Biosynthetic ganciclovir triphosphate: Its isolation and characterization from ganciclovir-treated herpes simplex thymidine kinase-transduced murine cells

A method is described for the preparation of ganciclovir triphosphate (GCV- TP) using murine colon cancer cells (MC38) transduced with the herpes simpl ex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral -tk contain required viral and host enzymes needed for complete cellular sy nthesis of this potent antiviral metabolite. Dose response studies showed o ptimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-t k cells to 300 muM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cel ls. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme in cluded methanolic extraction and anion-exchange chromatography to recover t he target triphosphate. Mass spectral analysis and selective enzyme degrada tion provided structural confirmation of the purified product. Biological a ctivity of the purified GCV-TP was demonstrated by competitive inhibition e xperiments using human DNA polymerase a and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement wit h previous reports. The GCV-TP obtained was further used to enzymatically p repare GCV mono- and diphosphate in high yield. This method provides an eas ily scalable means of preparing milligram amounts of the triphosphates of p harmacologically active acyclic nucleosides like ganciclovir.