Imaging proliferation in vivo with [F-18]FLT and positron emission tomography.

Positron emission tomography (PET) is now regularly used in the diagnosis and staging of cancer. These uses and its ability to monitor treatment response would be aided by the development of imaging agents that can be used to measure tissue and tumor proliferation. We have developed and tested [F-18]FLT (3'-deoxy-3'-fluorothymidine); it is resistant to degradation, is retained in proliferating tissues by the action of thymidine kinase 1 (TK), and produces high-contrast images of normal marrow and tumors in canine and human subjects.

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.

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.

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.

Routine synthesis of L-[18F]6-fluorodopa with fluorine-18 acetyl hypofluorite.

The synthesis of L-[18F]6-fluorodopa (2.4-10.6 mCi) was done by passing gaseous [18F]acetyl hypofluorite through a solution of L-methyl-N- acetyl-[beta-(3-methoxy-4-acetoxyphenyl)]alaninate in acetic acid at room temperature followed by the hydrolysis of the intermediate products with concentrated hydriodic acid. The desired fluorodopa isomer was isolated in 8% EOB radiochemical yield by high performance liquid chromatography in an overall synthesis time of 100 min.

5-Fluoro-1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl) uracil trapping in Morris hepatoma cells expressing the herpes simplex virus thymidine kinase gene.

UNLABELLED: The planning and individualization of gene therapy with suicide genes such as herpes simplex virus thymidine kinase (HSV-tk) necessitates the assessment of the enzyme activity expressed in the tumor. This can be done by uptake measurements of specific substrates for HSV-tk. Due to the molecular structure of 5-fluoro-1-(2'-deoxy-fluoro-beta-D-ribofuranosyl)uracil (FFUdR), it may be a substrate for both the mammalian thymidine kinase and HSV-tk. METHODS: Using a HSV-tk-expressing rat hepatoma cell line and a control cell line (bearing the empty vector) the uptake of 3H-FFUdR was determined with increasing incubation periods. Furthermore, measurements with graded mixtures of HSV-tk-expressing cells and control cells were made. To elucidate the mechanism of FFUdR transport into cells, a series of inhibition/competition experiments was performed with challenge inhibitors of the nucleoside and the nucleobase transport systems. RESULTS: The uptake studies with tritiated FFUdR revealed a 14- to 19-fold higher accumulation in the HSV-tk-expressing cell line compared to the control cell line. While the 3H-FFUdR uptake was 3- to 4-fold higher than the 3H-ganciclovir uptake in the HSV-tk-expressing cells, it was also higher in control cells (5-fold). Furthermore, FFUdR accumulation was linearly correlated with the amount of HSV-tk-expressing cells. FFUdR uptake and growth inhibition by therapeutic doses of ganciclovir were highly correlated, with r = 0.96. Inhibition/competition experiments showed that FFUdR is transported mainly by the equilibrative and the concentrative nucleoside transporter but not by the nucleobase transport systems. CONCLUSION: The FFUdR uptake is an indicator of the HSV-tk activity in tumor cells and can be used as a prognostic marker during gene therapy with HSV-tk. The relative merits of ganciclovir and FFUdR as specific substrates for HSV-tk will need to be further explored in vivo.

Routine synthesis of carbon-11-carboxyl-labeled L-dopa.

Carbon-11-carboxyl-labeled L-dopa has been synthesized by the modified Bucherer-Strecker method. The reaction mixture was first purified by chiral HPLC followed by deprotection using hydriodic acid. The entire procedure was performed in a remotely operated system which gave the product in 28% radiochemical yield (decay corrected) in an overall synthesis time of 55-60 min.

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.

Carbon-11-thymidine and FDG to measure therapy response.

UNLABELLED: This study was performed to determine if PET imaging with 11C-thymidine could measure tumor response to chemotherapy early after the initiation of treatment. Imaging of deoxyriboneucleic acid biosynthesis, quantitated with 11C-thymidine, was compared with measurements of tumor energetics, obtained by imaging with 18F-fluorodeoxyglucose (FDG). METHODS: We imaged four patients with small cell lung cancer and two with high-grade sarcoma both before and approximately 1 wk after the start of chemotherapy. Thymidine and FDG studies were done on the same day. Tumor uptake was quantified by standardized uptake values (SUVs) for both tracers by the metabolic rate of FDG and thymidine flux constant (K(TdR)) using regions of interest placed on the most active part of the tumor. RESULTS: In the four patients with clinical response to treatment, both thymidine and FDG uptake markedly declined 1 wk after therapy. Thymidine measurements of SUV and K(TdR) declined by 64% +/- 15% and 84% +/- 33%, respectively. FDG SUV and the metabolic rate of FDG declined by 51% +/- 9% and 63% +/- 23%, respectively. In the patient with metastatic small cell lung cancer who had disease progression, the thymidine SUV decreased by only 8% (FDG not done). In a patient with abdominal sarcoma and progressive disease, thymidine SUV was essentially unchanged (declined by 3%), whereas FDG SUV increased by 69%. CONCLUSION: Images show a decline in both cellular energetics and proliferative rate after successful chemotherapy. In the two patients with progressive disease, thymidine uptake was unchanged 1 wk after therapy. In our limited series, K(TdR) measurements showed a complete shutdown in tumor proliferation in patients in whom FDG showed a more limited decrease in glucose metabolism.

Feasibility of imaging pentose cycle glucose metabolism in gliomas with PET: studies in rat brain tumor models.

UNLABELLED: The feasibility of imaging pentose cycle (PC) glucose utilization in human gliomas with PET was explored in two rat glioma models by means of glucose radiolabeled in either the carbon-1 (C-1) or carbon-6 (C-6) position. METHODS: In vitro, monolayers of T-36B-10 glioma, tissue slices of intracerebral glioma grafts or slices of normal brain were fed [1-14C]glucose or [6-14C]glucose, and the generated [14C]CO2 was trapped to quantitate the ratio of [14C]CO2 from 14C-1 versus 14C-6. In vivo, rats bearing grafts of either T-36B-10 or T-C6 rat gliomas at six subcutaneous sites received simultaneous intravenous injections of either [1-11C]glucose and [6-14C]glucose, or [1-14C]glucose and [6-11C]glucose. Tumors were excised between 5 and 55 min postinjection to quantify tracer uptake while arterial plasma was collected to derive time-activity input curves. RESULTS: In vitro, the C-1/C-6 ratio for CO2 production from T-36B-10 monolayers was 8.8 +/- 0.4 (s.d.), in glioma slices it was 6.1 +/- 2.1 and in normal brain slices it was 1.1 +/- 0.7. PC metabolism in T-36B-10 was 1.8% +/- 0.5 of total glucose utilization. In vivo, tumor radioactivity levels normalized by plasma isotopic glucose levels showed that retained C-1 relative to C-6 radiolabeled glucose was significantly lower in both gliomas, 4.9% lower in T-36B-10 (p < 0.01) and 4.7% lower in T-C6 (p < 0.01). In an additional group of rats bearing T-36B-10 gliomas and exposed to 10 Gy of 137Cs irradiation 4 hr before isotope injection, the C-1 level was 5.6% lower than that for C-6 (p < 0.05). These results were analyzed with a model of glucose metabolism that simultaneously optimized parameters for C-1 and C-6 glucose kinetics by simulating the C-1 and C-6 tumor time-activity curves. The rate constant for loss of radiolabeled carbon from the tumors, k4, was higher for C-1 than for C-6 in all groups of rats (19% higher for T-36B-10 unirradiated, 32% for T-36B-10 irradiated and 32% for T-C6 unirradiated). CONCLUSION: Mathematical modeling, Monte Carlo simulations and construction of receiver-operator-characteristic curves show that if human gliomas have a similar fractional use of the PC, it should be measurable with PET using sequential studies with [1-11C]glucose and [6-11C]glucose.

Noninvasive detection of hypoxic myocardium using fluorine-18-fluoromisonidazole and positron emission tomography.

Fluoromisonidazole (FMISO) is metabolically trapped in viable cells as a function of reduced cellular pO2. Therefore [18F]-FMISO is potentially useful for evaluating patients with hypoxic but viable myocardium. The goal of this study was to investigate [18F]FMISO uptake in ischemic myocardium non-invasively using positron emission tomography (PET). Studies were performed in 10 open-chest dogs subjected to either complete (Group 1, n = 5) or partial (Group 2, n = 5) occlusion of the left anterior descending coronary artery. The tracer was administered by intravenous bolus following the onset of ischemia and serial PET images were acquired for the next 4 hr. In Group 1, viability was assessed using histochemical staining (nitroblue tetrazolium, NBT) and 99mTc-pyrophosphate (Tc-PYP). In Group 2, viability was assessed using measurements of regional wall motion, histochemical staining and histology (two animals). In each study, PET images obtained at times between 2 and 4 hr postinjection showed specific enhancement of tracer activity in the distal anterior wall and apex of the left ventricle. At 4 hr, the tissue-to-blood pool count ratio was significantly higher in ischemic regions; 1.8 +/- 0.4 for Group 1 and 1.6 +/- 0.2 for Group 2 versus 1.0 +/- 0.1 in nonischemic regions. Postmortem tissue sampling of Group 1 hearts showed significant FMISO retention in samples without evidence for infarction, either by NBT or Tc-PYP deposition, as well as in more severely ischemic regions. In Group 2 animals, FMISO was retained in myocardial regions with reduced blood flow (microspheres), which exhibited improved contraction following reperfusion. We conclude that PET imaging of [18F]FMISO is a promising technique for the noninvasive identification of viable hypoxic myocardium.

Radiosynthesis of 3'-deoxy-3'-[(18)F]fluorothymidine: [(18)F]FLT for imaging of cellular proliferation in vivo.

A reliable radiosynthesis of 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) has been developed based on [(18)10 mCi (370 MBq) of radiochemically pure [(18)1 Ci/micromol (37 GBq/micromol) at EOS within 100 min and in 13% radiochemical yield (end of bombardment (EOB); 7% end of synthesis (EOS)). [(18)F]FLT has been designed as a new positron emission tomography imaging agent for visualizing cellular proliferation in vivo based on the metabolism of thymidine.

Development of labeled thymidine analogs for imaging tumor proliferation.

We have sought nucleoside analogs suitable for labeling with F-18 that could be used to image tumor proliferation with positron emission tomography (PET). The following three thymidine analogs were labeled with tritium and screened for their catabolism and biodistribution in vivo in mice: 5-fluoro-1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)uracil (FFUdR), 5-fluoro-1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-uracil (FFaraU) and 5-methyl-1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)uracil (FTdR). We found that all three compounds were stable to degradation in vivo and when incubated in blood. Of the three analogs tested, only FFUdR showed preferential retention in rapidly proliferating tissues, such as the spleen and implanted tumors, and it attained tissue to blood ratios of 2.1 at 2 h.

Radiosyntheses of labeled beta-pseudothymidine ([C-11]- and [H-3]methyl) and its biodistribution and metabolism in normal and tumored mice.

In order to develop labeled probes for measuring DNA synthetic rates in vivo we investigated [H-3]- and [C-11]methyl labeled beta-pseudothymidine (2a), and report on their radiosyntheses from methyl iodide. We find methylation is rapid and regioselective on N-1 of the acylurea moiety of 2'-deoxy-beta-D-pseudouridine (1a), in the presence of N,N-diisopropylethylamine and N,N-dimethylformamide at 60 degrees C. Although yields are low (11% [C-11]-decay corrected and 4.4% [H-3]), the method is simple and high specific activity tritiated methyl iodide can be used. In contrast to the rapid degradative de-glycosylation of thymidine in blood, beta-pseudothymidine is stable. However, based on biodistribution and metabolite studies, the anticipated uptake of [H-3]methyl-beta-pseudothymidine into mouse DNA of proliferating tissues (e.g. spleen, thymus and duodenum) and implanted tumors was not observed.

Radiosynthesis of 6-[C-11]-D-glucose.

Availability of 6-[C-11]-D-glucose will permit positron emission tomography (PET) investigations of glucose utilization derived from the pentose shunt which supports biosynthesis in tissues. The first radiosynthesis of 6-[C-11]-D-glucose is described. As much as 1 mCi of 6-[C-11]-D-glucose, sufficient for animal studies, is obtained from [C-11]CO2 after 100 min with a 16% radiochemical yield (EOB). The radiosynthesis has many attractive features. The method uses [C-11]CH3I and combines a Wittig reaction and a stereoselective OsO4 catalyzed alkene hydroxylation. The OsO4 hydroxylation of the [C-11]-labeled alkene (9) is accomplished in less than 10 min with high stereoselectivity (94:6) in favor of the 6-[C-11]-D-gluco-isomer. HPLC purification (C-18) of the protected labeled sugar removes the undesired 6-[C-11]-L-ido-sugar at an early stage and avoids the use of an expensive low-capacity ion-exchange HPLC column. OsO4, a highly toxic reagent, is removed in the process by adsorption and inactivation on polymer-bound triphenylphosphine.

An improved HPLC system for the analysis and purification of organic amine radiopharmaceuticals.

This paper describes the separation of lipophilic amine radiopharmaceuticals on normal phase silica using a reversed phase type eluant. This class of compound usually gives very poor peak symmetry on reversed phase columns. However, excellent separation of a number of amines of interest to Positron Emission Tomography, such as spiperone and its derivatives, has been achieved on this system.

Incorporation of a phosphonium analogue of choline into the rat brain as measured by magnetic resonance spectroscopy.

A clear understanding of choline metabolism is important in our goal to modify demyelination and remyelination in multiple sclerosis. To develop a technique capable of measuring metabolic changes in the brain, we have studied the incorporation of a phosphonium analogue of choline (P-choline) in tissue extracts of rats. After feeding adult rats a choline-deficient diet supplemented with P-choline, the analogue was not detectable by in vivo volume-localized 1H spectroscopy. However, in vitro 31P measurements of brain extracts revealed an 11% incorporation of P-choline into phosphatidylcholine. We report that P-choline incorporates preferentially into the lipid pool over the lipid precursor pool and we provide evidence that the choline peak resolved by in vivo 1H spectroscopy is only composed of small molecular weight choline-containing compounds.