US20050048027A1 - Non-neurotoxic plasminogen activating factors for treating stroke - Google Patents

Non-neurotoxic plasminogen activating factors for treating stroke Download PDF

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US20050048027A1
US20050048027A1 US10/494,004 US49400404A US2005048027A1 US 20050048027 A1 US20050048027 A1 US 20050048027A1 US 49400404 A US49400404 A US 49400404A US 2005048027 A1 US2005048027 A1 US 2005048027A1
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activating factor
plasminogen activating
stroke
plasminogen
dspa
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Mariola Sohngen
Wolfgang Sohngen
Wolf-Dieter Schleuning
Robert Medcalf
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Paion Deutschland GmbH
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Priority to US12/196,785 priority Critical patent/US8119597B2/en
Priority to US13/370,706 priority patent/US20130039902A1/en
Priority to US14/151,390 priority patent/US20140199287A1/en
Priority to US15/079,550 priority patent/US20170051268A1/en
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Definitions

  • the invention pertains to the therapeutic use of non-neurotoxic plasminogen activators especially from the saliva of Desmodus rotundus (DSPA) preferentially for the treatment of stroke.
  • DSPA Desmodus rotundus
  • Ischaemic insults are characterized in a reduction or interruption of the blood circulation in the brain due to a lack of arterial blood supply. Often this is caused by thrombosis of an arteriosclerotic stenosed vessel or by arterio arterial, respecitively, cardial embolisms.
  • Haemorrhagic insults are based inter alia on the perforation of brain supplying arterias damaged by arterial hypertonia. However, only approximately 20% of all cerebral insults are caused by haemorrhagic insults. Thus, stroke due to thrombosis is much more relevant.
  • necrosis In comparison to other tissue ischaemias the ischaemia of the neuronal tissue is widely accompanied by necrosis of the effected cells.
  • necrosis In comparison to other tissue ischaemias the ischaemia of the neuronal tissue is widely accompanied by necrosis of the effected cells.
  • the higher incidence of necrosis in neuronal tissue can be explained with the new understanding of the phenomenon “excitotoxicity” which is a complex cascade comprising a plurality of reaction steps. The cascade is initiated by ischaemic neurons affected by a lack of oxygen which then lose ATP instantaneously and depolarize. This results in an increased postsynaptic release of the neurotransmitter glutamate which activates membrane bound glutamate receptors regulating cation channels. However, due to the increased glutamate release glutamate receptors become over activated.
  • Glutamate receptors regulate voltage dependent cation channels which are opened by a binding of glutamate to the receptor. This results in a Na + and Ca 2+ influx into the cell massively disturbing the Ca 2+ dependent cellular metabolism. Especially the activation of the Ca 2+ dependent catabolic enzymes could give reason to the subsequent cell death (Lee, Jin-Mo et al., “The changing landscape of ischaemic brain injury mechanisms”; Dennis W. Zhol “Glutamate neurotoxicity and diseases of the nervous system”).
  • the re-opening of the closed vessel has priority in the therapy of acute cerebral ischaemia.
  • the re-opening can be performed by different means.
  • PROCAT a study with prourokinase
  • the naturally occurring fibrinolysis is based on the proteolytic activity of the serine protease plasmin which originates from its inactive precursor by catalysis (activation).
  • the natural activation of plasminogen is catalyzed by the plasminogen activators u-PA (urokinase type plasminogen activator) and t-PA (tissue plasminogen activator) occurring naturally in the body.
  • u-PA urokinase type plasminogen activator
  • t-PA tissue plasminogen activator
  • t-PA forms a so called activator complex together with fibrin and plasminogen.
  • the catalytic activity of t-PA is fibrin dependent and is enhanced in its presence approximately 550-fold.
  • fibrinogen can stimulate t-PA mediated catalysis of plasminogen to plasmin—even though to a smaller extent. In the presence of fibrinogen the t-PA activity is only increases 25-fold. Also the cleavage products of fibrin (fibrin degradation products (FDP)) are stimulating t-PA.
  • FDP fibrin degradation products
  • the therapy with streptokinase has severe disadvantages since it is a bacterial protease and therefore can provoke allergic reactions in the body. Furthermore, due to a former streptococci infection including a production of antibodies the patient may exhibit a so called streptokinase resistance making the therapy more difficult. Besides this, 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 )) indicated an increased mortality risk and a higher risk of intracerebral bleeding (intracerebral haemorrhage, ICH) after treating patients with streptokinase. These trials had to be terminated early.
  • Urokinase also a classical fibrinolytic agent—can be applicated. In contrast to streptokinase it does not exhibit antigenic characteristics since it is an enzyme naturally occurring in various body tissues. It is an activator of plasminogen and independent of a co-factor. Urokinase is produced in kidney cell cultures.
  • rt-PA tissue type plasminogen activator
  • ECASS European Acute Stroke Trial
  • the thrombolytic treatment of stroke was also subject of a clinical trial conducted by the National Institute of Neurologic Disorder and Stroke (so called NINDS rtPA Stroke Trial) in the USA.
  • NINDS rtPA Stroke Trial concentrated on the effect of intravenous rt-PA treatment within only three hours after the onset of the symptoms. Patients were examined three months after the treatment. Due to the observed positive effects of this treatment on the viability of patients, rt-PA treatment within these limited time frame of three hours was recommended although the authors found a higher risk for ICH.
  • rt-PA acute cerebral ischaemia approved by the Food and Drug Administration (FDA) in the USA.
  • FDA Food and Drug Administration
  • plasmin is the effector of neurotoxicity (Chen Z L and Strickland S, 1997: Neuronal Death in the hippocampus is promoted by plasmin-catalysed degradation of laminin. Cell: 91, 917-925).
  • FIG. 9 A summarizing outline of the time depending neurotoxic effect of t-PA is given in FIG. 9 . Therein also the increased toxicity of the recombinant t-PA compared to endogenic t-PA becomes evident. This is probably due to rt-PA being able to enter into tissue in higher concentrations.
  • DSPA Desmodus rotundus Plasminogen Activator
  • DSPA is a plasminogen activator with a high homology (resemblance) to t-PA. Therefore—and in addition to the disillusionment resulting from the neurotoxic side effects of t-PA—there were no further expectations, for DSPA being a suitable drug for stroke treatment.
  • a further new treatment focuses neither on thrombus nor on blood thinning or anti coagulation but attempts to increase the vitality of cells damaged by the interruption of blood supply (WO 01/51613 A1 and WO 01/51614 A1).
  • aminoglycosides or chloramphenicol are applied.
  • citicholin directly after the onset of stroke.
  • citicholin is cleaved to cytidine and choline. The cleavage products form part of the neuronal cell membrane and thus support the regeneration of damaged tissue (U.S. Pat. No. 5,827,832).
  • a competitive inhibition (antagonistic action) of the glutamate receptor of NMDA type is possible e.g. with 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate (APH).
  • a non competitive inhibition can be achieved e.g. by substances binding to the phencyclidine side of the channels. Such substances can be phencyclidine, MK-801, dextrorphane or cetamine.
  • the central idea of the invention is the use of a plasminogen activator in the treatment of stroke, of which the mature enzyme exhibits an activity, which is selectively increased by fibrin manifold, namely more than the 650-fold.
  • the use of the plasminogen activators according to the invention is based on the following findings: Due to tissue damage in the brain caused by stroke the blood brain barrier is damaged or destroyed. Thus, fibrinogen circulating in the blood can enter into the neuronal tissue of the brain. There, it activates t-PA which—indirectly by activating the glutamate receptor or plasminogen—results in further tissue damage. In order to avoid this effect the invention suggests the use of a plasminogen activator which is highly fibrin selective and—as an inversion of the argument—has a reduced potential to be activated by fibrinogen.
  • this plasminogen activator is not—or compared to t-PA at least substantially less—activated by fibrinogen entering from the blood into neuronal tissue as a result of damaged blood brain barrier, since t-PA's activator fibrin cannot enter the neuronal tissue due to its size.
  • the plasminogen activators according to the invention therefore are non-neurotoxic.
  • non-toxic plasminogen activators are used, which comprise at least one element of the so called cymogene triade.
  • a comparable triade is known from the catalytic center of serine proteases of the chymotrypsine family consisting of three interacting amino acids aspartate 194, histidine 40 and serine 32.
  • this triade does not exist in t-PA which belongs also to the family of chymotrypsine like serine proteases.
  • the directed mutagenesis of native t-PA for the purpose of introducing at least one of the above amino acids at a suitable position results in a reduced activity of the pro-enzyme (single chain t-PA) and to an increased activity of the mature enzyme (double chain t-PA) in the presence of fibrin. Therefore, the introduction of at least one amino acid of the triade—or of an amino acid with the respective function in the triade—can increase the cymogenity of t-PA (i.e. the ratio between the activity of the mature enzyme an the activity of the pro-enzyme). As a result the fibrin specificity is remarkably increased. This is due to conformational interaction between the introduced amino acid residue and/or amino acid residues of the wild type sequence.
  • mutants comprise a substitution of Arg275 to R275E in order to prevent cleavage by plasmin at the cleavage site Aug275-Ile276, thereby converting the single chain t-PA to the double chain form.
  • the mutant site R275E alone leads to a 6,900 fold increase of the fibrin specificity of t-PA (K Tachias, Madison E L 1995: Variants of Tissue-type Plasminogen Activator Which Display Substantially Enhanced Stimulation by Fibrin, in: Journal of Biological Chemistry 270, 31: 18319-18322).
  • the positions 305 and 292 of t-PA are homologous to the positions His40 and Ser32 of the known triade of the chymotryptic serine proteases. By the corresponding substitutions introducing histidine or respectively serine, these amino acids can interact with the aspartate477 of t-PA resulting in a functional triade in the t-PA mutants (Madison et al., 1993).
  • t-PA mutants can be used for the treatment of stroke according to the invention because they show no or—compared to wild type t-PA—a significantly reduced neurotoxicity due to their increased fibrin specificity.
  • F305H; F305H; A292S alone or in combination with R275E we incorporate the publications of Madison et al., (1993) and Tachias and Madison (1995) hereby are fully incorporated by reference.
  • Plasminogen activators belong to the group of serine proteases of the chymotrypsin family and therefore comprise the conserved amino acid Asp194, which is responsible for the stability of the catalytic active conformation of the mature proteases. It is known that Asp194 interacts with His40 in the cymogenic form of serine proteases. After the cymogene is activated by cleavage this specific interaction is interrupted and the side chain of the Asp194 rotates about 170° in order to form a new salt bridge with Ile16. This salt bridge essentially contributes to the stability of the oxyanione pocket of the catalytic center of the mature serine proteases. It is also present in t-PA.
  • the Asp194 mutants of the plasminogen activators show a high increase of activity in presence of fibrin which enables their use according to the invention.
  • a mutant t-PA is used, in which Asp194 is substituted by glutamate (D194E) or respectively by asparagine (D194N).
  • D194E glutamate
  • D194N asparagine
  • the activity of t-PA is reduced 1 to 2000 fold in the absence of fibrin, whereas in the presence of fibrin, an increase of activity by a factor of 498,000 to 1,050,000 can be achieved.
  • These mutants can further comprise a substitution of Arg15 to R15E, which prevents the cleavage of the single chain t-PA at the peptide bond Arg15-Ile16 by plasmin, leading to the double chain form of t-PA.
  • This mutation alone increases the activation of t-PA by fibrin by the factor 12,000.
  • the publications of Strandberg and Madison (1995) are fully incorporated by reference.
  • An increase of the fibrin dependency of plasminogen activators can also be achieved by the introduction of point mutations in the so called “autolysis loop”.
  • This element is known from trypsine; it can also be found as a homologous part in serine proteases and is especially characterized by three hydrophobic amino acids (Leu, Pro and Phe).
  • the autolysis loop in plasminogen activators is responsible for the interaction with plasminogen. Point mutations in this area can have the effect that the protein-protein interaction between plasminogen and plasminogen activators cannot be effectively formed any longer. These mutations are only functionally relevant in the absence of fibrin.
  • t-PA is used showing point mutations in the positions 420 to 423. If these residues are substituted by directed mutagenesis this increases the fibrin dependency of t-PA is increased by a factor up to 61,000 (K Song-Hua et al.).
  • Song-Hua et al. examined the point mutations L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.
  • a modified tissue plasminogen activator with an amino acid sequence according to SEQ ID No. 1 ( FIG. 13 ) is used.
  • This modified t-PA differs from the wild type t-PA by the exchange of the hydrophobic amino acids in the position 420 to 423 in the autolysis loop as follows: His420, Asp421, Ala422 and Cys423.
  • This t-PA preferentially contains a phenyl alanine at the position 194.
  • the position 275 can be occupied by glutamate.
  • the position 194 is occupied by phenyl alanine.
  • a modified urokinase can be used according to the invention.
  • the urokinase according to the invention can comprise the amino acid sequence according to SEQ ID No. 2 ( FIG. 14 ) in which the hydrophobic amino acids of the autolysis loop are substituted by Val420, Thr421, Asp422 and Ser423.
  • the urokinase is carrying an Ile275 and a Glu194. This mutant shows—in comparison to wild type urokinase—a 500-fold increased fibrin specificity.
  • the plasminogen activator (DSPA) from the saliva of the vampire bat also shows a highly increased activity in the presence of fibrin—in specific a 100,000-fold increase.
  • DSPA comprises four different proteases, which fulfill an essential function for the vampire bat, namely an increased duration of bleeding of the wounds of pray (Cartwright, 1974). These four proteases (DSPA ⁇ 1, DSPA ⁇ 2, DSPA ⁇ , DSPA ⁇ ) display a high similarity (homology) to each other and to the human t-PA. They also show similar physiological activities, leading to a common classification under the generic term DSPA.
  • DSPA is disclosed in the patents EP 0 352 119 A1 and of U.S. Pat. Nos. 6,008,019 and 5,830,849 which are hereby fully incorporated by reference for purpose of disclosure.
  • DSPA ⁇ so far is the best analyzed protease from this group. It has an amino acid sequence with a homology greater than 72% in comparison to the known human t-PA amino acid sequence (Krfordschmar et al, 1991). However, there are two essential differences between t-PA and DSPA. Firstly all DSPA has full protease activity as a single chain molecule, since it is—in contrast to t-PA—not converted into a double chain form (Gardell et al., 1989; Krrienschmar et al., 1991). Secondly, the catalytic activity of DSPA is nearly absolutely dependent on fibrin (Gardell et al., 1989; Bringmann et al., 1995; Toschie et al., 1998).
  • DSPA ⁇ 1 For example the activity of DSPA ⁇ 1 is increased 100,000 fold in the presence of fibrin whereas the t-PA activity is only increased 550 fold. In contrast, DSPA activity is considerably less strongly induced by fibrinogen, since it only shows a 7 to 9 fold increase (Bringmann et al., 1995). In conclusion, DSPA is considerably more dependent of fibrin and much more fibrin specific as wild type t-PA which is only activated 550-fold by fibrin.
  • DSPA is an interesting candidate for the development of a thrombolytic agent.
  • the therapeutic use of DSPA as a thrombolytic agent was restricted to the treatment of myocardinal infarction in the past, because—due to the contribution of t-PA to the glutamate induced neurotoxicity—no justified hopes existed, that a plasminogen activator which is related to t-PA could reasonably be used for a treatment of acute stroke.
  • DSPA has no neurotoxic effects even though it shows a high resemblance (homology) to t-PA and even though the physiological effects of the molecules are comparable to a large extent.
  • the above conclusion led to the idea that DSPA after all may be successfully used as a thrombolytic agent for the therapy of stroke without causing severe risks of neuronal tissue damage.
  • DSPA can also be used later than 3 hours after the onset of stroke symptoms.
  • a further teaching of the present invention that evolved from the above findings is the option to modify or produce further plasminogen activators in such a way that they reveal the essential characteristics of DSPA, especially the lack of the neurotoxicity of t-PA.
  • the basis for this is the investigated relationship between structure and biochemical effects, making it possible to transform neurotoxic plasminogen activators into non-neurotoxic plasminogen activators and thereby to produce non-neurotoxic plasminogen activators on the basis of known or newly discovered neurotoxic plasminogen activators.
  • the new teaching is based on in vivo comparative examinations of the neurodegenerative effect of t-PA on one side and of DSPA on the other side which are performed by using the so called kainic acid model and a model for the examination of NMDA induced lesion of the striatum.
  • the kainic acid model (also kainic acid injury model) is based on the stimulation of the neurotoxic glutamate cascade by the external application of kainic acid (KA) as an agonist of the glutamate receptor of the kainic acid type (KA type) and of the NMDA and AMPA glutamate receptors.
  • KA kainic acid
  • t-PA deficient mouse stem as an experimental model it was possible to show that the sensitivity of the laboratory animals against kainic acid only reached the level of wild type mice after a supplementary application of external t-PA.
  • an infusion of an equimolar concentration of DSPA under the same experimental conditions does not restore the sensitivity to kainic acid (KA).
  • the lacking neurotoxicity of DSPA and of the other non-neurotoxic plasminogen activators offer the special advantage in stroke treatment that the use of these plasminogen activators—in contrast to the wild type t-PA—is not limited to a short maximum period of only 3 hours after the onset of stroke. In contrary, the treatment can be initiated later—for example after 6 hours or even later, since there is nearly no risk of stimulating excitotoxic responses.
  • First clinical trials with DSPA prove a safe treatment of patients even in a time range of over 6 to 9 hours after the onset of stroke symptoms.
  • DSPA as well as further non-neurotoxic plasminogen activators show no tissue damaging side effects.
  • Neuroprotective agents inhibiting the glutamate receptor competitively or non-competitively can be used.
  • Useful combinations are e. g. with the known inhibitors of the glutamate receptors of the NMDA type, the kainic acid type or the quisqualate type, as for example APV, APH, phencyclidine, MK-801, dextrorphane or cetamine.
  • cations especially Zn-ions, block the cation channel regulated by the glutamate receptor and can therefore reduce neurotoxic effects.
  • non-neurotoxic plasminogen activators can be combined with at least one further therapeutic agent or with a pharmaceutically tolerable carrier.
  • the combination with a therapeutic agent which supports the reduction of tissue damage by vitalizing the cells is especially advantageous, since it contributes to the regeneration of already damaged tissue or serves for the prevention of further stroke incidents.
  • Advantageous examples are combinations with antibiotics as quinones, anticoagulants as heparin or hirudin as well as with citicholine or acetylsalicylic acid.
  • a combination with at least one thrombin inhibitor can also be advantageous.
  • thrombomodulin and thrombomodulin analogs like for example solulin, triabin or pallidipin can be used.
  • Further combinations with anti-inflammatory substances are advantageous, since they influence the infiltration by leucocytes.
  • mice were anaesthetised, then transcardially perfused with PBS and the brains removed.
  • the hippocampus region was removed, transferred to eppendorf tubes and incubated in an equal volume (w/v) (approx. 30-50 ⁇ m) of 0.5% NP-40 lysis buffer containing no protease inhibitors (0.5% NP-40, 10 mM Tris-HCl pH 7.4, 10 mM NaCL, 3 mM MgCl2, 1 mM EDTA).
  • the brain extracts were homogenized by means of a hand-held glass homogeniser and left on ice for 30 minutes. The samples were then centrifuged and the supematant was removed. The amount of proteins present was determined (Bio-Rad-reagent).
  • the proteolytic activity in the samples and the brain tissue extracts was determined by zymographic analysis according to the method of Granelli, Piperno and Reich (1974).
  • the samples with recombinant proteins (up to 100 nM) or the brain tissue extracts (20 ⁇ g) were subjected to a (10%) SDS-PAGE under non-reducing conditions.
  • the gels were removed from the plates, washed in 1% triton X 100 for 2 hours and then overlaid onto an agarose gel containing polymerized fibrinogen and plasminogen (Granelli, Piperno and Reich, 1974).
  • the gels were incubated at 37° C. in a humified chamber until proteolysed zones appeared.
  • the kainic acid injury model was based on studies of Tsirka et al. (1995).
  • the animals were injected intraperitoneally (i. p.) with atropine (4 mg/kg) and then anaesthetised with an i. p. injection of sodium pentobarbitol (70 mg/kg).
  • mice were placed in a stereotaxic frame and a micro-osmotic pump (Alzet model 1007D, Alzet Calif. USA) containing 100 ⁇ l of either PBS or recombinant human t-PA (0.12 mg/ml, 1.85 ⁇ M) or DSPA ⁇ 1 (1.85 ⁇ M) was implanted subcutaneously between the shoulder blades.
  • the pumps were connected via sterile tubes to a brain cannula and inserted through a burr opening made through the skull at coordinates bregma ⁇ 2.5 mm, midiolateral 0.5 mm and dorsoventral 1.6 mm in order to introduce the liquid near the midline.
  • the cannula was fixed at the desired position and the pumps were allowed to infuse the respective solutions at a rate of 0.5 ⁇ l per hour for a total of 7 days.
  • mice Two days after infusion of the proteases the mice were reanaesthetised and again placed in the stereotaxic frame. Afterwards 1.5 nmol of kainic acid (KA) in 0.3 ⁇ l, PBS was 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 delivered for a duration of 30 seconds. After the kainic acid treatment the injection needle remained at these coordinates for further 2 minutes in order to prevent a reflux of the liquid.
  • KA kainic acid
  • the animals were anaesthetised and transcardially perfused with 30 ml PBS followed by 70 ml of a 4% paraformaldehyd solution, post fixed in the same fixative followed by incubation in 30% sucrose for further 24 hours. Coronal sections (40 ⁇ m) of the brain were then cut on a freezing microtome and either counter-stained with thionin (BDH, Australia) or processed for immunohistochemical examination as described below.
  • the quantification of neuronal loss in the CA1-CA3 hippocampal subfields was performed as previously described (Tsirka et al., 1995; Tsirka et al., 1996). Five consecutive parts of the dorsal hippocampus from all treatment groups were prepared taking care that the parts indeed comprised the place of the CA-injection and lesion area. The hippocampal subfields (CA1-CA3) of these sections were traced by means of camera lucida drawings of the hippocampus. The entire lengths of the subfields was measured by comparison to 1 mm standards traced under the same magnification.
  • the lengths of tissue with viable pyramidal neurons (having normal morphology) and lengths of tissue devoid of neurons (no cells present, no thionin staining) was determined.
  • the lengths, representing intact neurons and neuronal losses over each hippocampal subfield were averaged across sections and the standard deviations were determined.
  • Wild type mice (c57/Black 6) were anaesthetised and placed in a sterertaxic frame (see above). Mice then received an unilateral injection of 50 nmol NMDA in the left stratum, injected alone or in combination with either 46 ⁇ M rt-PA or 46 ⁇ M DSPA ⁇ 1. As controls t-PA and DSPA were also injected alone (both at a concentration of 46 ⁇ M). The injection coordinates were: bregma ⁇ 0.4 mm, midiolateral 2.0 mm and dorsoventral 2.5 mm.
  • mice were anaesthetised and perfused transcardially with 30 ml PBS followed by 70 ml of a 4% paraformaldehyd solution, post fixed in the same fixative for 24 hours with followed by incubation in 30% sucrose for further 24 hours. Brains were then cut (40 ⁇ m) on a freezing microtome and mounted onto gelatin coated glass slides.
  • the quantification of the striatal lesion volume was performed using the method described by Callaway et al. (2000). Ten consecutive coronal sections spanning the lesioned area were prepared. The lesioned area was visualised using the Callaway method and the lesion volume was quantified by the use of a micro computer imaging device (MCID, Imaging Research Inc., Brock University, Ontario, Canada).
  • Immunohistochemistry was performed using standard methodologies. Coronal sections were immersed in a solution of 3% H 2 O 2 and 10% methanol for 5 minutes followed by an incubation in 5% normal goat serum for 60 minutes. The sections were incubated over night either with an anti-GFAP antibody (1:1,000; Dako, Carpinteria, Calif., USA) for the detection of astrocytes, with an anti-MAC-1 antibody (1:1,000; Serotec, Raleigh, N.C., USA) for the detection of microglia or with polyclonal anti-DSPA antibodies (Schering A G, Berlin). After rinsing, the sections were incubated with the appropriate biotinylated secondary antibodies (Vector Laboratories, Burlingame, Calif., USA).
  • t-PA ⁇ / ⁇ were infused for seven days with either t-PA or DSPA (see above). Mice were then transcardially perfused with PBS and the brains removed. The ipsilateral and contralateral hippocampal regions were isolated as well as a region of the cerebellum (taken as a negative control). Tissue samples (20 ⁇ g) were subjected to SDS-PAGE and zymographic analysis according to the description in the methods section. As can be seen in FIG.
  • 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 infused proteases not only retained their activity in the brain but had also diffused within the hippocampal region. As a control, no activity could be detected in the extract prepared from the cerebellum.
  • t-PA ⁇ / ⁇ mice are characteristically resistant to kainic acid (KA) mediated neurodegeneration.
  • KA kainic acid
  • intrahippocampal infusion of rt-PA completely restores the sensitivity to KA-mediated injury.
  • cort-PA ⁇ / ⁇ mice infused with PBS were resistant to subsequent challenge with KA.
  • infusion of recombinant t-PA restored sensitivity to KA treatment.
  • infusion of the same concentration of DSPA into the hippocampal region did not alter the sensitivity of the animals to KA.
  • the concentration of t-PA used for the infusion was based on the concentration described by Tsirka et al. (1995) (100 ⁇ l of 0.12 mg/ml [1.85 ⁇ M]).
  • the KA-injury experiments were repeated using a 10-fold lower of t-PA (0.185 ⁇ M) and a 10-fold higher amount of DSPA (18.5 ⁇ M).
  • DSPA does not lead to an increase of sensitivity to KA.
  • t-PA and DSPA were also examined in a model of neurodegeneration in wild type mice.
  • DESAFIB is a preparation of soluble fibrin monomeres gained by the cleavage of highly pure human fibrinogen with the protease batroxobin. Batroxobin cleaves the Arg 16 -Gly 17 -binding in the A ⁇ -chain of fibrinogen and thereby releases fibrinopeptid A. The resulting des-AA-fibrinogen representing fibrin I monomers is soluble in the absence of the peptide Gly-Pro-Arg-Pro. The concentration of Lys-plasminogen was varied from 0.0125 up to 0.2 ⁇ M in the presence of DESAFIB and from 0.9 to 16 ⁇ M in absence of the co-factor.
  • Indirect chromogen standard tests were performed according to the publications cited above. Probes of 100 ⁇ l total volume containing 0.25-1 ng enzyme, 0.2 ⁇ M Lys-plasminogen and 0.62 mM spectrocyme PL were used. The tests were performed either in the presence of buffer, 25 ⁇ g/ml DESAFIB, 100 ⁇ g/ml cyanogen bromide fragments of fibrinogen (American Diagnostica) or 100 ⁇ g/ml of the stimulatory 13 amino acid peptide P368. The analysis were performed in microtiter-plates and the optic density was determined at a wave length of 405 nm every 30 seconds for 1 hour in a “Molecular Devices Thermomax”. The reaction temperature was 37° C.

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US20020089179A1 (en) * 2000-12-30 2002-07-11 Levon Guyumjan Hose coupling apparatus and method
US20060142195A1 (en) * 2001-11-02 2006-06-29 Paion Gmbh Non-neurotoxic plasminogen activating factors for treating of stroke

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060142195A1 (en) * 2001-11-02 2006-06-29 Paion Gmbh Non-neurotoxic plasminogen activating factors for treating of stroke
US20090004176A1 (en) * 2001-11-02 2009-01-01 Paion Deutschland Gmbh Non-neurotoxic plasminogen activating factors for treating of stroke
US20090263373A1 (en) * 2001-11-02 2009-10-22 Mariola Sohngen Non-neurotoxic plasminogen activating factors for treating of stroke
US8071091B2 (en) 2001-11-02 2011-12-06 Paion Deutschland Gmbh Non-neurotoxic plasminogen activating factors for treating stroke
US8119597B2 (en) 2001-11-02 2012-02-21 Paion Gmbh Non-neurotoxic plasminogen activating factors for treating of stroke
US20060135425A1 (en) * 2003-05-02 2006-06-22 Paion Deutschland Gmbh Intravenous injection of plasminogen non-neurotoxic activators for treating cerebral stroke
US20100272704A1 (en) * 2007-10-18 2010-10-28 Soehngen Mariola Novel patient subgroups for thrombolysis

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