AU2012202855B2

AU2012202855B2 - GLP-1 and exendin related invention

Info

Publication number: AU2012202855B2
Application number: AU2012202855A
Authority: AU
Inventors: Martin Behe; Thomas Behr; Burkhard J. Goke; Martin Gotthardt
Original assignee: Philipps Universitaet Marburg
Current assignee: Philipps Universitaet Marburg
Priority date: 2004-09-03
Filing date: 2012-05-16
Publication date: 2015-02-26
Anticipated expiration: 2025-08-26
Also published as: AU2012202855A1

Abstract

The invention relates to peptides derived from GLP-1 (Glucagon-like Peptide 1) and exendin-3 and/or exendin-4, which bind to GLP-1 receptors and may be used either marked or unmarked for the production of a means for diagnosis and therapy of benign and pernicious diseases in which GLP-1 receptor expression plays a role.

Description

I AUSTRALIA Patents Act 1990 PHILIPPS-UNIVERSITAT MARBURG COMPLETE SPECIFICATION STANDARD PATENT Invention Title: GLP- 1 and exendin related invention The following statement is a full description of this invention including the best method of performing it known to us:lA Patent application Inventors PD Dr. Martin Gotthardt Dr. Martin Behe 5 Teichmannsgsrten 6 Fichtenweg 8 35274 Kirchhain 35043 Marburg Prof. Dr. Thomas Behr Prof. Dr. Burkhard J. Goke Forsthausstr. 2 Tassilostr.14 10 35043 Marburg-Cappel 82131 Gauting 15 Invention concerning GLP-1 and exendin This is a divisional of AU2005279537, the entire contents of which are incorporated herein by reference. 20 The invention at hand concerns a method for the production of a pharmacon for the depiction and therapy of primarily gastroenteropancreatic tumors, but also other benign and malignant diseases of different organ systems, based upon the incretin hormone GLP-1 and the analogs thereof.

2 Description and introduction of the general field of the invention In the localization of gastroenteropancreatic neuroendocrine tumors, somatostatin receptor scintigraphy (SRS) is the most important diagnostic method next to 5 ultrasound. The principle here is the specific depiction of tumors with the help of radioactively labeled peptides, which are absorbed by the tumor cells. Then, with the help of gamma cameras, the accumulation of the radioactivity in the tumor tissue can be verified visually. When a type of tumor possesses one of the necessary receptors for SRS, e.g. for the somatostatin analog octreotide*, 10 verification of these tumors is unproblematic. If corresponding receptors are not expressed, however, they evade scintigraphic verification. In addition to localization diagnosis, radioactive labeled peptides also allow for an approach to the treatment of tumors in which one can implement a specific receptor-directed radiopeptide therapy through labeling a somatostatin analog, e.g. octreotide*, with 15 an appropriate radionuclide (a- or 9- emitter). The fact that the corresponding radionuclides are bound chemically by the peptide (e.g. through complexation with a metal chelator which was previously bound to that peptide) in such a way that they will indeed be absorbed by the tumor cells but can no longer be discharged, results in a high specific accumulation in the tumor tissue. 20 However, a whole series of neuroendocrine tumors (NET), among them insulinomas and small cell bronchial carcinomas, do not express the necessary subtypes of the somatostatin receptor which are essential for SRS or radiopeptide therapy with the somatostatin analog octreotide*. A substantial percentage of 25 insulinomas in particular are not detectable by scintigraphic diagnosis. In small cell bronchial carcinomas SRS also does not constitute an appropriate method as, although primary tumors are often visible, metastases are not able to be shown due to loss of receptor expression. Consequently they are inaccessible for radiopeptide therapy, which presents an interesting additional or alternative 30 therapy method. Therefore the need exists for an appropriate peptide, which will be absorbed by the previously mentioned tumors.

3 The incretin hormone glucagon-like peptide-1 (GLP-1), as well as its analogs exendin-3 and exendin-4 (from the saliva of the gila monster Heloderma horridum and Heloderma suspectum), are peptides, for which insulinomas and small cell bronchial carcinomas - along with many other kinds of tumors - express 5 receptors. Insulinomas originate from the insulin producing p-cells in the islet of Langerhans in the pancreas, in which GLP-1 as well as exendin-3 and exendin-4 elicit a postprandial insulin secretion. 10 Technical state of the art For the utilization of glucagon-like peptide-1 (GLP-1) in scintigraphy it is necessary to label the peptide. The method for that and for the labeling of proteins with radionuclides is known to the expert and documented in numerous patent applications (e.g. DE 690 18 226 T2) and scientific publications. The peptides 15 described there for application in diagnostic imaging and for the exchange of therapeutically effective molecules in pathological tissue are normally inserted on the N-terminal end through an amino in the peptide. The peptides must be further modified concurrently regarding stabilization. 20 The GLP-1 used in the US 2003/0232761A1 and its derivative GLP-1(7-37) are, for example, modified on the N-terminal end by one amino. Hence, the N-terminal end of GLP-1 is no longer available to bond a GLP-1 receptor, therefore both receptor bonding and internalization is inadequate in these peptides. The latter are thus inappropriate for utilization in radiopeptide therapy of insulinomas and small 25 cell bronchial carcinomas. As experience shows, a mutation, such as a substitution of an amino acid within the sequence of peptides of GLP-1 and exendin 3 or exendin 4, and their possible modifications through a therapeutic or a signalizing molecule most often causes damage to the peptide structure, obstructing any further bonding to the receptor. 30 A further method for the modification of GLP-1, without affecting the N-terminus, is not known at this time. Furthermore, no GLP-1 derivatives are known which are appropriate, either labeled or unlabeled, for use in the radiotherapy of insulinomas and small cell bronchial carcinomas.

4 Summary of the Invention The task of the invention at hand, therefore, is to eliminate the deficiency of the technological state of the art and make peptides available which can be labeled and 5 still bond the GLP-1 receptor with this labeling and be utilized in the production of an agent for the diagnosis and therapy of diseases, in which the expression of the GLP-1 receptor plays a role. Described herein are derivatives of GLP-1, exendin-3 and exendin-4, which are 10 modified by an amino at the C-terminus and bond via the N-terminus at the GLP-1 receptor as well as chimeric peptides of GLP-1 with exendin-3 or exendin-4. These peptide derivatives as well as the chimeric peptides are being unlabeled or labeled in order to be utilized in the production of an agent for diagnostic and therapy of benign or malignant diseases in which GLP-1 receptor expression plays a role. 15 In a related aspect described herein are peptide derivatives of exendin-3, wherein: (i) the N-terminus of the peptide derivatives binds to the GLP-1 receptor; (ii) the peptide derivatives comprise an amino acid sequence over 80% identical to the amino acid sequence of exendin-3: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu 20 Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser; (iii) the peptide derivatives comprise an attachment group A, which is located at the C-terminus, and the attachment group A is an amino acid with a free amino group, or is an organic group comprising a free amine; and 25 (iv) a chelator is coupled onto the attachment group A and suitable for labeling with a radionuclide, an MRI contrast agent, a fluorescent pigment, or a chemotherapeutic agent. Through these peptide derivatives of GLP-1, exendin-3 and exendin-4 as well as 30 chimeric peptides of GLP-1, exendin-3 or exendin-4 the production of a means for scintigraphic applications is carried out which will be utilized for diagnostic and therapy of GLP-1 receptor expressing tumors, including NET (especially from insulinomas) and small cell bronchial carcinomas.

4A This is possible for the first time, as the peptide derivatives based on the current invention are modified by an amino at the C-terminus, making thus available the N terminus for bonding to the GLP-1 receptor. 5 Through the bonding of the peptide derivatives based on the current invention, for instance radioactively labeled, and chimeric peptides of GLP-1, exendin-3 and exendin-4 to the GLP-1 receptor, the representation of GLP-1 receptor expressing tumors is possible, enabling hereby a considerable improvement of the patients' medical care. NET are, above all, gastroenteropancreatic NET, such as insulinomas, 10 for which to date no non-invasive method with sufficient sensitivity is available or of small cell bronchial carcinomas localized in the area of the lung in which case the specific differentiation between inflammable processes and tumors or metastases is neither possible by means of a non-invasive method.

5 Furthermore, by means of the peptide derivatives and the chimeric peptides, both based in the current invention, the density of insulin producing cells within the pancreas as well as the expression of GLP-1 receptors in vivo and in vitro are visualized. This is for instance in the representation of GLP-1 receptor expressing 5 cells in the case of the diabetes mellitus a in vivo representation, as these are the cells which also secrete insulin. The representation of the GLP-1 receptor density within the pancreas is particularly important in the case of patients with diabetes mellitus during and after therapy with pharmaceuticals. Additionally the distribution of GLP-1 receptors in malignant and benign tissues is 10 represented. The articulated questions are hereby both of clinical as well as of scientific nature, as there is to date no all inclusive data available concerning the arrangement of GLP-1 receptors in human beings. Thus, the advantage of the current invention is that peptide derivatives of GLP-1 15 (glucagon-like peptide-1), exendin-3 and exendin-4 as well as chimeric peptides of GLP-1, exendin-3 or exendin-4 are utilized for the production of an agent, especially for the receptor-oriented specific representation and therapy, particularly of NET, in the case at hand especially of insulinomas and small cell bronchial carcinomas. 20 A GLP-1 receptor scintigraphy is particularly applicable in the diagnosis of small cell bronchial carcinomas, allowing for the first time the specific detection of metastases within lymph nodes (lymph nodes changed by inflammation versus lymph nodes attacked by metastases). The application of peptide derivatives from (glucagon-like peptide-1), exendin-3 25 und exendin-4, as well as chimeric peptide from GLP-1, exendin-3 or exendin-4 according to the current invention remains an agent of diagnostic and therapy for all malignant and benign diseases in which GLP-1 receptor expression plays a role, in particular in the following: as a contrast agent in Magnetic Resonance Imaging (MRI); as a radioactive agent in scintigraphy (SPECT, Single Photon 30 Emission Computed Tomography) as well as in radiopeptide therapy; in PET (Positron Emissions Tomography); in receptor-mediated chemotherapy; and in optical diagnostic. Optical diagnostic here means the stimulation of a fluorescent molecule by a particular wave length, inducing a successive light emission of a different wave length. It is the emitted wave length which is detected.

6 An expert can easily choose the kind of labeling at the C-terminus of the peptide derivative from GLP-1 (glucagon-like peptide-1), exendin-3 and exendin-4 as well as the chimeric peptides from GLP-1, exendin-3 or exendin-4 depending on the desired application: for example, for scintigraphy or radiotherapy from radioactive nuclides; for 5 contrast agent in Magnetic Resonance Imaging (MRI) from gadolinium; and for endoscopic or scientific examinations from fluorescent pigments. According to the current invention malignant diseases are those in which the affected tissue shows changes in its level of differentiation as compared to healthy tissue, 10 invasive growth or a spreading of its tissue into the blood stream or lymphatic system. All neuroendocrine tumors fall into this category, in particular those of the gastrointestinal tract; especially insulinomas, bronchial carcinomas, pancreatic carcinomas and all other malignant diseases which are connected to the over expression of GLP-1 receptors. 15 According to the current invention benign diseases are those characterized by the fact the affected tissue does not significantly lose its level of differentiation, shows no invasive growth and does not have any tissue metastasis into the blood stream or lymphatic system. This includes, for example, diabetes mellitus, but also eating 20 disorders of disorders of the psyche. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other 25 element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general 30 knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. Characterization of peptide derivatives and chimeric peptides 35 Surprisingly it was found that peptide derivatives from GLP-1, exendin-3 and exendin 4, as well as chimeric peptides from GLP-1, exendin-3 or exendin-4, which are 6A modified via an amino at the C-terminus, bond at the N-terminus to the GLP-1 receptor. They even shown a high degree of affinity to the GLP-1 receptor, as do natural peptides. Experiments with tumor carrying hairless mice show a specific uptake in GLP-1 receptors of positive tumor tissue.

7 Peptide derivatives as well as chimeric peptides according to the current invention are unlabelled or, via a chelator at the C-terminus amino, labeled as an agent for application in the diagnostic and therapy of benign and malignant diseases in which GLP-1 receptor expression plays a role. The type of labeling here consists 5 mainly of a radiometal, a MRI contrast agent, a fluorescent chromophore or a chemotherapeutic agent. The process and method of labeling are well known to an expert (e.g. DE 690 18 226 T2), which take place, for example, through the coupling of radionuclides, 10 non-magnetic metals and other MRI contrast agents or fluorescent pigments; this means that the bonding of the receptors or the internalization of the peptide derivatives, as well as the chimeric peptides according to the current invention, are not impaired and the GLP-1 receptor bonding N-terminus remains free (unlinked). 15 The sequences of amino acids of the original peptides: GLP-1: 20 H-His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 25 Exendin-3: H-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 30 Pro-Ser-NH 2 38 39 8 Exendin-4: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Val-Arg-Leu-Phe-ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro 5 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Pro-Ser-NH 2 38 39 According to the current invention the following peptide derivatives of GLP-1 (1 10 37), exendin-3 and exendin-4 are produced: GLP-1(x-y)A'1 3 7 Exendin-3 (z-k)A' 40 15 Exendin-4 (z-k)A' 40 Whereby: x= amino acids 1-36 of the GLP-1 amino acid sequence 20 y= amino acids 2-37 of the GLP-1 amino acid sequence z= amino acids 1-38 of the exendin-3 or exendin-4 amino acid sequence k= amino acids 2-39 of the exendin-3 or exendin-4 amino acid sequence A = Attachment group consisting of one or more amino acid or its derivative as a 25 signal molecule, or to bond signal molecules or to stabilize them. The preference is for A to be located at the C-terminus and the amino is preferably lysine or alternatively another amino acid with a free amino, e.g. ornithine or an organic group with a free amino onto which a chelator is coupled for the labeling with radionuclides or a MRI contrast agent, fluorescent pigments or a 30 chemotherapeutic agent.

9 Chelators which can be used include DTPA (diethylenetriaminepentaacetic acid), alternatively N,N-Bis(2-[bis(carboxymethyl)amino]-ethyl)glycine), alternatively DOTA (1,4,7,1 0-tetraazacyclododecane-1,4,7,1 0-tetra-acetic acid), HYNIC (6 hydrazinopyridin-3-carbonic acid), MAG3 (mercaptoacetyl-glycylglycylglycine), N4 5 (1,4,8,11-tetraazaundecane) and all known derivatives of the above named chelators. The exponent indicates at which position the attachment group alternatively can be found within the amino acid sequence. 10 GLP-1 (x-y)A 1

-

37 Here GLP-1 derivatives of different lengths are included, whereby x can accept the numbers 1 to 36, which is smaller than y, which accepts the numbers 2 to 37. A is the attachment group which can be placed at any position, preferably however at the C-terminus and is one higher than y. Preferably, the attachment 15 group is the amino lysine. Exendin-3 (z-k)A'- 0 Here exendin-3 derivatives of different lengths are included, whereby z can accept the numbers 1 to 38, which is smaller than k, which accepts the numbers 2 to 39. 20 A is the attachment group which can be placed at any position, preferably, however, at the C-terminus and is one higher than y. Preferably, the attachment group is the amino lysine. Exendin-4 (z-k)A 40 25 Here exendin-4 derivatives of different lengths are included, whereby z can accept the numbers 1 to 38, which is smaller than k, which accepts the numbers 2 to 39. A is the attachment group which can be placed at any position, preferably, however, at the C-terminus and is one higher than y. Preferably, the attachment group is the amino lysine. 30 The following peptide derivatives are particularly preferred: 1. MC 10: (DTPA-Lys 37 ) GLP1 (7-36) amide 2. MC 13: (DTPA-Lys 40 ) exendin-3 amide 3. MC 11: (DTPA-Lys 40 ) exendin-4 amide 10 The synthesis takes place, for example, in the company Peptide Specialty Laboratories GmbH according to the Merrifield method and purification via HPLC. 5 MC 10 (DTPA-Lys 37 ) GLP1 (7-36) amide consists of the amino acids 7-36 of GLP 1, carrying at the C-terminal end as an amino acid with a free amino, preferably lysine at position 37 as well as the chelator DTPA. MC 13 (DTPA-Lys") exendin-3 amide consists of the complete amino acid 10 sequence of exendin-3, carrying at the C-terminal end as an amino acid with a free amino, preferably lysine at position 40 as well as the chelator DTPA. MC 11 (DTPA-LysQ) exendin-4 amide consists of the complete amino acid sequence of exendin-3, carrying at the C-terminal end as an amino acid with a 15 free amino, preferably lysine at position 39 as well as the chelator DTPA. According to the current invention the following chimeric peptides from GLP-1 (1 37) and exendin-3 or exendin-4 are produced: GLP-1(x-y) exendin-3(z-k)A'- 5 20 GLP-1(x-y) exendin-4(z-k)A' Exendin-3(z-k) GLP-1(x-y)A' " Exendin-4(z-k) GLP-1(x-y)A' 7 25 Here the following applies: x= amino acids 1-36 of the GLP-1 amino acid sequence y= amino acids 2-37 of the GLP-1 amino acid sequence z= amino acids 1-38 of exendin-3 or exendin-4 amino acid sequence k= amino acids 2-39 of exendin-3 or exendin-4 amino acid sequence 30 A = Attachment group consisting of one or more amino acid or its derivative as a signal molecule, or to bond signal molecules or to stabilize them. The preference is for A to be located at the C-terminus and the amino is preferably lysine or 11 alternatively another amino acid with a free amino, e.g. ornithine or an organic group with a free amino onto which a chelator is coupled for the labeling with radionuclides or a MRI contrast agent, fluorescent pigments or a chemotherapeutic agent. 5 Chelators which can be used include DTPA (diethylenetriaminepentaacetic acid), alternatively N,N-Bis(2-[bis(carboxymethyl)amino]-ethyl)glycine), alternatively DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid), HYNIC (6 hydrazinopyridin-3-carbonic acid), MAG3 (mercaptoacetyl-glycylglycylglycine), N4 (1,4,8,11-tetraazaundecane) and all known derivatives of the above named 10 chelators. The exponent indicates at which position the attachment group alternatively can be found within the amino acid sequence. 15 GLP-1(x-y) exendin-3(z-k)A 7 ' Here chimeric peptides from GLP-1 and exendin-3 are included, in which the amino acids 1 to 36 come from GLP-1 and then after that the amino acids 1 to 39 from exendin-3. A is the attachment group which can be placed at any position, preferably, however, at the C-terminus, and is one higher than the number of 20 amino acids from GLP-1 and exendin-3, preferably the amino lysine. GLP-1(x-y) exendin-4(z-k)Al 7 Here chimeric peptides from GLP-1 and exendin-3 are included, in which the amino acids 1 to 36 come from GLP-1 and then after that the amino acids 1 to 39 25 from exendin-4. A is the attachment group which can be placed at any position, preferably, however, at the C-terminus, and is one higher than the number of amino acids from GLP-1 and exendin-4, preferably the amino lysine. Exendin-3(z-k) GLP-1(x-y)A'-7s 30 Here chimeric peptides from exendin-3 and GLP-1 are included, whereby z can accept the numbers 1 to 38, but is smaller than k, which can accept the numbers 2 to 39. A is the attachment group which can be placed at any position, preferably, 12 however, at C-terminus and is one higher than y. Preferably, the attachment group is the amino lysine. Exendin-4(z-k) GLP-1(x-y)A'-" 5 Here chimeric peptides from exendin-4 and GLP-1 are included, whereby z can accept the numbers 1 to 38, but is smaller than k, which can accept the numbers 2 to 39. A is the attachment group which can be placed at any position, preferably, however, at C-terminus and is one higher than y. Poeferably, the attachment group is the amino lysine. 10 Exemplary for a chimeric GLP-1 (x-y) exendin-3(z-k)A- 7 5 or GLP-1(x-y) exendin 4(z-k)A 75 peptide is MC12, consisting of GLP-1 (7-36) exendin (33-39) Lys amide (Synthesis takes place in the company Peptide Specialty Laboratories GmbH 15 according to the Merrifield method and purification via HPLC.). MC 12: (Ser 37 , Gly , Ala'9, Pro 40 , Pro*4, Pro42, Ser 4 3 , DTPA-Lys44 amide) GLP1 (7-36) MC 12 consists of the complete amino acid sequence of GLP-1 (7-36), carrying at 20 the C-terminal end an amino, preferably lysine at position 44 as well as the chelator DTPA, as well as a chain of 7 amino acids from exendin (33-39) Lys amide. Thus there is a chimeric peptide GLP-1 (7-36) exendin (33-39)Lys amide. It is clear to the expert that hereby peptide derivatives of different lengths from 25 GLP-1, exendin-3 and exendin-4, as well as chimeric peptides of different lengths from GLP-1, exendin-3 or exendin-4 exist, which contain various combinations of the amino acid sequences from GLP-1, exendin-3 and exendin-4 on which they are based. 30 The peptide derivatives from GLP-1, exendin-3 and exendin-4, as well as chimeric peptides from GLP-1, exendin-3 or exendin-4 based on the current invention, which are modified by an amino at the C-terminus and bond via the N terminus at the GLP-1 receptor, also include molecules which are distinguishable at one or more positions from the peptide GLP-1, exendin-3 and exendin-4 13 described above and have a high degree of homology to those sequences. Homology here means a sequence identity of at least 40 %, in particular an identity of 60 %, preferably over 80 % and especially preferably over 90 %. The deviation to the amino acid sequences described above could arise through 5 deletion, substitution and/or insertion Furthermore, the chimeric peptides from GLP-1, exendin-3 or exendin-4 according to the current invention are also produced without any modifications to the C-terminus. They are particularly used in the production of an agent for the 10 therapy of diabetes. GLP-1(x-y) exendin-3(z-k) GLP-1(x-y) exendin-4(z-k) Exendin-3(z-k) GLP-1(x-y) 15 Exendin-4(z-k) GLP-1(x-y) MC 20: (Ser , Gly3, Ala 3 , Pro 3 , Pro 37 , Pro 3 , Ser 9 ) exendin GLP1 (7-36) MC 20 consists of the complete amino acid sequence of GLP-1 (7-36), carrying at the C-terminal end a chain of 7 amino acids from exendin (33-39). Thus there is 20 an unmodified chimeric peptide GLP-1 (7-36) exendin (33-39). Labeling of the peptide derivatives and the chimeric peptides Peptide derivatives as well as chimeric peptides according to the current invention are dissolved in a suitable stabilizing buffer, for example, in order to stabilize 25 metals, preferably in 0.5 M sodium acetate pH 5.4 with a concentration of approx. 10- M. Alternatively, for the stabilization of fluorescent pigments a buffer of ammonium acetate is preferable; for the stabilization of chemotherapeutic agent and contrast agents a physiological buffer is preferable. The labeling occurs at the attachment group A through the coupling of 30 radionuclides, MRI contrast agents, fluorescent pigments or chemotherapeutic agent. Different methods are applied according to whether the application will be in vitro or in vivo.

14 The following are used as radionuclides for covalent or complex coupling: Nuclide Procedure in t 1

/

2 [h] Emitted Energy [keV] Type of which applied radiation coupling F-18 PET 1,8 p+ 634 covalent Cu-64 PET 12.7 p + 1673 complex Cu-67 therapy 61.8 - 391 complex y 184 Ga-67 SPECT 79.2 y 93/184/300 complex Ga-68 PET 1.1 2921 complex Y-86 PET 14.8 1 1220 complex y 1076/1153 Y-90 therapy 64.1 2280 complex Tc-99m SPECT 6 y 140 complex In-111 SPECT 67.2 y 171/245 complex 1-123 SPECT 13.2 y 158 covalent 1-124 PET 101 P 2137/1534 covalent y 602 1-131 therapy 192 y 364 covalent 1p 606 Lu-177 therapy 158 y 208 complex @~ 112/208 Re-186 therapy 88.8 y 137 complex p~ 1071 Re-1 88 therapy 17 y 155/477/632 complex @~_ _1965/2120 Pt-193m therapy 104 y 135 complex auger e Pt-195m therapy 96 y 98 complex auger e Ac-225 therapy 240 y 99, 150 complex At-211 therapy 7.2 y 687 complex augere covalent Bi-213 therapy 0.76 y 440 complex Sm-153 therapy 46 y 103 complex Er-169 therapy 226 p 100 complex 15 PET (Positron Emissions Tomography), SPECT (Single Photon Emissions Computed Tomography) Fluorescent pigments/ chromophores such as the following were used: 5 Fluorescein, Rhodamin, Coumarin, BODIPY, Pyrene (Cascade Blue), Lucifer Yellow, Phycobiliprotein, Cyanin, AlexaFluoro, Oregon Green, Texas Red and their derivatives. Chelators which can be used include DTPA (diethylenetriaminepentaacetic acid), 10 alternatively N,N-Bis(2-[bis(carboxymethyl)amino]-ethyl)glycine), alternatively DOTA (1,4,7,1 0-tetraazacyclododecane-1,4,7,1 0-tetra-acetic acid), HYNIC (6 hydrazinopyridin-3-carbonic acid), MAG3 (mercaptoacetyl-glycylglycylglycine), N4 (1,4,8,11-tetraazaundecane) and all known derivatives of the above named chelators. 15 MRI contrast agents which can be used include: gadolinium, manganese, iron, europium, copper, nickel, chrome, prasodymium, dysprosium or holmium or their compounds, but also the negative MRI contrast agents such as perfluorocarbone, as well as isotopes such as such as F-1 9, H-1, P-31, Na-1 9 for MRI spectroscopy. 20 Negative MRI contrast agents according to the current invention are those which obliterate the MRI signal or greatly weaken it, i.e. not amplifying it. Chemotherapeutic agents which can be used include: alkylating agents, 25 intercalators, antimetabolite, enzyme inhibitors and blockers and spindle poisons (for example alkylsulfonate, ethylimine, nitrosomonas (N. ureae), nitrogen mustard derivatives, folic acid analogs, purine analogs, pyrimidine analogs, podophyllin derivatives, taxane, vincaalcaloide, anthracycline, other cytostatic antibiotics, platinum compounds, campthotecin derivatives, different hormones, 30 growth factors, interferons or interleukins), otherwise the chemotherapeutic agents described in "Onkologie 2004/05", authors Preiss, Dornhoff, Hagmann, Schmieder, published by Zuckschwerdtverlag, at pp. 230-287, but also all other cytostatic or cytotoxic substances.

16 Depending on the type of application in which the protein derivatives and chimeric proteins according to the current invention are used, and the agent produced with the above mentioned proteins for diagnostic and therapy of benign and malignant 5 diseases, in which GLP-1 receptor expression plays a role, the labeling reaction will be carried out in two variations. Labeling for in vitro application in radiotherapy 3 pL of the peptide derivative or the chimeric peptide according to the invention 10 which has been dissolved in a suitable stabilizing buffer, preferably 0.5 M sodium acetate pH 5.4 with a concentration of approx. 10~3 M, are added to 500 pL 0.5 M sodium acetate pH 5.4 for the purpose of labeling. The pH-value is between 3 and 6. Then 185 MBq "'InCl 3 (Tyco, Petten, The Netherlands) in 0.1 M HCI 500 pL is added and incubated for 30 minutes at 37'C. Finally 3 pL 10~3 M solution natInCl 3 is 15 added, followed by a further incubation for 30 min in order to saturate all bond sites. Quality control is carried out via a HPLC column: Column: CC 250/4.6 Nucleosil 120-5 C18 (Machery-Nagel, Oenisingen, Switzerland) 20 Gradient: 0->5 min 100% 0.05 M NH 4 00CCH 3 , pH 5.4 (buffer A); 5->25 min 100% buffer A ->50% buffer A/50% acetone nitrile. Quality control for an in vitro application is fulfilled with a labeling yield of over 98 Thus a radioactive labeled agent is available for diagnostic and therapy of benign 25 and malignant diseases, in which GLP-1 receptor expression plays a role, which can, for example, which can be employed in cell and tissue cultures of pancreatic cells. Labeling for in vivo application in radiotherapy 30 3 pL of the peptide derivative or the chimeric peptide according to the invention which has been dissolved in a suitable stabilizing buffer, preferably 0.5 M sodium acetate pH 5.4 with a concentration of approx. 10-3 M, are added to 500 pL 0.5 M sodium acetate pH 5.4 for the purpose of labeling. Finally 185 MBq "'InCl 3 (Tyco, 17 Petten, The Netherlands) in 0.1 M HCI 500 pL is added and incubated for 30 minutes at 370C. Quality control is carried out via a HPLC-Column: Column: CC 250/4.6 Nucleosil 120-5 C18 (Machery-Nagel, Oenisingen, Switzerland) 5 Gradient: 0->5 min 100% 0.05 M NH 4 00CCH 3 , pH 5.4 (buffer A); 5->25 min 100% buffer A ->50% buffer A/50% acetone nitrile. Quality control for an in vivo application is fulfilled with a labeling yield of over 98 10 Thus a radioactive labeled agent is available for diagnostic and therapy of benign and malignant diseases, in which GLP-1 receptor expression plays a role, which can, for example, be employed to detect tumors in patients. The term "patient" refers to humans and vertebrates alike. Thus, the agent can be 15 applied both in human and veterinary medicine. The therapeutically and diagnostically effective agent based on the current invention is given to patients as part of an acceptable pharmaceutical composition in one of the following forms: oral, rectal, parenteral, intravenous / intraarterial, intramuscular, subcutaneous, intrathecal, intracisternal, intracranial, intravaginal, intraperitoneal, intravascular, 20 local (powder, ointment or drops) or spray form (aerosol). The required dose is to be determined by a doctor in each individual case of diagnostic and therapy of benign and malignant diseases, in which GLP-1 receptor expression plays a role. 25 Internalization study The internalization study shows, in exemplary manner, the transport into the cell of the peptide derivatives and chimeric proteins, both in vitro radioactively labeled, 30 according to the current invention. In a 6 well plate 100,000 GLP-1 receptor transfected CHO cells are sown. The cells grow until they are confluent. Then 4 groups are formed: 18 Group 1: complete bonding, washed with PBS 100,000 cpm 'in (10~1 Mol) labeled peptide is added to 2 mL medium and incubated for 1 h at 37 0 C. Then it is washed 3x with PBS and the cells are 5 separated with 20 mM MOPS (3-Morpholinopropanesulfonic-acid) + 0.1% Triton X-100 (ph7.4). The uptake into the cells is measured with a y-counter. The number of cells is measured by the protein content, using the protein assay kit from Bio Rad (Munich, Germany), based on the Bradford method. The results are given in proteins cpm/pg. 10 Group 2: non-specific bonding, washed with PBS 20 pL of a 10-3 M GLP-1 solution and 100,000 cpm "'In labeled peptide are added to 2 mL medium and incubated for 1 h at 37 0 C. Then it is washed 3x with PBS and the cells are separated with 20 mM MOPS (3 15 Morpholinopropanesulfonic-acid) + 0.1% Triton-X-100 (ph7.4). The uptake into the cells is measured with a y-counter. The number of cells is measured by the protein content, using the protein assay kit from Bio-Rad (Munich, Germany), based on the Bradford method. The results are given in proteins cpm/pg. 20 Group 3: complete bonding, washed with acid 20 pL of a 10- M GLP-1 solution and 100,000 cpm "'In labeled peptide are added to 2 mL medium and incubated for 1 h at 37 0 C. Then it is washed 1x with 0.1 M sodium acetate buffer pH 4 and 2x with PBS and the cells are separated with 20 mM MOPS (3-Morpholinopropanesulfonic-acid) + 0.1% Triton-X-100 25 (ph7.4). The uptake into the cells is measured with a y-counter. The number of cells is measured by the protein content, using the protein assay kit from Bio-Rad (Munich, Germany), based on the Bradford method. The results are given in proteins cpm/pg. 30 Group 4: non-specific bonding, washed with acid 20 pL of a 10-3 M GLP-1 solution and 100,000 cpm "'In labeled peptide are added to 2 mL medium and incubated for 1 h at 37 0 C. Then it is washed 1x with 0.1 M sodium acetate buffer pH 4 and 2x with PBS and the cells are separated 19 with 20 mM MOPS (3-Morpholinopropanesulfonic-acid) + 0.1% Triton-X-100 (ph7.4). The uptake into the cells is measured with a y-counter. The number of cells is measured by the protein content, using the protein assay kit from Bio-Rad (Munich, Germany), based on the Bradford method. The results are given in 5 proteins cpm/pg. Evaluation: %IDsB= Res3.- Res4.*100 Res.- Re s2. 10 %ldsB = % of internalization of the specific bonding %ldsB MC10 75±5 MC11 70±7 MC12 73±9 The results show that a good transport has taken place into the cells. 15 Bonding studies Bonding studies show the specific bonding which takes place via the vivo-labeling radioactive labeled peptide derivatives and chimeric proteins according to the current invention onto the GLP-1 receptor. In a 6-well plate 100,000 GLP-1 receptor transfected CHO cells are sown. The 20 cells grow until they are confluent. Then 2 mL 100,000 cpm "'in labeled peptide is added. In order to test the bonding it is then blocked with 20 pL of a 10- M GLP-1 solution. % Blocking MC10 80±3 MC11 85±3 MC12 77±6 20 The in vivo bio-distribution can, for example, be shown in rodents such as hairless mice. For this purpose GLP-1 transfected CHO cells are injected into hairless mice. After approx. 3 to 5 weeks tumors approx. 300 mg in size had grown. The mice are then injected in a tail vein with 37 MBq "'In labeled peptide according to 5 the invention and are measured after 4 h under a y-camera. In the course of this procedure there was quick clearance via the kidneys and an uptake in the kidneys. There was also a high uptake in the GLP-1 receptor positive tumor, whereas the GLP-1 receptor negative tumor barely showed any uptake. There was also a slight uptake in the pancreas; other organs showed no 10 visible uptake. Ex vivo bio-distribution studies are conducted in groups of 4 mice each, in which 555 kBq In-111 labeled MC10 is injected into the tail vein. 1, 4 und 24 h p.i. all the mice are killed and their organs removed. 15 The uptake of radioactivity is measured and the organs are weighed. The % of injected dose pro gram of organ weight is calculated. The results are as follows: Organ 1 h Stadev 4 h Stadev 24 h Stadev Blood 0.01 0.00 0.00 0.00 0 0.00 Liver 0.03 0.01 0.01 0.01 0.01 0.00 Stomach 0.11 0.10 0.13 0.0 0 0 0.06 Spleen 0.0: 0.02 0.01 0.00 0.01 0.00 Pancreas 0.581 0.50 062 0.28 0.3 7 0.2 Kidneys 7.9 3.62 7.41 3.56 4.85 3.05 Intestine 0.12 0.06 0.07 0.06 0.0 0.03 Lungs 0.80 0.56 0.3 0.17 0.22 0.07 Heart 0.02 0.01 0.01 0.01 0.00 0.00 Bones 0.02 0.04 0.01 0.00 01 0.01 Muscle 0.01 0.00 0.00 0.00 00 0.00 rumor - 0.03 0.031002 0.01 001 0.00 rumor+ 0.42 0.1910.1 0.30 02 0.16 Mean value from the bio-distribution with 4 mice pro group in % i.D./g Stadev: 20 standard deviation.

Claims

1. Peptide derivatives of exendin-3, wherein: (i) the N-terminus of the peptide derivatives binds to the GLP- 1 receptor; 5 (ii) the peptide derivatives comprise an amino acid sequence over 80% identical to the amino acid sequence of exendin-3: His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val Arg-Leu-Phe-lle-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser; (iii) the peptide derivatives comprise an attachment group A, which is located 10 at the C-terminus, and the attachment group A is an amino acid with a free amino group, or is an organic group comprising a free amine; and(iv) a chelator is coupled onto the attachment group A and suitable for labeling with a radionuclide, an MRI contrast agent, a fluorescent pigment, or a chemotherapeutic agent. 15

2. Peptide derivatives according to claim 1, wherein said peptide derivatives comprise an amino acid over 90% identical to the amino acid sequence of the peptide exendin-3.

3. Peptide derivatives according to claim 2, comprising the amino acid sequence of 20 exendin-3.

4. Peptide derivatives according to any one of claims 1 to 3, wherein the attachment group A is lysine. 25

5. Peptide derivatives according to any one of claims 1 to 3, wherein the attachment group A is ornithine.

6. Peptide derivatives according to any one of claims 1 to 5, wherein the chelator is selected from the group consisting of DTPA (Diethylenetriaminepentaacetic acid), N,N 30 Bis(2- [bis(carboxymethyl)amino] -ethyl)glycine), DOTA (1,4,7,10-Tetraazacyclodo decane-1,4,7,10-tetra-acetic acid), HYNIC (6-Hydrazinopyridin-3 -carbonic acid), MAG3 (mercaptoacetyl-glycylglycylglycine), N4 (1,4,8,11-tetraazaundecane) and derivatives thereof. 22

7. Peptide according to any one of claims 1 to 6, wherein the labeling is a coupling of radionuclides, MRI contrast agent, fluorescent pigments, and/or a chemotherapeutic agent. 5

8. Peptide derivatives according to claim 7, wherein the chosen fluorescent pigments are selected from the group consisting of Fluorescein, Rhodamine, Coumarin, BODIPY, Pyrene (Cascade Blue), Lucifer Yellow, Phycobiliprotein, Cyanine, AlexaFluor, Oregon Green, Texas Red, and their derivatives. 10

9. Peptide derivatives according to claim 7, wherein the radionuclide is selected from the group consisting of F-18, Cu-64, Cu-67, Ga-67, Ga-68, Y-86, Y-90, Tc-99m, In-111, 1-123, 1-124, 1-13 1, Lu-177, Re-186, Re-188, Pt-193m, Pt-195m, Ac-225, At 211, Bi-213, Sm-153, and Er-169. 15

10. Peptide derivatives according to claim 7, wherein the MRI contrast agent is selected from the group consisting of gadolinium, manganese, iron, europium, copper, nickel, chrome, prasodymium, dysprosium or holmium or their compounds or perfluorocarbone or F-19, H-1, P-31, and Na-19. 20

11. Peptide derivatives according to claim 7, wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents, ethylimine, nitrosourea, nitrogen mustard derivatives, folic acid analogs, purine analogs, pyrimidine analogs, podophyllin derivatives, taxane, vincaalcaloide, anthracycline, cytostatic antibiotics, platinum compounds, campthotecin derivatives, hormones, growth factors, interferons, 25 interleukins, cytostatic substances, and cytotoxic substances.

12. Peptide derivatives according to any one of claims 7 or 9, wherein the labeling via the coupling of radionuclides takes place through the saturation of the bonding sites with "InCL3. 30

13. Use of peptide derivatives according to any one of claims 1 to 12 for the manufacture of an agent for diagnosis or therapy of diseases, in which the GLP-1 receptor expression plays a role. 35

14. Use of peptide derivatives according to any one of claims 1 to 12 in order to determine the density of insulin producing cells in a tissue. 23

15. Use of peptide derivatives according to claims 1 to 12 in order to determine the expression of GLP-1 receptors or their density.

16. Agent for the diagnosis or therapy of diseases, in which GLP-1 receptor 5 expression plays a role, wherein the agent comprises a labeled peptide derivative according to any one of claims 1 to 12.

17. Agent for the diagnosis and therapy of diseases, in which GLP-1 receptor expression plays a role, wherein it comprises an unlabeled peptide derivative from 10 exendin-3 according to any one of claims 1 to 6.

18. Agent according to claim 16 wherein the labeling contains a coupling of radionuclides, MRI contrast agents, fluorescent pigments, and/or chemotherapeutic agents. 15

19. Use of an agent according to any one of claims 16 to 18 for diagnosis and therapy of diseases in which GLP-1 receptor expression plays a role.

20. Use of an agent according to any one of claims 16 to 18 for diagnosis and 20 therapy of neuroendocrine tumors (NET), insulinomas, or small cell bronchial carcinomas.

21. Use of an agent according to claim 16 in scintigraphy, PET, SPECT, MRI, optical diagnostic, receptor-mediated chemotherapy, receptor-mediated, cytostatic or 25 cytotoxic therapy, or radiopeptide therapy.

22. Peptide derivatives according to any one of claims 1 to 12; the use according to any one of claims 13 to 15 and claims 19 to 21; and the agent according to any one of claims 16 to 18, wherein the chelator is not a maleimidopropinamide moiety.