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Tetrodotoxin conjugate and medicine composition having same

A technology of tetrodotoxin and conjugates, which can be used in drug combinations, medical preparations containing active ingredients, antitoxins, etc.

Inactive Publication Date: 2004-01-07
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no other detailed and systematic studies on TTX vaccines

Method used

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  • Tetrodotoxin conjugate and medicine composition having same
  • Tetrodotoxin conjugate and medicine composition having same
  • Tetrodotoxin conjugate and medicine composition having same

Examples

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preparation example Construction

[0022] In the method for preparing tetrodotoxin conjugates of the present invention, the linker is selected from formaldehyde and glutaraldehyde, preferably formaldehyde.

[0023] In the method for preparing tetrodotoxin conjugates of the present invention, the buffer system used in step 1) can be selected from: disodium hydrogen phosphate-citric acid buffer solution (pH2.2-8.0), citric acid-hydrogen Sodium oxide-hydrochloric acid buffer (pH2.2-6.5), citric acid buffer (pH3.0-6.6), acetate buffer (pH3.6-5.8), phosphate buffer (pH5.8-8.0), Barbie Sodium-hydrochloric acid buffer (pH6.8-9.6), Tris-HCl buffer (pH7.1-8.9), boric acid-borax buffer (pH7.4-9.0), sodium carbonate-sodium bicarbonate buffer (pH9. 2-10.8); wherein the preferred buffer system is citric acid buffer (pH3.0-6.6), acetate buffer (pH3.6-5.8), phosphate buffer (pH5.8-8.0), particularly preferably phosphate buffer Saline system and Tris-HCl buffer. Those skilled in the art know that an appropriate buffer system...

Embodiment 1

[0029] Example 1. Preparation of Chinese Limulus Hemocyanin

[0030] Hemolymph collection: Chinese horseshoe crabs were caught in the South China Sea and the Beibu Gulf. Select a healthy female horseshoe crab that has just gone out to sea and is active, and bend its body to the abdomen at an angle of nearly 90°. Hemolymph fluid was extracted from the light blue blood sinus under sterile conditions, and anticoagulated with 0.125% maleicamine and limulus blood at a ratio of 1:2 (v / v).

[0031]Protein separation by ultracentrifugation: centrifuge the blood sample obtained above (10000g, 30 minutes, 10°C), discard the white cell precipitate; further centrifuge the obtained supernatant (80000g, 2 hours, 10°C), discard the supernatant; Wash and collect the precipitate with % NaCl, centrifuge again (10000g, 30 minutes, 10°C), discard a little precipitate; take 100ml of blue supernatant and dialyze against 3000ml normal saline for 18 hours, centrifuge again (10000g, 1 hour, 10°C), D...

Embodiment 2

[0033] The preparation of embodiment 2 tetrodotoxin conjugates

[0034] 1. Preparation of TTX-TTH

[0035] At room temperature, the carrier protein TTH was weighed and dissolved in 20 mM PBS (pH 6.8), and the concentration was determined by ultraviolet spectrophotometry and corrected to 5 mg / ml. Take 2ml of a solution containing 10mg of protein, add 1mg of TTX (dissolved in 1ml of water), and 1ml of PBS to wash the TTX ampoule, combine and mix well. Wherein, when the weight ratio of TTX to TTH is 1:10, the molar ratio thereof is about 940:1.

[0036] To the resulting combined solution, 170 μl of 37% formaldehyde was added dropwise in total to reach a final concentration of 1.5%; after mixing, it was fixed in a water bath shaker, and reacted with slow shaking at 30°C for 72 hours. The resulting reaction solution was dialyzed against 1000 ml of 20 mM PBS (pH 7.2) at 4° C. under stirring for 2 days, during which the solution was changed 6 times to remove uncoupled toxins.

[0...

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Abstract

The present invention relates to a conjugate of tetrodotoxin (TTX), it contains tetrodotoxin, conjunction agent and carrier covalently-coupled with tetrodotoxin by means of conjunction agent, and thedescribed carrier is selected from Chinese limulus blood blue protein or tetanus toxin or fragment with correspondent biological function. Said invention also relates to the method for preparing saidconjugate, and also relates to the medicine composition containing said conjugate and its application for preventing tetrodotoxism.

Description

field of invention [0001] The present invention relates to a conjugate of tetrodotoxin (hereinafter referred to as TTX), which comprises tetrodotoxin, a linker and a carrier covalently coupled to tetrodotoxin through the linker, and the carrier is selected from Chinese limulus hemocyanin (hereinafter referred to as TTH), or tetanus toxoid (hereinafter referred to as TT), or fragments thereof having equivalent biological functions. The present invention also relates to a method for preparing the tetrodotoxin conjugate of the present invention. The present invention also relates to a pharmaceutical composition containing the tetrodotoxin conjugate, and the use of the tetrodotoxin conjugate in preparing a pharmaceutical composition for preventing tetrodotoxin poisoning. Background of the invention [0002] TTX is a small molecule and highly toxic neurotoxin produced by puffer fish and other marine organisms. It blocks sodium ion channels, blocks nerve conduction, causes paraly...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K39/385A61P39/02
Inventor 徐勤惠荣康泰
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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