CN109651496A - A kind of echidnotoxin stock solution, its composition and the method for stablizing echidnotoxin - Google Patents
A kind of echidnotoxin stock solution, its composition and the method for stablizing echidnotoxin Download PDFInfo
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- CN109651496A CN109651496A CN201811385662.XA CN201811385662A CN109651496A CN 109651496 A CN109651496 A CN 109651496A CN 201811385662 A CN201811385662 A CN 201811385662A CN 109651496 A CN109651496 A CN 109651496A
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- echidnotoxin
- stock solution
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- cobratide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention relates to a kind of echidnotoxin stock solution, its composition and the methods for stablizing echidnotoxin.The echidnotoxin main component is selected from the purification of Cobratide or Cobratide.The echidnotoxin stock solution pH value is 6.5~7.5.The echidnotoxin stock solution stability is good, almost unchanged in the content 12h of contained echidnotoxin under room temperature, is conducive to the holding of content and pharmacological activity in the storage and application process of echidnotoxin and combinations thereof, to guarantee clinical drug effect.
Description
Technical field
The invention belongs to biology and field of medicaments, in particular to a kind of echidnotoxin stock solution, its composition and stabilizations
The method of echidnotoxin.
Background technique
Before more than 2,000 years, the Shennong's Herbal of the Western Han Dynastry is described with snake come the method as medicine.Work " this of Li Shizhen (1518-1593 A.D.)
Careless detailed outline " in also mention " pallas pit viper can control withered, tribute disease pain in the chest and abdomen between brothers' internal organs of rising on one's feet ".The poison gland of snake can be secreted complicated
Mixture, including protein, polypeptide, enzyme and some small-molecule substances, with certain biological activity.From 20th century
Just, treatment of the snake venom in terms of pain just has research, and gradually has echidnotoxin Cobroxin, Nyloxin, Cobratide
(Cobratide) etc. it is found.Cobroxin, Nyloxin, Cobratide etc. not only have analgesic effect, but also without morphine etc.
It is additive." Chinese Pharmacopoeia " is recorded, and Cobratide is white powder, is the Nervous toxicity egg isolated and purified from southern cobra-venom
It is white；It is calculated by dry product, 35% must not be less than containing neural toxalbumin.The pharmacopeia such as identification, inspection, the content assaying method of Cobratide
On it is also on the books.
However, Cobratide belongs to protein, stability is poor, is easy denaturation, and deposit is difficult, and it is various to be that snake venom neurotoxin is made
The pharmaceutical preparation of dosage form, for one of clinical major technology bottleneck.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of echidnotoxin stock solution, the echidnotoxin main component is selected from section
The purification of rich peptide or Cobratide, pH value are 6.5~7.5, and preferentially include the phosphate buffer solution of pH=6.5~7.5.
Further preferably, the echidnotoxin stock solution includes the phosphate buffer solution of pH=6.8, specifically, described
Phosphate buffer solution can be by as follows at being grouped as: 0.2mol/L dipotassium hydrogen phosphate solution: 0.2mol/L sodium hydroxide solution: water
=25%:12%:63%.
As another preferred solution, the echidnotoxin stock solution includes the phosphate buffer solution of pH=7.0；
Further preferably, the echidnotoxin stock solution is by certain density echidnotoxin solution and pH=7.0 phosphate
Buffer is formulated in the ratio of 1:9.
Further preferably, the concentration of echidnotoxin is 300~350 μ g/mL in echidnotoxin solution, and is with 300 μ g/mL
It is good.
The present invention also provides the compositions comprising the echidnotoxin stock solution.The composition can be used as drug, in drug
Between product and quality testing sample or test sample.
When as drug or drug centre product, suitable pharmaceutic adjuvant, including but not limited to talcum powder, titanium dioxide are added
Silicon, magnesium silicate, glucose, lactose, silica gel, poly- D-sorbite, starch, cellulose, carbomer, povidone, poloxamer, poly- second
Glycol, cyclodextrin, dimeticone etc..
The present invention also provides a kind of methods of stable echidnotoxin: the PH of echidnotoxin being adjusted to 6.5~7.5, and preferential
Using the phosphate buffer solution of pH=6.5~7.5, the phosphate buffer solution of pH=6.8 or 7.0 is further preferentially used.
Echidnotoxin stock solution stability of the invention is good, under room temperature, in the content 12h of contained echidnotoxin almost
It is constant, be conducive to the holding of content and pharmacological activity in the storage and application process of echidnotoxin and combinations thereof.The deposit
It, can storage-stable half a year under the conditions of -20 DEG C of liquid.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
One, the specific embodiment of echidnotoxin stock solution
(1) embodiment
The echidnotoxin stock solution of embodiment 1:pH=6.5
Preparation method: preparing the echidnotoxin solution of 300 μ g/mL with ultrapure water, will with the phosphate buffer of pH=6.5
Echidnotoxin solution is diluted to 30 μ g/mL to get echidnotoxin stock solution.
The echidnotoxin stock solution of embodiment 2:pH=6.8
Preparation method: preparing the echidnotoxin solution of 300 μ g/mL with ultrapure water, will with the phosphate buffer of pH=6.8
Echidnotoxin solution is diluted to 30 μ g/mL to get echidnotoxin stock solution.
The echidnotoxin stock solution of embodiment 3:pH=7.0
Preparation method: preparing the echidnotoxin solution of 300 μ g/mL with ultrapure water, will with the phosphate buffer of pH=7.0
Echidnotoxin solution is diluted to 30 μ g/mL to get echidnotoxin stock solution.
The echidnotoxin stock solution of embodiment 4:pH=7.5
Preparation method: preparing the echidnotoxin solution of 300 μ g/mL with ultrapure water, will with the phosphate buffer of pH=7.5
Echidnotoxin solution is diluted to 30 μ g/mL to get echidnotoxin stock solution.
(2) measurement of echidnotoxin stock solution stability
1. high-efficient liquid phase chromatogram condition
Chromatographic column: Dikma Plastil ODS column (4.6mm × 150mm, 5um)；The selection of mobile phase: with 0.1% trifluoro
Aqueous acetic acid is mobile phase A, and trifluoracetic acid-acetonitrile-water (1 ︰, 500 ︰ 500) is Mobile phase B, 20 μ L of sample introduction.
Condition of gradient elution: elution original state Mobile phase B is 8%, keeps 2min, Mobile phase B increases in 8min
100%, 8min is kept, then return to original state in 6min, keeps 10min.36 DEG C of column temperature；Flow velocity is every 1mL.min-1；Inspection
Survey wavelength is 277nm.Number of theoretical plate should be greater than 3000 with the peak calculating of echidnotoxin.
2. Stability Determination
The echidnotoxin solution of 300 μ g/mL is prepared with ultrapure water, uses pH=5, pH=6.5, pH=6.8, pH=respectively
7.0, echidnotoxin solution is diluted to 30 μ g/mL by the phosphate buffer of pH=7.5, pH=9, is placed at room temperature for.Respectively at 0,
2, it 4,8,10,12, takes for 24 hours a little, echidnotoxin content is measured under 277nm using high performance liquid chromatography, initial concentration is set to
100%, the content of different time echidnotoxin is calculated with calibration curve method.
The result shows that echidnotoxin stability in the phosphate buffer of pH=6.5~7.5 is good, damage in content value 12h
Lose small, the variation minimum especially in the content value 12h in the phosphate buffer of pH=7.0 (see Table 1 for details).
Stability of 1 echidnotoxin of table in different pH solution
Two, the specific embodiment of the composition of the stock solution containing echidnotoxin
Embodiment 1: the composition of the stock solution containing echidnotoxin.
Preparation method: taking the echidnotoxin stock solution of pH=7, other reagents are then added as needed and are configured to containing snake venom
The detection sample or test sample of the drug of albumen.
Embodiment 2: the pharmaceutical composition of the stock solution containing echidnotoxin.
Preparation method: the echidnotoxin stock solution of pH=7 is taken, then adds suitable pharmaceutic adjuvant, including but not limited to
Talcum powder, silica, magnesium silicate, polyester acid magnesium, glucose, lactose, silica gel, poly- D-sorbite, starch, cellulose, card wave
Nurse, povidone, poloxamer, polyethylene glycol, dextrin, cyclodextrin, dimeticone etc. are configured to the pharmaceutical composition containing echidnotoxin
Object.
Three, stablize the specific embodiment of the method for echidnotoxin
The echidnotoxin solution that 350 μ g/mL are prepared with ultrapure water, with the phosphate buffer of pH=7.0 by echidnotoxin
Solution is diluted to 35 μ g/mL, -20 DEG C of storages.
Although the present invention is described in detail referring to the foregoing embodiments, those skilled in the art should manage
Solution: it is still possible to modify the technical solutions described in the foregoing embodiments, or to part of technical characteristic into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The spirit and scope of scheme.
Claims (10)
1. a kind of echidnotoxin stock solution, the echidnotoxin main component is selected from the purification of Cobratide or Cobratide, special
Sign is that pH value is 6.5~7.5.
2. echidnotoxin stock solution according to claim 1, it is characterised in that the phosphate comprising pH=6.5~7.5 is slow
Rush solution.
3. echidnotoxin stock solution according to claim 2, it is characterised in that the phosphate-buffered comprising pH=6.8 is molten
Liquid.
4. echidnotoxin stock solution according to claim 2, it is characterised in that the phosphate-buffered comprising pH=7.0 is molten
Liquid.
5. echidnotoxin stock solution according to claim 4, it is characterised in that by echidnotoxin solution and pH=6.5~
7.5 phosphate buffers are formulated in the ratio of 1:9.
6. echidnotoxin stock solution according to claim 5, it is characterised in that echidnotoxin is dense in echidnotoxin solution
Degree is 300~350 μ g/mL.
7. echidnotoxin stock solution according to claim 6, it is characterised in that echidnotoxin is dense in echidnotoxin solution
Degree is 300 μ g/mL.
8. a kind of composition, it is characterised in that include the described in any item echidnotoxin stock solutions of claim 1~7.
9. a kind of method of stable echidnotoxin, the echidnotoxin main component is selected from the purification of Cobratide or Cobratide,
It is characterized in that its PH is adjusted to 6.5~7.5.
10. the method for stable echidnotoxin according to claim 9, it is characterised in that using the phosphoric acid of pH=6.5~7.5
Salt buffer solution adjusts pH value.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811385662.XA CN109651496A (en) | 2018-11-20 | 2018-11-20 | A kind of echidnotoxin stock solution, its composition and the method for stablizing echidnotoxin |
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| CN201811385662.XA CN109651496A (en) | 2018-11-20 | 2018-11-20 | A kind of echidnotoxin stock solution, its composition and the method for stablizing echidnotoxin |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007740A2 (en) * | 2000-07-24 | 2002-01-31 | Bayer Aktiengesellschaft | Snake toxin and use thereof as a pharmaceutical |
| CN101381408A (en) * | 2007-09-06 | 2009-03-11 | 北京赛生药业有限公司 | Cobratide extraction method, cobratide extracted thereby and formulation containing cobratide |
| CN104327176A (en) * | 2014-10-24 | 2015-02-04 | 张庆宇 | Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin |
| CN104645312A (en) * | 2013-11-21 | 2015-05-27 | 贵州益佰制药股份有限公司 | Compounded analgesic preparation containing cobratide and oxycodone |
| CN105566482A (en) * | 2014-10-10 | 2016-05-11 | 贵州益佰制药股份有限公司 | Convenient and fast cobratide separation extraction technology |
-
2018
- 2018-11-20 CN CN201811385662.XA patent/CN109651496A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007740A2 (en) * | 2000-07-24 | 2002-01-31 | Bayer Aktiengesellschaft | Snake toxin and use thereof as a pharmaceutical |
| CN101381408A (en) * | 2007-09-06 | 2009-03-11 | 北京赛生药业有限公司 | Cobratide extraction method, cobratide extracted thereby and formulation containing cobratide |
| CN104645312A (en) * | 2013-11-21 | 2015-05-27 | 贵州益佰制药股份有限公司 | Compounded analgesic preparation containing cobratide and oxycodone |
| CN105566482A (en) * | 2014-10-10 | 2016-05-11 | 贵州益佰制药股份有限公司 | Convenient and fast cobratide separation extraction technology |
| CN104327176A (en) * | 2014-10-24 | 2015-02-04 | 张庆宇 | Extracting method of high-purity cobratoxin and pharmaceutical composition containing high-purity cobratoxin |
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| TA01 | Transfer of patent application right |
Effective date of registration: 20211224 Address after: Room 301, building 15, No. 439, Changhong Road, Yuhuatai District, Nanjing, Jiangsu 210012 Applicant after: NANJING ANGGU PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Address before: 211100 No.18 Zhilan Road, gaoxinyuan, Jiangning District, Nanjing City, Jiangsu Province Applicant before: NANJING ANGFENG PHARMACEUTICAL TECHNOLOGY Co.,Ltd. |
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Application publication date: 20190419 |