Uridine Excess does not Interfere with the Antiretroviral Efficacy of Nucleoside Analogue Reverse Transcriptase Inhibitors

Using human hepatocyte cultures (HepG2 cells), we have discovered that uridine supplementation may be used as a novel strategy to prevent or treat depletion of mitochondrial DNA (mtDNA) by pyrimidine nucleoside analogue reverse transcriptase inhibitors (NRTIs), for example, zalcitabine [1]. In this model, uridine increased mtDNA levels, and abrogated mitochondrial toxicity and its consequences for the entire cell. Others have also found beneficial effects of uridine supplementation in models of zidovudine-related haematopoietic toxicity, both in vitro and in animals [2,3]. Depending on the system studied, uridine was effective at concentrations of 50–200 μM. Competition of uridine or its metabolites with NRTIs is the most plausible explanation for the protective effect, whereby various steps of mitochondrial nucleotide utilization may also be involved. For these and other reasons, it is important to know if the high concentrations of uridine could interfere with the antiretroviral activity of the NRTIs, for example, by competition at the level of HIV reverse transcriptase. To our surprise, we could detect only minor influences of uridine on NRTI-mediated HIV inhibition. We have examined the potential interference in three systems: in the human CD4 lymphocytic T-cell line MT-2, in a modified HeLa indicator cell line carrying a HIV-Tat-responsive reporter gene and the CCR5 HIV co-receptor [4], and in primary human peripheral blood mononuclear cells (PBMCs). Virus replication was monitored by counting syncytia in the T-cell line as described [5], or by an X-Gal assay in the modified HeLa cells. The X-4 tropic HIV-1IIIB isolate was used for the syncytium assay, whereas the R-5 tropic HIV1BaL-isolate was used in the two other systems. Virus production in PBMCs was quantified on the modified HeLa cells. NRTIs were added to the replication systems in decreasing concentrations to establish dose–response curves. In parallel, uridine was either not added, or given in concentrations of 62, 185 or 615 μM. Each experiment was done in triplicate. EC50 and EC90 values of individual dose–response curves were calculated by statistical fitting with the Origin (v7.0.) program. Mean inhibitory concentrations among the different uridine concentrations were compared by Friedman’s trend analysis at the 0.05 significance level. As illustrated in Figure 1, none of the NRTIs was essentially compromised in its efficacy by uridine at a 30to 200000-fold molar excess over the EC50 concentration. A