Hydroxypyridinones as a Very Promising Platform for Targeted Diagnostic and Therapeutic Radiopharmaceuticals.

Hydroxypyridinones (HOPOs) have been used in the chelation therapy of iron and actinide metals. Their application in metal-based radiopharmaceuticals has also been increasing in recent years. This review article focuses on how multidentate HOPOs can be used in targeted radiometal-based diagnostic and therapeutic radiopharmaceuticals. The general structure of radiometal-based targeted radiopharmaceuticals, a brief description of siderophores, the basic structure and properties of bidentate HOPO, some representative HOPO multidentate chelating agents, radiopharmaceuticals based on HOPO multidentate bifunctional chelators for gallium-68, thorium-227 and zirconium-89, as well as the future prospects of HOPO multidentate bifunctional chelators in other metal-based radiopharmaceuticals are described and discussed in turn. The HOPO metal-based radiopharmaceuticals that have shown good prospects in clinical and preclinical studies are gallium-68, thorium-227 and zirconium-89 radiopharmaceuticals. We expect HOPO multidentate bifunctional chelators to be a very promising platform for building novel targeted radiometal-based diagnostic and therapeutic radiopharmaceuticals.

Synthesis and opioid receptor binding of indium (III) and [111In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors.

Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to µ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [111In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/µmol). Their log D7.4 values were -2.21 to -1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [111In]-labeled DPN-DOTA conjugates for imaging trials.

Alpha-induced production and robust radiochemical separation of 43Sc as an emerging radiometal for formulation of PET radiopharmaceuticals.

Scandium-43 is an emerging PET radiometal that was produced by α-particle bombardment on natural CaCO3 target via natCa (α,p) 43Sc and natCa (α,n) 43Ti→43Sc reactions using K-130 cyclotron at VECC. A robust radiochemical procedure based on selective precipitation of 43Sc as Sc(OH)3 was developed for separation of the radioisotope from the irradiated target. The overall yield of the separation process was >85% and it was obtained in a form suitable for preparation of target specific radiopharmaceuticals for PET imaging of cancer.

Development and Biodistribution of a Nerve Growth Factor Radioactive Conjugate for PET Imaging.

PURPOSE: The purpose of these studies was to develop a nerve growth factor (NGF) radiometal-chelator conjugate to determine the biodistribution and brain uptake of NGF by positron emission tomography/computerized tomography (PET-CT). PROCEDURES: Purified NGF from llama seminal plasma was conjugated with FITC, and the chelator NOTA or DFO. NGF conjugates were evaluated for bioactivity. NOTA- and DFO-conjugated NGF were radiolabeled with gallium-68 or zirconium-89 ([68 Ga]GaCl3, half-life = 68 min; [89Zr]Zr(oxalate)4, half-life = 3.3 days). [89Zr]Zr-NGF was evaluated for biodistribution (0.5, 1, or 24 h), PET imaging (60 min), and brain autoradiography in mice. RESULTS: Cell-based in vitro assays confirmed that the NGF conjugates maintained NGF receptor-binding and biological activity. Zirconium-89 and gallium-68 radiolabeling showed a high efficiency; however, only[89Zr]Zr-NGF was stable in vitro. Biodistribution studies showed that, as with most small proteins < 70 kDa, [89Zr]Zr-NGF uptake was predominantly in the kidney and was cleared rapidly with almost complete elimination of NGF at 24 h. Dynamic PET imaging from 0-60 min showed a similar pattern to ex vivo biodistribution with some transient liver uptake. Interestingly, although absolute brain uptake was very low, at 24 h after treatment, cerebral cortex uptake was higher than any other brain area examined and blood. CONCLUSIONS: We conclude that conjugation of DFO to NGF through a thiourea linkage allows effective radiolabeling with zirconium-89 while maintaining NGF bioactivity. Following intravenous administration, the radiolabeled NGF targets non-neuronal tissues (e.g., kidney, liver), and although absolute brain uptake was very low, the brain uptake that was observed was restricted to the cortex.

Production of Co-58m in a siphon-style liquid target on a medical cyclotron.

We present the production of 58mCo on a small, 13 MeV medical cyclotron utilizing a siphon style liquid target system. Different concentrated iron(III)-nitrate solutions of natural isotopic distribution were irradiated at varying initial pressures and subsequently separated by solid phase extraction chromatography. The radio cobalt (58m/gCo and 56Co) was successfully produced with saturation activities of (0.35 ± 0.03) MBq µA-1 for 58mCo with a separation recovery of (75 ± 2) % of cobalt after one separation step utilizing LN-resin.

Methods for the Determination of Transition Metal Impurities in Cyclotron-Produced Radiometals.

Cyclotron-produced radiometals must be separated from the irradiated target and purified from other metal impurities, which could interfere with the radiolabeling process. We compared different chromatographic and colorimetric methods to determine the amount of transition metals in radioactive samples. Besides commercially available colorimetric tests, 4-(2-pyridylazo)resorcinol and xylenol orange were used as a non-selective metal reagents, forming water-soluble chelates with most of the transition metals immediately. We compared the applicability of pre- and post-column derivatization, as well as colorimetric determination without separation. The studied chromatographic and colorimetric analyses are not suitable to completely replace atomic spectroscopic techniques for the determination of metal contaminants in radioactive samples, but they may play an important role in the development of methods for the purification of radiometals and in their routine quality control.

Improved Sc-44 production in a siphon-style liquid target on a medical cyclotron.

In order to use new and promising radiometals for molecular imaging, it is important that they can be obtained as inexpensively and easily as possible. This often requires a cyclotron with solid target hardware or a radionuclide generator, which are not widely available for rarely used radionuclides. Here, we investigate the improved production of 44Sc with a siphon-style liquid target system and compare to our previous work with a simple liquid target. A metal salt solution with a high concentration of natural abundance Ca(NO3)2 (0.14 g/cm3) was irradiated with a medical cyclotron (12 MeV protons; 20 µA). 44Sc was produced via the natCa(p,x)44Sc reaction. As the pressure increase during irradiation was reduced in the siphon-style target, it was possible to irradiate with a higher proton beam current (20 µA) than with the simple liquid target system (7.9 µA). In addition, the saturation yield per µA of 44Sc was increased by a factor of 3.18 ± 0.05 (6.2 ± 0.1 MBq/µA with the siphon target versus 1.94 ± 0.08 MBq/µA with the simple target). This results in an overall increase in 44Sc activity by a factor of 11.

The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules.

The aluminium-[18F]fluoride ([18F]AlF) radiolabelling method combines the favourable decay characteristics of fluorine-18 with the convenience and familiarity of metal-based radiochemistry and has been used to parallel gallium-68 radiopharmaceutical developments. As such, the [18F]AlF method is popular and widely implemented in the development of radiopharmaceuticals for the clinic. In this review, we capture the current status of [18F]AlF-based technology and reflect upon its impact on nuclear medicine, as well as offering our perspective on what the future holds for this unique radiolabelling method.

Characterization and Stabilization of a New 64Cu-Labeled Anti-EGFR Antibody NCAB001 for the Early Detection of Pancreatic Cancer with Positron Emission Tomography.

Early diagnosis of pancreatic cancer using current imaging modalities remains challenging. We have developed a new approach to identify tumor lesions ≥ 3 mm in the pancreas by positron emission tomography (PET) with a new intraperitoneally administered 64Cu-labeled anti-epidermal growth factor receptor (EGFR) antibody (encoded as NCAB001), called 64Cu-NCAB001 ipPET. Generally, in clinical research, a radiometal-antibody complex must be prepared immediately before use at the imaging site. To make 64Cu-NCAB001 ipPET available to daily clinical practices in a sustainable way, the NCAB001-chelator conjugate and 64Cu-NCAB001 must be characterized and stabilized. NCAB001 was manufactured under cGMP conditions. NCAB001 was conjugated with a bifunctional chelator (p-SCN-Bn-PCTA), and the antibody-chelator conjugate (PCTA-NCAB001) was characterized by LC/MS and ELISA. Thereafter, to effectively manufacture 64Cu-NCAB001, we developed a new formulation to stabilize PCTA-NCAB001 and 64Cu-NCAB001. An average of three PCTA chelators were conjugated per molecule of NCAB001. The relative binding potency of PCTA-NCAB001 was comparable to cetuximab. The formulation consisting of acetate buffer, glycine, and polysorbate-80 stabilized PCTA-NCAB001 for a year-long storage. Additionally, this formulation enabled the stabilization of 64Cu-NCAB001 for up to 24 h after radiolabeling with a sufficient radioactivity concentration for clinical use. These results may accelerate the future use of 64Cu-NCAB001 ipPET in clinical settings for the early diagnosis and treatment of pancreatic cancer.

Accelerator Production of Scandium Radioisotopes: Sc-43, Sc-44, and Sc-47.

Scandium radioisotopes are increasingly considered viable radiolabels for targeted molecular imaging (Sc-43, Sc-44) and therapy (Sc-47). Significant technological advances have increased the quantity and quality of available radioscandium in the past decade, motivated in part by the chemical similarity of scandium to therapeutic radionuclides like Lu-177. The production and radiochemical isolation techniques applied to scandium radioisotopes are reviewed, focusing on charged particle and electron linac initiated reactions and using calcium and titanium as starting materials.

Radiolabeled Nanoparticles for Cancer Diagnosis and Therapy.

Cancer remains as one of the major causes of death worldwide. The emergence of nanotechnology has opened new avenues for the development of nanoparticle (NP)- based diagnostic and therapeutic tools. NPs of different chemical composition, size, shape and surface decoration can be prepared using a wide variety of synthetic strategies. Subsequent radiolabelling with positron or gamma emitters results in potential diagnostic agents which may offer improved selectivity and/or specificity for the target organ or tissue, enabling the acquisition of images with higher signal-to-contrast ratio. Incorporation of alpha or beta emitters leads to therapeutic agents with application in the field of radiotherapy. Here, we first describe the different labeling strategies reported so far for the incorporation of radionuclides into NPs. Recent advances in the use of nanoparticulate constructs both in the diagnostic and therapeutic arenas are then discussed and examples of their application are briefly discussed.

Targeted Therapy Towards Cancer-A Perspective.

Radionuclide antibody conjugates (RACs) and antibody-drug conjugates (ADCs) can function as biotherapeutic missiles in order to target cancer cells and destroy them. The advent of new technology platforms consisting of imaging modalities, drug design and radiochemistry will facilitate the personalised approach for cancer patient treatment programmes. The utilisation of radionuclides and cytotoxic drugs conjugated to biovectors can deliver a cytotoxic drug payload with the ability to emit alpha and/or beta particles in the vicinity of the tumour by binding onto the cancer cells surface antigens initiating cell death. This perspective aims to provide an insight into targeted therapies in the treatment of various cancerous disease states including breast cancer, prostate bone metastases, lymphoma and leukaemia.

Synthetic Peptide Drugs for Targeting Skin Cancer: Malignant Melanoma and Melanotic Lesions.

BACKGROUND: Peptides play decisive roles in the skin, ranging from host defense responses to various forms of neuroendocrine regulation of cell and organelle function. Synthetic peptides conjugated to radionuclides or photosensitizers may serve to identify and treat skin tumors and their metastatic forms in other organs of the body. In the introductory part of this review, the role and interplay of the different peptides in the skin are briefly summarized, including their potential application for the management of frequently occurring skin cancers. Special emphasis is given to different targeting options for the treatment of melanoma and melanotic lesions. Radionuclide Targeting: α-Melanocyte-stimulating hormone (α-MSH) is the most prominent peptide for targeting of melanoma tumors via the G protein-coupled melanocortin-1 receptor that is (over-)expressed by melanoma cells and melanocytes. More than 100 different linear and cyclic analogs of α-MSH containing chelators for 111In, 67/68Ga, 64Cu, 90Y, 212Pb, 99mTc, 188Re were synthesized and examined with experimental animals and in a few clinical studies. Linear Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2 (NAP-amide) and Re-cyclized Cys- Cys-Glu-His-D-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2 (Re[Arg11]CCMSH) containing different chelators at the N- or C-terminus served as lead compounds for peptide drugs with further optimized characteristics. Alternatively, melanoma may be targeted with radiopeptides that bind to melanin granules occurring extracellularly in these tumors. Photosensitizer targeting: A more recent approach is the application of photosensitizers attached to the MSH molecule for targeted photodynamic therapy using LED or coherent laser light that specifically activates the photosensitizer. Experimental studies have demonstrated the feasibility of this approach as a more gentle and convenient alternative compared to radionuclides.

17AAG-induced internalisation of HER2-specific Affibody molecules.

The geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) is known to induce internalisation and degradation of the otherwise internalisation-resistant human epidermal growth factor receptor 2 (HER2) receptor. In the present study, 17-AAG was used to increase internalisation of the HER2-specific Affibody molecule ABY-025. The cellular redistribution of halogen-labelled 211At-ABY-025 and radiometal-labelled 111In-ABY-025 following treatment with 17-AAG was studied. 17-AAG treatment of SKOV-3 human ovarian carcinoma and SKBR-3 human breast carcinoma cells to some extent shifted the localisation of 111In-ABY-025 from the cell surface to intracellular compartments in the two cell lines. ABY-025 labelled with the high-linear energy transfer α emitter 211At was also internalised to a higher degree; however, due to its physiological properties, this nuclide was excreted faster. The results indicate that 17-AAG may be used to facilitate cell-specific intracellular localisation of a suitable cytotoxic or radioactive agent coupled to ABY-025 in HER2-overexpressing cells.

A comparative evaluation of the chelators H4octapa and CHX-A″-DTPA with the therapeutic radiometal (90)Y.

OBJECTIVES: To compare the radiolabeling performance, stability, and practical efficacy of the chelators CHX-A″-DTPA and H4octapa with the therapeutic radiometal (90)Y. METHODS: The bifunctional chelators p-SCN-Bn-H4octapa and p-SCN-Bn-CHX-A″-DTPA were conjugated to the HER2-targeting antibody trastuzumab. The resulting immunoconjugates were radiolabeled with (90)Y to compare radiolabeling efficiency, in vitro and in vivo stability, and in vivo performance in a murine model of ovarian cancer. RESULTS: High radiochemical yields (>95%) were obtained with (90)Y-CHX-A″-DTPA-trastuzumab and (90)Y-octapa-trastuzumab after 15min at room temperature. Both (90)Y-CHX-A″-DTPA-trastuzumab and (90)Y-octapa-trastuzumab exhibited excellent in vitro and in vivo stability. Furthermore, the radioimmunoconjugates displayed high tumoral uptake values (42.3±4.0%ID/g for (90)Y-CHX-A″-DTPA-trastuzumab and 30.1±7.4%ID/g for (90)Y-octapa-trastuzumab at 72h post-injection) in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. Finally, (90)Y radioimmunotherapy studies performed in tumor-bearing mice demonstrated that (90)Y-CHX-A″-DTPA-trastuzumab and (90)Y-octapa-trastuzumab are equally effective therapeutic agents, as treatment with both radioimmunoconjugates yielded substantially decreased tumor growth compared to controls. CONCLUSIONS: Ultimately, this work demonstrates that the acyclic chelators CHX-A″-DTPA and H4octapa have comparable radiolabeling, stability, and in vivo performance, making them both suitable choices for applications requiring (90)Y.

Production of Y-86 and other radiometals for research purposes using a solution target system.

INTRODUCTION: Diagnostic radiometals are typically obtained from cyclotrons by irradiating solid targets or from radioisotope generators. These methods have the advantage of high production yields, but require additional solid target handling infrastructure that is not readily available to many cyclotron facilities. Herein, we provide an overview of our results regarding the production of various positron-emitting radiometals using a liquid target system installed on a 13 MeV cyclotron at TRIUMF. Details about the production, purification and quality control of (89)Zr, (68)Ga and for the first time (86)Y are discussed. METHODS: Aqueous solutions containing 1.35-1.65 g/mL of natural-abundance zinc nitrate, yttrium nitrate, and strontium nitrate were irradiated on a 13 MeV cyclotron using a standard liquid target. Different target body and foil materials were investigated for corrosion. Production yields were calculated using theoretical cross-sections from the EMPIRE code and compared with experimental results. The radioisotopes were extracted from irradiated target material using solid phase extraction methods adapted from previously reported methods, and used for radiolabelling experiments. RESULTS: We demonstrated production quantities that are sufficient for chemical and biological studies for three separate radiometals, (89)Zr (Asat = 360 MBq/µA and yield = 3.17 MBq/µA), (86)Y (Asat = 31 MBq/µA and yield = 1.44 MBq/µA), and (68)Ga (Asat = 141 MBq/µA and yield = 64 MBq/µA) from one hour long irradiations on a typical medical cyclotron. (68)Ga yields were sufficient for potential clinical applications. In order to avoid corrosion of the target body and target foil, nitrate solutions were chosen as well as niobium as target-body material. An automatic loading system enabled up to three production runs per day. The separation efficiency ranged from 82 to 99%. Subsequently, (68)Ga and (86)Y were successfully used to radiolabel DOTA-based chelators while deferoxamine was used to coordinate (89)Zr.

Separation of cyclotron-produced 44Sc from a natural calcium target using a dipentyl pentylphosphonate functionalized extraction resin.

Significant interest in 44Sc as a radioactive synthon to label small molecules for positron emission tomography (PET) imaging has been recently observed. Despite the efforts of several research groups, the ideal 44Sc production and separation method remains elusive. Herein, we propose a novel separation method to obtain 44Sc from the proton irradiation of calcium targets based on extraction chromatography, which promises to greatly simplify current production methodologies. Using the commercially available Uranium and Tetravalent Actinides (UTEVA) extraction resin we were able to rapidly (<20min) recover >80% of the activity generated at end of bombardment (EoB) in small ~1M HCl fractions (400µL). The chemical purity of the 44Sc eluates was evaluated through chelation with DOTA and DTPA, and by trace metal analysis using microwave induced plasma atomic emission spectrometry. The distribution coefficients (Kd) of Sc(III) and Ca(II) in UTEVA were determined in HCl medium in a range of concentrations from zero to 12.1M. The 44Sc obtained with our method proved to be suitable for the direct labeling of small biomolecules for PET imaging, with excellent specific activities and radiochemical purity.

Cross-sections for (p,x) reactions on natural chromium for the production of (52,52m,54)Mn radioisotopes.

The production of positron-emitting isotopes of manganese is potentially important for developing contrast agents for dual-modality positron emission tomography and magnetic resonance (PET/MR) imaging, as well as for in vivo imaging of the biodistribution and toxicity of manganese. The decay properties of (52)Mn make it an excellent candidate for these applications, and it can easily be produced by bombardment of a chromium target with protons or deuterons from a low-energy biomedical cyclotron. Several parameters that are essential to this mode of production­target thickness, beam energy, beam current, and bombardment time­depend heavily on the availability of reliable, reproducible cross-section data. This work contributes to the routine production of (52g)Mn for biomedical research by contributing experimental cross-sections for natural chromium ((nat)Cr) targets for the (nat)Cr(p,x)(52g)Mn reaction, as well as for the production of the radiocontaminants (52m,54)Mn.

(44g)Sc production using a water target on a 13MeV cyclotron.

INTRODUCTION: Access to promising radiometals as isotopes for novel molecular imaging agents requires that they are routinely available and inexpensive to obtain. Proximity to a cyclotron center outfitted with solid target hardware, or to an isotope generator for the metal of interest is necessary, both of which can introduce significant hurdles in development of less common isotopes. Herein, we describe the production of 44Sc (t1/2=3.97 h, Eavg,ß⁺=1.47MeV, branching ratio=94.27%) in a solution target and an automated loading system which allows a quick turn-around between different radiometallic isotopes and therefore greatly improves their availability for tracer development. Experimental yields are compared to theoretical calculations. METHODS: Solutions containing a high concentration (1.44-1.55g/mL) of natural-abundance calcium nitrate tetrahydrate (Ca(NO3)2·4 H2O) were irradiated on a 13MeV proton-beam cyclotron using a standard liquid target. (44g)Sc was produced via the 44Ca(p,n)(44g)Sc reaction. RESULTS: (44g)Sc was produced for the first time in a solution target with yields sufficient for early radiochemical studies. Saturation yields of up to 4.6 ± 0.3 MBq/µA were achieved using 7.6 ± 0.3 µA proton beams for 60.0 ± 0.2 minutes (number of runs n=3). Experimental data and calculation results are in fair agreement. Scandium was isolated from the target mixture via solid-phase extraction with 88 ± 6% (n=5) efficiency and successfully used for radiolabelling experiments. The demonstration of the production of 44Sc in a liquid target greatly improves its availability for tracer development.