Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor.

We studied the tumor uptake of [67Ga]citrate, [59Fe]citrate, and 125I-labeled transferrin (TF) by the in vitro growth form of EMT-6, a sarcoma-like mammary tumor of BALB/c mice. In analyzing the binding experiments, we developed a new mathematical model based on a formulation originally used to express the interaction of hormones with specific tissue receptors. The uptake of both carrier-free 67Ga and 59Fe by tumor cells was mediated by kinetically identical TF receptors. We also studied teric acid extracts of the stroma of EMT-6 tumors grown both in vivo and in vitro. Chromatography of these extracts on Sephacryl S-200 SF demonstrated that the cellular stroma contained specific TF-binding macromolecules. On the basis of these findings, we proposed the "transferrin receptor hypothesis" for the mechanism of 67Ga uptake by tumors. According to this view, a tumor-associated TF receptor is the functional unit responsible for the affinity of gallium for certain neoplasms. This receptor was also active in the uptake of iron by tumors.

Pharmacological alteration of the lung vascular response to radiation.

The role of endothelial cell damage in the development of radiation injury in the lung was investigated in rats. Vascular permeability-surface area product (PS) was measured as an indicator of the degree of endothelial cell damage in lungs of rats exposed to single dose hemithorax irradiation. Hemithorax irradiation was chosen to simulate clinical radiotherapy, in which only a portion of the lung is irradiated. In addition, it provided a control lung to compare to the irradiated lung. Radiation is postulated to lead to activation of several different biochemical pathways that result in lung injury and fibrosis. Many of these pathways can be specifically blocked with drugs. Thirteen different drugs were studied. Dexamethasone, indomethacin, cromolyn, cyproheptadine, Vitamin D3, theophylline, and diethylcarbamazine were all effective at reducing lung PS on the irradiated side. Dexamethasone, Vitamin D3, and indomethacin also significantly reduced lung PS in the unirradiated lungs and in sham-irradiated rats. Captopril, cobra venom factor, penicillamine, trapidil, epsilon-amino caproic acid, and dapsone had no significant effect on lung PS after hemithorax irradiation. We conclude that the major pathways involved in early post-radiation lung injury involve prostaglandin, leukotriene, and histamine release from macrophages and mast cells. Complement activation, proteolytic enzymes, and neutrophil migration do not seem to be important mediators of early post-radiation lung injury.

Radioprotection in rat spinal cord with WR-2721 following cerebral lateral intraventricular injection.

The capacity of WR-2721 to provide radioprotection in central nervous system (CNS) tissue was assessed in F-344 rats irradiated with Cs-137 to the cervical spinal cord 45 min following injection of either 0.33 mg (0.60 X LD50) of WR-2721 or carrier solution in the right lateral cerebral ventricle. The radiation dose groups were 20, 26, 32, or 38 Gy; the dose rate was 1.48 Gy/min. Following irradiation, the time in weeks to forelimb and hindlimb paralysis was measured and statistical significance was assessed by means of the log rank sum test. The median times in weeks to forelimb paralysis in control vs. WR-2721-treated rats were, respectively, 20 vs. 22 at 38 Gy, 19 vs. 31 at 32 Gy (p less than 0.01), 23 vs. 28 at 26 Gy (p less than 0.01), and 49 vs. 60 at 20 Gy (p less than 0.01). The median times to hindlimb paralysis in control vs. WR-2721-treated rats were respectively, 20 vs. 29 at 38 Gy (p less than 0.001), 20 vs. 35 at 32 Gy (p less than 0.01), 23 vs. 34 at 26 Gy (p less than 0.001), and 58 vs. 65 at 20 Gy (p less than 0.01). From these results, we calculated the DMF for forelimb paralysis to be 1.3 and for hindlimb paralysis, 1.6. Histological studies from selected spinal cords from symptomatic killed rats showed petechial hemorrhages, rare microvascular thrombi, and scattered microinfarcts in both gray and white matter. In the white matter columns, there were scattered microfoci of demyelination. The histological findings did not differ between the control and WR-2721-treated groups, but were worse in the higher dose groups. These data indicate that WR-2721 has the capacity to be radioprotective in CNS tissues, when it is administered by a route that bypasses the blood-brain barrier.

Comparative biodistribution and radioprotection studies with three radioprotective drugs in mouse tumors.

The organ level biodistribution and tumor radioprotective properties of three drugs have been compared: WR-2721 (NSC 296961), WR-3689 (NSC 327729), and WR-77913 (NSC 318809). The three drugs have similar distribution patterns in normal mouse tissues. At 30 minutes after intraperitoneal injection, highest levels of 35S from radiolabeled protector are found in kidney and submandibular salivary gland, with lowest levels in brain and moderately low values in tumor and skin. Three of four tumors examined take up less WR-3689 than the other two protectors. For the three protectors, the dose modifying factors for the RIF-1 tumor irradiated in vivo and assayed in vitro are 1.5-1.7, but do not vary as predicted by differential uptake of drug into this neoplasm. In RIF-1, WR-3689 is taken up most avidly, but the three drugs tend to be equally protective.

Use of steroids to suppress vascular response to radiation.

A quantitative measure of the vascular permeability surface area product (PS) for albumin has been made using a double isotope technique. PS was significantly elevated in irradiated rat lung, heart, skin, and muscle, between 19 and 26 days following 18 or 25 Gray thorax irradiation. Administration of dexamethasone from 2 days before irradiation through the day of measurement suppressed the expected increase in PS in lung, heart, and muscle, but not in skin. Shorter periods of steroid administration were not as effective in suppressing this response to radiation exposure. Increased vascular permeability following radiation may be an essential element in the development of radiation fibrosis. We hypothesize that the ability to suppress this response could result in a long term reduction in the incidence of fibrosis.

Hypoxia mediated binding of misonidazole in non-malignant tissue.

Binding of 3H-misonidazole in hypoxic nonmalignant tissue was investigated in the gerbil stroke model. Cerebral infarcts were produced in male Mongolian gerbils by ligating the right common carotid artery and the severity of the lesions was quantified by scoring the animals' symptoms. The uptake of [H-3]misonidazole in the right cerebral hemisphere and the ratios of right:left hemispheral uptake correlated positively with the severity of the stroke when measured 6 to 10 hours after carotid ligation. Autoradiographs of the gerbil brains with severe infarcts showed heavy label, which was uniformly distributed, on the affected side. We conclude that the gerbil stroke model is useful for studying hypoxia in vivo. There is variability between animals that closely correlates with stroke index, but more importantly the hypoxia may be more homogeneous over regions of the brain within one animal. A reliable model of homogeneous induced hypoxia in vivo will be useful for evaluating radiolabeled drugs which may be used for quantitation of hypoxia in tumors by nuclear imaging.

Radiolabelled fluoromisonidazole as an imaging agent for tumor hypoxia.

Fluoromisonidazole labeled with H-3 or F-18 has been tested as a quantitative probe for hypoxic cells in vitro and in rodent and spontaneous dog tumors in vivo. In V-79, EMT-6(UW), RIF-1, and canine osteosarcoma cells in vitro, the binding of 50 microM [H-3]Fluoromisonidazole was 50% inhibited by 1000-2000 ppm O2, relative to binding under anoxic conditions. After a 3 hr incubation with labeled drug, the anoxic/oxic binding ratios ranged from 12 to 27 for the four cell types. Retention of [H-3]fluoromisonidazole 4 hr after injection was greater in large KHT tumors (400-600 mm3) with an estimated hypoxic fraction greater than 30%, than in smaller tumors (50-200 mm3) with an estimated hypoxic fraction of 7-12%. RIF-1 tumors, with an estimated hypoxic fraction of 1.5%, retained the least label, with tumor: blood ratios ranging from 1.7 to 1.9. Spontaneous dog osteosarcomas were imaged with a time of flight positron emission tomograph for up to 5 hr following injection of [F-18] fluoromisonidazole. Analysis of regions of interest in images allowed creation of dynamic tissue time activity curves and calculation of tissue uptake in cpm/gram. These values were compared to radioactivity in plasma. In all cases, retention in some tumor regions exceeded that in plasma and in normal tissue, such as muscle or brain, by 3 to 5 hr post injection. Uptake of fluoromisonidazole in tumors was heterogeneous, with ratios of maximum to minimum uptake as high as 4 in different regions of interest in the same tumor. Tumor:plasma values ranged from 0.28 to 2.02. The oxygen dependency of fluoromisonidazole retention was similar in a variety of cell types and was 50% inhibited by O2 levels in the transition between full radiobiological hypoxia and partial sensitization. The quantitative regional imaging of [F-18] fluoromisonidazole in spontaneous canine tumors at varying times post-injection lays the basis for imaging and modeling of oxygen-dependent drug retention in different regions of human neoplasms.

Toxicity and biodistribution of the radioprotectors, WR2721, WR77913, and WR3689, in the CNS following intraventricular or intracisternal administration.

The radioprotective capacity of the phosphorothioate compounds, WR2721, WR77913, and WR3689, in the CNS is being evaluated following injection of the drugs into the lateral cerebral ventricle or the cisterna magna of F-344 rats. This approach circumvents the blood-brain barrier and permits an assessment of the CNS toxicity and regional distribution of these compounds. Following intraventricular injection in 150-200 gm female rats, the LD50 doses for WR2721, WR77913, and WR3689 were respectively 0.60 +/- 0.07 mg (S.E.), 2.36 +/- 0.13 mg, and 3.56 +/- 0.26 mg. Following intracisternal injection the LD50 doses were 0.71 +/- 0.18 mg, 4.12 +/- 1.09 mg and 3.03 +/- 0.68 mg, respectively. WR 2721 produced lethargy, unsteady gait, and dishevelment but these signs all resolved completely within 1-3 days in survivors. In addition to these signs, WR77913 and WR3689 produced severe convulsions. At high doses, following intraventricular administration, all three drugs were associated with cerebral and diencephalic periventricular necrosis and ipsilateral necrosis of the lateral hippocampus. Biodistribution studies were performed with [S-35]-labeled derivatives of the drugs and tissue sampling. The three drugs demonstrated similar patterns. Forty-five minutes following either the intraventricular or intracisternal route of drug delivery the highest drug concentrations were in the brainstem, cerebellum, and cervical cord. Additional studies with autoradiography revealed that intraventricular injection was associated with high drug uptake in the cerebral white matter, the periventricular diencephalon, and the periaqueductal mesencephalon. The biodistribution and toxicity data together suggest that the drugs can be ranked, WR3689 greater than WR77913 greater than WR2721, according to the level of drug thiol that can be achieved in the CNS tissues with intraventricular or intracisternal injection. Tissue levels achievable with WR2721 following these two routes of administration are as high as levels others have reported as radioprotective in rodent skin and gut.

Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole.

Fluoromisonidazole (FMISO) has been shown to bind selectively to hypoxic cells in vitro and in vivo at radiobiologically significant oxygen levels. When labeled with the positron emitter fluorine-18 (F-18), its uptake in tissue can be detected quantitatively with high precision by positron emission transaxial tomography (PETT). This paper presents the first experiences with PETT imaging of [F-18]FMISO uptake in human malignancies, and describes the development of this technique as a tool for the non-invasive assessment of tumor hypoxia. Eight patients with selected cancers were imaged prior to primary radiotherapy, and 3 returned for follow-up scans, for a total of 11 imaging studies. Six of eight pre-radiotherapy studies revealed retention of [F-18]FMISO in tumors that significantly exceeded plasma concentrations by 2 hr after drug injection; all five patients with head and neck primaries had such "positive" scans. An analytic method for the interpretation of [F-18] FMISO PETT images is presented, defining hypoxic elements within a tumor volume as regions with a threshold regional tumor:plasma [F-18]FMISO ratio of greater than or equal to 1.4 by 2 or more hours after injection. Toward the end of a course of fractionated radiotherapy, three repeat studies in patients with initially positive scans showed no tumor accumulation of drug above the threshold ratio of 1.4, suggesting reoxygenation had occurred. Pharmacokinetic and dosimetry data support continued use of [F-18]FMISO as a safe hypoxia probe. Two imaging protocols have been developed for human studies; a long protocol allows for more complete biodistribution and dosimetry information, and a shorter protocol facilitates increased patient accrual by applying a simple, clinically expedient imaging procedure. When correlated with tumor outcome, [F-18]FMISO PETT imaging may be developed as a predictor of tumor response to conventional radiotherapy. The implications of this technique in addressing persistent questions of tumor hypoxia in human oncology is discussed.

Quantifying regional hypoxia in human tumors with positron emission tomography of [18F]fluoromisonidazole: a pretherapy study of 37 patients.

PURPOSE: To assess pretreatment hypoxia in a variety of tumors using positron emission tomography (PET) after injection of the hypoxia-binding radiopharmaceutical [18F]fluoromisonidazole ([18F]FMISO). METHODS AND MATERIALS: Tumor fractional hypoxic volume (FHV) was determined in 21 nonsmall cell lung cancer patients, 7 head and neck cancer patients, 4 prostate cancer patients, and 5 patients with other malignancies by quantitative PET imaging after injection of [18F]FMISO (0.1 mCi/kg). The FHV was defined as the proportion of pixels in the imaged tumor volume with a tissue:blood [18F] activity ratio > or = 1.4 at 120-160 min postinjection. A FHV > 0 was taken as evidence for tumor hypoxia. RESULTS: Hypoxia was observed in 36 of 37 tumors studied with FMISO PET imaging; FHVs ranged from 0 to 94.7%. In nonsmall cell lung cancers (n = 21), the median FHV was 47.6% and the range, 1.3 to 94.7%. There was no correlation between tumor size and FHV. In the seven head and neck carcinomas, the median FHV was 8.8%, with a range from 0.2 to 18.9%. In the group of four prostate cancers, the median and range were 18.2% and 0 to 93.9%, while in a group of five tumors of different types the median FHV was 55.2% (range: 21.4 to 85.8%). CONCLUSIONS: Hypoxia was present in 97% of the tumors studied and the extent of hypoxia varied markedly between tumors in the same site or of the same histology. Hypoxia also was distributed heterogeneously between regions within a single tumor. These results are consistent with O2 electrode measures with other types of human tumors. The intra- and intertumor variability indicate the importance of making oxygenation measures in individual tumors and the necessity to sample as much of the tumor volume as possible.

The potential role of external beam radiation in preventing restenosis after coronary angioplasty.

The potential role of radiation in the prevention of coronary artery restenosis after angioplasty has generated much recent interest. Animal research and pilot clinical efforts have focused primarily on intraluminal methods of radiation delivery. This article reviews the experience to date with external beam radiation in restenosis prevention and suggests issues that should be considered from the standpoint of both external beam and intravascular radiotherapy. External beam radiation can certainly play an effective role in clinical studies of coronary artery restenosis, and a multicenter randomized trial of external beam radiation after coronary angioplasty has been initiated.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids.

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.

Evaluation of oxygenation status during fractionated radiotherapy in human nonsmall cell lung cancers using [F-18]fluoromisonidazole positron emission tomography.

PURPOSE: Recent clinical investigations have shown a strong correlation between pretreatment tumor hypoxia and poor response to radiotherapy. These observations raise questions about standard assumptions of tumor reoxygenation during radiotherapy, which has been poorly studied in human cancers. Positron emission tomography (PET) imaging of [F-18]fluoromisonidazole (FMISO) uptake allows noninvasive assessment of tumor hypoxia, and is amenable for repeated studies during fractionated radiotherapy to systematically evaluate changes in tumor oxygenation. METHODS AND MATERIALS: Seven patients with locally advanced nonsmall cell lung cancers underwent sequential [F-18]FMISO PET imaging while receiving primary radiotherapy. Computed tomograms were used to calculate tumor volumes, define tumor extent for PET image analysis, and assist in PET image registration between serial studies. Fractional hypoxic volume (FHV) was calculated for each study as the percentage of pixels within the analyzed imaged tumor volume with a tumor:blood [F-18]FMISO ratio > or = 1.4 by 120 min after injection. Serial FHVs were compared for each patient. RESULTS: Pretreatment FHVs ranged from 20-84% (median 58%). Subsequent FHVs varied from 8-79% (median 29%) at midtreatment, and ranged from 3-65% (median 22%) by the end of radiotherapy. One patient had essentially no detectable residual tumor hypoxia by the end of radiation, while two others showed no apparent decrease in serial FHVs. There was no correlation between tumor size and pretreatment FHV. CONCLUSIONS: Although there is a general tendency toward improved oxygenation in human tumors during fractionated radiotherapy, these changes are unpredictable and may be insufficient in extent and timing to overcome the negative effects of existing pretreatment hypoxia. Selection of patients for clinical trials addressing radioresistant hypoxic cancers can be appropriately achieved through single pretreatment evaluations of tumor hypoxia.

Synthesis of an (iodovinyl)misonidazole derivative for hypoxia imaging.

Nitroimidazoles undergo a bioreduction in viable hypoxic tissue, resulting in trapping within these tissues, as demonstrated by misonidazole. A radioiodinated analogue of misonidazole (IVM, (E)-5-(2-Nitroimidazolyl)-4-hydroxy-1-iodopent-1-ene, 3) has been synthesized by halodestannylation, for evaluation as an imaging agent for hypoxia. A key step in the synthetic sequence involves the use of the Lewis acid BF3.Et2O to promote the nucleophilic ring opening of glycidyl tosylate with (E)-1-lithio-2-(tributylstannyl)ethylene. Direct comparison of IVM versus F-MISO (2) another misonidazole type hypoxic cell marker, in several in vitro cell culture studies, indicates that IVM behaves in analogous fashion to F-MISO and has promise as a hypoxia imaging agent for SPECT.

A transferrin-mediated uptake of gallium-67 by EMT-6 sarcoma. II. Studies in vivo (BALB/c mice): concise communication.

The EMT-6 sarcoma-like tumor of BALB/c mice can be grown as a solid subcutaneous transplantable tumor in vivo or as a monolayer culture in vitro. We have studied the uptake of gallium-67 by this tumor growing subcutaneously on the backs of 6-week-old BALB/c mice. After i.v. administration of Ga-67 citrate, tumor uptakes were as high as any others reported for mouse tumors. Also, for unknown reasons, there was appreciable reduction in tumor uptake with increasing amounts of Ga-67 citrate, even in the microcurie range. Furthermore, when mouse serum is prelabeled with Ga-67 and then injected, the EMT-6 uptake is greater than with Ga-67 administered as citrate (p less than 0.02). We believe that the finding of avid Ga-67 uptake in vivo helps to establish this unique in vivo/in vitro tumor system as a valid experimental model for studies regarding the mechanism of Ga-67 accumulation by neoplastic tissue.

A transferrin-mediated uptake of gallium-67 by EMT-6 sarcoma. I. Studies in tissue culture.

We have studied the in vitro uptake of gallium-67 by exponentially growing EMT-6 sarcoma cells in long-term tissue culture. In this system, the addition of transferrin to the medium was required before an appreciable cellular uptake of Ga-67 occurred. The transferrin effect was complex, with an initial stimulation to a peak cell-to-medium ratio of 8--10:1 at low concentrations of transferrin (0.2 mg/ml), followed by a gradual decline in uptake as transferrin in the medium was increased further. EMT-6 tumor-cell uptake of Ga-67 was probably mediated by a specific cellular receptor for transferrin. Scatchard analysis of the EMT-6 cellular binding of human transferrin labeled with iodine-125 indicated a cellular receptor with affinity for transferrin of 5 X 10(6) l/mole and abundance of 500,000 receptors per cell. Over the experimental range of transferrin concentration in the medium, the observed uptake of Ga-67 was closely correlated with the degree of formation of Ga-67-labeled transferrin and the fraction of transferrin bound to the cellular receptor (N = 69, r = 0.86, p less than 0.0001).

Synthesis and characterization of congeners of misonidazole for imaging hypoxia.

Misonidazole is a known hypoxic cell sensitizer that binds covalently in hypoxic cells. Its congeners labeled with 77Br, 75Br, or 18F, are likely candidates for imaging hypoxia. We have synthesized and tested [82Br]-4-bromomisonidazole, [3H]-4-bromomisonidazole, [3H]fluoromisonidazole and [3H]misonidazole as prototype radiopharmaceuticals and have compared their uptake in normal and malignant tissues. The higher lipophilicity of brominated misonidazole increased its concentration in the hypoxic portion of tumors at 2 hr, but high blood levels contributed to excessive background, incompatible with imaging. Hydrogen-3-fluoromisonidazole diffused into tumors at a slower rate than misonidazole but it also cleared from normal tissues so that after 2 hr tumor-to-blood ratios favorable for imaging were achieved. In the compounds that were studied, fluorine at the end of the alkyl chain is more stable in vivo than bromine on the imidazole ring. Our results indicate that [18F] fluoromisonidazole may be a useful tracer for imaging hypoxia at approximately 4 hr after injection.

Enhanced binding of the hypoxic cell marker [3H]fluoromisonidazole in ischemic myocardium.

The 2-nitroimidazole fluoromisonidazole is metabolically trapped in viable hypoxic cells in inverse proportion to PO2. This attribute suggests that [18F]fluoromisonidazole may be useful for imaging hypoxic tissue using positron emission tomography. To examine this potential, we studied the pharmacokinetics and biodistribution of [3H]fluoromisonidazole in six open chest dogs. In two normal dogs, plasma and urine samples were collected over a 4-hr period following i.v. injection of the drug. In four animals, regional myocardial ischemia was produced 2 hr prior to drug injection by occlusion of the circumflex coronary artery and maintained during the 4-hr sampling period. In all animals, postmortem samples of myocardium and other organs were obtained and tissue, plasma, and urine tritium activity were determined by liquid scintillation counting. In areas of reduced flow, [3H]fluoromisonidazole accumulated in myocardium in inverse proportion to myocardial blood flow measured by microspheres, indicating enhanced binding in hypoxic tissues. Maximum tissue concentrations in ischemic myocardium were two- to three-fold greater than in normal myocardium and plasma. Plasma clearance data indicate the drug is rapidly distributed into the total-body water, clears from the body with a half-life of 275 +/- 50 min, and undergoes minimal metabolism by 4 hr. We conclude [18F]fluoromisonidazole may be a suitable agent for radionuclide imaging of hypoxic myocardium.

A radiosynthesis of fluorine-18 fluoromisonidazole.

A new preparation of [18F]fluoromisonidazole [1H-1-(3-[18F] fluoro-2-hydroxypropyl)-2-nitroimidazole] is presented as a two-step, two-pot reaction sequence. The method is useful for the production of 20-30 mCi quantities of this compound from [18F]fluoride, available in 40% radiochemical yield at end of bombardment (EOB) with a specific activity (nca) of greater than 650 Ci/mmol (EOB) and a synthesis time of approximately 140 min. The key feature of the reaction scheme is the preparation of a new fluoroalkylating agent, [18F]epifluorohydrin.

Characterization of [18F]fluoroetanidazole, a new radiopharmaceutical for detecting tumor hypoxia.

UNLABELLED: Fluorinated derivatives of etanidazole are being explored as probes for tumor hypoxia. Our research group has synthesized [18F]fluoroetanidazole (FETA) and now reports the oxygen dependency of binding to cells in vitro, the biodistribution of the tracer in tumor-bearing mice and the analysis of metabolites in their plasma and urine. METHODS: Four cultured rodent cell lines (V79, 36B10, EMT6 and RIF1) were incubated with [18F]FETA for various times under graded O2 concentrations. We also compared the biodistributions of [18F]FETA and [18F]fluoromisonidazole (FMISO) at 2 and 4 h postinjection in C3H mice bearing KHTn tumors (130-430 mg). Reverse-phase high-performance liquid chromatography was used to distinguish metabolites from parent drugs in urine and plasma of mice injected with [18F]FETA or [18F]FMISO. RESULTS: In cells labeled in vitro, O2 levels of 600-1300 ppm inhibited binding by 50% relative to uptake under anoxic conditions (<10 ppm). These inhibitory values are not statistically different from those reported for [18F]FMISO in the same cell lines (700-1500 ppm). In the biodistribution studies, uptake in heart, intestine, kidney and tumor was similar for both tracers 4 h after injection, whereas retention of [18F]FETA in liver and lung was significantly lower. Less uptake of [18F]FETA in liver suggests that this nitroimidazole is metabolized less than [18F]FMISO. The brain-to-blood ratios indicate that [18F]FETA readily crosses the blood-brain barrier. High-performance liquid chromatography of urine demonstrated that 10% of [18F]FETA-derived activity was in metabolites at 2 h postinjection, with 15% in metabolites by 4 h; comparable values for [18F]FMISO were 36% and 57%, respectively. CONCLUSION: We conclude from these data that [18F]FETA holds promise as a new hypoxia tracer in patients, having oxygen dependency of binding similar to [18F]FMISO in vitro and displaying less retention in liver and fewer metabolites in vivo.