CN100523185C - Agkistrodonacutus thrombin preparation method and uses - Google Patents
Agkistrodonacutus thrombin preparation method and uses Download PDFInfo
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- CN100523185C CN100523185C CNB2005100851739A CN200510085173A CN100523185C CN 100523185 C CN100523185 C CN 100523185C CN B2005100851739 A CNB2005100851739 A CN B2005100851739A CN 200510085173 A CN200510085173 A CN 200510085173A CN 100523185 C CN100523185 C CN 100523185C
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Abstract
The invention discloses a preparation method for Hemocoagulase of Agkistrodon acutus peculiar to China, which comprises dissolving, centrifuging at low temperature, dialyzing, DEAE-Sepharose FF chromatography, and Sephadex G25 chromatography. The product comprises A and B subunits with molecular weight as 15.0KD and 14.2KD respectively, and the amino acid sequences of first to twenty-five sites are DCSSGWSSYEGHCYKVFKQSKTWTA and DCPSDWSSYECHCYKPDEPKTWEDA respectively. This product can be used to haemostasis in clinic.
Description
Technical field
The present invention relates to a kind of Agkistrodonacutus thrombin preparation method and purposes.Be meant especially a kind of from the agkistrodon acutus snake venom of special product of China the preparation method of the snake venom thrombin-like enzyme with anastalsis of separation and purification.
Background technology
Snake venom thrombin-like enzyme (Thrombin-like enzymes is that a class of separate purifying from snake venom has the active proteolytic enzyme of thrombin-like TLEs), can be directly the former formation scleroproein of hydrolysis of fibrin and impel blood coagulation specifically.Abroad with domestic approved production to be used for hematostatic Thrombin-like enzyme preparation mainly be reptilase, be the Thrombin-like enzyme that is purified into from hole pallas pit viper (Bothrops Atrox) snake venom.By trade(brand)name " reptilase " haemostatic medicament (Reptilase) of the plain tall and big pharmaceutical factory development of Switzerland Ba Sai, the Thrombin-like enzyme that from the exclusive hole agkistrodon halyx pallas venom of Brazil, refines exactly, China uses clinically widely from millions of reptilases of Switzerland's import every year.China does not also have similar medicine can substitute reptilase so far.Reptilase is the protein that a kind of molecular weight is about 42000Da, and the mechanism of its anastalsis is the fibrinogenic A-chain of hydrolyzable in blood, and the scleroproein I monomer of Xing Chenging can further aggregate into mixture thus.Compare with Fibrinogen, this scleroproein I monomer and mixture thereof are more vulnerable to the effect of zymoplasm and plasmin, also easily adhere to the damaged position of blood vessel simultaneously.Therefore, in the part of blood vessel injury,, can rapidly the hydrolysis of the monomeric B-chain of scleroproein I be removed because the concentration of zymoplasm is higher, be cross-linked to form physiological fibrin clot and produce anastalsis, the scleroproein I in circulation then can be removed by plasmin.But reptilase is the haemostatic medicament of separation and purification from the viper venom of Brazilian hole, and (mere formality is offered the big pharmacy of China, 1996,7 (3): 137) in the not distribution of this snake kind of China.
Agkistrodon acutus is the common snake kind in China South China, and is venin-derived abundant, and contains abundant TLEs.TLEs for the agkistrodon acutus snake venom, Taiwan's scholars Ouyang, C. and Cheng, H.C. etc., and unit such as Chinese University of Science and Technology had once carried out a large amount of research, but there is no the research report of the inside and outside anastalsis of animal body, the isolating TLEs also fiber eliminating enzyme character with present clinical use is similar mostly.Scholars such as Xiao Changhua reported once that point kiss snake venom had four kinds of TLEs components at least, wherein had two kinds of TLEs that the short anastalsis of coagulating is arranged in animal body.Scholars such as pipe rose-tinted clouds reported once also that the component of agkistrodon acutus snake venom can shorten the clotting time of animal, but did not see the report of further development research.Scholar such as Wei Wenli, Sun Jiajun once attempted utilizing the TLEs of agkistrodon acutus snake venom and agkistrodon halyx pallas venom to be used for the external local hemostasis of animal, but the used point kiss snake venom TLEs of these scholars has four kinds at least, and contain other foreign proteins, the purpose of its experimental design mainly is to want to utilize the point kiss snake venom TLEs hydrolysis characteristics different with B β chain to Fibrinogen A α chain with agkistrodon halyx pallas venom TLEs, form a kind of more near the grumeleuse of physiological status, stop blooding in hemorrhage part, but do not prove that these TLEs itself have anastalsis when using separately.The report of comprehensive domestic research does not still have the such Thrombin-like enzyme of similar reptilase of the autonomous research of China to ratify to be used for clinical as hemostatic drug at present.
The separation and purification of the sTLE of bibliographical information at present generally all adopts anion-exchange chromatography and molecular sieve layer analysis method to carry out.Adopt this processing method, the production cycle is long, yield is low, purity is relatively poor.
Summary of the invention
Technical problem to be solved by this invention is to avoid above-mentioned deficiency of the prior art, and proposes a kind of preparation method and purposes of new Agkistrodon acutus hemocoagulase atrox.
The preparation method of a kind of Agkistrodon acutus hemocoagulase atrox of production provided by the present invention, this method may further comprise the steps:
A. the thick poison of agkistrodon acutus is centrifugal with the phosphoric acid buffer dissolving, dialysis, and through the DEAE-Sepharose chromatography, the NaCl solution gradient wash-out of different solutions is collected and is obtained component.
B. dialyse again through the DEAE-Sepharose chromatography, obtain purified purity Agkistrodon acutus hemocoagulase atrox.
C. receive the purified Agkistrodon acutus hemocoagulase atrox, after the desalination of SephadexG25 chromatography column, obtain the Agkistrodon acutus hemocoagulase atrox bulk drug.
D. the Agkistrodon acutus hemocoagulase atrox bulk drug of receiving gained adds vehicle filtration, packing and lyophilize, makes the injection Agkistrodon acutus hemocoagulase atrox.
A kind of Agkistrodon acutus hemocoagulase atrox that separation and purification obtains from the distinctive agkistrodon acutus snake venom of China is made up of A and two subunits of B, has the blood coagulation of promotion and anastalsis.
Further, the molecular weight of Agkistrodon acutus hemocoagulase atrox A and two subunits of B is 15.0KD and 14.2KD.The aminoacid sequence that A subunit N end is first to 25 is DCSSGWSSYEGHCYKVFKQSKTWTA, and the aminoacid sequence that B subunit N end is first to 25 is DCPSDWSSYECHCYKPDEPKTWEDA.
Further, the anastalsis that has of Agkistrodon acutus hemocoagulase atrox is meant that this enzyme can be used for hemorrhage hemostasis of Mammals and people's the hemorrhage hemostasis of all kinds clinically.
A kind of pharmaceutical composition that has hemostatic function clinically, this pharmaceutical composition contains the Agkistrodon acutus hemocoagulase atrox with anastalsis.
Further, have the pharmaceutical composition of hemostatic function clinically, except that containing Agkistrodon acutus hemocoagulase atrox, also contain pharmaceutically acceptable carrier and/or vehicle with anastalsis.
The present invention has following advantage:
1, the preparation technology in enormous quantities that preparation technology of the present invention is with short production cycle, bulk drug yield height, purity is good.The yield of bulk drug is about 0.5%, and purity is more than 95%.
2, Agkistrodon acutus hemocoagulase atrox is two subunit class hemocoagulases of separation and purification from the distinctive agkistrodon acutus snake venom of China, and production cost is low, meets China's actual conditions.
3, prepared as stated above Agkistrodon acutus hemocoagulase atrox characteristics are as follows: the first, and Agkistrodon acutus hemocoagulase atrox is as a kind of new hemostatic drug, and it is strong to have anastalsis, the advantage that toxicity is extremely low.From present animal and human's body I phase, II phase, III phase clinical study result, still not observing Agkistrodon acutus hemocoagulase atrox has tangible toxic action.The second, measure through flight mass spectrum, this enzyme molecular weight is about 29300-29500Dalton, respectively by two polypeptide chains of about 15.0KD and 16.2KD with the glycoprotein that disulfide linkage is formed by connecting, contain 204 amino acid.Animal experiment shows that this enzyme can shorten the clotting time, has good anastalsis, and its mechanism of action also is by the former A-chain of hydrolysis of fibrin, forms scleroproein and plays anastalsis.The more important thing is that this enzyme toxicity is very low, can not survey LD in the acute toxicity test
50That passes through structural identification physico-chemical property and enzymatic property etc. studies have shown that reptilase is a strand, and Agkistrodon acutus hemocoagulase atrox is a kind of Thrombin-like enzyme that is different from reptilase fully, is a kind of brand-new protein haemostatic medicament.
The comparison of Agkistrodon acutus hemocoagulase atrox and reptilase
| Point kiss Pallas pit viper hemocoagulase reptilase |
| Chinese Brazilian agkistrodon acutus (Agkistrodon Acutus) hole pallas pit viper (Bothrops atrox) molecular weight 29.3-29.5KD 39.0-43.0KD peptide chain of originating constitutes 15.0KD, 14.2KD two peptide chain single peptide chain amino acid constitutes 204, Asp and Lys content are the highest 231, the higher isoelectric point (pl) 5.51-5.64 6-7 of Asp and Glu content |
Comparison shows that of above-mentioned Agkistrodon acutus hemocoagulase atrox and reptilase, Agkistrodon acutus hemocoagulase atrox are that a kind of structure is different from reptilase fully, but have the new snake venom thrombin-like enzyme of anastalsis.This product only has hemostatic function, does not influence thrombogen quantity and platelet counts in the blood, does not have the platelet aggregation effect in normal blood vessels, does not therefore have thrombosis danger.Agkistrodon acutus hemocoagulase atrox is that purity is more than 95% through the zymoplasm that separates and extract, and does not contain neurotoxin and other toxin.Its toxicologic study shows that this product is a kind of safe haemostatic medicament.Through studies show that of pharmacy, pharmacodynamics, toxicology, pharmacokinetics, the Agkistrodon acutus hemocoagulase atrox that the present invention relates to have toxicity little, significant promotion Blood clotting and anastalsis are arranged, be applicable to the prevention and the treatment of various types of hemorrhagic diseasess clinically.
Description of drawings
Fig. 1 is technological process of production figure of the present invention.
Embodiment
Below in conjunction with description of drawings the specific embodiment of the present invention.
Technical scheme of the present invention be at first from the distinctive agkistrodon acutus snake venom of China the purity of separation and purification greater than more than 97%, molecular weight is the Agkistrodon acutus hemocoagulase atrox of 29.2KD, and definite constitutional features is: the aminoacid sequence that A subunit N end is first to 25 is DCSSGWSSYEGHCYKVFKQSKTWTA, and the aminoacid sequence that B subunit N end is first to 25 is DCPSDWSSYECHCYKPDEPKTWEDA.And adopt the pharmacodynamic experiment proof to have hemostatic function.
As shown in Figure 1
1. the processing of thick malicious sample
Take by weighing snake venom 30g, be dissolved in 200ml 0.01M phosphoric acid buffer (pH7.4), 5000rpm * 30min is centrifugal at 4 ℃, supernatant liquor is poured in the dialysis tubing (molecular weight cut-off is 8000-10,000), in 4 ℃ of refrigerators, place same damping fluid dialysis 24 hours, wherein propose twice of dialyzate.
2.DEAE-Sepharose FF chromatography
Chromatography column (3.6 * 40cm
3) use fully balance of 0.01M phosphoric acid buffer (PH7.4), the snake venom after the dialysis injects in the post with the speed of 15ml/min with constant flow pump.Last sample finishes, with containing the above-mentioned phosphoric acid buffer wash-out of 0.02M NaCl, be eluted to the about 3100ml of volume after, carry out the straight line gradient elution with the above-mentioned phosphoric acid buffer of 0.02-0.1M NaCl, the about 20ml/min of flow velocity writes down also collection albumen elution peak.
3. dialysis
The elution fraction of collecting is qualitative through the human plasma coagulation activity, HPLC and SDS-PAGE identify errorless after, collect the component that contains target component,, dialysed 24 hours as dialyzate with the phosphoric acid buffer that contains 0.02M NaCl, wherein propose twice of dialyzate.
4.DEAE-Sepharose FF chromatography
Behind the last sample, respectively with containing 0.04,0.06,0.08 and 1.0M NaCl phosphoric acid buffer wash-out, elution fraction is qualitative through the human plasma coagulation activity, HPLC and SDS-PAGE identify, determines that the peak of 0.08M NaCl wash-out is target components.
5.Sephadex G25 chromatography desalination
Chromatography column (7.0 * 100cm
3) fully wash balance with water for injection.Sample on the snake venom component, the about 800ml of volume, with the water for injection wash-out, flow velocity 30ml/min collects elution peak.
6. vacuum freezedrying
Snake venom component solution after the desalination, aseptic subpackaged behind 0.22 μ m filtering with microporous membrane, after the lyophilize, promptly get the Agkistrodon acutus hemocoagulase atrox bulk drug.
7. the characterized of Agkistrodon acutus hemocoagulase atrox
The visible two protein subunit district bands of A.SOS-polyacrylamide gel electrophoresis, the A molecular weight subunit is 15.0KD, the B molecular weight subunit is 14.2KD.
B. identify through high performance liquid chromatography that adopt BIOSEP SEC chromatography column, Agkistrodon acutus hemocoagulase atrox is single symmetrical peak, calculates according to the peak area normalization method, Agkistrodon acutus hemocoagulase atrox content is greater than 97%.
C. through determined amino acid: it is DCSSGWSSYEGHCYKVFKQSKTWTA that Agkistrodon acutus hemocoagulase atrox A subunit N holds the 1st to 25 aminoacid sequence, and the aminoacid sequence that B subunit N end is first to 25 is DCPSDWSSYECHCYKPDEPKTWEDA.
The Agkistrodon acutus hemocoagulase atrox production process equipment comprises separation and purification, quality monitoring equipment.
Separation and purification equipment: after mainly adopting dextrane gel filling glass columns such as homemade glass chromatography column and G25, G75, can repeatedly use repeatedly.Its technico-economical comparison is to prepare highly purified Agkistrodon acutus hemocoagulase atrox bulk drug, and prolongs the life cycle of gel, to reduce cost.
Quality monitoring equipment: mainly adopt high performance liquid chromatography and electrophoresis system to detect, adopt analysis mode high performance liquid phase and conventional electrophoresis loading amount to carry out, because high performance liquid phase and electrophoresis are equipment commonly used in the present pharmaceutical grade protein research, its technico-economical comparison guarantees the purity quality of product to reach the Agkistrodon acutus hemocoagulase atrox bulk drug that can detect unimodal and single district band.The purity of Agkistrodon acutus hemocoagulase atrox bulk drug, as Fig. 5, HPLC purity: 〉=95%.
Embodiment one
The 10g agkistrodon acutus is thick malicious with after the phosphoric acid buffer dissolving, at the centrifugal 30min of 5000rpm, after centrifuged supernatant was dialysed 24 hours in dialysis tubing, last DEAE-Sepharose chromatography column chromatography, with 0.02M NaCl phosphoric acid buffer wash-out, carry out gradient elution with 0.02-0.1M NaCl phosphoric acid buffer more earlier, flow velocity is 20ml/min, collect the albumen elution peak, reinstall dialysis tubing and dialysed 24 hours.Solution after the collection dialysis is at last DEAE-Sepharose chromatography column chromatography, with 0.04,0.06,0.08 and behind the 1.0M NaCl phosphoric acid buffer gradient elution, adopt human plasma to solidify activation, HPLC and SOS-PAGE identify, determine that 0.08M NaCl wash-out part (volume is 200ml) is Agkistrodon acutus hemocoagulase atrox.
The Agkistrodon acutus hemocoagulase atrox of above-mentioned collection is identified through SDS-PAGE, is that two subunits are formed, and the A molecular weight subunit is 15.0KD, and the B molecular weight subunit is 14.2KD, identifies that through HPLC purity is 98%.Add vehicle at last, it is 3500 that packing, filtration sterilization, lyophilize, gland make the injection Agkistrodon acutus hemocoagulase atrox.
Embodiment two
The 15g agkistrodon acutus is thick malicious with after the phosphoric acid buffer dissolving, at the centrifugal 30min of 5000rpm, after centrifuged supernatant was dialysed 24 hours in dialysis tubing, last DEAE-Sepharose chromatography column chromatography, at 0.02M NaCl phosphoric acid buffer wash-out, carry out gradient elution with 0.02-0.1M NaCl phosphoric acid buffer again, flow velocity is 20ml/min, collect the albumen elution peak, reinstall dialysis tubing and dialysed 24 hours.The solution of collecting after dialysing is gone up DEAE-Sepharose chromatography column chromatography again, with 0.04,0.06,0.08 and behind the 1.0M NaCl phosphoric acid buffer gradient elution, adopt human plasma to solidify activation, HPLC and SOS-PAGE identify, determine that 0.08M NaCl wash-out part (volume is 230ml) is Agkistrodon acutus hemocoagulase atrox.
The Agkistrodon acutus hemocoagulase atrox of above-mentioned collection is accredited as two subunits through the SDS-PAGE electrophoresis to be formed, and the A molecular weight subunit is 15.0KD, and the B molecular weight subunit is 14.2KD, identifies that through HPLC purity is 98%.Add vehicle at last, it is 5000 that packing, filtration sterilization, lyophilize, gland make the injection Agkistrodon acutus hemocoagulase atrox.
Test method and result
Test (one)
This research is adopted Japan rabbit and mouse Agkistrodon acutus hemocoagulase atrox to be carried out pharmacodynamic indexs such as blood coagulation system, fibrinolytic system, bleeding time, platelet counts, platelet function and whole blood viscosity and is studied.
One, test objective
Observe Agkistrodon acutus hemocoagulase atrox through a vein shot to Japan rabbit and mouse hematostatic pharmacodynamic action, for clinical application provides the pharmacodynamics foundation.
Two, test materials
1, be subjected to the reagent thing: Agkistrodon acutus hemocoagulase atrox is provided lot number: 20010110 by KangChen Medicine Development Co., Ltd.Activity unit: every bottle of lyophilisate that contains an activity unit (1U/ bottle), face with preceding water for injection injected dissolving in the bottle.
2, reference substance: reptilase is the plain tall and big pharmaceutical factory of Basel, SUI, every bottle of activity unit, and lot number: 8834111 Ke Shi units/bottle, face with preceding water for injection injected dissolving in the bottle.
3, animal
(1), kunming mice, purchase in animal portion of epidemiological study institute conformity certification number: the capital is moving is betrothed to (1999) one No. 038, body weight: 17-22g, male and female have concurrently.
(2), Japan rabbit, purchase the animal center in the Department Of Medicine, Peking University, conformity certification number: the capital is moving is betrothed to 2000 No. 209, and body weight is that male and female have concurrently about 2.0 kilograms.
Three, test method
Test by " new drug (Western medicine) preclinical study governing principle compilation (pharmacy, pharmacology, toxicology) " requirement P.106-107.
1, mouse is cut the mensuration in tail bleeding time
Get small white mouse and be divided into 5 groups at random, that is: high (0.5U/kg), in (0.25u/kg), low (0.125u/kg) 3 dosage groups; 1 positive controls (reptilase 0.5u/kg) and 1 solvent control group (physiological saline).Behind the intravenous administration 30 minutes, cut off afterbody fast in mouse tail point about 0.5 centimeters that makes progress, start the watch clocks immediately, draws effusive blood with filter paper, the calculating in bleeding time from cut tail begin to no hemorrhage till.
2, whole blood clotting time and whole blood clot dissolution test
Get Japan rabbit whole blood 1ml and add in the glass test tube, place 37 ℃ of water-baths, whether the test tube that regularly tilts solidifies to observe blood, and record adds the time that test tube to blood all solidifies from blood.When the judgement that blood all solidifies was spent the inclination test tubes with 90, blood did not flow and is standard.Treat again test tube to be continued to place 37 ℃ of water-baths 24 hours after blood all solidifies, observe whole blood clot dissolution situation.
3, the mensuration of plasma prothrombin (PT)
0.1 37 ℃ of water bath heat preservation 1min of anticoagulate plasma, adding 0.2m people's thrombokinase is opened the aggegation detector simultaneously, detects the time that grumeleuse forms.
4, Fibrinogen (FIB) Determination on content
Turbidimetry uses ACL-100 U.S. Ku Erte Blood coagulation instrument to measure
5, activated partial thromboplastin time (APTT)
In the blood plasma of 0.1ml citrate anticoagulation, add the 0.1ml extracting solution of human placenta, put 37 ℃ of water-bath incubations 2 minutes, add 0.1ml 25mmol/Lcacl
2,, open the time that the aggegation detector checks that blood clot forms simultaneously to cause the aggegation process.
6. the mensuration of euglobulin setting time and euglobulin lysis time (ELT)
Get whole blood 1ml and add the test tube mixing of putting into 0.1ml 3mM structure rafter acid sodium solution in advance, centrifugal 10 minutes separated plasmas of 3000rpm/min.Get conical centrifuge tube, adding distil water 7.5ml, the about 0.12ml of 1% acetic acid, making pH is 4.5, puts in the ice bath.Get 0.5ml blood plasma and be added in the above-mentioned centrifuge tube, mixing continued to put in the ice bath 10 minutes, and euglobulin is fully separated out.The centrifugal 5min of 3000r/min, the supernatant liquor that inclines is inverted centrifuge tube on filter paper, inhales and removes residual liquid.Add borate buffer (pH9.0) 0.5ml in precipitation, put in 37 ℃ of water-baths, stirring makes it to dissolve fully gently.Add 0.025mol/Lcacl
2Solution 0.5ml starts stopwatch record setting time.Put in 37 ℃ of water-baths, observe the consoluet time of grumeleuse.
7, the mensuration of white corpuscle, red corpuscle and oxyphorase
Get the 1ml whole blood put into immediately the cuvette that fills the 10ml cell diluent add a cover the back shake well.With automatic blood cell count miriam (Celltac, MEK-5108K: counting white corpuscle (WBC), red corpuscle (RBC) and oxyphorase (HB) Japanese NIHONKOHOEN company).
8, platelet counts (PLT):
Get 10 μ l blood sample to be measured, after being diluted to 500 times with diluent (physiological saline), to going in the plastics detection cup special to shake up, be put into detection place of instrument, make blood sample reach the height of specified requirement, start instrument count (Guojia Light ﹠ Electric Co., Ltd., Shanghai produces JXJ-300 type laser blood cell counts instrument).
Mean platelet volume (MPV):
Get 10 μ l blood sample to be measured, put into and contain the 10ml diluent and contain Nacll17.47g for every liter, Kcl10.22g, Na2Hpo4.35g, Kcll0.28g, Licl10.43g, EDTA-Na20.36g. are prepared into the suspension of 1001 times of dilutions, fully shake up.At Baker, calculate platelet count on the Series-810 type thrombocyte numeration instrument.
Thrombocytocrit (PCT):
Get normal sorption blood plasma, rabbit brain powder leach liquor, 0.02mol/Lcacl
2Each 0.1ml of solution, mixing, 37 ℃ of incubations 1 minute.In above-mentioned solution, add in advance temperature to 37 ℃ examined serum 0.1ml, start the stopwatch record clotting time (s), repeat 2 times and average.
9, extracorporeal platelet aggregation test
Get blood 1ml from rabbit heart, (0.1ml, mixing in test tube 3mM) at the centrifugal 5min of 500rpm/min, are got the 0.3ml upper plasma and are added behind the film phonograph seal as platelet rich plasma (PRP) to add existing liquor sodii citratis.Remaining blood plasma is got the 0.2ml upper plasma as platelet poor plasma (PPP) again at the centrifugal 10min of 3000rpm/min.After the blood plasma preheating, transfer 100% with PPP, get phosphoric acid buffer 20ul or reptilase or Agkistrodon acutus hemocoagulase atrox 20ul and add among the PRP and be incubated 3min, add the variation (platelet aggregation determinator) of measuring platelet aggregation rate behind the ADP 10ul again.
10, thrombus is counted A
2(TXA
2) assay
Thromboxane A
2(TXA
2) be the arachidonic meta-bolites of thrombocyte.Measure its content and can be used as the index of estimating platelet function.But TXA
2Instability is metabolized to stable TXB very soon in the time of 37 ℃
2So usually to measure TXB
2Content replaces TXA
2Content.This experiment is adopted
125The TXB of I mark
2Radioimmunoassay method is measured, and test kit is purchased in University Of Suzhou thrombus research department.Get rabbit whole blood 1.5ml adding 0.2ml INDOMETHACIN-EDTA.Na is arranged
2Test tube in mixing, at the centrifugal 15min of 3500rpm/min (4 ℃), sucking-off blood plasma is preserved at-20 ℃ of refrigerators, according to the form below operation during mensuration.
| Reagent blank zero standard standard 1-8 sample |
| Buffer A 200 200---buffer B 100---100 standards---200-sample---, 200 γ-globulins 100 100 100 antiserums-100 100 100125I-thromboxane B 2Cultivate 24h 25% precipitation agent 500 500 500 500 for 100 100 100 100 4 ℃ |
Complete soln adds good back mixing, at room temperature places 15min.At the centrifugal 20min of 3500rpm/min (4 ℃), get the cpm value that the supernatant liquor of 2 pipes wherein measures and manage as T.On γ-automatic measuring instrument, measure the cpm value of each pipe precipitation part, and the establishing criteria curve is calculated TXB
2Concentration.
11, the mensuration of whole blood viscosity
Get whole blood 1ml from rabbit heart, add 1% heparin 0.1ml anti-freezing.With blood viscometer (VISCONIC ED type) be determined at shear rate be 1,5,30 and 200 o'clock whole blood viscosity change.
Four, dosage, medication and blood extracting method
1, dosage: the dosage of Japan rabbit auricular vein injection Agkistrodon acutus hemocoagulase atrox is respectively 0.12u/kg, 0.06u/kg, 0.03u/kg.The mouse tail vein injection dosage is respectively 0.5U/kg, 0.25U/kg and 0.12U/kg.
2, medication: Japan rabbit adopts the auricular vein drug administration by injection, and mouse is adopted the tail intravenously administrable, and is consistent with the clinical application approach.
3, rabbit blood extracting method: rabbit back of the body position is fixing, jugular vein or heart extracting blood under the etherization state.
Five, positive control drug
Select for use present widely used reptilase as positive control drug, produce by the plain tall and big pharmaceutical factory of Basel, SUI.
Experimental result
1, Agkistrodon acutus hemocoagulase atrox is cut the influence in tail bleeding time to mouse
Mouse tail vein injection Agkistrodon acutus hemocoagulase atrox 0.5u/kg, 0.25u/kg, 0.125u/kg and reptilase 0.50u/kg, 30min measures mouse and cuts the tail bleeding time after the administration.The result shows that Agkistrodon acutus hemocoagulase atrox 0.5u/kg, 0.25u/kg, 0.125u/kg can obviously shorten mouse and cut the tail bleeding time, and obvious dose-effect relationship is arranged.Compare with the physiological saline control group, there were significant differences.Positive control drug reptilase 0.50u/kg also obviously shortens mouse and cuts the tail bleeding time.The results are shown in Table 1.
Table 1 Agkistrodon acutus hemocoagulase atrox intravenously administrable is cut the influence in tail bleeding time to mouse
Compare with the blank group * P＜0.001.
2, Agkistrodon acutus hemocoagulase atrox is to the influence of rabbit whole blood setting time
Each dosage group of Agkistrodon acutus hemocoagulase atrox (0.12,0.06,0.03u/kg) all can make whole blood clotting time obviously shorten.Medicine peaks in the time of 30 minutes in the onset in 10 minutes of quiet notes, and the maximum percentage that high, medium and low dosage group and reptilase group whole blood clotting time shorten is respectively 47.95%, 45.92% and 34.97% and 41.35%.The effect of medicine continues to after the administration about 6 hours, level before administration returns to administration after 12 hours gradually.The positive control drug reptilase also can obviously shorten whole blood clotting time, and action intensity and time length and Agkistrodon acutus hemocoagulase atrox are more or less the same.The results are shown in Table 2.Place 37 ℃ of water-baths to continue 24 hours whole sample hoses after whole blood solidifies, the clot dissolution phenomenon all do not occur.
Table 2 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit whole blood clotting time
Remarks: 1, the whole blood clotting time unit that measures in the table is to calculate second, and data are whole blood clotting time LVFS (%) in the bracket.
2, * p＜0.05, * * p＜0.01 is with self is relatively before the administration.
3, Agkistrodon acutus hemocoagulase atrox is 0.12,0.06 and during 0.03U/kg to the Agkistrodon acutus hemocoagulase atrox intravenously administrable dosage that influences of rabbit plasma thrombogen, and the rabbit plasma thrombogen is not had influence.The results are shown in Table 3.
Table 3 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit plasma prothrombin (PT)
Determination data is represented with s in the table
4, Agkistrodon acutus hemocoagulase atrox influencing that the Agkistrodon acutus hemocoagulase atrox vein gives that dosage is 0.12,0.06, during 0.03U/kg, APTT not had obvious influence to rabbit activated partial thromboplastin time (APTT).The results are shown in Table 4.
Table 4 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit part thrombokinase
Data are represented with s in the table
5, Agkistrodon acutus hemocoagulase atrox is to the influence of scleroproein (Fib) content
Each dosage group of Agkistrodon acutus hemocoagulase atrox compares fibrinogenic content with the photograph medicine does not have obvious influence, drug-induced regular the variation do not occur, the results are shown in Table 5.
Table 5 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit fibrinogen content
Data are represented with mg/dl in the table.
6, Agkistrodon acutus hemocoagulase atrox solidifies euglobulin and the influence of dissolution time (ELT)
Agkistrodon acutus hemocoagulase atrox the results are shown in Table 6-7 to euglobulin setting time and dissolved influence.
Agkistrodon acutus hemocoagulase atrox 0.06 and 0.03U/kg dosage under, ELT has slight downtrending.ELT also has slight decline in positive control reptilase group, but the effect of the Agkistrodon acutus hemocoagulase atrox of three dosage and reptilase does not all have the clear regularity variation.
Table 6 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit euglobulin setting time
Data are with a minute expression in the table.
Table 7 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit euglobulin lysis time
Data are with a hour expression in the table.
7, Agkistrodon acutus hemocoagulase atrox to platelet counts, volume of platelets, thrombocytocrit influence Agkistrodon acutus hemocoagulase atrox 0.12,0.06 and 0.03u/kg dosage under, compare no evident regularity variation before the platelet counts of each time point after the administration, volume of platelets, thrombocytocrit and the administration, the results are shown in Table 8-10.
Table 8 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit platelet count
Unit in the table: X10
-9/ L.
Table 9 is quiet ± and arteries and veins injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit volume of platelets
Unit is in the table: fI
Table 10 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit thrombocytocrit
Data are represented with % in the table.
8, Agkistrodon acutus hemocoagulase atrox is to the influence of extracorporeal platelet aggregation function
Table 11 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit platelet aggregation rate
Data are represented with (%) in the table.
9, Agkistrodon acutus hemocoagulase atrox generates TXA to thrombocyte
2Influence
TXB is adopted in this experiment
2Radioimmunological kit is measured TXB in the blood plasma with radio immunoassay
2Content.The result shows that each dosage group of Agkistrodon acutus hemocoagulase atrox and positive control drug reptilase discharge TXA to thrombocyte
2Function do not have obvious influence, the results are shown in Table 12.
Table 12 intravenous injection Agkistrodon acutus hemocoagulase atrox is to Japan rabbit TXB
2Influence
Data are represented with pg/ml in the table.
10, Agkistrodon acutus hemocoagulase atrox is to the influence of rabbit whole blood viscosity.
Be respectively 1,5,30,200 o'clock at shear rate, the whole blood viscosity of Agkistrodon acutus hemocoagulase atrox different time points after administration of high dose group (0.12U/kg) is low slightly before than administration.The positive control drug reptilase shear rate be 30 and 200 o'clock whole blood viscosity low slightly before than administration.Agkistrodon acutus hemocoagulase atrox is 0.06 and whole blood viscosity is not had obvious influence during 0.03U/kg.The results are shown in Table 13,14,15,16.
Table 13 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit whole blood viscosity
Data unit in the table: L/s.
Table 14 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit whole blood viscosity (mPaS)
Data unit in the table: L/s.
Table 15 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit whole blood viscosity (mPaS)
Data unit in the table: L/s
Table 16 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit whole blood viscosity (mpaS)
Data unit in the table: L/s.
11, Agkistrodon acutus hemocoagulase atrox is to the influence of red corpuscle (RBC), oxyphorase (Hg) and white corpuscle (WBC).
Agkistrodon acutus hemocoagulase atrox does not have obvious influence 0.12,0.06 with during three dosage of 0.03U/kg to RBC, Hb and WBC.The positive control drug reptilase does not have influence to RBC, Hb and WBC yet.Relatively, have a declining tendency over time before the RBC that each group is measured in table 17 and table 18 and Hb and the administration, have nothing to do with drug effect, the reason of its decline may be lost blood relevant with the animal artificial property that causes of repeatedly drawing blood.
Table 17 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit red blood cell count(RBC)
X10
12
Table 18 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the erythrocytic influence of Japan rabbit oxyphorase
g/L。
Table 19 intravenous injection Agkistrodon acutus hemocoagulase atrox is to the influence of Japan rabbit white blood cell count(WBC)
Six, conclusion:
1, Agkistrodon acutus hemocoagulase atrox has the effect in remarkable shortening bleeding time.The Agkistrodon acutus hemocoagulase atrox of intravenous injection 0.50,0.25 and three dosage of 0.125U/kg makes mouse cut the tail bleeding time significantly to shorten.
2, Agkistrodon acutus hemocoagulase atrox has the effect that promotes blood coagulation.The Agkistrodon acutus hemocoagulase atrox of intravenous injection 0.12,0.06 and three dosage of 0.03U/kg can make the rabbit whole blood setting time shorten, and works in 10 minutes after the administration, peaks in 30 minutes, and effect continued to penetrate more than 6 hours.Intensity that the Agkistrodon acutus hemocoagulase atrox promoting blood solidifies and time are substantially parallel with the positive control drug reptilase.
3, Agkistrodon acutus hemocoagulase atrox is 0.12,0.06 with thrombogen, activated partial thromboplastin time and plasma fibrinogen content are not had obvious influence during 0.03U/kg dosage.
4, Agkistrodon acutus hemocoagulase atrox is 0.12,0.06 with euglobulin lysis time is not had obvious influence during 0.03U/kg dosage.Explanation Agkistrodon acutus hemocoagulase atrox under this dosage does not have obvious effect to fibrinolytic system.
5, Agkistrodon acutus hemocoagulase atrox 0.12,0.06 and during 0.03U/kg dosage to hematoblastic quantity with thrombocytocrit, volume of platelets, thrombocyte discharge TXA2 and the extracorporeal platelet aggregation function does not have obvious influence.
6, Agkistrodon acutus hemocoagulase atrox is behind high dosage 0.12U/kg intravenously administrable, and whole blood viscosity has downtrending.Positive control drug reptilase whole blood viscosity when 0.06U/kg also has slight downtrending.
Test (two)
The injection Agkistrodon acutus hemocoagulase atrox is to the validity of surgical incision anastalsis and the II clinical trial phase of security.
1, test objective
Preliminary assessment injection Agkistrodon acutus hemocoagulase atrox is to surgical incision hemorrhage clinical safety and validity, determines to provide foundation for III clinical trial phase research and design and dosage.
2, observation index
1) security is observed: vital sign, routine blood test, routine urinalysis, liver function (gpt, total bilirubin), renal function (blood urea nitrogen, creatinine), ionogen (K, Na, Cl, P), blood sugar, blood fat (TG, TC), blood coagulation phase (PT, TT, APTT, FIB), whole blood viscosity, Electrocardioscopy.
2) health giving quality is observed: operative incision bleeding stopping period, otch amount of bleeding, otch unit surface amount of bleeding.
3, dosage regimen
The low dose therapy group: injection Agkistrodon acutus hemocoagulase atrox 2U/ props up;
The high-dose therapy group: injection Agkistrodon acutus hemocoagulase atrox 3U/ props up;
Placebo: 0.8% dextran is formed.
Usage: select corresponding numbering medicine according to random number, each 1, after the dissolving of 2ml water for injection, vein is slowly injected or is instiled.
4, health giving quality evaluation
Estimate curative effect according to operative incision bleeding stopping period, otch amount of bleeding, otch unit surface amount of bleeding.
5, statistical analysis technique
Statistic data collection: purpose analytic set (ITT); Meet scheme collection (PP); Security date set (SS).
Statistical method: descriptive statistic and inferential statistics.
Statistical presentation: mainly adopt tabulated form.
Statistical software: adopt DAS database and DAS (ver1.0) software analysis.
6, be subjected to examination to go into the group situation and reach the crowd's data of respectively organizing
Group 180 examples are gone in 5 tame research centres altogether, high-dose therapy group 60 examples wherein, low dose therapy group 60 examples, placebo 60 examples.The high-dose therapy group is finished 58 examples, rejects 2 examples; The low dose therapy group is finished 58 examples, rejects 2 examples; Placebo is finished 58 examples, rejects 2 examples.High-dose therapy group, low dose therapy group and placebo are rejected case load comparing difference no statistical significance.Analysis of comparable shows, two groups of patients are at the comparing difference no statistical significance of aspects such as demographic characteristics, vital sign, the inspection of research previous experiments chamber, surgery situation.
7, efficacy result
Agkistrodon acutus hemocoagulase atrox high-dose therapy group, low dose therapy group and placebo compare, and the operative incision bleeding stopping period obviously shortens, and otch amount of bleeding and unit surface amount of bleeding all obviously are less than placebo, and statistics all has significant differences.Agkistrodon acutus hemocoagulase atrox high-dose therapy group and low dose therapy group compare, otch bleeding stopping period, otch amount of bleeding, unit surface amount of bleeding difference not statistically significant.
8, security observations
Trial drug all has no adverse effects to vital sign, routine blood test, routine urinalysis, liver function (gpt, total bilirubin), renal function (blood urea nitrogen, creatinine), ionogen (K, Na, Cl, P), blood sugar, blood fat (TG, TC), blood coagulation phase (PT, TT, APTT, FIB), whole blood viscosity, electrocardiogram(ECG etc.In process of the test, do not have bad incident and serious adverse reaction and take place.
9, conclusion:
High low dose therapy group of injection Agkistrodon acutus hemocoagulase atrox and low dose therapy group all have a clear and definite anastalsis for operative incision is hemorrhage; No obvious toxic-side effects; High-dose therapy group and low dose therapy group curative effect and security there was no significant difference; Do not observe tangible dose-effect relationship.
In sum, the I phase clinical tolerance Journal of Sex Research result of Agkistrodon acutus hemocoagulase atrox shows, body temperature, breathing, pulse, blood pressure, routine blood test, liver function, renal function, ionogen, electrocardiogram(ECG etc. are not seen the change that clinical meaning is arranged after the administration, do not see in the test that serious adverse reaction takes place.The human pharmacokinetics result of study shows that its transformation period is 2.5 hours.In the clinical II of the randomized, double-blind of finishing 180 examples, placebo test, high-dose therapy group (3U) 60 examples wherein, low dose therapy group (2U) 60 examples, placebo (0.8% dextran) 60 examples.Test-results shows: for operative incision bleeding stopping period, otch amount of bleeding, otch unit surface amount of bleeding, Agkistrodon acutus hemocoagulase atrox high-dose therapy group and low dose therapy group all obviously are better than placebo, but high-dose therapy group and low dose therapy group compare there was no significant difference; Whole 180 routine patients there is no adverse events and take place, to two the treatment groups of observations demonstration and the placebo there was no significant difference of each safety indexes.Illustrate that to sum up Agkistrodon acutus hemocoagulase atrox has good validity and security.
Claims (3)
1, a kind of Agkistrodon acutus hemocoagulase atrox, it prepares by the following method:
A. getting the thick poison of agkistrodon acutus dissolves with phosphoric acid buffer, the centrifugal 30min of 5000rpm, get supernatant dialysis 24 hours, last DEAE-Sepharose chromatography column, with 0.02M NaCl phosphoric acid buffer wash-out, carry out the density gradient wash-out with 0.02～0.1M NaCl phosphoric acid buffer more earlier, flow velocity is 20ml/min, collect albumen elution peak component, and dialysed again 24 hours;
B. with DEAE-Sepharose chromatography column on the solution after the above-mentioned dialysis,, collect albumen elution peak component with 0.08MNaCl phosphoric acid buffer wash-out;
C. the solution that step B is obtained SephadexG25 chromatography column desalination;
D. with the hemocoagulase solution after the desalination through 0.22 μ m filtering with microporous membrane, and lyophilize,
Described hemocoagulase molecular weight is about 29.3～29.5KD, contain 204 amino acid, iso-electric point is 5.51～5.64, have two subunits of A, B, wherein the A subunit is about 15.0KD, it is DCSSGWSSYEGHCYKVFKQSKTWTA that A subunit N holds the 1st to 25 aminoacid sequence, and the B subunit is about 16.2KD, and it is DCPSDWSSYECHCYKPDEPKTWEDA that B subunit N holds the 1st to 25 aminoacid sequence.
2, a kind of pharmaceutical composition that contains the described Agkistrodon acutus hemocoagulase atrox of claim 1.
3, pharmaceutical composition as claimed in claim 2, it also contains pharmaceutically acceptable carrier and/or vehicle.
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