Exendin-4 derivatives as selective glucagon receptor agonists

Abstract

The present invention relates to glucagon receptor agonists and their medical use, for example in the treatment of severe hypoglycemia.

Claims

1. A peptidic compound having the formula (I):
Tza-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X10-Ser-Lys-Gln-X14-Glu-Ser-Arg-Arg-Ala-Gln-X21-Phe-Ile-Glu-Trp-Leu-Leu-Ala-X29-Gly-Pro-Glu-Ser-Gly-Ala-Pro-Pro-Pro-Ser-R.sup.1(I) wherein, X10 represents an amino acid residue selected from Tyr, Leu, Val, Ile, Phe, Phenylglycine, 1-Naphthylalanine, 2-Fluorophenylalanine, Cyclohexylglycine and tert-Leucine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents an amino acid residue selected from Gly and Thr; and R.sup.1 represents OH or NH.sub.2; or a salt or solvate thereof.

2. The peptidic compound of claim 1, wherein R.sup.1 represents OH.

3. The peptidic compound of claim 1, wherein, X10 represents Leu; X14 represents an amino acid residue selected from Leu and Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents an amino acid residue selected from Gly and Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

4. The peptidic compound of claim 1, wherein, X10 represents Tyr; X14 represents an amino acid residue selected from Leu and Nle; X21 represents Glu; X29 represents an amino acid residue selected from Gly and Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

5. The peptidic compound of claim 1, wherein, X10 represents 1-Naphthylalanine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

6. The peptidic compound of claim 1, wherein, X10 represents Cyclohexylglycine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

7. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Tyr, Leu, Val, Ile, Phenylglycine, 1-Naphthylalanine, 2-Fluorophenylalanine and Cyclohexylglycine; X14 represents Leu; X21 represents an amino acid residue selected from Asp and Glu; X29 represents an amino acid residue selected from Gly and Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

8. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Tyr, Leu, Ile, Phe, 1-Naphthylalanine, Cyclohexylglycine and tert-Leucine; X14 represents Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

9. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Leu, Phe, 1-Naphthylalanine, 2-Fluorophenylalanine and Cyclohexylglycine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents Asp; X29 represents Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

10. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Tyr, Leu, Val, Ile, Phenylglycine, 1-Naphthylalanine, Cyclohexylglycine and tert-Leucine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents Glu; X29 represents an amino acid residue selected from Gly and Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

11. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Tyr, Leu, Val, Ile, Phe, Phenylglycine, 1-Naphthylalanine, 2-Fluorophenylalanine, Cyclohexylglycine and tert-Leucine; X14 represents an amino acid residue selected from Leu and Nle; X21 represents an amino acid residue selected from Asp and Glu; X29 represents Thr; and R.sup.1 represents OH; or a salt or solvate thereof.

12. The peptidic compound of claim 1, wherein, X10 represents an amino acid residue selected from Tyr, Leu and Val; X14 represents Leu; X21 represents Glu; X29 represents Gly; and R.sup.1 represents OH; or a salt or solvate thereof.

13. The peptidic compound of claim 1, wherein the peptidic compound is selected from the compounds of SEQ ID NO: 3-25 or a salt or solvate thereof.

14. The peptidic compound of claim 1, wherein the peptidic compound is selected from the compounds of SEQ ID NO:3, 5, 6, 9, 15, 20, 23, 24 and 25 or a salt or solvate thereof.

15. A pharmaceutical composition comprising the peptidic compound of claim 1 and at least one pharmaceutically acceptable carrier.

16. A method for treating hypoglycemia or type 2 diabetes mellitus in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of at least one peptidic compound of claim 1.

17. A pharmaceutical composition comprising at least one peptidic compound of claim 1 or a physiologically acceptable salt or solvent thereof.

18. A method for treating hypoglycemia in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of at least one peptidic compound of claim 1.

19. The method of claim 18, wherein the at least one peptidic compound and the at least one other compound are administered simultaneously, separately, or sequentially.

20. The method of claim 18, wherein the at least one peptidic compound is administered parenterally.

21. A method for treating hypoglycemia in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of at least one peptidic compound of claim 1 and a therapeutically effective amount of at least one other compound useful for treating hypoglycemia.

Description

LEGENDS TO THE FIGURES

(1) FIG. 1

(2) Blood glucose excursions after subcutaneous administration of GCG or SEQ. ID 5 in terminally anaesthetized rats. Values are meanSEM, n=6-8 rats.

(3) FIG. 2

(4) Blood glucose excursions after subcutaneous administration of GCG or SEQ. ID 6 in terminally anaesthetized rats. Values are meanSEM, n=6-8 rats.

(5) FIG. 3

(6) Effect of subcutaneous SEQ. ID 5 and human glucagon on blood glucose in dog

(7) FIG. 4

(8) Effect of subcutaneous and intramuscular SEQ. ID 5 on blood glucose in dog

(9) FIG. 5

(10) Effect of subcutaneous SEQ. ID 5 vs. SEQ. ID 6 on blood glucose in dog

METHODS

(11) Abbreviations employed are as follows: 2F-Phe 2-Fluorophenylalanine 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 Chg Cyclohexylglycine CTC 2-Chlorotrityl chloride 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 GCG Glucagon GLP-1 Glucagon related peptide 1 HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium 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 Nal 1-Naphthylalanine PBS phosphate buffered saline PEG polyethylene glycole Phg Phenylglycine RP-HPLC reversed-phase high performance liquid chromatography s.c. subcutaneous TFA trifluoroacetic acid Tle tert-Leucine TRIS Tris(hydroxymethyl)-aminomethan Trt trityl Tza 4-Thiazolylalanine UV ultraviolet
General Synthesis of Peptidic Compounds
Materials:

(12) For solid phase peptide synthesis preloaded Fmoc-Ser(tBu)-Wang resin was used. Fmoc-Ser(tBu)-Wang resin was purchased from Novabiochem with a loading of 0.3 mmol/g.

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

(14) 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-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-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.

(15) In addition, the following special amino acids were purchased from the same suppliers as above: Fmoc-L-Tza-OH, Fmoc-L-Phg-OH, Fmoc-L-Nal-OH, Fmoc-L-2F-Phe-OH, Fmoc-L-Chg-OH, Fmoc-L-Tle-OH

(16) The solid phase peptide syntheses were performed on a Prelude Peptide Synthesizer (Protein Technologies Inc) 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.

(17) 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 RP-HPLC purification procedure.

(18) General Preparative HPLC Purification Procedure:

(19) The crude peptides were purified either on an Akta 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+0.1% 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.

(20) Solubility and Stability Testing of Exendin-4 Derivatives:

(21) 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).

(22) For solubility testing, the target concentration was 10 mg/mL pure compound. Therefore, solutions from solid samples were prepared in different buffer systems with a concentration of 10 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.

(23) 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).

(24) For solubility testing, analytical Chromatography was performed with a Waters UPLC system on a Waters ACQUITY UPLC CSH C18 1.7 m (1502.1 mm) at 50 C. with a gradient elution at a flow rate of 0.5 mL/min and monitored at 210-225 nm. The gradients were set up as 20% B (0-3 min) to 75% B (3-23 min) followed by a wash step at 98% B (23.5-30.5) and a equilibration period (31-37 min at 20% B). Buffer A=0.5% trifluoracetic acid in water and B=0.35%) trifluoracetic acid in acetonitrile. Optionally, the LC was coupled to an Waters LCT Premier ESI-TOF mass spectrometer using the positive ion mode.

(25) For stability testing, the target concentration was 1.0 mg/mL pure compound in a pH 7.3 TRIS buffer (50 mM) containing m-cresol (30 mM), sodium chloride (85 mM) and polysorbate 20 (8 M). The solution was stored for 14 days at 50 C. After that time, the solution was analysed by UPLC.

(26) For stability testing, UPLC was performed on an Waters Acquity UPLC H-Class system with a Waters Acquity UPLC BEH130 C18 1.7 m column (2.1100 mm) at 40 C. with a gradient elution at a flow rate of 0.5 mL/min and monitored at 215 and 280 nm. The gradients were set up as 10% B to 90% B over 19.2 min and then 90% B for 0.8 min. Buffer A=0.1% formic acid in water and B=0.1% formic acid in acetonitrile.

(27) For determination of the amount of the remaining peptide, the peak areas of the target compound at t0 and t14 were compared, resulting in % Remaining peptide, following the equation
% Remaining peptide=[(peak area peptide t14)100]/peak area peptide t0.

(28) The % Normalized purity is defined by the % Relative purity at day 14 in relation to the % Relative purity at t0 following the equation
% Normalized purity=[(% Relative purity t14)100)]/% Relative purity t0

(29) The % Relative purity at t0 was calculated by dividing the peak are of the peptide at t0 by the sum of all peak areas at t0 following the equation
% Relative purity t0=[(peak area t0)100]/sum of all peak areas t0

(30) Likewise, the % relative purity t14 was calculated by dividing the peak are of the peptide at t14 by the sum of all peak areas at t14 following the equation
% Relative purity t14=[(peak area t14)100]/sum of all peak areas t14

(31) The potential difference between % Normalized purity and % Remaining peptide reflects the amount of peptide which did not remain soluble upon stress conditions.

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

(33) Anion Chromatography:

(34) Instrument: Dionex ICS-2000, pre/column: Ion Pac AG-18 250 mm (Dionex)/AS18 2250 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.

(35) In Vitro Cellular Assays for Glucagon Receptor Efficacy:

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

(37) 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 (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 665/620 nm. In vitro potency of agonists was quantified by determining the concentrations that caused 50% activation of maximal response (EC50).

(38) Blood Glucose Profile in Anesthetized Rats:

(39) The method aimed to study a test compound on the process of hepatic glycogenolysis. The rats had free access to food until the start of the experiment. It can be stated that the rise of blood glucose after administration of glucagon (GCG) or GCG-mimetic, and which lasted for about 60 to 90 minutes, was the result of the GCG- or GCG-mimetic-induced breakdown of hepatic glycogen. The effect of GCG-mimetic on hepatic glycogenolysis and the subsequent hyperglycemic peak in the blood was compared to the effect obtained with a subcutaneous bolus injection of GCG at a dose of 30 g/kg.

(40) Blood glucose levels were assayed in anaesthetized male Wistar rats as described previously (Herling et al. Am J Physiol. 1998; 274:G1087-93). Rats were anaesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg) and ketamine (10 mg/kg) and tracheotomized. Anesthesia was maintained for up to 5 hours by subcutaneous infusion of pentobarbital sodium (adjusted to the anesthetic depth of the individual animal; about 24 mg/kg/h). Body temperature was monitored with a rectal probe thermometer, and temperature was maintained at 37 C. by means of a heated surgical table. Blood samples for glucose analysis (10 l) were obtained from the tip of the tail every 15 minutes. The rats were allowed to stabilize their blood glucose levels after surgery for up to 2 hours. Then, GCG as reference compound, or the test compound were administered subcutaneously. For GCG a dose of 30 g/kg was used to induce hepatic glycogenolysis. The test compound SEQ. ID 5 was administered in doses of 10, 20 and 30 g/kg, and the test compound SEQ. ID 6 was administered in doses of 10 and 30 g/kg.

(41) Blood Glucose Profile in Normoglycemic Beagle Dogs:

(42) Male normoglycemic Beagle dogs were fasted overnight before and during the entire experiment. The animals were randomized to groups of n=6 per group. At time point 0 min the animals were treated with single doses of the test compound or native human glucagon as reference compound. The injection solutions were prepared freshly prior to the experiment. The test compound was administered as a single injection via three different routes (s.c., i.m. and i.v.) at doses of 1-100 g/kg. Blood sampling is performed consecutively via puncture of the jugular vein (vena jugularis) before drug administration (=0 min) and thereafter up to 240 min. Blood glucose was determined enzymatically (hexokinase method) from whole blood, insulin was analyzed from K-EDTA plasma with a dog-specific ELISA assay.

EXAMPLES

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

Example 1

Synthesis of SEQ ID NO: 25

(44) The solid phase synthesis was carried out on preloaded Fmoc-Ser(tBu)-Wang resin. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Fmoc-Tza-OH and in position 10 Fmoc-Tle-OH were used in the solid phase synthesis protocol. 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 (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.1% TFA).

(45) Finally, the molecular mass of the purified peptide was confirmed by LC-MS.

Example 2

Synthesis of SEQ ID NO: 24

(46) The solid phase synthesis was carried out on preloaded Fmoc-Ser(tBu)-Wang resin. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Fmoc-Tza-OH and in position 10 Fmoc-Chg-OH were used in the solid phase synthesis protocol. 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 (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.1% TFA).

(47) Finally, the molecular mass of the purified peptide was confirmed by LC-MS.

Example 3

Synthesis of SEQ ID NO: 5

(48) The solid phase synthesis was carried out on preloaded Fmoc-Ser(tBu)-Wang resin. The Fmoc-synthesis strategy was applied with HBTU/DIPEA-activation. In position 1 Fmoc-Tza-OH was used in the solid phase synthesis protocol. 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 (Sunfire, Prep C18) using an acetonitrile/water gradient (both buffers with 0.1% TFA).

(49) Finally, the molecular mass of the purified peptide was confirmed by LC-MS.

(50) In an analogous way, the peptides SEQ ID NO: 3-36 were synthesized, see table 2.

(51) TABLE-US-00006 TABLE 2 list of synthesized peptides and comparison of calculated vs. found molecular weight. SEQ ID calc. mass found mass 3 4259.68 4259.3 4 4229.66 4229.8 5 4279.67 4279.7 6 4323.73 4323.6 7 4259.68 4259.8 8 4273.71 4273.7 9 4215.63 4216.0 10 4293.70 4295.1 11 4357.80 4358.2 12 4273.71 4272.8 13 4293.70 4293.7 14 4273.71 4274.3 15 4259.68 4259.7 16 4273.71 4273.4 17 4323.73 4323.4 18 4311.69 4311.3 19 4343.70 4343.3 20 4293.70 4293.5 21 4311.69 4311.4 22 4343.76 4343.4 23 4285.72 4285.2 24 4299.69 4299.0 25 4273.65 4273.0 26 4242.64 4242.5 27 4212.61 4212.5 28 4262.56 4262.2 29 4306.68 4306.6 30 4242.64 4240.1 31 4256.66 4256.5 32 4276.65 4276.2 33 4340.75 4340.2 34 4256.66 4256.6 35 4242.64 4242.5 36 4256.66 4254.1

Example 4

Chemical Stability and Solubility

(52) Solubility and chemical stability of peptidic compounds were assessed as described in Methods. The results are given in Table 3.

(53) TABLE-US-00007 TABLE 3 Chemical stability and solubility Stability (pH7.3, Stability (pH7.3, Solubility 50 C., 2 w) 50 C., 2 w) SEQ ID (pH7.4) [mg/ml] [% NormalizedPurity] [% Remaining Peptide] 2 <0.2 70 n/a 1 >10.0 37 33 3 >10.0 94 92 6 >10.0 92 94 5 >10.0 96 88 9 >10.0 83 70 15 >10.0 86 75 20 >10.0 90 90 23 >10.0 94 92 24 >10.0 95 90 25 >10.0 89 82

Example 5

In Vitro Data on GLP-1 and Glucagon Receptor

(54) Potencies of peptidic compounds at the GLP-1 and glucagon receptors were determined by exposing cells expressing human glucagon receptor (hGLUC 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.

(55) The results for Exendin-4 derivatives with activity at the human GLP-1 receptor (hGLP-1 R) and the human glucagon receptor (hGLUC R) are shown in Table 4.

(56) TABLE-US-00008 TABLE 4 EC50 values of exendin-4 peptide analogues at GLP-1 and Glucagon receptors (indicated in pM) EC50 hGLP-1 R EC50 hGLUC R SEQ ID NO [pM] [pM] 1 0.4 >10000000 2 56.6 1.0 3 44333.3 1.8 4 3300.0 0.7 5 2190.0 0.6 6 9300.0 0.5 7 4190.0 2.4 8 5800.0 2.2 9 12200.0 2.2 10 45000.0 6.0 11 11700.0 0.9 12 20000.0 1.0 13 32100.0 3.1 14 52900.0 1.2 15 34500.0 2.2 16 19700.0 0.9 17 6940.0 1.0 18 25800.0 3.1 19 6640.0 0.7 20 38900.0 3.8 21 49700.0 1.4 22 8570.0 1.0 23 50700.0 3.5 24 8310.0 0.7 25 23100.0 0.8

Example 6

Comparison Testing

(57) A selection of exendin-4 derivatives comprising the artificial amino acid 4-thiazolylalanine in position 1 has been tested in comparison to corresponding compounds that have histidine in position 1. Histidine at position 1 is essential for the activation of the receptor in glucagon but also in many related peptides including GLP-1 and exendin-4. Therefore it is surprising that the artificial amino acid 4-thiazolylalanine leads to an even higher activation of the receptor compared to identical compounds that have the natural histidine at position 1. Furthermore, the activation of the GLP-1 receptor which counterregulates the glucagon effect is surprisingly reduced by the introduction of the artificial amino acid 4-thiazolylalanine. This leads to even more selective glucagon receptor agonists with a higher GCG/GLP-1 activity ratio. The reference pair compounds and the corresponding EC50 values at GLP-1 and Glucagon receptors (indicated in pM) are given in Table 5.

(58) TABLE-US-00009 TABLE 5 Comparison of exendin-4 derivatives comprising the artificial amino acid 4-thiazolylalanine in position 1 vs. exendin-4 derivatives having the natural amino acid histidine in position 1. EC50 values at GLP-1 and Glucagon receptors are indicated in pM. SEQ ID Amino acid EC50 EC50 NO in position 1 hGLP-1R hGlucagon-R Ratio 2 His 56.6 1.0 57:1 3 Tza 44333.3 1.8 24630:1 26 His 1240.0 9.4 132:1 4 Tza 3300.0 0.7 4714:1 27 His 80.8 1.3 62:1 5 Tza 2190.0 0.6 3650:1 28 His 52.4 1.0 52:1 6 Tza 9300.0 0.5 18600:1 29 His 145.0 0.9 161:1 7 Tza 4190.0 2.4 1746:1 30 His 1180.0 6.5 182:1 8 Tza 5800.0 2.2 2636:1 31 His 941.0 4.1 230:1 10 Tza 45000.0 6.0 7500:1 32 His 18700.0 12.0 1558:1 11 Tza 11700.0 0.9 13000:1 33 His 159.0 1.1 145:1 12 Tza 20000.0 1.0 20000:1 34 His 363.0 1.4 259:1 15 Tza 34500.0 2.2 15682:1 35 His 934.0 7.1 132:1 15 Tza 19700.0 0.9 21888:1 36 His 358.5 1.4 256:1

Example 7

Effect of SEQ. ID 5 and SEQ. ID 6 on Glucose Release in Anesthetized Rats after S.C. Injection

(59) During the 2 hr pre-treatment period blood glucose stabilized at a level of about 6 mmol/l, representing normal fed values in rats. GCG at the dose of 30 g/kg caused a rapid rise of blood glucose, which peaked after 30 minutes at blood glucose levels of about 10 to 11 mmol/l. The test compound SEQ. ID 5 at doses of 10, 20 and 30 g/kg subcutaneously caused a dose-dependent increase of blood glucose, which peaked 30, 45 and 90 min after injection, respectively. The dose of 20 g/kg of SEQ. ID 5 demonstrated a nearly comparable shape of blood glucose excursion compared to 30 g/kg GCG (FIG. 1).

(60) The test compound SEQ. ID 6 at doses of 10 and 30 g/kg caused a dose-dependent increase of blood glucose, which peaked 30 and 60 min after injection, respectively. The dose of 10 g/kg of SEQ. ID 6 demonstrated a more powerful blood glucose excursion compared to 30 g/kg GCG (FIG. 2).

Example 8

Effect of SEQ. ID. 5 and SEQ. ID 6 on Glucose Release in Normoglycemic Beagle Dogs after S.C. Injection

(61) In animals and humans injection of glucagon leads to a rapid recruitment of hepatic glycogen which is immediately broken down to glucose. This results in an acute but short lasting increase in blood glucose. In normoglycemic Beagle dogs subcutaneous (s.c.) injection of 1 g/kg human glucagon leads to rapid increase of blood glucose by 2-3 mmol/L within 15 min. s.c. injection of SEQ. ID 5 and SEQ. ID 6 mimicked the effect of human glucagon on blood glucose. In the dog the net total glucose response (change in blood glucose AUC(0-240 min) from baseline) after injection of 1 g/kg s.c. SEQ. ID 5 was similar to that of 1 g/kg s.c. human glucagon. Blood glucose response to SEQ. ID 5 increased depending on the dose until a peak increase of 3.5-4 mmol/L was reached with 10 g/kg s.c. (FIG. 3). Beyond this higher doses of s.c. SEQ. ID 5 did no longer result in higher glucose excursion. In dog the onset of glucose response s.c. SEQ. ID 5 was similar to that of human glucagon while the duration of the glucose response was slightly longer. SEQ. ID 5 was active through all parenteral routes as subcutaneous, intramuscular and intravenous injections resulted in rapid and transient blood glucose increase. There was no difference in activity and blood glucose time-action profile between subcutaneously and intramuscularly injections SEQ. ID 5 in dogs (FIG. 4).

(62) With respect to induction of a blood glucose response SEQ. ID 5 and SEQ. ID 6 were similarly active in normoglycemic dogs (FIG. 5).

(63) TABLE-US-00010 TABLE10 Sequences SEQ. ID sequence 1 H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A- V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P- P-P-S-NH2 2 H-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 3 Tza-S-Q-G-T-F-T-S-D-L-S-K-Q-Nle-E-S- R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 4 Tza-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A- P-P-P-S-OH 5 Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A- P-P-P-S-OH 6 Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 7 Tza-S-Q-G-T-F-T-S-D-V-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 8 Tza-S-Q-G-T-F-T-S-D-I-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 9 Tza-S-Q-G-T-F-T-S-D-V-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A- P-P-P-S-OH 10 Tza-S-Q-G-T-F-T-S-D-Phg-S-K-Q-L-E-S- R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 11 Tza-S-Q-G-T-F-T-S-D-1Nal-S-K-Q-L-E- S-R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 12 Tza-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 13 Tza-S-Q-G-T-F-T-S-D-F-S-K-Q-Nle-E-S- R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 14 Tza-S-Q-G-T-F-T-S-D-I-S-K-Q-Nle-E-S- R-R-A-Q-E-F-1-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 15 Tza-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R- R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 16 Tza-S-Q-G-T-F-T-S-D-L-S-K-Q-Nle-E-S- R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 17 Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-Nle-E-S- R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 18 Tza-S-Q-G-T-F-T-S-D-2FPhe-S-K-Q-Nle- E-S-R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E- S-G-A-P-P-P-S-OH 19 Tza-S-Q-G-T-F-T-S-D-1Nal-S-K-Q-Nle- E-S-R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E- S-G-A-P-P-P-S-OH 20 Tza-S-Q-G-T-F-T-S-D-Chg-S-K-Q-Nle-E- S-R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 21 Tza-S-Q-G-T-F-T-S-D-2FPhe-S-K-Q-L-E- S-R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 22 Tza-S-Q-G-T-F-T-S-D-1Nal-S-K-Q-L-E- S-R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 23 Tza-S-Q-G-T-F-T-S-D-Chg-S-K-Q-L-E-S- R-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 24 Tza-S-Q-G-T-F-T-S-D-Chg-S-K-Q-Nle-E- S-R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 25 Tza-S-Q-G-T-F-T-S-D-Tle-S-K-Q-Nle-E- S-R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S- G-A-P-P-P-S-OH 26 H-S-Q-G-T-F-T-S-D-L-S-K-Q-Nle-E-S-R- R-A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 27 H-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A-P- P-P-S-OH 28 H-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A-P- P-P-S-OH 29 H-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P- P-P-S-OH 30 H-S-Q-G-T-F-T-S-D-V-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P- P-P-S-OH 31 H-S-Q-G-T-F-T-S-D-I-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P- P-P-S-OH 32 H-S-Q-G-T-F-T-S-D-Phg-S-K-Q-L-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH 33 H-S-Q-G-T-F-T-S-D-1Nal-S-K-Q-L-E-S- R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G- A-P-P-P-S-OH 34 H-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R-R- A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P- P-P-S-OH 35 H-S-Q-G-T-F-T-S-D-L-S-K-Q-L-E-S-R-R- A-Q-D-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P- P-P-S-OH 36 H-S-Q-G-T-F-T-S-D-L-S-K-Q-Nle-E-S-R- R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A- P-P-P-S-OH