Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH.The authors acknowledge funding support from Cancer Research UK (CRUK; C19212/A16628; C19212/A911376), the National Institute for Health Research Cambridge Biomedical Research Centre and the School of Clinical Medicine at the University of Cambridge, the CRUK and Engineering and Physical Sciences Research Council (EPSRC) Cancer Imaging Centre in Cambridge and Manchester. E.M.S. is a recipient of funding from the European Union Seventh Framework Programme (FP7/2007-2013) under the Marie Curie Initial Training Network METAFLUX and has support from the Calouste Gulbenkian Foundation, Champalimaud Foundation, Ministerio de Saude and Fundacao para a Ciencia e Tecnologia, Portugal.This is the author accepted manuscript. The final version is available from American Association for Cancer Research via http://dx.doi.org/10.1158/0008-5472.CAN-15-085

New strategies for the introduction of 18F into peptides for imaging with Positron Emission Tomography

PET is a potent imaging technique that uses positron emitting radionuclides such as 18F, which is used either in the form of [18F]FDG or attached to another biomolecule for diagnosis of disease and monitoring of treatment. The major disadvantage of labelling molecules with 18F is their time-consuming, multiple step preparation. This thesis presents two different novel strategies that both aim to decrease the number of steps required to prepare l8F labelled peptides. The first strategy involved using the metal ruthenium as a binding site for 18F in a one-step labelling technology involving halide anion exchange. The second approach involved a chemoselective reaction between the hydrazine functionalised compounds HYBA or HYNIC and a labelled aromatic aldehyde to form a hydrazone. In the second strategy, a peptide is synthesised with a HYBA or HYNIC molecule attached to it. Following this, F labelled benzaldehyde is reacted with the peptide to give a 1 + 1 labelling strategy. Before using this methodology for labelling with l8F, the chemoselectivity of the hydrazone formation was thoroughly tested by conducting competition reactions. The competition reactions involved a reaction between an aromatic aldehyde and HYBA in the presence of a competing amine. The reaction was found to be selective between the aldehyde and HYBA, even with increased equivalents of the competing amine present in the reaction mixture. Rate monitoring reactions were carried out in the presence of the competing amine, benzylamine, to see how quickly the hydrazone product formed at room temperature and at 50°C. The formation of the hydrazone was found to be quick and clean, even at room temperature. Amino acid synthons, two containing HYBA and two containing HYNIC, were synthesised to be incorporated into the peptide ‘nanogastrin’ for chemoselective labelling. These hydrazine containing peptides were then to be radiolabelled with [18F]fluorobenzaldehyde. Early indications are that this approach to radiolabelling works as theorised

New strategies for the introduction of 18F into peptides for imaging with Positron Emission Tomography

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Four one-act plays: Double demon,

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