Abstracts
2000 Digestive Disease Week

# 2297 19FNMR Spectroscopic Detection and Kinetics of Conversion of 5-FC to 5-FU in Human Colon Cancer in Vivo and in Vitro: A Novel Approach for Detecting Adenoviral Cytosine Deaminase Gene Transfer.
Allen N. Gustin, Pal Kiss, Gabriel A. Elgavish, Laszlo Lenard, Donald J. Buchsbaum, Selwyn M. Vickers, Birmingham, AL

Introduction: Fluorinated pyrimidines can be detected by NMR spectroscopy with good discrimination. 19F is a 100% abundant isotope and can be detected with NMR sensitivity of 83% relative to proton. 19FNMR spectroscopy was evaluated for its ability to monitor the enzymatic conversion of the inactive prodrug 5-Fluorocytosine (5-FC) to the active chemotherapeutic drug 5-Fluorouracil (5-FU) following infection of human colon adenocarcinoma cells (LS174T) with an adenoviral vector encoding the cytosine deaminase (CD) gene in vitro and in vivo. Methods: LS174T cells were transfected in vitro with a replication incompetent adenovirus encoding the CD gene (AdCMVCD) at various MOIs (50,75,100 and 150). Once infected, cells were evaluated by in vitro conversion assay by TLC and by NMR spectroscopy. In vitro conversion evaluated cell lysate for conversion of tritiated 5-FC to 5-FU after 18 h incubation by TLC. Infected cells were placed in vials or injected subcutaneously into flanks of nude mice. Each vial or mouse was placed in a 19FNMR surface coil probe and then inserted in a vertical 8.4 Tesla wide-bore magnet of a Bruker 360 AM NMR spectrometer. After shimming, 500 ml of 5-FC (0.058 mmoles) was added and spectra (64 scans each) were acquired with a 5min total scan time for each spectrum, using a 5 sec repetition delay. Cells in flanks of nude mice were evaluated similarly. 5-FC (500 ml, 0.058 mmoles) was injected subcutaneously at site of tumor cells. Mice were positioned for direct contact between tumor cells and detection coil. Results: In vitro TLC conversion assay demonstrated higher MOIs resulted in higher percentage of conversion of 5-FU (MOI 100, 73.6 +/- 2.1% versus MOI 50, 62.0 +/- 1.8% p<0.05). These data were consistent with 5-FU conversion detected by 19FNMR. In vitro 19FNMR showed production of 5-FU was faster at high MOIs (time for 50% decrease in 5-FC signal with 50% increase in 5-FU signal was 45min and 130min for MOIs 150 and 75, respectively p<0.05). In vivo 19FNMR analysis corroborated the in vitro data with similar trends in conversion rates. Conclusion: Increasing rates of 5-FU conversion correlate with increasing MOI of AdCMVCD. 19FNMR provides a noninvasive method to evaluate in vivo efficacy of CD gene transfer in tumors and allows for determination of optimal time points for combined therapy with radiation.