Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of highly active antiretroviral therapy (HAART) recommended for the treatment of human immunodeficiency virus (HIV) infection (7,20), and the use of NRTI-containing combination therapy has significantly decreased the morbidity and mortality associated with HIV disease in treated patients (18,19). NRTIs share a common mechanism of action. All undergo intracellular activation to the NRTI triphosphate (NRTI-TP) form, after which they compete with endogenous deoxynucleotide triphosphates for binding to the viral reverse transcriptase (RT) enzyme, and incorporation of the monophosphate (MP) into the nascent DNA. Since NRTIs lack a substituent capable of supporting further DNA elongation, the incorporation of the NRTI-MP results in the termination of chain elongation and inhibition of reverse transcription.Many patients whose viral replication is effectively controlled by combination antiretroviral therapy ultimately experience virologic failure because of the development of antiretroviral resistance (for reviews, see references 23 and 24). A 6-year survey of viral genotypes in France found that almost 80% of clinical HIV samples collected until 2002 had mutations conferring resistance to NRTIs (29). Primary infection with resistant strains is also being increasingly recognized as a clinical problem in some countries (10,14,26,31). NRTI resistance results from mutational changes within the RT gene. The resulting resistance mechanisms fall into two main categories (for reviews, see references 4 and 8). One group of RT mutations acts to increase the rate of RT-catalyzed phosphorolysis, i.e., the RT-catalyzed excision of the incorporated NRTI-MP from the chain-terminated DNA. These mutations include M41L, D67N, K70R, L210W, T215Y, and K219Q and are sometimes referred to collectively as thymidine analogue mutations (TAMs). The accumulation of these mutations confers high-level resistance to zidovudine and affects viral sensitivity to other NRTIs, including stavudine, tenofovir, and abacavir (4). The other mechanism by which mutations in RT can cause resistance to NRTIs is by altering the discrimination between deoxynucleoside triphosphate substrates and NRTI-TP inhibitors by the substrate binding site of RT. Examples of such mutations include the M184V mutation, which is found frequently in patients experiencing virologic failure during treatment with lamivudine-containing HAART (6,15). This mutation causes high-level resistance to lamivudine and (in combination with other mutations) reduces sensitivity to didanosine, zalcitabine, and abacavir (32). Other mutations

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 Ki values for dOTC-TP obtained against human DNA polymerases α, β, and γ 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 μM, rising to only 2.53 and 2.5 μM 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 [3H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 μM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 μM, 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.

An established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous uridine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug-free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII-or AvaI-digested total cellular DNA.

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