CN1849133B - Intravenous injection of plasminogen non-neurotoxic activators for treating cerebral stroke - Google Patents

Abstract

The invention relates to the use of non-neurotoxic plasminogen-activating factors, e.g. from Desmodus rotundus (DSPA) or genetically modified plasminogen-activating factors especially of human origin, for producing a therapeutic agent that is to be injected for the therapeutic treatment of stroke in humans.

Description

Be used for the treatment of the intravenous injection of the non-neurotoxic plasminogen activator of apoplexy

The present invention relates to the non-neurotoxic plasminogen activator in intravenous application, in particular for the plasminogen activator and the plasminogen activator (DSPA) that is derived from Desmodus rotundus saliva through genetic modification of treatment people syndrome similar to wind disease.By these plasminogen activator treatment apoplexy of the known utilization of International Patent Application PCT/EP02/12204, the content of this patent disclosure is incorporated herein by reference in full.

The Clinical symptoms of apoplexy and biochemistry

The different clinical pictures that clinical symptoms is relevant are summarised as term " apoplexy ".According to pathogeny separately, these clinical pictures may at first be divided into so-called ischemic and hemorrhagic damage.

The feature of ischemia injury (ischemia) is to cause sanguimotor minimizing or interruption in the brain because of the arterial blood supply is deficient.This usually is that the thrombosis of the blood vessel that narrowed down by arteriosclerosis causes, or cause by arterial thrombosis or heart thromboembolism.

Hemorrhagic damage is owing to mainly too high and damage forms the perforation that brain is supplied tremulous pulse by arterial pressure.Yet, in all brain injury, only have about 20% to cause by hemorrhagic damage.Therefore, the apoplexy that is caused by thrombosis is more relevant.

Compare with other tissue ischemias, nervous tissue's ischemia generally is accompanied by the necrosis of influenced cell.The downright bad incidence rate that improves in the nervous tissue can be used the new understanding of " excitatory toxicity " phenomenon is explained, this phenomenon is a kind of complicated cascade reaction that comprises numerous reactions steps.This cascade reaction loses ATP rapidly by anoxybiotic ischemic neuron and depolarization causes.It causes the increase of postsynaptic release neurotransmitters glutamic acid, and this neurotransmitter activates the film of regulation and control cationic channel in conjunction with glutamate receptor.Yet, because the increase that this glutamic acid discharges makes glutamate receptor by excessive activation.

The voltage dependent form cationic channel that the glutamate receptor regulation and control are opened with combining of receptor by glutamic acid.It causes Na ⁺And Ca ²⁺Thereby a large amount of cells that flow into disturb Ca ²⁺Dependent form material cellular metabolism (comprising energy metabolism).Ca ²⁺The activation of dependent form catabolic enzymes especially can be explained cell death (Lee, Jin-Mo etc., " the The changing landscape of ischemic brain injury mechanisms " that takes place subsequently; Dennis W.Zhol " Glutamat neurotoxicity and diseases of the nervoussystem ").

Although the neurotoxicity mechanism of glutamic acid mediation is not also understood fully, people agree its death of having facilitated the neurocyte after the cerebral ischemia to a great extent (Jin-Mo Lee, etc.).

The treatment of apoplexy

Except that the safety and stable physiological parameter that guarantee important function, in the treatment of acute cerebral ischemia, at first to dredge the blood vessel of sealing again.This purpose is undertaken by diverse ways.Simple machinery is dredging method again, and for example the PTCA art under the myocardial infarction situation does not obtain gratifying effect up to now yet.Have only the fibrinolysis of success just can make patient's situation obtain gratifying improvement.

The fibrinolysis of self is that this enzyme is formed through catalysis (activation) by its unactivated precursor plasminogen based on the proteolytic activity of serine protease fibrinolysin.The natural activation of plasminogen is to be undertaken by plasminogen activator u-PA (urokinase type plasminogen activator) of self and t-PA (tissue plasminogen activator).Opposite with u-PA, t-PA forms a so-called activator complex with fibrin and plasminogen.Therefore, the catalytic activity of t-PA is that fibrin relies on, and strengthens about 550 times in the presence of fibrin.Except that fibrin, even Fibrinogen also can stimulate the catalytic action that the plasminogen by t-PA mediation changes to fibrinolysin-in very little scope.In the presence of Fibrinogen, the t-PA activity only strengthens 25 times.Fibrinous cleaved products (fibrin degradation product (FDP) (FDP)) also can stimulate the t-PA activity.

Known therapeutics approach

A) streptokinase

The early stage trial of acute apoplexy being carried out thrombolytic treatment can trace back to nineteen fifties.Use streptokinase, a kind of fibrinolytic agent that is derived from the β Hemolytic streptococcus, the extensive first clinical trial of carrying out just began in nineteen ninety-five.The complex that it can be fibrinolysin with other plasminogen molecular conversions that streptokinase and plasminogen form.

, therefore adopt the therapy of streptokinase to have major defect because streptokinase is thereby that a kind of bacterialprotease can excite allergy in body.Thereby also may occur so-called streptokinase resistance under the situation that has the streptococcal infection that corresponding antibodies produces before makes that this therapy is more difficult and carries out.In addition, Europe (Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter AcuteStroke Trial of Italy (MAST-I)) and Australia (Australien StreptokinaseTrial (AST)) clinical trial show, with presenting the mortality rate risk of rising and higher intracerebral hemorrhage (intracerebral hemorrhage, ICH) risk behind the streptokinase treatment patient.These tests have to just be terminated in early days.

B) urokinase

As selection, also can adopt urokinase-its " classical " fibrinolytic protein lytic agent that also is a kind of.Opposite with streptokinase, because it is a kind of natural enzyme that is present in a large amount of body tissues, therefore, it does not show antigenic characteristic.It is a kind of plasminogen activator, and does not rely on cofactor.Urine agent enzyme is produced by the nephrocyte culture.

C) recombinant t-PA (rt-PA)

Obtained with tissue-type plasminogen activator-so-called rt-PA-(referring to EP 0 093 619, US 4,766,075) the thromboclastic rich experience of therapeutic of carrying out that originates from the reorganization hamster cell.Except acute myocardial infarction was main indications, the nineties had been finished a series of clinical researches with t-PA in worldwide, obtained part and can't understand and the result of contradiction.In the acute apoplexy test in so-called Europe (ECASS), after appearring in patient, the apoplexy symptom use rt-PA to treat in intravenous in 6 hours.Check mortality rate and Barthel index deformity or independent living ability index after 90 days as patient.Do not report the remarkable improvement of viability, although reported-the not rising of remarkable-mortality rate.Therefore can infer that the thrombolytic treatment that the patient who selects individually according to medical history is separately carried out with rt-PA at once may be favourable after apoplectic seizure.Yet, do not recommend rt-PA generally use in 6 hours after apoplectic seizure, because observe after apoplectic seizure danger (C.Lewandowski C and Wiliam Barsan, 2001:Treatment of AcuteStrocke that application in the several hrs only can increase intracerebral hemorrhage (ICH); In:Annals of Emergency Medcine 37:2; S.202ff).

The thrombolytic treatment of apoplexy also was clinical research (the so-called NINDS rtPA apoplexy test) problem that American National Instrument Institute of NeurologicDisorder and Stroke carries out afterwards.The emphasis of this test concentrates on and occurs after the symptom in 3 hours on the effect with the rt-PA intravenous therapy, and this effect is based on the health status of patient after three months.Owing to observe this treatment, therefore be recommended in time period of three hours interiorly to treat, although the author has also found higher ICH risk at this with rt-PA to the blodynamic good effect of patient.

(ECASS II Trial:Alteplase Thrombolysis for AcuteNoninterventional Therapy in Ischemic Stroke (ATLANTIS)) investigated after the apoplectic seizure whether the good effect with the rt-PA treatment can reappear in three hours in the treatment in six hours in other two researchs.Yet owing to do not observed the improvement of clinical symptoms or any reduction of mortality rate outside the ICH risk that raises, this problem can not obtain affirmative acknowledgement (ACK).

Publish first in 1997 according to portion, the summary of upgrading March calendar year 2001 about various apoplexy tests, all thromboembolism thing treatment (urokinase, streptokinase, rt-PA or recombinaton urokinase) is particularly because ICH causes after the apoplexy in first 10 days significantly higher mortality rate, although the treatment in six hours has reduced dead or disabled patient's sum after apoplectic seizure.Therefore, do not recommend the extensive use of thromboembolism thing in the treatment apoplexy.

Even before, such result makes some other author provide pure satire speech: stroke patient must be selected live (SCRIP 1997:2265,26) dead or maimedly.

But, use the therapy of rt-PA to remain the Therapeutic Method of the acute cerebral ischemia of the unique approval of U.S. food Drug Administration (FDA) up to now.Yet restriction is to use rt-PA in 3 hours after apoplexy.

The recombinant plasminogen activator is sold with title Alteplase or with similar formulations Reteplase at present.The latter is the short t-PA segment with therapeutic activity of a kind of half-life.Therapeutic dose, Alteplase is about 70-100mg, Reteplase is 2 * 560mg, mainly use Reteplase for Alteplase and use (Mutschler: " Arzneimittelwirkungen " by injecting fast for twice with about 30 minutes interval by drop, the 8th edition, the 512-513 page or leaf).

The side effect of t-PA

Neurotoxicity and excitatory toxicity

Approval to rt-PA is in 1996.Before in nineteen ninety-five, first part of statement of the reason of the neurotoxicity of t-PA or excitatory toxicity effect disclosed, wherein the violent effect that produces when t-PA being applied to the apoplexy treatment outside three hours provides explanation, produces self property t-PA of the excitatory toxicity that participates in the glutamic acid mediation according to its described Hippocampus microglia and neurocyte.This conclusion is to derive from the research about t-PA deficiency and wild-type mice to draw, and wherein the glutamic acid agonist is injected into their Hippocampus respectively.The glutamic acid that the t-PA deficient mice is used external source (inthrathekal) shows significantly higher resistance (Tsirka SE etc., Nature, Vol.377,1995, " Exzitoxin-induced neuronaldegeneration and seizure are mediated by tissue plasminogen activator ").These results were confirmed in 1998, and the downright bad nervous tissue of almost twice amount appearred in proof such as Wang in the t-PA deficient mice when intravenous injection t-PA at that time.Under the situation of wild-type mice, the passive result of this of external source t-PA only is about 33% (Wang etc., 1998, Nature, " Tissue plasminogenactivator (t-PA) increases neuronal damage after focal cerebral ischemiain wild type and t-PA deficient mice ").

Nicole etc. has delivered further result of study (Nicole O, the Docagne F Ali C to the excitatory toxicity that is caused by t-PA at the beginning of calendar year 2001; Margaill I; Carmeliet P; MacKenzie ET, VivienD and Buisson A, 2001:The proteolytic activity of tissue-plasminogenactivator enhances NMDA receptor-mediated signaling; In:Nat Med 7,59-64).They can prove that the t-PA that is discharged by the depolarization cortical neuron separates with the NR1 subunit interaction of so-called NMDA type glutamate receptor and with it.This modification causes the rising of receptor active, and it causes after using glutamic acid agonist NMDA and bigger disorganization occurs by the excitatory toxicity that causes.Therefore, t-PA shows neurotoxicity by activating NMDA type glutamate receptor.During the apoplexy because blood brain barrier collapse in the affected tissue regions, the t-PA that dissolubility plasma protein sample Fibrinogen and treatment are used is able to contact with nervous tissue, has been shown its excitatory toxicity effect there by the activation glutamate receptor by the activated t-PA of Fibrinogen.

Although t-PA has the mortality rate effect of neurotoxic side effects and rising, it has still obtained the medicine mandate approval of FDA.This is in default of harmless and effective substitute and very practical cost effectiveness analysis.Yet there is demand just like continuing for the safety therapy pastly.If can not get rid of thrombolytic effect fully, just under the situation of the new thrombolytic drug of exploitation, go out to consider that the neurotoxicity problem is (referring to as a.a.O. such as Wang; Lewandowski and Barsan 2001a.a.O.).

For this reason, although all thrombolytic drugs all may be suitable in principle, stopped the further research of carrying out for exploitation apoplexy treatment novel drugs to the known thrombolytic drug that comprises DSPA (Desmodus rotundus plasminogen activator).For example with regard to DSPA, just pointed out its spreading in early days to this indication with property (Medan P; Tatlisumak T; Takano K; Carano RAD; Hadley SJ; Fisher M:Thrombolysis with recombinant Desmodus salivaplasminogen activator (rDSPA) in a rat embolic stroke model; In:Cerebrovasc Dis 1996:6; 175-194 (the 4th edition International Symposium onThrombolic Therapy in Acute Ishemic Stroke)).DSPA is a plasminogen activator a kind of and t-PA height homology (similar), so that when recognizing the neurotoxic side effects of t-PA, also DSPA is not entertained hope.

Alternate therapeutics approach

At present, the research of alternate therapeutics approach for example concentrates on anticoagulant, for example the active substance in heparin, aspirin or ancrod-Malaysia rattle snake (Malayischen Grubenotter) venom.Yet especially the clinical research (International Stroke Trial (IST) and Trial of ORG 10172 in Acute Stroke Trea tment (TOAST)) of two check heparin effects does not demonstrate the remarkable improvement of mortality rate or the prevention that apoplexy is sent out again.

The focus of alternative new therapy is neither concentrate on also non-dilute blood of thrombosis or anti-agglomeration effect, but attempts improving because of interrupting the vitality (WO 01/51613 A1 and WO01/51614A1) of blood supply damaged cells.In order to reach this purpose, used the antibiotic that is derived from quinones, aminoglycoside or chloromycetin.Based on similar reason, further suggestion applies citicoline immediately after apoplectic seizure.Citicoline is cut into cytidine and choline in vivo.Cleaved products forms the part of neuronal cell film and therefore supports the regeneration (US 5,827,832) of damaged tissues.

In the recent period to the research of safe treatment based on new discovery, that is, the lethal effect of apoplexy partly is only to be caused indirectly by the blood supply of interrupting and directly caused by excitatory toxicity or neurotoxicity in the presence of the glutamate receptor of excessive activation.T-PA strengthens this effect (referring to above-mentioned).Therefore a kind of prescription that reduces excitatory toxicity is to use so-called neuroprotective.They can use separately or unite use to reduce the neurotoxicity effect with fibrinolytic agent.They can or for example directly reduce excitatory toxicity or slacken excitatory toxicity (a.a.O. such as Jin-Mo Lee) indirectly by blocking voltage dependent form sodium or calcium channel as glutamate receptor antagonists.

Is possible at this with the competitive inhibition (antagonism) that for example 2-amino-5-phosphono valerate (APV) or 2-amino-5-phosphono enanthate (APH) produce NMDA type glutamate receptor.Can realize that noncompetitive suppresses with the material that for example is incorporated into passage phencyclidine (Phencyclidin) end.Such material can be phencyclidine, MK-801, dextrorphan or ketamine (ketamin).

Up to now, adopt the treatment of neuroprotective also not show desired success, may must combine with thrombolytic agent because neuroprotective will show their protective effect.This is applicable to other active substance (referring to Figure 10).

Yet, even the combination of t-PA and neuroprotective also only causes limited damage.Defective is, does not avoid the neurotoxicity of employed fibrinolytic agent.

The plasminogen activator of non-neurotoxic

Know the plasminogen activator that is used for the treatment of apoplexy by International Patent Application PCT/EP02/12204, the activity of its enzyme is strengthened manyfold by the fibrin highly selective, promptly greater than 650 times.This disclosed content all is incorporated herein by reference.

The feature of plasminogen activator is based on following knowledge with using, the neurotoxicity that is tissue plasminogen activator (t-PA) is owing to the following fact, that is exactly a blood brain barrier because the brain inner tissue damage that causes because of apoplexy sustains damage or destroys, so, circulation fibrin proper energy enters the cranial nerve tissue in blood, it has activated t-PA there, has caused further tissue injury indirectly by activating glutamate receptor or plasminogen activation.(referring to above-mentioned).

For fear of this effect, use a kind of plasminogen activator, it has shown a kind of fibrin selectivity of increase, and as a kind of opposite result, but shown the Fibrinogen reactivity that reduces.Therefore these plasminogen activators can't, perhaps with t-PA Comparatively speaking degree than the lowland, the activator fibrin of t-PA entered the Fibrinogen activation of nervous tissue from blood because blood brain barrier is impaired, because can not enter nervous tissue because it is big or small and insoluble.These plasminogen activator right and wrong are neurovirulent.

A) through the plasminogen activator of genetic modification

According to a preferred embodiment of the present invention, used non-toxicity plasminogen activator, it comprises at least a so-called proenzyme triplet element.Similar known triplet is the catalytic center from the serine protease of chymotrypsin protein enzyme family, and it is that aspartic acid 194, histidine 40 and serine 32 are formed by three interactional aminoacid.Yet this triplet is not present among the t-PA that belongs to chymotrypsin protein enzyme serine protease family.But, known suitable site to natural t-PA imports at least one above-mentioned amino acid whose site directed mutagenesis and causes original enzyme activity in the presence of fibrin (strand t-PA) to weaken with maturase activity (double-stranded t-PA) strengthening.Therefore, at least one aminoacid in the triplet, or have amino acid whose importing corresponding to the function in the triplet, can strengthen the proenzyme (Zymogenit of t-PA

T) (ratio of maturase activity and original enzyme activity just).The result causes fibrin-specific to significantly improve.This is to be interacted by the conformation between amino acid residue that is imported and/or the wild-type sequence amino acid residue to cause.

Known, replace Phe305 (F305H) and make 20 times of proenzyme enhancings with His among the t-PA, and simple F305H variant has just made 5 times of proenzyme enhancings (ELMadison, Kobe A, Gething M-J with the mutation that Ser replaces Ala 292 (A292S); Sambrook JF, Goldsmith EJ 1993:ConvertingTissue Plasminogen Activator to a Zymogen:A regulatory Triad ofAsp-His-Ser; Science:262,419-421).When having fibrin, these t-PA mutant activity strengthen respectively 30000 times (F305H) and 130000 times (F305H, A292S).In addition, these two mutants also comprise Arg275 are replaced into R275E to prevent that fibrinolysin is in the cutting of cleavage site Arg275-Ile276 and change strand t-PA into double chain form.Single mutant R275E makes the fibrin-specific of t-PA strengthen 6900 times of (K Tachias, Madison EL 1995:Variants of Tissue-typePlasminogen Activator Which Display Substantially Enhanced Stimulationby Fibrin, in:Journal of Biological Chemistry 270,31:18319-18322).

The site 305 of t-PA and 292 and chymase in the site His40 and the Ser32 homology of known triplet of serine protease.By the corresponding replacement of carrying out with histidine or serine respectively, the generation known triplet function (Madison etc., 1993) in the t-PA mutant thereby these aminoacid can interact with the aspartic acid 477 of t-PA.

According to the present invention because these t-PA mutants do not have because of its enhanced fibrin-specific or-compare-only have significantly reduced neurotoxicity with wild-type t-PA, therefore they can be used for the treatment of apoplexy.Be the open simple F305H of t-PA that has addressed; F305H; The A292S mutant or with the combinatorial mutagenesis of R275E, introduce the publication of Madison etc. (1993), and with the hereby incorporated by reference of Tachias and Madison (1995).

As an alternative, the enhancing of plasminogen activator fibrin-specific can be passed through the point mutation realization of Asp194 (or aspartic acid of homologous site).Plasminogen activator belongs to serine protease group in the chymotrypsin protein enzyme family, and therefore it comprises the conserved amino acid Asp194 of the catalytic activity conformational stability of being responsible for the maturation protein enzyme.Known Asp194 and His40 interaction in the proenzyme of serine protease.Activity by proenzyme cuts this interaction and is blocked, the Asp194 side chain rotate about 170 ° to form a new salt bridge with Ile16.This salt bridge participates in the stability in oxygen anion cave (Oxyanion-Tasche) of the catalysis triplet of ripe serine protease.It also is present among the t-PA.

The point mutation of Asp194 at first hinders the formation or the stability of serine stretch protein enzyme catalysis conformation.However, the plasminogen activator of this sudden change demonstrates remarkable enhanced activity (particularly comparing with ripe wild type) in the presence of their cofactor is fibrinous, this can only be interpreted as allowing with fibrinous interaction conformation change (the L Strandberg of promotion catalytic activity, Madison EL, 1995:Variants ofTissue-type Plasminogen Activator with Substantially Enhanced Responseand Selectivity towards Fibrin co-factors, in:Journal of BiologicalChemistry 270,40:2344-2349).Asp194 mutant its active height in the presence of fibrin that shows plasminogen activator thus strengthens, and this makes its application as the plasminogen activator of non-neurotoxic become possibility.

In a preferred implementation of the plasminogen activator of this class non-neurotoxic, used the t-PA mutant, Asp194 is replaced by glutamic acid (D194E) or agedoite (D194N) in this mutant.T-PA activity in these mutants reduces 1-2000 doubly when no fibrin, and its increased activity can reach 498000-1050000 doubly when fibrin exists.These mutants can further comprise the replacement of Arg15 to R15E, and it prevents that fibrinolysin from forming the t-PA of double chain form at the cutting single-chain t-PA of peptide bond Arg15-Ile16 place.This single sudden change strengthens 12000 times with fibrin to the activation of t-PA.For being disclosed in the t-PA sudden change of site 194 and 15, introduce Strandberg and Madison (1995) in full as a reference.

Also can strengthen plasminogen activator to fibrinous dependency by in so-called " self-dissolving ring (Autolyse-Schleife) ", importing point mutation.The known trypsin that comes from of this aminoacid segment; In the homology segment of serine protease, also can find this composition, and be feature with three hydrophobic amino acids (Leu, Pro and Phe) especially.Self-dissolving ring in the plasminogen activator is responsible for the interaction with plasminogen.Point mutation in this zone makes no longer valid formation protein protein interaction between plasminogen and plasminogen activator.These sudden changes only are that function is relevant when lacking fibrin.On the contrary when having fibrin, they are responsible for the active enhancing of plasminogen activator (K Song-Hua, Tachias K, Lamba D, BodeW, madison EL, 1997:Identifikation of a Hydrophobic exocite on TissueType Plasminogen Activator That Modulates Specificity for Plasminogen, In:Journal of Biological Chemistry 272; 3,181-1816).

In a preferred implementation, use in the site 420 to 423 t-PA with point mutation.If these residues replace through the orientation point sudden change, it makes the fibrin dependency of t-PA strengthen until 61000 times (K Song-Hua etc.).Song-Hua etc. have detected point mutation L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.For open purposes according to the present invention is incorporated herein by reference these publications in full.

According to further favourable embodiment, use modified tissue plasminogen activator with aminoacid sequence shown in the SEQ ID Nr.1 (accompanying drawing 13).The t-PA that this is modified and the difference of wild-type t-PA be, the replacement of the hydrophobic amino acid in 420 to 423 in site is as follows in its self-dissolving ring: His420, Asp421, Ala422 and Cys423.This t-PA preferably 194 has a phenylalanine in the site.Further, site 275 can be glutamic acid.Advantageously, site 194 is a phenylalanine.

Further, can use modified urokinase according to the present invention.According to the present invention, urokinase can have according to the aminoacid sequence shown in the SEQ ID Nr.2 (accompanying drawing 14), and wherein the hydrophobic amino acid of self-dissolving ring is replaced by Val420, Thr421, Asp422 and Ser423.Advantageously, urokinase has an Ile275 and a Glu194.This mutant has the fibrin-specific of comparing 500 times of enhancings with the wild type urokinase.

These two kinds of mutant-urokinases and t-PA-all carried out the sxemiquantitative analysis of experiments, and compare with wild-type t-PA and all to show enhanced fibrin-specific.

B) be derived from the plasminogen activator (DSPA) of vampire (Desmodus rotundus)

The plasminogen activator (DSPA) that is derived from vampire saliva also shows 100000 times of highly reinforcing activity-enhancing when fibrin exists, therefore it can be used to non-toxicity plasminogen activator.Term DSPA comprises four kinds of different protease, and the primary demand that they finish vampire is the prolongation (Cartwright, 1974) of prey wound bleeding time.These four kinds of protease (DSPA α 1, DSPA α 2, DSPA β, DSPA γ) present the similarity (homology) of consistent height with respect to people t-PA.They also show similar or consistent physiologically active, and they reasonably are summarized under the generic term DSPA.DSPA is disclosed in patent EP 0 352 119 A1 and US 6 008 019 and 5 830 849, therefore for satisfying openly they is incorporated herein by reference in full.

Up to now, DSPA α 1 is the protease of exhaustive analysis in this group.Its aminoacid sequence and known people t-PA aminoacid sequence have the homology (Kr greater than 72%

Tzschmar etc., 1991).Yet, the difference of two internals is arranged between t-PA and DSPA.At first, DSPA shows as complete active molecule as single chain molecule for peptide substrates, and it is opposite with t-PA, does not change double chain form (Gardell etc., 1989 into; Kr Tzschmar etc., 1991).Secondly, the catalytic activity of DSPA almost completely depends on fibrin (Gardell etc., 1989; Bringmann etc., 1995; Toschi etc., 1998).For example, in the presence of fibrin, 100000 times of the increased activity of DSPA the α 1 and activity of t-PA only strengthens 550 times.On the contrary, Fibrinogen to DSPA is active induce significantly a little less than, its activity only strengthens 7-9 doubly (Bringmann etc., 1995).DSPA significantly more depends on fibrin and fibrin-specific more than only being activated 550 times wild-type t-PA by fibrin.

Because its fibrinolysis and greatly similar to t-PA, DSPA become a kind of interesting material standed in the exploitation of thromboembolism reagent.However, DSPA is restricted to treatment to myocardial infarction in the past as the medical application of thromboembolism reagent, because t-PA participates in the neurotoxicity of glutamate induction, people can not reasonable prediction and the highly proximate plasminogen activator of t-PA can be successfully applied to the treatment of acute apoplexy.

Surprisingly, similar with t-PA to a great extent to t-PA highly similar (homology) and its physiological effect even DSPA demonstrates, DSPA does not but have the neurotoxicity effect yet.Above-mentioned conclusion causes producing a kind of like this understanding, and perhaps DSPA can successfully not cause the grave danger of neural tissue injury as thromboembolism reagent in the apoplexy treatment.This means especially, and DSPA also can use after 3 hours the apoplexy symptom occurring.

DSPA does not have the evidence of neuron excitotoxicity

Not having neurovirulent new knowledge for this plasminogen activator is based on based on one side t-PA and contrast experiment in the body of the neural degeneration effect of DSPA on the other hand.This experiment is by means of so-called " kainic acid-model " and the model that is used to detect the inductive striatal damage of NMDA carries out.

Kainic acid model (being also referred to as the kainic acid damage model) based on by applications kainic acid (KA) as the agonist of kainic acid type (KA type) glutamate receptor and NMDA and the AMPA glutamate receptor toxicity glutamic acid cascade reaction that excites nerve.As test model, only can show that after adding external source t-PA laboratory animal just can reach the level of wild-type mice to the sensitivity of kainic acid with t-PA deficient mice brain stem.On the contrary, the DSPA of molar concentration such as infusion can not recover sensitivity to kainic acid (KA) under the same experimental conditions.Reach a conclusion, the neurotoxicity effect of t-PA can not be substituted by DSPA.Figure 15 (table 1) is seen in these results' summary.

Show based on the quantitative study of this model, even 10 times DSPA concentration increases and can not recover the sensitivity that the t-PA deficient mice is handled KA, just caused the inductive tissue injury of KA and reduce by 10 times t-PA concentration.Reach a conclusion thus, with regard to the neural degeneration after impelling KA to handle stimulated, DSPA had low 100 times the activity (simultaneously referring to accompanying drawing 11 and 12) at least than t-PA.

In second neural degeneration model, with wild-type mice compared t-PA and DSPA impel that NMDA dependent form is neurodegenerative may effect.For this purpose, inject NMDA separately, perhaps with t-PA or DSPA combination injection NMDA (as the agonist of NMDA type glutamate receptor) to wild-type mice.This model can finally can cause neural degeneration and destroyed plasma proteins flows into because of blood brain barrier condition under detect the effect (Chen etc., 1999) of these protease.

The operation of this model makes the injection of NMDA cause the repeatability damage of mouse striaturn.The joint injection of t-PA and NMDA makes amount of damage increase at least 50%.On the contrary, the enhancing that enlarges of the damage that can not cause causing by NMDA with the common injection of DSPA α 1.Even in the presence of the plasma proteins in can being spread in the damage field that is caused by NMDA, DSPA can not cause neurodegenerative enhancing yet.Figure 16 (table 2) is seen in these results' summary.

First clinical test results shows that these results also are applicable to the treatment of people's syndrome similar to wind disease.Confirmed successfully to pour into back patient significantly improve (improving 8 some NIHSS or HIHSS value 0 to 1).Accompanying drawing 17 (table 3) has shown this situation.

In further test during the check intravenous administration, whether t-PA and DSPA can see through impaired blood brain barrier and increase histologic lesion in the brain.To this, NMDA stereotaxis (stereotaktisch) is expelled to mice is used for producing tissue injury at striatum, 6 or 24 hours intravenouss are used t-PA or DSPA in NMDA injection back.Compare with negative control, when injecting back 24 hours infusion t-PA at NMDA, laboratory animal demonstrates by NMDA injection injured tissues area has increased about 30%, and do not cause the increase of so a kind of tissue injury with DSPA, although can confirm that it enters into the zone (referring to accompanying drawing 18,19) of damaged tissues by a kind of antibody staining method.Inject back 6 hours with corresponding manner intravenous injection t-PA or DSPA at NMDA, do not determine the increase of damaged tissues area yet.This may owing to, when injection t-PA or DSPA, blood-brain barrier still has enough barrier actions.

These results have shown that DSPA mainly shows as a kind of inert protein enzyme in the central nervous system of mammal (also can be the people), opposite with t-PA, can't cause through KA or the inductive neurovirulent enhancing of NMDA.This neurovirulent shortage makes DSPA and the desired opposite thrombolytic agent that becomes a kind of suitable acute apoplexy of treatment.

The treatment potential of non-neurotoxic plasminogen activator

The neurotoxicity of DSPA and other non-neurotoxic plasminogen activator lacks feature (referring to above-mentioned) provides special advantage for the apoplexy treatment, and the longest after the use of these plasminogen activators-opposite with wild-type t-PA-the be not limited to apoplectic seizure is time period of 3 hours.On the contrary, treatment can begin later on-for example after 6 hours or slower, and can not cause the danger of excitatory toxicity reaction as t-PA.The clinical trial of carrying out with DSPA first proved after the apoplectic seizure in addition surpass 6 or 9 hours time range to patient's safe treatment.

This timeless treatment selection scheme particular importance of carrying out with the non-neurotoxic activator is not because its has the patient of the acute apoplexy symptom that safe enough ground treats for the first time can treat apoplectic seizure the time in time free from all inhibitionsly.This group patient is so far owing to disadvantageous risk assessment is excluded outside the thrombolytic therapy that adopts plasminogen activator.Therefore, eliminate thromboembolism reagent and be used for the taboo that apoplexy is treated.

Using of activator of plasminogen

Opposite with established Apoplexy treating medicine preparation rt-PA, also do not have to obtain to be familiar with reliably with the equal plasminogen activator treatment of non-neurotoxic apoplexy for possible method of application.

Therefore, the object of the present invention is to provide a kind of favourable method of application of the activator of plasminogen for these non-neurotoxics.

According to the present invention, intravenous is used plasminogen activator and is treated apoplexy, and under the situation that fibrin exists, its active increase is above 650 times.

Vein gives these non-neurotoxic plasminogen activator and is determined in clinical trial with the treatment apoplexy.Wherein, DSPA-in these patients, only causes some smaller side effect as this example-intravenous administration organizing fibrinolytic agent.

The result of these clinical researches is unexpected, because well-known, intravenous is used t-PA relevant with the grave danger of cerebral hemorrhage with other common fibrinolytic agent (referring to above-mentioned).

In order to reduce ICH, the strategy of newly-developed is no longer used these material intravenouss, but by conduit near the intra-arterial approach directly is applied to endovascular thrombosis.For this reason, the experiment of this reality has been used for the urokinase (PROCAT is as a kind of research relevant with pro-urokinase) that recombinant produces.Because this method of application significantly reduces accumulated dose, thus realized dose-dependent side effect minimizing-cerebral hemorrhage also can reduce like this.

Yet the significant advantage that these intra-arterial are used is destroyed by two shortcomings that may exist.At first, the prerequisite of this treatment is to need the time to carry out patient's preparation, but only this can not realize in 3 hours given in the treatment of apoplexy.People be sure of to reach a lower accumulated dose on the other hand, yet higher drug level arrives at terminal arteries with the part.Since under the apoplexy situation, the cause that the blood vessel endothelium barrier function weakens, the tissue around medicine also will arrive at high local concentrations.Will produce undesired side effect like this.

Than, if intravenous is used, drug concentrations will be diluted by venous blood flow.Therefore, if medicine has the potentiality of disorganization, as t-PA, that intra-arterial injection is exactly problematic (Forth, Henschler, Rummel, Starke: " Pharmakologie und Toxikologie ", sixth version, 1992,29 pages).

Yet this limitation-time range that intra-arterial is used complicate is narrow-and the side effect of damaged tissue-be not present in using at plasminogen activator according to the present invention.Therefore, this intra-arterial is used because its undeniable advantage (referring to above-mentioned) has formed the very useful a kind of method of application of non-neurotoxic plasminogen activator in principle.Therefore, clearly can follow the preferred method of application that this approach is sought these medicines.

Yet the applicant also selects to adopt controversial intravenous to use the plasminogen activator of non-neurotoxic, and being proved that it is surprising is favourable.

In addition, method of application of the present invention also is different from the conventional therapy convention, in order to have the pharmaceutical grade protein of high immunological competence, main use intramuscular injection or intravenous drip, its purpose is to reduce the danger (Mebs: " Gifttiere ", second edition, 2000) of anaphylactic shock.

The applied plasminogen activator of the present invention is opposite with self t-PA or come from the foreign protein (for example DSPA) of animal or the oneself protein of genetic modification, because its structural difference has new epitope.The anaphylaxis problem of following-especially when using high therapeutic dose, normally necessary such as using at intravenous-also be present in other equally contain for example fibrinolytic of streptokinase of foreign protein.

In a particularly advantageous embodiment, plasminogen activator used according to the invention can give by a kind of mode of injecting (vein fast injection) fast, also can be used as a kind of independent vein fast injection mode that contains whole therapeutic doses and gives.

In the scope of clinical research, confirm, even surprising low therapeutic dose intravenous is used also and can be produced favourable result.For example, between 90 μ g/kg and 230 μ g/kg, can reach preferred therapeutic outcome when dosage.When dosage can reach particularly preferred therapeutic outcome between 62.5 to 90 μ g/kg.In the middle of the patient who is investigated, the interval of apoplexy and drug administration is between 3 to 9 hours.Can determine the therapeutic effect (referring to accompanying drawing 20-29) of onset by suitable detection method.

DSPA and other non-neurotoxic plasminogen activator itself do not show neurotoxic side effects.Yet, being the tissue injury that restriction as far as possible has self glutamic acid to cause, they are treated in apoplexy with neuroprotective reagent use in conjunction is favourable.Can use competitiveness or noncompetitive to suppress the neuroprotective reagent of glutamate receptor.Effectively combination is to use for example known NMDA type, kainic acid type or Quisqualic Acid type glutamate receptor inhibitor, for example APV, APH, phencyclidine, MK-801, dextrorphan or ketamine.

Further, with the cation coupling be favourable, thereby because cation especially the cationic channel that can block by glutamate receptor regulation and control of Zn ion can reduce the neurotoxicity effect.

In further favourable embodiment, the non-neurotoxic plasminogen activator can with at least a further therapeutic agent or with the pharmaceutical acceptable carrier coupling.Especially advantageously with the coupling that helps avoid the therapeutic agent of tissue injury by activating cell, with the regeneration that impels tissue in damaged condition or preventing the Secondary cases apoplexy.Advantageously with antibiotic such as quinones, anticoagulant such as heparin or hirudin and with citicoline or acetysalicylic coupling.

With the coupling of at least a thrombin inhibitor also be favourable.Preferably, also can use thrombomodulin and thrombomodulin analog for example Solulin, Triabin or Pallidipin.Further the coupling with anti-inflammatory substance is favourable, because they influence leukocyte infiltration.

Below by discrete treatment examples general method of application of the present invention and preparation.

Embodiment

The comparative study of t-PA and DSPA:

A. method

1, animal

Wild-type mice (c57/Black 6) and t-PA deficient mice (t-PA-/-mice) (c57/Black6) (Carmeliet etc., 1994) by Dr.Peter Carmeliet, Leuven, Belgien provides.

2, cerebral tissue albumen extracting

Carry out the mensuration (Granelli Piperno and Reich, 1974) of proteolytic activity in the cerebral tissue behind t-PA or DSPA α 1 infusion with enzyme spectrum analysis.Hippocampus is carried out anesthetized mice behind seven days infusions, use PBS then, get brain through the heart perfusion.Downcut the hippocampus, change the Eppendorf pipe over to and be incubated at 0.5%NP-40 lysis buffer (0.5%NP-40,10mM Tris-HCl pH7.4,10mM NaCl, the 3mM MgCl that equal-volume (w/v) (about 30-50 μ m) does not conform to protease inhibitor ₂, 1mM EDTA) in, with manual type glass refiner homogenate brain extract and be placed on 30 minutes on ice.Centrifugal subsequently sample is obtained supernatant.Detect the protein content (Bio-Rad reagent) that exists.

3, the enzyme spectrum analysis of protease

According to the method usefulness enzyme spectrum analysis test sample of Granelli-Piperno and Reich (1974) or the proteolytic activity in the cerebral tissue extract.Under non-reduced condition, the sample that contains recombinant protein (100nmol at the most) or cerebral tissue extract (20 μ g) carried out 10% SDS-PAGE.Gel is taken out from dish, and washing is 2 hours in 1%Triton * 100, covers afterwards on the agarose gel that contains polymer fiber proteinogen and plasminogen (Granelli, Piperno and Reich, 1974).Under 37 ℃, glue incubation in wet pans can be discerned up to the Proteolytic enzyme band.

4, t-PA, DSPA infusion and the injection of kainic acid subsequently in Hippocampus

The kainic acid damage model is based on the research (1995) of Tsirka etc.To animal intraperitoneal (i.p.) injecting atropine (4mg/kg), afterwards by lumbar injection pentobarbital sodium (70mg/kg) anesthesia.Then mice is placed the three-dimensional positioning framework (stereotaktischen Rahmen) of brain, and contain 100 μ l PBS or recombined human t-PA (0.12mg/ml with one, 1.85 μ M) or the little of DSPA α 1 (1.85 μ M) ooze between the subcutaneous implantation scapula of pump (Alzet model 1007D, Alzet CA.USA).In order near center line, to import liquid, pump is connected with brain casing pipe needle through sterile tube, and is bregma-2.5mm from passing through coordinate, the skull perforate of middle side (midolateral) 0.5mm and back of the body veutro 1.6mm is inserted.The trocar is fixed on suitable site, and opens pump, poured into corresponding solution totally 7 days with the flow velocity of 0.5 μ l per hour.

Infusion protease two days later, anesthetized mice and being placed on again in the three-dimensional positioning framework of brain once more.Then, be dissolved in 1.5nmol kainic acid (KA) among the 0.3 μ l PBS to the one-sided injection of Hippocampus.Coordinate is: bregma-2.5mm, middle side 1.7mm and back of the body veutro 1.6mm.Excitotoxin (KA) was injected with 30 seconds.After kainic acid is handled, injection needle was kept 2 minutes at this coordinate again for preventing that liquid from flowing backwards.

5, brain research

After the KA injection 5 days, anesthetized animal and with 30ml PBS through the heart perfusion, then with the paraformaldehyde solution of 70ml 4% through the heart perfusion, with fixing 24 hours in the identical fixative, further incubation 24 hours in 30% sucrose subsequently.The crown brain sheet of cutting (40 μ m) on freezing microtome afterwards, then or with thionine (BDH, Australia) redye or as the following immunohistochemistry of carrying out detect and handle.

6, neuronic loss rate in the quantitative Hippocampus

As carrying out quantitative (Tsirka etc., 1995 as described in the forefathers to neurone loss in the Hippocampus CA1-CA3 subdomain; Tsirka etc., 1996).Prepare five serial section of dorsal part Hippocampus from all processed group, described section guarantees to have KA injection and damage field.Hippocampus subdomain (CA1-CA3) to these sections carries out photographing scanning mapping (Camera lucida Zeichnungen).Compare to determine the subdomain total length with the 1mm standard of mapping under the identical amplification several times.Measured and contained the length of organizing of organizing length and impassivity unit (not containing cell, no thionine dyeing) that vigor cone neurone (being normal morphology) is arranged.Represent the length of complete neuron of each Hippocampus subdomain and neurone loss to get brain sheet meansigma methods to these, and the basis of calculation is poor.

7, use or do not use the interior NMDA excitatory toxicity damage of striatum of t-PA or DSPA

Anesthesia wild-type mice (c57/Black 6) also is placed in the three-dimensional positioning framework of brain (referring to above-mentioned).Unite with the one-sided injection of 50nmol NMDA mice striatum revealed separately or with 46 μ M rt-PA or 46 μ M DSPA α 1.Inject t-PA and DSPA α 1 (concentration is 46 μ M) in contrast separately.The injection coordinate is: bregma-0.4mm, midolateral 2.0mm and back of the body veutro 2.5mm.Shifted solution (whole processed group cumulative volumes are 1 μ l) in 5 minutes with 0.2 μ l/ minute speed, after the injection syringe needle was kept 2 minutes in position so that liquid return drops to minimum again.Anesthetized mice after 24 hours pours into through heart with 30ml PBS, then pours into through heart with 70ml 4% paraformaldehyde solution, fixes 24 hours with identical fixative, afterwards further incubation 24 hours in 30% sucrose.Cutting brain sheet (40 μ m) on freezing microtome then, and place on the microscope slide of gelatin bag quilt.

8, to lesion volume behind the injection NMDA quantitatively

The method of describing with (2000) such as Callaway finish to the striatal damage volume quantitatively.10 continuous coronal sections of damage field are crossed in preparation.Method according to Callaway makes damage field visual, and by use a micro computer imaging device come the amount damage volume (MCID, Imaging Research Inc., Brock University, Ontario, Canada).

9, immunohistochemistry

Finish immunohistochemistry according to standard method.With crown section at 3%H ₂O ₂Soaked in/10% methanol 5 minutes, afterwards incubation 60 minutes in 5% normal goats serum.To cut into slices with anti--GFAP antibody (1: 1000; Dako, Carpinteria, CA, USA) overnight incubation to be detecting astrocyte, or with resisting-MAC-1 antibody (1: 1000; Serotec, Raleigh, NC, USA) overnight incubation is with the detection microglia or with resisting-DSPA polyclonal antibody (Schering AG, Berlin) overnight incubation.After the rinsing, section resisted with suitable biotinylation two (CA USA) is hatched together for Vector Laboratories, Burlingame.Then with 3,3 '-Diaminobebzidin/0.03%H ₂O ₂(CA USA) is hatched 60 minutes for Vector Laboratories, Burlingame with avidin/biotin one complex before the final dyeing.Then section is placed on the microscope slide of gelatin bag quilt, dry, dewater and seal up coverslip.

10, behind intravenous administration t-PA or the DSPA by NMDA injection the expansion of inductive tissue injury

In order to induce striatal tissue injury, with mice stereotactic injection NMDA.The NDMA injection was injected t-PA or DSPA (100 μ l by tail vein after six hours; 10mg/kg).Inject 100 μ l0.9%NaCl in contrast and pour into PBS subsequently.Again through after 24 hours with animal kill determination lesion volume.

Nearly the test group of 15 mices is injected with NDMA in test for the second time, and the NDMA injection is infusion t-PA or DSPA after 24 hours, and measures the expansion of corresponding tissue injury.Exist DSPA to use anti--DSPA antibody to crown section statining in the brain in order to prove according to standard method.

B. result

1, the t-PA of infusion or DSPA through t-PA-/-Hippocampus of mice distributes, and keeps its proteolytic activity

Initial test all kept their proteolytic activity to design in order to confirm DSPA and t-PA at 7 days between flush phase.Be this purpose, the t-PA of aliquot and DSPA (100nmol) were hatched 7 days in 37 ℃ and 30 ℃ of water-baths.For detecting proteolytic activity, 5 times of serial dilutions with probe under non-reduced condition carry out SDS-PAGE, come the evaluating protein hydrolysing activity with enzyme spectrum analysis.Compare with portion 7 days aliquot t-PA of freezing preservation and DSPA.As seeing, under 25 ℃ and 37 ℃, hatch and only cause faint DSPA or t-PA loss of activity during this period of time from accompanying drawing 1.

2, behind the infusion t-PA-/-can identify t-PA and DSPA activity in the Hippocampus extract of mice

The protease that at first must confirm institute's infusion appears at by in the infusion animal brain, and also still keeps their proteolytic activity in this compartment.For this reason with t-PA or DSPA infusion of t-PA-/-mice 7 days (referring to above-mentioned).Pour into mice with PBS through heart then, and the excision brain.Separate the Hippocampus homonymy and to lateral areas and cerebellum district (as negative control).(20 μ g) carries out SDS-PAGE with tissue sample, and makes enzyme spectrum analysis according to the explanation of method part.As seeing that t-PA and DSPA activity all have been detected with the lateral areas Hippocampus, but the lateral areas is also being detected some activity from accompanying drawing 2.This shows that the protease that is injected has not only kept their activity at brain, but also has diffused into hippocampus.In contrast, from the extract of cerebellum preparation, do not detect activity.

3, the immunohistochemistry evaluation of DSPA

For further confirming that DSPA has diffused into hippocampus really, behind the infusion DSPA to t-PA-/-the crown brain section of mice carries out immunohistochemical analysis.Detected DSPA-antigen at hippocampus, wherein the dyeing in infusion zone is the darkest.This result confirms that the DSPA that is injected is soluble and appears at Hippocampus really.

4, the DSPA infusion can not recover the interior neural degeneration that is relied on by kainic acid of body

T-PA-/-neurodegenerative resistance that mice performance relies on kainic acid.Yet the infusion of rt-PA in Hippocampus recovers the sensitivity to the damage of kainic acid-dependence fully.For judging whether DSPA can substitute t-PA in this model, with little ooze pump with t-PA or DSPA be infused to t-PA-/-the mice Hippocampus in.12 mices have all been tested for two groups.To animal injection kainic acid, be the phase of resting subsequently after 2 days.Kill animals after 5 days is got brain and is handled (referring to above-mentioned).In contrast, handle with KA before also to t-PA-/-mice carried out PBS injection (n=3).

Prepare crown brain section, and detect neuron with the Nissl staining.Shown in accompanying drawing 4a and 4b, with the dabbling t-PA-of PBS/-mice has resistance to KA.Yet infusion reorganization t-PA has recovered the sensitivity to the KA processing.On the contrary, the DSPA of hippocampus infusion same concentrations does not change the sensitivity of animal to KA.

Those results' quantitatively is based on the data available from 12 mices of each group.There are two to observe slight neural degeneration 12 mices of injecting DSPA.Its reason it be unclear that, may be irrelevant with the existence of DSPA.Synthetic data has been considered to observed this slight effect down in these two animals.All KA is handled responsive with all 12 mices that t-PA handles.These results show, when the t-PA of molar concentrations such as infusion or DSPA α 1, have only the t-PA administration to cause recovery to the inductive neurodegenerative sensitivity of KA.

5, the DSPA infusion does not cause microglial activation

The t-PA-that produces by the t-PA infusion/-reparation of mice KA sensitivity also causes microglial activation (Rogove etc., 1999).For determining with Mac-1 antibody the crown section of mice to be carried out immunohistochemical staining to determine activated microglia in the degree of t-PA-or DSPA infusion and the processing of KA subsequently back microglial activation.The showed increased that behind the t-PA infusion recovery of KA sensitivity is caused the Mac-1 positive cell.In mice, do not observe this phenomenon with the DSPA infusion.Therefore, KA handles the back does not cause microglia in the existence of DSPA activation.

6, the DSPA of mice hippocampus and t-PA titration

The t-PA concentration that is used for infusion is based on (1995) described concentration such as Tsirka (100 μ l0.12mg/ml[1.85 μ M]).Repeat the KA damage test with the t-PA (0.185 μ M) of low 10 times of amounts and the DSPA (18.5 μ M) of high 10 times of amounts.Lower t-PA concentration still can be recovered the sensitivity (n=3) to the KA processing.Especially DSPA that it should be noted that 10 times of KA processing back infusion concentration raisings only causes few neuronic loss.These data show that advantageously DSPA does not cause the enhancing to KA sensitivity.

7, t-PA and DSPA are to the neurodegenerative effect of NMDA-dependent form in the wild-type mice

Also detected t-PA and the DSPA effect in wild-type mice neural degeneration model.Injection t-PA causes the enhancing (Nicole etc., 2001) of the neural degeneration effect that caused by glutamic acid analog NMDA definitely in these mouse striaturns.

Injecting cumulative volume to wild-type mice striatum district in the presence of t-PA or DSPA (each 46 μ M) is the NMDA of 1 μ l.Get brain after 24 hours, and lesion size is carried out quantitatively (referring to above-mentioned) according to the method (Callaway etc., 2000) of Callaway.As seeing, inject NMDA separately and handle at all and produce repeatable damage in the mice (n=4) from accompanying drawing 7.When uniting when using t-PA and NMDA, that lesion size improves is about 50% (P＜0.01, n=4).Obviously opposite is that the common injection of the DSPA of NMDA and same concentrations is compared the increase that does not cause lesion size with independent injection NMDA.

Injection t-PA or DSPA do not cause detectable neural degeneration separately.To lack the result of (2001) such as effect and Nicole consistent for t-PA when individually dosed.These data show, the existence of DSPA even in the neural degeneration event procedure, also do not strengthen neural degeneration.

For confirming that the DSPA injection has diffused into hippocampus really, with DSPA antibody immunohistochemical analysis has been carried out in crown section, detection shows that DSPA has entered the striatum district really.

Test the activated dynamic analysis of carrying out of plasminogen with indirect chromogen

According to Madison E.L., Goldsmith E.J., Gerard R.D., Gething M.-J., Sambrook J.F. (1989) Nature 339 721-724; Madison E.L., Goldsmith E.J., Gerard R.D., Gething M.J., Sambrook J.F. and Bassel-Duby R.S. (1990) Proc.Natl.Acad.Sci U.S.A 87,3530-3533 and Madison E.L., GoldsmithE.J., Gething M.J., (Chme265,21423-21426 carries out the active indirect chromogen test to t-PA with substrate Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) for Sambrook J.F. and Gerard R.D. (1990) J.Biol..Having or do not having in the presence of the cofactor DESAFIB (American Diagnostica) and all test.DESAFIB is the SFM preparation that the high-purity human fibrinogen is cut acquisition with protease reptilase (Batroxobin).Thereby the Arg16-Gly17 binding site of reptilase cutting in Fibrinogen A α-chain discharges fibrinopeptide A.When peptide Gly-Pro-Arg-Pro did not exist, the monomeric des-AA-Fibrinogen of representative fibrin I that is produced was soluble.The concentration change scope of Lys-plasminogen is 0.0125-0.2 μ M when DESAFIB exists and is 0.9-16 μ M when no cofactor exists.

The indirect chromogen test of different stimulated thing infra.

Finish the indirect chromogen test of standard according to the above-mentioned publication of quoting.Used and contained 0.25-1ng enzyme, 0.2 μ M Lys-plasminogen and 0.62 μ M Spectrozym PL and cumulative volume is the probe of 100 μ l.Test is carried out existing under buffer, 25 μ g/ml DESAFIB, 100 μ g/ml Fibrinogen Bromine cyanide. segments (American Diagnostica) or 100 μ g/ml zests, the 13 amino acid peptide P368.In microtitration plate, analyze, and continue in " Molecular Devices Thermomax ", to detect in per 30 seconds in 1 hour an optical density (OD) in wavelength 405nm place.Reaction temperature is 37 ℃.

8, DSPA also can not cause the increase of neural tissue injury when intravenous is used

In the striatum of mice, tissue injury induces by injection NMDA, and next 6 or 24 hours intravenouss are used t-PA or DSPA after its injection.Compare with negative control, when NMDA-injected back 24 hours venoclysis t-PA, laboratory animal had been enlarged about 30% by the inductive damaged tissue of NMDA-injection; Comparatively speaking, DSPA can't cause the increase (referring to accompanying drawing 18) of such tissue injury.By proving that with anti--crown section of DSPA-antibody staining the DSPA that injects back 24 hours intravenous administrations at NMDA can enter into (referring to accompanying drawing 19) in the injured tissues zone.And inject back 6 hours correspondingly intravenous administration t-PA or DSPA at NMDA-, can not detect the increase of damaged tissue.Its reason may be still can have enough barrier actions at this moment blood brain barrier of using t-PA or DSPA.Therefore, DSPA can not demonstrate neurovirulent side effect yet when intravenous is used.

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Claims (12)

1. Vampire (Desmodus rotundus) but plasminogen activating factors be used for the treatment of purposes in the therapeutic agent that the intravenous of apoplexy uses in preparation, plasminogen activating factors is used with dosage from 62.5 to 230 μ g/kg.
2. 2. according to the purposes of the plasminogen activating factors of claim 1, wherein said plasminogen activating factors is DSPA α 1.
3. 3. according to the purposes of the plasminogen activating factors of claim 1 or 2, therapeutic dose from 62.5 to 90 μ g/kg wherein.
4. 4. according to the purposes of claim 1, wherein the tissue plasmin activity factor contains the aminoacid sequence shown in the with good grounds Seq.ID No.1.
5. 5. according to each the purposes of plasminogen activating factors of claim 1-4, it is characterized in that the drop infusion.
6. 6. according to each the purposes of plasminogen activating factors of claim 1-4, it is characterized in that injecting fast.
7. 7. according to the purposes of the plasminogen activating factors in the claim 6, it is characterized in that single injects fast.
8. 8. according to each the purposes of plasminogen activating factors of claim 1～4, be used for apoplectic seizure and treat people's syndrome similar to wind disease after 3 hours.
9. 9. according to each the purposes of plasminogen activating factors of claim 1～4, be used for treating people's syndrome similar to wind disease after 6 hours in apoplectic seizure.
10. 10. according to each the purposes of plasminogen activating factors of claim 1～4, be used for treating people's syndrome similar to wind disease after 9 hours in apoplectic seizure.
11. 11., be used for the treatment of uncertain stroke patient of apoplectic seizure time according to each the purposes of plasminogen activating factors of claim 1～4.
12. 12. according to each the purposes of plasminogen activating factors of claim 1～4, it is avoiding being used for the apoplexy treatment under the neurovirulent situation of wild-type t-PA.

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