RESUMO
Gene-directed enzyme prodrug therapy (GDEPT) uses tumor-tropic vectors to deliver prodrug-converting enzymes such as nitroreductases specifically to the tumor environment. The nitroreductase NfsB from Escherichia coli (NfsB_Ec) has been a particular focal point for GDEPT and over the past 25 years has been the subject of several engineering studies seeking to improve catalysis of prodrug substrates. To facilitate clinical development, there is also a need to enable effective non-invasive imaging capabilities. SN33623, a 5-nitroimidazole analogue of 2-nitroimidazole hypoxia probe EF5, has potential for PET imaging exogenously delivered nitroreductases without generating confounding background due to tumor hypoxia. However, we show here that SN33623 is a poor substrate for NfsB_Ec. To address this, we used assay-guided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologues. Introduction of the rational substitutions F70A and F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labeling of E. coli expressing the engineered enzyme. Serendipitously, the F70A and F108Y substitutions also substantially improved activity with the anticancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.
Assuntos
Monitoramento de Medicamentos/métodos , Proteínas de Escherichia coli/metabolismo , Etanidazol/análogos & derivados , Hidrocarbonetos Fluorados/metabolismo , Imagem Molecular/métodos , Nitroimidazóis/uso terapêutico , Nitrorredutases/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Pró-Fármacos/uso terapêutico , Antineoplásicos/uso terapêutico , Técnicas Biossensoriais/métodos , Hipóxia Celular/fisiologia , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Etanidazol/química , Etanidazol/metabolismo , Terapia Genética/métodos , Células HCT116 , Humanos , Hidrocarbonetos Fluorados/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Nitroimidazóis/farmacologia , Nitrorredutases/genética , Pró-Fármacos/metabolismo , Engenharia de ProteínasRESUMO
PURPOSE: Hypoxia contributes to radiotherapy resistance and more aggressive behaviour of several types of cancer. This study was designed to evaluate the repeatability of intratumour uptake of the hypoxia tracer [18F]EF5 in paired PET/CT scans. METHODS: Ten patients with newly diagnosed head and neck cancer (HNC) received three static PET/CT scans before chemoradiotherapy: two with [18F]EF5 a median of 7 days apart and one with [18F]FDG. Metabolically active primary tumour volumes were defined in [18F]FDG images and transferred to co-registered [18F]EF5 images for repeatability analysis. A tumour-to-muscle uptake ratio (TMR) of 1.5 at 3 h from injection of [18F]EF5 was used as a threshold representing hypoxic tissue. RESULTS: In 10 paired [18F]EF5 PET/CT image sets, SUVmean, SUVmax, and TMR showed a good correlation with the intraclass correlation coefficients of 0.81, 0.85, and 0.87, respectively. The relative coefficients of repeatability for these parameters were 15%, 17%, and 10%, respectively. Fractional hypoxic volumes of the tumours in the repeated scans had a high correlation using the Spearman rank correlation test (r = 0.94). In a voxel-by-voxel TMR analysis between the repeated scans, the mean of Pearson correlation coefficients of individual patients was 0.65. The mean (± SD) difference of TMR in the pooled data set was 0.03 ± 0.20. CONCLUSION: Pretreatment [18F]EF5 PET/CT within one week shows high repeatability and is feasible for the guiding of hypoxia-targeted treatment interventions in HNC.
Assuntos
Etanidazol/análogos & derivados , Radioisótopos de Flúor , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Hidrocarbonetos Fluorados , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Hipóxia Tumoral , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos ProspectivosRESUMO
BACKGROUND: There is increasing evidence that tumor hypoxia plays a significant role in the chemoresistance of melanoma, but to our knowledge, real-time tumor oxygenation during isolated limb infusion (ILI) has not been studied. We sought to demonstrate the feasibility of measuring real-time alterations in tissue oxygenation. METHODS: Consecutive patients with histologically confirmed in-transit melanoma were enrolled onto a prospective single-arm pilot study and administered the hypoxia marker drug EF5. All patients were treated with ILI. Optical spectroscopy readings were obtained at three locations: two discrete target lesions and one normal skin control. Measurements were taken at 11 predefined time points during ILI. RESULTS: A total of six patients were enrolled onto this pilot study. Intratumor and normal skin optical spectroscopy readings were found to have discrete inflection points throughout the duration of therapy, corresponding with established time points. Baseline hypoxia as measured by both optical spectroscopy and EF5 immunofluorescence was variable, but on the basis of optical spectra, tumors appeared to become more hypoxic compared to normal skin after tourniquet application. The optical hypoxia signature was variable between patients while hemoglobin absorption increased. CONCLUSIONS: To our knowledge, this is the first use of real-time optical spectroscopy to evaluate oxygenation and perfusion within melanoma lesions during regional chemotherapy. We report our development of this new noninvasive means of assessing tumor vascular function, which has the potential to be a powerful tool for noninvasive examination of the melanoma tumor microenvironment.
Assuntos
Etanidazol/análogos & derivados , Hidrocarbonetos Fluorados , Hipóxia/diagnóstico , Indicadores e Reagentes , Melanoma/patologia , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Estudos de Viabilidade , Feminino , Seguimentos , Humanos , Hipóxia/metabolismo , Masculino , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Projetos Piloto , Prognóstico , Estudos ProspectivosRESUMO
PURPOSE: In order to improve the treatment of squamous cell carcinoma of the head and neck, precise information on the treated tumour's biology is required and the prognostic importance of different biological parameters needs to be determined. The aim of our study was to determine the predictive value of pretreatment PET/CT imaging using [(18)F]FDG, a new hypoxia tracer [(18)F]EF5 and the perfusion tracer [(15)O]H2O in patients with squamous cell cancer of the head and neck treated with radiochemotherapy. METHODS: The study group comprised 22 patients with confirmed squamous cell carcinoma of the head and neck who underwent a PET/CT scan using the above tracers before any treatment. Patients were later treated with a combination of radiochemotherapy and surgery. Parametric blood flow was calculated from dynamic [(15)O]H2O PET images using a one-tissue compartment model. [(18)F]FDG images were analysed by calculating standardized uptake values (SUV) and metabolically active tumour volumes (MATV). [(18)F]EF5 images were analysed by calculating tumour-to-muscle uptake ratios (T/M ratio). A T/M ratio of 1.5 was considered a significant threshold and used to determine tumour hypoxic subvolumes (HS) and hypoxic fraction area. The findings were finally correlated with the pretreatment clinical findings (overall stage and TNM stage) as well as the outcome following radiochemotherapy in terms of local control and overall patient survival. RESULTS: Tumour stage and T-classification did not show any significant differences in comparison to the patients' metabolic and functional characteristics measured on PET. Using the Cox proportional hazards model, a shorter overall survival was associated with MATV (p = 0.008, HR = 1.108), maximum [(18)F]EF5 T/M ratio (p = 0.0145, HR = 4.084) and tumour HS (p = 0.0047, HR = 1.112). None of the PET parameters showed a significant effect on patient survival in the log-rank test, although [(18)F]EF5 maximum T/M ratio was the closest (p = 0.109). By contrast, tumour blood flow was not correlated with any of the clinical endpoints. There were no statistically significant correlations among [(18)F]FDG SUVmax, [(18)F]EF5 T/M ratio and blood flow. CONCLUSION: Our study in a limited number of patients confirmed the importance of MATV in the prognosis of locally advanced squamous cell carcinoma of the head and neck. It is of interest that high uptake of the hypoxia tracer [(18)F]EF5 showed a stronger correlation with a poor clinical outcome than [(18)F]FDG uptake. This confirms the importance of hypoxia in treatment outcome and suggests that [(18)F]EF5 may act as a surrogate marker of radioresistance.
Assuntos
Quimiorradioterapia , Etanidazol/análogos & derivados , Fluordesoxiglucose F18 , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/diagnóstico , Hidrocarbonetos Fluorados , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Adulto , Idoso , Feminino , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Multimodal , Prognóstico , Análise de Sobrevida , Adulto JovemRESUMO
OBJECTIVE: Atherosclerotic plaques with large lipid cores and inflammation contain regions of hypoxia. We examined the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide ([18F]EF5), a specific marker of hypoxia labeled for positron emission tomography, in mouse atherosclerotic plaques. METHODS AND RESULTS: Atherosclerotic mice of 2 different genetic backgrounds (low-density lipoprotein receptor-/- apolipoprotein B100/100 and insulin-like growth factor II/low-density lipoprotein receptor-/- apolipoprotein B100/100) were first fed a Western diet to induce development of plaques with variable phenotypes and then injected with [18F]EF5. C57BL/6N mice served as controls. Aortas were dissected for biodistribution studies, autoradiography, histology, and immunohistochemistry. Uptake of [18F]EF5 was significantly higher in the aortas of mice with large atherosclerotic plaques than in the C57BL/6N controls. Furthermore, autoradiography demonstrated, on average, 2.0-fold higher [18F]EF5 uptake in atherosclerotic plaques than in the adjacent normal vessel wall. Hypoxia in plaques was verified by using an EF5 adduct-specific antibody and pimonidazole. The blood clearance of [18F]EF5 was slow, with blood radioactivity remaining relatively high up to 180 minutes after injection. CONCLUSIONS: Large atherosclerotic plaques in mice contained hypoxic areas and showed uptake of [18F]EF5. Despite its slow blood clearance, the high uptake of [18F]EF5 in plaques suggested that plaque hypoxia is a potential target for identifying high-risk plaques noninvasively.
Assuntos
Aorta/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Etanidazol/análogos & derivados , Radioisótopos de Flúor , Hidrocarbonetos Fluorados , Hipóxia/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Análise de Variância , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteína B-100/deficiência , Apolipoproteína B-100/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autorradiografia , Modelos Animais de Doenças , Etanidazol/farmacocinética , Feminino , Radioisótopos de Flúor/farmacocinética , Genótipo , Hidrocarbonetos Fluorados/farmacocinética , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/patologia , Imuno-Histoquímica , Fator de Crescimento Insulin-Like II/deficiência , Fator de Crescimento Insulin-Like II/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitroimidazóis , Fenótipo , Compostos Radiofarmacêuticos/farmacocinética , Receptores de LDL/deficiência , Receptores de LDL/genética , Distribuição TecidualRESUMO
PURPOSE OF THE REPORT: The aim of this study was to evaluate the distribution of hypoxia using 18F-EF5 as a hypoxia tracer in cervical cancer patients with PET/MRI. We investigated the association between this 18F-EF5-PET tracer and the immunohistochemical expression of endogenous hypoxia markers: HIF1α, CAIX, and GLUT1. PATIENTS AND METHODS: Nine patients with biopsy-proven primary squamous cell cervix carcinoma (FIGO 2018 radiological stages IB1-IIIC2r) were imaged with dual tracers 18F-EF5 and 18F-FDG using PET/MRI (Int J Gynaecol Obstet. 2019;145:129-135). 18F-EF5 images were analyzed by calculating the tumor-to-muscle ratio to determine the hypoxic tissue (T/M ratio >1.5) and further hypoxic subvolume (HSV) and percentage hypoxic area. These 18F-EF5 hypoxic parameters were correlated with the size and localization of tumors in 18F-FDG PET/MRI and the results of hypoxia immunohistochemistry. RESULTS: All primary tumors were clearly 18F-FDG and 18F-EF5 PET positive and heterogeneously hypoxic with multiple 18F-EF5-avid areas in locally advanced cancer and single areas in clinically stage I tumors. The location of hypoxia was detected mainly in the periphery of tumor. Hypoxia parameters 18F-EF5 max T/M ratio and HSV in primary tumors correlated independently with the advanced stage (P = 0.036 and P = 0.040, respectively), and HSV correlated with the tumor size (P = 0.027). The location of hypoxia in 18F-EF5 imaging was confirmed with a higher hypoxic marker expression HIF1α and CAIX in tumor fresh biopsies. CONCLUSIONS: The 18F-EF5 imaging has promising potential in detecting areas of tumor hypoxia in cervical cancer.
Assuntos
Hipóxia Tumoral , Neoplasias do Colo do Útero , Hipóxia Celular , Etanidazol , Feminino , Radioisótopos de Flúor , Humanos , Hidrocarbonetos Fluorados , Hipóxia/diagnóstico por imagem , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Neoplasias do Colo do Útero/diagnóstico por imagemRESUMO
PURPOSE: The primary purpose of this study was to assess the biodistribution and radiation dose resulting from administration of (18)F-EF5, a lipophilic 2-nitroimidazole hypoxia marker in ten cancer patients. For three of these patients (with glioblastoma) unlabeled EF5 was additionally administered to allow the comparative assessment of (18)F-EF5 tumor uptake with EF5 binding, the latter measured in tumor biopsies by fluorescent anti-EF5 monoclonal antibodies. METHODS: (18)F-EF5 was synthesized by electrophilic addition of (18)F(2) gas, made by deuteron bombardment of a neon/fluorine mixture in a high-pressure gas target, to an allyl precursor in trifluoroacetic acid at 0° then purified and administered by intravenous bolus. Three whole-body images were collected for each of ten patients using an Allegro (Philips) scanner. Gamma counts were determined in blood, drawn during each image, and urine, pooled as a single sample. PET images were analyzed to determine radiotracer uptake in several tissues and the resulting radiation dose calculated using OLINDA software and standard phantom. For three patients, 21 mg/kg unlabeled EF5 was administered after the PET scans, and tissue samples obtained the next day at surgery to determine EF5 binding using immunohistochemistry techniques (IHC). RESULTS: EF5 distributes evenly throughout soft tissue within minutes of injection. Its concentration in blood over the typical time frame of the study (â¼3.5 h) was nearly constant, consistent with a previously determined EF5 plasma half-life of â¼13 h. Elimination was primarily via urine and bile. Radiation exposure from labeled EF5 is similar to other (18)F-labeled imaging agents (e.g., FDG and FMISO). In a de novo glioblastoma multiforme patient, focal uptake of (18)F-EF5 was confirmed by IHC. CONCLUSION: These results confirm predictions of biodistribution and safety based on EF5's characteristics (high biological stability, high lipophilicity). EF5 is a novel hypoxia marker with unique pharmacological characteristics allowing both noninvasive and invasive measurements.
Assuntos
Neoplasias Encefálicas/metabolismo , Etanidazol/análogos & derivados , Radioisótopos de Flúor , Glioblastoma/metabolismo , Hidrocarbonetos Fluorados/metabolismo , Hidrocarbonetos Fluorados/farmacocinética , Transporte Biológico , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Hipóxia Celular , Etanidazol/metabolismo , Etanidazol/farmacocinética , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Radiometria , Distribuição Tecidual , Imagem Corporal TotalRESUMO
Mechanical overloading of the temporomandibular joint (TMJ) and biochemical changes, like inflammation and hypoxia, contribute to cartilage degeneration and pain associated with osteoarthritis (OA). Yet, how overloading contributes to early dysregulation of chondrocytes is not understood, limiting the development of diagnostics and treatments for TMJ OA. Hypoxia-inducible factors (HIF)-1α/2α in chondrocytes were evaluated at Days 8 and 15 in a rat TMJ pain model induced by jaw loading (1 h/day for 7 days) using immunohistochemistry and compared between cases that induce persistent (3.5 N), acute (2 N), or no (0 N) sensitivity. Hypoxia was measured on Day 8 by immunolabeling of the tracer EF5 and 18 F-EF5 PET imaging. To assess the role of tumor necrosis factor (TNF) in painful TMJ loading, intra-articular etanercept was given before loading. Orofacial sensitivity was evaluated during and after loading. Facial grimace, TNF-α, HIF-2α, and hypoxia levels in the TMJ were measured after loading. HIF-2α was elevated (P = .03) after 3.5 N loading at Day 8, but HIF-1α was unchanged. EF5 uptake increased on Day 8 in the 3.5 N group (P < .048) by tissue assay and 18 F-EF5 PET. At Day 8, both HIF-2α (P = .01) and EF5 uptake (P = .005) were correlated with loading magnitude. Etanercept attenuated sensitivity (P < .01) and the facial grimace on Day 7 (P = .01). It also reduced (P < .01) HIF-2α and EF5 uptake on Day 8; but TNF-α levels were not different from controls at that time. Findings suggest that TMJ loading that induces persistent sensitivity upregulates the catabolic factor HIF-2α and reduces oxygen levels in the cartilage, which may be TNF-driven.
Assuntos
Etanercepte/administração & dosagem , Hipóxia/etiologia , Osteoartrite/tratamento farmacológico , Manejo da Dor/métodos , Articulação Temporomandibular , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Etanidazol/análogos & derivados , Etanidazol/farmacocinética , Feminino , Hidrocarbonetos Fluorados/farmacocinética , Injeções Intra-Articulares , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas de Escherichia coli/administração & dosagem , Genes Reporter , Neoplasias/diagnóstico por imagem , Nitrorredutases/administração & dosagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Antineoplásicos Alquilantes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas de Escherichia coli/genética , Etanidazol/administração & dosagem , Etanidazol/análogos & derivados , Etanidazol/farmacocinética , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Células HCT116 , Humanos , Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/farmacocinética , Imidazóis/administração & dosagem , Indicadores e Reagentes/administração & dosagem , Indicadores e Reagentes/farmacocinética , Camundongos , Imagem Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Compostos de Mostarda Nitrogenada/farmacologia , Compostos de Mostarda Nitrogenada/uso terapêutico , Nitrorredutases/genética , Medicina de Precisão/métodos , Estudo de Prova de Conceito , Compostos Radiofarmacêuticos/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Triazóis/administração & dosagem , Hipóxia Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In radiotherapy, tumor hypoxia is the main factor responsible for treatment resistance, and the development of radiosensitizers that can overcome this is imperative. However, many drugs that are effective in vitro and in vivo fail in clinical trials, and thus it is necessary to develop an animal model that can be used for the correct evaluation of pharmacokinetics and activity. Developing chicken eggs are commonly used in various research fields such as anticancer drug sensitivity tests and cardiotoxicity tests. We examined whether the radiosensitizing activity of etanidazole, as a hypoxic cell radiosensitizer, could be evaluated using tumor-bearing chick embryo. Following the transplantation of mouse mammary carcinoma EMT6 cells on day 11, a solid tumor was formed on day 15 and an evaluation of the time-course of the tumor revealed that the tumor weight was the highest on day 18. The maximum dose of etanidazole that did not affect tumor growth and fetal survival was 1.0mg and the maximum X-ray dose was 8Gy. Etanidazole was intravenously administered 10min prior to single dose X-ray irradiation. A significant tumor growth inhibitory effect was confirmed with 1.0mg of etanidazole in combination with 8Gy X-ray. In the case of mouse colon cancer colon26 cells, the combination of 3.0mg of etanidazole and 2Gy X-ray showed 2.79 times higher radiosensitizing activity than that observed for the control group. These results demonstrate that it is possible to evaluate the activity of radiosensitizers using tumor-bearing chick embryo.
Assuntos
Neoplasias da Mama/patologia , Avaliação Pré-Clínica de Medicamentos , Etanidazol/farmacologia , Radiossensibilizantes/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Embrião de Galinha , Camundongos , Raios XRESUMO
BACKGROUND AND PURPOSE: The usefulness of hyperbaric oxygen (HBO) and normobaric hyperoxia in acute ischemic stroke is being reexplored because both improve outcome in experimental cerebral ischemia. However, even the basic mechanisms underlying oxygen therapy are poorly understood. We investigated the effect of both oxygen therapies on tissue hypoxia and on the transcription factor hypoxia-inducible factor-1 alpha. METHODS: Mice were subjected to filament-induced middle cerebral artery occlusion for 2 hours. Twenty-five minutes after filament introduction, mice breathed normobaric air, normobaric 100% O(2) (normobaric hyperoxia), or 100% O(2) at 3 ata (HBO) for 95 minutes. Hypoxic regions were mapped on tissue sections after preischemic infusion of the in vivo hypoxia marker EF-5. Hypoxia-inducible factor-1 alpha protein was measured after 2-hour middle cerebral artery occlusion using immunofluorescence and immunoblotting. Vascular endothelial growth factor expression was analyzed using in situ mRNA hybridization. RESULTS: Severity of ischemia did not differ among groups. HBO (35.2+/-10.4 mm(2)) significantly reduced the area of EF-5-stained hypoxic regions in focal cerebral ischemia compared with normobaric hyperoxia (46.4+/-11.2 mm(2)) and air (49.1+/-8 mm(2), P<0.05, analysis of variance). Topographically, EF-5 fluorescence was decreased in medial striatum and in cortical ischemic border areas. Immunohistochemistry and immunoblotting revealed lower hypoxia-inducible factor-1 alpha protein in the ischemic hemisphere of HBO-treated mice. Moreover, mRNA in situ hybridization showed lower expression of vascular endothelial growth factor in HBO and normobaric hyperoxia groups. CONCLUSIONS: Measurement of extrinsic and intrinsic markers of hypoxia revealed that HBO improves penumbral oxygenation in focal ischemia. Modification of the transcription factor hypoxia-inducible factor-1 alpha and its downstream targets may be involved in effects of HBO.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Oxigenoterapia Hiperbárica , Hipóxia Encefálica/etiologia , Hipóxia Encefálica/fisiopatologia , Fator 1 Induzível por Hipóxia/metabolismo , Animais , Encéfalo/metabolismo , Isquemia Encefálica/complicações , Isquemia Encefálica/etiologia , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Regulação para Baixo , Etanidazol/análogos & derivados , Etanidazol/farmacocinética , Imunofluorescência , Hidrocarbonetos Fluorados/farmacocinética , Hipóxia Encefálica/metabolismo , Indicadores e Reagentes/farmacocinética , Infarto da Artéria Cerebral Média/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/uso terapêutico , Consumo de Oxigênio , RNA Mensageiro/metabolismo , Coloração e Rotulagem , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
UNLABELLED: The aim of this study was to evaluate 2-(2-nitro-(1)H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) labeled with (18)F-fluorine to image hypoxia in patients with squamous cell carcinoma of the head and neck (HNSCC). METHODS: Fifteen patients with HNSCC were studied. Measurement of tumor blood flow was followed by an (18)F-EF5 PET/CT scan. On a separate day, (18)F-FDG PET/CT was performed to determine the metabolically active tumor volume. In 6 patients, dynamic (18)F-EF5 images of the head and neck area were acquired, followed by static images acquired at 1, 2, and 3 h after injection. In the remaining 9 patients, only static images were obtained. (18)F-EF5 uptake in tumors was compared with that in neck muscle, and the (18)F-EF5 findings were correlated with the (18)F-FDG PET/CT studies. RESULTS: A total of 13 primary tumors and 5 lymph node metastases were evaluated for their uptake of (18)F-EF5. The median tumor-to-muscle (18)F-EF5 uptake ratio (T/M) increased over time and was 1.38 (range, 1.1-3.2) 3 h after tracer injection. The median blood flow in tumors was 36.7 mL/100 g/min (range, 23.3-78.6 mL/100 g/min). Voxel-by-voxel analysis of coregistered blood flow and (18)F-EF5 images revealed a distinct pattern, resulting in a T/M of 1.5 at 3 h to be chosen as a cutoff for clinically significant hypoxia. Fourteen of 18 tumors (78%) had subvolumes within the metabolically active tumor volumes with T/M greater than or equal to 1.5. CONCLUSION: On the basis of these data, the potential of (18)F-EF5 to detect hypoxia in HNSCC is encouraging. Further development of (18)F-EF5 for eventual targeting of antihypoxia therapies is warranted.
Assuntos
Carcinoma de Células Pequenas/diagnóstico por imagem , Carcinoma de Células Pequenas/metabolismo , Etanidazol/análogos & derivados , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/metabolismo , Hidrocarbonetos Fluorados/farmacocinética , Oxigênio/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Adulto , Idoso , Hipóxia Celular , Etanidazol/farmacocinética , Feminino , Radioisótopos de Flúor/farmacocinética , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Radiofarmacêuticos/farmacocinética , Sensibilidade e Especificidade , Adulto JovemRESUMO
PURPOSE: To study the changes in hypoxia resulting from mild temperature hyperthermia (MTH) in a subcutaneous xenograft model using dual-tracer immunohistochemical techniques. MATERIALS AND METHODS: HT29 tumors were locally heated at 41 degrees C. Changes in tumor hypoxia were investigated by pimonidazole and EF5. Pimonidazole was given 1h preheating, EF5 at various times during or after treatment, 1h later the animals were sacrificed. Blood vessels were identified by CD31 staining, and perfusion by Hoechst 33342 injected 1 min pre-sacrifice. RESULTS: The overall hypoxic fraction was significantly decreased by MTH during and immediately after heating. However, MTH induced both increases and decreases in tumor hypoxia in different parts of the tumor. Specifically, MTH decreased hypoxia in the regions with relatively well-perfused blood vessels, but increased hypoxia in regions that were poorly perfused. At 24-h post heating, newly formed hypoxic regions surrounded previously-hypoxic foci, which in turn surrounded pimonidazole-stained debris. Quantitative analysis did not evince changes in tumor oxygenation due to MTH at 24h post-treatment. CONCLUSION: In this xenograft model, the effect of MTH on tumor oxygenation was variable, both spatially and kinetically. Overall tumor oxygenation was improved during and after heating, but the effect was short-lived.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Hipertermia Induzida , Imuno-Histoquímica/métodos , Oxigênio/metabolismo , Radiossensibilizantes/farmacologia , Adenocarcinoma/irrigação sanguínea , Análise de Variância , Animais , Hipóxia Celular , Neoplasias Colorretais/irrigação sanguínea , Etanidazol/análogos & derivados , Etanidazol/farmacologia , Feminino , Hidrocarbonetos Fluorados/farmacologia , Camundongos , Nitroimidazóis/farmacologia , Transplante HeterólogoRESUMO
RTOG 95-02 assessed patient tolerance to hypoxic cell radiosensitizer, etanidazole (SR-2508), combined with radiosurgery. Patients had primary or metastatic brain tumors and previously localized or whole brain irradiation. The toxicity is reported in three groups of patients according to the tumor size. Etanidazole doses of 12g/m2 combined with radiosurgery were well tolerated.
Assuntos
Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirurgia , Etanidazol/uso terapêutico , Radiossensibilizantes/uso terapêutico , Radiocirurgia/métodos , Adulto , Neoplasias Encefálicas/secundário , Terapia Combinada , Humanos , Recidiva Local de Neoplasia , Dosagem Radioterapêutica , Taxa de Sobrevida , Resultado do TratamentoRESUMO
The binding kinetics of the hypoxia marker EF5 can be quantified by uptake of (14)C-labeled drug or calibrated flow cytometry using antibodies specific for drug adducts. Maximum EF5 binding is cell-line dependent and varies directly with drug exposure (area under the curve; concentration integrated over time) but inversely with pO(2) from 0 to >100 mmHg. For pimonidazole, binding is reported to be independent of the cell line and drug AUC, being zero above 10 mmHg, with an easily discriminated increase at lower pO(2). The basis for these kinetic differences is unknown, but the main experimental variable distinguishing the two marker techniques is antibody concentration ([Ab] - pimonidazole << EF5). In this study, EF5 and pimonidazole binding kinetics were compared as a function of pO(2) and antibody concentration in cells of two rat (9L and R3230) and two human (HT1080 and SiHa) cancer cell lines. For both markers, binding varied directly with AUC at all pO(2). The dynamic range of observed binding (maximum change from 0 to 76 mmHg oxygen) decreased with antibody concentration. The pO(2) dependence of binding for pimonidazole, but not EF5, varied dramatically with antibody concentration. Thus the data presented herein do not support the reported binding kinetics of pimonidazole. In particular, it is shown that the common use of antibody concentrations much lower than antigen concentrations can lead to unreliable estimations of adduct level and hence pO(2).
Assuntos
Hipóxia Celular , Etanidazol/análogos & derivados , Citometria de Fluxo/métodos , Hidrocarbonetos Fluorados/farmacologia , Nitroimidazóis/farmacologia , Radiossensibilizantes/farmacologia , Animais , Área Sob a Curva , Calibragem , Linhagem Celular Tumoral , Etanidazol/farmacologia , Humanos , Cinética , Microscopia de Fluorescência/métodos , Oxigênio/química , Oxigênio/metabolismo , Ratos , Fatores de TempoRESUMO
Tissue hypoxia results from the interaction of cellular respiration, vascular oxygen carrying capacity, and vessel distribution. We studied the relationship between tumor vasculature and regions of low pO(2) using quantitative analysis of binding of the 2-nitroimidazole EF5 given to patients intravenously (21 mg/kg) approximately 24 h preceding surgery. We describe new computer algorithms for determining EF5 binding as a function of radial distance from individual blood vessels and converting this value to tissue pO(2). Tissues from six human brain tumors were assessed. In a hemangiopericytoma, a WHO Grade 2 and WHO Grade 3 glial brain tumor, all tissue pO(2) values calculated by EF5 binding were >20 mmHg (described as "physiologically oxygenated"). In these three tumors, EF5 binding gradients (measured as a function of distance from each observed vessel) were low, with small positive and negative values averaging close to zero. Much lower tissue oxygen levels were found, including near some vessels, in glioblastomas. Gradients of EF5 binding away from vessels were larger in glioblastomas than in the low-grade tumors, but positive and negative values again averaged to near zero. Based on these preliminary data, we hypothesize a new paradigm for tumor blood flow in human brain tumors whereby in-flowing and out-flowing blood patterns may have contrasting effects on average tissue EF5 (and by inference, oxygen) gradients. Our studies also imply that neither distance to the nearest blood vessel nor distance from each observed blood vessel provide reliable estimates of tissue pO(2).
Assuntos
Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Hipóxia/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Animais , Neoplasias Encefálicas/metabolismo , Etanidazol/análogos & derivados , Etanidazol/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Hidrocarbonetos Fluorados/metabolismo , Camundongos , Pessoa de Meia-Idade , Oxigênio/metabolismoRESUMO
PURPOSE: (19)F magnetic resonance spectroscopy (MRS) was used to non-invasively detect EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] adducts in the Shionogi tumour model of prostate cancer to evaluate hypoxia. MATERIAL AND METHODS: (19)F MRS signal of EF5 in Shionogi mouse tumours was acquired using a 2 cm diameter solenoid volume coil with a 7.05 T Bruker scanner. MRS signal was observed in mouse tumours longitudinally following intraperitoneal (IP) injection of EF5. Another mouse group was injected intravenously (IV) with EF5, and in vivo MRS signal was obtained two hours after injection. This data was compared with the ex vivo percentage of hypoxic cells present in the corresponding excised tumours, determined by flow cytometry of bound EF5. RESULTS: Longitudinal (19)F MRS signal attributable to EF5 began to decline within five hours of EF5 administration. Flow cytometry comparisons yielded an inverse correlation (p-value < 0.006) between the MRS signal and tumour hypoxic cell percentage. The tumours exhibited an average cell viability of 34 +/- 26%. CONCLUSIONS: The results confirmed that MRS of EF5 in mice is an unsuitable technique for the determination of EF5 binding as a measure of tumour hypoxia.
Assuntos
Etanidazol/análogos & derivados , Hidrocarbonetos Fluorados , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos , Animais , Hipóxia Celular , Modelos Animais de Doenças , Feminino , Radioisótopos de Flúor , Espectroscopia de Ressonância Magnética , Masculino , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Neoplasias Hormônio-Dependentes/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , CintilografiaRESUMO
PURPOSE: Tumor hypoxia contributes to radiation resistance. A noninvasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia-targeted therapies. 18F-pentafluorinated etanidazole (18F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by 18F-EF5 PET with clinical outcomes after radiation therapy remains limited. METHODS AND MATERIALS: Our study prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment 18F-EF5 PET imaging. Depending on the level of 18F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dichloroacetate followed by repeated 18F-EF5 PET. The hypoxic subvolume of tumor was defined as the proportion of tumor voxels exhibiting higher 18F-EF5 uptake than the 95th percentile of 18F-EF5 uptake in the blood pool. Tumors with a hypoxic subvolume ≥ 10% on baseline 18F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess the correlation between imageable hypoxia and clinical outcomes after treatment. RESULTS: At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with stereotactic ablative radiation therapy and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while dichloroacetate did not result in a significant change under our protocol conditions. Tumors with imageable hypoxia had a higher incidence of local recurrence at 12 months (30%) than those without (0%) (P < .01). CONCLUSIONS: Noninvasive hypoxia imaging by 18F-EF5 PET identified imageable hypoxia in about 40% of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia.
Assuntos
Etanidazol/análogos & derivados , Radioisótopos de Flúor , Hidrocarbonetos Fluorados , Recidiva Local de Neoplasia/diagnóstico por imagem , Neoplasias/radioterapia , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Radioterapia Conformacional , Hipóxia Tumoral , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Neoplasias/mortalidade , Modelos de Riscos Proporcionais , Estudos ProspectivosRESUMO
PURPOSE: EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels, and prognostic significance of hypoxia in primary head and neck squamous cell tumors. METHODS AND MATERIALS: Twenty-two patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analyses of EF5 immunofluorescence was carried out, and these data were compared with patient outcome. RESULTS: EF5 immunostaining showed substantial intra- and intertumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria based on the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5-binding levels with event-free and overall survival irrespective of the pattern of cellular binding or treatment regimen. Patients with tumors containing EF5-binding regions corresponding to severe hypoxia (< or =0.1% oxygen) had a shorter event-free survival time than patients with pO(2) values greater than 0.1% (p = 0.032). Nodal status was also predictive for outcome. CONCLUSIONS: These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO(2) and provide support for the development of noninvasive hypoxia positron emission tomographic studies with fluorine 18-labeled EF5.