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Improved ziconotide

A technology of ziconotide and penetrating peptide, which is applied in the field of fusion peptides, can solve the problems that the analgesic effect and shelf life cannot reach clinical application, cannot carry out good quality control, cannot carry out large-scale promotion, etc., and achieve Long duration of drug effect, excellent analgesic effect, and low clinical risk

Active Publication Date: 2019-01-18
SHENZHEN RUIJIAN BIOSCI TECH LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] At present, there are technical methods in the prior art to use cell-penetrating peptides to assist conotoxins to cross the blood-brain barrier. For example, ziconotide is packaged in virus particles, and TAT polypeptide is linked to the surface of virus particles. The virus particles Ziconotide can be delivered through the blood-brain barrier. However, the preparation process of this method is complicated, and the virus packaging process cannot perform good quality control, and it is difficult to apply it on a large scale in the industry.
As another example, the N-terminus of the TAT peptide and conotoxin is used as a linking unit to prepare a fusion polypeptide. The fusion polypeptide can pass through the blood-brain barrier and is suitable for intravenous injection. However, the fusion polypeptide connected to the N-terminal Through intravenous injection, the analgesic effect and existence time in the body cannot meet the requirements of clinical application, and large-scale promotion cannot be carried out.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 different types of improved ziconotide

[0043] Four different types of improved ziconotide were prepared and named as protected polypeptides MVIIA-a, MVIIA-b, MVIIA-c, and MVIIA-d, respectively. At the same time, ziconotide was prepared and named MVIIA as a control. In this experiment, the F-moc automatic solid-phase synthesis method was adopted, and the specific steps are as follows:

[0044] Synthesis of polypeptides: Protected polypeptides and their derivatives were assembled on resin using a model of the 433A automatic synthesizer (ABI, Foster City, CA). The peptide resin was incubated in suspension for 2.5 hours at room temperature to deprotect the group. The suspension system is composed of 10 ml of TFA, 0.75 g of phenol, 0.25 ml of 1,2-ethanedithiol, 0.5 ml of sulfide anisole and 0.5 ml of water. (Wat methoxycarbonyl (Fmoc), a common alkoxycarbonyl amino protecting group). The resin was separated from the peptide deprotected m...

Embodiment 2

[0047] Example 2: Chemical properties and structural characterization of different types of improved ziconotide

[0048] 1. Chemical properties of MVIIA and its variants

[0049] The buffer-treated linear peptides were treated for 24-48 hours at 4°C, and then analyzed by high-performance liquid chromatography. It was found that the folding of the linear peptides resulted in the appearance of a major peak and several minor peaks. The buffer system includes 1mM glutathione, 0.1mM oxidized glutathione, 1mM EDTA, and 0.2mg / mL linear peptide, and the pH of the solution is 7.9. The major product was purified and evaluated by analytical reversed-phase high-performance liquid chromatography, and the purity of the peptide was determined to be greater than 98%. Determination was performed with an Ultraflex III TOF / TOF mass spectrometer (Bruker). The prepared polypeptide sequence is shown in Table 1, and its one-step oxidative folding HPLC analysis profile is shown in figure 1 shown. ...

Embodiment 3

[0058] Embodiment 3: Electrophysiological experiments of different types of improved ziconotide

[0059] In order to further study the electrophysiological effects of different types of improved ziconotide and the inhibitory effect on calcium ion (CaV2.2) channels, the following experiments were carried out:

[0060] HEK293T cells (capable of expressing SV40 large T antigen) were cultured in DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum, 1% penicillin and streptomycin. The incubator environment is 37°C, 5% CO 2 . Dr. Diane Lipscombe provided the alpha of the rat CaV2.2 channel 1B Splice variant e37a, auxiliary subunit α 2 δ 1 and beta 3 Plasmids (Addgene plasmid #26569, #26575, #26574). Then three kinds of plasmids (3μg), 0.4μg enhanced green fluorescent protein gene and liposome were transiently transfected into HEK293T cells. 24 hours after transfection, the cells were seeded on glass slides and incubated in an incubator (37°C, 5% CO 2 ) for at l...

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Abstract

The invention relates to improved ziconotide. A C end of ziconotide is linked with an N end of cytomembrane penetrating peptide through two glycines so as to form fused peptide, so that the deficiencies that ziconotide cannot penetrate through a blood brain barrier, an intramuscular injection manner cannot be realized, the surgical operation risk is high, the infection risk is high, and the like are overcome. The improved ziconotide is suitable for an intravenous, abdominal or nasal delivery manner, convenient to operate and low in clinic risk; by utilizing the intravenous, abdominal or nasaldelivery manner, the pharmacodynamic time of the improved ziconotide in a human body is long, the analgesic effect is excellent, the side effect is small, and the improved ziconotide is suitable for large-scale clinic application. The improved ziconotide is easy to prepare, and the quality can be controlled in the preparation process and the preparation course, so that the improved ziconotide is suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drugs, in particular to a fusion polypeptide of ziconotide. Background technique [0002] Ziconotide (trade name PrialtTM, Elan Pharmaceuticals) is the first conotoxin drug approved by the US Food and Drug Administration (FDA) in 2004. Its target action site is N-type voltage-gated calcium ion channel, and it is the first-line drug for subarachnoid space (intrathecal) compound analgesia. Ziconotide is the artificial synthesis of the hydrophilic polypeptide ω-MVIIA in the venom peptide of the Pacific fish-eating snail—the conch snail—and it is the first new type of non-morphine analgesic used in clinical practice. Its molecular formula is C 102 h 172 N 36 o 32 S 7 , the structural formula: [0003] H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys- Cys-NH 2 . [0004] Ziconotide can be used clinically to treat postherpetic neuralgia, phan...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/17A61K47/42A61P29/00
CPCA61K38/00A61P29/00C07K14/43504C07K2319/10
Inventor 李书鹏周强
Owner SHENZHEN RUIJIAN BIOSCI TECH LTD
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