PROTEIN-POLYMER CONJUGATES

• US 20090053177A1
• Filed: 08/15/2008
• Published: 02/26/2009
• Est. Priority Date: 08/16/2007
• Status: Active Grant

• Current Assignee: PharmaEssentia Corporation
• Original Assignee: PharmaEssentia Corporation
• Inventors: Lin, Ko-Chung
• Granted Patent: US 8,143,214 B2
• Time in Patent Office: Days
• US Class Current: 424/85.700
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: A61P 31/20 for DNA viruses

Abstract

This invention relates to protein-polymer conjugates described in the specification. Also disclosed are a method for preparing a protein-polymer conjugate and its use in treating hepatitis B virus or hepatitis C virus infection.

Claims

1. A substantially pure conjugate comprising at least one polymer moiety, a protein moiety, and a linker, wherein the at least one polymer moiety is attached to the linker;

the nitrogen atom of the N-terminus of the protein moiety is bonded to the linker;

the linker is a covalent bond, C_1-10 alkylene, C_2-10 alkenylene, or C_2-10 alkynylene; and

the protein moiety is an interferon-α

moiety, a human growth hormone moiety, or an erythropoietin moiety.

2. The conjugate of claim 1, wherein the protein moiety is a modified interferon-α

_2b moiety containing 1-4 additional amino acid residues at the N-terminus.

3. The conjugate of claim 2, wherein the purity of the conjugate is 90% or higher.

4. A protein-polymer conjugate of formula I:

wherein

each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C_1-5 alkyl, C_2-5 alkenyl, C_2-5 alkynyl, aryl, heteroaryl, C_3-8 cycloalkyl, or C_3-8 heterocycloalkyl;

each of A₁ and A₂, independently, is a polymer moiety;

each of G₁, G₂, and G₃, independently, is a bond or a linking functional group;

P is a protein moiety;

m is 0 or an integer of 1-10; and

n is an integer of 1-10.

5. The conjugate of claim 4, wherein G₃ is a bond and P is a protein moiety in which the amino group at the N-terminus is bonded to G₃.

6. The conjugate of claim 5, wherein each of A₁ and A₂ is an mPEG moiety having a molecular weight of 10-30 kD.

7. The conjugate of claim 6, wherein each of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atom as shown in formula I.

8. The conjugate of claim 7, wherein P is a modified interferon moiety containing 1-4 additional amino acid residues at the N-terminus.

9. The conjugate of claim 8, wherein n is 2.

10. The conjugate of claim 9, wherein the conjugate is

in which mPEG has a molecular weight of 20 kD and IFN is an interferon-α

_2b moiety.

11. A compound of formula II

wherein

X is a reactive group;

each of A₁ and A₂, independently, is a polymer moiety;

or one of A₁ and A₂ is a polymer moiety, and the other is a reactive group;

each of G₁, G₂, and G₃, independently, is a bond or a linking functional group;

each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C_1-5 alkyl, C_2-5 alkenyl, C_2-5 alkynyl, aryl, heteroaryl, C_3-8 cycloalkyl, C_3-8 heterocycloalkyl, or -G₄-X′

, G₄ being a bond or a linking functional group and X′

being a reactive group; and

each m and n, independently, is 0 or an integer of 1-10;

provided that if -G₃-X is —

CO(O)—

N-succinimidyl, then n is an integer of 1-10.

12. The compound of claim 11, wherein each of A₁ and A₂, independently, is a polymer moiety;

each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C_1-5 alkyl, C_2-5 alkenyl, C_2-5 alkynyl, aryl, heteroaryl, C_3-8 cycloalkyl, or C_3-8 heterocycloalkyl; and

X is CH(═

O) or a leaving group.

13. The compound of claim 12, wherein X is CH(═

O) and G₃ is a bond.

14. The compound of claim 13, wherein each of A₁ and A₂ is an mPEG moiety having a molecular weight of 12-30 kD.

15. The compound of claim 11, wherein each of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atom as shown in formula I; and n is an integer of 1-10.

16. A method of preparing a protein-polymer conjugate of formula I:

wherein each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C_1-5 alkyl, C_2-5 alkenyl, C_2-5 alkynyl, aryl, heteroaryl, C_3-8 cycloalkyl, or C_3-8 heterocycloalkyl;

each of A₁ and A₂, independently, is a polymer moiety;

each of G₁, G₂, and G₃, independently, is a bond or a linking functional group;

P is a protein moiety, in which the amino group at the N-terminus is attached to G₃;

m is 0 or an integer of 1-10; and

n is an integer of 1-10;

said method comprising;

coupling an N-terminus free protein H—

P, P being a protein moiety, with a di-polymer branched molecule of formula II;

wherein R₁, R₂, R₃, R₄, R₅, A₁, A₂, G₁, G₂, G₃, m and n are as defined above, and X is a leaving group;

or

coupling an N-terminus free protein H—

P, P being a protein moiety, with a di-polymer branched molecule of formula II;

wherein R₁, R₂, R₃, R₄, R₅, A₁, A₂, G₁, G₂, and m are as defined above, G₃ is a bond, n is 0 or an integer of 1-9, and X is CHO; and

then reducing the coupling product to form the protein-polymer conjugate.

17. The method of claim 16, wherein each of A₁ and A₂ is an mPEG moiety having a molecular weight of 20 kD.

18. The method of claim 17, wherein each of G₁ and G₂ is

wherein O is attached to A₁ or A₂, and NH is attached to a carbon atom as shown in formula I.

19. The method of claim 18, wherein P is a modified interferon moiety containing 1-4 additional amino acid residues at the N-terminus.

20. The method of claim 19, wherein P is

IFN being an interferon-α

_2b moiety, the N-terminus of which is bonded to the carbonyl group.

21. The method of claim 20, wherein G₃ is a bond and X is CH(═

O).

22. A method of treating hepatitis C virus infection or hepatitis B virus infection comprising administering to a subject in need thereof an effective amount of a protein-polymer conjugate of formula I:

wherein

each of R₁, R₂, R₃, R₄, and R₅, independently, is H, C_1-5 alkyl, C_2-5 alkenyl, C_2-5 alkynyl, aryl, heteroaryl, C_3-8 cycloalkyl, or C_3-8 heterocycloalkyl;

each of A₁ and A₂, independently, is a polymer moiety;

each of G₁, G₂, and G₃, independently, is a bond or a linking functional group;

P is an interferon moiety;

m is 0 or an integer of 1-10; and

n is an integer of 1-10.

23. A method of treating hepatitis C virus infection or hepatitis B virus infection comprising administering to a subject in need thereof an effective amount of a substantially pure conjugate comprising at least one polymer moiety, a protein moiety, and a linker, wherein the at least one polymer moiety is attached to the linker;

the nitrogen atom of the N-terminus of the protein moiety is bonded to the linker;

the linker is a covalent bond, alkylene, alkenyl, or alkynyl; and

the protein moiety is an interferon-α

_2b moiety.