Pharmacokinetics and the bystander effect in CD：：UPRT/5-FC bi-gene therapy of glioma

• Published in: Chinese medical journal Vol. 122; no. 11; pp. 1267 - 1272
• Main Authors: Shi, De-zhi, Hu, Wei-xing, Li, Li-xin, Chen, Gong, Wei, Dong, Gu, Pei-yuan
• Format: Journal Article
• Language: English
• Published: China Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China 05.06.2009
• Subjects: Animals; Antimetabolites - pharmacokinetics; Antimetabolites - therapeutic use; Cell Line; Cytosine Deaminase - genetics; Cytosine Deaminase - physiology; Flucytosine - pharmacokinetics; Flucytosine - therapeutic use; Genetic Therapy - methods; Glioma - drug therapy; Glioma - therapy; Humans; Magnetic Resonance Imaging; Male; Pentosyltransferases - genetics; Pentosyltransferases - physiology; Rats; Rats, Sprague-Dawley; UPRT/5-FC双基因; 临床试验; 脑胶质瘤; 药物动力学; bystander effect; pharmacokinetics; glioma; cytosine deaminase; magnetic resonance spectroscopy; uracil phosphoribosyltransferase
• ISSN: 0366-6999; 2542-5641
• DOI: 10.3760/cma.j.issn.0366-6999.2009.11.006

Background 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 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/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 ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT 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-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT 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 ^19F-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-CD：：UPRT cells, the survival rate was （79.55±0.88）% （P 〈0.01）. As the C6-CD：：UPRT ratio increased, the survival rate of the cells decreased. ^19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD：：UPRT in the mixture increased. Conclusions ^19F-MRS studies indicated that C6-CD：：UPRT cells could effectively express CD and UPRT enzymes. The CD：：UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD：：UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and ^19F-MRS can monitor transferred CD：：UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.