Dual GLP-1/glucagon receptor agonists derived from exendin-4

Abstract

The present invention relates to dual GLP-1/glucagon receptor agonists and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as for reduction of excess food intake.

Claims

1. A peptidic compound having the formula (I):
H.sub.2N-His-X2-X3-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-X14-X15-Glu-Glu-Ala-X19-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Aib-X28-X29-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R.sup.1  (I) or a salt or solvate thereof, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is an amino acid residue selected from the group consisting of Gin and His, X14 is an amino acid residue with a functionalized —NH.sub.2 side chain group selected from the group consisting of Lys, Orn, Dab, and Dap, wherein the —NH.sub.2 side chain group is functionalized by —Z—C(O)—R.sup.5, wherein Z is a linker comprising 1-5 amino acid linker groups selected from the group consisting of γ-glutamate (γE) and AEEAc and combinations thereof in all stereoisomeric forms, R.sup.5 is a moiety comprising up to 50 carbon atoms and heteroatoms selected from the group consisting of N and O, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is an amino acid residue selected from the group consisting of Thr, D-Ala, and Gly, and R.sup.1 is NH.sub.2 or OH.

2. The compound or salt or solvate thereof of claim 1, wherein R.sup.1 is NH.sub.2.

3. The compound or salt or solvate thereof of claim 1, wherein the peptidic compound has a relative activity of at least 0.1% compared to that of natural glucagon at the glucagon receptor.

4. The compound or salt or solvate thereof of claim 1, wherein the peptidic compound exhibits a relative activity of at least 0.1% compared to that of GLP-1(7-36)-amide at the GLP-1 receptor.

5. The compound or salt or solvate thereof of claim 1, wherein X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group —Z—C(O)R.sup.5, wherein Z is a group selected from the group consisting of γE, γE-γE, AEEAc-AEEAc-γE, and AEEAc-AEEAc-AEEAc, and R.sup.5 is a group selected from the group consisting of pentadecanyl, heptadecanyl, and 16-carboxy hexadecanyl.

6. The compound or salt or solvate thereof of claim 5, wherein X14 is Lys wherein the —NH.sub.2 side chain group is functionalized with a group —Z—C(O)R.sup.5, wherein Z is a group selected from the group consisting of γE, γE-γE, AEEAc-AEEAc-γE, and AEEAc-AEEAc-AEEAc, and R.sup.5 is a group selected from the group consisting of pentadecanyl and heptadecanyl.

7. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is an amino acid residue selected from the group consisting of Thr, D-Ala, and Gly, and R.sup.1 is NH.sub.2.

8. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-octadecanoylamino-butyryl- and (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is Ala, X28 is an amino acid residue selected from the group consisting of Ala and Lys, X29 is an amino acid residue selected from the group consisting of D-Ala and Gly, and R.sup.1 is NH.sub.2.

9. The compound or salt or solvate thereof of claim 1, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is Gln, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is an amino acid residue selected from the group consisting of Thr, D-Ala, and Gly, and R.sup.1 is NH.sub.2.

10. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the groups consisting of (S)-4-Carboxy-4-octadecanoylamino-butyryl- and (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is Glu, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala and Lys, X29 is an amino acid residue selected from the group consisting of D-Ala and Gly, and R.sup.1 is NH.sub.2.

11. The compound or salt or solvate thereof of claim 1, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is an amino acid residue selected from the group consisting of Thr, D-Ala, and Gly, and R.sup.1 is NH.sub.2.

12. The compound or salt or solvate thereof of claim 1, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is Ala, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is an amino acid residue selected from Thr, D-Ala, and Gly, and R.sup.1 is NH.sub.2.

13. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is Gln, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-octadecanoylamino-butyryl- and (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is Val, X28 is Ala, X29 is Gly, and R.sup.1 is NH.sub.2.

14. The compound or salt or solvate thereof of claim 1, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is Ala, X29 is an amino acid residue selected from the group consisting of D-Ala and Gly, and R.sup.1 is NH.sub.2.

15. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is Gln, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by the group (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl, X15 is Asp, X19 is Ala, X28 is Ser, X29 is an amino acid residue selected from the group consisting of Thr and Gly, and R.sup.1 is NH.sub.2.

16. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-octadecanoylamino-butyryl- and (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is Ala, X28 is Lys, X29 is an amino acid residue selected from the group consisting of D-Ala and Gly, and R.sup.1 is NH.sub.2.

17. The compound or salt or solvate thereof of claim 1, wherein X2 is an amino acid residue selected from the group consisting of D-Ser and Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-hexadecanoylamino-butyryl-, (S)-4-Carboxy-4-octadecanoylamino-butyryl-, (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, and [2-(2-{2-[2-(2-{2-[2-(2-Octadecanoylamino-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetylamino}-ethoxy)-ethoxy]-acetyl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is an amino acid residue selected from the group consisting of Ala and Val, X28 is an amino acid residue selected from the group consisting of Ala, Lys, and Ser, X29 is Gly, and R.sup.1 is NH.sub.2.

18. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is an amino acid residue selected from the group consisting of Gln and His, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by a group selected from the group consisting of (S)-4-Carboxy-4-octadecanoylamino-butyryl- and (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is an amino acid residue selected from the group consisting of Glu and Asp, X19 is Ala, X28 is an amino acid residue selected from the group consisting of Ala and Lys, X29 is D-Ala, and R.sup.1 is NH.sub.2.

19. The compound or salt or solvate thereof of claim 1, wherein X2 is D-Ser, X3 is Gln, X14 is Lys wherein the —NH.sub.2 side chain group is functionalized by the group (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-, X15 is Asp, X19 is Ala, X28 is an amino acid residue selected from the group consisting of Ala and Ser, X29 is an amino acid residue selected from the group consisting of Gly and D-Ala, and R.sup.1 is NH.sub.2.

20. The compound of claim 1, selected from any one of the compounds of SEQ ID NOs: 6-27, or a salt or solvate thereof.

21. The compound of claim 20, selected from any one of the compounds of SEQ ID NOs: 6-22 and 24-27, or a salt or solvate thereof.

22. The compound, salt or solvate of claim 1, consisting of the amino acid sequence of SEQ ID NO: 6, or a salt or solvate thereof.

23. The compound, salt or solvate of claim 1, consisting of the amino acid sequence of SEQ ID NO: 7, or a salt or solvate thereof.

24. The compound, salt or solvate of claim 1, consisting of the amino acid sequence of SEQ ID NO: 9, or a salt or solvate thereof.

25. A solvate of a compound of claim 1.

26. A hydrate of a compound of claim 1.

27. A pharmaceutical composition comprising one or more compounds of claim 1 or a salt or solvate thereof as an active ingredient and at least one pharmaceutically acceptable carrier.

28. The pharmaceutical composition of claim 27, further comprising at least one additional therapeutically active agent, wherein the additional therapeutically active agent is selected from the group consisting of: insulin and insulin derivatives selected from the group consisting of: insulin glargine, insulin glusiline, insulin detemir, insulin lispro, insulin degludec, insulin aspart, basal insulin and analogues thereof, pegylated insulin, recombinant human insulin, polysialated insulins, long-acting insulin, NN1045, insulin in combination with pramlintide, PE0139, fast-acting and short-acting insulins, insulin hydrogel, oral insulin, inhalable insulin, transdermal insulin and sublingual insulin, and insulin derivatives which are bonded to albumin or another protein by a bifunctional linker; GLP-1, GLP-1 analogues, and GLP-1 receptor agonists selected from the group consisting of: lixisenatide, exenatide, exendin-4, ITCA 650, AC-2993, liraglutide, semaglutide, taspoglutide, albiglutide, dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide, HM-11260C, CM-3, ORMD-0901, NN-9924, NN-9926, NN-9927, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, xtenylated exenatide, xtenylated glucagon, and polymer bound derivatives thereof; dual GLP-1/GIP receptor agonists; dual GLP-1/glucagon receptor agonists; protein YY.sub.3-36 (PYY3-36); pancreatic polypeptide; glucagon receptor agonists; GIP receptor agonists or antagonists; ghrelin receptor antagonists or inverse agonists; xenin; dipeptidyl peptidase IV (DPP-IV) inhibitors; sodium glucose cotransporter 2 (SGLT2) inhibitors; dual SGLT2/SGLT1 inhibitors; biguanides; thiazolidinediones; dual peroxisome proliferator-activated receptor (PPAR) agonists; sulfonylureas; meglitinides; alpha-glucosidase inhibitors; amylin; pramlintide; G protein-coupled receptor 119 (GPR119) agonists; GPR40 agonists; GPR120 agonists; GPR142 agonists; systemic or low-absorbable transmembrane G protein-coupled receptor 5 (TGR5) agonists; bromocriptine mesylate; inhibitors of 11-beta-hydroxysteroid dehydrogenase (HSD); activators of glucokinase; inhibitors of diacylglycerol acyltransferase (DGAT); inhibitors of protein tyrosinephosphatase 1; inhibitors of glucose-6-phosphatase; inhibitors of fructose-1,6-bisphosphatase; inhibitors of glycogen phosphorylase; inhibitors of phosphoenol pyruvate carboxykinase; inhibitors of glycogen synthase kinase; inhibitors of pyruvate dehydrogenase kinase; alpha2-antagonists; C-C motif receptor (CCR-2) antagonists; modulators of glucose transporter-4; somatostatin receptor 3 agonists; 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA)-reductase inhibitors; fibrates; nicotinic acid and derivatives thereof; nicotinic acid receptor 1 agonists; PPAR-alpha, gamma, or alpha/gamma agonists or modulators; PPAR-delta agonists; acyl-CoA cholesterol acyltransferase (ACAT) inhibitors; cholesterol absorption inhibitors; bile acid-binding substances; ileal bile acid transporter (IBAT) inhibitors; microsomal triglyceride transfer protein (MTP) inhibitors; modulators of proprotein convertase subtilisin/kinexin type 9 (PCSK9); low-density lipoprotein (LDL) receptor up-regulators by liver selective thyroid hormone receptor β agonists; high-density lipoprotein (HDL)-raising compounds; lipid metabolism modulators; phospholipase A2 (PLA2) inhibitors; apolipoprotein A1 (ApoA-1) enhancers; thyroid hormone receptor agonists; cholesterol synthesis inhibitors; and omega-3 fatty acids and derivatives thereof; substances for the treatment of obesity selected from the group consisting of: sibutramine, tesofensine, tetrahydrolipstatin, cannabinoid-1 (CB-1) receptor antagonists, melanin-concentrating hormone-1 (MCH-1) antagonists, melanocortin 4 (MC4) receptor agonists or partial agonists, neuropeptide Y5 (NPY5) or NPY2 antagonists, NPY4 agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the 5HT2c receptor, combinations of bupropione/naltrexone, combinations of bupropione/zonisamide, combinations of bupropione/phentermine, combinations of pramlintide/metreleptin, and combinations of phentermine/topiramate; lipase inhibitors; angiogenesis inhibitors; H3 antagonists; Agouti-related protein (AgRP) inhibitors; triple monoamine uptake inhibitors; methionine aminopeptidase type 2 (MetAP2) inhibitors; nasal formulation of the calcium channel blocker diltiazem; antisense molecules against production of fibroblast growth factor receptor 4; and prohibitin targeting peptide-1; and drugs for influencing high blood pressure, chronic heart failure, or atherosclerosis selected from the groups consisting of: angiotensin II receptor antagonists, angiotensin-converting-enzyme (ACE) inhibitors, endothelin-converting-enzyme (ECE) inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, and thrombocyte aggregation inhibitors.

29. A method for the treatment of hyperglycemia, type 1 diabetes, type 2 diabetes, obesity or any combination of these individual disease components, the method comprising administering to a patient in need of such treatment an effective amount of one or more compounds, salts or solvates of claim 1.

30. The method of claim 29, for the treatment of hyperglycemia or type 2 diabetes.

31. A method for the simultaneous treatment of diabetes and obesity which comprises administering to a patient in need of such treatment an effective amount of one or more compounds, salts or solvates of claim 1.

32. A method for the treatment of hyperglycemia, type 2 diabetes, type 1 diabetes, or obesity or any combination of these individual disease components, the method comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition of claim 27.

33. The method of claim 32, for the treatment of hyperglycemia or type 2 diabetes.

34. A method for the simultaneous treatment of diabetes and obesity, the method comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition of claim 27.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1. Body weight development during 4 weeks of subcutaneous treatment with SEQ ID NO: 6 and SEQ ID NO: 7, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(2) FIG. 2. Relative body weight change in % during 4 weeks of subcutaneous treatment with SEQ ID NO: 6 and SEQ ID NO: 7, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(3) FIG. 3. Determination of total fat mass measured by nuclear magnetic resonance (NMR), two days before and after 4 weeks of treatment with SEQ ID NO: 6 and SEQ ID NO: 7, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(4) FIG. 4. Acute effect of s.c. administration of compound SEQ ID NO: 6 and SEQ ID NO: 7 50 μg/kg on blood glucose in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(5) FIG. 5. Body weight development during 4 weeks of subcutaneous treatment with SEQ ID NO: 9, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(6) FIG. 6. Relative body weight change in % during 4 weeks of subcutaneous treatment with SEQ ID NO: 9, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(7) FIG. 7. Determination of total fat mass measured by nuclear magnetic resonance (NMR), two days before and after 4 weeks of treatment with SEQ ID NO: 9, 50 μg/kg bid in female high-fat fed C57BL/6 mice. Data are mean+SEM.

(8) FIG. 8. Acute effect of s.c. administration of compound SEQ ID NO: 9, 50 μg/kg bid on blood glucose in female high-fat fed C57BL/6 mice. Data are mean+SEM.

METHODS

Abbreviations Employed are as Follows

(9) AA amino acid AEEAc (2-(2-aminoethoxyl)ethoxy)acetyl cAMP cyclic adenosine monophosphate Boc tert-butyloxycarbonyl BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate BSA bovine serum albumin tBu tertiary butyl DCM dichloromethane Dde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl DIC N,N′-diisopropylcarbodiimide DIPEA N,N-diisopropylethylamine DMEM Dulbecco's modified Eagle's medium DMF dimethyl formamide DMS dimethylsulfide EDT ethanedithiol FA formic acid FBS fetal bovine serum Fmoc fluorenylmethyloxycarbonyl HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HBSS Hanks' Balanced Salt Solution HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HOBt 1-hydroxybenzotriazole HOSu N-hydroxysuccinimide HPLC High Performance Liquid Chromatography HTRF Homogenous Time Resolved Fluorescence IBMX 3-isobutyl-1-methylxanthine LC/MS Liquid Chromatography/Mass Spectrometry Mint monomethoxy-trityl Palm palmitoyl PBS phosphate buffered saline PEG polyethylene glycole PK pharmacokinetic RP-HPLC reversed-phase high performance liquid chromatography Stea stearyl TFA trifluoroacetic acid Trt trityl UV ultraviolet γE-Glutamate

(10) General Synthesis of Peptidic Compounds

(11) Materials

(12) Different Rink-Amide resins (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin, Merck Biosciences; 4-[(2,4-Dimethoxyphenyl)(Fmoc-amino)methyl]phenoxy acetamido methyl resin, Agilent Technologies) were used for the synthesis of peptide amides with loadings in the range of 0.2-0.7 mmol/g.

(13) Fmoc protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, his Biotech, Bachem, Chem-Impex International or MATRIX Innovation. The following standard amino acids were used throughout the syntheses: Fmoc-L-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

(14) In addition, the following special amino acids were purchased from the same suppliers as above: Fmoc-L-Lys(ivDde)-OH, Fmoc-L-Lys(Mmt)-OH, Fmoc-Aib-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Ala-OH, Boc-L-His(Boc)-OH (available as toluene solvate) and Boc-L-His(Trt)-OH.

(15) The solid phase peptide syntheses were performed for example on a Prelude Peptide Synthesizer (Protein Technologies Inc) or similar automated synthesizer using standard Fmoc chemistry and HBTU/DIPEA activation. DMF was used as the solvent. Deprotection: 20% piperidine/DMF for 2×2.5 mM Washes: 7×DME Coupling 2:5:10 200 mM AA/500 mM HBTU/2M DIPEA in DMF 2× for 20 min. Washes: 5×DME

(16) In cases where a Lys-side-chain was modified, Fmoc-L-Lys(ivDde)-OH or Fmoc-L-Lys(Mmt)-OH was used in the corresponding position. After completion of the synthesis, the ivDde group was removed according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DME The Mmt group was removed by repeated treatment with 1% TFA in dichloromethane. The following acylations were carried out by treating the resin with the N-hydroxy succinimide esters of the desired acid or using coupling reagents like HBTU/DIPEA or HOBt/DIC.

(17) All the peptides that have been synthesized were cleaved from the resin with King's cleavage cocktail consisting of 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% EDT. The crude peptides were then precipitated in diethyl or diisopropyl ether, centrifuged, and lyophilized Peptides were analyzed by analytical HPLC and checked by ESI mass spectrometry. Crude peptides were purified by a conventional preparative RP-HPLC purification procedure.

(18) Alternatively, peptides were synthesized by a manual synthesis procedure: 0.3 g Desiccated Rink amide MBHA Resin (0.66 mmol/g) was placed in a polyethylene vessel equipped with a polypropylene filter. Resin was swelled in DCM (15 ml) for 1 h and DMF (15 ml) for 1 h. The Fmoc group on the resin was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test (quantitative method) was used for the conformation of removal of Fmoc from solid support. The C-terminal Fmoc-amino acid (5 equiv. excess corresponding to resin loading) in dry DMF was added to the de-protected resin and coupling was initiated with 5 equivalent excess of DIC and HOBT in DME The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). Kaiser test on peptide resin aliquot upon completion of coupling was negative (no colour on the resin). After the first amino acid attachment, the unreacted amino group, if any, in the resin was capped used acetic anhydride/pyridine/DCM (1:8:8) for 20 minutes to avoid any deletion of the sequence. After capping, resin was washed with DCM/DMF/DCM/DMF (6/6/6/6 time each). The Fmoc group on the C-terminal amino acid attached peptidyl resin was deprotected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min. The resin was washed with DMF/DCM/DMF (6:6:6 time each). The Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.

(19) The remaining amino acids in target sequence on Rink amide MBHA Resin were sequentially coupled using Fmoc AA/DIC/HOBt method using 5 equivalent excess corresponding to resin loading in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6:6:6 time each). After each coupling step and Fmoc deprotection step, a Kaiser test was carried out to confirm the completeness of the reaction.

(20) After the completion of the linear sequence, the ε-amino group of lysine used as branching point or modification point was deprotected by using 2.5% hydrazine hydrate in DMF for 15 min×2 and washed with DMF/DCM/DMF (6:6:6 time each). The γ-carboxyl end of glutamic acid was attached to the ε-amino group of Lys using Fmoc-Glu(OH)—OtBu with DIC/HOBt method (5 equivalent excess with respect to resin loading) in DMF. The mixture was rotated on a rotor at room temperature for 2 h. The resin was filtered and washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on the glutamic acid was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min (25 ml each). The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.

(21) If the side-chain branching also contains one more γ-glutamic acid, a second Fmoc-Glu(OH)—OtBu used for the attachment to the free amino group of γ-glutamic acid with DIC/HOBt method (5 equivalent excess with respect to resin loading) in DMF. The mixture was rotated on a rotor at room temperature for 2 h. Resin was filtered and washed with DMF/DCM/DMF (6×30 ml each). The Fmoc group on the γ-glutamic acid was de-protected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 and 15 min (25 mL). The resin was washed with DMF/DCM/DMF (6:6:6 time each). A Kaiser test on peptide resin aliquot upon completion of Fmoc-deprotection was positive.

(22) Palmitic Acid & Stearic Acid attachment to side chains of Glutamic acid: To the free amino group of γ-glutamic acid, palmitic acid or stearic acid (5 equiv.) dissolved in DMF was added and coupling was initiated by the addition of DIC (5 equiv.) and HOBt (5 equiv.) in DMF. The resin was washed with DMF/DCM/DMF (6:6:6 time each).

(23) Final Cleavage of Peptide from the Resin:

(24) The peptidyl resin synthesized by manual synthesis was washed with DCM (6×10 ml), MeOH (6×10 ml) and ether (6×10 ml) and dried in vacuum desiccators overnight. The cleavage of the peptide from the solid support was achieved by treating the peptide-resin with reagent cocktail (80.0% TFA/5% thioanisole/5% phenol/2.5% EDT, 2.5% DMS and 5% DCM) at room temperature for 3 h. Cleavage mixture was collected by filtration and the resin was washed with TFA (2 ml) and DCM (2×5 ml). The excess TFA and DCM was concentrated to small volume under nitrogen and a small amount of DCM (5-10 ml) was added to the residue and evaporated under nitrogen. The process was repeated 3-4 times to remove most of the volatile impurities. The residue was cooled to 0° C. and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the supernatant ether was removed and fresh ether was added to the peptide and re-centrifuged. The crude sample was preparative HPLC purified and lyophilized. The identity of peptide was confirmed by LCMS.

(25) Analytical HPLC/UPLC

(26) Method A: detection at 210-225 nm column: Waters ACQUITY UPLC® CSH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+0.5% TFA: ACN+0.35% TFA (flow 0.5 ml/min) gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 2:98 (23.5 min) to 2:98 (30.5 min) to 80:20 (31 min) to 80:20 (37 min) optionally with mass analyser: LCT Premier, electrospray positive ion mode

(27) Method B: detection at 210-225 nm column: Aries prep XBC 18 (4.6×250 mm, 3.6 μm), Temp: 25° C. solvent: H.sub.2O+0.1% TFA: ACN+0.1% TFA (flow 1 ml/min) gradient: Equilibration of the column with 2% buffer B and elution by a gradient of 2% to 70% buffer B during 15 min.

(28) General Preparative HPLC Purification Procedure

(29) The crude peptides were purified either on an Akta Purifier System, a Jasco semiprep HPLC System or a Agilent 1100 HPLC system. Preparative RP-C18-HPLC columns of different sizes and with different flow rates were used depending on the amount of crude peptide to be purified. Acetonitrile+0.1% TFA (B) and water+0.1% TFA (A) were employed as eluents. Product-containing fractions were collected and lyophilized to obtain the purified product, typically as TFA salt.

(30) Solubility and Stability-Testing of Exendin-4 Derivatives

(31) Prior to the testing of solubility and stability of a peptide batch, its purity (HPLC-UV) was determined.

(32) For solubility testing, the target concentration was 10 mg pure compound/ml. Therefore, solutions from solid samples were prepared in different buffer systems with a concentration of 10 mg/mL compound based on the previously determined % purity. HPLC-UV was performed after 2 h of gentle agitation from the supernatant, which was obtained by 20 min of centrifugation at 4500 rpm.

(33) The solubility was then determined by comparison of a 0.2 μL-injection with the UV peak areas obtained with a stock solution of the peptide at a concentration of 1.2 mg/mL in DMSO (based on % purity), injecting various volumes ranging from 0.2-2 μl. This analysis also served as starting point (t0) for the stability testing.

(34) For stability testing, an aliquot of the supernatant obtained for solubility was stored for 7 days at 40° C. After that time course, the sample was centrifuged for 20 min at 4500 rpm and 0.2 μL of the supernatant were analysed with HPLC-UV.

(35) For determination of the amount of the remaining peptide, the peak areas of the target compound at t0 and t7 were compared, resulting in “% remaining peptide”, following the equation
% remaining peptide=[(peak area peptide t7)×100]/peak area peptide t0.

(36) The stability is expressed as “% remaining peptide”.

(37) As HPLC/UPLC method Method A has been used, detecting at 214 nm.

(38) In Vitro Cellular Assays for GLP-1, Glucagon and GIP Receptor Efficacy

(39) Agonism of compounds for the receptors was determined by functional assays measuring cAMP response of HEK-293 cell lines stably expressing human GLP-1, GIP or glucagon receptor.

(40) cAMP content of cells was determined using a kit from Cisbio Corp. (cat. no. 62AM4PEC) based on HTRF (Homogenous Time Resolved Fluorescence). For preparation, cells were split into T175 culture flasks and grown overnight to near confluency in medium (DMEM/10% FBS). Medium was then removed and cells washed with PBS lacking calcium and magnesium, followed by proteinase treatment with accutase (Sigma-Aldrich cat. no. A6964). Detached cells were washed and resuspended in assay buffer (1×HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cellular density determined. They were then diluted to 400000 cells/ml and 25 μl-aliquots dispensed into the wells of 96-well plates. For measurement, 25 μl of test compound in assay buffer was added to the wells, followed by incubation for 30 minutes at room temperature. After addition of HTRF reagents diluted in lysis buffer (kit components), the plates were incubated for 1 hr, followed by measurement of the fluorescence ratio at 665/620 nm. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC50).

(41) Bioanalytical Screening Method for Quantification of Exendin-4 Derivatives in Mice and Pigs

(42) Mice were dosed 1 mg/kg subcutaneously (s.c.). The mice were sacrificed and blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours post application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-life were calculated using WinonLin Version 5.2.1 (non-compartment model).

(43) Female Göttinger minipigs were dosed 0.1 mg/kg subcutaneously (s.c.). Blood samples were collected after 0.25, 0.5, 1, 2, 4, 8, 24, 32, 48, 56 and 72 hours post application. Plasma samples were analyzed after protein precipitation via liquid chromatography mass spectrometry (LC/MS). PK parameters and half-life were calculated using WinonLin Version 5.2.1 (non-compartment model).

(44) Gastric Emptying and Intestinal Passage in Mice

(45) Female NMRI-mice of a body weight between 20 and 30 g are used. Mice are adapted to housing conditions for at least one week.

(46) Mice are overnight fasted, while water remains available all the time. On the study day, mice are weighed, single-caged and allowed access to 500 mg of feed for 30 min, while water is removed. At the end of the 30 min feeding period, remaining feed is removed and weighed. 60 min later, a coloured, non-caloric bolus is instilled via gavage into the stomach. The test compound/reference compound or its vehicle in the control group is administered subcutaneously, to reach Cmax when coloured bolus is administered. After another 30 min, the animals are sacrificed and the stomach and the small intestine prepared. The filled stomach is weighed, emptied, carefully cleaned and dried and reweighed. The calculated stomach content indicates the degree of gastric emptying. The small intestine is straightened without force and measured in length. Then the distance from the gastric beginning of the gut to the tip of the farthest traveled intestinal content bolus is measured. The intestinal passage is given as relation in percent of the latter distance and the total length of the small intestine. Comparable data can be obtained for both female and male mice.

(47) Statistical analyses are performed with Everstat 6.0 by 1-way-ANOVA, followed by Dunnetts or Newman-Keuls as post-hoc test, respectively. Differences are considered statistically significant at the p<0.05 level. As post hoc test Dunnet's Test is applied to compare versus vehicle control, only. Newman-Keul's Test is applied for all pairwise comparisons (i.e. versus vehicle and reference groups).

(48) Automated Assessment of Feed Intake in Mice

(49) Female NMRI-mice of a body weight between 20 and 30 g are used. Mice are adapted to housing conditions for at least one week and for at least one day single-caged in the assessment equipment, when basal data are recorded simultaneously. On the study day, test product is administered subcutaneously close to the lights-off phase (12 h lights off) and assessment of feed consumption is directly started afterwards. Assessment included continued monitoring (every 30 min) over 22 hours. Repetition of this procedure over several days is possible. Restriction of assessment to 22 hours is for practical reasons to allow for reweighing of animals, refilling of feed and water and drug administration between procedures. Results can be assessed as cumulated data over 22 hours or differentiated to 30 min intervals. Comparable data can be obtained for both female and male mice.

(50) Statistical analyses are performed with Everstat 6.0 by two-way ANOVA on repeated measures and Dunnett's post-hoc analyses. Differences are considered statistically significant at the p<0.05 level.

(51) Acute and Chronic Effects after Subcutaneous Treatment on Blood Glucose and Body Weight in Female Diet-Induced Obese (DIO) C57BL/6 Mice

(52) C57BL/6 Harlan mice are housed in groups in a specific pathogen-free barrier facility on a 12 h light/dark cycle with free access to water and standard or high-fat diet. After prefeeding on high-fat diet, mice are stratified to treatment groups (n=8), so that each group has similar mean body weight. An age-matched group with ad-libitum access to standard chow is included as standard control group. Before the experiment, mice are subcutaneously (s.c.) injected with vehicle solution and weighed for 3 days to acclimate them to the procedures.

(53) 1) Acute effect on blood glucose in fed female DIO mice: initial blood samples are taken just before first administration (s.c.) of vehicle (phosphate buffer solution) or the exendin-4 derivatives (dissolved in phosphate buffer), respectively. The volume of administration is 5 mL/kg. The animals have access to water and their corresponding diet during the experiment. Blood glucose levels are measured at t=0 h, t=1 h, t=2 h, t=3 h, t=4 h, t=6 h and t=24 h (method: Accu-Check glucometer). Blood sampling is performed by tail incision without anaesthesia.

(54) 2) Chronic effect on body weight in female DIO mice: mice are treated twice daily s.c. in the morning and in the evening, respectively, at the beginning and the end of the light phase with either vehicle or exendin-4 derivatives for 4 weeks. Body weight is recorded daily. Two days before start of treatment and on day 26, total fat mass is measured by nuclear magnetic resonance (NMR).

(55) Statistical analyses are performed with Everstat 6.0 by repeated measures two-way ANOVA and Dunnetts post-hoc analyses (glucose profile) and 1-way-ANOVA, followed by Dunnetts post-hoc test (body weight, body fat). Differences versus vehicle-treated DIO control mice are considered statistically significant at the p<0.05 level.

(56) Effects of 4 Weeks of Treatment on Glucose, HbA1c and Oral Glucose Tolerance in Female Diabetic Dbdb-Mice

(57) 8 week old, female diabetic dbdb-mice of mean non-fasted glucose value of 14.5 mmol/l and a body weight of 37-40 g are used. Mice are individually marked and are adapted to housing conditions for at least one week.

(58) 7 days prior to study start, baseline values for non-fasted glucose and HbA1c are determined, 5 days prior to study start, mice are assigned to groups and cages (5 mice per cage, 10 per group) according to their HbA1c values to ensure even distribution of lower and higher values between groups (stratification).

(59) Mice are treated for 4 weeks, by twice daily subcutaneous administration in the morning and the afternoon. Blood samples from the tail tip are obtained for HbA1c on study day 21 and oral glucose tolerance is assessed in the 4th week.

(60) An oral glucose tolerance test is done in the morning without prior extra compound administration to majorly assess the effect of chronic treatment and less of acute compound administration. Mice are fasted for 4 hours prior to oral glucose administration (2 g/kg, t=0 min) Blood samples are drawn prior to glucose administration and at 15, 30, 60, 90, 120, and 180 min Feed is returned after the last blood sampling. Results are represented as change from baseline, glucose in mmol/l and HbA1c in %.

(61) Statistical analyses are performed with Everstat Version 6.0 based on SAS by 1-way-ANOVA, followed by Dunnett's post-hoc test against vehicle-control. Differences are considered statistically significant at the p<0.05 level.

(62) Glucose Lowering in Non-Fasted Female Diabetic dbdb-Mice

(63) Female diabetic dbdb-mice of mean non-fasted glucose value of 20-22 mmol/l and a body weight of 42 g+/−0.6 g (SEM) are used. Mice are individually marked and are adapted to housing conditions for at least one week.

(64) 3-5 days prior to study start mice are assigned to groups and cages (4 mice per cage, 8 per group) according to their non-fasted glucose values to ensure even distribution of lower and higher values between groups (stratification). On the study day, mice are weighed and dosed (t=0) Immediately prior to compound administration feed is removed while water remains available, and a first blood sample at a tail incision is drawn (baseline). Further blood samples are drawn at the tail incision at 30, 60, 90, 120, 240, 360, and 480 min.

(65) Statistical analyses are performed with Everstat Version 6.0 based on SAS by 2-way-ANOVA on repeated measures, followed by Dunnett's post-hoc test against vehicle-control. Differences are considered statistically significant at the p<0.05 level.

EXAMPLES

(66) The invention is further illustrated by the following examples.

Example 1

Synthesis of SEQ ID NO: 9

(67) The solid phase synthesis as described in Methods was carried out on Novabiochem Rink-Amide resin (4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 mesh, loading of 0.23 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on resin according to literature (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Hereafter Palm-Glu-Glu-OSu was coupled to the liberated amino-group employing DIPEA as base. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method A).

(68) Deconvolution of the mass signals found under the peak with retention time 12.61 min revealed the peptide mass 4581.5 which is in line with the expected value of 4581.1.

Example 2

Synthesis of SEQ ID NO: 5

(69) The manual synthesis procedure as described in Methods was carried out on a desiccated Rink amide MBHA Resin (0.66 mmol/g). The Fmoc-synthesis strategy was applied with DIC/HOBt-activation. In position 14 Fmoc-Lys(ivDde)-OH and in position 1 Boc-His(Boc)-OH were used. The ivDde-group was cleaved from the peptide on resin according to a modified literature procedure (S. R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603), using 4% hydrazine hydrate in DMF. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 10.46 min revealed the peptide mass 4450.5 which is in line with the expected value of 4451.9.

(70) In an analogous way, the peptides listed in Table 3 were synthesized and characterized.

(71) TABLE-US-00009 TABLE 3 list of synthesized peptides and comparison of calculated vs. found molecular weight SEQ Monoisotopic or ID NO calc. Mass found mass average mass 6 4604.2 4603.4 average 7 4590.1 4589.4 average 8 4489.1 4488.4 average 9 4581.1 4581.5 average 10 4522.1 4521.3 average 11 4578.3 4578.3 monoisotopic 12 4592.3 4592.3 monoisotopic 13 4480.1 4479.3 average 14 4451.9 4450.5 average 15 4594.3 4594.28 monoisotopic 16 4592.3 4592.28 monoisotopic 17 4609.2 4608.9 average 18 4638.3 4638.25 monoisotopic 19 4635.38 4635.30 monoisotopic 20 4739.4 4739.40 monoisotopic 21 4767.5 4767.40 monoisotopic 22 4783.5 4783.50 monoisotopic 24 4649.4 4649.39 monoisotopic 25 4516.3 4548.37 monoisotopic 26 4658.4 4658.42 monoisotopic 27 4672.4 4672.38 monoisotopic 28 4069.5 4068.6 average 29 4633.2 4632.0 average 30 4647.2 4646.2 average 31 4638.2 4637.60 average 32 4624.2 4623.55 average 33 4621.37 4621.35 monoisotopic 34 4110.0 4110.1 monoisotopic

(72) In an analogous way, the following peptides of Table 4 can be synthesized:

(73) TABLE-US-00010 TABLE 4 List of peptides that can be synthesized in an analogous way. SEQ ID NO 23

Example 3: Stability and Solubility

(74) Solubility and stability of peptidic compounds were assessed as described in Methods. The results are given in Table 5.

(75) TABLE-US-00011 TABLE 5 Stability and solubility Stability solubility [mg/ml] SEQ ID NO pH 4.5 pH 7.4 pH 4.5 pH 7.4 6 100.0 92.0 >8 >8 7 100.0 90.0 2.2 >8 9 77.0 93.0 2.2 >8 12 85.6 90.6 2.0 >8 14 94.7 96.3 >8 >8 19 96.0 96.0 >8 >8 24 81.8 95.4 >8 7 26 92.7 96.6 6.8 6.7

Example 4: In Vitro Data on GLP-1, Glucagon and GIP Receptor

(76) Potencies of peptidic compounds at the GLP-1, glucagon and GIP receptors were determined by exposing cells expressing human glucagon receptor (hGlucagon R), human GIP receptor (hGIP-R) or human GLP-1 receptor (hGLP-1R) to the listed compounds at increasing concentrations and measuring the formed cAMP as described in Methods.

(77) The results are shown in Table 6:

(78) TABLE-US-00012 TABLE 6 EC50 values of exendin-4 derivatives at GLP-1, Glucagon and GIP receptors (indicated in pM) SEQ ID NO EC50 hGLP-1R EC50 hGlucagon-R EC50 hGIP-R 6 2.6 17.7 1538.5 7 1.5 9.0 908.0 8 1.8 4.8 538.0 9 1.8 11.8 3600.0 10 6.5 140.0 982.0 11 1.2 5.1 560.0 12 2.8 66.0 335.5 13 2.7 6.8 2540.0 14 2.1 24.7 5360.0 15 1.8 12.3 5500.0 16 1.8 35.4 7780.0 17 2.4 47.1 7080.0 18 3.6 10.3 27500.0 19 2.0 17.0 7170.0 20 4.3 88.4 20200.0 21 1.0 9.1 2450.0 22 1.8 6.4 4160.0 24 2.4 76.5 14100.0 25 2.1 43.2 8320.0 26 2.0 40.6 5530.0 27 2.1 55.4 3220.0

Example 5: Comparison Testing

(79) A selection of inventive exendin-4 derivatives comprising a functionalized amino acid in position 14 has been tested versus corresponding compounds having in this position 14 a ‘non-functionalized’ amino acid with otherwise identical amino acid sequence. The reference pair compounds and the corresponding EC50 values at GLP-1, Glucagon and GIP receptors (indicated in pM) are given in Table 7. As shown, the inventive exendin-4 derivatives show a superior activity in comparison to the compounds with a ‘non-functionalized’ amino acid in position 14.

(80) Furthermore, a selection of inventive exendin-4 derivatives comprising an Aib in position 27 has been tested versus corresponding compounds having in this position a lysine residue as in native exendin-4 and otherwise identical amino acid sequence. The reference pair compounds and the corresponding EC50 values at GLP-1, Glucagon and GIP receptors (indicated in pM) are given in Table 8. As shown, the inventive exendin-4 derivatives show a reduced activity on the GIP receptor compared to the corresponding derivatives with Lys at position 27 as in native exendin-4.

(81) TABLE-US-00013 TABLE 7 Comparison of exendin-4 derivatives comprising a non-functionalized amino acid in position 14 vs. exendin-4 derivatives comprising a functionalized amino acid in position 14 and otherwise identical amino acid sequence. EC50 values at GLP-1, Glucagon and GIP receptors are indicated in pM. (K = lysine, L = leucine, E-x53 = (S)- 4-Carboxy-4-hexadecanoylamino-butyryl-, E-x70 = (S)-4-Carboxy- 4-octadecanoylamino-butyryl-, E-E-x53 = (S)-4-Carboxy-4-((S)-4- carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-)) SEQ ID EC50 hGLP- EC50 residue NO 1R EC50 hGlucagon-R hGIP-1 in position 14 28 3.8 1040.0 343000.0 L 9 1.8 11.8 3600.0 K(γE-γE-x53) 13 2.7 6.8 2540.0 K(γE-x70) 14 2.1 24.7 5360.0 K(γE-x53) 34 0.8 338.0 26000.0 L 32 1.9 5.5 565.0 K(γE-γE-x53)

(82) TABLE-US-00014 TABLE 8 Comparison of exendin-4 derivatives comprising an Aib in position 27 vs. exendin-4 derivatives comprising a Lys in position 27 and otherwise identical amino acid sequence. EC50 values at GLP-1, Glucagon and GIP receptors are indicated in pM. SEQ ID EC50 hGLP- EC50 residue in NO 1R EC50 hGlucagon-R hGIP-1 position 27 29 0.9 2.6 112.0 K 7 1.5 9.0 908.0 Aib 30 0.8 5.1 77.8 K 6 2.6 17.7 1538.5 Aib 31 1.3 7.4 344.7 K 16 1.8 35.4 7780.0 Aib 32 1.9 5.5 565.0 K 9 1.8 11.8 3600.0 Aib 33 1.0 1.4 70.8 K 11 1.2 5.1 560.0 Aib

Example 6: Pharmacokinetic Testing in Mice

(83) Pharmacokinetic profiles were determined as described in Methods. Calculated T.sub.1/2 and Cmax values are shown in Table 9.

(84) TABLE-US-00015 TABLE 9 Pharmacokinetic profiles of exendin-4 derivatives in mice. SEQ ID NO T.sub.1/2 [h] Cmax [ng/ml] 9 3.8 6560.0

Example 7: Acute and Chronic Effects of SEQ ID NO: 6 and of SEQ ID NO: 7 after Subcutaneous Treatment on Blood Glucose and Body Weight in Female Diet-Induced Obese (DIO) C57BL/6 Mice

(85) 1) Glucose Profile

(86) After blood sampling to determine the blood glucose baseline level, fed diet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQ ID NO: 6, 50 μg/kg of SEQ ID NO: 7 or phosphate buffered solution (vehicle control on standard or high-fat diet) subcutaneously. At predefined time points, more blood samples were taken to measure blood glucose and generate the blood glucose profile over 24 h (see FIG. 4)

(87) 2) Body Weight

(88) Female obese C57BL/6 mice were treated for 4 weeks twice daily subcutaneously with 50 μg/kg SEQ ID NO: 6, 50 μg/kg SEQ ID NO: 7 or vehicle. Body weight was recorded daily, and body fat content was determined before the start and after 4 weeks of treatment. Treatment with 50 μg/kg SEQ ID NO: 6 or 50 μg/kg SEQ ID NO: 7 showed a decrease in daily body weight when compared to vehicle DIO control mice (Table 10, FIGS. 1 and 2). These changes resulted from a decrease in body fat, as shown by the absolute changes in body fat content (Table 10, FIG. 3).

(89) TABLE-US-00016 TABLE 10 Weight change in DIO mice over a 4-week treatment period (mean ± SEM) Overall weight Example (Dose) change (g) Body fat change (g) Control standard diet +0.86 ± 0.3 +0.73 ± 0.2 Control high-fat diet +4.40 ± 0.6 +2.95 ± 0.6 SEQ ID NO: 6 (50 μg/kg bid) −6.08 ± 0.6 −4.64 ± 0.4 SEQ ID NO: 7 (50 μg/kg bid) −7.25 ± 0.7 −5.12 ± 0.6

Example 8: Acute and Chronic Effects of SEQ ID NO: 9 after Subcutaneous Treatment on Blood Glucose and Body Weight in Female Diet-Induced Obese (DIO) C57BL/6 Mice

(90) 1) Glucose Profile

(91) After blood sampling to determine the blood glucose baseline level, fed diet-induced obese female C57BL/6 mice were administered 50 μg/kg of SEQ ID NO: 9 or phosphate buffered solution (vehicle control on standard or high-fat diet) subcutaneously. At predefined time points, more blood samples were taken to measure blood glucose and generate the blood glucose profile over 24 h.

(92) SEQ ID NO: 9 demonstrated a decrease in blood glucose compared to DIO control (FIG. 8, mean±SEM).

(93) 2) Body Weight

(94) Female obese C57BL/6 mice were treated for 4 weeks twice daily subcutaneously with 50 μg/kg SEQ ID NO: 9 or vehicle. Body weight was recorded daily, and body fat content was determined before the start and after 4 weeks of treatment.

(95) Treatment with 50 μg/kg SEQ ID NO: 9 showed a decrease in daily body weight when compared to vehicle DIO control mice (Table 11, FIGS. 5 and 6). These changes resulted from a decrease in body fat, as shown by the absolute changes in body fat content (Table 11, FIG. 7).

(96) TABLE-US-00017 TABLE 11 Weight change in DIO mice over a 4-week treatment period (mean ± SEM) Overall weight Example (Dose) change (g) Body fat change (g) Control standard diet +0.94 ± 0.4 +2.56 ± 0.4 Control high-fat diet +3.83 ± 0.5 +5.00 ± 0.5 SEQ ID NO: 9 (50 μg/kg bid) −6.56 ± 1.0 −5.65 ± 0.9

(97) TABLE-US-00018 TABLE 12 Sequences SEQ. ID sequence 1 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I- E-W-L-K-N-G-G-P-S-S-G-A-P-P-P-S-NH2 2 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I- A-W-L-V-K-G-R-NH2 3 H-S-Q-G-T-F-T-S-D-Y-S-K-Y-L-D-S-R-R-A-Q-D-F-V- Q-W-L-M-N-T-OH 4 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(γE- x53)-E-F-I-A-W-L-V-R-G-R-G-OH 5 Y-A-E-G-T-F-I-S-D-Y-S-I-A-M-D-K-I-H-Q-Q-D-F-V- N-W-L-L-A-Q-K-G-K-K-N-D-W-K-H-N-I-T-Q-OH 6 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-E-E- E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P- S-NH2 7 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P- S-NH2 8 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-D-E-E- A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S- NH2 9 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P- S-NH2 10 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-E-E-E- A-V-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S- NH2 11 H-S-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-E- A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S- NH2 12 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-A-dAla-G-P-S-S-G-A-P- P-P-S-NH2 13 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-D-E-E- A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S- NH2 14 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x53)-D-E-E- A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S- NH2 15 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-S-G-G-P-S-S-G-A-P-P-P- S-NH2 16 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-E-E- E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P- S-NH2 17 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-V-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P- S-NH2 18 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-S-T-G-P-S-S-G-A-P-P-P- S-NH2 19 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-K-G-G-P-S-S-G-A-P-P-P- S-NH2 20 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E- x53)-D-E-E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S- G-A-P-P-P-S-NH2 21 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E- x70)-D-E-E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S- G-A-P-P-P-S-NH2 22 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- AEEAc-x70)-D-E-E-A-A-R-L-F-I-E-W-L-Aib-A-G-G- P-S-S-G-A-P-P-P-S-NH2 23 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(AEEAc-AEEAc- E- x99)-D-E-E-A-A-R-L-F-I-E-W-L-Aib-A-G-G-P-S-S- G-A-P-P-P-S-NH2 24 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-K-dAla-G-P-S-S-G-A-P- P-P-S-NH2 25 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-x70)-D-E-E- A-A-R-L-F-I-E-W-L-Aib-K-dAla-G-P-S-S-G-A-P-P- P-S-NH2 26 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-Aib-K-dAla-G-P-S-S-G-A-P- P-P-S-NH2 27 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-E-E- E-A-A-R-L-F-I-E-W-L-Aib-K-dAla-G-P-S-S-G-A-P- P-P-S-NH2 28 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-L-D-E-E-A-A-R-L- F-I-E-W-L-Aib-A-G-G-P-S-S-G-A-P-P-P-S-NH2 29 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S- NH2 30 H-dSer-H-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-E-E- E-A-A-R-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S- NH2 31 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-E-E- E-A-A-R-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S- NH2 32 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E- E-A-A-R-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S-   NH2 33 H-S-Q-G-T-F-T-S-D-L-S-K-Q-K(γE-γE-x53)-D-E-E- A-A-R-L-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S- NH2 34 H-dSer-Q-G-T-F-T-S-D-L-S-K-Q-L-D-E-E-A-A-R-L- F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S-NH2