Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine

Abstract

The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability of approximately 77%, with a high proportion (approximately 16.5% of the levels in serum) found in the cerebrospinal fluid.

FIG. 1

Chemical structures of the enantiomers of dOTC.

FIG. 2

Reverse-phase (C₁₈) HPLC elution profile of a 10% trichloroacetic acid extract of activated PBMCs incubated for 24 h with 2.5 μM [³H]dOTC (specific activity, 2.3 Ci/mmol). The column was equilibrated in the isocratic running buffer as described in Materials and Methods before application of the test sample. For this particular experiment, the retention times of the parent nucleoside (13.169 min) and its MP (10.163 min), DP (17.466 min), and TP (36.955 min) derivatives were determined by using chemically synthesized standards.

FIG. 3

Intracellular levels of dOTC or 3TC and their metabolites in PHA-P- and IL-2-stimulated PBMCs following continuous exposure of the cells to ³H-labeled nucleoside for up to 24 h. In this experiment the cells were incubated in the presence of 2.5 μM [³H]dOTC (specific activity, 2.3 Ci/mmol) (A) or 1 μM [³H]3TC (specific activity, 12 Ci/mmol) (B) for the indicated lengths of time. The data presented for dOTC are the averages of two or more independent experiments, while the data presented for 3TC represent data from one experiment.