Venin-sourced demulcent CTXn, and purification method and applications thereof

A technology of cytotoxin and purification method, applied in the field of biopharmaceuticals, to achieve strong analgesic effect, significant analgesic effect, and obvious dose-effect relationship

Inactive Publication Date: 2010-06-02
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Whether there is CTXn in Zhoushan cobra venom in other parts of mainla...

Method used

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  • Venin-sourced demulcent CTXn, and purification method and applications thereof
  • Venin-sourced demulcent CTXn, and purification method and applications thereof
  • Venin-sourced demulcent CTXn, and purification method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Determination of CTXn analgesic effect by acetic acid writhing method

[0039] 1) Collect Zhoushan cobra venom samples outside;

[0040] 2) Carry out the separation and purification of CTXn to the aforementioned Zhoushan cobra venom samples,

[0041] (1) Gel filtration first: Using Sephacryl S-100HR filler, the Zhoushan cobra venom sample was subjected to molecular sieve filtration through the AKTA explorer 100 rapid purification process development system to obtain I, II and III toxic components, respectively, with G-50 Prepacked column HiPrep 26 / 10 Desalting desalting, freeze-dried, ready for use;

[0042] (2) Through preliminary screening, select component III for the next step of "ion exchange";

[0043] (3) Ion exchange: Using CM Sepharose FF filler, component III was ion exchanged through the AKTA explorer 100 rapid purification process development system to obtain a single toxic polypeptide CTXn( figure 1 ), were desalted with G-50 prepacked column ...

Embodiment 2

[0054] Example 2: Determination of the analgesic effect of CTXn by hot plate method

[0055] Take a rat (male, weighing 180-220g) and place it on the metal hot plate (surface temperature: 55±0.5°C) of the UGO Pain Tester, start timing until the rat licks its hind paw or withdraws its paw, then stop timing. This period of time is the pain response latency of the rat. In the experiment, the basic pain threshold (latency period) of the rats was measured before administration, and if the latency period was greater than 30s, it was eliminated. Rats in each group were tested for latency after administration. In order to avoid scalding the hind feet of the rats, 55s was determined as the interruption time.

[0056] The results show that CTXn high (2mg / kg), middle (1mg / kg), low (0.5mg / kg) 3 dosage groups all can significantly improve the pain response threshold (table 2) of rats to noxious thermal stimulation, the The results suggest that CTXn has a good analgesic effect on somatic...

Embodiment 3

[0060] Example 3: Effects of different doses of CTXn on pain sensitivity in morphine-addicted rats

[0061] There was no significant difference in the pain domain of the rats in each group before administration (P>0.05). After 8 consecutive days of morphine position preference training between the morphine group and each dose group of CTXn, the time for the morphine-addicted rats to lift their feet on the pain tester was significantly shorten. From d10 to d18, the rats in the morphine group showed hyperalgesia and had a strong response to pain; while the CTXn groups showed that snake venom had a strong analgesic effect on the pain sensitization of morphine-addicted rats, which was dose-dependent. The analgesic effect is significant after administration for 8 consecutive days. Medium and high doses of CTXn (1-2 mg / kg) have stronger analgesic effects on hyperalgesia after morphine addiction than low doses of CTXn (0.5 mg / kg) (Table 3). The results showed that the snake venom C...

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Abstract

The invention relates to a venin-sourced demulcent CTXn, and a purification method and applications thereof. The invention is characterized in that (1) the molecular weight of the CTXn is 6697.429 Da; (2) and the amino acid sequence of the CTXn is described as the sequence list SEQ ID No.1. The preparation method comprises the following steps of: using a Zhoushan cobra venom sample; carrying out the CTXn separation and purification on the Zhoushan cobra venom sample; primary-screening to select a component III for next step of ion exchange; carrying out the ion exchange on the component III through a quick-purification technique exploitation system by using a filler to obtain a single-virulence polypeptide CTXn, desalting with a G-50 prepacked column, and freeze-drying; and measuring the molecular weight and the amino acid sequence of the CTXn. The CTXn of the invention has favorable demulcent function on somatalgia and visceralgia caused by nociceptive stimulus as well as pains caused by addiction to morphine, has the characteristics of obvious drug effect and low toxic or side effect, is beneficial to large-scale production, and has wide application prospects in the aspect of analgesic development.

Description

technical field [0001] The invention relates to a cytotoxin CTXn derived from snake venom for analgesia, a purification method and application thereof. A cytotoxin derived from the poison of Zhoushan cobra (Naja atra Cantor) is mainly used, which is suitable for analgesia, especially involving the new pharmaceutical application of the cytotoxin for treating acute and chronic pain. It belongs to the technical field of biopharmaceuticals. Background technique [0002] Since the 1970s, NT has conducted in-depth research on the basic research of analgesia, and its analgesic mechanism may be related to various systems such as cholinergic and endogenous opioid peptides. Compared with the traditional analgesic drug morphine, the analgesic effect of NT has the characteristics of less dosage, longer analgesic time and no addiction, etc. It is effective for intractable pain, neuropathic pain and malignant tumor pain. In view of the high neurotoxicity and lethal activity of NT, it is...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/36A61K38/17A61P29/00
Inventor 孔天翰刘先国崔超伟信文君董伟华崔跃黄绍仲维高唐娅
Owner 广州医学院
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