Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists

Abstract

The present invention relates to exendin-4 derivatives and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as reduction of excess food intake.

Claims

1. A peptidic compound of formula (I):
R.sup.1—Z—R.sup.2   (I) or a salt or solvate thereof, wherein Z is a peptide moiety of formula (II):
Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Ile-Gln-X14-X15-Lys-Arg-Ala-Ala-Aib-Glu- Phe-Ile-Glu-Trp-Leu-Lys-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-X40   (II) wherein: X14 is an amino acid residue selected from Met, Leu, and Nle, X15 is an amino acid residue selected from Glu and Asp, X40 is absent or is Lys, R.sup.1 is NH.sub.2, and R.sup.2 is OH or NH.sub.2.

2. The compound, salt, or solvate of claim 1, which is a glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist.

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

4. The compound, salt or solvate of claim 1, wherein the peptidic compound has a relative activity of at least 0.04% compared to that of natural GIP at the GIP receptor.

5. The compound, salt or solvate of claim 1, wherein the peptidic compound exhibits a relative activity of at least 0.07% compared to that of GLP-1(7-36) at the GLP-1receptor.

6. The compound, salt or solvate of claim 1, wherein X14 is an amino acid residue selected from Met, Leu, and Nle, X15 is an amino acid residue selected from Glu and Asp, and X40 is absent.

7. The compound, salt or solvate of claim 1, wherein X14 is an amino acid residue selected from Met, Leu, and Nle, X15 is Glu, and X40 is absent or is Lys.

8. The compound, salt or solvate of claim 1, wherein X14 is Leu, X15 is an amino acid residue selected from Glu and Asp, and X40 is absent or is Lys.

9. The compound, salt or solvate of claim 1, wherein X14 is Nle, X15 is an amino acid residue selected from Glu and Asp, and X40 is absent or is Lys.

10. The compound, salt or solvate of claim 1, wherein the compound is of any one of SEQ ID NOs: 8-12 or a salt or solvate thereof.

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

12. The pharmaceutical composition of claim 11, together with at least one pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 11, further comprising at least one additional therapeutically active agent, wherein the additional therapeutically active agent is selected from the group consisting of: insulin and insulin derivatives selected from the group consisting of insulin glargine, insulin glusiline, insulin detemir, insulin lispro, insulin degludec, insulin aspart, basal insulin and analogues thereof, pegylated insulin, recombinant human insulin, polysialated insulins, long-acting insulin, NN1045, insulin in combination with pramlintide, PE0139, fast-acting and short-acting insulins, insulin hydrogel, oral insulin, inhalable insulin, transdermal insulin and sublingual insulin, and insulin derivatives which are bonded to albumin or another protein by a bifunctional linker; GLP-1; GLP-1 analogues; GLP-1 receptor agonists selected from the group consisting of lixisenatide, exenatide, ITCA 650, AC-2993, liraglutide, semaglutide, taspoglutide, albiglutide, dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide, HM-11260C, CM-3, ORMD-0901, NN-9924, NN-9926, NN-9927, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, TT-401, BHM-034, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, xtenylated exenatide, xtenylated glucagon, and polymer bound derivatives thereof; dual GLP-1/GIP receptor agonists; dual GLP-1/glucagon receptor agonists; protein YY.sub.3-36 (PYY3-36); pancreatic polypeptide; glucagon receptor agonists; GIP receptor agonists or antagonists; ghrelin antagonists or inverse agonists; xenin; dipeptidyl peptidase IV (DPP-IV) inhibitors; sodium glucose cotransporter 2 (SGLT2) inhibitors; dual SGLT2/SGLT1 inhibitors; biguanides; thiazolidinediones; dual peroxisome proliferator-activated receptor (PPAR) agonists; sulfonylureas; meglitinides; alpha-glucosidase inhibitors; amylin and pramlintide; G protein-coupled receptor 119 (GPR119) agonists; GPR40 agonists; GPR120 agonists; GPR142agonists; systemic or low-absorbable transmembrane G protein-coupled receptor 5(TGR5) agonists; bromocriptine mesylate; inhibitors of 11-beta-hydroxysteroid dehydrogenase (HSD); activators of glucokinase; inhibitors of diacylglycerol acyltransferase (DGAT); inhibitors of protein tyrosinephosphatase 1; inhibitors of glucose-6-phosphatase; inhibitors of fructose-1,6-bisphosphatase; inhibitors of glycogen phosphorylase; inhibitors of phosphoenol pyruvate carboxykinase; inhibitors of glycogen synthase kinase; inhibitors of pyruvate dehydrogenase kinase; alpha2-antagonists; C-C motif receptor (CCR-2) antagonists; modulators of glucose transporter-4; somatostatin receptor 3 agonists; 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA)-reductase inhibitors; fibrates; nicotinic acid and derivatives thereof; nicotinic acid receptor 1 agonists; PPAR-alpha, gamma, or alpha/gamma agonists or modulators; PPAR-delta agonists; acyl-CoA cholesterol acyltransferase (ACAT) inhibitors; cholesterol absorption inhibitors; bile acid-binding substances; ileal bile acid transporter (IBAT) inhibitors; microsomal triglyceride transfer protein (MTP) inhibitors; modulators of proprotein convertase subtilisin/kinexin type 9(PCSK9); low-density lipoprotein (LDL) receptor up-regulators by liver selective thyroid hormone receptor β agonists; high-density lipoprotein (HDL)-raising compounds; lipid metabolism modulators; phospholipase A2 (PLA2) inhibitors; apolipoprotein A1 (ApoA-1) enhancers; thyroid hormone receptor agonists; cholesterol synthesis inhibitors; omega-3 fatty acids and derivatives thereof; substances for the treatment of obesity selected from the group consisting of sibutramine, tesofensine, tetrahydrolipstatin, cannabinoid-1 (CB-1) receptor antagonists, melanin-concentrating hormone-1 (MCH-1) antagonists, melanocortin 4(MC4) receptor agonists and partial agonists, neuropeptide Y5 (NPY5) or NPY2antagonists, NPY4 agonists, beta-3-agonists, leptin or leptin mimetics, agonists of the 5-hydroxy tryptophan 2c (5HT2c) receptor, combinations of bupropione/naltrexone, combinations of bupropione/zonisamide, combinations of bupropione/phentermine, combinations of pramlintide/metreleptin, and combinations of phentermine/topiramate; and lipase inhibitors; angiogenesis inhibitors; H3 antagonists; Agouti-related protein (AgRP) inhibitors; triple monoamine uptake inhibitors; methionine aminopeptidase type 2,(MetAP2) inhibitors; nasal formulation of the calcium channel blocker diltiazem; antisense molecules against production of fibroblast growth factor receptor 4; prohibitin targeting peptide-1; and drugs for influencing high blood pressure, chronic heart failure, or atherosclerosis selected from the group consisting of angiotensin II receptor antagonists, angiotensin-converting-enzyme (ACE) inhibitors, endothelin-converting-enzyme (ECE) inhibitors, diuretics, beta-blockers, calcium antagonists, centrally acting hypertensives, antagonists of the alpha-2-adrenergic receptor, inhibitors of neutral endopeptidase, and thrombocyte aggregation inhibitors.

14. The pharmaceutical composition of claim 11, together with at least one additional therapeutically active agent, wherein the at least one additional therapeutically active agent is selected from the group consisting of a GLP-1 agonist, an insulin, an insulin analogue, and a gastrointestinal peptide.

15. A solvate of a compound of claim 1.

16. A hydrate of a compound of claim 1.

17. A method of treating a disease or disorder comprising administering to a patient in need thereof the pharmaceutical composition of claim 11, wherein the disease or disorder is selected from the group consisting of hyperglycemia, type 2 diabetes, type 1 diabetes, and obesity.

18. The method of claim 17, wherein the disease or disorder is selected from the group consisting of hyperglycemia, type 2 diabetes, and obesity.

19. The method of claim 17, wherein the method delays the progression of impaired glucose tolerance (IGT) to type 2 diabetes or the progression of type 2 diabetes to insulin-requiring diabetes.

20. The method of claim 17, wherein the method regulates appetite or induces satiety.

21. A method of treating hyperglycemia, type 2 diabetes, or obesity in a patient, the method comprising administering to the patient an effective amount of at least one compound, salt or solvate of formula I according to claim 1 and an effective amount of at least one additional compound for treating hyperglycemia, type 2 diabetes, or obesity.

22. The method of claim 21, wherein the effective amounts of the at least one compound, salt or solvate of formula I and of the at least one additional compound are administered to the patient simultaneously.

23. The method of claim 21, wherein the effective amounts of the at least one compound, salt or solvate of formula I and of the at least one additional compound are administered to the patient sequentially.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1. Effect of acute s.c. administration of compounds SEQ ID NO: 8 and SEQ ID NO: 9 at 100 μg/kg on 24 h profile of blood glucose of diabetic db/db mice. Data are mean+SEM.

(2) FIG. 2. Effect of acute s.c. administration of compounds SEQ ID NO: 8 and SEQ ID NO: 9 at 100 μg/kg on 24 h profile of blood glucose of diabetic db/db mice, represented as change from baseline. Data are mean+SEM.

METHODS

(3) Abbreviations employed are as follows: AA amino acid cAMP cyclic adenosine monophosphate Boc tert-butyloxycarbonyl BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate BSA bovine serum albumin tBu tertiary butyl Dde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl ivDde 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl DIC N,N′-diisopropylcarbodiimide DIPEA N,N-diisopropylethylamine DMEM Dulbecco's modified Eagle's medium DMF dimethyl formamide EDT ethanedithiol FBS fetal bovine serum Fmoc fluorenylmethyloxycarbonyl HATU 0-(7-azabenzotriazol-1-yl)-N,N,N′-tetramethyluronium hexafluorophosphate HBSS Hanks' Balanced Salt Solution HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HOBt 1-hydroxybenzotriazole HOSu N-hydroxysuccinimide HPLC High Performance Liquid Chromatography HTRF Homogenous Time Resolved Fluorescence IBMX 3-isobutyl-1-methylxanthine LC/MS Liquid Chromatography/Mass Spectrometry Palm palmitoyl PBS phosphate buffered saline PEG polyethylene glycole PK pharmacokinetic RP-HPLC reversed-phase high performance liquid chromatography TFA trifluoroacetic acid Trt trityl UPLC Ultra Performance Liquid Chromatography UV ultraviolet
General Synthesis of Peptidic Compounds
Materials:

(4) 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 0.3-0.4 mmol/g.

(5) Fmoc protected natural amino acids were purchased from Protein Technologies Inc., Senn Chemicals, Merck Biosciences, Novabiochem, Iris Biotech, Nagase or Bachem.

(6) The following standard amino acids were used throughout the syntheses: Fmoc-L-Ala-OH, Fmoc-L-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.

(7) In addition, the following special amino acids were purchased from the same suppliers as above: Fmoc-L-Lys(ivDde)-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, Fmoc-L-Nle-OH, Fmoc-L-Met(O)—OH, Fmoc-L-Met(O.sub.2)—OH, Fmoc-(S)MeLys(Boc)-OH, Fmoc-(R)MeLys(Boc)-OH, Fmoc-(S)MeOrn(Boc)-OH and Boc-L-Tyr(tBu)-OH.

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

(9) All the peptides that had 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 HPLC purification procedure.

(10) Analytical HPLC/UPLC

(11) Method A: Detection at 215 nm

(12) column: Aeris Peptide, 3.6 μm, XB-C18 (250×4.6 mm) at 60° C. solvent: H.sub.2O+0.1% TFA:ACN+0.1% TFA (flow 1.5 ml/min) gradient: 90:10 (0 min) to 90:10 (3 min) to 10:90 (43 min) to 10:90 (48 min) to 90:10 (49 min) to 90:10 (50 min)
Method B: Detection at 220 nm column: Zorbax, 5 μm, C18 (250×4.6 mm) at 25° C. solvent: H.sub.2O+0.1% TFA: 90% ACN+10% H.sub.2O+0.1% TFA (flow 1.0 ml/min) gradient: 100:0 (0 min) to 98:2 (2 min) to 30:70 (15 min) to 5:95 (20 min) to 0:100 (25 min) to 0:100 (30 min) to 98:2 (32 min) to 98:2 (35 min)
Method C1: Detection at 210-225 nm, Optionally Coupled to a Mass Analyser Waters LCT Premier, Electrospray Positive Ion Mode column: Waters ACQUITY UPLC® BEH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+1% FA:ACN+1% FA (flow 0.5 ml/min) gradient: 95:5 (0 min) to 95:5 (1.80 min) to 80:20 (1.85 min) to 80:20 (3 min) to 60:40 (23 min) to 25:75 (23.1 min) to 25:75 (25 min) to 95:5 (25.1 min) to 95:5 (30 min)
Method C2: Detection at 210-225 nm, Optionally Coupled to a Mass Analyser Waters LCT Premier, Electrospray Positive Ion Mode column: Waters ACQUITY UPLC® BEH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+1% FA:ACN+1% FA (flow 0.6 ml/min) gradient: 95:5 (0 min) to 95:5 (1 min) to 65:35 (2 min) to 65:35 (3 min) to 45:55 (23 min) to 25:75 (23.1 min) to 25:75 (25 min) to 95:5 (25.1 min) to 95:5 (30 min)
Method C3: Detection at 210-225 nm, Optionally Coupled to a Mass Analyser Waters LCT Premier, Electrospray Positive Ion Mode column: Waters ACQUITY UPLC® BEH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+1% FA:ACN+1% FA (flow 1 ml/min) gradient: 95:5 (0 min) to 95:5 (1 min) to 65:35 (2 min) to 65:35 (3 min) to 45:55 (20 min) to 2:98 (20.1 min) to 2:98 (25 min) to 95:5 (25.1 min) to 95:5 (30 min)
Method C4: detection at 210-225 nm, optionally coupled to a mass analyser Waters LCT Premier, electrospray positive ion mode column: Waters ACQUITY UPLC® BEH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+1% FA:ACN+1% FA (flow 1 ml/min) gradient: 95:5 (0 min) to 95:5 (1.80 min) to 80:20 (1.85 min) to 80:20 (3 min) to 60:40 (23 min) to 2:98 (23.1 min) to 2:98 (25 min) to 95:5 (25.1 min) to 95:5 (30 min)
Method D: Detection at 214 nm column: Waters X-Bridge C18 3.5 μm 2.1×150 mm solvent: H.sub.2O+0.5% TFA:ACN (flow 0.55 ml/min) gradient: 90:10 (0 min) to 40:60 (5 min) to 1:99 (15 min)
Method E: Detection at 210-225 nm, Optionally Coupled to a Mass Analyser Waters LCT Premier, Electrospray Positive Ion Mode column: Waters ACQUITY UPLC® BEH™ C18 1.7 μm (150×2.1 mm) at 50° C. solvent: H.sub.2O+1% FA:ACN+1% FA (flow 0.9 ml/min) gradient: 95:5 (0 min) to 95:5 (2 min) to 35:65 (3 min) to 65:35 (23.5 min) to 5:95 (24 min) to 95:5 (26 min) to 95:5 (30 min)
General Preparative HPLC Purification Procedure:

(13) The crude peptides were purified either on an Äkta Purifier System or on a Jasco semiprep 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+0.05 to 0.1% TFA (B) and water+0.05 to 0.1% TFA (A) were employed as eluents. Alternatively, a buffer system consisting of acetonitrile and water with minor amounts of acetic acid was used. Product-containing fractions were collected and lyophilized to obtain the purified product, typically as TFA or acetate salt.

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

(15) Prior to the testing of solubility and stability of a peptide batch, its content was determined. Therefore, two parameters were investigated, its purity (HPLC-UV) and the amount of salt load of the batch (ion chromatography).

(16) For solubility testing, the target concentration was 1.0 mg/mL pure compound. Therefore, solutions from solid samples were prepared in different buffer systems with a concentration of 1.0 mg/mL compound based on the previously determined content. HPLC-UV was performed after 2 h of gentle agitation from the supernatant, which was obtained by 20 min of centrifugation at 4000 rpm.

(17) The solubility was then determined by comparison with the UV peak areas obtained with a stock solution of the peptide at a concentration of 2 mg/mL in pure water or a variable amount of acetonitrile (optical control that all of the compound was dissolved). This analysis also served as starting point (t0) for the stability testing.

(18) For stability testing, an aliquot of the supernatant obtained for solubility was stored for 7 days at 25° C. or 40° C. After that time course, the sample was centrifuged for 20 min at 4000 rpm and the supernatant was analysed with HPLC-UV.

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

(20) The amount of soluble degradation products was calculated from the comparison of the sum of the peak areas from all observed impurities reduced by the sum of peak areas observed at t0 (i.e. to determine the amount of newly formed peptide-related species). This value was given in percentual relation to the initial amount of peptide at t0, following the equation:
% soluble degradation products={[(peak area sum of impurities t7)−(peak area sum of impurities t0)]×100}/peak area peptide t0

(21) The potential difference from the sum of “% remaining peptide” and “% soluble degradation products” to 100% reflects the amount of peptide which did not remain soluble upon stress conditions following the equation
% precipitate=100−([% remaining peptide]+[% soluble degradation products])

(22) This precipitate includes non-soluble degradation products, polymers and/or fibrils, which have been removed from analysis by centrifugation.

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

(24) Anion Chromatography

(25) Instrument: Dionex ICS-2000, pre/column: Ion Pac AG-18 2×50 mm (Dionex)/AS18 2×250 mm (Dionex), eluent: aqueous sodium hydroxide, flow: 0.38 mL/min, gradient: 0-6 min: 22 mM KOH, 6-12 min: 22-28 mM KOH, 12-15 min: 28-50 mM KOH, 15-20 min: 22 mM KOH, suppressor: ASRS 300 2 mm, detection: conductivity.

(26) As HPLC/UPLC method, method D or E has been used.

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

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

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

(30) Bioanalytical Screening Method for Quantification of Exendin-4 Derivatives in Mice

(31) 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).

(32) Glucose Lowering in Female Diabetic dbdb-Mice

(33) Female diabetic dbdb-mice (BKS.Cg−+Leprdb/+Leprdb/OlaHsd) 10 weeks of age at study start were used. Mice were habituated to feeding and housing conditions for at least 2 weeks. 7 days prior to study start, HbA1c were determined to allocate mice to groups, aiming to spread low, medium and high HbA1c-values and in consequence the group-means (n=8), as equally as possible. On the day of study, food was removed, directly before sampling for baseline glucose assessment (t=0 min). Immediately afterwards, compounds or vehicle (phosphor buffered saline, PBS) were administered subcutaneously, 100 μg/kg, 10 ml/kg. Afterwards, blood samples were drawn by tail tip incision at 15, 30, 60, 90, 120, 150, 180, 240, 360, 480 min and 24 h. Food was re-offered after the 480 min-sampling.

(34) Data were analysed by 2-W-ANOVA on repeated measurements, followed by Dunnett's test as post-hoc assessment, level of significance p<0.05.

EXAMPLES

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

Example 1

Synthesis of SEQ ID NO: 8

(36) The solid phase synthesis was carried out on Rink-resin with a loading of 0.38 mmol/g, 75-150 μm from the company Agilent Technologies. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method B). Deconvolution of the mass signals found under the peak with retention time 14.76 min revealed the peptide mass 4126.2 which is in line with the expected value of 4125.6.

Example 2

Synthesis of SEQ ID NO: 9

(37) The solid phase synthesis was carried out on Rink-resin with a loading of 0.29 mmol/g, 75-150 μm from the company Agilent Technologies. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method C1). Deconvolution of the mass signals found under the peak with retention time 15.70 min revealed the peptide mass 4107.28 which is in line with the expected value of 4107.59.

Example 3

Synthesis of SEQ ID NO: 10

(38) The solid phase synthesis was carried out on Rink-resin with a loading of 0.29 mmol/g, 75-150 μm from the company Agilent Technologies. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. The peptide was cleaved from the resin with King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified via preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μM) using an acetonitrile/water gradient (both buffers with 0.1% TFA). The purified peptide was analysed by LCMS (Method C1). Deconvolution of the mass signals found under the peak with retention time 15.74 min revealed the peptide mass 4107.50 which is in line with the expected value of 4107.59.

(39) In an analogous way, the following peptides SEQ ID NO: 8-12 were synthesized and characterized, see table 4.

(40) TABLE-US-00009 TABLE 4 list of synthesized peptides and comparison of calculated vs. found molecular weight. SEQ ID calc. Found Rt NO Mass mass [min] Method 8 4125.6 4126.2 14.76 B 9 4107.6 4107.3 15.70 C1 10 4107.6 4107.5 15.74 C1 11 4235.8 4235.8 20.03 A 12 4093.6 4094.2 19.76 A

Example 4

Chemical Stability and Solubility

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

(42) TABLE-US-00010 TABLE 5 Chemical stability and solubility Stability Stability Temperature Solubility Solubility SEQ ID (pH 4.5) (pH 7.4) for (pH 4.5) (pH 7.4) NO [%] [%] stability [μg/ml] [μg/ml] 1 100 77 25° C. 933 >1000 8 99 92 25° C. >1000 >1000 9 100 100 40° C. 875 892 10 100 100 40° C. 960 >1000

Example 5

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

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

(44) The results for Exendin-4 derivatives with activity at the human GIP (hGIP R), human GLP-1 receptor (hGLP-1 R) and human glucagon receptor (hGLUC R) are shown in Table 6. All compounds are full agonists of the GIP and GLP-1 receptors.

(45) TABLE-US-00011 TABLE 6 EC50 values of exendin-4 peptide analogues at GLP-1, GIP and Glucagon receptors (indicated in pM) EC50 EC50 EC50 SEQ ID hGIP R hGLP-1 R hGLUC R NO [pM] [pM] [pM] 8 14.4 1.5 >1000000 9 7.9 1.2 155000 10 12.4 1.4 193000 11 14.1 1.6 129000 12 17.9 2.6 17900

Example 6

Pharmacokinetic Testing

(46) Pharmacokinetic profiles were determined as described in Methods. Calculated T.sub.1/2 and c.sub.max values are shown in Table 7.

(47) TABLE-US-00012 TABLE 7 Pharmacokinetic profiles of exendin-4 derivatives. SEQ ID NO T.sub.1/2 [h] Cmax [ng/ml] 8 0.3 734

Example 7

SEQ ID NO: 8 and SEQ ID NO: 9 on Glucose Lowering in Non-Fasted Female Diabetic dbdb-Mice

(48) Female dbdb-mice, received 100 μg/kg of SEQ ID NO: 8, SEQ ID NO: 9 or phosphate buffered saline (vehicle control) subcutaneously, at time 0 min. Both compounds immediately lowered glucose values (baseline on average at 29 mmol/l), with SEQ ID NO: 8 and SEQ ID NO: 9 reaching the maximal effect of ˜12 mmol/l and ˜16 mmol/I glucose reduction, respectively.

(49) SEQ ID NO: 8 and SEQ ID NO: 9 reached a statistical significant reduction of glucose compared to vehicle control from t=30 min until t=240 min, and from t=15 min until t=360 min respectively (p<0.05, 2-way-ANOVA on repeated measures, followed by Dunnett's post-hoc test; FIGS. 1 and 2).

(50) TABLE-US-00013 TABLE 6  sequences SEQ ID NO sequences 1 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G- G-P-S-S-G-A-P-P-P-S-NH2 2 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G- R-NH2 3 H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K(((S)-4-Carboxy-4- hexadecanoylamino-butyryl-))-E-F-I-A-W-L-V-R-G-R-G 4 Y-A-E-G-T-F-I-S-D-Y-S-I-A-M-D-K-I-H-Q-Q-D-F-V-N-W-L-L-A-Q-K- G-K-K-N-D-W-K-H-N-I-T-Q 5 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 6 Y-G-E-G-T-F-T-S-D-L-S-I-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 7 Y-A-E-G-T-F-T-S-D-V-S-I-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G-R 8 Y-Aib-E-G-T-F-T-S-D-L-S-I-Q-M-E-K-R-A-A-Aib-E-F-I-E-W-L-K-A- G-G-P-S-S-G-A-P-P-P-S-NH2 9 Y-Aib-E-G-T-F-T-S-D-L-S-I-Q-L-E-K-R-A-A-Aib-E-F-I-E-W-L-K-A-G- G-P-S-S-G-A-P-P-P-S-N H2 10 Y-Aib-E-G-T-F-T-S-D-L-S-I-Q-Nle-E-K-R-A-A-Aib-E-F-I-E-W-L-K-A- G-G-P-S-S-G-A-P-P-P-S-NH2 11 Y-Aib-E-G-T-F-T-S-D-L-S-I-Q-L-E-K-R-A-A-Aib-E-F-I-E-W-L-K-A-G- G-P-S-S-G-A-P-P-P-S-K-NH2 12 Y-Aib-E-G-T-F-T-S-D-L-S-I-Q-L-D-K-R-A-A-Aib-E-F-I-E-W-L-K-A-G- G-P-S-S-G-A-P-P-P-S-NH2