Amphiphilic Polymer-Protein Conjugates and Methods of Use Thereof

Abstract

Compositions and methods for transporting biologically active proteins and polypeptides, particularly across the blood-brain barrier, are provided.

1 . A method for delivering a therapeutic agent across the blood-brain barrier into the central nervous system of an animal comprising administering to said animal a conjugate comprising said therapeutic agent conjugated to an amphiphilic block copolymer by a cleavable linker moiety. 
     
     
         2 . The method of  claim 1 , wherein said therapeutic agent is a protein. 
     
     
         3 . The method of  claim 2 , wherein said amphiphilic block copolymer is a copolymer comprising at least one poly(oxyethylene) segment and at least one poly(oxypropylene) segment. 
     
     
         4 . The method of  claim 3 , wherein said amphiphilic block copolymer has a formula selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein x, y, z, i, and j have values from about 2 to about 800; wherein in formulas (IV) and (V), for each R 1  and R 2  pair one is hydrogen and the other is a methyl group; wherein in formula (VI), R* is an alkylene of about 2 to about 6 carbons, a cycloalkylene of about 5 to about 8 carbons, or phenylene; R 1  and R 2  are either both represent hydrogen or one represents hydrogen and the other represents methyl; R 3  and R 4  are either both are hydrogen or one is hydrogen and the other is methyl; if both of R 3  and R 4  represent hydrogen, then one of R 5  and R 6  represents hydrogen and the other is methyl; if one of R 3  and R 4  represents methyl, then both R 5  and R 6  represent hydrogen; and A represents 
       
         
           
           
               
               
           
         
       
     
     
         5 . The method of  claim 4 , wherein said amphiphilic block copolymer is of the formula: 
       
         
           
           
               
               
           
         
       
       wherein x, y, and z have values from about 2 to about 800. 
     
     
         6 . The method of  claim 5 , wherein x, y, and z have values from about 5 to about 80. 
     
     
         7 . The method of  claim 6 , wherein x+z=10 and y=68. 
     
     
         8 . The method of  claim 3 , wherein said amphiphilic block copolymer has a hydrophilic-lipophilic balance (HLB) of less than or equal to 20. 
     
     
         9 . The method of  claim 8 , wherein said amphiphilic block copolymer has an HLB of less than or equal to 16. 
     
     
         10 . The method of  claim 9 , wherein said amphiphilic block copolymer has an HLB of less than or equal to 12. 
     
     
         11 . The method of  claim 10 , wherein said amphiphilic block copolymer has an HLB of less than or equal to 8. 
     
     
         12 . The method of  claim 3 , wherein the molecular weight of said poly(oxypropylene) segments of said amphiphilic block copolymer is greater than or equal to 1500 Da. 
     
     
         13 . The method of  claim 12 , wherein said molecular weight is greater than or equal to 2000 Da. 
     
     
         14 . The method of  claim 13 , wherein said molecular weight is greater than or equal to 2500 Da. 
     
     
         15 . The method of  claim 14 , wherein said molecular weight is greater than or equal to 3000 Da. 
     
     
         16 . The method of  claim 1 , wherein said cleavable linker moiety linker comprises a disulfide bond. 
     
     
         17 . The method of  claim 16 , wherein said cleavable linker moiety linker is selected from the group consisting of:
 —OC(O)NH(CH 2 ) 2 NHC(O)(CH 2 ) 2 SS(CH 2 ) 2 C(O)NH—;   —OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 N═CH—; and   —OC(O)NH(CH 2 ) 2 SS(CH 2 ) 2 NH—.   
     
     
         18 . The method of  claim 1 , wherein said cleavable linker moiety linker comprises a recognition site for a protease. 
     
     
         19 . The method of  claim 18 , wherein said protease is selected from the group consisting of endosomal cathepsins, cathepsin B, lysosomal proteases, and colagenase. 
     
     
         20 . The method of  claim 18 , wherein said recognition site for a protease is a sequence selected from the group consisting of GFLG; GLG; GGGLGPAGGK; and KALGQPQ. 
     
     
         21 . The method of  claim 2 , wherein said protein is selected from the group consisting of cytokines; growth factors such as, without limitation, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF); amyloid beta binders; enkephalin; modulators of α-, β-, and/or γ-secretases; Glial-derived neutrotrophic factor (GDNF); vasoactive intestinal peptide; acid alpha-glucosidase (GAA); acid sphingomyelinase; iduronate-2-sultatase (I2S); α-L-iduronidase (IDU); β-Hexosaminidase A (HexA); Acid β-glucocerebrosidase; N-acetylgalactosamine-4-sulfatase; and α-galactosidase A. 
     
     
         22 . A method for delivering a protein across the blood-brain barrier into the central nervous system of an animal comprising administering to said animal a conjugate comprising said protein conjugated to a fatty acid by a cleavable linker moiety. 
     
     
         23 . The method of  claim 22 , wherein said fatty acid is selected from the group consisting of linoleic acid and alpha-linoleic acid.