Method for increasing the serum half-life of a biologically active molecule

Abstract

A method is provided for preparing a biologically active molecule having an increased serum half-life. The method involves conjugating a polymer such as polyethylene glycol to the biologically active molecule. Also provided are polypeptide drugs having an increased serum half-life, e.g., human urokinase plasminogen activator (human “uPA” or “hUPA”) or a fragment or derivative thereof. Pharmaceutical compositions containing such molecules and methods of using them to treat uPA-mediated and uPA receptor-mediated disorders are also provided.

1 . A method for preparing a biologically active molecule having an increased serum half-life, comprising coupling the molecule to a polymer by reacting an aldehyde functionality on the molecule with a hydrazide or semicarbazide group on the polymer under reaction conditions effective to promote formation of a conjugated molecule, wherein the molecule is bound to the polymer through a hydrazone or semicarbazone linkage.  
     
     
         2 . The method of  claim 1 , wherein the biologically active molecule is a polypeptide.  
     
     
         3 . The method of  claim 2 , wherein the polypeptide has a molecular weight of less than about 50,000.  
     
     
         4 . The method of  claim 3 , wherein the polypeptide has a molecular weight of less than about 30,000.  
     
     
         5 . The method of  claim 4 , wherein the polypeptide has a molecular weight of less than about 10,000.  
     
     
         6 . The method of  claim 5 , wherein the polypeptide is uPA 1-48  or an active portion thereof.  
     
     
         7 . A method for conjugating uPA 1-48  to polyethylene glycol, comprising: (a) providing activated polyethylene glycol in the form of polyethylene glycol hydrazide or semicarbazide; (b) providing uPA 1-48  having adjacent amino and alcohol groups at the N-terminus thereof; (c) oxidatively cleaving between the adjacent amino and alcohol groups to yield an aldehyde functionality in place thereof; and (d) reacting the aldehyde-containing uPA 1-48  provided in step (c) with the polyethylene glycol hydrazide or semicarbazide under reaction conditions effective to promote formation of PEGylated polypeptide, wherein the polypeptide is bound to polyethylene glycol through a hydrazone or semicarbazone linkage.  
     
     
         8 . A modified polypeptide comprising a biologically active polypeptide conjugated to a pharmaceutically acceptable polymer, wherein said modified polypeptide exhibits a proportional increase in serum half-life in an individual which is greater than any proportional decrease in its biological activity.  
     
     
         9 . The modified polypeptide of  claim 8 , wherein the increase in half-life is at least about three fold.  
     
     
         10 . The modified polypeptide of  claim 8 , wherein the biologically active polypeptide is a urokinase or derivative thereof.  
     
     
         11 . The modified polypeptide of  claim 10 , wherein the urokinase is uPA 1-48  or an active portion thereof.  
     
     
         12 . The modified polypeptide of  claim 11 , wherein uPA 1-48  is linked to the polymer through a hydrazone or semicarbazone linkage.  
     
     
         13 . The modified polypeptide of  claim 12 , wherein the polymer is linked at the N-terminus of uPA 1-48 .  
     
     
         14 . The modified polypeptide of  claim 11 , wherein the polymer is polyethylene glycol.  
     
     
         15 . The modified polypeptide of  claim 14 , wherein the polyethylene glycol has an average molecular weight of about 5,000 to about 50,000 daltons.  
     
     
         16 . The modified polypeptide of  claim 15 , wherein the polyethylene glycol has an average molecular weight of about 10,000 to about 40,000 daltons.  
     
     
         17 . The modified polypeptide of  claim 16 , wherein the polyethylene glycol has an average molecular weight of about 15,000 to about 30,000 daltons.  
     
     
         18 . A pharmaceutical composition comprising the modified polypeptide of  claim 11 , in combination with a pharmaceutically acceptable carrier or excipient.  
     
     
         19 . A pharmaceutical composition comprising the modified polypeptide of  claim 14 , in combination with a pharmaceutically acceptable carrier or excipient.  
     
     
         20 . A method of treating a uPA-mediated disorder or a uPA receptor-mediated disorder in a patient in need thereof, comprising administering to said patient a pharmaceutically acceptable composition comprising a therapeutically effective amount of a conjugate of uPA 1-48  and a polymer.  
     
     
         21 . The method of  claim 20 , wherein the molar amount of the conjugate which is administered is less than the molar amount of the nonconjugated uPA 1-48  necessary to provide a therapeutic effect.