Radiation Reveal: Moving from research engagement to involvement.

Here, we report on the process of a highly impactful and successful creative, collaborative, and multi-partner public engagement project, Radiation Reveal. It brought together ten young adults aged 17-25-year-olds with experience of radiotherapy with researchers at Cancer Research UK RadNet City of London across three 2-hour online workshops. Our aims were to 1) initiate discussions between young adults and radiation researchers, and 2) identify what people wish they had known about radiotherapy before or during treatment. These aims were surpassed; other benefits included peer support, participants' continued involvement in subsequent engagement projects, lasting friendships, creation of support groups for others, and creation and national dissemination of top ten tips for medical professionals and social media resources. A key learning was that this project required a dedicated and (com)passionate person with connections to national cancer charities. When designing the project, constant feedback is also needed from charities and young adults with and without radiotherapy experience. Finally, visually capturing discussions and keeping the door open beyond workshops further enhanced impact. Here, we hope to inform and inspire people to help project the patient voice in all we do.

Toward Bifunctional Chelators for Thallium-201 for Use in Nuclear Medicine.

Auger electron therapy exploits the cytotoxicity of low-energy electrons emitted during radioactive decay that travel very short distances (typically <1 µm). 201Tl, with a half-life of 73 h, emits ∼37 Auger and other secondary electrons per decay and can be tracked in vivo as its gamma emissions enable SPECT imaging. Despite the useful nuclear properties of 201Tl, satisfactory bifunctional chelators to incorporate it into bioconjugates for molecular targeting have not been developed. H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa are multidentate N- and O-donor chelators that have previously been shown to have high affinity for 111In, 177Lu, and 89Zr. Herein, we report the synthesis and serum stability of [nat/201Tl]Tl3+ complexes with H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa. All ligands quickly and efficiently formed complexes with [201Tl]Tl3+ that gave simple single-peak radiochromatograms and showed greatly improved serum stability compared to DOTA and DTPA. [natTl]Tl-pypa was further characterized using nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and X-ray crystallography, showing evidence of the proton-dependent presence of a nine-coordinate complex and an eight-coordinate complex with a pendant carboxylic acid group. A prostate-specific membrane antigen (PSMA)-targeting bioconjugate of H4pypa was synthesized and radiolabeled. The uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive and PSMA-negative prostate cancer cells was evaluated in vitro and showed evidence of bioreductive release of 201Tl and cellular uptake characteristic of unchelated [201Tl]TlCl. SPECT/CT imaging was used to probe the in vivo biodistribution and stability of [201Tl]Tl-pypa-PSMA. In healthy animals, [201Tl]Tl-pypa-PSMA did not show the myocardial uptake that is characteristic of unchelated 201Tl. In mice bearing DU145 PSMA-positive and PSMA-negative prostate cancer xenografts, the uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive tumors was higher than that in DU145 PSMA-negative tumors but insufficient for useful tumor targeting. We conclude that H4pypa and related ligands represent an advance compared to conventional radiometal chelators such as DOTA and DTPA for Tl3+ chelation but do not resist dissociation for long periods in the biological environment due to vulnerability to reduction of Tl3+ and subsequent release of Tl+. However, this is the first report describing the incorporation of [201Tl]Tl3+ into a chelator-peptide bioconjugate and represents a significant advance in the field of 201Tl-based radiopharmaceuticals. The design of the next generation of chelators must include features to mitigate this susceptibility to bioreduction, which does not arise for other trivalent heavy radiometals.

[68Ga]Ga-THP-Pam: A Bisphosphonate PET Tracer with Facile Radiolabeling and Broad Calcium Mineral Affinity.

Calcium minerals such as hydroxyapatite (HAp) can be detected noninvasively in vivo using nuclear imaging agents such as [18F]NaF (available from cyclotrons), for positron emission tomography (PET) and 99mTc-radiolabeled bisphosphonates (BP; available from 99mTc 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 [68Ga]Ga-THP-Pam. Radiolabeling with 68Ga 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. [68Ga]Ga-THP-Pam shows high stability in human serum. The calcium mineral binding of [68Ga]Ga-THP-Pam was compared in vitro to two other 68Ga-BPs which have been successfully evaluated in humans, [68Ga]Ga-NO2APBP and [68Ga]Ga-BPAMD, as well as [18F]NaF. Interestingly, we found that all 68Ga-BPs have a high affinity for a broad range of calcium minerals implicated in vascular calcification disease, while [18F]NaF is selective for HAp. Using healthy young mice as a model of metabolically active growing calcium mineral in vivo, we compared the pharmacokinetics and biodistribution of [68Ga]Ga-THP-Pam with [18F]NaF as well as [68Ga]NO2APBP. These studies revealed that [68Ga]Ga-THP-Pam has high in vivo affinity for bone tissue (high bone/muscle and bone/blood ratios) and fast blood clearance (t1/2 < 10 min) comparable to both [68Ga]NO2APBP and [18F]NaF. Overall, [68Ga]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.

Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose.

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4- were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4- in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4- resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4- led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4- after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4- caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

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 THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrations, the bifunctional THPMe-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 THPMe-NCS-huA33, followed after 24 h by 8-10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 µg, 7 MBq) and a 68Ga-only negative control (8-10 MBq of 68Ga3+). Imaging was performed 25 h after antibody administration (1 h after 68Ga3+ 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" 68Ga3+ in the tumour was found (12.9 %ID/g) even without prior administration of THPMe-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the 68Ga-only experiment was repeated using THPMe (20 µg, 1 h after 68Ga3+ administration) to clear circulating 68Ga3+, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THPMe as a 68Ga clearing agent in imaging applications with gallium citrate.

Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of αvß3 Integrin Expression with Gallium-68.

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (68Ga) 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-HP9) that can coordinate up to three Ga3+ ions. This derivative has been conjugated to a trimeric peptide (RGD3) containing three peptide groups that target the αvß3 integrin receptor. The resulting dendritic compound, HP9-RGD3, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP3-RGD and HP3-RGD3 that contain only a single metal binding site. PET scanning and biodistribution studies show that [68Ga(HP9-RGD3)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress αvß3 integrin than [68Ga(HP3-RGD)] and [68Ga(HP3-RGD3)]. However, concomitant nontarget organ retention of [68Ga(HP9-RGD3)] 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, [68Ga(HP3-RGD3)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [68Ga(HP3-RGD3)] enables clear delineation of αvß3 integrin receptor expression in vivo.

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

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 αvß3 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.

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

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.

Towards Effective Targeted Alpha Therapy for Neuroendocrine Tumours: A Review.

This review article explores the evolving landscape of Molecular Radiotherapy (MRT), emphasizing Peptide Receptor Radionuclide Therapy (PRRT) for neuroendocrine tumours (NETs). The primary focus is on the transition from ß-emitting radiopharmaceuticals to α-emitting agents in PRRT, offering a critical analysis of the radiobiological basis, clinical applications, and ongoing developments in Targeted Alpha Therapy (TAT). Through an extensive literature review, the article delves into the mechanisms and effectiveness of PRRT in targeting somatostatin subtype 2 receptors, highlighting both its successes and limitations. The discussion extends to the emerging paradigm of TAT, underlining its higher potency and specificity with α-particle emissions, which promise enhanced therapeutic efficacy and reduced toxicity. The review critically evaluates preclinical and clinical data, emphasizing the need for standardised dosimetry and a deeper understanding of the dose-response relationship in TAT. The review concludes by underscoring the significant potential of TAT in treating SSTR2-overexpressing cancers, especially in patients refractory to ß-PRRT, while also acknowledging the current challenges and the necessity for further research to optimize treatment protocols.

In Vitro and Preclinical Systematic Dose-Effect Studies of Auger Electron- and ß Particle-Emitting Radionuclides and External Beam Radiation for Cancer Treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO4- and [123I]I- and ß--particle emitter [188Re]ReO4-. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter-expressing organs was extrapolated to reference human adult models using OLINDA/EXM. RESULTS: [99mTc]TcO4- and [123I]I- reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4- reduced survival fraction in hNIS-expressing and parental cells. [123I]I- required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO4- and [188Re]ReO4-, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO4- and [123I]I- (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4- to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I- in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- for the first time. We further demonstrate the tumor-controlling effects of [123I]I- and [188Re]ReO4- in comparison with external beam radiation therapy.

Second Symposium of the European Working Group on the Radiobiology of Molecular Radionuclide Therapy.

Molecular radionuclide therapy is a relatively novel anticancer treatment option using radiolabeled, tumor-specific vectors. On binding of these vectors to cancer cells, radioactive decay induces DNA damage and other effects, leading to cancer cell death. Treatments, such as with [177Lu]Lu-octreotate for neuroendocrine tumors and [177Lu]Lu-PSMA for prostate cancer, are now being implemented into routine clinical practice around the world. Nonetheless, research into the underlying radiobiologic effects of these treatments is essential to further improve them or formulate new ones. The purpose of the European Working Group on the Radiobiology of Molecular Radiotherapy is to promote knowledge, investment, and networking in this area. This report summarizes recent research and insights presented at the second International Workshop on Radiobiology of Molecular Radiotherapy, held in London, U.K., on March 13 and 14, 2023. The symposium was organized by members of the Cancer Research U.K. RadNet City of London and the European Working Group on the Radiobiology of Molecular Radiotherapy.

Marshalling the Potential of Auger Electron Radiopharmaceutical Therapy.

Auger electron (AE) radiopharmaceutical therapy (RPT) may have the same therapeutic efficacy as α-particles for oncologic small disease, with lower risks of normal-tissue toxicity. The seeds of using AE emitters for RPT were planted several decades ago. Much knowledge has been gathered about the potency of the biologic effects caused by the intense shower of these low-energy AEs. Given their short range, AEs deposit much of their energy in the immediate vicinity of their site of decay. However, the promise of AE RPT has not yet been realized, with few agents evaluated in clinical trials and none becoming part of routine treatment so far. Instigated by the 2022 "Technical Meeting on Auger Electron Emitters for Radiopharmaceutical Developments" at the International Atomic Energy Agency, this review presents the current status of AE RPT based on the discussions by experts in the field. A scoring system was applied to illustrate hurdles in the development of AE RPT, and we present a selected list of well-studied and emerging AE-emitting radionuclides. Based on the number of AEs and other emissions, physical half-life, radionuclide production, radiochemical approaches, dosimetry, and vector availability, recommendations are put forward to enhance and impact future efforts in AE RPT research.

The chicken chorioallantoic membrane as a low-cost, high-throughput model for cancer imaging.

Mouse models are invaluable tools for radiotracer development and validation. They are, however, expensive, low throughput, and are constrained by animal welfare considerations. Here, we assessed the chicken chorioallantoic membrane (CAM) as an alternative to mice for preclinical cancer imaging studies. NCI-H460 FLuc cells grown in Matrigel on the CAM formed vascularized tumors of reproducible size without compromising embryo viability. By designing a simple method for vessel cannulation it was possible to perform dynamic PET imaging in ovo, producing high tumor-to-background signal for both 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and (4S)-4-(3-18F-fluoropropyl)-L-glutamate (18F-FSPG). The pattern of 18F-FDG tumor uptake were similar in ovo and in vivo, although tumor-associated radioactivity was higher in the CAM-grown tumors over the 60 min imaging time course. Additionally, 18F-FSPG provided an early marker of both treatment response to external beam radiotherapy and target inhibition in ovo. Overall, the CAM provided a low-cost alternative to tumor xenograft mouse models which may broaden access to PET and SPECT imaging and have utility across multiple applications.

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

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 ß- 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.

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

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 [201Tl]Tl(I) to chelatable [201Tl]Tl(III) and identify challenges in stable chelation of thallium to support future synthesis of effective [201Tl]-labelled radiopharmaceuticals. METHODS: A plasmid pBR322 assay was carried out to determine the DNA damaging properties of [201Tl]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 [201Tl]Tl(III) radiolabelling efficacy and complex stability. RESULTS: Isolated plasmid studies demonstrated that [201Tl]Tl(III) can induce single and double-stranded DNA breaks. Iodo-beads, iodogen and trichloroisocyanuric acid enabled more than 95% conversion from [201Tl]Tl(I) to [201Tl]Tl(III) under conditions compatible with future biomolecule radiolabelling (mild pH, room temperature and post-oxidation removal of oxidising agent). Although chelation of [201Tl]Tl(III) was possible with EDTA, DTPA and DOTA, only radiolabeled DOTA showed good stability in serum. CONCLUSIONS: Decay of [201Tl]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 [201Tl]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 [201Tl]Tl(III) complexes is required.

In vitro proof of concept studies of radiotoxicity from Auger electron-emitter thallium-201.

BACKGROUND: Auger electron-emitting radionuclides have potential in targeted treatment of small tumors. Thallium-201 (201Tl), a gamma-emitting radionuclide used in myocardial perfusion scintigraphy, decays by electron capture, releasing around 37 Auger and Coster-Kronig electrons per decay. However, its therapeutic and toxic effects in cancer cells remain largely unexplored. Here, we assess 201Tl in vitro kinetics, radiotoxicity and potential for targeted molecular radionuclide therapy, and aim to test the hypothesis that 201Tl is radiotoxic only when internalized. METHODS: Breast cancer MDA-MB-231 and prostate cancer DU145 cells were incubated with 200-8000 kBq/mL [201Tl]TlCl. Potassium concentration varied between 0 and 25 mM to modulate cellular uptake of 201Tl. Cell uptake and efflux rates of 201Tl were measured by gamma counting. Clonogenic assays were used to assess cell survival after 90 min incubation with 201Tl. Nuclear DNA damage was measured with γH2AX fluorescence imaging. Controls included untreated cells and cells treated with decayed [201Tl]TlCl. RESULTS: 201Tl uptake in both cell lines reached equilibrium within 90 min and washed out exponentially (t1/2 15 min) after the radioactive medium was exchanged for fresh medium. Cellular uptake of 201Tl in DU145 cells ranged between 1.6 (25 mM potassium) and 25.9% (0 mM potassium). Colony formation by both cell lines decreased significantly as 201Tl activity in cells increased, whereas 201Tl excluded from cells by use of high potassium buffer caused no significant toxicity. Non-radioactive TlCl at comparable concentrations caused no toxicity. An estimated average 201Tl intracellular activity of 0.29 Bq/cell (DU145 cells) and 0.18 Bq/cell (MDA-MB-231 cells) during 90 min exposure time caused 90% reduction in clonogenicity. 201Tl at these levels caused on average 3.5-4.6 times more DNA damage per nucleus than control treatments. CONCLUSIONS: 201Tl reduces clonogenic survival and increases nuclear DNA damage only when internalized. These findings justify further development and evaluation of 201Tl therapeutic radiopharmaceuticals.

One-step, kit-based radiopharmaceuticals for molecular SPECT imaging: a versatile diphosphine chelator for 99mTc radiolabelling of peptides.

Radiotracers labelled with technetium-99m (99mTc) enable accessible diagnostic imaging of disease, provided that radiotracer preparation is simple. Whilst 99mTc radiopharmaceuticals for imaging perfusion are routinely prepared from kits, and regularly used in healthcare, there are no 99mTc-labelled receptor-targeted radiopharmaceuticals in widespread clinical use. This is in part due to the multistep radiosyntheses required for the latter. We demonstrate that the diphosphine, 2,3-bis(diphenylphosphino)maleic anhydride (BMA), is an excellent platform for preparation of kit-based, receptor-targeted 99mTc-labelled radiotracers: its conjugates are simple to prepare and can be easily labelled with 99mTc using one-step, kit-based protocols. Here, reaction of BMA with the αvß3-integrin receptor targeted cyclic peptide, Arg-Gly-Asp-DPhe-Lys (RGD), provided the first diphosphine-peptide conjugate, DP-RGD. DP-RGD was incorporated into a "kit", and addition of a saline solution containing 99mTcO4- to this kit, followed by heating, furnished the radiotracer [99mTcO2(DP-RGD)2]+ in consistently high radiochemical yields (>90%). The analogous [ReO2(DP-RGD)2]+ compound was prepared and characterised, revealing that both [99mTcO2(DP-RGD)2]+ and [ReO2(DP-RGD)2]+ consist of a mixture of cis and trans geometric isomers. Finally, [99mTcO2(DP-RGD)2]+ exhibited high metabolic stability, and selectively targeted αvß3-integrin receptors, enabling in vivo SPECT imaging of αvß3-integrin receptor expression in mice.

Validation of the plasmid study to relate DNA damaging effects of radionuclides to those from external beam radiotherapy.

INTRODUCTION: The biological consequences of absorbed radiation doses are ill-defined for radiopharmaceuticals, unlike for external beam radiotherapy (EBRT). A reliable assay that assesses the biological consequences of any radionuclide is much needed. Here, we evaluated the cell-free plasmid DNA assay to determine the relative biological effects of radionuclides such as Auger electron-emitting [67Ga]GaCl3 or [111In]InCl3 compared to EBRT. METHODS: Supercoiled pBR322 plasmid DNA (1.25 or 5 ng/µL) was incubated with 0.5 or 1 MBq [67Ga]GaCl3 or [111In]InCl3 for up to 73 h or was exposed to EBRT (137Cs; 5 Gy/min; 0-40 Gy). The induction of relaxed and linear plasmid DNA, representing single and double strand breaks, respectively, was assessed by gel electrophoresis. Chelated forms of 67Ga were also investigated using DOTA and THP. Topological conversion rates for supercoiled-to-relaxed (ksrx) or relaxed-to-linear (krlx) DNA were obtained by fitting a kinetic model. RESULTS: DNA damage increased both with EBRT dose and incubation time for [67Ga]GaCl3 and [111In]InCl3. Damage caused by [67Ga]GaCl3 decreased when chelated. [67Ga]GaCl3 proved more damaging than [111In]InCl3; 1.25 ng/µL DNA incubated with 0.5 MBq [67Ga]GaCl3 for 2 h led to a 70% decrease of intact plasmid DNA as opposed to only a 19% decrease for [111In]InCl3. For both EBRT and radionuclides, conversion rates were slower for 5 ng/µL than 1.25 ng/µL plasmid DNA. DNA damage caused by 1 Gy EBRT was the equivalent to damage caused by 0.5 MBq unchelated [67Ga]GaCl3 and [111In]InCl3 after 2.05 ± 0.36 and 9.3 ± 0.77 h of incubation, respectively. CONCLUSIONS: This work has highlighted the power of the plasmid DNA assay for a rapid determination of the relative biological effects of radionuclides compared to external beam radiotherapy. It is envisaged this approach will enable the systematic assessment of imaging and therapeutic radionuclides, including Auger electron-emitters, to further inform radiopharmaceutical design and application.