Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue

Abstract

An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective concentration (EC(50)) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 microM compared to an EC(50) of 5 microM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1,000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37 degrees C and a half-life of 28.3 min in an MT-2 cell extract at 37 degrees C. The antiviral activity (>10 x the EC(50)) and the metabolic stability in MT-2 cell extracts (>35 x) and plasma (>2.5 x) were also sensitive to the stereochemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 microg-eq/ml compared to 0.2 microg-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available prodrug tenofovir DF (Viread).

FIG. 1.

Structure.

FIG. 2.

Routes of metabolism.

FIG. 3.

Radiochromatograms labeled with ¹⁴C from plasma (A), aggregated red blood cells (B), and PBMCs (C), obtained after incubation of 17.4 μM GS 7340 with whole human blood for 1 h at 37°C.

FIG. 4.

Metabolism of [¹⁴C]GS 7340 in PMBCs after 1-h incubation in whole human blood at 37°C (mean ± SD; n = 3 samples of whole blood).

FIG. 5.

Formation of tenofovir and metabolites in MT-2 cells during a 24-h incubation with 10 μM [¹⁴C]GS 7340 (mean ± SD; n = 3 samples of MT-2 cells).

FIG. 6.

Mean concentration versus time profiles for tenofovir, GS 7340, and GS 7339 in plasma and tenofovir in PBMCs after oral administration of GS 7171 (10 mg-eq/kg tenofovir) to dogs (mean ± SD for samples from four dogs).

FIG. 7.

Tenofovir in PBMCs and plasma (AUC_{0-24 h}) after subcutaneous administration of tenofovir and after oral administration of tenofovir DF, GS 7339, GS 7340, and GS 7171 (10 mg-eq/kg) to dogs (mean ± SD, n = 5 dogs).