Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters

Abstract

Purpose: To compare side-by-side the uptake of sorafenib and sunitinib in vitro by human uptake solute carriers of the SLC22A and SLCO families, the transport by and inhibition of efflux ATP-binding cassette (ABC) transporters, and the role of ABCB1 in the plasma pharmacokinetics and brain penetration of these agents.

Experimental design: Uptake of [(3)H]sorafenib or [(3)H]sunitinib was assessed in Xenopus laevis oocytes or mammalian cells transfected with cDNAs coding for human OATP1A2, OATP1B1, OATP1B3, OCT1, OAT2, OAT3, OCTN1, or OCTN2. Efflux and inhibition experiments were conducted in cells transfected with human ABCB1, ABCG2, ABCC2, or ABCC4. In vivo pharmacokinetic studies were done in knockout mice lacking Abcb1-type transporters.

Results: Intracellular uptake was not appreciably affected by any of the studied solute carriers and was minute relative to the respective prototypical substrates. Sorafenib and sunitinib showed concentration-dependent (1 and 10 micromol/L), low to moderate affinity for ABCB1 but were not affected by the other ABC transporters. Both agents inhibited all tested ABC transporters. The absence of Abcb1 had no affect on plasma pharmacokinetics, but brain penetration was moderately increased by 1.9- and 2.9-fold for sorafenib and sunitinib, respectively, in knockout animals versus controls.

Conclusions: Unlike other tyrosine kinase inhibitors, sorafenib and sunitinib do not appear to rely on active transport to enter the cell nor are they high-affinity substrates for ABC efflux transporters. Based on these characteristics, these two drugs may be less susceptible to transporter-mediated alterations in systemic exposure and transporter-related resistance mechanisms.

Fig. 1

Uptake of sorafenib and sunitinib by solute carriers in vitro. Accumulation of sorafenib (A) and sunitinib (B) by Xenopus laevis oocytes expressing OATP1A2, OATP1B1, OATP1B3 and OCT1 or HEK293 cells expressing OAT1, OAT3, OCTN1 and OCTN2. Oocytes or HEK293 cells were incubated with sorafenib 0.35 -1.5 μM or sunitinib 0.15 - 0.45 μM for 1 h. Data represent the mean and standard deviations of 9-27 observations and are expressed as percent of water injected control; a single control bar is shown for all experiments combined. Prototypical substrates for each transporter were evaluated with each experiment as a positive control (black bar).

Fig. 2

Transport of sorafenib and sunitinib by ABC transporters in vitro. (A) Transcellular transport of sorafenib and sunitinib in LLC-PK1 cells expressing ABCB1. Cells were incubated with 1μM drug for 1, 2, 3 and 4 h. Data represent 6 observations and are expressed as ABCB1-mediated Papp (B to A) / Papp (A to B) ratio. * p<0.05 versus ABCB1 (A to B) (B) Accumulation of sorafenib and sunitinib in Saos-2 cells overexpressing ABCG2 or ABCC4 or MDCKII cells overexpressing ABCC2. Cells were incubated with 1 μM drug for 4 h. Data represent the mean and standard deviations of 6-9 observations from 2-3 independent experiments and are expressed as percent control; a single control bar is shown for all experiments combined. Prototypical substrates for each transporter were evaluated with each experiment as a positive control (black bar). * p<0.05 versus control.

Fig. 3

Inhibition of ABCB1 and ABCG2 function by sorafenib and sunitinib in vitro. Sorafenib (A) and sunitinib (B) decreased the efflux of hoechst 33342 in Saos-2 cells expressing human ABCG2; and sorafenib (C) and sunitinib (D) decreased the efflux of calcein in LLC-PK1 cells expressing ABCB1. Cells were incubated with increasing drug concentrations for 1 h. Flow cytometry was used to assess of hoechst 33342 and calcein cellular efflux. Data are the mean and standard deviation of two observations; representative figures of 2-3 independent experiments are shown. The lines represent the fit of a maximum effect model to the data.

Fig. 4

Inhibition of ABCC2 and ABCC4 function by sorafenib and sunitinib in vitro. Sorafenib inhibited the efflux of docetaxel in MDCKII cells overexpressing ABCC2 (A) and PMEA in Saos-2 cells overexpressing ABCC4 (B). Cells were incubated with 20 μM sorafenib or sunitinib or 50 μM MK571 for 15 min, followed by coincubation with 5 μM docetaxel or 1 μM PMEA for 4 h. Data represent the mean and standard deviation of 6-9 observations from 2-3 independent experiments and are expressed as % change of cellular accumulation of the prototypical substrates docetaxel or PMEA.

Fig. 5

Effect of Sorafenib on ATPase hydrolysis by ABCC2. Sorafenib inhibits vanadate sensitive baseline and maximal ATPase activity of human ABCC2 expressed in membrane vesicles. Increasing concentrations of sorafenib were incubated with vesicles for 10 minutes. Data are a representative figure of 2-3 experiments performed in duplicate.

Fig. 6

Role of ABCB1 in the plasma pharmacokinetics and brain penetration of sorafenib and sunitinib in mice. Plasma concentration-time curves for sorafenib (A) and sunitinib (C) in wild-type and Abcb1 knockout mice. Brain penetration of sorafenib (B) and sunitinib (C) in wild-type and Abcb1 knockout mice. Sorafenib 40mg/kg or sunitinib 20mg/kg was administered to mice, plasma samples were obtained at 1, 2 and 4 h after treatment, and whole brain tissue homogenate were collected at 4h. Brain penetration was determined as the brain concentration at 4 h divided by the plasma AUC0-4h. Data are the mean and standard deviations from 2-3 independent experiments (6-9 observations per time point).