Background: A direct liquid chromatography tandem mass spectrometry (LC/MS/MS) assay was recently developed and validated to measure the active intracellular triphosphorylated anabolites of NRTIs in PBMC. The aim was to verify the relevance of this assay in samples coming from d4T-and ddI-treated patients and to approach the inter-patient variability (IPV) of the level measured.

Methods: Naïve adult patients were enrolled in an efficacy and tolerance open pilot study in Dakar, Senegal of d4T (40/30-mg BID), ddI (400/250-mg qd), and efavirenz (600 mg qd) regimen (IMEA012-ANRS1206). A substudy was conducted to assess d4T-TP and ddA-TP intracellular concentration (ICC) at M6. Delay between dosing to sampling was recorded. PBMCs were obtained by ficoll on EDTA. Limits of LC/MS/MS quantification (LOQ) are 61 and 53 fmol/sample for d4T-TP and ddA-TP, respectively. d4T and ddI plasma concentrations (PC) were simultaneously measured (LOQ: 0.5 ng/mL). Viral load (VL) and CD4 cells count were available at baseline and M6.

Results: All samples were available for the 28/40 first patients (12 male). At baseline, CDC stage C=46%, and mean values±sd: weight=54±8 kg, VL=5.5±0.4log copies/mL and CD4=134±86/mm³. At M6, mean values±sd: VL=2.3±1.0log copies/mL, CD4=226±153/mm³. VL remained>200 copies/mL in 6 patients. d4T-TP and ddA-TP ICC were above the LOQ in 25/28 and 26/28 patients respectively (median [range] d4T-TP and ddA-TP: 31 [0-99] and 8 [0-23] fmol/10⁶cells). 2 of the 3 d4T-TP undetectable patients had discontinued their treatment according to medical assessment, confirmed by high VL and undetectable d4T and ddI PC. In the 17 samples collected 2-5 hours after last dosing, d4T-TP IC median (range) was 37 (11-81) and d4T PC 281 (47-670) ng/mL. In the 22 samples collected 10-16 hours after last dosing, ddA-TP IC median (range) was 9.8 (0-23) and ddI PC 29 (2-162). ICC IPV (CV%) were 59 and 48 for ddA-TP and d4T-TP, respectively. Significant correlation was found between ICC and PC for d4T-TP vs d4T (r=0.6, p=0.005), not for ddI vs ddA-TP. In the 22/28 patients with VL<200 copies/mL at M6, d4T-TP and ddA-TP ICC median (range) were 34 (0-99) and 8 (0-23) fmol/10⁶ cells, respectively.

Conclusions: ICC were quantified for 25 of 26 patients for both ddA-TP and d4T-TP with the exception of 2 non-compliant patients. Further evaluation is needed to evaluate the impact of IPV on this assay. However, results support additional PK/PD studies of intracellular anabolites in order to define the position of this assay in ddI and/or d4T treatment monitoring.