WO2023231573A1 - 肽衍生物异构体及其组合物和用途 - Google Patents

肽衍生物异构体及其组合物和用途 Download PDF

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Publication number
WO2023231573A1
WO2023231573A1 PCT/CN2023/087204 CN2023087204W WO2023231573A1 WO 2023231573 A1 WO2023231573 A1 WO 2023231573A1 CN 2023087204 W CN2023087204 W CN 2023087204W WO 2023231573 A1 WO2023231573 A1 WO 2023231573A1
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agents
acid
isomer
acceptable salt
pharmaceutically acceptable
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PCT/CN2023/087204
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English (en)
French (fr)
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丁文锋
孙新林
陈雪
赵文豪
观富宜
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深圳市维琪科技股份有限公司
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Publication of WO2023231573A1 publication Critical patent/WO2023231573A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • 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
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to peptide derivative isomers, compositions containing these isomers and their uses.
  • the peptide derivative represented by the following formula (i) shows beneficial biological properties, but there is still a lack of research on the structure-activity relationship of this peptide derivative.
  • the compound of formula (i) has 3 chiral centers and therefore has 8 isomers.
  • the structural formula is shown in Table 1 below.
  • the compound of formula (i) has three chiral centers, two of which are located at hydroxyproline residues, and there are eight isomers in total. Research has found that the efficacy and activity of different isomers of the compound of formula (i) are significantly different.
  • the present invention aims to provide a peptide derivative isomer with repairing and anti-aging effects and muscle contraction inhibition effects.
  • the present invention provides an isomer of a peptide derivative represented by formula (I), a mixture of its stereoisomers, or its cosmetically acceptable salt, or its pharmaceutically acceptable salt,
  • the isomers of the peptide derivative of formula (I) and the cosmetic compositions or pharmaceutical compositions containing these isomers provided by the present invention can increase the activity of human skin fibroblasts, significantly increase cell adhesion, and promote collagen expression. Thereby increasing skin elasticity and/or skin firmness, it can be used to prevent or even treat skin sagging; it can treat, prevent or repair skin aging or photoaging; it can also inhibit muscle contraction and can be used to treat or care for conditions and disorders caused by muscle contraction. or disease; may also be used in products to reduce, prevent or treat wrinkles.
  • the invention also includes all suitable isotopic variants of the compounds of the invention.
  • Isotopic variants of the compounds of the invention are here understood to mean compounds in which at least one atom within the compound of the invention is replaced by another atom of the same atomic number, but which has an atomic mass different from that found in nature. The atomic mass usually or predominantly present.
  • isotopes that may be incorporated into the compounds of the invention are: those of hydrogen, carbon, nitrogen, oxygen, such as 2 H (deuterium), 3 H (tritium), 13 C, 14 C, 15 N, 17 O or 18 O .
  • isotopic variants of the compounds of the invention may be advantageous, for example, to examine the mechanism of action or the distribution of the active compound in vivo; due to the relative simplicity of their preparation and detectability, especially compounds labeled with 3 H or 14 C isotopes are suitable for this purpose.
  • isotopes e.g., deuterium
  • modifications of the compounds may also constitute preferred embodiments of the invention.
  • the present invention can be prepared by methods known to the person skilled in the art, for example by the methods described further below and described in the examples, by using the respective reagents and/or corresponding isotopic modifications of the starting substances. Isotopic variants of inventive compounds.
  • the invention also includes prodrugs of the compounds of the invention.
  • prodrug as used herein means compounds which may themselves be biologically active or inactive, but which, during their residence time in the body, react (e.g., metabolism or hydrolysis) to produce a compound of the invention.
  • the compounds of formula (I) may form monovalent or polyvalent, homogeneous or mixed salts with acids, for example with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; or with suitable carboxylic acids, for example aliphatic monovalent or mixed salts.
  • acids for example with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; or with suitable carboxylic acids, for example aliphatic monovalent or mixed salts.
  • Dicarboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, glycolic acid, succinic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, phthalic acid, citric acid, lactic acid or Tartaric acid; or with aromatic carboxylic acids such as benzoic acid or salicylic acid; or with aromatic-aliphatic carboxylic acids such as mandelic acid or cinnamic acid; or with heteroaromatic carboxylic acids such as nicotinic acid; or with aliphatic or aromatic Sulfonic acid such as methanesulfonic acid or toluenesulfonic acid.
  • the synthesis of the isomers of the peptide derivative represented by formula (I) of the present invention, or the cosmetically acceptable salts thereof, or the pharmaceutically acceptable salts thereof, can be carried out according to conventional methods known in the art, for example Solid-phase synthesis, liquid-phase synthesis, or a combination of solid-phase and liquid-phase methods can also be performed by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin. to prepare.
  • a method for obtaining the isomer represented by formula (I) includes the following steps:
  • Boc- ⁇ -Ala-OH condenses with HOSu to form Boc- ⁇ -Ala-OSu;
  • Boc- ⁇ -Ala-OSu reacts with hydroxyproline under weakly alkaline conditions to generate Boc- ⁇ -Ala-hydroxyproline;
  • the hydroxyproline is (2S,4R)-Hyp, (2S,4S)-Hyp, (2R,4S)-Hyp or (2R,4R)-Hyp.
  • Another aspect of the present invention provides a cosmetic or pharmaceutical composition, including an effective amount of the isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable amount thereof.
  • the adjuvant is selected from: a collagen synthesis stimulator, an agent that regulates PGC-1 ⁇ synthesis, an agent that modulates the activity of PPAR ⁇ , an agent that increases or decreases the triglyceride content of adipocytes, and stimulates or delays adipocytes.
  • the formulation of the cosmetic or pharmaceutical composition is selected from: cream, oil, milk, balm, foam, lotion, gel, liniment, serum, soap, shampoo, conditioner, serum , ointment, mousse, pomade, powder, rod, 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 tablets include: sugar-coated tablets.
  • the cosmetically or pharmaceutically effective amounts of the peptide derivative isomers of the invention to be administered, as well as their dosage, will depend on a number of factors, including age, condition of the patient, severity of the condition or disease, route and frequency of administration, and to be used Specific properties of isomers.
  • Cosmetically or pharmaceutically effective amount means an amount of one or more isomers of the invention that is non-toxic but sufficient to provide the desired effect.
  • the isomers of the invention are used in cosmetic or pharmaceutical compositions of the invention at a cosmetically or pharmaceutically effective concentration to achieve the desired effect; in a preferred form, at 0.00000001 relative to the total weight of the composition % (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), and even more preferably between 0.0001% (by weight) and 5% (by weight).
  • Another aspect of the invention provides a cosmetically or pharmaceutically acceptable delivery system or sustained release system to achieve better penetration of the active ingredient and/or improve its pharmacokinetic and pharmacodynamic properties, It contains an effective amount of an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or the above cosmetically acceptable salt. or pharmaceutical compositions.
  • delivery system refers to a diluent, adjuvant, excipient or carrier with which the isomers of the invention are administered, selected from: water, oil or surfactant, including petroleum sources, animal sources, plant sources , or those of synthetic origin, such as and without limitation peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate esters, sorbitan esters, ether sulfate esters, sulfate esters, betaines, glucosides, maltosides , fatty alcohols, nonoxynol ethers, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerin, digitonin and the like.
  • sustained release is used in its conventional sense and refers 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 release of the compound throughout the period of time.
  • Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant liposomes, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges substances, 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 having an internal structure of reverse micelles.
  • Sustained release systems may be prepared by methods known in the art and may be administered, for example, by topical or transdermal administration, including adhesive patches, non-adhesive 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 a specific body site, and preferably should Relatively constant amounts of these isomers of the invention are released.
  • topical or transdermal administration including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patch
  • systemic administration such as, and without limitation, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a specific body site, and preferably should Relatively constant amounts of these isomers of the invention are released.
  • the amount of isomer included in the sustained release system will depend, for example, on the site at which the composition is to be administered, the release rate of the isomer of the invention, kinetics and duration of radiation, and the nature of the condition, disorder and/or disease to be treated and/or cared for.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained release system in the preparation of cosmetic compositions or pharmaceutical compositions for the treatment or care of skin or mucous membranes.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained release system in the preparation of cosmetic compositions or pharmaceutical compositions for increasing skin elasticity and/or skin firmness use.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained-release system in the preparation of a cosmetic combination for increasing collagen production, improving fibroblast activity and/or increasing cell adhesion Use in medicines or pharmaceutical compositions.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained release system in the preparation of cosmetic compositions or pharmaceutical compositions for treating, preventing or repairing skin aging or photoaging. use.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained release system in the preparation of a cosmetic composition or pharmaceutical combination for the treatment or care of a condition, disorder or disease caused by muscle contraction.
  • the disorder or disease is dystonia, specifically, the dystonia is focal dystonia; dystonias include, but are not limited to, facial spasm, torsional dystonia, cervical dystonia, or strabismus. Cervical dystonia, laryngeal dystonia or spasmodic dysphonia, oromandibular dystonia, limb dystonia such as writer's cramp or musician's cramp or foot dystonia, nocturnal bruxism, hemifacial spasm, tics and/or strabismus; segments dystonia, Major syndrome, multifocal dystonia, hemidystonia, dopamine-responsive dystonia, and Segawa dystonia.
  • Another aspect of the present invention provides an isomer of the peptide derivative represented by the above formula (I), a mixture of its stereoisomers, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof , or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic or pharmaceutically acceptable delivery system or sustained release system in the preparation of products for reducing, preventing or treating wrinkles the use of.
  • the term “skin” is understood to mean the layers constituting it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes. In the present invention, the term “skin” includes the scalp.
  • treatment refers to the administration of an isomer of a peptide derivative of the present invention to reduce or eliminate a disease or disorder, or to reduce or eliminate one or more symptoms associated with such a disease or disorder.
  • treatment also encompasses the ability to reduce or eliminate the physiological consequences of the disease or condition.
  • care includes the prevention of disease and/or disorder.
  • prevention refers to the ability of the peptide derivative isomers of the present invention to prevent, delay, or hinder the occurrence or progression of a disease or condition before it occurs.
  • aging refers to the changes that the skin undergoes as we age (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 changes that are externally visible and/or perceptible by touch, such as and without limitation: the development of discontinuities in the skin (such as wrinkles, fine lines, expression lines, stretch lines, streaks, grooves, lines, unevenness or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of recovery from deformation, loss of resilience), skin sagging (such as sagging cheeks, bags under the eyes, or Double chin, etc.), changes in skin color (such as scars, redness, eye bags, or hyperpigmentation areas such as age spots or freckles, etc.), abnormal differentiation, hyperkeratinization, elastosis, keratosis, alopecia, orange Dermoid skin, loss of collagen structure, and other histological changes in
  • photoaging refers to the 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 hypopigmentation. Regular, abnormal and/or hyperkeratinization.
  • the compound of formula (i) has 3 chiral carbon atoms, 2 of which are located in the hydroxyproline residue, and there are 8 isomers in total. There is no research on the isomers of the compound of formula (i) in the prior art. After studying it, the present invention found that only 4 of the isomers have the effect of repairing and anti-aging and inhibiting muscle contraction.
  • the peptide derivative isomers of the present invention are easy to synthesize and highly safe. They can increase the activity of skin fibroblasts, significantly increase cell adhesion, promote collagen expression, thereby increasing skin elasticity and/or skin firmness. It can be used to prevent or even treat skin sagging; to treat, prevent, or repair skin aging or photoaging; it can also inhibit muscle contraction, and can be used to treat or care for conditions, disorders, or diseases caused by muscle contraction; it can also be used to reduce, prevent, or in products that treat wrinkles. Among them, isomer A has the best technical effect.
  • Figure 3 is a graph showing the effect of test samples on collagen content.
  • HOBt 1-hydroxybenzotriazole
  • THF tetrahydrofuran
  • DCM dichloromethane
  • DCC N,N'-dicyclohexylcarbodiimide
  • NMM N-methylmorpholine
  • piperidine piperidine
  • HOSu N-Hydroxysuccinimide
  • EDC.HCl 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • EtOAc ethyl acetate
  • Fmoc 9-fluorenylmethoxycarbonyl
  • Boc tert-butoxycarbonyl
  • Bzl benzyl Base
  • ⁇ -Ala ⁇ -alanine
  • Hyp hydroxyproline
  • Dab 2,4-diaminobutyric acid
  • step 1.2 Place 124g of DCC in a 500mL beaker, add 200mL of DCM, stir and dissolve, slowly add it in portions to the solution obtained in step 1.1, remove the ice bath, and react at room temperature for 5 hours. Use TLC analysis to follow the reaction until the reaction is complete.
  • the muscle nicotinic acetylcholine receptor (PDB Code: 4ZJS) was selected as the target protein, and the eight isomers of the compound of formula (i) were molecularly docked with the target protein, and the scoring function was used to evaluate the results.
  • the results are shown in Table 3 below.
  • Fetal calf serum Gibco
  • DMEM medium Gibco
  • penicillin streptomycin
  • MTT Methicillin
  • Microplate reader (MD, USA), CO2 incubator (Shanghai Yiheng), ultra-clean workbench (Suzhou Purification).
  • HSF Human skin fibroblasts
  • test concentrations are 50ppm and 100ppm;
  • Isomer B test concentration is 50ppm, 100ppm;
  • test concentration is 50ppm, 100ppm;
  • Isomer D test concentrations are 50ppm and 100ppm;
  • Isomer E test concentration is 50ppm, 100ppm;
  • Isomer F test concentration is 50ppm, 100ppm;
  • test concentrations are 50ppm and 100ppm;
  • Isomer H test concentration is 50ppm, 100ppm;
  • Blank control group PBS.
  • MTT method is a method to detect cell survival and growth.
  • the measured OD value is directly proportional to cell activity.
  • the results of the effect of test samples on HSF cell proliferation are shown in Figure 1.
  • the results show that compared with the blank control group, the peptide derivative isomers of the present invention have no toxic effect on HSF cells within the range of 100 ppm.
  • the peptide derivatives of the present invention isomer A, isomer B, isomer C and isomer D can improve Cell activity, promote HSF cell proliferation.
  • isomer A can increase HSF cell activity at a concentration of 50 ppm, with statistical differences compared with the blank control group. As the concentration increases, the effect of promoting cell proliferation increases.
  • Isomer A can significantly increase cell activity and significantly promote HSF cell proliferation at a concentration of 100 ppm.
  • the peptide derivative isomers of the present invention not only have no toxic effect on fibroblasts, but can also increase the activity of fibroblasts and promote their proliferation, thereby increasing skin elasticity and/or skin tightness, and can be used for To prevent or even treat skin sagging, isomer A has the best technical effect.
  • Fetal calf serum Gibco
  • DMEM medium Gibco
  • penicillin streptomycin
  • MTT Methicillin
  • Microplate reader (MD, USA), CO2 incubator (Shanghai Yiheng), ultra-clean workbench (Suzhou Purification).
  • HaCaT Human keratinocytes
  • test concentrations are 50ppm and 100ppm;
  • Isomer B test concentration is 50ppm, 100ppm;
  • test concentration is 50ppm, 100ppm;
  • Isomer D test concentrations are 50ppm and 100ppm;
  • Isomer E test concentration is 50ppm, 100ppm;
  • Isomer F test concentration is 50ppm, 100ppm;
  • test concentrations are 50ppm and 100ppm;
  • Isomer H test concentration is 50ppm, 100ppm;
  • Blank control group PBS.
  • the purpose of this experiment is to select HaCaT cuticle cells, plate them in a 96-well plate coated with the test sample, incubate and after a period of external force, evaluate the impact of the test sample on cell adhesion, thereby determining the effectiveness of the present invention.
  • Can isomers improve cell elasticity.
  • Discard the original culture medium add 90 ⁇ L of fresh culture medium and 10 ⁇ L of 5 mg/mL MTT to each well, and incubate for 3 h in a 37°C, 5% CO2 incubator. Discard the solution and add 150 ⁇ L of DMSO. Use a microplate reader to read the reference OD values at 490nm and 630nm wavelengths.
  • cells with strong adhesiveness can remain on the 96-well plate.
  • the adhesion of cells can be reflected by quantitative MTT analysis of living cells on the plate. The more viable cells maintained on the 96-well plate, the greater the measured OD value, indicating that the adhesion of the cells is stronger and the elasticity of the cells is stronger.
  • the peptide derivative isomers of the present invention can improve cell adhesion ability, significantly increase intercellular adhesion and cell-extracellular matrix adhesion, thereby increasing skin elasticity and/or skin firmness, and can be used for prevention and even treatment. sagging skin.
  • isomer A exhibits a strong cell adhesion-promoting effect at a lower concentration and has the best effect.
  • Fetal bovine serum (Gibco), DMEM medium (Gibco), penicillin, streptomycin, MTT (Sigma), collagen I ELISA kit (Yaibo), RIPA lysis buffer, BCA protein kit (Beyotime).
  • Microplate reader MD, USA
  • CO2 incubator Shanghai Yiheng
  • ultra-clean workbench Suzhou Purification
  • incubator Shanghai Yiheng
  • HSF Human skin fibroblasts
  • Blank control group PBS
  • UV group UV radiation, plus PBS.
  • Collagen is the most abundant protein found in connective tissue and plays an important role in plumping and firming the skin. In an ultraviolet overexposure environment, elastase activity increases significantly and elastin is hydrolyzed; collagen synthesis is also inhibited. In this experiment, a test sample is used to treat cells after ultraviolet radiation, and the collagen I content in the corresponding cells is detected to determine whether the isomer of the present invention can promote the synthesis of collagen.
  • the peptide derivative isomers of the present invention can promote collagen expression, increase collagen content, thereby increasing skin elasticity and/or skin firmness, and can be used to prevent or even treat skin sagging, treat, prevent or repair skin. Aging or photoaging. Among them, isomer A has the best technical effect.
  • HW-400S constant temperature smooth muscle tank BL-420E biological function experimental system, tension transducer.
  • Acetylcholine chloride (Shanghai Yuanye), chloral hydrate (Shanghai McLean).
  • test concentrations are 100ppm and 200ppm;
  • Isomer B test concentrations are 100ppm and 200ppm;
  • Isomer C test concentration is 100ppm, 200ppm;
  • Isomer D test concentrations are 100ppm and 200ppm;
  • Isomer E test concentrations are 100ppm and 200ppm;
  • Isomer F test concentration is 100ppm, 200ppm;
  • Isomer G test concentrations are 100ppm and 200ppm;
  • Isomer H test concentration is 100ppm, 200ppm.
  • the purpose of this experiment is to use rat ileal muscle as the experimental subject, administer the test sample after acetylcholine stimulation, observe the rhythm of the ileal muscle, and evaluate the impact of the test sample on muscle contraction, so as to determine the peptide derivative of the present invention.
  • Can isomers inhibit muscle contraction.
  • the order of adding acetylcholine was changed, and the test sample was first treated, and then stimulated with acetylcholine, to explore whether the peptide derivative isomer of the present invention could inhibit the agonistic effect of acetylcholine on smooth muscle after treatment.
  • the experimental rats were fasted and water-free for 12 hours before the experiment. Inject 10% chloral hydrate intraperitoneally at a dose of 0.1mL/25g. After the rat is anesthetized, the abdominal cavity is cut open to expose the enlarged cecum. A section of ileum is taken out from the ileocecal intestine and washed in benchtop solution that has been pre-oxygenated for 10 minutes. Cut several 4cm long sections of ileum and incubate them in 4°C benchtop liquid for later use; partially ligate and tie them with cotton thread at both ends of the ileum to form a ring;
  • the results of experiment 1 show that compared with isomer E, isomer F, isomer G and isomer H, the peptide derivatives of the present invention are isomer A, isomer B, isomer C and isomer H.
  • Construct D has a higher inhibitory rate on the contraction of ileal smooth muscle. After being stimulated by acetylcholine, a high-level signal peak will be generated. After being treated with 100 ppm of the peptide derivative isomer of the present invention, the high-level signal peak will be significantly weakened, and the muscle tension will be reduced, resulting in an inhibitory effect; as the test concentration increases, the muscle will be affected. The inhibitory effect on contraction is enhanced in a dose-dependent manner. Among them, the inhibition rate of isomer A at 100 ppm is 47.06%, and the inhibition rate at 200 ppm is as high as 64.71%. It has the best effect of inhibiting muscle contraction.
  • the results of experiment 2 show that compared with those treated with desktop solution, the muscle contraction rates obtained after being treated with isomer E, isomer F, isomer G, and isomer H and then stimulated by acetylcholine are higher. There is no detailed difference between the muscle contraction rate obtained after acetylcholine stimulation after treatment with benchtop solution; however, after treatment with isomer A, isomer B, isomer C, and isomer D, and then given acetylcholine stimulation, the contraction rate can be significantly reduced.
  • the muscle contraction tension of acetylcholine inhibits the stimulating effect of acetylcholine on smooth muscles, and has a good inhibitory effect on muscle contraction; the muscle contraction rate after treatment with desktop solution and stimulation with acetylcholine is as high as 191.67%, and the muscle contraction rate after treatment with acetylcholine is as high as 191.67% after treatment with isomer A of the present invention.
  • the muscle contraction rate was only 41.67%.
  • the muscle contraction rate after stimulation with acetylcholine was 60.28%.
  • the muscle contraction rate after stimulation with acetylcholine was 65.35%.
  • the muscle contraction rate was 65.35%.
  • the muscle contraction rate after stimulation with acetylcholine was 50.24%; among them, isomer A of the present invention has the best effect on inhibiting muscle contraction.
  • Acetylcholine as a neurotransmitter, can directly stimulate acetylcholine receptors, triggering a signaling cascade to cause muscle contraction.
  • muscle contraction can be inhibited and conditions, disorders, or diseases caused by muscle contraction can be treated or treated.
  • isomers of the peptide derivatives of the present invention have an obvious effect of inhibiting muscle contraction, and therefore can be used to treat or care for conditions, disorders or diseases caused by muscle contraction; treat, prevent or repair skin aging or photoaging; It can also be used in products to reduce, prevent or treat wrinkles.
  • isomer A has the best technical effect.
  • 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 was then added to the mixture. Finally, phase C is added to obtain a microemulsion composition containing isomer A.

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Abstract

式(I)的肽衍生物异构体、其立体异构体的混合物、或其盐、或它们的组合物、以及它们的用途。该式(I)肽衍生物的异构体、含有这些异构体的组合物，能够增加人皮肤成纤维细胞的活性，显著增加细胞黏附，促进胶原蛋白表达，从而增加皮肤弹性和/或皮肤紧致度。

Description

肽衍生物异构体及其组合物和用途 技术领域
本发明涉及一种肽衍生物异构体、以及含有这些异构体的组合物及其用途。
背景技术
据研究，下式(i)所示的肽类衍生物(分子式为C19H29N5O4)显示出有益的生物学性质，但仍缺乏对该肽类衍生物的构效关系研究。
式(i)的化合物有3个手性中心，因此有8个异构体，结构式见下表1。
表1肽衍生物异构体的名称及结构式

然而，未见式(i)化合物异构体的研究报道。不同异构体可能具有截然不同的功效活性，因此有必要对式(i)化合物的8种异构体进行研究，阐明其立体结构与生物活性之间的关系，以弥补化妆品领域皮肤活性物研发中对于异构体研究方面的缺失。
发明内容
式(i)化合物有3个手性中心，其中2个位于羟脯氨酸残基，共有8种异构体。经研究发现，式(i)的化合物不同的异构体的功效活性具有显著差别。本发明旨在提供一种具有修复抗衰功效以及抑制肌肉收缩作用的肽衍生物异构体。
鉴于此，本发明提供一种式(I)所示的肽衍生物异构体，其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，
式(I)中，X是：
本发明提供的式(I)肽衍生物的异构体、含有这些异构体的美容组合物或药物组合物，能够增加人皮肤成纤维细胞的活性，显著增加细胞黏附，促进胶原蛋白表达，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛；治疗、预防或修复皮肤老化或光老化；还能够抑制肌肉收缩，可用于治疗或护理由肌肉收缩导致的病况、失调或疾病；亦可用于减少、预防或治疗皱纹的产品中。
本发明还包括本发明化合物的所有合适的同位素变体。本发明化合物的同位素变体此处理解为是指这样的化合物：其中在本发明化合物内至少一个原子被替换为相同原子序数的另一个原子，但所述另一原子的原子质量不同于自然界中通常或主要存在的原子质量。可掺入本发明化合物中的同位素的实例是：氢、碳、氮、氧的那些，例如2H(氘)、3H(氚)、13C、14C、15N、17O或18O。本发明化合物的特定的同位素变体(特别是其中已经掺入一种或多种放射性同位素的那些)可能有利于，例如检查在体内的作用机理或活性化合物的分布；由于相对简单的可制备性和可检测性，尤其是用3H或14C同位素标记的化合物适用于该目的。另外，由于化合物的更强的代谢稳定性，同位素(例如氘)的掺入可以产生特定的治疗益处，例如体内半衰期的延长或所需活性剂量的降低；因此，在某些情况下，本发明化合物的这种改性还可构成本发明的优选实施方案。通过本领域技术人员已知的方法，例如通过在下文中进一步描述的方法和在实施例中所述的方法，通过使用各自的试剂和/或起始物质的相应的同位素改性物，可制备本发明化合物的同位素变体。
此外，本发明还包括本发明化合物的前药。术语“前药”在本文中意指这样的化合物：其本身可以是生物学上有活性的或无活性的，但是在它们在身体内的停留时间期间，其反应 (例如代谢或水解)生成本发明的化合物。
式(I)化合物可以与酸形成单价或多价的、均质或混合的盐，例如与无机酸如盐酸、氢溴酸、硫酸或磷酸；或者与合适的羧酸，例如脂族的一元或二元羧酸如甲酸、乙酸、三氟乙酸、三氯乙酸、丙酸、乙醇酸、琥珀酸、富马酸、丙二酸、马来酸、草酸、邻苯二甲酸、柠檬酸、乳酸或酒石酸；或者与芳族羧酸如苯甲酸或水杨酸；或者与芳族-脂族羧酸如扁桃酸或肉桂酸；或者与杂芳族羧酸如烟酸；或者与脂族或芳族磺酸如甲磺酸或甲苯磺酸。优选的为皮肤病学适合的盐。特别优选的为与乙酸和/或三氟乙酸和/或乳酸形成的盐。
本发明式(I)所示的肽衍生物异构体，或其美容上可接受的盐、或其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，例如固相合成法、液相合成法或固相与液相结合的方法，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、或植物来源的蛋白质的控制水解来制备。
可选地，一种获得式(I)所示的异构体的方法包括以下步骤：
(1)Boc-β-Ala-OH与HOSu缩合生成Boc-β-Ala-OSu；
(2)Boc-β-Ala-OSu在弱碱性条件下与羟脯氨酸反应生成Boc-β-Ala-羟脯氨酸；
(3)Fmoc-(S)-Dab(Boc)-OH与苄胺缩合生成Fmoc-(S)-Dab(Boc)-NH-Bzl；
(4)步骤(3)的产物脱除Fmoc保护基后，与Boc-β-Ala-羟脯氨酸缩合生成Boc-β-Ala-羟脯氨酸-(S)-Dab(Boc)-NH-Bzl；
(5)步骤(4)的产物脱除Boc保护基，纯化冻干得到式(I)所示的肽衍生物异构体，或其美容上可接受的盐、或其药学上可接受的盐；
可选地，所述羟脯氨酸是(2S,4R)-Hyp、(2S,4S)-Hyp、(2R,4S)-Hyp或(2R,4R)-Hyp。
本发明的另一方面，提供一种美容或药物组合物，包括有效量的上述的式(I)所示的肽衍生物异构体，其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。
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。
待施用的美容上或药学上有效量的本发明的肽衍生物异构体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 异构体的具体性质。
“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异构体的量。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异构体；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。
本发明的另一方面，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式(I)所示的肽衍生物异构体，其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”是指与本发明的异构体一起施用的稀释剂、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异构体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异构体。在该缓释系统中包含的异构体的量将取决于例如该组合物将被给予的部位、本发明的异构体的释 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。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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在制备用于治疗或护理皮肤或粘膜的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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在制备用于增加皮肤弹性和/或皮肤紧致度的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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在制备用于增加胶原蛋白生成、提高成纤维细胞的活性和/或增加细胞黏附的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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在制备用于治疗或护理由肌肉收缩导致的病况、失调或疾病的美容组合物或药物组合物中的用途，其中，所述失调或疾病是张力障碍。
本发明的实施方案，所述失调或疾病是张力障碍，具体地，所述张力障碍为局灶性肌张力障碍；张力障碍包括，但不限于脸痉挛、扭转性张力障碍、颈张力障碍或斜颈症、喉张力障碍或痉挛性发音困难、口下颌张力障碍、肢体张力障碍诸如书写痉挛或音乐家痉挛或脚部张力障碍、夜间磨牙、半侧颜面痉挛、抽搐症和/或斜视；节段性肌张力障碍、梅杰氏综合征、多灶性张力障碍、偏侧肌张力障碍、多巴胺反应性肌张力障碍和濑川张力障碍。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物异构体，其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在制备用于减少、预防或治疗皱纹的产品中 的用途。
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”，指的是给予本发明的肽衍生物异构体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”，指的是本发明的肽衍生物异构体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。
本发明相对于现有技术所取得的有益效果包括：
1、式(i)化合物有3个手性碳原子，其中2个位于羟脯氨酸残基，共有8种异构体。现有技术没有对式(i)化合物的异构体进行研究，本发明对其研究后发现，只有其中4种异构体具有修复抗衰功效以及抑制肌肉收缩作用，这4种异构体分别是式(i)化合物(2S,4R)-N-S构型(异构体A)、式(i)化合物(2S,4S)-N-S构型(异构体B)、式(i)化合物(2R,4S)-N-S构型(异构体C)以及式(i)化合物(2R,4R)-N-S构型(异构体D)， 即本发明所述的肽衍生物异构体。
2、本发明所述的肽衍生物异构体，合成方便，安全性高，能够增加皮肤成纤维细胞的活性，显著增加细胞黏附，促进胶原蛋白表达，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛；治疗、预防或修复皮肤老化或光老化；还能够抑制肌肉收缩，可用于治疗或护理由肌肉收缩导致的病况、失调或疾病；亦可用于减少、预防或治疗皱纹的产品中。其中，异构体A的技术效果最优。
附图说明
为了更清楚地说明本发明的技术方案，下面将对本发明的描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为测试样品对HSF细胞增殖的影响结果图。*表示给药组与空白对照组相比具有统计学差异，p<0.05(n＝5)。**表示给药组与空白对照组相比差异显著，p<0.01(n＝5)。
图2为测试样品对HaCaT细胞黏附的影响结果图。*表示给药组与空白对照组相比具有统计学差异，p<0.05(n＝5)。**表示给药组与空白对照组相比差异显著，p<0.01(n＝5)。
图3为测试样品对胶原蛋白含量的影响结果图。###表示非紫外辐射的空白对照组与UV组(UV+PBS)相比，差异极为显著，p<0.001(n＝3)。*表示给药组(UV+样品)与UV组(UV+PBS)相比，具有统计学差异，p<0.05(n＝3)。**表示给药组(UV+样品)与UV组(UV+PBS)相比，差异显著，p<0.01(n＝3)。
具体实施方式
为使本发明的所述目的、特征和优点能够更加明显易懂，下面结合附图和实施例对本发明作进一步详细的说明。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在本发明中，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。
HOBt：1-羟基苯并三唑；THF：四氢呋喃；DCM：二氯甲烷；DCC：N,N'-二环己基碳二亚胺；NMM：N-甲基吗啉；piperidine：哌啶；HOSu：N-羟基丁二酰亚胺；EDC.HCl： 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐；EtOAc：乙酸乙酯；Fmoc：9-芴基甲氧羰基；Boc：叔丁氧羰基；Bzl：苄基；β-Ala：β-丙氨酸；Hyp：羟脯氨酸；Dab：2,4-二氨基丁酸；
HOSu：-OSu：
(2S,4R)-Hyp：-(2S,4R)-Hyp-：
(2S,4S)-Hyp：-(2S,4S)-Hyp-：
(2R,4S)-Hyp：-(2R,4S)-Hyp-：
(2R,4R)-Hyp：-(2R,4R)-Hyp-：
(S)-Dab：-(S)-Dab-：
实施例1制备Boc-β-Ala-OSu
1.1将94.6g的Boc-β-Ala-OH，69g的HOSu加入3L圆底瓶中，用300mL DCM搅拌溶解，置于冰浴中，降温到13℃-15℃，搅拌5min。
1.2将124g的DCC置于500mL烧杯中，加入200mL DCM搅拌溶解，缓慢分次加入至1.1步骤所得溶液中，撤去冰浴，室温反应5h，利用TLC分析跟踪反应至反应完全。
1.3反应完后过滤，滤液浓缩后加入300mL石油醚，析出白色固体，过滤得到固体，用石油醚洗涤3次，干燥得到140g产物(理论值143.1g，收率98％)。
实施例2制备H-(S)-Dab(Boc)-NH-Bzl
2.1制备Fmoc-(S)-Dab(Boc)-NH-Bzl
将220g的Fmoc-(S)-Dab(Boc)-OH，54g的HOBt，溶于800mL无水THF，冰浴搅拌5min，滴加123g含DCC的THF溶液。滴加完毕后，继续搅拌5min，分次加入53g苄胺，200mL NMM，撤去冰浴，室温反应3h，利用TLC分析跟踪反应至反应完全。
过滤除掉形成的脲衍生物，并用THF洗涤3次，滤液浓缩得到浅黄色固体，加入2.5L DCM溶解，用饱和碳酸氢钠溶液洗涤7次，硫酸氢钾溶液洗涤3次，饱和氯化钠溶液洗涤3次，加入200g无水硫酸钠干燥3h，过滤浓缩得到白色固体。
2.2脱去Fmoc保护基
加入950mL piperidine/DCM(含20％piperidine)将2.1步骤所得固体溶解，室温搅拌反应3h，脱去Fmoc保护基，蒸发除掉DCM，得到黄色粘性固体。加入3L石油醚浸泡12h，打浆5h，抽滤得到固体，用石油醚洗涤9次。加入1.5L乙酸乙酯搅拌溶解所得固体，过滤不溶物，分别用饱和碳酸氢钠溶液洗涤7次，饱和氯化钠溶液洗涤3次，加入240g无水硫酸钠干燥1h，浓缩得到140g产物。
实施例3制备H-β-Ala-(2S,4R)-Hyp-(S)-Dab-NH-Bzl.HCl
3.1制备Boc-β-Ala-(2S,4R)-Hyp-OH
将5g的(2S,4R)-Hyp溶于50mL水中，加入11.6g碳酸氢钠，搅拌降温到10℃以下。将11g的Boc-β-Ala-OSu溶于THF后滴加入体系中。滴加完后撤去冰浴，室温反应6h，利用TLC分析跟踪反应至反应完全。
反应完后，浓缩，调节pH值到2，用THF:EtOAc(V:V＝1:1)萃取6次，合并有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥。抽滤，滤液浓缩得到半固态糖浆状物，用石油醚打浆得到10.3g白色固体。
3.2制备Boc-β-Ala-(2S,4R)-Hyp-(S)-Dab(Boc)-NH-Bzl
将2.9g的H-(S)-Dab(Boc)-NH-Bzl，1.0g的HOBt，用50mL THF溶解，加入20mL NMM，2.9g的EDC.HCl经THF溶解后缓慢加入上述反应体系，继续搅拌5min。2.7g的Boc-β-Ala-(2S,4R)-Hyp-OH溶于50mL THF，所得溶液分步滴加入反应体系，滴加完毕，撤去冰浴，常温反应6h，利用TLC分析跟踪反应至反应完全。过滤不溶物，蒸发掉THF得到黄色油状物。
用200mL乙酸乙酯将上述油状物溶解，过滤不溶物，转至分液漏斗，用饱和碳酸氢钠溶液洗涤8次，5％的硫酸氢钾洗涤7次，饱和氯化钠溶液洗涤3次，加入15g无水硫酸钠干燥1h，浓缩得到黄色油状固体。
3.3脱去Boc保护基
取128g甲醇加入1L圆底瓶中，冰水浴搅拌10min，缓慢滴加314g乙酰氯，滴加完毕，继续低温反应1h。将制备好的溶液加入到上述3.2步骤浓缩得到黄色油状固体中，析出白色固体，滴加完毕后继续浸泡反应1h。用乙酸乙酯洗涤2次，异丙醚洗涤2次，得到4.5g白色固体，粗产物纯度87.4％。
3.4纯化
取4.5g上述3.3步骤所得粗产物，溶于200mL纯水中，过滤得到微黄色液体，用强阴离子树脂调节pH值至6.19，过滤。将过滤后的样品通过反相HPLC纯化处理，纯化梯度如下：
收集馏分，浓缩冻干，得到纯度99.138％的异构体H-β-Ala-(2S,4R)-Hyp-(S)-Dab-NH-Bzl.HCl。
实施例4制备H-β-Ala-(2S,4S)-Hyp-(S)-Dab-NH-Bzl.HCl
4.1制备Boc-β-Ala-(2S,4S)-Hyp-OH
将5g的(2S,4S)-Hyp溶于50mL水中，加入11.6g碳酸氢钠，搅拌降温到10℃以下。将11g的Boc-β-Ala-OSu溶于THF后滴加入体系中。滴加完后撤去冰浴，室温反应6h，利用TLC分析跟踪反应至反应完全。
反应完后，浓缩，调节pH值到2，用THF:EtOAc(V:V＝1:1)萃取6次，合并有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥。抽滤，滤液浓缩得到半固态糖浆状物，用石油醚打浆得到9.6g白色固体。
4.2制备Boc-β-Ala-(2S,4S)-Hyp-(S)-Dab(Boc)-NH-Bzl
将2.9g的H-(S)-Dab(Boc)-NH-Bzl，1.0g的HOBt，用50mL THF溶解，加入20mL NMM，2.9g的EDC.HCl经THF溶解后缓慢加入上述反应体系，继续搅拌5min。2.7g的Boc-β-Ala-(2S,4S)-Hyp-OH溶于50mL THF，所得溶液分步滴加入反应体系，滴加完毕，撤去冰浴，常温反应6h，利用TLC分析跟踪反应至反应完全。过滤不溶物，蒸发掉THF得到黄色油状物。
用200mL乙酸乙酯将上述油状物溶解，过滤不溶物，转至分液漏斗，用饱和碳酸氢钠溶液洗涤8次，5％的硫酸氢钾洗涤7次，饱和氯化钠溶液洗涤3次，加入15g无水硫酸钠干燥1h，浓缩得到黄色油状固体。
4.3脱去Boc保护基
取128g甲醇加入1L圆底瓶中，冰水浴搅拌10min，缓慢滴加314g乙酰氯，滴加完毕，继续低温反应1h。将制备好的溶液加入到上述4.2步骤浓缩得到黄色油状固体中，析出白色固体，滴加完毕后继续浸泡反应1h。用乙酸乙酯洗涤2次，异丙醚洗涤2次，得到4.2g白色固体，粗产物纯度65.2％。
4.4纯化
取4.2g上述4.3步骤所得粗产物，溶于200mL纯水中，过滤得到微黄色液体，用强阴离子树脂调节pH值至6.19，过滤。将过滤后的样品通过反相HPLC纯化处理，纯化梯度如下：

收集馏分，浓缩冻干，得到纯度99.214％的异构体H-β-Ala-(2S,4S)-Hyp-(S)-Dab-NH-Bzl.HCl。
实施例5制备H-β-Ala-(2R,4S)-Hyp-(S)-Dab-NH-Bzl.HCl
5.1制备Boc-β-Ala-(2R,4S)-Hyp-OH
将5g的(2R,4S)-Hyp溶于50mL水中，加入11.6g碳酸氢钠，搅拌降温到10℃以下。将11g的Boc-β-Ala-OSu溶于THF后滴加入体系中。滴加完后撤去冰浴，室温反应6h，利用TLC分析跟踪反应至反应完全。
反应完后，浓缩，调节pH值到2，用THF:EtOAc(V:V＝1:1)萃取6次，合并有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥。抽滤，滤液浓缩得到半固态糖浆状物，用石油醚打浆得到10.8g白色固体。
5.2制备Boc-β-Ala-(2R,4S)-Hyp-(S)-Dab(Boc)-NH-Bzl
将5.3g的H-(S)-Dab(Boc)-NH-Bzl，1.8g的HOBt，用50mL THF溶解，加入20mL NMM，2.9g的EDC.HCl经THF溶解后缓慢加入上述反应体系，继续搅拌5min。4.9g的Boc-β-Ala-(2R,4S)-Hyp-OH溶于50mL THF，所得溶液分步滴加入反应体系，滴加完毕，撤去冰浴，常温反应6h，利用TLC分析跟踪反应至反应完全。过滤不溶物，蒸发掉THF得到黄色油状物。
用200mL乙酸乙酯将上述油状物溶解，过滤不溶物，转至分液漏斗，用饱和碳酸氢钠溶液洗涤8次，5％的硫酸氢钾洗涤7次，饱和氯化钠溶液洗涤3次，加入15g无水硫酸钠干燥1h，浓缩得到黄色油状固体。
5.3脱去Boc保护基
取128g甲醇加入1L圆底瓶中，冰水浴搅拌10min，缓慢滴加314g乙酰氯，滴加完毕，继续低温反应1h。将制备好的溶液加入到上述5.2步骤浓缩得到黄色油状固体中，析出白色固体，滴加完毕后继续浸泡反应1h。用乙酸乙酯洗涤2次，异丙醚洗涤2次，得到4.8g白色固体，粗产物纯度86.4％。
5.4纯化
取4.8g上述5.3步骤所得粗产物，溶于200mL纯水中，过滤得到微黄色液体，用强阴离子树脂调节pH值至6.24，用150mL纯水洗涤树脂，过滤。将过滤后的样品通过反相HPLC纯化处理，纯化梯度如下：
收集馏分，浓缩冻干，得到纯度99.466％的异构体H-β-Ala-(2R,4S)-Hyp-(S)-Dab-NH-Bzl.HCl。
实施例6制备H-β-Ala-(2R,4R)-Hyp-(S)-Dab-NH-Bzl.HCl
6.1制备Boc-β-Ala-(2R,4R)-Hyp-OH
将5g的(2R,4R)-Hyp溶于50mL水中，加入11.6g碳酸氢钠，搅拌降温到10℃以下。将11g的Boc-β-Ala-OSu溶于THF后滴加入体系中。滴加完后撤去冰浴，室温反应6h，利用TLC分析跟踪反应至反应完全。
反应完后，浓缩，调节pH值到2，用THF:EtOAc(V:V＝1:1)萃取6次，合并有机相，用饱和氯化钠溶液洗涤，无水硫酸钠干燥。抽滤，滤液浓缩得到半固态糖浆状物，用石油醚打浆得到11.3g白色固体。
6.2制备Boc-β-Ala-(2R,4R)-Hyp-(S)-Dab(Boc)-NH-Bzl
将6.5g的H-(S)-Dab(Boc)-NH-Bzl，2.3g的HOBt，用50mL THF溶解，加入20mL NMM，2.9g的EDC.HCl经THF溶解后缓慢加入上述反应体系，继续搅拌5min。6.1g的Boc-β-Ala-(2R,4R)-Hyp-OH溶于50mL THF，所得溶液分步滴加入反应体系，滴加完毕，撤去冰浴，常温反应6h，利用TLC分析跟踪反应至反应完全。过滤不溶物，蒸发掉THF得到黄色油状物。
用200mL乙酸乙酯将上述油状物溶解，过滤不溶物，转至分液漏斗，用饱和碳酸氢钠溶液洗涤8次，5％的硫酸氢钾洗涤7次，饱和氯化钠溶液洗涤3次，加入15g无水硫酸钠 干燥1h，浓缩得到黄色油状固体。
6.3脱去Boc保护基
取128g甲醇加入1L圆底瓶中，冰水浴搅拌10min，缓慢滴加314g乙酰氯，滴加完毕，继续低温反应1h。将制备好的溶液加入到上述6.2步骤浓缩得到黄色油状固体中，析出白色固体，滴加完毕后继续浸泡反应1h。用乙酸乙酯洗涤2次，异丙醚洗涤2次，得到5.8g白色固体，粗产物纯度88.9％。
6.4纯化
取5.8g上述6.3步骤所得粗产物，溶于120mL纯水中，过滤得到微黄色液体，用强阴离子树脂调节pH值至6.19，120mL纯水洗涤树脂，过滤。将过滤后的样品通过反相HPLC纯化处理，纯化梯度如下：
收集馏分，浓缩冻干，得到纯度99.025％的异构体H-β-Ala-(2R,4R)-Hyp-(S)-Dab-NH-Bzl.HCl。
实施例7
所获得的这些肽衍生物异构体通过ESI-MS测定分子量，通过旋光仪检测旋光度(d＝0.5dm，钠光谱589.3nm)，计算比旋光度，测试结果见下表2。
表2肽衍生物异构体的分子量及比旋光度

实施例8计算化合物异构体与受体的相互作用
选择以肌肉烟碱型乙酰胆碱受体(PDB Code：4ZJS)作为靶点蛋白，将式(i)化合物的8种异构体分别与靶点蛋白进行分子对接，并利用打分函数对结果进行评估，结果见下表3。
表3异构体与受体的分子对接评分结果
由表3结果可知，异构体A、异构体B、异构体C、异构体D能够更好地与受体进行结合，其中异构体A的评分结果最优。
实施例9细胞增殖实验
9.1试剂与材料
胎牛血清(Gibco)、DMEM培养基(Gibco)、青霉素、链霉素、MTT(Sigma)。
9.2仪器
酶标仪(美国MD)、CO2培养箱(上海一恒)、超净工作台(苏州净化)。
9.3细胞株
人皮肤成纤维细胞(HSF)。
9.4待测样品
给药组：
异构体A，测试浓度为50ppm、100ppm；
异构体B，测试浓度为50ppm、100ppm；
异构体C，测试浓度为50ppm、100ppm；
异构体D，测试浓度为50ppm、100ppm；
异构体E，测试浓度为50ppm、100ppm；
异构体F，测试浓度为50ppm、100ppm；
异构体G，测试浓度为50ppm、100ppm；
异构体H，测试浓度为50ppm、100ppm；
空白对照组：PBS。
9.5实验方法
取冻存的HSF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2000个/孔接种在96孔板中，待细胞贴壁后，按照倍比稀释法，分别加入给药组和对照组样品，补充培养基至200μL，置于37℃、5％CO2培养箱中孵育72h。
之后每孔加入22μL 5mg/ml MTT，继续于37℃、5％CO2培养箱中孵育4h。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。
9.6结果
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。
测试样品对HSF细胞增殖的影响结果见图1。结果显示，与空白对照组相比，本发明的肽衍生物异构体在100ppm范围内对HSF细胞均没有毒性作用。与异构体E、异构体F、异构体G、异构体H相比，本发明的肽衍生物异构体A、异构体B、异构体C、异构体D能够提高细胞活性，促进HSF细胞增殖。其中，异构体A在50ppm浓度下就能够增加HSF细胞活性，与空白对照组相比具有统计学差异。随着浓度升高，促细胞增殖作用随之增强， 异构体A在100ppm浓度下能够明显提高细胞活性，显著促进HSF细胞增殖。
由此可知，本发明的肽衍生物异构体对成纤维细胞不仅没有毒性作用，而且还能提高成纤维细胞的活性，促进其增殖，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，其中异构体A的技术效果最优。
实施例10促细胞黏附实验
10.1试剂与材料
胎牛血清(Gibco)、DMEM培养基(Gibco)、青霉素、链霉素、MTT(Sigma)。
10.2仪器
酶标仪(美国MD)、CO2培养箱(上海一恒)、超净工作台(苏州净化)。
10.3细胞株
人角质形成细胞(HaCaT)。
10.4待测样品
给药组：
异构体A，测试浓度为50ppm、100ppm；
异构体B，测试浓度为50ppm、100ppm；
异构体C，测试浓度为50ppm、100ppm；
异构体D，测试浓度为50ppm、100ppm；
异构体E，测试浓度为50ppm、100ppm；
异构体F，测试浓度为50ppm、100ppm；
异构体G，测试浓度为50ppm、100ppm；
异构体H，测试浓度为50ppm、100ppm；
空白对照组：PBS。
10.5实验目的
本实验的目的是通过选择HaCaT角质层细胞，铺板在测试样品包被好的96孔板中，孵育且经一段时间的外力作用后，评价测试样品对细胞黏附的影响，以此确定本发明的异构体能否改善细胞弹性。
10.6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％CO2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。顺时针翻转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％CO2培养箱中孵育3h。弃去溶液，加入150μL的DMSO。使用酶标仪读取490nm和630nm波长下的参比OD值。
10.7结果
在经过三维力作用之后，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，即可反映细胞的黏附作用。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，细胞的弹性也越强。
测试样品对HaCaT细胞黏附的影响结果见图2。结果显示，与空白对照组相比，异构体E、异构体F、异构体G、异构体H没有表现出促HaCaT细胞黏附的作用，而本发明的肽衍生物异构体A、异构体B、异构体C、异构体D能够促进HaCaT细胞黏附。其中，异构体A在50ppm浓度下就能够促进HaCaT细胞黏附，与空白对照组相比具有统计学差异。随着浓度增大，促细胞黏附的作用随之增强，异构体A在100ppm浓度下能够明显提高细胞黏附能力，促进HaCaT细胞黏附。
由此可知，本发明的肽衍生物异构体能够提高细胞黏附能力，显著增加细胞间黏附和细胞-细胞外基质的黏附，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛。其中，异构体A在更低浓度下便表现出较强的促细胞黏附作用，效果最优。
实施例11胶原蛋白含量测试
11.1试剂与材料
胎牛血清(Gibco)、DMEM培养基(Gibco)、青霉素、链霉素、MTT(Sigma)、胶原蛋白I ELISA试剂盒(伊艾博)、RIPA裂解液、BCA蛋白试剂盒(碧云天)。
11.2仪器
酶标仪(美国MD)、CO2培养箱(上海一恒)、超净工作台(苏州净化)、恒温箱(上海一恒)。
11.3细胞株
人皮肤成纤维细胞(HSF)。
11.4待测样品
给药组：
异构体A，测试浓度为100ppm；
异构体B，测试浓度为100ppm；
异构体C，测试浓度为100ppm；
异构体D，测试浓度为100ppm；
异构体E，测试浓度为100ppm；
异构体F，测试浓度为100ppm；
异构体G，测试浓度为100ppm；
异构体H，测试浓度为100ppm；
空白对照组：PBS；
UV组：UV辐射，加PBS。
11.5实验目的
以非紫外辐射的溶剂对照组(PBS)作为空白对照，进行紫外辐射后，胶原蛋白I含量降低50％以上即表明紫外建模成功。继而以紫外辐射(UV)组的胶原蛋白I含量为对照，分析给药后各组的胶原蛋白I含量变化趋势，评价本发明的异构体对胶原蛋白表达的影响。
11.6实验方法
取冻存的HSF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。
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％左右时建模。空白对照组加800μL培养基，不进行UVA照射；UV组和给药组，加入适量PBS反复洗至无色后，加入800μL PBS，置于UV灯下照射，灯源和培养瓶间距15cm。经照射后，弃去PBS，UV组加入PBS溶液和培养基，给药组加入培养基和倍比稀释药物至1200μL。空白对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育48h。
培养结束后使用细胞刮刮掉细胞，吹打混匀。使用RIPA提取液提取蛋白，并通过BCA蛋白试剂盒检测蛋白含量，将蛋白定到1mg/mL。按照胶原蛋白I ELISA试剂盒操作说明书进行操作。15min内用酶标仪在450nm处依序测量各孔的OD值。
11.7结果
胶原蛋白是结缔组织中发现的最丰富的蛋白质，对于皮肤饱满和紧致具有重要作用。在紫外线过暴环境中，弹性蛋白酶活性大幅增加，弹性蛋白水解；胶原蛋白的合成也受到抑制。本实验采用测试样品处理经紫外线辐射后的细胞，检测相应细胞中的胶原蛋白I含量，以确定本发明的异构体是否能够促进胶原蛋白的合成。
测试样品对胶原蛋白含量的影响结果见图3。结果显示，与空白对照组相比，UV组的胶原蛋白含量大幅降低，表明成功建立了紫外模型；与UV组相比，100ppm的异构体E、异构体F、异构体G、异构体H以及异构体B均没有表现出促进胶原蛋白表达的作用，而异构体A、异构体C、异构体D能够促进胶原蛋白的合成，特别是100ppm的异构体A能够明显促进胶原蛋白表达，增加经紫外辐射后的胶原蛋白含量。相比之下，100ppm的异构体A具有更优的促胶原蛋白生成的作用。
由此可知，本发明的肽衍生物异构体能够促进胶原蛋白表达，增加胶原蛋白含量，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，治疗、预防或修复皮肤老化或光老化。其中，异构体A的技术效果最优。
实施例12肌肉收缩抑制实验
12.1实验动物
10周龄雌性SD大鼠，重量220g-260g。
12.2实验仪器
HW-400S恒温平滑肌槽、BL-420E生物机能实验系统、张力换能器。
12.3实验试剂
氯化乙酰胆碱(上海源叶)、水合氯醛(上海麦克林)。
12.4待测样品
异构体A，测试浓度为100ppm、200ppm；
异构体B，测试浓度为100ppm、200ppm；
异构体C，测试浓度为100ppm、200ppm；
异构体D，测试浓度为100ppm、200ppm；
异构体E，测试浓度为100ppm、200ppm；
异构体F，测试浓度为100ppm、200ppm；
异构体G，测试浓度为100ppm、200ppm；
异构体H，测试浓度为100ppm、200ppm。
12.5实验目的
本实验的目的是通过以大鼠回肠肌作为实验对象，在乙酰胆碱刺激后，给药观察测试样品对回肠肌的律动情况，评价测试样品对肌肉收缩的影响，以此确定本发明的肽衍生物异构体能否抑制肌肉收缩。进一步地，改变乙酰胆碱加入顺序，先用测试样品处理，再用乙酰胆碱刺激，探究本发明肽衍生物异构体处理后是否能够抑制乙酰胆碱对平滑肌的激动作用。
12.6实验方法
将实验大鼠实验前禁食不禁水12小时。按0.1mL/25g剂量腹腔注射10％水合氯醛，待大鼠麻醉后，剪开腹腔，露出膨大盲肠，距回盲部肠处取出一段回肠，在预先通氧10min的台式液中洗干净，剪取数段4cm长回肠孵育于4℃台式液中备用；在回肠两端分别用棉线部分结扎并打结使之成一环；
打开HW-400S恒温平滑肌槽、BL-420E生物机能实验系统，将张力换能器连接至通道1，调节孵育温度为37℃，仪器预热0.5小时，通氧10min以上。肠段一端用棉线固定在进气支架口，另一端与张力换能器相连，适当调节换能器的位置与高度，使离体肠段置于平滑肌槽中央，开始记录肠活动情况；
实验①：待肌肉收缩稳定后，加入终浓度5μg/mL的乙酰胆碱，直至肌肉活动出现高位而稳定的波形，平衡5分钟后，分别加入待测样品。每更换一种受试药物，换新的回肠段。重复4次实验，仪器记录相关数据，根据下列公式计算回肠平滑肌收缩抑制率：回肠平滑肌收缩抑制率＝(乙酰胆碱刺激后收缩力-待测样品给药后收缩力)/乙酰胆碱刺激后收缩力×100％。
实验②：待肌肉收缩稳定后，加入台式液或者测试样品(测试浓度200ppm)，平衡5分钟以上，波形稳定后，加入终浓度1μg/mL的乙酰胆碱，记录瞬时波形。重复4次实验，仪器记录相关数据，根据下列公式计算乙酰胆碱促回肠平滑肌收缩率：乙酰胆碱促回肠平滑肌收缩率＝(乙酰胆碱刺激后收缩力-待测样品处理时收缩力)/待测样品处理时收缩力×100％。
实验数据以均数标准差(Mean±SD)表示。
12.7实验结果
实验①测试样品的回肠平滑肌收缩抑制率结果见下表4。
表4测试样品的回肠平滑肌收缩抑制率(％)
实验①结果表明，与异构体E、异构体F、异构体G、异构体H相比，本发明的肽衍生物异构体A、异构体B、异构体C、异构体D对回肠平滑肌的收缩抑制率更高。给予乙酰胆碱刺激后，会产生一段高位信号峰，给予100ppm本发明的肽衍生物异构体处理后，高位信号峰明显减弱，肌肉张力减小，产生抑制作用；随着测试浓度升高，对肌肉收缩的抑制作用随之增强，呈一定剂量依赖性。其中，异构体A在100ppm的抑制率为47.06％，200ppm的抑制率高达64.71％，其抑制肌肉收缩的效果最优。
通过实验②得到的乙酰胆碱促回肠平滑肌收缩率结果见下表5。
表5乙酰胆碱促回肠平滑肌收缩率(％)
实验②结果表明，与台式液处理的相比，经异构体E、异构体F、异构体G、异构体H处理后，再给予乙酰胆碱刺激，得到的肌肉收缩率均较高，与台式液处理经乙酰胆碱刺激后得到的肌肉收缩率没有明细差异；而经异构体A、异构体B、异构体C、异构体D处理后，再给予乙酰胆碱刺激，能明显减小乙酰胆碱的促肌肉收缩张力，抑制乙酰胆碱对平滑肌的激动作用，具有较好的抑制肌肉收缩效果；给予台式液处理经乙酰胆碱刺激后肌肉收缩率高达191.67％，而给予本发明异构体A处理经乙酰胆碱刺激后肌肉收缩率仅为41.67％，给予异构体B处理经乙酰胆碱刺激后肌肉收缩率为60.28％，给予异构体C处理经乙酰胆碱刺激后肌肉收缩率为65.35％，给予异构体D处理经乙酰胆碱刺激后肌肉收缩率为50.24％；其中，本发明的异构体A抑制肌肉收缩作用最优。
抑制面部表情肌的收缩可以有效地改善眼角纹、法令纹等面部皱纹。乙酰胆碱作为神经递质，能直接激动乙酰胆碱受体，触发信号级联反应而引起肌肉收缩。通过抑制乙酰胆碱释放或阻止乙酰胆碱与其受体结合，可以抑制肌肉收缩，治疗或护理由肌肉收缩导致的病况、失调或疾病。由此可知，本发明的肽衍生物异构体具有明显的抑制肌肉收缩的作用，因此可用于治疗或护理由肌肉收缩导致的病况、失调或疾病；治疗、预防或修复皮肤老化或光老化；亦可用于减少、预防或治疗皱纹的产品中。其中，异构体A的技术效果最优。
实施例13含异构体A的微米乳液组合物的制备
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，得到含异构体A的微米乳液组合物。
实施例14含异构体D的精华液的制备
将处方量的透明质酸钠加入水中，搅拌使混合均匀，然后加热到80～85℃，保温搅拌使其分散均匀。温度降到40℃以下，加入甘油、芦荟胶、异构体D、维生素C、辛甘醇和1，2-己二醇，搅拌均匀。用15％三乙醇胺调溶液的pH值至5.5左右，即得。
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 (14)

  1. 式(I)所示的肽衍生物异构体，其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，
    式(I)中，X是：
  2. 根据权利要求1所述的式(I)所示的肽衍生物异构体，其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)所示的肽衍生物异构体与酸形成的、单价或多价的、均质或混合的盐，所述酸为无机酸和/或有机酸。
  3. 根据权利要求2所述的式(I)所示的肽衍生物异构体，其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，所述酸包括：脂族的饱和和/或不饱和的一元和/或二元羧酸、芳族羧酸、芳族-脂族羧酸、杂芳族羧酸、脂族和/或芳族磺酸。
  4. 根据权利要求2所述的式(I)所示的肽衍生物异构体，其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，所述酸包括：盐酸、氢溴酸、硫酸、磷酸、甲酸、乙酸、三氟乙酸、三氯乙酸、丙酸、乙醇酸、琥珀酸、富马酸、丙二酸、马来酸、草酸、邻苯二甲酸、柠檬酸、乳酸、酒石酸、苯甲酸、水杨酸、扁桃酸、肉桂酸、烟酸、甲磺酸和/或甲苯磺酸；
    可选地，所述酸是乙酸、三氟乙酸和/或乳酸。
  5. 一种制备权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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的方法，其特征在于，所述方法包括以下步骤：
    (1)Boc-β-Ala-OH与HOSu缩合生成Boc-β-Ala-OSu；
    (2)Boc-β-Ala-OSu在弱碱性条件下与羟脯氨酸反应生成Boc-β-Ala-羟脯氨酸；
    (3)Fmoc-(S)-Dab(Boc)-OH与苄胺缩合生成Fmoc-(S)-Dab(Boc)-NH-Bzl；
    (4)步骤(3)的产物脱除Fmoc保护基后，与Boc-β-Ala-羟脯氨酸缩合生成Boc-β-Ala-羟脯氨酸-(S)-Dab(Boc)-NH-Bzl；
    (5)步骤(4)的产物脱除Boc保护基，纯化冻干得到式(I)所示的肽衍生物异构体，或其美容上可接受的盐、或其药学上可接受的盐；
    可选地，所述羟脯氨酸是(2S,4R)-Hyp、(2S,4S)-Hyp、(2R,4S)-Hyp或(2R,4R)-Hyp。
  6. 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1-4任一项所述的式(I)所示的肽衍生物异构体，其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紫外线和/或B紫外线的有机或无机光防护剂或其混合物。
  7. 根据权利要求6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。
  8. 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1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7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。
  9. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗或护理皮肤或粘膜的美容组合物或药物组合物中的用途。
  10. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于增加皮肤 弹性和/或皮肤紧致度的美容组合物或药物组合物中的用途。
  11. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于增加胶原蛋白生成、提高成纤维细胞的活性和/或增加细胞黏附的美容组合物或药物组合物中的用途。
  12. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗、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。
  13. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗或护理由肌肉收缩导致的病况、失调或疾病的美容组合物或药物组合物中的用途；
    可选地，所述失调或疾病是张力障碍；
    可选地，所述张力障碍为局灶性肌张力障碍；优选所述张力障碍包括，但不限于脸痉挛、扭转性张力障碍、颈张力障碍或斜颈症、喉张力障碍或痉挛性发音困难、口下颌张力障碍、肢体张力障碍诸如书写痉挛或音乐家痉挛或脚部张力障碍、夜间磨牙、半侧颜面痉挛、抽搐症和/或斜视；节段性肌张力障碍、梅杰氏综合征、多灶性张力障碍、偏侧肌张力障碍、多巴胺反应性肌张力障碍和濑川张力障碍。
  14. 权利要求1-4任一项所述的式(I)所示的肽衍生物异构体，其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6或7所述的美容或药物组合物，或权利要求8所述的美容上或药学上可接受的递送系统或缓释系统在制备用于减少、预防或治疗皱纹的产品中的用途。
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