Non-neurotoxic plasminogen activating factors for treating of stroke

Abstract

The invention pertains to the use and production of non-neurotoxic plasminogen activating factors e.g. of Desmodus rotundus (DSPA) for the therapeutic treatment of stroke in humans in order to provide a new therapeutic concept for treating stroke in humans.

1 . A method of treating stroke in a mammal, comprising administering to the mammal a therapeutically effective amount of a plasminogen activating factor (PAF), wherein the PAF's activity is enhanced by more than 650 fold in the presence of fibrin.  
     
     
         2 . The method of  claim 1 , wherein the plasminogen activating factor comprises at least a part of a cymogene triade, wherein the part of the cymogene triade comprises at least a histidine residue or serine residue which interact with an aspartate residue.  
     
     
         3 . The method according to  claim 2 , wherein the serine residue, if present, is located at a position at least partially homologous to 292, the histidine residue, if present, is located at a position at least partially homologous to 305, and the aspartate residue is located at a position at least partially homologous to 447 of t-PA.  
     
     
         4 . The method according to  claim 3 , wherein the plasminogen activating factor is t-PA mutant t-PA/R275E; t-PA/R275E, F305H; or t-PA/R275E, F305H, A292S.  
     
     
         5 . The method according to  claim 1 , wherein the plasminogen activating factor has a point mutation at Asp194, or at a aspartate at a position homologous to Asp194, wherein the point mutation reduces catalytically active conformation stability of the plasminogen activating factor in the absence of fibrin.  
     
     
         6 . The method according to  claim 5 , wherein Asp194 is substituted by glutamate or asparagine.  
     
     
         7 . The method according to  claim 1 , wherein the plasminogen activating factor comprises at least one mutation in its autolysis loop, wherein the mutation reduces the functional interactions between plasminogen and plasminogen activating factor in the absence of fibrin.  
     
     
         8 . The method according to  claim 7 , 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.  
     
     
         9 . The method according to  claim 8 , wherein at least one mutation is L420A, L420E, S421G, S421E, P422A, P422G, P422E, F423A or F423E.  
     
     
         10 . The method according to  claim 1 , wherein the plasminogen activating factor is a cymogene comprising at least one point mutation preventing the catalysis by plasmin.  
     
     
         11 . The method according to  claim 10 , wherein at least one point mutation is located at position 15 or 275 of t-PA, or at a homologous position.  
     
     
         12 . The method according to  claim 11 , wherein glutamate is at position 15 or 275.  
     
     
         13 . The method according to  claim 1 , wherein the plasminogen activating factor is vampire bat saliva plasminogen activating factor (DSPA).  
     
     
         14 . The method according to  claim 1 , wherein the PAF is administered at least 3 hours after onset of a stroke.  
     
     
         15 . The method according to  claim 1 , wherein the PAF is administered at least 6 hours after onset of a stroke.  
     
     
         16 . The method according to  claim 1 , wherein the PAF is administered at least 9 hours after onset of a stroke.  
     
     
         17 . The method according to  claim 1 , wherein stroke onset in the patient is not exactly determined.  
     
     
         18 . A tissue plasminogen activating factor (PAF) with an autolysis loop comprising His420, Asn421, Ala422 and Cys423.  
     
     
         19 . The PAF according to  claim 18 , further comprising a point mutation at position 194, which reduces catalytically active conformation stability of the PAF when fibrin is absent.  
     
     
         20 . The PAF according to  claim 19 , wherein position 194 comprises Phe194.  
     
     
         21 . The PAF according to  claim 18 , further comprising at least one point mutation that prevents plasmin catalysis.  
     
     
         22 . The PAF according to  claim 21 , wherein the point mutation is Glu275.  
     
     
         23 . A plasminogen activating factor having an amino acid sequence according to SEQ ID NO:1.  
     
     
         24 . A modified urokinase having enzymatic activity in presence of fibrin enhanced by more than 500-fold compared to wild type urkinase.  
     
     
         25 . A urokinase comprising an autolysis loop comprising Val420, Thr421, Asp422 and Ser423.  
     
     
         26 . The urokinase according to  claim 25 , further comprising a point mutation at position 194, wherein the point mutation reduces the stability of the catalytic active conformation of the urokinase in absence of fibrin.  
     
     
         27 . The urokinase according to  claim 26 , wherein the point mutation is Glu194.  
     
     
         28 . The urokinase according to  claim 24  or  25 , further comprising at least one point mutation that prevents plasmin catalysis.  
     
     
         29 . The urokinase according to  claim 28 , wherein the point mutation is Ile275.  
     
     
         30 . A urokinase having an amino acid sequence according to SEQ ID NO:2.  
     
     
         31 . A pharmaceutical composition comprising urokinase according to  claim 24 ,  25  or  30 .  
     
     
         32 . Method of conducting fibrinolysis comprising administering an effective amount of urkinase according to one of the claims  24 ,  25  or  30 .  
     
     
         33 . A method of treating stroke in a mammal, comprising administering to the mammal a therapeutically effective amount of the urkinase of  claim 24 ,  25  or  30 .  
     
     
         34 . A pharmaceutical composition containing a plasminogen activating factor according to  claim 18  or  23 .  
     
     
         35 . The pharmaceutical composition according to  claim 34 , further comprising at least one additional pharmaceutically active component or its pharmaceutically acceptable salt.  
     
     
         36 . The pharmaceutical composition according to  claim 35 , wherein at least one component is a neuroprotective agent.  
     
     
         37 . The pharmaceutical composition according to  claim 36 , wherein the neuroprotective agent is a glutamate receptor antagonist.  
     
     
         38 . The pharmaceutical composition according to  claim 37 , wherein the glutamate receptor antagonist is a competitive or non-competitive antagonist.  
     
     
         39 . The pharmaceutical composition according to  claim 35 , wherein at least one component is a thrombin inhibitor.  
     
     
         40 . The pharmaceutical composition according to  claim 39 , wherein the thrombin inhibitor is thrombomodulin, thrombomodulin analogues, triabin, pallidipin or solulin.  
     
     
         41 . The pharmaceutical composition according to  claim 35 , wherein the at least one component is an anticoagulant agent.  
     
     
         42 . The pharmaceutical composition according to  claim 41 , wherein the anticoagulant agent is hirudin, heparin, acetylsalicylic acid or ancrod.  
     
     
         43 . The pharmaceutical composition according to  claim 35 , wherein at least one component is an anti-inflammatory substance.  
     
     
         44 . The pharmaceutical composition according to  claim 35 , wherein at least one component is an antibiotic agent.  
     
     
         45 . The pharmaceutical composition according to  claim 35 , wherein at least one component is citicholine.  
     
     
         46 . A method for manufacturing 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: 
 (a) introducing at least a part of a cymogenic triade;    (b) substituting Asp194 or a homologous aspartate for reducing the stabilization of the catalytic active conformation in the absence of fibrin;    (c) substituting the hydrophobic amino acid residues in the autolysis loop or in homologous peptide sections to that; or    (d) introducing a mutation in the cymogene for preventing the catalysis of the cymogene by plasmin.    
     
     
         47 . A drug produced according to the method of  claim 46.