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A 3-step methodology for the synthesis of 1,5-benzothiazepin-4(5H)-one dipeptidomimetics has been elaborated via an Ugi-4CR followed by a S-trityl deprotection and an intramolecular Cu(i)-catalyzed Ullmann condensation with moderate to good yields. In silico and NMR conformational studies showed that the lowest energy conformers stabilize γ- and β-turn structures.

The overexpression of neuropeptide receptors observed in many cancers provides an attractive target for tumor imaging and therapy. Bombesin is a peptide exhibiting a high affinity for the gastrin releasing peptide (GRP) receptor, which is overexpressed by a variety of tumors such as breast or prostate cancer. In the present study, we have evaluated if the bombesin analogue [N(alpha)-histidinyl acetate]bombesin(7-14), radiolabeled with the novel [99mTc(OH(2))(3)(CO)(3)]+, has the potential to be used as a diagnostic radiopharmaceutical. Receptor saturation studies, carried out on the GRP receptor-expressing PC-3 human prostate cancer cell line, revealed for [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) K(d) values in the subnanomolar range. Competitive binding assays, using the cold rhenium(I)-labeled analogue as a surrogate for the 99mTc-conjugate, also showed high affinity binding. Incubation of the radioconjugate with PC-3 cells resulted in a rapid temperature- and time-dependent specific internalization. At 37 degrees C more than 70% was internalized within the first 15 min and remained constant up to 2 h. Despite the weak proteolytic stability of [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) in vitro, biodistribution studies, performed in PC-3 tumor-bearing mice, showed low uptake in the tumor (0.89 +/- 0.27% ID/g 30 min pi) but high uptake into the pancreas (7.11 +/- 3.93% ID/g 30 min pi), a GRP receptor-positive organ. Blockade experiment (coinjection of 300 microg bombesin/mouse with the radioligand) showed specificity of the uptake. Despite the low tumor uptake, tumor-to-blood ratios of 2.0 and 2.7 and tumor-to-muscle ratios of 8.9 and 8.0 were obtained at 30 min and 1.5 h postinjection, respectively. The promising results merit the future in vivo investigation of 99mTc/188Re-tricarbonyl-labeled bombesin analogues.

4-([18F]fluoro)benzoyl-neurotensin(8-13) (18FB-Arg8-Arg9-Pro10-Tyr11- Ile12-Leu13-OH, 1) and two analogs stabilized in one and two positions (18FB-Arg8Psi(CH2NH)Arg9-Pro10-Tyr11- Ile12-Leu13-OH, 2, 18FB-Arg8Psi(CH2NH)Arg9-Pro10-Tyr11-Tle12-Leu13-OH, 3) were synthesized in a radiochemical yield of 25 - 36% and a specific activity of 5 - 15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumors, overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). All analogs exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumors, measured by quantitative receptor autoradiography, HT-29 and WiDr cells, and to sections of tumors derived from these cell lines in mice. The radiotracers were internalized in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca2+ of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumors, only a moderate uptake of the radioligands into the studied tumors was observed, presumingly due to degradation in vivo and fast elimination by the kidneys. In comparison with the other analogs, the specific tumor uptake expressed as tumor-to-muscle relation was highest for the radioligand 3. The blood clearance of 3 was reduced by co-injection of peptidase inhibitors. The catabolic pathways of the radiofluorinated peptides were elucidated. The results suggest that the high binding affinity to NTR1 and the stabilization against proteolytic degradation are not yet sufficient for tumor imaging by PET.

4-([18F]fluoro)benzoyl-neurotensin(8-13) (18FB-Arg8-Arg9-Pro10-Tyr11- Ile12-Leu13-OH, 1) and three analogues stabilized in one, two and three positions (18FB-Arg8Psi(CH2NH)Arg9-Pro10-Tyr11- Ile12-Leu13-OH, 2, 18FB-Arg8Psi(CH2NH)Arg9-Pro10-Tyr11-Tle12-Leu13-OH, 3, 18FB-Arg8Psi(CH2NH)Arg9-Pro10-DmTyr11-Tle12-Leu13-OH, 4) were synthesized in a radiochemical yield of 25 - 36% and a specific activity of 5 - 15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumours, overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). The analogues exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumours, measured by quantitative receptor autoradiography (IC50: 1 0.42±0.05 nM, 2 0.91±0.20 nM, 3 4.1±0.60 nM) , HT-29 (IC50: 24.0±4.2 nM) and WiDr cells and to sections of tumours derived from these cell lines in mice. The radiotracers were internalised in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca2+ of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumours, only a moderate uptake of the radioligands into the studied tumours was observed, presumingly due to degradation in vivo and fast elimination by the kidneys of rats and by the liver of mice. The pathway of the fast enzymatic degradation of the non-stabilized or not sufficiently stabilized 18F-labeled peptides was elucidated. The main catabolic product is the 18FB-Arg8Psi(CH2NH)Arg9-Pro10. In vivo, in comparison with the other analogues, the specific tumour uptake expressed as tumour-to-muscle relation was highest for the radioligands 3 and 4. The blood clearance of the analogues was reduced by co-injection of peptidase inhibitors. The 4-[18F]fluorobenzoyl label on the N-terminal arginine, the introduction of one reduced peptide bond and two bulky amino acid residues (Tle, DmTyr), are tolerable for a high in vitro affinity to the neurotensin receptor 1 (NTR1) and the ability to be internalised in receptor-positive tumour cells. Only the double and threefold-stabilized (3, 4) exhibited a remarkable specific tumour uptake which was, however, not sufficient for tumour imaging in the abdomen. Additional efforts are required to improve molecule stabilization against enzymatic attack and to enable receptor-based tumour imaging. This work was partially supported by the BIOMED grant BMH4-CT98-3198 of the EC.

4-HO-Phenylacetyl-[Lys-Psi(CH2=NH)-Arg-Phe11-t-Leu12-]NT(10-13) is a Neurotensin (NT) derivative with a Ki of 25 nM and is a good candidate for labelling with radio iodine. A fast and efficient radio iodination procedure by electrophilic substitution is proposed. 0.1 µmol of peptide together with 200 MBq of Na125I in 1 ml of PBS of pH 7.4 were added to an Iodogen coated vial. Labelling was performed at 20°C for 5 min. QC by HPLC and Sep Pak showed labelling yields >98%. The pure n.c.a. (75 GBq/mmol) compound was obtained after semipreparative HPLC purification followed by recovery from a RPC-18 Bonda Pak column. Overall synthesis yields were over 80%. Biodistribution of 150 kBq of the n.c.a. compound in HT29 bearing nude mice revealed tumour uptake to ±5% of the I.D./g 10 min p.i. which could be inhibited for 75% by competition with native NT. The in vitro half-life of the pure peptide in human blood is 67 h. Due to its excellent biological half-life and its acceptable biological properties, the proposed 123I labelled peptide is a good candidate as a new peptidergic SPECT tracer for NT receptor expressing pathologies.

The potential of radiolabelled neurotensin ana-logues for in vivo imaging of neurotensin (NT) receptor overexpressing tumours by PET was studied. Methods: Three newly synthesised 18F-radiolabelled NT(8-13) agonists, characterised by HPLC, mass spectrometry, and NMR were used for in vitro binding and internalisation stud-ies with HT-29 or WiDr cells. Ca2+ mobilisation was investigated using FLIPR measurements. Biodistribution studies were performed in nude mice bearing HT-29 or WiDr tumours without and with blocking of the NT receptors. Biokinetic studies were done in Wistar rats using PET. The specificity of the peptide binding to the tu-mour cells was studied in receptor blocking ex-periments. Results: The KD's of the NT analogues were in the nanomolar range. The Ca2+ mobilisation ef-fect of the compounds were between 99% and 129% compared to NT with EC50 between 17nM and 120nM. The tumour uptake of the peptides was moderate and was highest for the double stabi-lised peptide {4-[18F]fluoro-benzoyl-Arg-Psi-(CH2-NH)-Arg-Pro-Phe11-T-Leu12-Leu-OH (FB-P3)} (3.5% ID/g tissue 10 min p.i.). It was specifically accumulated in vitro and also in vivo in the tumours (70% inhibition by NT(8-13)). In nude mice the tumour was clearly visible with PET if the location was distant from the excre-tory organs. Conclusion: The 18F-labeled stabilised NT analogues are NT agonists and potential radiotrac-ers for imaging of neurotensin receptor ex-pressing tumours. However, structural optimisa-tion is still needed. (supported by the EC, Contract No. BMH4-CT98-3198)

The tridecapeptide Neurotensin (NT) exhibits a high specific affinity to receptors located on various tumours like small cell lung carcinoma or human colon carcinoma. The purpose of this study was to evaluate the potential of using succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for the specific radiolabelling of a non-lysine containing oligopeptide. This was demonstrated using the C-terminal bioactive hexapeptide NT(8-13) as well as its partially reduced stabilised congeners [Arg8ps(CH2NH)Arg9]NT(8-13), [Arg8ps(CH2NH)Arg9, Tle12]NT(8-13). The radiosynthesis of [18F]SFB started from ethyl trimethylammonium benzoate via a standard three-step procedure. Finally O-(N-succinimidyl) N,N,N',N'-tetramethyluronium tetrafluoroborate was used as activating agent to give [18F]SFB in 40 % overall radiochemical yield related to [18F]F- within 100 min (Wester et al. Nucl. Med. Biol. 1996, 23, 365). Our labelling experiments revealed that [18F]SFB reacts with selected N-terminal Arg-peptides with moderate to good chemoselectivities in aqueous buffered solutions (phosphate-borate buffer; pH 7.1 - 8.3). To shorten the period of time necessary for a complete consumption of [18F]SFB we found it advisable to heat the mixture to 40 - 50 °C for 20 min. The desired products were obtained after HPLC purification in radiochemical yields of 10 to 43 % (80 - 120 min) related to [18F]SFB. The conditions mentioned above allow the active ester to react specifically with the a-amino group of the N-terminal arginine unit. The radioactive product was identified by chromatographic comparison with a reference compound. The reactions carried out with nonradioactive SFB were shown to give the corresponding 4-FB peptides of which their structural identity was confirmed by proton NMR and MS studies. The labelled peptides display in vitro affinities to the Neurotensin receptor NTR1 in the nanomolar range (KD values: 1-9 nM). The accumulation of radioactivity in tumour tissue of tumour bearing mice in vivo is still moderate, demonstrated by tumour/blood and tumour/muscle relations of 2-8 after 30 min (calculated from % D/g tissue). This work is supported by the European Community (BMH4-CT98-3198)

A major impact in diagnosis and treatment of cancer with peptide based radiopharmaceuticals is expected. Among others neurotensin is considered to be a promising candidate. However, most neurotensin analogues, which bind to the neurotensin receptor have a too short biological half live due to catabolism. Therefore, stabilized fragments have been prepared and labeled with the newly developed [Tc(CO)3](+)-moiety. A single histidine or a (N alpha-His)-Ac group coupled to the N-terminus of the neurotensin fragments were used as a bidentate or a tridentate ligand respectively, which coordinate the metal carbonyl efficiently. Affinity and binding studies of the 99mTc(I) radiolabeled neurotensin fragments revealed a behavior influenced by catabolism and properties of the metal complex.