CN108273067A - Glutamic acid stabilizer and preparation method for snake venom enzyme preparation - Google Patents
Glutamic acid stabilizer and preparation method for snake venom enzyme preparation Download PDFInfo
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- CN108273067A CN108273067A CN201810115677.8A CN201810115677A CN108273067A CN 108273067 A CN108273067 A CN 108273067A CN 201810115677 A CN201810115677 A CN 201810115677A CN 108273067 A CN108273067 A CN 108273067A
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- glutamic acid
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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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4806—Hydrolases (3) acting on peptide bonds (3.4) from animals other than mammals, e.g. snakes
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to pharmaceutical fields.The preparation method of glutamic acid stabilizer of the present invention for snake venom enzyme preparation comprises the steps of：Pharmaceutical grade glutamic acid 14.713g is weighed, appropriate water for injection is added to be heated to boiling, is 4.5 8.0 with pharmaceutical grade arginine or pharmaceutical grade lysine tune pH value, benefit injects water to 200ml, is made into the stabilizer of the concentration of the 0.5M in terms of glutamic acid.The advantages of this stabilizer：1. molecular weight is small；2. there is no antagonism with main ingredient snake venom enzyme preparation or having synergistic effect；3. without UV absorption, the measurement of preparation HPLC purity is not interfered with；4. two groups of amino acid it is electrically charged on the contrary, not influenced on the purity detecting of preparation SDS electrophoresis；5. not interfering main ingredient titration, main ingredient can be made accurately to feed intake, improve accuracy safety and the curative effect of clinical application.
Description
Technical field
The invention belongs to pharmaceutical fields.
Background technology
Chinese Patent Application No. 201410348725.X discloses a kind of " adjuvant of stabilizing pharmaceutical composition and auxiliary containing this
The pharmaceutical composition of agent ", the patent application provide a kind of adjuvant of stabilizing pharmaceutical composition and the pharmaceutical composition containing the adjuvant
Object, especially a kind of high stability using Defibrase as the pharmaceutical composition of main ingredient.In original Defibrase powder needle and injection group
It is added to enzyme activity protective agent in side, pharmaceutical active ingredient is Defibrase, and other non-active ingredients are stabilizer in prescription
With excipient dextran and enzyme activity protective agent, enzyme activity protective agent is small molecule enzyme activity protective agent or macromolecular enzyme activity
Protective agent or small molecule enzyme activity protective agent and the protectant combination of macromolecular enzyme activity are produced with guarantee, stored, sold
The stabilization of Defibrase potency in journey reduces production rate of charge, ensure that the titer plateaus of finished product, improves the peace of clinical application
Full property and curative effect.
Other than the above patent, the patent in terms of not retrieving in relation to reptilase formulation stabilizer agent and document.However snake
Toxenzyme is active protease, that is, can just be prepared into the medicament for various disease using its activity.General reptilase
All be unstable, still more its dosage is typically all Gamma Magnitude, micro reptilase easily by temperature, pH value, polymerization,
The influence of many factors such as degradation and oxidation, enzyme activity is unstable.Lead to the snake from the links such as production, transport, sale
The decline of toxenzyme preparation potency, therefore in the production process of snake venom enzyme preparation, in order to ensure that preparation potency meets quality standard, respectively
Manufacturer takes different measure according to the experience of oneself, still cannot get a satisfactory effect, only suffers from and do not find
Good stabilizer.Due to the unstable or protective agent of potency or the factor of pH value adjustment agent, seriously affect by prescription
The risk that the effect of dosage rational use of medicines and many unknown adverse reactions generate.
Existing technical solution is to add the people of 0.01-5% in drug prescription in the production process of snake venom enzyme preparation
Protective agent of the partial hydrolysis gelatin or gelatin hydrolysate of blood albumin and 0.001%-0.5% (W/V) as enzyme activity.Certain people's blood
Pure albumen and gelatin or partial hydrolysis gelatin and gelatin hydrolysate have protective effect to enzyme activity really, but are risen to the pharmacology of reptilase
Adverse reaction, these substances have expansion to reptilase, cause reptilase decrement to feed intake, it is reasonable by prescribed dose to seriously affect
The effect of medication, does not meet the requirement of GMP yet；More fatal is them in finished product HPLC purity detectings and SDS- polyacrylamides
When glue gel electrophoretic determination, non-compliant requirement is not inconsistent standardization and is just far from being titer plateaus, let alone improves and face
The safety of bed medication and curative effect.
Invention content
The purpose of the present invention is to provide a kind of preparation methods for keeping snake venom enzyme preparation safer, more effective, more stable.
Another object of the present invention is to provide this glutamic acid stabilizers.
The preparation method of glutamic acid stabilizer of the present invention for snake venom enzyme preparation comprises the steps of：It weighs
Pharmaceutical grade glutamic acid 14.713g adds appropriate water for injection to be heated to boiling, with pharmaceutical grade arginine or pharmaceutical grade lysine tune PH
Value is 4.5-8.0, and benefit injects water to 200ml, is made into the stabilizer of the concentration of the 0.5M in terms of glutamic acid.
In order to find a kind of charge balance agent of suitable snake venom enzyme preparation, the applicant has spent the time for many years, has done repeatedly
Experiment, has put into substantial contribution and has finally just found charge balance agent described herein --- stabilizer.This stabilizer is a kind of
Brand-new compound small-molecular peptides, are formed by acidic amino acid and basic amino acid reaction bonded, and forefathers did not use, do not have
It named, so the application is referred to as systems stabilisation.The advantages of this stabilizer：It is by two groups of needed by human body 1. molecular weight is small
Combination of amino acids；2. there is no antagonism with main ingredient snake venom enzyme preparation or having synergistic effect；3. without UV absorption, to preparation
The measurement of HPLC purity does not interfere with；4. two groups of amino acid it is electrically charged on the contrary, there is no shadow to the purity detecting of preparation SDS electrophoresis
It rings；5. not interfering main ingredient titration, main ingredient can be made accurately to feed intake, improve accuracy safety and the curative effect of clinical application；⑥
It is not required to add other non-human required pH value adjustment agent in preparation, safety higher keeps preparation component single, convenient for mirror
It is fixed, more meet the requirement of GMP.
Glutamic acid stabilizer of the present invention is mainly used in snake venom enzyme preparation.
The present invention has surmounted the thinking of the prior art, uses a systems stabilisation as stabilizer.With the big phase diameter of the prior art
Front yard, crucial effect are to achieve unexpected effect --- it does not need excess and feeds intake and with splendid stability.
Experimental data：
Contrast experiment：
Prepare contrast medium 1：
Defibrase freeze drying powder injection (10U/ bottles of specification)
Defibrase total amount 10000U
Dextran 40 10.0g
Benefit injects water to 1000ml
Preparation method is：Weigh in the balance and take 10.0g dextran for injection 40, add appropriate water for injection boil be dissolved to it is clear
Clearly, it is cooled to room temperature spare.Above-mentioned Dextran 40 liquid and Defibrase liquid 10000U mixing and are added into injection in graduated cylinder
Water to 1000ml, aseptic filtration is sub-packed in by 1.0ml/ bottles in 3ml cillin bottles, half tamponade, is sent into freeze drying box, is freezed to -35
DEG C hereinafter, vacuumize, 1 DEG C -5 DEG C of the heating of shelf setting per hour, highest preset temperature is no more than 28 DEG C, reaches the default highest temperature
It is kept for 3 hours after degree, you can total head plug, outlet roll lid.
Detection：Every bottle of throwing 10U, actual measurement only 5.58U, undesirable after freeze-drying.
7.0 glutamic acid of 10ml 0.5M pH values-arginic stabilizer is added in above-mentioned contrast medium 1, preparation method is same
Contrast medium 1.
Detection：Every bottle of throwing 10U surveys 10.22U and meets the requirements after freeze-drying.
Prepare contrast medium 2：
Defibrase liquid drugs injection (10U/ bottles of specification)
Defibrase total amount 10000U
Benefit injects water to 1000ml
Preparation method：By Defibrase 10000U in graduated cylinder and mend inject water to 1000ml, aseptic filtration is pressed
1.0ml/ bottles are sub-packed in 2ml peaces and cut open in bottle.
Detection：Every bottle of throwing 10U, actual measurement only have 10.34U, temporarily meet the requirements.
0.5M PH5.0 glutamic-lysine stabilizer 10ml, the complete phase of preparation method are added in above-mentioned contrast medium 2
Together.
Detection：Every bottle of throwing 10U, surveys 10.25U, meets the requirements.
Prepare contrast medium 3：
Injection-use reptilase (Ba Quting) (1KU/ bottles of specification)
Hemagglutinase total amount 100KU
Mannitol 1.0g
Benefit injects water to 100ml
Preparation method：It weighs in the balance and takes 1.0g injection mannitol, add appropriate water for injection to boil dissolving, be cooled to room temperature
It is spare.By above-mentioned mannitol and blood clotting enzyme solution 100KU in 100ml graduated cylinders mixing and mend inject water to 100ml, degerming
Filter, is sub-packed in by 1.0ml/ bottles in 3ml cillin bottles, half tamponade, is sent into freeze drying box, freezes to -35 DEG C hereinafter, vacuumizing, often
1 DEG C -5 DEG C of hour shelf setting heating, highest preset temperature is no more than 28 DEG C, reaches and keeps 3 hours after default maximum temperature, i.e.,
Can total head plug, outlet, roll lid.
Detection：Every bottle of throwing 1KU, surveys 0.51KU after freeze-drying, undesirable.
0.5M PH6.5 glutamic-lysine systems stabilisation 2.0ml are added in above-mentioned contrast medium 3, preparation method is the same as right
Than agent 3.
Detection：Every bottle of throwing 1KU, surveys 1.09KU after freeze-drying, meets the requirements.
Prepare contrast medium 4：
Batroxobin injection (5BU/ bottles of specification)
Batroxobin total amount 500BU
Benefit injects water to 100ml
Preparation method：For the accurate amount for measuring Batroxobin 500BU in 100ml graduated cylinders, benefit injects water to 100ml, removes
Bacterium is filtered, 1.0ml/ bottles of packing.
Detection：Every bottle of throwing 5BU, surveys 5.23BU, temporarily meets the requirements.
In above-mentioned contrast medium 4,0.5MPH5.5 glutamic acid-arginine systems stabilisation 2.0ml is added, preparation method is the same as right
Than agent 4.
Detection：Every bottle is still thrown 5BU, is surveyed 5.14BU, is met the requirements.
1 accelerated stability of table is tested
In conclusion be not added with this systems stabilisation from the point of view of contrast experiment, lost in freeze-drying process it is larger, it is undesirable,
Liquid drugs injection can temporarily meet the requirement of potency, but stability is undesirable；Only add this systems stabilisation, freeze drying powder injection and
Liquid drugs injection could meet the requirement of stability；This systems stabilisation is not only suitable for Defibrase, also is adapted for other snake venom enzyme preparations.
Specific implementation mode
Embodiment 1：
Defibrase freeze drying powder injection (10U/ bottles of specification)
Systems stabilisation is prepared：Weighing pharmaceutical grade glutamic acid 14.713g adds appropriate water for injection to heat, with pharmaceutical grade arginine
It is 7.0 to adjust pH value, and benefit injects water to 200ml, is made into that the concentration of the 0.5M in terms of glutamic acid is spare to take 10ml practical.Preparation method
With contrast medium 1.
Embodiment 2：
Defibrase liquid drugs injection (10U/ bottles of specification)
Defibrase total amount 10000U
0.5M PH5.0 glutamic-lysine systems stabilisations 10ml
Systems stabilisation is prepared：Weighing pharmaceutical grade glutamic acid 14.713g adds appropriate water for injection to heat, with pharmaceutical grade lysine
It is 5.0 to adjust pH value, and benefit injects water to 200ml, and it is spare to be made into the concentration of the 0.5M in terms of glutamic acid.Preparation method is the same as contrast medium 2.
Embodiment 3：
Injection-use reptilase (Ba Quting) (1KU/ bottles of specification)
Systems stabilisation is prepared：Weighing pharmaceutical grade glutamic acid 14.713g adds appropriate water for injection to be heated to boiling, and is relied with pharmaceutical grade
Propylhomoserin tune pH value is 6.5, and benefit injects water to 200ml, and it is spare to be made into the concentration of the 0.5M in terms of glutamic acid, and 2.0ml is practical.It prepares
Method is the same as contrast medium 3.
Embodiment 4：
Batroxobin injection (5BU/ bottles of specification)
Batroxobin total amount 500BU
0.5M PH5.5 glutamic acid-arginine systems stabilisation 2.0ml
Benefit injects water to 100ml
Systems stabilisation is prepared：Weighing pharmaceutical grade glutamic acid 14.713g adds appropriate water for injection to heat, with pharmaceutical grade arginine
It is 5.5 to adjust pH value, and benefit injects water to 200ml, and it is spare to be made into the concentration of the 0.5M in terms of glutamic acid, takes 2.0ml practical.Preparation side
Method is the same as contrast medium 4.
Note：Hemagglutinase (Ba Quting) and Batroxobin, are purchased from market.Hemagglutinase first adds appropriate water for injection to dissolve, Ba Qu
Enzyme, which is first freeze-dried, to be concentrated into right amount, then abandons all auxiliary materials in former preparation with special process is de-, since quantity limits experiment
It measures less.
Claims (2)
1. a kind of preparation method of glutamic acid stabilizer for snake venom enzyme preparation, it is characterised in that comprise the steps of：Claim
Pharmaceutical grade glutamic acid 14.713g is taken, appropriate water for injection is added to be heated to boiling, with pharmaceutical grade arginine or pharmaceutical grade lysine tune
PH value is 4.5-8.0, and benefit injects water to 200ml, is made into the stabilizer of the concentration of the 0.5M in terms of glutamic acid.
2. a kind of glutamic acid stabilizer as described in claim 1 being used for snake venom enzyme preparation, it is characterised in that by the above method
It is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810115677.8A CN108273067B (en) | 2018-02-06 | 2018-02-06 | Glutamic acid stabilizer for snake venom enzyme preparation and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810115677.8A CN108273067B (en) | 2018-02-06 | 2018-02-06 | Glutamic acid stabilizer for snake venom enzyme preparation and preparation method thereof |
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| Publication Number | Publication Date |
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| CN108273067A true CN108273067A (en) | 2018-07-13 |
| CN108273067B CN108273067B (en) | 2020-12-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201810115677.8A Active CN108273067B (en) | 2018-02-06 | 2018-02-06 | Glutamic acid stabilizer for snake venom enzyme preparation and preparation method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361717A (en) * | 2007-08-08 | 2009-02-11 | 北京赛生药业有限公司 | Batroxobin freeze-dry powder injection and preparation method thereof |
| CN104083756A (en) * | 2014-07-22 | 2014-10-08 | 张庆宇 | Auxiliary agent for stabilizing pharmaceutical composition and pharmaceutical composition containing auxiliary agent |
-
2018
- 2018-02-06 CN CN201810115677.8A patent/CN108273067B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101361717A (en) * | 2007-08-08 | 2009-02-11 | 北京赛生药业有限公司 | Batroxobin freeze-dry powder injection and preparation method thereof |
| CN104083756A (en) * | 2014-07-22 | 2014-10-08 | 张庆宇 | Auxiliary agent for stabilizing pharmaceutical composition and pharmaceutical composition containing auxiliary agent |
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| Publication number | Publication date |
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| CN108273067B (en) | 2020-12-08 |
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Address after: 650500 No. 789, Lanmao Road, majinpu sub district office, Kunming high tech Zone, Kunming, Yunnan Patentee after: KUNMING LONGJIN PHARMACEUTICAL Co.,Ltd. Address before: No. 789, Lanmao Road, majinpu, Chenggong District, Kunming, Yunnan 650000 Patentee before: KUNMING LONGJIN PHARMACEUTICAL Co.,Ltd. |