Novel in Vitro Method Reveals Drugs That Inhibit Organic Solute Transporter Alpha/Beta (OSTα/β)

Abstract

Drug interactions with the organic solute transporter alpha/beta (OSTα/β) are understudied even though OSTα/β is an important transporter that is expressed in multiple human tissues including the intestine, kidneys, and liver. In this study, an in vitro method to identify novel OSTα/β inhibitors was first developed using OSTα/β-overexpressing Flp-In 293 cells. Incubation conditions were optimized using previously reported OSTα/β inhibitors. A method including a 10 min preincubation step with the test compound was used to screen for OSTα/β inhibition by 77 structurally diverse compounds and fixed-dose combinations. Seven compounds and one fixed-dose combination (100 μM final concentration) inhibited OSTα/β-mediated dehydroepiandrosterone sulfate (DHEAS) uptake by >25%. Concentration-dependent OSTα/β inhibition was evaluated for all putative inhibitors (atorvastatin, ethinylestradiol, fidaxomicin, glycochenodeoxycholate, norgestimate, troglitazone, and troglitazone sulfate). Ethinylestradiol, fidaxomicin, and troglitazone sulfate yielded a clear concentration-inhibition response with IC₅₀ values <200 μM. Among all tested compounds, there was no clear association between physicochemical properties, the severity of hepatotoxicity, and the degree of OSTα/β inhibition. This study utilized a novel in vitro method to identify OSTα/β inhibitors and, for the first time, provided IC₅₀ values for OSTα/β inhibition. These data provide evidence that several drugs, some of which are associated with cholestatic drug-induced liver injury, may impair the function of the OSTα/β transporter.

Keywords: SLC51A/B; basolateral efflux; bile acid; cholestasis; drug-induced liver injury; inhibition.

Fig. 1.

Chemical diversity of the compounds tested for OSTα/β inhibition. The chemical space of test compounds (blue) and FDA-approved drugs (grey) was described using a principal component analysis (PCA) of ten selected molecular descriptors. The first two principal components (PCs), PC1 and PC2, which together account for 69% of the variability in the dataset, are plotted in this graph.

Fig. 2.

Time dependence of [³H]-dehydroepiandrosterone sulfate (DHEAS) transport in OSTα/β-overexpressing cells (OSTab). OSTab and Mock cells were incubated with DHEAS (300 nCi/ml; 4 µM final concentration) in extracellular fluid (pH 7.4) at 37°C for designated times. Background levels derived from Mock cells were subtracted, and uptake values were normalized to total cell protein. Each value represents the mean ± SD from two independent experiments, performed in triplicate. The inset shows DHEAS uptake during the early time points (5 s, 10 s and 0.5 min).

Fig. 3.

The effect of preincubation on the inhibition of OSTα/β-mediated transport of probe substrates. OSTab and Mock cells were preincubated with inhibitor (100 µM) for 10 min (Method 1, black), co-incubated with inhibitor and substrate during the uptake phase (Method 2, grey), or both preincubated and co-incubated, as described for Methods 1 and 2 (Method 3, white); the probe substrate, [³H]-dehydroepiandrosterone sulfate (DHEAS) or [³H]-taurocholate (TCA) (300 nCi/ml; 20 µM final concentration; 30 s uptake), was added in extracellular fluid (pH 7.4) at 37°C. Background levels derived from Mock cells were subtracted, and uptake measurements were normalized to total cell protein and uptake in vehicle treated cells. Each value represents the mean ± SEM from three independent experiments. ***, p< 0.001; **, p<0.005; *, p<0.05, significantly different than substrate uptake in control group. TLCAS, taurolithocholic acid sulfate.

Fig. 4.

The inhibitory effect of test compounds or fixed-dose combinations on OSTα/β-mediated dehydroepiandrosterone sulfate (DHEAS) uptake in OSTα/β-overexpressing (OSTab) cells. A) Compounds or fixed-dose combinations inhibiting DHEAS transport by >50% were denoted as strong inhibitors (n = 3), and those that inhibited between 25% and 50% of the DHEAS transport were designated as moderate inhibitors (n = 5). B) The compounds, illustrated in groups, were previously reported OSTα/β substrates or inhibitors (black), bile acids elevated in cholestasis (dark grey), classical hepatotoxic compounds (light grey), and compounds associated with cholestatic DILI from the Drug-Induced Liver Injury Network (DILIN) database (only compounds inhibiting DHEAS transport by >20% (white) are shown). The inhibition was studied using Method 1. OSTab and Mock cells were preincubated with putative inhibitor at a concentration of 100 μM for 10 min; the probe substrate, [³H]-DHEAS, was added in extracellular fluid (300 nCi/ml; 4 µM final concentration; pH 7.4) and 30-s uptake was measured at 37°C; inhibition was calculated as described in Materials and Methods. Each value represents the mean ± SEM from three independent experiments. **p< 0.0001; *p<0.0005; ^§p<0.05; ^#p<0.01 significantly different than substrate uptake in control group.

Fig. 5.

Concentration-dependent inhibitory effect of ethinylestradiol, fidaxomicin, troglitazone sulfate, norgestimate, atorvastatin, glycochenodeoxycholate and troglitazone on OSTα/β-mediated DHEAS transport. OSTab and Mock cells were preincubated with inhibitors for 10 min (Method 1; 0–200 μM) prior to the 30-s uptake with [³H]-dehydroepiandrosterone sulfate (DHEAS; 200 nCi/ml; 4 μM) at 37°C. Data are expressed as percentage of vehicle control; each value represents the mean ± SD of two independent experiments, each performed in triplicate. For troglitazone, only one experiment is shown (mean ± range).