The present invention relates to exendin-4 peptide analogues which selectively bind and activate the glucagon receptor and comprise a chelating moiety capable of binding a metal ion. Preferred metal ions are radionuclides, e.g. detectable by positron emission tomography (PET) or single photon emission computed tomography (SPECT). The obtained compounds are useful for visualizing cells overexpressing the glucagon receptor, in particular in the liver, as well as a method of detecting and treating neuroendocrine tumors characterized by an overexpression of the glucagon receptor. The invention includes a method of production of suitable agents.

BACKGROUND OF THE INVENTION

Exendin-4 is a 39 amino acid peptide which is produced by the salivary glands of the Gila monster (Heloderma suspectum) (Eng,J. et al., J. Biol. Chem., 267:7402-05,1992). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1 ) receptor, whereas it does not activate significantly the glucagon receptor.

The amino acid sequence of exendin-4 is shown as SEQ ID NO: 1

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2

Glucagon is a 29-amino acid peptide which is released into the bloodstream

when circulating glucose is low. Glucagon's amino acid sequence is shown as SEQ ID NO: 2.

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

Positron emission tomography (PET) is a routinely used nuclear medicine imaging technique, capable of producing three dimensional images of subjects. After injection of a suitable radioactive tracer containing a positron-emitting radionuclide, a pair of orthogonal gamma rays is detected resulting from annihilation of a positron. Three dimensional images can be obtained after computational reconstruction; correct anatomical localization is frequently ensured by simultaneous recording of a CT X-ray scan.

Another nuclear medicine tomographic imaging technique to provide three dimensional images is single photon emission computed tomography (SPECT). This method is based on detection of gamma rays emitted by a suitable radioisotope.

These methods are generally utilized to examine tissues and to monitor physiological processes e.g. by using 18-fluorodeoxy glucose for monitoring of metabolic activity. Alternatively, a marker radioisotope can be attached to a specific ligand to create a radioligand displaying specificity to certain tissues or receptors, for example GPCR's.

Specific detection of a single receptor type by PET or SPECT requires a selective interaction of the tracer ligand with the receptor of interest. Selective GLP-1 receptor agonists with an imaging moiety have been described (Marti, B. et al. WO2006024275; Selvaraju, R.K. et al, Journal of Nuclear Medicine, 2013, 54, 1-6; Eriksson, O. et al, J Clin Endocrinol Metab, 2014, 99(5):1519 -1524).

The present invention comprises imaging ligands that selectively interact with the glucagon receptor (GCG-R).

The peptides of this invention contain 4-Thiazolylalanine in position 1.

The use of 4-Thiazolylalanine in position 1 of a synthetic peptide has been described in WO07140284 for GLP-1 receptor agonists. Surprisingly and conversely 4-Thiazolylalanine in the present invention provides highly active glucagon receptor agonists with strongly reduced activity at the GLP-1 receptor when compared to peptides that carry the natural histidine at position 1 (native glucagon).

The compounds of the present invention therefore are well suited for the investigation of the glucagon receptor in vivo using imaging technologies, such as PET or SPECT.

BRIEF SUMMARY OF THE INVENTION

Provided herein are exendin-4 analogs which potently and selectively bind and activate the glucagon receptor and comprise a chelating moiety capable of binding a metal ion, making the molecule suitable for imaging studies, for example PET studies. All the compounds carry the artificial amino acid 4-Thiazolylalanine at position 1. This surprisingly results in a higher selectivity towards the glucagon receptor versus the GLP1 receptor when identical compounds are compared to each other differing only at position 1 (Tza in position 1 instead of His). The present invention therefore provides highly selective glucagon receptor agonists which are well suited for the investigation of the glucagon receptor in vivo using an imaging technology, for example the PET technology.

The invention provides a peptidic compound having the formula (I).

Tza-X2-X3-Gly-Thr-Phe-X7-Ser-Asp-X10-Ser-X12-X13-X14-X15-X16-X17- X18-Ala-X20-X21 -Phe-lle-Glu-Trp-Leu-Leu-X28-X29-Gly-Pro-X32-Ser-Gly- Ala-Pro-Pro-Pro-Ser-X40-R1 (I)

X2 represents an amino acid selected from Ser and d-Ser,

X3 represents an amino acid selected from Gin and His,

X7 represents an amino acid selected from Thr and Aib,

X10 represents an amino acid residue selected from Tyr, Leu, Val, lie, Phe,

Phenylglycine, Thr , 2-Fluorophenylalanine, Cyclohexylglycine and tert-Leucine

X12 represents an amino acid selected from Lys, Arg and Cys(VS-DO3A), X13 represents an amino acid selected from Gin, Tyr and Cys(VS-D03A), X14 represents an amino acid residue selected from Leu, Nle and Cys(VS- D03A),

X15 represents an amino acid selected from Glu and Asp,

X16 represents an amino acid selected from Ser, Glu, Aib and Cys(VS- D03A),

X17 represents an amino acid selected from Arg, Gin, Lys, Ala and Cys(VS- D03A),

X18 represents an amino acid selected from Arg, Lys and Ala,

X20 represents an amino acid selected from Gin, Glu, Aib, Lys and Cys(VS- D03A),

If X16 is Glu and X20 is Lys, the sidechains of X16 and X20 may form a cyclic ring via a lactam,

X21 represents an amino acid residue selected from Asp and Glu,

X28 represents an amino acid selected from Ala and β-Ala,

X29 represents an amino acid residue selected from Gly and Thr,

X32 represents an amino acid selected from Glu and Ser,

X40 represents an amino acid selected from Cys(VS-D03A), Cys(VS-N02A), Cys(mal-DOTA), Cys(mal-NOTA), Cys(mal-NODAGA), Lys (DOTA), Lys (N OTA), Lys (PEG- DOTA) and Lys(VS-D03A),

X40 may be absent if one of the amino acids X12, X13, X14, X16, X17 or X20 is Cys(VS-D03A),

wherein DOTA, NOTA, DO3A, NO2A or NODAGA may be unloaded or loaded with a metal ion selected from Gd3+, Ga3+, Cu2+, (Al-F)2+, Y3+, Tc ln3+, Lu3+ and Re3+,

R1 represents OH or NH2;

or a metal complex or a salt or a solvate thereof.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

X2 represents an amino acid selected from Ser and d-Ser,

X3 is Gin,

X7 represents an amino acid selected from Thr and Aib,

X10 represents an amino acid residue selected from Tyr, Leu, lie,

X12 is Lys,

X13 is Gin,

X14 represents an amino acid residue selected from Leu and Nle, X15 represents an amino acid selected from Glu and Asp,

X16 represents an amino acid selected from Ser and Glu,

X17 represents an amino acid selected from Arg and Gin,

X18 is Arg,

X20 represents an amino acid selected from Gin and Lys,

If X16 is Glu and X20 is Lys, the sidechains of X16 and X20 may form a cyclic ring via a lactam,

X21 represents an amino acid residue selected from Asp and Glu, X28 is Ala,

X29 represents an amino acid residue selected from Gly and Thr, X32 is Glu,

X40 is Cys(VS-DO3A),

wherein D03A, may be unloaded or loaded with a metal ion selected from Gd3+, Ga3+, Cu2+, (Al-F)2+, Y3+, Tc3+, ln3+, Lu3+ and Re3+,

R1 represents OH or NH2;

or a metal complex or a salt or a solvate thereof.

Specific examples of a peptidic compound of formula (I) are the compounds of SEQ ID NO: 3 to 90 as well as salts or solvates thereof.

Specific examples of a peptidic compound of formula (I) are the compounds of SEQ ID NO: 6, 8, 13, 14, 35, 36, 49, 50, 60, 61 , 79, 80, 85 and 86 as well as salts or solvates thereof.

A further embodiment of the invention provides a peptidic compound having the formula (I), wherein

DOTA, NOTA, D03A, N02A or NODAGA is unloaded.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal ion selected from Gd3+, Ga3+, Cu2+, (Al-F)2+, Y3+, Tc3+, ln3+, Lu3+ and Re3+.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal ion Ga3+.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal ion Gd3+.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, DO3A, NO2A or NODAGA is loaded with a metal radionucleotide ion (Cu-64)2+, (Ga-68)3+, (Al-F-18)2+, (Y-86)3+.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, DO3A, NO2A or NODAGA is loaded with one metal

radionucleotide ion (Ga-67)3+, (Tc-99)3+, (ln-111 )3+.

A further embodiment of the invention provides a peptidic compound having the formula (I) wherein

DOTA, NOTA, DO3A, NO2A or NODAGA is loaded with one metal

radionucleotide ion selected from (Cu-67)2+, (Y-90)3+, (ln-111 )3+, (Lu-177)3+,

(Re-186)3+ and (Re-188)3+.

Preferred compounds are the peptides with SEQ ID No. 3 to 90 listed in table 1 or a metal complex or a salt or a solvate thereof.

Table 1 : Sequences

SEQ. ID Sequence

H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G- 1 G-P-S-S-G-A-P-P-P-S-NH2

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

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-C(mal-DOTA)-NH2

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-C(mal-NOTA)-NH2

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-C(mal-NODAGA)-NH2

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-C(VS-D03A)-NH2

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-C(mal-DOTA(Ga))-NH2

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-C(VS-D03A(Ga))-NH2

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-C(mal-NOTA(Ga))-NH2

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-C(mal-NODAGA(Ga))-NH2

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-Bal-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

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-Bal-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-Aib-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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-Aib-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-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-DOTA)-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-NOTA)-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-NODAGA)-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-DOTA(Ga))-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-NOTA(Ga))-NH2

Tza-S-Q-G-T-F-Aib-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-C(mal-NODAGA(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-S-R-A-A-Q-D-F-l-E-W-L-L-A-G-G-P-S-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-S-R-A-A-Q-D-F-l-E-W-L-L-A-G-G-P-S-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-Q-R-A-Q-D-F-l-E-W-L-L-A-G-G-P-S-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-Q-R-A-Q-D-F-l-E-W-L-L-A-G-G-P-S-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(D0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(D0TA(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(N0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(PEG-N0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(PEG-N0TA(Ga))-NH2

Tza-S-Q-G-T-F-Aib-S-D-L-A-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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-Aib-S-D-L-A-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-C(VS-D03A(Ga))-NH2

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-C(VS-D03A)-NH2

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-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-C(VS-D03A)-Q-L-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-C(VS-D03A)-L-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-C(VS-D03A)-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-C(VS-D03A)-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-C(VS-D03A)-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R-A-C(VS-D03A)-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(D0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(D0TA(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-T-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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-T-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-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-Aib-S-D-L-S-K-Q-Nle-E-S-R-R-A-K-D-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-Aib-S-D-L-S-K-Q-Nle-E-S-R-R-A-K-D-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(N0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-Aib-R-R-A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-Aib-R-R-A-Q-E-F-I-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-S-H-G-T-F-T-S-D-Y-S-K-Q-L-E-Aib-R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-H-G-T-F-T-S-D-Y-S-K-Q-L-E-Aib-K-K-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-Aib-K-K-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-S-H-G-T-F-T-S-D-I-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E(*)-Q-R-A-K(*)-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E(*)-Q-R-A-K(*)-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

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-C(VS-N02A)-NH2

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-C(VS-N02A(Ga))-NH2

Tza-S-Q-G-T-F-Aib-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-C(VS-N02A)-NH2

Tza-S-Q-G-T-F-Aib-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-C(VS-N02A(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

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-K(D0TA)-NH2

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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(VS-N02A)-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(VS-N02A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(mal-N0TA)-NH2

Tza-S-Q-G-T-F-T-S-D-I-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-C(mal-N0DAGA)-NH2

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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-2F-Phe-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-C(VS-D03A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-N02A)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-N02A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(mal-NOTA)-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(mal-NODAGA)-NH2

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-C(VS-D03A)-OH

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-C(VS-D03A(Ga))-OH

Tza-S-Q-G-T-F-T-S-D-Y-S-R-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-K(DOTA)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-A-R-A-Q-D-F-l-E-W-L-L-A-T-G-P-S-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-A-R-A-Q-D-F-l-E-W-L-L-A-T-G-P-S-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-A-R-A-Q-D-F-I-E-W-L-L-A-T-G-P-S-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A(Ga))-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(mal-N0TA)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(mal-N0DAGA)-NH2

Tza-s-Q-G-T-F-T-S-D-Y-S-K-Q-L-D-E-Q-R-A-Q-E-F-l-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-N02A)-NH2

Tza-S-H-G-T-F-T-S-D-Y-S-K-Q-L-D-Aib-R-R-A-Q-E-F-I-E-W-L-L-A-T-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

H-S-Q-G-T-F-T-S-D-Y-S-K-Q-L-E-S-R-R-A-Q-E-F-l-E-W-L-L-A-G-G-P-E-S-G-A-P-P-P-S-C(VS-D03A)-NH2

In sequence 60 and 61 , asterisk ("*") indicates the formation of a lactam bridge between Glu16 and Lys20.

The compounds of the invention are capable of specifically binding to the glucagon receptor. The compounds of the invention are glucagon receptor agonists as determined by the observation that they are capable of stimulating intracellular cAMP formation upon binding at the receptor for glucagon. The compounds exhibit at least a relative activity of 0.1 %, preferably 0.5%, more preferably 1.0% and even more preferably 10.0% compared to that of natural glucagon at the glucagon receptor.

The compounds of the invention also activate the GLP1 receptor as determined by the observation that they are capable of stimulating intracellular cAMP formation upon binding at the receptor for GLP1. The activity of a given compound of this invention (expressed by its activity relative to the activity of GLP1 at the GLP1 receptor) is below 1 %, more preferably below 0.5% and even more preferably below 0.1 % compared to the activity of the same compound at the glucagon receptor (expressed by its activity relative to the activity of glucagon at the glucagon receptor).

Surprisingly, it was found that peptidic compounds of the formula I with

4-Thiazolylalanine at position 1 showed increased glucagon receptor activation and strongly increased selectivity towards the activity on the GLP-1 receptor compared to derivatives having a histidine at this position. Histidine is the naturally occurring amino acid in glucagon at position 1 and has been shown to be important for the activation mechanism of the glucagon receptor (Unson, C.G. et al, Arch. Biochem. Biophys., 300, 747-750, 1993).

Further, the compounds of the invention preferably have a favourable stability at acidic or physiological pH values, e.g., at pH 4.5 or at pH 7.4 at 4°C, 25°C or 40°C. Preferably, the purity of the compounds in these buffers after 7 days at 25 °C is greater than 80% and after 14 days greater than 60%.

Furthermore, the compounds of the invention contain a chelating moiety capable of binding a metal ion, making the molecule suitable for imaging studies, for example PET or SPECT studies. The chelating moiety represents a non-cyclic or cyclic structure containing electron pair donating elements to ensure strong binding to the metal cation. Strong chelation is a prerequisite for use as an imaging modality in order to prevent leaching of the radioisotope, which may result in systemic toxicity, increased background signal and reduction of the signal at the area of interest. Choice of an optimal chelating moiety depends on the nature of the complexed radiometal. Exemplifying frequently used chelating moieties and their corresponding names are listed in Scheme 1 , more examples can e.g. be found in Wadas T.J. et al, Chem.Rev. 2010, 110, 2858-2902.

DOTAM

Scheme 1

In certain embodiments, i.e. when the compound of formula (I) comprises genetically encoded amino acid residues, the invention further provides a nucleic acid (which may be DNA or RNA) encoding said compound, an expression vector comprising such a nucleic acid, and a host cell containing such a nucleic acid or expression vector.

In a further aspect, the present invention provides a composition comprising a compound of the invention in admixture with a carrier. In preferred embodiments, the composition is a pharmaceutically acceptable composition and the carrier is a pharmaceutically acceptable carrier. The compound of the invention may be in the form of a metal complex, e.g. a gallium(lll) complex, a salt, e.g. a

pharmaceutically acceptable salt, or a solvate, e.g. a hydrate. In still a further aspect, the present invention provides a composition for use in a method of medical treatment including diagnostic treatment, particularly in human medicine.

Compounds of this invention and formulation thereof may primarily be used to visualize the glucagon receptor in living subjects and relevant tissues, preferably using the PET technology.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The amino acid sequences of the present invention contain the conventional one letter and three letter codes for naturally occurring amino acids, as well as generally accepted three letter codes for other amino acids, such as Nle

(Norleucine).

Furthermore, the following codes were used for the amino acids shown in Table 2.

Table 2: Unnatural amino acids

The term„native exendin-4" refers to native exendin-4 having the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 1 ).

The invention provides peptidic compounds as defined above.

The peptidic compounds of the present invention comprise a linear backbone of amino carboxylic acids linked by peptide, i.e. carboxamide bonds. Preferably, the amino carboxylic acids are a-amino carboxylic acids and more preferably L-a-amino carboxylic acids, unless indicated otherwise. The peptidic compounds comprise a backbone sequence of 39 or 40 amino carboxylic acids.

For the avoidance of doubt, in the definitions provided herein, it is generally intended that the sequence of the peptidic moiety differs from native exendin-4 at least at one of those positions which are stated to allow variation. Amino acids within the peptide moiety can be considered to be numbered consecutively from 1 to 40 in the conventional N-terminal to C-terminal direction. Reference to a „position" within peptidic moiety should be constructed accordingly, as should reference to positions within native exendin-4 and other molecules, e.g., in exendin-4, His is at position 1 , Gly at position 2, ... , Met at position 14, ... and Ser at position 39.

In a further aspect, the present invention provides a composition comprising a compound of the invention as described herein, a metal comnplex, or a salt or solvate thereof, in admixture with a carrier.

The invention also provides a composition wherein the composition is a pharmaceutically acceptable composition, and the carrier is a pharmaceutically acceptable carrier.

Peptide synthesis

The skilled person is aware of a variety of different methods to prepare peptides that are described in this invention. These methods include but are not limited to synthetic approaches and recombinant gene expression. Thus, one way of preparing these peptides is the synthesis in solution or on a solid support and subsequent isolation and purification. A different way of preparing the peptides is gene expression in a host cell in which a DNA sequence encoding the peptide has been introduced. Alternatively the gene expression can be achieved without utilizing a cell system. The methods described above may also be combined in any way.

A preferred way to prepare the peptides of the present invention is solid phase synthesis on a suitable resin. Solid phase peptide synthesis is a well established methodology (see for example: Stewart and Young, Solid Phase Peptide

Synthesis, Pierce Chemical Co., Rockford, III., 1984; E. Atherton and R. C.

Sheppard, Solid Phase Peptide Synthesis. A Practical Approach, Oxford-IRL Press, New York, 1989). Solid phase synthesis is initiated by attaching an N-terminally protected amino acid with its carboxy terminus to an inert solid support carrying a cleavable linker. This solid support can be any polymer that allows coupling of the initial amino acid, e.g. a trityl resin, a chlorotrityl resin, a Wang resin or a Rink amide resin in which the linkage of the carboxy group (or carboxamide for Rink resin) to the resin is sensitive to acid (when Fmoc strategy is used). The polymer support must be stable under the conditions used to deprotect the a-amino group during the peptide synthesis.

After the first amino acid has been coupled to the solid support, the a-amino protecting group of this amino acid is removed. The remaining protected amino acids are then coupled one after the other in the order represented by the peptide sequence using appropriate amide coupling reagents, for example BOP, HBTU, HATU or DIC (Ν,Ν'-diisopropylcarbodiimide) / HOBt (1 -hydroxybenzotriazol), wherein BOP, HBTU and HATU are used with tertiary amine bases. Alternatively, the liberated N-terminus can be functionalized with groups other than amino acids, for example carboxylic acids, etc.

Finally the peptide is cleaved from the resin and deprotected. This can be achieved by using King's cocktail (D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The raw material can then be purified

by chromatography e.g. preparative RP-HPLC, if necessary.

The synthesized peptide is then further modified by attaching a side chain which contains a chelating moiety capaple of integration a metal ion, for example Ga3+. In those cases where the attachment point in the peptide backbone is a lysine the side chain can be linked by the reaction with a suitable amidation group, e.g. a hydroxyl succinimide ester, or with a vinylsulfone group via Michael addition. In other cases, when the attachment side is a thiol of a cysteine, the side chain can be connected by the reaction with a maleimide functionality or with a vinylsulfone group via Michael addition. The raw material can then be deprotected as necessary and purified by chromatography, e.g. preparative RP-HPLC. The side chains attached to the compounds of this invention are summarized in table 3.

For the compounds of the present invention the building blocks listed in table 4 were used. With the exception of the building blocks VS-D03A and VS-N02A these building blocks were commercially available. The synthesis of VS-D03A is described in Example 1 , the synthesis of VS-N02A can be performed in an analogous way.

Table 3: Side chains

Table 4: Relevant side chain building blocks

Side chain building block Building block structure

mal-DOTA building block

mal-NOTA building block

mal-NODAGA building

block

H O

DOTA building block

NOTA building block

SPDP-dPEG8-NHS ester

O H

VS-N02A building block

VS-D03A building block

OH The complexing moiety at the side chain of the peptide can further be charged with a suitable metal ion, e.g. Ga3+. To achieve this, the peptide with the side chain is heated with a suitable salt of the desired cation in a suitable solvent. The raw material can then be purified by chromatography, e.g. preparative RP-HPLC or SPE, if necessary.

CLAIMS

A peptidic compound having the formula (I):

Tza-X2-X3-Gly-Thr-Phe-X7-Ser-Asp-X10-Ser-X12-X13-X14-X15-X16-X17- X18-Ala-X20-X21 -Phe-lle-Glu-Trp-Leu-Leu-X28-X29-Gly-Pro-X32-Ser-Gly- Ala-Pro-Pro-Pro-Ser-X40-R1 (I)

X2 represents an amino acid selected from Ser and d-Ser,

X3 represents an amino acid selected from Gin and His,

X7 represents an amino acid selected from Thr and Aib,

X10 represents an amino acid residue selected from Tyr, Leu, Val, lie, Phe, Phenylglycine,Thr , 2-Fluorophenylalanine, Cyclohexylglycine and tert- Leucine

X12 represents an amino acid selected from Lys, Arg and Cys(VS-D03A), X13 represents an amino acid selected from Gin, Tyr and Cys(VS-D03A), X14 represents an amino acid residue selected from Leu, Nle and Cys(VS- D03A),

X15 represents an amino acid selected from Glu and Asp,

X16 represents an amino acid selected from Ser, Glu, Aib and Cys(VS- D03A),

X17 represents an amino acid selected from Arg, Gin, Lys, Ala and Cys(VS- D03A),

X18 represents an amino acid selected from Arg, Lys and Ala,

X20 represents an amino acid selected from Gin, Glu, Aib, Lys and Cys(VS- D03A),

If X16 is Glu and X20 is Lys, the sidechains of X16 and X20 may form a cyclic ring via a lactam,

X21 represents an amino acid residue selected from Asp and Glu,

X28 represents an amino acid selected from Ala and β-Ala,

X29 represents an amino acid residue selected from Gly and Thr,

X32 represents an amino acid selected from Glu and Ser,

X40 represents an amino acid selected from Cys(VS-D03A), Cys(VS- N02A), Cys(mal-DOTA), Cys(mal-NOTA), Cys (mal-NODAGA), Lys (DOTA),

Lys (N OTA), Lys (PEG- DOTA) and Lys(VS-D03A),

X40 may be absent if one of the amino acids X12, X13, X14, X16, X17 or X20 is Cys(VS-D03A),

wherein DOTA, NOTA, D03A, N02A or NODAGA may be unloaded or loaded with a metal ion selected from Gd3+, Ga3+, Cu2+, (Al-F)2+, Y3+, Tc3+, ln3+, Lu3+ and Re3+,

R1 represents OH or NH2;

or a metal complex or a salt or a solvate thereof.

2. A compound of formula (I) according to claim 1 , wherein

X2 represents an amino acid selected from Ser and d-Ser,

X3 is Gin,

X7 represents an amino acid selected from Thr and Aib,

X10 represents an amino acid residue selected from Tyr, Leu, lie,

X12 is Lys,

X13 is Gin,

X14 represents an amino acid residue selected from Leu and Nle,

X15 represents an amino acid selected from Glu and Asp,

X16 represents an amino acid selected from Ser and Glu,

X17 represents an amino acid selected from Arg and Gin,

X18 is Arg,

X20 represents an amino acid selected from Gin and Lys,

If X16 is Glu and X20 is Lys, the sidechains of X16 and X20 may form a cyclic ring via a lactam,

X21 represents an amino acid residue selected from Asp and Glu,

X28 is Ala,

X29 represents an amino acid residue selected from Gly and Thr,

X32 is Glu,

X40 is Cys(VS-D03A),

wherein D03A, may be unloaded or loaded with a metal ion selected from

Gd 3+ , ga 3+ , with Cu 2+ , (Al-F) 2+ , the Y 3+ , with Tc 3+ , the ln 3+ , Lu , 3+ And Re 3+ ,

R1 represents OH or NH2;

or a metal complex or a salt or a solvate thereof.

3. A compound of formula (I) according to claim 1 , selected from the

compounds of SEQ ID NO: 3 - 90 as well as a metal complex, salts or solvates thereof.

4. A compound of formula (I) according to any one of claims 1 to 3, selected from the compounds of SEQ ID NO: 6, 8, 13, 14, 35, 36, 49, 50, 60, 61 , 79, 80, 85 and 86 as well as a metal complex, salts or solvates thereof.

5. A compound of formula (I) according to any one of claims 1 to 4, wherein DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal ion Ga3+.

A compound of formula (I) according to any one of claims 1 to 4, wherein DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal ion Gd3+.

7. A compound of formula (I) according to any one of claims 1 to 4, wherein DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal radionucleotide ion (Cu-64)2+, (Ga-68)3+, (Al-F-18)2+, (Y-86)3+.

8. A compound of formula (I) according to any one of claims 1 to 4, wherein DOTA, NOTA, D03A, N02A or NODAGA is loaded with a metal radionucleotide ion (Ga-67)3+, (Tc-99m)3+, (ln-111 )3+.

9. A compound of formula (I) according to any one of claims 1 to 4, wherein DOTA, NOTA, DO3A, NO2A or NODAGA is loaded with a metal radionucleotide ion selected from (Cu-67)2+, (Y-90)3+, (ln-111 )3+, (Lu- 177)3+, (Re-186)3+ and (Re-188)3+.

10. A pharmaceutical composition comprising at least one compound

according to any one of claims 1 - 9 or a physiologically acceptable salt or solvents of any of them.

1 1 .The compound for use according to claim 10 which is present as an

active agent in a pharmaceutical composition together with at least one pharmaceutically acceptable carrier.

12. A compound according to any one of claims 1 - 9 for visualization of the glucagon receptor in living subjects and relevant tissues.

13. A compound of any one of claims 1 - 9 for use in medicine, particularly in human medicine.

14. A compound according to claim 6 for visualization of the glucagon receptor in living subjects and relevant tissues by MRI.

15. A compound according to claim 7 for visualization of the glucagon receptor in living subjects and relevant tissues by PET.

16. A compound according to claim 8 for visualization of the glucagon receptor in living subjects and relevant tissues by SPECT.

17. A compound according to claim 9 for use in radiotherapy.

18. The compound for use according to any one of claim 1 to 9 for treating hypoglycemia, increase blood glucose levels, or as adjunctive therapy with insulin.

19. A method for treating hypoglycemia in a patient, the method comprising administering to the patient an effective amount of at least one compound of formula (I) according to any one of claims 1 to 9.

20. The method according to claim 19 which is administered parenterally.