Biochemical interaction of anti-HCV telaprevir with the ABC transporters P-glycoprotein and breast cancer resistance protein

Abstract

Background: The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 are involved in the intestinal absorption and renal excretion of various substrate drugs. Their activities affect sub-therapeutic drug concentrations and excretion of natural transporter substrates. The new oral anti-HCV drug telaprevir has dramatically improved the efficacy of hepatitis-C virus (HCV) treatment, and recent studies have suggested a possible pharmacological interaction between telaprevir and P-gp. We studied the kinetics of in vitro interactions between telaprevir and P-gp and BCRP to understand the molecular basis of that interaction.

Findings: The effect of telaprevir on P-gp- and BCRP-mediated transport was evaluated by an in vitro vesicle transporter assay using different transport substrates, and the kinetics of transporter inhibition was determined. The results showed that telaprevir could inhibit P-gp- and BCRP-mediated transport in the in vitro vesicle transport assay, with each IC50 values of ≈ 7 μmol/L and ≈ 30 μmol/L, respectively. Analyses of Lineweaver-Burk plots showed that telaprevir was likely to be a competitive inhibitor against P-gp and BCRP. Photoaffinity labeling experiments were employed to observe competitive inhibition by telaprevir using iodoarylazidoprazosin (IAAP) as a binding substrate for P-gp and BCRP. These experiments revealed that telaprevir inhibited [125I]-IAAP-binding with P-gp and BCRP.

Conclusion: Telaprevir competitively inhibited P-gp and BCRP, and P-gp-mediated transport was more sensitive to telaprevir compared with BCRP-mediated transport. These data suggest that telaprevir represses the transporter functions of P-gp and BCRP via direct inhibition.

Figure 1

Effect of telaprevir on the intravesicular transport of vincristine sulfate (VCR) by P-gp. A. The ability to transport VCR in telaprevir-absent or -present conditions was determined by measuring the radioactivity taken up in membrane vesicles. B. Analyses of Lineweaver–Burk plots of inhibition of VCR uptake by P-gp. The VCR concentration was 100 nmol/L (A) and 50, 100, and 200 nmol/L (B), and of telaprevir was 0, 1, 10, and 100 μmol/L (A) and 20 μmol/L (B), respectively. Membrane vesicles from K562/MDR cells were mixed with each concentration of VCR, telaprevir, and 3 mmol/L of ATP in the incubation medium as described in the Methods section. VCR uptake is shown for parental K562 (white column for ATP-absent; gray column for ATP-present) and K562⁄MDR (dark-gray column for ATP-absent; black column for ATP-present) (A) and the inverse is shown for no inhibitor (open rhombus) and with telaprevir (black circle) (B). Results are means ± SD of triplicate (A) or quadruplicate (B) determinations.

Figure 2

Effect of telaprevir on the intravesicular transport of estrone 3-sulfate (E1S) and MTX by BCRP. A. The ability to transport E1S at telaprevir-absent or -present conditions was determined by measuring the radioactivity of [³H]-E1S taken up in membrane vesicles. A similar experiment was done for MTX. B. Analyses of Lineweaver–Burk plots of inhibition of E1S and MTX uptake by BCRP. E1S concentration was 50 nmol/L (A) and 50, 100, and 200 nmol/L (B), and of telaprevir was 0, 1, 10, and 100 μmol/L (A) and 50 μmol/L (B), respectively. MTX concentration was 100 nmol/L (A), and 100, 200 and 400 nmol/L (B). Membrane vesicles of K562/BCRP cells were used. The procedures were almost identical to those shown in Figure 1.

Figure 3

Effect of telaprevir on photoaffinity labeling of P-gp and BCRP with [¹²⁵I]-IAAP. K562/MDR- or K562/BCRP-vesicles were mixed with the indicated concentrations of telaprevir for 5 min. Vesicles were pre-incubated with 5 nmol/L [¹²⁵I]-IAAP (2200 Ci/mmol) for 10 min, and then illuminated with a UV lamp (365 nm) for 30 min. IAAP-labeled protein was solubilized by Laemmli SDS buffer directly (for the P-gp assay) or after immunoprecipitation (for the BCRP assay), and subjected to SDS-PAGE. Signals were visualized and analyzed with a FLA7000 system. The black arrowheads are P-gp or BCRP. Verapamil and FTC (typical inhibitors of P-gp or BCRP, respectively) were used as controls. The binding of [¹²⁵I]-IAAP with P-gp and BCRP was calculated from standard curve analysis using a FLA7000 Bioimage Analyzer (Fuji Film, Tokyo, Japan) and Multi-Gauge software (Fuji Film).