Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
Development of a 18F-labeled prostate specific membrane antigen (PSMA) agent for PET imaging of prostate carcinoma (#322)
Subhani Okarvi1, Ibrahim AlJammaz1
1 King Faisal Specialist Hospital and Research Centre, Cyclotron and Radiopharmaceuticals, Riyadh, Saudi Arabia
Despite the advancement in tumor diagnosis and treatment, prostate cancer is still one of the main causes of cancer-related deaths among men. Recent developments of PSMA tracers have opened a new scope in molecular imaging of this disease. Although 68Ga labeled PSMA have shown great clinical effectiveness for targeting prostate cancer, short half-life of 68Ga and limited amount of 68Ga elution from generator reduced doses for larger patients group. The aim of this study was to develop a new PSMA ligand label with relatively longer half-life PET isotope 18F thus allowing more doses preparation.
The PSMA binding ligand, Glu-NH-CO-NH-Lys was prepared by standard solid-phase peptide synthesis technique and coupled to an aminooxyacetic acid linker group. We have used 4-[18F] fluorobenzaldehyde ([18F]FB-CHO) as a synthon to prepare 18F-PSMA ligand. Aminooxy-functionalized PSMA targeting molecule was mixed with ([18F]FB-CHO) in acidic buffer in MeOH (pH 4) at 90°C for 15 min.
The PSMA targeting ligand functionalized with aminooxy moiety was radiolabeled efficiently with 18F in high labeling efficiency and high radiochemical and metabolic stability. 18F-PSMA ligand exhibited high binding affinity and specificity towards PSMA-expressing LNCaP prostate cancer cell line, with the binding affinity of 13.79 ± 2.08 nM. In nude mice implanted with LNCaP xenografts, 18F-PSMA ligand showed rapid clearance from the blood and other nontarget organs and tissues. The uptake and retention of 18F-PSMA ligand in all the major body organs (excluding kidneys) was found to be low to moderate (below 5% injected dose/gram). The tumor uptake was 2.93% injected dose/gram at 1 h post-injection, with good tumor to blood and tumor to muscle ratios.
A new and effective 18F-labeled PSMA ligand against PSMA tumor expression has been successfully synthesized via chemoselective oxime formation between an aminooxy-functionalized PSMA and 18F-FB-CHO. Initial favorable in vitro and in vivo characteristics warrant further preclinical studies of this potential PET radiotracer.
Keywords: PSMA, PET, Tumor imaging, radiolabeling, new tracer
[11C]Osimertinib as TKI-PET tracer for the imaging of T790M mutated EGFR (#297)
Antonia A. Högnäsbacka1, Alex Poot1, Marion Chomet1, Wissam Beaino1, Robert C. Schuit1, Esther Kooijman1, Mariska Verlaan1, Maxime Schreurs1, Danielle J. Vugts1, Guus A. M. S. van Dongen1, Albert D. Windhorst1
1 Amsterdam UMC, Radiology and Nuclear Medicine, Amsterdam, Netherlands
Tyrosine kinase inhibitors (TKIs) are highly effective for the treatment of non-small cell lung cancer expressing mutated epidermal growth factor receptor (EGFR). Biopsies are repeatedly required to determine the mutational status and next course of treatment due to the development of treatment resistance. Osimertinib is a 3rd generation TKI able to inhibit the T790M resistance mutation, as well as the primary mutations L858R and Del19. We developed [11C]Osimertinib as a TKI-PET tracer as a non-invasive option for the diagnosis and treatment monitoring of NSCLC expressing mutated EGFR.
Precursors were reacted with [11C]CH3I, resulting in two isotopologs of [11C]Osimertinib (Fig 1). Three NSCLC cell lines expressing different stages of mutation development were chosen to evaluate the tracers: A549 (wild-type EGFR), HCC827 (primary mutation Del19) and H1975 (resistance mutation T790M). The in vivo stability and the tumor targeting potential of the tracers was evaluated and compared in xenografted female nu/nu mice at 5, 15 and 45 minutes post injection. Blood, heart, lungs, liver, kidney, head, muscle, skin, brain and tumors were collected, weighed and counted for radioactivity accumulation and the percentage injected dose per gram of tissue (%ID/g) was calculated for both tracers. The tracer uptake in tumors and brain was compared in HCC827 xenografted mice by PET/CT.
The metabolism of the tracers mainly resulted in polar radioactive metabolites. [11C-methylindole]Osimertinib decreased to 10% within 5 minutes, remaining at that level, while [11C-dimethylamine]Osimertinib decreased to 35% within 5 minutes and decreased to 16% during the remaining 40 minutes. Further analysis of the apolar metabolite fraction showed that the tracers did not metabolize as expected based on the osimertinib metabolism. Whether this is due to species specific metabolism, or due to the low concentration is unknown.
Two isotopologs of Osimertinib were designed, synthesized and studied in vivo. The metabolism, the biodistribution and PET/CT of both tracers are comparable. Future experiments are planned to elucidate the specificity of the tracers as well as their targeting potential.
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska–Curie grant agreement No 675417.
 Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, et al. 2005 'Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain', PLOS Medicine, 2, 3, e73
 Jänne, PA, Yand, JCH, Kim, DW, Planchard, D et al. 2015, 'AZD9291 in EGFR Inhibitor–Resistant Non–Small-Cell Lung Cancer', N Engl J Med, 372, 1689-1699
 Dickinson, PA, Cantarini, MV, Collier, J, Frewer, P et al. 2016, 'Metabolic Disposition of Osimertinib in Rats, Dogs, and Humans: Insights into a Drug Designed to Bind Covalently to a Cysteine Residue of Epidermal Growth Factor Receptor', Drug Metabolism and Disposition, 44, 8, 1201-1212
Keywords: Osimertinib, carbon-11, EGFR, NSCLC
PET study of L- and D-[methyl-11C]alanine in a mouse prostate cancer model (#392)
Pilar Castellnou1, Luka Rejc1, Unai Cossío1, Vanessa Gómez-Vallejo1, Albert D. Windhorst2, Alex Poot2, Arkaitz Carracedo3, Jordi Llop1
1 CIC biomaGUNE, Radiochemistry and Nuclear Imaging Group, San Sebastián, Spain
Besides accelerated glucose metabolism, cancer cells present increased uptake of amino acids and incorporation into protein synthesis. Because of this, radiolabelled amino acids are excellent alternatives to 2-[18F]Fluoro-2-deoxy-d-glucose (FDG) as PET tracers for oncology imaging. Here we report the fully automated, enantioselective radiosynthesis of L- and D-[methyl-11C]alanine using phase transfer catalyst of Schiff bases  (Figure 1) and their investigation in a mouse model of prostate cancer using Positron Emission Tomography (PET).
The preparation of L- and D-[methyl-11C]alanine was carried out using a GE FXCpro synthesis module. [11C]CH3I was reacted with the precursor in the presence cesium hydroxide and the phase transfer catalyst (PTC-1 and PTC-2 for L- and D- enantiomers, respectively; Figure 1). The crude was purified by HPLC and the collected fraction was hydrolyzed under acidic conditions (100°C, 9 min). The solution was finally neutralized to yield ready-to-inject radiotracer. PET-CT studies (60 min, dynamic acquisition) were carried out in a mouse model of prostate cancer, driven by the conditional deletion of the tumor suppressor PTEN (Phosphatase and Tensin homolog) in the prostate epithelia. Finally, animals were sacrificed and excised prostates were submitted to PET-CT imaging (10 min).
Both enantiomers could be obtained in a non-decay corrected radiochemical yield of ca. 10% with respect to [11C]CH3I in less than 40 min from the end of bombardment, including purification and reformulation. The radiochemical purity and enantiomeric excess values were >99% and >90%, respectively. PET studies showed that whereas L-[methyl-11C]alanine presents an elevated uptake in prostate, liver and brown fat (Figure 2a), D-[methyl-11C]alanine accumulates rapidly in the kidneys due to the elevated amount of DAO (D-Amino acid Oxidase) enzyme which is present in this organ (Figure 2c). Ex vivo PET studies confirmed the high uptake of L-[methyl-11C]alanine (Figure 2b) in the prostate in comparison with D-[methyl-11C]alanine (Figure 2d).
Both L- and D-[methyl-11C]alanine can be synthesized in high yields using a fully automated and enantioselective process. PET studies in a mouse model of prostate cancer showed a clearly different biodistribution for the enantiomers, with high uptake of L-[methyl-11C]alanine in the prostate, liver and brown fat and fast accumulation of D-[methyl-11C]alanine in the kidneys.
AcknowledgmentPart of the project was funded by the Spanish Ministry of Economy and Competitiveness (project ID: CTQ2017-87637-R).
 Filp U., Pekosak A., Poot A. J., Windhorst A. D., 2016, Enantioselective synthesis of carbon-11 labeled L-alanine using phase transfer catalyst of Schiff bases.,Tetrahedron, 72, 6551-6557.
Keywords: Amino acids, PET Imaging, Asymmetric Radiosynthesis, Prostate Cancer
PET imaging of neurotensin receptor-positive tumors with 68Ga-labeled antagonists: « the chelate makes again the difference ». (#153)
Emma Renard1, Mathieu Moreau1, 2, Pierre-Simon Bellaye2, Franck Denat1, Victor Goncalves1
1 Institut de Chimie Moléculaire de l’Université de Bourgogne, ICMUB UMR CNRS 6302, Université Bourgogne Franche-Comté, Dijon, France
Neurotensin receptor 1 (NTSR1) is involved in the development and progression of numerous cancers. A small molecule NTSR1 antagonist, named [177Lu]Lu-IPN01087 (previously [177Lu]Lu-3BP227) is currently evaluated in phase I/II clinical trials for the targeted therapy of metastatic pancreatic adenocarcinoma.1 The identification of a PET companion diagnostic would facilitate the selection of patients eligible for NTSR1-targeted radiotherapy. In this study, we evaluated the influence of the nature of the chelator on the biodistribution of NTSR1 antagonists.
Seven compounds were synthesized, based on the structure of the NTSR1 antagonist SR142948A, using different chelators for gallium-68 (NOTA, (R)-NODAGA, DOTA, DOTAGA and THP). The compound 3BP-227 was used as a reference. These compounds were metalated with natGa and their affinity for NTSR1 was determined on a competition assay on CHO cells overexpressing NTSR1. The compounds were radiolabeled with 68Ga, partition coefficients were measured and their biodistribution was evaluated on nude mice bearing a subcutaneous xenograft of HT-29 cells. PET-MRI images were recorded between 1 h and 2 h post-injection (6 MBq). Two hours post-injection, mice were sacrificed, organs were collected, weighed and γ-counted.
The seven antagonists were obtained through a convergent synthesis in 21 steps. Ki values were in the nanomolar range, demonstrating high affinity for NTSR1. The nature and number of chelating agents did not have an influence on affinity. The NOTA/NODAGA-based and THP-based compounds were radiolabeled with 68Ga in 5 min at 37°C, whereas 10 min at 95°C were required for the DOTA/DOTAGA-based compounds to achieve molar activities of ca. 20 MBq/nmol at time of administration. Good radiochemical yields (58-79%) and purities (>95%) were obtained after purification. PET images and ex vivo biodistribution showed significant differences between compounds. The THP-compound showed signal in liver, spleen and lungs whereas the other compounds showed a good tumor uptake (4-7 %ID/g) with a renal elimination pathway. For the NOTA/NODAGA-based compounds, signal was observed in intestines.
The influence of the nature and the number of chelators on a series of NTSR1 antagonists was evaluated, side-by-side, on a preclinical model of NTSR1-positive tumor. This led us to the identification of a lead compound combining both high tumor uptake and fast clearance from non-targeted organs. The evaluation of this tracer on a more relevant orthotopic model of pancreatic cancer is underway.
Financial support was provided by the French National Research Agency (ANR) under the programs AAP Générique 2017 (project ZINELABEL, ANR-17-CE18-0016-01), the CNRS and the Université de Bourgogne. This work is also part of the project Pharmaco-imagerie et agents théranostiques supported by the Conseil Régional de Bourgogne Franche-Comté through the Plan d’Action Régional pour l'Innovation (PARI) and by the European Union through the PO FEDER-FSE 2014/2020 Bourgogne program. The authors thank the "Plateforme d'Analyse Chimique et de Synthèse Moléculaire de l'Université de Bourgogne” (http://www.wpcm.fr) for access to analytical instrumentation.
 Baum, R. P.; Singh A.; Schuchardt, C.; Kulkarni, H.; Klette, I.; Wiessala, S.; Osterkamp, F.; Reineke, U.; Smerling, C. 2019, 177Lu-3BP-227 for Neurotensin Receptor 1-Targeted Therapy of Metastatic Pancreatic Adenocarcinoma: First Clinical Results, J. Nucl. Med., 59, 809-814.
Keywords: neurotensin, positron emission tomography, gallium-68
In ovo PET and MR imaging of surface-modified nanoparticles (#612)
Jessica Loeffler3, 1, Nina Eberhardt1, Richard Fiedler5, Markus Lau5, Marco Raabe4, Andrea B. F. Koch1, Ellen Scheidhauer1, Alireza Abaei2, 3, Hendrik Herrmann1, Christoph Solbach1, Tanja Weil4, Mika Lindén5, Ambros J. Beer1, Volker Rasche2, 3, Gordon Winter1
1 Ulm University, Department of Nuclear Medicine, Ulm, Germany
The Hen's egg test chorioallantoic membrane (HETCAM) model is a promising alternative to animal experiments with regard to the 3R principle of animal welfare. High biocompatibility and biodegradability of nanoparticles (NPs) as well as easy adaptation of the surface with target ligands or compounds to optimize bioavailability increase the interest in NPs for medical applications. In the present work nanoparticles including nanodiamonds (NDs), mesoporous silica -(MSN) and superparamagnetic iron oxide - nanoparticles (SPIONs) were analyzed regarding in ovo biodistribution and tumor accumulation.
NDs (Ø 100 nm, Microdiamant, IOM Leipzig, Germany) were coated with PEGylated HSA and the chelator p-SCN-Bn-deferoxamine (DFO, Macrocyclics, USA). SPION were modified using PEI (polyethylenimine) while MSNs were surface modified with a fragment of CD47 and p-SCN-Bn-deferoxamine. Stability of the [89Zr]-labeling was tested in sodium chloride, cell culture medium and human serum using thin layer chromatography and was monitored for 4 days. Tumor xenografts based on cancer cell lines LNCaP C4-2, PC-3, HeLa, TZM-bl and AR42J were established in the HETCAM model and the NPs were intravenously injected. MRI (BioSpec 117/16, Bruker) and PET (Focus120, Concorde Microsystems Inc.) measurements were performed after particle injection in ovo between embryo development day (EDD) 12 and EDD 16.
All particles showed a labeling efficiency of at least 76 %. The [89Zr]-labeling for all nanoparticles was stable in sodium chloride (0.9 %), cell culture medium and human serum during a 4-day observation period. In human serum, the labeling of the MSNs remained stable, while for the SPIONs the data after 2 days indicated instability, as the labeling stability decreased to 35 %. While MSNs and NDs were measured in PET and MR, SPIONs were only used in MRI. In T2 weighted MR images, after SPION injection, a signal reduction could be detected in the liver and heart region of the embryos, indicating a respective accumulation. PET imaging of the radiolabeled NDs and MSNs revealed accumulation in the heart and liver. Surface modified NPs predominantly accumulated in organs that will later form the reticuloendothelial system.
The HETCAM model was used to assess biodistribution of different NPs with various surface modifications. NPs were successfully radiolabeled and analyzed combining high-resolution MRI and highly-sensitive PET imaging. The ability of in ovo assessment of the biodistribution of nanoparticles has the potential to further optimize the surface functionalization prior to performing animal trials, thus enabling further reduction of animal experiments.
PET-MRI superposition and 3D visualization of [89Zr]-labeled ND biodistribution
Biodistribution analysis using PET imaging of [89Zr]-labeled nanodiamonds in the HET-CAM xenograft model showed a clear locally restricted enrichment (a). The superposition of the PET data with the high-resolution anatomical information of the MR imaging made it possible to identify the enrichment region as the liver and heart of the embryo (b).
Keywords: PET imaging, MRI, Nanoparticles, HETCAM, in ovo
Multi-modal PET and MR imaging in the Hen’s egg test chorioallantoic membrane (HET-CAM) model for initial in vivo testing of target-specific radioligands (#581)
Gordon Winter1, Andrea B. F. Koch1, Jessica Loeffler1, 3, Christoph Solbach1, Hao Li3, Gerhard Glatting1, Ambros J. Beer1, Volker Rasche2, 3
1 Universität Ulm, Nuclear Medicine, Ulm, Germany
Non-invasive testing of the biodistribution is important in drug development. In contrast to animal studies, the Hen’s egg test chorioallantoic membrane (HET-CAM) model do not rise legal authorization issues if sacrificed before hatching, and thus is an attractive alternative for initial biodistribution studies regarding the 3R principle in animal welfare. Due to an undeveloped immune response, vascularized tumor xenografts can be established on the CAM. The applicability of the HET-CAM tumor model for biodistribution assessment of radiolabeled compounds after systemic injection was tested.
After six days of incubation at 37.8°C, silicone rings were placed on the membrane of the opened chicken eggs (n=20). Xenografts were established using the PCa cell lines LNCaP C4-2 (1x106 cells; PSMA+) and PC-3 (7.5x105 cells; PSMA-) with Matrigel (40%, v/v). MR and PET imaging was performed starting on day 12. High-resolution anatomical imaging was provided by using a small animal MR (BioSpin 117/16, Bruker) based on the protocol of Zuo et al. [1, 2]. 150µl of [68Ga]PSMA-11 solution was injected intravenously followed by a dynamic 60min PET scan (Focus 120, Concorde Microsystems Inc.). Tumors have been excised after measurement for quantitative assessment by gamma counting (COBRA II, Perkin Elmer). MRI and PET data were fused by fiducial registration using the 3Dslicer software .
MR imaging was successful in all (n=20) chicken embryos and detailed visualization of the tumor growth could be provided. A successful injection into a CAM vein was achieved for 65% of the eggs. Accumulation of [68Ga]PSMA-11 was observed in the PSMA-positive tumor and chick embryo in the PET images (Fig. 1). In direct comparison, the PC-3 tumors showed lower accumulation of [68Ga]PSMA-11 as the LNCaP C4-2 tumors: 0.26 ± 0.71 %ID (LNCaP C4-2) vs. 0.07±0.05 %ID (PC-3); (n=20) yielding a ratio of 4.2±4.0 (PSMA-specific/non-specific). In the longitudinal PET measurement, the LNCaP C4-2 activity increased over time by 8.5±2.6 % due to progressing accumulation of the [68Ga]PSMA-11 in the PSMA positive tumor (Fig.2).
The suggested technique appears as an efficient approach to initial biodistribution assessment under almost in vivo conditions only limited by the only short time available for tumor growth monitoring and technical challenges of the injection into the rather small CAM veins.
The successful use of the HET-CAM model for an initial evaluation of the biodistribution of radiolabeled compounds by combining the highly sensitive PET and high-resolution MRI techniques was demonstrated. These results prove the potential of the HETCAM model as an attractive alternative to established small animal models for the assessment on the target specificity.
AcknowledgmentThe authors received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667192, and by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Center 1279 – The exploration of the Human Peptidome. Further the general support of the MoMAN imaging center of Ulm University is acknowledged.
 Zuo, Z. et al. Scientific reports 2017, 7, 46690.
 Zuo, Z. et al. NMR in biomedicine 2015, 28 (4), 440-7.
 Fedorov, A. et al. Magn Reson Imaging. 2012;30:1323-41
Keywords: HET-CAM, MRI, PET, xenograft
Exchange of Ile12 by Tle12 in NT(8-13) based radiotracers: Effects on internalization, stability and tumor uptake (#53)
Panagiotis Kanellopoulos1, 2, Berthold A. Nock1, Eric P. Krenning3, Theodosia Maina1
1 NCSR “Demokritos”, Molecular Radiopharmacy, INRASTES, Athens, Greece
The neurotensin subtype 1 receptor (NTS1R) is overexpressed in ductal pancreatic adenocarcinoma (PDAC). As it is absent from healthy pancreas or pancreatitis, NTS1R has been fairly regarded as a promising molecular target for PDAC . In the present study, we investigated the effects of Ile12/Tle12-substitution in the neurotensin(8-13) sequence on the cell internalization, in vivo stability and tumor targeting of resulting radiotracers. For this purpose, we used the 99mTc-DT5 ([99mTc-N4-βAla7,Dab8]NT(7-13), Dab=diaminobutyric acid) and 99mTc-DT6 (99mTc-[Tle12]DT5) pair as an example .
The two analogs were incubated in human NTS1R-positive colon adenocarcinoma WiDr cells at 37oC for 1 h; the amount of membrane bound (treatment with acid buffer) and internalized (treatment with 1 M NaOH) fractions was determined. Each of 99mTc-DT5 or 99mTc-DT6 was intravenously (iv) administered in healthy mice alone or with phosphoramidon (PA) and/or lisinopril (Lis) coinjection. Blood samples collected at 5 min pi were analyzed by radio-HPLC. The biodistribution of 99mTc-DT5 and 99mTc-DT6 was compared in SCID mice bearing WiDr xenografts at 4 h pi. The radiopeptides were administered without or with PA+Lis coinjection. Results were calculated as percentage of injected dose/gram of tissue (%ID/g).
The Ile12/Tle12-substitution had a negative effect on cell-internalization (99mTc-DT6: 0.8±0.4% vs 99mTc-DT5:3.2±0.3%), as previously observed for other NT-analogs . On the other hand, this modification drastically improved in vivo stability, with 99mTc-DT6 remaining ~60% intact at 5 min pi vs ~1% of 99mTc-DT5. Notably, by the PA+Lis combination the in vivo stability increased to ~80% for both compounds. Tumor values for 99mTc-DT6 were 1.7±0.7%ID/g, increasing to 3.5±0.3%ID/g during PA+Lis treatment. While for 99mTc-DT5 the original tumor values were 0.9±0.1%ID/g, they remarkably increased to 12.3±2.7%ID/g by the PA+Lis combination. The positive effect of higher in vivo stability of Ile12/Tle12-substituted analog on tumor targeting was apparent, despite its poorer cell internalization. However, during in situ stabilization by PA+Lis, the non-modified and better internalizing 99mTc-DT5 indisputably showed superior tumor uptake.
In accordance to previous reports , the current study on Ile12/Tle12-substituted NT-analogs has demonstrated that in situ stabilization of biodegradable peptide-radioligands is a valid alternative to structural modifications towards enhanced tumor-targeting. This approach warrants further validation in clinical practice.
 Reubi JC, et al. Gut, 42(4), 546-550 (1998)
 Maina T, et al. Eur. J. Nucl. Med. Mol. Imaging, 34(11), 1804-1814 (2007)
 Mascarin A, et al. Chem. Med. Chem., 11(1), 102-107 (2016)
 Nock BA et al. J. Nucl. Med., 55(1), 121-127 (2014)
Keywords: Pancreatic Cancer, PDAC, Neurotensin, 99mTc-radiotracer
Effects of different-length PEG spacers on the biological responses of 99mTc-DB1 mimics in GRPR-positive preclinical models (#52)
Panagiotis Kanellopoulos1, 2, Emmanouil Lymperis1, Aikaterini Kaloudi1, Marion de Jong3, Eric P. Krenning4, Berthold A. Nock1, Theodosia Maina1
1 NCSR “Demokritos”, INRASTES - Molecular Radiopharmacy, Athens, Greece
The overexpression of gastrin-releasing peptide receptors (GRPRs) in prostate cancer provides the rationale for delivering clinically useful radionuclides to tumor sites using peptide carriers . Radiolabeled GRPR-antagonists have shown higher tumor uptake and faster background clearance than agonists. In addition, they are safer for human use because they do not activate the GRPR upon binding . We herein compare the biological profile of three 99mTc-DB1  mimics, carrying different-length PEG linkers, in GRPR-positive cells and animal models.
An acyclic tetraamine chelator was coupled to the potent GRPR-antagonist DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt  via different length PEGx spacers (x= 2, DB7; x= 3, DB13; and x= 4, DB14). Competition GRPR-binding assays were performed in human GRPR-expressing prostate cancer PC-3 cell membranes. Cell-association tests were conducted by 1 h incubation of 99mTc-DB7/13/14 in PC-3 cells at 37oC. In vivo radioligand stability was studied by HPLC analysis of blood samples collected 5 min after injection of the radioligands without or with coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) in mice . Biodistribution patterns at 4 h post-injection (pi) were compared in mice bearing PC-3 xenografts without or with PA-coinjection.
DB7/13/14 displayed single-digit nanomolar affinities for the human GRPR. The uptake in PC-3 cells was comparable and consistent with a radioantagonist profile showing a slight trend 99mTc-DB7 (2.6±0.5%) >99mTc-DB13 (2.0±0.5%) >99mTc-DB14 (1.6±0.2%); n=3, (P<0.0001 only for 99mTc-DB7 vs. 99mTc-DB14). The radiotracers were found ≈70% intact in mouse blood with this percentage rising >94% after coinjection of PA. Their 4 h pi tumor uptake without or with PA-coinjection followed the trend: 99mTc-DB7, 4.5±1.2%ID/g to 6.1±1.1%ID/g > 99mTc-DB13, 4.1±0.8%ID/g to 5.8±1.2%ID/g > 99mTc-DB14, 3.7±1.0%ID/g to 4.0±0.3%ID/g. Thus, introduction of PEGx spacers in the 99mTc-DB motif led to good in vivo stability. A marginal decrease in cell uptake and tumor localization was observed by increasing the PEGx length from x= 2 to x= 4. Treatment of mice with PA stabilized all three radiotracers in mice circulation and consistently enhanced their tumor uptake.
The present study has shown that increase of PEG-spacer length in the 99mTc-DB7/13/14 series had little effect on GRPR-affinity, specific uptake in PC-3 cells, in vivo stability and tumor uptake. A significant change on in vivo stability and tumor uptake was observed only after treatment of mice with PA, thereby corroborating previous findings .
 Markwalder, R.; Reubi, J. C. Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res. 1999, 59 (5), 1152-1159.
 Maina, T.; Nock, B. A.; Kulkarni, H.; Singh, A.; Baum, R. P. Theranostic prospects of gastrin-releasing peptide receptor-radioantagonists in oncology. PET Clin. 2017, 12 (3), 297-309.
 Nock, B. A. Charalambidis, D.; Sallegger, W.; Waser, B.; Mansi, R.; Nicolas, G. P.; Ketani, E.; Nikolopoulou, A.; Fani, M.; Reubi, J.C.; Maina T. New gastrin releasing peptide receptor-directed [99mTc]Demobesin 1 mimics: Synthesis and comparative evaluation. J Med Chem. 2018, 61(7), 3138-3150.
 Nock, B. A.; Maina, T.; Krenning, E. P.; de Jong, M. "To serve and protect": Enzyme inhibitors as radiopeptide escorts promote tumor targeting. J. Nucl. Med. 2014, 55 (1), 121-127.
Keywords: Gastrin-Releasing Peptide Receptor, GRPR-antagonist, 99mTc-radiotracer, PEG-spacer