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Chapter One introduces molecular irnaging and the modalities available for oncological irnaging. The radioisotopes and imaging agents for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) are discussed together with the bifunctional chelator approach for radio labelling of biomolecules. Finally, the chemistry and radioisotopes of copper are described, and copper bis(thiosemicarbazonato) complexes introduced in the context of PET irnaging. Chapter Two describes the synthesis and characterisation of novel carboxylate- and maleirnide- functionalised bis(thiosemicarbazonates) and their conjugation to biologically active molecules. Radiolabelling of a chelator-bombesin conjugate demonstrated site-specific labelling at room temperature and preliminary in vitro and in vivo studies confirmed its potential as an imaging agent. Bioconjugation to a model protein and subsequent radiolabelling was also investigated. Chapter Three introduces molecular irnaging of hypoxia with a focus on CuATSM. An overview of the currently accepted mechanism of hypoxia selectivity is presented. The emphasis is placed on the relationship between oxygenation status, uptake and retention which display cell- and tumour- line dependency. Chapter Four presents the synthesis of copper bis(thiosemicarbazonates), radiolabelled either at the metal (64CU) or at the ligand e8F or 123n for mechanistic studies. The physicochemical characteristics of the copper complexes were measured and the complexes evaluated for their in vitro hypoxia selectivity. Chapter Five describes in vitro and in vivo studies of the orthogonally radiolabelled complexes, inclusive of control experiments with [64Cu]CuATS~, the radiolabelled proligand and [64CU]CU2+ salts. In vitro cellular assays, as well as in vivo biodistribution studies including dynamic PET and SPECT were performed. Stability studies contrasting the in vitro and in vivo behaviour were carried out. The collective data suggest that the currently proposed redox trapping mechanism might not provide a full understanding of the factors governing biodistribution and tumour uptake. Chapter Six contains full experimental details for the work described in this thesis.

This study investigated several somatostatin analogues labeled with copper-64 for imaging and targeted therapy of SSTr positive cancer. Among three new cross-bridged bifunctional chelators coupled to Y3-TATE, 64Cu-CB-TE2A-Y3-TATE had the most favorable tumor targeting properties. The introduction of ionizable linker groups could not remedy the slow clearance from the kidney, and other modifications will be necessary to resolve this issue. The emerging idea of using the copper-64-labeled somatostatin antagonist 64Cu-CB-TE2A-sst2-ANT as a tumor targeting agent will require further experimentation. This radiopharmaceutical showed promising initial results in a biodistribution study in male Lewis rats, however, it should be compared to 111In-DOTA-sst2-ANT in the same model. Nuclear localization of copper-64 from two somatostatin analogues differing in their chelate stability strengthened the hypothesis of copper-64 dissociation from the bifunctional chelator prior to trafficking to the nucleus. However, the increased nuclear uptake of copper-64 from the less stable 64Cu-TETA-Y3-TATE did not result in a significant effect on cell killing of A427-7 cells. In experiments with [64Cu]copper acetate and the EGFR-antibody 64Cu-DOTA-cetuximab, the tumor suppressor protein p53 was identified as a mediator of the nuclear transport of copper. 64Cu-DOTA-cetuximab was also utilized in five cervical cancer cell lines with a wide range of EGFR expression. EGFR quantification by saturation receptor binding, and EGFR function as determined via internalization of 64Cu-DOTA-cetuximab closely followed the expression pattern of these cell lines found via EGFR mRNA profiling. This constitutes a first step in the evaluation of cetuximab for the treatment, and of 64Cu-DOTA-cetuximab for the imaging of advanced cervical cancer, as EGFR expression on the tumor cell surface clearly can be quantified and visualized with this experimental system. Copper-64 has been used in this study to probe the basic biochemical process of intracellular copper trafficking, and for the targeting of cell surface receptors via radiolabeled peptides and antibodies, providing an example of the powerful combination of radiopharmaceutical chemistry and cell biology.

Le travail de recherche présenté dans ce mémoire, concerne la synthèse et l'étude des propriétés de coordination du 64Cu par une nouvelle famille de ligands acycliques de type polyaminophosphonatés à fort potentiel en imagerie médicale, en particulier pour la tomographie par émission de positron TEP. Ce sujet a nécessité l'utilisation de méthodes analytiques telles que la potentiométrie, la spectrophotométrie et la spectrométrie de masse en mode electrospray pour les études thermodynamiques et des techniques de cinétique rapide a écoulement bloqué pour la détermination des vitesses de formation et de dissociation des complexes de Cu(II).Nous avons synthétisé quatre ligands (L1-L4) qui forment des complexes de formule CuL de forte stabilité et avec une bonne sélectivité pour le Cu(II) par rapport à d'autres métaux tels que Ni(II), Zn(II), Co(II) et Ga(III). La cinétique de complexation de Cu(II) s'est avérée rapide et elle suit un mécanisme à caractère dissociatif de type Eigen-Wilkins. Les complexes CuL formés avec cette série de ligand se caractérisent par une bonne inertie cinétique vis-à-vis de la démétallation et ils sont capables d'être réduit en présence de réducteurs biologiques tout en restant stables.Les complexes de lanthanides formés avec cette série de ligands sont très stables en milieu physiologique, en particulier ceux formés avec le ligand L1. Les complexes GdL1, GdL2 et GdL3 révèlent des relaxivités très intéressantes qui les désignent comme de futurs agents de contraste à fort potentiel en IRM.Le ligand L1 tetraphosphonaté a été fonctionnalisé par la présence de groupement réactif ce qui a permit le marquage de biomolécules, dont l'anticorps B28-13 anticorps anti-tenascineC pour la détection de tumeurs cancéreuses. / The research work presented in this report, concerns the synthesis and the study of the coordination properties of the 64Cu by new acyclic chelators for PET imaging.We have synthesized four ligands (L1-L4) based on a pyridine scaffold, functionnalized by a varying number of methanephosphonate functions. A combination of potentiometry, spectrophotometry and electrospay mass spectrometry was used to study the coordination properties of Cu(II) with this ligands. The kinetic studies of the formation and dissociation of Cu(II) complexes have been carried out with stopped-flow techniques.L1 was shown to display a very high affinity towards Cu(II) and selectivity over Ni(II), Zn(II), Co(II) and Ga(III). The chelation of the Cu(II) by the four ligands is fast and efficient and it follows a dissociative Eigen-Wilkins mechanism.The complexes of lanthanides formed with this series of ligands are very stable in physiological conditions in particular those formed with L1. The complexes GdL1, GdL2 and GdL3 show a very high relaxivity which pointed them as potential contrast agents for MRI.The ligand L1 was activated by a side chain containing a reactive NHS carbamate function in the para position of the pyridine. The labeling was further performed on B28-13, a mouse monoclonal antibody specifically recognizing tenascin-C protein in human cancer.

Les pathologies modifiant l’intégrité de la matrice cartilagineuse (arthrose, arthrite, pathologies des disques intervertébraux, chondrosarcome…) sont caractérisées par un diagnostic tardif dû à l’absence d’un marqueur spécifique du cartilage. Notre unité de recherche développe depuis quelques années un radiotraceur à base de 99mTc capable de se lier aux protéoglycanes, un des constituants essentiels du cartilage. Son utilisation en tomographie par émission monophotonique (TEMP) a permis de mettre en évidence sa capacité à cibler spécifiquement les protéoglycanes du cartilage. Les travaux présentés dans cette thèse s’inscrivent dans la continuité de cette étude. De nouveaux radiotraceurs à base de cuivre-64 ont été synthétisés avec pour objectif de réaliser une imagerie fonctionnelle et quantifiable du cartilage en tomographie par émission de positons (TEP). La structure de base de ces radiotraceurs combine une partie vectrice ciblant spécifiquement les protéoglycanes du cartilage (fonction ammonium quaternaire) et une partie chélatante affine de l’ion cuivrique (polyazamacrocycle). Nos études préliminaires montrent que ces radiotraceurs pourraient permettre un diagnostic précoce et un suivi longitudinal des pathologies du cartilage. / Pathologies altering the integrity of the cartilaginous matrix (osteoarthritis, arthritis, intervertebral discs diseases, chondrosarcoma…) are characterized by a delayed diagnosis due to the absence of a specific marker of cartilage. Since several years, our research unit developed a radiotracer based on 99mTc able of binding proteoglycans, one of the essential constituents of the cartilage. Its use in single photon emission tomography (SPECT) had enabled to demonstrate its ability to specifically target the cartilage proteoglycans. Works reported in this thesis are a follow-up to this study. New radiotracers based on copper-64 were synthesized with the aim to achieve functional and quantifiable cartilage imaging in positron emission tomography (PET). The basic structure of these radiotracers combines a carrier function specifically targeting cartilage proteoglycan (quaternary ammonium function) and a chelating moiety for cupric ion (polyazamacrocycle). Our preliminary studies show that these radiotracers could allow early diagnosis and a longitudinal follow-up of cartilage diseases.

This study investigated several somatostatin analogues labeled with copper-64 for imaging and targeted therapy of SSTr positive cancer. Among three new cross-bridged bifunctional chelators coupled to Y3-TATE, 64Cu-CB-TE2A-Y3-TATE had the most favorable tumor targeting properties. The introduction of ionizable linker groups could not remedy the slow clearance from the kidney, and other modifications will be necessary to resolve this issue. The emerging idea of using the copper-64-labeled somatostatin antagonist 64Cu-CB-TE2A-sst2-ANT as a tumor targeting agent will require further experimentation. This radiopharmaceutical showed promising initial results in a biodistribution study in male Lewis rats, however, it should be compared to 111In-DOTA-sst2-ANT in the same model. Nuclear localization of copper-64 from two somatostatin analogues differing in their chelate stability strengthened the hypothesis of copper-64 dissociation from the bifunctional chelator prior to trafficking to the nucleus. However, the increased nuclear uptake of copper-64 from the less stable 64Cu-TETA-Y3-TATE did not result in a significant effect on cell killing of A427-7 cells. In experiments with [64Cu]copper acetate and the EGFR-antibody 64Cu-DOTA-cetuximab, the tumor suppressor protein p53 was identified as a mediator of the nuclear transport of copper. 64Cu-DOTA-cetuximab was also utilized in five cervical cancer cell lines with a wide range of EGFR expression. EGFR quantification by saturation receptor binding, and EGFR function as determined via internalization of 64Cu-DOTA-cetuximab closely followed the expression pattern of these cell lines found via EGFR mRNA profiling. This constitutes a first step in the evaluation of cetuximab for the treatment, and of 64Cu-DOTA-cetuximab for the imaging of advanced cervical cancer, as EGFR expression on the tumor cell surface clearly can be quantified and visualized with this experimental system. Copper-64 has been used in this study to probe the basic biochemical process of intracellular copper trafficking, and for the targeting of cell surface receptors via radiolabeled peptides and antibodies, providing an example of the powerful combination of radiopharmaceutical chemistry and cell biology.

Coordination of transition metals to peptides, either through the incorporation of unnatural chelating groups or amino acid ligating side-chains, expands the utility of peptides for biological studies. The first part of this project describes induction of α-helical secondary structure in pentapeptides upon side-chain coordination of inert transition metal ions. The second part of this project describes the syntheses of biologically active peptide species that contain a macrobicyclic hexaamine ligand that can complex radioactive metal ions for diagnostic imaging purposes. / Short peptide sequences do not form thermodynamically stable α-helices in water. The capacity of two metal clips, cis-[Ru(NH3)4(solvent)2]2+ and cis [Pd(en)(solvent)2]2+ to induce α-helicity in peptides that are five amino acids long, Ac HARAH NH2 and Ac MARAM-NH2 has been explored. In all cases at pH < 5, the metal ions bind to the side-chains of amino acid residues at positions i, i+4 of the pentapeptides resulting in formation of bidentate macrocyclic species. Circular dichroism and 1H nuclear magnetic resonance data indicate that the metal complexes of Ac-MARAM-NH2 are highly α helical in water, and in the most spectacular case, coordination of Ac-MARAM-NH2 to cis-[Ru(NH3)4(solvent)2]2+ results in up to 80% α-helicity. In contrast, metal complexes of Ac-HARAH-NH2 exhibit significantly less α-helicity in water. / 64Cu-radiolabelled peptides have been investigated for their ability to target specific tissue or cell types. These peptides require a chelating group that binds copper ions strongly. Macrobicyclic hexaamine ligands, based on the compound commonly referred to as “sarcophagine”, have demonstrated extremely high stability under biological conditions. Here we describe the synthesis of diaminosarcophagine chelators with carboxylate groups for conjugation to peptides. These new chelators have been attached to the N-terminus or lysine side-chain of biologically-active peptides, including Tyr3 octreotate, Lys3-bombesin and an integrin targeting peptide. Spectroscopic and voltammetric studies of these species suggest that the conjugated sarcophagine group retains the high metal binding affinity and structural properties of the parent species, diaminosarcophagine. These are among the first sarcophagine-peptide compounds that have been properly characterised. The new sarcophagine-peptide conjugates can be easily radiolabelled with 64Cu2+ over a wide pH range at ambient temperature.