and active agents such as the peptides of the present invention.

[0348] (Example 8) The following compositions in Example 8 are examples of emulsions, creams, or rubs.

[0349] [Table 2]

[0350] The active ingredient may be ETIOLINE® [glycerin (and) ethylene glycol (and) bearberry leaf extract and Mitracarpus scaber extract]. ETIOLINE® is a skin whitening ingredient marketed by SEDERMA (International Publication No. 98 / 05299, November 19, 1996).

[0351] It will be understood that ETIOLINE can be replaced with any active agent, such as drugs that aid in muscle health, development, or recovery.

[0352] This formulation can be made according to the procedure generally outlined in Example 7. The composition may be an emulsion, cream, or lab comprising one or more active agents, including water, carbomers such as Ultrez 10, sugar alcohols such as glycerin, alcohols such as phenova (phenoxyethanol and mixed parabens), fatty acid esters such as ethylhexyl palmitate, fatty alcohols such as cetearyl alcohol, lactic acid esters such as myristyl lactate, sorbates such as polysorbate 20 and / or potassium sorbate, polymeric emulsifiers such as acrylates (C10-30 alkyl acrylates), and crosslinked polymers, siloxanes such as cyclomethicone, sodium hydroxide, muscle health agents, or agents for treating stretch marks such as siegesbeckia orientalis extract (Darutoside), and peptides.

[0353] (Example 9) The following composition in Example 9 is an example of an emulsion or cream. In this embodiment, the emulsion or cream is an anti-stretch mark cream.

[0354] [Table 3]

[0355] The active ingredient may be Darutoside (Tsukushimenamomi extract). Darutoside is a molecule marketed by SEDERMA for the treatment of stretch marks. It should be understood that Darutoside may be replaced with any active ingredient, such as any other drug that aids in muscle health, development, or recovery.

[0356] This emulsion or rub is prepared in the following manner: Phase A: Disperse Ultrez 10 (carbomer) in water and allow it to swell for 20 minutes, then add Phase B and heat to 75°C. Separately, heat Phase C to 75°C. Mix the two phases under stirring and homogenize, add Phase D, neutralize with Phase E, cool to 30°C, then add Phases F and G and adjust the pH to -6 with NaOH.

[0357] In embodiments of the present invention, the composition may be an emulsion, cream, or gel, preferably a gel containing at least one or more agents, such as water, a carbomer such as Ultrez 10, a sugar alcohol such as glycerin, an alcohol such as Phenova (phenoxyethanol and mixed parabens), a sorbate such as polysorbate 20 and / or potassium sorbate, a polymeric emulsifier such as acrylate (C10-30 alkyl acrylate), a siloxane such as cyclomethicone, sodium hydroxide, peptides, and an agent for muscle health, recovery, or development, or an extract of Imperata Cylindrica (root), or a suitable moisturizer such as an agent containing water, glycerin, PEG-8, and carbomer (MOIST 24). In embodiments, the gel is a moisturizing gel.

[0358] (Example 10) The following composition in Example 10 is an example of a gel. In this embodiment, the gel is a moisturizing gel.

[0359] [Table 4]

[0360] (Example 11) This formulation can be prepared by following the procedure generally outlined in Example 11.

[0361] The active ingredient may be MOIST-24® [Imperata cylindrica (root) extract (and) water (and) glycerin (and) PEG-8 (and) carbomer]. It will be understood that MOIST-24 may be replaced with any other drug, for example, any drug that aids in muscle health, development, or recovery.

[0362] In embodiments of the present invention, the composition may be an emulsion, cream, or lab comprising water, a carbomer such as Ultrez 10, a sorbate such as polysorbate 20, polysorbate 60, and / or potassium sorbate, an alcohol such as Phenova (phenoxyethanol and mixed parabens), butylene glycol (1,3-butanediol), lanolin alcohol, and / or cetearyl alcohol, a polydimethylsiloxane such as sorbitan stearate, dimethicone, isotridecyl isononanoate, caprylic / capric triglyceride, cetyl ester, sodium hydroxide, water, an agent for muscle health, recovery, and / or development, or an anti-aging agent such as an agent comprising butylene glycol, water, laureth 3, hydroxyethylcellulose, and acetyl-dipeptide-1-cetyl ester (CALMOSENSINE®).

[0363] The following compositions in Example 11 are examples of creams or rubs.

[0364] [Table 5]

[0365] This formulation can be prepared according to the procedure generally outlined in Example 9. The active ingredient may be CALMOSENSINE® [butylene glycol (and) water (and) Laureth-3 (and) hydroxyethylcellulose (and) acetyl-dipeptide-1-cetyl ester]. Calmosensine® is an analgesic peptide provided by SEDERMA (International Publication No. 98 / 07744 of February 26, 1998). It will be understood that CALMOSENSINE may be substituted with any other drug, for example, any drug that aids in muscle health, development, or recovery.

[0366] In embodiments of the present invention, the composition may be an emulsion, cream, or lab comprising one or more active agents such as water, a carbomer such as Ultrez 10, a sugar alcohol such as glycerin, a sorbate such as potassium sorbate, steareth such as Steareth 10, a fatty alcohol or phosphoric acid, or a mixture of fatty alcohols and phosphoric acid such as cetearyl alcohol, dicetyl phosphate, and Cereth 10 phosphate, or a combination thereof, ethylhexyl disuccinate, mixed parabens, sorbitan stearate, sodium hydroxide, water, peptides, and an agent for muscle health, recovery, and / or development, or an agent for mature skin such as an agent containing Trifolium Pratense (clover) petal extract (and) glycerin (and) butylene glycol (and) lecithin (TEROCARE®).

[0367] (Example 12) The following compositions in Example 12 are examples of creams or rubs.

[0368] [Table 6]

[0369] This formulation can be prepared according to the procedure generally outlined in Example 9. The activator may be STEROCARE® [Trifolium repens (clover) petal extract (and) glycerin (and) butylene glycol (and) lecithin]. Sterocare® is provided by SEDERMA as an active ingredient for mature skin (French Patent Application Publication No. 2769502, April 14, 2000; International Publication No. 99 / 18927, April 22, 1999). It will be understood that STEROCARE® may be substituted with any other agent, for example, any agent that aids in muscle health, development, or recovery.

[0370] (Example 13) The following compositions in Example 13 are examples of love or tonic.

[0371] [Table 7]

[0372] (Example 14) The following compositions in Example 14 are examples of creams or rubs.

[0373] [Table 8]

[0374] The agents may be deferoxamine and / or berberine. These are used as skin thickening agents. Deferoxamine and berberine can be replaced with other active agents, such as those for muscle health, recovery, and development.

[0375] (Example 15) The following compositions in Example 15 are examples of gels or labs.

[0376] [Table 9]

[0377] This gel can be prepared in the following manner: Homogenize part B and pour it into part A. Heat part (A+B) to 75°C. Heat parts C and D to 75°C. Pour part C into part (A+B) while stirring in a spiral motion, then pour part D into part (A+B+C). Add parts F and E. Pour in part G at approximately 35°C.

[0378] The activators may be rutin and / or Bowman-Burk inhibitors (BBIs). These drugs are used for tissue regeneration. Rutin and Bowman-Burk inhibitors (BBIs) can be replaced with other activators, such as drugs for muscle health, recovery, and development.

[0379] (Example 16) Puromycin ELISA Research Description Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. It is a structural analogue of aminoacyl-tRNA and can therefore be incorporated into an elongating peptide chain via the formation of a peptide bond. However, while aminoacyl-tRNA contains a hydrolyzable ester bond, puromycin does not have one in the equivalent position. Therefore, the binding of puromycin to an elongating peptide chain prevents the formation of a new peptide bond by the next aminoacyl-tRNA. As a result, puromycin binding leads to the termination of peptide elongation and the release of the puromycin-bound and cleaved peptide from the ribosome. At very high concentrations, puromycin effectively blocks the translation elongation phase and therefore inhibits protein synthesis. However, at very low concentrations, it does not suppress the overall translation rate, and the rate at which puromycin-labeled peptides are formed reflects the overall protein synthesis rate. This latter property makes puromycin a potential tool for measuring changes in protein synthesis rates.

[0380] method 1. Dispense 100 μl of coating buffer into each well of the 96-well ELISA plate, filling the required number of wells. Dilute and mix the protein extract to 5 μg / μl (in the same buffer solution used for the sample). Immediately dispense 1 μL into each well of the plate. 2. Incubate the plate at 4°C for 16-24 hours. 3. Shake the contents vigorously. Wash once with 200 μl of PBS. Add 4,200 μl of blocking solution (5% BSA in PBS). Incubate at room temperature for 4-6 hours. 5. Dilute the puromycin antibody in blocking solution and incubate at 4°C overnight. Wash twice with 8,200 μl of PBS. 9. Add 100 μl of secondary antibody diluted 1:1000 to the blocking solution. Incubate at room temperature for 1 hour. Wash four times with 10,200 μl of PBS. Shake the contents vigorously. Add 11,100 μl of TMB substrate and incubate for 15-20 minutes. 12. Add stop solution and read the plate at 450 nm.

[0381] result Figure 10 illustrates the increase in protein synthesis.

[0382] (Example 17) toxicity method 100 μl of HSkMC cells were placed in a 96-well plate pre-coated with collagen, with 1 × 10 cells per well. 4 The cells were seeded at a concentration of 1.5%. The cells were allowed to adhere overnight at 37°C and 5% CO2. The next day, the growth medium was replaced with 100 μl of cell differentiation medium, and the cells were returned to the incubator at 37°C and 5% CO2. The cell differentiation medium was changed every two days for six days until the HSkMC cells had completely differentiated into myotubes. On the seventh day, the cell differentiation medium was replaced with 100 μl of basal medium, and the cells were starved overnight at 37°C and 5% CO2.

[0383] On the day of the MTT assay, the starvation medium was removed and replaced with 100 μl of basal medium containing the peptide of SEQ ID NO: 1. Cells were incubated at 37°C and 5% CO2 for 20 minutes during this treatment. At this point, the treatment medium was removed and replaced with 110 μl of MTT solution, and incubated at 37°C and 5% CO2 for 2 hours. After this incubation step, the MTT solution was removed and replaced with 110 μl of DMSO. The plate was covered with tin foil and placed in a shaker at room temperature for 5 minutes. The plate was read using a spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, CA 94089 USA) set to 570 nm, and the cytotoxic effect of the peptide of SEQ ID NO: 1 was evaluated based on the optical density of the sample. The results are illustrated in Figure 11.

[0384] (Example 18) This set of experiments was designed to understand the mechanism of action of the peptide of Sequence ID No. 1.

[0385] Phosphoacetyl-CoA method The phosphoacetyl-CoA carboxylase (Ser79) sandwich ELISA kit is a solid-phase sandwich enzyme immunosorbent assay (ELISA) that detects the endogenous level of acetyl-CoA carboxylase (ACC) phosphorylated at Ser79. A rabbit antibody against phosphoACC (Ser79) was coated into microwells. After incubation with cell lysates, phosphoACC proteins were captured by the coated antibody. After thorough washing, ACC mouse detection mAbs were added to detect the captured ACC proteins. The bound detection antibody was then recognized using an anti-mouse IgG HRP-conjugated antibody. TMB, an HRP substrate, was added to induce color development. The magnitude of the absorbance for color development was proportional to the amount of ACC phosphorylated at Ser79. These results are illustrated in Figure 12.

[0386] Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. It is a key enzyme in fatty acid biosynthesis and oxidation. In rodents, the 265 kDa ACC1 (ACCα) form is mainly expressed in lipid synthesis tissues, while the 280 kDa ACC2 (ACCβ) is the major isoform in oxidative tissues. However, in humans, ACC2 is the dominant isoform in both lipid synthesis and oxidative tissues. Phosphorylation by AMPK at Ser79 or PKA at Ser1200 inhibits the enzymatic activity of ACC. ACC is a potential target for anti-obesity drugs.

[0387] PhosphoAkt1 method The Phospho-Akt1(Ser473) Sandwich ELISA Kit is a solid-phase sandwich enzyme immunosorbent assay (ELISA) for detecting endogenous levels of the Phospho-Akt1(Ser473) protein. Rabbit Phospho-Akt(Ser473) mAbs were coated into microwells. After incubation with cell lysates, the Phospho-Akt(Ser473) protein was captured with the coated antibody. After thorough washing, Akt1 mouse antibody was added to detect the captured Phospho-Akt1(Ser473) protein. The bound detection antibody was then recognized using an anti-mouse IgG HRP-conjugated antibody. The HRP substrate, TMB, was added to induce color development. The magnitude of the absorbance for this color development is proportional to the amount of Phospho-Akt1(Ser473) protein present.

[0388] The results are illustrated in Figure 13.

[0389] Akt, also known as PKB or Rac, plays a crucial role in regulating survival and apoptosis. This protein kinase is activated by insulin and various growth and survival factors and functions in the Wartmannin-sensitive pathway involving PI3 kinase. Akt is activated by PDK1-mediated activation loop phosphorylation at phospholipid binding and Thr308, as well as phosphorylation at the carboxyl terminus of Ser473. PDK2, previously unknown, which contributes to Akt phosphorylation at Ser473, has been identified as a mammalian target (mTOR) of rapamycin within a rapamycin-insensitive complex involving rictor and Sin1. Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad, a forkhead transcription factor, c-Raf, and caspase 9. PTEN phosphatase is a major negative regulator of the PI3 kinase / Akt signaling pathway. LY294002 is a specific PI3 kinase inhibitor. Another important Akt function is the regulation of glycogen synthesis through the phosphorylation and inactivation of GSK-3α and β. Akt can also play a role in insulin-stimulated glucose transport. In addition to its roles in survival and glycogen synthesis, Akt is also involved in cell cycle regulation by preventing the phosphorylation and degradation of cyclin D1 mediated by GSK-3β and by negatively regulating the cyclin-dependent kinase inhibitors p27 Kip1 and p21 Waf1 / Cip1. Akt also plays a crucial role in cell proliferation by directly phosphorylating mTOR within the rapamycin-sensitive complex containing raptor. More importantly, Akt phosphorylates and inactivates tuberine (TSC2), an mTOR inhibitor, within the mTOR-raptor complex.

[0390] PhosphoAMPKα method The phospho-AMPKα (Thr172) sandwich ELISA kit is a solid-phase sandwich enzyme immunosorbent assay (ELISA) that detects the endogenous level of AMPKα when phosphorylated at Thr172. AMPKα rabbit antibody was coated into microwells. After incubation with cell lysates, AMPKα (phospho and non-phospho) was captured with the coated antibody. After thorough washing, phospho-AMPKα (Thr172) mouse detection antibody was added to detect phosphorylation of the captured AMPKα protein at Thr172. Next, an anti-mouse IgG HRP-conjugated antibody was used to recognize the bound detection antibody. The HRP substrate, TMB, was added to induce color development. The magnitude of the absorbance for this color development was proportional to the amount of AMPKα phosphorylated at Thr172. The results are illustrated in Figure 14.

[0391] AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in regulating energy homeostasis. AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3). The kinase is activated by an increase in the AMP / ATP ratio resulting from cellular and environmental stresses such as heat shock, hypoxia, and ischemia. The accessory proteins STRAD and MO25, along with the associated tumor suppressor LKB1, phosphorylate AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation. AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinases and biological significance of these phosphorylation events remain unclear. The β1 subunit is posttranslationally modified by myristoylation and multisite phosphorylation, including Ser24 / 25, Ser96, Ser101, Ser108, ​​and Ser182. Phosphorylation of the β1 subunit at Ser108 is required for AMPK enzyme activation, while phosphorylation at Ser24 / 25 and Ser182 affects AMPK localization. Several mutations within the AMPKγ subunit have been identified, most of which are located at the putative AMP / ATP binding site (CBS or Bateman domain). Mutations at these sites result in reduced AMPK activity and cause glycogen accumulation in cardiac or skeletal muscle. Evidence of accumulation indicates that AMPK not only regulates fatty acid and glycogen metabolism but also modulates protein synthesis and cell proliferation via the EF2 and TSC2 / mTOR pathways, as well as blood flow via eNOS / nNOS.

[0392] conclusion The peptide with sequence number 1 acts via the AMPK pathway. In skeletal muscle, AMPK activation (phosphorylation) leads to phosphorylated ACC, which promotes fatty acid oxidation. This is a pathway very different from the insulin pathway, as shown in the gene expression profile within skeletal muscle cells described below.

[0393] (Example 19) Gene expression research method We searched for the top 50 genes showing excessive and underexpression compared to negative controls for insulin and the peptide of Sequence ID No. 1.

[0394] 2 ml of HSKMC cells were placed in a 6 cm plate pre-coated with collagen, with 2 x 10 cells per plate. 5 The cells were seeded at a concentration of 1.5 ml. The cells were allowed to adhere overnight at 37°C and 5% CO2. The next day, the growth medium was replaced with 2 ml of cell differentiation medium, and the cells were returned to the incubator at 37°C and 5% CO2. The cell differentiation medium was changed every two days for six days until the HSkMC cells had completely differentiated into myotubes. On the seventh day, the cell differentiation medium was replaced with 2 ml of basal medium, and the cells were starved overnight at 37°C and 5% CO2.

[0395] On the day of the experiment, the starvation medium was removed and replaced with 2 ml of basal medium containing 0.5 μg / ml of peptide SEQ ID NO: 1. In parallel, untreated cells were tested. The cells were incubated at 37°C and 5% CO2 for 20 minutes during this treatment. At this point, the treatment medium was removed and the cells were scraped off in 1 ml of PBS. The cell suspension was pelleted in a microcentrifuge at 1500 rpm for 5 minutes, and the supernatant was removed. Immediately, the cells were flash-frozen in liquid nitrogen and transferred to a -80°C freezer for storage until shipment to the CRO.

[0396] To determine gene expression, Agilent Single Color experiments were performed by Elda Biotech using the Agilent G2565CA microarray scanner system. RNA was extracted from cells and then hybridized to an Agilent Human Gene Expression Microarray. The following kits were used: Qiagen Rneasy, Agilent RNA 6000 Nano, Lowlnput QuickAmp labeling, RNA Spike In (monochromatic), Hi RPM GE Hybridisation Kit Large, and Gene Expression Wash Pack. The experiments were conducted using a SurePrint G3 Human Gene Expression v3 8x60K microarray. Each sample was triplicate. Samples passed RNA quality and quantity checks. Subsequent data analysis was performed using the Bioconductor package "limma".

[0397] Using the normexp method and an offset of 50, the raw intensity data was background-corrected and quartile-normalized, resulting in a log 2 expression scale. Using ComBat, batch-effect correction was applied based on the number of replicas revealed by principal component analysis to adjust for separation. Empirical Bayesian analysis was then applied. Differential expression was calculated based on comparison with untreated samples and evaluated using moderated t-statistics. To correct for multiple tests, the resulting p-values ​​were corrected using the Benjamini and Hochberg methods. Differential expression was not detected under an adjusted p-value threshold of 0.05; instead, a raw p-value threshold of 0.001 was used. Genes with absolute multiplicity changes greater than 1.3 are listed below.

[0398] List of significantly upregulated and downregulated genes, and their ploidy levels (microarray gene expression).

[0399] Genes upregulated in response to insulin treatment: ABCA9、ACIN1、ADAP2、AKIRIN2、ALG3、AMN、ANKS3、ANP32A-IT1、ARGLU1、ARHGEF35、ARPC5L、ASAP1-IT1、ATP5H、ATP8、AURKAPS1、BMS1P6、BOD1L1、BRD3、 BROX、C12orf65、C14orf169、C20orf96、C21orf59、C4orf33、C5orf24、C5orf58、C6orf47、CACNA1A、CASC15、CATSPER2P1、CBX5、CCDC102B、CCNL1、CCNT2 -AS1、CD86、CDK11B、CENPC、CFAP36、CHD3、CIAO1、CLASRP、CMTM8、COPG2IT1、CRHR1-IT1、CWC22、DKFZP586B0319、DKFZp686M1136、DPP9-AS1、DRAP1、DRD 4、DYX1C1、EIF4G3、EIF5、EPSTI1、EXOC1、FAM127C、FAM155A-IT1、FAM173A、FAM212A、FAM71F1、FASTKD1、FIP1L1、FLJ11292、FLJ13773、FTSJ3、GABP1-A S1、GADD45B、GATA2、GBP2、GOLGA2P6、GOLGA6L4、GPATCH11、GPR135、GRIN2D、GSK3A、HEBP2、HMGB1、HMGB3P1、HNRNPA1L2、HOXB-AS1、HOXC9、HOXD8、ID1、 IER2、IFI44、IFT172、IGF1R、IGF2BP2、KANK3、KCTD3、KIAAI654、K1Z、KLF17、KRTAP19-2、KRTAP4-11、LDHAL6A、LINC00083、LINC00504、LRRC37A2、MALAT 1、MAP3K10、MAP3K3、MBNL1、MIDN、MIR22HG、MIR612、MPV17L2、MRE11A、MTA2、MYO1C、MZF1、NAMPT、NBPF8、ND4L、NF1P2、NIPBL、NOL8、NOP58、NPIPB5、NR2C1、OLA1、PAK1IP1、PCDHGA2、PDCD6IP、PDGFRB、PFDN4、PGBD2、PGF、PGM5P2、PMAIP1、PNPLA8、POU2F1、PP12719、PPIG、PP6R1、PRKAG2、PRO2852、PRPF40A、PRR4, PSMA4, PTMA, RALBP1, RC3H2, RCC1, REP15, RHOB, RN7SL1, ROM1, RPLP1, RPS6KC1, SAMD11, SFSWAP, SH3KBP1, SLC25A34, SLFN5, SMARCC1, SMCR6, SMIM11, SNAI1, SNAR-A3, SNAR-B2, SNAR-D, SNAR-F, SNAR-G2, SNAR-H, SNHG9, SNORA10, SNORD97, SNO RD99, SOCS3, SOX8, SRP19, SRSF11, SSC5D, STAR, TAB2, TAF3, TCEAL7, TCF7L1, TFPI2, THOC2, TINAGL1, TNFAIP8L1, TOPORS, T RANK1, TRAPPC10, TXLNG, UBE2Q2P1, UBN2, VASP, VHL, VPS37D, YBX3, ZBTB2, ZCCHC17, ZMYND11, ZNF503-AS2, ZSWIM4, ZSWIM6,

[0400] Genes downregulated in response to insulin treatment: ABHD2, ACTA2, ACVR1, ADAMTS9, ADCY6, ADD3, ADPRHL2, AGTRAP, ALAD, AMZ2P1, ANXA2P1, AP2B1, ARMCX6, ATP5A1, ATP5B, ATP5G1, ATP6V0E1, ATRAID, AXL 、BFAR、BOD1、BRF2、C1orf43、C5orf15、C9orf78、CA12、CAV1、CD47、CD82、CD 9、CDC123、CDC42BPA、CDC42EP4、CHST14、CHST3、CHSY3、CLMP、CLPTM1L、CLU、 CNPPD1、COLGALT1、COPG1、COPZ2、CRISPLD2、CRTC3、CSRP1、CTSLP8、CYB5D2 、CYB5R1、DAG1、DANCR、DEK、DENND4C、DFNA5、DHRS1、DLST、DNAJB9、DNASE1L1 、DNASE2、DPAGT1、EBF2、EI24、EIF2B4、EIF3I、EIF3K、EIF3L、EMC3、ENDOD1、 ENO2、ENPP4、EPRS、ESYT1、ESYT2、EXT1、EZH1、FAF1、FAM57A、FAM96A、FAS、FH OD1、FKBP14、FLII、FLNB、FN3KRP、G6PD、GABARAP、GALM、GARS、GBA、GBAP1、G DE1、GJA1、GOLM1、GPRC5A、GPX3、GREM1、GSTM2、HDDC2、HEPH、HEXB、HLA-A、HL A-DMA、HLA-DPA1、HLA-DPB1、HLA-DRB4、HOOK2、HTRA2、IDH1、IDS、IFIT3、IG F2R、IGFBP6、IL10RB、IL11RA、ILK、ITGA3、ITGAE、JKAMP、KIF3B、KLHDC2、LAM A2, LAPTM4B, LASP1, LUM, MAGED1, MAN2A1, MAN2B2, MANBA, MANSC1, MAP2K1, MFSD5, MKNK1, MLEC, MMGT1, MPHOSPH8, MRPL17, MRPL37, MRPS18A, MTFR1L, MT HFD1L、MUL1、MYD88、NBAS、NDRG1、NDUFA9、NIF3L1、NLRP1、NME6、NPC1、NSMC E1、NUDT2、OAT、OLFML3、OXA1L、P4HA2、P4HTM、PAMR1、PARP6、PCYOX1、PDE6D、PDLIM1, PDXP, PEA15, PEAR1, PEF1, PELO, PGAM1, PGM1, PGRMC1, PHB2, PIGH, PLEKHG4, PLP2, PLS3, PPAP2B, PPIAL4B, PP L, PRKAB1, PRMT5, PRNP, PRPF8, PRPS1, PRRC1, PSAP, PSMB8, PSMD10, PTGS1, QSOX2, RAB11FIP5, RAF1, RARG, RBM23, RER 1, RHBDD2, RP9, RPL29P2, RPL5, RPRD1B, RPUSD4, RTCB, SCAMP2, SCG2, SDC4, SDHB, SEC22C, SEC23A, SEC63, SENP3, SEP1 5, SERINC1, SERINC3, SF3B2, SFXN3, SGCE, SIAE, SLBP, SLC12A4, SLC1A1, SLC35B1, SLC35E2, SLC35E3, SLC39A1, SLC39 A7, SMPD1, SNORA70F, SNX11, SNX19, SPPL2A, SPRED2, SRPR, STAU1, STEAP1B, STIM1, STK38, STX18, SYPL1, SYT11, TAF1 1, TAPBPL, TBC1D22B, TCN2, TCTA, TES, TFG, TFRC, TGFB3, TM7SF2, TM9SF1, TMBIM1, TMCO1, TMCO3, TMED10, TMEM138, TM EM179B, TMEM185A, TMEM216, TMEM50A, TNS1, TNS3, TPI1P2, TRAM2, TRIM16L, TSPAN9, TUB, TUBA1A, UBAC2, UBE2E3, UNC 50, USO1, USP24, VCL, VOPP1, VPS25, VPS33B, WAS, WBP1, WNT5B, ZCCHC24, ZFP91, ZMAT3, ZMYM6NB, ZNF384, ZNF667-AS1,

[0401] Genes upregulated in response to SEQ ID NO: 1 treatment ACAA2, ACSL1, ACVR1, ADAR, ADD3, ADIPOR2, ADPRM, AGTR1, AKR1B10, AKR1B15, ALAD, ALG14, AMZ2, AMZ2P1, ANKHD1, APOL6, ASCC1, ASH2L, ATF5, ATG3, ATP 5A1、ATP5C1、ATP5J2、ATP6V0E1、ATP6V1B2、ATXN7L3B、B2M、B4GALT5、BBS4、 BIRC5、BLMH、BOD1、BRK1、BST1、BTF3、C11orf73、C16orf58、C17orf62、C19or f52、C1orf43、C4orf3、C5orf15、C5orf58、C7orf25、C8orf33、CALM1、CCNB1 IP1、CCNG2、CD47、CDCA8、CFDP1、CHAMP1、CHCHD3、CHURC1、CISD3、CKAP5、CLM P、CLTA、COA1、COG3、COG8、COPS8、COX14、CPD、CSTF1、CSTF2T、CTCF、CYB5R3 、CYCS、CYTB、DCTN6、DCTPP1、DDX21、DDX5、DDX60、DECR1、DERL1、DFNA5、DHX1 5, DHX32, DRG1, DSTYK, EFNB3, EMCN, EMG1, ENDOD1, ENPP2, ERICH1, ETFA, EZH1, F8A2, FAM96A, FH, FOS, FOSB, FUCA2, FUNDC2, GABARAPL2, GADD45B, GALNT 15、GAS1、GATS、GBF1、GBP1、GDE1、GGA3、GJA1、GLB1、GLUL、GOLM1、GPR89B、G RAMD3、GSTA4、GTF2E2、H2AFZ、HES1、HEXB、HIBADH、HIST1H4F、HIST2H2AA4、H MGN3、HMGN4、HNRNPM、HOOK2、HSPB8、IDH1、IER3、IFI44L、IFIT1、IFT88、IMM P2L、IMPA2、INTS12、KAT7、KCTD10、KDELR3、KIAA0196、KIF2C、KIFAP3、KLF10 、KLHL18、LAP3、LAPTM4B、LDHA、LDLR、LETMD1、LMAN2L、LMO4、LOXL4、LRP10、 LSM1、LTBP2、LYPD6B、LYSMD2、MAD2L1BP、MANBA、MAT2A、MCL1、MEDAG、MFAP1、MIF-AS1、MIOS、MKKS、MMGT1、MRFAP1、MROH5、MRPL20、MRPL33、MYD88、NDN、N DRG1、NDUFV2、NKAP、NOLC1、NT5C3B、NT5E、OAS1、OAT、OAZ2、ODF3L2、OLFML1 、OLFML2B、OSBP、OSTC、OTUD1、OXCT1、PAIP2、PARL、PARP9、PATL1、PCED1A、P DE1A、PEAR1、PELO、PFKL、PFKM、PGAM1、PGK1、PGM1、PIGH、PLEKHJ1、POGK、POL D2, POLR2B, POLR3F, PPA2, PPAP2B, PPAPDC2, PPL, PPP2CB, PPP2R2A, PQLC2L, PRC1, PRDM4, PSMA2, PSMA5, PSMD10, PSME4, PTGS1, PTP4A1, PTTG2, RAB31, R AB32、RAB9BP1、RABEP1、RAC1、RASL11B、RCN1、RHOA、RIOK1、RNASEL、RNF121 、RNF146、RNLS、RPA2、RPL15、RPL21P44、RPP38、RTCB、SEC22C、SEC23IP、SENP 2、SEP15、SERP2、SERTAD4、SETX、SFRP1、SFT2D1、SGCE、SIAE、SLBP、SLC25A1 2、SLC25A25、SLC30A9、SLC35B1、SLC44A1、SLC9A3R1、SMAD7、SMIM19、SNAPC 5、SNHG3、SNORD116-19、SNORD96A、SNX19、SRRD、SRSF2、STARD7、STK16、STO M、STOML2、SUCLA2、SUCLG1、SYNGR4、SYT11、TASP1、TCP1、TFRC、TGFBR2、TIMM 17A、TIMMDC1、TINF2、TMCO1、TMEM173、TMEM203、TMEM230、TMEM50A、TMEM50 B、TMEM60、TMX2、TNC、TNS3、TOMM22、TOR1A、TOR3A、TP53I3、TPBG、TPI1、TRIB 1、TRIL、TUBB3、TUG1、TUSC3、UBE2C、UBE2E3、UBE2L3、UGCG、UNC119、UQCRFS 1、USP18、USP24、USP34、VAMP7、VDAC3、VIM-AS1、VOPP1、WARS、WDR36、WDR48、WDR61, WDYHV1, WLS, WRB, XPNPEP1, XRCC6BP1, YIPF6, ZFP91, ZMAT2, ZNF157, ZNF281, ZNF384, ZNF426, ZNF564, ZNF696, ZNF75D, ZSCAN32,

[0402] Genes downregulated by SEQ ID NO: 1 treatment ABLIM1、ACTR2、ADAM20、AHSA2、ALDH1L2、AMN、APC、APOLD1、ARGLU1、ARPC5、ASAP1-IT1、ATXN2、ATXN3、AURKAPS1、BCL2L11、BCLAF1、BRD1、BTG3、C14orf169、C1QTNF5、C20orf141、C20orf96、C5orf28、C6orf203、CACNA2D1、CCDC102B、CCDC125、CCDC6、CCNG1、CCNL1、CD46、CEACAM19、CENPC、CHML、CIRBP、CN2、COL1A1、COL4A1、CRYBG3、CWC22、DDX3X、DEND4B、DHRS3、DIAPH2、DLG1、DNAJB4、DNAJC3、DOCK6、DPEP3、DSE、DST、DYNLT3、DZIP3、EDIL3、EGLN1、EIF4G3、ELF2、ELP2、EPC1、FAM111A、FAM177A1、FBXO32、FERMT2、FGF7、FNIP1、FOSL2、FOXN2、FRYL、GABPB1-AS1、GABRE、GLS、GOLGA6L4、GOLGA8R、GOLIM4、GPATCH11、GPR135、GTF2H5、HCG11、HCG18、HIF1A、HNRNPA1、HOOK3、HOXC6、HSP90AA1、HSP90AA2P、HSPA1B、HSPA4、HSPH1、HTT、IGF2R、ITGB3、JMJD4、KCNMB2-AS1、KCTD3、KDM2A、KIAA1143、KIZ、KRAS、KRT8P12、LCE1D、LINC01506、LOX、LPGAT1、LPP、LRP12、LRRC3C、MACF1、MAMDC2、MAP1B、MAP2K3、MAP3K10、MATR3、MBNL1、MBTPS2、MDM2、MDM4、MED15、METTL15、MGC24103、MUSTN1、MYH3、MYH8、MYLPF、MYO6、MZF1、NAB1、NADK、NADK2、NBEAL1、NCKAP5、ND6、NEK7、NKTR、NR2C1、NRN1、OFD1、PAPOLA、PAWR、PAXBP1、PBRM1、PCDHGA2、PDE1C、PDZD8、PEAK1、PFDN2、PGBD2、PHF1、PJA2、PLOD3、POLR2J2、PPME1、PPP1R14A、PPP1R16B、PPP1R2, PPP1R3B, PRO2852, PRPF40A, PRUNE2, PTPN11, PUM2, PXK, RAB12, RAB2A, RBM10, RCN2, REEP3, REST, RHOBTB3, RHOQ, RIOK3, RNF19B, RNF215, RNPC3, ROBO1, RO CK1, RPL26L1, SAMD14, SDPR, SEPT7P2, SERP1, SFSWAP, SGIP1, SH3KBP1, SKI, SLC35A5, SLC46A3, SLC7A5, SMARCC1, SMARCC2, SMN1, SNAR-H, SNORA16A, SNORD89, SOX6, SPHAR, SREK1, SRSF11, STT3B, STX2, TAB2, TFPI, THBS1, THOC2, TINAGL1, TMA16, TMEM106C, TMEM26-AS1, TNFAIP8, TOB1, TTC28, TXLNG, UBE2D1, UBE2Q2P1, UBL3, UCHL 5, UPF2, UPF3A, USP15, USP33, UTRN, UVSSA, WHAMMP1, WNT3, YBX3, YWHAZ, ZBTB2, ZFC3H1, ZMYND11, ZNF22, ZNF37A, ZNF429, ZNF525, ZNF532, ZNF626, ZRANB2, ZSWIM8,

[0403] (Example 20) Proteomics method Protein levels were obtained using LC / MS / MS. Statistical analysis of protein counts revealed proteins with low and high abundances. Only absolute protein level multiplier changes greater than 0.379 were retained. For all of these proteins, microarray analysis revealed the corresponding transcript multiplier changes.

[0404] Mass spectrometry was performed using MSBioworks. Cell pellets were lysed in 100 μL of modified RIPA buffer by sonication. Quantification was performed using Qubit fluorescence spectroscopy. 20 μg of each sample was separated on a 4–12% bis-tris gradient gel using MOPS buffer. The gel was stained with Coomassie, and each lane was cut into 20 equally sized segments.

[0405] The gel samples were processed using robots (ProGest, DigiLab) according to the following protocol: The samples were washed with 25 mM ammonium bicarbonate followed by acetonitrile. After reduction with 10 mM dithiothreitol at 60°C, alkylation was performed with 50 mM iodoacetamide in RT. • The food was digested using trypsin (Promega) at 37°C for 4 hours. The supernatant was directly analyzed after quenching with formic acid without further processing.

[0406] The digests were analyzed by nanoLC / MS / MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded onto a capture column and eluted at 350 nL / min on a 75 μm analysis column. Both columns were packed with Luna CI 8 resin (Phenomenex). A 30-minute gradient was used (10 hours total per sample). The mass spectrometer was operated in data-dependent mode, and MS and MS / MS were performed using Orbitrap at resolutions of 70,000 FWHM and 17,500 FWHM, respectively. Fifteen of the most abundant ions were selected for MS / MS. Data were retrieved using a local copy of Mascot with the following parameters: Enzyme: Trypsin; Database: Swissprot Human (forward and reverse with common contaminating sequences); Fixed modification: Carbamide methyl (C); Variable modification: Oxidation (M), acetyl (protein N-terminus), Pyro-Glu (N-terminal Q), deamidation (NQ); Mass value: Monoisotopic Peptide mass tolerance: 10 ppm; Fragment mass tolerance: 0.02 Da; Maximum miscutting: 2 For validation, filtering, and the creation of a non-redundant list per sample, Mascot DAT files were sent to Scafold software. The data consisted of filtered 1% protein and peptide level false discovery rates (FDRs), requiring at least two unique peptides per protein. Upregulated and downregulated proteins were then estimated.

[0407] mass spectrometry The digests were analyzed by nanoLC / MS / MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded onto a capture column and eluted at 350 nL / min on a 75 μm analysis column, both columns packed with Luna CI 8 resin (Phenomenex). A 30-minute gradient was used (10 hours total per sample). The mass spectrometer was operated in data-dependent mode, and MS and MS / MS were performed using Orbitrap at resolutions of 70,000 FWHM and 17,500 FWHM, respectively. Fifteen of the most abundant ions were selected for MS / MS.

[0408] (Example 21) QPCR for target specific genes and their ploidy levels Experimental Procedure Quantitative PCR was performed using a TaqMan probe-based method to quantify target mRNA expression compared to the expression of the B2M housekeeping gene. A master mix containing primers / probes and Taqman® Gene Expression Master Mix (ABI Biosystems, CA, USA) was added to 1 μl of cDNA template. The final volume of 10 μl was pipettered in double-barrel into the wells of a 96-well Lightcycler plate (Sarstedt, Numbrecht, Germany), and real-time PCR was performed on a Roche Lightcycler 96 (Roche Diagnostics, Basal, Switzerland) real-time thermal cycler. The threshold cycle (Ct) for each well was calculated using the instrument software. Data analysis was based on the ΔΔCt method with raw data normalized by the B2M housekeeping gene incorporated on the plate. Results are expressed as a multiplicative change relative to the control.

[0409] QPCR results (The treatment method remained the same, and the concentration of SEQ ID NO: 1 also remained at 0.5 ug / ml)

[0410] [Table 10]

[0411] (Example 22) Exploring the effects of synthetic peptide (SEQ ID NO: 1) on blood glucose management in mouse models of obesity and diabetes. Experimental Procedure Animal description: Species: KK.Cg-Ay / J (KKAY mouse) ·Supplied by: Jackson Laboratories • Age in weeks: 12 weeks ·Gender: Male • Randomization: Based on baseline fasting blood glucose

[0412] Rearing and feeding: • Acclimatization: 5 days or more • Rearing: Mice are kept in a 12-hour light-dark cycle. Depending on the size, limit the number of mice per cage to four or fewer. Ventilated cage rack system • Feed: Standard rodent diet and water (continuous supply (ad libitum))

[0413] design: • Route of administration: Subcutaneous injection • Dosage: 10 mL / kg • Dosage levels: Investigational drug: 100 μM / kg, Metformin: 250 mg / kg • Number per group: 10 • Total number of animals: 30

[0414] [Table 11]

[0415] Oral glucose tolerance test in KKAY mice In the OGTT, baseline glucose and insulin levels were collected from mice 30 minutes prior to the test (baseline). The test drug was administered immediately afterward. 30 minutes later, at 0 minutes, all mice were administered a glucose solution (2 g / kg) via forced oral administration. Following the additional glucose administration, blood glucose levels were measured according to the schedule below.

[0416] [Table 12]

[0417] The results are illustrated in Figures 18 to 20. Figure 18 illustrates the fasting blood glucose levels of KKAY mice after a 5-day treatment with the peptide of Sequence ID No. 1. Figure 19 illustrates the fasting blood glucose levels of KKAY mice after a 7-day treatment with the peptide of Sequence ID No. 1. Figure 20 illustrates the body weight of KKAY mice after a 13-day treatment with the peptide of Sequence ID No. 1.

[0418] conclusion Treatment with the peptide of SEQ ID NO: 1 significantly reduced blood glucose levels after a 7-day treatment period. Treatment with the peptide of SEQ ID NO: 1 did not cause weight gain, unlike many other anti-diabetic treatments. SEQ ID NO: 1 reduced weight in the treated group compared to the untreated group.

[0419] (Example 23) Antioxidant assay (DPPH radical capture activity assay) The DPPH test was performed using the method described in Lin SY, Wang J, Zhao P, Pang Y, Ye HQ, Yuan Y, Liu JB and Jones G, Optimized antioxidant peptides fractions preparation and secondary structure analysis by MIR. Int J Biol Macromol 59: 151-157 (2013).

[0420] The DPPH radical was dissolved in ethanol at a final concentration of 0.1 mM. The peptide and ascorbic acid (positive control) were dissolved in desalted water to obtain the test concentrations. In a 96-well plate, the samples were prepared as follows: 100 μL of 0.1 mM DPPH solution, 100 μL of peptide solution, and 100 μL of ethanol were mixed in each well. Ascorbic acid was prepared as follows: 100 μL of 0.1 mM DPPH solution, 100 μL of ascorbic acid, and 100 μL of ethanol were mixed in each well. The blank was prepared as follows: 100 μL of 0.1 mM DPPH solution and 200 μL of ethanol were mixed in each well. Three wells were used, one for each patient condition.

[0421] The plates were incubated in the dark at 25°C for 30 minutes. Then, the absorbance at 517 nm was measured using a plate reader.

[0422] The DPPH radical scavenging activity (%) is as follows: DPPH radical capture activity (%) =[1-(absorbance in sample / absorbance in blank)]×100 The calculation was done as follows.

[0423] The data is presented as a percentage ± standard deviation of the mean DPPH radical scavenging activity.

[0424] (Example 24) Inflammatory response assay TNFα is secreted by macrophages in response to stimulation by endotoxins such as lipopolysaccharide (LPS). TNFα is thought to be involved in systemic inflammation, and dysregulation of TNFα production is thought to be involved in many diseases. The Biolegend assay is a sandwich ELISA kit designed for the accurate quantification of human TNFα derived from cell culture supernatant, serum, or plasma.

[0425] THP-1 monocytes were seeded at a rate of 10,000 cells per well in RPMI containing 10% fetal bovine serum (FCS), 1% Pen / strep, 1% L-glutamine, and 100 nM PMA in a 96-well plate, and differentiated for 72 hours prior to the experiment. After differentiation, the cells were incubated for 24 hours with 100 ng / ml, 10 ng / ml, or 1 ng / ml of synthetic peptide, respectively. Following treatment, the cells were stimulated with 10 ng / ml LPS for 5 hours, and the quantity of TNFα in the supernatant was determined using the Biolegend assay ELISA kit.

[0426] Results were calculated as a percentage relative to the untreated control. An increase in optical density readings indicates an increase in the amount of TNFα released into the cell culture supernatant. All experiments were prepared in a double-row configuration on three plates (six wells per state). Significance was determined by Student's t-test. * Compared to the control, p<0.05. ** Compared to the control, p<0.01. *** The calculation was performed using p<0.001) in comparison to the control. Equal portions

[0427] The preceding description details preferred embodiments of the present invention. Those skilled in the art, having examined these descriptions, will anticipate numerous modifications and variations in their implementation. These modifications and variations are intended to be covered within the scope of the accompanying claims.

Claims

1. A composition containing the peptide of SEQ ID NO:

1.

2. The composition according to claim 1, which is a pharmaceutical composition containing the peptide in a therapeutically effective amount in combination with an appropriate pharmaceutical carrier.

3. The composition according to claim 1 or 2, formulated for oral or parenteral administration to a patient.

4. The composition according to claim 1, wherein the peptide is a modified peptide.

5. The composition according to claim 4, wherein the peptide is modified to extend its plasma half-life.

6. The composition according to claim 4 or 5, wherein the peptide is modified by modification of the side chain, introduction of a protecting group, incorporation of up to five non-natural amino acids, cyclization, conjugation with a conjugation partner, covalent bonding with a binding partner, fusion with a fusion partner, PEGylation, amidation, lipidation, use of a crosslinking agent, or by imposing conformational constraints on the peptide.

7. The composition according to claim 6, wherein the peptide is modified with a modifying group selected from the group consisting of polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, poly(N-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyol, and polyvinyl alcohol, colomic acid or other carbohydrate-based polymers, amino acid polymers, and biotin derivatives.

8. A cosmetic composition according to any one of claims 1 to 7.

9. The composition according to any one of claims 1 to 8, wherein the composition is a formulation for a topical composition.

10. The composition according to claim 9, formulated for topical application to human skin in formulations selected from the group comprising creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, water-alcohol solutions, water-glycol solutions, cosmetics, personal care products, hydrogels, liniments, serums, soaps, dusting powders, pastes, semi-solid formulations, liniments, serums, shampoos, conditioners, ointments, any rinse formulations, talc, mousse, powders, sprays, aerosols, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, patches, gel patches, bandages, adhesive systems, water-in-oil emulsions, oil-in-water emulsions, and silicone emulsions.

11. The composition according to any one of claims 8 to 10, wherein the peptide has cell proliferation-promoting activity for use in delaying or inhibiting the onset of skin aging.

12. The composition according to any one of claims 8 to 10, wherein the peptide has cell proliferation-promoting activity for use in the treatment or care of one or more visible signs of wrinkles, stretch marks and dark circles under the eyes, dryness, fine lines, age spots, red spots, and sagging skin, as well as conditions caused by sun exposure, stress, pollution, and diet, including sunburn.

13. The composition according to any one of claims 1 to 7, wherein the peptide has glucose transport-promoting activity, for use in a method for treating or preventing diabetes or prediabetes.

14. The composition according to claim 13, wherein the diabetes is type 2 diabetes.

15. The composition according to claim 13 or 14, wherein the composition is to be administered together with one or more agents consisting of insulin, insulin analogs, insulin derivatives, glucagon-like peptide 1, glucagon-like peptide 2, or derivatives or analogs of glucagon-like peptide 1 or glucagon-like peptide 2.

16. A medical device comprising the composition according to any one of claims 1 to 10.

17. Peptide of SEQ ID NO:

1.

18. The modified peptide according to claim 17.

19. The peptide according to claim 18, which is modified with a protecting group.

20. The peptide according to claim 18, which is modified with an N-terminal protecting group or a C-terminal protecting group.

21. The peptide according to claim 18, which is modified by replacing at least one L-amino acid with a D-isomer.

22. The peptide according to claim 18, modified with a modifying group selected from polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polypropylene glycol, polyoxyethylated polyol and polyvinyl alcohol, colomic acid, or other carbohydrate-based polymers, amino acid polymers, and biotin derivatives.

23. The peptide according to claim 18, wherein at least one amino acid is modified by replacing it with an amino acid analog.

24. The peptide according to claim 18, which is modified to increase the resistance of the modified peptide to protein degradation by mammalian gastrointestinal proteases.

25. A peptide or modified peptide according to any one of claims 17 to 24 for use as a pharmaceutical.

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