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

Abstract

The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)-saquinavir, ritonavir, nelfinavir, and indinavir-interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1(+) CEM/VBL100 and MRP1(+) CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy.

FIG. 1

Calcein-AM flux in multidrug-resistant CEM cells. Calcein flux was measured as described in Materials and Methods. Relative fluorescence units (F) (mean ± standard deviation) are shown. Panel A shows the rate of accumulation of calcein in MDR1⁺ CEM/VBL100 (broken line) and wild-type CEM/WTB (solid line) cells. The effects of reserpine on the calcein flux in CEM/VBL100 and CEM/WTB cells are shown in panels B and C, respectively. Calcein fluorescence of CEM/VBL100 cells was significantly enhanced when incubated with the MDR1 antagonist reserpine (panel B, solid line) compared to CEM/VBL100 cells incubated with saline (panel B, broken line). By contrast, the calcein fluorescence of wild-type CEM/WTB cells was not different when incubated in the presence of either reserpine (hatched line) or saline (solid line). The P values indicate the statistical differences between the slopes of calcein fluorescence in CEM/WTB and in CEM/VBL100 cells (A) or between cultures incubated with reserpine and controls (B and C).

FIG. 2

Effects of various HIV PIs on calcein accumulation in MDR1⁺ CEM/VBL100 cells. The rates of accumulation of calcein in CEM/VBL100 cells incubated with 5 μM (each) saquinavir (A), ritonavir (B), indinavir (C), or nelfinavir (D) are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence in cultures incubated with HIV PIs and in controls. A statistically significant increase in calcein fluorescence was seen in the presence of ritonavir, nelfinavir, and indinavir, suggesting that they all inhibit MDR1. Significant differences were also observed with saquinavir, albeit at higher (50 μM) concentrations (data not shown).

FIG. 3

Effects of various HIV PIs on calcein accumulation in MDR1⁺ NB1643Dox^r cells. The rates of accumulation of calcein in NB1643Dox^r cells incubated with 5 μM (each) ritonavir, nelfinavir, indinavir, or saquinavir are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.

FIG. 4

Flow cytometry of calcein-AM-labeled CEM/VM-1-5 cells. MRP1⁺ CEM/VM-1-5 cells were incubated with calcein-AM in the absence or presence of 50 μM saquinavir, ritonavir, indinavir, or nelfinavir and analyzed by flow cytometry, as described in Materials and Methods. Means (± standard deviations) of three independent experiments are shown. The P values indicate the statistical differences between mean calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.