RESUMEN
The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2-4 kDa discrete, branched PEGylation reagents on mCC49 Fab' (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab' (Fab'-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG 12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 ((124)I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab' with Mal-dPEG-A (Fab'-A) reduced the binding affinity of the non PEGylated Fab' by 30%; however, in vivo, Fab'-A significantly lengthened the blood retention vs Fab'-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab'-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A.
Asunto(s)
Neoplasias del Colon/diagnóstico , Fragmentos Fab de Inmunoglobulinas/química , Neoplasias Experimentales/diagnóstico , Polietilenglicoles/química , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Rayos X/métodos , Animales , Antígenos de Neoplasias/inmunología , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Radioisótopos de Yodo/química , Ratones , Ratones Desnudos , Estructura Molecular , Imagen Multimodal/métodosRESUMEN
The functions of parathyroid hormone-related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock-in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp(Delta/Delta)) was developed. Pthrp(Delta/Delta) mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, beta-catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/Ink4a, P21, GSK-3beta, Il-6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down-regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment.
Asunto(s)
Apoptosis , Huesos/citología , Genes Letales , Hematopoyesis , Señales de Localización Nuclear/deficiencia , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Animales , Western Blotting , Huesos/patología , Condrocitos/citología , Condrocitos/patología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas para Inmunoenzimas , Masculino , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
BACKGROUND: 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) is widely used in diagnostic cancer imaging. However, the use of 18F-FDG in PET-based imaging is limited by its specificity and sensitivity. In contrast, anti-TAG (tumor associated glycoprotein)-72 monoclonal antibodies are highly specific for binding to a variety of adenocarcinomas, including colorectal cancer. The aim of this preliminary study was to evaluate a complimentary determining region (CDR)-grafted humanized CH2-domain-deleted anti-TAG-72 monoclonal antibody (HuCC49deltaCH2), radiolabeled with iodine-124 (124I), as an antigen-directed and cancer-specific targeting agent for PET-based imaging. METHODS: HuCC49deltaCH2 was radiolabeled with 124I. Subcutaneous tumor implants of LS174T colon adenocarcinoma cells, which express TAG-72 antigen, were grown on athymic Nu/Nu nude mice as the xenograft model. Intravascular (i.v.) and intraperitoneal (i.p.) administration of 124I-HuCC49deltaCH2 was then evaluated in this xenograft mouse model at various time points from approximately 1 hour to 24 hours after injection using microPET imaging. This was compared to i.v. injection of 18F-FDG in the same xenograft mouse model using microPET imaging at 50 minutes after injection. RESULTS: At approximately 1 hour after i.v. injection, 124I-HuCC49deltaCH2 was distributed within the systemic circulation, while at approximately 1 hour after i.p. injection, 124I-HuCC49deltaCH2 was distributed within the peritoneal cavity. At time points from 18 hours to 24 hours after i.v. and i.p. injection, 124I-HuCC49deltaCH2 demonstrated a significantly increased level of specific localization to LS174T tumor implants (p=0.001) when compared to the 1 hour images. In contrast, approximately 50 minutes after i.v. injection, 18F-FDG failed to demonstrate any increased level of specific localization to a LS174T tumor implant, but showed the propensity toward more nonspecific uptake within the heart, Harderian glands of the bony orbits of the eyes, brown fat of the posterior neck, kidneys, and bladder. CONCLUSIONS: On microPET imaging, 124I-HuCC49deltaCH2 demonstrates an increased level of specific localization to tumor implants of LS174T colon adenocarcinoma cells in the xenograft mouse model on delayed imaging, while 18F-FDG failed to demonstrate this. The antigen-directed and cancer-specific 124I-radiolabled anti-TAG-72 monoclonal antibody conjugate, 124I-HuCC49deltaCH2, holds future potential for use in human clinical trials for preoperative, intraoperative, and postoperative PET-based imaging strategies, including fused-modality PET-based imaging platforms.
Asunto(s)
Adenocarcinoma/diagnóstico por imagen , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antineoplásicos/farmacología , Antígenos de Neoplasias/inmunología , Neoplasias del Colon/diagnóstico por imagen , Glicoproteínas/inmunología , Tomografía de Emisión de Positrones , Adenocarcinoma/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antineoplásicos/farmacocinética , Neoplasias del Colon/inmunología , Fluorodesoxiglucosa F18 , Humanos , Radioisótopos de Yodo , Ratones , Ratones Desnudos , Radiofármacos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Animals sample sensory stimuli for longer periods when they must perform difficult discrimination tasks, implying that the brain's ability to represent stimuli improves as a function of time. Although it is true in other senses, few studies have examined whether increasing sampling time improves olfactory discrimination. In the experiments reported here, odor sampling time was controlled with the goal of testing whether odor concentration affected a honeybee's ability to learn, recognize, and discriminate odors. Increasing sampling time during conditioning and testing improved a honeybee's ability to learn, recognize, and differentiate low-concentration (0.0002 M) odors. For intermediate-concentration (0.02 M) odors, both acquisition and recognition improved when stimulus duration was longer, but discrimination was unaffected. Having longer to sample a high-concentration (2.0 M) stimulus also improved acquisition, but it did not affect the ability to recognize or differentiate odors. Differences in time to respond to the conditioned and novel odors during the test period depended on the difficulty of the discrimination task. The results suggest that the sensory coding of molecular identity takes longer for low-concentration odors.
Asunto(s)
Aprendizaje por Asociación/fisiología , Abejas/fisiología , Discriminación en Psicología/fisiología , Odorantes , Reconocimiento en Psicología/fisiología , Animales , Relación Dosis-Respuesta a Droga , Modelos Logísticos , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Estimulación Química , Factores de TiempoRESUMEN
PURPOSE: To determine whether bevacizumab and ranibizumab remain confined within the vitreous cavity after intravitreal injection and to determine the pharmacokinetic properties of these agents within the vitreous cavity. METHODS: Radiolabeling with I-124 was completed using a modified Iodogen method. After testing for radiochemical purity, three anesthetized Dutch-belted rabbits underwent intravitreal injection with I-124 bevacizumab, and three underwent it with I-124 ranibizumab. All rabbits were imaged with a Micro PET-CT scanner on days 0, 2, 5, 7, 14, 21, 28, and 35. RESULTS: The intravitreally placed radiolabeled agents were found to be contained within the vitreous cavity for the duration of the study with no extravasation into the central nervous system or elsewhere. I-124 bevacizumab was detectable until day 28, whereas I-124 ranibizumab was detectable until day 21. The kinetic model appears to represent a two-compartment model, and the average retention times for bevacizumab and ranibizumab after correction for radioactive decay were found to be 4.2 days and 2.8 days, respectively. CONCLUSIONS: There was no significant escape of bevacizumab and ranibizumab from the vitreous cavity after intravitreal injection. After correction for radioactive decay, both agents remained detectable until 28 and 21 days, respectively, with retention properties that validated those methods reported in previous studies.
Asunto(s)
Inhibidores de la Angiogénesis/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Radioisótopos de Yodo , Cuerpo Vítreo/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Bevacizumab , Extravasación de Materiales Terapéuticos y Diagnósticos/prevención & control , Inyecciones Intravítreas/métodos , Masculino , Modelos Animales , Modelos Biológicos , Tomografía de Emisión de Positrones/métodos , Conejos , Ranibizumab , Glándula Tiroides/diagnóstico por imagen , Glándula Tiroides/metabolismo , Distribución Tisular , Tomografía Computarizada por Rayos X/métodos , Cuerpo Vítreo/diagnóstico por imagenRESUMEN
The cranial neural crest (CNC) undergoes complex molecular and morphological changes during embryogenesis in order to form the vertebrate skull, and nearly three quarters of all birth defects result from defects in craniofacial development. The molecular events leading to CNC differentiation have been extensively studied; however, the role of the cAMP-dependent protein kinase [protein kinase A (PKA)] during craniofacial development has only been described in palate formation. Here, we provide evidence that strict PKA regulation in postmigratory CNC cells is essential during craniofacial bone development. Selective inactivation of Prkar1a, a regulatory subunit of the PKA holoenzyme, in the CNC results in perinatal lethality caused by dysmorphic craniofacial development and subsequent asphyxiation. Additionally, aberrant differentiation of CNC mesenchymal cells results in anomalous intramembranous ossification characterized by formation of cartilaginous islands in some areas and osteolysis of bony trabeculae with fibrous connective tissue stabilization in others. Genetic interaction studies revealed that genetic reduction of the PKA catalytic subunit C(alpha) was able to rescue the phenotype, whereas reduction in Cbeta had no effect. Overall, these observations provide evidence of the essential role of proper regulation of PKA during the ossification of the bones of the skull. This knowledge may have implications for the understanding and treatment of craniofacial birth defects.