Background: A significant number of early virologic failures result from 2 triple nucleoside analog (NA) regimens; 1 using didanosine (ddI), lamivudine (3TC), and tenofovir (TFV), and the other using abacavir (ABC), 3TC, and TFV. This study comprehensively evaluated the pharmacologic factors that may lead to virologic failures, including intracellular drug–drug interaction, changes in endogenous nucleotide levels, and efflux transporter expression.

Methods: U937 and CEM cells were used to determine whether an intracellular drug–drug interaction existed in either combinations:  ddI, 3TC, and TFV, or TFV, ABC, and 3TC. Nucleoside-resistant variants were developed using serial passage with increasing nucleoside exposure. U937 and CEM were treated alone, in dual or triple combination at 5 and 20 µM. The intracellular NA triphosphate anabolites (ddNTP) and their respective endogenous triphosphate levels were also determined using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Western analysis for MRP2 and MRP4 expression was evaluated after nucleoside treatment.

Results: Cells treated with 5 µM of ABC, TFV, and 3TC produced intracellular ratios of carbovir triphosphate (CBV-TP)/ deoxyguanosine triphosphate (dGTP), TFV-diphosphate (DP)/deoxyadenosine triphosphate (dATP), and 3TC-TP/deoxycytidine triphosphate (dCTP) of 82.5%, 88.3%, and 79.6%, respectively; 17-day treatment produced intracellular ratios of CBV-TP/dGTP, TFV-DP/dATP, and 3TC-TP/dCTP were 84.8%, 95.7%, and 67.3%, respectively. Cells treated with the combination of TFV and ABC in concentration escalating studies, both intracellular TFV-DP and CBV-TP were reduced significantly (p <0.001) in a concentration-dependent manner. Similarly, cells treated with 5 µM of ddI, TFV, and 3TC used in dual or triple combination revealed a reduction of intracellular ddNTP by 10 to 22%. When 20 µM TFV was used in combination with increasing concentrations of ddI, reduction in TFV-DP and dideoxyadenosine triphosphate (ddATP) levels were 25% and 40%, respectively (p <0.001). TFV at 20 µM increased endogenous dGTP and dATP levels by 2.4- and 2.7-fold, respectively (p <0.01). Intracellular ddNTP was significantly lower in U937^TFV and U937^ABC when compared to U937 (p <0.001).

Conclusions: These findings suggest competitive inhibition between TFV and ABC and TFV and ddI. Moreover, long-term exposure with either TFV or ABC can increase expression of both MRP2 and MRP4 corresponding to reduced intracellular levels of ddNTP. TFV at supraphysiologic concentrations was shown to increase endogenous dGTP and dATP levels. These findings taken together may provide significant insights into pharmacologic mechanisms leading to clinical virologic failures.