In light of the increasing interest in the syntheses of fluorocarbohydrates as well as in their radiolabelled analogues for use in positron emission tomography (PET), a two- step synthesis of 2-fluoro-2-deoxy-ß-D-allose (2-FDpA) has been developed. The present synthesis employed electrophilic fluorination of 3,4,6-tri-O-acetyl-D-glucal in anhydrous HF (aHF) solvent using F2 and AcOF and was more rapid and efficient than the existing synthesis of 2-deoxy-2-fluoro-D-allose, with a total synthesis time of approximately 45 min, and less laborious. The synthesis proved to be higly regio- and Stereosective, which is often hard to achieve from electrophilic fluorinations. The synthetic route to 2-FDßA was used to obtain the 18F-Iabelled analogue, 2-[18F]FDßA, for the first time with the anticipation that the labelled compound will be of use as a diagnostic agent for the detection and assessment of different tumours as well as for monitoring D-allose metabolism. The overall decay-corrected radiochemical yields (RCY) of the products resulting from radiofluorination of TAG in aHF with [18F]F2 and [18F]AcOF were 33 ±3% and 9 ±2%, respectively, with respect to [18FJF2. The RCY of 33 ±3% is the highest reported for direct fluorinations of TAG using [18FJF2 in any solvent. The radiochemical purities of 2-[18F]FDßA were 96 ±3% and 91 ±8% as determined by radio-HPLC and radio-TLC, respectively. Preliminary in vivo studies using normal rats showed significant differences between the uptake of 2-[18F]FDßA and 2-[18F]FDG, the most commonly used PET radiotracer for detection of various types of cancers. In addition, an animal study with a Polynoma Middle T mouse showed retention of 2-[18F]FDßA in the tumour. The 18F-Iabelling technique was also used as a mechanistic probe for the synthesis of 2-FDßA in the present study. / Thesis / Master of Science (MSc)

Le travail présenté dans ce manuscrit s’inscrit dans une stratégie de vectorisation d’agents diagnostiques et thérapeutiques, vers les protéoglycanes du chondrosarcome, via une structure chimique de reconnaissance : un ammonium quaternaire. Nous nous sommes intéressés aux deux aspects de la prise en charge du chondrosarcome : l’aspect diagnostique par imagerie scintigraphique et l’aspect thérapeutique. Pour l’imagerie, nous avons validé l’intérêt du radiotraceur 99m Tc-NTP 15-5 dans le diagnostic précoce, le staging et le suivi de la réponse au traitement dans un modèle de chondrosarcome de rat. Ce dernier démontre une accumulation spécifique et rapide au niveau du tissu tumoral, et ce, dès les stades précoces de la pathologie. Actuellement en phase de transfert vers la clinique pour la pathologie de l’arthrose, nos résultats ont permis de proposer une extension de l’application au chondrosarcome. Pour la thérapie, nous avons confirmé, in vitro, que la greffe du vecteur ammonium quaternaire, sur un agent alkylant, le melphalan, ne modifiait pas son activité cytotoxique. In vivo, les résultats obtenus avec le dérivé ammonium quaternaire du melphalan, ont mis en évidence un ralentissement net de la croissance tumorale associée à une diminution considérable du syndrome myélodysplasique associé au traitement. Ces résultats prouvent tout l’intérêt de la vectorisation pour l’amélioration de l’index thérapeutique et nous ont permis de valider la preuve de concept pour la vectorisation d’agent cytotoxique. Une autre approche de thérapie a consisté à greffer le vecteur sur des dérivés du cyclophosphamide clivables en hypoxie. Parmi les composés synthétisés, l’un d’entre eux a montré, in vitro, un différentiel de cytotoxicité hypoxie/normoxie intéressant. Son évaluation in vivo dans le modèle de xénogreffe de chondrosarcome HEMC-SS a démontré une inhibition importante de la croissance tumorale. En nous intéressant à l’environnement hypoxique de la tumeur, nous offrons ainsi une approche encore plus spécifique de la thérapie. L’ensemble de ces travaux offrent un véritable espoir pour le management du chondrosarcome, pathologie certes rare mais néanmoins redoutable. / Our strategy consists in using the quaternary ammonium function, that exhibits a high affinity for proteoglycans of extracellular matrix, as a selective carrier to chondrosarcoma of (i) radioisotopes for a targeted in vivo imaging or (i) cytotoxics for a targeted therapeutic approach. For diagnosis application, a radiotracer radiolabeled with 99m Tc ( 99m Tc-NTP 15-5) was designed and tested in vivo in an orthotopic Swarm chondrosarcoma model. For therapeutic application, a quaternary ammonium derivative of melphalan, Melphalan-AQ, was synthesized and assessed in vitro (cytotoxic activity, cell cycle distribution) and in vivo in the orthotopic Swarm chondrosarcome model (antitumor efficacy, toxicity). Since chondrosarcoma is characterized by hypoxia, research was focused on the vectorization of hypoxia-activated prodrugs. For diagnosis, 99m Tc-NTP 15-5 radiotracer highly accumulated in tumoral tissue at very early stage of pathology while no palpable nor measurable tumor could be assessed. For therapy, chondrosarcoma bearing rats treated with Melphalan-AQ showed a significant tumor growth inhibition with associated heamatological side effects being significantly reduced as compared to Melphalan. Among all phosphoramidate-AQ derivatives synthetised, one was selected on the basis of cytotoxic activity improvement under hypoxia respectively to normoxia. In vivo, this compound demonstrated a significant tumoral growth inhibition in the hypoxic HEMC-SS chondrosarcoma model. This work underlines the potential of the proteoglycans vectorization strategy developed in our lab, for a targeted management of chondrosarcoma including: (i) the first and only radiopharmaceutical able to provide in vivo a specific diagnosis and staging of the tumoral pathology of cartilage and (ii) a promising targeted antineoplastic approach exploiting both the chondrogenic and hypoxic features of chondrosarcoma.

Dentre as diversas aplicações dos radioisótopos, a utilização dos radiotraçadores é considerada uma das mais importantes, no diagnóstico de funcionamento dos equipamentos de processos, em plantas de indústrias químicas e petroquímicas. Os radiotraçadores são utilizados em procedimentos analíticos para obtenção de dados qualitativos e quantitativos de sistemas, em estudos de transferências físicas e físico-químicas. Na produção de radioisótopos gasosos utilizados como traçadores em processos industriais, destacam-se o 41Ar e 79Kr, gases nobres (inertes) que possuem baixa reatividade com os demais elementos químicos. O 41Ar é um emissor gama de alta energia (1,29 MeV) e apresenta elevada porcentagem de transformações com essa energia, o que resulta em quantidades relativamente pequenas necessárias em relação a outras para uma detecção eficaz, mesmo em componentes com grandes espessuras. Atualmente, a produção de radioisótopos gasosos em reatores nucleares de pesquisa é realizada em pequenas quantidades (bateladas), por meio de ampolas de quartzo contendo o gás natural 40Ar ou 78Kr. Nesse sentido, o objetivo desse estudo é desenvolver um sistema de irradiação capaz de produzir em escala contínua, o radioisótopo gasoso 41Ar, dentre outros, com atividade de 7,4x1011 Bq (20 Ci) por ciclo de irradiação, por meio do Reator IEA-R1 de 4,5 MW, fluxo de nêutrons térmicos médio de 4,71 x 1013 ncm-2s-1, para suprir uma demanda existente em empresas de END e inspeções, e pelo próprio Centro de Tecnologia das Radiações, no IPEN/CNEN-SP. O sistema de irradiação (SI) é constituído por uma cápsula de irradiação em alumínio, linhas de transferência, válvulas agulhas, conexões anilhadas, conectores rápidos, manovacuômetro, sistema de vácuo, dewar de liquefação, blindagem em chumbo, cilindros de armazenamento e transporte (CAT), dentre outros. O SI foi aprovado nos testes de estanqueidade e estabilidade (testes de formação de bolhas, pressurização, evacuação e com equipamento leak detector SPECTRON 600 T). Na produção experimental para obtenção de 1,07x1011 Bq (2,9 Ci) de 41Ar, distribuíram-se dosímetros de alanina em diversos componentes e dispositivos do SI. Além disso, determinaram-se as taxas de exposição na parede da blindagem em chumbo, ao concentrar o gás radioativo liquefeito e no CAT, após a transferência do 41Ar, pelo medidor de radiação portátil Teletector ® Probe 6150 AD-t/H. / Among the various applications of radioisotopes, the use of radiotracers is considered the most important in diagnosing operation and troubleshooting of industrial process plants in chemical and petrochemical companies. The radiotracers are used in analytical procedures to obtain qualitative and quantitative data systems, in physical and physicochemical studies transfers. In the production of gaseous radioisotopes used as tracers in industrial process measurements, argon-41 (41Ar) and krypton-79 (79Kr) have low reactivity with other chemical elements. 41Ar is a transmitter range with high-energy (1.29MeV) and a high percentage of this energy transformation (99.1%), resulting in relatively small quantities required in relation to the other, for an efficient detection, even in large thicknesses components. Nowadays, the production of gaseous radioisotopes in nuclear research reactors is performed in small quantities (batches), through quartz ampoules containing natural gas 40Ar or 78Kr. In this sense, the aim of this study is to develop an irradiation system for gaseous radioisotope production in continuous scale, applied in industrial applications of emission tomography and flow measurement. The irradiation system may produce 41Ar with activity of 7.4x1011Bq (20Ci) per irradiation cycle, through the Reactor IEA-R1 with 4.5MW and average thermal neutron flux of 4.71x1013 ncm-2s-1 to meet an existing demand in NDT and inspections companies, and even needed by the Radiation Technology Centre, at IPEN/CNEN-SP. The irradiation system consists of an aluminium irradiation capsule, transfer lines, needle valves, stripy connections, quick connectors, manometer, vacuum system, dewar, lead shielding, storage and transport cylinders, among other components. The irradiation system was approved in the leakage and stability tests (bubble test, pressurization, evacuation and with leak detector equipment SPECTRON 600 T). In the experimental production, alanine dosimeters were distributed into various components of the irradiation system, obtaining 1.07x1011Bq (2.9Ci) of 41Ar. In addition, exposure rates were determined in the lead shielding wall, in which the liquefied radioactive gas was concentrated, and in the storage and transport cylinders after 41Ar was transferred, by the portable radiation meter Teletector ® Probe 6150 AD-t/H.

Dentre as diversas aplicações dos radioisótopos, a utilização dos radiotraçadores é considerada uma das mais importantes, no diagnóstico de funcionamento dos equipamentos de processos, em plantas de indústrias químicas e petroquímicas. Os radiotraçadores são utilizados em procedimentos analíticos para obtenção de dados qualitativos e quantitativos de sistemas, em estudos de transferências físicas e físico-químicas. Na produção de radioisótopos gasosos utilizados como traçadores em processos industriais, destacam-se o 41Ar e 79Kr, gases nobres (inertes) que possuem baixa reatividade com os demais elementos químicos. O 41Ar é um emissor gama de alta energia (1,29 MeV) e apresenta elevada porcentagem de transformações com essa energia, o que resulta em quantidades relativamente pequenas necessárias em relação a outras para uma detecção eficaz, mesmo em componentes com grandes espessuras. Atualmente, a produção de radioisótopos gasosos em reatores nucleares de pesquisa é realizada em pequenas quantidades (bateladas), por meio de ampolas de quartzo contendo o gás natural 40Ar ou 78Kr. Nesse sentido, o objetivo desse estudo é desenvolver um sistema de irradiação capaz de produzir em escala contínua, o radioisótopo gasoso 41Ar, dentre outros, com atividade de 7,4x1011 Bq (20 Ci) por ciclo de irradiação, por meio do Reator IEA-R1 de 4,5 MW, fluxo de nêutrons térmicos médio de 4,71 x 1013 ncm-2s-1, para suprir uma demanda existente em empresas de END e inspeções, e pelo próprio Centro de Tecnologia das Radiações, no IPEN/CNEN-SP. O sistema de irradiação (SI) é constituído por uma cápsula de irradiação em alumínio, linhas de transferência, válvulas agulhas, conexões anilhadas, conectores rápidos, manovacuômetro, sistema de vácuo, dewar de liquefação, blindagem em chumbo, cilindros de armazenamento e transporte (CAT), dentre outros. O SI foi aprovado nos testes de estanqueidade e estabilidade (testes de formação de bolhas, pressurização, evacuação e com equipamento leak detector SPECTRON 600 T). Na produção experimental para obtenção de 1,07x1011 Bq (2,9 Ci) de 41Ar, distribuíram-se dosímetros de alanina em diversos componentes e dispositivos do SI. Além disso, determinaram-se as taxas de exposição na parede da blindagem em chumbo, ao concentrar o gás radioativo liquefeito e no CAT, após a transferência do 41Ar, pelo medidor de radiação portátil Teletector ® Probe 6150 AD-t/H. / Among the various applications of radioisotopes, the use of radiotracers is considered the most important in diagnosing operation and troubleshooting of industrial process plants in chemical and petrochemical companies. The radiotracers are used in analytical procedures to obtain qualitative and quantitative data systems, in physical and physicochemical studies transfers. In the production of gaseous radioisotopes used as tracers in industrial process measurements, argon-41 (41Ar) and krypton-79 (79Kr) have low reactivity with other chemical elements. 41Ar is a transmitter range with high-energy (1.29MeV) and a high percentage of this energy transformation (99.1%), resulting in relatively small quantities required in relation to the other, for an efficient detection, even in large thicknesses components. Nowadays, the production of gaseous radioisotopes in nuclear research reactors is performed in small quantities (batches), through quartz ampoules containing natural gas 40Ar or 78Kr. In this sense, the aim of this study is to develop an irradiation system for gaseous radioisotope production in continuous scale, applied in industrial applications of emission tomography and flow measurement. The irradiation system may produce 41Ar with activity of 7.4x1011Bq (20Ci) per irradiation cycle, through the Reactor IEA-R1 with 4.5MW and average thermal neutron flux of 4.71x1013 ncm-2s-1 to meet an existing demand in NDT and inspections companies, and even needed by the Radiation Technology Centre, at IPEN/CNEN-SP. The irradiation system consists of an aluminium irradiation capsule, transfer lines, needle valves, stripy connections, quick connectors, manometer, vacuum system, dewar, lead shielding, storage and transport cylinders, among other components. The irradiation system was approved in the leakage and stability tests (bubble test, pressurization, evacuation and with leak detector equipment SPECTRON 600 T). In the experimental production, alanine dosimeters were distributed into various components of the irradiation system, obtaining 1.07x1011Bq (2.9Ci) of 41Ar. In addition, exposure rates were determined in the lead shielding wall, in which the liquefied radioactive gas was concentrated, and in the storage and transport cylinders after 41Ar was transferred, by the portable radiation meter Teletector ® Probe 6150 AD-t/H.

Les maladies cardiovasculaires sont la première cause de mortalité dans le monde, et la maladie coronaire est responsable de la majorité de ces décès. L’infarctus du myocarde et l’AVC sont principalement causés par la rupture de plaques d’athérome vulnérables. Malgré les récentes avancées dans la prise en charge des patients, aucun outil non invasif n’est actuellement disponible en pratique clinique pour le diagnostic de ces lésions. Le laboratoire UMR_S1039 Radiopharmaceutiques Biocliniques a développé un agent d’imagerie innovant ciblant la molécule d’adhésion cellulaire VCAM-1, impliquée dans le processus inflammatoire qui caractérise les plaques vulnérables. 99mTc-cAbVCAM1-5 appartient à une nouvelle classe de radiotraceurs dérivés d’anticorps de camélidés possédant uniquement une chaine lourde dont le domaine variable est appelé nanobody. Les lésions athéromateuses aortiques ont été visualisées avec succès par imagerie non invasive (SPECT) chez un modèle de souris déficient pour le gène ApoE. L’objectif de ma thèse a été double : finaliser la caractérisation préclinique de cAbVCAM1-5 et transférer ce radiotraceur vers la clinique. Dans un premier temps, la sensibilité de 99mTc-cAbVCAM1-5 a été démontrée avec succès par l’utilisation de thérapies de référence (statine, ézétimibe). Ces résultats prometteurs nous ont permis d’évaluer le potentiel effet anti-athérogène de nouveaux agents thérapeutiques et d’initier le transfert en clinique de cet agent d’imagerie. cAbVCAM1-5 et l’intermédiaire de réaction utilisé pour son radiomarquage ont été produits selon les bonnes pratiques de fabrication (BPF). Ces deux produits ont été évalués et validés. De plus la méthode de radiomarquage a été optimisée dans la perspective de son transfert vers la clinique. L’étude de toxicité étendue à dose unique est en cours. A l’issue de cette thèse, le laboratoire INSERM U1039 dispose donc de tous les éléments nécessaires pour une demande d’autorisation de phase clinique I/IIa. / Cardiovascular diseases (CVD) are the leading causes of death worldwide, and Coronary Artery Disease (CAD) is responsible for the majority of CVD deaths. Myocardial infarction and stroke are mainly caused by the rupture of vulnerable atherosclerotic plaques. Despite recent advances in treatment and detection of CAD, no non-invasive tool is currently available for the diagnosis of vulnerable plaques. The laboratory UMR_S1039 Radiopharmaceutiques Biocliniques has recently developed a new radiotracer (99mTc-cAbVCAM1-5) targeted to the inflammatory marker Vascular Cell Adhesion Molecule 1 (VCAM-1) expressed in vulnerable plaques. 99mTc-cAbVCAM1-5 belongs to a new class of radiotracers derived from single domain antibodies (sdAb), or nanobodies, that are composed of the single variable domain of the heavy chain antibodies of camelidae. Aortic plaques have been successfully visualized noninvasively by SPECT in an Apolipoprotein E (ApoE) deficient mouse model of atherosclerosis. The objectives of my thesis were to complete the preclinical characterization of cAbVCAM1-5 and to transfer this innovative tool to the clinic. The sensitivity of 99mTc-cAbVCAM1-5 imaging has been successfully demonstrated using gold standard therapies (statin, ezetimibe). Those good results allowed us to employ this imaging agent for the evaluation of new therapeutics and to initiate the clinical translation of cAbVCAM1-5. The productions of cAbVCAM1-5 and of the tricarbonyl employed for its radiolabeling have been performed according to good manufacturing practices (GMP). Both products have been evaluated and validated. Furthermore, the radiolabeling method has been optimized to facilitate its clinical transfer. Single dose toxicity study is currently ongoing. At the end of this PhD fellowship, the U1039 Laboratory therefore possesses all required products and knowledge for the onset of a first in human clinical trial.

Le cancer du sein est la première cause de cancer chez la femme. Au moment du diagnostic, 30 % des patientes présente une maladie métastatique avec atteinte ganglionnaire loco-régionale ou d’organes à distance. De plus, 20 % des femmes diagnostiquées sans métastases récidiveront dans les 5 ans. Les métastases sont traitées par chimiothérapie ou thérapie ciblée, en fonction des résultats histologiques du site primitif. Cependant, 20 à 45 % des métastases ont un phénotype différent de celui du site primitif. Pouvoir phénotyper les différentes métastases permettrait de traiter les patientes plus efficacement, en accord avec les caractéristiques moléculaires de ces lésions. Toutes les métastases n’étant pas accessibles à la biopsie, un phénotypage en imagerie moléculaire serait donc d’un grand intérêt. La médecine nucléaire est, à ce jour, la seule technique d’imagerie moléculaire disponible en pratique clinique. Dans le cadre de cette thèse, l’objectif a été de réaliser l’imagerie phénotypique des tumeurs métastatiques du sein en ciblant la mésothéline et VCAM-1 dans des modèles pré-cliniques de cancer du sein. La première partie de ce travail a été consacrée à l’évaluation pré-clinique de nanobodies ciblant la mésothéline, une glycoprotéine à ancre GPI très faiblement exprimée à l’état physiologique mais surexprimée dans de nombreux cancers dont le cancer du sein. De nombreuses thérapies ciblant la mésothéline sont actuellement en cours d’essais cliniques. Un agent d’imagerie ciblant cette protéine permettrait d’identifier les patientes susceptibles d’en bénéficier. Au cours de ces travaux de thèse, le nanobody 99mTc-A1 a été entièrement validé pour l’imagerie des tumeurs exprimant la mésothéline. La seconde partie de ces travaux de thèse a été consacrée à l’imagerie phénotypique des tumeurs du sein exprimant VCAM-1, protéine impliquée dans la dissémination métastatique pulmonaire et osseuse. Un agent d’imagerie ciblant VCAM-1 pourrait servir d’outil pour la caractérisation de l’agressivité de la maladie métastatique. Au cours de ces travaux, l’utilisation d’un nanobody, 99mTc-cAbVCAM1-5, a été validée pour l’imagerie des tumeurs du sein exprimant VCAM-1. / Breast cancer is the leading cause of cancer among women. At the time of diagnosis, 30 % of patients have developed a metastatic disease either with regional lymph node colonization or distant organs colonization. Metastatic breast cancers are treated by chemotherapy or targeted therapy, according to histological results of primary site. However, 20 to 45 % of metastases have a different phenotype from the primary tumor. Getting access to metastases phenotype would therefore allow treating patients more accurately, in accordance with molecular characteristics of these lesions. Because metastases are not always accessible to biopsy, the use of molecular imaging could be of great interest. Nuclear medicine is the only molecular imaging technic available in clinical practice. The objective of this thesis was to perform the phenotypic imaging of metastatic breast cancer by targeting mesothelin and VCAM-1. The first part of this work was dedicated to the preclinical evaluation of mesothelin-targeting nanobodies. Mesothelin is a GPI-anchored membran protein. While its expression is restricted to a mesothelial cells in normal conditions, mesothelin is overexpressed in several cancers, including breast cancer. Several mesothelin-targeting therapies are currently ongoing clinical transfer. Identifying mesothelin-expressing metastases would allow to select patients who could benefit from those therapies. During this thesis, the nanobody-derived radiotracer 99mTc-A1 has been fully validated for the imaging of mesothelin expressing tumors. The second part of this work was dedicated to the nuclear imaging of VCAM-1 expressing breast cancer lesions. VCAM-1 is a membrane-associated protein involved in the metastatic spread of breast tumor cells. An imaging agent targeting at VCAM-1 could be a suitable tool to evaluate the role of VCAM-1 as a marker of tumor aggressiveness. In the present study, the nanobody 99mTc-cAbVCAM1-5 has been validated for the imaging of VCAM-1 expression in breast cancer.

The dopamine D2 receptor exists in vitro in two states of agonist affinity: a high-affinity state mediating dopamine’s physiological effects, and a physiologically-inert low-affinity state. Our primary goal was to determine the in vivo relevance of this two-affinity-state model for the agonist PET radiotracer [11C]-(+)-PHNO, developed for measurement of the D2 high-affinity state. Our second goal was to characterize the regional D2 versus D3 pharmacology of [3H]-(+)-PHNO binding and assess its utility for measuring drug occupancy at both receptor subtypes. Using ex vivo dual-radiotracer experiments in conscious rats, we showed that, contrary to the two-affinity-state model, the binding of [11C]-(+)-PHNO and the antagonist [3H]-raclopride were indistinguishably inhibited by D2 partial agonist (aripiprazole), indirect agonist (amphetamine) and full agonist ((-)-NPA) pretreatment. Furthermore, ex vivo [11C]-(+)-PHNO binding was unaffected by treatments that increase in vitro high-affinity state density (chronic amphetamine, ethanol-withdrawal), whereas unilateral 6-OHDA lesion, which increases total D2 receptor expression, similarly increased the ex vivo binding of [11C]-(+)-PHNO and [3H]-raclopride. These results do not support the in vivo validity of the two-affinity-state model, suggesting instead a single receptor state for [11C]-(+)-PHNO and [3H]-raclopride in conscious rat. Importantly, we also demonstrated that the increased amphetamine-sensitivity of the agonist radiotracers [11C]-(+)-PHNO and [11C]-(-)-NPA, commonly seen in isoflurane-anaesthetized animals and cited as evidence for the two-affinity-state model, is due to the confounding effects of anaesthesia. Using in vitro and ex vivo autoradiography in rat and the D3 receptor-selective drug SB277011, we found that [3H]-(+)-PHNO binding in striatum and cerebellum lobes 9 and 10 was due exclusively to D2 and D3 receptor binding, respectively, but in other extra-striatal regions to a mix of the two receptor subtypes. Surprisingly, the D3 contribution to [3H]-(+)-PHNO binding was greater ex vivo than in vitro. Also surprising, several antipsychotic drugs, at doses producing 80% D2 occupancy, produced insignificant (olanzapine, risperidone, haloperidol) or small (clozapine, ~35%) D3 occupancy, despite similarly occupying both receptor subtypes in vitro. These data reveal a significant discrepancy between in vitro and ex vivo measures of dopamine receptor binding and suggest that the D3 occupancy is not necessary for the therapeutic effect of antispychotic drugs.

The dopamine D2 receptor exists in vitro in two states of agonist affinity: a high-affinity state mediating dopamine’s physiological effects, and a physiologically-inert low-affinity state. Our primary goal was to determine the in vivo relevance of this two-affinity-state model for the agonist PET radiotracer [11C]-(+)-PHNO, developed for measurement of the D2 high-affinity state. Our second goal was to characterize the regional D2 versus D3 pharmacology of [3H]-(+)-PHNO binding and assess its utility for measuring drug occupancy at both receptor subtypes. Using ex vivo dual-radiotracer experiments in conscious rats, we showed that, contrary to the two-affinity-state model, the binding of [11C]-(+)-PHNO and the antagonist [3H]-raclopride were indistinguishably inhibited by D2 partial agonist (aripiprazole), indirect agonist (amphetamine) and full agonist ((-)-NPA) pretreatment. Furthermore, ex vivo [11C]-(+)-PHNO binding was unaffected by treatments that increase in vitro high-affinity state density (chronic amphetamine, ethanol-withdrawal), whereas unilateral 6-OHDA lesion, which increases total D2 receptor expression, similarly increased the ex vivo binding of [11C]-(+)-PHNO and [3H]-raclopride. These results do not support the in vivo validity of the two-affinity-state model, suggesting instead a single receptor state for [11C]-(+)-PHNO and [3H]-raclopride in conscious rat. Importantly, we also demonstrated that the increased amphetamine-sensitivity of the agonist radiotracers [11C]-(+)-PHNO and [11C]-(-)-NPA, commonly seen in isoflurane-anaesthetized animals and cited as evidence for the two-affinity-state model, is due to the confounding effects of anaesthesia. Using in vitro and ex vivo autoradiography in rat and the D3 receptor-selective drug SB277011, we found that [3H]-(+)-PHNO binding in striatum and cerebellum lobes 9 and 10 was due exclusively to D2 and D3 receptor binding, respectively, but in other extra-striatal regions to a mix of the two receptor subtypes. Surprisingly, the D3 contribution to [3H]-(+)-PHNO binding was greater ex vivo than in vitro. Also surprising, several antipsychotic drugs, at doses producing 80% D2 occupancy, produced insignificant (olanzapine, risperidone, haloperidol) or small (clozapine, ~35%) D3 occupancy, despite similarly occupying both receptor subtypes in vitro. These data reveal a significant discrepancy between in vitro and ex vivo measures of dopamine receptor binding and suggest that the D3 occupancy is not necessary for the therapeutic effect of antispychotic drugs.

La tomographie par émission de positons (TEP) est une technique d'imagerie médicale largement utilisée en oncologie. Cette technique peut, par exemple, fournir des informations sur la localisation de tumeurs dans le corps humain, et le développement de nouvelles méthodes pour la production automatisée de radiotraceurs spécifiques reste un domaine d’actualité. Dans ce contexte, un nouveau type de précurseurs silylés pouvant être fluorés en dernière étape a été développé. La fluoration de ce nouveau motif imidazole-silylé par du [19F]F- puis par du [18F]F- a été validée sur différents bioconjugués. Le greffage de ce motif a ensuite été testé selon plusieurs méthodes. En particulier, une des voies de synthèse a pu montrer que la fluoration de bioconjugués portant ce motif et greffés sur une résine était possible au [19F]F-. Enfin, le produit radiomarqué au [18F]F- a pu être détecté au cours d’essais préliminaires, permettant ainsi de valider la stratégie choisie. / Positron Emission Tomography (PET) is a medical imaging technique widely used in oncology. For example, this technique can provide information on the localization of growing tumors in the human body, and the development of news methods for the automated production of specific radiotracers is still required. In this context, a new silylated precursor, which can be fluorinated in the last step, has been developed. The fluorination of this new imidazole-silylated unit with [19F]F- and with [18F]F- has been validated on various bioconjugates. The grafting of this moiety has been tested following different strategies. In particular, one of the synthetic pathway showed that the fluorination of bioconjugates with resin grafted moiety was possible with [19F]F-. Finally, the [18F] radiolabeled product was observed during preliminary [18F]F- fluorination experiments, thus validating the chosen strategy.

The growing understanding of tumor biology and the identification of tumor specificgenetic and molecular alterations, such as the overexpression of human epidermal growthfactor receptor 2 (HER2), opens up for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools usedto identify patients that are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising non-invasive method to visualize and characterize the expression of such targets. This thesis, based on five papers, is focused on the development of radiolabeled Affibody molecules for imaging of HER2-expression in malignant tumors. Affibody molecules, which represent a rather novel class of affinity proteins developed by combinatorial protein engineering of the protein A derived Z-domain, display manyfeatures that make them promising tracers for molecular imaging applications. The aim of the work presented here was to further develop the tracer format for improved in vivo properties and flexibility in the choice of radionuclide. In paper I, the development of an assay that enables quantitative studies of the internalization rate and cellular processing of high affinity Affibody molecules is described. The assay was applied to a HER2-binding Affibody variant that was efficiently retained by HER2-expressing cells, although characterized by a slow internalization rate. This may have implications for the choice of label for Affibody molecules since high affinity to the target may be equally, or more, important for good imaging quality than residualizing properties of the radiolabel. In paper II, a HER2-binding Affibody molecule and the monoclonal antibody trastuzumab were labeled with positron emitting 124I, for a head-to-head in vivocomparison of the two tracer formats. The effects of tracer size and presence of an Fc region on the biodistribution profile were investigated. In paper III, a HER2-binding Affibody molecule was site-specifically labeled with radiocobalt and evaluated in vitro and in vivo.A head-to-head in vivo comparison with the well-studied 111In-labeled counterpart was performed, revealing promising potential for the cobalt-labeled molecule as a PET-tracerfor visualization of HER2. Paper IV describes the in vitro and in vivo evaluation of a panel of Affibody molecules with different C-terminal peptide-based chelators for the coordination of 99mTc. Even small changes in the C-terminal sequence had appreciable impact on the biodistribution of the Affibody molecules and by optimizing the design of the chelator, the kidney uptake of 99mTc could be significantly reduced. Finally, in paper V we describe the development of a HER2-targeting Affibody variant equipped with a Sel-tag for site-specific labeling with the short-lived positron emitter 11C. This novel Affibody tracer could be used to image HER2-expressing tumors in vivo within one hour after injection. Taken together, Affibody molecules show great promise as targeting tracers for radionuclide molecular imaging of HER2. Careful design and optimization of the tracer protein is important and can be used to improve the biodistribution and targeting properties of Affibody molecules. / QC 20110922