WO2013037267A1 - Variant of liraglutide and conjugate thereof - Google Patents

Variant of liraglutide and conjugate thereof Download PDF

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Publication number
WO2013037267A1
WO2013037267A1 PCT/CN2012/080758 CN2012080758W WO2013037267A1 WO 2013037267 A1 WO2013037267 A1 WO 2013037267A1 CN 2012080758 W CN2012080758 W CN 2012080758W WO 2013037267 A1 WO2013037267 A1 WO 2013037267A1
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Prior art keywords
liraglutide
variant
gly
ala
glu
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PCT/CN2012/080758
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French (fr)
Chinese (zh)
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潘俊锋
刘建
马亚平
袁建成
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深圳翰宇药业股份有限公司
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Publication of WO2013037267A1 publication Critical patent/WO2013037267A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • 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 invention relates to liraglutide variants and conjugates thereof. Background technique
  • Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist developed by Novo Nordisk, Denmark, with GLP in terms of molecular structure, biological activity, target of action and immunogenicity. -1 is similar.
  • the molecular structure of liraglutide is similar to that of GLP-1 (7-36), except that liraglutide replaces lysine at position 34 of lignin with arginine and increases at position 26 A palmitoyl fatty acid side chain.
  • Peptide drugs generally have short in vivo half-life, poor physical and chemical stability, and are easily degraded by various proteases in the body.
  • liraglutide As a subcutaneous injection, liraglutide has a half-life of about 12 to 14 hours. It needs to be used once a day. It can reduce blood sugar, but it imposes a heavy burden on the body, mind and economy, and limits the patient's medication. Compliance. Therefore, it is important to structurally modify liraglutide and develop new dosage forms to prolong its plasma cycle and increase its systemic drug exposure. Summary of the invention
  • the present invention provides a liraglutide variant and a conjugate thereof which have an extended half-life and can effectively lower blood sugar concentration.
  • the present invention first provides a liraglutide variant having an amino acid sequence of:
  • amino acid sequence of the liraglutide variant is:
  • amino acid sequence of the liraglutide variant is:
  • amino acid sequence of the liraglutide variant is:
  • amino acid sequence of the liraglutide variant is:
  • the present invention also provides a method for preparing a liraglutide variant, comprising the following steps: A) 2-CTC resin is washed 2 to 3 times with DMF, and swollen with DMF for 30 minutes;
  • the invention also provides the use of the liraglutide variant in the manufacture of a medicament for lowering blood glucose.
  • the invention also provides the use of the liraglutide variant in the manufacture of a medicament for reducing body weight.
  • the invention also provides a liraglutide variant conjugate comprising the PEG modifying group and the liraglutide variant.
  • the liraglutide variant conjugate provided by the modification of the PEG modifying group can prolong the half life of the liraglutide while retaining the biological activity of the liraglutide, and is not easily degraded by the protease in the body. Increased stability can reduce the number of medications for patients.
  • the molecular weight of the PEG modifying group is between 2 kDa and 20 kDa.
  • one or more PEG modifying groups are conjugated to the liraglutide variant, and the PEG modifying groups may be the same or different.
  • the liraglutide allosteric site at which the PEG modifying group can be modified includes:
  • the PEG modifying group can be conjugated to the liraglutide variant via a Cys residue to achieve specific modification in the form of a thioether;
  • the PEG-modified group used is mPEG-propionaldehyde.
  • Liraglutide variants contain glutamic acid, aspartic acid, glutamic acid and aspartic acid, which are very active modification sites.
  • the first method is to modify the valley with mPEG-NH2 as a raw material. a chain of amino acids or aspartic acid to give Fmoc-Asp(mPE-NH)-OH or
  • the second method is to use allyl ester-protected glutamic acid and aspartame After the peptide chain is assembled, the allyl ester is directly removed, and then coupled with mPEG-NH2 to obtain a PEG modified product; 4) C-terminal gas group modification: The PEG modifying group is PEG-NH2, and PEG is used.
  • -NH2 is coupled with a C-terminal carboxyl group under the action of dicyclohexylcarbodiimide or 1-(3-didecylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain a PEG modified product;
  • the PEG modifying group is selected from the following structures:
  • n represents an integer between 40 and 120;
  • R is selected from H, (C1-C30)alkyl, cyclo(C6-C30)alkyl or phenyl (C6-C30) Lys-PEG2 represents a two-branched PEG with lysine as the nucleus;
  • Glu-PEG2 represents a bi-branched PEG with glutamic acid as the nucleus;
  • Tris-PEG3 represents a tri-branched PEG;
  • Biotin-PEGll represents the end Connect 11 units of PEG to biotin.
  • the PEG modifying group is selected from the group consisting of PEG or mPEG.
  • the present invention also provides a method of preparing the liraglutide variant conjugate, comprising the steps of:
  • the buffer solution is selected from the group consisting of acetic acid-sodium acetate buffer solution, sodium phosphate buffer solution, sodium hydrogencarbonate, EDTA-NH4AC buffer solution or EDTA-sodium phosphate buffer solution.
  • the molar ratio of the PEG modifying group to the liraglutide variant is 3 to 5; the reaction time of the PEG modifying group with the liraglutide variant It is 0.5 ⁇ 12 hours.
  • the invention also provides the use of the liraglutide variant conjugate in the manufacture of a medicament for lowering blood glucose.
  • the invention also provides the use of the liraglutide variant conjugate in the manufacture of a medicament for reducing body weight.
  • the liraglutide variant provided by the present invention retains the biological activity of liraglutide, prolongs the half-life, and provides patients with more choice of drugs.
  • the invention provides a liraglutide variant conjugate according to the modification of the PEG modifying group on the basis of providing the liraglutide variant, and prolongs the biological activity of the liraglutide while prolonging
  • the half-life is not easily degraded by the protease in the body, the stability is improved, and the number of times of administration of the patient can be reduced.
  • the preparation method of the liraglutide variant and the conjugate thereof provided by the invention has the advantages of reducing blood sugar and reducing body weight. The effect is good, and provides more choices for lowering blood sugar medicine and lowering weight preparation, prolonging half-life, improving stability, reducing production cost, and facilitating the reduction of patient burden.
  • Figure 1 is a flow chart showing the solid phase synthesis process of the liraglutide variant of the present invention.
  • Figure 2 is a graph showing the effect of liraglutide and liraglutide variant 5 of the present invention on intracellular cAMP activity in vitro.
  • Figure 3 is a graph showing the effect of liraglutide variant 5 and its conjugate of the present invention on intracellular cAMP activity in vitro.
  • Figure 4 is a graph showing the effect of the hypoglycemic effect of liraglutide and liraglutide variant 5 of the present invention.
  • Figure 5 is a graph showing the effect of the hypoglycemic effect of liraglutide and liraglutide variant 5 conjugate of the present invention.
  • Figure 6 is a graph showing the effect of liraglutide variant 5 of the present invention and its conjugate on body weight. detailed description
  • the present invention discloses a liraglutide variant and a conjugate thereof, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the preparation and use of the liraglutide variants and conjugates thereof of the present invention have been described in terms of preferred embodiments, and it is apparent to those skilled in the art that the methods and methods described herein can be practiced without departing from the scope of the invention.
  • Application The techniques of the present invention are implemented and applied in the context of variations or modifications and combinations.
  • conjugate refers to a product formed by the covalent or non-covalent attachment of a polypeptide or polypeptide variant to a modifying group as described herein, including but not limited to the above. example of.
  • PEG modifying group includes PEG (polyethylene glycol) and polyethylene glycol derivatives as generally described. Since PEG is a polymer and is composed of molecules of different degrees of polymerization within a certain distribution range, the average molecular weight is generally used to indicate the molecular weight of the polymer, and specifically may be a number average molecular weight or a weight average molecular weight.
  • Biotin-PEGn-MAL and MAL-PEG(4)-[PEG(4)-OMe] 3 were purchased from Kangpide Biomedical Technology Co., Ltd., PEG5000, mPEGioooo-MAL, mPEG 20000 -MAL, PEG ⁇ -MAL, Biotin-PEG ⁇ -MAL, Lys-PEG 2 -MAL, Tris-PEG 3 -MAL, PEG 4 . . . . -MAL, mPEG 5 . . . -CH 2 CH 2 CHO, SC-mPEG 1() . . . And mPEG 5 . . . -NH 2 is purchased from Paige Biomedical (Suzhou) Co., Ltd.
  • Example 1 Solid phase synthesis of liraglutide variants.
  • Step 1 2-FTC resin with DMF Wash 2 ⁇ 3 times, swell with DMF for 30 minutes;
  • Step 2 Weigh N-terminal Fmoc-protected amino acid, DIEA as activator, activate for 3 ⁇ 5 minutes, add reaction column for 1 ⁇ 3 hours;
  • Step 3 Use The Fmoc protecting group was removed by a 1:4 piperidine and DMF mixture for 20 minutes. The ninhydrin method was used to detect whether Fmoc was completely removed.
  • Step 4 Repeat first Steps to the third step, according to the amino acid sequence of the liraglutide variant, the corresponding amino acids are coupled one by one, wherein lysine adopts Fmoc-Lys(Alloc)-OH until the last amino acid in the sequence The coupling was completed; the fifth step: removing the lysine Alloc side chain protecting group, coupling Palmitoyl-Glu-OtBu; the obtained peptide resin was washed 3 times with DCM, adding 12 eq of phenylsilane, reacting for 5 minutes, adding 0.8 eq Pd (PPh 3) 4, reacted for 60 minutes with ninhydrin detection method, the resin Color, indicating that Fmoc has been removed; adding 5 eq of Palmitoyl-Glu-OtBu, 5 eq PyBOP, 6 eq HOBt, lOeq DIEA, coupled for 2 hours, shrinking,
  • the liraglutide variants synthesized by the above methods are:
  • the first position of the N-terminus is the liraglutide variant of Cys 1 , and the amino acid sequence is:
  • the 4th position of the N-terminus is the liraglutide variant 2 of Cys, and the amino acid sequence is: 3 ⁇ 4IV-3 ⁇ 4lV-ui9- ⁇ 9-s ⁇ 3-n9i- ⁇ i-J9S--i9S- A-dsv--i9S--iilI-9ilI--iilI- ⁇ 9-ni9-3 ⁇ 4lV-siH Z
  • the 6th position at the C-terminus is the liraglutide variant of D-Ala 12, and the amino acid sequence is:
  • the 1 molar ratio of PEG5000 to liraglutide to 10 is 1 based on the results of several experiments, and the liraglutide variant 1 can be completely modified.
  • Example 3 Preparation of a PEG5000 conjugate of liraglutide variant 2.
  • liraglutide variant 2 5 mg was dissolved in 8 mL of 20 mM sodium phosphate buffer (pH 6.5), and weighed 20 by the ratio of PEG5000 to liraglutide 2 in a molar ratio of 3:1. Mg PEG5000, added to the above solution, properly shake the hook to dissolve PEG5000 and mix well with liraglutide variant 2, react at 25 °C for 2 hours, then use an excess of 0.5 M cysteine solution The reaction was terminated and finally placed at -20 ° C for separation and purification. The reaction was detected with a Waters Model 2695 High Performance Liquid Chromatography and purified to give the target compound.
  • Example 4 Preparation of mPEG 10000 conjugate of liraglutide variant 3.
  • Biotin-PEGn-MAL added to the above solution, properly shaken to dissolve Biotin-PEGn-MAL and mix well with liraglutide variant 4, react at 20 ° C for 2.5 hours, then use an excess of 0.5
  • the reaction was terminated with an M cysteine solution, finally placed at -20 ° C, and then isolated and purified.
  • the reaction was detected and purified using a Waters Model 2695 High Performance Liquid Chromatography to obtain the target compound.
  • Liraglutide was synthesized according to the amino acid sequence of liraglutide according to the synthesis method provided in Example 1.
  • the amino acid sequence of liraglutide is:
  • the cells were collected, the cells were sonicated, and the protein content of the cells was determined by BCA assay.
  • the cAMP enzyme-linked immunoassay kit set up standard groups of different concentrations to establish a standard curve. After the reaction is completed, the light absorption value is measured at a wavelength of 450 nm by a microplate reader, and the light absorption value is read on the standard curve. The corresponding cAMP concentration is calculated, and the cAMP concentration in the sample is finally calculated. The results obtained are shown in Figure 2.
  • Liraglutide increases the cAMP content in PC-12 cells in a dose-dependent manner, increasing the cAMP maximum (E) to 161.2 ⁇ 7.2 pmol/100 g (protein), and the EC value is 1.7 ⁇ 10. M.
  • the effect of liraglutide variant 5 on cAMP in PC-12 cells in vitro is very similar to liraglutide. Among them, liraglutide variant 5 increased the maximum cAMP (E was 152.4 7.8 pmol/100 (protein); EC value was 2.1 ⁇ 10-M. This indicates that cysteine (mercapto) was introduced at the 11th position of the N-terminus. Liraglutide variant 5 retains the biological activity of liraglutide itself.
  • Example 17 Effect of liraglutide variant 5 and its conjugate on intracellular cAMP activity in vitro.
  • PC-12 cells were cultured for 48 hours, the culture solution was discarded, and washed twice with buffer, and 1 mL of buffer containing 1% bovine serum albumin was added to liraglutide variant 5, liraglutide.
  • the PEG2000 conjugate of allosteric 5, the PEG5000 conjugate of liraglutide variant 5, and the PEG20000 conjugate of liraglutide variant 5 were each incubated with a concentration of 100 ⁇ M for half an hour, Discard the medium and add hydrochloric acid to stop the degradation of cAMP by the enzyme.
  • the cells were collected, sonicated, and the protein content of the cells was determined by BCA assay.
  • the instructions of the cAMP enzyme-linked immunoassay kit set up standard groups of different concentrations to establish a standard curve. After the reaction is completed, the light absorption value is measured at a wavelength of 450 nm by a microplate reader, and the light absorption value is read on the standard curve. The corresponding cAMP concentration is calculated, and the cAMP concentration in the sample is finally calculated.
  • PEG2000 to PEG5000 PEGylation modification hardly affects the activity of liraglutide variant 5, while PEG20000 reduces the biological activity of liraglutide variant 5 by about 20%.
  • Example 18 Liraglutide and Liraglutide Variant 5 Hypoglycemic effect aging test.
  • mice Male Kunming mice (body weight 27 ⁇ 32 g), the mice were not fasted and banned before the experiment, and were randomly divided into 3 groups, 6 in each group.
  • the blood glucose level at different time points is the ordinate and the time is the abscissa.
  • the aging curve of hypoglycemic effect is established, and the biological half-life of the hypoglycemic effect of liraglutide and liraglutide variant 5 is calculated.
  • the biological half-lives of the liraglutide variant and liraglutide after cysteine replacement were 11.5 ⁇ 0.2 hours and 11.0 ⁇ 0.2 hours, respectively. This indicates that liraglutide variant 5 and liraglutide have comparable biological half-lives and are slightly prolonged.
  • Example 19 Ageing test for hypoglycemic effect of liraglutide and liraglutide variant 5 conjugate. Male Kunming mice (body weight 22 ⁇ 25 g) were taken. The mice were not fasted and banned before the experiment.
  • the aging curve of hypoglycemic effect is established, and three PEG conjugates of liraglutide variant 5 and liraglutide variant 5 are calculated.
  • the biological half-life of hypoglycemic effects are shown in Figure 5.
  • Liraglutide variant 5, PEG2000 conjugate of liraglutide variant 5, PEG5000 conjugate of liraglutide variant 5 and liraglutide variant The biological half-lives of the PEG 20000 conjugate of Body 5 were 11.5 ⁇ 0.2 hours, 20.4 ⁇ 0.5 hours, 48.3 ⁇ 0.8 hours, and 121.1 ⁇ 4.0 hours, respectively (P > 0.05).
  • the guinea pig body weight loss was about 3%, 5%, 9%, and 12%, respectively.
  • the results showed that after administration of the same dose of liraglutide variant 5 PEG conjugate, its effect on reducing the body weight of guinea pigs was stronger than that of liraglutide variant 5, in which the liraglutide variant 5 PEG20000 The effect of the conjugate on reducing the body weight of guinea pigs was the most significant.

Abstract

Provided in the present invention is a variant of liraglutide and a preparation method thereof, wherein the amino acid sequence of the variant of liraglutide is X1-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-X9-Trp-X10-Val-Arg-Gly-Arg-X11; wherein X1 is His or Cys; X2 is Gly or Cys; X3 is Thr or Cys; X4 is Ser or Cys; X5 is Ser or Cys; X6 is Ser or Cys; X7 is Glu or Cys; X8 is Ala or deleted; X9 is Ala or Cys; X10 is Leu or Cys or D-Ala; X11 is Gly or Cys or Gly-Gly. Also provided in the present invention is a conjugate of the variant of liraglutide and a preparation method thereof. The variant of liraglutide and conjugate thereof provided in the invention retain biological activity of liraglutide, prolong the half-life, and are advantageous for relieving the patient's burden.

Description

利拉鲁肽变构体及其缀合物 本申请要求于 2011 年 9 月 14 日提交中国专利局、 申请号为 201110271343.8、 发明名称为"利拉鲁肽变构体及其缀合物 "的中国专利申请的 优先权， 其全部内容通过引用结合在本申请中。 技术领域  Liraglutide variants and conjugates thereof The present application claims to be filed on September 14, 2011, the Chinese Patent Office, Application No. 201110271343.8, entitled "Lilaglutide Variants and Conjugates" Priority of Chinese Patent Application, the entire contents of which is incorporated herein by reference. Technical field
本发明涉及利拉鲁肽变构体及其缀合物。 背景技术  The present invention relates to liraglutide variants and conjugates thereof. Background technique
利拉鲁肽是由丹麦诺和诺德公司研制的胰高血糖素样肽 -1 ( GLP-1 ) 受体 激动剂， 在分子结构、 生物活性、 作用靶点及免疫原性等方面与 GLP-1相似。 利拉鲁肽的分子结构与 GLP-l(7-36)类似， 不同之处在于： 利拉鲁肽将 GLP-1 的第 34位赖氨酸替换为精氨酸， 并在第 26位增加了一个棕榈酰脂肪酸侧链。  Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist developed by Novo Nordisk, Denmark, with GLP in terms of molecular structure, biological activity, target of action and immunogenicity. -1 is similar. The molecular structure of liraglutide is similar to that of GLP-1 (7-36), except that liraglutide replaces lysine at position 34 of lignin with arginine and increases at position 26 A palmitoyl fatty acid side chain.
多肽类药物普遍存在体内半衰期较短， 物理、化学稳定性较差， 易被体内 各种蛋白酶降解的特性。 利拉鲁肽作为一种皮下注射制剂， 半衰期约 12~14h, 需每天一次使用， 能起到良好的降低血糖作用， 但给患者身体、 心理和经济带 来较大的负担， 限制了患者用药依从性。 因此， 对利拉鲁肽进行结构改造及开 发新的剂型， 从而延长其血浆周期和增加其系统性药物暴露， 具有重要意义。 发明内容  Peptide drugs generally have short in vivo half-life, poor physical and chemical stability, and are easily degraded by various proteases in the body. As a subcutaneous injection, liraglutide has a half-life of about 12 to 14 hours. It needs to be used once a day. It can reduce blood sugar, but it imposes a heavy burden on the body, mind and economy, and limits the patient's medication. Compliance. Therefore, it is important to structurally modify liraglutide and develop new dosage forms to prolong its plasma cycle and increase its systemic drug exposure. Summary of the invention
为解决现有技术中存在的技术问题，本发明提供一种利拉鲁肽变构体及其 缀合物， 延长了半衰期， 可有效地降低血糖浓度。  In order to solve the technical problems existing in the prior art, the present invention provides a liraglutide variant and a conjugate thereof which have an extended half-life and can effectively lower blood sugar concentration.
本发明首先提供一种利拉鲁肽变构体， 其氨基酸序列为：  The present invention first provides a liraglutide variant having an amino acid sequence of:
Xl-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Ly s(N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-X9-Trp-X10-Val-Arg-Gly-Arg- XII； 其中： XI为 His或 Cys; X2为 Gly或 Cys; X3为 Thr或 Cys; X4为 Ser或 Cys; X5为 Ser或 Cys; X6为 Ser或 Cys; X7为 Glu或 Cys; X8为 Ala或删除； X9为 Ala或 Cys; X10为 Leu或 Cys或 D-Ala; Xll为 Gly或 Cys或 Gly-Gly。 采用上述技术方案， 利拉鲁肽变构体保留了利拉鲁肽的生物学活性， 可有 效地降低血糖， 且延长了半衰期， 为患者提供了更多的药物选择余地。 Xl-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Ly s(N-£-(Na-Palmitoyl- Ly-glutamyl))-Glu-Phe-Ile-X9-Trp-X10-Val-Arg-Gly-Arg-XII; wherein: XI is His or Cys; X2 is Gly or Cys; X3 is Thr or Cys; X4 is Ser or Cys; X5 is Ser or Cys; X6 is Ser or Cys; X7 is Glu or Cys; X8 is Ala or deleted; X9 is Ala or Cys; X10 is Leu or Cys or D-Ala; X11 is Gly or Cys or Gly-Gly. Using the above technical solution, the liraglutide variant retains the biological activity of liraglutide, can effectively lower blood sugar, and prolongs the half-life, providing patients with more choice of drugs.
作为本发明的进一步改进， 所述利拉鲁肽变构体的氨基酸序列为：  As a further improvement of the present invention, the amino acid sequence of the liraglutide variant is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly, 所述 X5为 Cys。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly, the X5 is Cys.
作为本发明的进一步改进， 所述利拉鲁肽变构体的氨基酸序列为：  As a further improvement of the present invention, the amino acid sequence of the liraglutide variant is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys (N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gl y, 所述 X8删除。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys (N-£-(Na-Palmitoyl-Ly-glutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gl y, the X8 deletion.
作为本发明的进一步改进， 所述利拉鲁肽变构体的氨基酸序列为：  As a further improvement of the present invention, the amino acid sequence of the liraglutide variant is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly- Arg-Gly, 所述 X10为 D-Ala。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Ly -glutamyl))-Glu-Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly-Arg-Gly, said X10 is D-Ala.
作为本发明的进一步改进， 所述利拉鲁肽变构体的氨基酸序列为：  As a further improvement of the present invention, the amino acid sequence of the liraglutide variant is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly , 所述 X 11为 Gly-Gly。  His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly, wherein X 11 is Gly-Gly.
相应的， 本发明还提供一种利拉鲁肽变构体的制备方法， 包括如下步骤： A ) 2-CTC树脂用 DMF洗涤 2~3次， 用 DMF溶胀 30分钟；  Correspondingly, the present invention also provides a method for preparing a liraglutide variant, comprising the following steps: A) 2-CTC resin is washed 2 to 3 times with DMF, and swollen with DMF for 30 minutes;
B )称取 N端 Fmoc保护的氨基酸， 加入有机碱 DIEA, 活化 3~5分钟， 力口入反应柱反应 1-3小时；  B) Weigh the N-terminal Fmoc-protected amino acid, add the organic base DIEA, activate for 3 to 5 minutes, and force into the reaction column for 1-3 hours;
C )用体积比为 1:4的哌啶和 DMF混合液（ DBLK )脱除 Fmoc保护基 20 分钟， 用茚三酮方法检测 Fmoc是否脱除完全， 树脂有颜色， 表明 Fmoc已脱 除；  C) The Fmoc protecting group was removed by a 1:4 molar ratio of piperidine and DMF (DBLK) for 20 minutes. The ninhydrin method was used to detect whether Fmoc was completely removed, and the resin was colored, indicating that Fmoc had been removed;
D )重复上述步骤 A、 B和 C, 按照所述利拉鲁肽变构体的氨基酸序列， 逐个偶联相应的氨基酸， 其中， 赖氨酸采用 Fmoc-Lys(Alloc)-OH, 直到序列中 最后一个氨基酸偶联结束；  D) repeating the above steps A, B and C, according to the amino acid sequence of the liraglutide variant, the corresponding amino acids are coupled one by one, wherein the lysine adopts Fmoc-Lys(Alloc)-OH until the sequence The last amino acid coupling is over;
E )脱除赖氨酸 Alloc侧链保护基， 偶联上 Palmitoyl-Glu-OtBu, 得到的肽 树脂用 DCM洗 3次，加入 12eq苯基硅烷，反应 5分钟，加入 0.8eq Pd(PPh3)4, 反应 60分钟， 用茚三酮检测， 树脂有颜色， 表明 Fmoc已脱除； 加入 5eq Palmitamide-Glu-OtBu, 5eq HOAt, 5eq HATU, lOeq TMP, 偶联 2小时， 收 缩， 抽干得到利拉鲁肽变构体树脂； E) removal of the lysine Alloc side chain protecting group, coupled with Palmitoyl-Glu-OtBu, the obtained peptide The resin was washed 3 times with DCM, 12 eq of phenylsilane was added, reacted for 5 minutes, 0.8 eq Pd(PPh3)4 was added, and the reaction was carried out for 60 minutes. The reaction was carried out with ninhydrin. The resin was colored, indicating that Fmoc had been removed; 5 eq of Palmitamide- was added. Glu-OtBu, 5eq HOAt, 5eq HATU, lOeq TMP, coupled for 2 hours, contracted, and drained to obtain liraglutide allosteric resin;
F )将得到的利拉鲁肽变构体树脂用 TFA裂解， 反应 1-3小时， 将多肽从 树脂上裂解下来， 同时脱除侧链保护基；  F) cleavage of the obtained liraglutide allosteric resin with TFA, and reacting for 1-3 hours to cleave the polypeptide from the resin while removing the side chain protecting group;
G )对裂解下来的多肽溶液用乙醚沉淀， 得到粗肽， 然后用 C8色语柱， 用常规的流动相洗脱， 收集组分， 冻干得到所需要的利拉鲁肽变构体；  G) precipitating the cleaved polypeptide solution with diethyl ether to obtain a crude peptide, and then using a C8 color column, eluting with a conventional mobile phase, collecting the components, and lyophilizing to obtain the desired liraglutide variant;
H )用 HPLC方法检测其纯度和含量，用二级质谱和 Edman降解分析其序 列。  H) The purity and content were determined by HPLC, and the sequence was analyzed by secondary mass spectrometry and Edman degradation.
本发明还提供所述利拉鲁肽变构体在制备用于降低血糖的药物中的用途。 本发明还提供所述利拉鲁肽变构体在制备用于降低体重的药物中的用途。 本发明还提供一种利拉鲁肽变构体缀合物， 包含所述 PEG修饰基团和所 述利拉鲁肽变构体。  The invention also provides the use of the liraglutide variant in the manufacture of a medicament for lowering blood glucose. The invention also provides the use of the liraglutide variant in the manufacture of a medicament for reducing body weight. The invention also provides a liraglutide variant conjugate comprising the PEG modifying group and the liraglutide variant.
采用上述技术方案， 通过 PEG修饰基团的修饰， 提供的利拉鲁肽变构体 缀合物， 在保留利拉鲁肽生物学活性的同时， 大大延长了半衰期， 不易被体内 的蛋白酶降解， 提高了稳定性， 可以减少患者的用药次数。  By adopting the above technical scheme, the liraglutide variant conjugate provided by the modification of the PEG modifying group can prolong the half life of the liraglutide while retaining the biological activity of the liraglutide, and is not easily degraded by the protease in the body. Increased stability can reduce the number of medications for patients.
作为本发明的进一步改进， 所述 PEG修饰基团的分子量在 2 kDa~20 kDa 之间。  As a further improvement of the present invention, the molecular weight of the PEG modifying group is between 2 kDa and 20 kDa.
作为本发明的进一步改进， 一个或者多个 PEG修饰基团缀合到利拉鲁肽 变构体上， 所述 PEG修饰基团可以相同， 也可以不同。 所述 PEG修饰基团可 以修饰的利拉鲁肽变构体位点包括：  As a further improvement of the present invention, one or more PEG modifying groups are conjugated to the liraglutide variant, and the PEG modifying groups may be the same or different. The liraglutide allosteric site at which the PEG modifying group can be modified includes:
1 ) Cys的修饰： PEG修饰基团可以通过 Cys残基缀合到利拉鲁肽变构体 上， 以硫醚的形式实现特异性修饰；  1) Modification of Cys: The PEG modifying group can be conjugated to the liraglutide variant via a Cys residue to achieve specific modification in the form of a thioether;
2 ) N端氨基的修饰： 采用的 PEG修饰基团为 mPEG-丙醛， mPEG-丙醛的 一个重要特性就是在酸性条件（pH=5.0 ) 下， 与 α-氨基的耦合具有很高的选 择性；  2) Modification of N-terminal amino group: The PEG-modified group used is mPEG-propionaldehyde. An important property of mPEG-propionaldehyde is that it has a high selectivity for coupling with α-amino group under acidic conditions (pH=5.0). Sex
3 )侧链羧基的修饰： 利拉鲁肽变构体含有谷氨酸、 天冬氨酸， 谷氨酸和 天冬氨酸是很有活性的修饰位点。 第一种方法是以 mPEG-NH2为原料修饰谷 氨酸或天冬氨酸侧链， 得到 Fmoc-Asp(mPE-NH)-OH或 3) Modification of side chain carboxyl groups: Liraglutide variants contain glutamic acid, aspartic acid, glutamic acid and aspartic acid, which are very active modification sites. The first method is to modify the valley with mPEG-NH2 as a raw material. a chain of amino acids or aspartic acid to give Fmoc-Asp(mPE-NH)-OH or
Fmoc-Glu(mPE-NH)-OH , 再以这种氨基酸 PEG修饰物为原料直接合成利拉鲁 肽 PEG侧链修饰物； 第二种方法是选用烯丙酯保护的谷氨酸和天冬氨酸， 肽 链组装完毕后直接脱除烯丙酯， 再与 mPEG-NH2偶联， 得到 PEG修饰产物； 4 ) C端氣基的修饰： 采用的 PEG修饰基团为 PEG-NH2, 用 PEG-NH2在 二环己基碳二亚胺或者 1-(3-二曱基氨基丙基) -3-乙基碳化二亚胺盐酸盐作用 下与 C端羧基偶联， 得到 PEG修饰产物；  Fmoc-Glu(mPE-NH)-OH, and directly synthesize liraglutide PEG side chain modification using this amino acid PEG modification; the second method is to use allyl ester-protected glutamic acid and aspartame After the peptide chain is assembled, the allyl ester is directly removed, and then coupled with mPEG-NH2 to obtain a PEG modified product; 4) C-terminal gas group modification: The PEG modifying group is PEG-NH2, and PEG is used. -NH2 is coupled with a C-terminal carboxyl group under the action of dicyclohexylcarbodiimide or 1-(3-didecylaminopropyl)-3-ethylcarbodiimide hydrochloride to obtain a PEG modified product;
5 )组氨酸侧链咪唑基的修饰。  5) Modification of the histidine side chain imidazolyl group.
作为本发明的进一步改进， 所述 PEG修饰基团选自以下结构：  As a further improvement of the present invention, the PEG modifying group is selected from the following structures:
(CH3)2CH-(OCH2CH2)n- , CH3(CH2)m-(OCH2CH2)n- , R-(OCH2CH2)n- , Lys-PEG2 , Glu-PEG2 , Tris-PEG3 , Biotin-PEGll 。 其中， m表示 1~6之 间的整数； n表示 40~120之间的整数； R选自 H、（ C1-C30 )烷基、环（ C6~C30 ) 烷基或苯基 ( C6-C30 )烷基； Lys-PEG2表示以赖氨酸为核的二分支型 PEG; Glu-PEG2表示以谷氨酸为核的二分支型 PEG; Tris-PEG3表示三分支型 PEG; Biotin-PEGll表示末端连接生物素的 11个单元 PEG。 (CH3)2CH-(OCH2CH2)n-, CH3(CH2)m-(OCH2CH2)n-, R-(OCH2CH2)n-, Lys-PEG2, Glu-PEG2, Tris-PEG3, Biotin-PEGll. Wherein m represents an integer between 1 and 6; n represents an integer between 40 and 120; R is selected from H, (C1-C30)alkyl, cyclo(C6-C30)alkyl or phenyl (C6-C30) Lys-PEG2 represents a two-branched PEG with lysine as the nucleus; Glu-PEG2 represents a bi-branched PEG with glutamic acid as the nucleus; Tris-PEG3 represents a tri-branched PEG; Biotin-PEGll represents the end Connect 11 units of PEG to biotin.
作为本发明的进一步改进， 所述 PEG修饰基团选自 PEG或 mPEG。  As a further improvement of the present invention, the PEG modifying group is selected from the group consisting of PEG or mPEG.
相应的， 本发明还提供一种制备所述利拉鲁肽变构体缀合物的方法， 包括 如下步骤：  Accordingly, the present invention also provides a method of preparing the liraglutide variant conjugate, comprising the steps of:
A )称取一定量的所述利拉鲁肽变构体， 溶于适当的緩沖溶液中；  A) weighing a certain amount of the liraglutide variant, dissolved in a suitable buffer solution;
B )按一定的 PEG修饰基团与利拉鲁肽变构体摩尔比， 称取 PEG修饰基团， 加入上述緩沖溶液中， 适当摇匀使 PEG修饰基团溶解并与利拉鲁肽变构体反 应； B) according to a certain PEG modification group and liraglutide variant molar ratio, weigh the PEG modification group, add to the above buffer solution, shake properly to dissolve the PEG modification group and decorate with liraglutide Body reaction
C )用过量的 Cys溶液终止反应， RP-HPLC检测反应并纯化得到目标化合物。  C) The reaction was stopped with an excess of Cys solution, and the reaction was detected by RP-HPLC and purified to obtain the target compound.
作为本发明的进一步改进， 所述緩沖溶液选自醋酸 -醋酸钠緩沖溶液、 磷 酸钠盐緩沖溶液、 碳酸氢钠、 EDTA-NH4AC緩沖溶液或 EDTA-磷酸钠盐緩沖 溶液。  As a further improvement of the present invention, the buffer solution is selected from the group consisting of acetic acid-sodium acetate buffer solution, sodium phosphate buffer solution, sodium hydrogencarbonate, EDTA-NH4AC buffer solution or EDTA-sodium phosphate buffer solution.
作为本发明的进一步改进， 所述 PEG修饰基团与所述利拉鲁肽变构体的 摩尔比为 3~5；所述 PEG修饰基团与所述利拉鲁肽变构体的反应时间为 0.5~12 小时。 本发明还提供所述利拉鲁肽变构体缀合物在制备用于降低血糖的药物中 的用途。 As a further improvement of the present invention, the molar ratio of the PEG modifying group to the liraglutide variant is 3 to 5; the reaction time of the PEG modifying group with the liraglutide variant It is 0.5~12 hours. The invention also provides the use of the liraglutide variant conjugate in the manufacture of a medicament for lowering blood glucose.
本发明还提供所述利拉鲁肽变构体缀合物在制备用于降低体重的药物中 的用途。  The invention also provides the use of the liraglutide variant conjugate in the manufacture of a medicament for reducing body weight.
与现有技术相比， 本发明的有益效果是： 本发明提供的利拉鲁肽变构体保 留了利拉鲁肽的生物学活性， 延长了半衰期， 为患者提供了更多的药物选择余 地； 本发明在提供利拉鲁肽变构体的基础上， 通过 PEG修饰基团的修饰， 提 供的利拉鲁肽变构体缀合物，在保留利拉鲁肽生物学活性的同时，延长了半衰 期， 不易被体内的蛋白酶降解， 提高了稳定性， 可以减少患者的用药次数； 本 发明提供的利拉鲁肽变构体及其缀合物的制备方法筒便，降血糖和降体重的效 果好， 为降低血糖药物和降低体重药物的制备提供了更多的选择，延长了半衰 期， 提高了稳定性， 降低了生产成本， 有利于患者负担的减轻。 附图说明  Compared with the prior art, the beneficial effects of the present invention are: The liraglutide variant provided by the present invention retains the biological activity of liraglutide, prolongs the half-life, and provides patients with more choice of drugs. The invention provides a liraglutide variant conjugate according to the modification of the PEG modifying group on the basis of providing the liraglutide variant, and prolongs the biological activity of the liraglutide while prolonging The half-life is not easily degraded by the protease in the body, the stability is improved, and the number of times of administration of the patient can be reduced. The preparation method of the liraglutide variant and the conjugate thereof provided by the invention has the advantages of reducing blood sugar and reducing body weight. The effect is good, and provides more choices for lowering blood sugar medicine and lowering weight preparation, prolonging half-life, improving stability, reducing production cost, and facilitating the reduction of patient burden. DRAWINGS
图 1为本发明利拉鲁肽变构体的固相合成工艺流程图。  Figure 1 is a flow chart showing the solid phase synthesis process of the liraglutide variant of the present invention.
图 2为本发明利拉鲁肽与利拉鲁肽变构体 5在体外对细胞内 cAMP活性的影 响。  Figure 2 is a graph showing the effect of liraglutide and liraglutide variant 5 of the present invention on intracellular cAMP activity in vitro.
图 3为本发明利拉鲁肽变构体 5及其缀合物在体外对细胞内 cAMP活性的影 响。  Figure 3 is a graph showing the effect of liraglutide variant 5 and its conjugate of the present invention on intracellular cAMP activity in vitro.
图 4为本发明利拉鲁肽与利拉鲁肽变构体 5降血糖作用实效试验。  Figure 4 is a graph showing the effect of the hypoglycemic effect of liraglutide and liraglutide variant 5 of the present invention.
图 5为本发明利拉鲁肽与利拉鲁肽变构体 5缀合物降血糖作用实效试验。 图 6为本发明利拉鲁肽变构体 5及其缀合物对体重的影响。 具体实施方式  Figure 5 is a graph showing the effect of the hypoglycemic effect of liraglutide and liraglutide variant 5 conjugate of the present invention. Figure 6 is a graph showing the effect of liraglutide variant 5 of the present invention and its conjugate on body weight. detailed description
本发明公开了一种利拉鲁肽变构体及其缀合物 ,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 利拉鲁肽变构体及其缀合物的制备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所述的方法和应用进 行改动或适当变更与组合， 来实现和应用本发明技术。 The present invention discloses a liraglutide variant and a conjugate thereof, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The preparation and use of the liraglutide variants and conjugates thereof of the present invention have been described in terms of preferred embodiments, and it is apparent to those skilled in the art that the methods and methods described herein can be practiced without departing from the scope of the invention. Application The techniques of the present invention are implemented and applied in the context of variations or modifications and combinations.
为了使本领域的技术人员更好地理解本发明的技术方案， 下面结合具体 实施例对本发明作进一步的详细说明。  In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.
说明书和权利要求书中所使用的缩写的含义列于下表中：  The meanings of the abbreviations used in the description and claims are listed in the following table:
Figure imgf000008_0001
Figure imgf000008_0001
本文中使用的术语 "缀合物"是指多肽或多肽变体与本文所述的修饰基团共 价或非共价连接后形成的产物， 所述修饰基团包括但不限于上文所述的例子。 本文中使用的术语 "PEG修饰基团"包括一般意义上所说的 PEG (聚乙二醇 ) 和聚乙二醇衍生物。 由于 PEG是聚合物， 是由一定分布范围内的不同聚合度 的分子构成，一般用平均分子量来表示聚合物的分子量， 具体来说可以是数均 分子量或重均分子量。 Biotin-PEGn-MAL和 MAL-PEG(4)-[PEG(4)-OMe]3购自 康肽德生物医药技术有限公司， PEG5000、 mPEGioooo-MAL, mPEG20000-MAL、 PEG誦 -MAL、 Biotin-PEG誦 -MAL、 Lys-PEG2-MAL、 Tris-PEG3-MAL、 PEG4。。。。-MAL、 mPEG5。。。-CH2CH2CHO、 SC-mPEG1()。。。和 mPEG5。。。-NH2均购 自派格生物医药 （苏州 )有限公司。 实施例一 利拉鲁肽变构体的固相合成。 The term "conjugate" as used herein, refers to a product formed by the covalent or non-covalent attachment of a polypeptide or polypeptide variant to a modifying group as described herein, including but not limited to the above. example of. The term "PEG modifying group" as used herein includes PEG (polyethylene glycol) and polyethylene glycol derivatives as generally described. Since PEG is a polymer and is composed of molecules of different degrees of polymerization within a certain distribution range, the average molecular weight is generally used to indicate the molecular weight of the polymer, and specifically may be a number average molecular weight or a weight average molecular weight. Biotin-PEGn-MAL and MAL-PEG(4)-[PEG(4)-OMe] 3 were purchased from Kangpide Biomedical Technology Co., Ltd., PEG5000, mPEGioooo-MAL, mPEG 20000 -MAL, PEG诵-MAL, Biotin-PEG诵-MAL, Lys-PEG 2 -MAL, Tris-PEG 3 -MAL, PEG 4 . . . . -MAL, mPEG 5 . . . -CH 2 CH 2 CHO, SC-mPEG 1() . . . And mPEG 5 . . . -NH 2 is purchased from Paige Biomedical (Suzhou) Co., Ltd. Example 1 Solid phase synthesis of liraglutide variants.
使用 2-氯三苯曱基氯树脂（2-CTC树脂）类型的固相载体， 选用 Fmoc保 护氨基策略合成利拉鲁肽巯基变体， 其步骤为： 第一步： 2-CTC树脂用 DMF 洗涤 2~3次， 用 DMF溶胀 30分钟； 第二步： 称取 N端 Fmoc保护的氨基酸， DIEA为激活剂， 活化 3~5分钟， 加入反应柱反应 1~3小时； 第三步： 用体积 比为 1： 4的哌啶和 DMF混合液脱除 Fmoc保护基 20分钟， 用茚三酮方法检 测 Fmoc是否脱除完全， 树脂有颜色， 表明 Fmoc已脱除； 第四步： 重复第一 步至第三步的过程，按照所述利拉鲁肽变构体的氨基酸序列，逐个偶联相应的 氨基酸， 其中， 赖氨酸采用 Fmoc-Lys(Alloc)-OH, 直到序列中最后一个氨基酸 偶联结束；第五步：脱除赖氨酸 Alloc侧链保护基，偶联上 Palmitoyl-Glu-OtBu; 得到的肽树脂用 DCM洗 3次， 加入 12eq苯基硅烷， 反应 5分钟， 加入 0.8eq Pd(PPh3)4, 反应 60分钟， 用茚三酮方法检测， 树脂有颜色， 表明 Fmoc已脱 除； 加入 5eq Palmitoyl-Glu-OtBu, 5eq PyBOP, 6eq HOBt, lOeq DIEA, 偶联 2小时， 收缩， 抽干得到利拉鲁肽变构体肽树脂； 第六步： 得到的利拉鲁肽变 构体肽树脂用 TFA裂解， 反应 1~3小时， 将多肽从树脂上裂解下来， 同时脱 除侧链保护基； 第七步： 对裂解下来的多肽溶液用乙醚沉淀， 得到粗肽， 然后 用 C8色语柱， 用常规的流动相洗脱， 收集组分， 冻干得到所需要的利拉鲁肽 巯基变体； 第八步： 用 HPLC方法检测其纯度和含量， 用二级质谱和 Edman 降解分析其序列。 The use of a solid phase carrier of the type 2-chlorotriphenylhydrazine chlororesin (2-CTC resin) and the Fmoc-protected amino group strategy to synthesize liraglutide thiol variants are as follows: Step 1: 2-FTC resin with DMF Wash 2~3 times, swell with DMF for 30 minutes; Step 2: Weigh N-terminal Fmoc-protected amino acid, DIEA as activator, activate for 3~5 minutes, add reaction column for 1~3 hours; Step 3: Use The Fmoc protecting group was removed by a 1:4 piperidine and DMF mixture for 20 minutes. The ninhydrin method was used to detect whether Fmoc was completely removed. The resin was colored, indicating that Fmoc had been removed. Step 4: Repeat first Steps to the third step, according to the amino acid sequence of the liraglutide variant, the corresponding amino acids are coupled one by one, wherein lysine adopts Fmoc-Lys(Alloc)-OH until the last amino acid in the sequence The coupling was completed; the fifth step: removing the lysine Alloc side chain protecting group, coupling Palmitoyl-Glu-OtBu; the obtained peptide resin was washed 3 times with DCM, adding 12 eq of phenylsilane, reacting for 5 minutes, adding 0.8 eq Pd (PPh 3) 4, reacted for 60 minutes with ninhydrin detection method, the resin Color, indicating that Fmoc has been removed; adding 5 eq of Palmitoyl-Glu-OtBu, 5 eq PyBOP, 6 eq HOBt, lOeq DIEA, coupled for 2 hours, shrinking, and draining to obtain liraglutide allosteric peptide resin; Step 6: The liraglutide variant peptide resin is cleaved with TFA, and the reaction is carried out for 1 to 3 hours to cleave the polypeptide from the resin while removing the side chain protecting group; Step 7: Precipitating the cleaved polypeptide solution with diethyl ether, The crude peptide is obtained, then eluted with a conventional mobile phase using a C8 color column, and the components are collected and lyophilized to obtain the desired liraglutide thiol variant; Step 8: HPLC method is used to detect the purity and content, The sequence was analyzed by secondary mass spectrometry and Edman degradation.
采用上述方法合成的利拉鲁肽变构体有：  The liraglutide variants synthesized by the above methods are:
1 ) N端第 1位为 Cys的利拉鲁肽变构体 1 , 氨基酸序列为： 1) The first position of the N-terminus is the liraglutide variant of Cys 1 , and the amino acid sequence is:
Cys-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-OH;  Cys-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) ))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-OH;
2 ) N端第 4位为 Cys的利拉鲁肽变构体 2, 氨基酸序列为： ¾IV-¾lV-ui9-^9-s^3-n9i-^i-J9S--i9S- A-dsv--i9S--iilI-9ilI--iilI-^9-ni9-¾lV-siH Z2) The 4th position of the N-terminus is the liraglutide variant 2 of Cys, and the amino acid sequence is: 3⁄4IV-3⁄4lV-ui9-^9-s^3-n9i-^i-J9S--i9S- A-dsv--i9S--iilI-9ilI--iilI-^9-ni9-3⁄4lV-siH Z
Figure imgf000010_0001
Figure imgf000010_0001
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8S.080/ZT0ZN3/X3d .9ζ.εο/ειοζ OAV His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala8S.080/ZT0ZN3/X3d .9ζ.εο/ειοζ OAV His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala
-Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Cys-Val-Arg-Gly-Ar g-Gly-OH; -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Cys-Val-Arg-Gly-Ar g-Gly-OH;
10 ) C端第 8位为 Cys的利拉鲁肽变构体 10, 氨基酸序列为：  10) The 8th position at the C-terminus is the liraglutide variant of Cys 10, and the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Cys-Trp-Leu-Val-Arg-Gly-A rg-Gly-OH; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) ))-Glu-Phe-Ile-Cys-Trp-Leu-Val-Arg-Gly-A rg-Gly-OH;
11 ) C末端添加 Gly的利拉鲁肽变构体 11 , 氨基酸序列为：  11) Adding Cly's liraglutide variant at the C-terminus 11 , the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-L-Y-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly-OH; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-LY -glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly-OH;
12 ) C端第 6位为 D-Ala的利拉鲁肽变构体 12, 氨基酸序列为：  12) The 6th position at the C-terminus is the liraglutide variant of D-Ala 12, and the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly- Arg-Gly-OH; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Ly -glutamyl))-Glu-Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly-Arg-Gly-OH;
13 ) C端第 13位缺失 Ala的利拉鲁肽变构体 13 , 氨基酸序列为：  13) The 13th position of the C-terminus is deleted from the liraglutide variant of Ala 13 , and the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Lys(N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-OH。 实施例二 利拉鲁肽变构体 1的 PEG5000缀合物的制备。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Lys (N-£-(Na-Palmitoyl-Ly-glutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-OH. Example 2 Preparation of a PEG5000 conjugate of liraglutide variant 1.
取 5 mg 利拉鲁肽变构体 1溶解于 8 mL 20 mM的磷酸钠盐緩沖液 (pH 6.5),按 PEG5000与利拉鲁肽变构体 1摩尔比为 3: 1的量称取 20 mg PEG5000, 加入到上述溶液中， 适当摇勾而使 PEG5000溶解并与利拉鲁肽变构体 1混合 均匀， 在 25°C条件下反应 2小时， 然后用过量的 0.5 M半胱氨酸溶液终止反 应， 最后放置于 -20°C下为分离纯化备用。 用 Waters 2695型高效液相色语仪检 测反应并纯化得到目标化合物。 PEG5000与利拉鲁肽变构体 1摩尔比为 3: 1 是根据多次实验结果总结出来的， 可以使利拉鲁肽变构体 1修饰完全。 实施例三 利拉鲁肽变构体 2的 PEG5000缀合物的制备。 Take 5 mg of liraglutide variant 1 dissolved in 8 mL of 20 mM sodium phosphate buffer (pH 6.5), weigh 20% of PEG5000 and liraglutide variant 1 molar ratio of 3:1 Mg PEG5000, added to the above solution, properly shake the hook to dissolve PEG5000 and mix well with liraglutide variant 1, react at 25 ° C for 2 hours, then use an excess of 0.5 M cysteine solution The reaction was terminated and finally placed at -20 ° C for separation and purification. The reaction was detected with a Waters Model 2695 high performance liquid chromatography and purified to give the desired compound. The 1 molar ratio of PEG5000 to liraglutide to 10 is 1 based on the results of several experiments, and the liraglutide variant 1 can be completely modified. Example 3 Preparation of a PEG5000 conjugate of liraglutide variant 2.
取 5 mg 利拉鲁肽变构体 2溶解于 8 mL 20 mM的磷酸钠盐緩沖液 (pH 6.5),按 PEG5000与利拉鲁肽变构体 2摩尔比为 3 : 1的量称取 20 mg PEG5000, 加入到上述溶液中， 适当摇勾而使 PEG5000溶解并与利拉鲁肽变构体 2混合 均匀， 在 25 °C条件下反应 2小时， 然后用过量的 0.5 M半胱氨酸溶液终止反 应， 最后放置于 -20°C下为分离纯化备用。 用 Waters 2695型高效液相色语仪检 测反应并纯化得到目标化合物。 实施例四 利拉鲁肽变构体 3的 mPEG 10000缀合物的制备。  5 mg of liraglutide variant 2 was dissolved in 8 mL of 20 mM sodium phosphate buffer (pH 6.5), and weighed 20 by the ratio of PEG5000 to liraglutide 2 in a molar ratio of 3:1. Mg PEG5000, added to the above solution, properly shake the hook to dissolve PEG5000 and mix well with liraglutide variant 2, react at 25 °C for 2 hours, then use an excess of 0.5 M cysteine solution The reaction was terminated and finally placed at -20 ° C for separation and purification. The reaction was detected with a Waters Model 2695 High Performance Liquid Chromatography and purified to give the target compound. Example 4 Preparation of mPEG 10000 conjugate of liraglutide variant 3.
称取 10 mg 利拉鲁肽变构体 3溶解到水中， 用碳酸氢钠调 pH=7~8 , 加入 Weigh 10 mg of liraglutide variant 3 dissolved in water, adjust pH with sodium bicarbonate = 7~8, add
3当量的 mPEGioooo-MAL,在室温下搅拌 1~5小时，然后用过量的 0.5 M半胱氨 酸溶液终止反应， 最后放置于 -20°C下为分离纯化备用； 用 Waters 2695型高效 液相色语仪检测反应并纯化得到目标化合物， 纯度为 98.5%。 最终产物以 MALDI-TOF-MS (基质辅助激光解吸电离飞行时间质谱）确定分子量。 实施例五 利拉鲁肽变构体 4的 mPEG20000缀合物的制备。 3 equivalents of mPEGioooo-MAL, stir at room temperature for 1-5 hours, then terminate the reaction with an excess of 0.5 M cysteine solution, and finally place at -20 °C for separation and purification; use Waters 2695 high-performance liquid phase The reaction was detected by a color detector and purified to give the target compound with a purity of 98.5%. The final product was molecular weight determined by MALDI-TOF-MS (matrix assisted laser desorption ionization time of flight mass spectrometry). Example 5 Preparation of mPEG20000 conjugate of liraglutide variant 4.
称取 10 mg 利拉鲁肽变构体 4溶解到 EDTA-N Ac緩沖溶液中， 调 ρΗ=6.9 , 加入 5当量的 mPEG^ooo-MAL , 在室温下搅拌 1 ~5小时， 然后用过 量的 0.5 M半胱氨酸溶液终止反应， 最后放置于 -20°C下为分离纯化备用； 用 Waters 2695型高效液相色语仪检测反应并纯化得到目标化合物。 最终产物以 MALDI-TOF-MS确定分子量。 实施例六 利拉鲁肽变构体 4的 PEG30000缀合物的制备。  Weigh 10 mg of liraglutide variant 4 into EDTA-N Ac buffer solution, adjust ρΗ=6.9, add 5 equivalents of mPEG^ooo-MAL, stir at room temperature for 1-5 hours, then use excess The reaction was terminated by a 0.5 M cysteine solution, and finally placed at -20 ° C for separation and purification; the reaction was detected by a Waters Model 2695 high performance liquid chromatography and purified to obtain the target compound. The final product was determined by MALDI-TOF-MS. Example 6 Preparation of a PEG30000 conjugate of liraglutide variant 4.
取 10 mg利拉鲁肽变构体 4溶解于 5 mL的磷酸钠盐緩沖液 (pH=6.5) , 按 PEG30ooo-MAL与 PEG3。。。。-MAL摩尔比为 4: 1的量称取 40 mg PEG3。。。。-MAL, 加入上述溶液中， 适当摇匀而使 PEGsoooo-MAL溶解并与利拉鲁肽变构体 4混 合均匀， 在 20°C条件下反应 2.5小时， 然后用过量的 0.5 M半胱氨酸溶液终止 反应， 最后放置于 -20 °C ,然后分离纯化。 用 Waters 2695型高效液相色谱仪检 测反应并纯化得到目标化合物。 最终产物以 MALDI-TOF-MS确定分子量。 实施例七 利拉鲁肽变构体 4的 Biotin-PEG3∞∞缀合物的制备。 10 mg of liraglutide variant 4 was dissolved in 5 mL of sodium phosphate buffer (pH = 6.5) according to PEG 30 ooo-MAL and PEG 3 . . . . The amount of -MAL molar ratio of 4:1 was weighed 40 mg of PEG 3 . . . . -MAL, added to the above solution, properly shaken to dissolve PEGsoooo-MAL and mix well with liraglutide variant 4, react at 20 ° C for 2.5 hours, then use an excess of 0.5 M cysteine The solution was quenched and finally placed at -20 ° C and then isolated and purified. The reaction was detected by a Waters Model 2695 high performance liquid chromatography and purified to give the target compound. The final product was determined by MALDI-TOF-MS. Example 7 Preparation of a Biotin-PEG 3 conjugate of liraglutide variant 4.
取 10 mg利拉鲁肽变构体 4溶解于 5 mL的磷酸钠盐緩沖液 (pH=6.5), 按 Biotin-PEG3oooo-MAL与利拉鲁肽变构体 4摩尔比为 4: 1的量称取 40 mg Biotin-PEG3oooo-MAL, 加入上述溶液中， 适当摇匀而使 Biotin-PEG30000-MAL 溶解并与利拉鲁肽变构体 4混合均匀，在 20°C条件下反应 2.5小时， 然后用过 量的 0.5 M半胱氨酸溶液终止反应，最后放置于 -20 °C,然后分离纯化。用 Waters 2695型高效液相色语仪检测反应并纯化得到目标化合物。 最终产物以 10 mg of liraglutide variant 4 was dissolved in 5 mL of sodium phosphate buffer (pH=6.5), and the molar ratio of Biotin-PEG 3 oooo-MAL to liraglutide was 4:1. Weigh 40 mg of Biotin-PEG 3 oooo-MAL, add to the above solution, and shake well to dissolve Biotin-PEG 30000 -MAL and mix well with liraglutide variant 4 at 20 °C. The reaction was allowed to proceed for 2.5 hours, and then the reaction was stopped with an excess of 0.5 M cysteine solution, finally placed at -20 ° C, and then isolated and purified. The reaction was detected with a Waters Model 2695 high performance liquid chromatography and purified to give the desired compound. Final product
MALDI-TOF-MS确定分子量。 实施例八利拉鲁肽变构体 4的 Biotin-PEGu缀合物的制备。 The molecular weight was determined by MALDI-TOF-MS. Example 8 Preparation of a Biotin-PEG u conjugate of liraglutide variant 4.
取 10 mg利拉鲁肽变构体 4溶解于 5mL的磷酸钠盐緩沖液 (pH=6.5), 按 Biotin-PEGn-MAL与利拉鲁肽变构体 4摩尔比为 4: 1的量称取 20 mg  10 mg of liraglutide variant 4 was dissolved in 5 mL of sodium phosphate buffer (pH = 6.5), and the molar ratio of Biotin-PEGn-MAL to liraglutide 4 was 4:1. Take 20 mg
Biotin-PEGn-MAL, 加入上述溶液中， 适当摇匀而使 Biotin-PEGn-MAL溶解 并与利拉鲁肽变构体 4混合均匀，在 20°C条件下反应 2.5小时， 然后用过量的 0.5 M半胱氨酸溶液终止反应，最后放置于 -20 °C,然后分离纯化。用 Waters 2695 型高效液相色语仪检测反应并纯化得到目标化合物。 最终产物以 Biotin-PEGn-MAL, added to the above solution, properly shaken to dissolve Biotin-PEGn-MAL and mix well with liraglutide variant 4, react at 20 ° C for 2.5 hours, then use an excess of 0.5 The reaction was terminated with an M cysteine solution, finally placed at -20 ° C, and then isolated and purified. The reaction was detected and purified using a Waters Model 2695 High Performance Liquid Chromatography to obtain the target compound. Final product
MALDI-TOF-MS确定分子量为 4689.2。 实施例九利拉鲁肽变构体 6的二分支型 PEG2缀合物的制备。 The molecular weight determined by MALDI-TOF-MS was 4689.2. Example 9 Preparation of a bi-branched PEG 2 conjugate of liraglutide variant 6.
称取 10 mg 利拉鲁肽变构体 6溶解到 EDTA-磷酸盐緩沖溶液中， 调 pH=6.5 , 加入 5当量的 Lys-PEG2-MAL (分子量为 20kDa )，在室温下搅拌 1~5 小时， 用 Waters 2695型高效液相色谱仪检测反应， 反应结束后， 加入过量的 0.5 M半胱氨酸溶液以终止反应， 并纯化得到目标化合物。 最终产物以 Weigh 10 mg of liraglutide variant 6 into EDTA-phosphate buffer solution, adjust pH=6.5, add 5 equivalents of Lys-PEG 2 -MAL (molecular weight 20kDa), stir at room temperature for 1~5 The reaction was detected by a Waters Model 2695 high performance liquid chromatography. After completion of the reaction, an excess of 0.5 M cysteine solution was added to terminate the reaction, and the target compound was purified. Final product
MALDI-TOF-MS确定分子量。 实施例十 利拉鲁肽变构体 7三分支型 PEG3缀合物的制备。 The molecular weight was determined by MALDI-TOF-MS. Example 10. Preparation of ten liraglutide variant 7 tri-branched PEG 3 conjugate.
称取 10 mg 利拉鲁肽变构体 7溶解到 EDTA-磷酸盐緩沖溶液中， 调 pH=6.5 ,加入 5当量的 Tris-PEG3-MAL (分子量为 30kDa ), 在室温下搅拌 1~5 小时， 用 Waters 2695型高效液相色谱仪检测反应， 反应结束后， 加入过量的 0.5 M半胱氨酸溶液以终止反应， 并纯化得到目标化合物。 最终产物以 Weigh 10 mg of liraglutide variant 7 and dissolve it into EDTA-phosphate buffer solution, adjust pH=6.5, add 5 equivalents of Tris-PEG 3 -MAL (molecular weight 30kDa), stir at room temperature for 1~5 The reaction was detected by a Waters Model 2695 high performance liquid chromatography. After completion of the reaction, an excess of 0.5 M cysteine solution was added to terminate the reaction, and the target compound was purified. Final product
MALDI-TOF-MS确定分子量。 实施例十一 利拉鲁肽变构体 7的 PEG(4)-[PEG(4)-OMe]3缀合物的合成。 取 10 mg利拉鲁肽变构体 7溶解于 15 mL的磷酸钠盐緩沖液 (pH=6.5),按 MAL-PEG(4)-[PEG(4)-OMe]3与肽摩尔比为 5: 1的量称取 40 mg The molecular weight was determined by MALDI-TOF-MS. Synthesis of the PEG(4)-[PEG(4)-OMe] 3 conjugate of Example 11 liraglutide variant. 10 mg of liraglutide variant 7 was dissolved in 15 mL of sodium phosphate buffer (pH=6.5), and the molar ratio of MAL-PEG(4)-[PEG(4)-OMe] 3 to peptide was 5. : 1 amount weighed 40 mg
MAL-PEG(4)-[PEG(4)-OMe]3 , 加入上述溶液， 适当摇匀而使 PEG溶解并与肽 混合均匀， 在 20°C条件下反应 3.0小时， 然后用过量的 0.5 M半胱氨酸溶液终 止反应，最后放置于 -20°C,然后分离纯化。用 Waters 2695型高效液相色语仪检 测反应并纯化得到目标化合物。 最终产物以 MALDI-TOF-MS确定分子量为 5100.6。 实施例十二 利拉鲁肽变构体 8的 PEG40000缀合物的制备。 MAL-PEG(4)-[PEG(4)-OMe] 3 , add the above solution, shake well to dissolve PEG and mix well with the peptide, react at 20 ° C for 3.0 hours, then use an excess of 0.5 M The cysteine solution was quenched and finally placed at -20 ° C and then isolated and purified. The reaction was detected with a Waters Model 2695 high performance liquid chromatography and purified to give the desired compound. The final product was determined to have a molecular weight of 5100.6 by MALDI-TOF-MS. Example 12 Preparation of a PEG40000 conjugate of liraglutide variant 8 .
称取 10 mg 利拉鲁肽变构体 8溶解到 EDTA-磷酸盐緩沖溶液中， 调 pH=6.5 ,加入 5当量的 PEG40000-MAL,在室温下搅拌 1~5小时，用 Waters 2695 型高效液相色谱仪检测反应， 反应结束后， 加入过量的 0.5 M半胱氨酸溶液以 终止反应， 用 RP-HPLC纯化得到目标化合物。 最终产物以 MALDI-TOF-MS 确定分子量。 实施例十三利拉鲁肽变构体 11的 mPEG5000缀合物的制备（ N端修饰）。 称取 5 mg利拉鲁肽变构体 11溶解到水中， 加入到醋酸-醋酸钠緩沖溶液 调 pH=5.0, 加入 3当量的 mPEG5000-CH2CH2CHO, 在室温下搅拌 2.5小时， 然 后用过量的 0.5 M半胱氨酸溶液终止反应， 最后放置于 -20°C下为分离纯化备 用； 用 Waters 2695型高效液相色语仪检测反应并纯化得到目标化合物。 最终 产物以 MALDI-TOF-MS确定分子量。 实施例十四 利拉鲁肽变构体 12的 mPEG 10000缀合物的制备 ( N端修 饰）。 称取 8 mg利拉鲁肽变构体 12溶解到水中， 用 0.2 M PBS调 pH=6.8, 加 入 5当量 SC-mPEG10000 ( mPEG-琥珀酰亚胺碳酸酯 ) 50mg, 在室温反应 3小 时， 然后用过量的 0.5 M半胱氨酸溶液终止反应， 最后放置于 -20°C下为分离 纯化备用， 2小时后用 HiPrep 26/10脱盐柱脱盐处理， 所用緩沖液体系为 pH 为 8.5的 25 mM Tris。 脱盐以后采用 Waters 2695型高效液相色谱仪纯化得到 目标化合物。 实施例十五 利拉鲁肽变构体 13的 mPEG5000缀合物的制^ C端修饰）。 取 5 mg 利拉鲁肽变构体 13溶解于 10 mL 20mM的磷酸钠盐緩沖液 (pH 6.0), 按 mPEG5。。。-NH2与利拉鲁肽变构体摩尔比为 4: 1的量称取 20 mg mPEG50oo-NH2 , 加入到上述溶液， 适当摇匀而使 mPEG50oo-NH2溶解并与利拉 鲁肽变构体 13混合均匀，在室温条件下反应 3.5小时，然后用过量的 0.5 M半 胱氨酸溶液终止反应， 最后放置于 -20 °C下为分离纯化备用。 用 Waters 2695 型高效液相色谱仪检测反应并纯化得到目标化合物。 实施例十六 利拉鲁肽与利拉鲁肽变构体 5在体外对细胞内 cAMP活性的 影响。 Weigh 10 mg of liraglutide variant 8 and dissolve it into EDTA-phosphate buffer solution, adjust pH=6.5, add 5 equivalents of PEG 40000 -MAL, stir at room temperature for 1-5 hours, and use Waters 2695 type efficiently. The reaction was detected by a liquid chromatograph. After completion of the reaction, an excess of 0.5 M cysteine solution was added to terminate the reaction, and purification was carried out by RP-HPLC to give the title compound. The final product was molecular weight determined by MALDI-TOF-MS. Example 13 Preparation of mPEG5000 conjugate of liraglutide variant 11 (N-terminal modification). Weigh 5 mg of liraglutide variant 11 into water, add to acetic acid-sodium acetate buffer solution to adjust pH=5.0, add 3 equivalents of mPEG 5000 -CH 2 CH 2 CHO, stir at room temperature for 2.5 hours, then The reaction was stopped with an excess of 0.5 M cysteine solution, and finally placed at -20 ° C for separation and purification; the reaction was detected by a Waters Model 2695 high performance liquid chromatography and purified to obtain the target compound. The final product was determined by MALDI-TOF-MS. Example 14 Preparation of mPEG 10000 conjugate of liraglutide variant 12 (N-terminal modification). Weigh 8 mg of liraglutide variant 12 into water, adjust pH=6.8 with 0.2 M PBS, add 5 equivalents of SC-mPEG 10000 (mPEG-succinimide carbonate) 50 mg, and react at room temperature for 3 hours. Then the reaction was stopped with an excess of 0.5 M cysteine solution, and finally placed at -20 ° C for separation and purification. After 2 hours, it was desalted by HiPrep 26/10 desalting column. The buffer system used was pH 8.5. mM Tris. After desalting, the target compound was purified by Waters Model 2695 high performance liquid chromatography. Example 15 Preparation of the mPEG5000 conjugate of liraglutide variant 13 for the C-terminal modification). 5 mg of liraglutide variant 13 was dissolved in 10 mL of 20 mM sodium phosphate buffer (pH 6.0), followed by mPEG 5 . . . -NH 2 and liraglutide allosteric molar ratio of 4: 1 weighed 20 mg mPEG 50 oo-NH 2 , added to the above solution, shake properly to make mPEG 50 oo-NH 2 dissolve and benefit The larupeptide variant 13 was uniformly mixed, and reacted at room temperature for 3.5 hours, and then the reaction was terminated with an excess of 0.5 M cysteine solution, and finally placed at -20 ° C for separation and purification. The reaction was detected by a Waters Model 2695 high performance liquid chromatography and purified to give the desired compound. Example 16 Effect of Liraglutide and Liraglutide Variant 5 on Intracellular cAMP Activity in Vitro.
根据实施例一中提供的合成方法，按照利拉鲁肽的氨基酸序列合成利拉鲁 肽。 利拉鲁肽的氨基酸序列为：  Liraglutide was synthesized according to the amino acid sequence of liraglutide according to the synthesis method provided in Example 1. The amino acid sequence of liraglutide is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-Lys(N-£-(N-a-Palmitoyl-L-Y-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly -Arg-Gly-OH; 将 PC-12细胞（肾上腺嗜铬细胞瘤细胞 )培养 48小时， 弃去培 养液， 并用緩沖液清洗 2~3次， 加入 1 mL含有 1%牛血清蛋白的緩沖液， 将 利拉鲁肽和利拉鲁肽变构体 5分别与浓度为 100 μΜ 的 ΙΒΜΧ共同孵育半小 时， 弃去培养基， 加入盐酸终止酶对 cAMP (环腺苷酸） 的降解。 收集细胞， 超声裂解细胞， 按 BCA检测法测定细胞蛋白含量。 按 cAMP酶联免疫试剂盒 说明进行操作，设立不同浓度的标准品组以建立标准曲线，反应完成后在酶标 仪于 450 nm波长下测定光吸收值， 用该光吸收值在标准曲线上读出对应的 cAMP浓度， 最后计算样品中 cAMP浓度。 所得结果如图 2所示， 利拉鲁肽呈剂量依赖式地增加 PC- 12细胞内 cAMP含 量，增加 cAMP最大值（ E )为 161.2士 7.2 pmol/100 g(蛋白）， EC 值为 1.7x10 M。利拉鲁肽变构体 5在体外对 PC-12细胞内 cAMP的影响与利拉鲁肽非常相似。 其中利拉鲁肽变构体 5增加 cAMP最大值（E 为 152.4士 7.8 pmol/100 (蛋 白）； EC 值为 2.1x10— M。 说明在 N端第 11位引入半胱氨酸（巯基）的利拉鲁 肽变构体 5保留了利拉鲁肽本身的生物学活性。 实施例十七 利拉鲁肽变构体 5及其缀合物在体外对细胞内 cAMP活性的 影响。 Ala-Lys(N-£-(Na-Palmitoyl-LY-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; PC-12 cells (adrenal gland) Pheochromocytoma cells were cultured for 48 hours, the culture solution was discarded, and washed twice with buffer, and 1 mL of buffer containing 1% bovine serum albumin was added to transform liraglutide and liraglutide into a variant. 5 Incubate with 100 μΜ of cesium for half an hour, discard the medium, and add hydrochloric acid to stop the degradation of cAMP (cyclic adenosine). The cells were collected, the cells were sonicated, and the protein content of the cells was determined by BCA assay. According to the instructions of the cAMP enzyme-linked immunoassay kit, set up standard groups of different concentrations to establish a standard curve. After the reaction is completed, the light absorption value is measured at a wavelength of 450 nm by a microplate reader, and the light absorption value is read on the standard curve. The corresponding cAMP concentration is calculated, and the cAMP concentration in the sample is finally calculated. The results obtained are shown in Figure 2. Liraglutide increases the cAMP content in PC-12 cells in a dose-dependent manner, increasing the cAMP maximum (E) to 161.2 ± 7.2 pmol/100 g (protein), and the EC value is 1.7×10. M. The effect of liraglutide variant 5 on cAMP in PC-12 cells in vitro is very similar to liraglutide. Among them, liraglutide variant 5 increased the maximum cAMP (E was 152.4 7.8 pmol/100 (protein); EC value was 2.1×10-M. This indicates that cysteine (mercapto) was introduced at the 11th position of the N-terminus. Liraglutide variant 5 retains the biological activity of liraglutide itself.Example 17 Effect of liraglutide variant 5 and its conjugate on intracellular cAMP activity in vitro.
将 PC-12细胞培养 48小时， 弃去培养液， 并用緩沖液清洗 2~3次， 加入 l mL含有 1%牛血清蛋白的緩沖液， 将利拉鲁肽变构体 5、 利拉鲁肽变构体 5 的 PEG2000缀合物、 利拉鲁肽变构体 5的 PEG5000缀合物、 利拉鲁肽变构体 5的 PEG20000缀合物分别与浓度为 100 μΜ 的 ΙΒΜΧ共同孵育半小时，弃去 培养基，加入盐酸终止酶对 cAMP的降解。 收集细胞，超声裂解细胞，按 BCA 检测法测定细胞蛋白含量。按 cAMP酶联免疫试剂盒说明进行操作，设立不同 浓度的标准品组以建立标准曲线， 反应完成后在酶标仪于 450 nm波长下测定 光吸收值，用该光吸收值在标准曲线上读出对应的 cAMP浓度，最后计算样品 中 cAMP浓度。  PC-12 cells were cultured for 48 hours, the culture solution was discarded, and washed twice with buffer, and 1 mL of buffer containing 1% bovine serum albumin was added to liraglutide variant 5, liraglutide. The PEG2000 conjugate of allosteric 5, the PEG5000 conjugate of liraglutide variant 5, and the PEG20000 conjugate of liraglutide variant 5 were each incubated with a concentration of 100 μM for half an hour, Discard the medium and add hydrochloric acid to stop the degradation of cAMP by the enzyme. The cells were collected, sonicated, and the protein content of the cells was determined by BCA assay. According to the instructions of the cAMP enzyme-linked immunoassay kit, set up standard groups of different concentrations to establish a standard curve. After the reaction is completed, the light absorption value is measured at a wavelength of 450 nm by a microplate reader, and the light absorption value is read on the standard curve. The corresponding cAMP concentration is calculated, and the cAMP concentration in the sample is finally calculated.
所得结果如图 3所示 ， 实验结果表明， 利拉鲁肽变构体 5剂量依赖式地 增加 PC12细胞内 cAMP含量， 其增加 cAMP最大值（E ) 为 103.9士 1.5  The results are shown in Figure 3. The results of the experiment showed that the liraglutide variant 5 dose-dependently increased the cAMP content in PC12 cells, which increased the cAMP maximum (E) to 103.9 ± 1.5.
_9  _9
pmol/100 (蛋白）， EC 值为 1.3x10 M。 PEG化修饰呈分子量（ 2000-20000 ) -依赖式地平行右移量效关系曲线，降低利拉鲁肽变构体 5生物活性。随着 PEG 分子量的增大， 它们 EC 值分别为 1.1x10 , 1.2x10 ，和 1.3x10 M。 其中 PEG2000 到 PEG5000 PEG化修饰几乎不影响利拉鲁肽变构体 5的活性， 而 PEG20000则降低了利拉鲁肽变构体 5的生物活性约 20%。 实施例十八利拉鲁肽与利拉鲁肽变构体 5降血糖作用时效试验。 Pmol/100 (protein) with an EC value of 1.3x10 M. The PEGylation modification showed a molecular weight (2000-20000)-dependently parallel right shift dose-effect curve, which reduced the biological activity of liraglutide variant 5. As the molecular weight of PEG increases, their EC values are 1.1x10, 1.2x10, and 1.3x10 M, respectively. Among them, PEG2000 to PEG5000 PEGylation modification hardly affects the activity of liraglutide variant 5, while PEG20000 reduces the biological activity of liraglutide variant 5 by about 20%. Example 18 Liraglutide and Liraglutide Variant 5 Hypoglycemic effect aging test.
雄性昆明小鼠（体重 27~32 g ) , 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（ 10 mL/kg )、 利拉鲁肽（ 0.6 mg/kg )和利拉鲁肽变构体 5 ( 0.6 mg/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 5降血糖作用的生物半衰期。经半胱氨酸置换后 的利拉鲁肽变构体与利拉鲁肽的生物半衰期分别为 11.5±0.2小时和 11.0±0.2 小时。 表明利拉鲁肽变构体 5与利拉鲁肽具有相当的生物半衰期， 略有延长。 实施例十九利拉鲁肽与利拉鲁肽变构体 5缀合物降血糖作用时效试验。 取雄性昆明小鼠（体重 22~25 g ) , 实验前小鼠不禁食禁水，随机分为 4组， 每组 6只。 分别皮下单次注射利拉鲁肽变构体 5 ( 10 g/kg )、 利拉鲁肽变构体 5的 PEG2000缀合物 （ 10 g/kg )、 利拉鲁肽变构体 5的 PEG5000缀合物 （ 10 g/kg )和利拉鲁肽变构体 5的 PEG20000缀合物（ 12 g/kg )。 注射后 0, 1 , 2 , 4, 8 , 12, 18, 24, 36, 48, 72, 96, 120, 148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，计算出利拉鲁肽变构体 5和利拉 鲁肽变构体 5的三种 PEG缀合物降血糖作用的生物半衰期。结果如图 5所示， 利拉鲁肽变构体 5、 利拉鲁肽变构体 5的 PEG2000缀合物、 利拉鲁肽变构体 5 的 PEG5000缀合物和利拉鲁肽变构体 5的 PEG20000缀合物的生物半衰期分 别为 11.5±0.2小时、 20.4 ±0.5小时、 48.3 ±0.8小时和 121.1±4.0小时 (P > 0.05)。 等效剂量的时效关系研究结果表明， 聚乙二醇化（ PEG20000 )利拉鲁肽变构 体 5延长利拉鲁肽变构体 5降血糖作用时间达 11倍左右。 实施例二十 利拉鲁肽变构体 5及其缀合物对体重的影响。 Male Kunming mice (body weight 27~32 g), the mice were not fasted and banned before the experiment, and were randomly divided into 3 groups, 6 in each group. A single injection of the same volume of normal saline (10 mL / kg), liraglutide (0.6 Mg/kg) and liraglutide variant 5 (0.6 mg/kg). 0, 1 , 4, 8, 12, 14, 16 hours after the injection, blood was collected from the tail tip, and blood glucose was measured using the Johnson & Johnson Stable Blood Glucose Meter and supporting test paper. The blood glucose level at different time points is the ordinate and the time is the abscissa. The aging curve of hypoglycemic effect is established, and the biological half-life of the hypoglycemic effect of liraglutide and liraglutide variant 5 is calculated. The biological half-lives of the liraglutide variant and liraglutide after cysteine replacement were 11.5 ± 0.2 hours and 11.0 ± 0.2 hours, respectively. This indicates that liraglutide variant 5 and liraglutide have comparable biological half-lives and are slightly prolonged. Example 19 Ageing test for hypoglycemic effect of liraglutide and liraglutide variant 5 conjugate. Male Kunming mice (body weight 22~25 g) were taken. The mice were not fasted and banned before the experiment. They were randomly divided into 4 groups, 6 in each group. Separate subcutaneous injection of liraglutide variant 5 (10 g/kg), liraglutide variant 5 PEG2000 conjugate (10 g/kg), liraglutide variant 5 PEG5000 Conjugate (10 g/kg) and PEG20000 conjugate of liraglutide variant 5 (12 g/kg). After the injection, 0, 1 , 2, 4, 8 , 12, 18, 24, 36, 48, 72, 96, 120, 148 hours of blood collection at the tip of the tail, blood glucose was measured using the Johnson & Johnson Stable Blood Glucose Meter and supporting test paper. The blood glucose values at different time points are plotted on the ordinate and the time is plotted on the abscissa. The aging curve of hypoglycemic effect is established, and three PEG conjugates of liraglutide variant 5 and liraglutide variant 5 are calculated. The biological half-life of hypoglycemic effects. The results are shown in Figure 5. Liraglutide variant 5, PEG2000 conjugate of liraglutide variant 5, PEG5000 conjugate of liraglutide variant 5 and liraglutide variant The biological half-lives of the PEG 20000 conjugate of Body 5 were 11.5 ± 0.2 hours, 20.4 ± 0.5 hours, 48.3 ± 0.8 hours, and 121.1 ± 4.0 hours, respectively (P > 0.05). The time-effect relationship study of equivalent doses showed that PEGylated (PEG20000) liraglutide variant 5 prolonged the hypoglycemic effect of liraglutide variant 5 by about 11 times. Example 20 Effect of liraglutide variant 5 and its conjugate on body weight.
雄性健康豚鼠 20只， 每只体重 300 g左右， 分 5组， 即生理盐水组、 利 拉鲁肽变构体 5、利拉鲁肽变构体 PEG2000缀合物、利拉鲁肽变构体 5 PEG5000 缀合物和利拉鲁肽变构体 5 PEG20000缀合物。 隔天分别对相应组别的豚鼠皮 下注射生理盐水（1 mL/kg)、 利拉鲁肽变构体 5 (0.6 mg/kg)、 利拉鲁肽变构体 PEG2000缀合物 (0.6 mg/kg)、 利拉鲁肽变构体 5 PEG5000缀合物 (0.6 mg/kg)和 利拉鲁肽变构体 5 PEG20000缀合物 (0.6 mg/kg) , 以时间为横坐标， 豚鼠体重 为纵坐标作图， 如图 6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 5、 利拉鲁肽变构体 5 PEG2000缀合物、 利拉鲁肽变构体 5 PEG5000缀合物和利拉鲁肽变构体 5 PEG20000缀合物 5周后， 豚鼠体重降低分别为约 3%、 5%、 9%和 12%、。 结 果可以看出给予同等剂量的利拉鲁肽变构体 5 PEG缀合物后， 其降低豚鼠体 重作用效果比利拉鲁肽变构体 5强， 其中以利拉鲁肽变构体 5 PEG20000缀合 物降低豚鼠体重作用效果最显著。 以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不 能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通 技术人员来说，在不脱离本发明构思的前提下，还可以做出若干筒单推演或替 换， 都应当视为属于本发明的保护范围。 20 healthy male guinea pigs, each weighing about 300 g, divided into 5 groups, namely saline group, liraglutide variant 5, liraglutide variant PEG2000 conjugate, liraglutide variant 5 PEG5000 conjugate and liraglutide variant 5 PEG20000 conjugate. The corresponding groups of guinea pigs were injected subcutaneously with normal saline (1 mL/kg), liraglutide variant 5 (0.6 mg/kg), and liraglutide variant PEG2000 conjugate (0.6 mg/). Kg), liraglutide variant 5 PEG5000 conjugate (0.6 mg/kg) and liraglutide variant 5 PEG20000 conjugate (0.6 mg/kg), with time as the abscissa, guinea pig body weight For the ordinate plot, as shown in Figure 6, the guinea pigs in the saline group continued to grow in weight. The same dose of liraglutide variant 5, liraglutide variant 5 PEG2000 conjugate, liraglutide variant 5 PEG5000 conjugate and lira were administered twice in a row compared to the saline group. After 5 weeks of the glutamate allosteric 5 PEG20000 conjugate, the guinea pig body weight loss was about 3%, 5%, 9%, and 12%, respectively. The results showed that after administration of the same dose of liraglutide variant 5 PEG conjugate, its effect on reducing the body weight of guinea pigs was stronger than that of liraglutide variant 5, in which the liraglutide variant 5 PEG20000 The effect of the conjugate on reducing the body weight of guinea pigs was the most significant. The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention can be deduced or substituted without departing from the spirit and scope of the invention.

Claims

权 利 要 求 Rights request
1. 一种利拉鲁肽变构体， 其特征在于： 氨基酸序列为：  A liraglutide variant characterized in that the amino acid sequence is:
Xl-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Ly s(N-£-(N-a-Palmitoyl-L-y-glutamyl))-Glu-Phe-Ile-X9-Trp-X10-Val-Arg-Gly-Arg- Xll ; Xl-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Ly s(N-£-(Na-Palmitoyl- Ly-glutamyl))-Glu-Phe-Ile-X9-Trp-X10-Val-Arg-Gly-Arg-Xll;
其中： XI为 His或 Cys; X2为 Gly或 Cys; X3为 Thr或 Cys; X4为 Ser 或 Cys; X5为 Ser或 Cys; X6为 Ser或 Cys; X7为 Glu或 Cys; X8为 Ala或 删除； X9为 Ala或 Cys; X10为 Leu或 Cys或 D-Ala; Xll为 Gly或 Cys或 Gly-Gly。  Wherein: XI is His or Cys; X2 is Gly or Cys; X3 is Thr or Cys; X4 is Ser or Cys; X5 is Ser or Cys; X6 is Ser or Cys; X7 is Glu or Cys; X8 is Ala or deleted; X9 is Ala or Cys; X10 is Leu or Cys or D-Ala; X11 is Gly or Cys or Gly-Gly.
2. 根据权利要求 1所述的利拉鲁肽变构体， 其特征在于： 氨基酸序列为： 2. The liraglutide variant according to claim 1, wherein the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly, 所述 X5为 Cys。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Cys-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly, the X5 is Cys.
3. 根据权利要求 1所述的利拉鲁肽变构体， 其特征在于： 氨基酸序列为： His-Ala-Glu-Gly- The liraglutide variant according to claim 1, wherein the amino acid sequence is: His-Ala-Glu-Gly-
Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys(N-c-(N-a-Pa lmitoyl-L-y-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly , 所述 Χ8 删除。 Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Lys(Nc-(Na-Palmitoyl-Ly-glutamyl))-Glu-Phe-Ile- Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, the Χ8 deletion.
4. 根据权利要求 1所述的利拉鲁肽变构体， 其特征在于： 氨基酸序列为： His-Ala-Glu-Gly- The liraglutide variant according to claim 1, wherein the amino acid sequence is: His-Ala-Glu-Gly-
Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-c-(N- a-Palmitoyl-L-γ- glutamyl))-Glu-Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly-Arg-Gly, 所述 XI 0为 D-Ala。 Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Nc-(N-a-Palmitoyl-L-γ- glutamyl))-Glu -Phe-Ile-Ala-Trp-D-Ala-Val-Arg-Gly-Arg-Gly, the XI 0 is D-Ala.
5. 根据权利要求 1所述的利拉鲁肽变构体， 其特征在于： 氨基酸序列为： 5. The liraglutide variant according to claim 1, wherein the amino acid sequence is:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala -Lys(N-£-(N-a-Palmitoyl-Lfglutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly, 所述 Xll为 Gly-Gly。 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-£-(Na-Palmitoyl-Lfglutamyl) )) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Ar g-Gly-Gly, said X11 is Gly-Gly.
6. —种制备权利要求 1所述的利拉鲁肽变构体的方法， 其特征在于： 包 括如下步骤： 6. A method of preparing a liraglutide variant according to claim 1, wherein: Including the following steps:
A ) 2-氯三苯曱基氯树脂用 DMF洗涤 2~3次， 用 DMF溶胀 30分钟； B )称取 N端 Fmoc保护的氨基酸， 加入有机碱 DIEA, 活化 3~5分钟， 力口入反应柱反应 1-3小时；  A) 2-Chlorotriphenylphosphonium chloride resin is washed 2 to 3 times with DMF, and swollen with DMF for 30 minutes; B) Weigh N-terminal Fmoc-protected amino acid, add organic base DIEA, activate for 3 to 5 minutes, force in The reaction column is reacted for 1-3 hours;
C )用体积比为 1:4的哌啶和 DMF混合液脱除 Fmoc保护基 20分钟， 用 茚三酮法检测 Fmoc是否脱除完全；  C) The Fmoc protecting group was removed by a 1:4 mixture of piperidine and DMF for 20 minutes, and the ninhydrin method was used to detect whether Fmoc was completely removed;
D ) 重复上述步骤 A、 B和 C, 按照所述利拉鲁肽变构体的氨基酸序列， 逐个偶联相应的氨基酸， 其中， 赖氨酸采用 Fmoc-Lys(Alloc)-OH, 直到序列中 最后一个氨基酸偶联结束；  D) repeating the above steps A, B and C, according to the amino acid sequence of the liraglutide variant, the corresponding amino acids are coupled one by one, wherein the lysine adopts Fmoc-Lys(Alloc)-OH until the sequence The last amino acid coupling is over;
E )脱除赖氨酸 Alloc侧链保护基， 偶联上 Palmitoyl-Glu-OtBu, 得到利拉 鲁肽变构体树脂；  E) removing the lysine Alloc side chain protecting group, coupled with Palmitoyl-Glu-OtBu, to obtain a liraglutide variant resin;
F )将得到的利拉鲁肽变构体树脂用 TFA裂解， 反应 1-3小时， 将多肽从 树脂上裂解下来， 同时脱除侧链保护基；  F) cleavage of the obtained liraglutide allosteric resin with TFA, and reacting for 1-3 hours to cleave the polypeptide from the resin while removing the side chain protecting group;
G )对裂解下来的多肽溶液用乙醚沉淀， 得到粗肽， 然后用 C8色语柱， 用常规的流动相洗脱， 收集组分， 冻干得到所需要的利拉鲁肽变构体；  G) precipitating the cleaved polypeptide solution with diethyl ether to obtain a crude peptide, and then using a C8 color column, eluting with a conventional mobile phase, collecting the components, and lyophilizing to obtain the desired liraglutide variant;
H )用 HPLC方法检测其纯度和含量，用二级质谱和 Edman降解分析其序 列。  H) The purity and content were determined by HPLC, and the sequence was analyzed by secondary mass spectrometry and Edman degradation.
7. 根据权利要求 1至 5中任一项所述的利拉鲁肽变构体在制备用于降低 血糖的药物中的用途。  The use of the liraglutide variant according to any one of claims 1 to 5 for the preparation of a medicament for lowering blood sugar.
8. 根据权利要求 1至 5中任一项所述的利拉鲁肽变构体在制备用于降低 体重的药物中的用途。  The use of the liraglutide variant according to any one of claims 1 to 5 for the preparation of a medicament for reducing body weight.
9. 一种利拉鲁肽变构体缀合物， 其特征在于： 包含 PEG修饰基团和权利 要求 1至 5中任一项所述的利拉鲁肽变构体。  A liraglutide variant conjugate comprising: a PEG modifying group and the liraglutide variant according to any one of claims 1 to 5.
10. 根据权利要求 9所述的利拉鲁肽变构体缀合物， 其特征在于： 所述 PEG修饰基团通过 Cys、 N端氨基、 侧链羧基、 C端^^或组氨酸侧链咪唑基 缀合到利拉鲁肽变构体上。  The liraglutide variant conjugate according to claim 9, wherein the PEG modifying group passes through a Cys, an N-terminal amino group, a side chain carboxyl group, a C-terminal group or a histidine side. The chain imidazolyl group is conjugated to the liraglutide variant.
11. 根据权利要求 9所述的利拉鲁肽变构体缀合物， 其特征在于： 所述 PEG修饰基团的分子量在 2 kDa~20 kDa之间。  The liraglutide variant conjugate according to claim 9, wherein the PEG modifying group has a molecular weight of between 2 kDa and 20 kDa.
12. 根据权利要求 9所述的利拉鲁肽变构体缀合物， 其特征在于： 所述 PEG修饰基团选自以下结构： （CH3)2CH-(OCH2CH2)n- , 12. The liraglutide variant conjugate according to claim 9, wherein: The PEG modifying group is selected from the group consisting of: (CH3)2CH-(OCH2CH2)n- ,
CH3(CH2)m-( OCH2CH2)n- , R-(OCH2CH2)n- , Lys-PEG2 , CH3(CH2)m-( OCH2CH2)n- , R-(OCH2CH2)n- , Lys-PEG2 ,
Glu-PEG2 , Tris-PEG3 , Biotin-PEGll ；  Glu-PEG2, Tris-PEG3, Biotin-PEGll;
其中， m表示 1~6之间的整数； n表示 40~120之间的整数； R选自 H、 ( C1~C30 )烷基、 环 （ C6~C30 )烷基或苯基 ( C6-C30 )烷基； Lys-PEG2表 示以赖氨酸为核的二分支型 PEG; Glu-PEG2表示以谷氨酸为核的二分支型 PEG; Tris-PEG3表示三分支型 PEG; Biotin-PEGll表示末端连接生物素的 11 个单元 PEG。  Wherein m represents an integer between 1 and 6; n represents an integer between 40 and 120; R is selected from H, (C1 to C30) alkyl, ring (C6-C30) alkyl or phenyl (C6-C30) Lys-PEG2 represents a two-branched PEG with lysine as the nucleus; Glu-PEG2 represents a bi-branched PEG with glutamic acid as the nucleus; Tris-PEG3 represents a tri-branched PEG; Biotin-PEGll represents the end Connect 11 units of PEG to biotin.
13. 根据权利要求 9所述的利拉鲁肽变构体缀合物， 其特征在于： 所述 PEG修饰基团选自 PEG或 mPEG。  13. The liraglutide variant conjugate according to claim 9, wherein: the PEG modifying group is selected from the group consisting of PEG or mPEG.
14. 一种制备权利要求 9所述的利拉鲁肽变构体缀合物的方法， 其特征在 于： 包括如下步骤：  14. A method of preparing the liraglutide variant conjugate of claim 9, characterized by: comprising the steps of:
A )称取一定量的所述利拉鲁肽变构体， 溶于适当的緩沖溶液中；  A) weighing a certain amount of the liraglutide variant, dissolved in a suitable buffer solution;
B )按一定的 PEG修饰基团与利拉鲁肽变构体摩尔比， 称取 PEG修饰基 团， 加入上述緩沖溶液中， 适当摇匀使 PEG修饰基团溶解并与利拉鲁肽变构 体反应；  B) according to a certain PEG modification group and liraglutide variant molar ratio, weigh the PEG modification group, add to the above buffer solution, shake properly to dissolve the PEG modification group and decorate with liraglutide Body reaction
C )用过量的 Cys溶液终止反应， RP-HPLC检测反应并纯化得到目标化合 物。  C) The reaction was stopped with an excess of Cys solution, and the reaction was detected by RP-HPLC and purified to obtain the target compound.
15. 根据权利要求 14所述的利拉鲁肽变构体缀合物的方法， 其特征在于： 所述緩沖溶液选自醋酸 -醋酸钠緩沖溶液、 磷酸钠盐緩沖溶液、 碳酸氢钠、 The method according to claim 14, wherein the buffer solution is selected from the group consisting of acetic acid-sodium acetate buffer solution, sodium phosphate buffer solution, sodium hydrogencarbonate,
EDTA-NH4AC緩沖溶液或 EDTA-磷酸钠盐緩沖溶液。 EDTA-NH4AC buffer solution or EDTA-sodium phosphate buffer solution.
16. 根据权利要求 14所述的利拉鲁肽变构体缀合物的方法， 其特征在于： 所述 PEG修饰基团与所述利拉鲁肽变构体的摩尔比为 3~5； 所述 PEG修饰基 团与所述利拉鲁肽变构体的反应时间为 0.5~12小时。  The method of claim 14, wherein the molar ratio of the PEG modifying group to the liraglutide variant is 3 to 5; The reaction time of the PEG modifying group with the liraglutide variant is 0.5 to 12 hours.
17.根据权利要求 9所述的利拉鲁肽变构体缀合物在制备用于降低血糖的 药物中的用途。  17. Use of a liraglutide variant conjugate according to claim 9 for the manufacture of a medicament for lowering blood glucose.
18.根据权利要求 9所述的利拉鲁肽变构体缀合物在制备用于降低体重的 药物中的用途。  18. Use of a liraglutide variant conjugate according to claim 9 for the manufacture of a medicament for reducing body weight.
PCT/CN2012/080758 2011-09-14 2012-08-30 Variant of liraglutide and conjugate thereof WO2013037267A1 (en)

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CN112955216A (en) * 2018-09-12 2021-06-11 奎亚培格制药公司 Releasable GLP-1 conjugates
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