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Method for preparing ziconotide

A ziconotide and linear peptide technology, applied in the field of polypeptide drug preparation, can solve the problems of low total product yield and purity, difficulty in separation and purification, and many impurities in ziconotide, so as to reduce the difficulty of purification and improve product yield. efficiency, broad practical value and application prospects

Inactive Publication Date: 2013-09-18
CHENGDU SHENGNUO BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is that the ziconotide prepared by the existing method has many impurities, difficult separation and purification, and low overall yield and purity of the product.

Method used

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  • Method for preparing ziconotide
  • Method for preparing ziconotide
  • Method for preparing ziconotide

Examples

Experimental program
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preparation example Construction

[0042] The preparation method of ziconotide comprises: preparing ziconotide linear peptide resin by solid-phase peptide synthesis method, acid hydrolyzing ziconotide linear peptide resin to obtain ziconotide linear peptide crude product, ziconotide linear peptide After the crude product is oxidized, the crude product of ziconotide is obtained, and finally the crude product of ziconotide is purified to obtain the pure product of ziconotide; the steps of preparing the linear peptide resin of ziconotide by the solid-phase peptide synthesis method are as follows: starting from the amino resin, passing through solid-phase peptide synthesis The phase-coupled synthesis method sequentially inserts the Fmoc-protected amino acids of the corresponding fragments in the following sequences to prepare the linear peptide resin of ziconotide:

[0043] R-Cys(Trt)-X(Boc)-X(Boc)-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-

[0044] Arg(Pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(Trt)-Cys(Trt)-

[0045] Y(tBu)-Ser(tBu...

Embodiment 2

[0091] The preparation of embodiment 2 ziconotide linear peptide crude product

[0092] Take the ziconotide linear peptide resin prepared in Example 1, add a cleavage reagent with a volume ratio of TFA:water:EDT=95:5:5 (cleavage reagent 10mL / g resin), stir evenly, and react with stirring at room temperature for 3 hours , the reaction mixture was filtered using a sand core funnel, the filtrate was collected, the resin was washed 3 times with a small amount of TFA, the combined filtrate was concentrated under reduced pressure, anhydrous ether was added to precipitate, and anhydrous ether was used to wash the precipitate 3 times, and the white powder was obtained by draining. It is the crude product of ziconotide linear peptide with a purity of 69.7%.

Embodiment 3

[0093] The preparation of embodiment 3 ziconotide crude product

[0094]The ziconotide linear peptide crude product obtained in Example 2 was dissolved with 1% ammonium acetate solution and made into a solution of about 0.2 mg / mL, adjusted to pH 7.5 with dilute ammonia water, stirred and reacted, and the reaction process was monitored by high performance liquid chromatography After the reaction was completed, the pH was adjusted to 3.5 with acetic acid, concentrated under reduced pressure at 40°C to obtain a concentrated solution of crude product of ziconotide with a purity of 63.1%.

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Abstract

The invention belongs to the technical field of polypeptide drugs, and particularly relates to a method for preparing ziconotide, and the method is used for solving the technical problems of difficult separation and purification and low total product yield and purity in the existing preparation methods. The method comprises the following steps of: preparation of ziconotide linear peptide resin based on a solid phase polytide method, acidolysis for obtaining a ziconotide linear peptide crude product, oxidation for obtaining a ziconotide crude product and purification for obtaining a ziconotide purified product, wherein the solid phase polytide method comprises the following steps of: preparing ziconotide linear peptide resin by sequentially connecting corresponding protected amino acid or protected amino acid fragment in the following sequence starting from amino resin through a solid phase coupling synthesis method: R-Cys(Trt)-X(Boc)-X(Boc)-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg(Pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(Trt)-Cys(Trt)-Y(tBu)-Ser(tBu)-Cys(Trt)-Arg(Pbf)-Z(tBu)-Lys(Boc)-Cys(Trt)-amino resin, wherein R is Fmoc, Boc or H, X is Lys-Gly, Y is Thr-Gly, and Z is Ser-Gly. The invention provides a novel method for shortening the production period and improving the product purity and the product yield.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drug preparation methods, in particular to a preparation method of ziconotide. Background technique [0002] Ziconotide has the following structure: [0003] [0004] Ziconotide is a synthetic ω-conotoxin MVIIA, which is a polycationic polypeptide composed of 25 amino acids, including 6 cysteine ​​residues connected by 3 disulfide bonds. The molecular weight is 2639.12. It is a specific and selective N-type voltage-sensitive calcium channel blocker, and has no obvious affinity for other ion channels. Ziconotide is 100 to 1000 times more potent than morphine and is a highly specific painkiller. [0005] This product is a polypeptide containing 3 disulfide bonds and 25 amino acids. It is the first neuron-specific N-type voltage-sensitive calcium channel blocker that is clinically used. This product binds to the N-type calcium channel on the primary nociceptive afferent nerve located in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/06C07K1/04C07K1/02
Inventor 郭德文童光彬文永均韩玉
Owner CHENGDU SHENGNUO BIOPHARM
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