Downregulation of Organic Anion Transporting Polypeptide (OATP) 1B1 Transport Function by Lysosomotropic Drug Chloroquine: Implication in OATP-Mediated Drug-Drug Interactions

Abstract

Organic anion transporting polypeptide (OATP) 1B1 mediates the hepatic uptake of many drugs including lipid-lowering statins. Decreased OATP1B1 transport activity is often associated with increased systemic exposure of statins and statin-induced myopathy. Antimalarial drug chloroquine (CQ) is also used for long-term treatment of rheumatoid arthritis and systemic lupus erythematosus. CQ is lysosomotropic and inhibits protein degradation in lysosomes. The current studies were designed to determine the effects of CQ on OATP1B1 protein degradation, OATP1B1-mediated transport in OATP1B1-overexpressing cell line, and statin uptake in human sandwich-cultured hepatocytes (SCH). Treatment with lysosome inhibitor CQ increased OATP1B1 total protein levels in HEK293-OATP1B1 cells and in human SCH as determined by OATP1B1 immunoblot. In HEK293-FLAG-tagged OATP1B1 stable cell line, co-immunofluorescence staining indicated that intracellular FLAG-OATP1B1 is colocalized with lysosomal associated membrane glycoprotein (LAMP)-2, a marker protein of late endosome/lysosome. Enlarged LAMP-2-positive vacuoles with FLAG-OATP1B1 protein retained inside were readily detected in CQ-treated cells, consistent with blocking lysosomal degradation of OATP1B1 by CQ. In HEK293-OATP1B1 cells, without pre-incubation, CQ concentrations up to 100 μM did not affect OATP1B1-mediated [(3)H]E217G accumulation. However, pre-incubation with CQ at clinically relevant concentration(s) significantly decreased [(3)H]E217G and [(3)H]pitavastatin accumulation in HEK293-OATP1B1 cells and [(3)H]pitavastatin accumulation in human SCH. CQ pretreatment (25 μM, 2 h) resulted in ∼1.9-fold decrease in Vmax without affecting Km of OATP1B1-mediated [(3)H]E217G transport in HEK293-OATP1B1 cells. Pretreatment with monensin and bafilomycin A1, which also have lysosome inhibition activity, significantly decreased OATP1B1-mediated transport in HEK293-OATP1B1 cells. Pharmacoepidemiologic studies using data from the U.S. Food and Drug Administration Adverse Event Reporting System indicated that CQ plus pitavastatin, rosuvastatin, and pravastatin, which are minimally metabolized by the cytochrome P450 enzymes, led to higher myopathy risk than these statins alone. In summary, the present studies report novel findings that lysosome is involved in degradation of OATP1B1 protein and that pre-incubation with lysosomotropic drug CQ downregulates OATP1B1 transport activity. Our in vitro data in combination with pharmacoepidemiologic studies support that CQ has potential to cause OATP-mediated drug-drug interactions.

Keywords: FDA Adverse Event Reporting System (FAERS); chloroquine (CQ); drug−drug interactions (DDIs); human sandwich-cultured hepatocytes (SCH); lysosomotropic drug; organic anion transporting polypeptide (OATP).

Figure 1

Characterization of OATP1B1 antibody and optimization of OATP1B1 immunoblot protocol. HEK293-OATP1B1 and -Mock cells were seeded at 1.1 × 10⁵ cells/well in 24-well plates and cultured for 48 h prior to being harvested for immunoblot. Whole cell lysates (WCL) of HEK293-OATP1B1 cells were prepared either by adding ice-cold lysis buffer directly onto the culture plate after aspirating culture medium without trypsinization or by lysing the cells after trypsinization and subsequent washing. Immunoblot of OATP1B1 was performed with the custom-generated OATP1B1 antibody. GAPDH was used as the loading control. Representative images from n = 3 experiments are shown.

Figure 2

Effects of CQ on total protein levels of OATP1B1 and Na,K-ATPase in HEK293-OATP1B1 cells and on OATP1B1 total protein levels in human SCH. HEK293-OATP1B1 cells were seeded in 24-well plates at 1.1 × 10⁵ cells/well, and were cultured for 48 h prior to treatment. Human SCH were cultured as described in the Experimental Section. WCL was prepared by directly lysing the cells in the culture plate without trypsinization. Representative immunoblot images of OATP1B1 (A) and Na,K-ATPase (B) in WCL of HEK293-OATP1B1 pretreated with CQ (25 or 100 μM) or CTL for indicated times are shown. GAPDH were used as the loading control for A and B. OATP1B1 and Na,K-ATPase protein levels determined by densitometry were normalized to levels of GAPDH. Fold changes of total protein levels of OATP1B1 in A and Na,K-ATPase in B (CQ vs CTL) are expressed as mean ± SD (n = 4 for both A and B). (C) Immunoblot of OATP1B1 and β-actin in WCL of human SCH pretreated with CQ (10 μM) or CTL for 5 h. OATP1B1 protein levels determined by densitometry were normalized to levels of β-actin. Fold changes of total protein levels of OATP1B1 (CQ vs CTL) are expressed as mean ± SD of n = 3 donor hepatocytes. Representative images are shown.

Figure 3

Colocalization of FLAG-OATP1B1 and LAMP-2 in HEK293-FLAG-OATP1B1 cells. Coimmunofluorescence staining of FLAG-OATP1B1 (red) and LAMP-2 (green) was performed in HEK293-FLAG-OATP1B1 cells treated with vehicle CTL or 25 μM CQ for 2 and 5 h as described in the Experimental Section. White arrow heads indicate the accumulation of FLAG-OATP1B1 inside the LAMP-2-positive vacuoles. Nuclei were counterstained with DAPI (blue). Images were taken using Olympus FV1000 confocal microscope. Representative images from the same experiments are shown (3 and 2 separate experiments for 2 h and 5 h treatment, respectively).

Figure 4

Pretreatment effect and direct inhibition of CQ on OATP1B1-mediated transport. Cells were seeded at 1.1 × 10⁵ cells/well in 24-well plates and cultured for 48 h (A, B, C, and E) or 72 h. (D) Accumulation of [³H]E₂17G (1 μM, 2 min) or [³H]pitavastatin accumulation (1 μM, 0.5 min) was determined in HEK293-OATP1B1 or -FLAG-OATP1B1 cells, as indicated in the figures. (A) Model-estimated fold change and associated SE in [³H]E₂17G accumulation vs CTL treatment in HEK293-OATP1B1 cells at each indicated pretreatment concentration and time (pre-incubation). (B) Model-estimated fold change and associated SE in [³H]pitavastatin accumulation vs CTL treatment in HEK293-OATP1B1 cells at each indicated pretreatment concentration and time (pre-incubation). (C) Model-estimated fold change and associated SE in [³H]E₂17G accumulation vs CTL treatment in HEK293-FLAG-OATP1B1 cells at each indicated pretreatment concentration and time (pre-incubation). (D) Model-estimated fold change and associated SE in [³H]E₂17G accumulation in the presence of 5–100 μM CQ or 25 μM rifampicin (Rif) vs CTL in HEK293-OATP1B1 cells without CQ pretreatment (co-incubation). (E) Model-estimated fold change and associated SE in [³H]E₂17G accumulation vs co-incubation control. Following pretreatment in culture medium containing 100 μM CQ (pre+co-incubation) or vehicle control (co-incubation) for 5 h, HEK293-OATP1B1 cells were rinsed three times with HBSS, and the [³H]E₂17G accumulation was determined in the presence of 100 μM CQ. A generalized linear mixed model as described in the Experimental Section was fit to data in A–E (n = 3 in triplicate for all panels of A–E). To account for multiple comparisons, p-values were adjusted based on Bonferroni's method. * indicates a statistically significant difference (adjusted p < 0.05) vs CTL.

Figure 5

Effects of monensin and bafilomycin A1 on OATP1B1-mediated [³H]E₂17G transport. HEK293-OATP1B1 were seeded at 1.1 × 10⁵ cells/well in 24-well plates for up to 72 h. (A) Model-estimated fold change and associated SE in [³H]E₂17G accumulation vs CTL at each indicated monensin (5 μM) pretreatment time. (B) Model-estimated fold change and associated SE in [³H]E₂17G accumulation vs CTL at each indicated bafilomycin A1 (0.5 μM) pretreatment time. A generalized linear mixed model as described in the Experimental Section was fit to data (n = 3 in triplicate for both A and B). * indicates a statistically significant difference (p < 0.05) vs CTL.

Figure 6

Effects of CQ on OATP1B1-mediated [³H]E₂17G transport after prolonged treatment in HEK293-OATP1B1 cells. HEK293-OATP1B1 cells were seeded at 0.8 × 10⁵ cells/well in 24-well plates, and cultured for 48 h prior to treatment. Cells were preincubated with CQ-free (CTL) or 25 μM CQ-containing medium for 2 h. At the end of pre-incubation, the culture medium was removed. CTL medium preincubated cells were cultured in CQ-free medium (CTL, black bar) and 25 μM CQ-preincubated cells were cultured in medium containing 1.5 μM CQ (grey bar), for indicated times up to 24 h. [³H]E₂17G accumulation (1 μM, 2 min) was determined at indicated time points (n = 4 in triplicate). Fold change and SE in [³H]E₂17G accumulation vs CTL at each indicated time points were estimated by generalized linear mixed models, as described in the Experimental Section. * indicates a statistically significant difference (p < 0.05) vs CTL.

Figure 7

Effect of CQ pretreatment on the accumulation of [³H]pitavastatin in human SCH. (A) Time-dependent accumulation of [³H]pitavastatin (1 μM) in human SCH. Data represent the mean ± SD in triplicate from a single hepatocyte donor. (B) Model-estimated fold change and associated SE in [³H]pitavastatin accumulation vs CTL in human SCH pretreated with 10 μM CQ or CTL for 5 h. Following pretreatment in culture medium containing CQ (10 μM) or control, human SCH were rinsed three times with HBSS, and [³H]pitavastatin accumulation (1 μM, 0.5 min) was determined. Fold change and SE were estimated by a generalized linear mixed model, as described in the Experimental Section (n = 3 in triplicate). * indicates a statistically significant difference (p < 0.05) vs CTL.

Figure 8

Effects of CQ on kinetic parameters of OATP1B1-mediated [³H]E₂17G transport. HEK293-OATP1B1 and -Mock cells were seeded at 1.1 × 10⁵ cells/well in 24-well plates and were cultured for 48 h prior to experiment. The concentration-dependent accumulation of [³H]E₂17G (0.1–40 μM, 2 min) was determined in HEK293-OATP1B1 and -Mock cells pretreated with CTL or CQ (25 μM, 2 h). Values of [³H]E₂17G accumulation in Mock cells were subtracted from those in HEK293-OATP1B1 cells. V_max and K_m values were determined as described in the Experimental Section. Solid and dashed lines represent the best fits of the Michaelis–Menten equation to the data of CTL (black circles) and CQ (25 μM, 2 h) pretreatment (white circles), respectively. Representative graph of three independent experiments in triplicate is shown. Student's t test was conducted to compare the V_max and K_m values between CQ and CTL pretreatment. * indicates a statistically significant difference (p < 0.05; CQvs CTL).