Non-neurotoxic plasminogen activating factors for treating stroke

Abstract

The invention concerns the use and the production of non-neurotoxin 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 . A method for treating stroke comprising administered to a subject in need of such treatment, a therapeutically effective amount of a plasminogen activating factor, wherein the the plasminogen activating factor has an enhanced fibrin specificity compared to wild-type t-PA.  
     
     
         2 . The method according to  claim 1 , wherein the plasminogen activating factor comprises at least a histidine or serine residue forming together with an aspartic acid residue at least a part of a zymogen triade.  
     
     
         3 . The method according to  claim 2 , wherein the serine residue is located at a position at least partly homologous to position 292 of t-PA, the histidine residue is located at a position at least partly homologous to position 305 of t-PA and the aspartic acid residue is located at a position at least partly homologous to position 447 of t-PA.  
     
     
         4 . The factor according to  claim 3 , wherein the plasminogen activating factor is selected from the group consisting of: t-PA/R275E; t-PA/R275E, F305H; t-PA/R275E, F305H, A292S.  
     
     
         5 . The method according to  claim 1 , wherein the plasminogen activating factor contains a point mutation at Asp194 or of an aspartic acid residue in a homologous position, which reduces stability of the catalytically active conformation of the plasminogen activating factor in the absence of fibrin.  
     
     
         6 . The method according to  claim 5 , wherein Asp194 is substituted by glutamatic acid or asparagine.  
     
     
         7 . The method according to  claim 6 , wherein t-PA carries a substitution at Asp194 to Glu194 or Asn194.  
     
     
         8 . The method according to  claim 1 , wherein the plasminogen activating factor comprises at least one mutation in its autolysis loop, which reduces functional interactions between plasminogen and plasminogen activating factor in the absence of fibrin.  
     
     
         9 . The method according to  claim 8 , wherein at least one mutation in the autolysis loop affects the amino acids at positions 420 to 423 of wild-type t-PA or at positions homologous thereof.  
     
     
         10 . The method according to  claim 9 , wherein the mutation is selected from the group consisting of: L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A and F423E.  
     
     
         11 . The method according to  claim 1 , wherein the plasminogen activating factor is a zymogen comprising at least one point mutation preventing catalysis by plasmin.  
     
     
         12 . The method according to  claim 11 , wherein the point mutation is located at position 15 or 275 of t-PA or at a position homologous thereto.  
     
     
         13 . The method according to  claim 12 , wherein glutamatic acid is at position 15 or 275.  
     
     
         14 . The method according to  claim 1 , wherein the plasminogen activating factor is isolated from the saliva of the vampire bat (DSPA).  
     
     
         15 . The method according to  claim 1 , wherein said plasminogen activating factor is administered to a human at least 3 hours after onset of a stroke in said human.  
     
     
         16 . The method according to  claim 1 , wherein said plasminogen activating factor is administered to a human at least 6 hours after onset of a stroke in said human.  
     
     
         17 . The method according to  claim 1 , wherein said plasminogen activity factor is administered to a human at least 9 hours after onset of a stroke in said human.  
     
     
         18 . The method according to  claim 1 , wherein onset of stroke in said subject is temporally not exactly determined.  
     
     
         19 . The method according to  claim 1 , wherein said plasminogen activating factor does not exhibit neurotoxicity of wild-type t-PA.  
     
     
         20 . An isolated tissue plasminogen activating factor comprising an autolysis loop comprising His420, Asn421, Ala422 and Cys423.  
     
     
         21 . The isolated tissue plasminogen activating factor according to  claim 20 , wherein said factor further comprises a point mutation at position 194, which reduces stability of the catalytically active conformation of the plasminogen activating factor in the absence of fibrin.  
     
     
         22 . An isolated tissue plasminogen activating factor according to  claim 21 , wherein said point mutation Phe194.  
     
     
         23 . An isolated tissue plasminogen activating factor according to  claim 20 , wherein said factor comprises at least one point mutation which prevents catalysis by plasmin.  
     
     
         24 . An isolated tissue plasminogen activating factor according to  claim 23 , wherein said point mutation is Glu275.  
     
     
         25 . An isolated tissue plasminogen activating factor consisting of an amino acid sequence as shown in Seq. ID No. 1.  
     
     
         26 . An isolated urokinase comprising an autolysis loop comprising Val420, Thr421, Asp422 and Ser423.  
     
     
         27 . The isolated urokinase according to  claim 26 , wherein said urokinase has a point mutation at position 194, which reduces stability of the catalytic active conformation of the urokinase in absence of fibrin.  
     
     
         28 . The isolated urokinase according to  claim 27 , wherein said point mutation is Glu194.  
     
     
         29 . The isolated urokinasee according to  claim 26 , wherein said urokinase has at least one point mutation which prevents catalysis by plasmin.  
     
     
         30 . The isolated urokinase according to  claim 29 , wherein said point mutation is Ile275.  
     
     
         31 . An isolated urokinase consisting of an amino acid sequence as shown in Seq. ID No. 2.  
     
     
         32 . A pharmaceutical composition comprising a plasminogen activating factor according to  claim 1  and at least one additional pharmaceutically active component or a pharmaceutically acceptable salt thereof.  
     
     
         33 . The pharmaceutical composition according to  claim 32 , wherein said component is a neuroprotective agent.  
     
     
         34 . The pharmaceutical composition according to  claim 33 , wherein said component is a glutamate receptor antagonist.  
     
     
         35 . The pharmaceutical composition according to  claim 34  wherein said component is a competitive or non-competitive antagonist.  
     
     
         36 . The pharmaceutical composition according to  claim 33 , wherein said component is a thrombin inhibitor.  
     
     
         37 . The pharmaceutical composition according to  claim 33 , wherein said component is an anticoagulant agent.  
     
     
         38 . The pharmaceutical composition according to  claim 33 , wherein said component is an anti-inflammatory agent.  
     
     
         39 . The pharmaceutical component according to  claim 33 , wherein said composition is an antibiotic agent.  
     
     
         40 . The pharmaceutical composition according to  claim 33 , wherein said component is citicholine.  
     
     
         41 . (Cancelled).  
     
     
         42 . A method for the production of a non-neurotoxic plasminogen activating factor comprising at least one of the following steps: 
 introducing a mutation in at least a part of a zymogen triade of a plasminogen activating factor;    introducing a substitution mutation at Asp194 or a homologous aspartic acid residue to reduce stabilization of the catalytic active conformation in the absence of fibrin;    introducing a substitution mutation in the hydrophobic amino acid residues in the autolysis loop or in homologous regions thereof; and    introducing a mutation in a zymogen to prevent catalysis of the zymogen by plasmin.    
     
     
         43 . A drug produced by the process according to  claim 42 .  
     
     
         44 . The pharmaceutical composition according to  claim 36 , wherein said thrombin inhibitor is selected from the group consisting thrombomodulin, thrombomodulin analogues, triabin, pallidipin and solulin.  
     
     
         45 . The pharmaceutical composition according to  claim 37 , wherein said anticoagulant is selected from the group consisting hirudin, heparin, acetylsalicylic acid and ancrod.