Semiautomated radiosynthesis and biological evaluation of [18F]FEAU: a novel PET imaging agent for HSV1-tk/sr39tk reporter gene expression.
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2'-deoxy-2'-[(18)F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil ([(18)F]FEAU) is a promising radiolabeled nucleoside designed to monitor Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene expression with positron emission tomography (PET). However, the challenging radiosynthesis creates problems for being able to provide [(18)F]FEAU routinely. We have developed a routine method using a commercial GE TRACERlab FX-FN radiosynthesis module with customized equipment to provide [(18)F]FEAU. All radiochemical yields are decay corrected to end-of-bombardment and reported as means +/- SD. Radiofluorination (33 +/- 8%; n = 4), bromination (85 +/- 8%; n = 4), coupling reaction (83 +/- 6%; n = 4), base hydrolysis steps, and subsequent high-performance liquid chromatography purification afforded purified [(18)F]FEAU beta-anomer in 5 +/- 1% overall yield (n = 3 runs) after approximately 5.5 h and a beta/alpha-anomer ratio of 7.4. Radiochemical/chemical purities and specific activity exceeded 99% and 1.3 Ci/micromol (48 GBq/micromol), respectively. In cell culture, [(18)F]FEAU showed significantly (P < 0.05) higher accumulation in C6 cells expressing HSV1-tk/sr39tk as compared to wild-type C6 cells. Furthermore, [(18)F]FEAU showed slightly higher accumulation than 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butylguanine ([(18)F]FHBG) in cells expressing HSV1-tk (P < 0.05), whereas [(18)F]FHBG showed significantly higher (P < 0.05) accumulation than [(18)F]FEAU in HSV1-sr39tk-expressing cells. micro-PET imaging of mice carrying tumor xenografts of C6 cells stably expressing HSV1-tk or HSV1-sr39tk are consistent with the cell uptake results. The [(18)F]FEAU mouse images also showed very low gastrointestinal signal with predominant renal clearance as compared to [(18)F]FHBG. The routine radiosynthesis of [(18)F]FEAU was successfully semiautomated using a commercial module along with customized equipment to provide the beta-anomer in modest yields. Although further studies are needed, early results also suggest [(18)F]FEAU is a promising PET radiotracer for monitoring HSV1-tk reporter gene expression. (+info)
Tracking cellular stress with labeled FMAU reflects changes in mitochondrial TK2.
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A radioimmunoassay for 1-beta-D-arabinofuranosyluracil with reference to cross-reactivity of 1-beta-D-arabinofuranosylcytosine with an antibody.
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Antibodies directed against 1-beta-D-arabinofuranosyluracil have been produced in rabbits by immunization with a conjugate of 1-(5-O-succinyl-beta-D-arabinofuranosyl)uracil with human serum albumin. Two of four antibodies so obtained showed high specificity for 1-beta-D-arabinofuranosyluracil and allowed the development of a sensitive and reliable radioimmunoassay for this substrate. On the other hand, one antibody had a high affinity for 1-beta-D-arabinofuranosylcytosine. The binding of 1-beta-D-arabinofuranosylcytosine to this antibody was practically constant between pH 5.2 and 9.0, whereas 1-beta-D-arabinofuranosyluracil binding was affected drastically by pH. The pH-binding profile for 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil was reminiscent of the specificity of ara-C-specific antibodies, which we previously obtained after immunization of rabbits with 1-(5-O-succinyl-beta-D-arabinofuranosyl)cytosine as a hapten. (+info)
Imaging of HSV-tk Reporter gene expression: comparison between [18F]FEAU, [18F]FFEAU, and other imaging probes.
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Biodistribution, cellular uptake and DNA-incorporation of the 2'-fluoro stabilized 5-iodo-2'-deoxyuridine analog 5-iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (FIAU).
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AIM: 5-Iodo-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil (FIAU) has been used for non-invasive monitoring of gene therapy and as an antiviral agent experimentally and in patients. However, FIAU metabolism in tumor cells is largely unknown. Here, the biological characteristics of FIAU in human leukemia and lymphoma cells in vitro and in a xenotransplant severe combined immunodeficient (SCID)-mouse model were investigated. METHODS: The susceptibility of FIAU to glycosidic bond cleavage by thymidine phosphorylase (TP) and its phosphorylation by human thymidine kinase 1 (hTK1) were examined. Cellular uptake and DNA-incorporation were determined in the leukemia cell line HL60 and the lymphoma cell line DoHH2. Biodistribution, in vivo stability of FIAU and expression of proliferation marker(67)Ki and thymidylate synthase were assessed in SCID-mice bearing HL60 xenotransplants. Cellular distribution of FIAU was imaged by microautoradiography. RESULTS: FIAU proved to be stable against degradation by TP and was phosphorylated by hTK1. Significant cellular uptake in DoHH2 and in HL60 cells was observed. The majority of intracellular [(131)I]FIAU was DNA incorporated. In vivo, moderate dehalogenation of [(131)I]FIAU was observed. Biodistribution studies showed a tumor uptake of 1.8+/-0.4% ID/g after 30 min. The half-life of [(131)I]FIAU in blood was 43+/-2 min. Microautoradiography showed a modest accumulation of [(125)I]FIAU in proliferating cells of small intestine, spleen and tumor. CONCLUSION: Despite phosphorylation by the hTK, efficient incorporation into the DNA and high in vivo stability, FIAU accumulates only moderately and transiently in proliferating cells, suggesting that FIAU is probably not appropriate for imaging of proliferation. (+info)
Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction.
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We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous. (+info)
Monitoring the efficacy of adoptively transferred prostate cancer-targeted human T lymphocytes with PET and bioluminescence imaging.
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