WO2018137400A1 - 胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤药物中的应用 - Google Patents

胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤药物中的应用 Download PDF

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WO2018137400A1
WO2018137400A1 PCT/CN2017/113636 CN2017113636W WO2018137400A1 WO 2018137400 A1 WO2018137400 A1 WO 2018137400A1 CN 2017113636 W CN2017113636 W CN 2017113636W WO 2018137400 A1 WO2018137400 A1 WO 2018137400A1
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ultraviolet light
use according
skin damage
protease inhibitor
induced skin
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PCT/CN2017/113636
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French (fr)
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殷卫海
张铭超
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上海交通大学
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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • the invention relates to the use of a cystine protease inhibitor in the field of pharmacy. Specifically, the use of a cystine protease inhibitor for the preparation of a medicament for treating or preventing ultraviolet light-induced skin damage.
  • Ultraviolet rays in the sun are the main cause of human skin damage caused by ultraviolet radiation.
  • Ultraviolet radiation can cause various pathological effects on the skin, such as redness, peeling, inflammation, ulceration, and various skin diseases, and it will produce a large amount of active oxidation.
  • the substance causes DNA damage in the subcutaneous cells and accelerates skin aging.
  • 90% of human skin aging is due to ultraviolet radiation, and the most serious disease caused by ultraviolet light is skin cancer.
  • Skin cancer patients account for 40% of all cancer patients, and there are more than 3 million new cases of skin cancer every year worldwide, and the Skin Cancer Foundation also points out that one in five Americans will have skin at some stage in their lives. cancer.
  • the ozone layer responsible for blocking ultraviolet rays in the atmosphere is continuously thinning.
  • Some experts have predicted that the thickness of the ozone layer is reduced by 1% and the intensity of ultraviolet radiation is increased by 2%. Therefore, the prevention and treatment of ultraviolet damage to the skin has significant socio-economic and clinical value for people to improve the health of the skin and prevent the occurrence of major skin diseases such as skin cancer.
  • UV skin damage has not received enough attention.
  • Many skin-injured patients will only go to the clinic after receiving particularly strong skin lesions, and in recent years, China has even UV-induced skin worldwide
  • the number of patients with disease and skin cancer is gradually increasing. Therefore, effective methods and treatments for detecting skin UV damage, as well as low-cost prophylactic drugs or preparations, are of great significance for the monitoring of skin health, the diagnosis and treatment of skin diseases, especially skin cancer.
  • the present inventors have found for the first time in the experiment that a cystine protease inhibitor has an obvious protective effect in preventing ultraviolet light-induced skin damage.
  • the present invention has been completed based on the above findings through further experiments.
  • the cysteine in the present invention is an amino acid which is common in living organisms and can be transformed from methionine (methionine, essential amino acid) in the body, and can be transformed with cystine.
  • Cysteine is a naturally occurring amino acid
  • cysteine is a reducing agent
  • cystine is an amino acid drug. Therefore, the cystatin protease inhibitor of the present invention includes both a cystine protease inhibitor and an inhibitor capable of inhibiting cysteine protease activity in vivo, and in the embodiment of the priority document Chinese Patent Application No.
  • cystine protease inhibitor has a biological function, and in cells in which only the natural amino acid, cysteine, is present, those skilled in the art will recognize that this effect is due to inhibition of the activity of the cysteine protease.
  • cystine is an amino acid formed by a disulfide bond between two cysteines
  • the effect of the prior art on a cystine protease inhibitor actually includes by inhibiting the enzymatic activity of cysteine protease.
  • the action, that is, the cystine protease inhibitor described in the present invention includes a cystine protease inhibitor and a cysteine protease inhibitor which inhibit the activity of cysteine protease.
  • cysteine protease inhibitors There are currently no specific cystine protease inhibitors on the market, and all, very small amounts of commercially available cystine protease inhibitors are also cysteine protease inhibitors.
  • the cysteine protease inhibitor E-64 which has been used by the inventors, is also one of the most commonly used cysteine protease inhibitors. This is the only cystin protease inhibitor used by the inventors. It was used in the Chinese patent application filed on January 26, 2017 (application number 201710061509.0). The only cystin protease inhibitor in the relevant assay is also the only cystin protease inhibitor used in the corresponding relevant test in this patent application. Therefore, the Chinese patent application filed on January 26, 2017 (Application No. 201710061509.0) and the "Caspase inhibitors" referred to in this patent application include cystatin proteases and cysteine enzymes. Inhibitor.
  • One of the objects of the present invention is to provide a use of a cystine protease inhibitor for the preparation of a medicament, a health care product or a medicament for treating or preventing ultraviolet light-induced skin damage.
  • cysteine protease inhibitor as a sole component in the manufacture of a medicament for preventing or treating ultraviolet light-induced skin lesions is disclosed.
  • cysteine protease inhibitor as a sole component in the manufacture of a medicament for preventing or treating ultraviolet light-induced skin lesions is disclosed.
  • cystine protease inhibitor as an active ingredient in the preparation of a medicament for preventing or treating ultraviolet light-induced skin damage.
  • cysteine protease inhibitor as an active ingredient in the preparation of a medicament for preventing or treating ultraviolet light-induced skin damage.
  • cystine protease inhibitor as the sole active ingredient in the preparation of a medicament for preventing or treating ultraviolet light-induced skin damage.
  • cysteine protease inhibitor as an active ingredient in the preparation of a medicament for preventing or treating ultraviolet light-induced skin damage.
  • the medicament, nutraceutical or agent is a composition further comprising a pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipient comprises a pharmaceutically acceptable carrier, diluent or excipient
  • the dosage form of the composition is selected from the group consisting of various external application preparations, tablets, capsules, granules, pills, suspensions, syrups, various enteric preparations, and injections. .
  • Cystatin inhibitors also include cystine protease inhibitors and cysteine protease inhibitors that inhibit the activity of cysteine proteases.
  • the indicator of ultraviolet light-induced skin damage is a decrease in epidermal stromal cells, an increase in the stratum corneum, and an increase in skin thickness.
  • the indicator of ultraviolet light-induced skin damage is an increase in apoptosis signal.
  • the indicator of ultraviolet light-induced skin damage is an increase in cell necrosis signal.
  • the indicator of ultraviolet light-induced skin damage is an increase in skin or cell autofluorescence.
  • the ultraviolet light-induced skin damage is a person whose skin is exposed to ultraviolet light.
  • the invention finds that the application of the cystine protease inhibitor in the preparation of a medicament for preventing and treating ultraviolet light-induced skin damage, and the basis for clinical prevention and treatment of ultraviolet light-induced skin damage can effectively prevent and treat ultraviolet light-induced skin damage, Give patients effective treatment.
  • Figure 1 shows that the autofluorescence intensity of mouse skin tissue is positively correlated with UV-induced skin damage.
  • Figure 2 shows a representation of a cystine protease inhibitor (also a cysteine protease inhibitor) E-64 that inhibits ultraviolet light-induced increase in skin autofluorescence.
  • Figure 3 shows a quantitative map of the inhibition of ultraviolet light-induced skin autofluorescence by a cystine protease inhibitor (also a cysteine protease inhibitor) E-64.
  • Figure 4 shows an immunoglobulin signature of a cystine protease inhibitor (also a cysteine protease inhibitor) E-64 that inhibits UV-induced reduction of keratin 1 content.
  • Figure 5 shows a quantitative map of inhibition of UV light induced keratin 1 content by a cystine protease inhibitor (also a cysteine protease inhibitor) E-64.
  • Figure 6 shows that UV light dose-dependently increases apoptosis necrosis after 24 hours of UV damage to B16 cells.
  • Figure 7 shows the UV-induced increase in green fluorescence of B16 cells after 0.5 to 3 hours of UV-damaged B16 cells.
  • Figure 8 shows that the cysteine protease inhibitor (also a cysteine protease inhibitor) E-64 was pretreated for 1 hour, and after UV light was irradiated to B16 cells for 0.5-3 hours, the autofluorescence of B16 cells was detected, and E was found. Treatment with -64 prevents UV-induced death of B16 cells.
  • keratin 1 As used herein, “keratin 1”, “keratin 1”, “KRT1” are interchangeable, and include keratin which can be detected using keratin 1 antibody recognition, which may be a single substance or a mixture;
  • keratin 1 comprises a keratin 1 fragment in which a partial amino acid sequence is deleted, and the keratin 1 fragment of the deleted partial amino acid sequence is N of keratin 1
  • a keratin 1 fragment of either 1 to 644 amino acids is deleted at the terminus or C-terminus.
  • Keratin 1 fragment lacking a partial amino acid sequence As used herein, “keratin 1 degradant” is interchangeable, which means that the N-terminus or C-terminus of keratin 1 is deleted by any one of 1 to 644 amino acids. Keratin 1 fragment.
  • cystpase inhibitors As used herein, “Caspase inhibitors”, “cystase inhibitors” are interchangeable and include cystine protease inhibitors and cysteine protease inhibitors that inhibit cysteine enzyme activity.
  • cystatin protease inhibitors including cysteine protease inhibitors
  • mice male C57 mice were used to pretreat the drug for several hours after the skin of the mouse.
  • the UVC, irradiated mice weighed between 15-25 g. After the completion of the UV radiation, the mice were housed in an animal room under conditions of 22-24 ° C for 12 hours of light/dark circulation and were free to eat water.
  • Non-invasive imaging of UV-irradiated skin using a laser confocal microscope within 24 hours.
  • the mice were sacrificed three days later, and the damaged skin tissue was taken for H&E (hematoxylin-eosin staining).
  • the skin tissue of the mouse was imaged by a laser confocal microscope, wherein the excitation wavelength of the laser confocal microscope was 440-600 nm.
  • Paraffin section of skin tissue soak the skin tissue in 4% paraformaldehyde solution for 24 hours, then dip the tap water, distilled water, gradient alcohol, xylene, etc. according to the method of paraffin sectioning. Paraffin wax, made into paraffin sections.
  • H&E staining of skin paraffin sections Dewaxed paraffin sections by dipping xylene, then immersed in gradient alcohol, distilled water, hematoxylin staining for 10 minutes, tap water rinse for 30 minutes, distilled water for 30 seconds, 95% ethanol for 10 seconds, Yihongfu Dyeing for 30 seconds, washing with 70% alcohol twice, sequentially dipping gradient alcohol, xylene, and neutral resin to seal.
  • Photographing and quantifying the staining results photographing the above two stains.
  • the indicators such as epidermal keratinization thickness and skin thickness were quantified.
  • the ear tissue was taken, placed in a weighed EP tube, added to the lysate, placed on ice, ground, added to the lysate, centrifuged, and the supernatant was stored in a -80 degree refrigerator. .
  • Standard concentrations of protein were prepared according to the instructions of the BCA kit.
  • the prepared gel was fixed in an electrophoresis apparatus, and protein samples were separately added. Electrophoresis, transfer film, milk at room temperature, TBST wash and add primary antibody overnight. The HRP-labeled secondary antibody was added after washing with TBST. Finally, the nitrocellulose membrane was developed with a color developing solution, and photographed by exposure in an imaging system.
  • Example 2 The skin fluorescence was enhanced under ultraviolet irradiation, and the fluorescence intensity was positively correlated with skin damage.
  • the present invention determined that the autofluorescence intensity of mouse skin tissue is positively correlated with ultraviolet light-induced skin damage (Fig. 1).
  • a cystine protease inhibitor (also a cysteine protease inhibitor) E-64 has the ability to prevent UV skin damage, which is significantly better than other proteases. Inhibitor. Among them, E-64 (CAS No.: 66701-25-5) can inhibit UV-induced skin autofluorescence ( Figures 2 and 3) and significantly prevent UV-induced skin damage.
  • the inventors also screened a variety of other family protease inhibitors, such as threonine protease inhibitors, serine protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, AEBSF), etc., and found them. There is no significant protective effect on UV-induced skin autofluorescence and skin damage.
  • Cysteine protease inhibitor can significantly inhibit ultraviolet light-induced reduction of skin keratin 1
  • the reduction of keratin 1 is an important mechanism of UV-induced skin damage.
  • the cystatin inhibitor also a cysteine protease inhibitor
  • E-64 inhibits UV-induced reduction of keratin 1 content ( Figures 4 and 5).
  • Example 5 Cell Level Experiment: Ultraviolet light induced cell autofluorescence enhancement with a positive correlation between fluorescence and damage.
  • B16 cells were cultured at 37 ° C in a 5% carbon dioxide atmosphere. After treating the cells with the drug for a while, the cells are treated with ultraviolet light and then returned to the normal culture medium for culture.
  • UV-treated cells were imaged using a laser confocal microscope.
  • the excitation wavelength of the laser confocal microscope is 440-600 nm.
  • the apoptosis-induced necrosis signal of the ultraviolet-treated cells was detected by flow cytometry using an apoptosis necrosis detection kit.
  • Figure 7 shows the UV-induced increase in green fluorescence of B16 cells after 0.5 to 3 hours of UV-damaged B16 cells. This autofluorescence rise is positively correlated with cell death.
  • the inventors screened a large number of drugs and found that the cystine protease inhibitor (also a cysteine protease inhibitor) E-64 has a protective effect against ultraviolet light-induced cell damage (Fig. 8). The inventors have found that other family protease inhibitors have no significant protective effect on UV-induced cell damage.
  • the experiment further found that a variety of inhibitors of caspase family and cysteine proteases such as Cathepsin family, including CA 074, Odanacatib, cysteine protease inhibitor hydrochdrochloride, etc., can effectively reduce UV damage of the skin.
  • Cathepsin family including CA 074, Odanacatib, cysteine protease inhibitor hydrochdrochloride, etc.
  • cysteine protease inhibitors can protect against UV-induced skin damage.
  • the specific form of the damage is that the epidermal stromal cells are reduced and the stratum corneum is increased. Increased skin thickness. The apoptotic signal increases and the necrotic signal increases. Increased skin or cell autofluorescence.
  • cystine protease inhibitors (including cysteine protease inhibitors) have important application value in the preparation of drugs for preventing and treating ultraviolet light-induced skin damage.

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Abstract

胱氨酸蛋白酶抑制剂在制备防治紫外光诱导的皮肤损伤药物中的应用。其中，胱氨酸蛋白酶抑制剂，包括可以抑制半胱氨酸蛋白酶活力的胱氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂均可以显著减少紫外光诱导的皮肤损伤，例如细胞死亡和组织损伤。

Description

胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤药物中的应用
相关申请的交叉引用
本发明要求2017年1月26日提交的中国专利申请(申请号201710061509.0)的优先权，其全部内容通过引用并入本文。
技术领域
本发明涉及胱氨酸蛋白酶抑制剂在药学领域中的应用。具体是胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤药物中的应用。
背景技术
阳光中的紫外线是人的皮肤受到紫外线辐射损伤的主要原因，紫外线辐射对于皮肤会产生多种病理作用，例如皮肤红肿，脱皮，炎症，溃烂，以及多种皮肤疾病等，并会产生大量活性氧化物，造成皮下细胞的DNA损伤，加速皮肤衰老。事实上，人体皮肤衰老90％的原因是由于紫外线照射，而紫外线诱导的最严重的疾病是皮肤癌，已有研究证明，90％以上的皮肤癌是由阳光中的紫外线照射引起的。皮肤癌患者占所有癌症患者中的40％，全世界每年有300多万新发皮肤癌病例，而皮肤癌基金会也指出，五分之一的美国人在一生中的某个阶段会得皮肤癌。随着人类工业化的进展，大气层中负责阻挡紫外线的臭氧层在不断变薄，有专家曾预测，臭氧层厚度减少1％，紫外线辐射强度增加2％。因此，对于皮肤紫外线损伤的预防以及治疗对于人们提高皮肤的健康水平、防止皮肤癌等重大皮肤疾病的产生，都有着重大的社会经济意义和临床价值。
而在我国，由于人口压力巨大，诊断和就医困难，紫外皮肤损伤尚未得到足够的重视，往往很多皮肤受创患者只有受到特别强的皮损后才会门诊就医，而使得近年来，我国乃至于全世界紫外创伤导致的皮肤 病和皮肤癌患者数量逐渐增多。因此，有效的皮肤紫外损伤的检测方法和治疗手段，以及低成本的预防性药物或制剂，对于皮肤健康的监测、皮肤疾病特别是皮肤癌的诊断和治疗，意义重大。
发明内容
本发明人在实验中首次发现：胱氨酸蛋白酶抑制剂(cystine protease inhibitor)在防治紫外光诱导的皮肤损伤中具有明显的保护作用。基于上述发现经过进一步实验从而完成了本发明。
本发明中的半胱氨酸是一种生物体内常见的氨基酸，可由体内的蛋氨酸(甲硫氨酸，人体必需氨基酸)转化而来，可与胱氨酸互相转化。半胱氨酸是一种天然产生的氨基酸，半胱氨酸是一种还原剂，胱氨酸为氨基酸类药物。因此本发明中的胱氨酸蛋白酶抑制剂中既包括胱氨酸蛋白酶抑制剂，也包括能够抑制体内的半胱氨酸蛋白酶活性的抑制剂，并且优先权文件中国专利申请号201710061509.0的实施例中明确记载胱氨酸蛋白酶抑制剂具有生物功能，在只存在天然氨基酸即半胱氨酸的细胞中，本领域技术人员可以认识到该作用是因为由于抑制了半胱氨酸蛋白酶的活力造成的。
另外，由于胱氨酸是两个半胱氨酸经过二硫键形成的氨基酸，因此现有技术中针对胱氨酸蛋白酶抑制剂的作用事实上也包括通过抑制半胱氨酸蛋白酶的酶活性发挥作用，即本发明中所述的胱氨酸蛋白酶抑制剂包括抑制半胱氨酸蛋白酶活力的胱氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂。
现在市场上没有任何特异性的胱氨酸蛋白酶抑制剂，所有的、极少量市场上可以购买到的胱氨酸蛋白酶抑制剂都同时是半胱氨酸蛋白酶抑制剂。发明人一直使用的胱氨酸蛋白酶抑制剂E-64同时也是最常用的半胱氨酸蛋白酶抑制剂之一。这是发明人唯一使用过的胱氨酸蛋白酶抑制剂，它是被使用在于2017年1月26日提交的中国专利申请书(申请号201710061509.0)中 相关试验中的唯一胱氨酸蛋白酶抑制剂，也是被使用在本专利申请书中对应的相关试验中的唯一胱氨酸蛋白酶抑制剂。所以，在2017年1月26日提交的中国专利申请书(申请号201710061509.0)以及本专利申请书中所指的“胱氨酸蛋白酶抑制剂”包括胱氨酸蛋白酶抑制剂和半胱氨酸酶抑制剂。
本发明的目的之一是提供了胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤的药物、保健品或试剂中的用途。
在一个实施例中，公开了胱氨酸蛋白酶抑制剂(cystine protease inhibitor)作为唯一成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
在一个实施例中，公开了半胱氨酸蛋白酶抑制剂(cysteine protease inhibitor)作为唯一成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
进一步的胱氨酸蛋白酶抑制剂作为活性成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
进一步的半胱氨酸蛋白酶抑制剂作为活性成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
进一步的，胱氨酸蛋白酶抑制剂作为唯一活性成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
进一步的半胱氨酸蛋白酶抑制剂作为活性成分在制备预防或治疗紫外光诱导的皮肤损伤药物中的应用。
在一个实施例中，所述药物、保健品或试剂是一种组合物，其还包括药学上可接受的药用辅料。
其中，所述药用辅料包括药学上可接受的载体、稀释剂或赋形剂、
在一个实施例中，所述组合物的剂型选自各种外用涂抹制剂、片剂、胶囊剂、颗粒剂、滴丸剂、混悬剂、糖浆剂、各种肠溶制剂及注射剂所组成的组。
胱氨酸蛋白酶抑制剂还包括抑制半胱氨酸蛋白酶活力的胱氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂。
在本发明中，所述紫外光诱导皮肤损伤的指标为表皮基质细胞减少、角质层增加和皮肤厚度增加。
在本发明中，所述紫外光诱导皮肤损伤的指标为细胞凋亡信号增加。
在本发明中，所述紫外光诱导皮肤损伤的指标为细胞坏死信号增加。
在本发明中，所述紫外光诱导皮肤损伤的指标为皮肤或者细胞自荧光增加。
进一步的，其中所述紫外光诱导的皮肤损伤，为皮肤受到紫外光辐射的人。
本发明发现胱氨酸蛋白酶抑制剂在制备防治紫外光诱导的皮肤损伤药物中的应用，为临床预防和治疗紫外光诱导的皮肤损伤建立基础，可以有效的预防和治疗紫外光诱导的皮肤损伤，使患者得到有效的治疗。
附图说明
图1示出小鼠皮肤组织的自荧光强度与紫外光诱导的皮肤损伤呈正相关。
图2示出胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64抑制紫外光诱导的皮肤自荧光上升的代表图。
图3示出胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64抑制紫外光诱导的皮肤自荧光上升的定量图。
图4示出胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64抑制紫外光诱导的角蛋白1含量的降低的免疫蛋白印记图。
图5示出胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64抑制紫外光诱导的角蛋白1含量的降低的定量图。
图6示出紫外光损伤B16细胞24小时后，紫外光剂量依赖性地增加细胞凋亡坏死。
图7示出紫外损伤B16细胞0.5-3小时后，紫外光诱导B16细胞绿色荧光上升图。
图8示出胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64预处理1小时，紫外光再照射B16细胞0.5-3小时后，检测B16细胞的自发荧光，发现E-64的处理可以防止紫外光诱导的B16细胞的死亡。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
如本文所用，“角蛋白1”、“keratin 1”、“KRT1”可互换，其是包括能够使用角蛋白1抗体识别检测的角蛋白，其可以是单一物质，也可以是混合物；在本发明中，角蛋白1包括其缺失部分氨基酸序列的角蛋白1片段，所述缺失部分氨基酸序列的角蛋白1片段为角蛋白1的N 端或C端缺失1至644个氨基酸中任一种的角蛋白1片段。
如本文所用，“缺失部分氨基酸序列的角蛋白1片段”、“角蛋白1降解物”可互换，其是指角蛋白1的N端或C端缺失1至644个氨基酸中任一种的角蛋白1片段。
如本文所用，“胱氨酸蛋白酶抑制剂”、“胱氨酸酶抑制剂”可互换，其包括抑制半胱氨酸酶活力的胱氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂。
实施例1实验方法
发明人进行了大量的实验，确定了胱氨酸蛋白酶抑制剂(包括半胱氨酸蛋白酶抑制剂)可以保护紫外光诱导的皮肤损伤。
根据本发明，使用雄性C57小鼠，将药物预处理在小鼠皮肤后若干小时。UVC、照射的小鼠重量在15-25g范围之间。紫外线辐射完成后，小鼠在动物房中进行饲养，条件为22-24℃，12小时的明/暗循环，并可自由进食取水。
24小时内，使用激光共聚焦显微镜对紫外线照射的皮肤进行无创成像。其后三天牺牲小鼠，取损伤皮肤组织，进行H&E(苏木精&伊红染色)检测。小鼠的皮肤组织用激光共聚焦显微镜成像，其中激光共聚焦显微镜的激发波长为440-600nm。
皮肤组织的储存：皮肤取出后，取出适量组织浸泡于4％多聚甲醛，用于制作石蜡切片，剩余组织用铝箔纸包裹好，使用液氮冷冻，之后转移到-80℃冰箱长期保存。
皮肤组织石蜡切片：将皮肤组织浸泡4％多聚甲醛溶液24h，然后依照石蜡切片的制作方法依次浸自来水、蒸馏水、梯度酒精、二甲苯、 石蜡，制作成石蜡切片。
皮肤石蜡切片的H&E染色：将石蜡切片浸二甲苯脱蜡，然后依次浸于梯度酒精、蒸馏水，苏木素染色10分钟，自来水流水冲洗30分钟，蒸馏水浸泡30秒，95％乙醇10秒，伊红复染30秒，70％酒精洗涤2次，依次浸梯度酒精、二甲苯，中性树脂封片。
对染色结果拍照、量化：对上述两种染色进行拍照。并针对表皮角质化厚度，表皮厚度等指标进行量化。
免疫蛋白印迹实验(Western blot)：
1.蛋白样品制备
用水合氯醛过量麻醉动物后，取耳朵组织，放入称重的EP管中，加入裂解液，置于冰上，研磨，加入裂解液，离心取上清分装后存于-80度冰箱。
2.蛋白浓度测定
按照BCA试剂盒的说明书，配制标准浓度蛋白。
3.SDS聚丙烯酰胺凝胶电泳
将配制好的凝胶固定在电泳装置中，分别将蛋白样品加入。电泳、转膜，牛奶室温封闭、TBST洗涤后加入一抗过夜。TBST洗后加入HRP标记的二抗。最后用显色液对硝酸纤维膜进行显色，在成像系统中曝光拍照。
实施例2紫外照射下皮肤荧光增强，且荧光强度与皮肤损伤呈正相关。
本发明通过以上实验，确定了小鼠皮肤组织的自荧光强度与紫外光诱导的皮肤损伤呈正相关(图1)
实施例3蛋白酶抑制剂的筛选
在所有的蛋白酶抑制剂中，由于组织内蛋白酶数量巨大，种类繁 多，发明人在经过大量的筛选后，最终发现1种胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64具有防止紫外皮肤损伤的能力，其明显好于其它的蛋白酶抑制剂。其中E-64(CAS号：66701-25-5)这个药物可以抑制紫外光诱导的皮肤自荧光上升(图2和图3)，并显著防止紫外光诱导的皮肤损伤。
发明人同时筛查了多种其他家族蛋白酶抑制剂，如苏氨酸蛋白酶抑制剂、丝氨酸蛋白酶抑制剂(4-(2-氨乙基)苯磺酰氟盐酸盐，AEBSF)等，发现它们对紫外光诱导的皮肤自荧光上升以及皮肤损伤没有明显保护作用。
实施例4半胱氨酸蛋白酶抑制剂可以显著抑制紫外光诱导的皮肤角蛋白1的降低
角蛋白1的降低是紫外光诱导的皮肤损伤的一个重要机制。胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64可以抑制紫外光诱导的角蛋白1含量的降低(图4和图5)。
实施例5细胞水平的实验：紫外光诱导细胞自发荧光增强，且荧光和损伤呈正相关。
根据本发明，使用B16细胞，培养在37℃，5％的二氧化碳环境中。使用药物处理细胞一段时间后，使用紫外光处理细胞后，重新放回普通培养液中培养。
1小时，使用激光共聚焦显微镜对紫外光处理过的细胞进行成像。其中激光共聚焦显微镜的激发波长为440-600nm。
24小时后，使用细胞凋亡坏死检测试剂盒，用流式细胞仪检测紫外光处理过的细胞凋亡坏死信号。
紫外光损伤B16细胞24小时后，细胞的自荧光可以作为细胞损伤的 标志物(图6)，紫外光剂量越高，细胞自荧光越强，细胞凋亡坏死数量越多，即紫外光剂量依赖性地增加细胞凋亡坏死。
图7示出紫外损伤B16细胞0.5-3小时后，紫外光诱导B16细胞绿色荧光上升图。该自发荧光上升和细胞死亡成正相关性。
发明人筛选了大量药物，发现胱氨酸蛋白酶抑制剂(同时也是半胱氨酸蛋白酶抑制剂)E-64，对紫外光诱导的细胞损伤具有保护作用(图8)。发明人发现其它家族蛋白酶抑制剂对紫外光诱导的细胞损伤没有显著保护作用。
实验进一步发现，多种针对caspase家族以及Cathepsin家族等半胱氨酸蛋白酶的抑制剂，包括CA 074,Odanacatib,cysteine protease inhibitor hydrochdrochloride等，都能够有效地减少皮肤的紫外损伤。
由以上实验可以得出，半胱氨酸蛋白酶抑制剂可以保护紫外光诱导的皮肤损伤。其中损伤的具体形式为，表皮基质细胞减少，角质层增加。皮肤厚度增加。细胞凋亡信号增加，坏死信号增加。皮肤或者细胞自荧光增加。
因此，胱氨酸蛋白酶抑制剂(包括半胱氨酸蛋白酶抑制剂)在制备防治紫外光诱导的皮肤损伤药物中有重要的应用价值。
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求的限制。本申请中引用的所有文献均完整地并入本文作为参考。

Claims (10)

  1. 胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤的药物、保健品或试剂中的用途。
  2. 如权利要求1所述的用途，其中，所述药物、保健品或试剂是一种组合物，其还包括药学上可接受的药用辅料。
  3. 如权利要求2所述的用途，其中，所述药用辅料包括药学上可接受的载体、稀释剂或赋形剂、
  4. 如权利要求2所述的用途，其中，所述组合物的剂型选自各种外用涂抹制剂、片剂、胶囊剂、颗粒剂、滴丸剂、混悬剂、糖浆剂、各种肠溶制剂及注射剂所组成的组。
  5. 如权利要求1至4任一项所述的用途，其中，胱氨酸蛋白酶抑制剂还包括抑制半胱氨酸酶活力的胱氨酸蛋白酶抑制剂和半胱氨酸蛋白酶抑制剂。
  6. 如权利要求1至4任一项所述的用途，其中所述紫外光诱导皮肤损伤的指标为表皮基质细胞减少、角质层增加和皮肤厚度增加。
  7. 如权利要求1至4任一项所述的用途，其中，所述紫外光诱导皮肤损伤的指标为细胞凋亡信号增加。
  8. 如权利要求1至4任一项所述的用途，其中，所述紫外光诱导皮肤损伤的指标为细胞坏死信号增加。
  9. 如权利要求1至4任一项所述的用途，其中，所述紫外光诱导皮肤损伤的指标为皮肤或者细胞自荧光增加。
  10. 如权利要求1至4任一项所述的用途，其中，所述受到紫外光诱导的皮肤损伤的受试者为皮肤受到紫外光辐射的人。
PCT/CN2017/113636 2017-01-26 2017-11-29 胱氨酸蛋白酶抑制剂在制备治疗或预防紫外光诱导的皮肤损伤药物中的应用 WO2018137400A1 (zh)

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