Bifunctional compounds comprising insulin peptides and EGF(A) peptides

1. A fusion protein comprising:

an insulin peptide, an Epidermal Growth Factor-like domain (A) (EGF(A)) peptide, a spacer and a substituent, wherein:

(i) said insulin peptide is human insulin (SEQ ID NOs 2 and 3) or an analogue of human insulin,

(ii) said EGF(A) peptide is an analogue of the EGF(A) domain of LDL-R (293-332) according to SEQ ID NO:1,

(iii) said spacer is a peptide linker comprising segments of (GAQP)n or (GQAP)n, wherein n=1-20, and connecting the N-terminal of the insulin analogue B-chain with the C-terminal of the EGF(A) analogue, and

(iv) said substituent is of formula (I): Acy-AA2 m -AA3 p -, wherein

Acy is a fatty diacid comprising from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue and m is an integer in the range from 1 to 10, and

AA3 is a neutral, alkyleneglycol-containing amino acid residue and p is an integer in the range from 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may appear in any order,

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The fusion protein according to claim 1 , wherein said EGF(A) analogue comprises the mutation 301L.

3. The fusion protein according to claim 2 , wherein said EGF(A) analogue further comprises a mutation selected from 309R, [309R, 312E] or [309R, 312E, 321E].

4. The fusion protein according to claim 1 , wherein said EGF(A) analogue sequence comprises a mutation selected from 301L, 309R, 312E and 321E.

5. The fusion protein according to claim 1 , wherein the insulin peptide is human insulin or an analogue/derivative of human insulin comprising up to 12 mutations.

6. The fusion protein according to claim 1 , wherein said insulin analogue comprises the mutation desB30.

7. The fusion protein according to claim 1 , wherein said spacer comprises (GAQP)n, wherein n is 2-10.

8. The fusion protein according to claim 1 , wherein said substituent is attached via a Lys/K amino acid residue in the insulin sequence within said fusion protein.

9. The fusion protein according to claim 8 , wherein said substituent is attached via a Lys/K amino acid residue B29K in the insulin sequence of said fusion protein.

10. The fusion protein according to claim 1 , wherein at least one acyl moiety comprises a fatty diacid group selected from 1,16-hexadecanedioic acid, 1,18-octadecanedioic acid and 1,20-eicosanedioic acid.

11. The fusion protein according to claim 1 , wherein said fusion protein is selected from the group consisting of compounds 1-24:

(i) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]2-Insulin(B3E, B29K(hexadecanedioyl-gGlu-2×OEG), desB30) (Chem. 1),

(ii) EGF(A)(301L, 309R, 312E, 321E)-[GAQP]10-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.2),

(iii) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]2-Insulin(B3E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.3),

(iv) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]2-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.4),

(v) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]2-Insulin(B3E, B29K(octadecanedioyl-gGlu-OEG), desB30) (Chem.5),

(vi) EGF(A)(301L, 309R, 312E, 32 1E)-[GAQP]2-Insulin(B3E, B29K(eicosanedioyl-gGlu-2×EG), desB30) (Chem.6),

(vii) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]2-Insulin(B29K(eicosanedioyl-gGlu-2×OEG), desB30) (Chem.7),

(viii) EGF(A)(301L, 309R, 312E, 321 E)-[GQAP]2-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.8),

(ix) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]3-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.9),

(x) EGF(A)(301L, 309R, 312E, 321E)-[GAQP]3-Insulin(A14E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 10),

(xi) EGF(A)(301L, 309R, 312E, 321E)-[GAQP]4-Insulin(A14E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 11),

(xii) EGF(A)(301L, 309R, 3 12E, 321 E)-[GAQP]4-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 12),

(xiii) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]6-Insulin(A14E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 13),

(xiv) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]6-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 14),

(xv) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]6-Insulin(A14E, B29K(octadecanedioyl-gGlu-OEG), desB30) (Chem. 15),

(xvi) EGF(A)(301L, 309R, 3 12E, 321E)-[GAQP]6-Insulin(A14E, B29K(eicosanedioyl-gGlu-2×OEG), desB30 (Chem.16),

(xvii) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]8-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 17),

(xviii) EGF(A)(301L, 309R, 3 12E, 321 E)-[GAQP]12-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 18),

(xix) EGF(A)(301L, 309R, 3 12E, 321 E)-[GAQP]19-Insulin(B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem. 19),

(xx) EGF(A)(301L, 309R, 3 12E, 321 E)-[GAQP]19-Insulin(A14E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.20),

(xxi) EGF(A)(301L, 309R, 3 12E, 321E)-[GAQP]19-Insulin(B3E, B29K(octadecanedioyl-gGlu-2×OEG), desB30) (Chem.21),

(xxii) EGF(A)(301L, 309R, 3 12E, 321 E)-[GAQP]19-Insulin(B29K(eicosanedioyl-gGlu-2×OEG), desB30) (Chem.22),

(xxiii) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]19-Insulin(A14E, B29K(eicosanedioyl-gGlu-2×OEG), desB30) (Chem.23), and

(xxiv) EGF(A)(301L, 309R, 312E, 321 E)-[GAQP]19-Insulin(B3E, B29K(eicosanedioyl-gGlu-2×OEG), desB30) (Chem.24).

12. A pharmaceutical composition, comprising a therapeutically effective amount of a fusion protein according to claim 1 , together with a pharmaceutically acceptable excipient.

13. A method of treatment of a disease comprising administering to a subject in need thereof of a pharmaceutically effective amount of the composition of claim 12 , wherein the disease is selected from the group consisting of Type 1 diabetes, Type 2 diabetes, impaired glucose tolerance, hyperglycemia or diabetic dyslipidemia.

14. The method according to claim 13 , wherein the subject is in need of treatment of a disease selected from the group consisting of Type 1 diabetes, Type 2 diabetes or diabetic dyslipidemia.