CN1403156A - Application of human urokininogenase in preparing medicine for preventing and treating cerebral infraction - Google Patents
Application of human urokininogenase in preparing medicine for preventing and treating cerebral infraction Download PDFInfo
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Abstract
THe present invention discloses one new application of human urokininogenase in pharmaceutical engineering. Specially, human urokininogenase is used as active component in preparing injection for preventing and treating cerebral infraction.
Description
(1) technical field
The present invention relates to the new purposes of a kind of biochemical in pharmaceutical engineering.More specifically say to be exactly the application of Human Urinary Kallidinogenase in the medicine of preparation treatment and prevention of brain infraction.The invention still further relates to the pharmaceutical composition that contains the Human Urinary Kallidinogenase.
(2) background technology
When certain branch of brain tremulous pulse because of vascular lesion (as arteriosclerosis, arteritis), or thrombosis, or thromboembolism, make arterial lumen narrow, even inaccessible, cause the acute brain blood supply insufficiency, cause local brain tissue ischemia, anoxia and necrose, a series of clinical symptoms occurs, the cerebral infarction of meaning.Up to now, international medical community does not still have the method that can stop cerebral tissue to necrose when ischemia, modern medicine has plenty of increases the brain blood flow alleviating local brain tissue ischemia, anoxia, or with certain chemical substance brain cell, to slow down, to alleviate its downright bad process.
Once there was people (as nineteen twenty-six Frey) to notice that the Human Urinary Kallidinogenase has the effect of blood vessel dilating in history.But because the extraction of the collection of raw material urine, enzyme, refining difficulty, the Human Urinary Kallidinogenase fails to obtain industrialized utilization always.Moreover, usually destroyed after the enzyme preparation oral administration at digestive tract, also limited its medical usage.Have report (diagnosis and treatment and new drug Vol.30[10] 199-212; 1993) say that the preparation method of kininogen enzyme intravenous formulations is being inquired into by Japanese Sanwa Chemical Co., Ltd.
At present, the pharmaceutical preparation of preparing treatment or prevention of brain infraction with the Human Urinary Kallidinogenase does not appear in the newspapers.
(3) summary of the invention
Kininogenase is the natural a kind of glycoprotein that is present in the Urina Hominis, molecular weight is about 54,000 dalton (gel filtration), the strand that it is made up of 238 amino acid residues, N--end and C-terminal amino acid residue are respectively isoleucine and serine, contain 5 pairs of S-S keys in the molecule, be respectively Cys7-Cys150, Cys26-Cys42, Cys29-Cys196, Cys161-Cys175 and Cys186-Cys211, isoelectric point, IP is about 4.0.Contain 14.4% sugar in the molecule, binding site lays respectively at Asn78, Asn84 and Asn141, its sugar consist of mannose 3%, galactose 1.7%, fucose 0.8%, N-acetyl-glucosamine 5.0%.Primary structure (aminoacid composition sequence) is formula as follows
The invention provides a kind of Human Urinary Kallidinogenase's new medical use: be active component with Human Urinary Kallidinogenase, be equipped with the pharmacy acceptable carrier, be made into the preparation of drug administration by injection, in order to treatment and prevention of brain infraction.
The processing step of Human Urinary Kallidinogenase's extraction and purification is that urine (male's freshly voided urine is good) is not higher than 0.05M Na2HPO4-NaH2PO4 to the water ultrafiltration to electric conductance with ultrafilter membrane, 0.1M NaCl, the buffer of pH7.4; The DEAE-Sepharose post is through 0.05M Na2HPO4-NaH2PO4,0.1M NaCl, the buffer balance of pH7.4.With DEAE-Sepharose post on the concentrated urine, with the level pad flushing, use 0.05MNa2HPO4-NaH2PO4,0.3M NaCl, the buffer solution elution of pH7.4 is collected eluting peak; The Aprotinin-Sepharose post is through 0.05M Na2HPO4-NaH2PO4, the level pad balance of pH8.0, to go up the Aprotinin-Sepharose post behind the DEAE-Sepharose post eluting peak accent pH8.0, wash with level pad, use 0.1M HAc-NaAc, the buffer solution elution of pH4.0 is collected eluting peak; Aprotinin-Sepharose post eluting peak is to 0.05M Na2HPO4-NaH2PO4,0.1M NaCl, the buffer dialysis of pH7.0 8 hours, 60 ℃ of heating of water bath with thermostatic control 10 hours, ultrafiltration and concentration; Sephadex G-100 post is through the normal saline balance, and with Sephadex G-100 post on the solution that heated, the normal saline eluting is collected eluting peak; Eluting peak is through the degerming of 0.22um membrane filtration, and ultrafiltration and concentration can get Human Urinary Kallidinogenase's highly finished product.
Human Urinary Kallidinogenase after the purification can mix with the pharmacy acceptable carrier, makes the medicine of multiple dosage form.
Said pharmacy carrier comprises medicinal diluent, stabilizing agent, excipient, as glycine, mannitol, pH7.0 phosphate buffered solution, albumin, dextran, glucose, lactose, sodium chloride, sorbitol, citric acid, gelatin, water for injection, normal saline etc., can use wherein one or more simultaneously.Preferably be combined as glycine, mannitol, pH7.0 phosphate buffered solution.
Medicine of the present invention can be made into and be suitable for used for intravenous injection, intramuscular injection usefulness, subcutaneous injection dosage form, comprises dosage forms such as powder pin, liquid drugs injection, transfusion.
Preferred administering mode is intravenous drip and/or intravenous injection.Medicine effective dose scope is 0.0001～0.05PNA/kg, and preferred consumption is 0.002～0.011PNA/kg.The dosage specification of powder pin or liquid drugs injection or infusion preparation is every (bottle) 0.1～0.2PNA unit, and preferred specification is 0.15PNA unit/(bottle).
PNA is defined as: at 37 ℃, under the condition of pH8.0, hydrolysis substrate Val-Leu-Arg-PNA discharged the free PNA of 1 μ mol in 1 minute, was 1PNA unit.
The pharmacodynamic study that the present invention carried out is shown, make Prevention Processing for the cerebral infarction that AA (arachidonic acid) causes with the Human Urinary Kallidinogenase, have the infarct size of dwindling, suppress MDA (malonaldehyde) generation, reduce effects such as cell LDH (lactic acid dehydrogenase) spills; The Cerebral Ischemia injury recovery phase, continuous application Human Urinary Kallidinogenase's treatment can alleviate cerebral edema, increases and organizes ATP (adenosine triphosphate) to stock, and brain cell is had certain protective role.
(4) beneficial effect of the present invention
Show that at the pharmacodynamic study that the present invention carried out the present invention can obtain beyond thought beneficial effect to treatment and prevention of brain ischemia, cerebral infarction, the result is as follows in the record zoopery.
Test 1 Human Urinary Kallidinogenase to the convalescent therapeutical effect of rabbit acute cerebral ischemia/reperfusion injury
Experimental technique
Get the male large ear rabbit of 1.8～2.2kg, overnight fasting before the art is freely drunk water.Back fixation, preparation acute cerebral ischemia/reperfusion injury model.Quiet notes pentobarbital sodium (30mg/kg) also cooperates local anaesthesia (1% procaine 3～5ml), operation.Intravenous injection heparin (1000u/kg) anticoagulation.The right femoral artery intubate enters ventral aorta, and intubate connects transducer respectively by the threeway syringe needle, and the reason of delivering a child is led instrument with monitoring blood pressure and storage blood device (height 40mmHg, i.e. 54.4cmH more
2The O post).Separate bilateral common carotid arteries, threading is standby.Postoperative was stablized 30 minutes.Open the threeway syringe needle to about 1～2 minute of the quick blood-letting of storage blood device, reach 40mmHg, close bilateral common carotid arteries with the bulldog clamp folder simultaneously, continue after 10 minutes, the bilateral bulldog clamp of loosening rapidly, the about 350mmHg of pressurization will store blood device inner blood and inject ex vivo interior (about 2 minutes) simultaneously, and oxygen uptake was simultaneously observed 30 minutes.The auricular vein following different pharmaceutical that instils then, capacity is about 2.5ml/kg, keeps and instils 30 minutes, remove arterial cannulation, and sew up the incision, feed separately, press following grouping intravenous drip various dose once a day Human Urinary Kallidinogenase or reference substance, continuous three days later on.
Experiment grouping (every group of n=6)
1. ischemic control group (I/R): normal saline
2. Human Urinary Kallidinogenase I organizes: 2 * 10
-3PNA/kg
3. Human Urinary Kallidinogenase I/II organizes: 5 * 10
-3PNA/kg
4. Human Urinary Kallidinogenase II organizes: 10 * 10
-3PNA/kg
5.PGE
1Group: PGE
1300ug/kg
Other gets 4 animals, does not do any special handling, as the normal control group.
Observation index
1. SM is 3 days, and drug withdrawal is after 24 hours, gets blood 2ml (anticoagulant) from internal jugular vein on 4th, measures LDH and CPK activity on automatic biochemical analyzer.Intracardiac injection 10%KCl puts to death animal fast then.
2. win full brain, get the left hemisphere first half and claim weight in wet base, 90 ℃ of oven dry, thoroughly dry weight is surveyed in the oven dry back, calculates water content.Brain water content=(weight in wet base-dry weight)/weight in wet base * 100%.Use exsiccata then, Na is surveyed in acid digestion back
+, Ca
2+Content (atomic absorption spectrophotometer mensuration).Left hemisphere is latter half of puts into-196 ℃ of freezing survey of liquid nitrogen MDA (thiobarbituricacid method), cAMP (adenosine cyclophosphate) and cGMP (cyclic GMP) rapidly.Get right hemisphere first half liquid nitrogen freezing survey ATP (HPLC method).Latter half of 10% formalin solution of putting into is immediately fixed, section, H-E dyeing, light microscopy checking.(X ± SD) expression, the q check takes statistics to learn and handles result of the test between the variance analysis group with mean ± standard deviation.
Experimental result
1. the change of brain water and sodium content: ischemic control group and normal control group are relatively, brain water content, sodium content obviously increase, and middle dosage and heavy dose of Human Urinary Kallidinogenase organize brain water content and reduce (relatively being respectively p＜0.05 and p＜0.01 with the ischemic control group).Low dose of Human Urinary Kallidinogenase's group and PGE
1Group brain water content and ischemic control group no significant difference.Though treatment is respectively organized the cerebral tissue sodium content a little less than the ischemic control group, not statistically significant (P＞0.05).
2. the change of cerebral tissue ATP and cyclic nucleotide content: after the cerebral ischemia, the normal animal of cerebral tissue ATP content reduces by 32% (p＜0.01).Human Urinary Kallidinogenase's various dose group ATP content increases by 11.9% (p＞0.05), 23.6% (p＜0.05) and 18.8% (p＜0.05) respectively than matched group; CAmp content reduces by 11.2% (p＞0.05), 26.5% (p＜0.01) and 32% (p＜0.01) respectively; Yet cGmp content increases by 2 times, 3 times and 4 times respectively.PGE
1Group ATP content increases by 17.3% (p＜0.05); CAmp content reduces by 9.3%, and cGmp content increases by 22.5%, but equal not statistically significant (p＞0.05).
3. cerebral veins, venae cerebri hemase activity change; After active increase by the 76.7% and 226%. heavy dose of Human Urinary Kallidinogenases' treatments of cerebral ischemia matched group cerebral veins, venae cerebri blood LDH, CPK, active 21% (p＜0.05) that reduces of LDH.Other each treatment group LDH are active to compare no difference of science of statistics with the ischemic control group.Each dosage group CPK activity of Human Urinary Kallidinogenase reduces by 1.4%, 7.9% and 10.6% than the ischemic control group, but not statistically significant (p＞0.05).PGE
1Group CPK activity reduces by 17% (p＜0.01) than the ischemic control group.
4. cerebral tissue calcium and lipid peroxide contents change: behind the cerebral tissue ischemia, and cerebral tissue lipid peroxidation end-product MDA and organize calcium content to increase by 62% and 147% respectively.Though each treatment group cerebral tissue MDA and calcium content and the comparison of ischemic control group have reduction, not statistically significant.
5. morphological change: Nissl dyeing Mus brain section, cell is arranged can divide six layers, I layer (molecular layer): a small amount of neurocyte is arranged, and does not have difference between each group.II layer (external granular layer): the ischemia/reperfusion group can't be distinguished the II layer, and PGE1 group II layer and the boundary of III layer are unclear, and Urina Hominis kininogen I group and Human Urinary Kallidinogenase II group II layer are more obvious, and it is thick to account for cortex 1/10.III layer (external pyramidal layer): this layer of ischemia/reperfusion group is not obvious, PGE
1Organize thinlyyer, pyramidal cell is arranged.Urina Hominis kininogen I group and Human Urinary Kallidinogenase II group are thicker, and pyramidal cell is arranged.IV layer (internal granular layer): no significant difference between each group.V layer (inner cone layer): the thick cortex 1/5 that accounts for has giant pyramidal cells, zero difference between each group.VI layer (many types of layer): zero difference between each group.
Discuss
This experiment is closed and losing blood property hypotension by rabbit bilateral common carotid arteries folder, low pressure, low perfusion, successfully duplicated the acute cerebral ischemia animal model, observe the 3rd day the damage performance of operation back, find that brain water, sodium content increase, the ATP stockage reduces, and cyclic nucleotide content increases, cerebral veins, venae cerebri blood LDH and CPK are active significantly to raise, and cerebral tissue lipid peroxide and calcium content increase.Morphological examination discovery cerebral cortex II layer and III layer are not obvious, can't distinguish.
The increase of brain water content is the important indication of reflection cerebral edema.Brain cell Na behind the brain tissue impairment
+Water retention.Therefore, it is generally acknowledged cerebral tissue Na after the edema
+Increase K
+Reduce.The cerebral tissue hypoxic-ischemic causes the peroxidating of cell membrane lipid, and permeability of cell membrane increases, and endocellular enzyme is spilt.Therefore, brain cell LDH and CPK go into blood and increase, and cerebral veins, venae cerebri blood LDH and CPK are active to be increased.
This laboratory observation arrives, and the Human Urinary Kallidinogenase has certain curative effect to cerebral ischemia reperfusion injury recovery process.Above-mentioned observation index has improvement in various degree, and especially high dose group alleviates the cerebral tissue edema, has increased cerebral tissue ATP content, reduces LDH activity in the venous blood.Though the Human Urinary Kallidinogenase has improvement to the cerebral tissue lipid peroxidation, not statistically significant.PGE
1Effect with blood vessel dilating has increased cerebral tissue energy ATP and has stocked, and reduces cerebral veins, venae cerebri blood CPK activity, and other indexs do not have obvious improvement.Thereby infer that the kallikrein Human Urinary Kallidinogenase not only brings into play curative effect by the effect of its blood vessel dilating, and may be by the damaging action of its anti-Medulla Leporis seu Oryctolagi ischemia/reperfusion injury recovery process of performance that interacts in renin-angiotensin system.
Conclusion
The Cerebral Ischemia injury recovery phase, continuous application Human Urinary Kallidinogenase's treatment can alleviate cerebral edema, increases and organizes ATP to stock, and brain cell is had certain protective role.
Test the influence of 2 Human Urinary Kallidinogenases to the rabbit cerebral ischemia reperfusion injury
Experimental technique
Get the female large ear rabbit of 1.8--2.2kg, overnight fasting before the art is freely drunk water.Back fixation, 3% chlorine alkane add 97% nitrogen and add the anesthesia of 3% oxygen, light insulation (25 ℃ of room temperatures) again.Tracheal intubation artificial respirator control breathing, 1000u/kg heparin intravenous injection anticoagulation.The right femoral artery intubate enters ventral aorta, and intubate connects transducer respectively by the threeway syringe needle, and the reason of delivering a child is led instrument with the monitoring blood pressure more, connects storage blood device (height 40mmHg, i.e. 54.4cm H
2The O post), the right femoral vein intubate is with the loading thing.It is standby to separate the bilateral common carotid arteries threading.Postoperative was stablized 30 minutes, opened the threeway syringe needle to the storage quick blood-letting of blood device (about 1～2 minute, reach 40mmHg BP), closed two common carotid artery with the bulldog clamp folder simultaneously.Continue after 10 minutes, the bilateral bulldog clamp of loosening rapidly, pressurization (about 300mmHg) simultaneously will be store blood device inner blood and be injected in the ex vivo (about 2 minutes), and oxygen uptake is simultaneously observed and was finished experiment in 30 minutes.
Experiment grouping (every group of n=10)
Laboratory animal is divided into following four groups, respectively at cerebral ischemia began to instil in preceding 10 minutes various dose Human Urinary Kallidinogenase or reference substance (capacity 2.5ml/kg PBS), keeps perfusion 30 minutes, finishes in instillation and finishes experiment in back 10 minutes.
1. ischemic control group: use simple PBS
2. Human Urinary Kallidinogenase I organizes: 2.0 * 10
-3PNA/kg
3. Human Urinary Kallidinogenase II organizes: 10.0 * 10
-3PNA/kg
4.Nicardipine group: 10ug/kg
Other gets 4 animals, only does anesthesia and the not sham-operation processing of ischemia, is called " 5.sham group ".
Observation index
1. animal dead number;
2. blood pressure before the cerebral ischemia and when pouring into 30 minutes again;
3. arterial cannulation blood-letting when finishing experiment, blood sampling, centrifugal separation plasma is measured blood glucose, lactate, LDH activity, MDA content.
4. win full brain, get left hemisphere and put liquid nitrogen freezing rapidly, the first half liquid nitrogen grinding, the preparation homogenate down of 6% trichloroacetic acid is measured and is organized ATP content.Right hemisphere first half claims weight in wet base, and dry weight is surveyed in 90 ℃ of oven dry behind the finish-drying, computation organization/dry weight ratio, reflection degree of cerebral edema.Half tissue prepares homogenate (1: 10W/V), organize MDA content with thiobarbituricacid mensuration with PBS behind the right hemisphere.
Experimental result
In the cerebral ischemia 10 minutes/pour into during 30 minutes again, each treated animal mortality rate is: ischemic control group 4/10, Human Urinary Kallidinogenase I group 4/10, Human Urinary Kallidinogenase II group 3/10, Nicardipine group 3/10, sham operated rats 0/4.Brain I/R damage, blood glucose appears in animal to be increased, and contents of mda and LDH activity all increase.Ischemic control group and sham operated rats compare, and These parameters increases by 46.46%, 211.45%, 60.79% and 397.13% (the P value is all less than 0.01) respectively.The These parameters of each group of treatment all is in various degree to be improved, but compares with the ischemic control group, and blood glucose only Nicardipine group reduces, and statistical significance (P＜0.001) is arranged.Blood plasma lactic acid salt is reduced in each group and shows as, and Human Urinary Kallidinogenase II organizes and the Nicardipine group all has significance (P＜0.01) with the ischemic control group difference, and Human Urinary Kallidinogenase II group is than Human Urinary Kallidinogenase I group obvious (P＜0.01).Though active each the treatment group of contents of mda and LDH is more lower slightly than ischemic control, not statistically significant (P＞0.05).
Cerebral Ischemia damage (ischemic control group and sham operated rats are relatively), cerebral tissue ATP content reduces (74.71%, P＜0.01), and (weight in wet base/dry weight ratio increases by 36.71% to the cerebral tissue edema, P＜0.01) and brain MDA generate to increase (+52.83%, P＜0.01).Each treatment group is in various degree improves (each group is all remarkable), alleviate the reduction (P＜0.01) of brain ATP content, Human Urinary Kallidinogenase II group is better than Human Urinary Kallidinogenase I group (P＜0.01), and each group of weight in wet base/dry weight ratio all slightly reduces than the ischemic control group, but not statistically significant (P＞0.05).
Suppress the generation of brain MDA, Human Urinary Kallidinogenase II group is than ischemic control group, Human Urinary Kallidinogenase I group and Nicardipine group effect stronger (P＜0.05).Suppress cerebral tissue MDA and generate, only Human Urinary Kallidinogenase II group relatively has statistical significance (P＜0.05) with the ischemic control group, and Human Urinary Kallidinogenase II group also has significance (P＜0.05) with Human Urinary Kallidinogenase I group and Nicardipine group difference.
Conclusion: the Urina Hominis kininogen does not have certain curative effect to alleviating the damage of rabbit Cerebral Ischemia, shows as the increase that alleviates the blood plasma lactic acid salt level, keeps cerebral tissue ATP to stock, and alleviates the cerebral tissue level of lipid peroxidation.
Test the influence of 3 Human Urinary Kallidinogenases to the rabbit cerebral infarction
Experimental technique
Animal selection and art pre-treatment, anesthesia, respiratory control are all same with experiment 2.Separate left common carotid artery, approach the internal carotid artery bifurcated from External Carotid Artery for Intubation, the bulldog clamp folder closes common carotid artery, inject arachidonic acid (AA) 0.5mg/kg (capacity 0.05ml/kg) fast from intubate, go into cerebral circulation from internal carotid artery, remove bulldog clamp then, aforesaid operations is finished (folder is plucked in administration) in 5 seconds.Injected behind the AA 15 minutes, and opened breast, left atrium intubate 10mmHg pressure is perfusion warm saline (37 ℃) 20ml down, thereupon India ink5.0ml (during perfusion under nearly ventral aorta bulldog clamp).Broken end is got brain, is fixed in the interior crosscut of 10% formaldehyde and becomes 5, and tangent plane is taken pictures, and the machine Flame Image Process is calculated infarct area (white area/[black region ten white area]) as calculated.With back 15 minutes (opening the front) of injection, collect blood sample from arterial cannulation respectively before the AA injection, measure MDA content and LDH vigor.Experiment is divided into 6 groups, respectively at making preceding 30 minutes auricular veins of cerebral infarction model following various dose medicine (capacity is 2.5ml/kgPBS) that instils.After instillation finishes, analogue formation immediately.
Experiment grouping (every group of n=10)
1. infraction group: simple PBS
2. Human Urinary Kallidinogenase I organizes: 2.0 * 10
-3PNA/kg
3. Human Urinary Kallidinogenase I/II organizes: 5.0 * 10
-3PNA/kg
4. Human Urinary Kallidinogenase II organizes: 10.0 * 10
-3PNA/kg
5.PGE
1(prostaglandin) group: 300ug/kg
6. oral group: preceding 2 hours oral agents of art are irritated 56u/kg of stomach
Experimental result
Infarct size: this experiment causes in the brain behind internal carotid artery injection arachidonic acid extensively blocks, and infraction group cerebral infarction district accounts for 1/3rd of full brain area.Each group control property administration of treatment can both make model dwindle infarct size (compare with the infraction group, be p＜0.01).The strong and weak order of effect is Human Urinary Kallidinogenase II group, Human Urinary Kallidinogenase I/II group, PGE
1Group, oral group and Human Urinary Kallidinogenase I group.Human Urinary Kallidinogenase II group cerebral infarct size only is 39.72% of an infraction group infarct size, and its effect that reduces infarct size obviously is better than other each treatment groups.
Contents of mda: AA causes that the mechanism of infraction comprises number of mechanisms such as stimulating vasospasm, injured blood vessel endothelium, the gathering of promotion platelet activation.Wherein AA promotes lipid peroxidation, measures end-product MDA content, and the infraction group increases about 2.4 times (p＜0.01) than sham operated rats.Each group of treatment, there is not (p＞0.05) the obvious effect except that oral group, Human Urinary Kallidinogenase I group, Human Urinary Kallidinogenase II group, Human Urinary Kallidinogenase I/II group and PGE1 group all obviously suppress MDA and generate, contents of mda is low respectively by-21.97% than the infraction group,-40.81% ,-38.12% and-30.94% (p all＜0.01).Human Urinary Kallidinogenase I/II group, Human Urinary Kallidinogenase II group and PGE
1It is also remarkable to organize more oral group of difference.
Blood plasma LDH activity: blocking tissue's cell ischemic injuries, endochylema endoenzyme LDH spills and discharges into blood circulation.Infraction group blood plasma LDH activity increases about 3 times (p＜0.01) than sham operated rats.Though oral group of blood plasma LDH is active in the infraction group, difference not statistically significant (p＞0.05); Human Urinary Kallidinogenase I group, Human Urinary Kallidinogenase II group, Human Urinary Kallidinogenase I/II group and PGE
1Group is than infraction group blood plasma LDH activity low respectively by-14.745% (p＜0.05) ,-15.77% (p＜0.05) ,-24.23% (p＜0.01) and-23.65% (p＜0.01).Human Urinary Kallidinogenase II group and oral group difference be remarkable (p＜0.05) also.
Conclusion: Human Urinary Kallidinogenase's Prevention Processing has the infarct size of dwindling, suppresses the MDA generation, reduces effects such as cell LDH spills, PGE for the cerebral infarction that AA causes
1Similar effect is also arranged.The effect of oral agents is not observed in this experiment.
Test the influence (experimentation) of 4 Human Urinary Kallidinogenases to focal cerebral ischemia in rats
Experimental technique
Get male rat, lumbar injection chloral hydrate 350mg/kg anesthesia, right lateral position is fixed.The surgical exposure zygomatic arch, bite away zygomatic arch with rongeur, cut off fascia, expose Nie's precoila, with little stretching device phosphorus shape bone and lower jawbone spacing are strutted, at the bottom of skull, open one 2cm * 2cm cranium window, tear cerebral dura mater, expose middle cerebral artery, blow with high frequency electric knife, with blocking blood flow, cause local cerebral ischemia, the layer-by-layer suture otch.Carry out the sublingual vein administration after 30 minutes, steam again and raise.Room temperature is strict controlled in 24～25 ℃.After 24 hours animal is carried out the nervous symptoms scoring, broken end is got brain, measures brain water content.
Other gets male rat, and grouping and administration are the same, and behind blocking-up one side middle cerebral artery 24 hours, broken end was got brain, and the reference literature method is measured cerebral infarct size.
Test grouping (every group of n=10)
1. sham operated rats: sublingual vein injecting normal saline
2. mesencephalic arteries blocking-up group: intravenous injection normal saline
3～5. mesencephalic arteries blocking-up+intravenous injection Human Urinary Kallidinogenases
17.5PNA * 10
-3/ kg intravenous injection group
8.75PNA * 10
-3/ kg intravenous injection group
3.5PNA * 10
-3/ kg intravenous injection group
6. mesencephalic arteries blocking-up+intravenous injection nimodipine 0.5mg/kg organizes
Observation index
Cerebral infarct size is measured: will peel off complete brain and put into the cuvette that 4 ℃ of refrigerators fill normal saline, remove olfactory bulb, cerebellum and low brain stem after 10 minutes, be cut to five along coronalplane, put into red tetrazolium (TTC) dyeing liquor immediately, lucifuge temperature in 37 ℃ of water-baths was incubated 30 minutes.It is fixing that taking-up brain sheet is put into 10% formalin.Normal structure is a rose; Ischemic tissue is white in color.Measure ischemic areas with the weight method of quadrature, calculate the percentage ratio that ischemic area accounts for full brain area.
Nervous symptoms scoring determination methods and standard:
(1) mention the Mus tail, normal mice two forelimbs are extended straight forward and symmetry.The operation Mus, the forelimb shoulder inward turning and the interior receipts of ischemia brain hemisphere offside are observed its degree difference and are chosen as 0～4 fen.
(2) tractive two limbs, normal rat muscular strength symmetry, the offside muscle of anterior limb of operation back cerebral ischemia hemisphere is unable, observes its degree difference and is chosen as 0～3 fen.
(3) push away two shoulders, normal rat bilateral shoulder resistance symmetry, the offside shoulder resistance of operation back cerebral ischemia hemisphere descends; Observe its degree difference and be chosen as 0～3 fen.
By above standard, full marks are 10 minutes.Mark is high more, illustrates that disordered brain function is serious more.
The mensuration of brain water content: block back 24 hours execution Mus in mesencephalic arteries, take out full brain.After peeling off complete brain decerebellation and low brain stem, weigh (weight in wet base), in the rearmounted case 120 ℃ about 18 hours roasting to constant weight, weigh again (dry weight).Brain water content=(cutaneous horn weight-brain stem is heavy)/cutaneous horn heavy * 100%; The t method of inspection.
Experimental result
The administration group is blocked back 30 minutes intravenous injection Human Urinary Kallidinogenases at mesencephalic arteries, can alleviate the cerebral ischemia, cerebral edema and the neurobehavioral symptom that cause because of the blocking-up of rat mesencephalic arteries, and dose-effect relationship is arranged; Positive controls (nimodipine) also has good therapeutical effect.
(5) specific embodiment
The invention will be further described by the following examples.
Embodiment 1 extracts and the purification Human Urinary Kallidinogenase
Fresh male's urine is not higher than 0.05MNa2HPO4-NaH2PO4 to the water ultrafiltration to electric conductance, 0.1M NaCl, the buffer of pH7.4 with the ultrafilter membrane of 10,000 Da.
The DEAE-Sepharose post is through 0.05M Na2HPO4-NaH2PO4,0.1M NaCl, the buffer balance of pH7.4.With DEAE-Sepharose post on the concentrated urine, with the level pad flushing, use 0.05MNa2HPO4-NaH2PO4,0.3M NaCl, the buffer solution elution of pH7.4 is collected eluting peak.
The Aprotinin-Sepharose post is through 0.05M Na2HPO4-NaH2PO4, the level pad balance of pH8.0, to go up the Aprotinin-Sepharose post behind the DEAE-Sepharose post eluting peak accent pH8.0, wash with level pad, use 0.1M HAc-NaAc, the buffer solution elution of pH4.0 is collected eluting peak.
Aprotinin-Sepharose post eluting peak is to 0.05M Na2HPO4-NaH2PO4,0.1M NaCl, the buffer dialysis of pH7.0 8 hours, 60 ℃ of heating of water bath with thermostatic control 10 hours, ultrafiltration and concentration.
Sephadex G-100 post is through the normal saline balance, and with Sephadex G-100 post on 60 ℃ of Human Urinary Kallidinogenase's solution that heated in 10 hours, the normal saline eluting is collected eluting peak.
Eluting peak is through the degerming of 0.22um membrane filtration, and ultrafiltration and concentration becomes Human Urinary Kallidinogenase's highly finished product.
Human Urinary Kallidinogenase's freeze-dried powder that embodiment 2 preparation injection for intravenous are used
Get Human Urinary Kallidinogenase 150PNA, glycine (medicinal) 5 grams, mannitol (medicinal) 7.5 grams, add pH7.0 to 1 liter of 0.02M phosphate buffered solution, stir evenly in 1000 peace bottles of back packing.Through lyophilizing sealing by fusing, aseptic filtration, make Human Urinary Kallidinogenase's freeze-dried powder.
Embodiment 3 preparation Human Urinary Kallidinogenase liquid drugs injections
Get Human Urinary Kallidinogenase's highly finished product of 150PNA, add the injection water to 1000ml, transfer pH to 6.8, stir evenly and aseptic filtration, in 1000 peace bottles of packing, sealing by fusing makes Human Urinary Kallidinogenase's liquid drugs injection.
Embodiment 4 preparation Human Urinary Kallidinogenase infusion preparations
Get Human Urinary Kallidinogenase's highly finished product of 150PNA, add normal saline, transfer pH to 6.8, stir evenly and aseptic filtration, in 1000 infusion bottles of packing, add a cover, make Human Urinary Kallidinogenase's transfusion to 250000ml.
Relate to some english abbreviations in the description, explain as follows: I/R acute cerebral ischemia/reperfusion injury PBS phosphate buffered solution MDA malonaldehyde LDH lactate dehydrogenase A TP adenosine triphosphate CPK E.C. 2.7.1.73 PGE1 prostaglandin cAMP adenosine cyclophosphate cGMP cyclic GMP AA arachidonic acid HPLC high performance liquid chromatography
Claims (7)
1. medicament, be used for the treatment of with prevention of brain and block, this medicament comprises the Human Urinary Kallidinogenase as active component, kininogenase is the natural a kind of glycoprotein that is present in the Urina Hominis, molecular weight is about 54,000 dalton (gel filtration), forms a strand by 238 amino acid residues, N-end and C-terminal amino acid residue are respectively isoleucine and serine, contain 5 pairs of S-S keys in the molecule, be respectively Cys7-Cys150, Cys26-Cys42, Cys29-Cys196, Cys161-Cys175 and Cys186-Cys211, isoelectric point, IP is about 4.0, contains 14.4% sugar in the molecule, binding site lays respectively at Asn78, Asn84 and Asn141, its sugar consist of mannose 3%, galactose 1.7%, fucose 0.8%, N-acetyl-glucosamine 5.0%, primary structure (aminoacid composition sequence) as shown in the formula
2. application Human Urinary Kallidinogenase according to claim 1 prepares the method for treatment and prevention of brain infraction medicine, it is characterized in that, is equipped with one or more pharmacy acceptable carriers.
3. the described pharmacy acceptable carrier of claim 2 is glycine, mannitol, the combination of pH7.0 phosphate buffered solution.
4. the dosage form of claim 2 and 3 defined pharmaceutical compositions is the powder pin.
5. the dosage form of claim 2 and 3 defined pharmaceutical compositions is a liquid drugs injection.
6. the dosage form of claim 2 and 3 defined pharmaceutical compositions is transfusion.
7. claim 4,5,6 described pharmaceutical preparatioies, PNA is defined as " under 37 ℃, pH8.0 condition; it is 1PNA unit that 1 minute hydrolysis substrate Val-Leu-Arg-PNA discharges the free PNA of 1umol ", the specification of powder pin or liquid drugs injection or infusion preparation is 0.1～0.2PNA unit/(bottle).
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021167834A CN1183966C (en) | 2002-05-13 | 2002-05-13 | Application of human urokininogenase in preparing medicine for preventing and treating cerebral infraction |
| AU2003234976A AU2003234976A1 (en) | 2002-05-13 | 2003-05-12 | The use of the human urinary kininogenase in the manufacture of a medicine for treating and preventing cerebral embolism |
| PCT/CN2003/000345 WO2003094935A1 (en) | 2002-05-13 | 2003-05-12 | The use of the human urinary kininogenase in the manufacture of a medicine for treating and preventing cerebral embolism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021167834A CN1183966C (en) | 2002-05-13 | 2002-05-13 | Application of human urokininogenase in preparing medicine for preventing and treating cerebral infraction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1403156A true CN1403156A (en) | 2003-03-19 |
| CN1183966C CN1183966C (en) | 2005-01-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021167834A Expired - Lifetime CN1183966C (en) | 2002-05-13 | 2002-05-13 | Application of human urokininogenase in preparing medicine for preventing and treating cerebral infraction |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN1183966C (en) |
| AU (1) | AU2003234976A1 (en) |
| WO (1) | WO2003094935A1 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006047948A1 (en) * | 2004-11-03 | 2006-05-11 | Techpool Bio-Pharma Co., Ltd. | The use of human urinary kallidinogenase for the manufacture of a medicine for the treatment of sepsis |
| WO2006047949A1 (en) * | 2004-11-03 | 2006-05-11 | Techpool Bio-Pharma Co., Ltd. | The use of human urinary kallidinogenase for the manufacture of a medicine for the treatment of acute coronary syndromes |
| CN1318090C (en) * | 2004-11-29 | 2007-05-30 | 广东天普生化医药股份有限公司 | Medicine composition for improving stability of human urine kininogenase |
| CN100388950C (en) * | 2006-09-05 | 2008-05-21 | 广东天普生化医药股份有限公司 | Application of human urokinase-type peptidase in preparing medicine for treating diabetes combined cerebral infraction |
| WO2009039704A1 (en) * | 2007-09-27 | 2009-04-02 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A method for producing human kallikrein 1 |
| CN101879308A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating acute renal failureacute renal failure |
| CN101879309A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating diabetic foot ulcers |
| CN101879310A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating diabetic nephropathy |
| CN101134953B (en) * | 2007-07-02 | 2011-02-09 | 广东天普生化医药股份有限公司 | Recombinant human pancreas kininogenase |
| CN101134106B (en) * | 2007-07-02 | 2012-01-11 | 广东天普生化医药股份有限公司 | Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction |
| CN108969758A (en) * | 2018-09-07 | 2018-12-11 | 广东天普生化医药股份有限公司 | Purposes of the human urokinase-type peptidase in preparation treatment thromboangiitis obliterans object |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101134105B (en) * | 2007-07-02 | 2011-04-20 | 广东天普生化医药股份有限公司 | Pharmaceutical composition containing recombination human pancreatic kininogenase for treating and/or preventing cerebral infarction |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0546533A2 (en) * | 1991-12-10 | 1993-06-16 | Sanwa Kagaku Kenkyusho Co., Ltd. | Medical use of kininogenase |
-
2002
- 2002-05-13 CN CNB021167834A patent/CN1183966C/en not_active Expired - Lifetime
-
2003
- 2003-05-12 AU AU2003234976A patent/AU2003234976A1/en not_active Abandoned
- 2003-05-12 WO PCT/CN2003/000345 patent/WO2003094935A1/en not_active Application Discontinuation
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006047949A1 (en) * | 2004-11-03 | 2006-05-11 | Techpool Bio-Pharma Co., Ltd. | The use of human urinary kallidinogenase for the manufacture of a medicine for the treatment of acute coronary syndromes |
| CN1292793C (en) * | 2004-11-03 | 2007-01-03 | 广东天普生化医药股份有限公司 | Application of kininogenase of human urine in preparing medication for treating pyemia |
| CN1308035C (en) * | 2004-11-03 | 2007-04-04 | 广东天普生化医药股份有限公司 | Application of human urine kininogenase for preparing medicine to treat acute coronary syndromes |
| WO2006047948A1 (en) * | 2004-11-03 | 2006-05-11 | Techpool Bio-Pharma Co., Ltd. | The use of human urinary kallidinogenase for the manufacture of a medicine for the treatment of sepsis |
| CN1318090C (en) * | 2004-11-29 | 2007-05-30 | 广东天普生化医药股份有限公司 | Medicine composition for improving stability of human urine kininogenase |
| CN100388950C (en) * | 2006-09-05 | 2008-05-21 | 广东天普生化医药股份有限公司 | Application of human urokinase-type peptidase in preparing medicine for treating diabetes combined cerebral infraction |
| CN101134953B (en) * | 2007-07-02 | 2011-02-09 | 广东天普生化医药股份有限公司 | Recombinant human pancreas kininogenase |
| CN101134106B (en) * | 2007-07-02 | 2012-01-11 | 广东天普生化医药股份有限公司 | Pharmaceutical composition containing human urine kininogenase for treating cerebral infarction |
| WO2009039704A1 (en) * | 2007-09-27 | 2009-04-02 | Shanghai Wanxing Biopharmaceuticals, Co., Ltd. | A method for producing human kallikrein 1 |
| CN101879310A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating diabetic nephropathy |
| CN101879309A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating diabetic foot ulcers |
| CN101879308A (en) * | 2010-06-13 | 2010-11-10 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating acute renal failureacute renal failure |
| CN101879308B (en) * | 2010-06-13 | 2012-04-18 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating acute renal failureacute renal failure |
| CN108969758A (en) * | 2018-09-07 | 2018-12-11 | 广东天普生化医药股份有限公司 | Purposes of the human urokinase-type peptidase in preparation treatment thromboangiitis obliterans object |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003234976A1 (en) | 2003-11-11 |
| CN1183966C (en) | 2005-01-12 |
| WO2003094935A1 (en) | 2003-11-20 |
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