Effect of protease inhibitors on nucleoside analogue phosphorylation in vitro

Combination antiretroviral therapy for human immunodeficiency virus (HIV) infection now involves both nucleoside analogues and protease inhibitors. Since intracellular phosphorylation is essential for the activity of all the nucleoside analogues this study was designed to investigate interactions with protease inhibitors at the intracellular level which may alter antiviral efficacy.

PHA-stimulated PBMCs (3 x 10[6] cell/plate) and U937 cells (4 x 10[6] cells/plate) were incubated with either radiolabelled zidovudine (ZDV), stavudine (d4T), zalcitabine (ddC), lamivudine (3TC) or didanosine (ddI) in the presence and absence of the protease inhibitors, indinavir, ritonavir, and saquinavir (0.1-10 microM) for 24 h. Cells were extracted overnight prior to analysis by radiometric h.p.l.c. Intracellular phosphates were standardised to pmol per million cells.

None of the three protease inhibitors tested had any significant effect on the intracellular phosphorylation of the five nucleoside analogues. It is particularly important to focus on the active triphosphate anabolites and data for control vs ritonavir (10 microM) incubations in U937 cells were as follows: ZDVTP, 0.19 +/- 0.02 vs 0.21 +/- 0.2 pmol/10(6) cells (mean +/- s.d.; n = 5); d4TTP, 0.30 +/- 0.13 vs 0.27 +/- 0.26; 3TCTP, 0.32 +/- 0.12 vs 0.26 +/- 0.19; ddCTP, 0.07 +/- 0.04 vs 0.06 +/- 0.02, ddATP, 0.014 +/- 0.003 vs 0.018 +/- 0.006 pmol/10(6) cells.

The protease inhibitors, indinavir, ritonavir and saquinavir have no effect on the enzymes responsible for phosphorylation. Combining protease inhibitors and nucleoside analogues should not lead to any intracellular interactions in vivo.