CN1432406A - Application of snake venom protein hydrolase in preparing medicine - Google Patents
Application of snake venom protein hydrolase in preparing medicine Download PDFInfo
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- CN1432406A CN1432406A CN 02158007 CN02158007A CN1432406A CN 1432406 A CN1432406 A CN 1432406A CN 02158007 CN02158007 CN 02158007 CN 02158007 A CN02158007 A CN 02158007A CN 1432406 A CN1432406 A CN 1432406A
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Abstract
The present invention is the new application of snake venom protein hydrolase in preparing hemostatic with blood coagulating effect. Specifically, from pallas pit viper venom, snake venom protein hydrolase including fibrinogen degrading enzyme and pallas pit viper thrombase is extracted and separated. The present invention features that through small dosage injection, fibrinogen is bleeding part is degraded to release fibrillarin peptide A, fibrillarin is polymerized to speed the formation of thrombocyte hemostatic plug, to shorten bleeding period and to reduce blood loss. Compared with available great dosage application, the present invention has the advantages of fast hemostasis, low cost, etc.
Description
Technical field
The present invention relates to the new purposes of venom proteolytic enzyme at pharmaceutical field.
Background technology
At present, venom proteolytic enzyme is just made anticoagulant with heavy dose clinically and is used, and with thrombus, suppresses thrombosis, microcirculation improvement.Be used for thrombotic disease, the treatment of cerebral infarction, thromboangiitis obliterans, femoral artery thromboembolism, pulmonary infarction etc., dosage is each intravenous injection 5-10 Defibrase unit (the Defibrase standard substance that provide with Nat'l Pharmaceutical ﹠ Biological Products Control Institute are measured the enzyme unit of the gained of tiring), during this dosage range, Fibrinogen in the blood is consumed in a large number, blood viscosity obviously reduces, thereby suppresses thrombosis or make thromboembolism, reaches the purpose of treatment thrombotic disease.The local medicine of past some province's approval of China such as Ahylysantinfarctase, refining Svate, Jiangsu and Zhejiang Provinces Svate, northeast agkistrodon halys ussuriensis Ahylysantinfarctase, agkistrodon acutus venom defibrase and injection thereof, though its title difference, but main component unanimity, all belong to this class venom proteolytic enzyme, for with a kind of medicine, playing National Drug Administration in 2000 is unified management, is Defibrase and injection Defibrase with its title is unified, and has worked out unified office and issued drug standard (standard No. is respectively WS
1-XG-031-2000 and WS
1-XG-032-2000).This zymoid molecular weight ranges is 28000 to 42000Dalton (sds polyacrylamide gel electrophoresis method mensuration).
Summary of the invention
The objective of the invention is for now only as the preparation anticoagulant this class venom proteolytic enzyme a kind of new purposes is provided, promptly in pharmaceutical field in order to the preparation hemostatic agent; And develop its high-purity venom proteolytic enzyme in order to the preparation hemostatic agent.
The present invention makes the Defibrase in the venom proteolytic enzyme hemorrhage of a per injection 0.0005-1 Defibrase unit; Purity is reached venom proteolytic enzyme (as Halase) more than 95% to be made a per injection 1-2 thrombin unit (a thrombin unit is equivalent under 37 ± 0.5 ℃ of conditions, make the jolting in 60 ± 20 seconds of 0.2ml normal man's blood plasma, the reptilase amount of white wadding net occurs) hemorrhage.
The used snake venom of the present invention is preferably gathered from agkistrodon shedaoensis Agkistrodon Shedaoensis (zhan), Jiangsu and Zhejiang Provinces Agkistrodon halys, Agkistrodon acutus Agkistrodon acutus (Guenther) or Changbai Mountain agkistrodon halys ussuriensis Agkistrodonhalys Agkistrodon halyss such as (ussriensis Emelianor), again through extract or separate, refining obtaining, (drug standard number is respectively WS for Defibrase of producing as National Drug Administration's approval and injection Defibrase
1-XG-031-2000 and WS
1-XG-032-2000) [its main constituent molecular weight ranges is 28000 to 42000Dalton (sds polyacrylamide gel electrophoresis method mensuration)] reduces dosage and makes injection, and dosage is by the administration of each 0.0005-1 Defibrase unit; Also can be molecular weight be positioned at this scope, purity up to the Agkistrodon halys thrombin more than 95% (as Halase Heamocoagulase Agkistrodon) and injection [as injection Halase Heamocoagulase Agkistrodon for Injection, its molecular weight is 29000-29500Dalton (a flight mass spectrum method mensuration)], dosage is by a per injection 1-2 thrombin unit.Do not consume the Fibrinogen in the blood when utilizing its low dose of administration in a large number, but with fibrinogen degradation, discharge fibrinopeptide A, and aggregate into fibrin polymer, quicken the formation of blood vessel breakage platelet tampon, obviously shorten the bleeding time, reduce blood loss, play this characteristic of anastalsis, with this class venom proteolytic enzyme be used for reducing bleed or the various medical conditions of hemostatic under hemorrhage and hemorrhage.
With venom proteolytic enzyme with 0.0005-1 Defibrase unit/time or 1-2 thrombin unit/time make injection (aqueous injection or injectable powder) as hemorrhage; Also effective analgesic composition in this class venom proteolytic enzyme and analgesics antibacterial example hydrochloric acid tramadol, GATIFLOXACIN and the snake venom etc. can be united and make vein or intramuscular injection with compound hemostatic analgesia or hemostatic and antibacterial injection water injection or injectable powder.
The present invention has opened up another purposes of this class venom proteolytic enzyme and has promptly made hemorrhage, have quick, the effective advantage of hemostasis, and processing technology is simple, and cost is low.
Description of drawings
Fig. 1 is the curve chart of various dose snake venom Defibrase of the present invention to the influence in mice bleeding time
The snake venom Defibrase can make the bleeding time of mice obviously shorten in 0.0001-0.01 Defibrase unit/kg scope as shown in Figure 1, and when dosage increases to 3.0 Defibrase units/kg, has then obviously prolonged the bleeding time of mice.Have blood coagulation resisting function when showing that this enzyme is heavy dose of and using, and have Blood clotting during low dose of the use.And the dose-difference of its Blood clotting and blood coagulation resisting function is apart from bigger, and about 500 to 1000 times, this has just guaranteed its safety as blood coagulation and blood coagulation resisting function drug use.
Following zoopery further proves the feasibility of venom proteolytic enzyme as coagulant.
Table 1. venin for injection Defibrase to the influence of the former content of rabbit fibrin (FIB) (mg/dl, X ± SD, n=6)
| Before group dosage (U/kg) administration after the administration after the administration in 0.5 hour after the administration in 1 hour 3 hours |
| Matched group (feminine gender) Ns 318.6 ± 37.82 312.4 ± 45.04 309.8 ± 58.36 338.2 ± 47.35 |
| 0.0005 336.42 ± 68.08 341.6 ± 57.37 320.42 ± 53.76 298.7 ± 90.28 sample sets 0.0015 306.4 ± 78.53 298.4 ± 51.38 284.8 ± 58.74 256.42 ± 42.81 0.0045 329.42 ± 37.88 331.7 ± 63.48 318.8 ± 54.04 279.8 ± 37.82 |
Table 2. venin for injection Defibrase is to rabbit platelet function (TXB
2) influence (pg/ml, X ± SD, n=6)
| Before group dosage (U/kg) administration after the administration after the administration in 0.5 hour after the administration in 1 hour 3 hours |
| Matched group (feminine gender) NS 418.64 ± 40.32 340.01 ± 118.86 429.84 ± 39.68 412.84 ± 87.33 |
| 0.0005 381.66 ± 75.83 422.54 ± 38.96 449.63 ± 20.82 418.65 ± 30.64 sample sets 0.0015 419.84 ± 38.42 413.87 ± 20.32 455.86 ± 13.48 398.44 ± 49.63 0.0045 385.66 ± 62.32 392.38 ± 52.44 440.28 ± 30.64 439.63 ± 37.58 |
Table 3. high-purity Agkistrodon halys thrombin to mice cut the tail bleeding time influence (X ± SD, n=10)
Annotate: compare * * p＜0.01 with negative control group
| Group dosage (the bleeding time (second) of μ/kg) |
| Matched group (feminine gender) 63.42 ± 11.32 |
| 0.5 31.62 ± 11.02** sample sets, 0.25 35.11 ± 10.38**, 0.125 38.02 ± 11.12** |
Table 4. high-purity Agkistrodon halys thrombin to the influence of venous hemorrhage time of rabbit ear edge (X ± SD, n=6)
Annotate: compare * * p＜0.01, p＜0.05 with negative control group
| Group dosage (the bleeding time (min) of μ/kg) |
| Matched group (feminine gender) 6.08 ± 0.52 |
| 0.20 3.02 ± 0.38** sample sets, 0.10 3.31 ± 0.45**, 0.05 3.90 ± 0.52* |
Table 1, the 2nd has further illustrated that from mechanism of coagulation the snake venom Defibrase can play Blood clotting really in suitable dosage range, table 1 data show that the snake venom Defibrase is when suitable dose, with negative control group (injection normal saline) mutually specific energy obviously reduce the rabbit blood fibrinogen content, point out it to promote fibrinogen degradation, discharge fibrinopeptide A, and aggregate into fibrin polymer, and play the hemostatic purpose; Table 2 shows this product when suitable dose, can obviously increase TXB2 content, i.e. platelet increasing aggregation capability, and play the hemostatic purpose.Table 3, the 4th, highly purified reptilase-Halase show that to the situation that influences in mice and rabbit bleeding time it can obviously shorten the bleeding time equally when low dosage, reach the hemostasis purpose.
The specific embodiment
Embodiment 1, takes by weighing agkistrodon shedaoensis Defibrase 5000 units, adds 5000g such as excipient dextran or mannitol, adds injection water to 50000ml, and dissolving is filtered the back fill in 50000 cillin bottles, lyophilizing.Vein, intramuscular injection, 1 of per injection.
Embodiment 2, take by weighing Jiangsu and Zhejiang Provinces Agkistrodon halys Defibrase 25 units, add 5000g such as diluent dextran or mannitol, mixing, and equivalent is sub-packed in 50000 cillin bottles.Vein, intramuscular injection, each 1.
Embodiment 3, take by weighing Agkistrodon halys Defibrase 5000 units, are dissolved in the 50000ml water for injection, filter, and fill is in 50000 cillin bottles then.Vein, intramuscular injection, each 1.
Embodiment 4, accurately take by weighing Changbai Mountain agkistrodon halys ussuriensis snake venom Defibrase (about 5000 units), add the injection water to 50ml, dissolving, and it is standby to measure its back of tiring.Measure 20% human albumin's injection 20ml, 6% Dextran-20 sodium chloride injection 83ml, mannitol 30g adds injection water 850ml, stirs, and regulating pH value with hydrochloric acid solution is 6.0 to 6.5.Precision is measured Defibrase reserve liquid an amount of (being equivalent to Defibrase 100 units approximately), joins in the above-mentioned auxiliary material liquid, stirs, and adds the injection water to 1000ml.0.22 μ m membrane filtration after measuring it and tiring, is sub-packed in the cillin bottle of cleaning sterile by the amount of every 0.1U in clean environment, lyophilization becomes sterilized powder.Vein, intramuscular injection, 1 of per injection.
Embodiment 5, take by weighing purity and be 96% 1000 units of Agkistrodon halys thrombin raw material medicine (as Halase) and excipient Dextran 10 g, put in the appropriate vessel, after adding an amount of water for injection dissolving mixing, add water for injection to 500ml, with 0.22 micron membrane filtration, packing in clean environment, make every bottle to contain 1 unit of Halase, lyophilization becomes the lyophilizing sterilized powder.Vein, intramuscular injection, 1 of per injection.
Embodiment 6, take by weighing purity and be 97% 1000 units of Agkistrodon halys thrombin raw material medicine (as Halase) and excipient dextran 4.0g, put in the appropriate vessel, after adding an amount of water for injection dissolving mixing, regulating pH value with sodium hydroxide solution is 6 to 7, adds the injection water to 500ml, membrane filtration with 0.22 micron, packing in clean environment makes every bottle to contain 1 unit of Halase, and lyophilization becomes the lyophilizing sterilized powder.Vein, intramuscular injection, 1 of per injection.
Embodiment 7, getting the snake venom crude product dissolves with PBS, centrifugal, get centrifugal back supernatant and dialyse, the solution that dialysis is held back DEAE-Sepharose CL-6B column chromatography, elution fraction is dialysed again behind the chromatography, the solution that dams is collected sodium chloride-PBS elution fraction once more through DEAE-Sepharose CL-6B column chromatography, reduces phlegm and internal heat former through the SephadexG-75 column chromatography, the desalination of reuse SephadexG-25 column chromatography promptly gets high-purity reptilase crude drug.
The conclusive evidence method: (1) gets this product, measures according to the SDS-polyacrylamide gel electrophoresis, and its molecular weight subunit is 16000 (± 2000) Dalton and 14000 (± 2000) Dalton.
(2) get this product, water is made the solution of 1 units per ml, as need testing solution; Get two of small test tubes, each adds 0.2 milliliter of human plasma, puts in 37 ± 0.5 ℃ the water-bath insulation 2 minutes, adds 0.2 milliliter of need testing solution, puts in 37 ± 0.5 ℃ the water-bath and observes, and solution should condense in two minutes.Add 1 milliliter of 5mol/L urea liquid, jolting, clot dissolution is settled solution.
Embodiment 8, take by weighing snake venom Defibrase 5000 units and tramadol hydrochloride 5000 grams (or 2500 grams), add 5 grams such as excipient dextran or mannitol, add injection water to 50000ml, and dissolving is filtered the back fill in 50000 cillin bottles, lyophilizing.Vein, intramuscular injection, 1 of per injection.
Embodiment 9, take by weighing snake venom Defibrase 5000 units and tramadol hydrochloride 5000 grams (or 2500 grams), add about 3 grams such as excipient dextran or mannitol, mixing, and equivalent is sub-packed in 50000 cillin bottles.Vein, intramuscular injection, each 1.
Embodiment 10, take by weighing snake venom Defibrase 5000 units and tramadol hydrochloride 5000 grams (or 2500 grams), are dissolved in the 50000ml water for injection, filter, and fill is in 50000 cillin bottles then.Vein, intramuscular injection, each 1.
Embodiment 11, accurately take by weighing snake venom Defibrase (about 5000 units) and tramadol hydrochloride 5000 grams (or 2500 grams), add the injection water to 50ml, dissolving, and it is standby to measure its back of tiring.Measure 20% human albumin's injection 20ml, 6% Dextran-20 sodium chloride injection 83ml, mannitol 2 grams add injection water 850ml, stir, and regulating pH value with hydrochloric acid solution is 6.0 to 6.5.Precision is measured Defibrase reserve liquid an amount of (being equivalent to Defibrase 100 units approximately), joins in the above-mentioned auxiliary material liquid, stirs, and adds the injection water to 1000ml.0.22 μ m membrane filtration after measuring it and tiring, is sub-packed in the cillin bottle of cleaning sterile by the amount of every 0.1U in clean environment, lyophilization becomes sterilized powder.Vein, intramuscular injection, 1 of per injection.
Embodiment 12, take by weighing 1000 units of high-purity Agkistrodon halys thrombin raw material medicine (as Halase) and tramadol hydrochloride 5000 grams (or 2500 grams), add 2 grams such as excipient dextran or mannitol, put in the appropriate vessel, add an amount of water for injection dissolving mixing after, add water for injection to 500ml, membrane filtration with 0.22 micron, packing in clean environment makes every bottle to contain 1 unit of Halase, and lyophilization becomes the lyophilizing sterilized powder.Vein, intramuscular injection, 1 of per injection.
Embodiment 13, take by weighing purity and be 95% 1000 units of Agkistrodon halys thrombin raw material medicine (as Halase) and tramadol hydrochloride 5000 grams (or 2500 grams), add 2 grams such as excipient dextran or mannitol, put in the container, after adding 350ml water for injection dissolving mixing, regulating pH value with sodium hydroxide solution is 6 to 7, add the injection water to 500ml, membrane filtration with 0.22 micron, packing in clean environment, make every bottle to contain 1 unit of Halase, lyophilization becomes the lyophilizing sterilized powder.Vein, intramuscular injection, 1 of per injection.
Claims (5)
1, the application of venom proteolytic enzyme in pharmacy, it is characterized in that with extraction from pit viper venom with separate, refined proteolytic enzymes makes the hemorrhage that the per injection amount is a 0.0005-1 Defibrase unit or a 1-2 thrombin unit.
2, the application of venom proteolytic enzyme according to claim 1 in pharmacy, it is characterized in that described enzyme be from pit viper venoms such as agkistrodon shedaoensis, Jiangsu and Zhejiang Provinces Agkistrodon halys, Agkistrodon acutus or Changbai Mountain agkistrodon halys ussuriensis, extract or separate, refining and get, its molecular weight ranges is 28000 to 42000Dalton (the sds polyacrylamide gel electrophoresis method is measured), comprise Defibrase and injection Defibrase, above-mentioned enzyme is made vein or intramuscular injection with injection powder injection or injection, dosage is used for all kinds of hemostasis by the administration of each 1-0.0005 Defibrase unit.
3, application in the pharmacy of venom proteolytic enzyme according to claim 1, the purity that it is characterized in that enzyme is up to (HPLC method mensuration) more than 95%, molecular weight is 29000 to 29500Dalton (flight mass spectrum method mensuration), two the peptides companies (subunit) that are respectively 16000 ± 2000Dalton and 14000 ± 2000Dalton (the sds polyacrylamide gel electrophoresis method is measured) by molecular weight connect into a molecule with disulfide bond, as Halase (Heamocoagulase Agkistrodon) and injection Halase (Heamocoagulase Agkistrodon for Injection), above-mentioned high-purity enzyme is made vein or intramuscular injection with injection powder injection or injection, dosage is used for all kinds of hemostasis by each 1-2 thrombin unit's administration.
4, the application in the pharmacy of venom proteolytic enzyme according to claim 1 is characterized in that this fermentoid is made vein or intramuscular injection with injectable powder or injection, and dosage is used for all kinds of hemostasis by the administration of each 0.01-0.1 Defibrase unit.
5,, it is characterized in that and to contain this zymoid vein or intramuscular injection compound injection (aqueous injection or injectable powder) with medication combined making such as analgesics, antibacterial according to the application in the pharmacy of claim 1 or 2 or 3 described venom proteolytic enzymes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100336903C (en) * | 2004-07-27 | 2007-09-12 | 沈阳斯佳科技发展有限公司 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
| CN1972706B (en) * | 2004-06-24 | 2012-03-21 | 东菱药品工业株式会社 | Malignant tumor-inhibiting preparation comprising des A fibrin |
| WO2022109758A1 (en) * | 2020-11-26 | 2022-06-02 | 沈喆景 | Application of proteolytic enzyme of pit viper in treatment of alzheimer's disease |
-
2002
- 2002-12-20 CN CN 02158007 patent/CN1432406A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1972706B (en) * | 2004-06-24 | 2012-03-21 | 东菱药品工业株式会社 | Malignant tumor-inhibiting preparation comprising des A fibrin |
| CN100336903C (en) * | 2004-07-27 | 2007-09-12 | 沈阳斯佳科技发展有限公司 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
| WO2022109758A1 (en) * | 2020-11-26 | 2022-06-02 | 沈喆景 | Application of proteolytic enzyme of pit viper in treatment of alzheimer's disease |
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