Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin

An extraction method and high-purity technology, which can be used in rectal drug preparations, oral absorption, and oral administration. It can solve problems such as poor compliance and slow onset of action, and achieve short drug effect onset time, significant analgesic effect, and analgesic effect. long lasting effect

Active Publication Date: 2015-02-04
张庆宇
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Problems solved by technology

Zhu Tianxin, Yuan Caijun, Ren Wanqiong. Large-scale preparation of cobra neurotoxin and its analgesic effect[J]. West China Pharmaceutical Journal, 2007, 22(3): 247-249. Mouse hot plate test, rat electric shock The results of the screaming test and the mouse writhing test are basically the same, showing that CNT has a strong analgesic effect, and has the characteristics of slow onset), Cobotide single preparation can only be administered by intramuscular injection, poor drug compliance of patients and other defects
[0007] The only commercially available analgesic drug containing cobotide orally is a compound preparation - Keluoqu Tablets, which is composed of cobotide, tramadol hydrochloride and ibuprofen in a ratio of 1:150:300, and its components are: Each tablet contains 160 μg of cobotide calculated as neurotoxic protein, and contains tramadol hydrochloride (C 16 h 25 NO 2 HCl) 25mg, containing ibuprofen (C 13 h 18 o 2 ) 50 mg; cobotide plays analgesic effect mainly by inhibiting the release of acetylcholine; tramadol hydrochloride exerts analgesic effect by binding to central opioid receptors; ibuprofen plays analgesic effect by inhibiting cyclooxygenase The three play an analgesic synergistic effect through different mechanisms of action; however, due to individual differences in patients and the complexity of physiological and biochemical indicators and lesions in individual patients, the interaction between drugs in the compound preparation will also cause some unexpected effects. Side effects that are contrary to the treatment goals, there are certain risks and limitations of medication

Method used

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  • Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin
  • Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin
  • Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin

Examples

Experimental program
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Effect test

Embodiment 1

[0075] 1) Take 8 grams of crude cobra venom (purchased from Yujiang Cobra Farm in Jiangxi) and dissolve it in 80 ml of NaAC-HAC solution with a pH of 5.050 mmol / L. After fully dissolving, centrifuge at low temperature (temperature: 0°C--10°C, speed: 3600 rpm, time: 30 minutes), take the supernatant, and wait for the column;

[0076] 2) Sp sepharose FF on the supernatant of snake venom, use PH 5.050mmol / L NaAC-HAC buffer with a salt gradient of 0.0-1.0mol / L NaCl for a total of 3000 ml of linear salt gradient elution, and the buffer to be eluted with a salt gradient When it reaches 0.7mol / L NaCl, collect the cobotide components according to the recorded spectrum, the cobotide neurotoxic protein peak is 320ml in total, see the chromatography results figure 1 ; Equipment setting parameters: ultraviolet detection parameters: 0.2A280nm; recorder parameters: 200mv 2cm / h; constant flow pump flow rate: 85ml / h; Low temperature dialysis at 4-10°C for 6 hours, wait for sample loading;

...

Embodiment 2

[0082] 1) Take 8 grams of crude cobra venom and dissolve it in 80 milliliters of 50 mmol / L Tris-HCl solution with pH 7.4. After fully dissolving, centrifuge at low temperature (temperature: 0°C--10°C, speed: 3600 rpm, time: 30 minutes), take the supernatant, and wait for the column;

[0083] 2) Sp sepharose FF on the supernatant of snake venom, use PH 7.4 50mmol / L Tris-HCl salt gradient buffer solution with a salt gradient of 0.0-1.0mol / L NaCl for a total of 3000 ml of linear salt gradient elution to collect Cobotide neurotoxic protein The total peak is 305ml; equipment setting parameters: UV detection parameters: 0.2A 280nm; recorder parameters: 200mv 2cm / h; constant flow pump flow rate: 85ml / h;

[0084] Subsequent steps are the same as in Example 1. Freeze-drying can obtain 378 mg of high-purity cobotide finished product, with a neurotoxic protein content of 78.20%; one SDS-PAGE electrophoresis is consistent with the cobotide reference substance; HPLC purity is 99.32%, and t...

Embodiment 3

[0086] 1) Take 8 grams of crude cobra venom and dissolve it in 80 milliliters of pH 6.850mmol / LTris-HCl solution. After fully dissolving, centrifuge at low temperature (temperature: 0°C--10°C, speed: 3600 rpm, time: 30 minutes), take the supernatant, and wait for the column;

[0087] 2) Sp sepharose FF on the supernatant of snake venom, use PH 6.8 50mmol / L Tris-HCl salt gradient buffer solution with a salt gradient of 0.0-1.0mol / L NaCl for a total of 3000 ml of linear salt gradient elution to collect Cobotide neurotoxic protein The total peak is 337ml; equipment setting parameters: UV detection parameters: 0.2A 280nm; recorder parameters: 200mv 2cm / h; constant flow pump flow rate: 85ml / h;

[0088] Subsequent steps are the same as in Example 1. Freeze-drying can obtain 362 mg of high-purity cobotide finished product, with a neurotoxic protein content of 80.35%; one SDS-PAGE electrophoresis is consistent with the cobotide reference substance; HPLC purity is 98.96%, and the chrom...

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Abstract

In consideration of physical and chemical properties that the cobratoxin is complex in component and cobratide is basic polypeptide, has molecular weight of about 7000 Dalton ad is relatively resistant to acid and alkali, and by considering the separating mechanism, the application characteristic, the long-term use stability, the cost controllability and the achievement of an extracting goal of each chromatographic column packing, the invention provides an extracting method of high-purity cobratoxin. The extracting method disclosed by the invention is stable in process, easily available in chromatography packing, low in comprehensive cost, good in operation condition adaptability and high in yield of neurotoxin. The invention further provides high-purity cobratoxin obtained by the method provided by the invention. An injection cobratide preparation prepared by the high-purity cobratoxin is less in impurities irrelevant with the treatment effect, quick to become effective in treatment effect and stable in curative effect. The invention further provides several pharmaceutical compositions taking the high-purity cobratoxin as an effective constituent formulated with preparation auxiliary materials needed for preparing a preparation as well as cobratide preparations with different dosage forms, which are suitable for being orally taken, absorbed by an oral cavity or rectal administration.

Description

technical field [0001] The application of the present invention relates to a method for extracting high-purity cobra venom neurotoxin and a pharmaceutical composition using the toxin as a single active ingredient. Cobotide is a preparation for oral administration, oral absorption and rectal administration as the main drug, and belongs to the field of biological and biochemical pharmacy. Background technique [0002] Neurotoxin (NT) is an important active component in snake venom, which is mainly distributed in rattlesnake venoms of Cobras, Sea Snakes and Viperidae. The purified trace cobra venom neurotoxin has a central analgesic effect similar to that of morphine, and is used for the analgesic treatment of intractable pain, malignant tumor pain and joint pain. The analgesic effect of NT is different from that of opioids, which is characterized by high potency, long duration, no tolerance and addiction. It is a new type of analgesic with great potential. Studies have shown...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C07K1/18C07K1/16A61K38/17A61P25/04
CPCA61K38/1703C07K14/46
Inventor 张庆宇
Owner 张庆宇
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