WO2023015533A1 - 一种合成肽以及它们的美容组合物或药用组合物及用途 - Google Patents

一种合成肽以及它们的美容组合物或药用组合物及用途 Download PDF

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WO2023015533A1
WO2023015533A1 PCT/CN2021/112345 CN2021112345W WO2023015533A1 WO 2023015533 A1 WO2023015533 A1 WO 2023015533A1 CN 2021112345 W CN2021112345 W CN 2021112345W WO 2023015533 A1 WO2023015533 A1 WO 2023015533A1
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Prior art keywords
agents
acceptable salt
agent
cosmetic
optionally
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English (en)
French (fr)
Inventor
丁文锋
赵文豪
孙新林
陈雪
彭晏
观富宜
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Shenzhen Winkey Pharmaceutical R&d Co Ltd
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Shenzhen Winkey Pharmaceutical R&d Co Ltd
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Priority to KR1020247007789A priority Critical patent/KR20240042649A/ko
Priority to US18/682,510 priority patent/US20240352069A1/en
Priority to PCT/CN2021/112345 priority patent/WO2023015533A1/zh
Priority to CN202180069751.7A priority patent/CN116829169B/zh
Priority to EP21953150.6A priority patent/EP4385516A4/en
Publication of WO2023015533A1 publication Critical patent/WO2023015533A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

  • the present invention relates to peptides capable of promoting the proliferation of fibroblasts in the skin or mucous membranes, as well as cosmetic or pharmaceutical compositions containing these peptides and their use in the treatment or care of the skin or mucous membranes.
  • the skin is the largest organ of the human body. It covers the body surface and plays an important barrier role for the body. It can resist mechanical damage, buffer external shocks, avoid damage to tissues and organs in the body, prevent the invasion of harmful substances from the outside, and resist oxidation and UV damage. It can also prevent the loss of various nutrients, water, and electrolytes in the body, and plays an important role in maintaining the stability of the internal environment of the human body.
  • Skin consists of epidermis, dermis and subcutaneous tissue. The outermost layer is the epidermis, which is mainly composed of keratinocytes, melanocytes, and Langerhans cells.
  • the dermis is located below the epidermis and contains dermal cells, collagen fibers, elastic fibers, matrix, hair, sebaceous glands, sweat glands, blood vessels, etc.
  • the dermis is tightly connected to the epidermis by a basement membrane.
  • Fibroblasts are large in size and diverse in shape, mostly protruding spindle-shaped or star-shaped flat structures.
  • the cytoplasm contains rich and developed rough endoplasmic reticulum, Golgi apparatus, and free ribosomes, so it has a strong ability to synthesize and secrete proteins. Function, can secrete extracellular matrix components such as procollagen, fibronectin and collagenase.
  • Fibroblasts use amino acids such as proline and lysine to synthesize proalpha polypeptide chains on the nucleosomes of the rough endoplasmic reticulum, and the polypeptide chains are transported to the Golgi apparatus to form procollagen molecules. ).
  • Procollagen molecules are released extracellularly by exocytosis. Under the catalysis of procollagen peptidase, the tail segment of the pre-alpha polypeptide chain of the procollagen molecule is removed to become the procollagen molecule (tropocollagen).
  • Procollagen molecules are arranged in parallel rows and combined into collagen fibrils with periodic horizontal striations. Collagen fibrils combine with each other to form collagen fibers. In addition to producing collagen fibers, fibroblasts can also produce elastic fibers.
  • the main component of collagen fibers is type I collagen, which has strong tensile strength and gives skin toughness.
  • Elastic fibers are composed of elastin and fibroin aggregates that give the skin its elasticity. Collagen fibers, elastic fibers, and extracellular matrix filled between fibers and cells form a support for the skin.
  • the skin's morphology, physiology, and its mechanical properties undergo significant changes.
  • the function of fibroblasts declines, collagen fibers and elastic fibers are damaged and broken, and the balance between the production and degradation of important biomolecules such as collagen in the skin is biased towards the degradation process with age, resulting in dermis Thinning and tissue destruction are progressive and cause relaxation of the dermis followed by wrinkling.
  • Prolonged exposure to sunlight or environmental pollutants can accelerate the skin aging process.
  • Ultraviolet rays can inhibit the synthesis of collagen and fibronectin in fibroblasts, and after ultraviolet radiation can cause high expression of matrix metalloproteinases (MMPs), causing serious damage to collagen in the dermis, resulting in the destruction of dermal integrity, skin strength and Decreased elasticity leads to accelerated aging of the skin.
  • MMPs matrix metalloproteinases
  • Non-pathogenic bacteria or opportunistic pathogenic bacteria will invade the skin, induce infection, and may even have a major impact on physical health. Therefore, accelerating the repair of skin wounds is of great significance in diseases with refractory wounds and postoperative repair.
  • Wound repair is a precise, complex, time-phased dynamic process involving network regulation of various cells such as cell proliferation, migration, degradation of extracellular matrix, angiogenesis, and epithelial tissue remodeling.
  • Fibroblasts are the main cells in wound repair. Mature fibroblasts are in a quiescent state, and their cell body volume is reduced, showing a long spindle shape, rough endoplasmic reticulum and Golgi apparatus are reduced, and mature fibroblasts are called fibroblasts. Fibroblasts and fibroblasts can transform into each other under certain conditions.
  • fibroblasts Under the stimulation of trauma and other factors, some fibroblasts re-transform into immature fibroblasts, and their synthetic and secretory functions are restored accordingly, stimulating the generation of new extracellular matrix, and a large amount of extracellular matrix is combined with new capillaries to form granulation tissue to fill the defect organize.
  • the proliferation and migration of fibroblasts are crucial to the formation of granulation tissue and the repair of wounds.
  • fibroblasts are mainly involved in the proliferation phase and tissue remodeling phase of wound repair.
  • fibroblasts proliferate, migrate and form granulation tissue in large numbers, which mainly play the role of resisting infection and filling wounds.
  • fibroblasts play an important role in the process of skin aging and wound repair.
  • the development of products that can promote the proliferation of fibroblasts can enhance the activity of fibroblasts and increase the content of collagen, elastin, glycosaminoglycans and other ingredients. Synthesize and secrete to compensate for the loss of extracellular matrix such as collagen and elastin with aging or exposure to sunlight and/or exposure to environmental pollutants, delay aging; fill the vacancy of necrotic tissue area after skin injury, and promote wound repair , is of great significance.
  • the inventors of the present invention have surprisingly found that the proliferation and activity of fibroblasts can be regulated by certain synthetic peptides through a large number of experimental studies.
  • the present invention aims to provide a peptide capable of promoting proliferation of fibroblasts in skin or mucous membranes.
  • These peptides and cosmetic compositions or pharmaceutical compositions containing these peptides can improve or prevent the treatment or care of conditions, disorders or diseases of the skin or mucous membranes by promoting the proliferation of fibroblasts, thereby improving the skin barrier function or treating, preventing or treating Repair skin aging or photoaging, or for re-epithelialization or healing of skin or mucous membranes.
  • the present invention provides a peptide represented by general formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt , characterized by:
  • R is selected from: H, alkyl, alkenylalkyl, carboxyalkyl, esterylalkyl, alkylmercapto, carboxyalkylmercapto, or
  • n is selected from: 1 or 2;
  • R 1 is selected from: H or R 4 -CO-, wherein R 4 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • R 3 is selected from: -NR 5 R 6 or -OR 5 , wherein each R 5 and R 6 are independently selected from each other: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • the groups R1 and R3 are bound to the amino-terminus (N-terminus) and carboxy-terminus (C-terminus) of these peptide sequences, respectively.
  • alkyl in the "alkyl”, “alkenylalkyl”, “carboxyalkyl”, “ester-based alkyl”, “alkylmercapto”, “carboxyalkylmercapto” refers to a group with 1-24 carbons Atom (optionally having 1-16 carbon atoms; optionally having 1-14 carbon atoms; optionally having 1-12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbons atom) saturated aliphatic straight-chain or branched alkyl; optionally selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl Base, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl;
  • alkenyl in the "alkenyl” and “alkenylalkyl” refers to having 2-24 carbon atoms (optionally having 2-16 carbon atoms; optionally having 2-14 carbon atoms; optionally having 2-12 carbon atoms, optionally having 2, 3, 4, 5, or 6 carbon atoms), straight-chain or branched alkenyl, said "alkenyl” having one or more carbon-carbon double bonds, Optionally have 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; said "alkenyl” is bonded to the rest of the molecule through a single bond; optionally selected from: vinyl, oil alkenyl, or linoleyl;
  • substituted groups in groups of the present invention indicate that the specified group may be in one or more of the available positions, preferably in 1, 2, or 3 positions, more preferably in 1 or Two positions, still more preferably one position, are substituted with one or more substituents.
  • R 2 is selected from: H, alkyl, alkenylalkyl, carboxyalkyl, esterylalkyl, alkylmercapto or carboxyalkylmercapto.
  • R is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, capryl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl acyl or linoleoyl.
  • R is selected from H, acetyl, lauroyl, myristoyl or palmitoyl.
  • R 1 is H, acetyl or palmitoyl.
  • R 2 is selected from: H, -CH 3 , -CH 2 CH 3 , -CH 2 CH ⁇ CH 2 , -CH 2 COOH, -CH 2 COOC(CH 3 ) 3 , -S-CH 2 CH 3 , -S-(CH 2 ) 5 CH 3 , -S-CH 2 COOH or
  • R 2 is selected from: H, -CH 3 , -CH 2 CH 3 , -CH 2 CH ⁇ CH 2 , -CH 2 COOH, -CH 2 COOC(CH 3 ) 3 , -S - CH2CH3 , -S-( CH2 ) 5CH3 or -S - CH2COOH .
  • R and R are independently selected from each other: H, methyl , ethyl, hexyl, dodecyl or hexadecyl;
  • R is H and R is selected from: H, methyl, ethyl, hexyl, dodecyl and hexadecyl;
  • R 3 is -OH or -NH 2 .
  • the peptide represented by formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, is selected from the following peptides (1)- (46):
  • the peptide represented by formula (I) of the present invention may exist as a stereoisomer or a mixture of stereoisomers; for example, these amino acids contained therein may have the configuration of L-, D-, or independently of each other racemic.
  • isomeric mixtures as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and which isomers are present.
  • Preferred structures of the peptides represented by formula (I) of the present invention are pure isomers, ie, enantiomers or diastereomers.
  • -Phe- is mentioned in the present invention, it should be understood that -Phe- is selected from -L-Phe-, -D-Phe-, or a mixture of both, and is racemic or non-racemic.
  • the preparation methods described in this document enable a person skilled in the art to obtain each stereoisomer of the peptides of the invention by selecting the amino acid with the correct configuration.
  • compositions refers to a salt approved for use in animals, and more specifically in humans, including metal salts of peptides represented by formula (I),
  • the metals include, but are not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum, etc.; including salts formed by the peptide represented by formula (I) and an organic base, and the organic base includes , but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine, etc.; including peptides and inorganic acids shown in formula (I) or salts of organic acids including, 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 acid, oleic acid, trifluoroacetic
  • Peptides and Chemical Approaches to the Synthesis of Peptides and Proteins (1997), CRC, Boca Raton, FL, USA], synthesis in solution, solid phase synthesis, and synthesis in solution Combination of methods or enzymatic synthesis [Kullmann W. (Kullmann W.), "Proteases as catalysts for enzymic syntheses of opioid peptides” (1980), Biochemical Journal (J. Biol. Chem.), 255, 8234-8238].
  • Peptides can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins with animal, fungal, or preferably plant origin, which releases peptide fragments containing at least the desired sequence.
  • a method for obtaining peptides of general formula (I) comprises the following steps:
  • the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising combining an amino acid having a protected N-terminus and a free C-terminus with a free N-terminus and a Amino acid coupling of the C-terminus bound to the polymer carrier; elimination of the group protecting the N-terminus; and repeating this sequence as many times as required to thereby obtain a peptide of the desired length, followed by cleavage of the desired length from the original polymer carrier Synthetic peptides.
  • the functional groups of the side chains of the amino acids remain fully protected with temporary or permanent protecting groups throughout the synthesis and can be deprotected simultaneously or orthogonally with the process of cleavage of the peptide from the polymeric support.
  • solid phase synthesis can be performed by a convergent strategy in which dipeptides or tripeptides are coupled to a polymeric support or to dipeptides or amino acids previously bound to a polymeric support.
  • Convergent synthesis strategies are well known to those skilled in the art and are described in Lloyd-Williams P., Albericio F. and Giralt E. "Convergent solid phase peptide synthesis", (1993), Tetrahedron 49:11065- 11133 in.
  • the method may comprise the additional steps of deprotecting the N-terminus and C-terminus and/or cleaving the peptide from the polymer support in a non-determined order using standard conditions and methods known in the art, which may subsequently be modified. terminal functional groups.
  • Optional modifications of the N-terminus and C-terminus can be carried out on the peptide of general formula (I) bound to a polymeric support, or after the peptide has been cleaved from the polymeric support.
  • the peptides of the present invention can be administered in the form of compositions containing them by any means that cause contact between these peptides and the site of action in mammals (preferably, the site of action in humans) in order to promote fibroblasts. Proliferation, increase collagen production.
  • another aspect of the present invention provides a cosmetic composition or a pharmaceutical composition, comprising an effective amount of the above-mentioned peptide of formula (I), or its stereoisomer, or a mixture of stereoisomers, or A cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and at least one excipient or optionally a cosmetically or pharmaceutically acceptable adjuvant.
  • These compositions can be prepared by conventional methods known to those skilled in the art ["Harry's Cosmetology", Seventh Edition, (1982), Wilkinson J.B. (Wilkinson J.B.), Moore R.J. ( Moore R.J., Longman House, Essex, UK].
  • the adjuvant is selected from: a collagen synthesis stimulator, an agent that regulates the synthesis of PGC-1 ⁇ , an agent that regulates the activity of PPAR ⁇ , an agent that increases or decreases the triglyceride content of adipocytes, stimulates or delays the adipocyte Differentiation agents, lipolytic agents or lipolysis stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinases Inhibitors, melanin synthesis stimulators or inhibitors, whitening or depigmenting agents, hyperpigmentation agents, self-tanning agents, antiaging agents, NO-synthase inhibitors, 5 ⁇ -reductase inhibitors, lysyl hydroxylation Enzyme and/or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and/or anti-air pollution agents,
  • these additional ingredients should not unacceptably alter the benefits of the peptides of the invention.
  • the nature of these additional ingredients may be synthetic or natural such as plant extracts, or from a biotechnological process, or from a combination of a synthetic and a biotechnological process. Additional examples can be found in "CTFA International Cosmetic Ingredient Dictionary & Handbook", 12th Edition (2008).
  • a biotechnological method is understood as any method for producing the active ingredient, or a part of the active ingredient, in an organism, or in a part of an organism.
  • the above-mentioned cosmetic composition or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by biotechnological methods, which is a collagen synthesis stimulating
  • a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by biotechnological methods which is a collagen synthesis stimulating
  • ascorbic acid and its derivatives such as ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate and ascorbyl ⁇ - and ⁇ -glucosides, retinol and retinol derivatives such as retinoic acid, retinol Xanthal, Retinyl Acetate or Retinyl Palmitate, Vegetable Extracts such as Aloe Vera and Centella Asiatica, Carnitine, Carnosine, Creatine, Asiatic Acid, Madecassic Acid, Centella Asiatica Grass glycosides, asiatica saponin, centella
  • the aforementioned cosmetic or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by biotechnological methods, which is an elastase-inhibiting Agents such as, without limitation, [INCI: Glycine Soja (Soybean) Protein], [INCI: Glycine Soja (Soybean) Protein, Oxidoreductase] or -Age [INCI: Hydrolyzed Rice Bran Hydrolyzed] sold by Pentapharm/DSM Glycerin, wild soybean (soybean) protein, oxidoreductase], Juvenesce sold by Coletica/Engelhard/BASF [INCI: Ethoxydiglycol and Caprylic Triglycerides, Retinol, Ursolic Acid, Vitamin K1, Ilox Ilomastat], MicromerolTM [INCI: Apple (Pyrus Malus) Extract], Heather Extract [INCI: Calluna Vulgaris Extract], [
  • the above-mentioned cosmetic or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by biotechnological methods, which is an active carbonyl species Substance scavengers, free radical scavengers and/or anti-glycation agents, such as and without limitation, carnosine and its derivatives, GHK [INCI: tripeptide-1] and its salts and/or derivatives, or sold by Lipotec [ INCI: hydrolyzed wheat protein, hydrolyzed soybean protein, tripeptide-1] or PreventheliaTM [INCI: diaminopropionyl tripeptide-33], etc.
  • Substance scavengers, free radical scavengers and/or anti-glycation agents such as and without limitation, carnosine and its derivatives, GHK [INCI: tripeptide-1] and its salts and/or derivatives, or sold by Lipotec [ INCI: hydrolyzed wheat protein, hydro
  • the above-mentioned cosmetic composition or pharmaceutical composition of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one extract, which is an anti-wrinkle agent and/or an anti-aging agent, such as and not limited to, grape, Extracts of Rosa canina, Curcuma longa, Iris pallida, Theobroma cacao, Ginkgo biloba, Leontopodium Alpinum or Dunaliella salina, etc., or At least one synthetic compound or product which is an anti-wrinkle or anti-aging agent such as and without limitation, [INCI: Palmitoyl Pentapeptide-3], Matrixyl [INCI: Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide], EssenskinTM [INCI: Calcium Hydroxymethionine], Renovage [INCI: Teprenone] or [INCI: Palmitoyl Oligopeptide], sold by Pentapharm/D SM [INCI: Five Peptide-3], Syn [INCI:
  • the aforementioned cosmetic or pharmaceutical compositions of the present invention may contain a cosmetically or pharmaceutically effective amount of at least one synthetic compound, plant extract, or product obtained by biotechnological methods, which stimulates healing Agents, agents that assist healing, agents that stimulate re-epithelialization, and/or agents that assist re-epithelialization, such as, and not limited to, Centella asiatica extract, Rosa moschata extract, Echinacea angustifolia Echinacea angustifolia extract, Symphytum officinal extract, Equisetum arvense extract, Hypericum perforatum extract, Mimosa tenuiflora extract, Aloe vera vera) extract, Epithelizing sold by Provital [INCI: Calendula (Calendula officinalis), Hypericum, Matricaria (Chamomilla recutita), Rosemary (Rosmarinus officinalis)], LS9028 sold by Laboratories Serobi GmbH/Cognis [ INC
  • the aforementioned cosmetic or pharmaceutical compositions of the present invention may contain or may be co-administered with analgesic and/or anti-inflammatory compounds in order to reduce swelling and swelling associated with sensitive skin and the healing process. irritation, or to treat overgrown scars or scars.
  • analgesic and/or anti-inflammatory compounds in order to reduce swelling and swelling associated with sensitive skin and the healing process. irritation, or to treat overgrown scars or scars.
  • emphasis may be placed on steroid-type compounds such as hydrocortisone, non-steroid-type compounds such as acetaminophen or acetylsalicylic acid, or natural extracts or essential oils with inherent analgesic and anti-inflammatory activities .
  • the formulation of said cosmetic or pharmaceutical composition is selected from the group consisting of creams, oils, milks, balms, foams, lotions, gels, liniments, serums, soaps, shampoos, conditioners, serums , ointment, mousse, pomade, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, syrup, elixir, polysaccharide film, jelly or gelatin;
  • the capsules include: soft capsules, hard capsules, optionally gelatin capsules;
  • the tablet includes: sugar-coated tablet.
  • compositions of the present invention may include transdermal enhancers that increase the absorption of the peptides of the present invention, such as and not limited to dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, nitrogen Ketone (1-dodecylazepan-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol or polyethylene glycol, etc.
  • transdermal enhancers that increase the absorption of the peptides of the present invention, such as and not limited to dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, nitrogen Ketone (1-dodecylazepan-2-one), alcohol, urea, ethoxydiglycol, acetone, propylene glycol or polyethylene glycol, etc.
  • the peptide represented by formula (I) of the present invention or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the Cosmetic or pharmaceutical compositions that can be prepared by iontophoresis, sonophoresis, electroporation, microelectropatch, mechanical compression, osmotic pressure gradient, dressing therapy, microinjection or needle-free injection with the aid of pressure (e.g. by Injections under oxygen pressure), or any combination thereof, are administered to the localized area to be treated in order to achieve greater penetration of the peptides of the invention.
  • the area of administration will be determined by the nature of the condition, disorder and/or disease to be treated and/or cared for.
  • the peptide represented by formula (I) according to the present invention may be incorporated into any form of functional food or fortified food, such as, and not limited to, dietary bars or compressed or non-compressed powders. These powders can be dissolved in water, juice, soda, dairy products, soy derivatives, or can be incorporated into meal bars.
  • the peptides of the invention can be formulated with common excipients or adjuvants used in oral compositions or food supplements, such as and not limited to, fatty components, aqueous components, humectants, Preservatives, thickeners, flavorings, fragrances, antioxidants, or coloring agents.
  • common excipients or adjuvants used in oral compositions or food supplements, such as and not limited to, fatty components, aqueous components, humectants, Preservatives, thickeners, flavorings, fragrances, antioxidants, or coloring agents.
  • the peptides of the invention have variable solubility in water depending on the nature of their sequence or any possible modification in the N-terminus and/or C-terminus.
  • the peptides of the present invention can thus be incorporated into compositions by aqueous solutions, and those that are insoluble in water can be dissolved in cosmetically or pharmaceutically acceptable conventional solvents such as and not limited to ethanol, propanol, isopropanol , propylene glycol, glycerin, butylene glycol, or polyethylene glycol, or any combination thereof.
  • the cosmetically or pharmaceutically effective amount of the peptides of the invention to be administered, as well as their dosage, will depend on many factors, including age, the state of the patient, the severity of the condition or disease, the route and frequency of administration, and the nature of the peptide to be used. specific nature.
  • Cosmetically or pharmaceutically effective amount means a non-toxic amount of one or more peptides of the invention which is sufficient to provide the desired effect.
  • the peptides of the invention are used in the cosmetic or pharmaceutical compositions of the invention at a cosmetically or pharmaceutically effective concentration to obtain the desired effect; in a preferred form, at 0.00000001% ( By weight) and 20% by weight, preferably between 0.000001% by weight and 15% by weight, more preferably between 0.0001% by weight and 10% by weight by weight), and even more preferably between 0.0001% by weight and 5% by weight.
  • a cosmetically or pharmaceutically acceptable delivery system or sustained release system in order to achieve better penetration of the active ingredient and/or to improve its pharmacokinetic and pharmacodynamic properties, which Comprising an effective amount of the peptide represented by the above-mentioned formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the above-mentioned cosmetic or pharmaceutical compositions;
  • delivery system refers to a diluent, adjuvant, excipient or carrier with which the peptides of the invention are administered, selected from the group consisting of: water, oil or surfactants, including petroleum sources, animal sources, plant sources, or Those of synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty Alcohol, Nonoxynol, Poloxamer, Polyoxyethylene, Macrogol, Dextrose, Glycerin, Digitonin and the like.
  • Those of ordinary skill in the art know the diluents that can be used in the different delivery systems in which the peptides of the invention can be administered.
  • sustained release is used in the conventional sense to refer to a delivery system that provides a gradual release of the compound over a period of time, and preferably, but not necessarily, a relatively constant level of compound release over the entire period of time.
  • Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant liposomal vesicles, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges Cyclodextrins, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.
  • Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.
  • Sustained release systems can be prepared by methods known in the art and can be administered, for example, by topical or transdermal administration, including adhesive patches, non-adherent patches, occlusive patches, and microelectronic patch; or by systemic administration such as and without limitation, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into specific body parts, and preferably should A relatively constant amount of these peptides of the invention is released.
  • the amount of peptide contained in the sustained release system will depend, for example, on the site where the composition is to be administered, the release kinetics and duration of the peptide of the invention, and the condition, disorder and/or condition to be treated and/or cared for. the nature of the disease.
  • Another aspect of the present invention provides a cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, which contains an effective amount of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above; selected from, but not limited to: talc, bentonite, dioxide Silicon, starch, or maltodextrin, etc.
  • Another aspect of the present invention provides a cosmetic product comprising an effective amount of the above-mentioned peptide represented by formula (I), or its stereoisomer, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof , or a pharmaceutically acceptable salt thereof, or the aforementioned cosmetic or pharmaceutical composition;
  • the cosmetic is selected from the group consisting of: concealer, foundation, makeup remover, makeup remover milk, eye shadow, lip stick, lip gloss, lip balm or lip powder.
  • Another aspect of the present invention provides a fabric, non-woven fabric or medical device, which comprises an effective amount of the above-mentioned peptide represented by formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or A cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the aforementioned cosmetic or pharmaceutical composition.
  • the peptides of the invention are released due to biodegradation of the fabric or non-woven fabric or the anchoring system of the medical device or by friction with the body, by body moisture, by the pH of the skin or by body temperature.
  • woven and nonwoven fabrics can be used to make clothes that come into direct contact with the body.
  • fabrics, nonwovens, clothing, medical devices and means of immobilizing peptides to them include the above-mentioned delivery systems and/or sustained release systems, which can be found in the literature and are known in the prior art [Schaab C.K. (1986 ) "Impregnating Fabrics With Microcapsules (using microcapsules to impregnate fabrics)", HAPPI May 1986; Nelson G. (2002) “Application of microencapsulation in textiles (microencapsulation in textiles)” Int.J.Pharm.242: 5562; “Biofunctional Textiles and the Skin” (2006) Curr.Probl.Dermatol.v.33, edited by Hipler U.C. and Elsner P. S.
  • Preferred fabrics, nonwovens, clothing or medical devices are bandages, gauze, t-shirts, socks, pantyhose, underwear, girdles, gloves, diapers, sanitary napkins, dressings, bedspreads, wipes, adhesive patches patch, non-adhesive patch, occlusive patch, microelectronic patch or mask.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable Acceptable salts, or cosmetic or pharmaceutical compositions described above, or cosmetically or pharmaceutically acceptable delivery systems or sustained release systems described above, or cosmetically or pharmaceutically acceptable solid organic polymers or solid inorganic supports described above , or the above-mentioned cosmetics, or the use of the above-mentioned fabric, non-woven fabric or medical device in the preparation of a cosmetic or pharmaceutical composition for the treatment or care of the skin or mucous membranes.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable Acceptable salts, or cosmetic or pharmaceutical compositions described above, or cosmetically or pharmaceutically acceptable delivery systems or sustained release systems described above, or cosmetically or pharmaceutically acceptable solid organic polymers or solid inorganic supports described above , or the above-mentioned cosmetics, or the above-mentioned fabrics, non-woven fabrics or medical devices in the preparation of cosmetic compositions or pharmaceutical compositions for treating or nursing body surface wounds, burns, skin ulcers, reducing scar generation, or accelerating scar repair the use of.
  • Another aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable Acceptable salts, or cosmetic or pharmaceutical compositions described above, or cosmetically or pharmaceutically acceptable delivery systems or sustained release systems described above, or cosmetically or pharmaceutically acceptable solid organic polymers or solid inorganic supports described above , or the above-mentioned cosmetics, or the above-mentioned fabrics, non-woven fabrics or medical devices in the preparation for promoting fibroblast proliferation, increasing collagen production, for improving skin barrier function, or for re-epithelialization of skin or mucous membranes or Use in a healing cosmetic or pharmaceutical composition.
  • One aspect of the present invention provides a peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetically or pharmaceutically acceptable delivery system or sustained-release system, or the above-mentioned cosmetically or pharmaceutically acceptable solid organic polymer or solid inorganic support, Or the above-mentioned cosmetics, or the use of the above-mentioned fabrics, non-woven fabrics or medical devices in the preparation of cosmetic compositions or pharmaceutical compositions for treating, preventing or repairing skin aging or photoaging.
  • the treatment, prevention or repair of skin aging or photoaging is the reduction, prevention or treatment of facial wrinkles.
  • Another aspect of the present invention provides a method for treating or caring for skin or mucous membranes, the method comprising administering a cosmetically or pharmaceutically effective amount of the above-mentioned peptide represented by formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the aforementioned cosmetic or pharmaceutical composition.
  • Another aspect of the present invention provides a method for treating or nursing body surface wounds, burns, skin ulcers, reducing scar production, and accelerating scar repair, the method comprising administering a cosmetically or pharmaceutically effective amount of the above-mentioned formula (I ), or a stereoisomer thereof, or a mixture of stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the aforementioned cosmetic or pharmaceutical composition.
  • a method for promoting fibroblast proliferation, increasing collagen production, for improving skin barrier function, or for re-epithelialization or healing of skin and/or mucous membranes comprising administering A cosmetically or pharmaceutically effective amount of the peptide represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt, or the aforementioned cosmetic or pharmaceutical compositions.
  • Another aspect of the present invention provides a method for treating, preventing or repairing skin aging or photoaging, the method comprising administering a cosmetically or pharmaceutically effective amount of the above-mentioned peptide represented by formula (I), or its stereo isomers, or mixtures of stereoisomers, or cosmetically acceptable salts thereof, or pharmaceutically acceptable salts thereof, or the aforementioned cosmetic or pharmaceutical compositions.
  • the treatment, prevention or repair of skin aging or photoaging is the reduction, prevention or treatment of facial wrinkles.
  • compositions of cosmetic or pharmaceutical compositions may be applied to the skin and/or mucous membranes, or administered orally or parenterally as required for the treatment and/or care of a condition, disorder and/or disease.
  • the frequency of administration or dosing can vary widely depending on the needs of each subject, with suggested administration or dosing ranging from 1 time per month to 10 times per day, preferably from 1 time per week to 4 times per day, More preferably from 3 times a week to 3 times a day, even more preferably 1 or 2 times a day.
  • the term “skin” is understood to mean the layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both inclusive. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, among others. In the present invention, the term “skin” includes the scalp.
  • treatment refers to the administration of the peptides according to the present invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with such a disease or condition.
  • treatment also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or disorder.
  • the term "care” includes the prevention of diseases and/or conditions.
  • prevention refers to the ability of the peptides of the invention to prevent, delay, or impede the onset or development of a disease or condition before it occurs.
  • aging refers to the changes the skin undergoes as it ages (natural aging), or through exposure to sunlight (photoaging) or exposure to environmental pollutants such as chemical dirt or pollutants, tobacco smoke, etc. changes, and includes all externally visible and/or perceptible changes by touch, such as and not limited to: the development of discontinuities in the skin (such as wrinkles, fine lines, expression lines, stretch marks, striae, furrows wrinkles, unevenness or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of ability to recover from deformation, loss of resilience), sagging of the skin (such as sagging cheeks, bags under the eyes, or double chin, etc.), changes in skin color (such as scarring, redness, bags under the eyes, or areas of hyperpigmentation such as age spots or freckles), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange Dermoid skin, loss of collagen structure, and other histologic
  • photoaging refers to premature aging of the skin due to long-term exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, such as and not limited to: sagging, sagging, color changes or hyperpigmentation Regular, abnormal and/or hyperkeratosis.
  • Ala represents NH2 -CH( CH3 )-COOH
  • Ala- represents NH2 -CH( CH3 )-CO-
  • -Ala represents -NH-CH( CH3 )-COOH
  • -Ala- Represents -NH-CH(CH 3 )-CO-.
  • Ac- is used in the present invention to denote the acetyl group (CH 3 -CO-), and the abbreviation “Palm-” is used to denote the palmitoyl group (CH 3 -(CH 2 ) 14 -CO-).
  • the peptide described in the present invention is obtained by artificial design, is easy to synthesize, can promote the proliferation of fibroblasts, improve the skin barrier function, is beneficial to promote wound healing, treat or care for body surface wounds, burns, skin ulcers, reduce scar generation, and accelerate scarring Restorative, and also useful for treating, preventing and/or repairing skin aging and/or photoaging.
  • Fig. 4 is a graph showing the results of the scratch experiment on HaCaT cells.
  • Fig. 5 is a graph showing the repairing effect of peptide (1) to peptide (5) on light-damaged HaCaT cells.
  • Fig. 6 is a graph showing the repairing effect of peptide (1) and peptide (2) on photodamaged NIH3T3 cells.
  • Wang Resin Wang resin; DMF: N,N-dimethylformamide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; Ac 2 O: acetic anhydride; DIPEA: diisopropylethylamine ; Fmoc: 9-fluorenylmethoxycarbonyl; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; TIS: triisopropylsilane; EDT: 1,2-ethanedisulfide Alcohol; DMSO: Dimethyl Sulfoxide; Ac: Acetyl; Palm: Palmitoyl; Ala: Alanine; Pro: Proline; Phe: Phenylalanine; Arg: Arginine; Met: Methionine acid; Gly: glycine; Ile: isoleucine; Cys: cysteine; Thr: threonine; Asn: asparagine; Pb
  • the swollen resin was de-Fmoc twice with 500ml 20% piperidine/DMF, the first reaction was 5 minutes, the second reaction was 8 minutes, and the solvent was drained for 3 minutes. Wash the resin 7-8 times with DMF, and remove the solvent.
  • the degree of substitution of the resin was detected to be 0.5, and the calculated synthesis scale was 24.9 mmol.
  • the N-terminal Fmoc group was deprotected, and 24.74 g of activated Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 equiv.) Coupled to the peptidyl resin, the reaction continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the N-terminal Fmoc group was deprotected and 32.37 g of activated Fmoc-Met-OH (87.15 mmol; 3.5 equiv.) was coupled to On the peptidyl resin, the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the resin was washed with DMF, 100 ml each time, the resin and the solvent were uniformly mixed, stirred for 2 minutes, and the solvent was drained. The resin was washed several times with DMF until the solvent filtered out with suction was clear and transparent.
  • Fmoc-Linker-Amide Resin was de-Fmoc twice with 500ml 20% piperidine/DMF, the first reaction was 5 minutes, the second reaction was 8 minutes, and the solvent was drained for 3 minutes. 500ml DMF wash resin 4 times, 500ml DCM wash resin 2 times. Aspirate off the solvent.
  • the N-terminal Fmoc group was deprotected and 34.78 g of activated Fmoc-Thr(tBu)-OH (87.5 mmol) was coupled to the peptidyl group in the presence of 14 g HOBt and 20 ml DIC using DMF as solvent On the resin, the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the resin was washed 4 times with 600 ml DMF for 2 min each, 2 times with 500 ml DCM and the solvent was aspirated off.
  • the N-terminal Fmoc protecting group was removed.
  • the R 1 palmitoyl group was introduced onto the peptidyl resin obtained in Example 3.
  • the R 1 acetyl group was introduced onto the peptidyl resin obtained in Example 3.
  • the peptidyl resins obtained in Examples 3, 4, and 5 were shrunk three times with 500 ml, 300 ml, and 200 ml of methanol. Vacuum dry.
  • Example 6 The crude peptide obtained in Example 6 was dissolved with methanol and water, and filtered with a filter membrane with a pore size of 0.45 ⁇ m.
  • Mobile phase A pure acetonitrile
  • mobile phase B 0.1% acetic acid aqueous solution
  • the filtered sample was injected and purified, the fractions were collected, concentrated and freeze-dried to obtain the following peptides with a purity of over 90%:
  • Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2 namely peptide (9);
  • Example 7 The peptide obtained in Example 7 was oxidized.
  • Example 7 The peptide obtained in Example 7 was added to a 250ml pear-shaped bottle, dissolved in 80ml of purified water, 8g of guanidine hydrochloride and 20ml of DMSO were added, stirred for 30h with the open mouth, and the reaction process was monitored by HPLC. After oxidation, add 4 ml of acetic acid to dissolve the precipitate, and vacuum filter it with a 0.22 ⁇ m filter membrane.
  • Example 8 The filtrate obtained in Example 8 was purified by high performance liquid chromatography.
  • Mobile phase A pure acetonitrile
  • mobile phase B 0.1% acetic acid aqueous solution
  • the mercapto group of the side chain of Cys8 of the peptide obtained in Example 7 was modified with mercaptoethane.
  • Mobile phase A pure acetonitrile
  • mobile phase B 0.1% acetic acid aqueous solution
  • the swollen resin was de-Fmoc twice with 500ml 20% piperidine/DMF, the first reaction was 5 minutes, the second reaction was 8 minutes, and the solvent was drained for 3 minutes. Wash the resin 7-8 times with DMF, and remove the solvent.
  • the degree of substitution of the resin was detected to be 0.5, and the calculated synthesis scale was 24.9 mmol.
  • the N-terminal Fmoc group was deprotected, and 24.74 g of activated Fmoc-Thr(tBu)-OH (62.25 mmol; 2.5 eq. ) was coupled to the peptidyl resin, and the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the N-terminal Fmoc group was deprotected and 32.37 g of activated Fmoc-Met-OH (87.15 mmol; 3.5 equivalents) were coupled to On the peptidyl resin, the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the resin was washed with DMF, 100 ml each time, the resin and the solvent were uniformly mixed, stirred for 2 minutes, and the solvent was drained. The resin was washed several times with DMF until the solvent filtered out with suction was clear and transparent.
  • Fmoc-Linker-Amide Resin was de-Fmoc twice with 500ml 20% piperidine/DMF, the first reaction was 5 minutes, the second reaction was 8 minutes, and the solvent was drained for 3 minutes. 500ml DMF wash resin 4 times, 500ml DCM wash resin 2 times. Aspirate off the solvent.
  • the N-terminal Fmoc group was deprotected and 34.78 g of activated Fmoc-Thr(tBu)-OH (87.5 mmol) was coupled to the peptide in the presence of 14 g HOBt and 20 ml DIC using DMF as solvent On the base resin, the reaction was continued for 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the resin was washed 4 times with 600 ml DMF for 2 min each, 2 times with 500 ml DCM and the solvent was aspirated off.
  • the N-terminal Fmoc protecting group was removed.
  • the R 1 palmitoyl group was introduced onto the peptidyl resin obtained in Example 18.
  • the R 1 acetyl group was introduced onto the peptidyl resin obtained in Example 18.
  • the peptidyl resins obtained in Examples 18, 19 and 20 were cleaved from the polymer support.
  • the peptidyl resins obtained in Examples 18, 19 and 20 were shrunk three times with 500 ml, 300 ml, and 200 ml of methanol. Vacuum dry.
  • Example 21 The crude peptide obtained in Example 21 was dissolved with methanol and water, and filtered through a filter membrane with a pore size of 0.45 ⁇ m.
  • Mobile phase A pure acetonitrile
  • mobile phase B 0.1% acetic acid aqueous solution
  • the filtered sample was injected and purified, the fractions were collected, concentrated and freeze-dried to obtain the following peptides with a purity of over 90%:
  • PBS Phosphate buffered saline
  • MTT thiazolium blue
  • DMSO dimethylsulfoxide
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • Microplate reader (MD, USA), CO 2 incubator (Shanghai Yiheng), ultra-clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).
  • HaCaT Human keratinocytes
  • NIH3T3 mouse skin fibroblasts
  • Administration groups peptide (1), peptide (2), peptide (3), peptide (4), peptide (5), the concentrations were 12.5ppm, 25ppm, 50ppm, 100ppm respectively.
  • Control group PBS blank control.
  • MTT method is a method to detect cell survival and growth.
  • the measured OD value is directly proportional to cell activity. The larger the OD value, the higher the cell activity.
  • PBS Phosphate buffered saline
  • MTT thiazolium blue
  • DMSO dimethylsulfoxide
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • Microplate reader (MD, USA), CO 2 incubator (Shanghai Yiheng), ultra-clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).
  • HaCaT Human keratinocytes
  • Administration group peptide (1), peptide (2), the concentrations were 12.5ppm, 25ppm, 50ppm, 100ppm respectively. Dissolve with PBS solution.
  • Control group PBS blank control.
  • the cryopreserved HaCaT human cuticle cells were cultured, subcultured at a ratio of 1:2 to about 5 passages, and the cells with better growth were selected as the experimental objects.
  • the sample to be tested was added to a 96-well plate at a rate of 20 ⁇ L/well, and dried overnight in a constant temperature oven at 37°C.
  • On the second day after digesting the well-growing HaCaT cells, seed the plate at a density of 10,000 HaCaT cells/well, supplement the medium to 200 ⁇ L, and incubate for 3 hours at 37°C and 5% CO 2 incubator. After the culture is over, take out the culture plate and continue to replenish the culture medium until the liquid level just overflows, and seal it with a parafilm to ensure that there are no air bubbles. Turn over for 20 minutes under the action of a three-dimensional force.
  • the original medium was discarded, and 90 ⁇ L of fresh medium and 10 ⁇ L of 5 mg/mL MTT were added to each well, and placed in a 37° C., 5% CO 2 incubator for 3 h. Discard the solution and add 150 ⁇ L of DMSO. Set the detection wavelength to 490nm, the reference wavelength to 630nm, and use a microplate reader to read the OD value.
  • HaCaT cells By selecting HaCaT cells, plate them in a drug-coated 96-well plate, and after incubation and a period of three-dimensional force, the cells with strong adhesion can be maintained on the 96-well plate.
  • MTT quantitative analysis on the live cells on the plate, That is, the effect of the drug on cell adhesion can be evaluated to determine whether the peptide of the present invention can improve cell elasticity. After three-dimensional force action, the more living cells remained on the 96-well plate, the greater the measured OD value, indicating the stronger the adhesion of the cells.
  • PBS Phosphate buffered saline
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • HaCaT Human keratinocytes
  • Administration group 100 ppm of peptide (1), 100 ppm of peptide (2), 50 U/mL of epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • Control group PBS blank control.
  • the cryopreserved HaCaT cells were cultured, subcultured to about 5 passages according to the ratio of 1:2, and the cells with better growth were selected as the experimental objects.
  • Cells were seeded in a 12-well plate at a density of 200,000 cells/well, with 2 mL of cell suspension in each well. After the cells adhered to the wall, the medium was replaced with 0.5% to 1% fetal bovine serum to maintain the cells for 24 hours to synchronize them. change. Scratch with a sterilized pipette tip, wash off the dropped cells with PBS, add the sample to be tested, continue to incubate in a 37°C, 5% CO 2 incubator, and place it under an optical microscope after 24 hours to analyze the wound healing area.
  • the cell scratch migration test can reflect the proliferation and repair ability of the cells.
  • the results are shown in Figure 4.
  • the cell scratches in the normal control group are still obvious, and the scratch spacing is normal; compared with the control group, the cell proliferation and migration in the EGF positive group is obvious. , the spacing was significantly shortened; while the scratch spacing of peptide (1) and peptide (2) was shorter than that of the normal control group, and cell migration tracks appeared, it can be known that the peptide of the present invention can promote HaCaT human keratin formation Cell proliferation and migration, has the effect of skin repair.
  • PBS Phosphate buffered saline
  • MTT thiazolium blue
  • DMSO dimethylsulfoxide
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • Microplate reader (MD, USA), CO 2 incubator (Shanghai Yiheng), ultra-clean bench (Suzhou Purification), biological inverted microscope (Chongqing Optoelectronics).
  • HaCaT Human keratinocytes
  • NIH3T3 mouse skin fibroblasts
  • the concentrations of peptide (1), peptide (2), peptide (3), peptide (4), and peptide (5) were 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm, respectively.
  • peptide (1) can significantly increase cell activity at 50 and 100ppm; peptide (2) can significantly increase NIH3T3 cell activity at each dose of 12.5ppm-100ppm. This shows that both peptide (1) and peptide (2) can repair photodamaged NIH3T3 cells within a certain concentration range, improve their activity, and have good skin repair ability.
  • Microplate reader (MD, USA), CO 2 incubator (Shanghai Yiheng), ultra-clean bench (Suzhou Purification).
  • Peptide (2) tested at a concentration of 100 ppm.
  • Operate according to the ELISA operating instructions move all reagents to room temperature and equilibrate for at least 30 minutes, prepare reagents according to the instructions, and set aside. Set up wells for standard products and wells for samples to be tested, add 100 ⁇ L of standard products or samples to be tested in each well, shake gently, cover with plate stickers, and incubate at 37°C for 2 hours. 4 replicate wells were set up for each sample. Discard the liquid, spin dry, and wash the plate 5 times. Add 100 ⁇ L of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 h.
  • Inflammatory factors such as TNF- ⁇ , IL-6 and IL-8 can cause excessive immune response of the body, leading to skin ulceration, redness and swelling, etc. Therefore, inhibiting inflammatory factors such as TNF- ⁇ , IL-6 and IL-8 can achieve Anti-inflammatory repair effect.
  • the inflammation model was established by stimulating RAW264.7 cells with 200ng/mL LPS, and the anti-inflammatory repairing effect of the peptide of the present invention was explored.
  • HPLC chromatographic separation conditions are as follows:
  • test solution Take an appropriate amount of this product and add water to dissolve and dilute to make a solution containing 1 mg per 1 ml.
  • Dipalmitoylphosphatidylcholine was weighed out and dissolved in chloroform. The solvent was evaporated under vacuum until a thin layer of phospholipids was obtained, which was hydrated by treatment with an aqueous peptide solution of the desired concentration at 55°C to give multilamellar liposomes.
  • the multi-lamellar liposomes are homogenized under high pressure to obtain smaller and uniform single-lamellar liposomes.
  • phase B According to the prescribed dosage, weigh the ingredients of Phase B and add them to the container. Next, add phase D to phase B and homogenize with continuous stirring. Phase A is then added to the mixture. Finally, phase C is added to obtain a microemulsion composition containing peptide (2).

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Abstract

通式(I)的肽、其立体异构体、立体异构体的混合物、或其美容上可接受的盐或其药学上可接受的盐、或它们的美容组合物或药用组合物、以及它们用于皮肤或粘膜的病状、病症或疾病的治疗或护理的用途。

Description

一种合成肽以及它们的美容组合物或药用组合物及用途 技术领域
本发明涉及能够在皮肤或粘膜中促进成纤维细胞增殖的肽、以及含有这些肽的美容组合物或药物组合物以及它们在治疗或护理皮肤或粘膜中的用途。
背景技术
皮肤是人体最大的器官，被覆于体表，对机体发挥着重要的屏障作用，可以抵御机械损伤，缓冲外部冲击，避免体内组织器官受损，防止外界有害的物质入侵，抗氧化，抗紫外线损伤，还可防止机体内各种营养物质、水分、电解质的流失，对保持人体内环境的稳定起着重要作用。皮肤由表皮层、真皮层和皮下组织构成。最外层是表皮，主要由角质形成细胞、黑色素细胞、朗格汉斯细胞等构成。真皮层位于表皮层下方，含有真皮细胞、胶原纤维、弹性纤维、基质、毛发、皮脂腺、汗腺、血管等。真皮通过基底膜与表皮紧密连接。
真皮中最主要的细胞是成纤维细胞。成纤维细胞体积大，形态多样，多为突起的纺锤形或星形的扁平结构，细胞质中含有丰富而发达的粗面内质网、高尔基体以及游离核糖体，因此具有强大的合成分泌蛋白质的功能，可以分泌前胶原、纤连蛋白和胶原酶等细胞外基质成分。成纤维细胞利用脯氨酸和赖氨酸等氨基酸，在粗面内质网的核蛋白体上合成前α多肽链(proalpha polypeptide chain)，多肽链输送到高尔基体后，组成前胶原分子(procollagen)。前胶原分子通过胞吐作用释放到细胞外。在前胶原肽酶催化下，前胶原分子的前α多肽链的尾段被除去，成为原胶原分子(tropocollagen)。原胶原分子成行平行排列，结合成具有周期性横纹的胶原原纤维。胶原原纤维互相结合形成胶原纤维。成纤维细胞除了可以制造胶原纤维外，还可以制造弹性纤维。胶原纤维中主要成分为Ⅰ型胶原，其抗拉力强，赋予皮肤韧性。弹性纤维由弹性蛋白和丝状蛋白聚合而成，赋予皮肤弹性。胶原纤维、弹性纤维以及充填于纤维和细胞之间的细胞外基质对皮肤形成支撑作用。
随着年龄增长，皮肤的形态学、生理学及其机械性质会发生显著的变化。在皮肤自然老化过程中，成纤维细胞功能下降，胶原纤维和弹性纤维受损、断裂，皮肤中胶原等重要生物分子的产生和降解之间的平衡随着年龄而偏向于降解过程，从而导致真皮进行性地变薄和组织破坏，并且引起真皮松弛，随后形成皱纹。长时间暴露于阳光或环境污染物会加速皮肤老 化过程。紫外线会抑制成纤维细胞中胶原和纤连蛋白的合成，并且紫外线辐射后可使基质金属蛋白酶(MMP)高表达，对真皮层的胶原蛋白造成严重损害，导致真皮完整性被破坏，皮肤强度和弹性降低，以致皮肤加速老化。
因此，通过促进成纤维细胞增殖，增强其活性，增加胶原蛋白、弹性蛋白、糖胺多糖等成分的合成和分泌，可以改善随着老化或暴露于阳光和/或暴露于环境污染物而损失的柔韧性、弹性和紧实度等皮肤机械性质，修复受损皮肤，减少皱纹。
皮肤作为人体第一道防线，容易受到外界损伤，从而失去屏障功能，导致抵抗力变弱，非致病菌或条件致病菌会侵入皮肤，诱发感染，甚至可能对身体健康造成重大影响。因此，加快皮肤创面修复，在伤口难愈的疾病以及术后修复中均具有十分重要的意义。
创面修复是一个精确复杂、分时相的动态过程，涉及细胞增殖、迁移、胞外基质降解、血管新生和上皮组织重塑等多种细胞的网络调控。成纤维细胞是创面修复的主要细胞。成熟的成纤维细胞处于静止状态，其胞体体积减小，呈长梭形，粗面内质网和高尔基体减少，成熟状态的成纤维细胞被称为纤维细胞。成纤维细胞与纤维细胞在一定条件下可以互相转变。在外伤等因素刺激下，部分纤维细胞重新转变为幼稚的成纤维细胞，其合成及分泌功能随之恢复，激发新的细胞外基质生成，大量细胞外基质与新生毛细血管复合形成肉芽组织填补缺损组织。在修复过程中，成纤维细胞的增殖与迁移对肉芽组织的形成和创面的修复是至关重要的。研究表明，成纤维细胞主要参与创面修复的增殖期与组织重塑期。在增殖期，成纤维细胞大量增殖、迁移并形成肉芽组织，主要起到抵抗感染、添补创面等作用。在组织重塑期，由成纤维细胞分化而来的肌成纤维细胞、内皮细胞及巨噬细胞发生凋亡，最终真皮组织由细胞外基质蛋白所填补，达到创面修复，维持皮肤结构和功能完整性的目的。
综上所述，成纤维细胞在皮肤老化、创面修复过程中发挥着重要作用，开发能够促进成纤维细胞增殖的产品，增强成纤维细胞活性，增加胶原蛋白、弹性蛋白、糖胺多糖等成分的合成和分泌，以补偿随着老化或暴露于阳光和/或暴露于环境污染物而损失的胶原蛋白、弹性蛋白等细胞外基质，延缓衰老；填补皮肤损伤后坏死组织区域的空缺，促进创面修复，具有重要意义。
发明内容
本发明的发明人通过大量实验研究，出人意料地发现成纤维细胞的增殖和活性可以通过 某些合成肽进行调节。本发明旨在提供一种能够在皮肤或粘膜中促进成纤维细胞增殖的肽。这些肽及含有这些肽的美容组合物或药物组合物，可以通过促进成纤维细胞增殖而改善或预防皮肤或粘膜的病况、病症或疾病的治疗或护理，从而改善皮肤屏障功能或治疗、预防或修复皮肤老化或光老化，或用于皮肤或粘膜的再上皮化或愈合。
鉴于此，本发明提供一种通式(Ⅰ)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于：
Figure PCTCN2021112345-appb-000001
式(I)中，
R 2选自：H、烷基、烯基烷基、羧基烷基、酯基烷基、烷基巯基、羧烷基巯基、或
Figure PCTCN2021112345-appb-000002
式(I)和式(i)中，
n选自：1或2；
R 1选自：H或R 4-CO-，其中R 4选自：取代的或未取代的烷基、取代的或未取代的烯基；
R 3选自：-NR 5R 6或-OR 5，其中各个R 5和R 6彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
基团R 1和R 3对应地结合至这些肽序列的氨基末端(N-末端)和羧基末端(C-末端)。
所述“烷基”、“烯基烷基”、“羧基烷基”、“酯基烷基”“烷基巯基”、“羧烷基巯基”中的烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链 或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
所述“烯基”、“烯基烷基”中的烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子、可选具有2、3、4、5、或6个碳原子)的直链或支链烯基，所述“烯基”具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述“烯基”是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
所述“酯基烷基”中的酯基为R(C＝O)OR′基团，其中，R、R′各自独立地为单键、烷基或亚烷基；
如在本技术领域中所理解的，在上述的基团中可以有一定程度的取代。因此，在本发明中的任何基团中都可以有取代。在本文件中提及的在本发明的基团中的取代的基团指示规定的基团可以在一个或多个可用的位置、优选在1、2、或3个位置、更优选在1或2个位置、还更优选在1个位置上被一个或多个取代基取代。这些取代基包括例如并且不局限于：C 1-C 4烷基；羟基；C 1-C 4烷氧基；氨基；C 1-C 4氨基烷基；C 1-C 4羰氧基；C 1-C 4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C 1-C 4烷基磺酰基；硫醇；C 1-C 4烷硫基；C 6-C 30芳氧基如苯氧基；-NR b(C＝NR b)NR bR c，其中R b和R c是独立地选自：H、C 1-C 4烷基、C 2-C 4烯基、C 2-C 4炔基、C 3-C 10环烷基、C 6-C 18芳基、C 7-C 17芳烷基、具有三至十元的杂环基、或氨基的保护基。
可选地，式(I)中，当n为2时，R 2选自：H、烷基、烯基烷基、羧基烷基、酯基烷基、烷基巯基或羧烷基巯基。
可选地，R 1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基或亚油酰基。
可选地，R 1选自H、乙酰基、月桂酰基、肉豆蔻酰基或棕榈酰基。
可选地，R 1是H、乙酰基或棕榈酰基。
可选地，R 2选自：H、-CH 3、-CH 2CH 3、-CH 2CH＝CH 2、-CH 2COOH、-CH 2COOC(CH 3) 3、-S-CH 2CH 3、-S-(CH 2) 5CH 3、-S-CH 2COOH或
Figure PCTCN2021112345-appb-000003
可选地，当n为2时，R 2选自：H、-CH 3、-CH 2CH 3、-CH 2CH＝CH 2、-CH 2COOH、-CH 2COOC(CH 3) 3、-S-CH 2CH 3、-S-(CH 2) 5CH 3或-S-CH 2COOH。
可选地，R 5和R 6彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
可选地，R 5是H并且R 6选自：H、甲基、乙基、己基、十二烷基以及十六烷基；
可选地，R 3是-OH或-NH 2。
式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，选自下列肽(1)-(46)：
Figure PCTCN2021112345-appb-000004
(2)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
Figure PCTCN2021112345-appb-000005
Figure PCTCN2021112345-appb-000006
(8)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH；
(9)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2；
(10)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH；
(11)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2；
Figure PCTCN2021112345-appb-000007
Figure PCTCN2021112345-appb-000008
Figure PCTCN2021112345-appb-000009
Figure PCTCN2021112345-appb-000010
Figure PCTCN2021112345-appb-000011
Figure PCTCN2021112345-appb-000012
(42)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2；
(43)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
(44)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2；
(45)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
(46)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2。
本发明的式(I)所示的肽可以作为立体异构体或立体异构体的混合物存在；例如，其所包含的这些氨基酸可以具有L-、D-的构型、或彼此独立地是外消旋的。因此，有可能获得同分异构混合物以及外消旋混合物或非对映混合物、或纯的非对映异构体或对映异构体，这取决于不对称碳的数量和存在什么同分异构体或同分异构混合物。本发明的式(I)所示的肽的优选的结构是纯的同分异构体，即，对映异构体或非对映异构体。
例如，当本发明所述-Phe-时，应理解-Phe-选自-L-Phe-、-D-Phe-、或两者的混合物，是外消旋的或非外消旋的。在本文件中描述的制备方法使本领域的普通技术人员能够通过选择具有正确构型的氨基酸来获得本发明的肽的每种立体异构体。
术语“美容上可接受的盐或药学上可接受的盐”指被认可的在动物，并且更确切地说在 人类中使用的一种盐，包括式(I)所示的肽的金属盐，所述金属包括，但不局限于：锂、钠、钾、钙、镁、锰、铜、锌或铝等；包括式(I)所示的肽与有机碱形成的盐，所述有机碱包括，但不局限于：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪等；包括式(I)所示的肽与无机酸或有机酸形成的盐，所述有机酸包括，但不局限于：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸(pamoate)或葡萄糖酸等；所述无机酸包括，但不局限于：盐酸、硫酸、硼酸或碳酸。
本发明式(I)的肽、或其立体异构体或其美容上可接受的盐、或药学上可接受的盐的合成可以根据现有技术中已知的常规方法来进行，如使用固相肽合成的方法[斯图尔特J.M.(StewartJ.M.)和杨J.D.(Young J.D.)，“固相肽合成”(“Solid Phase Peptide Synthesis”)，第二版，(1984)，皮尔斯化学公司(Pierce Chemical Company)，罗克福德(Rockford)，伊利诺伊州(Illinois)；Bodanzsky M.，Bodanzsky A.“肽合成的实践(The practice of PeptideSynthesis)”，(1984)，施普林格出版公司(Springer Verlag)，纽约；劳埃德-威廉姆斯·P.(Lloyd-Williams P.)、阿贝利希欧·F.(Albericio F.)、吉拉尔特·E.(Giralt E.)“肽和蛋白质合成的化学方法(Chemical Approaches to the Synthesis of Peptides andProteins)”，(1997)，CRC，博卡拉顿(Boca Raton)，佛罗里达州，美国]、在溶液中合成、固相合成和在溶液中合成的方法的组合或酶合成[库尔曼·W.(Kullmann W.)，：“蛋白酶作为催化剂用于阿片肽的酶合成(Proteases as catalysts for enzymic syntheses of opioidpeptides)”，(1980)，生物化学杂志(J.Biol.Chem.)，255，8234-8238]。肽还可以通过以产生所希望的序列为目标的生物技术方法、或通过具有动物、真菌、或优选地植物来源的蛋白质的控制水解来制备，这释放含有至少所希望的序列的肽片段。
例如，一种获得具有通式(Ⅰ)的肽的方法包括以下步骤：
-将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和受保护的或与固体载体结合的C-末端的氨基酸偶联；
-消除保护N-末端的基团；
-重复该偶联顺序和消除保护N-末端的基团，直到获得所希望的肽序列；
-消除保护C-末端的基团或从该固体载体裂解。
优选地，C-末端与一种固体载体结合并且该方法是在固相上进行，包括将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和与一种聚合物载体结合的C-末端的 氨基酸偶联；消除保护N-末端的基团；并且重复此顺序所需要的次数以便因此获得具有所希望的长度的肽，接着从最初的聚合物载体裂解所合成的肽。
在整个合成中这些氨基酸的侧链的官能团用临时或永久的保护基团保持充分地保护，并且可以与从该聚合物载体裂解肽的过程同时地或正交地脱保护。
可选地，固相合成可以通过将二肽或三肽偶联到聚合物支持体上或偶联到先前与聚合物支持体结合的二肽或氨基酸上的汇集策略(convergent strategy)来进行。汇集合成策略是本领域技术人员广为人知的并描述于Lloyd-Williams P.，Albericio F.and Giralt E.“Convergent solid phase peptide synthesis(汇集固相肽合成)”,(1993),Tetrahedron 49:11065-11133中。
该方法可以包括如下另外的步骤：使用本领域已知的标准条件和方法对N-末端和C-末端脱保护和/或以非确定的次序从聚合物支持体裂解肽，随后可以修饰所述末端的官能团。可以对与聚合物支持体结合的通式(Ⅰ)的肽进行N-末端和C-末端的任选的修饰，或在肽已从聚合物支持体裂解后进行N-末端和C-末端的任选的修饰。
可以通过引起这些肽与哺乳动物体内的作用部位(优选人体内的作用部位)之间相接触的任何手段、并且以含有它们的组合物的形式，来给予本发明的这些肽以便促进成纤维细胞增殖、增加胶原蛋白生成。
为此，本发明的另一方面，提供一种美容组合物或药用组合物，包括有效量的上述式(Ⅰ)的肽、或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，以及至少一种赋形剂或任选的美容上或药学上可接受的佐剂。这些组合物可以通过本领域技术人员已知的常规方法来制备[“哈利化妆品学(Harry’s Cosmeticology)”，第七版，(1982)，威尔金森·J.B.(Wilkinson J.B.)、摩尔·R.J.(Moore R.J.)编辑，朗文出版社(Longman House)，埃塞克斯郡(Essex)，英国]。
可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、 有机溶剂、液体推进剂、皮肤调理剂、保湿剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素或着色剂、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、角质剥离剂、角质层分离剂、抗微生物剂、抗真菌剂、抑真菌剂、灭菌剂、抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、热休克蛋白、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗皮炎剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、粘合剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子生长因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物，其条件是它们与组合物的组分在物理上和化学上相容，且特别是与本发明的组合物中包含的通式(Ⅰ)的肽相容。此外，这些另外成分的性质不应不可接受地改变本发明的肽的益处。这些另外成分的性质可以是合成的或天然的如植物提取物、或来自一种生物技术方法、或来自一种合成方法和一种生物技术方法的组合。另外的实例可以见于“CTFA国际化妆品成分字典和手册(CTFA International Cosmetic Ingredient Dictionary&Handbook)”，第12版(2008)。在本发明的背景下，生物技术方法应理解为用于在有机体内、或在有机体的一部分内产生该活性成分、或该活性成分的一部分的任何方法。
可选地，本发明的上述美容组合物或药物组合物，可以含有美容上或药学上有效量的至 少一种合成化合物、植物提取物、或通过生物技术方法获得的产物，其是胶原合成刺激剂，例如并且不限于，抗坏血酸及其衍生物例如棕榈酸抗坏血酸酯、抗坏血酸磷酸镁、抗坏血酸磷酸钠和抗坏血酸α-和β-葡糖苷，视黄醇及视黄醇衍生物例如视黄酸、视黄醛、醋酸视黄酯或棕榈酸视黄酯，蔬菜提取物例如芦荟属和积雪草属的提取物，肉碱，肌肽，肌酸，亚洲积雪草酸，羟基积雪草酸，羟基积雪草苷，积雪草皂苷，积雪草提取物，烟酰胺，变胞藻黄素，葡聚糖，诸如染料木黄酮和大豆黄素的大豆提取物和大豆异黄酮，芸香苷，白杨素，桑色素，槟榔子生物碱，毛喉素，桦木酸，车前属提取物，TGF-β，银杏提取物，谷氨酰胺，乙醇酸，由Sederma/Croda销售的[INCI：棕榈酰五肽-4]、Matrixyl[INCI：棕榈酰四肽-7、棕榈酰低聚肽]或[INCI：苯甲酸C12-15烷基酯、俞酸甘油酯(Tribehenin)、神经酰胺2、PEG10油菜籽甾醇、棕榈酰低聚肽]，由Pentapharm/DSM销售的-Coll[INCI：棕榈酰三肽-5]、[INCI：刺槐豆(Locust Bean)(长角豆(Ceratonia Siliqua))胶]或BeauActiveTM MTP[INCI：水解乳蛋白]，由Biotechmarine销售的ThalassineTM[INCI：藻类(Algae)提取物]，EquiStatTM[INCI：苹果果实提取物、野大豆籽提取物]或由Coletica/Engelhard/BASF销售的Juvenesce[INCI：乙氧基二甘醇和辛酸甘油三酯、视黄醇、熊果酸、维生素K1、伊洛马司他]，由Silab销售的SMS Anti-[INCI：由番荔枝(Annona Squamosa)纯化的低聚肽]或Papilactyl[INCI：水、油莎草(CyperusEsculentus)块茎提取物]，由Atrium Innovations/Unipex Group销售的ChroNOIineTM[INCI：甘油、水(水(Aqua))、葡聚糖、己酰基四肽-3(Caprooyl Tetrapeptide-3)]、[INCI：三肽-1]或4[INCI：水、葡聚糖、乙酰基四肽-2]，由Solabia销售的BioLyse T.A.[INCI：原低聚肽，由L-赖氨酸和L-精氨酸构成]或[INCI：精氨酸/赖氨酸多肽]，由Gattefosse销售的In-Tense[INCI：辛酸/癸酸甘油三酯、金钮扣(SpilanthesAcmella)花提取物]，由Mibelle Biochemistry销售的PhytoCellTecTM Malus Domestica[INCI：苹果(Malus Domestica)果实细胞培养物、黄原酸胶、甘油、卵磷脂、苯氧基乙醇、水]，由Evonik Goldschmidt销售的Phytosphingosine SLC[INCI：水杨酰植物鞘氨醇]，由Soliance销售的Dakaline[INCI：甜扁桃(Prunus Amygdalus)仁提取物、光滑果榆绿木(AnogeissusLeiocarpus)树皮提取物]，由Vincience销售的[INCI：六肽-9]、GP4G[INCI：卤虫(Artemia)提取物]、D’OrientineTM[INCI：枣椰子(Phoenix Dactylifera)(海枣(Date))籽提取物]或EderlineTM[INCI：苹果(Pyrus Malus)(苹果(Apple))果实提取物]，或由Provital销售的Homeostatine[INCI：扁浒苔(Enteromorpha Compressa)、刺云实(Caesalpinia Spinosa)]。
可选地，本发明的上述美容组合物或药物组合物，可含有美容上或药学上有效量的至少一种合成化合物、植物提取物、或通过生物技术方法获得的产物，其是弹性蛋白酶抑制剂，例如且不限于，由Pentapharm/DSM销售的[INCI：野大豆(Glycine Soja)(大豆)蛋白]、[INCI：野大豆(大豆)蛋白，氧化还原酶]或-Age[INCI：水解米糠蛋白、野大豆(大豆)蛋白，氧化还原酶]，由Coletica/Engelhard/BASF销售的Juvenesce[INCI：乙氧基二甘醇和辛酸甘油三酯、视黄醇、熊果酸、维生素K1、伊洛马司他(Ilomastat)]、MicromerolTM[INCI：苹果(Pyrus Malus)提取物]、石南提取物[INCI：帚石南(Calluna Vulgaris)提取物]、[INCI：水解马铃薯蛋白]或FlavagrumTMPEG[INCI：PEG-6异硬脂酸酯、橙皮素月桂酸酯]，由Laboratoires Sérobiologiques/Cognis销售的TP LS8657[INCI：豌豆(Pisum Sativum)提取物]，由SEPPIC销售的Sepilift DPHP[INCI：二棕榈酰羟脯氨酸]，由Rahn销售的[INCI：醇、水、甘油、水解米蛋白、枸骨叶冬青(Ilex Aquifolium)提取物、熊果酸钠，齐墩果酸钠]，由Gattefosse销售的Age Defense 2[INCI：胡桃(Juglans Regia)(胡桃(Walnut))子提取物]，由IEB和Atrium销售的IP 2000[INCI：葡聚糖、三氟乙酰三肽-2]，由Solabia销售的Radicaptol[INCI：丙二醇、水、西番莲花(PassifloraIncarnata Flower)提取物、黑茶镳子(Ribes Nigrum)(黑醋栗)叶提取物、葡萄(VitisVinifera)(葡萄(grape))叶提取物]或由Soliance销售的ViaPureTMBoswellia[INCI：Olivanum(齿叶乳香树)提取物]等。
可选地，本发明的上述美容组合物或药物组合物，可含有美容上或药学上有效量的至少一种合成化合物、植物提取物、或通过生物技术方法获得的产物，其是活性羰基类物质清除剂、自由基清除剂和/或抗糖化剂，例如并且不限于，肌肽及其衍生物、GHK[INCI：三肽-1]及其盐和/或衍生物、或由Lipotec销售的[INCI：水解小麦蛋白、水解大豆蛋白、三肽-1]或PreventheliaTM[INCI：二氨基丙酰三肽-33]等。
可选地，本发明的上述美容组合物或药物组合物，可含有美容上或药学上有效量的至少一种提取物，其是抗皱剂和/或抗老化剂，例如并且不限于，葡萄、犬蔷薇(Rosa canina)、姜黄(Curcuma longa)、香根鸢尾(Iris pallida)、可可树(Theobroma cacao)、银杏、高山火绒草(LeontopodiumAlpinum)或盐生杜氏藻(Dunaliellasalina)等的提取物、或至少一种合成化合物或产物，其是抗皱剂或抗老化剂，例如并且不限于，由Sederma/Croda销售的[INCI：棕榈酰五肽-3]、Matrixyl[INCI：棕榈酰四肽-7、棕榈酰低聚肽]、EssenskinTM[INCI：羟基甲硫氨酸钙]、Renovage[INCI：替普瑞酮]或[INCI：棕榈酰低聚肽]，由Pentapharm/D SM销售的 [INCI：五肽-3]、Syn[INCI：二肽二氨基丁酰苄基酰胺二乙酸盐]、-Coll[INCI：棕榈酰三肽-5]、Phytaluronate[INCI：刺槐豆(长角豆)胶]或[INCI：野大豆(大豆)蛋白、氧化还原酶]，由Laboratoires Sérobiologiques/Cognis销售的MyoxinolTM[INCI：水解咖啡黄葵(Hibiscus Esculentus)提取物]、SyniorageTM[INCI：乙酰基四肽-11]、DermicanTM[INCI：乙酰基四肽-9]或DN-AGETMLS[INCI：翅荚决明(Cassia Alata)叶提取物]，由Exsymol销售的Algisum[INCI：甲基硅烷醇甘露糖醛酸酯]或Hydroxyprolisilane[INCI：甲基硅烷醇羟脯氨酸酯天冬氨酸酯(Methylsilanol Hydroxyproline Aspartate)]，由Lipotec销售的[INCI：乙酰基六肽-8]、SNAP-7[INCI：乙酰基七肽-4]、SNAP-8[INCI：乙酰基八肽-3]、[INCI：五肽18]、InylineTM[INCI：(建议)乙酰基六肽-30]、[INCI：水解小麦蛋白、水解大豆蛋白、三肽-1]、PreventheliaTM[INCI：二氨基丙酰基三肽-33]、DecorinylTM[INCI：三肽-10瓜氨酸]、TrylagenTM[INCI：假交替单胞菌(Pseudoalteromonas)发酵提取物、水解小麦蛋白、水解大豆蛋白、三肽-10瓜氨酸、三肽-1]；[INCI：乙酰基四肽-5]、Peptide AC29[INCI：乙酰基三肽-30瓜氨酸]、Lipochroman-6[INCI：二甲基甲氧基色原烷醇]、ChromabrightTM[INCI：二甲基甲氧基色满基棕榈酸酯]、VilasteneTM[INCI：盐酸赖氨酸、卵磷脂、三肽-10瓜氨酸]、dGlyAGETM[INCI：盐酸赖氨酸、卵磷脂、三肽-9瓜氨酸]或[INCI：假交替单胞菌发酵提取物]，由InstitutEuropeen de BiologieCellulaire销售的[INCI：三肽-1、葡聚糖]，由Vincience销售的IS[INCI：六肽-9]、OrsirtineTM GL[INCI：稻(Oryza Sativa)(稻米)提取物]、D′OrientineTM IS[INCI：枣椰子(海枣)籽提取物]、PhytoquintescineTM[INCI：单粒小麦(Einkorn)(一粒小麦(Triticum monococcum))提取物]或QuintescineTM IS[INCI：二肽-4]，由InfinitecActivos销售的BONT-L Peptide[INCI：棕榈酰六肽-19]，由Seppic销售的DeepalineTM PVB[INCI：棕榈酰水解小麦蛋白]或DPHP[INCI：二棕榈酰羟脯氨酸]，由Gattefossé销售的Expression[INCI：千日菊(Acmella oleracea)提取物]、In-Tense[INCI：金钮扣花提取物]或Age Defense 2[INCI：胡桃(Juglans Regia)(胡桃(Walnut))子提取物]，由Biotechmarine销售的ThalassineTM[INCI：藻类提取物]，由Atrium Innovations/Unipex Group销售的ChroNOlineTM[INCI：己酰基四肽-3]或Thymulen-4[INCI：乙酰基四肽-2]，由Coletica/Engelhard/BASF销售的EquiStat[INCI：苹果果实提取物、野大豆籽提取物]或Juvenesce[INCI：乙氧基二甘醇和辛酸甘油三酯、视黄醇、熊果酸、维生素K1、伊洛马司他]，由Mibelle Biochemistry销售的Ameliox[INCI：肌肽、生育酚、水飞蓟(SilybumMarianum)果 实提取物]或PhytoCellTec Malus Domestica[INCI：苹果果实细胞培养物]，由Silab销售的Bioxilift[INCI：茴芹(Pimpinella Anisum)提取物]或SMS Anti-[INCI：番荔枝籽提取物]，Ca 2+通道拮抗剂例如并且不限于，阿尔维林(alverine)、锰盐或镁盐、某些仲胺或叔胺、视黄醇及其衍生物、和艾地苯醌(idebenone)及其衍生物，辅酶Q10及其衍生物，乳香酸及其衍生物，GHK及其衍生物和/或盐，肌肽及其衍生物，DNA修复酶例如并且不限于，光解酶或T4内切核酸酶V，或氯离子通道拮抗剂等。
可选地，本发明的上述美容组合物或药物组合物，可含有美容上或药学上有效量的至少一种合成化合物、植物提取物、或通过生物技术方法获得的产物，其是刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂和/或协助再上皮化的剂，例如并且不限于，积雪草(Centellaasiatica)提取物、麝香玫瑰(Rosa moschata)提取物、狭叶松果菊(Echinacea angustifolia)提取物、聚合草(Symphytum officinal)提取物、问荆(Equisetum arvense)提取物、贯叶金丝桃(Hypericum perforatum)提取物、细花含羞草(Mimosa tenuiflora)提取物、芦荟(Aloe vera)提取物、由Provital销售的Epithelizing[INCI:金盏菊(Calendula officinalis)、金丝桃、母菊(Chamomilla recutita)、迷迭香(Rosmarinus officinalis)]、由Laboratories Serobiologiques/Cognis销售的LS9028[INCI:水解酪蛋白、水解酵母蛋白、盐酸赖氨酸]、由Coletica/Engelhard/BASF销售的[INCI:玉蜀黍(Zea mays)(玉米)谷粒提取物]、钙粘蛋白、整联蛋白、选择蛋白、透明质酸受体、免疫球蛋白、成纤维细胞生长因子、结缔组织生长因子、血小板衍生的生长因子、血管内皮生长因子、表皮生长因子、胰岛素样生长因子、角质细胞生长因子、集落刺激因子、转化生长因子-β、肿瘤坏死因子-α、干扰素、白介素、基质金属蛋白酶、蛋白质酪氨酸磷酸酶受体、由Lipotec销售的[INCI:假交替单胞菌发酵提取物]或[INCI:三肽-10瓜氨酸]、或其混合物。
此外，本发明的上述美容组合物或药物组合物可以包含止痛化合物和/或抗炎化合物或可以与止痛化合物和/或抗炎化合物共同给予，以便减少与敏感性皮肤和愈合过程有关的肿胀和刺激，或者用于治疗过度生长的疤痕或瘢痕。在所述化合物中，可以着重强调的是类固醇型化合物例如氢化可的松、非类固醇型化合物例如对乙酰氨基酚或乙酰水杨酸、或具有固有的止痛和抗炎活性的天然提取物或精油。
可选地，所述美容或药物组合物的制剂选自：霜剂、油、奶、香膏、泡沫、洗剂、凝胶、擦剂、浆液、皂、洗发精、润发乳、血清、软膏、摩丝、润发油、粉末、杆剂、笔剂、喷雾 剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、糖浆剂、酏剂、多糖薄膜、胶冻或明胶；
可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
可选地，所述片剂包括：糖衣片剂。
本发明的美容组合物或药物组合物可以包括增加本发明的肽吸收的透皮促进剂，例如并且不限于二甲亚砜、二甲基乙酰胺、二甲基甲酰胺、表面活性剂、氮酮(1-十二烷基氮杂环庚-2-酮)、醇、脲、乙氧基二甘醇、丙酮、丙二醇或聚乙二醇等。
此外，本发明的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或所述的美容或药物组合物，可以通过离子电渗疗法、超声促渗、电穿孔、微电贴剂、机械压迫、渗透压梯度、包扎疗法、显微注射或借助于压力的无针注射(例如通过氧气压力的注射)、或其任何组合而被施用至有待治疗的局部区域，以便实现本发明的肽的更大渗透。该施用区域将是由有待治疗和/或护理的病状、病症和/或疾病的性质来确定。
此外，本发明所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述的美容或药物组合物可以被结合至任何形式的功能性食品或强化食品中，例如并且不局限于，膳食条或压缩或非压缩粉末中。这些粉末可以溶解于水、果汁、苏打水、乳制品、大豆衍生物、或可以被结合至膳食条中。本发明的这些肽可以与用于口服组合物或食品补充剂的常见赋形剂或佐剂一起配制，例如并且不局限于，在食品工业中常见的脂肪组分、水性组分、保湿剂、防腐剂、增稠剂、调味剂、香味剂、抗氧化剂、或着色剂。
本发明的肽根据它们的序列的性质或N-末端和/或C-末端中的任何可能的修饰，在水中具有可变的溶解度。因此本发明的肽可以通过水溶液掺入组合物中，且不溶于水的那些可溶解于美容上或药学上可接受的常规溶剂中，所述溶剂例如并且不限于乙醇、丙醇、异丙醇、丙二醇、甘油、丁二醇或聚乙二醇或其任何组合。
待施用的美容上或药学上有效量的本发明的肽以及它们的剂量将依赖于许多因素，包括年龄、患者的状态、病症或疾病的严重性、施用的途径和频率以及待使用的肽的具体性质。
“美容上或药学上有效量”意指无毒性的但足以提供希望的效果的本发明的一种或多种肽的量。在本发明的美容组合物或药物组合物中以获得希望的效果的美容上或药学上有效的 浓度使用本发明的肽；在一个优选形式中，相对于组合物的总重量，在0.00000001％(按重量计)和20％(按重量计)之间，优选在0.000001％(按重量计)和15％(按重量计)之间、更优选在0.0001％(按重量计)和10％(按重量计)之间，并且甚至更优选在0.0001％(按重量计)和5％(按重量计)之间。
本发明另一方面，提供一种美容上或药学上可接受的递送系统或缓释系统，以便实现有效成分的更好渗透和/或改进它的药物代谢动力学和药效动力学特性，其包含有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述的美容或药物组合物；
术语“递送系统”是指与本发明的肽一起施用的稀释剂、佐剂、赋形剂或载体，它们选自：水、油或表面活性剂、包括石油来源、动物来源、植物来源、或合成来源的那些，例如并且不限于花生油、大豆油、矿物油、芝麻油、蓖麻油、聚山梨醇酯、脱水山梨糖醇酯、醚硫酸酯、硫酸酯、甜菜碱、葡萄糖苷、麦芽糖苷、脂肪醇、壬苯醇醚、泊洛沙姆、聚氧乙烯、聚乙二醇、右旋糖、甘油、毛地黄皂苷和类似物。本领域的普通技术人员已知在可以给予本发明的肽的不同递送系统中可以使用的稀释剂。
术语“缓释”以常规含义使用，指提供化合物在一段时间内逐渐释放的化合物的递送系统，且优选地但不是必须地，在整个时间段内具有相对恒定的化合物释放水平。
递送系统或缓释系统的实例是脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳米粒子。优选的递送系统或缓释系统是脂质体和微米乳液，更优选具有反胶束的内部结构的油包水型微米乳液。
缓释系统可以通过现有技术中已知的方法来制备，并且可以例如通过以下方式来给予：通过局部或经皮给药，包括粘附贴剂、非粘附贴剂、封闭贴剂、以及微电子贴剂；或通过全身给药例如并且不局限于，口服或胃肠外途径，包括鼻、直肠、皮下植入或注射、或直接植入或注射至特定身体部位中，并且优选地应该释放相对恒定量的本发明的这些肽。在该缓释系统中包含的肽的量将取决于例如该组合物将被给予的部位、本发明的肽的释放动力学和持续时间、以及有待治疗和/或护理的病状、病症和/或疾病的性质。
本发明另一方面，提供一种美容上或药学上可接受的固体有机聚合物或固体无机支撑 体，其包含有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述的美容或药物组合物；其选自，但不局限于：滑石、膨润土、二氧化硅、淀粉、或麦芽糖糊精等。
本发明另一方面，提供一种化妆品，其包含有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述的美容或药物组合物；
可选地，所述化妆品选自：遮瑕膏、粉底、卸妆液、卸妆乳、眼影、唇棒、唇彩、护唇膏或唇粉。
本发明另一方面，提供一种织物、无纺织物或医学装置，其包含有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述的美容或药物组合物。由于织物或无纺织物或医学装置的锚定系统的生物降解或通过与身体的摩擦、通过身体水分、通过皮肤的pH或通过体温来释放本发明的肽。此外，织物和无纺织物可用于制造与身体直接接触的衣服。
织物、无纺织物、衣服、医学装置以及将肽固定到它们的方式的实例包括上述的递送系统和/或缓释系统，可以在文献中找到且是现有技术已知的[Schaab C.K.(1986)“Impregnating Fabrics With Microcapsules(利用微胶囊浸渍织物)”，HAPPI May 1986；Nelson G.(2002)“Application of microencapsulation in textiles(微囊化在纺织品中的应用)”Int.J.Pharm.242：5562；“Biofunctional Textiles and the Skin(生物功能性纺织品和皮肤)”(2006)Curr.Probl.Dermatol.v.33，Hipler U.C.和Elsner P.编辑。S.Karger A.G.，Basel，Switzerland；Malcom R.K.；McCullagh S.D.，Woolfson A.D.，Gorman S.P.，Jones D.S.和Cuddy J.(2004)“Controlled release of a model antibacterial drug from a novel self lubricating silicone biomaterial(来自新颖的自润滑硅酮生物材料的抗菌药物模型的控制释放)”J.Cont.Release97：313320]。优选的织物、无纺织物、衣服或医学装置是绷带、纱布、t-恤衫、短袜、裤袜、内衣、腰带、手套、尿布、卫生巾、敷裹物、床罩、擦拭巾、粘附贴剂、非粘附贴剂、封闭贴剂、微电子贴剂或面膜。
本发明的另一个方面，提供一种上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物，或上述的美容上或药学上可接受的递送系统或缓释系统，或上述的美容上或药学上可接受的 固体有机聚合物或固体无机支撑体，或上述的化妆品，或上述的织物、无纺织物或医学装置在制备用于治疗或护理皮肤或粘膜的美容组合物或药用组合物中的用途。
本发明的另一个方面，提供一种上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物，或上述的美容上或药学上可接受的递送系统或缓释系统，或上述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或上述的化妆品，或上述的织物、无纺织物或医学装置在制备用于治疗或护理体表创伤、烧伤、皮肤溃疡、减少疤痕产生、或加快疤痕修复的美容组合物或药物组合物中的用途。
本发明的另一个方面，提供一种上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物，或上述的美容上或药学上可接受的递送系统或缓释系统，或上述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或上述的化妆品，或上述的织物、无纺织物或医学装置在制备用于促进成纤维细胞增殖、增加胶原蛋白生成、用于改善皮肤屏障功能、或用于皮肤或粘膜的再上皮化或愈合的美容组合物或药物组合物中的用途。
本发明的一个方面，提供一种上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物，或上述的美容上或药学上可接受的递送系统或缓释系统，或上述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或上述的化妆品，或上述的织物、无纺织物或医学装置在制备用于治疗、预防或修复皮肤老化或光老化的美容组合物或药物组合物中的用途。
可选地，所述皮肤老化或光老化的治疗、预防或修复是减少、预防或治疗面部皱纹。
本发明另一个方面，提供一种用于治疗或护理皮肤或粘膜的方法，该方法包括给予美容上或药学上有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物。
本发明另一个方面，提供一种用于治疗或护理体表创伤、烧伤、皮肤溃疡、减少疤痕产生、加快疤痕修复的方法，该方法包括给予美容上或药学上有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物。
本发明另一个方面，提供一种用于促进成纤维细胞增殖、增加胶原蛋白生成、用于改善 皮肤屏障功能、或用于皮肤和/或粘膜的再上皮化或愈合的方法，该方法包括给予美容上或药学上有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物。
本发明另一个方面，提供一种用于治疗、预防或修复皮肤老化或光老化的方法，该方法包括给予美容上或药学上有效量的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物。
皮肤老化或光老化的治疗、预防或修复是减少、预防或治疗面部皱纹。
含有本发明的上述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或上述美容或药物组合物的组合物可以被施用至皮肤和/或粘膜、或根据需要被口服地或胃肠外给予以便治疗和/或护理一种病状、病症和/或疾病。
施用或给药频率可以变化很大，这取决于每个受试者的需要，其中建议的施用或给药范围从每月1次至每天10次，优选从每周1次至每天4次，更优选从每周3次至每天3次，甚至更优选每天1次或2次。
定义
为了便于理解本发明，对在本发明所使用的一些术语和表述的含义说明如下：
在本发明中，术语“皮肤”应理解为是构成它的多个层，从最上层或角质层至最下层或皮下组织，两个端点都包括在内。这些层由不同类型的细胞组成，如角质形成细胞、成纤维细胞、黑色素细胞、和/或脂肪细胞等。在本发明中，术语“皮肤”包括头皮。
术语“治疗”，指的是给予根据本发明的肽以减轻或消除一种疾病或病症、或减少或消除与这种疾病或病症相关的一种或多种症状。术语“治疗”还涵盖了减轻或消除该疾病或病症的生理后果的能力。
术语“护理”包括疾病和/或病症的预防。
术语“预防”，指的是本发明的肽在一种疾病或病症出现前防止、延迟、或阻碍其出现或发展的能力。
术语“老化”指的是皮肤随着年龄的增长经历的变化(自然老化)，或通过暴露于阳光(光老化)或暴露于环境污染物，如化学污垢或污染物、烟草烟雾等而经历的变化，并且包括所有外在可见的和/或通过触摸可感知的变化，例如并且不局限于：皮肤上的不连续性的 发展(如皱纹、细纹、表情纹、拉伸纹、条纹、沟纹、不平整或粗糙、毛孔尺寸增大、水分损失、弹性损失、紧致性损失、平滑性损失、变形恢复能力损失、回弹性损失)、皮肤下垂(如脸颊下垂、眼睛下方出现眼袋、或出现双下巴等)、皮肤颜色的变化(如瘢痕、变红、眼袋、或出现色素过度沉着区域如老年斑或雀斑等)、异常分化、过度角质化、弹性组织变性、角化症、脱发、橘皮样皮肤、胶原结构损失，以及角质层、真皮、表皮、血管系统(例如出现蜘蛛静脉或毛细血管扩张症)或靠近皮肤的那些组织的其他组织学变化。
术语“光老化”指的是由于皮肤长期暴露于紫外线辐射而导致的皮肤过早老化，它呈现出与自然老化相同的生理特征，例如并且不局限于：松弛、下垂、颜色改变或色素沉着不规则、异常和/或过度角质化。
在本说明书中，用于氨基酸的缩写遵循IUPAC-IUB生化命名委员会(IUPAC-IUBCommission of Biochemical Nomenclature)在欧洲生物化学杂志(Eur.J.Biochem.1984,138:9-37)中所指定的规则。
因此，例如，Ala表示NH 2-CH(CH 3)-COOH，Ala-表示NH 2-CH(CH 3)-CO-，-Ala表示-NH-CH(CH 3)-COOH，并且-Ala-表示-NH-CH(CH 3)-CO-。因此，表示肽键的连字符消除了当位于该符号的右侧时的氨基酸(在此用常规非离子化形式来表示)1-羧基中的OH，并且消除了当位于该符号的左侧时的氨基酸2-氨基中的H；两种修饰可以应用于同一个符号(见表1)。
表1氨基酸残基的结构以及它们的单字母和三字母缩写符号
Figure PCTCN2021112345-appb-000013
Figure PCTCN2021112345-appb-000014
缩写“Ac-”在本发明中用来表示乙酰基(CH 3-CO-)，并且缩写“Palm-”用来表示棕榈酰基(CH 3-(CH 2) 14-CO-)。
本发明相对于现有技术所取得的有益效果包括：
本发明所述的肽通过人工设计得到，合成方便，能够促进成纤维细胞增殖，改善皮肤屏障功能，有利于促进伤口愈合，治疗或护理体表创伤、烧伤、皮肤溃疡、减少疤痕产生、加快疤痕修复，亦可用于治疗、预防和/或修复皮肤老化和/或光老化。
附图说明
图1是肽(1)～肽(5)对HaCaT细胞活性的影响图。*表示给药组与空白对照组相比具有统计学差异，p<0.05(n＝4)。**表示给药组与空白对照组相比差异显著，p<0.01(n＝4)。
图2是肽(1)～肽(5)对NIH3T3细胞活性的影响图。*表示给药组与空白对照组相比具有统计学差异，p<0.05(n＝4)。**表示给药组与空白对照组相比差异显著，p<0.01(n＝4)。
图3是肽(1)及肽(2)对HaCaT细胞黏附的影响图。*表示给药组与空白对照组相比具有统计学差异，p<0.05(n＝4)。**表示给药组与空白对照组相比差异显著，p<0.01(n＝4)。***表示给药组与空白对照组相比差异极为显著，p<0.001(n＝4)。
图4是HaCaT细胞划痕实验结果图。
图5是肽(1)～肽(5)对光损伤HaCaT细胞的修复作用图。###表示与非辐射的正常对照组相比，差异极为显著，p<0.001(n＝4)。*表示与辐射后的对照组相比，具有统计学差异，p<0.05(n＝4)。**表示与辐射后的对照组相比，差异显著，p<0.01(n＝4)。***表示与辐射后的对照组相比，差异极为显著，p<0.001(n＝4)。
图6是肽(1)及肽(2)对光损伤NIH3T3细胞的修复作用图。###表示与非辐射的正常对照组相比，差异极为显著，p<0.001(n＝4)。*表示与辐射后的对照组相比，具有统计学差异，p<0.05(n＝4)。**表示与辐射后的对照组相比，差异显著，p<0.01(n＝4)。***表示与辐射后的对照组相比，差异极为显著，p<0.001(n＝4)。
图7是肽(2)对TNF-α分泌的影响图。###表示与control组相比，差异极为显著，p<0.001(n＝4)。***表示与LPS组相比，差异极为显著，p<0.001(n＝4)。
图8是肽(2)对IL-6分泌的影响图。###表示与control组相比，差异极为显著，p<0.001(n＝4)。***表示与LPS组相比，差异极为显著，p<0.001(n＝4)。
图9是肽(2)对IL-8分泌的影响图。##表示与control组相比，差异显著，p<0.01(n＝4)。**表示与LPS组相比，差异显著，p<0.01(n＝4)。
具体实施方式
为了更好地理解本发明，下面结合实施例及附图对本发明作详细的说明，然而，应当理解的是，这些实施例及附图仅用作说明目的，并且不旨在限制本发明的范围。
缩写
用于氨基酸的缩写遵循IUPAC-IUB的生物化学命名委员会在Eur J.Biochem.(1984)138：9-37和J.Chem(1989)264：633-673中指定的规则。
Wang Resin：王树脂；DMF：N,N-二甲基甲酰胺；DCM：二氯甲烷；DIC：二异丙基碳二亚胺；Ac 2O：乙酸酐；DIPEA：二异丙基乙胺；Fmoc：9-芴基甲氧羰基；piperidine：哌啶；HOBt：1-羟基苯并三氮唑；TFA：三氟乙酸；TIS：三异丙基硅烷；EDT：1,2-乙二硫醇；DMSO：二甲基亚砜；Ac：乙酰基；Palm：棕榈酰基；Ala：丙氨酸；Pro：脯氨酸；Phe：苯丙氨酸；Arg：精氨酸；Met：甲硫氨酸；Gly：甘氨酸；Ile：异亮氨酸；Cys：半胱氨酸；Thr：苏氨酸；Asn：天冬酰胺；Pbf：2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基；Trt：三苯甲 基(triphenylmethyl)或三苯甲基(Trityl)；tBu：叔丁基；Amide Resin：一种多肽合成用的起始树脂；Fmoc-linker：4-[(2,4-二甲氧基苯基)(Fmoc-氨基)甲基]苯氧乙酸。
实施例1
Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Cys(Trt)-Thr(tBu)-Thr(tBu)-Asn(Trt)-Wang Resin的制备。
1.1树脂的溶胀
称取Wang Resin 35g(49.7mmol)于2L固相合成反应柱中。加入DMF 100ml完全浸没树脂后通入氮气，开启氮气搅拌鼓泡，5min，后抽干溶剂5min。继续加入DMF 100ml，完全浸没树脂后通入氮气，30min，树脂完全溶胀，抽干溶剂。再加入DMF 100ml，完全浸没树脂后通入氮气，开启氮气搅拌鼓泡，抽干溶剂5min，溶胀结束。
1.2脱Fmoc
溶胀后的树脂用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。用DMF洗涤树脂7～8次，抽走溶剂。
1.3投料反应
称取53.38g的Fmoc-Asn(Trt)-OH(89.46mmol；1.8当量)，14.51g的HOBt加入干燥500ml圆底磨口烧瓶中。加入DMF使其溶解，放入±5℃度冰箱冷藏10min。加入20.78ml DIC活化10min，避免水汽。将活化后的氨基酸加入脱Fmoc保护后的树脂中反应2h，控制反应温度在25℃～35℃，抽走反应液。用DMF洗涤树脂，每次用量100ml，树脂和溶剂均匀混合后计时搅拌2min，抽干溶剂。用DMF洗涤树脂多次，直至抽滤出的溶剂澄清透明。
检测树脂替代度为0.5，计算合成规模为24.9mmol。
对N-末端Fmoc基团进行脱保护，并且在存在10.09g HOBt和14.46ml DIC的情况下，使用DMF作为溶剂，将活化后的24.74gFmoc-Thr(tBu)-OH(62.25mmol；2.5当量)偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在10.09g HOBt和14.46ml DIC的情况下，使用DMF作为溶剂，顺序地偶联24.74g的Fmoc-Thr(tBu)-OH(62.25mmol；2.5当量)；36.46g的Fmoc-Cys(Trt)-OH(62.25mmol；2.5当量)；22g的Fmoc-Ile-OH(62.25mmol；2.5当量)；18.51g的Fmoc-Gly-OH(62.25mmol；2.5当量)。
对N-末端Fmoc基团进行脱保护，并且在存在14.13g HOBt和20.24ml DIC的情况下， 使用DMF作为溶剂，将活化后的32.37gFmoc-Met-OH(87.15mmol；3.5当量)偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在14.13g HOBt和20.24ml DIC的情况下，使用DMF作为溶剂，顺序地偶联56.54g的Fmoc-Arg(Pbf)-OH(87.15mmol；3.5当量)；33.76g的Fmoc-Phe-OH(87.15mmol；3.5当量)；29.40g的Fmoc-Pro-OH(87.15mmol；3.5当量)以及随后27.13g的Fmoc-Ala-OH(87.15mmol；3.5当量)。
在该合成之后，用DMF洗涤树脂，每次用量100ml，树脂和溶剂均匀混合后计时搅拌2min，抽干溶剂。用DMF洗涤树脂多次，直至抽滤出的溶剂澄清透明。
实施例2
Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Cys(Trt)-Thr(tBu)-Thr(tBu)-Asn(Trt)-Linker-AmideResin的制备。
2.1 Fmoc-Linker-Amide Resin的制备
称取Amide Resin树脂50g(60mmol)于2L固相合成反应柱中。400ml DMF洗涤树脂一次，500ml DCM溶胀树脂20min，抽走溶剂。400ml DMF洗涤树脂3次。
称取Fmoc-linker 80.9g(150mmol)、HOBt 24.3g(180mmol)于干燥500ml广口三角瓶中。加约150ml DMF溶剂溶解后置于冰水浴中冷却2min，加DIC 30ml(195mmol)活化3min，避免水汽。将活化后的Fmoc-linker加入溶胀后的树脂中反应2h，抽走反应液。用400ml DMF洗涤树脂3次，每次2min。
加入Ac 2O 28ml(300mmol)、DIPEA 10.7ml(60mmol)、DMF 400ml混合液密封3h后，用DMF 500ml洗涤树脂四次，DCM 500ml洗涤树脂二次。抽走溶剂。
2.2脱Fmoc
Fmoc-Linker-Amide Resin用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。500ml DMF洗涤树脂4次，500ml DCM洗涤树脂2次。抽走溶剂。
2.3投料反应
称取52.21g的Fmoc-Asn(Trt)-OH(87.5mmol)，14g的HOBt加入干燥500ml广口三角瓶中。加入DMF使其溶解，置于冰水浴中冷却。加20ml DIC活化3min，避免水汽。将活化后的氨基酸加入脱保护后树脂中反应2h，抽走反应液。用500ml DMF洗涤树脂2次， 每次2min，400ml DCM洗涤树脂2次，抽走溶剂。
对N-末端Fmoc基团进行脱保护，并且在存在14g HOBt和20ml DIC的情况下，使用DMF作为溶剂，将活化后的34.78gFmoc-Thr(tBu)-OH(87.5mmol)偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在14g HOBt和20ml DIC的情况下，使用DMF作为溶剂，顺序地偶联34.78g的Fmoc-Thr(tBu)-OH(87.5mmol)；51.25g的Fmoc-Cys(Trt)-OH(87.5mmol)；30.92g的Fmoc-Ile-OH(87.5mmol)；26.01g的Fmoc-Gly-OH(87.5mmol)；32.50g的Fmoc-Met-OH(87.5mmol)；56.77g的Fmoc-Arg(Pbf)-OH(87.5mmol)；33.90g的Fmoc-Phe-OH(87.5mmol)；29.52g的Fmoc-Pro-OH(87.5mmol)以及随后27.24g的Fmoc-Ala-OH(87.5mmol)。
在该合成之后，用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例3
去除N-末端Fmoc保护基团。
将在实施例1和实施例2中所获得的这些肽基树脂的N-末端Fmoc基团脱保护，用500ml20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。用600ml DMF洗涤树脂4次，每次2min，500mlDCM洗涤树脂2次，抽走溶剂。
实施例4
将R 1棕榈酰基基团引入至实施例3中所获得的肽基树脂上。
在存在1.53g的HOBt(10mmol；10当量)和1.54mL的DIC(10mmol；10当量)的情况下，将预先溶解于1ml DMF中的2.56g的棕榈酸(10mmol；10当量)添加至1mmol的实施例3中所获得的肽基树脂上。允许它们反应15h，在此之后用600mlDMF洗涤树脂4次，每次2min，500mlDCM洗涤树脂2次，抽走溶剂。
实施例5
将R 1乙酰基基团引入至实施例3中所获得的肽基树脂上。
在存在25当量DIPEA的情况下、使用5mL的DMF作为溶剂，用25当量的Ac 2O处理1mmol的实施例3中所获得的肽基树脂。允许它们反应30min，在此之后用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例6
将实施例3、4、以及5中所获得的肽基树脂从聚合物支撑体上裂解。
用甲醇500ml，300ml，200ml收缩实施例3、4、以及5中所获得的肽基树脂三次。真空抽干。
称取1g所获得的干燥的实施例3、4、以及5的肽基树脂，用7ml的裂解液(TFA：EDT：苯甲醚：苯甲硫醚：TIS：H 2O＝87.5：2.5：2.5：2.5：2.5：2.5)处理，持续2.5h。用沙芯漏斗抽滤，1ml三氟乙酸洗树脂，将滤液缓慢滴加到冰冻的64ml异丙醚中，有大量白色固体析出，离心后用异丙醚洗6次。在真空下干燥最终的沉淀物。
实施例7
采用高效液相色谱仪进行制备纯化。
用甲醇与水溶解实施例6中所获得的粗肽，用孔径为0.45μm滤膜过滤。
纯化制备条件：
流动相A：纯乙腈；流动相B：0.1％醋酸水溶液；
检测波长：215nm；
色谱柱：C18-20*150mm；
洗脱梯度：
时间(min) 流速(ml/min) A％ B％
0 40 10 90
3 40 12 88
63 40 37 63
64 40 10 90
75 40 10 90
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到以下纯度超过90％的肽：
1、H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH；
2、H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2；
3、Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH，即肽(8)；
4、Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2，即肽(9)；
5、Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH，即肽(10)；
6、Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2，即肽(11)。
实施例8
将实施例7中所获得的肽进行氧化处理。
在250ml的梨形瓶中加入实施例7中所获得的肽，用80ml纯化水溶解，分别加入8g盐酸胍和20ml DMSO，敞口搅拌30h，通过HPLC监控反应过程。氧化完后加入4ml醋酸将析出物溶解，用0.22μm滤膜真空抽滤。
实施例9
采用高效液相色谱仪对实施例8所获得的滤液进行纯化。
纯化制备条件：
流动相A：纯乙腈；流动相B：0.1％醋酸水溶液；
检测波长：215nm；
色谱柱：C18-20*150mm；
洗脱梯度：
时间(min) 流速(ml/min) A％ B％
0 40 8 92
5 40 8 92
50 40 53 47
53 40 8 92
65 40 8 92
将滤液进样纯化，收集馏分，浓缩冻干，得到以下纯度超过95％的肽：
Figure PCTCN2021112345-appb-000015
Figure PCTCN2021112345-appb-000016
实施例10
将实施例7中所获得的肽的Cys8的侧链巯基进行烯丙基修饰。
取120mg实施例7中所获得的肽，溶于40ml水中，氮气保护，加入5倍量的溴丙烯，室温条件下反应，通过HPLC监控反应过程，直至反应完全，得到以下粗肽：
Figure PCTCN2021112345-appb-000017
Figure PCTCN2021112345-appb-000018
实施例11
将实施例7中所获得的肽的Cys8的侧链巯基进行溴乙酸叔丁酯修饰。
取120mg实施例7中所获得的肽，溶于40ml水中，氮气保护，加入5倍量的溴乙酸叔丁酯，室温条件下反应，通过HPLC监控反应过程，直至反应完全，得到以下粗肽：
Figure PCTCN2021112345-appb-000019
Figure PCTCN2021112345-appb-000020
实施例12
将实施例7中所获得的肽的Cys8的侧链巯基进行巯基乙烷修饰。
称取120mg实施例7中所获得的肽，溶于40ml水中，氮气保护，加入5倍量巯基乙烷，室温条件下反应，通过HPLC监控反应过程，直至反应完全，得到以下粗肽：
Figure PCTCN2021112345-appb-000021
Figure PCTCN2021112345-appb-000022
实施例13
将实施例7中所获得的肽的Cys8的侧链巯基进行己硫醇修饰。
称取120mg实施例7中所获得的肽，溶于40ml水中，氮气保护，加入5倍量己硫醇，室温条件下反应，通过HPLC监控反应过程，直至反应完全，得到以下粗肽：
Figure PCTCN2021112345-appb-000023
Figure PCTCN2021112345-appb-000024
实施例14
将实施例7中所获得的肽的Cys8的侧链巯基进行溴乙酸修饰。
称取120mg实施例7中所获得的肽，溶于40ml水中，氮气保护，加入5倍量溴乙酸，室温条件下反应，通过HPLC监控反应过程，直至反应完全，得到以下粗肽：
Figure PCTCN2021112345-appb-000025
Figure PCTCN2021112345-appb-000026
实施例15
采用高效液相色谱仪对实施例10-14所获得的产物进行纯化。
纯化制备条件：
流动相A：纯乙腈；流动相B：0.1％醋酸水溶液；
检测波长：215nm；
色谱柱：C18-20*150mm；
洗脱梯度：
时间(min) 流速(ml/min) A％ B％
0 40 11 89
48 40 35 65
56 40 38 62
59 40 48 52
60 40 80 20
70 40 80 20
进样纯化，收集馏分，浓缩冻干，得到纯度超过80％的肽。
实施例16
Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Met-Thr(tBu)-Thr(tBu)-Asn(Trt)-Wang Resin的制备。
16.1树脂的溶胀
称取Wang Resin35g(49.7mmol)于2L固相合成反应柱中。加入DMF 100ml完全浸没树脂后通入氮气，开启氮气搅拌鼓泡，5min，后抽干溶剂5min。继续加入DMF 100ml，完全浸没树脂后通入氮气，30min，树脂完全溶胀，抽干溶剂。再加入DMF 100ml，完全浸没树脂后通入氮气，开启氮气搅拌鼓泡，抽干溶剂5min，溶胀结束。
16.2脱Fmoc
溶胀后的树脂用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。用DMF洗涤树脂7～8次，抽走溶剂。
16.3投料反应
称取53.38g的Fmoc-Asn(Trt)-OH(89.46mmol；1.8当量)，14.51g的HOBt加入干燥500ml圆底磨口烧瓶中。加入DMF使其溶解，放入±5℃度冰箱冷藏10min。加入20.78ml DIC活化10min，避免水汽。将活化后的氨基酸加入脱Fmoc保护后的树脂中反应2h，控制反应温度在25℃～35℃，抽走反应液。用DMF洗涤树脂，每次用量100ml，树脂和溶剂均匀混合后计时搅拌2min，抽干溶剂。用DMF洗涤树脂多次，直至抽滤出的溶剂澄清透明。
检测树脂替代度为0.5，计算合成规模为24.9mmol。
对N-末端Fmoc基团进行脱保护，并且在存在10.09g HOBt和14.46ml DIC的情况下，使用DMF作为溶剂，将活化后的24.74g Fmoc-Thr(tBu)-OH(62.25mmol；2.5当量)偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在10.09g HOBt和14.46ml DIC的情况下，使用DMF作为溶剂，顺序地偶联24.74g的Fmoc-Thr(tBu)-OH(62.25mmol；2.5当量)；23.12g的Fmoc-Met-OH(62.25mmol；2.5当量)；22g的Fmoc-Ile-OH(62.25mmol；2.5当量)；18.51g的Fmoc-Gly-OH(62.25mmol；2.5当量)。
对N-末端Fmoc基团进行脱保护，并且在存在14.13g HOBt和20.24ml DIC的情况下，使用DMF作为溶剂，将活化后的32.37gFmoc-Met-OH(87.15mmol；3.5当量)偶联至肽基 树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在14.13g HOBt和20.24ml DIC的情况下，使用DMF作为溶剂，顺序地偶联56.54g的Fmoc-Arg(Pbf)-OH(87.15mmol；3.5当量)；33.76g的Fmoc-Phe-OH(87.15mmol；3.5当量)；29.40g的Fmoc-Pro-OH(87.15mmol；3.5当量)以及随后27.13g的Fmoc-Ala-OH(87.15mmol；3.5当量)。
在该合成之后，用DMF洗涤树脂，每次用量100ml，树脂和溶剂均匀混合后计时搅拌2min，抽干溶剂。用DMF洗涤树脂多次，直至抽滤出的溶剂澄清透明。
实施例17
Fmoc-Ala-Pro-Phe-Arg(Pbf)-Met-Gly-Ile-Met-Thr(tBu)-Thr(tBu)-Asn(Trt)-Linker-AmideResin的制备。
17.1 Fmoc-Linker-Amide Resin的制备
称取Amide Resin树脂50g(60mmol)于2L固相合成反应柱中。400ml DMF洗涤树脂一次，500ml DCM溶胀树脂20min，抽走溶剂。400ml DMF洗涤树脂3次。
称取Fmoc-linker 80.9g(150mmol)、HOBt 24.3g(180mmol)于干燥500ml广口三角瓶中。加约150ml DMF溶剂溶解后置于冰水浴中冷却2min，加DIC 30ml(195mmol)活化3min，避免水汽。将活化后的Fmoc-linker加入溶胀后的树脂中反应2h，抽走反应液。用400ml DMF洗涤树脂3次，每次2min。
加入Ac 2O 28ml(300mmol)、DIPEA 10.7ml(60mmol)、DMF 400ml混合液密封3h后，用DMF 500ml洗涤树脂四次，DCM 500ml洗涤树脂二次。抽走溶剂。
17.2脱Fmoc
Fmoc-Linker-Amide Resin用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。500ml DMF洗涤树脂4次，500ml DCM洗涤树脂2次。抽走溶剂。
17.3投料反应
称取52.21g的Fmoc-Asn(Trt)-OH(87.5mmol)，14g的HOBt加入干燥500ml广口三角瓶中。加入DMF使其溶解，置于冰水浴中冷却。加20ml DIC活化3min，避免水汽。将活化后的氨基酸加入脱保护后树脂中反应2h，抽走反应液。用500ml DMF洗涤树脂2次，每次2min，400ml DCM洗涤树脂2次，抽走溶剂。
对N-末端Fmoc基团进行脱保护，并且在存在14g HOBt和20ml DIC的情况下，使用DMF作为溶剂，将活化后的34.78g Fmoc-Thr(tBu)-OH(87.5mmol)偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在14g HOBt和20ml DIC的情况下，使用DMF作为溶剂，顺序地偶联34.78g的Fmoc-Thr(tBu)-OH(87.5mmol)；32.50g的Fmoc-Met-OH(87.5mmol)；30.92g的Fmoc-Ile-OH(87.5mmol)；26.01g的Fmoc-Gly-OH(87.5mmol)；32.50gFmoc-Met-OH(87.5mmol)；56.77g的Fmoc-Arg(Pbf)-OH(87.5mmol)；33.90g的Fmoc-Phe-OH(87.5mmol)；29.52g的Fmoc-Pro-OH(87.5mmol)以及随后27.24g的Fmoc-Ala-OH(87.5mmol)。
在该合成之后，用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例18
去除N-末端Fmoc保护基团。
将在实施例16和实施例17中所获得的这些肽基树脂的N-末端Fmoc基团脱保护，用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例19
将R 1棕榈酰基基团引入至实施例18中所获得的肽基树脂上。
在存在1.53g的HOBt(10mmol；10当量)和1.54mL的DIC(10mmol；10当量)的情况下，将预先溶解于1ml DMF中的2.56g的棕榈酸(10mmol；10当量)添加至1mmol的实施例18中所获得的肽基树脂上。允许它们反应15h，在此之后用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例20
将R 1乙酰基基团引入至实施例18中所获得的肽基树脂上。
在存在25当量DIPEA的情况下，使用5mL的DMF作为溶剂，用25当量的Ac 2O处理1mmol的实施例18中所获得的肽基树脂。允许它们反应30min，在此之后用600ml DMF洗涤树脂4次，每次2min，500ml DCM洗涤树脂2次，抽走溶剂。
实施例21
将实施例18、19以及20中所获得的肽基树脂从聚合物支撑体上裂解。
用甲醇500ml，300ml，200ml收缩实施例18、19以及20中所获得的肽基树脂三次。真空抽干。
称取1g所获得的干燥的实施例18、19以及20的肽基树脂，用7ml的裂解液(TFA：EDT：苯甲醚：苯甲硫醚：TIS：H 2O＝87.5：2.5：2.5：2.5：2.5：2.5)处理，持续2.5h。用沙芯漏斗抽滤，1ml三氟乙酸洗树脂，将滤液缓慢滴加到冰冻的64ml异丙醚中，有大量白色固体析出，离心后用异丙醚洗6次。在真空下干燥最终的沉淀物。
实施例22
采用高效液相色谱仪进行制备纯化。
用甲醇与水溶解实施例21中所获得的粗肽，用孔径为0.45μm滤膜过滤。
纯化制备条件：
流动相A：纯乙腈；流动相B：0.1％醋酸水溶液；
检测波长：215nm；
色谱柱：C18-20*150mm；
洗脱梯度：
时间(min) 流速(ml/min) A％ B％
0 40 10 90
4 40 12 88
64 40 32 68
65 40 10 90
75 40 10 90
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到以下纯度超过90％的肽：
43、H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH，即肽(2)；
44、H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2，即肽(42)；
45、Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH，即肽(43)；
46、Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2，即肽(44)；
47、Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH，即肽(45)；
48、Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2，即肽(46)。
实施例23
本发明通式(I)中的其他化合物可以通过类似的方法制备。
所获得的这些肽通过ESI-MS测定其分子量，部分化合物的测试结果见下表2。
表2质谱法测定分子量
Figure PCTCN2021112345-appb-000027
Figure PCTCN2021112345-appb-000028
实施例24细胞增殖实验
24.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、噻唑蓝(MTT)(Sigma)、二甲基亚砜(DMSO)(Sigma)、高糖培养基(DMEM)(Gibco)、胎牛血清(Gibco)。
24.2仪器
酶标仪(美国MD)、CO 2培养箱(上海一恒)、超净工作台(苏州净化)、生物倒置显微镜(重庆光电)。
24.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库，小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。
24.4待测样品
给药组：肽(1)、肽(2)、肽(3)、肽(4)、肽(5)，浓度分别为12.5ppm、25ppm、50ppm、100ppm。
对照组：PBS空白对照。
24.5实验方法
a)取处于指数生长期状态良好的细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×10 5个/mL，制成细胞悬液；
b)适当稀释取2000个/孔细胞悬液接种于96孔板上，200μL/孔，置恒温CO 2培养箱中培养24小时。
c)换液，加入待测样品，20μL/孔，培养24小时；
d)将MTT试剂加入96孔板中，20μL/孔，培养箱中反应4小时；
e)吸去上清液，加入DMSO，150μL/孔，平板摇床上振摇5分钟；
f)5分钟后使用酶标仪读取490nm和630nm波长下的参比OD值，并计算细胞活力：
细胞活力＝(给药孔OD-调零孔OD)/(对照孔OD-调零孔OD)×100％
24.6结果
按照平行设计原则，至少重复三次实验，实验数据以均数标准差(Mean±SD)表示，使用SPSS 17.0统计软件进行数据分析。
MTT法是一种检测细胞存活和生长的方法，测得的OD值与细胞活性成正比，OD值越大，表明细胞活性越高。
肽(1)～肽(5)对HaCaT细胞活性的影响见图1。结果表明，肽(1)～肽(5)均能在一定程度上提高HaCaT细胞的活性，其中肽(2)与肽(3)对HaCaT细胞活性的影响更大，在12.5ppm-100ppm各剂量均能改善HaCaT细胞的活性，促进其增殖。
肽(1)～肽(5)对NIH3T3细胞活性的影响见图2。结果表明，肽(1)～肽(5)均能在一定程度上提高NIH3T3细胞的活性，其中肽(3)对NIH3T3细胞活性的影响更大，在12.5ppm-100ppm各剂量均能改善NIH3T3细胞的活性，促进其增殖。
实施例25促细胞黏附作用测试
25.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、噻唑蓝(MTT)(Sigma)、二甲基亚砜(DMSO)(Sigma)、高糖培养基(DMEM)(Gibco)、胎牛血清(Gibco)。
25.2仪器
酶标仪(美国MD)、CO 2培养箱(上海一恒)、超净工作台(苏州净化)、生物倒置显微镜(重庆光电)。
25.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库。
25.4待测样品
给药组：肽(1)、肽(2)，浓度分别为12.5ppm、25ppm、50ppm、100ppm。用PBS溶液溶解。
对照组：PBS空白对照。
25.5实验方法
取冻存的HaCaT人角质层细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。
将待测样品按照20μL/孔加入至96孔板中，37℃恒温烘箱干燥过夜。于第二天将长势较好的HaCaT细胞消化后，以HaCaT细胞1万/孔密度种板，并将培养基补至200μL，于37℃、5％CO 2培养箱中孵育3h。培养结束后，将培养板取出并继续补充培养基至液面刚好溢出，用封口膜封闭，以保证没有气泡。在一个三维力的作用下翻转20min。弃去原有培养基，每孔加入90μL新鲜培养基和10μL 5mg/mL的MTT，置于37℃、5％CO 2培养箱中孵育3h。弃去溶液，加入150μL的DMSO。设置检测波长490nm，参比波长630nm，使用酶标仪读取OD值。
25.6结果
通过选择HaCaT细胞，铺板在药物包被好的96孔板中，孵育且经一段三维力作用后，黏附性强的细胞能保持在96孔板上，通过对板上活细胞进行MTT定量分析，即可评价药物对细胞黏附的影响，以此确定本发明的肽能否改善细胞弹性。在经过三维力作用之后，保持在96孔板上的活细胞越多，测得的OD值越大，表明细胞的黏附作用越强。
实验结果如图3所示，与正常对照组相比，肽(1)和肽(2)均能够提高HaCaT细胞的黏附能力，从而有利于提高其弹性。
实施例26划痕实验
26.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、高糖培养基(DMEM)(Gibco)、胎牛血清(Gibco)。
26.2仪器
光学显微镜、CO 2培养箱。
26.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库。
26.4待测样品
给药组：100ppm的肽(1)、100ppm的肽(2)、50U/mL的表皮生长因子(EGF)。
对照组：PBS空白对照。
26.5实验方法
取冻存的HaCaT细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。将细胞以20万个/孔的密度接种于12孔板，每孔2mL细胞悬浮液，待细胞贴壁长满后，换含0.5％～1％胎牛血清的培养基维持细胞24h使其同步化。用灭菌移液枪头进行划痕，PBS洗去掉落细胞，加入待测样品，继续于37℃、5％CO 2培养箱中孵育，24h后置于光学显微镜下分析伤口愈合区域。
26.6结果
细胞的划痕迁移实验能反应细胞的增殖修复能力，结果如图4所示，正常对照组的细胞划痕仍旧明显，划痕间距正常；与对照组相比，EGF阳性组的细胞增殖迁移明显，间距明显缩短；而肽(1)、肽(2)的划痕间距均较正常对照组细胞划痕间距缩短，并出现细胞迁移轨迹，由此可知，本发明的肽能够促进HaCaT人角质形成细胞增殖和迁移，具有皮肤修复作用。
实施例27光损伤修复实验
27.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、噻唑蓝(MTT)(Sigma)、二甲基亚砜(DMSO)(Sigma)、高糖培养基(DMEM)(Gibco)、胎牛血清(Gibco)。
27.2仪器
酶标仪(美国MD)、CO 2培养箱(上海一恒)、超净工作台(苏州净化)、生物倒置显微镜(重庆光电)。
27.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库，小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。
27.4待测样品
肽(1)、肽(2)、肽(3)、肽(4)、肽(5)，浓度分别为12.5ppm、25ppm、50ppm、100ppm。
27.5实验方法
取处于指数生长期状态良好的细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落， 计数1～4×10 5个/mL，制成细胞悬液。
将细胞以1万个/孔接种到96孔板中，待细胞长满至约80％时，建立UVB光损伤模型。加入50μL培养基，不进行UVB照射，以此作为空白对照组；实验组加入适量PBS反复洗至无色后，加入50μL PBS，以80J/cm 3UVB灯下照射，灯源和培养瓶间距15cm，实验组经照射后，弃去PBS，加入培养液和倍比稀释药物至200μL，继续于37℃、5％CO 2培养箱中孵育24h。
24h后，每孔加入22μL 5mg/mL的MTT，置于培养箱孵育4h后，弃去原溶液，加入150μL/孔的DMSO。5min后使用酶标仪读取490nm和630nm波长下的参比OD值。
27.6结果
通过建立UVB光损伤细胞模型，紫外光辐射后给药24h，使用MTT法检测细胞增殖活性，以此评价本发明的肽对光损伤细胞的修复能力。
HaCaT细胞光损伤修复结果见图5。结果显示，与非辐射的正常对照组相比，UVB辐射后细胞活性明显降低，提示光损伤模型建模成功。给药后，肽(1)～肽(5)均能在一定程度上提高光损伤HaCaT细胞的活性，其中肽(2)和肽(3)对细胞活性的提高作用更好。由此表明，本发明的肽能够修复光损伤的HaCaT细胞，提高其活性，具有良好的皮肤修复能力。
NIH3T3细胞光损伤修复结果见图6。结果显示，与未辐射的正常组相比，辐射后的NIH3T3细胞活性显著降低，提示光损伤模型建模成功。给药后，肽(1)在50、100ppm能够显著提升细胞活性；肽(2)在12.5ppm-100ppm各剂量均能显著提升NIH3T3细胞活性。由此表明，肽(1)及肽(2)在一定浓度范围内均能够修复光损伤的NIH3T3细胞，提高其活性，具有良好的皮肤修复能力。
实施例28抗炎修复实验
28.1试剂与材料
LPS、地塞米松(DXM)、ELISA试剂盒。
28.2仪器
酶标仪(美国MD)、CO 2培养箱(上海一恒)、超净工作台(苏州净化)。
28.3细胞株
小鼠单核巨噬细胞(RAW264.7细胞)。
28.4待测样品
肽(2)，测试浓度为100ppm。
28.5实验方法
a)取处于指数生长期状态良好的RAW264.7细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×10 6个/mL，制成细胞悬液；
b)适当稀释取100000个/孔细胞悬液接种于6孔板上，待细胞长满至80％左右时，建立LPS刺激炎症模型。设置空白对照组(control)、LPS组、地塞米松组(DXM)、多肽组。空白对照组加入1000μL培养基；LPS组加入200ng/mL LPS，补加培养基至1000μL；地塞米松组加入200ng/mL LPS及50ng/mL地塞米松，补加培养基至1000μL；多肽组加入200ng/mL LPS及肽(2)，补加培养基至1000μL(多肽最终测试浓度为100ppm)。继续于37℃、5％CO 2培养箱中孵育48h；
c)3000rmp离心10min收集细胞上清获得样本液；
d)按照ELISA操作说明书进行操作：将各种试剂移至室温平衡至少30min，按照说明书方法配制试剂，备用。分别设标准品孔，待测样品孔，每孔分别加标准品或待测样本100μL，轻轻摇匀，覆上板贴，37℃孵育2h。每个样品设4个复孔。弃去液体，甩干，洗板5次。每孔加生物素标记抗体工作液100μL，覆上新的板贴，37℃孵育1h。弃去孔内液体，甩干，洗板5次，每次浸泡1min，200μL/孔，甩干。每孔加辣根过氧化物酶标记亲和素工作液100μL，覆上新的板贴，37℃孵育1h。弃去孔内液体，甩干，洗板5次，每次浸泡1min，200μL/孔，甩干。依序每孔加底物溶液100μL，37℃避光显色15-30min。依序每孔加终止溶液100μL，终止反应。在反应终止后15min内用酶标仪在450nm处依序测量各孔的OD值。
28.6实验结果
炎症因子如TNF-α、IL-6以及IL-8等能够引起机体的免疫过度应答，导致肌肤的溃烂、红肿等问题，因此抑制TNF-α、IL-6以及IL-8等炎症因子可以达到抗炎修复的功效。通过200ng/mL LPS刺激RAW264.7细胞建立炎症模型，探究本发明的肽的抗炎修复作用。
实验结果如图7-9所示，经过LPS刺激，TNF-α、IL-6以及IL-8细胞因子明显上调；给药后，100ppm的肽(2)明显下调TNF-α、IL-6以及IL-8的表达，其抗炎效果与阳性药物地塞米松相当，说明肽(2)具有明显的抗炎修复作用。
实施例29稳定性试验研究
1、将肽H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH(下称为参比肽A)样品和本发明肽(1)、肽(2)样品分别溶于水中，在常温条件下放置，通过HPLC考察各样品0天、8天、18天的纯度变化情况，结果见下表3：
表3参比肽A水溶液及肽(1)、肽(2)水溶液的稳定性研究
试验条件 参比肽A水溶液(纯度) 肽(1)水溶液(纯度) 肽(2)水溶液(纯度)
溶液0天 88.030％ 98.443％ 99.291％
常温8天 80.880％ 98.362％ 99.122％
常温18天 72.693％ 96.703％ 98.045％
由表3结果可知，参比肽A水溶液暴露于空气中，在0天时，纯度即大为下降，在水溶液中常温放置18天后，纯度降至72.693％。相对于参比肽A水溶液，肽(1)、肽(2)水溶液在室温条件下放置18天，其纯度没有发生明显变化。
2、参比肽A粉末及肽(1)粉末、肽(2)粉末的稳定性研究
将参比肽A粉末、肽(1)粉末、肽(2)粉末分别放置在25℃、40℃，通过HPLC考察其纯度随时间的变化情况，结果见下表4：
表4参比肽A粉末及肽(1)粉末、肽(2)粉末的稳定性研究
Figure PCTCN2021112345-appb-000029
由表4结果可知，参比肽A粉末在40℃条件下，放置5天后，其纯度大为降低，仅为57.503％。相比之下，肽(1)粉末、肽(2)粉末在40℃条件下，放置20天后，其纯度没有发生明显变化。
3、肽(3)-(7)粉末的稳定性研究
将肽(3)-(7)的粉末样品置于高湿(RH92.5％，25℃)条件下，放置10天，通过HPLC考察各样品的纯度变化情况，结果见下表5：
表5肽(3)-(7)粉末的稳定性研究
样品编号 0天(纯度) 高湿10天(纯度)
肽(3) 97.605％ 96.251％
肽(4) 96.017％ 93.962％
肽(5) 97.482％ 94.603％
肽(6) 96.235％ 95.719％
肽(7) 98.541％ 97.863％
由表5结果可知，肽(3)-(7)在高湿(RH92.5％，25℃)条件下，放置10天，其纯度没有发生明显变化。
该实施例29中，HPLC色谱分离条件如下：
色谱柱：十八烷基键合硅胶为填充剂4.6*250mm，5μm
流动相A：乙腈(含0.09％三氟乙酸)
流动相B：0.1％三氟乙酸
稀释液：水
流速：1.0ml/min；
柱温：35℃；
检测波长：215nm；
进样量：10μl；
运行时间：32min
梯度洗脱表：
Figure PCTCN2021112345-appb-000030
Figure PCTCN2021112345-appb-000031
供试品溶液：取本品适量加水溶解稀释配成每1ml含1mg的溶液。
实施例30含肽(1)的脂质体的制备
Figure PCTCN2021112345-appb-000032
将二棕榈酰基磷脂酰胆碱称重且溶于氯仿。于真空下蒸发溶剂，直至得到磷脂薄层，将此层于55℃以所需浓度的肽水溶液处理而水化，得到多室脂质体。将多室脂质体通过高压均质处理，得到尺寸更加小而均一的单室脂质体。
实施例31含肽(2)的微米乳液组合物的制备
Figure PCTCN2021112345-appb-000033
按照处方用量，称取B相的成分加于容器中。接着，将D相添加至B相且于连续搅拌下均质化。随后将A相添加至混合物。最后，添加C相，得到含肽(2)的微米乳液组合物。
以上内容是结合具体的优选实施方式对本发明所做的进一步详细的说明，但是不表示本发明的具体实施是局限于这些说明。对于本发明所属领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或是替换，都应视为属于本发明的保护范围。

Claims (18)

  1. 式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，
    Figure PCTCN2021112345-appb-100001
    式(I)中，
    R 2选自：H、烷基、烯基烷基、羧基烷基、酯基烷基、烷基巯基、羧烷基巯基、或
    Figure PCTCN2021112345-appb-100002
    式(I)和式(i)中，
    n选自：1或2；
    R 1选自：H或R 4-CO-，其中R 4选自：取代的或未取代的烷基、取代的或未取代的烯基；
    R 3选自：-NR 5R 6或-OR 5，其中各个R 5和R 6彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
    所述“烷基”、“烯基烷基”、“羧基烷基”、“酯基烷基”“烷基巯基”、“羧烷基巯基”中的烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
    所述“烯基”、“烯基烷基”中的烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子、可选具有2、3、4、5、或6个碳原 子)的直链或支链烯基，所述“烯基”具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述“烯基”是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
    所述“酯基烷基”中的酯基为R(C＝O)OR′基团，其中，R、R′各自独立地为单键、烷基或亚烷基；
    可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C 1-C 4烷基；羟基；C 1-C 4烷氧基；氨基；C 1-C 4氨基烷基；C 1-C 4羰氧基；C 1-C 4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C 1-C 4烷基磺酰基；硫醇；C 1-C 4烷硫基；C 6-C 30芳氧基如苯氧基；-NR b(C＝NR b)NR bR c，其中R b和R c是独立地选自：H、C 1-C 4烷基、C 2-C 4烯基、C 2-C 4炔基、C 3-C 10环烷基、C 6-C 18芳基、C 7-C 17芳烷基、具有三至十元的杂环基、或氨基的保护基。
  2. 根据权利要求1所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，式(I)中，当n为2时，R 2选自：H、烷基、烯基烷基、羧基烷基、酯基烷基、烷基巯基或羧烷基巯基。
  3. 根据权利要求1或2所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，R 1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基或亚油酰基；
    可选地，R 1选自H、乙酰基、月桂酰基、肉豆蔻酰基或棕榈酰基；
    可选地，R 1是H、乙酰基或棕榈酰基。
  4. 根据权利要求1或2所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，R 2选自：H、-CH 3、-CH 2CH 3、-CH 2CH＝CH 2、-CH 2COOH、-CH 2COOC(CH 3) 3、-S-CH 2CH 3、-S-(CH 2) 5CH 3、-S-CH 2COOH或
    Figure PCTCN2021112345-appb-100003
    Figure PCTCN2021112345-appb-100004
    可选地，当n为2时，R 2选自：H、-CH 3、-CH 2CH 3、-CH 2CH＝CH 2、-CH 2COOH、-CH 2COOC(CH 3) 3、-S-CH 2CH 3、-S-(CH 2) 5CH 3或-S-CH 2COOH。
  5. 根据权利要求1或2所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，R 5、R 6彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
    可选地，R 5是H并且R 6选自：H、甲基、乙基、己基、十二烷基或十六烷基；
    可选地，R 3是-OH或-NH 2。
  6. 根据权利要求1-5任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，选自下列肽(1)-(46)：
    (1)
    Figure PCTCN2021112345-appb-100005
    (2)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
    (3)
    Figure PCTCN2021112345-appb-100006
    (4)
    Figure PCTCN2021112345-appb-100007
    (5)
    Figure PCTCN2021112345-appb-100008
    (6)
    Figure PCTCN2021112345-appb-100009
    (7)
    Figure PCTCN2021112345-appb-100010
    (8)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH；
    (9)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2；
    (10)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-OH；
    (11)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Cys-Thr-Thr-Asn-NH 2；
    (12)
    Figure PCTCN2021112345-appb-100011
    (13)
    Figure PCTCN2021112345-appb-100012
    (14)
    Figure PCTCN2021112345-appb-100013
    (15)
    Figure PCTCN2021112345-appb-100014
    (16)
    Figure PCTCN2021112345-appb-100015
    (17)
    Figure PCTCN2021112345-appb-100016
    (18)
    Figure PCTCN2021112345-appb-100017
    (19)
    Figure PCTCN2021112345-appb-100018
    (20)
    Figure PCTCN2021112345-appb-100019
    (21)
    Figure PCTCN2021112345-appb-100020
    (22)
    Figure PCTCN2021112345-appb-100021
    (23)
    Figure PCTCN2021112345-appb-100022
    (24)
    Figure PCTCN2021112345-appb-100023
    (25)
    Figure PCTCN2021112345-appb-100024
    (26)
    Figure PCTCN2021112345-appb-100025
    (27)
    Figure PCTCN2021112345-appb-100026
    (28)
    Figure PCTCN2021112345-appb-100027
    (29)
    Figure PCTCN2021112345-appb-100028
    (30)
    Figure PCTCN2021112345-appb-100029
    (31)
    Figure PCTCN2021112345-appb-100030
    (32)
    Figure PCTCN2021112345-appb-100031
    (33)
    Figure PCTCN2021112345-appb-100032
    (34)
    Figure PCTCN2021112345-appb-100033
    (35)
    Figure PCTCN2021112345-appb-100034
    (36)
    Figure PCTCN2021112345-appb-100035
    (37)
    Figure PCTCN2021112345-appb-100036
    (38)
    Figure PCTCN2021112345-appb-100037
    (39)
    Figure PCTCN2021112345-appb-100038
    (40)
    Figure PCTCN2021112345-appb-100039
    (41)
    Figure PCTCN2021112345-appb-100040
    (42)H-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2；
    (43)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
    (44)Palm-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2；
    (45)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-OH；
    (46)Ac-Ala-Pro-Phe-Arg-Met-Gly-Ile-Met-Thr-Thr-Asn-NH 2。
  7. 根据权利要求1-6任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，其特征在于，
    所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽的金属盐，所述金属包括：锂、钠、钾、钙、镁、锰、铜、锌或铝；
    可选地，所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽与有机碱形成的盐，所述有机碱包括：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪；
    可选地，所述美容上可接受的盐或药学上可接受的盐包括式(I)所示的肽与无机酸或有机酸形成的盐，所述有机酸包括：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸或葡萄糖酸；
    可选地，所述无机酸包括：盐酸、硫酸、硼酸或碳酸。
  8. 一种美容或药物组合物，其特征在于，包括有效量的权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，以及至少一种赋形剂和任选的美容上或药学上可接受的佐剂；
    可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增 白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、有机溶剂、液体推进剂、皮肤调理剂、保湿剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素或着色剂、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、角质剥离剂、角质层分离剂、抗微生物剂、抗真菌剂、抑真菌剂、灭菌剂、抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、热休克蛋白、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗皮炎剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、粘合剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子生长因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物。
  9. 根据权利要求8所述的美容或药物组合物，其特征在于，所述美容或药物组合物的制剂选自：霜剂、油、奶、香膏、泡沫、洗剂、凝胶、擦剂、浆液、皂、洗发精、润发乳、血清、软膏、摩丝、润发油、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、糖浆剂、酏剂、多糖薄膜、胶冻或明胶；
    可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
    可选地，所述片剂包括：糖衣片剂。
  10. 一种美容上或药学上可接受的递送系统或缓释系统，其特征在于，包含有效量的权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述的美容或药物组合物；
    所述美容上或药学上可接受的递送系统或缓释系统选自：脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳米粒子；可选为脂质体或微米乳液，可选具有反胶束的内部结构的油包水型微米乳液。
  11. 一种美容上或药学上可接受的固体有机聚合物或固体无机支撑体，其特征在于，包含有效量的权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述的美容或药物组合物；
    所述美容上或药学上可接受的固体有机聚合物或固体无机支撑体选自：滑石、膨润土、二氧化硅、淀粉或麦芽糖糊精。
  12. 一种化妆品，其特征在于，包含有效量的权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述的美容或药物组合物；
    可选地，所述化妆品选自：遮瑕膏、粉底、卸妆液、卸妆乳、眼影、唇棒、唇彩、护唇膏或唇粉。
  13. 一种织物、无纺织物或医学装置，其特征在于，包含有效量的权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述的美容或药物组合物；
    可选地，所述织物、无纺织物或医学装置选自：绷带、纱布、t-恤衫、短袜、裤袜、内衣、腰带、手套、尿布、卫生巾、敷裹物、床罩、擦拭巾、粘附贴剂、非粘附贴剂、封闭贴剂、微电子贴剂或面膜。
  14. 权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述美容或药物 组合物，或权利要求10所述的美容上或药学上可接受的递送系统或缓释系统，或权利要求11所述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或权利要求12所述的化妆品，或权利要求13所述的织物、无纺织物或医学装置在制备用于治疗或护理皮肤或粘膜的美容组合物或药用组合物中的用途。
  15. 权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述美容或药物组合物，或权利要求10所述的美容上或药学上可接受的递送系统或缓释系统，或权利要求11所述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或权利要求12所述的化妆品，或权利要求13所述的织物、无纺织物或医学装置在制备用于治疗或护理体表创伤、烧伤、皮肤溃疡、减少疤痕产生、或加快疤痕修复的美容组合物或药物组合物中的用途。
  16. 权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述美容或药物组合物，或权利要求10所述的美容上或药学上可接受的递送系统或缓释系统，或权利要求11所述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或权利要求12所述的化妆品，或权利要求13所述的织物、无纺织物或医学装置在制备用于促进成纤维细胞增殖、增加胶原蛋白生成、用于改善皮肤屏障功能、或用于皮肤或粘膜的再上皮化或愈合的美容组合物或药物组合物中的用途。
  17. 权利要求1-7任一项所述的式(I)所示的肽，或其立体异构体、或立体异构体的混合物、或其美容上可接受的盐、或其药学上可接受的盐，或权利要求8或9所述美容或药物组合物，或权利要求10所述的美容上或药学上可接受的递送系统或缓释系统，或权利要求11所述的美容上或药学上可接受的固体有机聚合物或固体无机支撑体，或权利要求12所述的化妆品，或权利要求13所述的织物、无纺织物或医学装置在制备用于治疗、预防或修复皮肤老化或光老化的美容组合物或药物组合物中的用途。
  18. 根据权利要求17所述的用途，其中，皮肤老化或光老化的治疗、预防或修复是减少、预防或治疗面部皱纹。
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CN116747148A (zh) * 2023-07-24 2023-09-15 山东福瑞达生物股份有限公司 一种适用于敏感肌的抗衰化妆品组合物
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CN117838562A (zh) * 2023-12-29 2024-04-09 花安堂生物科技集团有限公司 一种皮肤舒缓修护组合物及其制备方法和应用
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CN116440248A (zh) * 2023-03-31 2023-07-18 广州市尊爱日用化妆品有限公司 含肽和羟丙基四氢吡喃三醇外用制剂组合物及其制备方法
CN116440248B (zh) * 2023-03-31 2024-03-12 广州市尊爱日用化妆品有限公司 含肽和羟丙基四氢吡喃三醇外用制剂组合物及其制备方法
WO2024217505A1 (zh) * 2023-04-20 2024-10-24 深圳市维琪科技股份有限公司 肽及其组合物和用途
CN116747148A (zh) * 2023-07-24 2023-09-15 山东福瑞达生物股份有限公司 一种适用于敏感肌的抗衰化妆品组合物
CN117106021A (zh) * 2023-10-25 2023-11-24 深圳市维琪科技股份有限公司 六肽及其组合物和用途
CN117106021B (zh) * 2023-10-25 2023-12-26 深圳市维琪科技股份有限公司 六肽及其组合物和用途
CN117304265A (zh) * 2023-11-29 2023-12-29 深圳市维琪科技股份有限公司 一种皮肤护理多肽、组合物及其应用
CN117304265B (zh) * 2023-11-29 2024-01-30 深圳市维琪科技股份有限公司 一种皮肤护理多肽、组合物及其应用
CN117417408A (zh) * 2023-12-14 2024-01-19 深圳市维琪科技股份有限公司 一种抗衰六肽及其组合物和用途
CN117417408B (zh) * 2023-12-14 2024-02-13 深圳市维琪科技股份有限公司 一种抗衰六肽及其组合物和用途
CN117838562A (zh) * 2023-12-29 2024-04-09 花安堂生物科技集团有限公司 一种皮肤舒缓修护组合物及其制备方法和应用
CN117838562B (zh) * 2023-12-29 2026-04-03 花安堂生物科技集团有限公司 一种皮肤舒缓修护组合物及其制备方法和应用

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