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 homologue 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 - 19 . (canceled) 
     
     
         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 . (canceled) 
     
     
         24 . (canceled) 
     
     
         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 . (canceled) 
     
     
         30 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 .