9-(2-phosphonylmethoxyethyl)guanine (PMEG) is an acyclic nucleoside phosphonate analogue that has demonstrated significant anticancer activity in vitro and in animal model systems. The phosphonate (P-C) bond is catabolically stable and resistant to phosphoesterase and nucleotidase activities. As with other nucleoside analogues, PMEG must be phosphorylated intracellularly by kinases to its active form, PMEG diphosphate (PMEGpp), which is a substrate for nuclear DNA polymerases. Thus, the primary mechanism of action in replicating cells is via DNA synthesis inhibition. The lack of a 3’-hydroxyl moiety makes PMEG a potent DNA chain terminator. However, the clinical development of PMEG has been limited by its poor permeability and transporter-mediated accumulation in target tissues of toxicity. To address these limitations, PMEG was modified to generate a membrane-permeable prodrug, GS-9219. HPLC analysis demonstrated that [14C]GS-9219 is stable for 24 hr in tissue culture medium supplemented with 10% fetal bovine serum. After GS-9219 entered human leukemia cell lines or peripheral blood mononuclear cells, PMEG was generated through two enzymatic steps. PMEG was then subsequently phosphorylated by intracellular kinases resulting in the formation of its active metabolite, PMEGpp. The major intracellular forms from GS-9219 were PMEGpp and an intermediate metabolite, with lesser amounts of PMEGp. Pharmacokinetic studies showed that the uptake of GS-9219 was linear and proportional to the external concentrations (1-10 μM) in a time-dependent manner. Comparisons of the cellular metabolism of PMEG and GS-9219 in ML-1 cells demonstrated the accumulation of PMEGpp was 4-5-fold greater at 24 hr of incubation with GS-9219 compared to that generated by [3H]PMEG. The greater accumulation of PMEGpp in GS-9219 treatment was associated with the greater pharmacodynamic potency of the prodrug, as determined by DNA synthesis inhibition, growth inhibition and loss of clonogenicity. The half-life of PMEGpp retention in cells was at least 12 hr, dependent on cell type. In summary, our results indicate that GS-9219 is a membrane-soluble prodrug that effectively delivers PMEGpp into cancer cell lines.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA