Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T12:24:34Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T12:24:34Z (GMT). No. of bitstreams: 0 / O teste de frequência de micronúcleos (FMN) in vitro é uma das metodologias de escolha no desenvolvimento de testes de segurança toxicológica. Para o seu desenvolvimento no trabalho foram realizadas modificações da técnica convencional, relativas ao substrato de cultivo das células e à sua coloração. As culturas celulares foram desenvolvidas diretamente nas lâminas e a coloração foi realizada com laranja de acridina (AO) ao invés da coloração segundo Giemsa. Foram utilizados controles positivos com potenciais clastogênico (mitomicina C, benzo[a]pireno) e aneugênico (colchicina), recomendados pela OCDE (Organização para Cooperação e Desenvolvimento Econômico). Como moléculas-teste, foram utilizados compostos cuja associação a isótopos radioativos compõem radiofármacos produzidos pelo IPEN. DOTATATO e Ubiquicidina foram testados em diferentes concentrações proporcionais às concentrações máximas utilizadas em pacientes adultos. Para tanto, foram realizadas diluições correspondentes às concentrações 0,1X, 1X e 10X e culturas de células CHO-KI foram expostas a estas concentrações para ensaios de citotoxicidade e FMN. Nenhuma das concentrações induziu citotoxicidade significativa. Para análise de FMN, foram contabilizadas todas as células mononucleadas e multinucleadas até atingir a contagem de 1000 células binucleadas, com ou sem micronúcleos. Desta maneira foi possível analisar a frequência de micronúcleos e o índice de proliferação (CBPI). As concentrações dos fármacos em teste (0,1X, 1X e 10X) não induziram agressão às células. Nenhuma das concentrações revelou citotoxicidade ou genotoxicidade, ou ainda qualquer alteração no ciclo celular em comparação aos controles, comprovando sua segurança conforme os parâmetros exigidos pelas normas internacionais. Os resultados mostraram também uma boa concordância entre a comparação das leituras realizadas por analistas independentes, com pequenas discrepâncias discutíveis, e boa correlação com resultados obtidos com a coloração convencional. Desta maneira as modificações realizadas na técnica de FMN mostraram potencial para cumprir todos os quesitos como teste pré-clínico. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-10-09T14:48:07Z No. of bitstreams: 0 / Made available in DSpace on 2017-10-09T14:48:07Z (GMT). No. of bitstreams: 0 / Os radiofármacos são definidos como preparações farmacêuticas contendo um radionuclídeo em sua composição, são administrados intravenosamente em sua maioria, e, portanto, o cumprimento dos princípios de Boas Práticas de Fabricação (BPF) é essencial e indispensável à tais produtos. A validação de limpeza é um requisito das BPF e consiste na evidência documentada que demonstra que os procedimentos de limpeza removem os resíduos a níveis pré-determinados de aceitação, garantindo que não haja contaminação cruzada. Uma simplificação da validação dos processos de limpeza é admitida, e consiste na escolha de um produto, denominado de \"pior caso\" ou worst case, para representar a limpeza de todos os equipamentos da mesma linha de produção. Uma das etapas da validação de limpeza é o estabelecimento e validação do método analítico para quantificação do resíduo. O objetivo deste estudo foi estabelecer o pior caso para a validação de limpeza dos equipamentos de produção de reagentes liofilizados-RL para marcação com 99mTc, avaliar a utilização do teor de carbono orgânico total (COT) como indicador de limpeza dos equipamentos utilizados na fabricação dos RL, validar o método para determinação de CONP (carbono orgânico não purgável/volátil) e realizar testes de recuperação com o produto escolhido como pior caso. A escolha do produto pior caso baseou-se no cálculo de um índice denominado \"índice para pior caso - Worst Case Index (WCI)\", utilizando informações de solubilidade dos fármacos, dificuldade de limpeza dos equipamentos e taxa de ocupação dos produtos na linha de produção. O produto indicado como pior caso entre os RL foi o MIBI-TEC. Os ensaios de validação do método foram realizados utilizando-se um analisador de carbono modelo TOC-Vwp acoplado a um amostrador automático modelo ASI-V, ambos da marca Shimadzu&reg e controlados por software TOC Control-V Shimadzu&reg. Foi utilizado o método direto de quantificação do CONP. Os parâmetros avaliados na validação do método foram: conformidade do sistema, robustez, linearidade, limites de detecção (LD) e de quantificação (LQ), precisão (repetibilidade e precisão intermediária), e exatidão (recuperação) e foram definidos como: 4% acidificante, 2,5 mL de oxidante, tempo de integração da curva de 4,5 minutos, tempo de sparge de 3,0 minutos e linearidade na faixa de 40-1000 μgL-1, com coeficiente de correlação (r) e soma residual dos mínimos quadrados (r2) > 0,99 respectivamente. LD e LQ para CONP foram 14,25 ppb e 47,52 ppb, respectivamente, repetibilidade entre 0,11 4,47%; a precisão intermediária entre 0,59 a 3,80% e exatidão entre 97,05 - 102,90%. A curva analítica para Mibi mostrou-se linear na faixa de 100-800 μgL-1, com r e r2 > 0,99, apresentando parâmetros similares aos das curvas analíticas de CONP. Os resultados obtidos neste estudo demonstraram que a abordagem do pior caso para validação de limpeza é um meio simples e eficaz para diminuir a complexidade e morosidade do processo de validação, além de proporcionar uma redução nos custos envolvidos nestas atividades. Todos os resultados obtidos nos ensaios de validação de método CONP atenderam as exigências e especificações preconizadas pela norma RE 899/2003 da ANVISA para considerar a metodologia validada. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-11-23T10:55:22Z No. of bitstreams: 0 / Made available in DSpace on 2017-11-23T10:55:22Z (GMT). No. of bitstreams: 0 / O desempenho de uma prática segura e eficiente de um serviço de medicina nuclear depende, entre outros fatores, de um programa de controle de qualidade completo, principalmente em se tratando dos instrumentos medidores de atividade dos radionuclídeos, os ativímetros. Um programa de controle de qualidade completo deve incluir a calibração de todos os instrumentos de medição utilizados no procedimento. No entanto, no Brasil, a atual norma que estabelece os requisitos de proteção radiológica para serviços de medicina nuclear (SMN), não inclui, ainda, a calibração do ativímetro. Considerando que estes instrumentos, por diversas razões, são de difícil remoção para envio a um serviço de calibração, o propósito deste trabalho foi desenvolver uma metodologia de calibração de medidores de atividades que possa ser aplicada \"in situ\", para o principal radiofármaco utilizado atualmente, o 99mTc. Foram definidos os parâmetros de influência que devem ser levados em conta durante a calibração, assim como uma logística de transporte dos radiofármacos. Um programa de controle de qualidade foi aplicado aos ativímetros do Laboratório de Calibração de Instrumento (LCI). Neste trabalho foram desenvolvidas três metodologias diferentes de calibração, considerando a logística disponível e também a origem da fonte de referência. Na primeira metodologia poderá ser aplicada nos casos em que o LCI envia uma fonte de referência ao SMN. Na segunda o SMN envia uma fonte previamente medida ao LCI que determinará sua atividade real. A terceira metodologia foi aplicada para calibração dos ativímetros pertencentes ao setor de produção de radiofármacos do IPEN. Neste caso a fonte de referência foi enviada ao LCI após uma medição prévia pelo setor de produção. Foi possível aplicar as metodologias em alguns instrumentos pertencentes a clínicas e ao setor de produção. Em todos eles foram encontrados coeficientes de calibração diferentes entre si. A maior variação encontrada foi de 5%, indicando que a medição com este ativímetro está menor em 5% do que é necessário aplicar no paciente. Verificou-se que a troca de recipientes deixa um resíduo que não tem sido considerado nas medições clínicas, podendo acrescentar uma diferença de até 3% nas medições. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Title: Environmental Effects of 223 Ra Radiopharmaceuticals Author: Tereza Krmelová Branch: Environmental chemistry Type of thesis: Diploma thesis Advisor: Doc. Ing. Stanislav Smrček, CSc Abstract: In thesis was studied the possibility of extracting the nanoparticles of titanium dioxide or hydroxyapatite with bounded 223 Ra by root system of tested plant species Avena sativa and Zea mays as a model for phytoremediation technologies. The thesis obtains data to assess the potential of residues radioactivity and nanomaterials entering the food chain. There was also verified an experiment of 223 Ra phytoextraction, in the form of nitrate, in effects on addition of EDTA, which was conducted in Bachelor thesis. This experiment was repeated because of its results, which were inconsistent with generally described phytoextraction efficiency improvements after adding the complexing agents. In this work was primary studied translocation of radioactive material from the root to shoot. Experimentally was confirmed the extraction of nanoparticles with bounded 223 Ra and translocation into shoot. In the case of Avena sativa, capturing of nanoparticles hydroxyapatite with bounded 223 Ra was 53 % of which 88 % of activity was recovered in roots and 12 % in shoots. Capturing nanoparticles of titanium dioxide with bounded 223...

Cancer is a leading cause of morbidity and mortality in the developed world. While much has been learned about these diseases in the last few decades, one of the main barriers to widespread advancement is the heterogeneity of cancer biology. A growing body of evidence supports the idea that certain protein receptors are overexpressed on the surface of tumor cells as compared to normal tissues. These extracellular biomarkers provide a unique opportunity to selectively target the tumor with both imaging and therapeutic modalities. The research in this dissertation focuses on targeting proteins on the tumor cell surface with peptidomimetic ligands. Following a description of various extracellular receptors, chapter one discusses targeting ligands designed to specifically and selectively bind these receptors. It reviews recent literature on targeted alpha-particle therapy and ends with an explanation of the advantages of peptide ligands. Three distinct approaches to imaging and therapeutic modalities are then discussed in subsequent chapters. First, a peptide ligand was designed to target radionuclides to malignant melanoma cells in an effort to develop companion radiotherapeutics and diagnostic imaging agents. The second research project describes the synthesis of a novel antagonist peptide ligand with conjugated near infrared dye, and its utility for real-time intraoperative guidance during pancreatic adenocarcinoma resection. Finally, the last chapter describes how the relatively new field of immunomodulatory effectors may be enhanced by their derivatization with peptide targeting ligands.

Metastatic and primary bone cancers are devastating diseases of paediatric and adult humans because of the morbidity associated with bone pain. Controlling bone pain from multiple metastatic sites can be difficult in end-stage cancers using conventional therapies. Bone-seeking radiopharmaceuticals have been successful in this area as radiation can be delivered with moderate selectivity to the target. Unfortunately, targeted radiotherapy using radiopharmaceuticals have been relegated to palliative therapy as myelosuppression largely limits the radiation dose to sub-therapeutic levels. Efforts to overcome this therapeutic limitation include autologous bone marrow transplants in combination with chemotherapy-radiosensitization and the development of new radiopharmaceuticals. Development work using laboratory rodent models has been complicated by dosimetric limitations because of size and inherent problems with human xenografted tumour models in rodents. To address this need we studied naturally occurring canine osteosarcoma as a translational model for human bone cancer. Central to our hypothesis was that naturally occurring canine osteosarcoma would serve as an investigational model for comparing the pharmacokinetics (biodistribution), dosimetry, toxicity, and therapeutic effect of 153Sm-EDTMP, 188Re-HEDP, 186Re-HEDP, and a novel ligand, polyethyleneiminomethyl phosphonic acid (PEI-MP). Data collected from existing radiopharmaceuticals was then compared to PEI-MP labelled with 99mTc, 153Sm, and 186Re. This innovative and unique study allowed for the evaluation of various radiopharmaceuticals in a naturally occurring animal model of bone cancer, documenting the pharmacokinetics and dosimetry of a novel radiolabelled-ligand (PEI-MP). Benefits resulting from the successful completion of the study would allow more rapid transfer of rodent preclinical data into a naturally occurring cancer model more resembling to the human diseases and would thus more likely identify problems with pharmacokinetics and toxicity before proceeding to expensive clinical trials. The expected outcomes of the study were originally formulated based on limited previous published data in dogs. For instance, no data exists describing the pharmacokinetics or toxicity of 188Re-HEDP and 186Re-HEDP in the dog. The study was conducted in two phases. The first phase deals with the evaluation of 153Sm-EDTMP, 188Re-HEDP, and 186Re-HEDP in the dog model. Phase-two was the development of a novel ligand (PEI-MP) in the dog model of osteosarcoma, which has the characteristics of an ideal ligand. Pharmacokinetic results for 153Sm-EDTMP in normal dogs (n=4) for blood were similar to published reports for dogs and human. When compared statistically to human data the majority of results were the same, lending credence to the hypothesis that dogs could serve as models for human radiopharmaceutical research. Normal dogs and the osteosarcoma dog did differ from human pharmacokinetics in the urine elimination phase (t½-â). This can most likely be explained by the tumour burden in the human research populations or due to the fact that most humans were aged and likely to have some renal disease. Certainly, the trend in dogs with osteosarcoma was to have a prolonged urine elimination phase (t½-â) compared to normal dogs which supported the hypothesis that the biodistribution and pharmacokinetics results from dogs were similar to human published data. Statistical comparisons were also made between normal dogs receiving 188Re-HEDP and 153Sm-EDTMP. The prolonged urine elimination phase (t½-â) and increased blood retention of 188Re-HEDP was most likely a reflection of prolonged bone washout and soft issue retention. This could be attributed to the differences between the antiresorptive capability of bisphosphonate ligands e.g., EDTMP (lexidronam) with a greater than 100-fold antiresorptive capability than HEDP (etidronate). Additional observational biodistribution studies using macro- and micro-autoradiography techniques were also performed in canine tissue and Sprague-Dawley rats. Results from the studies showed heterogeneous uptake within tumours in dogs. In rats, localization of 153Sm-EDTMP in red marrow areas would lead to a high radiation dose to blood producing elements. In addition, high uptake was documented at the metaphyseal growth plate confirming the likelihood of a delay or cessation of growth if 153Sm-EDTMP were used in growing children. Phase-one of the clinical trial in dogs with naturally occurring osteosarcoma identified only mild toxicity at the dosage rate of 37 MBq/kg (1 mCi/kg) for both 153Sm-EDTMP and188Re-HEDP. In addition, a pilot trial was conducted in dogs receiving 153Sm-EDTMP which also received a carboplatin infusion at the time of the radiopharmaceutical administration followed by another 3 cycles of carboplatin at 3 weekly intervals. No differences in toxicity were noted between the carboplatin group and dogs receiving only 153Sm-EDTMP. As a part of the 188Re-HEDP clinical trial, 3 dogs with osteosarcomas received weekly dose of 188Re-HEDP at 37MBq/kg for 4 weeks in which only mild toxicity was noted. Unfortunately, there was no cessation in growth of the tumours, with all dogs showing progression. The median survival time for both radiopharmaceuticals was 4 months, significantly shorter than the 10-month median survival time for amputation and chemotherapy. Interestingly six dogs that had 99mTc-MDP and 153Sm-EDTMP showed scans of tumours that had consistently lower 99mTc-MDP uptake ratios (normal bone compared to cancerous bone) compared to solely 153Sm-EDTMP. In contrast, this was not evident for uptake ratios between 188Re-HEDP and 99mTc-MDP scans. Once again, this finding highlights the differences between the antiresorptive capabilities of the bisphosphonates ligands. Interestingly, another three dogs were scanned with 99mTc-MDP , 153Sm-EDTMP, and 99mTc-PEI-MP (10-30 kDa) showed a variation in uptake between scans of the same tumours. More importantly, the uptake ratios of 99mTc-MDP and 153Sm-EDTMP scans showed wide variation with a coefficient of variance of 52% and 39% respectively. However, the range in uptake from the 99mTc-PEI-MP (10-30 kDa) scan was narrow with a coefficient of variance of only 6%. This could be attributed to more consistent uptake ratio of the unique ligand PEI-MP and its hypothesized mechanism of action: enhanced permeability and retention (EPR) in tumour vasculature. This requires further investigation with larger groups. In phase-two, the pharmacokinetic result for the novel ligand PEI-MP was initially studied labelled with 99mTc. Various molecular weights were tested in normal dogs and compared to previously published results in baboons. Results from the dog studies were found to be similar to those from the primate study. As in the primate study, molecular weight and charge played a significant role in 99mTc-PEI-MP pharmacokinetics. Increasing the size of the macromolecules and altering their charge resulted in marked changes in their pharmacokinetics and biodistribution. The PEI-MP molecular weight of 10-30 kDa and 20-30 kDa were the most promising and fulfilled the hypothesized criteria of an ideal radiopharmaceutical. In keeping with the aims of the study, the 20-30kDa polymer was considered more desirable because of its faster clearance. However, because of the limitations imposed by the percentage yield of the different molecular weights of the ligand during filtration, we decided to label the 10-30kDa molecular weight MW-fraction with 153Sm. Unexpectedly, the 153Sm-PEI-MP 10-30 kDa had a prolonged urine elimination phase (t½-â) associated with increased liver uptake when compared to 99mTc-PEI-MP10-30 kDa. To explain this, computer modelling for blood plasma (ECCLES) was done which predicted that there would be some chemical dissociation of the 153Sm from the PEI-MP polymer in blood. This is due to interaction between the radiopharmaceutical and citrate, forming 153Sm-citrate. The ECCLES model for blood plasma also predicted that the anionic MW-fraction, PEI-MP 10-30kDa, would be a poor ligand complexed to 166Ho, 212Pb, 213Pb, and 89Sr, but was expected to be effective when complexed to 186Re or 188Re, based on their close proximity to 99mTc on the periodic table. As a preliminary study 186Re was complexed to 20-30 kDa (n=2) and 30-50 kDa (n=1) MW-fractions and tested in dogs. The results were similar to 99mTc-PEI-MP 10-30 kDa. The biodistribution data and pharmacokinetic data were also used to do comparative dosimetry between radiopharmaceuticals. Not surprisingly, the dosimetry data confirmed the high red marrow dose for 153Sm-EDTMP and the increased soft-tissue dose of the radionuclides complexed to HEDP. The radiation dose to the tumour for all radiopharmaceuticals fell within the range of 26Gy to 44Gy. This is well within the range used to treat canine osteosarcoma using external beam radiotherapy. When compared to external beam radiotherapy, the probable lack of tumour response in our clinical trial relates to the heterogenous distribution of the radiopharmaceutical in the tumour and the inherent resistance of osteosarcoma cells to continuous low-dose radiation delivery (CLDR) inherent in radionuclide -particle decay. The study met the majority of outcomes with the exception of labelling PEI-MP with 153Sm. This was due to the interaction of the 153Sm-PEI-MP complex with citrate ions in blood. Rapid deterioration of the Rhenium-188 generator also led to earlier than expected curtailment of the 188Re-HEDP therapeutic trial although sufficient data was available to be used in a comparative study. / Thesis (PhD)--University of Pretoria, 2013. / Internal Medicine / unrestricted

Technetium-99m (99mTc) radiopharmaceuticals are widely used for diagnostic imaging of heart, kidney, and liver disease, and cancer. Evolution from perfusion type tracers to targeted agents however has proven difficult. 99mTc labeled antibodies for imaging specific disease biomarkers would be of great interest, however the disparity between the isotopes half-life (6 hours) and the long circulation time of most antibodies (multiple days) has been a significant barrier. Furthermore, the conjugation of bifunctional 99mTc-chelate complexes to small molecules often has a detrimental impact on targeting. The use of bioorthogonal chemistry derived from tetrazines and trans-cyclooctene derivatives, along with pretargeting has the potential to overcome these issues and create a new generation of targeted 99mTc radiopharmaceuticals. Initially, the synthesis of three generations of imidazole based tridentate chelates linked to a tetrazine was completed. These new ligands were labeled with 99mTc under mild conditions (60 °C, 20 min, pH 3.5) with modest to good radiochemical yields ranging from 31 to 83%. Biodistribution studies revealed that compound 14, which contains a polyethylene glycol 5 (PEG5) linker had the best clearance from non-target tissues. Compound 14 was also used successfully in a pretargeting strategy along with a transcyclooctene (TCO) derivative of the bone targeting bisphosphonate, alendronate (ALN). One hour following the administration of TCO-ALN to BALB/c mice, compound 14 was injected intravenously where uptake at sites of high calcium turn over (i.e. the joints) was observed. At 6 hours post injection, for example, uptake reached as high as 20.1 ± 4.91 and 16.1 ± 4.84 %ID/g in the knee and shoulder, respectively. Pretargeted imaging studies were performed subsequently with a TCO-functionalized huA33 antibody in mice bearing SW122 xenografts. The TCO-huA33 antibody was injected 24 hours before the administration of two radiolabeled tetrazines at high and low specific activities. At 6 hours post injection tumour uptake was minimal, with tumour: blood ratios <1 in all cases. Blood clearance studies determined that the tetrazines were being cleared rapidly, with a blood residence half-life of 1.3-2.1 minutes. The hypothesis is that the low concentration of the antibody (owing to its high molecular weight), combined with the rapid clearance of the tetrazine and significant off-target uptake resulted in unfavorable kinetics and low tumor binding. Studies of the clearance pathway of 14 were investigated with clinically approved hepatobiliary transport inhibitors to help understand the mechanism of clearance, which could in turn be used to optimize the pharmacokinetics of the tetrazine ligands. A range of different inhibitors of key clearance pathways were evaluated with limited success. However, co-administration of 14 with ALN resulted in a 75% decrease in gall bladder uptake of 14 (216 ± 75.9 to 33.6 ± 3.93 %ID/g). Pretargeting studies of 14 with TCO-ALN in the presence of excess ALN revealed that ALN did not hinder the uptake of TCO-ALN in the bone, with all organs and tissues having the same uptake with TCO-ALN or TCO-ALN + ALN (knee: 20.1 ± 4.91 and 14.9 ± 2.43 %ID/g, respectively). There was also a concomitant decrease in gall bladder uptake (91.5 ± 17.1 to 28.8 ± 2.63 %ID/g). Further work on improving the distribution of the tetrazine ligands involved investigating the effect of the chelate. The core chelate found in 14 without the tetrazine moiety (compound 11a) was labeled with 99mTc to produce 11b in a 31% radiochemical yield. Biodistribution studies of 11b and 14 at 6 hours post injection demonstrated that the imidazole-based 99mTc-chelate was a major factor in the rapid and significant uptake and retention in the liver and gallbladder. A new triazole based chelate with optimal clearance from Kluba and coworkers was synthesized in 45% yield and successfully labeled with 99mTc (compound 23a). Biodistribution studies were performed where at 6 hours post injection, 23a had five times lower uptake in all non-target organs compared to 11b. The synthesis of a tetrazine derivative of 23a (compound 32) unfortunately demonstrated high hepatobiliary uptake compared to the original triazole chelate (gall bladder: 228 ± 251 and 8.77 ± 0.73 %ID/g, large intestine: 85.5 ± 83.5 and 6.88 ± 0.30 %ID/g, respectively). This particular derivative had a lipophilic linker as a result of the synthetic challenges faced during the preparation of a more hydrophilic triazole-tetrazine derivative. In addition to pretargeting applications, the 99mTc-tetrazine was used as a reagent to create multimodal imaging agents. Nanoscale gas vesicle (GV) ultrasound contrast agents were functionalized with TCO via an amide coupling to lysine residues. TCO-GVs were then radiolabeled by adding compound 6 where the desired product, a new multimodal probe, was obtained in 59% radiochemical yield. SPECT imaging and biodistribution studies in mice were completed where the labeled GV’s showed uptake in the gall bladder (120 ± 29.1 %ID/g), liver (16.8 ± 7.50 %ID/g), lungs (3.26 ± 1.53 %ID/g), small intestines (14.5 ± 5.30 %ID/g), and spleen (5.47 ± 2.71 %ID/g) at 120 min post injection. In addition to radiolabelling, the TCO-GVs were also functionalized with a near IR-tetrazine dye to produce a multimodal ultrasound/photoacoustic (US/PA) imaging agent in a 68% yield. / Thesis / Doctor of Philosophy (PhD)

No description available.

A utilização de radiofármacos no diagnóstico de doenças do organismo humano tem aumentado de forma significativa nas últimas décadas. O crescente desenvolvimento de novos reagentes liofilizados (RL) para a preparação de radiofármacos, embora proporcionem uma maior variedade para o mercado de radiofármacos, deixa evidente uma das lacunas na pesquisa radiofarmacêutica: a identificação de produtos de degradação. No presente trabalho, foram identificados os principais produtos de degradação dos RL de ácido 2,3-Dimercaptosuccínico (DMSA) e Etilenodicisteína Dietil Éster (ECD) utilizando as técnicas de cromatografia líquida de alta eficiência com Detecção por Arranjo de Diodos (HPLC-DAD) e cromatografia líquida acoplada à espectrometria de massas de múltiplos estágios (LC-MSn). Realizou-se o estudo de degradação forçada do RL de DMSA e do RL de ECD nas condições de estresse hidrolítico, fotolítico, oxidativo e termodegradação. As análises foram realizadas em equipamento HPLC-DAD Shimadzu e espectrômetro de massas Bruker Daltonics. Todas as análises foram desenvolvidas utilizando coluna cromatográfica Shim-Pack VP-ODS (150 mm x 4,6 mm; 5 &mu;m). O DMSA apresentou tempo de retenção de 5,58 minutos e m/z 204,8. A hidrólise ácida do DMSA não apresentou produtos de degradação. O perfil de degradação do DMSA após hidrólise alcalina apresentou três picos cromatográficos com características mais apolares que o DMSA. No espectro de fragmentação do íon de m/z 204,8 (MS2) pode-se observar a presença do fragmento de m/z 172,9, correspondente ao aduto sodiado de ácido mercaptosuccínico (MSA); e o fragmento de m/z 139,0 (MS3), correspondente ao aduto sodiado do ácido fumárico. O íon estanho (Sn) apresentou-se coordenado ao DMSA em todos os produtos de degradação após hidrólise alcalina do RL de DMSA. As amostras submetidas à hidrólise neutra não apresentaram degradação. Nos estudos de fotólise do DMSA, o íon de m/z 267,1 pode ser identificado como o ácido diacetil dimercaptosuccínico (BATSA). O íon de m/z 127,1 foi associado ao ácido hidroximetil fosfônico e observado nos estudos de oxidação. A termodegradação do DMSA e do RL de DMSA, não apresentou uma relação de decaimento da concentração do DMSA em função do tempo. Quanto ao RL de ECD, foi observado o ECD protonado em 5,55 minutos (m/z 325,6). As análises por LC-MSn do ECD sob hidrólise alcalina mostraram que o pico com tempo de retenção de 1,71 minutos foi identificado como o íon protonado do EC ([M+H]+) em m/z 269,2. Os picos com tempo de retenção de 3,34 e 3,69 minutos foram identificados como o íon protonado do ECD na forma monoéster (ECDM). A degradação alcalina do RL de ECD apresentou os íons de m/z 441,9 (ECD-Sn) e m/z 737,9 ([ECD2+Sn]-C2H2-2H). ECD monoester monoácida (ECDM) de m/z 295,2; ECD oxidado de m/z 323,5; ECD oxidado com duas pontes dissulfeto de m/z 389,1 e dímero de ECD de m/z 645,9 foram observados da degradação oxidativa. Conclui-se que as análises por HPLC-DAD e LC-MSn podem ser utilizadas no estudo de estabilidade de RL, identificando suas impurezas e produtos de degradação. / The use of radiopharmaceuticals in the diagnosis of diseases of the human organism has increased significantly in the last decades. The increasing development of new lyophilized reagents (LR) for the preparation of radiopharmaceuticals, although providing a greater variety of radiopharmaceuticals to the market, makes evident a gap in the radiopharmaceutical research: identification of degradation products. In the present work, the main degradation products of the 2,3-Dimercaptosuccinic acid (DMSA) and Ethylenedicysteine Diethyl Ester (ECD) LR were identified, using the techniques of high performance liquid chromatography with diode array detection (HPLC -DAD) and liquid chromatography multiple-stage mass spectrometry (LC-MSn). The study of forced degradation of DMSA and ECD LR was performed in the hydrolytic, photolytic and oxidative stress conditions and thermodegradation. Analyses were performed using an HPLC-DAD Shimadzu and mass spectrometer Bruker Daltonics equipment. All analyzes were carried out using Shim-Pack VP-ODS (150 mm x 4.6 mm; 5 &mu;m) chromatographic column. DMSA showed a retention time of 5.58 minutes and m/z 204.8. Acid hydrolysis of DMSA showed no degradation products. The degradation profile of DMSA after alkaline hydrolysis presented three chromatographic peaks with more nonpolar characteristics than DMSA. In the fragmentation spectrum of the ion m/z 204.8 (MS2), the presence of a fragment of m/z 172.9 was observed, corresponding to sodium adduct of mercaptosuccinic acid (MSA); and a fragment of m/z 139.0 (MS3), corresponding to sodium adduct of fumaric acid. The tin ion was coordinated to DMSA in all degradation products after alkaline hydrolysis of DMSA LR. The samples submitted to neutral hydrolysis showed no degradation. In the studies of photolysis of DMSA, the ion m/z 267.1 can be related to diacetyl dimercaptosuccinic acid (BATSA). The ion of m/z 127.1 observed in the studies of oxidation can be related to phosphonic hydroxymethyl acid. The thermodegradation of DMSA and its LR did not show a relationship between the decrease in the DMSA concentration and the time. The protonated ECD was observed in 5.55 minutes (m/z 325.6). Analysis by LC-MSn of ECD under alkaline hydrolysis showed that the peak with retention time of 1.71 minutes could be identified as the protonated ion of EC ([M+H]+) at m/z 269.2. The peaks with retention times of 3.34 and 3.69 minutes were identified as the protonated ion of ECD in its monoester form (ECDM). The alkaline degradation of ECD LR presented the m/z 441.9 (ECD-Sn) and m/z 737.9 ([ECD2+Sn]-C2H2-2H) ions. ECD monoester monoacid (ECDM) of m/z 295.2; ECD oxidized of m/z 323.5; ECD oxidized with two disulfide bonds of m/z 389.1 and ECD dimer of m/z 645.9 were observed in the oxidative degradation. It was concluded that the analysis by HPLC-DAD and LC-MSn can be used in the stability of LR, identifying and quantifying its impurities and degradation products.

Nenhuma / 3-Desoxi-3-[18F]Fluor-Timidina (18FLT) é um análogo radioativo do nucleosídeo timidina usado desde 1998 em exames de tomografia por emissão de pósitrons (PET) para diagnóstico de vários tipos de tumores, como de mama, pulmonar, colorretal, cerebral, entre outros. Seu uso amplia a cobertura dos exames PET em diagnóstico de câncer, já que existem limitações da [18F]Fludesoxiglicose (18FDG), o radiofármaco PET mais usado no mundo atualmente. Para implementação da produção de 18FLT, na Unidade de Pesquisa e Produção de Radiofármacos do Centro de Desenvolvimento da Tecnologia Nuclear (UPPR/CDTN), o módulo de síntese de 18FDG foi adaptado usando-se 3 protocolos nos quais se variou a concentração de etanol (0; 8 e 10% V/V). Também foram desenvolvidos testes de Controle de Qualidade, como pH, determinação de catalisador, determinação de etanol e acetonitrila, pureza química e radioquímica, entre outros. A formulação foi avaliada in vitro quanto a sua segurança, pelos ensaios de viabilidade metabólica (MTT) e toxicidade clonogênica, e quanto a sua eficácia para a detecção de tumores pelo ensaio de interação (Binding). Os rendimentos corrigidos obtidos da síntese de 18FLT foram 6,52%; 253% e 233% para o 1, o 2 e o 3 protocolo, respectivamente. O produto do protocolo 3 (Etanol 8%) apresentou menor citotoxicidade no teste in vitro MTT do que o do segundo (Etanol 10%). Além disso, a formulação de 18FLT e as impurezas químicas presentes na mesma não afetaram a clonogenicidade das células testadas. Com base nestes resultados o protocolo 3 foi escolhido como a síntese padrão de 18FLT. A interação da 18FLT com as linhagens celulares foi saturável, com ligação específica maior que 90%, atestando a eficácia do radiofármaco na detecção de tumores. A afinidade do radiofármaco 18FLT por células de glioblastoma humano (U87MG) foi da ordem de 0,24 &#956;mol/L (Kd). Por fim, constatou-se que quantidades adicionais de timidina e clorotimidina, duas impurezas presentes na formulação, reduziram significativamente a interação de 18FLT (IC50 ~ 1 - 34 &#956;mol/L) com as células tumorais, o que pode reduzir sua eficácia para o diagnóstico. Assim, sugere-se que um valor máximo seja estabelecido para a concentração destas impurezas como um dos critérios de Pureza Química de 18FLT, baseando-se no ensaio de eficácia de interação. Portanto, a UPPR/CDTN está apta a fornecer rotineiramente o radiofármaco 18FLT para estudos não clínicos e clínicos, de acordo com os requisitos de Boas Práticas de Fabricação de Medicamentos exigidos pela ANVISA. / 3-Deoxy-3-[18F]Fluorothymidine (18FLT) is a Thymidine radioactive analog that is being used since 1998 for cancer diagnostics by Positron Emission Tomography (PET) for several types of cancer, as breast, lung, colorectal, brain, among others. Its use extends coverage of PET scans in diagnosis of cancers in certain organs and tissues, since there are limitations of [18F] Fludeoxyglucose (18FDG), which is the most used PET radiopharmaceutical in the world today. An 18FDG synthesis module was adapted to implement 18FLT production in Research and Production Unit of Radiopharmaceuticals from Center of Nuclear Technology Development (UPPR/CDTN). Three protocols were used varying the concentration of ethanol (0%, 8% and 10%). Quality control tests were also developed, as pH, catalyst determination, ethanol and acetonitrile determination, chemical and radiochemical purity, amongst others. The formulation was evaluated in vitro for its safety, using the metabolic viability (MTTs assay) and clonogenic toxicity assays, and for its effectiveness in tumors, using the binding test. The corrected yields were 6.52%; 253%; and 233% for the first, second and third synthesis protocols, respectively. The third protocol (Ethanol 8%) was found to be less cytotoxicity comparing to the second one (Ethanol 10%). Furthermore, 18FLT chemical impurities and the entire formulation did not affect the clonogenicity of the cells in Clonogenic Assay. Based on these results the protocol 3 was chosen as the standard 18FLT synthesis. The interaction of 18FLT with the cell lines was saturable, with specific binding higher than 81%, confirming the effectiveness of the radiopharmaceutical in tumor detection. The affinity between 18FLT and human glioblastoma cells (U87MG) was found to be 0.24 &#956;mol/L (Kd). Finally, it was found that additional quantities of thymidine and chlorothymidine reduced significantly 18FLT interaction (IC50 ~ 1 - 34 &#956;mol/L) with tumor cells, which can reduce its effectiveness in PET diagnosis. For this reason it is suggested that a maximum value must be set for the concentration of these impurities in the 18FLT Chemical Purity quality control test based in efficacy binding assays. Therefore, CDTN is able to routinely provide 18FLT radiopharmaceutical for clinical and non clinical studies, according to the Brazilian Good Manufacturing Practices for Radiopharmaceuticals required by ANVISA.