Background:  Studies have reported 3-fold higher zidovudine (ZDV) -triphosphate concentrations in unfractionated peripheral blood mononuclear cells (PBMC) from patients with severe CD4-depletion versus patients with higher CD4 cells. Similar trends were also reported for lamivudine (3TC) -triphosphate. Such compartmentalization of triphosphate concentrations in different cell types may result in cell-specific or tissue-specific potency issues. The objective of this study was to compare ZDV- and 3TC-triphosphate concentrations in unfractionated PBMC versus CD4-depleted and CD4-purified cells from HIV-seronegative adults receiving ZDV and 3TC.

Methods:  Volunteers had taken 300 mg of ZDV + 150 mg of 3TC orally twice daily for at least 7 days. We collected 60 mL of blood 2 or 5 hours post-observed dose and PBMC were divided into unfractionated PBMC, CD4 purified cells, and CD4 depleted PBMC using CD4 magnetic immuno-beads. ZDV- and 3TC-triphosphate concentrations were determined in each cell fraction with liquid chromatrography-mass spectrometry. Triphosphates were compared across cell types with paired t-tests.

Results:  Participants included 6 men and 2 women. Cell processing was completed within 3.1 to 3.5 hours and all cell fractions were >95% viable just before lysing. The median (range) percentage of CD4⁺ cells in each fraction were 99% (99 to 100) for CD4 purified cells, 63% (53 to 69) for unfractionated PBMC, and 14% (4 to 24) for CD4 depleted PBMC. Median (range) ZDV-triphosphate concentrations were 8.0 (5.3 to 10.3) fmol/million cells in CD4-purified cells, 26.5 (12.9 to 42.2) in unfractionated PBMC, and 34.2 (16.4 to 52.2) in CD4-depleted PBMC (p <0.001 CD4 purified cells versus other fractions). 3TC-triphosphate concentrations were 4.6 (2.3 to 6.7) pmol/million cells in CD4 purified cells, 4.8 (3.5 to 8.8) in unfractionated PBMC, and 6.8 (4.0 to 13.1) in CD4 depleted PBMC (p <0.03 CD4 depleted PBMC versus other fractions).

Conclusions:  Compared with unfractionated PBMC, CD4-depleted PBMC had 1.3-fold higher ZDV-triphosphates and 1.4-fold higher 3TC-triphosphates in HIV-seronegative adults, which does not completely explain the magnitude of triphosphate differences seen in patients with depleted CD4 cells. An important finding was that ZDV-triphosphate concentrations were significantly decreased in CD4 purified cells. Triphosphate compartmentalization could lead to HIV sanctuary sites for cells with low triphosphates or toxicity for cells with high triphosphates.