Biosynthetic Ganciclovir Triphosphate: Its Isolation and Characterization from Ganciclovir-Treated Herpes Simplex Thymidine Kinase-Transduced Murine Cells

Author： Agbaria R. Candotti F. Kelley J.A. Hao Z. Johns D.G. Cooney D.A. Blaese R.M. Ford H.

Publisher： Elsevier

ISSN： 0006-291X

Source： Biochemical and Biophysical Research Communications, Vol.289, Iss.2, 2001-11, pp. : 525-530

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Abstract

A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 μM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10⁶ cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase α and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.