RESUMEN
The aim of the study was to evaluate real time in vivo molecular imaging of somatostatin receptors (sstrs) using a handheld miniaturized confocal laser scan microscope (CLM) in conjunction with fluorescein-labeled octreotate (OcF) in healthy mice and murine models of neuroendocrine tumors. For CLM a small rigid probe (diameter 7 mm) with an integrated single line laser (488 nm) was used (optical slice thickness 7 mum; lateral resolution 0.7 mum). OcF was synthesized via Fmoc solid-phase peptide synthesis and purified by HPLC showing high-affinity binding to the sstr2 (IC(50) 6.2 nmol). For in vitro evaluation, rat and human pancreatic cancer cells were used and characterized with respect to its sstr subtype expression and functional properties. For in vivo confocal imaging, healthy mouse pancreatic islet and renal tubular cells as well as immunoincompetent nude mice harboring sstr-expressing tumors were evaluated. Incubation of sstr-positive cells with OcF showed a specific time- and dose-dependent staining of sstr-positive cells. CLM showed rapid internalization and homogenous cytoplasmatic distribution. After systemic application to mice (n = 8), specific time-dependent internalization and cytoplasmatic distribution into pancreatic islet cells and tubular cells of the renal cortex was recorded. After injection in tumor-harboring nude mice (n = 8), sstr-positive cells selectively displayed a cell surface and cytoplasmatic staining. CLM-targeted biopsies detected sstr-positive tumor cells with a sensitivity of 87.5% and a specificity of 100% as correlated with ex vivo immunohistochemistry. CLM with OcF permits real-time molecular, functional, and morphological imaging of sstr-expressing cell structures, allowing the specific visualization of pancreatic islet cells and neuroendocrine tumors in vivo.
Asunto(s)
Islotes Pancreáticos/metabolismo , Microscopía Confocal/métodos , Tumores Neuroendocrinos/metabolismo , Receptores de Somatostatina/análisis , Animales , Unión Competitiva , Línea Celular Tumoral , Fluoresceínas/química , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Islotes Pancreáticos/citología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Confocal/instrumentación , Miniaturización , Imagen Molecular , Tumores Neuroendocrinos/patología , Octreótido/química , Octreótido/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND STUDY AIMS: Although various improvements in tissue imaging modalities have recently been achieved, in-vivo molecular and subsurface imaging in the field of gastroenterology remains a technical challenge. In this study we evaluated a newly developed, handheld, miniaturized confocal laser microscopy probe for real-time in-vivo molecular and subsurface imaging in rodent models of human disease. MATERIALS AND METHODS: The minimicroscope uses a 488-nm, single line laser for fluorophore excitation. The optical slice thickness is 7 microm, the lateral resolution 0.7 microm. The range of the z-axis is 0-250 microm below the tissue surface. Imaging was performed using different fluorescent staining protocols; 5-carboxyfluorescein-labeled octreotate was synthesized for targeted molecular imaging. RESULTS: Cellular and subcellular details of the gastrointestinal tract could be visualized in vivo at high resolution. Confocal real-time microscopy allowed in-vivo identification of tumor vessels and liver metastases, as well as diagnosis of focal hepatic inflammation, necrosis, and associated perfusion anomalies. Somatostatin-receptor targeting permitted in-vivo molecular staining of AR42-J-induced carcinoma and pancreatic islet cells. CONCLUSIONS: Confocal mini-microscopy allows rapid in-vivo molecular and subsurface imaging of normal and pathological tissue in the gastrointestinal tract at high resolution. Because this technology is applicable to humans, it might impact on future in-vivo microsocpic and molecular diagnosis of diseases such as cancer and inflammation.
Asunto(s)
Neoplasias Gastrointestinales/patología , Inflamación/patología , Microscopía Confocal/instrumentación , Animales , Modelos Animales de Enfermedad , Diseño de Equipo , Femenino , Fluoresceínas , Colorantes Fluorescentes , Inmunohistoquímica , Islotes Pancreáticos/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Microscopía Confocal/métodos , Miniaturización , Octreótido , Neoplasias Pancreáticas/patología , Receptores de SomatostatinaRESUMEN
The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.
Asunto(s)
Glucosa/farmacología , Gliburida/farmacología , Islotes Pancreáticos/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Unión Competitiva , Células COS , Chlorocebus aethiops , Glucosa/química , Glucosa/farmacocinética , Gliburida/química , Gliburida/farmacocinética , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Estructura Molecular , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Ratas , Receptores de Droga/genética , Receptores de Droga/metabolismo , Receptores de Sulfonilureas , Factores de TiempoRESUMEN
PURPOSE: The new beta2 radioligand (R,R)(S,S) 5-(2-(2-[4-(2-[18F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol ([18F]FE-fenoterol; [18F]FEFE), a fluoroethylated derivative of racemic fenoterol, was evaluated in vivo and ex vivo using a guinea pig model. METHODS: Dynamic PET studies over 60 min with [(18)F]FEFE were performed in nine Hartley guinea pigs in which a baseline (group 1, n=3), a predose (group 2, n=3; 2 mg/kg fenoterol 5 min prior to injection of [18F]FEFE) or a displacement study (group 3, n=3; 2 mg/kg fenoterol 5 min post injection of [18F]FEFE) was conducted. RESULTS: In all animal groups, the lungs could be visualised and semi-quantified separately by calculating uptake ratios to non-specific binding in the neck area. Premedication with non-radioactive fenoterol and displacement tests showed significant reduction of lung uptake, by 94% and 76%, respectively. CONCLUSION: These data demonstrate specific binding of the new radioligand to the pulmonary beta2-receptors in accordance with ex vivo measurements. Therefore, [18F]FEFE seems to be suitable for the in vivo visualisation and quantification of the pulmonary beta2-receptor binding in this animal model.
Asunto(s)
Fenoterol/análogos & derivados , Pulmón/diagnóstico por imagen , Pulmón/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animales , Estudios de Factibilidad , Fenoterol/farmacocinética , Cobayas , Tasa de Depuración Metabólica , Modelos Animales , Especificidad de Órganos , Cintigrafía , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
Pancreatic islet cell mass (PICM) is a major determinant of the insulin secretory capacity in humans. Currently, the only method for accurate assessment of the PICM is an autopsy study. Thus, development of a technique allowing the non-invasive quantification of PICM is of great interest. The aim of this study was to develop such a non-invasive technique featuring novel fluorine- and (99m)Tc-labelled glibenclamide derivatives. Despite the structural modifications necessary to introduce fluorine into the glibenclamide molecule, all derivatives retained insulin stimulating capacity as well as high affinity binding to human SUR1 when compared to the original glibenclamide. Contrastingly, the lipophilicity of the fluorine-labelled derivatives was altered depending on the particular modification. In the human PET-study a constant but weak radioactive signal could be detected in the pancreas using a fluorine-labelled glibenclamide derivative. However, a reliable assessment and visualisation of the PICM could not be obtained. It can be assumed that the high uptake of the fluorine-labelled tracer e.g. into the the liver and the high plasma protein binding leads to a relatively low signal-to-noise ratio. In case of the presented fluorine-labelled glibenclamide based compounds this could be the result of their invariably high lipophilicity. The development of a (99 m)Tc-labelled glibenclamide derivative with a lower lipophilicity and differing in vivo behaviour, glibenclamide based compounds for non-invasive imaging of the pancreatic islet cell mass may be possible.
Asunto(s)
Diabetes Mellitus/diagnóstico por imagen , Radioisótopos de Flúor , Gliburida/análogos & derivados , Hipoglucemiantes , Islotes Pancreáticos/diagnóstico por imagen , Radiofármacos , Tecnecio , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Gliburida/síntesis química , Gliburida/farmacocinética , Humanos , Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacocinética , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Imagen por Resonancia Magnética , Tomografía de Emisión de Positrones , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores de Droga/metabolismo , Receptores de SulfonilureasRESUMEN
18F-labeled non-sulfonylurea hypoglycemic agent (S)-2-(2-[(18)F]fluoroethoxy)-4-((3-methyl-1-(2-piperidin-1-yl-phenyl)-butylcarbamoyl)-methyl)-benzoic acid ([(18)F]repaglinide), a derivative of the sulfonylurea-receptor (SUR) ligand repaglinide, was synthesized as a potential tracer for the non-invasive investigation of the sulfonylurea 1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. [(18)F]Repaglinide could be obtained in an overall radiochemical yield (RCY) of 20% after 135 min with a radiochemical purity higher than 98% applying the secondary labeling precursor 2-[(18)F]fluoroethyltosylate. Specific activity was in the range of 50-60 GBq/micromol. Labeling was conducted by exchanging the ethoxy-moiety into a 2-[(18)F]fluoroethoxy group. To characterize the properties of fluorinated repaglinide, the affinity of the analogous non-radioactive (19)F-compound for binding to the human SUR1 isoform was assessed. [(19)F]Repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (K(D)) of 134 nM. Biological activity was proven via insulin secretion experiments on isolated rat islets and was comparable to that of repaglinide. Finally, biodistribution of [(18)F]repaglinide was investigated in rats by measuring the concentration of the compound in different organs after i.v. injection. Pancreatic tissue displayed a stable accumulation of approximately 0.12% of the injected dose from 10 min to 30 min p.i. 50% of the radioactive tracer could be displaced by additional injection of unlabeled repaglinide, indicating that [(18)F]repaglinide might be suitable for in vivo investigation with PET.