WO2021147740A1 - Mapk/erk通路抑制剂在拮抗皮肤老化与辐射性皮肤早衰中的应用 - Google Patents

Mapk/erk通路抑制剂在拮抗皮肤老化与辐射性皮肤早衰中的应用 Download PDF

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WO2021147740A1
WO2021147740A1 PCT/CN2021/071561 CN2021071561W WO2021147740A1 WO 2021147740 A1 WO2021147740 A1 WO 2021147740A1 CN 2021071561 W CN2021071561 W CN 2021071561W WO 2021147740 A1 WO2021147740 A1 WO 2021147740A1
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skin
mapk
aging
skin aging
hair
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PCT/CN2021/071561
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English (en)
French (fr)
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张亮
于瑶
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中国科学院上海营养与健康研究所
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Priority to CN202180002094.4A priority Critical patent/CN113853218B/zh
Publication of WO2021147740A1 publication Critical patent/WO2021147740A1/zh

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • 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
    • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the medical field, in particular to the application of MAPK/ERK pathway inhibitors in antagonizing skin aging and radiation-induced premature skin aging.
  • the aging of skin epithelial tissues is the most significant feature of human aging.
  • the main manifestations of skin aging include wrinkle formation, reduced healing/regeneration capacity, albinism/thinning of hair, and atrophy of hair follicles.
  • Epithelial stem cells in the skin mainly including hair follicle stem cells and epidermal stem cells
  • DNA damage is generally considered to be a major factor leading to the aging/premature aging of many tissues.
  • progeria patients with mutations in genes related to genome stability and their corresponding mouse genetic models typical skin aging phenotypes such as hair loss and albinism will appear earlier.
  • UV ultraviolet
  • IR ionizing radiation
  • the purpose of the present invention is to provide a substance and method that can effectively prevent, antagonize or delay skin aging.
  • the first aspect of the present invention provides a use of a MAPK/ERK pathway inhibitor to prepare a composition or preparation for preventing or treating skin aging (or anti-skin aging).
  • the composition includes a pharmaceutical composition and a daily chemical composition.
  • the daily chemical composition includes shampoo, shower gel, skin care products, hair care products, and/or cosmetics.
  • composition is administered transdermally or transdermally.
  • the composition is a topical composition.
  • composition or preparation is used for one or more (or all) applications selected from the following group:
  • the skin aging has one or more indicators selected from the following group:
  • the epithelial stem cells are selected from the group consisting of hair follicle stem cells, epidermal stem cells, skin epithelial stem cells, or a combination thereof.
  • the skin aging further includes: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers .
  • the representative senescent cell markers or markers include (but are not limited to): SA- ⁇ -galactosidase, P16, cell proliferation ability.
  • the representative young cell markers or markers include (but are not limited to): H3K9me3, collagen gene COL17A1.
  • the skin aging includes: skin aging caused by DNA damage, radiation-induced skin aging, natural skin aging, and/or skin photoaging.
  • the skin aging is induced by an inducing factor selected from the group consisting of ionizing radiation, gamma-ray irradiation, ultraviolet light, radiotherapy, excessive sun exposure, chemical mutagens, and natural proliferation of skin cells.
  • an inducing factor selected from the group consisting of ionizing radiation, gamma-ray irradiation, ultraviolet light, radiotherapy, excessive sun exposure, chemical mutagens, and natural proliferation of skin cells.
  • the skin aging is caused by age factors.
  • the MAPK/ERK pathway inhibitor inhibits the phosphorylation of MEK1/2.
  • the MAPK/ERK pathway inhibitor inhibits the phosphorylation of ERK1/2.
  • the MAPK/ERK pathway inhibitor is a specific inhibitor of phosphorylation of ERK1/2.
  • the MAPK/ERK pathway inhibitor is selected from the following group: Trametinib, U0126, MEK162, SCH772984, lixertinib, AZD0364, KO-947, HH2710, PD0325901, PD184352, Swiss Refametinib, carbitinib, AS-701255, AS-701173, Pimasertib, RDEA436, RO4987655, RG7420, or a combination thereof.
  • the MAPK/ERK pathway inhibitor is selected from the group consisting of U0126, Trametinib, MEK162, SCH772984 or a combination thereof.
  • the MAPK/ERK pathway inhibitor is trametinib, SCH772984, or a pharmaceutically acceptable salt thereof.
  • the administration mode of the MAPK/ERK pathway inhibitor includes oral or parenteral administration.
  • the non-oral administration includes (but is not limited to) smearing.
  • the second aspect of the present invention provides a composition or preparation that can be used to prevent or treat skin aging.
  • the composition or preparation includes: (a) a physiologically acceptable carrier; and (b) an effective amount of Active ingredient, said active ingredient is MAPK/ERK pathway inhibitor.
  • the MAPK/ERK pathway inhibitor is selected from the following group: small molecule compounds, antibodies, nucleic acids, or combinations thereof.
  • the MAPK/ERK pathway inhibitor is selected from the group consisting of U0126, Trametinib, MEK162, SCH772984 or a combination thereof.
  • the MAPK/ERK pathway inhibitor is inhibited by trametinib or a pharmaceutically acceptable salt thereof.
  • composition or preparation also contains (c) other anti-aging active ingredients.
  • the other anti-aging active ingredients are selected from the following group: microRNA31/miR-31 inhibitors (see CN2019111758754).
  • the active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required under aseptic conditions.
  • composition or preparation further contains a skin penetration enhancer.
  • the physiologically acceptable carrier is selected from the group consisting of water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, nanogels, and cellulose. Its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water or combinations thereof.
  • the dosage form of the composition or preparation is a topical dosage form.
  • the dosage form of the composition or preparation includes ointment, cream, patch, lotion, spray, microinjection and the like.
  • the method of administration of the composition or formulation is selected from the group consisting of topical application, transdermal application and transdermal application.
  • the composition contains 0.05-10 mg/ml (or mg/g), preferably 0.1-5 mg/ml (or mg/g), more preferably 0.2-2 mg/ml (or mg/g) Trametinib.
  • composition or preparation is a liquid preparation prepared by dissolving Trametinib in a solvent.
  • the solvent is selected from the following group: water, C1-C6 alcohol, DMSO or a combination thereof.
  • the C1-C6 alcohol is selected from the following group: ethanol, propylene glycol, propanol, butanol, glycerin, or a combination thereof.
  • the solvent contains 50 parts by weight of ethanol, 30 parts by weight of water, 20 parts by weight of propylene glycol and 0-5 parts by weight (preferably 2-4 parts by weight) of DMSO.
  • the third aspect of the present invention provides a method for in vitro screening of potential substances for anti-skin aging, including the steps:
  • test group and control group wherein a test substance is added to the detection system in the test group, and a control substance is added to the detection system in the control group, and the control substance includes a negative control substance;
  • the experimental conditions of the test group and the control group are the same or substantially the same.
  • the level of the MAPK/ERK pathway is the phosphorylation level of MEK1/2 and/or the phosphorylation level of ERK1/2.
  • the level of the MAPK/ERK pathway is the activity level and expression level of MAPK/ERK kinase (including protein level or mRNA level).
  • step (1) after or before step (1), the method further includes the following steps:
  • step (1a) treatment is performed by a method selected from the following group to form the DNA damage-inducing environment: irradiation treatment, chemical mutagens, high temperature, violent shaking, pathological induction Stress, or a combination thereof.
  • the irradiation treatment is selected from the group consisting of ionizing radiation, ⁇ -ray irradiation, UVA/UVB irradiation, radiotherapy, excessive sun exposure, or a combination thereof.
  • the detection system is a cell system.
  • the cells include skin cells, preferably epithelial cells, more preferably epithelial stem cells.
  • the method further includes the steps:
  • step (3) compare the DNA damage amplitude D1 in the test group with the DNA damage amplitude D0 in the control group, if the ratio of (D0-D1)/D0 is ⁇ 5%, preferably ⁇ 10 %, preferably ⁇ 20%, preferably ⁇ 50%, more preferably ⁇ 80%, and most preferably ⁇ 100%, it indicates that the test substance is a potential anti-aging substance.
  • the fourth aspect of the present invention provides a method for preventing or treating skin aging, including the steps of: administering a MAPK/ERK pathway inhibitor, or a composition or preparation containing a MAPK/ERK pathway inhibitor to a subject in need.
  • composition or preparation includes an inhibitor of the MAPK/ERK pathway as an active ingredient.
  • the MAPK/ERK pathway inhibitor is selected from the group consisting of U0126, Trametinib, MEK162, SCH772984 or a combination thereof.
  • the skin aging includes skin aging caused by DNA damage and radiation-induced premature skin aging.
  • the skin aging is induced by an inducing factor selected from the following group: ionizing radiation, ⁇ -ray irradiation, ultraviolet light, radiotherapy, excessive sun exposure, chemical mutagens, or a combination thereof.
  • the administration is before, during or after being induced by the inducing factor.
  • the application is applied to unwound skin, or applied without skin wounds (including topical, oral, or injection (such as intravenous, intramuscular) application).
  • a method for diagnosing/detecting skin aging including the steps:
  • sample to be tested is skin cells or skin tissue
  • the sample to be tested is skin epithelial tissue or skin epithelial cells.
  • the “significantly higher” refers to the increase in the expression level of the corresponding miRNA-31 (that is, the ratio of (L1-L0)/L0) ⁇ 10% compared with the negative control sample , Preferably ⁇ 20%, preferably ⁇ 50%, preferably ⁇ 100%, more preferably ⁇ 150%, and most preferably ⁇ 200%.
  • the level of the MAPK/ERK pathway is the phosphorylation level of MEK1/2 and/or the phosphorylation level of ERK1/2.
  • the level of the MAPK/ERK pathway is the activity level and expression level of MAPK/ERK kinase (including protein level or mRNA level).
  • the level of the MAPK/ERK pathway is the phosphorylation level of ERK1/2.
  • a level of MAPK/ERK pathway or use of its detection reagents for preparing detection reagents or detection kits for evaluating skin aging is provided.
  • the skin is human skin.
  • kit further contains instructions, and the instructions describe the method described in the fifth aspect of the present invention.
  • Figure 1 shows the establishment of a local skin premature aging model induced by local ionizing radiation. These include:
  • FIG. 1A Schematic diagram of the experimental procedure of the local premature skin aging model in mice induced by local ionizing radiation (localized skin premature aging mice). Yellow is the irradiated area. noRad: non-irradiated area; Rad: irradiated area, the same below.
  • Figure 2 shows that the oral MAPK/ERK pathway inhibitor Trametinib effectively antagonizes the skin aging phenotype in the antibody.
  • d0 Day 0 after trauma.
  • d9 The 9th day after trauma.
  • FIG. 3 shows that the application of MAPK/ERK pathway inhibitor Trametinib effectively antagonizes the skin aging phenotype in the antibody.
  • the mice received one-time local irradiation at around 7 weeks of age (P50).
  • the drug Trametinib or control solvent
  • the way of drug treatment is to smear the drug on the back every 3 days, a total of 5 times for 15 days. After five days of irradiation, wax hair removal was performed to induce hair regrowth.
  • noRad non-irradiated area; Rad: irradiated area.
  • +T Trametinib treatment group, the same below.
  • a round of phenotyping was performed before P83 epilation again. The second round of phenotypic analysis was performed at P111.
  • FIG. 4 shows that the application of MAPK/ERK pathway inhibitor Sch772984 effectively antagonizes the skin aging phenotype in the antibody.
  • the mice received one-time local irradiation at around 7 weeks of age (P50).
  • the drug (Sch772984 or control solvent) treatment was started.
  • the way of drug treatment is oral administration once every 3 days, a total of 5 times for 15 days. After five days of irradiation, wax hair removal was performed to induce hair regrowth.
  • noRad non-irradiated area
  • Rad irradiated area.
  • +S solvent treatment group. +sch Sch772984 treatment group, the same below.
  • a round of phenotyping was performed before P83 epilation again. The second round of phenotypic analysis was performed at P111.
  • Figure 5 shows a schematic diagram of the MAPK/ERK signaling pathway.
  • Figure 6 shows that a brief application of MAPK/ERK signaling pathway inhibitors can safely and effectively antagonize DNA-damaging premature skin aging.
  • A Schematic diagram of the experimental procedure for the intervention of Trametinib (+T) or solvent control (+S) surface application in the mouse skin premature aging model induced by local irradiation.
  • the mice received one-time local irradiation at around 7 weeks of age (P50).
  • the drug Trametinib or control solvent
  • the way of drug treatment is to smear the drug on the back every 3 days, a total of 1, 2 or 3 times. After five days of irradiation, wax hair removal was performed to induce hair regrowth.
  • noRad non-irradiated area
  • Rad irradiated area.
  • the inventor found for the first time that the activation of the MAPK/ERK signal pathway is closely related to the depletion of skin hair follicle stem cells stimulated by DNA damage and the subsequent premature skin aging phenotype.
  • Tests have shown that treatment with targeted drugs that antagonize the MAPK/ERK signaling pathway can significantly inhibit a series of skin aging/premature aging phenotypes in vivo, including: loss of skin hair follicle stem cells, hair albinism, decreased hair regeneration ability, and epidermal healing Injury function declines.
  • the research of the present invention unexpectedly discovered for the first time that the MAPK/ERK signaling pathway has a key role in promoting skin aging and premature aging, which is completely in line with the concept that the signaling pathway has a role in promoting skin cell growth/survival. different. More importantly, the research of the present invention shows that inhibiting the activity of the MAPK/ERK pathway is an effective means to antagonize skin aging and radiation-induced premature skin aging.
  • the intervention methods developed on this basis can be applied to the prevention and treatment of radiative skin premature aging and the delay of natural skin aging, and have broad application prospects.
  • the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Sequence identity compares two alignments along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) Sequence, and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein Sequence.
  • skin aging and “skin aging” are used interchangeably to refer to the aging of skin epithelial tissues.
  • the term includes normal skin aging, as well as premature or too rapid skin aging (ie, premature skin aging).
  • Skin epithelial stem cells (mainly including hair follicle stem cells and epidermal stem cells) are the basis of skin epithelial tissue homeostasis maintenance and wound repair.
  • Epithelial stem cells include hair follicle stem cells, epidermal stem cells, other stem cells belonging to skin epithelial tissues, or a combination thereof.
  • epidermal stem cells maintain the dynamic balance of epithelial tissue, while hair follicle stem cells support hair growth and regeneration, and can repair epithelium under traumatic conditions.
  • stem cells There are some poorly defined stem cells in the skin epithelial tissue, such as the stem cells at the junction of hair follicles and epidermis, collectively referred to as skin epithelial stem cells.
  • skin epithelial stem cells As a lifelong self-renewing tissue, the aging of skin epithelial tissue is closely related to the failure of its stem cells, but the specific molecular mechanism leading to this type of stem cell failure is not fully understood.
  • the skin aging has one or more indicators selected from the group consisting of: (1) loss of skin epithelial stem cells; (m) albinism of hair; (n) decreased ability of hair regeneration; and ( o) Decreased epithelial callus function.
  • skin aging also includes: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers.
  • Said representative markers or markers of senescent cells include (but are not limited to): SA- ⁇ -galactosidase, P16, cell proliferation ability; said representative markers or markers of young cells include (but Not limited to): H3K9me3, collagen COL17A1.
  • DNA damage is generally considered to be a major factor leading to tissue aging/premature aging.
  • tissue aging/premature aging In skin tissues, both exogenous and endogenous DNA damage inducing factors can cause significant premature aging.
  • typical skin aging phenotypes including hair loss and albinism will appear early.
  • ultraviolet light (UV) in the sun is a well-known cause of DNA damage, and it can also significantly accelerate skin aging, leading to the common symptoms of photoaging in the skin.
  • ionizing radiation commonly used in tumor radiotherapy is also a strong inducer of DNA damage, which can induce premature aging of skin cells in the radiotherapy area, and cause long-term skin callus dysfunction, which can lead to the development of refractory ulcers.
  • the risk of cancer is one of the most common complications in tumor radiotherapy.
  • DNA damage is the direct cause of skin epithelial and epithelial stem cell failure. Inhibiting the failure of skin epithelial stem cells caused by DNA damage has broad application prospects in skin anti-aging beauty and the prevention and treatment of radiation skin damage. However, there is no specific treatment that has entered the clinical stage.
  • the present invention provides a technical approach for inhibiting the aging effect of DNA damage on skin epithelial stem cells by targeting miR-31 inhibitors, thereby effectively combating skin aging and premature aging.
  • each group of mice was subjected to ⁇ -ray local irradiation treatment, the specific steps are as in Example 1, and then, in order to test the hair regeneration ability of mice , Wax treatment was used to induce hair regrowth, and then the skin phenotype was observed for about 28 days (4 weeks).
  • the experimental flow chart is shown in Figure 1A.
  • active ingredient or “active ingredient for skin anti-aging” refers to antagonists or inhibitors of MAPK/ERK pathway inhibitors.
  • MAPK/ERK pathway and “MAPK/ERK signaling pathway” are used interchangeably, referring to mitogen-activated protein kinase/extracelluar regulated protein kinase (MAPK/ ERK) Cascade path.
  • MAPK/ERK pathway mitogen-activated protein kinase/extracelluar regulated protein kinase (MAPK/ ERK) Cascade path.
  • the schematic diagram of the MAPK/ERK pathway can be seen in Figure 5.
  • the mitogen-activated protein kinase (MAPK) signaling pathway is related to cellular events such as growth, differentiation, and stress response.
  • MAPK mitogen-activated protein kinase
  • Four parallel MAPK channels have been identified so far: ERK1/ERK2, JNK, p38 and ERK5. These channels are a linear kinase cascade because MAPKKK phosphorylates and activates MAPKK, and MAPKK phosphorylates and activates MAPK. So far, seven MAPKK homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK 6 and MKK7) and four MAPK families (ERK1/2, JNK, p38 and ERK5) have been identified. The activation of these channels regulates the activity of various substrates through phosphorylation. MEK catalyzes the phosphorylation of MAPK substrates ERK1 and ERK2.
  • the MAPK/ERK pathway in the present invention refers to a cascade pathway composed of
  • representative MAPK/ERK pathway inhibitors include (but are not limited to): small molecule compounds, antibodies, nucleic acids, or combinations thereof.
  • Preferred examples include compounds and antibodies that specifically target proteins of the MAPK/ERK pathway (especially MAPK/ERK kinase).
  • MAPK/ERK inhibitor and “MAPK/ERK pathway inhibitor” are used interchangeably and refer to molecules that can interfere with MAPK activity, and especially ERK activity.
  • the MAPK/ERK inhibitor may be an anti-MAPK/ERK antibody, which specifically binds to the MAPK/ERK protein and inhibits its activity.
  • MAPK/ERK inhibitors can be inhibitory nucleic acids.
  • the inhibitory nucleic acid may be an aptamer that specifically binds to the MAPK/ERK protein and inhibits its activity.
  • Other inhibitory nucleic acids can bind to the MAPK/ERK transcript and inhibit its translation or degrade it.
  • inhibitory nucleic acids may be antisense nucleic acids, morpholino oligonucleotides, inhibitory RNA molecules, such as siRNA or microRNA or ribozymes.
  • the MAPK/ERK inhibitor may be a small molecule that binds to MAPK/ERK and inhibits its activity. Such small molecule inhibitors of MAPK/ERK can be obtained through well-known screening procedures or molecular modeling methods whose purpose is to determine the compound that binds to the active site of the MAPK/ERK kinase domain.
  • Some representative inhibitors of the MAPK/ERK signaling pathway can be found in WO2008079814, WO2008140553, WO2009064675, etc.
  • the MAPK/ERK inhibitor described in the present invention is a MEK inhibitor selected from the group consisting of U0126, GSK1120212 (Trametinib), MEK162, SCH772984, lixertinib, AZD0364, KO-947, HH2710, Or a combination. More preferably, the MAPK/ERK inhibitor is GSK1120212 (Trametinib), MEK162, or SCH772984.
  • a representative MAPK/ERK signaling pathway inhibitor is Trametinib; CAS: 871700-17-3; Molecular weight: 615.39; Chinese chemical name: N-[3-[3-cyclopropyl-5 -[(2-Fluoro-4-iodophenyl)amino]-3,4,6,7-tetrahydro-6,8-dimethyl-2,4,7-trioxopyrido[4,3 -d]pyrimidine-1(2H)-yl]phenyl]acetamide; chemical formula: C 26 H 23 FIN 5 O 4 .
  • the English name is TRAMETINIB, also known as GSK1120212, GSK 212 or JTP 74057. The inhibitor is disclosed in WO 2005/121142.
  • a representative MAPK/ERK signaling pathway inhibitor is SCH772984:CAS:942183-80-4, purchased from Selleck (S7101), chemical formula C 33 H 33 N 9 O 2 , is a specific ERK1/2 small Molecular inhibitors.
  • MAPK/ERK signaling pathway inhibitors include (but are not limited to): PD0325901 (Pfizer) (disclosed in WO 02/06213); PD184352 (Pfizer); Refametinib ( Also known as RDEA119 or Bay 86-9766); Carbitinib, also known as XL518, and commercially available from Roche under the trade name; AS-701255 (Merck Serono) ); AS-701173 (Merck Serono); Pimasertib, also known as AS-703026 or MSC1936369B (Merck Serono); RDEA436 (Ardea Biosciences )); RO4987655, also known as RG7167 (Roche); and/or RG7420, also known as GDC-0623 (Roche).
  • the present invention provides a composition or preparation (including cosmetics, daily chemicals, etc.) that can be used to prevent or treat skin aging.
  • the composition or preparation includes: (a) an acceptable carrier (including pharmaceutically acceptable A carrier, a physiologically acceptable carrier, a cosmetically acceptable carrier); and (b) an effective amount of active ingredient, said active ingredient being a MAPK/ERK pathway inhibitor.
  • the effective amount of the active ingredient can vary with the mode of use and the degree of skin aging.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • an effective amount or “effective dose” refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.
  • pharmaceutically acceptable ingredients are suitable for humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents.
  • physiologically acceptable carrier refers to carriers for active ingredients, including: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, Cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate , Vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, or combinations thereof.
  • the choice of carrier should match the mode of administration, which are well
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, and sustained-release agent.
  • it can be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition should be manufactured under aseptic conditions.
  • the effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (for example, through clinical trials).
  • the factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration The way and so on.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg/kg animal body weight (preferably 0.0001 mg-10 mg/kg animal body weight), satisfactory effects can be obtained.
  • several divided doses can be given every day, or the dose can be reduced proportionally.
  • composition or product is a cosmetic or daily chemical product containing the active ingredient of the present invention (targeting miR-31 inhibition).
  • composition or preparation preferably also contains a skin penetration enhancer.
  • the dosage form of the composition or preparation is a topical dosage form.
  • the dosage forms of the cosmetic composition or preparation include (but are not limited to): solid preparations, liquid preparations, gel preparations, and semi-solid preparations. Some preferred dosage forms are ointments, creams, patches, lotions, sprays, microinjections, solutions, shampoos, etc.
  • representative products include (but are not limited to): shampoo, shower gel, skin care products, hair care products, and facial masks.
  • the method of administration of the composition or formulation is selected from the group consisting of topical application, transdermal application and transdermal application.
  • the present invention has unexpectedly confirmed for the first time that irradiation stimulation leads to depletion of skin epithelial stem cells by activating the MAPK/ERK signaling pathway, which in turn leads to subsequent skin aging. Therefore, the MAPK/ERK signaling pathway can be used as a target for preventing or treating skin aging and premature aging. target.
  • MAPK/ERK pathway inhibitors effectively fight or delay skin aging (especially premature aging).
  • MAPK/ERK pathway inhibitors can effectively prevent the depletion of stem cells, thereby preventing subsequent skin aging.
  • MAPK/ERK signaling pathway inhibitors can be applied in different ways (oral or smeared) to achieve the same effect of antagonizing skin aging and radiation-induced premature skin aging.
  • C57BL/6 wild-type mice 7-week-old C57BL/6 wild-type mice were used as experimental mice to make an animal model of premature skin aging induced by local irradiation. All C57BL/6 wild-type mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • ⁇ -ray irradiation was performed according to standard experimental procedures in the Institute of Radiation Medicine, Fudan University School of Medicine.
  • the upper and lower lead plates are the same, the lead plates are perpendicular to the radiation source, and the two lead plates are each left vertically aligned.
  • a small hole with a diameter of 3 cm the upper half of the mouse is placed in the small hole, the lead plate covers the head and lower body of the mouse, and the limbs of the mouse are properly fixed with tape and then irradiated.
  • the irradiation dose is 10Gy. All irradiation experiments used 7-week-old C57BL/6 wild-type mice.
  • Trametinib (CAS: 871700-17-3), purchased from Selleck (S2673), chemical formula C 26 H 23 FIN 5 O 4 .
  • Trametinib powder was dissolved in DMSO to make a 15mg/ml stock solution. When oral, it was diluted in Corn oil (Sigma, C8267) at a dilution ratio of 1:25, and each mouse was dosed at 1mg/kg. Orally, once every three days, a total of five times. The control group was given the same solvent orally.
  • Trametinib (CAS: 871700-17-3), purchased from Selleck (S2673) with chemical formula C 26 H 23 FIN 5 O 4 .
  • it When applying local medicine, it is diluted in 50% ethanol, 30% water and 20% propylene glycol according to a 1:25 dilution ratio. (Sangon, A501754-0500) In the solvent, each mouse was applied at a dose of 1 mg/kg every three days for a total of five times. The control group was coated with the same solvent.
  • mice After the mice were killed with a carbon dioxide lethal device, their hair was shaved and the back skin was removed, the dermal fat tissue was removed with a scalpel, washed with PBS, and placed in Trypsin-EDTA (ThermoFisher, 25200056) for overnight digestion at 4°C. Scrape the cells with a blade on the next day, collect the cell suspension, and place it in PFE (PBS+1%FBS+1 ⁇ M EDTA) staining solution on ice for CD34 (1:100, eBioscience, 11-0341-82), CD49f (1:100, biolegend, 313602) dyeing. After staining, wash the cells and add the living cell dye Helix NP NIR (biolegend, 425301).
  • PFE PBS+1%FBS+1 ⁇ M EDTA
  • the stained cells were detected on a flow analyzer (BD, CytoFLEX LX).
  • Flow cytometry detected the CD34 + CD49f + live cell population as hair follicle stem cells.
  • Flow cytometry software FlowJo 10.0 was used to analyze the percentage of hair follicle stem cells in each group, and the ratio was compared with the percentage of untreated parts of wild-type mice.
  • mice After the mice were anesthetized with isoflurane, their hair was shaved, and the back was punched with an 8mm punch. Put on a surgical towel (3M, SP1107) to prevent the wound from deforming. Take it off after three days. Take pictures before and 0, 4, 7, 9, 12 and 15 days after the wound. Calculate the wound area according to the scale. Within each group The ratio of the wound area of mice at different time points to the 0-day area of their wounds was taken to obtain the percentage of wounds. The data of each group was recorded until the callus was completely finished.
  • mice After the mice were killed, the hair was removed with depilatory cream, and the fatty tissue was removed with a scalpel after disinfection with 75% alcohol.
  • punch a hole with a 1mm punch to obtain a skin block After washing in PBS, punch a hole with a 1mm punch to obtain a skin block, add 1-2ul matrigel (Corning, 356237) in the middle of each hole, attach the skin block and place 1mg/ml fibronectin (Millipore, FC010) -1mg) Coated 24-well plate (Corning, CLS3527) coated in culture. After 7 days of incubation, take pictures under a microscope, and use the image statistics software Imagej to measure the area of cells that migrated from the edge of the skin block with the skin block as the center point. The area of each group was ratioed to the area of the wild-type untreated part.
  • the method of repeated hair regeneration ability test is to repeat the process by performing a second wax depilation after the first hair regeneration is completed.
  • the camera Canon
  • the hair detector Hair and Scalp
  • Sch772984 (CAS: 942183-80-4), purchased from Selleck (S7101), chemical formula C 33 H 33 N 9 O 2 , is a specific ERK1/2 small molecule inhibitor.
  • the Sch772984 powder was dissolved in DMSO to prepare a 15mg/ml stock solution. When oral, it was diluted in corn oil (Sigma, C8267) at a dilution ratio of 1:25, and each mouse was dosed at 2mg/kg. Orally, once every three days, a total of five times. The control group was given the same solvent orally.
  • the premature skin aging research model was established in mice by induction of local ionizing radiation.
  • the method is as follows: referring to Figure 1A, an area of about 2 cm in diameter on the upper back of adult mice about 7 weeks old was irradiated with one-time ⁇ -ray localized irradiation, and hair regeneration was initiated by wax depilation after 5 days.
  • mice received local irradiation at around 7 weeks of age (P50). Wax hair removal (Wax) was performed five days later to induce hair regrowth. After 28 days (4 weeks), observe the skin phenotype after the first round of hair regrowth (one round phenotype), or induce the second round of hair regrowth through wax depilation again, and observe the second round about 4 weeks later. The skin phenotype after the completion of hair regrowth (second-round phenotype).
  • mice can show a series of typical premature skin phenotypes: mainly include:
  • Oral MAPK/ERK signaling pathway inhibitor (Trametinib) antagonizes skin aging and radiation-induced premature skin aging
  • a short-term drug treatment was introduced into the premature skin aging model to verify the effectiveness of inhibiting the MAPK/ERK signal pathway in antagonizing skin aging.
  • the test method is shown in Figure 2A.
  • the mice received one-time local irradiation at around 7 weeks of age (P50).
  • oral treatment of drugs (Trametinib or control solvent) was started at the same time.
  • the way of drug treatment is oral administration once every 3 days, a total of 5 times for 15 days.
  • wax hair removal was performed to induce hair regrowth.
  • 28 days (4 weeks) observe the skin phenotype after the first round of hair regrowth (one round phenotype), or induce the second round of hair regrowth through wax depilation again, and observe the second round about 4 weeks later.
  • the skin phenotype after the completion of hair regrowth (second-round phenotype).
  • the first round of hair regrowth was completed after 3 weeks of drug withdrawal (ie 4 weeks after irradiation). At this time, samples were taken to analyze the back skin phenotype of a part of the mice (a round of phenotype). Another part of the mice was waxed again to induce the second hair regeneration, and the back skin phenotype was analyzed 4 weeks later (the second round phenotype mainly represents the ability of repeated hair regeneration).
  • Example 3 Applying MAPK/ERK signaling pathway inhibitor (Trametinib) antagonist of skin aging and radiation-induced premature skin aging
  • Example 4 Oral MAPK/ERK signaling pathway inhibitor ( Sch772984 ) antagonizes skin aging and radiation-induced premature skin aging
  • Example 2 The experimental method and steps of Example 2 were repeated, except that the oral Trametinib was replaced with Oral Sch772984 (the summary of the experimental steps is shown in Figure 4A).
  • Example 5 A gradient metered topical application to inhibit skin aging and radiative skin premature aging in the MAPK/ERK pathway inhibitor (Trametinib) antagonist
  • Trametinib (CAS: 871700-17-3), purchased from Selleck (S2673) with chemical formula C 26 H 23 FIN 5 O 4 , is a MEK small molecule inhibitor used in the aforementioned patent application.
  • Trametinib powder was dissolved in DMSO to prepare a 15mg/ml stock solution, and the back hair of the mouse was shaved with a razor.
  • the local application was diluted in 50% ethanol, 30 at a 1:25 dilution ratio.
  • %water and 20% propylene glycol (Sangon, A501754-0500) solvents each mouse was applied at a dose of 1 mg/kg every three days for a total of 1, 2 or 3 times.
  • the control group was coated with the same solvent.
  • MEK1/2 inhibitor Trametinib as an example, we further tested the skin anti-aging effect of Trametinib paint under different application times on the basis of the foregoing examples.
  • the experimental design is similar to that of the aforementioned Example 3.
  • the radiation-induced premature skin aging mouse model established in the aforementioned Example 1 was administered with different times of skin smearing, and its subsequent premature skin aging phenotype was followed up (Figure 6A).
  • the MAPK/ERK signaling pathway is a highly evolutionarily conserved classic cell growth and differentiation regulatory signaling pathway. Its core is a protein kinase cascade composed of three protein kinases: MAPKKK (RAF), MAPKK (MEK1/2), and MAPK (ERK).
  • RAF RAF
  • MEK1/2 MAPK
  • ERK MAPK
  • Cells usually receive stimulation signals through surface receptors (receptor tyrosine kinases (RTKs), integrins, ion channels, etc.), and through docking proteins (SHC, GRB2, CRK, etc.) and guanylate exchange factors (SOS, C3G, etc.) to activate small molecule GTP binding proteins (RAS, RAP1, etc.), and then activate the above-mentioned core cascade pathway.
  • RTKs receptor receptor tyrosine kinases
  • SHC surface receptors
  • GRB2 CRK
  • SOS guanylate exchange factors
  • the MAPK/ERK signaling pathway is generally considered to promote cell proliferation and survival. It is a broad cancer-promoting signaling pathway, and its excessive activation is directly related to the occurrence and development of a variety of tumors. For example, activating mutations of RAF can promote the occurrence of melanoma and metastatic non-small cell lung cancer.
  • the BCR/ABL fusion gene promotes the occurrence of chronic myeloid leukemia by activating the MAPK/ERK signaling pathway, as well as the occurrence and development of breast cancer and prostate cancer. It is closely related to the abnormal activation of MAPK/ERK signal.
  • a variety of cells can significantly activate the MAPK/ERK signaling pathway under the stimulation of ionizing radiation. This activation has the effect of promoting cell survival after irradiation, and is considered to be a protective mechanism for cells to resist radiation.
  • the MAPK/ERK signaling pathway is also a classic growth-promoting/carcinogenic pathway.
  • the abnormal activation of the MAPK/ERK pathway mediated by the mutation of the RAS gene can cause the skin stem cells to become cancerous and thus induce the occurrence of squamous cell carcinoma.
  • increasing the activity of the MAPK/ERK signaling pathway will make cancerous skin stem cells have higher proliferation and migration capabilities, thereby exacerbating the malignancy of skin cancer.
  • the MAPK/ERK pathway has the effect of promoting healing. Physical trauma can significantly activate the MAPK/ERK pathway, while reducing its activity slows down wound healing.
  • UV stimulation can also significantly activate the MAPK/ERK signaling pathway in epidermal cells and lead to epidermal hyperplasia, but human skin exhibits a significantly down-regulated MAPK/ERK signaling pathway activity during the aging process.
  • the MAPK/ERK pathway is functionally related to the natural aging or photoaging of the skin in the body.
  • the research results of the present invention unexpectedly confirmed for the first time that inhibiting the MAPK/ERK pathway is an effective means to antagonize skin aging and radiation-induced skin damage.
  • the inventors also proved that inhibiting the MAPK/ERK pathway within a short time after irradiation can effectively alleviate the phenotype of premature skin aging caused by irradiation.
  • This method circumvents the possible side effects of long-term inhibition of the MAPK/ERK pathway, and provides a practical means to prevent and treat symptoms such as premature skin aging and long-term healing impairment caused by ionizing radiation in the clinic.

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Abstract

一种MAPK/ERK通路抑制剂的用途，其被用于制备预防或治疗皮肤衰老的组合物或制剂。还提供了一种可用于预防或治疗皮肤衰老的组合物或制剂以及体外筛选用于预防或治疗皮肤衰老的潜在物质的方法。

Description

MAPK/ERK通路抑制剂在拮抗皮肤老化与辐射性皮肤早衰中的应用 技术领域
本发明涉及医学领域，具体地涉及MAPK/ERK通路抑制剂在拮抗皮肤老化与辐射性皮肤早衰中的应用。
背景技术
皮肤上皮组织(包括表皮、毛囊、皮脂腺、汗腺等)的衰老是人类衰老最显著的特征。皮肤衰老的主要表现包括皱纹形成、愈伤/再生能力下降、毛发白化/稀疏、毛囊萎缩等。皮肤中的上皮干细胞(主要包括毛囊干细胞和表皮干细胞)是皮肤上皮组织稳态维护与创伤修复的基础。
作为一种终身不断自我更新的组织，皮肤上皮组织的衰老与其干细胞的衰竭密切相关，但导致这种干细胞衰竭的具体分子机制尚不完全清楚。
DNA损伤被普遍认为是导致多个组织衰老/早衰的一个主要因素。在基因组稳定性相关基因突变的早衰症患者及其对应的小鼠遗传模型中，毛发脱落与白化等典型皮肤衰老表型都会提早出现。
在正常人的皮肤组织中，外源辐射性刺激是DNA损伤的一个主要来源。其中，阳光中的紫外线(UV)是众所周知的DNA损伤诱因，同时能够加速皮肤衰老并导致皮肤的光老化。此外，肿瘤放疗中常用的电离辐射(IR)也能够诱发大量的DNA损伤，导致放疗区域的皮肤细胞早衰，进而导致长期性的皮肤愈伤功能障碍，是肿瘤放疗过程中最常见的并发症之一,目前尚无有效治疗手段。
近年来，有研究进一步证实了DNA损伤能够直接诱导皮肤表皮与毛囊干细胞的衰竭。因此，抑制DNA损伤所导致的皮肤上皮干细胞衰竭在皮肤抗衰老美容与放射性皮肤损伤的防治中都有着广阔的应用前景。但目前缺乏令人满意的皮肤抗衰老方法，也没有进入临床阶段的特效疗法。
因此，本领域急需开发能有效防止、拮抗或延缓皮肤衰老的物质。
发明内容
本发明的目的就是提供了一种能有效防止、拮抗或延缓皮肤衰老的物质和方法。
本发明的第一方面，提供了一种MAPK/ERK通路抑制剂的用途，用于制备预防或治疗皮肤衰老(或抗皮肤衰老)的组合物或制剂。
在另一优选例中，所述的组合物包括药物组合物、日用化学品组合物。
在另一优选例中，所述的日用化学品组合物包括洗发水、沐浴露、护肤品、护发产品、和/或化妆品。
在另一优选例中，所述的组合物为经皮施用的、或透皮施用的。
在另一优选例中，所述的组合物为外用组合物。
在另一优选例中，所述的组合物或制剂用于选自下组的一种或多种(或全部)应用：
(a)降低毛发白化比率或减少白化毛发的产生；
(b)提高皮肤愈伤能力或促进创伤愈合；
(c)促进角质细胞的迁移；
(d)促进上皮干细胞功能和/或减缓上皮干细胞的减少；和
(e)提高毛发再生能力。
在另一优选例中，所述的皮肤衰老具有选自下组的一个或多个指标：
(l)皮肤上皮干细胞丢失；
(m)毛发白化；
(n)毛发再生能力下降；和
(o)上皮愈伤功能下降。
在另一优选例中，所述的上皮干细胞选自下组：毛囊干细胞、表皮干细胞、皮肤上皮组织的干细胞、或其组合。
在另一优选例中，所述的皮肤衰老还包括：一种或多种代表性的衰老细胞标记物或标志的增多，和/或一种多种代表性的年轻细胞标记物或标志的减少。
在另一优选例中，所述的代表性的衰老细胞标记物或标志包括(但并不限于)：SA-β-半乳糖苷酶、P16、细胞增殖能力。
在另一优选例中，所述的代表性的年轻细胞标记物或标志包括(但并不限于)：H3K9me3、胶原蛋白基因COL17A1。
在另一优选例中，所述的皮肤衰老包括：DNA损伤导致的皮肤衰老、辐射性皮肤早衰、皮肤自然衰老、和/或皮肤光老化。
在另一优选例中，所述的皮肤衰老是由选自下组的诱导因素诱导的：电离辐射、γ射线辐照、紫外线、放射治疗、过度暴晒、化学诱变剂、皮肤细胞自然增殖 所导致的皮肤自然衰老或其组合。
在另一优选例中，所述的皮肤衰老是年龄因素导致的。
在另一优选例中，所述的MAPK/ERK通路抑制剂抑制MEK1/2的磷酸化。
在另一优选例中，所述的MAPK/ERK通路抑制剂抑制ERK1/2的磷酸化。
在另一优选例中，所述的MAPK/ERK通路抑制剂是ERK1/2的磷酸化的特异性抑制剂。
在另一优选例中，所述的MAPK/ERK通路抑制剂选自下组：曲美替尼(Trametinib)、U0126、MEK162、SCH772984、lixertinib、AZD0364、KO-947、HH2710、PD0325901、PD184352、瑞法替尼(Refametinib)、卡比替尼、AS-701255、AS-701173、匹玛舍替(Pimasertib)、RDEA436、RO4987655、RG7420、或其组合。
在另一优选例中，所述的MAPK/ERK通路抑制剂选自下组：U0126、曲美替尼(Trametinib)、MEK162、SCH772984或其组合。
在另一优选例中，所述的MAPK/ERK通路抑制剂为曲美替尼、SCH772984、或其药学上可接受的盐。
在另一优选例中，所述的MAPK/ERK通路抑制剂给药方式包括：口服或非口服。
在另一优选例中，所述的非口服包括(但不限于)涂抹。
本发明的第二方面，提供了一种可用于预防或治疗皮肤衰老的组合物或制剂，所述的组合物或制剂包括：(a)生理上可接受的载体；和(b)有效量的活性成分，所述的活性成分为MAPK/ERK通路抑制剂。
在另一优选例中，所述的MAPK/ERK通路抑制剂选自下组：小分子化合物、抗体、核酸、或其组合。
在另一优选例中，所述的MAPK/ERK通路抑制剂选自下组：U0126、曲美替尼(Trametinib)、MEK162、SCH772984或其组合。
在另一优选例中，所述的MAPK/ERK通路抑制剂抑制为曲美替尼或其药学上可接受的盐。
在另一优选例中，所述的组合物或制剂还含有(c)其他抗衰老的活性成分。
在另一优选例中，所述的其他抗衰老的活性成分选自下组：microRNA31/miR-31抑制剂(参见CN2019111758754)。
在另一优选例中，所述的活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
在另一优选例中，所述的组合物或制剂还含有透皮促进剂。
在另一优选例中，所述生理上可接受的载体选自下组：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纳米凝胶、纤维素及其衍生物、明胶、滑石、固体润滑剂、硫酸钙、植物油、多元醇、乳化剂、润湿剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水或其组合。
在另一优选例中，所述的组合物或制剂的剂型为外用(topical)剂型。
在另一优选例中，所述的组合物或制剂的剂型包括膏剂、乳霜、贴剂、涂剂、喷剂、微针剂等。
在另一优选例中，所述的组合物或制剂的施用方式选自下组：局部施用、透皮施用和经皮施用。
在另一优选例中，所述的组合物含有0.05-10mg/ml(或mg/g),较佳地0.1-5mg/ml(或mg/g)，更佳地0.2-2mg/ml(或mg/g)的Trametinib。
在另一优选例中，所述的组合物或制剂是将Trametinib溶于溶剂而制备的液态制剂。
在另一优选例中，所述的溶剂选自下组：水、C1-C6醇、DMSO或其组合。
在另一优选例中，所述的C1－C6醇选自下组：乙醇、丙二醇、丙醇、丁醇、甘油、或其组合。
在另一优选例中，所述的溶剂含有50重量份的乙醇、30重量份的水、和20重量份的丙二醇和0-5重量份(较佳地2-4重量份)的DMSO。
本发明的第三方面，提供了一种体外筛选用于抗皮肤衰老的潜在物质的方法，包括步骤：
(1)提供测试组和对照组，其中，在测试组中向检测体系中加入测试物，在对照组中向检测体系中加入对照物,所述对照物包括阴性对照物；
(2)分别检测测试组和对照组中MAPK/ERK通路的水平；如果阴性组相比，测试组的MAPK/ERK通路的水平显著降低，则表示所述测试物是抗皮肤衰老的潜在物质。
在另一优选例中，除了添加的测试物和对照物不同之外，所述的测试组和对照组的实验条件是相同的或基本相同的。
在另一优选例中，所述的MAPK/ERK通路的水平是MEK1/2的磷酸化水平和/或ERK1/2的磷酸化水平。
在另一优选例中，所述的MAPK/ERK通路的水平是MAPK/ERK激酶的活性水平、表达水平(包括蛋白水平或mRNA水平)。
在另一优选例中，所述的步骤(1)之后或之前还包括步骤：
(1a)分别将测试组和对照组的检测体系暴露于DNA损伤诱导环境下，从而诱导细胞衰老或诱导DNA损伤。
在另一优选例中，在步骤(1a)中，用选自下组的方法进行处理，从而形成所述的DNA损伤诱导环境：辐照处理、化学诱变剂、高温、剧烈震荡、诱导病理应激、或其组合。
在另一优选例中，所述的辐照处理选自下组：电离辐射、γ射线辐照、UVA/UVB辐照、放射治疗、过度暴晒、或其组合。
在另一优选例中，所述的检测体系为细胞体系。
在另一优选例中，所述的细胞包括皮肤细胞，较佳地为上皮细胞，更佳地为上皮干细胞。
在另一优选例中，所述方法还包括步骤：
(3)分别检测测试组和对照组中上皮干细胞耐受DNA损伤能力；如果与对照组相比，测试组的上皮干细胞耐受DNA损伤能力显著提高，则提示所述测试物是抗皮肤衰老的潜在物质。
在另一优选例中，在步骤(3)中，比较测试组中DNA损伤幅度D1和对照组中DNA损伤幅度D0,如果(D0-D1)/D0的比值≥5％，较佳地≥10％，较佳地≥20％，较佳地≥50％，更佳地≥80％，最佳地≥100％，则提示所述测试物是抗皮肤衰老的潜在物质。
本发明的第四方面，提供了一种预防或治疗皮肤衰老的方法，包括步骤：给需要的对象施用MAPK/ERK通路抑制剂、或含MAPK/ERK通路抑制剂的组合物或制剂。
在另一优选例中，所述的组合物或制剂包括：MAPK/ERK通路的抑制剂作为活性成分。
在另一优选例中，所述的MAPK/ERK通路抑制剂选自下组：U0126、曲美替尼(Trametinib)、MEK162、SCH772984或其组合。
在另一优选例中，所述的皮肤衰老包括DNA损伤导致的皮肤衰老、辐射性皮肤早衰。
在另一优选例中，所述的皮肤衰老是由选自下组的诱导因素诱导的：电离辐射、γ射线辐照、紫外线、放射治疗、过度暴晒、化学诱变剂、或其组合。
在另一优选例中，所述的施用是在受到所述诱导因素诱导之前、之中或之后施用。
在另一优选例中，所述的施用是施用于无伤口的皮肤，或在未出现皮肤伤口的情况下施用(包括外用、口服、或注射(如静脉内、肌内)等施用)。
在本发明的第五方面，提供了一种诊断/检测皮肤衰老的方法，包括步骤：
(a)提供一待测样本，所述待测样本为皮肤细胞、或皮肤组织；和
(b)检测所述待测样本中MAPK/ERK通路水平的L1，并与参考值L0进行相比，如果待测样本的MAPK/ERK通路的水平显著高于参考值，则提示所述待测样本的皮肤衰老进程快和/或皮肤衰老程度高，其中所述参考值L0是正常衰老的皮肤细胞中MAPK/ERK通路的水平。
在另一优选例中，所述的待测样本为皮肤上皮组织或皮肤上皮细胞。
在另一优选例中，所述的“显著高于”是指：与阴性对照样本相比，相应miRNA-31的表达水平的提高幅度(即(L1-L0)/L0的比值)≥10％，较佳地≥20％，较佳地≥50％，较佳地≥100％，更佳地≥150％，最佳地≥200％。
在另一优选例中，所述的MAPK/ERK通路的水平是MEK1/2的磷酸化水平和/或ERK1/2磷酸化水平。
在另一优选例中，所述的MAPK/ERK通路的水平是MAPK/ERK激酶的活性水平、表达水平(包括蛋白水平或mRNA水平)。
在另一优选例中，所述的MAPK/ERK通路的水平是ERK1/2的磷酸化水平。
在本发明的第六方面，提供了一种MAPK/ERK通路的水平或及其检测试剂的用途，用于制备用于评估皮肤衰老的检测试剂或检测试剂盒。
在另一优选例中，所述的皮肤为人的皮肤。
在另一优选例中，所述的试剂盒还含有说明书，所述的说明书中记载了本发明第五方面中所述的方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了建立局部电离辐照诱导的局部皮肤早衰模型。其中包括：
1A.局部电离辐照诱导的小鼠皮肤局部早衰模型(局部皮肤早衰小鼠)的实 验流程示意图。黄色为辐照区域。noRad：未辐照区；Rad：辐照区，下同。
1B.局部皮肤早衰小鼠的一轮表型分析-背皮毛发代表性照片。比例尺1cm。
1C.局部皮肤早衰小鼠的一轮表型分析-背皮全层创伤愈合速率统计图。横轴：愈伤天数，纵轴：愈合率。**：P<0.01,n＝3,双尾t-test。
1D.局部皮肤早衰小鼠的一轮表型分析-背皮皮肤组织块(Exo-plant)的角质细胞迁移愈伤速率实验。左图：显微镜照片，黑色为皮肤组织块本体，白色线指示迁移角质细胞层的前沿。比例尺500μm。右图：迁移角质细胞层的面积统计：**：P<0.01,n＝3,双尾t-test。
1E.局部皮肤早衰小鼠的一轮表型分析-背皮不同区域中的上皮毛囊干细胞相对比率统计(以noRad区为基准)。**：P<0.01,n＝3,双尾t-test。
1F.局部皮肤早衰小鼠的二轮表型分析-背皮毛发代表性照片。可见辐照区新生毛发明显稀疏，导致粉色的表皮部分裸露。比例尺1cm。上述所有图中的误差条(Error Bar)代表标准误(Standard Error)。
图2显示了口服MAPK/ERK通路抑制剂Trametinib有效地拮抗体内皮肤衰老表型。其中包括：
2A.在前述局部辐照诱导的小鼠皮肤早衰模型中进行药物(Trametinib或溶剂对照)拮抗衰老效果测试的实验流程示意图(皮肤早衰药物拮抗实验)。noRad：未辐照区；Rad：辐照区。+S溶剂处理组。+T：Trametinib处理组，下同。
2B.皮肤早衰药物拮抗实验中的二轮表型分析-背皮代表性照片。IR：接受了局部辐照的局部皮肤早衰小鼠。Ctrl：无辐照对照小鼠。可见IR+S组的辐照区(Rad)毛发基本完全白化，同时因毛发明显稀疏而暴露出粉色表皮。而IR+T组的小鼠的辐照区仅有少部分毛发白化，且无表皮裸露现象。比例尺1cm。
2C.皮肤早衰药物拮抗实验中IR组小鼠的二轮表型分析-背皮不同区域毛发相对密度统计。以noRad+S组为基准。**：P<0.01,n＝3,双尾t-test。
2D.皮肤早衰药物拮抗实验中IR组小鼠的背皮辐照区域(Rad)的二轮表型分析-毛发白化百分比统计。**：P<0.01,n＝3,双尾t-test。
2E.皮肤早衰药物拮抗实验中IR组小鼠的一轮表型分析-背皮不同区域上皮中的毛囊干细胞相对比例统计。**：P<0.01,n＝3,双尾t-test。
2F.皮肤早衰药物拮抗实验中IR组小鼠的一轮表型分析-背皮不同区域全层愈伤愈合过程的代表性照片。d0：创伤后第0天。d9：创伤后第9天。
2G.对F图所代表的愈伤过程的愈合率统计分析。横轴代表愈伤天数**： P<0.01,n＝3,*：P<0.05,双尾t-test。
2H.皮肤早衰药物拮抗实验中IR组小鼠的一轮表型分析-来自不同背皮区域的皮肤组织块的体外角质细胞迁移实验(Exo-plant)的代表性照片。白色虚线代表迁移的角质细胞层的前沿，左下角黑色区域为皮肤组织块本体。比例尺500μm。
2I.对H图所代表的体外角质细胞迁移实验中的角质细胞层相对覆盖面积的统计分析。**：P<0.01,n＝3,双尾t-test。
所有图中的误差条(Error Bar)代表标准误(Standard Error)。
图3显示了涂抹MAPK/ERK通路抑制剂Trametinib有效地拮抗体内皮肤衰老表型。其中包括：
A.在前述局部辐照诱导的小鼠皮肤早衰模型中进行Trametinib或溶剂对照表面涂抹干预的实验流程示意图。小鼠在7周龄(P50)左右小鼠接受一次性局部辐照。同时开始进行药物(Trametinib或对照溶剂)处理。药物处理的方式为每3天后背部涂抹给药一次，总计5次共15天。辐照五天后进行蜡脱毛以诱导毛发再生。noRad：未辐照区；Rad：辐照区。+S溶剂处理组。+T：Trametinib处理组，下同。一轮表型在P83再次脱毛之前进行。二轮表型分析在P111进行。
B.一轮表型，皮肤组织块体外愈伤能力分析。统计量为皮肤组织块(exoplant)在体外培养第6天时的角质细胞层迁移面积相对大小。类似于类似于前述图二I。
C.一轮表型，体内全层创伤愈合速率分析(n＝3)，类似于前述图二G。**与*均指Rad+S与Rad+T的差异。
D.一轮表型，上皮毛囊干细胞比例分析。类似于前述图二E。
E.二轮表型，+S/+T干预的辐照早衰模型小鼠(LIR)与正常对照小鼠(Ctrl)的背皮照片。类似于前述图二B。
F.二轮表型，上述E图中Rad区的白发比例统计(n＝3)，类似于前述图二D。
G.二轮表型，上述E图中LIR小鼠Rad/noRad区的相对毛发密度统计(n＝3)，类似于前述图二C。
**：P<0.01,*：P<0.05,双尾t-test。所有Error bar代表标准误(standard error)
图4显示了涂抹MAPK/ERK通路抑制剂Sch772984有效地拮抗体内皮肤衰老表型。其中包括：
A.在前述局部辐照诱导的小鼠皮肤早衰模型中进行Sch772984或溶剂对 照口服给药的实验流程示意图。小鼠在7周龄(P50)左右小鼠接受一次性局部辐照。同时开始进行药物(Sch772984或对照溶剂)处理。药物处理的方式为每3天口服给药一次，总计5次共15天。辐照五天后进行蜡脱毛以诱导毛发再生。noRad：未辐照区；Rad：辐照区。+S溶剂处理组。+sch：Sch772984处理组，下同。一轮表型在P83再次脱毛之前进行。二轮表型分析在P111进行。
B.一轮表型，皮肤组织块体外愈伤能力分析。统计量为皮肤组织块(exoplant)在体外培养第6天时的角质细胞层迁移面积相对大小。类似于类似于前述图二I。
C.一轮表型，上皮毛囊干细胞比例分析。类似于前述图二E。
D.二轮表型，+S/+sch干预的辐照早衰模型小鼠(LIR)与正常对照小鼠(Ctrl)的背皮照片。类似于前述图二B。
E.二轮表型，上述E图中Rad区的白发比例统计(n＝3)，类似于前述图二D。
F.二轮表型，上述E图中LIR小鼠Rad/noRad区的相对毛发密度统计(n＝3)，类似于前述图二C。
**：P<0.01,*：P<0.05,双尾t-test。所有Error bar代表标准误(standard error)。
图5显示了MAPK/ERK信号通路的示意图。
图6显示了MAPK/ERK信号通路抑制剂的短暂涂抹给药即可安全有效拮抗DNA损伤性皮肤早衰。其中：
A.在前述局部辐照诱导的小鼠皮肤早衰模型中进行Trametinib(+T)或溶剂对照(+S)表面涂抹干预的实验流程示意图。小鼠在7周龄(P50)左右小鼠接受一次性局部辐照。同时开始进行药物(Trametinib或对照溶剂)处理。药物处理的方式为每3天后背部涂抹给药一次，总计1次、2次或3次。辐照五天后进行蜡脱毛以诱导毛发再生。noRad：未辐照区；Rad：辐照区。
B.上述A中小鼠在一轮表型时间点的背皮照片。x1，x2，x3代表+T涂抹次数。比例尺1cm。
C.上述B中+Tx3小鼠的背皮切片中免疫细胞丰度相对统计(与+S组对比，n＝3)。n.s.:无统计学显著差异(no significance)。双尾t-test。所有Error bar代表标准误(standard error)。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次发现MAPK/ERK信 号通路的激活与DNA损伤刺激下的皮肤毛囊干细胞耗竭和后续的皮肤早衰表型密切相关。试验表明，用拮抗MAPK/ERK信号通路的靶向药物进行处理，可显著抑制了一系列的体内皮肤衰老/早衰表型，主要包括：皮肤毛囊干细胞丢失、毛发白化、毛发再生能力下降、表皮愈伤功能下降。因此，本发明的研究首次出乎意料地发现了MAPK/ERK信号通路具有促进皮肤衰老与早衰的关键作用，这与本领域中普通认为的该信号通路具有促进皮肤细胞生长/生存作用的观念截然不同。更重要的是，本发明的研究表明，抑制MAPK/ERK通路活性是一种拮抗皮肤老化与辐射性皮肤早衰的有效手段。以此为基础研发的干预手段可应用于辐射性皮肤早衰的防治与自然皮肤衰老的延缓，具有广泛的应用前景。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
皮肤衰老与DNA损伤
如本文所用，术语“皮肤衰老”、“皮肤老化”可互换使用，指皮肤上皮组织的衰老。此外，所述术语包括正常的皮肤衰老，以及过早或过快速的皮肤衰老(即皮肤早衰)。
皮肤上皮组织(包括上皮、毛囊、皮脂腺、汗腺)的衰老是人类衰老最显著的特征。主要表现为皱纹形成、愈伤/再生能力下降、毛发白化/稀疏、毛囊萎缩等。皮肤上皮干细胞(主要包括毛囊干细胞和表皮干细胞)是皮肤上皮组织稳 态维护与创伤修复的基础。上皮干细胞包括毛囊干细胞、表皮干细胞、其它属于皮肤上皮组织的干细胞、或其组合。其中表皮干细胞维持着上皮组织动态平衡，而毛囊干细胞支持着毛发生长与再生，并能够在创伤条件下修复上皮。皮肤上皮组织里面还存在一些不好明确定义的干细胞，例如毛囊和表皮交界处的干细胞，统称皮肤上皮干细胞。作为一种终身不断自我更新的组织，皮肤上皮组织的衰老与其干细胞的衰竭密切相关，但导致这种干细胞衰竭的具体分子机制尚不完全清楚。
在本发明的一个优选例中，所述的皮肤衰老具有选自下组的一个或多个指标：(l)皮肤上皮干细胞丢失；(m)毛发白化；(n)毛发再生能力下降；和(o)上皮愈伤功能下降。
另外，皮肤衰老还包括：一种或多种代表性的衰老细胞标记物或标志的增多，和/或一种多种代表性的年轻细胞标记物或标志的减少。所述的代表性的衰老细胞标记物或标志包括(但并不限于)：SA-β-半乳糖苷酶、P16、细胞增殖能力；所述的代表性的年轻细胞标记物或标志包括(但并不限于)：H3K9me3、胶原蛋白COL17A1。
DNA损伤被普遍认为是导致组织衰老/早衰的一个主要因素。在皮肤组织中，外源和内源性的DNA损伤诱导因素都能够导致显著的早衰现象。在基因组稳定性相关基因突变的早衰症患者及其对应的小鼠遗传模型中，包括毛发脱落与白化在内的典型皮肤衰老表型都会提早出现。另一方面，阳光中的紫外线(UV)是众所周知的DNA损伤诱因，同时也能够显著加速皮肤衰老，导致皮肤中常见的光老化症状。除了紫外线之外，肿瘤放疗中常用的电离辐射(IR)也是很强的DNA损伤诱因，能够诱导放疗区域皮肤细胞早衰，并导致长期性的皮肤愈伤功能障碍，进而带来发生难愈性溃疡的风险，是肿瘤放疗中最常见的并发症之一。近年来，有研究进一步证实了DNA损伤是促使皮肤上皮与上皮干细胞衰竭的直接诱因。抑制DNA损伤所导致的皮肤上皮干细胞衰竭在皮肤抗衰老美容与放射性皮肤损伤的防治中都有着广阔的应用前景。但目前尚无进入临床阶段的特效治疗手段。
本发明提供了一种通过靶向miR-31的抑制剂，来抑制DNA损伤对皮肤上皮干细胞的的促衰老效应从而有效对抗皮肤衰老和早衰的技术途径。
疾病模型构建
在本发明的一个优选实施例中，为了模拟射线导致的皮肤衰老和DNA损伤， 对各组小鼠进行γ射线局部辐照处理，具体步骤如实施例1,然后，为了检验小鼠毛发再生能力，使用了蜡脱毛(Wax)处理以诱导毛发再生，之后28天(4周)左右观测其皮肤表型。实验流程表如图1A所示。
MAPK/ERK通路抑制剂
在本发明中，术语“活性成分”或“用于皮肤抗衰老的活性成分”指MAPK/ERK通路抑制剂的拮抗剂或抑制剂。
如本文所用，术语“MAPK/ERK通路”、“MAPK/ERK信号通路”可互换使用，指丝裂原活化蛋白激酶/细胞外调节蛋白激酶(Motigen-activated proteinkinase/extracelluar regulated protein kinase,MAPK/ERK)级联通路。MAPK/ERK通路的示意图可参见图5。
促分裂原活化蛋白激酶(MAPK)信号传导途径与细胞事件例如生长、分化和应激反应有关。迄今为止已鉴定四个平行的MAPK通道：ERK1/ERK2、JNK、p38和ERK5。这些通道是线性激酶级联，因为MAPKKK磷酸化并活化MAPKK，以及MAPKK磷酸化并活化MAPK。迄今为止，已鉴定七种MAPKK同系物(MEK1、MEK2、MKK3、MKK4/SEK、MEK5、MKK 6和MKK7)和四个MAPK家族(ERK1/2、JNK、p38和ERK5)。这些通道的活化通过磷酸化调节着多种底物的活性。MEK催化MAPK底物ERK1和ERK2的磷酸化。优选地，本发明中的MAPK/ERK通路指由RAF-MEK1/2-ERK1/2组成的级联通路。
在本发明中，代表性的MAPK/ERK通路抑制剂包括(但并不限于)：小分子化合物、抗体、核酸、或其组合。优选的例子包括特异性靶向MAPK/ERK通路的蛋白(尤其是MAPK/ERK激酶)的化合物和抗体。
如本文所用，术语“MAPK/ERK抑制剂”、“MAPK/ERK通路抑制剂”可互换使用，指能够干扰MAPK活性，并且特别是ERK活性的分子。MAPK/ERK抑制剂可以是抗MAPK/ERK抗体，其特异性地结合MAPK/ERK蛋白并抑制其活性。此外，MAPK/ERK抑制剂可以是抑制性核酸。抑制性核酸可以是特异性结合MAPK/ERK蛋白并抑制其活性的适配体。其他抑制性核酸可以结合MAPK/ERK转录物，并抑制其翻译或使其降解。典型地，此类抑制性核酸可以是反义核酸、吗啉代寡核苷酸、抑制性RNA分子，例如siRNA或微小RNA或核酶。此外，MAPK/ERK抑制剂可以是结合MAPK/ERK并抑制其活性的小分子。MAPK/ERK的此类小分子抑制剂可以通过熟知的筛选程序或分子建模方法获得，所述分子建模方法的目的是确定与MAPK/ERK激酶结构域的活性位点结合的化合物。
一些代表性的MAPK/ERK信号通路的抑制剂可参见WO2008079814、 WO2008140553、WO2009064675等。
优选地，本发明中所述的MAPK/ERK抑制剂是选自以下的MEK抑制剂：U0126、GSK1120212(曲美替尼(Trametinib))、MEK162、SCH772984、lixertinib、AZD0364、KO-947、HH2710、或其组合。更佳地地，所述MAPK/ERK抑制剂是GSK1120212(曲美替尼(Trametinib))、MEK162，或SCH772984。
一种代表性的MAPK/ERK信号通路抑制剂是曲美替尼(Trametinib)；CAS:871700-17-3；分子量：615.39；中文化学名：N-[3-[3-环丙基-5-[(2-氟-4-碘苯基)氨基]-3，4，6，7-四氢-6,8-二甲基-2,4,7-三氧代吡啶并[4，3-d]嘧啶-1(2H)-基]苯基]乙酰胺；化学式：C 26H 23FIN 5O 4。英文名称为TRAMETINIB，又名GSK1120212、GSK 212或JTP 74057。该抑制剂公开于WO 2005/121142。
一种代表性的MAPK/ERK信号通路抑制剂是SCH772984:CAS:942183-80-4,购买自Selleck(S7101),化学式C 33H 33N 9O 2，是一种特异性的ERK1/2小分子抑制剂。
其他一些合适的MAPK/ERK信号通路抑制剂包括(但并不限于)：PD0325901(辉瑞公司(Pfizer))(WO 02/06213中披露)；PD184352(辉瑞公司)；瑞法替尼(Refametinib)(也称为RDEA119或Bay 86-9766)；卡比替尼，也称为XL518，并且可从罗氏公司(Roche)以商品名商购获得；AS-701255(默克雪兰诺公司(Merck Serono))；AS-701173(默克雪兰诺公司)；匹玛舍替(Pimasertib)，也称为AS-703026或MSC1936369B(默克雪兰诺公司)；RDEA436(阿尔代亚生物科学公司(Ard eaBiosciences))；RO4987655，也称为RG7167(罗氏公司)；和/或RG7420，也称为GDC-0623(罗氏公司)。
组合物或制剂
本发明提供了一种可用于预防或治疗皮肤衰老的组合物或制剂(包括化妆品、日化用品等)，所述的组合物或制剂包括：(a)可接受的载体(包括药学上可接受的载体、生理上可接受的载体、化妆品上可接受的载体)；和(b)有效量活性成分，所述的活性成分为MAPK/ERK通路抑制剂。
在本发明中，所述的活性成分的有效量可随使用的模式和皮肤衰老程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
术语“生理上可接受的载体”指用于活性成分的载体，包括：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2021071561-appb-000001
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
本发明的药物组合物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的活性成分每天以约0.00001mg-50mg/kg动物体重(较佳的0.0001mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
在本发明中，另一类组合物或产品是含本发明活性成分(靶向miR-31的抑制)的化妆品或日化用品。
在本发明中，所述的组合物或制剂还优选地含有透皮促进剂。
在另一优选例中，所述的组合物或制剂的剂型为外用(topical)剂型。
在本发明中，所述的化妆品组合物或制剂的剂型包括(但并不限于)：固体制剂、液体制剂、凝胶制剂、半固体制剂。一些优选的剂型膏剂、乳霜、贴剂、 涂剂、喷剂、微针剂、溶液剂、洗发等。
在本发明中，代表性的产品(或日化用品)包括(但并不限于)：洗发水、沐浴露、护肤品、护发产品、面膜。
在另一优选例中，所述的组合物或制剂的施用方式选自下组：局部施用、透皮施用和经皮施用。
本发明的主要优点包括：
(a)本发明首次意外地证实，辐照刺激通过激活MAPK/ERK信号通路导致皮肤上皮干细胞耗竭，进而导致后续的皮肤衰老，因此MAPK/ERK信号通路可作为预防或治疗皮肤衰老与早衰的标靶。
(b)MAPK/ERK通路抑制剂有效对抗或延缓皮肤的衰老(尤其是早衰)。
(c)MAPK/ERK通路抑制剂可有效防止干细胞的耗竭，进而防止后续的皮肤衰老。
(d)在辐照等导致皮肤早衰的情况下，早期抑制MAPK/ERK通路的激活即可有效防止后续的皮肤衰老，无须长期持续用药，因此可避免长期抑制MAPK/ERK通路，具有安全性。
(e)本发明首次意外地证实,不同MAPK/ERK信号通路抑制剂都可实现同样拮抗皮肤老化与辐射性皮肤早衰的效果。
(f)应用形式广泛,不同MAPK/ERK信号通路抑制剂的施用方式(口服或者涂抹)都可实现同样拮抗皮肤老化与辐射性皮肤早衰的效果。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料和方法
1)动物模型
使用7周龄C57BL/6野生型小鼠作为实验小鼠来制作局部辐照诱导皮肤早衰的动物模型。所有C57BL/6野生型小鼠购买自上海斯莱克实验动物有限责任公司。
2)γ射线局部辐照
γ射线辐照在复旦大学医学院放射医学研究所按标准实验步骤操作。进行局部辐照时，将麻醉后的小鼠置于辐照仓中的上下两层铅板中间的空隙，上下铅板相同，铅板垂直于放射源，两块铅板各自留有垂直对齐的直径3厘米的小孔，将小鼠上半部置于小孔中，铅板覆盖小鼠头部和下半身，用胶带适当固定小鼠四肢后进行辐照。辐照剂量10Gy。所有辐照实验均利用7周龄C57BL/6野生型小鼠。
3)Trametinib口服处理
Trametinib(CAS:871700-17-3)，购买自Selleck(S2673)，化学式C 26H 23FIN 5O 4。在小鼠实验中，将Trametinib粉末溶解于DMSO中，制成15mg/ml储备液，口服时按照1：25稀释比稀释于Corn oil(Sigma,C8267)中，每只小鼠按照1mg/kg剂量口服，每三天一次，共计五次。对照组口服同等溶剂。
4)Trametinib涂抹处理
Trametinib(CAS:871700-17-3)，购买自Selleck(S2673)化学式C 26H 23FIN 5O 4。将Trametinib粉末溶解于DMSO中，制成15mg/ml储备液，用剃毛刀剃去小鼠背部毛发，局部涂药时按照1：25稀释比稀释于50％乙醇,30％水和20％丙二醇(Sangon,A501754-0500)溶剂中，每只小鼠按照1mg/kg剂量每三天涂一次，共计五次。对照组涂抹同等溶剂。
5)小鼠背皮毛囊干细胞的流式分析
小鼠用二氧化碳致死装置处死后剃毛并取背皮，用手术刀去除真皮面脂肪组织后用PBS清洗，并置于Trypsin-EDTA(ThermoFisher,25200056)中4℃过夜消化。第二天用刀片刮取细胞，收集细胞悬液，置于PFE(PBS+1％FBS+1μM EDTA)染色液中于冰上进行CD34(1：100,eBioscience,11-0341-82)，CD49f(1：100,biolegend,313602)染色。染色结束后清洗细胞并加入活细胞染料Helix NP NIR(biolegend,425301)。染色后的细胞在流式分析仪(BD,CytoFLEX LX)上检测。流式检测CD34 +CD49f +活细胞群为毛囊干细胞，用流式分析软件FlowJo 10.0分析每组内毛囊干细胞所占的百分比，并与野生型小鼠未处理部位的百分比取比值。
6)小鼠背皮愈伤实验
小鼠用异氟烷麻醉后剃去毛发，背部用8mm打孔器进行打孔造创。贴手术巾(3M,SP1107)防止伤口变形，三天后取下，分别在造创前、造创后0、4、7、9、12和15天进行拍照，根据比例尺计算伤口面积，每组内不同时间点的小鼠创口面积与各自造创0天面积取比值，得到愈伤百分比。各组数据记录至愈伤完全结束。
7)体外培养的皮肤组织块(Exo-plant)的角质细胞迁移愈伤实验
小鼠处死后用脱毛膏去除毛发，75％酒精消毒后用手术刀去除脂肪组织。于PBS中清洗后用1mm打孔器打孔获得皮肤块，每孔中间加入1-2ul matrigel(Corning,356237)，贴附皮肤块并置于1mg/ml纤连蛋白(fibronectin)(Millipore,FC010-1mg)包被过的24孔板(Corning,CLS3527)包被中培养。培养7天后于显微镜下拍照，以皮肤块为中心点，用图像统计软件Imagej测量从皮肤块边沿迁出的细胞面积。各组面积分别与野生型未处理部位面积取比值。
8)小鼠毛发再生能力测试
用蜡去毛的方法完全移除现有毛发，以此激活毛发再生，并追踪拍照观测其再生速率。毛发反复再生能力测试的方法是在毛发首次再生完成后再次进行二次蜡脱毛以重复此过程。毛发再生过程中分别用相机(Canon)和毛发检测仪(Hair and Scalp)拍照记录。
9)Sch772984口服给药
Sch772984(CAS:942183-80-4),购买自Selleck(S7101),化学式C 33H 33N 9O 2，是一种特异性的ERK1/2小分子抑制剂。在小鼠实验中，将Sch772984粉末溶解于DMSO中，制成15mg/ml储备液，口服时按照1：25稀释比稀释于玉米油(Sigma,C8267)中，每只小鼠按照2mg/kg剂量口服，每三天一次，共计五次。对照组口服同等溶剂。
实施例1
皮肤早衰研究模型的建立
在本实施例中，通过局部电离辐照诱导在小鼠中建立了皮肤早衰研究模型。方法如下：参见图1A，对7周龄左右的成年小鼠上背部直径约2cm的区域进行一次性γ射线局部辐照，并在5天后通过蜡去毛启动毛发再生。
具体地，小鼠在7周龄(P50)左右小鼠接受局部辐照。五天后进行蜡脱毛(Wax)以诱导毛发再生。之后28天(4周)左右观测其第一轮毛发再生完成后的皮肤表型(一轮表型)，或再次通过蜡脱毛诱导第二轮毛发再生，并在之后4周左右观测其第二轮毛发再生完成后的皮肤表型(二轮表型)。
结果表明，小鼠上背部辐照区能够表现出一系列典型的皮肤早衰表型：主要包括：
1)新生毛发明显白化(图1B)。
2)皮肤愈伤能力减退。包括体内创伤愈合速率下降(图1C)和体外培养的皮肤组织块(Exo-plant)的角质细胞迁移速率下降(图1D)。
3)体内毛囊干细胞数量下降(图1E)。
4)毛发反复再生能力衰竭(图1F)。
实施例2
口服MAPK/ERK信号通路抑制剂(Trametinib)拮抗体内皮肤老化与辐射性皮肤早衰
为了验证抑制MAPK/ERK信号通路是一种拮抗体内皮肤老化与辐射性皮肤早衰的有效手段，使用了一种FDA批准的MEK1/2小分子抑制剂Trametinib作为范例。
在本实施例中，在皮肤辐照早衰模型中引入了短期药物处理，以便验证抑制MAPK/ERK信号通路对拮抗皮肤老化的有效性。
试验方法如图2A所示。小鼠在7周龄(P50)左右小鼠接受一次性局部辐照。在进行辐照的当天，同时开始进行药物(Trametinib或对照溶剂)口服处理。药物处理的方式为每3天口服给药一次，总计5次共15天。辐照五天后进行蜡脱毛以诱导毛发再生。之后28天(4周)左右观测其第一轮毛发再生完成后的皮肤表型(一轮表型)，或再次通过蜡脱毛诱导第二轮毛发再生，并在之后4周左右观测其第二轮毛发再生完成后的皮肤表型(二轮表型)。
结果：
在停药3周后(即辐照4周后)小鼠的第一轮毛发再生完成。在此时采样分析一部分小鼠的背皮表型(一轮表型)。对另一部分小鼠再次蜡去毛以诱导第二次毛发再生，并在4周后分析其背皮表型(二轮表型，主要代表毛发反复再生能力)。
结果表明，与溶剂对照处理组的辐照区皮肤相比，Trametinib处理组的辐照区皮肤表现出了显著缓解的皮肤衰老表型，主要体现在：
1)毛发白化现象显著缓解(图2B,2D)。
2)毛发再生能力衰退导致的毛发稀疏现象显著缓解(图2B,2C)。
3)毛囊干细胞数量耗竭现象显著缓解(图2E)。
4)体内皮肤愈伤能力衰退现象显著缓解(图2F，2G)。
5)皮肤组织块体外培养(Exo-plant)实验中的角质细胞迁移能力减退现象显著缓解(图2H，2I)。
实施例3 涂抹MAPK/ERK信号通路抑制剂(Trametinib)拮抗体内皮肤老化与辐射性皮肤早衰
重复实施例2的方法和步骤，区别在于仅仅将口服性给药替换成了皮肤表面涂抹给药，这个实验步骤概要如图3A所示。
结果显示局部涂抹Trametinib组的辐照区皮肤与涂抹溶剂对照处理组的辐照区皮肤相比表现出了显著缓解的皮肤衰老表型，与前述口服给药的效果类似。
具体包括：
1)皮肤愈伤能力下降表型显著缓解(图3B,3C)；
2)皮肤毛囊干细胞耗竭现象显著缓解(图3D)；
3)皮肤毛发白化与毛发稀疏表型显著缓解(图3E-G)。
实施例4 口服MAPK/ERK信号通路抑制剂( Sch772984)拮抗体内皮肤老化与辐射性皮肤早衰
重复实施例2的实验方法和步骤，不同之处在于，将口服Trametinib替换成了口服Sch772984(实验步骤概要如图4A所示)。
结果显示口服Sch772984组的辐照区皮肤与口服溶剂对照处理组的辐照区皮肤相比表现出了显著缓解的皮肤衰老表型，与前述口服Trametinib的效果类似。
具体表现为：1)皮肤愈伤能力下降表型显著缓解(图4B)；
2)皮肤上皮毛囊干细胞比例下降现象显著缓解(图4C)；
3)皮肤毛发白化与毛发稀疏表型显著缓解(图4D-F)。
实施例5 一种梯度计量局部涂抹抑制MAPK/ERK通路抑制剂(Trametinib)拮抗体内皮肤老化与辐射性皮肤早衰
5.1材料和方法
1)Trametinib梯度剂量局部涂抹给药
Trametinib(CAS:871700-17-3)，购买自Selleck(S2673)化学式C 26H 23FIN 5O 4，是在前述专利申请中使用过的MEK小分子抑制剂。在小鼠实验中，将Trametinib粉末溶解于DMSO中，制成15mg/ml储备液，用剃毛刀剃去小鼠背部毛发，局部涂药时按照1：25稀释比稀释于50％ethanol,30％water和20％propylene glycol(Sangon,A501754-0500)溶剂中，每只小鼠按照1mg/kg剂量每三天涂一次，共计涂抹1次、2次或3次。对照组涂抹同等溶剂。
5.2研究结果：
MAPK/ERK信号通路抑制剂的短暂涂抹给药即可安全有效拮抗DNA损伤性皮肤早衰。
本发明人在前述实施例中证明了MAPK/ERK信号通路抑制剂在为期约两周内的多次口服或皮肤涂抹给药均可显著拮抗DNA损伤性皮肤早衰。为了优化这种治疗方法的临床实用性与安全性，我们这里探讨了进一步压缩给药时间窗口的可行性。以MEK1/2抑制剂Trametinib为例，我们在前述实施例的基础上进一步测试了Trametinib涂剂在不同涂抹给药次数下的皮肤抗衰效果。实验设计与前述实施例3类似，对前述实施例1中建立的辐射性皮肤早衰小鼠模型进行了不同次数的皮肤涂抹给药，并追踪观察其随后的皮肤早衰表型(图6A)。
本实施例的结果显示，辐照后当天内的一次性涂抹给药即可显著缓解辐照区皮肤的毛发白化与稀疏表型，而后续更多的给药次数能小幅提升疗效(图6B)。此外，我们还通过免疫荧光染色对这些小鼠的皮肤切片进行了检查，发现Trametinib涂剂处理并未显著改变辐照区皮肤中的免疫细胞数量(图6C)，提示其并无诱发炎症反应的风险。因此，我们的结果显示MAPK/ERK信号通路抑制剂的短暂乃至一次性涂抹给药即可安全有效的拮抗DNA损伤性皮肤早衰，具有极好的临床实用性。
讨论
MAPK/ERK信号通路是一条高度进化保守的经典细胞生长与分化调控信号通路。其核心为由MAPKKK(RAF)、MAPKK(MEK1/2)、MAPK(ERK)三种蛋白激酶依次组成的蛋白激酶级联通路。细胞通常通过表面受体(受体酪氨酸激酶(RTKs)、整合素、离子通道等)来接受刺激信号，并通过对接蛋白(SHC、GRB2、CRK等)与鸟苷酸交换因子(SOS、C3G等)来激活小分子GTP结合蛋白(RAS，RAP1等)，进而激活上述核心级联通路。激活的ERK二聚物可在胞质中调控靶蛋白，也可在细胞核内通过磷酸化来调控多种转录因子活性。
MAPK/ERK信号通路通常被认为具有促进细胞增殖和存活的作用，是一种广泛的促癌信号通路，其过度激活与多种肿瘤的发生与发展直接相关。例如：RAF的激活性突变能够促进黑色素瘤和转移性非小细胞肺癌的发生，BCR/ABL融合基因通过激活MAPK/ERK信号通路促进慢性粒细胞白血病的发生，乳腺癌和前列腺癌的发生发展也与MAPK/ERK信号的异常激活密切相关。此外，多种细胞都能够在电离辐射刺激下显著激活MAPK/ERK信号通路，这种激活具有促进细胞在辐照后存活的作用，被认为是一种细胞抵抗辐射的保护性机制。
在皮肤中，MAPK/ERK信号通路同样是一条经典的促生长/致癌通路。RAS基因的突变所介导的MAPK/ERK通路的异常激活能够使皮肤干细胞癌变从而诱导鳞状细胞癌的发生。在皮肤癌的发展进程中，提高MAPK/ERK信号通路活性会使得癌变的皮肤干细胞具有更高的增殖和迁移能力，从而加剧皮肤癌的恶性程度。在正常皮肤组织中，MAPK/ERK通路具有促进愈伤的作用。物理创伤能够显著激活MAPK/ERK通路，而降低其活性则减缓伤口愈合。紫外线刺激同样能够显著激活表皮细胞中的MAPK/ERK信号通路进而导致表皮增生，但人类皮肤在衰老过程中却表现出显著下调的MAPK/ERK信号通路活性。在本发明之前，目前尚无报道MAPK/ERK通路与皮肤衰老的关系，也无明确证据显示MAPK/ERK通路与体内皮肤的自然衰老或光老化存在功能性关联。
本发明的研究结果首次意外地证实了：抑制MAPK/ERK通路是一种拮抗皮肤老化与辐射性皮肤损伤的有效手段。同时，本发明人也证明了在辐照后短时间内抑制MAPK/ERK通路即可有效缓解辐照导致的皮肤早衰表型。这种方法规避了长期抑制MAPK/ERK通路可能带来的副作用，为在临床上防治电离辐射导致的皮肤早衰与长期性愈伤能力障碍等症状提供了切实可行的手段。
之前的研究显示DNA损伤是皮肤表皮和毛囊干细胞衰老与早衰的直接诱因，而电离辐射是人类皮肤中常见的DNA损伤诱因之一。因此，本发明的研究提示，辐照刺激通过激活MAPK/ERK信号通路导致皮肤上皮干细胞耗竭，进而导 致后续的皮肤衰老。因此，只要在辐照刺激后早期抑制MAPK/ERK通路的激活即可有效防止干细胞的耗竭进而防止后续的皮肤衰老，无须长期持续用药。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种MAPK/ERK通路抑制剂的用途，其特征在于，用于制备预防或治疗皮肤衰老(或抗皮肤衰老)的组合物或制剂。
  2. 如权利要求1所述的用途，其特征在于，所述的组合物包括药物组合物、日用化学品组合物。
  3. 如权利要求1所述的用途，其特征在于，所述的组合物或制剂用于选自下组的一种或多种应用：
    (a)降低毛发白化比率或减少白化毛发的产生；
    (b)提高皮肤愈伤能力或促进创伤愈合；
    (c)促进角质细胞的迁移；
    (d)促进上皮干细胞功能和/或减缓上皮干细胞的减少；和
    (e)提高毛发再生能力。
  4. 如权利要求3所述的用途，其特征在于，所述的上皮干细胞选自下组：毛囊干细胞、表皮干细胞、皮肤上皮组织的干细胞、或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老包括：DNA损伤导致的皮肤衰老、辐射性皮肤早衰、皮肤自然衰老、皮肤光老化。
  6. 如权利要求1所述的用途，其特征在于，所述的皮肤衰老是由选自下组的诱导因素诱导的：电离辐射、γ射线辐照、紫外线、放射治疗、过度暴晒、化学诱变剂、皮肤细胞自然增殖所导致的皮肤自然衰老或其组合。
  7. 如权利要求1所述的用途，其特征在于，所述的MAPK/ERK通路抑制剂选自下组：曲美替尼(Trametinib)、U0126、MEK162、SCH772984、lixertinib、AZD0364、KO-947、HH2710、PD0325901、PD184352、瑞法替尼(Refametinib)、卡比替尼(Cobimetinib)、AS-701255、AS-701173、匹玛舍替(Pimasertib)、RDEA436、RO4987655、RG7420、LY3214996、优立替尼(Ulixertinib或VRT752271)、LTT462、JSI-1187、司美替尼(Selumetinib)、比美替尼(Binimetinib)或其组合。
  8. 如权利要求1所述的用途，其特征在于，所述的MAPK/ERK通路抑制剂抑制包括：曲美替尼、SCH772984、或其药学上可接受的盐。
  9. 一种可用于预防或治疗皮肤衰老的组合物或制剂，其特征在于，所述的组合物或制剂包括：(a)生理上可接受的载体；和(b)有效量的活性成分，所述的活性成分为MAPK/ERK通路抑制剂。
  10. 一种体外筛选用于预防或治疗皮肤衰老的潜在物质的方法，其特征在于，包括步骤：
    (1)分别向检测体系中加入待检测的物质或阴性对照物；和
    (2)分别检测加入了待检测的物质的样本和加入了阴性对照物的样本中MAPK/ERK通路的水平；如果与加入了阴性对照物的样本相比，加入了待检测的物质的样本的MAPK/ERK通路的水平降低，则是潜在的预防或治疗皮肤衰老疾病的物质。
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