RESUMO
A liquid chromatography-tandem mass spectrometry assay was developed and qualified for the multiplexed quantitation of a small molecule stimulator of soluble guanylate cyclase (sGC) and its target engagement biomarker, 3',5'-cyclic guanosine monophosphate (cGMP), in ocular tissues and plasma from a single surrogate matrix calibration curve. A surrogate matrix approach was used in this assay due to the limited quantities of blank ocular matrices in a discovery research setting. After optimization, the assay showed high accuracy, precision, and recovery as well as parallelism between the surrogate matrix and the biological matrices (rabbit plasma, vitreous, and retina-choroid). This assay provided pharmacokinetic and target engagement data after intravitreal administration of the sGC stimulator. The nitric oxide-sGC-cGMP pathway is a potential target to address glaucoma. Increasing sGC-mediated production of cGMP could improve aqueous humor outflow and ocular blood flow. The sGC stimulator showed dose-dependent exposure in rabbit vitreous, retina-choroid, and plasma. The cGMP exhibited a delayed yet sustained increase in vitreous humor but not retina-choroid. Multiplexed measurement of both pharmacokinetic and target engagement analytes reduced animal usage and provided improved context for interpreting PK and PD relationships.
Assuntos
GMP Cíclico , Guanilato Ciclase , Animais , Guanilato Ciclase/metabolismo , Óxido Nítrico , Coelhos , Transdução de Sinais , Guanilil Ciclase Solúvel/metabolismoRESUMO
Nanomedicine is a highly demanded discipline. Liposomes have seen an increased attention due to their physicochemical properties that allow them to act as nanocarriers of drugs and also of radioisotopes that can be used to diagnose and treat cancer. In order to obtain a novel permeability cancer imaging agent based on 99mTc-labeled liposomes, we describe microwave-assisted synthesis of stearyl 6-(benzylidenehydrazinyl) nicotinamide lipid, which was included in two formulations: nanometric hydrazinonicotinic acid (HYNIC) liposome and its PEGylated coated analogue, HYNIC-PEG liposome. Radiolabeling with 99mTc via stearyl 6-(benzylidenehydrazinyl) nicotinamide was found to be easy, reproducible, and stable, revealing high radiochemical purity (94 ± 1.7%) for both liposomal formulations. Biodistribution at 4 h and 24 h and scintigraphic images at 4 h were performed in normal and melanoma-bearing C57BL/6 mice. Biodistribution studies at 4 h showed tumor uptake of 99mTc-HYNIC liposome and 99mTc-HYNIC-PEG liposome (1.1 ± 0.6 and 2.5 ± 0.4, respectively) and also at 24 h p.i. (1.8 ± 0.5 and 3.0 ± 1.1, respectively). Scintigraphic images showed appreciable tumor uptake in melanoma tumor-bearing mice with both liposomal formulations. Our results show that 99mTc stearyl 6-(benzylidenehydrazinyl) nicotinamide liposomes can be used as diagnostic noninvasive in vivo tumor-targeting agents capable of evaluating tumor permeability and development who can be used in personalized chemotherapy planning.
Assuntos
Melanoma Experimental/metabolismo , Niacinamida/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/farmacocinética , Animais , Linhagem Celular Tumoral , Lipossomos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Niacinamida/administração & dosagem , Niacinamida/química , Permeabilidade/efeitos dos fármacos , Cintilografia/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Tecnécio/administração & dosagem , Tecnécio/química , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologiaRESUMO
About 95 % of people diagnosed with glioblastoma die within five years. Glioblastoma is the most aggressive central nervous system tumour. It is necessary to make progress in the glioblastoma treatment so that advanced chemotherapy drugs or radiation therapy or, ideally, two-in-one hybrid systems should be implemented. Tyrosine kinase receptors-inhibitors and boron neutron capture therapy (BNCT), together, could provide a therapeutic strategy. In this work, sunitinib decorated-carborane hybrids were prepared and biologically evaluated identifying excellent antitumoral- and BNCT-agents. One of the selected hybrids was studied against glioma-cells and found to be 4â times more cytotoxic than sunitinib and 1.7â times more effective than 10 B-boronophenylalanine fructose complex when the cells were irradiated with neutrons.
Assuntos
Antineoplásicos , Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma , Preparações Farmacêuticas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Compostos de Boro , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Humanos , Camundongos , FenilalaninaRESUMO
BACKGROUND: There is increasing interest in the development of newborn screening tests to identify children at risk of fetal alcohol spectrum disorder (FASD) in order to provide these children with early intervention. Phosphatidylethanol (PEth) has emerged as a potential universal newborn screening candidate. METHODS: The aim of this report was to present the results of a study designed to compare PEth levels in 1,140 postpartum women and their newborn infants in Montevideo, Uruguay, and Sao Paulo, Brazil. Self-report alcohol use during pregnancy data was collected, along with both maternal and newborn dried blood spot samples for PEth analysis. RESULTS: The average age and parity of the women in the sample were 26 years of age and 2.3 pregnancies. For the Uruguay sample (n = 611), 45.8% of postpartum women had PEth levels ≥ 8 ng/ml with a mean positive PEth of 43.6 ng/ml. In contrast, 86.8% of the newborns had PEth levels ≥ 8 ng/ml, with a mean positive PEth of 77.4 ng/ml. For the Brazil sample (n = 529), 33.2% of women had PEth levels ≥ 8 ng/ml with a mean positive PEth of 31 ng/ml. In contrast, 76.9% of the Brazil newborns had PEth levels ≥ 8 ng/ml and 43.9% with a mean positive PEth of 61.1 ng/ml. PEth levels were significantly higher in newborns compared with their postpartum mothers in both the Uruguay and Brazil samples. Self-reported third-trimester alcohol was 6% in the Uruguay sample and 9.1% in the Brazil sample compared with positive maternal PEth levels in 45.8% and 33.2%, respectively. CONCLUSIONS: Clinicians may want to consider newborn PEth screening in high-risk populations where prenatal alcohol use is common. The mechanism underlying significantly higher PEth levels in newborns compared with their mothers is not known.
Assuntos
Consumo de Bebidas Alcoólicas/sangue , Transtornos do Espectro Alcoólico Fetal/sangue , Glicerofosfolipídeos/sangue , Complicações na Gravidez/sangue , Efeitos Tardios da Exposição Pré-Natal/sangue , Adulto , Brasil , Teste em Amostras de Sangue Seco , Intervenção Educacional Precoce , Feminino , Transtornos do Espectro Alcoólico Fetal/diagnóstico , Humanos , Recém-Nascido , Masculino , Triagem Neonatal , Período Pós-Parto/sangue , Gravidez , Efeitos Tardios da Exposição Pré-Natal/diagnóstico , Autorrelato , UruguaiRESUMO
Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructose in the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructose by proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructose in Controls was 14.1 ± 1.5 pmol·mm-1·min-1. SGLT inhibition with phlorizin reduced Jfructose to 4.9 ± 1.4 pmol·mm-1·min-1 ( P < 0.008), whereas removal of Na+ diminished Jfructose by 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructose from 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm-1·min-1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructose by proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Carboidratos da Dieta/administração & dosagem , Frutose/administração & dosagem , Transportador de Glucose Tipo 2/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Transporte de Sódio-Glucose/metabolismo , Administração Oral , Animais , Metabolismo dos Carboidratos/efeitos dos fármacos , Transportador de Glucose Tipo 2/genética , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas de Transporte de Sódio-Glucose/genéticaRESUMO
Preconditioning with a low dose of endotoxin confers unparalleled protection against otherwise lethal models of sepsis. The mechanisms of preconditioning have been investigated extensively in isolated immune cells such as macrophages. However, the role of tissue in mediating the protective response generated by preconditioning remains unknown. Here, using the kidney as a model organ, we investigated cell type-specific responses to preconditioning. Compared with preadministration of vehicle, endotoxin preconditioning in the cecal ligation and puncture mouse model of sepsis led to significantly enhanced survival and reduced bacterial load in several organs. Furthermore, endotoxin preconditioning reduced serum levels of proinflammatory cytokines, upregulated molecular pathways involved in phagocytosis, and prevented the renal function decline and injury induced in mice by a toxic dose of endotoxin. The protective phenotype involved the clustering of macrophages around S1 segments of proximal tubules, and full renal protection required both macrophages and renal tubular cells. Using unbiased S1 transcriptomic and tissue metabolomic approaches, we identified multiple protective molecules that were operative in preconditioned animals, including molecules involved in antibacterial defense, redox balance, and tissue healing. We conclude that preconditioning reprograms macrophages and tubules to generate a protective environment, in which tissue health is preserved and immunity is controlled yet effective. Endotoxin preconditioning can thus be used as a discovery platform, and understanding the role and participation of both tissue and macrophages will help refine targeted therapies for sepsis.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Sepse/prevenção & controle , Animais , Arginina/metabolismo , Carga Bacteriana , Quimera , Citocinas/sangue , Modelos Animais de Doenças , Masculino , Metaboloma , Camundongos , Camundongos Knockout , Fagocitose , Sepse/sangue , Succinatos/metabolismo , Taxa de Sobrevida , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , TranscriptomaRESUMO
Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2-/- mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2-/- proximal tubules and Slc27a2-/- or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide-treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.
Assuntos
Apoptose/genética , Transporte Biológico/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Animais , Apoptose/efeitos dos fármacos , Atrofia , Células Cultivadas , Células Epiteliais/fisiologia , Ácidos Graxos não Esterificados/farmacologia , Feminino , Fibrose , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Ácido Palmítico/farmacologia , Proteinúria/induzido quimicamente , Proteinúria/genética , Proteinúria/patologia , RatosRESUMO
New 1,7-closo-carboranylanilinoquinazoline hybrids have been identified as EGFR inhibitors, one of them with higher affinity than the parent compound erlotinib. The comparative docking analysis with compounds bearing bioisoster-substructures, demonstrated the relevance of the 3D aromatic-boron-rich moiety for interacting into the EGFR ATP binding region. The capability to accumulate in glioma cells, the ability to cross the blood-brain barrier and the stability on simulated biological conditions, render these molecules as lead compounds for further structural modifications to obtain dual action drugs to treat glioblastoma.
Assuntos
Boro/análise , Receptores ErbB/antagonistas & inibidores , Glioma/tratamento farmacológico , Quinazolinas/uso terapêutico , Compostos de Anilina , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/química , Quinazolinas/químicaRESUMO
Pretargeted imaging, based on the highly reactive process between [1,2,4,5]tetrazines with trans-cyclooctene (TCO), appears as an attractive strategy to overcome disadvantages associated with traditional radioimmunoconjugates. To be successful, the radiolabeled component should react in vivo with the conjugated antibody and the non reactive excess clear fast from the organism. Herein, we explore the in vivo effects of hydrophilic linker incorporation into [1,2,4,5]tetrazine systems bearing a 6-hydrazinonicotinyl (HYNIC) moiety for technetium-99m coordination. Incorporation of a polypeptide chain containing hydrophilic aminoacids, resulted in a derivative with renal clearance. Pretargeted bevacizumab imaging was used as proof of concept.
RESUMO
INTRODUCTION: Rituximab was the first monoclonal antibody approved for the treatment of B-cell non-Hodgkin lymphoma (NHL) expressing CD20 antigen. This antibody has also the potential to be used as a specific fluorescent and radiolabel agent for targeting NHL. OBJECTIVE: To radiolabel rituximab with technetium-99m (99mTc) or Cy7 and evaluate both probes as potential imaging agents for NHL. METHODS: Rituximab was derivatized with the trifluoroacetyl hydrazino protected form of succinimidyl ester of HYNIC and radiolabeled with 99mTc. Radiochemical stability and in vitro cell assays were evaluated. Biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) were performed. Raji cells were transfected with luciferase for bioluminescent NHL imaging up to 21 days. Rituximab was labeled with Cy7 for in vivo noninvasive fluorescence imaging up to 96 h. RESULTS: Radiolabeling was carried out in a fast, reproducible, easy, and stable way with high radiochemical purity and did not interfere with epitope recognition. Biodistribution and SPECT/CT studies showed high liver and discrete tumor uptake. Bioluminescence and fluorescence studies helped us evaluate rituximab-Cy7 in Raji subcutaneous engraftment in BALB/c nude mice. CONCLUSIONS: Our results support the potential use of rituximab labeled either with 99mTc or Cy7 as a molecular imaging tool for staging, restaging, and guiding surgical excision of tumors, which merits further evaluation.
Assuntos
Carbocianinas , Linfoma não Hodgkin/diagnóstico por imagem , Imagem Molecular/métodos , Rituximab , Tecnécio , Animais , Antígenos CD20/metabolismo , Carbocianinas/farmacocinética , Linhagem Celular Tumoral , Usos Diagnósticos de Compostos Químicos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Rituximab/química , Rituximab/metabolismo , Rituximab/farmacocinética , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tecnécio/farmacocinética , Distribuição TecidualRESUMO
The reported new anilinoquinazoline-icosahedral borane hybrids have been evaluated as glioma targeting for potential use in cancer therapy. Their anti-glioma activity depends on hybrids' lipophilicity; the most powerful compound against glioma cells, a 1,7-closo-derivative, displayed at least 3.3â times higher activity than the parent drug erlotinib. According to the cytotoxic effects on normal glia cells, the hybrids were selective for epidermal growth factor receptor (EGFR)-overexpressed tumor cells. These boron carriers could be used to enrich glioma cancer cells with boron for cancer therapy.
Assuntos
Boranos/química , Inibidores de Proteínas Quinases/química , Tiazóis/química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/toxicidadeRESUMO
Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99mTc. The derivatizations via a 5'-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice.
Assuntos
Aptâmeros de Nucleotídeos/farmacocinética , Moléculas de Adesão Celular/antagonistas & inibidores , Corantes Fluorescentes/farmacocinética , Linfoma/diagnóstico , Melanoma/diagnóstico , Impressão Molecular , Inibidores de Proteínas Quinases/farmacocinética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/química , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Neoplasias Experimentais/diagnóstico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a (99m)Tc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with (99m)Tc at an efficiency of >95% and was radiochemically stable. (99m)Tc-HYNIC tetrazine reacted with the TCO-CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of (99m)Tc-HYNIC-tetrazine for tumor imaging with pretargeted mAbs.
Assuntos
Química Click , Imagem Molecular/métodos , Sondas Moleculares/química , Compostos de Organotecnécio/química , Tetrazóis/química , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Ciclo-Octanos/química , Humanos , Camundongos , Sondas Moleculares/farmacocinética , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/metabolismo , Compostos de Organotecnécio/farmacocinética , Tetrazóis/farmacocinética , Distribuição TecidualRESUMO
BACKGROUND: Breast cancer is the second leading cause of cancer death worldwide. Nanotechnology approaches can overcome the side effects of chemotherapy as well as improve the efficacy of drugs. Dendrimers are nanometric size polymers which are suitable as drug delivery systems. To the best of our knowledge, studies on the application of PAMAM G4.5 (polyamidoamine half generation 4) dendrimers as potential drug delivery systems in breast cancer have not been reported. In this work we developed a PAMAM G4.5 dendrimer containing FITC (fluorescein isothiocyanate) dye to study their uptake by murine breast cancer cells and BALB/c mice breast tumors. RESULTS: We performed a reaction between FITC and PAMAM G4.5 dendrimers which were previously derivatized with piperazine (linker molecule), characterized them by (1)H NMR (proton nuclear magnetic resonance) spectroscopy and MALDI-TOF (matrix-assisted laser desorption/ionization- time-of-flight) mass spectrometry. The experimental data indicated that 2 FITC molecules could be bound covalently at the PAMAM G4.5 dendrimer surface, with 17 FITC molecules probably occluded in PAMAM dendrimers cavity. PAMAM-FITC dendrimer (PAMAM G4.5-piperazinyl-FITC dendrimer) size distribution was evaluated by DLS (dynamic light scattering) and TEM (transmission electron microscopy). The nanoparticle hydrodynamic size was 96.3 ± 1.4 nm with a PdI (polydispersion index) of 0.0296 ± 0.0171, and the size distribution measured by TEM was 44.2 ± 9.2 nm. PAMAM-FITC dendrimers were neither cytotoxic in 4T1 cells nor hemolytic up to 24 h of incubation. In addition, they were uptaken in vitro by 4T1 cells and in vivo by BALB/c mice breast tumors. PAMAM G4.5-piperazinyl-FITC dendrimer intracellular distribution was observed through histologic analysis of the tumor by laser confocal microscopy. CONCLUSION: These results indicate that PAMAM G4.5 dendrimers enter tumor tissue cells, being good candidates to be used as antitumor drug delivery systems for breast cancer treatment and diagnosis.
Assuntos
Antineoplásicos/administração & dosagem , Dendrímeros/metabolismo , Portadores de Fármacos/metabolismo , Fluoresceína-5-Isotiocianato/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Nylons/metabolismo , Animais , Antineoplásicos/farmacocinética , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Fluoresceína-5-Isotiocianato/farmacocinética , Corantes Fluorescentes/farmacocinética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Medical imaging is commonly required in breast cancer (BC) clinical trials to assess the efficacy and/or safety of study interventions. Despite the lack of definitive epidemiological data linking imaging radiation with cancer development in adults, concerns exist about the risks of imaging radiation-induced malignancies (IRIMs) in subjects exposed to repetitive imaging. We estimated the imaging radiation dose and IRIM risk in subjects participating in BC trials. MATERIALS AND METHODS: The imaging protocol requirements in 10 phase III trials in the adjuvant and advanced settings were assessed to estimate the effective radiation dose received by a typical and fully compliant subject in each trial. For each study, the excess lifetime attributable cancer risk (LAR) was calculated using the National Cancer Institute's Radiation Risk Assessment Tool, version 3.7.1. Dose and risk calculations were performed for both imaging intensive and nonintensive approaches to reflect the variability in imaging performed within the studies. RESULTS: The total effective imaging radiation dose was 0.4-262.2 mSv in adjuvant trials and 26-241.3 mSv in metastatic studies. The dose variability resulted from differing protocol requirements and imaging intensity approaches, with computed tomography, multigated acquisition scans, and bone scans as the major contributors. The mean LAR was 1.87-2,410/100,000 in adjuvant trials (IRIM: 0.0002%-2.41% of randomized subjects) and 6.9-67.3/100,000 in metastatic studies (IRIM: 0.007%-0.067% of subjects). CONCLUSION: IRIMs are infrequent events. In adjuvant trials, aligning the protocol requirements with the clinical guidelines' surveillance recommendations and substituting radiating procedures with equivalent nonradiating ones would reduce IRIM risk. No significant risk has been observed in metastatic trials, and potential concerns on IRIMs are not justified. IMPLICATIONS FOR PRACTICE: Medical imaging is key in breast cancer (BC) clinical trials. Most of these procedures expose patients to ionizing radiation, and the risk of second cancer development after imaging has prompted recent concerns and controversy. Using accepted calculation models, the number of malignancies were estimated that were potentially attributable to the imaging procedures performed during a patient's participation in BC clinical trials. The results show that for patients participating in metastatic trials, the risk of imaging radiation-induced malignancies is negligible. In adjuvant trials, some second cancers due to imaging could be expected, and measures can be taken to reduce their risk.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Doses de Radiação , Radiografia/efeitos adversos , Neoplasias da Mama/patologia , Ensaios Clínicos como Assunto , Feminino , Humanos , Cintilografia , Fatores de RiscoRESUMO
GLUT's (facilitative glucose transporters) over-expression in tumor cells has allowed the detection of several cancer types, using a glucose analogue ((18)F-FDG) with PET images, worldwide. New glucose analogs radiolabeled with (99m)Tc could be a less-expensive and more accessible alternative for diagnosis using SPECT imaging. d-Glucose ((99m)Tc-IDAG) and 2-d-deoxyglucose ((99m)Tc-AADG) organometallic complexes were proposed and studied as potential (18)F-FDG surrogates. The glucose complexes were prepared and evaluated as potential cancer imaging agents, in a melanoma tumor model. Iminodiacetic acid (IDA) and aminoacetate (AA) moieties were chosen as chelating system for radiolabeling with (99m)Tc. Tumor uptake of the formed complexes was evaluated in B16 murine cell line in vitro and in vivo in melanoma bearing C57BL/6 mice. In vitro and in vivo studies were conducted with (18)F-FDG in order to compare the uptake of (99m)Tc-glucose complexes in the tumor model. IDAG and AADG compounds were synthesized and radiolabeled with (99m)TcO4(-) to obtain the (99m)Tc-IDAG and (99m)Tc-AADG complexes in high yield and stability. In vitro cell studies showed maximum uptake at 60 min for complexes, (99m)Tc-IDAG and (99m)Tc-AADG, with 6% and 2%, respectively. Biodistribution studies showed high tumor uptake one hour post-injection, reaching tumor-to-muscle ratios of 12.1 ± 3.73 and 2.88 ± 1.40 for (99m)Tc-IDAG and (99m)Tc-AADG, respectively. SPECT and micro-SPECT-CT images acquired after the injection of (99m)Tc-IDAG showed accumulation in tumor sites, suggesting that this glucose complex would be a promising candidate for cancer imaging.
Assuntos
Glucose/química , Glucose/farmacocinética , Melanoma Experimental/diagnóstico , Compostos de Organotecnécio/análise , Compostos de Organotecnécio/farmacocinética , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Compostos de Organotecnécio/síntese química , Compostos de Organotecnécio/química , Distribuição TecidualRESUMO
Nitric oxide (NO) regulates renal function. Luminal flow stimulates NO production in the thick ascending limb (TAL). Transient receptor potential vanilloid 4 (TRPV4) is a mechano-sensitive channel activated by luminal flow in different types of cells. We hypothesized that TRPV4 mediates flow-induced NO production in the rat TAL. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. Increasing luminal flow from 0 to 20 nl/min stimulated NO from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min (n = 5; P < 0.05). The TRPV4 antagonists, ruthenium red (15 µmol/l) and RN 1734 (10 µmol/l), blocked flow-induced NO production. Also, luminal flow did not increase NO production in the absence of extracellular calcium. We also studied the effect of luminal flow on NO production in TALs transduced with a TRPV4shRNA. In nontransduced TALs luminal flow increased NO production by 47 ± 17 AU/min (P < 0.05; n = 5). Similar to nontransduced TALs, luminal flow increased NO production by 39 ± 11 AU/min (P < 0.03; n = 5) in TALs transduced with a control negative sequence-shRNA while in TRPV4shRNA-transduced TALs, luminal flow did not increase NO production (Δ10 ± 15 AU/min; n = 5). We then tested the effect of two different TRPV4 agonists on NO production in the absence of luminal flow. 4α-Phorbol 12,13-didecanoate (1 µmol/l) enhanced NO production by 60 ± 11 AU/min (P < 0.002; n = 7) and GSK1016790A (10 ηmol/l) increased NO production by 52 ± 15 AU/min (P < 0.03; n = 5). GSK1016790A (10 ηmol/l) did not stimulate NO production in TRPV4shRNA-transduced TALs. We conclude that activation of TRPV4 channels mediates flow-induced NO production in the rat TAL.
Assuntos
Alça do Néfron/metabolismo , Óxido Nítrico/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Ativação Enzimática , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Aim: ChiTn, a mouse/human chimeric anti-Tn monoclonal antibody, was radiolabeled with iodine-131 (131I) and technetium-99m (99mTc) to assess its biodistribution and internalization in Tn-expressing (Tn+) and wild-type (Tn-) LL/2 lung cancer cells. Results: Selective accumulation and gradual internalization of ChiTn were observed in Tn+ cells. Biodistribution in mice with both Tn+ or Tn- lung tumors indicated that the uptake of radiolabeled ChiTn within tumors increased over time. Dual-labeling experiments with 99mTc and 131I showed different biodistribution patterns, with 99mTc exhibiting higher values in the liver, spleen, and kidneys, while 131I showed higher uptake in the thyroid and stomach. However, tumor uptake did not significantly differ between Tn+ and Tn- tumors. To improve tumor targeting, Losartan, an antihypertensive drug known to enhance tumor perfusion and drug delivery, was investigated. Biodistribution studies in Losartan-treated mice revealed significantly higher radiolabeled ChiTn uptake in Tn+ tumors. No significant changes were observed in the uptake of the control molecule IgG-HYNIC™99mTc. Conclusions: These findings demonstrate the enhanced tumor targeting of radiolabeled ChiTn in Losartan-treated mice with Tn-expressing lung tumors. They highlight the potential of ChiTn as a theranostic agent for cancer treatment and emphasize the importance of Losartan as an adjunctive treatment to improve tumor perfusion and drug delivery.
Assuntos
Anticorpos Monoclonais , Radioisótopos do Iodo , Losartan , Neoplasias Pulmonares , Animais , Camundongos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Losartan/farmacologia , Losartan/farmacocinética , Losartan/administração & dosagem , Distribuição Tecidual , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/farmacocinética , Tecnécio , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Linhagem Celular Tumoral , Feminino , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
BACKGROUND: Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake. OBJECTIVE: The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging. METHODS: Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice. RESULTS: Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies. CONCLUSIONS: We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.