Pharmacokinetics and the bystander effect in CD::UPRT/5-FC bi-gene therapy of glioma / 中华医学杂志(英文版)

ABSTRACT

<p><b>BACKGROUND</b>Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CDUPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CDUPRT/5-FC has not been verified in vivo and in vitro. Before the CDUPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.</p><p><b>METHODS</b>C6 and C6-CDUPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CDUPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CDUPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CDUPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA).</p><p><b>RESULTS</b>(19)F-MRS on samples from C6-CDUPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CDUPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CDUPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CDUPRT in the mixture increased.</p><p><b>CONCLUSIONS</b>(19)F-MRS studies indicated that C6-CDUPRT cells could effectively express CD and UPRT enzymes. The CDUPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CDUPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CDUPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.</p>