WO2022022225A1 - 具有抗衰老作用的多肽rv3和rv4及其应用 - Google Patents

具有抗衰老作用的多肽rv3和rv4及其应用 Download PDF

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WO2022022225A1
WO2022022225A1 PCT/CN2021/104281 CN2021104281W WO2022022225A1 WO 2022022225 A1 WO2022022225 A1 WO 2022022225A1 CN 2021104281 W CN2021104281 W CN 2021104281W WO 2022022225 A1 WO2022022225 A1 WO 2022022225A1
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polypeptide
elegans
aging
nematodes
results
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French (fr)
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徐寒梅
王栋
让•马丁内斯
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南京安吉生物科技有限公司
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Priority to JP2022521563A priority Critical patent/JP7367205B2/ja
Priority to EP21850783.8A priority patent/EP4190798A4/en
Priority to US17/767,397 priority patent/US20240076313A1/en
Priority to AU2021319252A priority patent/AU2021319252A1/en
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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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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 invention belongs to the field of biological medicine, and more particularly, relates to a polypeptide with anti-aging activity and its application.
  • Aging is a process in which the body goes through a period of sexual maturity, the self-renewal and repair capabilities of cells are weakened, the structure and function of tissues and organs degenerate, and eventually die. Risk increases. With the aging of the global population, various degenerative diseases of the elderly and the huge medical expenses incurred have become more and more serious social problems. Maintaining the healthy state of the elderly population is a key link in reducing the social and economic burden brought about by population aging. Therefore, it is of great significance to explore measures and methods for intervening in aging and aging-related diseases that are simple, economical, effective, safe and suitable for promotion.
  • Aging-related diseases generally refer to diseases whose incidence increases with aging.
  • aging-related diseases are mainly considered to be cardiovascular disease, tumor, rheumatism, osteoporosis, cataract, type 2 diabetes, hypertension and Alzheimer's disease.
  • cardiovascular disease mainly considered to be cardiovascular disease, tumor, rheumatism, osteoporosis, cataract, type 2 diabetes, hypertension and Alzheimer's disease.
  • aging-related diseases are homologous to the aging process, and the aging process itself is the basic risk factor for many aging-related diseases.
  • Social responsibility has also prompted biologists to realize that prolonging lifespan alone cannot alleviate the heavy socioeconomic burden brought about by aging, and only prolonging healthy lifespan has practical significance.
  • C. elegans As a classic model animal for studying aging, C. elegans has the following advantages: firstly, its relatively short lifespan, only 2-3 weeks under standard laboratory conditions, makes lifespan survival analysis possible; secondly, in controlled It is easy to obtain a large number of genetically identical animals under environmental conditions; third, the transparency of the worm body facilitates the direct observation of how cells and tissues change with age; Research to aid in anti-aging.
  • a major advantage of the C. elegans model is the ease with which genetic methods can be used to provide biological information, allowing the identification of a large number of genetic mutations that can alter lifespan.
  • C. elegans lifespan exhibits remarkable plasticity and may be affected by environmental, nutritional, and genetic mutations, and even under controlled conditions, individual lifespan may vary, revealing stochastic factors in aging. Therefore, C. elegans can be used as a model animal to study the anti-aging activity of drugs.
  • anti-aging drugs By far the most common form of anti-aging is the use of anti-aging drugs.
  • Most of the anti-aging drugs used clinically are synthetic drugs, such as vitamin E, which can promote cell division and inhibit the generation of oxygen free radicals, procaine preparations can prolong cell lifespan, and amidopyrone can delay brain aging, etc.
  • Oxidative stress delaying the decline of body function caused by aging, thereby prolonging the lifespan of nematodes
  • Metformin as an activator of adenylate-activated protein kinase (AMPK)
  • AMPK adenylate-activated protein kinase
  • PAL-12 A six-membered peptide
  • resveratrol analogs and other drugs are also favored for their anti-aging effects.
  • the present invention provides a polypeptide with anti-aging activity and application thereof.
  • the polypeptide of the present invention can effectively prolong the lifespan of Caenorhabditis elegans, has good anti-aging effect and has great development prospects.
  • the polypeptide with anti-aging activity is characterized in that X 1 X 2 is AF or KA.
  • Said polypeptide with anti-aging activity is characterized in that its amino acid sequence is TAFAA (named RV3) or TKAAA (named RV4).
  • one or more pharmaceutically acceptable adjuvants can also be added to the above-mentioned medicines, and the adjuvants include conventional diluents, fillers, adhesives, wetting agents, absorption enhancers, surface active agents in the pharmaceutical field. agents, lubricants and stabilizers.
  • the medicine of the present invention can be made into various forms such as injection, freeze-dried powder injection, tablet or granule.
  • the medicines in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.
  • the polypeptide of the present invention has a novel structure, the basic unit of which is composed of natural amino acids, and is easy to synthesize, separate and purify;
  • the polypeptide of the present invention has good safety, weak adverse reactions and toxic side effects, does not affect the growth and development of Caenorhabditis elegans, and does not affect the reproductive ability of the nematodes;
  • the anti-aging effect of the polypeptide involved in the present invention performs well in the Caenorhabditis elegans model, which is embodied in significantly improving the motor behavior of the nematode, delaying the decline of the motor ability in the aging process of the nematode, prolonging half the survival days of the nematode, increasing the Stress stress capacity of nematodes and prolong lifespan of nematodes.
  • FIG. 1 Survival curves of experiments on the effect of polypeptides on C. elegans lifespan. It can be seen from the figure that the RV4 polypeptide group has the effect of significantly prolonging the lifespan of C. elegans.
  • Figure 2 Results of the effect of polypeptides on the body length and width of Caenorhabditis elegans. Among them, picture A is the result of body length, and picture B is the result of body width. The results are expressed as Mean ⁇ SEM. Compared with the blank group, n.s. means no significant difference, *P ⁇ 0.05 means significant difference, **P ⁇ 0.01 means very significant difference. There was no significant difference between the polypeptide group and the blank group, indicating that the polypeptides RV3 and RV4 did not affect the normal growth and development of C. elegans, and the polypeptides had good safety.
  • Figure 3 Results of the effect of polypeptides on the number of broods in Caenorhabditis elegans. The results are expressed as Mean ⁇ SEM. Compared with the blank group, n.s. means no significant difference, *P ⁇ 0.05 means significant difference, **P ⁇ 0.01 means very significant difference. There was no significant difference between the polypeptide group and the blank group, indicating that the polypeptides RV3 and RV4 did not affect the normal reproductive ability of C. elegans, and the polypeptides had good safety.
  • Figure 4 Results of the effect of polypeptides on the motor behavior of Caenorhabditis elegans.
  • picture A is the result of head swing
  • picture B is the result of body bending
  • picture C is the result of pharyngeal pump frequency.
  • the results are expressed as Mean ⁇ SEM.
  • n.s. means no significant difference
  • *P ⁇ 0.05 means significant difference
  • **P ⁇ 0.01 means very significant difference.
  • RV4 polypeptide group had significant differences, indicating that the polypeptide RV4 could enhance the muscle movement of C. elegans and improve the locomotor behavior of C. elegans.
  • FIG. 5 Survival curves of acute heat stress experiments in C. elegans. Picture A is the survival curve of Day4 acute heat stress experimental nematodes, and picture B is the survival curve of Day8 acute heat stress experimental nematodes. It can be seen from the figure that both the RV3 and RV4 peptide groups can significantly improve the ability of nematodes to acute heat stress, indicating that the peptide drug can prolong the life cycle of nematodes in a thermal environment.
  • Figure 6 Motility rating results of acute heat stress experiments in C. elegans.
  • picture A is the exercise rating result of Day4 acute heat stress experimental nematodes
  • B picture is the exercise ability rating result of Day8 acute heat stress experimental nematodes.
  • the proportion of A-grade nematodes in the RV3 and RV4 peptide groups at Day4 is higher than that in the blank group
  • the proportion of C-grade nematodes in the RV3 and RV4 peptide groups at Day8 is lower than that of the blank group, which further shows that
  • the polypeptide drug can enhance the ability of Caenorhabditis elegans acute heat stress, that is, the ability of motor behavior under heat environment.
  • Fig. 7 The detection results of the acute oxidative stress experiment of Caenorhabditis elegans. Picture A is the detection result of the acute oxidative stress test on Day4, and picture B is the detection result of the acute oxidative stress test on Day8. The results are expressed as Mean ⁇ SEM. Compared with the blank group, *P ⁇ 0.05 is a significant difference, and **P ⁇ 0.01 is a very significant difference. It can be seen from the figure that the survival rate of nematodes in the RV4 polypeptide group is significantly higher than that in the blank group, which indicates that the polypeptide drug can prolong the life cycle of nematodes in an oxidative environment.
  • Figure 8 Motility rating results of acute oxidative stress experiments in C. elegans.
  • picture A shows the exercise rating results of the nematodes in the acute oxidative stress experiment on Day 4
  • picture B shows the exercise ability grading results in the nematodes in the acute oxidative stress experiment on Day 8.
  • the proportion of nematodes in the RV3 and RV4 peptide groups in grade A is higher than that of the blank group, which further indicates that the peptide drug can enhance the ability of C. elegans acute oxidative stress;
  • the Day4 acute oxidative stress experiment there was no difference in exercise between the peptide group and the blank group.
  • Figure 9 Results of motility rating of C. elegans at different ages.
  • picture A is the grading result of the nematode motility at the time point of Day4
  • picture B is the grading result of the movement ability of the nematodes at the time point of Day8
  • picture C is the grading result of the movement ability of the nematodes at the time point of Day12.
  • the proportion of C-level nematodes in the RV3 and RV4 peptide groups on Day8 and Day12 was significantly lower than that in the blank group, which means that the peptides can delay the muscle aging of C. elegans. Increases the mobility of the nematode, thereby extending the lifespan of the nematode.
  • Figure 10 The results of motility testing of C. elegans at different ages. Among them, picture A is the test result of nematode motility at the time point of Day4, picture B is the test result of the movement ability of nematodes at the time point of Day8, and picture C is the test result of the movement ability of nematodes at the time point of Day12. The results are expressed as Mean ⁇ SEM. Compared with the blank group, *P ⁇ 0.05 is a significant difference, and **P ⁇ 0.01 is a very significant difference.
  • RV4 polypeptide can delay the muscle aging of C. elegans, increase The motility of nematodes can delay the decline of nematode motility, thereby prolonging the lifespan of nematodes.
  • Polypeptide RV3 TAFAA and peptide RV4: TKAAA were synthesized by Jiangsu Synthetic Polypeptide Drug Discovery and Evaluation Engineering Center with purities of 97.30% and 93.85%, respectively.
  • Escherichia coli OP50 culture conditions: shaking incubator, 220rpm, 37°C
  • RV3 Thr-Ala-Phe-Ala-Ala, 10 nM
  • RV4 Thr-Lys-Ala-Ala-Ala, 10 nM
  • Synchronization of Caenorhabditis elegans Select an NGM petri dish with a moderate nematode density, pick L4 larvae with a picking needle, transfer them to a blank NGM petri dish, pick about 20 larvae in total, and wait until they develop into adults and give birth After the first batch of eggs, all the adults were picked away. After about 12 hours, the eggs hatched into larvae, and the synchronized L1-stage larvae were obtained.
  • Preliminary preparation for the C. elegans lifespan experiment Rinse the NGM medium with 1 ml of M9 buffer, collect the buffer containing the L1 stage larvae in an EP tube, place in a chromatographic cabinet at 4°C for 5 minutes, and centrifuge at 1500 x g/4°C for 3 minutes. Discard the supernatant, resuspend in 100 ⁇ l of buffer, and count the nematodes under the microscope, then dilute the nematode concentration to 30 worms/10 ⁇ l; Observe and count to ensure that the number of nematodes is about 30, and then place the NGM petri dish in a constant temperature incubator for cultivation.
  • Caenorhabditis elegans lifespan experiment After the L1 stage nematodes developed to the L4 stage, 100 ⁇ l of 15.6 ⁇ g/ml 5-FUDR was added to inhibit the oviposition of C. elegans; when the nematodes developed into adults, the drug was started and divided into control groups , 10nM administration group, this time was recorded as the first day of the lifespan experiment; after that, daily administration, observed and recorded the number of nematodes that survived, died and unexpectedly died, and observed and recorded the state of nematodes until the last nematode died. The criteria for judging death were: C.
  • the number of days of death in the RV4 polypeptide group was relatively delayed, and the longest lifespan of nematodes was 33 days, 5 days longer than that in the blank group; the number of days of death in the RV3 group was relatively delayed compared with the blank group, and the nematodes had a longer lifespan than the blank group.
  • the longest lifespan was 30 days, 2 days longer than the blank group, see Table 1 and Figure 1 for details.
  • the half of the death days of the RV4 administration group had a very significant difference, indicating that the RV4 polypeptide has the effect of significantly prolonging the lifespan of C. elegans, which can prolong the lifespan by 4 days; No significant difference. See Table 2 and Figure 1 for details, and the experimental results are statistically significant.
  • the detection method was as follows: 20 Caenorhabditis elegans were picked from each group, picked on a new NGM solid medium, and then 50 ⁇ l of NaN 3 was added dropwise at a concentration of 0.4M, after most of the worms are rigid, take pictures and record them under an inverted microscope, and then use the ruler tool of Photoshop to measure the body length and width of C. elegans. One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
  • Reproductive ability detection of Caenorhabditis elegans Collect the larvae at L1 stage after synchronization, spread them on NGM solid medium inoculated with Escherichia coli OP50, and divide them into Control group and 10nM administration group, which are placed in a constant temperature and humidity incubator Cultured to L4 stage larvae.
  • Detection of motor behavior of C. elegans larvae at L1 stage after synchronization were plated on NGM solid medium inoculated with Escherichia coli OP50, divided into Control group and 10nM administration group, placed in a constant temperature and humidity incubator Culture for 48h. Then, the following three indicators of motor behavior were detected: head swing frequency, 20 nematodes were picked from each group to a new clean medium, and after 1 hour of adaptation and recovery, an appropriate amount of M9 buffer was added and observed under an inverted microscope.
  • RV4 polypeptide group Compared with the blank group, the RV4 polypeptide group had significant difference. This means that RV4 polypeptide can significantly enhance the muscle movement of Caenorhabditis elegans and improve the motor behavior of nematodes; RV3 polypeptide has no significant difference compared with the blank group. See Table 5 and Figure 4 for details, and the experimental results are statistically significant.
  • Acute heat stress experiment of C. elegans Take C. elegans on the 4th day of continuous administration, and then place them in a 35°C constant temperature incubator for stress stress experiments. Record the life and death of the nematodes every 4 hours until the last one The nematodes died, and the criteria for judging death were the same as in the lifespan experiment; at the same time, at the 24th hour after the start of the experiment, the motor behavior of the surviving C. elegans was rated, and the mobility was divided into three grades, A, B, and C.
  • a nematode that can crawl autonomously is defined as A, a nematode that starts to move only after the pick needle touches the head is defined as C, and a state between the two is defined as B; take the nematodes on the 8th day of continuous administration and place them at 35°C In a constant temperature incubator, the life and death of the nematodes were recorded every 2 hours until the last death, and the criteria for judging death and rating criteria for mobility were the same as the heat stress experiment on the fourth day above. Kaplan-Meier statistical analysis was performed on the data, and the data results were expressed as Median ⁇ SE. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
  • Acute oxidative stress experiment of C. elegans Take C. elegans on the 4th and 8th day of continuous administration, soak in 30mM H2O2 for 4h, and then transfer to NGM solid medium for recovery after 24h , record the number of survival and death of C. elegans under a microscope, and rate the motor behavior of the surviving nematodes. The criteria for judging death and mobility are the same as those in the acute heat stress experiment.
  • One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
  • a nematode that can crawl autonomously is defined as A
  • C a nematode that starts to move only after the pick needle touches the head
  • B a state between the two
  • a nematode that can crawl autonomously is defined as A
  • C a nematode that starts to move only after the pick needle touches the head
  • B a state between the two
  • a nematode that can crawl autonomously is defined as A
  • C a nematode that starts to move only after the pick needle touches the head
  • B a state between the two
  • a nematode that can crawl autonomously is defined as A
  • C a nematode that starts to move only after the pick needle touches the head
  • B a state between the two
  • C. elegans exercise ability aging experiment take C. elegans on the 4th, 8th and 12th day of continuous administration, respectively, to detect the nematode's motor behavior, including head swing frequency and pharyngeal pump frequency, the detection method is the same Kinetic force detection in Example 4.
  • the motility of the surviving nematodes was rated, and the rating standard was the same as the motility rating in Example 5.
  • One-way ANOVAY statistical analysis was performed on the data, and the data results were expressed as Mean ⁇ SEM. Compared with the Control group, *P ⁇ 0.05 was a significant difference, and **P ⁇ 0.01 was a very significant difference.
  • C C. elegans that can crawl autonomously is defined as A
  • C nematodes that start to move only after the pick needle touches the head is defined as C
  • B B
  • the results of the nematode motility rating on Day8 and Day12 showed that in the RV3 and RV4 peptide groups, the proportion of nematodes in grade C was lower than that in the blank group; in the RV4 peptide group, the proportion of nematodes in grade A was higher than that in the blank group.
  • the polypeptide can delay the muscle aging of C. elegans, increase the motor behavior of the nematodes, thereby prolonging the lifespan of the nematodes, as shown in Table 16 and Fig. 9 for details.
  • the test results of nematode motility on Day4, Day8 and Day12 showed that the number of head swings between the RV4 polypeptide group and the blank group was significantly different, and the effect on the frequency of the pharyngeal pump was significantly different on the fourth day.
  • the frequency of pharyngeal pump was not significantly higher than that of the blank group, which means that the polypeptide can delay the muscle aging of C. elegans, increase the motor behavior of C.
  • the lifespan of nematodes; RV3 polypeptide group and blank group had no significant difference. See Table 17 and Figure 10 for details, and the experimental results are statistically significant.

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Abstract

提供一种能够延长秀丽隐杆线虫寿命的多肽，其具有氨基酸序列TX 1X 2AA(X 1X 2：任意氨基酸，优选为AF/KA)或其药学上可以接受的盐，或在该多肽的序列基础上，进行一个或多个氨基酸被删除、置换或添加后仍具有抗衰老活性或其药学上可以接受的盐。该多肽具有延长秀丽线虫寿命，抵抗衰老的作用，同时具有增强运动行为能力，提高压力应激能力和改善衰老进程中运动能力衰退的作用。

Description

具有抗衰老作用的多肽RV3和RV4及其应用 技术领域
本发明属于生物药物领域，更具体地说，涉及一种具有抗衰老活性的多肽及其应用。
背景技术
衰老是机体在经过性成熟时期后，细胞的自我更新和修复能力减弱，组织器官的结构和功能退化，并最终走向死亡的过程，其特征表现在压力应激能力减退，平衡状态打破和罹患疾病风险增加。随着全球人口老龄化，多种老年退行性疾病以及随之产生的巨大的医疗费用已成为越来越严重的社会问题。保持老年人群健康状态是减轻人口老龄化带来的社会负担和经济负担的关键环节。因此，探索简便易行，经济有效，安全性高并适宜推广的干预衰老及衰老相关疾病的措施和方法意义重大。
在早期的衰老研究中，科学家认为纯粹的衰老研究应与衰老相关疾病严格区分开来。衰老相关疾病一般指伴随衰老其发病率增加的疾病。目前认为的衰老相关疾病主要为心血管疾病、肿瘤、风湿、骨质疏松、白内障、二型糖尿病、高血压及老年痴呆等。但随着衰老研究的推进，人们发现多种衰老相关疾病的发生发展与衰老进程具有同源性，衰老进程本身是众多衰老相关疾病的基本风险因子。社会责任也促使生物学家意识到，单独的延长寿命并不能减轻老龄化所带来的沉重的社会经济负担，只有延长健康寿命才具有现实意义。因此，如何降低衰老相关疾病，提高老年人晚年生存质量，延长健康寿命成为衰老研究的热点所在。目前，健康寿命并无严格的指标衡量，一般认为衰老干预手段能够在延长寿命的同时，提高机体的应激能力，降低衰老相关疾病的发生发展，改善衰老相关性退化，就可称为延长健康寿命。
秀丽隐杆线虫作为研究衰老的经典模式动物，其具有如下优势：首先，寿命相对较短，在标准实验室条件下仅2-3周，使整个寿命存活分析成为可能；其次，在受控的环境条件下容易获得大量遗传上相同的动物；第三，虫体透明方便直接观察细胞和组织是如何随年龄变化的；第四，对线虫的细胞组织，神经元连接和全基因组的深入了解有助于抗衰老的研究。秀丽线虫模型的一个主要优点是可以很容易地利用遗传方法来提供生物学信息，从而确定了大量可以改变寿命的基因突变。最后，秀丽线虫的寿命表现出显著的可塑性，并且可能受到环境条件、营养条件以及基因突变的影响，即使是在受控条件下，个体寿命也可能发生变化，从而揭示衰老的随机因素。因此，秀丽隐杆线虫可以作为研究药物抗衰老活性的模型动物。
目前为止，最普遍的抗衰老方式是使用抗衰老药物。临床上使用的延缓衰老的药物大多 是合成类药物，如维生素E可促进细胞分裂及抑制氧自由基生成、普鲁卡因制剂能延长细胞寿命、酰胺吡酮可延缓脑衰老等；阿司匹林通过对抗氧化应激，延缓衰老引起的机体功能下降，从而延长线虫寿命；二甲双胍作为腺苷酸活化蛋白激酶(AMPK)激活剂，也可改善认知障碍，对延缓衰老也有一定的作用；PAL-12(一个六元肽)、白藜芦醇类似物等药物的抗衰老作用也备受青睐。
发明内容
1.要解决的问题
本发明提供一种具有抗衰老活性的多肽及其应用，本发明的多肽能有效地延长秀丽隐杆线虫的寿命，具有良好的抗衰老效果并具有很大的开发前景。
2.技术方案
为了解决上述问题，本发明所采用的技术方案如下：
一种具有抗衰老活性的多肽，其特征在于：多肽的氨基酸序列为X-TX 1X 2AA或其药学上可以接受的盐；X＝H、乙酰基或丙酰基；X 1和X 2：任意氨基酸。
所述的一种具有抗衰老活性的多肽，其特征在于X 1X 2为AF或者KA。
所述的一种具有抗衰老活性的多肽，其特征在于其氨基酸序列为TAFAA(命名为RV3)或TKAAA(命名为RV4)。
所述的多肽在制备抗衰老药物或保健品中的应用。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的辅料，所述辅料包括药学领域常规的稀释剂、填充剂、粘合剂、湿润剂、吸收促进剂、表面活性剂、润滑剂和稳定剂等。
本发明的药物可以制成注射液、冻干粉针剂、片剂或颗粒剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。
3.有益效果
相比于现有技术，本发明的有益效果为：
(1)本发明的多肽结构新颖，组成基本单位均为天然氨基酸，容易合成、分离和纯化；
(2)能够有效延长秀丽隐杆线虫的寿命，具有抗衰老的活性；
(3)本发明的多肽安全性好，不良反应和毒副反应弱，不影响秀丽隐杆线虫的生长发育，不影响线虫的生殖能力；
(4)本发明涉及的多肽抗衰老效果在秀丽隐杆线虫模型中表现良好，具体表现在显著提高线虫的运动行为能力，延缓线虫衰老进程中运动能力的衰退，延长线虫的半数生存天数，提高线虫的压力应激能力，延长线虫的寿命。
附图说明
图1多肽对秀丽隐杆线虫寿命影响实验的生存曲线。图中可以看出，RV4多肽组具有显著延长秀丽线虫寿命的效果。
图2多肽对秀丽隐杆线虫体长体宽的影响结果。其中，A图为体长结果，B图为体宽结果。结果以Mean±SEM表示，与空白组对比，n.s.为没有显著性差异，*P<0.05为显著性差异，**P<0.01为非常显著性差异。多肽组与空白组相比没有显著性差异，说明多肽RV3和RV4不影响秀丽线虫正常的生长发育，该多肽具有良好的安全性。
图3多肽对秀丽隐杆线虫育雏数的影响结果。结果以Mean±SEM表示，与空白组对比，n.s.为没有显著性差异，*P<0.05为显著性差异，**P<0.01为非常显著性差异。多肽组与空白组相比没有显著性差异，说明多肽RV3和RV4不影响秀丽线虫正常的生殖能力，该多肽具有良好的安全性。
图4多肽对秀丽隐杆线虫运动行为能力的影响结果。其中，A图为头部摆动结果，B图为身体弯曲结果，C图为咽泵频率结果。结果以Mean±SEM表示，与空白组对比，n.s.为没有显著性差异，*P<0.05为显著性差异，**P<0.01为非常显著性差异。RV4多肽组与空白组相比具有显著性差异，说明多肽RV4能够增强秀丽线虫的肌肉运动，提高线虫的运动行为能力。
图5秀丽隐杆线虫的急性热应激实验的生存曲线。其中A图为Day4急性热应激实验线虫的生存曲线，B图为Day8急性热应激实验线虫的生存曲线。图中可以看出，RV3和RV4多肽组均能显著提高线虫急性热压力应激的能力，说明该多肽药物能够延长线虫在热环境下的生命周期。
图6秀丽隐杆线虫的急性热应激实验的运动力评级结果。其中A图为Day4急性热应激实验线虫的运动力评级结果，B图为Day8急性热应激实验线虫的运动力评级结果。图中可以看出Day4时间点RV3和RV4多肽组中A等级的线虫占比要高于空白组，Day8时间点RV3和RV4多肽组中C等级的线虫占比要低于空白组，这进一步说明该多肽药物能够增强秀丽线虫急性热应激的能力，即在热环境下的运动行为能力。
图7秀丽隐杆线虫的急性氧化应激实验的检测结果。其中A图为Day4急性氧化应激实验的检测结果，B图为Day8急性氧化应激实验的检测结果。结果以Mean±SEM表示，与空白组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。图中可以看出，RV4多肽组线虫的存活率显著高于空白组，这说明该多肽药物能够延长线虫在氧化环境下的生命周期。
图8秀丽隐杆线虫的急性氧化应激实验的运动力评级结果。其中A图为Day4急性氧化应激实验线虫的运动力评级结果，B图为Day8急性氧化应激实验线虫的运动力评级结果。图 中可以看出Day8时期急性氧化应激实验中，RV3和RV4多肽组中处于A等级的线虫占比要高于空白组，这进一步说明该多肽药物能够增强秀丽线虫急性氧化应激的能力；Day4急性氧化应激实验中，多肽组和空白组没有运动力上的差异性。
图9不同年龄段的秀丽线虫的运动能力评级结果。其中A图为Day4时间点对线虫运动能力评级结果，B图为Day8时间点对线虫运动能力评级结果，C图为Day12时间点对线虫运动能力评级结果。图中可以看出，Day8和Day12时间点RV3和RV4多肽组与空白组相比，处于C等级的线虫占比要明显低于空白组，这意味着，该多肽能够延缓秀丽线虫的肌肉衰老，增加线虫的行动能力，从而延长线虫的寿命。
图10不同年龄段的秀丽线虫的运动能力检测结果。其中A图为Day4时间点对线虫运动能力检测结果，B图为Day8时间点对线虫运动能力检测结果，C图为Day12时间点对线虫运动能力检测结果。结果以Mean±SEM表示，与空白组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。图中可以看出，RV4多肽组与空白组相比，头部摆动次数显著高于空白组，且咽泵频率也高于空白组，这意味着，RV4多肽能够延缓秀丽线虫的肌肉衰老，增加线虫的运动行为能力，延缓线虫行动力的衰退，从而延长线虫的寿命。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。以下所述，仅是本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员均可能利用以下揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修饰或等同变化，均落在本发明的保护范围内。
多肽RV3：TAFAA和多肽RV4：TKAAA由江苏省合成多肽药物发现与评价工程中心合成，纯度分别为97.30％，93.85％。
实施例1
多肽对秀丽隐杆线虫模型的寿命影响实验
1.实验材料
大肠杆菌OP50，培养条件：振荡培养箱，220rpm，37℃培养
野生型秀丽隐杆线虫，培养条件：恒温恒湿培养箱，20℃，湿度45％-55％
5-FUDR：15.6μg/ml
NaN 3：0.4M
H 2O 2：30mM
RV3：Thr-Ala-Phe-Ala-Ala，10nM
RV4：Thr-Lys-Ala-Ala-Ala，10nM
2.实验方法
秀丽隐杆线虫的同步化：选取线虫密度适中的NGM培养皿，用挑针挑取L4期的幼虫，转移到空白的NGM培养皿，一共挑取20只左右，待它们发育到成虫并且产下第一批卵后，将成虫全部挑走，大约12h后，卵孵化成幼虫，即可得到同步化后的L1期幼虫。
秀丽隐杆线虫寿命实验的前期准备：用1ml M9缓冲液冲洗NGM培养基，收集含有L1期幼虫的缓冲液于EP管中，4℃层析柜放置5min，1500 x g/4℃离心3min，弃掉上清，100μl缓冲液重悬混匀后，于显微镜下计数线虫，然后稀释线虫浓度为30worms/10μl；取已接种大肠杆菌OP50的NGM培养皿，加入10μl的线虫稀释液，最后显微镜下观察计数，保证线虫数目在30只左右，然后将NGM培养皿置于恒温培养箱中培养。
秀丽隐杆线虫寿命实验：待L1期线虫发育到L4期，加入100μl的15.6μg/ml的5-FUDR，用来抑制秀丽线虫产卵；待线虫发育为成虫，开始给药，分为control组，10nM给药组，此时记为寿命实验的第1天；此后每天给药，每天观察记录存活、死亡和意外死亡的线虫数，观察记录线虫状态，直到最后一只线虫死亡。判断死亡的标准是：秀丽线虫对强光或敲击平板没有反应，高倍镜下咽部肌肉没有运动，最后用挑虫针敲击线虫的头部，若依然没有反应即可判断为死亡。死亡的线虫需要挑出培养皿，大肠杆菌OP50耗尽时及时添加。对数据进行Kaplan-Meier统计分析，数据结果以Median±SE表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
(1)秀丽隐杆线虫寿命实验结果记录：
表1 秀丽隐杆线虫的剩余数的记录结果
Figure PCTCN2021104281-appb-000001
Figure PCTCN2021104281-appb-000002
RV4多肽组的死亡出现天数和空白组相比要相对推迟，且线虫的最长寿命为33天，比空白组多5天；RV3组的死亡出现天数和空白组相比要相对推迟，线虫的最长寿命为30天，比空白组多2天，详见表1和图1。
(2)秀丽隐杆线虫的半数死亡天数：
表2 秀丽隐杆线虫的半数死亡天数的检测结果
Figure PCTCN2021104281-appb-000003
注：以上结果用Median±SE表示；与Control组比，*P<0.05，**P<0.01
RV4给药组的半数死亡天数与空白组相比，具有非常显著性差异，说明RV4多肽具有显著延长秀丽线虫寿命的作用，能够延长寿命4天；RV3多肽的半数死亡天数与空白组相比，没有显著性差异。详见表2和图1，实验结果具有统计学意义。
实施例2 多肽对秀丽隐杆线虫的生长发育影响实验
1.实验材料
同实施例1。
2.实验方法
秀丽隐杆线虫的同步化：同实施例1。
秀丽隐杆线虫的生长发育实验：收集同步化至L1期的幼虫，铺在已接种大肠杆菌OP50的NGM固态培养基上，分为Control组，10nM给药组，置于恒温恒湿培养箱中进行培养72h， 检测线虫成虫时期的体长和体宽，检测方法如下：每组分别挑取秀丽隐杆线虫20只，挑至新的NGM固态培养基上，然后滴加50μl NaN 3，浓度为0.4M，待大部分虫体僵直后，于倒置显微镜下拍照记录，然后用Photoshop的标尺工具，即可测量秀丽线虫的体长和体宽。对数据进行One-way ANOVAY统计分析，数据结果以Mean±SEM表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
表3 多肽对秀丽隐杆线虫的体长体宽的影响
Figure PCTCN2021104281-appb-000004
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
多肽组与空白组相比，秀丽线虫的体长和体宽均没有显著性差异，这意味着，RV3和RV4多肽均不影响秀丽隐杆线虫正常的生长发育，说明此多肽具有良好的安全性。详见表3和图2，实验结果具有统计学意义。
实施例3 多肽对秀丽隐杆线虫的生殖能力影响实验
1.实验材料
同实施例1。
2.实验方法
秀丽隐杆线虫同步化：同实施例1。
秀丽隐杆线虫的生殖能力检测：收集同步化后L1期的幼虫，铺在已接种大肠杆菌OP50的NGM固态培养基上，分为Control组，10nM给药组，置于恒温恒湿培养箱中培养至L4期幼虫。每组分别挑取1只L4期幼虫于新的NGM固态培养基上，并保证在该线虫的产卵期内，每天将线虫转移到新的培养基上，含有虫卵的培养基继续在培养箱中培养24h，然后对每个平板上的幼虫进行计数相加，直到该线虫的产卵结束，相加后的数据即该线虫的子代总数目。每组重复记录10只线虫的子代数目。对数据进行One-way ANOVAY统计分析，数据结果以Mean±SEM表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
表4 多肽对秀丽隐杆线虫的育雏数的影响
Figure PCTCN2021104281-appb-000005
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
多肽组与空白组相比，秀丽线虫产生的子代总数没有显著性差异，这意味着，RV3和RV4多肽不影响秀丽隐杆线虫正常的生殖能力，说明此多肽具有良好的安全性。详见表4和图3，实验结果具有统计学意义。
实施例4
多肽对秀丽隐杆线虫的运动行为能力影响实验
1.实验材料
同实施例1。
2.实验方法
秀丽隐杆线虫同步化：同实施例1。
秀丽隐杆线虫的运动行为能力检测：同步化后L1期的幼虫，铺在已接种大肠杆菌OP50的NGM固态培养基上，分为Control组，10nM给药组，置于恒温恒湿培养箱中培养48h。然后检测以下三种运动行为能力的指标：头部摆动频率，每组挑取20只线虫至新的干净的培养基上，使其适应恢复1h后，加入适量M9缓冲液，在倒置显微镜下观察记录30s内，线虫头部从一侧摆到另一侧再摆回来的次数；身体弯曲频率，每组挑取20只线虫至新的干净的培养基上，使其适应恢复1h后，在倒置显微镜下观察记录30s内，线虫的身体弯曲次数，线虫向前爬行一个波长的距离记为一次身体弯曲；咽泵运动频率，每组挑取20只线虫至已接种大肠杆菌OP50的培养基上，使其适应恢复1h后，在倒置显微镜下放大到能够看清线虫的咽泵运动，每只线虫录像30s，然后用PotPlayer以0.3倍速慢放录像，记数线虫的咽泵次数。对数据进行One-way ANOVAY统计分析，数据结果以Mean±SEM表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
表5 多肽对秀丽隐杆线虫的运动行为能力的影响
Figure PCTCN2021104281-appb-000006
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
RV4多肽组和空白组相比，具有显著性差异。这意味着，RV4多肽能够显著增强秀丽隐杆线虫的肌肉运动，提高线虫的运动行为能力；RV3多肽与空白组相比，没有显著性差异。详见表5和图4，实验结果具有统计学意义。
实施例5
秀丽隐杆线虫模型的压力应激实验
1.实验材料
同实施例1。
2.实验方法
秀丽隐杆线虫的同步化：同实施例1。
秀丽线虫的压力应激实验前期准备：同实施例1的寿命实验。
秀丽线虫的急性热应激实验：取连续给药第4天的秀丽隐杆线虫，然后置于35℃恒温培养箱中进行压力应激实验，每隔4h记录一次线虫的生死，直到最后一只线虫死亡，判断死亡的标准同寿命实验；同时，在实验开始后的第24h，对存活的秀丽线虫的运动行为能力进行评级，行动力分为三个等级，A、B、C，判断标准如下：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B；取连续给药第8天的线虫，置于35℃恒温培养箱中，每隔2小时记录一次线虫的生死，直到最后一次死亡，判断死亡标准和行动力评级标准同上述第4天的热应激实验。对数据进行Kaplan-Meier统计分析，数据结果以Median±SE表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
秀丽线虫的急性氧化应激实验：分别取连续给药第4天和第8天的秀丽隐杆线虫，置于30mM的H 2O 2中浸泡4h，然后转移到NGM固态培养基上恢复24h后，于显微镜下记录秀丽线虫的存活数和死亡数，并对存活线虫的运动行为能力进行评级，判断死亡标准和行动力评级标准同急性热应激实验。对数据进行One-way ANOVAY统计分析，数据结果以Mean±SEM表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
(1)秀丽隐杆线虫热应激实验结果记录：
表6 热应激Day4秀丽线虫的剩余数的记录结果
Figure PCTCN2021104281-appb-000007
Figure PCTCN2021104281-appb-000008
表7 热应激Day8秀丽线虫的剩余数的记录结果
Figure PCTCN2021104281-appb-000009
Day4急性热应激实验结果显示，RV3和RV4多肽组的死亡出现时间和空白组相比没有差异，但RV3多肽组的线虫最长存活时间为32h，比空白组多4h；Day8急性热应激实验结果显示，RV4多肽组的死亡出现时间和空白组相比要相对推迟，且两个给药组的线虫最长存活时间为18h，比空白组多4h，详见表6、表7和图5。
(2)秀丽隐杆线虫热应激实验的半数死亡时间：
表8 热应激Day4秀丽线虫的半数死亡时间的检测结果
Figure PCTCN2021104281-appb-000010
注：以上结果用Median±SE表示；与Control组比，*P<0.05，**P<0.01
表9 热应激Day8秀丽线虫的半数死亡时间的检测结果
Figure PCTCN2021104281-appb-000011
注：以上结果用Median±SE表示；与Control组比，*P<0.05，**P<0.01
Day4急性热应激实验结果显示，RV3多肽组的半数死亡时间与空白组相比，具有显著性差异，而RV4多肽组没有差异性；Day8急性热应激实验结果显示，RV3和RV4多肽组的半数死亡时间与空白组相比，均具有显著性差异，说明RV3和RV4多肽均能显著提高线虫急性热压力应激的能力，在35℃的热压力应激下，给药组的半数死亡时间要比空白组多4-6h。详 见表8、表9和图5，实验结果具有统计学意义。
(3)秀丽隐杆线虫热应激实验的运动能力评级：
表10 热应激Day4秀丽线虫的运动力评级检测结果
Figure PCTCN2021104281-appb-000012
注：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B
表11 热应激Day8秀丽线虫的运动力评级检测结果
Figure PCTCN2021104281-appb-000013
注：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B
Day4热应激运动力评级结果显示，RV3和RV4多肽组中，处于A等级的线虫占比要高于空白组；Day8热应激运动力评级结果显示，给药组中处于C等级的线虫要低于空白组，这说明该多肽药物能够增强秀丽线虫急性热应激的能力，详见表10、表11和图6。
(4)秀丽隐杆线虫氧化应激实验的检测结果：
表12 氧化应激Day4秀丽线虫的存活率检测结果
Figure PCTCN2021104281-appb-000014
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
表13 氧化应激Day8秀丽线虫的存活率检测结果
Figure PCTCN2021104281-appb-000015
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
Day4和Day8氧化应激实验结果显示，RV4多肽组与空白组相比，线虫的存活率具有显著性差异，这说明RV4多肽药物能够显著增强秀丽线虫急性氧化应激的能力。详见表12、表13和图7，实验结果具有统计意义。
(5)秀丽隐杆线虫氧化应激实验的运动能力评级：
表14 氧化应激Day4秀丽线虫的运动力评级检测结果
Figure PCTCN2021104281-appb-000016
注：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B
表15 氧化应激Day8秀丽线虫的运动力评级检测结果
Figure PCTCN2021104281-appb-000017
注：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B
Day8氧化应激运动力评级结果显示，RV3和RV4多肽组中，处于A等级的线虫占比要高于空白组，这说明该多肽药物能够增强秀丽线虫急性氧化应激的能力；Day4氧化应激运动力评级结果中，多肽组和空白组没有运动力上的差异性，详见表14、表15和图8。
实施例6
秀丽隐杆线虫模型的运动能力衰老实验
1.实验材料
同实施例1。
2.实验方法
秀丽隐杆线虫的同步化：同实施例1。
秀丽线虫的运动能力衰老实验前期准备：同实施例1的寿命实验。
秀丽线虫的运动能力衰老实验：分别取连续给药第4天，第8天和第12天的秀丽隐杆线虫，检测线虫的运动行为能力，包括头部摆动频率和咽泵频率，检测方法同实施例4中的运动力检测。同时对存活线虫的运动力进行评级，评级标准同实施例5中的运动力评级。对数据进行One-way ANOVAY统计分析，数据结果以Mean±SEM表示，与Control组对比，*P<0.05为显著性差异，**P<0.01为非常显著性差异。
3.实验结果
(1)秀丽线虫不同生存天数的运动能力评级结果：
表16 不同年龄段的秀丽线虫的运动能力评级结果
Figure PCTCN2021104281-appb-000018
Figure PCTCN2021104281-appb-000019
注：线虫能够自主爬行定义为A，线虫仅在挑针触碰头部后开始挪动定义为C，介于两者状态之间的定义为B
Day8和Day12时间点对线虫运动能力评级结果显示，RV3和RV4多肽组中，处于C等级的线虫占比要低于空白组；RV4多肽组中位于A等级的线虫占比要高于空白组，这意味着，该多肽能够延缓秀丽线虫的肌肉衰老，增加线虫的运动行为能力，从而延长线虫的寿命，详见表16和图9。
(2)秀丽线虫不同生存天数的运动能力检测结果：
表17 不同年龄段的秀丽线虫的运动能力检测结果
Figure PCTCN2021104281-appb-000020
注：以上结果用Mean±SEM表示；与Control组比，*P<0.05，**P<0.01
Day4,Day8和Day12时间点对线虫运动能力检测结果显示，RV4多肽组与空白组相比，头部摆动次数具有显著性差异，而对咽泵频率的影响在第4天时具有显著性差异，在第8天和第12天，咽泵频率非显著性地高于空白组，这意味着，该多肽能够延缓秀丽线虫的肌肉衰老，增加线虫的运动行为能力，延缓线虫行动力的衰退，从而延长线虫的寿命；RV3多肽组和空白组相比没有显著差异性。详见表17和图10，实验结果具有统计意义。

Claims (6)

  1. 一种具有抗衰老活性的多肽，其特征在于：多肽的氨基酸序列为X-TX 1X 2AA或其药学上可以接受的盐；其中X＝H、乙酰基或丙酰基；X 1和X 2：任意氨基酸。
  2. 根据权利要求1所述的一种具有抗衰老活性的多肽，其特征在于X 1X 2为AF或者KA。
  3. 根据权利要求1或2中所述的一种具有抗衰老活性的多肽，其特征在于其氨基酸序列为TAFAA或TKAAA。
  4. 根据权利要求1至3任意一项所述的多肽在制备抗衰老药物或保健品中的应用。
  5. 根据权利要求4所述的应用，其特征在于所述的多肽中还加入一种或多种药学上可接受的辅料，所述辅料包括药学领域常规的稀释剂、填充剂、粘合剂、湿润剂、吸收促进剂、表面活性剂、润滑剂和稳定剂。
  6. 根据权利要求5所述的应用，其特征在于所述的多肽制成注射剂、冻干粉针剂、片剂或颗粒剂。
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