RESUMEN
We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of ¹8F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-ß-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[¹8F]fluoroethoxy)ethyl)-N-methylhydroxylamine (¹8F-FENMA) yielding the ¹8F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin α(v)ß3. On the other hand, ¹8F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the ¹8F-prosthetic group.
Asunto(s)
Diagnóstico por Imagen/métodos , Integrina alfaVbeta3/metabolismo , Péptidos Cíclicos/síntesis química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Animales , Ácidos Carboxílicos/química , Estabilidad de Medicamentos , Radioisótopos de Flúor/química , Halogenación , Hidroxilaminas/química , Marcaje Isotópico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Osteosarcoma/diagnóstico , Osteosarcoma/metabolismo , Péptidos Cíclicos/sangre , Péptidos Cíclicos/farmacocinética , Propionatos/química , Radiofármacos/sangre , Radiofármacos/farmacocinética , Estirenos/química , Distribución Tisular , Trasplante HeterólogoRESUMEN
Three strategies for chemoselective labeling of RGD peptides with (18)F have been compared. Aminooxy [(18)F]fluorobenzaldehyde conjugation provided 40 +/- 12% decay-corrected radiochemical yield using a fully automated method. An one-pot protocol for 'click labeling' of the RGD scaffold with 2-[(18)F]fluoroethylazide afforded 47 +/- 8% decay-corrected radiochemical yield. Attempted conjugation with 3-[(18)F]fluoropropanethiol led to extensive decomposition and was therefore found unsuitable for labeling of the RGD peptide investigated. The results suggest that 'click labeling' of RGD peptides provides an attractive alternative to aminooxy aldehyde condensation, however, 2-[(18)F]-fluoroethylazide may be too small to allow separation of large (18)F-labeled RGD peptides from their precursors.
Asunto(s)
Azidas/química , Radioisótopos de Flúor/química , Marcaje Isotópico/métodos , Oligopéptidos/química , Oligopéptidos/síntesis químicaRESUMEN
Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.
Asunto(s)
Péptidos/química , Coloración y Etiquetado/métodos , Acetatos/química , Sitios de Unión , Tampones (Química) , Radioisótopos de Flúor , Concentración de Iones de Hidrógeno , Hidroxilaminas/química , Maleimidas/química , Péptidos/metabolismo , Tomografía de Emisión de Positrones , Radioquímica , Especificidad por Sustrato , Temperatura , Factores de TiempoRESUMEN
Achieving high-yielding, robust, and reproducible chemistry is a prerequisite for the (18)F-labeling of peptides for quantitative receptor imaging using positron emission tomography (PET). In this study, we extend the toolbox of oxime chemistry to include the novel prosthetic groups [(18)F]-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethoxy)acetaldehyde, [(18)F]5, and [(18)F]-4-(3-fluoropropoxy)benzaldehyde, [(18)F]9, in addition to the widely used 4-[(18)F]fluorobenzaldehyde, [(18)F]12. The three (18)F-aldehydes were conjugated to the same aminooxy-bearing RGD peptide and the effect of the prosthetic group on biodistribution and tumor uptake studied in mice. The peptide conjugate [(18)F]7 was found to possess superior in vivo pharmacokinetics with higher tumor to blood, tumor to liver, tumor to muscle, and tumor to lung ratios than either [(18)F]10 or [(18)F]13. The radioactivity from the [(18)F]7 conjugate excreted more extensively through the kidney route with 79%id passing through the urine and bladder at the 2 h time point compared to around 55%id for the more hydrophobic conjugates [(18)F]10 and [(18)F]13. The chemical nature of a prosthetic group can be employed to tailor the overall biodistribution profile of the radiotracer. In this example, the hydrophilic nature of the ethylene glycol containing prosthetic group [(18)F]5 clearly influences the overall excretion pattern for the RGD peptide conjugate.
Asunto(s)
Aldehídos/química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacocinética , Animales , Unión Competitiva , Carcinoma Pulmonar de Lewis/diagnóstico por imagen , Línea Celular Tumoral , Radioisótopos de Flúor , Interacciones Hidrofóbicas e Hidrofílicas , Integrinas/metabolismo , Ratones , Neovascularización Patológica/diagnóstico por imagen , Péptidos Cíclicos/metabolismo , Polietilenglicoles/química , Tomografía de Emisión de PositronesRESUMEN
The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent complexity of the process renders full automation a challenge and leads to protracted synthesis times. Here we describe a new (18)F-labeled prosthetic group based on nicotinic acid tetrafluorophenyl ester. Reaction of [(18)F]fluoride at 40 degrees C with the trimethylammonium precursor afforded 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) directly in 60-70% yield. [(18)F]F-Py-TFP was conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding assay for the integrin alpha(v)beta(3) was established to explore competition with peptide and peptidomimetic prepared from F-Py-TFP with (125)I-echistatin. The nonradioactive conjugates were found to possess high binding affinities with calculated K(i) values in the low nanomolar range.
Asunto(s)
Ácidos Nicotínicos/síntesis química , Radiofármacos/síntesis química , Unión Competitiva , Línea Celular Tumoral , Radioisótopos de Flúor , Humanos , Técnicas In Vitro , Integrina alfaVbeta3/metabolismo , Marcaje Isotópico , Ácidos Nicotínicos/química , Ácidos Nicotínicos/metabolismo , Oligopéptidos/química , Ensayo de Unión Radioligante , Radiofármacos/química , Radiofármacos/metabolismoRESUMEN
OBJECTIVES: The purpose of this study was to evaluate the feasibility of noninvasive imaging of angiotensin II (AT) receptor upregulation in a mouse model of post-myocardial infarction (MI) heart failure (HF). BACKGROUND: Circulating AT levels do not reflect the status of upregulation of renin-angiotensin axis in the myocardium, which plays a central role in ventricular remodeling and evolution of HF after MI. Appropriately labeled AT or AT receptor blocking agents should be able to specifically target AT receptors by molecular imaging techniques. METHODS: AT receptor imaging was performed in 29 mice at various time points after permanent coronary artery ligation or in controls using a fluoresceinated angiotensin peptide analog (APA) and radiolabeled losartan. The APA was used in 19 animals for intravital fluorescence microscopy on a beating mouse heart. Tc-99m losartan was used for in vivo radionuclide imaging and quantitative assessment of AT receptor expression in 10 mice. After imaging, hearts were harvested for pathological characterization using confocal and 2-photon microscopy. RESULTS: No or little APA uptake was observed in control animals or within infarct regions on days 0 and 1. Distinct uptake occurred in the infarct area at 1 to 12 weeks after MI; the uptake was at maximum at 3 weeks and reduced markedly at 12 weeks after MI. Ultrasonographic examination demonstrated left ventricular remodeling, and pathologic characterization revealed localization of the APA tracer with collagen-producing myofibroblasts. Tc-99m losartan uptake in the infarct region (0.524 +/- 0.212% injected dose/g) increased 2.4-fold as compared to uptake in the control animals (0.215 +/- 0.129%; p < 0.05). CONCLUSIONS: The present study demonstrates the feasibility of in vivo molecular imaging of AT receptors in the remodeling myocardium. Noninvasive imaging studies aimed at AT receptor expression could play a role in identification of subjects likely to develop heart failure. In addition, such a strategy could allow for optimization of anti-angiotensin therapy in patients after MI.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Microscopía Fluorescente , Infarto del Miocardio/complicaciones , Miocardio/metabolismo , Receptores de Angiotensina/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Remodelación Ventricular , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Animales , Sitios de Unión , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Estudios de Factibilidad , Colorantes Fluorescentes/metabolismo , Insuficiencia Cardíaca/diagnóstico por imagen , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/fisiopatología , Losartán/metabolismo , Masculino , Ratones , Microscopía Confocal , Microscopía Fluorescente/métodos , Microscopía de Fluorescencia por Excitación Multifotónica , Microscopía por Video , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Miocardio/patología , Radiofármacos/metabolismo , Tecnecio , Factores de Tiempo , Tomografía Computarizada de Emisión de Fotón Único/métodos , Microtomografía por Rayos XRESUMEN
Targeting the molecular pathways associated with angiogenesis offers great potential in detecting disease pathology using in vivo imaging technologies. Initiation of angiogenesis requires activation and migration of endothelial cells in order for neovascularization to proceed. Endothelial cells associate with the extracellular matrix through specific interactions with a variety of cell adhesion receptors known as integrins. Peptides containing the tripeptide sequence RGD are known to bind with high affinity to the alphavbeta3 and alphavbeta5 integrins associated with angiogenesis. We present herein the synthesis and in vitro binding affinity of the RGD-containing peptide NC-100717 and a range of molecular probes derived from this intermediate.
Asunto(s)
Proteínas Angiogénicas/farmacología , Endotelio Vascular/fisiología , Neovascularización Fisiológica , Oligopéptidos/química , Péptidos Cíclicos/farmacología , Proteínas Angiogénicas/química , Endotelio Vascular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/fisiología , Humanos , Integrina alfaVbeta3/efectos de los fármacos , Integrina alfaVbeta3/fisiología , Modelos MolecularesRESUMEN
[(18)F]Fluorothiols are a new generation of peptide labeling reagents. This article describes the preparation of suitable methanesulfonyl precursors and their use in no-carrier-added radiosyntheses of (18)F-fluorothiols. The preparations of (3-[(18)F]fluoropropylsulfanyl)triphenylmethane, (2-[2-[2-(2-[(18)F]fluoroethoxy)ethoxy]ethoxy]ethylsulfanyl)triphenylmethane, and 4-[(18)F]fluoromethyl-N-[2-triphenylmethanesulfanyl)ethyl]benzamide starting from the corresponding methanesulfonyl precursors were investigated. Following the removal of the triphenylmethane protecting group, the (18)F-fluorothiols were reacted with the N-terminal chloroacetylated model peptide ClCH(2)C(O)-LysGlyPheGlyLys. The corresponding radiochemical yields of (18)F-labeled isolated model peptide, decay-corrected to (18)F fluoride, were 10%, 32%, and 1%, respectively. These results indicate a considerable potential of (18)F-fluorothiols for the chemoselective labeling of peptides as tracers for positron emission tomography (PET).