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 EC50 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.
References
1.
CaneP., ChrystieI., DunnD.Time trends in primary resistance to HIV drugs in the United Kingdom: multicentre observational study.BMJ2005; 331: 1368.
2.
LooT.W., ClarkeD.M.Recent progress in understanding the mechanism of P-glycoprotein-mediated drug efflux.J Membr Biol2005; 206: 173–185.
3.
JannehO., OwenA., ChandlerB.Modulation of the intracellular accumulation of saquinavir in peripheral blood mononuclear cells by inhibitors of MRP1, MRP2, P-gp and BCRP.AIDS2005; 19: 2097–2102.
4.
WilliamsG.C., LiuA., KnippG., SinkoP.J.Direct evidence that saquinavir is transported by multidrug resistance-associated protein (MRP1) and canalicular multispecific organic anion transporter (MRP2).Antimicrob Agents Chemother2002; 46: 3456–3462.
5.
SrinivasR.V., MiddlemasD., FlynnP., FridlandA.Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters.Antimicrob Agents Chemother1998; 42: 3157–3162.
6.
MaffeoA., BellomiF., SolimeoI.P-glycoprotein expression affects the intracellular concentration and antiviral activity of the protease inhibitor saquinavir in a T cell line.New Microbiol2004; 27: 119–126.
7.
OwenA., ChandlerB., BrayP.G.Functional correlation of P-glycoprotein expression and genotype with expression of the human immunodeficiency virus type 1 coreceptor CXCR4.J Virol2004; 78: 12022–12029.
8.
OwenA., JannehO., HartkoornR.C.In vitro synergy and enhanced murine brain penetration of saquinavir coadministered with mefloquine.J Pharmacol Exp Ther2005; 314: 1202–1209.
9.
KimptonJ., EmermanM.Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated β-galactosidase gene.J Virol1992; 66: 2232–2239.
10.
ReedL.J., MuenchJ.A simple method of estimating fifty per cent endpoints.Am J Hyg1938; 27: 493–497.
11.
CrivellatoE., CandussioL., RosatiA.M., Bartoli-KlugmannF., MallardiF., DecortiG.The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1.J Histochem Cytochem2002; 50: 731–734.
12.
OwenA., ChandlerB., FordJ., KhooS., BackD.Differential expression of human immunodeficiency virus coreceptors, by CEM, CEMVBL, and CEM E1000 cells.J Infect Dis2003; 187: 874–875; author reply 875–876.
13.
KhooS.H., HoggardP.G., WilliamsI., MeadenE.R., NewtonP., WilkinsE.G.Intracellular accumulation of human immunodeficiency virus protease inhibitors.Antimicrob Agents Chemother2002; 46: 3228–3235.
14.
JonesK., BrayP.G., KhooS.H.P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance?AIDS2001; 15: 1353–1358.
