Conjugates of an EPO Moiety and a Polymer

Abstract

Conjugates of an EPO moiety and one or more non-peptidic water-soluble polymers are provided. Typically, the non-peptidic water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising such conjugates, methods of making conjugates, and methods of administering compositions comprising such conjugates to a patient.

1 . A method of preparing a conjugate of erythropoietin (EPO), said method comprising:
 reacting a polymer comprising the structure:   
       
         
           
           
               
               
           
         
         
           wherein: 
           POLY is a polyalkylene oxide, 
           Q is an optional linking group having a length of from one to 10 atoms, 
           m is an integer ranging from 0 to 20, 
           Z is selected from the group consisting of alkyl, substituted alkyl, aryl and substituted aryl, and 
           X is a leaving group, 
         
         with erythropoietin (EPO), under conditions effective to promote reaction of one or more amino sites on EPO with said polymer to thereby form a biologically active conjugate comprising the structure: 
       
       
         
           
           
               
               
           
         
       
       where “—NH-EPO” represents an amino group of EPO. 
     
     
         2 . The method of  claim 1 , wherein said reacting comprises combining an aqueous solution of said polymer with an aqueous solution of said EPO to form a polymer-EPO reaction mixture. 
     
     
         3 . The method of  claim 2 , wherein the pH of said polymer-EPO reaction mixture is in a range from about 5 to about 8. 
     
     
         4 . The method of  claim 2 , wherein said polymer is combined at a 10-fold or greater molar excess relative to said EPO. 
     
     
         5 . The method of  claim 4 , wherein said polymer is combined at a 20-fold or greater molar excess relative to said EPO. 
     
     
         6 . The method of  claim 2 , wherein said reacting further comprises, after said combining, stirring said reaction mixture for 1-24 hours at a temperature ranging from about −10° C. to about 40° C. 
     
     
         7 . The method of  claim 6 , wherein said temperature is ambient. 
     
     
         8 . The method of  claim 1 , wherein said method comprises, prior to said reacting, protecting amino groups of said EPO with a cyclic dicarboxylic acid anhydride protecting agent to form an amino-protected EPO. 
     
     
         9 . The method of  claim 8 , wherein said protecting agent is a maleic or citraconylic anhydride. 
     
     
         10 . The method of  claim 9 , wherein said protecting agent is dimethylmaleic anhydride. 
     
     
         11 . The method of  claim 8 , further comprising, after said reacting, deprotecting said amino groups of said amino-protected EPO. 
     
     
         12 . The method of  claim 11 , effective to form a conjugate of EPO wherein a minority of said conjugate species have a polyalkylene oxide covalently attached at lysine 52. 
     
     
         13 . The method of  claim 1 , further comprising, after said reacting, purifying said conjugate. 
     
     
         14 . The method of  claim 1 , wherein X is selected from the group consisting of chlorine, bromine, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, hydroxyl, 1-imidazolyl, and p-nitrophenyloxy. 
     
     
         15 . The method of  claim 1 , wherein said polymer comprises the structure: 
       
         
           
           
               
               
           
         
         where n ranges from about 200 to about 1400, and 
       
       said conjugate comprises the structure: