WO2023035632A1 - 一种活性肽及其相关用途 - Google Patents

一种活性肽及其相关用途 Download PDF

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WO2023035632A1
WO2023035632A1 PCT/CN2022/090011 CN2022090011W WO2023035632A1 WO 2023035632 A1 WO2023035632 A1 WO 2023035632A1 CN 2022090011 W CN2022090011 W CN 2022090011W WO 2023035632 A1 WO2023035632 A1 WO 2023035632A1
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cells
peptide according
polypeptide
biologically active
group
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PCT/CN2022/090011
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English (en)
French (fr)
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李钧翔
傅晓蕾
徐�明
夏春鑫
康思宁
陆益
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禾美生物科技(浙江)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to the field of skin repair and improvement, in particular to an active peptide and related uses thereof.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • adipocytes a type of pluripotent stem cells derived from the mesoderm in early development, and are currently one of the most studied and important adult stem cells.
  • MSCs are widely found in various tissues throughout the body, and can be cultured and expanded in vitro. Under specific conditions, they can differentiate into various cells including nerve cells, osteoblasts, chondrocytes, muscle cells, and adipocytes, and can be continuously passaged It still has multilineage differentiation potential after culture and cryopreservation.
  • MSCs derived from umbilical cord have the characteristics of convenient collection, no ethical controversy, low immunogenicity, fast self-renewal, stable doubling rate and strong proliferation ability. Therefore, umbilical cord MSCs are suitable for clinical research and application, and are the first choice for cell therapy and regenerative medicine.
  • MSCs are safe and effective for the treatment of a variety of diseases, including graft-versus-host disease, spinal cord injury, autoimmune disease, cardiovascular disease, bone and cartilage damage repair, Crowe's disease, etc.
  • diseases including graft-versus-host disease, spinal cord injury, autoimmune disease, cardiovascular disease, bone and cartilage damage repair, Crowe's disease, etc.
  • Grace disease diabetes and its complications, etc.
  • 11 mesenchymal stem cell drugs have been approved for the clinical treatment of the above diseases; however, the mechanism by which MSCs play a role in the treatment of diseases is still unclear, and further research and exploration are still needed.
  • MSCs mesenchymal stem cells
  • the MSC secretome is the sum of biologically active molecules secreted by MSCs, including cytokines, chemokines, growth factors and other active components, which regulate many physiological processes such as repair, regeneration and functional recovery of damaged tissues.
  • the active peptides found in nature and the human body have some defects, such as relatively long peptides that make them unstable in vivo, and the purification process and synthesis process are complicated, the cost of large-scale production is high, and the skin penetration is poor. Therefore, in order to further improve In order to become druggable and improve commercial value, it is necessary to carry out structural modification of related active peptides.
  • the inventors of the present disclosure obtained an active peptide of LQ-17 through research and development, and then obtained an active peptide of EQ-9 through structural modification. Its activity is equivalent to that of LQ-17, but because it is a truncated body, the production cost can be further reduced, and unexpected technical effects have been achieved.
  • the purpose of the present disclosure is to provide a biologically active polypeptide and its preparation method and application.
  • the present disclosure provides a biologically active polypeptide whose amino acid sequence conforms to the following general formula: R1-AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -AA 9
  • AA 1 is selected from E, D or bond
  • AA 2 is selected from S, T or a bond
  • AA 3 is selected from E, D, K
  • AA 4 is selected from T, A, S, I
  • AA 5 is selected from R, K, E
  • AA 6 is selected from I, L, A
  • AA 7 is selected from L, I, V
  • AA 8 is selected from L, I, V
  • AA 9 is selected from Q, E, D or a bond
  • R1 is selected from the group consisting of H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanonyl, cyclohexanecarboxy, capryl, capryl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl and the group consisting of linoleoyl.
  • the amino acid sequence of the biologically active polypeptide is selected from the group consisting of ESETRILLQ, ESETKILLQ, DSDAKILIQ, DSDAKLLIQ, ETDTRILVE, ESEARIILE, DSESKLVID, ESETEAVLQ, ESKIRILLQ, ETRILL, SETRIL.
  • the present disclosure provides a method for preparing the biologically active polypeptide, which can be artificially synthesized by genetic engineering, can be directly obtained from cells by separation and purification, and can be directly prepared by chemical synthesis.
  • the present disclosure provides the application of the biologically active polypeptide in the preparation of food, health products, medicine or cosmetics with anti-inflammatory function.
  • the present disclosure provides the application of the biologically active polypeptide in the preparation of food, health products or medicines with anti-aging function.
  • the present disclosure provides the application of the biologically active polypeptide in the preparation of food, health product or medicine with antioxidant function.
  • the present disclosure provides a product, including the biologically active polypeptide or its derivative; the biologically active polypeptide derivative refers to hydroxylation on the amino acid side chain group, amino terminal or carboxyl terminal of the biologically active polypeptide , carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation and other modifications to obtain polypeptide derivatives without affecting their biological activity.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a cosmetically or pharmaceutically effective amount of at least one active peptide according to the foregoing and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant.
  • the dosage form of the pharmaceutical composition includes cream, lotion, water, gel, oil, powder, slime, wax-based, patch, film or freeze-dried.
  • the excipients include: hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyethylene glycol, polyacrylamide, polyacrylic acid, polyvinyl alcohol, glucose Polysaccharides, ammonia, lactic acid, citric acid, pyrrolidone carboxylic acid, glycerin, urea, sorbitol, amino acids, lanolin, petrolatum.
  • the adjuvants include: solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, flavoring agents, surfactants, water, ions or Non-ionic emulsifiers, fillers, chelating agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments.
  • active peptides of the present disclosure can also be formulated with excipients and adjuvants commonly used in oral compositions or food supplements, such as but not limited to fat ingredients, aqueous ingredients, humectants, preservatives, conditioners, etc. commonly used in the food industry. agents, flavorings, fragrances, antioxidants and coloring agents.
  • bioactive peptides of the present disclosure may be used in combination with additional ingredients including, but not limited to: antioxidants, reactive carbonyl species scavengers, antiglycation agents, antihistamines, antiviral agents, antiparasitic agents, Emulsifiers, emollients, anti-wrinkle and/or anti-aging agents, etc.
  • the inventors conducted extensive research on the function of biologically active peptides with the structure R1-AA 1 -AA 2 -AA 3 -AA 4 -AA 5 -AA 6 -AA 7 -AA 8 -AA 9 , carried out a large number of experiments, and Determine the structural domain that plays an important role in its biological activity, and finally obtain a bioactive peptide with good activity, low production cost, good stability and multiple functions of anti-aging, anti-oxidation and anti-inflammatory response.
  • FIG 1 Chicken embryo safety experiment
  • Figure 8 UVA damage repair experiment of the modified bioactive peptide
  • LC-MS liquid chromatography-mass spectrometry was used to screen peptides with a molecular weight less than 30kDa, combined with ribosome mapping technology to screen nearly a thousand bioactive peptides, and then based on machine learning and biophysical protein - Peptide interaction prediction method (HSM PPI prediction) further screened to obtain LQ-17, and truncate on the basis of it to obtain truncated VQ-13, EQ-9, LT-12 sequences as follows:
  • Embodiment 1 Safety research-chicken embryo test
  • the experiment was carried out with reference to the relevant standards and procedures of chicken embryo chorioallantoic membrane test (SN/T 2329-2009) for eye irritation and corrosiveness of cosmetics.
  • the evaluation criteria are as follows:
  • HaCaT cells were recovered and passaged. After the cells were in good condition under the microscope, the cells were collected, the cell concentration was adjusted to 0.5-1.0*10 5 cells/mL, and 96-well plating was carried out at 100 ⁇ L/well. After culturing in an incubator (37°C, 5% CO 2 ) until the cell confluency is about 80%, a certain concentration of the test substance is added and incubated for 24 hours. The supernatant was removed, and the cell viability was measured using a CCK8 detection kit. See Figure 2 for the results. The ability of EQ-9 to promote cell survival was significantly stronger than that of LQ-17.
  • Example 3 Detection of the ability to protect cells from UVA damage
  • LQ-17 and EQ-9 can significantly improve the survival rate of cells under ultraviolet stimulation conditions.
  • LQ-17 and EQ-9 significantly increased the content of extracellular type I collagen under the condition of ultraviolet stimulation.
  • Embodiment 4 UVA damage repair ability test experiment
  • Human foreskin fibroblast HFF cells were resuscitated and passaged. After the cells were in good condition under the microscope, the cells were collected and added to a 96-well plate at a cell density of 2.0-2.5*10 4 cells/well. Cultivate in an incubator for 24 hours (37°C, 5% CO 2 ), discard the medium, add fresh medium, induce with a certain amount of UVA, and control the total irradiation to 5J/cm 2 . Discard the supernatant, add a medium containing a certain test substance, and use the corresponding kit to measure the cell viability after culturing for 24 hours.
  • Embodiment 5 Quantitative detection experiment of MMP-9 expression
  • Human foreskin fibroblast HFF cells were resuscitated and passaged. After the cells were in good condition under the microscope, the cells were collected and added to a 96-well plate at a cell density of 2.0-2.5*10 4 cells/well. Cultivate in an incubator for 24 hours (37°C, 5% CO 2 ), discard the medium, add fresh medium, induce with a certain amount of UVA, and control the total irradiation to 5J/cm 2 . Discard the supernatant, add a medium containing a certain test substance, collect the cell culture medium after culturing for 24 hours, centrifuge at 1000g for 10 minutes at 4°C, and use the corresponding kit to measure the MMP-9 protein content.
  • EQ-9 and LQ-17 could significantly reduce the expression of MMP-9 in cells after ultraviolet damage (MMP-9 belongs to the family of matrix metalloproteinases and can degrade extracellular collagen).
  • EQ-9 bioactive peptide is a promising anti-aging ingredient with good safety.
  • -17 has the same effect.
  • EQ-9 is significantly better than LQ-17; the length of EQ-9 bioactive peptide is shorter than LQ-17, but it has the same or better activity than LQ-17.
  • other truncated bodies such as: VQ-13, LT-12 have poor comprehensive effects.
  • the inventors conducted detailed structural and functional studies on EQ-9, and obtained the following active peptides through modification.
  • the modified bioactive peptide was subjected to UVA damage repair experiment (experimental procedure is the same as in Example 3), and the results are shown in FIG. 8 .
  • the EQ-9 biologically active peptide is modified as follows:
  • Polypeptides are synthesized by solid-phase synthesis, and the brief steps are as follows: first synthesize Fmoc-AA1-Wang resin, treat the resin with 20% piperidine solution dissolved in dimethylformamide (DMF) to unblock the amino groups, and then Treat with Fmoc-AA2 and 1-hydroxybenzotriazole (HOBt) to obtain Fmoc-AA2-AA1-Wang resin (Note: AA1 and AA2 represent amino acids). Repeat the above process to generate the final Fmoc-peptide-Wang resin. Finally, the amino group is unblocked and treated with palmitic acid and 1-hydroxybenzotriazole (HOBt) to obtain palm-peptide-Wang resin. Crude peptides were liberated from the resin using TFA in dichloromethane water and subsequently purified by HPLC to ensure >95% purity. Finally, the peptides were lyophilized into polymorphic crystalline powders.
  • DMF dimethylformamide
  • HOBt 1-hydroxybenz
  • the EQ-9 peptide after the above transformation is carried out in the UVA damage repair ability test experiment described in Example 4 (experimental steps are basically the same), wherein: 1) 10ppm EQ-9; 2) 4ppm EQ-9; 3) 10ppm EQ-9 liposome; 4) 10ppm EQ-9 palmitization modification; 5)
  • EK-10-pal the EQ-9 derivative with a lysine added at the C-terminus is called EK-10-pal, The concentration is 10ppm;
  • Palmitoylation of 10ppm EQ9 can significantly improve the anti-aging effect, even a dose of 4ppm can have a certain effect;
  • 10ppm EQ9 can also significantly improve the anti-aging effect through liposome encapsulation

Abstract

本公开涉及皮肤修复与改善领域，具体涉及一种活性肽及其相关用途，本公开获得的活性肽具有缓解衰老，抗氧化、抗炎性等优点，并能够有效的缓解由于紫外线照射带来的皮肤损伤。

Description

一种活性肽及其相关用途
相关申请的交叉引用
本公开要求于2021年09月09日提交中国专利局的申请号为“CN202111064688.6”名称为“一种活性肽及其相关用途”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及皮肤修复与改善领域，具体涉及一种活性肽及其相关用途。
背景技术
间充质干细胞(mesenchymal stem cells,MSCs)是一类来源于发育早期中胚层的多能干细胞，是目前研究最多、最重要的成体干细胞之一。MSCs广泛存在于全身多种组织中，并可在体外培养扩增，可在特定条件下分化为包括神经细胞、成骨细胞、软骨细胞、肌肉细胞、脂肪细胞在内的多种细胞，连续传代培养和冷冻保存后仍具有多向分化潜能。与其他来源的MSCs相比，来源于脐带的MSCs具有采集方便、无伦理争议、免疫原性低、自我更新快、倍增速度稳定和增殖能力强等特点。因此，脐带MSCs适合用于临床研究和应用，是细胞治疗和再生医学的首选。
根据www.ClinicalTrials.gov网站统计的数据表明，截至2018年10月,全球已注册705项基于MSCs的临床试验,80％以上的临床试 验处于临床Ⅰ期和Ⅱ期,15％左右处于临床Ⅲ期阶段。目前的实验结果表明：MSCs用于多种疾病的治疗是安全和有效的,这些疾病类型包括移植物抗宿主病、脊髓损伤、自身免疫性疾病、心血管疾病、骨及软骨损伤修复、克罗恩病、糖尿病及其并发症等。目前,全球已有11种间充质干细胞药物获批用于上述疾病的临床治疗；然而,MSCs在疾病治疗过程中发挥作用的机制尚不明确,仍需深入的研究和探索。
大量的证据表明，间充质干细胞(MSC)可以通过旁分泌或自分泌发挥治疗作用，其疗效与血管生成、免疫调节及细胞凋亡有关。MSC分泌组即MSC所分泌的生物活性分子的总和，包括细胞因子、趋化因子和生长因子等多种活性成分，对损伤组织的修复、再生及功能恢复等许多生理过程具有调控作用。在创伤治疗领域，基于MSC分泌组研制的制剂极有可能成为替代MSC治疗的新疗法，具有广阔的发展前景(王强等，间充质干细胞分泌组:创伤愈合的替代疗法，中国生物工程杂志2017,Vol.37 Issue(4):104-109)，然而目前基于间充质干细胞(MSC)分泌肽的研究仍处于比较初级的阶段，目前的研发成果难以满足消费者的多样化需求。此外，目前关于MSC分泌组正式用于临床之前，还需要更多的体内实验和体外实验，获得商业化前景更好的生物活性肽。
然而，自然界及肌体内发现的活性肽存在某些缺陷，比如肽段比较长导致其在体内不稳定，并且纯化工艺和合成工艺复杂，规模化生产成本高，透皮性差，因此，为了进一步提高成药性和提升商业化价 值，需要对相关活性肽进行结构改造。
本公开的发明人基于上述思路，在研发获得一个LQ-17活性肽的基础上，经过结构改造又获得EQ-9活性肽。其活性与LQ-17相当，但是由于属于截断体，生产成本可以进一步降低，取得了预料不到的技术效果。
发明内容
本公开的目的在于提供一种生物活性多肽及其制备方法和应用。
本公开的目的可以通过以下技术方案来实现：
本公开提供一种生物活性多肽，其氨基酸序列符合以下通式：R1-AA 1-AA 2-AA 3-AA 4-AA 5-AA 6-AA 7-AA 8-AA 9
其中，
AA 1选自E、D或是键
AA 2选自S、T或是键
AA 3选自E、D、K
AA 4选自T、A、S、I
AA 5选自R、K、E
AA 6选自I、L、A
AA 7选自L、I、V
AA 8选自L、I、V
AA 9选自Q、E、D或是键
R1选自由H、乙酰基、叔丁酰基、己酰基、2-甲基己酮基、环己烷羧基、辛酰基、癸酰基、月桂酰、肉豆蔻酰、棕榈酰、硬脂酰、 油酰基和亚油酰基组成的组。
在一些实施方式中，所述符合上述通式的生物活性多肽，其中AA 5为R、AA 6为I、AA 7为L、AA 8为L。
在一些实施方式中，所述生物活性多肽氨基酸序列选自ESETRILLQ、ESETKILLQ、DSDAKILIQ、DSDAKLLIQ、ETDTRILVE、ESEARIILE、DSESKLVID、ESETEAVLQ、ESKIRILLQ、ETRILL、SETRIL组成的组。
本公开提供了所述生物活性多肽的制备方法，可以通过基因工程的方法人工合成，可以从细胞中通过分离纯化的方法直接获得，更直接通过化学合成制备。
本公开提供了所述生物活性多肽在制备具有抗炎功能的食品、保健品、药物或化妆品中的应用。
本公开提供了所述生物活性多肽在制备具有抗衰老功能的食品、保健品或药物中的应用。
本公开提供了所述生物活性多肽在制备具有抗氧化功能的食品、保健品或药物中的应用。
本公开提供了一种产品，包括所述生物活性多肽或其衍生物；所述生物活性多肽的衍生物，是指在生物活性多肽的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰，得到的多肽衍生物，同时不影响其生物学活性。
本公开提供了一种包括美容上或药学上有效量的至少一种根据 前述的活性肽以及至少一种赋形剂或美容上或药学上可接受的佐剂的药物组合物。
在一些实施方式中，所述药物组合物的剂型包括膏霜、乳液、水剂、凝胶、油剂、粉剂、泥类、蜡基类、贴类、膜类或冻干类。
在一些实施方式中，所述赋形剂包括：羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、聚乙二醇、聚丙烯酰胺、聚丙烯酸、聚乙烯醇、葡聚糖、氨、乳酸、柠檬酸、吡咯烷酮羧酸、甘油、尿素、山梨糖醇、氨基酸、羊毛脂、矿脂。
在一些实施方式中，所述佐剂包括：增溶剂，增稠剂，胶凝剂，柔软剂，抗氧化剂，悬浮剂，稳定剂，发泡剂，调味剂，表面活性剂，水，离子或非离子乳化剂，填料，螯合剂，螯合剂，防腐剂，维生素，阻滞剂，润湿剂，精油，染料，颜料。
此外，本公开的活性肽还可与常用于口服组合物或食品补充物的赋形剂和佐剂配制，诸如且不限于食品工业中常用的脂肪成分、水性成分、保湿剂、防腐剂、调质剂、调味剂、香料、抗氧化剂和着色剂。
本公开的生物活性肽可与另外成分联用，所述另外成分包括但不限于：抗氧化剂、反应性羰基物类清除剂、抗糖化剂、抗组织胺剂、抗病毒剂、抗寄生剂、乳化剂、润肤剂、防皱剂和/或抗老化剂等。
发明人对结构为R1-AA 1-AA 2-AA 3-AA 4-AA 5-AA 6-AA 7-AA 8-AA 9的生物活性肽的功能进行了广泛研究，开展了大量实验，并确定对其生物学活性起重要作用的结构域，最终获得活性好、生产成本低、稳 定性好的具有显著抗衰老、抗氧化以及抗炎症反应多重功效的生物活性肽。
附图说明：
图1：鸡胚安全性实验；
图2：细胞活率测定实验；
图3：保护细胞免受UVA损伤能力检测实验(细胞活率指标)；
图4：保护细胞免受UVA损伤能力检测实验(I型胶原蛋白含量指标)；
图5：保护细胞免受UVA损伤能力检测实验(胞内ATP含量指标)；
图6：UVA损伤修复能力检测实验(细胞活率指标)；
图7：UVA损伤修复能力检测实验(MMP-9蛋白含量指标)；
图8：改构后的生物活性肽UVA损伤修复实验；
图9：不同修饰策略的EQ-9多肽UVA损伤修复实验；
实施例：
定义
尽管本公开的广义范围所示的数字范围和参数近似值，但是实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的 任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
本公开涉及的氨基酸在此用标准三字母或单字母缩写表示。
本公开相关多肽的获得采用以下流程：
以MSC分泌组为实验材料，通过LC-MS液相色谱质谱联用仪筛选分子量小于30kDa的多肽，结合核糖体图谱技术筛选获得近千种生物活性肽，再根据基于机器学习和生物物理的蛋白质-肽相互作用预测方法(HSM PPI prediction)进一步筛选获得LQ-17，并在其基础上进行截断获得截短体VQ-13、EQ-9、LT-12序列如下：
表1：LQ-17截短体
No. name sequence
1 LQ-17 LSDQVPDTESETRILLQ
2 VQ-13 VPDTESETRILLQ
3 EQ-9 ESETRILLQ
4 LT-12 LSDQVPDTESET
实施例1：安全性研究-鸡胚试验
该实验参照化妆品眼刺激性腐蚀性的鸡胚绒毛尿囊膜试验(SN/T 2329-2009)相关标准和步骤进行，实验结果采用刺激评 分法(IS)，结果为：IS＝0.35,无刺激性。评价标准如下：
表2：
刺激评分 刺激性分类
IS＜1 无刺激性
1≤IS＜5 轻刺激性
5≤IS＜9 中度刺激性
IS≤10 强刺激性/腐蚀性
实验结果表明：使用剂量高达500ppm的EQ-9没有安全问题，参见图1。
实施例2：细胞活力检测实验
HaCaT细胞复苏传代，镜检细胞状态良好后，收集细胞，调整细胞浓度为0.5-1.0*10 5个/mL，按照100μL/孔进行96孔铺板。培养箱培养(37℃，5％CO 2)至细胞融合度约80％后添加一定浓度的受试物，孵育24h。移去上清液，使用CCK8检测试剂盒测定细胞活率。结果参见图2。EQ-9促进细胞存活的能力明显强于LQ-17。
实施例3：保护细胞免受UVA损伤能力检测实验
人包皮成纤维细胞HFF细胞及永生化人角质形成细胞HaCaT细胞复苏传代，镜检细胞状态良好后，收集细胞，按照2.0-2.5*10 4个/孔细胞密度加入96孔板。培养箱培养24h(37℃，5％CO 2)，弃去培养基，加入含有一定浓度受试物的培养基，用 一定计量的UVA诱导，控制照射总量相同且介于5-15J/cm 2。弃去上清，加入含有一定受试物的培养基，培养24h后使用相应试剂盒测定细胞活率、胶原蛋白含量和ATP含量(参见图3-图5)。结果表明：
1.LQ-17和EQ-9可显著地提高细胞在紫外刺激条件下的存活率。
2.LQ-17和EQ-9显著地提高了紫外刺激条件下受试细胞外I型胶原蛋白的含量。
3.紫外刺激条件下，仅EQ-9能够显著增加细胞内ATP含量。
实施例4：UVA损伤修复能力测试实验
人包皮成纤维细胞HFF细胞复苏传代，镜检细胞状态良好后，收集细胞，按照2.0-2.5*10 4个/孔细胞密度加入96孔板。培养箱培养24h(37℃，5％CO 2)，弃去培养基，加入新鲜培养基，用一定计量的UVA诱导，控制照射总量为5J/cm 2。弃去上清，加入含有一定受试物的培养基，培养24h后使用相应试剂盒测定细胞活率。
实验结果表明(参见图6)：EQ-9和LQ-17可以修复UVA引起的损伤，增加细胞活力。
实施例5：MMP-9表达的定量检测实验
人包皮成纤维细胞HFF细胞复苏传代，镜检细胞状态良好后，收集细胞，按照2.0-2.5*10 4个/孔细胞密度加入96孔板。培养箱培养24h(37℃，5％CO 2)，弃去培养基，加入新鲜培养基，用一定计量的UVA诱导，控制照射总量为5J/cm 2。弃去上清，加入含有一定受试物的培养基，培养24h后收集细胞培养基，在4℃下1000g离心10min，使用相应试剂盒测定MMP-9蛋白含量。
实验结果表明(参见图7)：EQ-9和LQ-17可显着降低紫外损伤后细胞的MMP-9表达量(MMP-9属于基质金属蛋白酶家族，可降解细胞外胶原蛋白)。
综合以上实验结果表明：EQ-9生物活性肽是一种很有前途的抗衰老成分，安全性好，在促进细胞生长、保护细胞免受UVA损伤以及增加胶原蛋白I的产生方面的作用与LQ-17效果相当，在UVA刺激后增加细胞内ATP含量方面，EQ-9显著优于LQ-17；EQ-9生物活性肽长度比LQ-17短，却与其具有相同或更优的活性，生产成本更低，具有巨大的商业潜力。相比之下，其他截短体诸如：VQ-13、LT-12综合效果较差。
实施例6：EQ-9肽改造与生物活性研究
发明人针对EQ-9进行了详尽的结构和功能研究，通过改构获得以下活性肽。
表3：改构衍生肽汇总
No. Name Sequence
1 LQ-17 LSDQVPDTESETRILLQ
2 EQ-9 ESETRILLQ
3 EQ-9-1 ESETKILLQ
4 DQ-9 DSDAKILIQ
5 DQ-9-2 DSDAKLLIQ
6 EE-9-1 ETDTRILVE
7 EE-9-2 ESEARIILE
8 DD-9 DSESKLVID
9 EQ-9-2 ESETEAVLQ
10 EQ-9-3 ESKIRILLQ
11 EL-6 ETRILL
12 SL-6 SETRIL
将上述改构后的生物活性肽进行UVA损伤修复实验(实验步骤同实施例3)，结果如图8。
LQ-17截短实验结果显示，相较于VQ-13和EQ-9，LT-12几乎完全失去了活性，表明被截去的RILLQ对于活性有重要影响。对EQ-9进一步截短后发现，EL-6保留了较好的生物活性，而SL-6则表现出活性的丧失，进一步证明RILL区域的重要性。对EQ-9进行不同的点突变后，其生物活性都受到了一定程度的影响。比较EQ-9和EQ-9-1，将R突变为K后，多肽活性出现下降。比较EQ-9和EQ-9-2，将RIL突变为EAV后，多肽活性完全丢失，甚至表现出抑制效果。由此可见，发明人通过功能性 实验验证了EQ-9生物活性肽的核心功能域，结合其他的具有生物学功能的LQ-17截短体，综合判断以EQ-9效果最佳。
实施例7：EQ-9生物活性肽棕榈酰化修饰效果
将EQ-9生物活性肽进行如下改造：
7.1棕榈酰化构造
多肽是通过固相合成的方式合成的,简要步骤如下：首先合成Fmoc-AA1-Wang树脂，用溶解于二甲基甲酰胺(DMF)中的20％哌啶溶液处理树脂以解封氨基，然后用Fmoc-AA2和1-羟基苯并三唑(HOBt)处理得到Fmoc-AA2-AA1-Wang树脂(注：AA1和AA2代表氨基酸)。重复上述过程，生成最终的Fmoc-peptide-Wang树脂。最后解封氨基，用棕榈酸和1-羟基苯并三唑(HOBt)处理得到palm-peptide-Wang树脂。使用TFA二氯甲烷水从树脂中释放粗肽，随后通过HPLC纯化，保证其纯度>95％。最后，将肽冷冻干燥为多晶型结晶粉末。
7.2脂质体包裹递送
将不同摩尔浓度的枯草菌脂肽钠、大豆卵磷脂、1,3-丙二醇、甘油、1,2-戊二醇、1,2-己二醇等用乙醚溶解，逐渐滴加入含有1mg/mL多肽(EQ-9)的水溶液中，维持水溶液温度在50-60℃之间，持续搅拌2h，促使乙醚挥发。镜检，确定脂质体的形状和大小。取样，测定包封率。将符合要求的脂质体进行后续实验。
将经过上述改造后的EQ-9肽进行实施例4所述的UVA损伤 修复能力测试实验(实验步骤基本相同)，其中：1)10ppm的EQ-9；2)4ppm EQ-9；3)10ppm EQ-9脂质体；4)10ppm EQ-9棕榈化修饰；5)将EQ-9-pal的基础上，C端增加一个赖氨酸的EQ-9衍生物称为EK-10-pal，浓度为10ppm；
实验结果(如图9所示)如下：
1)10ppmEQ9棕榈酰化可以显著提高抗衰功效，即使4ppm的剂量也可以起到一定的效果；
2)10ppmEQ9通过脂质体包裹也可以显著提高抗衰功效；
3)相比之下，EK10-pal在该体外细胞模型中，并没有抗衰功效；
以上描述地仅是可选的实施方式，其只作为示例而不限制实施本公开所必需特征的组合。所提供的标题并不意指限制本公开的多种实施方式。术语例如“包含”、“含”和“包括”不意在限制。此外，除非另有说明，没有数词修饰时包括复数形式，以及“或”、“或者”意指“和/或”。除非本文另有定义，本文使用的所有技术和科学术语的意思与本领域技术人员通常理解的相同。

Claims (10)

  1. 一种生物活性多肽，其氨基酸序列符合以下通式：
    R1-AA 1-AA 2-AA 3-AA 4-AA 5-AA 6-AA 7-AA 8-AA 9
    其中，
    AA 1选自E、D或是键
    AA 2选自S、T或是键
    AA 3选自E、D、K
    AA 4选自T、A、S、I
    AA 5选自R、K、E
    AA 6选自I、L、A
    AA 7选自L、I、V
    AA 8选自L、I、V
    AA 9选自Q、E、D或是键
    R1选自由H、乙酰基、叔丁酰基、己酰基、2-甲基己酮基、环己烷羧基、辛酰基、癸酰基、月桂酰、肉豆蔻酰、棕榈酰、硬脂酰、油酰基和亚油酰基组成的组。
  2. 权利要求1所述的生物活性肽，其中AA 5为R、AA 6为I、AA 7为L、AA 8为L。
  3. 权利要求1所述的生物活性肽，其氨基酸序列选自ESETRILLQ、ESETKILLQ、DSDAKILIQ、DSDAKLLIQ、ETDTRILVE、ESEARIILE、DSESKLVID、ESETEAVLQ、ESKIRILLQ、ETRILL、 SETRIL组成的组。
  4. 权利要求1-3任一项所述的分泌肽在制备抗衰老功能的食品、化妆品、保健品或药物中的应用。
  5. 权利要求1-3任一项所述的分泌肽在制备抗紫外损伤功能的食品、化妆品、保健品或药物中的应用。
  6. 权利要求1-3任一项所述的分泌肽在制备抗炎功能的食品、化妆品、保健品或药物中的应用。
  7. 一种合成权利要求1-3任一项所述多肽的衍生物，其所述衍生物为在权利要求1-3所述多肽的氨基酸侧链基团上、氨基端或羧基端进行羟基化、羧基化、羰基化、甲基化、乙酰化、磷酸化、酯化或糖基化等修饰，得到的多肽衍生物，同时不影响其生物学活性。
  8. 一种药物组合物，其含有权利要求1-3所述的生物活性肽、权利要求6所述的生物活性肽衍生物，以及至少一种赋形剂或美容上或药学上可接受的佐剂。
  9. 权利要求8所述的药物组合物，所述赋形剂包括：羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素、聚乙二醇、聚丙烯酰胺、聚丙烯酸、聚乙烯醇、葡聚糖、氨、乳酸、柠檬酸、吡咯烷酮羧酸、甘油、尿素、山梨糖醇、氨基酸、羊毛脂、矿脂。
  10. 权利要求8所述的药物组合物，所述佐剂包括：增溶剂，增稠剂，胶凝剂，柔软剂，抗氧化剂，悬浮剂，稳定剂，发泡剂，调味剂，表面活性剂，水，离子或非离子乳化剂，填料，螯合剂，螯合剂，防腐剂，维生素，阻滞剂，润湿剂，精油，染料，颜料。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115340592A (zh) * 2021-09-09 2022-11-15 禾美生物科技(浙江)有限公司 一种活性肽及其相关用途
CN117327153A (zh) * 2023-10-10 2024-01-02 广州研智化妆品有限公司 一种活性肽与植物外泌体抗衰化妆品的制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114195884B (zh) * 2021-10-27 2024-03-29 禾美生物科技(浙江)有限公司 一种重组人源胶原蛋白及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102015764A (zh) * 2008-02-29 2011-04-13 利普泰股份公司 包含金属蛋白酶抑制剂的化妆品组合物或药物组合物
CN102395598A (zh) * 2009-04-17 2012-03-28 利普泰股份公司 用于治疗和/或护理皮肤和/或毛发的肽、及其在化妆用组合物或药物组合物中的用途
CN102711790A (zh) * 2009-10-23 2012-10-03 利普泰股份公司 用于治疗和/或护理皮肤、粘膜和/或毛发的肽、及其在化妆用组合物或药物组合物中的用途
US20180141998A1 (en) * 2015-04-23 2018-05-24 Nantomics, Llc Cancer neoepitopes
CN113288857A (zh) * 2021-05-24 2021-08-24 启迪禾美生物科技(嘉兴)有限公司 脐带间充质干细胞活性肽分泌物、制备方法、应用及皮肤微生态调节剂

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG2012096699A (en) * 2012-12-31 2014-07-30 Agency Science Tech & Res Amphiphilic linear peptide/peptoid and hydrogel comprising the same
CN108794603B (zh) * 2018-06-29 2020-06-09 浙江辉肽生命健康科技有限公司 一种生物活性多肽tvtmlmttil及其制备方法和应用
CN110526957B (zh) * 2019-07-01 2020-11-13 启迪禾美生物科技(嘉兴)有限公司 细胞渗透增强肽、组合物及其应用
CN112646042A (zh) * 2019-10-10 2021-04-13 陕西麦科奥特科技有限公司 活性多肽化合物
CN113248573B (zh) * 2021-05-18 2022-03-22 深圳市旷逸生物科技有限公司 改善皮肤生理特性的活性肽与间充质干细胞外泌体在药品或化妆品中的应用
CN115340592A (zh) * 2021-09-09 2022-11-15 禾美生物科技(浙江)有限公司 一种活性肽及其相关用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102015764A (zh) * 2008-02-29 2011-04-13 利普泰股份公司 包含金属蛋白酶抑制剂的化妆品组合物或药物组合物
CN102395598A (zh) * 2009-04-17 2012-03-28 利普泰股份公司 用于治疗和/或护理皮肤和/或毛发的肽、及其在化妆用组合物或药物组合物中的用途
CN102711790A (zh) * 2009-10-23 2012-10-03 利普泰股份公司 用于治疗和/或护理皮肤、粘膜和/或毛发的肽、及其在化妆用组合物或药物组合物中的用途
US20180141998A1 (en) * 2015-04-23 2018-05-24 Nantomics, Llc Cancer neoepitopes
CN113288857A (zh) * 2021-05-24 2021-08-24 启迪禾美生物科技(嘉兴)有限公司 脐带间充质干细胞活性肽分泌物、制备方法、应用及皮肤微生态调节剂

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115340592A (zh) * 2021-09-09 2022-11-15 禾美生物科技(浙江)有限公司 一种活性肽及其相关用途
CN117327153A (zh) * 2023-10-10 2024-01-02 广州研智化妆品有限公司 一种活性肽与植物外泌体抗衰化妆品的制备方法

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