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The [18F]fluorocyclobutyl group has the potential to be a metabolically stable prosthetic group for PET tracers. The synthesis of the radiolabeling precursor cis-cyclobutane-1,3-diyl bis(toluene-4-sulfonate) 8 was obtained from epibromohydrin in 7 steps (2% overall yield). The radiolabeling of this precursor 8 and its conjugation to L-tyrosine as a model system was successfully achieved to give the new nonnatural amino acid 3-[18F]fluorocyclobutyl-L-tyrosine (L-3-[18F]FCBT) [18F]17 in 8% decay-corrected yield from the non-carrier-added [18F]fluoride. L-3-[18F]FCBT was investigated in vitro in different cancer cell lines to determine the uptake and stability. The tracer [18F]17 showed a time dependent uptake into different tumor cell lines (A549, NCI-H460, DU145) with the best uptake of 5.8% injected dose per 5 105 cells after 30 min in human lung carcinoma cells A549. The stability of L-3-[18F]FCBT in human and rat plasma and the stability of the non-radioactive L-3-FCBT in rat hepatocytes were both found to be excellent. These results show that the non-natural amino acid L-3-[18F]FCBT is a promising metabolically stable radiotracer for positron emission tomography.

Objectives: The aim was to investigate whether fluorocyclobutyl rings can be introduced into targeting probes to improve metabolic stability, while maintaining its binding affinity, using tyrosine as a model system for the LAT transporters. Methods: The precursor, cis-cyclobutane-1,3-diol ditosylate, its corresponding F-19 reference compound trans-3-fluorocyclobutanol (FCB), along with the cis-(3-fluorocyclobutyl)-tyrosine (3FCBT), were synthesized using standard organic chemistry methodologies. The non-radioactive 3FCBT was tested in competition and efflux stimulation cell assays using A549 human lung carcinoma cells with [3H]-D-Tyrosine. The metabolic stability of reference compound 3FCBT was studied in both rat hepatocytes and human plasma. Radiosynthesis methods using standard radiofluorination of the prosthetic group [18F]FCB and its conjugation to tyrosine gave the desired 3[18F]FCBT after chromatographic purification. In vitro studies were performed in A549 cells using 3[18F]FCBT and incubated at 37°C for 10, 20, 30 and 60 minutes with and without inhibitors fluoroethyl-tyrosine (FET) and non-radioactive 3FCBT. Results: The syntheses of cis-cyclobutane-1,3-diol ditosylate, trans-3-fluorocyclobutanol (FCB), along with the cis-(3-fluorocyclobutyl)-tyrosine (3FCBT) were established. 3FCBT was shown to block the uptake of [3H]-D-tyrosine in the competition cell assay and could stimulate the release of 3H]-D-Tyrosine from the cell in an efflux stimulation cell assay. 3FCBT showed very high stability in both rat hepatocytes (> 95%) and human plasma (> 95%). The unoptimized radiosynthesis gave the desired 3[18F]FCBT, via the prosthetic group [18F]FCB, in moderate yield (12%) with high radiochemical purity (> 99%). The cell uptake showed an increase of 3[18F]FCBT over time and reached a plateau of 5.87% after 30 minutes. Conclusions: The radiosynthesis of the prosthetic group [18F]FCB and its conjugation to tyrosine to give 3[18F]FCBT were successfully established. The introduction of 3[18F]FCBT into the LAT-targeting vector D-Tyr was characterized by a significant in vitro uptake in A549 cells and was actively transported into these cells. The encouraging results warrant further investigations of this tracer in the in vivo setting.

Introduction: The matrix protein tenascin-c (TN-C) represents an interesting target for molecular imaging in oncology due to it´s high abundance in a variety of human tumors such as lung, breast and brain tumors [1]. First radiolabeled anti-TN-C antibodies could successfully proof the concept of tumor-TN-C targeting [2]. However, a persistently high blood level hampers their use for targeted imaging. Aptamers, a class of rapidly clearing high affinity oligonucleotides, have been introduced successfully to molecular imaging by the application of the Tc-99m labeled TN-C Targeting Aptamer-1 (TTA-1) [3]. Here we report on the radiolabeling, binding affinity and biodistribution of this novel targeting aptamer labeled with metal PET isotopes. Experimental: Labeling with Ga(III), Y(III), and In(III) was performed in a one pot reaction after conjugation of a DOTA-type chelating moiety to TTA-1-MAG2. In-111 was included in the study as a surrogate for Ga and Y in long term stability and affinity tests. The labeling was carried out in the presence of the aptamer-chelator conjugate and the respective radiometal in acetate buffer. Degradation was investigated in human plasma employing PAGE. Affinity for human TN-C was determined in a nitrous cellulose filter binding assay. Athymic mice, bearing the human U251 tumor cell line, were injected with the radiolabeled TTA-1 derivatives. Results and Discussion: Addition of the radiometal salts yielded the respective labeled aptamer conjugates in high yields (60 – 80 %). Ultrafiltration of the products led to > 95 % of pure material as determined by PAGE and HPLC methods. The biological stability in human plasma ranged from 68 % (In-111) to 95 % (Y-86) of intact material after 6 hr. Binding affinity for human TN-C revealed a KD of 1 nM (In-111). A maximum tumor uptake of 2.0 % ID/g (Ga-68), 1 hr p.i. and T/Non Tumor ratios of 5.1 (T/blood), 0.2 (T/kidney), 0.7 (T/liver) 1 hr p.i. proved to be adequate to image the U251 tumor xenograft in mice. Tumor visualization was possible for both Ga-68 and Y-86 labeled TTA-1. Conclusion: The introduction of a DOTA-type chelating moiety could be successfully used to radiolabel the aptamer probe with In, Ga and Y. The stability against nuclease degradation along with a significant tumor accumulation, high T/ blood levels and the imaging capabilities in PET-scans make these compounds promising candidates for further evaluation as multi-tumor imaging agents. Acknowledgement: 1. R. Chiquet-Ehrismann, Cell 47, 131 (1986) 2. P. Riva, Cancer 73, 1076 (1994) 3. B.J. Hicke, J. Nuc. Med. 47, 668-678 (2006).

Aim: Aptamers (synthetic oligonucleotides), can be generated by a combinatorial approach (SELEX1) and are promising probes for radiodiagnostic imaging and therapy due to their high affinity and target specificity. The aptamer, TTA1 is characterized by a low nanomolar affinity and a high selectivity for the human matrix protein tenascin-C. Tenascin-C concentration in normal adult matrix tissue is rather low, whereas it is overexpressed in the stroma of a variety of malignant tumors2, making this target potentially suitable as a multi tumor imaging agent. The aim of this study was to evaluate the pharmacokinetic properties of 99mTc(I) and 188Re(I) carbonyl radiometal chelates attached to TTA1 in tumor animal models in vivo. Material and Methods: [Carboxymethyl-(2-ethylsulfanyl-ethyl)-amino]-acetic acid, an appropriate chelating unit for coordination of 99mTc(I) or 188Re(I), was conjugated to the aptamer and a 99mTc(I) carbonyl precursor was synthesized3. Labeling of the aptamer with the 99mTc(I) carbonyl precursor was achieved in 15 min at 100°C. Synthesis of the 188Re(I) carbonyl labeled aptamer was performed in a one pot reaction at 60°C for 30 min. Both, binding affinity for human tenascin-C and in vitro stability in human plasma were measured. The biodistribution and elimination of the 99mTc-tracer was evaluated in a human U251 xenograft NMRI nude mouse model. Results: The radiochemical purity of the products was >95 % after purification by spin dialysis. Reaction yields ranged from 65 % to 72 % for the 99mTc(I) carbonyl labeled aptamer and from 25 % to 35 % for the 188Re(I) carbonyl labeled aptamer. The specific activity of the Tc(I) carbonyl labeled aptamer was 37 MBq/nmol and the stability in human blood plasma proved to be 65 % after incubation at 37°C for 24h. The binding affinity of the compound against human tenascin-C lies in the low nanomolar range. Significant tumor uptake was observed in the U251 tumor xenograft model after i.v. injection of the 99mTc(I) carbonyl complex. Conclusion: Aptamers can be labeled with both 99mTc(I) and 188Re(I) carbonyls in acceptable yields and good purity. Further investigations are ongoing to fully characterize the potential of the compounds for their use as diagnostic and therapeutic agents. Lit: 1. C. Tuerk, L. Gold, Science, 249, 505-510, 1990; 2. H. P. Erickson, M. A. Bourdon, Annu. Rev. Cell Biol., 5, 71-92, 1989; 3. R. Alberto, R. Schibli, A. P. Schubiger, J. Am. Chem. Soc. 121,6076 - 6077, 1999

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Background: Intraluminal beta-irradiation has been shown to decrease neointimal proliferation after angioplasty in experimental models. The purpose of this study was to test the technical feasibility and biological effects of 186Re-labeled stents. Methods and results: Thirty-four New Zealand White rabbits were fed a 0.5% cholesterol diet before balloon angioplasty and insertion of Palmaz stents in the infrarenal aorta. The animals were killed 7 weeks after stent implantation. Two of 34 animals died prematurely (aortic leak, pneumonia). Control stents (n=7) were compared with 186Re stents (2.6 MBq [n=6], 8.1 MBq [n=5], 16 MBq [n=6], and 25.3 MBq [n=8]). Stent application was successful in all cases. No thrombus occlusion was observed. After 7 weeks, neointima formation was 2.2±0.2 mm2 in the control group. In the treatment groups, a dose-dependent neointima reduction was detectable (0.5±0.5 mm2 [2.6 MBq], 0.4±0.4 mm2 [8.1 MBq], and 0 mm2 [16.0 MBq, 25.3 MBq]). No induction of neointimal formation was observed at the edges of the stents. Radiation resulted in delayed reendothelialization. Conclusions: 186Re stents were capable of reducing neointima formation in a dose-dependent fashion. 186Re stents did not cause late thrombosis or neointimal induction at the stent margins in the observation period of 7 weeks.

OBJECTIVES: Restenosis is a major problem occuring after angioplasty, atherectomy and implantation of stents. It has been shown, that external beam teletherapy (X-rays) and intravascular brachytherapy (e.g. 192Iridium ribbon seeds, 188Re filled catheters, P-32-coated stents) prevents restenosis by inhibition of the proliferation of medial smooth muscle cells. The aim of this study was to investigate the potential of 186Rhenium labeled stents to prevent restenosis in an animal model. METHODS: New Zealand White rabbits were fed a 0.5% cholesterol diet four weeks prior to intervention. 186Re (T = 3.8 d) labeled Palmaz-stents with an activity of 25.6 ± 2.5 MBq (n = 11) were placed in the infrarenal aorta of rabbits after balloon denudation. Animals with implanted unlabeled stents served as controls (n = 11). Whole body scintigrams were obtained after 1, 4, 24 hours and after 7 and 14 days to determine the bleaching of 186Re from the stents in vivo. Seven weeks later, the animals were sacrified and morphometry and immunohistology was performed. RESULTS: More than 80% of the 186Re remained on the stent 14 days after implantation as determined by ROI analysis of the scintigrams. The neointimal area inducted seven weeks after implantation of the unlabeled stents was 2.2 ± 0.2 mm2. No neointima was detectable in the aorta of animals with implanted radiolabeled stents. No intraluminal accumulation of fibrin and fibroblasts directly at the stent struts implies that the delivered dose can even be reduced. CONCLUSION: Arterial implantation of 186Re labeled stents was feasible and stable in vivo. The activity of 25.6 ± 2.5 MBq totally inhibited neointimal formation. In further studies we will reduce the stent activity in order to determine the therapeutic window of 186Re labeled stents