Site-specific insulin conjugate

Patent metadata

Field	Value
Publication number	US-2016000931-A1
Application number	US-201414770214-A
Country	US
Kind code	A1
Filing date	Feb 26, 2014
Priority date	Feb 26, 2013
Publication date	Jan 7, 2016
Grant date	—

Abstract

Provided are an insulin conjugate having improved insulin receptor binding affinity and increased activity, in which a non-peptidyl polymer and an immunoglobulin Fc region are site-specifically linked to an amino acid residue of the insulin beta chain excluding the N-terminus thereof via a covalent bond, a long-acting formulation including the same, and a preparation method thereof. The insulin conjugate of the present invention is used to provide an insulin formulation which exhibits a remarkably increased in vivo activity of the peptide.

First claim

1 . An insulin conjugate, wherein insulin and an immunoglobulin Fc region are linked to each other via a non-peptidyl polymer linker selected from the group consisting of polyethylene glycol, polypropylene glycol, an ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof, and one end of the non-peptidyl polymer is linked to an amino acid residue of an insulin beta chain excluding the N-terminus thereof and the other end thereof is linked to the immunoglobulin Fc region.

2 . The insulin conjugate of claim 1 , wherein the non-peptidyl polymer is linked to any one of the amino acid residues at positions 20 to 29 of the insulin beta chain.

3 . The insulin conjugate of claim 1 , wherein the non-peptidyl polymer is linked to any one of the amino acid residues at positions 25 to 29 of the insulin beta chain.

4 . The insulin conjugate of claim 1 , wherein the non-peptidyl polymer is linked to the lysine residue at position 29 of the insulin beta chain.

5 . The insulin conjugate of claim 1 , wherein the amino acid residue of the insulin beta chain, to which the non-peptidyl polymer is linked, has an amine group or a thiol group.

6 . The insulin conjugate of claim 1 , wherein the insulin is a native insulin, or a variant which is prepared by any one method of substitution, addition, deletion, and modification of some amino acids of native insulin or by a combination thereof, an insulin derivative, an insulin agonist, or a fragment thereof.

7 . The insulin conjugate of claim 1 , wherein both ends of the non-peptidyl polymer are linked to an amine group or a thiol group of the side chain of the amino acid residue of an immunoglobulin Fc region and the insulin beta chain, respectively.

8 . The insulin conjugate of claim 7 , wherein the amino acid is a naturally occurring or non-naturally occurring amino acid.

9 . The insulin conjugate of claim 1 , wherein the immunoglobulin Fc region is aglycosylated.

10 . The insulin conjugate of claim 1 , wherein the immunoglobulin Fc region is composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 domains.

11 . The insulin conjugate of claim 10 , wherein the immunoglobulin Fc region further includes a hinge region.

12 . The insulin conjugate of claim 1 , wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.

13 . The insulin conjugate of claim 12 , wherein each domain of the immunoglobulin Fc region is a hybrid of domains of a different origin derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

14 . The insulin conjugate of claim 12 , wherein the immunoglobulin Fc region is a dimer or a multimer composed of single-chain immunoglobulins composed of domains of the same origin.

15 . The insulin conjugate of claim 12 , wherein the immunoglobulin Fc region is an IgG4 Fc region.

16 . The insulin conjugate of claim 12 , wherein the immunoglobulin Fc region is a human aglycosylated IgG4 Fc region.

17 . The insulin conjugate of claim 1 , wherein the non-peptidyl polymer binds to the amine group or thiol group of the side chain of the amino acid residue of the insulin beta chain to form a peptide, hemithioacetal, imine or thiodioxopyrrolidinyl bond.

18 . The insulin conjugate of claim 1 , wherein both ends of the non-peptidyl polymer each independently have a reactive group selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group, and a succinimide derivative.

19 . The insulin conjugate of claim 18 , wherein the succinimide derivative is succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, or succinimidyl carbonate.

20 . The insulin conjugate of claim 18 , wherein both ends of the non-peptidyl polymer have a butyraldehyde reactive group or a succinimidyl valerate reactive group, respectively.

21 . A long-acting insulin formulation having improved in vivo duration and stability, comprising the insulin conjugate of claim 1 .

22 . The long-acting insulin formulation of claim 21 , wherein the formulation is used for the treatment of diabetes.

23 . A preparation method of the insulin conjugate of claim 1 , comprising the steps of:
(1) covalently linking a non-peptidyl polymer to an amino acid residue of the insulin beta chain excluding the N-terminus thereof; (2) isolating an insulin complex, in which the non-peptidyl polymer is covalently linked to the amino acid residue of the insulin beta chain excluding the N-terminus thereof, from the reaction mixture of (1); and (3) covalently linking an immunoglobulin Fc region to the other end of the non-peptidyl polymer of the isolated complex so as to produce an insulin conjugate, in which the immunoglobulin Fc region and insulin are linked to each end of the non-peptidyl polymer.

24 . The method of claim 23 , wherein the non-peptidyl polymer binds to the amine group or thiol group of the side chain of the amino acid residue of the insulin beta chain to form a peptide, hemithioacetal, imine or thiodioxopyrrolidinyl bond.

25 . The method of claim 23 , wherein both ends of the non-peptidyl polymer each independently have an aldehyde derivative, a maleimide derivative, or a succinimide derivative as a reactive group.

26 . The method of claim 23 , wherein both ends of the non-peptidyl polymer are linked to an amine group or a thiol group of the side chain of the amino acid residue of the insulin beta chain excluding the N-terminus thereof and an immunoglobulin Fc region, respectively.

27 . The method of claim 23 , wherein both ends of the non-peptidyl polymer each independently have a butyraldehyde group or a succinimidyl valerate reactive group.

28 . The method of claim 23 , wherein Step (1) is performed under an alkaline environment of pH 9.0±2.

29 . The method of claim 23 , wherein a molar ratio of the insulin and the non-peptidyl polymer in Step (1) is 1:1.5 to 1:10.

30 . The method of claim 23 , wherein a molar ratio of the insulin complex and the immunoglobulin Fc region in Step (3) is 1:1 to 1:10.

Assignees

• Hanmi Pharm Ind Co Ltd

Inventors

• Jang Myung Hyun
• Kim Dae Jin
• Hwang Sang Youn
• Kim Hyun Uk
• Jung Sung Youb
• Kwon Se Chang

Classifications

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61K47/6811

  the drug being a protein or peptide, e.g. transferrin or bleomycin · CPC title

• A61K47/6849 Primary

  the antibody targeting a receptor, a cell surface antigen or a cell surface determinant · CPC title

• C07K14/62 Primary

  Insulins · CPC title

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title