PT1622639E - Intravenous injection of plasminogen non-neurotoxic activators for treating cerebral stroke - Google Patents

Description

1 ΡΕ1622639

INTRAVENOUS INJECTION OF NON-NEUROXOTIC PLASMINOGEN ACTIVATORS FOR TREATMENT OF CEREBRAL VASCULAR ACCIDENT " The invention relates to the intravenous administration of non-neurotoxic plasminogen activators, in particular promoters of genetically modified plasminogen and plasminogen activators of saliva of Desmodus rotundus (DSPA) for the treatment of stroke in humans. Treatment of stroke with these plasminogen activators is known from the application of the international patent PCT / EP02 / 12204, to which reference is made throughout its scope.

Clinic and biochemistry of stroke

Under the term " stroke " different disease frames are gathered, which resemble their clinical symptomatology. A first differentiation of these disease pictures into the so-called ischemic lesions and hemorrhagic lesions from the respective pathogens is possible.

In the ischemic lesions (ischemia) this is a decrease or interruption of the irrigation of the brain due to lack of arterial blood supply. This is often caused by a thrombosis of a vessel stenosed by atherosclerosis, but also by arterio-arterial or cardiac embolisms.

The hemorrhagic lesions on the other hand have origin among others in a perforation of the arteries that irrigate the brain damaged by arterial hypertonia. Of all brain injuries, however, only about 20% are caused by this form of bleeding, so strokes caused by strokes have a much greater significance. Neuronal tissue ischemia implies - in comparison to ischemia of other tissues - a special proportion of necrosis of the affected cells. The incidence of increased necrosis can be explained on the basis of foreground, by the phenomenon of the so-called excitotoxi-city, which is a complex cascade of a multiplicity of reaction steps. This is therefore triggered by ischemic neurons suffering from lack of oxygen, rapidly losing ATP and depolarizing. This leads to an increased post-synaptic release of the neurotransmitter glutamate, which in turn activates membrane-bound glutamate receptors that regulate the cation channels. As a consequence of the high release of glutamate, the glutamate receptors are however hyper-excited. 3 ΡΕ1622639

Glutamate receptors, on the other hand, regulate voltage-dependent cation channels, which are opened by the binding of glutamate to its receptors. Thus a Na + and Ca 2+ entry stream is initiated into the cell, which leads to a massive disturbance of Ca2 + -dependent cellular metabolism - including cellular energy metabolism. For the subsequent cell death, the activation of Ca-dependent catabolic enzymes (Lee, Jin-Mo et al., &Quot; The changing landscape of ischemic brain injury mechanisms "; Dennis W. Zhol " Glutamat neurotoxicity and desires of the nervous system ").

Even though the mechanism of glutamate-mediated neurotoxicity is still not currently known in detail, there appears to be agreement that this phenomenon contributes substantially to neuronal cell death after cerebral ischemia (Jin-Mo Lee et al.).

Treatment of stroke

In the treatment of acute cerebral ischemia it is in the foreground, together with the safeguarding of vital functions and the stabilization of physiological parameters, first the intention of a reopening of the closed vessel. There are different approaches to this end. The purely mechanical reopening has not yet achieved satisfactory results, for example PTCA in cardiac infarction. Only with successful fibrinolysis 4 ΡΕ1622639 has a sufficient improvement in the condition of the patients been achieved so far. The body's own fibrinolysis is based on the proteolytic activity of plasmin serine protease, which originates from catalysis (activation) of the inactive plasminogen precursor. Natural activation of plasminogen occurs through plasminogen activators of the u-PA (plasminogen activator-urokinase type) and t-PA (Tissue Plasminogen Aktivator). The latter form - unlike u-PA - together with fibrin and plasminogen a so-called activator complex. The catalytic activity of t-PA is therefore dependent on fibrin and an increase of approximately 550-fold is observed in the presence of fibrin. Along with fibrin, fibrinogen can also stimulate t-PA-mediated catalysis of plasmin to plasminogen - even in a much smaller proportion. Thus there is an increase in t-PA activity only 25-fold in the presence of fibrinogen. Fibrin degradation products (FDP) also stimulate the activity of t-PA.

Known Therapeutic Preparations a) Streptokinase

The earliest preparations for the thrombolytic treatment of acute stroke date back to the 1950s. However only from 1995 were 5 ΡΕ1622639 the first large-scale clinical trials with streptokinase, a beta-hemolytic streptococcus fibrinolytic performed. Streptokinase forms with plasminogen a complex, which has the ability to convert other plasminogen molecules into plasmin. Treatment with streptokinase is however associated with substantial disadvantages, since streptokinase as a bacterial protease can trigger allergic reactions in the body. There may also be a so-called streptokinase resistance due to infection by prior streptococcus with the formation of respective antibodies, which hampers a treatment. In addition, European trials (Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter Acute Stroke Trial of Italy (MAST-I)) and Australia (Australien Streptokinase Trial (AST)) indicated a mortality risk in such a way (intracerebral hemorrhage (ICH) following the treatment of patients with streptokinase, that these studies have even had to be discontinued prematurely. b) Urokinase

In an alternative therapeutic approach, urokinase - also a classic "fibrinolytic" - is administered, which unlike streptokinase does not exhibit antigenic properties, since it is an enzyme of the organism present in numerous tissues. It represents a 6 ΡΕ1622639 plasminogen activator independent of cofactors. Urokinase is produced in renal cell culture. c) recombinant t-PA (rt-PA)

There is considerable experience in the area of therapeutic thrombolysis with tissue-type plasminogen activator - the so-called rt-PA - (see EP 0 093 619, U.S. Patent No. 4 766 075), which is produced in recombinant hamster cells. A series of clinical trials with partially unsolved and discrepant results were performed with t-PA - along with the main indication acute myocardial infarction - worldwide in the 1990s. Thus they were first treated in the so-called European Acute Stroke Trial (ECASS), treated patients within a time period of 6 hours after the onset of stroke symptoms intravenously with rt-PA and determined after 90 days the mortality rate as well as Barthel's index as a measure of incapacity or functional independence in patients' daily lives. It was found that there was no significant improvement of functionality in daily life, but rather a - even if not significant - increase in mortality. This allowed the conclusion that a thrombolytic treatment with rt-PA could possibly be advantageous for patients chosen individually according to their respective clinical history, immediately after the onset of stroke. Based on the observed increased risk of intracerebral hemorrhage (ICH) after only a few hours 7 ΡΕ1622639 following the onset of stroke, a generalized use of rt-PA within the time period investigated six hours after onset was discouraged of stroke (thus C. Lewandowski C and Wiliam Barsan, 2001: Treatment of Acute Strocke, in Annals of Emergency Medicine 37: 2, pp. 202 et seq.). The thrombolytic treatment of stroke was later also the subject of a clinical trial of the National Institute of Neurological Disorder and Stroke (so-called NINDS rtPA Stroke Trial) in the USA. However, the investigation of the effect of an intravenous rt-PA treatment within a period of time of only three hours after the onset of symptoms had the patient's health status at the forefront after three months. Although the authors also found an increased risk for ICH, however, treatment with rt-PA within this limited time period of three hours is recommended based on the positive effects also recognized in this study of this treatment in functionality in the daily life of patients .

In two other studies (ECASS II Trial; Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke (ATLANTIS) was investigated whether this positive statement about the effect of rt-PA treatment within three hours after the onset of stroke also However, this question could not be answered in a positive way, since with a treatment ΡΕ1622639 over this period of time, in addition to the increased risk of ICH, no improvement in clinical symptoms was observed or a decrease in mortality.

According to a synthesis of all " Stroke Trials " first published in 1997 and updated in March 2001, all treatments with thrombolytics (urokinase, streptokinase, rt-PA or recombined urokinase) led, in particular, to ICH, to a significantly increased mortality within the first ten days after while a treatment over a period of six hours reduced the total number of the sick or dead or disabled patients. A wide use of thrombolytics in the treatment of stroke is thus discouraged.

These results have already led other authors to the rather sarcastic assertion that patients with stroke would now have the choice of dying or surviving disabled (SCRIP 1997: 2265, 26). However, rt-PA treatment is currently the only method of treatment authorized by the US Food and Drug Administration (FDA) for acute cerebral ischemia. This is however limited to an administration of rt-PA within three hours after the onset of stroke. 9 ΡΕ1622639 Recombinant plasminogen activator is currently available on the market under the designation Altepla and the analogue preparation Reteplase. The latter is a fragment of t-PA with therapeutic effect, with a lower half-life. The therapeutic dose of Alteplase is approximately 70-100 mg, that of Reteplase is twice 560 mg, wherein Alteplase is essentially administered through an intravenous infusion, and Reteplase is instead administered through two injections in boluses separated by approximately 30 min. (Mustschler: " Arzneimittelwir-kungen " (" Effects of Drugs "), 8. Editing, pp. 512-513).

Side effects of t-PA

Neurotoxicity and excitotoxicity

Immediately before, especially in 1995, the first indications for the cause of the neurotoxic or excitotoxic effects of t-PA were known, which also led to a hypothesis explaining the dramatic effects of t-PA -PA in the treatment of stroke outside the treatment time period of three hours. Thereafter, the microglial cells and neuronal cells of the hippocampus produce t-PA from the body, which participates in glutamate-mediated excitotoxicity. This finding came about through a comparison of t-PA-deficient mice and wild-type mice, in which glutamate agonists were injected into the hippocampus respectively. Rats deficient in t-PA 10 ΡΕ1622639 demonstrated a markedly increased 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 the year 1998, when Wang et al. were able to demonstrate in t-PA deficient rats via intravenous injection of t-PA almost a doubling of necrotic neuronal tissue. This negative effect of external t-PA was however in wild type mice 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 ").

Nicole et al. published more results on the stimulation of excitotoxicity through t-PA at the beginning of 2001 (Nicole O; Docagne F Ali C; Margaill I; Carmeliet P; MacKenzie ET; Vivien D Buisson A, 2001: The proteolytic activity of tissue- plasminogen activator enhances NMDA receptor-mediated signaling; in: Nat Med 7, 59-64). They were able to prove that the t-PA released by the depolarized cortical neurons interacts with the so-called NR1 subunit of the NMDA-type glutamate receptor and dissociation. This modification leads to an increase in receptor activity, which in turn is responsible for increased tissue damage following NMDA administration of glutamate agonist through induced excitotoxicity. T-PA thus acts neurotoxically through the activation of the NMDA-type glutamate receptor. Once 11 ΡΕ1622639 that in the event of a stroke there is failure of the blood-brain barrier in the affected tissue region, soluble plasma proteins such as fibrinogen and therapeutically administered t-PA come into contact with the neuronal tissue where stimulated t-PA by fibrinogen develops its excitotoxic effect through the activation of the glutamate receptor.

That despite these indications of the neurotoxic side effects of t-PA and despite increased t-PA-proven mortality, there has nevertheless been a legal authorization of the drug by the FDA, it may be explained only by the lack of innocuous and effective alternatives - and a very pragmatic cost-benefit analysis. However, the need for safe treatments remains, as in the development of new thrombolytics - not to depart from thrombolysis altogether - the problem of neurotoxicity has to be considered (for example, Wang et al. Lewandowski and Barsan 2001).

For this reason, no further investigations of known thrombolytic agents, including DSPA (Desmodus rotundus plasminogen activator), have been carried out to develop new therapy for stroke, although in principle all thrombolytics may be appropriate and therefore example in the case where DSPA has already been mentioned in a first publication about its possible added value for this indication (Medan P; 12 ΡΕ1622639

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 Thrombolytic Therapy in Acute Ishemic Stroke). Precisely in the case of DSPA, however, it is a plasminogen activator with high homology (resemblance) to t-PA, so that - along with the disillusionment about the neurotoxic side effects of t-PA - in the DSPA no more hopes.

Alternative therapeutic preparations Investigation of alternative therapeutic preparations is currently targeted at anticoagulants such as heparin, aspirin or ancrod, the active ingredient in viper venom of the Crotalinae subfamily of Malaysia. Two clinical studies, including the International Stroke Trial (IST) and Trial of ORG 10172 in Acute Stroke Treatment (TOAST), among others, do not indicate a significant improvement in mortality and a a new stroke.

Another novel type of treatment method does not affect either the thrombus or the liquefaction of the blood or anticoagulants, but rather attempts to increase the vitality of the injured cells by interrupting the blood supply (WO 01/51613 AI and WO 01/51614 A1). For this purpose 13 ΡΕ1622639 administered quinolone, aminoglycoside or chloramphenicol antibiotics. Due to a similar reason it is further proposed to initiate immediately after stroke the administration of citicoline, which is dissociated in the body into cytidine and choline. These dissociation products are components of the neuronal cell membrane and may thus favor regeneration of the injured tissue (US Patent 5,827,832).

Further efforts in the search for safe treatment methods are based on foreground, that a part of the fatal consequences of stroke are due only indirectly to interruption of blood supply but to excitotoxicity or neurotoxicity with glutamate receptor involvement hyper-excited, which in turn is intensified especially also by t-PA (see above). One approach to decrease this excitotoxicity is thus the administration of so-called neuroprotectors. These can be used alone or in combination with fibrinolytics, so as to minimize their neurotoxic effects. They may lead to an attenuation of excitotoxicity, either directly for example as glutamate receptor antagonists or indirectly through blocking of voltage-dependent sodium or calcium channels (Jin-Mo Lee et al, cited work)

A competitive inhibition (antagonization) of the NMDA type glutamate receptor is possible for example with 14 ΡΕ1622639 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonohepta-noate (APH). A noncompetitive inhibition can be achieved for example with substances which bind to the phenocyridine side of the channels, such as phencyclidine, MK-801, dextrorphan or ketamine.

To date neuroprotective treatments have not, however, had the desired success, possibly because they have to be combined with thrombolytics in order to develop their protective effect. This is valid in the same way for other active ingredients (see Figure 10).

Even with a combination of t-PA and a neuro-protector, however, a success in limiting lesions is possible. The disadvantages of the neuro-toxicity of the fibrinolytic agent used per se are however not avoided.

Non-neurotoxic plasminogen activators

From the international patent application WO 03/037363, which is referred to throughout its scope, plasminogen activators are known for the treatment of stroke, the enzymatic activity of which is increased in a highly selective manner through fibrin in a multiplicative manner, namely in more than 650 times. The constitution and use of these plasminogen activators are based on the knowledge that the neurotoxicity of the tissue plasminogen activator (t-PA) originates, that as a consequence of tissue destruction in the brain caused by stroke, the blood-brain barrier is damaged or destroyed and fibrinogen circulating in the blood can thus enter the neuronal tissue of the brain. It activates t-PA, which - either through the activation of the glutamate receptor or through the activation of plasminogen - leads to further tissue damage (see above).

To avoid this effect a plasminogen activator is used which has increased fibrin selectivity and - in inverse conclusion - a lower ability to be activated by fibrinogen. As a consequence, these plasminogen activators are not activated - or in a significantly smaller size compared to t-PA - when the fibrinogen is passed from the blood into the neuronal tissue in sequence of the damaged blood-brain barrier, since its fibrin activator lacks the ability to enter into the neuronal tissue due to its size and insolubility. These plasminogen activators are thus non-neurotoxic. a) Genetically modified plasminogen activators

Thus non-neurotoxic non-neurotoxic plasminogen activators are used, which have at least one element of the so-called zymogen triad. A comparable triad is known from the catalytic center of serine proteases of the chymotrypsin family, which consists of the three amino acids interacting aspartate 194, histidine 40 and serine 32. This triad is not present in tPA, however, which belongs to the family of serine proteases of the chymotrypsin type. It is however known that targeted mutagenesis of native t-PA for introducing at least one of these amino acids into appropriate positions leads to a decrease in the activity of this proenzyme (single chain t-PA) and an increase in the activity of the mature enzyme (two-chain t-PA) in the presence of fibrin. Thus the introduction of at least one of the amino acids of the triad - or of amino acids assuming their triad function - leads to an increase in t-PA zymogeneity (ratio of mature enzyme activity to proenzyme activity) with clear increase in fibrin specificity through interaction in the conformation between the introduced amino acid residues and / or amino acid residues of the wild type sequence.

Thus, it is known that mutagenesis of native t-PA for replacement of Phe305 to His (F305H) as well as Ala 292 to Ser (A292S) leads to a 20 fold increase in zymogeneity, while already with the F305H variant alone it is obtained a 5-fold increase (El Madison, Kobe A, Gething MJ, Sambrook JF, Goldsmith EJ 1993: 17 ΡΕ1622639

Converting Tissue Plasminogen Activator to a Zymogen: A Regulatory Triad of Asp-His-Ser, Science: 262, 419-421). These mutant t-PAs exhibit an increase in activity in the presence of fibrin 30,000-fold (F305H) or 130,000-fold (F305H, A292S). Both mutants also present a substitution of Arg275 for R275E to avoid dissociation of single chain t-PA to the two-chain form at the Arg275-Ile276 dissociation site through plasmin. This R275E mutant alone increases the specificity for t-PA fibrin in 6,900-fold (K Tachias, Madison EL 1995: Variants of Tissue-type Plasminogen Activator Which Displays Substantially Enhanced Stimulation by Fibrin in: Journal of Biological Chemistry 270, 31: 18319-18322).

Positions 305 and 292 of t-PA are homologous to positions His40 and Ser32 of the known triad of serine chymotrypsin proteases. Through corresponding substitution with histidine or serine, these amino acids may interact with t-PA aspartate477, so that the known triad may be functionally formed in mutant t-PA (Madison et al 1993).

These mutant t-PAs can be used for the treatment of stroke because they exhibit because of their specificity for either increased fibrin or - as compared to the wild type t-PA - only clearly diminished neurotoxicity. For the purpose of the disclosure of the aforementioned t-PA mutants F305H; 18 ΡΕ1622639 F305H, A292S alone or in combination with R275E reference is made in full to the publications of Madison et al. 1993 as well as Tachias and Madison 1995. The increase in fibrin specificity of plasminogen activators is also alternatively possible through a point mutation of Aspl94 (or an aspartate in the homologous position). Plasminogen activators belong to the serine protease group of the chymotrypsin family and exhibit the conserved amino acid Aspl94 which is responsible for the stability of the catalytic active conformation of the mature proteases. It is known that Aspl94 in the serine protease zymogens interacts with His40. By activating dissociation of the zymogen these interactions become impossible and the side chain of the Aspl94 rotates approximately 170 ° to form a new saline bridge with the Ilel6. This saline bridge plays a role in the stability of the oxyanion pocket of the catalytic triad of mature serine protease. It is also present in t-PA.

A point mutation of Aspl94 makes initially impossible the formation or stability of the catalytic conformation of serine proteases. However, mutated plasminogen activators exhibit in the presence of their cofactor fibrin - especially also in comparison with the mature wild-type form - a clear increase in activity, which is only explicable, because the interactions with fibrin allow a change of the conformation that 19 ΡΕ1622639 enables catalytic activity (L Strandberg, Madison EL, 1995: Variants of Tissue-type Plasminogen Activator with Substantially Enhanced Response and Selectivity towards Fibrin Cofactors, in: Journal of Biological Chemistry 270, 40: 2344-23449 ). Thus Aspl94 mutants of plasminogen activators exhibit a high increase in activity in the presence of fibrin, which enables their use as a non-neurotoxic plasminogen activator.

A preferred example for such a non-neurotoxic plasminogen activator is t-PA, which Aspl94 was replaced by glutamate (D194E) or asparagine (D194N). Thus in the absence of fibrin the t-PA activity is reduced by a factor of 1-2000, whereas instead in the presence of fibrin an increase in activity by a factor of 498,000 to 1,050,000 can be achieved. These mutants may furthermore contain a substitution of Argl5 for R15E, which prevents the dissociation of single chain t-PA in the two-chain t-PA at the Argl5-Ilel6 peptide bond through fibrin. Through this mutation alone, the activation of t-PA by fibrin is already increased by a factor of 12,000. For the purposes of disclosing mutations at positions 194 as well as 15 of t-PA reference is made in full to the publication of Strandberg and Madison 1995.

In addition, an increase in fibrin dependence of plasminogen activators can be achieved by introducing point mutations in the so-called " ring-autolysis ". This segment of amino acids is known from trypsin; it is however present as a homologous segment also in the serine proteases and is especially characterized by three hydrophobic amino acids (Leu, Pro and Phe). Ring-autolysis in plasminogen activators is responsible for the interaction with plasminogen. Point mutations in this region may lead to no more functionally formed protein / protein interactions between plasminogen and plasminogen activator. These mutations are functionally relevant however only in the absence of fibrin. In the presence of fibrin, on the contrary, they are responsible for an increased activity of plasminogen activators (K Song-Hua, Tachias K, Lamba D, Bode W, Madison EL, 1997: Identifikation 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 mutations at positions 420-423 is used. If these residues are replaced by specific point mutations, the fibrin dependence of t-PA increases 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 hereby referred to throughout its scope for use in accordance with the invention.

In another advantageous embodiment, the modified tissue plasminogen activator 21 ΡΕ1622639 is modified with an amino acid sequence according to SEQ ID NO: 1 (Figure 13), which is distinguished from wild-type Î ± -PA by performing a exchange of the hydrophobic amino acids at positions 420-423 in the ring-autolysis, which is occupied as follows: His420, Asp421, Ala422 and Cys423. This t-PA preferably has a phenylalanine at position 194. In addition position 275 may be occupied by a glutamate. Advantageously position 194 is occupied by phenylalanine.

In addition a modified urokinase may be used. This urokinase may have the amino acid sequence according to SEQ. ID No. 2 (Figure 14), in which the hydrophobic amino acids of the ring-autolysis are replaced by Val420, Thr421, Asp422 and Ser423. Advantageously this urokinase has an Ile275 as well as a Glul94. This mutant displays a specificity for fibrin increased by 500-fold compared to wild-type urokinase.

Both mutants - both urokinase and t-PA - were investigated in semi-quantitative tests and exhibited increased fibrin specificity compared to wild-type t-PA. b) Desmodus rotundus plasminogen activators (DSPA)

A high increase in activity in the presence of 22 ΡΕ1622639 fibrin, in particular an increase of 100,000-fold, is further verified also with the plasminogen activator of bat-vampire saliva (Desmodus rotun-dus), which can therefore be used as activator non-neurotoxic plasminogen. Under the designation DSPA four different proteases meeting a fundamental vampire bat need are grouped, namely the time of increased hemorrhage from the wounds on the prey wounds caused by these animals (Cartwright, 1974). These four proteases (DSPAal, DSPAa2, DSPAp, DSPAY) similarly exhibit high similarity (homology) with human t-PA. They also present similar or similar physiological activities, which justify their grouping under the generic name DSPA. DSPA is the subject of EP 0 352 119 AI as well as of U.S. Patent 6,008,019 and 5,830,849 which are hereby referred to throughout their scope for disclosure purposes. DSPAai is the protease best investigated to date in this group. It has in its amino acid sequence a homology of above 72% relative to the amino acid sequence of the known human t-PA (Krátzschmar et al., 1991). However, there are two essential differences between t-PA and DSPA. First and foremost the DSPA represents as a single chain relative to peptide substrates a fully active molecule, which, like t-PA, is converted into a two-chain form (Gardell et al., 1989; Kratzsmar et al., 1991). Second, the catalytic activity of DSPA exhibits a virtually complete dependence on fibrin (Gardell et al., 1989; Bringmann et al., 1995; Toschi et al., 1998). Thus for example the activity of DSPA in the presence of fibrin is increased 100,000 fold, whereas the activity of tPA only increases by approximately 550 fold. The activity of DSPA is on the contrary less strongly induced by fibrinogen; shows only a 7-9 fold increase (Bringmann et al., 1995). DSPA is thus considerably more fibrin-dependent and fibrin-specific than wild-type t-PA, which is only activated at approximately 550-fold by fibrin.

Due to its fibrinolytic properties and its great similarity to t-PA, DSPA is indeed an interesting candidate for the development of a thrombolytic. Because of the participation of t-PA in glutamate-dependent neurotoxicity there was therefore no legitimate hope of successfully using a closely related t-PA plasminogen activator in the treatment of acute stroke.

It has now surprisingly been shown, however, that DSPA despite its high similarity (homology) with t-PA and despite the extensive agreement of its physiological action with t-PA, unlike it has no neurotoxic effects. With this observation implies the recognition that DSPA can ultimately be used as a thrombolytic agent in the treatment of stroke, without an additional risk of injury to the neuronal tissue. This means in particular that DSPA can also be used later than three hours after the onset of stroke symptoms.

Experimental evidence of the absence of neurotoxicity of DSPA The recognition of the absence of neurotoxicity of these plasminogen activators is based essentially on in vivo comparison investigations of the neurodegenerative effect of tPA on the one hand and DSPA on the other hand, which were performed with the help of the so-called "kainic acid model", as well as a model for investigation of NMDA-induced striatum lesions. The kainic acid model (also the kainic acid injury model) is based on the fact that the neurotoxic glutamate cascade is stimulated by administration of external kainic acid (KA) as a kainic acid glutamate receptor agonist (KA-type) as well as the NMDA and AMPA glutamate receptors. By using a lineage of t-PA-deficient rats as an experiment model it could thus be shown that the sensitivity of the test animals to kainic acid only reached after additional administration of external t-PA the level of mice of the type wild. On the contrary an infusion of an equimolar concentration of DSPA under the same experimental conditions was not able to restore sensitivity to kainic acid (KA). The effects of t-PA could not therefore be replaced by the DSPA. A summary of these results is shown in Fig. 15 (Table 1).

Quantitative investigations in this model showed that even a 10-fold increase in DSPA concentration did not restore the sensitivity of t-PA deficient mice to KA treatment, however, however, a 10-fold lower t-PA concentration already led to to tissue injuries induced by KA. It is concluded that in the stimulation of neurodegeneration after treatment with KA, DSPA exhibits at least 100-fold lower activity than t-PA (see also Figures 11 and 12).

In the second model of neurodegeneration, the possible effects of t-PA as well as DSPA on the stimulation of NMDA-dependent neurodegeneration in wild-type mice were compared. For this, wild-type mice were injected with NMDA (as an NMDA-type glutamate receptor agonist) alone or in combination with tPA or DSPA. This model allows the investigation of the effect of these proteases under conditions, even though a neurodegeneration occurs and causes an entrance of plasma proteins due to the collapse of the blood-brain barrier (Chen et al., 1999).

In working with this model the injection of NMDA led to reproducible lesions in the striatum of mice. The 26 ΡΕ1622639 volume of these lesions was increased by concomitant injection of t-PA and NMDA by at least 50%. In contrast, co-injection with DSPAα did not lead to any worsening of the increase in lesions caused by NMDA injection. Even in the presence of plasma proteins, which due to NMDA-dependent neurodegeneration could diffuse into the lesion area, DSPA did not lead to any increase in neurodegeneration. A summary of these results is shown in Fig. 16 (Table 2).

Early results from clinical studies show the transmissibility of these results also for the treatment of stroke in humans. Thus it is verified in patients after successful perfusion clear improvements (improvement in 8 points NIHSS or value HIHSS 0-1). FIG. 17 (Table 3) makes this evident.

In another experiment it has been tested whether t-PA and DSPA when administered intravenously can penetrate through the damaged blood-brain barrier and increase the tissue lesions in the brain. For this purpose, NMDA stereotactic injections were made in rat striatum to generate tissue lesions and administered intravenously t-PA or DSPA 6 or 24 hours after NMDA injection. Compared to a negative control, the test animals infused with a t-PA infusion 24 hours after NMDA injection showed a 30% increase in tissue area injured by NMDA injection, whereas DSPA did not cause such an increase in tissue injury , although its penetration into the lesioned tissue area through antibody staining (see Figure 18, 19) could have been demonstrated. With the corresponding intravenous administration of t-PA or DSPA 6 h after NMDA injection, no increase in the injured tissue area has yet been found. This is possibly due to the fact that the blood-brain barrier has a sufficient barrier function at this time of administration of the t-PA or DSPA.

These results show that DSPA represents in the central nervous system of a mammal - or humans as well - an essentially inert protease and - unlike t-PA - causes no increase in neurotoxicity induced by KA or NMDA. This absence of neurotoxicity causes that against all expectations the DSPA is an appropriate thrombolytic for the treatment of the acute cerebrovascular accident.

Therapeutic potential of non-neurotoxic plasminogen activators

Because of the absence of neurotoxicity of DSPA as well as of the other non-neurotoxic plasminogen activators (see above), it is especially advantageous for the treatment of stroke that the use of these plasminogen activators - other than t-PA of the type wild - is not limited only to the narrow time window of up to three hours after the onset of stroke. On the contrary, the treatment can also occur in a later period - so after six hours or even later - without being confronted - as in t-PA - with the risk of a stimulus of excitotoxicity. The first clinical investigations with the DSPA prove even the inconvenient treatment of patients in a period of time of more than 6 or 9 hours after the beginning of the symptoms.

This temporally unlimited treatment option with non-neurotoxic plasminogen activators is therefore so significant because it is possible for the first time to treat patients with acute stroke without hesitation for whom the onset of stroke could not be determined temporarily with sufficient security. This group of patients was so far excluded for reasons of precaution and assessment of the risk of thrombolysis with plasminogen activators. Thus a fundamental contraindication to the permissible use of a thrombolytic agent in stroke is suppressed.

Administration of plasminogen activators

Contrary to the rt-PA therapy for already established stroke, there is still no proven knowledge about a possible mode of administration for the treatment of stroke with non-neurotoxic plasminogen activators. 29 ΡΕ1622639

The object of the invention is thus to provide an advantageous mode of administration for these non-neurotoxic plasminogen activators.

According to the invention plasminogen activators, whose activity is increased by more than 650 fold in the presence of fibrin, are administered intravenously for the treatment of stroke. Intravenous administration of these non-neurotoxic plasminogen activators for the treatment of stroke has been confirmed in clinical studies, where DSPA - as an example for this group of fibrinolytics - was given intravenously and only caused few side effects. These results from clinical studies were not expected as it was known that intravenous administration of t-PA and other usual fibrinolytics is associated with a high risk of cerebral hemorrhage (see above).

In order to reduce intra-cerebral hemorrhages, strategies have recently been developed to administer these substances intravenously, but intra-arterially through a catheter directly next to the intravascular thrombus. Experience in this regard already exists with recombinantly produced urokinase (PROCAT as a pro-urokinase study). Since this mode of administration allows a significant reduction of the total dose, a decrease in dose-dependent side effects is thus achieved - as well as cerebral hemorrhages.

The enormous advantages of intra-arterial administration have, however, two possible disadvantages. Thus, it presupposes, on the one hand, a long prepaid preparation of the patient, which is not possible in the time span of the time window of only 3 hours available for the treatment of stroke. On the other hand, a lower total dose can thus be achieved. Locally however a higher concentration of the drug arrives at the arterial end vessels. With the damaged vascular endothelium barrier function due to stroke, the drug therefore also arrives in the surrounding tissues at high local doses. It can then cause undesirable side effects.

In contrast to intravenous administration, the concentration of the drug is diluted by the venous blood stream. Thus intra-arterial injection of drugs that damage tissues such as t-PA is problematic (Forth, Henschler, Ruminei, Starke: " Pharmakologie und Toxikologie, 6th edition, 1992, p29).

The limitations which hinder intra-arterial administration as the narrow temporal window and the side effects damaging the tissues, however, do not exist in the plasminogen activators used according to the invention. Thus intra-arterial administration represents, because of its undisputed advantages (see above), a very promising mode of administration of success essentially for non-neurotoxic plasminogen activators. Thus one would expect to resort to this path in the search for an advantageous mode of administration of this therapy. Notwithstanding the applicant, non-neurotoxic plasminogen activators have also been used for the previously problematic intravenous administration, which has in fact proved surprisingly advantageous.

The mode of administration according to the invention furthermore diverges from a usual therapeutic practice in which proteins with an increased immunogenic potential are generally administered intramuscularly or by intravenous injection. Thus it is also possible to reduce the risk of an anaphylactic shock (Mebs: " Gifttiere " Poisonous Animals "), 2nd edition, 2000).

The plasminogen activators used according to the invention are - unlike the organism's own t-PA - or proteins foreign to the organism of animal origin (see eg DSPA) or genetically modified proteins of the organism which they present due to their alterations new epitope types. The problem of allergic reactions - especially in the administration of high therapeutic doses as are usually required for intravenous administration 32 ΡΕ1622639 also exists for other fibrinolytics consisting of foreign proteins such as streptokinase.

In a particularly advantageous embodiment, the plasminogen activators used according to the invention are administered by bolus injection (intravenous rapid injection), which can also be administered as a single intravenous rapid injection with the total therapeutic dose.

In the context of clinical studies it has been proven that the intravenous administration also surprisingly of lower therapeutic doses leads to advantageous results. Preferred therapeutic results were achieved for example at dosages between 90 pg / kg and 230 pg / kg. Especially advantageous therapeutic doses were between 62.5 to 90 pg / kg. In the patients tested, the time period between the stroke and the administration of the therapy comprised between 3 and 9 hours. Through appropriate investigative methods the onset of a therapeutic action could be determined (see Figures 20 to 29). DSPA as well as the remaining non-neurotoxic plasminogen activators do not in themselves have any neurotoxic side effects. On the contrary, it may be advantageous to administer them for the treatment of stroke in combination with a neuroprotector, in order to limit as much as possible the tissue damage otherwise caused by the body's own glutamate 33 ΡΕ1622639. To this end neuroprotectors can be used which competitively or non-competitively inhibit the glutamate receptor. Convenient combinations are possible for example with the known NMDA, kainic acid type or quisqualate type glutamate receptor inhibitors, eg with APV, APH, phencyclidine, MK-801, dextrorphan or ketamine.

A combination with cations may also be advantageous since the cations, especially the Zn ions, block the cation channels regulated by the glutamate receptor and may thus reduce the neurotoxic effects.

In another advantageous embodiment, non-neurotoxic plasminogen activators may be combined with at least one other therapeutically or with a pharmaceutically acceptable adjuvant. Especially advantageous is the combination with a therapy, which helps prevent tissue damage by vitalizing the cells, contributes to a regeneration of already injured tissue or serves to prevent subsequent strokes. Advantageous may be eg combinations with antibiotics such as quinolones, anticoagulants such as heparin or hirudin as well as citicoline or aspirin.

Advantageous may furthermore also be a combination with at least one thrombin inhibitor. Preferably, thrombomodulin, thrombomodulin analogues, such as, for example, soluline, triabin or palladium may be used. Combinations with anti-inflammatory substances, which influence leukocyte infiltration, are also advantageous.

Thereafter the type of administration or the mode of administration according to the invention is elucidated on the basis of concrete treatment examples

TREATMENT EXAMPLES

COMPARATIVE INVESTIGATIONS OF T-PA AND DSPA A. Methods 1. Animals

Wild-type mice (c57 / Black 6) and t-PA deficient mice (t-PA - / - mice) (c57 / Black 6) (Carmeliet et al., 1994) were made available by

Dr. Peter Carmeliet, Leuven. 2. Protein Extract from Brain Tissue Determination of proteolytic activity in brain tissue after infusion of t-PA or DSPAα occurred by zymographic analysis (Granelli-Piperno and Reich, 1974). After a 7 day infusion time in the hippocampus, the rats were anesthetized, then perfused transcardially with PBS and the brains were removed. The hippocampal region was removed, added to tubes 35 ΡΕ1622639

Eppendorf and incubated with the same volumes (w / v) (approx. 30-50 μ) of 0.5% NP-40 lysis buffer without protease inhibitors (0.5% NP-40, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA). The extracts from the brain were homogenized through a hand glass homogenizer and left on ice for 30 min. The samples were then centrifuged and the supernatant removed. The amounts of protein present were determined (Bio-Rad reagent). 3. Zymographic analysis of proteases Proteolytic activity in samples or extracts of brain tissue was determined by zymographic analysis according to the method of Granelli-Piperno and Reich (1974). Samples of the recombinant protein (up to 100 nmol) or the brain tissue extract (20 μg) were subjected to 10% SDS-PAGE under non-reducing conditions. The gels were removed from the plates, washed for two hours with 1% Triton x 100 and then placed on an agarose gel with polymerized fibrinogen and plasminogen (Granelli-Piperno and Reich, 1974). The gels were incubated at 37øC in a humid chamber until proteolyzed zones were recognizable. 4. Intra-hippocampal infusion of t-PA, DSPA and subsequent injection with kainic acid The lesion-kainic acid model is based on the preparation of Tsirka et al. (1995). 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 stereotactic support, such that a microosmotic pump (Alzet model 1007D, Alzet CA. USA) with 100 μM PBS or recombinant human t-PA (0.12 mg / ml); 1.85 μΜ) or DSPAα (1.85 μΜ) could be implanted subcutaneously between the shoulder blades. The pumps were connected through sterile tubes to a cerebral cannula and through an opening in the skull placed in the bregma -2.5 mm, 0.5 mm mid-lateral and dorsoventral 1.6 mm coordinates, in order to introduce the liquid near of the midline. The cannulas were then glued to the desired position and the pumps were opened so as to infuse the corresponding solutions at a flow rate of 0.5 μm per hour over seven days.

Two days after the protease infusion the rats were again anesthetized and introduced into the stereotactic support. 1.5 nmol kainic acid in 0.3 μl PBS was then injected unilaterally into the hippocampus. The coordinates were: bregma 2.5 mm, medial-lateral 1.7 mm and dorsoventral 1.6 mm. Excitoxin (KA) was introduced for 30 sec. After treatment with kainic acid the needle remained for a further 2 min at these coordinates in order to prevent a backflow of the liquid. 5. Brain examination

Five days after injection of KA the animals were anesthetized 37 ΡΕ1622639 and transcardially perfused with 30 ml PBS, followed by 70 ml of a 4% paraformaldehyde solution, fixed for 24 hours in the same fixative, followed by an incubation in 30% sucrose for an additional 24 hours. Coronal sections (40 μm) of the brain were then cut with a freezing microtome, or stained with thionine (BDH, Australia) or prepared for immunohistochemical examination. 6. Quantification of neuronal loss rate in the hippocampus The quantification of neuronal loss in the CA1-CA3 hippocampal subfields was performed as previously described (Tsirka et al., 1995; Tsirka et al., 1996). Five cuts were prepared following the dorsal hippocampus of all treated groups, where the cuts effectively covered the KA injection site and the lesion region. The hippocampal sub-fields (CA1-CA3) of these cuts were followed through reproductions of the lucid camera of the hippocampus. The total length of these subfields was measured in comparison with 1 mm patterns, which were followed with the same increase. The lengths of tissue sections with vital pyramidal neurons (with normal morphology) and the length of tissue sections without neurons (no cells present, no thionine staining) were measured. Lengths, intact neurons and neuronal losses in the region of subfield of the hippocampus, the means between the sections were determined and the standard deviation was determined. 38 ΡΕ1622639

7. Intra-striatal NMDA lesions-excitotoxicity with or without t-PA or DSPA

Wild-type rats (c57 / Black6) were anesthetized and placed on stereotactic support (see above). The rats then received an injection

unilateral in the left stratum with 50 nmol NMDA alone or in combination with 4 6 μΜ rt-PA or 46 μΜ DSPAα. As a control t-PA and DSPA were also injected alone at a concentration of 46 μΜ as controls. The injection coordinates were: bregma -0.4 mm, mid-lateral 2.0 mm and dorsoventral 2.5 mm. The solutions (1 μl total volumes for all treatments) were administered in a time period of 5 minutes at 0.2 μg / min, where the needle remained at the injection site 2 min after injection to minimize backflow of liquid. After 24 hours the rats were anesthetized and perfused transcardially with 30 ml PBS, followed by 70 ml of a 4% paraformaldehyde solution, fixed for 24 hours in the same fixative, followed by incubation in 30% sucrose for a further 24 hours. Coronal sections (40 μm) were then cut with a freezing microtome, and placed on gelatin coated slides.

8. Quantification of the injured volume after NMDA injection The quantification of the striatal injured volume was 39 ΡΕ1622639 performed according to the method described by Callaway et al. (2000). 10 coronal sections were prepared one after the other, covering the area of the lesion. The injured region was visualized according to the Callaway method and the lesion volume was quantified using a Micro Computer Imaging Device (MCID, Imaging Research Inc., Brock University, Ontario, Canada). 9. Immunohistochemistry Immunohistochemistry was performed according to standard methods. Coronal sections were immersed in 3% H2C solution > 2/10% methanol for 5 minutes, followed by incubation with 5% normal goat serum for 60 minutes. The sections were then incubated overnight or with an anti-GFAP antibody (1: 1,000; Dako, Carpinteria, Ca, USA) for astrocyte testing with an anti-MAC-1 antibody (1: 1,000, Serotec, Raleigh, NC, USA) for microglia or anti-DSPA polyclonal antibodies (Schering AG, Berlin). After washing the cuts were incubated with appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA). Subsequent incubation with an avidin / biotin complex (Vector Laboratories, Burlingame, CA, USA) was then continued for 60 minutes prior to final staining with 3,3'-diaminobenzidine / 0.03% H2O2. The cuts were then placed on gelatin coated slides, dried, dehydrated and covered with permount. 40 ΡΕ1622639

10. Testing for increased tissue damage induced by NMDA injection by intravenous administration with t-PA and DSPA

For induction of tissue lesions in the striatum, the mice were injected stereotactically with NMDA. 6 hours after NMDA injection, t-PA or DSPA (100 μΐ, 10 mg / kg) was given by injection into the caudal vein. As a negative control 100 μl 0.9% NaCl was injected and subsequently infused with PBS. After another 24 hours the animals were killed and the size of the neuronal tissue injury was determined. In a second experimental model the test groups with up to 15 mice were injected correspondingly with NMDA, t-PA or DSPA 24 h after injection of i.v. infused NMDA and the increase in tissue injury was correspondingly determined. For evidence of DSPA in brain tissue, sections were stained and stained with anti-DSPA antibodies according to standard methods. B. Results 1. Infusion of t-PA or DSPA is distributed by the hippocampus of t-PA - / - mice and retains their proteolytic activity

The first experiments were conceptualized to confirm that both DSPA and t-PA retain their proteolytic activity over the 7 days of infusion. To this end, aliquots of t-PA 41 ΡΕ1622639 and DSPA (100 nmol) were incubated at both 37øC and also at 30øC for seven days in a water bath. To determine proteolytic activity, 5-fold serial dilutions of the samples were subjected to SDS-PAGE under non-reducing conditions and the proteolytic activity measured by zymographic analyzes. Respectively an aliquot of t-PA and DSPA that remained frozen during these seven days, were used as control. As is apparent from Figure 1, there was only a slight loss of DSPA or t-PA activity, both in incubation at 25 ° C and also at 37 ° C during this time period. 2. The activity of t-PA and DSPA is also found after infusion into hippocampal extracts in t-PA - / -

First it had to be determined that the proteins administered with the infusion were actually present in the brains of the treated animals and also retained their proteolytic activity there. For this, t-PA- / - mice received infusions with t-PA or DSPA for seven days (see above). The rats were then killed with transcardial perfusion with PBS and the brains were removed. The ipsilateral and contralateral hippocampal regions were isolated, as was a region of the cerebellum (as a negative control). Tissue samples (20æg) were subjected to SDS-PAGE and zymographic analysis according to the description in the method part. As shown in Figure 2, the activities of both t-PA and 42 ΡΕ1622639 DSPA were measured in the ipsilateral region of the hippocampus, where in addition some activity was measured on the contralateral side. This demonstrated that the infused proteases not only retain their activity in the brain but have also been distributed throughout the hippocampus region. In the control, no activity was demonstrated in the prepared extracts of the cerebellum.

3. DSPA immunohistochemistry test

To prove that the DSPA had actually been distributed throughout the hippocampus region, t-PA - / - mice rats were immunohistochemically assessed after infusion of DSPA. DSPA antigens were tested in the hippocampus region, where the area surrounding the infusion site showed the strongest staining. This result confirms that the infused DSPA is soluble and is actually present in the hippocampus. 4. Infusion of DSPA does not reestablish sensitivity to kainic acid-dependent neurodegeneration In vivo

T-PA- / - mice are resistant to kainic acid-dependent neurodegeneration. In contrast, an infusion of rt-PA into the hippocampus leads to a complete reestablishment of sensitivity to the cainic acid dependent lesion. To answer the question, if DSPA can replace t-PA in this effect, t-PA - / - mice received infusions with t-PA ΡΕ1622639 PA or DSPA in the hippocampus through a miniosmotic pump. Twelve rats were used for both groups. Two days later the animals received injections of kainic acid with a subsequent rest period. Five days later the animals were killed. Brains were removed and worked up (see above). As a control, t-PA - / - mice were infused with PBS prior to KA treatment (n = 3).

Coronal brain sections were prepared and neurons were detected by Nissl staining. It became evident that the t-PA - / - mice after infusion of PBS were resistant to KA. In contrast, infusions with recombinant t-PA led to the reestablishment of sensitivity to KA treatment. In contrast, infusions with the same concentration of DSPA did not alter the sensitivity of these animals to KA (see Figures 4a and 4b) in the hippocampus region.

A quantification of these results occurred with the use of 12 animals in each group. In 2 of the 12 mice infused with DSPA we observed some neurodegeneration of little importance. The reason for this is still unclear and possibly independent of the presence of DSPA. The combined data took into account the minor effect that was observed in both these animals. All 12 mice treated with t-PA were sensitive to treatment with KA. These results show that with an infusion of t-PA or DSPA in equimolar concentrations, only the addition of t-PA leads to a restoration of sensitivity to KA-induced neurodegeneration. 5. Infusion of DSPA does not cause any microglia activation Sensitivity reestablishment to KA in t-PA - / - mice after infusion of t-PA has been shown to result in microglial activation (Rogove et al., 1999). In order to determine the size of the microglia activation after infusion of t-PA or DSPA and subsequent treatment with KA, coronal sections of the mice were subjected to immunohistochemical staining for activated microglia cells using the Mac-1 antibody . Restoration of the sensitivity to KA after infusion of t-PA resulted in a clear increase of Mac-1-positive cells. This was not observed in rats given infusions of DSPA. Thus the presence of DSPA does not lead to activation of the microglia cells after treatment with KA. The concentration of t-PA that was used in the infusion is based on the concentration described by Tsirka et al (1995) (100 μΐ of 0.12 mg / ml [ 1.85 μΜ] We repeated KA loading experiments using a 10-fold lower (0.185 μ quantidade) amount of t-PA and a higher amount of DSPA (18.5 μΜ) The concentration 45 ΡΕ1622639 of lower t-PA still had the ability to restore sensitivity to KA treatment (n = 3). Of particular interest was that infusion of the 10-fold higher concentration of DSPA only caused a small neuronal loss after treatment with KA And the effect of t-PA and DSPA on NMDA-dependent neurodegeneration in wild-type rats

The effects of t-PA and DSPA were further investigated in a model of neurodegeneration in wild-type mice. Injection of t-PA into the striatum of these mice demonstrates an increase in neurodegenerative effects caused by NMDA analogue to glutamate (Nicole et al., 2001).

For this, injections of NMDA in a total volume of Ιμΐ in the striatum region were given in wild-type rats in the presence of t-PA or DSPA (respectively 46 μΜ). After 24 hours the brains were removed and lesion size was quantified according to the Callaway method (Callaway et al, 2000) (see above). As shown in Figure 7, injection of NMDA alone caused reproducible lesion in all treated rats (n = 4). If t-PA and NMDA were, however, injected together, the lesion size increased by approximately 50% (P < 0.01, n = 4). In apparent opposition, co-injections of NMDA and 46 ΡΕ1622639 same concentrations of DSPA did not cause any increase in lesion size compared to NMDA alone.

Injections of t-PA or DSPA alone did not lead to verifiable neurodegeneration. The lack of effect of t-PA on single administrations is consistent with the results of Nicole et al. (2001). These data show that the presence of DSPA does not further enhance neurodegeneration even during a neurodegenerative process.

In order to confirm that the injection of DSPA had actually been distributed in the hippocampus region, immunohistochemical investigations were performed in coronal sections using the antibody for DSPA. Investigations show that DSPA actually penetrated the striatal region.

Kinetic analysis of plasminogen activation by indirect chromogen test

Indirect chromogen tests of t-PA activity used the Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) substrates and were performed according to Madison EL, 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. Natl. Acad. Know. U.S.A. 87, 3530-3533 as well as

Madison E.L., Goldsmith E.J., Gething M.J., Sambrook J.F., and 47 ΡΕ1622639

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 dissociation of highly purified human fibrinogen by the batroxobin protease. Batroxobin dissociates the Arg16-Gly17 linkage into the A? Chain of fibrinogen and thereby releases fibrinopent A. The resulting des-AA fibrinogen in the form of fibrin I monomers is soluble in the presence of the Gly-Pro-Arg-Pro peptide. The concentration of Lys-plasminogen was varied in the presence of DESAFIB of 0.0125 to 0.2 μ in the absence of the co-factor of 0.9 to 16 μΜ.

Indirect chromogen test in the presence of different stimulators

Indirect chromogen standard tests were performed according to the publications cited above. We used 100 μg total volume preparations with 0.25-1 ng enzyme, 0.2 μg Lys-plasmogen and 0.62 mM Spectrozym PL. Tests were performed in the presence of either buffer, 25æg / ml DESAFIB, 100æg / ml cyanogenic fibrinogen bromide fragments (American Diagnostica) or 100æg / ml of the 13 amino acid peptide stimulator P368. The analyzes were performed on microtiter plates and the optical density was measured at wavelengths of 405 nm for 1 h every 30 sec in a " Molecular Devices Thermomax ". The reaction temperature was 37 ° C. 48 ΡΕ1622639 δ. DSPA also does not induce in intravenous administration any increase of neuronal tissue lesions

Tissue lesions were induced in rat striatum by NMDA injection and administered intravenously 6 or 24 hours later t-PA or DSPA. Compared to a negative control, the animals tested showed an approximately 30% increase in tissue area damaged by NMDA injection in the iv infusion of t-PA 24 hours after NMDA injection, whereas DSPA did not cause such an increase in tissue injury (see Figure 18). By staining coronal sections with an anti-DSPA antibody it could be shown that DSPA administered intravenously 24 hours after NMDA injection had penetrated the damaged tissue areas (see Figure 19). In the corresponding intravenous administration of t-PA or DSPA 6 h after NMDA injection no increase in the damaged tissue area has yet been verified. This is possibly due to which blood-brain barrier at this time of administration of t-PA or DSPA still possessed a sufficient barrier function. Therefore DSPA does not present any neurotoxic side effect even in intravenous administration.

Lisbon, March 22, 2013

Claims (7)

The use of DSPAai for the production of a therapy for intravenous administration for the treatment of stroke in humans after three hours after the onset of stroke, characterized by a dosage of between 62.5 to 230 pg / kg. The use of DSPAI according to claim 1 for the therapeutic treatment of stroke in humans after six hours after the onset of stroke. The use of DSPAI according to claim 1 for therapeutic treatment of stroke in humans after nine hours after the onset of stroke. The use of DSPA3 according to claim 1 for the therapeutic treatment of stroke in humans between three hours and nine hours after the onset of stroke. The use of DSPAIâ "¢ according to one of the preceding claims, characterized by an intravenous infusion. 2 ΡΕ1622639 Use of DSPAal according to previous claims, characterized by a bolus. Use of DSPAal according to previous claims, characterized by a bolus injection (single bolus). Lisbon, March 22, 2013 one of the injection of one of the simple