2-(2-nitro-1h-imidazol-1-yl)-n-(2-2-3-3-3-pentafluoropropyl)acetamide and Hypoxia

In the past 25 y, a large amount of clinical experience with hypoxia PET tracers has accumulated. This article discusses recent improvements in image acquisition protocols and tracer pharmacology that have resulted in improved understanding of the underlying physiologic processes. The widespread clinical adoption of hypoxia PET tracers will depend largely on their utility in treatment prescription and in outcome monitoring. The establishment and validation of hypoxia-directed treatment protocols are still under development, and it is envisaged that the design and use of future hypoxia PET tracers will develop as part of this process.

Topics: Etanidazole; Humans; Hydrocarbons, Fluorinated; Hypoxia; Nitroimidazoles; Positron-Emission Tomography; Radioactive Tracers; Triazoles

We have established basic methods, using quantitative measures of EF5 binding, to estimate the actual pO2 of cells and tissues. In situations where the tissue can be dissociated into single cells, or for cell cultures, we can measure the distribution of cellular binding rates using flow cytometry and these can be compared with cells treated under pO2S controlled by the spinner vial or thin-film methods in vitro. The flow cytometer is calibrated by staining V79 cells treated with EF5 under "standard" conditions. For intact tissues treated with EF5 in vivo, we need to correct for possible variations in drug exposure (AUC). Frozen sections are stained for EF5 binding and are analyzed by a sensitive (cooled) CCD camera with linear output vs fluorescence [figure: see text] input. The camera has very consistent sensitivity, but the entire optical system, including the camera, can be calibrated by an absolute fluorescence standard (dye in hemocytometer). This system can also be used to measure the fluorescence of the flow cytometer standards, providing a direct link between the two assays. We can measure the maximum binding rate using the tissue cube method, but need to assume an "average" oxygen dependence of binding for intact tissues. The best-fit approximation for existing data is an inverse relationship between binding and pO2, with binding decreasing 50-fold between 0.1 and 10% oxygen. Using these methods, we routinely estimate the minimum pO2 (maximum binding) in experimental rodent and human tumors. In normal tissue models, an excellent correlation is found between near-maximal binding (severe hypoxia) and apoptosis (heart infarct and ductus arteriosus). Some normal tissues (e.g., skeletal muscle) are refractory to both cellular disaggregation and cube calibration methods. To extend the tissue imaging measurements to a complete two- or three-dimensional analysis of the distribution of tissue pO2s requires a substantial additional investment of imaging methods, which are currently being implemented.

Topics: Animals; Biochemistry; Etanidazole; Flow Cytometry; Humans; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Mice; Neoplasms; Oxygen; Pressure; Time Factors

Pharmacokinetic studies were performed on the first 28 patients enrolled in a phase I trial to determine the ability of EF5 [2-(2-nitro-1-H-imidazolI-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] to detect hypoxia in human tumors in the absence of patient toxicity.. EF5 was made in purified form and formulated for intravenous injection by the National Cancer Institute. After obtaining consent from the patients, EF5 was administered and blood samples were drawn at various times over approximately 48 h. For most patients it was possible to collect total urine at approximately 8-h intervals. EF5 in plasma and urine was analyzed by high-performance liquid chromatography.. EF5's plasma concentration followed a simple exponential decay following infusion. The plasma half-life was 11.7 +/- 2.6 h (+/- SD) and was not affected by drug dose (9 to 28 mg/kg), fractional urine recovery, patient weight or gender. Absolute plasma values suggested even biodistribution of the drug throughout the soft tissue with a volume of distribution equal to 0.56 l/ kg. Despite the relatively high lipid partition coefficient (logP = 0.6), EF5 was excreted primarily (up to 70%) via kidney clearance. No drug metabolites (e.g. retaining the 2-nitroimidazole chromophore) were detected in either plasma or urine. No toxicity was found at drug doses adequate to detect tumor hypoxia.. Currently held paradigms of 2-nitroimidazole metabolism (e.g. clearance rate and toxicity as affected by octanol/ water partition coefficient) are discussed. The results reported herein suggest that EF5 is biologically stable with predictable pharmacokinetics. EF5's consistent half-life and clearance properties will allow quantitative analysis of EF5 binding relative to tissue oxygen levels.

Topics: Area Under Curve; Body Weight; Chromatography, High Pressure Liquid; Etanidazole; Half-Life; Humans; Hydrocarbons, Fluorinated; Hypoxia; Maximum Tolerated Dose; Metabolic Clearance Rate; Neoplasms; Predictive Value of Tests; Radiation-Sensitizing Agents; Sex Factors; Tissue Distribution

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

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Etanercept; Etanidazole; Female; Hydrocarbons, Fluorinated; Hypoxia; Injections, Intra-Articular; Osteoarthritis; Pain Management; Rats; Rats, Sprague-Dawley; Temporomandibular Joint; Tumor Necrosis Factor-alpha

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.. 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.. 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.. 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.

Topics: Aged; Antineoplastic Agents, Alkylating; Etanidazole; Feasibility Studies; Female; Follow-Up Studies; Humans; Hydrocarbons, Fluorinated; Hypoxia; Indicators and Reagents; Male; Melanoma; Melphalan; Middle Aged; Neoplasm Staging; Pilot Projects; Prognosis; Prospective Studies

The syntheses of new nitroimidazole compounds using silicon-[(18)F]fluorine chemistry for the potential detection of tumor hypoxia are described. [(18)F]silicon-based compounds were synthesized by coupling 2-nitroimidazole with silyldinaphtyl or silylphenyldi-tert-butyl groups and labeled by fluorolysis or isotopic exchange. Dinaphtyl compounds (6, 10) were labeled in 56-71% yield with a specific activity of 45 GBq/μmol, however these compounds ([(18)F]7 and [(18)F]11) were not stable in plasma. Phenyldi-tert-butyl compounds were labeled in 70% yield with a specific activity of 3 GBq/μmol by isotopic exchange, or in 81% yield by fluorolysis of siloxanes with a specific activity of 45 GBq/μmol. The labeled compound [(18)F]18 was stable in plasma and excreted by the liver and kidneys in vivo. In conclusion, the fluorosilylphenyldi-tert-butyl (SiFA) group is more stable in plasma than fluorosilyldiphenyl moiety. Thus, compound [(18)F]18 is suitable for further in vivo assessments.

Topics: Animals; Fluorine Radioisotopes; Humans; Hypoxia; Nitroimidazoles; Positron-Emission Tomography; Rats; Rats, Wistar; Silicon; Tissue Distribution

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.. 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.. 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.

Topics: Analysis of Variance; Animals; Aorta; Apolipoprotein B-100; Atherosclerosis; Autoradiography; Disease Models, Animal; Etanidazole; Female; Fluorine Radioisotopes; Genotype; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Insulin-Like Growth Factor II; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitroimidazoles; Phenotype; Positron-Emission Tomography; Radiopharmaceuticals; Receptors, LDL; Tissue Distribution

The endothelin-1 antagonist, Atrasentan (ABT-627) was used to modify perfusion in the human tumor xenograft model, HT29, growing in nude mice. Atrasentan produced a significant increase in perfusion, as measured in vivo by Gd-DTPA DCE-MRI. Changes in tumor hypoxia were assessed by comparing the binding of two hypoxia tracers, pimonidazole and EF5 given before and after Atrasentan administration. In vehicle-treated controls, the distribution of EF5 and pimonidazole was very similar. However, Atrasentan treatment was associated with decreased uptake of the second hypoxia tracer (EF5), relative to the first (pimonidazole). Although Atrasentan had no independent effect on the growth of HT29 tumors, Atrasentan combined with 20 Gy radiation led to a modest but significant increase in tumor growth delay compared to radiation alone.

Topics: Adenocarcinoma; Animals; Atrasentan; Colonic Neoplasms; Combined Modality Therapy; Etanidazole; Gadolinium DTPA; HT29 Cells; Humans; Hydrocarbons, Fluorinated; Hypoxia; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Nitroimidazoles; Perfusion; Pyrrolidines; Radiation-Sensitizing Agents; Radiotherapy; Treatment Outcome; Xenograft Model Antitumor Assays

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).

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Algorithms; Animals; Brain Neoplasms; Etanidazole; Glioblastoma; Humans; Hydrocarbons, Fluorinated; Hypoxia; Mice; Middle Aged; Oxygen

We have identified the presence of the hypoxia marker EF5 in the stage 4/5 chick heart fields. This suggests that cardiac cell differentiation occurs in a relatively anaerobic environment. Monocarboxylate transporter (MCT) studies in adult cardiac myocytes have demonstrated that MCTs catalyze proton-linked pyruvate and lactate transport activity. 5A11/Basigin is an ancillary protein that targets MCTs to the plasma membrane for their function. MCT-4 expression is most evident in cells with a high glycolytic rate associated with hypoxic energy production. Subsequent to the immunohistochemical localization of EF5 in the early heart field, we continued in our analysis during stages 5 to 12 for the expression of indicators of cellular glycolytic metabolism in the developing heart, such as MCT-4, MCT-1, and 5A11 (Basigin/CD147). Our observations indicate that MCT-4 and 5A11/Basigin are expressed early, in a differential left-right pattern, in the bi-lateral plate mesoderm, as the cardiac compartment is forming. At stage 11, MCT-4/5A11 continues to be highly expressed in the myocardial wall of the looping heart, but not in the dorsal mesocardium. RT-PCR analyses for MCT-1, -4, and 5A11 indicate that MCT-4 and 5A11 are expressed throughout precardiac, embryonic, and fetal stages in the heart. MCT-1 is first detected in the heart on embryonic day 3 and then remains expressed throughout development to hatching. These results indicate that cardiac precursor cells are equipped for differentiating in a hypoxic environment using anaerobic metabolism for energy production.

Topics: Animals; Avian Proteins; Basigin; Biomarkers; Cell Differentiation; Chick Embryo; Etanidazole; Hydrocarbons, Fluorinated; Hypoxia; Monocarboxylic Acid Transporters; Myocytes, Cardiac

The oxygen status of skin is a controversial topic. Skin is radiosensitive, suggesting it is well-oxygenated. However, it can be further sensitized with nitroimidazole drugs, implying that it is partially hypoxic. Skin oxygen levels are difficult to measure with either electrodes or the hypoxia-monitoring agent (3)H-misonidazole. For the latter, binding has previously been reported to be high in murine skin, but this could be attributed to either non-oxygen-dependent variations in nitroreductase activity, drug metabolism, and/or actual oxygen gradients. We obtained tumor and skin from patients given EF5, a 2-nitroimidazole tissue hypoxia monitor. We performed immunohistochemical studies using highly specific monoclonal antibodies for the hypoxia-dependent production of EF5 tissue adducts. Some tissue sections were counterstained using either Ki67 for proliferation or CD31 for vessels. We found that the human dermis is well-oxygenated, the epidermis is modestly hypoxic and portions of some sebaceous glands and hair follicles are moderately to severely hypoxic. Normal and irradiated skin had similar oxygenation patterns. Control studies demonstrated that these observations are not due to tissue variations in nitroreductase activity. The importance of the highly heterogeneous distribution of oxygen in skin requires further study, but recent investigations suggest that skin hypoxia may have important clinical ramifications including mediating cellular transformation.

Topics: Aged; Antibodies, Monoclonal; Dermis; Etanidazole; Female; Fluorescence; Humans; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Indicators and Reagents; Male; Neoplasms; Nitroreductases; Oxygen; Partial Pressure; Skin; Staining and Labeling; Tissue Distribution

Topics: Antineoplastic Agents; Brain Neoplasms; Etanidazole; Glioma; Humans; Hydrocarbons, Fluorinated; Hypoxia; Necrosis; Oxygen; Prognosis; Reproducibility of Results

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.

Topics: Animals; Apoptosis; Chick Embryo; DNA Primers; Etanidazole; Gene Expression; Heart; Hydrocarbons, Fluorinated; Hyperoxia; Hypoxia; Immunohistochemistry; Indicators and Reagents; Morphogenesis; Myocardium; Quail; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O(2) followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2-4) as compared with control mice (1-3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.

Topics: Animals; Disease Models, Animal; Etanidazole; Female; Humans; Hydrocarbons, Fluorinated; Hypoxia; Indicators and Reagents; Luminescent Proteins; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Mice; Mice, SCID; Microcirculation; Microscopy, Fluorescence; Oxygen; Uterine Cervical Neoplasms

We investigated whether increasing levels of tissue hypoxia, measured by the binding of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] or by Eppendorf needle electrodes, were associated with tumor aggressiveness in patients with previously untreated glial brain tumors. We hypothesized that more extensive and severe hypoxia would be present in tumor cells from patients bearing more clinically aggressive tumors. Hypoxia was measured with the 2-nitroimidazole imaging agent EF5 in 18 patients with supratentorial glial neoplasms. In 12 patients, needle electrode measurements were made intraoperatively. Time to recurrence was used as an indicator of tumor aggression and was analyzed as a function of EF5 binding, electrode values and recursive partitioning analysis (RPA) classification. On the basis of EF5 binding, WHO grade 2 tumors were characterized by modest cellular hypoxia (pO2s approximately 10%) and grade 3 tumors by modest-to-moderate hypoxia (pO2s approximately 10%- 2.5%). Severe hypoxia (approximately 0.1% oxygen) was present in 5 of 12 grade 4 tumors. A correlation between more rapid tumor recurrence and hypoxia was demonstrated with EF5 binding, but this relationship was not predicted by Eppendorf measurements.

Topics: Aged; Brain Neoplasms; Electrodes; Etanidazole; Glioma; Humans; Hydrocarbons, Fluorinated; Hypoxia; Indicators and Reagents; Middle Aged; Needles; Neoplasm Recurrence, Local; Radiography; Time Factors

Postnatal constriction of the full-term ductus arteriosus produces hypoxia of the muscle media. This is associated with anatomic remodeling (including smooth muscle death) that prevents subsequent reopening. We used late-gestation fetal and neonatal lambs to determine which factors are responsible for the postnatal hypoxia. Hypoxia [measured by 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide technique] and cell death (measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique) were observed in regions of the constricted ductus wall within 4 h after delivery. Although there was a decrease in ductus luminal flow during the first 6 h after delivery (measured by Doppler transducer), the amount of oxygen delivered to the ductus lumen (3070 +/- 1880 micromol O2 x min(-1) x g(-1)) far exceeded the amount of oxygen consumed by the constricted ductus (0.052 +/- 0.021 micromol O2 x min(-1) x g(-1), measured in vitro). Postnatal constriction increased the effective oxygen diffusion distance across the ductus wall to >3x the limit that can be tolerated for normal tissue homeostasis. This was owing to both an increase in the thickness of the ductus (fetus, 1.12 +/- 0.20 mm; newborn, 1.60 +/- 0.17 mm; p < 0.01) and a marked reduction in vasa vasorum flow (fetus, 0.99 +/- 0.44 mL x min(-1) x g(-1); newborn, 0.21 +/- 0.08 mL x min(-1) x g(-1); p < 0.01). These findings suggest that hypoxic cell death in the full-term ductus is caused primarily by changes in vasa vasorum flow and muscle media thickness and can occur before luminal flow has been eliminated. We speculate that in contrast with the full-term ductus, the preterm ductus is much less likely to develop the degree of hypoxia needed for vessel remodeling inasmuch as it only is capable of increasing its oxygen diffusion distance to 1.3x the maximally tolerated limit.

Topics: Animals; Animals, Newborn; Antibodies, Monoclonal; Cell Death; Cell Hypoxia; Ductus Arteriosus; Etanidazole; Gestational Age; Heart; Humans; Hydrocarbons, Fluorinated; Hypoxia; In Situ Nick-End Labeling; Indicators and Reagents; Models, Biological; Oxygen; Regional Blood Flow; Sheep; Vasa Vasorum

There is a great deal of clinical and experimental interest in determining tissue hypoxia using non-invasive imaging methods. We have developed EF5, 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, with both invasive and non-invasive hypoxia detection in mind. EF5 and other 2-nitroimidazoles are used to detect hypoxia, because the rate of their bioreductive metabolism is inversely dependent on oxygen partial pressure. Such metabolism leads to the formation of covalent adducts within the metabolizing cells. Previously, we have described the invasive detection of these adducts by highly specific monoclonal antibodies after tissue biopsy. In this report, we demonstrate the synthesis of 18F-labeled EF5, [18F]-2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, in greater than 10% yield by direct fluorination of the newly synthesized precursor 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,3,3-trifluoroallyl)-acetamide by [18F]-F2 in trifluoroacetic acid. Our objective was to optimize the electrophilic fluorination of the fluorinated alkene bond with fluorine gas, a new method of 18F-labeling of polyfluorinated molecules. Previous biodistribution studies in mice have demonstrated uniform access of EF5 to all tissues with bioelimination dominated by renal excretion. When [18F]-EF5 was injected into a rat followed by urine collection and analysis, we found no detectable metabolism to other radioactive compounds. Thus, [18F]-EF5 should be well suited for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Topics: Animals; Chromatography, High Pressure Liquid; Etanidazole; Fluorine Radioisotopes; Humans; Hydrocarbons, Fluorinated; Hypoxia; Indicators and Reagents; Radiopharmaceuticals; Tomography, Emission-Computed

Clinical trials utilizing strategies to manipulate tumor oxygenation, blood flow and angiogenesis are under way, although limited quantitative information exists regarding basic tumor pathophysiology. The current study utilized murine KHT fibrosarcomas, spontaneous mammary carcinomas and first-generation spontaneous transplants to examine heterogeneity in vascular structure and function, to relate these changes to the distribution of tumor hypoxia and to determine whether fundamental relationships among the different pathophysiological parameters exist. Three methods were included: (i) immunohistochemical staining of anatomical and perfused blood vessels, (ii) cryospectrophotometric measurement of intravascular oxyhemoglobin saturations and (iii) fluorescent detection of the EF5 hypoxic marker. While a distinct pattern of decreasing oxygenation with increasing distance from the tumor surface was observed for KHT tumors, striking intertumor variability was found in both spontaneous and first-generation transplants, with a reduced dependence on tumor volume. EF5 hypoxic marker uptake was also much more heterogeneous among individual spontaneous and first-generation tumors compared to KHT. Although mammary carcinomas demonstrated fewer anatomical blood vessels than fibrosarcomas, the proportion of perfused vessels was substantially reduced in KHT tumors, especially at larger tumor volumes. Vascular morphology, tissue histological appearance and pathophysiological parameters differed substantially between KHT tumors and both spontaneous and first-generation tumors. Such differences in vascular structure and function are also likely to correlate with altered response to therapies targeted to the vascular system. Finally, spontaneous differentiation status, tumor morphology, vascular configuration and function were well preserved in first-generation transplanted tumors, suggesting a close relationship between vascular development and function in early-generation transplants and spontaneous tumor models.

Topics: Animals; Borates; Calcium Compounds; Disease Models, Animal; Etanidazole; Female; Hydrocarbons, Fluorinated; Hypoxia; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; Oxygen; Perfusion; Radiation-Sensitizing Agents

After birth, the full-term ductus arteriosus actively constricts and undergoes extensive histologic changes that prevent subsequent reopening. These changes are thought to occur only if a region of intense hypoxia develops within the ductus wall after the initial active constriction. In preterm infants, indomethacin-induced constriction of the ductus is often transient and is followed by reopening. Prostaglandins and nitric oxide both play a role in inhibiting ductus closure in vitro. We hypothesized that combined inhibition of both prostaglandin and nitric oxide production (with indomethacin and N-nitro-L-arginine (L-NA), respectively) may be required to produce the degree of functional closure that is needed to cause intense hypoxia. We used preterm (0.67 gestation) newborn baboons that were mechanically ventilated for 6 d: 6 received indomethacin alone, 7 received indomethacin plus L-NA, and 16 received no treatment (control). Just before necropsy, only 25% of control ductus and 33% of indomethacin-treated ductus were closed on Doppler examination; in contrast, 100% of the indomethacin-plus-L-NA-treated ductus were closed. Control and indomethacin-treated baboons developed negligible-to-mild ductus hypoxia (EF5 technique). Similarly, there was minimal evidence of ductus remodeling. In contrast, indomethacin-plus-L-NA-treated baboons developed intense hypoxia in regions where the ductus was most constricted. The hypoxic muscle strongly expressed vascular endothelial growth factor, and proliferating luminal endothelial cells filled and occluded the lumen. In addition, cells in the most hypoxic regions were undergoing DNA fragmentation. In conclusion, preterm newborns are capable of remodeling their ductus, just like the full-term newborn, if they can reduce their luminal blood flow to a point that produces intense ductus wall hypoxia. Combined prostaglandin and nitric oxide inhibition may be necessary to produce permanent closure of the ductus and prevent reopening in preterm infants.

Topics: Animals; Animals, Newborn; Bisbenzimidazole; Blood Pressure; Cardiovascular Agents; DNA Fragmentation; Ductus Arteriosus; Endothelial Growth Factors; Enzyme Inhibitors; Etanidazole; Fetus; Fluorescent Dyes; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Indicators and Reagents; Indomethacin; Lymphokines; Nitric Oxide; Nitroarginine; Papio; Prostaglandins; Respiratory Physiological Phenomena; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia-ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90-150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90-150 min of hypoxia-ischemia was associated with intense immunofluorescence in the hemisphere ipsilateral to the carotid occlusion, with a pattern similar to that reported previously for the histological damage seen in this model. This study provides a sensitive method for the evaluation of the level of oxygen depletion in brain tissue after neonatal hypoxia-ischemia at times much earlier than any method demonstrates apoptotic or necrotic cell death Since the level of in vivo formation of macromolecular adducts of EF5 depends on the degree of oxygen depletion in a tissue, intracellular

Topics: Acute Disease; Animals; Animals, Newborn; Antibodies, Monoclonal; Brain; Cell Hypoxia; Cerebral Cortex; Chronic Disease; Corpus Striatum; Etanidazole; Female; Functional Laterality; Heart Defects, Congenital; Hippocampus; Hydrocarbons, Fluorinated; Hypothalamus; Hypoxia; Indicators and Reagents; Ischemic Attack, Transient; Male; Mice; Oxidation-Reduction; Rats; Rats, Inbred WKY; Thalamus

E2F1+/- mice subjected to 2 h middle cerebral artery occlusion developed an infarct of 77.0 +/- 3.2 mm3 (mean +/- s.e.m., n = 15) in the ischemic hemisphere after 24 h reperfusion. A significantly smaller infarct of 58.8 +/- 4.8 mm3 (n = 15; p < 0.01) was found in E2F1-/- animals. Both deficient and normal mice had similar cerebral angioarchitecture and intra-ischemic decreases in regional blood flow. Similar areas of hypoxia in both groups of ischemic animals were demonstrated directly by immunohistochemical detection of nitroimidazole adducts. It was concluded that all animals received the same ischemic insult, yet the subsequent damage was different in the mutant mice. This is the first indication that the E2F1 gene plays a role in ischemic death of post-mitotic neurons.

Topics: Animals; Brain Ischemia; Carrier Proteins; Cell Cycle Proteins; Cerebral Infarction; Cerebrovascular Circulation; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Etanidazole; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors

The presence and significance of tumor hypoxia has been recognized since the 1950's. Hypoxic cells in vitro and in animal tumors in vivo are documented to be three times more resistant to radiation-induced killing compared to aerobic cells. There is now evidence that tumor hypoxia is treatment-limiting in many human cancers. One common way to describe the extent of hypoxia in individual and groups of tumors is the "hypoxic fraction." This measurement infers that cells are present in only two radiobiologically significant states: oxygenated and hypoxic. In this paper, we demonstrate the qualitative and quantitative presence of hypoxic tumor cells using the oxygen dependent metabolism of the 2-nitroimidazole, EF5. Two assumptions concerning the calculation and interpretation of the hypoxic fraction are considered. The first is the use of multiple animals to describe the radiation response at a given radiation dose. We hypothesize that the presence of intertumor variability in radiation response due to hypoxia could negatively influenced the characterization of the change in slope required to calculate the hypoxic fraction. The studies presented herein demonstrate heterogeneity of radioresponse due to hypoxic fraction within and between tumor lines. The 9L subcutaneous tumor studied in air-breathing rats demonstrates a 2 log variation in surviving fraction at 17 Gy. The Morris 7777 hepatoma, in contrast, showed little variability of radiation response. Our second question addresses the limitations of using the "hypoxic fraction" to describe the radiation response of a tumor. This calculated value infers that radiobiological hypoxia is a binary measurement: that a tumor contains two cell populations, aerobic cells with maximal radiosensitivity and hypoxic cells with maximal radioresistance. The classic work of Thomlinson and Gray, however, implies the presence of an oxygen gradient from tumors vessel through the tissues. In both the 9L and Q7 tumors, flow cytometric analysis of EF5 binding demonstrates a continuous range of cellular pO2 levels. These studies suggest that: 1) there is extensive intertumor variability of radiation response in certain tumor lines; 2) the variability in radiation response between individual tumors in a group may affect the ability to describe a particular tumor type's "hypoxic fraction" and 3) The oxygen status of tumor cells is a continuum. This realization affects the ability to apply a binary concept such as the "hypoxic fract

Topics: Animals; Benzimidazoles; Cell Hypoxia; Cell Survival; Etanidazole; Fluorescent Dyes; Glioma; Humans; Hydrocarbons, Fluorinated; Hypoxia; Liver Neoplasms, Experimental; Neoplasms, Experimental; Oxygen; Radiobiology; Rats; Rats, Inbred F344; Tumor Stem Cell Assay

Topics: Animals; Antibodies, Monoclonal; Chickens; Etanidazole; Growth Plate; Humans; Hydrocarbons, Fluorinated; Hypoxia; Immunohistochemistry; Injections, Intravenous; Liver; Microscopy, Fluorescence; Myocardium; Papio; Rats; Reference Values; Regional Blood Flow; Sensitivity and Specificity; Tissue Distribution

Topics: Animals; Animals, Newborn; Antibodies, Monoclonal; Brain Ischemia; Colonic Neoplasms; Etanidazole; Humans; Hydrocarbons, Fluorinated; Hypoxia; Hypoxia, Brain; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Myocardial Infarction; Rats; Rats, Sprague-Dawley; Transplantation, Heterologous; Tumor Cells, Cultured

The characteristic reduction and binding of nitroimidazoles to cellular macromolecules in the absence of oxygen allows their use for detection and characterization of hypoxia. The biodistribution of a new nitroimidazole, EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide), in mice bearing EMT6 tumors is described. Detection methods based on radioactivity and monoclonal antibody techniques are compared for liver and tumor. All nonexcretory tissues demonstrated similar levels of radioactivity at 0.5 hr postinjection of drug, demonstrating equivalent access of EF5 to all tissues. At 24 hr, when unbound drug has been cleared, the tissues with the highest binding are the liver, esophagus, bladder and tumor. Typically, liver tissue contains the highest level of radioactivity at this time. Examination of tumor and liver tissue by use of fluorescence microscopy and Cy3-bound monoclonal antibodies specific for EF5 adducts showed that the patterns of binding in tumor are considerably more heterogeneous than those of liver. Histograms of fluorescence intensity, with use of these antibodies, demonstrate average and maximal binding higher in tumors than in the liver. This divergence from the radioactivity data was determined to be unrelated to sampling error, differential antibody access or staining efficiency of liver vs. tumor tissue. A possible cause is the scavenging of radioactive drug metabolites by liver. The data presented herein suggest that EF5 is useful as a hypoxia detector and that monoclonal antibody detection methods can give detailed information on the distribution of EF5 binding. This technology may allow an accurate estimation of the oxygenation and/or nitroreductase levels in both tumor and normal tissues.

Topics: Animals; Antineoplastic Agents; Etanidazole; Hydrocarbons, Fluorinated; Hypoxia; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Oxygen; Tissue Distribution

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Topics: Carcinoma; Colonic Neoplasms; Endothelial Growth Factors; Etanidazole; Gene Expression Regulation, Neoplastic; Humans; Hydrocarbons, Fluorinated; Hypoxia; In Situ Hybridization; Indicators and Reagents; Lymphokines; Neovascularization, Pathologic; Organoids; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors