WO2020037714A1

WO2020037714A1 - Application of trail mutants in preparation of medicines for treating acnes, and preparation

Info

Publication number: WO2020037714A1
Application number: PCT/CN2018/104064
Authority: WO
Inventors: 陈守春; 闫娟; 徐琦; 胡海洋; 魏利佳; 黄先洲; 丁奕
Original assignee: 成都华创生物技术有限公司
Priority date: 2018-08-23
Filing date: 2018-09-05
Publication date: 2020-02-27
Also published as: CN109125709A; CN109125709B

Abstract

The present invention provides an application of TRAIL mutants in preparation of medicines for treating acnes, and a preparation. TRAIL mutant proteins comprise TRAIL-Mu3, TRAIL-MuR5, TRAIL-MuR6, TRAIL-MuR5S4TR, and TRAIL-MuR6S4TR. Experimental researches prove that the five TRAIL mutants can inhibit proliferation of sebocytes in primary culture in vitro and induce apoptosis thereof, and biological activities of the mutants are greatly stronger than that of an isotretinoin control drug. The mutant proteins can be made into various forms, such as creams, ointments, gels, sprays, and masks. Animal experiments prove that the five TRAIL mutant preparations can evidently inhibit and relieve skin lesion of an acne animal model, and can well treat acnes of a human body.

Description

TRAIL突变体在制备治疗痤疮药物中的应用及一种制剂Application of TRAIL mutant in preparing medicine for treating acne and a preparation thereof

技术领域Technical field

本发明涉及药物技术领域，尤其涉及多种不同结构的TRAIL突变体在制备治疗痤疮药物中的应用及一种制剂。The invention relates to the technical field of medicines, in particular to the application of a variety of TRAIL mutants with different structures in the preparation of a medicine for treating acne and a preparation thereof.

背景技术Background technique

1.痤疮的发病与危害1. The incidence and harm of acne

痤疮是一种毛囊皮脂腺的慢性炎症性皮肤病，是皮肤科的常见病、多发病，尤其在***男、女的发病率最高。国内调查，在12～15岁年龄组中约有85％的个体在不同时期发病。国外报告，青春发育期的痤疮发生率女性达到90.6％，男性达100％。痤疮对青少年的心理和社交的影响超过了哮喘和癫痫。鉴于痤疮的流行广泛，而本病又主要在面部，出现粉刺、丘疹、囊肿，甚至个别患者形成瘢痕疙瘩，损毁人的面容，给人带来痛苦和烦恼，影响患者的生活质量。Acne is a chronic inflammatory skin disease of the hair follicle sebaceous glands. It is a common and frequently-occurring disease in dermatology, especially in adolescent men and women. According to domestic surveys, about 85% of individuals in the 12 to 15-year-old age group developed disease at different times. It is reported abroad that the incidence of acne in adolescence reaches 90.6% in women and 100% in men. Acne affects adolescents more psychologically and socially than asthma and epilepsy. In view of the widespread epidemic of acne, and the disease is mainly on the face, acne, pimples, cysts, and even individual patients form keloids, damage people's faces, bring pain and annoyance, and affect the quality of life of patients.

痤疮在不同患者人群中的表现有差异，可根据皮损性质将痤疮分为3度和4级，其中轻度(I级)：仅有粉刺；中度(II级)：炎性丘疹；中度(III级)：脓疱；重度(IV级)：结节、囊肿。痤疮的治疗应该根据其分级选择相应治疗药物和手段。外用维A酸类药物是目前轻度痤疮的单独一线药物，中重度痤疮的联合用药以及痤疮维持治疗的首选药物。目前常用的外用维A酸类药物包括第一代维A酸类药物如0.025％～0.1％的全反式维A酸乳霜或凝胶和异维A酸凝胶，第三代维A酸类药物如阿达帕林凝胶和他扎罗汀等。由于痤疮的慢性过程和易复发的临床特点，无论哪一级痤疮，均没有一劳永逸的特效疗法。The performance of acne in different patient populations is different. Acne can be divided into 3 degrees and 4 grades according to the nature of the skin lesions. Mild (grade I): acne only; moderate (grade II): inflammatory pimples; moderate Degree (grade III): pustules; severity (grade IV): nodules, cysts. The treatment of acne should be based on the classification of the appropriate treatment drugs and methods. Topical retinoic acid drugs are currently the first-line drugs for mild acne, a combination of moderate and severe acne, and the drug of choice for acne maintenance treatment. Topical retinoids commonly used today include first-generation retinoids such as 0.025% to 0.1% all-trans retinoid creams or gels, isotretinoin gels, and third-generation retinoids. Drugs such as adapalene gel and tazarotene. Due to the chronic course of acne and the clinical characteristics of easy recurrence, no matter what level of acne there is, there is no one-size-fits-all cure.

痤疮的主要病理变化为：(1)皮脂腺分泌过多；(2)毛囊漏斗部角化过度与脱屑减少，形成黑头粉刺；(3)痤疮丙酸杆菌移生进入毛囊；(4)机体的炎症免疫反应。The main pathological changes of acne are: (1) Excessive secretion of sebaceous glands; (2) Excessive keratinization and desquamation of the funnel of the hair follicle, resulting in blackheads; (3) Propionibacterium acnes migrates into the hair follicle; (4) Inflammatory immune response.

2.TRAIL与痤疮发病和病理2.TRAIL and acne pathogenesis and pathology

肿瘤坏死因子相关凋亡诱导配体(Tumor necrosis factor-related apoptosis-inducing ligand，TRAIL)为肿瘤坏死因子(Tumor necrosis factor，TNF)超家族的成员，其基因序列分别于1995年由Wiley等人和1996年由Pitti等人独立克隆获得，后者将其命名为凋亡素2配体(Apo2 Ligand，Apo2L)。后来的研究证实，Apo2L与TRAIL实质上是同一种蛋白质，因此习惯上可将其称为Apo2L/TRAIL。TRAIL的功能首先是作为生物体先天性或获得性免疫的调节剂，其次在细胞外源性凋亡途径中作为免疫监视发挥抗肿瘤作用。TRAIL的最大优点是可以选择性地诱导多种肿瘤细胞凋亡而对正常细胞几乎没有毒性。研究资料表明，无论在体外还是体内，Apo2L/TRAIL对于各种来源的人肿瘤细胞系，包括结(直)肠癌、肺癌、乳腺癌、前列腺癌、胰腺癌、肾癌、中枢神经***肿瘤、甲状腺癌、淋巴瘤、白血病以及多发性骨髓瘤等都具有诱导凋亡的作用。Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the Tumor necrosis factor (TNF) superfamily, and its gene sequences were obtained in 1995 by Wiley et al. And Independently cloned in 1996 by Pitti et al., Which named it apoptin 2 ligand (Apo2 Ligand, Apo2L). Subsequent research confirmed that Apo2L and TRAIL are essentially the same protein, so it is customarily called Apo2L / TRAIL. The function of TRAIL is firstly as a regulator of organisms' innate or adaptive immunity, and secondly, it plays an anti-tumor role as immune surveillance in the exogenous apoptosis pathway of cells. The biggest advantage of TRAIL is that it can selectively induce apoptosis in a variety of tumor cells with little toxicity to normal cells. Research data show that Apo2L / TRAIL is effective for human tumor cell lines of various origins, including colon (rectal) cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, central nervous system tumor, Thyroid cancer, lymphoma, leukemia, and multiple myeloma can all induce apoptosis.

异维A酸是被FDA批准的，唯一能够改变痤疮病情的药物。A.M Nelson等通过研究发现，虽然异维A酸(13-cis RA)治疗痤疮的全部作用机制尚不清楚，但13-cis RA诱导参与凋亡的脂细胞中的关键基因。单独应用重组人TRAIL(rhTRAIL)蛋白处理可增加SEB-1脂细胞的TUNEL阳性染色，而TRAIL siRNA显著降低了TUNEL阳性SEB-1脂细胞对13-cis RA治疗应答的百分比。与基线相比，异维A酸治疗1周后痤疮患者皮肤中TRAIL表达增加，且TRAIL表达局限于皮脂腺。与脂细胞不同，TRAIL蛋白在正常人表皮角质形成中对13-cis RA的反应没有增加，rhTRAIL也没有诱导角质形成细胞凋亡。实验研究表明，TRAIL是13-cis RA脂细胞特异性凋亡作用的关键。Isotretinoin is the only drug approved by the FDA to change the condition of acne. A.M. Nelson and other researchers found that although the full mechanism of action of isotretinoin (13-cisRA) in the treatment of acne is unknown, 13-cisRA induces key genes in adipocytes involved in apoptosis. Treatment with recombinant human TRAIL (rhTRAIL) protein alone increased TUNEL-positive staining of SEB-1 adipocytes, while TRAIL siRNA significantly reduced the percentage of TUNEL-positive SEB-1 adipocytes responding to 13-cisRA treatment. Compared with baseline, TRAIL expression in acne patients' skin increased after 1 week of isotretinoin treatment, and TRAIL expression was limited to sebaceous glands. Unlike adipocytes, TRAIL protein does not increase its response to 13-cisRA in normal human epidermal keratinogenesis, and rhTRAIL does not induce keratinocyte apoptosis. Experimental studies have shown that TRAIL is the key to the specific apoptosis of 13-cis RA lipid cells.

B.C.Melnik根据已有的研究提出以下假说。这个假说认为痤疮是一种PI3K-AKt-MTORC1驱动的皮脂腺毛囊过度增殖性疾病，伴有TRAIL介导的死亡信号受损。已有实验表明，抗痤疮剂异维A酸可增强TRAIL诱导的死亡信号，从而调整寻常性痤疮皮脂腺毛囊存活和死亡信号的紊乱平衡。假说认为，TRAIL是痤疮发病机制中的潜在关键因素。TRAIL不仅可以终止痤疮脂细胞的促存活信号，还通过诱导总蛋白和I型转谷酰胺酶参与晚期表皮分化。TRAIL诱导的Caspase激活诱导调节表皮增殖和分化的关键转录因子P63的降解。B.C. Melnik proposed the following hypothesis based on existing research. This hypothesis considers acne to be a PI3K-AKt-MTORC1-driven sebaceous glands follicle hyperproliferative disease with impaired TRAIL-mediated death signals. Previous experiments have shown that the anti-acne agent isotretinoin can enhance TRAIL-induced death signals, thereby adjusting the disordered balance of sebaceous hair follicle survival and death signals in acne vulgaris. It is hypothesized that TRAIL is a potentially critical factor in the pathogenesis of acne. TRAIL not only stops the pro-survival signal of acne fat cells, but also participates in late epidermal differentiation by inducing total protein and type I transglutaminase. TRAIL-induced Caspase activation induces degradation of the key transcription factor P63, which regulates epidermal proliferation and differentiation.

3.TRAIL不能用于痤疮治疗3.TRAIL cannot be used for acne treatment

尽管rhTRAIL能够诱导体外传代皮脂腺细胞SEB-1的凋亡，但rhTRAIL用于痤疮的治疗仍存在无法逾越的障碍。其原因如下：(1)现有的永生化皮脂腺细胞(包括SEB-1和SZ95细胞)均为SV40大T抗原基因转染细胞，不能完全代表自然生长状态下的皮脂腺细胞，永生化皮脂腺细胞培养被视为痤疮发病机制关键特征不恰当的模型。(2)rhTRAIL在体外诱导SEB-1细胞凋亡的作用比较弱。经TUNEL染色分析，在SEB-1细胞中，12ng/ml和100ng/ml的rhTRAIL使SEB-1细胞的凋亡率分别增加到23％和35％，均未达到半数抑制的效果。(3)缺乏动物模型的支持。痤疮的动物模型已经比较成熟，常用的痤疮动物模型包括兔耳药物外擦模型、大鼠耳皮内丙酸杆菌注射模型、豚鼠腹部人角质蛋白皮内注射模型等。(4)rhTRAIL的理化特性和药代动力学特性缺陷使其并不适合于皮肤外用给药。由于毛囊、皮脂腺的主要结构均位于真皮层，rhTRAIL不能直接透过表皮作用于皮脂腺和毛囊的深部，故其在实际应用中不能发挥作用。活性的rhTRAIL为非共价结合的三聚体形式，为带正电荷的碱性蛋白质，而皮肤表面为PH5～6的酸性环境，蛋白在该条件的稳定性差。rhTRAIL生物半衰期短，其作用严格依赖于其与细胞表面的受体DR4和DR5的结合。以上的不足和缺陷决定了rhTRAIL单独用于痤疮的治疗仍然存在巨大障碍。Although rhTRAIL can induce the apoptosis of sebaceous gland cells SEB-1 in vitro, there are still insurmountable obstacles for rhTRAIL in the treatment of acne. The reasons are as follows: (1) The existing immortalized sebaceous gland cells (including SEB-1 and SZ95 cells) are all SV40 large T antigen gene transfected cells, which cannot completely represent the sebaceous gland cells in the natural growth state. Immortalized sebaceous gland cell culture Models considered inappropriate for key features of acne pathogenesis. (2) The role of rhTRAIL in inducing apoptosis of SEB-1 cells is weak. According to TUNEL staining analysis, in SEB-1 cells, 12ng / ml and 100ng / ml rhTRAIL increased the apoptotic rate of SEB-1 cells to 23% and 35%, respectively. (3) Lack of animal model support. Animal models of acne are relatively mature. Commonly used animal models of acne include a rabbit ear external rubbing model, a rat model of propionibacterium intradermal injection, and a model of guinea pig abdominal human keratin intradermal injection. (4) The physical and chemical characteristics and pharmacokinetic characteristics of rhTRAIL make it unsuitable for external skin administration. Because the main structures of the hair follicles and sebaceous glands are located in the dermis, rhTRAIL cannot directly affect the sebaceous glands and hair follicles through the epidermis, so it cannot play a role in practical applications. Active rhTRAIL is a non-covalently bound trimer form, a positively charged basic protein, and the skin surface is an acidic environment with a pH of 5 to 6, and the protein has poor stability in this condition. rhTRAIL has a short biological half-life, and its effect depends strictly on its binding to the cell surface receptors DR4 and DR5. The above deficiencies and shortcomings determine that rhTRAIL still has huge obstacles in the treatment of acne alone.

发明内容Summary of the Invention

针对TRAIL用于痤疮治疗具有的现实缺陷和不足，本申请在原已申请专利保护的5种TRAIL突变体的结构、制备方法和肿瘤药物用途领域外进行了拓展，将上述突变体蛋白用于治疗痤疮的药物中。因为，首先，上述5种TRAIL突变体具有高于野生型rhTRAIL的体内外生物活性，与细胞表面的DR4/DR5受体具有更强的亲和力，能够在细胞膜表面聚集和重分布，有利于其发挥作用。其次，上述蛋白突变体均具有细胞膜穿透功能，有助于克服细胞和组织屏障，可作用于真皮层的皮脂腺和毛囊结构，发挥抑制皮脂腺细胞功能的作用。第三，上述蛋白理化性质更为稳定，生物代谢半衰期更长，更有利于发挥作用。Aiming at the practical defects and deficiencies of TRAIL for acne treatment, this application extends the structure, preparation method, and tumor drug use of the five patented TRAIL mutants that were originally patented, and uses the above mutant proteins for the treatment of acne Of drugs. Because, first of all, the above five TRAIL mutants have higher biological activity in vitro and in vivo than wild-type rhTRAIL, have stronger affinity with DR4 / DR5 receptors on the cell surface, and can aggregate and redistribute on the cell membrane surface, which is conducive to their exertion. effect. Secondly, the above-mentioned protein mutants all have cell membrane penetrating function, which is helpful to overcome the cell and tissue barriers, and can act on the structure of the sebaceous glands and hair follicles of the dermis and play a role in inhibiting the function of the sebaceous gland cells. Third, the physicochemical properties of the above-mentioned proteins are more stable, and the half-life of biological metabolism is longer, which is more conducive to the function.

鉴于此，本申请提供了TRAIL突变体在制备治疗痤疮药物中的应用。In view of this, the present application provides the application of a TRAIL mutant in the preparation of a medicine for treating acne.

进一步地，所述TRAIL突变体为：TRAIL穿膜肽样突变体蛋白TRAIL-Mu3(WO2015103894A1)、及TRAIL-MuR5(WO2016138618A1)、TRAIL-MuR6(WO2016138625A1)和TRAIL穿膜肽融合蛋白TRAIL-MuR5S4TR(WO2017066962A1)和TRAIL-MuR6S4TR(WO2017066963A1)等5种TRAIL突变体。Further, the TRAIL mutant is: TRAIL transmembrane peptide-like mutant protein TRAIL-Mu3 (WO2015103894A1), and TRAIL-MuR5 (WO2016138618A1), TRAIL-MuR6 (WO2016138625A1), and TRAIL transmembrane peptide fusion protein TRAIL-MuR5S4TR ( WO2017066962A1) and TRAIL-MuR6S4TR (WO2017066963A1) and other five TRAIL mutants.

进一步的，上述TRAIL突变体在体外能抑制原代培养皮脂腺细胞的增殖并诱导凋亡，其抑制活性大大强于野生型rhTRAIL和阳性对照药异维A酸。Further, the above-mentioned TRAIL mutant can inhibit the proliferation and induce apoptosis of primary cultured sebaceous gland cells in vitro, and its inhibitory activity is much stronger than that of wild-type rhTRAIL and the positive control drug isotretinoin.

本申请还提供了一种制剂，所述制剂包括TRAIL突变体和药学上可接受的辅料。The application also provides a formulation comprising a TRAIL mutant and a pharmaceutically acceptable excipient.

进一步的，上述制剂的剂型乳霜剂、软膏剂、凝胶剂、喷剂、面膜等多种形式，本申请同时优化并完善了相关制剂的制备工艺。Further, the above-mentioned formulations are in the form of creams, ointments, gels, sprays, masks, etc., and the present application simultaneously optimizes and perfects the preparation process of the related formulations.

进一步地，所述TRAIL突变体在所述制剂中的质量百分含量为0.1％～1.0％。Further, the mass percentage content of the TRAIL mutant in the preparation is 0.1% to 1.0%.

进一步地，所述TRAIL突变体在所述制剂中的质量百分含量为0.25％～0.5％。Further, the mass percentage content of the TRAIL mutant in the preparation is 0.25% to 0.5%.

进一步地，所述制剂适用于一天两次对患者施用。Further, the formulation is suitable for administration to a patient twice a day.

本申请提供了TRAIL突变体在制备治疗痤疮药物中的应用。实验结果表明：TRAIL突变体在体外能抑制原代培养皮脂腺细胞的增殖并诱导凋亡，其抑制活性大大强于野生型rhTRAIL和阳性对照药异维A酸；本申请提供的制剂，在多种不同痤疮动物模型上具有明显的抑制和减轻痤疮的作用，而且在不少于30例痤疮人体试验中优于复方异维A酸乳膏具有良好的治疗作用。This application provides the application of a TRAIL mutant in the preparation of a medicine for treating acne. Experimental results show that the TRAIL mutant can inhibit the proliferation and induce apoptosis of primary cultured sebaceous gland cells in vitro, and its inhibitory activity is much stronger than that of wild-type rhTRAIL and the positive control drug isotretinoin; Different acne animal models have obvious effects of inhibiting and alleviating acne, and have better therapeutic effect than compound isotretinoin cream in no less than 30 cases of acne human trials.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1 TRAIL-Mu3和TRAIL-MuR5S4TR乳膏的生产工艺；Figure 1 Production process of TRAIL-Mu3 and TRAIL-MuR5S4TR cream;

图2 TRAIL-Mu3和TRAIL-MuR5S4TR凝胶的生产工艺。Figure 2 Production process of TRAIL-Mu3 and TRAIL-MuR5S4TR gel.

具体实施方式detailed description

为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但应当理解，这些描述只是为了进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。In order to further understand the present invention, the preferred embodiments of the present invention are described below with reference to the examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the invention.

实施例1 TRAIL-Mu3和TRAIL-MuR5S4TR乳膏的制备Example 1 Preparation of TRAIL-Mu3 and TRAIL-MuR5S4TR cream

1.处方Prescription

TRAIL-Mu3TRAIL-Mu3	5g5g
尿囊素Allantoin	10g10g
维生素EVitamin E	20g20g
凡士林Vaseline	125g125g
单甘油脂肪酸酯Monoglycerol fatty acid ester	25g25g
甘油glycerin	62.5ml62.5ml
丙二醇Propylene glycol	62.5ml62.5ml
十二烷基硫酸钠Sodium dodecyl sulfate	10g10g
硬脂醇Stearyl alcohol	90g90g
羟苯乙酯Hydroxyphenethyl	1g1g
纯化水purified water	适量Right amount
制成production	1000g1000g

2.制备工艺2. Preparation process

2.1油相的制备(4种成分)2.1 Preparation of oil phase (4 ingredients)

取硬脂醇、凡士林及单甘油脂肪酸加热熔融，加入维生素E，置于油相罐中，边搅拌边加热至80℃，使之完全熔融并搅拌均匀，得油相备用。Take stearyl alcohol, petroleum jelly and monoglycerin fatty acid to heat and melt, add vitamin E, place in oil phase tank, heat to 80 ° C while stirring, make it completely melt and stir well, and get oil phase for future use.

2.2水相的制备(5种成分)2.2 Preparation of the aqueous phase (5 ingredients)

在水相罐中加入甘油、丙二醇、十二烷基硫酸钠及水加热，加入尿囊素溶解，加入羟苯乙酯边搅拌边加热至80℃，直至溶解，得水相备用。Add glycerin, propylene glycol, sodium lauryl sulfate and water to the water phase tank for heating, add allantoin to dissolve, add hydroxyphenethyl ester and heat to 80 ° C while stirring until it dissolves.

2.3乳化2.3 Emulsification

将TRAIL-Mu3或TRAIL-MuR5S4TR加入适量纯化水中，并保存备用。将水相加入油相中快速搅拌(转速900r/min)15min，待温度降至20℃时加入TRAIL-Mu3或TRAIL-MuR5S4溶液，快速(转速900r/min)搅拌15min，混匀，分装即成。Add TRAIL-Mu3 or TRAIL-MuR5S4TR to an appropriate amount of purified water and save it for future use. Add the water phase to the oil phase and stir rapidly (rotation speed 900r / min) for 15min. When the temperature drops to 20 ° C, add TRAIL-Mu3 or TRAIL-MuR5S4 solution, stir quickly (rotation speed 900r / min) for 15min. to make.

3.工艺流程图3.Process flow chart

工艺流程图如说明书附图1所示。The process flow is shown in Figure 1 of the specification.

按上述处方和生产工艺，分别制得TRAIL-Mu3乳膏和TRAIL-MuR5S4TR乳膏各95支(10g/支)，有效成分含量为0.5％。According to the above prescription and production process, 95 TRAIL-Mu3 creams and TRAIL-MuR5S4TR creams (10 g / branch) were prepared respectively, and the effective ingredient content was 0.5%.

实施例2 TRAIL-Mu3和TRAIL-MuR5S4TR凝胶的制备Example 2 Preparation of TRAIL-Mu3 and TRAIL-MuR5S4TR gels

1.处方Prescription

TRAIL-Mu3TRAIL-Mu3	5g5g
卡波姆940Carbomer 940	10g10g
维生素EVitamin E	50g50g
乙醇Ethanol	50g50g
丙二醇Propylene glycol	50g50g
聚山梨醇酯80 Polysorbate 80	2g2g
尼泊金Nipagin	1g1g
氢氧化钠Sodium hydroxide	4g4g
纯化水purified water	加至Add to

Zh	1000g1000g
制成production	1000g1000g

2.制备工艺2. Preparation process

将卡波姆与聚山梨醇酯80和300ml纯化水混合，将氢氧化钠用100ml纯化水溶解后加入上液搅匀，再将尼泊金溶于乙醇后缓慢加入搅匀，再加入丙二醇，搅匀。在上述混合物中加入预先溶解于100ml水的5g的TRAIL-Mu3或TRAIL-MuR5S4TR，加水至总量共1000g，搅匀，即得。Carbomer is mixed with polysorbate 80 and 300 ml of purified water. Sodium hydroxide is dissolved in 100 ml of purified water and added to the top liquid to mix well. After dissolving nipagin in ethanol, slowly add and mix, then add propylene glycol. Stir well. Add 5 g of TRAIL-Mu3 or TRAIL-MuR5S4TR dissolved in 100 ml of water to the above mixture, add water to a total of 1000 g, and stir to obtain.

3.工艺流程图3.Process flow chart

工艺流程如说明书附图2所示。The process flow is shown in Figure 2 of the specification.

按上述处方和生产工艺，分别制得TRAIL-Mu3凝胶和TRAIL-MuR5S4TR凝胶各98支(10g/支)，有效成分含量为0.5％。According to the above prescription and production process, 98 TRAIL-Mu3 gels and TRAIL-MuR5S4TR gels (10 g / branch) were prepared respectively, and the effective ingredient content was 0.5%.

实施例3 TRAIL-Mu3和MuR5S4TR等5种蛋白对原代培养人皮脂腺细胞的生长抑制作用Example 3 Inhibitory effect of 5 proteins including TRAIL-Mu3 and MuR5S4TR on the growth of human cultured sebaceous gland cells

1.人皮脂腺细胞的分离和原代培养1. Isolation and primary culture of human sebaceous gland cells

从皮肤科手术患者的正常人头皮组织中分理出皮脂腺细胞。正常人头部皮肤新鲜标本依次用PBS、70％酒精清洗一次，置于

培养液中(加2％胎牛血清、抗生素混合物)。去除皮下脂肪，将皮肤切成0.5cm×0.5cm大小。加中性蛋白酶，置于4℃冰箱内过夜。目视显微镜下剥离表皮层，用镊子分离15个以上皮脂腺，置于具有胶原涂层的细胞培养皿(6cm直径，皿内

培养液约为1.25ml)，置于5％CO ₂培养箱中37℃培养，2～3天更换一次培养液，待细胞长满后进行传代或进行TRAIL-Mu3和TRAIL-MuR5S4TR增殖抑制活性检测。 Sebaceous gland cells were isolated from normal human scalp tissue in patients undergoing dermatological surgery. Fresh specimens of normal human head skin were washed once with PBS and 70% alcohol, and placed in

Medium (add 2% fetal calf serum, antibiotic mixture). The subcutaneous fat was removed, and the skin was cut into a size of 0.5 cm × 0.5 cm. Add neutral protease and place in a refrigerator at 4 ° C overnight. The epidermal layer was peeled off under a visual microscope, and more than 15 sebaceous glands were separated with tweezers and placed in a cell culture dish with a collagen coating (6 cm diameter, inside the dish

The culture medium is about 1.25ml), and cultured in a 5% CO ₂ incubator at 37 ° C. The culture medium is replaced once every 2 to 3 days. After the cells are full, passaging or TRAIL-Mu3 and TRAIL-MuR5S4TR proliferation inhibition activity detection .

2.CCK8检测TRAIL-Mu3和MuR5S4TR等5中蛋白对皮脂腺细胞的抑制作用2.CCK8 detects the inhibitory effect of proteins in TRAIL-Mu3 and MuR5S4TR5 on sebaceous gland cells

(1)接板：取常规培养处于对数生长期的人皮脂腺细胞，0.25％的胰蛋白酶将培养瓶中的细胞消化，用Sebomed基础培养液稀释细胞，配置成单个细胞悬液，充分混匀后调整细胞浓度至5×10 ⁵/ml，以每孔100ul的量接种于96孔板，置37℃、5％CO ₂饱和湿度培养箱中培养。 (1) Connection plate: take human sebaceous gland cells in logarithmic growth phase, digest cells in culture flask with 0.25% trypsin, dilute cells with Sebomed basic culture solution, configure as a single cell suspension, and mix well After adjusting the cell concentration to 5 × 10 ⁵ / ml, inoculate 100ul per well into a 96-well plate, and place it in a 37 ° C, 5% CO ₂ saturated humidity incubator to culture.

(2)加药：接板6h后，用倒置显微镜观察细胞完全贴壁后，分别在细胞培养板中加入100ul Sebomed培养基配制的不同浓度的异维A酸或TRAIL-Mu3或MuR5S4TR等蛋白溶液。上述药物的上样起始浓度均为1ug/ml，进行10个梯度稀释上样。每个浓度设双复孔，继续培养48h。(2) Dosing: After connecting the plate for 6 hours, observe that the cells are completely adhered with an inverted microscope, and add 100ul Sebomed medium to different concentrations of isotretinoin or TRAIL-Mu3 or MuR5S4TR protein solutions in cell culture plates. . The initial loading concentrations of the above drugs were 1 ug / ml, and 10 gradient dilution loadings were performed. Double wells were set for each concentration and the culture was continued for 48 h.

(3)测活：培养48h后，超净台中操作培养板。吸弃旧的培养液，分别向每孔加入18ul Sebomed培养基和20ul CCK溶液，置37℃、5％CO ₂饱和湿度下继续培养1h。取出培养板，在离心机中1000rpm离心5分钟，置酶联检测仪上，设定检测波长为490/630nm，检测各孔吸光度(OD)值，按下列公式计算每种药物对皮脂腺细胞的生长抑制率。 (3) Activity measurement: After 48 hours of incubation, operate the culture plate in ultra-clean Taichung. The old culture solution was aspirated, and 18 ul of Sebomed medium and 20 ul of CCK solution were added to each well, and cultured at 37 ° C and 5% CO ₂ saturation humidity for 1 hour. Remove the culture plate, centrifuge at 1000 rpm for 5 minutes in the centrifuge, set on the enzyme linked detector, set the detection wavelength to 490 / 630nm, detect the absorbance (OD) value of each well, and calculate the growth of sebaceous gland cells by each drug according to the following formula Inhibition rate.

皮脂腺细胞生长抑制率＝[(Ac-As)/(Ac-Ab)]×100％Growth inhibition rate of sebaceous gland cells = [(Ac-As) / (Ac-Ab)] × 100%

Ac：样品的OA/RLU(细胞+CCK8+待测化合物)Ac: OA / RLU of the sample (cell + CCK8 + test compound)

As：阴性对照的OA/RLU(细胞+CCK-8)As: OA / RLU of negative control (cell + CCK-8)

Ab：阳性对照的OA/RLU(细胞+CCK-8)Ab: OA / RLU of positive control (cell + CCK-8)

运用软件Graphpad Prism 5并采用计算公司log(inhibitor)vs normalized response-Variable slope进行IC50曲线拟合并计算出各自的IC ₅₀值。 Using the software Graphpad Prism 5 and the calculation company log (inhibitor) vs normalized response-Variable slope to perform IC50 curve fitting and calculate their respective IC ₅₀ values.

3.结果3. Results

本实验测试了异维A酸、TRAIL-Mu3、MuR5S4TR6等6个样品对原代培养人皮脂腺细胞的生长抑制作用，实验结果如下表所示：In this experiment, six samples including isotretinoin, TRAIL-Mu3, and MuR5S4TR6 were tested to inhibit the growth of human sebaceous gland cells in primary culture.

表1.不同样品对皮脂腺细胞的生长抑制作用(ug/ml)Table 1. Growth inhibition of sebaceous gland cells by different samples (ug / ml)

异维A酸和TRAIL-Mu3、MuR5S4TR等5个TRAIL类蛋白具有抑制皮脂腺细胞生长的作用，且5种TRAIL类蛋白的抑制活性比异维A酸强。初步机理研究显示，上述样品对皮脂腺细胞的生长抑制是通过诱导皮脂腺细胞凋亡而实现的。Isotretinoin and five TRAIL proteins such as TRAIL-Mu3 and MuR5S4TR can inhibit the growth of sebaceous gland cells, and the inhibitory activity of the five TRAIL proteins is stronger than isotretinoin. Preliminary mechanism studies have shown that the above-mentioned samples inhibit the growth of sebaceous gland cells by inducing apoptosis of sebaceous gland cells.

实施例4 TRAIL-Mu3和TRAIL-MuR5S4TR乳膏对兔耳实验性角化模型的治疗作用Example 4 Therapeutic effect of TRAIL-Mu3 and TRAIL-MuR5S4TR cream on experimental keratosis model of rabbit ear

1.健康雄性新西兰兔(白色)64只，平均体重(2.5±0.2)kg，由四川实验动物中心提供，煤焦油原液(成都凯宜德公司)。1. 64 healthy male New Zealand rabbits (white), average weight (2.5 ± 0.2) kg, provided by Sichuan Experimental Animal Center, coal tar stock solution (Chengdu Kaiyide Company).

空白对照组8只不做任何处理，常规喂饲。余下56只兔，在兔耳内侧耳管开口处2.0×2.0cm范围内，左耳给予煤焦油原液0.5ml/次，1次/d，同时右耳作为自身对照给予纯净水0.5ml/次，1次/d，连续给药2周，予常规饲料。造模2周后，在涂药处作肉眼观察及组织学检查，以证实模型是否成功。每日除给药外，应肉眼观察局部给药处皮肤的变化、耳厚薄、硬度、粗糙程度、毛囊口有无黑色角栓等。Eight of the blank control group were given no treatment and were fed conventionally. In the remaining 56 rabbits, the left ear was given coal tar stock solution 0.5ml / time, once / d within the range of 2.0 × 2.0cm in the ear canal opening of the inner ear of the rabbit, while the right ear was given 0.5ml / time of pure water as its own control. 1 time / d, continuous administration for 2 weeks, conventional diet. After 2 weeks of modeling, visual observation and histological examination were performed at the application site to confirm the success of the model. In addition to daily administration, the skin changes at the local administration site, ear thickness, hardness, roughness, and whether there are black horn plugs at the mouth of the hair follicles should be observed with the naked eye.

2.证实模型成功后，空白对照组(组1)不给药，余下56只兔随机分为7组，分别为组2(异维酸A乳膏)、组3(TRAIL-Mu3乳膏低剂量组，有效成分含量0.25％)、组4(TRAIL-Mu3乳膏中剂量组，0.5％)、组5(TRAIL-Mu3乳膏高剂量组，1％)、组6(MuR5S4TR乳膏低剂量组，0.25％)、组7(MuR5S4TR乳膏中剂量组，0.5％)、组8(MuR5S4TR乳膏大剂量组)，分别在模型耳侧涂上上述药物0.25g/次，2次/d，连续2周。2. After confirming the success of the model, the blank control group (group 1) was not administered, and the remaining 56 rabbits were randomly divided into 7 groups, which were group 2 (isoretinoin A cream) and group 3 (TRAIL-Mu3 cream low). Dose group, active ingredient content 0.25%), group 4 (TRAIL-Mu3 cream medium dose group, 0.5%), group 5 (TRAIL-Mu3 cream high dose group, 1%), group 6 (MuR5S4TR cream low dose Group, 0.25%), group 7 (MuR5S4TR cream medium-dose group, 0.5%), group 8 (MuR5S4TR cream high-dose group), respectively, the above medicine was coated with the above drug 0.25g / times, 2 times / d, 2 consecutive weeks.

3.按下表观察各组病理学组织变化，并每日观察各组动物临床症状，治疗组与阳性药物组均出现不同程度症状改善。症状改善包括表皮的变薄、毛囊内角化物的减少，毛囊漏斗部的恢复。3. Observe the pathological changes of each group according to the following table, and observe the clinical symptoms of animals in each group daily. The treatment group and the positive drug group showed different degrees of improvement. Symptom improvement includes thinning of the epidermis, reduction of keratin in the hair follicle, and recovery of the funnel of the hair follicle.

表2.兔耳痤疮模型病理组织学分级变化Table 2. Histopathological grade changes of rabbit ear acne model

4.异维A酸乳膏、TRAIL-Mu3乳膏各剂量组、MuR5S4TR乳膏各剂量组均能明显改善兔耳实验性痤疮模型的症状和病理变化，TRAIL-Mu3乳膏及MuR5S4TR乳膏中，高剂组明显优于异维A酸乳膏组。4. Isotretinoin cream, TRAIL-Mu3 cream in each dose group, and MuR5S4TR cream in each dose group can significantly improve the symptoms and pathological changes of experimental acne models in rabbit ears. TRAIL-Mu3 cream and MuR5S4TR cream The high-dose group was significantly better than the isotretinoin cream group.

实施例5 TRAIL-Mu3和TRAIL-MuR5S4TR乳膏对痤疮的治疗作用Example 5 Therapeutic effect of TRAIL-Mu3 and TRAIL-MuR5S4TR cream on acne

1.诊断标准Diagnostic criteria

纳入病例诊断标准参照《临床皮肤病学》，即皮损表现为白头粉刺、黑头粉刺、炎性丘疹、化脓丘疹，部分可见结节、囊肿、瘢痕形成，无自觉症状或局部有压痛，可伴有面部油脂分泌过多，毛孔扩大；好发于颜面部，尤其是前额、颊部、鼻部、下颌，其次是胸部和背部。多见于青年男女，各种皮损可同时存在。The diagnostic criteria of the cases were referred to "Clinical Dermatology", that is, the skin lesions were whiteheads, blackheads, inflammatory pimples, and pimples. Some nodules, cysts, and scars were visible. There were no conscious symptoms or local tenderness. Excessive secretion of facial oils and enlarged pores occur on the face, especially the forehead, cheeks, nose, and jaw, followed by the chest and back. More common in young men and women, various skin lesions can exist at the same time.

2.病例纳入2. Case inclusion

共收集病例90例，随机分成两个治疗组(治疗组1和治疗组2)和对照组，其中治疗组1 30例，年龄18～39岁，病程4～58个月；治疗组2 30例，年龄19～37岁，病程4～56个月；对照组30例，年龄19-38岁，病程3～50个月。三组年龄、性别、病情及病情综合分级等情况无显著性差异(P＞0.05)。A total of 90 cases were collected and randomly divided into two treatment groups (treatment group 1 and treatment group 2) and control group, of which 30 cases were in the treatment group, aged 18 to 39 years, and the course of disease was 4 to 58 months; 2 cases were treated in the treatment group 30 , Aged 19 to 37 years, with a course of 4 to 56 months; 30 patients in the control group, aged 19 to 38 years, with a course of 3 to 50 months. There were no significant differences in age, gender, disease status, and comprehensive classification of the three groups (P> 0.05).

3.给药方法3. Method of administration

治疗组1和2分别给予0.5％的TRAIL-MuR5S4和TRTRAIL-Mu3乳膏，每日2次，充分洁面后涂抹患处，厚涂，防止短时干燥，拭去；对照组给予0.1％的复方异维A酸乳膏，每日2次，用法同治疗组，疗程均为4周。Treatment groups 1 and 2 were given 0.5% of TRAIL-MuR5S4 and TRTRAIL-Mu3 creams twice a day, respectively. After the face was fully cleansed, apply the affected area, thickly to prevent short-term drying, and wipe away; the control group was given 0.1% Vitamin A acid cream, twice daily, the same usage as the treatment group, the course of treatment is 4 weeks.

4.疗效判定4. Efficacy determination

按照痤疮综合分级***，以表格形式详细记录治疗前和治疗后1天、2天、3天、4天、5天、1周、2周、3周、4周各区皮损分值。According to the comprehensive acne grading system, the scores of the skin lesions before and after treatment were recorded in detail in the form of a table in one, two, three, four, five, one, two, three, and four weeks.

5.疗效标准5. Efficacy criteria

(1)痊愈：皮损消退，或仅留色素沉着，疗效指数＞90％(1) Healing: the skin lesions subsided, or only pigmentation remained, and the efficacy index was> 90%

(2)显效：皮损大部分消退，疗效指数在＜90％与＞60％之间(2) Significant effect: most of the skin lesions have subsided, and the efficacy index is between <90% and> 60%

(3)有效：皮损部分消退，疗效指数在＜60％与＞20％之间(3) Effective: The skin lesions have partially subsided, and the therapeutic index is between <60% and> 20%

(4)无效：皮损消退不明显，疗效指数＜20％(4) Ineffective: the skin lesions do not regress significantly, and the curative effect index is less than 20%

疗效指数：分别计算治疗前后的总分值，再用下列公式计算而得。Efficacy Index: Calculate the total score before and after treatment, and then use the following formula to calculate.

疗效指数＝(治疗前总积分－治疗后总积分)/治疗前总积分×100％Efficacy Index = (Total Points Before Treatment-Total Points After Treatment) / Total Points Before Treatment × 100%

痊愈率＝痊愈例数/病例总数×100％Recovery rate = number of cured cases / total number of cases × 100%

有效率＝(痊愈例数+显效例数+有效例数)/病例总数×100％Effective rate = (number of cured cases + number of significant cases + number of effective cases) / total number of cases × 100%

6.评分方法6. Scoring method

参照1997年，Doshi等人提出的痤疮综合分级***(Global Acne Grading System，GAGS)。该法根据皮脂腺单位的密度、分布及大致面积，把好发部位分为6个区：Ⅰ区(前额)：2；Ⅱ区(右颊)：2；Ⅲ区(左颊)：2；Ⅳ区(鼻部)1；Ⅴ区(下颌)：1；Ⅵ区(胸或后背)：3，数值为不同区域分值。同时将皮损严重程度分为5级，每级按皮损程度评分。无皮损：0；粉刺：1；丘疹：2；脓疱：3；结节、囊肿：4。总分值：先将每一区最严重皮损类型的分值与分区因素分值相乘即得每区分值，6个分区分值之和即为患者总的分值。Refer to the Global Acne Grading System (GAGS) proposed by Doshi et al. In 1997. According to the density, distribution and approximate area of sebaceous gland units, the method divides the hair-prone areas into 6 areas: area Ⅰ (forehead): 2; area Ⅱ (right cheek): 2; area Ⅲ (left cheek): 2; IV Area (nose) 1; area Ⅴ (mandible): 1; area Ⅵ (chest or back): 3, values are scores for different areas. At the same time, the severity of skin lesions was divided into 5 grades, and each grade was scored according to the degree of skin lesions. No skin lesions: 0; acne: 1; pimples: 2; pustules: 3; nodules, cysts: 4. Total score: First, multiply the score of the most severe skin lesion type in each zone with the score of the zone factor to obtain each score. The sum of the scores of the 6 zones is the total score of the patient.

7.观察结果7. Observation results

观察结果见表3。The observation results are shown in Table 3.

表3.两组痤疮治疗效果Table 3. Acne treatment effect in two groups

结论：TRAIL-Mu3和TRAIL-MuR5S4TR乳膏对痤疮具有良好的治疗作用，其作用优于复方异维A酸乳膏。Conclusion: TRAIL-Mu3 and TRAIL-MuR5S4TR creams have a good therapeutic effect on acne, and their effects are better than that of compound isotretinoin cream.

实施例6 TRAIL-Mu3和TRAIL-MuR5S4TR凝胶对痤疮的治疗作用Example 6 Therapeutic effect of TRAIL-Mu3 and TRAIL-MuR5S4TR gel on acne

1.诊断标准Diagnostic criteria

纳入病例，诊断标准参照《临床皮肤病学》，即皮损表现为白头粉刺、黑头粉刺、炎性丘疹、化脓丘疹，部分可见结节、囊肿、瘢痕形成，无自觉症状或局部有压痛，可伴有面部油脂分泌过多，毛孔扩大；好发于颜面部，尤其是前额、颊部、鼻部、下颌，其次是胸部和背部。多见于青年男女，各种皮损可同时存在。Included cases, the diagnostic criteria refer to "Clinical Dermatology", that is, skin lesions manifested as whiteheads, blackheads, inflammatory pimples, pimples, some nodules, cysts, scar formation can be seen, no conscious symptoms or local tenderness. It is accompanied by excessive secretion of facial oils and enlarged pores. It occurs on the face, especially the forehead, cheeks, nose, and jaw, followed by the chest and back. More common in young men and women, various skin lesions can exist at the same time.

2.病例纳入2. Case inclusion

共收集病例93例，随机分成两个治疗组(治疗组1和治疗组2)和对照组，其中治疗组1 32例，年龄16-35岁，病程6-47个月；治疗组2 31例，年龄17-37岁，病程6-45个月；对照组30例，年龄19-36岁，病程3-40个月。三组年龄、性别、病情及病情综合分级等情况无显著性差异(P＞0.05)。A total of 93 cases were collected and randomly divided into two treatment groups (treatment group 1 and treatment group 2) and control group, of which 32 cases were in the treatment group, aged 16-35 years, and the course was 6-47 months; 31 cases were in the treatment group 2 , Aged 17-37 years, with a course of 6-45 months; 30 patients in the control group, aged 19-36 years, with a course of 3-40 months. There were no significant differences in age, gender, disease status, and comprehensive classification of the three groups (P> 0.05).

3.给药方法3. Method of administration

治疗组1和治疗组2分别给予0.25％的TRAIL-MuR5S4和TRTRAIL-Mu3凝胶，每日2次，充分洁面后涂抹患处，厚涂，防止短时干燥，拭去；对照组给予0.1％的复方异维A酸凝胶，每日2次，用法同治疗组，疗程均为4周。The treatment group 1 and the treatment group 2 were given 0.25% of TRAIL-MuR5S4 and TRTRAIL-Mu3 gels twice a day. After the face was cleansed, the affected area was applied, thickly to prevent short-term drying, and wiped off. The control group was given 0.1%. Compound isotretinoin acid gel was used twice a day in the same way as the treatment group, and the course of treatment was 4 weeks.

4.疗效判定4. Efficacy determination

5.疗效标准5. Efficacy criteria

6.评分方法6. Scoring method

参照1997年，Doshi等人提出的痤疮综合分级***(Global Acne Grading System，GAGS)。该法根据皮脂腺单位的密度、分布及大致面积，把好发部位分为6个区：Ⅰ区(前额)：2；Ⅱ区(右颊)：2；Ⅲ区(左颊)：2；Ⅳ区(鼻部)1；Ⅴ区(下颌)：1；Ⅵ区(胸或后背)：3，数值为不同区域分值。同时将皮损严重程度分为5级，每级按皮损程度评分。无皮损：0；粉刺：1；丘疹：2；脓疱：3；结节、囊肿：4。Refer to the Global Acne Grading System (GAGS) proposed by Doshi et al. In 1997. According to the density, distribution and approximate area of sebaceous gland units, the method divides the hair-prone areas into 6 areas: area Ⅰ (forehead): 2; area Ⅱ (right cheek): 2; area Ⅲ (left cheek): 2; IV Area (nose) 1; area Ⅴ (mandible): 1; area Ⅵ (chest or back): 3, values are scores for different areas. At the same time, the severity of skin lesions was divided into 5 grades, and each grade was scored according to the degree of skin lesions. No skin lesions: 0; acne: 1; pimples: 2; pustules: 3; nodules, cysts: 4.

总分值：先将每一区最严重皮损类型的分值与分区因素分值相乘即得每区分值，6个分区分值之和即为患者总的分值。Total score: First, multiply the score of the most severe skin lesion type in each zone with the score of the zone factor to obtain each score. The sum of the scores of the 6 zones is the total score of the patient.

7.观察结果7. Observation results

观察结果见表4。The observation results are shown in Table 4.

表4.两组痤疮治疗效果Table 4. Two groups of acne treatment effect

结论：0.25％的TRAIL-Mu3和TRAIL-MuR5S4TR凝胶对痤疮具有良好的治疗作用，其作用优于复方异维A酸凝胶。Conclusion: 0.25% of TRAIL-Mu3 and TRAIL-MuR5S4TR gels have a good therapeutic effect on acne, and their effect is better than that of compound isotretinoin gel.

以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。The above are only the preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to this. Any person skilled in the technical field is within the technical scope disclosed by the present invention, according to the technical solution of the present invention. The equivalent replacement or change of the invention concept should be covered by the protection scope of the present invention.

Claims

TRAIL突变体在制备用于治疗痤疮药物中的应用。Application of TRAIL mutant in preparing medicine for treating acne.
权利要求1所述应用，其特征在于，所述TRAIL突变体为：TRAIL穿膜肽样突变体蛋白TRAIL-Mu3、TRAIL-MuR5和TRAIL-MuR6及TRAIL穿膜肽融合蛋白TRAIL-MuR5S4TR和TRAIL-MuR6S4TR。The application according to claim 1, wherein the TRAIL mutants are: TRAIL transmembrane peptide-like mutant proteins TRAIL-Mu3, TRAIL-MuR5 and TRAIL-MuR6, and TRAIL transmembrane peptide fusion proteins TRAIL-MuR5S4TR and TRAIL- MuR6S4TR.
权利要求1或2所述应用，其特征在于，TRAIL突变体在体外能抑制原代培养皮脂腺细胞的增殖并诱导凋亡。The use according to claim 1 or 2, characterized in that the TRAIL mutant can inhibit the proliferation and induce apoptosis of primary cultured sebaceous gland cells in vitro.
一种制剂，其特征在于，包括TRAIL突变体和药学上可接受的辅料。A preparation characterized by comprising a TRAIL mutant and a pharmaceutically acceptable excipient.
权利要求4所述制剂，其特征在于，所述制剂的剂型为乳霜剂、软膏剂、凝胶剂、喷剂或面膜。The preparation according to claim 4, characterized in that the dosage form of the preparation is a cream, an ointment, a gel, a spray or a mask.
权利要求4所述制剂，其特征在于，所述TRAIL突变体在所述制剂中的质量百分含量为0.1％～1.0％。The preparation according to claim 4, wherein the mass percentage content of the TRAIL mutant in the preparation is 0.1% to 1.0%.
权利要求6所述制剂，其特征在于，所述TRAIL突变体在所述制剂中的质量百分含量为0.25％～0.5％。The preparation according to claim 6, characterized in that the mass percentage content of the TRAIL mutant in the preparation is 0.25% to 0.5%.
权利要求4-7任一项所述制剂，其特征在于，适用于一天两次对患者施用。The preparation according to any one of claims 4 to 7, characterized in that it is suitable for administration to a patient twice a day.