Background:  Tenofovir is an acyclic nucleotide analog which has potent in vitro and in vivo activity against HIV.  Tenofovir disoproxil (TDF), an oral prodrug of tenofovir, is approved for the treatment of HIV.  After oral administration of TDF, only tenofovir is observed in plasma.  Since tenofovir has low cellular permeability we sought to synthesize a novel prodrug of tenofovir which is orally bioavailable, stable in plasma and selectively metabolized inside cells.

Methods: GS 7340 was tested in a variety of in vitro and in vivo analyses.  For PK studies, dogs and rhesus monkeys were dosed orally.  Plasma and PBMCs were collected over 24 hours, and analyzed for tenofovir and GS 7340 by RP-HPLC.  Tissue distribution of ¹⁴C-labeled GS 7340 was examined in dogs.  Anti-HIV activity was determined using the MT-2 lymphoid cell line, mitogen-stimulated primary lymphocytes, and primary macrophages. Anti-HBV activity was assessed using the HepG2 2.2.15 hepatoblastoma cell line.

Results:  After incubation in whole blood, GS 7340 was stable in plasma but selectively metabolized to tenofovir and the mono- and diphosphate species inside PBMCs. The bioavailability of tenofovir after oral administration of GS 7340 in dogs and rhesus monkeys was approximately 20%. After a single oral dose of GS 7340 in dogs and monkeys, the concentration of tenofovir in PBMCs (AUC_0-24) was increased by >38-fold as compared with TDF.  Efficient accumulation in lymphoid tissues was also observed at 24 hours.  In vitro, the EC₅₀ against HIV was approximately 5 nM in all cell types examined; 5- to 10-fold more potent than TDF and 500- to 1000-fold more potent than tenofovir. EC₅₀ against HBV was approximately 10 nM.
Conclusions: GS 7340 has potent antiviral activity against HIV and HBV in a variety of cell types.  GS 7340 is orally bioavailable, stable in plasma and selectively hydrolyzed inside lymphatic tissue.  The high concentrations of tenofovir observed in lymphatic tissues is expected to result in increased potency and may effect low level, on-going viral replication in these tissues during HAART.