Inhibition of Human Hepatic Bile Acid Transporters by Tolvaptan and Metabolites: Contributing Factors to Drug-Induced Liver Injury?

Abstract

Tolvaptan is a vasopressin V(2)-receptor antagonist that has shown promise in treating Autosomal Dominant Polycystic Kidney Disease (ADPKD). Tolvaptan was, however, associated with liver injury in some ADPKD patients. Inhibition of bile acid transporters may be contributing factors to drug-induced liver injury. In this study, the ability of tolvaptan and two metabolites, DM-4103 and DM-4107, to inhibit human hepatic transporters (NTCP, BSEP, MRP2, MRP3, and MRP4) and bile acid transport in sandwich-cultured human hepatocytes (SCHH) was explored. IC(50) values were determined for tolvaptan, DM-4103 and DM-4107 inhibition of NTCP (∼41.5, 16.3, and 95.6 μM, respectively), BSEP (31.6, 4.15, and 119 μM, respectively), MRP2 (>50, ∼51.0, and >200 μM, respectively), MRP3 (>50, ∼44.6, and 61.2 μM, respectively), and MRP4 (>50, 4.26, and 37.9 μM, respectively). At the therapeutic dose of tolvaptan (90 mg), DM-4103 exhibited a C(max)/IC(50) value >0.1 for NTCP, BSEP, MRP2, MRP3, and MRP4. Tolvaptan accumulation in SCHH was extensive and not sodium-dependent; intracellular concentrations were ∼500 μM after a 10-min incubation duration with tolvaptan (15 μM). The biliary clearance of taurocholic acid (TCA) decreased by 43% when SCHH were co-incubated with tolvaptan (15 μM) and TCA (2.5 μM). When tolvaptan (15 μM) was co-incubated with 2.5 μM of chenodeoxycholic acid, taurochenodeoxycholic acid, or glycochenodeoxycholic acid in separate studies, the cellular accumulation of these bile acids increased by 1.30-, 1.68-, and 2.16-fold, respectively. Based on these data, inhibition of hepatic bile acid transport may be one of the biological mechanisms underlying tolvaptan-associated liver injury in patients with ADPKD.

Keywords: bile acids; drug-induced liver injury; hepatic transporters.

FIG. 1.

Chemical structures of tolvaptan, DM-4103, and DM-4107.

FIG. 2.

Effect of tolvaptan, DM-4103, and DM-4107 on NTCP-mediated transport or BSEP-mediated transport of TCA. NTCP-overexpressing cells were co-incubated with tolvaptan (0–50 μM), DM-4103 (0–75 μM), or DM4107 (0–200 μM) and [³H]TCA (2 µCi/ml; 1 µM) for 5 min at 37°C (A-C); BSEP vesicles were co-incubated with tolvaptan (0–50 μM), DM-4103 (0–75 μM), or DM4107 (0–200 μM) and [³H]TCA (2 µCi/ml; 2 µM) for 2 min at 37°C (D-F). NTCP-mediated uptake of TCA was calculated by subtracting substrate uptake in the absence of sodium from substrate uptake in the presence of sodium. BSEP-mediated transport was calculated by subtracting TCA uptake in the presence of AMP from TCA uptake in the presence of ATP. Values are expressed as percentage of vehicle control; each value represents the mean ± SD from 3 independent experiments. The curve represents the best fit line using nonlinear regression techniques. See “Materials and Methods” section for further details.

FIG. 3.

Effect of tolvaptan and DM-4103 on BSEP-mediated transport of TCA. Uptake of TCA (2, 5, 10, 20, or 30 µM) was determined in the presence of 0 µM (•), 10 µM (○), 25 µM (▴), or 50 µM (Δ) tolvaptan (A) or 0 µM (•), 2 µM(○), 5 µM (▴), or 20 µM (Δ) of DM-4103 (B). TCA uptake was measured after an incubation of 2 min at 37°C in the presence or absence of inhibitor. BSEP-mediated transport was calculated by subtracting TCA uptake in the presence of AMP from TCA uptake in the presence of ATP; each value represents the mean ± SD from 3 independent experiments. The curves represent the best fit model using nonlinear regression techniques. See “Materials and Methods” section for further details.

FIG. 4.

Effect of tolvaptan, DM-4103, and DM-4107 on MRP2-mediated transport of E₂17G, MRP3-mediated transport of E₂17G, and MRP4-mediated transport of DHEAS. MRP2 (A–C), MRP3 (D–F), or MRP4 (G–I) vesicles were co-incubated with tolvaptan (0–50 μM), DM-4103 (0–50 μM for MRP2 and MPR3, and 0–20 μM for MRP4), or DM-4107 (0–200 μM) and [³H]E₂17G (2 µCi/ml; 50 µM) (A–C) for 2 min, [³H]E₂17G (2 µCi/ml; 10 µM) (D–F) for 5 min, or [³H]DHEAS (2 µCi/ml; 2 µM) (G–I) for 2 min at 37°C. MRP-mediated transport was calculated by subtracting substrate uptake in the presence of AMP from substrate uptake in the presence of ATP. Values are expressed as percentage of vehicle control; each value represents the mean ± SD from 3 independent experiments. If the inhibitor did not reduce substrate transport by >50% at the highest tested concentration, nonlinear regression to estimate an IC₅₀ value was not conducted. The curve represents the best fit line using nonlinear regression techniques. See “Materials and Methods” section for further details.

FIG. 5.

Time- and temperature-dependent accumulation of tolvaptan in SCHH. Accumulation of tolvaptan in cells + bile (black bars) and cells (white bars) was measured by incubating SCHH with 15 µM of tolvaptan for 5, 10, or 20 min at 37°C, or for 10 min at 4°C (A). Accumulation of tolvaptan, and the generated metabolites DM-4103 and DM-4107 in cells + bile (black bars) and cells (white bars) was measured by incubating SCHH with 15 µM of tolvaptan for 10 min at 37°C (B). *Cellular concentrations were calculated using the total mass of tolvaptan, DM-4103, or DM-4107 in cells (white bars) and hepatocyte fluid volume as described in “Materials and Methods” section; each value represents the mean ± SD from n = 1 liver in triplicate.

FIG. 6.

Accumulation of TCA, CDCA, TCDCA, and GCDCA in the presence and absence of tolvaptan in SCHH. Accumulation of d₈-TCA (2.5 µM) and d₅-CDCA (2.5 µM) (A), or TCDCA (2.5 µM) and GCDCA (2.5 µM) (B) in cells + bile (black bars) and cells (white bars) was measured after incubating SCHH with designated bile acids in the presence or absence of tolvaptan (15 µM) for 10 min at 37°C; each value represents the mean ± SD from n = 1 liver in triplicate.