Compounds as peptidic trigonal GLP1/glucagon/GIP receptor agonists

Abstract

The present invention relates to trigonal GLP-1/glucagon/GIP 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 compound of the formula I:
H.sub.2N-His-Aib-His-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Leu-X14-Glu-Glu-Gln-Arg-Gln-X20-Glu-Phe-Ile-Glu-Trp-Leu-Lys-Ala-X29-Gly-X31-Pro-Ser-Aib-Lys-Pro-Pro-Pro-Lys-R.sup.1I wherein: 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 ZC(O)R.sup.5, wherein Z is a linker in all stereoisomeric forms and R.sup.5 is a moiety comprising up to 50 carbon atoms and heteroatoms selected from N and O, X20 is an amino acid residue selected from Aib and Lys, X29 is an amino acid residue selected from D-Ala and Gly, X31 is an amino acid residue selected from His and Pro, R.sup.1 is NH.sub.2 or OH, or a salt or solvate thereof.

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

3. The compound of claim 1, or the salt or solvate thereof, which has a relative activity of at least 5% compared to that of natural glucagon at the glucagon receptor.

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

5. The compound of claim 1, or the salt or solvate thereof, which exhibits a relative activity of at least 4% compared to that of GIP at the GIP receptor.

6. The compound of claim 1, wherein X14 is Lys, wherein the NH.sub.2 side chain group is functionalized with the group ZC(O)R.sup.5, wherein: Z is a group selected from gGlu, gGlu-gGlu, gGlu-AEEAc-gAAA-, gGlu-gGlu-AEEAc, AEEAc-AEEAc-gGlu and AEEAc-AEEAc-AEEAc; and R.sup.5 is a group selected from pentadecanyl and heptadecanyl; or a salt or solvate thereof.

7. The compound of claim 1, wherein X14 is Lys, wherein the NH.sub.2 side chain group is functionalized with a group ZC(O)R.sup.5, wherein: Z is a group selected from gGlu, gGlu-gGlu, gGlu-AEEAc-gAAA- and gGlu-gGlu-AEEAc; and R.sup.5 is a group selected from pentadecanyl and heptadecanyl: or a salt or solvate thereof.

8. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by (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-[(4 S)-4-Carboxy-4-hexadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl, (2-{2-[2-(2-{2-[(4 S)-4-Carboxy-4-octadecanoyl amino-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-; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

9. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (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, and (2-{2-[2-(2-{2-[(4S)-4-Carboxy-4-octadecanoylamino-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

10. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-; X20 is selected from Lys and Aib; X29 is an amino acid residue selected from D-Ala and Gly; X31 is an amino acid residue selected from His and Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

11. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl- and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X20 is Lys; X29 is an amino acid residue selected from D-Ala and Gly; X31 is an amino acid residue selected from His and Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

12. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl- and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X20 is Lys; X29 is an amino acid residue selected from D-Ala and Gly; X31 is His; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

13. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl- and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X20 is Lys; X29 is Gly; X31 is an amino acid residue selected from His and Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

14. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl- and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X20 is Aib; X29 is an amino acid residue selected from D-Ala and Gly; X31 is an amino acid residue selected from His and Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

15. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by a group selected from (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl- and (S)-4-Carboxy-4-octadecanoylamino-butyryl-; X20 is Aib; X29 is an amino acid residue selected from D-Ala and Gly; X31 is Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

16. The compound of claim 1, wherein: X14 is Lys, wherein the NH.sub.2 side chain group is functionalized by (S)-4-Carboxy-4-((S)-4-carboxy-4-hexadecanoylamino-butyrylamino)-butyryl-; X20 is Aib; X29 is D-Ala; X31 is an amino acid residue selected from His and Pro; and R.sup.1 is NH.sub.2; or a salt or solvate thereof.

17. The compound of claim 1, selected from the compounds of SEQ ID NOs: 6-27, or salts or solvates thereof.

18. The compound of claim 1, wherein the compound is the compound of SEQ ID NO: 6, or a salt or solvate thereof.

19. The compound of claim 1, wherein the compound is the compound of SEQ ID NO: 9, or a salt or solvate thereof.

20. The compound of claim 1, wherein the compound is the compound of SEQ ID NO: 11, or a salt or solvate thereof.

21. A pharmaceutical composition comprising the compound of claim 1, or a salt or solvate thereof.

22. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt or solvate thereof, which is present as an active agent together with at least one pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 21, further comprising at least one additional therapeutically active agent.

24. The pharmaceutical composition of claim 23, wherein the at least one additional therapeutically active agent is selected from the group consisting of: insulin and insulin derivatives; GLP-1, GLP-1 analogues and GLP-1 receptor agonists; DPP-4 inhibitors; SGLT2 inhibitors; dual SGLT2/SGLT1 inhibitors; biguanides, thiazolidinediones, dual PPAR agonists, sulfonylureas, meglitinides, alpha-glucosidase inhibitors, amylin and amylin analogues; GPR119 agonists, GPR40 agonists, GPR120 agonists, GPR142 agonists, systemic or low-absorbable TGR5 agonists; bromocriptine mesylate, inhibitors of 11-beta-HSD, activators of glucokinase, inhibitors of DGAT, inhibitors of protein tyrosine-phosphatase 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 dehydrokinase, alpha 2-antagonists, CCR-2 antagonists, SGLT-1 inhibitors, modulators of glucose transporter-4, somatostatin receptor 3 agonists; lipid lowering agents; active substances for the treatment of obesity; gastrointestinal peptides; lipase inhibitors, angiogenesis inhibitors, H3 antagonists, AgRP inhibitors, triple monoamine uptake inhibitors, MetAP2 inhibitors, nasal formulation of the calcium channel blocker diltiazem, antisense molecules against production of fibroblast growth factor receptor 4, prohibitin targeting peptide-1; and drugs for influencing high blood pressure, chronic heart failure or atherosclerosis.

25. A method for the treatment of a disease or disorder in a patient, the method comprising administering to the patient an effective amount of the compound of claim 1, or a salt or solvate thereof, wherein the disease or disorder is glucose intolerance, insulin resistance, pre-diabetes, increased fasting glucose, hyperglycemia, type 2 diabetes, hypertension, dyslipidemia, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or any combination of these individual disease components.

26. The method of claim 25, wherein the disease or disorder is control of appetite, feeding and calorie intake, increase of energy expenditure, prevention of weight gain, promotion of weight loss, reduction of excess body weight, obesity, or morbid obesity.

27. The method of claim 25, wherein the disease or disorder is hepatosteatosis.

28. The method of claim 25, wherein the disease or disorder is selected from hyperglycemia, type 2 diabetes, obesity, and combinations thereof.

29. The method of claim 25, wherein the disease or disorder is both type 2 diabetes and obesity.

30. The method of claim 25, wherein the disease or disorder is diabetes.

31. The method of claim 25, wherein the disease or disorder is obesity.

32. The method of claim 25, wherein the disease or disorder is atherosclerosis.

33. A method for reducing the intestinal passage, increasing the gastric content and/or reducing the food intake of a patient, the method comprising administering to the patient an effective amount of the compound of claim 1, or a salt or solvate thereof.

34. A method for reducing blood glucose levels and/or reducing HbA1c levels of a patient, the method comprising administering to the patient an effective amount of the compound of claim 1, or a salt or solvate thereof.

35. A method for reducing body weight of a patient, the method comprising administering to the patient an effective amount of the compound of claim 1, or a salt or solvate thereof.

36. A method for the treatment of a disease or disorder in a patient, the method comprising administering to the patient the pharmaceutical composition of claim 23, wherein the compound, or the salt or solvate thereof, and the additional active ingredient are administered to the patient simultaneously, wherein the disease or disorder is glucose intolerance, insulin resistance, pre-diabetes, increased fasting glucose, hyperglycemia, type 2 diabetes, hypertension, dyslipidemia, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or any combination of these individual disease components.

37. A method for the treatment of a disease or disorder in a patient, the method comprising administering to the patient the pharmaceutical composition of claim 23, wherein the compound, or the salt or solvate thereof, and the additional active ingredient are administered to the patient sequentially, wherein the disease or disorder is glucose intolerance, insulin resistance, pre-diabetes, increased fasting glucose, hyperglycemia, type 2 diabetes, hypertension, dyslipidemia, arteriosclerosis, coronary heart disease, peripheral artery disease, stroke, or any combination of these individual disease components.

38. A method for the treatment of non-alcoholic liver-disease (NAFLD) or non-alcoholic steatohepatitis (NASH), the method comprising administering to the patient an effective amount of the compound of claim 1, or a salt or solvate thereof.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1: Comparison of Dynamic Debye plots for peptide formulations exhibiting reversible self-association (attractive interactions) and repulsive virial interactions

(2) FIG. 2. SEQ ID NO: 6, Blood glucose profile following first treatment in fed DIO mice

(3) FIG. 3. SEQ ID NO: 6, Body mass in DIO mice

(4) FIG. 4. SEQ ID NO: 6, Body mass change in DIO mice

(5) FIG. 5. SEQ ID NO: 6, Whole body fat mass change in DIO mice

(6) FIG. 6. SEQ ID NO: 6, Feed consumption estimate in DIO mice

(7) FIG. 7. SEQ ID NO: 6, Terminal liver mass in DIO mice

(8) FIG. 8. SEQ ID NO: 6, Terminal plasma triglycerides in fed DIO mice

(9) FIG. 9. SEQ ID NO: 6, Plasma LDL in fed DIO mice

(10) FIG. 10. SEQ ID NO: 11, Blood glucose profile following first treatment in fed DIO mice

(11) FIG. 11. SEQ ID NO: 11, Body mass in DIO mice

(12) FIG. 12. SEQ ID NO: 11, Whole body mass change in DIO mice

(13) FIG. 13. SEQ ID NO: 11, Whole body fat mass change in DIO mice

(14) FIG. 14. SEQ ID NO: 11, Feed consumption estimate in DIO mice

(15) FIG. 15. SEQ ID NO: 11, Terminal liver mass in DIO mice

(16) FIG. 16. SEQ ID NO: 11, Terminal plasma triglycerides in fed DIO mice

(17) FIG. 17. SEQ ID NO: 11, Plasma LDL in fed DIO mice

(18) FIG. 18. SEQ ID NO: 11, Terminal plasma insulin in fed DIO mice

(19) FIG. 19. SEQ ID NO: 11, Terminal plasma glucose in fed DIO mice

(20) FIG. 20. SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, Blood glucose profile in fed db/db mice

(21) FIG. 21. SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, Blood Glucose Area Under the Curve in fed db/db mice

(22) FIG. 22. SEQ ID NO: 7, Blood glucose profile in fed db/db mice

(23) FIG. 23. SEQ ID NO: 7, Blood glucose Area Under the Curve in fed db/db mice

(24) FIG. 24. SEQ ID NO: 11, Blood glucose profile in fed db/db mice

(25) FIG. 25. SEQ ID NO: 11, Blood Glucose Area Under the Curve in fed db/db mice

(26) FIG. 26. SEQ ID NO: 11, Terminal serum triacylglycerol concentration in fed db/db mice

(27) FIG. 27. SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 26, Blood glucose profile in fed db/db mice

(28) FIG. 28. SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 26. Blood Glucose Area Under the Curve in fed db/db mice

(29) FIG. 29. Food intake and body weight monitoring data (SEQ ID NO: 11, SEQ ID NO: 6, Liraglutide)

(30) FIG. 30. Aortic plaque area data (SEQ ID NO: 11, SEQ ID NO: 6, Liraglutide)

(31) FIG. 31. Serum total cholesterol and LDL-cholesterol data (SEQ ID NO: 11, SEQ ID NO: 6, Liraglutide)

METHODS

(32) Abbreviations employed are as follows:

(33) AA amino acid

(34) AEEAc (2-(2-aminoethoxy)ethoxy)acetyl

(35) Aib alpha-amino-isobutyric acid

(36) cAMP cyclic adenosine monophosphate

(37) Boc tert-butyloxycarbonyl

(38) BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

(39) BSA bovine serum albumin

(40) tBu tertiary butyl

(41) dAla D-alanine

(42) DCM dichloromethane

(43) Dde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl

(44) ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl

(45) DIC N,N-diisopropylcarbodiimide

(46) DIPEA N,N-diisopropylethylamine

(47) DMEM Dulbecco's modified Eagle's medium

(48) DMF dimethyl formamide

(49) DMS dimethylsulfide

(50) EDT ethanedithiol

(51) FA formic acid

(52) FBS fetal bovine serum

(53) Fmoc fluorenylmethyloxycarbonyl

(54) gAAA gamma-amino adipic acid

(55) gGlu gamma-glutamate (yE)

(56) HATU O-(7-azabenzotriazol-1-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate

(57) HBSS Hanks' Balanced Salt Solution

(58) HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate

(59) HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

(60) HOBt 1-hydroxybenzotriazole

(61) HOSu N-hydroxysuccinimide

(62) HPLC High Performance Liquid Chromatography

(63) HTRF Homogenous Time Resolved Fluorescence

(64) IBMX 3-isobutyl-1-methylxanthine

(65) LC/MS Liquid Chromatography/Mass Spectrometry

(66) Mmt monomethoxy-trityl

(67) Palm palmitoyl

(68) PBS phosphate buffered saline

(69) PEG polyethylene glycole

(70) PK pharmacokinetic

(71) RP-HPLC reversed-phase high performance liquid chromatography

(72) Stea stearyl

(73) TFA trifluoroacetic acid

(74) Trt trityl

(75) UV ultraviolet

(76) General Synthesis of Peptidic Compounds

(77) Materials

(78) 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.

(79) Fmoc protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, Iris 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.

(80) 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.

(81) Furthermore, the building blocks (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid and Boc-L-His(Trt)-Aib-OH can be applied. Both building blocks were synthesized separately.

(82) 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 22.5 min. Washes: 7DMF. Coupling 2:5:10 200 mM AA/500 mM HBTU/2M DIPEA in DMF 2 for 20 min. Washes: 5DMF.

(83) 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., 1998, 39, 1603), using 4% hydrazine hydrate in DMF. The Mmt group was removed by repeated treatment with AcOH/TFE/DCM (1/2/7) for 15 minutes at RT, the resin then repeatedly washed with DCM, 5% DIPEA in DCM and 5% DIPEA in DCM/DMF.

(84) 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.

(85) 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.

(86) 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 swollen 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 of the next Fmoc-amino acid was initiated with 5 equivalent excess of DIC and HOBT 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). 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.

(87) 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.

(88) After the completion of the linear sequence, the -amino group of lysine used as branching point or modification point was deprotected by using 2.5% hydrazine hydrate in DMF for 15 min2 and washed with DMF/DCM/DMF (6:6:6 time each). The -carboxyl end of glutamic acid was attached to the -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 (630 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.

(89) If the side-chain branching also contains one more -glutamic acid, a second Fmoc-Glu(OH)-OtBu used for the attachment to the free amino group of -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 (630 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). 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.

(90) Palmitic acid and stearic acid attachment to side chains of glutamic acid:

(91) To the free amino group of -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).

(92) Final cleavage of peptide from the resin:

(93) The peptidyl resin synthesized by manual synthesis was washed with DCM (610 ml), MeOH (610 ml) and ether (610 ml) and dried in vacuum desiccators overnight.

(94) 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 (25 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.

(95) In addition, a different route for the introduction of the lysine side chain is used, applying a prefunctionalized building block where the side chain is already attached to the lysine (e.g. (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid) as coupling partner in the peptide synthesis. 0.67 mmol of peptide resin bearing an amino-group is washed with 20 ml of dimethylformamide. 2.93 g of (2S)-6-[[(4S)-5-tert-butoxy-4-[[(4S)-5-tert-butoxy-4-(hexadecanoylamino)-5-oxo-pentanoyl]amino]-5-oxo-pentanoyl]amino]-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid is dissolved in 20 ml of dimethylformamide together with 310 mg of hydroxybenzotriazol hydrate and 0.32 ml of diisopropylcarbodiimide. After stirring of 5 minutes the solution is added to the resin. The resin is agitated for 20 h and then washed 3 times with 20 ml of dimethylformamide each. A small resin sample is taken and subjected to the Kaiser-test and the Chloranil-test (E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem. 1970, 34, 595-598; Chloranil-Test: T. Vojkovsky, Peptide Research 1995, 8, 236-237). This procedure avoids the need of a selective deprotection step as well as the selective attachment of the side chain building blocks on a very advanced synthesis intermediate.

(96) Analytical HPLC/UPLC

(97) Method A: Detection at 210-225 nm column: Waters ACQUITY UPLC CSH C18 1.7 m (1502.1 mm) at 50 C. solvent: H.sub.2O+0.05% TFA: ACN+0.035% 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 analyzer: LCT Premier, electrospray positive ion mode

(98) Method B: Detection at 214 column: Waters ACQUITY UPLC CSH C18 1.7 m (1502.1 mm) at 50 C. solvent: H.sub.2O+0.05% TFA: ACN+0.035% 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 analyzer: Agilent 6230 Accurate-Mass TOF, Dual Agilent Jet Stream ESI

(99) Method C: Detection at 214 nm column: Waters ACQUITY UPLC CSH C18 1.7 m (1502.1 mm) at 50 C. solvent: H.sub.2O+0.1% TFA: ACN+0.1% 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 (38 min) optionally with mass analyzer: Agilent 6230 Accurate-Mass TOF, Agilent Jet Stream ESI

(100) Method D: Detection at 220 nm

(101) column: Waters ACQUITY BEH C18 (2.1100 mm1.7 m), Temp: 40 C. Aries peptide XB C18 (4.6250 mm3.6 m), Temp: 40 C. solvent: H.sub.2O+0.1% formic acid (buffer A): ACN+0.1% b formic acid (flow 1 ml/min) (buffer B) gradient: Equilibration of the column with 2% buffer B and elution by a gradient of 2% to 70% buffer B during 15 min??

(102) Method E: Detection at 215 nm column: Waters ACQUITY UPLC CSH C18 1.7 m (1502.1 mm) at 50 C. solvent: H.sub.2O+0.05% TFA: ACN+0.035% TFA (flow 0.5 ml/min) gradient: 80:20 (0 min) to 80:20 (3 min) to 25:75 (23 min) to 5:95 (23.5 min) to 5:95 (25.5 min) to 80:20 (26 min) to 80:20 (30 min)

(103) General Preparative HPLC Purification Procedure

(104) 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.

(105) Solubility Assessment of Exendin-4 Derivatives

(106) Prior to the solubility measurement of a peptide batch, its purity was determined through UPLC/MS.

(107) For solubility testing the target concentration was 10 mg pure compound/ml. Therefore solutions from solid samples were prepared in a buffer system with a concentration of 10 mg/mL compound based on the previously determined % purity:

(108) Solubility buffer system A) Acetate Buffer pH 4.5, 100 mM sodium acetate trihydrate, 2.7 mg/ml m-Cresol

(109) Solubility buffer system B) Phosphate Buffer pH 7.4, 100 mM sodium hydrogen phosphate, 2.7 mg/ml m-Cresol

(110) Solubility buffer system C) Citrate Buffer pH 6.0, citric acid 100 mM, 2.7 mg/mL m-cresol

(111) UPLC-UV was performed after 1 hour of gentle agitation from the supernatant, which was obtained after 15 min of centrifugation at 2500 RCF (relative centrifugal acceleration).

(112) The solubility was determined by the comparison of the UV peak area of 2 L-injection of a buffered sample diluted 1:10 with a standard curve of a reference peptide with known concentration. The different UV extinction coefficients of sample and reference peptide were calculated based on the different amino acid sequences and considered in the concentration calculation.

(113) Chemical Stability Assessment of Exendin-4 Derivatives

(114) Prior to the chemical stability measurement of a peptide batch, its purity was determined through UPLC/MS. For stability testing the target concentration was 1 mg pure compound/ml. Therefore solutions from solid samples were prepared in a buffer system with a concentration of 1 mg/mL compound based on the previously determined % purity:

(115) Chemical stability buffer system A) 25 mM acetate buffer pH 4.5, 3 mg/mL L-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85% Chemical stability buffer system B) 25 mM phosphate buffer pH 6.0, 3 mg/mL L-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

(116) Peptide solutions were filtered through 0.22 M pore size and filled into aliquots under aseptic conditions. At starting point, UPLC-UV was performed by injection of 2 l of the undiluted sample.

(117) For chemical stability testing, aliquots were stored for 28 days at 5 and 40 C. After this time course the samples were centrifuged for 15 min at 2500 RCF. Then 2 l of the undiluted supernatant were analysed with UPLC-UV.

(118) The chemical stability was rated through the relative loss of purity calculated by the equation:
[(purity at starting point)(purity after 28 days at X C.)]/(purity at starting point)]*100%

(119) X=5 or 40 C.

(120) The purity is calculated as

(121) [(peak area peptide)/(total peak area)]*100%

(122) Dynamic Light Scattering (DLS) for the Assessment of Physical Stability

(123) A monochromatic and coherent light beam (laser) is used to illuminate the liquid sample. Dynamic Light Scattering (DLS) measures light scattered from particles (1 nmradius1 m) that undergo Brownian motion. This motion is induced by collisions between the particles and solvent molecules that themselves are moving due to their thermal energy. The diffusional motion of the particles results in temporal fluctuations of the scattered light [Pecora, R. Dynamic Light Scattering: Applications of Photon Correlation Spectroscopy, Plenum Press, 1985].

(124) The scattered light intensity fluctuations are recorded and transformed into an autocorrelation function. By fitting the autocorrelation curve to an exponential function, the diffusion coefficient D of the particles in solution can be derived. The diffusion coefficient is then used to calculate the hydrodynamic radius R.sub.h (or apparent Stokes radius) through the Stokes-Einstein equation assuming spherical particles. This calculation is defined in ISO 13321 and ISO 22412 [International Standard ISO13321 Methods for Determination of Particle Size Distribution Part 8: Photon Correlation Spectroscopy, International Organisation for Standardisation (ISO) 1996; International Standard ISO22412 Particle Size AnalysisDynamic Light Scattering, International Organisation for Standardisation (ISO) 2008].

(125) In case of polydisperse samples, the autocorrelation function is the sum of the exponential decays corresponding to each of the species. The temporal fluctuations of the scattered light can then be used to determine the size distribution profile of the particle fraction or family. The first order result is an intensity distribution of scattered light as a function of the particle size. The intensity distribution is naturally weighted according to the scattering intensity of each particle fraction or family. For biological materials or polymers the particle scattering intensity is proportional to the square of the molecular weight. Thus, small amount of aggregates/agglomerates or presence or a larger particle species can dominate the intensity distribution. However this distribution can be used as a sensitive detector for the presence of large material in the sample. The intensity distribution can be converted into a volume or mass distribution of the particle sizes using the Mie theory under certain assumptions. In contrast to the intensity distribution, the mass distribution is best used for comparative purposes and should never be considered absolute (due to the underlying assumptions).

(126) The DLS technique produces distributions with inherent peak broadening. The polydispersity index % Pd is a measure of the width of the particle size distribution and is calculated by standard methods described in ISO13321 and ISO22412 [International Standard ISO13321 Methods for Determination of Particle Size Distribution Part 8: Photon Correlation Spectroscopy, International Organisation for Standardisation (ISO) 1996; International Standard ISO22412 Particle Size AnalysisDynamic Light Scattering, International Organisation for Standardisation (ISO) 2008].

(127) DLS Interaction Parameter (k.sub.D)

(128) The DLS interaction parameter (k.sub.D) is a measure to describe inter-particle interactions, where the particles are folded proteins or peptides [Sandeep Yadav et al. (2009) J Pharm Sc, Vol 99(3), pp 1152-1168; Brian D. Connolly et al. (2012) Biophysical Journal Volume 103, pp 69-78].

(129) The parameter k.sub.D is derived from the concentration dependence of the diffusion coefficient D, which is given by an expansion in powers of the concentration c:
D(c)=D.sub.0(1+k.sub.Dc+k.sub.iDc.sup.2+k.sub.jDc.sup.3+ . . . )

(130) Neglecting the higher order terms, i.e. k.sub.iD=k.sub.jD= . . . =0, the data can be fitted linearly and k.sub.D is obtained from the slope of the curve D=D.sub.0(1+k.sub.Dc) and D.sub.0. D.sub.0 is the diffusion coefficient at zero concentration. The parameter k.sub.D can be used to describe interaction of protein or peptide molecules or oligomers with their-self and their environment in solution and is theoretically related to the virial coefficient B.sub.22 as for example described by Harding and Johnson, where M is the molar mass, k.sub.s the first order concentration coefficient of sedimentation velocity and u the partial specific volume [Harding S E, Johnson P. (1985) Biochem J, 231, pp 543-547].
k.sub.D=2B.sub.22Mk.sub.su

(131) Positive B.sub.22 values indicate samples that favor salvation over self-association, while negative B.sub.22 values indicate samples that prefer self-association. From a pragmatic point of view, k.sub.D is analogous in its meaning to B.sub.22 and gives information on the net-forces between the molecules. High values indicate strong net-repulsive interactions, while low values indicate net-attractive forces. Therefore, k.sub.D can be used for relative, qualitative comparison (see FIG. 1).

(132) For every peptide solution, the hydrodynamic radius R.sub.h and the diffusion constant D (related via the Stokes-Einstein equation) were determined as an average over triplicates. Both parameters were determined at different peptide concentrations (e.g., R.sub.h1 and D.sub.1: 1 mg/ml and R.sub.h5 and D.sub.5: 5 mg/ml) in the same buffer system. The difference of these parameters between low and high peptide concentration is a surrogate for the DLS interaction parameter k.sub.D. R.sub.h5<R.sub.h1 or D.sub.5>D.sub.1 correspond to k.sub.D>0 and therefore to repulsive inter-particle interactions that result in improved physical (or colloidal) stability.

(133) DLS buffer system A) 25 mM acetate buffer pH 4.5, 3 mg/mL L-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

(134) DLS buffer system B) 25 mM phosphate buffer pH 6.0, 3 mg/mL L-Methionine, 2.7 mg/mL m-cresol, 18 mg/mL Glycerol 85%

(135) DLS Method A: DLS measurements were performed on a W130i apparatus (Avid Nano Ltd, High Wycombe, UK) and using a low-volume disposable cuvette (UVette, Eppendorf AG, Hamburg, Germany). The data were processed with i-Size 3.0 provided by Avid Nano. Parameters of the particle size distribution were determined with non-negatively constrained least squares (NNLS) methods using DynaLS algorithms. Measurements were taken at 25 C. with a 660 nm laser light source and at an angle of 90.

(136) DLS Method B: DLS measurements were performed on a Nanosizer ZS (Malvern Instruments, Malvern, UK) and using disposible UV cuvettes (Brand macro, 2.5 mL and Brand semi-micro 1.5 mL, Brand GmbH+Co KG, Wertheim, Germany). The data were processed with Malvern Zetasizer software Version 7.10 or 7.01. Parameters of the particle size distribution were determined with non-negatively constrained least squares (NNLS) methods. Measurements were taken at 25 C. with a 633 nm laser light source in NIBS (Non-Invasive Back-Scatter) mode at an angle of 173.

(137) DLS Method C: DLS measurements were performed on a DynaPro Plate Reader II (Wyatt Technology, Santa Barbara, Calif., US) and using one of the following black, low volume, and non-treated plates: polystyrene 384 assay plate with clear bottom (Corning, N.Y., US), polystyrene 96 assay plate with clear bottom (Corning, N.Y., US), cyclo olefin polymer (COP) 384 assay plate with clear bottom (Aurora, Mont., US), or polystyrene 384 assay plate with clear bottom (Greiner Bio-One, Germany). The data were processed with the Dynamics software provided by Wyatt Technology. Parameters of the particle size distribution were determined with non-negatively constrained least squares (NNLS) methods using DynaLS algorithms. Measurements were taken at 25 C. with an 830 nm laser light source at an angle of 158.

(138) ThT Assay for the Assessment of Physical Stability

(139) Low physical stability of a peptide solution may lead to amyloid fibril formation, which is observed as well-ordered, thread-like macromolecular structures in the sample, which eventually may lead to gel formation. Thioflavin T (ThT) is widely used to visualize and quantify the presence of misfolded protein aggregates. [Biancalana et al. (2010) Biochimica et Biophysica Acta. 1804 (7): 1405-1412]. When it binds to fibrils, such as those in amyloid aggregates, the dye displays a distinct fluorescence signature [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284]. The time course for fibril formation often follows the characteristic shape of a sigmoidal curve and can be separated into three regions: a lag phase, a fast growth phase, and a plateau phase.

(140) The typical fibril formation process starts with the lag phase in which the amount of partially folded peptide turned into fibrils is not significant enough to be detected. The lag-time corresponds to the time the critical mass of the nucleus is built. Afterwards, a drastic elongation phase follows and fibril concentration increases rapidly.

(141) Investigations were carried out to determine fibrillation tendencies under stress conditions by shaking at 37 C. within Fluoroskan Ascent FL.

(142) For the tests in Fluoroskan Ascent FL, 200 L sample were placed into a 96 well mictrotiter plate PS, flat bottom, Greiner Fluotrac No. 655076. Plates were sealed with Scotch Tape (Quiagen). Samples were stressed by continuous cycles of 10 s shaking at 960 rpm and 50 s rest period at 37 C. The kinetic was monitored by measuring fluorescence intensity every 20 minutes.

(143) Peptides were diluted in a buffer system to a final concentration of 3 mg/ml. 20 L of a 10.1 mM ThT solution in H2O were added to 2 mL of peptide solution to receive a final concentration of 100 M ThT. For each sample eight replicates were tested.

(144) Tht buffer system A) 100 mM Acetate pH 4.5 including m-cresol (100 mM Natriumacetat trihydrat, pH adjustment using 2N CH3COOH, 2.7 mg/mL m-cresol) Tht buffer system B) 100 mM citrate buffer pH 6.0

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

(146) 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.

(147) cAMP content of cells was determined using a kit from Cisbio Corp. (cat. no. 62AM4PEJ) 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 (1HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and cellular density determined. They were then diluted to 400000 cells/ml and 25 l-aliquots dispensed into the wells of 96-well plates. For measurement, 25 l 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 665/616 nm. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC50).

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

(149) Mice were dosed 1 mg/kg subcutaneously (s.c.). The mice were sacrified 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).

(150) Female Gttinger minipigs were dosed 0.05 mg/kg, 0.075 mg/kg or 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).

(151) Acute and Chronic Effects After Subcutaneous Treatment on Blood Glucose, Body Mass, Whole Body Fat Content, and Feed Consumption in Female Diet-Induced Obese (DIO) C57BL/6 Mice

(152) Female C57BL/6NHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed with shipped cage mates in shoebox caging with wood chip bedding until day 38 of the predose phase. At the study start mice were between 25-26 weeks old.

(153) Mice were housed under vivarium conditions that included a 12 h light/dark cycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26 C. and a relative humidity between 30-70%. All animals had free access to water and a high fat diet (TD97366) for 16 weeks prior to pharmacological intervention (dosing phase). Feed was replaced with fresh feed weekly until and for the last time on day 38 of the predose phase. During the subsequent dosing phase, approximately 50% of the remaining feed were removed, replaced with fresh feed, and pellets were mixed evenly once per week.

(154) On predose day 38, obese DIO mice were assigned to treatment groups (n=8) to match mean body masses between all DIO groups. An age-matched group with ad libitum access to a rodent maintenance diet (Teklad Global Diets Rodent 2014, pelleted) was included in the study as a lean control group. In the predosing phase from day 32 through 38, all study animals were treated with vehicle (Phosphate Buffered Saline, PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) once daily (s.c. approximately 0.2 mL/mouse).

(155) On day 37 of the predose phase, the test article was diluted with PBS to a concentration of 100 g/mL and aliquots of this stock solution were stored at approximately 60 C. Stock aliquots were thawed for weekly use and thereafter stored in a refrigerator at approximately 4 C. The injected test article solution was prepared fresh once on each dosing day by diluting stock solution with PBS to achieve the desired concentration.

(156) Mice were treated twice daily with a s.c. injection of PBS-vehicle or the test article for 28 days. The morning dosing was initiated and completed between 06:00 and 07:30 AM and the afternoon dosing between 2:00 and 3:30 PM. On day 28 of the dosing phase only the morning dose was administered. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(157) 1) Acute effect on blood glucose profiles in non-fasting, female DIO mice:

(158) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at hour 0 prior to the first s.c. dose of PBS-vehicle or test article and 1, 2, 3, 4, 6, and 24 hours post-dose in non-anesthetized animals. The 24 hour blood collection was performed prior to dosing on day 2. Between the 6 and 24 hour blood sample the afternoon dose was administered. Glucose measurements were performed in whole blood and in duplicate or triplicate using Aviva glucometers.

(159) 2) Chronic effect on body mass in non-fasting, female DIO mice:

(160) Body mass was measured daily approximately between 06:00-07:30 AM from day 32 through 38 of the predosing phase and throughout the 28 days of the dosing phase. During the dosing phase mice were treated twice daily with either a s.c. injection of PBS-vehicle or test article.

(161) 3) Chronic effect on whole body fat mass in non-fasting, female DIO mice:

(162) To determine whole body fat mass Quantitative Nuclear Magnetic Resonance (QNMR) measurements were performed on predosing day 37 and on day 26 of the dosing phase. During the dosing phase mice were treated twice daily with either a s.c. injection of PBS-vehicle or test article.

(163) 4) Effect on feed consumption in female DIO mice:

(164) Feed consumption was based on the daily assessment of the feeder weights of each cage between 06:00-07:30 AM. Each cage housed four mice and feed consumption was calculated throughout the 28 days of the dosing phase. During the dosing phase mice were treated twice daily with either a s.c. injection of PBS-vehicle or test article.

(165) 5) Terminal plasma parameters in non-fasting, female DIO mice:

(166) On day 28 blood was collected prior to any other inlife activities for determination of plasma insulin concentrations. Then the morning dose was administered and necropsy was performed 4 hours post-dose. For this purpose animals were anesthetized with isoflurane and blood was collected by orbital bleeding.

(167) 5) Terminal liver mass in non-fasting, female DIO mice:

(168) On day 28 and 4 hours after the morning dose, blood was collected from mice under isoflurane anesthesia as described above. Then mice were killed and livers were collected and weighed.

(169) 6) Quantification of liver lipids:

(170) Liver aliquots were incubated with dichlormethane:methanol (2:1). The lipohilic and lipohobic phase were separated by adding dH.sub.2O and subsequent centrifugation. The bottom lipophilic phase was collected and the procedure repeated with the remaining lipophobic layer and liver tissue. Next the lipophilic phases were combined and the solvent evaporated. At 60 C. and continuous shaking samples were then incubated with 2-propanol. Total cholesterol, triacylglycerol and phospholipid concentrations were quantified enzymatically with a commercial kit according to the manufacturer's instructions.

(171) 7) Statistical analyses:

(172) Statistical analyses were performed with Sigmaplot 12.5. Two-tailed T-Tests were used to compare groups of DIO-vehicle mice (in general n=8) with DIO-test article treated mice (in general n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Lean-Vehicle group data are depicted in the Figures but were used as a reference dataset for the non-obese state.

(173) Acute Effects After Subcutaneous Treatment on Blood Glucose Concentrations in Non-Fasting, Female, Diabetic db/db Mice

(174) Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obese BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed in shoebox caging with wood chip bedding until day 15 of the predose phase. At the study start mice were approximately 12 weeks old.

(175) Mice were housed under vivarium conditions including a 12 h light/dark cycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26 C. and a relative humidity between 30-70%. All animals had free access to water and Purina Fomulab Diet 5008.

(176) On predose day 9 blood glucose and body mass (approximately between 08:00-10:00 AM) as well as HbA1c measurements were performed. On day 15 of the predose phase animals were assigned to treatment groups (n=8) and to new cages to match mean HbA1c and body masses between all db/db groups. An age-matched lean group was included in the study as a healthy, lean reference.

(177) Prior to day 1 of the dosing phase, the test article was diluted with Phosphate Buffered Saline (PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) to a concentration of 1 mg/mL and aliquots of this stock solution were stored at approximately 60 C. On day 1 of the dosing phase the stock aliquot was thawed and the injected test article solution was prepared fresh by diluting it with PBS to achieve the desired concentration.

(178) On Day 1 of the dosing phase db/db mice were either treated once with a s.c. injection of PBS-vehicle or 30 g/kg test article. The lean reference group was treated once with a s.c. injection of PBS-vehicle. The dosing was initiated and completed between 08:00 and 10:00 AM. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(179) 1) Acute effect on blood glucose profiles in non-fasting animals:

(180) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at minute 30 and 0 prior to any other inlife activities. At minute 0 mice received a s.c. dose of either PBS-vehicle or 30 g/kg test article. Further blood samples were harvested at hour 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements were performed in whole blood using AlphaTRAK glucometers. If the glucose concentrations of two measurements differed by more than 20 mg/dL a third value was recorded. The Area Under the Curve (AUC) for blood glucose was calculated via the trapezoid method and for the duration of the 24 post-dose hours.

(181) 2) Statistical analyses:

(182) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. A One Way Analysis of Variance and multiple comparisons (Dunnett's Method) were performed comparing the group of diabetic, obese db/db vehicle mice (n=8) with each diabetic, obese db/db test article treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Non-diabetic, lean-vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese, non-diabetic state.

EXAMPLES

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

Example 1

(184) Synthesis of SEQ ID NO: 6

(185) 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 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the methods. Hereafter Palm-gGlu-gGlu-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. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (RP18 XSelectCSH-5 m 50150 mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B).

(186) Deconvolution of the mass signals found under the peak with retention time 8.737 min revealed the peptide mass 4932.68 which is in line with the expected value of 4932.67.

Example 2

(187) Synthesis of SEQ ID NO: 11

(188) 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 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu-gGlu-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. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 ODB 5 m 30250 mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B).

(189) Deconvolution of the mass signals found under the peak with retention time 9.995 min revealed the peptide mass 4863.67 which is in line with the expected value of 4863.63.

Example 3

(190) Synthesis of SEQ ID NO: 20

(191) 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 0.43 mmol/g. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 14 Fmoc-Lys(Mmt)-OH and in position 1 Boc-His(Trt)-OH were used in the solid phase synthesis protocol. The Mmt-group was cleaved from the peptide on resin as described in the Methods. Hereafter Palm-gGlu(OSu)-OtBu (CAS 204521-63-1) 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. 1990, 36, 255-266). The crude product was purified via preparative HPLC on a Waters column (Sunfire Prep C18 ODB 5 m 30250 mm) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B).

(192) Deconvolution of the mass signals found under the peak with retention time 8.837 min revealed the peptide mass 4763.670 which is in line with the expected value of 4763.617.

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

(194) TABLE-US-00006 TABLE 3 list of synthesized peptides and comparison of calculated vs. found molecular weight Monoisotopic SEQ ID calc. or average Retention NO Mass found mass mass time (min) 6 4932.7 4932.7 monoisotopic 8.737 7 4946.7 4946.7 monoisotopic 8.699 8 4892.7 4892.7 monoisotopic 9.376 9 4906.7 4906.7 monoisotopic 9.505 10 4849.6 4849.7 monoisotopic 9.758 11 4863.6 4863.7 monoisotopic 9.995 12 4889.6 4889.7 monoisotopic 8.971 13 4903.6 4903.6 monoisotopic 9.184 14 5077.7 5077.8 monoisotopic 8.599 15 5091.8 5091.8 monoisotopic 8.839 16 4734.6 4734.7 monoisotopic 9.589 17 4762.6 4762.7 monoisotopic 10.278 18 4720.6 4720.6 monoisotopic 9.992 19 4748.6 4748.3 monoisotopic 10.822 20 4763.6 4763.7 monoisotopic 8.837 21 4791.6 4791.7 monoisotopic 9.674 22 5094.8 5094.8 average n.a. 23 5108.9 5107.2 average n.a. 24 5011.8 n.a. n.a. n.a. 25 5025.8 n.a. n.a. n.a. 26 4997.8 n.a. n.a. n.a. 27 5011.8 n.a. n.a. n.a.

Example 4

Stability

(195) Peptide samples were prepared in Chemical stability buffer system A and the stability was assessed as described in Methods. The results are given in Table 4.

(196) TABLE-US-00007 TABLE 4 stability Chemical stability [relative purity loss SEQ ID 28 days 40 C.] NO (%) at pH 4.5 6 7.1 7 8.1 8 10.3 9 6.9 10 10.6 11 8.8 12 11.9 13 10.5 14 8.1 15 7.2 22 9.5 23 8.6

Example 5

Solubility

(197) Peptide samples were prepared in solubility buffer system A and solubility was assessed as described in Methods. The results are given in Table 5.

(198) TABLE-US-00008 TABLE 5 Solubility SEQ ID Solubility [mg/ml] NO at pH 4.5 6 >9.7 8 9.5 9 >9.3 10 >9.8 11 >9.8

Example 6

Stability as Assessed by DLS Interaction Parameter

(199) The hydrodynamic radius R.sub.h of peptide samples was determined at different peptide concentrations (1 mg/ml and 5 mg/ml) in DLS buffer system A using DLS method C as described in Methods as surrogate for the DLS interaction parameter k.sub.D. The results are given in Table 6.

(200) TABLE-US-00009 TABLE 6 hydrodynamic radius R.sub.h at peptide concentrations of 1 mg/ml and 5 mg/ml. A decrease of R.sub.h at the higher peptide concentration indicates higher physical stability due to repulsive inter-particle interactions. R.sub.h1 [nm] R.sub.h5 [nm] Delta R.sub.h [nm] = SEQ. ID c = 1 mg/ml c = 5 mg/ml R.sub.h5 R.sub.h1 6 2.5 1.7 0.9 8 3.0 2.6 0.4 9 2.8 2.6 0.2 12 2.9 2.6 0.3 13 2.8 2.5 0.3 14 3.0 2.9 0.1 15 3.0 2.6 0.4

Example 7

Stability was Assessed in the ThT Assay

(201) Lag time in hours in Thioflavin T (ThT) assay of peptide samples was determined in ThT buffer system A as described in Methods. The results are given in Table 7.

(202) TABLE-US-00010 TABLE 7 Lag time in hours in Thioflavin T (ThT) assay SEQ ID Increase in Fl Lag time before NO at pH 4.5 increase [h] 6 NO >45 7 NO >45 8 NO >45 9 NO >45 10 NO >45 11 NO >45 12 NO >45 13 NO >45 14 NO >45 15 NO >45 16 NO >45 17 NO >45 18 NO >45 19 NO >45 20 NO >45 21 NO >45 22 NO >45 23 NO >45

Example 8

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

(203) 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-1 R) to the listed compounds at increasing concentrations and measuring the formed cAMP as described in Methods.

(204) The results are shown in Table 8:

(205) TABLE-US-00011 TABLE 8 EC50 values of exendin-4 derivatives at human GLP-1, Glucagon and GIP receptors (indicated in pM) SEQ ID EC50 NO EC50 hGLP-1R hGlucagon R EC50 hGIP R 6 1.4 2.3 2.1 7 1.7 3.2 2.6 8 1.3 2.0 1.6 9 1.6 2.9 2.6 10 2.3 1.6 1.1 11 2.9 2.1 1.5 12 1.8 2.0 1.9 13 2.8 2.1 2.7 14 0.9 3.4 1.2 15 1.1 4.5 1.8 16 7.1 4.3 3.7 17 6.4 5.3 7.3 18 5.3 2.8 2.0 19 6.1 3.1 4.0 20 4.8 4.2 1.9 21 3.4 1.9 2.6 22 1.0 5.9 1.1 23 1.4 6.8 1.5

Example 9

Comparison Testing

(206) A selection exendin-4 derivatives carrying (among others) a His in position 1, Leu in position 13, Glu in position 15, Gln in position 19, an Aib amino acid in position 34, Pro at position 32 and Lys at position 35 and 39 has been tested versus corresponding compounds having in these positions the amino acid residues of native exendin-4 or other amino acids. The reference pair compounds and the corresponding EC50 values at human GLP-1, Glucagon and GIP receptors (indicated in pM) are given in Table 9. As shown, the inventive exendin-4 derivatives show an improved activity on the GIP receptor compared to the corresponding derivatives carrying the amino acids as in native exendin-4 or other amino acids, keeping their GLP-1 receptor and glucagon receptor activity.

(207) TABLE-US-00012 TABLE 9 Comparison of exendin-4 derivatives carrying a His in position 1, Leu in position 13, Glu in position 15, Gln in position 19, an Aib amino acid in position 34, Pro at position 32 and Lys at position 35 and 39 vs. exendin-4 derivatives comprising at these positions the amino acid residues of native exendin-4 (Lys27, Ser32, Gly34, Ala35, Ser39) or other amino acids. EC50 values at human GLP-1, Glucagon and GIP receptors are indicated in pM. SEQ ID EC50 hGLP- EC50 EC50 residue NO 1R hGlucagon-R hGIP-1 differences 28 2.5 1.9 39.7 Gln3, Gln13, Asp15, Ala19, Ser32, Gly34, Ala35, Ser39 21 3.4 1.9 2.6 His3, Leu13, Glu15, Gln19, Pro32, Aib34, Lys35, Lys39 29 15.1 1.2 23.0 Tyr1, Gln3, Gln13, Asp15, Ala19, Ser28, Ser32, Gly34, Ala35, Ser39 21 3.4 1.9 2.6 His1, His3, Leu13, Glu15, Gln19, Ala28, Pro32, Aib34, Lys35, Lys39

Example 10

Acute and Chronic Effects of SEQ ID NO: 6 After Subcutaneous Treatment on Blood Glucose, Body Mass, Whole Body Fat Content, Feed Consumption, Terminal Liver Weight and Terminal Plasma Parameters in Fed, Female Diet-Induced Obese (DIO) C57BL/6 Mice

(208) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(209) 1) Blood glucose profile in morning-fed animals

(210) Animals had unlimited access to water and feed during the experiment. Blood glucose concentrations were determined on day 1 of the dosing phase at hour 0 prior to the first s.c. injection of PBS-vehicle or 30 g/kg SEQ ID NO: 6 and then 1, 2, 3, 4, 6, and 24 hours post-dose. Between the 6 and 24 hour blood sample the afternoon dose was administered.

(211) SEQ ID NO: 6 treated animals demonstrated a pronounced decrease in blood glucose concentrations over 24 hours. In contrast, no such change in blood glucose concentrations was observed in DIO control mice (FIG. 2).

(212) 2) Body mass

(213) Twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 6 induced a consistent decrease in body mass over the 28 days of the treatment phase compared to the vehicle DIO group (FIG. 3). Chronic, twice daily treatment with SEQ ID NO: 6 resulted in a statistically significantly more pronounced body mass reduction within the 28 days of the dosing phase compared to DIO animals treated with vehicle (FIG. 4, Table 10).

(214) 3) Whole Body Fat Mass

(215) Whole body fat mass measurements were performed on predosing day 37 and on day 26 of the dosing phase.

(216) In parallel to the pronounced body mass loss, twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 6 resulted in a statistically significantly more pronounced whole body fat mass reduction during the 28 days of the dosing phase compared to DIO-Vehicle animals (FIG. 5, Table 10).

(217) 4) Feed Consumption

(218) Each cage housed four mice and feed consumption was estimated throughout the 28 days of the dosing phase.

(219) After dosing start, twice-daily, chronic treatment with 30 g/kg SEQ ID NO: 6 suppressed feed intake, however mice habituated to the pharmacological effects within approximately ten days. Thereafter, the estimated feed consumption was comparable between the SEQ ID NO: 6 and DIO-Vehicle group (FIG. 6).

(220) 5) Terminal Liver Mass

(221) On day 28 mice were euthanized 4 hours after the morning dose and livers were collected.

(222) Twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 6 resulted in a statistically significant reduction in liver mass on day 28 of the dosing phase compared to DIO animals treated with vehicle (FIG. 7, Table 10).

(223) 6) Terminal plasma triglycerides and LDL

(224) On day 28 and 4 hours after the morning dose, blood was collected from anesthetized, non-fasting mice by orbital bleeding. Twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 6 resulted in a statistically significant reduction in non-fasting plasma triglycerides (FIG. 8, Table 10) and plasma LDL concentrations (FIG. 9, Table 10) compared to DIO animals treated with vehicle.

(225) 7) Statistical Analyses

(226) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. Two-tailed T-Tests were used to compare groups of DIO-vehicle mice (n=8) with DIO-test article treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Lean-Vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese state.

(227) TABLE-US-00013 TABLE 10 Effects resulting from 28 days subcutaneous treatment with SEQ ID NO: 6 in fed, female diet-induced obese (DIO) C57BL/6 mice. DIO-SEQ DIO-Vehicle ID NO: 6 twice-daily twice-daily Parameter PBS 30 g/kg Body mass change +3.76 0.40 5.51 0.64 (g) P < 0.00001 Whole body fat mass change +2.89 0.27 4.68 0.67 (g) P < 0.00001 Terminal liver mass 2.13 0.08 1.10 0.04 (g) P < 0.00001 Terminal plasma triglyceride 0.64 0.08 0.28 0.04 (mmol/L) P < 0.001 Terminal plasma LDL 1.28 0.06 0.70 0.06 (mmol/L) P < 0.00001 Data are means SEM. n = 8/group.

Example 11

Acute and Chronic Effects of SEQ ID NO: 11 After Subcutaneous Treatment on Blood Glucose, Body Mass, Whole Body Fat Content, Feed Consumption, Terminal Liver Weight and Terminal Plasma Parameters in Fed, Female Diet-Induced Obese (D10) C57BL/6 Mice

(228) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(229) Mice were treated twice daily with a s.c. injection of PBS-vehicle or 30 g/kg SEQ ID NO: 11 for 28 days, except on day 1 and 28 when mice received a single dose. The morning dosing was initiated and completed between 06:00 and 07:30 AM and the afternoon dosing between 2:00 and 3:30 PM. On day 1 and day 28 of the dosing phase only the morning dose was administered. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(230) 1) Blood Glucose Profile in Morning-Fed Animals

(231) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at hour 0 prior to the first s.c. dose of PBS-vehicle or 30 g/kg SEQ ID NO: 11 and 1, 2, 3, 4, 6, and 24 hours post-dose in non-anesthetized animals. The 24 hour blood collection was performed prior to dosing on day 2. Glucose measurements were performed in whole blood and in duplicate or triplicate using Aviva glucometers

(232) SEQ ID NO: 11 treated animals demonstrated a pronounced decrease in blood glucose concentrations that persisted over 24 hours. In contrast, no such change in blood glucose concentrations was observed in DIO control mice (FIG. 10).

(233) 2) Body Mass

(234) Body mass was measured daily approximately between 06:00-07:30 AM from day 32 through 38 of the predosing phase and throughout the 28 days of the dosing phase. During the dosing phase, except on day 1 and 28, animals were treated twice daily with either a s.c. injection of PBS-vehicle or 30 g/kg SEQ ID NO: 11. Chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 11 induced a consistent decrease in body mass over the 28 days of the treatment phase compared to the vehicle DIO group (FIG. 11). Chronic, twice daily treatment with SEQ ID NO: 11 resulted in a statistically significantly more pronounced body mass reduction within the 28 days of the dosing phase compared to DIO animals treated with vehicle (T-Test, Two-Tailed P<0.001, FIG. 12, Table 11).

(235) 3) Whole Body Fat Mass

(236) To determine whole body fat mass Quantitative Nuclear Magnetic Resonance (QNMR) measurements were performed on predosing day 37 and on day 26 of the dosing phase. During the dosing phase mice were treated twice daily with either a s.c. injection of PBS-vehicle or 30 g/kg SEQ ID NO: 11.

(237) In parallel to the pronounced body mass loss, twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 11 resulted in a statistically significantly more pronounced whole body fat mass reduction during the 28 days of the dosing phase compared to DIO animals treated with vehicle (T-Test, Two-Tailed P<0.001, FIG. 13, Table 11).

(238) 3) Feed Consumption

(239) Feed consumption was estimated based on the daily assessment of the feeder weights between 06:00-07:30 AM. Each cage housed four mice and feed consumption was determined throughout the 28 days of the dosing phase. During the dosing phase mice were treated twice daily with either a s.c. injection of PBS-vehicle or 30 g/kg SEQ ID NO: 11.

(240) After dosing start, treatment with 30 g/kg SEQ ID NO: 11 suppressed feed intake, however mice habituated to the pharmacological effects within approximately 15 days. After day 15 in the dosing phase, the estimated feed consumption was similar between SEQ ID NO: 11 and vehicle treated DIO mice (FIG. 14).

(241) 4) Terminal Liver Mass

(242) On day 28 blood was collected, then the morning dose was administered and necropsy was performed 4 hours post-dose. For this purpose animals were euthanized by isoflurane anesthesia, blood was collected by orbital bleeding followed by cervical dislocation, decapitation, bilateral thoracotomy, exsanguination, or vital organ removal to ensure death following last blood collection. Then the liver was collected and liver mass was recorded.

(243) Twice-daily, chronic treatment of DIO animals with 30 g/kg SEQ ID NO: 11 resulted in a statistically significant reduction in liver mass on day 28 of the dosing phase compared to DIO animals treated with vehicle (T-Test, Two-Tailed P<0.001, FIG. 15, Table 11).

(244) 5) Terminal Plasma Triglycerides, LDL, Insulin and Glucose

(245) On day 28 blood was collected, then the morning dose was administered and necropsy was performed four hours post-dose. For this purpose non-fasting animals were euthanized by isoflurane anesthesia and blood was collected by orbital bleeding for measurement of plasma parameters.

(246) Four hours after the last dose on day 28 day of the dosing phase compared to DIO animals treated with vehicle chronic treatment of DIO animals with 30 g/kg SEQ ID

(247) NO: 11 resulted in a statistically significant reduction in non-fasting plasma triglyceride (T-Test, Two-Tailed P<0.05, FIG. 16, Table 11), LDL (T-Test, Two-Tailed P<0.0001, FIG. 17, Table 11), insulin (T-Test, Two-Tailed P=0.001, FIG. 18, Table 11) and glucose concentrations (T-Test, Two-Tailed P<0.00001, FIG. 19, Table 11).

(248) 6) Statistical Analyses

(249) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. Two-tailed T-Tests were used to compare groups of DIO-vehicle mice (n=8) with DIO-compound treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Lean-Vehicle group data are depicted in the Figures but served as a reference dataset for the non-obese state.

(250) TABLE-US-00014 TABLE 11 Effects resulting from a single dose with SEQ ID NO: 11 in fed, female diet-induced obese (DIO) C57BL/6 mice. Example DIO-Vehicle DIO-SEQ ID NO: 11 Dose twice-daily twice-daily PBS 30 g/kg Body mass change +4.49 0.40 7.74 0.56 (g) P < 0.00001 Whole body fat mass change +3.03 0.28 6.84 0.46 (g) P < 0.00001 Terminal liver mass +2.39 0.21 +1.07 0.04 (g) P < 0.0001 Terminal plasma triglyceride +0.70 0.15 +0.34 0.06 (mmol/L) P < 0.05 Terminal plasma LDL +1.58 0.12 +0.84 0.05 (mmol/L) P < 0.0001 Terminal plasma insulin +129.35 21.81 +39.59 4.74 (g/L) P = 0.001 Terminal plasma glucose +7.21 0.26 +4.91 0.08 (mmol/L) P = 0.00001 Data are means SEM. n = 8/group.

Example 12

Acute and Chronic Effects of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 After Subcutaneous Treatment on Blood Glucose in Fed, Female Diabetic db/db Mice

(251) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(252) Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obese BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed in disposable shoebox caging with wood chip bedding until day 15 of the predose phase. At the study start mice were approximately 12 weeks old.

(253) Mice were housed under vivarium conditions including a 12 h light/dark cycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26 C. and a relative humidity between 30-70%. All animals had free access to water and Purina Fomulab Diet 5008.

(254) On predose day 9 blood glucose and body mass (approximately between 08:00-10:00 AM) as well as HbA1c measurements were performed. On day 15 of the predose phase animals were assigned to treatment groups (n=8) and to new cages to match mean HbA1c and body masses between all db/db groups. An age-matched lean group was included in the study as a healthy, lean reference.

(255) Prior to day 1 of the dosing phase, the test article was diluted with Phosphate Buffered Saline (PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) to a concentration of 1 mg/mL and aliquots of this stock solution were stored at approximately 60 C. On day 1 of the dosing phase the stock aliquot was thawed and the injected test article solution was prepared fresh by diluting it with PBS to achieve the desired concentration.

(256) On Day 1 of the dosing phase db/db mice were either treated once with a s.c. injection of PBS-vehicle or 30 g/kg test article. The lean reference group was treated once with a s.c. injection of PBS-vehicle. The dosing was initiated and completed between 08:00 and 10:00 AM. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(257) 3) Blood Glucose Profile in Morning-Fed Animals

(258) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at minute 30 and 0 prior to any other inlife activities. At minute 0 mice received a s.c. dose of either PBS-vehicle or 30 g/kg SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9. Further blood samples were harvested at hour 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements were performed in whole blood using AlphaTRAK glucometers. If the glucose concentrations of two measurements differed by more than 20 mg/dL a third value was recorded. The Area Under the Curve (AUC) for blood glucose was calculated via the trapezoid method and for the duration of the 24 post-dose hours.

(259) Single treatment of diabetic, non-fasting db/db mice with 30 g/kg SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 within 6 hours normalised hyperglycemia to the non-fasting blood glucose concentrations observed in non-obese, lean reference mice. Twenty four hours post-dose mean blood glucose concentrations of all treated animals were at or close to baseline (FIG. 20). Single treatment of diabetic db/db mice with 30 g/kg SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 resulted in a statistically significant reduction in the blood glucose AUC compared to the Diabetic-Vehicle group (One-Way ANOVA, Dunnet's Method, P<0.001 all treatment groups versus Diabetic-Vehicle group, FIG. 21, Table 12).

(260) 4) Statistical Analyses

(261) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. A One Way Analysis of Variance and multiple comparisons (Dunnett's Method) were performed comparing the group of diabetic, obese db/db vehicle mice (n=8) with each diabetic, obese db/db compound treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Non-diabetic, lean-vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese, non-diabetic state.

(262) TABLE-US-00015 TABLE 12 Effects resulting from 28 days subcutaneous treatment with SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 in fed, female diabetic db/db mice. Example db/db- db/db-SEQ db/db-SEQ db/db-SEQ Vehicle ID NO: 6 ID NO: 8 ID NO: 9 Dose once once once PBS 30 g/kg 30 g/kg 30 g/kg Blood Glucose AUC 646.38 45.04 376.58 23.20 406.72 34.41 422.08 37.43 (mmol/L for 24 hours) P < 0.001 P < 0.001 P < 0.001 Data are means SEM. n = 8/group.

Example 13

Acute Effects of SEQ ID NO: 7 After Subcutaneous Treatment on Blood Glucose in Fed, Female Diabetic db/db Mice

(263) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(264) Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obese BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed in disposable shoebox caging with wood chip bedding until day 15 of the predose phase. At the study start mice were approximately 12 weeks old.

(265) Mice were housed under vivarium conditions including a 12 h light/dark cycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26 C. and a relative humidity between 30-70%. All animals had free access to water and Purina Fomulab Diet 5008.

(266) On predose day 9 blood glucose and body mass (approximately between 08:00-10:00 AM) as well as HbA1c measurements were performed. On day 15 of the predose phase animals were assigned to treatment groups (n=8) and to new cages to match mean HbA1c and body masses between all db/db groups. An age-matched lean group was included in the study as a healthy, lean reference.

(267) Prior to day 1 of the dosing phase, the test article was diluted with Phosphate Buffered Saline (PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) to a concentration of 1 mg/mL and aliquots of this stock solution were stored at approximately 60 C. On day 1 of the dosing phase the stock aliquot was thawed and the injected test article solution was prepared fresh by diluting it with PBS to achieve the desired concentration.

(268) On Day 1 of the dosing phase db/db mice were either treated once with a s.c. injection of PBS-vehicle or 30 g/kg test article. The lean reference group was treated once with a s.c. injection of PBS-vehicle. The dosing was initiated and completed between 08:00 and 10:00 AM. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(269) 5) Blood Glucose Profile in Morning-Fed Animals

(270) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at minute 30 and 0 prior to any other inlife activities. At minute 0 mice received a s.c. dose of either PBS-vehicle or 30 g/kg SEQ ID NO: 7. Further blood samples were harvested at hour 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements were performed in whole blood using AlphaTRAK glucometers. If the glucose concentrations of two measurements differed by more than 20 mg/dL a third value was recorded. The Area Under the Curve (AUC) for blood glucose was calculated via the trapezoid method and for the duration of the 24 post-dose hours.

(271) Single treatment of diabetic, non-fasting db/db mice with 30 g/kg SEQ ID NO: 7 within 6 hours normalised hyperglycemia to the non-fasting blood glucose concentrations observed in non-obese, lean reference mice. Twentyfour hours post-dose the mean blood glucose concentration of SEQ ID NO: 7 treated animals was at baseline (FIG. 22). Single treatment of diabetic db/db mice with 30 g/kg SEQ ID NO: 7 resulted in a statistically significant reduction in the blood glucose AUC compared to the Diabetic-Vehicle group (One-Way ANOVA, Dunnet's Method, P<0.001 SEQ ID NO: 7 versus Diabetic-Vehicle group, FIG. 23, Table 13).

(272) 6) Statistical Analyses

(273) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. A One Way Analysis of Variance and multiple comparisons (Dunnett's Method) were performed comparing the group of diabetic, obese db/db vehicle mice (n=8) with diabetic, obese db/db compound treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Non-diabetic, lean-vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese, non-diabetic state.

(274) TABLE-US-00016 TABLE 13 Effects resulting from 28 days subcutaneous treatment with SEQ ID NO: 7 in fed, female diabetic db/db mice. Example db/db- db/db-SEQ Vehicle ID NO: 7 Dose once PBS 30 g/kg Blood Glucose AUC 705.52 49.31 480.59 21.92 (mmol/L for 24 hours) P < 0.001 Data are means SEM. n = 8/group.

Example 14

Acute Effects of SEQ ID NO: 11 After Subcutaneous Treatment on Blood Glucose in Fed, Female Diabetic db/db Mice

(275) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(276) Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obese BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed in disposable shoebox caging with wood chip bedding until day 15 of the predose phase. At the study start mice were approximately 12 weeks old.

(277) On day 15 of the predose phase animals were assigned to treatment groups (n=8) and to new cages to match mean HbA1c and body masses between all db/db groups. An age-matched lean group was included in the study as a healthy, lean reference.

(278) Prior to day 1 of the dosing phase, the test article was diluted with Phosphate Buffered Saline (PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) to a concentration of 1 mg/mL and aliquots of this stock solution were stored at approximately 60 C. On day 1 of the dosing phase the stock aliquot was thawed and the injected test article solution was prepared fresh by diluting it with PBS to achieve the desired concentration.

(279) On Day 1 of the dosing phase db/db mice were either treated once with a s.c. injection of PBS-vehicle or 30 g/kg test article. The lean reference group was treated once with a s.c. injection of PBS-vehicle. The dosing was initiated and completed between 08:00 and 10:00 AM. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(280) Blood Glucose Profile in Morning-Fed Animals

(281) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at minute 30 and 0 prior to any other inlife activities. At minute 0 mice received a s.c. dose of either PBS-vehicle or 30 g/kg SEQ ID NO: 11. Further blood samples were harvested at hour 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements were performed in whole blood using AlphaTRAK glucometers. If the glucose concentrations of two measurements differed by more than 20 mg/dL a third value was recorded. The Area Under the Curve (AUC) for blood glucose was calculated via the trapezoid method and for the duration of the 24 post-dose hours.

(282) Single treatment of diabetic, non-fasting db/db mice with 30 g/kg SEQ ID NO: 11 within 6 hours normalised hyperglycemia to the non-fasting blood glucose concentrations observed in non-obese, lean reference mice. Twentyfour hours post-dose mean blood glucose concentrations of SEQ ID NO: 11-treated animals were still below baseline concentrations (FIG. 24). Single treatment of diabetic db/db mice with 30 g/kg SEQ ID NO: 11 resulted in a statistically significant reduction in the blood glucose AUC compared to the Diabetic-Vehicle group (One-Way ANOVA,

(283) Dunnet's Method, P<0.001 SEQ ID NO: 11 Versus Diabetic-Vehicle group, FIG. 25, Table 12).

(284) Terminal Serum Triacylglycerol Concentrations in Morning-Fed Animals

(285) Animals had unlimited access to water and feed during the experiment. At the terminal study endpoint animals were deeply anesthetized with isoflurane, an orbital blood sample was harvested, and serum prepared for triacylglycerol determination. Twenty-four hours post-dose, mean serum triacylglycerol concentrations of SEQ ID NO: 11-treated animals were statistically significantly below those displayed by animals of the Diabetic-Vehicle group (One-Way ANOVA, Dunnet's Method, P=0.007 SEQ ID NO: 11 versus Diabetic-Vehicle group, FIG. 26).

(286) Statistical Analyses

(287) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Sigmaplot 12.5. A One Way Analysis of Variance and multiple comparisons (Dunnett's Method) were performed comparing the group of diabetic, obese db/db vehicle mice (n=8, except n=7 for the tracylglycerol analysis) with the group of diabetic, obese db/db compound treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Non-diabetic, lean-vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese, non-diabetic state.

(288) TABLE-US-00017 TABLE 14 Effects resulting from 28 days subcutaneous treatment with SEQ ID NO: 11 in fed, female diabetic db/db mice. Example db/db- db/db-SEQ Vehicle ID NO: 11 Dose once PBS 30 g/kg Blood Glucose AUC 763.59 39.53 391.06 24.25 (mmol/L for 24 hours) P < 0.001 Terminal serum 192.13 23.32 108.88 9.89 triacylglycerol P = 0.007 concentration (mg/dL) Data are means SEM. n = 7-8/group.

Example 15

Acute Effects of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 26 After Subcutaneous Treatment on Blood Glucose in Fed, Female Diabetic db/db Mice

(289) Animals, Study Design (Predosing Phase, Dosing Phase), Pharmacological Intervention:

(290) Female healthy, lean BKS.Cg-(lean)/OlaHsd and diabetes-prone, obese BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice were ordered group housed from Envigo RMS Inc., shipped group housed and remained group housed in disposable shoebox caging with wood chip bedding until day 15 of the predose phase. At the study start mice were approximately 12 weeks old.

(291) Mice were housed under vivarium conditions including a 12 h light/dark cycle (light phase 04:00 AM-4:00 PM), room temperatures between 23-26 C. and a relative humidity between 30-70%. All animals had free access to water and Purina Fomulab Diet 5008.

(292) On predose day 9 blood glucose and body mass (approximately between 08:00-10:00 AM) as well as HbA1c measurements were performed. On day 15 of the predose phase animals were assigned to treatment groups (n=8) and to new cages to match mean HbA1c and body masses between all db/db groups. An age-matched lean group was included in the study as a healthy, lean reference.

(293) Prior to day 1 of the dosing phase, the test article was diluted with Phosphate Buffered Saline (PBS, Gibco, without CaCl.sub.2 and MgCl.sub.2) to a concentration of 1 mg/mL and aliquots of this stock solution were stored at approximately 60 C. On day 1 of the dosing phase the stock aliquot was thawed and the injected test article solution was prepared fresh by diluting it with PBS to achieve the desired concentration.

(294) On Day 1 of the dosing phase db/db mice were either treated once with a s.c. injection of PBS-vehicle or 30 g/kg test article. The lean reference group was treated once with a s.c. injection of PBS-vehicle. The dosing was initiated and completed between 08:00 and 10:00 AM. The applied volume was 5 ml/kg and the dose was adjusted to the most recent body mass recording of each individual.

(295) 1) Blood Glucose Profile in Morning-Fed Animals

(296) Animals had unlimited access to water and feed during the experiment. On day 1 of the dosing phase approximately 5 L of blood were collected via tail clips at minute 30 and 0 prior to any other inlife activities. At minute 0 mice received a s.c. dose of either PBS-vehicle or 30 g/kg SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 26. Further blood samples were harvested at hour 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 24 post-dose. Glucose measurements were performed in whole blood using AlphaTRAK glucometers. If the glucose concentrations of two measurements differed by more than 20 mg/dL a third value was recorded. The Area Under the Curve (AUC) for blood glucose was calculated via the trapezoid method and for the duration of the 24 post-dose hours.

(297) Single treatment of diabetic, non-fasting db/db mice with 30 g/kg SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 26 within 6 hours normalised hyperglycemia to the non-fasting blood glucose concentrations observed in non-obese, lean reference mice. Twentyfour hours post-dose mean blood glucose concentrations of all treated animals were at or close to baseline (FIG. 27). Single treatment of diabetic db/db mice with 30 g/kg SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 26 resulted in a statistically significant reduction in the blood glucose AUC compared to the Diabetic-Vehicle group (One-Way ANOVA, Dunnet's Method, P<0.001 all treatment groups versus Diabetic-Vehicle group, FIG. 28, Table 15).

(298) 2) Statistical Analyses

(299) In the Figures data are depicted as meansSEM. Statistical analyses were performed with Everst@t 6.0.12. A One Way Analysis of Variance and multiple comparisons (Dunnett's Method) were performed comparing the group of diabetic, obese db/db vehicle mice (n=8) with each diabetic, obese db/db compound treated mice (n=8). When the difference in the mean values of the two groups was greater than 0.05 they were considered statistically significantly different. Non-diabetic, lean-vehicle group data are depicted in the Figures serving as a reference dataset for the non-obese, non-diabetic state.

(300) TABLE-US-00018 TABLE 15 Effects resulting from subcutaneous treatment with SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 26 in fed, female diabetic db/db mice. Example Blood Glucose AUC Dose (mmol/L for 24 hours) db/db-Vehicle 671.39 15.42 PBS db/db-SEQ ID NO: 10 413.77 37.33 once 30 g/kg P < 0.001 db/db-SEQ ID NO: 12 334.37 25.07 once 30 g/kg P < 0.001 db/db-SEQ ID NO: 13 373.42 13.99 once 30 g/kg P < 0.001 db/db-SEQ ID NO: 16 321.96 29.27 once 30 g/kg P < 0.001 db/db-SEQ ID NO: 26 416.25 30.62 once 30 g/kg P < 0.001 Data are means SEM. n = 8/group.

Example 16

(301) Anti-Atherosclerotic Activity of the Trigonal GLP-1R/GCGR/GIPR Agonists in ApoE KO Mice

(302) The apolipoprotein E (ApoE) knockout (KO) mouse model is widely used to investigate atherosclerosis. These mice spontaneously develop atherosclerotic lesions that are morphologically similar to those observed in humans (Meir & Leitersdorf 2004, Arterioscler Thromb Vasc Biol 24: 1006-1014, Rosenfeld et al. 2000, Arterioscler Thromb Vasc Biol 20: 2587-2592).

(303) Animals

(304) Male ApoE KO mice (B.129P2-apoetm1Unc/J) were randomly allocated to control or treatment groups (N=15 per group); wild-type mice (C57BL6/J) received vehicle treatment and acted as a second, healthy control.

(305) Study Procedure

(306) ApoE KO and wild-type mice received constant 16 weeks infusion using subcutaneous osmotic minipumps (ALZET) filled with either vehicle (sterile acetate buffer, pH 4.5), Peptides SEQ ID NO. 6 & SEQ ID NO. 11 (150 g/kg/day) or with Liraglutide (600 g/kg/day). Body weight and food intake was monitored throughout the study on a weekly basis.

(307) Blood Lipid Parameters

(308) Blood samples for blood lipid analysis were drawn before treatment and at weeks 7 and 16 to analyze total cholesterol, low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol (not shown).

(309) Quantification of Atherosclerotic Plaque Formation

(310) The aorta was dissected and aortic atherosclerotic plaque formation was measured as absolute and relative plaque area (% oilred stained area of total aortic surface area) using quantitative and automated image analysis.

(311) The results are shown in the figures:

(312) FIG. 29: Food intake and body weight monitoring data

(313) FIG. 30: Aortic plaque area data

(314) FIG. 31: Serum total cholesterol and LDL-cholesterol data

(315) In male ApoE KO mice chronic 4 months treatment with the peptides SEQ ID NO. 6 & SEQ ID NO. 11 at a dose of 150 g/kg/day led to a significant reduction of atherosclerotic plaques by 63% and 73% relative to vehicle control. Anti-atherosclerotic efficacy was accompanied by a marked reduction in LDL-cholesterol.

(316) In contrast, a 4-fold higher dose of the pure GLP-1 receptor agonist liraglutide reduced aortic plaques formation significantly but only by 37% with LDL-cholesterol lowering being far less pronounced.

(317) TABLE-US-00019 TABLE8 Sequences SEQ. ID Sequence 1 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 2 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(gGlu-Palm)-E- F-I-A-W-L-V-R-G-R-G-OH 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-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 5 Y-A-E-G-T-F-I-S-D-Y-S-1-A-M-D-K-1-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-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-K-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 7 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-K-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 8 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-K-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 9 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-K-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2 10 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 11 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-Aib-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2 12 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-Aib-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 13 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-Palm]-E-E-Q-R- Q-Aib-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K-NH2 14 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E- E-Q-R-Q-K-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 15 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E- E-Q-R-Q-K-E-F-1-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K- NH2 16 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Palm]-E-E-Q-R-Q-Aib- E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2 17 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Stea]-E-E-Q-R-Q-Aib- E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K-NH2 18 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Palm]-E-E-Q-R-Q-Aib- E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 19 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Stea]-E-E-Q-R-Q-Aib- E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 20 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Palm]-E-E-Q-R-Q-K- E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 21 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-Stea]-E-E-Q-R-Q-K-E- F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K-NH2 22 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E- E-Q-R-Q-K-E-F-I-E-W-L-K-A-G-G-H-P-S-Aib-K-P-P-P-K-NH2 23 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E- E-Q-R-Q-K-E-F-I-E-W-L-K-A-dAla-G-H-P-S-Aib-K-P-P-P-K- NH2 24 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E- E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K- NH2 25 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E- E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-dAla-G-P-P-S-Aib-K-P-P-P-K- NH2 26 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-gGlu-AEEA-Palm]-E- E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K- NH2 27 H-Aib-H-G-T-F-T-S-D-L-S-K-L-K[gGlu-AEEA-gAAA-Palm]-E- E-Q-R-Q-Aib-E-F-I-E-W-L-K-A-G-G-P-P-S-Aib-K-P-P-P-K- NH2 28 H-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K[gGlu-Stea]-D-E-Q-R-A-K- E-F-I-E-W-L-K-A-G-G-P-S-S-G-A-P-P-P-S-NH2 29 Y-Aib-Q-G-T-F-T-S-D-L-S-K-Q-K[gGlu-Stea]-D-E-Q-R-A-K-E- F-I-E-W-L-K-S-G-G-P-S-S-G-A-P-P-P-S-NH2