RESUMO
Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.
Assuntos
Endopeptidases/metabolismo , Neoplasias Hepáticas Experimentais , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Tecnécio , Animais , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Compostos de Organotecnécio/farmacologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Tecnécio/química , Tecnécio/farmacocinética , Tecnécio/farmacologiaRESUMO
Invasive Candida albicans infections are a serious health threat for immunocompromised individuals. Fluconazole is most commonly used to treat these infections, but resistance due to the overexpression of multidrug efflux pumps is of grave concern. This study evaluated the ability of five synthetic organotellurium compounds to reverse the fluconazole resistance of C. albicans clinical isolates. Compounds 1 to 4, at <10 µg/ml, ameliorated the fluconazole resistance of Saccharomyces cerevisiae strains overexpressing the major C. albicans multidrug efflux pumps Cdr1p and Mdr1p, whereas compound 5 only sensitized Mdr1p-overexpressing strains to fluconazole. Compounds 1 to 4 also inhibited efflux of the fluorescent substrate rhodamine 6G and the ATPase activity of Cdr1p, whereas all five of compounds 1 to 5 inhibited Nile red efflux by Mdr1p. Interestingly, all five compounds demonstrated synergy with fluconazole against efflux pump-overexpressing fluconazole-resistant C. albicans clinical isolates, isolate 95-142 overexpressing CDR1 and CDR2, isolate 96-25 overexpressing MDR1 and ERG11, and isolate 12-99 overexpressing CDR1, CDR2, MDR1, and ERG11 Overall, organotellurium compounds 1 and 2 were the most promising fluconazole chemosensitizers of fluconazole-resistant C. albicans isolates. Our data suggest that these novel organotellurium compounds inhibit pump efflux by two very important and distinct families of fungal multidrug efflux pumps: the ATP-binding cassette transporter Cdr1p and the major facilitator superfamily transporter Mdr1p.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Testes de Sensibilidade Microbiana , Compostos de Organotecnécio/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Radiolabeled gold nanoparticles may function simultaneously as radiotherapy and thermal ablation systems. The gastrin-releasing peptide receptor (GRP-r) is overexpressed in prostate cancer, and Lys(3) -bombesin is a peptide that binds with high affinity to the GRP-r. HIV Tat(49-57) is a cell-penetrating peptide that reaches the DNA. In cancer cells, (177) Lu shows efficient crossfire effect, whereas (99m) Tc that is internalized in the cancer cell nuclei acts as an effective system of targeted radiotherapy because of the biological Auger effect. The aim of this research was to evaluate the in vitro potential of (99m) Tc-labeled and (177) Lu-labeled gold nanoparticles conjugated to Tat(49-57)-Lys(3) -bombesin peptides ((99m) Tc/(177) Lu-AuNP-Tat-BN) as a plasmonic photothermal therapy and targeted radiotherapy system in PC3 prostate cancer cells. Peptides were conjugated to AuNPs (5 nm) by spontaneous reaction with the thiol group of cysteine (Cys). The effect on PC3 cell viability after laser heating of the AuNP-Tat-BN incubated with the cancer cells was conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm (0.65 W/cm(2) ). For the (99m) Tc/(177) Lu-AuNP-Tat-BN to be obtained, the (177) Lu-DOTA-Gly-Gly-Cys and (99m) Tc-HYNIC-octreotide radiopeptides were first prepared and added simultaneously to a solution of AuNP-Tat-BN. (99m) Tc/(177) Lu-AuNP-Tat-BN (20 Bq/cell) was incubated with PC3 cells, and the effect on the cell proliferation was evaluated after 3 days. Fluorescence images of (99m) Tc/(177) Lu-AuNP-Tat-BN internalized in nuclei of PC3 were also obtained. After laser irradiation, the presence of AuNP-Tat-BN caused a significant increase in the temperature of the medium (46.4 vs 39.5 °C of that without AuNP) resulting in a significant decrease in PC3 cell viability down to 1.3%. After treatment with (99m) Tc/(177) Lu-AuNP-Tat-BN, the PC3 cell proliferation was inhibited. The nanosystem exhibited properties suitable for plasmonic photothermal therapy and targeted radiotherapy in the treatment of prostate cancer.
Assuntos
Bombesina/análogos & derivados , Núcleo Celular/efeitos dos fármacos , Nanopartículas Metálicas/química , Compostos de Organotecnécio/química , Compostos Radiofarmacêuticos/química , Bombesina/química , Bombesina/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos da radiação , Sobrevivência Celular , Humanos , Lasers , Masculino , Compostos de Organotecnécio/farmacologia , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos/farmacologiaRESUMO
BACKGROUND: The gastrin-releasing peptide receptor (GRP-r) is overexpressed in prostate and breast cancers. technetium-99m-bombesin (Tc-BN) has been reported as a radiopharmaceutical with specific cell GRP-r binding. The HIV Tat (49-57)-derived peptide has been used to deliver a large variety of molecules to cell nuclei. A new hybrid radiopharmaceutical of type Tc-N2S2-Tat(49-57)-Lys-BN (Tc-Tat-BN) internalized in cancer cell nuclei could act as an effective system of targeted radiotherapy using Auger and internal conversion electron emissions near DNA. AIM: The aim of this study was to assess the in-vitro nucleus internalization kinetics of Tc-Tat-BN in GRP r-positive cancer cells and to evaluate the subcellular-level radiation-absorbed dose associated with the observed effect on cancer cell DNA proliferation. METHODS: Tc-Tat-BN in-vitro internalization kinetics were evaluated in human prostate cancer PC-3 cells and breast carcinoma cell lines MCF7 and MDA-MB231. Nuclei from cells were isolated using a nuclear extraction kit. Total disintegration in each subcellular compartment was calculated by the integration of experimental time-activity kinetic curves. Nucleus internalization was corroborated by confocal microscopy images using immunofluorescently labelled Tat-BN. The PENELOPE code was used to simulate and calculate the absorbed dose by the contribution of Auger and internal conversion electrons in the cytoplasm and nucleus using geometric models built from immunofluorescent cell images. A cell proliferation kit was used to evaluate DNA concentration after cancer cell incubation with Tc-Tat-BN. RESULTS: The results showed that 59.7, 61.2 and 41.5% of total disintegration per unit of Tc-Tat-BN activity (1 Bq) bound to the cell occurred in the nucleus of PC-3, MCF7 and MDA-MB231, respectively. The Tc-Tat-BN absorbed doses delivered to nuclei were 0.142 mGy/decay (PC-3), 0.434 mGy/decay (MCF7) and 0.276 mGy/decay (MDA-MB231). Tc-Tat-BN produced a significant decrease in PC-3 (52.98%), MCF7 (45.71%) and MDA-MB231 (35.80%) cellular proliferation with respect to untreated cells. CONCLUSION: The hybrid radiopharmaceutical could be potentially useful as a therapeutic agent for prostate and breast cancers.
Assuntos
Bombesina/análogos & derivados , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Compostos de Organotecnécio/farmacocinética , Neoplasias da Próstata/patologia , Tecnécio/farmacocinética , Bombesina/farmacocinética , Bombesina/farmacologia , Neoplasias da Mama/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Masculino , Compostos de Organotecnécio/síntese química , Compostos de Organotecnécio/farmacologia , Neoplasias da Próstata/metabolismo , Ligação Proteica , Radiometria , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Bombesina/metabolismo , Frações SubcelularesRESUMO
PURPOSE: To investigate inflammatory (zymosan) and infectious (Staphylococcus aureus) processes in experimental models in rats using technetium-99m-labeled ceftizoxime (CFT). METHODS: Male Wistar rats were used for the development of the inflammatory (zymosan) and infectious (S. aureus) processes in the medullary cavity of the left tibia. Sterile saline was used for the control group. At 48 h after induction of the processes, the animals were anesthetized and scintigraphic images were acquired at 1, 2, 4, and 6 h after intravenous injection of 0.1 ml of 99mTc-CFT (55 MBq). Quantitative analysis of the scintigraphic images was performed by counting the radioactivity in the regions of interest. Samples of tibia were taken for histopathological examination. RESULTS: The images showed that 99mTc-CFT presented higher tropism to infectious foci than with the inflammatory site. The average value of the target/nontarget ratio of the 99mTc-CFT was significantly higher in the infected (2.40+/-0.22) than in the inflamed tibia (1.50+/-0.05) and the control group (1.05+/-0.04) for all of the investigated times. The histological data showed a similar inflammatory response for both the S. aureus and zymosan groups. CONCLUSION: The 99mTc-CFT presented a high tropism and retention for an infected region in this model of osteomyelitis, thereby constituting an interesting strategy to distinguish aseptic from septic sites.
Assuntos
Ceftizoxima/análogos & derivados , Compostos de Organotecnécio/farmacologia , Osteomielite/diagnóstico por imagem , Osteomielite/diagnóstico , Cintilografia/métodos , Animais , Proteínas Sanguíneas/química , Ceftizoxima/farmacologia , Modelos Animais de Doenças , Inflamação , Masculino , Compostos Radiofarmacêuticos/farmacologia , Ratos , Ratos Wistar , Sepse/diagnóstico , Sepse/diagnóstico por imagem , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/diagnóstico por imagem , Fatores de Tempo , Zimosan/metabolismoRESUMO
The synthesis and evaluation of a series of oxotechnetium and oxorhenium complexes containing a nitroaromatic moiety as potential radiopharmaceuticals for targeting tumour hypoxia is presented. 99mTc labelling was performed in high yield (>85%) and radiochemical purity (>90%). Their structure was corroborated by means of the rhenium complexes. Reduction potentials were in the range for bioreducible compounds. 99mTc complexes III-VI were selected for "in vivo" experiments in view of the results of cytotoxicity studies. Biodistribution in normal animals was characterized by high initial blood, lung and liver uptake, fast blood and soft tissue depuration and preferential excretion via the hepatobiliary system. Initial tumour uptake was moderate but tumour/muscle ratios for complexes III and IV, were favourable at all time points. Although the results are encouraging further development is still necessary in order to achieve higher tumour uptake and lower gastrointestinal activity.