Simultaneous LC-MS/MS method for the quantification of 5-flucytosine, ganciclovir, and valganciclovir to support combined prodrug-activated gene therapy.

In prodrug-activated gene therapy (PAGT), a prodrug is converted into a cytotoxic metabolite only in cells transduced with viral or bacterial genes. Recent studies have explored the combined use of cytosine deaminase/5-flucytosine (CD/5-FC) and herpes simplex virus-derived thymidine kinase/ganciclovir (HSV-TK/GCV) PAGT systems to enhance anticancer efficacy. This study aimed to develop a simultaneous analytical method for 5-FC, GCV, and valganciclovir (VGC), the prodrug of GCV, using LC-MS/MS and to investigate the pharmacokinetic differences following their coadministration in Sprague-Dawley (SD) rats. The developed simultaneous analytical method met FDA guidelines and demonstrated good reproducibility and reliability. Using this method, the pharmacokinetic characteristics of the three compounds (5-FC, GCV, and VGC) were assessed in SD rats. Following intravenous and oral administration of VGC, 61.44% of VGC converted to GCV, and the bioavailability of GCV improved from 9.78% to 61.38%. In vitro studies were conducted to evaluate the metabolic pathways and conversion rates. Slow elimination of VGC in the microsomes and plasma suggests that its primary metabolic pathway is not mediated by cytochrome P450 enzymes or plasma esterases. Although certain pharmacokinetic parameters differed between the single and coadministration groups, the differences in the maximum plasma concentration (Cmax) were small, and no significant differences were observed in area under the plasma concentration-time curve (AUC). Considering that renal excretion is the major elimination pathway for both 5-FC and GCV, the extent of drug-drug interactions appears to be minimal, suggesting that their established maximum doses can be applied without safety concerns. Our study results may provide a basis for future studies on drug-drug interactions and dosage optimization.