Synergistic biomolecule-polymer conjugates

Abstract

The synergistic biomolecule-polymer conjugates are the long-acting, in vivo controlled continuous-release and hybrid synergy systems of biomolecules that provide increased biological activities and enhanced pharmacological properties for achieving greater therapeutic efficacies.

1 . A synergistic biomolecule-polymer conjugates comprising the formula:
   (P n -L n -R—C) x -M OR [(P-L) n R—C] x -M
   
       wherein M is a biologically active molecule; wherein x is the number of cleavable-linkages polymers coupled to a biomolecule and x≧1; wherein n is n≧2; wherein P is a polymer or polymer lipid; wherein Pn are multiple polymeric arms or pieces that their types and sizes may be the same or different; wherein L is a functional linkage moiety comprising at least one cleavable linkage and at least one permanent linkage; or Ln are all releasable linkages; wherein R is a non-cleavable spacer connected between polymer and biomolecule; C is a coupling group capable of attaching to a biomolecule. 
     
     
         2 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the conjugated polymer is a permanent-cleavable-linkages branched polymers (Type Ia). 
     
     
         3 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the conjugated polymer is a all-cleavable-linkages branched polymers (Type Ib). 
     
     
         4 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the conjugated polymer is a permanent-cleavable-linkages linear polymers (Type IIa). 
     
     
         5 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the conjugated polymer is a all-cleavable-linkages linear polymers (Type IIb). 
     
     
         6 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the permanent linkage is selected from the group consisting of amide, carbamate, carbamide, imide, amine, urea, ether, urethane, sulfide, thiourea, thiocarbamate, thiocarbamide, and dithiocarbamate.; and the releasable linkage is selected from the group consisting of carboxylic ester, carbonate, sulfonic ester, phosphoric ester, acylimidazo, carbamate-imidazo, and disulfide linkage. 
     
     
         7 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein said cleavable linkage is in vivo cleavable, blood plasma cleavable, enzymes degradable, pH-induced hydrolyzable, pH induced self-cleavable, physiologically cleavable, in vivo substance induced cleavable, biochemical reaction induced cleavable, or chemically cleavable, and wherein said enzymes selected from the group consisting of Esterases, phosphatases and sulfatases. 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein P is PEG polymer having molecular weight in the range from about 50 to about 40,000. 
     
     
         11 . (canceled) 
     
     
         12 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein C is a coupling moiety selected from the group consisting of active ester, mixed anhydride, alkyl aldehyde, aromatic aldehyde, maleimide, haloacetyl, carboxylic acid, hydroxyl, isocyanate, isothiocyanate, carbonyl, thiol, disulfide, amino, hydroxyl, hydrazide, and hydrazine, wherein said active ester is selected from the group consisting of N-hydroxysuccinimide ester, p-nitophenyl ester, N-succinimidyl carbonate, p-nitrophenyl carbonate, acylimidazo or trichlorophenylcarbonate. 
     
     
         13 . (canceled) 
     
     
         14 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein the R is amino acid selected from the group consisting of lysine, serine, threonine, cysteine, tyrosine, histidine, arginine, glutamic acid or aspartic acid; wherein the nonprotein amino acid is selected from the group consisting of homocysteine, homoserine or ornithine. 
     
     
         15 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein said n is two (n=2) and said the Type 1 synergistic cleavable-branched polymer conjugates comprising the formula: 
       
         
           
           
               
               
           
         
       
       wherein L 1  is a permanent linkage and wherein L 2  is a cleavable linkage for the permanent-cleavable-linkages two-arm branched polymer (Type Ia); wherein L 1  and L 2  both are cleavable linkages for the all-cleavable-linkages two-arm branched polymer (Type Ib). 
     
     
         16 . (canceled) 
     
     
         17 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein P 1  and P 2  are methoxy poly(ethylene glycol); wherein R is lysine connected with methoxy poly(ethylene glycol) having structures represented by: 
       
         
           
           
               
               
           
         
       
       wherein  a  bond is a permanent linkage selected from the group consisting of amide, carbamate, carbamide, imide, amine, thiocarbamate, thiocarbamide, urethane and dithiocarbamate; wherein  a  bond is connected to either α or ε-amino group; wherein  b  bond is a cleavable linkage selected from the group consisting of carboxylic ester, carbonate, and carboxyl-imidazo, wherein the free amino further connects spacers comprising activated moieties selected from the group consisting of N-hydroxysuccinimide ester, p-nitophenyl ester, N-succinimidyl carbonate, p-nitrophenyl carbonate, acylimidazo, aldehyde, maleimide, haloacetyl, carboxylic acid, hydroxyl, isocyanate, isothiocyanate, carbonyl, thiol, disulfide, amino, hydroxyl, hydrazide, and hydrazine. 
     
     
         18 . (canceled) 
     
     
         19 . The synthetic methods used for preparing the permanent-cleavable-linkages polymers, all-cleavable-linkages polymers and asymmetric branched arms polymers comprising: the cleavable linkages formed on carboxyl group via activation, coupling, and acylation procedures using mPEG-OH or mPEG derivative, the permanent linkages formed on amino groups via acylation, coupling, and condensation with mPEG electrophilic derivatives, the insertion and activation of a spacer moiety and the procedures of protection, de-protection used for the synthesis of the cleavable-permanent-mixed linkages. 
     
     
         20 . The synergistic biomolecule-polymer conjugates of  claim 17 , wherein the permanent-cleavable linkages branched PEG derivative includes Lys(α-10K mPEG, carbamate; 20K mPEG Ester) ε-Succinimidyl Suberate, Compound 5. 
     
     
         21 . The synergistic biomolecule-polymer conjugates of  claim 17 , wherein the permanent-cleavable linkages branched PEG derivative includes Lys(α-10K mPEG, Amide; 20K mPEG, Ester) ε-Succinimidyl Suberate, Compound 6. 
     
     
         22 . The synergistic biomolecule-polymer conjugates of  claim 17 , wherein the permanent-cleavable linkages branched PEG derivative includes Lys(ε-10K mPEG, carbamate; 20K mPEG, Ester) α-Succinimidyl Suberate, Compound 7. 
     
     
         23 . The synergistic biomolecule-polymer conjugates of  claim 17 , wherein the permanent-cleavable linkages branched PEG derivative is Lys(ε-10K mPEG, amide; 20K mPEG, Ester) α-Succinimidyl Suberate, Compound 8. 
     
     
         24 . The synergistic biomolecule-polymer conjugates of  claim 17 , wherein the permanent-cleavable linkages branched PEG derivative is 3-Maleimidopropionyl Lys(α-10K mPEG, carbamate)-20K mPEG Ester, Compound 9. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . A synergistic biomolecule-polymer conjugate of  claim 1 , wherein said M is interferons including interferon-α, interferon-β, interferon-γ, interferon gamma-1b and interferon-λ (interferon lambda). 
     
     
         28 . A synergistic biomolecule-polymer conjugate of  claim 1 , wherein said M is Interferon-α including Interferon-α-2a, Interferon-α-2b, Interferon-α-1, interferon alfacon-1 and consensus interferon. 
     
     
         29 . A synergistic biomolecule-polymer conjugate of  claim 1 , wherein said M is interferon-α-2b; wherein R is lysine, aspartic acid or glutamic acid as structures described in  claim 17 ,  41 , or  42 , respectively; wherein P1 and P2 are mPEG and each one has a molecular weight ranging from about 50 to 40,000; wherein L1 bond is amide, carbamate or amine linkage and L2 bond is carboxylic ester or carbonate linkage. 
     
     
         30 . A synergistic biomolecule-polymer conjugate of  claim 1 , wherein said M is interleukins or cytokines; wherein R is lysine, aspartic acid or glutamic acid as structures described in  claims 17 ,  41 , and  42 , respectively; wherein P1 and P2 are mPEG and each one has a molecular weight ranging from about 50 to 40,000; wherein L1 bond is amide, carbamate or amine linkage and L2 bond is carboxylic ester or carbonate linkage. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The synergistic interferon α-30kPEG (10k mPEG carbamate, 20k mPEG ester) ε-Suberate conjugate comprising one single component, x=1. 
     
     
         34 . The synergistic interferon α-30kPEG (10k mPEG carbamate, 20k mPEG ester) ε-Suberate conjugate of  claim 33 , wherein the conjugate comprising a mixture having composition x=1 to 2 or x=1 to 3. 
     
     
         35 . The synergistic interferon α-30kPEG (10k mPEG carbamate, 20k mPEG ester) conjugate in any one of  claim 33  or  34 , wherein the interferon α is interferon α-2b. 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . A synergistic biomolecule-polymer conjugate of  claim 1 , wherein the synergistic biomolecule-polymer conjugate is for prophylactic treatment; wherein said M is blood clotting factors VII, VIIa, VIII or IX. 
     
     
         40 . A method of treatment comprising administering to a subject in need thereof an effective amount of a synergistic biomolecule-polymer conjugate of  claim 1 . 
     
     
         41 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein P 1  and P 2  are methoxy poly(ethylene glycol); wherein R is aspartic acid connected with methoxy poly(ethylene glycol) having structures represented by: 
       
         
           
           
               
               
           
         
       
       wherein the permanent linkage selected from the group consisting of amide, carbamate, carbamide, imide, amine, thiocarbamate, thiocarbamide, urethane and dithiocarbamate; wherein the cleavable linkage selected from the group consisting of carboxylic ester, carbonate, and carboxyl-imidazo; wherein x is an activated ester for coupling protein or further connects spacers for protein conjugation that comprise activated moieties selected from the group consisting of N-hydroxysuccinimide ester, p-nitophenyl ester, N-succinimidyl carbonate, p-nitrophenyl carbonate, acylimidazo, aldehyde, maleimide, haloacetyl, carboxylic acid, hydroxyl, isocyanate, isothiocyanate, carbonyl, thiol, disulfide, amino, hydroxyl, hydrazide, and hydrazine. 
     
     
         42 . The synergistic biomolecule-polymer conjugates of  claim 1 , wherein P 1  and P 2  are methoxy poly(ethylene glycol); wherein R is glutamic acid connected with methoxy poly(ethylene glycol) having structures represented by: 
       
         
           
           
               
               
           
         
       
       wherein the permanent linkage selected from the group consisting of amide, carbamate, carbamide, imide, amine, thiocarbamate, thiocarbamide, urethane and dithiocarbamate; wherein the cleavable linkage selected from the group consisting of carboxylic ester, carbonate, and carboxyl-imidazo; wherein x is an activated ester for coupling protein or further connects spacers for protein conjugation that comprise activated moieties selected from the group consisting of N-hydroxysuccinimide ester, p-nitophenyl ester, N-succinimidyl carbonate, p-nitrophenyl carbonate, acylimidazo, aldehyde, maleimide, haloacetyl, carboxylic acid, hydroxyl, isocyanate, isothiocyanate, carbonyl, thiol, disulfide, amino, hydroxyl, hydrazide, and hydrazine.