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

Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC genetherapy,5-FU will be mostly converted into nontoxic β-alanine without uracil phosphoribosyltransferase （UPRT）.UPRTcatalyzes the conversion of 5-FU to 5-fluorouridine monophosphate,which directly kills CD::UPRT-expressing cells andsurrounding cells via the bystander effect.But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has notbeen verified in vivo and in vitro.Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial,it is essentialto monitor the transgene expression and function in vivo.Thus,we developed a preclinical tumor model to investigate thefeasibility of usingF-magnetic resonance spectroscopy (F-MRS) and optical imaging to measure non-invasive CDand 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 by19F-MRS.Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FCand theirF-MRS spectra recorded.Then the pharmacokinetics of 5-FC was proved.Mixtures of C6 and C6-CD::UPRTcells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded.To determinethe mechanism of the bystander effect,the culture media from cell comprising 25% and 75% C6-CD::UPRT cells wereexamined byF-MRS.A comparative study of mean was performed using analysis of variance （ANOVA）.ResultsF-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signalscorresponding to 5-FC,5-FU and fluorinated nucleotides （F-Nuctd）.For the C6 mixture,only the 5-FC peak was detected.Invivo serialF-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection.The5-FU concentration reached a maximum at about 50 minutes.The F-Nuctd signal appeared after about 1 hour,reached amaximum at around 160 minutes,and was detectable for several hours.At a 10% ratio of C6-CD::UPRT cells,thesurvival rate was （79.55±0.88）% （P <0.01）.As the C6-CD::UPRT ratio increased,the survival rate of the cells decreased.F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT inthe mixture increased.ConclusionsF-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 genetherapy is suitable for 5-FC to F-Nuctd metabolism; andF-MRS can monitor transferred CD::UPRT gene expressionand catalysis of substrates noninvasively,dynamically and quantitatively.