MXPA05011761A - Intravenous injection of plasminogen non-neurotoxic activators for treating cerebral stroke - Google Patents

Abstract

The invention relates to the use of plasminogen non-neurotoxic activators factors, for example from Desmodus rotundus (DSPA) or genetically modified plasminogen activator factors of human origin for producing an injection therapeutic agent for therapeutically treating human cerebral stroke.

Description

INTRAVENOUS INJECTION OF NON-NEUTROTOX PLASMINOGEN ACTIVATORS FOR VASCULAR ACCIDENT TREATMENT CEREBRAL DESCRIPTION OF THE INVENTION The invention relates to the intravenous application of activators of non-neurotoxic plasminogens, particularly of genetically modified plasminogen activators and plasminogen activators of the Desmodus rotundus saliva (DSPA) for the treatment of stroke in humans. The treatment of cerebral vascular accident with these plasminogen activators is known from the international patent application PCT / EP02 / 12204 whose manifestation is referred to in full. Clinical and biochemical characteristic of cerebral vascular accident The term "cerebral vascular accident" encompasses different clinical pictures that resemble clinical symptoms. A first differentiation of these clinical pictures in so-called ischemic insults and hemorrhagic insults can be made on the basis of the respective pathogenesis. Ischemic insults (ischemia) are a decrease or interruption of blood flow in the brain due to - an insufficient supply of arterial blood. This is frequently caused by a thrombosis of a vessel with arteriosclerotic stenosis due to a thrombosis, but also by cardiac arterio-arterial emboli. The hemorrhagic insults, in contrast, are due, among other causes, to a perforation of the arteries, which feed the brain, due to arterial hypertension. Of all the brain insults, only approximately 20% is caused by this form of bleeding, so that cerebral vascular accidents caused by thrombosis have by far the greatest importance. Ischemia of neuronal tissue is accompanied in comparison with other ischemias of tissue - particularly important of the necrosis of affected cells. The higher incidence of necrosis can be explained, according to more recent understanding, due to the phenomenon of so-called excitotoxicity, which is a complex cascade of a large number of reactive stages. This is initiated, according to this, because ischemic neurons, suffering from low oxygen deficiency, rapidly lose ATP and depolarize. This causes an increased postsynaptic delivery of the neurotransmitter glutamate, which in turn activates neuronal glutamate receptors that are located in the membrane and that regulate the cation channels. As a result of the increased emission of glutamate, glutamate receptors are, however, overactivated. The glutamate receptors, in turn, regulate cations channels dependent on electrical charge, which open when glutamate binds to its receptors. This initiates an entry of Na + and Ca2 + into the cell, which produces a massive disruption of Ca2 + -dependent cellular metabolism, including energy metabolism. Responsible for the cell necrosis that follows could be particularly catabolic enzymes that depend on the activation of Ca2 + (Lee, Jin-Mo et al., "The changing landscape of ischemic brain injury mechanisms"; Dennis W. Zhol "Glutamat neurotoxicity and deseases of the nervous system "). Even though the mechanism of neurotoxicity mediated by the glutamate mechanism is still not understood in detail, there seems to be unanimity, however, that this phenomenon contributes considerably to neuronal cell necrosis after cerebral ischemia (Jin-Mo Lee et al.) Cerebral vascular accident therapy In the therapy of acute cerebral ischemia, besides the assurance of vital functions and the stabilization of physiological parameters, the effort is concentrates primarily on the re-opening of the closed vessel. This objective is pursued through different approaches. The purely mechanical reopening has not yet achieved satisfactory results, such as PTCA in a cardiac infarction. Only with successful fibrinolysis could it be shown, until now, a sufficient improvement of the patient's condition. Physiological fibrinolysis is based on the proteolytic activity of the serine protease plasmin that is generated by catalysis (activation) of a previous inactive stage of plasminogen. The natural activation of plasminogen is carried out by the endogenous plasminogen activators u-PA (urokinase-type plasminogen activator) and t-PA (tissue plasminogen activator). The latter form - in contrast to u-PA - together with fibrin and plasminogen, a so-called activating complex. The catalytic activity of t-PA depends, accordingly, on fibrin and suffers in the presence of fibrin an increase of approximately 550 times. In addition to fibrin, fibrinogen can also stimulate t-PA mediated catalysis from plasmin to plasminogen - but in a substantially smaller volume. The activity of t-PA in the presence of fibrinogen suffers an increase of only 25 times. The cleavage products of fibrin (fibrin degradation products (FDP)) also stimulate the activity of t-PA. Known proposals for therapy a) Streptokinasis Previous proposals for thrombolytic treatment of acute cerebral vascular accident began as early as the 1950s. But only since 1995 have large-scale clinical trials been carried out with streptokinase, a fibrinolytic obtained from beta-streptococci hemolysers. Streptokinase forms a complex together with plasminogen that is capable of transforming other plasminogen molecules into plasmin. Streptokinase therapy, however, is associated with essential disadvantages, since streptokinase, as a bacterial protease, can induce allergic reactions in the body. A so-called streptokinase resistance can also occur due to a previous streptococcal infection with the corresponding formation of antibodies, which makes therapy difficult. In addition, clinical studies in Europe. { Multicener Acute Stroke Trial of Europe (MAST-E), Multicenter Acute Stroke Trial of Italy (MAST-I)) and Australia. { Australian Streptokinase Trial (AST)) suggested an increased risk of mortality and the considerable risk of intracerebral bleeding (intracerebral hemorrhage, IHC) after treatment of patients with streptokinase, which was even necessary finish the studies prematurely. b) urokinase In an alternative therapy proposal, urokinase - also a "classic" fibrinolytic - is applied, which, unlike streptokinase, does not have antigenic properties, since it is an endogenous enzyme, present in many tissues. It is a plasminogen activator that is independent of cofactors. Urokinase is produced in kidney cell cultures. c) recombinant t-PA (rt-PA) There are extensive experiences in the area of therapeutic thrombolysis with the tissue-type plasminogen activator - the so-called rt-PA - (see EP 0 093 619, patent publication 4 766 075 ), which is produced in recombinant hamster cells. A series of clinical studies with t-PA - in addition to the main indication of acute myocardial infarction - was performed in the 1990s worldwide, with partially unfulfilled and contradictory results. First, patients were treated, in the so-called European Acute Stroke Trial (European trial of acute cerebral vascular accident, ECASS, for its acronym in English) intravenously with rt-PA within a period of six hours after the start of the symptoms of the stroke and was analyzed after 90 days the death rate as well as the Barthel index as a measure for incapacitation or ability to lead an independent life of patients. There was no significant improvement in the ability to live, but a significant - although not significant - increase in mortality. This allowed the conclusion that a thrombolytic treatment with rt-PA, immediately after the start of the stroke, of patients selected in a focused manner on an individual basis according to their respective clinical history could be possibly advantageous. Due to the increased risk, which was observed, of an intracerebral hemorrhage (ICH) after only a few hours after the onset of the stroke, however, a generalized application of rt-PA within the investigated time of six hours afterwards was not advisable. of the beginning of the cerebral vascular accident (as Le An- dowski C and Wiliam Barsan, 2001: Treatment of Acute Stroke; in: Annals of Emergency Medicine 37: 2; p.202 ss). The thrombolytic treatment of the stroke was later also the object of a clinical study by the National Institute of Neuropathic Disorder and Stroke (so-called NINDS rtPA Stroke Trial) in the United States. This focused, however, on the investigation of the effect of a treatment with intravenous rt-PA, within a period of only three hours after the onset of symptoms, on the patient's state of health. Although the authors determined a higher risk of ICH, treatment with rt-PA is still recommended within this limited period of three hours, thanks to the positive effects, also detected by the study, on the independent living capacity of the patients . In two other studies (ECASS II Trial; Alteplase Thrombolysis for Acute Noninterventional The in Ischemic Stroke (ATLANTIS)) was analyzed if this positive conclusion about the effect of rt-PA treatment within three hours after the start of the stroke could also be confirmed for a treatment in the six-month period. hours. This question, however, could not be answered in a positive way, because for one treatment in this period, in addition to the increased risk of ICH, it was not possible to observe an improvement in clinical symptoms or a decrease in mortality. According to a summary, first published in 1997 and updated in March 2001, of all the "stroke trials", all thrombolytic treatments (urokinase, streptokinase, rt-PA or recombinant urokinase) produced, particularly due to ICH, a significantly greater mortality within the first ten days after the stroke, while in a treatment within 6 hours the total number of dead or disabled patients was reduced. Therefore, a broad application of thrombolytics for a stroke treatment is not recommended. These results have already led other authors to the rather sarcastic assertion that stroke patients are now facing the option of dying or surviving as disabled (SCRIP 1997: 2265, 26). However, rt-PA therapy now represents the only treatment method authorized by the Food and Drug Administration (FDA) in the United States for acute cerebral ischemia. This is limited, however, to an application of rt-PA within three hours after the onset of stroke. A recombinant plasminogen activator is currently offered on the market under the name Al teplase, as well as an analogous preparation Reteplase. The latter is a fragment of therapeutically active t-PA with a low half-period. The therapeutic dose for Alteplase is approximately 70-100 mg, for Reteplase twice 560 mg, with Alteplase being administered essentially by drop infusion, Reteplase, in contrast, by bolus injection, twice with a interval of approximately 30 min. (Mutscher: "Arzneimittel irkungen", 8th edition, p.512-513). Side effects of t-PA Neurotoxicity and excitotoxicity The authorization of rt-PA was given in 1996.

Immediately before, namely, in 1995, first indications were known for the cause of the respectively excitotoxic neurotoxic effects of t-PA that provide an approach to an explanation for the dramatic effects of t-PA in the treatment of vascular accident. brain after the three-hour period for treatment. According to this, the microglia cells and neuronal cells of the hippocampus produce endogenous t-PA that participates in the glutamate-mediated excitotoxicity. This conclusion was produced by a comparison of mice with t-PA deficiency and wild-type mice that respectively received injections of glutamate agonists in the hippocampus. Mice with deficiency of t-PA showed a clearly superior resistance against externally applied glutamate (intrathecal) (Tsirka SE et al., Nature, Vol. 377, 1995, "Exzitoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator" ). These results were confirmed in 1998, when Wang et al. were able to check in mice with t-PA deficiency by intravenous injection of t-PA almost a duplication of necrotic neural tissue. This negative effect of external t-PA was found in wild type mice, in contrast, in only approximately 33% (Wang et al., 1998, Nature, "Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischemia in wild type and t-PA deficient mice "). Subsequent results on the stimulation of excitotoxicity by t-PA were published by Nicole et al. at the beginning of the year 2001 (Nicole 0; Docagne F Ali C; Margaill 1; Carmeliet P; MacKenzie ET; Vivien D und Buisson A, 2001: The proteolytic activity of tissue-plasminogen activator enhancers NMDA receptor-mediated signaling; in: Nat Med 7.59-64). They were able to verify that t-PA emitted by depolarized cortical neurons interacts with the so-called NR1 subunit of the NMDA-type glutamate receptor and separates them. This modification produces an increase in the activity of the receptor, which in turn is responsible for a greater tissue injury after the application of the glutamate agonist NMDA through an induced excitotoxicity. t-PA acts in a neurotoxic manner, therefore, by activating the NMDA-type glutamate receptor. As the blood-brain barrier collapses in a stroke in the affected tissue region, soluble plasma proteins, such as fibrinogens, as well as t-PA applied in the course of therapy, make contact with the neuronal tissue, where t-PA stimulated by fibrinogen develops its excitotoxic activity through the activation of the glutamate receptor. That, notwithstanding these indications about the neurotoxic side effects of t-PA and notwithstanding the proven increase in mortality due to t-PA, the pharmacological legal authorization was carried out by the FDA, probably explained only by the lack of risk-free and effective alternatives - and a very pragmatic cost-utility analysis. There is, however, still a requirement for safe therapies, and in the development of new thrombolytics - to the extent that it will not be necessary to completely distance oneself from thrombolysis - the problem of neurotoxicity must be taken into account (thus, for example, Wang et al, oc: Le andowski and Barsan, oc). Therefore, no further investigations of known thrombolytics have been carried out, including DSPA (Desmodus rotundus Plasminogen Activator, for its acronym in English) for the development of a new therapeutic agent for stroke, although in principle any thrombolytic agent could be appropriate and, for example, in the case of DSPA, early publications point to its possible suitability for this indicative field (Medan P; Tatlisumak T; Takano K; Carano RAD; Hadley SJ; Fisher M: Thrombolysis with recombinant Desmodus saliva plasminogen activator (rDSPA) in a rat embolic stroke model; in: Cerebrovasc Dis 1996: 6; 175-194 (4th International Symposium on Thrombolic Therapy in Acute Ishemic Stroke)). Just DSPA is, however, a plasminogen activator with a high homology (similarity) to t-PA, so that - concurrently with the disappointment about the neurotoxic side effects of t-PA - no further hopes were placed on DSPA . Alternative proposals for therapies The exploration of alternative proposals for therapy is currently focused, for example, on anticoagulants such as heparin, aspirin or ancrod, the active substance obtained from the venom of the Malayan viper. Two clinical studies, carried out, among others, with a focus also on the effect of heparin. { International Stroke Trial (IST) and Trial of ORG 10112 in Acute Stroke Treatment (TOAST)), however, give no indication of a significant improvement in mortality and an inhibition of a new stroke. Another novel method of treatment is part of either the thrombus or the liquidification of blood or anticoagulation, but attempts to increase the vitality of the cells injured by interruption of blood flow (WO 01/51613 Al and WO 01/51614 Al). For this purpose antibiotics of the group of quinones, aminoglycosides or chloramphenicol are applied. For a similar reason it is also proposed to start immediately after the stroke with the administration of citicoline, which is separated in the body in cytidine and choline. These cleavage products are components of the neuronal cell membrane and can thus support the regeneration of injured tissue (US patent publication 5 827 832). More recent efforts in the search for safe methods are based on new discoveries in the sense that part of the fatal consequences of the stroke should be attributed only to the interruption of blood flow, but to excitotoxicity and neurotoxicity with participation of the overexcited receptor. of glutamate, which in turn is increased particularly by t-PA (see above). A proposal to reduce this excitotoxicity is, therefore, the application of so-called neuroprotectors. These can be used alone or in combination with fibrinolytics, to minimize the neurotoxic effect of these. They can produce in this or directly, for example as antagonists of the glutamate receptor, or indirectly by blocking the sodium channels and calcium-dependent loading, a reduction in excitotoxicity (Jin-Mo Lee et al., o.c). A competitive inhibition (antagonization) of the NMDA-type glutamate receptor is possible, for example, with 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate (APH). Non-competitive inhibition can be achieved, for example, with substances that bind to the phencyclidine side of the channels, such as phencyclidine, MK-801, dextrorphan or ketamine. Treatments with neuroprotective agents, however, have not yet had the desirable success, possibly because they would have to be combined with thrombolytic agents to develop their protective activity. This is true in the same way also of the other active substances (see Fig. 10). Even by a combination of t-PA and a neuroprotective factor, however, success is eventually achieved only in the sense of a limitation of the lesion. The disadvantages of the neurotoxicity of the fibrinolytic agent itself, however, can not be avoided with it. Non-neurotoxic plasminogen activators International patent application PCT / EP02 / 12204, whose manifestation is referred to in all its contents, is known activators of plasminogen for the treatment of cerebral vascular accident whose highly selective enzymatic activity is magnified by fibrin by a multiple, namely, for more than 650 times. The characteristic and the application of these plasminogen activators are based on the discovery that the neurotoxicity of the tissue plasminogen activator (t-PA) must be attributed to the fact that as a consequence of the destruction of tissue caused due to the stroke, it is damaged or destroyed In the brain, the blood-brain barrier and the fibrinogen circulating in the blood can penetrate, therefore, in the brain's neuronal tissue. There, it activates t-PA which - indirectly by activating the glutamate receptor or by activating plasminogen - causes additional tissue damage (v.a.). In order to avoid this effect, a plasminogen activator is used that shows a higher selectivity of fibrin and - by inverse conclusion - a lower activation capacity by fibrinogen. From the above it is concluded that these plasminogen activators are not activated by passing fibrinogen from the blood to the neuronal tissue as a consequence of the damaged blood-brain barrier, or in a clearly lower degree compared to t-PA, since its activating fibrin because of its size and insolubility can not penetrate neuronal tissue. These plasminogen activators, therefore, are not neurotoxic. a) genetically modified plasminogen activators For this purpose, for example, non-toxic plasminogen activators having at least one element of a so-called zymogen triad are used. A comparable triad is known from the catalytic center of serine proteases of the chymotrypsin family which consists of the three interactive amino acids aspartate 194, histidine 40 and serin 32. This triad, however, does not exist in the t-PA which belongs to the family of serine protease type chymotrypsin. But it is known that oriented mutagenesis of native t-PA to introduce at least one of these amino acids at convenient positions leads to a decrease in the activity of the proenzyme (t-PA of a chain) and an increase in the activity of the mature enzyme (two-chain t-PA) in the presence of fibrin. Therefore, the introduction of at least one amino acid of the triad - of amino acids that take the corresponding function in the triad - leads to an increase in the zymogenic characteristic of t-PA (ratio of the activity of the mature enzyme to the activity of proenzyme) with clear increase in fibrin specificity, through reciprocal conformational effects between the introduced amino acid radicals and / or the amino acid residues of the wild-type sequence. It is known, for example, that the mutagenesis of native t-PA to replace Phe305 with His (F305H), as well as Ala292 with Ser (A292S), leads to a twenty-fold increase in the zymogen characteristic, whereas only with the variant F305H an increase of 5 times more is already produced (EL Madison, Kobe A, Gething MJ, Sambrook JF, Goldsmith EJ 1993: Converting Tissue Plasminogen Activator to Zymogen: A regulatory Triad of Asp-His-Ser; Science: 262, 419-421). These mutants of t-PA show in the presence of fibrin an activity increase of 30,000 times more (F305) respectively 130,000 times more (F305H, A292S). Mutant abos additionally have a substitution of Arg275 with R275E, to prevent the separation of t-PA from a chain in the form of two chains at the Aug275-lle276 separation point by plasmin. Only this mutant R275E increases the fibrin specificity of t-PA 6,900 times more (1 Tachias, Madison EL 1995: Variants of Tissue-type Plasminogen Activator Which Display Substantially Enhanced Stimulation by Fibrin, in: Journal of Biological Chemistry 270, 31: 18319-18322). The positions 305 and 292 of the t-PA are homologous to the His40 and Ser32 positions of the known triad of chymotryptic serine proteases. By replacing corresponding with histidine respectively serin, these amino acids can interact with aspartate 477 of t-PA, so that the known triad can functionally form the t-PA mutant. (Madison et al 1993). These t-PA mutants can be used for the treatment of cerebral vascular accident, since due to their increased fibrin specificity they do not have neurotoxicity, or - in comparison with the wild-type t-PA - only a clearly diminished neurotoxicity. For demonstration purposes of the t-PA F305H mutants; F305H, A292S alone or in combination with R275E, reference is made to the publications of Madison et al. 1993 as well as Tachias and Madison 1995. The increase of the fibrin specificity of plasminogen activators is possible as an alternative by point mutation of Aspl94 (or of an aspartate in a homologous position). Plasminogen activators belong to the group of serine proteases of the chymotrypsin family and have coincidentally the conserved amino acid Aspl94, which is responsible for the stability of the catalytically active conformation of the mature protease. It is known that Aspl94 interacts in the zymogens of serine proteases with His40. By the separation that activates the zymogen these interactions become impossible, and the side chain of the Aspl94 turns approximately 170 °, to then form a new salt bridge with the Ilelß. This salt bridge participates in the stability of the oxyanion bag of the catalytic triad of the mature serine protease. It is also present in the t-PA. A point mutation of Aspl94 first makes it impossible to form the stability of the catalytic conformation of serine proteases respectively. However, mutant plasminogen activators show in the presence of their cofactor fibrin - just also in comparison with the mature form of the wild type - an increase in their notorious activity, which can be explained only because the reciprocal effect with fibrin allows a catalytic activity What facilitates conformational changes (L Strandberg, Madison EL, 1995: Variants of Tissue-type Plasminogen Activator with Substantially Enhanced Response and Selectivity to Ards Fibrin Co-factors, in: Journal of Biological Chemistry 270, 40: 2344-23449). Therefore, Aspl94 mutants of plasminogen activators show a significant increase in activity in the presence of fibrin, which allows their use as a non-neurotoxic plasminogen activator. A preferred example for such non-toxic plasminogen activator represents t-PA, whose Aspl94 is substituted with glutamate (D194E) respectively asparagine (D194N). With this, the activity of t-PA by the factor 1-2000 is reduced in the absence of fibrin, while in the presence of fibrin an increase in activity can be achieved by the factor 498,000 to 1,050,000. These mutants may also contain a substitution of Argl5 with R15E, which prevents the separation of t-PA from one strand in the t-PA of two strands in the Argl5-Ilel6 peptide bond by plasmin. With only this mutation the activation of t-PA by fibrin is increased by the factor of 12,000. For purposes of manifestation of mutations at positions 194 as well as 15 of t-PA full reference is made to the publication of Strandberg and Madison 1995. An increase in fibrin dependence of plasminogen activators can also be achieved by introduction of point mutations in the so-called "autolysis loop". This amino acid section is known from trypsin; it is present, however, also as a homologous section in serine proteases and is characterized in particular by three hydrophobic amino acids (Leu, Pro and Phe). The link of autolysis in plasminogen activators is responsible for the interaction with plasminogen. Point mutations in this area can have as a consequence that the reciprocal effects of protein to protein between plasminogen and plasminogen activator can no longer be formed functionally. These mutations, however, are functionally relevant only in the absence of fibrin. In the presence of fibrin, on the contrary, they are responsible for an increase in the activity of plasminogen activators (K Song-Hua, Tachias K, Laniba D, Bode W, Madison EL, 1997: Identification of a Hydrophobic exocyte on Tissue Type Plasminogen Activator That Modulates Specificity for Plasminogen, in: Journal of Biological Chemistry 272; 3, 181-1816). In a preferred form a t-PA with point mutation at positions 420-423 is employed. If these radicals are replaced with targeted point mutations, the fibrin dependence of t-PA is increased by a factor of up to 61,000 (K Song-Hua et al.). Song-Hua et al. investigated the point mutations L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E. The publication is included for the inventive use by reference to complete content. In another advantageous embodiment, the tissue plasmin activator modified with an amino acid sequence according to SEQ is used. ID No. 1 (Fig. 13). This modified t-PA differs from the wild type t-PA by the exchange of the hydrophobic amino acid at positions 420-423 in the autolysis loop, which are being occupied as follows: His420, Asp421, Ala422 and Dys423. This t-PA preferably has a phenylalanine at position 194. In addition, position 275 can be occupied with a glutamate. Advantageously, position 194 is occupied with phenylalanine. Additionally, a modified urokinase can be introduced inventively. This inventive urokinase can have the amino acid in accordance with SEQ. ID No. 2 (Fig. 14), in which hydrophobic amino acids of the autolysis loop are substituted with Val420, Thr421, Asp422 and Ser423. Advantageously, this urokinase has an Ile275 as well as a Glul94. This mutant shows, in comparison with the wild-type urokinase, a fibrin specificity increased 500 times more. Both mutants - both urokinase and t-PA - were analyzed in semi-quantitative assays and resulted in increased fibrin specificity compared to wild-type t-PA. b) Desmodus rotundus plasminogen activator (DSPA) A strong increase in activity in the presence of fibrin - namely, a 100,000-fold increase - also shows the plasminogen activator (DSPA) of vampire saliva [Desmodus rotundus] ), which can therefore be used preferentially. Under the term DSPA, four different proteases are summarized here, they fulfill a basic requirement of the vampire, namely, the duration of prolonged bleeding from wounds caused by these animals (Cartwright, 1974). These four proteases (DSPAcii, DSPAa2, DSPAß, DSPA?) Coincidentally have a great similarity (homology) with human t-PA. They also have similar or coincidental physiological activities, which justify summarizing them under the general term DSPA. DSPA is the subject of EP 0 352 119 Al, as well as US 6 008 019 and 5 830 849 patents, which are presently included by reference for demonstration purposes. DSPActi is the protease of this group that has been better investigated to date. It has a homology of 72% in its amino acid sequence with respect to amino acid sequences of known human t-PA (Kratzschmar et al., 1991). However, between t-PA and DSPA there are two important differences. Firstly, DSPA, as an individual chain, represents a totally active molecule against peptide substrates, which is not transformed - like t-PA - into a two-chain form (Gardell et al, 1989; Kratzschmar et al., 1991) . Second, the catalytic activity of the DSPA shows an almost complete dependence on fibrin (Gardell et al., 1989; Bringmann et al., 1995; Toschí et al., 1998). Thus, for example, increases the activity of DSPAai in the presence of fibrin 100,000 times more, while the activity of t-PA only increases approximately 550 times more. The DSPA activity is, however, induced, essentially less strongly by fibrinogen; it only experiences an increase of 7 to 9 times more (Bringmann et al., 1995). DSPA is, therefore, much more dependent on fibrin and fibrin-specific than wild type t-PA, which is activated only 550 times more by fibrin. Thanks to its fibrinolytic properties and its great resemblance to t-PA, DSPA represents an interesting candidate for the development of a thrombolytic agent. Due to the participation of t-PA in glutamate-dependent neurotoxicity, there was no justifiable hope of successfully using a plasminogen activator related to t-PA for the treatment of acute stroke. Now surprisingly, despite its great resemblance (homology) to t-PA and despite a broad coincidence of its physiological activity with t-PA, in contrast to this, DSPA has no neurotoxic effects. This note is immediately accompanied by the recognition that DSPA can then be used as a thrombolytic agent for stroke therapy, without this being related to an additional risk of neuronal tissue damage. This means in particular that DSPA can still be used later than three hours after the onset of stroke symptoms. Experimental verification of neurotoxicity lacking in DSPA The discoveries about the lack of neurotoxicity of these plasminogen activators are based essentially on in vivo comparative research of the neurodegenerative activity of t-PA on the one hand, and of the DSPA on the other, with the help of the so-called "acid model" "Cainic" as well as a model to investigate the NMDA-induced injury of striatum. The model of cainic acid (also model of injury of cainic acid) is based on the neurotoxic cascade of glutamate is stimulated by the application of external cainic acid (KA) as a glutamate receptor agonist of the cainic acid type (KA-Typ) as well as the NMDA and AMPA glutamate receptors. By using a population of mice with t-PA deficiency as a test model it could be shown by this, that the sensitivity of the test animals against caynic acid reached only by additional administration of external t-PA the level of the mice of wild type. In contrast, an infusion of an equimolar concentration of DSPA under the same test conditions could not reestablish the sensitivity to kainic acid (KA). The activity of t-PA could not be replaced, therefore, by DSPA. A summary of these results is shown in Fig. 15 (table 1) - Quantitative investigations in this model show that even a 10-fold increase in the concentration of DSPA did not restore the sensitivity of mice with T-PA deficiency against KA treatment, while at a ten-fold lower concentration of t -PA caused tissue injuries. It follows that DSPA has activity at least 100 times lower in neurodegenerative activity after treatment with KA than t-PA (see also figures 11 and 12). In a second neurodegeneration model, the potential effects of t-PA as well as DSPA on the increase of neurodegenerative activity against NMDA in wild-type mice were compared. For this, wild-type NMDA mice (as NMDA-type glutamate receptor agonist) were injected alone or in combination with t-PA or DSPA. This model allows the analysis of the effect of these proteases under conditions, where neurodegeneration occurs in all forms and there is an entry of plasma proteins due to the rupture of the blood-brain barrier (Chen et al., 1999). In work with this model, NMDA injection caused reproducible lesions in the striatum of mice. He The volume of these lesions was increased by a combined injection of t-PA and NMDA by at least 50%. On the contrary, an injection combined with DSPActi did not lead to any increase in the increase of the lesion caused by the NMDA injection. Even in the presence of plasma proteins that could spread to the lesion area as a result of neurodegeneration based on NMDA, DSPA did not lead to any increase in neurodegeneration. A summary of these results is shown in Fig. 16 (table 2). First results of clinical studies show that the results can also be transferred to the treatment of stroke in humans. In patients with successful perfusion, for example, clear improvements can be seen (8 points NIHSS or NIHSS score 0 - 1 improvements). This is clearly shown in Fig 17 (table 3). In another trial it was verified whether t-PA and DSPA can pass the injured blood-brain barrier in the case of intravenous application and increase tissue injury in the brain. For this, mice were injected stereotactically with NMDA to produce tissue lesions in the striatum and applied 6, respectively 24 hours after the injection of NMDA t-PA or DSPA in intravenous form. In comparison with a negative control, the test animals showed in the case of an infusion of t-PA 24 hours after the NMDA injection an approximately 30% increase in the tissue region injured by the NMDA injection, whereas DSPA did not cause such an increase in tissue lesions, even though its penetration into the injured tissue region checked by antibody staining (see Fig. 18, 19). In a corresponding intravenous application of t-PA or DSPA 6 h after the NMDA injection, an increase in the injured tissue region could not yet be detected. This is possibly due to the fact that the blood-brain barrier possessed at this time of the application of t-PA respectively DSPA still sufficient barrier function. These results demonstrate that DSPA in the central nervous system of a mammal - therefore also of the human - represents an essentially inert protease and - in contrast to t-PA - does not cause an increase in neurotoxicity induced with KA or NMDA. This lack of neurotoxicity converts DSPA, contrary to expectations, into an appropriate thrombolytic for the treatment of an acute stroke. Therapeutic potential of non-neurotoxic plasminogen activators Thanks to the absence of neurotoxicity of DSPA, as well as of the other non-neurotoxic plasminogen activators (see above), it is advantageous Particularly for the treatment of a cerebral vascular accident the fact that the use of these plasminogen activators - unlike the wild-type t-PA - is not limited to the narrow temporal period of up to three hours after the onset of stroke. Rather, the treatment can also be performed at a later time - thus, for example, without more also after six hours or even later, without the risk - as in the case of t-PA - of an increase in excitotoxicity I inhibited it. First clinical analyzes with the DSPA prove a patient-friendly treatment even in a period of more than six, respectively, nine hours after the onset of symptoms. This treatment option without time limit with non-neurotoxic plasminogen activators is just as important because this makes it possible for the first time to treat patients of an acute cerebral vascular accident without problem, where it can not be ascertained with sufficient certainty temporarily. beginning of the cerebral vascular accident. This group of patients has been excluded, until now, for reasons of caution and risk estimation, of thrombolysis with plasminogen activators. This eliminates an essential contraindication for the authorized application of a thrombotic in a stroke. Application of plasminogen activators Contrary to the therapeutic agent rt-PA of stroke already established, there is no safe knowledge for the treatment of cerebral vascular accidents with non-neurotoxic plasminogen activators about a possible form of administration. The object of the invention is, therefore, the provision of an advantageous administration form for these non-neurotoxic plasminogen activators. Inventively apply plasminogen activators whose activity in the presence of fibrin is increased by more than 650 times, in an intravenous form for the treatment of a stroke. The intravenous administration of these non-neurotoxic plasminogen activators for the treatment of stroke has already been confirmed in clinical investigations in which DSPA - as an example of this group of fibrinolytics - was administered to patients intravenously and caused only a few side effects . These results of the clinical studies came as not expected, since it was known that the intravenous application of t-PA and other usual fibrinolytics is associated with a high risk of cerebral hemorrhage (see above). For the reduction of intra-cerebral hemorrhages, strategies have recently been developed for not applying these substances intravenously, but intra-arterially through a probe directly near the intravascular thrombus. There are already experiences about this with urokinase of recombinant production (PORCAT as a study with Pro-urokinase). As this form of administration allows a clear reduction of the total dose, a reduction of the dose-dependent side effects is achieved - that is, also of cerebral hemorrhages. The considerable advantages of the intra-arterial application, however, are confronted with two possible disadvantages. On the one hand it presupposes a prolonged patient preparation that is not possible for the treatment of stroke in the period of only 3 hours available. On the other hand, it is true that a lower total dose can be achieved. Locally, however, a higher concentration of the therapeutic agent reaches the arterial end vessels. Due to the injured barrier function of the vascular endothelium as a result of a cerebral vascular accident, therefore, the therapeutic agent arrives, also at locally high concentrations, the surrounding tissue. There it can, then, cause effects undesirable side effects In intravenous application, on the contrary, the concentration of the therapeutic agent is diluted by the flow of venous blood. Intra-arterial injection is, therefore, problematic in the case of therapeutic agents that injure tissue, such as t-PA (Forth, Henschler, Rummel, Starke: "Pharmakologie und Toxikologie", 6th edition, 1992, p. ). The limitation that hinders an intra-arterial application, due to the narrow period of time and the side effects harmful to the tissue, do not exist, however, in the case of the plasminogen activators used inventively. The intra-arterial application represents, thanks to its indisputable advantages (see above) in principle a promising administration form for non-neurotoxic plasminogen activators. It would have been evident, therefore, to take this route in the search for an advantageous form of administration for these therapeutic agents. However, the applicant has also decided for the non-neurotoxic plasminogen activators in favor of the rather problematic intravenous application which was, however, surprisingly advantageous. With the form of inventive management, it also deviates from a usual therapeutic practice in which proteins with a higher immunological potential are applied usually in intramuscular form or by intravenous drip injection. In this way it is possible to reduce the risk of an allergic shock (Mebs: "Gifttiere", 2nd edition, 2000). Inventively applied plasminogen activators are - in contrast to endogenous t-PA - either foreign proteins of animal origin (see, for example, DSPA) or genetically modified endogenous proteins which, due to their structural changes, have novel epitopes. The problem, associated with this, of allergic reactions - particularly in the case of administration of high therapeutic doses, as is usually required in the case of intravenous application - must also be addressed in other fibrinolytics consisting of exogenous proteins such as, for example, , streptokinase. In a particularly advantageous embodiment, the inventively applied plasminogen activators are administered by a bolus injection (rapid intravenous injection) which can also be administered as a single intravenous rapid injection of the full therapeutic dose. It has been proven in the framework of clinical studies that also the intravenous administration of surprisingly low therapeutic doses has advantageous results. Preferred therapeutic results were achieved in this, for example, with dosages between 90 μg / kg and 230 μg / kg. Therapeutically particularly advantageous doses were in this at 62.5 to 90 μg / kg. The period between the stroke and the administration of the therapeutic agent was found in the investigated patients between 3 and 9 hours. By means of appropriate analysis methods, the onset of the therapeutic effect could be determined (Figures 20 to 29). DSPA itself and the other non-neurotoxic plasminogen activators show no neurotoxic side effects. Still, it may be advantageous to administer them, for the treatment of stroke, in combination with a neuroprotector to thereby limit the tissue lesions caused in other ways by the endogenous glutamate. For this, neuroprotectors that inhibit the glutamate receptor in a competitive or non-competitive way can be used. Suitable combinations in this are, for example, with the known inhibitory substances of NMDA-type glutamate receptors, of the kainic acid or quisqualate type, for example, with APV, APH, phencyclidine, MK-801, dextrorphan or ketamine.

Also advantageous may be a combination with cations, since cations, particularly Zn ions, can block the cation channel regulated by the glutamate receptor and thus reduce the neurotoxic effects. In another advantageous embodiment, the non-neurotoxic plasminogen activators can be combined with at least one different therapeutic agent or with a pharmacologically compatible carrier. Particularly advantageous in this case is the combination with a therapeutic agent that helps to avoid injury to the tissue by revitalizing the cells, which contribute to the regeneration of the tissue already injured or which serves to prevent subsequent cerebral vascular accidents. Advantages may be advantageous, for example combinations with antibiotics such as quinones, anticoagulants such as heparin or hirudin, as well as citicoline or aspirin. Advantageously, it can also be the combination with at least one thrombus inhibitor. Preferably, thrombomodulin, thrombomodulin analogs such as, for example, Solulin, Triabin or Pallidipin can be used. Also combinations with anti-inflammatory substances are convenient, what influence the infiltration of leukocytes. The type of application respectively 'the administration form according to the invention through concrete examples of therapy. Examples of therapy Comparative investigations of t-PA and DSPA A. Methods 1. Animals Wild type mice (c57 / Black 6) and mice with t-PA deficiency (t-PA - / - mice) (c57 / Black6) (Carmeliet et al., 1994) were supplied by Dr. Peter Carmeliet, Leuven, Belgium. 2. Brain tissue protein extraction The determination of proteolytic activity in brain tissue after infusion of t-PA or DSPAal was performed by zymography analysis (Granelli-Piperno and Reich, 1974). After a 7-day infusion period to the hippocampus the mice were anesthetized, and then transcardiac perfusion was performed with PBS and the brains were removed. The hippocampal region was removed, placed in Eppendorf vessels and incubated in equal volumes (w / v) (approximately 30-50 μl) with 0.5% NP-40 lysis buffer without protease inhibitors (0.5% NP-40). , 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 M MgCl 2, 1 M EDTA). Brain extracts were homogenized by a Glass homogenizer and left for 30 min on ice. The samples were then centrifuged and the supernatant was removed. The amount of protein was determined present (Bio-Rad reagent). 3. Analysis of proteases by zymography Proteolytic activity in brain tissue samples or extracts was determined by zymography analysis in accordance with the method of Granelli-Piperno and Reich (1974). For this, samples of the recombinant protein (up to 100 nmol) or brain tissue extract) 20 μg) were subjected to 10% SDS-PAGE under non-reductive conditions. The gel was removed from the plates, washed for 2 hours in 1% Triton XlOO and then placed on an agar sheet with fibrinogen and polymerized plasminogen (Granelli-Pipero and Reich, 1974). The gel was incubated at 37 ° C in a humid oven until proteolyzed zones could be observed. 4. Intra-Hippocampal Infusion of t-PA, DSPA followed by injection of cainic acid The injury model with cainic acid is based on the proposal of Tsirka et al. (nineteen ninety five) . The animals were injected intraperitoneally (i.p.) with atropine (4 mg / kg), then anesthetized with an i.p. of sodium pentobarbital (70 mg / kg). They were then placed in a stereotactic frame, so that an osmotic minipump (Alzet model 1007D, Alzet CA, USA) could be implanted subcutaneously with 100 μl PBS or recombinant human t-PA (0.12 mg). / ml; 1.85 μM) or DSPAal (1.85 μM). The pumps were connected by sterile tubes with a cerebral cannula and inserted through an opening in the head in the coordinates bregma -2.5 mm, medial-lateral 0.5 mm and dorsoventral 1.6 mm, to introduce the liquid near the center line. The cannula was then fixed with glue in the desirable position and the pumps were opened, to infuse the corresponding solutions with a flow rate of 0.5 μl per hour for seven days. Two days after the protease infusion the mice were anesthetized again and placed in the stereotactic frame. Then 1.5 nmol of cainic acid in 0.3 μl PBS was injected into the hippocampus unilaterally. The coordinates were: bregma 2.5 mm, medial-lateral 1.7 mm and dorsoventral 1.6 mm. The excitotoxin (KA) was introduced for a period of 30 seconds. After the treatment with cainic acid, the needle stayed another two minutes in these coordinates, to avoid the return of the liquid. 5. Brain analysis Five days after the injection of KA, the animals were anesthetized and a transcardiac perfusion was applied with 30 ml of PBS, followed by 70 ml of a 4% solution of paraformaldehyde, remained fixed in the same fixative for 24 hours, followed by incubation in % sucrose for another 24 hours. Coronal sections (40 μm) of the brain were then sectioned into a freezing microtome, either stained with contrast with thionin (BDH, Australia) or prepared for immunohistochemical analysis. 6. Quantification of the rate of neuronal loss within the hippocampus The quantification of neuronal loss in the CA1-CA3 subfields of the hippocampus was performed as previously described (Tsirka et al., 1995, Tsirka et al., 1996). Five consecutive sections of the dorsal hippocampus of all treated groups were prepared, with the sections actually comprising the injection site with KA and the injured area. The subfields (CA1-CA3) of the hippocampus of these sections were traced by lucid chamber drawings of the hippocampus. The total length of these subfields was measured compared to 1 mm standard, which was plotted with the same increase. The length of tissue sections was determined with vital pyramidal neurons (with normal morphology) and the length of the sections without neurons (without the presence of cells, without staining with thionin). The lengths that represented intact neurons and the neuronal losses in the area of each subfield of the hippocampus were averaged between the sections and the standard deviation was determined. 7. Lesions by NMDA excitotoxicity in the striatum with or without t-PA or DSPA Wild-type mice (c57 / Black6) were anesthetized and then placed in a stereotactic frame (see above). The mice then received a unilateral injection into the left striatum with 50 nmol of NMDA alone or in combination with 46 μM rt-PA or 46 μM DSPAal. As a control, t-PA and DSPAal were also injected only at a concentration of 46 μM as control. The coordinates of the injections were: bregma -.04 mm, half-lateral 2.0 mm and dorsoventral 2.5 mm. The solutions (1 μl of the total volume for all treatments) were transferred during a period of 5 minutes to 0.2 μl / min, while the needle remained for another 2 minutes at the site of the injection after the injection, to minimize the return of the liquid. After 24 hours the mice were anesthetized and a transcardiac perfusion was applied with 30 ml of PBS, followed by 70 ml of a 4% solution of paraformaldehyde, a subsequent fixation for 24 hours in the same fixative, followed by an incubation in 30 minutes. % sucrose for another 24 hours. Coronal sections (40 μm) were sectioned in a freezing microtome and placed on glass slides coated with gelatin. 8. Quantification of the volume of injury after injection with NMDA The quantification of injured volume in the striatum was performed by the method described by Callaway et al (2000). For this, 10 consecutive coronal sections were prepared, which comprised the injured region. The injured region was visualized by the Callaway method and the lesion volume was quantified by using a microcomputer display device (MCID, Imaging Research Inc., Brock University, Ontario, Canada). 9. Immunohistochemistry Immunohistochemistry was performed with standard methods. Coronal sections were immersed in a solution of 3% H202 / 10% methanol for 5 minutes, followed by incubation with 5% normal goat serum for 60 min. The sections were then incubated overnight either with an anti-GFAP antibody (1: 1,000; Dako, Caprinteria, Ca, USA:) to check the presence of astrocytes; or with an an i-MAC-1 antibody (1: 1,000; Serotec, Raleigh, NC, USA) to check for the presence of microglia or with polyclonal anti-DSPA antibodies (Schering AG, Berlin). After washing the sections were incubated with the corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA). Then a final incubation was carried out in a Avidin / biotin complex (Vector Laboratories, Burlingame, CA, USA) for 60 minutes before final staining with 3, 3 'diaminobenzidine / O .03% H202. The sections were then transferred to gelatin-coated slides, dried, dehydrated and capped with Permount. 10. Test of increase of tissue lesions induced with NMDA injection by intravenous application of t-PA and DSPA For the induction of tissue lesions in the striatum, mice were injected stereotactically with NMDA. 6 hours after the injection t-PA or DSPA (100 μl, 10 mg / kg) were applied by tail vein injection. As a negative control, 100 μl of 0.9% NaCl was injected and infusion was then made of PBS. After another 24 h the animals were killed and the magnitude of the neuronal tissue damage was determined. In a second type of assay, groups of tests were injected with up to 15 mice corresponding to NMDA, i.V. with t-PA or DSPA 24 after injection with NMDA and the increase in tissue injury was determined accordingly. To check the DSPA in brain tissue, coronary sections were stained by an anti-DSPA antibody according to standard methods. B. Results 1. Infusion of t-PA or DSPA is distributed by the hippocampus of t-AP V- mice and retains its proteolytic activity. The first tests were designed to confirm that both DSPA and also t-PA maintain their proteolytic activity during the period of 7 days of the infusion. For this purpose, aliquots of t-PA and DSPA (100 nmol) were incubated at 37 ° C as well as at 30 ° C for seven days in the naiad bath. To determine the proteolytic activity, a 5-fold serial dilution of the samples was subjected to SDS-PAGE under non-reductive conditions and the proteolytic activity was measured by zymography analysis. Respectively an aliquot of t-PA and DSPA, which remained frozen during these seven days, were used as control. As can be seen in Figure 1, only a small loss of DSPA or t-PA activity occurred, both in the incubation at 25 ° C and at 37 ° C during this period. 2. Activity of t-PA and DSPA can also be found in hippocampal extracts of t-PA - / - mice after infusion. First, it had to be determined that the proteases administered with the infusion were present in the brain of the treated animals and also that they maintained their proteolytic activity there. For this, t-PA - / - mice received infusions with t-PA for seven days o DSPA (see precedent). The mice were then sacrificed by transcardiac perfusion with PBS and the brains were separated. The ipsilateral and contralateral regions of the hippocampus were isolated, as well as a region of the cerebellum (as a negative control). Tissue tests (20 μg) were subjected to SDS-PAGE and zymography analysis in accordance with the description in the method section. As can be seen in Figure 2, both t-PA and DSPA activities were measured in the ipsilateral area of the hippocampus, and some activity was also measured on the contralateral side. This showed that the infused proteases not only maintain their activity in the brain, but also diffuse into the hippocampal region. In the control, no activity was found in the extract prepared from the cerebellum. 3. Immunohistochemical testing of DSPA To prove that DSPA has actually been dispersed throughout the hippocampal region, coronal sections of brain from t-PA - / - mice were analyzed immunohistologically after infusion of DSPA. In this, the presence of DSPA antigen was confirmed in the hippocampus region, and the environment of the infusion site showed the most intense dyeing. This result confirms that the infused DSPA is soluble and that it is actually present in the hippocampus. 4. Infusion of DSPA does not re-establish in vivo the sensitivity with respect to the neurodegeneration dependent on cainic acid. Mice t-PA - / - are resistant to the neurodegeneration dependent on cainic acid. On the contrary, an infusion of rt-PA in the hippocampus leads to a complete reestablishment of sensitivity to cainic acid-dependent lesions. To answer the question, if DSPA can substitute t-PA for this effect, t-PA - / - mice received infusions with t-PA and DSPA in the hippocampus by means of an osmotic minipump. For both groups, 12 mice were used. Two days later the animals received injections of cainic acid followed by a resting phase. Five days later, the animals were sacrificed. The brains were separated and prepared (see above). As a control, t-PA - / - mice were also infused before treatment with KA with PBS (n = 3). Coronal brain sections were prepared and the neurons were detected by staining with the Nissl technique. It was clear that the t-PA - / - mice were resistant to KA after infusion with PBS. But the infusions with recombinant t-PA caused the reestablishment of the sensitivity towards the treatment with KA. Contrary to this, the infusions in the region The hippocampus of the same DSPA concentrations did not modify the sensitivity of the animals against KA (see Fig. 4a and 4b). A quantification of these results was done using 12 mice in each group. In 2 of the 12 mice infused with DSPA we observed some small neurodegenerations. The cause of this is not yet clear and possibly independent of the presence of DSPA. The combined data consider the small effect observed in these two animals. All 12 animals treated with t-PA were sensitive for treatment with KA. These results show that, in an infusion with t-PA or DSPAal in equimolar concentrations, only the administration of t-PA leads to the re-establishment of the sensitivity with respect to the neurodegeneration induced with KA. 5. Infusion of DSPA does not cause activation of itticroglia The reestablishment of KA sensitivity of t-PA - / - mice after infusion of t-PA proves verifiably in a microglia activation (Rogove et al., 1999). To determine the magnitude of the activation of microglia after infusion of t-PA or DSPA followed by treatment with KA, coronal sections of the mice were subjected to immunohistochemical staining for activated microglia cells using the Mac-1 antibody. The reestablishment of KA sensitivity after Infusion of t-PA resulted in a clear increase of Mac-1 positive cells. This was not observed with mice that had received an infusion of DSPA. Therefore, the presence of DSPA does not lead to activation of microglia cells after treatment with KA. 6. Volumetric analysis with DSPA and t-PA in the hippocampal region of mice The concentration of t-PA, which was used for the infusion, was based on the concentration described by Tsirka et al (1995) (100 μl of 0.12 mg / ml [1.85 μM]). We repeated the experiments of exposure to KA using a ten-fold lower amount of t-PA (0.185 μM) and a higher amount of DSPA (18.5 μM). The lower concentration of t-PA still had the capacity to re-establish the sensitivity with respect to the KA treatment (n = 3). Of particular interest was that the infusion of the 10-fold higher concentration of DSPA caused only a small neuronal loss after treatment with KA. These data indicate in a reinforced way that DSPA does not increase the sensitivity for KA. 7. Effects of t-PA and DSPA on NMDA-dependent neurodegeneration in wild-type mice The effects of t-PA and DSPA were further analyzed in a neurodegeneration model of wild-type mice. The injection of t-PA into the striatum of these mice leads verifiably to an increase in neurodegenerative effects induced by the glutamate analogue, NMDA (Nicole et al., 2001). For this, NMDA injections were administered in a total volume of 1 μl in the striatum region of wild-type mice in the presence of t-PA or DSPA (46 μM respectively). After 24 hours, the brains were separated and the magnitude of the lesion was quantified according to the Callaway method (Callaway et al., 2000) (see precedent). As can be seen in Figure 7, injection with NMDA alone produced a reproducible lesion in all treated mice (n = 4). However, if injected t-PA and NMDA in combination increased the magnitude of the lesion by approximately 50% (P <; 0.01, n = 4). In sharp contrast to this, the combined injection of NMDA and the same concentration of DSPA did not cause an increase in the size of the lesion compared to NMDA alone. Injections with t-PA or DSPA alone did not lead to verifiable neurodegeneration. The absence of t-PA effect when administered is only consistent with the results of Nicole et al. (2001). These data demonstrate that the presence of DSPA does not further increase neurodegeneration even during a neurodegenerative event. In order to confirm, that the injection of DSPA effectively diffused in the hippocampal region, it was performed immunohistochemical investigations on coronal sections using the DSPA antibody. Investigations showed that DSPA did indeed penetrate the striatum region. Kinetic analysis of plasminogen activation by the indirect chromogenic assay Chromogenic indirect assays of t-PA activity use the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were prepared in accordance with the Madison EL method , Goldsmith EJ, Gerard RD, Gething M.-J., Sambrook JF (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. Nati Acad. Sci. EE. UU 87, 3530-3533, as well as Madison EL., Goldsmith EJ. , Gething M.J., Sambrook J.F. and Gerard R.D. (1990) J. BioL Chem. 265, 21423-21426. Tests were performed both in the presence and absence of the cofactor DESAFIB (American Diagnostica). DESAFIB, a preparation of soluble fibrin monomers was obtained by the separation of very pure human fibrinogen by batroxobin protease. Batroxobin separates the Arg16-Gly17 bond in the Aa chain of fibrinogen and releases fibrinopeptide A in this. The resulting des-AA-fibrinogen in the form of fibrin I monomers is soluble in the presence of the peptide Gly-Pro-Arg-Pro. The concentration of Lys-plasminogen was varied in the presence of DESAFIB from 0.0125 to 0.2 μM, in the absence of the cofactor from 0.9 to 16 μM. Indirect chromogenic assays in the presence of different stimulators Indirect chromogenic standard assays were carried out in accordance with the publications cited above. For this, preparations of 100 μl total volume were used with 0.25-1 ng of enzyme, 0.2 μM of Lys-plasminogen and 0.62 μM of Spectrozy PL. Assays were performed in the presence of either buffer, 25 μg / ml DESAFIE, 100 μg / ml fragments of cyanogen bromide of fibrogene (American Diagnostica) or 100 μg / ml of peptide 13 amino acid P368 stimulant. The analyzes were performed on microtiter plates and the optical density was measured at 405 nm wavelength for 1 h every 30 sec. in a "Thermomax" molecular device. The reactive temperature was 37 ° C. 8. DSPA does not cause increased neuronal tissue damage even in the case of intravenous application. Tissue lesions were induced in the striatum of mice and 6, respectively, 24 hours after t-PA or DSPA were applied intravenously. In comparison with a negative control, the test animals showed in the case of an i.V. of t-PA 24 hours after the injection with NMDA an increase of approximately 30% of the tissue region injured by the NMDA injection, whereas DSPA did not cause such an increase in tissue damage (see Fig. 18). It was possible to verify by staining coronal sections with an anti-DSPA antibody that the DSPA applied intravenously 24 hours after the injection of NMDA had penetrated into the regions of injured tissue (see Fig. 19). In a corresponding intravenous application of t-PA or DSPA 6 h after the injection of NMDA an increase in the injured tissue region could not yet be detected. This is possibly explained because the blood-brain barrier possessed at this time of the application of t-PA respectively DSPA still sufficient barrier function. DSPA, therefore, also shows no neurotoxic side effects in the case of an intravenous application. References: Baranes D, Lederfein D, Huang YY, Chen M, Bailey CH, Kandel ER. 1998. Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway. Neuron 21: 813-25. Bringmann P, Gruber D, Liese A, Toschi L, Kratzchmar J, Schleuning WD, Donner P. 1995. 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Claims (45)

1. Use of a factor of activation of plasminogen, whose activity is increased in more than 650 times by the presence of fibrin, for the production of a therapeutic agent for intravenous application for the treatment of stroke in humans.

2. Use of a plasminogen activation factor according to claim 1, characterized by the plasminogen activation factor of Desmodus rotundus (DSPA) or its pharmaceutically compatible salt.

3. Use of a plasminogen activating factor according to claim 2, characterized by a dosage between 62.5 to 230 μg / kg.

4. Use of a plasminogen activation factor according to claim 3, characterized by a therapeutic dosage between 62.5 and 90 μg / kg.

5. Use of a plasminogen activation factor according to claim 1, characterized in that the plasminogen activation factor comprises at least one histidine or serine radical, which together with an aspartate radical forms a part of a zymogen triad.

6. Use of a plasminogen activation factor according to claim 5, characterized in that the serine radical is located in a position that is at least homologous. with respect to position 292, the histidine radical in one which is relative to position 305 and the aspartate radical in one which is relative to position 447 of t-PA.

7. Use of a plasminogen activation factor according to claim 6, characterized in that the plasminogen activation factor is selected from the group of the following t-PA mutants: t-PA / R275E; t-PA / R275E, F305H; t-PA / R275E, F305H, A292S.

8. Use of a plasminogen activation factor according to claim 1, characterized in that the plasminogen activation factor has a point mutation of Aspl94 or an aspartate in homologous position that decreases the stabilization of the catalytically active conformation of the activation factor of plasminogen in the absence of fibrin.

9. Use according to claim 8, characterized in that Aspl94 is substituted with glutamate or asparagine.

10. Use according to claim 9, characterized by t-PA with the Aspl94 substitution with Glul94 or Asnl94.

11. Use of a plasminogen activation factor according to claim 1, characterized in that the plasminogen activation factor has at least one mutation in its autolysis loop that reduces the formation of a functional reciprocal effect between plasminogen and plasminogen activating factor in the absence of fibrin.

12. Use according to claim 11, characterized by at least one mutation in the autolysis loop at the position of amino acid 420 to 423 of the wild-type t-PA or in a position homologous thereto.

13. Use according to claim 12, characterized by a mutation selected from the group of the following mutants: L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.

14. Use of a plasminogen activation factor according to claim 1, characterized in that the plasminogen activating factor has at least one point mutation as a zymogen that prevents plasmin catalysis.

15. Use of a plasminogen activation factor according to claim 14, characterized in that the point mutation is located in positions 15 or 275 of t-PA or in a position homologous to it.

16. Use according to claim 15, characterized by glutamate in position 15 or 275.

17. Use according to claim 1, characterized by a factor of tissue plasminogen activation with a loop of autolysis with His420, Asn421r Ala422 and Cys423.

18. Use according to claim 1, characterized by a point mutation in position 194, which decreases the stabilization of the catalytically active conformation of the plasminogen activating factor in the absence of fibrin.

19. Use according to claim 18, characterized by a tissue plasminogen activation factor with Phel94.

20. Use according to claim 1, characterized by a plasminogen activation factor with at least one point mutation that prevents plasmin catalysis.

21. Use according to claim 20, characterized by a tissue plasminogen activation factor with Glu 275.

22. Use according to claim 1, characterized by a tissue plasminogen activation factor with an amino acid sequence according to SEQ. ID No. 1.

23. Use according to claim 1, characterized by a urokinase with an autolysis loop with Fal420, Thr421, Asp422 and Ser423.

24. Use according to claim 23, characterized by a urokinase with a point mutation in position 194 that decreases the stabilization of the catalytically active conformation of urokinase in the absence of fibrin.

25. Use according to claim 24, characterized for a urokinase with Glul94.

26. Use according to claim 23 to 25, characterized by a urokinase with at least one point mutation that inhibits plasmin catalysis.

27. Use according to claim 1, characterized by a urokinase with an amino acid sequence according to SEQ. ID No. 2.

28. Use of a plasminogen activation factor according to one of the preceding claims, characterized by a drip infusion.

29. Use of a plasminogen activating factor according to one of the preceding claims, characterized by a bolus injection.

30. Use of a plasminogen activating factor according to one of the preceding claims, characterized by a single bolus injection (single Bolus).

31. Use of a urokinase with Ile275 for the production of a drug for the treatment of stroke, characterized in that urokinase is applied intravenously.

32. Use of a plasminogen activating factor according to one of the preceding claims for the therapeutic treatment of stroke in humans after having spent three hours since the beginning of the stroke.

33. Use of a plasminogen activation factor according to one of the preceding claims for the therapeutic treatment of stroke in humans after six hours have elapsed since the onset of stroke.

34. Use of a plasminogen activating factor according to one of the preceding claims for the therapeutic treatment of stroke in humans after nine hours have elapsed since the onset of stroke.

35. Use of a plasminogen activating factor according to one of the preceding claims for the therapeutic treatment of stroke patients with a temporally uncertain onset of stroke.

36. Use of a plasminogen activating factor according to one of the preceding claims for cerebral vascular accident therapy, wherein the neurotoxicity caused by the wild type of t-PA is avoided.

37. Pharmaceutical composition for use according to one of the preceding claims containing one of the plasminogen activation factors mentioned in one of the preceding claims and at least one Therapeutically active additional component or its pharmaceutically compatible salt.

38. Pharmaceutical composition according to claim 37, characterized by a neuroprotective agent.

39. Pharmaceutical composition according to claim 38, characterized by a glutamate receptor antagonist.

40. Pharmaceutical composition according to claim 39, characterized by a competitive or non-competitive antagonist.

41. Pharmaceutical composition according to claim 37, characterized by at least one thrombin inhibitor, preferably from the group of the following substances: thrombomodulin, thrombomodulin analogs, Triabin, Pallidipin or Solulin.

42. Pharmaceutical composition according to claim 37, characterized by at least one anticoagulant, preferably selected from the group of the following anticoagulants: hirudin, heparin, aspirin or Ancrod.

43. Pharmaceutical composition according to claim 37, characterized by anti-inflammatory substances.

44. Pharmaceutical composition according to claim 37, characterized by an antibiotic.

45. Pharmaceutical composition according to claim 37, characterized by citicoline.