Lack of Evidence for In Vivo Transformation of Zidovudine Triphosphate to Stavudine Triphosphate in Human Immunodeficiency Virus-Infected Patients

Abstract

The in vivo and in vitro determination of significant intracellular stavudine (d4T) triphosphate (d4TTP) concentrations in human immunodeficiency virus (HIV)-infected subjects and NS-1 cells treated with zidovudine (ZDV) has recently been reported. This study was conducted to corroborate these findings with in vivo samples from HIV-infected subjects taking ZDV and in vitro CEM(SS) cells incubated with different ZDV concentrations. Previously, we have reported on our validated high-performance liquid chromatography coupled with tandem mass spectrometry methodology for the simultaneous determination of d4TTP, lamivudine triphosphate, and ZDV triphosphate (ZDVTP) concentrations. Using this methodology, we monitored the d4TTP concentration in more than 100 samples from HIV-infected subjects treated with d4T. In addition, we simultaneously monitored the concentrations of d4TTP and ZDVTP in more than 500 samples from HIV-infected individuals who were taking ZDV. Finally, we performed in vitro studies by incubating CEM(SS) cells with 10 microM, 50 microM, and 100 microM ZDV and monitored the formation of d4TTP at 24 and 48 h. We could measure d4TTP concentrations from HIV-infected individuals with a limit of quantitation (LOQ) of 2.7 fmol/10(6) cells (total injection, 54 fmol). In the in vivo studies, we measured the d4TTP concentrations among patients receiving d4T treatment, but the samples from patients taking ZDV did not provide d4TTP concentrations above the LOQ. Furthermore, in vitro samples did not produce any signal for d4TTP, despite the detection of substantial ZDVTP concentrations in CEM(SS) cells. Thus, contrary to the previous report, we found no evidence for the in vivo or in vitro transformation of ZDVTP to d4TTP in HIV-infected subjects or CEM(SS) cells.