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1.
Mol Pharm ; 14(12): 4353-4361, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29028357

RESUMEN

The folate receptor (FR) has been established as a promising target for imaging and therapy of cancer (FR-α), inflammation, and autoimmune diseases (FR-ß). Several folate based PET radiotracers have been reported in the literature, but an 18F-labeled folate-PET imaging agent with optimal properties for clinical translation is still lacking. In the present study, we report the design and preclinical evaluation of folate-PEG12-NOTA-Al18F (1), a new folate-PET agent with improved potential for clinical applications. Radiochemical synthesis of 1 was achieved via a one-pot labeling process by heating folate-PEG12-NOTA in the presence of in situ prepared Al18F for 15 min at 105 °C, followed by HPLC purification. Specific binding of 1 to FR was evaluated on homogenates of KB (FR-positive) and A549 (FR-deficient) tumor xenografts in the presence and absence of excess folate. In vivo tumor imaging with folate-PEG12-NOTA-Al18F was compared to imaging with 99mTc-EC20 using nu/nu mice bearing either KB or A549 tumor xenografts. Specific accumulation of 1 in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folate. Radiosynthesis of 1 was accomplished within ∼35 min, affording pure radiotracer 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ∼100% radiochemical purity after HPLC purification and a specific activity of 35.8 ± 15.3 GBq/mmol. Further in vitro and in vivo examination of 1 demonstrated highly specific FR-mediated uptake in FR+ tumor, with Kd of ∼0.4 nM (KB), and reduced accumulation in liver. Given its facile preparation and improved properties, the new radiotracer, folate-PEG12-NOTA-Al18F (1), constitutes a promising tool for identification and classification of patients with FR overexpressing cancers.


Asunto(s)
Receptores de Folato Anclados a GPI/metabolismo , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Células A549 , Compuestos de Aluminio/química , Compuestos de Aluminio/farmacocinética , Animales , Evaluación Preclínica de Medicamentos , Femenino , Fluoruros/química , Fluoruros/farmacocinética , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacocinética , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Ácido Fólico/farmacocinética , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacocinética , Compuestos Heterocíclicos con 1 Anillo , Humanos , Marcaje Isotópico/métodos , Células KB , Ratones , Ratones Desnudos , Neoplasias/patología , Compuestos de Organotecnecio , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Radiofármacos/química , Distribución Tisular , Microtomografía por Rayos X/métodos , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Mol Pharm ; 13(5): 1520-7, 2016 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-27054811

RESUMEN

UNLABELLED: Folate-receptor-targeted PET radiotracers can potentially serve as versatile imaging agents for the diagnosis, staging, and prediction of response to therapy of patients with folate-receptor (FR)-expressing cancers. Because current FR-targeted PET reagents can be compromised by complex labeling procedures, low specific activities, poor radiochemical yields, or unwanted accumulation in FR negative tissues, we have undertaken to design an improved folate-PET agent that might be more amenable for clinical development. For this purpose, we have synthesized a folate-NOTA-Al(18)F radiotracer and examined its properties both in vitro and in vivo. METHODS: Radiochemical synthesis of folate-NOTA-Al(18)F was achieved by incubating (18)F(-) with AlCl3 for 2 min followed by heating in the presence of folate-NOTA for 15 min at 100 °C. Binding of folate-NOTA-Al(18)F to FR was quantitated in homogenates of KB and Cal51 tumor xenografts in the presence and absence of excess folic acid as a competitor. In vivo imaging was performed on nu/nu mice bearing either FR+ve (KB cell) or FR-ve (A549 cell) tumor xenografts, and specific accumulation of the radiotracer in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folic acid. Image quality of folate-NOTA-Al(18)F was compared with that of (99m)Tc-EC20, a clinically established folate-targeted SPECT imaging agent. RESULTS: Total radiochemical synthesis and purification of folate-NOTA-Al(18)F was completed within 37 min, yielding a specific activity of 68.82 ± 18.5 GBq/µmol, radiochemical yield of 18.6 ± 4.5%, and radiochemical purity of 98.3 ± 2.9%. Analysis of FR binding revealed a Kd of ∼1.0 nM, and micro-PET imaging together with ex vivo biodistribution analyses demonstrated high FR-mediated uptake in an FR+ tumor and the kidneys. CONCLUSIONS: Folate-NOTA-Al(18)F constitutes an easily prepared FR-targeted PET imaging agent with improved radiopharmaceutical properties and high specificity for folate receptor expressing tumors. Given its improved properties over (99m)Tc-EC20 (i.e., higher resolution, shorter image acquisition time, etc.), we conclude that folate-NOTA-Al(18)F constitutes a viable alternative to (99m)Tc-EC20 for use in identification, diagnosis, and staging of patients with FR-expressing cancers.


Asunto(s)
Radioisótopos de Flúor/química , Receptores de Folato Anclados a GPI/metabolismo , Ácido Fólico/administración & dosificación , Ácido Fólico/química , Compuestos Heterocíclicos/química , Neoplasias/diagnóstico , Radiofármacos/química , Células A549 , Animales , Femenino , Compuestos Heterocíclicos con 1 Anillo , Humanos , Células KB , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Tomografía de Emisión de Positrones/métodos , Radioquímica/métodos , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón Único/métodos
3.
Mol Imaging Biol ; 23(2): 241-249, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33098025

RESUMEN

PURPOSE: In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES: ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS: Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS: [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.


Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Antígeno B7-H1/metabolismo , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Radiometría/métodos , Radiofármacos/farmacocinética , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Radioisótopos de Flúor , Radioisótopos de Galio , Humanos , Inmunoterapia/métodos , Macaca mulatta , Ratones , Imagen Molecular/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismo , Radiofármacos/administración & dosificación , Distribución Tisular , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Cancer Res ; 65(4): 1471-8, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15735035

RESUMEN

Positron emission tomography (PET) provides an effective means of both diagnosing/staging several types of cancer and evaluating efficacy of treatment. To date, the only U.S. Food and Drug Administration-approved radiotracer for oncologic PET is (18)F-fluoro-deoxyglucose, which measures glucose accumulation as a surrogate for malignant activity. Engineered antibody fragments have been developed with the appropriate targeting specificity and systemic elimination properties predicted to allow for effective imaging of cancer based on expression of tumor associated antigens. We evaluated a small engineered antibody fragment specific for the HER2 receptor tyrosine kinase (C6.5 diabody) for its ability to function as a PET radiotracer when labeled with iodine-124. Our studies revealed HER2-dependent imaging of mouse tumor xenografts with a time-dependent increase in tumor-to-background signal over the course of the experiments. Radioiodination via an indirect method attenuated uptake of radioiodine in tissues that express the Na/I symporter without affecting the ability to image the tumor xenografts. In addition, we validated a method for using a clinical PET/computed tomography scanner to quantify tumor uptake in small-animal model systems; quantitation of the tumor targeting by PET correlated with traditional necropsy-based analysis at all time points analyzed. Thus, diabodies may represent an effective molecular structure for development of novel PET radiotracers.


Asunto(s)
Inmunoconjugados , Fragmentos de Inmunoglobulinas , Neoplasias Ováricas/diagnóstico por imagen , Radiofármacos , Receptor ErbB-2/inmunología , Animales , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/metabolismo , Radioisótopos de Yodo/química , Marcaje Isotópico/métodos , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Distribución Tisular , Trasplante Heterólogo
5.
J Nucl Med ; 58(11): 1852-1857, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28588151

RESUMEN

Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1-binding Affibody molecule NOTA-ZPD-L1_1 with 18F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1-binding Affibody ligand ZPD-L1_1 was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of 18F-AlF. In vivo studies were performed in PD-L1-positive, PD-L1-negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by γ-counting biodistribution. Immunohistochemical staining with an antibody specific for anti-PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-ZPD-L1_1 was labeled, with a radiochemical yield of 15.1% ± 5.6%, radiochemical purity of 96.7% ± 2.0%, and specific activity of 14.6 ± 6.5 GBq/µmol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1-positive tumor, whereas the PD-L1-negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1-positive (percentage injected dose per gram [%ID/g] = 2.56 ± 0.33) and -negative (%ID/g = 0.32 ± 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.


Asunto(s)
Antígeno B7-H1/metabolismo , Radioisótopos de Flúor , Tomografía de Emisión de Positrones/métodos , Radiofármacos , Marcadores de Afinidad , Animales , Anticuerpos Monoclonales , Autorradiografía , Femenino , Radioisótopos de Flúor/farmacocinética , Humanos , Inmunohistoquímica , Marcaje Isotópico/métodos , Masculino , Ratones SCID , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/metabolismo , Compuestos Organometálicos , Radiofármacos/farmacocinética , Resonancia por Plasmón de Superficie , Distribución Tisular
6.
Science ; 357(6350): 507-511, 2017 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-28705990

RESUMEN

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cardiomegalia/inducido químicamente , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Imidazoles/farmacología , Piridinas/farmacología , Animales , Bencimidazoles , Glucemia/efectos de los fármacos , Ayuno , Glucógeno/metabolismo , Hipoglucemia/inducido químicamente , Imidazoles/efectos adversos , Imidazoles/química , Insulina/farmacología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Piridinas/efectos adversos , Piridinas/química
7.
ILAR J ; 57(2): 212-220, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-28053073

RESUMEN

It is well understood that the biopharmaceutical industry must improve efficiency along the path from laboratory concept to commercial product. In vivo imaging is recognized as a useful method to provide biomarkers for target engagement, treatment response, safety, and mechanism of action. Imaging biomarkers have the potential to inform the selection of drugs that are more likely to be safe and effective. Most of the imaging modalities for biopharmaceutical research are translatable to the clinic. In vivo imaging does not require removal of tissue to provide biomarkers, thus reducing the number of valuable preclinical subjects required for a study. Longitudinal imaging allows for quantitative intra-subject comparisons, enhancing statistical power, and further reducing the number of subjects needed for the evaluation of treatment effects in animal models. The noninvasive nature of in vivo imaging also provides a valuable approach to alleviate or minimize potential pain, suffering or distress.


Asunto(s)
Alternativas al Uso de Animales , Diagnóstico por Imagen/métodos , Animales , Biomarcadores , Humanos , Modelos Animales , Investigación
8.
J Nucl Med ; 45(7): 1237-44, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15235072

RESUMEN

UNLABELLED: Time-dependent PET imaging can be an important tool in the assessment of radiotracer performance in murine models. We have performed a quantitative analysis of PET images of (124)I, acquired on a clinical PET system using a small-animal phantom. We then compared the recovered activity concentrations with the known activity concentration in the phantom spheres. The recovery coefficients found from the phantom data were applied to in vivo (124)I anti-HER2/neu C6.5 diabody PET data and compared with necropsy biodistribution data from the same tumor-bearing immunodeficient mouse. METHODS: The small-animal phantom consisted of a 4 x 8 cm water-filled acrylic cylinder with hollow spheres filled with water ranging in volume from 0.0625 to 1.0 mL and activity concentration of 27 +/- 2 kBq/mL. The background activity concentrations varied from 0 to 0.05 to 0.10 of the spheres. Data were acquired at 0, 5, and 10 cm from the scanner longitudinal axis. Recovery coefficients were theoretically calculated for spheres of different volume, background-to-target concentrations, and distance from the scanner's longitudinal axis. The theoretic recovery coefficients were applied to the maximum sphere activity concentration measured from the PET images, thus obtaining a recovered activity concentration to be compared with the known activity concentration of the spheres. RESULTS: The mean recovered activity concentration for the phantom spheres was 25 +/- 2 kBq/mL. The (124)I diabody PET image of a mouse with a tumor xenograft was then analyzed using the techniques described. The tumor percentage injected dose per gram estimated from the murine PET image (4.8 +/- 0.4) compared well with those obtained from necropsy studies (5.1). CONCLUSION: This study indicates the feasibility of performing quantitative imaging on murine (124)I antibody fragment PET images using a large-bore clinical scanner, which enables high-throughput studies to evaluate the performance of PET tracers in a timely and cost-effective manner by imaging multiple animals simultaneously. Tracers deemed promising by this screening method can then be further evaluated using traditional necropsy studies. Our group is currently conducting time-dependent (124)I diabody PET and necropsy comparative studies with larger numbers of mice.


Asunto(s)
Interpretación de Imagen Asistida por Computador/métodos , Radioisótopos de Yodo/farmacocinética , Modelos Biológicos , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/metabolismo , Tomografía Computarizada de Emisión/métodos , Animales , Anticuerpos Monoclonales/farmacocinética , Carga Corporal (Radioterapia) , Simulación por Computador , Femenino , Humanos , Tasa de Depuración Metabólica , Ratones , Ratones SCID , Especificidad de Órganos , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Dosis de Radiación , Protección Radiológica/métodos , Radiometría/instrumentación , Radiometría/métodos , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Técnica de Sustracción , Distribución Tisular , Tomografía Computarizada de Emisión/instrumentación , Tomografía Computarizada por Rayos X/instrumentación , Tomografía Computarizada por Rayos X/métodos , Recuento Corporal Total/métodos
9.
Int J Mol Imaging ; 2011: 405840, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21629847

RESUMEN

Annexin A5 has been used for the detection of apoptotic cells, due to its ability to bind to phosphatidylserine (PS). Four different labeled Annexin A5 adducts were evaluated in rhesus monkey, with radiolabeling achieved via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Of these adducts differing conjugation methods were employed which resulted in nonspecific radiolabeling (AxA5-I), or site-specific radiolabeling (AxA5-II). A nonbinding variant of Annexin A5 was also evaluated (AxA5-II(NBV)), conjugation here was site specific. The fourth adduct examined had both specific and nonspecific conjugation techniques employed (AxA5-II(mDOTA)). Blood clearance for each adduct was comparable, while appreciable uptake was observed in kidney, liver, and spleen. Significant differences in uptake of AxA5-I and AxA5-II were observed, as well as between AxA5-II and AxA5-II(NBV). No difference between AxA5-II and AxA5-II(mDOTA) was observed, suggesting that conjugating DOTA nonspecifically did not affect the in vivo biodistribution of Annexin A5.

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