Neutron Capture Enhances Dose and Reduces Cancer Cell Viability in and out of Beam During Helium and Carbon Ion Therapy.

PURPOSE: Neutron capture enhanced particle therapy (NCEPT) is a proposed augmentation of charged particle therapy that exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with 2 different targeted neutron capture agents (NCAs). METHODS AND MATERIALS: Human glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a polymethyl methacrylate phantom either laterally adjacent to or within a 100 × 100 × 60 mm spread out Bragg peak (SOBP). The effect of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in 6 independent experiments. Neutron fluence within the phantom was characterized by quantifying the neutron activation of gold foil. RESULTS: Cells placed inside the treatment volume reached 10% survival by 2 Gy of carbon or 2 to 3 Gy of helium in the presence of NCAs compared with 5 Gy of carbon and 7 Gy of helium with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of carbon or helium (to the treatment volume), compared with no detectable effect on cells without NCA. The mean thermal neutron fluence at the center of the SOBP was approximately 2.2 × 109 n/cm2/Gy (relative biological effectiveness) for the carbon beam and 5.8 × 109 n/cm2/Gy (relative biological effectiveness) for the helium beam and gradually decreased in all directions. CONCLUSIONS: The addition of NCAs to cancer cells during carbon and helium beam irradiation has a measurable effect on cell survival and growth in vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumors, by targeting this dose to microinfiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumor tissue within the treatment volume, with a reduction in radiation doses to off-target tissue.

Kinetic isotope effects and synthetic strategies for deuterated carbon-11 and fluorine-18 labelled PET radiopharmaceuticals.

The deuterium labelling of pharmaceuticals is a useful strategy for altering pharmacokinetic properties, particularly for improving metabolic resistance. The pharmacological effects of such metabolites are often assumed to be negligible during standard drug discovery and are factored in later at the clinical phases of development, where the risks and benefits of the treatment and side-effects can be wholly assessed. This paradigm does not translate to the discovery of radiopharmaceuticals, however, as the confounding effects of radiometabolites can inevitably show in preliminary positron emission tomography (PET) scans and thus complicate interpretation. Consequently, the formation of radiometabolites is crucial to take into consideration, compared to non-radioactive metabolites, and the application of deuterium labelling is a particularly attractive approach to minimise radiometabolite formation. Herein, we provide a comprehensive overview of the deuterated carbon-11 and fluorine-18 radiopharmaceuticals employed in PET imaging experiments. Specifically, we explore six categories of deuterated radiopharmaceuticals used to investigate the activities of monoamine oxygenase (MAO), choline, translocator protein (TSPO), vesicular monoamine transporter 2 (VMAT2), neurotransmission and the diagnosis of Alzheimer's disease; from which we derive four prominent deuteration strategies giving rise to a kinetic isotope effect (KIE) for reducing the rate of metabolism. Synthetic approaches for over thirty of these deuterated radiopharmaceuticals are discussed from the perspective of deuterium and radioisotope incorporation, alongside an evaluation of the deuterium labelling and radiolabelling efficacies across these independent studies. Clinical and manufacturing implications are also discussed to provide a more comprehensive overview of how deuterated radiopharmaceuticals may be introduced to routine practice.

Fluorine-18 Radiolabelling and Photophysical Characteristics of Multimodal PET-Fluorescence Molecular Probes.

Positron emission tomography (PET)-fluorescence imaging is an emerging field of multimodality imaging seeking to attain synergy between the two techniques. The probes employed in PET-fluorescence imaging incorporate both a fluorophore and radioisotope which enable complementary information to be obtained from both imaging techniques via the administration of a single agent. Fluorine-18 is the most commonly used radioisotope in PET imaging and consequently many novel attempts to radiofluorinate various fluorophores have transpired over the past decade. In this Minireview, the most relevant fluorine-18 labelled PET-fluorescence probes have been classified into four groups as per the implemented fluorophore: 1) boron-dipyrromethene (BODIPY) dyes, 2) cyanine dyes, 3) alternative organic fluorophores and 4) organometallics, such as quantum dots (QDs) and rhenium complexes. The biological, radiochemical and photophysical properties of each probe have been systematically compared to aid future endeavours in PET-fluorescence chemistry.

Rhenium(i) complexation-dissociation strategy for synthesising fluorine-18 labelled pyridine bidentate radiotracers.

A novel fluorine-18 method employing rhenium(i) mediation is described herein. The method was found to afford moderate to high radiochemical yields of labelled rhenium(i) complexes. Subsequent thermal dissociation of the complexes enabled the radiosynthesis of fluorine-18 labelled pyridine bidentate structures which could not be radiofluorinated hitherto. This rhenium(i) complexation-dissociation strategy was further applied to the radiosynthesis of [18F]CABS13, an Alzheimer's disease imaging agent, alongside other 2,2'-bipyridine, 1,10-phenanthroline and 8-hydroxyquinoline labelled radiotracers. Computational modelling of the reaction mechanism suggests that the efficiency of rhenium(i) activation may be attributed to both an electron withdrawal effect by the metal center and the formation of an acyl fluoride intermediate which anchors the fluoride subsequent to nucleophilic addition.

[18 F]Ethenesulfonyl Fluoride as a Practical Radiofluoride Relay Reagent.

Fluorine-18 is the most utilized radioisotope in positron emission tomography (PET), but the wide application of fluorine-18 radiopharmaceuticals is hindered by its challenging labelling conditions. As such, many potentially important radiotracers remain underutilized. Herein, we describe the use of [18 F]ethenesulfonyl fluoride (ESF) as a novel radiofluoride relay reagent that allows radiofluorination reactions to be performed in minimally equipped satellite nuclear medicine centres. [18 F]ESF has a simple and reliable production route and can be stored on inert cartridges. The cartridges can then be shipped remotely and the trapped [18 F]ESF can be liberated by simple solvent elution. We have tested 18 radiolabelling precursors, inclusive of model and clinically used structures, and most precursors have demonstrated comparable radiofluorination efficiencies to those obtained using a conventionally dried [18 F]fluoride source.

A rapid MS/MS method to assess the deuterium kinetic isotope effect and associated improvement in the metabolic stability of deuterated biological and pharmacological molecules as applied to an imaging agent.

The deuterium kinetic isotope effect has been known for a period of 40 years, but it is only relatively recently that new drug entities (NDEs) incorporating deuterium demonstrating beneficial pharmacokinetics, pharmacodynamics, and toxicology have arrived to market. Determination of the precise location to deuterate and subsequently any evaluation for a kinetic isotope effect (KIE) is challenging. Typically, such an evaluation would be performed in an in vitro metabolic assay (e.g. liver microsomes) in separate reaction media for both the deuterated and non-deuterated analogues. Here, we have devised an approach whereby we incubate a 1:1 ratio of both the deuterated and protio-form of an imaging agent together in the same liver microsomal assay and determine the relative rate of consumption of both moieties, based upon specific MS-MS transitions unique to both molecules without the need for liquid chromatography-mass spectrometry (LC-MS) separation and quantification. Any deviation of the ratio of the MS transitions from the initial starting point indicated an observable KIE. A site specific deuteration of PBR111, a neuroinflammation imaging agent, was chosen for a proof-of-concept study. Based upon prior mechanistic knowledge of PBR111, two locations were selected for deuteration; an active and inactive site, to corroborate that there was no significant KIE for the inactive site and confirm the efficacy of the developed methodology.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Synthesis, bioconjugation and stability studies of [18 F]ethenesulfonyl fluoride.

Fluorine-18 labelled prosthetic groups (PGs) are often necessary for radiolabelling sensitive biological molecules such as peptides and proteins. Several shortcomings, however, often diminish the final yield of radiotracer. In an attempt to provide higher yielding and operationally efficient tools for radiolabelling biological molecules, we describe herein the first radiochemical synthesis of [18 F]ethenesulfonyl fluoride ([18 F]ESF) and its Michael conjugation with amino acids and proteins. The synthesis of [18 F]ESF was optimised using a microfluidic reactor under both carrier-added (c.a.) and no-carrier-added (n.c.a.) conditions, affording, in a straightforward procedure, 30-50% radiochemical yield (RCY) for c.a. [18 F]ESF and 60-70% RCY for n.c.a. [18 F]ESF. The conjugation reactions were performed at room temperature using 10 mg/mL precursor in aqueous/organic solvent mixtures for 15 min. The radiochemical stability of the final conjugates was evaluated in injectable formulation and rat serum, and resulted strongly substrate dependent and generally poor in rat serum. Therefore, in this work we have optimised a straightforward synthesis of [18 F]ESF and its Michael conjugation with model compounds, without requiring chromatographic purification. However, given the general low stability of the final products, further studies will be required for improving conjugate stability, before assessing the use of this PG for PET imaging.

Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22.

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1-7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1-7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

A Fluorine-18 Radiolabeling Method Enabled by Rhenium(I) Complexation Circumvents the Requirement of Anhydrous Conditions.

Azeotropic distillation is typically required to achieve fluorine-18 radiolabeling during the production of positron emission tomography (PET) imaging agents. However, this time-consuming process also limits fluorine-18 incorporation, due to radioactive decay of the isotope and its adsorption to the drying vessel. In addressing these limitations, the fluorine-18 radiolabeling of one model rhenium(I) complex is reported here, which is significantly improved under conditions that do not require azeotropic drying. This work could open a route towards the investigation of a simplified metal-mediated late-stage radiofluorination method, which would expand upon the accessibility of new PET and PET-optical probes.

Sulfur - fluorine bond in PET radiochemistry.

The importance of the sulfur-fluorine bond is starting to increase in modern medicinal chemistry literature. This is due to a better understanding of the stability and reactivity of this moiety depending on the various oxidation states of sulfur. Furthermore, several commercial reagents used for mild and selective fluorination of organic molecules are based on the known reactivity of S-F groups. In this review, we will show how these examples are translating into the 18F field, both for use as stable tags in finished radiopharmaceuticals and as mildly reactive fluoride-relay intermediates. Finally, we also discuss current opportunities where examples of non-radioactive S-F applications/chemistry may be translated into future 18F radiochemistry applications.

In Vivo Evaluation of Radiofluorinated Caspase-3/7 Inhibitors as Radiotracers for Apoptosis Imaging and Comparison with [18F]ML-10 in a Stroke Model in the Rat.

PURPOSE: The first biological evaluation of two potent fluorine-18 radiolabelled inhibitors of caspase-3/7 was achieved in a cerebral stroke rat model to visualize apoptosis. PROCEDURES: In vivo characteristics of isatins [(18)F]-2 and [(18)F]-3 were studied and compared by µPET to previously described 1-[4-(2-[(18)F]fluoroethyl)benzyl]-5-(2-methoxymethylpyrrolidin-1-ylsulfonyl)isatin ([(18)F]-1) and to 2-(5-[(18)F]fluoropentyl)-2-methyl-malonic acid ([(18)F]ML-10) used as a reference radiotracer in a rat stroke model. RESULTS: [(18)F]-2 and [(18)F]-3 were radiolabelled with high radiochemical purity and high specific radioactivity. Radioactivity uptakes in ischemic and contralateral brain regions were weak for the three radiolabelled isatins and lower for [(18)F]ML-10. In µPET, time activity curves showed significant uptake differences between both regions of interest for [(18)F]-1 after 45 min. No differences were observed for [(18)F]ML-10. CONCLUSIONS: Radiolabelled isatins are more promising radiotracers to image apoptosis than [(18)F]ML-10 in this stroke animal model without craniectomy. In particular, [(18)F]-1 presented significant uptake in apoptotic area 45 min after administration.

Radiosynthesis and 'click' conjugation of ethynyl-4-[(18)F]fluorobenzene--an improved [(18)F]synthon for indirect radiolabeling.

Reproducible methods for [(18)F]radiolabeling of biological vectors are essential for the development of new [(18)F]radiopharmaceuticals. Molecules such as carbohydrates, peptides and proteins are challenging substrates that often require multi-step indirect radiolabeling methods. With the goal of developing more robust, time saving, and less expensive procedures for indirect [(18)F]radiolabeling of such molecules, our group has synthesized ethynyl-4-[(18)F]fluorobenzene ([(18)F]2, [(18)F]EYFB) in a single step (14 ± 2% non-decay corrected radiochemical yield (ndc RCY)) from a readily synthesized, shelf stable, inexpensive precursor. The alkyne-functionalized synthon [(18)F]2 was then conjugated to two azido-functionalized vector molecules via CuAAC reactions. The first 'proof of principle' conjugation of [(18)F]2 to 1-azido-1-deoxy-ß-D-glucopyranoside (3) gave the desired radiolabeled product [(18)F]4 in excellent radiochemical yield (76 ± 4% ndc RCY (11% overall)). As a second example, the conjugation of [(18)F]2 to matrix-metalloproteinase inhibitor (5), which has potential in tumor imaging, gave the radiolabeled product [(18)F]6 in very good radiochemical yield (56 ± 12% ndc RCY (8% overall)). Total preparation time for [(18)F]4 and [(18)F]6 including [(18)F]F(-) drying, two-step reaction (nucleophilic substitution and CuAAC conjugation), two HPLC purifications, and two solid phase extractions did not exceed 70 min. The radiochemical purity of synthon [(18)F]2 and the conjugated products, [(18)F]4 and [(18)F]6, were all greater than 98%. The specific activities of [(18)F]2 and [(18)F]6 were low, 5.97 and 0.17 MBq nmol(-1), respectively.

Novel Fluorinated 8-Hydroxyquinoline Based Metal Ionophores for Exploring the Metal Hypothesis of Alzheimer's Disease.

Zinc, copper, and iron ions are involved in amyloid-beta (Aß) deposition and stabilization in Alzheimer's disease (AD). Consequently, metal binding agents that prevent metal-Aß interaction and lead to the dissolution of Aß deposits have become well sought therapeutic and diagnostic targets. However, direct intervention between diseases and metal abnormalities has been challenging and is partially attributed to the lack of a suitable agent to determine and modify metal concentration and distribution in vivo. In the search of metal ionophores, we have identified several promising chemical entities by strategic fluorination of 8-hydroxyquinoline drugs, clioquinol, and PBT2. Compounds 15-17 and 28-30 showed exceptional metal ionophore ability (6-40-fold increase of copper uptake and >2-fold increase of zinc uptake) and inhibition of zinc induced Aß oligomerization (EC50s < ∼5 µM). These compounds are suitable for further development as drug candidates and/or positron emission tomography (PET) biomarkers if radiolabeled with (18)F.

Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents.

This study reports the synthesis, [(123)I]radiolabeling, and biological profile of a new series of iodinated compounds for potential translation to the corresponding [(131)I]radiolabeled compounds for radionuclide therapy of melanoma. Radiolabeling was achieved via standard electrophilic iododestannylation in 60-90% radiochemical yield. Preliminary SPECT imaging demonstrated high and distinct tumor uptake of all compounds, as well as high tumor-to-background ratios compared to the literature compound [(123)I]4 (ICF01012). The most favorable compounds ([(123)I]20, [(123)I]23, [(123)I]41, and [(123)I]53) were selected for further biological investigation. Biodistribution studies indicated that all four compounds bound to melanin containing tissue with low in vivo deiodination; [(123)I]20 and [(123)I]53 in particular displayed high and prolonged tumor uptake (13% ID/g at 48 h). [(123)I]53 had the most favorable overall profile of the cumulative uptake over time of radiosensitive organs. Metabolite analysis of the four radiotracers found [(123)I]41 and [(123)I]53 to be the most favorable, displaying high and prolonged amounts of intact tracer in melanin containing tissues, suggesting melanin specific binding. Results herein suggest that compound [(123)I]53 displays favorable in vivo pharmacokinetics and stability and hence is an ideal candidate to proceed with further preclinical [(131)I] therapeutic evaluation.

Derivatives of imidazotriazine and pyrrolotriazine C-nucleosides as potential new anti-HCV agents.

Previous investigations identified 2'-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.

Lanthanoid "bottlebrush" clusters: remarkably elongated metal-oxo core structures with controllable lengths.

Large metal-oxo clusters consistently assume spherical or regular polyhedral morphologies rather than high-aspect-ratio structures. Access to elongated core structures has now been achieved by the reaction of lanthanoid salts with a tetrazole-functionalized calixarene in the presence of a simple carboxylate co-ligand. The resulting Ln19 and Ln12 clusters are constructed from apex-fused Ln5O6 trigonal bipyramids and are formed consistently under a range of reaction conditions and reagent ratios. Altering the carboxylate co-ligand structure reliably controls the cluster length, giving access to a new class of rod-like clusters of variable length.

Optimization of nucleophilic ¹8F radiofluorinations using a microfluidic reaction approach.

Microfluidic techniques are increasingly being used to synthesize positron-emitting radiopharmaceuticals. Several reports demonstrate higher incorporation yields, with shorter reaction times and reduced amounts of reagents compared with traditional vessel-based techniques. Microfluidic techniques, therefore, have tremendous potential for allowing rapid and cost-effective optimization of new radiotracers. This protocol describes the implementation of a suitable microfluidic process to optimize classical (18)F radiofluorination reactions by rationalizing the time and reagents used. Reaction optimization varies depending on the systems used, and it typically involves 5-10 experimental days of up to 4 h of sample collection and analysis. In particular, the protocol allows optimization of the key fluidic parameters in the first tier of experiments: reaction temperature, residence time and reagent ratio. Other parameters, such as solvent, activating agent and precursor concentration need to be stated before the experimental runs. Once the optimal set of parameters is found, repeatability and scalability are also tested in the second tier of experiments. This protocol allows the standardization of a microfluidic methodology that could be applied in any radiochemistry laboratory, in order to enable rapid and efficient radiosynthesis of new and existing [(18)F]-radiotracers. Here we show how this method can be applied to the radiofluorination optimization of [(18)F]-MEL050, a melanoma tumor imaging agent. This approach, if integrated into a good manufacturing practice (GMP) framework, could result in the reduction of materials and the time required to bring new radiotracers toward preclinical and clinical applications.

Ascertaining the suitability of aryl sulfonyl fluorides for [18F]radiochemistry applications: a systematic investigation using microfluidics.

Optimization of [(18)F]radiolabeling conditions and subsequent stability analysis in mobile phase, PBS buffer, and rat serum of 12 aryl sulfonyl chloride precursors with various substituents (electron-withdrawing groups, electron-donating groups, increased steric bulk, heterocyclic) were performed using an Advion NanoTek Microfluidic Synthesis System. A comparison of radiochemical yields and reaction times for a microfluidics device versus a conventional reaction vessel is reported. [(18)F]Radiolabeling of sulfonyl chlorides in the presence of competing nucleophiles, H-bond donors, and water was also assessed and demonstrated the versatility and potential utility of [(18)F]sulfonyl fluorides as synthons for indirect radiolabeling.

Multifunctional hybrid materials based on transparent poly(methyl methacrylate) reinforced by lanthanoid hydroxo clusters.

Three pentanuclear lanthanoid hydroxo clusters of composition [Ln(OH)(5)(abzm)(10)], where Ln = Eu, Tb, Ho and abzm = di(4-allyloxy)benzoylmethanide, have been prepared. The structures have been characterised by means of IR, Raman, elemental analyses and X-ray diffraction, showing a pyramidal square-based cluster core. The clusters (Tb and Ho) exhibit Curie-Weiss Law behaviour, displaying antiferromagnetic ordering at low temperatures. The emission properties of the Eu cluster demonstrate the abzm(-) ligand is an efficient antenna (λ(ex) = 420 nm) only for the sensitisation of Eu luminescence in the visible range, via energy transfer to the (5)D(0) state of the trivalent metal. The clusters have been reacted in the presence of methyl methacrylate and azobisisobutyronitrile to prepare reinforced polymers via radical polymerisation. The obtained materials exhibit swelling upon immersion into organic solvents up to ≈ 110% of their original size, in agreement with the presence of cluster-crosslinked polymeric chains. Also, no loss of transparency was observed in the preparation of the materials. The characteristic red emission of the Eu cluster in also retained in the polymeric material.