Publications by authors named "Samantha Y A Terry"

27 Publications

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Methods and techniques for in vitro subcellular localization of radiopharmaceuticals and radionuclides.

Nucl Med Biol 2021 Apr 22;98-99:18-29. Epub 2021 Apr 22.

Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, King's College London, London SE1 7EH, United Kingdom. Electronic address:

In oncology, the holy grail of radiotherapy is specific radiation dose deposition in tumours with minimal healthy tissue toxicity. If used appropriately, injectable, systemic radionuclide therapies could meet these criteria, even for treatment of micrometastases and single circulating tumour cells. The clinical use of α and β particle-emitting molecular radionuclide therapies is rising, however clinical translation of Auger electron-emitting radionuclides is hampered by uncertainty around their exact subcellular localisation, which in turn affects the accuracy of dosimetry. This review aims to discuss and compare the advantages and disadvantages of various subcellular localisation methods available to localise radiopharmaceuticals and radionuclides for in vitro investigations.
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http://dx.doi.org/10.1016/j.nucmedbio.2021.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610823PMC
April 2021

Targeted Auger electron-emitter therapy: Radiochemical approaches for thallium-201 radiopharmaceuticals.

Nucl Med Biol 2021 Apr 17;98-99:1-7. Epub 2021 Apr 17.

King's College London, School of Population Health and Environmental Sciences, Analytical, Environmental and Forensic Sciences, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom. Electronic address:

Introduction: Thallium-201 is a radionuclide that has previously been used clinically for myocardial perfusion scintigraphy. Although in this role it has now been largely replaced by technetium-99 m radiopharmaceuticals, thallium-201 remains attractive in the context of molecular radionuclide therapy for cancer micrometastases or single circulating tumour cells. This is due to its Auger electron (AE) emissions, which are amongst the highest in total energy and number per decay for AE-emitters. Currently, chemical platforms to achieve this potential through developing thallium-201-labelled targeted radiopharmaceuticals are not available. Here, we describe convenient methods to oxidise [Tl]Tl(I) to chelatable [Tl]Tl(III) and identify challenges in stable chelation of thallium to support future synthesis of effective [Tl]-labelled radiopharmaceuticals.

Methods: A plasmid pBR322 assay was carried out to determine the DNA damaging properties of [Tl]Tl(III). A range of oxidising agents (ozone, oxygen, hydrogen peroxide, chloramine-T, iodogen, iodobeads, trichloroisocyanuric acid) and conditions (acidity, temperature) were assessed using thin layer chromatography. Chelators EDTA, DTPA and DOTA were investigated for their [Tl]Tl(III) radiolabelling efficacy and complex stability.

Results: Isolated plasmid studies demonstrated that [Tl]Tl(III) can induce single and double-stranded DNA breaks. Iodo-beads, iodogen and trichloroisocyanuric acid enabled more than 95% conversion from [Tl]Tl(I) to [Tl]Tl(III) under conditions compatible with future biomolecule radiolabelling (mild pH, room temperature and post-oxidation removal of oxidising agent). Although chelation of [Tl]Tl(III) was possible with EDTA, DTPA and DOTA, only radiolabeled DOTA showed good stability in serum.

Conclusions: Decay of [Tl]Tl(III) in proximity to DNA causes DNA damage. Iodobeads provide a simple, mild method to convert thallium-201 from a 1+ to 3+ oxidation state and [Tl]Tl(III) can be chelated by DOTA with moderate stability. Of the well-established chelators evaluated, DOTA is most promising for future molecular radionuclide therapy using thallium-201; nevertheless, a new generation of chelating agents offering resistance to reduction and dissociation of [Tl]Tl(III) complexes is required.
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http://dx.doi.org/10.1016/j.nucmedbio.2021.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610824PMC
April 2021

[Ga]Ga-THP-Pam: A Bisphosphonate PET Tracer with Facile Radiolabeling and Broad Calcium Mineral Affinity.

Bioconjug Chem 2020 Aug 27. Epub 2020 Aug 27.

School of Biomedical Engineering & Imaging Sciences, King's College London, St Thomas' Hospital, London SE1 7EH, U.K.

Calcium minerals such as hydroxyapatite (HAp) can be detected noninvasively using nuclear imaging agents such as [F]NaF (available from cyclotrons), for positron emission tomography (PET) and Tc-radiolabeled bisphosphonates (BP; available from Tc generators for single photon emission computed tomography (SPECT) or scintigraphy). These two types of imaging agents allow detection of bone metastases (based on the presence of HAp) and vascular calcification lesions (that contain HAp and other calcium minerals). With the aim of developing a cyclotron-independent PET radiotracer for these lesions, with broad calcium mineral affinity and simple one-step radiolabeling, we developed [Ga]Ga-THP-Pam. Radiolabeling with Ga is achieved using a mild single-step kit (5 min, room temperature, pH 7) to high radiochemical yield and purity (>95%). NMR studies demonstrate that Ga binds via the THP chelator, leaving the BP free to bind to its biological target. [Ga]Ga-THP-Pam shows high stability in human serum. The calcium mineral binding of [Ga]Ga-THP-Pam was compared to two other Ga-BPs which have been successfully evaluated in humans, [Ga]Ga-NO2AP and [Ga]Ga-BPAMD, as well as [F]NaF. Interestingly, we found that all Ga-BPs have a high affinity for a broad range of calcium minerals implicated in vascular calcification disease, while [F]NaF is selective for HAp. Using healthy young mice as a model of metabolically active growing calcium mineral , we compared the pharmacokinetics and biodistribution of [Ga]Ga-THP-Pam with [F]NaF as well as [Ga]NO2AP. These studies revealed that [Ga]Ga-THP-Pam has high affinity for bone tissue (high bone/muscle and bone/blood ratios) and fast blood clearance ( < 10 min) comparable to both [Ga]NO2AP and [F]NaF. Overall, [Ga]Ga-THP-Pam shows high potential for clinical translation as a cyclotron-independent calcium mineral PET radiotracer, with simple and efficient radiochemistry that can be easily implemented in any radiopharmacy.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00401DOI Listing
August 2020

Three-dimensional super-resolution fluorescence imaging of DNA.

Sci Rep 2020 07 27;10(1):12504. Epub 2020 Jul 27.

Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London, UK.

Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.
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http://dx.doi.org/10.1038/s41598-020-68892-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385144PMC
July 2020

PET Imaging of Liposomal Glucocorticoids using Zr-oxine: Theranostic Applications in Inflammatory Arthritis.

Theranostics 2020 26;10(9):3867-3879. Epub 2020 Feb 26.

School of Imaging Sciences & Biomedical Engineering, King's College London, St. Thomas' Hospital, London, SE1 7EH, UK.

The encapsulation of Glucocorticoids (GCs) into long-circulating liposomes (LCLs) is a proven strategy to reduce the side effects of glucocorticoids and improve the treatment of inflammatory diseases, such as rheumatoid arthritis (RA). With the aim of supporting the development of GC-loaded LCLs, and potentially predict patient response to therapy clinically, we evaluated a direct PET imaging radiolabelling approach for preformed GC-LCLs in an animal model of human inflammatory arthritis. A preformed PEGylated liposomal methylprednisolone hemisuccinate (NSSL-MPS) nanomedicine was radiolabelled using [Zr]Zr(oxinate) (Zr-oxine), characterised and tracked using PET imaging in a K/BxN serum-transfer arthritis (STA) mouse model of inflammatory arthritis and non-inflamed controls. Histology and joint size measurements were used to confirm inflammation. The biodistribution of Zr-NSSL-MPS was compared to that of free Zr in the same model. A therapeutic study using NSSL-MPS using the same time points as the PET/CT imaging was carried out. The radiolabelling efficiency of NSSL-MPS with [Zr]Zr(oxinate) was 69 ± 8 %. PET/CT imaging of Zr-NSSL-MPS showed high uptake (3.6 ± 1.5 % ID; 17.4 ± 9.3 % ID/mL) at inflamed joints, with low activity present in non-inflamed joints (0.5 ± 0.1 % ID; 2.7 ± 1.1 % ID/mL). Importantly, a clear correlation between joint swelling and high Zr-NSSL-MPS uptake was observed, which was not observed with free Zr. STA mice receiving a therapeutic dose of NSSL-MPS showed a reduction in inflammation at the time points used for the PET/CT imaging compared with the control group. PET imaging was used for the first time to track a liposomal glucocorticoid, showing high uptake at visible and occult inflamed sites and a good correlation with the degree of inflammation. A subsequent therapeutic response matching imaging time points in the same model demonstrated the potential of this radiolabeling method as a theranostic tool for the prediction of therapeutic response - with NSSL-MPS and similar nanomedicines - in the treatment of inflammatory diseases.
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http://dx.doi.org/10.7150/thno.40403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086351PMC
April 2021

Manipulating the In Vivo Behaviour of Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

Int J Mol Sci 2020 Feb 22;21(4). Epub 2020 Feb 22.

School of Biomedical Engineering and Imaging Sciences, King's College London, Fourth Floor Lambeth Wing, St Thomas' Hospital, London SE1 7EH, UK.

Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THP as a high-affinity pair capable of combining in vivo. After confirming the ability of THP to bind Ga in vivo at low concentrations, the bifunctional THP-NCS was conjugated to a humanised huA33 antibody targeting the A33 glycoprotein. Imaging experiments performed in nude mice bearing A33-positive SW1222 colorectal cancer xenografts compared pretargeting (100 μg of THP-NCS-huA33, followed after 24 h by 8-10 MBq of Ga) with both a directly labelled radioimmunoconjugate (Zr-DFO-NCS-huA33, 88 μg, 7 MBq) and a Ga-only negative control (8-10 MBq of Ga). Imaging was performed 25 h after antibody administration (1 h after Ga administration for negative control). No difference between pretargeting and the negative control was observed, suggesting that pretargeting via metal chelation is not feasible using this model. However, significant accumulation of "unchelated" Ga in the tumour was found (12.9 %ID/g) even without prior administration of THP-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the Ga-only experiment was repeated using THP (20 μg, 1 h after Ga administration) to clear circulating Ga, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THP as a Ga clearing agent in imaging applications with gallium citrate.
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http://dx.doi.org/10.3390/ijms21041496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073083PMC
February 2020

In vitro cytotoxicity of Auger electron-emitting [Ga]Ga-trastuzumab.

Nucl Med Biol 2020 Jan - Feb;80-81:57-64. Epub 2019 Dec 13.

Department of Imaging Chemistry and Biology, School of Biomedical Engineering and Imaging Sciences, St. Thomas' Hospital, London, SE1 7EH, United Kingdom. Electronic address:

Introduction: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of Ga as a therapeutic radionuclide.

Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters Ga and In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2-negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography.

Results: Labelling efficiencies for [Ga]Ga-THP-trastuzumab and [In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78-92 GBq/μmol). At 4 nM total antibody concentration and 200 × 10 cells/mL, [Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population.

Conclusions: [Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.
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http://dx.doi.org/10.1016/j.nucmedbio.2019.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099941PMC
January 2021

Imaging DNA Damage Repair In Vivo After Lu-DOTATATE Therapy.

J Nucl Med 2020 05 22;61(5):743-750. Epub 2019 Nov 22.

CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, United Kingdom

Molecular radiotherapy using Lu-DOTATATE is a most effective treatment for somatostatin receptor-expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after Lu-DOTATATE therapy using SPECT imaging with In-anti-γH2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals. Six cell lines were exposed to external-beam radiotherapy (EBRT) or Lu-DOTATATE, after which the number of γH2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor-positive tumor xenografts were treated with Lu-DOTATATE or sham-treated and coinjected with In-anti-γH2AX-TAT, In-IgG-TAT control, or vehicle. Clonogenic survival after external-beam radiotherapy was cell-line-specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with Lu-DOTATATE, clonogenic survival decreased and γH2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between Lu-DOTATATE uptake and γH2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to Lu-DOTATATE irradiation. In-anti-γH2AX-TAT allows monitoring of DNA damage after Lu-DOTATATE therapy and reveals heterogeneous damage responses.
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http://dx.doi.org/10.2967/jnumed.119.232934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198382PMC
May 2020

A Clinical Feasibility Study to Image Angiogenesis in Patients with Arteriovenous Malformations Using Ga-RGD PET/CT.

J Nucl Med 2020 02 13;61(2):270-275. Epub 2019 Sep 13.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Arteriovenous malformations (AVMs) have an inherent capacity to form new blood vessels, resulting in excessive lesion growth, and this process is further triggered by the release of angiogenic factors. Ga-labeled arginine-glycine-aspartate tripeptide sequence (RGD) PET/CT imaging may provide insight into the angiogenic status and treatment response of AVMs. This clinical feasibility study was performed to demonstrate that Ga-RGD PET/CT imaging can be used to quantitatively assess angiogenesis in peripheral AVMs. Ten patients with a peripheral AVM (mean age, 40 y; 4 men and 6 women) and scheduled for endovascular embolization treatment were prospectively included. All patients underwent Ga-RGD PET/CT imaging 60 min after injection (mean dose, 207 ± 5 MBq). Uptake in the AVM, blood pool, and muscle was quantified as SUV and SUV, and a descriptive analysis of the PET/CT images was performed. Furthermore, immunohistochemical analysis was performed on surgical biopsy sections of peripheral AVMs to investigate the expression pattern of integrin αβGa-RGD PET/CT imaging showed enhanced uptake in all AVM lesions (mean SUV, 3.0 ± 1.1; mean SUV, 2.2 ± 0.9). Lesion-to-blood and lesion-to-muscle ratios were 3.5 ± 2.2 and 4.6 ± 2.8, respectively. Uptake in blood and muscle was significantly higher in AVMs than in background tissue ( = 0.0006 and = 0.0014, respectively). Initial observations included uptake in multifocal AVM lesions and enhanced uptake in intraosseous components in those AVM cases affecting bone integrity. Immunohistochemical analysis revealed cytoplasmatic and membranous integrin αβ expression in the endothelial cells of AVMs. This feasibility study showed increased uptake in AVMs with angiogenic activity, compared with surrounding tissue without angiogenic activity, suggesting that Ga-RGD PET/CT imaging can be used as a tool to quantitatively determine angiogenesis in AVMs. Further studies will be conducted to explore the potential of Ga-RGD PET/CT imaging for guiding current treatment decisions and for assessing response to antiangiogenic treatment.
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http://dx.doi.org/10.2967/jnumed.119.231167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100027PMC
February 2020

Call to arms: need for radiobiology in molecular radionuclide therapy.

Eur J Nucl Med Mol Imaging 2019 Jul 8;46(8):1588-1590. Epub 2019 May 8.

IRCM, Institut de Recherche en Cancérologie de Montpellier, F-34298, Montpellier, France.

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http://dx.doi.org/10.1007/s00259-019-04334-3DOI Listing
July 2019

In Vivo Characterization of 4 Ga-Labeled Multimeric RGD Peptides to Image αβ Integrin Expression in 2 Human Tumor Xenograft Mouse Models.

J Nucl Med 2018 08 6;59(8):1296-1301. Epub 2018 Apr 6.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center Nijmegen, Nijmegen, The Netherlands.

αβ integrins play an important role in angiogenesis and cell migration in cancer and are highly expressed on the activated endothelial cells of newly formed blood vessels. Here, we compare the targeting characteristics of 4 Ga-labeled multimeric cyclic arginine-glycine-aspartate (RGD)-based tracers in an αβ integrin-expressing tumor model and a tumor model in which αβ integrin is expressed solely on the neovasculature. Female BALB/c nude mice were subcutaneously injected with SK-RC-52 (αβ integrin-positive) or FaDu (αβ integrin-negative) tumor cells. Ga-labeled DOTA-(RGD), TRAP-(RGD), FSC-(RGD), or THP-(RGD) was intravenously administered to the mice (0.5 nmol per mouse, 10-20 MBq), followed by small-animal PET/CT imaging and ex vivo biodistribution studies 1 h after injection. Nonspecific uptake of the tracers in both models was determined by coinjecting an excess of unlabeled DOTA-(RGD) (50 nmol) along with the radiolabeled tracers. Imaging and biodistribution data showed specific uptake in the tumors for each tracer in both models. Tumor uptake of Ga-FSC-(RGD) was significantly higher than that of Ga-DOTA-(RGD), Ga-TRAP-(RGD), or Ga-THP-(RGD) in the SK-RC-52 model but not in the FaDu model, in which Ga-FSC-(RGD) showed significantly higher tumor uptake than Ga-TRAP-(RGD) Most importantly, differences were also observed in normal tissues and in tumor-to-blood ratios. All tracers showed sufficient targeting of αβ integrin expression to allow for tumor detection. Although the highest tumor uptake was found for Ga-FSC-(RGD) and Ga-THP-(RGD) in the SK-RC-52 and FaDu models, respectively, selection of the optimal tracer for specific diagnostic applications also depends on tumor-to-blood ratio and uptake in normal tissues; these factors should therefore also be considered.
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http://dx.doi.org/10.2967/jnumed.117.206979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175042PMC
August 2018

Ga-THP-PSMA: A PET Imaging Agent for Prostate Cancer Offering Rapid, Room-Temperature, 1-Step Kit-Based Radiolabeling.

J Nucl Med 2017 08 13;58(8):1270-1277. Epub 2017 Apr 13.

Division of Imaging Sciences and Biomedical Engineering, King's College London, London, United Kingdom; and

The clinical impact and accessibility of Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of Tc radiopharmaceuticals. Currently, chelating agents used in Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of Ga generator eluate to a cold kit. A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing Ga-THP-PSMA with Ga-HBED-CC-PSMA. Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40-60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of Ga radiopharmaceuticals.
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http://dx.doi.org/10.2967/jnumed.117.191882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175039PMC
August 2017

Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of αβ Integrin Expression with Gallium-68.

Bioconjug Chem 2017 02 14;28(2):481-495. Epub 2016 Dec 14.

King's College London , Division of Imaging Sciences and Biomedical Engineering, Fourth Floor Lambeth Wing, St Thomas' Hospital, London SE1 7EH, United Kingdom.

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP) that can coordinate up to three Ga ions. This derivative has been conjugated to a trimeric peptide (RGD) containing three peptide groups that target the αβ integrin receptor. The resulting dendritic compound, HP-RGD, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP-RGD and HP-RGD that contain only a single metal binding site. PET scanning and biodistribution studies show that [Ga(HP-RGD)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress αβ integrin than [Ga(HP-RGD)] and [Ga(HP-RGD)]. However, concomitant nontarget organ retention of [Ga(HP-RGD)] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [Ga(HP-RGD)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [Ga(HP-RGD)] enables clear delineation of αβ integrin receptor expression in vivo.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314429PMC
February 2017

Re-assessing gallium-67 as a therapeutic radionuclide.

Nucl Med Biol 2017 Mar 29;46:12-18. Epub 2016 Oct 29.

King's College London, Department of Imaging Chemistry and Biology, St. Thomas' Hospital, London, SE1 7EH, UK. Electronic address:

Introduction: Despite its desirable half-life and low energy Auger electrons that travel further than for other radionuclides, Ga has been neglected as a therapeutic radionuclide. Here, Ga is compared with Auger electron emitter In as a potential therapeutic radionuclide.

Methods: Plasmid pBR322 studies allowed direct comparison between Ga and In (1MBq) in causing DNA damage, including the effect of chelators (EDTA and DTPA) and the effects of a free radical scavenger (DMSO). The cytotoxicity of internalized (by means of delivery in the form of oxine complexes) and non-internalized Ga and In was measured in DU145 prostate cancer cells after a one-hour incubation using cell viability (trypan blue) and clonogenic studies. MDA-MB-231 and HCC1954 cells were also used.

Results: Plasmid DNA damage was caused by Ga and was comparable to that caused by In; it was reduced in the presence of EDTA, DTPA and DMSO. The A values (internalized activity of oxine complexes per cell required to kill 50% of cells) as determined by trypan blue staining was 1.0Bq/cell for both Ga and In; the A values determined by clonogenic assay were 0.7Bq/cell and 0.3Bq/cell for In and Ga respectively. At the concentrations required to achieve these uptake levels, non-internalized Ga and In caused no cellular toxicity. Qualitatively similar results were found for MDA-MB-231 and HCC1954 cells.

Conclusion: Ga causes as much damage as In to plasmid DNA in solution and shows similar toxicity as In at equivalent internalized activity per cell. Ga therefore deserves further evaluation for radionuclide therapy.

Advances In Knowledge And Implications For Patient Care: The data presented here is at the basic level of science. If future in vivo and clinical studies are successful, Ga could become a useful radionuclide with little healthy tissue toxicity in the arsenal of weapons for treating cancer.
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http://dx.doi.org/10.1016/j.nucmedbio.2016.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303015PMC
March 2017

In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow.

J Nucl Med 2017 02 4;58(2):214-220. Epub 2016 Aug 4.

Laboratory of Pediatric Oncology, Department of Pediatrics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites.

Methods: In vivo localization of In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics.

Results: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo.

Conclusion: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow-resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow-resident macrophages may provide a protective niche to leukemic cells.
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http://dx.doi.org/10.2967/jnumed.116.177741DOI Listing
February 2017

Monitoring Therapy Response of Experimental Arthritis with Radiolabeled Tracers Targeting Fibroblasts, Macrophages, or Integrin αvβ3.

J Nucl Med 2016 Mar 3;57(3):467-72. Epub 2015 Dec 3.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Unlabelled: Rheumatoid arthritis is an autoimmune disease resulting in chronic synovial inflammation. Molecular imaging could be used to monitor therapy response, thus enabling tailored therapy regimens and enhancing therapeutic outcome. Here, we hypothesized that response to etanercept could be monitored by radionuclide imaging in arthritic mice. We tested 3 different targets, namely fibroblast activation protein (FAP), macrophages, and integrin αvβ3.

Methods: Male DBA/1J mice with collagen-induced arthritis were treated with etanercept. SPECT/CT scans were acquired at 1, 24, and 48 h after injection of (111)In-RGD2 (integrin αvβ3), (111)In-anti-F4/80-A3-1 (antimurine macrophage antibody), or (111)In-28H1 (anti-FAP antibody), respectively, with nonspecific controls included. Mice were dissected after the last scan, and scans were analyzed quantitatively and were correlated with macroscopic scoring.

Results: Experimental arthritis was imaged with (111)In-28H1 (anti-FAP), (111)In-anti-F4/80-A3-1, and (111)In-RGD2. Tracer uptake in joints correlated with arthritis score. Treatment decreased joint uptake of tracers from 23 ± 15, 8 ± 4, and 2 ± 1 percentage injected dose per gram (%ID/g) to 11 ± 11 (P < 0.001), 4 ± 4 (P < 0.001), and 1 ± 0.2 %ID/g (P < 0.01) for (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2, respectively. Arthritis-to-blood ratios (in mice with arthritis score 2 per joint) were higher for (111)In-28H1 (5.5 ± 1; excluding values > 25), (111)In-anti-F4/80-A3-1 (10.4 ± 4), and (111)In-RGD2 (7.2 ± 1) than for control (111)In-DP47GS (0.7 ± 0.5; P = 0.002), (111)In-rat IgG2b (0.5 ± 0.2; P = 0.002), or coinjection of excess RGD2 (3.5), indicating specific uptake of all tracers in arthritic joints.

Conclusion: (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2 can be used to specifically monitor the response to therapy in experimental arthritis at the molecular level. Further studies, however, still need to be performed.
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http://dx.doi.org/10.2967/jnumed.115.162628DOI Listing
March 2016

Non-invasive molecular imaging of inflammatory macrophages in allograft rejection.

EJNMMI Res 2015 Dec 26;5(1):69. Epub 2015 Nov 26.

Department of Imaging Chemistry and Biology, Division of Imaging Sciences and Biomedical Engineering, King's College London, St. Thomas' Hospital, London, SE1 7EH, UK.

Background: Macrophages represent a critical cell type in host defense, development and homeostasis. The ability to image non-invasively pro-inflammatory macrophage infiltrate into a transplanted organ may provide an additional tool for the monitoring of the immune response of the recipient against the donor graft. We therefore decided to image in vivo sialoadhesin (Sn, Siglec 1 or CD169) using anti-Sn mAb (SER-4) directly radiolabelled with (99m)Tc pertechnetate.

Methods: We used a heterotopic heart transplantation model where allogeneic or syngeneic heart grafts were transplanted into the abdomen of recipients. In vivo nanosingle-photon emission computed tomography (SPECT/CT) imaging was performed 7 days post transplantation followed by biodistribution and histology.

Results: In wild-type mice, the majority of (99m)Tc-SER-4 monoclonal antibody cleared from the blood with a half-life of 167 min and was located predominantly on Sn(+) tissues in the spleen, liver and bone marrow. The biodistribution in the transplantation experiments confirmed data derived from the non-invasive SPECT/CT images, with significantly higher levels of (99m)Tc-SER-4 observed in allogeneic grafts (9.4 (±2.7) %ID/g) compared to syngeneic grafts (4.3 (±10.3) %ID/g) (p = 0.0022) or in mice which received allogeneic grafts injected with (99m)Tc-IgG isotype control (5.9 (±0.6) %ID/g) (p = 0.0185). The transplanted heart to blood ratio was also significantly higher in recipients with allogeneic grafts receiving (99m)Tc-SER-4 as compared to recipients with syngeneic grafts (p = 0.000004) or recipients with allogeneic grafts receiving (99m)Tc-IgG isotype (p = 0.000002).

Conclusions: Here, we demonstrate that imaging of Sn(+) macrophages in inflammation may provide an important additional and non-invasive tool for the monitoring of the pathophysiology of cellular immunity in a transplant model.
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http://dx.doi.org/10.1186/s13550-015-0146-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661159PMC
December 2015

New Tris(hydroxypyridinone) Bifunctional Chelators Containing Isothiocyanate Groups Provide a Versatile Platform for Rapid One-Step Labeling and PET Imaging with (68)Ga(3.).

Bioconjug Chem 2016 Feb 2;27(2):309-18. Epub 2015 Sep 2.

King's College London , Division of Imaging Sciences and Biomedical Engineering, Fourth Floor Lambeth Wing, St Thomas' Hospital, London SE1 7EH, United Kingdom.

Two new bifunctional tris(hydroxypyridinone) (THP) chelators designed specifically for rapid labeling with (68)Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, RGD. Each conjugate can be radiolabeled and formulated by treatment with generator-produced (68)Ga(3+) in over 95% radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60-80 MBq nmol(-1). Competitive binding assays and in vivo biodistribution in mice bearing U87MG tumors demonstrate that the new (68)Ga(3+)-labeled THP peptide conjugates retain affinity for the αvβ3 integrin receptor, clear within 1-2 h from circulation, and undergo receptor-mediated tumor uptake in vivo. We conclude that bifunctional THP chelators can be used for simple, efficient labeling of (68)Ga biomolecules under mild conditions suitable for peptides and proteins.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4759618PMC
February 2016

¹¹¹In-anti-F4/80-A3-1 antibody: a novel tracer to image macrophages.

Eur J Nucl Med Mol Imaging 2015 Aug 27;42(9):1430-8. Epub 2015 May 27.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands,

Purpose: Here, the expression of F4/80 on the cell surface of murine macrophages was exploited to develop a novel imaging tracer that could visualize macrophages in vivo.

Methods: The immunoreactive fraction and IC50 of anti-F4/80-A3-1, conjugated with diethylenetriaminepentaacetic acid (DTPA) and radiolabelled with (111)In, were determined in vitro using murine bone marrow-derived macrophages. In vivo biodistribution studies were performed with (111)In-anti-F4/80-A3-1 and isotype-matched control antibody (111)In-rat IgG2b at 24 and 72 h post-injection (p.i.) in SCID/Beige mice bearing orthotopic MDA-MB-231 xenografts. In some studies mice were also treated with liposomal clodronate. Macrophage content in tissues was determined immunohistochemically. Micro-single photon emission computed tomography (SPECT)/CT images were also acquired.

Results: In vitro binding assays showed that (111)In-anti-F4/80-A3-1 specifically binds F4/80 receptor-positive macrophages. The immunoreactivity of anti-F4/80-A3-1 was 75 % and IC50 was 0.58 nM. In vivo, injection of 10 or 100 μg (111)In-anti-F4/80-A3-1 resulted in splenic uptake of 78 %ID/g and 31 %ID/g, respectively, and tumour uptake of 1.38 %ID/g and 4.08 %ID/g, respectively (72 h p.i.). Liposomal clodronate treatment reduced splenic uptake of 10 μg (111)In-anti-F4/80-A3-1 from 248 %ID/g to 114 %ID/g and reduced (111)In-anti-F4/80-A3-1 uptake in the liver and femur (24 h p.i.). Tracer retention in the blood and tumour uptake increased (24 h p.i.). Tumour uptake of (111)In-anti-F4/80-A3-1 was visualized by microSPECT/CT. Macrophage density in the spleen and liver decreased in mice treated with liposomal clodronate. Uptake of (111)In-rat IgG2b was lower in the spleen, liver and femur when compared to (111)In-anti-F4/80-A3-1.

Conclusion: Radiolabelled anti-F4/80-A3-1 antibodies specifically localize in tissues infiltrated by macrophages in mice and can be used to visualize tumours. The liver and spleen act as antigen sink organs for macrophage-specific tracers.
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http://dx.doi.org/10.1007/s00259-015-3084-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502320PMC
August 2015

Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti-Fibroblast Activation Protein Antibody Correlates with Severity of Arthritis.

J Nucl Med 2015 May 9;56(5):778-83. Epub 2015 Apr 9.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Unlabelled: One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA.

Methods: SPECT and PET with (111)In- and (89)Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score.

Results: Both (111)In-28H1 and (89)Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of (111)In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis.

Conclusion: SPECT/CT imaging with the anti-FAP antibody (111)In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. (89)Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of (89)Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.
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http://dx.doi.org/10.2967/jnumed.114.152959DOI Listing
May 2015

Can 111In-RGD2 monitor response to therapy in head and neck tumor xenografts?

J Nucl Med 2014 Nov 27;55(11):1849-55. Epub 2014 Oct 27.

Department of Radiology and Nuclear Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.

Unlabelled: RGD (arginylglycylaspartic acid)-based imaging tracers allow specific imaging of integrin αvβ3 expression, proteins overexpressed during angiogenesis; however, few studies have investigated the potential of these tracers to monitor responses of antiangiogenic or radiation therapy. In the studies presented here, (111)In-RGD2 was assessed for its potential as an imaging tool to monitor such responses to therapies.

Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with (111)In ((111)In-RGD2), and biodistribution studies were performed in mice with subcutaneous FaDu or SK-RC-52 xenografts after treatment with either antiangiogenic therapy (bevacizumab or sorafenib) or tumor irradiation (10 Gy). Micro-SPECT imaging studies and subsequent quantitative analysis were also performed. The effect of bevacizumab, sorafenib, or radiation therapy on tumor growth was determined.

Results: The uptake of (111)In-RGD2 in tumors, as determined from biodistribution studies, correlated well with that quantified from micro-SPECT images, and both showed that 15 d after irradiation (111)In-RGD2 uptake was enhanced. Specific or nonspecific uptake of (111)In-RGD2 in FaDu or SK-RC-52 xenografts was not affected after antiangiogenic therapy, except in head and neck squamous cell carcinoma 19 d after the start of sorafenib therapy (P < 0.05). The uptake of (111)In-RGD2 followed tumor volume in studies featuring antiangiogenic therapy. However, the uptake of (111)In-RGD2 in FaDu xenografts was decreased as early as 4 h after tumor irradiation, despite nonspecific uptake remaining unaltered. Tumor growth was inhibited after antiangiogenic or radiation therapy.

Conclusion: Here, it is suggested that (111)In-RGD2 could allow in vivo monitoring of angiogenic responses after radiotherapy and may therefore prove a good clinical tool to monitor angiogenic responses early after the start of radiotherapy in patients with head and neck squamous cell carcinoma. Despite clear antitumor efficacy, antiangiogenic therapy did not alter tumor uptake of (111)In-RGD2, indicating that integrin expression was not altered.
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http://dx.doi.org/10.2967/jnumed.114.144394DOI Listing
November 2014

Imaging integrin αvβ3 on blood vessels with 111In-RGD2 in head and neck tumor xenografts.

J Nucl Med 2014 Feb 9;55(2):281-6. Epub 2014 Jan 9.

Department of Nuclear Medicine, Radboud UMC, Nijmegen, The Netherlands.

Unlabelled: Arginine-glycine-aspartic acid (RGD)-based imaging tracers allow specific imaging of integrin αvβ3, a protein overexpressed during angiogenesis, leading to the possibility that it might serve as a tool to stratify patients for antiangiogenic treatment. However, these tracers have generally been characterized in xenograft models in which integrin αvβ3 was constitutively expressed by the tumor cells themselves. In the studies presented here, the use of (111)In-RGD2 as a tracer to image only integrin αvβ3 expression on blood vessels in the tumor was determined using tumor xenografts in which tumor cells were integrin αvβ3-negative.

Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with (111)In ((111)In-RGD2), and biodistribution studies were performed in squamous cell carcinoma of the head and neck (HNSCC) xenograft mouse models to determine the optimal peptide dose to image angiogenesis. Next, biodistribution and imaging studies were performed at the optimal peptide dose in 3 HNSCC mouse models, FaDu, SCCNij3, and SCCNij202. Immunohistochemical analysis of tumor vascular and cell surface expression of integrin αvβ3 and correlation analysis of vascular integrin αvβ3 and autoradiography were completed.

Results: All 3 HNSCC xenografts expressed integrin αvβ3 on the vessels only. The optimal peptide dose of (111)In-RGD2 was 1 μg or less for specific integrin αvβ3-mediated uptake of the tracer. SPECT/CT imaging showed clear uptake of the tracer in the periphery of the tumors, corresponding with well-vascularized areas of the tumor. Within the tumor, (111)In-RGD2 autoradiography coincided with vascular integrin αvβ3 expression, as determined immunohistochemically. Integrin αvβ3-mediated uptake was also detected in nontumor tissues, which, through immunohistochemical analysis, proved positive for integrin αvβ3.

Conclusion: (111)In-RGD2 allows the visualization of integrin αvβ3 in xenograft models in which integrin αvβ3 is expressed only on the neovasculature, such as in the HNSCC tumors. Thus, (111)In-RGD2 allows specific visualization of angiogenesis in tumor models lacking constitutive tumoral integrin αvβ3 expression but may be less useful for this purpose in many tumors in which tumor cells express integrin αvβ3.
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http://dx.doi.org/10.2967/jnumed.113.129668DOI Listing
February 2014

Radiolabeled imaging probes targeting angiogenesis for personalized medicine.

Curr Pharm Des 2014 ;20(14):2293-307

Department of Nuclear Medicine, Radboud University Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

Angiogenesis is essential for tumor growth and inhibiting angiogenesis has become an important therapeutic strategy in clinical oncology. Nonetheless, the mechanisms behind anti-angiogenic therapeutics as well as resistance to these drugs remain unclear. With a lack of validated genetic or molecular biomarkers for anti-angiogenic responsiveness, novel methods to identify responsive patients are required. Non-invasive nuclear imaging would allow the elucidation of the basic drug mechanisms as well as resistance routes and aid the personalization of anti-angiogenic therapy by enabling target expression quantification prior to and during treatment. This review focuses on the development of radiolabeled probes to image four key proteins expressed during angiogenesis, namely vascular endothelial growth factor and its receptor, integrin αv β3, the extracellular domain of fibronectin and matrix metalloproteases, and how these probes can be utilized for personalized anti-angiogenic therapy.
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http://dx.doi.org/10.2174/13816128113196660665DOI Listing
January 2015

Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide.

Contrast Media Mol Imaging 2013 May-Jun;8(3):238-45

Department of Biochemistry, Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands.

Integrin αv β3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F in a simple one-pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g(-1)) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p <0.001) and (111) In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g(-1) ; p < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with (18)F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g(-1)). The αv β3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with (18)F-labeled NODAGA-E-[c(RGDfK)]2 . In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with (18)F using a direct aqueous, one-pot method and it accumulated specifically in αv β3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.
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http://dx.doi.org/10.1002/cmmi.1523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034708PMC
November 2013

Relationship between chromatin structure and sensitivity to molecularly targeted auger electron radiation therapy.

Int J Radiat Oncol Biol Phys 2012 Jul 14;83(4):1298-305. Epub 2012 Feb 14.

CR-UK/MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology, University of Oxford, Oxford, UK.

Purpose: The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated.

Methods And Materials: Chromatin structure was altered in MDA-MB-468 and 231-H2N human breast cancer cells by suberoylanilide hydroxamic acid (SAHA), 5-aza-2-deoxycytidine, or hypertonic treatment. The extent and duration of chromatin structural changes were evaluated using the micrococcal nuclease assay. DNA damage (γH2AX assay) and clonogenic survival were evaluated after exposure to (111)In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of (111)In-DTPA-hEGF after chromatin modification was investigated in cell fractionation experiments.

Results: Chromatin remained condensed for up to 20 minutes after NaCl and in a relaxed state 24 hours after SAHA treatment. The number of γH2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 μM) compared with IR alone (16 ± 0.6 and 14 ± 0.3 vs. 12 ± 0.4 and 11 ± 0.2, respectively). More γH2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to (111)In-DTPA-hEGF (6 MBq/μg) plus SAHA vs. (111)In-DTPA-hEGF alone (11 ± 0.3 and 12 ± 0.7 vs. 9 ± 0.4 and 7 ± 0.3, respectively). 5-aza-2-deoxycytidine enhanced the DNA damage caused by IR and (111)In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 μM) vs. IR alone (0.6% ± 0.01 and 0.3% ± 0.2 vs. 5.8% ± 0.2 and 2% ± 0.1, respectively) and after (111)In-DTPA-hEGF plus SAHA compared to (111)In-DTPA-hEGF alone (21% ± 0.4% and 19% ± 4.6 vs. 33% ± 2.3 and 32% ± 3.7). SAHA did not affect (111)In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer γH2AX foci per cell after IR and (111)In-DTPA-hEGF compared to controls but did not significantly alter clonogenic survival.

Conclusions: Chromatin structure affects DNA damage and cell survival after exposure to Auger electron radiation.
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http://dx.doi.org/10.1016/j.ijrobp.2011.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175060PMC
July 2012

Mechanisms of the formation of radiation-induced chromosomal aberrations.

Mutat Res 2010 Aug 27;701(1):23-6. Epub 2010 Mar 27.

Bute Medical School, Bute Medical Buildings, University of St Andrews, St Andrews KY16 9TS, UK.

Although much is now known about the mechanisms of radiation-induction of DNA double-strand breaks (DSB), there is less known about the conversion of DSB into chromosomal aberrations. In particular the induction and 'rejoining' of chromatid breaks has been a controversial topic for many years. However, its importance becomes clear in the light of the wide variation in the chromatid break response of human peripheral blood lymphocytes from different individuals when exposed to ionizing radiation, and the elevation of the frequency of radiation-induced chromatid breaks in stimulated peripheral blood lymphocytes of around 40% of breast cancer cases. A common assumption has been that chromatid breaks are merely expansions of initiating DSB, although the classic 'breakage-first' hypothesis (Sax, Ref. 44) was already challenged in the 50's by Revell [30] who maintained that chromatid breaks were formed as a result of an incomplete exchange process initiated by two interacting lesions of an unspecified nature. Here we argue that both these models of chromatid break formation are flawed and we suggest an alternative hypothesis, namely that a radiation-induced DSB initiates an indirect mechanism leading to a chromatid break. This mechanism we suggest involves the nuclear enzyme topoisomerase IIalpha and we present evidence from topoisomerase IIalpha expression variant human cell lines and from siRNA treatment of human cells that supports this hypothesis.
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http://dx.doi.org/10.1016/j.mrgentox.2010.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175058PMC
August 2010

Suppression of topoisomerase IIalpha expression and function in human cells decreases chromosomal radiosensitivity.

Mutat Res 2009 Apr 4;663(1-2):40-5. Epub 2009 Feb 4.

Bute Medical School, University of St Andrews, St Andrews, Scotland, UK.

The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIalpha. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIalpha expression. Here we report that suppression of topoisomerase IIalpha in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIalpha in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.
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http://dx.doi.org/10.1016/j.mrfmmm.2009.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175043PMC
April 2009