OP03 Selective extraction of medically-related radionuclides from proton-irradiated thorium targets
V. Radchenko1, J. W. Engle1, C. Roy2, J. Griswold2, M. F. Nortier1, E. R. Birnbaum1, M. Brugh1, S. Mirzadeh2, K. D. John1, M. E. Fassbender1
1Los Alamos National Laboratory, Los Alamos, New Mexico, USA; 2Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
Correspondence: V. Radchenko – Los Alamos National Laboratory, Los Alamos, New Mexico, USA
Background: Clinicians rely on nuclear medicine for the treatment of numerous diseasesimpacting millions of patients annually. Recently, Targeted Radiotherapy (TR) has been successfully advanced with the US FDA approval of several radionuclide based drugs]. Combinations of several types of radionuclide emissions for therapy (i.e., α-therapeutic agent combined with β- therapeutic) could lead to even more effective treatment options. One of the limiting factors in the development of TR as a widely adopted treatment option is the availability of select radionuclides with optimum emission properties (both in volume and periodicity of delivery), which poses a challenge due to the fact that different radionuclides typically require different target materials and/or nuclear reaction pathways for their formation.
Materials and
methods: We already published a successful strategy for the isolation of 225/227Ac from irradiated thorium targets [5]. We also published the recovery of Pa isotopes [6] from proton irradiated thorium. In this work, we propose the isolation of several other medically related radionuclides namely 103Ru, 223/225Ra, 111Ag from the same target material.
Results: Several methods based on ion exchange chromatography and solid phase extraction show promise for the co-extraction of 103Ru and Ra isotopes from thorium irradiated targets. Anion exchange in HCl media proved to be an efficient method for the isolation of 103Ru, while a combination of cation exchange resin/citrate and DGA resin/HNO3 is suitable for Ra isotopes separation.
Discussion/conclusion: Production yields for the proposed radionuclides were evaluated by comparison of actual product yields with calculated (predicted) yields. Radiochemical strategies for co-extraction of 103Ru and 223/225Ra isotopes based on ion exchange and solid phase extraction chromatography will be discussed.
OP04 Comparison of [68Ga]FSC(succ-RGD)3 and [68Ga]NODAGA-RGD for PET imaging of αvβ3 integrin expression
Chuangyan Zhai1, Gerben M. Franssen2, Milos Petrik3, Peter Laverman2, Clemens Decristoforo1
1Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria; 2Department of Radiology & Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands; 3Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic
Correspondence: Chuangyan Zhai – Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria
Background: The arginine-glycine-aspartic (RGD) peptide sequence serves as a high-affinity antagonist of the integrin αvβ3 receptor that plays an important role in tumor angiogenesis. Recently we reported [68Ga]FSC(succ-RGD)3, a trimeric RGD peptide, exhibited excellent targeting properties for αvβ3 integrin expression and significant improved tumor uptake compared to monomeric [68Ga]NODAGA-RGD.(1) Here we report the PET imaging properties of [68Ga]FSC(succ-RGD)3 in different xenograft tumor model and compared them with [68Ga]NODAGA-RGD.
Materials and methods: The PET imaging properties of [68Ga]FSC(succ-RGD)3 were studied in nude mice bearing M21 human melanoma xenografts and human glioblastoma U87MG xenograft tumor. A parallel PET imaging of 68GaNODAGA-RGD in same mouse bearing U87MG xenograft tumor was performed as a comparison.
Results: The static PET image of [68Ga]FSC(succ-RGD)3 in nude mice showed highly visualized tumors of M21 (positive) whereas nonvisualized tumor of M21-L (negative) tumor xenografts 1 h post injection confirming receptor-specific activity accumulation. The dynamic PET images of [68Ga]FSC(succ-RGD)3 showed rapid clearance of [68Ga]FSC(succ-RGD)3 from the circulation while the tumor remained clearly visible. A direct comparison of [68Ga]FSC(succ-RGD)3 with [68Ga]NODAGA-RGD in nude mice bearing U87MG xenograft tumor using PET/CT resulted comparable target/background ratio (tumor/kidneys ratio = 1.3 and 1.6, tumor/muscle ratio = 4.9, 5, respectively, 90 min post injection). The time activity curves from dynamic PET data showed an increase of the activity concentration of [68Ga]FSC(succ-RGD)3 in tumor firstly, then remained almost constant whereas that of [68Ga]NODAGA-RGD decreased quickly. The significant enhanced tumor uptake (3.8 vs. 1.6 % ID/g) in addition to the slower washout rate from tumor for [68Ga]FSC(succ-RGD)3 not only allows the PET imaging at late time points, but also provides the possibility to achieve the same image contrast using less radioactivity as well as detect low-level integrin expression.
Discussion/conclusion: [68Ga]FSC(succ-RGD)3 shows the advantages in the respect of delayed imaging, reduced radiation dose as well as monitoring low-level integrin expression in tissues in comparison to [68Ga]NODAGA-RGD, therefore it is a promising agent for integrin αvβ3 receptor imaging.
OP05 A new NPY-Y1R targeting peptide for breast cancer PET imaging
Ait-Mohand Samia1, Dumulon-Perreault Véronique2, Guérin Brigitte1,2
1Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et Sciences de la Santé, Université de Sherbrooke, QC, Canada J1H5N4; 2 Centre d’Imagerie Moléculaire de Sherbrooke (CIMS), CR-CHUS, Sherbrooke, QC, Canada J1H5N4
Correspondence: Guérin Brigitte – Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et Sciences de la Santé, Université de Sherbrooke, QC, Canada J1H5N4
Background: NPY-Y1 receptor (NPY-Y1R) is a promising target for breast cancer imaging. Previously, our group prepared and tested a series of truncated NPY analogs derived from BVD-15 (; [Pro30, Tyr32, Leu34]NPY(28-36)-NH2) for 64Cu-labeling). Unfortunately the biological half-live of the most potent tracer, [Lys(64Cu/DOTA)4]BVD15, when injected in mouse plasma was shorter than 15 minutes. In this study, we improved the design of BVD15 in order to increase its stability in vitro and in vivo and maintain its targeting capability.
Materials and methods: Modifications of the peptide backbone, the chelator and the use of D- and non-naturel amino acids were proposed to improve the peptide tracer stability. The peptides were synthesized on solid phase and conjugated to NOTA chelator. Binding studies on MCF-7 human breast cancer cells (2) were performed after each structural modification to make sure that the potency and the selectivity of the new NOTA-peptide conjugates to NPY-Y1R were maintained. Once active compounds were identified, they were radiolabeled with 64Cu for performing plasma stability, cellular uptake, internalization, and blocking studies on MCF-7 cells in order to rapidly identify the promising candidates for in vivo studies.
Results: A 64Cu/NOTA-BVD15 derivative presenting a very low Ki (9 nM) and showing a very high stability in plasma up to 20 h and in vivo for 30 minutes has been identified. Cell assays showed a constant uptake and internalization over the whole experiment. The internalized fraction after 2h was ~20%. The radiopeptide uptake was blocked in presence of an excess of unlabeled peptide.
Discussion/conclusion: We have identified a new 64Cu-labeled peptide presenting a good stability and an excellent affinity to NPY-Y1R. On the basis of the cellular results, the 64Cu/NOTA-BVD15 derivative appears to have a potential for the targeting of NPY-Y1R positive tumors.
OP06 The influence of multivalency on CCK 2 receptor targeting
D. Summer1, A. Kroess1, C. Rangger1, H. Haas2, P. Laverman3, F. Gerben3, E. von Guggenberg1, C.Decristoforo1
1Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria; 2Division of Molecular Biology/Biocenter, Medical University Innsbruck, Innsbruck, Austria; 3Department of Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands
Correspondence: D. Summer – Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria
Background: Multivalency has shown to enhance accumulation of receptor targeted radiopharmaceuticals, due to the avidity effect and increased apparent tracer concentration. For metabolically unstable peptides multimerisation may also delay metabolic processes like oxidation and enzymatic cleavage leading to loss of binding affinity. The aim of this study was to show the benefit of multimeristation on the targeting behaviour using a CCK2-receptor targeting, metabolically unstable Minigastrin peptide (MG11).
Materials and methods: Fusarinine C, a cyclic siderophore bearing three amino residues, was chosen as chelating agent and up to three peptide sequences (MG11= D-Glu1, desGlu2-6]-minigastrin) were conjugated by using maleimide chemistry. Radiotracers were labelled with 68Ga as well as with 89Zr following standard radiolabeling protocols. For in vitro characterisation stability studies, determination of the partition coefficient and cell uptake studies were performed. In vivo experiments included biodistribution studies (1h p.i) and static micro PET/CT imaging and were carried out in tumour xenograft bearing balb/c nude mice.
Results: Peptide conjugates could be labelled with 68Ga (5-15min RT, pH4-5) and 89Zr (60min, RT, pH7) achieving radiochemical yields of more than 98%. All complexes showed excellent stabilities in the presence of EDTA, DTPA, FeCl3 and in human serum after certain timepoints. Partition coefficient (logP) values ranging from -1 to -3 revealed a hydrophilic character of the radiotracers. As expected a decrease of hydrophilicity correlated with increasing grade of multimerisation. The cell uptake ranged from 10 to 15% per mg protein and could be reduced significantly by blocking with human minigastrin indicating specific receptor binding of all conjugates. Biodistribution studies showed a tumour uptake ranging from 5% (monomer) to approximately 10% ID/g (multimer) one hour post injection. Tumour to organ ratio was decreased by multimerisation and especially the kidney retention was increased significantly by the di- and trimeric radiotracers. Static animal imaging one and two hours after injection of 68Ga labeled conjugates confirmed the outcome of the biodistribution studies. 89Zr labeled counterparts showed very similar results but after 24 hours post injection the trimeric bioconjugate showed much better tumour imaging ability than the corresponding mono- and dimer.
Discussion/conclusion: Though these results are preliminary multimerisation seems to correlate with higher tumour uptake leading to better late timepoint imaging but high kidney retention seems to be a major limitation. Stability studies are ongoing.
OP07 SPECT Imaging of αvβ3 Expression by [99mTc(N)PNP43]- Bifunctional Chimeric RGD Peptide not Cross-Reacting with αvβ5
Cristina Bolzati1, Nicola Salvarese1,2, Fiorenzo Refosco1, Laura Meléndez-Alafort2, Debora Carpanese2, Antonio Rosato2,3, Michele Saviano4, Annarita Del Gatto5, Daniela Comegna5, Laura Zaccaro5
1IENI-CNR, Padua, Italy; 2DiSCOG-University of Padua, Padua, Italy; 3IOV Padua, Padua, Italy; 4IC-CNR, Bari, Italy; 5IBB-CNR, Naples, Italy
Correspondence: Cristina Bolzati – IENI-CNR, Padua, Italy
Background: Recently a new bifunctional chimeric RGD peptide (RGDechi), comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain, has been reported1. In vitro and in vivo biological studies evidenced that this chimeric peptide selectively binds to αvβ3 integrin and does not cross-react with αvβ52. In order to obtain an optimal SPECT radiotracer, a series of [99mTc(N)PNP] labelled peptides has been prepared and their pharmacological properties investigate.
Materials and methods: RGDechi-hCit (1) and three truncated peptide derivatives [RGDechi1_17 (2), RGDechi1_16 (3) and RGDechi1_14 (4)] lacking the two, three and five C-terminal amino acids, were synthetized in solid phase by Fmoc chemistry and conjugated with a cysteine linked to the Lys1 side chain to allow the labeling with [99mTc(N)PNP43]-synthon (PNP43 = (CH3)2P(CH2)2N(C2H4OCH3)(CH2)2P(CH3)2). In vitro stability and pharmacological parameters of the corresponding compounds, 99m
Tc1-4, were assessed. Challenges with an excess of glutathione and cysteine and Log P values were also investigated. Furthermore, radiolabeled peptides (99m
Tc1-4) were applied to study in vivo stability and the pharmacokinetic profiles on tumor bearing mice.
Results: All 99mTc-compounds were obtained with RCY > 90%. Log P values demonstrate the hydrophilic nature of the radiolabeled peptides ranging from -2.96 to -2.12. No significant variations in RCPs of the complexes were detected in challenge experiments with an excess (10 mM) of glutathione and cysteine. In general, a high in vitro stability was observed after incubation in human and mice sera as well as in mice liver homogenate; a slight degradation of 99m
Tc1-4 was found in kidneys homogenate. Cell uptake assays showed that, excluding 99m
Tc4 compound, 99m
Tc1-3 radiolabeled peptides accumulate selectively in cells expressing αvβ3 integrin and does not accumulate in cell expressing moderate levels of αvβ5 and undetectable levels of αvβ3 integrins. In agreement with in vitro findings, biodistribution studies showed that the 99m
Tc1-3 radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing αvβ3 and fails to accumulate in those expressing αvβ5 integrin.
Discussion/conclusion:
99mTc-labeled RGDechi, RGDechi1_17 and RGDechi1_16 chimeric peptides can be used for highly selective αvβ3 expression imaging by SPECT technology. Among the tested compounds, 99m
Tc2 possess the best distribution profile and highest localization in tumor expressing αvβ3. This research was supported by MIUR through PRIN 20097FJHPZ-004 and FIRB “RINAME”2010-RBAP114AMK, by Programma Operativo Nazionale Ricerca e Competitivita PON 01_02388 and by Italian Association for Cancer Research AIRC IG 13121.
OP09 New dienophiles for the inverse-electron-demand Diels-Alder reaction and for pretargeted PET imaging
Emilie Billaud1, Muneer Ahamed1, Frederik Cleeren1, Elnaz Shahbazali2, Tim Noël2, Volker Hessel2, Alfons Verbruggen1, Guy Bormans1
1Laboratory of Radiopharmacy, KU Leuven, Leuven, Belgium; 2Micro Flow Chemistry & Process Technology, Chemical Engineering and Chemistry Department, TU Eindhoven, Eindhoven, The Netherlands
Correspondence: Emilie Billaud – Laboratory of Radiopharmacy, KU Leuven, Leuven, Belgium
Introduction: In cancer research, pretargeted PET imaging has emerged as an effective two-step approach that combines the affinity and selectivity of antibodies with the rapid pharmacokinetics and favorable dosimetry of smaller molecule radiolabeled with short-lived radionuclides. This approach can be based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) “click” reaction between tetrazines and trans-cyclooctene (TCO) derivatives. Our project aims to develop new [18F]TCO-dienophiles with high reactivity for the IEDDA reaction, improved in vivo stability and favorable pharmacokinetics. New dienophiles were synthesized using an innovative continuous-flow micro-photochemistry process, and their reaction kinetics with a tetrazine were determined. In vivo stability studies of the most promising 18F-radiolabeled-TCO-derivative ([18F]trans-EB-70) was investigated, and its potential for pretargeted PET imaging was assessed.
Materials and Methods:
Organic chemistry Fluoro-cis-cyclooctene derivatives and mesylate precursors for radiofluorination were synthesized in 5-8 steps. Structures of intermediates and final compounds were confirmed by NMR and HRMS. Photochemistry
Trans-for-cis isomerization was performed using a microfluidic setup. Kinetics Reactions between dienophiles and 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine in MeOH at 25°C were monitored by UV-spectrophotometry at 290 nm (pseudo-first order conditions). 18
F-radiolabeling (semi-automated) Nucleophilic substitution of mesylate trans-EB-77 using [18F]KF,K222 was achieved in MeCN at 90°C for 15 min. [18F]trans-EB-70 was purified by HPLC. In vivo stability [18F]trans-EB-70 was evaluated in healthy NMRI mice, by ex vivo biodistribution (2, 10, 30, 60 min p.i.). In vitro pretargeting Prostate tumor slices (LNCaP and PC-3 cells) were successively incubated with a prostate-specific membrane antigen (PSMA) inhibitor modified with 3-(4-(trifluoromethyl)phenyl)-6-phenyl-1,2,4,5-tetrazine, and [18F]trans-EB-70. Direct incubation with the corresponding [18F]”preclicked”-compound, and blocking experiments (2-(phosphonomethyl)pentane-1,5-dioic acid) were performed.
Results Fluoro-cis-cyclooctene derivatives and mesylate precursors were synthesized in 3-35% overall yields. The trans-for-cis micro-photoisomerization reached yields of 48%. Reaction kinetics of the new dienophiles are fast, with k2 ranging from 475.6±32.8 to 1913.0±195.9 M-1.s-1. Radiosynthesis of [18F]trans-EB-70 was achieved in 60 min, with 12% radiochemical yield (decay-corrected), a radiochemical purity >99% (for at least 2h), and 70-188 GBq.μmol-1 specific activity. Biodistribution of [18F]trans-EB-70 in mice demonstrated absence of in vivo defluorination and a fast clearance via urinary and hepatobiliary systems. Regarding in vitro experiment, the binding on LNCaP tumor slices (expressing PSMA receptors) subjected to pretargeting was PSMA-specific and slightly inferior to the binding of [18F]”preclicked”-compound. No significant binding was observed in PC-3 cells (negative control).
Discussion/conclusion: We demonstrated that [18F]trans-EB-70 is a suitable dienophile for the IEDDA “click” reaction and for pretargeting applications. Therefore, [18F]trans-EB-70 will be investigated further in pretargeted μPET experiments.
Research support: SBO MIRIAD (IWT Flanders)
OP10 New complexing agent for Al18F-labelling of heat-sensitive biomolecules: Synthesis and preclinical evaluation of Al18F-RESCA1-HAS
Cleeren F1, Lecina J1, Koole M2, Verbruggen A1, Bormans G1
1Laboratory for Radiopharmacy, University of Leuven, Leuven, Belgium; 2Department of Nuclear Medicine and Molecular Imaging, University of Leuven, Leuven, Belgium
Correspondence: Cleeren F – Laboratory for Radiopharmacy, University of Leuven, Belgium
Introduction: The Al18F-labelling strategy involves formation of aluminium mono[18F]fluoride ({Al18F}2+) which is trapped by a suitable chelator –mostly bound to a biomolecule- in aqueous medium.1At this moment however, the need for elevated temperatures (100-120 °C) limits its widespread use. Therefore, we designed new restrained complexing agents (RESCAs) for use of this strategy at moderate temperature. RESCA1 is an acyclic pentadentate ligand with a N2O3 coordinative set that is able to complex {Al18F}2+ efficiently at 25 °C. To evaluate the stability and kinetic inertness of the chelate in vivo, RESCA1 was conjugated to human serum albumin (HSA) and labelled with {Al18F}. The Al18F-labelled conjugate was monitored in vivo for 6 h p.i.
Materials and methods: HSA was reacted with RESCA1-TFP. RESCA1-HSA (7.5 mg, 110 nmol) in 750 μl sodium acetate buffer (0.1 M, pH 4.5) was added to a freshly prepared {Al18F}2+ solution (1.4 GBq, 50 nmol AlCl3, 12 min, RT). The product was purified using a PD-10 column and RCP was determined with SEC-HPLC. To test stability in serum, Al18F-RESCA1-HSA in 100 μl PBS was added to rat serum (900 μl), kept at 37°C and monitored up to 4 h. Ex vivo biodistribution was studied at 1 h, 3 h and 6 h p.i. of Al18F-RESCA1-HSA (2-7.5 MBq) in healthy female rats. Small-animal whole-body PET imaging was performed using a FOCUS 220 tomograph.
Results: RESCA1-HSA was obtained with a chelator-to-protein ratio of 3, estimated by ESI-TOF-HRMS analysis. Al18F-RESCA-HSA was prepared in high RCY (>70%) and purity (>95%) in < 30 min. 91% of product was still intact in rat serum after 4 h incubation. Distribution in rats showed high retention in blood with 5.42 ± 0.23% ID/g, 4.93 ± 0.22% ID/g and 3.66 ± 0.06% ID/g at 1h, 3h and 6 h respectively from which the blood biological half-life was calculated to be 8.6 h. No significant increase in bone uptake was observed, indicating excellent in vivo stability of the Al18F-labelled construct.
Discussion/conclusion: We successfully labelled for the first time a heat-sensitive biomolecule via the Al18F-method in one radiolabelling step. Al18F- RESCA1-HSA showed excellent stability and favourable properties for PET blood pool imaging applications.
OP11 A novel versatile precursor efficient for F-18 radiolabelling via click-chemistry
B. Lugatoa1, S. Stucchia1, E.A. Turollaa1, L. Giulianoa1, S.Toddea1, P. Ferraboschib2
1Department of Medicine and Surgery, Tecnomed Foundation, University of Milano-Bicocca, Milan, Italy; 2Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy
Correspondence: B. Lugatoa – Department of Medicine and Surgery, Tecnomed Foundation, University of Milano-Bicocca, Milan, Italy
Introduction In the last years, the Cu(I)-catalyzed Huisgen [3+2] cycloaddition between terminal alkynes and azides emerged as a powerful tool in F-18 radiolabelling of biomolecules such as peptides, because of its regioselectivity, mild aqueous organic conditions, reduced reaction times, and high yields.(1).A weak point of the method is the lack of suitable commercially available, stable precursors.(2) In this paper we report the synthesis and F-18 radiolabelling of a new, versatile, easy to handle, and stable azido precursor useful for click-chemistry.
Materials and methods: Reagents and solvents were purchased from Sigma-Aldrich. [18F]Fluoride was produced with an IBA Cyclone 18/9 cyclotron. Radioactive synthesis were carried out on a fully automated radiosynthesis module (GE TracerLab FX-FN Pro), and analyzed by analytical RP-HPLC with UV and radiochemical detectors. Non-radioactive compounds were fully characterized by NMR, ESI-MS and IR.
Results: A series of bifunctional precursors, bearing the azido moiety and different leaving groups (e.g. tosylate, mesylate, iodo), coupled to a short polyetyleneglycol chain (to improve their stability and hydrophilicity) were designed and successfully prepared following a ten-step synthetic pathway. A protection-deprotection strategy of functional groups achieved the precursors and the fluorinated reference with good yield and purity. Precursors were radiolabelled with F-18 and then coupled to propargylglycine as alkyne counterpart. [18F]Fluoride was purified following standard procedure, and nucleophilic displacement of the iodo leaving group took place at 100° C, in 20 min. The resulting labelled azide was successfully purified using a Sep-Pak tC18 cartridge (51% radiochemical yield not decay corrected, 93% radiochemical purity). The purified azide was then conjugated to propargylglycine, showing 52% conversion within 30 min at room temperature. Purification and formulation have still to be optimized.
Discussion/conclusion: A new optimized precursor useful for F-18 radiolabelling and click-chemistry was prepared. It demonstrated to be effective in radiolabelling non-protected alkynyl-modified aminoacids, by a fully automated synthetic procedure. Good results, in terms of radiochemical yield and purity, were obtained from the iodo-derivative precursor. Purification and formulation of the final cycloaddition product are in progress.
OP12 A general applicable method to quantify unidentified UV impurities in radiopharmaceuticals
R.P. Klok1, M.P.J. Mooijer1, N.H. Hendrikse1,2, A.D. Windhorst1
1Department of Radiology & Nuclear Medicine, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Clinical Pharmacology & Pharmacy, VU University Medical Center, Amsterdam, The Netherlands
Introduction: Radiopharmaceuticals are released for administration by a quality control procedure against pre-set specifications. One of these release specifications is the chemical purity of the drug product, determined with High Pressure Liquid Chromatography (HPLC) and UV detection. In the European Pharmacopeia (EP) hardly any specifications are given for the chemical purity of a radiopharmaceutical. When unknown impurities are present in the chromatogram, the decision if the radiopharmaceutical can be released, is very frequently based on unclear parameters like ‘no unidentified UV signals present’. There is a need for an objective specification in order to have a save and reliable release.
Aim: The purpose of the presented work is to define a generally applicable method to define tolerances for unidentified impurities in radiopharmaceuticals.
Materials and methods: A retrospective analysis was performed on HPLC analysis results of [11C]Flumazenil, [11C]PIB, [11C]Erlotinib, [11C]DPA713, [18F]PK209 and [18F]FES. Quantification of the carrier signal in the UV chromatogram was determined by use of calibration curves, utilizing Chromeleon® 6.8. Unidentified impurities were semi-quantified utilizing the surface area in the UV chromatogram relative to the quantified carrier signal. Based on the EP. monography of [11C]Flumazenil and [18F]FET, the specification for unidentified UV impurities was determined to be 0.22 pmol/injection volume for a single unidentified impurity and 0.88 pmol/injection volume for the total of unidentified impurities. This specification was tested for over 500 batches of the radiopharmaceuticals and compared to the less specific parameter ‘no unidentified UV signals present’.
Results: In a pilot assessment we encountered in 5-10% cases unidentified UV impurities leading to rejection of the batch, based on the specification ‘no unidentified UV signals present’. Of these rejected batches 25% was also rejected with the new defined specification. Reason for this reduced number of rejections is that with the new specification the presence of unidentified impurities is evaluated objectively. The analysis of the full database is currently on-going.
Discussion/conclusion: With this method the amount of unidentified impurities can be estimated in an optimal and objective way, utilizing EP limits of [11C]Flumazenil and [18F]FET, without operator variability.
OP13 Development of [18F]Fluoro-C-glycosides to radiolabel peptides
Collet C.1,2, Petry N.1,3, Chrétien F.1,3, Karcher G.1,2,4, Pellegrini-Moïse N.1,3, Lamandé-Langle S.1,3
1Université de Lorraine, F-54500 Vandoeuvre les Nancy – France; 2NancycloTEP, Plateforme d’imagerie expérimentale, 54500 Vandoeuvre les Nancy – France; 3CNRS, UMR 7565 SRSMC, F-54506 Vandoeuvre les Nancy – France; 4CHU de Nancy-Brabois, F-54511 Vandoeuvre les Nancy – France
Correspondence: Collet C. – Université de Lorraine, F-54500 Vandoeuvre les Nancy – France
Introduction: The 18F-labeling of peptides for PET applications has been used for many years.1 However, the sensitivity of these peptides does not allow their direct radiolabelling under harsh conditions, except few recent examples. A solution is to use a prosthetic group, an easily radiolabeled small molecule, subsequently coupled in mild condition to the peptide. In continuation of our previous work,2 we therefore propose to develop and use new C-glycoside-based prosthetic groups. The use of sugar derivatives as prosthetic group would improve bioavailability and pharmacokinetic properties of peptides.
Materials and methods: These C-glycoside derivatives should have a good leaving group thus allowing easy substitution by fluorine-18, i.e. triflate. A copper catalyzed azide alkyne cycloaddition (CuAAC) was then used for coupling these carbohydrates with a peptide. Some model peptides containing a cysteine residue as RGDC and c(RGDfC) are used. The high nucleophilicity of the thiol function can thus be exploited to prepare S-propargylated derivatives. The fully automated radiosynthesis of these [18F]fluoro-glycopeptides was performed on an AllInOne® (Trasis) synthesizer.
Results: Triflated precursors of these C-glucosides prosthetic groups and the non-radioactive references were synthesized in alpha and beta configuration. Fluoride-18 radiolabeling was optimized and the automated radiosynthesis of [18F]fluoro-glycopeptides with some model peptides (RGDC, c(RGDfC)) was presented.
Discussion/conclusion: The synthesis and radiosynthesis of 6-[19F/18F]fluoro-C-glycosides displaying a three carbon arm terminated with an azide group were optimized. CuAAC of fluoro-C-glycosides with RGD derivative peptides gave [19F/18F]fluoro-glycopeptides in good yields.
OP14 A Microfluidic Approach for the 68Ga-labeling of PSMAHBED-CC and NODAGA-RGD
Sarah Pfaff1,2, Cecile Philippe1, Markus Mitterhauser1,3, Marcus Hacker1, Wolfgang Wadsak1,2
1Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria; 2Department of Inorganic Chemistry, University of Vienna, Vienna, Austria; 3LBI for Applied Diagnostics, Vienna, Austria
Correspondence: Sarah Pfaff – Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Medical University of Vienna, Vienna, Austria
Introduction: In nuclear medicine a remarkably high demand of 68Ga-radiotracers has emerged during the last decade. For a variety of non-68Ga-containing radiotracers a microfluidic approach for their syntheses could be established, enabling an enhancement of yields due to high surface-to-volume ratios1,2. In this proof-of-principle study, the 68Ga-radiolabeling of PSMAHBED-CC and NODAGA-RGD using a microfluidic approach was evaluated. Furthermore, adding TWEEN 20 (a surfactant suitable for in vivo applications) and its impact on the radiochemical yield was explored.
Materials and methods: The syntheses of 68Ga-PSMAHBED-CC and 68Ga-NODAGA-RGD (both precursors from ABX) were performed using an Advion NanoTek LF microfluidic device. The system incorporates a flow-through reactor that consists of a silica capillary (l=2 m, Ø100 μm, V=15.6 μL). 68Ga3+ was obtained from a 68Ge/68Ga-generator (3.7 GBq; Obninsk) according to a fractionized protocol. The precursor and 68Ga3+ were loaded in two storage loops and distinct volumes thereof were pushed through the reactor with different flowrates (30, 50, 80 μL/min) at different temperatures (25, 50, 80, 100, 120, 150°C) using NaOAc and HEPES for pH adjustment, respectively. Additionally, the influence of TWEEN 20 as a surfactant, to reduce known adsorption effects in microfluidic tubing, was investigated.
Results: Accomplished experiments revealed feasibility of 68Ga-labeling of PSMAHBED-CC and NODAGA-RGD using a microfluidic device. All temperatures and flowrates resulted in mean radiochemical yields in a range of 20-55%. Syntheses at higher temperatures and flowrates proved to be more efficient. For instance, HEPES buffered syntheses at 100°C and flowrate of 80 μL/min yielded 68Ga-PSMAHBED-CC in 42.6 ± 22.1 % (n=10) and 68Ga-NODAGA-RGD in 49.7 ± 32.5 % (n=7). Applying the same parameters, TWEEN 20 could strikingly improve the yield of 68Ga-PSMAHBED-CC to 70.8 ± 16.8 % (n=13).
Discussion/conclusion: This study provides the proof-of-principle of 68Ga-labeling in a microfluidic “flow-through” system. The application of TWEEN 20 led to drastically increased yields of 68Ga-PSMAHBED-CC due to its surfactant nature. As a result, this microfluidic approach will be pursued to increase availability of 68Ga-radiopharmaceuticals according to the dose-on-demand principle.
OP16 Surprising reactivity of astatine in the nucleophilic substitution of aryliodonium salts: application to the radiolabeling of antibodies
François Guérard1, Yong-Sok Lee2, Sébastien Gouard1, Kwamena Baidoo3, Cyrille Alliot1,4, Michel Chérel1, Martin W. Brechbiel3, Jean-François Gestin1
1Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Unité INSERM 892 - CNRS 6299, Nantes 44007, France; 2Center for Molecular Modeling, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892, USA; 3Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 4Arronax GIP, Nantes 44817, France
Correspondence: François Guérard – Centre de Recherche en Cancérologie Nantes-Angers (CRCNA), Unité INSERM 892 - CNRS 6299, Nantes 44007, France
Introduction: Aryliodonium salts have recently emerged as versatile precursors for the synthesis of 18F-radiolabeled compounds for PET imaging.1,2 However, little is known about the applicability of these reagents for labeling with the heaviest radiohalogens iodine and astatine, both useful for nuclear imaging and/or therapy.3 In this study, we aimed at probing the reactivity of radio-iodide (125I) and astatide (211At) towards diaryliodonium salts in order to assess their usefulness for radiolabeling biomolecules of interest in nuclear medicine.
Materials and methods: First, parameters of radio-iodination and astatination reaction (solvent, temperature, duration and counter-ion of iodonium) were studied on model compounds. Bifunctional iodonium salts were then designed, allowing the synthesis of [125I]-SIB and [211At]-SAB, two prosthetic groups widely used for radio-iodination and astatination of biomolecules. Both [125I]-SIB and [211At]-SAB were conjugated to the multiple myeloma targeting mAb 9E7.4 (anti-CD138). Conjugation yields and resulting immunoreactivity were compared with the conventional arylstannane chemistry approach.
Results: Initial reaction parameters studies highlighted a striking difference of reactivity between radio-iodide and astatide that could not be anticipated from the trends observed within the halogen series. Not only the astatination reaction was highly efficient at much lower temperature than iodination, but it appeared also solvent and counter-ion independent (not iodination). Thermochemical studies highlighted a large difference of activation energy in acetonitrile between both halogens with Ea = 23.5 kcal/mol and 17.2 kcal/mol for radio-iodination and astatination, respectively. Quantum chemical calculations support the hypothesis that astatination occurs via a monomeric iodonium complex whereas iodide occurs via a dimeric complex which requires more energy for the reaction to proceed. This explains the large reactivity difference observed. Radiolabeling of an antibody with specifically designed iodonium salts outperformed conventional arylstannane chemistry approaches in terms of global efficiency (radiochemical yields >90%, conjugation yields ≈ 75%, and simpler purification: no HPLC needed) with excellent preservation of immunoreactivity of the IgG with both radionuclides and less concerns regarding the toxicity of precursors and side products.
Discussion/conclusion: In comparison with the conventional arylstannane approach, aryliodonium salts appear as more efficient precursors for preparation of radio-iodinated and astatinated compounds. Furthermore, they allow simpler purification procedures (easy transfer to automation), with the additional advantage of a much lower toxicity, which is of primary importance for human use. Most of all, the unexpected reactivity of astatine we unveiled highlights that a lot is still to be discovered about the chemistry of this radioelement which remains to date largely unexplored.
OP17 64Cu-NOTA-pertuzumab F(ab')2 fragments, a second-generation probe for PET imaging of the response of HER2-positive breast cancer to trastuzumab (Herceptin)
Lam K1, Chan C1, Reilly RM1,2,3
1Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada; 2Department of Medical Imaging, University of Toronto, Toronto, Canada; 3Toronto General Research Institute, University Health Network, Toronto, Canada
Correspondence: Lam K – Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada
Introduction: SPECT/CT imaging with 111In-BzDTPA-pertuzumab detected trastuzumab (Herceptin)-mediated HER2 downregulation in human breast cancer xenografts in mice and was correlated with a good response to treatment. This agent is now being studied in a Phase I/II clinical trial sponsored by OICR (PETRA; ClinicalTrials.gov identifier: NCT01805908). Our objective was to develop and characterize a second-generation positron-emitting analogue for PET/CT imaging, 64Cu-NOTA-pertuzumab F(ab')2, to provide greater sensitivity, more accurate radiotracer quantitation and a lower radiation absorbed dose.
Materials and methods: To determine the optimal dose, mice with subcutaneous HER2-overexpressing tumours were injected with 5, 50, 100 or 200 μg of 64Cu-NOTA-F(ab')2 (2.2±0.6 MBq) and sacrificed at 24 h p.i. for biodistribution. To determine the normal tissue distribution, pharmacokinetics and radiation dosimetry of 64Cu-NOTA-F(ab')2, non-tumour bearing Balb/c mice were administered 50 μg of 64Cu-NOTA-F(ab')2 (2.9±0.3 MBq) and sacrificed at select time points. Three groups of mice bearing HER2-overexpressing tumours were injected with 64Cu-NOTA-F(ab')2 (50 μg; 10.6±0.4 MBq) with or without administration of pertuzumab (1 mg) 24 h before, or with 64Cu-NOTA-F(ab')2 prepared from nonspecific human IgG (50 μg; 8.2±1.9 MBq) to demonstrate specificity. Mice were imaged with PET/CT at 24 and 48 h p.i. and sacrificed for biodistribution.
Results: The 50 μg dose of 64Cu-NOTA-F(ab')2 showed the highest tumour to blood ratio, followed by the 100, 5, and 200 μg doses (18.2±7.2, 15.4±0.8, 14.0±5.0, and 10.9±1.8, respectively). In non-tumour bearing mice, only the kidney retained significant radiotracer uptake at 24 h p.i. (49.1±5.9 %ID/g). Initial estimates for t
½
α and t
½
β were 1.2 h and 6.0 h, respectively. The projected total body radiation absorbed dose to humans was 0.02 mSv/MBq, half that estimated for 111In-BzDTPA-pertuzumab. Tumour to normal tissue contrast of PET/CT images appeared similar between the 24 and 48 h p.i. imaging time points. Tumour accumulation of 64Cu-NOTA-F(ab')2 was significantly higher in mice administered 64Cu-NOTA-F(ab')2 without pertuzumab blocking (8.4±3.4 %ID/g) relative to mice pre-injected with 1 mg of pertuzumab (3.9±0.5 %ID/g; P < 0.05), and mice administered 64Cu-NOTA-F(ab')2 prepared from non-specific human IgG (2.7±0.5 %ID/g; P < 0.05).
Discussion/conclusion:
64Cu-NOTA-F(ab')2 is HER2 specific and can visualize HER2-overexpressing tumours at 24 or 48 h p.i. with PET/CT imaging. The lower projected radiation absorbed doses for 64Cu-NOTA-F(ab')2 compared to 111In-BzDTPA-pertuzumab may make this a more attractive imaging agent to detect trastuzumab-mediated HER2 downregulation in breast cancer. Supported by grants from the OICR Smarter Imaging and High Impact Clinical Trials (HICT) Programs.
OP18 Development of radiohalogenated analogues of a avb6-specific peptide for high LET particle emitter targeted radionuclide therapy of cancer
Salomé Paillas1, John Marshall2, Jean-Pierre Pouget3, Jane Sosabowski1
1Centre for Molecular Oncology, Barts Cancer Institute, Charterhouse Square, Queen Mary University of London, London, UK; 2Centre for Tumour Biology, Barts Cancer Institute, Charterhouse Square, Queen Mary University of London, London, UK; 3Institut de Recherche en Cancérologie de Montpellier, Inserm U1194, Montpellier, France
Correspondence: Salomé Paillas – Centre for Molecular Oncology, Barts Cancer Institute, Charterhouse Square, Queen Mary University of London, UK
Introduction: Targeted radionuclide therapy (TRT) of solid tumors has a limited efficacy mainly because these tumours have high radioresistance and take up limited amounts of radiolabeled vectors. Strategies to overcome this include the use of small peptides combined with radionuclides that emit highly cytotoxic particles, namely high linear energy transfer (LET) particles such as alpha particles and Auger electrons. We have chosen to target the epithelial-specific integrin αvβ6, which is weak or absent on normal tissues but is upregulated on many cancers where it is strongly associated with reduced survival. One targeting vector is a 20 mer peptide, A20FMDV2 which we have previously used to image αvβ6-positive tumours with SPECT (In-111) or PET (F-18, Ga-68). The peptide showed extremely high kidney retention when radiolabelled with radiometals, but this was not seen with the F-18 analogue. Due to anticipated dose-limiting toxicity in kidney for radiometal-based TRT, we have developed a peptide suitable for radiohalogenation with the Auger electron emitter I-125 and the alpha particle emitter At-211.
Materials and methods: The 20 mer high affinity αvβ6-targeting peptide, A20FMDV2 used in previous work has been derivatised to contain a trimethylstannyl benzamide moiety so that it can be radiolabelled with I-125 and At-211. The peptide has been radiolabelled with I-125 and radioligand binding and internalization assays have been carried out in cells that overexpress αvβ6 and compared with that of the 111In-DTPA-A20FMDV2 studied previously. Clonogenic assays have been carried out on both the non-radiolabelled peptide and the radiolabelled analogue in both αvβ6-positive and negative cells to determine the effect of high LET irradiation.
Results: Radiolabelling efficiency with I-125 was >91 % and the analogue showed high binding affinity and rapid internalisation. Clonogenic assays showed that the non-labelled peptide alone was able to inhibit cell growth and that this effect was not seen on αvβ6-negative cells. This effect was enhanced when radiolabelled with a high LET particle emitter.
Discussion/conclusion: The αvβ6-specific peptide when radiolabelled with a high-LET particle emitter shows promise as an agent for targeted radionuclide therapy.
OP19 Ligand Specific Efficiency (LSE) as a guide in tracer optimization
Emmanuelle Briard1, Yves P. Auberson1, John Reilly2, Mark Healy2, David Sykes3
1Novartis Institutes for Biomedical Research, Basel, Switzerland; 2Novartis Institutes for Biomedical Research, Cambridge, USA, 3University of Nottingham, Nottingham, UK
Correspondence: Emmanuelle Briard – Novartis Institutes for Biomedical Research, Basel, Switzerland
Introduction: Successful radiotracers result from a favorable combination of target density, ligand affinity, nonspecific binding and permeation. All these parameters can be measured independently and the interplay between some of them is well known: for instance, Bmax/Kd > 10. Importantly, the required affinity of a tracer is also correlated to nonspecific binding: An increased affinity is beneficial only if the nonspecific binding remains constant. This work aimed at identifying an index taking into account the relationship between these two parameters, to guide optimization from the early stage of tracer development projects.
Materials and methods: Similarly to the Ligand Efficiency (LE) index, we explored the usefulness of the Ligand Specific Efficiency index (LSE), which we defined as the ratio between affinity (expressed as e.g. pIC50 or pKd), and the logarithmic value of the experimental non-specific binding measurement, CHI(IAM).[Jiang] LSE provides a measure of affinity, normalized to non-specific binding. It shows how efficient the ligand is at binding to the desired target, compared to all other non-specific binding partners.
Results: A series of well-described PET tracers was evaluated to set the LSE threshold. This analysis showed that an LSE > 5 and preferably LSE > 5.4 is required for a successful tracer. This concept was applied to the development of a prostacyclin receptor (IPR) tracer. Our chemical starting point was Ro1138452,[Clark] which we selected based on encouraging overall properties, including a high affinity for IPR (Ki = 0.23 nM). In contrast, its CHI(IAM) value of 58 clearly indicated a high tendency for non-specific binding. Despite such high non-specific binding, Ro1138452 has a LSE value of 5.1, which is close to the minimum value that would be expected for a successful PET tracer. This raised hope that some improvement in binding specificity would allow the use of a close derivative for imaging purposes. The use of LSE during the IPR tracer optimization will be presented.
Discussion/conclusion: LSE is based on a rather intuitive concept, in the sense that a good PET tracer candidate should have the optimum balance of affinity and binding specificity. It is a convenient index to evaluate and compare molecules based on measured, rather than in silico values, and is applicable independently of target and chemotype. It is a useful index from the beginning of a project, facilitates the selection of the most promising scaffold and guides their optimization.
OP23 The radiosynthesis of an 18F-labeled triglyceride, developed to visualize and quantify brown adipose tissue activity
Andreas Paulus1, Wouter van Marken Lichtenbelt1,Felix Mottaghy2,3, Matthias Bauwens1,3
1Research School NUTRIM, Maastricht University, Maastricht, Netherlands; 2Division of Nuclear Medicine, Uniklinikum Aachen, Aachen, Germany; 3Department of Medical Imaging, Division of Nuclear Medicine, MUMC, Maastricht, Netherlands
Correspondence: Matthias Bauwens – Research School NUTRIM, Maastricht University, Maastricht, Netherlands
Introduction: Brown adipose tissue (BAT) is an interesting type of tissue that receives major interest as a target to combat obesity. It was (re-)discovered in 2009, when PET-CT studies with [18F]-FDG showed its presence and metabolic activity even in adult humans [1]. It is however difficult to quantitatively assess this metabolic activity using [18F]-FDG, as BAT mainly uses lipids as an energy source. Radiolabeled fatty acids, such as [18F]-FTHA, can provide useful information, but are not optimal considering the lipid uptake mechanism BAT is not based on singular fatty acids, but instead triglycerides (in lipoproteins). As such, we aim to develop a radiolabeled triglyceride, hoping this will shed more light on the lipid burning of BAT, thus allowing to calculate the impact of BAT on the total metabolism of the human body.
Methods: A fatty acid (C16) BODIPY-Fl dye is radiolabeled with F-18 using an F-18/F-19 exchange reaction of the boron-fluoride core of the BODIPY dye to yield a bimodal PET/fluorescent imaging tool. BODIPY-C16 is esterified with 1,3 – Diolein and assistance of thionylchloride yielding the resulting fluorescent triglyceride (TG). In vitro experiments with BODIPY-C16 are conducted to investigate the applicability the dual-modality imaging probe.
Results: BODIPY-C16 could be radio-labeled in a Lewis Acid assisted F-18/F-19 exchange reaction in a reasonable yield (66%, not corrected for decay) and a final purity after C18 Sep-Pak purification of more than 97%. Esterification of BODIPY-C16 resulted in a BODIPY-TG with a yield of more than 90% within 30 minutes. First in vitro experiments showed specific uptake of BODIPY-C16 in BAT and WAT as the compound was located within the lipid droplets of the cell. Direct radiolabeling of the BODIPY-TGL was also successful (60% yield, >97% purity).
Discussion/conclusion: First experiments implied radiolabeled BODIPY-C16 is a promising BAT PET tracer with the opportunity to resolve its metabolic character on a sub-cellular level due to its dual-modality. In vivo imaging of radiolabeled BODIPY-C16 and BODIPY-TGL, incorporation into micelles as well as further in vitro experiments will be conducted in the near future to investigate the applicability of this tracer in lipid metabolism imaging of BAT.
OP24 Influence of the fluorescent dye on the tumor targeting properties of dual-labeled HBED-CC based PSMA inhibitors
Baranski, Ann-Christin1, Schäfer, Martin1, Bauder-Wüst, Ulrike1, Haberkorn, Uwe2, Eder, Matthias1, Kopka, Klaus1
1Division of Radiopharmaceutical Chemistry, German Cancer Research Center (dkfz), Heidelberg, Germany; 2Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany
Correspondence: Baranski, Ann-Christin – Division of Radiopharmaceutical Chemistry, German Cancer Research Center (dkfz), Heidelberg, Germany
Introduction: Image-guided cancer surgery using fluorescence imaging has high clinical impact and already shows potential to improve the outcome of oncological surgery (1). As first clinical experiences with the 68Ga-labeled PSMA-targeting inhibitor Glu-urea-Lys-Ahx-HBED-CC (PSMA-11) demonstrated high and specific tracer uptake in prostate cancer lesions a fluorescence dye conjugate of PSMA-11 might represent a promising bimodal tracer. The combination of preoperative staging by means of PET/CT and PET/MRI, followed by image-guided surgery will further improve the accuracy of detecting PSMA-positive tumor lesions by merging the strengths of both techniques. Therefore, various fluorescent dyes were conjugated to the inhibitor PSMA-11 to determine the impact of the dye conjugation on the ligand’s in vitro and in vivo characteristics.
Materials and methods: The optical-dye labeled tracer PSMA-HBED-CC-FITC, PSMA-HBED-CC-AlexaFluor488 and PSMA-HBED-CC-IRDye800CW were synthesized based on PSMA-11. The binding properties were analyzed in a competitive cell binding assay followed by internalization experiments in human PSMA expressing LNCaP cells. Biodistribution studies were performed in LNCaP tumor-bearing mice (BALB/c nu/nu) to determine specific tumor uptake and pharmacokinetic properties.
Results: Comparative cell binding experiments revealed a high affinity to PSMA expressing cell lines for all conjugates, which is in line with the values obtained with the reference 68Ga-PSMA-11. The radiolabeled fluorescent-dye conjugates showed specific cell uptake and were effectively internalized into the PSMA expressing cell line LNCaP. First in vivo results indicated slightly varying pharmacokinetic properties depending on the fluorescent dye. The FITC- and AlexaFluor488-conjugates revealed a higher tumor uptake compared to 68Ga-PSMA-11, while a minor, but still satisfying uptake was detected for the IRDye800CW-conjugate.
Discussion/conclusion: Conjugation of a fluorescent dye to the well-established imaging agent PSMA-11 showed rather minor dye-dependent impact on cell binding properties, tumor uptake and the pharmacokinetic characteristics. In order to further improve the biodistribution profile of the IRDye800CW-conjugate, structural optimization will be done. These first preclinical results emphasize the potential of a dual-labeled PSMA inhibitor to serve as a multimodal imaging agent, enabling sensitive pre-, intra- and post-therapeutic identification of metastases with one and the same molecule.
OP25 [18F]MEL050 as a melanin PET tracer : fully automated radiosynthesis and evaluation for the detection of pigmented melanoma in mice pulmonary metastases
Chaussard M1,2, Hosten B1,2,4, Vignal N1,2,4, Tsoupko-Sitnikov V1,2, Hernio N1,5, Hontonnou F1,5, Merlet P1,2,5, Poyet JL3,5, Sarda-Mantel L1,2,5, Rizzo-Padoin N1,2,4
1Unité Claude Kellershohn, IUH, Hôpital Saint-Louis, Paris, F-75010, France; 2GH Saint-Louis Lariboisière F. Widal AP-HP, Paris, F-75010, France; 3Inserm U1160, Hôpital Saint-Louis, Paris, F-75010 France; 4Université Paris Descartes, Faculté de Pharmacie, Paris, F-75006, France; 5Université Paris Diderot, Faculté de médecine, Paris, F-75010, France
Correspondence: Chaussard M – Unité Claude Kellershohn, IUH, Hôpital Saint-Louis, Paris, F-75010, France
Introduction: Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. Early detection of melanoma is the best way to reduce mortality. Several radiolabeled imaging probes have been evaluated for melanoma imaging. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, using for the first time, the AllInOne radiosynthesis platform (Trasis, Ans, Belgium), and to validate this PET radiotracer in vivo in a mouse model of melanoma.
Materials and methods: [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Briefly, [18F]MEL050 was prepared from a bromo-precursor, using no-carrier-added 18F-KF-Kryptofix 222 (dimethylformamide, 150°C, 6 min), followed by preparative HPLC purification (C18 column (300x7.8 mm, 7 μm), isocratic elution with acetonitrile/20 mM ammonium bicarbonate (20:80, v/v), 3.0 ml/min) and cartridge-reformulation (Sep-Pak® Plus C18 environmental) of the collected fraction. Radiochemical and chemical purity, stability and specific activity measurements were monitored using analytical HPLC. Chemical identity of the labeled compound [18F]MEL050 was assessed by co-injection with non-radioactive standard MEL050. Experimental model of pulmonary metastatic melanoma was obtained by IV injection of B16-F10 cells in NMRI mice. Mice (n=8) were imaged 15 days after inoculation, using INVEON microPET/CT device (Siemens), after IV injection of 0.36±0.04MBq/g of [18F]MEL050. Dynamic and static acquisitions were acquired from time of injection to 2h after tracer injection. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined using ROIs manually drawn on 1h-post injection PET images, and correlated to ex-vivo findings.
Results: The fully automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 60 min, with an end-of-synthesis yield of 20-26% (n=12). Isocratic semi-preparative HPLC allowed efficient separation of [18F]MEL050 from the reaction mixture. The radiotracer was consistently produced with radiochemical purity higher than 99%. The specific activity was in the range of 177-325GBq/μmol and the product stability was maintained at RCP>98% over 6 h. PET/CT images retrieved known biodistribution of [18F]MEL050 in mice, and allowed clear visualization of <1mm lung tumours with [18F]MEL050 %/ID/g Max of 4.7±2.6%.
Discussion/conclusion: We successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne radiosynthesis platform, including HPLC separation and formulation. In vivo PET/CT validation of the radiotracer was obtained in a mouse model of metastatic pigmented melanoma, showing high specific [18F]MEL050 uptake in sub-millimetric lung tumours.
OP26 Design and Preclinical Evaluation of Novel Radiofluorinated PSMA Targeting Ligands Based on PSMA-617
J. Cardinale1, M. Schäfer1, M. Benešová1, U. Bauder-Wüst1, O. Seibert2, F. Giesel2, U. Haberkorn2, M. Eder1, K. Kopka1
1DKFZ, Division of Radiopharmaceutical Chemistry, Heidelberg, Germany; 2DKFZ, Clinical Cooperation Unit Nuclear Medicine, Heidelberg, Germany
Correspondence: J. Cardinale – DKFZ, Division of Radiopharmaceutical Chemistry, Heidelberg, Germany
Introduction: Urea-based inhibitors of the prostate-specific membrane antigen (PSMA) are well known and promising candidates for the diagnosis (1, 2) and therapy of prostate cancer. The aim of the project was the development of F-18 labeled PSMA ligands based on the theranostic compound PSMA-617. The compounds evaluated during the preliminary experiments showed a high uptake in non-target organs caused by their relatively high lipophilicity. Therefore we currently reduce this lipophilicity by the addition of charged amino acids to the linker region of our PSMA inhibitors (3).
Materials and methods: The PSMA binding motif Glu-NH-CO-NH-Lys was synthesized by a well-established method (4) using solid phase chemistry and subsequently an amino acid linker was built up by fmoc-based solid phase peptide synthesis (SPPS). The non-radioactive reference compounds were also prepared analogically and eventually conjugated by 6-fluoronicotinic acid. After separation from the resin and deprotection the peptidomimetics were labeled using the TFP-ester of 6-[18F]fluoronicotinic acid as prosthetic group. The Ki values of all compounds were determined by competitive binding assays on PSMA-positive LNCaP cells against 68Ga-PSMA-10 (5) using the respective cold reference compounds. Additionally, the cellular internalization of the radiofluorinated ligands was determined.
Results: All 18F-labeled PSMA-inhibitors presented in this study showed low nanomolar affinities towards PSMA, usually paired with high internalization ratios of more than 15 %, shown in vitro. Among those PSMA-1007 showed an outstandingly high internalization ratio of about 70 % while the K
i
was in the typical range (6 nM). Hence PSMA-1007 was further evaluated in vivo. The organ distribution showed a high and specific tumor uptake of 8.0±2.4 %ID/g. Finally, the PSMA-targeting potential of PSMA-1007 was further demonstrated by dynamic μPET experiments.
Discussion/conclusion: Based on our experience with PSMA-617 and preliminary results we developed a series of radiofluorinated PSMA inhibitors with high affinities and internalization ratios. Among those a promising candidate for further translation into the clinic has already been found. This candidate – namely PSMA-1007 – is currently transferred into first-in-man studies. Nevertheless further optimization of the lead compound is still ongoing.
OP27 A novel radiolabeled peptide for PET imaging of prostate cancer: 64Cu-DOTHA2-PEG-RM26
Mansour Nematallah1, Paquette Michel1, Ait-Mohand Samia1, Dumulon-Perreault Véronique2, Lecomte Roger1,2, Guérin Brigitte1,2
1Département de médecine nucléaire et radiobiologie, Faculté de médecine et sciences de la santé, Université de Sherbrooke, QC, Canada J1H5N4; 2Centre d’Imagerie Moléculaire de Sherbrooke (CIMS), CR-CHUS, Sherbrooke, QC, Canada J1H5N4
Correspondence: Guérin Brigitte – Département de médecine nucléaire et radiobiologie, Faculté de médecine et sciences de la santé, Université de Sherbrooke, QC, Canada J1H5N4
Introduction: The Gastrin-Releasing Peptide Receptor (GRPR) is overexpressed in a wide variety of prostate cancers, hence its interest as a potential biomarker. As such, previous work used different radiolabeled GRPR-binding peptides to specifically target tumors in vivo (1, 2). Recently, we synthesized a novel bifunctional chelator bearing hydroxamic acid arms, called DOTHA2, for which our group demonstrated fast and stable complexation to 64Cu (3). The goal of this study was to develop a GRPR antagonist, D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM26), conjugated to the novel [64Cu]-DOTHA2 to visualize prostate tumor by PET imaging.
Material and methods: DOTHA2-PEG-RM26 was synthesized on solid support. The inhibition constant (K
i) was measured on PC3 cells. Dynamic PET images were acquired during 60 minutes after bolus administration on nu/nu male mice bearing PC3 tumors. Biodistribution studies were performed at different time points, using balb/c male mice and nu/nu male mice bearing PC3 tumors. To assess specific binding, a cohort received 500 nmol/kg of unlabeled peptide 15 minutes prior tracer injection for both PET and dissection studies.
Results: The K
i of Cu-DOTHA2-PEG-RM26 was in the low nanomolar range (0.68 nM). DOTHA2-PEG-RM26 complexed 64Cu with fast kinetics at room temperature. The radiopeptide showed high stability, low residual activity in various tissues and fast clearance in normal mice. Small animal blocking experiments showed a significant uptake drop compared to tracer by biodistribution in the GRPR-rich pancreas for both balb/c and nu/nu mice (respectively p< 0.05 and p < 0.01 at 30 min). A significant uptake decrease from 5.3±0.8%ID/g to 3.4±1.4%ID/g was also observed in PC3 tumors when unlabeled peptide was added in biodistribution experiments at 30 minutes, whereas a significantly reduced tumor uptake was also assessed by PET imaging from 20 to 60 minutes (p<0.05) post-injection.
Discussion/conclusion: The use of [64Cu]-DOTHA2-PEG-RM26 is promising for visualizing prostate tumors. These preliminary data suggest that DOTHA2 can be used to develop many other peptide- and protein-derived PET tracers.
OP29 Biodistribution of [18F]Amylovis®, a new radiotracer PET imaging of β-amyloid plaques
Fernandez-Maza L1, Rivera-Marrero S2, Prats Capote A3, Parrado-Gallego A1, Fernandez-Gomez I1, Balcerzyk M1, Sablon-Carrazana M2, Perera-Pintado A3, Merceron-Martinez D2, Acosta-Medina E4, Rodriguez-Tanty C2
1Centro Nacional de Aceleradores. Universidad de Sevilla, CSIC, Junta de Andalucia, Sevilla, Spain; 2Centro de Neurociencias (CNEURO), La Habana, Cuba; 3Centro de Isótopos (CENTIS), Mayabeque, Cuba; 4Centro de Estudios Avanzados de Cuba (CEAC), La Habana, Cuba
Correspondence: Fernandez-Maza L – Centro Nacional de Aceleradores. Universidad de Sevilla, CSIC, Junta de Andalucia, Sevilla, Spain
Aim: [18F]-2-(3-fluoropropyl)-6-methoxynaphtalene ([18F]Amylovis®) is a new naphthalene-derivative for detecting β-amyloid plaques in Alzheimer’s disease. The aim of the study is the assessment of the animal biodistribution of this new radiotracer.
Material and methods: [18F]Amylovis® was synthesized by nucleophilic substitution of the tosyl group of the precursor. Thirty five healthy male Balb/C mice of 10-12 weeks were divided into 6 groups of 5 animals each and injected with similar doses of [18F]Amylovis® through a lateral tail vein. Blood samples were collected and the animals were sacrificed at 5, 15, 30, 45, 70 and 180 minutes. Organs of interest were removed and washed with saline. Radioactivity of blood, plasma, urine, faeces, brain, cerebellum, heart, liver, stomach, spleen, bowel, colon, left kidney, muscle, bone and tail was measured in a well counter. To assess protein binding, plasma samples were diluted with acetonitrile and centrifuged at 4000 g. Pellets of proteins and supernatants were separated and their radioactivity measured in a well counter. RadioTLC analysis of plasma were performed for the same purpose in silicagel 60 and mobile phase of acetonitrile/water (95/5). 20μL of each supernatant was analysed by HPLC-RP using a C18 column and acetonitrile/water (75/25) as mobile phase to identify plasma metabolites. Pharmacokinetic parameters (AUC, t1/2, Cmax, Cl, Vss) were calculated using non-compartmental analysis (NCA). Dynamic PET/CT images of healthy and transgenic APPSwe/PS1dE9 mice were acquired for 2.5 h after i.v. administration. Immunohistochemistry of control and transgenic mice brains were performed to identify β-amyloid plaques.
Results: [18F]Amylovis® crossed blood brain barrier. PET/CT images showed significant differences between healthy and transgenic mice, expressed in Cortex to cerebellum SUV ratio, with maximum difference at 30 minutes. Postmortem studies of immunohistochemistry showed also differences in healthy vs transgenic mice (amyloid positive). Plasma T1/2 of 37 min. No significant protein binding was observed. Renal and hepatic pathways were the main excretion routes. Some amount of in vivo degradation metabolites appeared in blood from 1 h post-administration.
Conclusion: [18F]Amylovis® could be a promising PET radiotracer for amyloid plaques visualization.
OP30 Synthesis and preclinical evaluation of [11C]-BA1 PET tracer for the imaging of CSF-1R
Bala Attili, Muneer Ahamed, Guy Bormans
Laboratory for Radiopharmacy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
Correspondence: Bala Attili – Laboratory for Radiopharmacy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
Introduction: Colony stimulating factor-1R (CSF-1R) is also called as Feline McDonough Sarcoma (FMS), is a type-III kinase receptor. FMS widely expressed and considered to regulate development, maintenance and functioning of mononuclear phagocyte lineage such as monocytes, macrophages, dendritic cells, langerhans cells, microglia and osteoclasts1. Over expression of FMS have been implicated in number of disease states including cancer, rheumatoid arthritis, Crohn’s disease and Bone disorders. FMS also known to play a key role in microglia differentiation and activation and assuming that it was key mediator in neuroinflammatory process.
Materials and methods: All the chemicals and reagents used in the experiments were obtained from commercial sources and used without any further purification. Synthesis of 5-cyano-N-(4-(4-methylpiperazine-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)furan-2-carboxamide and 5-cyano-N-(2-(4-methylpiperidine-1-yl)-4-(piperazine-1-yl)phenyl)furan-2-carboxamide molecules were done according to literature methods available with slight modifications for radiolabeling experiments3. The [11C]-methyl triflate reacts with the precursor in presence of a base at room temperature for 2 minutes gives the carbon-11 radiolabeled [11C]BA-1 which was purified by using semi-preparative reversed-phase high pressure liquid chromatography (RP-HPLC). The peak corresponding the reference compound will be collected and checked for the purity using analytical RP-HPLC. Baseline biodistribution study was performed at 2, 10, 30 and 60 min. respectively. Blocking experiment (10 mg/kg cold compound) was performed at 30 min time point in healthy female adult mice n=3 and compared with vehicle treated batch. In vitro autoradiography experiments were carried on rat brain slices by incubating tracer and cold blocking solution. MicroPET imaging studies were performed on a Focus™ 220 microPET scanner with female rats.
Results: Reference and precursor molecules were synthesized with comparable purities and yields reported in the literature, the radiochemical yield (Alkylation yield with [11C]CH3I) 60 % and radiochemical purity of 98 % and specific activity (n=5) 247.3 GBq/μmol. Biodistribution study shows higher tracer uptake into the brain % ID 4 at 2 min time point, main route of excretion via renal and hepatobiliary circulation. High lung uptake was observed with % ID 14 at 2 min. Blocking with cold compound did not observe any blocking effect in brain but we observed blocking in peripheral organs like liver and spleen, also observed slight blocking in pancreas and kidneys. We did not observed any blocking with in vitro autoradiography experiments. Baseline microPET scans suggests good uptake of tracer into brain with SUV 1.2 but with blocking we did not observed any blocking effect despite we observe higher uptake in brain with SUV 2.
Discussion/conclusion: We successfully synthesized, [11C]-BA1. In preclinical evaluation we did not observe any significant blocking effect in the brain, we are currently looking for other high affinity molecules for CSF-1R.
OP31 In vivo imaging of the MCHR1 in the ventricular system via [18F]FE@SNAP
C. Philippe1, M. Zeilinger1, T. Scherer2, C. Fürnsinn2, M. Dumanic1, W. Wadsak1, M. Hacker1, M. Mitterhauser1,3
1Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Radiopharmacy and Experimental Nuclear Medicine, Medical University of Vienna, Vienna, Austria; 2Department of Medicine III, Division of Endocrinology and Metabolism, Medical University of Vienna, Vienna, Austria; 3Ludwig Boltzmann Institute for Applied Diagnostics, Vienna, Austria
Correspondence: C. Philippe – Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine, Radiopharmacy and Experimental Nuclear Medicine, Medical University of Vienna, Austria
Introduction: The melanin concentrating hormone receptor 1 (MCHR1) is predominately expressed in the lateral hypothalamus and is playing a key role in energy homeostasis and obesity. Recently, it has been shown that the MCHR1 is expressed in the ependymal cells of the 3rd ventricle where it is involved in the regulation of cilia beat frequency [1]. This beating facilitates cerebrospinal fluid circulation, which is crucial for brain function, as defects in ventricular cilia result in hydrocephalus. Our aim was to investigate the potential of the MCHR1 ligand [18F]FE@SNAP [2] for PET-imaging of the MCHR1 in the ventricular system.
Materials and methods:
In vivo experiments were conducted in naïve male Sprague Dawley rats. For small-animal PET/CT/MR experiments, rats were anesthetized by isoflurane. 25min after [18F]FE@SNAP iv injection (47.8±1MBq; SA: 19.7±6GBq/μmol), vehicle (baseline group, n=3) or 15mg/kg SNAP-7941 (blocking group, n=3) were administered through the tail vein. 75min after tracer injection, the rats were sacrificed. Radioactivity concentrations in brain were calculated and expressed as SUVs. In another set of experiments, [18F]FE@SNAP (51.3±26MBq; SA: 36.1±28GBq/μmol) as well as vehicle (baseline group, n=3) or 15mg/kg SNAP-7941 (blocking group, n=3) were injected into conscious freely moving rats via a permanent catheter implanted into the jugular vein, thus excluding an influence of anaesthesia. 45min after tracer application, rats were sacrificed, the brain was removed and cut for ex vivo autoradiography. Subsequently, brain slices were put on Phosphor Imager plates for exposure and analyzed with a Cyclone Phosphor Imager the following day. Regions of interest (ROIs) were drawn for the hypothalamic region, the ventricle and a non-target region. ROIs resulted in normalized DLU/mm2); ratios of ventricle/non-target were calculated.
Results: PET/CT/MR experiments: the SUV in the ventricular system was 0.73±0.11 for the baseline group and 0.34±0.07 for the blocking group, which represents a significant reduction. Ex vivo autoradiography showed a distinct uptake in the ventricular, which was significantly reduced in the blocking group.
Conclusion: [18F]FE@SNAP evinced specific uptake in the ventricular system of naïve rats regardless their state of consciousness and is therefore a suitable imaging agent for cilia beat function.
OP32 Synthesis of the first carbon-11 labelled P2Y12 receptor antagonist for imaging the anti-inflammatory phenotype of activated microglia
B. Janssen1, D.J. Vugts1, G.T. Molenaar1,2, U. Funke1,2, P.S. Kruijer2, F. Dollé3, G. Bormans4, A.A. Lammertsma1, A.D. Windhorst1
1Department of Radiology & Nuclear Medicine, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands; 2BV Cyclotron VU, Amsterdam, The Netherlands; 3CEA, Institut d’Imagerie BioMédicale, Service Hospitalier Frédéric Joliot, Orsay, France; 4Laboratory for Radiopharmacy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
Correspondence: B. Janssen – Department of Radiology & Nuclear Medicine, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
Introduction: Activated microglia are a hallmark of neuroinflammation (NI), which in turn is associated with neurodegenerative diseases. The P2Y12 receptor (P2Y12R) is upregulated in the anti-inflammatory phenotype of activated microglia, and is not expressed on macrophages and other brain cells. Therefore, P2Y12R could be an interesting new target for PET imaging of microglial activation in NI. Recently, a series of P2Y12R antagonists with high binding affinity was reported. Based on this series, the purpose of the present study was to label urea derivative 5 (IC50 = 6 nM) with carbon-11.
Materials and methods: The synthesis of sulfonylurea 5, as reference, and azetidine amine 3 and sulfonyl azide 6, as precursors for the radiosynthesis of [11C]5, is depicted below. Carbon-11-labelling was performed via a rhodium(I)-mediated [11C]CO carbonylation reaction [4,5]. [11C]5 was evaluated using in vitro autoradiography of healthy mouse brains and ex vivo biodistribution and radiometabolite analyses in healthy male Wistar rats.
Results: Compound 5 and precursors 3 and 6 were successfully synthesised. [11C]5 was obtained in a radiochemical yield of 10±2 % (corrected for decay, calculated from [11C]CO2 (n=6)), and high (radio)chemical purity (≥98%) and specific activity (79±32 GBq·μmol-1 (n=6)). In in vitro autoradiography studies of the healthy mouse brain, [11C]5 could be blocked (81%) with ticagrelor, indicating specific binding to P2Y12R. However, rapid metabolism in rat plasma was observed with only 30±4% (n=3) of [11C]5 left at 45 min p.i.. In addition, ex vivo biodistribution revealed that [11C]5 did not enter the rat brain.
Discussion/conclusion: [11C]5 is not suitable for in vivo studies, but can still be used in vitro to validate P2Y12R as a target for imaging the anti-inflammatory phenotype of activated microglia.
Acknowledgement: 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).
OP33 Radiosynthesis of a selective HDAC6 inhibitor [11C]KB631 and in vitro and ex vivo evaluation
Koen Vermeulen1, Muneer Ahamed1, Michael Schnekenburger2, Mathy Froeyen3, Dag Erlend Olberg4, Marc Diederich2, Guy Bormansa1
1Lab of Radiopharmacy, KU Leuven, Leuven, Belgium; 2Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Luxembourg, Grand Duchy of Luxembourg; 3Laboratory for Medicinal Chemistry, Rega Institute of Medical Research, KU Leuven, Leuven, Belgium; 4School of Pharmacy, University of Oslo and Norwegian Medical Cyclotron Centre, Oslo, Norway
Correspondence: Koen Vermeulen – Lab of Radiopharmacy, KU Leuven, Leuven, Belgium
Introduction: HDAC6 has been reported as a regulator in numerous diseases ranging from different kinds of cancers (ovarian, breast, prostate,..) to neurological deficiencies (Alzheimer’s disease, Huntington’s disease, amyotrophic sclerosis,..) [1,2]. Still many characteristics and functions of HDAC6 in these pathologies are unrevealed. Hence, we aim to develop a selective HDAC6 PET tracer to visualize the dynamics of HDAC6 in normal and disease states. Recently, Lu and coworkers synthesized [11C]KB631, a highly potent (IC50=1.4 nM) and selective (3700-fold selectivity against HDAC1) PET tracer for HDAC6 imaging in the brain, which showed similar pharmacokinetic properties as tubastatin A [3]. However, [11C]KB631 showed low brain bioavailability [4]. In this regard we opted to use this tracer as a more peripheral imaging agent. The radiosynthesis was redesigned and preliminary results were obtained with a biodistribution study and autoradiography experiments in brain and tumor slices.
Materials and methods: [11C]KB631 was synthesized through methylation of the corresponding precursor (300 μg) with [11C]-MeI in anhydrous DMSO (250 μL) at 100 °C for 4 min. The biodistribution was studied in NMRI-mice at 2, 10, 30 and 60 min (n = 3/time point) post injection (p.i.). Organs and tissues were harvested and radioactivity was counted in a gamma-counter. Autoradiography studies were carried out with [11C]KB631 on wild-type Wistar rat brain tissue and PC3/LNCaP prostate tumor slices. Slices were incubated with 18.5 MBq/400μL (brain) or 18.5 MBq/250 μL (tumor) of tracer with/without 100 μM of cold authentic reference compound (KB631) or non-structural related pan-HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA) [5].
Results: Based on prep HPLC integration, the methylation yield was 55 % with a radiochemical purity of 97 % and a specific activity of 48 GBq/μmol. Biodistribution studies indicated low brain uptake (<0.1 %ID at 2, 10, 30 and 60 min) and renal and hepatobiliary excretion. Autoradiography experiments showed regional binding. Binding in brain/tumor slices was highly displaceable in the presence of 100 μM non-labeled reference KB631 (up to 90% for brain, PC3 and LNCaP) or 100 μM SAHA (73% brain, 39% PC3 and 59% LNCaP).
Discussion/conclusion: We successfully radiolabeled and evaluated a potential carbon-11 labeled radiotracer for in vitro and ex vivo visualization of HDAC6. However, biodistribution studies indicated low brain uptake, peripheral potency still needs to be further examined. Autoradiography studies showed regional and displaceable binding. Furthermore, radiometabolite studies followed by μPET on a mice tumor model will be performed.
OP34 Improving metabolic stability of fluorine-18 labelled verapamil analogues
Raaphorst RM1, Luurtsema G2, Lammertsma AA1, Elsinga PH2, Windhorst AD1
1Department of Radiology & Nuclear Medicine, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Correspondence: Raaphorst RM – Department of Radiology & Nuclear Medicine, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
Introduction:
(R)
-[11C]verapamil is widely used as a PET tracer for investigating P-glycoprotein (P-gp) function in patients with epilepsy, Alzheimer´s disease and other neurodegenerative diseases [1]. Recently, we have developed the fluorine-18 analogues (R)-N-[18F]fluoroethylverapamil (1) and (R)-O-[18F]fluoroethylnorverapamil (2), potentially enabling P-gp studies in centres without an on-site cyclotron. These analogues showed specific P-gp substrate behaviour, but metabolic stability was poor. The purpose of the present study was to assess whether deuterated analogues would have better metabolic stability.
Materials and methods: To a dried 18F/K2.2.2/K2CO3 complex, 2-bromoethyl-d
4
-tosylate in DMF was added and reacted for 10 min at 90°C to obtain 2-bromo-[18F]fluoroethane-d
4
. This was distilled at 100 °C through an AgOTf column at 200 °C into a cooled (0 °C) vial containing 1.5 mg of (R)-normethyl verapamil (1a) and 3 mg of K2CO3 in ACN. While stirring, this reaction mixture was heated at 120 °C for 15 min and purified by HPLC, resulting in 1b. Tracer 2b, 3b and 4b were obtained by direct fluorination of precursors 2a, 3a and 4a and only 2b required final Boc-deprotection with TFA. 1b and 2b were administered to Wistar rats, and the level of labelled metabolites was measured in blood plasma and brain samples.
Results: 1b, 2b, 3b and 4b were obtained in a radiochemical yield of 3, 6, 5 and 10%, respectively, a purity >98% and a specific activity >80 GBq·μmol-1. Results of the metabolite analysis are presented. The deuterated analogues showed improved metabolic stability compared with the non-deuterated compounds.
Discussion/conclusion: Labelling of tracers 1b-4b was successful. Although, both 1b and 2b showed significantly increased metabolic stability, total intact tracer levels at 15 min are still lower than desired. The resulting effect of increased metabolic stability for PET imaging will be evaluated in P-gp KO mice. To determine the effect of a deuterated N-methyl group, tracer 3b and 4b were designed and still need to be evaluated for metabolic stability.
OP36 Development of a novel PET tracer for the activin receptor-like kinase 5
Lonneke Rotteveel1, Uta Funke1, Peter ten Dijke3, Harm Jan Bogaard2, Adriaan A. Lammertsma1, Albert D. Windhorst1
1Department of Radiology & Nuclear medicine, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Pulmonary Medicine, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands; 3Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
Correspondence: Lonneke Rotteveel – Department of Radiology & Nuclear medicine, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands
Introduction: Pulmonary arterial hypertension (PAH) is a disease in which pulmonary arterial obstruction increases vascular resistance, leading to right ventricular failure [1]. Inhibition of transforming growth factor-β (TGF-β) signalling via activin-receptor-like kinase 5 (ALK5) prevents progression and development of pulmonary hypertension [2]. To further understand the role of ALK5 in PAH, the purpose of this study was to synthesize a carbon-11 labeled ALK5 tracer (IC50 = 5.5 nM) [3] and to assess its potential as a positron emission tomography (PET) ligand for measuring ALK5 expression and activity in vivo.
Materials and methods: The [11C]ALK5 tracer was synthesized by a carboxylation reaction. The radiolabeling was carried out by heating [11C]CO2, the precursor molecule, isobutyl iodide and BEMP in DMSO for 10 minutes at 75°C. The tracer was evaluated using biodistribution and metabolite studies in Wistar rats (n=4 per time point at 15 and 60 min). In addition, specific binding was assessed using autoradiography on ALK5 expressing MDA-MB-231 tumor sections.
Results: The [11C]ALK5 tracer was synthesized with a yield of 18±6 %, a specific activity of 116±31 GBq·μmol-1 and a purity of > 95 %. The tracer showed a normal biodistribution ex vivo and a moderate stability in vivo. The autoradiograms presented binding of the tracer to the tumor sections. Pretreatment of the tumor sections with ALK5 blocking agents (EW-7197, SB-431542 and the ALK5 inhibitor) decreased the binding of the tracer significantly.
Conclusion: The ALK5 tracer was synthesized successfully, and initial in vitro and ex vivo studies indicate its potential as a putative tracer of ALK5, warranting further in vivo evaluation.
Acknowledgment: CVON is acknowledged for funding of this project and the BV Cyclotron VU for providing [11C]CO2.
OP37 SPECT imaging and biodistribution studies of 111In-EGF-Au-PEG nanoparticles in vivo
Lei Song, Sarah Able, Nadia Falzone, Veerle Kersemans, Katherine Vallis
CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK
Correspondence: Lei Song – CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK
Introduction: Radiolabelled antibodies and peptides hold promise for molecular radiotherapy but are often limited by low payload resulting in delivery of inadequate amounts of radioactivity to tumour tissue and, therefore, modest therapeutic effect. We have developed PEGylated epidermal growth factor (EGF)-gold nanoparticles (NP) with a high indium-111 (111In) payload (111In-EGF-Au-PEG NPs) as a prototypic NP-based theranostic radiopharmaceutical.
Materials and methods: EGF-Au-PEG NPs were prepared via an interaction between gold and the disulphide bonds of EGF followed by PEGylation by mPEG-thiol (MW: 800, 2000, 6000) and characterised by SEC-HPLC, DLS and zeta potential. The targeting property of NPs with various PEG MWs was investigated by confocal imaging following exposure of MDA-MB-468 (1.3 x 106 EGFR/cell) and MDA-MB-231/H2N (105 EGFR/cell) cells to Cy3-EGF-Au-PEG NPs. 111In-EGF-Au-PEG (MW: 6000) and 111In-EGF-Au NPs were chosen for SPECT imaging and biodistribution studies using MDA-MB-468 xenograft-bearing mice.
Results: Successful PEGylation was confirmed by DLS and zeta potential measurements, showing the hydrodynamic sizes of NPs were 18.5, 19.4, 24.8 and 32.5 nm; the zeta potentials in water were -24, -15, -14 and -9 mV for EGF-Au and EGF-Au-PEG with MWs of 800, 2000 and 6000, respectively. SEC-HPLC showed that the retention time of EGF-Au-PEG NPs was shorter than EGF-Au NPs as PEGylation resulted in larger NPs. Confocal imaging demonstrated that both EGF-Au and EGF-Au-PEG NPs were efficiently bound and internalised by MDA-MB-468 cells. In vivo SPECT studies in mice bearing MDA-MB-468 xenografts revealed high tumour uptake in animals that received 111In-EGF-Au-PEG (MW: 6000) compared to 111In-EGF-Au. The tumour/muscle radioactivity ratios for 111In-EGF-Au-PEG and 111In-EGF-Au were 7.2 and 2.5.
Conclusion: An 111In-labelled EGF-Au-PEG nanosystem was developed. It enabled targeted delivery of a high 111In payload to an EGFR-positive cancer model that can be potentially exploited for molecularly targeted radiotherapy.
OP38 Melanoma targeting with [99mTc(N)(PNP3)]-labeled NAPamide derivatives: preliminary pharmacological studies
Davide Carta1, Nicola Salvarese2, Wiebke Sihver3, Feng Gao3, Hans Jürgen Pietzsch3, Barbara Biondi4, Paolo Ruzza4, Fiorenzo Refosco2, Cristina Bolzati2
1DSF, University of Padua, Via Marzolo 5, 35131 Padova, Italy; 2IENI-CNR, Corso Stati Uniti 4, 35127 Padova, Italy; 3Institute of Radiopharmaceutical Cancer Research, HZDR, Bautzner Landstrasse 400, 01328 Dresden, Germany; 4ICB-CNR, Via Marzolo 1, 35131 Padova, Italy
Correspondence: Cristina Bolzati – IENI-CNR, Corso Stati Uniti 4, 35127 Padova, Italy; 3Institute of Radiopharmaceutical Cancer Research, HZDR, Bautzner Landstrasse 400, 01328 Dresden, Germany
Introduction: Malignant melanoma is the most lethal form of skin cancer and the most commonly diagnosed malignancy among young adults with an increasing incidence. Hence, the development of new melanoma-specific pharmaceutical for diagnosis and/or therapy is a subject of great interest and intense research. The purpose of this study was to examine the effect of cyclization on the biological profile of [99mTc(N)(PNP3)]-labeled α-MSH peptide analogs (PNP3 = N,N-bis(dimethoxypropylphosphinoethyl)methoxyethylamine).
Method: A lactam bridge-cyclized H-Cys-Ahx-βAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11-Pro-Val-NH2 (NAP―NS2) and the corresponding linear H-Cys-Ahx-βAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their MC1R binding affinity were determined in B16/F10 melanoma cells. In vitro stability and pharmacological parameters of [99mTc(N)(NAP―NS1)(PNP3)]+ (1) and [99mTc(N)(NAP―NS2)(PNP3)]+ (2) were assessed. Challenges with an excess of glutathione and cysteine and Log P values were also investigated. Furthermore, 1 and 2 were applied to study in vivo stability and the pharmacokinetic profiles on healthy rats.
Results: 1 and 2 were obtained in high yield (RCY > 90%). Log P values demonstrate the hydrophilic nature of the radiolabelled peptides: -1.43 for 1; - 2.087 for 2. No significant variations in RCPs of both the complexes were observed in challenge experiments with an excess (10 mM) of glutathione and cysteine. A high in vitro stability was observed for both complexes after incubation in human and rat sera as well as in rat liver homogenate; a fast degradation of 2 was detected in kidneys homogenate. 1 retains a high receptor affinity (Kd=7.1 ± 0.5 nM). Biodistribution data of 1 display a favorable pharmacokinetic profiles characterized by a fast blood clearance and elimination from normal tissues. A rapid excretion via the renal pathway was observed according to the high hydrophilic character of the radio-conjugate. The effect of the cyclization on the pharmacokinetic profile of 2 was reflected in a reduction of the blood clearance and of the elimination from the other organs, in particular, from excretory organs such as liver and kidneys.
Conclusion: Compared with the linear peptide, cyclization negatively affects the biological properties of NAP-NS2 peptide by reducing its binding affinity for MCR1R (Ki: 0.9±0.3 nM for NAP_NS1; 7.1±2.4 nM for NAP_NS2) and decreasing the overall excretion rate of the corresponding [99mTc(N)(PNP3)]-labeled peptide from the body as well as its stability. Thus only the linear [99mTc(N)(PNP3)-labeled peptide is suitable for further investigations in tumor bearing animals. This research was supported by MIUR through PRIN 20097FJHPZ-004 and FIRB “RINAME”2010-RBAP114AMK.
OP39 [68Ga]NODAGA-RGD: cGMP synthesis and data from a phase I clinical study
Roland Haubner1, Armin Finkensted2, Armin Stegmair1,3, Christine Rangger1, Clemens Decristoforo1, Heinz Zoller2, Irene J. Virgolini1
1Department of Nuclear Medicine, Medical University of Innsbruck, Innsbruck, Austria; 2Department of Internal Medicine, Medical University of Innsbruck, Austria; 3FH Gesundheit/University of Applied Sciences Tyrol, Innsbruck, Austria.
Correspondence: Roland Haubner –Department of Nuclear Medicine, Medical University of Innsbruck, Innsbruck, Austria
Introduction: Preclinical studies demonstrated that [68Ga]NODAGA-RGD allows imaging of integrin αvβ3 expression using PET. Here we present all data (quality control, toxicological study, and dosimetry estimation) necessary to initialize clinical studies, the establishment of the remote controlled synthesis, and data from the phase I clinical study.
Methods: Labelling was carried out in a remote controlled synthesis unit under clean room conditions. Quality control included TLC, HPLC, GC, pH control, Ge-breakthrough, half-life, endotoxin content, and control of sterility. Storage stability after 4 h was studied and dose estimations based on animal data were carried out using OLINDA. Sprague-Dawley rats were used for an extended single dose toxicity study. The phase I clinical study included 9 patients with hepatocellular carcinoma (HCC). Static scans at 5, 30, and 60 min p.i. including 5 bed positions each were performed using the Discovery 690 PET/CT. Blood was sampled 30 and 60 min p.i. and urine 60 min p.i. and used for stability studies via HPLC.
Results: [68Ga]NODAGA-RGD could be produced in high radiochemical yield and radiochemical purity (HPLC and TLC >99%). The pH in the final isotonic saline formulation was approx. 6. Ethanol content was between 2.5 and 3.0% v/v. No detectable 68Ge-germanium was found. LAL test revealed 0.7 EU/ml. Sterility tests showed that all samples met the specifications according to Ph. Eur.. No radiolysis of the tracer was found in the formulated solution. The single dose toxicity study showed that the compound was well tolerated in animals. This was confirmed by the clinical study where no severe side effects were observed. High metabolic stability of [68Ga]NODAGA-RGD was found based on the analysis of blood and urine samples. The static scans showed rapid tracer elimination from the body with low background activity in almost all organs. The calculated effective dose was 21.5±5.4 μSv/MBq. Unfortunately, the investigated tumors did not show increased tracer accumulation indicating no or low integrin αvβ3 expression.
Conclusion: This study revealed that [68Ga]NODAGA-RGD can be easily produced under GMP conditions and met the requirements for the clinical use. The phase I clinical study with patients bearing HCC did not allow identification of the lesions but demonstrated rapid elimination from the body, high metabolic stability and low radiation burden. The low tracer accumulation in the tumor might be related to low receptor expression, thus further studies are needed to verify the integrin αvβ3 imaging properties.
OP44 Implementation of a GMP-grade radiopharmacy facility in Maastricht
Ivo Pooters1, Maartje Lotz1, Roel Wierts1, Felix Mottaghy1,2, Matthias Bauwens1,3
1Department of Radiology and Nuclear Medicine, MUMC+, Maastricht, The Netherlands; 2Nuclear Medicine, Uniklinikum Aachen, Aachen, Germany; 3Research School NUTRIM, Maastricht University, Maastricht, The Netherlands
Correspondence: Matthias Bauwens – Department of Radiology and Nuclear Medicine, MUMC+, Maastricht, The Netherlands
Introduction: In the Netherlands, a modified “light” version of the European GMP, i.e. GMP-z, for the (kit-) production of registered radiopharmaceuticals for individual patients in hospital pharmacies is in effect. However, GMP-z does not allow to synthesize radiopharmaceuticals for clinical trials or therapeutic applications, in which case the European GMP applies. At the MUMC, a growing interest in performing in-house clinical trials with new radiopharmaceuticals and in the synthesis of radiotherapeuticals led to the decision of upgrading the current Radiopharmacy facility. Several restrictions applied: radiopharmaceuticals will not be sold to third parties and the surface area of the entire facility is limited to 55 m2 (including the production lab, QC lab and separate sluices for personnel and goods). Additionally, the facility had to allow both preparation of routine 99mTc-compounds as well as GMP-grade radiopharmaceuticals. This study examines the time line of implementation for such a small scale facility and may be relevant to other hospitals considering to comply with this regulation.
Materials and methods: A team was founded late 2012, including a radiochemist, QA officer, radiopharmacist, radiation safety officer, clinical physicist and a construction project leader (totaling nearly 5 fulltime-equivalents). User requirements for the facility, hotcells and laboratory equipment were determined. Preliminary building plans were drawn up and several expert companies chosen, specialized in HVAC, cleanroom construction and area and radiation monitoring.
Results: For the hotcells, a successful Factory Acceptance Test was performed in February 2015. Construction of the new facility started end of May 2015, starting with the planned installation of the hotcells for which removal of a section from the outer wall was necessary. The second phase of the reconstruction (setting up of a GMP-compliant cleanroom) is scheduled to start January 2016, leading to full operability in April 2016 and, upon inspection by the Dutch Inspectorate (IGZ), GMP-compliant production is expected by the end of 2016.
Discussion/conclusion: It is essential to have a high degree of in-house expertise when starting a radiopharmacy building project. Completely documented construction planning, based on a solid list of requirements, takes 2-3 years to build up and is a necessity to allow timely and high-quality construction.
OP45 Setting up a GMP production of a new radiopharmaceutical
Forsback, Sarita1, Bergman Jörgen1, Kivelä Riikka2
1Radiopharmaceutical Chemistry Laboratory, Turku PET Centre, University of Turku, Turku, Finland; 2Turku University Hospital, Hospital Pharmacy, Turku, Finland
Correspondence: Forsback, Sarita – Radiopharmaceutical Chemistry Laboratory, Turku PET Centre, University of Turku, Turku, Finland
Introduction: Good Manufacturing Practice (GMP) sets strict requirements for the manufacturing conditions, synthesis and quality of radiopharmaceuticals. Robust production and quality control methods for radiopharmaceuticals are essential for diverse and effective clinical utilization of Positron Emission Tomography (PET). This presentation describes the set up procedure of a new radiopharmaceutical in Turku PET Centre (TPC). All issues other than directly related to GMP such as toxicology, labeling chemistry, analytical methods or documentation for the authorities (i.e. IMPD) are not discussed here.
Materials and Methods: Quality standards and product specification are expressed in EU (Annex 15 Qualification and Validation, revision into operation 1 Oct 2015) and European Pharmacopoeia (8th Edition). The setup of a new radiopharmaceutical at TPC requires existence of the following: Established quality system, Documentation system, Competent personnel, Classified clean rooms, Qualified equipment, System for material management. Initially the specifications for critical materials and primary packing materials of the new radiopharmaceutical must be defined. At TPC specifications for new radiopharmaceutical are: Appearance, Identification, Radioactivity, Radionuclidic identity, Radionuclidic purity, Radiochemical purity, Chemical purity, Residual solvents, Content of ethanol, pH, Sterility, Bacterial endotoxins, Shelf life.
Results: After ensuring that the production of a new radiopharmaceutical is robust and repeatable and all analytical methods including sterility and endotoxin tests are validated, the process can be validated according to a written validation plan. In addition bioburden (number of bacteria living on the drug solution before sterilization) must be determined. The aseptic processing of operators must also be confirmed by performing media fills (the performance of an aseptic manufacturing procedure using a sterile microbiological growth medium in place of the drug solution). At TPC the process validation includes three consecutive process validation batches which shall fulfill all specifications for the given radiopharmaceutical. This also qualifies the operator for production. Additional batches must be done in order to qualify more operators. Finally, process validation and documentation is compiled. Process validation report, method description for preparation and quality control and master batch record are written.
Discussion/conclusion: After all documentation has been accepted by QA, the new radiopharmaceutical is ready for clinical production and all changes to the process must be performed via change control process.
OP48 In vitro and in vivo evaluation of 68-gallium labeled Fe3O4-DPD nanoparticles as potential PET/MRI imaging agents
M. Karageorgou1, M. Radović2, C. Tsoukalas1, B. Antic2, M. Gazouli3, M. Paravatou-Petsotas1, S. Xanthopouls1, M. Calamiotou4, D. Stamopoulos4,5, S. Vranješ-Durić2, P. Bouziotis1
1Radiochemical Studies Laboratory, INRASTES, NCSR “Demokritos”, Athens, Greece; 2Vinča” Institute of Nuclear Sciences, Laboratory for Radioisotopes, University of Belgrade, Belgrade, Serbia; 3Department of Basic Medical Science, Laboratory of Biology, School of Medicine, NKUA, Athens, Greece; 4Department of Solid State Physics, NKUA, Athens, Greece; 5INN, NCSR “Demokritos”, Athens, Greece
Correspondence: M. Karageorgou – Radiochemical Studies Laboratory, INRASTES, NCSR “Demokritos”, Athens, Greece
Introduction: The combination of different imaging modalities has received increasing interest over the past decade, as it enables to overcome the limitations of a single imaging modality and ensures enhanced interpretation of diseases and abnormalities in vivo. Dual modality PET/MRI imaging agents, such as radiolabeled magnetic nanoparticles, are promising candidates for a number of diagnostic and therapeutic applications (i.e. MRI-magnetic hyperthermia and radiotherapy). The aim of the present study is to evaluate the efficacy of 68Ga labeled Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) coated with DPD phosphonate, as potential PET/MRI imaging agents.
Materials and methods: SPIONs coated with biocompatible DPD-phosphonate were radiolabeled with positron-emitting Gallium-68 to quantify the accumulation of the nanoparticles in vivo. In vitro stability studies, in PBS, saline and human serum, were performed to evaluate their aqueous solubility in vivo. In vivo biodistribution study was performed in 9 healthy mice at 30, 60 and 120 min post-injection.
Results:
68Ga-Fe3O4-DPD SPIONs presented high radiolabeling yield (95%) and proved stable in vitro. The in vivo study exhibited significant liver and spleen uptake at all examined time points in healthy mice, whereas minor fractions attained in other organs. A small fraction of radiolabeled nanoparticles presented in bones is indicative of high affinity of phosphonate to bone tissue. The biodistribution profiles between 68Ga-Fe3O4-DPD SPIONs and free 68Ga-acetate were also compared, indicating different pharmacokinetic behavior for 68Ga-acetate, with no target tissue and excretion via the kidneys.
Conclusion:
68Ga-Fe3O4-DPD SPIONs demonstrated high radiolabeling efficiency and in vitro stability and satisfactory in vivo behavior. Cytotoxicity studies to explore the potential toxic effects of the nanoparticles, as well as biodistribution studies in tumor models, are in progress.
OP49 Fast PET imaging of inflammation using 68Ga-citrate with Fe-containing salts of hydroxy acids
A. S. Lunev1,2, A. A. Larenkov1, K.A. Petrosova1, O. E. Klementyeva1, G. E. Kodina1
1Burnasyan Federal Medical Biophysical Center of FMBA Russia, Moscow, Russia; 2Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow, Russia
Correspondence: A. S. Lunev – Burnasyan Federal Medical Biophysical Center of FMBA Russia, Moscow, Russia
Introduction:
68Ga-citrate is one of the radiotracers for PET imaging of inflammation/infection. Gallium (like iron) has high affinity to blood transport proteins (e.g. transferrin) due to their similar physical/chemical properties [1]. One of the functions of transport proteins is delivery iron (gallium too) to inflammation/infection foci for inclusion in metabolic pathways related with eradication of pathology [2]. But excessive gallium bindings with proteins are cause of slow blood clearance, long accumulation time in foci (24-72 h) and exception of application possibility of the short-lived 68Ga (T½ = 1,13 h) unlike 67Ga (T½ = 78,26 h). Injection of additional nonradioactive chemical agents (e.g. Fe3+ containing salts of hydroxy acids: citrate, tartrate, lactate, etc.) competing with gallium to the protein joining (blocking of its metal binding capacity) is one of the ways to solve formulated problem. It can be used for correction of 68Ga-citrate pharmacokinetics for increasing of the blood clearance, accumulation in foci and fast imaging.
Materials and methods.
68Ga-citrate without/with extra injection of Fe3+ containing salts (citrate, tartrate, lactate, malate, and ascorbate) was injected mice with modeled lung infection (was got using intra lung injection of cell suspension of E. coli; acute phase of infection was evoluted for 3-4 days). PET/X-RAY Genisys4 (Sofie Bioscience, USA) was used for noninvasive PET imaging with subsequent reconstruction of imaging and their analysis (value of clearance, distribution volume). Scanning time is 10 min.
Results: I. v. injection of Fe3+ containing salts of hydroxy acids blocked the metal-binding capability of transferrin serum and others and allowed decreasing gallium-68 radioactivity in blood significantly and increasing accumulation in inflammation (3-5 time). It allowed receiving more informative PET-images of inflammation early (for 30-60 min after injection). Pharmacokinetic parameters proved it.
Discussion/conclusion. There was no statistically significant difference between 68Ga-citrate accumulation for different inflammation model because PET imaging is indication of pathological processes and isn't their identification.
