Liraglutide variant and conjugate thereof

A technology for liraglutide and allosteric forms, which is applied in the field of liraglutide allosteric forms and their conjugates, can solve the problems of short half-life of polypeptide drugs, limiting patient medication compliance, poor physical and chemical stability, etc. , to achieve the effect of retaining biological activity, reducing the frequency of medication, and being difficult to degrade.

Active Publication Date: 2012-01-18
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Polypeptide drugs generally have short half-life in vivo, poor physical and chemical stability, and are easily degraded by various proteases in the body.
As a subcutaneous injection preparation, liraglutide has a half-life of about 12-14 hours and needs to be used once a day. It can effectively lower blood sugar, but it brings a great burden on the body, psychology and economy of patients, which limits the use of drugs for patients. compliance

Method used

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  • Liraglutide variant and conjugate thereof
  • Liraglutide variant and conjugate thereof
  • Liraglutide variant and conjugate thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Solid-phase synthesis of variants of liraglutide.

[0060] Using 2-chlorotrityl chloride resin (2-CTC resin) as a solid phase carrier, Fmoc protected amino groups were used to synthesize liraglutide sulfhydryl variants. The steps are as follows: Step 1: 2-CTC resin with DMF Wash 2~3 times, swell with DMF for 30 minutes; Step 2: Weigh the N-terminal Fmoc-protected amino acid, use DIEA as the activator, activate for 3~5 minutes, add the reaction column to react for 1~3 hours; Step 3: Use The mixture of piperidine and DMF with a volume ratio of 1:4 removes the Fmoc protecting group for 20 minutes. Use the ninhydrin method to detect whether the Fmoc is completely removed. The color of the resin indicates that the Fmoc has been removed; the fourth step: repeat the first step From the third step to the process, according to the amino acid sequence of the liraglutide variant, the corresponding amino acids are coupled one by one, wherein the lysine uses Fmoc-Lys(All...

Embodiment 2

[0095] Example 2 Preparation of PEG5000 conjugate of liraglutide variant 1.

[0096] Dissolve 5 mg of liraglutide variant 1 in 8 mL of 20 mM sodium phosphate buffer (pH 6.5), and weigh 20 mg PEG5000, added to the above solution, shake properly to dissolve PEG5000 and mix well with liraglutide variant 1, react at 25°C for 2 hours, and then terminate with excess 0.5 M cysteine ​​solution The reaction was finally placed at -20°C for separation and purification. The reaction was detected by Waters 2695 high performance liquid chromatography and purified to obtain the target compound. The molar ratio of PEG5000 to liraglutide variant 1 is 3:1, which is concluded based on the results of multiple experiments, and can completely modify liraglutide variant 1.

Embodiment 3

[0097] Example 3 Preparation of PEG5000 conjugate of liraglutide variant 2.

[0098] Dissolve 5 mg of liraglutide variant 2 in 8 mL of 20 mM sodium phosphate buffer (pH 6.5), and weigh 20 mg PEG5000, added to the above solution, shake properly to dissolve PEG5000 and mix well with liraglutide variant 2, react at 25°C for 2 hours, and then terminate with excess 0.5 M cysteine ​​solution The reaction was finally placed at -20°C for separation and purification. The reaction was detected by Waters 2695 high performance liquid chromatography and purified to obtain the target compound.

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Abstract

The invention provides a liraglutide variant and a preparation method thereof. An amino acid sequence of the liraglutide variant is X1-Ala-Glu-X2-Thr-Phe-X3-X4-Asp-Val-X5-X6-Tyr-Leu-X7-Gly-Gln-Ala-X8-Lys(N-epsilon-(N-alpha-Palmitoyl-L-gamma-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 is deleted; X9 is Ala or Cys; X10 is Leu, Cys or D-Ala; and X11 is Gly, Cys or Gly-Gly. The invention also provides a conjugate of the liraglutide variant and a preparation method thereof. The liraglutide variant and the conjugate thereof provided by the invention keep bioactivity of liraglutide; the half life is prolonged; and the burden of a patient is favorably relieved.

Description

technical field [0001] The present invention relates to liraglutide variants and conjugates thereof. Background technique [0002] Liraglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist developed by Novo Nordisk, Denmark. It is similar to GLP in terms of molecular structure, biological activity, target and immunogenicity. -1 similar. The molecular structure of liraglutide is similar to that of GLP-1(7-36), the difference is that liraglutide replaces the 34th lysine of GLP-1 with arginine, and adds a palmitoyl fatty acid side chain. [0003] Polypeptide drugs generally have the characteristics of short half-life in vivo, poor physical and chemical stability, and are easily degraded by various proteases in the body. As a subcutaneous injection preparation, liraglutide has a half-life of about 12-14 hours and needs to be used once a day. It can lower blood sugar well, but it brings great physical, psychological and economic burdens to patients, which limits the us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/20C07K1/06C07K1/04C07K1/107A61K38/26A61K47/48A61P3/10A61P3/04
CPCC07K1/04A61K47/48C07K1/107C07K1/06A61K38/26A61K47/48038C07K14/605A61K38/00A61K47/542A61P3/04A61P3/10
Inventor 潘俊锋刘建马亚平袁建成
Owner HYBIO PHARMA
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