Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMPA, in blood

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HI V. In the cells, PMPA is efficiently phosphorylated by intracellular kinase s to produce PMPApp, the pharmacologically active metabolite. Despite its d emonstrated antiviral potency, PMPA has limited cell permeability presumabl y resulting from the presence of two negative charges on the phosphonyl gro up. To enhance intracellular concentrations of PMPA, we developed a prodrug , selectively metabolized inside cells. GS-7340 (9-[(R) under bar -2-[[[[(( S) under bar -1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-metho xy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as t he intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in periph eral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was e xamined following exposure of whole blood to PMPA or GS-7340 at concentrati ons similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in p lasma, produced high levels of PMPA and its phosphorylated metabolites in P BMC but not in RBC. No intact prodrug was present in PBMC. The only other s pecies present in PBMC was monoalaninyl PMPA. The levels of PMPA and the ph osphorylated metabolites were over 20 times greater than those after incuba tion with PMPA. The dog and human blood data were similar. The intracellula r levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10- fold concentration range indicating a lack of saturability of uptake and ph osphorylation. Since PMPApp is the species responsible for antiviral activi ty of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.