CN1651079A - Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method - Google Patents
Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method Download PDFInfo
- Publication number
- CN1651079A CN1651079A CN 200410101538 CN200410101538A CN1651079A CN 1651079 A CN1651079 A CN 1651079A CN 200410101538 CN200410101538 CN 200410101538 CN 200410101538 A CN200410101538 A CN 200410101538A CN 1651079 A CN1651079 A CN 1651079A
- Authority
- CN
- China
- Prior art keywords
- buffer
- lyophilized injectable
- injectable powder
- column
- grams
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A freeze dried powder injection for promoting blood circulation and preventing thrombosis is prepared from fresh leech and fresh earthworm through extracting hirudin and vermis kinase, ultrafiltration, chromatography, reverse osmosis for concentrating, adding Tyr and medical additive, and freeze drying.
Description
Affiliated technical field
The invention belongs to technical field of traditional Chinese medicine pharmacy, be specifically related to a kind of Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and preparation method thereof.
Background technology
Pheretima is a huge earthworm section animal Pheretima aspergillum, popular Pheretimatschiliensis, the whole body of William's hair earthworm or blind Pheretimatschiliensis short of money, has the heat clearing away arresting convulsion, collateral dredging, relieving asthma, diuretic actions, be used for unconsciousness due to high fever, infantile convulsion, arthralgia, numb limbs and tense tendons, hemiplegia, dyspnea and cough due to lung-heat, the oliguria edema, hypertension, the use history in existing thousands of years, nineteen eighty-three Mihara etc. finds that first the Lumbricus water extract has the effect of direct solution fibrin and plasminogen activation, and called after " Lumbrukinase ", after healthy people uses this product, the fibrinolytic that the fibrin plate method records the peripheral blood euglobulin obviously increases, euglobulin lysis time obviously shortens, think that this extract is the novel thrombolytic agent, Pheretima is rich in Lumbrukinase, Lumbrukinase is the serine protease with kinases and fibrinolysin dual-use function, be rich in acidic amino acid, isoelectric point, IP 3-5, relative molecular mass are 2.0 * 10
4-4.0 * 10
4, fibrinolytic is strong, and pharmacological action is extensive, and oral effective, untoward reaction is light; Hirudo is China's Chinese medicine, and is just on the books in the Shennong's Herbal before 1800.The traditional Chinese medical science thinks and the curative effect that it has removing blood stasis, removing blood stasis, stimulates the menstrual flow is mainly used in treatment abdominal mass disease, mass in the abdomen, blood stasis, amenorrhea and traumatic injury that west Hirudo also commonly used is sucked blood to treat some disease.From Hirudo and salivary gland thereof, extracted the various active composition, hirudin is wherein active the most remarkable and study at most a kind of composition, the small protein that it is made up of 65-66 aminoacid (polypeptide), hirudin has extremely strong inhibitory action to thrombin, be up to now the natural special inhibitor of the strongest thrombin of finding, animal experiment and clinical research show, hirudin is anticoagulation efficiently, antithrombotic forms, and further blood stasis phenomenons such as the thrombin activation of prevention catalyzed by thrombin and platelet response, in addition, it can also the inductive fibroblasts proliferation of Trombin inhibiting and thrombin to the stimulation of endotheliocyte.Compare with heparin, not only consumption is few for it, can not cause bleeding, also do not rely on the endogenous cofactor, heparin then has the danger that causes bleeding, and Antithrombin III often reduces in the pathogenic process of disseminated inravascular coagulation, this will limit the curative effect of heparin, adopt Hirudo to have effect preferably, hirudin is the up-and-coming anticoagulant blood stasis dispelling of a class medicine, and it can be used for treating the especially treatment of phlebothrombosis and the blood coagulation of diffusivity blood vessel of various thrombus diseases; Also can be used for the formation of prevention of arterial thrombosis behind the surgical operation, behind the prevention thrombus or the formation of thrombosis behind the revascularization, improve extracorporeal circulation of blood and blood dialysis, hirudin is more stable, trypsin and Chymotrypsin do not destroy its activity, and some hydrolysis fragment of hirudin still has the effect of Trombin inhibiting;
Thrombin is one of principal element that causes thrombosis, and the formation of thrombosis is the main cause that causes cardiovascular and cerebrovascular disease.Cardiovascular and cerebrovascular disease is one of principal disease of present harm humans life and health, and according to The World Health Organization's statistics, annual nearly 1,700 ten thousand people in the whole world die from cardiovascular and cerebrovascular disease, just have 1 to die from cardiovascular and cerebrovascular disease among promptly global per 3 dead persons.Expect the year two thousand twenty, because of cardiovascular and cerebrovascular disease death number will increase by 50% than this numeral, up to 2,500 ten thousand people.China more than 60 years old population surpassed 100,000,000, promptly China has above need of 5,000,000 people prevent and treat and treat cardiovascular and cerebrovascular disease with anticoagulant every year, and this class disease develops to rejuvenation more than 5% every year for this ages sickness rate.If calculate by 1% number, the radix of sickness rate can be bigger.
Patent " manufacture method and products thereof of the ejection preparation of treatment cardiovascular and cerebrovascular disease " (application number 03148281.3) discloses uses the process that Hirudo, Pheretima prepare ejection preparation, this patent is used 105-136 ℃ of hot pressing 10-45 minute in the separation and purification process, under this violent condition, hirudin, Lumbrukinase will all lose activity.
Summary of the invention
For these reasons; it is solvent extraction hirudin, Lumbrukinase that the present invention adopts water; adopt ultrafiltration, anion exchange and cation exchange column chromatography method to carry out remove impurity; the reuse reverse osmosis membrane technology is removed the salts substances that leaching process produces; the purity of hirudin, Lumbrukinase is improved greatly, add the enzymatic protective reagent tyrosine that we find in experimentation, prolonged hirudin, Lumbrukinase keeps the active time; be prepared into lyophilized injectable powder, have better pharmacological action.
Tyrosine is a kind of primary amino acid, and we dredge in the process of the logical lyophilized injectable powder of blood in research, add tyrosine, and the activity of the hirudin in the said preparation, Lumbrukinase is had protective effect, prolongs its active retention time greatly.
The invention provides the lyophilized injectable powder that a kind of Pheretima, Hirudo are made, wherein hirudin, the Lumbrukinase content in active component is not less than 90%.
The present invention also provides a kind of preparation method of said medicine.
One. process recipes
(1) crude drug weight proportion of the present invention is:
Hirudo 6-7.5 weight portion, Pheretima 2.5-4 weight portion;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that the hollow fiber column of 50000-100000 carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration adds water to original volume, repeatable operation 3-5 time during to the 1/3-1/2 of original solution volume, merge permeate, be concentrated into the solution that relative density is 1.05-1.10 in the time of 20 ℃;
(4) spissated filtrate being transferred pH value with buffer is 6.0-7.0, ionic strength is that 0.01mol/l passes through cation exchange column, wash 4-6 column volume eluting doubly with buffer, collect eluent, it is that 6.0-7.0 passes through anion-exchange column that the reuse buffer is transferred pH value, be that the buffer of 6.0-7.0 is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that the buffer of 6.0-7.0 adds 0.2-0.5mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) the eluent concentrating and desalinating obtains semi-finished product;
(6) semi-finished product are added enzymatic protective reagent tyrosine and pharmaceutic adjuvant, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain lyophilized injectable powder.
Wherein, permeate is concentrated in the step (3), is to adopt reverse osmosis membrane to concentrate.
Buffer is a kind of of phosphate buffer and tris-HCI buffer (Tris-HCL) in the step (4); Cation exchange column is to be selected from a kind of in CM-cross-linking dextran, CM-agarose, CM-cellulose, SP-cross-linking dextran, SP-agarose, the SP-cellulose; Anion-exchange column is to be selected from a kind of in DEAE-cross-linking dextran, DEAE-agarose, DEAE-cellulose, Q-cross-linking dextran, Q-agarose, the Q-cellulose.
Two. check and analysis
Adopt high performance liquid chromatography that hirudin, Lumbrukinase (in xanthine) are detected.
1. experimental apparatus: waters600E type high performance liquid chromatograph, 996PDA detector, chromatographic column: Japanese TOSOHTSK2000SW 7.5*300
2. experiment medicine: lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
2. chromatographic condition: work station: (compound method is according to the main raw and auxiliary material quality control standard of Chinese biological goods version in 2000 for the phosphate buffer solution of millennium chromatogvaph mobile phase: 0.01mol/L, pH value 7.2, Chinese biological standard of articles committee compiles, Chemical Industry Press, the P13-14 configuration).
3. sample preparation: get the moving dry powder injection of the present invention, add the water for injection dissolving fully, use the peptide purification centrifuge tube, carry out centrifugalize, the extracting centrifugal liquid body is sample.
4. go up sample: the sample of getting above-mentioned processing is drawn quantitative liquid with microsyringe, injects injector and detects.
5. result: through the content that accounts for active component that calculates hirudin, Lumbrukinase is 91.3%.
Three. lyophilized injectable powder of the present invention is than the detection of living
1. experimental principle: the activity of natural hirudin, Lumbrukinase detects country's detection method of publishing standard not as yet, root
According to the detection method (Chinese biological goods rules version appendix 1 in 2000) of thrombin activity,
Is proportional the combination according to hirudin, Lumbrukinase with thrombin, consumes a thrombin unit
Be equal to the principle of an anticoagulation unit (ATU), adopt test tube method (serial dilution)
The anticoagulation unit of detection hirudin, Lumbrukinase tires.
2. the fibrinogenic normal saline solution of experiment material: 5g/L, injection normal saline (the proud medicine in sky, Guangdong
Development corporation, Ltd. laboratory experiment chamber provides)
(Anhui Mulberry protozoa technical research institute buys the lyophilization thrombin, and every bottle of content is
500IU, the time spent is diluted to every milliliter with normal saline and contains thrombin 0.5IU)
3. testing sample: this patent lyophilized injectable powder (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
4. detection method: get 8 in 10 * 80mm test tube, in every test tube, add normal saline 0.2ml (first pipe
Do not add), in first pipe, add testing sample 0.2ml; Add 0.2ml in second pipe, fill
Absorption 0.2ml adds tee pipe after dividing mixing, and example pushes away in proper order, until being added to the 7th pipe, and will
Unnecessary 0.2ml mixed liquor is discarded, and this moment, each test tube all was 0.2ml, but concentration successively
Be 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64, the 8th pipe is not because of containing Hirudo
Plain, Lumbrukinase, as experimental control, after application of sample finishes, be put into 37 ℃ of water-baths make sample with
Thrombin action 5-10min adds 5g/l fibrinogen solution 0.2 again in each test tube
Ml puts into 37 ℃ of water-baths again, observed and recorded 1-24 hour result, and calculate.Meter
The calculation method is: solidify or sedimentary test tube is a no antithrombin activity (feminine gender), do not solidify
Or there is not test tube that precipitation produces for having antithrombin activity.
5. specific activity calculates: adopt Chinese biological goods rules (an one) micromethod, with BCATM Protein Assay
Measure the protein content of separation component, calculate every milli according to the antithrombin activity measurement result
Restrain proteinic antithrombase specific activity.Find natural hirudin, Lumbrukinase body according to experiment
Measurement result has positive correlation in outer measurement result and the body.
6. experimental result: the ratio of lyophilized injectable powder of the present invention is lived and is 203.1IU/mg.
Four. the active retention time of different preparations is relatively
The experiment medicine: lyophilized injectable powder of the present invention is experimental group 1, adopt that extraction and purification process of the present invention is prepared half
The lyophilized injectable powder that finished product does not add the tyrosine preparation is experimental group 2 (the proud drug development in sky, Guangdong
The company limited laboratory provides)
Experimental technique: different preparations were placed 0 month under the same conditions, 3 months, 6 months, 12 months, 18
Individual month, 24 months, to carry out surveying according to the method for above-mentioned experiment three than biopsy, testing result sees Table
1.
The ratio of the different preparations of table 1 is lived relatively
24 months
The ratio that the ratio that the ratio that the ratio that 0 month ratio was lived 3 months was lived 6 months was lived 12 months was lived 18 months is lived
Group is than living
(IU/mg) (IU/mg) (IU/mg) (IU/mg) (IU/mg)
(IU/mg)
Experimental group 1 203.1 202.0 198.7 190.4 185.6 179.9
Experimental group 2 203.2 185.3 161.9 130.2 112.7 107.3
Conclusion: lyophilized injectable powder of the present invention kept 80% activity in the time of 24 months, and did not add the lyophilizing of tyrosine
Injectable powder only remained about 50% activity in the time of 24 months, prove absolutely in the technology of the present invention to add enzyme
The significance of protective agent tyrosine.
Five. toxicological experiment research
1. acute toxicity testing
Laboratory animal: healthy mice 18-22g,, male and female half and half.
Experiment medicine: lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
Experimental technique: according to 500 times, 1000 times, 1500 times, 2000 times, 2500 times, 3000 of human body administration
Doubly, 4000 times dosage tail vein gives mice, negative control group gives the physiology of matched doses
Saline, animal are freely taken the photograph vital movement and the death condition that the diseases caused by retention of fluid food is observed animal, 10d after the administration
The mice of survival is slaughtered, got the variation of every index of its its peripheral blood of whole blood assay, employing changes
(The Fourth Military Medical University's journal, 667-669) is asked LD50 at 2002 the 7th phases to good karber's method.
Experimental result: the LD50 of lyophilized injectable powder of the present invention is 117.9g/kg, and for the clinical application amount 2947 times show
The toxicity of lyophilized injectable powder of the present invention is lower, and clinical application belongs to safety.
2. long term toxicity test
Laboratory animal: healthy rat 300-400 gram,, male and female half and half.
Experiment medicine: lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
Experimental technique: lyophilized injectable powder of the present invention is given according to 10 times of human body administrations, three dosage of 63 times, 375 times
The rat tail vein injection, blank group gives the normal saline of matched doses, continuous 30 days, detects
Every index.
Experimental result: the general behavior performance of each administration group, food ration, body weight gain rate, hematology, serum biochemistry
Indexs such as inspection, organ coefficient, histopathological examination compare with the blank group, all do not have
Notable difference illustrates that lyophilized injectable powder of the present invention is in clinical practice amount 10-375 multiple dose scope
In, rat there is not obvious toxic-side effects.
Six. pharmacology embodiment
Embodiment 1
The experiment of anticoagulant
Laboratory animal: SD rat, body weight 200～250g, male and female half and half.
Experiment medicine: SHUXUETONG ZHUSHEYE (Mudanjiang Youbo Pharmaceutical Co., Ltd.)
Lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
Normal saline
Experimental technique: get rat, be divided into lyophilized injectable powder group of the present invention, SHUXUETONG ZHUSHEYE group, normal saline group,
Dosage 40mg/kg, normal saline etc. hold inequality 2 times/day, use after administration next day 1 time
1% pentobarbital sodium (dosage: intraperitoneal injection of anesthesia 0 5ml/100g).Press document (Zhu Yidong master
Compile disseminated inravascular coagulation Beijing: People's Health Publisher, 1982; 279 293) method is multiple
System rat anticoagulant model.At once get blood 3ml from carotid artery behind the duplicating model, 38% sodium citrate
Platelet rich plasma (PRP) and platelet poor plasma (PPP) are made in anticoagulant (1: 10), and it is little to calculate blood
Plate coagulation rate, platelet aggregation rate is measured with SPA-4 type platelet aggregation instrument, and short poly-agent is with two
Adenosine phosphate, (ADP), concentration 10 μ mol/L.Testing index has one minute aggregation rate (A
1), maximum agglutination rate (Amax) and maximum gathering time (Tmax).See Table 2:
The comparison of the different preparation agglutinate rate of blood platelet of table 2
Number of animals A
1Amax Tmax
Group
(only) (%) (%) (s)
Normal saline group 10 28 ± 7 26 ± 9 64 ± 29
SHUXUETONG ZHUSHEYE group 10 13 ± 10
*15 ± 10
*60 ± 28
Lyophilized injectable powder group 10 8 ± 3 of the present invention
*7 ± 4
*55 ± 21
Annotate: compare with the normal saline group
*P＜0.01,
*P＜0.05
Embodiment 2
Utilize the thrombosis method to measure to the thrombotic influence of rat.
Experiment medicine: normal saline (blank group)
SHUXUETONG ZHUSHEYE (Mudanjiang Youbo Pharmaceutical Co., Ltd.)
Lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
30 of the rat of laboratory animal: body weight 300-400 gram, male and female are regardless of.Be divided into three groups, 10 every group.
Experimental technique: with the rat administration, dosage 40mg/kg, normal saline etc. hold inequality, will be big after the administration
(pentobarbital sodium 30-40mg/kg, ip), dorsal position is fixed, and separates trachea, inserts in white mouse anesthesia
Go into a plastic bushing, and separate right common carotid artery and left external jugular vein, in the stage casing of polyethylene tube
Put into a long silk thread of 6 centimetres,, be full of polyethylene tube with heparin-saline solution, when
After one end of polyethylene tube inserts left external jugular vein, inject anticoagulant heparin accurately by polyethylene tube,
And then the other end of polyethylene tube inserted right common carotid artery, and open bulldog clamp, blood is from the right side
Common carotid artery flow to polyethylene tube, returns left external jugular vein, open blood flow Herba Clinopodii after 15 minutes,
Take out silk thread rapidly and weigh, gross weight deducts silk thread weight and promptly gets thrombus weight.With the blank group and
The animal wet weight of thrombus of other two groups of administration groups carries out record, calculates, and is as shown in table 3:
The thrombus weight of each administration group of table 3
The number of animals wet weight of thrombus
Group
(only) (mg)
Normal saline group 10 35.12 ± 0.91
SHUXUETONG ZHUSHEYE group 10 24.38 ± 0.77
*
Lyophilized injectable powder group 10 11.16 ± 0.09 of the present invention
*
Annotate: compare with the normal saline group
*P＜0.01,
*P＜0.05
Embodiment 3
Influence to the inductive thrombosis death of mice ADP
Experiment medicine: normal saline (blank group)
SHUXUETONG ZHUSHEYE (Mudanjiang Youbo Pharmaceutical Co., Ltd.)
Lyophilized injectable powder of the present invention (Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides)
Experimental technique: get 60 of healthy mices, male and female half and half are divided into 3 groups at random: normal saline group, lyophilized powder of the present invention
Injection group, commercially available SHUXUETONG ZHUSHEYE group tail every day intravenously administrable, dosage 57mg/kg, continuous 7
D, 1h tail vein injection ADP48.0mL/kg after the last administration, record animal dead feelings
Condition.See Table 4:
Table 4 is respectively organized preparation the influence of the inductive thrombosis death of mice ADP is compared
An a group sum/death toll/mortality rate
Normal saline group 20 20 100%
SHUXUETONG ZHUSHEYE group 20 14 70.0%
Lyophilized injectable powder group 20 8 40.0% of the present invention
Embodiment 4
Utilization is measured lyophilized injectable powder of the present invention and to the influence of rat myocardial infarction situation in body-centered flesh infarction method.
Experiment medicine: SHUXUETONG ZHUSHEYE (Mudanjiang Youbo Pharmaceutical Co., Ltd.)
(Tianzhijiao Medication Development Co., Ltd., Guangdong's laboratory provides lyophilized injectable powder of the present invention
2. laboratory animal: 20 of male rat 250-300 grams are divided into two groups.
3. experimental technique: get 20 of male rats, after the urethane 0.65g/kg abdominal cavity light anaesthesia, back of the body position is fixing, uses
More than half rubber bulb is connected to artificial respirator, practices artificial respiration, rat chest unhairing,
Sterilization is cut about 2 centimetres of skin along left mid-clavicular line, at the 4th or the 5th intercostal passivity branch
From basic unit, open the thoracic cavity, cut off pericardium, gently press the right side thorax, extrude heart, at tremulous pulse
After Coronary vein place joint is pricked left coronary artery between circular cone and the left auricle, heart is put back to the thoracic cavity, fast
Speed is sewed up the thoracic cavity, stops the artificial respiration, with two kinds of preparations 10 rats are experimentized respectively,
It is as shown in table 5 to obtain data computation:
The pharmacology of table 5 liang group preparation relatively
Group obvious effective rate effective percentage inefficiency total effective rate
SHUXUETONG ZHUSHEYE 18.3% 54.0% 27.7% 72.3%
Lyophilized injectable powder of the present invention 34.1% 58.2% 7.7% 92.3%
Conclusion: can learn that from testing us lyophilized injectable powder of the present invention is compared with SHUXUETONG ZHUSHEYE and had better
Pharmacological action.
Four. preparation embodiment
Embodiment 1
(1) crude drug weight proportion of the present invention is:
Hirudo 6000 grams, Pheretima 4000 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 50000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/3 o'clock of the original solution volume, and repeatable operation 3 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.05 solution;
(4) spissated filtrate being transferred pH value with phosphate buffer is 6.0, ionic strength is that 0.01mol/l passes through CM-cross-linking dextran cation exchange column, wash 4 times column volume eluting with buffer, collect eluent, it is 6.0 by DEAE-cross-linking dextran anion-exchange column that the reuse buffer is transferred pH value, be that 6.0 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.0 buffer adds 0.2mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 9.6 grams;
(6) semi-finished product 9.6 grams are added enzymatic protective reagent tyrosine 10 grams and pharmaceutic adjuvant mannitol 30.4 grams, after water for injection dissolving fully, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 2
(1) crude drug weight proportion of the present invention is:
Hirudo 7500 grams, Pheretima 2500 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 100000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/2 o'clock of the original solution volume, and repeatable operation 5 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.10 solution;
(4) spissated filtrate being transferred pH value with tris-HCI buffer (Tris-HCL) buffer is 7.0, ionic strength is that 0.01mol/l passes through CM-agarose cation exchange column, wash 6 times column volume eluting with buffer, collect eluent, it is 7.0 by DEAE-agarose anion-exchange column that the reuse buffer is transferred pH value, be that 7.0 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 7.0 buffer adds 0.5mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 9.2 grams;
(6) semi-finished product 9.2 grams are added enzymatic protective reagent tyrosine 9 grams and pharmaceutic adjuvant sucrose 31.8 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 3
(1) crude drug weight proportion of the present invention is:
Hirudo 6500 grams, Pheretima 3500 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 80000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/3 o'clock of the original solution volume, and repeatable operation 4 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.08 solution;
(4) spissated filtrate being transferred pH value with phosphate buffer is 6.5, ionic strength is that 0.01mol/l passes through CM-cellulose cation exchange column, wash 5 times column volume eluting with buffer, collect eluent, it is 6.5 by DEAE-cellulose anion-exchange column that the reuse buffer is transferred pH value, be that 6.5 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.5 buffer adds 0.3mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 9.4 grams;
(6) semi-finished product 9.4 grams are added enzymatic protective reagent tyrosine 9.5 grams and pharmaceutic adjuvant lactose 31.1 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 4
(1) crude drug weight proportion of the present invention is:
Hirudo 7000 grams, Pheretima 3000 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 50000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/3 o'clock of the original solution volume, and repeatable operation 5 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.09 solution;
(4) spissated filtrate being transferred pH value with tris-HCI buffer (Tris-HCL) buffer is 6.3, ionic strength is that 0.01mol/l passes through SP-cross-linking dextran cation exchange column, wash 4 times column volume eluting with buffer, collect eluent, it is 6.3 by Q-cross-linking dextran anion-exchange column that the reuse buffer is transferred pH value, be that 6.3 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.3 buffer adds 0.35mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent concentrating and desalinating obtains semi-finished product 9.2 grams;
(6) semi-finished product 9.2 grams are added enzymatic protective reagent tyrosine 9.2 grams and pharmaceutic adjuvant mannitol, sucrose 31.6 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 5
(1) crude drug weight proportion of the present invention is:
Hirudo 7200 grams, Pheretima 2800 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 100000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/3 o'clock of the original solution volume, and repeatable operation 3 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.07 solution;
(4) spissated perphosphate filtrate being transferred pH value with buffer is 6.8, ionic strength is that 0.01mol/l passes through SP-agarose cation exchange column, wash 4 times column volume eluting with buffer, collect eluent, it is 6.8 by Q-agarose anion-exchange column that the reuse buffer is transferred pH value, be that 6.8 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.8 buffer adds 0.40mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 9.2 grams;
(6) semi-finished product 9.2 grams are added enzymatic protective reagent tyrosine 10 grams and pharmaceutic adjuvant sucrose, lactose 30.8 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 6
(1) crude drug weight proportion of the present invention is:
Hirudo 6800 grams, Pheretima 3200 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 80000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/2 o'clock of the original solution volume, and repeatable operation 4 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.06 solution;
(4) spissated filtrate being transferred pH value with tris-HCI buffer (Tris-HCL) buffer is 6.2, ionic strength is that 0.01mol/l passes through SP-cellulose cation exchange column, wash 5 times column volume eluting with buffer, collect eluent, it is 6.2 by Q-cellulose anion-exchange column that the reuse buffer is transferred pH value, be that 6.2 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.2 buffer adds 0.45mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 9.3 grams;
(6) semi-finished product 9.3 grams are added enzymatic protective reagent tyrosine 9.5 grams and pharmaceutic adjuvant mannitol, lactose 31.2 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 7
(1) crude drug weight proportion of the present invention is:
Hirudo 6200 grams, Pheretima 2800 grams;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 50000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/2 o'clock of the original solution volume, and repeatable operation 5 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.09 solution;
(4) spissated filtrate being transferred pH value with phosphate buffer is 6.4, ionic strength is that 0.01mol/l passes through CM-cross-linking dextran cation exchange column, wash 6 times column volume eluting with buffer, collect eluent, it is 6.4 by Q-cellulose anion-exchange column that the reuse buffer is transferred pH value, be that 6.4 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.4 buffer adds 0.25mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent concentrating and desalinating obtains semi-finished product 9.5 grams;
(6) semi-finished product 9.5 grams are added enzymatic protective reagent tyrosine 10 grams and pharmaceutic adjuvant mannitol, sucrose, lactose 30.5 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 1000 bottles of lyophilized injectable powders.
Embodiment 8
(1) crude drug weight proportion of the present invention is:
70 kilograms of Hirudos, 30 kilograms of Pheretimas;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that 80000 hollow fiber column carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration added water to original volume to 1/3 o'clock of the original solution volume, and repeatable operation 3 times merges permeate, was concentrated into relative density in the time of 20 ℃ and is 1.09 solution;
(4) spissated filtrate being transferred pH value with tris-HCI buffer (Tris-HCL) buffer is 6.9, ionic strength is that 0.01mol/l passes through SP-cellulose cation exchange column, wash 4 times column volume eluting with buffer, collect eluent, it is 6.9 by DEAE-cross-linking dextran anion-exchange column that the reuse buffer is transferred pH value, be that 6.9 buffer is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that 6.9 buffer adds 0.4mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) eluent reverse osmosis membrane concentrating and desalinating obtains semi-finished product 92.3 grams;
(6) semi-finished product 92.3 grams are added enzymatic protective reagent tyrosine 95 grams and pharmaceutic adjuvant mannitol 312.7 grams, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain 10000 bottles of lyophilized injectable powders.
Annotate: fresh Pheretima of the present invention is put into clear water hungry a couple of days, and Pheretima is spued, and impurity carries out extraction separation again in the body.
Claims (7)
1. the logical lyophilized injectable powder of a thin blood is characterized in that the content that wherein hirudin, Lumbrukinase account for active component is not less than 90%.
2. the preparation method of the logical lyophilized injectable powder of thin blood according to claim 1 is characterized in that it being to extract hirudin from bright Hirudo, extracts Lumbrukinase from bright Lumbricus, and adding enzymatic protective reagent tyrosine and pharmaceutic adjuvant are prepared into lyophilized injectable powder behind the purification.
3. the preparation method of the logical lyophilized injectable powder of thin blood according to claim 2, its feature may further comprise the steps:
(1) the crude drug weight proportion is:
Hirudo 6-7.5 weight portion, Pheretima 2.5-4 weight portion;
(2) get bright Hirudo, Pheretima, add water for injection, flush away impurity is used the electrical meat mincer fragmentation, the fragmentation of reuse ultrasonic low temperature; Homogenate is sub-packed in the container with lid, freezed 24 hours in-30 ℃ of refrigerator-freezers, reuse flowing water thaws, and freezes once more, freezes repeatedly, melts 5 times, after thawing for the last time, uses refrigerated centrifuger centrifugation, and it is standby to draw supernatant;
(3) be that the hollow fiber column of 50000-100000 carries out ultrafiltration with the supernatant molecular cut off, ultrafiltration adds water to original volume, repeatable operation 3-5 time during to the 1/3-1/2 of original solution volume, merge permeate, be concentrated into the solution that relative density is 1.05-1.10 in the time of 20 ℃;
(4) spissated filtrate being transferred pH value with buffer is 6.0-7.0, ionic strength is that 0.01mol/l passes through cation exchange column, wash 4-6 column volume eluting doubly with buffer, collect eluent, it is that 6.0-7.0 passes through anion-exchange column that the reuse buffer is transferred pH value, be that the buffer of 6.0-7.0 is washed 3 times of column volumes with pH value respectively, eluent discards, be that the sodium chloride that the buffer of 6.0-7.0 adds 0.2-0.5mol/l carries out eluting with pH value at last, collect eluent, be eluted to UV-detector and detect less than till absorbing;
(5) the eluent concentrating and desalinating obtains semi-finished product;
(6) semi-finished product are added enzymatic protective reagent tyrosine and pharmaceutic adjuvant, after water for injection adjustment concentration, divide to install in the cillin bottle, carry out lyophilization, roll lid, detect, obtain lyophilized injectable powder.
4. according to the described preparation method of claim 3, wherein step (4) buffer is a kind of of phosphate buffer and tris-HCI buffer (Tris-HCL).
5. according to the described preparation method of claim 3, wherein step (4) cation exchange column is a kind of in CM-cross-linking dextran, CM-agarose, CM-cellulose, SP-cross-linking dextran, SP-agarose, the SP-cellulose.
6. according to the described preparation method of claim 3, wherein step (4) anion-exchange column is a kind of in DEAE-cross-linking dextran, DEAE-agarose, DEAE-cellulose, Q-cross-linking dextran, Q-agarose, the Q-cellulose.
7. according to the described preparation method of claim 3, wherein concentrating of step (3) permeate is to adopt reverse osmosis membrane to concentrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410101538 CN1651079A (en) | 2004-12-23 | 2004-12-23 | Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410101538 CN1651079A (en) | 2004-12-23 | 2004-12-23 | Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1651079A true CN1651079A (en) | 2005-08-10 |
Family
ID=34869622
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410101538 Pending CN1651079A (en) | 2004-12-23 | 2004-12-23 | Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1651079A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008003259A1 (en) | 2006-06-28 | 2008-01-10 | Zhenguo Li | An extract for preventing or treating thrombotic diseases |
| WO2010029453A1 (en) * | 2008-09-10 | 2010-03-18 | Pt.Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| CN101961353A (en) * | 2010-10-12 | 2011-02-02 | 唐培克 | Earthworm extractive, preparation method and application thereof and earthworm extractive-containing medicinal composition |
| CN102139092A (en) * | 2011-03-21 | 2011-08-03 | 中山大学 | Leech freeze-dried powder injection solution for injection and preparation method thereof |
| CN102153645A (en) * | 2010-12-24 | 2011-08-17 | 南宁乙翔生物科技有限公司 | Method for removing heavy metal ions in hirudin by using electro-osmosis |
| CN101584853B (en) * | 2009-06-15 | 2012-12-19 | 南京中医药大学 | Earthworm protein polypeptide preparation |
| CN104888206A (en) * | 2015-06-19 | 2015-09-09 | 广西复鑫益生物科技有限公司平南分公司 | Combination drug containing hirudin for treating cardiovascular and cerebrovascular diseases |
| CN104958268A (en) * | 2015-06-19 | 2015-10-07 | 广西复鑫益生物科技有限公司平南分公司 | Preparation method of hirudin freeze-dried powder injection |
| CN105132398A (en) * | 2015-08-19 | 2015-12-09 | 江苏中邦制药有限公司 | Method for purifying 1umbrmonase in earthworms |
| CN106366184A (en) * | 2016-08-31 | 2017-02-01 | 天津瑞普生物技术股份有限公司 | Preparation method of hirudin crude extract |
| CN106526051A (en) * | 2016-04-15 | 2017-03-22 | 北京儒展生化药物研究中心 | Method for determining contents of high-molecular-weight proteins and small-molecular-weight impurities in lumbrukinase injection |
-
2004
- 2004-12-23 CN CN 200410101538 patent/CN1651079A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008003259A1 (en) | 2006-06-28 | 2008-01-10 | Zhenguo Li | An extract for preventing or treating thrombotic diseases |
| CN101340921B (en) * | 2006-06-28 | 2011-09-28 | 李振国 | Extract for preventing and treating embolus disease |
| US8252340B2 (en) | 2006-06-28 | 2012-08-28 | Mudanjiang Youbo Pharmaceutical Co., Ltd | Extract for treating thrombotic diseases |
| WO2010029453A1 (en) * | 2008-09-10 | 2010-03-18 | Pt.Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| AU2009290466B2 (en) * | 2008-09-10 | 2014-06-12 | Pt. Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| CN101584853B (en) * | 2009-06-15 | 2012-12-19 | 南京中医药大学 | Earthworm protein polypeptide preparation |
| CN101961353B (en) * | 2010-10-12 | 2013-11-06 | 唐培克 | Earthworm extractive, preparation method and application thereof and earthworm extractive-containing medicinal composition |
| CN101961353A (en) * | 2010-10-12 | 2011-02-02 | 唐培克 | Earthworm extractive, preparation method and application thereof and earthworm extractive-containing medicinal composition |
| CN102153645A (en) * | 2010-12-24 | 2011-08-17 | 南宁乙翔生物科技有限公司 | Method for removing heavy metal ions in hirudin by using electro-osmosis |
| CN102139092A (en) * | 2011-03-21 | 2011-08-03 | 中山大学 | Leech freeze-dried powder injection solution for injection and preparation method thereof |
| CN104888206A (en) * | 2015-06-19 | 2015-09-09 | 广西复鑫益生物科技有限公司平南分公司 | Combination drug containing hirudin for treating cardiovascular and cerebrovascular diseases |
| CN104958268A (en) * | 2015-06-19 | 2015-10-07 | 广西复鑫益生物科技有限公司平南分公司 | Preparation method of hirudin freeze-dried powder injection |
| CN105132398A (en) * | 2015-08-19 | 2015-12-09 | 江苏中邦制药有限公司 | Method for purifying 1umbrmonase in earthworms |
| CN105132398B (en) * | 2015-08-19 | 2018-07-10 | 江苏中邦制药有限公司 | The purification process of Lumbrokinase in a kind of earthworm |
| CN106526051A (en) * | 2016-04-15 | 2017-03-22 | 北京儒展生化药物研究中心 | Method for determining contents of high-molecular-weight proteins and small-molecular-weight impurities in lumbrukinase injection |
| CN106366184A (en) * | 2016-08-31 | 2017-02-01 | 天津瑞普生物技术股份有限公司 | Preparation method of hirudin crude extract |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Johns et al. | Further fractionations of histones from calf thymus | |
| CN1651079A (en) | Shu xue tong (blood coursing free flowing) freeze dried powder snjection agent and its preparation method | |
| WO2021077794A1 (en) | Preparation and application of soluble tim-3 recombinant protein and mutant protein thereof | |
| CN116589533A (en) | Euphausia superba small molecule active peptide, and preparation method and application thereof | |
| CN1660145A (en) | New method for preparing injection of freeze-dried powder blood dredge | |
| CN109527270A (en) | Leech specific nutrient induces the extracting method of liquid and natural hirudin | |
| CN1198639C (en) | Immune enhancing compositions | |
| CN1891235A (en) | Pheretima injection preparation and its preparing method | |
| CN1720978A (en) | Pulse invigorating injection and method for preparing the same | |
| CN111100191A (en) | Active protein or polypeptide derived from human placenta and its application | |
| CN115124591B (en) | Spirulina platensis phycocyanin angiotensin converting enzyme inhibitory peptide and preparation method and application thereof | |
| CN1261159C (en) | Freeze dried powder injection of hirudin and its preparation method | |
| CN1255183C (en) | Myocardium peptide and its use | |
| CN101078009A (en) | Method for extracting single component batroxobin from Bothrops atrox poison | |
| CN113215214B (en) | Method for preparing ACE inhibitory peptide by enzymolysis of mulberry leaf protein | |
| CN102021160B (en) | Snake venom serine protease and coding gene and application thereof | |
| CN106879884B (en) | Method for improving royal jelly performance | |
| CN1912115A (en) | Venomous snake thrombin sample enzyme modified by polyethylene glycol | |
| CN1876016A (en) | Preparation method of compound kuhseng preparation for injection and medical use thereof | |
| CN1788775A (en) | Traditional Chinese medicine composition for improving immunity and preparation method and quality control method thereof | |
| CN1615994A (en) | Method for preparing Chinese medicine composition for treating ischemic brain apoplexy | |
| Dinter et al. | Suppression of the inhibitory action of guanidine on virus multiplication by some amino acids | |
| CN1853701A (en) | Effective part of Sang Medicine and preparation thereof | |
| CN1300171C (en) | Preparation of myocardium peptide | |
| CN118388590B (en) | Antarctic krill ACE (angiotensin converting enzyme) inhibitory peptide LFAGA, RDWPEGR, DWPEGR and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |