CN1088836A - Lepirudin 023 ludon and complex thereof are used for preparation prevention and treatment thrombotic disease medicine - Google Patents
Lepirudin 023 ludon and complex thereof are used for preparation prevention and treatment thrombotic disease medicine Download PDFInfo
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- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 19
- 238000011282 treatment Methods 0.000 title claims abstract description 16
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Abstract
The present invention relates to a kind of medicine and preparation method, particularly relate to the medicine that is used to prevent blood coagulation and treatment thrombotic disease that a kind of artificial synthesis with gene prepares lepirudin 023 ludon and hirudin and complex.
The present invention provides a kind of lepirudin 023 ludon and complex by 1: 20-1 in order to improve: 2 part by weight are made into the medicine that is used for anticoagulation and treatment thrombotic disease, and prepare pharmaceutical methods of the present invention.This method is simple, is easy to grasp synthesize, and has improved the drug effect of the medicine of preparation widely.
Description
The present invention relates to a kind of medicine and preparation method, particularly relate to the medicine that is used to prevent blood coagulation and treatment thrombotic disease that a kind of artificial synthesis with gene prepares lepirudin 023 ludon and hirudin and complex.
Hirudin is a kind of Acid polypeptide of the salivary gland secretion of Hirudo, has strong blood coagulation resisting function.Markwardt
(1)In nineteen fifty-five first from Hirudo medicinalis separation and purification hirudin.Natural hirudin has multiple isomer, is made up of 65 or 66 aminoacid, and structure is quite similar each other.Hirudin can form stable non-covalent bonded complex with thrombin, and thrombin is had special inhibitory action.Hirudin is the natural inhibitor of at present known the most strong thrombin
(2)
The medicine of at present clinical treatment thrombosis commonly used, such as urokinase, plasminogen activators (tPA), snake venom, Lumbrukinases etc. all are to carry out thromboembolism treatment after thrombosis, and the function of hirudin is an anticoagulation, the formation of anti-tampon, and side effect is little, so hirudin will become the novel drugs of prevention and treatment thrombus disease.External also in the research of being engaged in the hirudin clinicing aspect, but as yet not in wide clinical application.People are also synthesizing the derivant of making hirudin in addition, and complex etc. are used for the treatment and the prevention of thrombus disease.
The medical value of hirudin obtains people's generally attention, but because the hirudin natural origin is limited, its research and application is restricted.In recent years, the foundation of technique for gene engineering, making people make hirudin becomes possibility.Since 1986, external several laboratorys have successively been reported the result who expresses hirudin in escherichia coli and yeast cells.As: No. 91101687.2, Chinese patent application, " secretions of hirudin derivatives ", put down in writing, the secretions of this hirudin derivatives is that directly be positioned the to encode mode in dna fragmentation downstream of antibacterial signal peptide of the gene with the coding hirudin derivative makes up recombinant vector, conversionization escherichia coli secreting type mutant then, cultivate the antibacterial after transforming in culture medium, and obtain the method for hirudin derivative from culture medium, its hirudin derivative is:
-terminal amino acid sequence A-Thr-Yyr-Thr-Asp,
Wherein A represents Ala,
And for example: Chinese patent application No. 89100165.4 " hirudin derivatives " is described.They or be the host with escherichia coli or with yeast, when being the host with escherichia coli, because escherichia coli are harmful, yeast is safer when comparing as the preparation medicine with yeast, above-mentioned in addition several method for preparing hirudin derivative is complicated, and these methods resulting be a kind of simple hirudin or their derivant, do not have a kind of combination drug that is used for the treatment of and prevents thrombotic disease of making.
The objective of the invention is to overcome the shortcoming and defect of prior art, to have thrombolytic agent therapeutic effect and the range of application that enlarges hirudin itself now in order improving, thereby to provide a kind of lepirudin 023 ludon or lepirudin 023 ludon and its complex with 1: 20-1: 2 ratios are made into the medicine of anticoagulation and prophylactic treatment thrombosis and prepare the method for medicine of the present invention.
This method may further comprise the steps:
1, with the hirudin HV of synthetic
2Gene place the promoter of yeast α-factor gene and lead after the peptide, be configured to expression vector.
2,, and obtain the mutant strain of hirudin gene through mutation with the expression vector transformed yeast bacterial strain in the step (1).
3, the mutant yeast strain in the incubation step (2) in complete medium, and from culture fluid, obtain hirudin HV
2
4, the hirudin that obtains in the step (3), can obtain purity through purification repeatedly is 95% hirudin product, uses it for clinical experiment.
5, hirudin and Lumbrukinase are with 1: 20-1: coordination in 2 scopes, form anticoagulation, the combination drug of thrombus dissolving.
The present invention relates to the secreting, expressing of hirudin gene in the yeast mutation bacterial strain, and obtain being used for the antithrombotic medicine lepirudin 023 ludon of anticoagulation.Its content comprises the synthetic of hirudin gene, the structure of secreted expression carrier, express the mutation of the yeast strain of hirudin, expression of gene, the separation and purification of expression product, the production that hirudin is fairly large, hirudin preclinical pharmacology toxicological study, clinical practice, the preparation of hirudin complex, the type of dosage form of medicine etc.It is the synthetic of hirudin gene that the present invention has its tangible characteristics: Fig. 1, as shown in Figure 1, and the order of the amino of hirudin of the present invention and natural hirudin HV
2Identical, and adopt yeast genes to use the nucleotide sequence of code Design hirudin gene always.When making up the hirudin Yeast expression carrier, utilize yeast α-factor expression system promptly to utilize plasmid PYA2C, hirudin gene obtains secreting, expressing in the presence of α-factor gene startup and guiding peptide.And with 3 of cytochrome C-gene '-the end termination stops the expression of hirudin gene in proper order.5 ' the end of while at the hirudin gene of synthetic designed the guiding peptide end similar to unartificial yeast α-factor peptide C end coding in proper order, and the coding (see figure 3) of yeast intracellular protein enzyme YSCF action site order
Fig. 2 is the design of graphics of hirudin Yeast expression carrier so that the hirudin that makes expression correctly processed in cell, except that the 6th cruel propylhomoserin not by the Sulfation, the lepirudin 023 ludon that obtains is with natural identical.A stability problem that subject matter is plasmid in the yeast expression system, auxotrophic yeast strain often cause the output of exogenous gene expression product on the plasmid to descend because of back mutation takes place.Our conventional mutation by repeatedly is in conjunction with the selection under the 5Fu pressure, blocked the back mutation of yeast strain ura approach effectively, improve the stability of the mutant strain BJ-1995-SC-HF that contains expression plasmid pjy-Hl greatly, guaranteed the efficiently expressing of Hirudo.In the purge process of hirudin, utilize anion-cellulose to handle the supernatant of Yeast Cultivation liquid, not only played inspissation but also a separating effect has preferably been arranged.
Lepirudin 023 ludon provided by the invention or lepirudin 023 ludon and the preparation of complex composition are used to prevent blood coagulation and treatment thrombolytic agent, and it is composed as follows:
Simple hirudin
Hirudin and Lumbrukinase are by 1: 20-1: 2 part by weight are made into combination drug.
Fig. 3 is that the terminal order of the guiding peptide of synthetic water whitmania gene 5 ' end compares in proper order with yeast x factor peptide, describes the present invention below in conjunction with embodiment and accompanying drawing.
Hirudin HV involved in the present invention
2, its gene is by nucleotide sequence shown in Figure 1, adopts the solid phase phosphoramidite method synthetic.Full gene is 239bp altogether, and 14 oligonucleotide are synthetic, and each fragment is mixed the annealing splicing, and the gained full-length gene is inserted in the PUC18 plasmid, and its correctness is identified in order-checking.
The structure of embodiment 2 secreting type Yeast expression carriers
Fig. 2 demonstrates the program that the hirudin Yeast expression carrier makes up, with the cytochrome C encoding gene order among the full gene replacement of synthetic hirudin among the embodiment 1 plasmid PYA2C, promptly constitute α-factor expression order hirudin expression unit that (5 ' control region, promoter and guiding peptide coding)-hirudin gene-cytochromeC3 ' end termination is connected in series in proper order.This is expressed unit cut out with BamHl and put into escherichia coli-yeast shuttle plasmid PJH158, promptly constitute the expression vector PJH-Hl of hirudin gene.
Embodiment 3 expresses mutation and the expression of the yeast strain of hirudin
With the method transformed yeast BJ-1995-SC strain of the hirudin expression plasmid PJY-Hl described in the embodiment 2 according to people such as Ito, the conversion that obtains ties up to the purification that goes down to posterity on the incomplete culture medium of ura, handle through ultraviolet mutagenesis 25W ultraviolet sterilization lamp pipe 4 minutes and nitrosoguanidine mutagenesis, (0.5mg/ml 30 ℃ 20 minutes), and at 5Fu(10mmul/L)-not exclusively repeated screening on the culture medium, choosing at last can be at the preservation plasmid that it is fixed that rich medium is paid taxes, the yeast strain BJ-1995-SC-HF of hirudin secretory volume height (20ATu/ml).To complete medium, 30 ℃ of shaking tables were cultivated 36-48 hour with this inoculation, with enzyme activity anticoagulant of ChromozymTH chromogenic substrate survey supernatant (hirudin), surveyed the anticoagulation vigor of supernatant with the thrombin titrimetry.
The purification of embodiment 4 hirudins and fairly large production
The supernatant that obtains among the embodiment 3, through cellulose adsorption, gel filtration, ion exchange, purification steps such as high pressure liquid chromatography (HPLC) can obtain purity 95%, and amino acid N do not hold single lepirudin 023 ludon.The supernatant of the Yeast Cultivation liquid that 5-10 rises is transferred more than the PH7.0, concentrates with anionic cellulose absorption; The supernatant of the hirudin after concentrating filters through SephadexG-50, removes most of foreign protein; Hirudin partly is adsorbed onto on the DEAE-Celulose post, through the NaCl stepwise elution, collects merging hirudin part, and dialysis back lyophilizing remakes gel filtration one time; Use high-pressure liquid phase (HPLC) the C18 reversed phase column chromatography of half preparation amount to separate then,, can get purity and be 95% hirudin with the acetonitrile eluting of 10-60%.
Research of embodiment 5 pharmacological toxicologies and clinical practice
Decide with the hirudin that obtains among the embodiment 4 and to want pharmacodynamics and general pharmacology to learn research, acute toxicity, long term toxicity, specific toxicity experiment and pharmacokinetic.With regard to its main pharmacodynamics is the anticoagulation anti-thrombosis function of hirudin, no matter be experiment in vitro (as whole blood coagulation time, recalcification time, thrombin time etc.), or acute result of experiment in the body, all demonstrate hirudin anticoagulation and antithrombotic are formed with remarkable result.Thick, highly finished product with hirudin are made different preparations, are used for clinical experiment.Dosage with per kilogram of body weight patient medication 0.1-0.5mg has obvious treatment and preventive effect to the diffusion-type intravascular coagulation, to coronary artery thrombosis, and phlebothrombosis, cerebral thrombosis and thrombophlebitis also all have prevention and therapeutical effect.Surgery venous thrombosis operation back has directly promptly prevented the thromboembolism once more of vein blood vessel with hirudin.
Embodiment 6 hirudins and Lumbrukinase are formed antithrombotic thrombus dissolving combination drug
Lumbrukinase is a kind of new thrombolytic, now produces as a trial through the Ministry of Public Health approval.And the key property of hirudin is an anticoagulation, and antithrombotic forms, and the two combination is a kind of prevention and treats thrombosis and the ideal medicament of the disease that thrombosis causes.Lumbrukinase and hirudin are made into combination drug with the ratio of 20: 1 and 2: 1, with this medicine the thrombotic disease patient treated, and the patient of 70 kg body weight, medication every day 20-100mg receives tangible curative effect.With the patient of thromboembolism again behind the tPA thromboembolism treatment, use hirudin can make the state of an illness obtain in week taking a turn for the better separately at 1-3.
6, the dosage form of medicine
Prepared the injection injection, adopted subcutaneous or intramuscular injection, and be oral medicine as capsule.Also make the lyophilization product and be used for perfusion therapy, the external hirudin is used for preventing thromboembolism again behind the surgical operation.
Claims (2)
1, a kind of lepirudin 023 ludon and Lumbrukinase are by 1: 20-1: the medicine of anticoagulation that 2 weight ratios are formed and treatment thrombotic disease.
2, a kind ofly be used for the pharmaceutical methods of anticoagulation and treatment thrombotic disease, it is characterized in that by claim 1 preparation:
(1) with the hirudin NV of synthetic
2Gene place the promoter of yeast α-factor gene and lead after the peptide, be configured to expression vector;
(2), and obtain the mutant strain of hirudin gene through mutation with the expression vector transformed yeast bacterial strain in the step (1);
(3) mutant yeast strain in the incubation step (2) in complete medium, and from culture fluid, obtain hirudin HV
2;
(4) hirudin that obtains in the step (3), can obtain purity through purification repeatedly is 95% hirudin product;
(5) hirudin product that step (4) is obtained and Lumbrukinase are with 1: 20-1: 2 weight ratios are made into anticoagulation and treatment thrombotic disease medicine.
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| CN 92111829 CN1088836A (en) | 1992-12-30 | 1992-12-30 | Lepirudin 023 ludon and complex thereof are used for preparation prevention and treatment thrombotic disease medicine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328380C (en) * | 2001-09-24 | 2007-07-25 | 天津天士力制药股份有限公司 | High-efficiency expression recombinant hirudin and producing method thereof |
| CN101094867B (en) * | 2004-10-19 | 2011-08-24 | 隆萨股份公司 | Method for solid phase peptide synthesis |
| 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 |
| CN105816860A (en) * | 2015-12-03 | 2016-08-03 | 北京百奥药业有限责任公司 | Compound preparation of lumbrukinase and clopidogrel, and preparation method thereof |
| CN107083366A (en) * | 2017-05-19 | 2017-08-22 | 何向锋 | Express adoptive immunity cell of hirudin and its production and use |
-
1992
- 1992-12-30 CN CN 92111829 patent/CN1088836A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328380C (en) * | 2001-09-24 | 2007-07-25 | 天津天士力制药股份有限公司 | High-efficiency expression recombinant hirudin and producing method thereof |
| CN101094867B (en) * | 2004-10-19 | 2011-08-24 | 隆萨股份公司 | Method for solid phase peptide synthesis |
| CN102225966B (en) * | 2004-10-19 | 2012-12-26 | 隆萨股份公司 | Method for solid phase peptide synthesis |
| 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 |
| CN105816860A (en) * | 2015-12-03 | 2016-08-03 | 北京百奥药业有限责任公司 | Compound preparation of lumbrukinase and clopidogrel, and preparation method thereof |
| CN107083366A (en) * | 2017-05-19 | 2017-08-22 | 何向锋 | Express adoptive immunity cell of hirudin and its production and use |
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