Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

Interaction of 2,4-Diaminopyrimidine-Containing Drugs Including Fedratinib and Trimethoprim with Thiamine Transporters.

Inhibition of thiamine transporters has been proposed as a putative mechanism for the observation of Wernicke's encephalopathy and subsequent termination of clinical development of fedratinib, a Janus kinase inhibitor (JAKi). This study aimed to determine the potential for other JAKi to inhibit thiamine transport using human epithelial colorectal adenocarcinoma (Caco-2) and thiamine transporter (THTR) overexpressing cells and to better elucidate the structural basis for interacting with THTR. Only JAKi containing a 2,4-diaminopyrimidine were observed to inhibit thiamine transporters. Fedratinib inhibited thiamine uptake into Caco-2 cells (IC50 = 0.940 µM) and THTR-2 (IC50 = 1.36 µM) and, to a lesser extent, THTR-1 (IC50 = 7.10 µM) overexpressing cells. Two other JAKi containing this moiety, AZD1480 and cerdulatinib, were weaker inhibitors of the thiamine transporters. Other JAKi-including monoaminopyrimidines, such as momelotinib, and nonaminopyrimidines, such as filgotinib-did not have any inhibitory effects on thiamine transport. A pharmacophore model derived from the minimized structure of thiamine suggests that 2,4-diaminopyrimidine-containing compounds can adopt a conformation matching several key features of thiamine. Further studies with drugs containing a 2,4-diaminopyrimidine resulted in the discovery that the antibiotic trimethoprim also potently inhibits thiamine uptake mediated by THTR-1 (IC50 = 6.84 µM) and THTR-2 (IC50 = 5.56 µM). Fedratinib and trimethoprim were also found to be substrates for THTR, a finding with important implications for their disposition in the body. In summary, our results show that not all JAKi have the potential to inhibit thiamine transport and further establish the interaction of these transporters with xenobiotics.

Implications of efficient hepatic delivery by tenofovir alafenamide (GS-7340) for hepatitis B virus therapy.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR-2 (SLC19A3).

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH's, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (Km = 1.15 ± 0.2 mM) and other cationic compounds (MPP(+) and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug-drug and drug-nutrient interactions.

Contribution of the organic anion transporter OAT2 to the renal active tubular secretion of creatinine and mechanism for serum creatinine elevations caused by cobicistat.

Many xenobiotics including the pharmacoenhancer cobicistat increase serum creatinine by inhibiting its renal active tubular secretion without affecting the glomerular filtration rate. This study aimed to define the transporters involved in creatinine secretion, applying that knowledge to establish the mechanism for xenobiotic-induced effects. The basolateral uptake transporters organic anion transporter OAT2 and organic cation transporters OCT2 and OCT3 were found to transport creatinine. At physiologic creatinine concentrations, the specific activity of OAT2 transport was over twofold higher than OCT2 or OCT3, establishing OAT2 as a likely relevant creatinine transporter and further challenging the traditional view that creatinine is solely transported by a cationic pathway. The apical multidrug and toxin extrusion transporters MATE1 and MATE2-K demonstrated low-affinity and high-capacity transport. All drugs known to affect creatinine inhibited OCT2 and MATE1. Similar to cimetidine and ritonavir, cobicistat had the greatest effect on MATE1 with a 50% inhibition constant of 0.99 µM for creatinine transport. Trimethoprim potently inhibited MATE2-K, whereas dolutegravir preferentially inhibited OCT2. Cimetidine was unique, inhibiting all transporters that interact with creatinine. Thus, the clinical observation of elevated serum creatinine in patients taking cobicistat is likely a result of OCT2 transport, facilitating intracellular accumulation, and MATE1 inhibition.

Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620.

The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with l-alanine-isopropyl ester and phenol moieties attached to the 5'-phosphate that release the nucleoside monophosphate in hepatocytes and a 3'-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3'-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.

Evaluation of the effect of cobicistat on the in vitro renal transport and cytotoxicity potential of tenofovir.

A once-daily single-tablet antiretroviral regimen containing tenofovir (TFV) disoproxil fumarate, emtricitabine (FTC), elvitegravir (EVG), and cobicistat (COBI) is an approved combination for the treatment of patients infected with HIV. COBI and TFV have been reported to interact with distinct transporters in renal proximal tubules; while TFV is renally eliminated by a combination of glomerular filtration and tubular secretion via anion transporters OAT1, OAT3, and MRP4, COBI inhibits renal cation transporters, particularly MATE1, resulting in a measurable decrease in the tubular secretion of creatinine. To investigate the potential for a renal drug-drug interaction between TFV and COBI in vitro, the uptake of TFV in the presence and absence of COBI was determined in fresh human renal cortex tissue and in cells expressing the relevant renal transporters. At concentrations exceeding clinical protein-unbound plasma levels, COBI did not significantly inhibit the transport of TFV by the anion transporters OAT1, OAT3, and MRP4 (50% inhibitory concentrations [IC50s] of >15, 6.6, and 8.5 µM, respectively). Conversely, TFV had little or no effect on the cation transporters OCT2 and MATE1 (IC50 > 100 µM). Consistent with studies using individual transporters, no increase in the accumulation of TFV in freshly isolated human renal cortex tissue or renal proximal tubule cells (RPTECs) was observed in the presence of COBI. Finally, COBI alone or in combination with FTC and EVG did not affect the sensitivity to TFV of cultured primary RPTECs or cells coexpressing OAT1 and MRP4. These results illustrate that COBI and TFV interact primarily with distinct renal transporters and indicate a low potential for pharmacokinetic renal drug-drug interaction.

Transport of A1 adenosine receptor agonist tecadenoson by human and mouse nucleoside transporters: evidence for blood-brain barrier transport by murine equilibrative nucleoside transporter 1 mENT1.

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.

Cobicistat boosts the intestinal absorption of transport substrates, including HIV protease inhibitors and GS-7340, in vitro.

The experimental pharmacoenhancer cobicistat (COBI), a potent mechanism-based inhibitor of cytochrome P450 3A enzymes, was found to inhibit the intestinal efflux transporters P-glycoprotein and breast cancer resistance protein. Consistent with its transporter inhibition, COBI significantly increased the absorptive flux of potential candidates for clinical coadministration, including the HIV protease inhibitors atazanavir and darunavir and the lymphoid cell- and tissue-targeted prodrug of the nucleotide analog tenofovir, GS-7340, through monolayers of Caco-2 cells in vitro.

Pharmacokinetic/pharmacodynamic modeling of methylprednisolone effects on iNOS mRNA expression and nitric oxide during LPS-induced inflammation in rats.

PURPOSE: Increased expression of inducible nitric oxide synthase (iNOS) resulting in nitric oxide elevation represents an important component of inflammatory responses. We assess the effects of methylprednisolone (MPL) on these processes during endotoxin-induced acute inflammation and provide a mechanism-based model to quantitatively describe them. METHODS: Male Lewis rats were dosed with lipopolysaccharide (50 µg/kg LPS) alone or with methylprednisolone (10 and 50 mg/kg) and sacrificed at different time points. Plasma MPL, lung iNOS mRNA expression, plasma nitric oxide (NO) and other physiological factors were measured. Sodium nitrate (750 µmole/kg) was given to a separate cohort of rats to assess NO disposition kinetics. PK-PD modeling was performed with ADAPT 5. RESULTS: Disposition kinetics of plasma MPL and NO showed bi-exponential decline and were described by two-compartment models. LPS increased expression of iNOS mRNA in lung and increased plasma NO, while MPL dosing palliated this increase in a dose-dependent manner. These effects were well captured using tandem indirect response and precursor-pool models. CONCLUSION: The model provides a quantitative assessment of the suppression of NO production by MPL and shows that the major effects are at the transcriptional level by reducing expression of iNOS mRNA.

Discovery of an Atropisomeric PI3Kß Selective Inhibitor through Optimization of the Hinge Binding Motif.

A series of PI3Kß selective inhibitors derived from a novel 4-(1H-benzo[d]imidazol-1-yl)quinoline chemotype has been rationally designed. Crucial to achieving the desired selectivity over the other class I PI3K isoforms, including the challenging δ-isoform, was the identification of a subset of substituted pyridine hinge binders. This work led to the discovery of (P)-14, a highly selective and orally bioavailable PI3Kß inhibitor displaying an excellent pharmacokinetic profile in addition to great cellular potency in various PTEN-deficient tumor cell lines. Results from a dog toxicology study revealing structure-related, off-target ocular toxicity are also briefly discussed.

Atropisomerism by Design: Discovery of a Selective and Stable Phosphoinositide 3-Kinase (PI3K) ß Inhibitor.

Atropisomerism is a type of axial chirality in which enantiomers or diastereoisomers arise due to hindered rotation around a bond axis. In this manuscript, we report a case in which torsional scan studies guided the thoughtful creation of a restricted axis of rotation between two heteroaromatic systems of a phosphoinositide 3-kinase (PI3K) ß inhibitor, generating a pair of atropisomeric compounds with significantly different pharmacological and pharmacokinetic profiles. Emblematic of these differences, the metabolism of inactive ( M)-28 is primarily due to the cytosolic enzyme aldehyde oxidase, while active ( P)-28 has lower affinity for aldehyde oxidase, resulting in substantially better metabolic stability. Additionally, we report torsional scan and experimental studies used to determine the barriers of rotation of this novel PI3Kß inhibitor.

Beyond drug-drug interactions: effects of transporter inhibition on endobiotics, nutrients and toxins.

INTRODUCTION: Membrane transport proteins play a central role in regulating the disposition of endobiotics, dietary nutrients and environmental toxins. The inhibition of transporters by drugs has potential physiologic consequences. The full extent of the effect of drugs on the function of transporters is poorly understood because only a small subset of the hundreds of transporters expressed in humans - primarily those mediating the rate-determining step in the elimination of specific drugs - are assessed during clinical development. Areas covered: We provide a comprehensive overview of literature reports implicating the inhibition of transporters as the mechanism for off-target effects of drugs. Expert opinion: Transporter inhibition, the mechanism of action of many marketed drugs, appears to play an underappreciated role in a number of side effects including vitamin deficiency, edema, dyslipidemia, cholestasis and gout. Cell systems more broadly expressing transporter networks and methods like unbiased metabolomics should be incorporated into the screening paradigm to expand our understanding of the impact of drugs on the physiologic function of transporters and to allow for these effects to be taken into account in drug discovery and clinical practice.

Discovery of a Phosphoinositide 3-Kinase (PI3K) ß/δ Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kß Potency in a PI3Kδ-Selective Template by Targeting Nonconserved Asp856.

Phosphoinositide 3-kinase (PI3K) ß signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kß/δ inhibitors in which PI3Kß potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kß/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

Renal Transporter-Mediated Drug-Drug Interactions: Are They Clinically Relevant?

The kidney, through the distinct processes of passive glomerular filtration and active tubular secretion, plays an important role in the elimination of numerous endobiotics (eg, hormones, metabolites), toxins, nutrients, and drugs. Renal transport pathways mediating active tubular secretion and reabsorption in the proximal tubule are complex, involving apical and basolateral transporters acting in concert. Detailed studies of the molecular mechanisms of net active tubular secretion have established the involvement of multiple transporters with overlapping substrate specificity mediating competing secretion and reabsorption pathways. Although drug interactions arising from inhibition of renal transporters are rare relative to other mechanisms, they can involve commonly administered drugs (eg, cimetidine, metformin), may be underappreciated due to muted effects on plasma pharmacokinetics relative to tissue levels, can affect narrow-therapeutic-index medications (eg, antiarrhythmic, oncology medications), and may disproportionately affect sensitive populations where polypharmacy is common (eg, the elderly, diabetics). In particular, there is the potential for larger-magnitude interactions in subjects with reduced glomerular filtration rates due to the increased relative contribution of tubular secretion. The assessment of additional endpoints in drug-drug interaction studies including pharmacodynamics, positron emission tomography imaging, and metabolomics promises to expand our understanding of the clinical relevance of renal drug interactions.

Discovery of Orally Efficacious Phosphoinositide 3-Kinase δ Inhibitors with Improved Metabolic Stability.

Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

2,4,6-Triaminopyrimidine as a Novel Hinge Binder in a Series of PI3Kδ Selective Inhibitors.

Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.

Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium Current Inhibitor (Late INai), a Phase II Agent with Demonstrated Preclinical Anti-Ischemic and Antiarrhythmic Properties.

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Artemisinin derivatives: toxic for laboratory animals, safe for humans?

A discrepancy seems to prevail with regard to the toxicity and safety of the artemisinin family of antimalarials. While these compounds have been found to be virtually void of any serious side effects in humans, their neurotoxicity in animal models has raised concerns about their use. In this paper, we present selected examples of both pre-clinical and clinical studies dealing with adverse effects of artemisinin drugs. We suggest that the prolonged presence of artemisinins upon slow release from oil-based intramuscular formulations is the main cause of the observed toxicity in laboratory animals. In contrast, oral intake of these compounds, which is by far the most common formulation used for treatment of malaria patients, results in rapid clearance of these drugs and is thus unlikely to cause any toxicity in human subjects. Another plausible factor may be the relatively high doses of artemisinin compounds used in animal studies. In conclusion, the observation of the toxicity of artemisinin compounds in animals, but not in humans, is most likely due to different pharmacokinetic profiles after different routes of administrations.

Evaluation of the endothelin receptor antagonists ambrisentan, bosentan, macitentan, and sitaxsentan as hepatobiliary transporter inhibitors and substrates in sandwich-cultured human hepatocytes.

BACKGROUND: Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs). METHODS: Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3. RESULTS: The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x). CONCLUSIONS: Significant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.