EMIM 2019
To search for a specific ID please enter the hash sign followed by the ID number (e.g. #123).

PET/SPECT/CT & Multimodal | New Probes II

Session chair: Wiktor Szymansky (Groningen, Netherlands); Jason Lewis (New York, US)
 
Shortcut: PW13
Date: Thursday, 21 March, 2019, 12:45 p.m.
Room: ALSH | level 0,BOISDALE | level 0,CARRON | level +1,DOCHART | level +1
Session type: Poster

Contents

Click on an contribution to preview the abstract content.

644

68Ga and MMAF dual-labelled single-chain variable fragments (scFv) for PET imaging of HER2 overexpressed tumors (#328)

Ruisi Fu1, 2, Marta Braga2, Laruence Carroll2, Ioanna Stamati3, Gokhan Yahioglu3, Eric Aboagye2, Philip Miller1

1 Imperial College London, Department of Chemistry, London, United Kingdom
2 Imperial College London, Department of Surgery and Cancer, London , United Kingdom
3 Antikor, Stevenage Bioscience Catalyst, Stevenage, United Kingdom

Introduction

Single-chain variable fragments (scFv) are small (28 kDa) antibody fragments that have been demonstrated to display excellent targeting and pharmacokinetics for both molecular imaging and drug delivery.1 Loaded antibody fragments with drug ‘pay-loads’ can, however, alter their pharmacokinetics.2  Herein, we aim to develop a novel 68Ga-radiolabelled scFv (TCT1067) and drug loaded 68Ga/Monomethyl auristatin F(MMAF) TCT1067 dual conjugate and assess their uptake and biodistribution in HER2 overexpressed cell lines and  in vivo tumor models.

Methods

In this study, a lysine enriched HER2 specific scFv TCT1067 was modified with either TCO-PEG4 or both MMAF-PEG2 and TCO-PEG4. The conjugates were characterized with mass spectrometry and surface plasmon resonance. A bioorthogonal inverse electron demand Diels–Alder (iEDDA) cycloaddition reaction between a 68Ga-tetrazine derivative ([68Ga]GaDOTA-Tz) and trans-cyclooctene (TCO) modified scFv’s was developed for the radiolabeling. Radiolabeling of the tetrazine derivative was achieved following a previously published method.3 The HPLC purified scFv conjugates labeled with tetrazine compound [68Ga]GaDOTA-Tz (scheme 1) at 37 °C for 30 min. The radiolabeling efficiency was confirmed using Reverse Phase HPLC (RP-HPLC) and size exclusion chromatography (SEC).

Results/Discussion

The Mass spectrometry results confirmed the conjugation of three to five TCO-PEG4 groups and three to seven MMAF’s per modified TCT1067. All the conjugates displayed low pM to low nM binding affinity toward HER2. The DOTA-tetrazine was labelled with 68Ga in high radiochemical incorporation (>90%). After solid phase extraction, a >99% radiochemical purity was achieved. The non-decay corrected radiochemical yield ranged 30-40%. The iEDDA reaction of [68Ga]GaDOTA-Tz and TCO functionalized TCT1067 was confirmed by size exclusion HPLC. In vitro uptake with HER2 positive SKOV3 cell lines showed high uptake with both TCO and MMAF/TCO dual conjugates. (Figure 1A, 1D) Uptake with unlabeled TCT1067 blocking showed that the high uptake values are specific to HER2. Uptake of the radiolabeled TCT1067 are low in HER2 negative cell lines (MDA-MB-231, MDA-MB-468) and following the same trend as the HER2 expression levels. (Figure 1A, 1C). There is no uptake of [68Ga]GaDOTA-Tz. (Figure 1B)

Conclusions

We have developed conjugation and labelling strategies for the preparation of novel HER2 specific scFv TCO and MMAF/TCO dual conjugates. We have demonstrated highly specific uptake of the 68Ga labelled and 68Ga/MMAF dual-labelled scFv conjugates in HER2 overexpressed cancer cells.  In vivo uptake studies with HER2 positive and negative xenograft are currently ongoing and will be presented in due course.

References

[1]    Fu, R. et al. ChemMedChem 2018 Sep 23.

[2]    Al-Saden, N. et al. Mol. Pharm. 2018 15 (8), 3383–3393

[3]    Evans, H. L. et al. ChemComm. 2014 50, 9557-9560,

Acknowledgement

Ruisi Fu gratefully acknowledges the award of a PhD scholarship from the China Scholarship Council and support from Department of Chemistry, Imperial College London.

Scheme 1

Radiolabeling between TCO modified TCT1067 and [68Ga]GaDOTA-Tz are achieved  in PBS/10% EtOH at 37°C for 30min.

Figure 1

(A)In vitro uptake of radiolabeled conjugates. The HER2 positive SKOV3 always has the highest uptake followed by MCF7, MDA-MB-231 and MDA-MB-468. (B)Uptake of [68Ga]GaDOTA-Tz and [68Ga]GaDOTA-TCT1067 in HER2 positive SKOV3 cells. (C)Western blot of p185-HER2 of cell lines. (D)Uptake of [68Ga]GaDOTA-TCT1067 and uptake of [68Ga]GaDOTA-TCT1067 with 10x unlabeled TCT1067 in HER2 positive SKOV3 cells.

Keywords: Single-chain variable fragments, immuno-PET, Ga-68, bioorthogonal click, HER2
646

AlF-18-Labeling of new AMPTA-based chelators for applications in Positron Emission Tomography Imaging (#467)

Lisa Russelli1, Jonathan Martinelli2, Francesco De Rose1, Michael Herz1, Wolfgang Weber1, Lorenzo Tei2, Calogero D'Alessandria1

1 Klinikum rechts der Isar der TUM, Nuclear Medicine Department, Munich, Bavaria, Germany
2 University of Piemonte Orientale, Department of Science and Technological Innovation, Alessandria, Italy

Introduction

Positron emission tomography (PET) is a non-invasive molecular imaging technology constantly expanding, with a high demand for specific antibody-derived imaging probes. The need to develop stable tracers based on temperature sensitive molecules leads us to design three chelators based on the structure of 2-aminomethylpiperidine with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, 2-AMPDA-NHB and 2-AMPDA-HB). The labelling with AlF-18 at room temperature would allow the use of these systems with heat sensitive biomolecules.

Methods

Three different AMPTA-based chelators were designed and synthetized, 2-AMPDA-HB, 2-AMPDA-NHB and 2-AMPTA. All the chelators were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed at different pH (4 and 5) and temperature (rt, 37 and 80°C). The products were purified using Sep-Pak Alumina N Plus Light cartridges and eluted with a 0.9% NaCl solution. All the products were analyzed by radio-TLC and radio-HPLC and the stability of the tracers was investigated at 10, 30, 60, 120 and 240 minutes via incubation in three different solutions: human serum, PBS and 0.9% NaCl solutions.

Results/Discussion

For all chelators a radiolabeling efficiency between 50 and 78% was obtained at pH 5 and room temperature (50% 2-AMPDA-HB, 74% 2-AMPDA-NHB, 78% 2-AMPTA). A similar result was obtained at 37°C where the radiolabeling efficiency is 60% for 2-AMPDA-HB, 68% for 2-AMPDA-HB and 75% for 2-AMPTA. After the stability study good results were obtained on the chelators labeled at room temperature and pH 5. High stability in human serum was measured for the ligand 2-AMPDA-HB, with a 90% of AlF-18 complexed and 55% for the 2-AMPTA chelator after 120 min. In particular the stability after 240 minutes in human serum was respectively 68% of AlF-18 complexed for the chelator 2-AMPDA-HB and 33% for the 2-AMPTA.

Conclusions

In a preliminary screening of the reported chelators, promising results have been obtained in terms of radiolabeling efficiency at T-room and 37 °C, and good stability in physiological conditions. The selection of the best chelator will lead to the synthesis of the bifunctional derivate followed by the conjugation with temperature sensitive biomolecules, e.g. Fab fragments and/or nanobodies, the labeling with AlF-18 and then in vivo applications.

2-AMPTA derivatives chelators for the complexation of AlF-18.
Keywords: AlF-18 labeling, radiochemistry, chelators, PET imaging
647

111In-labeled VHH B9 for SPECT imaging of endogenous tumor hypoxia marker carbonic anhydrase IX (#77)

Sanne A M van Lith1, Fokko J Huizing2, Bianca A W Hoeben2, Martin Gotthardt1, Paul M P van Bergen en Henegouwen3, Johan Bussink2, Sandra Heskamp1

1 Radboudumc, Radiology and Nuclear Medicine, Nijmegen, Netherlands
2 Radboudumc, Radiation Oncology, Nijmegen, Netherlands
3 Utrecht University, Cell Biology, Utrecht, Netherlands

Introduction

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme that is upregulated by cells under hypoxic conditions. Hypoxia is present in the majority of solid tumors and is associated with poor outcome and radioresistance. Therefore, non-invasive imaging of CAIX could be of prognostic value and utilized for radiotherapy planning and treatment effect monitoring. The aim of this study was to validate and optimize SPECT imaging of CAIX expression in a head-and-neck squamous cell carcinoma model using an anti-CAIX variable domain of heavy chain antibody (VHH).

Methods

VHH B9 was conjugated with maleimide-DTPA and labeled with 111In. The binding affinity and internalization of [111In]InDTPA-B9 was analyzed using CAIX expressing SKRC-52 cells. Subsequently, a dose-escalation study was performed in athymic nude mice with subcutaneous SCCNij153 head-and-neck cancer xenografts. Targeting specificity was determined by blocking the specific uptake with unlabeled VHH B9, and by analyzing tumor uptake of an 111In-labeled irrelevant VHH R2. To reduce renal retention of [111In]InDTPA-B9, a subgroup of mice was co-injected with gelofusin. Tracer uptake at 4 hours after injection was determined by ex vivo radioactivity counting and SPECT/CT scans. Furthermore, autoradiography images of tumor sections were analyzed for correlation with CAIX immunohistochemistry.

Results/Discussion

DTPA-B9 was radiolabeled with 111In at a specific activity of 4 MBq/µg. [111In]InDTPA-B9 bound to SKRC-52 cells with high affinity (IC50 = 11.3 nm, Kd = 27.2 nM) and the internalization rate of the tracer was relatively low (Ke = 0.01). A protein dose of 5 µg resulted in the highest uptake in SCCNij153 tumors in vivo (1.05±0.14%ID/g), with tumor-to-blood and tumor-to-muscle ratios of 11.5 and 24.7, respectively (Figure 1). Unlabeled B9 reduced tumor uptake to 0.30±0.03%ID/g and irrelevant [111In]InDTPA-R2 showed tumor uptake of 0.20±0.14%ID/g. Gelofusin did not significantly alter renal retention of [111In]InDTPA-B9. Uptake of [111In]InDTPA-B9 in the SCCNij153 xenografts could be visualized with SPECT/CT and autoradiography (Figure 2). Furthermore immunohistochemistry and autoradiography images showed co-localization of [111In]InDTPA-B9 and CAIX expression.

Conclusions

[111In]InDTPA-B9 VHH shows specific targeting of CAIX-expression in head-and-neck cancer xenografts. In ongoing studies we will compare this tracer to other available CAIX tracers and we will assess its potential for treatment selection and monitoring of hypoxia responses to therapy.

Figure 1. In vivo dose-escalation
Biodistribution of [111In]InDTPA-B9 and negative control [111In]InDTPA-R2 in tumor, blood and muscle 4 hours post tracer injection.
Figure 2. SPECT and autoradiography
Uptake of [111In]InDTPA-B9 in a SCCNij153 tumor as visualized with (A) SPECT and (B) autoradiography. Note the similar spatial correlation of the signal in the two figures.  
Keywords: SPECT/CT imaging, Hypoxia, Carbonic anhydrase IX, VHH
648

Screening of small molecule compounds as PET tracers targeting oligomeric and aggregated α-synuclein (#455)

Sabrina Buss1, Kristina Herfert1, Laura Kuebler1, Andreas Maurer1, Maria Wahle1, Constanze Heinzel1, Felix Schmidt4, Andrei Leonov2, Sergey Ryazanov2, Armin Giese3, Christian Griesinger2, Bernd J. Pichler1

1 Eberhard Karls University , Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Tuebingen, Baden-Württemberg, Germany
2 Max Planck Institute for Biophysical Chemistry, Goettingen, Lower Saxony, Germany
3 Ludwig-Maximilians-University, Center for Neuropathology and Prion Research, Munich, Bavaria, Germany
4 MODAG GmbH, Wendelsheim, Bavaria, Germany

Introduction

Imaging α-synuclein (αSYN) pathology to distinguish synucleinopathies from other neurodegenerative disorders is challenging and relevant PET tracers are still missing. In our previous studies, we identified one compound, showing a very high affinity towards recombinant αSYN fibrils and good selectivity over Aß and tau. Although it showed favorable in vivo kinetics with fast washout from the brain after C-11 labeling, we identified one lipophilic metabolite in the brain, which was the demethylated form of the parent compound, confounding the in vivo quantification.

Methods

To test the metabolite and other derivatives, we performed a competition binding assay (CBA) and determined the binding affinity Ki. Saturation binding assays (SBA) and CBA were performed using recombinant human αSYN fibrils and a mixture of αSYN monomers, oligomers and fibrils. For SBA, fibrils were incubated with increasing concentrations (0.02nM–48nM) of the tritiated compound to obtain total binding. Non-specific binding was achieved using an excess of the cold compound. For CBA, fibrils were incubated with decreasing concentrations of competitor (1000nM–0.0002nM) in the presence of a fixed concentration of the tritiated lead compound (2nM). After vacuum filtration and washing, liquid scintillation counting was performed. Kd- and Ki-values were calculated using non-linear regression.

Results/Discussion

Our lead compound showed high affinity to pure αSYN fibrils (Kd<4nM) and to αSYN monomeric and oligomeric structures (Kd<5nM). Competition binding experiments revealed Ki-values between <0.1nM and1000 nM. We identified one compound with a very high competitive inhibition towards pure αSYN fibrils (Ki<0.1nM) as well as the mixture of αSYN monomers, oligomers and fibrils (Ki<0.1nM). Good competitive inhibition was seen for the identified lipophilic metabolite (Ki<1nM) and another compound (Ki<2nM) on αSYN fibrils.

Conclusions

In our screening assay, we identified three promising PET tracer candidates with a high affinity towards recombinant αSYN fibrils. In further experiments, we will test these compounds towards their selectivity over Aß and tau and evaluate their specificity and selectivity in human brain tissue with confirmed αSYN, Aß and tau pathology.

Keywords: alpha synuclein, PET tracer, synucleinopathies, fibrils
649

44Sc-AAZTA-PSMA: Synthesis, Characterization and Preclinical Evaluation of a new Pet Tracer (#442)

Ivan Hawala1, Simona Ghiani2, Dezso Szikra3, Gyorgy Trencsenyi3, Gabor Nagy3, Alessandro Maiocchi2, Silvio Aime1

1 Università degli studi di Torino, Dipartimento di Biotecnologie Molecolari e Scienze per la salute, Torino, Italy
2 Bracco Imaging Spa, Bracco Research Centre, Colleretto Giacosa (TO), Italy
3 Scanomed Ltd., Scanomed Ltd., Debrecen, Hungary

Introduction

PSMA is an extracellular hydrolase highly upregulated in metastatic and hormone-refractory prostate carcinomas1. Recently 44Sc (t1/2=3.97h) received much attention as potential radionuclide with favourable characteristics for PET imaging applications.

AAZTA was thoroughly studied as chelator for Gd3+ for MRI applications2. The excellent results of the equilibrium, kinetic, and labelling studies led to a preliminary assessment of the in vitro and in vivo behavior of 44Sc(AAZTA)- and two derivatives by assuming that the physicochemical properties of Sc(AAZTA) are maintained in its bioconjugates3.

Methods

The first aminoacid (Fmoc-L-2Nal-OH) was anchored on a 2-chlorotrytil chloride resin (loading: 1.63 mmol g-1). All solid phase synthesis steps were performed by standard Fmoc protocol. After the coupling of the ligand moiety, the fully protected peptide was cleaved from the resin as the next steps were carried out in solution. The best labelling condition for AAZTA-PSMA was found using HEPES buffer at pH 4: after 5 minutes reaction time at room temperature in the presence of low concentration of the ligand (1 µM), the RCP was >95% (Figure 1). Dynamic (0-90 min) and static (2.5 h) PET scans were acquired after the injection of AAZTA-PSMA (~18 MBq) in LNCaP tumour-bearing mice (n=5). A representative image is reported in Figure 2 showing high tumour uptake at 2.5 h from the administration.

Results/Discussion

The bioconjugate was synthesized combining solid phase peptide synthesis (SPPS) and solution chemistry and the desired high purity (97%) product was obtained with a chemical yield of 9%.

Subsequently, the radiolabelling with 44Sc was carried out using the radionuclide obtained from the irradiation of natural calcium target in cyclotron. Good radiochemical yields under mild conditions (pH 4, 298 K) were achieved.

Herein we report our preclinical results of a 44Sc-labeled PSMA binding motif based peptide for in vivo PET imaging of PSMA expression in a preclinical cancer model. Dynamic scans showed high tracer accumulation in the tumour regions already after 20 minutes and pharmacokinetic in different organs. Kidney is the main organ involved for the tracer clearance. Another focus of this work was also devoted to demonstrate that the ligand and spacer choice could has a strong impact on in vivo uptake of the probe.

Conclusions

The synthesis of AAZTA-PSMA consists of three main steps, namely i) the solid phase synthesis, ii) a solution chemistry step to carry out the PSMA binding motif conjugation and iii) a final purification by preparative HPLC.

This work have demonstrated the suitability of AAZTA-PSMA conjugate for the complexation with 44Sc and the feasibility of PETs images acquisitions for prostate cancer diagnosis.

References

1 Weineisen et al, EJNMMI Research 2014, 4:63

2 Manzoni et al, ChemMedChem 20127, 1084–1093

3 Nagy et al, AngewChemIntEd. 2017, 56, 1-6

Acknowledgement

All the radiolabellings and in vivo experiments were carried out by D.Szikra, G.Trencsényi, G.Nagy at Scanomed Ltd. (Debrecen, Hungary). The project is totally funded by Bracco Imaging Spa (Colleretto Giacosa, Italy).

Figure 1. Scandium 44 labelling of AAZTA-PSMA
Labelling of AAZTA-PSMA with 44Sc in 0.1 M HEPES buffer pH 4 at room temperature and different reaction time
Figure 2. Representative PET/MRI image
Static PET/MRI image of 44Sc-AAZTA-PSMA 2.5 h after injection
Keywords: PSMA, Scandium 44, PET, Synthesis, AAZTA
650

Development of 68Ga and 18F radiolabelled peptide probes for PET imaging of αvβintegrin expression in cancers and fibrotic diseases. (#395)

Juozas Domarkas1, 2, James Thompson2, 3, George Herbert1, 2, Benjamin P. Burke2, 4, Simon Hart3, Christopher Cawthorne1, 2, Alyn Morice3, Stephen J. Archibald1, 2, 4

1 University of Hull, School of Life Sciences, Hull, United Kingdom
2 University of Hull, Positron Emission Tomography Research Centre, Hull, United Kingdom
3 Hull York Medical School, Hull, United Kingdom
4 University of Hull, School of Mathematics and Physical Sciences, Hull, United Kingdom

Introduction

Expression of integrin αvβis upregulated in multiple cancers and in injured epithelium, making it an attractive target for cancer and fibrotic phenotype imaging.1,2 In tracer development, small peptides occupy an intermediate position between small molecules (difficult to develop, fast PK) and antibodies (easy to develop, slow PK).We have modified an αvβbinding peptideto allow for its radiolabelling with either 68Ga,177Lu or 18F. A library of tracers, both imaging and theragnostic, was developed with variable PK properties to provide a better understanding of SAR requirements.

Methods

The peptide core was prepared by manual solid phase peptide synthesis and derivatised to contain DO3A, a Ga3+/ Lu3+chelator, or azide/ alkyne groups, for the introduction of fluorinated prosthetic groups via Cu(I) “click” cycloadditions. Pharmacokinetic (PK) variability was generated by use of rigid and/or hydrophilic linkers between the peptide and selected radiotag. Affinity to αvβ6 was assessed by hot ligand uptake and/or ELISA assays. 68Ga3+ and 177Lu3+ were introduced by labelling in HEPES pH 3.5 buffer (90oC, 10 min). 18F labelled prosthetic groups were prepared from tosylated precursors and K222/K18F complex (95oC, 10-20 min). LogD4.7 values were measured using a shake-flask method. Naïve and tumour bearing mice were used to acquire PET/CT images and to study biodistribution.

Results/Discussion

All studied tracers showed low or sub nM affinity specific to αvβ(ELISA). Derivatisation of the N-terminus had little effect on binding affinity, with metal chelation further increasing it. Study of 68Ga/ 177Lu tracers showed high hydrophilicity (LogD7.4= ca.-3). 68Ga3+ chelates were stable in human serum at 37oC for 3 h, (177Lu3+ chelate was stable up to 12 h), but showed ~ 50% degradation in mouse urine 90 minutes post-injection. PET/CT imaging using the 68Ga tracer showed fast renal clearance and blockable tumour uptake (BxPC3, αvβ6xenograft) in addition to gut and submandibular gland (natural αvβexpressing organs) uptake. Preliminary investigation of an example 18F derivative showed reduced hydrophilicity and renal clearance accompanied with increased gut uptake and lack of tumour uptake. Preparation and assessment of fluorinated derivatives with a range of linkers is ongoing.

Conclusions

ELISA and hot ligand cell binding assay revealed that the selected peptide can be functionalised on the N-terminus without a loss of αvβ6 affinity. In vivo PET/CT imaging showed that it is also sufficiently stable to biodistribute to the periphery and selectively retain within αvβexpressing tumours/organs. These results warrant extension of the study to a larger set of radiolabelled tracers for better understanding of optimal properties.

References

 

  1. W. Guo and F. Giancotti, Nat. Rev. Mol. Cell Biol., 2004, 5(10), 816-826.
  2. A. Tatler and G. Jenkins, Proc. Am. Thorac. Soc., 2012, 9(3), 130-136.
  3. H. Wan,ADMET & DMPK, 2016, 4(1), 1-22.
  4. J. Hsiao, Y. Chang, Y. Chen, S. Hsieh, K. Hsu, C. Wang, S. Tsai and Y. Jin, Head & Neck, 2010, 32(2), 160-72.

Acknowledgement

The authors would like to thank Dr. Assem Allam for his generous contribution to the University of Hull Positron Emission Research Centre.

Figure 1

The structure of αvβtargeted cyclopeptide with DOTA chelator for 68Ga/177Lu chelation and 18F conjugated prosthetics via triazole unit.

Figure 2
PET/CT images of 68Ga tracer in BxPC3 tumour-bearing NSG mice, without (left) and with prior blocking with αvβantibody for 24 hours (right).
Keywords: Gallium-68, Lutetium-177, Flourine-18, Integrin αvβ6, Peptide
651

Positron Emission Tomography Imaging of Chemokine Receptor CXCR4 using 68Ga and ­18F Radiolabelled Tetraazamacrocyclic Antagonists (#313)

Isaline Renard1, 2, Juozas Domarkas1, 2, Sophie Poty4, Noemi Perujo Holland1, 2, Benjamin P. Burke2, 3, Franck Denat4, Christine Goze4, Christopher Cawthorne1, 2, Stephen J. Archibald1, 2, 3

1 University of Hull, School of Life Sciences, Hull, United Kingdom
2 University of Hull, Positron Emission Tomography Research Centre, Hull, United Kingdom
3 University of Hull, School of Mathematics and Physical Sciences, Hull, United Kingdom
4 Université de Bourgogne, Institut de Chimie Moléculaire, Dijon, France

Introduction

C-X-C chemokine receptor 4 (CXCR4) plays an important role in the cross-talk between cancer cells and their environment. The over-expression of CXCR4 is known in over 23 different tumour types, and is associated with poor prognosis and increased risk of metastasis. The development of CXCR4 imaging agents for the non-invasive imaging of CXCR4 could improve disease staging and treatment planning. In this study, two derivatives of AMD3100 were synthesised to accommodate 68Ga using different linkers and chelators, and subsequently tested in vivo for their CXCR4 affinity and specificity.1,2

Methods

AMD3100 was functionalised on the central phenyl moiety with diamino-polyethylene glycol 3 (PEG3). This derivative was reacted with two chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). The AMD3100-based ligands were labelled with 68Ga.2

PET imaging studies were performed on SCID-beige mice implanted with CXCR4 over-expressing tumours. The subjects were injected with approximately 2 MBq of tracer (i.v.) and then underwent 66 min. dynamic PET scans. CXCR4 specificity was confirmed by administration of a blocking dose of either AMD3100 (5 mg/kg) or Cu2CB-Bicyclam3 (4 mg/kg).

Results/Discussion

The radiotracers [68Ga]Ga-1 and [68Ga]Ga-2 showed significantly different uptake in vivo. PET imaging studies for [68Ga]Ga-1 showed similar liver and tumour uptake, whereas [68Ga]Ga-2 showed greater accumulation in the liver compared to the tumour. Biodistribution studies using both [68Ga]Ga-1 and [68Ga]Ga-2 with blocking doses of Cu2CB-Bicyclam3 and AMD3100 confirmed CXCR4 specificity. Metabolism studies were performed in order to investigate in vivo stability and showed [68Ga]Ga-2 to stay intact while [68Ga]Ga-1 was metabolised.

Conclusions

Two radiotracers [68Ga]Ga-1 and [68Ga]Ga-2 were successfully synthesised and their affinity for CXCR4 was assessed in vivo. [68Ga]Ga-1 and [68Ga]Ga-2 showed specific, blockable uptake in CXCR4 over-expressing tumour models by biodistribution and PET imaging studies. [68Ga]Ga-2 metabolism studies demonstrated stability in vivo. Derivatives are being synthesised to include 18F, and preliminary in vitro and in vivo studies are being undertaken.

References

  1. U. M. Domanska, R. C. Kruizinga, W. B. Nagengast, H. Timmer-Bosscha, G. Huls, E. G. E. de Vries and A. M. E. Walenkamp, Eur. J. Cancer, 2013, 49, 219–230.
  2. S. Poty, E. Gourni, P. Désogère, F. Boschetti, C. Goze, H. R. Maecke and F. Denat, Bioconjug. Chem., 2016, 27, 752–761.
  3. A. Khan, G. Nicholson, J. Greenman, L. Madden, G. McRobbie, C. Pannecouque, E. De Clercq, R. Ullom, D. L. Maples, R. D. Maples, J. D. Silversides, T. J. Hubin and S. J. Archibald, J. Am. Chem. Soc., 2009, 131, 3416–3417.

Acknowledgement

The authors acknowledge the support of the Daisy Appeal with a fellowship to BPB, and would like to thank Dr Assem Allam for studentship funding to NPH and his generous contribution to the University of Hull PET Research Centre.

Figure 1.
Dynamic PET scan of CXCR4 over-expressing tumour bearing animals injected with approximately 2 MBq of [68Ga]Ga-2 (left), [68Ga]Ga-1 (middle) and [68Ga]Pentixafor (right).
Keywords: CXCR4, PET imaging, Gallium-68, Fluorine-18
652

Biodistribution of manganese in healthy mice using 52Mn (#324)

George Firth1, Julia Blower1, Jayanta Bordoloi1, Jesper Fonslet2, Philip Blower1

1 King's College London, School of Biomedical Engineering & Imaging Sciences, London, United Kingdom
2 Technical University of Denmark, The Hevesy Lab, Roskilde, Denmark

Introduction

52Mn (β+, t1/2 = 5.6 days, Iβ+ = 30%) is a positron emitting radionuclide that has the potential to non-invasively study manganese trafficking on a whole-body scale to better understand the role of manganese in biological processes and health in vivo. This could help elucidate potential changes to the transport of manganese in a range of pathological diseases, including diabetes and Alzheimer's disease. In this project we address the whole body distribution of 52Mn, administered intravenously as MnCl2 in healthy BALB/c mice. The data will be used in later studies as a healthy control.

Methods

[52Mn]MnCl2 was administered i.v. at time 0 h, a dynamic PET scan followed from 0-1 h and PET/CT scans were then performed at 4, 24, 48 and 72 h post injection. The mice (n = 3) were then euthanised at 96 h to provide ex vivo biodistribution data.

Results/Discussion

Fast blood clearance was observed initially with an estimated biological half-life of 1.35 min. Activity at 1 h was localised primarily to the abdominal organs with small amounts of radioactivity observed in the heart and joints. Retention of 52Mn varied over time for each organ, with activity associated with the salivary glands increasing over time. Prominent uptake at 96 h was seen in the liver (8.21 ± 0.62 %ID/g), pancreas (22.99 ± 3.44 %ID/g), salivary glands (9.25 ± 0.18 %ID/g) and kidneys (20.87 ± 3.11 %ID/g). Maximum intensity projection (MIP) PET images at 1 and 4h also demonstrated heart and intestine uptake which decreased over time as 52Mn was excreted via the faeces. Modest brain uptake (2.30 ± 0.24 %ID/g) was observed at 96 h.

Conclusions

The research described sets the foundation for the study of 52Mn trafficking using PET, and will be developed further to investigate the importance of manganese in a range of diseases.

References

1. Graves SA et al. Novel Preparation Methods of 52Mn for ImmunoPET Imaging. Bioconjug Chem. 2015;26:2118-2124.

2. Gawne P et al. Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine(8-hydroxyquinoline) as an ionophore. Dalton Trans. 2018;47:9283-9293.

PET/CT images of 52Mn biodistribution at various time points

Figure 1. PET/CT images of 52Mn biodistribution at various time points: representative maximum intensity projection (MIP) and transverse slices of PET-CT images of a healthy BALB/c mouse injected with [52Mn]MnCl2 (1.5MBq, 100 μL) at 1 h, 4 h, 24 h, 48 h and 72 h post injection.

Keywords: Manganese-52, PET Metallomics, Manganese biodistribution
653

Imaging of apoptosis by PET/CT: a new F18-labeled Caspase-3 substrate (#279)

Federica Pisaneschi1, Brian J. Engel1, Seth T. Gammon1, Rajan Chaudhari2, Zhen Lu2, Hailing Yang2, Argentina Ornelas1, Victoria Yan1, Md Nasir Uddin1, Lindsay Kelderhouse1, Amer Najjar3, William Tong1, Shuxing Zhang2, David Piwnica-Worms1, Robert Bast2, Steven W. Millward1

1 UT MDAnderson Cancer Center, Cancer Systems Imaging, Houston, Texas, United States of America
2 UT MDAnderson Cancer Center, Department of Experimental Therapeutics, Houston, Texas, United States of America
3 UT MDAnderson Cancer Center, Department of Pediatrics - Research, Houston, Texas, United States of America

Introduction

Quantitative imaging of apoptosis in vivo could enable real-time monitoring of acute cell death pathologies such as traumatic brain injury (TBI) as well as the efficacy and toxicity of cancer chemotherapy and radiotherapy.  Here, we describe the development of a minimized F-18-labeled caspase-3 substrate, [18F]-TBD, for PET/CT imaging of apoptosis and its validation in a model of hepatotoxicity.

Methods

The minimized caspase 3 substrate,TBD, was designed by structural optimization of M808 irreversible caspase-3 inhibitor. [18F]-TBD was synthesized on the GE TracerLab automated module by click chemistry with [18F]-fluoroethylazide, using a TentaGel-derived azide stripping resin to remove the unreacted alkyne and enhance molar activity.1 [18F]-TBD was tested in vitro in cisplatin-treated ovarian cancer cells and in vivo in a Jo2 antibody-induced hepatotoxicity mouse model.

Results/Discussion

TBD showed a 14-fold improvement in substrate activity and significantly enhanced caspase selectivity relative to first-generation derivatives. Molecular modeling suggested that these improvements resulted from favorable interactions mediated by both the side chain of the P2 O-benzyl-threonine and the linker functionality between the scissile amide bond and the triazole. [18F]-TBD was obtained in 9.8±4.2% (n = 12) decay corrected end of synthesis radiochemical yield, 17.7-149.5 GBq/mmol (n = 12) molar activity and >99% radiochemical purity. The resulting radiotracer accumulated in cisplatin-treated ovarian cancer cells in a caspase- and cisplatin-dependent fashion. Using the Jo2 antibody-induced hepatotoxicity mouse model, [18F]-TBD showed 1.5-fold increased liver uptake in Jo2-treated mice compared with untreated controls. A chemical control tracer, [18F]-TBA, that could not be cleaved by caspase-3, showed no change in liver accumulation after induction of hepatocellular apoptosis.

Conclusions

These data suggest that [18F]-TBD imaging could provide an immediate pharmacodynamic readout of tumor apoptosis in vivo and enable rapid evaluation of treatment efficacy.2

References

1. Pisaneschi, F.; Kelderhouse, L. E.; Hardy, A.; Engel, B. J.; Mukhopadhyay, U.; Gonzalez-Lepera, C.; Gray, J. P.; Ornelas, A.; Takahashi, T. T.; Roberts, R. W.; Fiacco, S. V.; Piwnica-Worms, D.; Millward, S. W., Automated, Resin-Based Method to Enhance the Specific Activity of Fluorine-18 Clicked PET Radiotracers. Bioconjugate Chem. 2017, 28 (2), 583-589.

2. Engel, B. J.; Gammon, S. T.; Chaudhari, R.; Lu, Z.; Pisaneschi, F.; Yang, H.; Ornelas, A.; Yan, V.; Kelderhouse, L.; Najjar, A. M.; Tong, W. P.; Zhang, S.; Piwnica-Worms, D.; Bast, R. C.; Millward, S. W., Caspase-3 Substrates for Noninvasive Pharmacodynamic Imaging of Apoptosis by PET/CT. Bioconjugate Chem. 2018, 29 (9), 3180-3195.

Acknowledgement

This work was supported by UTMDACC startup funds (SWM), a UTMDACC Moonshot Knowledge Gap Pilot Project, 1R21CA181994-01 (SWM, ZL). MD Anderson’s Nuclear Magnetic Resonance Facility and Small Animal Imaging Facility (SAIF) are supported by the MD Anderson Cancer Center Support Grant CA016672 (Pisters).

[18F]-TBD accumulates and is retained in apoptotic livers of Jo2-treated mice.

a) Representative PET 7.5 min post i.v. injection of female athymic nude mice with [18F]-TBD (top) [18F]-TBA (bottom) in Jo2 treated (left) versus untreated (right) mice. b) Structures of [18F]-TBD and [18F]-TBA. c) Three-tissue compartment model used for dynamic PET analysis. d) Kinetic parameter k3 [18F]-TBD (left) or [18F]-TBA (right) treatment.

 

Keywords: apoptosis, caspase 3, PET imaging, click chemistry
654

Evaluation of novel 89Zr chelators and corresponding 89Zr-labeled immunoconjugates (#531)

Pierre Adumeau1, René Raavé2, Christian B. Jacobsen3, Gerwin Sandker2, Sandra Heskamp2, Otto Boerman2, Mark Rijpkema2, Floriane Mangin1, Michel Meyer1, Jean-Claude Chambron1, Mathieu Moreau1, Claire Bernhard1, Adrien Dubois1, Laurène Da Costa1, Victor Goncalves1, Franck Denat1

1 University of Burgundy, Institute of Molecular Chemistry of the University of Burgundy, Dijon, France
2 Radboud University Medical Center, Departement of Radiology and Nuclear Medicine, Nijmegen, Netherlands
3 Novo Nordisk A/S, Global Research Technologies, Måløv, Denmark

Introduction

For immunoPET with 89Zr, the current gold standard to label antibodies is desferrioxamine (DFO).1 However, preclinical studies have shown that the 89Zr-DFO complex is partly unstable in vivo, leading to 89Zr release and subsequent accumulation in mineral bone. This bone uptake may impede the detection of bone metastases, and hampers accurate estimation of the radiation dose to the bone marrow in dose planning for radioimmunotherapy. Therefore, there is a need for more stable 89Zr chelators.

Methods

We have synthesized new octacoordinating 89Zr-bifunctional chelating agents derivated from the DFO* chelator.2 These new chelators were synthesized by coupling different hydroxamic acid-bearing arms to DFO, followed by the introduction of an isothiocyanate moiety. The model antibody trastuzumab was conjugated to the NCS-derivated chelators and DFO-pPhe-NCS as a reference, and radiolabeled with 89Zr. The stability of the radiolabeled chelators and radiolabeled conjugates were evaluated in human plasma, and in PBS in presence of EDTA or DFO. The in vitro behavior of the most promising compounds was investigated more thoroughly using HER2-experessing SK-OV3 cells, and in vivo distribution was studied in mice with subcutaneous SK-OV3 xenografts by PET/CCT imaging and ex vivo tissue analysis.

Results/Discussion

The bifunctional chelators were conjugated efficiently to trastuzumab. Radiolabeling of the conjugates with 89Zr yielded the radioconjugates with high yield, purity and specific activity (RCY >95%, RCP >99%, SA >100 MBq/mg). When challenged with EDTA or DFO, the 89Zr-chelates and the corresponding radioconjugates displayed an improved stability compared to 89Zr-DFO and 89Zr-DFO-trastuzumab, with the best results obtained for the chelator dubbed cycloDFO*. The immunoreactive fraction and IC50 were similar for 89Zr-DFO-trastuzumab and 89Zr-cycloDFO*-trastuzumab. Internalisation after 2h was significantly higher for 89Zr-cycloDFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. Accumulation of 89Zr in bone was significantly lower for 89Zr-DFO-cyclo*-trastuzumab compared to 89Zr-DFO-trastuzumab in knee (3.6 ± 0.4% vs 5.9 ±0.6%), femur (2.2 ± 0.2% vs 3.4 ± 0.3%), and sternum (3.5± 0.4% vs 4.5 ±0.4%) at 72 h after injection. Uptake in the SK-OV3 tumor was similar for both antibody conjugates.

Conclusions

The new 89Zr-chelators and the associated radioconjugates show improved in vitro stability compared to DFO and 89Zr-DFO-trastuzumab. The radioconjugate derivated from the more promising chelator, 89Zr-cycloDFO*-trastuzumab, demonstrated a better in vivo stability compared to 89Zr-DFO-trastuzumab. Therefore, less radiation exposure to bone marrow and improved bone metastasis detection could be achieved using cycloDFO*.

References

1 S. Heskamp, R. Raavé, O. Boerman, M. Rijpkema, V. Goncalves, F. Denat, Bioconjugate Chem., 2017, 28, 2211-2223.

2 D. Vugts, C. Klaver, C. Sewing, A. Poot, K. Adamzek; S. Huegli, C. Mari, G. Visser, I. Valverde, G. Gasser, T. Mindt, G. van Dongen, Eur. J. Nucl. Med. Mol. Imaging, 2017, 44, 286-295

Acknowledgement

This project receives funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 116106. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA.

Keywords: ImmunoPET, 89Zr-chelates, DFO-derivatives, Trastuzumab