CN1939290A - Medical usage of colchicine and its derivative - Google Patents
Medical usage of colchicine and its derivative Download PDFInfo
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- CN1939290A CN1939290A CN 200510030151 CN200510030151A CN1939290A CN 1939290 A CN1939290 A CN 1939290A CN 200510030151 CN200510030151 CN 200510030151 CN 200510030151 A CN200510030151 A CN 200510030151A CN 1939290 A CN1939290 A CN 1939290A
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Abstract
A medical application of the colchicin and its derivatives in preventing and treating the diseases caused by intravascular thrombosis and embolism is disclosed.
Description
Technical field
The present invention relates to the medical usage of a kind of alkaloid and its derivant, more specifically refer to from the Colchicum plant, extract colchicine and its derivant.
Background technology
Colchicine (colchicine) has another name called Colchicine, COLC, Colchineos, Colcin, Colgout, Aquacolchin, NSC-757. are from Liliaceae Colchicum plant colchici cormus and seed etc. or from liliaceous plant, and a kind of alkaloid that extracts in Pseudobulbus Cremastrae Seu Pleiones and the Chinese narcissus is produced in Yunnan.Formal name used at school N-((7S)-5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo (a) heptalen-7-yl-acetamide, structural formula is C
22H
25NO
6, molecular weight is its chemical mechanism See Figure of 399.44Dolton:
Colchicine separated (Pelletier PS, Caventon J.Ann.Chim.Phys.1820 as far back as 1820; 14:69), be used to treat gout at first, after be found colchicine and can be combined in intracellular tubular proteins, suppress the mitosis of cell.After, it is found that colchicine can suppress the free of blood neutrophilic granulocyte, has the reaction of reducing inflammation curative effect.
Colchicine thirties is just as the instrument medicine of chromosome research.According to existing document description, colchicine and derivant thereof can act on the M phase of cell cycle, suppress cell mitogen, cause the nucleus textural anomaly and cause cell death.Dividing vigorous histiocyte is affected at first.This pharmacologically active of colchicine is used to treat tumor.On the tumor cell in vitro model, the cytostatic concentration of colchicine is that 0.5-1.3 μ M (0.2 μ g-0.6 μ g/ml) is (with reference to Reduction in S100 protein β subunit RNA in C6 rat glioma cells followingtreatment with anti-microtubular drugs.Robert Dunn, Charles Landry, David O ' Hanlon, James Dunn, Robert Allore, Ian Brown and Alexander Marks.The Journal of BiologicalChemistry.1987,262 (8): 3562-3566).On the experiment mice tumor model colchicine suppress the dosage of tumor be every day 1mg/kg (with reference to Evaluation of antivascular and antitumor effects of tubulin bindingagents in solid tumor therapy.Yukio Nihei, Manabu Suzuki, Akira Okano, Takashi Tsuji, Yukio Akiyama, Takashi Tsuruo, Sachiko Saito, Katsuyoshi Hori and Yasufumi Sato.Jpn.J.Cancer Res.1999,90:1387-1395).
At present, colchicine and derivant thereof are used to treat tumor and leukemia clinically, also are used for the treatment of acute gouty arthritis, diseases such as Cystic Fibrosis.Its therapeutic dose is: quiet, and each 1-2mg, the each 1mg of intravenous injection, once a day.Oral each 1mg.Quiet of colchicine derivative colchiceinamide or compound recipe colchiceinamide be 10mg once a day, oral each 5mg, every day 4 times.Untoward reaction is: oral administration can cause stomachache, diarrhoea, gastrointestinal reactions such as nausea and vomiting.Oral or intravenous administration can cause leukocyte and thrombocytopenia for a long time, and bone marrow depression and aplastic anemia, respiratory center suppress (with reference to modern clinical medicine grand ceremony, Luo Mingsheng, Gao Tianhui, Lao Jiahua chief editor, January calendar year 2001 front page, Sichuan science tech publishing house, 268-269 page or leaf; China's internal medicine, Chen Minzhang chief editor, People's Health Publisher, front page in 1999,3332-3333 page or leaf; Modern anti-tumor medicine is learned, Liao Zijun, Nan Kejun, Han Jun chief editor, Xi'an company of world book publishing house, front page in 2002,272-274 page or leaf).
Summary of the invention
The purpose of this invention is to provide a kind of tazettine and its derivant new medical use.
The present invention sets forth the pharmacologically active and the mechanism of action of colchicine and its derivant.Promoting promptly that the blood fibrinolytic system is active raises and promotes thromboembolism, thereby illustrates the various diseases that colchicine and derivant thereof can be used for treating and prevent to cause because of thrombosis in the blood vessel or thromboembolism.
The blood in human body in pipe comprises the tremulous pulse of all size diameter, and vein and blood capillary are under the normal physiological state, and tube chamber is open, and blood flow is unobstructed, with this nutrition and oxygen supply that guarantees each position internal organs of human body, keeps each position internal organs normal function running.Possesses a kind of clotting mechanism in the blood of human body, in case blood vessel wall occurs damaged, the activation of blood coagulation system, the Fibrinogen (fibrinogen) that finally causes solubility is converted into the fibrin (fibrin) of mutual linked network shape down in thrombin (thrombin) effect, at part generation blood coagulation.This physiology protection mechanism can prevent that loss of blood is too much, and helps the reparation of blood vessel wall.But under pathologic condition, blood vessel wall, especially directly the endothelial layer that contacts with blood can occur also can activating already contg thrombin and platelet in the blood unusually because of various factors.Its result causes Fibrinogen to be converted into fibrin, and is deposited on the blood vessel wall, thereby forms solid pathology thrombosis, causes the angiostenosis of diseased region, even stops up fully.In blood vessel, especially the thrombosis that forms in the vein with the blood flow walking, causes the blood vessel blockage in distal tubule chamber after can also coming off, and forms sudden thromboembolism.No matter be the formation of primary thrombus, or the sudden thromboembolism of far-end all can cause existing the internal organs blood of organizing at thrombosis position to supply blocking, cell death, infarction takes place in deterioration.The clinical typical disease that causes thus has myocardial infarction, and deep venous thrombosis and cerebral infarction are waited indefinitely.
Under normal circumstances can be removed behind the blood coagulation, to guarantee tissue repair by fibrinolysis mechanism.The fibrinolytic system that is grouped into by the Fibrinolytic various one-tenth of participation abbreviates fibrinolytic system as.Wherein most important composition be tissue plasminogen activator (tissue plasminogen activator, t-PA).T-PA is by the serine protease that is discharged into behind the vascular endothelial cell irriate in the blood, and the t-PA burst size has been controlled the speed of dissolving the thrombosis grumeleuse of being made up of fibrin.T-PA has highly affine fibrin, but not fibrinogenic biological nature, this characteristic has guaranteed that its effect has height locality and specificity.The t-PA that is adsorbed on the fibrin becomes fibrinolysin (plasmin) by transforming the plasminogen (plasminogen) that is adsorbed on equally on the fibrin, by fibrinolysin cutting thrombosis, the fibrin that makes mutual commissure is hydrolytic cleavage gradually, thereby removes thrombosis (with reference to the clinical new technique of thrombotic disease.Wang Hongli, Wang Xuefeng chief editor, January in 2003 the 1st edition, People's Medical Officer Press).
Therefore, thrombotic disease can be by pathologic promotion coagulation factor long-term existence, and is strong excessively, or the fibrinolytic system of human body is active crosses weak institute and cause.Therefore, the medicine that is used in the treatment thrombotic disease at present clinically is divided into two kinds, and a kind of passing through suppresses blood coagulation activity and antiplatelet aggregation, slows down thrombotic speed, represents medicine that heparin is arranged, vitamin K inhibitor and aspirin etc.Another kind of medicine is the fibrinolysis enhancing medicine, and by injecting a large amount of activator of plasminogens, the activation fibrinolysin is to reach the curative effect of quick thrombolytic.Represent medicine that recombiant protein t-PA is arranged.Other alternative medicine has streptokinase and urokinase.They are the bioprotein goods equally, by the activation plasminogen, reach thrombolytic purpose.But the general life-time service of last class medicine, the control thrombosis takes place and growth, then a class medicine generally when thrombosis causes that mortality is organized infarction the short time internal electronic monitoring use, reach the purpose of the life that gives emergency treatment to a patient.
The present invention's explanation, colchicine and derivant thereof can significantly improve vascular endothelial cell and discharge human body t-PA under the condition of low concentration, thereby promote thromboembolism to accelerate, therefore, colchicine and derivant thereof can be used for treating the various diseases that is caused by thromboembolism.
The present invention implements like this:
Materials and methods:
1.RT-PCR method is measured the expression of endothelial cell line tissue plasminogen activator
Human microvascular endothelial cell (mvec) is that the HMEC-1 cultivation after 48 hours, adds 5ng/ml respectively in the plate that contains the MCDB131 culture fluid that adds 10% calf serum; 2.5ng/ml, hatched 18 hours with the colchicine (Sigma company) of 0.5ng/ml, take off cell with trypsin, clean with phosphate buffer is centrifugal, extract the interior RNA always of cell.Do the expression that RT-PCR measures tissue plasminogen activator with following primer:
Primer 1:5 '-GTGTACACCAAGGTTACCAA-3 '
Primer 2: 5 '-GGAATACCTTCTGAGAGCCA-3 '
Do the expression that RT-PCR measures urokinase with following primer:
Primer 1:5 '-TCAAGTTCCATCGAACTGTG-3 '
Primer 2: 5 '-ATCAATGAAGCAGTGTGTGG-3 '
Above primer is ordered from Invitrogen company.
2. immune enzyme linked immunosorbent assay (ELISA) is measured the secretion colchicine of endothelial cell line tissue plasminogen activator:
Human microvascular endothelial cell (mvec) is that the HMEC-1 cultivation after 48 hours, adds 50ng/ml respectively in the plate that contains the MCDB131 culture fluid that adds 10% calf serum; 10ng/ml; 5ng/ml; The colchicine of 1ng/ml and 0.5ng/ml was hatched 24 hours, collected supernatant.Measure the concentration of tissue plasminogen activator in the supernatant samples with immune enzyme linked immunosorbent assay (test kit of American Diagnostic company).Cell is taken off counting with trypsin.Colchicine derivative:
Human microvascular endothelial cell (mvec) is that the HMEC-1 cultivation after 48 hours, adds 10ng/ml respectively in the plate that contains the MCDB131 culture fluid that adds 10% calf serum; 5ng/ml; 2.5ng/ml with the Demecolcine (Demecolcine, ICN company) of 1ng/ml, hatched 24 hours, collect supernatant.Measure the concentration of tissue plasminogen activator in the supernatant samples with immune enzyme linked immunosorbent assay (test kit of AmericanDiagnostic company).Cell is taken off counting with trypsin.
3. the active mensuration of tissue plasminogen activator in the laboratory animal blood plasma.
42 eight all big female C57BL/6 mices are divided into 7 groups, every group of 6 mices at random.Accept lumbar injection colchicine (being dissolved in normal saline) 40 μ g/kg respectively; 16 μ g/kg; 6.5 μ g/kg; 2.5 μ g/kg; 1 μ g/kg and 0.1 μ g/kg.Control mice is accepted the normal saline injection.Take a blood sample after 24 hours, with 3.8% sodium citrate anticoagulated whole blood, centrifugal (2500xg, 10 minutes) separate platelet poor plasma.The t-PA active concentration adopts mouse tissue activator of plasminogen active immne enzyme linked immunosorbent assay mensuration test kit (mouse t-PA activity ELISA assay kit) (Gentaur company) to measure in the blood plasma.Because that embedding is plasminogen activator inhibitor (PAI-1) on 96 orifice plates, have only free activated t-PA just possible in conjunction with onboard, so what measure is the t-PA active concentration.
4. tissue plasminogen activator's activity in the euglobulin method determination experiment animal blood slurry
25 16 all big female nude mices are divided into 5 groups, every group of 5 mices at random.Accept lumbar injection colchicine (being dissolved in normal saline) 40 μ g/kg respectively; 16 μ g/kg; 2.5 μ g/kg and 0.1 μ g/kg.Control mice is accepted the normal saline injection.Extract about 1ml blood sample by heart after 15 hours, with 3.8% sodium citrate anticoagulated whole blood, centrifugal (2500xg, 10 minutes) separate platelet poor plasma.(method is with reference to the modern pharmacology experimental technique to take the euglobulin clot lysis time method to measure in the blood plasma tissue plasminogen activator's activity.The Zhang Juntian chief editor, combined publication society of China Concord Medical Science University of Beijing Medical University, front page in 1998,1201 pages).
5. the molten observation of laboratory animal pulmonary infarction model bolt is (with reference to people's such as Stassen JM method: Stassen JM, Vanlinthout I, Lijnen HR and Collen D.A hamster pulmonary embolism model for thecvaluaton of the thrombolytic and pharmacokinetic properties of thrombolytic agents.Fibrinolysis, 1990 suppl 2,4:15-21 and Carmeliet P etc. are to the improvement of the method: Adenovirus-mediated transfer of tissue-type plasminogen activator augments thrombolysis in tissue-typeplasminogen activater-deficient and plasminogen activator-1-overexpressing mice.Blood, 1997,90 (4): 1527-1534).
125The preparation of the fibrin blood plasma grumeleuse of I labelling: use
125The Fibrinogen of I labelling, the mice platelet poor plasma (the same) of 3.8% sodium citrate anticoagulant preparation, and thrombin and CaCl
2Be mixed and made into.
21 ten all big female nude mices are divided into 3 groups, every group of 7 mices at random.Medication group mice is accepted lumbar injection colchicine (being dissolved in normal saline) 16 μ g/kg and 2.5 μ g/kg respectively, and control group mice is accepted the normal saline injection.After 12 hours, implement mouse anesthesia, with 25 μ l's
125The fibrin blood plasma grumeleuse of I labelling (contains and has an appointment 70000cpm's
125The human fibrinogen of I labelling and Mus blood plasma are mixed to be made) enter by the jugular vein injection and form pulmonary infarction in the body.The injection plasma clot placed the position, thoracic cavity to carry out pulmonary's isotope detection the gamma counter probe after 1 hour, as the molten preceding reference of bolt.The molten situation of continuous detecting bolt in the experimentation.Put to death mice after 16 hours, detect heart and the residual isotopic content of pulmonary.The isotopic mass of the isotopic mass contrast injection that disappears is as the molten percentage ratio of bolt.
6. colchicine is observed the toxicity of laboratory animal
24 seven all big female nude mices are divided into 3 groups, every group of 8 mices at random.Two groups of mices are accepted lumbar injection colchicine 40 μ g/kg and 16 μ g/kg every day, continuous 30 days respectively.Control group mice is accepted intraperitoneal injection of saline every day, continuous 30 days.Claimed a body weight in per two days.
Experimental result
1.RT-PCR experimental result shows that 5ng/ml and 2.5ng/ml colchicine can significantly increase the expression (Fig. 1) of endothelial cell tissue activator of plasminogen (t-PA).Under similarity condition, the expression of urokinase (u-PA) unaffected (Fig. 2).
2. immune enzyme linked immunosorbent assay experimental result shows, 50ng/ml, 10ng/ml, 5ng/ml and 1ng/ml colchicine can significantly increase the secretion (Fig. 3 A) of endothelial cell tissue activator of plasminogen (t-PA).In addition, observe used colchicine dosage in the experiment endotheliocyte is not had overt toxicity.10ng/ml; 5ng/ml and 2.5ng/ml Demecolcine also can significantly increase the secretion (Fig. 3 B) of endothelial cell tissue activator of plasminogen (t-PA).
3. colchicine significantly strengthens tissue plasminogen activator's activity in the laboratory animal blood plasma.Lumbar injection 6.5 μ g/kg and 2.5 μ g/kg colchicine can make tissue plasminogen activator's active concentration increase by 12 times and 16.8 times (Fig. 4) respectively in 24 hours.Euglobulin clot lysis time method result shows lumbar injection 40 μ g/kg, 16 μ g/kg; 2.5 μ g/kg and 0.1 μ g/kg colchicine after 15 hours the complete dissolution time of blood plasma euglobulin be respectively 108 ± 5 minutes, 55 ± 4 minutes, 37 ± 4 minutes and 110 ± 6 minutes, and matched group is 128 ± 5 minutes.
Table 1
| The injection natural law | Matched group body weight (g) | Inject 40 μ g/kg group body weight (g) | Inject 16 μ g/kg group body weight (g) |
| Zero day | 17.6±0.7 | 17.4±0.6 | 17.6±0.8 |
| Two days | 17.4±0.7 | 17.2±0.7 | 17.4±0.8 |
| Four days | 17.2±0.8 | 17.0±0.7 | 17.3±0.8 |
| Six days | 17.4±0.7 | 17.2±0.7 | 17.5±0.8 |
| Eight days | 18.7±0.7 | 18.5±0.7 | 18.7±0.8 |
| Ten days | 19.8±0.7 | 19.3±0.6 | 20.1±0.8 |
| 12 days | 20.5±0.9 | 20.2±0.7 | 20.6±0.7 |
| Fortnight | 21.7±0.7 | 21.2±0.6 | 21.9±0.8 |
| 16 days | 22.3±0.9 | 22.1±0.7 | 22.6±0.8 |
| 18 days | 22.8±0.9 | 22.4±0.7 | 23±0.9 |
| 20 days | 23.3±0.9 | 22.9±0.7 | 23.3±0.8 |
| 22 days | 23.9±0.7 | 23.5±0.8 | 24.1±0.7 |
| Two fortnights | 24.6±0.8 | 23.9±0.7 | 24.6±0.7 |
| 26 days | 25.1±0.9 | 24.7±0.9 | 25.2±0.9 |
| 28 days | 25.4±0.9 | 25.1±1.1 | 25.5±0.8 |
| 30 days | 26.9±1.2 | 26.4±1.3 | 27.1±0.9 |
4. colchicine accelerated tests animal pulmonary infarction dissolving.Lumbar injection colchicine 16 μ g/kg and 2.5 μ g/kg can cause 65% ± 7 and 87% ± 8 pulmonary infarction dissolving respectively after 16 hours.The control animals pulmonary infarction is dissolved as 32% ± 7 (Fig. 5).
5. continuous 30 day every day, lumbar injection colchicine 40 μ g/kg and 16 μ g/kg did not cause bleeding, and lost weight toxic reactions such as depilation and loss of appetite.Injection is the 30th day continuously, and the control group mice average weight is 26.9 ± 1.2g; Injection colchicine 40 μ g/kg group is 26.4 ± 1.3g; Injection colchicine 16 μ g/kg group is 27.1 ± 0.9g (table 1).
Experimental result shows that colchicine and derivant thereof can significantly strengthen the expression and the secretion of tissue plasminogen activator (t-PA) in quite low concentration range, thereby promotes fibrinolytic.And under similarity condition, the expression of urokinase (u-PA) is unaffected.Therefore the microdosage colchicine can be used for the treatment and the prevention of all thrombotic disease.
Colchicine and derivant or its pharmaceutically acceptable salt with the form of pharmaceutical composition separately or with materia medica on acceptable carrier or excipient, form compositions, compositions comprising but be not limited to low dose of aspirin, the compositions that Reopro etc. form.Pharmaceutically acceptable carrier refers to: one or more compatibility solids or liquid filling agent or excipient, they are suitable for the people uses, and enough purity and enough low toxicity must be arranged." compatibility " referred to herein as each component energy and chemical compound of the present invention and blending mutually between them in the compositions, and the drug effect of not obvious reduction chemical compound, medically acceptable carrier part example has sugar (as glucose, sucrose, lactose etc.) starch is (as corn starch, potato starch etc.) cellulose and common derivant thereof (are received as carboxylic formaldehyde cellulose, the acetaldehyde cellulose is received, cellulose ethanoate, the fine little element of little product) Polyethylene Glycol, gelatin, Pulvis Talci, stearic acid, magnesium stearate, calcium sulfate, vegetable oil (Oleum Glycines, Oleum sesami, Oleum Arachidis hypogaeae semen, the Fructus Canarii albi wet goods).It can also be chlorinating agent (as tween) wetting agent (as the dodecane sodium sulphate).The administering mode of chemical compound is depended in the selection of the carrier of compositions of the present invention.
Description of drawings
Fig. 1 is that RT-PCR measures endothelial cell tissue activator of plasminogen expression of results.
Cell is used 5ng/ml respectively; 2.5ng/ml and the colchicine of 0.5ng/ml is handled after 18 hours extraction RNA.Be RT-PCR with the t-PA special primer.The RT-PCR product is taken pictures through the agar plate electrophoresis, and tissue plasminogen activator's expression as shown in Figure 1.
Fig. 2 is that RT-PCR measures endotheliocyte urokinase expression of results.
Cell is used 5ng/ml respectively; 2.5ng/ml and the colchicine of 0.5ng/ml is handled after 18 hours extraction RNA.Be RT-PCR with the urokinase special primer.The RT-PCR product is taken pictures through the agar plate electrophoresis, and the urokinase expression as shown in Figure 2.
Fig. 3 is that immune enzyme linked immunosorbent assay is measured endothelial cell tissue activator of plasminogen secretion result.
Cell is used 50ng/ml respectively; 10ng/ml; 5ng/ml; The colchicine of 1ng/ml and 0.5ng/ml was handled after 24 hours, collected supernatant, measured the secretion of tissue plasminogen activator with immune enzyme linked immunosorbent assay.Counting cells.The concentration of tissue plasminogen activator is with per 10
6The t-PA secretory volume of individual cell is expressed among Fig. 3 A.
Cell is used 10ng/ml respectively; 5ng/ml; 2.5ng/ml; The Demecolcine of 1ng/ml was handled after 24 hours, collected supernatant, measured the secretion of tissue plasminogen activator with immune enzyme linked immunosorbent assay.Counting cells.The concentration of tissue plasminogen activator is with per 10
6The t-PA secretory volume of individual cell is expressed among Fig. 3 B.
Fig. 4 is the result of tissue plasminogen activator's active concentration in the immune enzyme linked immunosorbent assay determination experiment mice plasma.
Experiment mice is accepted lumbar injection colchicine (being dissolved in normal saline) 40 μ g/kg respectively; 16 μ g/kg; 6.5 μ g/kg; 2.5 μ g/kg; 1 μ g/kg and 0.1 μ g/kg.Take a blood sample after 24 hours, with 3.8% sodium citrate anticoagulated whole blood, centrifugal separation plasma.The active mouse tissue activator of plasminogen active immne enzyme linked immunosorbent assay mensuration test kit (mouse t-PA activity ELISA assay kit) (Gentaur company) that adopts of t-PA is measured in the blood plasma.The gained active concentration is the result show among Fig. 4.
Fig. 5 is the dissolved result of pulmonary infarction in the isotopic labeling body.Mice is accepted earlier lumbar injection colchicine (being dissolved in normal saline) 16 μ g/kg and 2.5 μ g/kg respectively.Anesthetized mice is incited somebody to action then
125The fibrin blood plasma grumeleuse of I labelling enters by the jugular vein injection and forms pulmonary embolism in the body.Put to death mice after 16 hours, detect heart and the residual isotopic content of pulmonary.The result is expressed among Fig. 5 after being converted into the molten percentage ratio of bolt.
Table 1 is the toxicity observed result of colchicine to laboratory animal.
Experiment mice is divided into 3 groups at random, every group of 8 mices.Two groups of mices are accepted lumbar injection colchicine 40 μ g/kg and 16 μ g/kg every day, continuous 30 days respectively.Control group mice is accepted intraperitoneal injection of saline every day.Claimed a body weight in per two days, the result lists table 1 in.
The specific embodiment
25 16 about 28 grams of all big female nude mice body weight are divided into 5 groups, every group of 5 mices at random.First group of mice accepted 1 property lumbar injection 40 μ g/kg colchicine (200 μ l are dissolved in normal saline, and concentration is 5.6 μ g/ml).Second group of mice accepted 1 property lumbar injection 16 μ g/kg colchicine (200 μ l are dissolved in normal saline, and concentration is 2.24 μ g/ml).The 3rd group of mice accepted 1 property lumbar injection 2.5 μ g/kg colchicine (200 μ l are dissolved in normal saline, and concentration is 0.35 μ g/ml).The 4th group of mice accepted 1 property lumbar injection 0.1 μ g/kg colchicine (200 μ l are dissolved in normal saline, and concentration is 0.014 μ g/ml).The 5th group of control mice accepted 1 property lumbar injection 200 μ l normal saline.Inject and use the Isofluran anesthetized mice after 15 hours, extract about 1ml blood sample, insert in the 1.5ml eppendorf centrifuge tube that contains 100 μ l3.8% sodium citrate by heart, mixing, room temperature centrifugal (2500xg, 10 minutes), separate platelet poor plasma, each get 0.55ml blood plasma approximately.Get 0.5ml blood plasma, take the euglobulin clot lysis time method to measure tissue plasminogen activator's activity in the blood plasma." modern pharmacology experimental technique " 1201 pages of described carrying out that concrete operations are edited according to Zhang Juntian fully.Experimental result shows lumbar injection 40 μ g/kg; 16 μ g/kg; 2.5 μ g/kg and 0.1 μ g/kg colchicine are respectively organized the complete dissolution time of mice plasma euglobulin and on average were respectively 108 ± 5 minutes after 15 hours, and 55 ± 4 minutes, 37 ± 4 minutes and 110 ± 6 minutes, and matched group is 128 ± 5 minutes.
Embodiment 2
The mixed mice platelet poor plasma of 3.8% sodium citrate anticoagulant preparation: 2 eight all big female nude mices are anaesthetized with Isofluran, extract about 1ml blood sample by heart, insert in the 1.5mleppendorf centrifuge tube that contains 100 μ l, 3.8% sodium citrate, mixing, centrifugal (the 2500xg of room temperature, 10 minutes), separate platelet poor plasma, the blood plasma of 2 mices of gained is mixed evenly standby.
125The human fibrinogen of I labelling: provide by Amersham company, be diluted to 70000cpm/ μ l with normal saline.
125The preparation of the fibrin blood plasma grumeleuse of I labelling: in the eppendorf of 0.5ml pipe, add the above-mentioned mice plasma of 21 μ l respectively, 1 μ l 70000cpm's
125The human fibrinogen of I labelling, and the 1M CaCl of the thrombin of the 0.07UI/ μ l of 0.5 μ l and 2.4 μ l
2Mix, and be drawn into immediately in the conduit of 6FG, place 37 ℃ to hatch 30 minutes, treat that grumeleuse forms the back room temperature and places standby.
21 ten about 20 grams of all big female nude mice body weight are divided into 3 groups, every group of 7 mices at random.First group of mice accepted disposable celiac and injects 16 μ g/kg colchicine (200 μ l, be dissolved in normal saline, concentration is 1.6 μ g/ml), second group of mice accepted disposable celiac injection colchicine 2.5 μ g/kg (200 μ l, be dissolved in normal saline, concentration is 0.25 μ g/ml), the 3rd group of control mice accepted disposable 200 μ l normal saline lumbar injections.With mouse anesthesia, jugular vein is exposed after 12 hours, will contain 25 μ l's
125The conduit of the fibrin blood plasma grumeleuse of I labelling is introduced in the jugular vein, the thromboembolism injection is entered form pulmonary infarction in the body.Change the heparin solution of tube injection 0.1ml (300U/ml) then.The injection plasma clot placed the position, thoracic cavity to carry out pulmonary's isotope detection the gamma counter probe after 1 hour, as the molten preceding reference of bolt.Put to death mice after 16 hours, detect heart and the residual isotopic content of pulmonary.The isotopic mass of the isotopic mass contrast injection that disappears is as the molten percentage ratio of bolt.Experimental result is seen Fig. 5.
Embodiment 3
42 eight about 18 grams of all big female C57BL/6 mice body weight are divided into 7 groups, every group of 6 mices at random.Accept disposable celiac respectively and inject 200 μ l colchicine solutions (being dissolved in normal saline).Inject 40 μ g/kg (concentration is 3.6 μ g/ml) for first group; Inject 16 μ g/kg (concentration is 1.45 μ g/ml) for second group; Inject 6.5 μ g/kg (concentration is 0.6 μ g/ml) for the 3rd group; Inject 2.5 μ g/kg (concentration is 0.225 μ g/ml) for the 4th group; Inject 1 μ g/kg (concentration is 0.09 μ g/ml) for the 5th group; Inject 0.1 μ g/kg (concentration is 0.009 μ g/ml) for the 6th group.The 7th group of control mice accepted the injection of 200 μ l normal saline.Extract about 200 μ l blood samples by the eye socket vein after 24 hours, as described in embodiment 1, with 3.8% sodium citrate anticoagulated whole blood, centrifugal (2500xg, 10 minutes) separate platelet poor plasma.The t-PA active concentration adopts mouse tissue activator of plasminogen active immne enzyme linked immunosorbent assay mensuration test kit (mouse t-PA activityELISA assay kit) (Gentaur company) to measure in the blood plasma.Concrete operations are fully according to the test kit requirement.Experimental result is seen Fig. 4.
Claims (5)
1, the medical usage of following colchicine of a kind of structural formula and its derivant is used in preparation prevention, treatment are caused the medicine of thrombotic disease by thrombosis or thromboembolism
2, the medical usage of colchicine according to claim 1 and its derivant is characterized in that comprising in myocardial infarction, deep venous thrombosis and cerebral infarction, cerebral thrombosis, the medicine at the thrombotic disease that preparation prevention, treatment are caused by thrombosis or thromboembolism and uses.
3, the medical usage of colchicine according to claim 1 and its derivant, it is characterized in that comprising low-dosage aspirin in the compositions that preparation is made up of colchicine and its derivant, low molecular weight heparin, Reopro and medically acceptable carrier are used in the medicine of excipient.
4, the medical usage of colchicine according to claim 3 and its derivant is characterized in that colchicine and its derivant and pharmaceutically acceptable vehicle excipients make tablet, injection or other dosage form.
5, the medical usage of colchicine according to claim 3 and its derivant, comprise colchicine and its derivant as main active, the medicine or the health promoting product of thrombotic disease that auxiliary activity composition or adding ingredient, preparation prevention, treatment are caused by thrombosis or thromboembolism and the disease relevant with thrombosis or thromboembolism.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150196514A1 (en) * | 2012-11-02 | 2015-07-16 | Murray And Poole Enterprises Limited | Method of treating cardiovascular events using colchicine concurrently with an antithrombotic drug |
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-
2005
- 2005-09-29 CN CN 200510030151 patent/CN1939290A/en active Pending
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