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WO2022266927A1 - 一种利拉鲁肽变构体及其制备方法、应用 - Google Patents

一种利拉鲁肽变构体及其制备方法、应用 Download PDF

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Publication number
WO2022266927A1
WO2022266927A1 PCT/CN2021/102059 CN2021102059W WO2022266927A1 WO 2022266927 A1 WO2022266927 A1 WO 2022266927A1 CN 2021102059 W CN2021102059 W CN 2021102059W WO 2022266927 A1 WO2022266927 A1 WO 2022266927A1
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liraglutide
amino acid
preparation
variant
resin
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French (fr)
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张宝乐
陶志强
尹传龙
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Hybio Pharmaceutical Co Ltd
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Hybio Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to the technical field of polypeptides, in particular to a variant of liraglutide and its preparation method and application.
  • Liraglutide (liraglutide) was developed by Novo Nordisk in Denmark. It was first launched in the European Union in July 2009, and it was launched in Japan and the United States in 2010. Belonging to the glucagon-like peptide-1 (GLP-1) receptor agonist hypoglycemic drugs, it can stimulate the secretion of natural insulin. Its molecular structure is similar to that of GLP-1, the difference is that liraglutide changes the 34th Lys of GLP-1 to Arg, and adds a palmitoyl fatty acid side chain at the 26th position.
  • GLP-1 glucagon-like peptide-1
  • Peptide drugs are easily degraded by proteases in the body, and liraglutide is also easily degraded by DPP-IV enzymes in the human body, resulting in a decrease in bioavailability.
  • liraglutide needs to be injected once a day, which brings great psychological and economic burdens to patients. Therefore, in order to improve medication compliance, it is necessary to modify its structure or develop new dosage forms to improve its biological activity or bioavailability.
  • the purpose of the present invention is to provide a variant of liraglutide and its preparation method and application.
  • the biological activity of the liraglutide variant is significantly improved, which is twice that of the original liraglutide preparation.
  • the present invention provides a variant of liraglutide, the 12th amino acid L-Ser is modified into D-Ser, and the amino acid sequence is shown in SEQ ID NO: 1:
  • the relative potency of the liraglutide variant provided by the present invention is 204% relative to the original research reference preparation, indicating that its biological activity is twice that of liraglutide, and it can be used to prepare hypoglycemic and weight loss drugs and has broad prospects .
  • the present invention also provides the application of the liraglutide variant in the preparation of hypoglycemic drugs.
  • the present invention also provides a preparation method of the liraglutide variant, comprising the following steps:
  • Step 1 Take Fmoc-Gly-resin and add it to the solid-phase reaction column, and swell it with DMF for 30 minutes; weigh the N-terminal protected amino acid, dissolve it in DMF, add a coupling reagent to activate it, add it to the reaction column, and react 1-3 Hour;
  • Step 2 extract the reaction solution, remove the Fmoc protecting group with a mixed solution of piperidine and DMF;
  • Step 3 Repeat the above steps 1 to 2, according to the sequence of the amino acid sequence shown in SEQ ID NO: 1 from the C-terminal to the N-terminal, couple the protected amino acids one by one until the last amino acid in the coupling main chain ends, and obtain the peptide resin;
  • Step 4 cracking the peptide resin to obtain crude peptides, and purifying the crude peptides to obtain refined peptides.
  • the type of resin is not particularly limited, and the types commonly used in the field are sufficient.
  • the resin is Wang resin.
  • the removal agent used to remove the N-terminal Fmoc protecting group in the present invention is preferably a mixed solution of piperidine and DMF , including but not limited to. In some specific embodiments, 20% piperidine in DMF solution is used to remove the Fmoc protecting group; the times of removal are two times, 5 min and 7 min respectively.
  • the coupling agent is DIC/Oxymap pure, and the activation time is 3-5 minutes.
  • step 3 the protected amino acid used for coupling the 12th amino acid of liraglutide is Fmoc-D-Ser(tBu)-OH, and the protected amino acid used for the 20th amino acid is Fmoc-Lys(Pal- Glu-OtBu)-OH.
  • the cracking agent used for cracking is composed of trifluoroacetic acid, purified water, phenol and EDT; the volume ratio of the trifluoroacetic acid, purified water, phenol and EDT is 87.5:5:5 : 2.5.
  • the purification is RP-HPLC purification.
  • the chromatographic columns used for purification include but are not limited to C18 chromatographic columns.
  • the 12th amino acid Ser is modified into a D-type amino acid, and the amino acid sequence is shown in SEQ ID NO:1.
  • the relative potency of the liraglutide variant provided by the present invention is 204% relative to the original research reference preparation, indicating that its biological activity is twice that of liraglutide, and it can be used to prepare hypoglycemic and weight loss drugs and has broad prospects .
  • Fig. 1 shows the dose-response curve graph of biological reference substance (BRPS) and system suitability sample (SSS);
  • Fig. 2 shows the dose-response curve of biological reference substance (BRPS) and liraglutide variant solution (TS);
  • Figure 3 shows a comparison curve of hypoglycemic effects of liraglutide and liraglutide variants
  • Figure 4 shows the comparison curve of the weight loss effect of liraglutide and liraglutide variants.
  • the present invention provides a variant of liraglutide and its preparation method and application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.
  • the reagents and instruments used in the present invention are all common commercial products and can be purchased in the market.
  • the 12th serine is modified into D-type serine, and the amino acid sequence is shown in SED ID NO: 1:
  • the present invention provides a preparation method of the liraglutide variant.
  • the present invention adopts Fmoc solid-phase synthesis strategy, and specific method is as follows:
  • Embodiment 2 biological activity assay experiment
  • sample solvent Take 1.42g of disodium hydrogen phosphate dihydrate, 14g of propylene glycol, and 5.5g of phenol, add water to dissolve to 1L, adjust the pH to 8.15 with 1mol/L sodium hydroxide solution, and filter through 0.2 ⁇ m.
  • Preparation of the liraglutide variant solution Dissolve the liraglutide variant in a solvent to a concentration of 6 mg/ml to obtain a liraglutide variant solution.
  • the liraglutide variant solution was serially diluted to 10 concentration points (from 3.75 nM to 0.0018 nM) using assay buffer. Each diluted sample was analyzed for cAMP content in duplicate wells, one cell negative control sample and one cell basal level sample were analyzed.
  • cAMP standard curve 8 concentration points were prepared from the theoretical cAMP concentration of the standard curve: 0.35nM, 0.69nM, 1.39nM, 2.78nM, 11.13nM, 44.5nM, 89.00nM, 178.00nM.
  • Concentration-response data (DeltaR) were fitted with a 4-parameter logistic model to obtain a cAMP standard curve.
  • the original preparation of liraglutide (JS68L85) was used as the system suitability sample (SSS), and the original preparation of liraglutide (JS68L68) was used as the biological reference sample (BRPS).
  • Biological reference substance (BRPS), system suitability sample (SSS) and liraglutide variant solution (TS) were serially diluted with analysis buffer to 10 concentration points (7.5nM—3.7 ⁇ 10 -3 nM ; 2 ⁇ compound), each concentration was paralleled to 2 wells, sample dilution was carried out at room temperature, and the reaction was carried out within one hour.
  • HEK293-GLP1R cells were provided by Shanghai Heyuan Biotechnology Co., Ltd. for cell resuscitation and subculture for 2-3 days.
  • the cells in the culture medium were taken out from the incubator and the cell confluence was visually inspected under a microscope (the cells accounted for 2% of the culture flask). area) is greater than 80% (cell generation is not greater than P15) can be used in the experiment; transfer the cultured cells into a biological safety cabinet, open the cell culture medium bottle, suck out the culture solution with a pipette gun and discard it.
  • HEK293-GLP1 cells were induced to produce cAMP by comparing liraglutide API solution (ie test sample TS), the amount of cAMP produced was represented by the signal ratio of 665nM/620nM, and a curve was fitted by multiple signal ratios of 665nM/620nM , and get the EC50.
  • BRPS biological reference substance
  • TS liraglutide variant solution
  • the data were collected, analyzed and organized by BioTek SynergyH1 microplate reader. The experimental results are shown in Table 2.
  • mice Male Kunming mice (body weight 26-31 g) were randomly divided into 3 groups, 6 mice in each group, without food or water deprivation before the experiment. Equal volumes of normal saline (10 mL/kg), liraglutide (0.6 mg/kg) and liraglutide variant samples (0.3 mg/kg) were injected subcutaneously in a single dose. Blood was collected at the tip of the tail at 0, 1, 4, 8, 12, 14, and 16 hours after the injection, and the blood glucose was tested using the Johnson & Johnson Wenhao blood glucose meter and supporting test strips.
  • the time-effect curve of the hypoglycemic effect was established, and the biological half-life of the hypoglycemic effect of liraglutide and liraglutide variants was calculated.
  • liraglutide variants have comparable biological half-lives to liraglutide.
  • the dose of liraglutide variants was halved, and the hypoglycemic effect was comparable to that of liraglutide, indicating that the hypoglycemic effect was better.
  • the guinea pigs of the corresponding groups were subcutaneously injected with normal saline (1mL/kg), liraglutide variant (0.6mg/kg), and liraglutide (0.6mg/kg) the next day, with time as the abscissa,
  • the body weight of the guinea pigs was plotted on the ordinate, as shown in Figure 4, during which the body weight of the guinea pigs in the saline group continued to increase.
  • the body weight of liraglutide variants and liraglutide guinea pigs given the same dose for two consecutive times decreased by about 7% and 3%, respectively.
  • the effect on reducing the body weight of guinea pigs is stronger than that of liraglutide.

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Abstract

本发明公开了一种利拉鲁肽变构体及其制备方法、应用。该利拉鲁肽变构体的第12位氨基酸Ser变构为D型氨基酸。本发明提供的利拉鲁肽变构体的生物活性是利拉鲁肽的两倍，可用于制备降糖、减肥药物。

Description

一种利拉鲁肽变构体及其制备方法、应用 技术领域
本发明涉及多肽技术领域，尤其涉及一种利拉鲁肽变构体及其制备方法、应用。
背景技术
利拉鲁肽(liraglutide)是由丹麦诺和诺德研制，于2009年7月首先在欧盟上市，2010年先后在日本和美国上市。属于胰高血糖素样肽-1(GLP-1)受体激动剂类降糖药，能够刺激天然胰岛素的分泌。其分子结构与GLP-1类似，不同之处在于，利拉鲁肽将GLP-1的第34位Lys变为Arg，并在第26位增加了一个棕榈酰脂肪酸侧链。
多肽药物易被体内蛋白酶降解，利拉鲁肽在人体内同样容易被DPP-IV酶降解，导致生物利用度降低。利拉鲁肽作为一种注射剂，需每天注射一次，给患者心理、经济带来很大的负担。因此，为了改善用药依从性，有必要对其结构进行改造或开发新的剂型，提高其生物活性或生物利用度。
发明内容
有鉴于此，本发明目的在于提供一种利拉鲁肽变构体及其制备方法、应用。该利拉鲁肽变构体的生物活性明显提高，为原研利拉鲁肽制剂的两倍。
本发明提供一种利拉鲁肽变构体，其第12位氨基酸L-Ser变构为D-Ser，氨基酸序列如SEQ ID NO:1所示：
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoy-L-γ-Glutamyl-))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH。
本发明提供的利拉鲁肽变构体相对原研参比制剂的相对效价为204％，说明其生物活性是利拉鲁肽的两倍，可用于制备降糖、减肥药物，具有广 阔的前景。
本发明还提供了所述利拉鲁肽变构体在制备降糖药物中的应用。
以及，所述的利拉鲁肽变构体在制备减肥药物中的应用。
本发明还提供了所述利拉鲁肽变构体的制备方法，包括以下步骤：
步骤1：取Fmoc-Gly-树脂加入固相反应柱内，DMF溶胀30分钟；称取N端保护氨基酸，用DMF溶解，加入偶联试剂活化，加入到所述反应柱内，反应1-3小时；
步骤2：抽取反应液，用哌啶和DMF的混合溶液脱除Fmoc保护基；
步骤3：重复上述步骤1～2，按照SEQ ID NO:1所示氨基酸序列C端到N端的顺序，逐一偶联保护氨基酸，直至偶联主链中最后一个氨基酸结束，获得肽树脂；
步骤4：将所述肽树脂裂解得到粗肽，粗肽纯化，获得精肽。
本发明对树脂的种类没有特殊限定，本领域常用的种类即可。在一些具体实施例中，树脂为Wang树脂。
逐一偶联氨基酸时，由于每个氨基酸都有保护基，需要脱除N端保护基再进行偶联，本发明脱除N端Fmoc保护基所采用的脱除剂优选哌啶和DMF的混合溶液，包括但不仅限于此。一些具体实施例中，采用20％哌啶DMF溶液脱除Fmoc保护基；所述脱除的次数为两次，分别为5min和7min。
一些实施方案中，步骤3中，所述偶联剂为DIC/Oxyma pure，活化时间为3～5min。
一些实施方案中，步骤3中，偶联利拉鲁肽第12位氨基酸采用的保护氨基酸为Fmoc-D-Ser(tBu)-OH，第20位氨基酸采用的保护氨基酸为Fmoc-Lys(Pal-Glu-OtBu)-OH。
一些实施方案中，步骤4中，所述裂解用的裂解剂由三氟乙酸、纯化水、苯酚和EDT组成；所述三氟乙酸、纯化水、苯酚和EDT的体积比为87.5：5：5：2.5。
本发明步骤4中，所述纯化为RP-HPLC纯化。纯化采用的色谱柱包括但不仅限于C18色谱柱。
本发明提供的利拉鲁肽变构体，其第12位氨基酸Ser变构为D型氨基酸，氨基酸序列如SEQ ID NO:1所示。本发明提供的利拉鲁肽变构体相对原研参比制剂的相对效价为204％，说明其生物活性是利拉鲁肽的两倍，可用于制备降糖、减肥药物，具有广阔的前景。
附图说明
图1示生物参照品(BRPS)和系统适用性样品(SSS)的剂量反应曲线图；
图2示生物参照品(BRPS)和利拉鲁肽变构体溶液(TS)剂量反应曲线图；
图3示利拉鲁肽和利拉鲁肽变构体降糖效果对比曲线图；
图4示利拉路途和利拉鲁肽变构体减重效果对比曲线图。
具体实施方式
本发明提供了一种利拉鲁肽变构体及其制备方法、应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的试剂、仪器皆为普通市售品，皆可于市场购得。
本发明中，英文缩写的含义如表1所示。
表1
缩写及英文 含义
DMF N,N-二甲基甲酰胺
Fmoc 9-芴氧羰基
DIC N,N-二异丙基碳二亚胺
OxymaPure 2-肟氰乙酸乙酯
CAMP 环磷酸腺苷
EDT 1,2-乙二硫醇
PBS缓冲液 磷酸缓冲盐溶液
EDTA 乙二胺四乙酸
本发明提供的利拉鲁肽变构体，其第12位的丝氨酸变构为D型丝氨酸，氨基酸序列如SED ID NO:1所示：
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-G ly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoy-L-γ-Glutamyl-))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH。
相应的，本发明提供了该利拉鲁肽变构体的制备方法。一些实施方案中，本发明采用Fmoc固相合成策略，具体方法如下：
(1)取Fmoc-Gly-Wang Resin树脂加入固相反应柱内，DMF溶胀30分钟；
(2)称取N端保护氨基酸，用DMF溶解，加入偶联试剂DIC/Oxyma pure，活化3-5分钟，加入反应柱内，反应1-3小时。用茚三酮监测反应，树脂透明，表明反应完全。
(3)抽取反应液，用20％(V/V)哌啶DMF混合溶液分两次脱Fmoc保护，时间分别为5分钟和7分钟，茚三酮检测反应，要求树脂显色。
(4)重复上述步骤，其中12位氨基酸用Fmoc-D-Ser(tBu)-OH，20位氨基酸用Fmoc-Lys(Pal-Glu-OtBu)-OH进行偶联，直至偶联完主链中所有的氨基酸。
(5)得到的肽树脂收缩干燥后，将合成的全保护肽树脂用TFA裂解2-4小时，裂解液用乙醚沉淀，经离心、干燥等步骤，得到粗肽。最后，粗肽用C8色谱柱进行制备纯化得到精肽。
下面结合实施例，进一步阐述本发明：
实施例1：利拉鲁肽变构体的固相合成
取替代度0.3mmol/g的Fmoc-Gly-Wang Resin树脂(50.0g)，加入固相反应柱内，DMF(300mL)洗涤2次，然后DMF(300mL)溶胀30分钟；称取N端保护氨基酸(3eqv)，用DMF(300mL)溶解，加入偶联试剂DIC(3.9qev)/Oxymapure(3.6eqv)，活化3-5分钟，加入反应柱内，反应1-3 小时。用茚三酮监测反应，树脂透明，表明反应完全。抽取反应液，用20％(V/V)哌啶DMF混合溶液(300mL)分两次脱Fmoc保护，时间分别为5分钟和7分钟，茚三酮检测反应，要求树脂显色。重复上述步骤，其中12位氨基酸用Fmoc-D-Ser(tBu)-OH(3eqv)，20位氨基酸用Fmoc-Lys(Pal-Glu-OtBu)-OH(3eqv)进行偶联，直至偶联完主链中所有的氨基酸，甲醇收缩，每次用量(300mL)，干燥后得到的肽树脂127.1g，将合成的全保护肽树脂用(三氟乙酸：纯化水：苯酚：EDT＝87.5：5：5：2.5，v/v)裂解2-4小时，裂解液用乙醚沉淀，沉淀、离心、干燥等步骤，得到粗肽62.1g，经反相制备纯化得到精肽9.8g。
实施例2：生物活性测定实验
样品溶媒的配制：取二水合磷酸氢二钠1.42g、丙二醇14g、苯酚5.5g，加水溶解至1L，用1mol/L氢氧化钠溶液调pH至8.15，经0.2μm过滤即得。
利拉鲁肽变构体溶液制备：使用溶媒溶解利拉鲁肽变构体至浓度为6mg/ml，得到利拉鲁肽变构体溶液。使用分析缓冲液梯度稀释利拉鲁肽变构体溶液至10个浓度点(从3.75nM至0.0018nM)。每个稀释样品以双复孔分析cAMP的含量，分析1份细胞阴性对照样品和1份细胞基底水平样品。
cAMP标准曲线：标准曲线的cAMP理论浓度配制8个浓度点：0.35nM、0.69nM、1.39nM、2.78nM、11.13nM、44.5nM、89.00nM、178.00nM。浓度-反应数据(DeltaR)以4参数logistic模型拟合，得到cAMP标准曲线。
以利拉鲁肽原研制剂(JS68L85)为系统适用性样品(SSS)，利拉鲁肽原研制剂(JS68L68)为生物参照样品(BRPS)。将生物参照品(BRPS)和系统适用性样品(SSS)及利拉鲁肽变构体溶液(TS)分别用分析缓冲液进行梯度稀释为10个浓度点(7.5nM—3.7×10 -3nM；2×化合物)，每个浓度平行2孔，样品稀释在常温进行，需要一个小时内进行反应。
细胞培养：由上海和元生物技术股份有限公司提供HEK293-GLP1R细胞进行细胞复苏传代培养2-3天，从培养箱中取出在培养基的细胞在显 微镜下目测细胞汇合度(细胞占培养瓶的面积)约大于80％时(细胞代次不大于P15)可使用于实验；将培养好的细胞转入生物安全柜中，打开细胞培养基瓶，用移液枪吸出培养液弃去，在每培养瓶(25cm 2)中，加入2-3cm的PBS缓冲液对细胞培养瓶底面进行清洗，取出PBS溶液，加入1-2mL的消化液(胰酶-0.25％EDTA)。在显微镜下观察细胞周围透亮趋于变圆时，加入培养基终止消化并混悬细胞，并将细胞悬液用离心机，转速1000rpm室温离心4min，去上清。加入分析缓冲液重悬细胞，采用细胞计数仪对重悬的细胞进行计数，再根据计数的结果对重悬的细胞液进行稀释制成实验用的细胞液(细胞浓度约4×10 5/mL；细胞活率在95％以上)。
通过比较利拉鲁肽API溶液(即测试样品TS)诱导HEK293-GLP1细胞产生cAMP，产生的cAMP的量值用665nM/620nM的信号比值代表，多个665nM/620nM的信号比值拟合出一条曲线，并得出EC50。
生物参照品(BRPS)和系统适用性样品(SSS)的实验结果见图1。
生物参照品(BRPS)和利拉鲁肽变构体溶液(TS)的剂量反应曲线见图2。
由图1可以看出剂量反应曲线平行，证明系统适应性很好。由图2结果可知，
比较生物参照品和利拉鲁肽变构体样品的EC50得出相对效价，计算剂量反应曲线的平行性以及利拉鲁肽变构体样品的相对效价以评估利拉鲁肽变构体样品的生物活性。相对效价(％)＝(EC50生物参照品/EC50测试样品或系统适用性样品)×100％，此处的EC50值来源于F检验中非线性拟合的结果。数据由BioTek SynergyH1酶标仪采集、经分析及整理，实验结果如表2所示。
表2
Figure PCTCN2021102059-appb-000001
Figure PCTCN2021102059-appb-000002
结果表明，本发明利拉鲁肽变构体相比原研参比制剂BRPS的相对效价为204％，说明本发明利拉鲁肽变构体生物活性是利拉鲁肽的两倍。
实施例3利拉鲁肽变构体降血糖效果
雄性昆明小鼠(体重26～31g)，实验前小鼠不禁食禁水,随机分为3组,每组6只。分别皮下单次注射等体积的生理盐水(10mL/kg)、利拉鲁肽(0.6mg/kg)和利拉鲁肽变构体样品(0.3mg/kg)。注射后0，1，4，8，12，14，16小时尾尖采血，使用美国强生公司稳豪型血糖仪及配套试纸检测血糖。以不同时间点的血糖值为纵坐标，时间为横坐标，建立降血糖作用的时效曲线，计算出利拉鲁肽与利拉鲁肽变构体降血糖作用的生物半衰期。如图3所示，利拉鲁肽变构体与利拉鲁肽具有相当的生物半衰期。利拉鲁肽变构体剂量减半，降糖效果和利拉鲁肽相当，表明降糖效果更佳。
实施例4利拉鲁肽变构体对体重的影响
雄性健康豚鼠15只，每只体重300g左右，分5组，即生理盐水组、利拉鲁肽变构体及利拉鲁肽。隔天分别对相应组别的豚鼠皮下注射生理盐水(1mL/kg)、利拉鲁肽变构体(0.6mg/kg)、利拉鲁肽(0.6mg/kg)，以时间为横坐标，豚鼠体重为纵坐标作图，如图4所示，期间生理盐水组豚鼠体重持续增长。与生理盐水组比较，连续2次给予同等剂量的利拉鲁肽变构体、利拉鲁肽豚鼠体重降低分别为约7％、3％。结果可以看出给予同等剂量的利拉鲁肽变构体，其降低豚鼠体重作用效果比利拉鲁肽强。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种利拉鲁肽变构体，其特征在于，其氨基酸序列如SEQ ID NO:1所示。
  2. 权利要求1所述的利拉鲁肽变构体在制备降糖药物中的应用。
  3. 权利要求1所述的利拉鲁肽变构体在制备减肥药物中的应用。
  4. 权利要求1所述的利拉鲁肽变构体的制备方法，其特征在于，包括以下步骤：
    步骤1：取Fmoc-Gly-树脂加入固相反应柱内，DMF溶胀30分钟；称取N端保护氨基酸，用DMF溶解，加入偶联试剂活化，加入到所述反应柱内，反应1-3小时；
    步骤2：抽取反应液，用哌啶和DMF混合溶液脱除Fmoc保护基；
    步骤3：重复上述步骤1～2，按照SEQ ID NO:1所示氨基酸序列C端到N端的顺序，逐一偶联保护氨基酸，直至偶联主链中最后一个氨基酸结束，获得肽树脂；
    步骤4：将所述肽树脂裂解得到粗肽，粗肽纯化，获得精肽。
  5. 根据权利要求4所述的制备方法，其特征在于，所述树脂为2-氯三苯甲基氯树脂或Wang树脂。
  6. 根据权利要求4所述的制备方法，其特征在于，步骤2中，用20％哌啶DMF溶液脱除Fmoc保护基；所述脱除的次数为两次，分别为5min和7min。
  7. 根据权利要求4所述的制备方法，其特征在于，步骤3中，所述偶联剂为DIC/Oxymapure，活化时间为3～5min。
  8. 根据权利要求4所述的制备方法，其特征在于，步骤3中，偶联利拉鲁肽第12位氨基酸采用的保护氨基酸为Fmoc-D-Ser(tBu)-OH，第20位氨基酸采用的保护氨基酸为Fmoc-Lys(Pal-Glu-OtBu)-OH。
  9. 根据权利要求4所述的制备方法，其特征在于，步骤4中，所述裂解用的裂解剂由三氟乙酸、纯化水、苯酚和EDTA组成；所述三氟乙酸、纯化水、苯酚和EDTA的体积比为87.5：5：5：2.5。
  10. 根据权利要求1～9任一项所述的方法，其特征在于，步骤4中，所述纯化为RP-HPLC纯化。
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