HK1092045B - Intravenous injection of plasminogen non-neurotoxic activators for treating cerebral stroke - Google Patents

Description

The invention relates to the intravenous application of non-neurotoxic plasminogen activators, in particular genetically modified plasminogen activators and plasminogen activators from the saliva of Desmodus rotundus (DSPA), for the treatment of stroke in humans.

Clinic and biochemistry of stroke

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Ischemic stroke (ischemia) is a reduction or interruption of blood flow to the brain due to a lack of arterial blood supply, often caused by a thrombosis of an arteriosclerotically stenosed vessel, but also by arterial or cardiac embolism.

In contrast, hemorrhagic strokes are caused by perforation of arteries damaged by arterial hypertension, but only about 20% of all cerebral strokes are caused by this form of bleeding, so that strokes caused by thrombosis are by far the most important.

Neural tissue ischemia is associated with necrosis of the affected cells to a greater extent than other tissue ischemias. The increased incidence of necrosis can be explained by the phenomenon of excitotoxicity, which is a complex cascade of many reaction steps. It is thus triggered by the rapid loss and polarization of ATP by the ischemic neurons suffering from oxygen deficiency. This leads to an increased depoynaptic release of the neurotransmitter glutamate, which in turn activates membrane-bound neuronal glutamate receptors, which regulate blood channels.

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Even if the mechanism of glutamate-mediated neurotoxicity is not yet fully understood, there seems to be agreement that this phenomenon contributes to a significant extent to neuronal cell death following cerebral ischemia (Jin-Mo Lee et al.).

Treatment of stroke

In the treatment of acute cerebral ischemia, in addition to ensuring vital functions and stabilizing physiological parameters, the focus is first on the reopening of the closed vessel. This goal is pursued with various approaches.

The natural activation of plasminogen is by the body's plasminogen activators u-PA (urokinase type plasminogen activator) and t-PA (tissue plasminogen activator). The latter, unlike u-PA, together with fibrin and plasminogenesis, forms a so-called activator complex. The catalytic activity of t-PA is therefore very much fibrin-dependent and undergoes a 550-fold increase in the presence of fibrin. Fibrin can also stimulate the degradation of plasmin by the t-finger product (e.g. F-PA) - although it also stimulates the degradation of plasmin by its 25-fold increase in the activity of the t-finger product (e.g. F-PA).

Known therapeutic approaches (a) Streptokinase

Early approaches to thrombolytic treatment of acute stroke date back to the 1950s, but it was not until 1995 that the first large-scale clinical trials were conducted with streptokinase, a fibrinolytic made from beta-hemolytic streptococci.

Streptokinase therapy, however, has significant disadvantages, as streptokinase as a bacterial protease can cause allergic reactions in the body. Also, so-called streptokinase resistance may exist in previous streptococcal infection with corresponding antibody formation, which makes treatment difficult. In addition, clinical trials in Europe (Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter Acute Stroke Trial of Italy (MAST-I)) and Australia (Australian Streptokinase Trial (AST)) suggested such an increased risk of mortality and the significantly increased risk of intracintral bleeding (including intra-abdominal haemorrhage, ICH) after patients were treated with streptokinase, that these trials should be discontinued early.

(b) Urokinase

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(c) recombinant t-PA (rt-PA) is used in the manufacture of

There is extensive experience in the field of therapeutic thrombolysis with the tissue-type plasminogen activator, rt-PA (see EP 0 093 619, US Patent No 4 766 075), produced in recombinant hamster cells. t-PA was used in the 1990s as the main indication for acute myocardial infarction, and in a number of clinical trials worldwide, some of which have not yet been fully understood and with conflicting results.This led to the conclusion that thrombolytic treatment of rt-PA patients individually selected according to their medical history could potentially be beneficial immediately after the onset of stroke. However, due to the observed increased risk of intracerebral haemorrhage (ICH) within a few hours of stroke onset, general use of rt-PA within the six hours after the onset of stroke was not recommended (see C. Lewandowski and Wiliam Barsan, 2001: Treatment of Stroke; in Emergency of Medcine: Annals of Emergency; p.202 et seq.)

The thrombolytic treatment of stroke was also later the subject of a clinical trial conducted by the National Institute of Neurologic Disorder and Stroke (NINDS rtPA Stroke Trial) in the USA, but the focus was on the effect of intravenous rtPA treatment within only three hours of the onset of symptoms on patients' health after three months. Although the authors also found an increased risk of ICH, rtPA treatment within this limited period of three hours is still recommended due to the positive effects of this treatment on patients' life expectancy also identified in the study.

Two other studies (ECASS II trial; Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke (ATLANTIS)) investigated whether this positive statement about the effect of rt-PA treatment within three hours of stroke onset could be confirmed for treatment within the six-hour period, but this question could not be answered positively because treatment during this period, in addition to the increased risk of ICH, did not show an improvement in clinical symptoms or a reduction in mortality.

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These findings have led other authors to the rather sarcastic statement that stroke patients now have a choice between dying or surviving disabled (SCRIP 1997: 2265, 26).

However, rt-PA therapy is currently the only treatment approved by the Food and Drug Administration (FDA) in the United States for acute cerebral ischemia, but is limited to rt-PA within three hours of the onset of the stroke.

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Side effects of t-PA Neurotoxicity and excitatotoxicity

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Further results on the stimulation of excitotoxicity by t-PA were published by Nicole et al. in early 2001 (Nicole O; Docagne F Ali C; Margaill I; Carmeliet P; MacKenzie ET; Vivien D and 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 demonstrate that t-PA secreted by depolarized cortical neurons interacts with and cleavages the so-called NR1 subunit of the NMDA type glutamate receptor, which leads to an increase in receptor activity, which in turn increases the activation of the glutamate receptor by inducing a proliferation of the activator in the bloodstream.

The fact that despite these indications of neurotoxic side effects of t-PA and despite the proven increase in mortality due to t-PA, a drug authorization by the FDA was obtained can probably only be explained by the lack of safe and effective alternatives - and a very pragmatic cost-benefit analysis.

For this reason, further studies of known thrombolytics including DSPA (Desmodus rotundus plasminogen activator) to develop a new stroke therapy were not undertaken, although in principle all thrombolytics could be suitable and, for example, in the case of DSPA, its potential suitability for this indication was already indicated in an initial publication (Medan P; Tatumaklis T; Takano K; Carano RAD; Hadley SJ; Fisher M: Thrombosis with recombinant Desmodus saliva plasminogen activator (rDSPA) in an embolic stroke model; in: Cerexovascovasc Disrupt 1996:6; 175-194 (4th International Symposium on Thrombolic Therapy in Acute Ishish Stroke t).

Alternative therapies

Research into alternative therapeutic approaches is currently focused on, for example, anticoagulants such as heparin, aspirin or ancrod, the active substance in the venom of the Malaysian pit viper.

Another novel treatment method, which does not target thrombus, blood thinning or anticoagulation, but tries to increase the vitality of cells damaged by interruption of blood supply (WO 01/51613 A1 and WO 01/51614 A1). It uses antibiotics from the group of quinones, aminoglycosides or chloramphenicol. For a similar reason, it is also proposed to start immediately after a stroke with the administration of citicoline, which is broken down in the body into cytidine and choline. These breakdown products are part of the neuronal cell membrane and can thus aid the regeneration of damaged tissue (US Patent No. 527 8832).

Recent efforts to find safe treatments are based on the new findings that part of the fatal consequences of stroke are due only indirectly to interrupted blood supply but directly to excitatory or neurotoxicity involving the overexcited glutamate receptor, which in turn is particularly enhanced by t-PA (see p.o.). An approach to reducing this excitatory toxicity is the application of so-called neuroprotective agents. These may be used alone or in combination with fibrinolytics to minimize their neurotoxic effect.

Competitive inhibition (antagonisation) of the NMDA type glutamate receptor is possible with, for example, 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate (APH). Non-competitive inhibition can be achieved, for example, by substances that bind to the phencyclidine side of the channels, such as phencyclidine, MK-801, dextrorphan or ketamine.

However, so far treatments with neuroprotective agents have not produced the desired results, possibly because they would have to be combined with thrombolytics to provide their protective effect.

However, even a combination of t-PA and a neuroprotective drug can only be a success in terms of harm reduction, but it does not eliminate the disadvantages of the neurotoxicity of the fibrinolytic drug itself.

Other, not chemically defined

From the international patent application WO 03/037363, the disclosure of which is fully referred to, plasminogen activators for the treatment of stroke are known, whose enzyme activity is increased by fibrin in a highly selective manner by many times, namely by more than 650 times.

The nature and use of these plasminogen activators is based on the understanding that the neurotoxicity of the tissue plasminogen activator (t-PA) is due to the fact that, as a result of tissue damage in the brain caused by stroke, the blood-brain barrier is damaged or destroyed and the fibrinogen circulating in the blood can thus enter the neuronal tissue of the brain, where it activates t-PA, which leads to further tissue damage (e.g. indirectly by activating the glutamate receptor or by activating plasminogen).

To avoid this effect, a plasminogen activator is used, which has increased fibrin selectivity and, conversely, reduced activation by fibrinogens. It follows that these plasminogen activators are not activated, or to a significantly reduced extent compared to t-PA, when fibrinogen is transferred from the blood to the neuronal tissue as a result of damage to the blood-brain barrier, since their activator fibrin cannot enter the neuronal tissue due to its size and insoluble nature. These plasminogen activators are therefore non-neurotoxic.

(a) Genetically modified plasminogen activators

For example, non-toxic plasminogen activators are used, which have at least one element of a so-called zymogen triad. A similar triad is known from the catalytic centre of serine proteases of the chymotrypsin family, which consists of the three interacting amino acids aspartate 194, histidine 40 and serine 32. This triad is not present in t-PA, which belongs to the family of chymotrypsin-like serine proteins. However, it is known that the targeted mutagenesis of native t-PA to introduce at least one of these amino acids into suitable positions leads to a reduction in the activity of the pro-enzyme (eosinophilic t-PA) and an increase in the activity of the enzyme (eosinophilic t-PA) - at least in the case of the introduction of the two types of the enzyme - to a significant increase in the activity of the pro-enzyme (eosinophilic t-PA) - by introducing a corresponding ratio of amine/tribromine in the presence of the most active amino acids (eosinophilic t-PA) - and the enzyme (t-PA) - in the presence of the most active amino acids (eosinophilic t-pa) - to a significant increase in the activity of the activity of the enzyme (t-PA) - in the presence of the two types of the amine/tribromine-pa - in the enzyme (t-pa) - in the case of the most active amino acids.

Thus, it is known that the mutagenesis of native t-PA to substitute Phe305 for His (F305H) and Ala 292 for Ser (A292S) results in a 20-fold increase in cytogenetic activity, whereas the F305H variant alone produces a 5-fold increase (EL Madison, Kobe A, Gething M-J, Sambrook JF, Goldsmith EJ 1993: Converting Tissue Plasminogen Activator to a Zymogen: A regulatory Triad of Asp-His-Ser; Science: 262, 419-421). These t-PA mutants, in the presence of fibrin, show an increase in cytogenetic activity of 30,000 t-plastic tissue (F305H) and 130,000 t-plastic tissue (F305H) respectively (FK2752A, which shows a dramatic increase in the activation of T-A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2A2

The positions 305 and 292 of t-PA are homologous to the positions His40 and Ser32 of the known triad of chymotryptic serine proteases.

These t-PA mutants can be used for stroke treatment because of their increased fibrin specificity, they have no or only a significantly reduced neurotoxicity compared to the wild t-PA. For the purpose of identifying the mentioned t-PA mutants F305H; F305H, A292S alone or in combination with R275E, full reference is made to the publications of Madison et al. 1993 and Tachias and Madison 1995.

Alternatively, the increase in fibrin specificity of plasminogen activators is also possible by a point mutation of Asp194 (or an aspartate at homologous position). Plasminogen activators belong to the group of serine proteases of the chymptrypsin family and correspondingly contain the conserved amino acid Asp194, which is responsible for the stability of the catalytically active conformation of the mature proteases. Asp194 is known to interact with His40 in the zymogens of the serine proteases. The activating cleavage of the zymogen makes these interactions impossible, and the side chains of Asp194 rotate about 170° with a new Ileucine T16 to form a new Ileucine T16 bridge. This is also due to the stability of the saline salt in the tricyclic acid chain of the serine protease.

A point mutation of Asp194 makes the formation or stability of the catalytic conformation of the serine proteases initially impossible. Nevertheless, the mutated plasminogen activators show a marked increase in activity in the presence of their co-factor fibrin - especially compared to the mature wild-type form - which can only be explained by the fact that the interactions with fibrin allow a conformational change that allows a catalytic activity (L Strandberg, Madison EL, 1995: Variants of Tissue-type Plasminogen Activator with Substantially Enhanced Response and Selectivity towards Fibrin Co-factors. in: Journal of Biological Chemistry 270, 40: 2344-23449).

A preferred example of such a non-neurotoxic plasminogen activator is t-PA, whose Asp194 is substituted by glutamate (D194E) or asparagine (D194N) respectively, which reduces t-PA activity by a factor of 1-2000 in the absence of fibrin, while in the presence of fibrin an increase in activity of a factor of 498,000 to 1,050,000 can be achieved. These mutants may also contain a substitution of argin15 to R15E, which prevents the splitting of the single t-PA into two-fold t-PA at the argin15-ile16 peptide link by plasmin. This mutation alone increases activation of t-PA by fibrin by a factor of 12,000.

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In a preferred form, a t-PA with point mutations is used in positions 420-423. If these residues are replaced by targeted point mutations, the fibrin dependence of the 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, S421 G, S421 E, P422A, P422G, P422E, F423A and F423E. The publication is hereby fully considered for use in accordance with the invention.

Another advantageous variant uses a modified tissue plasminogenin activator with an amino acid sequence according to SEQ. ID No.1 (Fig. 13) which differs from the wild-type t-PA by replacing the hydrophobic amino acids in positions 420-423 in the autolysis loop, which are occupied as follows: His420, Asp421, Ala422 and Cys423. This t-PA prefers a phenylalanine at position 194. In addition, position 275 can be occupied by a glutamate.

Furthermore, a modified urokinase can be used. This urokinase can have the amino acid sequence according to SEQ. ID No. 2 (Fig. 14) in which the hydrophobic amino acids of the autolysis loop are replaced by Val420, Thr421, Asp422, and Ser423.

Both mutants - both urokinase and t-PA - were studied in semi-quantitative tests and showed increased fibrin specificity compared to wild-type t-PA.

(b) Plasminogen activator from Desmodus rotundus (DSPA) and other products of the same heading as the product

A high increase in activity in the presence of fibrin, namely an increase of 100,000 times, is also shown by the plasma activator (DSPA) from the saliva of the vampire bat (Desmodus rotundus), which can therefore be used as a non-neurotoxic plasma activator. DSPA is the term used to describe four different proteases which fulfil a basic requirement of the vampire bat, namely the prolonged bleeding time of wounds caused by these animals in prey (Cartwright, 1974). These four uppermost proteases (DSPAα1, DSPAα2, DSPAγβ, DSPA04) show a consistent high similarity (homology) to human pathogens. They show or are similar in physiological activity. The term is taken in its entirety in relation to the US Pathogenesis Directive 89/398/EEC and the subject of the Directive 89/398/EEC.

DSPAα1 is the best-studied protease of this group, with an amino acid sequence homology of over 72% to the known human t-PA amino acid sequence (Kratzschmar et al., 1991). However, there are two major differences between t-PA and DSPA: first, DSPA is a fully active molecule, as a single chain to peptide substrates, which is not translated into a binary form (Gardell et al., 1989; Krätzschmar et al., 1991). Second, the catalytic activity of DSPA shows a near-absolute fibrin dependence (Gardell et al., 1989; Bringmann et al., 1995; Sofrin et al., 1995; Frin et al., 1995).

Although DSPA is an interesting candidate for thrombolytic development due to its fibrinolytic properties and its close similarity to t-PA, due to its involvement in glutamate-dependent neurotoxicity, there were no legitimate hopes for the successful use of a closely related plasminogen activator for the treatment of acute stroke.

However, it has now been surprisingly shown that despite its high similarity (homology) to t-PA and despite the broad consistency of its physiological effects with t-PA, DSPA does not have any neurotoxic effects. This finding is directly linked to the finding that DSPA can be used as a thrombolytic for stroke therapy without an additional risk of damage to neuronal tissue.

Experimental evidence of the absence of neurotoxicity of DSPA

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The casein acid model (also casein acid injury model) is based on the fact that the neurotoxic glutamate cascade is stimulated by the application of external casein acid (KA) as an agonist of the casein acid type (KA) glutamate receptor and NMDA and AMPA glutamate receptors. Using a t-PA deficient mouse strain as a test model, it was shown that the sensitivity of the test animals to casein acid only with additional administration of external t-PAs reached the level of wild type mice.

Quantitative studies on this model showed that even a 10-fold increase in the concentration of DSPA did not restore the sensitivity of t-PA-deficient mice to KA treatment, while already a 10-fold decrease in t-PA concentration caused KA-induced tissue damage.

The second model of neurodegeneration compared the potential effects of t-PA and DSPA on the promotion of NMDA-dependent neurodegeneration in wild-type mice by injecting wild-type mice with NMDA (as an agonist of the NMDA-type glutamate receptor) alone or in combination with t-PA or DSPA, which allows the study of the effect of these proteases under conditions where neurodegeneration occurs anyway and an influx of plasma proteins is induced by the breakdown of the blood-brain barrier (Cro et al., 1999).

In this model, NMDA injection resulted in reproducible lesions in the striatum of the mice. The volume of these lesions was increased by at least 50% by a joint injection of t-PA and NMDA. By contrast, co-injection with DSPAα1 did not result in an increase in the size of the lesions caused by NMDA injection. Even in the presence of plasma proteins that could diffuse into the lesions area as a result of NMDA-dependent neurodegeneration, DSPA did not result in an increase in neurodegeneration. A summary of these results is shown in Fig. 16 (Table 2).

Initial results from clinical trials show the transferability of these results to the treatment of stroke in humans, with significant improvements in patients after successful perfusion (up to 8 points NIHSS or HIHSS score 0 - 1).

In another experiment, compared to a negative control, mice that were not given t-PA and DSPA 24 hours after NMDA showed an approximately 30% increase in the area of tissue damage caused by NMDA, while DSPA did not show any results on the extent of tissue damage caused by NMDA, and no evidence of increased antibodies in the area of blood damage caused by t-PA or DSPA was found (although this may not be the case with a new dose of DSPA 18 or DSPA 19).

Therapeutic potential of non-neurotoxic plasminogen activators

Due to the lack of neurotoxicity of DSPA and other non-neurotoxic plasminogen activators (NPAs), a particular advantage for stroke treatment is that, unlike the wild type t-PA, the use of these plasminogen activators is not limited to the narrow window of up to three hours after the onset of the stroke, but can be continued at a later time, i.e. six hours or more, without the risk of promoting exotoxicity as with t-PA.

This unlimited treatment option with non-neurotoxic plasminogen activators is particularly important because it will for the first time make it possible to treat acutely stroke patients without any concerns, in whom the onset of the stroke cannot be determined with sufficient certainty in time.

The applicant shall provide the technical documentation referred to in point (a) of Article 4 (1) of this Regulation.

Unlike the established stroke therapy drug rt-PA, there is no established evidence of a possible dose for the treatment of stroke with the non-neurotoxic plasminogen activators.

The purpose of the invention is therefore to provide a beneficial dosage form for these non-neurotoxic plasminogen activators.

According to the invention, plasminogen activators, whose activity is increased by more than 650 times in the presence of fibrin, are administered intravenously for the treatment of stroke.

The intravenous use of these non-neurotoxic plasminogen activators for stroke treatment has already been confirmed in clinical trials where DSPA, as an example of this group of fibrinolytics, was administered intravenously to patients and caused only minor adverse reactions.

These results from the clinical trials were unexpected as intravenous administration of t-PA and other common fibrinolytics was known to be associated with a high risk of cerebral haemorrhage (see above).

In order to reduce intra-cerebral bleeding, strategies have recently been developed to apply these substances intravenously rather than intravenously via a catheter directly near the intravascular thrombus.

The significant advantages of intra-arterial administration are however countered by two possible disadvantages: on the one hand, it requires time-consuming patient preparation, which is not possible for stroke treatment within the available time window of only 3 hours; on the other hand, although a lower total dose can be achieved, a higher concentration of the therapeutic drug reaches the arterial end vessels locally.

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However, the limitations of the narrow window of time and the tissue-damaging side effects which make intra-arterial application difficult do not exist in the plasminogen activators used in accordance with the invention. Therefore, due to their indisputable advantages (see above), intra-arterial application is in principle a very promising form of administration for non-neurotoxic plasminogen activators.

However, the notifier also used the rather problematic intravenous application for the non-neurotoxic plasminogen activators, which surprisingly proved to be beneficial.

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The plasminogen activators used in accordance with the invention are either animal-derived foreign proteins (e.g. DSPA) or genetically modified foreign proteins which have novel epitopes due to structural changes, as opposed to t-PA, which is normally administered intravenously, and the associated problem of allergic reactions, particularly when high therapeutic doses are used, is also present in other fibrinolytics based on foreign proteins, such as streptokinase.

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

Clinical studies have shown that intravenous administration at surprisingly low therapeutic doses also produces beneficial results. For example, preferential therapeutic results have been achieved at doses between 90 μg/kg and 230 μg/kg. Particularly beneficial therapeutic doses were 62.5 to 90 μg/kg. The time between stroke and administration of the therapeutic was between 3 and 9 hours in the patients studied. Appropriate test methods were used to determine the onset of a therapeutic effect (see Figures 20 to 29).

While DSPA and the other non-neurotoxic plasminogen activators themselves do not exhibit neurotoxic side effects, it may be beneficial to use them in combination with a neuroprotective drug to treat stroke in order to minimise tissue damage caused by the body's own glutamate, and to do so with competitive or non-competitive inhibitors of the glutamate receptor. Useful combinations are possible, for example, with the known inhibitors of the NMDA, ketone acid or quisquamate type of glutamate receptors, such as APV, APB, phenylpropion, AP-801, dextrorphan or MKB.

Similarly, a combination with cations may be beneficial, as cations, especially Zn ions, may block the glutamate receptor-regulated cation channel and thus reduce neurotoxic effects.

In another advantageous embodiment, the non-neurototoxic plasminogen activators may be combined with at least one other therapeutic agent or with a pharmaceutically safe carrier. Particularly advantageous is the combination with a therapeutic agent that helps to prevent tissue damage by revitalizing the cells, contributes to the regeneration of already damaged tissue or prevents subsequent strokes. For example, combinations with antibiotics such as quinones, anticoagulants such as heparin or hirudin, and with citicoline or aspirin may be advantageous.

In addition, a combination with at least one thrombin inhibitor may be beneficial, preferably thrombomodulin, thrombomodulin analogues such as solulin, triabin or pallidipin, and combinations with anti-inflammatory agents that affect leukocyte infiltration.

The following is intended to illustrate the application and/or dosage according to the invention by means of specific therapeutic examples.

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Wild type mice (c57/Black 6) and t-PA-deficient mice (c57/Black 6) (Carmeliet et al., 1994) were provided by Dr. Peter Carmeliet, Leuven, Belgium.

Protein extraction from brain tissue

The hippocampal region was removed, given to Eppendorf vessels and in equal volumes (w/v) (approximately 30-50 μl) with 0.5% NP-40 lysophore without protease inhibitors (0.5% NP-40, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA) and the remaining samples were incubated by hand-operated glass homogeniser and then subjected to 30 minutes of ice-proofing.

The results of the tests were published in the Official Journal of the European Communities.

The proteolytic activity in the samples or extracts of the yeast tissue was determined by zymographic analysis according to the method of Granelli-Piperno and Reich (1974) whereby the samples of the recombinant protein (up to 100 nmol) or the yeast tissue extract (20 μg) were subjected to a 10% SDS PAGE under non-reducing conditions. The gels were removed from the plates, washed for two hours in 1% Triton X100 and then placed on an agar gel with polymerized fibrinogen and plasminogen (Granelli-Piperno and Reich, 1974).

Intrahippocampal infusion of t-PA, DSPA and subsequent injection of cainic acid

The cacinic acid injury model is based on the approach of Tsirka et al. (1995). The animals were injected intraperitoneally (i.p.) with atropine (4 mg/kg), then i.p. with sodium pentobarbital (70 mg/kg). They were then placed in a stereotactic setting, so that a microosmotic pump (Alzet model 1007D, Alzet, USA) containing 100 μl PBS or recombinant human t-PA (0.12 mg/ml; 1.85 μM) or DSPAα1 (1.85 μM) could be implanted subcutaneously between the shoulder blades. The pumps were connected by a sterile tube with a red tube and connected by an opening at the coastal midrib, approximately 0.5 mm (0.5 mm) per day, to the central canal, and after 1.6 hours, they were placed in a position to absorb the fluid at a rate of 0.5 μm per day and 1.6 μm per hour.

Two days after the protease infusion, the mice were re-anesthetised and placed in the stereotactic range. 1.5 nmol of caesium in 0.3 μl PBS were then injected unilaterally into the hippocampus. The coordinates were bregma 2.5 mm, medial-lateral 1.7 mm and dorsoventral 1.6 mm. The excitotoxin (KA) was administered for 30 sec. After the caesium treatment, the needle was left at these coordinates for another 2 min to prevent re-flow of fluid.

5. Brain examination

Five days after the KA injection, the animals were sedated and infused transcardially with 30 ml PBS, followed by 70 ml of a 4% paraformaldehyde solution, fixed in the same fixative for 24 hours, followed by incubation in 30% sucrose for another 24 hours.

Quantifying the rate of neuronal loss within the hippocampus

The quantification of neuronal loss in the CA1-CA3 hippocampus subfields was performed as described above (Tsirka et al. 1995; Tsirka et al. 1996). Five consecutive sections of the dorsal hippocampus from all treated groups were prepared, with the sections actually covering the site of KA injection and the area of the lesion. The hippocampus subfields (CA1-CA3) of these sections were tracked using camera lucida drawings of the hippocampus. The total length of these subfields was measured compared to 1 mm standards tracked at the same magnification. The lengths of the pyramidal tissue sections (with vitals) and the lengths of the tissue sections without morphine (without neurons) were determined using the standard and non-invasive neuron loss between the hippocampus and the neural tube.

Intra-striatal NMDA excitoxicity lesions with or without t-PA or DSPA

The mice were then given a unilateral injection into the left stratum of 50 nmol NMDA alone or in combination with 46 μM rt-PA or 46 μM DSPAα1 as a control, and t-PA and DSPAα1 alone were injected as controls at a concentration of 46 μM. The injection coordinates were: bregma mid-0.4 mm, 2.0 mm lateral and 2.5 mm dorsov. The solutions (1 μl total volume for treatments) were administered over a period of 5 minutes at 0.2 μl/ min, with the needles being followed by a further 2 min injection at the injection site to minimise the backscatter.

8. Quantification of lesion volume after NMDA injection

The quantification of striatal lesion volume was performed using the method described by Callaway et al. (2000), which prepared 10 consecutive corona sections covering the area of the lesion, visualized the affected region using the Callaway method, and quantified the lesion volume using a Micro Computer Imaging Device (MCID, Imaging Research Inc., Brock University, Ontario, Canada).

Immunohistochemistry

The sections were then incubated overnight with either an anti-GFAP antibody (1:1,000; Dako, Carpinteria, Ca, USA) for detection of astrocytes, with an anti-MAC-1 antibody (1:1,000; Serotonin, Raleigh, Dara, NC) for detection of microglia or polyclonal anti-DSPA antibodies (Schering AG, Berlin, USA). After washing, the sections were incubated with the appropriate biotamped secondary antibodies (Vector Laboratories, Burlingame, USA). The sections were then incubated in a dehydrogenated A/B.

Test for NMDA-induced tissue damage amplification by intravenous application of t-PA and DSPA

To induce tissue damage in the striatum, mice were stereotactically injected with NMDA. 6 hours after NMDA injection, t-PA or DSPA (100 μl; 10 mg/kg) were applied by injection into the tail vein. As a negative control, 100 μl 0.9% NaCl was injected and then PBS infused. After another 24 h, the animals were killed and the extent of neuronal tissue damage determined.

B. Results Infusion of t-PA or DSPA is distributed through the hippocampus of t-PA-/ mice and maintains their proteolytic activity.

The first trials were designed to confirm that both DSPA and t-PA retained their proteolytic activity over the 7-day duration of the infusion. To this end, aliquots of t-PA and DSPA (100 nmol) were incubated in a water bath at both 37°C and 30°C for seven days. To determine the proteolytic activity, serial 5-fold dilutions of the SDS-PAGE samples were subjected to non-reducing conditions and the proteolytic activity was measured by zymographic analyses. Each aliquot of t-PA and DSPA that remained frozen over these seven days was used as a control. As shown in Figure 1, there was only a small loss or reduction in t-PAC activity at both 37°C and 25°C.

2. t-PA and DSPA activity is also detected in hippocampal t-PA extracts from mice -/- after infusion

The ipsilateral and contralateral hippocampal regions were isolated, as well as a region of the cerebellum (as a negative control). T-PA-/- mice were given seven days of t-PA or DSPA infusions (see below) and the brains were removed. The ipsilateral and contralateral hippocampal regions were isolated, as well as a region of the cerebellum (as a negative control). Female samples (20 μg) were subjected to an SDS-PAGE and cymographic analysis as described on page 2.

Immunohistochemical evidence of DSPA

To demonstrate that DSPA had indeed spread through the hippocampus region, coronal brain sections of t-PA-/ mice following DSPA infusion were immunohistochemically analysed, detecting DSPA antigens in the hippocampus region, with the most intense staining around the infusion site, confirming that the infused DSPA is soluble and indeed present in the hippocampus.

4. DSPA infusion does not restore sensitivity to caic acid-dependent neurodegeneration in vivo

In order to answer the question of whether DSPA can replace t-PA in this effect, t-PA-/- mice were infused with t-PA or DSPA into the hippocampus by means of a miniosmotic pump. For both groups, 12 mice were used. Two days later, the animals received injections of caic acid during a subsequent rest period. Five days later, the animals were killed. The brains were removed and processed (see also as control t-PA-/- mice, KABS was infused with P=n3).

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

These results were quantified using 12 mice in each group. In 2 of the 12 mice infused with DSPA, we observed some minor neurodegenerations. The reason for this is still unclear and possibly independent of the presence of DSPA. The combined data account for the minor effect seen in these two animals. All 12 t-PA treated mice were sensitive to KA treatment. These results show that when t-PA or DSPAα1 is infused at equimolar concentrations, only administration of t-PA leads to a restoration of sensitivity to KA-induced neurodegeneration.

5. DSPA infusion does not cause microglia activation

To determine the extent of microglia activation after t-PA or DSPA infusion and subsequent KA treatment, coronal sections of mice were immunohistochemically stained for activated microglia cells using the Mac-1 antibody. The restoration of sensitivity after t-PA infusion resulted in a clear increase in Mac-1 positive cells. This was not observed in the mice that received DSPA infusions.

Titration of DSPA and t-PA in the hippocampus region of mice

The concentration of t-PA used for 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 KA load experiments using a 10-fold lower t-PA (0.185 μM) and a higher DSPA (18.5 μM). The lower t-PA concentration was still able to restore sensitivity to KA treatment (n=3). Of particular interest was that infusion of the 10-fold increased DSPA concentration caused only slight neuronal loss after KA treatment. These data further indicate that DSPA does not increase sensitivity to KA.

Effect of t-PA and DSPA on NMDA-dependent neurodegeneration in wild-type mice

The effects of t-PA and DSPA have also been studied in a model of neurodegeneration in wild-type mice, where the injection of t-PA into the striatum of these mice has been shown to enhance the neurodegenerative effects induced by the glutamate analogue NMDA (Nicole et al., 2001).

In this study, NMDA injections with a total volume of 1 μl were administered into the striatum region of wild-type mice in the presence of t-PA or DSPA (46 μM each). After 24 hours, the brains were removed and the size of the lesions were quantified using the Callaway method (Callaway et al., 2000). As shown in Figure 7, NMDA injection alone caused a reproducible lesion in all treated mice (n=4). However, when t-PA and NMDA were co-injected, the size of the lesions increased by approximately 50% (P< 0.01, n=4).

The absence of action of t-PA alone is consistent with the results of Nicole et al. (2001) These data show that the presence of DSPA does not further increase neurodegeneration even during a neurodegenerative event.

To confirm that the injection of DSPA did indeed spread to the hippocampus region, immunohistochemical studies were performed on coronary sections using the DSPA antibody, which showed that DSPA did indeed enter the striatal region.

Kinetic analysis of plasminogens Activation by indirect chromogen assay

Indirect chromogenic tests of t-PA activity use the substrates Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica) and were performed in accordance with Madison E.L., Goldsmith E.J., Gerard R.D., Gething M.-J., Sambrook J.F. (1989) Nature 339 721-724; Madison E.L., Goldsmith E.J., Gerard R.D., Gething M.J., Sambrook J.F. and Bassel-Duby R.S. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3530-3533 and Madison E.L., Goldsmith E.J., Gething M.J., Sambrook A.F. and Gerard J.D. (1990) Biochemical 2626-2235, Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrinol Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fibrin Fib

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 chromogen testing in the presence of various stimulators

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8. DSPA does not cause neuronal tissue damage even when administered intravenously

Compared to a negative control, animals administered t-PA 24 hours after NMDA showed an approximately 30% increase in the area of tissue damaged by NMDA, whereas DSPA did not cause such an increase in tissue damage (see Figure 18). When coronary section staining with an anti-DSPA antibody was performed, it was demonstrated that the t-PA administered 24 hours after NMDA was administered had some effect on the affected areas (see Figure 19).

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Claims (7)

1. Use of DSPAα1 for producing a therapeutic agent for intravenous application for the treatment of stroke in humans after three hours have elapsed since the onset of the stroke, characterised by a dose of from 62.5 to 230 µg/kg.
2. Use of DSPAα1 according to claim 1 for the therapeutic treatment of stroke in humans after six hours have elapsed since the onset of the stroke.
3. Use of DSPAα1 according to claim 1 for the therapeutic treatment of stroke in humans after nine hours have elapsed since the onset of the stroke.
4. Use of DSPAα1 according to claim 1 for the therapeutic treatment of stroke in humans between three hours and nine hours after the onset of the stroke.
5. Use of DSPAα1 according to any of the preceding claims, characterised by an intravenous drip.
6. Use of DSPAα1 according to any of the preceding claims, characterised by a bolus injection.
7. Use of DSPAα1 according to any of the preceding claims, characterised by a single bolus injection.