Novel series of (177)Lu-labeled bombesin derivatives with amino acidic spacers for selective targeting of human PC-3 prostate tumor cells.

AIM: Bombesin (BBN) has demonstrated the ability to bind with high affinity and specificity to GRP receptor, overexpressed on human prostate cancer. A large number of BBN derivatives have been synthesized for this purpose but most of them exhibit high abdominal accumulation, which may represent a problem in their clinical use due to serious side effects to patients. In this study we describe the results of radiolabeling with lutetium-177, stability and in vivo studies of novel phenyl-glycine-extended bombesin derivatives. The spacers were inserted to improve bombesin in vivo properties and to reduce its target to non-tumor sites. METHODS: Preliminary studies were done to establish the ideal conditions for labeling bombesin derivatives. Chromatography systems were applied to determine free lutetium and the stability of the preparations was evaluated either after storing at 2-8 ºC or incubation in human serum at 37 ºC. In vivo experiments included biodistribution, pharmacokinetics and SPECT images and were performed in Balb-c and Nude mice bearing PC-3 xenografts. RESULTS: The derivatives were labeled with high yield and kept stable at 2-8 ºC and are metabolized by human serum enzymes. In vivo studies showed fast blood clearance of labeled peptides and rapid excretion, performed mainly by renal pathway. In addition, biodistribution and imaging studies showed low abdominal accumulation and significant and specific tumor uptake of (177)Lu-labeled derivatives. CONCLUSIONS: The derivative with longer spacer holds a higher potential as radiopharmaceutical for prostate tumor diagnosis and the derivatives with shorter spacers are potential radiopharmaceuticals for prostate tumor treatment.

Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice.

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Investigation of whole blood of SJL/J mice using neutron activation analysis

Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (São Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel eletrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of 60Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.

Simultaneous determination of five elements in whole blood of dystrophin-deficient mdx mouse by NAA

Concentrations of Br, Ca, Cl, K and Na in whole blood of dystrophin-deficient mouse [the Dmdmdx line] were determined using NAA, resulting in reference values that are relevant for clinical blood investigation. The comparison with human being whole blood values was also performed in order to establish possible indexes and similarities among the experimental and clinical applications.

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis.

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.

Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients.

Genetic damage caused by ionizing radiation and repair capacity of blood lymphocytes from 3 breast cancer patients and 3 healthy donors were investigated using the comet assay. The comets were analyzed by two parameters: comet tail length and visual classification. Blood samples from the donors were irradiated in vitro with a 60Co source at a dose rate of 0.722 Gy/min, with a dose range of 0.2 to 4.0 Gy and analyzed immediately after the procedure and 3 and 24 h later. The basal level of damage and the radioinduced damage were higher in lymphocytes from breast cancer patients than in lymphocytes from healthy donors. The radioinduced damage showed that the two groups had a similar response when analyzed immediately after the irradiations. Therefore, while the healthy donors presented a considerable reduction of damage after 3 h, the patients had a higher residual damage even 24 h after exposure. The repair capacity of blood lymphocytes from the patients was slower than that of lymphocytes from healthy donors. The possible influence of age, disease stage and mutations in the BRCA1 and BRCA2 genes are discussed. Both parameters adopted proved to be sensitive and reproducible: the dose-response curves for DNA migration can be used not only for the analysis of cellular response but also for monitoring therapeutic interventions. Lymphocytes from the breast cancer patients presented an initial radiosensitivity similar to that of healthy subjects but a deficient repair mechanism made them more vulnerable to the genotoxic action of ionizing radiation. However, since lymphocytes from only 3 patients and 3 normal subjects were analyzed in the present paper, additional donors will be necessary for a more accurate evaluation.

Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients

Genetic damage caused by ionizing radiation and repair capacity of blood lymphocytes from 3 breast cancer patients and 3 healthy donors were investigated using the comet assay. The comets were analyzed by two parameters: comet tail length and visual classification. Blood samples from the donors were irradiated in vitro with a 60Co source at a dose rate of 0.722 Gy/min, with a dose range of 0.2 to 4.0 Gy and analyzed immediately after the procedure and 3 and 24 h later. The basal level of damage and the radioinduced damage were higher in lymphocytes from breast cancer patients than in lymphocytes from healthy donors. The radioinduced damage showed that the two groups had a similar response when analyzed immediately after the irradiations. Therefore, while the healthy donors presented a considerable reduction of damage after 3 h, the patients had a higher residual damage even 24 h after exposure. The repair capacity of blood lymphocytes from the patients was slower than that of lymphocytes from healthy donors. The possible influence of age, disease stage and mutations in the BRCA1 and BRCA2 genes are discussed. Both parameters adopted proved to be sensitive and reproducible: the dose-response curves for DNA migration can be used not only for the analysis of cellular response but also for monitoring therapeutic interventions. Lymphocytes from the breast cancer patients presented an initial radiosensitivity similar to that of healthy subjects but a deficient repair mechanism made them more vulnerable to the genotoxic action of ionizing radiation. However, since lymphocytes from only 3 patients and 3 normal subjects were analyzed in the present paper, additional donors will be necessary for a more accurate evaluation