Rodrigo Teodoro

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies

INTRODUCTION Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.

Influence of colloid particle profile on sentinel lymph node uptake

INTRODUCTION Particle size of colloids employed for sentinel lymph node (LN) detection is not well studied. This investigation aimed to correlate particle size and distribution of different products with LN uptake. METHODS All agents (colloidal tin, dextran, phytate and colloidal rhenium sulfide) were labeled with (99m)Tc according to manufacturers instructions. Sizing of particles was carried out on electron micrographs using Image Tool for Windows (Version 2.0). Biodistribution studies in main excretion organs as well as in popliteal LN were performed in male Wistar rats [30 and 90 min post injection (p.i.)]. The injected dose was 0.1 ml (37 MBq) in the footpad of the left posterior limb. Dynamic images (0-15 min p.i.) as well as static ones (30 and 90 min) were acquired in gamma camera. RESULTS Popliteal LN was clearly reached by all products. Nevertheless, particle size remarkably influenced node uptake. Colloidal rhenium sulfide, with the smallest diameter (5.1 x 10(-3)+/-3.9 x 10(-3) microm), permitted the best result [2.72+/-0.64 percent injected dose (%ID) at 90 min]. Phytate displayed small particles (<15 microm) with favorable uptake (1.02+/-0.14%ID). Dextran (21.4+/-12.8 microm) and colloidal tin (39.0+/-8.3 microm) were less effective (0.55+/-0.14 and 0.06+/-0.03%ID respectively). Particle distribution also tended to influence results. When asymmetric, it was associated with biphasic uptake which increased over time; conversely, symmetric distribution (colloidal tin) was consistent with a constant pattern. CONCLUSION The results are suggesting that particle size and symmetry may interfere with LN radiopharmaceutical uptake.

Neurotensin(8-13) analogue: radiolabeling and biological evaluation using different chelators.

INTRODUCTION Several strategies on the development of radiopharmaceuticals have been employed. Bifunctional chelators seem to be a promising approach since high radiochemical yields as well as good in vitro and in vivo stability have been achieved. To date, neurotensin analogs have been radiolabeled using the (99m)Tc-carbonyl approach and none was described employing the bifunctional chelating agent technique. AIM The purpose of this study was to evaluate the radiochemical and biological behaviour of NT(8-13) analogue radiolabeled with (99m)Tc, using HYNIC and NHS-S-acetyl-MAG(3) as chelator agents. METHODS Radiolabeling, in vitro stability toward cysteine and glutathione, partition coefficient and plasma protein binding were assessed for both radioconjugates. Biodistribution in healthy Swiss mice were carried out in order to evaluate the biological behaviour of the radiocomplexes. RESULTS Radiochemical yields were higher than 97% and no apparent instability toward transchelant agents was observed for both radioconjugates. A higher lipophilic character was observed for the radioconjugate labeled via MAG(3). The chelators seem to have no effect on the percentage of the radioconjugate bound to plasma proteins. A similar biological pattern was observed for both radioconjugates. Total blood, bone and muscle values revealed a slightly slower clearance for the radiocomplex labeled via MAG(3). Moreover, a remarkable liver and intestinal uptake was observed for the radiocomplex labeled via MAG(3) even at the later time points studied. CONCLUSION The high radiochemical yields achieved and the similar in vivo pattern found for both radioconjugates make them potential candidates for imaging tumors using nuclear medicine techniques.

Parameters optimization defined by statistical analysis for cysteine-dextran radiolabeling with technetium tricarbonyl core

The objective of this study was the development of a statistical approach for radiolabeling optimization of cysteine-dextran conjugates with Tc-99m tricarbonyl core. This strategy has been applied to the labeling of 2-propylene-S-cysteine-dextran in the attempt to prepare a new class of tracers for sentinel lymph node detection, and can be extended to other radiopharmaceuticals for different targets. The statistical routine was based on three-level factorial design. Best labeling conditions were achieved. The specific activity reached was 5 MBq/μg.

Radiolabeled nano‐peptides show specificity for an animal model of human PC3 prostate cancer cells

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 (β‐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 %) was achieved with 1 hour of incubation, decreasing as time progressed. Specific binding fell to 1.05±0.35 %. 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 %ID/g at 7 h for B‐βAla‐BBN, with a region of interest (ROI) value of 3.9 % 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.

Size and specificity of radiopharmaceuticals for sentinel lymph node detection.

Background Biological performance of radiotracers for sentinel node detection analyzed in the light of molecular design and dimension is not widely available. Purpose To evaluate the effect of dextran molecular size and the presence of tissue-binding units (mannose) within the model of 99mTc-carbonyl conjugate for sentinel lymph node detection. Material and Methods Four dextran conjugates with and without mannose in the chemical backbone were included. All polymers were radiolabeled using the precursor [99mTc(OH2)3(CO)3]+. Radiolabeling conditions targeted the best radiochemical purity and specific activity for each radiopharmaceutical, and partition coefficients were also defined. Lymphoscintigraphy and ex-vivo biodistribution in popliteal lymph node, liver and kidneys were performed in Wistar rats. The effects of molecular weight and mannose presence were assessed by a two-level factorial design. Results Radiochemical purity was indirectly related to molecular weight and presence of mannose in the polymer structure. All products were able to detect popliteal lymph node, however, uptake was strongly influenced by use of mannose (4-fold higher). Excretion was similarly modulated by differences in molecular weight. Mannose-enhanced lymph node uptake and higher molecule size in the range under study benefitted lymphoscintigraphic performance. Conclusion Screening of radiopharmaceuticals for lymphoscintigraphy might improve with attention to the mentioned physico-chemical features of the molecule.

Estudos in vitro e in vivo de análogo da timidina marcada com complexo organometálico de tecnécio-99m para potencial uso em diagnóstico tumoral

Analogos da timidina tem sido marcados com diferentes radioisotopos devido ao seu potencial em monitorar a proliferacao incontrolavel de celulas. Considerando que o radioisotopo tecnecio-99m ainda mantem uma posicao privilegiada devido as suas propriedades quimicas e nucleares, este trabalho constituiu-se no desenvolvimento da marcacao da timidina com o 99mTc, mediante o emprego de compostos organometalicos. Os objetivos principais foram a sintese do precursor carbonil-tecnecio-99m, marcacao da timidina com este precursor, estudo da estabilidade, e avaliacoes radioquimicas e biologicas com animais sadios e portadores de tumor. A sintese do precursor organometalico e a marcacao da timidina com este precursor foi realizada com > 97% e > 94% de pureza radioquimica, respectivamente, obtendo-se tambem uma boa estabilidade em ate 6 h em temperatura ambiente. A transquelacao frente aos aminoacidos cisteina e histidina apresentou perdas entre 8 e 11% para concentracoes de ate 300 mM. Os ensaios de biodistribuicao em camundongos sadios indicaram que o complexo radiomarcado apresentou um rapido depuramento sanguineo e baixa captacao nos demais orgaos, com predominância de excrecao da droga pelo sistema urinario e hepatobiliar. A captacao tumoral foi de 0,28 e 0,18 %DI/g para tumor de pulmao e mama, respectivamente. Os resultados obtidos sugerem maiores investigacoes em outros analogos da timidina.