JP2012077090A - Intravenous injection of non-neurotoxic plasminogen activator for treating cerebral infarction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide intravenous injection of a non-neurotoxic plasminogen activator for treating cerebral infarction.SOLUTION: There is provided an intravenous application of non-neurotoxic plasminogen activating factors especially a genetically modified plasminogen activating factors of human origin, and plasminogen activator (DSPA) derived from the saliva of a Desmodus rotundus to therapeutically treat human cerebral stroke. A preferable embodiment is the use of a nontoxic plasminogen activator which contains at least one of the components of so-called zymogen triade.

Description

  The present invention treats non-neurotoxic plasminogen activator, in particular, genetically modified plasminogen activator and plasminogen activator (DSPA) from Desmodus rotundus saliva to treat human cerebral infarction In order to apply intravenously. Treatment of cerebral infarction with these plasminogen activators is known from International Patent Application No. PCT / EP02 / 12204, the entire disclosure of which is incorporated by reference.

(Clinical features and biochemistry of cerebral infarction)
Various clinical features are summarized in the term “cerebral infarction” associated with clinical symptoms. Depending on the pathogenesis, first of all, these clinical features can be differentiated between so-called ischemic attacks and hemorrhagic attacks.

  Ischemic stroke (ischemia) is characterized by a reduction or inhibition of blood circulation in the brain due to the absence of arterial blood supply. This is often caused by arteriosclerotic constricted vascular thrombus or by arterial individual arterial embolism.

  Hemorrhagic seizures are due, inter alia, to perforations in the brain-supplied arteries damaged by arterial hypertonia. However, about 20% of all brain attacks are only due to hemorrhagic attacks. Thus, thrombotic attacks are much more relevant.

  Compared to other tissue ischemia, neural tissue ischemia is extensively associated with necrosis of the affected cells. The high incidence of necrosis in neural tissue can be explained by a complex cascade involving multiple reaction steps, the newly elucidated “excitotoxicity”. This cascade is initiated by ischemic neurons that immediately lose ATP and depolarize when they suffer from oxygen deprivation. As a result, post-synaptic release of glutamate, a neurotransmitter that activates membrane-bound glutamate receptors that control cation channels, is increased. However, increased glutamate release results in overactivation of the glutamate receptor.

The glutamate receptor regulates voltage-gated cation channels that are opened by glutamate binding to the receptor. As a result, influx of Na ⁺ and Ca ²⁺ into the cell occurs, resulting in massive inhibition of Ca ²⁺ -dependent cell metabolism. In particular, the activation of Ca ²⁺ -dependent catabolism can cause subsequent cell death (Lee, Jin-Mo et al., “The changing landscape of ischemic brain injury mechanisms”; Dennis W. Zholit neurato diseases of the nervous system ").

  The mechanism of glutamate-mediated neurotoxicity has not yet been fully elucidated, but there is consensus that it contributes to a considerable extent to neuronal cell death after cerebral ischemia (Jin-Mo Lee, et al. .).

(Treatment of cerebral infarction)
In the treatment of acute cerebral ischemia, priority is given to resuming closed blood vessels in addition to maintaining biological functions and stabilizing physiological parameters. This resumption can be performed by various means. For example, mere mechanical resumption, such as PTCA after a heart attack, has so far not been satisfactory. Only by successful fibrinolysis can the patient's health be improved to an acceptable level. This can be done by topical application with a catheter (PROCAT, study with prourokinase). However, despite the first successful results, this method has not yet been officially approved as a drug treatment.

  Natural fibrinolysis is due to the proteolytic activity of plasmin, which is a serine protease and is generated from its inactive precursor plasminogen by catalysis (activation). The natural activation of plasminogen is caused by u-PA (urokinase-type plasminogen activator) and t-PA (tissue plasminogen activation) which are naturally occurring plasminogen activators in vivo. Catalyzed by a factor). In contrast to u-PA, t-PA forms a so-called activator complex with fibrin and plasminogen. Thus, the catalytic activity of t-PA is fibrin dependent and is enhanced about 550-fold when it is present. In addition to fibrin, fibrinogen, though to a lesser extent, can promote t-PA catalysis from plasminogen to plasmin. In the presence of fibrinogen, t-PA activity is only enhanced 25-fold. In addition, a fibrin cleavage product (fibrin degradation product (FDP)) promotes t-PA.

(Known treatment)
(A) Streptokinase)
Early attempts to treat thrombolysis of acute cerebral infarction date back to the 1950s. The first extensive clinical trials using streptokinase, a fibrinolytic factor derived from β-streptococci, has just begun in 1995. Streptokinase, together with plasminogen, forms a complex that catalyzes other plasminogen molecules to plasmin.

  Because streptokinase is a bacterial protease and can induce allergic reactions in the body, treatment with streptokinase is associated with significant disadvantages. Furthermore, if there is a previous infection with streptococci and antibody production, the patient may exhibit so-called streptokinase resistance, making treatment more difficult. In addition, clinical trials in Europe (European Multicenter Acute Cerebral Infarction Trial (MAST-E), Italian Multicenter Acute Cerebral Infarction Trial (MAST-I) and Australia (Australian Streptokinase Trial (AST)) The risk of death increased after the patient was treated with a higher risk of cerebral hemorrhage (hemorrhage in the brain, ICH), and these trials had to be terminated early.

(B) Urokinase)
Alternatively, urokinase, which is also a classic fibrinolytic factor, can be applied. In contrast to streptokinase, it is an enzyme that occurs naturally in various body tissues and therefore does not exhibit antigenic properties. It is an activator of plasminogen and does not depend on cofactors. Urokinase is produced in kidney cultured cells.

(C) Recombinant t-PA (rt-PA))
A wide range of experimental results on thrombolytic therapy is the so-called rt-PA (European Patent No. 0093619, US Pat. No. 4,766,075), a tissue-type plasminogen activator produced in recombinant hamster cells. No.) is available. In the 1990s, several global clinical trials using t-PA were conducted with acute myocardial infarction as the main indication, but with some inconsistent results that were partially unintelligible. In the so-called European acute cerebral infarction test (ECASS), rt-PA was administered intravenously within a time frame of 6 hours after the onset of cerebral infarction. After 90 days, mortality and Barthel index were examined as an index for survival independent of patient disability and treatment. Not only was there no significant improvement in survival, but it was not significant, but an increase in mortality was reported. Therefore, it can be concluded that it may be advantageous to treat thrombolytic treatment with rt-PA for patients individually selected according to their medical history immediately after the onset of cerebral infarction. However, the general use of rt-PA within the time frame of 6 hours after onset of cerebral infarction was not recommended. This is because application within this time increases the risk of intracerebral hemorrhage (ICH) (C. Lewandowski C and William Barsan, 2001: Treatment of Act Stroke; in: Anals of Emergency Medicine; .).

Thrombolytic treatment of cerebral infarction is also performed in the US by the National Institute of Neurological Disorder and
This was the subject of a clinical trial conducted by Stroke) (so-called NINDS rtPA cerebral infarction test). This study focused on the effect of intravenous rt-PA treatment only within 3 hours of the onset of symptoms. Patients were investigated 3 months after treatment. Because this treatment was observed to have a positive effect on patient survival, the authors found that the limited time of 3 hours despite noticing that the risk of ICH was higher In-frame rt-PA treatment was recommended.

  Two additional studies (ECASS II study: alteplase thrombolysis for acute non-interventional treatment in ischemic cerebral infarction (ATLANTIS)), positive effect of rt-PA treatment within 3 hours after onset of cerebral infarction Was repeated if treated within 6 hours. However, the answer to this question is not positive, as no improvement in clinical symptoms or any reduction in mortality was observed. The risk to ICH remains the same.

  According to a review of all cerebral infarction studies, first published in 1997 and revised in March 2001, all treatments with thrombolytic agents (urokinase, streptokinase, rt-PA, or recombinant urokinase) When thrombolytics are used within 6 hours after the onset of cerebral infarction, the total number of patients who die or become disabled is reduced while mortality within the first 10 days after cerebral infarction is significantly increased. As a result. This effect was mainly due to ICH. Therefore, the widespread use of thrombolytic agents to treat cerebral infarction was not recommended.

  It was because of this result that even before that, the mere irony of choosing whether a patient with cerebral infarction would die or survive was a disability (SCRIP 1997: 2265, 26).

  Nevertheless, to date, treatment with rt-PA is the only acute cerebral ischemia treatment approved by the Food and Drug Administration (FDA) in the United States. However, it is limited to applying rt-PA within 3 hours after the onset of cerebral infarction.

Recombinant plasminogen activators are currently marketed with similar drugs named alteplase or reteplase. The latter is a t-PA fragment with a short half-life and active as a therapeutic agent. The dose of alteplase is about 70-100 mg and for reteplase is 2 x 560 mg, but in these cases, alteplase is applied primarily by infusion, and reteplase is applied with two repeated bolus injections at approximately 30 minute intervals. is the ^{(Mutschler: "Arzneimittelwirkungen", 8} th Edition, pages 512-513).

(T-PA side effects)
(Neurotoxicity and excitotoxicity)
rt-PA approval was obtained in 1996. Prior to that in 1995, the first announcement on the negative side effects of t-PA became known and the dramatic effect when they were applied to the treatment of cerebral infarction out of the 3 hour time frame. This is the basis for explanation. Thus, hippocampal microglia and neuronal cells produce t-PA that contributes to glutamate-mediated excitotoxicity. This is concluded from comparative experiments between t-PA-deficient mice and wild-type mice when glutamate agonists were injected into the hippocampus of t-PA-deficient mice and wild-type mice, respectively. t-PA-deficient mice showed significantly higher resistance to glutamate applied externally (subarachnoidally) (Tsirka SE et al., Nature Vol. 377, 1995, “Excitoxin-induced neuronal degeneration and seizure area moderated by tissue plasminogen activator "). These results were confirmed in 1998 when Wang et al. Proved that intravenous injection of t-PA almost doubled the amount of necrotic tissue in t-PA deficient mice. This negative effect of external t-PA on wild-type mice was only about 33% (Wang et al., 1998, Nature, “Tissue plasminogen activator (t-PA) increases neuronal damage after focal).
cerebral ischaemia in wild type and t-PA defensive mice ").

In addition, further results on the promotion of excitotoxicity by t-PA were published by Nicole et al. In early 2001 (Nicore O., Docagne F Ali).
C: Marguell I; Carmelite P; MacKenzie ET, Vivien D and Buison A, 2001: The proteolytic activity of tissue-N They were able to demonstrate that t-PA released by depolarized cortical neurons can interact with the so-called NR1 subunit of the NMDA-type glutamate receptor leading to NR1 cleavage. This increases the receptor activity that causes higher tissue damage when applying the glutamate agonist NMDA. NMDA agonists induced excitotoxicity. Therefore, t-PA exhibits a neurotoxic effect by activating the NMDA type glutamate receptor. During cerebral infarction, the blood brain barrier is destroyed in the affected tissue area, so that soluble plasma proteins such as fibrinogen and therapeutically applied t-PA can come into contact with neural tissue, where t-PA is When stimulated by fibrinogen, it exhibits neurotoxic effects through activation of glutamate receptors.

  Despite being effective in increasing neurotoxic side effects and mortality, t-PA was approved by the FDA. This can only be explained by the fact that there are no other harmless and effective alternatives, ie, a very practical cost-benefit analysis. Thus, there is still a need for safe treatments. However, if they were still based on thrombolytic agents, i.e., if it was not possible to find an alternative to thrombolysis, neurotoxicity issues must be considered (see, e.g., Wang et al. aaO; see Lewandowski and Barson 2001 aaO).

Thus, basically all thrombolytic agents had the potential to be compatible, but known thrombolytic agents such as DSPA (Desmodus rotundus plasminogen activator) were used for cerebral infarction. Further studies to develop new drugs are over.
In particular, in the case of DSPA, the possibility of adapting to this medical application has been previously pointed out (Medan P; Tatsumakak T; Takano K; Carano RAD; rDSPA) in a rat embryo stroke model; in: Cerebrovasc Dis 1996: 6; 175-194 (4 ^th International Symposium on Thrombological Therapy in IPS). Minogen activation factor It. Therefore, (in addition to disappointing due to adverse events neurotoxicity t-PA,) DSPA is expected that an agent suitable for treating a cerebral infarction was no longer.

(Alternative treatment)
Alternative therapies are currently focused on anticoagulants such as, for example, heparin, aspirin, or ancrod, an active substance derived from the venom of Malayan pit viper. Two additional clinical trials have been conducted to investigate the effects of heparin (the ORG 10172 trial in the International Cerebral Infarction Trial (IST) and the Treatment of Acute Cerebral Infarction (TOAST)) with significant mortality improvement or cerebral infarction Does not show prevention.

  Newer therapies attempt to increase the vitality of damaged cells by blocking the blood supply without paying attention to thrombus or blood thinning or anticoagulation (WO 01 / 51613A1). And WO 01/51614 A1). To accomplish this, an antibiotic selected from the group of quinones, aminoglycosides, or chloramphenicol is applied. For the same reason, it is recommended to apply citicoline first immediately after the onset of cerebral infarction. In the body, citicoline is cleaved into cytidine and choline. This cleavage product forms part of the membrane of nerve cells and helps regenerate damaged tissue (US Pat. No. 5,827,832).

  Recent research on safe therapies has shown that some of the fatal consequences of cerebral infarction are only indirectly caused by the inhibition of blood supply and are directly overactivated Based on new findings of excitotoxicity or neurotoxicity involving glutamate receptors. This effect is enhanced by t-PA (see above). Therefore, thinking about reducing excitotoxicity is trying to employ so-called neuroprotective agents. They can be used separately or in combination with fibrinolytic agents to minimize neurotoxic effects. They can, for example, reduce excitotoxicity directly as glutamate receptor antagonists or indirectly by inhibiting voltage-dependent sodium or calcium channels (Jin-Mo Lee et al. A. AO)).

  Competitive inhibition (antagonism) of the NMDA type glutamate receptor is made possible by, for example, 2-amino-5-phosphonovaleric acid (APV) or 2-amino-5-phosphonoheptanoic acid (APH). Non-competitive inhibition can be performed, for example, by a substance that binds to the phencyclidine side of the channel. Such materials can include phencyclidine, MK-801, dextrophan, ketamine, and the like.

  So far, treatment with neuroprotective agents has to be used in combination with thrombolytic agents in order to show a protective effect, and has not had the expected success. The same applies to other substances (see FIG. 10).

  Even when t-PA and a neuroprotective agent are used in combination, the damage can only be limited, and the disadvantage of the fibrinolytic agent such as neurotoxicity cannot be avoided.

(Non-neurotoxic plasminogen activator)
Plasminogen activator for the treatment of cerebral infarction has its enzyme activity increased very selectively by many times, ie more than 650 times, by fibrin, from international patent application PCT / EP02 / 12204 Are known. The disclosure of this document is fully incorporated into the reference.

  The nature and administration of these plasminogen activators are related to the neurotoxicity of tissue plasminogen activator (t-PA), the function of the blood brain barrier as a result of tissue destruction mainly caused by cerebral infarction It is based on the knowledge that it is due to the fact that fibrinogen circulating in the blood is able to penetrate into the nervous tissue of the brain when it is damaged or destroyed. Fibrinogen then activates t-PA, which indirectly leads to further tissue damage by activating glutamate receptors or plasminogen (see above).

  In order to prevent this effect, a plasminogen activator is applied that shows high selectivity for fibrin and vice versa and is less likely to be activated by fibrinogen. Thereby, these plasminogen activators are not activated, or compared to t-PA, even if fibrinogen from the blood penetrates into the nerve tissue as a result of damage to the blood brain barrier, The plasminogen activator should only be activated to a very low degree because the activator fibrin cannot enter the nerve tissue due to its insolubility. Thus, these plasminogen activators are non-neurotoxic.

(A) Genetically modified plasminogen activator)
According to a preferred embodiment of the present invention, a non-toxic plasminogen activator is used which comprises at least one component of a so-called zymogen triad. Equivalent triads are known from the catalytically active center of the serine proteases of the chymotrypsin family consisting of three interacting amino acids: aspartic acid 194, histidine 40 and serine 32. However, this triad is not present in t-PA belonging to the chymotrypsin-like serine protease family. Nevertheless, when site-specific mutagenesis of natural t-PA is carried out for the purpose of introducing at least one of the above-mentioned amino acids into an appropriate position, the proenzyme (single-chain t-PA) is present in the presence of fibrin. It is known that the activity decreases and the activity of the mature enzyme (double-stranded t-PA) increases. Therefore, by introducing at least one amino acid of the triad or an amino acid having each function in the triad, the zymogenicity of t-PA (that is, the ratio of the activity of the mature enzyme to the proenzyme) can be increased. . As a result, fibrin specificity is significantly increased. This is due to conformational interaction with the introduced amino acid residues and / or amino acid residues of the wild type sequence.

  Mutagenesis of native t-PA by replacing Phe305 with His (F305H) and Ala292 with Ser (A292S) leads to a 5-fold higher zymogenicity with the F305H mutant alone, whereas Zymogenicity is known to be 20 times higher (EL Madison, Kobe A, Gething M-J; Sambrook JF, Goldsmith EJ 1993: Converting Tissue Plasmogen Activator to AZ Science: 262, 419-421). In the presence of fibrin, these t-PA variants show a 30,000-fold (F305H) and 130,000-fold (F305H, A292S) increase in activity, respectively. These variants also contain a substitution of Arg275 to R275E to prevent cleavage by plasmin at the cleavage site of Arg275-Ile276, thereby changing the single-stranded t-PA to the double-stranded form. To do. The mutation site R275E alone increases the fibrin specificity of t-PA by 6,900 fold (K Tachias, Madison ELl 1995, Variant of Tissue-Hymantibine Biostimulative Biosubstance). 270, 31: 18319-18322).

  Positions 305 and 292 of t-PA are homologous to the known triads His40 and Ser32 of the chymotrypsin serine protease. Histidine or corresponding substitutions that introduce serine into each allow these amino acids to interact with aspartic acid 477 of t-PA, resulting in a functional triad in the t-PA mutant (Madison et al. al., 1993).

  Since these t-PA mutants have high fibrin specificity, they do not show any neurotoxicity or significantly lower neurotoxicity than wild-type t-PA. Therefore, the present invention is useful for treating cerebral infarction. Can be used. For purposes of disclosing the t-PA mutants F305H; F305H; A292S, alone or in combination with R275E, we have made Madison et al. (1993) and the publications of Tacias and Madison (1995) are hereby incorporated by reference in their entirety.

  Increasing the fibrin specificity of a plasminogen activator can also be achieved by a point mutation in Asp194 (or aspartic acid at a homologous position). Plasminogen activators belong to the serine protease group of the chymotrypsin family and thus contain Asp194, a conserved amino acid involved in the stability of the catalytically active steric structure of the mature protease. Asp194 is known to interact with His40 in the zymogen form of the serine protease. When the zymogen is activated by cleavage, this specific interaction is inhibited and the side chain of Asp194 rotates approximately 170 ° to form a new salt bridge with Ile16. This salt bridge contributes essential to the stability of the oxyanion pocket at the catalytic center of the mature serine protease. This is also present in t-PA.

  It is clear that the introduction of point mutations that replace Asp194 inhibits the formation or stability of the catalytic conformation of serine proteases, respectively. Nevertheless, it shows that mutant plasminogen activators significantly increase activity in the presence of their cofactor fibrin, especially when compared to mature wild type, This can only be explained by the fact that interaction with fibrin allows for a conformational change that promotes catalytic activity (L Strandberg, Madison EL, 1995: Variants of Tissue-type Platinum and Anti-substitutant). Selectivity towers Fibrin co-factors, in: Journal of Biological Chemistry 270, 40 2344-2349).

  In short, the Asp194 variant of the plasminogen activator shows a high increase in activity in the presence of fibrin and can thus be utilized by the present invention.

  In a preferred embodiment of the invention, a mutant t-PA in which Asp194 is replaced by glutamic acid (D194E) or by asparagine (D194N) is used. In these mutants, in the absence of fibrin, the activity of t-PA decreases by 1 to 2000 times, but in the presence of fibrin, an increase in activity of 498,000 to 1050,000 times can be achieved. In addition, these mutants contain a substitution of Arg15 to R15E, which prevents plasmin from cleaving the single-stranded t-PA at the peptide bond Arg15-Ile16, so that It becomes a chain type. This mutation alone enhances t-PA activation by fibrin by 12,000 times. The publication Strandberg and Madison (1995) is fully incorporated by reference because it discloses t-PA mutations at positions 194 and 15.

  The fibrin-dependent increase in plasminogen activator can also be realized by introducing point mutations into the so-called “autolytic loop”. This element is known from trypsin and can also be found in the homologous part of serine proteases and is characterized in particular by three hydrophobic amino acids (Leu, Pro and Phe). The autolytic loop in plasminogen activator is involved in the interaction with plasminogen. Point mutations in this region can have the effect that protein-protein interactions between plasminogen and plasminogen activator are no longer formed effectively. These mutations are functionally relevant only in the absence of fibrin. On the other hand, in the presence of fibrin, they are involved in increased activity of plasminogen activator (K Song-Hua, Tacias K, Lamba D, Board W, Madison EL, 1997: Identification of a Hydrophobic exociton on Tissue Type Plasminogen Activator That Modulations Specificity for Plasgenogen, in: Journal of Biological Chemistry 272; 3, 1811-1816).

  In a preferred embodiment, t-PA showing a point mutation from position 420 to position 423 is used. When these residues are replaced by site-directed mutagenesis, the fibrin dependence of t-PA increases up to 61,000-fold (K Song-Hua et al.). Song-Hua et al. Examined point mutations L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E. These publications are fully incorporated by reference to disclose their use according to the present invention.

  According to a further advantageous embodiment, a modified tissue plasminogen activator having the amino acid sequence set forth in SEQ ID NO: 1 (FIG. 14) is used. This modified t-PA differs from wild-type t-PA in that it exchanges hydrophobic amino acids 420 to 423 in the autolytic rub as follows: His420, Asp421, Ala422 and Cys423. This t-PA preferentially contains phenylalanine at position 194. In addition, position 275 may be occupied by glutamic acid. Preferably, position 194 is occupied by phenylalanine.

  Furthermore, modified urokinase can be used according to the present invention. The urokinase according to the present invention may comprise the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 13) in which the hydrophobic amino acids of the autolytic loop are substituted with Val420, Thr421, Asp422, and Ser423. Preferably, urokinase has Ile275 and Glu194. This mutant shows fibrin specificity 500 times higher compared to wild type urokinase.

  Both mutant urokinase and mutant t-PA were analyzed in a semi-quantitative test and showed higher fibrin specificity compared to wild-type t-PA.

(B) Desmodus rotundus-derived plasminogen activator (DSPA))
Plasminogen activator (DSPA) from the saliva of the vampire bat (Desmodus rotundus) also shows very high activity, specifically a 100,000-fold increase in the presence of fibrin. Thus, DSPA can be used preferentially by the present invention. The term DSPA includes four different proteases that satisfy the functions required for Desmodus rotundus, ie, prolonging bleeding from prey wounds (Cartwright, 1974). These four proteases (DSPAα1, DSPAα2, DSPAβ, DSPAγ) show high similarity (homology) to each other and to human t-PA. In addition, they show similar physiological activities and are generally classified into the generic name DSPA. DSPA is disclosed in European Patent No. 0352119A1 and US Pat. Nos. 6,008,019 and 5,830,849, which are hereby incorporated by reference in their entirety for the purposes of disclosure.

  To date, DSPAα1 is the protease that has been best analyzed in this group. It has an amino acid sequence with greater than 72% homology to the known human t-PA amino acid sequence (Kraetzschmar et al, 1991). However, there are two major differences between t-PA and DSPA. First, all DSPA does not change to double-stranded form, as opposed to t-PA, so it has full protease activity in single-stranded molecules (Gardell et al., 1989; Kraetzschmar et al. 1991). Second, the catalytic activity of DSPA is almost entirely dependent on fibrin (Gardell et al., 1989; Bringmann et al., 1995; Toschi et al., 1998). For example, DSPAα1 activity is increased 100,000-fold in the presence of fibrin, whereas t-PA activity is only increased 550-fold. In contrast, DSPA activity is not so strongly induced by fibrinogen because DSPA activity shows only a 7 to 9 fold increase (Bringmann et al., 1995). In short, DSPA is more fibrin-dependent and much more fibrin-specific than wild-type t-PA, which is only activated 550-fold by fibrin.

  DSPA has become an interesting candidate for developing thrombolytic agents because of its fibrinolytic properties and high similarity to t-PA. Nevertheless, the use of DSPA as a thrombolytic agent for treatment has been limited in the past to the treatment of myocardial infarction. Because t-PA contributes to glutamate-induced neurotoxicity, there is a reasonable hope that plasminogen activator related to t-PA can naturally be used for the treatment of acute cerebral infarction. Because there was not.

  Surprisingly, DSPA has a neurotoxic effect in spite of its high similarity (homology) to t-PA and despite the considerable physiological effects of its molecules. It was shown that there was no. The above conclusion ultimately led to the idea that DSPA could be successfully used in the treatment of cerebral infarction as a thrombolytic agent without causing a significant risk of nerve tissue damage. Of particular interest is the fact that DSPA can be used even more than 3 hours after the onset of cerebral infarction symptoms.

(Experimental evidence that DSPA is not neurotoxic)
This new teaching is based on in vivo comparative experiments of the neurodegenerative effects of t-PA on the one hand and DSPA on the other hand, using the so-called kainate model and the model for NMDA-induced damage in the striatum. .

  The kainic acid model (or kainic acid damage model) is a neurotoxic glutamate cascade by externally applying kainic acid (KA) as an agonist of kainic acid type (KA type) glutamate receptors and glutamate receptors of NMDA and AMPA. Based on stimulating. A t-PA deficient mouse strain is used as an experimental model to show that the sensitivity of experimental animals to kainic acid only reaches the level of wild-type mice after supplemental application of external t-PA. I was able to. On the other hand, even if an equimolar solution of DSPA is injected under the same experimental conditions, the sensitivity to kainic acid (KA) is not restored. It was concluded that the neurotoxic effect of t-PA is not induced by DSPA. A summary of these results is shown in FIG. 15 (Table 1).

  Quantitative experiments based on this model did not restore the sensitivity of t-PA-deficient mice to KA treatment even when the concentration of DSPA was increased 10-fold, but at 10-fold lower t-PA concentrations, KA induction It was revealed that tissue damage was already caused. This leads to the conclusion that DSPA has a neurotoxic ability at least 100 times lower than t-PA in terms of promoting neurodegeneration after KA treatment (see also FIGS. 11 and 12).

  In the second model of neurodegeneration, what is thought to be the effect of t-PA and DSPA on the promotion of NMDA-dependent neurodegeneration was compared to wild type mice. For this purpose, NMDA (as an agonist of NDMA type glutamate receptor) was injected into wild type mice alone or in combination with either t-PA or DSPA. This model allows comparison of the effects of these proteases under conditions that inevitably result in neuronal degeneration and plasma protein influx due to disruption of the blood brain barrier (Chen et al., 1999).

  While studying this model, injection of NMDA resulted in reproducible damage to the striatum of mice. Injection with a combination of t-PA and NMDA increased the volume of the injury site by at least 50%. In contrast, co-injection with DSPAα1 did not result in increased and expanded damage caused by NMDA injection. DSPA did not lead to increased neurodegeneration, even in the presence of plasma proteins that could freely diffuse into the damaged area induced by NMDA. A summary of these results is shown in FIG. 16 (Table 2).

  Initial results of clinical trials show that these results can also be transferred to human cerebral infarction treatment. It was discovered that significant improvements could be achieved in patients after successful perfusion (0 to 1 improvement in 8-point NIHSS or NIHSS score). This is shown in FIG. 17 (Table 3).

  Further experiments examined whether t-PA and DSPA could penetrate the damaged blood brain barrier when injected intravenously and, as a result, increase tissue damage in the brain. To address this issue, mice were stereotactically injected with NMDA to cause tissue damage to the striatum, and then t-PA or DSPA was applied intravenously 6 or 24 hours after NMDA injection. Compared to the negative control, 24 hours after NMDA injection, when t-PA was administered as an infusion, experimental animals showed an approximately 30% increase in the area of damaged tissue induced by NMDA injection. In contrast, the same treatment with DSPA detected the penetration of DSPA into the damaged tissue area by the antibody staining method, but no such increase in tissue damage occurred (see FIGS. 18 and 19). When t-PA or DSPA was applied intravenously in a similar manner 6 hours after NMDA injection, no increase in damaged tissue area was detected yet. Thereby, it can be explained that the blood brain barrier was still functioning as a sufficient barrier at the time of injection of t-PA or DSPA.

  These results indicate that DSPA constitutes an almost inactive protease in the mammalian (and therefore human) central nervous system and, in contrast to t-PA, the neurotoxicity induced by KA or NMDA. It indicates that no potentiating effect is produced. The lack of this neurotoxin, contrary to general expectations, makes DSPA a thrombolytic agent suitable for the treatment of acute cerebral infarction.

(Therapeutic ability of non-neurotoxic plasminogen activator)
The lack of neurotoxicity of DSPA and other non-neurotoxic plasminogen activators (see above) indicates that the use of these plasminogen activators is in contrast to wild-type t-PA. It offers a special advantage in the treatment of cerebral infarction that it is not limited to as short as 3 hours at most after the onset of cerebral infarction. On the contrary, there is almost no risk of stimulating an excitotoxic response, so treatment can be started even after a delay, for example after 6 hours or more. The first clinical trial with DSPA demonstrates that patients can be treated safely even in a time range of 6 to 9 hours after the onset of cerebral infarction.

  Of particular importance is the option of being able to be treated with non-neurotoxic activators without time limitations. This is because it becomes possible for the first time to safely treat a patient with acute cerebral infarction symptoms even if the diagnosis is delayed or the onset time of cerebral infarction cannot be determined with sufficient certainty. In the prior art, this group of patients was excluded from thrombolytic therapy with plasminogen activator because of the adverse risk assessment. Thus, the essential contraindications to the approved usage of thrombolytic agents for cerebral infarction are eliminated.

(Application of plasminogen activator)
In contrast to the already established cerebral infarction agent rt-PA, there is still no useful information available on possible applications for cerebral infarction treatment with non-neurotoxic plasminogen activator.

  Accordingly, it is an object of the present invention to provide an advantageous mode of application for these non-neurotoxic plasminogen activators.

  According to the present invention, a plasminogen activator whose activity is higher than 650 times in the presence of fibrin is applied intravenously to treat cerebral infarction.

  Intravenous administration of these non-neurotoxic plasminogen activators to treat cerebral infarction has already been evaluated in clinical trials, in which DSPA is a member of this group of fibrinolytic agents. As an example, these patients were applied intravenously, thereby causing only minor side effects.

  These results of clinical trials were unexpected because it was well known that intravenous application of t-PA and other conventional fibrinolytic agents was associated with a significant risk of cerebral hemorrhage. (See above).

  To alleviate intracerebral hemorrhage, efforts have recently been made to develop methods for applying these substances in the immediate vicinity of intravascular thrombi using catheters by intra-arterial routes rather than intravenously. Practical experience is already possible for recombinantly produced urokinase (PROKAT as a study with pro-urokinase). This mode of application has also been shown to reduce dose-dependent side effects because the total dosage can be significantly reduced, thus reducing cerebral hemorrhage.

  However, these important advantages of intra-arterial application are also countered by two possible drawbacks. First of all, this treatment needs time to be prepared by the patient, but it is not possible to realize it in a predetermined frame of only 3 hours in the treatment of cerebral infarction. On the other hand, this treatment can reliably achieve a lower total dosage. However, if the drug concentration is increased, the terminal arterial blood vessel is locally reached. In the case of cerebral infarction, the vascular endothelium causes the barrier function to be impaired. As a result, the drug substance locally reaches the surrounding tissue at a high concentration. This can cause undesirable side effects.

  In contrast, in the case of intravenous application, the concentration of the pharmaceutical substance is diluted by the blood flow of the vein. Therefore, intraarterial injection is problematic for drugs that can destroy tissue such as t-PA (Forth, Henscher, Rummel, Starke: “Pharmacology and Toxiology”, 6th Edition, 1992, page 29).

  However, these constraints that complicate intra-arterial application, namely the narrow time frame and tissue damage side effects, do not apply to the plasminogen activator applied by the present invention. Thus, because of its undeniable advantages (see above), intraarterial application, in principle, constitutes a very promising application mode for non-neurotoxic plasminogen activators. Thus, it would have been obvious that this route should be followed when searching for a preferred mode of applying these agents.

  Nevertheless, those who apply non-neurotoxic plasminogen activators have also chosen to rely on problematic intravenous applications, but surprisingly proved that it is advantageous It is.

  Furthermore, the application method according to the present invention is also mainly intended for intramuscular injection or intravenous administration, in order to reduce the risk of allergy / shock, in order to administer a protein having high immunogenic ability, contrary to the usual therapeutic action. Infusion is used (Mebs: “Gifttier”, 2th Edition, 2000).

  In contrast to t-PA which is a natural body substance, the plasminogen activator applied in accordance with the present invention is an animal-derived foreign protein (such as DSPA) or its structural It is one of the genetically modified in vivo proteins that presents new epitopes due to differences. A concomitant problem of allergic reactions, especially when applying high therapeutic dosages, as is generally required for intravenous applications, is another problem with foreign proteins such as streptokinase. It also applies to fibrinolytic agents.

  In a particularly useful embodiment, the plasminogen activator applied according to the present invention is administered in a manner called bolus injection (rapid intravenous injection), as a single intravenous rapid injection containing the entire therapeutic amount. It can also be administered.

  In reviewing clinical experiments, it has been discovered that even surprisingly low therapeutic doses can be applied intravenously to provide advantageous results. Preferred treatment results were obtained, for example, at doses of 90 μg / kg to 230 μg / kg. Particularly preferred treatment results here were obtained at doses of 62.5 μg / kg to 90 μg / kg. In the patients studied, the time taken from cerebral infarction to drug application was 3-9 hours. Appropriate testing methods could be used to determine the onset of therapeutic effect (see FIGS. 20 and 29).

  DSPA, like other non-neurotoxic plasminogen activators, shows no tissue damage side effects. However, in order to limit tissue damage induced by glutamic acid naturally present in the human body, it may be preferable to apply them in combination with a neuroprotective agent for treating cerebral infarction. Neuroprotective agents that competitively or non-competitively inhibit glutamate receptors can also be used. Useful combinations include, for example, with known inhibitors of NMDA, kainate, or quisqualate glutamate receptors, such as APV, APH, phencyclidine, MK-801, dextrorphan, or ketamine. It is a combination.

  In addition, cations, particularly Zn-ions, may block the cation channels regulated by glutamate receptors and thus suppress neurotoxic effects, so it may be preferred to combine with cations.

  In a further preferred embodiment, the non-neurotoxic plasminogen activator can be combined with at least one further therapeutic agent or a pharmaceutically acceptable carrier. Particularly suitable for use in combination with therapeutic agents that help suppress tissue damage by activating cells, as it contributes to the regeneration of already damaged tissue or helps prevent further cerebral infarction from occurring It is. Preferred examples are combinations with antibiotics such as quinones, anticoagulants such as heparin or hirudin, and citicoline or acetylsalicylic acid.

It may also be suitable to use in combination with at least one thrombin inhibitor. Preferentially, thrombomodulin and thrombomodulin analogues such as eg solulin, triabin or palidipine can also be used. Furthermore, it is preferable to use it together with an anti-inflammatory substance because it affects the infiltration by leukocytes.
For example, the present invention provides the following items.
(Item 1)
Use of a plasminogen activator, wherein the activity of the plasminogen activator is enhanced at least 650 times in the presence of fibrin to produce an intravenously applicable drug for treating cerebral infarction. Use of a plasminogen activator.
(Item 2)
Use of a plasminogen activator according to item 1, characterized by the use of Desmodus rotundus plasminogen activator (DSPA) or a pharmaceutically acceptable salt thereof.
(Item 3)
The use of a plasminogen activator according to item 1, wherein the plasminogen activator comprises at least a histidine residue or a serine residue which together with an aspartic acid residue forms at least part of a zymogen triad.
(Item 4)
The serine residue is located at least partially homologous to position 292 of t-PA, the histidine residue is located at least partially homologous to position 305 of t-PA, and 4. Use of a plasminogen activator according to item 3, wherein the aspartic acid residue is located at a position at least partially homologous to position 447 of t-PA.
(Item 5)
Item 4 wherein the plasminogen activator is selected from the group of the following t-PA variants: t-PA / R275E; t-PA / R275E, F305H; t-PA / R275E, F305H, A292S Use of the described plasminogen activator.
(Item 6)
The plasminogen activator has a point mutation of Asp194 or an aspartic acid at a homologous position, and the point mutation stabilizes the catalytically active conformation of the plasminogen activator in the absence of fibrin. Use of the plasminogen activator according to item 1, which reduces
(Item 7)
Item 7. The use according to item 6, wherein Asp194 is substituted by glutamic acid or asparagine.
(Item 8)
8. Use according to item 7, comprising replacement of ASP194 with Glu194 or Asn194.
(Item 9)
The plasminogen activator comprises at least one mutation in its autolytic loop, which mutation is functional between plasminogen and plasminogen activator in the absence of fibrin. Use of a plasminogen activator according to item 1, which reduces the interaction.
(Item 10)
9. Use according to item 8, wherein at least one mutation in the autolytic loop affects amino acid positions 420-423 or homologous positions of wild type t-PA.
(Item 11)
10. Use according to item 9, wherein the mutation is selected from the group consisting of the following mutants: L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.
(Item 12)
The use of a plasminogen activator according to item 1, wherein the plasminogen activator is a zymogen containing at least one point mutation that prevents catalysis by plasmin.
(Item 13)
12. Use of a plasminogen activator according to item 11, wherein the point mutation is located at position 15 or 275 of t-PA or a position homologous thereto.
(Item 14)
13. Use according to item 12, wherein glutamic acid is located at position 15 or 275.
(Item 15)
The use according to item 1, wherein the plasminogen activation comprises an autolytic loop comprising His420, Asn421, Ala422 and Cys423.
(Item 16)
Use according to item 1, characterized in that the plasminogen activator is characterized by a point mutation at position 194 that reduces the stability of the catalytically active conformation of the plasminogen activator in the absence of fibrin.
(Item 17)
16. Use according to item 15, wherein the plasminogen activator is characterized by Phe194.
(Item 18)
Use according to item 1, characterized in that the plasminogen activator is characterized by at least one point mutation that prevents catalysis by plasmin.
(Item 19)
18. Use according to item 17, wherein the plasminogen activator comprises Glu275.
(Item 20)
The use according to item 1, wherein the plasminogen activator comprises the amino acid sequence set forth in SEQ ID NO: 1.
(Item 21)
Item 21. The use according to Item 20, wherein the plasminogen activator is urokinase having an autolytic loop including Val420, Thr421, Asp422 and Ser423.
(Item 22)
21. Use according to item 20, characterized in that the urokinase is characterized by a point mutation at position 194 that reduces the stability of the catalytically active conformation of urokinase in the absence of fibrin.
(Item 23)
The use according to item 21, wherein said urokinase comprises Glu194.
(Item 24)
23. Use according to any of items 20 to 22, characterized in that the urokinase is characterized by at least one point mutation that prevents catalysis by plasmin.
(Item 25)
The use according to item 1, wherein the plasminogen activator comprises the sequence set forth in SEQ ID NO: 2.
(Item 26)
26. Use according to any one of items 1 to 25, characterized by infusion.
(Item 27)
27. Use according to any one of items 1 to 26, characterized by bolus injection.
(Item 28)
27. Use according to item 26, characterized by a single bolus injection.
(Item 29)
Use of urokinase comprising ILE275 for the manufacture of a medicament for treating cerebral infarction, wherein the urokinase is injected intravenously.
(Item 30)
30. Use according to any one of items 1 to 29, for the therapeutic treatment of cerebral infarction in a human at least 3 hours after the onset of cerebral infarction.
(Item 31)
31. Use of a plasminogen activator according to at least one of items 1 to 30 for the therapeutic treatment of cerebral infarction in a human at least 6 hours after the onset of cerebral infarction.
(Item 32)
32. Use of a plasminogen activator according to at least one of items 1 to 31 for the therapeutic treatment of cerebral infarction in a human at least 9 hours after the onset of cerebral infarction.
(Item 33)
33. Use of a plasminogen activator according to at least one of items 1 to 32 for therapeutic treatment of a cerebral infarction patient whose onset of cerebral infarction cannot be accurately determined in time.
(Item 34)
34. Use of a plasminogen activator according to at least one of items 1 to 33 for treating cerebral infarction while avoiding neurotoxicity of wild-type t-PA.
(Item 35)
35. A pharmaceutical composition comprising at least one plasminogen activator according to items 1 to 34 and at least one further pharmaceutically active ingredient or a pharmaceutically acceptable salt thereof.
(Item 36)
35. A pharmaceutical composition according to item 34, characterized by a neuroprotective agent.
(Item 37)
36. A pharmaceutical composition according to item 35, characterized by a glutamate receptor antagonist.
(Item 38)
37. A pharmaceutical composition according to item 36, characterized by a competitive antagonist or a non-competitive antagonist.
(Item 39)
35. A pharmaceutical composition according to item 34, characterized by at least one thrombin inhibitor preferentially selected from the group of the following substances: thrombomodulin, thrombomodulin analogues, triabin, paridipine or sollin.
(Item 40)
35. A pharmaceutical composition according to item 34, characterized by at least one anticoagulant preferentially selected from the group of the following anticoagulants: hirudin, heparin, acetylsalicylic acid or ancrod.
(Item 41)
35. Pharmaceutical composition according to item 34, characterized by an anti-inflammatory substance.
(Item 42)
35. A pharmaceutical composition according to item 34, characterized by an antibiotic.
(Item 43)
35. A pharmaceutical composition according to item 34, characterized by citicoline.

not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed. not listed.

In the following, the application method and prescription according to the present invention will be outlined with characteristic treatment examples.
(Comparison test of t-PA and DSPA)
(A. Method)
(1. animals)
Wild-type mice (c57 / Black6) and t-PA-deficient mice (t-PA-/-mice) (c57 / Black6) (Carmeliet et al., 1994) were obtained from Dr. Peter Carmeliet, Leuven, Belgium. obtained.

(2. Protein extraction from brain tissue)
Evaluation of proteolytic activity in brain tissue after infusion of either t-PA or DSPAα1 was performed by zymograph analysis (Granelli-Pipeno and Reich, 1974). After injection into the hippocampus for 7 days, the mice were anesthetized and perfused transcardially with PBS before the brain was excised. The hippocampal region was removed, transferred to an Eppendorf tube, and 0.5% NP-40 lysis buffer equivalent solution (w / v) (about 30-50 μl) (0.5% NP-40) containing no protease inhibitors. , 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ , 1 mM EDTA). The brain extract was homogenized with a manual glass homogenizer and left on ice for 30 minutes. Then, the sample was centrifuged and the supernatant was taken out. The amount of protein present was measured (Bio-Rad-reagent).

(3. Zymograph analysis of protease)
Proteolytic activity in samples and brain tissue extracts was measured by zymographic analysis according to the method of Granelli-Pipeno and Reich (1974). Samples containing recombinant protein (up to 100 nmol) or brain tissue extract (20 μg) were subjected to SDS-PAGE under non-reducing conditions (10%). The gel was removed from the plate, washed with 1% Triton × 100 for 2 hours, and then overlaid on an agarose gel containing polymerized fibrinogen and plasminogen (Granelli-Pipeno and Reich, 1974). The gel was incubated at 37 ° C. in a humidified chamber until a proteolytic zone appeared.

(4. Intrahippocampal injection of t-PA, DSPA followed by kainic acid injection)
The kainic acid damage model is described in Tsirka et al. (1995). Animals were injected intraperitoneally (ip) with atropine (4 mg / kg) followed by pentobarbital sodium (70 mg / kg) i.p. p. Anesthetized by injection. The mouse is then placed in a stereotaxic frame and a small osmotic pump containing either 100 μl PBS or recombinant human t-PA (0.12 mg / ml, 1.85 μM) or DSPAα1 (1.85 μM). (Alzet model 1007D, Alzet CA. USA) was implanted subcutaneously between the shoulder ribs. This pump is connected to the brain cannula via a sterile tube, and then the liquid is introduced in the vicinity of the midline, so that -2.5 mm in the Bregma direction, 0.5 mm in the inward and outward directions, and 1 in the dorsoventral direction. It was inserted through a jagged opening made in the skull at a coordinate of 6 mm. The cannula was fixed in the desired position and each solution was infused from the pump at a rate of 0.5 μl per hour for a total of 7 days.

  Two days after the protease injection, the mice were anesthetized again and placed in a fixed frame again. Thereafter, 1.5 nmol of kainic acid in 0.3 μl of PBS was unilaterally injected into the hippocampus. The coordinates were 2.5 mm in the Bregma direction, 1.7 mm in the inward and outward directions, and 1.6 mm in the dorsoventral direction. Excitotoxin (KA) was delivered for 30 seconds. After kainic acid treatment, the needle was left at these coordinates for an additional 2 minutes to prevent the liquid from flowing back.

(5. Brain processing method)
Five days after KA injection, the animals were anesthetized and transcardially perfused with 30 ml PBS followed by 70 ml 4% paraformaldehyde solution and postfixed with the same fixative for 24 hours, followed by 30% shoal. The mixture was further incubated for 24 hours. Then, a coronal section (40 μm) of the brain was cut out on a frozen microtome and either counterstained with thionine (BDH, Australia) or processed for immunohistochemical examination as described later.

(6. Determination of neuronal loss in the hippocampus)
Quantification of neuronal loss in the CA1-CA3 subhippocampal region was performed as previously described (Tsirka et al., 1995; Tsirka et al., 1996). Five consecutive portions of the dorsal hippocampus from all treatment groups were prepared with care that the site actually contained the CA injected location and the injured area. The hippocampal subregions (CA1-CA3) of these sections were traced by hippocampal camera seeder imaging. The total length of these subregions was measured relative to a 1 mm standard traced under the same magnification. The length of tissues with live cone neurons (with normal morphology) and the length of tissues without neurons (no cells and not thionine stained) were measured. Lengths indicating complete neuronal and neuronal loss across each hippocampal subregion were averaged over the entire section to determine the standard deviation.

(7. Intrastriatal NMDA excitotoxic injury with or without t-PA or DSPA)
Wild type mice (c57 / Black6) were anesthetized and placed in a stereotaxic frame (see above). The left striatum of the mouse was then injected unilaterally with 50 nmol of NMDA alone or in combination with 46 μM rt-PA or 46 μM DSPAα1. As controls, t-PA and DSPAα1 were also injected alone (both at a concentration of 46 μM). The injected coordinates were −0.4 mm in the Bregma direction, 2.0 mm in the inward / outward direction, and 2.5 mm in the dorsoventral direction. The solution (1 μl total volume for all treatments) was moved at a rate of 0.2 μl / min over 5 minutes and the needle was left for an additional 2 minutes after injection to prevent liquid backflow. After 24 hours, the mice were anesthetized, transcardially perfused with 30 ml PBS followed by 70 ml 4% paraformaldehyde solution, fixed after 24 hours with the same fixative, and then further 24 with 30% sucrose solution. Incubated for hours. Then, the brain was cut out (40 μm) on a frozen microtome and mounted on a glass slide coated with gelatin.

(8. Quantification of damaged volume after NMDA injection)
Callaway et al. Using the method described by (2000), the striatum damage volume was quantified. Ten consecutive coronal sections spanning the damaged area were prepared. Using the Callaway method, the damaged area was visualized and the damaged volume was quantified with a microcomputer imaging device (MCID, Imaging Research Inc., Block University, Ontario, Canada).

(9. Immunohistochemical method)
Immunohistochemistry was performed using standard methods. The coronal sections were soaked in a solution of 3% H ₂ O ₂ /10% methanol for 5 minutes and then incubated in 5% normal goat serum for 60 minutes. Sections were treated with anti-GFAP antibody (1: 1,000; Dako, Carpinteria, CA., USA) for detecting astrocytes, anti-MAC-1 antibody (1: 1,000 for detecting microglial cells). Serotec, Raleigh, NC, USA) or an anti-DSPA antibody (Schering AG, Berlin), which is a polyclonal antibody, was incubated overnight. After washing, the sections were incubated with the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA., USA). Thereafter, it was finally incubated with avidin / biotin complex for 60 minutes before visualization with 3,3′-diaminobebzidin / 0.03% H ₂ O ₂ . Then, the section was mounted on a gelatin-coated slide, dried and dehydrated, and a cover glass was placed on the par mount.

(10. Promotion of tissue damage induced by NMDA injection by intravenous administration of t-PA or DSPA)
In order to cause tissue damage in the striatum, mice were stereotaxic and injected with NMDA. Six hours after NMDA injection, t-PA or DSPA (100 μl; 10 mg / kg) was injected via the tail vein. As a control, 100 μl of 0.9% NaCl was injected followed by PBS. After 24 hours, the animals were killed and the damaged volume was measured.

  In the second experiment, a group of animals containing up to 15 mice was similarly injected with t-PA or DSPA injection solution 24 hours after NMDA injection, after which tissue damage was measured. As evidence that DSPA is present in the brain, coronal sections were stained with anti-DSPA antibody according to a conventional method.

(B. Results)
(1. When t-PA or DSPA is injected, it is dispersed in the hippocampus of t-PA − / − mice and retains proteolytic activity)
An initial experiment was designed to confirm that both t-PA and DSPA retain proteolytic activity for a 7 day infusion period. For this purpose, an equal volume of t-PA and DSPA (100 nmol) was incubated for 7 days at 37 ° C. and 30 ° C. in a hot water bath. In order to measure the proteolytic activity, SDS-PAGE was performed on a 5-fold serial dilution of the probe under non-reducing conditions, and the proteolytic activity was measured by zymographic analysis. Equivalent solutions of t-PA and DSPA that had been frozen for 7 days were evaluated as controls. As can be seen from FIG. 1, within this time, either t-PA or DSPA activity was only slightly lost, whether incubated at 25 ° C. or 37 ° C.

(2. Activity of t-PA or DSPA is restored in hippocampal extracts prepared from t-PA-/-mice after injection)
First, it was necessary to confirm that the injected protease was present in the brain of the injected animal and that it retained proteolytic activity while present in this compartment. To handle this point, either t-PA or DSPA was injected into t-PA − / − for 7 days (see above). The mice were then perfused transcardially with PBS and the brain was removed. The ipsilateral and contralateral hippocampal regions and a region of the cerebellum (as a negative control) were isolated. SDS-PAGE and zymographic analysis was performed on tissue samples (20 μg) as described in the method section. As can be seen from FIG. 2, both t-PA and DSPA activities were detected in the ipsilateral region of the hippocampus, while some activity was also detected on the contralateral side. This indicates that the injected protease not only retained activity in the brain, but was further dispersed within the hippocampal region. As a control, no activity could be detected in extracts prepared from cerebellum.

(3. Immunohistochemical evaluation of DSPA)
To further confirm that DSPA was indeed dispersed in the hippocampal area, coronal sections of t-PA − / − mouse brains were analyzed immunohistochemically after DSPA injection. DSPA antigen was detected in the hippocampal region with the most pronounced staining in the injection site region. This result confirms that the injected DSPA is soluble and actually present in the hippocampus.

(4. Infusion of DSPA does not restore in vivo neurodegeneration by kainic acid)
Characteristically, t-PA − / − mice are resistant to neurodegeneration mediated by kainic acid (KA). However, intra-hippocampal injection of rt-PA completely restores susceptibility to KA-mediated damage. In this model, t-PA or DSPA was injected into the hippocampus of t-PA − / − mice using a mini-osmotic pump to determine whether DSPA could substitute for t-PA. Twelve mice were tested for both groups. Two days later, the animals were allowed to recover by injecting kainic acid. After 5 days, the animals were killed and the brains removed and prepared (see above). As a control, t-PA − / − mice were injected with PBS before KA treatment (N = 3).

  Coronal sections of the brain were prepared and neurons were detected by Nissl staining. As shown in FIGS. 4a and 4b, t-PA − / − mice injected with PBS were resistant to subsequent induction by KA. However, injection of recombinant t-PA restored sensitivity to KA treatment. In contrast, infusion of the same concentration of DSPA into the hippocampal area did not change the sensitivity of the animals to KA.

  The quantification of these results was based on data obtained from 12 mice in each group. In 2 of 12 mice injected with DSPA, mild neurodegeneration was seen. The reason is not clear, but probably not related to the presence of DSPA. The summarized data also takes into account the trivial effects seen in these two animal cases. All 12 mice treated with t-PA were sensitive to KA treatment. These results indicate that when t-PA or DSPAα1 is injected at an equimolar concentration, the sensitivity to neurodegeneration induced by KA is restored only when t-PA is administered.

(5. DSPA injection does not result in microglial cell activation)
The restoration of KA sensitivity in t-PA − / − mice, brought about by t-PA injection, also leads to microglial activation (Rogove et al, 1999). To assess the extent of microglial activation after t-PA or DSPA injection and subsequent KA treatment, mice were immunized against activated microglial cells against coronal sections of mice using Mac-1 antibody. Histochemical staining was performed. As a result of recovering KA sensitivity after t-PA injection, the number of Mac-1-positive cells was clearly increased. This was not observed in mice injected with DSPA. Thus, the presence of DSPA does not result in activating microglial cells after KA treatment.

6. Titration of DSPA and t-PA in the hippocampal region of mice
The concentration of t-PA used for the injection was determined by Tsirka et al. (1995) (0.12 mg / ml 100 μl [1.85 μM]). The KA damage experiment was repeated with 10-fold lower t-PA (0.185 μM) and 10-fold higher DSPA amount (18.5 μM). Even lower t-PA concentrations could still restore sensitivity to KA treatment (n = 3). Of particular interest was the discovery that injection of 10-fold higher DSPA concentrations resulted in slight neuronal loss after KA treatment. These data strongly indicate that DSPA does not enhance sensitivity to KA.

(7. Effect of t-PA and DSPA on NMDA-dependent neurodegeneration in wild-type mice)
The effects of t-PA and DSPA were also examined in a model of neurodegeneration in wild type mice. Injection of t-PA into the striatum of these mice clearly resulted in an increase in neurodegenerative effects caused by NMDA, an analog of glutamate (Nicore et al., 2001).

  Wild type mice were injected with NMDA in the striatal region in the presence of t-PA or DSPA (46 μM each) in a total volume of 1 μl. After 24 hours, the brain was removed and the size of the lesion was quantified by the Callaway method (Callaway et al., 2000) (see above). As can be seen from FIG. 7, injection of NMDA alone resulted in reproducible damage in all treated mice (N = 4). Application of t-PA and NMDA together increased the size of the lesion by about 50% (P <0.01, n = 4). In sharp contrast, simultaneous injection of NMDA and the same concentration of DSPA did not increase the size of the lesion compared to NMDA alone.

  Injection of t-PA or DSPA alone did not lead to detectable neurodegeneration. The absence of t-PA effects when administered alone has been shown by Nicole et al. This agrees with the result of (2001). These data indicate that the presence of DSPA does not promote neurodegeneration, even during neurodegeneration.

  In order to confirm that the DSPA injection solution actually diffused into the hippocampal region, immunohistochemistry was performed on coronal sections using DSPA antibodies. As a result of the test, it was shown that DSPA actually entered the striatum region.

(Dynamic analysis of plasminogen activation by indirect chromogen test)
An indirect chromogenic test for t-PA activity was performed by Madisan E. et al. L. Goldsmith E .; J. et al. Gerard R., et al. D. , Gething M .; -J. Sambrook J .; F. (1989) Nature 339 721-724; Madison E. et al. LO. Goldsmith E .; J. et al. Gething M .; J. et al. Sambrook J .; F. and Bassel-Duby R.M. S. (1990) Proc. Natl. Acad. Sci. U. S. A 87, 3530-3533, and Madison E. et al. L. Goldsmith E .; J. et al. Gething M .; J. et al. Sambrook J .; F. and Gerard R.M. D. (1990) J. MoI. Biol. The substrates described in Chem 265, 21423-21426 were performed using Lys-plasminogen (American Diagnostica) and Spectrozyme PL (American Diagnostica). The test was conducted in the presence and absence of a cofactor, DESAFIB (American Diagnostica). DESAFIB is a soluble fibrin monomer preparation obtained by cleaving highly pure human fibrinogen with the protease batroxobin. Batroxobin cleaves the Arg ¹⁶ -Gly ¹⁷ bond in the Aα-chain of fibrinogen to release fibrinopeptide A. The resulting des-AA-fibrinogen representing fibrin I monomer is soluble without the peptide Gly-Pro-Arg-Pro. The concentration of Lys-plasminogen was varied from 0.0125 to 0.2 μM in the presence of DESAFIB and from 0.9 to 16 μM in the absence of cofactor.

(Indirect chromogen test in the presence of various stimulating substances)
An indirect chromogen standard test was performed according to the publication cited above. A total of 100 μl of probe containing 0.25 to 1 ng enzyme, 0.2 μM Lys-plasminogen, and 0.62 mM Spectrozyme PL was used. The test was performed in the presence of either buffer, 25 μg / ml DESAFIB, 100 μg / ml cyanogen bromide fragment of fibrinogen (American Diagnostica), or 100 μg / ml stimulating 13 amino acid peptide P368. The analysis was performed in a microtiter plate, and the optical density was measured every 30 seconds for 1 hour at a wavelength of 405 nm in “Molecular Devices Thermomax”. The reaction temperature was 37 ° C.

(8. DSPA also does not increase nerve tissue damage when applied intravenously)
In the striatum of mice, tissue damage was induced by intravenous application of t-PA or DSPA 6 or 24 hours after NMDA injection. Compared to the negative control, when t-PA was administered as an intravenous infusion 24 hours after NMDA injection, the experimental animals showed an increase in the damaged tissue area of about 30% caused by the NMDA injection. However, this was in contrast to DSPA, which did not so increase tissue damage (see FIG. 18). It was possible to detect that DSPA administered intravenously 24 hours after NMDA injection penetrated into the area of damaged tissue by staining of coronal sections with anti-DSPA antibody (Fig. 19). Similarly, when t-PA or DSPA was applied intravenously 6 hours after NMDA injection, an increase in the area of damaged tissue was not yet detected. This may be because the blood brain barrier was still providing sufficient barrier function at the time t-PA or DSPA was applied. Thus, DSPA does not exhibit neurotoxic side effects when applied intravenously.

Claims (1)

Invention described in the specification.