Caterina F. Ramogida

Tumour targeting with radiometals for diagnosis and therapy

Use of radiometals in nuclear oncology is a rapidly growing field and encompasses a broad spectrum of radiotracers for imaging via PET (positron emission tomography) or SPECT (single-photon emission computed tomography) and therapy via α, β(-), or Auger electron emission. This feature article opens with a brief introduction to the imaging and therapy modalities exploited in nuclear medicine, followed by a discussion of the multi-component strategy used in radiopharmaceutical development, known as the bifunctional chelate (BFC) method. The modular assembly is dissected into its individual components and each is discussed separately. The concepts and knowledge unique to metal-based designs are outlined, giving insight into how these radiopharmaceuticals are evaluated for use in vivo. Imaging nuclides (64)Cu, (68)Ga, (86)Y, (89)Zr, and (111)In, and therapeutic nuclides (90)Y, (177)Lu, (225)Ac, (213)Bi, (188)Re, and (212)Pb will be the focus herein. Finally, key examples have been extracted from the literature to give the reader a sense of breadth of the field.

Non-Innocent Ligand Behavior of a Bimetallic Ni Schiff-Base Complex Containing a Bridging Catecholate

The geometric and electronic structure of a bimetallic Ni Schiff-base complex and its one-electron oxidized form have been investigated in the solid state and in solution. The two salen units in the neutral complex 1 are linked via a bridging catecholate function. The one-electron oxidized form [1](+) was determined to exist as a ligand radical species in solution, with the electron hole potentially localized on the redox-active dioxolene, the phenolate ligands, or delocalized over the entire ligand system. Electrochemical experiments and UV-vis-NIR spectroscopy, in combination with density functional theory (DFT) calculations, provide insight into the locus of oxidation and the degree of delocalization in this system. The one-electron hole for [1](+) was determined experimentally to be localized on the dioxolene bridge with a small amount of spin density on the outer phenolate moieties predicted by the calculations. The resonance Raman spectrum of [1](+) (λ(ex) = 413 nm) in CH(2)Cl(2) solution clearly exhibited a new band at 1315 cm(-1) in comparison to 1, which is predicted to be a combination of dioxolene ring and C-O bond stretching modes, consistent with oxidation of the bridging moiety in [1](+). Analysis of the NIR bands for [1](+), in association with time-dependent DFT calculations, suggests that the low energy bands are ligand to ligand charge transfer transitions from the terminal phenolates to the central dioxolene unit. In combination, this data is consistent with a description of the overall electronic structure of [1](+) as a bridge-localized semiquinone ligand radical species. This is in contrast to the mixed-valence ground state description for many one-electron oxidized Ni salen monomer systems, and analysis in terms of intervalence charge transfer (IVCT) theory.

H4octapa-Trastuzumab: Versatile Acyclic Chelate System for 111In and 177Lu Imaging and Therapy

A bifunctional derivative of the versatile acyclic chelator H4octapa, p-SCN-Bn-H4octapa, has been synthesized for the first time. The chelator was conjugated to the HER2/neu-targeting antibody trastuzumab and labeled in high radiochemical purity and specific activity with the radioisotopes (111)In and (177)Lu. The in vivo behavior of the resulting radioimmunoconjugates was investigated in mice bearing ovarian cancer xenografts and compared to analogous radioimmunoconjugates employing the ubiquitous chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The H4octapa-trastuzumab conjugates displayed faster radiolabeling kinetics with more reproducible yields under milder conditions (15 min, RT, ~94-95%) than those based on DOTA-trastuzumab (60 min, 37 °C, ~50-88%). Further, antibody integrity was better preserved in the (111)In- and (177)Lu-octapa-trastuzumab constructs, with immunoreactive fractions of 0.99 for each compared to 0.93-0.95 for (111)In- and (177)Lu-DOTA-trastuzumab. These results translated to improved in vivo biodistribution profiles and SPECT imaging results for (111)In- and (177)Lu-octapa-trastuzumab compared to (111)In- and (177)Lu-DOTA-trastuzumab, with increased tumor uptake and higher tumor-to-tissue activity ratios.

Class III Delocalization and Exciton Coupling in a Bimetallic Bis‐ligand Radical Complex

The geometric and electronic structure of an oxidized bimetallic Ni complex incorporating two redox-active Schiff-base ligands connected via a 1,2-phenylene linker has been investigated and compared to a monomeric analogue. Information from UV/Vis/NIR spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, electrochemistry, and density functional theory (DFT) calculations provides important information on the locus of oxidation for the bimetallic complex. The neutral bimetallic complex is conformationally dynamic at room temperature, which complicates characterization of the oxidized forms. Comparison to an oxidized monomer analogue 1 provides critical insight into the electronic structure of the oxidized bimetallic complex 2. Oxidation of 1 provides [1˙](+), which is characterized as a fully delocalized ligand radical complex; the spectroscopic signature of this derivative includes an intense NIR band at 4500 cm(-1). Oxidation of 2 to the bis-oxidized form affords a bis-ligand radical species [2˙˙](2+). Variable temperature EPR spectroscopy of [2˙˙](2+) shows no evidence of coupling, and the triplet and broken symmetry solutions afforded by theoretical calculations are essentially isoenergetic. [2˙˙](2+) is thus best described as incorporating two non-interacting ligand radicals. Interestingly, the intense NIR intervalence charge transfer band observed for the delocalized ligand-radical [1˙](+) exhibits exciton splitting in [2˙˙](2+), due to coupling of the monomer transition dipoles in the enforced oblique dimer geometry. Evaluating the splitting of the intense intervalence charge transfer band can thus provide significant geometric and electronic information in less rigid bis-ligand radical systems. Addition of excess pyridine to [2˙˙](2+) results in a shift in the oxidation locus from a bis-ligand radical species to the Ni(III) /Ni(III) derivative [2(py)4](2+), demonstrating that the ligand system can incorporate significant bulk in the axial positions.

Non-innocent ligand behaviour of a bimetallic Cu complex employing a bridging catecholate

The geometric and electronic structure of a bimetallic Cu Schiff-base complex and its one-electron oxidized form have been investigated. The two salen units in the neutral complex 1 are linked via a bridging catecholate function, and the coupling between the two Cu(II) d(9) centres was determined to be weakly antiferromagnetic on the basis of solid-state magnetic studies (J = -3 cm(-1)), and variable-temperature electron paramagnetic resonance (EPR) (J = -3 cm(-1)). Theoretical calculations (DFT) were in agreement with the experimental results (J = -7 cm(-1)), and provided insight into the coupling mechanism for the neutral system. One-electron oxidation provided [1](+) which was observed to have limited stability in solution. The oxidized complex was determined to be a ligand radical species in solution, with the electron hole potentially localized on the redox-active dioxolene, the phenolate ligands, or delocalized over the entire ligand system. Electrochemical experiments and UV-vis-NIR spectroscopy, in combination with density functional theory (DFT) calculations, provided insight into the locus of oxidation and the degree of delocalization in this system. The ligand radical for [1˙](+) was determined experimentally to be localized on the dioxolene bridge with a small amount of spin density on the outer phenolate moieties predicted by the calculations. This assignment was aided via comparison to data for the Ni analogue (Inorg. Chem., 2011, 50, 6746). The resonance Raman spectrum of [1˙](+) (λ(ex) = 413 nm) in CH(2)Cl(2) solution clearly exhibited a new band at 1308 cm(-1) in comparison to 1, supporting semiquinone formation. Variable-temperature EPR on the three-spin system [1˙](+) did not provide definitive information on the coupling interaction, possibly due to a very small difference in energy between the S = 3/2 and S = 1/2 states and/or a very small zero-field splitting, in combination with significant line-broadening. The data is consistent with a description of the overall electronic structure of [1˙](+) as a bimetallic Cu(II) complex with a bridge-localized semiquinone ligand radical species.

Multi-isotope SPECT imaging of the 225Ac decay chain: feasibility studies

Effective use of the [Formula: see text] decay chain in targeted internal radioimmunotherapy requires the retention of both [Formula: see text] and progeny isotopes at the target site. Imaging-based pharmacokinetic tests of these pharmaceuticals must therefore separately yet simultaneously image multiple isotopes that may not be colocalized despite being part of the same decay chain. This work presents feasibility studies demonstrating the ability of a microSPECT/CT scanner equipped with a high energy collimator to simultaneously image two components of the [Formula: see text] decay chain: [Formula: see text] (218 keV) and [Formula: see text] (440 keV). Image quality phantoms were used to assess the performance of two collimators for simultaneous [Formula: see text] and [Formula: see text] imaging in terms of contrast and noise. A hotrod resolution phantom containing clusters of thin rods with diameters ranging between 0.85 and 1.70 mm was used to assess resolution. To demonstrate ability to simultaneously image dynamic [Formula: see text] and [Formula: see text] activity distributions, a phantom containing a [Formula: see text] generator from [Formula: see text] was imaged. These tests were performed with two collimators, a high-energy ultra-high resolution (HEUHR) collimator and an ultra-high sensitivity (UHS) collimator. Values consistent with activity concentrations determined independently via gamma spectroscopy were observed in high activity regions of the images. In hotrod phantom images, the HEUHR collimator resolved all rods for both [Formula: see text] and [Formula: see text] images. With the UHS collimator, no rods were resolvable in [Formula: see text] images and only rods  ⩾1.3 mm were resolved in [Formula: see text] images. After eluting the [Formula: see text] generator, images accurately visualized the reestablishment of transient equilibrium of the [Formula: see text] decay chain. The feasibility of evaluating the pharmacokinetics of the [Formula: see text] decay chain in vivo has been demonstrated. This presented method requires the use of a high-performance high-energy collimator.

Synthesis and characterization of lipophilic cationic Ga(III) complexes based on the H2CHXdedpa and H2dedpa ligands and their 67/68Ga radiolabeling studies

68 Ga is an attractive isotope for incorporation into a positron-emission tomography (PET) imaging agent, and is finding use as an alternative generator-produced isotope to 99mTc particularly in imaging of myocardial perfusion. We have synthesized six new chelating ligands based on our previously reported H2dedpa and H2CHXdedpa scaffolds (CHX = cyclohexyl, H2dedpa = 1,2-[[carboxypyridin-2-yl]methylamino]ethane). These ligands are designed to incorporate several lipophilic appendages at the secondary nitrogens, and upon coordination to 68Ga(III) will form lipophilic, cationic complexes designed to mimic the properties of other clinically relevant myocardial perfusion imaging agents. The non-radioactive Ga(III) complexes were prepared and characterized by NMR spectroscopy; each ligand retained its predicted hexadentate N4O2 binding to Ga(III). The radiolabeling properties of the six ligands were assessed using the longer-lived 68Ga surrogate, 67Ga. The absence of ‘free’ uncomplexed 67Ga in the HPLC radio-chromatograms indicated >99% radiochemical yields (10 minutes at ambient temperature, ligand concentrations of 10−4 M). However, the N,N′-benzyl functionalized derivatives displayed multiple peaks corresponding to the presence of additional 67Ga-complexes which complicated further study. Selected 67Ga-CHXdedpa complexes were tested for in vitro stability against the metal-binding protein apo-transferrin, and were found to be sufficiently stable (>80%) in a 2 h challenge assay, suggesting that alternative N,N′-alkylated derivatives which introduce more lipophilic character will be of interest in future studies.

89Zr for antibody labeling and in vivo studies – a comparison between liquid and solid target production

INTRODUCTION Zirconium-89 (89Zr, t1/2=78.4h) liquid target (LT) production offers an approach to introduce this positron-emitting isotope to cyclotron centres without the need for a separate solid target (ST) production set up. We compared the production, purification, and antibody radiolabeling yields of 89Zr-(LT) and 89Zr-(ST), and assessed the feasibility of 89Zr-(LT) for preclinical PET/CT. METHODS 89Zr-(ST) production was performed with an 89Y foil on a TR 19 cyclotron at 13.8MeV. For LT production; an aqueous solution of yttrium nitrate (Y(NO3)3·6H2O) was irradiated on a TR 13 cyclotron at 12MeV. 89Zr was purified from the ST or LT material with hydroxamate resin, and used to radiolabel p-SCN-Bn-Deferoxamine (DFO)-conjugated Trastuzumab. MicroPET-CT imaging was performed at 1, 3 and 5days post-injection of 89Zr-DFO-Trastuzumab from ST or LT with biodistribution analysis on day 5. RESULTS Irradiation of the ST yielded 2.88±1.07GBq/μA with a beam current of 14.0±3.8μA and irradiation time of 137±48min at end of bombardment while LT yielded 0.27±0.05GBq/μA with a beam current of 9.9±2.2μA and irradiation time of 221±29min. Radiolabeling of DFO-Trastuzumab with 89Zr-(ST) or 89Zr-(LT) was successful with purity>97% and specific activity>0.12MBq/μg (of antibody). MicroPET-CT imaging and biodistribution profiles showed similar uptake of 89Zr-(ST)-DFO-Trastuzumab and 89Zr-(LT)-DFO-Trastuzumab in tumor and all organs of interest. CONCLUSION 89Zr-(LT) was effectively used to prepare antibody bioconjugates with specific activities suitable for small animal imaging. PET imaging and biodistribution revealed similar behaviours between bioconjugates labeled with 89Zr produced from the two target systems. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE These results have important implications for the production of PET isotopes such as 89Zr to cyclotron facilities with only LT capabilities - such as most clinical centres - expanding the availability of 89Zr-immunoPET.

H4octox: Versatile Bimodal Octadentate Acyclic Chelating Ligand for Medicinal Inorganic Chemistry

H4octox, a versatile new octadentate acyclic chelating ligand, has been investigated as an alternative to the acyclic DTPA and the macrocyclic DOTA for trivalent metal ions useful in diagnostic medical imaging or therapeutic applications (Y3+, In3+, La3+, Gd3+, Lu3+). The synthesis of H4octox is straightforward in less steps and thus more economical than those of most previously reported chelators. Complex formation equilibria in the presence of Y3+, In3+, La3+, Gd3+, and Lu3+ revealed fast chelation and high metal-sequestering capacity. Quantitative labeling with 111In3+ was achieved within 15 min at room temperature at ligand concentrations as low as 10-7 M, exactly the properties required for the development of kit-based radiopharmaceuticals. In vitro serum stability studies and in vivo SPECT imaging confirmed excellent complex stability of [111In(octox)]-. Moreover, it is more lipophilic than most of the multidentate carboxylate- or picolinate-based chelators; it therefore shows more liver clearance and provides a complementary choice in the design of metal-based pharmaceuticals and in the tuning of their pharmacokinetic properties. Finally, H4octox showed a large fluorescence enhancement upon complexation with different metals, in particular, with Y3+ and Lu3+, which could be useful for non-radioactive fluorescent stability and cell studies as well as bimodal imaging. Excellent in vitro stability of [Y(octox)]- against transferrin and Fe3+ was confirmed employing this fluorescence.

Development of 225Ac Radiopharmaceuticals: TRIUMF Perspectives and Experiences

Background: The development of radiopharmaceuticals containing 225Ac for targeted alpha therapy is an active area of academic and commercial research worldwide. Objectives: Despite promising results from recent clinical trials, 225Ac-radiopharmaceutical development still faces significant challenges that must be overcome to realize the widespread clinical use of 225Ac. Some of these challenges include the limited availability of the isotope, the challenging chemistry required to isolate 225Ac from any co-produced isotopes, and the need for stable targeting systems with high radio-labeling yields. Results: Here we provide a review of available literature pertaining to these challenges in the 225Ac-radiopharmaceutical field and also provide insight into how performed and planned efforts at TRIUMF - Canada’s particle accelerator centre - aim to address these issues