CN100369629C - Compound hemostatic originated from venom - Google Patents
Compound hemostatic originated from venom Download PDFInfo
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- CN100369629C CN100369629C CNB2005100473326A CN200510047332A CN100369629C CN 100369629 C CN100369629 C CN 100369629C CN B2005100473326 A CNB2005100473326 A CN B2005100473326A CN 200510047332 A CN200510047332 A CN 200510047332A CN 100369629 C CN100369629 C CN 100369629C
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Abstract
The present invention relates to compound hemostatic originated from venom, which is characterized in that the medicine comprises the main active components: batroxobin with the purity of more than 97% and X activator of blood coagulation factors. The proportional range of the batroxobin and the X activator of blood coagulation factors is from 1: 1 to 1000: 1. The hemostatic of the present invention can ensure curative effects and simultaneously reduce the dosage of proteinase to the greatest extent, and thereby, the reaction probability of the occurrence of bad immune responses is reduced.
Description
Technical field:
The present invention relates to a kind of enzyme preparation, a kind of compound hemostatic specifically is provided, contained active ingredient is snake venom thrombin-like enzyme (batroxobin) and Stuart factor activator (FXA), and particularly separation purifying technique and the compound formulation prescription to FXA in this medicine carried out preferred.
Background technology:
Discovery adder snake venom such as Macfarlane had the very significant blood coagulation effect that makes in 1934, when its dilution 1000 times the time can also 17 seconds make the hemophilia patients'blood solidify (referring to The Lancet, Drs.Macfarlane﹠amp; Barnett:Haemostatic Possibilities ofSnake-venom, Nov, 3,1934,985-987).External more early stage areas used as the external hemorrhage (referring to The Lancet, Clinical and Labboratory Notes, Feb.22, what 1936,428), used at that time is full poison without separation and purification, as Stypven (Wellcome UK), how this product is as diagnostic reagent (referring to BloodReviews7,176-189,1993).
Austrian scholar Von Klobusitzky isolated multiple blood coagulation composition first from Brazilian spearhead Agkistrodon halys (Bothrops atrox) venom in 1963, found that they work at the varying level of blood coagulation.The blood coagulation composition of first separation and purification is Batroxobin, and it is a kind of serine proteinase enzyme (Thrombin-LikeEnzyme a---batroxobin), just said batroxobin (Stocker, K. ， ﹠amp; Egberg, n., 1973, Thromb.Diath.Haemorrh.1,361; Stocker, K. ， ﹠amp; Barlow, G.H., 1976, MethodEnzymol.45,214; Holleman, W.H. ， ﹠amp; Weiss, L.J., 1976, J.Biol.Chem.251.1663).Fibrinopeptide A in the mammiferous plasma fibrinogen of Batroxobin enzyme action makes it to change into fibrin, therefore in the external cohesion that can cause plasma fibrin, a Blood clotting (Funk, C., etc., 1975, J.Br.J.Haematol.21,43).But it does not activate labile factor and blood coagulation factor VIII (Niewiarowski etc., 1977, Trombin Res.10.863 in vivo; Niewiarowski etc., 1979, Biochemistry, 21,3437; Kirby etc., 1979, Biochemistry, 18,3564), so the side chain of the fibrin clot that is produced by its hydrolysis can not be crosslinked, easily, cause the reduction of body inner fibrin original content and show anticoagulant effect, thereby influence hemorrhage-coagulation process of animal by the plasmin enzymatic degradation.Also contain a kind of hemostasis composition in the Bothrops atrox snake venom, i.e. Stuart factor activator (FXA) (Nahas etc., 1964, Thromb.Diath.Haemorrh.12,355; Nahas etc., 1979, Thromb.Haemorrh, 41,314; Hofmann etc., 1983, Biochemistry, 65,201).It is through the DEAE anion exchange chromatography, the active ingredient that purification process such as gel filtration chromatography obtain, its molecular weight is 75000, reduction SDS-PAGE electrophoresis confirms, it is that 59000 heavy chain and molecular weight are formed (Hofmann etc. by 15000 light chain polymer by disulfide bond by molecular weight, 1987Biochemistry, 26,780).FXA can be converted into Stuart factor has amide active and blood coagulation activity Xa or intermedium.When FXA activates Stuart factor by Ca
2+Ion-activated, FXA does not have hydrolysing activity to synthetic substrate TAME, S-2238, S2337.When FXA and the RVV-X that derives from Vipera rnsselli (or Daboia russelli) are compared, find that both all can activate Stuart factor specifically, at the Arg of the heavy chain of Stuart factor
52-Ile
53Key cuts off, and discharges the bioactive peptide Xa of 52 residues.Different with RVV-X is that FXA also has two other restriction enzyme site: one is the N-terminal at heavy chain, produces factor X μ, and another is at the end of factor Xa α, the factor Xa γ of generation.
The Reptilase of external import (Chinese trade name: reptilase) or Hemocoagulase (referring to Handb Exp.Pharm.52Snake Venom and Blood Coagulation 1991, Reprinted by SigmaChemical Company) be the haemostatic medicament preparation of making by the batroxobin (batroxobin) of extraction from Brazilian spearhead Agkistrodon halys (Bothrops atrox).
Domestic have reptilase Counterfeit Item-Ba Quting, also is the enzyme hemorrhage that batroxobin (batroxobin) composition that extracts from Brazilian spearhead Agkistrodon halys is made.
Domestic also have the adder snake venom test kit of Stuart factor activating enzymes as diagnosis, is used for diagnosing whether Stuart factor lacks in the blood plasma.On April 5th, 2000 disclosed Chinese patent, the number of applying for a patent is CN1249338A, exercise question: a kind of method of high efficiency extraction Stuart factor activating enzymes.RVV-X behind the purification is mainly used in the research of scientific research fields such as tumor migration mechanism.
Another relevant patent is a disclosed Chinese patent on August 18th, 2004, and its number of applying for a patent is CN1520880A, exercise question: a kind of hemorrhage.To derive from the Main Ingredients and Appearance of Vipera russelli (or Daboiarusselli) RVV-X first as hemorrhage, and with snake venom thrombin-like enzyme as synergist.As everyone knows, RVV-X is a metalloproteases, and its molecular weight is 92880D, is respectively two light chains of 16400D, 19400D by the heavy chain of molecular weight 57600D and molecular weight and forms by disulfide bond.It is the best example of the blood coagulation outer activator of research.
In fact,, human body is easy to cause bad immune response as the large molecular weight protein medicine, as: exothermic reaction, produce antibody, form the big immune complex of one's share of expenses for a joint undertaking amount, glomerule is caused certain damage, thereby limited its using dosage.
Summary of the invention:
The purpose of this invention is to provide a kind of compound hemostatic, this hemorrhage can reduce the consumption of protease as far as possible when guaranteeing curative effect, thereby has reduced generation undesirable immune response reaction probability.
The invention provides a kind of compound hemostatic, it is characterized in that: the main active ingredient of described medicine is a purity greater than 97% batroxobin and Stuart factor activator, and the active proportion of batroxobin and Stuart factor activator is 1: 1～1000: 1.Preferable active proportion is 20: 1～500: 1.
In the compound hemostatic of the present invention, described batroxobin is the batroxobin that contains in Brazilian spearhead Agkistrodon halys, agkistrodon halys ussuriensis or the Agkistrodon acutus snake venom, or the batroxobin of the reorganization Brazil spearhead Agkistrodon halys, agkistrodon halys ussuriensis or the Agkistrodon acutus that obtain by genetic engineering.
In the compound hemostatic of the present invention, described Stuart factor activator is Stuart factor activator FXA or the genetic engineering reorganization FXA that contains in the Brazilian spearhead agkistrodon halyx pallas venom; Or derive from the Stuart factor composition of Agkistrodon halys Viperarusselli or Daboia russelli or its genetic engineering recombinant products.
In the compound hemostatic of the present invention; best is chosen as; batroxobin is the batroxobin of Brazilian spearhead Agkistrodon halys a---batroxobin; the Stuart factor activator is the Stuart factor activator FXA that contains in the Brazilian spearhead agkistrodon halyx pallas venom; wherein can also contain the aminoacid composition as the protease protective agent; the aminoacid first-selection is a glycine; can be excipient with mannitol, batroxobin+Stuart factor activator wherein: glycine: the amount of mannitol be 20～0.1: 1～0.01: 0.02～0.06 unit: gram: gram.
In the compound hemostatic of the present invention, the isolation and purification method of its Stuart factor activator is to adopt DEAE-Fast Flow series anionic exchanger resin to separate with Sephacryl series molecular sieve column to realize.Its vigor is identified and is detected by so-called chromogenic substrate method and realize.
The present invention compared with prior art mainly contains following difference.At first be that main pharmacodynamics composition source is different.The molecular weight of considering the Stuart factor activator is bigger, be not suitable as the main pharmacodynamics composition of medicine, so hemorrhage set forth in the present invention is the batroxobin that extracts from Brazilian spearhead agkistrodon halyx pallas venom or the genetic engineering recombinant batroxobin Main Ingredients and Appearance as hemorrhage, its drug effect is remarkable, clinical practice, and the Stuart factor activator as synergist be from Brazilian spearhead agkistrodon halyx pallas venom, extract or obtain by genetic engineering.Next is that both proportioning is different fully, and styptic activity is stronger than the former.Once more, both application dose differences, because styptic activity is stronger than the former, therefore, used dosage is low than the former, but haemostatic effect is better than the former.Be the pharmaceutical formulation difference at last, the pharmaceutical formulation of this hemorrhage contains protease protective agent glycine and excipient mannitol.
The specific embodiment:
1, the isolation and purification method of Stuart factor activator
Take by weighing Brazilian spearhead agkistrodon halyx pallas venom and be dissolved in 10mM Tris-HCl, pH 7.5 (containing 50mM NaC1), and dialysis, centrifugal, get anion-exchange column DEAE-cellulose chromatography on the supernatant, carry out gradient elution, collect active peak with above-mentioned buffer 0.05-0.3M NaCl.Active peak is directly gone up with the equilibrated Sephadex G-150 of same buffer (containing 75mMNaCl) chromatographic column chromatography, and collects active peak, and then goes up the DEAE-cellulose column chromatography.Collect active peak, lyophilizing is preserved under-20 ℃ of conditions.Product confirms that through native polyacrylamide gel electrophoresis its molecular weight is 75000D, and denaturing polyacrylamide gel electrophoresis confirms, it is that the heavy chain of 59000D and light chain that molecular weight is 14000-15000D are formed by disulfide-bonded by molecular weight.Consistent with bibliographical information (Hofmann etc., 1987 Biochemistry, 26,780).
2, the discrimination method of Stuart factor activator (chromogenic substrate method)
The Stuart factor activator can act on Stuart factor, the Xa that the latter discharges can make in chromogenic substrates such as S-2222 or S-2337 and produce p-nitroaniline, by under the 405nm wavelength, detecting the variation of absorbance, realize discriminating and vitality test to the Stuart factor activator.
3, the unit definition of Stuart factor activator
In following system, the variation that detects absorbance value and write down absorbance value in 10 minutes in 405nm wavelength place.
System:
1. buffer 1 (50mmol/L Tris-HCL pH=7.5,150mmol/L NaCl, 2.5mmol/L CaCl
2, (containing 1mmol/L Benzamidine hydrochioride)---100 μ l;
2. the FX of 0.5unit/ml---50 μ l;
3. testing sample---50 μ l;
Fully mix the back in 37 ℃ of insulations 10 minutes;
4. buffer 2 (200mmol/L Tris-HCL pH=8.3,150mmol/L NaCl, 10mmol/LEGTA)---250 μ l;
Abundant mixing;
⑤2.5mmol/ml S-2337——50μl；
Measure the variation of absorbance value in 10 minutes after being mixed rapidly.
Being defined in the vigor that absorbance value under the above-mentioned system is changed to 0.02 ± 0.005 Stuart factor activator is 1 unit.
Simultaneously, the Stuart factor activator also can adopt the external thrombotest method vigor that carries out to detect.
4, external thrombotest
Adopt the active assay method of like product to carry out external thrombotest, concrete experimental technique is: get people-citric acid anticoagulate plasma 0.2ml, add in the test tube of diameter 1cm, put in 37 ℃ of water-baths and be incubated 3 minutes, add the sample solution 0.2ml of 37 ℃ of preheatings, mixing timing immediately, in beginning in 40 seconds, check clotting of plasma situation, the record presetting period, measure 3 pipes simultaneously, 3 pipe presetting period errors should be less than 20 seconds.If the presetting period less than 40 seconds, is then suitably diluted need testing solution, write down the need testing solution concentration of solidifying in 60 ± 20 seconds, under these conditions, the enzyme amount that 0.2ml people-citric acid anticoagulate plasma was solidified at 60 seconds is defined as a unit.
5, procoagulant activity check in the body
Mice docking method is adopted in the test of body intravascular coagulation.Stuart factor activator and batroxobin are mixed with the solution of debita spissitudo respectively as need testing solution.
Get healthy mice, be divided into 7 groups at random, 5 every group, by dosage mouse tail vein injection 0.5ml in the table, and when injecting back 15 minutes, to cut off at distance mouse tail point 0.3cm place with shears, timing immediately after blood flows out voluntarily stops timing to blood coagulation.With the index of bleeding time shortening as the judgement haemostatic effect.Use normal saline as negative control simultaneously.
6, preparation prescription screening
The present invention also provides the preparation prescription of the haemostatic medicament with Stabilization, and this medicine contains batroxobin, FXA, excipient-mannitol and protease protective agent-glycine.
The external thrombotest of embodiment 1 Stuart factor activator (FXA)
Adopt the active assay method of like product to carry out external thrombotest, concrete experimental technique is: get people-citric acid anticoagulate plasma 0.2ml, add in the test tube of diameter 1cm, put in 37 ℃ of water-baths and be incubated 3 minutes, add the sample solution 0.2ml of 37 ℃ of preheatings, mixing timing immediately, in beginning in 40 seconds, check clotting of plasma situation, the record presetting period, measure 3 pipes simultaneously, 3 pipe presetting period errors should be less than 20 seconds.If the presetting period less than 40 seconds, is then suitably diluted need testing solution, write down the need testing solution concentration of solidifying in 60 ± 20 seconds, under these conditions, the enzyme amount that 0.2ml people-citric acid anticoagulate plasma was solidified at 60 seconds is defined as a unit.Calculating is tired, log.
The external thrombotest result of table 1 Stuart factor activator (FXA)
| Experiment number | The diluted sample multiple | Setting time | |||
| 1 | 2 | 3 | Average time | ||
| 1 | 100 | 12 | 12 | 12 | 12 |
| 2 | 300 | 25 | 26 | 25 | 25.3 |
| 3 | 400 | 50 | 51 | 49 | 50 |
| 4 | 450 | 56 | 58 | 57 | 57 |
| 5 | 500 | 60 | 60 | 60 | 60 |
| 6 | 550 | 63 | 64 | 64 | 63.6 |
| 7 | 800 | 86 | 85 | 85 | 85.3 |
This test determines that the vigor of this product is: 500u/ml.
The external thrombotest result of table 2 batroxobin (batroxobin)
| Experiment number | The diluted sample multiple | Setting time (second) | |||
| 1 | 2 | 3 | Average time | ||
| 1 | 100 | 7 | 7 | 7 | 7 |
| 2 | 500 | 25 | 25 | 25 | 25 |
| 3 | 1000 | 60 | 60 | 60 | 60 |
| 4 | 950 | 58 | 57 | 57 | 57.3 |
| 5 | 1050 | 64 | 64 | 64 | 64 |
| 6 | 1200 | 84 | 85 | 83 | 84 |
This test determines that the vigor of this product is: 1000u/ml
Embodiment 2 batroxobins (batroxobin) and Stuart factor activator are worked in coordination with external thrombotest
With concentration is that batroxobin (batroxobin) and the concentration of 500u/ml is that the required proportioning of 500u/ml Stuart factor activator according to the form below is prepared need testing solution, guarantees that the vigor theoretical value of whole solution is: 500u/ml.Positive control 1 is the batroxobin (batroxobin) of 1 unit, and positive control 2 is the Stuart factor activator of 1 unit, and negative control is a normal saline.
Table 3 class is coagulated inulinase (batroxobin) and the collaborative external thrombotest result of Stuart factor activator
| Experiment number | Extension rate | Sample ligand compares coefficient | Corner when solidifying (second) | |||
| 1 | 2 | 3 | Average time | |||
| Positive control 1 | - | - | 61 | 60 | 59 | 60 |
| Positive control 2 | - | - | 60 | 60 | 60 | 60 |
| 1 | 500 | 1∶1 | 60 | 59 | 58 | 59 |
| 2 | 500 | 10∶1 | 57 | 56 | 56 | 57.3 |
| 3 | 500 | 100∶1 | 50 | 52 | 52 | 51.3 |
| 4 | 500 | 500∶1 | 53 | 54 | 52 | 53 |
| 5 | 500 | 1000∶1 | 56 | 58 | 55 | 56.3 |
| 6 | 500 | 1100∶1 | 61 | 62 | 66 | 63 |
| 7 | 500 | 1200∶1 | 63 | 63 | 64 | 633 |
| 8 | 500 | 1300∶1 | 63 | 64 | 61 | 63.2 |
| Negative control | 64 | 64 | 63 | 63.7 |
Hemostasis trial in the independent and collaborative body of embodiment 3 batroxobins (batroxobin) and Stuart factor activator
According to the clinical research guideline, mice docking method is adopted in the test of body intravascular coagulation.We select the clinical consumption situation of association class thrombin (batroxobin): this scope of 20～0.1 units/60kg selects four points to do the body intravascular coagulation test of mice to batroxobin (batroxobin) and Stuart factor activator respectively, mice does corresponding the adjustment by people's dosage, is 8～0.04u/kg according to the relevant adjusted dosage of ratio.
Concrete grammar is as follows: get Stuart factor activator and batroxobin (batroxobin) and dispose the solution of debita spissitudo respectively as need testing solution.
Get healthy mice, be divided into 7 groups at random, 5 every group, by dosage mouse tail vein injection 0.5ml in the table, and when injecting back 15 minutes, to cut off at distance mouse tail point 0.3cm place with shears, timing immediately after blood flows out voluntarily stops timing to blood coagulation.With the index of bleeding time shortening as the judgement haemostatic effect.Use normal saline as negative control simultaneously, use the reptilase positive as positive control.The results are shown in following table
Annotate: "-" for shortening the bleeding time, "+" is for prolonging the bleeding time.
Table 3 batroxobin (batroxobin) body intravascular coagulation result of the test
| Dosage | The mice numbering | Meansigma methods | |||||
| Mice | 1 | 2 | 3 | 4 | 5 | ||
| Bleeding time | 8u/kg | 183 | 181 | 179 | 182 | 175 | 180 |
| (second) | 4u/kg | 165 | 175 | 176 | 169 | 164 | 169.8 |
| 0.4u/kg | 177 | 169 | 165 | 172 | 175 | 171.2 | |
| 0.04u/kg | 190 | 180 | 185 | 195 | 192 | 188.4 |
Table 4 Stuart factor activator body intravascular coagulation result of the test
| Dosage | The mice numbering | Meansigma methods | |||||
| Mice | 1 | 2 | 3 | 4 | 5 | ||
| Bleeding time (second) | 8u/kg | 180 | 175 | 183 | 185 | 180 | 180.6 |
| 4u/kg | 176 | 169 | 165 | 178 | 175 | 172.6 | |
| 0.4u/kg | 174 | 178 | 165 | 170 | 178 | 173 | |
| 0.04u/kg | 190 | 195 | 193 | 186 | 193 | 191.4 | |
Based on routine clinical consumption 1u, get Stuart factor activator and batroxobin (batroxobin) and dispose the solution of debita spissitudo respectively as need testing solution.Work in coordination with hemostatic test in the body.Positive reference substance is a reptilase, and negative control is a normal saline.It the results are shown in following table:
Table 7 batroxobin (batroxobin) (A) is worked in coordination with hemostasis trial result (dosage is 0.4u/kg) in the body with Stuart factor activator (B)
| Experiment number | Sample allocation ratio A: B | Setting time (second) | |||||
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | ||
| Positive control | 1 | 185.4 | 189.7 | 190.1 | 185 | 190.8 | 190.4 |
| 1 | 1∶1 | 172 | 175 | 183 | 184 | 186 | 180 |
| 2 | 10∶1 | 169.9 | 170.8 | 178 | 179.8 | 180.5 | 175.8 |
| 3 | 20∶1 | 149 | 155 | 156 | 158 | 162 | 156 |
| 4 | 100∶1 | 129.5 | 131 | 135.5 | 139 | 141 | 135.2 |
| 5 | 500∶1 | 145 | 148.5 | 159 | 161 | 162.5 | 155.2 |
| 6 | 600∶1 | 170 | 171.5 | 175 | 183 | 187.5 | 177.4 |
| 7 | 1000∶1 | 169 | 178 | 180 | 185 | 186 | 179.6 |
| Negative control | 277 | 281 | 285 | 287 | 288 | 283.6 |
Result of the test shows: when batroxobin and Stuart factor activator were made into the combination medicine-feeding preparation, the anastalsis of batroxobin had obtained collaborative, and anastalsis is improved significantly.Can find out significantly: batroxobin is 1: 1～1000: 1 with the anastalsis application dose scope ratio of Stuart factor activator, and wherein optimum application dose scope specific activity is 20: 1～500: 1.Compare with reptilase, its anastalsis is more obvious.
Hemostasis trial in the collaborative body of embodiment 4 agkistrodon halys ussuriensis batroxobins and Stuart factor activator
Experimental technique by embodiment 3 carries out.The results are shown in Table 8
Collaborative hemostasis trial result (dosage is 0.4u/kg) in table 8 agkistrodon halys ussuriensis batroxobin (A) and Stuart factor activator (B) body
| Experiment number | Sample allocation ratio A: B | Setting time (second) | |||||
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | ||
| Positive control | 1 | 188 | 185 | 194 | 192 | 189 | 189.6 |
| 1 | 10∶1 | 180 | 179 | 176 | 183 | 179 | 179.4 |
| 2 | 20∶1 | 165 | 162 | 166 | 168 | 160 | 164.2 |
| 3 | 100∶1 | 138 | 135 | 140 | 144 | 138 | 139.0 |
| 4 | 500∶1 | 158 | 160 | 155 | 159 | 162 | 158.8 |
| Negative control | 281 | 283 | 279 | 292 | 280 | 283.0 | |
Result of the test shows that agkistrodon halys ussuriensis batroxobin and Stuart factor activator had collaborative anastalsis at 20: 1～500: 1 in the scope equally.
Hemostasis trial in the collaborative body of embodiment 5 agkistrodon halys ussuriensis batroxobins and thrombin RVV-X
Experimental technique by embodiment 3 carries out.The results are shown in Table 9.
Hemostasis trial result (dosage is 0.4u/kg) in the collaborative body of table 9 agkistrodon halys ussuriensis batroxobin (A) and thrombin RVV-X (B)
| Experiment number | Sample allocation ratio A: B | Setting time (second) | |||||
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | ||
| Positive control | 1 | 194 | 189 | 188 | 190 | 193 | 190.8 |
| 1 | 10∶1 | 180 | 182 | 178 | 181 | 180 | 180.2 |
| 2 | 20∶1 | 160 | 162 | 160 | 165 | 163 | 162.0 |
| 3 | 100∶1 | 133 | 135 | 135 | 139 | 134 | 135.2 |
| 4 | 500∶1 | 162 | 160 | 163 | 158 | 155 | 159.6 |
| Negative control | 288 | 285 | 282 | 282 | 286 | 284.6 | |
Result of the test shows that agkistrodon halys ussuriensis batroxobin and thrombin RVV-X had collaborative anastalsis at 20: 1～500: 1 in the scope too.
Hemostasis trial in embodiment 6 Agkistrodon acutus batroxobins and the Stuart factor activator body
Experimental technique by embodiment 3 carries out.The results are shown in Table 10.
Hemostasis trial (dosage is 0.4u/kg) in table 10 Agkistrodon acutus batroxobin (A) and Stuart factor activator (B) body
| Experiment number | Sample allocation ratio A: B | Setting time (second) | |||||
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | ||
| Positive control | 1 | 185 | 188 | 186 | 184 | 190 | 186.6 |
| 1 | 10∶1 | 177 | 179 | 182 | 179 | 180 | 179.4 |
| 2 | 20∶1 | 165 | 163 | 169 | 166 | 163 | 165.2 |
| 3 | 100∶1 | 140 | 144 | 141 | 138 | 140 | 140.6 |
| 4 | 500∶1 | 163 | 160 | 154 | 159 | 160 | 159.2 |
| Negative control | 277 | 270 | 274 | 280 | 273 | 274.8 | |
Result of the test shows that Agkistrodon acutus batroxobin and Stuart factor activator had collaborative anastalsis at 20: 1～500: 1 in the scope equally.
Hemostasis trial in the collaborative body of embodiment 7 Agkistrodon acutus batroxobins and thrombin RVV-X
Experimental technique by embodiment 3 carries out.The results are shown in Table 9
See Table hemostasis trial (dosage is 0.4u/kg) in 9 Agkistrodon acutus batroxobins (A) and thrombin RVV-X (B) body
| Experiment number | Sample allocation ratio A: B | When solidifying (second) | |||||
| 1 | 2 | 3 | 4 | 5 | Meansigma methods | ||
| Positive control | 1 | 188 | 184 | 182 | 187 | 185 | 185.2 |
| 1 | 10∶1 | 180 | 185 | 187 | 184 | 181 | 179.4 |
| 2 | 20∶1 | 165 | 163 | 169 | 166 | 163 | 165.2 |
| 3 | 100∶1 | 132 | 130 | 134 | 133 | 140 | 134.4 |
| 4 | 500∶1 | 160 | 168 | 166 | 158 | 165 | 163.4 |
| Negative control | 280 | 279 | 282 | 284 | 282 | 281.4 | |
Result of the test shows that agkistrodon halys ussuriensis batroxobin and thrombin RVV-X had collaborative anastalsis at 20: 1～500: 1 in the scope equally.
Embodiment 8 stablizes the test of hemorrhage preparation prescription
Carry out proportioning test according to following table.
Table 11 prescription experiment sieving result
| Prescription is formed | Every milliliter of content | Prescription 1 | Prescription 2 | Prescription 3 |
| Hemocoagulase | 1.0U | + | + | + |
| The Stuart factor activator | 0.002U | + | + | + |
| The glycine mixture | 6mg | + | - | - |
| The glycine mixture | 8mg | - | + | - |
| The glycine mixture | 10mg | - | - | - |
| Mannitol | 50mg | + | + | + |
| Setting time (second) before the lyophilizing | 50 | 50 | 50 | |
| 30 days setting times (second) after the lyophilizing | 63 | 52 | 80 | |
| The setting time (second) that increases | 13 | 2 | 30 | |
From table data as can be seen, the activity ratio of preparation will stablizing of not adding after adding glycine, 2 the activity change minimum of wherein writing out a prescription, it is 2 the most stable promptly to write out a prescription, thereby determines with the glycine to be stabilizing agent, and its content is preferably 8mg/ml.
Claims (8)
1. compound hemostatic is characterized in that: the main active ingredient of described medicine is a purity greater than 97% batroxobin and Stuart factor activator, and the proportion of active batroxobin and factor Xa X activator is 20: 1～500: 1.
2. according to the described compound hemostatic of claim 1, it is characterized in that: described batroxobin is the batroxobin that contains in Brazilian spearhead Agkistrodon halys, agkistrodon halys ussuriensis or the Agkistrodon acutus snake venom, or the batroxobin of the reorganization Brazil spearhead Agkistrodon halys, agkistrodon halys ussuriensis or the Agkistrodon acutus that obtain by genetic engineering.
3. according to the described compound hemostatic of claim 1, it is characterized in that: described Stuart factor activator is Stuart factor activator FXA or the genetic engineering reorganization FXA that contains in the Brazilian spearhead agkistrodon halyx pallas venom; Or derive from the Stuart factor activator composition of Agkistrodon halys Vipera russelli or Daboia russelli or its genetic engineering recombinant products.
4. according to the described compound hemostatic of claim 1, it is characterized in that: described batroxobin is the batroxobin batroxobin of Brazilian spearhead Agkistrodon halys, the Stuart factor activator is the Stuart factor activator FXA that contains in the Brazilian spearhead agkistrodon halyx pallas venom, wherein also contains the aminoacid composition.
5. according to the described compound hemostatic of claim 4, it is characterized in that: described aminoacid is glycine.
6. according to the described compound hemostatic of claim 5, it is characterized in that: be excipient with mannitol.
7. according to the described compound hemostatic of claim 6, it is characterized in that: batroxobin+Stuart factor activator: glycine: the amount of mannitol is 20～0.1 units: 1～0.01 gram: 0.02～0.06 gram.
8. according to the described compound hemostatic of claim 1, it is characterized in that: the isolation and purification method of its Stuart factor activator is to adopt DEAE-Fast Flow series anionic exchanger resin to separate with Sephacryl series molecular sieve column to realize that its vigor is identified and detected by so-called chromogenic substrate method and realize.
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| CNB2005100473326A CN100369629C (en) | 2005-09-30 | 2005-09-30 | Compound hemostatic originated from venom |
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| CN100369629C true CN100369629C (en) | 2008-02-20 |
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| CN102798598A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting enzymatic activity of phospholipid-depending factor X activator |
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| CN107796793B (en) * | 2017-09-28 | 2020-04-10 | 中国科学技术大学 | FXa detection reagent and FXa detection method |
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| 立止血的研制历史及其应用概况. 康佐文等.蛇志，第12卷第4期. 2000 * |
| 立止血的药理与临床应用》，，1994年第5卷第4期. 李树昌等.中国药方，第5卷第4期. 1994 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102798598A (en) * | 2011-05-25 | 2012-11-28 | 兆科药业（合肥）有限公司 | Method for detecting enzymatic activity of phospholipid-depending factor X activator |
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