WR1065 mitigates AZT-ddI-induced mutagenesis and inhibits viral replication

Abstract

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.

Fig. 1

Comparison of the induced mutant frequency at the HPRT locus of human TK6 lymphoblastoid cells exposed for 3 days to vehicle alone, 150 μM WR1065, 100 μM AZT-ddI (equimolar), or 100 μM AZT-ddI (equimolar) plus 150 μM WR1065. Drug-induced mutant frequencies were determined by subtracting the mean mutant frequency measured in the vehicle-exposed group (mutant frequency = 2.8 × 10⁻⁶) from the mean observed mutant frequency measured in individual treatment groups. An asterisk (*) designates that the mean observed mutant frequency in AZT-ddI-exposed cells was significantly increased over the mean (vehicle-exposed) control cell value (P < 0.05), whereas the double asterisk (**) indicates that the mean observed mutant frequency in cells exposed to AZT-ddI plus WR1065 was significantly reduced compared with cells exposed to AZT-ddI only (P < 0.05) but was not significantly different from the mean observed value for vehicle-exposed cells. WR1065 reduced the mutagenic effects of AZT-ddI by at least 85%. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 2

Titration of Ad4p infectious virus yields by plaque assay in A549 cell monolayers. Cells in six-well plates were infected with 1000 PFU of Ad4p (RI-67) and treated with 0 or 100 μM WR1065. After 1 hr adsorption, cells were replenished with minimum essential medium containing 0 or 100 μM WR1065. At 7 days postinfection, infected cells were frozen and freeze-thawed preparations were titered by standard plaque assay. Titers shown are from Table III, experiment 1 except the mean virus yields are expressed as PFUs × 10⁶/ml.

Fig. 3

Dose response for intracellular concentration of WR1065 following treatment of primary mouse (A) or rat (B) lymphocytes for 1 or 2 hr with 0, 10, 50, or 100 μM amifostine converted to WR1065 (n = 2–4/species for each exposure group). Amifostine was dephosphorylated for 30 min with alkaline phosphatase to yield WR1065 for cell treatment. A high-pressure liquid chromatography method with electrochemical detection was used to measure WR1065, and the concentration of WR1065 per million cells in each sample was determined by interpolating the peak areas to a standard curve for WR1065 [Pamujula et al., 2004]. (A) Data from WR1065-exposed mouse lymphocytes; (B) data from WR1065-exposed rat lymphocytes.