Pharmaceutical formulation comprising incretin-insulin conjugates

1. An aqueous pharmaceutical formulation comprising (i) a therapeutically effective amount of an incretin-insulin conjugate which comprises an incretin peptide and an insulin molecule, (ii) a buffer, (iii) glycerin, (iv) phenol or m-cresol, and (v) L-arginine HCl,

wherein the pharmaceutical formulation has a pH of about 6.9-7.5;

wherein the incretin peptide has the structure:

X 1 X 2 X 3 GX 4 FTSDX 5 SX 6 YLDX 7 XsAAX 9 X 1 oFVX 11 WLLX 12 X 13 GPSSGAPPPSX 14

wherein

X 1 is His, Tyr, or absent;

X 2 is aminoisobutyric acid;

X 3 is Glu or Gln;

X 4 is Thr or Ile;

X 5 is Tyr or Lys acylated with a C 16 to C 20 alkyl group optionally via a gamma Glu linker;

X 6 is Ile or Arg;

X 7 is Lys, Arg, or Glu;

X 8 is Gln or Arg;

X 9 is Gln or aminoisobutyric acid;

X 10 is Glu or Asp;

X 11 is Asn, Gln, or Ala;

X 12 is Ala or Asp;

X 13 is Ala or Gly; and

X 14 is absent, Lys, Lys acylated with a C 16 to C 20 alkyl group optionally via a gamma Glu;

wherein the insulin molecule comprises an A chain polypeptide and a B chain polypeptide of human insulin, wherein the B chain is linked to said A chain through disulfide linkages and wherein the incretin peptide is linked to the insulin peptide via a linear chain spacer of 5 to 10 atoms, wherein the spacer comprises a disulfide linkage within the backbone of the spacer linear chain, wherein the linear chain spacer joining the incretin peptide to the insulin molecule comprises the general structure of:

wherein R 3 is H or CH 3 ;

wherein the cysteine of the linear chain spacer joining the incretin peptide to the insulin molecule is at the C-terminal end of the incretin peptide;

wherein the aqueous pharmaceutical formulation comprises about 1-100 mg/mL of the incretin-insulin conjugate, about 4-6 mg/mL of m-cresol or about 5.0 mg/mL of phenol about 6.3 mg/mL of the incretin-insulin conjugate, about 16 mg/mL of glycerin, about 10 mM sodium phosphate dibasic, and about 10.53 mg/mL of L-Arginine HCl.

2. The aqueous pharmaceutical formulation of claim 1 , wherein the incretin peptide is selected from the group consisting of:

(i) YX 2 EGTFTSDYSIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPS, wherein X 2 and X 9 are each independently aminoisobutyric acid (SEQ ID NO:4);

(ii) YX 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPS, wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:5);

(iii) YX 2 EGTFTSDYSIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPSX 14 , wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 14 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:6);

(iv) HX 2 EGTFTSDXsSRYLDERAAQEFVAWLLDAGPSGAPPPSK, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:7);

(v) HX 2 QGTFTSDXsSRYLDERAAQDFVQWLLDAGPSGAPPPSK, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:8);

(vi) HX 2 QGTFTSDX 5 SRYLDERAAQDFVQWLLDGGPSGAPPPSK, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:9);

(vii) HX 2 EGTFTSDXsSRYLDERAAQDFVQWLLDGGPSGAPPPSK, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:10); and

(viii) X 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSGAPPPS, wherein X 2 and X 9 are each independently aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:11).

3. The aqueous pharmaceutical formulation of claim 2 , wherein the incretin peptide is YX 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPS, wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu (SEQ ID NO:5).

4. The aqueous formulation of claim 1 , wherein the incretin-insulin conjugate is selected from one of:

(i) YX 2 EGTFTSDYSIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPSC, wherein X 2 and X 9 are each independently aminoisobutyric acid (SEQ ID NO:12);

(ii) YX 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPSC, wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:13);

(iii) YX 2 EGTFTSDYSIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPSX 14 C, wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 14 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:14);

(iv) HX 2 EGTFTSDX 5 SRYLDERAAQEFVAWLLDAGPSGAPPPSKC, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:15)

(v) HX 2 QGTFTSDXSSRYLDERAAQDFVQWLLDAGPSGAPPPSKC, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:16);

(vi) HX 2 QGTFTSDXSSRYLDERAAQDFVQWLLDGGPSGAPPPSKC, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:17);

(vii) HX 2 EGTFTSDXSSRYLDERAAQDFVQWLLDGGPSGAPPPSKC, wherein X 2 is aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:18); and

(viii) X 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSGAPPPSC, wherein X 2 and X 9 are each independently aminoisobutyric acid and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:19);

wherein the S group of the Cys residue of the incretin of any one of SEQ ID NO: 12-19 is conjugated to the alpha amino group of the N-terminal Phe residue of the B-chain polypeptide of an insulin heterodimer having an A-chain polypeptide of SEQ ID NO: 2 and a B-chain polypeptide of SEQ ID NO: 3 via a linker having the structure S-CH2-CH2-CH2 in a disulfide linkage.

5. The aqueous pharmaceutical formulation of claim 4 , wherein the incretin-insulin conjugate is YX 2 EGTFTSDX 5 SIYLDKQAAX 9 EFVNWLLAGGPSSGAPPPSC, wherein X 2 and X 9 are each independently aminoisobutyric acid, and X 5 is Lys acylated with a C 16 fatty acyl group via gamma-Glu spacer (SEQ ID NO:13) and wherein the S group of the Cys residue of the incretin of any one of SEQ ID NO: 12-19 is conjugated to the alpha amino group of the N-terminal Phe residue of the B-chain polypeptide of an insulin heterodimer having an A-chain polypeptide of SEQ ID NO: 2 and a B-chain polypeptide of SEQ ID NO: 3 via a linker having the structure S-CH2-CH2-CH2 in a disulfide linkage.

6. The aqueous pharmaceutical formulation of claim 1 , wherein the formulation comprises phenol.

7. The aqueous pharmaceutical formulation of claim 1 , wherein the incretin-insulin conjugate is compound 99, compound 100, compound 101, compound 102, compound 103, compound 104, compound 105, or compound 106.

8. The aqueous pharmaceutical formulation of claim 7 , wherein the incretin-insulin conjugate is compound 100.

9. The aqueous pharmaceutical formulation of claim 1 , wherein the formulation does not contain zinc.

10. The aqueous pharmaceutical formulation of claim 1 , wherein administration of the formulation results in a protracted pharmacokinetic profile as compared to an aqueous pharmaceutical formulation which does not contain L-Arginine HCl.

11. The aqueous pharmaceutical formulation of claim 1 , wherein the formulation is contained in a glass vial or an injection device.

12. A method of treating a patient or individual having a metabolic disease, comprising administering to the patient or individual an effective amount of the formulation of claim 1 to treat the metabolic disease in the patient or individual.

13. The method of claim 12 , wherein the metabolic disease is diabetes, non alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or obesity.

14. The method of claim 13 , wherein the diabetes is Type I diabetes, Type II diabetes, or gestational diabetes.