Polyphenolic insulin
Insulin complexes are stabilized using a ligand that increases binding interactions between the insulin monomers, and particularly between the B-chains of two adjacent insulin monomers. Particularly preferred ligands are polyphenols, and insulin complexes are typically in the R-state and may therefore also include metal cations and a small phenolic compound in a binding site that is distinct from the polyphenol binding site.
1 . A pharmaceutical composition, comprising:
an insulin complex comprising a plurality of insulin monomers, each insulin monomer comprising an A-chain and a B-chain coupled together via disulfide bonds;
a polyphenol non-covalently bound to an apical area of an interface formed by respective B-chains of a first and a second insulin monomer in the complex; and
wherein at least part of the apical area is solvent exposed.
2 . The composition of claim 1 , wherein the insulin complex comprises six or twelve insulin monomers, and/or wherein the insulin complex has a T 3 R 3 or R 6 conformation.
3 . The composition of claim 1 , wherein the insulin complex further comprises a divalent metal cation, and/or wherein the insulin complex further comprises a phenolic ligand in which two hydrogen bonds engage the phenolic hydroxyl group from the A 6 carbonyl oxygen and A 11 amide hydrogen.
4 . The composition of claim 1 , wherein at least one of the insulin monomers has one or more amino acid substitutions relative to a corresponding wildtype that increases serum half-life of the insulin complex, or wherein at least one of the insulin monomers has a chemical modification that increases serum half-life of the insulin complex.
5 . The composition of claim 1 , wherein the polyphenol binds to at least one amino acid side chain of an amino acid in a B-chain of the first insulin monomer and to at least one amino acid side chain of another amino acid in a B-chain of the second insulin monomer to thereby increase a binding interaction between the first and second insulin monomers.
6 . The composition of claim 1 , wherein the polyphenol binds to at least two distinct amino acid side chains of respective amino acids in a B-chain of the first insulin monomer and to at least one amino acid side chain in a B-chain of another amino acid in the second insulin monomer to thereby increase a binding interaction between the first and second insulin monomers.
7 . The composition of claim 1 , wherein the polyphenol binds to B 5 His and B 26 Tyr of the first insulin monomer and B 16 Tyr of the second insulin monomer.
8 . The composition of claim 1 , wherein the polyphenol is a flavonoid, a flavonol, a (flavan-3-ol), a flavan-4-ol, a flavanone, a flavone, a flavanol, a stilbenoid polyphenol, or a curcuminoid polyphenol.
9 . A method of producing a pharmaceutical composition comprising insulin, comprising:
providing an insulin complex comprising a plurality of insulin monomers, each insulin monomer comprising an A-chain and a B-chain coupled together via disulfide bonds;
combining the insulin complex with a polyphenol to produce a stabilized insulin complex, wherein the polyphenol in the stabilized insulin complex is non-covalently bound to an apical area of an interface formed by respective B-chains of a first and a second insulin monomer in the insulin complex; and
wherein at least part of the apical area is solvent exposed.
10 . The method of claim 9 , wherein the insulin complex comprises six or twelve insulin monomers, and/or wherein the insulin complex has a T 3 R 3 or R 6 conformation.
11 . The method of claim 9 , wherein at least one of the insulin monomers has one or more amino acid substitutions relative to a corresponding wildtype that increases serum half-life of the insulin complex, or wherein at least one of the insulin monomers has a chemical modification that increases serum half-life of the insulin complex.
12 . The method of claim 9 , wherein the polyphenol binds to at least one amino acid side chain of an amino acid in a B-chain of the first insulin monomer and to at least one amino acid side chain of another amino acid in a B-chain of the second insulin monomer to thereby increase a binding interaction between the first and second insulin monomers.
13 . The method of claim 9 , wherein the polyphenol binds to at least two distinct amino acid side chains of respective amino acids in a B-chain of the first insulin monomer and to at least one amino acid side chain of another amino acid in a B-chain of the second insulin monomer to thereby increase a binding interaction between the first and second insulin monomers.
14 . The method of claim 9 , wherein the polyphenol binds to B 5 His and B 26 Tyr of the first insulin monomer and B 16 Tyr of the second insulin monomer.
15 . The method of claim 9 , wherein the polyphenol a flavonoid, a flavonol, a (flavan-3-ol), a flavan-4-ol, a flavanone, a flavone, a flavanol, a stilbenoid polyphenol, or a curcuminoid polyphenol.
16 . A method of increasing storage stability of an insulin complex, comprising:
providing an insulin complex comprising a plurality of insulin monomers, each insulin monomer comprising an A-chain and a B-chain coupled together via disulfide bonds;
combining the insulin complex with a polyphenol to produce a stabilized insulin complex, wherein the polyphenol in the stabilized insulin complex is non-covalently bound to an apical area of an interface formed by respective B-chains of a first and a second insulin monomer in the insulin complex, and wherein the stabilized insulin complex has a storage stability that is greater than that of the insulin complex without the polyphenol; and
wherein at least part of the apical area is solvent exposed.
17 . The method of claim 16 , wherein the stabilized insulin complex has at least 10% increased storage stability as compared to a corresponding insulin complex without the polyphenol.
18 . The method of claim 16 , wherein the insulin complex further comprises a divalent metal cation and/or a phenolic ligand in which two hydrogen bonds engage the phenolic hydroxyl group from the A 6 carbonyl oxygen and A 11 amide hydrogen.
19 . The method of claim 16 , wherein at least one of the insulin monomers has one or more amino acid substitutions relative to a corresponding wildtype that increases serum half-life of the insulin complex or wherein at least one of the insulin monomers has a chemical modification that increases serum half-life of the insulin complex.
20 . The method of claim 16 , wherein the polyphenol binds to B 5 His and B 26 Tyr of the first insulin monomer and B 16 Tyr of the second insulin monomer, and wherein the polyphenol is a flavonoid, a flavonol, a (flavan-3-ol), a flavan-4-ol, a flavanone, a flavone, a flavanol, a stilbenoid polyphenol, or a curcuminoid polyphenol.