Non-neurotoxic plasminogen activating factors for treating of stroke

Abstract

The invention concerns the use and the production of non-neurotoxic plasminogen activating factors, derived, for example, from the common vampire Desmodus rotundus (DSPA), for therapeutic treatment of stroke in humans. The invention provides a novel therapeutic base for treating stroke in humans.

1 . Use of a plasminogen activating factor for the treatment of stroke wherein the activity of the plasminogen activating factor is enhanced by more than 650 fold in the presence of fibrin. 
     
     
         2 . Use of a plasminogen activating factor according to  claim 1  wherein the plasminogen activating factor comprises at least a histidine or serine residue forming together with an aspartade residue at least a part of a cymogene triade. 
     
     
         3 . Use of a plasminogen activating factor according to  claim 2  wherein the serine residue is located at a position at least partly homologous to position 292, the histidine residue is located at a position at least partly homologous to position 305 and the aspartate residue is located at a position at least partly homologous to position 447 of t-PA. 
     
     
         4 . Use of a plasminogen activating factor according to  claim 3  wherein the plasminogen activating factor is selected from the group of the following t-PA mutants: t-PA/R275E; t-PA/R275E, F305H; t-PA/R275E, F305H, A292S. 
     
     
         5 . Use of a plasminogen activating factor according to  claim 1  wherein the plasminogen activating factor carries a point mutation of the Asp194 or of an aspartate in a homologe position, leading to a reduced stability of the catalytically active conformation of the plasminogen activating factor in the absence of fibrin. 
     
     
         6 . Use according to  claim 5  wherein Asp194 is substituted by glutamate or asparagine. 
     
     
         7 . Use according to  claim 6  wherein t-PA carries a substitution of Asp194 to Glu194 or Asn194. 
     
     
         8 . Use of a plasminogen activating factor according to  claim 1  wherein the plasminogen activating factor comprises at least one mutation in its autolysis loop, which reduces the functional interactions between plasminogen and plasminogen activating factor in the absence of fibrin. 
     
     
         9 . Use according to  claim 8  wherein at least one mutation in the autolysis loop affects the amino acid positions 420 to 423 of wild type t-PA or homologous positions. 
     
     
         10 . Use according to  claim 9  wherein the mutation is selected from the group consisting of the following mutants: L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E. 
     
     
         11 . Use of a plasminogen activating factor according to  claim 1  wherein the plasminogen activating factor is a cymogene comprising at least one point mutation preventing the catalysis by plasmin. 
     
     
         12 . Use of a plasminogen activating factor according to  claim 11  wherein the point mutation is located at the position 15 or 275 of t-PA or at a position homologous to that. 
     
     
         13 . Use according to  claim 12  wherein glutamate is in the position 15 or 275. 
     
     
         14 . Use of a plasminogen activating factor according to  claim 1  wherein the plasminogen activating factor was isolated from the saliva of the vampire bat (DSPA). 
     
     
         15 . Use of a plasminogen activating factor according to at least one of the above claims for the therapeutic treatment of stroke in humans at least 3 hours after stroke onset. 
     
     
         16 . Use of a plasminogen activating factor according to at least one of the above claims for the therapeutic treatment of stroke in humans at least 6 hours after stroke onset 
     
     
         17 . Use of a plasminogen activating factor according to at least one of the above claims for the therapeutic treatment of stroke in humans at least 9 hours after stroke onset. 
     
     
         18 . Use of a plasminogen activating factor according to at least one of the above claims for the therapeutic treatment of stroke patients in which the onset of stroke is temporally not exactly determined. 
     
     
         19 . Use of a plasminogen activating factor according to at least one of the above claims for the therapy of stroke under avoidance of the neurotoxicity of wild type t-PA. 
     
     
         20 . Tissue plasminogen activating factor with an autolysis loop comprising His420, Asn421, Ala422 and Cys423. 
     
     
         21 . Tissue plasminogen activating factor according to  claim 20  characterized in a point mutation in position 194, which reduces the stability of the catalytically active conformation of the plasminogen activating factor in the absence of fibrin. 
     
     
         22 . Tissue plasminogen activating factor according to  claim 21  characterized in Phe194. 
     
     
         23 . Tissue plasminogen activating factor according to at least one of the  claim 20  to  22  characterized in at least one point mutation which prevents the catalysis by plasmin. 
     
     
         24 . Tissue plasminogen activating factor according to  claim 23  characterized in Glu275. 
     
     
         25 . Tissue plasminogen activating factor with an amino acid sequence according to Seq. ID No. 1. 
     
     
         26 . Urokinase with an autolysis loop comprising Val420, Thr421, Asp422 and Ser423. 
     
     
         27 . Urokinase according to  claim 26  characterized in a point mutation in position 194, which reduces the stability of the catalytic active conformation of the urokinase in absence of fibrin. 
     
     
         28 . Urokinase according to  claim 27  characterized in Glu194. 
     
     
         29 . Urokinase according to at least one of the  claims 26  to  28  characterized in at least one point mutation which prevents the catalysis by plasmin. 
     
     
         30 . Urokinase according to  claim 29  characterized in Ile275. 
     
     
         31 . Urokinase with an amino acid sequence according to Seq. ID No. 2. 
     
     
         32 . Pharmaceutical composition containing a plasminogen activating factor according to at least one of the above claims and at least one additional pharmaceutically active component or its pharmaceutically acceptable salt. 
     
     
         33 . Pharmaceutical composition according to  claim 32  characterized in a neuroprotective agent. 
     
     
         34 . Pharmaceutical composition according to  claim 33  characterized in a glutamate receptor antagonist. 
     
     
         35 . Pharmaceutical composition according to  claim 34  characterized in a competitive or non-competitive antagonist. 
     
     
         36 . Pharmaceutical composition according to  claim 33  characterized in at least one thrombin inhibitor, preferentially selected from the group of the following substances: thrombomodulin, thrombomodulin analogues, triabin, pallidipin or solulin. 
     
     
         37 . Pharmaceutical composition according to  claim 33  characterized in at least one anticoagulant agent, preferentially selected from the group of the following anticoagulant agents: hirudin, heparin, acetylsalicylic acid or ancrod. 
     
     
         38 . Pharmaceutical composition according to  claim 33  characterized in anti-inflammatory substances. 
     
     
         39 . Pharmaceutical compositions according to  claim 33  characterized in antibiotic agent. 
     
     
         40 . Pharmaceutical composition according to  claim 33  characterized in citicholine. 
     
     
         41 . Use of a pharmaceutical composition according to at least one of the  claims 36  to  45  according to at least one of the  claims 1  to  19 . 
     
     
         42 . Method for the production of a drug for the treatment of stroke containing one non-neurotoxic plasminogen activating factor comprising at least one of the following steps for the modification of the plasminogen activating factor:
 introduction of at least a part of a cymogenic triade;   substitution of Asp194 or a homologous aspartate for reducing the stabilisation of the catalytic active conformation in the absence of fibrin;   substitution of the hydrophobic amino acid residues in the autolysis loop or in homologous peptide sections to that;   introduction of a mutation in the cymogene for preventing the catalysis of the cymogene by plasmin.   
     
     
         43 . Drug, produced according to  claim 42 .