ABSTRACT
Purpose: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99mTc-HYNIC-ß-Ala-RGD. Methods: 99mTc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. Results: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. Conclusion: 99mTc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.
Objetivo: A angiogênese em resposta a fenômenos isquêmicos envolve vários mediadores como as integrinas, sendo que o tripeptídeo RGD possui uma seqüência de aminoácidos com reconhecimento para este alvo. O modelo animal de isquemia de pata traseira é simples e conveniente, porém não há uma padronização do procedimento de injeção e controle radioisotópico em membro desvascularizado, dificultando, portanto a interpretação de resultados. O objetivo deste estudo foi avaliar a neovascularização em modelo murino de isquemia de pata traseira através do radiotraçador 99mTc-HYNIC-ß-Ala-RGD. Métodos: O análogo 99mTc-HYNIC-RGD foi preparado usando coligantes. A isquemia foi induzida em ratos Wistar por dupla-ligação da artéria femoral comum na prega inguinal. Peptídeo RGD radiomarcado foi injetado após 2h, assim como 1, 3, 5, 7, 10 e 14 dias. A captação foi avaliada por imagem planar e estudos de biodistribuição. Resultados: A maior diferença de captação entre isquemia e pata controle foi obtida no 7º dia (2,62 ± 0,95), com decréscimo acentuado no 14º dia. Para o pertecnetato a razão no 7º dia foi 0,87 ± 0,23. A imagem cintilográfica confirmou as diferentes captações. Conclusões: O análogo 99mTc-HYNIC-RGD concentrou-se no tecido isquêmico na etapa em que a angiogênese é mais acentuada, e o estudo do pertecnetato confirmou a redução no fluxo sanguíneo. Desta maneira, este protocolo diagnóstico pode ser recomendado para modelos isquêmicos.
Subject(s)
Animals , Male , Rats , Hindlimb/blood supply , Ischemia/physiopathology , Neovascularization, Physiologic/physiology , Organotechnetium Compounds , Radiopharmaceuticals , Amino Acid Sequence , Conserved Sequence , Hindlimb/metabolism , Hindlimb , Ischemia , Oligopeptides , Organotechnetium Compounds/pharmacokinetics , Rats, Wistar , Radiopharmaceuticals/pharmacokinetics , Tissue DistributionABSTRACT
OBJECTIVES: Cancer has been investigated using various pre-targeting techniques or models focusing on radiobombesin analogues; however, both are not offered together. In this study, nano-bombesin labeling by a pre-targeting system was undertaken to develop an alternative approach for prostate tumor treatment. METHODS: A two-step pre-targeting system utilizing a combination of streptavidin (SA), biotinylated morpholino (B-MORF), biotinylated BBN (B-BBN) with two different spacers (b-Ala and PEG), and a radiolabeled cMORF was evaluated in vitro and in vivo. RESULTS: Final conjugation conditions consisted of a 1:1:2 ratio of SA:B-MORF:B-BBN, followed by addition of 99mTc-cMORF to compensate for free MORF. In vitro binding experiments with prostate cancer cells (PC-3) revealed that total binding was time-dependent for the Ala spacer but not for the PEG spacer. The highest accumulation (5.06 ± 1.98 percent) was achieved with 1 hour of incubation, decreasing as time progressed. Specific binding fell to 1.05 ± 0.35 percent. The pre-targeting biodistribution in healthy Swiss mice was measured at different time points, with the best responses observed for 7-h and 15-h incubations. The effector, 99mTc-MAG3-cMORF, was administered 2 h later. Strong kidney excretion was always documented. The greatest tumor uptake was 2.58 ± 0.59 percentID/g at 7 h for B-bAla-BBN, with a region of interest (ROI) value of 3.9 percent during imaging. The tumor/blood ratio was low due to the slow blood clearance; however, the tumor/muscle ratio was 5.95. CONCLUSIONS: The pre-targeting approach with a peptide was a viable concept. Further evaluation with modified sequences of MORF, including less cytosine, and additional test intervals could be worthwhile.
Subject(s)
Animals , Male , Mice , Bombesin/metabolism , Molecular Imaging/methods , Morpholines/pharmacokinetics , Nanoparticles , Prostatic Neoplasms/metabolism , Radioisotopes , Streptavidin/pharmacokinetics , Bombesin/analogs & derivatives , Bombesin , Cell Line, Tumor , Chromatography, High Pressure Liquid/methods , Disease Models, Animal , Mice, Nude , Organotechnetium Compounds , Prostatic Neoplasms , Random Allocation , Radioisotopes/chemistry , Time FactorsABSTRACT
Análogos da timidina têm sido marcados com diferentes radioisótopos devido ao seu potencial em monitorar a proliferação incontrolável de células. Considerando que o radioisótopo tecnécio-99m ainda mantém uma posição privilegiada devido às suas propriedades químicas e nucleares, este trabalho constituiu-se no desenvolvimento da marcação da timidina com o 99mTc, mediante o emprego de compostos organometálicos. Os objetivos principais foram a síntese do precursor carbonil-tecnécio-99m, marcação da timidina com este precursor, estudo da estabilidade, e avaliações radioquímicas e biológicas com animais sadios e portadores de tumor. A síntese do precursor organometálico e a marcação da timidina com este precursor foi realizada com > 97 por cento e > 94 por cento de pureza radioquímica, respectivamente, obtendo-se também uma boa estabilidade em até 6 h em temperatura ambiente. A transquelação frente aos aminoácidos cisteína e histidina apresentou perdas entre 8 e 11 por cento para concentrações de até 300 mM. Os ensaios de biodistribuição em camundongos sadios indicaram que o complexo radiomarcado apresentou um rápido depuramento sangüíneo e baixa captação nos demais órgãos, com predominância de excreção da droga pelo sistema urinário e hepatobiliar. A captação tumoral foi de 0,28 e 0,18 por centoDI/g para tumor de pulmão e mama, respectivamente. Os resultados obtidos sugerem maiores investigações em outros análogos da timidina.
Thymidine analogs have been labeled with different radioisotopes due to their potential in monitoring the uncontrollable cell proliferation. Considering that the radioisotope technetium-99m still keeps a privileged position as a marker due to its chemical and nuclear properties, this work was designed to develop a new technique of labeling of thymidine analog with 99mTc, by means of the organometallic compounds. The aims of this research were: synthesis of the organometallic precursor technetium-99m-carbonyl, thymidine labeling with this precursor, study of stability, and radiochemical e biological evaluation with healthy and tumor-bearing animals. The organometallic precursor and the labeling of thymidine with this precursor were resulted with a radiochemical pureness of > 97 percent and > 94 percent, respectively, with good radiochemical stability up to 6 h in room temperature. The cysteine and histidine challenge indicated losses between 8 and 11 percent for concentrations until 300 mM. The biodistribution assay in healthy mice revealed rapid blood clearance and low uptake by general organs with renal and hepatobiliary excretion. The tumor concentration was of 0.28 and 0.18 percentID/g for lung and breast cancer, respectively. The results imply more studies in other thymidine analogs.