Helen M. Betts

Cellular confocal fluorescence studies and cytotoxic activity of new Zn(II) bis(thiosemicarbazonato) complexes

We report the synthesis and characterisation of new, highly fluorescent, zinc complexes of bis(thiosemicarbazone) ligands incorporating extended aromatic backbones which are cytotoxic at levels comparable to cisplatin; cellular fluorescence imaging studies suggest these cause cell death by disruption of mitochondria.

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.

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.

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).

2-Bromo-6-[18F]fluoropyridine: two-step fluorine-18 radiolabelling via transition metal-mediated chemistry†

Novel radiolabelling methods are important for the development of new tracers for positron emission tomography. Direct nucleophilic fluorination of aromatic rings with [(18) F]fluoride is limited to activated substrates, restricting the application of this approach. Inspired by transition metal-mediated transformations, a fluorine-18 synthon was prepared to supplement the radiolabelling methods available for molecules unsuitable for direct labelling. 2-Bromo-6-[(18) F]fluoropyridine (denoted [(18) F]1) was prepared in high yield, and palladium-mediated cross-coupling reactions were exemplified. High incorporation of fluoride and efficient cross-coupling reactions demonstrate that compound [(18) F]1 holds promise as a new synthon for construction of fluorine-18-labelled molecules via transition metal-mediated reactions.

Automated radiosynthesis of GMP quality [18F]HX4 for PET imaging of hypoxia.

INTRODUCTION [(18)F]HX4 is a 2-nitroimidazole based investigational radiotracer for imaging hypoxia. METHODS A two-step, one-pot synthetic procedure was developed on a GE Tracerlab MX-FDG with a disposable cassette. Nucleophilic substitution of a nosylate group with [(18)F]fluoride was followed by solvent evaporation and acidic removal of the acetyl protecting group. HPLC purification in a bio-compatible solvent mixture was developed. RESULTS AND CONCLUSIONS Using starting activities of 80-110 GBq [(18)F]fluoride, GMP compliant [(18)F]HX4 was produced in non-decay corrected radiochemical yields of 12 ± 3% (n = 9) in 55 min including HPLC purification. No reformulation steps were required. The mean specific activity of the final product was 2450 GBq/μmol. Modifications to the process and final formulation were included to prevent decomposition of the product, and these changes resulted in an improved stability of the formulated [(18)F]HX4, with a shelf-life of at least 8h post-synthesis. The product consistently passed all required quality control tests to determine that the [(18)F]HX4 was suitable for clinical use. Using a 90 minute target bombardment, and 80-110 GBq starting [(18)F]fluoride, the method produced multiple patient doses.

A tolerability and patient acceptability pilot study of a novel antimicrobial urinary catheter for long-term use

We have developed a novel antimicrobial urinary catheter (AUC) impregnated with rifampicin, triclosan, and sparfloxacin and demonstrated that it has long‐term (∼84 days) protection against bacterial colonization in vitro. This study aimed to assess the safety and patient acceptability of this device in long‐term catheter users.