RESUMEN
PURPOSE: TRPC5 belongs to the mammalian superfamily of transient receptor potential (TRP) Ca2+-permeable cationic channels and it has been implicated in various CNS disorders. As part of our ongoing interest in the development of a PET radiotracer for imaging TRPC5, herein, we explored the radiosynthesis, and in vitro and in vivo evaluation of a new C-11 radiotracer [11C]HC070 in rodents and nonhuman primates. PROCEDURES: [11C]HC070 was radiolabeled utilizing the corresponding precursor and [11C]CH3I via N-methylation protocol. Ex vivo biodistribution study of [11C]HC070 was performed in Sprague-Dawley rats. In vitro autoradiography study was conducted for the rat brain sections to characterize the radiotracer distribution in the brain regionals. MicroPET brain imaging studies of [11C]HC070 were done for 129S1/SvImJ wild-type mice and 129S1/SvImJ TRPC5 knockout mice for 0-60-min dynamic data acquisition after intravenous administration of the radiotracer. Dynamic PET scans (0-120 min) for the brain of cynomolgus male macaques were performed after the radiotracer injection. RESULTS: [11C]HC070 was efficiently prepared with good radiochemical yield (45 ± 5%, n = 15), high chemical and radiochemical purity (> 99%), and high molar activity (320.6 ± 7.4 GBq/µmol, 8.6 ± 0.2 Ci/µmol) at the end of bombardment (EOB). Radiotracer [11C]HC070 has good solubility in the aqueous dose solution. The ex vivo biodistribution study showed that [11C]HC070 had a quick rat brain clearance. Autoradiography demonstrated that [11C]HC070 specifically binds to TRPC5-enriched regions in rat brain. MicroPET study showed the peak brain uptake (SUV value) was 0.63 in 129S1/SvImJ TRPC5 knockout mice compared to 1.13 in 129S1/SvImJ wild-type mice. PET study showed that [11C]HC070 has good brain uptake with maximum SUV of ~ 2.2 in the macaque brain, followed by rapid clearance. CONCLUSIONS: Our data showed that [11C]HC070 is a TRPC5-specific radiotracer with high brain uptake and good brain washout pharmacokinetics in both rodents and nonhuman primates. The radiotracer is worth further investigating of its suitability to be a PET radiotracer for imaging TRPC5 in animals and human subjects in vivo.
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Encéfalo , Tomografía de Emisión de Positrones , Animales , Humanos , Masculino , Ratones , Ratas , Encéfalo/metabolismo , Radioisótopos de Carbono/química , Mamíferos/metabolismo , Ratones Noqueados , Tomografía de Emisión de Positrones/métodos , Primates/metabolismo , Radiofármacos/farmacocinética , Ratas Sprague-Dawley , Distribución Tisular , Canales Catiónicos TRPC/metabolismoRESUMEN
PURPOSE: [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer's disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. METHODS: [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. RESULTS: Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer's disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 µM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. CONCLUSION: These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Carbolinas , Humanos , Ligandos , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Tomografía de Emisión de Positrones/métodos , Ratas , Proteínas tau/metabolismoRESUMEN
INTRODUCTION: Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a Ki of 0.91 nM that can suppress necroptosis in mouse and human cells. To develop a radiotracer for investigating the RIPK1 in vivo, we radiosynthesized [11C]TZ7774 and performed preliminary in vitro and in vivo evaluations in rodents and macaque. METHODS: Synthesis of the desmethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [11C]TZ7774 was achieved through TZ7790 reacting with [11C]methyl iodide via N-methylation. Ex vivo biodistribution of [11C]TZ7774 was performed in normal Sprague-Dawley rats. Characterization of [11C]TZ7774 in response to inflammation was performed using ex vivo biodistribution study in normal and LPS treated (10 mg/kg) C57BL/6 mice, and in vitro autoradiography and immunohistochemistry of the spleen. MicroPET brain study of [11C]TZ7774 in the macaque was also performed. RESULTS AND CONCLUSIONS: The radiosynthesis of [11C]TZ7774 was achieved with good radiochemical yield (30-40%, decay corrected to the end of bombardment (EOB)), high chemical purity (>90%), high radiochemical purity (>99%), and high molar activity (>207 GBq/µmol, decay corrected to EOB). Biodistribution studies in Sprague-Dawley rats showed [11C]TZ7774 has a high brain uptake of 0.53 (%ID/g) at 5 min post injection; pancreas, spleen, kidney, and liver also showed a relatively high initial uptake of 0.49, 0.41, 0.62, and 0.95 at 5 min respectively. Uptake of [11C]TZ7774 increased in LPS-treated C57BL/6 mice by 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. In vitro autoradiography study also revealed increased uptake of [11C]TZ7774 in the spleen of LPS-treated mice. Further characterization with immunohistochemistry confirmed increased expression of RIPK1 in red and white pulp of the spleen for mice pre-treated with LPS. MicroPET demonstrated that [11C]TZ7774 had good initial brain uptake in macaque with an (SUV) of â¼3.7 at 6-10 min, and quickly washed out from brain. These data confirm successful radiosynthesis of a RIPK1 specific radiotracer [11C]TZ7774. Our preliminary studies showed good response to LPS-induced inflammation in rodents and good uptake in macaque brain. [11C]TZ7774 has a potential to image RIPK1 related necroptosis and inflammatory processes.
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Enfermedades Neurodegenerativas , Tomografía de Emisión de Positrones , Animales , Encéfalo/metabolismo , Radioisótopos de Carbono , Inflamación/diagnóstico por imagen , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/metabolismo , Tomografía de Emisión de Positrones/métodos , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [18F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [3H]VAT, a tritiated counterpart of [18F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [3H]VAT with Kd = 6.5 nM and Bmax = 22.89 pmol/mg protein. In the [3H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [3H]VAT was observed using VAChT ligands, the Ki values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [3H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [3H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [3H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (-)-vesamicol, but not by the σ1 receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [18F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.
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Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
Nine new compounds targeting the transient receptor potential vanilloid-4 (TRPV4) were synthesized and their biological activities toward TRPV4 were determined using freshly isolated mouse skin macrophages through live cell Ca2+ imaging assay. Three compounds 4b, 4c, and 4i exhibited higher percentages of in vitro activation of TRPV4 as 48.1%, 59.3% and 33.5%, which are comparable to 56.4% activation response of the reported TRPV4 agonist GSK1016790A (3). The compound 4i was chosen for 11C-radiosynthesis using its phenol precursor 4g to reacted with [11C]methyl iodide. The radiosynthesis was achieved with good radiochemical yield (16 ± 5%), high chemical and radiochemical purity (>95%), and high molar activity (16-21 GBq/µmol, decay corrected to the end of bombardment, EOB n ≥ 4). Furthermore, the initial ex vivo biodistribution study in rats showed that [11C]4i had higher uptake in kidney, liver and small intestine compared to other tissues with rapid washout.
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Radiofármacos/farmacología , Canales Catiónicos TRPV/agonistas , Animales , Calcio/análisis , Radioisótopos de Carbono , Relación Dosis-Respuesta a Droga , Ligandos , Ratones , Estructura Molecular , Trazadores Radiactivos , Radiofármacos/síntesis química , Radiofármacos/química , Relación Estructura-Actividad , Distribución TisularRESUMEN
INTRODUCTION: Sphingosine-1-phosphate receptor 2 (S1PR2) activation exerts a critical role in biological abnormalities and diseases. A suitable radiotracer will advance our understanding of S1PR2 pathophysiology of diseases. The objective of this study is to evaluate the potential of iodine-125 labeled [125I]TZ6544 to be used for screening new compounds binding toward S1PR2, and assessing the changes of S1PR2 expression in the kidney of streptozotocin-induced diabetic rats. METHODS: [125I]TZ6544 was synthesized from borate precursor by copper (II)-catalyzed iodization reaction with [125I]NaI. [125I]TZ6544 was characterized using human recombinant S1PR2 cell membrane and biodistribution studies of [125]TZ6544 were performed on Wistar rats that were euthanized at 5 and 30 min post-injection. A rat model of diabetes was induced by IV injection of streptozotocin (55 mg/kg). In vitro autoradiography studies, immunostaining, and enzyme-linked immunosorbent assay (ELISA) analysis were performed in both diabetic and control rats. RESULTS: Radiosynthesis of [125I]TZ6544 was achieved successfully with good radiochemical yields of ~47% and high radiochemical purity of >99%. [125I]TZ6544 is a potent ligand in vitro for S1PR2 with Kd value of 4.31 nM. [125I]TZ6544 and [32P]-labeled endogenous S1P provided comparable IC50 values in radioactive competitive binding assays against known S1PR2 ligands. Compared to control, the kidney of diabetic rats had increased uptake of [125I]TZ6544, which could be reduced by a S1PR2 antagonist, JTE-013. Immunostaining and ELISA analysis confirmed that the diabetic rat had increased S1PR2 expression in the kidney. CONCLUSIONS: [125I]TZ6544 was synthesized successfully in high yields, and in vitro evaluation suggested [125I]TZ6544 has high potential to be used for screening new S1PR2 compounds and investigating the pathophysiology of S1PR2 functions. The availability of [125I]TZ6544 may facilitate the development of therapeutics and imaging agents targeting S1PR2. ADVANCES IN KNOWLEDGE: [125I]TZ6544 showed increased expression of S1PR2 in diabetic rat kidney and can be used to determine binding potency of S1PR2 compounds.
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Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/fisiopatología , Radioisótopos de Yodo/metabolismo , Riñón/patología , Radiofármacos/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Riñón/metabolismo , Ligandos , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Distribución TisularRESUMEN
The nonselective Ca2+ -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [125 I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [125 I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5â cell line and performed Ca2+ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An inâ vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [125 I]TZ66127 had binding specificity toward TRPC5. Altogether, [125 I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [125 I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5.
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Ensayo de Unión Radioligante , Radiofármacos/farmacología , Canales Catiónicos TRPC/antagonistas & inhibidores , Animales , Encéfalo/metabolismo , Calcio/análisis , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Radioisótopos de Yodo , Estructura Molecular , Tomografía de Emisión de Positrones , Radiofármacos/síntesis química , Radiofármacos/química , Ratas , Canales Catiónicos TRPC/química , Canales Catiónicos TRPC/metabolismoRESUMEN
The transient receptor potential channel subfamily member 5 (TRPC5) is a calcium permeable cation channel widely expressed in the brain. Accumulating evidence indicates that it plays a crucial role in psychiatric disorders including depression and anxiety. Positron emission tomography (PET) combined with a TRPC5 specific radioligand may provide a unique tool to investigate the functions of TRPC5 in animal disease models to guide drug development targeting TRPC5. To develop a TRPC5 PET radiotracer, the potent TRPC5 inhibitor HC608 was chosen for C-11 radiosynthesis through the N-demethyl amide precursor 7 reacting with [11C]methyl iodide. Under optimized conditions, [11C]HC608 was achieved with good radiochemical yield (25 ± 5%), high chemical and radiochemical purity (>99%), and high specific activity (204-377 GBq µmol-1, decay corrected to the end of bombardment, EOB). The in vitro autoradiography study revealed that [11C]HC608 specifically binds to TRPC5. Moreover, initial in vivo evaluation of [11C]HC608 performed in rodents and the microPET study in the brain of non-human primates further demonstrated that [11C]HC608 was able to penetrate the blood brain barrier and sufficiently accumulate in the brain. These results suggest that [11C]HC608 has the potential to be a PET tracer for imaging TRPC5 in vivo.
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Encéfalo/diagnóstico por imagen , Tomografía de Emisión de Positrones , Radiofármacos/química , Canales Catiónicos TRPC/análisis , Radioisótopos de Carbono , Humanos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
PURPOSE: A novel caspase-3 substrate-based probe [(18)F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors. METHODS: Uptake of [(18)F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis. RESULTS: The in vitro cell assays showed caspase-3-dependent uptake of [(18)F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [(18)F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [(18)F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC). CONCLUSIONS: [(18)F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [(18)F]-CP18 as a promising new PET imaging agent for apoptosis.
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Apoptosis , Glicopéptidos/farmacocinética , Imagen Molecular/métodos , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Animales , Caspasa 3/análisis , Caspasa 3/metabolismo , Caspasa 7/análisis , Caspasa 7/metabolismo , Línea Celular Tumoral , Glicopéptidos/química , Humanos , Modelos Lineales , Ratones , Radiofármacos/química , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We identified and validated [(18)F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells. PROCEDURES: CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [(18)F]-CP18 were assessed in vivo. [(18)F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity. RESULTS: In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [(18)F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [(18)F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice. CONCLUSIONS: We report the use [(18)F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.
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Apoptosis , Glicopéptidos/farmacocinética , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Análisis de Varianza , Animales , Caspasa 3/metabolismo , Línea Celular Tumoral , Dexametasona/efectos adversos , Glicopéptidos/química , Humanos , Modelos Lineales , Ratones , Radiofármacos/química , Radiofármacos/farmacocinética , Timo/química , Timo/efectos de los fármacos , Distribución TisularRESUMEN
OBJECTIVE: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains. METHODS: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and Aß rather than synthetic tau aggregates or Aß fibrils generated in vitro to measure the affinity and selectivity of [(18)F]T807 to tau and Aß. Brain uptake and biodistribution of [(18)F]T807 in mice were also tested. RESULTS: In vitro autoradiography results show that [(18)F]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (Kd) of [(18)F]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and Aß on adjacent sections demonstrated that [(18)F]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aß plaques. In vivo studies in mice demonstrated that [(18)F]T807 was able to cross the blood-brain barrier and washed out quickly. CONCLUSIONS: [(18)F]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD.
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Enfermedad de Alzheimer/diagnóstico , Encéfalo/diagnóstico por imagen , Radioisótopos de Flúor , Proteínas tau/química , Proteínas tau/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides/metabolismo , Animales , Autorradiografía , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Tomografía de Emisión de Positrones , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Distribución Tisular , Proteínas tau/genéticaRESUMEN
Senile plaques and neurofibrillary tangles are prominent neuropathological hallmarks in Alzheimer's disease and are considered to be targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. While there are a number of amyloid-ß positron emission tomography (PET) tracers currently in different stages of clinical development and commercialization, there have been very few reports on imaging agents selectively targeting tau aggregates. In search of [18F]-PET tracers that possess great binding affinity and selectivity toward tau tangles, we tested more than 900 compounds utilizing a unique screening process. A competitive autoradiography assay was set up to test compounds for binding to native tau tangles and amyloid-ß plaques on human brain tissue sections. In our in vitro assays, the 18F labeled compound [18F]-T808 displayed a high level of binding affinity and good selectivity for tau aggregates over amyloid-ß plaques. [18F]-T808 showed rapid uptake and washout in rodent brains. Our in vitro and preclinical in vivo studies suggest that [18F]-T808 possesses suitable properties and characteristics to be a specific and selective PET probe for imaging of paired helical filament tau in human brains.
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Radioisótopos de Flúor , Tomografía de Emisión de Positrones/métodos , Radiofármacos , Proteínas tau , Péptidos beta-Amiloides/metabolismo , Animales , Autorradiografía/métodos , Radioisótopos de Flúor/metabolismo , Humanos , Ratones , Ratones Endogámicos ICR , Placa Amiloide/diagnóstico por imagen , Placa Amiloide/metabolismo , Unión Proteica/fisiología , Radiofármacos/química , Radiofármacos/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismoRESUMEN
PURPOSE: To demonstrate that non-invasive bioluminescent imaging can monitor restricted expression from a nonreplicating adenovirus in which the cyclooxygenase-2 (COX-2) promoter drives firefly luciferase. PROCEDURES: Adenovirus in which the COX-2 promoter drives the firefly luciferase imaging gene was injected intratumorally into xenografts that express relatively low and relatively high levels of COX-2. Adenovirus that expresses Renilla Luciferase from the cytomegalovirus early promoter was co-injected, to normalize for injection, leakage, vascularization, etc. COX-2 restricted firefly luciferase and global Renilla Luciferase activities were measured by optical imaging techniques both in vivo and in isolated tissues. RESULTS: Dramatic reduction in hepatic luciferase expression after intravenous viral injection can be imaged non-invasively in living animals. Following intratumoral injection, luciferase levels in tumor xenografts that express differing endogenous COX-2 levels reflect the luciferase levels observed when these cells are infected in cell culture. Essentially no luciferase expression is observed in liver following intratumoral injection. CONCLUSION: Both tissue restricted expression and transcriptional redirection to tumors expressing COX-2 can be imaged non-invasively following injection of Adenovirus expressing firefly luciferase from the COX-2 promoter.
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Adenoviridae/aislamiento & purificación , Adenoviridae/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Neoplasias/metabolismo , Receptores Virales/metabolismo , Adenoviridae/genética , Animales , Línea Celular Tumoral , Ciclooxigenasa 2 , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/metabolismo , Mucosa Gástrica/metabolismo , Expresión Génica , Terapia Genética/métodos , Humanos , Inyecciones Intravenosas , Hígado/metabolismo , Hígado/virología , Luciferasas de Luciérnaga/análisis , Luciferasas de Luciérnaga/genética , Pulmón/metabolismo , Pulmón/virología , Proteínas de la Membrana , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/terapia , Neoplasias/virología , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Receptores Virales/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Estómago/virología , Transducción GenéticaRESUMEN
PURPOSE: To demonstrate the efficacy of repeated, non-invasive optical imaging of reporter gene expression in monitoring the ability of bi-specific recombinant molecules (i) to "transductionally untarget" adenovirus from Coxsackie and Adenovirus Receptor (CAR)-dependent infection of normal tissue and (ii) to "transductionally retarget" infection to specific target cells. PROCEDURES: sCAR-EGF is a recombinant, bi-specific molecule containing the soluble portion of CAR fused to Epidermal Growth Factor. The sCAR moiety binds to the virus and blocks CAR-dependent adenovirus infection. The EGF moity binds to cellular EGF receptors. We used non-invasive optical imaging of firefly luciferase to repeatedly monitor, in living animals, the ability of sCAR-EGF (i) to "transductionally untarget" systemically administered Ad.CMVfLuc, an adenovirus that constitutively expresses luciferase, from normal tissues and (ii) to "transductionally redirect" adenovirus infection in mice to xenograft tumors that express elevated epidermal growth factor (EGF) receptor levels. RESULTS: Systemic injection of sCAR-EGF "coated" adenovirus expressing firefly luciferase from the CMV early promoter, reduces expression of the reporter gene in the liver and facilitates expression of the reporter gene in tumor xenografts expressing high levels of the EGF-receptor. CONCLUSION: Both liver "untargeting" and tumor "retargeting" of adenovirus by recombinant sCAR-EGF can be imaged non-invasively using a luciferase reporter gene.
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Adenoviridae/genética , Luciferasas de Luciérnaga/análisis , Neoplasias/genética , Regiones Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Transcripción Genética/genética , Transgenes/genética , Adenoviridae/aislamiento & purificación , Adenoviridae/fisiología , Animales , Línea Celular Tumoral , Ciclooxigenasa 2 , Genes Reporteros/genética , Terapia Genética , Humanos , Inyecciones Intravenosas , Luciferasas de Luciérnaga/genética , Neoplasias Pulmonares/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias/virología , Especificidad de Órganos , Prostaglandina-Endoperóxido Sintasas/análisis , Transducción GenéticaRESUMEN
BACKGROUND: We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES). METHODS AND RESULTS: Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart. CONCLUSIONS: The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.
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Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , Guanina/análogos & derivados , Corazón/diagnóstico por imagen , Espiperona/análogos & derivados , Adenoviridae/genética , Animales , Células Cultivadas/metabolismo , Virus Defectuosos/genética , Radioisótopos de Flúor , Genes Reporteros , Terapia Genética , Vectores Genéticos/administración & dosificación , Herpesvirus Humano 1/genética , Inyecciones , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Cintigrafía , Radiofármacos , Ratas , Ratas Desnudas , Ratas Sprague-Dawley , Receptores de Dopamina D2/genética , Proteínas Recombinantes de Fusión/biosíntesis , Timidina Quinasa/genética , Transducción GenéticaRESUMEN
Gene therapy protocols are hampered by the inability to monitor the location, magnitude, and duration of ectopic gene expression following DNA delivery. Consequently, it is difficult to establish quantitative correlations and/or causal relationships between therapeutic gene expression and phenotypic responses in treated individuals. One approach to monitor "therapeutic gene" expression indirectly is to incorporate reporter genes that can be imaged in vivo into bicistronic transcription units, along with the therapeutic genes. Expression of the dopamine D2 receptor (D2R) and herpes simplex virus thymidine kinase (HSV1-TK) can both be monitored, in vivo, by positron-emission tomography (PET). We created ad.DTm, an adenovirus containing a cytomegalovirus (CMV) early promoter-driven transcription unit, in which the D2R gene is placed proximal to an encephalomyocarditis virus internal ribosomal entry site (IRES) and a modified HSV1-tk gene is placed distal to the IRES. Following intravenous ad.DTm injection into mice, correlated hepatic D2R and HSV1-sr39tk PET reporter gene expression was demonstrated. Repeated microPET scanning quantitated both D2R-dependent sequestration of a positron-emitting ligand and HSV1-TK-dependent sequestration of a positron-emitting product. It is possible, in living mice, to investigate noninvasively and to measure quantitatively and repeatedly correlated expression of two coding regions from a bicistronic transcription unit over a 3-month period following adenovirus delivery.