Background: Previous work has demonstrated that the multidrug resistance protein transporter MRP4 alters the efflux of nucleotide monophosphates. However, altered efflux has not been determined specifically for effects on accumulation of TP necessary for antiviral activity nor have the kinetics of mono- (MP) and di- (DP) phosphates been characterized. The aim of this study was to construct a complete kinetic model for the phosphorylation of ZDV and examine altered kinetics as a function of differential expression of MRP4.

Methods: The accumulation, kinetics, and persistence of ZDV and each nucleotide were determined in wild type CEM (CEM^wt) and mutant CEM (CEM^mut) cells overexpressing MRP4. Expression of MRP4 was determined by real time PCR in wt and mut CEMs and in human PBMCs. Accumulation experiments were done at clinically relevant ZDV concentrations over 24 hrs in the presence and absence of an MRP4 inhibitor (MK571). The kinetic parameters for ZDV and each nucleotide (MP, -DP, -TP) were determined during washout experiments in drug-free media.

Results: Semi-quantitative RT-PCR demonstrated a 5-fold difference in MRP4 expression for the CEM^mut vs CEM^wt. Initial experiments in human PBMCs showed at least a 2-fold difference in MRP4 expression. As previously shown, ZDV-MP was 9X higher in CEM^wt than CEM^mut at 24 hrs. However, ZDV, DP, and TP were also increased 8-, 5-, and 6-fold, respectively. Concurrent incubation with the MRP4 inhibitor increased the amount of ZDV-TP 1.5- to 2-fold in both CEM^wt and CEM^mut cells. Kinetic parameters were determined for ZDV and each nucleotide. Disappearance of ZDV and each nucleotide was biphasic (T½a and b) with no change in half-lives for ZDV but a higher T½a (6X) and b (22X) for ZDV-MP in CEM^wt versus CEM^mut. Moreover, there was a 3- to 5-fold faster T½a and a 2-fold higher T½b for ZDV-DP and -TP. T½b for ZDV-TP was 866 minutes in CEM^wt and 415 minutes in CEM^mut.

Conclusions: Increased MRP4 expression alters the kinetics of ZDV-TP as well as ZDV-MP with a correspondence between MRP4 expression, kinetics of phosphorylation, and extent of ZDV-TP accumulation. Inhibition of MRP4 activity increases ZDV-TP accumulation. A kinetic model has been developed that defines the efflux, and phosphorylation of ZDV analogous to the turnover of endogenous nucleotide pools and can be used to examine factors that determine intersubject variability in the lymphocytes of patients receiving nucleoside therapy.

Keywords: Transport; nucleosides; MRP4