Kallikrein Inhibitors and Anti-Thrombolytic Agents and Uses Thereof

Abstract

Methods, kits and compositions are described that include a non-naturally occurring kallikrein inhibitor and an anti-thrombolytic agent, e.g., an anti-fibrinolytic agent, for preventing or reducing blood loss and/or ischemia, e.g., ischemia associated with perioperative blood loss and cerebral ischemia, the onset of systemic inflammatory response, and/or reperfusion injury, e.g., reperfusion injury associated with cerebral ischemia or a focal brain ischemia, e.g., in patients subjected to invasive surgical procedures, especially procedures requiring cardiopulmonary bypass.

1 . A method for preventing or reducing blood loss cerebral ischemia in a patient comprising administering to the patient a non-naturally occurring kallikrein inhibitor polypeptide in combination with an anti-fibrinolytic agent. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the polypeptide comprises the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1),
 wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are each individually an amino acid or absent;   Xaa10 is an amino acid selected from the group consisting of: Asp and Glu;   Xaa11 is an amino acid selected from the group consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr;   Xaa13 is an amino acid selected from the group consisting of: Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln;   Xaa15 is an amino acid selected from the group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln;   Xaa16 is an amino acid selected from the group consisting of: Ala, Gly, Ser, Asp and Asn;   Xaa17 is an amino acid selected from the group consisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr;   Xaa18 is an amino acid selected from the group consisting of: His, Leu, Gln and Ala;   Xaa19 is an amino acid selected from the group consisting of: Pro, Gln, Leu, Asn and Ile;   Xaa21 is an amino acid selected from the group consisting of: Trp, Phe, Tyr, His and Ile;   Xaa22 is an amino acid selected from the group consisting of: Tyr and Phe;   Xaa23 is an amino acid selected from the group consisting of: Tyr and Phe;   Xaa31 is an amino acid selected from the group consisting of: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr;   Xaa32 is an amino acid selected from the group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val;   Xaa34 is an amino acid selected from the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu;   Xaa35 is an amino acid selected from the group consisting of: Tyr, Trp and Phe;   Xaa39 is an amino acid selected from the group consisting of: Glu, Gly, Ala, Ser and Asp;   Xaa40 is an amino acid selected from the group consisting of: Gly and Ala;   Xaa43 is an amino acid selected from the group consisting of: Asn and Gly;   Xaa45 is an amino acid selected from the group consisting of: Phe and Tyr; and wherein the polypeptide inhibits kallikrein.   
     
     
         6 . The method of  claim 5 , wherein Xaa10 is Asp. 
     
     
         7 . The method of  claim 5 , wherein Xaa11 is Asp. 
     
     
         8 . The method of  claim 5 , wherein Xaa13 is Pro, Xaa15 is Arg, Xaal  6  is Ala, Xaa17 is Ala, Xaa18 is His and Xaa19 is Pro. 
     
     
         9 . The method of  claim 5 , wherein Xaa21 is Trp. 
     
     
         10 . The method of  claim 5 , wherein Xaa31 is Glu. 
     
     
         11 . The method of  claim 5 , wherein Xaa32 is Glu. 
     
     
         12 . The method of  claim 5 , wherein Xaa34 is Ile. 
     
     
         13 . The method of  claim 5 , wherein Xaa35 is Tyr. 
     
     
         14 . The method of  claim 5 , wherein Xaa39 is Glu. 
     
     
         15 . The method of  claim 5 , wherein the polypeptide comprises: 
       
         
           
                 
                 
               
                   (amino acid residues 3-60 of SEQ ID NO: 2) 
                     
                 
                 
                 
               
                   Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly 
                     
                 
                     
                 
                   Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn 
                 
                     
                 
                   Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly 
                 
                     
                 
                   Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 
                 
                     
                 
                   Glu Glu Cys Lys Lys Met Cys Thr Arg Asp. 
                 
             
                
               
            
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         16 . The method of  claim 15 , wherein the polypeptide further comprises a Glu-Ala sequence prior to the Met residue. 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 1 , wherein the anti-fibrinolytic agent is selected from the group consisting of: tranexamic acid (Cyklokapron™), epsilon amino caproic acid (Amicar™), aprotinin (Trasyol™), Desmopressin (DDAVP), pirfenidone, and combinations thereof. 
     
     
         19 . The method of  claim 1 , wherein the anti-fibrinolytic agent is epsilon amino caproic acid (Amicar™). 
     
     
         20 . The method of  claim 1 , wherein the anti-fibrinolytic agent is aprotinin (Trasyol™). 
     
     
         21 - 26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the cerebral ischemia is a stroke. 
     
     
         28 . The method of  claim 27 , wherein the stroke is selected from the group consisting of an embolism associated stroke, a thrombus-associated stroke, a thromboembolitic stroke and a hemorrhage-associated stroke. 
     
     
         29 . The method of  claim 5 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2. 
     
     
         30 . The method of  claim 5 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:2. 
     
     
         31 . The method of  claim 5 , wherein the polypeptide consists of residues 3-60 of SEQ ID NO:2.