Is Stavudine Triphosphate a Natural Metabolite of Zidovudine?

Abstract

Our group has published evidence of the presence of intracellular stavudine triphosphate (d4T-TP) in patients treated with zidovudine (AZT) (1). In a recently published paper, Melendez et al. (6) present clinical data in complete contradiction with our finding, since they failed to detect d4T-TP in peripheral blood mononuclear cells (PBMCs) from more than 500 patients on AZT therapy. It is worth noting that we use a technique allowing direct determination of nucleoside reverse transcriptase inhibitor (NRTI) triphosphates, whereas Melendez et al. use an indirect method involving removal of NRTI phosphate before high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analysis. To explain these contradictory results, Melendez et al. suggest that in our experiments d4T-TP is produced artificially during the HPLC-MS-MS process. We would like to comment on this and other points raised by Melendez et al. Our demonstration of the presence of d4T-TP in PBMCs from patients on AZT therapy is based on a fully validated method (2) routinely used in our laboratory (7, 9, 10). We have never observed d4T-TP in any of hundreds of samples, processed in a number of clinical labs, from patients treated with any drug other than d4T or AZT. This therefore excludes the cross-contamination or interference alluded to by Melendez et al. The hypothesis that d4T-TP is produced artificially during the HPLC-MS-MS process is untenable because the spiking of cells with AZT-TP before processing does not lead to the production of any d4T-TP. The methodology used by our laboratory (especially ion pairing with dimethylhexylamine [DMH]) is not in question since it has been used successfully by many other groups (4, 5, 8). We note that Melendez did not succeed in using the same methodology; thus, unfortunately, a direct comparison could not be made. Since 2003, we have confirmed our initial findings with a larger number of individuals on AZT therapy by using new methods allowing direct and simultaneous determination of d4T-TP and AZT-TP in the same sample. In addition, we have demonstrated the presence of intracellular d4T-TP by using an indirect method similar to the one used by Melendez et al. These data are included in a paper in preparation. Finally, in their paper, Melendez et al. did not reference an independent study demonstrating the presence of d4T in plasma from patients treated with AZT (3), thus indirectly demonstrating the in vivo production of d4T from AZT. We have been able to confirm this observation recently (unpublished results). In conclusion, there is clearly a complete disagreement between our data and those reported by Melendez et al., but this discrepancy cannot simply be explained by crude analytical error or contamination due to our methodology. In addition, we cannot discount the possibility that the intracellular metabolism of AZT differs between the two study populations. We feel that this discrepancy should be investigated through an exchange of in vivo and artificially spiked samples between our two laboratories, so that they can be analyzed by the two techniques. We are willing to participate in such a study under the supervision of an independent laboratory.