Introduction In the past several years there has been a growing interest in the development of radiopharmaceuticals labeled with metallic radionuclides (Anderson et al. 1999). Of particular interest is the positron emitter Cu-64 (t½ = 12.7 h) for molecular imaging of small molecules as well as peptides and antibodies (Smith 2004). This has led us to the recent implementation of a solid target production facility using commercially available target irradiation station and chemistry modules. Routine production of Cu-64 was achieved with an average production yield of 0.32 mCi/μAh, however purification of Cu-64 has proven to be problematic; with several metallic contaminants compromising subsequent radiolabeling. We report in this work, the step by step procedure which led us to the successful production of low metal contaminant 64Cu with high specific activity and high labeling efficiency. Material and Methods Detailed implementation of our solid target was reported earlier (Poniger et al. 2012). A Nirta Solid Target from IBA was coupled to our 18/9 cyclotron using a 2-meter external beam line. A pneumatic solid target transfer system (STTS) designed by TEMA was use to deliver the irradiated target disks to a dedicated hotcell. Modules from IBA (Pinctada metal) were used for electroplating 64Ni onto a Ag disk and for acid dissolution and purification of the irradiated target. Typical irradiation parameters were 14.9 MeV at 35 μA for 5–6 hours with 64Ni plating’s ranging from 10–60 μm thickness at 6–12 mm. Radionuclidic purities were evaluated by gamma spectroscopy and traces of metallic impurities were determined by ICP-MS or ICP-AES. Labeling efficiency was evaluated by measuring the amount of 64Cu uptake per 20 μg of scFv-cage. Results and Conclusion Initial 64Cu purifications following the manufacturers recommended method resulted in high levels of Cu, Fe and Zn metal contaminants (see TABLE 1, ID 1). Note that little Ag contamination is observed nevertheless the 64Ni is plated directly on a Ag disk. After several productions, visual inspection of the module quickly revealed that the heater block used for heating the back of the Ag target disk was heavily corroded. Replacing the copper heater block with a PEEK heater block drastically reduced the levels of Cu and Fe contaminants. Unfortunately unusually high levels of Zn were still observed regardless of the stringent conditions and ultrapure reagents used during the processing (see TABLE 1, ID 5). In our quest for answers, ICP-MS analysis of the 64Ni plating solution as well as critical stock reagents such as Milli-Q water (18 MΩ cm−1) and 30% HCl TraceSelect Ultra (Sigma) was performed (see TABLE 1, ID 2,3,4). The results were surprising, with high level of Zn found not only in the 64Ni plating solution, but as well in the HCl TraceSelect Ultra. It was hypothesized that the Pinctada’s glass bottles (Kay, 2004) used to store the reagents, especially concentrated acidic solutions were the source of Zn contamination and all glass bottles were replaced by LDPE or PFA types. Our hypothesis was confirmed by subsequent ICP-MS analysis of fresh samples of HCl TraceSelect Ultra and the 64Ni plating solution prepared/stored in plastic containers (see TABLE 1, ID 6,7). We also confirmed by ICP-MS analysis that no contamination occurred when performing a non-radioactive dissolution/purification sequence on the Pinctada module using a blank PTFE target disk in conjunction with the change to plastic reagent storage bottles (see TABLE 1, ID 8). Initially the purification protocol was modified as described by Ometakova et al., 2012 to help reduce the co-elution of Zn contaminants with the 64Cu from the AG1-X8 resin. This change resulted in a significant amount of 64Cu eluting from the resin during the resin washing steps, so that protocol was abandoned and the protocol as described by Thieme et al., 2012 was adopted. By modifying the AG1-X8 resin washing protocol to this new method and eluting the 64Cu from with 0.1M HCl rather than Milli-Q water (see TABLE 1, ID 9), we were able to further reduce metal contaminants, especially Zn. During the course of these experiments, the true specific activity of 64Cu increased from as low as 12 mCi/μmol of Cu (n = 2, TABLE 1, ID 1) to 649 mCi/μmol of Cu (n = 7, TABLE 1, ID 5) and finally to 4412 mCi/μmol of Cu (n = 3, TABLE 1, ID 9). In the same time, the effective specific activity increased from 0.03 ± 0.02 mCi per 20 μg of scFv-cage, to 3.7 ± 0.3 mCi per 20 g of scFv-cage with 64Cu. In conclusion, a significant reduction in Cu, Fe and Zn contaminants was achieved when processing 64Cu using the Pinctada module: i) after replacement of the Cu heater block; ii) after elimination of glass reagent storage containers from the Pinctada module and procedures during preparation of the 64Ni plating solution and iii) after implementation of a new purification protocol (Thieme et al. 2012). Introduction of a 6M HCl wash-up cycle of the module prior to the dissolution procedure was also effective. However in recent 64Cu productions slightly elevated Ag levels have been observed and are under investigation (see TABLE 1, ID 9).

Introduction Copper-64 is an emerging radionuclide with applications in PET molecular imaging and/or internal therapy and it is typically produced by proton irradiation of isotopically enriched 64Ni electrodeposited on a suitable backing substrate. We recently reported a simple and efficient method for the preparation of nickel targets from electrolytic solutions of nickel chloride and boric acid [1]. Herein we report our recent research work on the analysis of metallic impurities in the copper-fraction of the radiochemical separation process. Material and Methods Nickel targets were prepared and processed as previously reported [1]. Briefly, the bath solution was composed of a mixture of natural NiCl2. 6H2O (135 mg/ml) and H3BO3 (15 mg/ml) and Ni was electrodeposited using a gold disk as cathode and a platinum wire as anode. The plating process was carried out at room temperature using 2 ml of bath solution (pH = 3.7) and a constant current density of 60 mA/cm2 for 1 hour. The unirradiated Ni targets were dissolved in 1–2 ml of concentrated (10M) HCl at 90 oC. After complete dissolution of the Ni layer, water was added to dilute the acid to 6M, and the solution was transferred onto a chromatographic column containing AG 1-X8 resin equilibrated with 6M HCl. The Ni , Co and Cu isotopes were separated by using the well-known chromatography of the chloro-complexes. The sample-fractions containing the Cu isotopes (15 ml, 0.1M HCl) were collected in plastic centrifuge tubes previously soaked in 1M HNO3 and rinsed with Milli-Q water (18 MΩ cm). Impurities of B, Co, Ni, Cu and Zn in these samples were determined by inductively coupled plasma-mass spectroscopy (ICP-MS) at the Department of Geosciences (Laboratory of Isotopic Studies) of the National University. Results and Conclusions The mass of Ni deposited in 1 h was 25.0 ± 1.0 mg (n = 3) and the current efficiency was > 75 % in all cases. The pH of the electrolytic solution tended to decrease along the electrodeposition process (3.71.6). The results of ICP-MS analysis of the Cu-fractions from the cold chromatography separation runs are shown in FIG. 1. We were particularly interested in the boron impurities as H3BO3 is used as buffer for electrodeposition of the Ni targets. Except for the Ni impurities that were deter-mined to be in the range of ppm (mg/l), all other analyzed metallic impurities were found to be in the range of ppb (µg/l), including boron. The Co, Ni, Cu and Zn impurities determined in the Cu-fraction in this work using Ni targets electrode-posited from a NiCl2 acidic solution, are in the same order of magnitude compared with that obtained when using targets prepared from an alkaline solution [2], with the advantage of the simplicity of the electrodeposition method from NiCl2 solutions, as the target material is already recovered in the chemical form of NiCl2, enabling a simpler, one step process to prepare a new plating solution when using enriched 64Ni target material for the production of 64Cu.

Introduction In the past several years there has been a growing interest in the development of radiopharmaceuticals labeled with metallic radionuclides (Anderson et al. 1999). Of particular interest is the positron emitter Cu-64 (t½ = 12.7 h) for molecular imaging of small molecules as well as peptides and antibodies (Smith 2004). This has led us to the recent implementation of a solid target production facility using commercially available target irradiation station and chemistry modules. Routine production of Cu-64 was achieved with an average production yield of 0.32 mCi/μAh, however purification of Cu-64 has proven to be problematic; with several metallic contaminants compromising subsequent radiolabeling. We report in this work, the step by step procedure which led us to the successful production of low metal contaminant 64Cu with high specific activity and high labeling efficiency. Material and Methods Detailed implementation of our solid target was reported earlier (Poniger et al. 2012). A Nirta Solid Target from IBA was coupled to our 18/9 cyclotron using a 2-meter external beam line. A pneumatic solid target transfer system (STTS) designed by TEMA was use to deliver the irradiated target disks to a dedicated hotcell. Modules from IBA (Pinctada metal) were used for electroplating 64Ni onto a Ag disk and for acid dissolution and purification of the irradiated target. Typical irradiation parameters were 14.9 MeV at 35 μA for 5–6 hours with 64Ni plating’s ranging from 10–60 μm thickness at 6–12 mm. Radionuclidic purities were evaluated by gamma spectroscopy and traces of metallic impurities were determined by ICP-MS or ICP-AES. Labeling efficiency was evaluated by measuring the amount of 64Cu uptake per 20 μg of scFv-cage. Results and Conclusion Initial 64Cu purifications following the manufacturers recommended method resulted in high levels of Cu, Fe and Zn metal contaminants (see TABLE 1, ID 1). Note that little Ag contamination is observed nevertheless the 64Ni is plated directly on a Ag disk. After several productions, visual inspection of the module quickly revealed that the heater block used for heating the back of the Ag target disk was heavily corroded. Replacing the copper heater block with a PEEK heater block drastically reduced the levels of Cu and Fe contaminants. Unfortunately unusually high levels of Zn were still observed regardless of the stringent conditions and ultrapure reagents used during the processing (see TABLE 1, ID 5). In our quest for answers, ICP-MS analysis of the 64Ni plating solution as well as critical stock reagents such as Milli-Q water (18 MΩ cm−1) and 30% HCl TraceSelect Ultra (Sigma) was performed (see TABLE 1, ID 2,3,4). The results were surprising, with high level of Zn found not only in the 64Ni plating solution, but as well in the HCl TraceSelect Ultra. It was hypothesized that the Pinctada’s glass bottles (Kay, 2004) used to store the reagents, especially concentrated acidic solutions were the source of Zn contamination and all glass bottles were replaced by LDPE or PFA types. Our hypothesis was confirmed by subsequent ICP-MS analysis of fresh samples of HCl TraceSelect Ultra and the 64Ni plating solution prepared/stored in plastic containers (see TABLE 1, ID 6,7). We also confirmed by ICP-MS analysis that no contamination occurred when performing a non-radioactive dissolution/purification sequence on the Pinctada module using a blank PTFE target disk in conjunction with the change to plastic reagent storage bottles (see TABLE 1, ID 8). Initially the purification protocol was modified as described by Ometakova et al., 2012 to help reduce the co-elution of Zn contaminants with the 64Cu from the AG1-X8 resin. This change resulted in a significant amount of 64Cu eluting from the resin during the resin washing steps, so that protocol was abandoned and the protocol as described by Thieme et al., 2012 was adopted. By modifying the AG1-X8 resin washing protocol to this new method and eluting the 64Cu from with 0.1M HCl rather than Milli-Q water (see TABLE 1, ID 9), we were able to further reduce metal contaminants, especially Zn. During the course of these experiments, the true specific activity of 64Cu increased from as low as 12 mCi/μmol of Cu (n = 2, TABLE 1, ID 1) to 649 mCi/μmol of Cu (n = 7, TABLE 1, ID 5) and finally to 4412 mCi/μmol of Cu (n = 3, TABLE 1, ID 9). In the same time, the effective specific activity increased from 0.03 ± 0.02 mCi per 20 μg of scFv-cage, to 3.7 ± 0.3 mCi per 20 g of scFv-cage with 64Cu. In conclusion, a significant reduction in Cu, Fe and Zn contaminants was achieved when processing 64Cu using the Pinctada module: i) after replacement of the Cu heater block; ii) after elimination of glass reagent storage containers from the Pinctada module and procedures during preparation of the 64Ni plating solution and iii) after implementation of a new purification protocol (Thieme et al. 2012). Introduction of a 6M HCl wash-up cycle of the module prior to the dissolution procedure was also effective. However in recent 64Cu productions slightly elevated Ag levels have been observed and are under investigation (see TABLE 1, ID 9).

Introduction Copper-64 is an emerging radionuclide with applications in PET molecular imaging and/or internal therapy and it is typically produced by proton irradiation of isotopically enriched 64Ni electrodeposited on a suitable backing substrate. We recently reported a simple and efficient method for the preparation of nickel targets from electrolytic solutions of nickel chloride and boric acid [1]. Herein we report our recent research work on the analysis of metallic impurities in the copper-fraction of the radiochemical separation process. Material and Methods Nickel targets were prepared and processed as previously reported [1]. Briefly, the bath solution was composed of a mixture of natural NiCl2. 6H2O (135 mg/ml) and H3BO3 (15 mg/ml) and Ni was electrodeposited using a gold disk as cathode and a platinum wire as anode. The plating process was carried out at room temperature using 2 ml of bath solution (pH = 3.7) and a constant current density of 60 mA/cm2 for 1 hour. The unirradiated Ni targets were dissolved in 1–2 ml of concentrated (10M) HCl at 90 oC. After complete dissolution of the Ni layer, water was added to dilute the acid to 6M, and the solution was transferred onto a chromatographic column containing AG 1-X8 resin equilibrated with 6M HCl. The Ni , Co and Cu isotopes were separated by using the well-known chromatography of the chloro-complexes. The sample-fractions containing the Cu isotopes (15 ml, 0.1M HCl) were collected in plastic centrifuge tubes previously soaked in 1M HNO3 and rinsed with Milli-Q water (18 MΩ cm). Impurities of B, Co, Ni, Cu and Zn in these samples were determined by inductively coupled plasma-mass spectroscopy (ICP-MS) at the Department of Geosciences (Laboratory of Isotopic Studies) of the National University. Results and Conclusions The mass of Ni deposited in 1 h was 25.0 ± 1.0 mg (n = 3) and the current efficiency was > 75 % in all cases. The pH of the electrolytic solution tended to decrease along the electrodeposition process (3.71.6). The results of ICP-MS analysis of the Cu-fractions from the cold chromatography separation runs are shown in FIG. 1. We were particularly interested in the boron impurities as H3BO3 is used as buffer for electrodeposition of the Ni targets. Except for the Ni impurities that were deter-mined to be in the range of ppm (mg/l), all other analyzed metallic impurities were found to be in the range of ppb (µg/l), including boron. The Co, Ni, Cu and Zn impurities determined in the Cu-fraction in this work using Ni targets electrode-posited from a NiCl2 acidic solution, are in the same order of magnitude compared with that obtained when using targets prepared from an alkaline solution [2], with the advantage of the simplicity of the electrodeposition method from NiCl2 solutions, as the target material is already recovered in the chemical form of NiCl2, enabling a simpler, one step process to prepare a new plating solution when using enriched 64Ni target material for the production of 64Cu.

Les polyazacycloalcanes sont largement utilisés pour l’élaboration de radiopharmaceutiques destinés à la médecine nucléaire. Ces structures, et plus particulièrement celles dérivées du cyclam, permettent une complexation idéale du cuivre et ainsi une application en imagerie TEP, avec l’utilisation du 64Cu, ou en radiothérapie grâce à l’isotope 67Cu. Le cyclam doit, en plus d’être N-fonctionnalisé par des bras coordinants, disposer d’une fonction supplémentaire permettant la bioconjugaison à une biomolécule pour un ciblage spécifique des cellules cancéreuses. Une première partie de cette thèse a porté sur la synthèse du cyclam monopicolinate C-fonctionnalisé par une fonction de bioconjugaison de type benzyle isothiocyanate. Cette synthèse, basée sur des travaux antérieurs du laboratoire, a nécessité la mise au point d’une méthode d’alkylation régiospécifique du cyclam C-fonctionnalisé par le biais de protections sélectives des atomes d’azote du macrocycle. Le ligand a ensuite été étudié in vitro et in vivo, par nos collaborateurs nantais du CRCINA, pour l’imagerie immuno-TEP du myélome multiple.La seconde partie de ce travail s’est consacrée à l’élaboration d’un dérivé polyfonctionnel du cyclam possédant deux fonctions permettant le ciblage des cellules tumorales. Ce composé a été synthétisé au sein du laboratoire brestois puis étudié, in vitro et in vivo, dans les locaux de la NECSA en Afrique duSud pour l’imagerie TEP du cancer du sein.Ces deux projets ont permis d’obtenir une preuve de concept en imagerie TEP ce qui confirme le potentieldes ligands dérivés de cyclam C-fonctionnalisés pour l’élaboration de radiopharmaceutiques à base de cuivre pour la médecine nucléaire. / Polyazacycloalkanes are wildly used in the conception of radiopharmaceuticals for nuclear medicine. These structures, and especially cyclam derivatives, provide ideal complexation properties of copper, which can be applied in nuclear medicine applications with the 64Cu isotope for PET imaging or with 67Cu for radiotherapy purpose. Cyclams derivatives have to be N-functionalized with coordinative arms, and moreover include an additional function especially introduced for the bioconjugation of a biomolecule in the aim to preferentially target cancer cells.The first project treated in this manuscript consisted of the synthesis of a monopicolinate cyclam C-functionalized with a benzyl isothiocyanate function for the bioconjugation. Based on precedent results obtained in the Lab, a regiospecific alkylation method has been developed for the synthesis of this ligand.This method implies the selective protection and deprotection of the macrocycle nitrogen atoms. This ligand, once obtained, has been studied in vitro and in vivo, by our collaborators of the CRCINA in Nantes, for multiple myeloma immuno-PET imaging.The second project of this work is dedicated to the conception of a radiopharmaceutical based on apolyfunctionnal cyclam which bear two different moieties allowing the targeting of cancer cells. This ligand has been synthesized in our Lab in Brest and studied, in vitro and in vivo, in the South African NECSA company for breast cancer PET imaging.These two projects were elaborated in the aim to obtain a proof of principle in PET imaging and to confirm the high potential of C-funcitonnalized cyclam derivatives for nuclear medicine applications.

O Laboratório de Metrologia Nuclear (LMN) do Instituto de Pesquisas Energéticas e Nucleares (IPEN) realizou a padronização primária dos radionuclídeos 64Cu e 166Ho. Esses radioisótopos são utilizados na Medicina Nuclear em investigações diagnósticas e em radioterapia. A atividade absoluta foi determinada utilizando o método de coincidências 4πβ(PC)- γ associado à técnica de extrapolação de eficiência. Para isto, foram utilizados um contador proporcional em geometria 4π acoplado ao um contador de cintilação NaI (Tl). Para a padronização do 64Cu foram utilizados dois sistemas para registro de dados: sistema TAC e sistema SCS. No sistema TAC é utilizado o módulo Time to Amplitude Converter (TAC) associado a um analisador multicanal, sendo que a eficiência do detector 4πPC foi variada por meio de absorvedores externos e foi aplicado fator de correção para as eficiências beta denominado fator Kawada. No sistema SCS (Software Coincidence System) o registro dos eventos foi feito por meio da digitalização dos pulsos, a eficiência do detector 4πPC foi variada por discriminação eletrônica e a atividade foi obtida pela extrapolação biparamétrica. A padronização do 166Ho em sistema de coincidências 4πβ(PC)- γ foi realizada utilizando o sistema de aquisição de dados por software, SCS (Software Coincidence System). Para determinação das probabilidade de emissão gama dos radionuclídeos foi utilizado um espectrômetro gama HPGe calibrado com ampolas padrão de 152Eu, 60Co, 133Ba, 137Cs e 241Am, calibradas no sistema primário 4πβ(PC)- γ. A probabilidade de emissão gama por decaimento do fóton de 1345,7 keV do 64Cu obtida foi de 0,472 (10) % apresentando um bom acordo com os dados da literatura. A probabilidade de emissão gama por decaimento do fóton gama de 1379,45 keV do 166Ho foi de 0,936 (4) %. Por meio destas medidas foi possível também a determinação das intensidades relativas de outros fótons emitidos por este radionuclídeo. Os resultados obtidos foram concordantes com a literatura dentro da incerteza experimental. / The procedure followed by the Nuclear Metrology Laboratory (LMN) at the Nuclear and Energy Research Institute (IPEN) for the primary standardization of 64Cu and 166Ho is described. These radionuclides are widely applied to Nuclear Medicine such as in diagnostic investigations and in radiotherapy. The absolute activity of both radionuclides were determined by the efficiency extrapolation method applied to 4πβ(PC)-γ coincidence counting system using a proportional counter in 4π geometry coupled with NaI(Tl) scintillation counter. The primary standardisation of 64Cu for the conventional coincidence counting, the events were registered using a Time to Amplitude Converter (TAC) system associated with a multi-channel analyzer and by a software coincidence counting system, SCS, a digital method for primary activity measurements that simplifies the setting of optimal coincidence parameters. For perfoming the standardization of 64Cu in the TAC system, the efficiency of the 4πβ(PC)-γ detector was varied using external absorbers and in order to correct the beta efficiency, the Kawada factor was applied. In the SCS counting, the events were registered by means of pulse digitalization and the efficiencies (εEC and εβ) were varied through eletronic discrimination. The biparametric method was used for determining the absolute activity and the result was in good agreement with the ones obtained with TAC system. The standardization of 166Ho in the primary system was performed using the software coincidence counting, SCS. The gamma-ray emission probability per decay of the main transitions of both radionuclides were determined using the results obtained from the primary method, the 4πβ(PC)-γ coincidence system, associated with a calibrated HPGe gamma-ray spectrometer with standard ampoules of 152Eu, 60Co, 133Ba, 137Cs e 241Am, previously standardized in the 4πβ(PC)-γ system. The gamma-ray emission probability per decay of the 1345,7 keV transition of 64Cu was 0,472 (10) %, in good agreement with the literature. The gamma-ray emission probability per decay of the 1379,45 keV transition of 166Ho was 0,936 (4) %. It were also determined the relative gamma ray emmission probability of other gamma rays for this nuclide . All the results obtained were in good agreement with the literature.

Wilson's disease is a hereditary disorder of copper metabolism, which causes copper accumulation in organism, especially in the liver, kidneys and brain. Current treatment is based on using low-molecular weight copper chelators and high doses of zinc salts. Unfortunately, they can induce some severe side effects due to systemic action. The aim of this thesis is to improve the treatment of Wilson's disease by using of polymeric drug delivery systems. The size of polymer particles in tens of microns should provide non-resorbability of the drug after oral administration. Synthetic microparticles of poly(glycidyl methacrylate-co- ethylene dimethacrylate), natural microcrystalline cellulose and cross-linked chitosan were used as polymer matrices. N,N-di(2-pyridylmethyl)amine, triethylenetetraamine and 8-hydroxyquinoline were selected as specific copper chelators, which can complex copper cations with high efficiency. The principle of the proposed treatment is that the polymeric carrier-bound chelator complex copper directly from the food in digestive tract of the organism. Because of non-resorbability, the entire complex should be eliminated from the body together with stools. This virtually eliminates systemic side effects. The ability of adsorption of copper and the stability of polymer complex under...