| Summary: | 碩士 === 國立陽明大學 === 放射醫學科學研究所 === 89 === Objectives: The ability to detect the localization and level of gene expression precisely and non-invasively would represent a big progress in the objective evaluation of gene therapy. The expression of transfected gene could be demonstrated in vivo by using an appropriate combination of “marker gene” and “marker substrate”. HSV1-tk gene is particularly promising as a marker gene for transfected gene detection when used with radiolabeled thymidine analogues as marker substrates.
18F-FUdR (18F- 5-fluoro-2’-deoxyuridine) is an analogue of thymidine. After phosphorylation by HSV-1 thymidine kinase, 18F-FdUMP acts as an irreversible inhibitor of thymidylate synthase forming stable trimer complex within the transduced cell. The cell or tissue accumulation of metabolites of 18F-FUdR reflects the HSV1-tk gene expression. In the literature, preparation of 18F-FUdR via direct radiofluorination of its deoxyuridine precursor gave low radiochemical yield and radiochemical purity due to low reactivity and poor regioselectivity. In our study, 18F-FUdR is synthesized from radiofluoro-destannylation of its Bu3SnUdR-(OAc)2 or Bu3SnUdR-(THP)2 organotin precursor. The regiospecific subsititution of tributyltin by radiofluorine increases both radiolabeling yield and radiochemical yield of 18F-FUdR significantly. The cell and animal model were then used to determine if 18F-FUdR is an effective marker substrate and a potent PET imaging agent in HSV1-tk gene therapy model.
Methods: The preparation of 18F-FUdR is achieved in a hot cell using the semi-automatic robotic synthesis module. The tributyltin precursor was first radiofluorinated with 18F-F2 then followed by acidic or basic hydrolysis and semi-preparative HPLC purification to obtain the 18F-FUdR product. In-vitro cellular uptake of 18F-FUdR in thymidine kinase gene transduced (NG4TL4-STK) and non-transduced (NG4TL4) murine sarcoma cell lines were determined. In-vivo biological characterization and PET imaging were performed after administration of 18F-FUdR into the tail vein of both NG4TL4-STK and NG4TL4 sarcoma-bearing FVB/N mice. 131I-FIAU was also prepared and was characterized on the same cell lines and animal model as a comparison.
Results: The radiochemical purity of 18F-FUdR prepared from its tributyltin precursor is higher than 97%, the radiochemical yield (EOB) is about 30~35% based on the radioactivity of 18F-F2, and the overall yield is about 50% based on the precursor used. The cellular uptake of 18F-FUdR in NG4TL4-STK and NG4TL4 cells are 51.9 and 34.2 pmol/105 cells after 4 hrs incubation at 37℃, while those of 131I-FIAU (carrier was added to attain the same specific activity as 18F-FUdR) are 27.0 and 13.1 pmol/105 cells, repectively. The biodistribution of 18F-FUdR showed that the tumor tissues retained the highest level than all other organs at 4 hrs after i.v. administration, while the gene transduced NG4TL4-STK tumor retained even more 18F- radioactivity (0.59 %ID/g) than non-transduced NG4TL4 tumor (0.39 %ID/g). Due to low normal tissue uptake, the PET images revealed highly specific localization of 18F-FUdR to NG4TL4-STK and NG4TL4 tumors at 4 hr post injection.
Conclusion: The semi-automatic robotic synthesis module for preparation of [F-18]FUdR from its organotin precursor was successfully established in this study. The radiolabeling yield and radiochemical yield are improved significantly. Both the cell uptake and biodistribution studies show that HSV1-tk(+) tumor retains more FUdR uptake than HSV1-tk(-) tumor due to HSV1-tk gene expression. However, the contribution of cellular proliferation in tumor FUdR accumulation is also obvious and can’t be ignored. Our study clearly demonstrated that 18F-FUdR is a promising radiotracer for nucleic acid metabolism to evaluate the proliferative potential of tumor, but may not be an ideal scintigraphic gene probe for HSV1-tk gene expression detection in NG4TL4-STK cell line and tumor.
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