High molecular weight derivatives of vitamin k-dependent polypeptides

Abstract

Modifications of vitamin K-dependent polypeptides that lead to enhanced protein function on a weight or molar basis and/or increase of protein lifetime in the circulation are described. Both objectives are important for using vitamin K-dependent polypeptides for pro- and anti-coagulation therapies, as well as for other uses in the circulation.

What is claimed is:  
     
         1 . An isolated vitamin K-dependent polypeptide linked to a polyethylene glycol (PEG) polymer.  
     
     
         2 . The polypeptide of  claim 1 , wherein said polypeptide is selected from the group consisting of factor VII, factor II, protein C, protein S, gas6, and bone matrix Gla protein.  
     
     
         3 . The polypeptide of  claim 1 , wherein said polypeptide and PEG polymer are directly linked.  
     
     
         4 . The polypeptide of  claim 1 , wherein said polypeptide and PEG polymer are indirectly linked.  
     
     
         5 . The polypeptide of  claim 1 , wherein said polypeptide is a protease selected from the group consisting of factor VIIa, factor IIa, and activated protein C.  
     
     
         6 . The polypeptide of  claim 5 , wherein said protease is further linked to an active-site inhibition reagent.  
     
     
         7 . The polypeptide of  claim 6 , wherein said active-site inhibition reagent is a chloromethylketone derivatized amino acid or peptide.  
     
     
         8 . The polypeptide of  claim 6 , wherein said PEG polymer is linked to said protease via said active-site inhibition reagent.  
     
     
         9 . The polypeptide of  claim 5 , wherein said protease is factor VIIa.  
     
     
         10 . An active-site inhibition reagent linked to a PEG polymer.  
     
     
         11 . The active-site inhibition reagent of  claim 10 , wherein said reagent is a chloromethylketone derivatized amino acid or peptide.  
     
     
         12 . The active-site inhibition reagent of  claim 10 , wherein said reagent is a phosphohalide derivative.  
     
     
         13 . An anticoagulant agent comprising two polypeptide monomers, wherein at least one of said polypeptide monomers is a vitamin K-dependent polypeptide, and wherein said polypeptide monomers are covalently linked.  
     
     
         14 . The anticoagulant agent of  claim 13 , wherein said polypeptide monomers are covalently linked via a bi-functional active-site inhibition reagent.  
     
     
         15 . The anticoagulant agent of  claim 13 , wherein said two polypeptide monomers are the same polypeptide.  
     
     
         16 . The anticoagulant agent of  claim 13 , wherein said two polypeptide monomers are different polypeptides.  
     
     
         17 . The anticoagulant agent of  claim 13 , wherein each of said two polypeptide monomers is a vitamin K-dependent polypeptide.  
     
     
         18 . The anticoagulant agent of  claim 17 , wherein each of said vitamin K-dependent polypeptides is a factor VIIa polypeptide.  
     
     
         19 . The anticoagulant agent of  claim 14 , wherein said bi-functional active-site inhibition reagent is linked to a PEG polymer.  
     
     
         20 . The anticoagulant agent of  claim 13 , wherein at least one of said polypeptide monomers is directly linked to a PEG polymer.  
     
     
         21 . A bifunctional active-site inhibition reagent comprising two covalently linked active-site inhibitors.  
     
     
         22 . The bifunctional active-site inhibition reagent of  claim 21 , wherein at least one of said active-site inhibitors is linked to a PEG polymer.  
     
     
         23 . A method of directly linking a vitamin K-dependent polypeptide to a PEG polymer, said method comprising incubating said PEG polymer with said vitamin K-dependent polypeptide for a time sufficient to link said PEG polymer to said vitamin K-dependent polypeptide, wherein said PEG polymer is reactive with amino groups or carbohydrate groups on said vitamin K-dependent polypeptide.  
     
     
         24 . A method of indirectly linking, a vitamin K-dependent polypeptide to a PEG polymer, said method comprising providing a PEG-modified, active-site inhibition reagent, wherein said PEG polymer is reactive with amino groups on said active-site inhibition reagent; and incubating said PEG-modified, active-site inhibition reagent with said vitamin K-dependent polypeptide for a time sufficient to link said PEG modified, active-site inhibition reagent to said vitamin K-dependent polypeptide.  
     
     
         25 . A method of making an anticoagulant agent comprising incubating a bifunctional active-site inhibition reagent and at least one vitamin K-dependent polypeptide in the presence of phospholipid for a time sufficient to link said bi-functional active site inhibition reagent and said vitamin K-dependent polypeptide.  
     
     
         26 . A pharmaceutical composition comprising an isolated vitamin K-dependent polypeptide linked to a PEG polymer and a pharmaceutically acceptable carrier.  
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein said polypeptide and PEG polymer are indirectly linked.  
     
     
         28 . The pharmaceutical composition of  claim 26 , wherein said polypeptide is a protease selected from the group consisting of factor VIIa, factor IIa, and activated protein C.  
     
     
         29 . The pharmaceutical composition of  claim 28 , wherein said protease is further linked to an active-site inhibition reagent.  
     
     
         30 . The pharmaceutical composition of  claim 29 , wherein said active-site inhibition reagent is a chloromethylketone derivatized amino acid or peptide.  
     
     
         31 . The pharmaceutical composition of  claim 29 , wherein said PEG polymer is linked to said protease via said active-site inhibition reagent.  
     
     
         32 . The pharmaceutical composition of  claim 28 , wherein said protease is factor VIIa.  
     
     
         33 . A pharmaceutical composition comprising an anticoagulant agent and a pharmaceutically acceptable carrier, said anticoagulant agent comprising two polypeptide monomers, wherein at least one of said polypeptide monomers is a vitamin K-dependent polypeptide, and wherein said polypeptide monomers are covalently linked.  
     
     
         34 . The pharmaceutical composition of  claim 33 , wherein said polypeptide monomers are covalently linked via a bi-functional active-site inhibition reagent.