Effect of ABCB1 genetic polymorphisms on the transport of rivaroxaban in HEK293 recombinant cell lines

Abstract

Direct oral anticoagulants (DOAC) are substrates for the ABCB1 transporter (also called P-glycoprotein), an active efflux pump. ABCB1 polymorphisms have been previously reported to influence the pharmacokinetics of several drugs such as immunosuppressants and tyrosine kinase inhibitors. Recently, in vivo studies have suggested that genetic variants might contribute to the inter-individual variability in DOAC plasma concentrations. Therefore, we evaluated the in vitro effect of the most common coding ABCB1 single nucleotide polymorphisms (SNP), 1236 C > T-2677G > T-3435C > T, and the coding ABCB1 1199 G > A SNP on the transport activity towards rivaroxaban. HEK293 cells were transfected to overexpress the ABCB1 wild-type (1236C-2677G-3435C, 1199 G) or variant proteins (1236C-2677G-3435T, 1236T-2677T-3435T or 1199 A). ABCB1 expression decreased the intracellular accumulation of rivaroxaban, when compared to control cells. This confirms the involvement of ABCB1 in the active transport of rivaroxaban. However, the ABCB1 1236 C > T-2677G > T-3435C > T and 1199 G > A SNPs had no significant influence on the intracellular accumulation of rivaroxaban when compared to the wild-type protein. These results suggest that the ABCB1 coding SNPs investigated in the present study are unlikely to contribute to the inter-individual variability in rivaroxaban plasma concentrations.

Figure 1

ABCB1 cell surface expression. Flow cytometry histograms of HEK293 cells transfected with (A) the empty pcDNA3.1 vector, ABCB1_CGC, ABCB1_CGT or ABCB1_TTT and (B) the empty pcDNA3.1 vector, ABCB1_1199G, ABCB1_1199A. Cells were incubated with a FITC anti-ABCB1 antibody (blue) or a matched isotypic control (red).

Figure 2

Impact of ABCB1 1236 C > T-2677G > T-3435C > T on the intracellular accumulation of rivaroxaban. Intracellular accumulation of rivaroxaban after 120 min of incubation (N = 3) at different concentrations in HEK_control, HEK_CGC, HEK_CGT and HEK_TTT. The absolute amount of rivaroxaban (in ng) was divided by the total amount of proteins in cell extracts (in mg). *Compared to control cells: *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Impact of ABCB1 1199 G > A on the intracellular accumulation of rivaroxaban. Intracellular accumulation of rivaroxaban after 120 min of incubation (N = 3) at different concentrations in HEK_control, HEK_1199G and HEK_1199A. The absolute amount of rivaroxaban (in ng) was divided by the total amount of proteins in cell extracts (in mg). *Compared to control cells: *p < 0.05, **p < 0.01, ***p < 0.001.