18F-FEAU as a radiotracer for herpes simplex virus thymidine kinase gene expression: in-vitro comparison with other PET tracers.

OBJECTIVE: The herpes simplex virus thymidine kinase (HSVtk) gene has frequently been applied as a reporter gene for monitoring transgene expression in animal models. In clinical gene therapy protocols, however, extremely low expression levels of the transferred gene are generally observed. Consequently, sensitive and selective radiotracers for imaging are required. This study describes the in-vitro evaluation of 2'-[18F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (18F-FEAU) as a candidate tracer for HSVtk imaging with positron emission tomography (PET). METHODS: In cellular accumulation experiments, the potential of 18F-FEAU as a PET tracer for HSVtk was compared to the known acyclic guanosine derivatives 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine (18F-FHPG) and 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG), and the thymidine derivatives 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT), 2'-deoxy-2'-[18F]fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) and 2'-deoxy-2'-[18F]fluoro-5-iodo-1-beta-D-arabinofuranosyluracil (18F-FIAU). For this purpose, C6 control cells and HSVtk-expressing C6tk cells were incubated with the different tracers for various periods of time and cellular uptake and initial uptake rates were analysed. The initial rate of tracer uptake was determined from the slope of the plot of tracer uptake versus incubation time. RESULTS: After 2 h of tracer incubation, the C6tk/C6 accumulation ratio was 1.6 for 18F-FLT, 2.4 for F-FMAU, 5.5 for 18F-FHPG, 10.3 for 18F-FIAU, 40.8 for 18F-FHBG and 84.5 for 18F-FEAU. The initial tracer uptake rate in C6tk cells was in the order FLT>FMAU>FEAU>FIAU>FHBG>FHPG, whereas the initial tracer uptake rate in C6 control cells was FLT>FMAU>FIAU>FEAU approximately = FHBG approximately = FHPG. The highest HSVtk specific uptake was observed for FEAU. CONCLUSION: This study indicates that the high uptake rate of FEAU together with its high selectivity make this tracer an excellent candidate as a PET tracer for HSVtk gene expression.

The human norepinephrine transporter in combination with 11C-m-hydroxyephedrine as a reporter gene/reporter probe for PET of gene therapy.

UNLABELLED: Although the herpes simplex virus thymidine kinase gene has been frequently applied as a reporter gene for monitoring gene transfection in animals, it has some intrinsic limitations for use in humans. In our search for a reporter gene that lacks these limitations, we have evaluated the feasibility of the human norepinephrine transporter (hNET) as a reporter gene in combination with the reporter probe 11C-m-hydroxyephedrine (mHED) for PET. METHODS: An adenoviral vector (AdTrack-hNET) containing the hNET gene as reporter gene and the enhanced green fluorescent protein (EGFP) as a substitute for a therapeutic gene was constructed. After COS-7, A2780, and U373 cells were transiently transduced with AdTrack-hNET, hNET protein expression, EGFP fluorescence, and cellular uptake of 11C-mHED were determined. In rats, U373 tumor xenografts were grown and transiently transduced with either AdTrack-hNET or an AdTrack-Luc control adenovirus. Intratumoral accumulation of 11C-mHED was determined by PET and ex vivo biodistribution. The tumors were subsequently examined for EGFP fluorescence. RESULTS: 11C-mHED uptake was positively correlated with AdTrack-hNET viral titer and hNET protein expression. However, large differences in transfection efficiency between cell lines were observed. The highest 11C-mHED uptake was found in hNET transfected U373 cells, in which tracer uptake was >70-fold higher than that in control cells. 11C-mHED accumulation could be inhibited by desipramine, a potent inhibitor of hNET. In all cell lines, 11C-mHED uptake was positively correlated with EGFP fluorescence, implying that imaging of hNET with 11C-mHED would enable monitoring of a coexpressed therapeutic gene. In the animal model, gene transfection efficiencies were very low, as determined by EGFP fluorescence. Still, a significantly higher 11C-mHED uptake in hNET transduced tumors than that in control tumors was demonstrated by ex vivo biodistribution studies. PET with a clinical camera could visualize 1 of 3 hNET transduced tumors, indicating that the transfection efficiency was near the detection limit. CONCLUSION: These results indicate that monitoring of gene therapy using the hNET/11C-mHED reporter gene/probe is feasible, but further investigation with regard to the sensitivity of the technique is required.

Scintigraphic imaging of HSVtk gene therapy.

The evolution of molecular biology has enabled the exploration of novel sophisticated gene-directed treating modalities for cancer. Suicide gene therapy - i.e. transfection of a so-called suicide gene that sensitizes target cells towards a prodrug - may offer an attractive approach to treat malignant tumors. For the development of effective clinical suicide gene therapy protocols, a non-invasive method to assay the extent, the kinetics and the spatial distribution of transgene expression is essential. This would allow investigators and physicians to assess the efficiency of experimental and therapeutic gene transfection protocols and would enable early prognosis of therapy outcome. Radionuclide imaging techniques like single photon emission computed tomography (SPECT) and positron emission tomography (PET), which can non-invasively visualize and quantify metabolic processes in vivo, are being evaluated for repetitive monitoring of transgene expression in living animals and humans. Transgene expression can be monitored directly by imaging the expression of the therapeutic gene itself, or indirectly using a reporter gene that is coupled to the therapeutic gene. Various radiopharmaceuticals have been developed and are now being evaluated for imaging of transgene expression. This review surveys the progress that has been made in the field of non-invasive nuclear imaging of transgene expression and focuses on the herpes simplex virus type 1 thymidine kinase (HSVtk) gene therapy approaches.

Synthesis and in vivo evaluation of 18F-desbromo-DuP-697 as a PET tracer for cyclooxygenase-2 expression.

UNLABELLED: Cyclooxygenase-2 (COX-2) overexpression has been observed in various pathologies, such as inflammation, cancer, ischemia, and Alzheimer's disease. As an initial step toward a noninvasive PET technique to assay COX-2 expression, this study describes the synthesis and preliminary evaluation of the radiolabeled COX-2 inhibitor (18)F-desbromo-DuP-697. METHODS: Desbromo-DuP-697 was radiolabeled by a nucleophilic aromatic substitution reaction of the nitro precursor with (18)F-fluoride. Biodistribution studies of the tracer were performed in a carrageenan-induced hyperalgesia rat model. Brain uptake was investigated with autoradiography. To confirm the results of the biodistribution, COX activity was determined by a peroxidase assay. RESULTS: Biodistribution studies showed specific binding of the tracer to COX-2 in heart, kidney, brain, and blood cells, but not in the inflamed paw, which was probably due to low COX-2 expression. In the brain, regional differences in tracer uptake were observed, with high uptake in cortical regions. (18)F-Desbromo-DuP-697 did not show any binding to COX-1. Nonspecific uptake was high in fat and intestines. CONCLUSION: Because of its ability to cross the blood-brain barrier, (18)F-desbromo-DuP-697 appears to be suitable for COX-2 imaging in the brain. Its high nonspecific uptake in the intestines may limit its use for imaging in the abdominal region.

Tumor imaging with 2 sigma-receptor ligands, 18F-FE-SA5845 and 11C-SA4503: a feasibility study.

UNLABELLED: Our objective was to study 2 radioligands for visualization of sigma-receptors with PET. METHODS: Two radioligands-sigma(1)-selective (11)C-1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine ((11)C-SA4503) and nonsubtype-selective 1-(4-2'-(18)F-fluoroethoxy-3-methoxyphenethyl)-4-(3-(4-fluorophenyl)propyl)piperazine ((18)F-FE-SA5845)-were evaluated for tumor imaging. RESULTS: Binding studies to rat glioma cells (C6) and human nonsmall cell lung cancer cells (N417) indicated interaction of (18)F-FE-SA5845 with 2 sites and interaction of (11)C-SA4503 with a single site. Specific binding of (18)F-FE-SA5845 was 93% +/- 2% and that of (11)C-SA4503 was 78% +/- 6% of the total cellular uptake of radioactivity. Uptake of the (18)F-labeled ligand, but not that of the (11)C-labeled ligand, appeared to be related to the growth phase of the cells. Biodistribution experiments in C6 tumor-bearing nude rats (Ham HSD RNU rnu) indicated tumor-to-plasma ratios of 13.3 for (11)C-SA4503 and 8.0 for (18)F-FE-SA5845 and tumor-to-muscle ratios of 5.0 for (11)C-SA4503 and 4.9 for (18)F-FE-SA5845, 60 min after injection, which were reduced to values ranging from 1.4 to 2.0 after pretreatment of animals with haloperidol (2 micromol/kg). Tumor uptake of (18)F-FE-SA5845 showed a negative correlation with tumor size (P < 0.0001), in contrast to that of (11)C-SA4503, suggesting that tissue binding of the former ligand is related to cellular proliferation. A study with (11)C-SA4503 in a human volunteer indicated high uptake in liver, kidney, and heart but relatively low background in thorax and lower abdomen. CONCLUSION: Both (18)F-FE-SA5845 and (11)C-SA4503 demonstrate specific binding to sigma-receptors in vivo and may be useful for the detection of pulmonary and abdominal tumors. However, the (18)F-labeled compound may be better for tumor staging than the (11)C-labeled drug.