Influence of prior exposure to zidovudine on stavudine phosphorylation in vivo and ex vivo

Intracellular phosphorylation of stavudine (d4T) and zidovudine (ZDV) was i nvestigated in peripheral blood mononuclear cells (PBMCs) isolated from ZDV -naive and ZDV-experienced human immunodeficiency virus (HIV)-positive pati ents. An in vivo study measured the amount of d4T triphosphate (d4TTP), whi le an ex vivo study assessed the capacity of cells to phosphorylate added d 4T. Endogenous dTTP was also measured. d4TTP and dTTP were determined in vi vo using a reverse transcriptase chain termination assay, In ex vivo studie s, d4T (1 muM) was incubated in resting and phytohemagglutinin-stimulated ( 10 mug ml(-1); 72 h) PBMCs for 24 h, After washing and methanol extraction, radiolabeled anabolites were detected by high-performance liquid chromatog raphy. d4TTP reached its highest level 2 to 4 h after dosing (0.21 +/- 0.14 pmol/10(6) cells; n = 27 [mean +/- standard deviation]). Comparison of ZDV -naive and ZDV-experienced individuals showed no significant difference in levels of d4TTP (ZDV naive, 0.23 +/- 0.17 pmol/10(6) cells [n = 7] versus Z DV experienced, 0.20 +/- 0.14 pmol/10(6) cells [n = 20]; P = 0.473) or the d4TTP/dTTP ratio (0.14 +/- 0.12 [n = 7] and 0.10 +/- 0.08 [n = 20], respect ively; p = 0.391), Ex vivo data demonstrated no significant difference in t he formation of d4TTP or total d4T phosphates in naive and experienced pati ents (0.086 +/- 0.055 pmol/10(6) cells in ZDV-naive patients [n = 17] versu s 0.081 +/- 0.038 pmol/10(6) cells in ZDV-experienced patients [n = 22]; P = 0.767), The ability of HIV-infected patients to phosphorylate d4T in vivo and ex vivo was unchanged with increasing exposure to ZDV.