Simon R. Bayly

In Vitro and In Vivo Evaluations of a Hydrophilic 64Cu-Bis(Thiosemicarbazonato)–Glucose Conjugate for Hypoxia Imaging

A water-soluble glucose conjugate of the hypoxia tracer 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM) was synthesized and radiolabeled (64Cu-ATSE/A-G). Here we report our initial biological experiments with 64Cu-ATSE/A-G and compare the results with those obtained for 64Cu-ATSM and 18F-FDG. Methods: The uptake of 64Cu-ATSE/A-G and 64Cu-ATSM into HeLa cells in vitro was investigated at a range of dissolved oxygen concentrations representing normoxia, hypoxia, and anoxia. Small-animal PET with 64Cu-ATSE/A-G was performed in male BDIX rats implanted with P22 syngeneic carcinosarcomas. Images of 64Cu-ATSM and 18F-FDG were obtained in the same model for comparison. Results: 64CuATSE/A-G showed oxygen concentration–dependent uptake in vitro and, under anoxic conditions, showed slightly lower levels of cellular uptake than 64Cu-ATSM; uptake levels under hypoxic conditions were also lower. Whereas the normoxic uptake of 64Cu-ATSM increased linearly over time, 64Cu-ATSE/A-G uptake remained at low levels over the entire time course. In the PET study, 64CuATSE/A-G showed good tumor uptake and a biodistribution pattern substantially different from that of each of the controls. In marked contrast to the findings for 64Cu-ATSM, renal clearance and accumulation in the bladder were observed. 64Cu-ATSE/A-G did not display the characteristic brain and heart uptake of 18F-FDG. Conclusion: The in vitro cell uptake studies demonstrated that 64Cu-ATSE/A-G retained hypoxia selectivity and had improved characteristics when compared with 64Cu-ATSM. The in vivo PET results indicated a difference in the excretion pathways, with a shift from primarily hepatointestinal for 64Cu-ATSM to partially renal with 64Cu-ATSE/A-G. This finding is consistent with the hydrophilic nature of the glucose conjugate. A comparison with 18F-FDG PET results revealed that 64Cu-ATSE/A-G was not a surrogate for glucose metabolism. We have demonstrated that our method for the modification of Cu-bis(thiosemicarbazonato) complexes allows their biodistribution to be modified without negating their hypoxia selectivity or tumor uptake properties.

Fluorescent copper(II) bis(thiosemicarbazonates): synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies.

Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radioassays show good stability in solution and about 25 % binding to protein over 1 h, which is comparable with the hypoxia selective tracer [(64)Cu(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1 does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu(II) complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (>1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn(II) analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.

Nitroimidazole conjugates of bis(thiosemicarbazonato)64Cu(II) – Potential combination agents for the PET imaging of hypoxia

Combination agents comprising two different pharmacophores with the same biological target have the potential to show additive or synergistic activity. Bis(thiosemicarbazonato)copper(II) complexes (e.g. (64)Cu-ATSM) and nitroimidazoles (e.g. (18)F-MISO) are classes of tracer used for the delineation of tumor hypoxia by positron emission tomography (PET). Three nitroimidazole-bis(thiosemicarbazonato)copper(II) conjugates were produced in order to investigate their potential as combination hypoxia imaging agents. Two were derived from the known bifunctional bis(thiosemicarbazone) H(2)ATSM/A and the third from the new precursor diacetyl-2-(4-N-methyl-3-thiosemicarbazone)-3-(4-N-ethylamino-3-thiosemicarbazone) - H(2)ATSM/en. Oxygen-dependent uptake studies were performed using the (64)Cu radiolabelled complexes in EMT6 carcinoma cells. All the complexes displayed appreciable hypoxia selectivity, with the nitroimidazole conjugates displaying greater selectivity than a simple propyl derivative used as a control. Participation of the nitroimidazole group in the trapping mechanism is indicated by the increased hypoxic uptake of the 2- vs. the 4-substituted (64)Cu-ATSM/A derivatives. The 2-nitroimidazole derivative of (64)Cu-ATSM/en demonstrated superior hypoxia selectivity to (64)Cu-ATSM over the range of oxygen concentrations tested. Biodistribution of the radiolabelled 2-nitroimidazole conjugates was carried out in EMT6 tumor-bearing mice. The complexes showed significantly different uptake trends in comparison to each other and previously studied Cu-ATSM derivatives. Uptake of the Cu-ATSM/en conjugate in non-target organs was considerably lower than for derivatives based on Cu-ATSM/A.

Metal-Based Anion Receptor Systems

Metal complexes play an important role in anion receptor chemistry. In the majority of examples metal centres are used as optical and/or electrochemical reporter groups in anion-sensing applications. Metal centres can also act as Lewis acidic anion-binding sites in their own right, and/or as structural components allowing the self-assembly of anion-binding domains. This review describes the development of metal-based receptors with regard to their anion-sensing properties, and is therefore divided into sections on electrochemical and optical anion sensing. Within these sections coverage has been given to the diverse range of metals and anion-binding groups that have been studied. Emphasis has been placed on recently described novel supramolecular, nanoscale and surface confined anion receptor systems that give added functionality. The last section describes metal-based anion receptors without reporter groups.

Towards nanomedicines: design protocols to assemble, visualize and test carbon nanotube probes for multi-modality biomedical imaging

Nanomedicine is an interdisciplinary field, still in its infancy, where an accurate scientific assessment of potential risks and benefits is urgently needed, as is the engagement of end users and the public in this facet of the nanotechnology debate. There is increasing interest in improving our understanding of the interactions between nanomaterials and living systems, with regard to both the underlying chemistry and the physics of effects on the nanoscale. Ultimately, such knowledge promises new vistas for designing the ‘smart’ medicines of the future, of which targeted personalized drugs are the holy grail. Imaging and therapeutic components, including metallic radioisotopes, semiconductor quantum dots and magnetic materials, may be used to construct ‘nanocarriers’ (by encapsulation or conjugation) by rapid and simple (covalent and supramolecular) chemistry. The biomedical functions of the resulting materials are as yet largely unexplored. Encapsulation in nanocarriers could achieve delivery of the reagents (imaging and therapeutic drugs) to the sites of action in the body, while minimizing systemic toxicity and enzymatic degradation. These functional systems have the potential to become a general solution in drug delivery. Here we review recent developments concerning the applications of nanoparticles, including carbon nanotubes, as synthetic scaffolds for designing nanomedicines. This article will also focus on how understanding and design at the molecular level could help interdisciplinary teams develop research towards new diagnostics and therapeutics both in the short and the long term.

Controlled axial coordination: solid-phase synthesis and purification of metallo-radiopharmaceuticals.

Solid-supported reagents show great potential for improving the synthesis of radiodiagnostic agents, in terms of radiochemical yield and purity, as well as convenience and safety. The preparation of the positron emission tomography (PET) imaging agent [F]-2-fluoro-2-deoxy-d-glucose has recently been demonstrated using a solid-bound substrate which is selectively cleaved from the solid support upon reaction with [F]-fluoride ions. Whilst this covalent approach is appropriate for the nucleophilic substitution chemistry of fluoride ions, its utility with metallonuclides is limited and the only examples to date have used Tc in conjunction with proligands attached to resins and gold surfaces. Conventional solid-phase synthesis is therefore limited by the requirement for proligands possessing a donor group which can both be covalently attached to the solid support and cleaved from it upon coordination to the desired metal ion. Herein, we report a novel strategy for solid-phase synthesis based on selective axial coordination of Zn substrates to 4-(dimethylamino)pyridine(DMAP)-functionalized polystyrene resin and exemplify its use in the preparation and purification of known and potential Cu and Tc radiopharmaceuticals. This strategy is applicable to a wide range of metallic radionuclides and is suitable for the macrocyclic ligand systems that are favored in nuclear medicine because of their high in vivo stability. Zn complexes of tetradentate ligands that are constrained in pseudo-square-planar conformations can potentially bind a fifth donor atom in an axial coordination site. Jahn–Teller distortion disfavors the coordination of a fifth donor atom in the axial site of analogous Cu complexes. A Zn precursor can be bound to polymer-supported DMAP by axial coordination. Upon transmetalation of the Zn complex with Cu, the coordinate bond to the solid support is broken and only the transmetalated complex is released from the resin (Scheme 1 a). Similarly, polystyrene-supported DMAP can be used to selectively bind the Zn complex from a solution-phase reaction mixture leaving only the radiolabeled complex in solution (Scheme 1b).

Orthogonal 18F and 64Cu labelling of functionalised bis(thiosemicarbazonato) complexes

The synthesis of three pairs of orthogonally labelled fluorinated Cu bis(thiosemicarbazonato) complexes is presented. These are the first examples of (18)F-labelled Cu(II)-complexes designed to serve as new hypoxia selective PET tracers and as mechanistic probes to study the mode of action of this class of markers. In vitro evaluation revealed that the fluorinated Cu-complex derived from amide coupling is suitable for in vivo work.

Organometallic Receptors for Charged and Neutral Guest Species

Due to the many advantageous physical and chemical properties they possess, organometallic molecules continue to play a crucial role in the development of synthetic receptors for cationic, anionic, and neutral guest species. This chapter is the first review to look exclusively at organometallic receptors as a single class. It provides an overview of the subject with reference to the nature of guest species and the type of organometallic group incorporated. Special emphasis is given to the properties that organometallic subunits impart to molecular receptors; whether they act as host-binding groups, redox-, photoactive reporter groups, or structural components. In particular, when these roles are combined, organometallic receptors display a range of functionality not available to purely organic structures. The final section of this chapter details recent advances toward the application of organometallic receptors as sensing devices, including their use in dendrimer, polymer, functionalized nanoparticle and self-assembled monolayer systems.