Antes de se iniciar os estudos clínicos de uma nova droga, é necessário realizar uma bateria de testes de segurança, para avaliar o risco humano. Os radiofármacos como qualquer outra nova droga, devem ser testados levando em conta sua especificidade, duração de tratamento e principalmente a toxicidade de ambas as partes, a molécula não marcada e a sua radioatividade em si, além das impurezas provindas da radiólise. Órgãos regulatórios como o Food and Drug Administration-EUA (FDA) e a Agência de Medicina Européia (EMEA), estabelecem guias para a regulamentação de produção e pesquisas de radiofármacos, No Brasil a produção de radiofármacos não era regulamentada até o final de 2009, quando foram estabelecidas pela Agência Nacional de Vigilância Sanitária (ANVISA) as resoluções nº 63, que visa as Boas Práticas de Fabricação de Radiofármacos e a nº 64 que visa o registro do radiofámaco. Para a obtenção do registro de radiofármacos são necessárias a comprovação da qualidade, segurança, eficácia e especificidade do medicamento. Para a segurança dos radiofármacos devem ser apresentados estudos de toxicidade aguda, subaguda e crônica como também a toxicidade reprodutiva, mutagênica e carcinogênica. Hoje o IPEN-CNEN/SP produz um dos radiofámacos mais importantes da medicina nuclear, o 18F-FDG, que é utilizado em muitas aplicações clínicas, em particular no diagnóstico e estadiamento de tumores. O objetivo deste trabalho foi avaliar a toxicidade sistêmica (aguda/subaguda) do radiofármaco 18F- FDG em um sistema teste in vivo, conforme preconiza a RDC nº 64, que servirá de modelo para os protocolos de toxicidade dos radiofármacos produzidos no IPEN. Os ensaios realizados foram: os testes de toxicidade aguda e de toxicidade subaguda, estudos de biodistribuição do 18F-FDG, ensaio cometa e toxicidade reprodutiva. Na toxicidade aguda, ratos sadios foram injetados com 18F- FDG e observados durante 14 dias enquanto na toxicidade subaguda os animais foram observados durante 28 dias. Os resultados não mostraram nenhuma evidência de toxicidade na exposição ao 18F-FDG na toxicidade aguda e na subaguda. A biodistribuição demonstrou resultados semelhantes aos da literatura, onde a bexiga é o órgão que mais recebe radiação. O ensaio cometa mostrou que a radiação do radiofármaco não foi significativa para gerar danos no DNA. Na toxicidade reprodutiva, casais de ratos expostos ao 18F-FDG geraram filhotes completamente normais e saudáveis. Por fim, o 18F-FDG não evidenciou nenhuma toxicidade. / Before starting clinical trials of a new drug, it is necessary to perform a battery of safety tests for assessing human risk. Radiopharmaceuticals like any new drug must be tested taking into account its specificity, duration of treatment and especially the toxicity of both parties, the unlabeled molecule and its radionuclide, apart from impurities emanating from radiolysis. Regulatory agencies like the Food and Drug Administration - USA (FDA) and the European Medicine Agency (EMEA), establish guidelines for the regulation of production and research of radiopharmaceuticals. In Brazil the production of radiopharmaceuticals was not regulated until the end of 2009, when were established by the National Agency for Sanitary Surveillance (ANVISA) resolutions No. 63, which refers to the Good Manufacturing Practices of Radiopharmaceuticals and No. 64 which seeks the registration of record radiopharmaceuticals. To obtain registration of radiopharmaceuticals are necessary to prove the quality, safety, efficacy and specificity of the drug . For the safety of radiopharmaceuticals must be presented studies of acute toxicity, subacute and chronic toxicity as well as reproductive, mutagenic and carcinogenic. Nowadays IPEN-CNEN/SP produces one of the most important radiopharmaceutical of nuclear medicine, the 18F-FDG, which is used in many clinical applications, particularly in the diagnosis and staging of tumors. The objective of this study was to evaluate the systemic toxicity (acute/ subacute) radiopharmaceutical 18F-FDG in an in vivo test system, as recommended by the RDC No. 64, which will serve as a model for protocols toxicity of radiopharmaceuticals produced at IPEN . The following tests were performed: tests of acute and subacute toxicity, biodistribution studies of 18F-FDG, comet assay and reproductive toxicity. In acute toxicity, healthy rats were injected with 18F-FDG and observed for 14 days while in subacute toxicity animals were observed for 28 days. The results showed no evidence of toxicity at exposure 18F-FDG in acute and subacute toxicity. The biodistribution showed similar results to the literature, where the bladder is the organ that receives the most radiation. The comet assay showed that the radiation from the radiopharmaceutical was not significant to generate DNA damage. In reproductive toxicity in coupled rats exposed to 18F-FDG generated completely normal and healthy puppies. Finally, the 18F-FDG did not show any toxicity.

Introduction The increasing demand for [18F]FDG for clinical PET-CT and the efficiencies associated with large production runs have encouraged endeavors to increase the amount of 18F− produced by cyclotrons in a single run. The amount of 18F− is determined by the saturation yield of the nuclear reaction, the irradiation time and the beam current striking the target. The saturation yield is a function of beam energy (typically fixed for PET cyclotrons), the enrichment of the H218O (typically > 97 %) and the efficiency of the target design. Target design has already been optimized on current systems. Diminishing gains in activity are achieved by extending the irradiation time much beyond 3 hrs, so the main focus has been to increase beam current onto the targets. Increasing the beam current requires: i) a cyclotron capable of producing the increased beam current; ii) targets that tolerate the beam current without appreciable loss in saturation yield; iii) sufficient shielding of the cyclotron and hot cells to accommodate the proportionally larger radiation dose rates during higher current irradiation and from the larger activities delivered to the hot cells. We reported [1] that the self-shielded targets fitted to our cyclotron can accommodate 100 µA currents without appreciable loss in saturation yield. We also identified the potential of routine production at 200 A (100 A per target in dual target irradiation mode), but had not establish its long-term viability in routine use. We present our experience in using 200 µA for routine production of 18F- since September 2012. Material and Methods Our PETtrace cyclotron was installed in 2002 and has been used for routine production of various 18F and 11C tracers since January 2003. It has been upgraded incrementally so that it is now equivalent to a current generation PETtrace 880 cyclotron, which is specified at a total beam current of 130 µA. The only components on our cyclotron currently not part of the standard PETtrace 880 cyclotron configuration are the self-shielded targets and a license which allows total target beam current of 200 µA. The self-shielded targets utilize a W/Cu alloy for the main body of the target surrounding the Havar foil to provide shielding from the Havar foil by a factor of about 10 and shielding of any remnant 18F- activity in the targets by a factor of about 100 [1]. The niobium target chamber is the same size as used in the standard GE Nb25 targets. However, it dispenses with the He cooling and the vacuum foil. Only the water foil is used, which is directly exposed to the vacuum in the chamber. Foil cooling is through the water in the target chamber. One of the issues that we previously identified [1] is beam stripping by gas molecules in the vacuum tank. The amount of beam that is stripped and which impacts on components in the cyclotron is proportional to the beam current. At high currents, this can result in a runaway condition, where the effects of the stripped beam deteriorate vacuum; this then results in more beam stripping and more severe effects. The effect of diffusion pump maintenance on vacuum system performance and on the reduction of beam stripping was investigated as part of this study. We have previously found that running the ion source gas at a low flow rate (2 sccm) when cyclotron is not used greatly reduces deterioration of ion source performance over time and with use [1]. This gas flow also appears to have a beneficial effect on the vacuum. Ion source gas flow when cyclotron is off has been employed throughout the evaluation period. [18F]FDG was produced with TRACERlab MXFDG modules or FASTlab modules using both Phosphate and Citrate cassettes. Stability studies of [18F]FDG were performed to ensure it met specifications over the specified expiry time. Our current stabilization regime did not have to be adjusted for the higher activities produced with the higher beam currents. [18F]FDG yields were calculated using input activity estimates from saturation yield and beam time and current and the non-decay corrected [18F]FDG activity measured at the end of synthesis. Thus yield calculations include target yield variations and losses in the transfer lines and not just synthesis yield. Results and Conclusion The flip-in probe to extraction foil transmissions as a function of ion source gas flow are given in TABLE 1. Transmission decreases with increasing ion source gas flow, as expected for a system with an internal ion source. In addition, diffusion pump maintenance had a positive impact on the transmission and this is of particular benefit at the higher beam currents where minimising beam stripping becomes more critical. The ion source output, however, decreases with decreasing ion source gas flow; hence ion source gas flow is a compromise between ion source output and probe to foil transmission. We currently use a gas flow of 5.5 sccm for our 200 µA runs. Over the period from 1st September 2012 to end of March 2014, a total of 419 [18F]FDG produc-tions were performed at total target beam currents ranging from 160 µA to 200 µA, with 227 production runs being performed at 200 µA. Beam times were typically 90 to 120 min, with some productions up to 180 min. The [18F]FDG yields are summarized in TABLE 2. The yields for the FASTlab phosphate and citrate cassettes have been listed separately in TABLE 2 as they are known to be different [2,3]. The yields obtained with the TRACERlab MXFDG are also shown. The yields at 200 µA total target current are not appreciably different from those at < 200 µA current, irrespective of the synthesis method. Consistency of yield is also not adversely impacted by the higher beam current. For a 180 min, 200 µA test production, the [18F]FDG activity produced using the FASTlab phosphate cassette was 763 GBq (20.6 Ci). Clinical productions with the FASTlab phosphate were limited to 130 min maximum beam time for 200 µA and achieved a maximum [18F]FDG activity of 656 GBq (17.7 Ci). The tolerance to a reduction in performance of the critical components to achieve high current operation (RF, ion source output and vacuum system) is reduced at high beam currents. The requirements for routine maintenance of ion source, targets and extraction system, however, have not increased with the increase in beam current from 160 µA to 200 µA. Extraction foil life and ion source maintenance intervals have remained at about 2000 Ah and >120 µAh, respectively. As more experience has been gained with the self-shielded targets, service interval is actually being extended from about 10,000 µAh to 20,000 µAh, despite the higher beam currents. Diffusion pump maintenance is currently recommended every 5 years, but a 2 year maintenance interval may be advantageous for 200 µA, given the observed deterioration over a 5 year period and the improvement in performance post service (Table 1). The more frequent service is associated with the additional costs of diffusion pump oil and an extra day of scheduled down-time. Typically, vacuum is sufficiently well established 24 h after opening of the vacuum tank to run 200 µA beams with the vacuum and beam conditioning that we employ. The targets generally have coped well with the 100 µA per target current (200 µA total beam current for dual target irradiation) over this 18 month period. However, currents of 80 µA to µ100 A per target in dual target irradiation mode reduce the tolerance to sudden increases in one of the target currents. There were 4 occasions (2 test beams and 2 production beams) when there were sudden increases of target current from 90 µA and 100 µA to about 150 µA. The rapid increase in heat deposited on the foil and target chamber and the resultant rapid pressure rise in the target chamber could not be withstood by the foil and target foil rupture ensued. This compared to 1 target foil issue over a similar period of time (18 months) at lower beam currents on the standard Nb25 target. Three separate causes were identified for these overshoots in target current: 1) behavior of control system when beam is allowed to continue past the set time; 2) large changes of set current of one of the two targets irradiated during a dual irradiation test beam and 3) an issue with DEE voltage regulation caused by the mechanical flap controls. These issues have been addressed by procedural changes (issues 1 and 2) and by fitting an available upgrade of the mechanical flap control mechanism (issue 3). The two target foil ruptures during production did not cause cancellation or delays to patient scanning, as the demand could be met by multi-ple productions and deliveries from the unaf-fected target. No unscheduled down-days occurred during the evaluation period. We have been able to achieve routine operation at 200 µA beam current through careful optimization of the critical components and parameters and a maintenance regime that we have detailed previously [1]. This maintenance scheme has not changed for the routine 200 µA operation. The safety margin, however, is reduced and so careful monitoring of the system is required to ensure that issues in one of the subsystems do not cause major events such as target foil ruptures. Our [18F]FDG yields have been maintained at the higher current and 200 µA allows large quantities of [18F]FDG to be produced routinely in a single run with relatively short beam times.

Introduction Activity measurement of the produced radionuclide prior its transport to further processing in the clean rooms indicates proper irradiation settings and target functioning. It is particularly true for shortlived radionuclides. Precise online activity measurement of the radionuclides transported from the target to the hot cells in a liquid phase was highly desirable in order to estimate compliance with the required value. In this paper, we present simple operational systems for activity measurement of the irradiated enriched (18O) water for 18F labelled PET radiopharmaceuticals and 81Rb aqueous solution for manufacturing radionuclide generator 81Rb/81mKr. Material and Methods Irradiated aqueous solution (2.5 ml of enriched water with 18F up to 200 GBq) is transported via capillary to a synthesis module. Due to spreading out the liquid product on measuring vial walls, measured activity may vary up to 12 %. In order to avoid this variability, we have introduced simple system based on the measurement of several loops of the transporting capillary. The product is then evenly distributed around GM tube positioned in the loops’ centre. Typical GM tube response is displayed on FIG. 1. The data are recorded and processed online. Maximum mean value of 20 consecutive values is calculated. The GM tube response was calibrated by precise activity measurement of the same product in a calibrated ionizing chamber (Atomlab). Calibration covers full range of the produced 18F activities. Radionuclide 81Rb for the 81Rb/81mKr generator is produced via proton irradiation of pressurized enriched 82Kr gas. The product deposited on the target walls is washed out by water and transported to a container in a hot cell for filtration before transfer to a clean lab. The solution activity in the container (7–25 GBq) is measured with a GM tube in constant geometry. Typical response of the GM tube to the measured activity of 81Rb is displayed on FIG. 2. For activity determination, the mean value of 200 consecutive readouts starting from the 120th readout following maximum is used. The calibration for the whole range of the produced activities was performed via precise measurement of the cumulative 81Rb activity concentration by standard γ-spectrometry using HPGe detector. Results and Conclusion A simple operational system for online activity measurement of 18F and 81Rb in aqueous solutions using GM tube was designed, calibrated and implemented. Long term statistics show that the measured activities do not differ from the values obtained on calibrated ionizing chamber (18F) or γ-spectrometer (81Rb) for more than ± 2.5 %. The method seems to be cheap and rapid for reliable estimate of the produced activities online.

Introduction In order to address the increasing demand for Fluorine-18 and the rising cost per mL of 18O enriched water, IBA developed improvements to their 18F- production systems. For this new design we started from scratch, with the main objectives of reducing the required enriched water volume and improving the cooling of the insert. A better cooling allows increasing the target current and thus the produced activity. Finally, we aimed to reduce the number of parts and improve the design of auxiliary components. Material and Methods Six Niobium conical inserts with different target chamber volumes were machined and tested. Only 4 of these were selected to create the new range of IBA 18F− targets shown in TABLE 1. The new Niobium target inserts have a complex shape with drilled channels on the outside of the chamber and a deep channel next to the beam strike area (FIG. 1, green circle) to ensure efficient cooling. The 18O water inlet lines are now directly inserted in the Niobium body (FIG. 1, blue circle) to improve 18F- quality (no more contact with small o-rings). In operation, a 35µm Havar® target window is used. All tests were performed using IBA Cyclone® 18 cyclotron. The targets were filled with different volumes of enriched 18O water (enrichment > 92 %) and irradiated with 18 MeV protons on target with beam currents up to 145 μA for 30 to 150 minutes, while the internal pressure rise of the target was recorded. For each target, a pressure-current curve was plotted and an optimum balance between target water fill volume, pressure and current has been determined, which maximises available activity after two hours, in each case. Results and Conclusion Radionuclidic impurities were measured and more than 100 FDG syntheses on various synthesizers confirmed the effectiveness of the new design. Increasing the current up to 145µA in Conical 16, the production reached 18 Ci in 2 hours, single beam, with a target pressure under 43 bar. Today, the use of these new targets for daily commercial production is increasing within the IBA Cyclone® installed base.

Antes de se iniciar os estudos clínicos de uma nova droga, é necessário realizar uma bateria de testes de segurança, para avaliar o risco humano. Os radiofármacos como qualquer outra nova droga, devem ser testados levando em conta sua especificidade, duração de tratamento e principalmente a toxicidade de ambas as partes, a molécula não marcada e a sua radioatividade em si, além das impurezas provindas da radiólise. Órgãos regulatórios como o Food and Drug Administration-EUA (FDA) e a Agência de Medicina Européia (EMEA), estabelecem guias para a regulamentação de produção e pesquisas de radiofármacos, No Brasil a produção de radiofármacos não era regulamentada até o final de 2009, quando foram estabelecidas pela Agência Nacional de Vigilância Sanitária (ANVISA) as resoluções nº 63, que visa as Boas Práticas de Fabricação de Radiofármacos e a nº 64 que visa o registro do radiofámaco. Para a obtenção do registro de radiofármacos são necessárias a comprovação da qualidade, segurança, eficácia e especificidade do medicamento. Para a segurança dos radiofármacos devem ser apresentados estudos de toxicidade aguda, subaguda e crônica como também a toxicidade reprodutiva, mutagênica e carcinogênica. Hoje o IPEN-CNEN/SP produz um dos radiofámacos mais importantes da medicina nuclear, o 18F-FDG, que é utilizado em muitas aplicações clínicas, em particular no diagnóstico e estadiamento de tumores. O objetivo deste trabalho foi avaliar a toxicidade sistêmica (aguda/subaguda) do radiofármaco 18F- FDG em um sistema teste in vivo, conforme preconiza a RDC nº 64, que servirá de modelo para os protocolos de toxicidade dos radiofármacos produzidos no IPEN. Os ensaios realizados foram: os testes de toxicidade aguda e de toxicidade subaguda, estudos de biodistribuição do 18F-FDG, ensaio cometa e toxicidade reprodutiva. Na toxicidade aguda, ratos sadios foram injetados com 18F- FDG e observados durante 14 dias enquanto na toxicidade subaguda os animais foram observados durante 28 dias. Os resultados não mostraram nenhuma evidência de toxicidade na exposição ao 18F-FDG na toxicidade aguda e na subaguda. A biodistribuição demonstrou resultados semelhantes aos da literatura, onde a bexiga é o órgão que mais recebe radiação. O ensaio cometa mostrou que a radiação do radiofármaco não foi significativa para gerar danos no DNA. Na toxicidade reprodutiva, casais de ratos expostos ao 18F-FDG geraram filhotes completamente normais e saudáveis. Por fim, o 18F-FDG não evidenciou nenhuma toxicidade. / Before starting clinical trials of a new drug, it is necessary to perform a battery of safety tests for assessing human risk. Radiopharmaceuticals like any new drug must be tested taking into account its specificity, duration of treatment and especially the toxicity of both parties, the unlabeled molecule and its radionuclide, apart from impurities emanating from radiolysis. Regulatory agencies like the Food and Drug Administration - USA (FDA) and the European Medicine Agency (EMEA), establish guidelines for the regulation of production and research of radiopharmaceuticals. In Brazil the production of radiopharmaceuticals was not regulated until the end of 2009, when were established by the National Agency for Sanitary Surveillance (ANVISA) resolutions No. 63, which refers to the Good Manufacturing Practices of Radiopharmaceuticals and No. 64 which seeks the registration of record radiopharmaceuticals. To obtain registration of radiopharmaceuticals are necessary to prove the quality, safety, efficacy and specificity of the drug . For the safety of radiopharmaceuticals must be presented studies of acute toxicity, subacute and chronic toxicity as well as reproductive, mutagenic and carcinogenic. Nowadays IPEN-CNEN/SP produces one of the most important radiopharmaceutical of nuclear medicine, the 18F-FDG, which is used in many clinical applications, particularly in the diagnosis and staging of tumors. The objective of this study was to evaluate the systemic toxicity (acute/ subacute) radiopharmaceutical 18F-FDG in an in vivo test system, as recommended by the RDC No. 64, which will serve as a model for protocols toxicity of radiopharmaceuticals produced at IPEN . The following tests were performed: tests of acute and subacute toxicity, biodistribution studies of 18F-FDG, comet assay and reproductive toxicity. In acute toxicity, healthy rats were injected with 18F-FDG and observed for 14 days while in subacute toxicity animals were observed for 28 days. The results showed no evidence of toxicity at exposure 18F-FDG in acute and subacute toxicity. The biodistribution showed similar results to the literature, where the bladder is the organ that receives the most radiation. The comet assay showed that the radiation from the radiopharmaceutical was not significant to generate DNA damage. In reproductive toxicity in coupled rats exposed to 18F-FDG generated completely normal and healthy puppies. Finally, the 18F-FDG did not show any toxicity.

This thesis has focussed on ¹⁸F-fluorination reactions activated by an organomediator, with the aim of broadening the scope of metal-free reactions in ¹⁸F-radiosynthesis. <b>Chapter 1</b> provides an introduction to positron emission tomography (PET) and ¹⁸Fradiochemistry, including radioisotope production and modes of activation in ¹⁸Fradiosynthesis. In <b>Chapter 2</b>, the concept of chirality and its relevance in the context of radiotracer design is introduced. The previously disconnected fields of organomediated asymmetric fluorination and ¹⁸F-radiosynthesis are merged for the first time via investigation of three distinct activation modes: chiral non-racemic secondary amine-mediated asymmetric &alpha;-¹⁸F-fluorination of aldehydes employing [¹⁸F]N-fluorobenzenesulfonimide; use of a phase transfer reagent for asymmetric ¹⁸F-fluorocyclisation and application of a chiral nonracemic ¹⁸F-fluorinating agent, chiral [¹⁸F]Selectfluor bis-triflate. Application of the first of these approaches to the radiosynthesis of the PET tracer (2S,4S)-4-[¹⁸F]fluoroglutamic acid with high d.r. is described. <b>Chapter 3</b> explores the use of hypervalent iodine reagents to mediate the oxidative fluorination of N-arylsulfonamides with nucleophilic fluoride via an umpolung approach. Preliminary studies on translation to radiosynthesis with [¹⁸F]fluoride are also disclosed. In <b>Chapter 4</b>, experimental data is provided for all compounds, as well as analytical and chiral HPLC traces for ¹⁸F-reactions.

Two α-amino acids labelled with 18F, 2-[18F]fluoro-L-tyrosine and 6-[18F]fluoro-L-dopa, are routinely produced at the CRC for PET clinical investigation in oncology and neurology. The first one is a tracer for the in vivo quantitative assessment of cerebral protein synthesis (tumor seeking agent) and the second one is a tracer for in vivo cerebral studies of presynaptic dopaminergic functions. Routine production of these two α-amino acids has been conducted in our laboratory, using a nucleophilic, multistep radiosynthesis approach. The no-carrier-added (nca) [18F]fluoride, which is available in large amounts from proton irradiation of 18O-enriched water in a cyclotron, allows the labelling of benzaldehyde derivatives by the substitution of a good leaving group. The synthesis of [18F]fluorobenzyl bromide derivative by reduction and bromination with gaseous HBr followed by the enantioselective alkylation under Phase Transfer Catalysis (PTC) of a benzophenone imine provides the protected radiopharmaceutical which is hydrolysed and purified by HPLC. Due to the fact that gaseous HBr is quite cumbersome to handle and that its complete automation is difficult, the first goal of our work is to evaluate new methods for the synthesis of the [18F]fluorobenzyl bromide derivatives starting from the labelled benzaldehyde derivatives. In order to propose a radiochemical reaction which can be easily automated, we have evaluated different reactions on solid (and non solid) supported reagents. Five reagents were tested. N-piperidinoaminomethylpolystyrene hydrobromide resin gives the the [18F]fluorobenzyl bromide derivatives as unique products in good yields. In the second part of our work, a new approach for the labelling of peptides from the key intermediate p-[18F]fluorobenzyl bromide has been evaluated using the concept of click chemistry. 1,3-dipolar cycloaddition provides fast access to a large number of five-membered heterocycles. We have used this cycloaddition to label protected p-ethynyl-L-phenylalanine with p-[18F]fluorobenzyl azide. This interesting labelling method will be applied to the labelling of a peptide containing this α-amino acid residue.

This thesis focuses on the development of a novel nucleophilic ¹⁸F-fluorination of (hetero)arenes and of a rapid screening experiment to facilitate the application of this reaction to complex heterocyclic targets of medicinal importance. <strong>Chapter 1</strong> introduces the use of molecules labelled with fluorine-18 as tracers in positron emission tomography and reviews methods for the preparation of [¹⁸F]fluoroarenes published prior to the start of the work in this thesis. <strong>Chapter 2</strong> describes the development of a novel method for the preparation of electronically-diverse [¹⁸F]fluoroarenes from aryl boronic esters and [¹⁸F]fluoride, mediated by a copper complex. Application of this ¹⁸F-fluorodeboronation to electron-rich radiotracers is demonstrated. Methods for the preparation of [¹⁸F]fluoroarenes published after the start of the work in this thesis are reviewed. <strong>Chapter 3</strong> outlines a rapid screening experiment for assessing the tolerance of the ¹⁸F-fluorodeboronation towards heterocycles, and the use of this method to guide the retro-radiosynthesis of heterocycle-rich, medicinally relevant molecules. <strong>Chapter 4</strong> contains synthetic procedures and characterisation data for compounds in Chapters 2 and 3.

No description available.

Antisense oligodeoxynucleotides (asODNs) show strong binding and high selectivity and can be constructed to recognize specific cellular targets such as gene regulated mRNA. Radiolabelled asODNs have the potential to image gene expression through mRNA targeting and could be a valuable tool in the early assessment of outcome to cancer treatment. We have explored the potential of in vivo imaging of p21 gene expression, using fluorine-18 labelled asODNs ([18F]asODNs) and in vitro techniques, recognizing the relationship between the expression of this gene and resistance of cancer cells to radiation therapy. Radiolabelling of fully phosphorothioated, 20-mer ODNs was performed using the [18F]-labelled prosthetic group, 4-N-[18F]fluorobenzyl-2-bromoacetamide ([18F]FBBA). [18F]FBBA was first synthesized in an automated synthesis unit, resulting in a modest radiochemical yield. Methods to improve the yield were investigated using a metal catalyst-assisted borohydride exchange resin. Alkylation of [18F]FBBA to ODN resulted in radiochemical yields of 40%. Cellular uptake and retention studies were performed in human carcinoma cells expressing p21+/+ (HCT116) and the p21 knock-out cell line, 80S4, using both [18F]-labelled antisense and random sequence ODNs. Nonradioactive FBBA-labelled ODNs were used to evaluate the antisense effectiveness and distribution of the FBBA-modified ODNs. In vitro studies demonstrated that FBBA did not interfere with the antisense effect of ODNs against p21 mRNA; however, the probes required a transfection agent to observe an antisense effect. Cell fractionation studies with [18F]ODNs revealed increasing accumulation of liposome-transfected [18F]asODN in the cytoplasm of HCT116 cells over time. A biocompatible spermine-grafted block copolymer (SP) was subsequently evaluated as a potential vector to improve the delivery of [18F]asODN into cells. SP was shown to direct [F]-labelled ODNs to the cytoplasm, whereas naked [F]ODNs remained sequestered in vesicles, and liposome-transfected [F]ODNs localized mostly in the nucleus. Selective uptake and retention of [18F]asODN was observed in p21+/+ cells only when the probe was transfected with SP. Based on these studies, it can be concluded that [18F]asODNs have the potential to image gene expression, however the focus may need to be directed to find an appropriate vector which can rapidly deliver [18F]-labelled asODNs to the target tissue in vivo.