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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.

Photoactivatable agents offer the prospect of highly selective cancer therapy with low side effects and novel mechanisms of action that can combat current drug resistance. 1,8‐Naphthalimides with their extended π system can behave as light‐harvesting groups, fluorescent probes and DNA intercalators. We conjugated N‐(carboxymethyl)‐1,8‐naphthalimide (gly‐R‐Nap) with an R substituent on the naphthyl group to photoactive diazido PtIV complexes to form t,t,t‐[Pt(py)2(N3)2(OH)(gly‐R‐Nap)], R=H (1), 3‐NO2 (2) or 4‐NMe2 (3). They show enhanced photo‐oxidation, cellular accumulation and promising photo‐cytotoxicity in human A2780 ovarian, A549 lung and PC3 prostate cancer cells with visible light activation, and low dark cytotoxicity. Complexes 1 and 2 exhibit pre‐intercalation into DNA, resulting in enhanced photo‐induced DNA crosslinking. Complex 3 has a red‐shifted absorption band at 450 nm, allowing photoactivation and photo‐cytotoxicity with green light., Green to go: Photoactive all‐trans diazido PtIV complexes with an axial 1,8‐naphthalimide ligand that can pre‐intercalate into DNA have been synthesised. They can be photoactivated with visible light to generate PtII DNA interstrand crosslinks and cytotoxic ROS, and are thus promising photo‐chemotherapeutic agents. Notably, a 4‐NMe2 substituent red‐shifted absorption of the naphthalimide ring, giving rise to its green‐light photo‐cytotoxicity.

The novel Pt(iv) complex trans,trans-[Pt(N3)2(Py)2(OH)(OCO-(PEG)2-NHCSNH-Ph-NCS)] (Pt4) conjugates to the side chain of lysine amino acids in proteins under mild conditions. Reaction with myoglobin generated a bioconjugate that was stable in the dark, but released a Pt(iv) prodrug upon visible light irradiation. A similar procedure was used to conjugate Pt4 to the antibody trastuzumab, resulting in the first photoactivatable Pt(iv)-antibody conjugate, demonstrating potential for highly selective cancer phototherapy., This platinum anticancer complex can be conjugated to proteins, including antibodies, under mild conditions and activated by visible light, providing a basis for highly selective cancer phototherapy.

Computed tomography (CT) is one of the most widespread imaging techniques in clinical use worldwide. CT contrast agents are administered to improve soft tissue contrast and highlight blood vessels. However, the range of CT contrast agents available in the clinic is limited and they suffer from short-circulation times and low k-edge values that result in the need for high doses for in vivo applications. Nanomaterials containing a mixture of electron-dense elements, such as BaYbF5 nanoparticles, have shown promise as more efficient CT contrast agents, but they require biocompatible coatings for biomedical applications. Here, we explore the use of a bifunctional PEG polymer (5 kDa) containing a terminal bisphosphonate (BP) anchor for efficient binding to the surface of BaYbF5 nanomaterials. The resulting PEG(5)-BP–BaYbF5 nanoparticles were synthesized and characterized using TEM, DLS, TGA, XRD and Z-potential measurements. Their in vitro stability was verified and their ability to produce CT contrast in a wide range of X-ray energies, covering preclinical and clinical scanners, was demonstrated. In vitro toxicity studies with PEG(5)-BP–BaYbF5 in the phagocytic pro-monocytic human cell line U937 did not identify toxic effects, even at high concentrations (30 mM). In vivo, PEG(5)-BP–BaYbF5 exhibited efficient CT contrast for angiography imaging, highlighting blood vessels and vascular organs, and long circulation times as expected from the PEG coating. However, at late time points (48 h), in vivo toxicity was observed. While the causes could not be completely elucidated, in vitro studies suggest that decomposition and release of Yb3+ and/or Ba2+ metal ions after decomposition of PEG(5)-BP–BaYbF5 may play a role. Overall, despite the promising CT contrast properties, our results suggest that BaYbF5 nanomaterials may suffer from significant long-term toxicities., PEG(5)-BP–BaYbF5 nanoparticles provide superior CT contrast and circulation time compared to clinically-used iodinated molecules, but suffer from unexpected in vivo toxicity.

Metals play vital roles in nutrients and medicines and provide chemical functionalities that are not accessible to purely organic compounds. At least 10 metals are essential for human life and about 46 other non-essential metals (including radionuclides) are also used in drug therapies and diagnostic agents. These include platinum drugs (in 50% of cancer chemotherapies), lithium (bipolar disorders), silver (antimicrobials), and bismuth (broad-spectrum antibiotics). While the quest for novel and better drugs is now as urgent as ever, drug discovery and development pipelines established for organic drugs and based on target identification and high-throughput screening of compound libraries are less effective when applied to metallodrugs. Metallodrugs are often prodrugs which undergo activation by ligand substitution or redox reactions, and are multi-targeting, all of which need to be considered when establishing structure–activity relationships. We focus on early-stage in vitro drug discovery, highlighting the challenges of evaluating anticancer, antimicrobial and antiviral metallo-pharmacophores in cultured cells, and identifying their targets. We highlight advances in the application of metal-specific techniques that can assist the preclinical development, including synchrotron X-ray spectro(micro)scopy, luminescence, and mass spectrometry-based methods, combined with proteomic and genomic (metallomic) approaches. A deeper understanding of the behavior of metals and metallodrugs in biological systems is not only key to the design of novel agents with unique mechanisms of action, but also to new understanding of clinically-established drugs., The vital roles of metals in nutrients and medicines are not accessible to purely organic compounds.

We have studied activation of the methyl C–H bonds in the cyclopentadienyl ligands of half-sandwich Rh(III) complexes [η5-CpXRh(N,N′)Cl]+ by observing the dependence of sequential H/D exchange on variations in CpX = Cp* (complexes 1 and 2), Me4PhCp (CpXPh, 3) or Me4PhPhCp (CpXPhPh, 4), and chelated ligand N,N′ (bpy, 1; phen, 2–4). H/D exchange was fastest in d4-MeOD (t1/2 = 10 min, 37 °C, complex 1), no H/D exchange was observed in DMSO/D2O, and d4-MeOD enhanced the rate in CD3CN. The proposed Rh(I)–fulvene intermediate was trapped by [4 + 2] Diels–Alder reactions with conjugated dienes and characterized. The Rh(I) oxidation state was confirmed by X-ray photoelectron spectroscopy (XPS). Influence of solvent on the mechanisms of activation and Diels–Alder adduct formation was modelled using DFT calculations with the CAM-B3LYP functional and CEP-31 g basis set, and influence on the reaction profile of the dimiine ligand and phenyl substituent using the larger qzvp basis set. The Rh(III)–OH intemediate is stabilised by H-bonding with methanol and a Cp* CH3 hydrogen. The Rh(I)(Me4fulvene) species, stabilised by interaction of methanol with a coordinated water, again by two H-bonds H2O–HOMe (1.49 Å) and fulvene CH2 (1.94 Å), arises from synchronous transfer of the methanol OH proton to a Rh(III)–OH ligand and Cp* methyl hydrogen to the methanol oxygen. Additionally, the observed trend in catalytic activity for complexes 1–4 was reproduced by DFT calculations. These complexes form a novel class of catalytic molecular motors with a tunable rate of operation that can be stalled in a given state. They provide a basis for elucidation of the effects of ligand design on the contributions of electronic, rotational and vibrational energies to each step in the reaction pathway at the atomic level, consideration of which will enhance the design principles for the next generation of molecular machines.

The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.\ud \ud

Mono-axial functionalised octahedral diazido Pt(iv) complexes trans, trans, trans-[Pt(py)2(N3)2(OR1)(OR2)] (OR1 = OH and OR2 = anticancer agent coumarin-3 carboxylate (cou, 2a), pyruvate dehydrogenase kinase (PDK) inhibitors 4-phenylbutyrate (PhB, 2b) or dichloroacetate (DCA, 2c)), and their di-axial functionalised analogues with OR1 = DCA and OR2 = cou (3a), PhB (3b), or DCA (3c) have been synthesised and characterised, including the X-ray crystal structures of complexes 2a, 3a, 3b and 3c. These complexes exhibit dark stability and have the potential to generate cytotoxic Pt(ii) species and free radicals selectively in cancer cells when irradiated. Mono-functionalised complexes 2a–2c showed higher aqueous solubility and more negative reduction potentials. Mono- and di-functionalised complexes displayed higher photocytotoxicity with blue light (1 h, 465 nm, 4.8 mW cm–2) than the parent dihydroxido complex 1 (OR1 = OR2 = OH) in A2780 human ovarian (IC50 0.9–2.9 μM for 2a–2c; 0.11–0.39 μM for 3a–3c) and A549 human lung cancer cells (5.4–7.8 μM for 2a–2c; 1.2–2.6 μM for 3a–3c) with satisfactory dark stability. Notably, no apparent dark cytotoxicity was observed in healthy lung MRC-5 fibroblasts for all complexes (IC50 > 20 μM). Significantly higher platinum cellular accumulation and photo-generated ROS levels were observed for the di-functionalised complexes compared with their mono-functionalised analogues when cancer cells were treated under the same concentrations.

We report the synthesis and characterization of novel pentamethylcyclopentadienyl (Cp*) iridium(III) complexes [(Cp*)Ir(4-methyl-4′-carboxy-2,2′-bipyridine)Cl]PF6 (Ir-I), the product (Ir-II) from amide coupling of Ir-I to dibenzocyclooctyne-amine, and its conjugate (Ir-CP) with the cyclic nona-peptide c(CRWYDENAC). The familiar three-legged ‘piano-stool’ configuration for complex Ir-I was confirmed by its single crystal X-ray structure. Significantly, copper-free click strategy has been developed for site-specific conjugation of the parent complex Ir-I to the tumour targeting nona-cyclic peptide. The approach consisted of two steps: (i) the carboxylic acid group of the bipyridine ligand in complex Ir-I was first attached to an amine functionalized dibenzocyclooctyne group via amide formation to generate complex Ir-II; and (ii) the alkyne bond of dibenzocyclooctyne in complex Ir-II underwent a subsequent strain-promoted copper-free cycloaddition with the azide group of the modified peptide. Interestingly, while complex Ir-I was inactive towards A2780 human ovarian cancer cells, complex Ir-II exhibited moderate cytotoxic activity. Targeted complexes such as Ir-CP offer scope for enhanced activity and selectivity of this class of anticancer complexes.

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.

The platinum((IV)) prodrug trans,trans,trans-[Pt(N(3))(2)(OH)(2)(py)(2)] (1) is stable and non-toxic in the dark, but potently cytotoxic to cancer cells when irradiated by visible light, including cisplatin-resistant cells. On irradiation with visible light, it generates reactive Pt(II) species which can attack DNA, and produces reactive oxygen species (ROS) and reactive nitrogen species (RNS) which exert unusual effects on biochemical pathways. We now show that its novel mechanism of action includes induction of immunogenic cell death (ICD). Treatment of cancer cells with 1 followed by photoirradiation with visible light induces calreticulin (CRT) expression at the surface of dying cancer cells. This is accompanied by release of high mobility group protein-1B (HMGB1) and the secretion of ATP. Autophagy appears to play a key role in this chemotherapeutically-stimulated ICD. The observed uneven distribution of ecto-CRT promotes phagocytosis, confirmed by the observation of engulfment of photoirradiated CT26 colorectal cancer cells treated with 1 by J774.A1 macrophages. The photoactivatable prodrug 1 has a unique mechanism of action which distinguishes it from other platinum drugs due to its immunomodulating properties, which may enhance its anticancer efficacy.

We report the synthesis, characterisation and cytotoxicity of six cyclometalated rhodium(III) complexes [Cp(X)Rh(C^N)Z](0/+), in which Cp(X) = Cp*, Cp(ph), or Cp(biph), C^N = benzo[h]quinoline, and Z = chloride or pyridine. Three x-ray crystal structures showing the expected “piano-stool” configurations have been determined. The chlorido complexes hydrolysed faster in aqueous solution, also reacted preferentially with 9-ethyl guanine or glutathione compared to their pyridine analogues. The 1-biphenyl-2,3,4,5,-tetramethylcyclopentadienyl complex [Cp(biph)Rh(benzo[h]quinoline)Cl] (3a) was the most efficient catalyst in coenzyme reduced nicotinamide adenine dinucleotide (NADH) oxidation to NAD(+) and induced an elevated level of reactive oxygen species (ROS) in A549 human lung cancer cells. The pyridine complex [Cp(biph)Rh(benzo[h]quinoline)py](+) (3b) was the most potent against A549 lung and A2780 ovarian cancer cell lines, being 5-fold more active than cisplatin towards A549 cells, and acted as a ROS scavenger. This work highlights a ligand-controlled strategy to modulate the reactivity and cytotoxicity of cyclometalated rhodium anticancer complexes.

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 &mu, g of THPMe-NCS-huA33, followed after 24 h by 8&ndash, 10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 &mu, g, 7 MBq) and a 68Ga-only negative control (8&ndash, 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 &ldquo, unchelated&rdquo, 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 &mu, g, 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.

150 years after Mendeleev created his periodic table of 63 elements, now is the time to explore its impact on our understanding of life itself and the discovery of new medicines and diagnostic agents. We illustrate that currently 76 elements have compounds either approved for clinical use or in clinical trials for therapy or diagnosis. Modern analytical techniques and the methods of modern cell and molecular biology, including proteomics and genomics, can now be used to guide the design and application of both essential and non-essential elements in therapy and diagnosis, including stable and radioactive elements. In particular, there is potential for the target sites and mechanisms of action of metallodrugs to be identified. However, the speciation of the elements in biological environments presents enormous challenges. The biological activity of metal coordination complexes is highly dependent on the metal plus its ligands, and also on both thermodynamics (strengths of metal-ligand bonds), and kinetics (timescales of the making and breaking of coordination bonds), as well as metal- and ligand-based redox processes. Notably, biological systems/metabolic pathways are inherently dynamic. Using examples from our recent research, we illustrate how ligands exert fine control over the chemical reactivity and biological activity of metal complexes. Elucidation of the spatial and temporal coordination chemistry of metals in biological systems is likely to add exciting new dimensions to the periodic table.

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.

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 67Ga as a therapeutic radionuclide. Methods: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, 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 [ 67Ga]Ga-THP-trastuzumab and [ 111In]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 3 cells/mL, [ 67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [ 111In]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% [ 67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [ 111In]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 67Ga 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: [ 67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga 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.

The clinical impact and accessibility of 68Ga 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 99mTc radiopharmaceuticals. Currently, chelating agents used in 68Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a 68Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of 68Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with 68Ga 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 68Ga-THP-PSMA with 68Ga-HBED-CC-PSMA. Results: 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 68Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Conclusion:68Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other 68Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of 68Ga radiopharmaceuticals.

Diazido Pt(IV) complexes with a general formula [Pt(N3)2(L)(L′)(OR)(OR′)] are a new generation of anticancer prodrugs designed for use in photoactivated chemotherapy. The potencies of these complexes are affected by the cis/trans geometry configuration, the non-leaving ligand L/L′ and derivatisation of the axial ligand OR/OR′. Diazido Pt(IV) complexes exhibit high dark stability and promising photocytotoxicity circumventing cisplatin resistance. Upon irradiation, diazido Pt(IV) complexes release anticancer active Pt(II) species, azidyl radicals and ROS, which interact with biomolecules and therefore affect the cellular components and pathways. The conjugation of diazido Pt(IV) complexes with anticancer drugs or cancer-targeting vectors is an effective strategy to optimise the design of these photoactive prodrugs. Diazido Pt(IV) complexes represent a series of promising anticancer prodrugs owing to their novel mechanism of action which differs from that of classical cisplatin and its analogues.

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 67Ga as a therapeutic radionuclide. Methods THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, 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 [67Ga]Ga-THP-trastuzumab and [111In]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 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]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% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]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 67Ga 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 [67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga 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.

The outstanding efficiency of the tris(hydroxypyridonone) ligand THPMe for radiolabelling PET radiotracers with 68Ga is surpassed by THPH., The prototype tris(1,6-dimethyl-3-hydroxypyridin-4-one) chelator for gallium-68, THPMe, has shown great promise for rapid and efficient kit-based 68Ga labelling of PET radiopharmaceuticals. Peptide derivatives of THPMe have been used to image expression of their target receptors in vivo in preclinical and clinical studies. Herein we describe new synthetic routes to the THP platform including replacing the 1,6-dimethyl-3-hydroxypyridin-4-one N1–CH3 group of THPMe with O (tris(6-methyl-3-hydroxypyran-4-one, THPO) and N1–H (tris(6-methyl-3-hydroxypyridin-4-one), THPH) groups. The effect of these structural modifications on lipophilicity, gallium binding and metal ion selectivity was investigated. THPH was able to bind 68Ga in extremely mild conditions (5 min, room temperature, pH 6, 1 μM ligand concentration) and, notably, in vivo, when administered to a mouse previously injected with 68Ga acetate. The 67Ga radiolabelled complex was stable in serum for more than 7 days. [68Ga(THPH)] displayed a log P value of –2.40 ± 0.02, less negative than the log P = –3.33 ± 0.02 measured for [68Ga(THPMe)], potentially due to an increase in intramolecular hydrogen bonding attributable to the N1–H pyridinone units. Spectrophotometric determination of the Ga3+/Fe3+ complex formation constants for both THPMe and THPH revealed their preference for binding Ga3+ over Fe3+, which enabled selective labelling with 68Ga3+ in the presence of a large excess of Fe3+ in both cases. Compared to THPMe, THPH showed significantly reduced affinity for Fe3+, increased affinity for Ga3+ and improved radiolabelling efficiency. THPO was inferior to both THPH and THPMe in terms of labelling efficiency, but its benzylated precursor Bn-THPO (tris(6-methyl-3-benzyloxypyran-4-one)) provides a potential platform for the synthesis of a library of THP compounds with tunable chemical properties and metal preferences.

Gallium-68, and indium-111 share the same group in the periodic table and can be harnessed for a range of applications in nuclear medicine, including scintigraphy, SPECT, PET and targeted radiotherapy. Indium-111 and gallium-67 are gamma-emitting radionuclides with long half-lives (67.3 h and 78.3 h, respectively), while gallium-68 is a positron-emitting radionuclide with a very short half-life (68 min). All three radiometals can be conjugated to biomolecular vectors using bifunctional chelators. Despite the chemical parallels between the three isotopes, their applications in nuclear medicine vary widely. Indium-111 is principally used in conjunction with vectors with longer in vivo pharmacokinetic profiles such as cells and monoclonal antibodies. In contrast, the use of gallium-67 has centred upon exploiting the biological behaviour of the free Ga3+ ion, specifically for the imaging of tumours and infection. Finally, gallium-68 typically serves as a radiolabel for small molecules with rapid pharmacokinetic profiles, such as peptides. Both indium-111 and gallium-67 emit Auger electrons as well as photons, leading to their potential use for radionuclide therapy. Chelators and bifunctional chelators for gallium and indium share common features but are not interchangeable. While gallium-67 and indium-111 were foundational nuclides for the field, they currently have diminished profiles, whereas the use of gallium-68 is burgeoning. The design of chelators for gallium has advanced markedly in the last decade, stimulated by the need for chelators that can be labeled with gallium-68 quickly under mild conditions using a radiopharmaceutical “kit”.

A range of macrocyclic and acyclic chelators have been reacted with the PET isotope, gallium-68, and their radiolabelling efficiencies have been compared. Structural data for complexes of HBED with Ga3+ are reported., Gallium-68 (68Ga) is a positron-emitting isotope used for clinical PET imaging of peptide receptor expression. 68Ga radiopharmaceuticals used in molecular PET imaging consist of disease-targeting biomolecules tethered to chelators that complex 68Ga3+. Ideally, the chelator will rapidly, quantitatively and stably coordinate 68Ga3+ at room temperature, near neutral pH and low chelator concentration, allowing for simple routine radiopharmaceutical formulation. Identification of chelators that fulfil these requirements will facilitate development of kit-based 68Ga radiopharmaceuticals. Herein the reaction of a range of widely used macrocyclic and acyclic chelators with 68Ga3+ is reported. Radiochemical yields have been measured under conditions of varying chelator concentrations, pH (3.5 and 6.5) and temperature (25 and 90 °C). These chelators are: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane macrocycles substituted with phosphonic (NOTP) and phosphinic (TRAP) groups at the amine, bis(2-hydroxybenzyl)ethylenediaminediacetic acid (HBED), a tris(hydroxypyridinone) containing three 1,6-dimethyl-3-hydroxypyridin-4-one groups (THP) and the hexadentate tris(hydroxamate) siderophore desferrioxamine-B (DFO). Competition studies have also been undertaken to assess relative complexation efficiencies of each chelator for 68Ga3+ under different pH and temperature conditions. Performing radiolabelling reactions at pH 6.5, 25 °C and 5–50 μM chelator concentration resulted in near quantitative radiochemical yields for all chelators, except DOTA. Radiochemical yields either decreased or were not substantially improved when the reactions were undertaken at lower pH or at higher temperature, except in the case of DOTA. THP and DFO were the most effective 68Ga3+ chelators at near-neutral pH and 25 °C, rapidly providing near-quantitative radiochemical yields at very low chelator concentrations. NOTP and HBED were only slightly less effective under these conditions. In competition studies with all other chelators, THP demonstrated highest reactivity for 68Ga3+ complexation under all conditions. These data point to THP possessing ideal properties for rapid, one-step kit-based syntheses of 68Ga-biomolecules for molecular PET imaging. LC-MS and 1H, 13C{1H} and 71Ga NMR studies of HBED complexes of Ga3+ showed that under the analytical conditions employed in this study, multiple HBED-bound Ga complexes exist. X-ray diffraction data indicated that crystals isolated from these solutions contained octahedral [Ga(HBED)(H2O)], with HBED coordinated in a pentadentate N2O3 mode, with only one phenolic group coordinated to Ga3+, and the remaining coordination site occupied by a water molecule.

Derivatives of 3,4-hydroxypyridinones have been extensively studied for in vivo Fe(3+) sequestration. Deferiprone, a 1,2-dimethyl-3,4-hydroxypyridinone, is now routinely used for clinical treatment of iron overload disease. Hexadentate tris(3,4-hydroxypyridinone) ligands (THP) complex Fe(3+) at very low iron concentrations, and their high affinities for oxophilic trivalent metal ions have led to their development for new applications as bifunctional chelators for the positron emitting radiometal, (68)Ga(3+), which is clinically used for molecular imaging in positron emission tomography (PET). THP-peptide bioconjugates rapidly and quantitatively complex (68)Ga(3+) at ambient temperature, neutral pH and micromolar concentrations of ligand, making them amenable to kit-based radiosynthesis of (68)Ga PET radiopharmaceuticals. (68)Ga-labelled THP-peptides accumulate at target tissue in vivo, and are excreted largely via a renal pathway, providing high quality PET images.

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 αvβ3 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 αvβ3 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 αvβ3 integrin receptor expression in vivo.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists, and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this chapter illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the chapter is the shift from single photon radionuclide imaging toward PET, and in turn the emerging shift from PET/CT toward PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo , and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short-lived (Ga-68) and long-lived (Zr-89) positron-emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabeled iron oxide-based nanoparticles.

The development of medical imaging is a highly multidisciplinary endeavor requiring the close cooperation of clinicians, physicists, engineers, biologists and chemists to identify capabilities, conceive challenges and solutions and apply them in the clinic. The chemistry described in this article illustrates how synergistic advances in these areas drive the technology and its applications forward, with each discipline producing innovations that in turn drive innovations in the others. The main thread running through the article is the shift from single photon radionuclide imaging towards PET, and in turn the emerging shift from PET/CT towards PET/MRI and further, combination of these with optical imaging. Chemistry to support these transitions is exemplified by building on a summary of the status quo, and recent developments, in technetium-99m chemistry for SPECT imaging, followed by a report of recent developments to support clinical application of short lived (Ga-68) and long-lived (Zr-89) positron emitting isotopes, copper isotopes for PET imaging, and combined modality imaging agents based on radiolabelled iron oxide based nanoparticles.