Effect of transporter modulation on the emergence of nelfinavir resistance in vitro

Background: HIV drug resistance is of increasing concern and could result from inadequate drug potency, poor therapy adherence and the existence of pharmacological sanctuary sites for viral replication. One contributing factor to the generation of such sites could be drug efflux transporters, which have been shown capable of effluxing HIV protease inhibitors from cells. Methods: In this 'proof-of-concept' study, the ability of the efflux transport inhibitor verapamil to modulate the intracellular accumulation of radiolabeled nelfinavir (NFV) and the antiviral effect of NFV was assessed in MT4 cells. Wild-type virus was then serially passaged with increasing concentrations of NFV with and without verapamil and resistance mutations monitored by sequencing of the viral protease gene. Results: The cellular accumulation ratio of ³H-NFV was 116.8 ±9.7 in controls and was significantly increased to 149.8 ±24.5 following incubation with verapamil (P<0.05, n=4). The EC ₅₀ of NFV was decreased in MT4 cells in the presence of verapamil from 8.5 ±1.3 nM to 4.4 ±0.8 nM (P<0.001, n=6). Of the 24 isolates passaged without verapamil, 21 carried the D30N mutation at detectable levels. Of the 23 passaged with verapamil, 14 carried the mutation (odds ratio=4.5; P<0.05). Conclusions: These results suggest that 'intracellular boosting' of PIs is achievable through inhibition of drug efflux proteins in vitro and that such boosting has the ability to enhance suppression of viral replication, slowing the emergence of resistance mutations.