Click on an contribution preview the abstract content.
Click on an contribution preview the abstract content.
Influence of Mrp4 on brain distribution and urinary excretion of 6-bromo-7-[11C]methyl-purine in mice (#172)
V. Zoufal1, S. Mairinger1, M. Krohn2, T. Wanek1, J. D. Schuetz3, J. Stanek1, 4, M. Sauberer1, T. Filip1, J. Pahnke2, O. Langer1, 4
1 AIT Austrian Institute of Technology GmbH, Health and Bioresources, Seibersdorf, Austria
6-Bromo-7-[11C]methylpurine ([11C]BMP) is a PET tracer which allows to measure multidrug resistance-associated protein 1 (Mrp1) function in the mouse brain . This PET tracer enters into cells, where it gets rapidly converted into its glutathione conjugate, which is actively effluxed by Mrp1. Mrp4 is another member of the MRP family expressed in different organs including the brain and the kidneys. The aim of this study was to assess the influence of Mrp4 on brain distribution and excretion of [11C]BMP in mice.
Female C57BL6 wild-type (WT), Mrp1(-/-) and Mrp4(-/-) mice (n=4-6 per group) underwent 90 min dynamic [11C]BMP PET scans. In addition, WT mice were scanned at 30 min after intraperitoneal pre-treatment with the MRP inhibitor MK571 (300 mg/kg). Radiolabelled metabolites of [11C]BMP were analyzed by radio-TLC in separate groups. Time-activity curves were obtained and the cerebral efflux rate (keff,brain) of radioactivity from brain was calculated using data from 17.5 - 80 min after radiotracer injection. Integration plot analysis was used to estimate the rate constant for excretion of radioactivity from the kidney into the urine (kurine) from 12.5 min - 65.0 min after radiotracer injection.
Keff,brain values were significantly lower (1-way ANOVA) in Mrp1(-/-) and Mrp4(-/-) mice as well as in MK571 pre-treated WT mice compared to untreated WT mice (keff,brain (h-1): WT: 1.37 ± 0.25; WT+MK571: 0.93 ± 0.16; Mrp1(-/-): 0.15 ± 0.01; Mrp4(-/-): 0.88 ± 0.03). PET data showed that [11C]BMP underwent predominantly renal excretion. Kurine was significantly lower in Mrp1(-/-) and Mrp4(-/-) mice compared to WT mice (kurine (min-1): WT: 1.17 ± 0.47, Mrp1(-/-): 0.26 ± 0.09, Mrp4(-/-): 0.42 ± 0.11). Pre-treatment of WT mice with MK571 completely abolished urinary excretion of radioactivity (kurine (min-1): WT+MK571: -0.009 ± 0.024). Radio-TLC analysis showed that at 15 min after radiotracer injection almost all radioactivity in the brain and urine consisted of the glutathione conjugate.
Our data confirm previous findings that [11C]BMP allows to measure Mrp1 function in the mouse brain. However, we found that keff,brain and kurine values were significantly reduced in Mrp4(-/-) mice, suggesting that Mrp4 contributes to brain distribution and urinary excretion of this radiotracer.
 Okamura T. et al, J Cereb Blood Flow
The research leading to these results has received funding from the Austrian Science Fund (FWF) project I 1609-B24 to O. Langer and the Deutsche Forschungsgesellschaft (DFG) project DFG PA930/9-1 to J. Pahnke.
Rate constant for excretion of radioactivity from the kidney into the urine (kurine)
This figure shows the rate constants for excretion of radioactivity from the kidney into the urine (kurine) in wild-type, Mrp1(-/-) and Mrp4(-/-) mice as well as in MK571 pre-treated WT mice. (mean ± S.D, *p < 0.01, one-way ANOVA)
Keywords: 6-bromo-7-[11C]methyl-purine, PET, Mrp4, brain distribution, urinary excretion
[18F]Fludarabine-PET as a promising tool in differentiation between CNS lymphoma and glioblastoma: comparative analysis with [18F]FDG in human xenograft models (#244)
N. Hovhannisyan1, S. Guillouet1, M. Ibazizene1, F. Fillesoye1, S. Valable2, B. Plancoulaine3, L. Barré1
1 ISTCT/LDM-TEP group, Normandie Univ, UNICAEN, CEA, CNRS, CHU Caen, Caen, France
[18F]Fludarabine ([18F]FDB) is a PET radiotracer , the specificity of which has been studied in several lymphoma models (follicular RL7, DOHH2; MM RPMI8226) [2-5]. The first-in-human study in two types of lymphoid malignancies (DLBCL, CLL) has consolidated the great potential of this innovative tool for lymphoma imaging in oncology [6,7]. The purpose of this study was to investigate whether [18F]FDB-PET can help differentiate between central nervous system (CNS) lymphoma and glioblastoma (GBM), considering also multimodal analyses with [18F]FDG, MRI and histology.
Nude rats were implanted with human MC116 lymphoma- (n=10) or U87 glioma-cells (n=5). Tumor growth was monitored by MRI, with T2-weighted sequence (RARE) for anatomical features and T1-weighted (EPI) with gadolinium (Gd) enhancement for BBB permeability assessment. For PET investigation, ~11 MBq [18F]FDB or [18F]FDG were injected via tail vein and PET dynamic images were acquired up to 90 min after radiotracer injection. Paired scans of the same rat with the two [18F]-labelled radiotracers were investigated. Initial volumes of interest (tumor and healthy contralateral or cerebellum tissue) were manually delineated on T2w image and set on co-registered PET image and tumour-to-background ratios (TBR) were calculated to semi-quantitatively assess the tracer accumulation in the tumour.
In lymphoma model, PET time-activity curves revealed a differential response of [18F]FDB between tumoral and healthy tissues with average TBR varying from 2.45 to 3.16 between 5 to 90 min post-injection. In contrast, [18F]FDG demonstrated similar uptake profiles for tumoral and normal regions with TBR varying from 0.84 to 1.06 between these two time points. In GBM model, the average TBRs were from 1.67 to 1.07 for [18F]FDB and from 1.08 to 1.65 for [18F]FDG. Therefore, inter-model comparisons showed significantly divergent responses (p<0.001) of [18F]FDB between lymphoma and GBM, while [18F]FDG demonstrated considerable overlap (p=0.03) between the groups. Tumour characterisation with histology (based mainly on Hoechst, CD68 and CD79), as well as with MRI were in overall better agreement with [18F]FDB-PET than [18F]FDG with regard to tumour selectivity. Furthermore, comparative analysis between [18F]FDB and [11C]Methionine is underway and will be presented.
The potential of [18F]FDB-PET to distinguish CNS lymphoma from GBM is quite evident and will be further investigated.
Keywords: [18F]Fludarabine-PET, Lymphoma, Imaging, CNS
Effect of Nanobody-based radiopharmaceuticals on kidney retention (#247)
M. Crauwels1, 2, S. Massa2, 3, C. Betti4, C. Martin4, S. Ballet4, M. D'Huyvetter1, S. Hernot1, V. Caveliers1, 5, T. Lahoutte1, 5, N. Devoogdt1, S. Muyldermans2, C. Xavier1
1 VUB, In Vivo Cellular and Molecular Imaging Laboratory, Jette, Brussels, Belgium
Development of [11C]osimertinib as TKI-PET tracer for the imaging of tumors expressing T790M mutated EGFR (#472)
A. Högnäsbacka1, M. Chomet1, D. J. Vugts1, G. A. M. S. van Dongen1, A. J. Poot1, A. D. Windhorst1
1 VU University Medical Center, Radiology and Nuclear Medicine, Amsterdam, Netherlands
NSCLC tumors expressing primary epidermal growth factor receptor (EGFR) mutations are highly sensitive to treatment with 1st/2nd generation tyrosine kinase inhibitors (TKIs). Unfortunately tumors will ultimately develop treatment resistance to these TKIs. The most frequent mutation causing treatment resistance is the T790M mutation. [1-3] Osimertinib is a third generation TKI able to inhibit the T790M mutation, as well as the primary mutations.  The aim of this project is to develop [11C]osimertinib as a TKI-PET tracer and evaluate its potential to image tumors expressing mutated EGFR.
[11C]Osimertinib was synthesized by methylation of the precursor AZ5104 using [11C]CH3I as shown in Fig. 1.
To determine the in vivo stability of [11C]Osimertinib a metabolite analysis was performed in non-tumor bearing female Nu/Nu mice. The animals were injected i.v. (via ocular plexus) with 17-24 MBq and sacrificed at 5, 30, and 60 minutes post injection followed by blood sample collection and in some cases brain collection.
[11C]Osimertinib was obtained in a radiochemical yield of 10-20% (decay corrected to end of bombardment), with a molar activity of over 169 GBq/µmol and a radiochemical purity of over 95%. The tracer was obtained as a sterile, pyrogen free and isotonic solution. A preliminary metabolite study showed that the tracer is quickly metabolized in blood but is stable in the brain.
[11C]Osimertinib as a 3rd generation PET tracer is ready for preclinical evaluation, both in vitro and in vivo. In addition its GMP compliant synthesis allows for clinical application of the tracer. By developing a TKI-PET tracer based on the structure of osimertinib a new non-invasive method to assess tumors expressing EGFR mutations sensitive towards treatment is established. The tracer would allow stratification of patients, monitoring of treatment response, as well as aid drug development by PET imaging.
1. Wee-Lee Yeo et al., J Thorac Oncol. 2010; 5(7): 1048–1053
2. Lecia V. Sequist et al., J Clin Oncol 2013; 31:3327-34
3. William Pao et al.,PLoS Med 2005; 2 (3): e73
4. Pasi A. Jänne et al., N Engl J Med 2014; 372:1689-99
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska–Curie grant agreement No 675417.
Keywords: PET, [11C]osimertinib, EGFR, NSCLC
Evaluating hepatobiliary transport with 18F labeled bile acids: the effect of radiolabel position and bile acid backbone on radiosynthesis, in vitro and in vivo performance (#193)
S. De Lombaerde1, K. Kersemans2, S. Neyt1, J. Verhoeven1, C. Vanhove3, F. De Vos1
1 Ghent University, Laboratory for Radiopharmacy, Ghent, Belgium
Bile acids are steroid derivatives that are produced in the hepatocytes of the liver and excreted in bile and intestines. They play an important role in lipid digestion in the intestines . Their highly efficient hepatobiliary transport can however be disrupted and cause a toxic accumulation of bile acids, by a xenobiotic inhibiting the bile acid transporters on the hepatocytes or in certain liver diseases [2–4]. In vivo determination of bile acid hepatobiliary transport with 18 fluorine labeled bile acids for Positron Emission Tomography (PET) can provide important insight in liver disease.
A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3, 7 or 12 OH-function is substituted with a fluorine atom (3α-[18F]FCA, 7β-[18F]FCA; 12β-[18F]FCA); a glycocholic- and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines; efflux by BSEP in membrane vesicles. PET-scans were performed in healthy wild-type FVB-mice (n=3 per group): hepatobiliary transport was monitored and compared to reference tracer 3β-[18F]FCA.
Compounds 3α-[18F]FCA, 3β-[18F]FGCA and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4-10 % n.d.c.) and high radiochemical purity (>99 %); 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized in a suitable yield: these compounds could not be included in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1 and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines and is hence less suited to monitor possible disturbances of bile acid transport in vivo.
A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as a PET-biomarker for hepatobiliary transport of bile acids.
 Hofmann a. F, Hagey LR. Bile Acids: Chemistry, Pathochemistry, Biology, Pathobiology, and Therapeutics. Cell Mol Life Sci 2008;65:2461–83.
 de Lima Toccafondo Vieira M, Tagliati CA. Hepatobiliary transporters in drug-induced cholestasis: a perspective on the current identifying tools. Expert Opin Drug Metab Toxicol 2014
 Jüngst C, Berg T, Cheng J, Green RM, Jia J, Mason AL, et al. Intrahepatic cholestasis in common chronic liver diseases. Eur J Clin Invest 2013;43:1069–83.
 Kubitz R, Dröge C, Stindt J, Weissenberger K, Häussinger D. The bile salt export pump (BSEP) in health and disease. Clin Res Hepatol Gastroenterol 2012;36:536–53.
Conceived 18F labeled bile acid analogues in the present study
Compounds 3α-[18F]FCA, 3β-[18F]FCDCA and 3β-[18F]FGCA on the upper row differ in bile acid backbone (cholic acid, chenodeoxycholic acid and glycocholic acid) or 3 α/β position of the radiolabel. The structures 12β-[18F]FCA and 7β-[18F]FCA have the same cholic acid backbone, but different position of the radiolabel (7 or 12).
Time-activity curves (TACs) of 3α-[18F]FCA, 3β-[18F]FCA, 3β-[18F]FGCA and 3β-[18F]FCDCA
Time-activity curves of the different 18F labeled bile acids in liver (red curve) and gallbladder and intestines (GBI; green curve) of wild-type FVB-mice. Uptake of the tracers was expressed as % injected dose (% ID) and normalized for a 20 g mouse. Data are mean ± SD (n = 3 per group).
Keywords: PET, liver, bile acid, fluoro 18 labeling
Assessment of hepatic ABCB1 and ABCG2 function with [11C]tariquidar and positron emission tomography (#140)
A. Traxl1, T. Wanek1, S. Mairinger1, J. Stanek1, C. Kuntner1, O. Langer1, 2, 3
1 AIT Austrian Institute of Technology GmbH, Center for Health & Bioresources / Biomedical Systems, Seibersdorf, Austria
Tariquidar is an inhibitor of the ABC transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), which is predominantly excreted via the hepatobiliary route. PET studies showed that [11C]tariquidar is transported by ABCB1 and ABCG2 at the rodent and human blood-brain barrier [1,2]. ABCB1 and ABCG2 are also expressed in the canalicular (bile-facing) membrane of hepatocytes, where they promote excretion of drugs and their metabolites into bile. Aim of this study was to investigate the influence of ABCB1 and ABCG2 on hepatobiliary excretion of [11C]tariquidar in mice.
Female wild-type, Abc1a/b(-/-), Abcg2(-/-) and Abc1a/b(-/-)Abcg2(-/-) mice (n=4-5 per group) with a FVB genetic background underwent two consecutive 60-min dynamic PET scans with [11C]tariquidar. A baseline scan was followed by a second scan in which unlabeled tariquidar (15 mg/kg) was administered intravenously 120-min prior radiotracer injection. To estimate the rate constant for transfer of radioactivity from liver via bile into intestine (kbile) a graphical analysis method (integration plot) was used.
In the baseline scan, kbile values in Abc1a/b(-/-) and Abcg2(-/-) mice were not significantly different from wild-type mice (kbile, Abc1a/b(-/-): 0.0027±0.0004 min-1, Abcg2(-/-): 0.0026±0.0008 min-1, wild-type: 0.0031±0.0011 min-1). However, in Abc1a/b(-/-)Abcg2(-/-) mice kbile was significantly, by 1.8-fold, lower than in wild-type mice (kbile, Abc1a/b(-/-)Abcg2(-/-): 0.0017±0.0004 min-1). Pretreatment with unlabeled tariquidar significantly reduced kbile values in wild-type, Abc1a/b(-/-) and Abcg2(-/-) mice, by 1.8-, 2.1- and 2.5-fold, respectively, compared to the baseline scan. In contrast, in Abc1a/b(-/-)Abcg2(-/-) mice kbile was not significantly different between the two scans (Figure 1).
Our findings suggest that the hepatobiliary excretion of [11C]tariquidar and/or its radiolabeled metabolites is mediated in mice by Abcb1a/b and Abcg2. Furthermore, our study provides evidence for a mutual functional compensation between Abcb1a/b and Abcg2 in the murine liver in promoting the hepatobiliary excretion of dual substrate drugs. Thus, [11C]tariquidar PET may be used to measure the transport activities of ABCB1 and ABCG2 in the liver and might also be applicable to investigate the influence of liver diseases, genetic polymorphisms or drugs on hepatic ABCB1 and ABCG2 function.
 Bankstahl et al. Drug Metab Dispos. 2013 Apr;41(4):754-62.
 Bauer et al. Clin Pharmacol Ther. 2016 Aug;100(2):131-41.
The research leading to these results has received funding from the Lower Austria Corporation for Research and Education (NFB) project LSC15-003 and from the Austrian Science Fund (FWF) project KLI 480-B30.
Rate constant for transfer of radioactivity from liver via bile into intestine (kbile) in wild-type, Abc1a/b(-/-), Abcg2(-/-) and Abc1a/b(-/-)Abcg2(-/-) mice before (baseline) and after administration of unlabeled tariquidar (15 mg/kg). ***p < 0.001, **p < 0.01, *p < 0.05, two-tailed unpaired t test and two-tailed paired t test.
Keywords: positron emission tomography, liver, ABCB1, ABCG2, [11C]tariquidar
Rapid nuclear imaging of tumour cell death in vivo using C2Am (#446)
A. A. Neves1, F. Bulat1, 2, L. T. L. Brandt1, B. Xie1, D. Soloviev1, D. - E. Hu1, D. Lewis1, F. Leeper2, K. M. Brindle1
1 University of Cambridge, CRUK Cambridge Institute, Cambridge, United Kingdom
Cell death is an important target for imaging the early response of tumours to treatment. We have described recently 99mTc-, 111In- and near-infrared fluorophore-labelled derivatives of C2Am for imaging tumour cell death in vivo using SPECT  and photoacoustic  imaging, respectively. We describe here the development of 68Ga- and 18F-labelled C2A derivatives for PET imaging.
We have developed novel bioconjugation routes for the production of PET derivatives of C2Am. A tris(hydroxypyridinone)-maleimide (THP-mal) chelate was conjugated to reduced C2Am, under physiological conditions, using the single cysteine in C2Am. C2Am-THP was later loaded with either cold 69Ga or 68Ga for biodistribution and imaging studies. We have also performed a one-pot two-step automated synthesis of a [18F]-N-fluoropentyl maleimide radiotracer, which was conjugated to C2Am for [18F]-based PET imaging.
99mTc-C2Am and 111In-C2Am showed favourable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in mouse spleen and liver at 24 h after probe administration. 99m Tc-C2Am [Fig.1] and 111In-C2Am generated tumour-to-muscle ratios in drug-treated tumour-bearing mice of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. A fluorophore-labelled C2Am derivative also showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumour cell death (<5%). There was a significant correlation (R=0.9) between fluorescently labelled C2Am binding and histologic markers of cell death (cleaved caspase-3); no such correlation was observed with a site-directed mutant of C2Am that does not bind phosphatidylserine (iC2Am). New results with 68Ga and 18F-labelled C2Am will be presented.
Given the favourable biodistribution profile of radiolabelled C2Am derivatives, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation, particularly as PET tracers. We have initiated GMP manufacture and toxicology studies required for a Phase 1 trial.
 Neves AA, Xie B, Fawcett S, Alam IS, Witney TH, de Backer MM, Summers J, Hughes W, McGuire S, Soloviev D, Miller J, Howat WJ, Hu DE, Rodrigues TB, Lewis DY, Brindle KM. Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I. (2017) J Nucl Med. 58:881-887.
 Xie B, Tomaszewski MR, Neves AA, Ros S, Hu DE, McGuire S, Mullins SR, Tice D, Sainson RCA, Bohndiek SE, Wilkinson RW, Brindle KM. Optoacoustic Detection of Early Therapy-Induced Tumor Cell Death Using a Targeted Imaging Agent. (2017) Clin Cancer Res. 23:6893-6903.
Cell death imaging using (99m)Tc-C2Am in the Emu-myc model
SPECT/CT fusion images were acquired before (left) and 24h after (right) treatment. Imaging was conducted 2h after injection of (99m)Tc-C2Am. Lymph node tumours are visible in the neck (1), axilla (2, right arrow) and thymus (2, left arrow).
Multimodal imaging agents: a new type of small bimodal imaging agents for PET/OI and SPECT/OI (#150)
I. Heing-Becker1, C. Grötzinger2, S. Prasad2, R. Haag1, K. Licha1
1 Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany
The multimodal imaging technique PET (or SPECT) with optical imaging (OI) offers significant advantages during the evaluation of new drug candidates and intraoperative imaging compared to classical approaches. Cyanine dyes are widely employed for OI and offer a versatile platform to introduce further moieties applicable for PET and bioconjugation. Using only one comparatively small molecule for two different imaging procedures, biodistribution and cellular uptake studies can be accomplished in one experimental setting, employing PET via a radiometal and subsequent OI via the fluorophore.
A bifunctional asymmetric indocarbocyanine dye (ICC) was synthesized from differently substituted indolenine precursors and modified to obtain an amine group and a carboxylic acid. This bifunctionality was used for the subsequent introduction of a DOTA-based chelator and for the conjugation to (bio)molecules. Spectroscopic characterization included absorption and fluorescence spectra. For a bimodal labelled peptide conjugate, the toxicity was tested as well as its functionality via cellular uptake studies.
A new type of small bimodal imaging agents for PET/OI or SPECT/OI was synthesized and can be used for the labelling of new drug candidates and disease markers. For a proof of concept, the model compound DOTA-ICC-TOC was successfully synthesized. DOTATOC, without the dye, employs an octapeptide that binds to the somatostatin receptor SSTR2 and is used for PET diagnostics via 68Ga radiolabelling and treatment via 177Lu radiolabelling. The metal-free form of DOTA-ICC-TOC showed no toxicity up to 10 µM and high SSTR2-receptor affinity being taken up at low concentrations via endocytosis. Its functionality was proven by colocalisation with the SSTR2 receptor in confocal microscopy using RIN1038 cells (transfected with SSTR2 and GFP). First radiolabelling experiments with DOTA-ICC-TOC indicate that 68Ga labelling via the DOTA-chelator can be performed, thereby enabling studies on tumour uptake and organ distribution in vivo.
The synthetic route to a new class of conjugatable small bimodal imaging agents for PET/OI and SPECT/OI based on an indocarbocyanine dye and a DOTA-chelator is presented. Our approach, exemplified by a bimodal labelled SSTR2 binding octapeptide, shows that labelling of a small peptide enabled maintaining high functionality and receptor binding and indicates suitability for radiolabelling.
 F. L. Thorp-Greenwood, M. P. Coogan, Dalton Trans. 2011, 40, 6129 – 6143.
 K. Licha, U. Resch-Genger, Comprehensive Biomedical Physics, Elsevier, Oxford, 2014, 85 – 109.
 I. Velikyan, B. Langström et al., Nucl. Med. Biol. 2010, 37, 265 – 275.
The Dahlem Research School is acknowledged for funding.
Keywords: Multimodal imaging, Cyanine dyes, DOTA
Synthesis and radiolabelling (124I) of PEG-stabilized gold nanorods (AuNRs) as boron drug delivery candidates with potential application in Boron Neutron Capture Therapy. (#123)
K. R. Pulagam1, J. Kumar2, V. Gómez-Vallejo1, L. Liz-Marzan2, J. Llop1
1 CIC biomaGUNE, Radiochemistry and Nuclear imaging Lab, San Sebastian, Spain
Boron neutron capture therapy (BNCT) is widely accepted as a promising cancer treatment. One limitation of BNCT is the need to develop drugs that are able to deposit a sufficient number of 10B atoms specifically in tumor cells. Here, we report the preparation and characterization of AuNRs functionalized with Poly(ethylene glycol) methyl ether thiol and boron-based molecule cobalt bis(dicarbollide), [3,3’-Co(1,2-C2B9H11)2]-, commonly known as COSAN. The resulting functionalized AuNRs were radiolabelled with 124I either on the surface of the gold core or covalently attached to the COSAN.
The cobalt-bis(dicarbollide) anion (COSAN) was treated with tetrahydropyran and sequentially reacted with potassium thioacetate (KSAc) and iodine to achieve THP ring opening and iodination of the cluster (Figure 1). Radiolabelling was achieved by palladium-catalyzed isotopic exchange using 124I on compound -. Final hydrolysis yielded the thio-compound, ready to be incorporated into AuNRs. The synthesis of AuNRs followed a reported seed-mediated method.1 Radiolabelled, boron-rich AuNRs were prepared using two strategies: (i) absorption of 124I- on the surface of the gold core followed by attachment of PEG-SH and COSAN derivatives; and (ii) functionalisation of CTAB-stabilized AuNRs with PEG-SH and (124I-- + -), to incorporate the radionuclide at the shell of the AuNRs (Figure 2).
The (protected) thiolated iodo-cobalt bis(dicarbollide) derivative - could be obtained in an overall yield of 70±6%. Radiolabelling by isotopic exchange (T=100ºC, t=15 min) resulted in conversion values in the range of 75-80%. COSAN could be efficiently incorporated into AuNRs (size: 10 x 30 nm) as demonstrated by X-ray photoelectron spectroscopy (XPS). Both labelling strategies resulted in good yields (>55%).
PEG stabilized and thiolated iodo-cobalt bis(dicarbollide) functionalized radiolabelled AuNRs were prepared. These could be appropriate boron-rich drug candidates with application in BNCT. Biodistribution studies in healthy and tumour-bearing animals are planned for the next future.
1. B. Nikoobakht, MA. El-Sayed, Chem Mater. 2003, 15, 1957.
This project was partially funded by Ministerio de Ciencia e Innovación (Grant number CTQ2009-08810).
Keywords: BNCT, Gold nanorods, Radiolabeling (I-124)
Micro-algae as radionuclide carrier for nuclear imaging of aquatic animals: a proof of concept (#22)
C. L. Schmidt1, S. Seegers2, P. Jatzek2, M. Gebert3, M. Rafecas2
1 Universität zu Lübeck, Isotopenlabor der Sektion Naturwissenschaften, Lübeck, Germany
Aquatic animals are used to study biological processes and to model human diseases, e.g. in cancer research or immunology . Zebrafishes and some invertebrate species are well-established models, also in biotechnology and environmental sciences, while aquaculture research focuses anyhow on fishes, mussels, etc. On the other hand, in-vivo imaging of aquatic animals, in particular radionuclide imaging, has proved to be difficult . The administration of the radiotracer is one of challenges. In this work we explore the use of micro-algae to transport radioactive iodine (125I) into mussels.
The target mussels, Mytilus edulis collected form the Baltic seashore, were kept in a tank under controlled conditions prior to the experiments. Chlamydomonas reinhardtii (SAG 83.81), Mychonastes homosphaera (SAG 6.95) and Chlorella sorokiniana (SAG 211-31) were used as radionuclide carriers. Chemical iodination with Chloramin-T was used for labeling. The concentrations of 125I-KI, Chloramin T, and reaction times were optimized. For feeding tests, mussels were placed in 600 ml of saltwater and defined amounts of labeled micro-algae added at different time points. A gamma-counter was used to estimate the 125I uptake in the micro-algae and the mussels, and the activity in saltwater samples at various times. A phosphor imager was used to visualize the activity distribution in the mussels.
Chlorella and Mychonastes cells were most successfully iodinated. The reaction conditions had significant effects on the labeling efficiency. Up to 45% of 125I was bound to the cells. Routinely 1 mg/ml Chloramin T and 30 min reaction time was used yielding ~30% incorporation. Uptake by the mussels showed a large variability. A step-wise feeding over 120 min, with the addition of cells not exceeding 8000 (Chlorella) or 51300 (Mychonastes) cells/ml  in 30 to 40-min-intervals, led to the best results: 70 to 180 kBq could be taken up within the 1st hour (Fig. 1). Phosphor-imager data showed 125I mainly localized within the digestive system, particularly in the stomach (Fig. 2). Approx. 80 min after the last feeding the mussels started to release significant amounts of activity: 30 to 40% of the activity was released within the following 4 h. Up to 30% of the activity remained in the animals after 24 h. A time dependent redistribution of activity within the mussels could be observed.
Feeding saltwater mussels with labeled micro-algae led to detectable 125I uptake in the mussels. Using labeled micro-algae as radionuclide carriers might offer new possibilities to investigate the metabolism of several aquatic animals. At present, the time required by the labelling and feeding procedures excludes the use of short-lived radioisotopes. Further work should focus on optimizing both procedures, to be adapted to the animal under study. Extrapolation to other tracers will be also the focus of future work, including testing other micro-algae species and aquatic animals.
 Schartl M. Beyond the zebrafish: diverse fish species for modeling human disease. Dis Model Mech 2014 Feb;7(2):181-92. doi: 10.1242/dmm.012245.
 Koba W, Jelicks LA, Fine EJ. MicroPET/SPECT/CT imaging of small animal models of disease. Am J Pathol 2013 Feb;182(2):319-24. doi: 10.1016/j.ajpath.2012.09.025.
 Riisgård HU, Egede PP, Saavedra IB. Feeding Behaviour of the Mussel, Mytilus edulis: New Observations, with a Minireview of Current Knowledge. J Mar Biol 2011. doi: 10.1155/2011/312459
The authors would like to thank Jan-Christer Schorch (Isotopenlabor der Sektion Naturwissenschaften, Universität zu Lübeck), Cindy Krause (Institute of Medical Engineering, Universität zu Lübeck) and Dirk Scheel (Fraunhofer-Institution for Marine Biotechnology and Cell Technology) for their valuable contribution.
Uptake of labeled Chlorella cells by a single mussel
a) Uptake in a single mussel. The arrows indicate the addition of 4.8x106 cells. b) Activity release into water from the mussel. Due to the measurements of activity within the mussel a 60 to 80 min delay lies between the last data point in a) and the first measurement in b). The images show the mussel (left), the 125I activity distribution at the end of the feeding, and 24h after feeding (right).
Activity distribution of iodine in a mussel fed with labeled Mychonastes homosphaera cells
Activity distribution in a mussel previously fed with 125I labeled Mychonastes homosphaera cells. Both sides of the mussels (top and bottom rows) were measured by the phosphor imager a, d) immediately after feeding, b, e) approximately 23 hours after feeding and c, f) approximately 25 hours after feeding.
Keywords: radionuclide imaging, Iodine-125, acuatic animals, micro-algae, bivalvia
A novel norepinephrine transporter tracer [18F]NS12137: nucleophilic radiosynthesis and preclinical CNS binding evaluation (#387)
F. R. López-Picón1, 2, A. K. Kirjavainen3, 4, S. Forsback3, 4, J. Takkinen1, 2, M. Haaparanta-Solin1, 2, D. Peters5, 6, O. Solin3, 4, 7
1 University of Turku, Preclinical Imaging, Turku PET Centre, Turku, Finland
Several psychiatric and neurodegenerative diseases are associated with malfunction of brain norepinephrine transporter (NET). However, current clinical evaluations of NET function are limited by the lack of sufficiently sensitive methods of detection. Therefore we have synthesized a novel radiotracer [18F]NS12137 for positron emission tomography (PET) and have demonstrated that it is highly specific for in vivo detection of NET-rich regions of rat brain tissue.
[18F]NS12137 was produced by nucleophilic radiofluorination. In vivo and ex vivo characterization of [18F]NS12137 was performed using adult and 14 to 16 days-old Sprague-Dawley rats. The specificity of [18F]NS12137 binding was demonstrated on the basis of competitive binding by nisoxetine, a known NET antagonist of high specificity.
[18F]NS12137 was successfully synthesized with a decay-corrected radiochemical yield of 18.6 ± 5.9% calculated from the initial 18F-activity. The radiochemical purity was>99 % and the molar activity (MA) of the tracer was>500 GBq/µmol, respectively. The initial in vivo brain uptake (0-20 min) of [18F]NS12137 in adult and immature rats, showed binding in the cortex, thalamus and pons. After washout (50-60 min), in the adult rats, the signal decreased. In contrast, in immature rats, signal was detected in the locus coeruleus (LC). In the autoradiographic (ARG) study, the binding in adult and immature rat brain was detected in the main brain areas, with the highest binding in NET rich areas such as the bed nucleus of the stria terminalis (BNST) and the LC. The specificity of [18F]NS12137 was tested using nisoxetine (5 mg/kg). After nisoxetine injection, the in vivo binding decreased in all brain regions, and in the ARG study, the binding was almost completely blocked in the NET rich areas
The novel NET tracer [18F]NS12137 was successfully synthesized with a high MA. In addition, [18F]NS12137 showed good brain uptake, specific binding to NET-rich brain areas, and a fast brain clearance. [18F]NS12137 is a promising NET-tracer with possible clinical utility.
Financial support of The Academy of Finland (grants 116084, 128591, and 266891), the Orion Research Foundation, the State Funding for University Level Health Research for the Turku University Hospital, and the University of Turku Foundation.
Keywords: Locus Coeruleus, [18F]NS12137, neurodegeneration, neuropsychiatric, Alzheimer, norepinehrine transporter, PET