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21st International Symposium on Radiopharmaceutical Sciences

Poster: S75

075 Evaluation of a Novel Tc-99m-Labeled HYNIC-Conjugated Lactam Bridge-Cyclized Alpha-MSH Peptide for Human Melanoma Imaging Liqin Liu, Yubin Miao College of Pharmacy, University of New Mexico, Albuquerque, New Mexico, United States Objectives We previously reported 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex {hydrazinonicotinamide-8aminooctanoic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} as a very promising imaging agent for melanoma in B16/F1 murine melanoma mouse model. The purpose of this study was to determine the human melanoma targeting property of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex in M21 human melanoma-xenografts to facilitate its potential application for human melanoma imaging. Methods HYNIC-Aoc-Nle-CycMSHhex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of the peptide was determined in M21 human melanoma cells (1,281 receptors/cell). The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-AocNleCycMSHhex was determined in M21 human melanoma-xenografted nude mice. Results The IC50 value of HYNIC-AocNle-CycMSHhex was 0.48 ± 0.01 nM in M21 human melanoma cells. The M21 human melanoma uptake of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex was 4.03 ± 1.25, 3.26 ± 1.23 and 3.36 ± 1.48% ID/g at 0.5, 2 and 4 h post-injection, respectively. Approximately 92% of injected dose cleared out the body via urinary system at 2 h post-injection. 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. The M21 human melanomaxenografted tumor lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h post-injection. Conclusions 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited favorable human melanoma imaging property, highlighting its potential as an imaging probe for human metastatic melanoma detection. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S76: Poster

21st International Symposium on Radiopharmaceutical Sciences

076 111 In-Labeled tetrapeptides with high affinity and selectivity for mu opioid receptors. John R. Lever1, 2, Emily Fergason-Cantrell1, T Carmack2, L Watkinson2, Fabio Gallazzi3 1 Radiology, University of Missouri, Columbia, Missouri, United States, 2Radiopharmaceutical Sciences Institute, University of Missouri, Columbia, Missouri, United States, 3Structural Biology Core, University of Missouri, Columbia, Missouri, United States Objectives To develop metabolically stable 111In-labeled tetrapeptides for SPECT imaging that bind selectively and with high affinity to the peripheral mu opioid receptors expressed by normal tissues and over-expressed by breast and lung cancers. The novel peptide H-Dmt-D-Ala-Phe-Orn-NH2 (DAPO; Dmt = 2´,6´-dimethyltyrosine) was designed for conjugation with DOTA and labeling with indium-111. Methods DAPO was synthesized using Fmoc/t-Bu chemistry and purified by reversed-phase HPLC. DAPODOTA was obtained using differentially protected Fmoc-Orn(Mtt)-OH and (t-Bu)3DOTA to couple DOTA to the delta-amino group of Orn, and purified by HPLC. In(III) / 111In complexation was accomplished in 0.4 M NH4OAc at 100 °C. The In(III) complex was purified using a C-18 extraction cartridge, while the 111In complex was purified by reversed-phase HPLC. Binding assays were performed using standard techniques, and biodistribution studies were conducted using male CD1 mice under Institutional approvals. Results DAPO, DAPO-DOTA and In(III)-DAPO-DOTA were obtained in high purity, and exhibited appropriate mass spectral characteristics. 111In-DAPO-DOTA was obtained in 70% isolated yield. In vitro, DAPO showed very high mu affinity (Ki 0.033 nM) accompanied by 970-fold selectivity over kappa sites and 1818-fold selectivity over delta sites. In(III)-DAPO-DOTA also showed high affinity for mu sites (Ki 0.48 nM), with 4892-fold selectivity over kappa sites and 277-fold selectivity over delta sites. 111In-DAPO-DOTA gave 90% specific binding to mu receptors in guinea pig brain membranes, with inhibition by the mu selective DAMGO (IC50 5.0 nM). No inhibition was observed using kappa-selective U69,593 or delta-selective DPDPE at a 100 nM concentration. In vivo, 111In-DAPO-DOTA exhibited a modest level of specific binding, ca. 25% as defined by naltrexone blockade, to mu opioid receptors in mouse gut 30 min after i.v. administration. Conclusions Novel tetrapeptides DAPO and In(III)-DAPO-DOTA display high binding affinity for mu opioid receptors in vitro, with excellent selectivity over the kappa and delta types. 111In-DAPO-DOTA labels mu sites in brain membranes in vitro, and exhibits a modest level of specific binding to mu sites in mouse gut in vivo. Although the level of specific binding is low, this study may well be the first to show in vivo labeling of mu sites by a peptide radioligand. Acknowledgements We thank the United States taxpayers for their support through National Institutes of Health, National Cancer Institute grant R21 CA174873. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S77

077 Synthesis and Biological Evaluation of a [Re/99mTc(CO)3]+-Labeled Somatostatin Receptor-Seeking Antagonist Lauren Radford1, George Makris2, Fabio Gallazzi3, Ashley Szczodroski4, Timothy Hoffman4, 1, Michael R. Lewis4, Silvia S. Jurisson1, Dionysia Papagiannopoulou2, Heather M. Hennkens5 1 Chemistry, University of Missouri, Columbia, Missouri, United States, 2Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3Structural Biology Core, University of Missouri, Columbia, Missouri, United States, 4Research Service, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 5 Research Reactor Center, Columbia, Missouri, United States Objectives Novel neuroendocrine cancer imaging agents can be prepared by [99mTc(CO)3]+ labeling of tridentate chelating scaffolds that are covalently attached to somatostatin receptor (SSTR)-targeting biomolecules. L-sst2ANT is a chelator-peptide bioconjugate molecule comprised of a novel tridentate [NSN]-type ligand [L; 3-(2aminoethylthio)-3-(1H-imidazol-4-yl)propanoic acid] and an antagonist peptide with high SSTR2 (subtype 2) affinity [sst2-ANT1; 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2]. We used L-sst2-ANT to chelate [Re/99mTc(CO)3]+ and present here the in vitro and in vivo evaluation of the Re/99mTc-labeled complexes. Methods The SSTR2 binding affinity of ReL-sst2-ANT was measured in AR42J cells. L-sst2-ANT was radiolabeled using the [99mTc(CO)3(H2O)3]+ intermediate2 by heating at 75 °C for 30 min. In vitro stability of the HPLC-purified 99mTcL-sst2-ANT was determined by challenges with histidine and cysteine at 1 mM concentrations in PBS at physiological conditions over 12 h. Biodistribution and microSPECT/CT imaging studies were performed on SCID mice bearing AR42J tumors. Biodistribution data were obtained at 1, 4, and 24 h post-injection (pi), and images were captured at 1 and 4 h pi. Results The 99mTcL-sst2-ANT radiocomplex was synthesized in high yield (>98%), characterized by HPLC coinjection with the Re analogue (15 nM IC50), and shown to be stable against histidine and cysteine challenges in vitro. Biodistribution data showed tumor uptake of 2.3% ID/g at 1 h pi that increased to 3.2% ID/g at 4 h and was significantly blocked by co-administration of somatostatin-14. Slow clearance from blood was observed, along with slow hepatic and renal excretion, attributed to the lipophilicity of the Tc(CO)3-complex. MicroSPECT/CT images verified the biodistribution findings. Conclusions The novel 99mTcL-sst2-ANT complex successfully targets SSTR2-expressing tumors via a receptormediated process. Further studies with structurally-modified complexes designed for improved biodistribution profiles are ongoing. Acknowledgements We thank the University of Missouri, American Chemical Society, and the Department of Energy for funding support and gratefully acknowledge the support provided by the VA Biomolecular Imaging Center at the Harry S. Truman VA Hospital and University of Missouri. References 1Ginj, M. et al (2006) Proc Natl Acad Sci USA, 103, 16436-16441. 2Alberto, R. et al (2001) J Am Chem Soc, 123, 3135-3136.

J Label Compd Radiopharm 2015: 58: S1- S411

S78: Poster

21st International Symposium on Radiopharmaceutical Sciences

078 PET imaging of changes in copper metabolism associated with Alzheimer's disease: potential new diagnostics Julia B. Torres, Erica Andreozzi, Antony Gee, Philip J. Blower Imaging Chemistry and Biology, King's College London, London, United Kingdom Objectives Growing evidence suggests that a dysregulation in brain copper metabolism occurs in Alzheimer's disease [1][2].The aim of this project is to investigate copper homeostasis in Alzheimer’s disease in vivo using PET imaging to evaluate its potential as a diagnostic marker of the disorder. Methods Brain copper metabolism was investigated in TASTPM mice and controls (n=4, male, 6-8 mo) using the 64 CuGTSM complex (Fig.1A), which is capable of crossing the blood-brain barrier and releasing copper bioreductively within cells.Animals were intravenously injected with 64CuGTSM and scanned at 0-30 min and 2425 h post-injection (see Fig.1B).64Cu brain uptake was quantified by atlas-based image analysis and texture analysis.After imaging, animals were sacrificed and organs were removed and gamma-counted. Regional brain distribution of 64Cu was evaluated via autoradiography and compared to amyloid-β plaque deposition in TASTPM mice. Results PET ROI analysis revealed significant differences in 64Cu biokinetics within the CNS between TASTPM mice and controls. Atlas-based quantification and texture analysis showed substantial variations in 64Cu brain regional distribution and PET signal heterogeneity between the two groups of mice. Regional 64Cu distribution in the brain did not correlate well with amyloid-β burden in the TASTPM model. Conclusions This work has demonstrated the potential of 64CuGTSM as a radiotracer for the imaging of brain copper metabolism. It has also identified significant differences in 64Cu brain regional uptake and PET signal heterogeneity between TASTPM and controls that need to be further examined to reveal the potential for application of this approach as a diagnostic tool for Alzheimer's disease. Acknowledgements The authors acknowledge financial support from the Alzheimer's Society, Alzheimer's Research UK, King's Health Partners and Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award. References [1] Bush A. I. et al. Neurotherapeutics. 5, 3, 421–432 (2008) [2] Kozlowski, H. et al. Coord Chem Rev, 256, 2129–2141 (2012)

Fig.1. Structure of 64CuGTSM (A); Sagittal PET/CT MIP image illustrating 64Cu distribution in the brain of TASTPM mouse injected with 64CuGTSM at 30 min post-injection (B).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S79

079 99m Tc/Re-tricarbonyl complexes with tridentate N-(phosphonomethyl)iminodiacetic acid: a novel approach to new bifunctional chelating agents Jeffrey Klenc, Andrew T. Taylor, Malgorzata Lipowska Radiology and Imaging Sciences, Emory University, Atlanta, Georgia, United States Objectives Aminopolycarboxylate ligands, such as nitrilotriacetic acid, tend to form stable mononuclear complexes with the M(CO)3 core (M = 99mTc, Re) and provide an avenue for radiotracer development. Since phosphonates can also act as coordination sites, we studied N-(phosphonomethyl)iminodiacetic acid (PMIDA) as a representative chelator for the 99mTc/Re-tricarbonyl core. The PMIDA amine, acetate and phosphonate donor centers allow insertion of the chelate at any position along a peptide sequence through formation of amide or ester links. Methods A matched pair of 99mTc/Re-tricarbonyl complexes was prepared by reaction of PMIDA with [M(CO)3(H2O)3]+. Re(CO)3(PMIDA) was characterized by 1H and 31P NMR and MS as a structural analog of the 99m Tc tracer. The pH dependent formation of two possible linkage isomers was assessed by HPLC and NMR solution studies to determine the relative amount of each formed Re(CO)3 isomer. Stability of the M(CO)3(PMIDA) complexes was tested at physiological pH for 24 h. Results HPLC analysis of 99mTc(CO)3(PMIDA) showed a single peak (>98%), and its identity was confirmed by co-injecting with its Re analog . No measurable decomposition at pH 7 was observed for up to 24h. At low pH, the Re reaction was complete within 15 min at 70°C and in 3h at 25 °C giving one major product (1) as detected by HPLC (90%). 1H NMR confirmed the presence of only the isomer 1 resulting from the symmetrical coordination of PMIDA via the two carboxylates and the amine group. However, at pH 7-10, the product was isolated as a mixture of two isomers with a ratio of 3:2. 1H NMR revealed the unsymmetrical coordination of PMIDA in the isomer 2 through the phosphonate rather than the second carboxylate. No changes in this ratio were observed for 3 days at pH 7-10. However, at low pH the ratio quickly changed to 9:1 within 1h. Conclusions The combination of acetate and phosphate donor groups provides a novel approach for the development of new bifunctional chelating agents for radiolabeling specific biomolecules. Acknowledgements This work was supported by the National Institute of Health grant R37 DK038842. References

J Label Compd Radiopharm 2015: 58: S1- S411

S80: Poster

21st International Symposium on Radiopharmaceutical Sciences

080 Synthesis, Characterization and In Vitro Evaluation of New 99mTc/Re-cyclized Octreotide Analogues Yawen Li1, Lixin Ma2, 6, Vikram Gaddam1, Fabio Gallazzi3, Heather M. Hennkens4, Michael Harmata1, Michael R. Lewis5, 6, Carol A. Deakyne1, Silvia S. Jurisson1, 4 1 Chemistry, University of Missouri, Columbia, Missouri, United States, 2Department of Radiology, University of Missouri, Columbia, Missouri, United States, 3Structural Biology Core, University of Missouri, Columbia, Missouri, United States, 4Research Reactor Center, University of Missouri, Columbia, Missouri, United States, 5 Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States, 6 Research Service, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States Objectives Our interest in developing 186/188Re- and 99mTc-based therapeutic and diagnostic radiopharmaceuticals led to investigation of 99mTc/Re-cyclized octreotide analogues.1-3 Tc-99m/Re-cyclized thiolated-DPhe-Cys-TyrDTrp-Lys-Thr-Cys-Thr(OH) (99mTc/Re-SDPhe-TATE) were of interest because the thiolate at the peptide Nterminus may improve the stability of 99mTc/Re-cyclized Tyr3-octreotate (99mTc/Re-TATE) without compromising its somatostatin receptor subtype 2 (sst2) binding affinity.1-3 Methods Purified linear thiolated-DPhe-Cys-Tyr-DTrp-Lys-Thr-Cys-Thr(OH) (SDPhe-TATE) was reacted with [TBA][ReOCl4] in anhydrous DMF under Ar to synthesize Re-SDPhe-TATE. LC-ESI-MS, 2D NMR experiments and electronic structure calculations were employed to elucidate the molecular structure of the Re-peptide complex. The sst2 binding affinity of the Re-SDPhe-TATE isomers was evaluated in AR42J cells. Linear SDPheTATE was cyclized with [99mTcO]3+ by ligand exchange from 99mTc-glucoheptonate. The stability of 99mTc-SDPheTATE was evaluated in vitro. Results Four isomers of Re-SDPhe-TATE were observed by LC-ESI-MS analysis; the isomers exhibited 0.13 to 1.5 μM sst2 IC50 values in AR42J cells. NMR studies suggest [ReO]3+ is coordinated to three thiolates and the amide of Cys2 in isomers 1 and 4, whereas [ReO]3+ is coordinated to three thiolates and the amide of Tyr3 in isomer 2. Quantum chemical calculations clarified the stereochemistry of Re-SDPhe-TATE and the possible peptide arrangements around the [ReO]3+ core. Four isomers were observed for 99mTc-SDPhe-TATE with comparable HPLC retention times to the Re-SDPhe-TATE isomers. About 100% and 62% of 99mTc-SDPhe-TATE remained intact in PBS buffer and 1 mM Cys solution under physiological conditions at 6 h, respectively. Conclusions The effect of substituting a thiolate for the N-terminal amine on the sst2 binding affinity and stability of 99mTc/Re-TATE was investigated by evaluating 99mTc/Re-SDPhe-TATE. The Re-SDPhe-TATE isomers are less potent than Re-TATE but possess greater sst2 binding affinities compared to the previously reported NS3 Recyclized octreotide analogues.1-3 The 99mTc-SDPhe-TATE isomers have improved stability compared to 99mTcTATE but are more susceptible to ligand exchange than the NS3 analogues with three cysteines.1-3 Acknowledgements We thank the University of Missouri Mass Spectrometry and NMR facilities and Harry S. Truman Memorial Veterans' Research Service. References [1] Bigott-Hennkens, H. M., et al. J Med Chem 2008, 51, 1223-30. [2] Dannoon, S. F., et al. Nucl Med Biol 2010, 37, 527-37. [3] Bigott-Hennkens, H. M., et al. Nucl Med Biol 2011, 38, 549-555.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S81

081 Development of fluorine-18 and gallium-68 labelled aprotinin Charlotte L. Denholt, Troels E. Jeppsen, Christina Schjøth-Eskesen, Jacob Madsen, Nic Gillings Department of Clinical Physiology, Nuclear Medicine and PET, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark Objectives Technetium-99m aprotinin is used for imaging of amyloid deposits in patients [1]. Aprotinin consists of 58 amino acids arranged in a single polypeptide chain. As potential sites for radiolabelling the aprotinin sequence contains four lysine residues, which do not contribute to the inhibitory properties [2].Here we present fluorine-18 and gallium-68 labelling of aprotinin for PET imaging. Methods [18F]SFB was synthesised as previously described with minor modifications [3]. Aprotinin was reacted with [18F]SFB in a HEPES buffer (pH 7.8) using various temperatures and reaction times. The crude product was purified using a PD-10 column eluted with 0.9% NaCl in H2O (aq). For gallium-68 labelling, aprotinin was NOTA-conjugated by reaction with 10 and 100 equivalents of p-SCN-BnNOTA (0.1M sodium carbonate buffer pH 9.5, at 37°C for 20 h). After purification, the NOTA-aprotinin conjugates were radiolabelled with 25-30 MBq [68Ga]Cl3 in 0.7 M NaOAc buffer (pH 4.1 and 5.2) for 20 minutes at room temperature. Labelling efficiencies for [18F]aprotinin and [68Ga]NOTA-aprotinin were monitored using radio-HPLC with an Aeris PEPTIDE® XB-C18 column (0.1% TFA in H2O (A): MeCN (B); gradient: 8-60% B, flow 1.5 ml/min) and protein precipitation. Both products were also analysed using SDS-PAGE with aprotinin as a reference on the ladder. Results [18F]aprotinin was produced with overall radiochemical yields of 30-50 % (decay-corrected). The most optimal radiolabelling condition was 60°C for 20 minutes. The radiochemical purity was 61-64% before purification and >93 % after purification. The radiochemical purity of [68Ga]NOTA-aprotinin was >95% using 0.7M NaOAc buffer at pH=5.2 and 100 eq of the p-SCN-Bn-NOTA. Using 10 eq of p-SCN-Bn-NOTA the radiochemical purity was 85-90%. SDS-PAGE showed only one band for both [18F]aprotinin and [68Ga]NOTA-aprotinin, corresponding to the aprotinin reference band in the ladder. Conclusions We have successfully radiolabelled aprotinin with fluorine-18 and gallium-68 in good radiochemical yields and purities. [18F]aprotinin and [68Ga]NOTA-aprotinin will be evaluated as candidate radiopharmaceuticals for PET imaging of amyloid deposits. Acknowledgements We would like to thank Dr Malene Brandt-Larsen and Mrs. Annette Cortsen for excellent technical assistance. References [1] Smyth, DR, et al (2005), Nucl Med Biol 32, 885-889. [2] Kiernan, JA, et al (1973), Histochemie 34, 77-84. [3] Tang, G, et al (2008), J Label Compd Radiopharm 51, 68-71.

J Label Compd Radiopharm 2015: 58: S1- S411

S82: Poster

21st International Symposium on Radiopharmaceutical Sciences

082 In Vitro and In Vivo Studies Describing 68Ga/64Cu/90Y radiolabeled bivalent [RGD-Glu-(DO3A)-6-Ahx-RM2] Targeting GRPR/αvβ3 Biomarkers on Prostate Cancer Cells. Zongrun Jiang1, 5, Rajendra P. Bandari1, 2, Tamila S. Reynolds6, Rebecca Schehr7, Jingli Xu3, Yubin Miao3, Tammy L. Rold1, 4, Silvia S. Jurisson5, Charles J. Smith1, 2 1 Research Service, Truman VA, Columbia, Missouri, United States, 2Department of Radiology, University of Missouri School of Medicine, Columbia, Missouri, United States, 3University of New Mexico College of Pharmacy, Albuquerque, New Mexico, United States, 4Department of Internal Medicine, University of Missouri School of Medicine, Columbia, Missouri, United States, 5Department of Chemistry , University of Missouri, Columbia, Missouri, United States, 6University of Texas El Paso, El Paso, Texas, United States, 7University of Missouri College of Veterinary Medicine, Columbia, Missouri, United States Objectives The use of multivalent ligands to target more than one cell-surface receptor is of great interest due to their advantages over monovalent ligands. The primary objective of this research was to develop a heterodimeric ligand, [RGD-Glu-(64Cu/68Ga/90Y-DOTA)-6-Ahx-RM2], that is capable of targeting both αvβ3 (integrin biomarker) and GRPR (gastrin releasing peptide biomarker) that are co-expressed on prostate tumors for PET imaging and/or therapy of prostate cancer. RGD, with the amino acid sequence [Arg-Gly-Asp], is a peptide used for targeting the αvβ3 integrin. RM2, with the amino acid sequence [D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2], is a bombesin analogue for targeting the GRPR. Methods [RGD-Glu-DOTA-6-Ahx-RM2] was purchased and metallated with natural Cu, Ga, and Y to produce [RGD-Glu-(natCu/natGa/natY-DOTA)-6-Ahx-RM2]. The metallated conjugates were purified by RP-HPLC (reversed-phase high-performance liquid chromatography) and characterized by ESI-MS (electrospray ionizationmass spectrometry). In vitro studies of the metallated ligands were conducted on U87-MG (αvβ3 positive) and PC-3 (GRPR positive) cells to determine the receptor binding affinity. Radiolabeling of the conjugate with 64Cu-, 68Ga-, or 90YCl3 produced [RGD-Glu-(64Cu/68Ga/90Y-DOTA)-6-Ahx-RM2]. The new tracers were evaluated in vivo in normal CF-1 mice and in the PC-3, tumor-bearing, severe combined immunodeficient (SCID) mice. Results The metallated conjugates were produced in high radiochemical yield and displayed very high binding affinity for GRPR (8.64 ± 2.16, 7.78 ± 2.42, and 5.65 ± 0.00 nM; Cu, Ga, Y, respectively) in in vitro assays. In vitro assays in human, glioblastoma U87-MG cells indicated moderate binding affinity for the αvβ3 integrin (308 ± 42.6, 307 ± 0.0, and 346 ± 5.3 nM; Cu, Ga, Y, respectively). In vivo studies for the 90Y-radiolabeled ligand in PC-3 tumor-bearsing SCID mice showed high tumor uptake (8.70 ±0.35 %ID/g) at 1h p.i. and good tumor retention (5.28 ±0.12%ID/g) at 24h p.i. Low accumulation and rapid clearance of tracer in normal tissues was observed. Conclusions Favorable pharmacokinetics for the 90Y-radiolabeled tracer in in vivo studies support further investigations for microPET imaging or therapy in animal models. In vivo studies for 64Cu/68Ga-radiolabeled tracers are underway and results will be presented. Acknowledgements Research funding was provided by the University of Missouri Department of Radiology and the Truman VA, Columbia, MO. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S83

083 PET - imaging using phage display derived bicyclic peptide scaffolds Matthias Eder1, Daniel Teufel2, Ulrike Bauder-Wüst 1, Silvia Pavan2, Ed Walker 2, Ursula Schierbaum3, Katerine van Rietschoten 2, Catherine Stace2, Helen Rapley2, Uwe Haberkorn3, Spencer Campbell2, Klaus Kopka 1 1 Radiopharmaceutical Chemistry, German Cancer Research Center, Heidelberg, Germany, 2Bicycle Therapeutics Limited, Cambridge, United Kingdom, 3CCU Nuclear Medicine, German Cancer Research Center, Heidelberg, Germany Objectives Phage display-derived bicyclic low molecular weight peptide scaffolds were found to bind with excellent affinity and specificity to protein targets [1]. Thus, radiolabeled with a positron emitter this strategy represents a highly attractive platform for the design of radiopharmaceuticals for PET imaging of various oncological entities and for therapeutic applications. In the present work, bicyclic peptides directed against the tumor associated matrix metalloproteinase 14 (MMP-14, also known as MT1-MMP) were developed and preclinically characterized with regard to their clinical potential as MMP-PET imaging agent. Methods Conjugation with tris-(bromomethyl)benzene via the three reactive cysteines of a partly randomized amino acid sequence and fusion to the phage gene-3-protein generated a library of bicyclic peptide conjugates with two peptide loops [1]. A subsequent affinity selection process yielded several binders with variants showing single digit nanomolar affinity and high specificity for MMP-14. The selected optimal variant was conjugated to DOTA and analyzed preclinically according to its cell binding and in vivo tumor-targeting properties. Organ distribution, dose study and µPET imaging was performed using HT1080 tumor-bearing nude mice. Results Besides high proteolytic stability, the selected compound DOTA-N144 revealed high specificity and nanomolar affinity (KD= 0.5 nM) as determined by fluorescence polarization competition assays for the extracellular hemopexin domain of MMP-14 and MMP-14-expressing tumor cells. Organ distribution at 30 min. p.i. of the optimal dose of 150 pmol showed a specific tumor uptake of 4.99 ± 0.21 %ID/g (0.51 ± 0.09 %ID/g for non-binding scrambled variant) and fast background clearance (< 1 %ID/g for all organs except kidneys) resulting in high imaging contrast in the µPET studies. Conclusions The phage display selection of bicyclic peptides was shown to be a highly flexible and attractive strategy for generating and developing peptidic drug leads. With the Bicyclic DOTA-compounds being roughly 2000 Da in size, organ distribution studies revealed MMP-14-selective tumor uptake in the xenograft model and rapid clearance from non-target crucial organs resulting in high imaging contrasts. Directed against tumor specific membrane-type biological targets such as MMP-14 and labeled with radiometals these molecules represent highly promising radiopharmaceuticals for future clinical PET imaging and radioendotherapy of various oncological diseases. Acknowledgements References [1] Heinis C, et al (2009) Nat Chem Biol, 5(7), 502-07.

J Label Compd Radiopharm 2015: 58: S1- S411

S84: Poster

21st International Symposium on Radiopharmaceutical Sciences

084 Monitoring Breast Cancer Resistance Protein (BCRP)-Mediated Functional Transport Activity using 68GaGalmydar Scott E. Harpstrite, Jothilingam Sivapackiam, Stephen Mattingly, Vijay Sharma Radiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, United States Objectives Radiopharmaceuticals capable of imaging the functional transport activity of breast cancer resistance protein (BCRP, ABCG2) are desired to assist in the stratification of chemotherapeutic choices. Galmydar, a fluorescent and moderately hydrophobic Ga(III) monocationic complex, was evaluated as a substrate capable of monitoring BCRP functional transport activity. To assess the sensitivity and specificity of Galmydar to probe BCRP-mediated efflux activity in cellulo, cellular accumulation was determined in transfected cells. To evaluate the potential for Galmydar to enable molecular imaging of BCRP-mediated functional transport activity at the blood brain barrier (BBB) in vivo, microPET/CT imaging was performed in gene-knockout mouse models. Methods For cellular accumulation studies, live-cell fluorescence microscopy and 67Ga-radiotracer uptake assays were performed. Galymdar, or its 67Ga-labeled counterpart, was incubated in HEK293 cells stably transfected with BCRP (HEK293/BCRP), or control plasmid, at 37°C under a continuous flux of 5% CO2 atmosphere, in the presence or absence of 5 µM Ko143, a potent BCRP antagonist. For live-animal imaging, microPET/CT brain imaging of 68Ga-Galmydar was performed in ABCG2-/- (BCRP knockout, KO) and mdr1a/1b-/-ABCG2-/- (PgpBCRP triple knockout, tKO) mouse models, 15 and 60 min post-intravenous injection. Results Cellular accumulation profiles of Galymdar in HEK293 cells, measured via live-cell fluorescence microscopy and 67Ga-radiotracer uptake assays, are inversely proportional to BCRP expression. BCRP antagonist Ko143 induces accumulation in HEK293/BCRP cells, observed in both methods, indicating target sensitivity and specificity. Finally, microPET/CT imaging shows higher initial penetration and retention of 68Ga-Galmydar in brains of KO and tKO mice compared to their age-matched WT counterparts. Conclusions Live-animal imaging data coupled with cellular accumulation data establishes Galmydar as a template scaffold for development of a PET tracer for imaging BCRP-mediated functional transport in vivo. Acknowledgements We thank Julie L. Prior for technical assistance. Financial assistance to this work was provided by grants from the National Institutes of Health in part by RO1 HL111163 (VS), R33 AG033328 (VS), American Health Assistance Foundation (A2007-383; VS), and P50AG05681 (PI: Morris, Pilot: VS). References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S85

085 [94mTc] labeled peptide targets Met in human gastric carcinoma (MKN-45, SNU-16) and glioblastoma (U87MG) cells and xenografts Elaine M. Jagoda1, Osama Elbuluk1, Mark Williams1, Karen Wong1, Biying Xu2, Lawrence P. Szajek3, Peter Choyke1 1 Molecular Imaging Program, National Cancer Institute, Bethesda, Maryland, United States, 2Imaging Probe Development Center, National Heart Lung and Blood Institute, Bethesda, Maryland, United States, 3PET Dept, CC, National Institutes of Health, Bethesda, Maryland, United States Objectives The overexpression of hepatocyte growth factor receptor (Met) in cancers has been linked to increased proliferation, progression and drug resistance.An imaging agent assessing Met status would aid in diagnosis, patient selection for Met-targeted therapies and monitor therapeutic responses. Previously, we described a peptide targeting Met labeled with [99mTc] (AH113018) which exhibited nM affinity and targeting to warrant further development for human use [1]. As PET may offer increased sensitivity compared to SPECT, this Met peptide (MetP) was labeled with the shorter lived PET isotope, [94mTc] (52.5 min;[94mTc]MetP) and evaluated in vitro and in vivo with high (MKN-45, MK), medium (SNU-16, SN) and low (U87-MG, U87) Met expressing cells and mouse xenografts. Methods [94mTc]MetP was synthesized with modification of a [99mTc]chelate kit method (GE Healthcare Life Sciences, Oslo, Norway) using [94mTc]O4- (NIH Cyclotron facility) [2] and then evaluated in cell binding and biodistribution/PET imaging studies with MK, SN or U87 cells or xenografts. Results [94mTc]MetP exhibited high affinity (nM) and provided estimates of Met expression consistent with known expression in MK, SN and U87 cells. [94mTc]MetP uptakes in MK xenografts were rapid with the highest uptakes observed in tumor and kidneys at all times. MK tumors had the highest tumor:muscle ratios (T:M) at all times, whereas SN and U87 T:M were lower (2 to 10 fold) (Table1, T1). MK and SN T:M were relatively stable from 60 to 120 min due to greater tumor retention compared to muscle (T1). MK T:M were lowered ~60% when blocked with MetP (50 µg) indicating specfic Met targeting in vivo (T1). In images MK and SN tumors were visualized whereas U87 tumors were not (Fig1); imaging T:M ratios were found comparable to the biodistribution T:M ratios. Conclusions [94mTc]MetP bound to Met with high affinity and had uptakes correlating with Met expression levels in vitro and in vivo. These results suggest that [94mTc]MetP may have potential to identify patients whose tumors express moderate-high levels of Met in tumors and therefore, who may benefit from Met-targeted therapies. Acknowledgements References [1] Jagoda EM, et al, (2011), J Nucl Med, 51,140P, [2] Szajek LP et al, (2003), Radiochim Acta, 91, 613.

J Label Compd Radiopharm 2015: 58: S1- S411

S86: Poster

21st International Symposium on Radiopharmaceutical Sciences

086 PET Imaging of Tenascin-C with a Radiolabeled Single-Strand DNA Aptamer Orit Jacobson, Xiaoyuan Chen National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, United States Objectives Tenascin C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that Tenascin C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting Tenascin C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral Tenascin C expression in vivo. In order to address the need for an agent to image and quantify Tenascin C, we report the development of a radioactive PET tracer based on a Tenascin C specific single stranded DNA aptamer (Tenascin C aptamer). Methods Tenascin C aptamer was radiolabeled with 18F and 64Cu was found to be stable in vitro and in vivo. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of Tenascin C aptamer were performed in comparison to a non-specific scrambled aptamer (Sc aptamer). Results The labeled Tenascin C aptamer provided clear visualization of Tenascin C-positive but not Tenascin Cnegative tumors. The uptake of Tenascin C aptamer was significantly higher than that of Sc aptamer in Tenascin Cpositive tumors. The labeled Tenascin C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. Conclusions Our data suggest that suitably labeled Tenascin C aptamer can be used as a PET tracer to image tumor expression of Tenascin C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring. Acknowledgements NIBIB/NIH Intramural Research Program. References

Representative coronal PET image of U87MG tumor mouse at 1 h post-injection of64Cu-NOTA-Tenascin Captamer.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S87

087 DFO* - An Octadentate Bifunctional Chelating Agent for the Development of Stable Zirconium-89 Based Molecular Imaging Probes Thomas L. Mindt2, Malay Patra1, Gilles Gasser1, Andreas Bauman2, Seraina Hügli1, Danielle J. Vugts3, Guus A. van Dongen3 1 Department of Chemistry, University of Zurich, Zurich, Switzerland, 2University of Basel, Radiopharmaceutical Chemistry, Basel, Switzerland, 3VU University Medical Center , Amsterdam, Netherlands Objectives Zirconium-89 (89Zr) is an emerging metallic radionuclide with promising characteristics for PET, in particular for immuno-PET. [1] A major limitation of the use of 89Zr in nuclear medicine is the lack of appropriate methods for the stable chelation of the radiometal. To date, 89Zr -based imaging probes are obtained exclusively through derivatives of desferrioxamine (DFO), a chelator which does not complete the octadentate coordination sphere of the radiometal. There is compelling preclinical evidence that the incomplete coordination of 89Zr by DFO is responsible for the observed instability of the chelate in vivo. The release of the radiometal from the tracer results in the non-specific accumulation of the radiometal in radiation sensitive bones, a potential safety issue for clinical applications. Methods Herein, we wish to report the rational design, DFT-calculation guided synthesis, and in vitro evaluation of the first octadentate bifunctional chelating agent (BFCA) for the development 89Zr-based radiopharmaceuticals with an improved stability profile. [2] Results In comparison to DFO, the novel chelating system (termed DFO*) exhibits similar radiolabeling characteristics suitable for applications to delicate biomolecules (e.g., antibodies) but a remarkably improved stability as demonstrated by, e.g., transmetallation challenging experiments.First results of the application of DFO* to the radiolabeling of antibodies will be presented. Conclusions The novel DFO* bifunctional chelating agent is a promising candidatefor the development of Zr89 labelled immuno PET agents. Acknowledgements This work is supported by the Swiss National Science Foundation. References [1] Fischer, G. et al. C. Molecules 2013, 18, 6469 [2] Patra M. et al.Chem. Commun. 2014, 50, 11523

J Label Compd Radiopharm 2015: 58: S1- S411

S88: Poster

21st International Symposium on Radiopharmaceutical Sciences

088 Influence of chelators on targeting properties of 111In and 68Ga labeled GRPR antagonist Anna Orlova, Zohreh Varasteh, Bogdan Mitran, Maria Rosestedt, Irina Velikyan, Ulrika Rosenström, Gunnar Lindeberg, Jens Sörensen, Mats Larhed, Vladimir Tolmachev Uppsala University, Uppsala, Sweden Objectives Gastrin-releasing peptide receptors (GRPR) are upregulated in prostate cancer and could serve as molecular targets for diagnostic imaging using radiolabeled bombesin (BN) analogues. We have previously evaluated a high-affinity, antagonistic BN analog (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) conjugated to NOTA via a diethylene glycol (PEG2-RM26).We aimed to study influence of chelators on the biodistribution of PEG2-RM26 with 68Ga and 111In. Methods A series of PEG2-RM26-based analogs with NOTA, NODAGA, DOTA, DOTAGA were synthesized and radiolabeled with 68Ga and 111In. Stability, specificity, affinity, cellular processing, and pharmacokinetics were studied. Results The conjugates were synthesized, stably labeled with 68Ga and 111In with retained binding specificity. IC50 were in low nanomolar range. Biodistribution studies showed rapid clearance from blood and normal organs including that’s with GRPR expression with renal excretion. In mice bearing PC-3 tumors conjugates demonstrated specific uptake in tumors. The influence of chelators on biodistribution of conjugates was essential and differed between 68Ga and 111In. 68Ga-labeled conjugates 2 h pi had radioactivity uptake in tumors higher than in normal organs. 68Ga-NOTA-PEG2-RM26 provided superior tumor-to-organ ratios. 111In-labeled conjugates with NOTA and NODAGA had radioactivity uptake in liver, kidneys, pancreas and intestinal tissue close to that in tumors. 111 In-DOTA-PEG2-RM26 provided superior tumor-to-organ ratios 4h pi (Figure 1). Conclusions NOTA was the superior chelator for 68Ga labeling of RM26 (NOTA>NODAGA>DOTA> DOTAGA) and DOTA was the superior chelator for 111In labeling (DOTA>DOTAGA>NOTA>NODAGA). Acknowledgements References

SPECT/CT images of GRPR expression in PC-3 tumor bearing mice 4 h pi

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S89

089 PET-MRI dual-modal iron oxide based nanoparticles for targeting asialoglycoprotein receptors and CD206 Bo Yeun Yang1, Sung-Hyun Moon1, Min Jeong Jeon2, Yun-Sang Lee1, Dong Soo Lee1, June-Key Chung1, Young Il Kim2, Jae M. Jeong1 1 Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2Institute of Radiation Medicine, SNUMRC, Seoul National University College of Medicine, Seoul, Korea (Republic of) Objectives PET-MRI hybrid system is emerging for diagnosis because of its high sensitivity and accuracy. In this study, we developed iron oxide nanoparticle (IO NP) based PET-MRI dual imaging probes for targeting asialoglycoprotein receptor and CD206 by an encapsulation method using specially designed amphiphiles [1]. Methods Functionalized amphiphiles were synthesized by conjugating SCN-Bn-NOTA, SCN-Bn-Mannose or Lactose with stearylamine. NOTA-IO-Man and NOTA-IO-Lac were prepared by incorporating IO NP with Tween 60 and the synthesized amphiphiles for targeting CD206 and asialoglycoprotein receptor, respectively. Size of the prepared NP was analyzed by DLS and TEM. Stability in human serum was checked after labeling with 68Ga. T2weighted MR (3.0 T) signals were measured using Ferumoxide as a control. After i.v. injection into mice, PET and MRI studies were performed. The presence of IO in tissue was.check by Prussian blue staining Results Surface modified IO NPs showed narrow size distribution (NOTA-IO-Man=10.5±1.3; NOTA-IOLac=10.7±1.62 nm). Both probes were labeled with 68Ga in high yield (>99%) and was stable in human serum. The calculated relaxivity coefficient values were higher than Ferumoxide (NOTA-IO-Man=449.9; Ferumoxide=180.38 mM-1S-1). According to PET, both NPs showed high liver uptakes, but 68Ga-NOTA-IO-Man showed higher spleen uptake as expected (Liver/Spleen ratio, 68Ga-NOTA-IO-Man=1.776; 68Ga-NOTA-IO-Lac=1.533). Decreased MR signals in livers were found after administration of NPs (Fig. 1). The MR signal decrease in spleen was more significant with 68Ga-NOTA-IO-Man than with 68Ga-NOTA-IO-Lac. Existence of ferric iron was confirmed in the extracted liver and spleen. Conclusions We developed PET-MRI imaging probes for targeting asialoglycoprotein receptor and CD206 using facile encapsulation method. Both of them showed specificity in both PET and MRI studies. PET was superior in quantification and MRI in resolution. Acknowledgements This research was supported by grants NRF-2013R1A2A1A05006227 and NRF2012M2A2A7035853. References [1] Lee YK, et al (2012) J Nucl Med, 53, 1462-70.

Figure 1. In vivo images of 68Ga-NOTA-IO-Man and 68Ga-NOTA-IO-Lac after i.v. injection into mice. a) PET and b) MRI.

J Label Compd Radiopharm 2015: 58: S1- S411

S90: Poster

21st International Symposium on Radiopharmaceutical Sciences

090 2,2'-Bipyridine Containing Peptides as β-Sheet Mimics Coordinated to Re/Tc(I) William L. Turnbull1, Jinqiang Hou2, André H. St. Amant2, Leonard Luyt1, 2 1 University of Western Ontario, London, Ontario, Canada, 2London Regional Cancer Program, London, Ontario, Canada Objectives Protein-protein interactions are often mediated by secondary structural elements such as α-helices and β-sheets [1]. The design of peptidomimetics to mimic these structural elements is essential to effectively target these interactions in the design of diagnostics and therapeutics. Previously we have shown that incorporation of Re/Tc(I) into a cyclic β-sheet peptidomimetic is possible without interfering with secondary structure [2]. The objective of this project is to design and synthesize a novel amino acid containing bipyridine to allow for the incorporation of a bipyridine chelator into a peptide by standard solid-phase methods. The bipyridine residue acts as a bidentate chelator for Re/Tc(I), as well as to form a β-sheet conformation by mimicking a turn region. Methods The bipyridine amino acid was synthesized by Ullmann coupling of 3-nitro-2-chloropyridine, followed by reduction to the diamine, addition of a succinic group to one of the amines, followed by protection of the free amine with an Fmoc group. Peptides were synthesized using Fmoc solid-phase peptide chemistry, and purified using RP-HPLC and analyzed by ESI-MS, 2D NMR, and CD spectroscopy. Peptides were coordinated with Re(CO)3+ and the resulting complexes purified by RP-HPLC and analyzed by ESI-MS and CD spectroscopy. Peptides were also radiolabelled with 99mTc(CO)3+ and analyzed by HPLC and the retention time compared to the Re standard. Results The bipyridine amino acid was synthesized in 4 steps with >95% purity. Bipyridine peptides were synthesized in >95% purity and characterized by ESI-MS. Further characterization by CD spectroscopy and 2D NMR suggests the presence of β-sheet-like secondary structure. Coordination of the peptide to Re(CO)3+ was performed in water using [Re(H2O)3(CO)3]OTf, followed by the addition of DMAP to form a [2+1] complex and the coordination confirmed by ESI-MS. Characterization by CD spectroscopy suggests the retention of the secondary structure upon complexation to Re. Coordination to 99mTc(CO)3+ was performed by reduction of 99m TcO4- to [99mTc(H2O)3(CO)3]+ and then following similar conditions as for Re(CO)3+. HPLC analysis showed a peak with a similar retention time as the Re standard. Conclusions Peptides containing bipyridine are able to mimic β-sheet secondary structure and retain this secondary structure upon coordination to Re(CO)3+. This method of forming a β-sheet mimic may have future value for the development of receptor targeted 99mTc(CO)3+ imaging probes. Acknowledgements Natural Sciences and Engineering Research Council; Ontario Graduate Scholarship to WT. References [1] Whitby, LR et al. (2012) Acc Chem Res, 45, 1698-1709. [2] Hickey, JL et al. (2014) Chem Eur J, DOI: 10.1002/chem.201404774.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S91

091 New zinc folate derivative useful to prepare Tc-99m radiopharmaceuticals with high specific activity Joan Suades, Jordi Borràs Department of Chemistry, Universitat Autonoma de Barcelona, Cerdanyola del Valles, Spain Objectives We previously reported that the transmetallation reaction between zinc bioconjugated derivatives and the technetium carbonyl [99mTc(H2O)3(CO)3]+ is a useful reaction to prepare technetium radiopharmaceuticals with high specific activity [1]. The objective of the present work is to prepare a new zinc bioconjugate derivative of folic acid (1) that can be used to prepare new technetium and rhenium radiopharmaceuticals with this biomolecule following this new approach. Methods The HMDA-folic acid compound (2) was prepared using previously reported methods. In a first step, the HMDA-Boc-Folic acid intermediate was synthesised by reaction between HMDA-Boc and folic acid (1) [2]. Next, the reaction between this compound and trifluoroacetic acid [3] yielded to compound (2). Finally, the zinc folate derivative (4) was prepared by means of the reaction between the succinimidyl ester of the zinc(II) dithiocarbamate of isonipecotic acid (3) and compound (2) using previously reported methods [1]. Results The activated ester group of the zinc complex (3) reacts with the amine group of the HMDA-folic acid compound (2), which was synthesised from folic acid (1) using previously reported methods [2, 3], to yield the target metal complex (4). This reaction is performed at room temperature and the zinc complex (4) is obtained as a yellow powder with a good yield (70 %) and it can be isolated from the reaction medium a cause of its low solubility. The IR and 1H NMR spectroscopic data agrees with the proposed structure and the ESI HRMS analysis shows in the negative mode a signal at 787.1803 (m/z) that it is consistent with the stoichiometry of the [ML]+ fragment of complex (4) (calculated for C32H39N10O6S2Zn: 787.1792 m/z). Studies of transmetallation reactions between complex (4) and [M(H2O)3(CO)3]+ (M= Re, 99mTc) are in course. Conclusions We have successfully synthesised a new bioconjugated zinc complex of folic acid useful to prepare radiopharmaceuticals with high specific activity. Acknowledgements Supported by BIO2012-39682-C02-02 and 2014SGR-00423. References [1] Lecina, J. et al (2012) Organometallics, 31, 5884-5893. [2] Baier, G. et al (2012) Biomacromolecules, 13, 2704-2715. [3] Schneider, R. et al (2005) Bioorg. Med. Chem., 13, 2799-2808.

Scheme of synthesis of the new zinc folate derivative

J Label Compd Radiopharm 2015: 58: S1- S411

S92: Poster

21st International Symposium on Radiopharmaceutical Sciences

092 1,4,7-triazacyclononane ligands as versatile platform for radiocopper-labeled agents Kritee Pant1, Holger Stephan1, Ralf Bergmann1, Jens Pietzsch1, Jörg Steinbach1, Bim Graham2, Leone Spiccia2 1 Institute of Radiopharmaceutical Cancer Research, HZDR, Dresden, Germany, 2Monash University, Melbourne, Victoria, Australia Objectives Pyridine containing bifunctional chelating agents (BFCA) based on 1, 4, 7 – triazacyclononane backbones (DMPTACN) are suitable platforms for copper (II) complexes. These ligands rapidly form highly stable CuII complexes that are resistant to metal leaching. The ligand structure enables convenient introduction of additional conjugatable groups like -COOH groups to allow coupling to various biomolecules. Since 64Cu is a highly useful PET radioisotope because of its intermediate half-life (64Cu, t1/2 -12.7 h) , these conjugatable DMPTACN chelators can be coupled with various vectors for e.g., proteins, dendritic polymers in order to understand their detailed in vivo pharmacokinetic properties with respect to their ADME behavior. The current objective of the work was to synthesize new maleimide and -SCN derivatives of DMPTACN ligands which could be employed to be conjugated to anti-inflammatory dendritic polyglcerol sulfate (dPGS) & neutral dPG derivatives in order to study the metabolic fate of these potentially therapeutic polymers using positron emission tomography. Methods DMPTACN was synthesized by a 10-step process using an established protocol. Two coupling groups such as maleimide (1) or isothiocyanate (2) have been attached for further conjugation. These ligands were then conjugated to the thiolated dPGS via Michael addition of 1 and also via direct labeling of 2 to dPGS amine to yield highly stable conjugates. 64Cu was produced following an established protocol with high specific activity. 64Culabeling of the conjugates were performed using [64Cu]CuCl2 at ambient temperature in aqueous buffer solution (0.1 M MES/NaOH) and resulted in a radiochemical purity of ≥99% within a few minutes. Challenge experiments were conducted in presence of EDTA or copper seeking superoxide dismutase (SOD) for evaluation of in vitro stability. Biodistribution and PET studies were conducted in male Wistar rats. Results DMPTACN-dPG/S conjugates form highly stable metal complexes with 64Cu under mild conditions (ambient temperature, aqueous solution) showing resistance to demetalation in vitro and high in vivo stability. Biodistribution data and PET experiments show a charge dependant excretion of the dPGS and dPG macromolecules. Conclusions DMPTACN ligands are a versatile platform which can be applied for labeling small molecules, proteins as well as polymers for detailed insight on their biodistribution profiles using 64Cu that allows studying relatively long biochemical processes and thus, prove to be an attractive candidate for PET imaging. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S93

093 Uptake of 89Zr-DFO by Gram-positive and Gram-negative bacteria and potential for infection imaging Nora C. Goscinski1, Timothy A. Wencewicz1, Suzanne E. Lapi2 1 Chemistry, Washington University in St. Louis, Saint Louis , Missouri, United States, 2Mallinckrodt Institute of Radiology, Washington University in St. Louis, Saint Louis, Missouri, United States Objectives Siderophores are small, Fe3+-binding molecules produced by bacteria to harvest iron from their environment. They are highly selective for Fe3+, and a given Fe3+-siderophore will be selectively taken up by the strain of bacteria that synthesized the molecule. Zirconium-89 is an emerging radiometal with chemistry similar to iron. Therefore, we are investigating 89Zr-siderophores as a promising class of imaging agents for assessment of bacterial infection. Methods 89Zr was added to cultures of Staphylococcus aureus and Pseudomonas aeruginosa alone or complexed with the siderophore p-SCN-deferoxamine (DFO). Cells were labeled in LB (Miller) broth, human serum and 0.9 % NaCl, as well as a low-iron medium (LB broth with 100 μM 2,2`-bipyridyl). Labeled cultures were incubated for various amounts of time, the cells isolated and cell-associated activity quantified. Results Chelation with DFO allowed 89Zr to be taken up by both Gram-negative (P. aeruginosa) and Grampositive (S. aureus) bacteria, while human cells (SKBR3) did not take up 89Zr-DFO at all, raising the possibility of low-background positron emission tomography imaging of bacterial infections in mammalian systems. Additionally, the Gram-positive and -negative bacteria exhibited different uptake behavior: S. aureus rapidly bound over 50% of applied 89Zr-DFO (internalizing a small fraction), while P. aeruginosa bound less. The media in which cells were grown and labeled greatly affected 89Zr-DFO uptake behavior, as did length of incubation and iron availability during growth. Experiments examining the dose- and time-dependence of the uptake are ongoing. Conclusions We have shown that 89Zr-DFO is rapidly taken up by bacteria and that the extent of this uptake and internalization depends on the type of bacteria exposed to the complex as well as the conditions under which the exposure occurs. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S94: Poster

21st International Symposium on Radiopharmaceutical Sciences

094 Linker modification of urea-based PSMA inhibitors for successful tumor-targeting in vivo Martina Benesova1, Martin Schäfer1, Ulrike Bauder-Wüst 1, Uwe Haberkorn2, Walter Mier2, Matthias Eder1, Klaus Kopka1 1 Division of Radiopharmaceutical Chemistry, German Cancer Research Center (dkfz), Heidelberg, Germany, 2 Department of Nuclear Medicine, University Hospital of Heidelberg, Heidelberg, Germany Objectives PSMA is a highly promising biological target for the treatment of PCa. Currently, our group and others [1-4] are working on new urea-based PSMA ligands suitable for endoradiotherapy of prostate cancer (PCa). This work summarizes an evaluated new series of PSMA ligands for both imaging and endoradiotherapy of PCa. Their in vivo tumor-targeting properties have been optimized by modification of the linker regions. By an effort to elucidate the structure-activity relationships (SAR) of up to 30 new PSMA ligands, the tailor-made candidate PSMA-617 has been identified with the putatively best in vivo characteristics. Methods The peptidomimetic structures were prepared by SPPS. 68Ga-labeling resulted in >97% RCY. The in vitro assays were performed with PSMA+ LNCaP cells. The biodistribution and µPET imaging studies were investigated in LNCaP-tumor bearing BALB/c nu/nu mice. First individual clinical diagnostic experience with 68 Ga-PSMA-617 is under way. Results All compounds revealed high PSMA inhibition potencies, those additionally showing high specific internalization in LNCAP cells indicated an improved tumor uptake in vivo. Obviously two cyclic building blocks including at least one aromatic moiety in the linker region is preferable in the design of PSMA ligands, resulting into the best candidate PSMA-617 (Ki = 2.34 ± 2.94 nM; internalization up to 17.67 ± 4.34 %IA/106 LNCaP cells). Organ distribution revealed specific uptake in LNCaP tumors (8.47 ± 4.09 %ID/g; 0.98 ± 0.32 %ID/g blocked) and in the kidneys (113.3 ± 24.4 %ID/g; 2.38 ± 1.40 %ID/g blocked) 1 h p.i.. PSMA-617 exhibited a rapid clearance from the kidneys to 2.13 ± 1.36 %ID/g at 24 h p.i.. First clinical experience with 68Ga-labeled PSMA-617 demonstrated clear visualization of PCa lesions with high T/B ratios already 1 h p.i.. Conclusions The overall characteristics of the here presented new PSMA ligands could be realized successfully by systematic chemical modification of the respective linker region. Tailor-made PSMA-617 exhibits high PSMAspecific tumor uptake, rapid background and fast kidney excretion and is, thus, ready for first-in-man theranostic studies. Acknowledgements Support by PhD Stipend from the Helmholtz International Graduate School for Cancer Research, grants from DFG (ED234/2-1), and Klaus Tschira Foundation. References [1] Banerjee SR, et al (2014) J Med Chem, 57(6), 2657-69. [2] Barrett JA, et al (2013) J Nucl Med, 54(3), 380-7. [3] Pavlicek J, et al (2014) Bioorg Med Chem Lett, 24(10), 2340-5. [4] Weineisen M, et al (2014), EJNMMI Research, 4:63.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S95

095 Dosimetry of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in rodents, pigs, non-human primates and human Irina Velikyan2, 1, Ramkumar Selvaraju2, Thomas Bulenga2, Daniel Espes3, Mark Lubberink1, Jens Sörensen1, Barbro Eriksson4, Sergio Estrada2, Olof Eriksson2 1 PET Centre, Uppsala University Hospital, Uppsala, Sweden, 2Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, Sweden, 3Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden, 4Department of Medical Sciences, Uppsala University, Uppsala, Sweden Objectives Positron emission tomography (PET) using [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 has potential for the quantitative imaging of diabetes and cancer. However, the radiation dose to the kidneys has been a concern for the possibility of repeated imaging studies in humans. Therefore, we investigated the dosimetry of [68Ga]Ga-DO3AVS-Cys40-Exendin-4 based on ex vivo and in vivo biodistribution data in rats, pigs, non-human primates (NHP) and a human. Methods Ex vivo organ distribution of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in rats (n=12) was conducted at 30, 60, and 80 min post injection. The dynamic uptake of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 in the abdomen was assessed by PET/CT scanning of pigs (n = 4, 0–60 min), NHP (cynomolgus; n=3; 0–90 min), and human (female; n=1; 0–40, 100, 120 min). The organ distribution data in each species was extrapolated to those of a human, assuming similar distribution pattern. Residence times were assessed by trapezoidal approximation of the kinetic data, and were used for the estimation of human organ and total body absorbed and total effective doses using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1). Results The extrapolated human whole body effective dose was 0.017±0.004 (rats), 0.014±0.004 (pigs), 0.017±0.004 (NHP), and 0.016 (human) mSv/MBq. The absorbed dose to the kidneys was the limiting parameter: 0.33±0.06 (rats), 0.28±0.05 (pigs), 0.65±0.11 (NHP), and 0.28 (human) mGy/MBq, which corresponded to the maximum possible yearly administered amounts of 455 (rat), 536 (pig), 231 (NHP), and 536 (human) MBq before reaching the yearly kidney limiting dose of 150 mGy. Conclusions More than 200 MBq of [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 can be administered yearly in a human thus allowing for 2-3 repeated examinations. This potentially enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma. Acknowledgements Olof Eriksson’s position was supported by ExoDiab. Ram Kumar Selvaraju’s position was supported by the Barndiabetesfonden and Diabetesfonden. Thomas N. Bulenga was supported by the Swedish Institute Scholarship. References

J Label Compd Radiopharm 2015: 58: S1- S411

S96: Poster

21st International Symposium on Radiopharmaceutical Sciences

096 Extrapolated dosimetry of two novel 68Ga-labelled imaging agents for fibrosis: feasibility of multiple examinations Irina Velikyan2, 1, Ulrika Rosenström2, Thomas Bulenga2, Olof Eriksson2, Gunnar Antoni2 1 PET Centre, Uppsala University Hospital, Uppsala, Sweden, 2Department of Medicinal Chemistry, Preclinical PET Platform, Uppsala University , Uppsala, Sweden Objectives Fibrosis is involved in many chronic diseases. It affects functionality of vital organs such as liver, lung, heart, and kidney. Two novel imaging agents for Positron emission tomography (PET) have pre-clinically demonstrated promising target binding and organ distribution characteristics [1]. However, the relevant disease monitoring in clinical setup would require multiple repetitive examinations per year. Thus it is of paramount importance to investigate the absorbed doses and total effective doses, and thus potential maximum number of examinations per year. Methods Two cyclic peptide analogues coupled via ethylene glycol linker to either 2-(4,7-bis(2-(tert-butoxy)-2oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NO2A-Col) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA-Col) were labelled with 68Ga. The resulting agents, [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col, were administered in the tail vein of male and female Sprague Dawley rats (N=24). Ex vivo organ distribution study was performed at 5, 10, 20, 40, 60, and 120 min time points. The resulting data was extrapolated for the estimation of human organ and total body absorbed and total effective doses using Organ Level Internal Dose Assessment Code software (OLINDA/EXM 1.1). Results Ex vivo organ distribution revealed fast blood clearance (half-life of 1.23±0.05 min) and washout from most of the organs with elimination half-life of 2.2±0.8 min for the fast phase and 27.1±7.4 min for the slow phase. Organ absorbed dose for kidneys was 0.1 mGy/MBq and would allow for the administration of over 1460 MBq per year for both [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col. The total effective dose was 0.0155/0.0156 (female/male) mSv/MBq and 0.0164/0.0158 (female/male) mSv/MBq, respectively for [68Ga]Ga-NO2A-Col and [68Ga]Ga-NODAGA-Col with possibility for the administration of 643 and 621 MBq per year. The statistically significant difference in the uptake of the agents in liver and spleen has been demonstrated previously [1] and was also reflected in this study with higher residence time and organ absorbed doses for [68Ga]Ga-NODAGA-Col. Conclusions The total effective dose would allow for at least six examinations per year that might be sufficient for the adequate disease monitoring in longitudinal studies and routine clinical setup. Acknowledgements References [1] Velikyan et al., Nucl. Med. Biol. 2014, 41, 728

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S97

097 Development of a GMP compliant synthesis of 68Ga-NODAGA-E[c(RGDyK)]2 Malene Brandt-Larsen1, Andreas Kjaer1, 2, Jacob Madsen1 1 PET & Cyclotron Unit, Rigshospitalet, Copenhagen, Denmark, 2Faculty of Health Sciences, Cluster for Molecular Imaging, University of Copenhagen, Copenhagen, Denmark Objectives The aim of this study was to develop a GMP compliant synthesis of 68Ga-NODAGA-E[c(RGDyK)]2 for human use. Methods Gallium-68 labelling of NODAGA-E[c(RGDyK)]2 acetate was performed using a Modular-Lab EAZY module (Eckert & Ziegler). The 68Ge/68Ga generator (IGG100, Eckert & Ziegler) was eluted with 6 ml 0.1M HCl. The eluate was concentrated on a Bond Elut SCX cartridge and eluted with 600 µl 5M NaCl/5.5M HCl (41:1). NODAGA-E[c(RGDyK)]2 (20 or 30 nmol) was labelled in 0.7M NaOAc buffer pH 5.4 containing 400 µl 50 % EtOH at 60oC for 400 s. The resulting 68Ga-NODAGA-E[c(RGDyK)]2 was formulated with saline or phosphate buffer. Results In our group 68Ga-NODAGA-E[c(RGDyK)]2 was previously synthesized using the fractionated method, either manually1 or automated using Modular-Lab tubing system, resulting in radiochemical purities higher than 96% (20 nmol peptide). Labelling peptides with the Modular-Lab EAZY module is performed using an acetone-free method2 without purification of the product. Therefore, from a radiochemical point of view this method requires high incorporation of gallium-68 in order to obtain sufficient radiochemical purity (>95%). From a radiopharmaceutical point of view the reagents and the cassettes need to be produced according to guideline ICH Q7A – GMP for APIs, since they contribute to the composition of the final product. All reagents were supplied in GMP quality. Using the ModularLab EAZY module the synthesis was completed in 14 minutes. The incorporation and thus also the radiochemical purity of the resulting product was high (> 98%), and there was no need to test for residual acetone in the final product. Conclusions Based on our previous findings in mice1 68Ga-NODAGA-E[c(RGDyK)]2 appears to be a very promising candidate for clinical translation into humans for PET imaging neo-angiogenesis in many common cancer types. 68Ga-NODAGA-E[c(RGDyK)]2 can be synthesised using the Modular-Lab EAZY module with very good radiochemical yield and purity in a GMP compliant way well-suited for clinical routine production. Acknowledgements References 1Oxboel, J.; Brandt-Larsen, M.; Schjoeth-Eskesen, C.; Myschetzky, R.; El-Ali; HH.; Kjaer, A. Nucl. Med. Biol. 2014, 41(3), 259-67 2 Mueller, D; Klette I, Baum R.P.; Gottschaldt M; Schultz M.K.; Breeman W.A.P.; Bioconjugate Chem. 2012, 23, 1712-1717

J Label Compd Radiopharm 2015: 58: S1- S411

S98: Poster

21st International Symposium on Radiopharmaceutical Sciences

098 A novel cyclopentadienyl tricarbonyl 99mTc complex containing 5,6-dimethoxyisoindoline motif – synthesis and evaluation of a radiotracer for imaging of sigma-2 receptors in cancer Yuanyuan Chen1, Winnie Deuther-Conrad2, Jörg Steinbach2, Peter Brust2, Boli Liu1, Hongmei Jia1 1 Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China, 2Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, Leipzig, Germany Objectives The sigma-2 (σ2) receptor may serve as a biomarker of the proliferative status of solid tumors [1]. Until now, there are few SPECT radiotracers for σ2 receptor imaging in clinic. In our previous work, cyclopentadienyl tricarbonyl 99mTc complexes possessing high affinity for σ2 receptors but low subtype selectivity were developed [2]. To improve the subtype selectivity of this ligand class, the herein presented novel [(Cp-R)M(CO)3](M = Re, 99m Tc) complexes were synthesized and evaluated. Methods The synthetic routes of 99mTc/Re complexes are shown in Scheme 1. The biological properties of the complexes were investigated by in vitro binding assays and uptake studies in DU145 and MCF-7 (human prostate cancer and breast adenocarcinoma, respectively) cell lines. The in vivo kinetics of the 99mTc-labeled radiotracer was investigated in ICR mice by biodistribution studies. Results In vitro the rhenium complex 3c showed moderate affinity for σ2 receptors (Ki(σ2) = 22.6 ± 0.5 nM) and subtype selectivity (Ki(σ1) = 295.5 ± 103.9 nM, Ki(σ2)/Ki(σ1) = 13.1). [99mTc]6 was prepared in 53 ± 8% (n = 5) radiochemical yield with radiochemical purity of >99%. The log D value at pH 7.4 of [99mTc]6 was 2.39 ± 0.05. The uptake of [99mTc]6 in DU145 and MCF-7 cells was time-dependent and could be blocked by haloperidol dosedependently. Biodistribution studies in mice showed low uptake of [99mTc]6 in muscle at 4 h postinjection. Administration of haloperidol 5 min prior to injection of [99mTc]6 significantly reduced the radiotracer accumulation at 1 h and 2 h postinjection in the brain, heart, lungs, spleen, and kidney. Conclusions These findings suggest that [99mTc]6 warrants further evaluation as a potential SPECT radiotracer for the investigation of σ2 receptors in cancer. Acknowledgements Supported by NSFC (21471019). References [1] Mach RH, et al (2013) J Med Chem, 56, 7137-7160. [2] Li Dan, et al (2013) J Label Compd Radiopharm, 56 (Suppl. 1), S383.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S99

099 Potential labeling strategies with NCA 197(m)Hg Martin Walther, Chao Wang, Ralf Bergmann, Hans-Jürgen Pietzsch, Jörg Steinbach Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Sachsen, Germany Objectives The decay properties of both nuclear isomers, like convenient half life 197mHg (T1/2 = 23.8 h, Eγ 133.98 keV, 33.5%) and 197Hg (T1/2 = 64.14 h, Eγ 77.4 keV, 18.7%), low energy gamma radiations for imaging and numerous Auger- and conversion electrons useful for therapy combined with unique chemical and physical properties of mercury and its compounds represent the motivation for this project. The no carrier added (NCA) radionuclide 197(m)Hg is accessible in sufficient quantity and quality for radiopharmaceutical research by irradiation of gold with protons using a cyclotron [1]. As the following logical step after examination of the production feasibility, the search for a suitable labeling tool was intensified. Methods Three different approaches to prepare a stable labeling unit at NCA level with 197(m)Hg were studied. The reactivity of the mercury(II) ions towards sulfur containing ligands (a), solvomercuration of alkenes (b) and electrophilic aromatic substitution (c) were investigated in this context. Prepared characteristic representatives of all three groups are shown in Figure 1. Results For all studied reactions the desired 197(m)Hg labeled compounds were detected. The mercury thiolate complexe (a) and the product of solvomercuration (b) show low stability in the presence of competing thiol ligands and therefore the suitability for radiopharmaceutical applications is not given. In contrast, diphenylmercury (c) as the simplest representative for symmetric diarylmercury compounds shows high stability against competing ligands. Conclusions As a basis for the development of a convenient labeling method different kinds of mercury compounds were prepared and characterized at NCA level. After nuclide production this was the required succeeding part of the evaluation of the cyclotron-based NCA 197(m)Hg regarding their suitability for diagnostics and therapy of tumors. First promising results of investigations concerning the development of mercury compounds stable in vivo will be reported. Acknowledgements References [1] Walther, M., Preusche, S., Bartel, S., Wunderlich, G., Freudenberg, R., Steinbach, J., Pietzsch, H.J., Theranostic mercury: 197(m)Hg with high specific activity for imaging and therapy (2014) Appl. Radiat. Isot. Submitted

J Label Compd Radiopharm 2015: 58: S1- S411

S100: Poster

21st International Symposium on Radiopharmaceutical Sciences

100 Synthesis of [123I]Iodoaniracetam, a SPECT agent for Brain Imaging Murthy R. Akula, David W. Blevins, George W. Kabalka, Dustin Osborne Radiology, University of Tennessee Medical Center, Knoxville, Tennessee, United States Objectives Piracetam is a nootropic of the racetam class of chemicals and known to be used for cognitive disorders, dementia, vertigo and dyslexia.1 Recently 99mTc-piracetam has been reported 2 as a brain imaging agent. Aniracetam is an aromatic analogue, lipophilic and more potent than piracetam. We wish to report the synthesis of [123I]iodoaniracetam (1) as a potential SPECT agent for brain evaluation. Methods Tin precursor 4 for the radioiododestannylation reaction was prepared in three steps starting from 3-iodo4-methoxybenzoic acid that was refluxed in thionyl chloride to obtain 3-iodo-4-methoxybenzoyl chloride, 2. NBenzoylation of pyrrolidin-2-one was carried out with benzoyl chloride 2 to afford iodoaniracetam 3. Deiodostannylation of 3 using hexabutylditin, (Ph3P)4Pd(0) in refluxing toluene afforded the tin precursor 4. Radioiodnation of tin precursor 4 was carried out with Na123I and peracetic acid as an oxidant. The formation of the labeled compound was monitored by radio-TLC and the product was purified on a C18 Sep-Pak. The radiochemical purity was determined using an Agilent Binary 1200 radio-HPLC. Results To no-carrier-added Na123I (0.5 mCi in 0.1 % aqueous NaOH) in a 2 mL Wheaton vial was added tin precursor 4 ( 100 µL of 4.8 X 10-2 M solution in H2O:THF=1:1). Peracetic acid (100 µL; 0.3 % THF solution) was added and the resulting mixure was stirred for 10 min at rt. The reaction was monitored by Bioscan radio-TLC and purified by C18 Sep-Pak eluting the product with Et2O to get 0.42 mCi of 1 (84 %). The solvent was removed under argon to suspend the product in a buffer. The radiochemical purity was assessed by HPLC to be ≥98 %. Conclusions A novel pyrrolidinone based and GABA related SPECT agent for brain imaging has been successfully synthesized in high radiochemical yield and purity. Initial studies have shown that there is brain uptake and further studies of bio-distribution are currently underway. Acknowledgements We wish to acknowledge the Molecular Imaging and Translational Research Program and University Health Systems for the support of this research. References [1] Ince Gunal et al. (2008) J Clin Pharm Ther. 33, 175-178. [2] Amin AM et al. (2013) Radiochemistry 55, 534-538

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S101

101 p-NO2-Bn-bisDFO: A new high denticity chelator for 89Zr radiochemistry Eric W. Price1, Melissa A. Deri1, 2, Lynn C. Francesconi2, Jason S. Lewis1 1 Department of Radiology & the Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 2Chemistry, Hunter College of the City University of New York, New York, New York, United States Objectives There has been a recent surge in interest to study new 89Zr chelators. The gold standard chelator desferrioxamine (DFO) is sufficient for 89Zr PET imaging, but 89Zr is leached from the chelator and localizes in the bone. An improved chelator may decrease bone uptake and dose to bone marrow in patients. Some examples of new chelators that have been recently studied for 89Zr are H6phospa, HOPO, DFO*, and cyclic DFO. 89Zr has a preference for oxygen donors, with hydroxamic acids being the best groups used to date. With the goal of enhancing 89Zr-chelate stability, we have been studying a new high denticity DFO derivative (12-coordinate) called p-NO2-Bn-bisDFO (Fig 1). Methods The chelator p-NO2-Bn-bisDFO was synthesized via peptide coupling between DFO-COOH (1 step from DFO-mesylate) and p-NO2-Bn-propylenediamine (4 steps). The chelator was purified by RP-HPLC and characterized by HRMS and NMR. The purified chelator p-NO2-Bn-bisDFO and DFO were then radiolabeled with 89 Zr and yields were confirmed to be >99% for both chelators by iTLC (30 min, RT). These two quantitatively radiolabeled chelators were then mixed with a 100 fold excess of EDTA at a variety of pH values, and analyzed by iTLC at various time points. EDTA competition solutions of both 89Zr-chelators at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 (n = 3 for each) were checked at times 0 h, 1 h, 3 h, 1 d, 3 d, 5 d, and 7 d. Results The new chelator p-NO2-Bn-bisDFO and DFO were found to both radiolabel with 89Zr in quantitative yields after 30 minutes at RT. EDTA competition experiments performed over a period of 7 days and at various pH values demonstrated the new chelator p-NO2-Bn-bisDFO to be substantially more stable than DFO. After 7 days the stability values for 89Zr-p-NO2-Bn-bisDFO at pH 7.5, 7.0, and 6.5 were found to be 95, 85, and 90 %, respectively. After 7 days the stability values for 89Zr-DFO were substantially lower at 24, 16, and 9 %, respectively. Conclusions The chelator p-NO2-Bn-bisDFO has shown superior stability to DFO with 89Zr in an EDTA variablepH competition. Synthesis of the bifunctional p-SCN-Bn-bisDFO is being attempted, and must be completed so that in vivo stability can be evaluated. Acknowledgements NSERC PDF (E.W.P), P30 CA008748 (J.S.L), NIH NRSA 1F31CA180360 and NSF IGERT 0965983 (M.A.D) for funding. References

Figure 1. Structure of p-NO2-Bn-bisDFO

J Label Compd Radiopharm 2015: 58: S1- S411

S102: Poster

21st International Symposium on Radiopharmaceutical Sciences

102 Nitroimidazole-containing acyclic chelates as potential PET imaging agents of hypoxia with 68Ga Caterina F. Ramogida1, 2, Cara L. Ferreira3, Jacqueline F. Cawthray1, Chris Orvig1, Michael J. Adam2 1 Chemistry, University of British Columbia, Vancouver, British Columbia, Canada, 2Nuclear Medicine, TRIUMF, Vancouver, British Columbia, Canada, 3Nordion, Vancouver, British Columbia, Canada Objectives Clinically important nitroimidazole (NI) probes, such as 18F-MISO, for PET imaging of hypoxia suffer from limited clearance and low tumor-to-background ratios due to their relative lipophilicity. The development of more hydrophilic agents labeled with β+-emitter 68Ga (an attractive alternative to 18F, produced in a commercially available 68Ge/68Ga generator system) would be of great interest. Herein, we report NI derivatives of recently reported acyclic Ga3+ chelate H2dedpa (N4O2) [1], to investigate specific targeting of hypoxic tumor cells. H2dedpa binds 68Ga quickly and under mild conditions with high thermodynamic stability and kinetic inertness. Methods A small library of NI-containing H2dedpa derivatives were synthesized (Figure 1), and screened for their ability to bind Ga3+. The compounds were radiolabeled with 67/68Ga, stability studies against human apo-transferrin were performed at 37°C, and lipophilicity (log P) of the tracers was measured. Cell uptake studies of the 68Gatracers were performed under hypoxic (0.5% O2) and normoxic conditions with LCC6HER-2 and HT-29 cell lines. Results All pro-ligands were quantitatively radiolabeled (RCY >99%) with 67/68Ga in 10 minutes at RT at ligand concentrations of 10-5 M. Moreover, the 67Ga-complexes exhibited excellent stability (>90% intact) against transchelation to human apo-transferrin after 2 hrs. The hydrophilic 68Ga-dedpa-NI tracers (log P = -2.2 – -2.7) exhibited excellent differentiation between hypoxic versus normoxic retention in vitro, with hypoxic/normoxic ratios as high as 7.2 after 60 min incubation. Conclusions A small library of H2dedpa-nitroimidazole compounds was successfully synthesized, which have the ability to label gallium isotopes quantitatively at RT in 10 minutes. All compounds showed excellent in vitro stability against apo-transferrin, and exhibited specific retention in hypoxic cells (compared to normoxic cells) in vitro. These properties suggest the compounds will be good candidates for further testing in vivo. Acknowledgements Nordion and NSERC CR&D, NSERC Discovery (CO), NSERC CGSD and UBC four-year fellowship (CFR) for funding. References [1] Boros E, et al (2010), J. Am. Chem. Soc., 132, 15726-15733.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S103

103 Simple, effective, and stable radioarsenic labeling of thiol-modified mesoporous silica nanoparticles Paul Ellison1, Feng Chen2, Todd E. Barnhart1, Robert J. Nickles1, Weibo Cai1, 2, Onofre T. DeJesus1 1 Department of Medical Physics, University of Wisconsin, Madison, Madison, Wisconsin, United States, 2 Department of Radiology, University of Wisconsin, Madison, Madison, Wisconsin, United States Objectives With several imaging and therapy isotopes of a variety of biologically relevant half-lives, radioarsenic has significant radiopharmaceutical potential. Mesoporous silica nanoparticles (MSN) are used for in vivo imaging and drug delivery and characterized by their high surface area, large pore volume, good biocompatibility, and ability to be conjugated to biological targeting vectors [1]. The present work utilizes thiol-functional-groupmodified MSN to stably bind to radioarsenic, with the resulting product well suited for in vivo PET imaging and drug delivery applications. Methods Radioarsenic (*As, *=71,72,74,76) was produced and radiochemically isolated [2]. MSN were synthesized, functionalized with thiol groups, physically characterized, and analyzed for thiol concentration [1]. MSN and *As were combined and labeling efficacy was monitored as a function of MSN-thiol concentration, contact time, pH, and arsenic oxidation state through autoradiography-visualized thin-layer chromatography [2]. After labeling, *As-MSN were centrifugally isolated and the stability of *As bound to thiolated-MSN was monitored in whole mouse serum over 24 hours at 37 °C. Results The radioseparation procedure isolated *As as 85% *As(III) and 15% *As(V) in 1 mL of 0.1 M HEPES, 0.5 M hydroxylamine, 25 mM EDTA at pH 7.5 with a decay-corrected radiochemical yield of (52 ± 8)%. The synthesized 60 - 80 nm thiol-functionalized MSN showed affinity for *As in a thiol-concentration, time, pH, and arsenic oxidation state dependent manner (Fig. 1). Optimal labeling conditions were observed at the highest studied MSN-thiol concentration (600 nmol/mL) at pH 9, with ~75% *As-MSN yield after 22 hours. When incubated with mouse serum, the isolated *As-MSN showed exceptional stability with >80% of *As bound after 24 hours and ~60% after 4 days. Conclusions The labeling of thiol-modified MSN with radioarsenic occurs with high yield and the resulting *AsMSN exhibits high in vitro serum stability. Applications of *As-MSN will take advantage of the versatility of MSN for passive and site-specific tumor targeting and drug delivery coupled with diagnostic or therapeutic radioarsenic isotopes yielding unique theranostic agents. Acknowledgements References [1] Chen, F. et al. (2013) ACS Nano, 7, 9027-9039. [2] Jahn, M. et al. (2010) Radiochim Acta, 98, 807-812.

J Label Compd Radiopharm 2015: 58: S1- S411

S104: Poster

21st International Symposium on Radiopharmaceutical Sciences

104 First 111In-labeled activity-based probe for SPECT imaging of uPA activity: in vivo study in human cancer xenografts David Thomae1, 2, Christel Vangestel1, 3, Jeroen Van Soom2, Leonie wyffels3, 1, Patrick Pauwels4, Pieter Van der Veken2, Sigrid Stroobants1, 3, Jurgen Joossens2, Koen Augustyns2, Steven Staelens1 1 Molecular Imaging Center Antwerp, University of Antwerp, Wilrijk, Belgium, 2University Antwerp Medicinal Chemistry, University of Antwerp, Wilrijk, Belgium, 3Nucelar Medicine , University Hospital Antwerp, Edegem, Belgium, 4Department of Pathology, University of Antwerp, Edegem, Belgium Objectives The urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as prognostic biomarkers for breast cancer with the highest level of evidence. Current methods for quantification of uPA do not discriminate between active and inactive uPA and require patient tumor material. Single Photon Emission Computed Tomography (SPECT) is a powerful tool to monitor disease stages, to study human biology and this technique is quantitative and non invasive which is a major advantage. In this report, we present the synthesis and the in vivo evaluation of a covalently binding uPA inhibitor [111In]MICA-401. Methods [111In]MICA-401 was prepared by complexation of [111In]InCl3 by the DOTA chelator (Scheme1). The radioligand was injected (1 mCi) in two different cancer xenografts (human breast cancer: MDA-MB-231 and human colorectal cancer: HT-29) with respectively high and low levels of uPA expression in vitro, and in a healthy control group (n = 5/group). SPECT imaging was performed 95h post injection (pi) followed by ex vivo biodistribution. Tumor uptake was correlated with human and murine uPA expression determined by ELISA and immunohistochemistry (IHC) on excised tumors. Results [111In]MICA-401 was synthesized in 49% radiochemical yield, in high radiochemical purity (> 95%) and with a specific activity above 9.25 Gbq/μmol. [111In]MICA-401 showed a comparable tracer distribution in the three groups. Tumor uptake of 5.68 ±1.41%ID/g for MDA-MB-231 and 5.43 ± 1.29%ID/g for HT-29 was obtained. Human uPA ELISA and IHC showed significant higher uPA expression in the MDA-MB-231 tumors, while staining for mouse uPA revealed more similar staining intensity for both tumors. It has been described that for an in vitro negative uPA cell line (HT29) to grow in vivo, it may need to recruit uPA expressed by neighboring stroma cells through its surface uPAR, which may explain the equal tumor uptake observed in both tumor models. Conclusions Our data clearly demonstrate that [111In]MICA-401 enables the imaging of uPA activity in vivo and may be a promising candidate for humans studies. Acknowledgements References [1] Hsiao J-K, et al. (2006) J Biomed Optics. 11, 034013 1-5.

Scheme 1: Radiosynthesis of [111In]MICA-401.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S105

105 An attractive method for radiolabeling antibodies with Tc-99m Gerd Wunderlich1, Anne Naumann1, Maik Schubert2, Hans-Jürgen Pietzsch2 1 Clinic of Nuclear Medicine, TU Dresden, Dresden, Germany, 2Inst.of Radiopharmaceutical Cancer Research, Helmholtz-Centre Dresden-Rossendorf, Dresden, Germany Objectives Radiolabeled Cetuximab (C225, Ab) is an attractive tool for tumor targeting and delivering of particles for therapy or imaging applications of EGFR positive tumors. The labeling of Ab with radionuclides requires suitable chelating agents for a stable binding of the radionuclides. Well known is the Ab labeling with In-111 (imaging) and Y-90 (therapy). The aim of the present study was to develop a sufficient radiolabeling technique of this Ab with Tc-99m for SPECT imaging. A second label with a fluorescent dye (Alexa 488) enables to track the uptake of the compound with fluorescent microscopy. Methods NOTA (2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic-acid) was linked to C225 and labeled with the [Tc99m]Tc(H2O)3(CO)3 complex that was made by a standard tricarbonylkit preparation [1]. For preparation of [Tc99m]Tc(CO)3-NOTA-C225-Alexa(488) (figure 1) and [Tc-99m]Tc(CO)3-NOTA-C225 1 nM of the modified antibody was incubated with up to 1 GBq [Tc-99m]Tc(H2O)3(CO)3 complex and was shaken for 2 h at 40°C. The product was isolated by gelfiltration and tested for yield and stability with ITLC (Silica gel impregnated glass fiber sheets, Varian) in 5% acetic acid. The cell membrane binding and cell uptake of the compound was detected with Cetuximab receptor positive A431 cells and Cetuximab negative MDA cells. For comparison the pure NOTA ligand and unmodified Cetuximab were labeled with [Tc-99m]Tc(H2O)3(CO)3. Results NOTA-C225-Alexa(488), NOTA-C225 and NOTA ligand were successfully labeled with [Tc99m]Tc(H2O)3(CO)3. Sufficient radiolabeling of Cetuximab was achieved and determined by ITLC. Yields: [Tc99m]Tc-NOTA-C225-Alexa(488) 25-30% and [Tc-99m]Tc-NOTA-C225 50-60%. After purification the labeled compound is stable in cell culture medium and phosphate buffered saline to 24 h with a release of about 20%. Maximum membrane uptake at A431cells is determined after 1 h followed by a partly internalization into the cells. The affinity constant was found Kd = 3.71 nM and Bmax = 35 nM. Already after 1 h the localisation of NOTAC225-Alexa(488) is visualized with fluorescence microscope at cell membrane. Conclusions NOTA-Cetuximab can be radiolabeled with Tc-99m which is an interesting approach for SPECT studies in Nuclear Medicine besides the Ab labeling with Ga-68 or Cu-64. Acknowledgements References [1] Alberto, R. et al. (1998) J. Am. Chem. Soc., 120, 7987-7988.

J Label Compd Radiopharm 2015: 58: S1- S411

S106: Poster

21st International Symposium on Radiopharmaceutical Sciences

106 Synthesis and evaluation of the first non-peptidic [99mTc]labeled ligand for the molecular imaging of inflammatory processes using S100A9 as novel target Felix Busch1, Sven Hermann1, Michael Schäfers1, 3, Johannes Roth2, Thomas Vogl2, Günter Haufe4, Andreas Faust1 1 European Institute for Molecular Imaging, University Münster, Münster, Germany, 2Institute of Immunology, University Münster, Münster, Germany, 3Department of Nuclear Medicine, University Hospital Münster, Münster, Germany, 4Organic Chemistry Institute, University Münster, Münster, Germany Objectives Inflammatory processes are associated with high expression of the alarmin S100A8/S100A9 released by activated phagocytes. We could show with a Cy5.5 labelled antibody against S100A9 that the protein is a valuable and sensitive molecular target for imaging inflammatory disorders [1]. We identified 3-quinolinecarboxamides as high affinity small molecule drug binding S100A9 proteins [2]. Coupling with a cyanine dye gave access to a specific optical tracer (Cy5.5-CES271) which was successfully evaluated in vitro and diverse in vivo models like atherosclerosis, myocardial infarction and ear inflammation [3]. Because of limitations of optical imaging methods due to tissue penetration also radionuclides like 99mTc should be attached to get a suitable SPECT-tracer. Methods We choose the Isolink® labelling strategy and replaced the cyanine dye in Cy5.5-CES271 with a bisimidazolyl-unit for labelling with 99mTc. For verification of the labelled tracer [99mTc]FEB054 the corresponding Re-complex was synthesized, characterized and compared using radio-HPLC. First in vivo biodistribution studies were done in WT-mice on a small animal SPECT/CT-system. Results The synthesis of the corresponding 3-quinoline carboxylic acid was done in six steps starting from 3nitrophthalic anhydride. The chelator unit was prepared by double reductive amination of an 2imidazolecarboxaldehyde with azido-PEG4-amine followed by reduction, substitution with aniline and coupling with the carboxylic acid yielding the precursor molecule. After treatment with fac[99mTc(CO)3(H2O)3)]+ we got [99mTc]FEB054 with a rcy of 22±5% (110 min) with 99% rcp after HPLC. In blood serum stability tests no decomposition was observed over a period of 120 minutes. The first in vivo biodistribution experiments showed a good tracer availability in the blood in the first 10 minutes with predominant hepato-biliary elimination within 20 minutes. Further studies with higher temporal resolution are ongoing to characterize tracer dynamics. Conclusions We synthesized the first non-peptidic SPECT-tracer [99mTc]FEB054 as radioactive alternative to Cy5.5-CES271. [99mTc]FEB054 has very good blood serum stability and a suitable blood half-life for first in vivo experiments with inflammatory disease models. For tuning the blood half-life also chelators with stronger or weaker protein binding will be evaluated. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG), CRC656 (MoBil, project A9). References [1] Vogl T, et al. (2014), Nat. Commun., 5, 4593. [2] Björk P, et al. (2009), PLoS Biol., 7, e97. [3] unpublished results.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S107

107 64 Cu-labelling and preclinical evaluation of NODAGA-cetuximab-F(ab’)2 Laura van Dijk2, 3, Cheng-Bin Yim1, Gerben Franssen3, Johannes Kaanders2, Johan Rajander1, Olof Solin1, Tove Grönroos1, Otto C. Boerman3, Johan Bussink2 1 Turku PET Centre, University of Turku and Åbo Akademi University, Turku, Finland, 2Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, Netherlands, 3Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, Netherlands Objectives Several radiolabeled cetuximab derivatives have been investigated for monitoring epidermal growth factor receptor (EGFR) expression in various solid tumors, including head and neck squamous cell carcinomas (HNSCC) [1,2]. In this study, 64Cu-labelled NODAGA-cetuximab-F(ab’)2 was developed and evaluated for imaging EGFR in mice. Methods Cetuximab-F(ab’)2 was conjugated with NODAGA and subsequently labelled with 64Cu. In vitro characterization of the radiotracer included plasma stability. Mice with subcutaneous HNSCC xenografts, UTSCC-8 (n = 5) or UT-SCC45 (n = 7) were studied with PET/CT at 24 h post-injection of 64Cu-cetuximab-F(ab’)2. Receptor specificity was verified by pre-injection of an excess unlabelled cetuximab. Ex vivo tracer distribution was determined after imaging, and tumors were analyzed by autoradiography and immunohistochemistry. Results 64Cu-cetuximab-F(ab’)2 was stable in murine plasma: > 80 % of the radioactivity was associated with the intact tracer after 26 h incubation. Accumulation of 64Cu-cetuximab-F(ab’)2 in the tumor was EGFR-mediated and SUVmax of UT-SCC-8 tumor was higher than that of UT-SCC-45; 1.5 ± 1.0 and 0.8 ± 0.2 (p<0.05), respectively (Fig. 1). Autoradiography and immunohistochemistry corroborated above findings and inferred a correlation between tracer uptake and EGFR expression in both tumors; r = 0.86 ± 0.06 (UT-SCC-8) and 0.90 ± 0.06 (UTSCC-45). Conclusions 64Cu-cetuxmab-F(ab’)2 is a promising PET tracer to determine expression of EGFR in vivo. Its viability to monitor the tumor response to EGFR-inhibitor treatments requires further investigations. Acknowledgements Supported by the Dutch Cancer Society (NKB-KUN 2010-4688) and Finnish Centre of Excellence in Molecular Imaging in Cardiovascular and Metabolic Research. References [1] Sihver W, et al. (2014) Pharmaceuticals, 7, 311-338. [2] van Dijk LK, et al. (2013) J Nucl Med, 54, 2118-2124.

J Label Compd Radiopharm 2015: 58: S1- S411

S108: Poster

21st International Symposium on Radiopharmaceutical Sciences

108 Radiolabeled PTK7 targeting aptamer: challenges and approaches Victoria Calzada1, Romina Castelli1, María F. Garcia1, Jessica Newton2, Marcelo Fernandez1, Joel Gonzalez1, Juan Pablo Gambini1, Pablo Cabral1, Thomas P. Quinn2, Hugo Cerecetto1 1 Radiopharmacy, Centro de Investigaciones Nucleares, Montevideo, Uruguay, 2Biochemistry , University of Missouri, Columbia, Missouri, United States Objectives A small 13 kDa DNA oligonucleotide aptamer, that binds protein tyrosine kinase 7 (PTK7) [1], was coupled with HYNIC and DOTA bifunctional chelators and labeled with 99mTc and 177Lu, respectively. The aptamer radiolabeling efficiencies and radiochemical stabilities were determined as were the in vitro tumor cell binding properties. Preliminary investigations into the pharmacokinetics of the radiolabeled aptamers were determined in by biodistribution and imaging studies. Methods NHS-HYNIC-Tfa and NHS-DOTA bifunctional chelators were conjugated to an amine terminated Aptamer at 25˚C as described before [2,3]. Preliminary physicochemical studies were performed by HPLC. Binding studies were analyzed with CCRF-CEM, Ramos and LS174T cancer cell lines. Confocal microscopy was also used to visualize cellular uptake of Alexa647 conjugated aptamer in CCRF-CEM cancer cells. In vivo, the biodistribution properties of the radiolabeled aptamer were determined in Balb-c normal mice injected with 37 MBq of 99mTc-HYNIC-aptamer and imaged at selected time points using a small animal gamma and X-ray camera (Bruker). Results Both, HYNIC and DOTA derivatizations of amine terminated aptamer, were performed at room temperature and under mild buffer conditions. The integrity of the aptamer conjugates was verified by HPLC and radiolabeling. Radiolabel conditions were performed at ≤ 37ºC with radiolabeling efficiencies of 54% and 29%, respectively. The radiolabeling yield of reaction did not increase with temperatures above 37˚C. In vitro studies showed that the aptamer bound the PTK7 expressing CCRF-CEM cell line as well as the Ramos and LS174T human cancer cell lines at similar levels. Finally, images of 99mTc-HYNIC-Aptamer in normal mice at 1, 2 and 20 hours post-injection, showed rapid clearance via the liver and kidneys. Conclusions Preliminary results demonstrated that the amine terminated DNA aptamer could be conjugated with radiometal chelators and stably radiolabeled at temperatures ≤ 37ºC. The labeled aptamer was able to bind several cancer cell lines. The data indicated challenges remain in the use of radiolabeled aptamers but advantages in aptamer targeting specificity and biodistribution highlight their potential as imaging and therapy agents. Acknowledgements University of Missouri, Mid-America Isotopes, CSIC, ANII, PEDECIBA Quimica, UdelaR. References [1] Shangguan D, et al (2007) Chembiochem, 8(6),603-6. [2] Calzada V, et al (2013) World J Nucl Med, 12(1), 27-32. [3] Calzada V, et al (2012) Curr Radiopharm, 5, 318-324, 2012.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S109

109 Metabolically-stable 68Ga-NOTA-bombesin derivative for targeting GRP receptor in prostate cancer xenografts Susan Richter1, Melinda Wuest1, Cody Bergman1, Stephanie Krieger2, Buck E. Rogers2, Frank Wuest1 1 Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri, United States Objectives Peptide receptor-based targeted molecular imaging and therapy of cancer is on the current forefront of nuclear medicine preclinical research and clinical practice. The frequent overexpression of gastrin-releasing peptide (GRP) receptor in prostate cancer prompted the development of radiolabeled bombesin derivatives as high affinity peptidic ligands for selective targeting of the GRP receptor. This study is aimed on the evaluation of a novel radiometal-complexed bombesin derivative for its targeting of GRP receptor in various prostate cancerxenografted mice with positron-emission-tomography (PET) along with the investigation of its metabolic stability in vivo in the presence and absence of a protease inhibitor. Methods Modified BBN(7-14) sequence NOTA-Bn-thiourea-Ava-QWAV-Sar-H-FA01010-Tle-CONH2 (NOTABBN2) and its natGa reference (Ga-NOTA-BBN2) were synthesized via Fmoc-based solid-phase peptide synthesis. Peptides were studied in a Ca2+ assay for antagonistic or agonistic behaviour and a competitive binding assay was performed to assess their binding affinity to the GRP receptor using PC3 cells. Ga-68 radiolabeling was carried out in sodium acetate buffer and SPE purification of 68Ga-NOTA-BBN2. The radiopharmacological profile of 68Galabeled bombesin was evaluated in PC3 and LNCaP tumour-bearing Balb/C mice involving metabolic stability studies in vivo (also involving a protease inhibtitor), biodistribution experiments and dynamic small animal PET studies. Results NOTA-BBN2 and natGa-NOTA-BBN2 demonstrate characteristic antagonistic behaviour and high binding affinity towards the GRP receptor (IC50 = 4.6 – 8.2 nM). Starting from 20 μg NOTA-BBN2, 68Ga-NOTABBN2 was obtained in RCY above 62% d.c. within 35±4 min including purification. LogD7.4 was determined to be -2.1. 68Ga-NOTA-BBN2 exhibits high resistance against degradation by peptidases in vivo with greater than 70% of the peptide found to be intact after 60 min. Application of protease inhibitor phosphoramidon did not have an enhancing effect on metabolic stability. PET studies displayed high tumor uptake in PC3- and LNCaP-BalB/C mice (SUV 0.69 vs. 0.79 at 5 min p.i. and 0.61 vs. 0.66 at 60 min p.i.). Conclusions The PET radiotracer 68Ga-NOTA-BBN2 represents a novel radiometal-labeled bombesin derivative for suitable targeting of GRP receptor in PC3 and LNCaP prostate cancer-bearing mice. A further increase of its remarkable metabolic stability profile in vivo and favourable tumor uptake by coadministration of protease inhibitor phosphoramidon was unsuccessful. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S110: Poster

21st International Symposium on Radiopharmaceutical Sciences

110 Exploration of 89Zr-Nano-Hydroxyapatite as a PET Tracer Stacy Queern, Suzanne E. Lapi, Tolulope Aweda Washington University of St. Louis, St. Louis, Missouri, United States Objectives 89Zr has a half-life (78.41hrs), which matches well to the extended biological half-life of antibodies and nanoparticles. Our goal was to use the natural 89Zr affinity to hydroxyapatite to prepare 89Zr-nano-hydroxyapatite (89Zr-nHAp) particles as potential imaging agents. Ideally, our particles would prevent translocation of 89Zr to bone in the body and the ability to functionalize n-HAp will allow the control of the 89Zr in the body making it an effective PET tracer. Methods 89Zr in 1M HEPES buffer (pH=7) was added to nHAp suspended in 1M HEPES where it was incubated and activity measured. Activity was measured for 89Zr–nHAp in serum for several times throughout a 7day incubation period. nHAp and 89Zr–nHAp particles were analyzed using SEM FEI Nova 230 (NRF). Acetylation of n-HAp (nHAp-Ace) was done via Wang et al [2] with minor modifications. Triethylamine was replaced with a NHS-fluorescein (NHS-F*). Labeling and stability studies were conducted for each compound formed in the acetylation process following same method as nHAP. Results The radiolabeling of 89Zr-nHAP had an average percent loss (PL) of 3±2 after ~2 hour incubation. The stability of 89Zr–nHAp had a PL of 9±4 after ~7days of incubation. The trials specific activity (SA) range was 8.820.2μCi/mg with PL range of 3.6-13.2%. The preliminary findings for the surface of the 89Zr–nHAp in comparison to nHAp via SEM showed no obvious structure damage with some aggregation. NHS-F* confirmed the acetylation of n-HAp giving 2.4(±0.8)×10-4 NHS-F*/mg of nHAp-Ace. Radiolabeling and stability of nHAp-Ace and its intermediate product had a PL of 7±5 and 5±1, respectively, after ~2 hour incubation. The stability of intermediate and final products after ~7days of incubation had a PL of 15±6 and 21±4, respectively. The trials SA range was 12.3-86.9μCi/mg with PL range of 8-23%. Conclusions nHAp was radiolabeled with 89Zr and the stability study showed minimal loss of activity in serum. The ability to label nHAp-Ace was not hindered by the functionalization, but the stability in serum had a larger PL relative to 89Zr-nHAp. The preliminary investigation of 89Zr-nHAp indicates its potential for the basis of a new class of imaging agents. Acknowledgements Special thanks to the St. Louis Cyclotron Facility for the production of Zr-89 and the NRF facility at WUSTL for the use of their equipment. References [1] Wang S, Wen S, Shen M, Guo R, Cao X, Wang J, et al. Amino-propyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay. Int J Nanomed 2011;6:3449e59.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S111

111 The application of metal free click-chemistry to imaging cancer targeting phage María F. Garcia1, Manankumar Shah2, Jessica Newton2, Pablo Cabral1, Susan L. Deutscher2, 3, Hugo Cerecetto1, Thomas P. Quinn2 1 Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay, 2 Biochemistry Department, University of Missouri, Columbia, Missouri, United States, 3Truman Veterans’ Administration Hospital, Columbia, Missouri, United States Objectives The application of metal-free click chemistry [1,2] to image tumor targeting phage via fluorescence and nuclear imaging were investigated. Tumor avid G3C12 phage, a recombinant filamentous phage particle that expresses a galectin-3 recognition peptide, was used to target Gal-3 expressed on breast carcinoma cells. In this study Cy5-tetrazine or 99mTc-HYNIC-tetrazine probes, were reacted with pretargeted G3C12 trans-cyclooctene (TCO) modified phage and examined for conjugation efficiencies, stability, Gal-3 affinity and cell binding. Methods Phage particles were modified with NHS-TCO [2]. Tetrazine-Cy5 cycloaddition reactions with TCOphage were performed in solution and excess tetrazine removed by Zeba spin columns. The HYNIC-tetrazine analogue was synthesized from 4-(1,2,4,5-tetrazine-3-yl)phenyl)methanamine and TFA protected HYNIC precursor. Labeling with 99mTc was performed using tricine as coligand. Click reactions on the phage surface were followed by a dot blot assay. Binding studies were performed with MB-231 and MB-435 cell lines. Results Increasing levels of phage TCO conjugation with respect to the α-amino groups on phage coat-protein pVIII was observed by incubating with increasing molar excess of TCO. The phage-TCO Cy5-tetrazine reaction was optimized using different stocks TCO conjugated phage after a 1 h reaction at 25 ˚C. There was binding of Cy5-tetrazine-TCO-phage to MB-435 and MB-231 cells as analyzed by confocal microscopy and FACS analyses. The HYNIC-tetrazine derivate was obtained with 40 % yield. Dot blot assays showed a radioactive signal with TCO conjugated phage but no signal in non-TCO conjugated phage. Conclusions The synthesis of TCO conjugated phage and the subsequent reaction with tetrazine-Cy5 fluorophore or 99mTc-HYNIC-tetrazine allowed efficient phage labeling and direct visualization of phage binding to cancer cells. These results underscore the use of metal free cycloaddition chemistry for visualization of pretargeted tumor avid phage as imaging probes. Acknowledgements Agencia Nacional de Innovación, Comisión Sectorial de Investigación Científica. References [1] Blackman M, et al (2008), J Am Chem Soc, 130, 13518-19. [2] Rossin R, et al (2010), Angew Chem, 122, 3447–50.

J Label Compd Radiopharm 2015: 58: S1- S411

S112: Poster

21st International Symposium on Radiopharmaceutical Sciences

112 Labelling and preclinical evaluation of NOTA cyclo-RGD dimers labelled with Ga-68 for imaging of the integrin receptors Dana Niculae, Catalin Tuta, Adrian Niculae Horia Hulubei National Institute for Physics and Nuclear Engineering, Magurele, Romania Objectives The αvβ3 integrin receptor, expressed on tumor cell membranes can be preferentially targeted by peptides containing the RGD sequence. DOTA-E-[c(RGDfK)2] and NOTA-SCN-Bn-E-[c(RGDyK)2] were labelled with Ga-68 and tested for radiolabelling yield, purity, stability, in vitro binding, ex vivo biodistribution, and in vivo imaging. Methods The radiolabelling was performed using an automated system with inline quality control. Ga-68 eluate from a tin oxide based Ge-68/Ga-68 generator, purified and concentrated to 600 MBq in 0.2 mL water on an anionic exchanger was used to label DOTA/NOTA-derivatised RGD peptides. Elution, concentration, labeling and purification procedures were performed in 25 min. The ex vivo biodistribution of was tested in tumor bearing animal models (Walker 256, Guerin, Melanoma and AR42J). The affinity of DOTA/NOTA cyclo-RGD dimers to tumor cell-surface receptors (HT29, AR42J, A431) was determined by real-time quantification. Results Nanomoles of peptides were labeled with high yields. NOTA-SCN-Bn-E-[c(RGDyK)2] was labelled at room temperature in less than 20 min and no postlabelling purification was required. The biodistribution pattern of 68 Ga-NOTA-SCN-Bn-E-[c(RGDyK)2] in Walker and melanoma models shows high tumor uptake. The blood clearance is fast, the renal elimination is more rapid than in the case of 68Ga-DOTA-E-[c(RGDfK)2]. High tumor to background ratios was observed. Binding to receptors is achieved in the first 3 min of incubation. PET/CT scan highlight the tumor uptake at 30 min postinjection. Conclusions The radiolabelling with Ga-68 of very promising candidates for imaging targets of interest in cancer diagnosis and therapy follow-up such as αvβ3 receptors, were successfully adapted on the automated module, reducing the reaction time and operator exposure. The preclinical biological evaluation of 68Ga-DOTA-E[c(RGDfK)2] and 68Ga-NOTA-SCN-Bn-E-[c(RGDyK)2] show a high and stable tumor uptake. Acknowledgements IAEA Vienna, CRP16500 PN II MEN - UEFISCDI, project 228 References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S113

113 Evaluation of In-House Manufactured 99mTc-ECDG in Baboons: Normal Biodistribution Jan Rijn Zeevaart1, Jenine Horn-Lodewyk2, Judith Wagener1, Sep Lamprecht2, Magritha Janse van Vuuren2, Anton Otto2 1 Radiochemistry, Necsa, Pretoria, South Africa, 2Nuclear Medicine, University of the Free State&Universitas Academic Hospital, Bloenfontein, South Africa Objectives In-House Manufactured (IHM) technetium-ethylenedicysteine-deoxyglucose (99mTc-ECDG) was developed as an effective tumour and infection glucose metabolism imaging agent. The aim of the study was to determine the normal biodistribution of the newly synthesised IHM 99mTc-ECDG in baboons. Methods Ethical approval was received from the Interfaculty Animal Ethics Committee of the Faculty of Health Sciences of the UFS, ETOVS Nr. 10/09. Seven healthy non-fasting baboons (Papio ursinus) were injected with the IHM 99mTc-ECDG. Planar wholebody imaging was performed with a gamma camera using recording times at 0, 1 and 2 hours post-injection. One baboon received a wholebody Single Photon Emission Computed Tomography at 0, 1 and 2 hours post-injection of the IHM 99mTc-ECDG to do anatomical and physiological organ localization. Regions of interest were drawn around the organs on the planar wholebody images for different time points to determine the biodistribution of the IHM 99mTc-ECDG. Results IHM 99mTc-ECDG was labelled successfully with a radiochemical purity of >95% as determined by HPLC. Biodistribution to the stomach and salivary glands was not visible on the scintigraphic images of IHM 99m Tc-ECDG at imaging time points, thus confirming non-significant amounts of free 99mTc-pertechnetate. The IHM 99mTc-ECDG kit’s biodistribution was to the heart, liver, gallbladder, joints and kidneys. IHM 99mTc-ECDG was excreted from the kidneys to the bladder. Conclusions The normal biodistribution of the IHM 99mTc-ECDG was effectively demonstrated in all seven baboons with no adverse effect recorded. The IHM 99mTc-ECDG visually demonstrated lower biodistribution to the brain as observed with 18F-FDG (gold standard). IHM 99mTc-ECDG is ready to be evaluated further in clinical trials. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S114: Poster

21st International Symposium on Radiopharmaceutical Sciences

114 Ultrasmall radioiodinated αMSH-C dots for melanoma imaging and therapy Xiuli Zhang1, Miriam Benezra2, Kai Ma3, Fabio Gallazzi4, Li Zhang2, Mohan Pauliah2, Uli Wiesner3, Thomas P. Quinn1, Michelle Bradbury2 1 Biochemistry, University of Missouri, Columbia, Missouri, United States, 2Radiology, Sloan Kettering Institute for Cancer Research, New York, New York, United States, 3Materials Science and Engineering, Cornell University, Ithaca, New York, United States, 4Research Cores, University of Missouri, Columbia, Missouri, United States Objectives Cornell dots (C dots) are core-shell silica nanoparticles with an average diameter of 7 nm[1]. C dots received FDA-investigational new drug approval for a first-in-human clinical trial [2]. In this study, a melanomatargeting alpha melanocyte stimulating hormone (αMSH) peptide analog was conjugated to the C dots [3]. The αMSH-C dots were synthesized and characterized for melanoma cell binding. Radioiodinated αMSH-C dots were examined for their biodistribution properties in melanoma bearing mice. Methods A cyclic αMSH peptide analog [3] was conjugated to PEG-silane and attached to the surface of the C dots. IC50 studies were performed with 125I-NDP peptide at various αMSH-C dot concentrations. The αMSH-C dots were radiolabeled by 125I-Bolton-Hunter reagent or preconjugated with Bolton-Hunter reagent then iodinated with 125 I using Iodogen™. Cell binding of the 125I-αMSH-C dots was examined in B16/F10. Biodistribution studies were performed in B16/F10 murine melanoma bearing C57 mice and M21 human melanoma xenografted nude mice. Results The IC50 value of αMSH-C dots was 0.86±0.03 nM. Specific cell binding of 1% and 1.3% was obtained for the 125I-αMSH-C dots at 2 h and 4 h incubations, while blocking studies at the same time points decreased cell binding to 0.5% and 0.8%. Biodistribution studies revealed that the B16/F10 tumor uptake of the 125I-αMSH-C dots was 4.31±0.61 % ID/g, 4.73±2.17 % ID/g and 4.73±0.77 % ID/g at 1 h, 4 h and 24 h post injection and uptake in M21 human melanoma xenografted nude mice was 5.81±1.29 % ID/g, 3.27±0.35 % ID/g and 5.68±1.41 % ID/g at 1 h, 4 h and 24 h post injection. Conclusions 125I radiolabeled αMSH-C dots exhibited rapid radiochemical formulation and receptor-mediated tumor uptake in melanoma cells. In vivo, 125I-αMSH-C dots were taken up by melanoma xenografts and were retained by tumors over a period of several days. Radiolabeled αMSH-C dots are a promising nanoparticle construct for in vivo melanoma imaging and therapy applications. Acknowledgements NIH R01 CA161280-02 References [1] Benezra M, et al (2011), J Clin Invest, 121, 2768-80. [2] Phillips E, et al (2014) Sci Transl Med, 6, 260ra149. [3] Miao Y, et al (2007) Front Biosci, 12, 4514-4524.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S115

115 A DOTA based bisphosphonate containing an albumin binding moiety for delayed body clearance in bone targeting radiotherapy Marian Meckel1, Nina Pfannkuchen1, Matthias Miederer2, Frank Roesch1 1 Institute of Nuclear Chemistry, Johannes Gutenberg-University, Mainz, Germany, 2Department of Nuclear Medicine, University Hospital, Mainz, Germany Objectives Radio-labeled bisphosphonates (BP) are commonly used in diagnosis and therapy of bone metastases. In many cases blood clearance is fast and only 30 - 50 % of the injected activity is retained in the skeleton. A longer blood circulation may enhance accumulation of the compounds in the bone metastases. Therefore, a modified macrocyclic BP derivative with an additional binding entity to human serum albumin (HSA) was synthesized and evaluated in vitro and in vivo. Methods The theranostic bisphosphonate BPAMD was compared to the novel albumin binding BP DOTAGA(428-D-Lys)MBP (ABBP). Both tracers were labeled with 68Ga and evaluated in binding studies to artificial bone material as well as to HSA. The compounds were further compared in in vivo μPET and ex vivo organ distribution studies in healthy Wistar rats over a time period of 3 h p.i.. Results Binding studies revealed a consistent affinity to apatite of both the albumin binding BP (81.3 ± 2.2 %) and the original compound (81.6 ± 0.5 %). [68Ga]ABBP showed a distinguished binding of 88.1 ± 5.9 % to HSA compared to 28.8 ± 1.1 % for [68Ga]BPAMD. In vivo μPET and ex vivo organ distribution studies resulted in significant longer blood concentration levels (SUVblood = 2.59 ± 0.72 and 0.06 ± 0.01, respectively). Skeletal accumulation of the modified compound increased strongly over time, while the accumulation of the non-modified BPAMD stayed constant. Ratios of the femur epiphyseal plate to the diaphysis showed to be more favorable for [68Ga]ABBP with a ratio of 2.9 compared to 1.9 for [68Ga]BPAMD. Conclusions The modification of BPAMD towards an albumin binding BP resulted in a novel compound which conserves the activity of both functional groups within one molecule. Thus, blood retention was significantly longer for the albumin binding DOTA-BP. The better bioavailability resulted in better ratios between high and low metabolic bone sections. Further studies with the therapeutic nuclide 177Lu have to determine the therapeutic potential of these novel tracers. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S116: Poster

21st International Symposium on Radiopharmaceutical Sciences

116 Dual Indium-111 and Fluorescent Labeling of iRGD-Targeted Undecylenic-acid –modified Thermally Hydrocarbonized Porous Silicon (UnTHCPSi) Particles for Drug Delivery System Evaluation In Vivo Mirkka Sarparanta1, 5, Chang-Fang Wang2, Ermei Mäkilä3, Maija Hyvönen4, Pirjo Laakkonen4, Jarno Salonen3, Jouni Hirvonen2, Hélder Santos2, Anu J. Airaksinen1 1 Laboratory of Radiochemistry, University of Helsinki, Helsinki, Finland, 2Division of Pharmaceutical Chemistry and Technology, University of Helsinki, Helsinki, Finland, 3Laboratory of Industrial Physics, University of Turku, Turku, Finland, 4Institute of Biomedicine, University of Helsinki, Helsinki, Finland, 5Memorial Sloan Kettering Cancer Center, New York, New York, United States Objectives Our aim was to develop and evaluate with SPECT/CT imaging and fluorescence microscopy a biocompatible porous silicon nanosystem for the delivery of the antiangiogenic drug sorafenib in a mouse model of prostate cancer. Methods The carboxylic acid residues on UnTHCPSi particle surface were modified with dibenzocyclooctynePEG4 using EDC/NHS coupling, followed by copper-free click conjugation of azido-modified Alexa Fluor 488, and DOTA [1]. The nanoparticles were radiolabeled with 111InCl3 in 0.1 M sodium acetate buffer (pH 6) at 85°C for 15 min, followed by conjugation of azidoalanine-modifed iRGD in deionized water at 37°C for 30 min. 111Inlabeled nanoparticles were administered to male NMRI nude mice bearing PC3-MM2 xenografts either intravenously (i.v.) or intratumorally (i.t.) and the mice were imaged on a Bioscan NanoSPECT/CT at 1 h and 26 h after administration followed by ex vivo biodistribution. The therapeutic efficacy of the nanosystem was assessed by two successive administrations of sorafenib-loaded nanoparticles complemented by determination of long-term tissue-level biodistribution using fluorescence microscopy to detect the Alexa Fluor 488 label. Results Modification of UnTHCPSi nanoparticles with iRGD, Alexa Fluor 488, and DOTA was corroborated with FTIR spectroscopy. The nanoparticles were radiolabeled at decay-corrected radiochemical yields ranging from 25– 40% (43.3–60.8 MBq/mg) for UnTHCPSi-iRGD and 48–72% (39.6–56.9 MBq/mg) for non-targeted UnTHCPSi, respectively. After i.v. administration both particle types were taken up by the organs of the MPS, the liver and the spleen. However, the iRGD-modification significantly improved the retention of the nanosystem at the tumor. A prolonged inhibitory effect compared to the free drug was achieved after intratumoral delivery for both particle types likely due to sustained release of the drug from the porous network. Conclusions In conclusion, the results demonstrate the feasibility of using multimodality approach for simultaneous interrogation of multiple aspects of PSi drug delivery system performance in vivo together with therapeutic efficacy. Acknowledgements The University of Helsinki Funds, the Academy of Finland (decisions 252215, 256394, 136805, and 272908), Biocentrum Helsinki, and the ERC (FP/2007–2013, grant no. 310892) for funding. References [1] Wang CF et al. (2012) Biomaterials, 35(4): 1257–1266.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S117

117 Synthesis and Evaluation of [2+1] Re(I)/99mTc(I) Complexes as Isostructural Luminescent Turn-on and Nuclear Imaging Probe Abdolreza Yazdani, Shannon Czorny, John Valliant Chemistry, McMaster University, Hamilton, Ontario, Canada Objectives The objective was to prepare [2+1] complexes of Re/Tc as complementary pairs of turn-on optical and nuclear probes. Bioorthogonal chemical reactions between radiolabeled tetrazines and biomolecules derivatized by strained alkenes such as trans-cyclooctene have been used recently to create highly effective molecular imaging probes.1,2 In addition, fluorophore-tetrazine derivatives were used to prepare highly sensitive turn-on optical probes for visualization of cellular processes where fluorescent emission increases upon reaction with the complementary TCO derived component.3,4 Combining these features, the goal was to develop a new Re-tetrazine complex as a turn-on luminophore that can be used for assessing cellular distribution and as an optical stain for margin determination during surgery which also has a 99mTc analogue that can be employed to prepare the complementary SPECT agent for whole body imaging. Methods To prepare the luminescent metal complex, [Re(CO)3(H2O)3]+ was combined with 2,2'-bipyridine (bipy) to give [Re(CO)3(bipy)Br] which in turn was converted to the desired tetrazine derivative at room temperature through the treatment with a functionalized imidazole conjugate. The corresponding 99mTc complex, [99mTc(CO)3(bipy)(L)]+ (L = imidazole-tetrazine), was obtained by adding [99mTc(CO)3(bipy)(H2O)]+ to the imidazole-tetrazine ligand and heating at 60°C for 30 min. The reactivity of the metal complexes were then assessed using various TCO derivatives. Results The complex [Re(CO)3bipyL]+ was synthesized in 55% yield and showed minimal luminescence prior to reaction with TCO. The corresponding 99mTc complex was synthesized in >85% yield using a convenient two step process where stability studies showed that the metal complex was resistant to ligand challenge and is suitable for in vivo imaging. The reaction between the Tc-complex and TCO was found to have a second rate order constant of 8.6 × 103 M-1S-1 at 37°C, which is adequate for use with pretargeting strategies. Conclusions Isostructural 99mTc and Re complexes of tetrazine derivatives were prepared as a unique class of isostructural and nuclear and optical turn-on probes. The reagents offer the opportunity to employ Tc or Re to create targeted multimodal probes using the presence of the tetrazine as a way to ligate the metal complexes to TCO functionalized biomolecules, including antibodies. Acknowledgements References [1] Li Z, et al. Chem. Commun. (2010) 46, 8043-8045. [2] Rossin R, et al. Angew. Chem. Int. Ed. (2010) 49, 3375-3378. [3] Carlson J C T, et al. Angew. Chem. Int. Ed. (2013) 52, 6917-6920. [4] Devaraj N K, et al. Accounts Chem. Res. (2011) 44, 816-827.

J Label Compd Radiopharm 2015: 58: S1- S411

S118: Poster

21st International Symposium on Radiopharmaceutical Sciences

118 Radiolabeling DNA aptamers with 68Ga for molecular cancer imaging Marlies Gijs1, 2, Sylvestre Dammicco1, Corentin Warnier1, An Aerts2, Nathalie Impens2, Sarah Baatout2, André Luxen1 1 Cyclotron Research Center, University of Liège, Liège, Belgium, 2Radiobiology Unit, Belgian Nuclear Research Center, Mol, Belgium Objectives Aptamers are small (5-15 kDa, 15–60 mer), synthetic, single-stranded nucleic acids (DNA or RNA) that can interact with a specific target at high affinity and specificity. Aptamers exhibit significant advantages relative to protein vectors of radiopharmaceuticals in terms of size, synthesis, modifications, possible targets and immunogenicity. In this study, a DNA aptamer (40-mer) was radiolabeled with 68Ga for molecular cancer imaging by position emission tomography (PET). Methods A disulfide-functionalized DNA aptamer was bioconjugated to the macrocylic bifunctional chelator MMA-NOTA (maleimido-mono-amide (1,4,7-triazanonane-1,4,7-triyl)triacetic acid) in 1 M ammoniumacetate buffer. The NOTA-aptamer bioconjugate was radiolabeled with pre-concentrated and purified 68Ga (Mueller method1) in 1 M HEPES buffer at room temperature. The radiochemical yield, purity and stability were determined by radio-TLC and radio-HPLC. Results Quantitative bioconjugation of the disulfide-functionalized aptamer with MMA-NOTA (75-fold molar excess) was achieved within 120 minutes at 40°C. ESI-MS analysis confirmed that a single chelator was attached per aptamer. Afterwards, this NOTA-aptamer bioconjugate was radiolabeled with purified and pre-concentrated 68 Ga with quantitative radiochemical yield, high radiochemical purity and high chelate stability. Under the tested conditions, the use of a 1470-fold molar excess (or 2.5 nmol of bioconjugate) resulted in a maximal expected specific activity of 51.1 MBq/nmol, which is approximately half of the theoretic specific activity. Conclusions Post-processing of generator-produced 68Ga and radiolabeling conditions for DNA aptamers were successfully optimized to obtain pure and stable 68Ga-radiolabeled aptamers in a fast and simple way without the need for post-labeling purification steps or additional manipulations. At the moment, in vivo studies using xenografted mice are ongoing to evaluate the 68Ga-labeled aptamer biodistribution profile and its potential as imaging radiopharmaceutical using micro-PET. Interestingly, the same bioconjugate could be used after the imaging procedure with other radiometals, such as 177Lu or 90Y, for radionuclide therapy. Acknowledgements M. Gijs is a recipient of a SCK•CEN - ULg PhD grant References 1. D. Mueller, I. Klette, R. Baum, M. Gottschaldt, M. Schultz and W. Breeman, Bioconjugate Chem., 2012, 23, 1712−1717

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S119

119 Design of a Trivalent 99mTc-Probe for High Avidity Receptor Targeting and Enhanced Target Uptake Yuki Mizuno, Chun-wei Jen , Hirofumi Hanaoka, Tomoya Uehara, Yasushi Arano Graduate School of Phrmaceutical Sciences, Chiba University, Chiba , Japan Objectives Generally most receptor targeted 99mTc-labeled probes contain an excess of unlabeled ligand in the reaction mixture, besides the desired 99mTc-complex. The presence of this unlabeled ligand can impair targetuptake of 99mTc-complex by competing for the receptor at the target site, resulting in lower target uptake. This can be alleviated by a purification step, however this adds additional practical considerations for ease of use in a radiopharmacy setting. Herein we describe a novel synthetic strategy that utilizes the multivalence of metal complexes and can provide a trivalent 99mTc-labeled complex from a monovalent receptor-targeting ligand. We hypothesize that the avidity of the trivalent 99mTc-complex of the present design would be higher than its unlabeled ligand present in the reaction solution and this could minimize the competitive inhibition by unlabeled ligand. In this study, we describe the evaluation and validity of this design strategy on the receptor-specific radiopharmaceutical that target the avb3 integrin. Methods An isonitrile ligand, CN-βAla-GlyGly-c(RGDfK) (L1), was synthesized as a monovalent targeting molecule. 99mTc-complexation with [99mTc(CO)3]+ results in a trivalent [99mTc(CO)(L1)3]+. For comparison, we also prepared 3-l-cys-propionic acid-c(RGDfK) (L2) as a conventional ligand which forms “1 to 1” complex with [99mTc(CO)3]+. The binding affinity to integrin αvβ3 positive cells of each Re-complex and ligand were determined by the competitive inhibition assay. The in vivo biodistribution of radioactivity at 1 h p.i. of each non-purified 99m Tc-labeling reaction solution (0.3 μCi, 5 nmol of unlabeled ligand/100 μL) was evaluated in tumor bearing nude mice. Results Radiochemical yield of [99mTc(CO)3(L1)3]+ obtained was >95%. In the affinity assays, [185/187Re(CO)3(L1)3]+ displayed 10 times lower IC50 than the monovalent ligand L1 did, while the monovalent complex [185/187Re(CO)3(L2)] and the monovalent ligand L2 displayed similar IC50. In the in vivo biodistribution studies, the tumor uptake of [99mTc(CO)3(L1)3]+ was 3.80 ± 0.57 %ID/g; significantly higher than that observed for [99mTc(CO)3(L2)], 1.14 ± 0.11%ID/g. Conclusions The multivalent design strategy based on the “1 to 3” radiolabeling reaction for 99mTc-complexes allowed the formation of 99mTc-labeled complex that displays high tumor uptake despite the presence of its unlabeled ligand. This strategy may enable the preparation of receptor-specific 99mTc-labeled complex displaying high in vivo target uptake without tedious post-labeling purification. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S120: Poster

21st International Symposium on Radiopharmaceutical Sciences

120 68 Ga-NOTA-UBI: A novel PET imaging probe detecting infection Jan Rijn Zeevaart1, Thomas Ebenhan2, Akin Orunmuyi2, Mike Sathekge2 1 Radiochemistry, Necsa, Pretoria, South Africa, 2Nuclear Medicine, University of Pretoria & Steve Biko Academic Hospital, Pretoria, South Africa Objectives Non-invasive diagnoses of infection represents a clinical challenge, however preclinical studies proved the ability of a NOTA (1,4,7-triazacyclononane-triacetic acid)-conjugated fragment (29-41) of ubiquicidin (UBI) complexed with Gallium-68 (68Ga-NOTA-UBI) to be an infection-selective imaging agent in rabbits [1]. As antimicrobial structures are almost unexploited for non-invasive molecular localization of infectious microorganisms 68Ga-NOTA-UBI PET was evaluated in a first-in-man study. Methods High-specific activity Gallium-68-radiolabeling of NOTA-UBI was prepared according to [2]. A 69 year old female patient with history of discitis of the lumbar spine (MRI confirmed L2/3 and L3/4 spondylo-discitis with significant erosion of the L3 and L4 vertebral bodies) was injected intravenously with 68Ga-NOTA-UBI (injected dose: 170 MBq, specific activity of 38 GBq/µmol, unstarved, oral contrast given). A whole body PET-CT (Siemens Biograph True Point, 40 slice CT) image was acquired at 60 minutes followed by 3D-image analysis, drawing volume-of-interests yielding the highest targeted activity concentration (SUVmax) to allow for target to non-target (T/R) ratio calculation. Results The sagittal image slides (Fig. 1) clearly define the elevated uptake in the spine representing bacterial localization despite the antibiotic treatment of the patient. A SUVmax value of 2.8 for the infectious areal was obtained resulting in moderate T/R ratios, amounting to 2.5 and 2.2 for soft and muscle tissues respectively. Conclusions Translation of 68Ga-NOTA-UBI successfully passed from the pre-clinical phase into clinical research not only adding value but also making it relevant from a patient care perspective. Acknowledgements References [1] Ebenhan TE, Zeevaart JR, et.al. J. Nucl. Med. 55 (2014) 308-314. [2] Ebenhan TE, et. al. Nucl. Med. Biol. 41 (2014) 390-400.

Sagittal PET (A) and fused PET-CT (B) image acquired 60 minutes after i.v. administration of 170 MBq 68GaNOTA-UBI. The arrows indicate the site of infection.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S121

121 The revival of 111In leukocyte labeling? Svend B. Jensen1, 2, Lotte Studsgaard S. Meyer 1, Aage K. Alstrup 3, Pia Afzelius4, Dirk A. Bender1, Henrik C. Schønheyder5, Ole L. Nielsen 6 1 Nuclear Medicine, Aalborg Universitetshospital, Aalborg, Denmark, 2Chemistry and Biochemistry, Aalborg University, Aalborg, Denmark, 3Nuclear Medicine and PET Centre, , Aarhus University Hospital, , Aarhus, Denmark, 4Department of Diagnostic Imaging , North Zealand Hospital, Hillerød, Denmark, 5Clinical Microbiology, Aalborg University Hospital, Aalborg, Aalborg, Denmark, 6Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark Objectives 111In-leuckocyte imaging has for many years been the golden standard for detection of infections. We are applying a pig model in a quest for finding superior osteomyelitis tracers. We did a head to head comparison of 7 common tracers with a potential for detection of osteomyelitis [1]. Here, we highlight the performance of 111Inleukocytes versus 18F-FDG. Methods Staphylococcus aureus inoculation was done into a week before the scan day using an established technique [2]. 18F-FDG and 111In-labeled leukocytes was produced by a standard procedure. SPECT/CT was performed on a Symbia T16 scanner. We applied the medium-energy collimators as suggested in [3]. PET/CTs were obtained using an integrated 64 PET/CT system. Furthermore, we conducted a systematic literature search on leukocyte scintigraphy in pigs using PubMed and Web of Science. Results 18F-FDG was found to be slightly better than 111In-leukocyte SPECT as an osteomylitis tracer [1]. In humans an 111In-leukocyte scan is normally conducted after 24-48 h. 111In-leukocyte scans were performed after only 5 h (+/- 1h) due to our conplex experimental setup (18h/ 7 different tracers)[1]. We found a lower labeling efficiency in pigs than in humans which we presumed to be related to a different composition of the pig blood compared to human. We’ll explore alternative explanations that may encompass non optimal scan time or simply a better performance of 18F-FDG than 111In-leukocyte. Conclusions We found 18F-FDG to be a slightly superior tracer for osteomylitis infection compared with 111Inleukocyte when the label leukocytes were injected 6 h prior to scan. We discuss and have seeked further explanations for this through a review of the literature on leukocyte SPECT in pigs. Acknowledgements Grant no. 0602-01911B (11-107077) Danish Council for Independent Research, . References [1] Nielsen OL et al, Am. J. Nucl. Med. Mol. Imaging (in press). [2[ Johansen LK et al, (2012), J Comp Pathol; 147: 343-353 and al (2013), J Invest Surg; 26: 149-153. [3] Jodal L et al (2014). J Nucl Med Technol; 42: 42-50.

J Label Compd Radiopharm 2015: 58: S1- S411

S122: Poster

21st International Symposium on Radiopharmaceutical Sciences

122 64 Cu-labeled human serum albumin for tumor imaging Hyunjung Kim1, 2, Choong Mo Kang1, 2, Hyun-Jung Koo1, 2, Gwang Il An3, Yearn Seong Choe1, 2, Joon Young Choi1, 2, Kyung-Han Lee1, 2, Byung-Tae Kim1, 2 1 Nuclear Medicine, Samsung Medical Center, Seoul, Korea (the Republic of), 2Sungkyunkwan University School of Medicine, Seoul, Korea (the Republic of), 3Korea Institute of Radiological and Medical Sciences, Seoul, Korea (the Republic of) Objectives Human serum albumin (HSA) is the most abundant protein in blood plasma and has long been known for its function as a drug carrier. In addition, it was reported that 111In-labeled rat serum albumin underwent lysosomal degradation and radioactivity was trapped by the catabolic sites of tumor-bearing mice [1, 2]. In this study, we prepared and evaluated a HSA-based PET imaging probe for tumor imaging. Methods A DOTA molecule was conjugated to a Cys residue of HSA, which was then purified using a PD-10 column. The number of DOTA molecules conjugated to HSA was calculated using the reported method [3]. The DOTA-conjugated HSA was labeled with 64Cu and purified using a PD-10 column. In vitro serum stability was determined by incubating the 64Cu-labeled probe with fetal bovine serum at 37 oC for 48 h and analyzing the incubation mixture at the indicated time points. Cell binding study was performed by incubating the 64Cu-labeled probe with U87MG cells at 37 oC for 42 h. U87MG tumor-bearing mice were injected with the probe and then underwent microPET imaging at 1, 16, 22, and 42 h after injection. At the end of the imaging, the mice were sacrificed and the major tissues were collected, weighed, and counted. Results The number of DOTA molecule conjugated to HSA was 0.9. 64Cu-labeled probe was prepared in 84.3% radiochemical yield with specific activity of 0.9 GBq/mg. Serum stability study showed that the probe was stable for 48 h. Cell binding of the probe increased in a time-dependent manner from 0.4% ID at 1 h to 69.8% ID at 42 h. MicroPET imaging showed high radioactivity accumulation in the liver, kidneys, and tumors, and ROI analysis of the tumor tissues revealed 3.8% ID/g at 16 h and 4.2% ID/g at 42 h. Biodistribution of the mice at 42 h demonstrated the tumor uptake of 1.5% ID/g, with the kidney uptake of 5.6% ID/g and the liver uptake of 10.1% ID/g. Conclusions We successfully prepared 64Cu-labeled HSA. Cell binding, microPET imaging, and biodistribution results showed that 64Cu-labeled probe has potential for tumor imaging. These results suggest that 64Cu-labeled HSA can be further used as a platform to improve tumor detection by conjugating with tumor targeting molecules. Acknowledgements Supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2011-0030164). References [1] Wunder A, et al (1997) Int J Oncol, 11, 497-507. [2] Stehle G, et al (1997) Crit Rev Oncol Hematol, 26, 77-100. [3] Cai W, et al (2006) Cancer Res, 66, 9673-9681.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S123

123 Development and evaluation of a new bifunctional chelator (DOTAPyr) for multimodal molecular imaging via PET, SPECT, NIR and MRI Sebastian Görres1, Yong Ook Kim2, Mustafa Diken4, Lukas Beckmann3, Nils Engelbogen3, Frank Roesch3, Detlef Schuppan2, Tobias L. Ross1, 3 1 Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany, 2Institute of Translational Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany, 3Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz, Mainz, Germany, 4TRON – Translational Oncology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Objectives For multimodal molecular imaging the challenge is to enable a wide range of imaging techniques by appropriate modifications to the molecule without altering the biochemical characteristics. The aim of this work was to develop a versatile chelator, which makes various modalities available only by replacing the central atom. The focus was on PET (Ga-68), NIR (Eu, Tb) and MRI (Gd). Methods As a matrix DO2A was chosen because of the desirable wide range of possible central atoms. To connect the chelator to biomolecules without lowering the dentate of the chelator, glutaric acid was linked at the 2 position. To allow NIR measurements via antenna effect, picolinic acid was introduced at N10 of DO2A. The new DOTAPyr and a peptide construct containing DOTAPyr were labelled with Eu and its photophysical properties were determined [1]. First radiolabelling of DOTAPyr was performed in 1.5M HEPES buffer at 100 °C. The Eupeptide is being evaluated in in vitro NIR studies. Labelling with Tb is under execution. In vivo PET imaging with Ga-68-labelled DOTAPyr and with the DOTAPyr containing peptide are planned. Results 2-(4,10-bis(carboxymethyl)-7-((6-carboxypyridin-2-yl)methyl)-1,4,7,10-tetraazacyclododecan-1yl)glutaric acid (DOTAPyr) could be synthesized successfully within 10 steps with a very good overall yield of 10%. Eu has already been successfully chelated and displays desired emissions in the range of 700 nm. The chelation of Tb is currently being carried out similarly. First radiolabelling with Ga-68 shows high labelling yields of about 80%. Conclusions The new chelator offers excellent facilities to apply a wide range of imaging modalities without changing the structure of the actual molecule or the need to attach additional structures. First biologically interesting constructs are already evaluated and show promising results. Acknowledgements References [1] Regueiro-Figueroa M., et al (2011) Inorg. Chem., 50, 4125-4141

J Label Compd Radiopharm 2015: 58: S1- S411

S124: Poster

21st International Symposium on Radiopharmaceutical Sciences

124 Optimization of labeling PSMAHBED with 68Ga and its quality control systems Elisabeth Eppard1, Tatjana Homann2, Anna de la Fuente2, Frank Roesch2 1 Nuclear Medicine, University Hospital Bonn, Bonn, Germany, 2Institute of Nuclear Chemistry, Johannes Gutenberg-University, Mainz, Germany Objectives Radiolabeling of the prostate-specific membrane antigen (PSMA) inhibitor, Glu-NH-CO-NH-Lys (Ahx), using the 68Ga chelator HBED-CC (PSMAHBED) allows imaging of lesions of prostate cancer due to the high expression of PSMA in prostate carcinoma cells. The aim of this work was the optimization of the labeling of the 68 Ga-PSMAHBED using a CEX post-processing, adaption on an automated module system, as well as the development of a TLC-based quality control system Methods Labeling was optimized for online ethanol post-processed 68Ga eluate. Influence of buffer (0.1-1 M), temperature (25-90°C) and tracer amount (0.1-0.7 nmol) was investigated. Additional acetone post-processed 68Ga eluate was investigated with regard to influence of temperature using 0.2 nmol PSMAHBED and 0.1 M ammonium acetate buffer (pH=5). Synthesis was established on an automated module system and optimized. For radio-TLC quality control various mobile phases were analyzed on silica gel 60 plates and results validated using HPLC. The most superior mobile phases were also applied on ITLC-SG-plates. Results Using optimized conditions labeling yields of > 95% could be obtained applying both post-processing methods within 10 min. Purification using STRATA-X is also possible if needed. The synthesis was effectively processed on an automated module system. For the TLC quality control on silica gel 60 plates four mobile phases with good separation properties and complementary Rf values could be identified. Only two of these systems showed the same separation qualities on ITLC-SG-plates (MeOH/NH4OAc (1:1); 5% NaCl/MeOH/25% NH3 (3:1:1)). With ITLC_SG analysis could be finished within 5 min. Conclusions Labeling of PSMAHBED was optimized for CEX post-processing that ensure high nuclide purity of final 68Ga-PSMAHBED. Using this approach, the synthesis is easily adapted by automated synthesis modules. The new quality control method allows the quality control in a short time using a fast, reliable, low cost radio TLCmethod with little equipment effort. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S125

125 Authentically radiolabelled Mn(II) complexes as bimodal PET/MR tracers Christian Vanasschen1, Marie Brandt1, Johannes Ermert1, Bernd Neumaier2, Heinz H. Coenen1 1 Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Jülich, Jülich, Germany, 2Institut für Radiochemie und Experimentelle Molekulare Bildgebung, Universitätsklinik Köln, Köln, Germany Objectives Hybrid PET/MR imaging will pave the way for a better understanding of physiological and disease mechanisms in preclinical and clinical settings. Authentic radiolabeling of MR contrast agents ensures a fast and simple access to such bimodal tracers. In this case, a ligand chelating a paramagnetic metal ion (e.g. Mn) spiked with the authentic PET isotope (such as 52gMn) leads to a labelled molecule which can be detected with both imaging modalities. Paramagnetic [55Mn(CDTA)]2- shows an excellent compromise between thermodynamic stability, kinetic inertness and MR contrast enhancement [1]. The aim of this work was a proof of principle study of cyclohexanediaminetetraacetic acid (CDTA) ligands as prosthetic groups for Mn-labelled PET/MR tracers. Methods N.c.a. 52gMn [t1/2: 5.6 d; Eβ+: 575.8 keV (29.6%)] was produced by proton irradiation of a natCr target and purified by cation-exchange chromatography [2]. CDTA radiolabeling with n.c.a. 52gMn2+ was performed in NaOAc buffer (1M, pH 6) at RT and monitored by radio-TLC as well as IC. Purification of the complex was performed by RP-HPLC and a test on stability in NaOAc buffer. A hydroxyalkyl functionalized CDTA ligand was synthesized starting from 3-cyclohexene-1-methanol in 5 steps. Results The quantitative formation of [52gMn(CDTA)]2- was observed already within 30 min at RT. The complex was stable for at least 48 h at 50 °C. If an isotopic 52g/55Mn2+ mixture was applied the first prototypical manganese based bimodal PET/MR tracer was obtained. Furthermore, the hydroxyalkyl functionalized CDTA ligand was synthesized with an overall yield of 18-25%. Conclusions Due to the hydrolytic stability and simple preparation of [52g/55Mn(CDTA)]2-, the CDTA ligand should be highly suitable for the preparation of manganese based PET/MR bimodal tracers. Acknowledgements The authors thank M. Buchholz for production of 52gMnCl2. References [1] Kalman, F. et al. (2012), Inorg. Chem., 51, 10065; [2] Buchholz, M. et al. (2013), Radiochim. Acta, 101, 491.

J Label Compd Radiopharm 2015: 58: S1- S411

S126: Poster

21st International Symposium on Radiopharmaceutical Sciences

126 Stability comparison study using gel electrophoresis and radio-HPLC Heesu Ahn, Jeongsoo Yoo Department of Molecular Medicine , Kyungpook National University School of Medicine, Daegu, Korea (the Republic of) Objectives 64Cu is a radiometal with a mid-long half life of 12 h, which is very suitable for long term imaging studies. However, copper being an important micronutrient, can easily bind to proteins like serum albumin and superoxide dismutase (SOD) present in the body. So there is an impending need to develop ultrastable bifunctional chelating agents (BFC). We have previously synthesized ultra stable chelate PCB-TE2A-NCS, conjugated with c(RGDyK), radiolabeled with 64Cu and confirmed its stability by using radio-HPLC.[1] However, this method is time consuming and relatively expensive. A new technique for in vitro stabilty determination was reported which was basically a protein challenge experiment and a serum stability assay.[2] So, we used these polyacrylamide gel electrophoresis techniques to evaluate the stability of peptide conjugated chelates synthesized in our lab. Methods 64Cu labeled PCB-TE2A-NCS-c(RGDyK) and ECB-TE2A-c(RGDyK) were incubated with human serum and human SOD for 1 hour at 37°C. After incubation, SDS-PAGE and native PAGE was performed respectively at 80 V followed 140 V. After electrophoresis, the gels were exposed on image plate for 2 hours and autoradiography was performed. Following autoradiography, the gels were stained with commassi blue dye which would allow us to confirm the position of protein bands on the gel. Results From this in vitro stability assay we found that 64Cu-PCB-TE2A-NCS-c(RGDyK) is more stable than 64 Cu-ECB-TE2A-c(RGDyK). This result is well matched with our previously reported in vivo stability data. Mainly, using this technique we can easily and efficiently compare the stabilty of both the radio tracers. Conclusions These in vitro stabilty assays can very well serve as a substitute for time consuming and expensive in vivo stability experiment using radio-HPLC. Acknowledgements Supported by R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A2A2A01012250, 2013M2A2A 6042317, 20090078235) and the BK21 Plus funded by the Ministry of Education, Korea (21A2013221 2094). References [1] Pandya DN, et al (2014), J. Med. Chem., 57, 7234–7243. [2] K. Zarschler, et al (2014), RSC Adv., 4, 10157–10164.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S127

127 Synthesis and in ovo evaluation of the neuropeptide cathelicidin 89Zr-CATH2 Pantelis Marsouvanidis1, Albert van Dijk2, John Kruijtzer3, Trijntje Cuperus2, Gerben visser2, Henk Haagsman2, Philip Elsinga1, Janine Doorduin1, Gert Luurtsema 1 NGMB, UMC Groningen, University of Groningen, Groningen, Groningen, Netherlands, 2Division of Molecular Host Defense, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, 3Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, Netherlands Objectives Host defense peptides (HDPs) exhibit a wide array of immunomodulatory functions and may so boost innate immunity. The chicken HDP analog cathelicidin 2 (CATH2) is a 26 amino acid long peptide that exhibits antibacterial and immunomodulatory properties in vitro. In ovo administration of CATH2 reduced bacteria-related mortality and morbidity in young birds. We aimed to determine the in ovo distribution of CATH2 over time, using PET/CT. For this purpose we developed a peptide chelator conjugate for labelling with 89Zr. Methods The lyophilized peptide conjugate was dissolved in HPLC-grade water at a final 10 mM concentration. Labeling was conducted by addition of 50 µL saline and 25-30 MBq of 89Zr-oxalate, followed by 5-7 µL of Na2CO3 2 M (pH 4.5). After incubation at room temperature, 150 µL HEPES 0.1 M was added for pH adjustment to 7. Finally, 100 µg of DFO-CATH2 was added followed by incubation for 30 min at room temperature. The mixture was eluted through an Oasis pre-activated column with a 20% formic acid solution of ethanol resulting in a final volume of ≈ 500 µL. The eluent was concentrated to a small volume (≈ 10-20 µL) under a gentle N2-flux at 350C, diluted with PBS and analyzed by RP-HPLC and TLC. 89Zr-CATH2 peptide (3 MBq) was injected into the amnion of 18-day old Ross308 chicken embryos. Static PET scans of 30 min were performed at 10 min, 4 h, 24 h and 48 h after injection, and were followed by a CT scan. After the last scan, ex vivo biodistribution was performed. Results 89Zr-DFO-CATH2 was obtained in almost quantitative yields as verified by RP-HPLC and ITLC N2-flux. Subsequent reconstitution with PBS did not effect the stability of the peptide. Highest uptake of 89Zr-DFO-CATH2 was found in the intestinal tract. At 4 h after injection uptake was mainly observed in the upper part of the intestinal tract, while at 48 h uptake was found in the lower part and additionally in the lungs. Conclusions We could successfully label DCATH-2 with 89Zr and the uptake of the radiolabelled peptide in chicken embryos over time could be monitored with PET/CT. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S128: Poster

21st International Symposium on Radiopharmaceutical Sciences

128 Synthesis of 99mTc(CO)3 labeled 2-(4-chloro)phenyl-imidazo[1,2-a]pyridine analog (99mTc-CB256) as a new TSPO-selective SPECT radiotracer Hong Jin Lee1, Ji Young Choi1, 2, Mara Perrone 3, Valentino Laquintana3, Jae Ho Jung1, Byung Seok Moon1, Annalisa Cutrignelli3, Angela Lopedota3, Massimo Franco3, Nunzio Denora3, Byung Chul Lee1, 4, Sang Eun Kim1, 2 1 Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, Korea (the Republic of), 2Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Korea (the Republic of), 3Department of Pharmacy – Drug Sciences, University of Bari “A. Moro”, Bari, Italy, 4Advanced Institutes of Convergence Technology, Suwon, Korea (the Republic of) Objectives The 18 kDa translocator protein (TSPO) is a mitochondrial protein associated with a wide number of biological processes including cell proliferation, apoptosis, steroidogenesis, and immunomodulation [1]. In this work, we report the radiosynthesis and TSPO-positive tumor cell binding assay of 99mTc-CB256 as a new TSPOselective SPECT imaging agent. Methods The synthesis of Re-CB256 and 99mTc-CB256 were carried out as described in Fig 1.99mTc(CO)3 incorporation to CB256 was performed according to literature [2] and purified by HPLC. In vitro tumor cellbinding assay of 99mTc-CB256 was evaluated in C6 rat glioma and U87-MG human glioblastoma cells in presence of PK11195 (0 ~ 103 nM), including measurement of log D, IC50 ([3], see Fig. 1), and in vitro stability. Results 99mTc-CB256 was synthesized in 75~85% of radiochemical yield (d.c.) with over 98% of radiochemical purity. The chemical identity of 99mTc-CB256 was confirmed by comparing retention time of 99mTc-CB256 (22.5 min) with that of Re-CB256 (22 min), including NMR and HRMS. The obtained 99mTc-CB256 was shown to be highly stable (>99%) in human serum for 4 h and had a relatively low lipophilicity (log D = 2.15 ± 0.02). In vitro time dependent tumor cell binding uptake of 99mTc-CB256 was shown 10.26 ± 0.23 and 7.88 ± 0.23 %ID in C6 and U87-MG cells at 60 min, respectively. Conclusions Our in vitro data indicated that 99mTc-CB256 can be considered as a new TSPO-positive cancer imaging agent and provides the foundation for further in vivo biological evaluation in tumor xenograft. Acknowledgements This study was supported by grant (2014M3C7A1046042, 2014R1A2A2A01007980, 2014M2A2A7045181, 2009-0078370) of South Korea. The University of Bari (Italy), the Italian Ministero dell’Università e della Ricerca (MIUR), and the Inter-University Consortium for Research on the Chemistry of Metal Ions in Biological Systems (C.I.R.C.M.S.B.) are gratefully acknowledged. References [1] Rupprecht V, et al (2010) Nat Rev Drug Discovery, 9, 971-88. [2] Lee BC, et al (2012) RSC Adv, 3, 782-92. [3] Nunzio Denora, et al. (2014), Med. Chem. Lett. , 5, 685−9. .

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S129

129 Evaluation of 99mTc-labelled FR-targeting Imaging Probe with Novel Linkage to Improve the Tumor Uptake Zhide Guo1, 2, Manli Song1, Mengna Gao1, Pu Zhang1, Linyi You1, Xianzhong Zhang1 1 Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, China, 2Department of Isotope, China Institute of Atomic Energy, Beijing, China Objectives In the present study, we reported the potential use of 99mTc-HYNIC-T-FA as FR-targeting imaging probe. The aim of this study is to evaluate a new molecule probes with novel linkage. Methods The folate derivatives were radiolabeled with 99mTc using tricine and TPPTS as coligands. To investigate their affinity properties, cell uptake, biodistribution and micro-SPECT/CT imaging were evaluated, respectively. Results Radioligands displayed high cell uptake. Pre-addition of excess folic acid resulted in a significant blocking effect. Biodistribution and imaging study demonstrated the preeminent properties of the folate conjugate with PEG backbone. The biodistribution result of 99mTc-HYNIC-D0-PEG-FA1 displayed considerable tumor uptakes compared with 99mTc-HYNIC-T-FA (18.01 ± 3.17 %ID/g vs. 9.67 ± 1.19%ID/g at 2 h p.i. and (15.04 ± 2.43 %ID/g vs. 8.14 ± 0.45 %ID/g at 4 h p.i.). The SPECT imaging showed that 99mTc-HYNIC-D0-PEG-FA1 had higher uptake in KB tumor than non-PEG linker. While, notable uptake observed in the kidneys was increased. Excellent tumorto-kidney ratio was achieved by injecting PMX before radioligands. Conclusions Construction of linkage is an effective approach to alter the tumor uptake and excretion kinetics. Acknowledgements This work was supported by the National Natural Science Foundation of China (21271030). References [1] Müller C, Schibli R. (2011) Journal of Nuclear Medicine, 52, 1-4. [2] Meszaros LK, Dose A, Biagini SC, Blower PJ. (2010) Inorganica Chimica Acta, 363, 1059-1069. [3] Müller C, Forrer F, Schibli R, Krenning EP, de Jong M. (2008) Journal of Nuclear Medicine, 49, 310-317.

The cell binding properties in KB cells. (A and B). The biodistribution results (C and D). SPECT/CT imaging of KB-tumor xenografts bearing athymic nude mice at 2 h (E).

Structure of the radioligands.

J Label Compd Radiopharm 2015: 58: S1- S411

S130: Poster

21st International Symposium on Radiopharmaceutical Sciences

130 Preparation and evaluation of 64Cu-labeled streptavidin/biotin-based RGD dimer for dual PET/optical imaging of αvβ3 receptor expression Choong Mo Kang1, 2, Hyunjung Kim1, 2, Hyun-Jung Koo1, 2, Gwang Il An3, Yearn Seong Choe1, 2, Joon Young Choi1, 2, Kyung-Han Lee1, 2, Byung-Tae Kim1, 2 1 Nuclear medicine, Samsung Medical Center, Seoul, Korea (the Republic of), 2Sungkyunkwan University School of Medicine, Seoul, Korea (the Republic of), 3Molecular Imaging Research Center, Korea Institute of Radiological & Medical Sciences, Seoul, Korea (the Republic of) Objectives Integrin αvβ3, which is a heterodimeric cell surface receptor, plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides, selective antagonists for integrin αvβ3, have thus been used for imaging of integrin αvβ3 expression. Previously, we have developed a dual PET/optical probe for imaging of vascular endothelial growth factor receptor 2 expression using a streptavidin/biotin complex [1]. In this study, we aimed to develop a 64Cu-labeled (AlexaFluor 680)streptavidin/biotin-based RGD dimer (64Cu-labeled dual probe) for PET/optical imaging of αvβ3 receptor expression. Methods DOTA-(AlexaFluor 680)streptavidin and biotin-PEG-RGD dimer were respectively prepared, and the mixture was stirred at room temperature for 1 h. The resulting mixture was then radiolabeled with 64Cu and purified using a spin column. For receptor binding assay, U87MG cancer cells were incubated with different concentrations of the unlabeled dual probe or RGDyK, and 125I-RGDyK was used as the radioligand. U87MG tumor-bearing mice were injected with 64Cu-labeled dual probe and subjected to PET/optical imaging. For the blocking study, the mice were co-injected with 64Cu-labeled dual probe and RGD dimer. At the end of the 20-h imaging, the mice were sacrificed and tissues of interest were collected, weighed, and counted. Immunofluorescence staining of the tumor tissues was performed to verify β3 expression. Results Unlabeled dual probe was shown to be free from biotin-PEG-RGD dimer when analyzed using a sizeexclusion HPLC column. The IC50 values of the unlabeled dual probe and RGDyK were 62.4 nM and 145.8 nM, respectively. MicroPET imaging showed high radioactivity accumulation in the liver, spleen and tumors, and ROI analysis of the tumors revealed 2.6% ID/g at 1 h and 5.4% ID/g at 20 h. In the blocking group, the tumor uptake decreased by 38% at 16 h. Optical images also showed strong fluorescence signals in the tumors. Biodistribution study of the mice at 20 h demonstrated the tumor uptake of 3.29% ID/g. Immunofluorescence staining of the tumor tissues indicated high level of β3 expression on the endothelial cells. Conclusions 64Cu-labeled dual probe was successfully prepared using a streptavidin/biotin platform. In vitro and in vivo results demonstrated that the 64Cu-labeled dual probe has potential for dual PET/optical imaging of integrin αvβ3 expression. Acknowledgements Supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2011-0030164). References [1] Kang CM, et al (2013) Biomaterials, 34, 6839-6845.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S131

131 68 Ga-labeled peptides for targeting phosphatidylserine (PS) in apoptosis Amanda Perreault, Susan Richter, Christian Foerster, Cody Bergman, Melinda Wuest, Frank Wuest Oncology, University of Alberta, Edmonton, Alberta, Canada Objectives Phosphatidylserine (PS), a membrane phospholipid that is externalized to the outer leaflet of the cell membrane during the early stages of apoptosis, is a promising target for an apoptosis-detecting molecular imaging agent. The most common PS-binding ligand used to identify apoptotic cells in vitro is annexin V. However, its use as an in vivo imaging agent is limited by its dependency on extracellular calcium and short blood half-life [1]. Here we report the application of a novel radiometric PS binding assay using 64Cu-labeled annexin V as radiotracer to investigate novel PS-binding peptides as potential leads for the development of an apoptosis-detecting molecular imaging agent. Methods Peptides were prepared using solid-phase peptide synthesis and subjected to a radiometric PS binding assay to assess their inhibitory potency towards PS. Selected peptides were radiolabeled with 68Ga. Results Annexin V displayed the highest inhibitory potency to compete with [64Cu]Cu-NOTA-annexin V binding to immobilized PS (IC50 52 nM). Several hexapeptides exhibited IC50 values in the range of 1-15 mM, whereas 14mer peptides based on PS-binding motif FNFRLKAGAKIRFG (PSBP-6 [1]) displayed more favorable inhibitory potencies (IC50 9-600 µM). Peptide PSBP-6 was conjugated with NOTA, followed by radiolabeling with 68 Ga. In vivo metabolic stability of [68Ga]NOTA-PSBP-6 in mice revealed that 36% of the tracer remained intact after 60 minutes. [68Ga]NOTA-PSBP-6 resides primarily in the blood plasma rather than blood cells or proteins, demonstrating a good blood distribution profile. Conclusions The novel radiometric binding assay with [64Cu]Cu-NOTA-annexin-V as radiotracer can be used as a versatile tool to screen compounds for their PS-binding potency. Favorable inhibitory potency of PSBP-6 has prompted us to investigate this peptide as a potential lead for the development of a PET radiotracer for Ca2+independent molecular imaging of PS as a biomarker of early apoptosis. Acknowledgements References [1] Xiong C, et al (2011) J Med Chem, 54, 1825-35.

J Label Compd Radiopharm 2015: 58: S1- S411

S132: Poster

21st International Symposium on Radiopharmaceutical Sciences

132 203 Pb-labeled radiotracer targeting metastatic melanoma. Izabela Tworowska1, 2, Nilesh Wagh2, Sanjay Thamake1, Jessica Reedy3, David Ranganathan1, Martin Brechbiel4, Ebrahim S. Delpassand1, Michael K. Schultz3 1 RadioMedix Inc, Houston, Texas, United States, 2RITA Foundation, Houston, Texas, United States, 3Radiology and Radiation Oncology Free Radical and Radiation Biology Program, University of Iowa, Iowa City, Iowa, United States, 4National Cancer Institute 9609 Medical Center Drive, Rm 1-E424 , Rockville, Maryland, United States Objectives The objectives of our studies were to develop a method for Pb-203 labeling of DOTA-glycoconjugates that target metabolic pathways in cancer cells; and to confirm the accumulation and retention of the Pb-203DOTA-glycoconjugates in vitro in metastatic melanoma cell lines. Methods A glucosamine-based glycoconjugate (DOTA-RMX-GC-08) was synthesized via a two-step process described previously.1 The compound was radiolabeled with Pb-203 (t1/2=51 h) obtained from Lantheus Medical Imaging (North Billerica,MA); and the National Cancer Institute (Rockville,MD). The radiolabeling reaction was carried out under mild conditions (0.5M NH4OAc, pH=5.6, T=95oC) for 30min., followed by rC18 Sep-Pak purification of final product. An alternative Pb-203 pre-purification step was explored to determine the potential for improvement on radiolabeling yields. The Pb203-labeled compound was analyzed using radio-HPLC prior to in vitro studies. In vitro competitive uptake of the agent was assessed using the human melanoma cancer cell lines A357(BRAF mutant); MeWo(BRAF WT); and murine metastatic melanoma cell line B16. Uptake and accumulation were evaluated at various time points using varying concentrations of GLUT competitors. Results [Pb-203]-RMX-GC-08 was obtained in 31% yield; with more than 95% purity based on rHPLC and LCMS data. Radiolabeling of agent proceeded with >98% RCY with achievable specific activity of greater than 5 MBq/nmole using pre-purification approach. [Pb-203]-glycoconjugates were stable in PBS, NH4OAC, and citrate buffers for up to 5 days at r.temp. The accumulation of [Pb-203]-glycoconjugate ranged from 5.26 to 11.54 %ID/mg protein with an incubation time of 3h in the tested cells. Pre-incubation of cancer cells with GLUT competitors glucose; glucosamine; cytochalasin B; fasentin; and phloretin inhibited uptake of the [Pb-203]agent and decreased accumulation of agent by a minimum 54%. Conclusions A method for labeling of DOTA-gluconjugates with SPECT isotope Pb-203 was developed and tested. The method allows for efficient radiolabeling of DOTA-conjugated GLUT targeted molecules and can be obtained in sufficient %RCP for preclinical applications. The [Pb-203]-conjugate was found to be radiochemically stable in standard buffers. In vitro studies demonstrate selective uptake and accumulation of [Pb-203]agent in melanoma cell lines. Further studies are required to extend the achievable specific activity and validate the in vivo tumor targeting properties of this class of radiotracers. Acknowledgements This work was supported by NIH/NCI grant 1R43CA186364-01. References 1.I.Tworowska, S.Zamanian, E.Delpassand, M.Schultz-J. Nucl. Med.2012,53,1568.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S133

133 Copolymer-Based Functional SPECT/MR Imaging Agent for Asialoglycoprotein Receptor Targeting Pu Zhang1, 2, Zhide Guo1, Deliang Zhang1, Chang Liu2, Xianzhong Zhang1, Manli Song1 1 Xiamen University, XiaMen, China, 2Beijing Normal University, College of Chemistry, Beijing, China Objectives The synergistic combination of single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) will provide us with accurate diagnosis. The aim of this study is to develop one copolymer-based agent that can be labeled with technetiumn-99m and gadolinium as SPECT/MR dual-modality imaging agent and targeting to asialoglycoprotein receptor (ASGPR) via galactose. Methods Monomers VNI (for 99mTc labeling), V2DTPA (for Gd labeling) and VLA ( for ASGPR targeting) were synthesized respectively. Then the copolymer P(VLA-co-VNI-co-V2DTPA) was polymerized and characterized. After labeled with technetium-99m and gadolinium simultaneously, the radiochemical purity (RCP), relaxivity (r1), toxicity, biodistribution and in vivo SPECT/MR imaging in mice were evaluated respectively (Figure 1). Results Results of biodistribution study showed that the liver uptake was 79.50±6.01 %ID/g and decreased significantly after blocking with GSA at 5 min after injection. SPECT images (Figure 2) also showed specific and high uptake in the liver. The blood vessel of the liver in the normal mice showed obvious enhancement of T1 contrast during 30 min after injection of Gd-P(VLA-co-VNI-co-V2DTPA). Conclusions The SPECT and MR imaging studies identify the multifunctional ability of the probe for multi-modal imaging. Acknowledgements National Natural Science Foundation of China (21271030) References [1] Yang WJ, et al. (2011) J Nucl Med, 52, 978–985. [2] Liu C, et al. (2014) Nucl Med Biol, 41, 587593.

Figure 1 DLS, HPLC and relaxivity measurements.

.

Figure 2 SPECT/CT and MR imaging

J Label Compd Radiopharm 2015: 58: S1- S411

S134: Poster

21st International Symposium on Radiopharmaceutical Sciences

134 Synthesis and biological evaluation of new bone-seeking [Re/99mTc(CO)3(κ3-L)] complexes with [NSN] and [NSO] bifunctional chelators George Makris1, Ioannis Pirmettis2, Minas S. Papadopoulos2, Dionysia Papagiannopoulou1 1 School of Pharmacy, Aristotle University Of Thessaloniki, Thessaloniki, Greece, 2INRASTES, NCSR “Demokritos”, Athens, Greece Objectives The aim of this work is the development of new 99mTc-bisphosphonates for bone imaging. For this purpose we introduced [1] a novel pharmacophore, 1-(3-aminopropylamino)-ethane-1,1-diyldiphosphonic acid, 1, suitable for 99mTc labeling (99mTc1). We report herein two new bone-seeking [Re/99mTc(CO)3(κ3-L)] complexes prepared by conjugating 1 with the potent imidazol-containing bifunctional chelators, 3-(2-aminoethylthio)-3-(1Himidazol-4-yl)propanoic acid in L1 and 2-(2-carboxyethylthio)-3-(1H-imidazol-4-yl)propanoic acid in L2 [1, 2]. Directly labeled 99mTc1 and organometallic tracers of the bifunctional approach, 99mTcL1 and 99mTcL2, were evaluated in vitro and in vivo. Methods Re/99mTc-complexes were synthesized by reacting [Re/99mTc(H2O)3(CO)3]+ with L1 and L2 in water. 99m Tc-complexes were identified by HPLC co-injection with the Re-analogues. 1 was labeled directly with 99m TcO3- in the presence of SnCl2. In vitro hydroxyapatite (HA) binding was determined in suspensions of 1, 3 and 5 mg/mL after 1 h at 37 oC. Biodistribution of 99mTc-complexes was studied in Balb/c mice at 5 min, 1 h and 4 h post-injection (p.i.). 99mTc1 and 99mTcMDP were used as standards. Results The spectroscopic analysis of Re-complexes indicated coordination via N imidazol, S thioether, N amine for ReL1 and via N imidazol, S thioether, O carboxylate for ReL2. 99mTcL1, 99mTcL2 and directly labeled 99mTc1 were synthesized in high yield. 99mTcL1 was stable against histidine/cysteine over 24 h, whereas 99mTcL2 was stable over 4 h. Tracers bound strongly to HA in vitro (% HA binding at 5 mg/mL: 49 for 99mTc1, 84 for 99mTcL1 and 82 for 99mTcL2). Biodistribution in mice demonstrated rapid accumulation and long retention time in the bone (% I.D./g at 1 h p.i. 9.73±0.47 for 99mTc1, 14.11±0.57 for 99mTcL1 and 13.37±1.55 for 99mTcL2). All 99mTc complexes exhibited fast blood/tissue clearance mainly via the urinary system. Conclusions 99mTcL1 and 99mTcL2 exhibited higher bone uptake compared to the directly labeled 99mTc1. In addition the higher target/ non target ratios calculated for 99mTcL1 are suitable for imaging applications and encourage us for further evaluation. Acknowledgements References [1] Makris G, et al (2014) Nucl Med Biol, 41, 623-624. [2] Makris G, et al (2012) Eur J Inorg Chem, 2012, 3132-3139.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S135

135 Characterization and Development of Two Phage Display Selected Anti-ErbB3 Peptides Mirel Cabrara3, Jessica Newton-Northup2, 1, Mercedes Gonzalez3, Thomas P. Quinn1, Susan L. Deutscher2, 1 1 Biochemistry, University of Missouri, Columbia, Missouri, United States, 2Research Service, Harry S. Truman Veterans Memorial Hospital, Columbia, Missouri, United States, 3Centro de Investigaciones Nucleares, Universidad de la República, Montevideo, Uruguay Objectives The development of an ErbB3 selective radiotracer would benefit breast cancer diagnosis and treatment. To this end, we performed a bacteriophage (phage) display to identify ErbB3 specific phage and targeting peptides. Methods Phage display was employed against purified ErbB3 extracellular domain (ECD). Affinity and specificity of the phage for ECD and human ErbB3 expressing cell lines was investigated. Phage were labeled with either NHS-Dylight680 for direct imaging or NHS-PEG-Biotin for three-step pretargeted imaging in SCID mice bearing MDA-MB-435 human breast carcinomas tumors was performed. Peptides displayed on phage MSP3 and MSP4 were synthesized as DOTA-conjugates and radiolabeled with 177LuCl3 (18.5 MBq) at pH of 4.5. Results MSP3 and MSP4 phage bound 9.3 fold and 20 fold more to ECD than wild type (WT) phage, respectively. Binding to ErbB3 expressing cell lines was 200X greater than WT phage. Optical imaging of phage labeled with Dylight680 in MDA-MB-435 breast tumor xenografted mice showed tumor accumulation of phage at 4 h postinjection. Ex vivo quantification of fluorescent signal at 4 h post-injection revealed MSP4 intensity of 2.55x106 RFU within the tumor, and was double that of the MSP3 signal. A three-step pretargeted protocol was performed using biotinylated phage, neutravidin, and ATTO-680 labeled biotin. The fluorescent signal from the regions of interest were 2.77x106 RFU and 3.42x106 RFU for MSP3 and MSP4, respectively, compared to that of 1.47x106 RFU for ATTO680-biotin only. 177Lu labeled DOTA-peptide conjugates of MSP3 and MSP4 were stable in both saline and mouse serum for up to 24 h. 177Lu-DOTA-MSP3 bound with a Kd of 6.8x10-12 M and 1.3x10-10 M for ErbB3-ECD and ErbB2-ECD, respectively. In comparison, 177Lu-DOTA-MSP4 had Kd values of 1.8x10-10 M and 7.3x10-11 M for ErbB3-ECD and ErbB2-ECD, respectively. Conclusions Phage MSP3 and MSP4 were used successfully for in vivo optical imaging of MDA-MB-435 xenografted tumors. Once synthesized as DOTA-conjugated peptides, both MSP3 and MSP4 had similar affinities for ErbB3, however, MSP3 possessed higher specificity for ErbB3 than MSP4. Acknowledgements This research was funded by ANII (MC), PEDECIBA (MC), VA Merit BX000964 (SD). References

J Label Compd Radiopharm 2015: 58: S1- S411

S136: Poster

21st International Symposium on Radiopharmaceutical Sciences

136 Imidazole fused phenanthroline (PIP) ligands for the preparation of multimodal Re(I) and 99m Tc(I) probes. Tamil Selvi Pitchumony, Samantha R. Slikboer, John Valliant Chemical Biology, McMaster University, Hamilton, Ontario, Canada Objectives [2+1] complexes of Re(I) and 99mTc(I) are an emerging class of compounds[1,2] that can be used to prepare optical and nuclear imaging probes using a single construct. The objective was to identify new metal complexes in this class that have superior optical and pharmacokineticproperties compared to conventional ligands. A new class of imidazole fused phenanthroline ligands (PIP) for the [2+1] system, where PIP is the bidentate ligand and N-methylimidazole (NMI) is the monodentate ligand, were developed. The PIP ligand was synthesized with various substituents in order to fine tune the physical properties of the corresponding Re(I) and Tc(I) complexes. Methods The new PIP ligands were synthesized by reacting 1,10-phenanthroline-5,6-dione, ammonium acetate, a diamine and glacial acetic acid in a one pot reaction at 125°C for 3 h. The Re(I) complexes were prepared in two steps starting from [Re(CO)5Cl] and PIP-R to give [Re(CO)3(PIP-R)Cl], which was then reacted with NMI to give the final product, [Re(CO)3(PIP-R)(NMI)]+. The corresponding 99mTc complexes were synthesized from [99mTc(CO)3(H2O)3]+ to yield [99mTc(CO)3(PIP-R)(NMI)] in two steps. The quantitative formation of the final product was confirmed by HPLC and comparison to the corresponding Re(I) standards. Results The Re(I) complexes were synthesized in excellent yields and all the complexes exhibit intense luminescence in the visible region (550 – 650 nm). The nature of the substituent on the PIP ligand played a role in the stability of the complexes where electron donating groups resulted in robust complexes while electron withdrawing groups rendered the axial position labile, thereby preventing the formation of the final compounds. In contrast, all 99mTc(I) complexes synthesized were stable for more than 12 hrs in saline and thus are suitable for in vivo imaging studies. Conclusions We have successfully developed a series of bidentate ligands which were used to prepare novel [2+1] Re(I) and 99mTc(I) complexes. The Re(I) complexes exhibit attractive optical properties while the 99mTc(I) analogues are robust consequently the PIP system is an attractive new approach for creating isostructural optical and nuclear molecular imaging probes. Acknowledgements Funding for this project was provided by NSERC (Canada) and OICR (Ontario). References [1] Pitchumony T et al (2013) Inorg. Chem., 52, 13521-13528. [2] Chan C Y et al (2014) Inorg. Chem. 53, 10862-10873.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S137

137 Target-Specific Radiolabeled Gold Nanoconjugates Francisco Silva2, Ajit zambre1, Maria Campello2, Lurdes Gano2, Amolak Singh1, Anandhi Upendran3, Paulo Antonio2, Raghuraman Kannan1 1 Radiology , University of Missouri, Columbia, Missouri, United States, 2Grupo de Ciências Radiofarmacêuticas, IST/C2TN, Bobadela, Portugal, 3iCATS, MU, Columbia, Missouri, United States Objectives Gold nanoparticles (AuNPs) is emerging as platform to deliver drugs selectively to tumor. Even though the in vivo applications of AuNPs are well recognized, synthesis of multifunctional nanoparticles with both peptide or antibody, and radioisotope is challenging. Surface attachment of radionuclide to AuNPs require chelating ligands that can irreversibly bind isotopes within short reaction time and exhibit high in vivo stability. The objective of the present investigation is to design and develop AuNPs surface functionalized with DOTA and DTDTPA chelators for complexation with radionuclide-Gallium and with a bioactive peptide (GE11 derivative) for targeted delivery to epidermal growth factor receptor (EGFr) overexpressing cancer cells. Methods AuNP-DTDTPA nanoparticles were synthesized based by reduction of HAuCl4 with NaBH4 in the presence of a dithiolated DTPA derivative (DTDTPA) as a stabilizer molecule. Loading of the bioactive peptide into these AuNPs was done afterwards by reaction with a GE11 peptide derivative (TA-GE11-DOTA). Radiolabeling with 67Ga was performed using two distinct routes: i) direct labeling of the GE11 peptide-containing AuNPs (Post-DTAu-GE11-DOTA); ii) pre-labeling approach, in which the GE11 peptide derivative was first labeled with 67Ga and then conjugated to AuNP-DTDTPA (Pre-DTAu-GE11-DOTA). Stability studies in physiological media and in the presence of apo-transferrin were performed for the 67Ga-labeled AuNPs, as well as cell uptake studies using EGFr-overexpressing cancer cells (A431). Results Both direct and pre-labeling approaches provided 67Ga-labeled AuNPs functionalized with a GE11 peptide derivative. Post-DTAu-GE11-DOTA displayed a lower capability to maintain suitable 67Ga coordination in physiological media and in the presence of apo-transferrin, compared with Pre-DTAu-GE11-DOTA. Both radiolabeled nanoconstructs display high internalization in A431 cells, with the highest internalization being observed for Post-DTAu-GE11-DOTA. However, blocking studies with EGF showed no significant difference in internalization for Post-DTAu-GE11-DOTA, while in the case of Pre-DTAu-GE11-DOTA about 30% decrease was observed. Conclusions AuNPs stabilized with DTDTPA and functionalized with TA-GE11-DOTA were successfully synthesized. Labeling approach confirmed that Gallium is irrversibly attached to AuNP. Acknowledgements The authors would like to thank Coulter Foundation (Kannan), US and Fundação para a Ciência e Tecnologia (FCT) (EXCL/QEQ-MED/0233/2012) for the financial support. References

J Label Compd Radiopharm 2015: 58: S1- S411

S138: Poster

21st International Symposium on Radiopharmaceutical Sciences

138 Synthesis, Characterization and Imaging of a 99mTc(I)-Tetrazine Tridentate Ligand Complex for Bioorthogonal Chemistry Zainab Ahmad, Megan Blacker, Aimen Zlitni, Holly Bilton, John Valliant Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada Objectives 99mTc remains the most widely used radionuclide in diagnostic medicine. However, Tc is limited to labeling targeting vectors that have rapid binding kinetics and clearance rates from non-target tissues, due to its 6 hr half-life.1,2 This precludes the use of 99mTc with larger biomolecules such as antibodies, which often require days for optimal target to non-target ratios. The objective is to overcome this limitation by making it possible to employ pre-targeting strategies based on the inverse electron demand Diels-Alder coupling reaction between radiolabeled tetrazines (99mTc-Tz) and transcyclooctene (TCO) labelled antibodies.3 Methods A new tridentate Tc(I) chelate-tetrazine derivative was synthesized and the optimal conditions (temperature and pH) to radiolabel the ligand were determined to prevent tetrazine degradation. The reactivity of the 99mTc-tetrazine product was assessed using a series of TCO derivatives including a TCO-labeled antiVEGFR2 antibody. In vitro and in vivo studies were conducted subsequently to evaluate the potential utility of the strategy. Results The Tc(I) bispyridyl tetrazine chelate was synthesized in 54% yield via a reductive amination reaction and the product was coupled to a commercially available tetrazine. The corresponding 99mTc complex was prepared in a one step labeling method and the product isolated in 83% radiochemical yield free from any residual ligand. The complex was stable in PBS for up to 4 hours and its logP value was less than 1. In vitro binding assays showed a significant increase in the amount of 99mTc-tetrazine binding to H520 VEGFR2(+) cells compared to A431 VEGFR2(-) cells, after pre-targeting with a TCO-anti-VEGFR2 antibody. In vivo SPECT imaging data showed a major difference in tissue distribution between 99mTc-tetrazine administered subsequently with the TCO-antibody, and when mixed prior to injection. Conclusions A new construct that allows for the preparation of 99mTc-labeled-tetrazine was developed. This construct and related analogues allow the use of 99mTc to assess the biodistribution of larger biomolecules such as antibodies, using a pre-targeting approach and bioorthogonal chemistry. Acknowledgements Funding for this project was provided by NSERC (Canada) and OICR (Ontario). References [1] M. K. Levadala et al., (2004), Synthesis (Stuttg). 11, 1759–1766. [2] M. R. Karver et al., (2011) Bioconjug. Chem. 22, 2263–70. [3] M. Bartholomä et al., (2009), Chem. Commun. (Camb). 7345, 493–512.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S139

139 Synthesis, In Vitro Evaluation, and In Vivo PET/CT of 64Cu-labeled CD11b-targeting Probe Haixun Guo1, 2 1 Radiology, University of Louisville, Louisville, Kentucky, United States, 2Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, Kentucky, United States Objectives CD11b is an integrin receptor which over-expressed on activated leukocytes and is an ideal target for PET imaging to monitor the innate immune response to infection and inflammation. This study is to develop a novel CD11b-targeting radioactive probe and to test it in both in vitro and in vivo for future PET imaging of infection and inflammation. Methods Metal chelator NOTA(1,4,7-triazacyclononane-1,4,7-triacetic acid) was used to modify the structure of CD11b-targeting compound L1 through PEG linker for radiolableing. The NOTA-PEG-L1 was labeled with 64 CuCl2 using 0.5 M NH4OAc (pH5.4) buffer according to our published procedure[1]. 64Cu-NOTA-PEG-L1 was purified by Radio-HPLC and its uptake in CD11b+ monocytic THP-1 cells was tested. PET/CT of 64Cu-NOTAPEG-L1 was performed in healthy C57BL/6 mice for testing its in vivo distribution. Results NOTA-PEG-L1 was successfully synthesized and characterized via NMR and LC-MS. 64Cu-labeling showed more than 98% radiochemical yield for reaction at 75oC for 1 h. The 64Cu-NOTA-PEG-L1 was successfully purified from the unlabeled compound and free 64Cu2+ via HPLC. The HPLC-purified 64Cu-NOTAPEG-L1 showed high uptake in THP-1 cells, and the uptake can be blocked by the unlabeled compound. The PET/CT of 64Cu-NOTA-PEG-L1 showed clear background except for the stomach, which is the excreting pathway of this compound. Conclusions We successfully synthesized a CD11b-targeting radioactive compound and evaluated it in both THP1 cells and healthy mice. The specific binding on THP-1 cells and clear background on PET/CT of this radioactive compound suggest its potential for future evaluation in infected and inflammatory animal models. Acknowledgements The authors would like to thank the UofL startup funds, and the staff supports of the Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases. References [1] Guo, H et al. Mol. Pharmaceutics, 2012, 9(8): 2322-2330

J Label Compd Radiopharm 2015: 58: S1- S411

S140: Poster

21st International Symposium on Radiopharmaceutical Sciences

140 Synthesis and In Vitro Bacteria Binding of 68Ga-labeled Yersiniabactin Haixun Guo1, 2, Jonathan Warawa2, 3, Matthew Lawrenz2, 3 1 Department of Radiology, University of Louisville, Louisville, Kentucky, United States, 2Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, Kentucky, United States, 3Department of Microbiology and Immunolog, University of Louisville, Louisville, Kentucky, United States Objectives This study is to synthesize novel 68Ga-labeled siderophores for studying the Y. pestis and K. pneumoniae iron uptake mechanism, and in vivo PET imaging of Y. pestis and K. pneumoniae infections. Methods Yersiniabactin was labeled with 68GaCl3 using 0.5 M NH4OAc (pH 4.5) buffer and purified via RadioRP-HPLC. The HPLC-purified 68Ga-siderophore was incubated with various strains of Y. pestis and K. pneumoniae and the bacteria-bound radioactivity was count via a Wizard2 gamma counter. Results 68Ga-Yersiniabactin was successfully synthesized and the radiochemical yield was more than 98% for 5 minutes reaction at room temperature. The 68Ga-Yersiniabactin was successfully separated from the unlabeled Yersiniabactin on HPLC. The 68Ga-Yersiniabactin showed significant higher K. pneumoniae uptake in irondepleted medium than in iron-containing medium. The 68Ga-Yersiniabactin also showed significantly higher Y. pestis uptake compared with a yersiniabactin uptake mutant. Conclusions 68Ga-Yersiniabactin was successfully synthesized and tested in various bacteria strains. The promising in vitro binding data suggests its potential for in vivo PET imaging of bacteria infection, especially for Y. pestis and K. pneumoniae. Acknowledgements The authors would like to thank the UofL startup funds. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S141

141 A Novel SPECT Metalloprobe 67Galmydar for Myocardial Perfusion Imaging Jothilingam Sivapackiam, Scott E. Harpstrite, Julie L. Prior, Vijay Sharma Department of Radiology, Washington University School of Medicine, St. Louis, Missouri, United States Objectives Coronary artery disease (CAD) is among the leading causes of death in the world. Myocardial perfusion imaging (MPI), a versatile tool in clinical diagnosis, plays an important role in the noninvasive assessment of the coronary artery disease. To achieve this objective, we have synthezised a cationic and moderately hydrophobic metalloprobe (67Galmydar), incorporated with gallium-67 (t1/2 = 78.28 h), a SPECT radioisotope. The agent shows demonstrates high first pass extraction into myocardium of normal mice and rats, while also undergoing transporter-mediated efflux pathways to facilitate excretion from neighboring tissues, such as liver to enable myocardial perfusion imaging. Methods 67Galmydar was synthesized from Schiff base ligand by following literature procedure, purified on C-18 column using radio-HPLC, and employed for bioassays. Pharmacokinetic studies in 4-6 weeks old FVB (WT) and mdr1a/1b(-/-) mice (KO), normal rats, metabolite analysis, and NanoSPECT imaging were performed using literature methods. Results 67Galmydar shows uptake and retention in heart and excretion from blood, liver, and lung in mice and rats. Following intravenous tail-vein injection in rats, preliminary metabolic studies indicate that pharmacokinetic profiles of 67Galmydar are mediated by the presence of the parental compound in the heart. NanoSPECT imaging demonstrates sustained retention in the heart and facile clearance from the liver, 60 min post tail-vein injection in rats. Importantly, 67Galmydar showed sensitivity to detect the major perfusion defect in rats, following ligation of the left anterior descending (LAD) coronary artery. Finally, Galmydar does not show any abnormal clinical pathology following administration of a single IV bolus (10,000 X the tracer dose) injection in rats. Conclusions 67Galmydar shows sustained retention in heart of rats and ability to monitor perfusion defect in vivo. Acknowledgements NIH RO1HL111163 (VS), R33AG033328 (VS), American Health A2007-383(VS) References Sharma, V. et. al. PlosOne 2014, 9, e109361; Sivapackiam, J. et. al. Dalton Transactions, 2010, 39, 5842; Harpstrite, S.E. et. al. J. Inorg. Biochem. 2007, 101, 1347; Sharma, V. J. Nucl. Med. 2005, 46, 354; Bioconjugate Chem 2004, 15, 1464.

J Label Compd Radiopharm 2015: 58: S1- S411

S142: Poster

21st International Symposium on Radiopharmaceutical Sciences

142 Development of ring substituted pyridine based chelates for fac-[MI(CO)3]+ (M=Re, 99mTc) based radiopharmaceuticals Thomas R. Hayes1, Patrice A. Lyon1, Charles Barnes2, Steven Trabue3, Paul D. Benny1 1 Chemistry, Washignton State University, Pullman, Washington, United States, 2Department of Chemistry, University of Missouri, Columbia, Missouri, United States, 3National Soil Tilth Laboratory, USDA, Ames, Iowa, United States Objectives While a number of chelates and strategies have been developed for the fac-[MI(OH2)3(CO)3]+ (M=Re, Tc) precursor, developing methods to improve the overall in vivo function and performance of these ligands remains a challenge. Since its discovery, the fac-[MI(CO)3]+ core has served as an essential platform in the development of new 99mTc based radiopharmaceuticals due to its easy availability and ease of substitution. The overall stability and lipophilic character of the CO ligands, however, impacts the pharmacokinetics and in vivo clearance of these complexes. Many of the ligand systems that are used with the fac-[MI(CO)3]+ core contain a pyridine moiety which offers an attractive target to tune the characteristics of the metal complex. Methods Complexes 2-6 were synthesized through a 2+1 approach using picolinic acid as a bidentate ligand. Re complexes were prepared by heating the respective monodentate pyridine ligand with 1. The complexes were characterized by NMR, IR, MS, elemental analysis and crystallography. 99mTc complexes were synthesized by reacting 1a with 1 mM pyridine ligand at 90 °C for 30 min, and characterized by comparison of γ-HPLC traces to the UV traces of the respective Re analogs. Stability was accessed by incubating purified 99mTC complexes with 1 mM of cysteine or histidine at 37 °C for 1h. After analysis of the monodentate data, tridentate ligands were synthesized and characterized to determine the utility of these changes in a tridentate ligand. Results Re complexes were synthesized in moderate to good yields (30-88%). Crystallography of the Re complexes showed that Re-Npy bond length correlated to pKa of the monodentate pyridine. Conversion to the 2+1 99m Tc complexes was accomplished for each complex and was proportionate to the pKa of the monodentate ligand used. Stability data showed that all of the substituted pyridine complexes tested (3a-6a) were more stable than pyridine complex 2a. Synthesis of tridentate pyridine based ligands with OMe and NMe2 substitution showed higher complexation efficiency at 10-6 M concentration than the dipicolylamine analog. Conclusions These results suggest that substituted pyridines can be used to tune the stability, binding and lipophilicity of fac-[MI(CO)3]+ complexes. Acknowledgements References 99m

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S143

143 Clickable bifunctional cross-bridged phosphonate-based chelator through alkyne and aldehyde modifications Zhengxin Cai1, Barbara Li2, Leon Wong2, Edward Wong2, Gary Weisman2, Carolyn J. Anderson1 1 Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 2Department of Chemistry, University of New Hampshire, Durham, New Hampshire, United States Objectives As cross-bridged macrocyclic chelators form stable chelates with copper, the only bottle neck for applying these chelators in radiopharmaceutical science is to incorporate bifunctionality without compromising the in vivo stability. Here, we describe the synthesis and application of the alkyne- or aldehyde-modified phosphonatebased cross-bridged macrocyclic chelators for conjugation with azide- or N-hydroxylamie-modified peptides. Methods The alkyne and aldehyde modified chelator 2 and 3 were synthesized efficiently from mono-phosphonate 1. (Figure 1) The somatostatin analogue, Y3-TATE, was conjugated with the two chelators. The bioconjugates were efficiently labeled with 64Cu under mild conditions. Binding and internalization assays were performed with sstr2-transfected HCT116 tumor cells. Mouse xenografts bearing the same tumor cells was used as the animal model. Results CB-TE1P1T-Y3-TATE (4) was obtained in 25% overall yield and was labeled with 64Cu at rt within 30 min in > 95% RCY. CB-TE1P1B-Y3-TATE (5) was labeled at 70 °C within 30 min in > 95% RCY. The Kd of 4 was 0.52 ± 0.10 nM, with a Bmax of 4800 ± 190 fmol/mg. The Kd of 5 was 0.62 ± 0.15 nM, with Bmax being 5348 ± 274 fmol/mg. The tumor uptake of 4 reached 7.54 ± 1.43%ID/g at 4 h p.i., 91% of which can be blocked by Y3TATE . The tumor uptake of 5 reached 11.55 ± 1.56%ID/g at 4 h p.i., which was blocked with Y3-TATE to 0.81 ± 0.08%ID/g. The SUV of 4 was 2.2 ± 0.5; while the tumor SUV of 5 was 2.6 ± 0.4. Conclusions Both bifunctional cross-bridged chelators 2 and 3 were synthesized efficiently, conjugated with Y3TATE, and radiolabeled with 64Cu under mild conditions in high-specific activity. Both tracers retained high affinity to sstr2. PET/CT imaging and biodistribution showed excellent tumor uptakes. We anticipate broad applications of these clickable chelators in labeling other biologically important molecules. Acknowledgements The PET/CT imaging at Molecular Imaging Center was supported in part by P30CA047904 (UPCI CCSG). References

Figure1. General synthetic scheme of 4 and 5

J Label Compd Radiopharm 2015: 58: S1- S411

S144: Poster

21st International Symposium on Radiopharmaceutical Sciences

144 Enhancing [64Cu]-NODAGA-Exendin-4 Uptake into GLP-1 Receptor Expressing Cells Using an Agoallosteric Modulating Quinoxaline Derivative: A Step Towards Improved β-Cell imaging. Gregory D. Bowden, Filippo Michelotti, Valerie Honndorf, Bernd J. Pichler Department of preclinical imaging and radiopharmacy, Eberhard Karls Universität Tübingen, Tübingen, Germany Objectives Radiolabelled Exendin-4 (Ex-4) is currently one of the benchmark tracers for GLP-1 receptor imaging despite showing high non-specific uptake in the kidneys. GLP-1R is selectively expressed on the surface of insulin producing β-cells, making it a good biomarker for β-Cell mass. The small molecule quinoxaline derivative “Compound 2” (C2) has been identified in the past as an ago-allosteric modulator of GLP-1R and has been shown to increase the binding affinities of Ex-4 and other orthosteric GLP-1R ligands.1 We hypothesized that this property of C2 could be used to selectively increase tracer uptake in an INS1 tumor model and could thus be exploited for better GLP-1R imaging with radiolabeled Ex-4. Methods Two pilot studies were carried out using xenographt INS-1 tumor models; the first was performed using Inveon mircoPET scanners while the second utilized a PET/MR insert in order to localize the pancreas. Typically, the treatment group of mice, carrying INS-1 tumors on their right shoulders, was injected with C2 (25 mg/Kg) 15min before the injection of [64Cu]-Ex-4 (100 μCi)(≈0.1 μg) to allow for C2 to interact with GLP-1R, while the control group received no C2. 1 mouse from each group was scanned dynamically for 1hr post tracer injection while the others were scanned statically for 10min (30min with the PET/MR insert) 1hr post injection. Results As expected, all groups showed high tracer uptake in the kidneys (60-80 %ID/CC). In the 1st study, the mean uptake of tracer into the INS-1 tumor in the control and treatment groups after 1hr were 2,66 ± 0,45 and 13,50 ± 2,27 % ID/CC respectively, showing a drastically improved uptake of the tracer into the tumor with the use of C2. The 2nd study showed similar results as well as increased tracer uptake into the pancreas. The dynamically scanned C2 treated mouse showed an enhanced time activity curve relative to a control. Conclusions The initial results of our study strongly suggest that the ago-allosteric modulating C2 greatly boosts the uptake of the [64Cu]-Ex-4 tracer into INS-1 Cells via GLP-1R. As this was only a pilot study with a small sample size, larger studies are currently being prepared to establish the statistical significance of this result. The quinoaxline derivative "C2" may thus one day prove to be a new tool for enhancing PET and SPECT images derived from peptide based GLP-1R radiotracers, improving the target-to-kidney signal ratio. Acknowledgements References 1. Teng, M. et al. Small molecule ago-allosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor. Bioorg. Med. Chem. Lett. 17, 5472–8 (2007).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S145

145 Isothiocyanate-functionalized bifunctional chelates for 99mTc and Re complexes for uPAR targeting in prostate cancer Benjamin B. Kasten1, Xiaowei Ma2, Kai Cheng2, Lihong Bu2, Zhen Cheng2, Paul D. Benny1 1 Department of Chemistry, Washington State University, Pullman, Washington, United States, 2Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, , Stanford University, Stanford, California, United States Objectives The objective was to explore an isothiocyanate (ITC) coupling strategy to incorporate either a tridentate chelate for the fac-[MI(CO)3]+ (M = Re, 99mTc) core or the respective complex into amine-containing biological targeting vectors. N-Boc-lysine (BocLys) was used as a model amine to explore coupling efficiencies through “click, then chelate” and “chelate, then click” methods with the ligand and metal complexes. This strategy was applied to a peptide (AE105) that targets the urokinase-type plasminogen activator receptor (uPAR), an emerging biomarker in prostate cancer (CaP) prognosis, to examine the effectiveness of the ITC coupling strategy with fac-[MI(CO)3]+ and understand the in vivo behavior. Methods 2,2´-dipicolylamine (DPA) was functionalized with a isothiocyanate pendant functional group in three steps from primary amine to an R-NCS group using CS2 and Boc2O. Complexation of ITC-DPA with fac[MI(CO)3]+ was performed at 40 or 70 °C in MeOH (Re) or pH 7.4 buffer (99mTc). Two different strategies, “Click, then chelate” and “chelate, then click”, were examined using lysine as a model to evaluate coupling conditions and efficiency. These approaches were also extended to the peptide AE105 utilizing ITC functional group to couple the chelate or complex to the terminal amine. In vitro assays in PC3 cells and in vivo biodistribution analysis in PC3 xenograft CaP models are currently under investigation. Results All compounds and complexes with Re were produced in 32-90% yields and fully characterized by chemical methods. 99mTc complexes were identified by comparative HPLC analysis with the Re analogs. Model chelate, then click studies with BocLys and 99mTc-DPA-ITC at 37 °C in both aqueous and organic conditions produced the final complex in 46-94% yields after 1 h, while yields were 91-96% for the click, then chelate route following heating at 70 °C. Radiolabeling yields for the functionalized AE105 peptide were >99% following heating at 70 °C with specific activities up to 103 mCi/µmol prior to purification. In vitro and in vivo biological studies with the radiolabled peptide using PC3 CaP models are currently in progress. Conclusions Isothiocyanate ligation strategy with DPA and fac-[MI(CO)3]+ complexes was found to be facile method to couple amine containing molecules. Both routes had reasonable yields and reaction times that demonstrated the potential and feasibility for labeling amine containing biomolecules. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S146: Poster

21st International Symposium on Radiopharmaceutical Sciences

146 Clickable cross-bridged chelator to prepare dimeric peptide for targeted PET imaging Dexing Zeng1, Marie-Caline Abadjian3, Erik C. Wiener1, 2, Douglas Grotjahn3, Carolyn J. Anderson1, 2 1 Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States, 2University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States, 3Department of Chemistry and Biochemistry, San Diego State University, San Diego, California, United States Objectives Compared to monomeric peptides, the dimeric peptides have shown significant improvements in binding affinity and pharmacokinetic performance. Preparation of such dimer usually requires complex organic chemistry and bioconjugation strategies. The objective of this study is to develop a novel click chemistry platform for facile preparation of chelator-conjugated dimeric peptides for PET imaging. Methods A methyl ester protected chelator, CB-TE2(AOMe)2A, was synthesized in a multi-step synthesis. Due to having two azido groups, it could be conjugated with two acetylene-cyclo(RGDyK) peptides via copper-catalyzed azide–alkyne cycloaddition (CuAAC), followed by methyl hydrolysis in (nBu)4N+OH- solution. The purified dimeric peptide was radiolabeled with 64Cu in NH4OAc buffer (pH = 8.0), and the resulting 64Cu-CB-TE2A(RGD)2 was evaluated for serum stability. Competition binding assays were performed to determine its binding affinity, and in vivo evaluation of the 64Cu labeled dimer was conducted in mice bearing 4T1 tumor xenografts. Data from the dimer were compared to those obtained from the mono-clicked analog CB-TE2A-RGD that was prepared from a chelator (CB-TE2(AOMe)1A) containing only one azido group Results The two clickable chelators were synthesized in an overall yield of 10%, and acetylene-cyclo(RGDyK) prepared from acetylene-NHS and cyclo(RGDyK) was conjugated in >95% yield. The dimeric peptide CB-TE2A(RGD)2 could be radiolabelled with 64Cu at 37 °C for 1 h, whereas 64Cu labeling of the monomer CB-TE2A-RGD needed relatively harsh conditions, 60 °C for 1 h. The IC50 of the peptide dimer and monomer were 1.0 and 2.8 nM respectively, and both 64Cu labeled radiotracers showed less than 2% 64Cu dissociation in human serum at 37 °C for 24 h. Compared to 64Cu-labeled monomer (64Cu)CB-TE2A-RGD, dimeric (64Cu)CB-TE2A-(RGD)2 showed higher tumor uptake and signal to background ratio (such as, tumor/muscle and tumor/blood). Conclusions A novel cross-bridged chelator for facile preparation of 64Cu-labeled peptide dimer was developed. Compared to 64Cu-labeled monomer, the 64Cu-labeled dimer demonstrated improved radiolabeling efficiency, in vitro binding and in vivo performance. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S147

147 Synthesis and comparative evaluation of a 6-carbon spacer ISO-1 vs. ISO-1 for imaging the sigma-2 receptor status of solid tumors Iljung Lee, Brian P. Lieberman, Robert H. Mach Department of Radiology/Nuclear Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States Objectives Sigma-2 receptors have been reported as an important biomarker of tumor cell proliferation [1]. Among the imaging agents for sigma-2 receptor, ISO-1 (N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2fluoroethoxy)-5-methylbenzamide) is in the clinical trial stage. Another group has reported that a 6-carbon spacer benzamide analogue had higher binding affinity and selectivity for sigma-2 receptors than a 4-carbon spacer [2]. In this study, we compared and evaluated ISO-1 as a 4 carbon spacer and a 6-carbon spacer ISO-1 (1) for tumor imaging as a sigma-2 receptor probe. Methods Compound 1 was synthesized from 2-(2-fluoroethoxy)-5-methylbenzoic acid and 6-(6,7-dimethoxy-3,4dihydroisoquinolin-2(1H)-yl)hexan-1-amine by modified known method [1]. Binding affinities of ISO-1 and compound 1 for sigma-2 receptors were measured using [1H]DTG and rat liver membrane homogenates in the presence of 100 nM (+)-pentazocine to block sigma-1 sites at room temperature for 120 min. The radiochemical synthesis of [18F]1 was carried out by labeling of the mesylate precursor in the present of K18F at 120 ºC for 15 min. The radiotracer, [18F]1 was injected into the mice (Female Balb/c) implanted with EMT-6 tumors at the right flanks. The mice were sacrificed at the specific time points (5, 60, and 120 min), and the organs were dissected, weighed and counted within the gamma counter. Results Compound 1 and the precursor for the radiolabeling were easily synthesized in 79% and 12% overall yield, respectively. The binding affinities of ISO-1 and compound 1 for sigma-2 receptors were 13.3 nM and 23.1 nM, respectively. Decay-corrected radiochemical yield of [18F]1 was 10-15% and specific activity was 37 GBq/µmol. Biodistribution of [18F]1 showed an uptake of 0.52, 1.55, and 1.19 %ID/g in the EMT-6 tumor and 2.31, 1.65, and 1.16 %ID/g in the blood at 5, 60, and 120 min post-injection. In contrast, [18F]ISO-1 displayed a 3.67, 1.14, and 0.64 %ID/g in the tumor and 2.49, 0.56, and 0.35 %ID/g in the blood at 5, 60, and 120 min post-injection [1]. Compound [18F]1 had lower tumor/muscle, tumor/skin, and tumor/blood ratios than [18F]ISO-1, and showed slow clearance of the blood sample. Conclusions These results show that increasing from a 4-carbon spacer to a 6-carbon spacer compound reduces binding affinity for sigma-2 receptors and decreases in vivo properties of ISO-1. Acknowledgements This study was supported by a grant from the U.S. Department of Energy. References [1] Tu Z. et al (2007) J. Med.Chem., 50, 3134-3204. [2] Ashford M. et al (2014) Org. Biomol. Chem., 12, 783-794

J Label Compd Radiopharm 2015: 58: S1- S411

S148: Poster

21st International Symposium on Radiopharmaceutical Sciences

148 (Pyridinyl-phenyl)selenoxides and (pyridinyl-phenyl)selenones as precursors for preparing [18F]fluoropyridines from [18F]fluoride ion Sureshbabu Dadiboyena, Fabrice G. Siméon, Shuiyu Lu, Lisheng Cai, Victor W. Pike Molecular Imaging Branch, National Institute of Mental Health, NIH, Bethesda, Maryland, United States Objectives The labeling of candidate radiotracers in pyridinyl moieties with no-carrier-added (NCA) fluorine-18 is often attractive for achieving resistance to radiodefluorination in vivo, and methods are keenly sought for this purpose.[1,2] Herein, we report the unprecedented use of (pyridinyl-phenyl)selenoxides and (pyridinylphenyl)selenones as precursors for the synthesis of [18F]fluoropyridines from NCA [18F]fluoride ion. Methods We prepared (pyridinyl-phenyl)selenoxides 2a–d and (pyridinyl-phenyl)selenones 3a–c from the corresponding diarylselenides 1a–d as illustrated in Figure 1. Treatment of the selenides 1a–d with t-BuOCl gave the selenoxides 2a–d, which upon further oxidation gave the desired selenones 3a–c. For radiochemistry, each selenoxide 2a–d or selenone 3a–c was treated with [18F]F−-K+-K 2.2.2 in DMF in a microfluidic apparatus (Advion; Nanotek) at a set temperature between 50 and 200 ºC for 188 s. Product [18F]fluoropyridines [18F]4a–d were identified by co-elution with reference compound on HPLC. Results Selenoxides 2a–d and selenones 3a–c were obtained from the corresponding (pyridinyl-phenyl)selenides 1a–d in high yields. (Pyridinyl-phenyl)selenones 3a–c reacted more readily than the corresponding (pyridinylphenyl)selenoxides 2a–c with NCA [18F]fluoride ion to give the desired [18F]fluoropyridines [18F]4a–c in low to moderate decay-corrected radiochemical yields. [18F]2-fluoropyridines bearing a 5-Me ([18F]4b) or 5-Ph group ([18F]4d) were accessible through this method (Figure 1). Conclusions (Pyridinyl-phenyl)selenoxides and especially (pyridinyl-phenyl)selenones may prove useful for labeling PET radiotracers with NCA fluorine-18 at pyridinyl groups. The scope of these new methods is being further explored. Acknowledgements Funded by CNRM-HJF and the IRP of the NIH (NIMH). References [1] Cai LS et al (2008) Eur J Org Chem, 2853. [2] Chun J-H and Pike VW (2012) Chem Commun 48, 9921.

Figure 1. Syntheses of selenoxides 2a–d, selenones 3a–c, and [18F]fluoropyridines [18F]4a–d.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S149

149 Diarylselenoxides as precursors to no-carrier-added [18F]fluoroarenes Fabrice G. Siméon, Shuiyu Lu, Victor W. Pike Molecular Imaging Branch, NIMH, NIH, Bethesda, Maryland, United States Objectives As a part of our ongoing efforts [1,2] to explore hypervalent compounds for the preparation of nocarrier-added (NCA) [18F]fluoroarenes from [18F]fluoride ion, we report here on the reactivity of diarylselenoxides as novel precursors. Methods Diarylselenoxides 2a–d were prepared by oxidation of the corresponding diarylselenides 1a–d with tbutyl hypochlorite. For radiochemistry, each selenoxide precursor 2a–d was treated with [18F]F−-K+-K2.2.2 for different times (≤ 188 s) and at set temperatures between 50 to 200 ºC in DMF in a microfluidic apparatus (Advion; Nanotek). To investigate effect of solvents, 2b was treated with NCA [18F]fluoride ion complex in acetonitrile, DMF, or DMSO. Finally, 2b was also treated with [18F]fluoride ion plus K2CO3 in DMF/H218O (86/14, v/v), in absence of cryptand. Each radioactive product was identified by its comobility with reference compound on HPLC. Results Diarylselenoxides 2a–d were obtained in 45–70% yields. Treatment of 2a–d with [18F]F−-K+- K 2.2.2 in DMF at 200 ºC produced [18F]3a–d in 83, 90, 59 and 2% decay-corrected radiochemical yields (RCYs), respectively. Radiofluorination of 2b in acetonitrile at 120 ºC and in DMSO at 200 ºC gave [18F]3b in 45% and 94% RCY, respectively. When 2b was treated with [18F]F--K+ in aqueous DMF at 130 ºC, [18F]3b was produced in 33% RCY. Conclusions Diarylselenoxides bearing a p-electron withdrawing group react rapidly and efficiently with [18F]fluoride ion to give the desired [18F]fluoroarenes in high RCYs. The reaction proceeds smoothly in various solvents and appears to tolerate partially aqueous conditions in absence of cryptand. These reactions may prove useful in PET radiotracer synthesis, mostly when it is desirable to retain a potentially nucleofugic group, such as nitro. The scope of the reaction is being further explored. Acknowledgements This work was funded by the IRP of NIH (NIMH). References [1] Chun J-H et al. (2011) Eur J Org Chem, 4439. [2] Chun J-H et al. (2013) Chem Commun, 49, 2151.

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J Label Compd Radiopharm 2015: 58: S1- S411

S150: Poster

21st International Symposium on Radiopharmaceutical Sciences

150 Applications of [18F]Hexafluorobenzene for Preparing Cyclized or Dimeric Radioligands Orit Jacobson, Dale O. Kiesewetter, Xiaoyuan Chen NIBIB, National Institutes of Health, Bethesda, Maryland, United States Objectives Hexafluorobenzene (HFB) was shown to react with free thiols in cyclic peptides to produce a perfluoroaromatic linkage between the sulfurs and provide unique changes in the biological properties.(1) Moreover, the synthesis of [18F]HFB has been reported. (2) We set out to evaluate [18F]HFB as a prosthetic group for modifying and labeling molecules that contain a disulfide bridge or to dimerize molecules that contain a free thiol. Methods [18F]HFB was prepared by a fluorine exchange reaction using an automated synthesis module and isolated by distillation. [18F]HFB was reacted with a monothio RGD peptide (2 eq.) or di-thiol T-140 peptide and TRIS in DMF at room temperature to provide dimeric RGD peptide or cyclized T-140 products. Authentic products were prepared by analogous procedures. In addition, [18F] fluoride exchange was conducted on a (perfluoro-1,4-phenylene)bis((4-chlorobenzyl)sulfane) at 90ᴼC in DMSO. PET imaging, using a Siemens Inveon scanner, was conducted on a U87MG xenograft model. Results [18F]HFB was obtained in 25 % radiochemical yield in an automated 50 min procedure using 0.5 mg of HFB (SA = 50 mCi /µmol). The direct exchange reaction on (perfluoro-1,4-phenylene)bis((4-chlorobenzyl)sulfane) proceeded in 33 % RCY. An internally cyclized T-140 peptide analog was prepared with an uncorrected yield of 12% after a 30 min incubation. A dimeric RGD peptide was obtained in an optimized uncorrected yield of 40% (SA 15 mCi/µmol, EOS) based on [18F]HFB, which showed prominent uptake in integrin αvβ3 positive U87MG tumor (see image). Conclusions The prosthetic group, [18F]HFB, can be incorporated into thiol-containing peptides with good efficiency to provide novel imaging agents. In addition, an 18F for 19F exchange reaction of the tetrafluorophenyl moiety was also achieved. Acknowledgements Funding was provided by the Intramural Research Program of NIBIB/NIH. References 1) A.M. Spokoyny, Y. Zou, J.J. Ling, H Yu, Y.S. Lin, B.L. Pentelute. J. Am. Chem. Soc. 2013, 135, 5946-5949. 2) E. Blom, F. Karimi, B. Langstrom J. Label Compd. Radiopharm, 2009, 52, 504-511.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S151

151 Novel approach to production of high specific-activity [18F]-Sodium Tetrafluoroborate Alex Khoshnevisan1, Jennifer Young1, Gareth Smith2, Alex Jackson2, Philip J. Blower1, Antony Gee1 1 Imaging Chemistry and Biology, King's College London, London, United Kingdom, 2GE Healthcare, Amersham, United Kingdom Objectives The tetrafluoroborate anion (BF4-) is a well-established substrate for the sodium-iodine symporter (NIS). Recently, radiolabelling of NaBF4 with fluorine-18 by isotopic exchange was reported [1], along with associated evaluation of biological activity in rodent models by PET/CT. While this highlighted the potential for clinical use in imaging thyroid disorder, the limitations of the radiolabelling method on the specific activity (SA) restrict the maximum image quality that can realistically be achieved in future applications. To that end, we sought an alternative route via nucleophilic addition to yield high SA [18F]-NaBF4. Methods Various [18F]-fluoride salts were prepared from [18F]-F- in H2O, dried azeotropically and reacted with BF3.OEt2 under anhydrous conditions in MeCN (Scheme 1). Incorporation of 18F- into the desired product was monitored by radioTLC using alumina strips and methanol as the mobile phase. Purification was effected using a neutral alumina or weak anion exchange, followed by QMA cartridge. Characterisation of the final product and determination of SA was effected using an ion chromatography apparatus with in-line radio-detector. Results Using 18F-K222/KF and K2CO3, or [18F]-TBAF as the fluoride source was found to be an unsuitable approach, due to product hydrolysis and precursor hydrolysis (leading to [19F]-NaBF4 formation) respectively. Reaction with [18F]-NaF gave the desired radiotracer with a high radiochemical conversion (58-92%). The final non-decay corrected radiochemical yield (RCY, 10-19%) and purity (RCP) were also high (> 98%). The SA of the [18F]-NaBF4 produced by this method was consistently greater than 38 GBq/µmol. Conclusions The approach described afforded the desired radiotracer at a substantially higher specific activity than previously reported. Considering known IC50 values for the in vitro inhibition of [18F]-NaBF4 NIS uptake by [19F]NaBF4 [2], by estimation the resulting tracer concentration in vivo for an injected patient dose using this method should now fall well outside that at which it is self-inhibiting. Acknowledgements Financial support from the EPSRC and GE Healthcare (Amersham, UK) References [1] Jauregui-Osoro et al. (2010), Eur. J. Nucl. Med. Mol. Imaging, 37, 11, 2108-2116; [2] Weeks et al. (2011), Nucl. Med. Commun., 32, 2, 98-105

Scheme 1 – Reported and existing [1] methods and SA values for [18F]-NaBF4 synthesis

J Label Compd Radiopharm 2015: 58: S1- S411

S152: Poster

21st International Symposium on Radiopharmaceutical Sciences

152 Cerenkov luminescence imaging with PET probes for brain function analysis Mikako Ogawa, Mutsumi Kosugi, Yasuhiro Magata Medical Photonics Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Objectives Optical imaging has advantages in its easy-to-use feature. However, brain imaging is not easy in optical molecular imaging, since the molecular sizes of fluorescent probes are usually too large to penetrate the bloodbrain-barrier. On the other hands, PET imaging is superior in quantitative analysis, and many brain imaging probes are available. Cerenkov radiation is electromagnetic radiation emitted when a charged particle (such as a positron) passes through a dielectric medium at a speed greater than the phase velocity of light in that medium. Here, we evaluated the feasibility of CLI for the analysis of brain functions using [18F]FDG, [11C]raclopride and [11C]β-CFT. Methods For [18F]FDG imaging, the mice were separated into two groups. For one group, the hair of the mice were shaved (w/o hair), and for other group, the skull was additionally removed (w/o hair & skull). [18F]FDG (3 MBq, iv) was injected to each group of the mice, and PET imaging was performed from 30 to 60 min after the injection. Then, Cerenkov luminescence imaging (CLI) was done for 10 min from just after the PET imaging. After in vivo CLI, the brain was removed and ex vivo CLI was performed. Then, the radioactivity of the brain was measured by a gamma-counter. Various fasting times were set for each mouse in order to produce various blood sugar levels. For [11C]raclopride imaging, the mice (w/o hair) were injected with 7.4 MBq of the tracer, and sequential 8-min CLI (followed by 2-min interval) was performed from 8-10 min after the injection. In vivo blocking study was done by treating the mice with haloperidol. Also, the images were obtained by [11C]β-CFT. Results In [18F]FDG studies, there was a good relationship between the radioactivity and CLI signal in removed brains (r=0.93). The good correlation was observed in w/o hair & skull group (r=0.92). The correlation was weaker in w/o hair group (r=0.74), but it thought to be acceptable for the analysis in mice. The striatum was successfully visualized by [11C]raclopride and [11C]β-CFT. Luminescence in the striatum was decreased by haloperidol treatment. Since the brain uptake of [11C]β-CFT was higher than [11C]raclopride, [11C]β-CFT produced more clearer images in CLI. Conclusions Optical imaging was possible with PET imaging probes. Although both quantitativity and sensitivity are inferior to PET imaging, it is possible to use CRI for brain function analysis in mice. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S153

153 Electrophilic fluorination and preclinical evaluation of a novel [18F]DPA-714 analogue, [18F]F-DPA. Thomas Keller1, Anna M. Krzyczmonik1, Sarita Forsback1, Anna Kirjavainen1, Francisco Lopez-Picon3, Fanny Cacheux2, Annelaure Damont2, Frédéric Dollé2, Merja Haaparanta-Solin3, Olof Solin1 1 Radiopharmaceutical Chemistry Laboratory, Turku PET Centre, University of Turku and Turku University Central Hospital, Turku, Finland, 2CEA, I2BM, Service hospitalier Frédéric Joliot, Orsay, France, 3MediCity/PET Preclinical Laboratory, Turku PET Centre, University of Turku, Turku, Finland Objectives Electrophilic 18F-fluorination can provide a fast and simple synthetic route to a range of target molecules inaccessible by nucleophilic labelling. [18F]Selectfluor is a versatile electrophilic fluorination reagent that can be produced in our facility with moderate to high SA [1]. We report here the synthesis of [18F]F-DPA, a novel TSPO-specific radioligand, by an electrophilic approach using post-target produced [18F]F2-derived [18F]Selectfluor. Methods [18F]F2 and [18F]Selectfluor were prepared according to previously reported methods [1,2]. Without purification, the crude stock of [18F]Selectfluor in Acetone-d6 was added to a vessel containing the stannylated precursor and 2 equivalents of AgOTf. The reaction mixture was stirred at a temperature of 45°C and samples for HPLC analysis were taken at 15 and 30 minutes after start of synthesis. Results The [18F]F-DPA target molecule was successfully synthesised by electrophilic substitution in 31-44% RCY decay corrected to EOB. [18F]F-DPA for preclinical evaluation in healthy SD rats was obtained with > 99% radiochemical purity and SA of 9 GBq/µmol. Ex vivo brain autoradiography showed a fast clearance for areas of non-specific uptake and TLC analysis of brain homogenate and blood plasma demonstrated a higher metabolic stability of [18F]F-DPA relative to [18F]DPA-714 with minimal or no defluorination being observed. Conclusions The initial labelling syntheses and preclinical evaluation were successful and yielded promising results. Hence [18F]F-DPA merits further preclinical evaluation in an animal model of neuroinflammation. Acknowledgements Funding was received from the European Union’s 7th Framework Programme for Research, grant number 316882 and from the Academy of Finland, grant number 266891. References [1] Teare H, et al (2010) Angew Chem Int Ed., 49, 6821-24 [2] Bergman J, Solin O. (1997) Nucl Med Biol, 24, 677-83

J Label Compd Radiopharm 2015: 58: S1- S411

S154: Poster

21st International Symposium on Radiopharmaceutical Sciences

154 Synthesis and evaluation of two novel 18F-labeled acetamidobenzoxazolone radioligands for PET imaging of translocator protein (18 kDa) Anjani K Tiwari1, 2, Masayuki Fujinaga1, Katsushi Kumata1, Yoko Shimoda1, Joji Yui1, Tomoteru Yamasaki1, Lin Xie1, Akiko Hatori1, Masanao Ogawa1, Kazunori Kawamura1, Ming-Rong Zhang1 1 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Dalhi, India Objectives Development of PET radioligand for translocator protein [18 kDa, TSPO] to study its role in activation of glial cells in the injured brain as well as in neurodegenerative diseases is one of the most attractive issues of PET imaging. Most of PET radioligands bind TSPO with high affinity in small animals; however, their specific bindings are sometimes reduced in human brain. Moreover, many radioligands exhibited mixed affinity with TSPO in “binder” and “non-binder” human subjects. We have developed 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide ([11C]MPMB) with a benzoxazolone moiety as a new PET ligand for TSPO.1 In this study we synthesized two 18F-fluoroalkyl analogues, [18F]FEBMP and [18F]FPBMP, and to evaluate their potentials for imaging of TSPO expression in brain in vivo. Methods Unlabeled FEBMP and FPBMP were synthesized in house.2 [18F]FEBMP and [18F]FPBMP were prepared by reacting desmethyl precursor with [18F]FEtBr and [18F]FPrBr, respectively. In vitro binding assay, biodistribution, in vitro autoradiography and PET imaging studies of these radioligands were carried out. Results [18F]FEBMP and [18F]FPBMP were prepared with radiochemical yields of 20-40% based on [18F]fluoroalkyl agent. They were found to specifically bind TSPO with high affinity (Ki = 6.6 ± 0.7 nM and 16.7 ± 2.5 nM). Biodistribution study indicated high accumulation of radioactivity in the TSPO-rich organs. In vitro autoradiography for ischemic rat brain showed that the ratios of radioactive signals in the ipsilateral side compared with the contralateral side were 3.1 for [18F]FEBMP and 2.1 for [18F]FPBMP. PET imaging of the rat brains with [18F]FEBMP revealed increased uptake in the ipsilateral side, showing high binding potential (BPND = 2.72 ± 0.27). In vitro autoradiography with [18F]FEBMP for postmortem human brains demonstrated that TSPO rs6971 polymorphism did not give significant effect on the binding sites. Conclusions [18F]FEBMP is a promising PET ligand for in vivo visualization of neuroinflammation generally occurred in neurodegenerative disorders. Acknowledgements References [1] Tiwari AK, et al (2014) J Neurochem, 129, 712. [2] Tiwari AK, et al (2014) Org Biomol Chem, 12, 9621.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S155

155 Automation of [18F]-radiotracers on the AllinOne Shihong Li, Alex Schmitz, Hsiaoju Lee, Robert H. Mach Radiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States Objectives To develop fully automated methods for producing [18F]-labeled radiotracers in a cGMP environment with good yields using the Trasis AllinOne module. Methods The synthesis of each [18F]-radiotracer required project-specific software programming which separated the syntheses into several automated steps (e.g., drying [18F]F-, radiolabeling, deprotection, HPLC purification, final formulation). Results In addition to the built-in nucleophilic [18F]-F-DOPA program, four new programs were developed for [18F]-ISO-11, [18F]-FTP2, [18F]-Fallypride, and [18F]-(2S,4R)4-Fluoroglutamine3. For the first three radiotracers, a one-step one-vessel synthesis was established; for [18F]-(2S,4R)4-Fluoroglutamine, a two-vessel two-step synthesis method was developed. The resulting automated production runs for these radiotracers showed reproducible good yields (Table) with run times ranging from 45-95 min. The purity and identity of each [18F]-radiotracer was confirmed by a separate analytical HPLC system. Conclusions The synthesizer offers a flexible automation method for the aforementioned radiotracers. All of our [18F]-radiotracer automation on the AllinOne displayed comparable yields and reliability in comparison to other synthesis methods. The advantages of this automated synthesis system include (i) the flexibility to rapidly modify all or part of the automation steps based on the needs of the radiotracer without hardware modification, (ii) the use of commercially available modular disposables, such as manifolds and vials, to eliminate the need for system cleaning, and (iii) built-in system control to ensure module readiness prior to synthesis. In addition, the ability to carry out multi-step two-vessel reactions and semi-prep HPLC purification followed by optional solid-phase extraction opens up this synthesis module for most of our clinical and research [18F]-radiotracer needs. Acknowledgements References [1] Tu Z et al (2011) Nucl. Med. Biol. 38, 725-739 [2] Tu Z et al (2007) J. Med. Chem. 50, 3194-3204. [3] Qu W et al (2011) J .Am. Chem. Soc. 133, 1122-1133.

A: One vessel, one step with HPLC and SPE; B: Two vessel, two step with HPLC and SPE

J Label Compd Radiopharm 2015: 58: S1- S411

S156: Poster

21st International Symposium on Radiopharmaceutical Sciences

156 Copper-mediated Late-Stage Radiofluorination of Electron-rich Arenes Allen F. Brooks1, Naoko Ichiishi2, Joseph Topczewski2, Yong-Woon Jung1, Melanie S. Sanford2, Peter J. Scott1, 3 1 Radiology, University of Michigan, Ann Arbor, Michigan, United States, 2Chemistry, University of Michigan, Ann Arbor, Michigan, United States, 3Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, United States Objectives Many highly sought-after 18F-radiotracers contain electron-rich arenes but there are few methods for accessing such tracers using nucleophilic fluoride.1 To address this, our group is developing Cu-mediated radiofluorination methods (Scheme 1), and herein we report new developments with our recently reported Cumediated radiofluorination of (mesityl)(aryl)iodonium salts using [18F]KF (Method A),2 including syntheses of numerous radiotracers of clinical interest. We also introduce a novel Cu-mediated method for the radiofluorination of electron-rich aryl iodides with [18F]AgF (Method B). Methods To an aq. solution of the appropriate fluoride salt (K18F or Ag18F) was added 18-crown-6 in MeCN and the mixture was azeotropically dried. The dried 18F was reconstituted in DMF and added to a mixture of the copper catalyst and precursor (iodonium salt or aryl iodide) in DMF. The reaction mixture was heated (Scheme 1) and then analyzed by TLC and/or HPLC. Results Numerous 18F-labeled arenes have been synthesized from diaryliodonium salts or aryl iodides (Scheme 1) in moderate to high radiochemical yields (RCY). Both methods are tolerant of electron-deficient, -neutral or -rich arenes. The high yields and mild conditions for fluorination of (mesityl)(aryl)iodonium salts make this method well-suited for synthesis of clinical radiotracers, and syntheses of [18F]FDOPA, [18F]MHPG and others are currently being qualified for clinical use in our lab. Preliminary data for the Cu-mediated late-stage fluorination of electron-rich aryl iodides with [18F]AgF suggest this method could change the 18F radiochemistry landscape by enabling direct radiolabeling of any aryl iodide. Extensive optimization studies are underway to improve RCY and explore reaction scope. Remarkably, no drop in RCY is observed in the presence of unprotected functional groups. Most notably, 3-[18F]fluorotyrosine is formed in 5% RCY without the need for any protecting groups. Conclusions Cu-mediated fluorination methods allow facile radiolabeling of electron-deficient, -neutral or -rich arenes starting from readily available aryl iodides or mesityl iodonium salts. Acknowledgements We acknowledge the NIH (GM073836 (MSS) and T32-EB005172 (PJHS)) and the Alzheimer’s Association (NIRP-14-305669 (PJHS)) for financial support. References [1] See Brooks AF, Topczewski JJ, Ichiishi N, Sanford MS, Scott PJH. Late-stage [18F]Fluorination: New Solutions to Old Problems. Chem. Sci., 2014;5:4545-4553 and references therein; [2] Ichiishi N, Brooks AF, Topczewski JJ, Rodnick ME, Sanford MS, Scott PJH. Copper Catalyzed [18F]Fluorination of (Mesityl)(Aryl)Iodonium Salts. Org. Lett., 2014;14:3224-3227.

Scheme 1: Copper-mediated Radiofluorination Reactions

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S157

157 Development of a novel 18F-labelled tetrazine with low lipophilicity Outi M. Keinänen1, Xiang-Guo Li2, Naveen K. Chenna3, Mirkka Sarparanta1, 4, Kerttuli Helariutta1, Tapani Vuorinen3, Anu J. Airaksinen1 1 Laboratory of Radiochemistry, University of Helsinki, Helsinki, Finland, 2Turku Positron Emission Tomography (PET) Centre, University of Turku and Turku University Hospital, Turku, Finland, 3Department of Forest Products Technology, Aalto University School of Chemical Technology, Espoo, Finland, 4Department of Radiology and Program in Molecular Pharmacology and Chemistry, Memorial Sloan Kettering Cancer Center, New York, New York, United States Objectives Phenyl substituted 1,2,4,5-tetrazines have exhibited the fastest reaction kinetics towards the most commonly used dienophiles in bioorthogonal reactions. We have developed an efficient synthesis method for preparation of a novel 18F-labelled phenyl tetrazine, (E/Z)-N-(4-(1,2,4,5-tetrazin-3-yl)benzyl)-2-(((5-fluoro-2,3,4trihydroxypentylidene)amino)oxy)acetamide ([18F]TAF, Fig. 1). Methods (4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine was functionalized with an aminooxy functional group which has high reactivity towards aldose sugars, aldehydes and ketones. 18F-labelling was achieved in mild reaction conditions with 5-deoxy-5-[18F]fluororibose-based conjugation[1],[2]. Reactivity of [19F]TAF was determined with several different dienophiles in methanol at RT: trans-cyclooctenol (TCO-OH), (1R,8S,9s)bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) and exo-5-norbornene-2-methanol (E5N2M). In vitro stability of [18F]TAF in PBS (pH 7.41) at RT and in mouse plasma (in 50% PBS) at 37 °C were assessed by HPLC. Lipophilicity of [18F]TAF was determined with the shake flask method at pH 7.41. Results [18F]TAF was synthesized with over 50% decay-corrected yield (n=5). The total duration of the synthesis was 2 hours and radiochemical purity was ˃99%. TAF has an imine double bond and exists as E/Z isomers (E:Z = 3:1). The second-order rate constants with TCO-OH, BCN-OH and E5N2M were 300±9 M-1s-1, 129±4 M-1s-1 and 0.35±0.04 M-1s-1, respectively. The logP values were -0.4315±0.0071 (E) and -0.0150±0.0174 (Z). The [18F]TAF isomers have no difference in their stability in PBS or mouse plasma (50% in PBS). The proportion of the intact [18F]TAF in PBS after 1 h and 3 h was >99% and >99%, respectively. The proportion of the intact [18F]TAF in mouse plasma (50% in PBS) after 1 h and 3 h was 96.3% and 86.2%, respectively. Conclusions [18F]TAF was successfully synthesized in mild reaction conditions with high radiochemical yield and purity. TAF showed fast reaction kinetics towards the most commonly used dienophiles for bioorthogonal chemistry and sufficient stability under physiological conditions. The results indicate that [18F]TAF is a promising candidate for in vivo bioorthogonal chemistry. Acknowledgements Supported by the Academy of Finland (272908) and Doctoral Program in Chemistry and Molecular Science (CHEMS). References [1] Li X. G. et al. (2012) Chemical communications, 48, 5247-5249 [2] Li X. G. et al. (2013) Chemical communications, 49, 3682-3684

Figure 1. Structure of [18F]TAF.

J Label Compd Radiopharm 2015: 58: S1- S411

S158: Poster

21st International Symposium on Radiopharmaceutical Sciences

158 Copper-mediated aromatic radiofluorination revisited: efficient production of PET-Tracers on a preparative Scale Johannes Zischler, Boris D. Zlatopolskiy, Philipp Krapf, Fadi Zarrad, Elizaveta A. Urusova, Elena Kordys, Heike Endepols, Bernd Neumaier Institute for Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne , Cologne, Germany Objectives Recently two novel methods for the copper-mediated aromatic nucleophilic radiofluorination of (mesityl)(aryl)iodonium salts (MAI)[1] and aryl pinacol boronates (ArBPin)[2] were published. Evaluation of these methods revealed that although both showed high efficacy in small scale experiments, they were inoperative for PET tracer production on a clinical scale. The aim of this work was the development of a versatile and practical procedure for the preparation of PET tracers via Cu-mediated radiofluorination. Methods Cu catalysts were unstable under conventional basic radiofluorination conditions ([18F]KF/K2.2.2 or 18crown-6, 2.5-3.5 mg K2CO3). Therefore, 18F– was eluted with only 0.06-0.1 mg K2CO3 (“low base” conditions). After azeotropic drying [18F]KF/K222 or 18-crown-6 complex was redissolved in a solution of the appropriate Cusalt and the corresponding ArBPin or iodonium salt precursor and the resulting solution was heated for 20 min. Furthermore, the radiofluorination of iodonium salts under "minimalist" conditions (no addition of base and cryptand, no azeotropic drying) was examined. [18F]Fluoride was eluted from the anion exchange resin with onium precursor in MeOH. The solvent was evaporated, the residue taken up in a solution of (MeCN)4CuOTf in DMF and heated. Results [[18F]Fluorobenzene, 4-[18F]fluoroanisole and 3-[18F]fluorobenzaldehyde were obtained under “low base” conditions in RCCs of 26–56% (from MAI) and 41–64% (from ArBPin), respectively. Under "minimalist" conditions, the model compounds were prepared from MAI in RCCs of 78–90%. Protected 6-[18F]fluorodopamine and 4-[18F]fluorophenylalanine were obtained in 71–94% RCCs. Subsequent hydrolysis of the protected intermediates and HPLC isolation afforded the PET tracers in RCYs of 46 and 66%, respectively. Additionally, [18F]DAA1106 was efficiently prepared in 60% RCY. The specific activity of 4-[18F]FPhe and [18F]DAA1106 amounted to 109 GBq/µmol and 66 GBq/µmol starting from 49.5 and 2.4 GBq, respectively. Conclusions We have established a robust “low base” protocol for the efficient copper-mediated preparation of [18F]arenes from diaryliodonium salts and aryl pinacol boronates. Furthermore, we have developed a novel radiofluorination method that combines the advantages of our “minimalist” approach with the exceptional capabilities of copper mediated aromatic nucleophilic radiofluorination. Acknowledgements References [1] N. Ichiishi et al , Org. Lett. 2014, 16, 3224-3227. [2] M. Tredwell et al, Angew. Chem. 2014, 126, 7885-7889.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S159

159 Radiosynthesis of [18F]cabozantinib and [18F]fluoroethyl-sunitinib: two RTK-inhibitors of VEGFR-2 Martin Schwebe, Ralf Bergmann, Jörg Steinbach, Jens Pietzsch, Torsten Kniess Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany Objectives Radiolabeled inhibitors of the angiokinase VEGFR-2 might be suitable probes for monitoring induction of angiogenesis and anti-angiogenic therapy response in vivo with PET. [1,2]. We selected two VEGFR-2 inhibitors, cabozantinib (IC50, 0.03 nM) and sunitinib (IC50, 9.0 nM), both bearing a fluorine substituent, as lead structures for 18F-radiolabeled PET tracers. Methods [18F]Cabozantinib is synthesized by a 3-step radiosynthesis with final condensation of 4[18F]fluoroaniline with an acyl chloride precursor. 4-[18F]Fluoroaniline is formed by substitution of 1,4dinitrobenzene with [18F]fluoride, subsequent reduction of the intermediate 4-[18F]fluoro-nitrobenzene with Pd/C and NaBH4. Since [18F]sunitinib is not accessible via direct nucleophilic 18F-substitution, we developed the 5fluoroethylated derivative (IC50, 9 nM) as well the corresponding radiolabeled analogue. Results 4-[18F]fluoroaniline was obtained in >60% rcy starting from [18F]fluoride after SPE purification. [18F]Cabozantinib was formed by reaction of 4-[18F]fluoroaniline with 10 mg of acyl precursor in THF at rt in >90% rcy. HPLC purification delivered [18F]cabozantinib in 95% purity and specific activity >20 GBq/µmol. Reaction of the methanesulfonyl-substituted sunitinib precursor with [18F]fluoride resulted in 8% 18F-incorporation. HPLC purification yielded [18F]fluoroethyl-sunitinib in 100 MBq scale. First in vitro investigations on VEGFR-2 expressing human A 2058 melanoma cell line showed cellular uptake of [18F]cabozantinib up to 790±100 %ID/mg protein at 60 min that could be significantly blocked by 46±3% by its non-radioactive counterpart (10 µM). For [18F]fluoroethyl-sunitinib the uptake reached 340±48 %ID/mg protein at 60 min. Stability tests in rat blood over 60 min revealed almost no metabolism for both radiotracers. Conclusions With the reliable radiosynthesis of [18F]cabozantinib and [18F]fluoroethyl-sunitinib two radiolabeled VEGFR-2 inhibitors with nano- and sub-nanomolar affinity and high in vivo stability are available. Acknowledgements References [1] Slobbe P. et al (2012) Drug Discov. Today, 17, 1175-1187 [2] Kniess T. (2012) Curr Pharm Des, 18, 2867-2874

J Label Compd Radiopharm 2015: 58: S1- S411

S160: Poster

21st International Symposium on Radiopharmaceutical Sciences

160 Rapid strain induced sydnone-alkyne cycloaddition reactions for bioorthogonal PET imaging Maruthi Kumar Narayanam1, 2, Jennifer M. Murphy1, 2 1 Crump Institute for Molecular Imaging, University of California Los Angeles, Los Angeles, California, United States, 2Department of Molecular & Medical Pharmacology, University of California Los Angeles, Los angeles, California, United States Objectives Extremely selective and high yielding bioorthogonal click chemistry reactions are widely studied and used as tools for radiolabelling biomolecules and small molecules for positron emission tomography (PET).1 Electron deficient sydnones undergo copper-free cycloaddition reactions with bicyclo-[6.1.0]-nonyne (BCN)2,3 which we have investigated in depth. Moreover, a new Cu-free cycloaddition reaction between sydnone derivatives and the biarylazacyclooctynone (BARAC) is described; to date, it is the fastest reported cycloaddition reaction with BARAC, with rates of 101 to 102 M-1s-1. We demonstrate efforts to develop an 18F-labeled sydnone, which can replace 18F-azides, for radiolabeling of biomolecules for PET imaging. Methods 3-Arylsydnones 1 were synthesized via intramolecular cyclization of N-nitroso-N-aryl amino acids with trifluoroacetic anhydride. The rate constant for the cycloaddition between sydnone and BARAC or BCN was determined under pseudo-first order conditions and followed via the exponential decay in UV absorbance over time (Fig. 1). Synthesis of the 18F-labeled sydnone and its applications are currently underway in our laboratory. Results The reactions of sydnones with BCN or BARAC proceed at room temperature, in aqueous media and without a transition-metal catalyst. The phenyl ring and C-4 substitutions on the sydnone significantly impact the reaction rate constants. We observed improved reaction rates with electron deficient sydnones and 4chlorosydnones; the cycloaddition reaction of phenyl sydnone with BARAC is nearly 104-fold faster than with BCN. Conclusions We report functionalized sydnones as 1,3-dipoles for metal-free cycloaddition partners with ringstrained alkynes. The highly stable sydnones display excellent reaction kinetics with BARAC, yielding 103-fold rate enhancement over azides. Efforts towards the use of 18F-labeled sydnones for in vivo PET imaging are under progress. Acknowledgements References [1] Thirumurugan P., et al (2013) Chem. Rev., 113, 4905-4979 [2] Wallace S., et al (2014) Chem. Sci., 5, 1742–1744 [3] Plougastel L., et al (2014) Chem. Comm., 50, 9376-9378.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S161

161 Automated concentration of 18F-fluoride into microliter volumes Mark S. Lazari1, 2, Maruthi Kumar Narayanam1, Jennifer M. Murphy1, R. Michael van Dam1, 2 1 Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California, United States, 2Department of Bioengineering, UCLA, Los Angeles, California, United States Objectives Rapidly concentrating large amounts of 18F-fluoride into µL volumes is needed for several emerging trends in radiochemistry. First, it enables production of clinically-relevant amounts of tracers using batch-mode microfluidic radiosynthesizers [1,2], which offer compact system size and high specific activity. Second, it can enable radiofluorination without azeotropic drying by dilution of the µL 18F-fluoride volume into conventional volumes of anhydrous solvent. We developed an automated system that efficiently traps 18F-fluoride from entire target volumes of 18O-H2O onto a micro strong anion-exchange cartridge (µSAX) and elutes the activity with µL volumes. As an example, we demonstrate the direct application of the eluted activity for the Ni-mediated radiosynthesis of model compound N-boc-5-[18F]fluoroindole [3] without azeotropic drying. Methods Figure 1 shows the fluidic diagram of the system. Trapping consisted of passing 0.42 – 620 mCi (16 MBq – 23 GBq) of 18F-fluoride in 18O-H2O through the µSAX, followed by acetonitrile (MeCN) and then N2 gas to dry. Trapping efficiency was compared after various preconditioning solutions (Table 1A). Elution was performed by passing two consecutive slugs through the µSAX. Elution efficiency was compared for various salts (Table 1B) and solvent fractions (Table 1C). For the example synthesis, activity eluted with the 80% MeCN solution (Table 1C) was collected in a vial prefilled with MeCN, pulled into a reaction vial containing the Ni-indole complex and hypervalent iodine oxidant (Figure 1), and instantly reacted at room temperature [3]. Radiochemical conversions (RCC) were determined by radio-TLC. Results Trapping and elution was completed in 5 – 10 min with efficiencies listed in Table 1 and recovered volume of 12.4 ± 0.4 µL (n = 4). RCC of the N-boc-5-[18F]fluoroindole was 54 ± 6% (n = 6), which is comparable to literature [3]. Conclusions Our concentrator system successfully delivered 18F-fluoride, which, when diluted with 500 µL MeCN, reduced water content to 0.5% for the successful reaction of N-boc-5-[18F]fluoroindole, and could also be used to deliver high levels of activity for microfluidic reactions. Acknowledgements References [1] Keng P.Y., et al (2012) Proc. Natl. Acad. Sci. U.S.A., 109, 690–695. [2] Lebedev A., et al (2013) Lab Chip., 13, 136–145. [3] Lee, E., et al (2012) J. Am. Chem. Soc., 134, 17456–17458.

J Label Compd Radiopharm 2015: 58: S1- S411

S162: Poster

21st International Symposium on Radiopharmaceutical Sciences

162 Site-specific Labelling of Active Site Inhibited Factor Seven (ASIS) with fluorine-18 by Oxime Coupling Troels E. Jeppesen1, Carsten Behrens2, Jesper B. Kristensen2, Carsten H. Nielsen1, 3, Lars C. Petersen2, Jacob Madsen1, Andreas Kjaer1 1 Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Denmark , Copenhagen Ø, Denmark, 2Novo Nordisk A/S, Måløv, Denmark, 3 Minerva Imaging ApS, Copenhagen, Denmark Objectives TF is implicated in cancer progression, angiogenesis and metastasis via TF/FVIIa-mediated activation of PARs. TF is therefore a potential target for assessment of cancer aggressiveness.[1] Active Site Inhibited Factor VIIa (ASIS) exhibits a five times higher affinity towards TF than Factor VIIa and is without pro-coagulant activity.[2] We have recently labelled ASIS with [18F]SFB, showing promising results.[3] The aim of this study was to label ASIS modified with a hydroxylamine specifically in the glycan side chain (ASIS-ONH2) with [18F]1 via an oxime coupling. Methods [18F]SFB was produced in a one-pot procedure as previously described[4] combined with a final HPLC purification step.[3] The product was further reacted with 4,4-Diethoxybutylamine, deprotected with TFA and purified by HPLC and SepPak. The resulting product ([18F]1 ) was evaporated to dryness, reacted with ASIS-ONH2 and purified with a PD-10 column (Figure 1). Site-specific labelled ASIS was evaluated by HPLC, SDS-PAGE and protein precipitation. Results 128.5-1605 MBq [18F]1 was prepared in 81-93% yield by HPLC (n=3). Coupling of [18F]1 to ASIS-ONH2 was achieved in 27-30% d.c. radiochemical yield (n=2). The overall synthesis time was 294 minutes. Analysis after purification indicated that more than 95% of the radioactivity was associated with ASIS, and that ASIS was intact after labelling. Conclusions A new method for site-specific labelled ASIS with 18F was developed. Further work is undertaken to shorten the synthesis-time of the prosthetic group ([18F]1), and to evaluate the tracer in vivo as an non-invasive agent for imaging of the aggressiveness of cancer. Acknowledgements Supported by the Innovation Fund Denmark and Novo Nordisk A/S References [1] W. Ruf, et al. (2011) J Thromb Haemost, 9, 306-315. [2] B. B. Sorensen, et al. (1997) J. Biol. Chem., 272, 11863-11868. [3] Unpublished results. [4] G. Tang, et al. (2010) J. Label Compd. Radiopharm, 53, 543-547.

Figure 1: Synthesis of [18F]1 and coupling to ASIS-ONH2. a) 1. K[18F]F-K222. 2. TPAH, 5 min, 120 °C. 3. TSTU, 10 min, 90°C. b) 4,4-Diethoxybutylamine, 5 min, r.t. c) TFA/H2O 10%, 5 min, r.t. d) ASIS-ONH2, aniline 1% vol., 1h, r.t.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S163

163 Molecular Imaging of Neuroinflamation and b-amyloid density in Alzheimer’s disease Johnny Vercouillie1, Caroline Hommet2, Karl Mondon2, Emilie Beaufils2, Nicolas Arlicot1, Serge Maia2, Denis Guilloteau2, Maria Joao Ribeiro2 1 CERRP, U930, Tours, France, 2CHRU Bretonneau, Tours, France Objectives Alzheimer Disease (AD) is the most common aetiology of dementia in the elderly. AD is characterized by the accumulation of β-amyloïd proteins within senile plaques and neurofibrillary tangles of hyperphosphorylated tau protein both being associated with neuronal death. Over the last decades increasing evidence has suggested that neuroinflammation plays a crucial role in the pathogenesis of AD. The translocator protein (TSPO) is up-regulated during neuroinflammation. Consequently, targeting TSPO with radiolabeled ligands for PET may be considered an attractive biomarker for neuroinflammation. In this work, we aim to look for the relationships between amyloid load (with 18F-Florbetapir) and the putative consequences of microglial activation (with 18F-DPA 714) in the clinical course of the AD. Methods Three groups of subjects, control, mild cognitive impairment (MCI) and AD, will be studied on PET with both radioligands. A 20 min image was acquired 50 min after the injection of a mean dose of 225 MBq of 18FFlorbetapir. A PET acquisition was performed over 59 min following iv injection of 18F-DPA-714 (mean activity: 235 MBq). Regions of interest were defined for frontal, posterior cingulate, precuneus, parietal, temporal and occipital cortex as also for cerebellum (used as reference region to obtain SUVr values) for each radioligand. Results The preliminary results show an increased uptake of both radioligands on the PET images obtained for the AD subjects when compared to control and MCI groups. Conclusions Our preliminary results show an increase density of β-amyloïd proteins associated to an activation of TSPO on AD patients when compared to controls. Acknowledgements The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 278850 (INMiND), French National Agency for Research (‘Investissements d’Avenir’ n°ANR-11-LABX-0018-01, IRON), PHAO 13/CH/NIDeCo, Thérèse Planiol Fundation and Cyclopharma Laboratories. References - James ML, Fulton RR, Vercouillie J, Henderson DJ, Garreau L, Chalon, S, Dolle F, Selleri S, Guilloteau D, Kassiou M. DPA-714, a New Translocator Protein–Specific Ligand: Synthesis, Radiofluorination, and Pharmacologic Characterization. J Nucl Med. 2008;49:814-22. - Arlicot N, Vercouillie J, Ribeiro MJ, Tauber C, Venel Y, Baulieu JL, Maia S, Corcia P, Stabin MG, Reynolds A, Kassiou M, Guilloteau D. Initial evaluation in healthy humans of [18F]DPA-714, a potential PET biomarker for neuroinflammation. Nucl Med Biol. 2012 May;39:570-8. - Corcia P, Tauber C, Vercouillie J, Arlicot N, Prunier C, Praline J, Nicolas G M, Venel Y, Hommet C, Baulieu JL, Cottier JP, Roussel C, Kassiou M, Guilloteau D, Ribeiro MJ. Molecular imaging of microglial activation in amyotrophic lateral sclerosis. PLoS One. 2012;7(12):e52941. - Ribeiro MJ, Vercouillie J, Debiais S, Cottier JP, Bonnaud I, Camus V, Banister S, Kassiou M, Arlicot N, Guilloteau D. Could (18) F-DPA-714 PET imaging be interesting to use in the early post-stroke period? EJNMMI Res. 2014;4:28.

J Label Compd Radiopharm 2015: 58: S1- S411

S164: Poster

21st International Symposium on Radiopharmaceutical Sciences

164 Synthesis of 18F-FDOPA via Nucleophilic Pathway on IBA's Synthera® Christophe Sauvage1, Neva Lazarova1, Marco Mueller2, David Goblet3 1 Radiopharma Solutions, IBA sa, Louvain-la-Neuve, Belgium, 2ABX, Radeberg, Germany, 3IBA, Louvain-LaNeuve, Belgium Objectives 18F-L-FDOPA (FDOPA) is a diagnostic PET agent used in the imaging of Parkinson disease, head and neck cancers and, most recently, of neuroendocrine tumors. A nucleophilic synthesis route of the tracer is achieved on IBA’s Synthera® platform. Methods The nucleophilic synthesis (Coenen et al [1]), using the ABX precursor (prod. number 1336), comprises of trapping of the fluoride/elution/drying, nucleophilic 18F-fluorination, oxidation, hydrolysis and solid phase purification. Results The fluorination of the precursor is accomplished on IFP 1 (Fig. 1 and 2), the intermediate is purified on a reversed-phase cartridge and eluted with m-CPBA (in ACN) to IFP 2 where the oxidation takes place. The removal of the protecting groups is performed with HCl, the crude mixture is diluted with a citrate buffer and then purified via solid phase extraction. The synthesis time is 90 minutes with n.d.c. yield of > 10%, radiochemical and chemical purity of > 95% and ee purity of > 98%. Conclusions A fully automated FDOPA production via the nucleophilic route with ABX precursor is achieved on IBA’s Synthera synthesizer with reliable and consistent yield, high radiochemical, chemical and enantiomeric purity. Acknowledgements References [1] F. M. Wagner, et al, J.Nucl.Med. 2009,50, 1724-1729.

Figure 1: IFP 1

Figure 2: IFP 2

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S165

165 18 F-Labeling and Radiopharmacological Evaluation of Novel Purinedione Multi-Eph Inhibitors Constantin Mamat1, Marc Pretze2, Neuber Christin1, Birgit Mosch1, Jörg Steinbach1, Jens Pietzsch1 1 Institut für Radiopharmazeutische Krebsforschung, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 2 Institut für Klinische Radiologie und Nuklearmedizin, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany Objectives The overexpression of various Eph receptors in tumors provokes the recent interest in highly affine inhibitors as attractive leads for the development of new targeted radioligands to image cancer [1]. Selective Ephtyrosine kinase inhibitors based on the purinedione skeleton have been explored in the past as potential probes for imaging of EphB4 [2] and a SNEW peptide for EphB2 [3]. However up to now, there is still no optimal radiotracer available. Herein, we report the synthesis, radiofluorination and biological evaluation of two novel purinedione derivatives as potential multi Eph inhibitor radioligands. Methods Based on known positions for affinity-related interactions of the lead structure with the receptor [4] two positions are favorable for the labeling with fluorine-18. Two precursors 1 and 3 as well as their reference compounds 2 and 4 were prepared. The radiolabeling was done in dry ACN at 100°C for 30 min. First cell association studies were performed using various Eph expressing melanoma cells (A375wt/mock, A375EphB4, A375EphB6, A375EphB4) and Eph-negative controls (HL-60). Results After labeling, both tracers [18F]2 and [18F]4 were obtained in 10 – 15 % RCY (n.d.c.) after HPLC separation (RCP: > 95%). Cell experiments in vitro revealed a substantial cell association of both [18F]2 and [18F]4 ranging from 40 to 50 %ID/mg protein at 120 min in all cell lines used. The lack of any significant difference between wild type, recombinant and control cells is indicative for cell association of, as expected, low selectivity, but also of low specificity. The latter is consistent with the observation that preincubation with 100 µM of nonradioactive compound did not result in substantial inhibition of cell association. Conclusions [18F]2 and [18F]4 were synthesized successfully and first in vitro experiments were accomplished showing substantial cell association for both tracers in various melanoma cells. However, the cell experiments revealed data on specificity of purinedione derivatives that are contradictory to data from literature [4]. These observations will be elucidated in ongoing studies. Acknowledgements References [1] Mosch, B. et al. (2010), J. Oncol., DOI: 10.1155/2010/135285; [2] Mamat, C. et al. (2012), ChemMedChem, 7, 1991–2003; [3] Pretze, M., et al. (2013) ChemMedChem, 8, 935–945; [4] Lafleur, K. et al. (2009) J. Med. Chem., 52, 6433–6446.

J Label Compd Radiopharm 2015: 58: S1- S411

S166: Poster

21st International Symposium on Radiopharmaceutical Sciences

166 18 F-Radiolabeling of Second Generation EphB4 Inhibitors Based on Bis-anilinopyrimidines Constantin Mamat1, Jens Wiemer2, Birgit Mosch1, Jens Pietzsch1, Jörg Steinbach1 1 Institut für Radiopharmazeutische Krebsforschung, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 2 Klinikum der LMU, Munich, Germany Objectives Ephrins and its Eph receptors are dysregulated in several human tumor entities including malignant melanoma. In this regard, the EphB4/ephrinB2 system seems to play a major role in melanoma angiogenesis [1]. Thus,we developed a fluorine-18-containing peptide [2] extracellularly binding to EphB4 and a small 18F-labeled molecule which intracellularly binds to the EphB4 kinase domain with high affinity [3] in the past. However, the results showed low binding/uptake in A375EphB4 melanoma cells in vitro and in vivo. Therfore, a “second generation” lead structure based on bis-anilinopyrimidines (IC50 = 1.3 nM) [4] was chosen for novel EphB4targeted radioligands. Methods The lead compound is based on two substructures (part A and B) which were synthesized independently. Two positions of the original inhibitor for the best position of the radiolabel were figured out using docking studies. Based on this, references 2 and 4 as well as precursors 1 and 3 were obtained. In order to introduce [18F]fluoride by ring opening, precursors 1 and 3 were prepared as azetidinium mesylates and lead to high RCYs. The radiolabeling was done in anhydrous acetonitrile for 30 min at 100°C. Afterwards, the EOE protecting group, which is mandatory for the successful introduction, was cleaved under acidic conditions. The subsequent purification should be easy done by cartridges due to the ionic nature of the precursors [5]. Results Interestingly, radiofluorination of the first precursor 1 did not lead to the desired tracer [18F]2. The delocalization of the positive charge over both aromatic rings might be the reason for this result. On the other hand, radiofluorination of diazaspirononane precursor 3 was successful and gives the desired [18F]4 in a radiochemical yield of 34% (n.d.c.) and high purity (>95%). Conclusions [18F]4 as novel potential EphB4-targeted radioligand based on the bis-anilinopyrimidine scaffold has been successfully synthesized and radiolabeled. Ongoing work is focused on the alternative preparation of radiotracer [18F]2 and on the biological evaluation of both radiotracers to be a suitable target for diagnostic applications. Acknowledgements References [1] Mosch, B. et al. (2010), J. Oncol., DOI: 10.1155/2010/135285, [2] Pretze, M., et al. (2013) ChemMedChem, 8, 935–945, [3] Mamat, C., et al. (2012) ChemMedChem, 7, 1991–3002, [4] Bardelle, C., et al. (2010) Bioorg. Med. Chem. Lett., 20, 6242–6245, [5] Grosse-Gehling, P., et al. (2011) Radiochim. Acta 99, 365– 373.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S167

167 Design and Synthesis of Novel F-18 PET probes for early detection of Alzheimer’s disease Sundaram S. Guruswami1, Paul Kotzbauer2, Nigel Cairns2, John R. Cirrito2, Jin-Moo Lee2, Vijay Sharma1 1 Radiology, Washington University School of Medicine, St Louis, Missouri, United States, 2Neurology, washington university school of medicine, St Louis, Missouri, United States Objectives Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by decline in cognitive functions. Currently five million people are affected in U.S. and is expected to be twenty two million by 2050. AD diagnosis at preclinical stages continues to be a challenging undertaking, and an unmet goal. One of the most exciting aspect of ongoing research in this area is neuroimaging. Development of sophisticated neuroimaging techniques could have a profound impact on improving early diagnosis, monitoring disease progression, and tracking response to treatment. While several PET tracers (PIB, Avid 45, GE-067, AZD4694) are under development, Avid45, GE-067 (Flutemetamol) and AZD4694 (Florbetaben) have been recently approved by FDA for Aβ imaging. These agents show high levels of nonspecific white matter retention and are unable to detect diffuse Aβ plaques, a preclinical manifestation of the disease. To supplement an existing armamentarium of FDA approved agents, we have discovered a small fluorescent molecule that shows characteristics of translatable imaging agents while also binding the diffuse plaques in addition to fibrillar plaques in neuropathologically confirmed AD tissues. Methods The fibril binding assay, multiphoton imaging, biodistribution, PET imaging in mice, immunohistochemistry in mice and humans have been performed using established procedures. Results The lead agent shows a concentration dependent and saturable binding with preformed Aβ1-42 aggregates (Kd=1.58±0.05nM). The F-18 counterpart demonstrates high first pass extraction into brains (8.86 % ID/g; 2 min) of normal mice, followed by clearance over 60 min. Additionally, the agent exhibits staining of fibrillar and diffuse plaques in the parenchymal regions of brain sections in live APP/PS1 mice, and distinct labeling of diffuse and fibrillar Aβ plaques in the hippocampal tissue of a neuropathologically confirmed AD patient. Preliminary multiphoton real-time imaging shows its ability to traverse the blood-brain barrier and label parenchymal Aβ plaques in brains of transgenic mice within minutes post-intravenous injection. Conclusions The lead agent demonstrates characteristic features of translatable imaging agents and provide a template scaffold for further optimization and development of Aβ-targeted probes. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S168: Poster

21st International Symposium on Radiopharmaceutical Sciences

168 Direct 18F-labelling of tosylated Pluronic® F127 and first in vivo animal PET imaging Johannes M. Postema, Jens P. Bankstahl, Tobias L. Ross Medizinische Hochschule Hannover, Hannover, Germany Objectives In the development of macromolecular structures as drug-delivery-systems, PET imaging becomes more and more important. As a result, there is a demand for suitable labelling strategies for these compounds. In 18 F-labelling, generally only multi-step approaches have been applied to macromolecules, so far. With this research we aimed at the efficient direct 18F-labelling of tosyl modified Pluronic® F127. This FDA approved Pluronic® F127 can be used to build large supramolecular structures for drug delivery or imaging purposes. Methods Tosyl modified Pluronic® F127 was produced via standard organic reactions starting from Pluronic® F127 and tosylchloride. 18F-Labelling studies of tosyl modified Pluronic® F127 were then performed in MeCN in the Kryptofix 2.2.2./K2CO3-system at a Temperature of 80 °C. The reaction progress was monitored by radioTLC. The 18F-labelled Pluronic® F127 macromolecule was applied to in vivo PET-imaging in mice. Results The modification of commercially available Pluronic® F127 with tosyl chloride proceeded with high yields. The direct 18F-labelling of tosyl modified Pluronic® F127 proceeded with a radiochemical yield of around 30% in 15 minutes. The new 18F-labelled Pluronic® F127 was applied for in vivo PET imaging in healthy mice where an uptake of the compound in the liver was seen. Conclusions We have successfully modified Pluronic® F127 with tosyl chloride. The tosylated Pluronic® F127 enables direct 18F-labelling under mild conditions in high radiochemical yields. The first in vivo PET imaging in healthy mice has shown that the compound is circulating and that it is taken up by the liver. Acknowledgements This work was funded by the People Program (Marie Curie Actions) of the EU FP7/20072013 (No. PITN-GA-2012-317019 ‘TRACE ‘n TREAT’). References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S169

169 Fast 18F-fluoroethylation without azeotropic drying in the radiosynthesis of cyclooxygenase-2 inhibitors Torsten Kniess, Markus Laube, Jens Pietzsch, Jörg Steinbach Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany Objectives 18F-Fluoroethylation is a basic approach in PET labeling chemistry and 2-[18F]fluoroethyl tosylate ([18F]FETs) is one of the mostly used agents. Usual protocols with [18F]FETs are covering the azeotropic drying of [18F]fluoride, nucleophilic substitution, purification and 18F-fluoroethylation within 60-90 min synthesis time. We developed a fast 18F-fluoroethylation avoiding azeotropic drying to yield e.g. 18F-fluoroethylated cyclooxygenase-2 (COX-2) inhibitors within 25 min. Methods Our approach is based on the finding that [18F]fluoride trapped on SAX cartridges can be completely eluted by a mixture of K222/K2CO3/acetonitrile/2% water and is sufficiently reactive for 18F-labeling. [1,2] [18F]Fluoride, trapped on the SAX cartridge is eluted with 0.7 mL K222/K2CO3/acetonitrile/H2O into a vial containing 20 µmol bis-tosylate precursor. The vial is heated 10 min at 100°C, than 20 µmol hydroxyl precursor and 40 µmol Cs2CO3 dissolved in 0.5 mL DMF are added. Additional heating for 10 min at 110°C yields the 18Ffluoroethylated COX-2 radiotracers, by almost complete consumption of [18F]FETs. We used three different precursors to build COX-2 inhibitors (Fig) as model compounds to elucidate 18F-fluoroethylation. Results By elution of the SAX cartridge (46 mg) with K222/K2CO3/acetonitrile/H2O (42 µmol, 21 µmol, 679 µL, 21 µL) the adsorbed activity could be tranferred nearly quantitatively (93-95%). [18F]FETs was formed in 79-88% rcy as confirmed by radio-TLC. Subsequent 18F-fluoroethylation of the corresponding hydroxyl precursors resulted in yields of 77-92% (n=7) in case of the cyclopentene (1), 54-65% (n=3) for the pyrazolo[1,5-b]pyridazine (2), and 44-70% (n=3) for the indomethacine (3). Conclusions The [18F]KF/K222/K2CO3/H2O complex, formed without azeotropic drying is highly reactive to form [18F]FETs in yields up to 88%. Hence the reaction time can be shortened resulting in fast 18F-fluoethylations with total radiochemical yields up to 92% as exemplified for three radiolabeled COX-2 inhibitors. Acknowledgements References [1] Wessmann S.H. et al., Nuklearmedizin, 2012, 51, 1-8 [2] Kolb H.C. et al., J.Label.Compd.Radiopharm.,2011, 54, S518

Figure: 18F-fluoroethylated COX-2 inhibitors

J Label Compd Radiopharm 2015: 58: S1- S411

S170: Poster

21st International Symposium on Radiopharmaceutical Sciences

170 Improved Radiosynthesis and Whole-body Biodistribution of PHF-tau Tracer [18F]T807 in Mice Ya-Yao Huang1, Chia-Ling Tsai1, Hao-Yu Hsieh2, Ling-Wei Hsin2, Kai-Yuan Tzen1, Chyng-Yann Shiue1 1 PET Center, National Taiwan University Hospital, Taipei, Taiwan, 2College of Medicine, National Taiwan University, Taipei, Taiwan Objectives [18F]T807 has been proved to be a promising PHF-tau tracer for studying AD in humans [1, 2]. However, the reported methods for [18F]T807 production were somewhat complicated [2,3] . In order to use this tracer for human studies, we have improved its synthesis using TracerLab FxN module and studied its whole-body biodistribution in mice using microPET before human studies are undertaken. Methods The [18F]T807 was synthesized in a FXFN module as reported previously with some modifications [3]. Briefly, fluorination of N-t-Boc protected precursor (1) was carried out with K[18F]/K2.2.2 in DMSO at 130 0C for 10-20 min to give the intermediate (2). A solution of 1N HCl(aq) was added to (2) and then the mixture was heated at 90 0C for 5-10 min . Following neutralizion, the crude product was purified with a semi-preparative HPLC (Phenomenex Luna(2) HILIC, 0.1% HCl, 3 mL/min). The fraction containing [18F]T807 (3) was collected, neutralized and passed through a 0.22μm Millipore filter into a sterile vial. For animal study, fasten male ICR mice (20–30g) were injected with a bolus of about 200 μCi of [18F]T807 (n=4). By using small-animal Argus PET/CT scanner, dynamic sinograms were produced and VOIs were defined on co-registrated PET/CT images. Timeactivity data of source organs were used to calculate the residence times and estimate the absorbed radiation dose using the OLINDA/EXM software. Results The radiochemical yield of [18F]T807 synthesized by this method was 13.2±3.9 % (EOS, n〉3) in a synthesis time of about 70 min from EOB. Both the chemical and radiochemical purity were 〉95 % with a specific activity of 151±52 GBq/μmol. Whole-body biodistribution in mice showed that the radioactivity in the gallbladder and lower large intestine increased with time suggesting the [18F]T807 may be eliminated via the hepatobiliary and renal systems. The effective dose of [18F]T807 in mice was 94±19 μSv/MBq (n=4). Conclusions With the improved radiosynthesis method, [18F]T807 could be reliably produced for preclinical and clinical studies. Additionally, whole-body biodistribution in mice showed that [18F]T807 is safe for human studies. Acknowledgements We are grateful to Mr. Pei-Yao Lin and Mr. Chi-Han Wu of Molecular Imaging Center of National Taiwan University for their technical assistances. References [1] Xia CF, et al (2013) Alzheimer’s Dement, 9, 666. [2] Chien DT, et al (2013) Alzheimer’s Dis, 34, 457. [3] Shoup TM, et al (2013) J Labelled Cpd Radiopharm, 56, 736.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S171

171 Interrogation of status of Thioflavin T in human epidermal carcinoma cells using fluorescence imaging Tenira Townsend, Kristen Binz, Vijay Sharma, Sundaram Guruswami Radiology, Washington University in St. Louis, St. Louis, Missouri, United States Objectives β-amyloid (Aβ) deposition is a hallmark of Alzheimer’s disease (AD). While several PET tracers (PIB, Avid-45, GE-067, AZD4694) are under development, Avid-45 and GE-067 (Flutemetamol) have been recently approved by the FDA for Aβ imaging. Among these agents, PIB is an analogue of Thioflavin T (ThT), a benzothiazole dye, used for postmortem neuropathological confirmation of AD. While showing high specificity to Aβ, ThT lacks ability to traverse the blood-brain barrier (BBB). However, the mechanistic aspects for the poor BBB penetration of ThT have not been investigated. P-glycoprotein (Pgp, ABCB1, 140kDa), located on the luminal surface of vascular endothelial cells of the brain, recognizes hydrophobic cations, restricts CNS drug delivery, and also mediates chemotherapeutic resistance. To interrogate whether or not ThT is recognized by Pgp as a transport substrate, cellular uptake profiles of ThT in human epidermal carcinoma cells are investigated. Methods Cellular uptake assays were used to determine status of ThT as a Pgp substrate. Human epidermal carcinoma cells (KB-8-5, Pgp+; KB-3-1, Pgp-) were incubated with ThT (10µM) in the presence and absence of LY335979 (LY), a Pgp antagonist. Cellular uptake of ThT was monitored via fluorescence microscopy. Images were analyzed using FIJI/Image J. Results Pgp+ cells show significantly lower ThT fluorescence signal compared to Pgp- cells. Upon treatment with LY, fluorescence signal in drug resistant Pgp+ KB-8-5 cells dramatically increases to levels equivalent to PgpKB-3-1 cells. Finally, fluorescence imaging also shows intracellular localization of ThT in the mitochondria of cells. Conclusions ThT cellular uptake profiles in human epidermal carcinoma cells are mediated by expression of Pgp. These data indicate that ThT is recognized by Pgp as a transport substrate. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S172: Poster

21st International Symposium on Radiopharmaceutical Sciences

172 Radiosynthesis of [18F]-FAZA on Trasis AllinOne for tumor hypoxia PET imaging in patients suffering from chordoma of the skull base and spine Emilie Da Costa Branquinho, Julien Fouque, Olivier Madar, Samuel Huguet, Keyvan Rezaï, Hamid Mammar, François Lokiec Radio-pharmacology, Institut Curie, Saint Cloud, France Objectives Hypoxic tumors are associated with poor radiotherapy outcome because of their increased resistance to ionizing radiation. PET imaging using a specific tumor hypoxia tracer may be relevant in predicting prognosis and selecting patients for a different treatment strategy.[1] The aim of this study is to synthesize [18F]-fluoroazomycin arabinose ([18F]-FAZA), a specific tumor hypoxia PET tracer, and use it as a tool for guiding dose escalation to the hypoxic fraction of the tumor, in patients with chordomas.[2] An additional dose of proton beam therapy will be provided to patients with a positive [18F]-FAZA PET scan. Methods [18F]-FAZA was prepared according to GMP regulations, with an AllinOne (Trasis) synthesizer through a two-step synthetic pathway: nucleophilic radiofluorination of a tosylate precursor followed by hydrolysis of the protecting groups (Figure 1). In a typical procedure, a solution of precursor AZA in DMSO was added to the dry 18 F residue and the reaction heated to afford the radiofluorinated intermediate [18F]-1. After cooling at 35°C, an aqueous solution of NaOH was added and the mixture allowed to stand for an additional 3 min. After neutralization, the solution was purified on an online semi-preparative HPLC. The purified fraction of [18F]-FAZA was then passed through an Oasis® HLB cartridge and the radiotracer eluted with ethanol and saline. The formulated product was filtered through a 0.22 µm PVDF membrane. Results Optimal labeling conditions were achieved at 120°C for 10 min, using 5 mg of precursor in 600 µL of DMSO. [18F]-FAZA was afforded at high radioactivity levels (400-420 GBq at EOB) within 52 min, in 11-13% radiochemical yield (n=9; decay-corrected) after HPLC purification and formulation. Radiochemical purity was greater than 99% (UPLC® and radio-TLC). Specific radioactivity was > 18.5 TBq/µmol at EOS. Conclusions The fully automated synthesis of [18F]-FAZA including labeling, HPLC purification and formulation was optimally carried-out at high radioactivity levels with an AllinOne synthesizer. The radiotracer was afforded in high specific activity and purity, ready for clinical use. Fifty-eight patients with chordoma of the skull base and spine will undergo [18F]-FAZA PET scan before proton beam therapy. Acknowledgements The authors would like to thank Trasis. References [1] Mammar, H. et al. (2012) Int. J. Radiat. Oncol. Biol. Phys., 84, 681-687. [2] EudraCT n° 2014003699-21.

Figure 1. Radiosynthesis of [18F]-FAZA.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S173

173 On the development of a novel non-peptidic 18F-labeled radiotracer for in vivo imaging of oxytocin receptors with PET Barbara Wenzel1, Jan Mollitor2, Winnie Deuther-Conrad1, Mathias Kranz1, Sladjana Dukic-Stefanowic1, Robert Günther1, Rodrigo Teodoro1, Friedrich-Alexander Ludwig1, Steffen Fischer1, René Smits2, Jörg Steinbach1, Alexander Hoepping2, Peter Brust1 1 Helmholtz-Zentrum Dresden-Rossendorf, Research Site Leipzig, Institute of Radiopharmaceutical Cancer Research, Leipzig, Germany, 2ABX, Radeberg, Germany Objectives The peptide oxytocin is synthesized in the hypothalamus and acts as neurotransmitter to regulate a diverse range of CNS functions. Its receptor (OTR) is expressed in specific brain areas related to psychiatric diseases. So far, a non-invasive investigation of the OTR in brain is hampered by a lack of suitable radiotracers. To develope a PET ligand with high affinity toward OTR, we synthesized a series of fluorinated non-peptidic small molecules and performed radiofluorination of a selected candidate to investigate its in vivo properties in mice and pigs. Methods Binding affinities of the compounds to the human OTR were determined by radioligand displacement studies. The radiosynthesis of [18F]ABX163 (KD = 12.3 nM) was performed via thermal and microwave heating (see scheme 1). Metabolism and organ distribution of the radiotracer were studied in female CD-1 mice. Dynamic PET scans were performed in mice (animal PET/MR; 60 min) and in one female piglet (PET; 120 min). Results Using microwave heating for the synthesis of [18F]ABX163 provided higher RCY in shorter reaction time compared to thermal heating (RCY 25.4 ± 3.1% (n=5); SA 35-160 GBq/µmol; RP > 97%). Both organ distribution and dynamic PET imaging studies revealed limited uptake of the radiotracer in mouse brain (SUVmean = 0.04). Besides, significant uptake in the pituitary gland was observed (SUV55 min p.i. = 0.85), which indicates target-specific binding of [18F]ABX163. By a dynamic PET study in pig, a mean SUV of 0.43 was estimated for the whole brain at 120 min p.i. A two fold higher uptake was observed in the olfactory bulb (SUV120 min p.i. = 0.73), a region with high expression of OTR. In vitro autoradiography studies on rat and pig brain slices revealed an interaction of [18F]ABX163 with several off target receptors. Conclusions Due to the low brain uptake and the insufficient selectivity, [18F]ABX163 is not suitable for imaging of OTR in living brain. Acknowledgements The authors thank the SAB (Sächsische AufbauBank) for funding this project. References

J Label Compd Radiopharm 2015: 58: S1- S411

S174: Poster

21st International Symposium on Radiopharmaceutical Sciences

174 Continuous-Flow Microfluidic [18F]Radiofluorination of Spirocyclic Iodonium(III) Ylides Samuel B. Calderwood1, Thomas L. Collier2, Véronique Gouverneur1, Steven H. Liang2, Neil Vasdev2 1 Chemistry Research Laboratory, University of Oxford, Oxford, Oxfordshire, United Kingdom, 2Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Boston, Massachusetts, United States Objectives Labeling of non-activated aromatic rings with [18F]fluoride is a challenging chemical transformation(1). Our laboratory discovered a novel methodology using spirocyclic iodonium(III) ylides to give 18F-arenes(2). Continuous-flow microfluidics offers significant advantages for preparation of PET tracers(3). The goal of this work is to automate our procedure for the synthesis of non-activated aromatic building blocks and radiopharmaceuticals. Methods On a commercially available microfluidic platform (NanoTek, Advion), the nucleophilic [18F]fluorination of 6,10-dioxaspiro[4.5]decane-7,9-dion-[1,1’-biphenyl-4-iodonium] ylide to yield 4-[18F]fluorobiphenyl (1) was used as a model reaction for proof of concept and optimisation (solvent, base loading, precursor loading, temperature). Precursors of 4-[18F]fluorobenzyl azide (2), 3-[18F]fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]FPEB) (3) and 5-[18F]fluorouracil (4) were subjected to these conditions. Radiochemical conversions (RCC) were determined by radioTLC and radioHPLC. Investigations toward isolation of the final products are underway via automated SPE or integration with a GE Tracerlab FXN. Results DMF was shown to be the most favourable solvent, with improved RCC from higher precursor loading. Dependence on tetraethylammonium bicarbonate loading was displayed; lower loading gave better RCC at lower temperatures, exceeding optimum loading lead to decreased RCC across the temperature range. No significant changes were observed on variation of flow rate or ratio of precursor to [18F]fluoride. Final conditions gave excellent RCC to 1 (95±1%, n=4). Application gave fair to good RCC for 2 (68±5%, n=3), 3 (68±5%, n=2) and 4[18F]fluoro-2,6-dimethoxypyrimidine (18±1%, n=3). SPE isolation of 2 gives a non-decay corrected RCY=24%. Conclusions Microfluidic radiofluorination of spirocyclic iodonium(III) ylides to yield 18F-arenes has been demonstrated. This methodology has been expanded to pharmaceutically relevant substrates, which show improvements over previously published procedures. Acknowledgements Financial support provided by MRC (SC), Advion (SC, NV) and Ground Fluor Pharmaceuticals (NV). We thank Dr B. Rotstein and Dr N. Stephenson for selected synthesis and discussion. References (1) Scott P. J. H. et al. (2014) Chemical Science, 5, 4545-4553; (2) Rotstein, B. H. et al. (2014) Nature Communications, 5, 4365; (3) Rensch, C. et al. (2013) Molecules, 18, 7930-7956

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S175

175 [18F]FP-DTBZ : Radiosynthesis on a TRACERLab module and its use in PET imaging of beta-cell mass in pig Frédéric Dollé, Géraldine Pottier, Bertrand Kuhnast, Sébastien Goutal, Duc Loc Nguyen, Stéphane Demphel, Stéphane Le Helleix, Fabien Caillé, Marie-Anne Peyronneau, Raphaël Boisgard CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France Objectives Pancreatic beta-cells are in charge of insulin secretion for the regulation of blood glucose level and the number of these cells is notably decreased in diabetes. Beta-cells expressed the VMAT2, an attractive target for in vivo imaging and quantifying the density of these cells. We herein report the preparation of the VMAT2 radiofluorinated ligand [18F]FP-DTBZ [1] on a TRACERLab module and the use of the radiotracer to evaluate the beta cell mass in normoglycemic minipigs using PET imaging. Methods Methods: Fluorine-18 labeling was performed using a TRACERLab FX-FN synthesizer (GEMS). It comprises i) Reaction of K[18F]F-Kryptofix®222 with the corresponding tosylate (4.0 mg), 160°C / 5 min, in DMSO (0.7 mL), ii) SepPak®Plus Alumina N cartridge and HPLC purification (Zorbax® SB-C-18) and iii) SepPak® cartridge-based reformulation. PET imaging (Dynamic acquisition, 60 min, pancreas placed in the center of the field of view, HR+ (Siemens)) was carried out on normoglycemic anesthetized Göttingen minipigs (inj. dose: 0.8 ± 0.2 MBq/kg). Arterial input function was measured and corrected from metabolites. Images were reconstructed using OSEM 2D and pancreatic uptake of the tracer, decay-corrected, was expressed as Standardized Uptake Value (SUV). Results [18F]FP-DTBZ (13-15 GBq batches, 35-40% ndc yields, 37-111 GBq/µmol) was routinely synthesized within 50-55 min from a 37 GBq [18F]fluoride batch. [18F]FP-DTBZ uptake in the pancreas was rapid with a maximum reached at 5 min post-injection. The mean SUV uptake in the pancreas at 60 min was 4.2 ± 0.9 with high differences between pigs, ranging from 6.2 to 3.0. Test-retest PET imaging provided similar SUV values. Conclusions The preparation of [18F]FP-DTBZ has been implemented on a TRACERLab FX-FN synthesizer. This radiotracer has been used to image the pancreas in normoglycemic Göttingen minipigs and will now be used in a model of diabetes. Acknowledgements IMIDIA (grant agreement n° 155005). References [1] Goswami R. et al (2006), Nucl. Med. Biol., 33, 685-694.

Chemical structure of [18F]FP-DTBZ and its uptake in pig pancreas, MIP images.

J Label Compd Radiopharm 2015: 58: S1- S411

S176: Poster

21st International Symposium on Radiopharmaceutical Sciences

176 Nucleophilic radiofluorination at the alpha-carbon of an acetamide motif: preparation of a novel fluorine18-labeled analogue of SSR180575 Annelaure Damont1, Fanny Cacheux1, Bertrand Kuhnast1, Frank Marguet2, Frédéric Puech2, Géraldine Pottier1, Raphaël Boisgard1, Frédéric Dollé1 1 CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France, 2Sanofi, Exploratory Research Unit, ChillyMazarin, France Objectives The TSPO 18 kDa, as early marker of neuroinflammation, has driving extensive efforts towards the development of dedicated radiolabeled ligands for PET imaging. The pyridazino-indole acetamide SSR180575 belongs to a promising TSPO ligands family and has already been labeled with carbon-11 [1,2]. Addition of a single fluorine atom at the acetamide position is proposed as the simplest way for developing a fluorine-18 counterpart dedicated to in vivo PET imaging. Methods Fluoro-SSR180575 (1) and its tosyloxy analogue 3 as precursor for [18F]labeling were both synthesized from SSR180575 in 2 steps via the alpha-hydroxy intermediate 2. Binding affinity of 1 for the TSPO was determined as well as selectivity versus the CBR. [18F]Fluoro-SSR180575 ([18F]-1) was prepared using standard labeling conditions on a TRACERLab FX-N Pro synthesizer. Preliminary in vivo imaging was performed using Inveon PET or PET/CT (Siemens) systems on the AMPA rat model of neuroinflammation for comparison with [11C]SSR180575 and [11C]PK11195. Results Fluoro-SSR180575 (1) and its tosyloxy analogue 3 were obtained in 29 % and 10 % yields, respectively. 1 exhibited a good affinity for the TSPO (Ki = 1.7 nM) and selectivity was retained (Ki CBR > 1 mM). After radiolabeling, up to 2.0 GBq (7 % ndc RCY) of ready-to-inject [18F]-1 were obtained in 55-60 min, starting from a 30 GBq [18F]fluoride batch. SRA ranged from 40 to 80 GBq/µmol. In vivo, [18F]-1 showed a high contrast (3.08 ± 0.41) between the lesioned area and the corresponding area in the intact contralateral hemisphere, 60 min p.i. Conclusions Fluoro-SSR180575 (1) and its tosyloxy derivative were synthesized in a straightforward manner. Labeling with fluorine-18 was rather efficient and represents an original introduction of fluorine-18 at the alpha position of an acetamide motif. MicroPET studies demonstrate the in vivo potential of this radiotracer to image neuroinflammation. Acknowledgements INMiND (HEALTH-F2-2011-278850) References [1] Chauveau F. et al. (2008), Eur. J. Nucl. Med. Mol. Imag., 35, 2304; [2] Thominiaux C. et al. (2010), J. Label. Compds Radiopharm., 53, 767.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S177

177 Development of High Affinity Ghrelin (1-8) Analogues for [18F] Imaging of GHSR-1a Carlie Charlton3, Jin Qiang Hou1, Rebecca McGirr2, Savita Dhanvantari3, 2, Ting-Yim Lee3, 2, Michael Kovacs3, 2, Leonard Luyt3, 1 1 London Regional Cancer Program, London, Ontario, Canada, 2Lawson Health Research Institute, London, Ontario, Canada, 3The University of Western Ontario, London, Ontario, Canada Objectives Ghrelin is a 28 amino acid peptide ligand for the growth hormone secretagogue receptor-1a (GHSR1a). GHSR-1a is known to be differentially expressed in prostate cancer. We have previously designed a ghrelin(119) analogue which binds to GHSR-1a and distinguishes between healthy, benign and cancerous prostate tissue ex vivo [1, 2, 3]. The objective is to design a focused library of ghrelin(1-8) analogues bearing unnatural modifications in order to obtain high affinity ligands for GHSR-1a and increased in vivo stability. These analogues contain [19F] small molecule side-chain modifications at position-3 to permit further development as PET imaging agents for GHSR-1a. Methods Ghrelin(1-8) analogues were synthesized by solid phase peptide synthesis. Diaminopropanoic acid-3 was functionalized with [19F] small molecules using an orthogonal protecting group. IC50 values were obtained using 125 I-ghrelin(1-28) (PerkinElmer) and the various analogues with HEK293/GHSR-1a cells. 6-Trimethylamino-2ethylnaphthoate was reacted with dry [18F-] at 120°C in DMSO. Tetrabutylammonium hydroxide (1 M) was added for ester hydrolysis followed by synthesis of N-hydroxysuccinimide ester using TSTU. The 2-N-succinimidyl-6[18F] fluoronaphthoate (SFN) prosthetic group was purified by RP-HPLC. The [18F] SFN was coupled to ghrelin(18) analogues at the position-3 free amine. Results Modifications of ghrelin(1-8) at positions 1, 3, 4 and 8 have lead to a library of ghrelin(1-8) analogues. IC50 values of these analogues have ranged from 1086 nM to 3.5 nM. Lead analogues have IC50 values of equal or greater affinity than human ghrelin (IC50= 8 nM). Synthesis of [18F] SFN was carried out manually affording a 23% (dcy) after RP-HPLC purification. Optimization of coupling procedures is underway and will be followed by in vivo studies in HT1080/GHSR-1a xenografts. Conclusions Ghrelin has been truncated from 28 amino acids to 8 N-terminal amino acids while maintaining equal or greater affinity to GHSR-1a. [18F] SFN has been successfully synthesized and will allow for ghrelin(1-8) PET imaging agents to be developed targeting GHSR-1a. Acknowledgements Prostate Cancer Canada, Canadian Institutes of Health Research References [1] Chen, L. et al., The Prostate, 2012, 72, 825-833. [2] McGirr, R., et al., Regulatory Peptides 2011, 172, 69-76. [3] Rosita, D, et al., J. Med. Chem., 2009, 52, 2196-2203.

J Label Compd Radiopharm 2015: 58: S1- S411

S178: Poster

21st International Symposium on Radiopharmaceutical Sciences

178 [18F]THK-523, [18F]T808 and [18F]T807: Radiosynthesis on a TRACERLab FX N Pro synthesizer Yann Bramoulle1, Nadja Van-Camp1, Karine Cambon1, Alexis-Pierre Bemelmans1, Philippe Hantraye1, Frédéric Dollé2 1 CEA, I2BM, Molecular Imaging Research Center, Fontenay-aux-Roses, France, 2CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France Objectives In contrast to Aβ deposition, neurofibrillary tangles (NFT) composed of hyperphosphorylated Tau correlate with cognition deficits at different stages of Alzheimer's disease. Recently, different Tau specific radioligands have been developed: [18F]THK-523 [1], [18F]T808 [2,3], [18F]T807 [4,5]. In order to compare the in vivo imaging properties of these radiotracers in dedicated animal models of taupathies, we decided to standardize as much as possible their radiosynthesis and to implement them on a TRACERLabTM FX N Pro synthesizer. Methods The FX N Pro synthesizer was used without any modification. Once prepared, the K[18F]FKryptofix®222 complex was reacted in DMSO with the protection-free appropriate precursor using dedicated conditions: [18F]THK-523: the tosyloxy derivative (7 mg), 110°C / 10 min; [18F]T808: the tosyloxy derivative (2 mg), 120°C / 5 min; [18F]T807: the nitro-derivative (0.7 mg), 130°C / 15 min. [18F]THK-523 and [18F]T807 were pre-purified on a SepPak®Plus Alumina N and C-18 cartridge respectively before HPLC purification. Concerning [18F]T808, before the pre-purification on a SepPak®Plus Alumina N cartridge, the reaction mixture was treated with aq. 3N NaOH and then neutralized with aq. 3N HCl. HPLC purifications were all performed on the same XTerra® column using the following conditions: [18F]THK-523: H2O/CH3CN/TFA : 80/20/0.5; [18F]T808: NH4OAc 0.1M pH 10/CH3CN : 60/40; [18F]T807: H2O/EtOH pH 2 : 84/16. The HPLC-collected fractions were then reformulated using a SepPak®Plus C-18 cartridge. Results Starting from 55 GBq of [18F]fluoride, 4.1 to 18.5 GBq (6-33% non-decay corrected yields) of [18F]THK523, [18F]T808 or [18F]T807, > 95% radiochemically pure and ready-to-inject (SRA 50-350 GBq/µmol at EOB), were obtained. The overall preparation times were 60 min and 75 min for [18F]THK-523 / [18F]T808 and [18F]T807, respectively. Conclusions The radiosynthesis of [18F]THK-523, [18F]T808 and [18F]T807 have been successfully implemented on a TRACERLabTM FX N Pro synthesizer. Acknowledgements References [1] Fodero-Tavoletti et al. (2011), Brain, 134, 1089; [2] Gao et al. (2014), Bioorg. Med. Chem. Lett., 24, 254; [3] Chien et al. (2014), J. Alzheimer’s Dis., 38, 171; [4] Shoup et al. (2013), J. label. Compds Radiopharm., 56, 736; [5] Chien et al. (2013), J. Alzheimer’s Dis., 34, 457.

Chemical structures of [18F]THK-523, [18F]T808 and [18F]T807

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S179

179 Fully automated radiosynthesis of 1-[18F]FETrp, a potential substrate for indoleamine 2,3-dioxygenase PET imaging Jean Henrottin1, 3, Christian Lemaire1, Astrid Zervosen1, Dominique Egrise2, Alain Plenevaux1, Serge Goldman2, André Luxen1 1 Cyclotron Research Center, Université de Liège (ULg), Liège, Belgium, 2Erasme Hospital, Université libre de Bruxelles (ULB), Brussels, Belgium, 3Chemistry Department, Université de Liège (ULg), Liège, Belgium Objectives Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial step in the catabolism of L-tryptophan along the kynurenine pathway and exerts immunosuppressive properties in inflammatory and tumoral tissues by blocking locally T-lymphocytes proliferation.1 Furthermore, 1-[19F]FETrp was reported to be a good and specific substrate of IDO.2 Thus, 1-(2-[18F]-fluoroethyl)-tryptophan (1-[18F]FETrp, 5)3 could be a highly desirable PET imaging radiotracer for the clinical detection of IDO expressing cells. Herein, we report a fully automated radiosynthesis of 5 and these in vitro and in vivo uptakes. Methods The one-pot two-steps fully automated n.c.a. radiosynthesis of DL-[18F]5, including HPLC purification and formulation on the same cassette, was carried out in a FASTlabTM synthesizer (GE Healthcare). Chiral separation and formulation of both isomers were also developed. In vitro and in vivo cellular uptakes were then investigated with these potential PET tracers of IDO. Results The tosylate precursor was labelled (5min, 140°C, radiochemical yield (RCY): 57% (d.c.), n=3). After hydrolysis (HBr 6N), HPLC purification and formulation, DL-[18F]5 was obtained (80min; global RCY: 30±4% (d.c., n=7); purity >98%; specific activity: 600±180 GBq/µmol (n=5)). The subsequent chiral separation of the enantiomers was performed by HPLC and both were formulated on a second FASTlabTM cassette. In vitro enzymatic assays reveal that L-[18F]5 is better substrate than D-[18F]5. In vitro cellular assays show a negligible uptake of D-[18F]5 and an uptake of the racemate varying from 30% to 50% of that of L-[18F]5. A high specificity of L-[18F]5 to IDO as compared to TDO expressing cells is also observed. Finally, our preliminary in vivo assays with mice bearing two different tumors (expressing or not IDO) confirm a higher uptake of L-[18F]5 in IDO expressing cells. Conclusions Herein, a fully automated radiosynthesis of DL-[18F]5 with good RCY and the chiral separation of these enantiomers were developed. In vitro and preliminary in vivo studies (currently under progress) show that L[18F]5 is a good and specific substrate of IDO allowing its potential use as PET radiotracer for cancer imaging. Acknowledgements We gratefully acknowledge the Région Wallonne (Keymarker Project-BioWin) and the Chemistry Department of ULg for the financial support. References [1] C. Uyttenhove, & al. Nat. Med. 2003,9, 1269–1274. [2] J. Henrottin, A. Zervosen, C. Lemaire, F. Sapunaric, S. Laurent, B. Van den Eynde, S. Goldman, A. Luxen. ACS Med. Chem. Lett. (submitted). [3] T. Sun, & al. Appl. Radiat. Isot. 2012,70, 676–680.

J Label Compd Radiopharm 2015: 58: S1- S411

S180: Poster

21st International Symposium on Radiopharmaceutical Sciences

180 The pyrazolo[1,5-a]pyrimidine F-DPA: synthesis, in vitro characterization and radiolabeling with fluorine18 using a nucleophilic approach Annelaure Damont, Fabien Caillé, Vincent Medran Navarrete, Fanny Cacheux, Bertrand Kuhnast, Frédéric Dollé CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France Objectives The TSPO ligand [18F]DPA-714 is today used to image neuroinflammation in vivo with PET [1] despite the in vivo formation of [18F]labeled small metabolites resulting from O-dealkylation [2]. Keeping the DPA-scaffold, a novel derivative, F-DPA (1), has been designed with a fluorine atom directly attached to the phenyl ring. The synthesis, in vitro binding and attempts of [18F]labeling using a nucleophilic approach, are reported herein. Methods F-DPA (1) was prepared in three steps and evaluated in vitro for TSPO affinity and selectivity versus the CBR. Two precursors for [18F]labeling were also designed: a nitro derivative (5) and a trimethylammonium salt (6). As difficulties were expected for [18F]labeling using conventional SNAr reaction, a diaryliodonium salt (7) [3] was also prepared. Radiofluorination was attempted using a TRACERLab FX N Pro synthesizer with [18F]fluoride and varying the counter-ion and/or the anion activation phase transfer catalyst as well as the solvent, temperature and reaction time. Results F-DPA (1) was obtained in 13 % yield and exhibited a good affinity (Ki = 1.7 nM) and selectivity (Ki CBR > 1 mM) for the TSPO. Synthetic intermediates 2, 3 and 4 were obtained in 50 %, 29 % and 32 % yields, respectively. The precursors for radiofluorination 5, 6 and 7 were generated in one step from 2, 3 and 4 respectively, with good yields. So far, the preparation of [18F]-1, using either 5, 6, 7 or 1 as precursor and [18F]fluoride under various reaction conditions, yielded a maximum of 3 % [18F]fluorine incorporation. Conclusions The preparation of [18F]F-DPA ([18F]-1) is rather challenging using [18F]fluoride and not yet reliable enough to proceed with in vivo evaluation. Further optimizations of the radiolabeling process are on-going considering the recent successful results reported in the literature with the use of aryliodonium ylides [4]. Such precursors will be designed in house for the preparation of [18F]-1. Acknowledgements INMiND (HEALTH-F2-2011-278850) References [1] Damont A et al. (2008), J. Label. Compds Radiopharm., 51, 286; [2] Peyronneau M-A et al. (2013), Drug. Metab. Dispos., 41, 122; [3] Pike VW et al. (1995) J. Chem. Soc. Chem. Commun., 2215; [4] Rotstein BH et al. (2014) Nature Commun., 5, 4365.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S181

181 Modified Resin-Supported Silicon-Based Precursors for Kit-Like F-18 Labeling Norio Yasui1, Sung Hoon Kim1, Dong Zhou2, Carmen S. Dence2, John Katzenellenbogen1 1 Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 2 Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri, United States Objectives Because of the high affinity of silicon for fluoride ion, silicon-based methods enable rapid and efficient F-18 fluoride incorporation under mild conditions.1–3 For this purpose, we developed novel class of precursors, silyl esters, which not only can produce F-18 fluorosilanes quickly using F-18 water directly from cyclotron, but can also be attached to a resin from which the F-18 labeled product is released after fluoride incorporation (label/release), unreacted, resin-retained precursor being eliminated simply by filtration. The challenge, however, is to avoid competitive hydrolysis of the resin-bound silyl ester, which would release the undesired silyl alcohol together with the F-18 labeled product. Herein, we report a modification of the resin-supported silyl ester which reduces formation of the silyl alcohol byproduct. Methods To avoid hydrolysis of resin-bound silyl ester, the ester carbonyl was sterically hindered by α,α-dimethyl substitution to retard nucleophilic attack by water but not block fluoride attack at silicon. The α-geminal dimethyl carboxylate linker, derived from 2,2-dimethyl-3-chloropropanoic acid, was substituted with N-Boc-2-amino ethanethiol, which was extended through an amide bond to an azide terminus for click loading onto resin, and the di-t-butyl silyl moiety with NHS ester was attached to the carboxylic acid from the corresponding chlorosilane. The intact system was clicked onto a dibenzo-aza-cyclooctyne (DBCO) modified resin. Results F-18 water, straight from cyclotron and used without drying, was mixed with DMSO (1/5) and added to the resin. The desired [18F]SiFB was released from the resin in 66% RCY at 50oC within 10 minutes. Even though the F-18 incorporation was somewhat slower than with resin without the geminal dimethyl substitution, [18F]SiFB was generated in practical yield without NHS ester hydrolysis. The amount silyl alcohol was significantly reduced,enabling much easier byproduct separation. Conclusions Introduction of a-geminal dimethyl groups onto the resin-bound silicon precursor reduced formation of undesired silyl alcohol while maintaining reactivity towards fluoride ion, enabling the development of a kit-like solid-supported F-18 fluorosilane labeling method. Our current goal is to avoid hydrolysis completely while maintaining the efficiency of F-18 fluoride incorporation by further modification of the silyl ester moiety. Acknowledgements Supported by DOE (DOE DE-SC0005434) and (DE-FG02-08ER64671). References [1] Kostikov, AP., et al. (2012) Bioconjug. Chem., 23, 106. [2] Mu, L., et al. (2008) Angew. Chem. Int. Ed. Engl, 47, 4922. [3] Liu, Z., et al. (2014) Angew. Chemie Int. Ed., 55, 1499

J Label Compd Radiopharm 2015: 58: S1- S411

S182: Poster

21st International Symposium on Radiopharmaceutical Sciences

182 Synthesis and evaluation of novel 18F-labelled P2X7 receptor antagonists Enrico R. Fantoni1, Bobbi Fleiss1, Simon Lovestone2, Antony Gee1 1 Department of Imaging Sciences and Biomedical Engineering, King's College London, London, United Kingdom, 2 Department of Psychiatry, Univeristy of Oxford, Oxford, United Kingdom Objectives P2X7 receptor expression is required to elicit neuroinflammation. Given the receptor’s prevalence in M2 anti-inflammatory activated microglia, we proposed that a P2X7 radiotracer could act as imaging agent for M2type neuroinflammation. Starting from the known antagonist A-804598, we developed and evaluated in vitro a library of novel 18F-labelled PET ligands targeting P2X7. Methods The radiotracers were synthesised via a new 4-step procedure and radiofluorinated by copper-catalysed alkyne-azide cycloadditions. We preformed radiotracer incubations with live BV-2 microglia of high vs. low P2X7 expression (M2 and M1 phenotype, respectively) in the presence or absence of Brilliant Blue G (BBG) blockade. Mouse brains were fixed in cold acetone, cut into coronal sections and mounted onto microscope slides. The slices were later incubated with the radiotracers in the presence or absence of BBG. Results We synthesised a library of P2X7 radiotracers with 9-33% DC RCY and >99% RCP (Figure 1). A preliminary tracer evaluation in BV-2 microglia delivered encouraging results. This prompted us to investigate the in vitro uptake in hypoxic ischemic mouse brain tissues showing heterogeneous tracer localisation (Figure 2). Conclusions The work carried out forms the basis for the development of a series of novel PET tracers targeting P2X7. Here, we report the synthesis of a small library of 18F tracers and their preliminary in vitro evaluation in cell and mouse tissue models of neuroinflammation. Acknowledgements This study represents independent research funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at the South London and Maudsley and the Guy's and St Thomas' NHS Foundation Trusts and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. References Donnelly-Roberts, D.L., et al, (2009) Neuropharmacology, 56, 223-9. Able, S.L., et al, (2011) Br. J. Pharmacol., 162, 405-14. Glaser, M. et al, (2007) Bioconjugate Chem., 18, 989-993.

Synthetic route to the library of P2X7 radiotracers

Tissue autoradiography with 0.2 nM of P2X7 tracer [18F]AFA. A: Co-incubation with 1mM BBG; B: Unblocked

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S183

183 Improving the SRA of [18F]-tracers with easy-to-implement solutions: the experience at the Service Hospitalier Frédéric Joliot Stéphane Le Helleix, Fabien Caillé, Daniel Gouel, Stéphane Demphel, Christophe Lechêne, Tony Catarina, Frédéric Dollé, Bertrand Kuhnast I2BM, Service Hospitalier Frédéric Joliot, CEA, Orsay, France Objectives Specific Radioactivity (SRA) is important in the development of radiotracers labelled with fluorine-18. Even if non-carrier-added produced, the theoretical SRA (63 TBq/µmol) is never reached and practical values are around 0.10/0.25 TBq/µmol. Based on the preparation of [19/18F]DPA-714, we investigated parameters influencing the SRA focusing on the target-holder and the Teflon® lines. Easy-to-implement solutions were deducted. Methods [19F]DPA-714 was synthesized with a TRACERLab FxFN, isolated and quantified. The conditions are : #1 and #2)HPLC-grade water or H2[18O]O, respectively, directly introduced in the H2[18O]O-recovery vial, #3)H2[18O]O transferred from the target-holder to the synthesizer via Teflon® lines, #4)H2[18O]O circulated in the target-holder, and #5)H2[18O]O circulated in the target-holder and exit. [18F]DPA-714 was synthesized according to [1] in 3 conditions: #6) without rinsing lines, #7)with a partial rinsing and #8)with a full rinsing of the target-holder and lines. Results All syntheses were conducted with the same synthesizer, reagents and cartridges so that the basal contribution in [19F]fluoride is considered constant and to be <10 nmol of [19F]DPA-714 (Tab1,#1,#2). The contribution of the target-holder and exit do not significantly increase the quantity of [19F]DPA-714 (Tab1,#4,#5). In the case of a standard synthesis (Tab1,#3) (circulation of H2[18O]O through the target-holder and lines) the quantity [19F]DPA-714 increases by a factor 100 demonstrating the high contribution of the lines. This result was expected but not in such an extent. The evolution of the SRA of [18F]DPA-714 (Tab2) confirms the observed trends in cold experiments. It results from a partial rinsing (Tab2,#7) a doubling of the SRA and an increase by a factor 5 (Tab2,#8) in the case of a full rinsing. The latter is consistent with published data (Tab2,#9). Conclusions A simple rinsing of the target-holder and Teflon® lines allows for maintaining elevated SRA. Acknowledgements References [1] Kuhnast B, et al (2012) Appl Radiat Isot, 70, 489-497.

J Label Compd Radiopharm 2015: 58: S1- S411

S184: Poster

21st International Symposium on Radiopharmaceutical Sciences

184 Customization of the TRACERLab FxN Pro for the simultaneous double synthesis of [18F]-labelled radiotracers Stéphane Demphel, Annelaure Damont, Frédéric Dollé, Bertrand Kuhnast I2BM, Service Hospitalier Frédéric Joliot, CEA, Orsay, France Objectives Medicinal chemistry generates large series of compounds for a dedicated target, for which their affinity and selectivity are evaluated in vitro. For the translation to in vivo imaging, the radiolabelling of only the best candidates is not always sufficient. Today, commercially available synthesizers are dedicated to the production of one radiotracer per run, which is not optimized for “medicinal radiochemistry”. Based on the preparation of [18F]DPA-714 and [18F]CfO-DPA-714 [1,2], two radiotracers targeting the Translocator Protein 18 kDa (TSPO), a TRACERLab FxN Pro (GEMS) synthesizer was customized to allow for the simultaneous synthesis of these two radiotracers, their purification and final formulation. Methods The TRACERLab FxN Pro synthesizer displays 14 vials to store the reagents. Vials 1 to 6 and 12 to 14 are used in nominal mode. Vials 7 to 9 are derivatized to be used for the formulation of the second radiotracer and 10, 11 are used to refill the dilution flask, in which radiotracers are collected from HPLC. After a classical radiosynthesis involving a mixture of both precursors (Tos-DPA-714, 3 mg and Tos-CfO-DPA-714, 4 mg) in DMSO (1 mL) reacted with the K[18F]K-K222 complex at 110°C for 10 min, the reaction mixture is purified by HPLC. [18F]DPA-714 is collected, classically formulated via a C18-based SPE cartridge and extracted from the hot cell as an ethanolic solution. Then the HPLC pump is stopped. The C18 cartridge is flushed with ethanol (5 mL, Vial 14) that is eliminated and reconditioned with water (10 mL, V7). The dilution flask is refilled with water (10 mL, V10). The HPLC pump is restarted and [18F]CfO-DPA-714 is collected and formulated with water (10 mL) and ethanol (2 mL) stored in vials 8 and 9 respectively. [18F]DPA-714 and [18F]CfO-DPA-714 are subjected to radioanalytical HPLC quality control. Results 1.1 GBq of [18F]DPA-714 and 0.37 GBq of [18F]CfO-DPA-714 (EOB) can be produced. Both radiotracers display chemical and racdiochemical purities higher than 95%. Specific radioactivities are between 90 and 185 GBq/µmol. Conclusions The TRACERLab FxN Pro can be customized for the double simultaneous synthesis of fluorine-18labelled radiotracers. Acknowledgements INMiND program (HEALTH-F2-2011-278850) References [1] Kuhnast et al. (2012) Appl Radiat Isot, 70, 489. [2] Damont et al. (2011) J Label Compd Radiopharm, 54, S461.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S185

185 The presence of oxygen stabilizes [F-18]FLT against radiolytic degradation Joseph Walsh2, Fanrong Mu1 1 PETNET, 6140 Bristol Parkway, California, United States, 2Eng. and Dev., PETNET, Culver City, California, United States Objectives To determine how the stability of [F-101718]FLT is impacted in the both the presence and absence of air Methods [F-18]FLT, prepared in 8% ethanol:92% phosphate buffer, was synthesized in high strength (mCi/mL) using an Explora RN synthesis module. The subsequent [F-18]FLT doses were placed in vials or syringes under an atmosphere of argon, air or devoid of any headspace. After aging, the radiochemical purity of [F-18]FLT was measured by radioHPLC. Peroxide strips were used to measure peroxide levels in sample solutions. Results At concentrations of greater than 50 mCi/mL, [F-18]FLT was stabilized against radiolytic decomposition through the introduction of air (~20% oxygen). Conversely, samples of [F-18] sparged with argon or devoid of headspace underwent facile degradation. The degradation products were predominantly faster eluting on radioHPLC than the parent compound. In air-stabilized samples, peroxide levels were detected in the range of 0.5 to 2.0 ppm. Unstabilized samples had no detectable peroxides. Radiolytic decomposition of [F-18]FLT was found to be induced or halted by the manipulation of air in the sample. Oxygen is believed to be the stabilizing molecule responsible for slowing the degradation of [F-18]FLT. The upper limit of [F-18]FLT stabilization using air was found to be between 130 and 197 mCi/mL in the above mentioned formulation. In comparison, ascorbic acid was also found to stabilize FLT against degradation up to 130 mCi/mL independent of the presence of oxygen in the same ethanol:phosphate formulation. Conclusions Oxygen stabilizes FLT against radiolytic degradation in buffered aqueous ethanol solutions. Removal of oxygen accelerates the decomposition of FLT. The presence of oxygen in stabilized samples leads to the increased presence of peroxide. Oxygen may stabilize other radiohphamaceuticals against degradation, but further exploration of this concept is needed. Acknowledgements The authors would like to also thank Vani Mocharla and Umesh Gangadharmath for their contributions to this project. References

J Label Compd Radiopharm 2015: 58: S1- S411

S186: Poster

21st International Symposium on Radiopharmaceutical Sciences

186 Regiospecific radiolabeling of Nanofitin on Ni Magnetic Beads with 18F-FBEM and in vivo PET studies Sylvestre Dammicco1, 2, Christian Lemaire1, Mathieu Cinier3, Alain Plenevaux1, André Luxen1 1 Cyclotron Research Center, Liege, Belgium, 2Chemistry Department, ULg, Liege, Belgium, 3Affilogic SAS, Nantes, France Objectives Nanofitins(NFs) are a small, single chain and cysteine-free protein able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (013) and temperature (Tm ~80°C). Their extreme stability, low cost and high tolerability for chemical coupling make NFs an interesting alternative to antibodies. The aim of this study was to develop the first synthesis of a radiolabeled NF since no in vivo biodistribution kinetic studies have been published. Methods A single cysteine has been incorporated in a model NF(H4) and its regioselective radiolabeling has been performed with 18F-4-fluorobenzamido-N-ethylamino-maleimide. The synthesis and purification of 18F-FBEM was completely implemented on a FastLab synthesizer. Histagged Cys-H4 dimer were trapped on Ni magnetic beads. After reduction of the disulfide bonds with TCEP and washing steps, 18F-FBEM was dissolved in PBS and added to the beads. After stirring for 20min, the unreacted 18F-FBEM was removed and washing steps were performed. In order to elute the radiolabeled 18F-FBEM-Cys-H4, imidazole buffer was added to the beads. After dilution with saline, the solution was ready for injection. In vivo biodistribution studies were assessed via 2h whole-body dynamic imaging of B6 male mice. Relative tracer retention of excised tissues was quantified. Results The RCY (DC) of 18F-FBEM-Cys-H4 was 53.8±7.3% based on starting 18F-FBEM. The radiochemical purity was >95% and the average specific activity was 60MBq/nmol. Biodistribution studies of H4 showed the most prominent uptake in the kidneys and liver revealing a good safety profile. Conclusions We successfully radiolabeled a Cys-tagged Nanofitin model with 18F-FBEM synthesized with a FastLab at high activity. A simple and new method of biomolecule radiolabeling with high yield on Ni magnetic beads was developed. This efficient procedure can be used for other NF or any histagged biomolecules. PET-CT studies on mice were conduct after injection of 18F-FBEM-Cys-H4 to know the biodistribution of this NF model. Acknowledgements We gratefully acknowledge the Région Wallonne and the Chemistry Department of Liege University for the financial support. We thank also Affilogic for providing NF. References Kiesewetter D.O.et al., Eur J Nucl Med Mol Imaging. 2012, 39(2), 300–308 ; Cheng Z.et al., J Nucl Med. 2012, 53, 1110-1118 ; Garg, S.et al., Bio. Chem. 2009, 20, 583–590

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S187

187 A fully automated, one-step and one Curie level radiosynthesis of nca [18F]UCB-H, a radiotracer for the PET imaging of SV2A Corentin Warnier, Christian Lemaire, Joël Aerts, Alain Plenevaux, André Luxen Cyclotron Research Centre, University of Liege, Liege, Belgium Objectives [18F]UCB-H has recently shown its potential as an efficient PET imaging agent for Synaptic Vesicle Proteins 2A. It is hence a powerful tool for the studying of neurotransmission processes and the yet uncompletely understood mechanisms of epilepsy. In this work, we present a new automated, high-activity, one-step radiosynthesis of [18F]UCB-H from a N-heteroaryl iodonium precursor (see Image 1). The automation on a All-InOne (Trasis S.A., Ans, Belgium) radiosynthesis unit includes the [18F]labeling step, purification and formulation of the final product. Methods The cold N-heteroaryl iodonium precursor 6 was obtained from commercial products with an overall yield of 30% (5 steps). Low activity labeling tests of 6 using the classical Kryptofix/K18F fluorination method yielded [18F]UCB-H with RCY's > 40% (10min, ACN, 125°C). No transition-metal based catalyst or reagent was used at any step of the cold and hot syntheses outlined herein. Results An automation sequence for this one-step radiosynthesis was developed, and high-activity labeling tests were carried out. The fully automated radiosynthesis, from [18F]fluoride trapping on QMA to the obtention of the injectable solution of [18F]UCB-H, lasts one hour and affords a RCY of 30%, non corrected for decay. 690 mCi of injectable [18F]UCB-H were obtained from 2.3 Ci of nca-[18F]fluoride, with a specific activity higher than 15 Ci/µmol. Preliminary data concerning radiochemical and chemical purity, specific activity of the labeled compound and solvent trace analysis in the final formulation clearly suggest that this production method will fulfill the standard GMP requirements. Conclusions In this work, we designed a cheap, efficient and metal-free cold synthesis of a N-heteroaryl iodonium precursor of [18F]UCB-H. We also successfully established highly efficient [18F]labeling conditions and transposed them to a radiosynthesis automation system, allowing for reproducible, high-yield, high-activity and high specific activity routine productions of injectable [18F]UCB-H. Acknowledgements We gratefully acknowledge the FRS-FNRS (FRIA grant) and the Walloon Region (RADIOSYNA project) for funding this work. References [1] Warnock, G. I. et al., J Nucl Med 2014, 55(8), 1336; [2] Bretin, F. et al., EJNMMI research 2013, 3(1), 35; [3] Lynch, B. A. et al. Proc Natl Acad Sci USA 2004, 101(26), 9861; [4] Mercier, J. et al. ChemMedChem 2014, 9(4), 693; [5] Bielawski, M. et al., ChemistryOpen 2014, 3, 19

Schematic representation of the overall synthesis of [18F]UCB-H

J Label Compd Radiopharm 2015: 58: S1- S411

S188: Poster

21st International Symposium on Radiopharmaceutical Sciences

188 Preclinical characterization of [18F]THK-5117 enantiomers as a PET probe for tau pathology in Alzheimer’s disease Tetsuro Tago1, 4, Shozo Furumoto1, 2, Nobuyuki Okamura3, Ryuichi Harada5, Hajime Adachi1, 4, Yoichi Ishikawa1, Kazuhiko Yanai1, 3, Ren Iwata1, Yukitsuka Kudo5 1 Cyclotron and Radioisotope Center, Tohoku University, Sendai, Miyagi, Japan, 2Flontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Miyagi, Japan, 3Tohoku University School of Medicine, Sendai, Miyagi, Japan, 4Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan, 5 Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan Objectives Deposition of senile plaques and neurofibrillary tangles, composed of abnormally aggregated amyloidβ and tau protein, respectively, are pathological hallmarks of Alzheimer’s disease (AD). Recently, we have developed [18F]THK-5117 for PET tau imaging [1]. THK-5117 has a chiral center and so two enantiomers exist. In this study, we evaluated preclinical characteristics of [18F]THK-5117 enantiomers for a further stereochemical optimization of it as a PET tau probe. Methods Binding characteristics of [18F](R)- and [18F](S)-THK-5117 for tau lesions were evaluated by autoradiography (ARG) with AD brain sections. Binding affinity and kinetics for AD brain homogenates were assessed by in vitro binding assays. The pharmacokinetics of 18F-labeled enantiomers was evaluated in normal mice by biodistribution studies. Radiometabolites in the normal mouse brain and blood were analyzed by radioTLC. Results Both enantiomers showed high binding selectivity for tau lesions over amyloid-β in ARG images. Kd values of (R)- and (S)-form for AD brain homogenates were determined to be 26 and 13 nM, respectively. Differences in binding kinetics (association and dissociation) were observed between enantiomers. Although both enantiomers showed comparable brain uptake of about 7 %ID/g at 2 min post-injection (p.i.), the 2 to 60 min ratios of (R)-form and (S)-form were 14 and 59, respectively. This pharmacokinetic difference in the normal mouse brain was assumed to be resulted from the stereospecificity in metabolism and clearance from blood. Furthermore, the radioactivity in the bone of (R)-form at 120 min p.i. (1.8 %ID/g) was much higher than that of (S)-form (0.60 %ID/g). Conclusions This study revealed that the chirality of [18F]THK-5117 affects its binding affinity for AD brain homogenates and pharmacokinetic characteristics in normal mice. Altogether, [18F](S)-THK-5117 had more favorable properties as a PET tau probe than [18F](R)-THK-5117. Acknowledgements Supported by the research fund from GE Healthcare, the SEI (Sumitomo Electric Industries, Ltd.) Group CSR Foundation, the Research Grant Program of the NEDO (09E51025a), JSPS KAKENHI (25293259), and Research Grants from the Ministry of Health, Labor, and Welfare of Japan. References [1]Okamura N, et al. (2013) J Nucl Med, 54, 1420-7.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S189

189 Radiosynthesis and in vitro evaluation of N-aryl-18F-fluorodeoxyglycoylamines as prodrugs for imaging the acidic interstitial microenvironment Robert R. Flavell, Charles Truillet, Tanushree Ganguly, Melanie Regan, Joseph Blecha, Michael Evans, Henry F. Van Brocklin, David Wilson Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, California, United States Objectives Solid tumors have a mildly acidic interstitial pH that ranges from 6.5 – 7.0. Several techniques have been developed to image this property (1). We reasoned that a pro-drug approach could be used for this purpose, in which 18F-FDG is conjugated with an acid labile protecting group. We selected aniline conjugation for this purpose on the basis of prior reports demonstrating acid-labile N-aryl-glycosylamines (Fig 1A, ref 2). We describe the radiosynthesis of N-aryl-18F-fluorodeoxyglycosylamine prodrugs and in vitro evaluation of uptake in a cell culture model. Methods Radiosynthesis was accomplished from 18F-FDG by mixing a solution of 500 mM aniline derivative, 500 mM acetic acid in a 1:1 ratio with 18F-FDG solution and reacting at 80° C for 30 minutes (Fig 1B). Uptake of the resulting prodrugs was assessed in a PC3 cells by incubating in RPMI at pH 7.4 or 6.5 and counting the cellassociated activity. Results The stability of N-aryl-18F-fluorodeoxyglycosylamines were investigated in phosphate buffer at pH 6.5 and 7.4 using HPLC, demonstrating increased decomposition to 18F-FDG at mildly acidic pH (Fig 1C). We found that electron donating substituents accelerated the decomposition. In a cell culture assay, we found marked reduction of uptake of the unsubstituted N-aryl-18F-fluorodeoxylgycosylamine 3 at pH 7.4 but not at pH 6.5, in comparison with unmodified 18F-FDG (Fig 1D). Conclusions We have demonstrated the radiosynthesis of several N-aryl-18F-fluorodeoxyglycosylamine pro-drugs, and found that the uptake of these compounds in cells is blocked at neutral pH but not at mildly acidic pH. We anticipate that these compounds will serve as useful pro-drug derivatives for imaging the acidic tumor microenvironment in vivo. Acknowledgements Dr. Flavell acknowledges support from NIH grant 5T32EB001631-10. References 1. Hashim AI et al., NMR Biomed. 2011 2. Bridiau N et al., Tetrahedron. 2007

J Label Compd Radiopharm 2015: 58: S1- S411

S190: Poster

21st International Symposium on Radiopharmaceutical Sciences

190 Iodonium Ylide Mediated Radiofluorination: Expanded Substrate Scope and Validation of a Radiopharmaceutical for Human Use Steven H. Liang, Benjamin H. Rotstein, Nickeisha Stephenson, Lu Wang, Jon Patteson, Lee Collier, Neil Vasdev Nuclear Medicine and Molecular Imaging, MGH/Harvard, Boston, Massachusetts, United States Objectives We have recently reported a radiofluorination method for non-activated arenes using spirocyclic iodonium ylide (SCIDY) precursors.[1] The goal of the present study was to expand the substrate scope of such reactions with new hypervalent iodine (III) ylides to include well-functionalized heterocycles and tertiary amines and demonstrate their suitability for routine clinical research studies. Methods The labeling precursors were prepared in 15-70% yield under ‘one-pot’ conditions using oxone in trifluoroacetic acid/dichloromethane (1:1) solution, followed by ligand exchange with 6,10-dioxaspiro[4.5]decane7,9-dione. Syntheses of select 18F-molecules were carried out in DMF at 80-120 °C and the radiopharmaceutical [18F]FPEB was automated and validated for human use. Results Twenty substrates with focus on nitrogen-containing drug molecules and radiopharmaceuticals were labeled in 15-80% conversions. Representative heterocycles include 4-, 5- and 6-[18F]fluoroindoles (10-36%), 4and 6-[18F]fluoroisoquinolines (73-80%), and a tertiary amine, 4-[18F]fluorobenzyl morpholine (44%). In order to demonstrate suitability of this method for human PET imaging studies, 3-[18F]fluoro-5-[(pyridin-3-yl)ethynyl] benzonitrile ([18F]FPEB; radiotracer for imaging mGluR5 in human) was synthesized in 20% (n = 3) uncorrected radiochemical yields with specific activities of 18 ± 1.4 Ci/μmol (666 ± 51.8 GBq/µmol), which featured a greater than ten-fold increase in the radiochemical yield and a greater than two-fold increase in specific activity than that of literature method [2] (Scheme 1). [18F]FPEB prepared by this method was validated for human use. Conclusions We have demonstrated new applications of SCIDY on fully-functionalized nitrogen-containing aromatics and the suitability of this technology is validated to prepare radiopharmaceuticals, including [18F]FPEB. Acknowledgements References 1. Rotstein et al. Nature Communications, 2014, 5, 4365; 2. Liang et al. Med. Chem. Commun. 2014, 5, 432.

18

F-labeled heterocyclic building blocks, fragments of drugs and radiopharmaceuticals

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S191

191 Structure activity relationships of 18F-labeled 7α-alkyl-estradiol derivatives Jun Toyohara1, Mayumi Okamoto2, Hiromitsu Shibayama3, Kyousuke Naka3, Yuya Kitagawa3, Kiichi Ishiwata1, Isao Shimizu3 1 Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2Research Institute for Science and Engineering, Waseda University, Tokyo, Japan, 3Faculty of Science and Engineering, Waseda University, Tokyo, Japan Objectives Over the past 30 years, several derivatives of 18F-labeled 17β-estradiol (E2) have been synthesized and evaluated [1]. The most successful ligand advanced to date is 16α-[18F]fluoro-17β-estradiol (16α-[18F]FES) [2]. On the other hand, several lines of evidence suggest that C-7α-substituted E2 derivatives were well tolerated by estrogen receptor (ER) [3, 4]. In line with this hypothesis, we have had an interest in the design and synthesis of 18 F-lableled C-7α-substituted E2 as a molecular probe to visualize ER. Previously, we successfully synthesized a 7α-(3-[18F]fluoropropyl) E2 and showed promising results for quantification of ER density in vivo, although extensive metabolism was observed in rodents [5]. Therefore, optimizations of the alkyl side chain lengths are needed to obtain suitable radioligand based on C-7α-substituted E2 pharmacophore. Methods From fluoromethyl (C1) to fluorohexyl (C6) derivatives of 7α-[18F]fluoroestradiol (7α-[18F]FES), except fluoropentyl derivative (C5), which has been synthesized by Frence et al., [3] were synthesized. In vitro binding to ERα isoform and in vivo biodistribution studies in mature female mice were carried out. Results The in vitro IC50 value of 7α-FES was highest in C1 derivative (0.22 nM) and gradually decreased to 17.12 nM (C6) depending on the alkyl side chain lengths. The in vitro IC50 value of C1 derivative was comparable to that of E2 (0.17 nM) and 16α-FES (0.14 nM). C1 derivative showed the highest uptake in ER-rich tissues such as uterus (8.67 ± 1.38 %ID/g at 30 min, n = 4). The uterus uptake was gradually decreased to 2.02 ± 0.49 %ID/g (C6, n = 5) depending on the alkyl chain lengths. The bone uptake, which indicates de-fluorination, was marked in C2 to C4 derivatives (C2: 6.29 ± 1.48 %ID/g at 30 min, n = 4; C3: 8.67 ± 2.53 %ID/g at 30 min, n = 4; C4: 8.31 ± 1.72 %ID/g at 30 min, n = 5). However, C1 and C6 derivatives showed the limited uptake in the bone (1.30 ± 0.20 %ID/g at 30 min, n = 4 and 2.71 ± 0.92 %ID/g at 30 min, n = 5, respectively). C1 derivative showed the same levels of uptake in uterus and bone in comparison with that of 16α-[18F]FES (uterus: 8.87 ± 3.70 %ID/g at 30 min, n = 5; bone: 1.00 ± 0.08 %ID/g at 30 min, n = 5). Conclusions The optimized derivative of 18F-labeled C-7α-alkyl-E2 was 7α-(1-[18F]fluoromethyl) E2. Acknowledgements Supported by Global COE program by MEXT in Japan. References [1] Anstead GM, et al (1997), Steroids, 62, 268-303. [2] Kiesewetter DO, et al (1984), J Nucl Med, 25, 1212-21. [3] French AN, et al (1993), Steroids, 58, 157-69. [4] Okamoto M, et al (2012), Steroids, 77, 845-9. [5] Okamoto M, et al (2013), J Label Cmpd Radiopharm, 56, S395.

J Label Compd Radiopharm 2015: 58: S1- S411

S192: Poster

21st International Symposium on Radiopharmaceutical Sciences

192 Radiosyntheis of [18F]RAGER, the First Small-Molecule PET Radioligand for In Vivo RAGE Quantification Brian P. Cary1, Allen F. Brooks1, Carole Quesada1, Phillip Sherman1, Peter J. Scott1, 2 1 Radiology, University of Michigan, Ann Arbor, Michigan, United States, 2Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, United States Objectives The Receptor for Advanced Glycation End-products (RAGE)1,2 has been implicated in the pathogenesis of Alzheimer's Disease (AD) because it mediates transport of plasma amyloid-β into the brain, and we have confirmed 4x higher levels of RAGE in post-mortem AD brain samples vs. normal controls (unpublished results). Exisitng radioligands for RAGE include a 99mTc antibody3 and 18F-labeled S100A4 protein.4 While these probes have shown some value, their preparation is cumbersome and they are too large to cross the blood-brain barrier (BBB). Our objective is to make in vivo RAGE quantification accessible to neuroscientific investigation with a small molecule PET probe. FPS-ZM1, reported by Deane et al., appeared like an ideal scaffold due to its low nanomolar inhibition of RAGE, reported BBB permeability, and amenability to nucleophilic radiofluorination.5 Methods Precursor 1 and reference standard 2 were synthesized by reductive amination with benzaldehyde and cyclohexylamine followed by acylation with the appropriately substituted benzoyl chloride. Precursor 1 was fluorinated with [18F]KF to yield [18F]RAGER (RAGE Radioligand), and the labeled product was purified by HPLC and reconstituted into ethanolic saline (Scheme 1). Results Reference standard 1 and precursor 2 have been prepared from readily available starting materials in 53% and 47% yield, respectively. Standard radiofluroination conditions yielded [18F]RAGER (15.6 mCi, 1% non decaycorrected yield, >99% radiochemical purity, >2 Ci/μmol specific activity, n = 2) in sufficient quantities for preclinical evaluation using in vitro autoradiography and in vivo rodent/primate microPET imaging. These studies are currently underway and will be reported in due course. Conclusions We have developed a concise synthesis of [18F]RAGER, the first small molecule PET radiotracer for RAGE, and are currently undertaking pre-clinical evaluation of the new radiotracer. Acknowledgements We acknowledge the NIH (T32- EB005172) and the Alzheimer’s Association (NIRP-14305669) for financial support. References [1] Schmidt, A. M., et al (1992) J. Biol. Chem., 267, 14987–14997. [2] Schmidt, A. M.; et al (2000) BBA Mol. Cell. Res., 1498, 99–111. [3] Tekabe, Y., et al (2010) J. Nucl. Med., 51, 92–97. [4] Wolf, S., et al (2011) Amino Acids, 41, 809–820. [5] Deane, R., et al (2012) J. Clin. Invest., 122, 1377–1392.

Scheme 1: Synthesis of Precursor (1), HPLC Reference standard (2), and [18F]RAGER

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S193

193 Evaluating the Metabolism Profile of the Novel AT1R Radioligand, [18F]FPyKYNE-Losartan, in Rat Kidney and Pig Plasma Maryam Hachem2, Tayebeh Hadizad2, Jean DaSilva1, 2 1 Radiologie, radio-oncologie et médecine nucléaire, Université de Montréal, Montréal, Quebec, Canada, 2 University of Ottawa Heart Institute, Ottawa, Ontario, Canada Objectives Angiotensin II Type 1 receptor (AT1R) expression is altered in renal and cardiovascular diseases. Structure-activity relationship studies and recent work reported that large prosthetic groups can be introduced at the imidazole 5-position with minimal changes in binding properties compared to Losartan (1). The novel derivative of the clinically used AT1 antagonist Losartan, [18F]FPyKYNE-Losartan, was produced via click chemistry (Scheme), and exhibited high binding selectivity for renal AT1R over AT2R in vivo in rat kidneys (2). The aim of this work was to determine the relative proportions of unchanged tracer and labeled metabolites in rat kidney and pig plasma using column-switch HPLC. Methods Sprague-Dawley rats were injected iv with 74-148 MBq of [18F]FPyKYNE-Losartan and sacrificed at -5 (control), 5, 10, 20 and 30 min post-injection (n=3 per group). Rat kidneys were homogenized in 80/20 ethanol/water (v/v) and centrifuged at 22,000 rpm for 15 min. The supernatant was evaporated and reconstituted in 1/99 acetonitrile/water (v/v). Three Yorkshire pigs were injected iv with 5 MBq/Kg of [18F]FPyKYNE-Losartan and plasma samples were analyzed at -5 (control), 1, 2, 5, 10, 20, and 40 min post-injection. The following week, the same pigs were injected with a saturating dose of the AT1 blocker Candesartan (10 mg/Kg) 15 min prior to tracer injection. Plasma samples were prepared by blood centrifugation at 4000 rpm for 5 min. Plasma protein binding was disrupted by adding urea. Samples were injected and analysed by column-switch HPLC with coincidence radiation detector. Results Rat kidney samples revealed the presence of hydrophilic labeled metabolite(s), eluting from the capture column, one hydrophobic metabolite (Rt 7.5 min after switch) and unchanged tracer. At 30 min post-injection, 39±20% hydrophilic metabolites, 3±2% hydrophobic metabolite and 58±19% unchanged [18F]FPyKYNE-Losartan (Rt 11 min after switch) were observed in rat kidney. Pig plasma exhibited only the presence of hydrophilic metabolite(s) and unchanged tracer until 20 min post-injection (6±4%). Blocking AT1Rs with Candesartan resulted in a faster metabolism of tracer (1±2% at 20 min). Conclusions [18F]FPyKYNE-Losartan metabolism to mostly hydrophilic labeled compounds in rat kidney, and the absence of labeled hydrophobic metabolites in pig plasma indicate minimal interference of labeled metabolites to AT1Rs, and support its use for renal AT1R evaluation with PET imaging. Acknowledgements We thank the radiochemistry staff, and granting agencies; CIHR (MOP-126079), and OPIC (MRI ORF #RE03-51). References (1) Carini DJ, et al. J Med Chem. 1991;34:2525-47. (2) Arksey N, et al. Bioorg. Med Chem. 2014;22:3931–3937.

J Label Compd Radiopharm 2015: 58: S1- S411

S194: Poster

21st International Symposium on Radiopharmaceutical Sciences

194 The development of new amino acid derivatives using click reaction and simple SPE purification method Cheol-min Yook1, 2, Sang Ju Lee1, Seung Jun Oh1, Hyun-Joon Ha2, Jong Jin Lee1 1 Nuclear medicine, Asan medical center, Seoul, Korea (the Republic of), 2Department of Chemistry, Hankuk University of Foreign Studies, Yongin, Kyunggi-Do, Korea (the Republic of) Objectives [18F]fluorocyclobutyl group is known as metabolically stable prosthetic group [1]. In addition, the unnatural amino acids including cyclobutyl group were studied for cancer imaging, such as prostate cancer and brain tumor, by Goodman group [2]. Cu(I)-catalyzed click reaction is very useful method for radiolabeling under the mild condition and purification without HPLC. Therefore, we developed new amino acid derivatives containing cyclobutane group using click reaction and SPE purification method. Methods [18F]fluoride was trapped on a QMA cartridge and eluted with 100 μL water + 1 mL methanol mixture containing 3.5 mg K2CO3 and 15 mg Kryptofix 2.2.2 into the reaction vessel. This mixture was completely dried under mild stream of nitrogen with acetonitrile (500 μL × 2) at 100oC. 3 mg of 3-ethynylcyclobutyl-4-methyl benzensulfonate precursor dissolved in 500 µL acetonitrile was added and [18F]fluorination was conducted at 120oC for 20 min. After [18F]fluorination, the reaction vial was connected to trapping vial using polyethylene tubing. 100 µL of ethanol was already filled in trapping vial and this vial was soaked in ice/acetone bath. During the distillation, the volatile product (1-ethynyl-3-[18F]fluorocyclobutane) was evaporated with acetonitrile and trapped in the trapping vial. The distilled 1-ethynyl-3-[18F]fluorocyclobutane was added into click reaction vial. And then 0.5 M CuSO4, 1.5 M Na ascorbate, and three kinds of non-protected azido-amino acid ((S)-2-amino-3azidopropanoic acid, (S)-2-amino-4-azidobutanoic acid, (S)-2-amino-5-azido-pentanoic acid) in ethanol were added. Click reaction was conducted at 50oC for 20 min. The reaction mixture was diluted with 0.1 N HCl and passed through Silica and C18 envir. cartridge to remove residual copper and lipophilic organic impurities. Lastly, final product was trapped on MCX cartridge to remove the large amount of organic and eluted with 2 ml of phosphate buffer and ethanol mixture. The [18F]-labeled triazol amino acid derivatives product was analyzed with HPLC. Results 1-ethynyl-3-[18F]fluorocyclobutane was obtained at a 51 ± 8.0% (n = 15) non decay corrected radiochemical yield and the radiochemical purity was 100% without any purification. Click reaction with the three kinds of azido-amino acid showed 39 ± 1.0% , 37 ± 3.6%, and 37 ± 1.3% (n = 3) of radiochemical yields (n.d.c), respectively, and radiochemical purity was 100%. Conclusions We developed three kinds of new [18F]-labeled amino acid derivatives using click reaction and simple SPE purification with high radiochemical yield and purity. Acknowledgements References [1] Dominic F. et al (2013), Bioorg. Med. Chem., 21, 643-652. [2] Yu W. et al (2009), Bioorg. Med. Chem., 17, 1982-1990.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S195

195 [18F]FLT prodrug strategy for in vivo detection of H2O2 using PET Valerie N. Carroll1, Brian W. Michel4, Joseph Blecha1, Melanie Regan1, Henry F. Van Brocklin1, Kayvan Keshari3, David Wilson1, Christopher W. Chang2 1 Department of Radiology and Biomolecular Imaging, University of California San Francisco, San Francisco, California, United States, 2Departments of Chemistry and Molecular and Cell Biology and the Howard Hughes Medical Institute, University of California Berkeley, Berkeley, California, United States, 3Radiology and Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 4Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado, United States Objectives Reactive oxygen species, including hydrogen peroxide (H2O2), have been shown to play an important role in the development and progression of cancer. Recently we have reported a new probe PC-[18F]FLT-1 which is capable of sensing endogenously produced H2O2 [1]. This prodrug strategy utilizes a novel carbonate ester linkage coupling [18F]FLT to a peroxide labile phenyl boronic acid moiety. In an effort to increase the kinetics of peroxide dependent liberation of [18F]FLT we have since extended this chemistry to include a napthyl boronic acid, PC[18F]FLT-2. We hypothesized that increased electron delocalization in the napthyl structure would lead to more rapid cleavage of the boronic acid moiety upon oxidation. Methods [18F]FLT was prepared according to previously reported techniques [2]. PC-[18F]FLT-1 and PC[18F]FLT-2 were then obtained by incubating with imidazole precursor 1 or 2 respectively in the presence of DMAP and TEA. Deprotection with 10% citric acid followed by HPLC purification gave the desired products. In vitro uptake of PC-[18F]FLT-2 was compared to PC-[18F]FLT-1 in UOK 262 renal carcinoma cells with and with out added extracellular H2O2. Results PC-[18F]FLT-2 was isolated in a 16.4% (n=1) radiochemical yield. A modest increase in cell associated activity from 1.45 ± 0.17% (0 μM added H2O2) to 2.24 ± 0.09% (100 μM added H2O2) was observed for PC-[18F]FLT-2. Under the same conditions, a greater than 10 fold increase in cell uptake was observed for PC-[18F]FLT-1 with the addition of 100 μM H2O2 at 1 h incubation. Conclusions Owing to a high uptake in the absence of peroxide, PC-[18F]-FLT-2 did not result in a notable increase in cell associated activity as a result of extra cellular H2O2. However, PC-[18F]FLT-1 continues to show promising characteristics for sensing H2O2 in vitro. Our current efforts focus on extending this technique to in vivo detection of H2O2 including tumor bearing animal models. Acknowledgements This work was supported by NIH R01 CA166766 References [1] Carroll V, et al (2014) Journal of the American Chemical Society, 136, 1742-45 [2] Martin S, et al (2002) Nuclear Medicine and Biology, 29,263-73

a) Synthesis of PC[18F]FLT-1 and PC-[18F]FLT-2. b) Cell uptake in the absence of peroxide. c) Peroxide dependent cell uptake.

J Label Compd Radiopharm 2015: 58: S1- S411

S196: Poster

21st International Symposium on Radiopharmaceutical Sciences

196 Phase-transfer catalytic,nucleophilic, asymmetric synthesis of 6-[18F]-L-DOPA with simplified cleavage of the protective group Victoria Orlovskaja2, Olga Fedorova2, Olga Kuznetsova2, Victor Maleev3, Yuri Belokon3, Arpine Geolchanyan4, Ashot Saghyan4, Linjing Mu1, Roger Schibli1, Simon M. Ametamey1, Raisa Krasikova2 1 BIO, Paul Scherrer Institute, Villigen-PSI, Switzerland, 2N.P. Bechtereva Institute of Human Brain, St. Petersburg, Russian Federation, 3A.N. Nesmeyanov Institute of Organoelement Compounds, Moscow, Russian Federation, 4SPC Armbiotechnology NAS, Yerevan, Armenia Objectives The automation of nucleophilic asymmetric synthesis of 6-[18F]-L-DOPA remains a challenge. Cleavage of the protective groups commonly employed in the synthesis of 6-[18F]-L-DOPA, such as 4,5methylenedioxy or 4,5-bis-methoxy, require harsh reaction conditions (57% aq. HI, 180-200oC, 20 min), which are not compatible with most synthesis modules. We addressed this problem by employing an 18F-alkylated achiral Schiff base NiII complex (See Image 1). The reaction was performed in the presence of (S)-NOBIN as a phasetransfer catalyst in dichloromethane (DCM) [1]. Recently the methoxymethhyl (MOM) group was suggested as protecting group, allowing much milder deprotection conditions (aq. HCl, 100oC, 5 min) [2]. However, this approach resulted in 6-[18F]-L-DOPA with insufficient enantiomeric purity. In the present study we re-evaluated this approach and tested the solvent effect on the enantiomeric purity of the product. Methods 18F-fluoroalkylation agent (II) was prepared in three steps (See Image 1) starting from customsynthesized 4,5-bis(methoxymethyl)-2-nitrobenzaldehyde (I) and purified on a Silica Light SPE cartridge. The alkylation was carried out in DCM, acetone and acetonitrile (MeCN) for 5 min at r.t . After hydrolysis and deprotection of the 18F-labelled complex, 6-[18F]-L-DOPA was purified by HPLC and analyzed on a chiral HPLC column [1]. Results From several solvents investigated, the use of MeCN allowed significantly improve stereoselectivity and led to 6-[18F]-L-DOPA in more than 99.5% enantiomeric purity. In addition, the MOM groups were easily cleaved in MeCN/ HClaq. mixture, consequently avoiding the necessity to remove the solvent before the deprotection. When 18F-alkylation was carried out in DCM, the content of L-isomer varied from 46 to 82%, while in acetone it was about 80%. Conclusions The presented method employing the MOM-protected benzaldehyde (I) as the precursor and MeCN as a solvent during the chiral alkylation step is advantageous for the automated synthesis of 6-[18F]-L-DOPA in high enantiomeric purity. Acknowledgements Supported by RFBR grant 14-03-31492 and SNF grant IZ73ZO_152360/1. References [1] Krasikova R, et al (2004) Nucl. Med. Biol., 31, 597-603; [2] Krasikova R, et al (2014) QJNMMI, 2014, 58, Suppl 1: 63-64.

Image 1

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S197

197 Synthesis of [18F]IDO5L: a novel potential PET probe for imaging of IDO-1 expression Xuan Huang, Haibin Tian Department of Cancer Imaging and Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States Objectives Indoleamine 2, 3-dioxygenase-1 (IDO1) is involved in immune escape of tumor cells and blockage of its activity can help tumor-bearing mice to reject tumors.[1] It is reported that patients with high level of IDO1 expression was correlated with later clinical phases, larger tumors and indicated a worse prognosis in various cancers.[2] We synthesized 18F-IDO5L, a potent IDO1 inhibitor(IC50 = 19 nM, Hela cell assay)[3], as a novel probe for the PET imaging to profile IDO1 expression. Methods 18F-IDO5L was synthesized using 3-chloro-4-[18F]fluoroaniline as intermediate. (Scheme 1) The first step involved the nucleophilic aromatic substitution of triflate precursor 1 by Kryptofix 222/K2CO3 activated [18F] in acetonitrile. After purified by C18 Sep-pak, the 18F-2 was reduced by NaBH4 under the catalysis Pd/C and constant stirring. The excess NaBH4 was quenched by HCl and Pd/C was removed by filteration. After dried at 100 °C under a stream of nitrogen, the residue 18F-3 was re-dissolved in methanol and coupled with the 4-Amino-Nhydroxy-1,2,5-oxadiazole-3-carboximidamidoyl chloride under base conditions. The filtered reaction mixture was then purified by HPLC to afford 18F-IDO5L. Results Typically, starting from 0.34 to 0.74 GBq [18F]fluoride, 35 to 77 MBq of purified [18F]IDO5L could be obtained in ~90 min. The overall three steps decay-corrected radiochemical yield was 18.2±2.1% (n=4) with the radiochemical purity exceeded 98 %. Specific activity was 11 -15 GBq/μmol at EOS. Identity of the [18F]IDO5L was confirmed in HPLC analysis by co-injection with IDO5L. Conclusions We developed a facile radiosynthesis of 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5oxadiazole-3-carboximidamide [18F]IDO5L as a novel IDO1 targeted tracer. PET imaging studies are currently being carried out in various cancer animal models in order to evaluate the in vivo potential of this IDO-1 inhibitor. Acknowledgements This project was supported by the American Cancer Society's Institutional Research Grant ACS-IRG project number 60-16318-99-19. References [1] X. Liu, et al (2010) Blood 115, 3520-30. [2] X.-Q. Liu, et al (2013) Leukemia & Lymphoma 55, 405-14. [3] E. W. Yue (2009) J. Med. Chem. 52, 7364-7.

J Label Compd Radiopharm 2015: 58: S1- S411

S198: Poster

21st International Symposium on Radiopharmaceutical Sciences

198 Synthesis of a New Thiol Reactive Bifunctional Agent [18F]FPyAM using Microfluidic System Murthy R. Akula, David W. Blevins, George W. Kabalka, Dustin Osborne Radiology, The University of Tennessee Medical Center, Knoxville, Tennessee, United States Objectives Sensitive peptides and protiens are radiofluorinated indirectly using bifunctional agents. Notable thiol reactive agents include [18F]FBAM, [18F]FBOM, [18F]FPyME. We report a microfluidic synthesis of a new thiol reactive bifunctional agent, [18F]FPyAM (5), utilizing a novel bicyclic triflate precursor 3. Methods Precursor 3 was synthesized in two steps from 6-bromonicotinaldehyde (2). The radiofluorination of 3 was performed in a micro-reactor of an Advion Microfluidic System. The condensation of aldehyde 4 and the previously known oxamine 11,2 was carried out in a vial using second concentrator module to obtain 5 (Figure). HPLC purification was performed on PerkinElmer 200. An Agilent Binary 1200 HPLC was used to get analytical data. Results [18F]Fluoride (~ 50 mCi) in DMF (400 µL) and DABCO triflate 3 (3.0 mg) in DMF (500 µL) were allowed to react in a micro-reactor (100 µ x 2 m) at 120 oC with a combined flow rate of 100 µL/min. The reactor outlet was immersed in a reaction vial in the second concentrator module containing precursor 1 (5 mg in 0.5 mL of MeOH and 0.5 mL of 2 N HCl). The mixture was heated at 75 oC for 15 min. The product was purified by semipreparative HPLC (Luna C18, 10 µ, 10 x 250 mm, 3 mL/min. A: CH3CN, B: 0.1 M HCOONH4; 0-5 min 40%A ; 5-15 min 40%A-70%A; 15-30 min 70% A. The peak at 25-27 min was collected) followed by C18 Sep-Pak separation to obtain 13-15 mCi of [18F]FPyAM. The radiochemical purity was determined to be ≥98%. [18F]FPyAM (10 mCi in 0.5 mL EtOH) was conjugated with GSH in PBS (pH 7.5) at room temperature for 10 min to obtain 7 mCi of GSH-[18F]FPyAM and the radiochemical purity was found to be ≧95%. Conclusions A new thiol reactive bifunctional agent, [18F]FPyAM was successfully synthesized ( n = 4) and conjugated with glutathione with high radiochemical yield and purity in 50 min. The advantage of DABCO triflate is that it does not produce volatile Me[18F] generally observed when trimethylammonium triflates are used. Acknowledgements We acknowledge the support of this research by the Molecular Imaging and Translational Research Programme. References [1] Brendt M, et al. (2007) Nucl Med Biol, 34, 5-15. [2] Akula M, et al. (2011) J Label Compd Radiopharma, 54, S533

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S199

199 Improved production of [18F]PF-05270430 for clinical imaging of PDE2A in brain Thomas Morley, Cedrik Massif, Caroline Papin, Vincent Carroll, David Alagille, Ronald M. Baldwin, Gilles Tamagnan Molecular NeuroImaging, New Haven, Connecticut, United States Objectives PET imaging of CNS phosphodiesterases (PDEs) has helped illuminate their role in memory and cognitive function.1 PDE type-2A (PDE2A) is highly localized in the limbic brain structures,2 which are associated with emotion, behavior and memory; therapeutic intervention of PDE2A has the potential for relieving the symptoms of cognitive decline. Only recently has a PDE2A-specific radiotracer, [18F]PF-05270430, been described to study this enzyme in vivo.3, 4 However, reported radiolabeling yields were low. To further evaluate [18F]PF05270430 in human, we developed a GMP-compliant radiosynthesis, with an improved yield, for use in clinical PET studies. Methods PF-05270430 and its tosylate precursor were synthesized as described.4 Reaction of the precursor (Figure 1) with [18F]fluoride in the presence of K2CO3 and Krytofix-222 were carried out on a GE TRACERlab® FXF-N synthesizer. Results Using tamyl alcohol as the labeling solvent,4 resulted in low (<2%) radiochemical yields (RCY). However, switching to DMSO gave substantial improvement in the RCY (up to 20%). After HPLC purification (Figure 1) with 55% acetonitrile eluent at pH 2, and formulation into normal saline with ascorbic acid, [18F]PF-05270430 was obtained in a decay corrected yield of 14 ± 3% (n = 10, 60 min production), radiochemical purity (RCP) >99.5% and specific activity of 210 ± 52 GBq/µmol (5.7 ± 1.4 Ci/μmol). RCP remained >99% for 8 h at room temperature. All quality control requirements for radiotracers produced under GMP, including appearance, chemical and radionuclidic identity and purity, pH, Kryptofix content, endotoxin, sterility and residual solvent content were met. Conclusions The production method of [18F]PF-05270430 has been optimized for a commercially available synthesis module, with an improved yield (up to 20%), and under conditions suitable for clinical use. Acknowledgements References [1] J. I. Andres et al, Curr. Top. Med. Chem. 2012; 12, 1224. [2] V. Lakics et al, Neuropharmacol. 2010; 59, 367. [3] L. Gomez et al, Bioorg. Med. Chem. Lett. 2013; 23, 6522. [4] L. Zhang et al, J. Med. Chem. 2013; 56, 4568.

J Label Compd Radiopharm 2015: 58: S1- S411

S200: Poster

21st International Symposium on Radiopharmaceutical Sciences

200 Automated Production of 18F-labeled Acyl Fluorides as 18F-fluorination Synthons Timothy R. DeGrado, Gregory Nathan, Huailei Jiang Mayo Clinic, Rochester, Minnesota, United States Objectives Presence of trace water and metal impurities in preparations of 18F-fluoride compromise radiofluorination efficiencies. Gaseous 18F-acyl fluorides represent a source of anhydrous, reactive 18F-fluoride. The objective of this project was to develop a high-yield, automated production of 18F-acetyl fluoride (18F-AcF) or 18 F-propanoyl fluoride (18F-PrF). Methods An automated module was developed to produce gaseous 18F-acyl fluorides via the reaction of 18Ffluoride with acetic anhydride or propanoyl anhydride for production of 18F-AcF or 18F-PrF, respectively. The module utilizes two sets of disposable columns: 1) An MP-64 columns (130-140 mg) for trapping of 18F-fluoride with recovery of 18O-water and reaction to produce 18F-acyl fluorides, and 2) purification columns of Porapak Q (1 g) and sodium sulfate (1 g). A cylindrical "gun revolver" geometry provides for up to 12 runs from a single setup of the module. Multiple output lines are accommodated. The purified 18F-AcF was trapped in anhydrous polar organic solvents such as acetonitrile, or on solid-phase extraction cartridges such as Oasis WAX, or in a cooled tubing loop at - 40 °C to - 80°C. An acetone rinse cycle was used between runs. Results 18F-AcF was produced in decay-corrected radiochemical yields of 93±5% in 20 min. Further reductions of production time are anticipated. Radiochemical purity was >99% by radio-GC. Radiochemical stability of 18F-AcF was >99% to at least 4 h post-production. 18F-AcF was readily transported in nitrogen through 15 m of 0.8 mm ID polypropylene tubing with low (0.64±0.12%) adsorption to the tubing. Following dissolution of 18F-AcF in acetonitrile containing the phase-transfer catalyst tetraethylammonium bicarbonate and various labeling precursors, both aliphatic and aryl radiofluorinations were achieved in medium to high yields. After measurement of the limit of detection for AcF, we estimated the specific activity to be >1.3 GBq/umol with a starting radioactivity of 1.5 GBq. Conclusions 18F-acyl fluorides represent a new paradigm for preparation and transport of anhydrous, reactive 18Ffluoride as raw material for radiofluorinations. The automated module opens the possibility for production of highly transportable 18F-acyl fluorides near to the cyclotron and highly efficient transport of 18F-fluoride in the gas phase. Furthermore, this overcomes limitations imposed by transport of 18F-fluoride in water and the required maintenance of isotope delivery lines. Acknowledgements The authors acknowledge support from the Dept. of Radiology, Mayo Clinic. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S201

201 Synthesis of PET tracers via SN2 radiofluorination under "minimalist" conditions Aymen M. Omrane, Boris D. Zlatopolskiy, Elizaveta A. Urusova, Raphael Richarz, Philipp Krapf, Bernd Neumaier Institute of Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne, Germany Objectives Recently we developed a novel procedure for the preparation of 18F-labeled arenes under “minimalist” conditions using only onium salts as radiolabeling precursors and [18F]fluoride. Neither azeotropic drying, nor base or any other additives were necessary. The aim of this work was to extend this method to the preparation of radiofluorinated aliphatic compounds via SN2 radiofluorination. Methods [18F]Fluoride was eluted from a QMA cartridge with appropriately protected Me3N+-substituted sulfonate ester precursors of 5-[18F]FDR, 6-[18F]Gal and [18F]FET in MeOH. MeOH was evaporated at 65–70 °C within 2–3 min, MeCN was added and the resulting solutions were heated to give the corresponding 18F-labeled intermediates. The latter was separated from the precursor by SPE and thereafter deprotected under acidic conditions. If necessary radiolabeled products were purified by SPE or HPLC. Conjugation of 5-[18F]FDR with the model compound, methyl 6-aminooxyhexanoate (6-Aohx-OMe), was optimized with respect to reaction conditions and precursor amount. Results [18F]F– was eluted from an anion exchange resin almost quantitatively. Under optimized conditions protected 5-[18F]FDR, 6-[18F]Gal and [18F]FET were prepared from the corresponding sulfonyl precursors in RCCs of >95%, 16% and 71%, respectively. After SPE purification followed by the deprotection of the intermediate with 1 M HCl (110 °C, 10 min) 5-[18F]FDR was obtained in 48% RCY and RCP >98%. No additional purification was needed. The content of D-Rib (60–80 µg) was sufficiently low to allow an efficient conjugation with 6-Aohx-OMe. The corresponding oxime was prepared in RCCs up to 93% and RCPs >96%. N-Boc-[18F]FET-OtBu was quantitatively deprotected with DCE/TFA (10 min; 80 °C) to give radiofluorinated amino acid in RCP >98%. Conclusions SN2 aliphatic radiofluorination under “minimalist” conditions is suitable for fast and simple preparation of 18F-labeled compounds. An important advantage is also the cationic trimethylammonium tag, which often allows an efficient SPE separation from the labeling precursor. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S202: Poster

21st International Symposium on Radiopharmaceutical Sciences

202 Preparation of [18F]fluorodopamine ([18F]FDA), [18F]-m-tyrosine ([18F]FMT) and 6-[18F]fluoro-(3,4dihydroxyphenyl)alanine ([18F]FDOPA) via nickel-mediated fluorination with [18F]fluoride Johannes Zischler, Boris D. Zlatopolskiy, Elizaveta A. Urusova, Bernd Neumaier Institute for Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne, Germany Objectives Recently, a procedure for the fast preparation of radiolabeled arenes via Ni-mediated oxidative fluorination with [18F]fluoride under exceptionally mild conditions was published. The aim of this work was to examine the applicability of the novel radiofluorination method for the synthesis of clinically relevant PET tracers. The following PET probes: 6-[18F]FDA, [18F]F-DOPA and [18F]FMT were chosen as model compounds. These biogenic aromatic amines and amino acids are usually obtained via cumbersome electrophilic radiofluorination or 18 19 F/ F isotopic exchange resulting in low specific activities. Methods The aryl nickel-complexes were radiolabeled via oxidative nucleophilic [18F]fluorination at RT for 10 min using [18F]KF/K2CO3/18-crown-6 and hypervalent iodine oxidant in MeCN. Protected intermediates were treated with 12 m HCl (10 min, 130 °C) to yield the desired tracers which were finally isolated via preparative HPLC. A comparison of c.a. [18F]FDOPA obtained via electrophilic radiofluorination and n.c.a. [18F]FDOPA was carried out in a rat model of hemi-Parkinson's disease. Results Under optimized reaction conditions the protected 18F-labeled compounds were obtained in RCCs of 5– 18%. Subsequent deprotection of the radiolabeled intermediates proceeded almost quantitatively. The one-pot synthesis yielded [18F]FDA and [18F]DOPA in isolated RCYs of 12 and 6%, respectively. Radiochemical purities amounted to >99% and specific activities to 60GBq/µmol ([18F]FDA) and 126 GBq/µmol ([18F]FDOPA). In a rat model of hemi-Parkinson's disease c.a and n.c.a. [18F]FDOPA were almost identical with respect to lesion detection, signal-to-noise ratio and brain distribution. Conclusions Ni-mediated radiofluorination of [18F]FDA, [18F]FDOPA and [18F]FMT demonstrated significant advantages over routinely applied synthesis methods with respect to SA, reaction time and reaction step economy. In the first study of n.c.a vs. c.a [18F]F-DOPA in vivo in rats did not reveal significant advantages of n.c.a vs. c.a. tracer. Acknowledgements References [1] E. Lee et al, J. Am. Chem. Soc. 2012, 134, 17456-17458.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S203

203 Development of peptidic fluorine-18 labeled PET tracers for in vivo determination of tissue transglutaminase activity Berend van der Wildt1, Benjamin Drukarch2, Esther J. Kooijman1, Cornelis A. Jongenelen2, Christian Buechold3, Ralf Pasternack3, Adriaan A. Lammertsma1, Albert D. Windhorst1 1 Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, Netherlands, 2Anatomy and Neurosciences, VU University Medical Center, Amsterdam, Netherlands, 3Zedira GmbH, Darmstadt, Germany Objectives Tissue transglutaminase (tTG) is an enzyme known for its protein crosslinking activity. It is associated with formation of neurotoxic plaques in diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease [1]. Z006 is a tetrapeptidic irreversible inhibitor of tTG [2] with high inhibitory potency and selectivity. The aim of this study was to develop fluorine-18 labeled analogs with the view of generating an in vivo tool to elucidate the role of tTG in the pathogenesis of various diseases. Methods [18F]tTG-036 and [18F]tTG-038 were synthesized in three and four reaction steps (fig. 1), respectively. For both tracers, 20-40 MBq was administered to healthy Wistar rats. Animals were sacrificed, organs were counted for activity and blood plasma was analyzed for metabolites. Metabolite characterisation was carried out by LC-MS/MS analysis of blood plasma. Results [18F]tTG-036 and [18F]tTG-038 were obtained in an average 22 and 13% decay corrected yield in 120 and 150 minutes, respectively, with radiochemical purities >98% and specific activities >100 GBq·µmol-1. Ex vivo biodistribution studies showed no extreme uptake in any peripheral organs and low brain uptake (<0.10% ID/g) for both tracers. Metabolism was fast, both tracers were metabolized completely within 15 minutes p.i. Remarkably, [18F]tTG-038 was converted to just one single non-polar metabolite which remained metabolically stable even at 60 minutes p.i. Following carrier added administration, this metabolite was determined by LC-MS/MS analysis as the [18F]tTG-038 saponified parent. This metabolite is an equipotent tTG inhibitor with an IC50 of 31 ± 4 nM compared to 72 ± 6 nM for parent tTG-038. Conclusions Both [18F]tTG-036 and [18F]tTG-038 were succesfully synthesized in good yields and with high specific activities. Low brain uptake, however, will be a limiting factor for brain PET studies. The [18F]tTG-038 metabolite is a potent inhibitor of tTG and requires further evaluation as tracer. Acknowledgements Research support by ‘AJ Coops Foundation’. Fluorine-18 was donated by BV Cyclotron VU References [1] Wilhelmus MMM, et al. (2008) European Journal of Pharmacology, 585, 464-472. [2] Verhaar R et al. (2011) Neurochemistry International, 58, 785-793.

J Label Compd Radiopharm 2015: 58: S1- S411

S204: Poster

21st International Symposium on Radiopharmaceutical Sciences

204 Targeting lysyl oxidase for molecular imaging in breast cancer Melinda Wuest2, Manuela Kuchar1, Sai Kiran Sharma2, 3, Susan Richter2, Monica Wang2, Larissa Vos2, John R. Mackey2, Frank Wuest2, 3, Reik Löser1 1 Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany, 2 Department of Oncology, University of Alberta, Edmonton, Alberta, Canada, 3Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada Objectives Lysyl oxidase (LOX, EC 1.4.3.13) and its family members LOX-like 1-4 are copper-dependent matrixmodifying enzymes [1]. The expression of LOX is elevated in many human cancers, including breast cancer and correlates with tissue hypoxia. The enzyme plays a critical role in breast cancer metastasis [2]. The goal of the current study was to target LOX with fluorescent and radiolabeled oligopeptides to visualize LOX in preclinical models of breast cancer. Methods mRNA expression of all 5 LOX family members was analyzed by gene expression microarray analysis on samples from 176 breast cancer patients. The peptidic substrate GGGDPKGGGGG was selected to target LOX [3]. The peptide was labeled with either FITC for confocal microscopy experiments or with the positron emitter fluorine-18 for molecular imaging in vivo using positron emission tomography (PET) (Figure 1). The preclinical breast cancer models utilized were the murine breast cancer cell line EMT-6 and xenografts of MCF-7 and MDAMB-231. Results Immunofluorescence with a LOX-specific antibody confirmed that LOX protein expression is enhanced in hypoxic EMT-6 cells. FITC-labeled oligopeptide binds to several cell compartments of EMT6 cells under hypoxic conditions. After injection of 18F-labeled oligopeptide, radioactivity uptake was visible in all three breast cancer models in vivo with SUV5min values of: 0.70±0.07 (n=3) in EMT-6, 0.57±0.01 (n=3) in MCF-7 and 0.68 (n=2) in MDA-MB-231. The following continuous washout of radioactivity led to SUV60min values of: 0.18±0.03 (n=3) in EMT-6, 0.14±0.02 (n=3) in MCF-7 and 0.13 (n=2) in MDA-MB-231. Tumor uptake was reduced by pre-dosing with the irreversible LOX inhibitor BAPN 4 h and 24 h prior to injection of the radiotracer. Conclusions These data support further investigations towards the development of LOX-binding peptides as molecular probes for imaging of LOX expression in breast cancer. Acknowledgements The access to the Alberta Cancer Foundation-supported CBCF Tumor Bank is well appreciated. References [1] Payne SL, et al (2007) J. Cell. Biochem.101, 1338-54. [2] Erler JT, et al (2006) Nature 440, 12226. [3] Nagan N and Kagan HM (1994) J. Biol. Chem. 269, 22366-71.

Figure 1. Peptidic LOX substrate with N-terminal modifications for confocal microscopy (R1 = Fluorescein-5thiourea with a pentanoyl residue as spacer) and PET studies (R2 = 18F-fluorobenzoyl residue), respectively.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S205

205 Synthesis, 18F-labeling and radiopharmacological characterization of a claudin-targeting peptide Reik Löser, Miriam Bader, Manuela Kuchar, Robert Wodtke, Ralf Bergmann, Jens Lenk, Cathleen Haase-Kohn, Johanna Pufe, Jörg Steinbach, Jens Pietzsch Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany Objectives The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumors and represents a promising target for both diagnosis and treatment of solid tumors of epithelial origin [1]. A suitable ligand to address Cld-4 in vivo seems to be the C-terminal fragment of the Clostridium perfringens enterotoxin cCPE(290-319) (1; Figure 1) [2]. Methods 1 and N-terminally modified (fluorobenzoylated and FITC-conjugated) as well as other analogs were synthesized by microwave-assisted solid-phase peptide synthesis (SPPS). Their affinity to a protein construct containing both extracellular loops of Cld-4 was studied by surface plasmon resonance (SPR). Labeling of 1 with fluorine-18 was achieved on solid phase using [18F]SFB and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents [3]. The stability of the resulting radiotracer was evaluated in different physiological media. Its cell binding was investigated using the HT29, A375 and A431 tumor cell lines. The in vivo behavior of 18F-labeled 1 was studied in NMRI nu/nu mice and Wistar rats by dynamic PET imaging and radiometabolite analyses, respectively. Furthermore, the binding of FITC-conjugated 1 was investigated by fluorescence microscopy. Results Among several approaches tried, sequential SPPS using three pseudoproline-dipeptide building blocks revealed as the most efficient one to afford 1 and its derivatives. Their affinities to the Cld-4 mimicking construct are in the low micromolar range. 18F-labeling was most advantageous when [18F]SFB was reacted with resin-bound 1 containing an N-terminal aminohexanoic spacer. The resulting radiotracer was sufficiently stable in cell supernatants and plasma. Its cell binding was time-dependent and higher to the Cld-4-positive A375 and A431 compared to the negative HT29 line. Results of confocal microscopy using FITC-1 and A431 cells are in accordance with these findings. 18F-labeled 1 is subject to substantial liver uptake and rapid metabolic degradation in vivo. Conclusions The synthesis and 18F-labeling of 1 was successfully established. Its binding to Cld-4 in vitro and in cellulo has been demonstrated. Initial radiopharmacological studies suggest the limited suitability of this peptide in its current form to target Cld-4 in vivo. Acknowledgements References [1] Neese A, et al (2012) Arch. Biochem. Biophys. 524, 64–70. [2] Ling J, et al (2008) J. Biol. Chem. 283, 30585–30595. [3] Kuchar M, et al (2012) Amino Acids 43, 1431-1443.

Figure 1. Structure of cCPE(290-319) (1)

J Label Compd Radiopharm 2015: 58: S1- S411

S206: Poster

21st International Symposium on Radiopharmaceutical Sciences

206 New strategy of a two-step radiosynthesis of [18F]fluoropyridine-based maleimide-containing prosthetic groups for labelling of peptides and proteins Melanie Cavani, Dirk Bier, Heinz H. Coenen Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Jülich, Jülich, Germany Objectives The use of thiol-reactive groups allows the introduction of fluorine-18 in a peptide or protein with cysteine residues, and different radiofluorinated maleimide-containing prosthetic groups have been described in the literature. All compounds were, however, prepared by a two- or three-step synthesis with an overall reaction time of at least 70 minutes. The aim of this work was to improve the radiosynthesis of maleimide-containing compounds with reduced reaction steps by using a new synthetic route via protection of the maleimide function. This was first done here with the example of 1-[3-(2-[18F]fluoropyridin-3-oxy)propyl]pyrrol-2,5-dione ([18F]FPyME) which is usually prepared in three steps [1]. Methods The first step for the preparation of the precursor was a Williamson reaction to form the ether from 3hydroxy-2-nitropyridine with 1,3-dibromopropane. In a parallel process maleimide was protected with 2,5dimethylfurane via a Diels-Alder-reaction. These two products were combined by N-alkylation to form the precursor. The radiosynthesis includes the introduction of fluorine-18 by nucleophilic substitution of the nitro group followed by deprotection of the maleimide function (see fig. 1). The reaction steps were optimized with regard to temperature, time and solvents. Results The optimal conditions for the n.c.a. radiofluorination were identified at a temperature of 80 °C in DMSO and a reaction time of 5 minutes, resulting in a radiochemical yield of about 29 ± 3 %. At higher temperatures the deprotection of the precursor and the labelled compound come to the fore and thereby the decomposition of the unprotected maleimide occurs due to the basic reaction conditions. The deprotection step was quantitatively carried out within 15 minutes. [18F]FPyME was isolated by HPLC to provide the pure prosthetic group which was directly used for effective peptide labelling. The overall synthesis time was about 60 minutes and the overall radiochemical yield was about 20 %. Conclusions The described synthetic route provides the possibility to gain a variety of further [18F]fluoropyridinebased maleimide-containing compounds in two steps only. In addition, this method offers to be performed as onepot synthesis. Acknowledgements References [1] de Bruin B. et al. (2006) Bioconjugate Chem., 16, 406-420.

Two-step radiosynthesis of [¹⁸F]FPyME.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S207

207 A new method for the two-step synthesis of n.c.a. 6-[18F]fluorotryptophan Dominique C. Schäfer, Philipp S. Weiss, Johnny Castillo Meleán, Johannes Ermert, Heinz H. Coenen Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Juelich, Jülich, Germany Objectives Objectives: Tryptophan was recently described as a possible radiotracer for tumor diagnostics [1,2]. Furthermore, the α-amino acid is the precursor of serotonin biosynthesis and takes part in neuronal functions which seem to be inhibited by fluorinated tryptophan [3]. Earlier approaches using the Balz-Schiemann reaction yielded [18F]fluorotryptophan in low radiochemical yields (RCY). Newer approaches, such as the isotopic exchange in the carbonyl activated aromatic system with subsequent reductive decarbonylation required three radiosynthetic steps, yielded [18F]fluorotryptophan in carrier-added form. The aim of this work was to develop a two-step synthesis of n.c.a. 6-[18F]fluorotryptophan with high RCY. Methods Methods: The concept of radiosynthesis is illustrated in Figure 1. The precursor 2 for 6[18F]fluorotryptophan was prepared from 6-bromo-indole. It includes the Schöllkopf’s amino acid auxiliary and a boronic ester group at the desired position in the aromatic ring. The radiosynthesis was performed by a novel copper(II) mediated 18F-fluorination [4] and a subsequent hydrolysis as a two-step reaction. Results Results: Preparation of the precursor was accomplished in a linear six-step synthesis with an overall yield of 23 %. The following n.c.a. 18F-fluorination, performed under modified conditions of the [Cu(OTf)2(py)4] mediated n.c.a. 18F-fluorination, gave radiochemical yields of 60-65 %. After separating the product, an acidic hydrolysis was performed with about 20 % RCY. Finally L-6-[18F]fluorotryptophan was purified by HPLC with an overall RCY of about 8 % and a molar activity of about 60 GBq/µmol within 95 min total reaction time. Conclusions Conclusions: The radiosynthesis of n.c.a. L-6-[18F]fluorotryptophan was performed in a two-step synthesis. The radiotracer was selectively labeled in 6-position with a high molar activity. It is now ready for preclinical evaluation studies. Acknowledgements References References: [1] Lin J. et al., (1988) J. Membran Biol., 104, 1-20. [2] Prendergast G.C., (2011) Nature, 478, 192-194. [3] Jacquot C. et al., (1992) Biochem. Pharm., 45, 1049-1056. [4] Gouverneur V. et al., (2014) Angew. Chem., 53, 7751-7755.

Figure 1: Pathway to 6-[18F]fluorotryptophan 3 starting from 6-bromo indole 1 (PG = protecting group).

J Label Compd Radiopharm 2015: 58: S1- S411

S208: Poster

21st International Symposium on Radiopharmaceutical Sciences

208 Radiosynthesis and preclinical evaluation of [18F]6-fluoro-marsanidine – potential α2A-adrenoceptor tracer. Anna Krzyczmonik1, Thomas Keller1, Francisco Lopez-Picon2, Sarita Forsback1, Anna Kirjavainen1, Aleksandra Wasilewska3, Mika Scheinin4, 5, Merja Haaparanta-Solin2, Franciszek Saczewski3, Olof Solin1 1 Radiopharmaceutical Chemistry Laboratory, Turku PET Centre, University of Turku , Turku, Finland, 2 MediCity/PET Preclinical Laboratory, Turku PET Centre, University of Turku, Turku, Finland, 3Department of Chemical Technology of Drugs, Medical University of Gdansk, Gdansk, Poland, 4Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Turku, Finland, 5Unit of Clinical Pharmacology, Turku University Hospital, Turku, Finland Objectives α2-adrenoceptors (AR) mediate many actions of adrenaline and noradrenaline and regulate their release. Brain α2-ARs are potential therapeutic targets in various neuropsychiatric disorders, such as depression, schizophrenia, and neurodegenerative diseases. Of the three α2-AR subtypes, only α2A and α2C are present in the human central nervous system. Recently, 6-fluoro-marsanidine was identified as a potential PET tracer candidate selective for α2A-ARs [1]. Methods Marsanidine was labelled by electrophilic 18F-fluorination. [18F]F2 and [18F]Selectfluor were produced by previously described methods [2,3]. [18F]Selectfluor derived from [18F]F2 was used directly in the labelling of marsanidine, without purification. The biodistribution of [18F]6-fluoro-marsanidine in SD rats and C57 Black mice was investigated in vivo with PET. Binding in the brain was assessed ex vivo by autoradiography. The extent of metabolism in the blood and brain tissue was analysed by TLC. Results The product was obtained with > 99% radiochemical purity, a radiochemical yield of 17% and a SA of 6.3 GBq/μmol (decay-corrected to EOB). Preclinical studies showed uptake of the tracer in the brain and proved that [18F]6-fluoro-marsanidine passes the blood-brain barrier. The parent tracer demonstrated relatively fast metabolism, with the formation of numerous labelled metabolites. Conclusions A method for synthesis of [18F]6-fluoro-marsanidine was developed. The selectivity of the tracer for the α2A-AR will be confirmed by further preclinical studies in mice devoid of α2A-AR. Acknowledgements This project has received funding from the European Union’s 7th Framework Programme for Research, grant number 316882, from National Science Centre (Poland), grant number 2011/01/B/NZ4/00520 and from the Academy of Finland, grant number 266891. References [1] Wasilewska A. et al. (2014) Eur J Med Chem 87:386-97 [2] Bergman J, Solin O. (1997) Nucl Med Biol, 24, 677-833 [3] Teare H. et al. (2010) Angew Chem Int Ed Engl 49:6821

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S209

209 A fluorine-18 labeled prodrug targeting excitatory amino acid transporters: development of nucleophilic and electrophilic radiosynthetic approaches Joseph Blecha1, ahmed Syed2, 3, Shorouk Dannoon1, John Gerdes2, 3, Henry F. Van Brocklin1 1 Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, California, United States, 2Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States, 3Department of Biomedical & Pharmaceutical Sciences, University of Montana, Missoula, Montana, United States Objectives Amyotrophic lateral sclerosis (ALS, Lou Gehrig’s Disease) is a neurodegenerative disorder marked by the progressive loss of motor neurons in the spinal cord with a hallmark decrease in concentration of the excitatory amino acid transporter 2 (EAAT2) in spinal cord, and also motor and sematosenory cortices1; the astrocytic transport protein for clearance of L-glutamate from the synaptic cleft. Based on a series of aspartylamide ligands2 that bind to EAAT2, we selected ligand 1 (Figure 1) to develop as a fluorine-18 prodrug PET imaging agent to quantify EAAT2 changes in ALS. Methods The preparation of radioligand 1 by the two fluorination approaches is shown in Figure 1. A low specific activity (LSA) route was initiated with tributyl tin fluorene precursor 2 for the electrophilic fluorination with 18F-F2 gas. Subsequent deprotection of the CBZ group with concentrated HBr followed by RP-HPLC gave 1 3,4. An alternate nucleophilic approach was undertaken to provide high specific activity (HSA) EAAT2 prodrug 1. Commercially available fluroenone 3 was labeled with [18F]fluoride ion followed by reduction to give the amino[18F]fluoro-fluorene. Coupling of the BOC protected aspartic acid methyl ester followed by TFA deprotection and RP-HPLC purification provided the desired EAAT2 agent. Results The LSA EAAT2 prodrug 1 was prepared in ~ 3 h with an average yield of 3% (decay corrected; n = 18) 3,4 . The HSA radioligand form was prepared in ~ 3 h with an average yield of 7.5% (decay corrected; n = 6). The range of specific activities for the LSA EAAT2 was 0.25- 3 Ci/ mmol and 1000- 1400 Ci/ mmol for HSA EAAT2. A streamlined radiosynthesis following the nucleophilic approach is being developed to automate the EAAT2 tracer preparation. Conclusions Two different fluorine-18 labeling strategies were successfully developed to prepare a tracer to evaluate EAAT2 densities in spinal and cerebral tissues. Acknowledgements The Robert Packard Center for ALS Research, Johns Hopkins University; The ALS Association; and NIH P30 NS055022. References [1] Rothstein JD, et al. (1992) New Eng J Med 326:1464. [2] Greenfield A, et al. (2005) Bioorg Med Chem Lett 15:4985. [3] Gerdes J, et al. (2012) J Nuc Med 53(S1): 1639. [4] Gerdes J, et al. (2012) J Cerebral Blood Flow Metab 32(S1): 26

Figure 1. Synthesis of PET prodrug imaging agent by two pathways.

J Label Compd Radiopharm 2015: 58: S1- S411

S210: Poster

21st International Symposium on Radiopharmaceutical Sciences

210 One-Step Regioselective Diaryl Iodonium Salt Preparation From a Koser Reagent and an Electron Rich Arene Sung Hoon Kim1, Dong Zhou2, Carmen S. Dence2, John Katzenellenbogen1 1 Chemistry, University of Illinois , Urbana, Illinois, United States, 2Mallinckrodt Institute of Radiology, Washington University Medical School, Saint Louis, Missouri, United States Objectives It is a still challenge to F-18 label an electron-rich aromatic ring. Recently developed labeling methodologies often use diaryliodonium salts as precursors, with either copper-mediated or direct substitution with F-18 fluoride ion. Most diaryliodonium salts for F-18 labeling are synthesized by the reaction of a Koser reagent (or other oxidized iodoarene) with a second arene bearing specific functional groups, such as a boronic acid or a trialkyl tin, but it often takes several steps to introduce these functional groups. We have modified a previous method1, 2 to develop a practical way to synthesize many diaryiodonium salts regioselectively in one step, simply by mixing a Koser reagent and an electron-rich arene. Methods 2-Fluoroestradiol, 5-fluorouracil, and 6-fluoro-m-tyrosine are all important PET imaging agents for diagnosing and monitoring cancer and CNS diseases. Despite its promising binding properties for the estrogen receptor, 2-fluoroestradiol in particular has not been studied by PET because of challenges in preparing it in F-18 labeled form. We found that treatment of estradiol-3-methyl ether with the mesitylene Koser reagent in CH2Cl2 at rt afforded the mesitylene-estradiol iodonium salt substituted at only the C2 position in 68-95% yield, remarkably shortening production of this useful diaryliodonium salt. Reaction of 1,3-diprotected uracil with the same Koser reagent also formed its corresponding iodonium salt substituted at only the C5 position in 72% yield, and similarly the protected m-tyrosine precursor produced the corresponding iodonium salt substituted at only the desired C6 position in 68% yield. Results A modification of Peter Scott’s method3 was employed for F-18 labeling. To improve F-18 incorporation, KHCO3 was used to elute F-18. In this way, we obtained a 20% yield of F-18 labeled 2-[18F]fluoroestradiol 3methyl ether (2-[18F]FES-OMe) using 2.2 mg precursor, 1 mg Cu(OTf)2, 0.4 mg KHCO3, and 18-crown-6 after heating for 20 min at 85 oC. Conclusions Electron-rich aryl-mesitylene iodonium salts were synthesized regioselectively in one step and in good to excellent yields by direct electrophilic substitution. This method remarkably simplifies the synthesis and increases the overall yield for preparing the compounds illustrated. A good yield of F-18 labeled of 2-[18F]FESOMe was also obtained using KHCO3 as a base. We are exploring the use of this method to prepare other PET imaging precursors. Acknowledgements Supported by DOE (DOE DE-SC0005434) and (DE-FG02-08ER64671) References [1] Dohi, T., et al. (2010) Tetrahedron, 66, 5775; [2] Merrit, E. A., et al. Angew. Chem. Int. Ed., (2009), 48, 9052; [3] Ichiishi, N., et al. (2014) OL, 16, 3224.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S211

211 [18F]Tau911, a candidate for PET imaging of the THK site in neurofibrillary tangles Lisheng Cai1, Baoxi Qu2, Bryan Hurtle1, Sureshbabu Dadiboyena1, Cheryl Morse1, Ramon Diaz-Arrastia2, Robert Innis1, Victor W. Pike1 1 Molecular Imaging Branch, National Institute of Mental Health, Bethesda, Maryland, United States, 2Center for Neuroscience and Regenerative Medicine, USUHS, Bethesda, Maryland, United States Objectives Through simple chemical approaches, we aimed to discover new ligands for neurofibrillary tangles (NFTs) as potential radioligands for PET imaging in tauopathies. We have previously found two binding sites on NFTs that are possible targets for PET radioligands [1]. They are dubbed the THK site and T807 site after their typical high-affinity ligands, [18F]THK-523 and [18F]T807, respectively. Because NFT and β-amyloid plaques are two hallmarks of Alzheimer’s Disease [2,3], binding affinity plus selectivity for binding to one of the sites on NFT versus the PIB binding site on β-amyloid should be evaluated for candidate PET radioligands. Methods New candidate ligands were generally synthesized by Pd-catalyzed coupling of aryl boronic acids or esters with aryl halides. All synthesized compounds were evaluated with in vitro binding assays using [3H]PIB, [3H]THK-523 and [3H]T807 as radioligands to determine affinity and selectivity for one of the biding sites in NFTs. One ligand, tau 911, was selected as being suitable for labeling with fluorine-18, and for autoradiography to evaluate its potential as a PET radioligand in vivo. Results Binding assays showed that tau911 binds selectively to the THK site with an affinity (16 nM) that is about three-fold higher than that of THK-523 in the same assay. Tau911 was labelled with fluorine-18 in high decaycorrected radiochemical yield (RCY; Figure 1). Autoradiography showed that [18F]tau911 selectively stained NFTs. Conclusions We identified a new naphthyl-pyridinyl scaffold for tau ligands. A radioligand based on this scaffold, [18F]Tau911, has higher binding affinity than the prototypical PET radioligand [18F]THK-523 and is promising for further evaluation. Acknowledgements Support from the IRP of NIH (NIMH), the Henry Jackson Foundation, and the Department of Defense (CNRM), and provision of human brain tissue by Dr. M. Herman (NIMH). References [1] Cai L et al. (2014) Neuroreceptor Mapping Meeting, poster P131. [2] Selkoe DJ (2001) Physiol Rev, 81, 741. [3] Chien DT et al. (2013) J Alzheimer's Dis, 34, 457.

J Label Compd Radiopharm 2015: 58: S1- S411

S212: Poster

21st International Symposium on Radiopharmaceutical Sciences

212 18 F-PARPi-FL as novel potential dual modality PET/Optical probe for PARP1 imaging Giuseppe Carlucci1, Brandon Carney 1, Christian Brand1, Susanne Kossatz1, Christopher Irwin1, Sean Carlin1, Edmund J. Keliher2, Wolfgang A. Weber1, 2, Thomas Reiner1, 2 1 Radiology, Memorial Sloan Kettering Cancer Center, New York, New York, United States, 2Weill Cornell Medical College, New York, New York, United States Objectives PARP1 is a DNA repair enzyme which is overexpressed in several malignancies. We previously described the development of PARPi-FL, a PARP1 targeted fluorescent small molecule which can be used for optical imaging and detection of glioblastoma. In the current study, we present the 18F radiolabeled, while otherwise structurally identical version of PARPi-FL for bimodal PET/optical imaging with the aims of combining the favorable biophysical properties of PARPi-FL with the deep tissue penetration of PET imaging agents. Methods 18F-PARPi-FL was obtained by the replacing of one of the boron-bound 19F with an 18F. The 18F/19F exchange was achieved in the presence of the Lewis acid SnCl4 at 35 °C for 30 min, based on earlier work [1, 2]. PET experiments and biodistribution studies were performed in U87 MG xenograted athymic nude mice. Wholebody images were acquired 30 minutes after injection of ∼200 μCi of 18F-PARPi-FL. Ex-vivo IVIS imaging and autoradiography were performed in U87 MG bearing mice as well as on healthy mice used as negative control. Results 18F-PARPi-FL was obtained with a radiochemical yield ∼30% and a radiochemical purity >98%. After RP-HPLC purification, the optimized specific activity was about ∼0.8 Ci/μmol. In athymic nude U87 MG xenografts (n=4), at 90 min post injection, the uptake of 18F-PARPi-FL was 0.8 ± 0.1 % ID/g. The agent’s uptake was correlated using both PET and optical imaging mdalities. PARP1 accumulation could be efficiently blocked after co-injection (30 min before) of non-labeled PARP1 inhibitor (Olaparib, 50 folds excess, 3.75 μmol). Measured uptake of 18F-PARPi-FL in blocked U87 MG bearing mice was 0.12 ± 0.02 % ID/g. PET imaging showed accumulation in the tumor (1h p.i.), as confirmed by ex-vivo phosphor autoradiography. Epifluorescence imaging showed that the accumulation of PARPi-FL was higher in tumor than and brain tissues. Conclusions This PARP1 dual modality PET/fluorescent imaging probe shows promising results as a bimodal imaging agent in preoperative and intraoperative guided imaging routines. Acknowledgements Supported by the Brain Tumor Center of Memorial Sloan-Kettering Cancer Center (TR), the Imaging and Radiation Sciences Program of MSKCC (TR) and the American Italian Cancer Foundation (GC). References [1] Keliher E.J., et al., 2014, ChemMedChem,. 9(7): p.1368-73 ; [2] Liu S., et al., 2013, Theranostics, 3(3): p. 181-9.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S213

213 Automated synthesis of a 18F-labeled lysine-urea-glutamate for PSMA imaging Vincent R. Bouvet1, 2, Melinda Wuest1, 2, Nancy Janzen3, John Valliant3, 4, Francois Benard5, Frank Wuest1, 2 1 Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Pharmacy, University of Alberta, Edmonton, Alberta, Canada, 3Chemistry, Mc Master University, Hamilton, Ontario, Canada, 4Medical Physics and Applied Radiation Sciences, Mc Master University, Hamilton, Ontario, Canada, 5Radiology, University of British Columbia, Vancouver, British Columbia, Canada Objectives Prostate Cancer (PCa) is the second most lethal cancer in men. An attractive target for molecular imaging of PCa is prostate-specific membrane antigen (PSMA).1 To date, lysine-urea-glutamates (LuG)-containing compound [18F]DCFPyl, a compound developed by Pomper et al. at John Hopkins Medical School, has entered into clinic as a 18F-labeled radiotracer for PSMA imaging.2 The goal of this study is the synthesis and radiopharmacological evaluation of a series of 18F-labeled urea-based compounds for PSMA imaging in PCa. Methods Three different LuG analogs (1-3) were prepared using triphosgene and/or activated carbamate methodologies. PSMA binding potency was performed in a competitive radiometric binding assay. Most potent compound 2 was labeled with 18F, and its pharmacokinetic profile was evaluated using small animal PET imaging. An automated synthesis of compound [18F]2 was further developed using a GE Tracerlab FXFDG automated synthesis unit (ASU). In vivo targeting of PSMA was analyzed with dynamic PET in LNCap and PC3 tumor bearing mice. Results Compounds 1 to 3 were synthesized with good to excellent yields (5 steps: ~40% ). All compounds displayed low nanomolar IC50 values toward PSMA inhibition. Radiolabeled compound [18F]2 was obtained in good radiochemical yields of >50% at high specific radioactivity. Automated synthesis of compound [18F]2 using a GE Tracerlab FXFDG ASU afforded the desired compound in radiochemical yields greater 30% within 60 min, including HPLC purification. In vivo tumor uptake reached SUV60min values of 0.91 in LNCap tumors (PSMA+) vs. 0.06 in muscle tissue. Almost no uptake was detected in PC3 (PSMA-) tumors (SUV60min = 0.11). Uptake of [18F]2 in LNCap tumors was blocked by co-injection of compound 2 (300 µg) resulting in SUV60min of 0.27. Conclusions Synthesis of the radiotracer [18F]2 was accomplished in good radiochemical yield and purity using an ASU. Favorable radiopharmacological profile and automated synthesis of [18F]2 allows for future clinical applications of this 18F-labeled PSMA imaging agent. Acknowledgements Research Support: This work was supported by the Medical Imaging Trial Network of Canada. (MITNEC) References [1] Osborne J.R. et al. 2013, Urologic Oncology, 31, 144. [2] Chen Y. et al. 2011, Clin. Cancer Res., 24, 7645.

J Label Compd Radiopharm 2015: 58: S1- S411

S214: Poster

21st International Symposium on Radiopharmaceutical Sciences

214 Synthesis of N-[4-(2'-[18F]fluoroethyloxybenzoyl)]pyrrolidin-2-one, a Potential New Brain Imaging Agent Murthy R. Akula, David W. Blevins, George W. Kabalka, Dustin Osborne Radiology, University of Tennessee Medical Center, Knoxville, Tennessee, United States Objectives Aniracetam and a slew of other pyrrolidin-2-one based nootropic drugs are derivatives of GABA. These agents are known to restore the cell membrane fluidity1and are used as cognitive enhancers and to treat dementia and dyslexia. Some of these agents are demonstrated to have neuroprotective and anticonvulsant properties2. We wish to report the synthesis of N-[4-(2'-[18F]fluoroethyloxybenzoyl)]pyrrolidin-2-one (1) as a PET tracer for brain imaging. Methods The tosylate precursor 4 was synthesized from N-trimethylsilylpyrrolidin-2-one (2) in three steps [Figure]. N-Benzoylation3 of 2 was carried out by refluxing it with 4-acetoxybenzoyl chloride in CHCl3. Deacetylation using 3 N NaOAc afforded phenol 3 which was alkylated with ethylene ditosylate and CsCO3 in DMF to obtain tosylate 4. Radiofluorination of 4 was performed in an Advion Nanotek System using a vial chemistry. The formation of the prodcut was monitored by Bioscan radio-TLC. The product was purified by semipreparative HPLC using PekinElmer 200 and C18 Sep-Pak. The product purity was assessed by analytical Agilent Binary 1200 radio-HPLC Results Cylcotron produced [18F]fluoride ( ~ 50 mCi) was converted to [18F]F-/K222/K2CO3 using a drying macro. The mixture of isotope (0.5 mL of acetonitrile ) and the tosylate 4 (10 mg in 0.5 mL acetonitrile) was heated at 90 o C. The reaction was monitored by radio-TLC every 5 min. and by 15 min there was no trace of unreacted isotope. The product was purified by semi-preparative HPLC (Luna C18, 10 µ, 10 x 250 mm, 3 mL/min; isocratic 30 % water in CH3CN), The required fraction was collected and passed through a C18 Sep-Pak followed by the elution with Et2O to obtain 17 mCi of N-[4-(2'-[18F]fluoroethyloxybenzoyl)]pyrrolidin-2-one (1). Conclusions We successfully synthesized a new brain PET imaging agent in high radiochemical yield (34 % n.d.c; n=4) and purity (≥97 %). Preliminary studies indicate that there is brain uptake which would warrant futher investigation of this tracer for brain and neurological disorders. Acknowledgements We wish to acknowledge the Molecular Imaging and Translational Research Program and University Health Systems, Knoxville for the support of this research. References [1] Malykh AG et al. (2010) Drugs 70, 287-312. [2] Ince Gunal D et al. (2008) J Clin Pharm Ther. 33, 287-312 [3] Schwarz G et al. (1981) Liebigs Ann. Chem. 1257-1270

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S215

215 Conversion of Pyridine N-Oxides to Trialkylammonium Precursors of 2-Fluoropyridines and Its Application to the Radiosynthesis of [18F]AV-1451 Hui Xiong, Adam Hoye, Kuo-Hsien Fan, Ximin Li, Jennifer Clemens, Carey L. Horchler, Nathaniel Lim, Giorgio Attardo Avid Radiopharmaceuticals, Philadelphia, Pennsylvania, United States Objectives 2-[18F]Fluoropyridines are commonly used motifs in PET imaging agents[1] that are prepared from suitably functionalized pyridine precursors. 2-Trialkylammonium pyridines have previously been radiolabeled with [18F], but these precursors often suffer from poor synthetic accessibility due to broad functional group incompatibility with known methods of their synthesis. To overcome this challenge, we investigated the viability of using pyridine N-oxides for the preparation of 2-trialkylammonium pyridines. To this end, we have demonstrated the scope and limitation of the indirect regiospecific conversion of pyridine N-oxides into 2fluoropyridines via their trialkylammonium salts under mild and metal-free conditions. These stable and isolable ammonium salt intermediates also serve as effective radiofluorination precursors. Methods 2-Trialkylammonium pyridines can be prepared from pyridine N-oxide by treatment with trifluoroacetic anhydride in a suitable solvent (e.g. CH2Cl2) and isolated by trituration; 2-Fluoropyridines can be prepared from the above described 2-trialkylammonium pyridine salts by treatment with fluoride source (e.g. TBAF) in a suitable solvent (e.g. DMF) and isolated by column chromatography. Results This method has been successfully applied to the synthesis of a diverse collection of 2-fluoropyridines, as well as to the radiosynthesis of [18F]AV-1451 (aka [18F]T807), a PET tracer currently under development for imaging tau.[2] It is noteworthy that this method was not applicable to 4-monosubstituted pyridine N-oxides. Conclusions The conversion of pyridine N-oxides to trialkylammonium precursors of 2-fluoropyridines was achieved under mild and metal-free conditions. Of considerable relevance to PET imaging research, this method gives access to both the non-radioactive 19F-fluoride standard and the stable, isolable trialkylammonium salt as the radiolabelling precursor. Finally, the utility of this method was showcased in the successful preparation of [18F]AV-1451. Acknowledgements References [1] Dollé, F. (2005) Curr. Pharm. Design, 11, 3221 [2] Xia, C.-F. et al (2013) Alzheimers Dement. 9, 666

Figure 1. Synthesis of trimethylammonium salts from pyridine N-oxides and their application in [18F] radiofluorination and the synthesis of 2-fluoropyridines.

J Label Compd Radiopharm 2015: 58: S1- S411

S216: Poster

21st International Symposium on Radiopharmaceutical Sciences

216 Improved synthesis and purification of meta-[18F]fluorobenzylguanidine (mFBG) for clinical use Amy L. Vavere1, Bao Hu2, Kiel D. Neumann3, Stephen G. DiMagno2, Scott E. Snyder1 1 Radiological Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, United States, 2Chemistry, University of Nebraska - Lincoln, Lincoln, Nebraska, United States, 3Ground Fluor Pharmaceuticals, Lincoln, Nebraska, United States Objectives Recent work has indicated that meta-[18F]fluorobenzylguanidine (mFBG) possesses advantages, including shorter uptake time and better resolution, over mIBG for the diagnosis and staging of neuroblastoma tumors.1 Current literature methods describe manual production of low-yielding fluorobenzylnitrile followed by reduction, multiple extractions, and gradient HPLC purification. Final overall yields after reformulation were 1011% with a synthesis time of 3 hours.2,3 Our goal for this work was to develop a facile, automated synthesis for clinical production with improved yields while avoiding the need for reformulation of the final product. Methods Using an IBA Synthera® automated synthesizer, [18F]fluoride ion was separated from target [18O]water using QMA resin and elution with a Kryptofix 2.2.2/potassium carbonate solution in acetonitrile and water. The solvent was removed under argon flow with reduced pressure and heating to 110°C. The diaryliodonium salt mFBG precursor, dissolved in dry acetonitrile and toluene, was added to the reactor for fluorination at room temperature followed by thermolysis at 120°C to generate the protected intermediate. Solvent removal with argon flow under reduced pressure was followed by deprotection (hydrochloric acid, 120°C) to afford the [18F]mFBG product. The solution was diluted with HPLC eluent (10% ethanol/10 mM HCl), and the compound was purified on a semi-prep Hamilton PRP-1 HPLC column. Results Fully automated production of meta-[18F]fluorobenzylguanidine was achieved using an IBA Synthera® in 53 minutes with purification. Average, final yield was 21% (n = 4, 29% decay-corrected) with radiochemical purity of > 97%. Isocratic HPLC purification in ethanol and HCl allowed easy preparation for injection by dilution and pH adjustment with buffer with no reformulation necessary. Conclusions Significant improvements in the preparation of mFBG were demonstrated by conversion from manual to automated synthesis and a reduction in synthetic steps. This method shows promise with a 2-fold improvement in yield, 2-fold reduction of the synthesis time, and simplification of the purification over previously reported methods. Optimization and quality control testing for patient use are currently underway. Acknowledgements Funding provided by The Hartwell Foundation, NIBIB 5 R01EB015536-03, and ALSAC-St. Jude Children’s Research Hospital. References [1] Zhang H, et al. (2014) Clin Cancer Res, 20(8): 2182-2191. [2] Garg, PK, et al. (1994) Nucl Med Biol, 21(1): 97-103. [3] Zhang et al., Eur J Nucl Med Mol Imaging (2014) 41:322-332.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S217

217 Stability of [18F]FMeNER-D2: correlation of in vivo behavior with in vitro enzymes Christina Rami-Mark1, 2, Nadine Eberherr1, Lukas Nics1, Rupert Lanzenberger3, Marcus Hacker1, Wolfgang Wadsak1, 2, Markus Mitterhauser1 1 Biomedical Imaging and Image Guided Therapy, Medical University of Vienna, Vienna, Austria, 2Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria, 3Department of Psychiatry and Psychotherapy, Medical University of Vienna, Vienna, Austria Objectives In neurodegenerative diseases dysregulation of the norepinephrine transporter (NET) is reported. For visualization of NET availability and occupancy in human brain, PET imaging is used. Amongst existing NETPET tracers, [18F]FMeNER-D2 has been showing best results in a clinical set-up so far[1,2]. However, using this ligand in clinical trials, an unexpected uptake behavior was observed, hampering a quantification of NET in cortical areas[3]. Assuming that this intense accumulation originates from an arising radio-metabolite, a deeper understanding of the metabolic fate of this NET-PET tracer is needed. Methods In vitro metabolic stability was assessed using a variety of human and murine enzymes; i.e. pooled human and rat liver microsomes, human and rat plasma, human CYP1A1, CYP3A4, CYP2D6, CYP1B1 and CES. Incubation of freshly prepared [18F]FMeNER-D2[4] in PBS (pH 7.4 at 37°C) containing the respective enzyme and cofactors was performed over 1h. Enzymatic reactions were stopped by adding ice-cold methanol at certain time points. The mixtures were centrifuged (23.000g, 4min, 4°C). Aliquots of the supernatant were analyzed by HPLC and TLC, as results % of test compound metabolized was obtained. A possible involvement of bone binding was examined. Therefore [18F]FMeNER-D2, as well as the mixture of enzymatic degradation (containing intact [18F]FMeNER-D2 + radiometabolites) were incubated in a mixture of PBS containing 1mg spongiosa or 1mg hydroxyapatite. The % bone binding was determined after centrifugation and washing of the pellet. Results Good metabolic stability was observed in human plasma (99.5% intact after 1h). Moreover, only 6.5±1.2% of the tracer were metabolized after 60min of human microsomal incubation. Incubation in human CYP single enzymes yielded only up to 5.8% radiometabolites. In contrast, low metabolic stability was observed in both rat plasma (20% degraded [18F]FMeNER-D2 after 1h) and microsomes (only 0.93±0.12% intact after 10min). Interestingly, defluorination did not take place, since no free F- was detected on TLC. Also no binding of [18F]FMeNER-D2 or its radioactive metabolites to bone or hydroxyapatite was observed. Conclusions The uptake to the non-target structures close to the brain is not due to peripheral metabolism. Hence, the formation of potential radio-metabolites by cerebral enzymes (i.e. MAO A, MAO B and catechyl-O-methyl transferase) will have to be adressed in the future. High metabolic instability was found in rat models, putting some constraints on their applicability as animal model. Acknowledgements References [1]Takano A, et al. (2008) EJNMMI 35:153-7. [2]Takano A, et al. (2008) NeuroImage. 42:474-82. [3]Vanicek T, et al. (2014) JAMA Psychiatry 71:1340-1349. [4]Rami-Mark et al. (2014) Nucl Med Biol 40:10491054.

J Label Compd Radiopharm 2015: 58: S1- S411

S218: Poster

21st International Symposium on Radiopharmaceutical Sciences

218 The development and imaging with human osteocalcin in female mice; [F-18] FB-Osteocalcin (FBO) Patrick Carberry1, John W. Castrillon1, Lori N. Khrimian2, Paula Mera2, Patricia F. Ducy3, Gerard Karsenty2, Chaitanya R. Divgi1 1 Radiology, Columbia University Medical Center, New York, New York, United States, 2Genetics and Development, Columbia University Medical Center, New York, New York, United States, 3Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States Objectives We have developed a novel F-18 imaging agent, [18F]-fluorobenzoyl osteocalcin 2 (FBO), and demonstrated its potential for imaging of osteocalcin receptors [1, 2]. Two cohorts of 4-month old female mice, wild-type and osteocalcin knockout, were imaged using a Siemen's Inveon rodent PET scanner. Methods The radiolabeling of [18F]-FBO 2 was achieved through coupling with N-succinimidyl-4[18F]fluorobenzoate (SFB) (Figure 1). [18F]-SFB [3] was purified through solid-phase extraction. Two cohorts of wild-type and osteocalcin knockout 4-month old female mice (n = 3 / each) were injected with 60-89 µCi [18F]FBO 2 (2.22-3.29 MBq). Whole body dynamic scans were acquired for 2 hours post-injection. Results The overall synthesis for [18F]-FBO 2 was achieved in a total of four steps, starting with the formation of [18F]-SFB [3]. The fluoride-18 labeling of prosthetic ligand [18F]-SFB was achieved in radiochemical yields (RCY) ranging from 9-22% with excellent radiochemical purity (95 ± 5%, n = 12, based on HPLC analyses). The coupling step of the prosthetic ligand to the N-terminus of (Glu 17, 21, 24) osteocalcin 1 (see Figure 1) was achieved in RCY = 7 ± 1% (n = 3) with purification via semi-prep HPLC to provide high RCP (97 ± 1%, n = 3). Qualitative analysis of rodent PET reveals the agent crosses the blood-brain barrier (BBB); auto-radiographic studies to examine specific binding to the hippocampus are underway. Conclusions A novel radiotracer, [18F]-FBO 2, is reported and evaluated for imaging of osteocalcin receptors. The desired labeled peptide was synthesized in four steps, with the use of [18F]-SFB as the prosthetic ligand. The tracer appears to cross the BBB and binds to both cerebral and muscular osteocalcin receptors. Acknowledgements This work was supported by a grant from Columbia Technology Ventures. References [1] Karsenty, G., et al, (2012), Nature, 481, 314-320. [2] Hosseini, S., et al, (2013), J. Biol. Chem., 288, 7885-7893. [3] Mäding, P., et al, (2005), Appl. Radiat. Isot., 63, 329-332.

Figure 1. Coupling of (Glu 17, 21, 24) osteocalcin 1 with [18F]-SFB to form [18F]-FBO 2.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S219

219 Radiosynthesis of fluorinated a7 ligands and their evaluation by biodistribution and µPET studies Pakorn Tangpong1, Aziz Ouach2, Franck Suzenet2, Sylvie Chalon3, Sophie Serrier3, Jean-Bernard Deloye4, Lucette Garreau1, Sylvain Routier2, Denis Guilloteau5, Johnny Vercouillie1 1 CERRP, U930, Tours, France, 2ICOA, Orléans, France, 3U930, Tours, France, 4Laboratoires Cyclopharma, Clermont-Ferrand, France, 5CHRU Bretonneau, Tours, France Objectives The alpha-7 nicotinic acetylcholine receptor (α7 nAChR) is founded in the central nervous system (CNS) and changes in its concentration has been reported in various disorders such as Alzheimer’s disease (AD), schizophrenia and depression. In vivo imaging of a7 nAChR should be helpful to understand physiopathology of these disorders and in this aim we developed fluorinated PET ligands (1,2). The present study emphasized for the development radiosynthesis processing by using microwave-assisted reaction method to enhance radiochemical yield and to shorten reaction times but also the biological evaluation of the tracers in rats. Methods The 18F- was prepared by nuclear reaction 18O(p,n)18F and then was transferred to synthesizer module GE TRACERlab®FX N Pro and was passed through a Sep-Pak® QMA cartridge. 18F was prepared according classical procedures prior adding the precursors diluted in DMSO. Solutions were heated using conventional heating or transferred into a 2 mL vial of the PETwave (CEM) for microwave assisted reaction. Radiotracers were evaluated in rats by biobistribution and µPET studies using methyllycaconitine, a selective alpha 7 receptor antagonist to evaluate non specific binding of the developed tracers. Results [18F]OA545, [18F]OA904 and [18F]OA1046 were respectively obtained in 7%, 12% and 17% (decaycorrected). Radiochemical purity was >98% of all products and specific activity in the range of 10-47GBq/µmol. The total synthesis times were 100 minutes for [18F]OA545, [18F]OA904 and 80 minutes for [18F]OA1046. Unfortunately, the tracers display a low blood brain barrier (BBB) uptake and a high non specific binding. Conclusions Using microwave synthesis reduces radiolabeling times and/or allows preparation of the tracers. New developments are currently ongoing to increase BBB uptake and to get higher signal to noise ratio. Acknowledgements This research was supported by grants from the Région Centre (IMAD, the ANR Malz program (ANR-10-MALZ-0004) and France Alzheimer. References 1-Pin F, Vercouillie J, Ouach A, Mavel S, Gulhan Z, Chicheri G, Jarry C, Massip S, Deloye JB, Guilloteau D, Suzenet F, Chalon S, Routier S. Design of alpha 7 Nicotinic Acetylcholine Receptor Ligands in Quinuclidine, Tropane and Quinazoline Series. Chemistry, Molecular Modeling, Radiochemistry, in vitro and in Rats Evaluations of a [18F] quinuclidine derivative. European Journal of Medicinal Chemistry. 2014, 82:214-24. 2- Routier S, Suzenet F, Pin F, Chalon S, Vercouillie J, Guilloteau D. 1,4-Disubstituted 1,2,3-Triazoles, methods for preparing same, and diagnostic therapeutic uses thereof. 2012. WO2012143526

J Label Compd Radiopharm 2015: 58: S1- S411

S220: Poster

21st International Symposium on Radiopharmaceutical Sciences

220 One-pot radiosynthesis of [18F]FEDAC as a clinically applicable PET ligand for imaging TSPO. Kazunori Kawamura1, Katsushi Kumata1, Kenji Furutsuka1, 2, Satoshi Shiomi1, 3, Tomoya Fujishiro1, 3, Ryuji Watanabe1, 3, Makoto Takei1, Hiroki Hashimoto1, Takehito Ito1, 2, Masanao Ogawa1, 2, Nobuyuki Igarashi1, 3, Masatoshi Mutou1, 3, Nobuki Nengaki1, 2, Kazuyoshi Nemoto1, Ming-Rong Zhang1 1 Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Chiba, Japan, 2SHI Accelerator Service, Tokyo, Japan, 3Tokyo Nuclear Services, Tokyo, Japan Objectives Translocator protein (18 kDa) (TSPO), a nucleus-encoded mitochondrial target transmembrane protein, has been indicated as an active participant in the modulation of mitochondrial function. PET using radiolabeled TSPO probes has allowed non-invasive and reliable investigation of TSPO in neuropathological damages of experimental animals and humans. Many PET probes for TSPO imaging have been developed. Among them, [18F]FEDAC has potent binding affinity and selectivity for TSPO [1], high signal to neuroinflammation [2], and high sensitivity and specificity for detection of fatty liver diseases progression [3]. We had previously synthesized [18F]FEDAC by reaction of desmethyl-precursor with [18F]fluoroethyl bromide at two steps using a modified 18Flabelling synthesizer developed in our institute [1-3]. In this study, we simplified the synthesis of [18F]FEDAC by direct 18F-fluorination using a typical 18F-labelling synthesizer to transfer the preparation of [18F]FEDAC to other institutes toward multicenter clinical study. Methods Tosylate-precursor for radiosynthesis of [18F]FEDAC was synthesized according to the procedures, as shown in Fig. 1. [18F]FEDAC was prepared by heating the tosylate-precursor with 18F- in DMSO at 110 ⁰C for 1015 min. Results Tosylate-precursor of [18F]FEDAC was successfully synthesized from desmethyl-precursor. [18F]FEDAC was obtained with sufficient radioactivity and suitable quality for injection in clinical application. The synthesis of [18F]FEDAC was reproduceble to achieve >740 MBq, >300 GBq/µmol and >97% of radiochemical purity within 70 min of an overall synthesis time. All other analytical results were in compliance with our in-house quality control and assurance specifications. Conclusions We successfully synthesized [18F]FEDAC by fluorination of the tosylate-precursor with 18F- using one-pot 18F-labelling synthesizer. This radioligand will be used in clinical study. Acknowledgements References [1] Yanamoto K, et al (2009), Bioorg Med Chem Lett, 19, 1707-10. [2] Yui J, et al (2010), J Nucl Med, 51, 1301-9. [3] Xie L, et al (2012), J Hepatol, 57, 1076-82.

Fig.1. Synthesis of [18F]FEDAC. Reagents (a). 1). THPO-CH2CH2Br, K2CO3/ DMF; 2) Tos-OH/ CH3OH; 3) (Tos)2O, 2,6-Lutidine / CH2Cl2

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S221

221 Novel 18F-labelled triazine derivatives for PET imaging of phosphodiesterase 2A Susann Schröder1, Barbara Wenzel1, Winnie Deuther-Conrad1, Rodrigo Teodoro1, Ute Egerland2, Mathias Kranz1, Steffen Fischer1, Norbert Höfgen2, Jörg Steinbach1, Peter Brust1 1 Department of Neuroradiopharmaceuticals (FWPN), Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Leipzig, Germany, 2BioCrea GmbH, Radebeul, Germany Objectives Phosphodiesterases (PDEs) are enzymes which degrade the second messengers cAMP and cGMP thereby affecting cellular functions. PDE2A is involved in the pathophysiology of Alzheimer´s disease and cancer. Therefore PDE2A inhibitors are suggested as potential therapeutics. Accordingly we aim to develop an 18F-labelled radioligand for PET imaging of PDE2A. Methods Based on a triazine compound [1] (TA1) novel fluoroalkylated derivatives (TA2-5, see Table 1) were synthesized and their affinity and selectivity towards PDE2A were determined. 18F-labelling of selected candidates was accomplished by nucleophilic substitution in acetonitrile using tosylate precursors. In vitro autoradiographic studies on rat brain sections were performed with [18F]TA3 and [18F]TA4 under control and blocking conditions. For PET/MR studies of [18F]TA3 in mice the radiosynthesis was performed in a TRACERlabTM FX F-N module. In vivo metabolism studies of [18F]TA3 and [18F]TA4 in mouse plasma and brain samples were carried out by conventional extraction procedure as well as by direct injection of the samples into a micellar HPLC system. Results [18F]TA3, [18F]TA4 and [18F]TA5 were successfully synthesized with labelling yields of 40 - 70%, radiochemical yields of 30 - 45% and specific activities of ≥ 60 GBq/µmol. In vitro autoradiographic experiments showed region-specific accumulation of [18F]TA3 (see Table 1) and [18F]TA4 with higher binding density in cortex and striatum than in cerebellum, which is consistent with the distribution pattern of PDE2A in rat brain. PET/MR studies of [18F]TA3 in mice exhibited a fast wash out of radioactivity from the striatum while constantly increasing uptake was observed in the cerebellum. Metabolism studies of [18F]TA3 and [18F]TA4 at 30 minutes p.i. revealed a significant brain concentration of radiometabolites (≥ 40%). Conclusions Due to the brain accumulation of radiometabolites, the new radioligands [18F]TA3 and [18F]TA4 are exclusively suitable for in vitro imaging of PDE2A. Further in vitro and in vivo characterization of the highly affine and selective PDE2A radioligand [18F]TA5 is currently in progress. Acknowledgements References [1] Stange et al.: Triazine Derivatives as Inhibitors of Phosphodiesterases; Patent WO2010/054253 A1.

Table 1: Structures and affinity data of TA1-5, autoradiographic image of [18F]TA3.

J Label Compd Radiopharm 2015: 58: S1- S411

S222: Poster

21st International Symposium on Radiopharmaceutical Sciences

222 Synthesis and evaluation of a novel 18F-labelled radiotracer for cyclooxygenase-2 (COX-2) imaging Jatinder Kaur1, 2, Ole Tietz1, Alison Marshall1, Monica Wang1, Atul Bhardwaj1, 2, Jenilee D. Way1, Melinda Wuest1, Frank Wuest1 1 Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Pharmacy and Pharmaceutical sciences, University of Alberta, Edmonton, Alberta, Canada Objectives Cyclooxygenase-2 (COX-2) is an enzyme expressed in a variety of disease states. The involvement of COX-2 in the development of human cancer makes it an interesting target for molecular imaging. The objective of this project is the translation of a successful fluorescence imaging agent into the corresponding PET imaging agent. Methods The radiotracer [18F]2 was designed on the basis of a successful fluorescence imaging agent (1) previously developed in our lab (Figure 1) [1]. [18F]2 was radiolabelled using 4-[18F]fluorobenzyl amine ([18F]FBA) as building block. Cell uptake studies with radiotracer [18F]2 were performed in COX-2 expressing HCA-7 cells. [18F]2 was further evaluated in HCA-7 tumor-bearing NIH-III mice using a dynamic small animal PET imaging. Results IC50 value of compound 2 against COX-2 was found to be 360 nM, compared to 190 nM for fluorescence agent (1) in the same assay. Radiosynthesis of [18F]2 was accomplished within 90 min in a decay-corrected radiochemical yield of 20%. Specific activity of radiotracer [18F]2 was determined to be >40 GBq/μmol. Cellular uptake of [18F]2 in COX-2 positive HCA-7 cells reached ~450 % radioactivity/mg protein after 60 min. Only pretreatment with 100 μM of 2 blocked uptake of [18F]2, but not treatment with celecoxib or rofecoxib (10 and 100 μM). Dynamic PET studies in HCA-7-bearing NIH-III mice revealed a tumor SUV5min of 0.32 clearing to SUV120min 0.18. Over time, no increase in tumor-to-muscle ratio was observed. Conclusions A novel and selective 18F-labelled COX-2 inhibitor has been synthesized. Although uptake of the radiotracer into HCA-7 cells in vitro was high, COX-2 specific binding could be not confirmed. Uptake into HCA7 tumors in vivo was low and comparable to reference tissue. By contrast, the fluorescence imaging agent (1) was shown to bind to COX-2 in HCA-7 cells in vitro. Acknowledgements This work was supported by the Dianne and Irving Kipnes Foundation, the Canadian Institute for Health Research (CIHR) and the Natural Sciences, Engineering Research Council of Canada (NSERC) and the Alberta Cancer Foundation. References [1] Bhardwaj A, et al (2014) ChemMedChem, 9, 109–116.

Structures of fluorescence imaging agent (1) and PET imaging agent ([18F]2).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S223

223 Synthesis and [18F]labeling of a fluoropyridinyl analogue of the TSPO ligand SSR180575, modified at the N,N-dimethylacetamide position Fanny Cacheux1, Fabien Caillé1, Bertrand Kuhnast1, Frank Marguet2, Frédéric Puech2, Géraldine Pottier1, Raphaël Boisgard1, Frédéric Dollé1, Annelaure Damont1 1 CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France, 2Exploratory Research Unit, Sanofi, ChillyMazarin, France Objectives The TSPO 18 kDa overexpression in response to inflammation within the CNS has attracted much attention for the development of specific radioligands for PET imaging. The indole acetamide [11C]SSR180575 [1,2] is a promising representative of such ligands and some modifications have already been made on its structure for the discovery of potent analogues labelable with F-18 [3,4]. Herein, the preparation of a novel derivative modified at the acetamide position is presented as well as its [18F]radiolabeling for preliminary in vivo PET evaluation. Methods The fluoropyridinyl analogue (1) of SSR180575 and the bromo precursor (2) for [18F]labeling were both synthesized from the acid derivative 3. Binding affinity of 1 for the TSPO was determined (competition with [3H]PK11195) as well as selectivity versus the CBR (competition with [3H]flunitrazepam). [18F]-1 was labeled using standard conditions on a TRACERLab FX-N Pro synthesizer. Results The fluoropyridinyl SSR180575 analogue (1) and the bromo precursor 2 were obtained in one step from compound 3 in 50 % and 35 % yields, respectively. Compound 1 exhibited excellent affinity for the TSPO (Ki = 0.46 nM) even better than the parent molecule (Ki = 0.5-0.9 nM), and good selectivity (Ki CBR > 1 mM). Radiolabeling was performed using K[18F]F-K222 at 160 °C for 10 min in DMSO. An average 2.2 GBq of ready-toinject [18F]-1, chemically and radiochemically pure (> 99 %), were obtained in 55-60 min, starting from a 48 GBq [18F]fluoride batch with a 70 ± 30 GBq/µmol SRA. Conclusions The fluoropyridinyl SSR180575 analogue (1) and the bromo precursor were efficiently prepared and [18F]labeling successfully afforded the production of [18F]-1 in reproducible batches. MicroPET studies with the AMPA rat model are on-going to evaluate the potential of this radiotracer to image neuroinflammation. Acknowledgements INMiND (HEALTH-F2-2011-278850) References [1] Chauveau F. et al. (2008), Eur. J. Nucl. Med. Mol. Imag., 35, 2304; [2] Thominiaux C. et al. (2010), J. Label. Compds Radiopharm., 53, 767. [3] Damont A. et al. (2011), J. Label. Compds Radiopharm., 54(S1), S34; [4] Damont A. et al. (2013), J. Label. Compds Radiopharm., 56(S1), S292.

J Label Compd Radiopharm 2015: 58: S1- S411

S224: Poster

21st International Symposium on Radiopharmaceutical Sciences

224 Strategies for Green Fluorine-18 Radiochemistry Megan N. Stewart1, 2, Brian G. Hockley2, Alexandra Sowa1, 2, Peter J. Scott2, 1 1 Medicinal Chemistry, The University of Michigan, Ann Arbor, Michigan, United States, 2Radiology, The University of Michigan, Ann Arbor, Michigan, United States Objectives Recently, aq. ethanol was used by our group in carbon-11 radiolabeling as an alternative to other organic solvents [1,2], moving our facility towards a green laboratory. Herein we extend the concept of green radiochemistry, and present greener approaches to fluorine-18 labeling of PET radiotracers. In addition to moving us towards a green sustainable lab, the work has two objectives: (1) to simplify QC testing of radiopharmaceuticals by eliminating the requirement for residual solvent analysis of hazardous solvents and, (2) to address fundamental questions pertaining to the reactivity of fluoride in green, polar protic solvents. Methods 18F was trapped on a QMA sep-pak to remove [18O]H2O, and eluted into a reaction vial using aq. K2CO3. A solution of kryptofix-222 (K222) in EtOH was added to the reaction vessel and 18F was dried by azeotropic evaporation. The appropriate precursor was then added in H2O/EtOH (or other green solvent) for nucleophilic 18Ffluorination and reaction progress was monitored by TLC and/or HPLC. Results Azeotropic Drying: The usual solution of K222 (15 mg/1 mL MeCN) used to azeotropically dry 18F was substituted with K222 (15 mg/1 mL EtOH). The change had no detrimental effect on reaction yields of [18F]FDG, [18F]FET, [18F]FAZA or [18F]flubatine (radiochemical yields (RCY) = 5 - 99%). Nucleophilic Fluorination: [18F]FDG was used as a model radiotracer to explore the feasibility of nucleophilic aliphatic 18F-labeling in varying concentrations of aq. ethanol. The highest 18F incorporation occurred when the EtOH:H2O ratio was 85:15, generating protected FDG in 63% RCY. To examine scope of the green methodology, [18F]FET, [18F]FAZA and [18F]flubatine, as well as an extensive range of model substrates were labeled by aliphatic or aromatic radiofluorination with varying degrees of success (3 - 72% RCY). Conclusions We report successful synthesis of fluorine-18 labeled radiopharmaceuticals using only ethanol and water for drying of fluoride and subsequent fluorination. This work challenges the longheld belief that nucleophilic fluorination reactions cannot take place in polar protic solvents, demonstrating that such reactions can in fact proceed in reasonable yields using aq. EtOH. Acknowledgements The authors wish to thank the University of Michigan (Rackham Merit Fellowship, the College of Pharmacy and the Interdepartmental Program in Medicinal Chemistry) for financial support of this work. References [1] Shao X, Schnau PL, Fawaz M, Scott PJH. Nucl. Med. Biol., 2013;40:109-116. [2] Shao X, Fawaz MV, Jang K, Scott PJH. Appl. Radiat. Isotop., 2014;89:125-129.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S225

225 In Vivo Albumin Labeling with 18F-AlF-NEB for Lymph Node Imaging Xiaoyuan Chen, Lixin Lang, Gang Niu National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, United States Objectives The ability to accurately and easily locate sentinel lymph nodes (SLNs) with non-invasive imaging methods would assist in tumor staging and patient management. For this purpose, we developed a lymphatic imaging agent by mixing fluorine-18 aluminum fluoride labeled NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid) conjugated truncated Evans blue (18F-AlF-NEB) and Evans blue (EB) dye. After local injection, both 18FAlF-NEB and EB form complexes with endogenous albumin in the interstitial fluid and allow for visualizing the lymphatic system. Methods PET and/or optical imaging of lymph nodes (LNs) was performed in three different animal models including hind limb inflammation model, orthotropic breast cancer model, and metastatic breast cancer model. Results For the hindlimb inflammation model, 18F-AlF-NEB PET imaging was performed on day 5 after turpentine injection. Due to the inflammatory stimulation, the left popliteal LNs had an obviously higher tracer uptake than the contralateral normal LNs. Corresponding T2-weighted MR images confirmed swelling of the popliteal LNs but not the sciatic LNs. For the tumor draining lymph node imaging, at 30 days after tumor inoculation, MDA-MB-435 tumor mice were scanned following intra-tumoral injection of 18F-AlF-NEB. Besides the tracer injection site, a satellite spot with high signal intensity was identified on PET images as accessory axillary LN. We also applied 18 F-AlF-NEB PET to image tumor metastatic LNs. Four weeks after inoculation of Fluc+ 4T1 cells via hock injection, obvious bioluminescence signal could be seen at the popliteal fossa. T2-weighted MR image also showed enlarged tumor-side popliteal LNs. There was dramatically higher tracer uptake in tumor draining popliteal LNs compared with the contralateral LNs. The contralateral LNs showed a similar trend but with much lower signal intensity. Conclusions The imaging agent we developed in this study can be used for visualization and detection of sentinel lymph nodes. After local injection, the imaging probe quickly formed complex with albumin within the interstitial fluid. Thus, the radioactive signal reflects the behavior of endogenous albumin, avoiding the usage of colloids, nanoparticles or polymers. The SLNs can be visualized by PET scans before surgery and then removed under the guidance of fluorescence signal and blue color deposit during surgery. The excellent imaging quality, easy preparation, multimodality, and biosafety guarantee the clinical translation to map SLNs and provide intraoperative guidance. Acknowledgements NIBIB/NIH Intramural Research Program. References

J Label Compd Radiopharm 2015: 58: S1- S411

S226: Poster

21st International Symposium on Radiopharmaceutical Sciences

226 Evaluation of the novel radiotracer 18F-DBT-10 for imaging the α7 nicotinic acetylcholine receptor in nonhuman primates Ming-Qiang Zheng1, Ansel Hillmer1, Matthias Scheunemann2, Daniel Holden1, Songye Li1, Shu-fei Lin1, david Labaree1, Winnie Deuther-Conrad2, Rodrigo Teodoro2, Richard E. Carson1, Peter Brust2, Henry Huang1 1 PET Center, Department of Diagnostic Radiology, Yale University, New Haven, Connecticut, United States, 2 Helmholtz-Zentrum Dresden – Rossendorf, Leipzig, Germany Objectives The α7 nicotinic acetylcholine receptor (α7AChR) is involved in cognition and thus a target for drug development in Alzheimer’s disease and schizophrenia. 18F-DBT-10 was a candidate radioligand for α7AChR (Kranz et al. J Nucl Med 2014; 55 (S1):1143). We performed PET experiments in rhesus monkeys to assess its kinetic and imaging characteristics. Methods DBT-10 affinities for human AChRs were determined in radioligand binding assays.18F-DBT-10 was prepared from its nitrophenyl precursor via nucleophilic displacement with K18F/Kryptofix-222 in DMSO at 140 °C for 10 min. Dynamic PET imaging sessions were carried out on the Focus-220 scanner for 240 min each in two monkeys. Metabolite analysis was performed with HPLC. Arterial plasma input functions were generated for compartmental modeling to derive regional distribution volume (VT). Results DBT-10 displayed high α7AChR binding affinity (Ki = 0.60 nM) in vitro and excellent selectivity over other AChRs. 18F-DBT-10 was prepared in 14.5 ± 4.6% radiochemical yield and >99% radiochemical purity (n = 5). Plasma free fraction of 18F-DBT-10 was 18 ± 2% (n = 4). Metabolism rate varied between the two animals, resulting in different tissue kinetics. Ex vivo analysis of brain tissues from one monkey found no radioactive metabolites at 120 min post-injection. 18F-DBT-10 showed high brain uptake and fairly rapid tissue kinetics, with peak uptake at 10-50 min in most regions (Fig. 1). Time-activity curves were fitted well with the 2-tissue model. Mean VT values were 58.0, 57.5, 54.9, 54.5, 52.0, 48.4, 39.9, and 34.8 cm3/mL, respectively, for the thalamus, insular, frontal and cingulate cortices, striatum, temporal cortex, hippocampus, occipital cortex, and cerebellum (n = 2). Pre-treatment with the selective α7AChR ligand ASEM (0.69 & 1.24 mg/kg) dose-dependently reduced 18FDBT-10 binding in all regions by 30% and 64%, respectively. Conclusions In rhesus monkeys 8F-DBT-10 displays high brain uptake, appropriate tissue kinetics, and high specific binding signals and thus is a promising agent for PET imaging of the α7AChR in humans. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S227

227 Microfluidic Synthesis of [18F]FPyTFP Using a Novel Bicyclic Triflate Precursor Murthy R. Akula, David W. Blevins, George W. Kabalka, Dustin Osborne Radiology, The University of Tennessee Medical Center, Knoxville, Tennessee, United States Objectives 18F-Prosthetic groups have been widely used to label sensitive biomoleclules such as peptides, proteins and nucleotides. Direct labeling of active ester based prostheic groups such as [18F]SFB has resulted in disappointingly low radiochemical yields1, because of harsh reaction conditions. We wish to report a rapid and high yielding microfludic synthesis of [18F]FPyTFP2,3 (1) using a new bicyclic ammonium triflate precursor 3 Methods Precursor 3 was synthesized in two steps by the SNAr reaction of DABCO with 2,3,5,6-tetrafluorophenyl 6-bromonicotinate, 2, in DCM, followed by bromide exchange with corresponding triflate using trimethylsilyl triflate4 [FIGURE] . Radiofluorination was performed in an Advion Microfluidic System. Cyclotron produced [18F]fluoride was trapped on QMA cartridge to remove water and released with a solution of TBA bicarbonate, and dried azeotropically three times with ACN. The optimum reaction conditions were obtained using Discovery Mode. The final product was purified on a preconditioned Oasis MCX Sep-Pak cartridge and the radiochemical purity was determined by both radio-TLC and radio-HPLC using Bioscan and an Agilent Binary 1200 respectively. Results Using the Batch Mode, anhydrous [18F]fluoride (~50 mCi) dissolved in 4:1=t.BuOH/ACN (450 µL) and the precursor 2 dissolved in 4:1=t.BuOH/ACN (10 mg, 1 mL) were loaded on to the pump 3 loop (400 µL) and the pump 1 loop (400 µL) respectively and allowed to react in the micro-reactor (100 µ x 2 m) at 80 oC with a combined flow rate of 80 µL/min. The crude reaction mixture did not show any trace of unreacted isotope by radio-TLC. Purification of crude prodcut using Oasis MCX catridge and eluting with 2 mL of ACN afforded 38 mCi of [18F]FPyTFP (RCY = 76 % n.d.c; n = 3 ) in a radiochemical purity of ≥97%. Conclusions [18F]FPyTFP has been rapidly synthesized from a novel labeling precursor in a very high radiochemical yield and purity. The advantage of new DABCO precursor is that it does not produce volatile Me[18F], typically observed using trimethylammonium triflates. Acknowledgements We wish to thank the Molecular Imaging and Translational Research Program and the University of Tennessee Health System for funding this research. References [1] Lang L and Eckelman W. (1994) Appl. Radiat. Isot, 45, 1155-1163. [2] Olberg DE. et al. (2010) J Med Chem, 53, 1732-40. [3] Cumming et al (2013) J Label Compd Radiopharma. 56, S447. [4] Shi YJ et al. (2006) Adv. Synth. and Catal. 348, 309 – 312

J Label Compd Radiopharm 2015: 58: S1- S411

S228: Poster

21st International Symposium on Radiopharmaceutical Sciences

228 Evaluation of precursors for the production of a potential Tau imaging agent, 18F-AV-1451 Kuo-Hsien Fan, Junichi Ogikubo, Aldo Cagnolini, Hui Xiong, Jennifer Clemens, Adam Hoye, Carey L. Horchler, Ximin Li, Tho Thieu, Shyamali Ghosh, Giorgio Attardo, Nathaniel Lim Avid Radiopharmaceuticals Inc., Philadelphia, Pennsylvania, United States Objectives Literature procedures described the use of AV-1524 (nitro precursor to 18F-AV-1451) (also known as T807P) or “BOC-T807P” in DMSO for the production of 18F-AV-1451 (also known as 18F-T807). Chien et al developed a process that required destruction of the unreacted T807P (by reduction to the corresponding amine using iron powder) [1] which provided an average uncorrected radiochemical yield (uRCY) of 16% in 93 minutes [29% decay corrected yield (DCY)]. As an alternative, Shoup et al reported a process using BOC-T807P wherein reduction of the nitro precursor was not needed [2]. This process afforded an average uRCY of 14% in 60 minutes (20% DCY). Although these routes afforded 18F-AV-1451 with >95% radiochemical purity, both present challenges for routine commercial production (e.g., adaptation onto a fully automated radiosynthesizer due to the required reduction step and low yield). Therefore, a variety of alternative precursors to 18F-AV-1451 were evaluated. Methods The evaluations were performed using a GE TRACERlab FXF-N radiosynthesizer. A precursor to 18F-AV1451 (“Precursor”) was dissolved in DMSO, and the resulting solution reacted with dried [18F]Fluoride/K222/K2CO3 at 110 oC or 130 oC. When applicable, the N-Boc-protecting group was removed by 3N HCl(aq) at 100 oC. The crude reaction mixture was purified by semi-prep HPLC and the isolated product was dissolved in the appropriate formulation medium thereafter. Results The halide Precursors, AV-1551 and AV-1578, were subjected to 20 minutes of heating at 130 oC and provided poor uRCY of 0.6% and 2%, respectively in 60 minutes (0.9% and 2.9% DCY). On the other hand, the trimethylammonium salt Precursor, AV-1622, provided 27% uRCY (39% DCY, 60 minute process) with a reaction time of 10 minutes at 130 oC and 5 minutes at 100 oC for protecting group removal. The unprotected trimethylammonium salt Precursor, AV-1731, provided a lower uRCY of 14% in 55 minutes (20% DCY). The labeling condition utilizing AV-1622 was further optimized to afford an uRCY of 33-36% in 50 minutes (45-49% DCY). Other N-Boc-protected ammonium salt Precursors (AV-1918 and AV-1932) provided similar yields under the same conditions utilized for AV-1622. Conclusions A manufacturing process suitable for the routine commercial production of 18F-AV-1451 on an automated radiosynthesizer was developed by using a trialkylammonium salt Precursor. This new manufacturing process provides a higher yield with a shorter synthesis time than previously reported methods. Acknowledgements References [1] Chien DT et al (2013) J. Alzheimer’s Disease 34, 457–468 [2] Shoup TM et al (2013) J. Label Compd. Radiopharm 56, 736-740

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S229

229 Readily accessible heteroaromatic silanes as fluoride acceptors in a mild 18F–19F isotopic exchange Christopher M. Waldmann1, Anton A. Toutov2, Robert H. Grubbs2, Jennifer M. Murphy1 1 Crump Institute for Molecular Imaging and Department of Molecular & Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States, 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States Objectives Silicon fluoride acceptors (SiFAs) are under study as new imaging agents useful for positron emission tomography (PET).[1] They are readily labeled with the radioisotope flourine-18 via a fast and mild 18F–19F isotopic exchange reaction (IEX).[2] The application of SiFA-based PET probes has been hampered by their high intrinsic lipophilicity, originating from bulky tert-butyl groups required for in vivo stabilization of the Si–18F bond. Herein, we present the first example of 18F–labeled N- and S-heteroaromatic SiFAs with improved lipophilicity. Furthermore, we demonstrate that simple cartridge-based purification is sufficient, which avoids the usual timeconsuming HPLC step. Methods N- and S-heteroarenes were directly silylated using a novel cross-dehydrogenative catalytic C–H functionalization reaction and subsequently transformed into the respective di-tert-butyl fluorosilanes (Figure 1). A "dry down"-free cartridge-based procedure was utilized to obtain reactive 18F- for the IEX reaction which was performed at ambient temperature in 10 mins. Radiolabeled heteroarylsilanes were subjected to C18-solid phase extraction and reformulated in PBS. In vitro stability was evaluated via HPLC. Results The conditions for the IEX were optimized using the substrate 2-(di-tert-butylfluorosilyl)-1-methyl-indole, which gave 84% ± 6% RCC (Figure 1). After incubating in PBS for 3 hours at 37°C, no evidence for hydrolysis of the Si–18F bond was observed. Conclusions Our results demonstrate the first example of an 18F-labeled heteroaromatic silane. The operational simplicity to produce highly pure 18F-labeled heteroarylsilanes in a rapid manner showcases the value and practicality of this method. We anticipate that the radiosynthetic ease of 18F–19F IEX along with the improved lipophilicity of the resulting heteroaryl silane render this new class of compounds useful for future PET imaging agents. Optimization of the IEX using microfluidic technology that will allow for lower precursor loading and, as a result, a higher specific activity is currently under investigation. Acknowledgements References [1] Wängler, C. et al. (2012) Appl. Sci., 2, 277-302 [2] Kostikov, A. P. et al. (2012), Nature Protocols, 7, 1956-1963.

J Label Compd Radiopharm 2015: 58: S1- S411

S230: Poster

21st International Symposium on Radiopharmaceutical Sciences

230 Automation of [18F]VAT Using the Eckert and Ziegler Modular-Lab, GE TRACERlab FX-N Module Xuyi Yue, Christopher Bognar, Gregory Gaehle, Stephen Moerlein, Xiang Zhang, Zhude Tu Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, Missouri, United States Objectives VAChT is a reliable biomarker for assessing the loss of cholinergic neuron which correlates to the cognitive dysfunction in dementia. The depletion in VAChT levels has been associated with the onset and severity of dementia progression. Currently most developed radioligands for VAChT imaging lack of selectivity over σ receptors, poor brain kinetics or rapid metabolism and are not appropriate for further clinical investigations. Our group designed and synthesized a spectrum of fluorinated carbonyl-containing VAChT ligands with high potency and good selectivity. One of the promising fluorinated analogues was successfully radiolabeled and the results in vivo evaluation in rats and non-human primates (NHP) are very promising. Here, we reported the automation of producing [18F]VAT and got it ready for human studies. Methods The radiosynthesis of [18F]VAT was accomplished by a two-step procedure in a Good Manufacturing Practice (GMP) facility. The first step was to make the [18F]fluoroethyl tosylate using an Eckert and Ziegler Modular-Lab by the reaction of ethylene glycol ditosylate with [18F]KF in acetonitrile. The second step was to make the [18F]VAT by transferring the pure [18F]fluoroethyl tosylate into the solution of corresponding phenol precursor in DMSO at base condition using a GE TRACERlab FX-N module. Upon HPLC purification, the radioactive HPLC collection was passed through the C-18 Sep-Pak cartridge. The trapped target [18F]VAT was eluted with ethanol, formulated with 0.9% saline solution, filtered into a dose vial, which was ready for quality control and clinical use. Results With the optimized condition, [18F]VAT was achieved in approximate 140 min with yield of 15.4% (decay corrected to end of synthesis), specific activity > 3.0 Ci/µmol (n = 3), average mass 0.33 µg/mL, the radiochemical purity was > 99% and chemical purity was > 90%. The residual solvents of the final dose formulation meet the requirement for human use. Conclusions [18F]VAT was successfully automated with high yield, good reproducibility and high quality. These promising results warrant to translate the tracer into clinical investigation upon the IND approval. Acknowledgements Research support from NIH/NINDS and NIH/NIMH Grants: MH092797, NS061025 and NS075527. References 248th ACS National Meeting & Exposition. August 10-14, 2014, San Francisco, CA.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S231

231 {Al18F}2+ approach: Studies of new chelators for radiolabelling biomolecules at mild conditions Joan Lecina, Frederik W. Cleeren, Emilie Billaud, Alfons Verbruggen, Guy Bormans KU Leuven, Leuven, Belgium Objectives The high temperatures required for the complexation of NODA- or NOTA-peptide derivatives1 makes the use of these chelators incompatible with heat sensitive biomolecules. Therefore, new chelators for aluminium fluoride complexation at mild conditions with a less rigid structure were evaluated as alternative of these wellknown macrocyclic ligands. Methods Aluminium fluoride production was carried out using a slight modification of previous reported methods1 and the complexation was carried out at different temperatures with an incubation time of 12 min. Radiochemical yields were calculated using autoradiography analysis of instant Thin Layer Liquid Chromatography iTLC strips. The stability of the chelators was evaluated by adding the Al18F-complex in either phosphate buffer saline (PBS, pH 7) or in rat serum at 37°C. Biodistribution was performed in wild type NMRI mice. Mice were sacrificed at 10 at 60 min p.i. and the activity in every organ/tissue was counted by a gamma-counter. Results Only the ligands that contain the fragment ethylendiamine-N,N’-diacetic acid in their structure showed a good reactivity to chelate {Al18F}2+. At 40°C the ligand containing a pyridine ring was less reactive than with a phenol ring. The stability test carried out in both PBS and in rat serum showed a clear difference in stability for the different {Al18F}-complexes. While the complex with yhe pyridine group was completely unstable, the other ligands presented higher stability giving in one case RCP% higher than 80% after 2 h of incubation. The biodistribution study performed for the most promising ligand showed a negligible bone uptake 10 and 60 min p.i. and revealed predominant hepatobiliary excretion. Conclusions The study carried out with the new ligands showed a direct relation between the structure and the functional groups involved in the coordination of the {Al18F}2+ fragment and the stability of the metal complexes. One of the ligands presented promising results for the labeling of heat sensitive biomolecules with a good reactivity at room temperature and a good in vivo stability. Acknowledgements References [1] Kristell L.S, et al (2014) J Nucl Med, 55, 2050-2056.

J Label Compd Radiopharm 2015: 58: S1- S411

S232: Poster

21st International Symposium on Radiopharmaceutical Sciences

232 Optimized Microwave Assisted 18F-DOPA Synthesis Using a Diaryliodonium Salt Precursor Alejandro Amor-Coarasa1, Kiel Neumann3, Stephen DiMagno2, John W. Babich1 1 Radiology, Weill Cornell Medical College, New York, New York, United States, 2University of Nebraska, Lincoln, , Nebraska, United States, 3Ground Fluor Pharmaceuticals Inc, Lincoln, , Nebraska, United States Objectives To reproducibly synthesize 18F-L-DOPA in higher yields than the previously reported 5-29 % (decay corrected (DC) for both electrophilic and nucleophilic fluorination) [1]. This is to be accomplished by using the ALP-DOPA diaryliodonium salt precursor in a multiple step microwave assisted radio-fluorination. Methods The 18F/K222/K2CO3 mixture was dried at 95 ºC in a conventional heater block with 25 mbar vacuum, 30 ml/min N2 and 300 rpm stirring for 25 minutes. Fluorination was optimized at 140 ºC for 8 min in a microwave (Initiator Oven, Biotage, USA). The solution was purified using a 3 ml silica cartridge, 5 ml hexane and 5 ml EtOAc elution. The EtOAc fraction containing the labeled protected compound was dried at 95 ºC in conventional heater with 25 mbar vacuum, 30 ml/min N2 and 300 rpm stirring for 15 minutes. Deprotection was performed in the microwave with 1.5 M HCl 1 ml solution at 140 ºC for 10 min to yield 18F-L-DOPA. For overall synthesis (A) and reaction flow (B) see figure .1 Results The total synthesis time for the optimized conditions was 60 min. The microwave reaction showed a fluoride reactivity of up to 97 % (only 3% unreacted 18F fluoride), however the highest obtained yield for 18F-ALPDOPA (fully protected form) production was 75.9 %, with ≈21 % of partially protected radiolabeled precursor and other radio-impurities. The optimized purification step recovers 75% of the trapped 18F-ALP-DOPA 98 % radiochemically pure. The microwave assisted deprotection was quantitative. The end product was >97% radiochemically pure 18F-L-DOPA and the total DC yield of the synthesis was 48.5±15.2 %. Conclusions Greater than 97% radiochemically pure 18F-L-DOPA was obtained using the ALP-Dopa diaryliodonium salt precursor in a microwave assisted synthesis. The DC yield obtained for the optimized conditions was 48.5±15.2 % (twice the yield reported, with no need for HI deprotection) in a 60 min synthesis (≈ 33% non-DC yield). Acknowledgements References 1. M. Pretze, C. Wängler, and B.Wängler; 6-[18F]Fluoro-L-DOPA: A Well-Established Neurotracer with Expanding Application Spectrum and Strongly Improved Radiosyntheses. Review Article; BioMed Research International; Volume 2014, Article ID 674063, 12 pages

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S233

233 Photoactivated F-18 Fluorination on a Novel BODIPY Derivative Shaohui Zhang1, Patrick L. Cavins2, Vamsidhar Akurathi1, Timothy E. Glass2, Alan B. Packard1 1 Division of Nuclear Medicine, Department of Radiology, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, United States, 2Department of Chemistry, University of Missouri, Columbia, Missouri, United States Objectives F-18 fluorination reactions are mostly achieved by heating the reaction mixture at high temperature and pressure. Here, we report a novel room temperature F-18 fluorination method using a BODIPY precursor that employs light as the source of activation energy. Methods A photoreactor (Fig. 1) was built using a PVC tube, a 470 nm blue LED light source, and an electric cooling fan to dissipate the heat generated by LED electronics. A BODIPY precursor with unique photoactivation properties was synthesized (Fig. 2). The reaction was carried in a borosilicate glass NMR tube attached to a thermometer to monitor the reaction temperature. The BODIPY precursor was dissolved with F-18/Kryptofix in an anhydrous solvent (acetonitrile or DMSO). The reaction mixture was then injected into the NMR tube and placed into the center of the photoreactor. The reaction vessel was kept in the photoreactor under 470 nm light irradiation at room temperature. The reaction conditions (e.g., precursor concentration, solvent, reaction time) were optimized. Results F-18 labeled BODIPY was synthesized in 20% decay-corrected yield at room temperature using a novel photoactivation method in a custom-built photoreactor. The optimal reaction conditions were found to be 1 mg/mL of precursor in anhydrous acetonitrile with a reaction time of 15 min. Additional studies are underway to evaluate different precursors as well as the biological properties of this F-18-labeled BODIPY derivative. Conclusions A novel room temperature F-18 fluorination method was developed using a BODIPY precursor that employs light as the source of activation energy. Additional studies are underway to evaluate different precursors as well as the biological properties of this F-18-labeled BODIPY derivative. Acknowledgements 5R01HL108107, DE-SC0002040, Children’s Hospital Radiology Foundation References

Figure 1. Photoreactor

J Label Compd Radiopharm 2015: 58: S1- S411

Figure 2. Reaction scheme

S234: Poster

21st International Symposium on Radiopharmaceutical Sciences

234 Development of a Continuous Flow Microwave Reactor and Its Application to the Radiochemistry Hiroyuki Kimura1, 2, Noriyuki Ohneda3, Hiro Odajima3, Masahiro Ono1, Hideo Saji1 1 Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan, 2Radioisotope Research Center, Kyoto University, Kyoto, Japan, 3SAIDA FDS Inc., Yaizu, Shizuoka, Japan Objectives Microwave technology has been successfully applied to enhance radiolabeling reactions. When microwave is used as a source of energy, the reactions proceed in a shorter time and at higher yields than those performed under the conventional thermal conditions. However, most commercially available reactors using microwave that already been developed are batch-types, and there are few flow-types. We have previously reported the development of a novel resonant-type microwave reactor powered by a solid-state device. [1] In this study, we have developed a novel continuous flow microwave reactor that is powered by a solid-state device. Methods The reaction tube (PTFE) was irradiated with microwave irradiation at 30W. The temperature of the reaction solution (MeCN) was measured using an infrared thermography through the open slit of the cavity. Additionally, we demonstrated the flow radiosynthesis of ethyl [18F]fluorobenzoate in MeCN at the flow rate of 2 ml/min. Results The use of the latest telecommunication power devices and resonance technology succeeded in generating uniform electromagnetic field (TM110) in the microwave cavity. Our newly developed microwave reactor was also equipped a real-time monitoring system, which allowed for several variables to be monitored simultaneously, including (1) the resonance frequency, (2) input power, (3) reflection power and (4) pressure. The reaction fluid is pressurized by a pump. The pressure is controlled using a pressure regulator. The solvent were heated immediately to 75.6 °C (1.1 MPa) and 91.9 °C (1.25 MPa) at the flow rate of 10 ml/min and 6 ml/min, respectively. The resonance frequency (steady state) is 2.43 GHz. The labeling efficiency of ethyl [18F]fluorobenzoate was low (< 10 %), but it was confirmed by radio-HPLC. Conclusions While further work will be necessary, the above preliminary results indicate that developed system has a potential as a continuous flow microwave reactor. Acknowledgements References [1] Hiroyuki Kimura, Yusuke Yagi, Noriyuki Ohneda, Hiro Odajima, Masahiro Ono, Hideo Saji. Journal of Labelled Compounds and Radiopharmaceuticals, 57 (12), 680-686 (2014)

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S235

235 Identification and in vivo evaluation of [18F]MNI-617 as a radioligand for PDE4 imaging in non human primate David Thomae1, Caroline Papin1, Thomas Morley1, David Alagille1, Olivier Barret2, Hsiaoju Lee1, Ronald M. Baldwin1, Gilles Tamagnan1, 2 1 Molecular NeuroImaging, New Haven, Connecticut, United States, 2bInstitute for Neurodegenerative Disorders, New Haven, Connecticut, United States Objectives [11C]-(R)-(-)-Rolipram is a selective PET radiotracer of phosphodiesterase 4 (PDE4), which suffers from the inherent very short half file of C-11 (20.3 min).1 The aim of this study was to develop radiotracers based on the structure of rolipram, but labeled with the longer lived radiohalogens F-18 (110 minutes) and I-123 (13.2 hours). Methods A small library of rolipram analogues was synthesized and evaluated in a competitive in vitro binding assay against [3H]-R-(-)-rolipram in mouse brain homogenates. The best PET tracer candidate, [18F]MNI-617 (Figure 1), was radiolabeled in a two-step synthesis from [18F]fluoride using a GE TRACERlab FXFN module and evaluated in vivo in non-human primate. Results Structural modifications of the 4-methoxy and the 3-cyclopentoxy substituents were not well tolerated by the PDE4 receptor resulting in large decrease in binding affinity for 8 out of the 15 compounds synthesized. Iodocompounds all showed a marked decrease in binding, making them unsuitable for a SPECT tracer. MNI-617 was the best candidate, with a five-fold increase in affinity for PDE4 (Kd = 0.26 nM) over R-(-)-rolipram (Kd = 1.6 nM). [18F]MNI-617 was obtained in a one-pot two-step process from the corresponding phenol precursor. After [18F]fluorination of diiodomethane with [18F]fluoride/K2CO3/Kryptofix-222, O-alkylation of the phenol by the resulting [18F]fluoroiodomethane afforded [18F]MNI-617 in 1.5% decay-corrected radiochemical yield from starting [18F]fluoride. The final product was obtained with radiochemical purity of 99% and specific activity of 140 GBq/µmol (3.8 Ci/μmol). PET imaging in rhesus monkey showed that [18F]MNI-617 rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4. Conclusions Fifteen analogs of R-(-)-rolipram were synthesized and evaluated in vitro. Lead compound MNI-617 was successfully radiolabeled with 18F using a two-step process on a commercial GE TRACERlab FXFN with good specific activity and radiochemical purity. [18F]MNI-617 was found to rapidly penetrate the brain, with SUVs of between 4 and 5 making it a promising candidate for PET imaging of PDE4 in brain. Acknowledgements References 1] Fujita, M. et al (2005) NeuroImage, 26, 1201; Parker, C. A. (2005) Synapse, 55, 270; DaSilva J. N. (2002) Eur. J. Nucl. Med. Mol. Imag., 29, 1680.

Figure 1. [18F]MNI-617 and [11C]Rolipram

J Label Compd Radiopharm 2015: 58: S1- S411

S236: Poster

21st International Symposium on Radiopharmaceutical Sciences

236 In vitro evaluation of new tau PET tracer candidates using [18F]T808 and [18F]T808 AD brain slice autoradiography binding inhibition studies Lieven Declercq1, Sofie Celen1, Thomas Tousseyn3, Diederik Moechars4, Jesus Alcazar5, Rik Vandenberghe6, Jose Ignacio Andres5, Fierens Katleen7, Ariza Manuela8, Koen Van Laere2, Alfons Verbruggen1, Guy Bormans1 1 Lab Radiopharmacy, KU Leuven, Leuven, Belgium, 2University Hospitals Leuven, Nuclear Medicine & Molecular Imaging, Leuven, Belgium, 3Translational Cell and Tissue Research, Leuven, Belgium, 4Janssen, Pharmaceuticals, Beerse, Belgium, 5Janssen, Pharmaceuticals, Toledo, Spain, 6University Hospitals Leuven, Neurology Department, Leuven, Belgium, 7Janssen Research & Development, Beerse, Belgium, 8Janssen Research & Development, Beerse, Belgium Objectives [18F]T808 and [18F]T807 (respectively AVE-680 and AVE-1451), have shown promising results as tracers for in vivo imaging of neurofibrillary tangles (NFTs). Biodistribution, radiometabolite analysis and autoradiography studies were performed with both tracers in order to acquire comparative preclinical data and establish T808 and T807 as benchmark compounds for the evaluation of recently reported tau tracers (PBB3, THK5105, THK-5117, BF-158, BF-170, lansoprazole and astemizole) and newly synthesized ligands in semiquantitative autoradiography studies. Methods [18F]T808 and [18F]T807 were synthesized according to a published procedure [1, 2]. We performed a biodistribution study in NMRI-mice at 2, 10, 30 and 60 min (n=4/time point) post injection (p.i.) of [18F]T808 and [18F]T807. Radiometabolites of [18F]T808 and [18F]T807 were quantified in plasma at 2, 10, 30 and 60 min p.i. and in brain at 10 and 60 min p.i. Semi-quantitative autoradiography studies were done with [18F]T808, [18F]T807 and [11C]PIB on human tissue sections of AD-brains and P301L transgenic mice models and their wild-type controls. Slices were incubated with 0.74 MBq/500 µL of tracer with/without 1 µM of cold reference compounds. Results Biodistribution studies confirmed the reported rapid brain uptake and washout. Analysis of brain homogenate extracts showed the presence of two polar radiometabolites and a fraction of intact tracer of 88% at 10 min and 34% at 60 min p.i. for T808. In plasma, 34% of radioactivity was present as intact tracer at 10 min p.i. and 26% at 60 min p.i. No radiometabolites were detected for [18F]T807 in brain or plasma. In the semi-quantitative autoradiography studies on human AD-slices we observed more than 50% tau-selective blocking of [18F]T808 and [18F]T807 in the presence of 1 µM of reported and newly synthesized ligands. Displacement of [11C]PIB was negligible. No specific binding to murine tau in the P301L transgenic mouse model was observed. Conclusions This research determined moieties needed for selective interaction with NFTs, furthermore, they provided a straightforward comparison of the currently reported tau tracers. As a result these data could be used to synthesize new highly selective and specific tau-tracers. Acknowledgements Janssen Research & Development Beerse and IMIR KU Leuven References [1] Zhang W et al. J Alzheimers Dis 2012; 31:601-612 [2] Shoup TM et al. J Labelled Comp Radiopharm 2013; 56:736-740

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S237

237 Optimisation of the synthesis and purification of 18F-4-fluorobenzaldehyde on a FASTlab cassette for labelling of macromolecules Julian Grigg1, Torgrim Engell2, Dimitrios Mantzilas2, Graeme McRobbie1, Imtiaz Khan1, Carina Wickmann2, Gareth Getvoldsen1, Helen Betts1, Knut Dyrstad2, James Nairne1, Mathias Glaser1, Peter Iveson1 1 GE Healthcare, Amersham, United Kingdom, 2GE Healthcare, Oslo, Norway Objectives A common method for the indirect incorporation of [18F]fluorine into 18F-PET tracers is to introduce 18 F via the intermediate 18F-4-fluorobenzaldehyde ([18F]FBA), especially for labelling macromolecules, such as, αvβ3-integrin (Fluciclatide), c-Met and HER2 Affibody [1-3]. A major challenge in the synthesis of [18F]FBA has been to achieve a high yield and understanding of the chemical impurities generated. We previously presented our early work on the radiosynthesis of [18F]FBA for coupling with the c-Met peptide [2]. We have since developed a general [18F]FBA method on the FASTlab synthesiser. Methods The optimisation work reported covers several steps on the FASTlab cassette. For each step the critical variables were identified and examined using Chemometrics. This strategy was used to define the composition of the [18F]fluoride eluent and for optimisation of the activation and fluorination steps (Figure 1). In addition, the selection of a Solid Phase Extraction method for [18F]FBA was based on derivatising the [18F]FBA reaction mixture with the Fluciclatide peptide precursor. Fluciclatide and by-products were analysed using HPLC-MS analysis to reveal the composition of crude [18F]FBA. Results The chemical impurity profile showed that during the formation of [18F]FBA multiple reactive by-products are formed which compete with FBA in the amino-oxy precursor conjugation step. The by-products have been identified and some fully characterised. The level of impurities in the [18F]FBA have been reduced by up to 95% while maintaining a sufficient radiochemical yield of [18F]FBA (> 65 %, decay corrected). Conclusions A method for manufacture of purified [18F]FBA in good yields with lower levels of chemical impurities has been developed on a FASTlab cassette. This [18F]FBA method has enabled the development of fully automated FASTlab manufacturing methods of peptide-based PET tracers for the αvβ3-integrin, c-Met and HER2 receptors. Acknowledgements References [1] Kenny L, et al (2011) Eur J Nucl Med, 38, 2186-97. [2] McRobbie G, et al (2013) EANM, P503. [3] Glaser M, et al (2013) J Nucl Med, 54, 1981-88.

Figure 1. Manufacture of purified [18F]FBA from [18F]fluoride on a FASTlab cassette.

J Label Compd Radiopharm 2015: 58: S1- S411

S238: Poster

21st International Symposium on Radiopharmaceutical Sciences

238 Mechanistic Studies on Forming [18F]Trifluoromethyl Groups from Gem-difluoroenol Ethers Allen F. Brooks1, Dylan J. Kahl2, Jimmy E. Jakobsson3, Dale Mallette1, Garrett M. Carpenter1, Patrick J. Riss3, Peter J. Scott1, 2 1 Radiology, University of Michigan, Ann Arbor, Michigan, United States, 2Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, United States, 3Chemistry, University of Oslo, Oslo, Norway Objectives We have recently shown that treating gem-difluoroenol ethers with 18F- in the presence of a proton source yields [18F]CF3 groups and have used this method to access radiotracers of interest including tau imaging agents based on lansoprazole [1,2]. While the method is adequate, yields of the CF3 product are lower than expected because of formation of significant quantities of the [18F]gem-difluoroenol ether as a side product (Scheme 1), offering scope for improvement. Key to optimizing formation of the desired CF3 product over unwanted enol ether is a detailed understanding of the reaction mechanism. Conducting mechanistic studies using deuterated reaction components (solvents, proton source) is the focus of the present study. Methods In a typical experiment, the enol ether precursor (~3 mg) and proton source (isopropanol (iPrOH), NH4Cl) in anhydrous DMSO (500 μL) were added to dry fluoride, and the reaction was heated at 90°C for 3 min. The reaction mixture was cooled, diluted (H2O) and analyzed by HPLC and NMR to determine CF3:CH=CF2 product ratio. Experiments were repeated using deuterated components (DMSO-d6, iPrOH-d8, ND4Cl), and additional analysis by deuterium NMR identified any sites of deuterium incorporation. Results A range of model substrates and lansoprazole analogs were tested in these experiments. Reversibility studies confirmed generation of the CF3 product is not reversible. In most cases, deuterium incorporation occurred when a deuterated proton source (iPrOH-d8, ND4Cl) was added to the reaction but not when DMSO-d6 was used as reaction solvent, consistent with our proposed addition followed by simple proton quench mechanism. However, in certain cases (e.g. lansoprazole), deuterium was found in the product when DMSO-d6 was used, suggesting a more complicated reaction pathway. These and other key findings have allowed optimization of the synthesis of [18F]CF3 groups and will be reported in this presentation. Conclusions The mechanistic insights gained from this research have improved the radiosynthesis of alkyl [18F]CF3 groups, critical for our intended translation of the method into cGMP compliant synthesis of fluorine-18 labeled radiotracers for clinical use. Acknowledgements NIH (T32- EB005172), Alzheimer’s Association (NIRP-14-305669), University of Michigan (College of Pharmacy, MRC, UROP). References [1] a) F. I. Aigbirhio and P. J. Riss, Chem. Commun., 2011, 47, 11873; b) P. J. Riss, V. Ferrari, L. Brichard, P. Burke, R. Smith and F. I. Aigbirhio, Org. Biomol. Chem., 2012, 10, 6980; [2] M. V. Fawaz, A. F. Brooks, M. E. Rodick, G. M. Carpenter, X. Shao, T. J. Desmond, P. Sherman, C. A. Quesada, B. G. Hockley, M. R. Kilbourn, R. L. Albin, K. A. Frey and P. J. H. Scott, ACS Chem. Neurosci., 2014, 5, 718.

Scheme 1: Generation of [18F]Trifluoromethyl Groups

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S239

239 Using Biotransformations for Radioligand Discovery and Validation – Preparative Liver Microsome Incubations Shed Further Light on the Impact of Radiometabolites on In Vivo Imaging Magnus Schou1, Nahid Amini2, Andreas Westermark2, Hanna Jacobson-Ingemyr2, Akihiro Takano2, Marie Svedberg2, Miklos Toth2, Lenke Tari2, Jenny Häggkvist2, Andrea Varrone2, Christer Halldin2 1 AstraZeneca, Stockholm, Sweden, 2Karolinska Institutet, Stockholm, Sweden Objectives In view of our long standing objective to increase understanding of the contribution of radioligand metabolites in CNS PET imaging, we have recently applied liver microsomes to identify radioligand metabolites in plasma [1]. The aim of this study was to perform preparative microsome incubations to examine the binding of [18F]FE-PE2I radiometabolites to the dopamine transporter (DAT) in vitro and in vivo. Methods [18F]FE-PE2I was incubated with monkey liver microsomes. Hydroxy- ([18F]1), desalkyl- ([18F]2) and combined hydroxy- and desalkyl [18F]FEPE2I ([18F]3) metabolites were isolated using HPLC and redissolved in saline. In vitro autoradiography was performed on whole hemisphere sections from the post mortem human brain. In vivo PET in rats (n=4) and non-human primates (NHP, n=2) were acquired on the nanoPET/MR and HRRT PET systems, respectively. Radiometabolites in brain (rat) and plasma (NHP) were studied using radio-HPLC. The simplified reference tissue model (SRTM) was used to quantify binding. Results [18F]1, [18F]2 and [18F]3 were obtained in 2-10% radiochemical yields and at >95% radiochemical purity. In vitro autoradiography demonstrated the corresponding rank order in specific binding to DAT: [18F]1 > [18F]2 > [18F]3. Following intravenous injection in rodents, [18F]1 readily entered brain and distributed highly into DAT-rich regions, with lower binding in the cerebellum (CER). A preliminary quantification showed a BPND of 2.2 in striatum (STR), similar to that of [18F]FE-PE2I. At 90 min after injection of [18F]1, >95% of the radioactivity in rat brain corresponded to [18F]1. Following intravenous injection of [18F]1 in NHP, high levels of specific binding were observed both in STR and substantia nigra (SN). BPND of [18F]1 was 1.0 in SN and 5.4-5.6 in the STR. In a displacement experiment with GBR12909 (5 mg/kg), radioactivity in STR and SN approached the CER, which was unaffected under these conditions. Conclusions Liver microsomes were essential for understanding the in vitro and in vivo binding of [18F]FE-PE2I radiometabolites in brain tissue and led to the discovery of a novel radioligand for dopamine transporter imaging, i.e. [18F]1. A major advantage with the liver microsome approach for radiometabolite synthesis is that costly and time-consuming syntheses of precursor and reference materials can be avoided. Acknowledgements The authors thank members of the PET group at Karolinska Institutet. References 1. Amini et al. (2013) Anal. Bioanal. Chem., 405, 1303-10

J Label Compd Radiopharm 2015: 58: S1- S411

S240: Poster

21st International Symposium on Radiopharmaceutical Sciences

240 Synthesis and Biological Evaluation of 18F-labeled FMTPD as a Focal Adhesion Kinase-Targeted Tumor Imaging Agent Yu Fang1, Dawei Wang1, Xingyu Xu1, Lingzhou Zhao1, Jinming Zhang2, Mingxia Zhao1, Xia Lu3, Chuanmin Qi1, Huabei Zhang1 1 Beijing Normal University, Beijing, China, 2General Hospital of PLA of China, Beijing, China, 3Anzhen Hospital, Beijing, China Objectives The focal adhesion kinase (FAK) is over-expressed in almost all kinds of tumor cells, and participates in many biological behaviors of tumor cells. Therefore, FAK is a potential target for the diagnosis and therapy of tumor [1]. The FAK inhibitors of ourselves which have strong inhibitory activity, were labelled with F-18 and evaluated in order to develop novel tumor imaging agent. Methods Figure 1 shows the synthesis of the [18F]-FMTPD. The biodistribution experiments were performed in Kunming mice with S180 tumor. A saline solution containing the 18F-labeled tracer (370 KBq per 100 μL) was injected directly into the tail. The mice ( n =5 for each time point) were sacrificed at 5, 15, 30, 60, and 120 min after injection. The organs of interest were removed and weighed, and radioactivity was measured with an automatic γcounter. The percent dose per gram of wet tissue was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. Results The radiochemical yields of the [18F]-FMTPD was 20% and the radiochemical purity of it was greater than 97% after purification by HPLC. Biodistribution study of the radiolabeled tracer in Kunming mice with S180 tumor shows that, the uptake of the [18F]-FMTPD in tumors was (5.32±0.30) %ID/g and (5.33±0.03) %ID/g at 30 min and 60min post-injection, respectively. The uptake in tumor was moderate and the blood background was relatively low. However, at 30 min and 60min post-injection, the uptake in muscle and bone was relatively high, which indicated that the [18F]-FMTPD had a certain defluorination phenomenon. Conclusions For the development of the FAK-targeted tumor imaging agent, further improvement should be undertaken in decreasing the uptake in muscle and avoiding the defluorination of the radiotracer in vivo. Acknowledgements This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (Grant No. 2014ZX09507007-003). References [1] Heinrich, T.; Seenisamy, J.; Emmanuvel, L.; Kulkarni, S.; Bomke, J.; Rohdich, F.; Greiner, H.; Esdar, C.; Krier, M.; Gradler, U.; Musil, D. J. Med. Chem. 2013, 56, 1160-1170.

Figure 1. The Synthesis of the [18F]-FMTPD

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S241

241 First radiosynthesis of 18F-labeled anle138b a potential tracer for imaging of neurodegenerative diseases associated with protein deposition in brain Fadi Zarrad, Boris D. Zlatopolskiy, Elizaveta A. Urusova, Bernd Neumaier Institute of Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne, Germany Objectives Anle138b [5-(3-bromophenyl)-3-(piperonyl)pyrazole] is a novel compound which binds selectively to pathological protein aggregates deposited in Parkinson’s, Alzheimer’s and prion diseases. The aim of this work was to prepare a 18F-labeled analog of anle138b. A feasible synthesis route consists of a 1,3 dipolar addition between the radiofluorinated phenyldiazomethane generated in situ and the 3-bromophenylacetylene as a key step. Methods The 1,3-dipolar cycloaddition between 4-[18F]fluorophenyldiazomethane prepared from the corresponding tosylhydrazone and 4-fluorophenylacetylene was studied as a model reaction. 6-[18F]Fluoro-3,4methylendioxybenzylidene tosylhydrazone ([18F]1) was prepared via the reaction of known 6-[18F]fluoropiperonal ([18F]2) with tosylhydrazide. After SPE purification [18F]1 was deprotonated with base and heated to give 18Flabeled diazomethane. The latter reacted in situ with 3-bromophenylacetylene to give 6’-[18F]anle138b. Results 4-[18F]Fluorobenzylidene tosylhydrazone was obtained in 62% RCY and with a RCP of 93% after SPE purification from 18F- via a one-pot two-step preparation procedure. The latter was used to generate 4[18F]fluorophenyl diazomethane which was allowed to react with the model alkyne to give the appropriate 18Flabeled 3,5-disubstituted pyrazole in 67% RCC. Under optimized reaction conditions (MeOH, 70°C, 10 min) [18F]1 was obtained from [18F]2 in 80% RCY and in 99% RCP. The highest RCCs of 6-[18F]anle138b (27–34%) were achieved using LiOtBu as a base in MeCN at 95 °C for 25 min. Conclusions An efficient method for the regioselective preparation of radiofluorinated 3,5-disubstituted pyrazoles was developed. It allowed to produce 6’-[18F]anle138b, a potential tracer for protein deposition in brain, in activity amounts sufficient for preclinical studies. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S242: Poster

21st International Symposium on Radiopharmaceutical Sciences

242 Construction of 18F-labeled molecules via metal-free cross-coupling reactions of radiofluorinated tosylhydrazones Fadi Zarrad, Boris D. Zlatopolskiy, Elizaveta A. Urusova, Bernd Neumaier Institute of Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne, Germany Objectives Cross-coupling reactions of tosylhydrazones are very powerful synthetic tools for the construction of C-C and C-X bonds. Despite this, they have not yet found application in radiochemistry. The aim of this study was to develop a simple procedure for the preparation of 18F-labeled tosylhydrazones and to evaluate the feasibility of metal-free cross-couplings of tosylhydrazones with boronic acids, alkenes and thiols for the preparation of radiofluorinated compounds. Methods 4-[18F]Fluorobenzylidene tosylhydrazone ([18F]1) was synthesized by the reaction of [18F]FBA with tosyl hydrazide. [18F]1 was allowed to react with phenyl-, 3-aminophenyl and 3-formylphenyl boronic acids; styrene and methyl acrylate; methyl 4-mercaptobenzoate and Boc-Cys-OMe to afford the corresponding radiofluorinated diarylmethanes [18F]2a-c, cyclopropanes [18F]3a,b and thioethers [18F]4a,b, respectively. The cross-coupling step for the preparation of [18F]2-4 was optimized with respect to base, solvent, reaction temperature and reaction time. Additionally, the feasibility of a one-pot preparation of [18F]1 and [18F]2a were studied. Results [18F]2 was obtained from [18F]FBA under optimized reaction conditions (MeOH, 70 °C, 10 min) in 91±2% RCY and in 97±1% RCP after SPE purification. Using acetyl chloride to neutralize excess of base after the [18F]FBA synthesis [18F]2 was prepared via a one-pot two-step reaction sequence in DMSO in 62±10% RCY. Highest RCYs of [18F]3a-c (28-75%) were achieved in 1,4-dioxane at 105 °C for 10 min using K2CO3 as a base. Disubstituted cyclopropanes were obtained in ca. 50% RCY by the reaction between [18F]2 and alkenes in toluene at 140 °C for 10 min in the presence of K2CO3 and TEAC. Finally, thioethers [18F]5a,b were prepared in 67±2% and 20±2% RCY, respectively (K2CO3/TBAB, pyridine, 100 °C, 10 min). Conclusions A simple and efficient method for the preparation of [18F]1 was developed. Subsequently, the broad versatility of the novel radiofluorinated building block was exemplified by the fast and simple preparation of various radiolabeled compounds. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S243

243 Radiosynthesis and preclinical evaluation of new Al18F-labelled Urea-based PSMA inhibitors for PET imaging of prostate cancer Frederik W. Cleeren1, Joan Lecina1, Emilie Billaud1, Muneer Ahamed1, bryan holvoet2, Karolien Goffin2, Christophe Deroose2, Alfons Verbruggen1, Guy Bormans1 1 Laboratory for Radiopharmacy, University of Leuven, Leuven, Belgium, 2Department of Nuclear Medicine and Molecular Imaging, University of Leuven, Leuven, Belgium Objectives Prostate-specific membrane antigen (PSMA) can be considered as a promising target for specific prostate cancer imaging and therapy. Two new Al18F-labelled urea-based PSMA inhibitors were developed and their biodistribution in mice was compared with that of 68Ga-HBEDD-CC-PSMA1. Methods The recently developed chelator mHEDDA (4-(((2-(tert-butoxy)-2-oxoethyl)(2-((2-(tert-butoxy)-2oxoethyl)(2-hydroxybenzyl)amino)ethyl)amino)methyl)benzoic acid) and NODA-MPAA2 were conjugated to the lysine-glutamate urea scaffold to form mHEDDA-PSMA and NODA-MPAA-PSMA. Both constructs were labeled with [Al18F]2+ at a concentration of 300 nM ( NaOAc 0.05 M, pH 4, 20% EtOH) by incubation for 12 min at 40°C or 110°C and the resulting complex was purified with HPLC (Platinum EPS C18, 3 µm, 4.6 mm x 100 mm, Grace or XBridge C18, 5 µm, 4.6 mm x 150 mm, Waters respectively). The resulting tracers were analysed with radioHPLC-HRMS on an Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm x 150 mm, Waters). The compounds Al18F-mHEDDA-PSMA, Al18F-NODA-MPAA-PSMA or 68Ga-HBED-CC-PSMA were injected via the tail vain (1-2 MBq) with or without coadministration of 2 mg/kg 2-PMPA. At 10 min or 60 min p.i (N=4), the animals were sacrificed. Blood and major organs were collected in tarred tubes and weighed. The radioactivity was measured using an automated γ-counter and calculated as % ID/g. Results Al18F-mHEDDA-PSMA and Al18F-NODA-MPAA were prepared in high radiochemical yields ( > 80%) and purity (>95%) and were stable (>95%) in saline for at least three hours. Identification of the [Al18F]-complexes was done by radio-LC-HRMS. Al18F-mHEDDA-PSMA, Al18F-NODA-MPAA and 68Ga-HBED-CC-PSMA all showed low bone uptake (0.74 ± 0.07% ID/g, 0.13 ± 0.02% ID/g and 0.39 ± 0.09% ID/g) and fast clearance from plasma and PSMA negative tissue. The retention at 60 min p.i. in organs with reported high expression of PSMA such as kidney (121.27 ± 10.82% ID/g, 15.68 ± 6.27% ID/g and 269.90 ± 47.23% ID/g) and spleen (4.38 ± 0.55% ID/g, 0.23 ± 0.07% ID/g and 2.72 ± 0.42% ID/g) was nearly completely blocked when 2-PMPA was coinjected to block PSMA specific binding indicating PSMA specific retention. Conclusions Two new Al18F-labelled urea-based PSMA inhibitors were successfully produced with high radioactive yields and purity. The first preclinical results of especially Al18F-mHEDDA-PSMA looks promising and further preclinical evaluation in mice bearing LNCaP and PC-3 tumors will be conducted soon. Acknowledgements References 1. Eder, M. et al. (2014) Bioconjug. Chem, 23, 688–697. 2. McBride, W. J.et al. (2012) Appl. Radiat. Isot. 70, 200–204

J Label Compd Radiopharm 2015: 58: S1- S411

S244: Poster

21st International Symposium on Radiopharmaceutical Sciences

244 Less is more: Preparation of 18F-labeled arenes without azeotropic drying, base and other additives Raphael Richarz, Philipp Krapf, Boris D. Zlatopolskiy, Elizaveta A. Urusova, Bernd Neumaier Institute for Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne, Germany Objectives Conventional 18F-labeling methods via aromatic nucleophilic substitution compromise time consuming azeotropic drying of aqueous solutions of [18F]fluoride salts. Furthermore additives like kryptands are often necessary to enhance the nucleophilicity of [18F]fluoride and to obtain satisfactory RCYs. Strong basic conditions hamper the application of base sensitive labeling precursors and preparation of base sensitive tracers. Alternative labeling strategies without azeotropic drying published so far are only limitedly suitable for the preparation of PET tracers on a preparative scale. The objective of this work was the development of a novel radiolabeling procedure from only radiofluorination precursor and [18F]fluoride, circumventing azeotropic drying, base and addition of other additives. Methods [18F]Fluoride was eluted from an anion exchange cartridge with onium precursors in different solvents. The resulting onium salts (anilinium-, diaryliodonium- oder triarylsulfonium-[18F]fluorides) were redissolved in an appropriate solvent and heated to afford the 18F-labeled products. Results Quantitative recovery of [18F]fluoride from the anion exchange resin was achieved with onium precursors in MeOH and EtOH. The alcohol could be completely removed at 70 °C within 3 min without the need for azeotropic drying. 2-, 3- and 4-[18F]Fluorobenzaldehydes (FBAs) were prepared in RCCs of 40–94% from the corresponding N,N,N-trimethylanilinium (2-, 4-[18F]FBAs) and (4-anisyl)iodonium (3-[18F]FBA) salts. 28 GBq 4[18F]FBA was produced from 32 GBq [18F]fluoride in RCP of >98% within 15 min. Furthermore, radiofluorinated building blocks, 4-[18F]fluoroiodobenzene and 2,3,5,6-tetrafluorophenyl-4-[18F]fluorobenzoate were synthesized in 66% and 25% RCC, respectively. The model peptide 4-[18F]fluorobenzoyl-βAla-Phe-OMe was obtained from the corresponding (4-anisyl)iodonium iodide precursor in 56% RCC. Conclusions We have demonstrated for the first time that 18F-labeled compounds could be efficiently prepared without azeotropic drying using only [18F]fluoride and onium salt precursor. The versatility and the wide scope of the novel radiofluorination procedure were demonstrated by syntheses of several useful 18F-labeled prosthetic groups and by the one-step preparation of a radiolabeled model peptide. Acknowledgements References R. Richarz, P. Krapf, F. Zarrad, E. A. Urusova, B. Neumaier and B. D. Zlatopolskiy, Org. Biomol. Chem., 2014,12, 8094-8099

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S245

245 Synthesis and pharmacological evaluation of hydrophilic [F-18]-labeled GBR12935 derivatives as novel PET imaging agents for dopamine transporter Li-Te Chang1, Zih-Rou Huang1, Ya-Yao Huang2, Kai-Yuan Tzen2, Chyng-Yann Shiue2, Ling-Wei Hsin1, 3 1 School of Pharmacy, National Taiwan University, Taipei, Taiwan, 2PET Center, Department of Nuclear Medicine, National Taiwan University Hospital, Taipei, Taiwan, 3Molecular Probes Development Core, Molecular Imaging Center, National Taiwan University, Taipei, Taiwan Objectives In searching for novel potential dopamine transporter (DAT) imaging agents with better pharmacokinetic properties, e.g. less nonspecific binding and higher metabolic stability, for positron emission tomography (PET), [F-18]-labeled (S)-(-)-1-[4-[2-(diphenylmethoxy)ethyl]piperazinyl]-3-(4-fluorophenyl)propan-3-ol ((S)-1) was prepared and evaluated as a PET imaging agent for DAT. Methods 3-Chloro-4`-fluoropropiophenone was reduced by BH3-SMe2 in the presence of (R)-2-methyl-CBSoxazaborolidine to afford the key intermediate, (S)-1-chloro-3-(4-fluoro-phenyl)propan-3-ol, with > 99.9% ee. 1[2-(Diphenylmethoxy)ethyl]piperazine was then alkylated with the chiral synthon in DMF, using N,N-diisopropylN-ethylamine and sodium iodide, to yield (S)-1 in 97% ee. The corresponding chiral diaryliodonium tosylates derivative of (S)-1 was prepared in similar procedures with modifications as the precursor for the radiosynthesis of [F-18]-labeled (S)-1 ([18F](S)-1). Results (S)-1 showed high and selective DAT binding affinity (Ki = 3.0 nM, Ki ratio SERT/DAT = 311) and much higher hydrophilicity (LogP = 3.41) than GBR12935. [18F](S)-1 was synthesized by treatment of the diaryliodonium tosylate precursor with [18F]-fluoride in the presence of K2CO3 and K222 in DMSO. The total time required for the one-pot two-step radiosynthesis including semi-preparative HPLC purification was 66 min and the overall radiochemical yield of [18F](S)-1 was 2.2%. In animal PET study, [18F](S)-1 demonstrated selective accumulation in the striatum regions of rat brains. Conclusions Based on the promising pharmacological and physicochemical profiles of (S)-1 and the preliminary in vivo results from animal PET study, [18F](S)-1 is a novel potential DAT imaging agent for PET and further studies are in progress. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S246: Poster

21st International Symposium on Radiopharmaceutical Sciences

246 Synthesis of deuterium-substituted [18F]fluoromethyl-PBR28 and its initial PET evaluation compared with [18F]fluoromethyl-PBR28 in a rat model of neuroinflammation Byung Seok Moon1, Jae Ho Jung1, Hyun Soo Park1, Marialessandra Contino2, Nunzio Denora2, Byung Chul Lee1, 3, Sang Eun Kim1, 4 1 Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, Korea (the Republic of), 2Department of Pharmacy – Drug Sciences, University of Bari “A. Moro”, Bari, Bari, Italy, 3Advanced Institutes of Convergence Technology, Suwon, Korea (the Republic of), 4Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Korea (the Republic of) Objectives Previously, [18F]fluoromethyl-PBR28 [(18F]1), sterically almost identical to [11C]PBR28, was developed for TSPO imaging [1] but [18F]1 had a little unstable in vivo due to probably de-fluorination. In present study, [18F]fluoromethyl-PBR28-d2 ([18F]1-d2) was designed and synthesized to develop as the enhanced metabolic stable radiotracer. Methods [18F]1-d2 was synthesized in two steps from CD2Br2, which involved the preparation of [18F]fluoromethyl bromide-d2, followed by the O-alkyation of desmethyl-PBR28. Tissue biodistribution was performed to evaluate whether [18F]1-d2 shows increasing in vivo stability compared to [18F]1 in the normal mice. Comparison of brain PET study with [18F]1-d2 and [18F]1 was carried out in the same neuroinflammatory rat model, including IC50, log D and in vitro stability. Results [18F]1-d2 was prepared in 12.4±5.1% of RCY with over 185 GBq/μmol of SA. IC50 and log D of two radiotracers showed similar values. In tissue biodistribution study, [18F]1-d2 was relatively stable in in vivo environment (%ID/g: 1.5±0.4 for [18F]1-d2 and 4.1±1.7 for [18F]1 in femur at 120 min). [18F]1-d2 was highly accumulated in the TSPO-enriched tissues such as lung and heart, whereas it exhibited comparatively low uptake and fast clearance in liver. In PET imaging studies, [18F]1-d2 was selectively accumulated in the ipsilateral striatum with high target-to-background ratio. Also, BPND of [18F]1-d2 was 1.4 times higher than that of [18F]1 and exhibited the high specific and selective binding for TSPO. Conclusions These results demonstrate that [18F]1-d2 hold promise as a novel neuroinflammation PET imaging agent in the field of brain degenerative disease. Acknowledgements Grants: 2014M3C7A1046042, 2012R1A1A2005887, 2009-0078370, HI12C0035 of South Korea. References [1] Moon BS, et al (2014) Bioconjugate Chem, 25, 442-50. Figure. Comparison of [18F]1 (A) and [18F]1-d2 (B), and TAC of two radiotracers (C) in a neuroinflammation rat model.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S247

247 A simplified production method of 18F-FMISO using a cation exchange resin for the deprotection reaction. Norifumi Abo1, Ken-ichi Nishijima1, Fei Feng2, Nagara Tamaki2, Yuji Kuge1 1 Central institute of isotope science, hokkaido university, Sapporo, Hokkaido, Japan, 2nuclear Medicine, Hokkaido University, Sapporo, Hokkaido, Japan Objectives In radiosynthesis of 18F-fluoromisonidazole (18F-FMISO), a most widely used PET tracer for hypoxia imaging, hydrochloric acid (HCl) is generally used in the deprotection reaction (Fig.1). Here we report an easy manufacturing deprotection method using a cation exchange resin aiming to simplify the synthetic processes of 18FFMISO. Methods 18F-NITTP was 18F-fluorinated from 1-(2’-nitro-1’-imidazolyl)-2-O-tetrahydropyranyl-3-Otoluensulfonyl-propandiol (NITTP) and [K/K222]18F. To the 50 µL of 18F-NITTP/CH3CN solution was added 1 M HCl 50 µL or 45 mg cation exchange resin, then heated at 100°C for 5 min. Afterwards, each reaction mixture was cooled to room temperature, then adding 1 M NaOH 50 µL to the HCl reaction vessel to neutralize it, or adding H2O 100 µL to the resin one. The rate of the deprotection reaction and the amount of K222 were determined by HPLC. Results Both HCl and resin methods achieved high deprotection rates; 94.5±0.2 % (n=3) in the HCl method and 91.8±2.1 % (n=3) in the resin one. The amount of K222 was under the detection limit in the resin method, while it was 2562 ± 80 ppm per 1µL in the HCl method. The resin method using cation exchange resin enables us oncolumn deprotection reaction. In addition, this makes it possible to simultaneously remove K222 from the reaction system. Thus, our resin method using a cation exchange resin may contribute to simplify the manufacturing processes and to develop automatic synthesis apparatus of 18F-FMISO. Conclusions A simplified synthetic process has been exploited by employing cation exchange resin in the deprotection step of 18F-FMISO synthesis. Our resin method enables us achieve on-column deprotection, as well as the removal of the Phase Transfer Catalyst K222 from the reaction system. Acknowledgements References

Figure 1: The fluorination reaction of NITTP and deprotection reaction of 18F-NITTP

J Label Compd Radiopharm 2015: 58: S1- S411

S248: Poster

21st International Symposium on Radiopharmaceutical Sciences

248 Synthesis, characterization, and in vivo evaluation of 18F-labeled 2-phenyl-imidazo[1,2-a]pyridine analog (18F-CB251) as a new TSPO PET radiotracer Byung Seok Moon1, Mara Perrone 2, Hyun Soo Park1, Valentino Laquintana2, Jae Ho Jung1, Annalisa Cutrignelli2, Angela Lopedota2, Massimo Franco2, Nunzio Denora2, Byung Chul Lee1, 3, Sang Eun Kim1, 4 1 Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam, Korea (the Republic of), 2Department of Pharmacy – Drug Sciences, University of Bari “A. Moro”, Bari, Italy, 3Advanced Institutes of Convergence Technology, Suwon, Korea (the Republic of), 4Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Korea (the Republic of) Objectives The 18 kDa TSPO has become an extremely attractive biomarker for activated microglial cells occurring in inflammatory neurodegenerative disease [1]. In this work, we report the synthesis, the characterization, and in vivo evaluation of a new TSPO PET imaging agent, [18F]CB251 ([18F]1), in a rat model of neuroinflammation. Methods [18F]1 was prepared by incorporating of F-18 into the tosylate precursor in a single-step. Comparison of neuroinflammation imaging with [18F]1 vs [11C]PBR28 was performed in the same rat model, including measurement of IC50, log D, in vitro stability, and tissue biodistribution (Fig. 1). Results [18F]1 was highly accumulated in the TSPO-enriched tissues such as the lung, heart and kidney, whereas it exhibited comparatively low uptake in liver and brain. In PET imaging studies, [18F]1 rapidly approached the highest target-to-background ratio at early imaging time and was selectively accumulated in the ipsilateral striatum. There was a greater (2.7 times) increase in ipsilateral striatum compared to contralateral (Fig. 1). The ratio of AUC in the ipsilateral and contralateral striatum of [18F]1 lesion was comparable to that of [11C]PBR28. In blocking experiment with the consecutive injection of flumazenil and PBR28, flumazenil did not intercept the uptake of [18F]1, whereas the uptake of the ipsilateral area significantly decreased after injection with PBR28. Conclusions Our results of in vitro TSPO-binding affinity, stability, in vivo tissues distribution, PET imaging studies presented here suggest that [18F]CB251 hold promise as a neuroinflammation PET imaging agent in the field of brain degenerative disease. Acknowledgements Grants: 2014M3C7A1046042, 2012R1A1A2005887, 2009-0078370, HI12COO35 of South Korea; Univ. Bari, the Italian MIUR and the Inter-University for Consortium for Research on the CMBS. References [1] Papadopoulos V, et al (2006) Trends Pharmacol Sci, 27, 402-9.

Fig. 1. Structure and brain PET/CT image of [18F]1 in LPSinduced neuroinflammatory rat model

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S249

249 Development of 18F-labeled PET Probes for C-MET Imaging Lihong Bu1, Xiaowei Ma1, Ao Zhang2, Baozhong Shen3, Zhen Cheng1 1 Radiology, Stanford University, Stanford, California, United States, 2Shanghai Institute of Materia Medica, Shanghai, China, 3The 4th hospital of Harbin Medical University, Harbin, China Objectives The important roles of mesenchymal epithelial transition factor (C-MET) in tumor biology provide strong rationales for development of C-MET targeted agents for cancer imaging and therapy. 18F labeled C-MET small inhibitors are thus highly desirable for in vivo imaging because of ideal physical properties of 18F for PET imaging. Recently, we explored 3,5-disubstituted and 3,5,7-trisubstituted quinolines as promising small molecule scaffolds to discover ATP-competitive C-MET inhibitors including AZC1 and AZC2. Our purpose here is to label AZC1 and AZC2 with 18F and further evaluate the resulting PET probes (18F-AZC1 and 18F-AZC2) for C-MET imaging in small animal tumor models. Methods 18F-AZC1 and 18F-AZC2 were synthesized using a one-step radiochemical synthesis. Their in vitro CMET targeting profiles were evaluated in C-MET positive human glioma cell lines (U87MG, U251MG, U373MG and Hs683) or negative human prostate cancer LnCap cells. The cell binding specificity and affinity and mouse serum stability of the PET probes were tested. The tumor imaging properties of the PET probes were then studied in U87MG or LnCap tumor mice (n=4). Results The PET probes were easily prepared with good radiochemical yield (5~20%, decay corrected) and high specific activity (9–15 GBq/nmol at the end of synthesis). 18F-AZC1 displayed C-MET targeting specificity but poor in vivo stability and performance for imaging of U87MG glioma. In comparison, 18F-AZC2 demonstrated high stability, binding specificity and affinity. It showed excellent tumor targeting and imaging properties and favorable biodistribution profile. High accumulation in U87MG tumors (4.28±1.04 %ID/g) and low uptake in LnCap tumors (1.31±0.53 % ID/g) (P=0.01) at 2h after injection were observed. The blocking study revealed that the U87MG tumor uptake of 18F-AZC2 could be significantly reduced to 1.79±0.44 %ID/g at 2h (P=0.02). Smallanimal PET/CT imaging demonstrated rapid tumor accumulation of 18F-AZC2, and the U87MG tumor was clearly delineated with good tumor–to–contralateral muscle ratio at 0.5h after administration of 18F-AZC2 intravenously. Conclusions Our work demonstrates that C-MET can be successfully imaged by small molecule based PET probe in vivo, and 18F-AZC2 developed is a promising C-MET targeted PET probe with high clinical translation potential. Acknowledgements This work was supported in part, by the U.S. Department of Energy (DE-SC0008397), NIH P50 CA114747, and the International Cooperative Program of the National Science Foundation of China (31210103913). References

J Label Compd Radiopharm 2015: 58: S1- S411

S250: Poster

21st International Symposium on Radiopharmaceutical Sciences

250 Evaluation of Trehalose Derivatives as Radiotracers Specific for Tuberculosis in Animal Models of Disease Gwendolyn A. Marriner1, Dale O. Kiesewetter2, François D'Hooge3, Seung S. Lee3, Omar Boutureira3, Ritu Raj3, Naseer Khan3, Laura E. Via1, Clifton E. Barry1, Ben G. Davis3 1 Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States, 2National Institute for Biomedical Imaging and Bioengineering , National Institutes of Health, Bethesda, Maryland, United States, 3Department of Chemstry, University of Oxford, Oxford, United Kingdom Objectives We are developing PET radiotracers specific to Mycobacterium tuberculosis (Mtb). In evaluating new treatments, [18F]-2-fluoro-2-deoxyglucose (FDG) has been shown to be a useful tool, but it is not highly correlated with bacterial burden. We are evaluating trehalose radiotracers which may be covalently incorporated in vivo into the Mtb cell wall to give a PET signal directly correlated to bacterial burden rather than reporting on inflammation or other host response. Methods We have designed several [18F]-labeled trehalose derivatives and are evaluating them in animal models of tuberculosis. Two of the probes, [18F]-6-fluoro-6-deoxytrehalose (6-FTre) and [18F]-epi-4-fluoro-4-deoxytrehalose (epi-4-FTre) are chemically synthesized in a method similar to [18F]-2-fluoro-2-deoxyglucose (FDG) from peracetylated precursors, and one probe, [18F]-2-fluoro-2-deoxytrehalose (2-FTre), is chemoenzymatically synthesized in a one-pot, three-enzyme cocktail from [18F]-FDG. All three were initially evaluated in Mtb-infected rabbits by acquiring PET/CT scans and blood and urine samples for metabolism analysis. The 2-FTre probe is also currently being evaluated in a non-human primate model of tuberculosis. Results In initial experiments in Mtb-infected rabbits, the 6-FTre probe was rapidly metabolized to [18F]-6-fluoro6-deoxyglucose. However, the epi-4-FTre was metabolically stable and did label lesions in the rabbit lung. A typical signal-to-noise for labeling was 2-3:1. The 2-FTre gave a slightly higher signal-to-noise and also appeared to give some differential labeling when compared to FDG. However, we saw potential probe metabolism and decided to change to a marmoset model of tuberculosis, which should express lower levels of trehalase than rabbits. The 2-FTre is currently under evaluation in a marmoset model of disease and appears to be labeling lesions selectively. No metabolism is observed in blood or urine of the marmosets. Conclusions The trehalose derived probes are demonstrate uptake in PET scans at the site of Mtb lesions. This technology will allow us to selectively label Mtb infection and monitor treatment in animals on experimental drug regimens. Acknowledgements Technical assistance from Emmanuel Dayao, Daniel Schimel, Danielle Weiner, Katherine Robbins, Michelle Sutphin and Michael Goodwin is gratefully acknowledged. This research was supported in part by the Intramural Research Program of the NIH, NIAID and by the Bill and Melinda Gates Foundation. References Chen, RY; Dodd, LE; Lee, M; Paripati, P; Hammoud, DA; Mountz, JM; Jeon, D; Zia, N; Zahiri, H; Coleman, MT; Carroll, MW; Lee, JD; Jeong, YJ; Herscovitch, P; Lahouar, S; Tartakovsky, M; Rosenthal, A; Somaiyya, S; Lee, S; Goldfeder, LC; Cai, Y; Via, LE; Park, SK; Cho, SN; Barry, CE 3rd. Sci. Trans. Med. 2014, 6(265), 265ra166. Backus KM, Boshoff HI, Barry CS, Boutureira O, Patel MK, D'Hooge F, Lee SS, Via LE, Tahlan K, Barry CE 3rd, Davis BG. Nat. Chem. Biol. 2011;7(4):228-35.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S251

251 Synthesis and First ex-vivo Results of [19F]-Fluoroethyl-resorufin for Imaging of Cerebral Amyloid Angiopathy in APP23 Mice Andreas Maurer, Christian Kesenheimer, Ramona Stumm, Florian C. Maier, Bernd J. Pichler Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University Tübingen, Tuebingen, Germany Objectives In an aging society, the study and treatment of neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease plays an increasingly important role in biomedical research. Cerebral Amyloid Angiopathy (CAA) is closely related in its biochemical pathway to Alzheimer’s disease (AD). In contrast to AD, fibrillary deposits of amyloid b are formed in the cortical vessels and not in the cerebral parenchyma [1]. AD and CAA can lead to pronounced cerebrovascular dysfunction, characterized by impaired neurovascular and metabolic regulation of cerebral blood flow. To date, there is only a styrylpyridine based PET in-vivo imaging agent available for the differentiation of vascular from neuritic amyloid b deposits [2] while [11C]-PIB, a widely used tracer for AD, is unable to discriminate the different types of plaques for the investigation of cerebral amyloid angiopathy [3]. We aim to develop a PET probe which allows us to selectively image CAA in comparison to parenchymal deposits. The tracer is based on the structure of resorufin and ethyl resorufin, published by Han et al. [4]. Methods Resorufin was alkylated by using [19F]-fluoroethyl-tosylate with potassium carbonate in dimethylformamide. In a preliminary study, we have prepared the brain slices of a 21-month old APP23 mouse and stained them with 0.05 % solutions of resorufin and its [19F]-fluoroethyl derivative respectively. Results Despite the dramatically reduced fluorescence of the fluoroethylated molecule, fluorescence microscopy was successfully used to evaluate its binding to CAA plaques. In ex-vivo studies we could show that [19F]fluoroethyl-resorufin exhibits a similar binding selectivity towards CAA Ab deposits as the parent compound resorufin. Through the alkylation at the phenolic group we increased the lipophilicity in comparison to the parent compound which enables the fluoroethylated compound to cross the blood brain barrier. This could already be confirmed in a first experiment with C57BL/6 wild type mice. Conclusions The synthesized resorufin derivative is a promising candidate for an application as a selective CAA PET tracer for the non-invasive imaging in neurodegenerative diseases. After demonstrating the binding of the non-radioactive compound, we are now establishing the automated synthesis of the F-18 PET tracer. Acknowledgements References [1] J. Klohs et al., Frontiers Aging Neurosci. 2014, 6, 32, 1-30. [2] Z. Zha et al., J. Med. Chem. 2011, 54, 8085-8098 [3] a) M. E. Gurol et al., Ann. Neurol. 2013, 73, 529-536. b) M. E. Gurol et al. Neurology 2012, 79, 320-326 c) J.C. Baron et al., J. Cereb. Blood Flow Metab. 2014, 1-6 [4] Han et al., Mol. Neurodegen. 2011, 6, 86

J Label Compd Radiopharm 2015: 58: S1- S411

S252: Poster

21st International Symposium on Radiopharmaceutical Sciences

252 The Automated Production of 18F-Labeled PET Molecular Imaging Probes using a Microfluidic Chip-based Synthesizer Mingwei Wang1, 2, Yong-Ping Zhang1, 2, Ying-Jian Zhang1, 2 1 Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Shanghai, China, 2Center for Biomedical Imaging, Fudan University, Shanghai, China Objectives The microfluidic-based synthesizers have been mainly investigated to optimize the reaction conditions of 18F-labeled PET tracers because of the intrinsic advantages over conventional methods. However, it meets some barriers for the batch production of 18F-radiopharmaceuticals. Herein, we aimed to develop the automated production of 18F-labeled PET imaging probes using the microfluidic chip-based synthesizer designed in our lab. Methods The microfluidic chip-based synthesizer, consisting of PDMS chip and microreactor, and semipreparative HPLC were used to run the automated preparation and purification of 18F-labeled probes, exemplified by 18F-fluoroacetate (18F-FAC), 2-(5-[18F]fluoropentyl)-2-methyl malonic acid (18F-ML-10) and 18F-FDG. Starting from the high activity of 18F ion (~ Curie level) in target water (2.4mL) and 1/10 precursor amount used before (Table 1), the batch production of 18F-FAC, 18F-ML-10 and 18F-FDG were performed using the corresponding control program based on the radiochemical route, respectively. The radiochemical yield and purity were measured. Results Our microfluidic chip-based synthesizer was proved again to be competent for the automated preparation of 18F-labeled PET imaging probes. The batch production of 18F-FAC, 18F-ML-10 and 18F-FDG were accomplished under the control program, respectively. As results, 100~200 mCi of 18F-FAC and 18F-ML-10 and 300~500 mCi of 18 F-FDG could be obtained, starting from the curie level of 18F ion and trace amount of precursor (Table 1). The synthesis time and radiochemical yield were comparable with that of conventional and commercially available modules. The radiochemical purity was all greater than 97%, qualified the requirement of radiopharmaceuticals. Conclusions The automated production of 18F-labeled PET molecular imaging probes, including 18F-FAC, 18FML-10 and 18F-FDG, was developed using our microfluidic chip-based synthesizer with the advantages of low consumption of precursor, low cost and good radiochemical yield. Acknowledgements Research was supported by SRF for ROCS, SEM and partly by National Natural Science Foundation of China (No. 11275050, No. 30700188). References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S253

253 Clinical grade [18F]FTC-146: Radiosynthesis of sigma-1 receptor ligand for human PET studies Bin Shen1, Jun Hyung Park1, Natasha C. Arksey1, C. R. McCurdy2, Frederick T. Chin1 1 Radiology, Stanford University, Stanford, California, United States, 2Department of BioMolecular Sciences, University of Mississippi, University, Mississippi, United States Objectives PET imaging with sigma-1 receptor (S1R) radioligands may provide useful insights into S1R-related diseases. The current clinical S1R radiotracer [11C]SA4503 (Ki = 4.6 nM [1]) displays significant affinity for S2R (Ki = 63 nM [1]) and vesicular acetylcholine transporters (VAChT; Ki = 50 nM [2]), which may confound imaging results for S1R. We previously reported the radiosynthesis of [18F]6-(3-fluoropropyl)-3-(2-(azepan-1yl)ethyl)benzo[d]thiazol-2(3H)-one ([18F]FTC-146) that demonstrated high affinity for S1R (Ki = 0.0025 nM) and superior selectivity for S1R over S2R (Ki = 364 nM) and VAChT (Ki = 450 nM). PET studies in wild-type and S1R knockout mice, and non-human primates showed that [18F]FTC-146 is highly specific and selective for S1Rs. Since [18F]FTC-146 presents excellent S1R imaging potential, we want to translate it for our clinical S1R-PET studies in normal and S1R-related disease subjects. Methods [18F]FTC-146 was synthesized via aliphatic nucleophilic substitution ([18F]fluoride/tosylate exchange) using TRACERlab FX FN. Briefly, tosylate precursor solution (2 mg in 1 mL anhydrous DMSO) was added to azeotropically dried 18F/K222/K2CO3 complex, heated to 150 oC for 15 min, and the crude product was then purified by semi-preparative HPLC. The [18F]FTC-146 HPLC fraction was formulated in 0.9% NaCl containing no more than 10% ethanol via sterile filtration. Quality control criteria were set and the tests were performed according to USP 823. Results [18F]FTC-146 was obtained in 1.8±0.6% radiochemical yield with a specific radioactivity of 5.0±1.7 Ci/μmol (n=4, decay-corrected to EOS). Both radiochemical and chemical purities were > 99%. Stability tests also showed that prepared doses were stable for 4 hours at ambient temperature. All other QC test results met specified criteria and the radiochemistry protocols were set for our future clinical studies. Conclusions We have performed four consecutive validation runs for preparing clinical-grade, highly S1Rselective radiotracer [18F]FTC-146. These validation runs and eIND application have been submitted for FDA review, and the first human study to examine tracer biodistribution in different S1R-related human disease subpopulations is planned for early 2015. Acknowledgements References [1] Lever JR, et al (2006) Synapse, 59, 350-8. [2] Ishiwata K, et al (2006) Nucl Med Biol, 33, 543-8.

J Label Compd Radiopharm 2015: 58: S1- S411

S254: Poster

21st International Symposium on Radiopharmaceutical Sciences

254 Microfluidic optimization and production of [18F]FP-TZTP Giancarlo Pascali, Lidia Matesic, Ivan Greguric LifeSciences, ANSTO, Sydney, New South Wales, Australia Objectives The study of Muscarinic receptor regulation has been related to Alzehimer’s Disease (AD) mechanism. In fact, loss of M2 subtype population has been reported in diseased conditions, and efficient M2 imaging agents would provide novel basis for diagnosis and follow-up of AD. The most promising PET imaging agent for M2 is currently [18F]FP-TZTP; this tracer was first produced using a two-step synthetic process, that was subsequently improved using one-step labelling of either a mesylate or tosylate precursor1,2. Due to our interest in AD imaging, we investigated the radiolabelling and production of [18F]FP-TZTP using a microfluidic approach. Methods The mesylate precursor was radiofluorinated using a non dried fluoride complex on an Advion NanoTek System (Figure 1). Precursor concentration, flow rates and temperature were optimized following a reported protocol3. The optimal radiofluorination process was coupled to automated HPLC/SPE purification and reformulation, to produce injectable solutions of [18F]FP-TZTP. Results Maximum incorporation RCY of 78% was obtained at 190°C at a total flow rate of 40µL/min (15.6 μL reactor). RCY was decreased by employing faster flow rates and lower temperatures, but also by increasing precursor concentration from 2 mg/mL to 10 mg/mL (16% RCY). The full production of [18F]FP-TZTP was initially attempted by on-line dilution with water prior trapping onto an OASIS cartridge, then followed by injection in a semi-prep HPLC. We realized that this pre-concentration step resulted in the degradation [18F]FPTZTP. Therefore, full production was performed by simple injection of the untreated reaction mixture. The final product was then obtained in 16±2% RCY in a 45 min procedure, with a specific activity of >300GBq/µmol (n=5). Conclusions During the radiolabelling of [18F]FP-TZTP, we found, for the first time in a microfluidic environment, that an increase in precursor quantity actually reduced the RCY, instead of plateauing out. In addition, we verified that on-line dilution of the reaction mixture with water contributed to the degradation of the product, most likely by defluorination. Using the best process, we produced [18F]FP-TZTP in sufficient quantities for preclinical imaging, obtaining a yield comparable to reported literature but a nearly doubled specific activity. Acknowledgements References [1] Kiesewetter, DO, et al (2003), Nuc Med Biol, 30, 73-77. [2] Van Oosten, ER, et al (2009), Appl Rad Isot, 67, 611-616. [3] Pascali, G, et al (2014), Nature Prot, 9, 2017-2029.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S255

255 Synthesis of [18F]Tetrafluoroborate via Radiofluorination of BF3 Huailei Jiang, Mukesh K. Pandey, Timothy R. DeGrado Mayo Clinic, Rochester, Minnesota, United States Objectives The sodium/iodide symporter (NIS) is an important target in thyroid disease and reporter gene imaging. Although [18F]tetrafluoroborate ([18F]TFB) has shown promise for NIS imaging [1], the synthesis method of isotopic exchange on unlabeled TFB gives suboptimal radiochemical yield and specific activity. The aim of our study was to produce [18F]TFB via direct radiofluorination on BF3 to enhance both yield and specific activity. Methods 18F-fluoride (37-74 MBq) in 2 mL deionized water was trapped on a QMA (46 mg, carbonate form) cartridge. The QMA cartridge was rinsed with 10 mL anhydrous acetone, and flushed with nitrogen for 2 min. A solution of BF3 (5 mL) predissolved in THF (0.5 mL)/petroleum ether (25 mL) was purified through a column of 200 mg MP-64 resin, and then passed through the QMA cartridge in 10 s to react with 18F-flouride. Both 18Fflouride and [18F]TFB remain on the QMA cartridge. The QMA cartridge was rinsed with a solution of 0.1 mL trifluoroacetic acid in 20 mL THF to remove the unreacted BF3 complex, and followed by another 20 mL of THF rinse. The QMA cartridge was further dried with nitrogen for 2 min, and then treated with 2 mL 0.9% saline to elute the product from the resin. The crude product solution was purified by 2 alumina-N SepPak cartridges to remove the unreacted 18F-fluoride. Results The radiochemical yield of [18F]TFB was 18±2% (n = 5) uncorrected in a synthesis time of 8 min. Radiochemical purity was >99% as shown on silica gel TLC (MeOH, Rf = 0.8) or anion chromatography HPLC (Dionex IC-2100, AS19 analytical column 4.7 x 150 mm, 35 mM KOH, 1 mL/min, Rt = 8.3 min). Specific activities of 78±20 MBq/mg (n = 5) were achieved despite the modest starting activities of 37-74 MBq. Conclusions A solid-phase supported synthesis of [18F]TFB was developed via radiofluorination of BF3. Scale up of the synthesis to higher starting radioactivity levels is in progress. Acknowledgements The work was supported by the Department of Radiology, Mayo Clinic. References [1]. Jauregui-Osoro MK et al. (2010) Eur J Nucl Med Mol Imaging, 37, 2108-2116.

J Label Compd Radiopharm 2015: 58: S1- S411

S256: Poster

21st International Symposium on Radiopharmaceutical Sciences

256 Synthesis of radiobrominated amino acid derivatives via silicon-bromine exchange reaction Shigeki Watanabe1, Keiichi Yamada2, Saki Watanabe2, Hiroyuki Oku2, Tomohisa Moriguch2, Kazuo Shinozuka2, Noriko S. Ishioka1 1 Quantum Beam Science Center, Japan Atomic Energy Agency, Takasaki, Japan, 2Graduate School of Science and Technology, Gunma University, Kiryu, Japan Objectives 76Br (half-life: 16.1 h) is of interest for in vivo imaging using PET. Therefore, 76Br labeled peptides which have high affinity to tumor cells are promising PET tracers for tumor imaging. Tin-halogen exchange reaction have been used for radiobromination. However, it is likely that synthesis of stannylated peptides is difficult because stannyl group is readily eliminated under acidic condition. Silicon-carbon bond is resistant to acid, leading us to employ silicon-bromine exchange reaction for the radiobromination. In this study, we are aiming to synthesize a radiobrominated phenylalanine derivative via the silicon-bromine exchange reaction as a feasible study. Methods 77Br (half-life: 57 h) was used instead of 76Br. 77Br was produced via the natSe(p,n)77Br reaction and then isolated by the dry distillation. A silylated phenylalanine derivative is synthesized as shown in Figure 1. Radiobromination was carried out at room temperature for 15 min in the presence of tert-butyl hypochlorite (TBHC). HPLC and TLC were used for characterization of the desired compound and evaluation of labeling efficiency. Results Efficient amount of 77Br (~ 10 MBq) was successfully harvested by the dry distillation in 66% recovery efficiency. Silicon-substituted precursor 2 was readily prepared from 1 in the presence of Rh complex in a good yield (56%). HPLC analysis showed that radiobromination was quantitatively accomplished within 15 min when using TBHC. TLC analysis indicated that the desired compound was synthesized in an excellent labeling yield ( >90%). Conclusions The silicon-bromine exchange reaction has shown to give high labeling efficiency for radiobromination of the amino acid derivative. The reaction will be available for preparation of radiobrominated peptides. Acknowledgements We are grateful to Dr. Satoshi Watanabe for the production of 77Br at the Japan Atomic Energy Agency. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S257

257 A F-18 Labeled Enantiomeric PET Radiotracer for σ1 Receptors Xuyi Yue1, Hongjun Jin1, Xiang Zhang1, Hui Liu1, Junfeng Li1, Hao Yang1, Hubert P. Flores2, Joel S. Perlmutter1, 2, Stanley M. Parsons3, Zhude Tu1 1 Mallinckrodt Institute of Radiology, Washington University in St. Louis, St. Louis, Missouri, United States, 2 Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, United States, 3 Department of Chemistry and Biochemistry, University of California, Santa Barbara, California, United States Objectives The σ1 receptor plays key roles in neuropsychiatric and neurodegenerative diseases. It is considered as an important biomarker for central nervous system disorders. Our group reported previously a promising σ1 selective radioligand [18F]TZ3108 which is a racemic compound. It’s well known that the σ1 receptor has stereoselective binding. Therefore, in the current study we focused on exploring the potent enantiomer of [18F]TZ3108 and characterizing its in vivo binding properties targeting the σ1 receptors in rats and nonhuman primates. Methods Racemic TZ3108 precursor and standard TZ388 were resolved by Chiralcel OD column. The binding affinity of the racemic, optical pure standards towards σ1, σ2, vesicular acetylcholine transporter (VAChT) were determined by in vitro competitive binding assays. The biodistribution of (-)-[18F]TZ388 was conducted in Sprague-Dawley rats at 5, 30, 60, 120 min (n = 3 for each group) p.i. and the tracer was preliminarily validated in adult male cynomolgus macaques. Results The ee values for both isomers of the precursor, standards were above 95% upon resolution by Chiralcel OD column. Competitive binding assay showed that (-)-TZ388 (Ki-σ1 = 0.031 ± 0.001 nM) was 150-fold more potent than the (+)-TZ388 (Ki-σ1 = 4.55 ± 2.00 nM). More importantly, (-)-TZ388 (Ki-σ2 = 6958 ± 809 nM, Ki-VAChT = 976 ± 87 nM) exhibited 200,000-fold selectivity over σ2 and 30,000-fold selectivity over VAChT. The radiolabeled (-)-[18F]TZ388 was obtained in 11% decay corrected yield (specific activity > 2 Ci/µmol) within approximately 70 min. The biodistribution showed the total brain uptake was obvious at 5 min (1.285 ± 0.062 %ID/g) and remained high at 120 min (0.802 ± 0.129 %ID/g). Kidney uptake indicated a rapid kidney excretion. The distribution of (-)-[18F]TZ388 in the brains of rats and monkeys is consistent with the reported distribution of the σ1 receptor. Conclusions We successfully resolved the racemic σ1 ligand TZ3108 to obtain the enantiomeric minus and plus isomers. The minus isomer (-)-TZ388 was over 150-fold potent than plus (+)-TZ388. In vivo evaluation of (-)[18F]TZ388 in rats and monkeys suggested (-)-[18F]TZ388 is a promising PET tracer for quantifying sigma-1 receptor in the brain. Acknowledgements Research support from NIH/NINDS and NIH/NIMH Grants: MH092797, NS061025 and NS075527. References

J Label Compd Radiopharm 2015: 58: S1- S411

S258: Poster

21st International Symposium on Radiopharmaceutical Sciences

258 Development of 18F-Labeled Peptides for Imaging EGFL7 in Prostate Cancer Lihai Yu2, Choi-Fong Cho3, John Lewis3, Ting-Yim Lee1, Michael Kovacs4, 1, Leonard Luyt1, 2 1 University of Western Ontario, London, Ontario, Canada, 2London Regional Cancer Program, London, Ontario, Canada, 3Oncology, University of Alberta, Edmonton, Alberta, Canada, 4Lawson Health Research Institute, London, Ontario, Canada Objectives Angiogenesis plays a critical role in the expansion and metastasis of tumours. Epidermal growth factorlike domain 7 (EGFL7), a protein secreted almost exclusively by endothelial cells, promotes endothelial cell sprouting during angiogenesis and is highly expressed in human tumours. We aim to develop peptide-based 18Flabeled PET imaging agents targeting EGFL7 to image angiogenesis and to allow for non-invasive monitoring of anti-angiogenic therapy. Methods Two peptides were discovered with high binding affinity to EGFL7 protein as determined by SPR analysis. One octapeptide (HMYFLLGH: KD = 13.2 nM) was discovered by high-throughput one-bead onecompound (OBOC) methodology. The other peptide (GLQQFSHVLLSG: KD = 14.3 nM) was based on a fragment of EGFL7 from within the dimerization region. Peptides as fluoride-containing analogues, [18F]1 and [18F]2, were prepared successfully by CuAAC click chemistry with high specific activity and radiochemical purity based upon HPLC analysis (Figure 1A). A series of biological studies in vitro and in vivo were carried out to evaluate their ability to target EGFL7. Results Cell uptake data shows that [18F]1 retained binding affinity to EGFL7 protein, as indicated by higher uptake in EGFL7-HT1080 cells than in control HT1080 cells, however, [18F]2 lacked specificity. Biodistribution data indicates [18F]1 has a higher uptake ratio of tumor to blood/muscle in EGFL7-HT1080 tumor models (1.4 for tumor/blood and 3.5 for tumor/muscle) than in control HT1080 tumor mice (1.0 for tumor/blood and 2.3 for tumor/muscle). In a prostate cancer model, [18F]1 was able to clearly image the LNCaP tumour at 60 min (Figure 1B). Conclusions Here we report on the de novo discovery of a peptide-based PET imaging agent capable of targeting EGFL7 in vivo. [18F]1 was successfully synthesized via CuAAC click chemistry and was determined to bind EGFL7 protein in vitro and in vivo. Further preclinical evaluation will determine the potential clinical application of this agent as a method of imaging angiogenesis. Acknowledgements Financial support from the Ontario Institute for Cancer Research (OICR). References

Figure 1. (A) Synthetic strategy for [18F]1 and [18F]2; (B) PET image of [18F]1 in LNCaP mouse at 60 min postinjection.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S259

259 Novel Microscale Automated System for F-18 Radiopharmaceutical Synthesis Joe O. Armstrong2, Jeanne M. Link1 1 Oregon Health and Science University, Portland, Oregon, United States, 2University of Washington, Seattle, Washington, United States Objectives The objective of this project was to create a microscale F-18 synthesis system capable of producing radiochemical yields comparable to commercial synthesis systems. The system was designed to be flexible enough to synthesize any one pot F-18 radiopharmaceutical using reduced amounts of reagents in order to decrease the cost of synthesis. Two model compounds were tested, 2-[18F]-2-fluoro-2-deoxy-D-glucose (FDG) and [18F]fluoromisonidazole (FMISO). Methods All reagents were from Sigma-Aldrich Chemical Co unless noted. A Chromafix F-18 cartridge (GE Healthcare) was used to separate F-18 from O-18 water. The F-18 was eluted from the cartridge in the opposite direction into the reaction vessel using 70 μL of 0.04M K2CO3 followed by 2.25 mg [2.2.2] Kryptofix in 500 μL acetonitrile. The solution was dried for 6.5 minutes at 90°C-100°C under vacuum and with argon flow. The precursor was added and the reaction vessel was closed off for the duration of the reaction. Radiochemical yields were assessed using Silica Gel TLC with 100% acetonitrile as a solvent. The TLC was placed into an EZ-Scan TLC scanner. Two peaks were observed in the analysis of the FMISO and FDG crude products. The two peaks, the labeled peak and the free F-18 peak, were used to determine radiochemical yield. Results The syntheses of FMISO and FDG were complete in approximately 21 minutes. The microscale system uses five valves, is less than 0.75 ft3 in size, and can synthesize FDG and FMISO in twelve programming steps with radiolabeled yields comparable to large-scale commercial synthesis units. The system can support up to eight additions following the F-18 labeling. The radiochemical yields shown in table 1 demonstrate the labeling capabilities of the system. Conclusions Research oriented groups can benefit from this unit’s simplicity and efficiency, while also maintaining low radiation exposure to the chemist. Acknowledgements NCI PO1 CA042045 and Xuehe Li, PhD References

Table 1. Model Compound Radiochemical Yields

J Label Compd Radiopharm 2015: 58: S1- S411

S260: Poster

21st International Symposium on Radiopharmaceutical Sciences

260 Synthesis of 18F-TH302 for PET hypoxic tumor imaging Dhanabalan Murali, Todd E. Barnhart, Robert J. Nickles, Onofre T. DeJesus Department of Medical Physics, University of Wisconsin, Madison, Wisconsin, United States Objectives 2-Nitroimidazoles undergo hypoxia-selective reduction in biological systems. TH302 (1, Fig. 1) is a potent and highly selective hypoxia-activated phosphoramidate mustard analog of 2-nitroimidazole.1 The goal of this work is to synthesize 18F labelled TH302 (2) and assess its suitability for PET imaging of hypoxic tumors. Methods TH302 was purchased from MedChem Express, NJ and used as received. [18F]F- prepared via (p,n) reaction of [18O]H2O was trapped on QMA, eluted with aq.CH3CN/K2CO3/Kryptofix and dried. Compd. 1 in DMSO was added to [18F]F- and stirred at rt. for 5-8 min, diluted with H2O and passed through C18 and washed with H2O. The cold standard for 2 is prepared as shown in scheme 1 and is characterized by NMR and mass spectroscopy. Analytical HPLC consisted of a C18 4.6x150mm 5u column and 28% aq.CH3CN mobile phase at 1 mL/min flow rate. Results Chloro and bromophosphoramidate mustards readily undergo nucleophilic displacement reactions in which the Cl or Br group is replaced by the nucleophile. So, initially, direct fluorination of 1 with [18F]F- was attempted for the synthesis of 2. About 50% of the radioactivity of the reaction mixture was trapped in C18. And almost all of the activity trapped in C18 was eluted with 1ml CH3CN. HPLC analysis of the CH3CN elute showed almost a single product with a retention time (RT) ca. 2 min. Under the same HPLC conditions TH302 eluted at 11 min. Based on the RT, it is suspected that the 18F fluorination product is compound 3 formed by the nucleophilic attack of [18F]F- at the imidazolyl CH2 and -OP(O)NR2 moiety acting as the leaving group. Current efforts are to definitively identify the reaction product and to synthesize compound 2 using more reactive 4 as the precursor for the 18F-fluorination step. Conclusions A precursor with more reactive leaving group such as –OTf is required for synthesis of 2 by direct nucleophilic fluorination. Other routes to prepare 2 such as those using 18F-labelled synthons will be explored. Acknowledgements References 1. J-X Juan, et al., J. Med. Chem. 2008, 51, 2412-2420

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S261

261 Synthesis and biological evaluation of [18F]DOPP for FAAH imaging Rajesh Kamble1, Maria Baron1, Zijing Li1, Shankar Vallabhajosula2, Alan Wilson3, Yu-Shin Ding1 1 NYUMC, New York, New York, United States, 2Cornell, New York, New York, United States, 3University of Toronto, Toronto, Ontario, Canada Objectives Fatty acid amide hydrolase (FAAH), an enzyme responsible for regulating endocannabinoid signaling, plays an important role in pain, addictions and neurological disorders. Although [11C]CURB has advanced to clinical trials, 18F-labeled radiotracers with longer half-life would present additional advantages for non-invasive clinical imaging of FAAH. Biodistribution and autoradiography studies of [18F]DOPP in rats and recent PET studies in monkeys showed promising results. It is therefore imperative to develop an efficient, reliable precursor synthesis. The modified precursor synthesis, radiosynthesis and in vivo microPET imaging in rats will be discussed. Methods The reported synthesis of the intermediate oxazoline from 3-cyanophenol gave unreliable and low yields (~10%). The procedure was thus modified: esterification of 3-hydroxybenzoic acid followed by treatment with ethanolamine and cyclization. [18F]DOPP was obtained in a three-step radiosynthesis using GE FXFN module. [18F]DOPP (< 1 mCi) was injected via tail-vein and dynamic imaging (Siemens Inveon) was carried out for 1 h. Blocking study was conducted in rats pretreated with URB597 (FAAH inhibitor, 2 mg/kg, IP, 30 min prior). Results The oxazoline intermediate was efficiently synthesized in reliable yields (>48%) via the modified procedure, leading to the FAAH precursor in an overall 21% yield. One-pot three-step radiosynthesis consistently afforded [18F]DOPP in approx. 14% yield (RCY) with high radiochemical purity (>99%) and high specific activity (~1.4 Ci/µmol). PET imaging shows high brain uptake, consistent with reported regional FAAH enzyme activity. Pretreatment with URB597 completely abolished binding in all brain regions, demonstrating exceptional specificity of the radiotracer for FAAH (images attached). Conclusions To our knowledge, this is the first microPET imaging study in rats using [18F]DOPP. Results indicate that [18F]DOPP is a promising PET ligand for FAAH imaging. Acknowledgements References

Three views of two rats after injection of [F-18]DOPP: Control (left), URB587 (right).

J Label Compd Radiopharm 2015: 58: S1- S411

S262: Poster

21st International Symposium on Radiopharmaceutical Sciences

262 Improved Syntheses of Reference Standard and Precursors for Tau Imaging Agent, [18F]AV-1451 Carey L. Horchler, Jennifer Clemens, Adam Hoye, Ximin Li, Hui Xiong, Giorgio Attardo Discovery Research, Avid Radiopharmaceuticals, Philadelphia, Pennsylvania, United States Objectives Imaging tau pathology is of great interest in the field of Alzheimer’s research. [18F]AV-1451 ([18F]T807) is emerging as a useful PET imaging agent for detecting tau in AD patients. In the course of developing this tracer, we have explored multiple precursors and scalable syntheses. Herein, we describe the optimized syntheses of [18F]AV-1451 reference standard, AV-1451 and two precursors, AV-1524 and AV-1622. Methods The first synthesis of [18F]AV-1451 reference standard relied on sequential Suzuki reactions followed by cyclization to the final gamma-carboline product. Standard coupling conditions and careful chromatographic purification afforded the necessary pure biaryl intermediate 1. Following the second Suzuki reaction, stringent control of reaction temperature and concentration allowed cyclization of 2 in good yield to afford AV-1451. A second synthesis commenced from 7-bromo-5H-pyrido[4,3-b]indole and provided AV-1451 in two synthetic steps utilizing a one-pot Suzuki coupling and deprotection as the final step. Our optimized synthesis of AV-1524 was accomplished in five synthetic steps featuring a protection/deprotection strategy to allow a more facile Suzuki coupling of the nitro pyridine moiety, easier purification, and introduction of a palladium scavenging step. Optimization of reaction conditions and workup methods also allowed for a scalable synthesis of AV-1622. This synthesis employed a novel synthetic method to introduce the N,N,N-trimethylpyridin-2-aminium moiety. Results Two syntheses of AV-1451 reference standard were developed. The overall yields of 40 and 60% were a marked improvement over the original synthesis (19% yield). The second route provided greater ease of purification. The reported synthesis of AV-1524 was amenable to scale up and had an improved overall yield (42%) relative to previously reported syntheses. AV-1622 was synthesized in four steps and 28% overall yield. Production of [18F]AV-1451 using this precursor has proven to be efficient and offers technical advantages over other known precursors of [18F]AV-1451. Conclusions Improved processes for AV-1451, AV-1524 and AV-1622, have been achieved. Original yields have been significantly improved to allow robust production of 20-50 gram batches of these compounds. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S263

263 Intricacies of 18F-labelling of exendin-4 via click chemistry Paula Lehtiniemi, Cheng-Bin Yim, Kirsi Mikkola, Pirjo Nuutila, Olof Solin Turku PET Centre, University of Turku and Åbo Akademi University, Turku, Finland Objectives A proof-of-concept study in type 1 diabetic patients and healthy subjects showed a correlation between beta cell mass and uptake of 111In-exendin-4, which could lead to further insight into the pathophysiology of diabetes [1]. Recent results with 18F-exendin-4 analogues demonstrated enhanced pharmacokinetic properties, as compared to radiometal-labelled counterparts [2]. This report describes in detail considerations during the synthesis of [18F]exendin-4 (Fig. 1). Methods Several conditions and solvents were investigated, which led to the following methodology. Alkyne tosylate 1 and the 18F-fluoride-Kryptofix complex were allowed to react in DMSO (5 min at 100 °C). [18F]2 was isolated by preparative HPLC on a C12 Proteo column using a methanol gradient. The collected intermediate was diluted and concentrated on an HLB cartridge, followed by elution with THF (300 µL). Peptidyl azide was added to react with [18F]2, in the presence of CuSO4/Na-ascorbate, for 10 min at room temperature. The reaction mixture was applied on a SepPak C8 cartridge (50 mg), washed with increasing ethanol concentrations, and [18F]exendin-4 was eluted with ethanol. Radiochemical analysis was performed by TLC and HPLC. Results The conversion of tosylate precursor was on average 50 %. Preparative isolation of [18F]2 was very effective with high purity (> 99 %). [18F]exendin-4 was obtained with a yield of approximately 25 % (from [18F]2, uncorrected) and a purity of 85-89 %. Conclusions Much of the difficulties during radiosynthesis is attributed to the very sticky nature of exendin-4. However, the present study showed the applicability of SepPak C8 for isolating [18F]exendin-4. Current studies are underway to improve overall yield and purity. Acknowledgements Supported by the Finnish Centre of Excellence in Molecular Imaging in Cardiovascular and Metabolic Research. References [1] Brom M, et al. (2014) Diabetologia, 57, 950-959. [2] Mikkola K, et al. (2013) Diabetologia, 56, S211-S212.

J Label Compd Radiopharm 2015: 58: S1- S411

S264: Poster

21st International Symposium on Radiopharmaceutical Sciences

264 Facile 18F radiosynthesis of olaparib derivatives as potential poly[ADP-ribose] polymerase 1 (PARP-1) imaging agents Dong Zhou1, Jinbin Xu1, Wenhua Chu1, Delphine L. Chen1, Kooresh . Shoghi1, John Katzenellenbogen2 1 Radiology, Washington University in Saint Louis, Saint Louis, Missouri, United States, 2Department of Chemistry, University of Illinois, Urbana, Illinois, United States Objectives Molecular imaging of poly[ADP-ribose] polymerase 1 (PARP-1) activity in vivo using positron emission tomography (PET) is urgently needed in research and in clinical trials in which PARP-1 is the target. Olaparib is a commonly used PARP-1 inhibitor, and it tolerates major modification without losing its PARP-1 inhibitory potency. The objective of this project is to develop a facile 18F radiosynthesis of olaparib derivatives as potential PARP-1 imaging agents. Methods A triazole derivative was radiosynthesized by a Cu(I)-catalyzed click reaction with 2-[18F]fluoroethyl azide ([18F]FEA), which was isolated with a Waters Oasis HLB cartridge after 18F incorporation reaction. A 4fluorobenzamide derivative was radiosynthesized by conjugation reaction with 4-[18F]fluorobenzoic acid ([18F]FBA) or N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). The radio-tracers were purified by HPLC. The potential of PARP-1 imaging was evaluated in cell uptake and animal imaging studies. Results Both derivatives of olaparib were readily radiosynthesized in high yields. The reactions were complete in 10 min, and HPLC purification afforded final products in good purity. As shown in Figure 1, the FBA derivative (18F]DZ-1) was taken up in PC3 and LNCaP cells and uptake was markedly blocked by olaparib, affording comparable results to those of WC-4-138,1 a known PARP-1 tracer. There results suggest that the uptake is specific and due to PARP-1 activity in these cells. The animal imaging study also indicated the potential of this tracer for imaging PARP-1 activity in cardiovascular injury. Conclusions Two derivatives of olaparib have been readily prepared in F-18 labeled form. Preliminary results demonstrated that both tracers deserve further evaluation for imaging PARP-1 activity in tumors and in cardiovascular injury. Acknowledgements DOE DE-SC0005434 and DE-SC0002032, NIH CA25836 References 1. Zhou, et al (2014) Bioorg. Med. Chem. 22, 1700.

Figure 1. Cell uptake and blocking studies in PC3 and LNCaP cells

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S265

265 A Perfluoroaryl-Cysteine SNAr Chemistry Approach towards F-18 labeling of Bioactive Peptides Jitendra K. Mishra1, Chi Zhang2, Alexander M. Spokoyny2, Bradley L. Pentelute2, Scott E. Snyder1 1 Radiological Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, United States, 2Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States Objectives Driven by the promising results obtained with perfluoroaryl-cysteine derivatization of peptides and proteins (e.g. for peptide stapling1 and selective cysteine modification on proteins2) and the broad application of radiolabeled peptides and proteins in radiopharmaceuticals,3 the goal of this study is to develop an efficient methodology to incorporate F-18 into these perfluoroaryl-cysteine fragments using SNAr Chemistry (19F-18F exchange). This technology could be used to label antibody fragments or other bioactive peptides. Methods [18F]Fluoride ion was separated from target [18O]water using QMA resin and elution with a solution of phase-transfer base (e.g. Cs2CO3, 1 mg) in acetonitrile and water (600 µL, v/v 5:1) This solution was collected in a vial sealed with a teflon-lined septum and was evaporated at 100 °C while argon gas was passed. When no liquid was visible, the vial was cooled to room temperature and 2-10 mg of precursor in 200 μL polar aprotic solvent was added. The reaction mixture was allowed to stand at various temperatures and the progress of reaction was monitored by TLC with radioisotope detection. After completion, the reaction was quenched using 1 mL of MeOH. Results The protected para-[18F]pentafluorocysteine analog was synthesized using an automated synthesis module. The reaction was optimized using different reaction conditions (Solvents: MeCN, DMF, DMSO; Phase transfer bases: Kryptofix, TEAB, Cs2CO3; Precursor concentration (200 µL solvent): 2-10mg; Temperature: 10-120 oC; Reaction time: 20-60 min). DMSO, Cs2CO3, rt, 5mg precursor and 20 min reaction gave us optimum results. Average final yield by TLC was 7% (EOS, n = 3). Conclusions We demonstrated a new, mild synthetic platform for [18F]perfluoroaryl cysteine production using a 19 18 F- F exchange reaction. The developed method is rapid, operates at room temperature in polar organic solvents and is amenable to the preparation of 18F-labeled peptides. Acknowledgements Funding provided by the ALSAC-St. Jude Children’s Research Hospital. References [1] Spokoyny, A. M. et al. (2013) J. Am. Chem. Soc., 135, 5946−5949, [2] Zhang, C. et al. Angew. Chem. Int. Ed. 2013, 52, 14001 –14005, [3] Wester, H. J. et al. (2007) Ernst Schering Foundation Workshop 64: 79-111.

J Label Compd Radiopharm 2015: 58: S1- S411

S266: Poster

21st International Symposium on Radiopharmaceutical Sciences

266 Rapid, microfluidic radiosynthesis of a [18F]F-Py-GnRH peptide Robin C. Cumming1, Julie L. Sutcliffe2, 3 1 Biomedical Engineering , University of California Davis, Davis, California, United States, 2Internal Medicine Division of Hematolgy/Oncology, University of California Davis, Sacramento, California, United States, 3Center for Molecular and Genomic Imaging, Davis, California, United States Objectives 18F-radiolabeled peptides have shown much promise as cancer imaging agents1. Unfortunately, 18Fpeptide radiolabeling procedures can be multi-step, lengthy processes that often require a prosthetic group for 18F incorporation. Therefore, developing a simplified, faster approach would be advantageous. Here we present a potential platform for the rapid, automated synthesis of 18F-peptides using a microfluidic device. We describe a proof of concept synthesis using a gonadotropin releasing hormone (GnRH) peptide analog (pGlu-HWSYkLRPG) and the [18F]F-Py-TFP prosthetic group. Methods A three pump microfluidic device was used to produce [18F]F-Py-GnRH (III) from [18F]fluoride in a twostep reaction (Schematic 1). Step one was the synthesis of [18F]F-Py-TFP (II) from [18F]fluoride2, and step two was the peptide radiolabeling. [18F]fluoride (13.8mCi) was azeotropically dried with acetonitrile (ACN), reconstituted in 4:1 tBuOH/ACN and loaded into pump 3. Pump 1 was loaded with precursor (I) (10 mg/mL) in 4:1 tBuOH/ACN, and Pump 2 was loaded with GnRH peptide (1 mg/mL) in a 0.2M NaHCO3 buffer pH 8.5. Reactions were monitored using radio high performance liquid chromatography (radioHPLC). Reaction 1 was done according to literature2, and reaction 2 was done at 50oC, 1:2 reaction 1 bolus:GnRH peptide ratio by volume, and flow rates of 12-30 μL/min. Results [18F]F-Py-GnRH peptide was obtained in 49-61% yield from [18F]fluoride within 5 minutes (n=3) as determined by radioHPLC. These results compare favorably with previously published results of a model peptide YGGFL (up to 28% in 8 min)2. Conclusions We have demonstrated a proof of concept platform for the rapid, automated 18F-radiolabeling of a biologically relevant peptide in high radiochemical yield (up to 61%) on a microfluidics device. Current work focuses on optimizing reaction conditions (temperature, flow rate, and ratio of reagents) and utilizing this approach for radiolabeling other peptides, including cRGDyK, A20FMDV2 and a bombesin fragment. Acknowledgements The authors would like to thank Lee Collier for technical advice. This research was supported by NIH 1RC4EB01 2836-01 and the Office of Science, US Department of Energy DE-SC0002061. References [1] Gambhir et al., Nat Rev Cancer; 2(9), 683-693, 2002. [2] Cumming et al., RSC Adv; (4) 4952949534, 2014.

Schematic 1: Two-step synthesis of 18F-Py-GnRH- peptide

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S267

267 Synthesis and biological evaluation of a novel α,α-diakyl amino acid PET tracer for brain tumor and prostate cancer imaging. Ahlem Bouhlel1, Dong Zhou1, Aixiao Li1, Liya Yuan2, Rebecca Sohn3, Zhi Hong Lu3, Jeffrey Arbeit3, Keith Rich2, Jonathan McConathy1, 4 1 Radiology, Washington University in St Louis, Saint Louis, Missouri, United States, 2Department of Neurosurgery, Washington University in St Louis, St Louis, Missouri, United States, 3Department of Surgery, Urological Division, Washington University in St Louis, St Louis, Missouri, United States, 4Division of Nuclear Medicine, Washington University in St Louis, Saint Louis, Missouri, United States Objectives The objective of this work is to develop novel 18F-labeled amino acids (AAs) with an optimal balance of transport by system L to maintain brain availability and by other neutral AA systems to increase tumor to normal tissue ratios and improve tumor imaging outside of the brain. System L is active at the blood-brain barrier (BBB), allowing substrates such as [18F]FET, [18F]FDOPA, and [11C]methionine to reach the entire tumor volume even if the BBB is not disrupted. Unlike some other AA transporters, system L is not concentrative, leading to relatively low tumor to brain ratios and poor visualization of most tumors outside of the brain. Methods The non-radioactive form of FAMPe (our target) was synthesized as a racemic mixture. Radiolabeling was performed from enantiomerically pure tosylate precursors in t-amyl alcohol at ~100°C for 10 min. After a C18 HPLC, deprotection and neutralization on a Dionex OnGuard II A cartridge, both tracers were obtained in a suitable form for animal studies. AA uptake assays were carried out in mouse DBT gliomas cells to evaluate the transport mechanism of (S)- and (R)-[18F]FAMPe. Biodistribution studies were conducted in male BALB/c mice with subcutaneous DBT gliomas at 5, 30 and 60 min after injection (n= 5). Additionally, small animal PET/CT studies were conducted in male BALB/c mice with intracranial DBT gliomas and a separate cohort of mice with disseminated IGR-CaP1 prostate cancer metastases. Results (S)- and (R)-[18F]FAMPe were obtained in 24-58% RCY (n= 8 runs) and over 99% radiochemical purity. Both tracers enter DBT gliomas cells in part through system L transporter and systems that recognize glutamine. (S)-[18F]FAMPe demonstrated rapid and high DBT tumor uptake over time (7.37 %ID/g at 30 min and 9.88 %ID/g at 60 min) and high tumor to brain ratios. At 60 min post-injection, tumor uptake was higher than all the other normal tissues evaluated, suggesting this tracer could be useful for neoplasms outside of the brain. The small animal PET/CT studies conducted in both DBT gliomas and metastatic prostate cancer models demonstrate good tumor visualization with (S)-[18F]FAMPe. Conclusions The novel PET tracers (R)- and (S)-[18F]FAMPe demonstrated good imaging properties for brain tumor imaging and may also be useful for detecting prostate cancer metastases. Future efforts include the development of analogues of (S)-[18F]FAMPe and assessing this class of tracers in other cancer models. Acknowledgements Grant Support: DE-SC0004832, K08CA154790, P50CA094056 References (1) Yu W, et al. J Med Chem. 2010; 53:876. (2) McConathy J, et al. J Med Chem. 2002; 45:2240. (3) McConathy J, et al. Nucl Med Biol. 2003; 30:477.

J Label Compd Radiopharm 2015: 58: S1- S411

S268: Poster

21st International Symposium on Radiopharmaceutical Sciences

268 Radiolabelling of a α-synuclein PET radioligand candidate using a copper-mediated nucleophilic fluorination reaction Christophe Plisson, Sac-Pham Tang, Jan Passchier Imanova, London, United Kingdom Objectives Parkinson's Disease (PD) is one of a number of neurodegenerative conditions, which may be collectively grouped as "protein deposition diseases", associated with the conversion of native soluble proteins into deposited aggregates. Abnormal accumulation of the protein α-synuclein, in combination with a misfolding process, leads to the formation of insoluble aggregates, also referred to as Lewy bodies, which are a definitive marker for the pathological diagnosis of Parkinson's Disease. The ability to monitor the progression of α-synuclein deposition in vivo will aid understanding of disease progression, including the onset of dementia in PD patients and in time may lead to improved diagnosis as well as support drug development initiatives. This work looks at the radiosynthesis of a α-synuclein binder as potential PET radioligand. Methods [18F]IMA201, a radiolabelled analogue of a molecule found to provide protection in cellular models of αsynuclein-mediated dysfunction, was prepared following the recently developed copper II-mediated 18Ffluorination methodology [1]. Typically 17 mg (0.047 µmol) of the 4,4,5,5-tetramethylpinacol phenylboronic ester precursor and 12 mg (0.017 µmol) of tetrakis(pyridine)copper(II) triflate dissolved in 0.6 mL of DMF were added to the azeotropically dried [18F]KF, K2,2,2/K2CO3 mixture. The reaction mixture was heated at 110°C for 15 min and intermittent introduction of oxygen was achieved by bubbling air through the reaction mixture since oxygen was proved to be beneficial for the reaction. [18F]IMA201 was purified by reversed-phase semi-preparative HPLC with acetonitrile and ammonium formate 100 mM pH 8 as eluent and reformulated in 10% ethanol/saline. Results [18F]IMA201 was synthesised in low but non-optimised radiochemical yields of ~200 MBq starting from an estimated 8-9 GBq of [18F]fluoride, which corresponds to approximately 2% non decay-corrected radiochemical yield. The total synthesis time was 70 min. The radiochemical purity was greater than 97% and the specific activity greater than 70 GBq/µmol at the end of synthesis. Conclusions A fully automated radiolabelling method has been successful in preparing a 18F-labelled putative marker of α-synuclein in radiochemical purities and radiochemical yields relevant for preclinical and clinical imaging. The evaluation of [18F]IMA201 specificity for α-synuclein fibrils in PD brain tissue using techniques such as saturation radioligand binding, quantitative autoradiography and immunohistochemistry is on-going. Acknowledgements References [1] Matthew Tredwell et al. (2014) Angew. Chem. Int. Ed., 53, 1-6.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S269

269 Modelling Reactions for F-18 Labeling of Chitosan Nanoparticles Ana Lima Oubina, Tünde Miklovicz, Anita Matolcsy, József Varga, Pál Mikecz Nuclear Medicine, Clinical Center, University of Debrecen, Debrecen, Hungary Objectives The aim of this work is to model F-18 labelling of chitosan based nanoparticles via 4[18F]fluorobenzaldehyde (FBA) on the amine group of one of its components, the glucosamine (GlcN). In order to study the reaction between FBA and GlcN, its derivatives (N-Acetyl-GlcN (NAGlcN), 1,3,4,6-Tetra-O-AcetylGlcN (TAGlcN) and glucose were also used. Methods [18F]FBA was synthesized from 4-Formyl-N,N,N-Trimethylanilinium triflate (5-7 mg in 1mL anhydrous DMSO) using Kriptofix 222 and K2CO3 (15/2,8 mg) in a 15-minute reaction at 70 oC following Speranza et al [1]. In order to decrease the precursor and 4-(Dimethylamino)benzaldehyde (DMABA) in the product, the amounts of reagents were decreased to 3,7, 5,1 and 1,4 mg as described by Mcrobbie et. al. [2]. The latter reaction lasted 5-10 minutes at 80 oC, and KHCO3 was used instead of K2CO3. To model the reaction on the amine group direct (L.Carroll et al.3) and reductive amination reactions (AbdelMagid et al.4) between [18F]FBA with GlcN and TAGlcN were used. NaBH3CN was employed as reducing agent. To study the possible side reactions with the hydroxyl groups the same reactions on NAGlcN and glucose were performed. Results [18F]FBA was obtained by a 50±6 % yield with >95% radiochemical purity. The precursor and DMABA content was significantly lower using the [2] method (25±10 and 17±13 µg/mL respectively). Direct reaction on GlcN or TAGlcN gave poor yield (<7%). In case of reductive amination, we found radiolabeled products in 58±7% and 24±2% (respectively) of the total activity determined by HPLC that expected to be Nlabeled. The reactions with N-Acetyl-GlcN and glucose gave different compounds with significantly lower yield. Conclusions Modelling reaction of reductive amination shows that the [18F]FBA reacts mainly with the NH2 group from GlcN and 1,3,4,6-Tetra-O-Acetyl-GlcN with high yields. The isolation of N-benzyl derivatives of GlcN and their subsequent analysis is in progress. In case of blocked amine group or its absence we observed low yield aldol side reactions. These results could be applied for radiolabeling chitosan based nanoparticles. Acknowledgements This work was supported by FP7-PEOPLE-2012-ITN (316882 RADIOMI project). References [1]Speranza et al., Applied Radiation and isotopes 67(2009)1664-1669 [2]G.McRobbie et al.,Eur J Nucl Med Mol Imaging (2013) 40 (Suppl 2):S89–S567 [3]L.Carroll et al., Med. Chem. Commun., 2013,4, 653 (2013) [4]F.Abdel-Magid et al., J. Org. Chem. 1996, 61, 3849-3862 3849

J Label Compd Radiopharm 2015: 58: S1- S411

S270: Poster

21st International Symposium on Radiopharmaceutical Sciences

270 A 18F Labeling Strategy for Near Infrared Fluorophores. Multi-Modal Probes for Reactive Oxygen Species Salma Al-karmi, Silvia A. Albu, Nancy Janzen, Alfredo Capretta, John Valliant Chemistry and Chemical Biology, McMaster University, Toronto, Ontario, Canada Objectives Reactive oxygen species (ROS) are highly reactive molecules derived from molecular oxygen. A homeostatic balance via redox regulation of ROS is required in order to prevent a pro-oxidative cellular state. "Oxidative stress" has been implicated in the initiation/progression of multiple diseases, particularly cancer [1]. The objective was to develop a simple radiolabeling strategy for ROS sensitive dyes that react specifically with . OH and O2.- based on an oxidative H-abstraction mechanism (Scheme 1) [2]. Methods A 19F NIR dye was synthesized in two steps from a commercial dye. The ROS sensing ability of this probe was assessed in vitro using PC3 prostate cancer cells while stability towards autooxidation was assessed using HPLC and by collecting fluorescence readings as a function of time. The ROS selectivity profile using chemical induction was performed to determine probe reactivity. The radiolabeling precursor was synthesized and radiolabeling performed using a two step process. Results The 19F NIR probe was synthesized in a 82% overall yield. Chemical induction experiments show that 1 exhibited selectivity towards .OH and O2.- radicals accompanied by a respective 200 and 21 fold increase in fluorescence intensity. Stability experiments demonstrated that 1 is not prone to autooxidation as compared with previously reported dihydroethidium ROS probes. 1 was shown to respond to ROS generated by PC3 cells (P ˂ 0.001). 2 was synthesized in 12% radiochemical yield (non-decay corrected) by substitution on a O-nitropyridine precursor. Conclusions An 18F labeled dual optical/nuclear dye that specifically responds to .OH and O2.- was developed and was evaluated as a ROS probe in PC3 cells. The method to create the probe is synthetically tractable and the agent exhibits numerous advantages compared with previously reported ROS probes.The radiolabeling strategy involves easily accessible and stable precursors which can be readily separated from the labeled product. Acknowledgements Funding for this project was provided by NSERC (Canada) and OICR (Ontario). References [1] K.A Gelderman et al., (2007), Antioxid. Redox Signaling, 9, 1541. [2] K. Kundu et al., (2009), Angew. Chem. Int. Ed., 48, 299

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S271

271 Production of [F-18]THK-5117 using Siemens Explora chemistry module Tobey J. Betthauser1, Patrick Lao1, Andrew T. Higgins1, Dhanabalan Murali1, Todd E. Barnhart1, Robert J. Nickles1, Sterling Johnson2, Nobuyuki Okamura4, Shozo Furumoto3, Bradley T. Christian1 1 Medical Physics, University of Wisconsin - Madison, Madison, Wisconsin, United States, 2Department of Medicine, University of Wisconsin - Madison, Madison, Wisconsin, United States, 3Cyclotron and Radioisotope Center, Tohoku University, Sendai, Japan, 4Department of Pharmacology, Tohoku University, Sendai, Japan Objectives The goal of this work was to modify the Siemens Explora FDG4 chemistry module and to create an automated routine using a one pot synthesis for the radiolabeling of [F-18]THK-5117, a radiotracer for tau imaging. Methods The Explora module was modified from its original factory setup to accommodate the adding of four separate reagents from small volume vessels. This was accomplished by converting trap lines into small volume reagent add lines. The micropump was then bypassed and a low flow N2 line was rerouted to purge the contents of the small volume reagent vials into the reaction vessel. A synthesis program was written using Siemens’ GUI based Explora GN software to perform the azeotropic distillation, nucleophilic substitution with the THK-5119 precursor, deprotection, quenching of the reaction, dilution and trapping of the [F-18]THK-5117 product on a Waters tC18 cartridge according to the reported methods.1 The cartridge was then manually eluted in three fractions and injected onto semi-preparative HPLC for separation of bulk impurities. Results [F-18]THK-5117 has been successfully synthesized with a mean specific activity of 3800±1700 mCi/umol and a mean total activity at end of synthesis of 51±30 mCi. Typical decay corrected yields were 26% using 2.0mg of precursor and 39% using 3.0mg of precursor. The radiolabeling of the [F-18]THK-5117 was completed within 45 minutes and a total synthesis time from beginning of synthesis through purification to final formulation was 75 minutes. Conclusions The Explora chemistry module can be modified to regularly produce [F-18]THK-5117 with specific activities on the order of 4000 mCi/umol with a starting F-18 activity of greater than 300 mCi. Decay corrected yields of 20% to 40% can be achieved with the amount of precursor being the largest factor in radiolabeling yield. This demonstrates that the Explora FDG4 system can be readily modified and programmed to produce [F-18]THK5117 for research applications to study the presence of tau in Alzheimer’s disease pathology. Acknowledgements References [1] Okamura N, et al (2013) The Journal of Nuclear Medicine, vol. 54, no. 8, pp. 1420-1427.

J Label Compd Radiopharm 2015: 58: S1- S411

S272: Poster

21st International Symposium on Radiopharmaceutical Sciences

272 PET imaging and ex vivo brain autoradiography in a mouse model of Alzheimers disease using the TSPO tracers [18F]DPA-714 and [18F]F-DPA Merja Haaparanta-Solin1, Francisco Lopez-Picon 1, Rana Almajidi1, Jatta Takkinen1, Thomas Keller2, Anna Krzyczmonik2, Sarita Forsback2, Anna Kirjavainen2, Fanny Cacheux3, Annelaure Damont3, Juha O. Rinne2, Frédéric Dollé3, Olof Solin2 1 Turku PET Centre, MediCity/PET Preclinical Laboratory, University of Turku, Turku, Finland, 2Turku PET Centre,Radiopharmaceutical Chemistry Laboratory and Turku University Central Hospital, University of Turku, Turku, Finland, 3CEA, I2BM, Service hospitalier Frédéric Joliot, Orsay, France Objectives We aimed to characterize and compare the TSPO binding PET tracers, [18F]DPA-714 and [18F]F-DPA [1], for in vivo and ex vivo detection of neuroinflammation in a mouse model of Alzheimer´s disease (AD). Methods We used the APP/PS1-21 mouse model of AD. For 60 minutes in vivo PET-CT scans, groups of mice were imaged with both [18F]DPA-714 and [18F]F-DPA. For the ex vivo brain autoradiography study, immediately after the in vivo scans, the mice were killed, the brain dissected, frozen and sliced (20 μm) with a cryostat, and then apposed to an imaging plate. Immunohistochemical and histological staining for amyloid and microglial activation were performed. Radiometabolites were determined from plasma and brain tissue using radioTLC. Age matched WT mice were used as controls. Results In vivo PET results showed increased [18F]DPA-714 and [18F]F-DPA uptake in cortex, thalamus and striatum of AD mice compared to the WT mice used as baseline. The ex vivo results demonstrated increased cortex, thalamus and striatum to cerebellum ratios. [18F]F-DPA, as compared to [18F]DPA-714, demonstrated much reduced formation of radiolabelled metabolites in brain and plasma. Conclusions This preclinical study demonstrates the validity of the novel 18F tracers [18F]DPA-714 and [18F]FDPA for the detection of neuroinflammation in a mouse model of AD. Due to its much slower formation of radiolabelled metabolites, [18F]F-DPA might be the preferred tracer for small animal imaging. Acknowledgements Funding was received from the European Union’s 7th Framework Programme for Research, grants number 316882 and HEALTH-F2-2011-2788850 (INMiND) and from the Academy of Finland, grant number 266891. References [1] Keller et al., Electrophilic fluorination and preclinical evaluation of a novel [18F]DPA-714 analogue, [18F]F-DPA, this Symposium.

Figure 1. Structural formulae of [18F]DPA-714 and [18F]F-DPA.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S273

273 Synthesis of [11C]GSK1482160 as a new potential PET radiopharmaceutical for targeting the P2X7 receptor Mingzhang Gao, Min Wang, Mark Green, Gary Hutchins, Qi-Huang Zheng Department of Radiology and Imaging Sciences, Indiana University, Indianapolis, Indiana, United States Objectives P2X7 is an adenosine triphosphate (ATP)-gated ion-channel, which is found in the immune, peripheral and central nervous systems, implicated in ATP-mediated cell death, regulation of receptor trafficking and inflammation, and associated with various cancer, neurological and cardiovascular disorders. GSK1482160 developed by GlaxoSmithKline is a potent P2X7 antagonist with excellent biological activity (IC50 3 nM for human P2X7) [1,2]. Here we report the synthesis of [11C]GSK1482160 as a new candidate PET agent for imaging of P2X7 receptor. Methods The authentic standards GSK1482160 and its isomer, as well as the radiolabeling precursors desmethylGSK1482160 and BOC-protected desmethyl-GSK1482160 were synthesized from L-pyroglutamic acid, methyl Lpyroglutamate and 2-chloro-3-(trifluoromethyl)benzylamine according to the literature methods [1,2] with modifications. [11C]GSK1482160 was prepared from either desmethyl-GSK1482160 or BOC-protected desmethylGSK1482160 with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in a home-built automated multi-purpose 11C-radiosynthesis module, allowing measurement of specific radioactivity during synthesis [3]. Results The overall chemical yields for GSK1482160 and GSK1482160 isomer were 21% in 3 steps and 59% in 4 steps, respectively. The overall chemical yields for desmethyl-GSK1482160 and BOC-protected desmethylGSK1482160 were 79% in 1 step and 35% in 2 steps, respectively. The decay corrected radiochemical yields for [11C]GSK1482160 from [11C]CO2 at EOB were 40-50% in 1-pot-1-step radiosynthesis using desmethylGSK1482160 and 30-40% in 1-pot-2-step radiosynthesis using BOC-protected desmethyl-GSK1482160, respectively. The [11C]GSK1482160 and [11C]GSK1482160 isomer were formed in an ~10:1 ratio from desmethylGSK1482160. The radiochemical purity of [11C]GSK1482160 is >98%, and the specific activity of [11C]GSK1482160 was 370-1110 GBq/µmol at EOB (92.5-277.5 GBq/µmol at EOS). Conclusions A facile synthetic route to the precursors desmethyl-GSK1482160 and BOC-protected desmethylGSK1482160, standards GSK1482160 and GSK1482160 isomer and a new PET tracer [11C]GSK1482160 has been developed. Acknowledgements Supported by the Indiana University Department of Radiology and Imaging Sciences and the Indiana State Department of Health (ISDH) Indiana Spinal Cord & Brain Injury Fund (ISDH EDS-A70-2-079612). References [1] Abdi MH, et al. (2010) Bioorg Med Chem Lett, 20, 5080-4. [2] Abberley L, et al. (2010) Bioorg Med Chem Lett, 20, 6370-4. [3] Wang M, et al (2012), Appl Radiat Isot, 70, 965-73.

J Label Compd Radiopharm 2015: 58: S1- S411

S274: Poster

21st International Symposium on Radiopharmaceutical Sciences

274 Radiolabeling of small peptides with L-[11C]alanine. Ulrike Filp, Aleksandra Pekošak, Alex J. Poot, Albert D. Windhorst Radiology and Nuclear Medicine, VU Medical Center, Amsterdam, Netherlands Objectives Peptides have become an important class of compounds in molecular imaging. Today’s radiolabeling methods use modified peptides, like the attachment of a chelator for radiometals or a prosthetic group for fluorine18. Some of these modified peptides have shown good results, however it could be favorable to have non-modified natural peptides in some cases. Unfortunately, there is currently no method available to label peptides with PET radionuclides without modifying them. Here we present a method for the radiolabeling of small peptides with L[11C]alanine. Methods L-[11C]alanine was synthesized via a phase-transfer catalyzed reaction of 1 with [11C]MeI in high yield and ee. Peptides were synthesized according to Fmoc-protocols on a Rink-amide resin. The peptide coupling reaction with L-[11C]alanine was first evaluated thoroughly with benzylamine as a model reaction (Scheme 1). These conditions were then to be applied to the coupling of H-Gly-Leu-Ala-Phe-NH2 4 in solution and with the peptide 4B on resin. Results An optimization study was performed for the synthesis of (S)-2-amino-N-benzylpropanoamide 3 (table 1, entries 1-3). Best results were obtained with 0.74 µL of benzylamine, 3.1 mg of BOP and >100 times excess of DIPEA at 50 ºC for 5-10 min. The coupling to 1 mg of 4 (in solution) and 2 mg of peptide 4B (on resin) is under investigation. Conclusions With the in-house established enantioselective synthesis of L-[11C]alanine, we developed a coupling strategy to benzylamine in good yields. We are currently investigating coupling methods for small peptides with a focus on peptides on resin. Acknowledgements RADIOMI funded by FP7-PEOPLE-2012-ITN. References Fasth KJ, Antoni G, Långström B (1988) J. Chem. Soc. Perkin. Trans. Ι, 3081-3084; Ooi T, Tayame E, Maruoka K (2003) Ang. Chem. 115, 599-602; White JB, Hausner SH, Sutcliffe JL (2012) App. Rad. Isotop. 70, 2720-2729; Fani M, Maecke HR (2012) Eur. J. Nucl. Med. Mol. Imaging 39, S11-S30.

Scheme 1. Labeling of small amines and peptides with L[11C]alanine.

Table 1. Reaction of 2 and 4 with BOP.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S275

275 Syntheses of 1-[11C]Methyl-4-aryloxy-1,2,3,6-tetrahydropyridines as In Vivo Metabolic Trapping Radiotracers for Monoamine Oxidase-A and –B Allen F. Brooks, Xia Shao, George W. Kropog, Phillip Sherman, Carole Quesada, Peter J. Scott, Michael R. Kilbourn Radiology, University of Michigan, Ann Arbor, Michigan, United States Objectives Imaging monoamine oxidase activity is of interest for studying psychiatric (MAO-A in depression) and neurodegenerative (MAO-B in astrogliosis) diseases. To utilize the concept of in vivo metabolic trapping, 1[11C]methyl-tetrahydropyridines were designed as substrates for MAO-A and -B, with selectivity for the isoform possible through selection of the aryl group connected via the ether link to the tetrahydropyridine group [1,2]. MAO-catalyzed oxidation and spontaneous hydrolysis of such ethers yields the non-toxic metabolite 1[11C]methyl-2,3-dihydropyridin-4(1H)-one, which is trapped within brain tissues. A novel method for the generation of N-[11C]methyl-tetrahydropyridines was developed utilizing a one-pot methylation and reduction sequence. Methods Performing the [11C]methylation of pyridines in ethanol [3] allowed for developing a one-pot synthesis, as the intermediate N-[11C]methylpyridiniums are rapidly reduced to yield the tetrahydropyridine MAO substrates. Two different radiotracers with phenyl (general MAO) [1] and coumarin (MAO-B selective) [2] ether groups were produced and evaluated. Results The [11C]methylation ([11C]CH3OTf in EtOH) and sodium borohydride reduction were performed using a TracerLab FXC-Pro system. The products were purified by reverse phase chromatography and reformulated from a C-18 extraction disk (10% EtOH in saline). In non-optimized yields, 7.3 mCi of the phenyl ether (0.8% yield from MeOTf, non-decay corrected; n=8) and 15.6 mCi of the coumarin ether (1.7% yield from MeOTf, non-decay corrected; n=10) were collected. The doses produced were assessed via standard quality control techniques and were appropriate for preclinical studies. Conclusions A facile method for the production of 1-[11C]methyl-tetrahydropyridines has been developed and utilized to produce two analogs for in vivo imaging of MAO-A and -B. Synthetic details as well as preclinical imaging data will be presented. Acknowledgements Supported by NIH grant R21NS075553 References [1] Kalgutkar AS, et. al. (1994) J Med Chem, 37, 944-949. [2] Long S, et. al. (2012) Chem Commun, 48, 7164-7166. [3] Shao X, et. al. (2013) Nucl Med Biol, 40, 109-116.

J Label Compd Radiopharm 2015: 58: S1- S411

S276: Poster

21st International Symposium on Radiopharmaceutical Sciences

276 Novel radiosynthetic approaches applying carbon-11 labeled isobutanol and isobutyl iodide Lonneke Rotteveel1, Uta Funke1, 2, Adriaan A. Lammertsma1, Albert D. Windhorst1, Alex J. Poot1 1 Radiology and Nuclear Medicine, VU University Medical Centre, Amstelveen, Netherlands, 2BV Cyclotron VU, Amsterdam, Netherlands Objectives The isobutyl moiety is often present in biologically active compounds. [1] [2] The purpose of this study was to improve the synthesis of [11C]isobutanol ([11C]i-BuOH) and [11C]isobutyl iodide ([11C]i-BuI) [3] and investigate the use of these synthons in four model reactions for [11C]etherification and [11C]alkylation. Methods [11C]i-BuOH and [11C]i-BuI were synthesized according to scheme 1 and purified by distillation. [11C]iBuI was then applied in two alkylation reactions on an oxygen and a sulfur nucleophile (scheme 2, I and II) and [11C]i-BuOH was applied in a nucleophilic aromatic substitution (scheme 2, III). Finally, an unprecedented cobaltcatalyzed cross-coupling reaction of phenylmagnesium bromide and [11C]i-BuI was performed (scheme 2, IV). Results The alkylation of thiophenol with [11C]i-BuI (specific activity > 50 GBq/µmol) went rapidly and with high conversion rates (scheme 2, I). However, alkylation of the phenol was less efficient, likely due to the moderate nucleophilicity of the phenolate and the formation of [11C]isobutylene (scheme 2, II). Ether formation from fluorobenzene and [11C]i-BuOH was accomplished, but high amounts of fluorobenzene were required (scheme 2, III). The cross-coupling reaction provided decent yields (scheme 2, IV). Conclusions The synthesis of [11C]i-BuOH and [11C]i-BuI was improved and the synthons were successfully used in four model reactions. In addition, the Cobalt-catalyzed cross coupling reaction provides a new synthetic strategy for carbon-11 labeling of aromatic isobutyl functions. Acknowledgements References [1] Wang J.L., et al, (2010) Bioorg. & Med. Chem. Lett., 20, 7159-63; [2] Zang MR., et al, (2006) J.Med. Chem., 4;49(9), 2735-2742; [3] Långström B., et al. (1986) Int. J. Rad. App. and Instr. 37 (11), 1141–1145.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S277

277 Initial evaluation of P2X7R antagonists [11C]A-740003 and [11C]SMW64-D16 as PET tracers of microglial activation in neuroinflammation Bieneke Janssen1, Dieter Ory2, Shane M. Wilkinson4, Danielle J. Vugts1, Esther J. Kooijman1, Joost Verbeek1, Uta Funke3, 1, Ger T. Molenaar3, 1, Perry S. Kruijer3, Adriaan A. Lammertsma1, Michael Kassiou4, 5, Guy Bormans2, Albert D. Windhorst1 1 Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, Netherlands, 2Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium, 3BV Cyclotron VU, Amsterdam, Netherlands, 4School of Chemistry, University of Sydney, Sydney, New South Wales, Australia, 5Brain and Mind Research Institute, Sydney, New South Wales, Australia Objectives Progression of neurodegenerative diseases is associated with neuroinflammation (NI), in particular the activation of microglia. Since the P2X7 receptor is upregulated in activated microglia [1], it is an interesting target for imaging of NI with PET. Two recently developed radiotracers, [11C]A-740003 [2] and [11C]SMW64-D16, were evaluated in vitro in a NI rat model and both in vitro and in vivo for binding to human P2X7 receptor (hP2X7R). Methods Both radiotracers were obtained by [11C]methylation (fig 1A). In vitro autoradiography (AR) was performed (n=2) on snap frozen brain slices of rats unilaterally injected with either an adenoviral-associated viral vector expressing hP2X7R (AAV-hP2X7R) or lipopolysaccharide (LPS) to induce NI. PET images were acquired on a Focus 220 PET scanner after i.v. injection of 45-55 MBq of either [11C]A-740003 (n=3) or [11C]SMW64-D16 (n=3) two weeks after stereotactic injection of AAV-hP2X7R. Results AR revealed about 1.5-fold increased binding in AAV-hP2X7R injected striatum compared with healthy striatum for both tracers (fig 1B). In LPS injected rats, about 2-fold decreased binding was observed for both tracers in inflamed compared with healthy striatum. PET images showed no brain uptake of [11C]A-740003. Although for [11C]SMW64-D16 slightly increased uptake in AAV-hP2X7R injected striatum was observed, differences in time activity curves were not significant. Conclusions Both [11C]A-740003 and [11C]SMW64-D16 bind to hP2X7R in vitro. Decreased binding in LPS injected striatum might indicate that P2X7R expression is downregulated in activated microglia in this particular NI model. Preliminary in vivo PET studies show some hP2X7R binding of [11C]SMW64-D16, but further studies are needed to assess whether the signal is high enough. Acknowledgements This research has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° HEALTH-F2-2011-278850 (INMiND). D. Ory is fellow of the Research Foundation Flanders (FWO). References [1] Monif M. et al. (2010), Int. J. Biochem. Cell Biol. 42: 1753-1756. [2] Janssen, B. et al. (2014), J. Labelled Compd. Radiopharm. 57: 509-516.

J Label Compd Radiopharm 2015: 58: S1- S411

S278: Poster

21st International Symposium on Radiopharmaceutical Sciences

278 Synthesis and in vitro evaluation of radiobrominated adenosine A1 receptor ligand [*Br]CPBPX Katharina Breunig, Dirk Bier, Marcus H. Holschbach, Annette Schulze, Heinz H. Coenen Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum Jülich, Jülich, Germany Objectives [18F]CPFPX (8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine) [1] is an established ligand for investigation of the adenosine A1 receptor (A1AR) in man using PET. The iodinated derivative [131I]CPIPX (8cyclopentyl-3-[(E)-3-[131I]iodoprop-2-en-1-yl]-1-propylxanthine) [2] has been synthesized as well and evaluated with regard to its binding profile. Since no radiobrominated A1AR ligand is known so far, the synthesis of the radiobrominated analogue [*Br]CPBPX (8-cyclopentyl-3-[(E)-3-[*Br]bromoprop-2-en-1-yl]-1-propylxanthine) was performed for closing the gap of lipophilicity in this xanthine series, which is important for systematic studies. Methods The applied radionuclide mixture of 76,77,82Br (*Br) was produced via natSe(p,xn)-reactions on the new target material NiSe at the BC1710 cyclotron of the Forschungszentrum Jülich and isolated as sodium [*Br]bromide in no-carrier-added form [3]. The radiosynthesis of [*Br]CPBPX was carried out via radiobromodestannylation of the tributyltin precursor by in situ oxidation of n.c.a. [*Br]bromide. The binding profile of the new ligand to the A1AR was investigated in vitro. For the determination of the logP value of [*Br]CPBPX, a new experimental procedure had to be developed, as the radioligand decomposes in water/octanol yielding a polar compound (probably free bromide). The conventional liquid-liquid extraction was followed by an additional thin layer chromatography of both phases, in order to separate hydrophilic from lipophilic components, and hence to allow the determination of the “true” logP value. Results [*Br]CPBPX was obtained after 0.5 min at RT in radiochemical yields of 54 ± 8 % with a molar activity of 8.6 GBq/µmol. The latter would be enhanced by using enriched selenium as target material. In competition studies a KI-value of 26 nM was determined for CPBPX (KI of CPFPX in relation to [3H]DPCPX 4.9 nM). Preliminary in vitro autoradiographic studies on rat brain slices show an increased accumulation of [*Br]CPBPX in areas with high A1AR density and a fraction of specific binding of ca. 20 %. Using the improved experimental procedure, a logP value of 3.4 was determined for [*Br]CPBPX. Conclusions [*Br]CPBPX closes the gap of lipophilicity of the known fluoro and iodo derivatives, but its affinity to the A1AR is more similar to the iodine analogue. With regard to the determination of experimental logP values of radiohaloalkylated ligands, the necessity of an improved procedure was revealed, as such components may release halide, falsifying the experimental results. Acknowledgements References [1] Holschbach M. H. et al. (2002) J. Med. Chem., 45, 5150-5156. [2] Sihver W. et al. (2003) Nucl. Med. Biol., 30, 661-668. [3] Breunig K. et al. Radiochim. Acta (in press).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S279

279 Use of phase-transfer catalysis for the enantioselective synthesis of [11C]alanine. Ulrike Filp, Aleksandra Pekošak, Alex J. Poot, Albert D. Windhorst Radiology and Nuclear Medicine, VU Medical Center, Amsterdam, Netherlands Objectives Amino acids and peptides have become an important class of compounds in molecular imaging. Unfortunately, no general and reliable methods for the synthesis of carbon-11 labeled amino acids are available. The aim of our study was to develop a reliable enantioselective radiochemical synthesis of [11C]alanine. Therefore, we studied a radiolabeling in which a new carbon-carbon bond is introduced enantioselective in the presence of a chiral phase-transfer catalyst. Methods All reactions were performed using [11C]MeI which was synthesized from [11C]CO2. Optimization of the alkylation reactions were done using Schiff base 1, base and chiral phase-transfer catalyst (A-D) in various organic solvents (Scheme 1). After deprotection of 2 under acidic conditions, the ee was determined using chiral HPLC of 3. Additionally, this radiolabeling method was also applied to synthesize H-[11C]Ala-Ala-OH dipeptides, starting from the dipeptide Schiff base as precursor. Results Optimal [11C]MeI incorporation and ee was achieved for reactions stirred at 5 ºC. Furthermore, 170 µM of CsOH is best, both solvent and catalyst were crucial to obtain enantiomerically pure [11C]alanine (RCY: >80%, ee >90%). Major results of a full optimization study to synthesize [11C]alanine and dipeptides are depicted in table 1. Conclusions We developed a reliable enantioselective synthesis of [11C]alanine and model dipeptides with good radiochemical yield and ee by performing a systematic study of alkylation in the presence of chiral phase-transfer catalysts. Acknowledgements RADIOMI is funded by FP7-PEOPLE-2012-ITN. References Ooi T (2007) Ang. Chem. Int. Ed. 46, 4222–4266; Fasth KJ, Antoni G, Långström B (1988) J. Chem. Soc. Perkin. Trans. Ι, 3081-3084.

Scheme 1. Enantioselective radiolabeling and chiral catalysts.

Table 1. Enantioselective synthesis of [11C]alanine and [11C]Ala-Ala-OH.

J Label Compd Radiopharm 2015: 58: S1- S411

S280: Poster

21st International Symposium on Radiopharmaceutical Sciences

280 Towards the Stereoselective Synthesis of [11C]Phenylalanine and Obtaining Carbon-11 Labeled Peptidic PET Tracers Aleksandra Pekošak, Ulrike Filp, Alex J. Poot, Albert D. Windhorst Radiology and Nuclear Medicine, VU Medical Center, Amsterdam, Netherlands Objectives Peptides and their receptors play a crucial role in cancer diagnosis and treatment. Furthermore, peptides are often used for molecular imaging of biological processes and receptor expression levels after labeling with a radionuclide. Unfortunately, this requires major adaptions to the peptide. Consequently, these modifications change the physical-chemical nature and possibly also the biological characteristics of the peptide. The general aim of this project is to establish a method for labeling peptides in a natural position. Therefore, an element of the peptide needs to be substituted by a radionuclide of the same element and with at 20 min t½, carbon-11 seems to be best suited for this. The aim of the current study is synthesize [11C]phenylalanine as a first step in to the native radiolabeling of peptides. Methods [11C]Benzyl iodide ([11C]BnI) was synthesized in 3 steps starting from PhMgBr 1 and [11C]CO2[1]. Synthesis of DL-[11C]Phe 7 and L-[11C]Phe 10 was investigated by reacting [11C]BnI 4 with precursor 5 in the absence or presence of phase-transfer catalyst 8[2][3].The general strategy is summarized in Scheme 1. Results With a total synthesis time of 10 min, [11C]BnI 4 (n>5) was obtained in high yields, purity and specific activity (RCY: 52±3%, purity: 95±3%, SA: 123±17 GBq/μmol). Subsequently, addition of strong base deprotonated 5, followed by alkylation with the [11C]benzyl group occurred on the α-position to yield of DL[11C]Phe 6 (81±18% (n=9) incorporation of [11C]benzyl) or L-[11C]Phe 9 (86±8% (n=8)). Final quantitative acidic deprotection resulted in DL-[11C]Phe 7 or L-[11C]Phe 10 with moderate enantioselectivity (ee 48±3% (n=3)) with the aid of the commercially avaliable catalyst 8. Conclusions [11C]BnI was obtained in high yield, radiochemical purity and specific activity. Furthermore, we achieved successful synthesis of [11C]Phe in high yield and moderate enantioselectivity. Currently our research focusses on increasing the enantioselectivity by the application of other catalysts. Acknowledgements Supported by the RADIOMI (FP7-PEOPLE-2012-ITN). References [1] Fasth KJ, et al. (1988) J. Chem. Soc., 3081-3084. [2] Kato K, et al. (2011) Bioorg. Med. Chem. Lett., 21, 2437-2440. [3] Ooi T, et al. (2007) Ang. Chem. Int. Ed., 46, 4222-4266.

Scheme 1: Radiochemical synthesis of DL-[11C]Phe and L-[11C]Phe.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S281

281 Synthesis of [11C]vemurafenib via a unique [11C]CO carbonylative Stille coupling to image V600E mutated B-Raf in cancer. Paul Slobbe, Albert D. Windhorst, Guus A. van Dongen, Alex J. Poot Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam, Noord-Holland, Netherlands Objectives Kinase pathways are of great importance in cancer treatment and kinase inhibitors have therefore found great application as anti-cancer drugs.[1] PET imaging with the TKI [11C]erlotinib was demonstrated to be clinically relevant in EGFR mutated non-small cell lung cancer and facilitates patient selection.[2,3] We aim to extend this concept to other kinases that express oncogenic mutations for which there is high clinical demand. Vemurafenib, applied in e.g. melanoma and colorectal cancer, selectively targets serine/threonine kinase B-Raf with a V600E mutation. Vemurafenib is however a very challenging molecule to label with a PET-nuclide. We took the challenge to apply an innovative Stille coupling with [11C]CO insertion, which has not been used before to obtain such heavily functionalized molecules in radiochemistry. Next to that, we planned a preclinical study in tumor bearing mice expressing wild-type or V600E mutated B-Raf. Methods Using precursor 1 and 2, Stille cross-coupling reactions were investigated. Catalysts (PdCl2Dppf, Pd(OAc)2 and PD2DBA3) and co-ligands (none, tri-phenyl phosphine, tri-o-tolylphosphine, triphenylarsine) were explored in THF at 100 °C. Melanoma xenografts Colo829 (V600E), A375 (V600E) and CHL-1 (wt) and colorectal cells HT-29 (V600E), SW-620 (V600E) and colo205 (wt) were selected for biodistribution and imaging studies and the sensitivity to vemurafenib treatment was determined. Results For the synthesis of [11C]vemurafenib a combination of Pd2DBA3 and AsPh3 as catalyst and co-ligand was most effective (table 1). Optimal labeling conditions resulted in an isolated yield of 368-559 MBq starting from 2025 GBq [11C]CO2 (scheme 1). Cellular assays indicated sensitivity in V600E mutated cells and this will be correlated to [11C]vemurafenib uptake. Conclusions A unique synthesis of [11C]vemurafenib has been established in moderate but sufficient yields for preclinical evaluation. [11C]Vemurafenib is currently under preclinical evaluation in tumor bearing mice. Acknowledgements References [1] Baselga J, et al. (2006) Science, 312, 1175-1178. [2] Memon AA, et al. (2009) Cancer Res, 69, 873-878. [3] Bahce I, et al. (2013) Clin Cancer Res, 19, 183-193.

J Label Compd Radiopharm 2015: 58: S1- S411

S282: Poster

21st International Symposium on Radiopharmaceutical Sciences

282 Improved Synthesis and Application of [11C]Benzyl Iodide as Synthon for PET Radiotracer Production Aleksandra Pekošak, Ulrike Filp, Alex J. Poot, Albert D. Windhorst Radiology and Nuclear Medicine, VU Medical Center, Amsterdam, Netherlands Objectives Over the last decades a large number of carbon-11 labeled radiopharmaceuticals has been developed and demonstrated significant potential utility and application. A promising radiolabeling synthon would be [11C]benzyl iodide ([11C]BnI)[1], since the benzyl group is widely present in biologically active compounds. Unfortunately, little attention has been devoted to the synthesis of [11C]BnI as radiolabeling synthon, which resulted in limited application in PET-tracer radiolabeling. Therefore, we have investigated the synthesis of [11C]BnI in order to significantly improve it and for the labeling of [11C]Clebopride[2], a dopamine D2 antagonist. Methods [11C]BnI was synthesized from [11C]CO2 and PhMgBr to obtain [11C]benzoic acid 2, which was reduced to [11C]benzyl alcohol 3 with LiAlH4 and iodinated with 57% HI to yield [11C]BnI 4. [11C]BnI was purified prior to further application in radiosynthesis. Subsequently, [11C]BnI 4 was reacted 5 to yield [11C]Clebopride 6 (see Figure 1). All reaction mixtures were analyzed by HPLC with on-line radioactivity detector and a UV detector. Results With a total synthesis time of 10 minutes, [11C]BnI 4 (n>5) was obtained in high yields, purity and specific activity (RCY: 52±3%, purity: 95±3%, SA: 123±17 GBq/μmol) as depicted in Table 1. Major improvements in the synthesis are reduced amounts of chemicals, temperature for trapping [11C]CO2 in Grignard reagent and addition of 57% HI, moreover home-made column for purification [11C]BnI. [11C]Clebopride 6 was synthesized within 5 min at 50°C (80±12% (n=6) incorporation of [11C]benzyl) in an isolated decay-corrected RCY of 10%, calculated from [11C]CO2, with a radiochemical purity >99 % in a total synthesis time of 29 min from EOB. Conclusions The synthesis of [11C]BnI was optimized significantly and [11C]BnI is now available as a reliable high quality carbon-11 labeling synthon, to be used in development of PET radiopharmaceuticals. Acknowledgements Supported by the RADIOMI (FP7-PEOPLE-2012-ITN). References [1] Fasth KJ, et al. (1988) J. Chem. Soc., 3081-3084. [2] Mach RH, et al. (1993) Nucl. Med. Biol., 20, 777-794.

Figure 1. Synthesis of [11C]BnI 4 and model PET-tracer [11C]Clebopride 6

Table 1. Incorporation according to HPLC and yields of intermediates and [11C]BnI

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S283

283 Carbon-11 Labeled Fumaric Acid Esters Scott Apana1, Dustin Kentala4, Mark Lane4, Scott Haller4, Jacob Hesterman3, Whitney Woodson3, Kelly Orcutt3, Jack Hoppin3, Ajay verma2, Marc S. Berridge1 1 3D Imaging LLC, Maumelle, Arkansas, United States, 2Biogen Idec, Cambridge, Massachusetts, United States, 3 InviCRO, Boston, Massachusetts, United States, 4MPI Research, Mattawan, Michigan, United States Objectives Fumarate and its esters have a variety of uses in the areas of food and medicine, most notably dimethyl fumarate is approved as a treatment of multiple sclerosis. In order to further study its distribution in-vivo, we sought to label fumarate esters, and especially dimethyl fumarate, in both the ester and carbonyl positions and proceed to PET imaging in rodents at the Translational Imaging Center (TIC) at MPI. Methods Ester labeling: [C-11]dimethyl fumarate labeled in the ester position was prepared by the reaction of [C11]methanol with methyl fumaryl chloride. The resulting [C-11]dimethyl fumarate was purified by C-18 cartridge. Monomethyl fumarate was obtained by reaction of [C-11]methanol with fumaryl dichloride. Low specific activity dimethyl fumarate was obtained by further reaction with methanol. Carbonyl: [C-11]carbon dioxide reacted with (Z)-3-lithiopropenoic acid methyl ester, freshly prepared by metalhalogen exchange of (Z)-3-bromopropenoic acid methyl ester. The resulting [C-11]methyl fumarate methylated to the diester with methyl iodide and methyl triflate. The resulting [C-11]dimethyl fumarate was purified by C-18 cartridge. Results [C-11]dimethyl fumarate was obtained in 20% chemical yield from carbon dioxide in 20 minutes (ester labeled) or 1.5% from carbon dioxide in 30 minutes (carbonyl labeled). Solubility of fumarate compounds and methylations of fumaryl chlorides and fumarate were surprisingly poor, leading to alterations in labeling strategy. Conclusions [C-11]Dimethyl fumarate was synthesized both in the ester position and the carbonyl position in sufficient yield to permit further work and PET scanning in rodents at the TIC. Acknowledgements Sponsorship and intellectual contributions of Biogen Idec are gratefully acknowledged. References

J Label Compd Radiopharm 2015: 58: S1- S411

S284: Poster

21st International Symposium on Radiopharmaceutical Sciences

284 A two-step, one-pot synthesis of indole-3-[1-11C]acetic acid ([11C]auxin) using tetraethylene glycol solvent So Jeong Lee1, 2, David Alexoff2, Colleen Shea2, Dohyun Kim2, Michael Schueller2, Joanna S. Fowler2, Wenchao Qu2 1 Department of Chemistry, Stony Brook University , Stony Brook, New York, United States, 2Brookhaven National Laboratory, Upton, New York, United States Objectives Objectives: Our original 2-step radiosynthesis of [11C]indole-3-acetic acid via the reaction of gramine and [11C]cyanide in DMSO followed by hydrolysis required purification of the intermediate[11C]indole-3acetonitrile ([11C]IAN) to remove DMSO which inhibited the basic hydrolysis step [1]. Here we investigated the use of the bis polyether tetraethylene glycol (TEG) to enhance the nucleophilicity of [11C]cyanide without interfering with the hydrolysis so that both steps could be carried out in one pot. Methods Methods: We optimized conditions (temperature, time, solvent) for both the nucleophilic [11C]cyanation step with H11CN in presence of K2CO3 in TEG and the basic hydrolysis of [11C]IAN to form the [11C]IAA. Optimal formation of the [11C]IAN occurred in 5 min at 145 °C in TEG. Hydrolysis to [11C]IAA occurred in 4.0 M NaOH for 8 min at 145 °C in the same reaction vessel. [11C]IAA was purified by HPLC and formulated by SPE [2]. Results Results: The [11C]cyanation of gramine to form [11C]IAN was rapid (5 min at 145 °C) and occurred in high yield (~80%) in TEG. TEG was also compatible with the hydrolysis of the [11C]IAN to [11C]IAA and both steps could be carried out in one pot (Figure 1). Total synthesis time to 40 min and the production cycle and formulation was completed 55 min. The overall decay-corrected RCY of [11C]IAA was 33.5 ± 9.5% (n = 5); radiochemical purity was > 98% and specific activity was 47 ± 12.5 GBq/mmol (n = 5). Conclusions Conclusions: We have developed a successful two-step, one-pot method for routine production of [11C]IAA for plant imaging research. The high yield [11C]cyanation using TEG as a nucleophilic substitution promoter in presence of potassium salts is potentially useful for other [11C]cyanation reactions for PET radiotracers containing the [11C]cyano group and functional groups derived therefrom. Acknowledgements Acknowledgements: Supported by the U. S. Department of Energy Office of Biological and Environmental Research (Contract DE-AC02-98CH10886). References References: [1] Reid AE, et al. (2011) JLCR, 54, 433-7. [2] Xu, Y., et al. (2014) Appl Radiat Isot, 91, 155-60.

Figure 1. Two-step, one-pot radiosynthesis of [11C]IAA using TEG.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S285

285 Simplified radiochemical synthesis of [11C]clozapine Patrick Lao1, Tobey J. Betthauser1, Andrew T. Higgins1, Robert J. Nickles1, Onofre T. DeJesus1, Dhanabalan Murali1, Todd E. Barnhart1, Jonathan Oler2, Patrick H. Roseboom3, Ned H. Kalin2, Bradley T. Christian1 1 Medical Physics, University of Wisconsin at Madison, Madison, Wisconsin, United States, 2Psychiatry, University of Wisconsin-Madison, Madison, Wisconsin, United States, 3Neurology, University of Wisconsin-Madison, Madison, Wisconsin, United States Objectives There is a renewed interest in the use of [11C]clozapine as a probe for imaging the proper transfection of designer receptors in designer receptors exclusively activated by designer drugs (DREADD) studies. Previously published methods for the radiochemical synthesis of [11C]clozapine requires hazardous solvents like chloroform, long reaction times, and elutions from cation exchange cartridges. A new approach simplifies the production of [11C]clozapine while improving specific activity for use in PET scans. Methods A reduced amount (0.25 mg) of the precursor norclozapine was prepared in as little as 300 µL of acetonitrile. N-methylation of norclozapine was done with [11C]methyl triflate, and the reaction took place at room temperature for five minutes. Excellent chemical separation was achieved on a semi-preparative reverse phase C18 column using 50/50 0.05 M sodium acetate/acetonitrile mobile phase. A 7 mL/min flow rate yielded a retention time of approximately 14 minutes. Organic solvents were removed via a C18 Seppak Light cartridge under positive nitrogen pressure and was confirmed with gas chromatography. Elution into final formulation was performed with a 10% ethanol solution. Results Compared to previous methods, the amount of norclozapine was reduced from 1.0 mg to 0.25 mg, and the solvent was switched from 1.0 mL chloroform to 0.3 mL acetonitrile. Production times were kept under 30 minutes. Chemical purity was consistently above 98%. Decay corrected radiochemical yields were 32.0-38.4%, which is lower than the previously reported value of 70%. Final batches of [11C]clozapine had specific activities of 5,500-13,500 mCi/umol, compared to previously published values of 2,500-3,500 mCi/umol. Conclusions A new, simplified method for the radiochemical synthesis of [11C]clozapine proves to be practical and time efficient. The production method uses a reduced amount of norclozapine and more readily available solvents. The N-methylation of norclozapine is performed under milder conditions and requires a shorter reaction time. Removal of organic solvents no longer requires an acetic acid-activated anion exchange cartridge and a cumbersome elution. Specific activities of final batches exceed that of previously reported methods. PET scans with [11C]clozapine in the rhesus monkey have shown high brain uptake. Acknowledgements References Bender D, Holschbach M, and Stocklin G. Synthesis of n.c.a. Carbon-11 labelled clozapine and its major metabolit clozapine-N-oxide and comparison of their biodistribution in mice. Nucl. Med. Biol., Vol. 21, No. 7, 1994; 921-925.

J Label Compd Radiopharm 2015: 58: S1- S411

S286: Poster

21st International Symposium on Radiopharmaceutical Sciences

286 Synthesis of a candidate 11C-labeled ligand for imaging brain oxytocin receptors in rhesus monkey Sanjay Telu, Jeih-San Liow, Sami Zoghbi, Alicia Woock, Robert Gladding, Robert B. Innis, Victor W. Pike Molecular Imaging Branch, NIMH, NIH, Bethesda, Maryland, United States Objectives Oxytocin is strongly implicated in important human behavior including social cognition, pair bonding, stress and fear [1]. A PET radioligand for imaging brain oxytocin receptors (OTRs) would be valuable for understanding the role of oxytocin in psychiatric disorders. We identified the antagonist, (2S,4EZ)-(2'chlorobiphenyl-4-yl)(2-(3-methyl-1,2,4-oxadiazol-5-yl)-4-(methylimino)pyrrolidin-1-yl)methanone ((2S,4EZ)-1; hOTR IC50 1 nM; logD 3.87), as a candidate for radioligand development [2]. The Z-isomer is more potent than the E-isomer. Our aim was to prepare [11C](2S,4Z)-1 for evaluation. Methods The labeling precursor desmethyl-(2S,4Z)-1, was prepared in 6 steps. [11C](2S,4Z)-1 was prepared by treating desmethyl-(2S,4Z)-1 and t-BuOK in THF with [11C]MeI at 70 °C for 5 min (Figure 1A). PET imaging with determination of arterial input function was performed after i.v. injection of [11C](2S,4Z)-1 into rhesus monkey at baseline and after attempted OTR block with another antagonist [3]. Distribution volumes (VT) were calculated and compared. Results [11C](2S,4Z)-1 was produced in ~9% decay-corrected overall yield with specific radioactivities of ~370 GBq/µmol. At baseline, [11C](2S,4Z)-1 rapidly entered monkey brain giving high radioactivity uptake followed by fast washout (Figure 1B). With receptor preblock, a global reduction of brain uptake was obtained (Figure 1B). VT decreased by 35-40% across all brain regions. Plasma free fraction (fp) values were 1.24 and 0.58% under baseline and preblock conditions, respectively. Conclusions [11C](2S,4Z)-1 demonstrated high initial brain uptake and fast washout without definite OTR-specific signal in monkey brain; the uniform reduction of VT could primarily be due to the decrease of fp under the preblock condition. This non-quantifiable signal indicates that OTR density in rhesus monkey brain may be very low and that a higher affinity PET radioligand will likely be required. Acknowledgements Support from the IRP of NIH (NIMH) References [1] Lindenberg A M et al. (2011) Nat Rev Neurosci, 12, 524. [2] Schwarz M et al. (2002) WO 02/102799 A2. [3] Bellenie BR et al. (2009) Bioorg Med Chem Lett, 19, 990.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S287

287 Direct and Efficient Cobalt Carbonyl-Mediated Aryl Acetylation using [11C]Methyl Iodide Kenneth Dahl1, Magnus Schou2, Christer Halldin1 1 Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Translational Science Centre, AstraZeneca, Stockholm, Sweden Objectives Aryl methyl ketones is a valuable class of carbonyl derivates that can serve as versatile synthetic building blocks for pharmacologically active molecules. In PET radiochemistry,11C-labelled aryl methyl ketones are readily available via Pd-mediated 11C-carbonylation with either 11CO or [11C]acetyl chloride.[1,2] A drawback with both these methodologies is that they are based on technically demanding multistep radiosyntheses starting from the radioactive precursor 11CO2. In addition, radioligands synthesized from 11CO2 are usually obtained at lower specific radioactivity compared to those obtained from in-target produced [11C]methane.[3] The aim of this study was thus to develop a cobalt carbonyl-mediated protocol to form 11C-labelled aryl methyl ketones using the well-established radioactive precursor 11CH3I as a labelling agent. Methods No-carrier-added, in-target produced [11C]methane was converted into 11CH3I as previously described.[3] 11 CH3I was trapped in a vial containing aryl halide and 1 ml anhydrous acetonitrile. After completed trapping, the solution was transferred to a sealed 4 ml reaction vessel pre-charged with Co2(CO)8 and the resulting mixture was heated at 130oC for 1 min using a single-mode microwave reactor equipped with temperature control. Radiochemical yields (RCY) were determined with HPLC. Results We selected [11C]acetophenone ([11C]1) as a model compound for exploration of the cobalt-mediated carbonylative coupling of aryl halides with 11CH3I under microwave irradiation (Figure 1). A series of experiments was thus conducted in which the RCY of [11C]1 was investigated in relation to substrate type and reaction temperature. Interestingly, when the less reactive bromo- and chlorobenzene were substrates, [11C]1 was obtained in higher yields, RCYs of 76% and 77±2% (n=3), respectively, than with iodobenzene (RCY 47%). The lower RCY with iodobenzene as substrate may partly be explained by the highly rapid competing diaryl ketone formation reaction.[4] Attempts to perform the reaction at different temperatures was not proven to significantly improve the RCY. Conclusions The high-yield radiolabeling of [11C]acetophenone demonstrates the potential of direct cobalt carbonyl-mediated synthesis for the preparation of 11C-labelled aryl methyl ketones using 11CH3I. Acknowledgements References [1] Karimi F, et al (2005) Eur. J. Org. Chem, 11, 2374-2378. [2] Arai T, (2009) Tetrahedron Letters, 50, 4788-4791. [3] Andersson J, et al (2009), 67, 106-110. [4] Enqvist PA, et al (2003) Org. Lett, 5, 4876-4878.

J Label Compd Radiopharm 2015: 58: S1- S411

S288: Poster

21st International Symposium on Radiopharmaceutical Sciences

288 Gas-Liquid Segmented Microfluidics: 11C-Carbonylation Reactions Made Easy Kenneth Dahl1, Magnus Schou2, Obaidur Rahman1, Christer Halldin1 1 Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Translational Science Centre, AstraZeneca, Stockholm, Sweden Objectives [11C]Carbon monoxide has many attractive features as a synthon in PET chemistry, including its facile production and high versatility in transition metal mediated 11C-carbonylation. The aim of this study was to develop a general platform where 11C-carbonylation reactions are performed in a gas-liquid segmented microfluidic system. Methods The microfluidic system (Figure 1) consists of the following: 1) mixing-tee (50 µm i.d.) to permit gas and liquid contact and facilitate µ-bubble formation. 2) 5 m long fused-silica glass capillary (200 µm i.d.). 3) backpressure regulator (BPR) to keep the pressure constant during the microfluidic reaction. No-carrier-added, in-target produced 11CO2 was converted into 11CO as previously described.[1] In a typical experiment, 11CO was trapped and concentrated on a silica column at -196oC, after which the accumulated 11CO was transferred into the micro-reactor using a micro flow controller (helium, 100-200 µl/ml). At the same time the premixed carbonylation reagents (iodobenzene, Pd-ligand and benzylamine in THF) was infused into the micro-reactor at a flow rate of 20 µl/min using a syringe pump. The product was finally collected in a vial connected to the micro-reactor outlet with a leaktight gas bag in series to receive volatile radioactive products (e.g. 11CO). Radiochemical yields (RCY) were determined with HPLC. Results Two different catalytic systems were applied, Pd-PPh3 and Pd-xantphos, for the 11C-aminocarbonylation of iodobenzene with benzylamine, while varying the reaction temperature and the gas flow rate. To our delight, at 100oC and gas flow rate of 100 µl/min using the standard Pd-ligand catalytic system, Pd(PPh3)4, [11C]Nbenzylbenzamide was obtained in a reproducible RCY of 95±1% (n=3). Attempts to perform the reactions at lower temperatures (80oC) or at higher gas flows (200 µl/min) resulted in a decreased TE and thereby a lower RCY. On the other hand, a RCY of 98% was obtained using Pd-xantphos already at room temperature (RT), thus further illustrating the superiority of the Pd-xantphos catalytic system for the 11C-aminocarbonylation reaction.[1] Conclusions A gas-liquid segmented microfluidic platform has been developed. The Pd-mediated 11Caminocarbonylation of [11C]N-benzylbenzamide proceeds smoothly and in excellent RCY. Acknowledgements References [1] Dahl K, et al (2013) Eur. J. Org. Chem, 7, 1228-1231. [2] Miller PW, (2010) Chem. Eur. J, 17, 460-463.

Figure 1. The gas-liquid microfluidic experimental setup

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S289

289 Carbon-11-labelling of two novel indole-2-carboxamides targeting the TSPO Frédéric Dollé1, Isaac Freelander2, Fabien Caillé1, Rajeshwar Narlawar2, Annelaure Damont1, Bertrand Kuhnast1, Géraldine Pottier1, Raphaël Boisgard1, Michaël Kassiou2 1 CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France, 2University of Sydney, Sydney, New South Wales, Australia Objectives TSPO density increases markedly in microglia after a neuronal insult, rendering this protein a useful marker of microglial activation [1-3]. Within a novel class of 1-benzylindole-2-carboxamides targeting the TSPO, two potent candidates were selected for carbon-11-labelling and future µPET imaging studies. Methods Both selected candidates (1,2) were prepared from commercially available ethyl 5-methylindole-2carboxylate through sodium hydride mediated N-benzylation, base-catalysed saponification, and amide coupling. The corresponding precursors for labelling were then synthesized by hydrogenolytic demethylation. Binding were evaluated through the displacement of [3H]PK11195 from rat kidney membrane TSPO [4]. Carbon-11 labelling was performed using a TRACERLab FX-C Pro synthesizer (GEMS), starting from [11C]CO2. Conditions comprised (1) trapping at -20°C of [11C]MeOTf in acetone containing the appropriate nor-derivative (0.9-1.1 mg) and a base (aq. NaOH); (2) heating at 110°C for 2 min ; (3) HPLC-purification (Waters Symmetry® C-18) and (4) SepPak®Plus C-18-based reformulation. Results Compounds 1 and 2 were synthesized in 45%-50% overall yields. The corresponding precursors were then obtained in 85-88% yields. 1 conformed to a two-site binding model (Ki = 2.67 ± 0.34 pM and 450.7 ± 0.1 nM), while 2 was a single-site binder (Ki = 0.46 ± 0.05 nM). Labelling with carbon-11 was straightforward and gave [11C]-1 and [11C]-2 in 15-20% decay-corrected yields (n=5), based on starting [11C]CO2. Chemical and radiochemical purities were greater than 95% and ready-to-inject batches were obtained in 40 minutes. Specific radioactivities ranged from 50 to 100 GBq/µmol. Conclusions Two novel TSPO ligands belonging to the indole-2-carboxamide series have been synthesized and labelled with carbon-11. Dynamic µPET studies in a rat model of neuroinflammation will be performed to demonstrate the potential of these compounds to in vivo image TSPO overexpression. Acknowledgements INMiND (HEALTH-F2-2011-278850) References [1] Gavish M. et al. (1999), Pharmacol. Rev., 51, 629-650. [2] Scarf A.M. et al. (2009), J. Med. Chem., 52, 581-592. [3] Trapani A. et al. (2013), Bioconjugate Chem., 24, 1415-1428 ; [4] Selleri S. et al. (2001), Bioorg. Med. Chem., 9, 2661-2671.

J Label Compd Radiopharm 2015: 58: S1- S411

S290: Poster

21st International Symposium on Radiopharmaceutical Sciences

290 Radiosynthesis and evaluation of [11C]BU99008 with ultra-high specific activity as a PET ligand for imaging I2-imidazoline receptors. Kazunori Kawamura1, Yoko Shimoda1, Joji Yui1, Masayuki Fujinaga1, Tomoteru Yamasaki1, Akiko Hatori1, Lin Xie1, Yiding Zhang1, Katsushi Kumata1, Masanao Ogawa1, 2, Yusuke Kurihara1, 2, Nobuki Nengaki1, 2, Hidekatsu Wakizaka3, Ming-Rong Zhang1 1 Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2 SHI Accelerator Service Ltd, Tokyo, Japan, 3Biophysics Program, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Objectives The I2-imidazoline receptor (I2R) is involved in various neuropsychiatric disorders, but the these function are unknown. In addition, selective I2R ligands promote food intake and may therefore alter eating behavior. The carrier dose of PET ligand may modify both binding and pharmacokinetics in small animal imaging studies. Ultra-high specific activity (SA) is a useful tool to investigate a receptor with low density and small region in the brain [1]. We recently reported that [11C]FTIMD with ultra-high SA (mean 4470 GBq/μmol) for imaging of I2Rs showed higher specific-binding than [11C]FTIMD with conventional SA (about 100 GBq/μmol) in the hypothalamus, which is highly expressed I2Rs [2]. More recently, Kealey et al. developed [11C]BU99008 as a more potent PET ligand for I2R imaging [3]. [11C]BU99008 displayed a relatively high brain penetration and specific binding in the porcine and rhesus brain [3,4]. In this study, to image I2Rs in extremely small region such as hypothalamus, we synthesized and evaluated [11C]BU99008 with ultra-high SA as a PET ligand. Methods [11C]BU99008 was prepared by methylation of the BU-precursor and [11C]methyl iodide. In case of ultrahigh SA, [11C]methyl iodide was produced by iodination of [11C]methane using the single-pass method [1,2]. Biodistribution and brain PET studies were conducted in rats. Results [11C]BU99008 with conventional and ultra-high SA were successfully synthesized in the range of SA at 55∼220 [n = 8] and 5400∼16600 [n = 7] GBq/μmol at the end of synthesis, respectivery, yielding a radioactivity suitable for injection. In PET studies, the radioactivity after injection of [11C]BU99008 with conventional or ultrahigh SA was highly accumulated in the hypothalamus. Pretreatment with BU224 (an high affinity I2R ligand; 1 mg/kg) significantly decreased the radioactivity (AUC30-60 min) of conventional or ultra-high SA in the hypothalamus to 76% or 86% of the correspoding control radioactivity (AUC30-60 min), respectively. Conclusions [11C]BU99008 with ultra-high SA showed high specific binding in rat brain. PET study using [11C]BU99008 with ultra-high SA would contribute to the detection of I2Rs expression in small regions or with small change. Acknowledgements References [1] Noguchi J, et al (2003), Nucl Med Biol, 30, 335-43. [2] Kawamura K, et al (2012), Nucl Med Biol, 39, 199-206. [3] Kealey S, et al (2013), J Nucl Med, 54, 139-44. [4] Parker CA, et al (2014), J Nucl Med, 55, 83844.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S291

291 Improved Radiosynthesis of the P-glycoprotein Tracer (R)-[11C]Verapamil Using A New HPLC Purification Method Ya-Yao Huang, Kai-Yuan Tzen, Chyng-Yann Shiue PET Center, National Taiwan University Hospital, Taipei, Taiwan Objectives (R)-[11C]Verapamil is a well characterized radiotracer for studying P-glycoprotein (P-gP) in animals and humans [1]. The purpose of this study was to develop a new HPLC purification method for the synthesis of this tracer before human studies are undertaken. Methods An analytical-HPLC condition for the separation of (R)-Verapamil and its precursor was first established and optimized for further semi-preparative HPLC purification. (R)-[11C]Verapamil was synthesized by methylation of its precursor (1.0 mg in acetone) with [11C]MeOTf at 60℃ for 3 min (Scheme 1) using TraceLab FxC module. The reaction mixture was then purified with a semi-preparative HPLC (YMC C8, sodium acetate (pH5.0)/EtOH (70/30), 4ml/min). The fraction containing the product was collected and passed through a Millipore filter into a sterile vial containing phosphate buffer (pH=7). The identity of the product was verified by analytical HPLC as compared with the authentic sample. Results Several chromatographic conditions were evaluated to optimize the chromatographic separation and resolution between (R)-[11C]Verapamil and its precursor. For (R)-[11C]Verapamil, YMC C8 column with ethanol as the mobile phase gave the fastest separation and the best resolution between the product and its precursor. With this improved purification method, we have produced (R)-[11C]Verapamil in RCY of 10% (based on radioactivity of [11C]MeOTf, EOS) in a synthesis time of about 50 min and specific activity of 5 GBq/μmol (EOB). The ethanol content in the formulated solution was 〈10 %. Conclusions By using this new improved HPLC purification method, we have produced (R)-[11C]Verapamil in reliable RCY and high radiochemical purity. Acknowledgements References [1] Wagner CC et al. (2009) J Nucl Med, 50, 1954.

Scheme 1. Radiosynthesis of (R)-[11C]Verapamil

J Label Compd Radiopharm 2015: 58: S1- S411

S292: Poster

21st International Symposium on Radiopharmaceutical Sciences

292 Direct radiolabelling with [11C]carbon disulfide: synthesis of thioureas and the progesterone receptor agonist Tanaproget Steven Kealey1, Tom Haywood1, Louis Allott2, Graham Smith2, Christophe Plisson3, Philip Miller1 1 Department of Chemistry, Imperial College London, London, United Kingdom, 2Institute of Cancer Research, Sutton, Surrey, United Kingdom, 3Imanova Ltd, London, United Kingdom Objectives Our group recently described the preparation of [11C]carbon disulfide and its reactivity with amines to form [11C]dithiocarbamate salts and esters [1]. Here we report an improved method for the production of 11CS2 and extend its reactivity with amines to the synthesis of symmetrical [11C]thioureas (Figure 1) as well as the progesterone receptor (PR) agonist [11C]Tanaproget. Methods 11CS2 was produced by passing 11CH3I in a He gas stream through a quartz tube containing a mixture of sulfur and sand at 500oC. The resultant 11CS2/He gas stream was delivered into a glass vial containing amine (10 mg) in MeCN or DMSO (400 µL) which was then heated for 10 min. An aliquot of the crude reaction mixture was analysed by HPLC to determine the radiochemical purity (RCP) of the [11C]thiourea product. We investigated the reactivity of 11CS2 with a series of primary aliphatic amines (R = methyl, cyclohexyl, benzyl) and aromatic amines (aniline) as well as di-nucleophiles (2-aminobenzyl alcohol, 1,2-diaminocyclohexane, acyclic Tanaproget) for the formation of cyclic species. Results Initially [11C]dithiocarbamate salts are formed at room temperature which convert to the corresponding symmetrical [11C]thioureas upon heating. Aliphatic amines react at 90oC in MeCN with >95% RCP. Aromatic amines required heating at 150oC in DMSO to achieve >95% RCP, while [11C]Tanaproget formed more slowly, with an RCP of 75% following 10 min at 150oC. Using these conditions, the synthesis of Tanaproget was scaled up, and following semi-preparative HPLC purification and reformulation, was successfully obtained in 0.5-1.0 GBq isolated yield with excellent RCP (>99%) and high chemical purity. Conclusions A straightforward and rapid method for producing 11CS2 from 11CH3I has been developed. 11CS2 has proven to be highly reactive with amines for the formation of a range of 11C-labelled organo-sulfur functionalities. Future PET studies will examine [11C]Tanaproget in vivo, for its potential to measure PR levels in breast cancer. Acknowledgements We are grateful to the EPSRC for funding (grant no. EP/L025140/1) References [1] Miller, P. W. and Bender, D., Chem. Eur. J., 2012, 18, 433.

Radiosynthesis of [11C]thioureas and [11C]Tanaproget

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S293

293 Robust [11C]methylation of a terminal alkyne: the BACE-1 PET tracer [11C]RO6807936 Idriss Bennacef1, Quang Truong2, Shawn P. Walsh2, Kerry Riffel1, cristian A. Salinas1, Mona Purcell1, Marie A. Holahan1, Hyking Haley1, Xiangjun Meng1, Jared Cumming2, Eric D. Hostetler1 1 Imaging, Merck & Co, West Point, Pennsylvania, United States, 2Lead Optimization Chemistry, Merck & Co., Kenilworth, New Jersey, United States Objectives The amyloid hypothesis as a cause for Alzheimer Disease postulates that an imbalance between production and clearance of amyloid beta peptide (Aβ) causes the formation of toxic Aβ species, among them plaques. The first enzyme in the Aβ cascade is the β -site APP Cleaving Enzyme-1 (BACE-1). Currently, the best approach to test the Aβ hypothesis resides in the administration in early AD subjects of BACE-1 inhibitors. PET would be an invaluable tool to assess BACE-1 target engagement in-vivo. [11C]RO6807936 was proposed as a radioligand suitable for imaging BACE-1 [1]. In the described procedure, [11C]RO6807936 was obtained by palladium-mediated [11C]methyl-destannylation to obtain the carbon 11-labelled methyl alkyne compound. However, in our hand this radiochemistry was low yielding and not sufficiently robust to allow for PET studies in non-human primate (NHP). Hence, we strived to obtain a robust and general method for the [11C]methylation of terminal alkynes. Methods Palladium derivative species were tested in an array for the nonradioactive methylation of methyl-alkyne using the terminal alkyne, a PdLn reagent, a base and a sub-stoichiometric amount of methyliodide. When translated to tracer level, the system consisting of Di(1-adamantyl)-N-butylphosphine, Pd(OAc)2, KF, [11C]CH3I, and terminal alkyne precursor of RO6807936 provided the desired radioligand robustly and in high yield. The in vivo properties of this tracer were evaluated in wild type and P-gp KO rat and NHP in baseline and in blocking experiment using a BACE-1 inhibitor. Results [11C]RO6807936 was obtained in good radiochemical yield, chemical and radiochemical purity and specific activity. The tracer enters the brain of rat (WT and P-gp KO) as well as NHP and displays a homogeneous distribution. [11C]RO6807936 does not appear to be a P-gp substrate in-vivo. Time-activity curves were fit using a two-tissue compartment model. Pre-administration of a selective BACE-1 inhibitor showed the BACE1 specificity of the radioligand. Conclusions By using the possibilities offered by array screening, we were able to discovere a robust system for [11C]methylation of a terminal alkyne. This chemistry was successfully applied to the radiosynthesis of [11C]RO6807936 and its binding to BACE-1 was confirmed in NHP. Acknowledgements References [1] Gobbi L et al. (2013) J. Label Compd Radiopharm, 56, S299.

J Label Compd Radiopharm 2015: 58: S1- S411

S294: Poster

21st International Symposium on Radiopharmaceutical Sciences

294 Preparation of 13N-labelled polysubstituted triazoles via Huisgen cycloaddition Sameer M. Joshi, Vanessa Gómez-Vallejo, Jordi Llop Radiochemistry, CIC biomaGUNE, San Sebastian, Spain Objectives We present the unprecedented synthesis of 13N-labelled phenyl azide and the preparation of polysubstituted triazoles via Huisgen cycloaddition using [13N]NO2-. Methods The synthesis of 13N-labelled triazoles was approached via a 4-step process (Figure 1) [1,2]. Nitrogen-13 was produced by 16O(p,α)13N nuclear reaction. The irradiated solution was reduced with cadmium to convert [13N]NO3- into [13N]NO2-. A solution containing aniline in diluted HCl was added and the formation of the 13Nlabelled diazonium salt was allowed to occur (1 min, T = 25°C). Then a solution of NaNO2, AcOH and hydrazine hydrate was added for the formation of 13N-labelled azide (1 min, T = 25°C). The 13N-labelled azide was extracted with CH2Cl2, the solvent was evaporated and acetylene derivatives (Figure 1) were added along with [(Icy)2Cu]PF6 in ACN. The reaction was run for 10 min at T = 25°C or 50°C. Results No carrier added 13N-labelled phenyl azide was prepared in overall corrected radiochemical yield of 55±9%. For the preparation of 13N-labelled polysubstituded triazoles, radiochemical conversion (RCC) values of 16%, 50%, 08%, and 71% were achieved for compounds 1-4 at 25ºC. RCYs were improved up to 52%, 98%, 65%, and 98%, respectively, at 50ºC. Conclusions The synthesis of 13N-labelled polysubstituted triazoles can be achieved by reaction of the 13N-labelled azide with acetylenes with excellent RCC values. This general strategy might be applied to the preparation of 13Nlabelled biologically active compounds for their in vivo investigation using PET. Acknowledgements All the work has been supported by the RADIOMI project (EU FP7-PEOPLE-2012-ITNRADIOMI). References [1] Siddiki A, Takale B, Telvekar V, 2013, Tetrahedron Lett. 54, 1294-1297. [2] Diez-Gonzalez S, Nolan S, 2008, Angew. Chem. Int. Ed. 47, 8881-8884.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S295

295 Synthesis and preclinical evaluation of two novel radioligands for PDE7 imaging in the brain David Thomae1, 2, Sijn Servaes1, Naiara Vazquez3, 1, Leonie wyffels4, Stefanie Dedeurwaerdere3, Pieter Van der Veken2, Jurgen Joossens2, Koen Augustyns2, Sigrid Stroobants1, 4, Steven Staelens1 1 Molecular Imaging Center Antwerp, University of Antwerp, Wilrijk, Belgium, 2University Antwerp Medicinal Chemistry, University of Antwerp, Wilrijk, Belgium, 3Department of Translational Neurosciences, University of Antwerp, Wilrijk, Belgium, 4Nucelar Medicine , University Hospital Antwerp, Edegem, Belgium Objectives PDE 7 regulates cyclic adenosine monophosphate (cAMP) which is a an intracellular second messenger. Regulation of cAMP concentration is essential for many physiological processes, such as cardiac and smooth muscle contraction and neurological function [1]. To date there is no radiotracer for PDE7 imaging described and detection of PDE7 is only possible by immunohistochemistry. In this report we describe the synthesis and the in vivo evaluation in mice of two potential radiotracers to image PDE7 in the brain. Methods We have synthesized two PDE7 inhibitors MICA-003 (PDE7 IC50= 17.0 nM), MICA-005 (PDE7 IC50= 1.7 nM) and their corresponding precursors for radiolabeling. [18F]MICA-003 and [11C]MICA-005 were injected in C57BL/6J mice and in vivo images were acquired by µPET imaging. Radiometabolite analysis in plasma and brain was performed to determine the stability of the radioligand. Results [18F]MICA-003 was synthesized by displacement of the tosylate with [18F]fluoride and was produced in high decay corrected radiochemical yield (40%). On the other hand [11C]MICA-005 was prepared by methylation of the tetrazole with [11C]CH3OTf in a 5% decay corrected radiochemical yield (Scheme1). μPET imaging revealed that both radioligands crossed the blood brain barrier and had a homogenous distribution over the brain which washes out after the initial peak uptake. [18F]MICA-003 was quickly metabolized. At 5 min post injection (p.i.) we observed the presence of a radiometabolite in the brain in a proportion of 25.7 ± 2.57%. [11C]MICA-005 was more stable in vivo but a metabolite was also detected in the brain at 15 min p.i. in a proportion of 15.5 ± 4.40%. Conclusions We were the first to describe the synthesis and the in vivo evaluation of two radioligands for PDE7 imaging. [18F]MICA-003 and [11C]MICA-005 showed uptake in the brain and presented favorable kinetics. Unfortunately the presence of metabolites in the brain make [18F]MICA-003 and [11C]MICA-005 not suitable for accurate PDE7 quantification in the brain. Acknowledgements References [1] Huai Q., et al (2003) Biochemistry, 42, 13220-13226.

Scheme 1: Radiosynthesis of [18F]MICA-003 and [11C]MICA-005.

J Label Compd Radiopharm 2015: 58: S1- S411

S296: Poster

21st International Symposium on Radiopharmaceutical Sciences

296 An Improved Radiolabelling Strategy for the 5-HT2A Agonist, [11C]Cimbi-36 Nic Gillings1, Szabolcs Lehel1, Matthias Herth1, Sebastian Leth-Petersen2, Jacob Madsen1, Charlotte GabelJensen3, Jesper L. Kristensen2 1 Department of Clinical Physiology, Nuclear Medicine & PET, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark, 2Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark, 3Department of Analytical Biosciences, University of Copenhagen, Copenhagen, Denmark Objectives We have previously reported the radiosynthesis of the potent 5-HT2A agonist, [11C]Cimbi-36, and its in vivo evaluation in pigs, non-human primates and humans [1-3]. During radioHPLC analysis of plasma samples a characteristic metabolite is seen which accounts for up to 80% of the radioactivity in late samples from human PET studies. This has been identified as the 5-O-glucuronide. Due to concerns that this metabolite may enter the brain and confound quantification of the PET data, we have explored alternative radiolabelling positions. Methods 2- or 5-O-([11C]methyl)Cimbi-36 was prepared by methylation of 2-or 5-O-desmethyl-Cimbi-36 (0.3 mg) with [11C]methyl triflate in acetone containing aqueous sodium hydroxide as base (see scheme). The crude product was purified by HPLC and the collected product fraction was sterile filtered and diluted with sterile PBS to give an injectable solution. Results [11C]Cimbi-36 was labelled in the 2- and 5-methoxy positions in similar yields to the original method. Pig plasma analysis revealed that the glucuronide metabolite is present when labelling on the 2-methoxy position but absent when labelling on the 5-methoxy position. 5-O-([11C]methoxy)Cimbi-36 was prepared by O-methylation in acetone without protection of the secondary amine (60:40, O:N-methylation) using aqueous sodium hydroxide as base. Other bases and solvents were explored and the best O-methyl selectivity was acheived using solid caesium carbonate in DMSO (8:1, O:N-methylation). Unfortunately these conditions led to several radiolabelled biproducts which reduced the overall radiochemical yield and confounded the HPLC purification. 2-3.5 GBq of 5-O([11C]methoxy)Cimbi-36 could be prepared with specific activities of 500-1000 GBq/µmol. Conclusions Preparation of the potent 5-HT2A agonist, [11C]Cimbi-36 has been acheived using an alternative labelling position and with a simplified synthesis strategy. The overall preparation time has been reduced and the new labelling position eliminates the formation of a potentially confounding radiometabolite in plasma. A head-tohead comparision with the original tracer is planned in human subjects in the near future. Acknowledgements References [1] Ettrup et al. (2011) Eur J Nucl Med Mol Imaging, 38(4), 681-693. [2] Finnema et al. (2014) Neuroimage, 84, 342-353. [3] Ettrup et al. (2014) J Cereb Blood Flow Metab, 1188-1196.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S297

297 Synthesis of [11CO]Raclopride and a Comparison with [11CH3]Raclopride in a Monkey PET Study Obaidur Rahman1, 2, Kenneth Dahl1, Akihiro Takano1, Naoki Kanegawa1, Nahid Amini1, Bengt Långström2, 3, Lars Farde1, Christer Halldin1 1 Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 2Bencar AB, Uppsala, Sweden, 3Department of Chemistry, Uppsala University, Uppsala, Sweden Objectives The objectives were to develop a method to synthesize [11CO]raclopride ([11CO]RAC) and to compare this radioligand with [11CH3]raclopride ([11CH3]RAC) in a monkey PET study with regards to brain kinetics, radiometabolism and protein binding. Methods Radiochemistry Pd(π-cinnamyl) chloride (13.9 µmol), xantphos ( 14.3 µmol) and 4,6-dichloro-2-iodo-3-methoxyphenol ( 20.2 µmol) were taken in a reaction vial and dissolved in anhydrous THF (500 µL). (S)-(-)-2-Aminomethyl-1ethylpyrrolidine (137.8 µL) was added and mixed properly. [11C]CO was produced and trapped in the reaction vial as described previously.1 The vial was heated at 100°C for 5 min. The product was purified by semi-preparative HPLC using Xbridge C18 column and formulated in PBS and 10% ethanol. The product identity and purity were confirmed by HPLC and LC-MS. Monkey PET Study Two female cynomolgus monkeys were used under gas anesthesia of sevoflurane. Dynamic PET measurements were performed in two experimental days for 63 min with HRRT (Siemens) PET camera after intravenous injection of [11CO]RAC and [11CH3]RAC, respectively, in the same day. The order of injection of the two PET radioligands was changed during the second experimental day. In the PET study, venous blood samples were taken for radiometabolite analysis and protein binding. Binding potential (BPND) of the putamen and caudate was calculated with SRTM using the cerebellum as reference region. Results [11CO]Raclopride was synthesized with 53-57% rcy and 95% rcp. The trapping efficiency of 11CO was 6065% and the specific radioactivity was 33 to 35 GBq/µmol after a synthesis time of 50 min. A comparison of [11CH3]RAC and [11CO]RAC binding in monkey brain was performed using PET. The percent difference of BPND in putamen was <3% and that in caudate was <9% for the two radioligands. The plasma protein binding were 85.7 ± 0.6% and 86.2 ± 0.3% for [11CH3]RAC and [11CO]RAC, respectively. Conclusions Raclopride was successfully 11C-labelled at the carbonyl position using a [11C]carbonylation reaction. The monkey PET study with [11CO]raclopride showed similar results as for [11CH3]raclopride. Acknowledgements The authors thank the members of the PET group involved in these experiments at Karolinska Institutet. This work was supported by the EU project Mindview, no. K831218133. References 1. K. Dahl, M. Schou, N. Amini, C. Halldin, Eur. J. Org. Chem. 2013, 1228.

J Label Compd Radiopharm 2015: 58: S1- S411

S298: Poster

21st International Symposium on Radiopharmaceutical Sciences

298 Radiosynthesis and evaluation of [11C]isradipine, a calcium channel antagonist, for PET neuroimaging Benjamin H. Rotstein1, Ali A. Bonab1, Eli Livni1, Roy H. Perlis2, Steven H. Liang1, Neil Vasdev1 1 Nuclear Medicine and Molecular Imaging, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, United States, 2Department of Psychiatry and Center for Human Genetic Research, Center for Experimental Drugs and Diagnostics, Massachusetts General Hospital, Boston, Massachusetts, United States Objectives In the interest of developing PET probes for imaging calcium channels in psychiatric conditions such as schizophrenia, we have prepared a carbon-11 isotopologue of a dihydropyridine Ca2+-channel antagonist, [11C]isradipine ([11C]2), using [11C]CH3I. We have evaluated the potential of this radiotracer by PET/CT neuroimaging in normal rats. Methods The precursor to [11C]2, carboxylic acid 1 (1 mg), was dissolved in DMF (80 µL) with substoichiometric tetrabutylammonium hydroxide (TBAOH). [11C]CH3I, prepared in the gas phase using a GE FXMeI module, was exposed to 1 using the “loop method”1 on a GE FXN module. The product [11C]2 was purified and reformulated in ethanolic saline. Dynamic PET/CT brain imaging was conducted in rats for 30 min immediately following tail-vein injection of [11C]2, at baseline and after pretreatment with 2 (2 mg/kg, i.p.) 30 min prior to radiotracer administration. Results [11C]Isradipine ([11C]PN 200-110, [11C]2) was previously prepared by treatment of 1 with [11C]diazomethane.2 This method required specialized apparatus and produced [11C]2 in relatively modest specific activity (0.7 Ci/µmol). Using the same precursor, we have developed a radiosynthesis of [11C]2 using [11C]CH3I. By this method, 135.0 ± 53.5 mCi of [11C]2 was isolated (n = 4), ready for injection (6.5 ± 1.6% n.d.c. RCY from [11C]CO2) in high specific activity (6.61 ± 1.11 Ci/µmol at EOS). The logD of [11C]2 was measured by the shakeflask method,3 and found to be 2.15 (n = 8), and is in the appropriate range of most brain penetrating radiotracers. Peak whole-brain uptake of [11C]2 in rats was 2 SUV at baseline, with substantial washout over 30 min. After pretreatment with 2, whole-brain uptake was significantly diminished at peak (61% blocked) and over the course of the imaging experiment. Conclusions We have developed a radiosynthesis of Ca2+-channel antagonist [11C]2 using [11C]CH3I and that yields the radiotracer in high specific activity for neuroimaging studies. Preliminary rodent PET imaging data with [11C]2 is promising for imaging this target in the central nervous system, warranting evaluation in higher species. Acknowledgements References [1] Wilson AA, et al. (2009) J. Labelled Compd. Radiopharm. 52, 490–492. [2] Crouzel C, et al. (1990) Int. J. Radiat. Appl. Instrum., Part A 41, 241–242. [3] Wilson AA, et al. (2001) Appl. Radiat. Isot. 54, 203– 208.

Figure 1. Radiosynthesis of [11C]isradipine

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S299

299 The synthesis of [11C]PBD150, a glutaminyl cyclase inhibitor for the early detection of Alzheimer’s Disease, via the selective methylation of a phenol in the presence of a thiourea. Allen F. Brooks, Isaac M. Jackson, Xia Shao, George W. Kropog, Phillip Sherman, Carole Quesada, Peter J. Scott Radiology, University of Michigan, Ann Arbor, Michigan, United States Objectives Glutaminyl cyclase (QC) is primarily expressed in the brain and is responsible for the formation of pyroglutamate at the N-terminus of several neuropeptides [1]. This modification stabilizes the peptide against proteolytic decomposition [2]. In Alzheimer’s Disease (AD) the expression of QC is upregulated and leads to the formation of pGlu-Aβ peptides that are more neurotoxic and resistant to removal than normal Aβ and acts as a seeding peptide, forming plaques with unmodified Aβ [3]. The QC inhibitor PBD150 (Ki = 60 nM) has been demonstrated in vitro and in vivo to reduce the formation of Aβ aggregates [4,5]; [11C]PBD150 could provide a PET radioligand for the detection of AD prior to Aβ aggregation. Methods The novel desmethyl precursor was prepared by forming the thiourea via the thioisocynate of the protected phenol. Initial attempts to prepare [11C]PBD150 resulted in an alternate methylated product, likely from the methylation of the thiourea given its nucleophilicity [6]. The [11C]methylation of the phenol was investigated under a variety of conditions with various bases, including preforming the TBA phenoxide, to select for labeling the phenol over the thiourea. Ultimately, treatment of the precursor with a saturated K2CO3 solution subsequently dried over Na2SO4 prior to labeling with [11C]MeOTf was found to be the best method for generating [11C]PBD150. Results The [11C]methylation of the precursor was performed with [11C]MeOTf. The products were purified by reverse phase chromatography (20% EtOH) and the 2 mL of buffer collected with product were added to 8 mL of saline to prepare the dose. An average yield of 62.3 mCi of product was collected (6.9% from MeOTf, non-decay corrected; radiochemical purity of 96.4%, n=4). The doses produced were appropriate for preclinical studies. Conclusions A method for the [11C]methylation of a phenol in the presence of thiourea has been developed. This synthesis and those of related analogs produced through methylation and carbon fixation will be presented. Acknowledgements NIH (T32- EB005172), Alzheimer’s Association (NIRP-14-305669) and the Michigan Research Community for financial support. References [1] Hfling C, et. al. (2014) Int J Devel Neuroscience, 36, 64-73. [2] Folkers K, et. al. (1970) Biochem Biophys Res Comm, 39, 110-113. [3] Schilling S, et. al. (2006) Biochemistry 45, 12393-12399. [4] Buchholz M, et. al. (2006) J Med Chem, 49, 664-677. [5] Schilling S, et. al. (2008) Nat Med, 14, 1106-1111. [6] Campopiano O, et. al. (2001) Encyclopedia of Reagents for Organic Synthesis, Thiourea, John Wiley & Sons, Ltd.

J Label Compd Radiopharm 2015: 58: S1- S411

S300: Poster

21st International Symposium on Radiopharmaceutical Sciences

300 Optimization of tumor targeting using liposome-carried radiotracer at various conditions Woonghee Lee1, Gwang Il An2, Jeongsoo Yoo1 1 Department of Molecular Medicine, Kyungpook National University, Daegu, Korea (the Republic of), 2Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul, Korea (the Republic of) Objectives In our previous work, we developed a facile method for preparation of a tri-modal tumor imaging agent using liposome platform.[1] By development of a radiotracer that emits strong Cerenkov luminescence light, optical and nuclear images were obtained from the single imaging component. Tumors were detected by all optical, nuclear and magnetic resonance imaging modalities. The liposome-carried radiotracer showed longer retention in tumor but cleared quickly from reticuloendothelial organs. However, we need to optimize uptake of liposomecarried radiotracer into tumor at various conditions. Methods Herein, we focused on optimization of liposome compositon, difference of tumor uptake based on liposome size (30 nm to 100 nm) and concentration, tumor size, effect of tumor positon, sex of mice and housing condition. Finally, we compared tumor uptake of liposome-carried radiotracer in several type of xenograft tumor models. Results A reduction in cholesterol showed decreasing tumor uptake, whereas an increase in lipids and polyethyleneglycol (PEG) showed enhanced tumor uptake. So, we could optimize the ratio of liposome composition. Existing size of liposome (100 nm) was more effective than smaller size of liposome and the effect of concentration was marginal. Tumor position, sex of mice and housing condition was not significant in tumor uptake, while smaller tumor size led to higher tumor uptake. Among several type of tumor models, tumor uptake of liposome was best in murine colon carcinoma (CT26) tumor model. Conclusions We could optimize liposome-carried radiotracer into tumor uptake at these various conditions and select best condition. Acknowledgements Supported by R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A2A2A01012250, 2013M2A2A 6042317, 20090078235) and the BK21 Plus funded by the Ministry of Education, Korea (21A2013221 2094). References [1] Kim, Jonghee, et al. (2014), ACS Medicinal Chemistry Letters, 5, 390-394.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S301

301 [11C]MADAM used as a model for understanding the metabolism of diphenyl sulfide positron emission tomography radioligands. Fabienne Gourand1, 2, Nahid Amini1, Shizeng Jia1, Sharon Stone-Elander3, Denis Guilloteau4, Louisa Barré2, Christer Halldin1 1 Karolinska Institutet, Department of Clinical Neuroscience, Section of Psychiatry, Stockholm, Sweden, 2CEA, DSV/I2BM, LDM-TEP Group, GIP Cyceron, Caen, France, 3Karolinska University Hospital, MicroPET and Clinical Neurosciences, Stockholm, Sweden, 4INSERM U930-Université François Rabelais de Tours, CHRU de Tours, Tours, France Objectives Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT), and therefore it is important to investigate their metabolism. In quantitative PET measurements, metabolite analysis in plasma is an essential prerequisite to determine the exact arterial input function. The aim of this project was to explore the metabolic profile of [11C]MADAM, a radioligand widely used for in vivo PET studies [1-3]. Methods The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the metabolic rate of the radioligand [11C]MADAM was examined both in vitro and in vivo by radioHPLC. RLM and HLM incubations were carried out at two different carrier concentration of 1 and 10µM. Urine samples were analysed by radio-HPLC after perfusion of [11C]MADAM/MADAM into rats. Results The metabolites produced in vitro were analyzed by UHPLC/Q-ToF-MS and were as a result of Ndemethylation, S-oxidation and benzylic hydroxylation (NHMADAM, m/z 259.13; SOMADAM, m/z 289.14; NHSOMADAM, m/z 275.12; hydroxyl-MADAM, m/z 289.14 and hydroxyl-NHMADAM, m/z 275.12). The presence of carrier greatly affected the metabolism rate of [11C]MADAM in RLM and HLM experiments. In the presence of carrier, the metabolism rate of [11C]MADAM decreased drastically, 80 ± 5 % of unchanged [11C]MADAM was present after 45 min of incubation with HLM in comparison to 58 ± 2% with 1µM of carrier and 35 ± 1% in the absence of carrier. In vivo studies were carried out and solutions containing [11C]MADAM /MADAM (25µg to 1mg) were administered into rats; no radiometabolite was observed in urine samples when more than 25µg of carrier was used. Conclusions The metabolic kinetics of the radioligand [11C]MADAM is dose-dependent and a good accordance is observed between the in vitro studies performed by RLM and HLM, and the in vivo data obtained from rat studies. Acknowledgements The authors sincerely thank the entire PET group at Karolinska Institutet for their support during this study and Siv Eriksson and Li Lu, for excellent technical assistance for in vivo studies. This project was partially funded by the DiMI: WP3, the European programme. References [1] Halldin C, Lundberg J, Sóvágó J, Gulyás B, Guilloteau D, Vercouillie J, Emond P, Chalon S, Tarkiainen J, Hiltunen J, Farde L (2005) Synapse, 58, 173-83. [2] Lundberg J, Odano I, Olsson H, Halldin C, Farde L (2005) J Nucl Med, 46,1505-15. [3] Gourand F, Emond P, Bergström JP, Takanao A, Gulyás B, Guilloteau D, Barré L, Halldin C (2014) Nucl Med Biol, 41, 501-506.

J Label Compd Radiopharm 2015: 58: S1- S411

S302: Poster

21st International Symposium on Radiopharmaceutical Sciences

302 Microdose study of 11C radiolabeled Donecopride, a multi-target-directed ligand for Alzheimer’s disease treatment. Fabien Fillesoye1, Meziane Ibazizene1, Martine Dhilly1, Auriane Maiza1, Cedric Lecoutey2, Christophe Rochais2, Patrick Dallemagne2, Fabienne Gourand1, Louisa Barré1 1 CEA,DSV/I2BM, LDM-TEP Group, GIP Cyceron, Caen, France, 2Université de Caen Basse-Normandie, EA4258-CERMN, Caen, France Objectives An interesting pharmacological approach to cure Alzheimer’s disease (AD) is the development of multi-target-directed ligands (MTDLs) [1]. Donecopride, a dual 5-HT4R partial agonist (Ki=10.4nM)/AChE inhibitor (IC50=16nM) was described to have the ability to display improvements in both cholinergic neurotransmission and inhibition of amyloid-β peptide (Aβ) accumulation and aggregation [2]. The main objective of our study was to study in vivo its behavior using positron emission tomography (PET). Methods Donecopride was labeled with [11C]CH3OTf. After [11C]-Donecopride administration in rat (control condition): BBB crossing, biodistribution, plasma and brain metabolism were evaluated. Blockade experiments were carried out with GR125487 (5-HT4R antagonist, 1mg/kg) and donepezil (AChE antagonist, 2.5mg/kg) respectively, injected alone or in association 30 min prior to [11C]-Donecopride. The role of P-glycoprotein (P-gp) transporter to regulate the BBB crossing of [11C]-Donecopride was investigated with Elacridar (inhibitor of P-gp; 5 mg/kg) injected 30 min prior to the radiotracer. Results [11C]-Donecopride was prepared with sufficient incorporation yield (60-70% at EOS), good purity (>99%) and specific radioactivity (>75GBq/µmol at EOS) and was stable over 2 hours in formulation solution. Evaluation of radioactive brain content indicated a relative stability of the parent compound (85% at 60 min); in contrast to plasma (60% radiometabolites at 60 min); in control conditions at 10 min p.i., no difference between brain structures was observed. Blockade studies with GR125487 and donepezil reduced [11C]-Donecopride uptake in some peripheral organs but not in brain structures. Although a low brain uptake was observed after injection of [11C]-Donecopride (0.13%ID at 2 min p.i.), its co-administration with unlabeled compound (therapeutic doses) induced a significant improvement of the radioactivity in the brain (2.5 times). The influence of dose administration could be mediated by P-gp transporters; this hypothesis has been studied with Elacridar where [11C]Donecopride brain uptake was 5.5 times greater than in control conditions. Conclusions These data provide important information on metabolism and distribution of Donecopride. Our study permits to identify that the BBB crossing is dose-dependent and [11C]-Donecopride uptake could be modulated by P-gp. Acknowledgements References [1] A. Cavali et al., (2008) J Med Chem, 51: 347-372.[2] C. Lecoutey et al., (2014) PNAS, 111, 38253830

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S303

303 Single valve module for production and efficient transfer of [11C]carbon monoxide to a disposable reaction vial Jonas Eriksson, Gunnar Antoni Uppsala University Hospital, PET centre, Uppsala, Sweden Objectives A method to confine [11C]CO at ambient pressure in small volume reaction vials using xenon as transfer gas has previously been presented.[1] The method utilize the high solubility of xenon to bubble the gas stream into an organic solvent in a closed vial without building up pressure. Although the solubility of [11C]CO is poor it cannot escape from the vessel and remains available for chemical transformations via equilibration between the liquid and gas phase. Here we present a new improved module based on the ‘xenon-method’ along with data on its efficiency. Methods One 2-position valve with 12 ports (Valco) connected all components. The xenon and helium flow was 1.5 and 20 mL/min, respectively. Zinc was used as reductor. The traps were cooled in liquid-N2 to concentrate the activity and heated with cable heaters to release it. The septum-equipped glass vial (0.9 mL) contained THF (0.2 mL). Pneumatic lifts were used to activate cooling and to lower the needle into the vial. The valve was toggled between position A and B during the process as follows: 1. System idle = A 2. [11C]CO2 transfer to CO2-trap = B 3. [11C]CO2 reduction to [11C]CO and transfer to CO-trap = A 4. [11C]CO transfer to reaction vial = B Results After releasing the [11C]CO2 from the CO2-trap 82% (decay corrected) was recovered in the vial as [11C]CO. The loss of activity was primarily due to absorption on the zinc. The process took 4 min counting from when [11C]CO2 was collected until [11C]CO was delivered to the vial. Conclusions This single valve module facilitates reliable and efficient use of [11C]CO in PET tracer production. Acknowledgements References [1] J. Eriksson et al J. Labelled Comp. Radiopharm. 2012, 55 (6), 223-228

J Label Compd Radiopharm 2015: 58: S1- S411

S304: Poster

21st International Symposium on Radiopharmaceutical Sciences

304 [11C] Carbonylation via Conversion of [11C]CO2 → [11C]CO Mediated by [11C]Silane Derivatives Carlotta Taddei, Salvatore Bongarzone, Abdul Karim Haji Dheere, Antony Gee Imaging Sciences and Biomedical Engineering, King's College London, London, United Kingdom Objectives Carbon monoxide (CO) is used in synthesising amides, esters, ketones and carboxylic acid anhydrides, which are common functional groups in many biologically active molecules.1 [11C]CO is traditionally obtained by the gas phase reduction of [11C]CO2 at elevated temperatures2 or by [11C]CO2 → [11C]CO conversion via electrochemical3 or chemical processes. The CO2 → CO chemical conversion can be achieved using silane derivatives4 through two steps: i) CO2 insertion in a silane derivative leading to a silacarboxylic acid intermediate followed by ii) CO release triggered by an activator. CO can be released to a second vial and coupled with an amine and an organic halide yielding the aminocarbonylation product (Figure 1). Here, this carbonylation method is applied to carbon-11 labelling by converting [11C]CO2 to [11C]CO and subsequently used to synthesise [11C] amide-containing molecules. This represents the first [11C]carbonylation methodology mediated by silane derivatives reported to date. Methods The [11C]carbonylation reaction was developed using a simple two-vial reaction set-up. In the first vial, [11C]CO2 → [11C]CO conversion was achieved by coupling [11C]CO2 and a silane derivative at low temperatures (Vial 1, Figure 1). Following the addition of a decarbonylating agent (activator) with heating, [11C]CO was liberated. Passage of [11C]CO to a second vial containing an amine and an organic halide led to the production of the corresponding [11C]amide derivative (Vial 2, Figure 1A). Results As model reaction, [11C]N-benzylbenzamide was obtained in a high radiochemical purity (>98%) and short synthesis time (~15 min from EOB), Figure 1B. Conclusions A new carbonylation method mediated by [11C]silacarboxylic acids has been successfully established. This methodology represents an attractive method to produce [11C]carbonyl-containing molecules, obviating the need for gas phase reduction infrastructure. Acknowledgements The authors acknowledge the financial support from the Medical Research Council (MRC), Biomedical Research Centre (BRC) and FP7-PEOPLE-2012-ITN (European Commission, RADIOMI). References [1] C. F. J. Barnard (2008) Organometallics, 27, 5402; A. Brennfuhrer, et al (2009) Angew. Chem. Int. Ed. Engl., 48, 4114. [2] S. K. Zeisler, et al (1997) Appl. Radiat. Isot., 48, 1091. [3] H. Ishida, et al (1985) Chem. Lett., 405. [4] S. D. Friis, et al (2011) J. Am. Chem. Soc., 133, 18114; C. Lescot, et al (2014) J. Am. Chem. Soc., 136, 6142.

Figure 1. [11C]CO2 → [11C]CO conversion followed by Pd-catalised aminocarbonylation (1A). Radio-HPLC analysis of the reaction mixture in Vial 2 (1B).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S305

305 Synthesis and biological evaluation of 2-amino-2-[11C]methyl-butanoic acid as a potential PET probe for tumor imaging Chie Suzuki1, 2, Atsushi B. Tsuji1, Koichi Kato3, 1, Hitomi Sudo1, Ming-Rong Zhang1, Yasushi Arano2, Tsuneo Saga1 1 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan, 3Department of Integrative Brain Imaging, National Center of Neurology and Psychiatry, Tokyo, Japan Objectives 2-Amino-2-methyl[1-14C]butanoic acid (isovaline, Iva) was previously reported to provide high tumor uptake, however, 11C-labeling procedure of [11C]Iva has not been reported. In the present study, we synthesized [11C]Iva and evaluated the potential as a PET probe for tumor imaging. Methods 11C-Methylation of benzophenone imine analog of tert-butyl 2-amino butyrate 1 and subsequent hydrolyses were employed for the synthesis of [11C]Iva (Scheme 1). Effects of electron transfer inhibitor (TMB), radical scavenger (TEMPO), and organocatalyst for electron transfer (o-phen) in addition to KOtBu and other bases were determined on 11C-methylation of using [11C]methyl iodide. In vitro cell uptake of [11C]Iva with and without inhibitors of various amino acid transporters was determined in SY small cell lung cancer cells at 37°C. Dynamic PET scan was performed for 90 min starting just after injection of [11C]Iva into SY tumor bearing mice. Results High radiochemical conversion (RCC) was obtained for the 11C-methylation of 1, when KOtBu was used as a base in the presence of TMB or TEMPO, suggesting that inhibition of radical generation induced by KOtBu improves the 11C-methylation of 1. Acid hydrolyses of 11C-methylated product proceeded almost quantitatively. [11C]Iva was obtained in 4.4 ± 1.7% radiochemical yield (decay uncorrected, referred to [11C]carbon dioxide) within 35 min from the end of bombardment. The uptake of [11C]Iva into SY cells increased time-dependently and was almost completely inhibited by an inhibitor of system L-amino acid transporter, but not by inhibitors of systems A and ASC. PET represented that [11C]Iva highly accumulated in SY tumors, but not in normal organs, except for pancreas, kidney, and bladder. Conclusions [11C]Iva was synthesized with high radiochemical yield via 11C-methylation assisted with KOtBu, and TMB or TEMPO. [11C]Iva was a substrate for L-type amino acid transporter. [11C]Iva could be a promising PET probe for noninvasive tumor imaging. Acknowledgements References

Scheme 1. Synthesis of [11C]Iva.

J Label Compd Radiopharm 2015: 58: S1- S411

S306: Poster

21st International Symposium on Radiopharmaceutical Sciences

306 Radiosynthesis of [11C]S44121, a first-in-class drug targeting the ryanodine receptor calcium release channel Frédéric Dollé1, Stéphane Demphel1, Bertrand Kuhnast1, Sandrine Moreau2, Wadad Saba1, Héric Valette1, François Lefoulon2 1 CEA, I2BM, Service hospitalier Frédéric Joliot, Orsay, France, 2Technologie Servier, Orléans, France Objectives Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca2+ from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle [1]. RyRs are the largest known ion channels and exist as three mammalian isoforms, RyR1, RyR2 and RyR3. Stabilisation of the RyR2 prevents aberrant activation of the channels during the resting phase of the cardiac muscle whereas loss of this stabilisation may be associated with cardiac arrhythmias, and/or sudden death in pathophysiological conditions such as heart failure. Among a new series of selective RyRs stabilizers codeveloped by Servier and Armgo, S44121 (1) [2] has been selected for PET studies and its labelling with carbon11 is reported herein. Methods Carbon-11-labelling of S44121 (1) was performed using a TRACERLab FX-C Pro synthesizer, starting from [11C]CO2 and comprised (1) trapping of [11C]MeI at -20°C in DMF (0.4 mL) containing the nor-derivative 2 (0.7-1.0 mg) and aq. 5N NaOH (7 µL) ; (2) heating (80°C, 5 min) ; (3) purification using semi-preparative reversed-phase HPLC (Hewlett Packard Zorbax® SB C18) and (4) SepPak®Plus C-18-based formulation for i.v. injection. Results [11C]S44121 ([11C]-1) was obtained in 9-13% ndc yields, based on starting [11C]CO2. Typically, starting from a 50 GBq [11C]CO2 batch, 4.5 to 6.5 GBq of [11C]-1, > 95% chemically and radiochemically pure, ready-toinject, were obtained in 50 min (including HPLC-purification and formulation). Specific radioactivities ranged from 55 to 75 GBq/µmol. No attempts were made to further optimize these reactions, the yields producing sufficient material for further evaluation. Worth mentioning, the conditions used herein led exclusively to the formation of the desired [11C]methoxy derivative (i.e. [11C]-1) without evidence of formation of side-product such as the [11C]methyl ester derivative. Conclusions S44121 (1) was isotopically labelled with carbon-11 at its methoxy site. PET-imaging (ECAT EXACT HR+, Siemens) is currently underway to evaluate the potential of this radiotracer to image the ryanodine receptors within the baboon heart. Acknowledgements CEA-I2BM intramural research programs and Servier References [1] Van Petegem F (2012) J. Biol. Chem., 287, 31624-31632 ; [2] Marks AR et al. (2009) Patent No. US 7,544,678 B2.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S307

307 Radiosynthesis and pharmacokinetic comparison of 1-N-[11C]methyl]-L- and -D-tryptophan Katsushi Kumata, Lin Xie, Joji Yui, Akiko Hatori, Yiding Zhang, Nobuki Nengaki, Masayuki Fujinaga, Tomoteru Yamasaki, Yoko Shimoda, Jun Maeda, Kazunori Kawamura, Ming-Rong Zhang Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Objectives The indole-containing small molecule 1-methyl-tryptophan (1MTrp) is known as a specific inhibitor targeted immune checkpoint protein indoleamine-2,3-dioxygenase (IDO)- switching off the effector T cells of the immune system to support tumor growth. 1MTrp has shown attractive antitumor activity without noticeable toxicity [1], and its phase I trial had been completed in 2014. However, 1MTrp exists as 2 stereoisomers of L and D. Most preclinical studies have employed the racemic mixture, thus leaving a long-standing debate in immunology and oncology, which stereoisomers have the potential of immune checkpoint inhibitor [2,3]. To remove the blindfold of 1MTrp effects and guide its immunotherapy development, we synthesized 1-N[11C]methyl]-L- and -D-tryptophan ([11C]L-1MTrp and [11C]D-1-MTrp) and compared their pharmacokinetics by PET. Methods [11C]L-1MTrp and [11C]D-1MTrp were synthesized by reaction of the corresponding Boc-Trp-OEt with [11C]CH3I at 80 °C for 5 min, followed by deprotection with 2 N HCl at 100 °C for 5 min, respectively. Pharmacokinetics of the L and D isomers were compared by dynamic PET scans and biodistribution study following the injection of the radioprobes in rats. Results [11C]L-1MTrp and [11C]D-1MTrp were obtained with radiochemical yields of 47.0 ± 6.3% (n = 80, based on [11C]CO2, EOS), radiochemical purity of > 98%, and specific activity of 47―130 GBq/μmol, showing high enantiomeric purity. PET imaging in rats revealed [11C]L-1MTrp had the highest accumulation of radioactivity (SUV 2.88 ± 0.03) in the pancreas with high IDO expression, while [11C]D-1MTrp showed the highest uptake (SUV 1.16 ± 0.03) in the kidney at 60 min after the radioprobes injection respectively. Ex vivo biodistribution results supported the PET images and indicated that uptake of L isomer in each organ was significantly higher than D isomer except in the kidney. Quite different tissue distribution was verified between the 2 isomers. Conclusions Both [11C]L-1MTrp and [11C]D-1MTrp are useful PET probes for tracking the pharmacokinetics of 1MT in vivo. Imaging the immune checkpoint inhibitors would enable us to take a new look at cancer immunotherapy and drug development. Acknowledgements References [1] Muller AJ, et al (2005) Nat Med, 11, 312. [2] Hou DY, et al (2007) Cancer Res, 67, 792. [3] Lob S, et al (2008). Blood 111, 2152.

J Label Compd Radiopharm 2015: 58: S1- S411

S308: Poster

21st International Symposium on Radiopharmaceutical Sciences

308 Exploration of analogs of [11C]dLop as potential probes for measuring increased P-gp function Lisheng Cai1, Yiyuan Peng2, Yepeng Xie2, Jinsoo Hong1, Jeih-San Liow1, Lora Deuitch1, Matthew Hall3, Robert Innis1, Victor W. Pike1 1 Molecular Imaging Branch, National Institute of Mental Health, Bethesda, Maryland, United States, 2Department of Chemistry, Jiangxi Normal University, Nanchang, China, 3CCR/LCB/MRS, National Cancer Institute, Bethesda, Maryland, United States Objectives We aimed to explore analogs of [11C]dLop as potential probes for measuring increased P-gp function, which is not possible with [11C]dlop itself (or similar radiotracers) because of very low uptake in normal human brain [1]. Promising analogs should be weaker P-gp substrates with some uptake into normal brain that can be reduced when P-gp activity is increased [2,3]. We set out to vary three regions of dLop to try to attenuate its P-gp substrate behavior, namely (a) the diaryl region; (b) the linker between the diaryl and piperidinyl group, and (c) the terminal chlorophenyl region. Methods We altered the original synthesis of dLop to give ready accessibility to cyano, amido, and monomethylamido analogs. Prepared compounds were subject to an FACS assay using fluorescent dyes as reporters with flow cytometry to assess substrate behavior towards P-gp in vitro. dLopA (Figure 1) was selected for labeling by 11 C-methylation of an amide precursor. [11C]dLopA was then studied in wild-type rats and in rats in which P-gp had been blocked with the inhibitor DCPQ. Results Cyano, amido, and monomethyl amido derivatives of dLop were obtained in useful yields. In vitro assay identified some compounds as weaker P-gp substrates than dLop, such as dLopA (Figure 1). PET imaging of [11C]dLop in wild-type and DCPQ-treated rats gave time-activity-curves that were almost overlapping with those from [11C]dLop. Conclusions Although the in vitro assay indicated that dLopA is weaker than dLop as a substrate for P-gp, early PET experiments with [11C]dLopA in rats do not show evidence of weak substrate behavior. Compounds showing significantly weaker P-gp substrate behavior in vitro are now being sought for radiolabeling and further investigation. Acknowledgements Support from the IRP of NIH (NIMH) and grant aid from NIH-China collaboration. References [1] Lazarova N et al. (2008) J Med Chem, 51, 6034. [2] Liow J-S et al. (2009) J Nucl Med, 50, 108. [3] Kreisl C et al. (2010) J Nucl Med, 51, 559.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S309

309 Development of electronegative LDL mimotope peptide based tracers for atherosclerosis PET imaging Soraya M. Kazuma1, 2, Marcela F. Cavalcante2, Dulcineia S. Abdalla2, Yongjian Liu1 1 Radiology, Washington University in St. Louis, Saint Louis, Missouri, United States, 2Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences/University of Sao Paulo, Sao Paulo, Brazil Objectives Atherosclerosis is chronic a inflammatory disease initiated with modified low-density lipoprotein (LDL) accumulation in artery wall. Electronegative LDL [LDL(-)] emerges as one of the modified forms of LDL and has an important role in the progression of atherosclerosis as an auto-antigen. A phage display selected peptide mimetic to LDL(-) epitope recognized by 1A3 monoclonal antibody which was obtained as a tool to investigate atherosclerosis. Herein, considering the peptide can target anti-LDL(-) antibodies in the plaque by mimicking immune relevant parts of LDL(-), we aim to develop LDL(-) mimotope peptide based PET agents for atherosclerosis imaging. Methods Mimotope peptide (P1) was conjugated to a DOTA chelator (DOTA-P1) for radiolabeling with both 68Ga and 64Cu. Structure of DOTA-P1 in simulated radiolabeling conditions was characterized by mass spectrometry. Specifically, 726 MBq 68Ga was incubated with 26.9 nmol of DOTA-P1 in pH 4.6 sodium acetate buffer under 95°C for 10 minutes. For 64Cu, 37 MBq was used to radiolabel 1 nmol of DOTA-P1 in pH 5.5 ammonium acetate solution under 45°C for 1 hour, followed by EDTA challenge. Both reactions were purified by C18 column and the radiochemical purities were measured with radio-HPLC and TLC. The stability of the radiolabeled DOTA-P1 was evaluated by incubating the tracer in rat serum for 1 h and determined by radio-HPLC. Initial in vivo evaluation of radiolabeled 68Ga-DOTA-P1 was performed by PET scan in male LDL receptor knockout mice (LDLr-/-, 10 weeks old) fed on chow diet. Results Mass spectrometry results under radiolabeling conditions showed circular structure stayed intact, which ensured the binding properties of the peptide. The specific activities for 68Ga and 64Cu radiolabeling were 27 MBq/nmol and 37 MBq/nmol, respectively. Serum stability study confirmed that 100% of compound was stable for in vivo applications. PET imaging of 68Ga-DOTA-P1 showed high renal clearance. Conclusions Preliminary results confirmed the DOTA conjugation with P1 peptide and intact peptide structure. The radiolabeling with 68Ga and 64Cu demonstrated high specific activities for trace amount administration in vivo. The incubation with rat serum confirmed the stable radiolabeling and initial PET imaging showed fast renal excretion. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S310: Poster

21st International Symposium on Radiopharmaceutical Sciences

310 Cu-64-ATSM PET scanning predicts degree and location of tumor hypoxia in dogs for hypoxia-directed therapy Jeffrey N. Bryan1, Deborah Tate1, Charles Maitz1, Sandra Axiak-Bechtel1, Joni Lunceford1, Carolyn Henry1, 2, Kimberly A. Selting1, Jimmy C. Lattimer1, Brian K. Flesner1, Lindsay Donnelly1, Brooke Fowler1, Chamisa Herrera1, Anastasia Glahn1, Amanda Collins3, Mary Varterasian3, David Tung3, Jeffrey Roix3, Linping Zhang3, Saurabh Saha3 1 Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States, 2University of Missouri, Ellis Fischel Cancer Center, Columbia, Missouri, United States, 3Biomed-Valley Discoveries, Kansas City, Missouri, United States Objectives Cu-64-ATSM is reduced and trapped in hypoxic cells, localizing for imaging with positron emission tomography (PET). Identification of hypoxic regions in tumors could allow patient selection to be optimized for hypoxia-directed medical, immunological, and radiation therapies. The hypothesis was that Cu-64-ATSM would localize with higher intensity in regions of hypoxia as measured by direct oximetry, and that these regions would be heterogeneous in proportion and distribution. Methods Dogs were injected with 74MBq (2mCi) of Cu-64-ATSM 45 minutes prior to scanning. Dogs were anesthetized and maintained on a propofol infusion breathing room air. Scans were acquired with a Philips C-PET+ with attenuation correction. Reconstructed images were evaluated using MIMfusion software. Maximum SUV and threshold volume were calculated. Direct oximetry was performed in select tumors using an OxyLite fiberoptic system. The relationships between direct oximetry and maximum SUV, maximum SUV and hypoxic volume, and tumor volume and hypoxic volume were evaluated using linear regression with trial-and-error transformation. P < 0.05 was considered significant. Results Tumor types evaluated included nerve sheath tumor (10), apocrine carcinoma (1), melanoma (3), and oral sarcoma (6). Maximum SUV ranged from 0.3-6.6. Measured oxygen tension ranged from 0.05-89.9mmHg. Inverse of SUV had a strong linear relationship with oxygen tension (R2=0.53, P=0.0048). Hypoxia <8mmHg was associated with an SUV>1.0. Hypoxic volume ranged from 0 to 100% of gross tumor volume (GTV). Maximum SUV was positively associated with hypoxic volume (R=0.674; P=0.0001), but not GTV (P=0.182). Tumor hypoxic volume was heterogeneous in location and distribution. Conclusions Cu-64-ATSM PET scanning predicts hypoxia patterns within tumors of dogs as measured by direct oximetry. Total tumor volume does not accurately predict degree of tumor hypoxia. Hypoxic regions are distributed heterogeneously within tumors and cannot be predicted by geometry. This approach is useful for planning hypoxia-directed therapy. Acknowledgements The authors acknowledge BioMed Valley Discoveries for funding of this project. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S311

311 Syntheses and Evaluation of Carbon-11- and Fluorine-18-Radiolabeled pan-Tropomyosin Receptor Kinases (Trk) Inhibitors for PET imaging. Vadim Bernard-Gauthier1, 2, Arturo Aliaga3, Antonio Aliaga4, Mehdi Boudjemeline4, Robert Hopewell4, Alexey Kostikov4, Pedro Rosa-Neto3, Alexander Thiel5, Frank Wuest1, Ralf Schirrmacher1 1 Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Medicine, McGill University, Montreal, Quebec, Canada, 3Douglas Mental Health University Institute, Montreal, Quebec, Canada, 4McConnell Brain Imaging Center, McGill University, Montreal, Quebec, Canada, 5Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada Objectives Tropomyosin receptor kinases (TrkA/B/C) are involved in the development of the nervous system, in neurological disorders as well as in neural and non-neural neoplasms. Hence, radiopharmaceuticals specifically targeting Trk would represent a powerful tool to non invasively investigate the specific roles of Trk receptors in those pathological conditions. We designed and evaluated the radiotracers [11C]GW441756 ([11C]1), and [18F]2 for Trk-PET imaging. Methods Inhibitor 1 and the des-methyl labeling precursor for the synthesis of [11C]1 were prepared following a strategy as reported by Wood et al.[1]. 1, a highly potent pan-Trk inhibitor, was labeled with 11C using [11C]MeI. A series of derivatives of 1 were rationally designed based on in silico predictions. Among those Trk ligands, the fluorinated inhibitor 2 was selected for further radiotracer development. [18F]2 was obtained via alkylation of the phenolic precursor with [18F]FETos. Inhibitor 2 Trk selectivity was assessed over a panel of cancer relevant kinase targets. [11C]1 and [18F]2 biodistribution was evaluated in rats with PET. In vitro autoradiography using rat brain and human neuroblastoma cryosections was also performed. Results In vitro autoradiographic studies in rat brain and human neuroblastoma cryosections confirmed that [11C]1 and [18F]2 specifically bind to Trk receptors. MicroPET studies revealed that binding of [11C]1 in the rodent brain is mostly nonspecific despite initial high brain uptake. However, specific binding in the lungs was confirmed. [18F]2 displayed high oxidative metabolic susceptibility and was unsuitable for Trk PET imaging. Conclusions The tracer [11C]1 is the first brain penetrant Trk ligand and the information gained in this study will guide further exploration of the 4-aza-2-oxindole scaffold as a lead for Trk PET tracer development[2]. Acknowledgements References 1. Wood, E. R.,et al. Discovery and in vitro evaluation of potent TrkA kinase inhibitors: Oxindole and aza-oxindoles. Bioorg. Med. Chem. Lett. 2006, 14, 953−957. 2. Bernard-Gauthier V, et al.Syntheses and Evaluation of Carbon-11- and Fluorine-18- Radiolabeled panTropomyosin Receptor Kinase (Trk) Inhibitors: Exploration of the 4-Aza-2-oxindole Scaffold as Trk PET Imaging Agents. ACS Chem. Neurosci, dx.doi.org/10.1021/cn500193f.

J Label Compd Radiopharm 2015: 58: S1- S411

S312: Poster

21st International Symposium on Radiopharmaceutical Sciences

312 Synthesis of a candidate brain-penetrant COX-2 PET radioligand as a potential probe for neuroinflammation Michelle Cortes, Prachi Singh, Cheryl Morse, Aneta Kowalski, Kimberly Jenko, Saurav Shrestha, Sami Zoghbi, M. Fujita, Robert B. Innis, Victor W. Pike Molecular Imaging Branch, National Institute of Mental Health, NIH, Bethesda, Maryland, United States Objectives Cyclooxygenase-2 (COX-2) is an inducible enzyme involved in prostaglandin synthesis and is present in brain. COX-2 expression increases in response to inflammatory conditions [1]. Therefore, a PET radioligand for imaging brain COX-2 might be useful for studying neuroinflammation. Such a radioligand does not yet exist due to candidate low affinity, high lipophilicity, or inability to enter brain [2]. 6-Methoxy-2-(4-(methylsulfonyl)phenyl)N-(thiophen-2-ylmethyl)pyrimidin-4-amine (which we dub MC1) is attractive for development as a PET radioligand due to its reported high affinity, selectivity for COX-2 and moderate lipophilicity (clogD = 2.80) [3]. In seeking a PET radioligand for imaging COX-2, we developed a method for preparing [11C]MC1 for evaluation in monkey with PET. Methods MC1 and a desmethyl precursor for radiolabeling were prepared by modifying published methods [3]. MC-1 was tested for affinity to human COX-2 and COX-1 with whole blood assays. [11C]MC1 was synthesized by treating precursor (1 mg) in DMF (0.08 mL) plus TBAH in methanol (1M; 3.0 μL) with [11C]MeI for 5 min, using an ‘autoloop method’ (Figure 1). [11C]MC1 was separated by reversed phase HPLC and formulated for intravenous injection. PET experiments were performed with [11C]MC1 administered intravenously to two rhesus monkeys at baseline and after attempted block of brain COX-2 with MC1 (3 mg/kg, i.v.). Radioligand arterial input function and plasma free fraction (fP) were measured to calculate total volume of distribution (VT) and VT/fP. Results MC1 showed high affinity (IC50 = 3 nM) and selectivity (< 1000-fold vs. COX-1) for binding to human COX-2. Pure [11C]MC1 was obtained in useful radiochemical yield and high specific activity. PET showed that [11C]MC1 entered monkey brain with radioactivity peaking at ~2.9 SUV at 2 min and reducing by ~60%, after 40 min. Pre-block experiments reduced brain VT by 47% and 40%, and VT/fP by ~ 20% and ~ 10%. fP was 0.71 and 0.82% at baseline, and 0.49 and 0.52% at preblock. Conclusions [11C]MC1 enters primate brain adequately. Further detailed evaluation of [11C]MC1 is required to verify COX-2 specific signal and assess its magnitude. Acknowledgements Support from the IRP of NIH (NIMH). References [1] Zarghi A and Arfaei S (2011) Iranian J Pharm Res, 10, 655. [2] Laube M et al. (2013) Molecules, 18, 6311. [3] Orjales A et al. (2008) Bioorg Med Chem, 16, 2183.

Figure 1. Radiosynthesis of [11C]MC1.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S313

313 A Microscale Radiosynthesis of [C-11]-Rosuvastatin Jeanne M. Link1, Jashvant Unadkat2 1 Diagnostic Radiology, Oregon Health & Science University, Portland, Oregon, United States, 2Pharmaceutics, University of Washington, Seattle, Washington, United States Objectives Rosuvastatin (Crestor) is used to treat high cholesterol. In order to evaluate whether rosuvastatin is a substrate for organic anion transporters (OATPs) and whether OATPs mediate hepatobiliary transport of rosuvastatin in rats, we developed a synthesis of 11C-rosuvastatin. Methods N-desmethylrosuvastatin (NRSV) was from Santa Cruz Biochemicals. Other labeling reagents were from Sigma-Aldrich Chem. About 0.3 mg of the NRSV was dissolved in 0.15 mL of anhydrous DMSO with heating. Then 0.05-0.25 mg of the NRSV was added to a 1.0 mL glass vial followed by 5 uL of tetrabutylphosphonium hydroxide (1/50 v:v in acetonitrile (ACN)). Carbon-11 labeled methyl iodide in 100 µL of anhydrous ACN and added to the precursor. The reaction was capped and heated for 5 minutes at 105-120oC. The product was purified by reversed-phase chromatography using a Luna C18 column (150 mm long X 4.6 mm diam from Phenomenex). Specific activity was assessed by C18 HPLC with MS (ES+) and radioactivity detection. The product was eluted with linear gradients of 15% ethanol in 0.2 mM HCOOH to 65% ethanol at 4.0 minutes, ramped to 80% ethanol at 5.0 minutes and held for 2.0 minutes at 80% ethanol. Results The reaction time was 35 min. with an average decay-corrected radiochemical yield of 54%. Starting with 3 to 11 GBq of 11CH3I the product was 97 ± 3% radiochemically pure and contained 0.1 to 2 nanomoles of rosuvastatin with a specific activity of 190 ± 320 GBq/µmol (range 38 to >1000) at the end of synthesis. A typical product chromatogram is shown in figure 1. Conclusions Carbon-11 rosuvastatin is easily synthesized and purified. Addition of base is required for radiolabeling. Acknowledgements NCI PO1 CA042045 and Steven C. Shoner. PhD References [1] He J, et al (2014) Molecular Pharmaceutics, 11, 2745-2754. [2] Link, J (1997) Nucl Med Biol, 24, 93-97.

Carbon 11 rosuvastatin chromatogram where the x axis is time and the y axis is the percent of the maximum signal, the chromatograms from top to bottom show radioactivity, UV from 260 to 350 nm, precursor mass and bottom is mass for rosuvastatin eluting at seven minutes

J Label Compd Radiopharm 2015: 58: S1- S411

S314: Poster

21st International Symposium on Radiopharmaceutical Sciences

314 Estimation of the unbound brain to plasma ratio for CNS drug candidates – comparing results obtained with PET microdosing and microdialysis in non-human primates Peter Johnström1, 2, Katarina Varnäs2, Linda Bergman2, Jonas Malmquist3, Christer Halldin2, Lars Farde1, 2 1 AstraZeneca Translational Science Centre at Karolinska Institutet, Stockholm, Sweden, 2Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden, 3AstraZeneca R&D, Södertälje, Sweden Objectives Early confirmation of brain exposure and target engagement in CNS drug development is key to success. We have compared the outcome of two methods assessing brain exposure, positron emission tomography (PET) [1] and microdialysis [2]. The methods were applied to AZD3241 a potent and selective myeloperoxidase (MPO) inhibitor expected to delay progression in patients with neurodegenerative brain disorders. Methods AZD3241 was radiolabelled by demanding 4-step radiochemistry starting from [11C]potassium cyanide and using microwave assisted heating. Brain exposure was measured with PET in non-human primates following administration of a microsdose of [11C]AZD3241 and repeated after co-administrated with a pharmacological dose. In a separate study, the concentration of AZD3241 in brain striatal regions was determined with microdialysis at two doses. Brain to plasma concentration ratios at steady-state were in both studies determined using compartmental analysis. Results [11C]AZD3241 was produced in good radiochemical yield. There was no evident difference in the total brain to plasma concentration ratio between microdose and pharmacological dose as measured by PET. Drug distribution in the putamen was dose-proportional for the dose range examined by microdialysis. The estimated value of the unbound brain to plasma partition coefficient from PET and microdialysis data were 1.1 (n=3) and 1.5 (n=4), respectively. Both methods confirmed adequate brain exposure supporting translation of AZD3241 to Phase 2a studies. Conclusions This study validates the potential of PET to have early impact in CNS drug development. However PET will require labelled drug candidates and as most drug molecules are not intended for rapid radiolabelling this can be challenging in some projects. In comparison to the invasive positioning of microdialysis probes in preselected brain regions, PET is non-invasive, will yield distribution data for several brain regions in the same experiment and can be translated to humans. Acknowledgements The authors would like to thank Kristina Claeson Bohnstedt, Bart Ploeger and Margareta Bielenstein at AstraZeneca, Jane Sutcliffe at Maccine Pte Ltd and members of the PET group at the Karolinska Institutet. References [1] Lee C-M & Farde L (2006) Trends Pharmacol Sci, 27, 310-6. [2] Hammarlund-Udenaes M (2000) Adv Drug Deliv Rev, 45, 283-294.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S315

315 Preclinical evaluation of the NET-PET-tracer [11C][email protected] Christina Rami-Mark1, 2, Cecile Philippe1, Chrysoula Vraka1, Lukas Nics1, Rupert Lanzenberger3, Helmut Spreitzer4, Marcus Hacker1, Markus Mitterhauser1, Wolfgang Wadsak1 1 Biomedical Imaging and Image Guided Therapy, Medical University of Vienna, Vienna, Austria, 2Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria, 3Department of Psychiatry and Psychotherapy, Medical University of Vienna, Vienna, Austria, 4Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria Objectives Recently the radiosynthesis of the novel PET tracer [11C][email protected] for the Norepinephrine Transporter (NET) was described[1].This study aimed at the preclinical evaluation of this NET-PET-tracer regarding its plasma stability, blood brain barrier (BBB) penetration and binding behavior in in vitro autoradiography. Methods Immobilized artificial membrane chromatography was done using a Redistech column to predict passive diffusion over the BBB[2]. Furthermore, lipophilicity was determined using ODP-chromatography[3] and total polar surface (tPSA) was calculated. Metabolic stability was assessed in pooled human and rat plasma by incubation at 37°C for 1h[1]. In vitro autoradiography was done on coronal rat brain slices (20µm) by incubation with 6-8nM of [11C][email protected] in TRIS buffer. After washing, slices were placed on phosphoimager films and % of specific binding was measured. Nissl staining and immunohistochemistry (IHC) was done to correlate hot areas. Further in vitro binding was tested using a cell based approach (hNET membranes,harvester). Results Lipophilicity for [email protected] (logD= 3.13) was in the range for ideal PET-Tracers and comparable to FMeNER-D2 (logD=2.73). BBB penetration seemed likely by passive diffusion: [email protected] tPSA: 35.58, Pm=0.55, compared to FMeNER-D2 (tPSA 39.72, Pm=0.48). Affinity and selectivity for [email protected] was high (kI NET: 3nM, SERT: ≤669 nM; DAT:≤10.000nM) and should allow quantification of NET. In vitro autoradiography (Fig1b) revealed specific binding of [11C][email protected] in NET rich regions, the same regions were visualized using [18F]FMeNER-D2. Interestingly, we observed concentration dependent binding enhancement for [11C][email protected] using 3 different blockers (Nisoxetin, FMeNER-D2, [email protected]), but binding displacement using same conditions for [18F]FMeNER-D2. Therefore, we used hNET membranes to test in vitro binding behavior. Hereby, binding displacement was observed in a concentration dependent manner(Fig.1a). Conclusions All tested preclinical parameters indicate that [11C][email protected] is a suitable NET-PET-tracer. The observed binding enhancement in in vitro autoradiography will be further elucidated in ex vivo autoradiography and in vivo µPET studies. Acknowledgements References [1]Mark,C.et al.(2013)Nucl Med Biol. 295-303. [2]Vraka,C.et al(2014)EJNNMI 41:442.[3]Donovan,C.et al (2002)J Chrom A 952:47-61.

J Label Compd Radiopharm 2015: 58: S1- S411

S316: Poster

21st International Symposium on Radiopharmaceutical Sciences

316 On the search for novel PET tracers for the Norepinephrine Transporter Christina Rami-Mark1, 2, Stefanie Foltin1, Chrysoula Vraka1, Catharina Neudorfer3, Alexander Hoepping4, Rupert Lanzenberger5, Helmut Spreitzer3, Marcus Hacker1, Markus Mitterhauser1, Wolfgang Wadsak1, 2 1 Biomedical Imaging and Image Guided Therapy, Medical University of Vienna, Vienna, Austria, 2Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria, 3Department of Drug and Natural Product Synthesis, University of Vienna, Vienna, Austria, 4ABX Advanced Biochemical Compounds, Radeberg, Germany, 5 Department of Psychiatry and Psychotherapy, Medical University of Vienna, Vienna, Austria Objectives The norepinephrine transporter (NET) is suspected to play a crucial role in a variety of psychiatric or neurodegenerative diseases. To elucidate the pathophysiological impact of the NET, specific and selective NETPET ligands are needed. Existing NET-PET tracers are based on reboxetine but display certain drawbacks in in vivo imaging. Hence, we aimed at the development and evaluation of several novel potential compounds as improved NET-PET tracers. Methods A substance library of 21 new candidate tracers – not based on reboxetine - for the NET was set up based on previously described lead structures [1-6]. Therefore, 3 benzo[d]imidazolone derivatives (FAPPI), 9 phenoxyisochromen derivatives (PHOXI), 3 indolinyl derivatives (DIMAP), 3 thiadiazole derivatives (HAPTHI) and 3 indolinole derivatives (MAPPI) have been designed, synthesized and tested in silico (tPSA, docking studies) and in vitro (binding affinity; lipophilicity; IAM-chromatography for BBB penetration prediction and HSAchromatography to determine plasma protein binding). Radiosynthesis of the most promising compounds is concurrently set-up. Results Amongst the portfolio of tested compounds, four very promising compounds were found displaying high affinity (<10nM), good selectivity and ideal range of permeability (<3.5) and logD (<4). Therefore, we started to set up radiolabelling for these favorable ligands by [11C]methylation at the amine group. Conclusions A database for potential NET-PET tracers was established, enabling a structure-activity relationship for further tracer development. Moreover, four of the 21 novel ligands displayed very promising properties, 11Cradiolabelling of these compounds is currently in progress. Acknowledgements References [1] Zhang, P. et al. (2008) Bioorg Med Chem. 18:6067-6070. [2] Neudorfer, C.et al. (2014) Molecules. accepted [3] Hudson, S. et al. (2008) Bioorg Med Chem Lett 18, 4495-4498. [4] Vu, A. et al. (2010) 53, 20512062. [5] O’Neill, D. et al. (2011) J Med Chem 54, 6824-6831. [6] McComas C. et al. (2008) Bioorg Med Chem Lett 18, 4929-4931.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S317

317 Efficient [13N]nitrate reduction catalyzed by Aspergillus niger nitrate reductase immobilized on positively charged carrier Eunice Sofia da Silva Fernandes1, Vanessa Gómez-Vallejo1, Jordi Llop1, Fernando López-Gallego2, 3 1 Molecular Imaging, CIC biomaGUNE, San Sebastian / Donostia, GUIPÚZCOA, Spain, 2Biofunctional Nanomaterials, CIC biomaGUNE, San Sebastian, Spain, 3IKERBASQUE - Basque Foundation for Science, Bilbao, Spain Objectives We present the unprecedented strategy for the reduction of [13N]NO3- to [13N]NO2- using an heterogeneous biocatalyst. Methods The design and fabrication of the heterogeneous biocatalyst started with the immobilization of A. niger nitrate reductase (eNR) on agarose beads activated by different chemistries. The immobilization was carried out by adding 200mg of the corresponding carrier to 1mL of soluble eNR (1-5U/mL) in 10mM sodium phosphate buffer at pH=7.5 under mild stirring at 25ºC. This heterogeneous biocatalyst was biochemically characterized. The radiochemical reaction (reduction from [13N]NO3- to [13N]NO2-) was performed in batch mode, where 6.72mU of immobilized eNR were used to reduce 90µl of radioactive solution (containing cyclotron produced [13N]NO3-) in the presence of NADPH (0.21mM) in mild conditions (25°C and pH 7.5) during 4 min. The radiochemical conversion was determined by radio-HPLC. To proof the potential use of the herein fabricated heterogeneous biocatalyst, we coupled the enzymatic reduction of [13N]NO3- to the synthesis of S-[13N]GSNO. The reaction conditions used to carry out this synthesis were the same reported before.1,2 Results We achieved the best results with 100% of immobilization yield and 25% of expressed activity by immobilizing eNR on agarose beads activated with tertiary amino group (Ag-DEAE). Such immobilized preparation was 12-fold more thermostable than the soluble enzyme. A radiochemical conversion above 92% was achieved when the eNR optimal heterogeneous biocatalyst was used to reduce [13N]NO3- into [13N]NO2-, and it could be re-used up to 7 times within one day without catalytic efficiency loss. Finally, to demonstrate the potential of this heterogeneous biocatalyst we proofed that it could be efficiently integrated into the two step chemoenzymatic cascade to synthesize S-[13N]GSNO with radiochemical conversion values up to 95% with respect to [13N]NO2-. Conclusions In this work we have applied for the first time one eukaryotic nitrate reductase immobilized on a solid carrier to a radiochemical process. This immobilized enzyme has been able to reduce [13N]NO3- to [13N]NO2- aided by NADPH as redox cofactor with high radiochemical conversions. The labeled precursor is suitable to approach further 13N-nitrosation reactions. Acknowledgements All the work has been supported by the RADIOMI project (EU FP7-PEOPLE-2012-ITNRADIOMI). References [1] Gómez-Vallejo V, et al. (2009) Bioorg. Med. Chem. Lett., 19, 1913. [2] Llop J, et al. (2009) Appl. Radiat. Isot., 67, 95.

J Label Compd Radiopharm 2015: 58: S1- S411

S318: Poster

21st International Symposium on Radiopharmaceutical Sciences

318 Transition Metal Mediated Kumada Cross Coupling of [11C]CH3I and Aryl Grignard Johan H. Dam Department of Nuclear Medicine, Odense University Hospital, Odense, Denmark Objectives Radiolabeling of small molecules with carbon-11 has to date been largely based on the SN2 reaction by amines or alkoxides on [11C]CH3I or [11C]CH3OTf. The chemical space for labeling by cross coupling is still being developed within radiochemistry.[1] Herein, it was sought to explore the Kumada cross coupling for incorporation of the [11C]methyl moiety. Methods A range of transition metals viz. Pd, Ni, Fe and Co and ligands DPPF, DPPP, PPh3, P(oTol)3, TMEDA were screened for their capability to mediate a model Kumada cross coupling between PhMgBr and [11C]CH3I in THF. The [11C]CH3I was prepared by standard gas phase reactions on the Tracerlab FXc Pro by extracting [11C]CH3I from Valve 17 to an external glass vial with 0.35 mL THF, 4 µmol catalyst and 100 µmol of PhMgBr(in THF), cooled to -20 °C. The trapping was complete within 2 minutes and the vial was heated to 60 °C over 2 minutes, purged with N2 for 2 minutes to discharge residual [11C]CH3I and quenched with 1 mL 70% ethanol. The radiochemical purity was assessed by RP-HPLC on a Zorbax C18 Stablebond. Results As a simple model system, many different combinations of transition metals and ligands produced the [11C]toluene to some extent. Of the different catalysts and ligands examined, only PdCl2DPPF, PdCl2(P(oTol)3)2 and CoCl2 with DPPP produced the carbon-11 labeled toluene in the higher yields of 67.9%, 67.0% and 66.5%, d.c., respectively. For the cobalt mediated cross coupling, addition of PhMgBr after trapping of the [11C]CH3I was necessary in order to obtain a high yield. Increasing the reaction time from 2 minutes to 4 minutes with PdCl2(P(oTol)3)2 did not increase the overall radiochemical yield. Lowering the reaction time to 1 minute reduced the radiochemical yield to 36.1% d.c. Conclusions The Kumada cross coupling was examined for coupling of [11C]CH3I with PhMgBr. Palladium with phosphine ligands, DPPF and P(oTol)3, was found to produce the [11C]toluene in slightly better yields. As such, the palladium mediated Kumada cross coupling represent an attractive method for fast insertion of a metabolically more stable radiolabel into small molecules. Acknowledgements References [1] Pretze, M. et al. (2011) Molecules, 16, 1129-1165.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S319

319 Synthesis of a novel 125I-labelled radiotracer for molecular imaging of cyclooxygenase-2 (COX-2) Ole Tietz1, James Dzandzi2, Atul Bhardwaj1, 3, John Valliant2, Frank Wuest1 1 Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada, 3Pharmacy and Pharmaceutical sciences, University of Alberta, Edmonton, Alberta, Canada Objectives Cyclooxygenase-2 (COX-2) is the key enzyme in prostaglandin synthesis and acts as an important mediator in a variety of diseases. The enzyme is membrane bound and located on the inside of the endoplasmic reticulum. Effective perfusion of inhibitors to the active site requires highly lipophilic drugs, which consequently display background accumulation in fatty tissues. The objective of this work is to identify a small molecule radiolabelled with longer-lived radioisotopes to enable longer imaging and better target to background ratios. Methods An iodine containing trifluoro-pyrimidine was synthesized on the basis of a previously reported lead structure [1], evaluated in a COX binding assay and further developed as a 125I-labelled radiotracer ([125I]1) (t½ (125I) = 59.4 days) (Figure 1). [125I]1 was prepared using an electrophilic iododestannylation reaction on a trimethylstannyl precursor. The reaction was optimized using the well established Iodobead and Iodogen coated vial methods and compared to a recently developed oxidant, fluorous derivative of chloramine-T (F-CAT), [2] in solution and coated on an Eppendorf vial. Results IC50 value for inhibitory potency of compound 1 was found to be 0.85 μM. Radiosynthesis of [125I]1 was accomplished within 30 min, including HPLC purification. Radiochemical yields (starting from Na125I compared to product recovered from HPLC) of reactions carried out in F-CAT coated vials were found to be 91 ± 4 % (n=3) compared to 81 ± 3 % (n=3) using Iodogen coated vials. The radiochemical yields in reactions using F-CAT in solution (49 %) or Iodobeads (32 %) were significantly lower. Radiochemical purity of [125I]1 was > 99 %. Conclusions The novel oxidant F-CAT is highly effective in iodo destaynnlation reactions. [125I]1 represents a potent and long-lived COX-2 selective radiotracer. The use of positron emitting isotopes, such as 123I and 76Br, might lead to the development of a longer-lived COX-2 PET radiotracer in the future. Acknowledgements This work was supported by the Dianne and Irving Kipnes Foundation, the Canadian Institute for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC). References [1] Tietz O, et al (2013) Org Biomol Chem, 11, 8052-8046. [2] Dzandzi JPK, et al (2014), (In Press).

Figure 1. Radiolabelling of trimethylstannyl precursor.

J Label Compd Radiopharm 2015: 58: S1- S411

S320: Poster

21st International Symposium on Radiopharmaceutical Sciences

320 Synthesis of Symmetrical Ureas via the Direct Incorporation of [11C]CO2 Abdul Karim Haji Dheere, Carlotta Taddei, Ran Yan, Antony Gee king's College London, London, United Kingdom Objectives Ureas are common structures in biologically active molecules. Therefore, the development of new methods to efficiently radiolabel these classes of compounds is of interest. Carbon-11 (11C) is obtained directly from a cyclotron in the form of [11C]CO2 and is generally converted in more reactive forms, such as [11C]CH3I. However, due to the short half-life of 11C, the development of methods to radiolabel compounds using [11C]CO2 directly from the cyclotron is of great interest. We hereby report a new development to our previously established method1 for the incorporation of [11C]CO2 into symmetrical aliphatic and aromatic amines. Methods [11C]CO2 was bubbled into a solution containing 1,8-diazabicycloundec-7-ene (DBU), and an amine. The solution was stirred at 50 oC for 1 minute before Mitsunobu reagents (diisopropyl azodicarboxylate and tributylphosphine) were added and stirred for a further 1 minute. Results [11C]CO2 was reacted with a range of primary and secondary amines to form 11C radiolabelled symmetrical ureas. Excellent radiochemical yields (RCY) over 70% were observed for the aromatic amines bearing both electron donating and electron withdrawing groups. Conclusions An efficient radiolabelling method that incorporates [11C]CO2 in aliphatic, aromatic and benzylic amines to produce 11C radiolabelled symmetrical ureas has been developed. The synthesis avoids the need of converting the short-lived 11C into other reactive forms, opening up the opportunity to radiolabel pharmacologically active molecules rapidly. Acknowledgements The authors acknowledge funding from the EPSRC (MRS7140) and the NIHR Biomedical Research Centre at Guy's & St Thomas' Hospital NHS Trust and King’s College London. References 1(a) A. K. Haji Dheere and A. Gee, J. Label. Compd. Radiopharm., 2013, 56, S90; (b) A. K. Haji Dheere, N. Yusuf and A. Gee, Chem. Commun., 2013, 49, 8193.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S321

321 Cellular uptakes of novel gold nanoparticle conjugates via radioactivity analysis and photoacoustic imaging Xia Shao1, Chao Tian1, Qian Wei2, Xueding Wang1 1 Radiology, University of Michigan, Ann Arbor, Michigan, United States, 2IMRA America. Inc, Ann Arbor, Michigan, United States Objectives Gold nanoparticles (AuNPs) have been attracting interest for fundamental science and biomedical applications. Surface modifications are essential. The novel AuNPs used here were produced by femtosecond laser ablation. The conjugation of functional ligands to the surface can be tunable between 0 and 100%. In this study the novel AuNPs have conjugated quantitatively with different density of RGD peptides and their cellular uptakes were measured via radioactivity analysis and photoacoustic imaging. Methods AuNPs were synthesized using femtosecond laser ablation of metal target and conjugated to RGD peptide in various concentration (RGD molecules per AuNP) radiolabeled by mixing with [125I]NaI. 125I-labeled and non-labeled AuNPs were added to the individual cell plate of DU145 cells. The cells were incubated. The cells containing 125I-labeled AuNPs were extracted using Trypsin and measured using a γ-counter. Photoacoustic measurements were performed with the cells containing non-radioactive AuNPs. Results Based on radioactivity, the cellular uptake of 125I-labeled conjugates increased when RGD molecules on the AuNP surface increased (Table 1). The photoacoustic signal approached the maximum at the certain density of RGD peptide and decreased. Conclusions Novel RGD-AuNP conjugates have been evaluated quantitatively with radioactivity analysis and photoacoustic imaging. The higher density of RGD gave higher cellular uptake based on radioactivity. The observation from photoacoustic imaging is significant to design AuNPs as photoacoustic contrast agents with the maximum performance. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S322: Poster

21st International Symposium on Radiopharmaceutical Sciences

322 Radiosynthesis of the auxin class plant growth hormone 4-chloro-3-indolyl[1-11C]acetic acid Paul Ellison, Dhanabalan Murali, Todd E. Barnhart, Andrea Jedele, Robert J. Nickles, Onofre T. DeJesus Department of Medical Physics, University of Wisconsin, Madison, Madison, Wisconsin, United States Objectives Auxins are a class of plant growth hormone implicated in stem elongation, response to light and gravity, and vasculature, lateral root, and organ formation. Recently, the principal auxin, indole-3-acetic acid (IAA) was radiolabeled with C-11 for PET imaging auxin transport in plants [1]. 4-Chloroindole-3-acetic acid (4-Cl-IAA) is another highly potent auxin naturally present in species of pea, bean, lentil and pine seeds. The present work aims to develop a radiosynthesis procedure for 4-chloro-3-indolyl[1-11C]acetic acid (4-Cl-[11C]IAA) in order to explore its role in plant physiology using PET imaging. Methods 4-Chlorogramine was synthesized from 4-chloroindole according to literature [3]. [11C]Methane was produced by proton-irradiating 90% N2, 10% O2, cryotrapped, purged, released, combined with high purity ammonia gas, then flowed through a 1000 °C platinum oven, forming [11C]cyanide. This gas was bubbled through DMSO containing 4-Cl-gramine and heated to 140 °C, forming 4-Cl-3-indolyl[1-11C]acetonitrile (4-Cl-[11C]IAN). This intermediate product was purified by diluting with water, trapping on a C18 Sep-Pak, rinsing with 99 : 1 then 95 : 5 :: 0.1% HCl : acetonitrile, and eluting with diethyl ether. The purified 4-Cl-[11C]IAN was hydrolyzed with base to form 4-Cl-[11C]IAA. The product was purified using preparative high-performance liquid chromatography (HPLC) using a C18 column with 3 mL/min of 35: 65 :: acetonitrile : 0.1% formic acid. Analytical HPLC was performed using a C18 column with a linear gradient over 20 minutes of 10 : 90 - 30 : 70 :: acetonitrile : 0.1% formic acid at 1 mL/min. Results [11C]Cyanide production and trapping in precursor solution was measured to be (55 ± 15)% of total activity, with (13 ± 7)% of trapped activity reacting to form 4-Cl-[11C]IAN (n=3). A hydrolysis and preparative HPLC yield of ~50% was observed, with overall decay-corrected synthesis yield of ~3.5%. Retention time of 4-ClIAA was observed to be 16.5 minutes and 10.2 minutes for preparative and analytical HPLC, respectively. End-ofbombardment decay-corrected specific activity was (26 ± 19) MBq/nmol (n=2). Conclusions The radiosynthesis and purification of 4-Cl-[11C]IAA is reported with non-optimized yields suitable for performing preliminary microPET imaging of chloroauxin transport and distribution in plants. Comparison of the PET-visualized auxin transport of IAA and 4-Cl-IAA may elucidate similarities and differences in the regulatory mechanisms of the two auxins. Acknowledgements References [1] Reid, A.E. et al. (2011) J Lab Compd Radiopharm, 54, 433-437. [2] Reinecke, D.M. (1999) Plant Growth Regul, 27, 3-13. [3] Somei, M. et al. (1981) Chem Pharm Bull, 29, 3145-3157.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S323

323 11 C ammonium thiocyanate: a novel 11C synthon for PET radiolabelling Tom Haywood1, Steven Kealey1, Christophe Plisson2, Philip Miller1 1 Chemistry, Imperial College London, London, United Kingdom, 2Imanova Limited, London, United Kingdom Objectives Our group has recently described the synthesis and reactivity of a new radiolabelling reagent [11C]carbon disulfide.[1] 11CS2 is an electrophile with reactivity similar to 11CO2. It has proven to be reactive towards amines, forming [11C]thioureas in excellent yields.[2] Herein we report a novel route to ammonium [11C]thiocyanate via the reaction of 11CS2 with NH3, and data on its nucleophilic substitution reactions for the formation of [11C] organo-thiocyante molecules. Methods 11CS2 was produced via the reaction of 11CH3I with sulfur at 500oC, delivered in a stream of He into MeCN(1 mL). NH3 solution (2M in MeOH, 100 μL) was added heated at 90oC for 10 minutes to form ammonium [11C]thiocyanate. Alkyl bromide precursors(10 mg) were added and the mixture, heated for a further 10 min. An aliquot was removed for analysis by radio-analytical HPLC. Results Ammonium [11C]thiocyanate forms quantitatively upon reaction of 11CS2 with NH3. The resulting [11C]thiocyanate ion displayed good reactivity with all alkyl bromides tested, forming the 11C-labelled phenacyl/benzyl-thiocyanates (Fig 1) in >95% radiochemical purity(Table 1) Conclusions A nucleophilic synthon, -S11CN, has been formed by reaction of 11CS2 with ammonia. This reagent provides a novel and fast approach to 11C-labelling of electrophiles, showing good functional group tolerance. Acknowledgements We are grateful to the EPSRC for funding References [1] Miller, P. W. and Bender, D., Chem. Eur. J., 2012, 18, 433; [2] Kealey, S. et al., ISRS abstract submission 2015

Fig 1. Formation of C-11 phenacylthiocyanate and benzylthiocyante

Table 1. RCP of C-11 thiocyanate products

J Label Compd Radiopharm 2015: 58: S1- S411

S324: Poster

21st International Symposium on Radiopharmaceutical Sciences

324 Radiosynthesis and Selective Purification of [11C]Palmitic Acid (PA) using a Single Disposable Cartridge James M. Kelly, Alejandro Amor-Coarasa, John W. Babich Radiology, Weill Cornell Medical College , New York, New York, United States Objectives To develop a rapid synthesis of [11C]PA for human use that requires minimal target irradiation times using simple disposable components. A miniature “gas capture” system was developed based on the use of zeolite 13X for gas trapping, phase-transition labeling and purification. Methods [11C]CO2 (200-300 mCi at EOB) was transferred to a cartridge containing three separate stages of zeolite 13X. The cartridge was vertically mounted on a plastic 3D-printed holder. 1 mL of a 0.25M solution of npentadecylmagnesium bromide in Et2O was pushed through the column bed and let react for 5 min at room temperature. Radio-impurities were selectively removed by venting the column to a waste vial, followed by 1 mL of a 0.5M NaOH solution, 2 x 10 mL air, 3 mL of a 2M HCl solution and 2 x 10 mL air. [11C]PA was eluted with 1 mL of a 2.5% solution of AcOH in EtOH and 2 x 10 mL air, filtered, collected, and reformulated for injection in a sterile vial. Results More than 25 mCi of injectable [11C]PA was synthesized in 8 minutes from a starting [11C]CO2 activity of 200-300 mCi with a radiochemical purity of 99% and specific activity of 250-350 mCi/µmol (Table 1). The product was found to be sterile and pyrogen free. In comparison to other reported automated and semi-automated syntheses of [11C]PA, the shorter synthesis time and target bombardment time and the USP- and Ph.Eur-compliant properties offer significant advantages to routine clinical production of [11C]PA. A larger starting activity would increase both the final dose activity and the specific activity of the tracer, however we consider the short irradiation time a logistical and operational (minimized exposure) improvement and an attractive feature of this method. Conclusions We have developed a disposable cassette for the rapid radiosynthesis of 1-[11C]PA that is approximately the size of a common mobile phone. Injectable patient doses (> 25 mCi, 99% radiochemical purity and 250-350 mCi/µmol specific activity) were prepared in 8 minutes from an EOB activity of 200-300 mCi. Acknowledgements Thanks to Dr. Shankar Vallabhajosula, Dr. Paresh Kothari, Sophie Hurez and Joseph Lipman for their support. References 1. M. A. Channing, N. Simpson, J. Label Compd. Radiopharm. 1993, 33, 541-546. 2. B. H. Mock, C. Brown-Proctor, M. A. Green, B. Steele, B. E. Glick-Watson, Q.-H. Zheng, Nucl. Med. Biol. 2011, 38, 1135-1142. 3. R. Iwata, T. Ido, M. Tada, Appl. Radiat. Isot. 1995, 46, 117-121.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S325

325 Synthesis of Radiolabeled Glycogen Synthase Kinase 3β Inhibitors for CNS imaging Andrew V. Mossine1, Erin L. Cole1, Allen F. Brooks1, Lei Li3, Franklin Aigbirhio3, Peter J. Scott1, 2 1 Radiology, University of Michigan, Ann Arbor, Michigan, United States, 2Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, United States, 3Wolfson Brain Imaging Centre, University of Cambridge, Cambridge, United Kingdom Objectives Glycogen Synthase Kinase-3β (GSK3β) is a multifunctional serine/threonine kinase whose dysregulation is associated with a number of neurodegenerative diseases. Although there is significant impetus to develop a GSK3β radiotracer for CNS imaging, previous attempts have been largely unsuccessful due to poor blood-brain barrier permeability [1,2]. As such, this study focuses on examining several unexplored GSK3β inhibitors to identify a brain permeable radiotracer for GSK3β. Four compounds ([11C]SB-216763 (Maleimide, ATP competitive, A), [18F]4FPB (HMK, irreversible inhibitor, B), and [18F]VP1.15 (ITDZ, substrate competitive, C and D)) are presented, each of which exhibits a different mode of interaction with GSK3β. Methods Precursors and reference standards were synthesized via modified literature procedures (A, C, and D), or purchased from commercial suppliers (B) [3,4]. [11C]SB-216763 was prepared according to the method reported by Li et al. [5]. Radiolabeling of the other radiotracers was performed according to the routes shown in Scheme 1. HPLC and TLC were used to confirm product identity and determine radiochemical yields. Results To prepare [11C]SB-216763, protection of the maleimide with a dimethoxybenzyl group was required to prevent pericyclic side reactions [5]. Treatment of the protected precursor with [11C]MeI followed by deprotection produced tracer A in 1% RCY (non-corrected based upon [11C]MeI). [18F]4FPB was prepared by Cu2+ catalyzed fluorination of the Bpin acetophenone (B) in 48% RCC, followed by bromination with NBS [6]. End stage 18F fluorination of VP1.15F (C, D) through a tosylethyl precursor proved unsuccessful, so alternate approaches (C and D) are currently being pursued to label VP1.15F [7]. Conclusions Precursors, reference standards and preliminary radiolabeling of four GSK3β tracers encompassing three unique scaffolds are presented. Optimization of radiolabeling conditions and preclinical evaluation of these agents in vivo is currently underway. Acknowledgements We acknowledge the US DOE/NIBIB (DE-SC0012484), NIH (T32-EB005172) and Alzheimer’s Association (NIRP-14-305669) for financial support. References [1] Cole, E. L. et al., (2014) Nucl. Med. Biol. 41, 507. [2] Vasdev, N., (2005) Bioorg Med Chem Lett 15, 5270. [3] Smith, D.G. et al., (2001) Bioorg Med Chem Lett, 11, 635. [4] Palomo, V. et al., (2012) J Med Chem, 55, 1645. [5] Li, L. et al., (2013) J Nucl Med 54 (Suppl 2), 98P. [6] Tredwell, M., (2014) Angew Chem Int Ed, 53, 7751. [7] Gilissen, C. et al. (1998) J Labelled Comp Radiopharm, 41, 491.

Scheme 1: Radiolabeling of four precursors belonging to three known GSK3β inhibitor scaffolds

J Label Compd Radiopharm 2015: 58: S1- S411

S326: Poster

21st International Symposium on Radiopharmaceutical Sciences

326 Evaluation of Bioorthogonal Reactive Pairs for Cathepsin B Imaging Patricia Edem, Nancy Janzen, Silvia A. Albu, Denis R. Beckford Vera, John Valliant McMaster Universtiy, Hamilton, Ontario, Canada Objectives Radiolabeled activity-based probes (ABPs) have shown promise as molecular imaging probes, targeting key proteases involved in cancer [1]. These probes have exhibited the ability to label active cathepsins in vitro, but have lower tumor uptake and tumor:non-tumor ratios in vivo, when compared to fluorescently labeled ABPs [2]. This can in part be attributed to the instability of the radiolabeled prosthetic groups used in vivo. To circumvent these issues a bioorthogonal approach can be employed. The objective was to apply the inverse electron demand Diels-Alder (IEDDA) reaction involving a transcylcooctene (TCO) tagged ABP and a radioiodinated tetrazine (Tz) (Figure 1). Methods The cathepsin B inhibitor, Z-Phe-Lys-CH2OCOPhMe2, (Ki = 52 nM) [1] was modified with a PEG-4 linked TCO through an amide bond at the epsilon nitrogen of the lysine, giving CaBInIX-2 in 64% yield. Its bioorthogonal partner, 3-(5-iodopyridin-2-yl)-6-(pyridin-2-yl)-1,2,4,5-tetrazine (125I-Tz) was prepared through an iododestannylation reaction with iodine-125 (RCY = 80%). Using a fluorogenic substrate, cathepsin B activity was measured in cell and tumour lysates. Cell assays were performed using the glioma cell line, u87MG, which exhibited high cathepsin B activity and the lower expressing breast cancer cell line, MDA-MB231. Results CaBInIX-2 exhibited similar affinity for the protease (Ki = 186 nM) as CaBInIV-3; an inhibitor tagged with a 125I-benzamide prosthetic group (Ki = 181 nM) [1]. The 125I-Tz exhibited higher binding to cells that were pre-treated with CaBInIX-2. Cells treated with the unfunctionalized inhibitor Z-Phe-Lys-CH2OCOPhMe2 or with (E)-cyclooct-4-enol showed much lower 125I-Tz binding, implying specificity. To confirm active protease labeling, SDS-PAGE was performed and bands corresponding to cathepsin B and the closely related cathepsin L were observed. Conclusions A cathepsin B inhibitor was derivatized with TCO and was able to undergo a ligation with its bioorthogonal partner, 125ITz, after binding to the enzyme. This tandem labeling strategy can be used to detect cathepsin B in vitro and in vivo, while also providing a platform amedable for PET imaging with 124I. Acknowledgements Funding for this project was provided by NSERC (Canada), CIHR, and the OICR (Ontario). References [1] P.E. Edem et al., (2014), J. Med. Chem. 57, 9564; G. Ren et al. (2011), PLoS One 6, e28029. [2] G. Blum et al., (2007) Nat. Chem. Biol. 3, 668. [3] H.C. Hang et al. (2006) ACS Chem. Biol. 1, 713; M.R. Pratt (2009) Chem. & Biol. 16, 1001.

Figure 1. Bioorthogonal pairs: CaBInIX-2/125I-Tz and ABP: CaBInIV-3

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S327

327 Radiosynthesis of [11C]Allyl Amides by Amidation of a [11C]Carboxylate Intermediateintermediate Allen F. Brooks, Peter J. Scott, Xia Shao Radiology, University of Michigan, Ann Arbor, Michigan, United States Objectives Bruton's tyrosine kinase (BTK) is a mediator of the B-cell-receptor signaling pathway implicated in the pathogenesis of B-cell cancers. Early-stage clinical trials found ibrutinib, a BTK inhibitor, showed antitumor activity in several types of B cell lymphoma. The drug recently has been approved by the FDA for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia1. Radiolabeled ibrutinib has the potential to function as a companion diagnostic for predicting response to ibrutinib therapy and also to support future drug discovery efforts around the BTK pathway. We report here a novel method for carbon-11 labeling ibrutinib. Methods The synthetic scheme was shown in Figure 1. Briefly, a solution of vinyl magnesium bromide in THF was sparged with [11C]CO2, followed by addition of oxalyl chloride to generate [11C]acryloyl chloride. The entire [11C]acryloyl chloride reaction mixture was then transferred into a vial containing precursor amine and DTBP in THF/DMF. The product was purified by preparative HPLC and reconstituted into ethanolic saline using a C-18 Sep-Pak to provide [11C]ibrutinib formulated for injection. Results [11C]Ibrutinib was prepared in a multistep synthesis at room temperature. Total synthesis time was 50 minutes from end of beam. In non-optimized conditions, total radiochemical yield was 1% based on [11C]CO2 (non-decay corrected) with a radiochemical purity of 90% determined by HPLC analysis. Conclusions Cancer therapy agent, ibrutinib, has been radiolabeled with carbon-11 for the first time. This novel radiosynthetic method can be extensively used to label various amides from the required Grignard reagent and amine. Biological evaluation and preclinical studies of [11C]ibrutinib are underway. Acknowledgements References (1) Davids, Matthew S.; Brown, Jennifer R. Future Oncology (2014), 10(6), 957-967.

Figure 1: 11C-labeling of ibrutinib.

J Label Compd Radiopharm 2015: 58: S1- S411

S328: Poster

21st International Symposium on Radiopharmaceutical Sciences

328 Rapid Synthesis of [11C]Urea derivatives by [11C]CO2 Incorporation: Application to novel mGluR1 brain imaging probes. Abdul Karim Haji Dheere, Salvatore Bongarzone, nisha singh, Antony Gee king's College London, London, United Kingdom Objectives A novel and rapid high-yielding synthetic methodology has previously been described for the synthesis of urea-containing 11C compounds.1 We applied this synthetic protocol to create a library of 11C labelled metabotropic glutamate receptor 1 (mGluR1) receptor ligands. Methods [11C]CO2 was bubbled into a solution containing 1,8-diazabicycloundec-7-ene and selected amines. After heating the solution at 50 oC for 1 minute, Mitsunobu reagents (diisopropyl azodicarboxylate and tributylphosphine) were added, yielding the desired labelled ligands (Figure 1). Results The 11C radiochemical synthesis has been successfully applied to labelling a library of urea-containing mGluR1 ligands with radiochemical yields > 20 % (corrected for decay), radiochemical purity of > 98 % and a total synthesis time of 20 minutes including purification. Conclusions A facile, efficient and rapid methodology to radiolabel molecules containing urea structures with [11C]CO2 has been developed. This has now been applied to the synthesis of novel mGluR1 PET radioligands. The candidate’s binding to mGluR1 receptors are currently being evaluated using autoradiography and homogenate binding assays. Acknowledgements The authors acknowledge financial support from EU-AIMS (IMI, European Commission) and the NIHR Biomedical Research Centre at Guy's & St Thomas' Hospital NHS Trust and King’s College London. References

Figure 1. The radiolabelling of mGluR1 ligands with [11C]CO2.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S329

329 Synthesis of C-11 and F-18 labeled NOP 46 - A potential marker for chronic pain Shunichi Oya1, John W. Castrillon1, Xiaoming Xu 2, Shi-Xian Deng2, Donald W. Landry2, Chaitanya R. Divgi1 1 Radiology, Columbia University, New York, New York, United States, 2Medicine, Columbia University, New York, New York, United States Objectives Chronic pain results in long-term hyperexcitability (LTH)1), associated with activation of protein kinase G‐1α (PKG) in the dorsal root ganglion (DRG) associated with the region of pain. The balanol derivative NOP462) has high affinity to activated PKG. The chemical structure of NOP46 has an anisole moiety, where a C11 labeled methyl group can be placed. NOP 46 also contains fluorine, which can be replaced with [18F]fluorine. NOP 46 thus may be labeled with either C11 or F18 without altering its original structure or biologic properties. Short half-life of [11C]NOP46 (1) permits multiple administrations of tracers in short intervals. Longer half-life of [18F]NOP46 (2) enables data acquisition over an extended time period. Radiolabeled NOP46 may provide a novel method to visualize chronic pain. Methods To produce [11C]NOP46, phenolic precursor with Boc and acetyl protection groups for amino groups (3) was prepared. C11 Methylation was carried out with [11C]methyl triflate using GE-FXc Pro synthesis module. To produce [18F]NOP46, nitro precursor (4) was synthesized. [18F]Fluorine was introduced using [18F]KF/K222/K2CO3 in DMSO. (Figure 1) Chemical identities of labeled products were determined by HPLC. In vitro uptake study of [11C]NOP46 was carried out in normal rats. Results [11C]NOP46 (1) was produced successfully after the protecting groups were removed by a treatment of 2N HCl. The HPLC confirmed the chemical identity. Radiochemical purity of [11C]NOP46 was >99% determined by a HPLC. Radiochemical yield was ~10 % (decay corrected). [18F]NOP46 (2) was produced by a radio fluorination with nitro leaving group. Radiochemical yield was 1~2 % (decay corrected). Preliminary rodent study showed no evidence of blood brain barrier transit of [11C]NOP46. Rodent PET demonstrates the route of excretion is primarily hepatobiliary. Conclusions [11C] and [18F]NOP46 were successfully prepared. Radiolabeled NOP46 may provide novel method to assess chronic pain. Acknowledgements Financial gift from MedTech Inc. is gratefully acknowledged. References [1] Sung YJ; et al. (2004) Neurol Res., 26, 195-203. [2] Ambron, R; et al. (2007) PCT int, Appl, WO 2007095586 A2 20070823.

Figure 1. Synthesis of C11 and F18 NOP46s

J Label Compd Radiopharm 2015: 58: S1- S411

S330: Poster

21st International Symposium on Radiopharmaceutical Sciences

330 Radiosynthesis and evaluation of the 5-HT2A inverse agonist: [11C]pimavanserin Valdemar L. Andersen2, Hanne Hansen1, Matthias Herth2, Agnete Dyssegaard1, Gitte Moos Knudsen1, Jesper L. Kristensen2 1 Neurobiology Research Unit, Rigshospitalet, Copenhagen, Denmark, 2Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark Objectives Positron emission tomography (PET) is a useful tool to investigate the role of the 5-HT2A receptor in various mental disorders. Pimavanserin is a high affinity 5-HT2A inverse agonist with a promising selectivity profile [1], suggesting a potential use as 5-HT2A PET-ligand. The structure of pimavanserin allows for both 11Cand 18F-labeling, either through N-methylation or labeling using [18F]fluorobenzaldehyde. In-vivo evaluation of [11C]pimavanserin should reveal its potential as a PET-ligand with potential for expansion as an 18F-labeled variant. Methods The precursor was synthesized in a three step reaction from (4-isobutoxyphenyl)methanamine and tertbutyl 4-((4-fluorobenzyl)amino)piperidine-1-carboxylate. (4-isobutoxyphenyl)methanamine was converted into its corresponding isocyanate using triphosgene and reacted with tert-butyl 4-((4-fluorobenzyl)amino)piperidine-1carboxylate to form the boc-protected precursor. Subsequent deprotection yielded the finished precursor. Carbon11 labeling was achieved using [11C]MeOTf in acetone at 60ºC (see image 1). Purification was performed by semipreparative HPLC and subsequently analyzed by analytical HPLC. Subsequent biological evaluation was performed in Danish Landrace pigs. Results Carbon-11 labeled pimavanserin was produced successfully. Radiochemical yields were between 1 and 1.7 GBq, radiochemical purity >98% and specific activities of 34-80 GBq/µmol. Upon injection into pigs the compound showed brain uptake with binding in cortex and cerebellum corresponding to 5-HT2A distributions [2,3,4]. Unexpectedly, high binding was also observed in thalamus. Binding was unable to be displaced by ketanserin suggesting a lack of 5-HT2A specificity. Conclusions [11C]pimavanserin was successfully labeled through N-methylation. Subsequent injection into pigs revealed brain uptake which could not be blocked by ketanserin, limiting its potential use as a 5-HT2A PET-ligand. Acknowledgements References [1] Vanover, et al (2006) Journal of Pharmacology and Experimental Therapeutics, 317, 910-918. [2] Ettrup, et al (2011) Synapse, 65, 136-145. [3] Hansen, et al. (2013) Synapse, 67, 328-337. [4] Debus et. al (2010) Nuclear medicine and biology 37, 487-495.

Labeling of [11C]pimavanserin.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S331

331 Synthesis of Dipeptide [11C]Phe-Phe-NH2, a High Affinity Ligand for the Substance P 1-7 Specific Binding Site Aleksandra Pekošak, Janja Škrinjar, Ulrike Filp, Alex J. Poot, Albert D. Windhorst Radiology and Nuclear Medicine, VU Medical Center, Amsterdam, Netherlands Objectives Peptides modified with non-proteinogenic amino acids have become increasingly important as they play special role in peptidomimetics with enhanced properties[1]. This may be illustrated by dipeptide [11C]PhePhe-NH2 (Ki=1.5nM) as a small and high activity ligand for the substance P1-7 specific binding site and its intriguing ability to reduce neuropathic pain[2]. Furthermore, labeled with a radionuclide the dipeptide can be used for further verification if it indeed binds to the desired brain regions for the treatment of neuropathic pain. The general aim of this project is to establish a regioselective method for labeling peptide [11C]Phe-Phe-NH2 without changing the original structure, which can be translated to variety of small peptides. Methods [11C]Benzyl iodide ([11C]BnI) 4 was synthesized in 3 steps starting from PhBrMg 1 and [11C]CO2[1]. Synthesis of DL-[11C]Phe-NH2 7 and [11C]Phe-Phe-NH2 9 was investigated by reacting 4 with precursor 5 or 8, respectively, in the presence of TBAF (see Scheme 1). Results With a total synthesis time of 10 minutes, [11C]BnI 4 (n>5) was obtained in high yields, purity and specific activity (RCY: 52±3%, purity: 95±3%, SA: 123±17 GBq/μmol). Subsequently, addition of strong base deprotonated precursor 5, followed by selective α-monoalkylation with the [11C]benzyl group to yield racemic DL[11C]Phe-NH2 6 (75±4% (n=2) incorporation of [11C]benzyl). Final quantitative acidic deprotection resulted in DL[11C]Phe-NH2 7. Under the same conditions predicted α-monoalkylation on precursor 8 showed simultaneous Nalkylation on C-terminal side, with incorporation of [11C]benzyl group 28±4% (n=2) for [11C]Phe-L-Phe-NH2 9 and 58±5% (n=2) for α- and N- di-alkylated product 10. Conclusions We achieved successful synthesis of DL-[11C]Phe-NH2 7 with carbon-11 in high yield (>75%). Current research focusses on selective α-monoalkylation of [11C]Phe-Phe-NH2 and small peptides. Nevertheless, this study has provided several valuable contributions to the native peptides radiolabeling with carbon-11. Acknowledgements Supported by RADIOMI (FP7-PEOPLE-2012-ITN). References [1] Park YS, et al. (2001) Bull. Korean Chem. Soc., 958-962 [2]Fransson R, et al. (2013) J. Med. Chem., 4953-4963 [3] Fasth KJ, et al. (1988) J. Chem. Soc., 3081-3084.

Scheme 1: Synthesis of DL-[11C]Phe-NH2 7 and [11C]Phe-Phe-NH2 9.

J Label Compd Radiopharm 2015: 58: S1- S411

S332: Poster

21st International Symposium on Radiopharmaceutical Sciences

332 Radiolabelling and initial in vivo evaluation of 11C N-methyl Linsitinib (OSI 906) targeting IGF-1 and insulin receptors Mikkel Holm Vendelbo1, Morten Busk2, Dirk A. Bender1 1 Nuclear Medicine & PET Center, Aarhus University Hospital, Aarhus, Denmark, 2Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark Objectives Tyrosine Kinase Receptor Class II receptors, Insulin- and IGF-I receptors, are increased in various cancer types, leading to enhanced tumor growth through the Akt/mTOR signaling pathway. We aimed to test the N-methylated analog of Linsitinib with respect to inhibition of Insulin- and IGF-I receptors. Methods N-methyl Linsitinib was prepared by N-methylation of commercially available Linsitinib using methyl iodide and excessive NaH in THF. Effect of inhibition was assessed with a 2-deoxy-2-[18F]fluoro-D-glucose (18FFDG) uptake assay and measurements of site specific phosphorylations on Akt and mTOR in non-stimulated and stimulated conditions. 11 C N-methyl Linsitinib was prepared by adoption of methylation condtions applied for the synthesis of the reference compound, but with the exception of using a mixture of THF and DMF as solvent. In vivo biodistribution of 11C N-methyl Linsitinib was studied by µPET/MR scanning in a healthy mouse. Results N-methyl Linsitinib profoundly decreased 18F-FDG uptake in MCF7, HCT8, HT29, and SW948 cancer cells, both in non-stimulated and stimulated condition. This was associated with substantially inhibition of Akt Thr308 and Ser473, and mTOR Ser2448 phosphorylation in non-stimulated condition and after 20 minutes of insulin or IGF-I stimulation. Compared to Linsitinib, N-methyl Linsitinib showed a somewhat weaker inhibitory effect. 11 C N-methyl Linsitinib was prepared in ca. 30% radiochemical yield within 40 minutes. Specific radioactivity was > 37 GBq/µmol. The radiochemical purity was limited to 90%, due to sentivity towards radiolysis. Excessive amounts of NaH are crucial to reduce the formation of the competitive O-methylation product. µPET/MR revealed that the tracer was highly accumulated in the liver and in skeletal muscle. Conclusions 11C N-methyl Linsitinib might serve as imaging tool targeting Insulin- and IGF-I receptors, but further in vivo evaluation in tumor bearing mice models ir needed. Acknowledgements References Eliot T. McKinley, Joseph E. Bugaj, Ping Zhao, et al.Clin Cancer Res 2011;17:3332-3340.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S333

333 Development of efficient construction of [11C]carbamate moiety using [11C]COCl2 Masayuki Fujinaga1, Masanao Ogawa1, 2, Katsushi Kumata1, Yoko Shimoda1, Kazunori Kawamura1, Ming-Rong Zhang1 1 Molecular Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan, 2 SHI Accelerator Service Co. Ltd., Tokyo, Japan Objectives [11C]Phosgene ([11C]COCl2) is an important 11C-labeling reagent for synthesizing various radioactive compounds containing a [11C]urea or [11C]carbamate moiety. We have developed several 11C-labeled lignads containing carbamate or unsymmetrical urea moiety using [11C]COCl2 and reported the synthesis of [11C]1 as a novel PET ligand for imaging of fatty acid amide hydrolase using [11C]COCl2 or [11C]CO2 [1]. However, the synthesis of [11C]1 had some problems in poor reactivity and reproducibility. In this study, we developed a method for synthesizing [11C]1-3 via 2 routes, starting from several phenol derivatives and base, to improve the reproducibility and versatility for constructing the [11C]carbamate moiety. Methods The alkylamine 10 was prepared from commercialy available materials. [11C]1-3 were synthesized by reaction of phenol derivatives with [11C]COCl2 in the presence of various bases, followed by reaction with amine 10, via 2 routes as shown in Scheme 1. Results In the case of route A, when Et3N, DMAP, pyridine, or DBU was used, [11C]1 was obtained with moderate yield but [11C]2 or [11C]3 could not be obtained because 8 or 9 was formed at low yields. Instead of these bases, when 1,2,2,6,6-pentamethylpiperidine was used, 7-9 were produced in 68%, 88%, and 91% yields, which were determined by analyzing the reaction mixtures using radio-HPLC. However, following reaction of 8 or 9 with 10 did not proceed. In the case of route B, when 1,2,2,6,6-pentamethylpiperidine was used, after [11C]COCl2 was trapped to the reaction mixture, immediate addition of amine 10 to the mixture accomplished the reaction. After the two-steps reaction, it was found that [11C]1-3 were produced with 92%, 82%, and 68% yields in the mixtures. Conclusions We succeeded in improvement of reaction efficiency of [11C]1-3. These results showed that utilization of 1,2,2,6,6-pentamethylpiperadine was more effective for constructing the [11C]carbamate moiety compared to other bases, such as Et3N, DBU, and DMAP. Acknowledgements References [1] Fujinaga M, et al. (2013) J Label Compd Radiopharm, 56, S92. [2] Kumata K, et al. (2015) ACS Chemical Neuroscience.

J Label Compd Radiopharm 2015: 58: S1- S411

S334: Poster

21st International Symposium on Radiopharmaceutical Sciences

334 Selective O-[11C]methylation of 1-(3-hydroxyphenyl)piperazine Szabolcs Lehel1, Matthias Herth1, Valdemar L. Andersen2, Nic Gillings1, Jacob Madsen1 1 Department of Clinical Physiology, Nuclear Medicine & PET, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark, 2Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark Objectives Phenylpiperazines are common moieties of biologically active molecules and drugs, for example phenylpiperazinyl-butoxyindoles, which are selective 5-HT7 receptor antagonists [1]. The preparation and evaluation of the first 11C-labelled phenylpiperazinyl-butoxyindoles has been published recently [2]. 1-(3[11C]Methoxyphenyl)piperazine (2) can serve as a precious 11C-labelled moiety to obtain a large series of compounds with minimal precursor synthesis and thus help to accelerate preclinical evaluation. A similar 2 step, one-pot reaction strategy has recently been described using reductive amination in the second reaction step [3]. This is complementary to traditional last-step labelling methods which usually imply a one-tracer, one-precursor strategy. This study aimed to optimise reaction conditions for selective O-[11C]methylation of 1-(3hydroxyphenyl)piperazine (1) by testing its reaction with [11C]MeOTf in several solvents, with various bases and at different temperatures (Figure 1.). Methods 1.0 mg of 1 was dissolved in 300μl of each solvent, acetone, acetonitrile, THF, DMSO and DMF, and different bases (aqueous alkali metal hydroxides/carbonates, tetrabutylammonium hydroxide and tetrabutylammonium fluoride in methanol) were added. The mixtures were pre-heated for several minutes at elevated temperature (40-120oC) then cooled down to room temperature. [11C]Methyl triflate was collected by bubbling through the solutions and the reaction mixtures were subsequently heated for 5 min at 40oC, quenched by addition of 0.5 mL water and analysed using radio-HPLC. Results According to our observations pre-heating of 1-(3-hydroxyphenyl)piperazine with base prior methylation with [11C]MeOTf is essential for selective O-[11C]methylation. The best O-methylation/N-methylation selectivity achieved was 6:1 by using acetonitrile as solvent and 1M TBAH in MeOH as base with reaction at 40oC. Conclusions 1-(3-Hydroxyphenyl)piperazine can be selectively [11C]methylated on the phenolic hydroxide-group without protecting the more reactive secondary amino-group of piperazine ring. Acknowledgements References [1] B. Volk et al 2008 (J. Med. Chem. 51, 2522), [2] H. Hansen et al. 2014 (J. Nucl. Med. 55, 640), [3] M. Herth et al 2014 (RSC Adv. 4, 21347)

Figure 1. [11C]Methylation of 1-(3-hydroxyphenyl)piperazine

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S335

335 Synthesis and preclinical evaluation of [11C]/[3H]Alisertib, an aurora kinase A (AURKA) inhibitor Joost Verbeek1, 2, Jeroen A. Goos2, 1, Annemieke Hiemstra2, Pien Delis-van Diemen2, Albert Geldof1, Otto Hoekstra1, Steven Stroud3, Daniel Bradley3, Gerrit Meijer2, Remond Fijneman2, Albert D. Windhorst1 1 Radiology and Nuclear medicine, VU University medical center, Amsterdam, Netherlands, 2Pathology, VU university medical center, Amsterdam, Netherlands, 3Takeda Pharmaceuticals International Co, Boston, Massachusetts, United States Objectives Survival of patients after resection of colorectal cancer liver metastasis (CRCLM) is less than 30%. Novel positron emission tomography (PET) tracers, which image the expression of prognostic biomarkers, may contribute to improving selection methods for curative surgery and, accordingly, prognosis. Aurora kinase A (AURKA) expression is associated with survival of patients after CRCLM resection and is thus a potential target for PET imaging Methods [3H]Alisertib was synthesized, starting from [3H]methyl nosylate, under similar conditions as [11C]alisertib, which was synthesized starting from [11C]CH3I in a two-step procedure as shown in figure 1. In vitro uptake of [3H]alisertib was measured in cancer cells with high (Caco2), moderate (A431, HCT116, SW480) and low (MKN45) AURKA expression (mRNA) levels, also after its downmodulation using small interfering RNA (siRNA) and after inhibiting P-glycoprotein (P-gp) activity. In vivo uptake and biodistribution of [11C]alisertib were measured, using nude mice, xenografted with A431, HCT116 or MKN45 cells, or P-gp knockout mice Results [3H]Alisertib was synthesized with an overall yield of 42% and [11C]alisertib with an overall yield of 23±9% (radiochemical purity ≥99%)(A). Uptake of [3H]alisertib in Caco2 cells was higher than in A431 cells, SW480 cells, HCT116 cells and MKN45 cells and uptake in A431 cells was higher than SW480 cells, HCT116 cells and MKN45 cells(B). Downmodulation of AURKA expression decreased [3H]alisertib uptake in Caco2 cells. P-gp inhibition increased [3H]alisertib uptake in Caco2 and MKN45 cells. In vivo stability after 90 minutes of [11C]alisertib was 94.7±1.3% and tumour-to-background ratios were 2.3±0.8 (A431), 1.6±0.5 (HCT116) and 1.9±0.5 (MKN45) at 70-90 minutes after injection(C). In brains of P-gp knockout mice [11C]alisertib uptake was increased Conclusions [11C]- and [3H]Alisertib have been successfully synthesized, appeared stable in vivo and can be used for the imaging of AURKA in cells with high expression levels, however, the exact mechanisms underlying its accumulation in cancer cells need to be further investigated Acknowledgements The authors would like to acknowledge I. de Greeuw and R. Vos for experimental assistance, and the BV cyclotron VU for providing [11C]CO2

J Label Compd Radiopharm 2015: 58: S1- S411

S336: Poster

21st International Symposium on Radiopharmaceutical Sciences

336 The introduction of an useful organic strong base, 1,8-bis(tetramethylguanidino)naphthalene for [11C]methylation: a preparation of [11C]raclopride So-Hee Kim1, Sang-Yoon Lee1, 2, Tatsuo Ido1 1 Neuroscience Research Institute, Gachon University, Incheon, Korea (the Republic of), 2Radiological Science, Gachon University, Incheon, Korea (the Republic of) Objectives Generally, strong inorganic bases (NaOH, KOH, NaH etc.) has been employed for C-11 methylation of phenolic alcohols ([11C]raclopride, [11C]DTBZ), but they are not perfectly dissolved in reaction media which cause lowering solubility of precursors, (S)-O-desmethylraclopride and 9-O-Desmethyl-DTBZ and gives erratic patterns of radiochemical yields. Proton Sponge (PS, Sigma Aldrich), 1,8-bis(tetramethylguanidino)naphthalene (TMGN), is an organic base (relatively strong base, pKa=25.1 in ACN), which may enhance the solubility of labeling precursors and reaction yields with the poor reactivity of phenolic alcohols in precursors. Methods The Radiolabeling procedure was carried out in conventional way. The [11C]CO2 and [11C]MeOTf were produced in same condition (55μA, 30 min bombardment) for all synthesis. We applied same synthesis recipes for preparations of [11C]raclopride but the preparation time of precursor solution (additional 30 min for NaOH reaction with vortex mixer). (S)-O-desmethylraclopride (1 mg) was dissolved in MEK (300 μl) with bases (0.5M NaOH 3 μl or Proton sponge 1 mg, 0.8 eq) before [11C]methylation by [11C]MeOTf. The final product was isolated from HPLC purification and SPE method (tC18) and dissolved with 10% EtOH solution into the sterile vial. Results The radiochemical yields from two different bases are 3.65±0.33 (n=10) and 4.85±0.03 (n=3) GBq for NaOH and PS respectively (Figure 1, Left). Total synthesis time with NaOH was 30 min longer than that with PS (30 min). Conclusions There was the significant difference in radiochemical yields between NaOH and PS by 30% which originates from precursor solubility in reaction mixture and PS is useful for [11C]O-methylation of [11C]raclopride (figure 1, Right). We also confirmed higher radiochemical yields for [11C]DTBZ (not shown) with PS as a base. Acknowledgements This research was supported by the Gachon University research fund of 2014 (GCU-20140136) References

Figure 1. Radiochemical yields from bases (Left) and precursor solubility test in comparison between NaOH and Proton Sponge (Right).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S337

337 A Simple Method for Evaluation of [11C]PBR28 Partitioning Within Blood Mark A. Green, Carla J. Mathias, Yen Ng, Michelle A. Keiski Radiology and Imaging Sciences, Indiana University, Indianapolis, Indiana, United States Objectives To evaluate the partitioning of [11C]PBR28 among the constituents of whole blood. The [11C]PBR28 radiopharmaceutical [1] is widely used for PET evaluation of neuroinflammation. However, a genetic polymorphism stratifies the population into high- mixed- and low-affinity [11C]PBR28 binders [2,3,4], complicating kinetic modeling and limiting applicability based on patient genotype. Methods Partitioning of [11C]PBR28 between the red cell, plasma, and platelet/WBC compartments of blood was evaluated after centrifugation of blood in a hematocrit tube. A Bioscan radiochromatogram scanner with highresolution collimator was then employed to assess the location of 11C within the blood sample. Blood was analyzed after i.v. administration of the tracer, and after mixing [11C]PBR28 with whole blood in vitro. Results For the majority of subjects studied, a very high concentration of [11C]PBR28 was detected at the RBC:Plasma interface (Figure 1A and 1B), apparently reflecting avid tracer accumulation by platelet binding sites in high- and mixed-affinity binders. This striking finding was not apparent in the blood of subjects subsequently shown by genetic testing to be low affinity binders (Figure 1C). Conclusions The partitioning of [11C]PBR28 within blood is complex, with a very high fraction of the radiopharmaceutical localized in the platelet compartment of blood from the high- and mixed-affinity binders. In the 23 subjects studied, all three later genotyped as low affinity binders were correctly predicted as such from the observed partitioning of [11C]PBR28 in their blood (Figure 1C). Acknowledgements Supported by the Indiana University Department of Radiology and Imaging Sciences, and the Indiana Spinal Cord and Brain Injury Research Fund. References [1] Briard E, et al. 2008 J Med Chem 51:17–30. [2] Owen DR, et al. 2011 J Nucl Med 52:24-32. [3] Kreisl WC, et al. 2013 J Cereb Blood Flow Metab 33:53-58. [4] Yoder KK, et al. 2013 J Nucl Med 54:1320-1322.

Figure 1. Partitioning of [11C]PBR28 between the red blood cell (RBC), platelet, and plasma fractions of blood. The RBC:Plasma interface is at the “0” position. A. Blood from a patient subsequently shown to be a high-affinity binder. B. Blood collected 5-minutes after i.v. administration of [11C]PBR28 to this high-affinity binder. C. Blood from a patient subsequently shown to be a low affinity binder. For the in vitro studies (A and C) 1-µL [11C]PBR28 dose solution was added to 1000-µL blood at 37°C before sample centrifugation in a hematocrit tube.

J Label Compd Radiopharm 2015: 58: S1- S411

S338: Poster

21st International Symposium on Radiopharmaceutical Sciences

338 GMP compliant synthesis of [11C]phenytoin by rhodium mediated [11C]carbon monoxide carbonylation performed in disposable glass vials Jonas Eriksson1, Alf Thibblin1, Joost Verbeek2, Albert D. Windhorst2, Gunnar Antoni1 1 Uppsala University Hospital, PET centre, Uppsala, Sweden, 2VU University Medical Center, Amsterdam, Netherlands Objectives Phenytoin is an antiepileptic drug in clinical use. Here we report an efficient GMP compliant method for the synthesis of [11C]phenytoin developed for an upcoming PET study in which the tracer will be used to investigate drug resistance in epilepsy patients. Methods [11C]Phenytoin was produced at Uppsala PET Centre using automated equipment. The chemistry was based on a previously described method with improvements.[1] Chloro(1,5-cyclooctadiene)rhodium(I) dimer (1.0 mg, 2.0 μmol) was dissolved in tetrahydrofuran (800 μL). A portion of the solution (200 μL) was then added to 1,2-bis(diphenylphosphino)ethane (0.6 mg, 1.5 μmol) and 2-azido-2,2-diphenylacetamide (2.0 mg, 7.9 μmol) in a septum-equipped disposable glass vial (0.9 mL). The mixture was degassed and shaken to form a clear reddish solution. [11C]CO2 was converted over heated Zn to [11C]CO, concentrated and transferred in xenon gas into the vial containing the reagent solution.[2] The solution was heated at 130°C for 5 min and then diluted with water prior to semi-prep HPLC purification. The eluent was removed by rotary evaporation followed by reformulation in phosphate buffer and sterile filtration. Results Two batches of [11C]phenytoin ready for injection containing 3.9 and 4.6 GBq were obtained from 28.1 and 30.1 GBq of [11C]CO, respectively. The total synthesis time from end of radionuclide production was 28 min. The radiochemical purity was >99.5% and the decay corrected yield based on [11C]CO was 28% and 31%. Conclusions This synthesis method for [11C]phenytoin include several improvements. Preparations were simplified as the reagents spontaneously formed a homogenous solution without preheating. Handling of [11C]CO according to the xenon-method[2] allowed use of disposable vials which made cleaning of the vessel superfluous while minimizing effects from surface chemistry and carry-over associated with stainless-steel autoclaves. [11C]CO was efficiently transferred to the reaction vial. In addition, the possibility to weigh up the precursor in the reaction vial contributed to the 53% reduction in precursor load compared to the previous method while the radiochemical yield was increased from 22±4% to 28-30%. Acknowledgements References [1] J. Verbeek et al EJNMMI Res. 2012, 2 (1), 36 [2] J. Eriksson et al J. Labelled Comp. Radiopharm. 2012, 55 (6), 223-228

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S339

339 Synthesis of N-succinimidyl-3-fluoro-4-[124I]-iodobenzoate (S-FIB), a novel protein iodination analogue Christina L. Brown, Destiny J. Lusinger, Scott Apana, Samantha Moores, Marc S. Berridge 3DImaging, LLC, Little Rock, Arkansas, United States Objectives Radioiodinated proteins are valuable for diagnosis and treatment. Direct iodination is simple, but the oxidant can damage the biological function of the molecule. Indirect iodination using conjugation chemistry reduces the risk of damaging the function of the protein. The Bolton-Hunter reagent is most common for indirect iodination, but is itself susceptible to oxidative damage. Yields are typically variable and low (5-35%). We report synthesis of N-succinimidyl-3-fluoro-4-[124I]-iodobenzoate (S-FIB), which is stabilized in formation and to autoradiolysis by the electron-donating fluorine. Methods 4-fluorobenzoic acid was reacted with iodine-124 and chloramine-T in dimethylformamide (DMF) to give intermediate 3-fluoro-4-[124I]iodobenzoic acid (FIBA). This was converted to S-FIB in 75-85% yield using Nhydroxysuccinimide (NHS) and N,N'-dicyclohexylcarbodiimide (DCC) in ethyl acetate. The resultant product was purified to provide S-FIB with >90% radiochemical purity. Results 4-fluorobenzoic acid was converted to FIBA in 65-80% yield using DMF as the reaction solvent, as compared to 30% using acetonitrile, or 0% in dimethylsulfoxide. Intermediate purification of FIBA was attempted, but did not improve yield of the esterfication. The esterification of FIBA with NHS ranged from 75-85% yield depending on solvent and temperature. The fluorinated ring enhances yield and stability, while the two step procedure avoids destruction of the succinimide under reaction conditions. Conclusions Synthesis of S-FIB proceeded quickly providing a stable reagent for indirect iodination of proteins and peptides. Synthesis and purification was accomplished in under 90 minutes. Results show S-FIB is a useful radioiodination reagent offering ease of synthesis and increased stability. S-FIB has successfully labeled proteins, and offers a new iodination alternative. Acknowledgements References

Iodination and esterification of N-succinimidyl-3-fluoro-4-[124I]-iodobenzoate (S-FIB)

J Label Compd Radiopharm 2015: 58: S1- S411

S340: Poster

21st International Symposium on Radiopharmaceutical Sciences

340 Radionuclide imaging of metastatic melanoma using68Ga-glyco-click-conjugates. Nilesh Wagh1, Sanjay Thamake2, David Ranganathan2, Jessica Reedy3, Ebrahim S. Delpassand2, Michael K. Schultz3, Izabela Tworowska2 1 RITA FOUNDATION, Houston, Texas, United States, 2RadioMedix Inc., Houston, Texas, United States, 3 Radiology and Radiation Oncology Free Radical and Radiation Biology Program, University of Iowa, Iowa City, Iowa, United States Objectives The objectives of our studies were to evaluate GLUT -targeting properties of radiolabeled glucosamine click conjugate; and their application for imaging of metastatic melanoma cancer cells. Methods 68Ga-RMX-GC-08 was synthesized with chemical yield of 31 %; and chemical purity of 95% as analyzed by rHPLC and LC-Mass Spectroscopy. 68Ga-radiolabeling of RMX-GC proceeded with radiochemical yield of 98.8%. Cellular studies confirmed GLUT specific accumulation of radiotracer in tested cancer cell lines with values ranging from 4.5-to-8 %ID/mg during the first 2h of in vitro incubation. In the presence of GLUT competitors such as glucose, glucosamine and cytochalasin B, the uptake of 68Ga-RMX-GC was decreased by 4080% compared to control studies. Preliminary biodistribution studies of RMX-GC-08 in B16 murine metastatic melanoma xenografts showed tumor-specific retention of agent (4.3 ± 0.7 %ID/g) at 1 h post-injection with tumorto-muscle ratio of 2.02. Results Synthesis and radiolabeling of 68Ga-RMX-GC-08 was performed using 68Ge/68Ga generator (1.85 GBq, ITG GmBH, Germany) according to protocol described previously.1 GLUT targeting properties of radiotracer were confirmed in vitro in time- and concentration-dependent uptake, internalization and competition studies using human melanoma cancer cell lines A357 (BRAF mutant); MeWo (BRAF WT); and murine metastatic melanoma cell line B16. Conclusions Preliminary studies of 68Ga-RMX-GC-08 confirmed GLUT specific accumulation of agent in tested melanoma cancer cells with highest values of accumulation observed in MeWo (BRAF WT) cells. Further studies are required to validate pharmacokinetic properties of this agent in vivo. Acknowledgements This work was supported by NIH/NCI grant 1R43CA186364-01 (RadioMedix-Houston TX; Viewpoint MT-Iowa City-IA) References 1. 68Ga-labeled click conjugate targeting GLUT1-transporters- I. Tworowska, S. Zamanian, E. Delpassand, and M. Schultz- J. Nucl. Med. 2012, 53, 1568

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S341

341 77 As Complexes for Potential PET and Radiotherapy Yutian Feng1, Anthony DeGraffenreid1, Matthew D. Gott1, Donald E. Wycoff1, Alan R. Ketring2, Cathy S. Cutler2, Silvia S. Jurisson1 1 Department of chemistry, University of missouri-columbia, Columbia, Missouri, United States, 2The MU Research Reactor, University of Missouri-Columbia, Columbia, Missouri, United States Objectives 72As is a positron emitter (t1/2 26 h) potentially available from a 72Se/72As generator, while 77As is a beta emitting radionuclide (t1/2 38.8 h) with nuclear properties suitable for radiotherapy. We are developing the no carrier added (NCA) chemistry to synthesize [77As] dithiolarylarsines. Methods 77As is produced by 76Ge(n, γ)77As from 76GeO2. 77As is separated from the Ge target in NCA concentrations (~nM) as arsenate (AsO43-). Radiolabeling involves reduction to the arsenite As(III) with a monothiol, with reducing agent concentration, temperature and reaction time optimized. A modified Bart reaction (Fig 1) was used to successfully incorporate the aryl group onto 77As(III), with solvent, copper catalyst, aryl donor, dithiol ligand, temperature and time optimized to form the dithiolarylarsine. Chromatography and macroscopic standards were used for evaluating products and yields. Fig 2 shows the crystal structure of p-ethoxyphenyl-1, 2ethanedithiolatoarsine. Results At the macroscopic level, various dithiolarylarsines have been synthesized and characterized. Using the macroscopic standards and chromatography, we evaluated various reaction conditions (e. g. solvent, aryl donor, dithiol ligand, temperature, time) to optimize the labeling yield. Conclusions NCA synthesis of dithiolarylarsines was accomplished with the best yield to date observed for [77As] p-ethoxyphenyl-1,2-ethanedithiolatoarsine at 57%. Further optimization studies are underway. Acknowledgements Department of Energy (DOE) DE-SC0010283 grant; Department of Chemistry, University of Missouri-Columbia; the MURR, University of Missouri-Columbia References [1] Jurisson, S. S., et al. Chemical Reviews 93.3 (1993): 1137-1156. [2] Cutler, Cathy S., et al. Chem. Rev 113.2 (2013): 858-883. [3] Hamilton, C. S. and Morgan J. F. Organic Reactions (1944).

J Label Compd Radiopharm 2015: 58: S1- S411

S342: Poster

21st International Symposium on Radiopharmaceutical Sciences

342 One-pot radiosynthesis of [11C]formic acid as labelling agent for heterocycles via cyclization reaction using a GE Tracerlab FX C Pro synthesis module Severin Mairinger1, Johann Stanek1, 2, Thomas Wanek1, Claudia Kuntner1, Oliver Langer1, 2 1 Health & Environment Department, AIT Austrian Institute of Technology GmbH, Seibersdorf, Austria, 2 Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria Objectives [11C]Formic acid ([11C]HCOOH) is a potentially useful labelling agent, especially for ring closure reactions. [11C]HCOOH is synthesized by reducing [11C]carbon dioxide ([11C]CO2) with lithium triethylborohydride (LiEt3BH) to LiEt3B-adduct followed by an aqueous hydrolyzation step [1]. Here we describe the automated radiosynthesis of [11C]HCOOH and its subsequent use in the synthesis of a [11C]benzimidazolylsubstituted central nervous system drug candidate using a TracerLab FX C Pro synthesis module. Methods [11C]CO2 was trapped on the Carbosphere column in the Tracerlab FX C Pro synthesis module and was then transferred into the reactor containing LiEt3BH in tetrahydrofuran cooled to -20°C. The mixture was allowed to react for 40 s, followed by the addition of water to obtain [11C]HCOOH. A 1,2-diaminobenzyl derivative dissolved in aq. hydrochloric acid was added, the reaction was carried out at 140°C for 10 min. The product was purified by HPLC separation. For PET experiments in rodents the product was reformulated in a mixture of 0.9% aq. saline/ethanol (9/1, v/v). The formation of [11C]methanol or [11C]formaldehyde as by-products was not observed as confirmed by analytical HPLC using an Aminex HPX-87H (300mm x 7.8 mm, Bio-Rad Lab., USA) column. Results [11C]HCOOH was almost quantitatively (>90%) produced by the reduction of [11C]CO2 with LiEt3BH, without formation of [11C]methanol or [11C]formaldehyde as by-products. The radiochemical yield of [11C]benzimidazole derivative was 11±6% (n=13) (based on [11C]CO2, decay corrected) in a total synthesis time of 36±3 min. Radiochemical purity was 99.2±0.6% (n=13) and specific activity at end of synthesis was 33.7±10.3 GBq/µmol (n=13). Conclusions We were able to synthesize a [11C]benzimidazole derivative via ring closure reaction with [11C]HCOOH in good radiochemical yield and purity. The presented method allows reliable automated radiosynthesis to label different benzimidazole derivates and may be also applicable to other ring closure reactions. Acknowledgements We would like to dedicate this work to the memory of Dr. Dirk Roeda from the Service Hospitalier Frédéric Joliot (Orsay, France), who passed away on July 19th, 2013. References [1] Roeda D et al Radiochim Acta 2004; 92:329–32.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S343

343 Evaluation of carbon-11 labelled H4R antagonists as potential PET tracers of neuroinflammation Uta Funke1, 2, Frank van der Aa2, Dieter Ory3, Athanasios Metaxas2, Rogier A. Smits4, Danielle J. Vugts2, Bieneke Janssen2, Ger T. Molenaar1, 2, Esther J. Kooijman2, Mariska Verlaan2, Perry S. Kruijer1, Rob Leurs5, Adriaan A. Lammertsma2, Guy Bormans3, Albert D. Windhorst2 1 BV Cyclotron VU, Amsterdam, Netherlands, 2Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, Netherlands, 3Pharmaceutical and Pharmacological Sciences, KU University of Leuven, Leuven, Belgium, 4Griffin Discoveries BV, Amsterdam, Netherlands, 5Medicinal Chemistry, VU University Amsterdam, Amsterdam, Netherlands Objectives As the histamine H4 receptor (H4R) mediates activation and chemotaxis of microglia [1,2], imaging H4R using PET may be a tool to visualize and quantify neuroinflammation (NI). In the present study the H4R antagonists [11C]JNJ7777120 (1, Fig. 1, left) and [11C]JNJ39594906 (2) were investigated as putative PET tracers. Methods 1 and 2 were synthesized by [11C]methylation of the corresponding desmethyl-precursors. In vitro autoradiography (AR) studies were performed on snap frozen brain slices of healthy rat or of NI induced by kainic acid (KA, i.p.) or lipopolysaccharide (LPS, stereotactic). Ex vivo biodistribution and in vivo PET studies were performed after i.v. injection of 1 and 2 in healthy rats (n≥4). In addition, two rats with induced NI (LPS, stereotactic, 5 days prior imaging) were scanned after i.v. administration of 1. Results AR images showed only non-specific binding of 1 (Fig. 1, right) and 2 in healthy rat brain, as both tracers could not be blocked by different H4R inhibitors. In NI brains, 1 showed slightly reduced binding in the hippocampus of KA and the right striatum of LPS treated animals. In vivo, 2 did not enter the healthy rat brain, whereas 1 uptake was high (SUV 3.5, 3 min p.i.) although it could not be blocked by thioperamide, an H3R/H4R antagonist. No difference could be observed in uptake of 1 in the LPS region of NI animals compared to healthy rat brain. Conclusions 2 is not suitable as a PET tracer due to its negligible uptake in brain. In vitro as well as in vivo, binding of 1 was not significantly different between healthy and inflamed rat brains. Further studies are needed to assess whether this is due to lack of H4R upregulation in these specific NI models or NI in general, or a failure of 1 to detect this because of relatively high non-specific binding. Acknowledgements This work was funded by the EU's 7th Framework Programme (FP7/2007-2013) under grant agreement n○ HEALTH-F2-2011-278850 (INMiND). Dieter Ory is fellow of the Research Foundation Flanders (FWO). References [1] Ferreira R, et al (2012) J Neuroinflammation, 9, 90. [2] Dong H, et al (2014) Mol Biol, 49, 1487.

Figure 1. Investigated PET tracers (left) and in vitro AR images of 1 binding on rat brain (right).

J Label Compd Radiopharm 2015: 58: S1- S411

S344: Poster

21st International Symposium on Radiopharmaceutical Sciences

344 SPECT/CT imaging and ex vivo biodistribution of peptide modified 111In-labelled Silicon nanoparticles in a rat model of myocardial infarction Sanjeev Ranjan1, Mónica Ferreira2, Sini Kinnunen3, Vimalkumar Balasubramanian2, Severi Muje1, Alexandra Correia2, Ermei Mäkilä4, Virpi Talman3, Jarno Salonen4, Jouni Hirvonen2, Heikki Ruskoaho3, Hélder Santos2, Anu J. Airaksinen1 1 Laboratory of Radiochemistry, University of Helsinki, Helsinki, Finland, 2Division of Pharmaceutical Chemistry and Technology, University of Helsinki, Helsinki, Finland, 3Division of Pharmacology and Pharmacotherapy, University of Helsinki, Helsinki, Finland, 4Laboratory of Industrial Physics, University of Turku, Turku, Finland Objectives Porous silicon nanoparticles (PSiNP) are promising carriers for targeted drug delivery. The present study aims to investigate the behaviour and biodistribution of the heart-targeted PSiNP in vivo. The PSiNP were modified with PEG and DOTA for radiolabeling, and further conjugated with targeting peptides (Pep-1-3). As a control, PSiNP without any peptide and a scrambled sequence of Pep-3 was used. Methods Functionalized PSiNP were radiolabeled with 111In with yield above 70%, and their stabilities were studied in buffers covering a physiologically relevant pH range and in 50% human plasma. A myocardial infarction model was induced by isoprenaline injection and [111In]PSiNP were administered iv in order to study their ability to target heart tissue. Results Peptide-modified [111In]PSiNP improved their accumulation in the heart, up to 4.5% of the injected dose (ID) with highest accumulation at 10 min as quantified by SPECT/CT images (Fig. 1). The ex vivo biodistribution studies revealed that at 4h, the [111In]PSiNP were mainly accumulated in liver and spleen. Conclusions Functionalized PSiNP were successfully labelled with 111In for in vivo studies. Peptide modified [111In]PSiNP get markedly enhanced accumulation into the heart. Acknowledgements This study was funded by the Academy of Finland, TEKES (no 40495/13) and University of Helsinki Research Funds.

Fig. 1: SPECT/CT image quantification of the heart showing the biodistribution of the intravenously administered [111In]PSiNP modified with different targeting peptides at 10 min, 20 min, and 4h time points. Values represent mean ± s.d. (n = 4/5).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S345

345 (Radio)pharmacological characterization of novel α-MSH derivatives Feng Gao1, Wiebke Sihver1, Ralf Bergmann1, Cathleen Haase-Kohn1, Jörg Steinbach1, Davide Carta2, Cristina Bolzati3, Andrea Calderan4, Jens Pietzsch1, Hans-Jürgen Pietzsch1 1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany, 2 Department of Pharmaceutical Sciences, University of Padua, Padua, Italy, 3IENI-CNR, Padua, Italy, 4ICB-CNR, Padua, Italy Objectives Melanocortin-1 receptor (MC1R) is well known to be overexpressed in melanoma. Thus, it has been a great interest in targeting this receptor for diagnosis of human metastasized melanoma. We aimed at investigating (radio)pharmacological properties of novel derivatives of the α-melanocyte-stimulating hormone (α-MSH) and selecting most promising candidates for further studies in melanoma models in vivo. Methods Linear and cyclic α-MSH derivatives (NAP-NS1(1), NOTA-NAP-NS1(2), natCu-NOTA-NAP-NS1(3), NAP-NS2(4), NOTA-NAP-NS2(5), natCu-NOTA-NAP-NS2(6), DPA-NAP-NS1(7) and Re-tricarbonyl-DPA-NAPNS1(8)) were investigated in competition assays in both murine B16F10 and human MeWo cells. In vitro stabilities of [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 were tested in phosphate buffer (pH=7.4) and human serum at 37°C for 1h and 24h. Transchelation and octanol/water partition coefficients of radiolabeled peptides were also investigated. Additionally, [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 with high radiochemical purities and specific activities were applied in saturation assays and kinetic studies. Results Linear α-MSH derivatives (1, 2, 3, 7 and 8) showed higher affinities on both murine and human cells than cyclic α-MSH derivatives (4, 5, 6). Linking the chelator to the peptide and coordinating the chelator-peptide with nat Cu or Re were accompanied by some loss of affinity. [64Cu]Cu-2, [64Cu]Cu-5 and 99mTc-tricarbonyl-7 were stable in phosphate buffer and serum at 37°C after incubation for 1h and 24h. No transchelation of radiolabeled peptides was observed in cysteine and histidine challenge experiments. LogD values suggested that [64Cu]Cu-2 (-2.30±0.01) and [64Cu]Cu-5 (-3.39±0.04) had higher hydrophilicity than 99mTc-tricarbonyl-7 (-0.43±0.01). Saturation studies in both cell lines resulted in Kd values (nM) in the lower nanomolar ranges for [64Cu]Cu-2 (B16F10: 1.7±0.2; MeWo: 2.6±0.5) and 99mTc-tricarbonyl-7 (B16F10: 6.0±0.5; MeWo: 4.5±0.8). But Bmax (fmol/mg protein) of [64Cu]Cu-2 on murine and human cells (B16F10: 46.6±3.9; MeWo: 16.6±1.6) was notably lower than that of 99mTc-tricarbonyl-7 (B16F10: 403.5±46.1; MeWo: 50.3±6.4). Kinetic study of [64Cu]Cu-2 in murine cells showed rapid cellular association and dissociation in vitro. Conclusions [64Cu]Cu-2 showed high stability, hydrophilicity, binding affinities and rapid cellular association and dissociation in vitro, which made it promising for further investigations in melanoma models. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S346: Poster

21st International Symposium on Radiopharmaceutical Sciences

346 Imaging atherosclerotic plaque inflammation with the CD80/CD86-specific 111In-DOTA-Belatacept Romana Meletta1, Adrienne Müller Herde1, Patrick Dennler2, Eliane Fischer2, Stefanie D. Krämer1, Roger Schibli1, 2 1

Department of Chemistry and Applied Biosciences, Center for Radiopharmaceutical Sciences, ETH Zurich, Zürich, Switzerland, 2Paul Scherrer Institut, Villigen, Switzerland

Objectives Atherosclerotic plaque vulnerability is related to underlying inflammatory processes with antigenpresenting cells (APCs) playing a central role. We recently identified the co-stimulatory molecules CD80 and CD86 expressed on APCs as promising targets for imaging plaque inflammation [1]. In a proof-of-principle study, the fusion protein Belatacept (Nulojix®), containing as binding motif the extracellular portion of CTLA-4, was labeled with indium-111. We analyzed the in vitro stability of this radiotracer and evaluated it in mice bearing CD80- and CD86-positive Raji xenografts and in a mouse model of atherosclerosis (ApoE KO). Methods Commercially available Belatacept was conjugated with p-SCN-Bn-DOTA, radiolabeled with In-111 and purified by size-exclusion chromatography (SEC). Stability was investigated after incubation in human or mouse plasma at 37 °C up to 72 h by SEC. CD1 nude mice bearing Raji xenografts and ApoE KO mice were injected i.v. with 10 MBq 111In-Belatacept (25 µg, baseline). Blockade animals received additionally unlabeled Belatacept (500 µg). In vivo and ex vivo SPECT/CT scans or biodistribution studies were performed 48 h post injection. Oil red o staining was performed with excised blood vessels to visualize plaque lipids. Results 111In-Belatacept was successfully produced in 73-78% radiochemical yield. 82% of labeled fusion protein remained intact after 72 h of incubation in plasma. In vivo, 111In-Belatacept accumulated in Raji xenografts and a reduced accumulation was determined under blockade conditions. These results were confirmed by biodistribution revealing a significant reduction in blockade animals. Ex vivo scans of the aortic arch and the carotids of ApoE KO mice showed a high accumulation in atherosclerotic plaques and a reduced signal under blockade conditions. Tracer accumulation and plaque localization determined by oil red o staining were consistent. Conclusions 111In-Belatacept accumulates in CD80- and CD86-positive tissue in vivo and binds specifically to plaques in a mouse model of atherosclerosis. This confirms CD80/CD86 as promising targets for the non-invasive imaging of atherosclerotic plaques. Acknowledgements References [1] Müller A., et al. (2014) Int J Cardiol, 174, 503-15.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S347

347 Studies on minimal required specific activities (SA) of 177Lu for labeling a typical clinical dose of 0.2 mg DOTATATE with 7.4 or 11.1 GBq 177Lu for Peptide Receptor Radionuclide Therapy (PRRT) Wouter A. Breeman, Erik de Blois, Jim Nieuwenhuizen Nuclear Medicine, Erasmus MC, Rotterdam, Netherlands Objectives PRRT of neuroendocrine tumors is performed with 177Lu-DOTA0,Tyr3-octreotate (177Lu-DT) at high SA, expressed in MBq per mass of DT. 177Lu is reactor-produced via (n,γ) reaction from enriched 176Lu. In high flux reactors target (176Lu) “burn up” after days of irradiation is considerable. Hence, assuming (by vendors of 177Lu) the mass of target remains constant during irradiation, is not valid ⇒SA of 177Lu at end of irradiation will be higher [1]. The presence of 175+176Lu reduces highest achievable SA in practice to 0.12 GBq 177Lu per nmol DT [2]. Pretherapeutic dosimetry measurements and PRRT should be performed with same amount of same substance [3]. A typical formulation of 177Lu-DT for PRRT contains 0.2 mg DT and 7.4 GBq 177Lu. Here we present an overview of our investigations with 177Lu-DT addressing the need to determine actual SA of 177 Lu and the requirements on SA of 177Lu for radiolabeling 0.2 mg DT with 7.4 or 11.1 GBq 177Lu. Methods To label DT at high SA it is essential to know the actual concentration of all reaction ingredients. Therefore methods were developed: 1st a method to quantify content and purity of DOTA-peptides [4], 2nd method to determine actual SA of 177Lu [4]. SA of 177Lu expressed as a function of time are calculated without the correction for decay of 177Lu. Whereas 177Hf, decay product of 177Lu does not incorporate in the DOTA-moiety under our labeling conditions ⇒ SA of 177Lu is underestimated [1,2], and underlines the need to determine SA of 177 Lu. Labelings were performed at various SA of 177Lu and monitored by ITLC and HPLC [4]. Results SA of 177Lu was always higher as stated by vendors (mean 30%, range 10-70% higher). During radiolabeling we could monitor co-introduction and tracing of impurities and eliminate these impurities, especially in buffers and quenchers. After labeling 0.2 mg DT with 7.4 GBq 177Lu at a SA of 177Lu of 350 GBq per mg Lu, at >99% incorporation of 177Lu and recovered 9 mol% of DT as DT and 91% as Lu-DT. After labeling 0.2 mg DT with 11.1 GBq 177Lu at a SA of 177Lu of 525 GBq per mg Lu and >99% incorporation of 177 Lu and recovered 11 mol% of DT as DT and 89% as Lu-DT. Even at a molar ratio (DT/Lu) of 1.05 we had full (>99%) incorporation of 177Lu. Conclusions DT can effectively be labeled at the preferred constant amount of a typical clinical of 0.2 mg DT with 7.4 GBq 177Lu at a SA of 177Lu of ≥350 GBq 177Lu per mg Lu and with 11.1 GBq 177Lu at a SA of 177Lu of ≥525 GBq 177Lu per mg Lu. Acknowledgements References [1] Breeman et al., Current Radiopharmaceuticals (A), in press [2] Breeman et al., Current Radiopharmaceuticals (B), in press [3] Kletting et al., Med Phys, 2012 [4] Breeman et al., J Radioanal Nucl Chem, 2014

J Label Compd Radiopharm 2015: 58: S1- S411

S348: Poster

21st International Symposium on Radiopharmaceutical Sciences

348 Quality of Animal Experiments – Problems and Pitfalls P. A. Schubiger1, Marianne I. Martic-Kehl2 1 Pharmacy, Swiss Federal Institute of Technology, Zurich, Switzerland, 2Collegium Helveticum, Swiss Federal Institute of Technology, Zürich, Switzerland Objectives In vivo experiments using animals with (mostly) induced diseases as human surrogate organisms are a crucial step in any drug development process including the development of radiopharaceuticals. Against the assumption that preclinical animal testing yields reliable and relevant data for the clinics, in practice, high discrepancies between so-called bench and bedside results have been observed. Methods With the aim of identifying problems and pitfalls In this project we assessed the quality of preclinical animal studies in the process of anti-angiogenic cancer drug development by performing a systematic review. We searched two major biomedical databases (Pubmed and Thomson Integrity) for original articles up to 2011. We collected 71 study- and experiment-specific parameters out of 232 original articles and abstracts describing 1358 single experiments and outcomes. According to an a priori defined list of quality parameters we assigned a quality score to each experiment. Results General experimental design and reporting features were frequently neglected in the investigated papers. Fulfilment of quality criteria in investigated articles (in %): Statement of potential conflict of interest (12%), Sample-size calculation (0.5%), Allocation concealment (0%), Randomization (41%), Blinded assessment of outcome (2%) Outcome definition was very variable between publications, it was therefore necessary to define a uniform outcome score. By comparing study quality with outcome, we found a trend towards higher fraction of undesirable outcome in high quality studies. Furthermore, if a drug was the main focus of a study (i.e. Hypothesis: Drug x is efficacious), authors were more likely to report desirable outcome, than if a drug served as a control intervention. A two-step cluster analysis revealed four clusters, which confirm the trend towards higher study quality leading to more undesirable outcome. Conclusions Our investigations have revealed rather low quality standards of preclinical animal study performance. One of the mainly neglected study design features was ‘blinded assessment of outcome’. Bearing this in mind, it is not particularly surprising that we also found what we refer to as ‘hypothesis bias’. If researchers are aware of the treatment of a test subject during data evaluation, they are biased towards confirming their hypothesis. For pre-clinical drug testing, it is also advisable to use orthotopic tumor models where tumors are inoculated at the organ of tumor cell origin. Acknowledgements We thank the OPO foundation and Dr. Richard Dähler for financial support. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S349

349 In vivo evaluation of syn- and anti-[123I]IVACBC as potential SPECT tumor imaging agents Weiping Yu, Mark M. Goodman Radiology, Emory University, Atlanta, Georgia, United States Objectives Radioiodinated amino acid ACBC analogues syn- and anti-1-amino-3-(2-[123I]iodoethenyl)cyclobutane-1-carboxylic acid (syn-[123I]IVACBC 1 and anti-[123I]IVACBC 2) were synthesized for SPECT tumor imaging in 5 human tumor xenografts. Methods The protected syn- and anti-IVACBC stannylated precursors for radiolabeling were prepared in a series of synthetic steps starting from allylbenzylether. Radioiodination was carried out with no-carrier-added [123I]NaIH2O2/H+ followed by hydrolysis with TFA and chromatographic purification. The tumor uptake and tissue distribution of syn- and anti-[123I]IVACBC (1 and 2) were examined using subcutaneous implanted human tumors (A549 lung, DU145 prostate, SKOV3 ovary, MDA MB468 breast and U87 brain) SCID mice xenografts. In the studies, the mice (n=5 each time point) were injected with tracer through tail vein and sacrificed at 15, 30, 60, and 120 min post injection. The uptake of radioactivity in tumors and 10 normal tissues were calculated and presented as percent of injected dose per gram tissue (%ID/g). Results Radioactivity uptake in tumor and the background tissue/organ (blood, muscle, brain and bone) at 60 min post injection were reported in the Table 1. Very low uptake was found in thyroid. Conclusions The biodistribution experiments showed a rapid and prolonged accumulation of radioactivity in tumors with slight higher uptake of anti-isomer 2 than that of syn-isomer 1. Anti-isomer 2 gave a higher tumor to muscle ratio than syn-isomer 1 in general. These results support the candidacy of syn- and anti-[123I]IVACBC as potential SPECT tumor imaging agents where the radios of tumor to background were greater than 1.5. Acknowledgements This study was supported by Nihon Mediphyiscs Co., Ltd., Japan. References Weiping Yu, et al, Bioorg & Med Chem Lett 18 (2008) 1264–1268.

J Label Compd Radiopharm 2015: 58: S1- S411

S350: Poster

21st International Symposium on Radiopharmaceutical Sciences

350 Radiochemical stability study of [methyl-11C]choline by HPLC method István Jószai, Nóra Papréti, Dezsö Szikra, Tünde Miklovicz, Pál Mikecz Department of Nuclear Medicine, University of Debrecen, Debrecen, Hungary Objectives In Hungary [18F]FDG, [11C]methionine and [11C]acetate-Na are accessible radiopharmaceuticals for human PET diagnostics. University of Debrecen in this year obtained marketing authorization for newer tracer, namely [methyl-11C]choline, which is planned to be used uniquely in the country for diagnosis of prostate cancer. The IMPD of the radiopharmaceutical contains the radiochemical stability study of the active ingredient. The aim of this work is to report the results of stability program of [methyl-11C]choline. Due to radiolytic decomposition of [methyl-11C]choline 11C labelled methylamines can be produced1. Methods The HPLC system used for the determination of degradation products of [methyl-11C]choline consisted of an HPLC pump (Jasco), a syringe-loading sample injector valve (Rheodyne), a radiometric detector (atomki, Hungary) and a UV-Vis detector (Jasco). Standards of choline and methylamines were purchased from Sigma. [methyl-11C]choline was synthesized by methylation of dimethylaminoethanol with [11C]methyl-iodide. Results For the determination of radiochemical purity of [methyl-11C]choline and study of radiolytic decomposition of the active ingredient an HPLC method was developed. Chromatographic conditions: Zorbax SCX 4.6x50mm, 5 µm column, radioactivity and UV-Vis (280 nm) detectors, flow rate 3 mL/min, 20 µL injected volume, 3 min measurement time, eluent: 20% acetonitrile, 80% buffer solution (56 mg/L dopamine, 25 mM ammonium-acetate, pH=5). 2 mg/mL standards of choline, monomethylamine, dimethylamine and trimethylamine were prepared. The method was able to separate choline from amines thus applicable to determine the radiochemical purity of the radiopharmaceutical. The critical resolution of choline and trimethylamine peaks was 1.2. According to stability results of [methyl-11C]choline batches with radioactive concentration of 3.5 GBq/mL showed radiochemical purity of 99.76% until the end of the shelf life. This was above the acceptable 95% RCP limit. Conclusions The developed HPLC method was applicable to determine the radiochemical stability of [methyl11 C]choline. The radiochemical purity of [methyl-11C]choline did not show any dramatic reduction with time until the end of the shelf life of the radiopharmaceutical at relatively high initial radioactive concentration. Consequently, there is no need for using additives for stabilization of [methyl-11C]choline. Acknowledgements References [1] Tayosawa, I. (1964) XI. Bull. Univ. Osaka Pref., Ser. B. 1-9.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S351

351 Synthesis and Evaluation of a new 18F-Synthon for copper-free click Labeling of Biomolecules Kathrin Kettenbach1, Benedikt Sandhöfer1, Frank Roesch1, Tobias L. Ross2 1 Institute of Nuclear Chemistry, Johannes Gutenberg-University , Mainz, Germany, 2Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany Objectives This work aims at the development of a copper-free 18F-clicklabeling strategy based on a strained cyclic alkyne for the labeling of biomolecule-azides. [18F]ADIBO [1] was modified with a polyethyleneglycolspacer to increase polarity of the 18F-synthon. With the new 18F-synthon the use of copper can be avoided, which makes the 18F-labeling of biomolecules for positron emission tomography (PET) application much easier. Methods The diethylenglycol-based precursor was obtained by acid functionalization through tert-butylacrylate. After deprotection, the intermediate was coupled to dibenzocyclooctyne(DBCO)-amine. Tosylate and mesylate served as leaving groups for fluorination. Different bases and base concentrations were tested for 18F-radiolabeling. The new 18F-synthon was isolated via HPLC and SPE and tested concerning its stability. We finally tested the new 18 F-synthon in copper-free click reactions with various biomolecule-azides, like an azido-cRGD and other azidefunctionalized peptides. Results The DBCO-based precursor for the 18F-labeling could be synthesized in good overall yields of 10 % over 4 steps. The 18F-labeling was optimized using different bases and reaction conditions, leading to excellent radiochemical yields of up to 85 % after 15 min. Furthermore, the separation and purification of the synthon were optimized using HPLC and SPE. The synthon can be prepared within a total reaction time of 60 min. With the copper-free click reaction we could already label several biomolecules (eg. azido-cRGD), with fluorine-18 in very high yields of ≥ 85% in 10 min at room temperature. Although an azide-functionalized PDGF-β ligand could be labeled in excellent yields of ≥ 90% after 3 min at 40 °C, which was not achievable via the copper-catalyzed cycloaddition. Conclusions This new 18F-PEG-DBCO enables the 18F-labeling of sensitive biomolecules under very mild conditions without the need of any copper species. High yields and fast reaction kinetics can be achieved while extensive separation and purification from copper is no longer necessary. Further investigations concerning optimization of the click reaction and application range for this new synthon, as well as in vivo evaluation of the new 18F-labeled biomolecules, are ongoing. Acknowledgements The authors thank the Max Planck Graduate Centre Mainz and the Ci3 cluster of excellence for financial support. References [1] S. Arumugam et al., Bioorg. Med. Chem. Lett., 21, 6987-6991

J Label Compd Radiopharm 2015: 58: S1- S411

S352: Poster

21st International Symposium on Radiopharmaceutical Sciences

352 Evaluation of the Chick Embryo as a Test System for Radiopharmaceuticals Stephanie Haller1, Simon M. Ametamey2, Roger Schibli1, Cristina Mueller1 1 Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, Villigen-PSI, Switzerland, 2 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland Objectives The chick embryo has been used as a test system in numerous areas of research, including radiopharmaceutical sciences [1-2]. The aim of this study was to investigate whether it can be used as a model to evaluate radiopharmaceuticals by PET. For this purpose, the in vivo behavior of [18F]fallypride, 3`aza-2`[18F]fluorofolic acid ([18F]-AzaFol [3]) and [18F]fluoride were compared in chick embryos and mice. Methods Chick embryos were cultivated ex ovo for 17-19 days before application of the radiotracers. About 10MBq of [18F]fluoride, [18F]fallypride or [18F]-AzaFol were injected into chick embryos 60-90 min before euthanasia in liquid nitrogen. PET acquisitions were performed with a benchtop PET scanner (Genisys4, Sofie Biosciences). The results were compared with those obtained with the mouse model. Results Bone uptake of [18F]fluoride was shown in both species. [18F]-AzaFol accumulated in kidneys and liver of the chick embryo and the mouse [3]. Radioactivity was not detected in the bones, indicating that [18F]-AzaFol is stable in vivo (Fig. 1A/B). [18F]Fallypride accumulated in the intestine of the chick embryo and the mouse besides binding to D2-receptors in the brain of the mouse. However, in both species, accumulation of radioactivity was seen also in the bones, which indicates in vivo defluorination (Fig. 1C/D). Conclusions It was demonstrated that the radiopharmaceuticals have comparable behavior in chick embryos and mice. Based on these results, the chick embryo may be a cost-effective alternative to the mouse for the first in vivo evaluation of novel radiopharmaceuticals. These promising results warrant further investigations. Acknowledgements References [1] Würbach L et al, (2012) Mol Imaging Biol, 14:688-98. [2] Warnock G et al, (2013) J Nucl Med, 54:1782-8. [3] Betzel T et al, (2013) Bioconjug Chem, 24:205-14.

Figure 1. PET images of chick embryos and mice after the injection of [18F]-AzaFol (A/B) and [18F]fallypride (C/D).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S353

353 44 Sc and 177Lu-labeling of DOTA-PSMA DKFZ-617 for dosimetry and therapy of prostate cancer Elisabeth Eppard1, Anna de la Fuente2, Stefan Kürpig1, Frank Roesch2, Markus Essler1 1 Nuclear Medicine, University Hospital Bonn, Bonn, Germany, 2Institute of Nuclear Chemistry, Johannes Gutenberg University, Mainz, Germany Objectives Radiolabeling of the prostate-specific membrane antigen (PSMA) inhibitor, Glu-NH-CO-NH-Lys (Ahx), using DOTA as chelator, a broad pool of radionuclides becomes available for labeling. These possible variations allow the visualization of the biological behavior over different periods of time depending on the halflife of the used radionuclide (68Ga t1/2 = 68 min; 44Sc t1/2= 3.9 h). Additionally, the usage of different imaging modalities or endoradiotherapies can be applied depending on the employed radionuclide. In this study DKFZPSMA-617 was radiolabeleld with 44Sc and 177Lu for theranostic application. Methods DKFZ-PSMA-617 was obtained from ABX (Radeberg, Germany). 44Sc was obtained from a 44Ti/44Sc generator in Mainz, where 44Ti decays with a half-life of 60 d to no-carrier-added (n.c.a) 44Sc. Radiolabeling with 44 Sc was performed in 3 mL 0.25 M ammonium acetate buffer with varying amount of ligand at 95°C. 177Lu was obtained from IDB Holland and labeling was performed in a gentisinic acid/sodium ascorbate solution with varying amount of ligand at 95°C. For both radiolabeled compounds stability studies, for example with serum and NaCl, were performed over a period of two half-lives. Quality control was performed using radioHPLC and radioTLC. Results 44Sc-DKFZ-PSMA-617 and 177Lu-DKFZ-PSMA-617 were effectively labeled at 95 °C. Subsequent cartridge-based solid-phase-extraction (C-18) resulting in a radiochemical purity of the final tracers of ≥98%. Radiochemical purity could be analyzed effectively using radioHPLC and radioTLC. For both tracers stability studies were successfully performed, for example 177Lu-DKFZ-PSMA-617 shows high stability in fetal calf serum over two half-lives. Conclusions The radiolabeling of DKFZ-PSMA-617 with the new generator-derived PET radionuclide 44Sc was investigated in detail with regard of reaction kinetics, purification and stability. Also radiolabeling of 177Lu-DKFZPSMA-617 was optimized with regard to therapeutic application and evaluated for its stability i.a. in fetal calf serum. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S354: Poster

21st International Symposium on Radiopharmaceutical Sciences

354 Synthesis and biodistribution of novel 99mTc labeled chitosan modified metronidazole analogs as a potential probe for tumor hypoxia imaging Liqin Liu, Zhuo Wang, Lili Chen Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing , China Objectives The identification and quantitative estimation of tumor hypoxia was an important factor in planning the therapeutic strategy for a better clinical outcome. We have reported that 99mTc-hydrazino-nicotinamidemetronidazole complex (99mTc-HYNIC-MN) had high tumor hypoxia tissues uptake in S180 tumor bearing mice [1]. Chitosan is a biocompatible polysaccharide with low toxicity, a naturally occurring polymer which has attracted significant scientific interest [2]. In this study, we prepared chitosan modified compound 99mTc-HYNICCS-MN and evaluated it as a potential tumor hypoxia imaging agent. Methods Chitosan modified metronidazole analogs (HYNIC-CS-MN) was prepared successfully according to multi-step reaction (Fig. 1). HYNIC-CS-MN was labeled with 99mTc at 100°C and pH 7 using tricine (50mg) and EDDA (1 mg) as coligands, stannous chloride as a reducing agent. Biodistribution studies were carried out in Kunming mice bearing S180 tumor. Results New complexes of HYNIC-CS-MN was synthesized and the structure was determined by 1HNMR and MS. HYNIC-CS-MN could be labeled easily and efficiently with 99mTc using tricine/EDDA as coligands in high yield (>93%). The tumor cell experiment showed that the 99mTc-HYNIC-CS-MN complexes had certain hypoxic selectivity. The biodistribution studies of 99mTc-HYNIC-CS-MN in Kunming mice bearing S180 tumor showed high tumor uptake, and relatively low accumulation in non-target organs, suggesting 99mTc-HYNIC-CS-MN would be a novel potential tumor hypoxia imaging agent. Conclusions The novel ligand HYNIC-CS-MN had been successfully synthesized and 99mTc-HYNIC-CS-MN was prepared in high yields using tricine/EDDA as coligands. 99mTc-HYNIC-CS-MN showed high tumor uptake, good retention and high target to non-target activity ratios in tumor-bearing mice, suggesting that it could be potentially useful for tumor hypoxia imaging agent. Acknowledgements The work was financially supported by the Fundamental Research Funds for the Central Universities of China (FRF-BR-12-017) and the National Natural Science Foundation of China (81301319). References [1] Liu LQ, et al (2011), J Radioanal Nucl Chem, 287, 847-852. [2] Kumar, MNVR, et al (2004), Chem Rev, 104, 6017-6084.

Figure 1. Synthesis of chitosan modified metronidazole analogs HYNIC-CS-MN. (i) (CH2CH2CO)2O, DMAP, CH3CN, rt; (ii) Chitosan, DMF/water, EDC, rt; (iii) Succinimidyl HYNIC, CH3CN/water, rt.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S355

355 New quantification method of radiochemical purity using Cerenkov radiation Yeong Su Ha, Jeongsoo Yoo Department of Molecular Medicine, Kyungpook National University School of Medicine, Daegu, Korea (the Republic of) Objectives Radiochemical purity (RCP) is usually determined by radio-TLC (Thin Layer Chromatography) scanner or radio-HPLC (High-Performance Liquid Chromatography) methods. Radio-TLC method is a simple technique for determining RCP, which takes few minutes to perform. However, the resolution between closely located spots are limited. Radio-HPLC method provides better resolution and accurate quantification data but is more complicated than radio-TLC; it takes longer time than radio-TLC method and needs expensive chromatograhpic column, and skills for handling HPLC instrument. Here, we report a new method of RCP determination, which utilizes 'Cerenkov radiation (CR)' emitted from the radionuclides. This new method for the measurement of RCP is simple, easy to handle but providing accurate quantification data in short time. Methods We chose I-131, I-124 and P-32 as charged particle emitted radionuclides for Cerenkov luminescence imaging (CLI). Various conditions such as the existance of fluorescence, the thickness of coating, the backing material of TLC plate and the refractive index of placed glass as medium onto a TLC plate were tested to increase CR signal by CLI. Then all imaging data were quantified and compared with those of conventional methods. Results TLC plates with fluorescence active solid phase showed positive CR signal. A thicker coating of solid phase on TLC plate had stronger CR signal than thinner coating. Glass supported TLC plates was the best for CLI. In order to increase CR signal, glass cover having higher refractive index than air was better. Quantification data of new method was well-matched with the results of conventional methods. Conclusions We screened various TLC plates for the best quantification method of RCP based on CR. The best results come from the TLC plate with fluorescence, thick coating, glass backing in our studies. The quantification value of the CR signal emitted from radionuclides can be used as new quantification method of RCP. Acknowledgements Supported by R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A2A2A01012250, 2013M2A2A 6042317, 20090078235) and the BK21 Plus funded by the Ministry of Education, Korea (21A2013221 2094). References

J Label Compd Radiopharm 2015: 58: S1- S411

S356: Poster

21st International Symposium on Radiopharmaceutical Sciences

356 Two Novel Nanosized Radiolabeled Analogues of Somatostatin for Neuroendocrine Tumor Imaging Blanca E. Ocampo-García1, Guillermina Ferro-Flores1, Emmanuel Orocio-Rodríguez1, Flor de María Ramírez2, Clara L. Santos-Cuevas1, Erika Azorin-Vega1 1 Materiales Radiactivos, Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Estado de Mexico, Mexico, 2 Química, Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Mexico Objectives To prepare two nanosized multimeric systems for neuroendocrine tumor imaging, 99mTc-PAMAMTyr3-Octreotide and 99mTc-AuNP-Tyr3-Octreotide, and to compare their in vitro uptake in SR-positive AR42J cancer cells as well as their biodistribution in athymic mice bearing AR42J tumors. Methods [Tyr3, Lys(Boc)5]-Octreotide was conjugated to the carboxylate groups of the PAMAM dendrimer (G3.5) [1]. 99mTc labeling was carried out by a direct method. 99mTc-Tyr3-Octreotide was conjugated to AuNPs (20 nm) by spontaneous reaction with the cysteine thiol group. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG. In vitro binding studies were performed in AR42J cancer cells. Biodistribution studies were accomplished in athymic mice with AR42J-induced tumors with blocked and unblocked receptors. Results Elemental analysis demonstrated that 26 Tyr3-Octreotide molecules were conjugated to one molecule of PAMAM. RP for both nanosized conjugates was >94% and showed recognition for SR in AR42J cells. The tissue distribution 2 h after 99mTc-PAMAM-Tyr3-Octreotide administration in mice showed specific tumor uptake (4.12 ± 0.57% ID/g) and high accumulation in pancreas (15.08 ± 3.11% ID/g) which expresses SR. No significant difference in the tumor uptake was found between both multimeric radioconjugates. However, the dendrimerpeptide conjugate showed a significant renal excretion (Fig. 1). Conclusions Both radiopharmaceuticals demonstrated properties suitable for use as target-specific agents for molecular imaging of tumors that overexpressed SR Acknowledgements IAEA, Contract 18358/RO References Astruc D et al (2010) Chem. Rev. 110, 1857–1959

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S357

357 Cerebral uptake of FET after tumor resection in a F98 rat glioma model Stefanie Geisler1, Johannes Ermert1, Marion Rapp2, Antje Willuweit1, Michael Christoph Sabel2, Heinz H. Coenen1, Nadim J. Shah1, Karl J. Langen1 1 Institut für Neurowissenschaften und Medizin, INM, Forschungszentrum Jülich, Jülich, Germany, 2 Neurochirurgische Klinik, Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany Objectives In comparison with morphological MRI, PET using the amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (FET) allows an improved delineation of tumor tissue from benign tissue reactions. However, unspecific uptake of FET was observed in non-neoplastic brain lesions and in areas of reactive astrogliosis in experimental studies. The surgical resection of a tumor might induce tissue responses that potentially could lead to an unspecific accumulation of FET, and might falsely be interpreted as residual tumor tissue. Therefore, the intracerebral FET uptake after tumor resection in a F98 rat glioma model was examined. Methods F98 gliomas were implanted into the cerebral cortex of 21 male Fischer 344 rats and resected after 7 days of tumor growth. After 2, 3, 7 or 14 days of surgery, FET was administered intravenously into the tail vein. Within 1 h after injection, coronal cryosections of the brains were taken and evaluated by FET autoradiography and histological stainings. Cerebral FET uptake was quantified by lesion to brain (L/B) or tumor to brain (T/B) ratios and compared with data on tumoral FET uptake in human glioblastomas. Results After tumor resection, all animals exhibited a slightly increased unspecific uptake of FET in the peritumoral area of resection. A maximum of unspecific FET uptake could be observed 3 days after surgery (L/B: 1.94 ± 0.26) which decreased significantly after 7-14 days (L/B: 1.57-1.39 ± 0.23-0.18; p<0.05). Compared to data on FET uptake in human glioblastomas, FET demonstrated a significant lower unspecific uptake in the vicinity of the surgical defect from the 7th day after surgery. At each time point, F98 rat tumors exhibited a significant higher tumoral FET uptake (4.4 ± 0.8; p<0.05) and could be separated from unspecific FET uptake in the vicinity of the resection area. Conclusions The results of this study demonstrate that the surgical resection of a tumor in rats causes a slightly increased uptake of FET in the vicinity of the resection area that can be clearly separated from tumor tissue. From the 7th day after surgery, unspecific FET uptake decreased significantly in rats and was considerably lower than that observed in human glioblastomas. Thus, it can be assumed that the correct identification of residual tumor tissue by FET PET and the planning of postoperative radiotherapy appear to be only little affected by unspecific FET uptake later than 7 days after tumor resection. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S358: Poster

21st International Symposium on Radiopharmaceutical Sciences

358 Good manufacturing practice compliant production of a 68Ga-labelled Affibody agent for breast cancer imaging: first-in-human Irina Velikyan1, 2, Anders Wennborg3, Joachim Feldwisch3, Anna Orlova2, Vladimir Tolmachev2, Henrik Lindman4, Jörgen Carlsson4, Jens Sörensen4 1 PET Centre, Uppsala University Hospital, Uppsala, Sweden, 2BMS, Department of Radiology, Oncology, and Radiation Sciences, Uppsala University, Uppsala, Sweden, 3Affibody AB, Solna, Sweden, 4Department of Radiology, Oncology, and Radiation Sciences, Uppsala University, Uppsala, Sweden Objectives The antibody based breast cancer treatment considers targeting human epidermal growth factor receptor type 2 (HER2). The introduction of a non-invasive and quantitative diagnostic method such as positron emission tomography (PET) for the pre-therapeutic determination of HER2 density would significantly improve patient management efficacy and treatment cost. The essential part of the PET methodology is the production of the imaging agent in compliance with good manufacturing practice (GMP) and good radiopharmacy practice (GRPP). Methods Affibody protein analogue, ABY025, was provided by Affibody AB. 68Ga was obtained from a 68 Ge/68Ga-generator. The eluate fractionation method was used for the production [1]. Time, temperature, radical scavenger, buffer, pH, product purification step, product formulation, and sterile filtration were optimized. Quality control was conducted using UV-Radio-HPLC. The patients received bolus intravenous administration. PET/CT study was performed twice with low (LD, 1 µg/kg) and high (HD, 6 µg/kg) protein doses. In each occasion dynamic acquisition during 0-45 minutes was followed by static images after 1, 2 and 4 hours. The study was approved by the Swedish Medicinal Products Agency (EU-nr 2012-005228-14) and local ethical committee. Results GMP-compliant, reliable and highly reproducible production with control over the product protein concentration and radioactivity was accomplished within one hour. The specific radioactivity could be kept similar throughout whole study due to the moderation of the radioactivity amount by the generator eluate pre-buffering. It was 6-fold higher for the LD preparation. Final product purification excluded contamination with 68Ge and provided radiochemical purity (RCP) of over 95%. Intrapatient comparison of the two protein doses allowed imaging optimization for the discrimination between HER2-positive and HER2-negative metastases. The HD decreased the uptake in the healthy tissue, in particular liver, improving image contrast and allowing detection of small metastases. The later imaging time points enhanced the contrast. Conclusions The production of 68Ga-ABY025 in two protein doses was developed and the resulting tracers were successfully used to investigate the influence of the protein dose on the biodistribution, pharmacokinetics, and dosimetry of the imaging agent as well as feasibility of the HER2 receptor expression quantification in patients affected by breast cancer. Acknowledgements References [1] Velikyan, I., et al., Bioconjugate Chem, 2004. 15(3): p. 554-60.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S359

359 Melanoma targeting with [99mTcN(PNP3)]-labeled α-MSH peptide analogs: Preliminary studies Feng Gao1, Davide Carta2, Nicola Salvarese3, Wiebke Sihver1, Hans-Jürgen Pietzsch1, Barbara Biondi4, Paolo Ruzza4, Fiorenzo Refosco5, Cristina Bolzati5 1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Dresden, Germany, 2 Department of Pharmaceutical Sciences, University of Padua, Padua, Italy, 3Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy, 4ICB-CNR, Padua, Italy, 5IENI-CNR, Padua, Italy Objectives The purpose of this study was to examine the effect of cyclization on the biological profiles of [99mTcN(PNP3)]-labeled α-MSH analogs. Methods The linear peptide H-Cys-Ahx-bAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) (1) and a corresponding lactam bridge-cyclized peptide, H-Cys-Ahx-bAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11Pro-Val-NH2 (NAP-NS2) (2), were synthesized, characterized by ESI-MS, and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16F10 melanoma cells. In vitro stability and pharmacological parameters of [99mTc(N)(NAP-NS1)(PNP3)]+ (1a) and [99mTc(N)(NAP-NS2)(PNP3)]+ (2a) were assessed. Challenges with an excess of glutathione and cysteine and LogD values were also investigated. Furthermore, 1a and 2a were applied to study in vivo stability and the pharmacokinetic profiles on healthy rats. Results 1a and 2a were obtained in high yield (RCY > 90%). LogD values demonstrated the hydrophilic nature of the radiolabeled peptides: -1.43 for 1a; - 2.09 for 2a. No significant variations in RCPs of both the complexes were observed. Both complexes showed high stability after incubation in human and rat sera as well as in rat liver homogenate. A fast degradation of 2a was detected in kidneys homogenate. 1a retained a high receptor affinity (Kd: 7.1±0.5 nM). Biodistribution of 1a displayed a favorable pharmacokinetic profile with fast blood clearance and elimination from normal tissues. Rapid renal excretion of 1a was observed due to the high hydrophilic character. The pharmacokinetic profile of 2a was reflected in reduction of the blood clearance and the elimination from the other organs; especially the kidneys showed restraint elimination. Conclusions Compared with the linear peptide 1, cyclization affected the pharmacological properties of 2 negatively by reducing its stability, its binding affinity to MC1Rs (Ki: 0.9±0.3 nM for 1; 7.1±2.4 nM for 2) and decreasing the overall excretion rate of the corresponding [99mTcN(PNP3)]-labeled peptide from the body. Thus, only the linear labeled peptide 1a will be considered for further investigations in tumor bearing mice. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S360: Poster

21st International Symposium on Radiopharmaceutical Sciences

360 Evaluation of various bifunctional chelators for the synthesis of radiometal-labeled CNS tracers Anett Schulz, Philipp Spang, Markus Piel, Frank Roesch Institut für Kernchemie, Johannes-Gutenberg Universität, Mainz, Germany Objectives Because of the strict requirements for BBB penetration, nearly all PET CNS tracers are labelled using non-metallic radionuclides (i.e. 11C and 18F). However, it would be desirable to make the favorable properties of radiometals, especially 68Ga, also available for neuroimaging with PET. Hence, different bifunctional chelators were synthesized and coupled to a well-established lipophilic lead compound. The properties of the resulting 68Galigand-compounds were examined systematically. Methods Different bifunctional chelators like HBED, NODAPA-OH and HB-TACN were synthesized in an asymmetrical manner, to obtain a phenolic moiety for coupling. The brain-penetrating lead compound was synthesized to contain a tosyl moiety as leaving group. Coupling of the chelator and the lead structure was performed via ether formation between the phenol group and the tosyl moiety. Afterwards the labeling reactions were examined and reaction parameters, such as pH, reaction temperature, buffer systems and precursor concentration, were optimized. For the 68Ga-labeled compounds stability studies in isotonic NaCl-, DTPA- and EDTA-solution and in human serum were performed. Finally, lipophilicities were determined both via HPLC and “shake flask” method. Results The syntheses of the bifunctional chelators were established in reaction sequences of 7 steps and finally coupled to the lead compound in yields of 5 to 79%. For the 68Ga-labeling reactions different parameters were examined, resulting in radiochemical yields of 87 to 97 % after optimization. During these optimizations, HBED and NODAPA-OH generally showed very fast kinetics with maximum yields after 1 minute, whilst for HB-TACN only slower kinetics were observed. The stability studies revealed a high stability of all compounds over 2 hours, showing > 95% of intact compound for HBED- and NODAPA-OH-derivatives and > 90% for the HB-TACNcompound. Lipophilicities of the compounds were determined at pH 7.4 with different methods, giving mean logD values ranging from 0.9 to 1.2. The logD value of the lipophilic lead compound is about 2.5. Conclusions Different bifunctional chelators were synthesized and coupled to a lipophilic lead compound. All derivatives were labeled in high yields and showed good stabilities and sufficient logD values. Due to these promising results first in vivo imaging studies are in preparation. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S361

361 Simple and highly efficient automatic synthesis of [18F]LBT-999 with protic solvent Cheol-min Yook1, Sang Ju Lee1, Jae Seung Kim1, Frédéric Dollé2, Seung Jun Oh1 1 Nuclear medicine, Asan medical center, Seoul, Korea (the Republic of), 2CEA, I2BM, Service Hospitalier Frédéric Joliot, Orsay, France Objectives [18F]LBT-999 is a cocaine analogue, structurally closely-related to [18F]FP-CIT [1], and a promising radiopharmaceutical for imaging with PET the dopamine transporter. [18F]FP-CIT can be manufactured with protic solvents and as such is widely used for clinical diagnosis of Parkinson’s disease in Korea since 2008. The process of preparation of [18F]LBT-999 was developed so far using the aprotic solvent DMSO [2,3]. The purpose of our study was to develop a new [18F]fluorination method of preparation of [18F]LBT-999 and to implement it on commercially available synthesizers for clinical application. Methods After trapping of 185 MBq/ 0.5 mL of aqueous [18F]Fluoride on a QMA cartridge, radioactivity was eluted with a KOMs/K222 mixture as buffer. As another method, we added TBAOH (6 µmol) to 185 MBq/0.5 mL of [18F]fluoride/H218O solution in the reactor without the use of any cartridge. After completely drying, [18F]fluorination was performed with 4 mg of the chlorinated derivative as precursor for labelling, dissolved in 100 µL of CH3CN and 1000 µL of t-amyl alcohol or DMSO, at 120°C or 140oC for 30 min for each condition. After [18F]fluorination, t-amyl alcohol was evaporated and DMSO removed using an SPE cartridge, respectively. The crude mixture was then purified with HPLC. HPLC purification conditions were 0.1 M ammonium acetate:CH3CN (40:60) with a flow rate of 4 mL/min. The processes were implemented on our synthesis modules according to GE TRACERLab manuals :TBAOH, w/o QMA on a FX module and KOMs/K222 on a MX module. Results The TBAOH (w/o QMA) method showed the highest [18F]fluorination yield with values as high as 90% in t-amyl alcohol. Using DMSO, the yield was much lower (18%). Interestingly, about 55% of [18F]fluorination yields were obtained with KOMs/K222 method in both solvent. Overall, 20% of radiochemical yields (n.d.c.) were obtained with a MX module. Lower yields (below 10%) were obtained with the FX module. Radiochemical purity was greater than 99% and specific radioactivity was about 110 GBq/µmol (3 Ci/µmol). Conclusions We developed a new and automatic synthesis method using a protic solvent for a reliable production of [18F]LBT-999. Our method showed high radiochemical yields and high reproducibility, and will enable [18F]LBT-999 to be used clinically and commercially. Acknowledgements References [1] Lee, S.J. et al (2007), Nucl. Med. Biol. 34, 345-351; [2] Dollé, F. et al (2006), J. Label. Compounds Radiopharm. 49, 687-698; [3] Dollé F et al. (2007), J. Label. Compounds Radiopharm. 50, 716-723.

J Label Compd Radiopharm 2015: 58: S1- S411

S362: Poster

21st International Symposium on Radiopharmaceutical Sciences

362 Preparation of [F-18]FLT using an ABT compact cyclotron and continuous flow microfluidics Hariprasad Gali2, Gregory Nkepang2, Wendy Galbraith2, Kelvin Hammond1, Vibhudutta Awasthi2, Lee Collier1 1 R&D, Advion Inc, Chester, New Hampshire, United States, 2Pharmaceutical Sciences, The University of Oklahoma College of Pharmacy, Oklahoma City, Oklahoma, United States Objectives The objective in this study was to determine if clinical doses of [F-18]FLT could be produced on a compact cyclotron using continuous-flow microfluidics. This combination can offer significant advantages for the preparation of PET radiotracers, including reduced cost of operation, lower starting activites, reduced precursor consumption, faster kinetics, rapid optimisation, high reproducibility, enhanced product yields and facile automation1. Methods Using the NanoTek microfluidic platform (Advion) and the compact cyclotron (BG-75, ABT), [F18]FLT was selected for the initial proof of concept. The compact cyclotron produces ~1 mCi / min of [F18]fluoride at beam current of 5 µA and produced the 70-80 mCi of [F-18]fluoride used in these studies in a volume of ~500 μl. The microfluidic method used in this study was the same as previously published2. The 3-NBoc-5′-O-dimethoxytrityl-3′-O-nosyl-thymidine precursor was used at a concentration of ~20 mg/ml (8.7 mg per synthesis). The reaction conditions used for the incorporation step were a flow rate of 200 µl/min through the reactor at 160°C using a 100 µm i.d. X 2 m reactor. The operating pressure of the reactor was between 200 and 225 psi to prevent boiling of the acetonitrile at the elevated temperatures. The intermediate formed was then hydrolyzed using 2N HCl at 100○C for 5 minutes, neutralized with 0.2M sodium phosphate, filtered through a 5 μm and then purified by HPLC. The product was then analyszed using standard QC techniques (radioTLC, radioHPLC, GC etc). Results Starting with 70-80 mCi of [18F]-fluoride, the radiochemical yield of [18F]FLT was 8 ± 1% (n=3, not decay corrected) after semi-prep HPLC, with a total synthesis time of 66 minutes. The QC of the purified product was found to be within the limits specified for human use. Conclusions This work has demonstrated the feasability of using microfluidics and a compact cyclotron to rapidly radiolabel and purify 3’-deoxy-3’-[18F]fluoro-thymidine (FLT) in sufficient quantity and purity for human clinical trial use. Further optimization of the system is underway. Acknowledgements References 1. Rensch, C. et al. (2013) Molecules, 18, 7930-7956. 2. Akula M et al, J Nucl Med. 2010; 51 (Supplement 2):1473

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S363

363 A 99mTc-labeled antibody for the dog EGF-receptor - a preclinical evaluation Christina Rami-Mark1, 2, Judit Fazekas3, 4, Josef Singer3, 1, Michael Willmann5, Wolfgang Wadsak1, 2, Erika JensenJarolim3, 4, Markus Mitterhauser1 1 Biomedical Imaging and Image Guided Therapy, Medical University of Vienna, Vienna, Austria, 2Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria, 3Department for Comparative Immunology and Oncology, Institute of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria, 4 Comparative Medicine, Messerli Research Institute, University of Veterinary Medicine Vienna, Vienna, Austria, 5 Department for Companion Animals and Horses, University of Veterinary Medicine, Vienna, Austria Objectives Overexpression of the epidermal growth factor receptor (EGFR) plays a pivotal role in various tumors[1]. Cetuximab is a chimeric antibody targeting EGFR, used as anticancer and antimetastatic treatment in humans. A caninized Cetuximab-derived antibody for the EGFR (CAN-225) was synthesized recently[1] and functionalized with DTPA for radiolabeling (Pichem, Austria), enabling in vivo studies in dog patients. Aim of this work was the in vitro testing of the tracer [email protected] regarding its binding properties to EGFR, plasma stability and autoradiography on canine EGFR+ tumors. Methods Stability of [email protected] was tested in dog serum for up to 4h at 37°C. Binding properties were determined using Western blot (nitrocellulose membranes were blotted with canine and human EGFR, Her-2 and BSA). Membranes were incubated with the radiolabeled antibody in milk powder for 1 h. After washing with TBST, membranes were placed on a phosphorimager film. For autoradiography, 20µm cryo-slices of 3 different canine EGFR+ tumors (IHC-verified) were incubated with [email protected] in TRIS + 1%BSA. After 1h incubation, the solution was discarded and the slices were washed in cold buffer and water. After drying, slices were placed on phosphor imager films for 12h. Results After 1h incubation in dog serum 60,5 ±1.8 % of the [email protected] was still intact, after 4h 30,2 ±2,3% remained unmetabolized. Western blot revealed only bands at EGFR binding pockets (human and canine EGFR), no binding to BSA or Her-2 was observed. In vitro autoradiography revealed uptake in EGFR specific tumor regions. Additionally, Nissl staining of the same slices was done to correlate hot areas. These findings were in concordance with IHC staining of EGFR. Conclusions Stability of the radiolabeled 99mTc-DTPA-CAN-225 proved to be sufficient. Binding properties were found to be conserved in Western blot and autoradiography on canine breast cancer samples. A step towards a clinical application of the radiolabeled antibody in dog patients is made. Acknowledgements References [1]Mitri, Z.et al.(2012)Chemother Res Pract:743193. [2]Singer, J.et al. (2014)Molecular cancer therapeutics13:1777-90.

J Label Compd Radiopharm 2015: 58: S1- S411

S364: Poster

21st International Symposium on Radiopharmaceutical Sciences

364 Facile synthesis of 6-[18F]FDOPA using electrophilic substitution reaction Yong-Hyun Cho2, Yun-Sang Lee1, Jae-Hyung Lee2, Yong-Suk Lee2, Hong-Jae Lee2, Jin-Eu Kim2, Keon Wook Kang1, Jae M. Jeong1 1 Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea (the Republic of), 2Nuclear Medicine, Seoul National University Hospital, Seoul, Korea (the Republic of) Objectives [18F]FDOPA has been synthesized using CFCl3 as a solvent for electrophilic substitution reaction because of high yield and convenience. However, many laboratories replaced CFCl3 with other solvents such as CHCl3, CDCl3, CH2Cl2, or CD2Cl2 since the effect of Montreal Protocol [1-3]. We tried to set up a procedure for the most simple and high yield using CDCl3 as a solvent. Methods We used an automated synthesizer Fx-Fn (GE, U.S.A.) for production. [18F]F2 gas was produced by 20 Ne(d,α)18F reaction in the presence of 0.1% F2 gas using a cyclotron and was passed through the reaction vial containing 30 mg precursor in 1 mL CDCl3 at room temperature. After purging for 10 min, solvent was evaporated by raising temperature to 130 °C and further 30 sec purging. Hydrolysis was performed using 0.6 mL HBr (48%) at 130 °C for 10 min. The reaction mixture was neutralized with 0.6 mL NH4OH (28%), and then was injected into the HPLC equipped with a semi-prep reverse phase column (Fig. 1). Results Synthesis time including purification was less than 35 min and final yield was 19.3 ± 3.1% (no decay corrected). Specific activity was 20.2 ± 4.6 GBq/ mmol. HPLC eluent was in 0.5% acetic acid. We added 0.5 mL 8.6% NaHCO3 to neutralize it before injection into the patients. [18F]FDOPA was stable for at least 2 h at room temperature. Conclusions We successfully developed a high yield, simple and fast electrophilic synthesis method for [18F]FDOPA. Acknowledgements This research was supported by grants NRF-2013R1A2A1A05006227 and NRF2012M2A2A7035853 funded by MEST. References [1] De Vries EFJ, et al (1999), Appl Radiat Isot, 51, 389-94. [2] Fűchtner F, et al (2008), Nuklearmedizin, 47, 62-4. [3] Forsback S, et al (2009), J Label Compd Radiopharm, 52, 286-8.

HPLC profiles of before (upper) and after (lower) hydrolysis.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S365

365 Preparation and Characterization of Dendrimer-Encapsulated Ytterbium Nanoparticles to produce a New Nano- Radio Pharmaceutical Navideh Aghaei Amirkhizi1, Soodeh Sadat Sadjadi2, Leila Moghaddam Banaem2, mitra Athari1, Fariba Johari Daha2 1 Department of Nuclear engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran (the Islamic Republic of), 2Nuclear Science and Technology Research Institute, Tehran, Iran (the Islamic Republic of) Objectives Preparation dendrimer encapsulated ytterbium nanoparticles to produce a nano- radio pharmaceutical therapeutic for solid tumors Methods Dendrimers are good candidates for preparing metal nanoparticles because they can structurally and chemically well-defined templates and robust stabilizers. Poly amidoamine (PAMAM) dendrimer-based multifunctional cancer therapeutic conjugates have been designed and synthesized in pharmaceutical industry. For preparation of dendimer-metal nanocomposite, A dendrimer solution containing an average of 55 Yb+3 ions per dendrimer was prepared . Prior to reduction, the pH of this solution was adjusted to 7.5 using NaOH. NaBH4 was used to reduce the dendrimer-encapsulated Yb+3 to the zerovalent metal. The pH of the resulting solution was then adjusted to 3, using HClO4, to decompose excess BH4-. the UV-Vis absorption spectra of the mixture were recorded to ensure the formation of Yb-G5-NH2 complex. High-resolution electron microscopy (HRTEM) and size distribution results provide additional information about dendimer-metal nanocomposite shape, size, and size distribution of the particles. The resulting mixture was irradiated in Tehran Research Reactor 2h. The specific activity was 7MBq. Radiochemical and chemical and radionuclide purity for quality control testes were carried. Gamma Spectroscopy and High-performance Liquid Chromatography HPLC, Thin-Layer Chromatography TLC were recorded. Results The results show the dendrimer-encapsulated ytterbium nanoparticles. The injection of resulting solution to solid tumor in mice shows that it could be resized the tumor. Conclusions Ytterbium encapsulated-dendrimer radiopharmaceutical could be introduced as a new therapeutic for the treatment of solid tumors. Acknowledgements References [1] Yong-Gu Kim, Sang-Keun Oh and Richard M. Crooks: Preparation and characterization of 1-2 nm dendrimer-encapsulated gold nanoparticles having very narrow size distributions, Chem. Matter.2004, 16, 167-172. [2] Mingqi Zhao and Richard M. Crooks: Intradendrimer exchange of metal nanoparticles, Chem. Mater. 1999, 11, 3379-3385.

J Label Compd Radiopharm 2015: 58: S1- S411

S366: Poster

21st International Symposium on Radiopharmaceutical Sciences

366 Preparation of 99mTc-BDTC complexes and evaluation as tumor marker Alyne E. Lafratta1, André G. Araújo Fernandes3, Victor M. Deflon2, Carlos A. Buchpiguel1, Fabio L. Navarro Marques1 1 Radiology, Medicine School - University of São Paulo, São Paulo, SP, Brazil, 2Chemistry, São Carlos Chemistry Institute - University of São Paulo, São Carlos, São Paulo, Brazil, 3Ciências Exatas e Tecnológicas, Universidade Estadual de Santa Cruz, Ilhéus, Bahia, Brazil Objectives Thiosemicarbazone and metal derivatives has been used for tumor therapy and, with radiometal, for diagnostic. In this work we have prepared two 99mTc complexes from 5-hydroxy-3-methyl-5-phenyl-pyrazol-1-(Sbenzyldithiocarbazate) (BDTC) under different reactional condition and evaluate them as marker for melanoma tumor cell. Methods 99mTc-BDTC was prepared by addition of 300 μg of BDTCin DMSO, 37-74 MBq of 99mTcO4- and 5 μg of SnCl2.2H2O at pH 7.2 reacted at 90oC for 5 min giving 99mTc-BDTC; the same preparation was done without heating giving [ReO(BDTC)2]-. Compounds were analyzed by HPLC (RP-C18, acetone:H2O:MeOH - 66:22:12), and by planar chromatography (PC) W3MM/acetone and W3MM/NaCl 0.9%. LogP were determined by partition coeficient in n-octanol/PBS. Uptake studies in tumor cells were performed in murine melanoma B16F10 and TM1M cells, at the time 15, 60 and 120 minutes. Washout was determined taking cell containing radiocomplex and changing medium free of radiocomplex at 15 and 60 min; at 120 min cells were separated from the medium and and the radioactivity was measured at this last one and the three medium fractions. Results Two complexes were obtained, one named 99mTc-BDTC with retention time at 2.3 min, in HPLC, and other named [99mTcO(BDTC)2]- with retention time of 10.17 min, in agreement with characterized structure [ReO(BDTC)2]-. PC confirm no existence of 99mTcO4- or 99mTcO2. LogP values were 1,41 and 1,01, respectively. A typical uptake value obtained for the compound 99mTc-BDTC and cell TM1M (15; 60; 120 min): 1.83±0.39; 0.83±0.22; 0.65±0.17; and for [99mTcO(BDTC)2]- and TM1M (15; 60; 120 min): 1.03±0.13; 0.75±0.06; 0.70±0.08. Statistical difference (t student - p<0,05) was observed for time comparisson between 15 min and 60 or 120 min; and for comparison among the two compounds at time of 15 min, for respective cell lines; now difference were observed between cell lines at correspondent times. Extrusion results were similar among the compounds and cells, with more than 60 % of the radioactivity left the cells after 15 min of incubation in a medium free of radiocomplexes. Conclusions We successfully prepared two new 99mTc-BDTC complex with high purity. Compounds showed low uptake in tumor cells and fast washout from them. In vivo biodistribution study must be performed to know full potentiality of these compounds. Acknowledgements We thanks CAPES for scholarship and FAPESP (14/22250-1) for funding support. References (1) Cipriani M. et al.Eur. J. Inorg. Chem. 2014, 4677–4689; (2) Manikandan R. et al. Inorganica Chimica Acta. 2014, 421: 80–90; (3) El-Gammal O.A. Mol. and Biomol. Spectr. 2015, 137: 207–219

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S367

367 Studying the Biological Potential of [99mTc(CO)3]-Dextran-Cysteine-Cysteine-Mannose (DCCM) as a feasible Molecular Radiopharmaceutical for Sentinel Node Detection Irfan U. Khan1, Abubaker Shahid1, Fayyaz Ahmad1, Ume-Kalsoom Dar2, Munir Ahmad1 1 Radiopharmacy and PET Radiochemistry Division, Institute of Nuclear Medicine and Oncology (INMOL), Lahore, Pakistan, 2Institute of Chemistry, University of the Punjab, Lahore, Pakistan

Objectives The objective of this work was to radiolabel and bioevaluate the technetium-99m labeled dextran dicysteine mannose (DCCM) - [99mTc(CO)3]-DCCM for sentinel lymph node detection. Methods Dextran dicysteine mannose was radiolabeled using the carbonyl method. Various parameters were studied such as in vitro stability at room temperature up to 5 h, protein binding and partition coefficient. Bioevaluation was performed in a rabbit model by developing images under a gamma camera at various time intervals. Biodistribution was performed in Wistar rat models (n = 3) by dissection and measurement of percent injected dose in various body organs, at 60 and 180 min post-injection intervals. Biodistribution was performed in two different groups of animals: in the first group, the radiolabeled compound was injected at a concentration of 200 lg/ml, thus delivering 10 lg radiolabeled compound at the site of injection; in the second group, the radiolabeled compound was injected at a concentration of 50 lg/ml, delivering 2.5 lg radiolabeled compound at the site of injection. Results Radiolabeling efficacy was 97.5 ± 1 % which remained quite stable till 5 h. Protein binding data show that 71.1 ± 5 % drug exhibited binding with blood proteins. Partition coefficient results show that our radiopharmaceutical is quite hydrophilic in nature. It can be inferred from the imaging data that sentinel node can be visualized within 30 min post-injection. Rat dissection data showed that when the radiolabeled compound was injected at a concentration of 50 lg/ml, at 60 min post-injection, ∼2.85 % of activity was retained in the sentinel node with a significantly less accumulation, e.g.,∼0.12 %, in the secondary node, which resulted in very high popliteal extraction (PE) value, e.g.,∼98 %. At 180 min post-injection, 2.46 ± 0.29 % was found to be retained in the sentinel node and PE (99.64 ± 0.23 %), thus resulting in almost complete washout from the secondary node (0.05 ± 0.01 %). Conclusions [99mTc(CO)3]-DCCM is specially designed for the SLN study. It can be concluded from the data that due to all potential and interesting properties of [99mTc(CO)3]-DCCM, it might prove a useful candidate for SLND in future clinical studies. Acknowledgements The authors highly acknowledge the efforts of radiopharmaceutics group at INMOL, Lahore, Pakistan, for performing this research work, especially the services of Mr. Khalid Mahmood, Senior Technologist, for assisting in the imaging and biodistribution studies. References 1. Mariani G, Moresco L, Viale G, Villa G, Bagnasco M, Canavese G, et al. Radioguided sentinel lymph node biopsy in breast cancer surgery. J Nucl Med. 2001;42:1198–215. 2. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol Rev. 2001; 53:283–318. 3. Chakera AH, Hesse B, Burak Z, Ballinger JR, Britten A, Caraco C, et al. EANM-EORTC general recommendations for sentinel node diagnostics in melanoma. Eur J Nucl Med Mol Imaging. 2009;36:1713– 42. 4. Wieler H, Bartenstein P, Becker HP, Bell E, Decker P, Jacob R, et al. Guideline for therapy of malign ant thyroid tumours. Nuklearmedizin. 2004;43:121–3. 5. Kogan MJ, Olmedo I, Hosta L, Guerrero AR, Cruz LJ, Albericio F. Peptides and metallic nanoparticles for biomedical applications. Nanomedicine (London). 2007;2(3):287–306. 6. Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small. 2005;1:325–7. 7. Goodman CM, McCusker CD, Yilmaz T, Rotello VM. Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjug Chem. 2004;15:897–900. 8. Wallace AM, Hoh CK, Vera DR, Darrah DD, Schulteis G. Lymphoseek: a molecular radiopharmaceutical for sentinel node detection. Ann Surg Oncol. 2003;10(5):531–8. 9. Mindt TL, Struthers H, Brans L, Anguelov T, Schweinsberg C, Maes V, et al. ‘‘Click to chelate’’: synthesis and installation of metal chelates into biomolecules in a single step. J Am Chem Soc. 2006;128:15096–7. 10. Alberto R, Ortner K, Wheatly N, Schibli R, Schubiger AP. Synthesis and properties of boranocarbonate: a convenient in situ co source for the aqueous preparation of [99mTc(OH2)3(CO)3]?. J Am Chem Soc. 2001;123:3135–6. 11. Alberto R, Schibli R, Egli A, Schubiger AP, Abram U, Kaden TA. Novel organometallic aqua complex of technetium for the labeling of biomolecules: synthesis of [99mTc(OH2)3(CO)3]? from [99mTcO4]- in aqueous solution and its reaction with a bifunctional ligand. J Am Chem Soc. 1998;120:7987–8. 12. Vera DR, Wallace AM, Hoh CK, Mattrey RF. A synthetic macromolecule for sentinel node detection: [99mTc]DTPAmannosyl-dextran. J Nucl Med. 2001;42:951–9. 13. Steer CJ, Ashwell G. Receptor-mediated endocytosis: mechanisms, biologic function, and molecular properties. 2nd ed. Philadelphia: WB Saunders; 1990. 14. Vera DR, Buonocore MH, Wisner ER, Katzberg RW, Stadalnik RC. A molecular receptor-binding contrast agent for magnetic resonance imaging of the liver. Acad Radiol. 1995;2:497–507.

J Label Compd Radiopharm 2015: 58: S1- S411

S368: Poster

21st International Symposium on Radiopharmaceutical Sciences

368 Synthesis and preclinical evaluation of the 99mTc-Bombesin-Folate heterobivalent peptide for simultaneous imaging of folate and gastrin-releasing peptide receptors Liliana Aranda Lara1, 2, Guillermina Ferro-Flores1, Flor de María Ramírez3, Blanca E. Ocampo-García1, Clara L. Santos-Cuevas1, Erika Azorin-Vega1, Keila Isaac-Olivé2 1 Materiales Radiactivos, Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Estado de Mexico, Mexico, 2 Fisica-Medica, Universidad Autonoma del Estado de Mexico, Toluca, Mexico, 3Quimica, Instituto Nacional de Investigaciones Nucleares, Ocoyoacac, Mexico Objectives The aim of this work was to synthesize Lys1(Folate)Lys3(99mTc-EDDA/HYNIC)-Bombesin (1-14) and to evaluate the in vitro and in vivo radiopharmaceutical potential for simultaneous detection of gastrin releasing peptide (GRP) and folate receptors overexpressed in ovarian and breast cancer cells. Methods Lys1Lys3(HYNIC)-Bombesin (1-14) was conjugated to the terminal carboxylic group of the (2S)-2-[[4[(2-amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid (folic acid) and purified by size exclusion HPLC. Chemical characterization was carried out by UV-Vis and FT-IR spectroscopy and MALDI-TOF mass spectrometry. 99mTc labeling was performed using ethylendiamine diacetic acid (EDDA)/tricine as coligands and SnCl2 as reducing agent. Radiochemical purity (RP) was determined by reversed-phase HPLC. In vitro binding studies were carried out in T47D (breast cancer) and CHO-K1 (ovarian cancer) cells. Biodistribution studies were accomplished in athymic mice with T47D- or CHO-K1-induced tumors with blocked and unblocked receptors. Molecular images (radioisotopic/X-ray imaging system, In vivo Xtreme, Bruker) in athymic mice with induced tumors were obtained at 2 h after radiopharmaceutical administration Results Spectroscopic studies and HPLC analyses indicated that the radioconjugate was obtained with high chemical and radiochemical purity (>94%). The in vitro results showed an important uptake of the radiopharmaceutical in the T47D cells and moderate uptake in CHO-K1 cells which was significantly inhibited by pre-incubation with cold folic acid or cold bombesin (Table 1). Biodistribution studies and images showed tumor uptakes of 1.94±0.41% I.D./g (CHO-K1) and 8.93±1.2% I.D./g (T47D) Conclusions Lys1(Folate)Lys3(99mTc-EDDA/HYNIC)-Bombesin may be useful as an imaging agent for breast tumors overexpressing GRPr as well as a SPECT agent to evaluate their metabolic rate (FR) [1] Acknowledgements This study was supported by CONACyT-Mexico (SEP-CB-2014-01-242443) References [1] Müller C and Schibli R, (2011) J. Nucl. Med. 52, 1-4

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S369

369 High output [18F]FDOPA production on AllInOne (Trasis) at commercial scale. Muhammad Otabashi1, Christian Cascione1, Christian Lemaire2, Jean-Luc Morelle1, Gautier Philippart1 1 Trasis SA, Ans, Belgium, 2Liege University, Liege, Belgium Objectives Effective commercial production of FDOPA requires producing large enough amounts, implying high yield at high activity. This objective, currently pursued by several groups, is not easily achieved. Lemaire et al have proposed a NCA enantioselective FDOPA synthetic route using a chiral phase-transfer catalyst (1,2). This method, optimized and implemented by Trasis on the AllInOne synthesizer, has proved to meet this goal Methods [18F]FDOPA, is prepared from the 6-Nitroveratraldehyde. This precursor reacts with dried [18F]fluoride to give 2-[18F]fluoro-4,5-dimethoxybenzaldehyde. This compound is purified on a tC18 cartridge, and then reduced to an alcohol with NaBH4. After this reduction, the hydroxyl group is iodinated with concentrated HI. The resulting iodinated compound is eluted with dichloromethane, purified and reacted with a Schiff's base in presence of a phase transfer catalyst. Alkylation reaction occurs and gives the protected [18F]FDOPA which is subsequently deprotected with concentrated HI at 160°C. Finally the crude solution is purified by HPLC to isolate the [18F]FDOPA and formulated for injection (figure 1). Results The average NDC yield gathered from over 10 users across the world is 38% at starting activities ranging from 100 to 400 GBq, with highest values around 45%. The radiochemical purity is higher than 99% and the enantiomeric purity always above 97%. The formulated product is stable over at least 16 hours Conclusions The FDOPA “Liege method”, automated and optimized by Trasis on an AllInOne synthesis module is appropriate for routine commercial scale purposes. Acknowledgements References 1. Lemaire, C., et al.,. J. Fluorine Chem., 2012. 138: p. 48-55. 2. Libert, L.,. Journal of Labelled Compounds and Radiopharmaceuticals, 2011. 54 (S1).

J Label Compd Radiopharm 2015: 58: S1- S411

S370: Poster

21st International Symposium on Radiopharmaceutical Sciences

370 Azeotropic drying free FDG synthesis and its application to a microfluidic platform Simon K. Lindner1, Christian Rensch2, Stephanie Neubaur1, Manuela Neumeier1, Ruben Salvamoser2, Victor Samper2, Stefan Riese3, Peter Bartenstein1 1 Department of Nuclear Medicine, University of Munich LMU, Munich, Bavaria, Germany, 2GE Global Research, Munich, Germany, 3GE Healthcare, Münster, Germany Objectives [18F]fluoride in an anhydrous condition is commonly considered to be a basic prerequisite for the 18Flabeling of PET radiopharmaceuticals. Azeotropic drying is the standard process for transferring aqueous [18F]fluoride into aprotic conditions and it requires the application of heat, a controlled gas stream, and reduced partial pressure to a reaction chamber. Implementing this method in microfluidics increases system complexity due to the challenges of the gas/liquid interface on a micro-scale or the integration of vapor permeable membranes. This study presents a very simple and time-saving cartridge-based drying technique that mitigates the challenges of the azeotropic drying process and reduces microfluidic hardware complexity. Based on the results of previous publications (1-4), a complete protocol utilizing protic solvents which tolerate high water content, is developed and applied to [18F]FDG synthesis. Subsequently, the method is transferred to a microfluidic synthesis platform. Methods Aqueous [18F]fluoride is trapped on a strong anion-exchange cartridge and dried with acetonitrile and air. Activity is eluted with a mixture of tetrabutylammonium phosphate dissolved in t-butanol and 1% water. The eluate is directly used for the radiolabeling of the precursor. Basic hydrolysis and final purification is done using a conventional solid phase extraction (SPE) protocol. After manual optimization, the process was transferred to a microfluidic synthesis platform. Results The manual process has been experimentally proven (n = 5) with an overall radiochemical yield (RCY) of 55% - 60% (d.c.) and a radiochemical purity (RCP) of >96%. The drying process is completed within 2 minutes reducing overall [18F]FDG synthesis time to less than 20 min. The applicability to a microfluidic platform has been demonstrated. Conclusions In this study, a new protocol for the synthesis of [18F]FDG in protic solvents and in the presence of water was established. The convenience of solvent exchange via a cartridge-based drying technique is presented as a significant advance in [18F]FDG synthesis protocols adapted for integration in microfluidic systems. Acknowledgements We gratefully acknowledge financial support by the German Federal Ministry of Education and Research and the Leading Edge Cluster “m4 – Personalized Medicine and Targeted Therapies”. References (1) Moon, D.H. et al., US patent US7847092B2. (2) Kim, D. W. et al., J. Am. Chem. Soc. 2006, 128(50), 16394-7. (3) Seo, J. W. et al., Bull. Korean Chem. Soc. 2011, 32(1), 71-76. (4) Wessmann, S. et al., Nuklearmedizin 2012, 51(1), 1-8.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S371

371 4S-(3-[18F]Fluoropropyl)-L-glutamic acid (FSPG): Biodistribution and tumor uptake in SLC7A11-KO mice Andre Mueller1, Norman Koglin1, Mathias Berndt1, Heribert Schmitt-Willich1, Hideyo Sato2, Volker Gekeler3, Christoph Schatz3, Andrew Stephens1, Matthias Friebe1, Ludger Dinkelborg1 1 Piramal Imaging, Berlin, Germany, 2Niigata University, Niigata, Japan, 3BayerHealthCare, Berlin, Germany Objectives The glutamate-cystine exchanger system xC- (a SLC7A11/SLC3A2 heterodimer) gains increasing interest in oncology, mediating oxidative stress and providing an additional growth and survival advantage for tumor cells. System xC- activity can be visualized with FSPG-PET, a 18F-labeled glutamate derivate.1 Knock-out (KO) of the SLC7A11 subunit in C57BL/6 mice was reported previously2. Aim of this study was to compare the FSPG biodistribution and tumor uptake in wildtype (wt) and in SLC7A11 KO mice and to investigate the physiological background of system xC- activity. Methods FSPG was radiolabeled via nucleophilic substitution of its N-Boc/OtBu-protected nosylate precursor as described previously1. 10 MBq in 100µl PBS were injected in wt C57BL/6, in SLC7A11-/- C57BL/6 mice and in the corresponding B16F10 tumor bearing animals. PET images were acquired from 50-60 min p.i. and quantitatively analyzed. After sacrificing the animals, pancreas tissue from wt and KO mice was removed and used to validate an IHC method for SLC7A11 detection with the NB300-318 antibody. Results C57BL/6 wt mice showed a similar biodistribution pattern as observed previously in rodents: fast washout and renal excretion with low uptake in most organs and tissues except pancreas, skin and the juvenile thymus. C57BL/6 SLC7A11 KO mice showed only kidney and bladder signal and no uptake in pancreas, skin and thymus. High B16F10 tumor uptake and good visualization was observed in wt and KO mice. IHC analysis revealed specific plasma membrane staining of mouse exocrine pancreas cells in wt tissue but no staining in KO tissue. Conclusions Analysis of FSPG biodistribution in KO mice confirmed uptake of FSPG in pancreas is caused by system xC- activity. Investigation of tumor-bearing KO mice further revealed that FSPG tumor-signal in the B16F10 model is derived from tumor-associated system xC- activity and not from potential additional uptake in tumor stroma. Elevated cystine plasma levels reported from KO mice (~2-fold increased) did not influence the FSPG tumor uptake indicating high efficiency of the transport mechanism and only a low to non-dependency of nutritional conditions. Therewith FSPG has the potential to be a robust and specific PET agent for system xCactivity, being further evaluated in clinical studies. Acknowledgements We are grateful to Joerg Jannsen, Eva Bickel, Ingo Horn, Marion Zerna, and Sabine Jabusch for their excellent technical assistance. References 1 Koglin et al., Clin.Cancer Res. 2011, 17:6000-11 2 Sato et al. J.Biol.Chem. 2005, 280:37423-29

J Label Compd Radiopharm 2015: 58: S1- S411

S372: Poster

21st International Symposium on Radiopharmaceutical Sciences

372 11 C-labeling and evaluation of E55888, a 5-HT7R agonistic PET tracer Matthias Herth1, Valdemar L. Andersen1, Hanne Hansen1, Szabolcs Lehel1, Jesper L. Kristensen2, Gitte Moos Knudsen1 1 Rigshospitalet, Copenhagen, Denmark, 2University of Copenhagen, Copenhagen, Denmark Objectives The serotonin 7 (5-HT7) receptor is the latest addition to the serotonin receptor family. It is implicated in various diseases, physiological functions and pathological conditions like thermoregulation, sleep physiology, memory, depression, anxiety or schizophrenia [1]. Several antagonistic tracers have recently been published [2,3,4]. This study aimed to develop the first 5-HT7 selective agonistic PET tracer. Methods The precursor and reference compound were synthesized by means of a Suzuki reaction. Radiolabeling succeeded with [11C]MeOTf (figure 1). Evaluation was conducted in Danish Landrace pigs. One baseline, one selfblock and one block study with SB-269970 were conducted. Results E55888 could successfully be synthesized and radiolabeled. E-55888 displayed a uniform brain distribution in vivo and could not be blocked applying a self-block or with a SB-269970, a known 5-HT7 selective compound (Figure 1). Conclusions [11C]E55888 is not a suitable, agonistic 5-HT7 PET tracer. Acknowledgements References [1] Zimmer et al. (2013) J. Label Compd. Radiopharm, 56, 105–113. [2] Herth et al. (2012) ACS Chem. Neurosci., 3, 1002−1007. [3] Hansen et al. (2014) J.Nuc.Med., 55, 640-646. [4] Herth et al. (2012) Bioorg. Med.Chem., 20, 4574–4581.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S373

373 Using anti CEA antibody [123I]HB8747 for luminal targeting of colorectal cancer Joost Verbeek3, 1, Meike de Wit 2, Charles Sio3, Iris verel3, Caren van Kammen3, Tilman Läppchen3, Albert D. Windhorst1, Katia Donato3, Pien Delis-van Diemen2, Gerrit Meijer2, Remond Fijneman2, Rolf Lamerichs3 1 Radiology and Nuclear medicine, VU University medical center, Amsterdam, Netherlands, 2Pathology, VU University medical center, Amsterdam, Netherlands, 3Minimally invasive healtcare, Philips research europe, Eindhoven, Netherlands Objectives Colonoscopy is the gold standard in the detection of colorectal cancer. However, it is a highly invasive technique and cannot distinguish between adenomas at low and high risk of progression, leading to overtreatment. Since colorectal adenomas are located at the luminal side of the colon, luminal targeting using a radiolabelled biomarker may provide a solution. This concept was tested in a mouse model in which colorectal tumors either or not express human CEA at their surface [1]. Methods The human anti-CEA antibody HB8747 was radiolabelled with Na[123I] using the Iodogen method. Immunoreactivity was assessed via a Lindmo assay. Next, [123I]HB8747 was administered rectally in human CEA positive or negative mice after a laxative prior to rectal administration. Tumor uptake was analyzed by in vivo SPECT/CT imaging, followed by analysis of biodistribution in the mice. Results [123I]HB8747 is synthesized in a yield of 34 ± 11% (N=9), and the immunoreactivity against human antiCEA expressing MKN45 cells was over 50%. Mice were administered 25 microgram 123I]HB8747 (4-7 MBq)rectally 60 minutes prior to the SPECT/CT scan (NanoSPECT-CT (Bioscan Inc., Washington, DC)), which was directly followed by biodistribution analysis. This resulted in CEA positive and CEA negative mice with varying tumour uptake. It appeared that the colon was not cleared completely in all tumour bearing mice, which may be due to compromised bowel function. The remaining faeces in the colon absorbed most of the administered [123I]HB8747. In addition, cleaning of the tumour during biodistribution was hampered by the rough tumour surface and faeces remaining’s near the tumour tissue may have led to false positives. Conclusions There was a variable tumour uptake of [123I]HB8747 in both CEA positive and negative mice, possibly due to incomplete bowel cleaning. We therefore could not determine whether luminal targeting in this model is feasible. Acknowledgements The authors would like to acknowledge Raquel Diaz-Lopez, Monique Berben, and Marije Jansen for experimental assistance. This study was funded by CTMM (grant 03O-101). References [1]Wilkinson RW et al. (2001) PNAS vol 98. No. 18, 10256-10260.

374 Comparative biodistribution of 99mTc-duramycin: influence of species, purification and kit composition Luca Palmieri1, 2, Filipe Elvas1, 2, Christel Vangestel2, Brian Gray3, Koon Pak3, Sigrid Stroobants1, 2, Steven Staelens2, Leonie wyffels1, 2

J Label Compd Radiopharm 2015: 58: S1- S411

S374: Poster

21st International Symposium on Radiopharmaceutical Sciences

1

Department of Nuclear Medicine, University Hospital Antwerp, Edegem, Belgium, 2Molecular Imaging Center Antwerp, University of Antwerp, Antwerp, Belgium, 3MTT, Inc., West Chester, Pennsylvania, United States

Objectives 99mTc-duramycin is a small radiopharmaceutical peptide that binds to phosphatidylethanolamine in dead and dying cells. In the current study we evaluated the influence of kit composition, purification and PEGylation of duramycin on the pharmacokinetic (PK) profile of 99mTc-duramycin in both mice and rats. Methods Four single-step kits were prepared (Table 1) and labeled by adding 20mCi of 99mTcO4- and heating to 80°C for 20min. RCP was assessed by radio-HPLC and TLC. 99mTc-duramycin was used without or with further purification (Oasis HLB Plus cartridge washed with H2O and eluted with 96% ethanol). The biodistribution profile of the different kits was evaluated in CD1 nude mice or Sprague-Dawley rats by iv injection of non purified or purified 99mTc-duramycin and sacrificing at 24h pi for ex-vivo biodistribution (n=3/condition). Results When using kit A, B and C 99mTc-duramycin was obtained with a RCP of 95% - 97%. HLB Plus cartridge purification increased RCP to >98%. For kit D RCP was only 87%. In vivo evaluation in mice demonstrated that for all the kits highest uptake was present in liver, spleen and kidneys. The impact of purification, PEGylation, kit composition and species is presented in Table 1. Conclusions HLB purification of kit prepared 99mTc-duramycin and reduction of kit ingredients had only a minor effect on the PK profile. PEGylation of duramycin on the other hand results in an improved PK profile with lower liver and spleen uptake in mice. The best PK profile was obtained in rats, which indicates species dependent uptake difference of 99mTc-duramycin. Acknowledgements References

Table 1. Composition and in vivo evaluation of kits (P=purified, NP= non purified).

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S375

375 Reproducibility of O-(2-[18F]fluoroethyl)-L-tyrosine kinetics in brain tumors and influence of dexamethasone therapy: A PET study in rat gliomas Carina Stegmayr, Antje Willuweit, Michael Schöneck, Christian Filss, Nadim J. Shah, Heinz H. Coenen, Johannes Ermert, Karl J. Langen Institut für Neurowissenschaften und Medizin, INM, Forschungszentrum Jülich, Jülich, Germany Objectives Previous studies have shown that uptake kinetics of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) in brain tumors correlates with the grade of malignancy. This study investigates the reproducibility of FET kinetics in rat gliomas and the influence of the commonly used dexamethasone (Dex) therapy. Methods Tumor cells were implanted in the striatum of 31 Fischer rats (15 rats with 9L gliosarcoma; 16 with F98 glioma). Dynamic FET PET (0-60 min post injection, p.i.) was performed after 10 days of tumor growth (baseline) and 48 h later (control) using animal PET (Inveon). Group 1 (n= 8 for 9L; n=10 for F98) received no treatment and group 2 (n= 7 for 9L; n=6 for F98) was injected with 8 mg/kg Dex intraperitoneal after baseline PET plus 4 mg/kg Dex 24 h and 1 h before control PET. Tumor to brain ratios (TBR) (18-60 min p.i.) and the slope of a linear fit of the time activity curve (15 to 60 min p.i.) were determined. Results The two tumor models showed different FET kinetics (9L slope: +0.17 ± 0.12 SUV/h; F98 slope: +0.76 ± 0.09 SUV/h; p < 0.001) and different TBR (9L: 1.87 ± 0.07; F98: 1.98 ± 0.07; p = 0.005). Both models showed no significant changes of slope in both groups. In all untreated gliomas, TBR slightly increased (9L: 1.87 ± 0.07 vs. 1.95 ± 0.08, p < 0.001; F98: 1.98 ± 0.07 vs. 2.04 ± 0.06, p = 0.002) from baseline to control scan, while the TBR in all Dex treated gliomas decreased (9L: 1.86 ± 0.03 vs. 1.65 ± 0.08, p < 0.001; F98: 1.99 ± 0.10 vs. 1.89 ± 0.15, p = 0.025). This decrease is due to enhanced FET accumulation in normal brain tissue after Dex treatment. Conclusions FET kinetics in cerebral gliomas show high reproducibility and little interference with Dex therapy. Thus, changes of FET kinetics may be considered reliable indicators of biological reactions occurring in brain tumors during radio/chemotherapy. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S376: Poster

21st International Symposium on Radiopharmaceutical Sciences

376 Radiochemical stability of 68Ga- and 177Lu-labeled high-affinity DOTATATE (DOTA-3-iodo-Tyr3octreotate) Dirk Mueller, Christian Breunig, Manfred Baehre, Andreas Odparlik Nuclear Medicine, University Hospital Halle(Saale), Halle(Saale), Germany Objectives Radiolabeled HA-DOTATATE (DOTA-3-iodo-Tyr3-octreotate) is increasingly used for diagnosis and treatment of neuroendocrine tumors (NET). HA-DOTATATE is not governed by patent restrictions, commercially available and therefore an alternative to other somatostatin-ligands. The objective of our study was to investigate the radiochemical stability of 68Ga- and 177Lu-labeled HA-DOTATATE in the clinical practice. Methods HA-DOTATATE was labeled with 68Ga using the NaCl based labeling procedure. In order to reduce radiolysis of 68Ga-HA-DOTATATE in the final product, ascorbic acid was added to the reaction mixture prior to the labeling. The labeling of HA-DOTATATE with 177Lu was carried out in 0.4M sodium acetate buffer at pH 5.5 under presence of 2,5-diydroxybenzoic acid (gentisic acid) as radical scavenger. A purification of the radiometallabeled HA-DOTATATE using a C-18 cartridge was not required in both cases. The radiochemical purity was determined by radio-HPLC (RP-18, gradient water-acetonitrile). The radiolabeled peptides were stored at room temperature. Results The radiolysis of the 68Ga-labeled peptide could not be observed and 68Ga-HA-DOTATATE is stable within four hours. Because of the halflife of 68Ga, the activity of the radiopharmaceutical after more than four hours is usually lower than the minimum patient dose. The radiolysis of 177Lu-HA-DOTATATE could be detected after 12h and was significantly observed after 24h. The concentration of the radiolabeled peptide was then lower than 95%. Within 48h the concentration of radiolysis products increased to 35% of the total activity. Conclusions Our results showed that 68Ga labeled HA-DOTATATE is stable for four hours at room temperature and 177Lu-HA-DOTATATE can be used within 12h after the labeling. Acknowledgements References Brogsitter C, Zöphel K, Hartmann H, Schottelius M, Wester HJ, Kotzerke J, Twins in spirit part II: DOTATATE and high-affinity DOTATATE--the clinical experience. Eur J Nucl Med Mol Imaging. 2014 41(6):1158-65 Mueller D, Klette I, Baum R P, Gottschaldt M, Schultz M K, Breeman W A P. Simplified NaCl Based 68Ga Concentration and Labeling Procedure for Rapid Synthesis of 68Ga Radiopharmaceuticals in High Radiochemical Purity. Bioconjug Chem. 2012 23(8):1712-7.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S377

377 Synthesis and evaluation of 99mTc labeled metronidazole analogs HYNIC-ADIBO-MN via copper-free click chemistry for tumor hypoxia imaging Liqin Liu, Yuan Yan Qin, Yang Yang Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing , China Objectives While conventional copper(I)-catalyzed click chemistry has become commonplace in drug development[1], the use of metal catalyst often limits the utility of the method. Copper ions are cytotoxic, can cause degradation of DNA molecules, and induce protein denaturation[2, 3]. In this study, we prepared new 99mTc labeled metronidazole analogs HYNIC-ADIBO-MN via metal-free click chemistry and evaluated 99mTc-HYNICADIBO-MN as a potential tumor hypoxia imaging agent. Methods Metronidazole analogs HYNIC-ADIBO-MN was synthesized successfully via copper-free click chemistry by multi–step reaction (Fig. 1). 99mTc-HYNIC-ADIBO-MN was prepared by labeled with 99mTc at 100°C and pH 7 using tricine (50mg) and EDDA (1 mg) as coligands. Biodistribution studies were performed in Kunming mice bearing S180 tumor. Results The new complexes of HYNIC-ADIBO-MN was synthesized and radiolabeled with 99mTc to form the 99m Tc-HYNIC-ADIBO-MN complex in high yield (>92%). The tumor cell experiment showed that the 99mTcHYNIC-ADIBO-MN complexes had certain hypoxic selectivity. The biodistribution studies of 99mTc-HYNICADIBO-MN in Kunming mice bearing S180 tumor showed relatively high tumor uptake, and low accumulation in non-target organs, suggesting 99mTc-HYNIC-ADIBO-MN would be a novel potential tumor hypoxia imaging agent. Conclusions The novel ligand HYNIC-ADIBO-MN had been successfully synthesized via copper-free click chemistry and 99mTc-HYNIC-ADIBO-MN was prepared in high yields. 99mTc-HYNIC-ADIBO-MN showed relatively high tumor uptake, good retention and relatively high target to non-target ratios in tumor-bearing mice, suggesting that it could be potentially useful for tumor hypoxia imaging agent. Acknowledgements The work was financially supported by the Fundamental Research Funds for the Central Universities of China (FRF-BR-12-017) and the National Natural Science Foundation of China (81301319). References [1] Sharpless, KB, et al (2006), Expert Opin Drug Discov, 1, 525-538. [2] Gaetke, LM, et al (2003), Toxicology, 189, 147-163. [3] Burrows, CJ, et al (1998), Chem Rev, 98, 1109-1152.

Figure 1. Synthesis of HYNIC-ADIBO-MN via copper-free click chemistry. (1) NH2OH·HCl, pyridine; (2) PPA, 125 °C; (3) LiAlH4, ether; (4) 6-Chloronicotinoyl Chloride, pyridine, CH2Cl2; (5) Pyridinium tribromide, rt; (6) tBuOK, THF, rt; (7) NH2NH2·H2O, 85 °C; (8) (BuOCO)2O/Et3N, DMF, rt; (9) MN-N3, rt; (10) 3 M HCl, rt.

J Label Compd Radiopharm 2015: 58: S1- S411

S378: Poster

21st International Symposium on Radiopharmaceutical Sciences

378 [99mTc]ZOL - First in vivo imaging in dogs Daniela Haeusler1, Maximilian Pagitz2, Merete Tschokert1, Michael Willmann2, Julia Sommer1, Sonja Kappel3, Wolfgang Wadsak1, Marcus Hacker1, Markus Mitterhauser1 1 Dept of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Vienna, Austria, 2 Dept for Companion Animals and Horses, Veterinary University of Vienna, Vienna, Austria, 3Dept of Surgery, Medical University of Vienna, Vienna, Austria Objectives Zoledronic acid (ZOL) is known for its antitumor characteristics [1] and significantly reduced lung metastases in an orthotopic osteosarcoma (OS) model [2]. Hence, we hypothesize that ZOL, labeled with 99mTc, will add value as a bone seeker -since the gold standards for bone imaging lack sensitivity for the specific imaging of lung metastases. Considering that lung metastases decrease the 5-year-survival rate of OS-patients to 25%, a specific tracer for their visualisation would be crucial. Methods ZOL was labeled [3] with 99mTc (n=4) and examined for bone binding [4] (n=4) and for in vitro autoradiography on 10µm cryo-tissues of lung metastases of human OS-patients (n=2). 20MBq/kg bw [99mTc]ZOL and [99mTc]MPD were administered to healthy dogs (Beagles, n=3 each); after 180min of in vivo distribution, a wholebody scan were performed. Results Successful radiolabeling (98±2%) allowed binding to hydroxyl apatite, amorphous calcium phosphate, human diaphysis and human cancellous bone (Fig 1a). [99mTc]ZOL was selectively displaced by unlabeled ZOL on tumor tissues (41.4±7%, p=0.0016, no uptake on control tissue). [99mTc]DPD was not displaced with unabeled DPD (3,1±4%). First in vivo imaging in dogs with [99mTc]ZOL and [99mTc]MPD revealed excellent bone uptake of [99mTc]ZOL (Fig 1a,b). Conclusions [99mTc]ZOL uptake on lung metastases tissues of human OS-patients was selective and displaceable. [99mTc]ZOL showed bone uptake in vitro and in vivo. Therefore, we hypothesize that [99mTc]ZOL will be a valuable and specific tracer for the imaging of OS with lung metastases. Since OS is a highly comparable disease in humans and dogs [5], [99mTc]ZOL will be evaluated in veterinary OS-patients with lung metastases. Acknowledgements This project is sponsored by the Austrian Science Fund (FWF-T553-B19) awarded to D. Haeusler. References [1] Gronich N, et al (2013) Nat Rev Clin Oncol, 10, 625-42. [2] Koto K, et al. (2009) Cancer Lett, 274, 271-278. [3] Haeusler D, et al. (2013) J Label Compd Radiopharm, 56, S423. [4] Mitterhauser M, et al. (2004) Bone, 34, 835-44. [5] Withrow SJ, et al. (2010) ILAR Journal, 51, 208-13.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S379

379 Synthesis and evaluation of two novel Tc-99m-labeled lactam bridge-cyclized melanocortin peptides for melanoma SPECT imaging Vania Teixeira1, Xiuli Zhang2, 3, Fabio Gallazzi4, Williams Porcal5, Juan Pablo Gambini6, Pablo Cabral1, Thomas P. Quinn2, 3 1 Radiofarmacia, Universidad de la Republica, Montevideo, Montevideo, Uruguay, 2Biochemistry Department, University of Missouri, Columbia, Missouri, United States, 3Truman Memorial Veterans’ Hospital, Columbia, Missouri, United States, 4Research Cores , University of Missouri, Columbia, Missouri, United States, 5Química Orgánica, Universidad de la República, Montevideo, Montevideo, Uruguay, 6Centro de Medicina Nuclear, Hospital de Clínicas., Universidad de la República, Montevideo, Montevideo, Uruguay Objectives The aims of this work were to synthesize and evaluate the melanoma targeting and imaging properties of two novel new 99mTc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues using a tetraamine system as a bifunctional chelating agent Methods The peptides Tetraamine-GGNle-CycMSHhex and Tetraamine-Ahx-Nle-CycMSHhex-GPV were synthesized using Fmoc solid phase synthesis [1]. Their melanocortin-1 receptor (MC1R) binding affinities were determined in B16/F1 melanoma cells. The peptides were labeled with 99mTc under alkaline conditions (pH 11.5) in presence of citrate and stannous chloride. Radiochemical purity, stability and the partition coefficient (Log P) were determined. In vitro B16/F1 cell binding, internalization and blocking studies were examined. In vivo SPECT/CT imaging studies were performed in B16/F1 melanoma tumor bearing C57 mice at 2 h post injection. Results The IC50 values of Tetraamine-GGNle-CycMSHhex and Tetraamine-Ahx-Nle-CycMSHhex-GPV were 1.7±0.05 nM and 0.7±0.04 nM, respectively. The radiochemical purities of 99mTc-(Tetraamine)-GGNle-CycMSHhex and 99mTc-(Tetraamine)-Ahx-Nle-CycMSHhex-GPV were over 90%, with Log P values of -0.42±0.05 and 1.17±0.19, respectively. Both compounds were stable and internalized by B16/F1 melanoma cells. In vivo SPECT/CT imaging studies showed tumor selective uptake for 99mTc-(Tetraamine)-Ahx-Nle-CycMSHhex-GPV, which was confirmed by competitive blocking. However, the 99mTc-(Tetraamine)-GGNle-CycMSHhex exhibited no tumor uptake. Conclusions Tetraamine-GGNle-CycMSHhex and Tetraamine-Ahx-Nle-CycMSHhex-GPV were synthesized and radiolabeled with 99mTc with high purity and yield. The radiolabeled peptides showed in vitro selective MC1R targeting on melanoma cells, however, in vivo SPECT/CT imaging showed tumor uptake for only the 99mTc(Tetraamine)-Ahx-Nle-CycMSHhex-GPV compound. Acknowledgements ANII, PEDECIBA-QUIMICA, CHLCC References 1-Guo H, et al (2010) J Nucl Med, 51, 418-26

J Label Compd Radiopharm 2015: 58: S1- S411

S380: Poster

21st International Symposium on Radiopharmaceutical Sciences

380 Reactivity and structure relations in Mn(I), Re(I) and Tc(I)-tricarbonyl complexes – impact on drug design. Hendrik G. Visser1, Andreas Roodt1, Marietjie Schutte1, Alice Brink1, Thembani Twala1, Gerdus Kemp2, Fabio Zobi3 1 Chemistry, University of the Free State, Bloemfontein, Free State, South Africa, 2Petlabs, Pretoria, South Africa, 3 Chemistry, University of Fribourg, Fribourg, Switzerland Objectives Objectives: 99mTc(I)-tricarbonyl complexes are investigated for their potential use as radiopharmaceuticals. Its third row congener, Re, also has applications as either therapeutic or diagnostic or both. “Cold” rhenium and manganese complexes were used as models for technetium. The objectives of this study was to investigate the effects of coordinated bidentate ligands on the rate of substitution of methanol or water by a range of entering ligands including halides, N and S-donating ligands, using various techniques including X-ray crystallography, UV-vis and stopped-flow spectroscopy. The typical metal complexes used were [M(CO)3(Bid)X]n (M = Mn/Re; Bid = N-N’, N-O’, O-O’ bidentate ligands as well as the protein, HEW lysozyme; X = MeOH/H2O and n = 0/+1) [1 - 6]. Methods Normal procedures were followed for all the complexes synthesized. Confirmation of structures were performed by using IR, UV-vis, elemental analysis, NMR and X-ray Crystallography. The kinetics were performed on a normal UV-vis spectrophotometer (slow reactions) or by using a stopped flow device with a dead time of only 2 ms(fast reactions). Results Results: In general, the rate of substitution decreases from Mn > Tc > Re down the group of the periodic table. This was also observed in our studies. Most importantly, an expected increase of about 20 000 times was observed for methanol substitution in [Re(CO)3(Bid)X]n just by changing the bidentate ligand (Bid) from N-N’ donor ligands to O-O’ type ligands. The intimate mechanism suggests an Id type mechanism, meaning that the dissociation of ligands bound to the central Tc/Re determines the rate of labelling. Conclusions Conclusions: The greater significance of our results is that bidentate ligands can labilize the metal center and that this work should be further expanded to explore other ligand systems, but even more significant is the increased affinity of Re(I) for hard nucleophiles like pyridine, indicitave of the influence of the bidentate ligands. This is also the first study to our knowledge where the effect of a protein coordinated to a metal was evaluated kinetically. Acknowledgements Acknowledgements: The UFS, SASOL, PETLabs, SA-NRF/THRIP for financial support. References [1] Brink, A. et al (2013) Inorg. Chem., 52, pp 8950–8961. [2] Brink, A. et al (2014) Inorg. Chem., 53, pp 12480–12488. [3] Schutte, M. et al (2012) Inorg. Chem., 51 , pp 11996–12006. [4] Schutte, M. et al (2011) Inorg. Chem., 50 , pp 12486–12498. [5] Schutte, M. et al (2015) Dalton Trans., 44, pp 3278-3288. [6] Zobi, F. et al (2007), Inorg. Chem, 46, pp. 10458-10460.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S381

381 In vivo imaging and characterization of [18F]DPA-714, a potential new TSPO ligand, in mouse brain and peripheral tissues using small-animal PET Caterina Vicidomini1, 2, Mariarosaria Panico1, 2, Adelaide Greco4, 2, Sara Gargiulo1, 2, Anna Rita Daniela Coda1, 2, Antonella Zanetti1, 2, Matteo Gramanzini1, 2, Giovanni N. Roviello1, Mario Quarantelli1, Bruno Alfano1, Bertrand Tavitian5, 3, Frédéric Dollé3, Annelaure Damont3, Marco Salvatore4, Arturo Brunetti4, 2, Sabina Pappata1 1 Institute of Biostructure and Bioimaging, CNR, Naples, Italy, 2Biotecnologie Avanzate, s.c.a.r.l, CEINGE, Naples, Italy, 3Service Hospitalier Frederic Joliot, CEA, I2BM, Orsay, France, 4Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy, 5Hospital European Georges Pompidou, Inserm U970, PARCC Universite Paris Descartes, Paris, France Objectives The translocator protein 18 kDa (TSPO), a biochemical marker of neuroinflammation, is highly expressed in the brain activated microglia and it is also expressed by peripheral inflammatory cells and normal peripheral tissues. Thus, development of radioligands for the TSPO may contribute to further understanding of the in vivo TSPO function in central and peripheral inflammatory processes and other pathologies. Here, we report the biodistribution, the specific binding and the radiometabolites of [18F]DPA-714, a promising fluorinated PET radiotracer, in normal mice using a microPET/CT scanner. Methods The in vivo biodistribution and kinetics of [18F]DPA-714 were measured in mice brain and peripheral tissues. Specific binding to TSPO sites was assessed using pharmacological competitive studies by means of saturation experiments performed by i.v. injection of 1 mg/kg of unlabeled DPA-714 or 3 mg/kg of unlabeled PK11195. A region of interest analysis was performed to generate time-activity curves in the brain, heart, lung, kidney, spleen and liver. Metabolites assay was performed in the plasma and peripheral organs by radio-HPLC. Results [18F]DPA-714 reached high concentration in lung, heart, kidney and spleen, tissues well known to be rich in TSPO sites. [18F]DPA-714 kinetics were faster in the lung and slower in the kidney. Pre-injection of unlabeled DPA-714 or PK11195 inhibited about 80% of [18F]DPA-714 uptake in the lung and heart (p<0.0005). The percentage of inhibition in the kidney was lower and achieved at later times only with DPA-714 (p<0.05) but not with PK11195. Sixty minutes after radiotracer injection only unmetabolized radioligand was found in the brain, lung, heart and spleen. Conclusions These results suggest that [18F]DPA-714 is a suitable PET ligand for imaging in mice brain and peripheral tissues since it binds with high specificity TSPO binding sites and it is almost unchanged at 60 minutes after radiotracer injection in the brain and TSPO-rich regions. Acknowledgements References [1] Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapère JJ, Lindemann P,et al. Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci. 2006;27(8):402-9. [2] Venneti S, Lopresti BJ, Wiley CA. Molecular imaging of microglia/macrophages in the brain. Glia. 2013;61(1):10-23. [3] Chen MK, Guilarte TR. Translocator protein 18 kDa (TSPO): molecular sensor of brain injury and repair. Pharmacol Ther. 2008; 118(1): 1–17. [4] Jacobs AH, Tavitian B; INMiND consortium. Noninvasive molecular imaging of neuroinflammation. J Cereb Blood Flow Metab. 2012;32(7):1393-415.

J Label Compd Radiopharm 2015: 58: S1- S411

S382: Poster

21st International Symposium on Radiopharmaceutical Sciences

382 Metabolism study of radiolabeled hydrazinocurcumin derivative Sarah Shin1, 2, Hyun-Jung Koo1, 2, Yearn Seong Choe1, 2, Joon Young Choi1, 2, Kyung-Han Lee1, 2, Byung-Tae Kim1, 2 1

Department of Nuclear Medicine, Samsung Medical Center, Seoul, Korea (the Republic of), 2Sungkyunkwan University School of Medicine , Seoul, Korea (the Republic of)

Objectives Curcumin is known to have diverse biological activity, however it is shown to undergo rapid metabolism in the liver and intestine wall via reduction of vinyl groups and conjugation reactions [1]. As an alternative, hydrazinocurcumin (HC) was developed and shown to have anti-angiogenesis activity [2]. However, metabolism of HC has not been fully understood. In this study, we radiolabeled a HC derivative with 18F ([18F]1), evaluated its metabolism, and compared the results with those of curcumin. Methods Non-radioactive ligand 1 was synthesized from fluoroethoxyethylcurcumin and NH2NH2, and fluoroethoxyethylcurcumin was synthesized in 2 steps using aldol condensation reactions. Radioligand [18F]1 was synthesized by 18F-labeling of the tosylate precursor followed by removal of protecting groups. For the in vitro metabolism study using alcohol dehydrogenase (ADH), which is known to be responsible for the reductive metabolism of curcumin, 1 and curcumin were incubated with ADH in the presence of NADPH at 37 oC for 1 h, and then the mixture was analyzed by HPLC. In vivo metabolism study was performed using ICR mice injected with [18F]1. At the indicated time points, mice were sacrificed, and samples of blood and liver were obtained and analyzed by HPLC. In order to identify the radiometabolites, the liver samples were incubated with βglucuronidase and analyzed by HPLC. Results [18F]1 was synthesized in good yield with high specific activity. In vitro reductive metabolism study demonstrated that curcumin was mostly converted into tetrahydrocurcumin, whereas HC derivative, 1 remained intact. In vivo metabolism study of [18F]1 showed that there were no major radiometabolite peaks in the blood samples, whereas two radiometabolite peaks (m1 and m2) as well as the parent peak appeared in the HPLC chromatograms of the liver samples. When the liver samples were incubated with β-glucuronidase, the radiometabolite m1 decreased and the parent peak ([18F]1) increased, indicating that m1 is [18F]1-glucuronide. Based on the HPLC data of the known metabolites of curcumin [3], it is likely that m2 is [18F]1-sulfate. Conclusions This study suggests that HC does not undergo reductive metabolism but undergoes phase II metabolism (conjugation), probably due to the presence of the hydrazine moiety, while curcumin undergoes both metabolic pathways. These results can be widely used in designing metabolism-resistant curcumin derivatives for tumor imaging. Acknowledgements Supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2011-0030164). References [1] Anand P, et al (2007) Mol Pharmaceut, 4, 807-818. [2] Shim JS, et al (2002) Bioorg Med Chem, 10, 2439-2444. [3] Ireson C, et al (2001) Cancer Res, 61, 1058-1064.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S383

383 Chemical Conversion of 11CO2 to 11CO via Silacarboxylic Acids: Applications in Palladium-Mediated Carbonylations Patrik Nordeman1, Stig D. Friis2, Thomas L. Andersen2, Hélène Audrain3, Mats Larhed1, Troels Skrydstrup2, Gunnar Antoni1 1 Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden, Uppsala, Sweden, 2The Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus, Denmark, 3Department of Nuclear Medicine and PET Center, Aarhus University Hospital, Aarhus, Denmark Objectives The objectives of this study were to investigate the chemical reduction of 11CO2 to 11CO by using 11Csilcarboxylic acids and to use this carbon monoxide releasing molecule in palladium-mediated 11C-carbonylative radiosynthesis. Methods The reaction setup consisted of one dewar and two vials. In the first vial 11CO2 was initially captured and converted into 11C-silacarboxylic acid whereafter the 11CO was released by the using tetrabutylammonium fluoride. The 11CO was then transported to the second vial in which the reaction was performed. The reactions using aromatic/heteroaromatic iodides were performed at 120 oC for 8 minutes whereafter the product was isolated by semi-prepartive radio-HPLC. Results The capture of 11CO2 by the silyl-lithium compound was quantitative. The conversion into 11Csilacarboxyic acids was >95% (RCP) as deducted by radio-HPLC. The overall conversion of 11CO2 to 11CO was >90% and the isolated radiochemical yields of synthesized compounds was 40-80% calculated from starting amount of 11CO2. In this way, starting from 270 mCi of 11CO2, 16-103 mCi of labeled product was isolated. The total synthesis time from EOB was less than 29 minutes. Conclusions In conclusion, we have developed a fast and effective method for the chemical conversion of 11CO2 to 11 CO at room temperature without the use of metal as reducing agent. The 11CO2 was first captured in a vial by a silyl-lithium compound and converted into a 11C-silacarboxylic acid which releases 11CO after addition of tetrabutylammonium fluoride. The trapping of 11CO2 was quantitative and the release of 11CO was >90%. The formed 11CO was then transported into a second vial were the reagents and Pd/Ligand was placed. The reactions for aryl iodides were performed at 120 oC for 8 minutes and the product were isolated by radio-HPLC. Acknowledgements References

Chemical conversion of 11CO2 to 11CO and subsequent radiosynthesis.

J Label Compd Radiopharm 2015: 58: S1- S411

S384: Poster

21st International Symposium on Radiopharmaceutical Sciences

384 Multiple Bacteriophage Selection Strategies for Improved Affinity of a Peptide Targeting ERBB2 Benjamin Larimer2, 1, Jeanne Quinn1, Kevin Kramer1, Susan L. Deutscher2, 1 1 Biochemistry, University of Missouri, Columbia, Missouri, United States, 2Research Service, Harry S. Truman Veterans Memorial Hospital, Columbia, Missouri, United States Objectives Due to the heterogeneity in ERBB2-expression between tumors and course of treatment, a non-invasive molecular imaging agent is needed to properly detect overall ERBB2 status. We previously obtained a peptide KCCYSL from bacteriophage (phage) display that bound with moderate affinity to ErBB2 expressing carcinomas and imaged a variety of tumors in vivo. Radiolabeled versions of the peptide bound breast, prostate, and ovarian human tumor xenografts in mice. Here, we developed and screened a novel microlibrary based on KCCYSL to select peptides with improved affinity and specificity for ERBB2. Methods One limitation of phage display selections is the difficulty in releasing the highest affinity phages from the target by incubation of acidic buffer. A microlibrary in fUSE5 phage was created by flanking the parent sequence with random amino acids. In an attempt to recover high affinity second-generation peptides from the ERBB2 microlibrary, two elution strategies from immobilized ERBB2, sonication and target elution, were undertaken in an attempt to affinity maturate the peptide. Three rounds of selection were performed. Affinity and specificity were analyzed by ELISA and confocal microscopy. Results Sonication resulted in an approximately 50-fold enhancement in recovered phage per round of selection in comparison to target elution with purified ERBB2. The binding affinity to ERBB2 of the sonication eluted phage displayed peptides ranged from 1.6±0.4 μM to 8.7±0.6 μM. For the target eluted peptides, the affinities ranged from 1.7±0.6 μM to 7.6±1.5 μM. For direct comparison, the binding affinity of KCCYSL was determined to be 4.4±1.2 μM. One peptide each from sonication (S13) and ERBB2 elution (E6) bound with a significantly improved dissociation constant (KD) (P < 0.05) than KCCYSL. S13 bound with 2.7 times higher affinity, while E6 had a 2.5 fold improvement over the first generation peptide. Conclusions Although both selections yielded peptides with significantly improved affinity in comparison to KCCYSL, the improvements were modest. These data suggest that the parental peptide binding cannot be drastically improved by additional amino acids flanking KCCYSL. Acknowledgements This research was funded in part by VA Merit BX000964 to SLD. References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S385

385 Improved Production of Zr-89 from Cyclotron using a Second-Generation Solution Target Mukesh K. Pandey1, Aditya Bansal1, John Byrne2, Alan B. Packard3, Timothy R. DeGrado1 1 Radiology, Mayo Clinic, Rochester, Minnesota, United States, 2Brigham and Women's Hospital, Boston, Massachusetts, United States, 3Children's Hospital, Boston, Massachusetts, United States Objectives To improve the production yield of Zr-89 in a solution target for an economical supply of Zr-89 using a low energy cyclotron. Methods Cyclotron production of Zr-89 has been achieved using solid and liquid targets[1,2]. The solution target approach provided modest levels of Zr-89 without the need for solid target infrastructure, but was limited by intarget salt precipitation and radiolytic gas production. We have redesigned our solution target with enhanced heat dissipation by incorporating 2 foils (0.2 mm Al and 25 μ Havar) with helium-cooling and water-cooled target regions. A 2 M solution of Y(NO3)3 was irradiated and nitric acid (1.25M) was included to minimize in-target precipitation[2]. Irradiations were performed on a PETtrace (~14MeV) cyclotron at 40-μA beam current for 2 h without venting of the target. Produced Zr-89 was purified by selective trapping of Zr-89 on hydroxamate resin (75 mg) followed by washing with 72 mL of 2N HCl and 20 mL of water to remove yttrium. The hydroxamate resin was wetted with 1.2 M KHPO4 (~0.2 mL) solution at pH 3.5 and allowed to stand for 30 min at room temperature. Finally, the Zr-89 was eluted from the hydroxamate resin with 0.3 mL 1.2 M KHPO4. For comparison to the conventional approach, 1M oxalic acid was also utilized to elute Zr-89 [1,2]. ICP-MS was used to measure the specific activity (SA) and concentrations of metal contaminants in the Zr-89-phosphate/oxalate solutions. Results The second-generation solution target produced Zr-89 without in-target precipitation and overpressure issues. The new target yielded a decay-corrected activity of 314±16MBq (8.5 ± 0.42 mCi) at end of beam with a SA of 1.11±0.87 GBq/μg (30±23 mCi/μg). The elution efficiency of Zr-89 with 1.2 M KHPO4 solution was found to be 70-75%, compared to >95% for 1 M oxalic acid. Both methods gave very small levels of metal contaminants (<3 μg Al, <2 μg Fe, <0.5 μg Y). Conclusions The second-generation solution target tripled the production yield of Zr-89 to 15.7±0.91MBq/μA.h from the previously reported 4.36±0.48 MBq/μA.h[2]. Elution of Zr-89 from the hydroxamate resin with phosphate buffer provides Zr-89 in a more biologically favorable medium. Acknowledgements This work is financially supported by DOE grant to TRD/ABP DE-SC0008947. References [1] Holland JP et al.(2009) Nucl. Med. Biol. 36:729–739. [2] Pandey MK, et al. (2014) Nucl. Med. Biol. 41(4): 309-316.

J Label Compd Radiopharm 2015: 58: S1- S411

S386: Poster

21st International Symposium on Radiopharmaceutical Sciences

386 82 Sr/82Rb production with an SR resin®: characterization and quantification by mass spectrometry of radiation-induced degradation of various 18-crown-6 crow-ethers Cyrille ALLIOT2, Manon DUPLEICHS3, Steffen HAPPEL4, Sandrine C. Huclier-Markai1 1 SUBATECH/ARRONAX, Nantes Cedex 3, France, 2ARRONAX/ INSERME U 892, Nantes, France, 3 ARRONAX, Nantes, France, 4TRISKEM International, Bruz, France Objectives In the context of processes of purification of the radionuclides, novel exchange resins can be used. However, no information exists on the stability of these resins under radiation. Taking into account the activities used in nuclear medicine, the radioactive dose potentially received by the resin can be important and can lead to a degradation of the resin components. Currently, the purification process of 82Sr used in 82Sr/82Rb generator, validated by the authorities, requires 3 steps and do not discard competitor cations like calcium and barium. However, an alternative purification that would necessitate a single step is under development. The process would involve using a resin known as SR resin ®(Triskem). The resin consists of impregnated Crown 18-crown-6 ether diluted in octanol. In order to further propose this resin; it is required to ensure the quality of this resin during the process of 82Sr purification Methods Methods of characterization of various 18-crown-6 crow-ethers by mass spectrometry (ESI-APCI/ITTOF) were developed and optimized. This work has three stages: qualitative analysis (choice of the method of ionization), quantitative analysis (method of ionization, LD, LQ, Internal standard, sensitivity) and application to irradiated resins Results The qualitative study established that the APCI mode is most favorable for the analysis of Crown ethers; this last reduces very significantly the interference due to the environment. Quantitative analysis evidenced that ESI was the most suitable ionization mode leading to a greater sensitivity. It can even be increased, if necessary, through a preliminary liquid-liquid extraction step. These two steps, once optimized, were used to study the leaching properties of non-irradiated and irradiated resins Conclusions The results obtained allowed to conclude that the determination of crown ether released by leaching from the non-irradiated resin is possible and that this crown ether concentration in the final 82Sr solution would be about 10 ng. Only a qualitative analysis on the irradiated resin was possible. The same peaks were observed compared with those obtained from the non-radiated resin. No significant degradation product was evidenced Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S387

387 Improved elution procedure of 3’-Deoxy-3’-[18F]fluorothymidine [18F]FLT for safer human injectable radiotracer Surendra Nakka, Mohammed H. Al Qahtani Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Center-Riyadh, Riyadh, Saudi Arabia Objectives Production of more suitable and safer human injectable [18F]FLT by lowering the ethanol content in final product. Optimizing the radiosynthesis method of [18F]FLT as an important Positron Emission Tomography (PET) radiotracer for imaging cellular proliferation in human cancers, to improve the final product characterizations becomes essential. Methods Automated radiosynthesis and purification of [18F]FLT were performed on TRACERlabMX with careful choice of reaction conditions, reagents and purification method. Results Production of [18F]FLT with presented modified parameters mainly the eluent solution (kryptofix or TBA HCO3), labelling temperature and extraction of product with various concentrations of ethanol could accomplish in collecting 70 % of the activity which then improved radiochemical yields (20-25 % decay corrected yields) and radiochemical purities (≥ 99%) with very negligible amounts of chemical impurities (thymidine, thymine and stavudine and cold [19F]FLT). Ethanol concentration is quantified by Gas chromatography with FID. The final formulated product [18F]FLT obtained with ethanol concentrations of 4.6- 5.0% compared to 10% on the published references. Conclusions The optimized parameters produced an efficient radiosynthesis method suitable for Good Manufacturing Practice (GMP) production and provided consistent yields and purities for clinical applications and more importantly safer for human use. Acknowledgements References

J Label Compd Radiopharm 2015: 58: S1- S411

S388: Poster

21st International Symposium on Radiopharmaceutical Sciences

388 A study on remote production of 43Sc from natCaO target via the natCa(α,x)-reaction Kotaro Nagatsu, Katsuyuki Minegishi, Ming-Rong Zhang Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Objectives Metallic radionuclides are widely used in nuclear medicine for both SPECT and PET imaging as well as for internal radiotherapy. Recently, radio-scandium is being focused as a potential radio-metal due to its favorable characteristic; particularly 43Sc has a moderate half-life of 3.9 h with enough emission rate of positron (88%), and 47Sc (β–, 3.3 d) is a candidate for internal radiotherapy. These radio-scandiums can be produced from respective Ca target by using accelerated alpha particles, namely 40Ca(α,x) or 44Ca(α,n)-channel. In this study, we attempted to produce 43Sc remotely from natCaO target as a pilot case. Methods A commercially available, 200 mg of natCaO (40Ca = 97 atom%) powder without further purification was loaded into a target vessel made of silicon carbide (SiC). The target vessel was covered by a 12.7 µm Nb foil with O-ring, and placed at a vertical irradiation station. The non-self-supported target powder was able to keep its thickness by the force of gravity without any solidification process [1]. Irradiations were performed by NIRS AVF930 cyclotron with 34 MeV alpha particles at 10 µA for 2 h. After the irradiation, 0.5N-HCl was directly introduced into the target vessel to dissolve CaO target. Then, 6%-NH3 solution was similarly introduced into the target vessel to adjust pH of mixed solution at around 9, and recovered to the hotcell through a Teflon tubing (~15 m) by means of N2 pressure. The recovered solution was loaded into a chelating resin (InertSep ME2, 120 mg, GL Sciences, Japan) to trap 43Sc. The resin was washed with 0.05N-HCl to remove parasitic Ca2+; then 43Sc was eluted by 0.5N-HCl as the final product. Results The yield of purified 43Sc was 1.04 GBq (28 mCi) which obtained within 1 h from EOB. Activity balance of the final product was reached to 75% of whole produced, and the radionuclidic purity of 43Sc showed 95% or higher till 17 h from EOB. By-produced nuclides found in the 43Sc product were, for example 44mSc(58.6 h, 0.0051% vs 43Sc), 44Sc(3.93 h, 3.2%), 46Sc(83.8 h, 0.0043%), and 47Sc(3.35 d, 0.034%) at 2 h from EOB. An estimation with a special emphasis on the production of 47Sc by using above result, the expected yield of 47Sc was 0.7 MBq (19 µCi)/µAh from enriched, i.e. 99 atom% 44Ca target. Conclusions 43Sc was remotely obtained by irradiation of natCaO that showed acceptable yield and purity, and this system can be applied to the production of 47Sc. Acknowledgements The authors are grateful to the NIRS cyclotron staff for their excellent operation of AVF-930. References [1] Nagatsu, K. et al. (2012) Nucl. Med. Biol. 39, 1281–1285

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S389

389 A feasibility study on 68Ge production from elemental natGa by in-situ extraction process Kotaro Nagatsu, Katsuyuki Minegishi, Ming-Rong Zhang Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan Objectives Germanium-68 (EC, 271 d) is a parent nuclide for 68Ga (β+/EC, 68 min), and the systemic elution device called 68Ge/68Ga generators available commercially are widely used. 68Ge can be produced via the 69 Ga(p,2n) and 71Ga(p,4n)-channels at a relatively high yield (e.g., 30 µCi+/µAh at 30 MeV+ protons, [1]) though, considerably long irradiation time is needed to obtain a practical yield due to its long half-life. Furthermore, Ga is a low m.p. metal (30°C) and molten Ga shows corrosivity that are regarded as major issues to perform secure irradiation. Thus, we attempted to irradiate an elemental Ga safely and easily by using a vertical irradiation system coupled with a target vessel made of an inert material, silicon carbide (SiC). The 68Ge produced in target Ga was remotely recovered by extraction into a portion of HCl/H2O2 media. Methods An elemental natGa piece (2.5 g, New Met Koch, UK) was placed in a SiC target vessel. The SiC vessel covered with a 50 µm Nb foil was set at a vertical irradiation port. Irradiations were carried out by NIRS AVF-930 cyclotron with 30 MeV protons at 20–23 µA for 1–2 h. Then, a portion of 1N-HCl/ 3%-H2O2 (1:1) was introduced into the target vessel, and let the solution go into-and-eject out the vessel repeatedly to stir the molten Ga for about 10 h at 40°C. Subsequently, the crude solution was transferred to hotcell through a Teflon tube (~15 m) by means of N2 pressure. The recovered solution was loaded into a cation exchange resin (AG50W-X8, Bio-Rad) to trap byproduced nuclides and partially dissolved Ga. Results The extraction of 68Ga was almost quantitative, which was reached to about 94% of total produced. Byproduced nuclides found in a crude sample were 67Ga(3.3 d), Ge-69(1.6 d), Zn-65(244 d), and Zn-69m(13.8 h). However, characteristic gammas from 67Ga and 69Ge were good indications to evaluate the behavior of both cold Ga and 68Ge, respectively; and we found that about 3% of target Ga was dissolved into the extract, and the extraction rate of Ge instantly. Conclusions Although the evaluation of this production system is still challenging, particularly for longer irradiation periods and at higher beam intensities, remote recovery of 68Ge by using a simple method with common solvents was achieved. Acknowledgements The authors are grateful to the NIRS cyclotron staff for their excellent operation of AVF-930. We also express our gratitude to Dr. Tadashi Nozaki and Dr. Akira Goto for fruitful discussions on the 68Ge/Ga generator, its application, and the visions for educational purpose. References [1] Adam-Rebels, R. et al. (2013) Radiochim. Acta/ DOI 10.1524/react2013.2057

J Label Compd Radiopharm 2015: 58: S1- S411

S390: Poster

21st International Symposium on Radiopharmaceutical Sciences

390 Application of Compton-suppressed Gamma-Ray Spectroscopy for Improved Quality Control for production of 82Sr. Suzanne V. Smith1, Lisa Muench1, Robert Harding2, Partha Chowdhury2, Christopher Lister2, Elizabeth A. McCutchan3, Gustavo Nobre3, Alejandro Sonzogni3 1 Collider-Accelerator , Brookhaven National Lab, Upton, New York, United States, 2Dept. of Phys. Appl. Phys, U. Mass Lowell, Lowell, USA 3National Nuclear Data Center, BNL, Upton, USA Objectives The radionuclide 82Sr produced at BNL, is used in the production of 82Sr/82Rb generators for PET imaging. Other radionuclides such as 83Rb and 85Sr are co-produced and must be quantified to ensure the 82Sr meets regulatory compliance. Accurate quantification of those impurities is challenging because their gamma energies overlap with those of 82Rb. HPGe detectors are relatively insensitive to the 82Sr signal and sensitive to Compton scattering which creates high background. This background can be drastically reduced by surrounding the HPGe crystal with an “anti-Compton” shield[1]. Here we compare the performance of standard and Compton-suppressed gamma-ray spectroscopy on the quantification of 83Rb in a mixed 83Rb/82Sr sample. Methods High purity 83Rb and 82Sr stock solutions were used to prepare a series of mixed 82Sr and 83Rb samples in a range of concentrations between 1 in 500 and 1 in 5000. The samples were counted using a standard HPGe detector and a Compton suppressed (30% n-type Ortec coaxial detector with a hybrid BGO/NaI suppression shield). Spectra were analyzed with GammaVision software [2] and the Radware package [3]. Results The 521, 530 and 553 keV gamma emissions used to quantify 83Rb lie directly in the Compton-background of the much stronger transition of 772 keV from 82Sr. Hence for the mixed 82Sr/83Rb samples the standard (unsuppressed) gamma spectrometry gave values for each 83Rb line with an uncertainty of > 30%. For the same sample, the application of the Compton-suppression detector resulted in a dramatic improvement in the uncertainty of their values to 5-10% Conclusions The quantification of 83Rb in a mixed 83Rb/82Sr sample was notably improved using a Comptonsuppressed HPGe detector by reducing the background in the region of the characteristic 83Rb lines. Uncertainty in peak fitting was reduced by a factor of at least 3 following the application of Compton-suppression methods. Further refinements in the method suggest even higher sensitivities can be achieved. This study shows that the application of Compton-suppressed Gamma-Ray Spectroscopy has potential to improve the quality control of a wide range of radionuclides. Future work will focus on the reduction of the positron annihilation peak at 511 keV in order to better quantify the 85Sr content using the characteristic 514 keV transition. Acknowledgements References [1] I-Yang Lee, Nucl. Phys. A 520, 641c (1990). [2] ORTEC Software App. (2013) Oakridge, TN. [3] Radware: http://radware.phy.ornl.gov/ www.radware.ornl.gov

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S391

391 Alternative Methods for the Production of Mo-99: non-Fission Based Solid-State, Mo-100 (g,n) Mo-99, and sub-Critical Solution-State, U-235 (n,f) Mo-99 David A. Rotsch1, Amanda Youker2, John Krebs2, Michael Kalensky2, Peter Tkac2, Thad Heltemes2, Dominique Stepinski2, James Byrnes2, James Jerden2, William Ebert2, Candido Pereira2, Martin Steindler2, Del Bowers2, Zaijing Sun2, Sergey Chemerisov2, Roman Gromov2, Vakhtang Makarashvili2, Bradley Micklich2, Charles Jonah2, Keith Woloshun3, Michael Holloway3, Frank Romero3, Jim Harvey4, Greg Dale3, George Vandegrift2 1 Nuclear Engineering, Argonne National Laboratory, Argonne, Illinois, United States, 2Argonne National Laboratory, Argonne, Illinois, United States, 3Los Alamos National Laboratory, Los Alamos, Mexico, 4NorthStar Medical Technologies, Madison, Wisconsin, United States Objectives Argonne National Laboratory (ANL), Los Alamos National Laboratory (LANL), and other national labs are supporting NorthStar Medical Technologies and SHINE Medical Technologies in their efforts to become US-domestic Mo-99 producers using two different methods. Irradiations were performed at Argonne's LowEnergy Accelerator Facility (LEAF) which houses a 30 MeV electron linac and a 3 MeV van de graaff generator. Methods NorthStar is utilizing the photonuclear reaction, Mo-100 (g,n) Mo-99, with enriched Mo-100 targets. Enriched-Mo-100 metallic disks are irradiated, dissolved, and then loaded onto a NorthStar Generator, RadioGenix™, valuable Mo-100 is then recycled for future targets. SHINE intends to produce Mo-99 by accelerator driven fission of a sub-critical aqueous solution of LEU uranyl sulfate. After irradiation, Mo-99 is isolated on a titania column and the bulk solution recycled for future runs. The Mo-99 is remotely washed, eluted, and transfer to a hot cell for further processing. The solution is concentrated and further purified using LEU-Modified Cintichem Processing. NaHSO4 has been used as a surrogate to test the newly designed system. Mo-99 spike trials have been performed to test each aspect of this large experiment. Results It was demonstrated that Mo-99 production using solid targets is possible and that all system monitoring equipment could survive the irradiation. Several Mo-100 enriched disks were irradiated, counted, dissolved, and purified using the RadioGenix™ system. Several methods for the recovery of Mo-100 have been tested and have demonstrated >95% Mo-100 recovery. Mo-99 spiked NaHSO4 solutions have been irradiated at ANL's LEAF facility to demonstrate the complete SHINE process. It was shown that remote processing was possible and gave yields >80%. Concentration and preparation of the solution for LEU-modified Cintichem gave >95% recovery and final purification gave >80% recovery. Conclusions Non-reactor-based Mo-99 production has been demonstrated. Acknowledgements Work supported by the U.S. Department of Energy, National Nuclear Security Administration's (NNSA's) Office of Defense Nuclear Nonproliferation, under Contract DE-AC02-06CH11357. Argonne national Laboratory is operated for the U.S. Department of Energy by UChicago Argonne, LLC. References

J Label Compd Radiopharm 2015: 58: S1- S411

S392: Poster

21st International Symposium on Radiopharmaceutical Sciences

392 A simplified conventional manual C18 Light Sep-Pak system for purification and reformulation of carbon11 PET tracers Qi-Huang Zheng, Barbara Glick-Wilson, Brandon Steele, Michael Shaffer, Larry Corbin, Mark Green Department of Radiology and Imaging Sciences, Indiana University, Indianapolis, Indiana, United States Objectives We have previously developed a multi-purpose fully automated 11C-radiosynthesis system for the routine production of carbon-11 PET tracers within our PET chemistry facility [1]. In this system, we use V-vial method for labeling reaction; semi-preparative HPLC, disposable solid-phase extraction (SPE) cartridges, or HPLC combined SPE for purification; either rotatry evaporation or Sep-Pak trap and release for reformulation. The module is constructed with many PTFE/silicone liners (septa) and Teflon tubing that limit the pressure range for gas (N2) push; therefore, we are unable to use C18 Light Sep-Pak due to its back pressure and instead a C18 Plus Sep-Pak is required in the automated device. Unfortunately, reliance on the larger C18 Plus Sep-Pak increases the volume of ethanol required for product recovery and undesirably raises the % ethanol in final formulation of radiotracer with sterile saline. In order to meet the requirements of USP Chapter 823 Radiopharmaceuticals for Positron Emission Tomography – Compounding, we develop a simplified conventional manual C18 Light Sep-Pak system connected to our automated 11C-radiosynthesis module for purification and reformulation of carbon-11 PET tracers, allowing radiopharmaceutical recovery and formulation with less ethanol. Methods The C18 Light Sep-Pak system is used for routine production of C11 tracers [11C]raclopride, [11C]PBR28 and [11C]PIB. The HPLC product fraction was collected in a recovery vial containing water (30 mL). The diluted tracer solution was then passed through a C18 Light Sep-Pak cartridge, and washed with water (3 × 10 mL). The cartridge was eluted with EtOH (3 × 0.4 mL) followed by saline (10 mL) to release the tracer. The eluted product was then sterile-filtered through a Millex-FG 0.2 µm membrance into a sterile intermediate product vial. Results The solution composition for [11C]raclopride, [11C]PBR28 and [11C]PIB is 5-10% ethanol in saline solution. The chemical purity of [11C]raclopride is >98%. The limit for the impurities has been established at 1 µg per administered [11C]PBR28 dose, or 10% of the permitted PBR28 mass. The limit for PIB precursor has been established at 1.28 µg per administered [11C]PIB dose, or 10% of the permitted PIB mass. Conclusions A simplified conventional manual C18 Light Sep-Pak system for purification and reformulation of carbon-11 PET tracers has been developed to meet the requirements of USP Chapter 823 and allowing final product formulation with ≤5% ethanol. Acknowledgements References [1] Mock BH, et al. (2005) J Label Compd Radiopharm, 48, S225.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S393

393 Separation of Cu-67 from an irradiated zinc target by electrolysis Donald E. Wycoff1, Ramesh Sharma2, Suzanne V. Smith2, Alan R. Ketring3, Cathy S. Cutler3, Silvia S. Jurisson1, 3 1 Chemistry, University of Missouri, Columbia, Missouri, United States, 2Collider Accelerator Department, Brookhaven National Laboratory, Upton, New York, United States, 3University of Missouri Research Reactor Center, University of Missouri, Columbia, Missouri, United States Objectives Cu-67 (2.57 d; 141 β-mean; 91, 93, 185 keV γ) [1] is of interest for therapeutic applications. The current process for the isolation of 67Cu from a 68Zn(p,2p)67Cu reaction uses three column separations. A more efficient separation of 67Cu from the zinc target would be useful for an improved production process. Methods The electrolysis of trace amounts of copper from zinc solutions was investigated [2]. A series of electrolysis experiments were performed by adding carrier added 64Cu (0.063 to 305 µg from a natCu(n,γ)64,66Cu reaction) to a zinc chloride cathode solution (0 to 5.2 M ZnCl2 and 0.005 to 3M HCl), in varying ionic strength (1.0 to 16.6 M). Room temperature electrolysis was performed in an H-cell with no applied potential, a Pt cathode and a Zn anode. Zinc ion migration from the cathode to anode cell compartments was followed with 65Zn. A natZn target was proton irradiated at the Brookhaven Linac Isotope Producer (BLIP) at Brookhaven National Laboratory (BNL), digested in HCl and its electrolysis evaluated. Results First order deposition of copper was observed with cathode solution concentrations of HCl between 0.01 and 0.33 M and the ionic strength below 7 M. Electrolysis half-lives were on the order of 29 to 71 minutes, with rates limited by the Pt wire surface area. With a Pt mesh electrode, more than 95 % of the total 64Cu added to the cathode solution was recovered from the platinum electrode after one hour of electrolysis. The half-life was less than 14 minutes. Zinc ion migration from cathode to anode cell compartments is dependent on the relative ionic strength of the solution in each compartment. Cathode solution ionic strength at 6.26 M and anode solution ionic strength at either 3.16 or 6.26 M resulted in migration of 0.39 and 0.04 % of the zinc ion, respectively, at 60 minutes. Electrolysis of a small portion of the proton irradiated zinc target enhanced the 67Cu radionuclidic purity with respect to 52,54,56Mn, 59Fe, 55,56,57,58Co, 56,57Ni, 62,65,69mZn, 66Ga and other nuclides. Conclusions Efficient removal of 64Cu from the cathode solution was found with HCl at 0.1 M, ZnCl2 at 1.2 M and a Pt mesh electrode. Zinc ion migration from the cathode to the anode compartment can be minimized by matching the relative ionic strength of the two solutions. Isolation of copper by electrolysis of a portion of the BNL irradiated zinc target with zero applied potential resulted in enhanced radionuclidic purity of 67Cu. Future studies will optimize the recovery of copper from larger portions of the zinc target. Acknowledgements DOE grants: DE-SC0010283 and DE-SC0007348 References [1] http://www.nndc.bnl.gov/ [2] Mirzadeh, S., et al (1992) Radiochim Acta, 57, 193-199.

J Label Compd Radiopharm 2015: 58: S1- S411

S394: Poster

21st International Symposium on Radiopharmaceutical Sciences

394 Investigation of Accelerator-Based Production of High Specific Activity 186Re Matthew D. Gott1, 2, Donald E. Wycoff1, Ethan R. Balkin3, Cathy S. Cutler2, Alan R. Ketring2, Michael Fassbender4, D. Scott Wilbur3, Silvia S. Jurisson1 1 Department of Chemistry, University of Missouri, Columbia, Missouri, United States, 2University of Missouri Research Reactor Center, Columbia, Missouri, United States, 3Department of Radiation Oncology, University of Washington, Seattle, Washington, United States, 4Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States Objectives 186Re is a beta-emitter with nuclear properties making it an ideal therapeutic surrogate for 99mTc. The drawback is the current reactor production pathway, 185Re(n, γ)186Re results in a low specific activity product [1]. Accelerator-based production yielding high specific activity 186Re will enable its use in therapeutic applications. The objective of this work is to evaluate W and Os-based target forms, accelerator-based production schemes, and develop methods to isolate 186Re and recover the W/Os target materials for reuse. Methods Metallic W and Os are hard; pressed pellets of these materials are brittle. Doping the metal with wax allowed for firm pressing. The wax volatilizes at high temperature leaving a pressed metal pellet. Targets of WS2 and OsS2 were also prepared and formed robust pressed pellets. Thermal stability for all targets was determined using short irradiations of increasing current. Production rate, yield, and purity were determined by gamma spectroscopy. Separation methods were developed to isolate 186Re from W and Os. Anion exchange isolated Re and recovered W [2]. Re is loaded in NaOH while W elutes; Re is eluted with HNO3. Liquid-liquid extraction isolated Re into the organic layer while Os and Ir remained in the aqueous [1]. Os is recovered by distillation of the volatile OsO4 species. Results Irradiation of Os-based targets produced nCi amounts of Re and µCi amounts of Ir. A liquid-liquid extraction method separated the Re from Ir and Os. Irradiation of W-based targets produced µCi amounts of Re. An anion exchange method separated Re from W. Future studies will use enriched 189Os and 186W targets to reduce unwanted radionuclides. Production rates, yields, and purity for each target form will be reported. Conclusions Potential production routes for accelerator-produced high specific activity 186Re were evaluated. Cyclotron irradiations of natOs-based targets established the feasibility of producing Re via the Os(p, αxn)Re reaction, and indicated higher proton energies are needed to reduce production of unwanted Ir and increase production of Re. A rapid separation method isolated Re from Ir and Os. Irradiation of W-based targets demonstrated the ability to produce 186Re via the (p, n) reaction. Acknowledgements The authors thank the MURR staff for conducting the irradiations. We acknowledge the support of the US DOE through the Office of Science, Nuclear Physics (DE-SC0007348) and trainee support from the NSF under IGERT award DGE-0965983 (M.D. Gott). References [1] Moustapha, ME, et al. (2006) Nucl Med Biol, 33(1), 81-89. [2] Gott, MD, et al. (2014) Radiochim. Acta, 102(4), 325-332.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S395

395 Design of a multi-functional automated 68Ga-synthesis program Karin M. Nielsen1, 2 1 Department of Nuclear Medicine, Aalborg University Hospital, Aalborg, Denmark, 2Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark Objectives In our development of potential bacteria-specific, 68Ga-tracers [1-2], we found it necessary to design a general, flexible 68Ga-synthesis program, that would accommodate our need for extensive variations in synthesis criteria as well as adapt to various purification methods. Additionally, program functions facilitating the evaluation of parameters during 68Ga-syntheses, e.g. enabling activity measurement of the generator eluate prior to synthesis or separating the waste of the pre- and post-purification procedures, were wanted. Methods The 68Ga-labelling program was designed in the Modular-Lab 4.3.2.0 software and employs the ModularLab PharmTracer synthesis module and IGG100 generator (All from Eckert & Ziegler). Special efforts were made to make the program adjustable to every type of 68Ga-synthesis, i.e. by changing specific 68Ga-labelling variables; the program will automatically switch between generator pre-purification methods (cationic vs. fractionated) and add a post-purification step of the reaction mixture, if needed. Furthermore, the overall synthesis time was reduced by designing parallel programs e.g. to perform the pre-conditioning of the post-purification cartridge during 68Gachelation. In addition to the use in pre-clinical 68Ga-tracer evaluation, the 68Ga-synthesis program has also been applied on routine clinical 68Ga-tracers, such as 68Ga-DOTATOC, 68Ga-PSMA-HBED-CC, as well as 68Ga-citrate which use a slightly different synthesis setup [3]. Results Some of the key features in the 68Ga-labelling program are presented in Table 1. During synthesis, the program interface shows the parameter values set for each 68Ga-labelling as well as information on the synthesis steps which are running and skipped. Subsequently, the data is saved in the audit-trail process of the program. The reaction time, radiochemical purity and yield, of the routine clinical 68Ga-traceres tested, were comparable to values reported for tracer-specific synthesis programs. Conclusions A multi-functional automated 68Ga-synthesis program has been designed to facilitate the optimization of 68Ga-labelling conditions during pre-clinical development. Acknowledgements Eckert & Ziegler Eurotope GmbH References [1] Nielsen KM, et al (2013), J Label Compd Radiopharm, 56, S199. [2] Nielsen KM, et al (2014), Q J Nucl Med Mol Im, 58 S1, 34-35. [3] Jensen SB, et al (2013), Nucl Med Commun, 34, 806-812.

Table 1: Some key features in the 68Ga-labelling program

J Label Compd Radiopharm 2015: 58: S1- S411

S396: Poster

21st International Symposium on Radiopharmaceutical Sciences

396 68 Ge quantification in eluate and after 68Ga-DOTA-peptide labelling for clinical application Erik de Blois, Max van Peski, Ho Sze Chan, Wout A. Breeman Nuclear Medicine, Erasmus MC, Rotterdam, Netherlands Objectives 68Ga-DOTA-peptides are applied clinically. According European Pharmacopeia maximum amount of 68 Ge is 0.001 % (expressed as activity 68Ge vs activity 68Ga). Suppose there is 100 MBq 68Ga, maximum amount 68 Ge according Pharmacopeia is 1 kBq. 68Ga-DOTA-peptide can be produced in different ways, either with robotic or manually. Here we compared 68Ge content in eluate with/without cation exchange (using NaCl technique) and with/without C18 solid phase extraction (SPE) and in final labelling product. We developed a method to quantify 68 Ge and compared 68Ge content in above described methods of 68Ga-DOTA-peptides production. Methods Pharmaceutical Grade EZAG 68Ge/68Ga generator was used as 68Ge source.68Ge content was determined in a Wizard 1480 3” automatic gamma counter (Perkin Elmer Life Sciences) via measurement of 68Ga (>1 day ingrowth) by using a calibration curve of 18F (range 185 kBq - <1 Bq), corrected for gamma yield (511 KeV) for 68 Ga [1]. 68Ge content in eluate was determined >1 day after elution of the 68Ge/68Ga generator. 68Ge content in cation (with NaCl method [2]) and C18 SPE purified samples was determined, also >1 day after cation or C18 purification. Results Lower level of detection (LLD) of 68Ge in here described technique was: 0.41 ± 0.06 Bq. 68Ge breakthrough of 1110 MBq EZAG generator (8 month after production, 400 MBq 68Ga in 6 mL eluate) we found 368 ± 8 Bq in 6 mL eluate (<10-4%). In eluate purified by cation and NaCl method 68Ge content was reduced to 1.1 ± 0.2 Bq (factor of >300, see Table 1). After radiolabelling and C18 SPE column purification 68Ge content was lowered by a factor of >600 (see Table 1). After radiolabelling performed with eluate purified with cation and NaCl method and C18 SPE column 68Ge content was below LLD (see Table 1). Conclusions 68Ge content can be quantified as low as 0.4 Bq 68Ge. In all described methods of labelling 68GaDOTA-peptides, 68Ge content is below 0.001% (expressed as activity 68Ge vs activity 68Ga) and thus within European Pharmacopeia specification. Acknowledgements References [1] Breeman W.A. et al., Eur J Nucl Med Mol Imaging, 2005, [2] Mueller D. et al., Bioconj Chem, 2012

Table 1: Quantification of 68Ge content in eluate and final product

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S397

397 Production of Platinum Radioisotopes at Brookhaven Linac Isotope Producer (BLIP) Suzanne V. Smith1, Lisa Muench1, Robert Harding2, Partha Chowdhury2, Christopher Lister2, Elizabeth A. McCutchan3, Gustavo Nobre3, Alejandro Sonzogni3 1 Collider Accelerator Department, Brookhaven National Laboratory, Upton, New York, United States, 2University of Massachusetts Lowell, Lowell, Massachusetts, United States, 3BNL, National Nuclear Data Center, Upton, New York, United States Objectives Research into platinum chemotherapeutics is experiencing resurgence due approval of new agents and findings on mechanisms of resistance.1-5 Monitor the pharmacokinetics of platinum agents using 188Pt (t1/2=10.2 d), 189 Pt (t1/2=10.87 hr), 191Pt(t1/2=2.802 d) or 195mPt (t1/2= 4.02 d) can be used to optimized use of current and design new agents.6 This study assesses the feasibility of producing these radioisotopes at BLIP. Methods Platinum foils were irradiated (at 193 MeV and 107 MeV) for 90 min at 113.9 uA.hr. The Pt isotopes were purified using reported method.7 The Pt was dissolved in hot aqua regia solution and then digested with concentrated HCl. The residue dissolved in 0.1 M HCl was converted to Pt(NH3)2I2 by the addition of KI and NH4OH. Addition of AgNO3 afforded conversion to the diaqua [Pt(NH3)2(H2O)2]2+ species. The samples were analyzed by conventional and Compton-suppressed gamma-ray spectrometry. The data along with published cross sections up to 70 MeV8 were correlated with theoretical calculations from the nuclear reaction model code EMPIRE for 10 to 193 MeV protons. Results The cross sections for the Au, Ir and Pt radionuclides present in each sample correlated well with published data and EMPIRE calculations. The use of Compton-suppressed gamma-ray spectroscopy provided significant reduction of the overall background leading to improved quantification of the gamma-ray emissions and higher sensitivity to weak production channels. [188Pt(NH3)2(H2O)2]2+ was isolated and found to be stable in solution at room temperature for over 20 days. Conclusions A range of Au, Pt, and Ir nuclides were produced at 107 and 193 MeV proton irradiations of a natural Pt foil at BLIP. A method for purification of Pt radionuclides was developed and found to be successful for the separation of 188Pt. EMPIRE calculations for viable nuclide reactions and their respective cross sections correlated with the present results and previously published data at lower energies. This study combined with the EMPIRE predictions, demonstrate that 188Pt, 189Pt, 191Pt, 193Pt or 195mPt can be preferentially produced at BLIP by optimizing the proton energy. Acknowledgements Work supported by the U.S. Department of Energy Office of Science, Office of Nuclear Physics, under award number DE-FG02-94ER40848 and ST5001030 and Contract No. DE-AC02-98CH10946. References [1] Wheate, N. J. et al (2010) Dalton Trans. 2010; 39: 8113–8127. [2] Kelland, L (2007) 7: 573-84 [3]Dasari, S; Tchounwou, (2014) P. B.; Eur. J. Pharm. 740, 364-378. [4] Siddik, Z. H. (2003) Oncogene, 22, 72657279. [5] Martin, L. P. Hamilton, T.C. and Schilder, R. J.; (2008) Clin. Cancer. Res. 14(5) 1291- 1295. [6] Bruijnincx, C.A. and Sadler, P. J. (2008) Curr. Opin. Chem. Biol. 12, 197-206. [7]Sathekge, M, Wagner J, Smith SV et al, Nuklearmedizin Nucl Med, (2013) 52, 227. [8]Tarkanyi, F, Ditroi, F, Takacs S et al (2004) Nucl Instr Methods Phys Res B226, 473 – 489.

J Label Compd Radiopharm 2015: 58: S1- S411

S398: Poster

21st International Symposium on Radiopharmaceutical Sciences

398 Automated and monitored liquid target transfer system Milan Vuckovic1, Long Hin (Julian) Fong1, Maurice Dodd2 1 Functional Imaging, BC Cancer Agency, Vancouver, British Columbia, Canada, 2TRIUMF, Vancouver, British Columbia, Canada Objectives Safety of personel is of a paramount importance in any nuclear facility. With an increased number of cyclotron runs required for the production of various radio-isotopes, the possibility of an inadvertent transfer of activity to the wrong location could be very problematic. The purpose of this project was to develop and implement a new, secure liquid target transfer system for radioisotopes. Methods Process of transfering target activity is run by and controlled using Opto 22 electronics and NI LabView software, employing Valco Instruments’ 14-positon selector valve, three radiation detectors[1] and one Helium flow detector (Omega Engineering, Inc.). Results Transfer of target contents is enabled only if two critical conditions are met: hot cell doors, including inner, Plexiglas lid, are properly closed and a chemist working in a clean room is ready to receive the transfer. After completion of a cyclotron run, an operator sends an audio signal to clean room. The chemist presses a button indicating that he’s ready to receive activity. Pressing the “ready” button does three things: starts a horn and strobe alarm, puts a selector valve in appropriate position and opens a safety valve that’s located inside a designated hot cell compartment. This way activity can only be transferred to the chosen location. All actions are logged and all analog signals can be later retrieved and graphed. Conclusions A safe and reliable way of transferring target contents to a number of hot cells was developed and is being used at BC Cancer Agency Cyclotron Facility. Acknowledgements NSERC, CIHR. References [1] Zeisler SK, et al (1994), Appl.Radiat.Isot., Vol. 45, No. 3, pp. 377-378.

Screenshot of a typical transfer of liquid target from cyclotron to designated hot cell

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S399

399 Development of a separation for the purification of Actinium-225 from bulk Thorium Jonathan Fitzsimmons1, Bryan Foley2, Bryna Torre3, Leonard Mausner1 1 Brookhaven National Lab, Upton, New York, United States, 2Fairmont State University , Fairmont, West Virginia, United States, 3University at Buffalo The State University of New York, Buffalo, New York, United States Objectives Actinium-225 (Ac-225) and its daughter (Bi-213) are used in radioimmunotherapy, however purification of Ac-225 from its parent isotope Thorium-229 (Th-229) will not produce enough to support clinical trials. The Brookhaven Linac Isotope Producer has the best characteristics to produce Ac-225 by high energy irradiation of Th. Spallation and fission reaction occur co-producing up to 400 other isotopes. The current purification method developed by ORNL involves 5-7 columns and would be difficult to scale up to production scale targets. A method to capture Ac-225 on a resin and elute the bulk of the Th could reduce the purification time and result in a more efficient purification. A complexing agent can be used to form a negatively charged complex with thorium that should not be retained on a cation column, while a positively charged actinium / lanthanum complex will be retained on the resin.1 We explored the development of a separation based on this approach. Methods Lanthanum (La) is chemically similar to actinium, safer to handle and used in the initial studies. The separation was developed with a representative solution containing 50 μgrams of: La, Cu, Pb, Zn, Co, Cr, Cd, Ni, Fe, Mn, Al, Ga, Ge, Sr, Be, Mg, Rb, Ba, Ce, Lu, and Zr, and the metals were tracked by ICP-OES. To develop the separation we examined: citrate or tartrate as the chelating agent, varied the pH of the load solution from 1.6 to 4.6, examined rinse steps with various molarities of HCl, HNO3 or H2SO4, evaluated separations involving different molarities of HCl and HNO3 solutions in different sequences and different concentrations of HCl to elute the La. Results Studies with citric or tartaric acids had minor differences in the metal elution profile. Cu, Pb, Zn, Co, Cr, Cd, Ni, Fe, Mn, Al, Ga, Sr, Be, Mg, Rb, Ba, Ce, Lu and La were all retained on the cation column from the load solution and pH 2.0 was optimal for retaining La. The optimal rinse steps were 3 bed volumes of: 2 M HCl, 3 M HCl, water, 2.5 M nitric acid. Eluting La from the column was achieved by eluting the column with greater than five bed volumes of 3.5- 6 M HCl. Conclusions Future studies will involve testing the separation steps with solutions containing the metals, Ac-225 and Th. Acknowledgements This project was supported in part by the DOE Office of Science, Nuclear Physics, Isotope Development and Production for Research and Applications, and the Office of Workforce Development for Teachers and Scientists (WDTS) under the Science Undergraduate Laboratory Internships Program (SULI). References [1] Chen Y; et al (1959) J. Chinese Chem Soc. 6, 1, p. 55-67.

J Label Compd Radiopharm 2015: 58: S1- S411

S400: Poster

21st International Symposium on Radiopharmaceutical Sciences

400 Solid phase synthesis of 62Cu-bis(thiosemicarbazonato) via transmetalation based on a new 62Cu/62Zn generator Zilin Yu1, David Parker2, Antony Gee1, Philip J. Blower1, 3 1 Division of Imaging Sciences and Biomedical Engineering, King's College London, London, United Kingdom, 2 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom, 3Division of Chemistry, King's College London, London, United Kingdom Objectives Bis(thiosemicarbazonato) complexes of copper radionuclides such as 61Cu (T1/2=3.3 h) and 60 Cu(T1/2=23.7 m) are promising agents for non-invasive imaging of hypoxia with positron emission tomography (PET). As these complexes have rapid in vivo pharmacokinetics, labelling with 62Cu (T1/2= 9 min) may allow repeat PET imaging studies in a single session, to determine the effect of an intervention such as carbogen breathing on hypoxic tumours. Fukumura(1) and Haynes (2) have reported 62Zn/62Cu generators using anion-exchange resin and cation-exchange Sep-Pak cartridges. In this study, we developed an optimised 62Cu generator based on Fukumura’s method with Chromabond PS-OH-(strong anion exchanger, OH–form) cartridge. A solid-phase method (3)based on transmetallation of the zinc bis(thiosemicarbazonato) complexes was applied to develop a suitably rapid and efficient synthesis of 62Cu-II-diacetyl-bis(N(4)-methylthiosemicarbazone)(62Cu-ATSM). Methods The target(copper foil, 0.5 mm thickness, 10 mm diameter) irradiated with 29 MeV protons at a beam current of 30 µA was dissolved in a equivalent volume mixture of concentrated H2O2 and HCl(50 mL). To isolate 62 Zn, the solution was diluted with 50 mL water and loaded on a PS-OH- cartridge. Excess HCl (2 M) was applied to remove metal contaminants. Parent radionuclide 62Zn was eluted with water(20 mL) and loaded on a CM plus cartridge. The 62Cu was eluted with glycine solution (3mL, 200 mM) once per hour. Radio-synthesis of 62Cu-ATSM was achieved by transmetalation of the natZn-ATSM-preloaded 4(dimethylamino)pyridine (DMAP) resin while the 62Cu passing through. Results The preliminary results have shown successful elution of ~305 MBq 62Cu with low levels of 62Zn breakthrough (<0.2%) and carrier (< 1ppm) using small volume (3 mL) of glycine solution after starting with ~450 MBq 62Zn. The 62Cu-ATSM was prepared in high radiochemical yield (>95%)and purity (>98 %). Free 62Cu and Zn-ATSM precursor were absent in the final product. Conclusions We successfully developed a new 62Cu generator with commercial available cartridges and combined it with the transchelation method to speed up the synthesis of 62Cu-ATSM. The new 62Cu generator was ready to elute in 2.5 h and synthesis of 62Cu-ATSM was instant. Acknowledgements The authors acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King’s College Hospital NHS Foundation Trust. References [1] Fukumura T, et al. (2006) Nucl Med Biol, 33(6), 821-827.[2] Haynes N. G, et al. (2000) J Nucl Med, 41, 309-314. [3] Betts M. H, et al. (2008), Angew. Chem. Int. Ed. 47, 8416 –8419

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S401

401 Promising anti-Gram-negative antimicrobial peptides (AMPs): Radiolabeling and biodistribution profiles Unai Cossío1, Vanessa Gómez-Vallejo1, Alessandro Pini3, Chiara Falciani2, Jordi Llop1 1 Molecular Imaging Unit, CIC biomaGUNE , San Sebastian, Spain, 2Setlance srl, Siena, Italy, 3Medical Biotechnology, University of Siena, Siena, Italy Objectives Radiolabeling and determination of the biodistribution pattern of two anti-Gram-negative antimicrobial peptides in healthy animals. Methods Two antimicrobial peptides were synthesized in tetra-branched form as previously reported.1 Both peptides had the same sequence of amino acids which included one terminal tyrosine, but one of them had a PEG chain (MW=5796 g/mol) intercalated between the branching core and the tyrosine residue.2 The radiolabeling was approached by electrophylic iodination on the tyrosine residues using a well-established method.3 Purification of the labeled peptides was achieved by solid phase extraction, and chemical and radiochemical purity was assessed by radio-HPLC. Biodistribution was determined at different time points after intravenous administration using dissection and gamma counting. With that aim, labeled peptides (100 µL, approx. 370 kBq, 9 nmol) were injected via the tail vein of BALB/c mice. At 1, 5, 30, 60 min and 2, and 24 hours after administration, animals were sacrificed by exsanguination followed by perfusion with saline solution, organs were harvested and the amount of radioactivity was determined using a gamma counter. Results were finally expressed as percentage of injected dose per gram of tissue. Results Labeling efficiencies close to 90% were achieved for both peptides when incubation was carried out for 10 minutes at room temperature. After purification, radiochemical purity exceeded 95% in all cases, and no chemical impurities were detected in the HPLC profile (UV detector, λ = 220 nm). Biodistribution data showed fast elimination via urine for both peptides. Interestingly, the PEG-functionalized peptide presented a higher initial uptake in the lungs and longer residence time in the blood, with %ID/g > 1.5% after 2 hours of injection vs. %ID/g < 1% for the non PEGylated peptide. Conclusions Both peptides showed a fast clearance from the body with elimination mainly via urine. The presence of the PEG chain results in a prolonged biological half-life and higher initial accumulation in the lungs. Metabolism studies are currently being carried out. Acknowledgements This work was funded by the European Commission, FP7-NMP-2013-LARGE-7, project reference 604434. References [1] Pini, A, et al (2010) FASEB J., 24(4):1015-22. [2] Falciani, C, et al (2014) AminoAcids 46(5):1403-7 [3] Markwell, M.A.K., (1982) Anal. Biochem., 125, 427-432.

J Label Compd Radiopharm 2015: 58: S1- S411

S402: Poster

21st International Symposium on Radiopharmaceutical Sciences

402 Production and In Vivo Evaluation of Radioactive MGF-198AuNPs for Prostate Cancer Treatment Amal Y. Al-Yasiri1, Cathy S. Cutler2, 1, L Watkinson4, T Carmack4, Charles J. Smith3, 4, Maryna Kuchuk2, Menka Khoobchandani3, Kattesh V. Katti3, Sudarshan Loyalka2, Kattesh V. Katti3, 5 1 NSEI, University of Missouri, Columbia , Missouri, United States, 2MURR, University of Missouri, Columbia, Missouri, United States, 3Department of Radiology, University of Missouri, Columbia, Missouri, United States, 4 Harry S. Truman Memorial Veterans Hospital, University of Missouri, Columbia , Missouri, United States, 5 Department of Physics, University of Missouri, Columbia, Missouri, United States Objectives The radioactive properties of 198Au(βmax=0.96MeV; t½=2.7d) make it an ideal candidate for use in radio-therapeutic applications.The goal of this research was to synthesize and evaluate radioactive gold nanoparticles using a nontoxic and antitumor phytochemical reducing agent, Mangiferin (MGF). The anti-oxidative and anti-cancer properties of MGF make it an excellent candidate to use to synthesize radioactive AuNPs Methods MGF-198AuNPs were synthesized by adding 3.1mg MGF to 4mL water while stirring and heating at 99100°C. Then, 0.0036mg 198Au and 1.307mg NaAuCl4 (390µCi-5mCi, 68.2µL) were mixed and added resulting in an immediate color change from pale yellow to red-purple. The solution was removed from heating but stirring was continued for an additional 1hr. The pH was then adjusted to7and made isotonic with NaOH and DPBS. A stability study was performed of MGF-198AuNPs out to seven days. Quality control included measuring the pH, λmax, and Radio-TLC every day for seven days. Normal CF1 mice were injected with MGF-198AuNPs (8µCi/100µL) via tail vein. Mice were euthanized (n=5) at 0.5, 1, 2, 4, 24hr post-injection. Organs were harvested, weighed and counted along with standards to determine the injected dose/organ and injected dose/gram. Results UV-vis spectroscopy measurements showed that λmax was in range of 530-535nm and Radio-TLC confirmed that over 97% of 198Au was present in the nanoparticulate form. Bio-distribution in mice showed that the majority of nanoparticles accumulated in the liver post injection with values remaining steady from 30min to 4hr post injection. Quantitatively, (87±2.6% of the injected dose/organ, at 30min, decreasing to 85.9±3.3% at 4hr), followed by spleen (6.8±2.6% at 30 min, increasing to 8.6±2.9% at 4hr). Low uptake of nanoparticles was found in the lungs (0.6±0.09% at 30min, decreasing to0.3±0.07% at 4hr). At 24hr post injection, 95±1% of the injected dose was in the liver and 2.9±0.6% in spleen and only 0.14±0.03% in the lungs. These results showed that MGF198 AuNPs are stable in vivo. Studies in PC3 tumor mice are underway and will be presented. Conclusions The results showed MGF could be utilized to synthesize MGF-198AuNPs that are stable in vitro and in vivo. Results will be presented on their distribution in normal and tumor bearing mice. Acknowledgements This research has been supported by NSEI, MURR, Green Technology institute/MU. AlYasiri has also been supported by University of Baghdad and NSEI References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S403

403 Radiosynthesis and Quality Control of Nanosized Resveratrol-Au-198 T M. Sakr1, 2, Kattesh V. Katti2, L Watkinson3, T Carmack3, Charles J. Smith3, Cathy S. Cutler2, Kattesh V. Katti2 1 Egyptian Atomic Energy Authority, Cairo, Egypt, 2MURR, University of Missouri (MU), Columbia, Missouri, United States, 3Harry S. Trauman Memorial Hospital, Columbia, Missouri, United States Objectives The preparation of Resveratrol-198Au nanoparticles in high radiochemical purity. RES-198AuNPs are of interest for treatment of neuro degenerative diseases and cancers. Methods Gold-198 Production and QC Gold foil target was irradiated in the reflector at MURR. Then, it was dissolved in Aqua Regia and dried to near dryness. The sample was re-dissolved in 0.05M HCl then dried twice. Finally, the dried sample was dissolved in nearly 200μL of 0.05M HCl. The radionuclidic and radiochemical purity of 198Au were checked by HpGe spectroscopy and Radio-TLC. Resveratrol-198AuNP Preparation and QC About 0.5mg Resveratrol was dissolved in 0.3ml absolute ethanol. Next 1477.5µg of gold (198Au + NaAuCl4) was added followed by 1.2ml Milli Q water while stirring for 2h until a dark ruby red color was obtained. The UV-Vis and radiochemical purity were assayed and the pH was adjusted to pH 77.5. The in-vitro stability was tested. After complete decay, the particle core diameter was checked using TEM. Normal female CF1 mice were injected via the tail vein with 100µCi in 100µL and euthanized at different time points. Organs were harvested, weighed and counted with standards to calculate the % ID/g and % ID/organ. Results Gold-198 was produced with 99.99% radionuclidic purity and with 99% radiochemical purity. RES198 AuNP were successfully prepared with high radiochemical purity (97%) in reproducible batches. They were stable out to 10 days in serum. Its λmax was at 540nm. The TEM showed a particle core diameter of 20-50nm. The biodistribution study showed liver and spleen initial average uptake of 55.63±4.48 and 63.95±26.66 %ID/g at 30min which increased to 61.47±8.14, and 69.8±33.12 %ID/g at 24h respectively, (Fig 1) with nearly no uptake in the other organs. While blood showed 1.61±0.49 %ID/g at 30min decreasing to 0.61±0.37 %ID/g at 24h confirming the in-vivo stability of RES-198AuNP Conclusions RES-198AuNP is a new nanosized radiopharmaceutical with potential applications in radiosynovectomy agent and/or as a radiotherapeutic agent for various cancers. Acknowledgements Research supported by IAEA and MURR. References Scarlati F, Sala G, et al 2007 Cancer Lett, 253, 124. Katti KV, Cutler C, et al 2012 PNAS, 109(31):12426. Katti KV, Cutler C, et al 2012 Wiley Interdiscip Rev Nanomed Nanobiotechnol, 4, 42. Chanda N, Kan P, et al 2010 Nanomedicine, 6, 201.

J Label Compd Radiopharm 2015: 58: S1- S411

S404: Poster

21st International Symposium on Radiopharmaceutical Sciences

404 Synthesis of radiolabelled boron-rich functionalized NPs as boron carriers for boron neutron capture therapy Kiran B. Gona, Jaya Lakshmi VNP Thota, Vanessa Gómez-Vallejo, Jordi Llop Molecular Imaging, CICbiomaGUNE, Donostia, san sebastian, GUIPÚZCOA, Spain Objectives To synthesize and radiolabel, water soluble, gold nanoparticles functionalized with cobaltbis(dicarbollide) (COSAN) derivatives as potential boron carriers with application in BNCT. Methods COSAN was treated with tetrahydropyran (THP) and then reacted with KSAc and ICl to achieve THP ring opening and iodination on one of the boron atoms of the cluster, respectively, as shown in Figure 1. Radiolabelling was achieved by isotopic exchange using either 124I or 125I. Final hydrolysis yielded the desired thioderivative. Gold NPs were prepared following a protocol of Brust and Schiffrin1. In brief, a mixture of the thiolated ligands was added to gold(III) salt solution in the presence of NaBH4. The thiolated ligands directly attached and self-organised on the growing gold nuclei, resulting in the formation of extremely small gold nanoparticles, which were dispersible and stable in water. Results The (protected) thiolated iodo-COSAN derivative could be obtained in overall yield of 76±6% after purification. Radiolabelling resulted in excellent yields for both 124I and 125I, with radiochemical conversions of 7080% when the reaction was conducted at 100ºC for 10 minutes. After quantitative hydrolysis and purification by solid phase extraction, the labeled thiolated derivative was diluted with the non-radioactive analog and NPs were prepared. Incorporation ratios were 20-60%. Conclusions Boron-rich functionalized and radiolabelled gold NPs were prepared. These could be appropriate boron-rich drugs with application in BNCT. Acknowledgements This project was partially funded by Ministerio de Ciencia e Innovación (Grant number CTQ2009-08810). References M. Brust, et al., J. Chem. Soc. Chem. Commun. 1994, 801.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S405

405 Strategies to Probe the Chemistry of Actinium Justin J. Wilson1, Maryline Ferrier1, Beau J. Barker1, Valery Radchenko1, Joel Maassen1, Enrique R. Batista2, Richard L. Martin2, Stosh A. Kozimor1, Marianne P. Wilkerson1, Kevin John1, Jon Engle1, Eva Birnbaum1 1 Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States, 2Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States Objectives The physical properties of 225Ac are well suited for targeted alpha therapy (TAT) [1]. However, the chemistry of Ac3+remains unexplored, hindering the design of chelating agents [2]. This work aims to characterize the properties of Ac3+ to enable the design of ligands for the delivery of 225Ac. Methods The 225Ac-labeling properties of a series of macrocyclic ligands (Fig. 1a) were investigated by radioTLC. Emission spectra of a fluorescent ligand (Ds-DOTAM, Fig. 1b) were acquired in the presence of different +3 metal ions, including Ac3+ in the form of 227Ac (t1/2 = 21.8 a), and probed by DFT. Results Radiolabeling of the nitrogen-rich ligands with 225Ac revealed the selective chelation of the 213Bi daughter isotope. These ligands rapidly and stably chelate Bi(III), suggesting their utility for 213Bi TAT. Treatment of DsDOTAM with other +3 metal ions revealed subtle shifts in the emission spectra and different quenching efficiencies. DFT calculations suggest that variations result in the electrostatic influence of the charged metal ion on the chromophore. The extent of this perturbation depends partly on the ionic radius of the metal. Conclusions The selective chelation of 213Bi in the presence of 225Ac by the macrocycles reflects the relative nitrogen donor atom affinity of Bi3+ and Ac3+, implicating strategies for the design of ligands for these ions. Fluorescence spectroscopy with DFT calculations has proven to be a valuable tool for probing the low concentrations of 227Ac3+ ions. Acknowledgements This work was funded by the U.S. DOE through the LANL LDRD Program and the Office of Science, Office of Nuclear Physics, via the Isotope Development and Production for Research and Applications subprogram, as well as a Seaborg Institute Fellowship (JJW). LA-UR-14-29484. References [1] Miederer, M, et al (2008) Adv Drug Delivery Rev, 60, 1371-82. [2] Deal, KA, et al (1999) J Med Chem, 42, 2988-92.

J Label Compd Radiopharm 2015: 58: S1- S411

S406: Poster

21st International Symposium on Radiopharmaceutical Sciences

406 Hypoxia imaging biomarker, Tc-99m-HL91, in injectable in situ forming thermosensitive implant at a murine tumor mode Bi-Fang Lee1, Nan-Tsing Chiu1, Xi-Zhang Lin2 1 Nuclear Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, 2Internal medicine, National Cheng-Kung University, Tainan, Taiwan Objectives Injectable in situ forming thermosensitive implant (IM) could be used for targeted molecular therapy of cancer. Tc-99m-HL91 (HL91) is a hypoxia imaging biomarker. It is to investigate HL91 imaging for IM therapy in a murine tumor model. Methods Cryo-TEM image was done. Mice were implanted with CT-26 colon tumors. When tumor volumes (tv) reached 300 mm3, pretreatment HL91-images were acquired. When tv reached 500mm3, the mice were divided into two groups: implant and controls. When tv reached 1000 mm3, H&E stain was done. Results IM was made of lipiodol, cholesterol, lecithin, dextrin, marcogol, hydroxystearate, vitamin E, cetyl alcohol, etc. Cryo-TEM showed co-existence of tubular vesicles and vesicles. Mice had no toxic effects after the injection of IM. H&E stain revealed necrosis of the tumor tissue. IM delayed tumor growth and enhanced the survival. Tumor hypoxia is associated with angiogenesis and tumor progression. Tumor hypoxia detected by Tc99m-HL91 imaging suggests a poor prognosis. Our results indicate that Tc-99m-HL91 imaging allows identifying tumor hypoxia and predicting success of IM therapy for cancer. Conclusions This study provides the first evidence that Tc-99m-HL91 can serve as an imaging biomarker for predicting the treatment responses of IM in a murine colon tumor model. Acknowledgements We thank Shang-Rung Wu for Cryo-TEM image. This work was supported in part by Grants from the National Science Council (NL1031046 ) and National Cheng Kung University Hospital (NCKUH 10306010), Taiwan. References Lee BF,et al. Cancer gene therapy 2012;19:255-262.

Cryo-TEM image.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S407

407 cGMP Kit Production of CycloSam® (Sm-153-DOTMP), A New Therapeutic Bone Agent R K. Frank1, Kelli Jay1, Jaime Simón1, Shannon L. Phillips1, George M. St. George1, Zechariah D. Sandlin1, Scot H. Ellebracht1, Leslie M. Swirsky1, Shannon L. Gonzales1, Richard E. Wendt, III2, Alan R. Ketring3 1 IsoTherapeutics Group LLC, Angleton, Texas, United States, 2Imaging Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States, 3Research Reactor, The University of Missouri, Columbia, Texas, United States Objectives We are developing a new therapeutic bone agent, CycloSam, based on Sm-153 and the macrocyclic chelant DOTMP. The objectives of this study were to design, manufacture, and test a cold kit for the radiopharmacy preparation of CycloSam. Methods A three-vial cold kit was designed to reproducibly prepare CycloSam in a radiopharmacy setting. SOPs for the cGMP manufacture and release of the three component vials were written. Multiple lots of these vials were manufactured and tested. In addition to release assays for each kit vial, the final CycloSam produced using this kit was evaluated in vivo. Batches of up to 150 mCi CycloSam were prepared. The formulated final product was tested for sterility and endotoxins. Its biodistribution was assessed in 240g Sprague-Dawley rats. HPLC and in vivo uptake and clearance were used to evaluate the shelf-life of the formulated product. Results The cGMP CycloSam kits consistently produced CycloSam with >99% radiochemical purity. Both the individual vials and the final CycloSam product were sterile and pyrogen free. Rat biodistribution experiments showed 42% bone uptake and a 45 hour effective half-life with practically no soft tissue accumulation. Based on preliminary results we have established a 24-hour shelf life for the final product. Human dosimetry estimates based on rat biodistribution were consistent with previously published results[1]. Conclusions Kits for the preparation of clinical grade CycloSam have been manufactured and successfully tested. These will allow for the easy and reproducible radiopharmacy preparation of this promising new therapeutic bone agent. Acknowledgements The authors gratefully acknowledge support from the National Cancer Institute, R43CA150601. References [1] J. Simón, R. K. Frank, D. K. Crump, W. D. Erwin, N. T. Ueno, and R. E. Wendt III, “A preclinical investigation of the saturation and dosimetry of 153Sm-DOTMP as a bone-seeking radiopharmaceutical,” Nucl. Med. Biol., vol. 39, no. 6, pp. 770–776, Aug. 2012.

J Label Compd Radiopharm 2015: 58: S1- S411

S408: Poster

21st International Symposium on Radiopharmaceutical Sciences

408 Treatment with Samarium-153-DOTMP in Dogs with Naturally Occurring Osteosarcoma for Therapeutic Benefit and as a Translational Model Kimberly A. Selting1, Jaime Simón2, Jimmy C. Lattimer1, Alan R. Ketring3, Sandra Axiak-Bechtel1, R K. Frank2, Richard E. Wendt, III4, Jeffrey N. Bryan1, Deborah Tate1, Charles Maitz1, Joni Lunceford1, Lindsay Donnelly1, Kevin Keegan1 1 Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States, 2IsoTherapeutics LLC, Angleton, Texas, United States, 3Research Reactor, University of Missouri, Columbia, Missouri, United States, 4MD Anderson Cancer Center, University of Texas, Houston, Texas, United States Objectives Evaluate 153Sm-DOTMP (CycloSam®) as a radiopharmaceutical targeted to bone tumors. DOTMP is a macrocyclic chelating agent with superior binding ratio and affinity to 153Sm compared to EDTMP (Quadramet™). The improved binding of radioisotope to chelant is predicted to result in less non-specific chelant binding and thus more efficient delivery of radiation to the affected bone and less systemic toxicity. Pilot investigations in dogs with spontaneously-occurring bone tumors have supported this. We endeavored to evaluate the use of this compound in a prospective clinical trial for dogs with osteosarcoma. Methods Dogs with osteosarcoma were recruited from the hospital population at the University of Missouri Veterinary Medical Teaching Hospital. All dogs were staged according to clinical practice with thoracic imaging and routine blood and biochemical evaluation. Dogs were assessed with both 99mTc-MDP and 18FDG-PET prior to 153 Sm-DOTMP injection at increasing dosages. Lameness was evaluated using motion detectors pre- and 1 month post injection, and 18FDG-PET was repeated 1 month after injection. Weekly hematologic and biochemical profiles were collected. Chemotherapy was administered after bone marrow recovery and dogs were monitored at regular intervals during and after treatment. Results An initial 7 dogs were treated at 1 mCi/kg with no resulting myelosuppression. Twelve additional dogs were treated at 1.5-2.3 mCi/kg with minimal myelosuppression. The prospective trial then opened and six dogs were enrolled (3 at 1.5 mCi/kg and 3 at 1.75 mCi/kg). Mild thrombocytopenia was noted but there was no other organ or systemic toxicity in any dog. One trial dog had almost complete resolution of pain and lameness. Conclusions CycloSam® administration to treat bone tumors may allow for earlier dosing with chemotherapy because of decreased myelosuppression, and shows promise for palliation of morbidity associated with bone tumors. Acknowledgements Research support: NCI R43CA150601 References

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S409

409 Synthesis and in vivo evaluation of 68Ga-probestin for renal cortical imaging by PET Gopal. Pathuri 1, Andria F. Hedrick 2, Godsfavour Umoru 1, Vibhudutta Awasthi 1, Benjamin D. Cowley 2, Hariprasad Gali 1 1 Pharmaceutical Sciences, The University of Oklahoma College of Pharmacy, Oklahoma City, OK, United States 2 Nephrology Section, Department of Internal Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States Objectives Aminopeptidase N (APN) is highly expressed in the epithelium of kidney proximal tubules. Our objective was to synthesize a novel gallium-68 labeled probestin (a known potent APN inhibitor) conjugate and evaluate its potential application for PET imaging of renal cortex. Methods Probestin conjugate containing a PEG2 linker and a DOTA chelator was synthesized using a manual solid phase peptide synthesis method. The 68Ga-labeling was achieved by reacting probestin conjugate (100 µg) with 68GaCl4- (54-110 MBq) in an acetate buffer (pH–4.5) at 90 °C for 10 min. Biodistribution of 68Ga-probestin was evaluated in healthy female Sprague Dawley rats (n = 4) at 1 hr p.i. Static abdominal PET/CT imaging was conducted to evaluate kidney uptake of 68Ga-probestin in two groups (n = 4) of healthy rats at 1 hr p.i. The first group was injected with 68Ga-probestin alone and the second group was co-injected with an excess of probestin to block the APN. Results 68Ga-probestin was obtained in a radiochemical purity >98% before HPLC purification. Biodistribution studies revealed an efficient clearance of 68Ga-probestin from circulation primarily through the renal-urinary pathway with 0.13 ± 0.01, 0.11 ± 0.07, and 14.3 ± 1.7 %ID/g remaining in blood, muscle, and kidney respectively. Over 76% of 68Ga-probestin uptake in kidney was blocked by co-injection of excess probestin. Conclusions The results indicate that 68Ga-probestin uptake is APN-specific and suggests it as a potential radiotracer for imaging renal cortex by PET. Acknowledgements The Oklahoma Center for the Advancement of Science and Technology (Project Number: HR13-210) and the NIH IDeA Networks of Biomedical Research Excellence grant P20GM103447 References

J Label Compd Radiopharm 2015: 58: S1- S411

S410: Poster

21st International Symposium on Radiopharmaceutical Sciences

410 Radiosynthesis and In Vitro Evaluation of [18F]FP-PEAQX as a Potential PET Radioligand for Imaging the GluN2A Subunit of the NMDA Receptor Selena Milicevic Sephton 1, Yves Auberson 2, Stjepko Cermak 1, Linjing Mu 1,3, Adrienne Müller Herde 1, Roger Schibli 1, Stefanie Dorthea Krämer 1, Simon M. Ametamey 1 1 Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland. 2Novartis Institute for Biomedical Research, Basel, Switzerland 3University Hospital Zurich, Zurich, Switzerland Objectives The fast excitatory neurotransmission of glutamate is primarily regulated by the N-methyl-D-aspartate receptors (NMDAR).1 Significant efforts have been made to develop PET radiotracer for imaging NMDAR,2 which is challenging due to the complex nature of the receptor. The quinoxaline derivative, FPPEAQX (Figure 1), exhibits a binding affinity of 2 nM towards the GluN2A subunit of the NMDAR and the purpose of this study was to establish its potential as a PET radiotracer. Methods Radiolabelling was accomplished in two steps from the tosylate precursor (Figure 1). Shake-flask method was employed to measure logD7.4 value. Autoradiography was performed on horizontal rat brain slices (20 mm, male, Wistar) and GluN2A antagonist NVP-AAM077, as well as GluN2B antagonist Eliprodil were used in excess as blocking agents. Results The radiosynthesis of [18F]FP-PEAQX was accomplished in 90 min from end of bombardment with a radiochemical yield of 10% (decay corrected) and >95% HPLC purity. Specific radioactivity ranged from 130-400 GBq/mmol. [18F]FP-PEAQX showed a logD7.4 value of –1.11±0.01 and good stability in both rat and human plasma as well as PBS over 180 min. Autoradiography showed activity distribution in agreement with the known distribution pattern of GluN2A (e.g. cortex and hippocampus).3 Blocking experiments with NVP-AAM077, showed a complete blocking, whereas with Eliprodil, no significant changes in radioactivity signals were observed. Conclusions [18F]FP-PEAQX showed high in vitro specificity towards GluN2A and therefore potential as PET radiotracer and due to its low logD7.4 value, it is expected to exhibit poor penetration across the BBB. The synthesis of more lipophilic derivatives is underway in our laboratory. References [1] Li F, et al (2009), New Engl J Med, 361, 302-303. [2] Sobrio F, et al (2010), Mini-Rev Med Chem, 10, 870-886. [3] Paoletti P, et al (2007), Curr Opin Pharmacol, 7, 39-47.

J Label Compd Radiopharm 2015: 58: S1- S411

21st International Symposium on Radiopharmaceutical Sciences

Poster: S411

411 Preliminary Evaluation of Dual 18 F- and 125I-Labeled Benzyloxybenzenes for Comparative Imaging of βAmyloid Plaques Yanping Yang 1, Mengchao Cui 2, Boli Liu 1, Hongmei Jia2 1 Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, China 2Beijing Normal University, Beijing, China Objectives Beyond the well-known planar and rigid Aβ binding probes, we recently reported radioiodinated flexible benzyloxybenzene analogs for lighting up the Aβ plaques [1]. Based on the novel benzyloxybenzene scaffold, we developed two ligands (7a and 12a) with fluoroethoxy groups and iodides para-substituted at both benzene rings in this study. Separate 18F and 125I radiolabeling can yield two pairs of radioisotopmers, enabling comparative PET/SPECT imaging with a single ligand. Methods Binding affinities were predicted by 3D-QSAR models, and then experimentally determined through in vitro competition binding assay using Aβ42 aggregates. Specific binding to Aβ plaques was verified by in vitro autoradiography. In vivo biological studies including biodistribution, dynamic PET/CT and SPECT/CT imaging, and metabolic stability were conducted in normal ICR mice (20-22 g, male). Results Ligands 7a and 12a displayed high binding affinities to Aβ42 aggregates (Ki = 19.5 and 23.9 nM), which agreed well with values predicted by the QSAR models. The high binding potency was verified by in vitro autoradiography on AD brain sections (Figure 1). All the desired ligands exhibited high initial brain uptakes (5.277.04 %ID/g at 2 min p.i.). However, 125I-labeled ligands showed much faster washout rates from the brain than corresponding 18F-labeled ligands. Dynamic microPET/CT and microSPECT/CT imaging data conformed the brain uptake pattern. In metabolic studies, no significant metabolism of [18F]7a and [18F]12a occurred in liver, while [125I]7a and [125I]12a rapidly degraded to two polar and hydrophilic metabolites, which could be absorbed by the intestine and excreted though the urine. The rapid metabolism in liver may be responsible for the faster clearance of these 125I-labeled compounds. Conclusions The preliminary results indicated that the 18F- and 125I-labeled benzyloxybenzene derivatives are promising candidates for PET/SPECT dual imaging of Aβ plaques in AD brains. Acknowledgements Supported by NSFC (21201019) References: Yang, Y. et al. J. Med. Chem. 57, 6030-6042 (2014).

J Label Compd Radiopharm 2015: 58: S1- S411

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21st International Symposium on Radiopharmaceutical Sciences Poster: S75 075 Evaluation of a Novel Tc-99m-Labeled HYNIC-Conjugated Lactam Bridge-Cyc...

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