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The rapid growth of demands for drug discovery has necessitated the ongoing pursuit of new methods for specific ligands screening and identification. This work combined receptor-affinity chromatography (RAC) with high-throughput sequencing techniques to rapidly screen and identify the specific ligands. By this method, immobilized angiotensin II type I receptor (AT 1 R) and endothelin receptor A (ET A R) based on RAC were utilized for lead screening from a DNA-encoded library. The specific ligands of AT 1 R (ligand A 1 , A 2 ) and ET A R (ligand B 1 , B 2 ) were synthesized after decoding by high-throughput sequencing techniques. The dissociation rate constants (k d ) of ligand A 1 , A 2 to AT 1 R and B 1 , B 2 to ET A R were 9.65 × 10 -4 , 31.1 × 10 -4 and 0.66, 1.22 s -1 by peak profiling assay. The association constant (K A ) to the receptors of four ligands was 5.4 × 10 6 , 3.3 × 10 6 and 1.6 × 10 6 , 2.2 × 10 5 by injection amount dependent method. The kinetic and thermodynamic parameters of the four specific ligands are similar to those of the positive drugs. This indicates that they are promising to drug candidates. The druggability of the four ligands through pharmacokinetic investigation by HPLC-MS/MS presented desired pharmacokinetic behavior including the fast absorption, the relatively slow elimination. These results, taking together, indicated that the RAC combined with high-throughput sequencing techniques can screen and identify the specific ligands according to various proteins, thus creating a general strategy for rapid discovery of promising drug candidates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Aprocitentan is an orally active, dual endothelin (ET) receptor antagonist developed for the treatment of hypertension in which, despite available treatments, a medical need exists for drugs with a new mechanism of action., Subjects and Methods: In this study, the single- and multiple-dose tolerability, safety, pharmacokinetics (PK), and pharmacodynamics of up to 600 mg (single doses) and 100 mg once a day (qd; multiple doses) of aprocitentan were investigated in healthy male and female subjects. The effect of age on the tolerability and PK parameters was investigated at a dose of 100 mg qd., Results: Aprocitentan was well tolerated across all doses. No serious adverse events (AEs) occurred. The most frequently reported AE was headache. Small increases in body weight were recorded in subjects receiving 100 mg qd. Plasma concentration-time profiles of aprocitentan were similar after single- and multiple-dose administration, and support a qd dosing regimen based on a half-life of 44 hours. After multiple doses, PK was dose proportional. Accumulation at steady state, reached by Day 8, was 3-fold. Only minor differences in exposure between healthy females and males, healthy elderly and adult subjects, and fed and fasted conditions were observed. Plasma ET-1 concentrations, reflecting ET B receptor antagonism, significantly increased with doses ≥25 mg. Time-matched analysis of electrocardiogram (ECG) parameters did not suggest drug-induced ECG effects. Exposure-response analysis indicated no QTc prolongations at plasma levels up to 10 µg/mL., Conclusion: Aprocitentan was well tolerated in healthy subjects with a PK profile favorable for qd dosing., Competing Interests: Disclosure JD is a fellow of the American College of Clinical Pharmacology. PNS and JD are current employees of Idorsia Pharmaceuticals Ltd and former employees of Actelion Pharmaceuticals Ltd. MM is a current employee of Idorsia Pharmaceuticals Ltd. MKK was the principal investigator of the study that was sponsored by Actelion Pharmaceuticals Ltd. The authors report no other conflicts of interest in this work.

Aim: The aim of the present study was to investigate whether increasing the bosentan dosing frequency from 2 mg kg -1 twice daily (b.i.d.) to 2 mg kg -1 three times daily (t.i.d.) in children with pulmonary arterial hypertension (PAH) (from ≥3 months to <12 years of age) would increase exposure., Methods: An open-label, prospective, randomized, multicentre, multiple-dose, phase III study was conducted. Patients (n = 64) were randomized 1:1 to receive oral doses of bosentan of 2 mg kg -1 b.i.d. or t.i.d. The main pharmacokinetic endpoint was the daily exposure to bosentan over 24 h corrected to the 2 mg kg -1 dose (AUC 0-24C ). The maximum plasma concentration corrected to the 2 mg kg -1 dose (C maxC ), the time to reach the maximum plasma concentration (t max ) and safety endpoints were also assessed., Results: The geometric mean [95% confidence interval (CI)] for AUC 0-24C was 8535 h.ng ml -1 (6936, 10 504) and 7275 h.ng ml -1 (5468, 9679) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.85 (0.61, 1.20)]. The geometric mean (95% CI) for C maxC was 743 ng ml -1 (573, 963) and 528 ng ml -1 (386, 722) for 2 mg kg -1 b.i.d. and t.i.d., respectively [geometric mean ratio (95% CI) 0.71 (0.48, 1.05)]. The median (range) for t max was 3.0 h (0.0-7.5) and 3.0 h (1.0-8.0) for 2 mg kg -1 b.i.d. and t.i.d., respectively. The proportions of patients who experienced ≥1 adverse event were similar in the b.i.d. (66.7%) and t.i.d. (67.7%) groups., Conclusions: There appeared to be no clinically relevant difference in exposure to bosentan, or in safety, when increasing the frequency of bosentan dosing from b.i.d. to t.i.d. Therefore, the present study provides no indication that the dosing recommendation should be changed, and 2 mg kg -1 b.i.d. remains the recommended dosing regimen for bosentan in paediatric PAH patients., (© 2017 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Endothelin (ET) receptor antagonists are potentially novel therapeutic agents in chronic kidney disease and resistant hypertension, but their use is complicated by sodium and water retention. In animal studies, this side effect arises from ET B receptor blockade in the renal tubule. Previous attempts to determine whether this mechanism operates in humans have been confounded by the hemodynamic consequences of ET receptor stimulation/blockade. We aimed to determine the effects of ET signaling on salt transport in the human nephron by administering subpressor doses of the ET-1 precursor, big ET-1. We conducted a 2-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. After sodium restriction, subjects received either intravenous placebo or big ET-1, in escalating dose (≤300 pmol/min). This increased plasma concentration and urinary excretion of ET-1. Big ET-1 reduced heart rate (≈8 beats/min) but did not otherwise affect systemic hemodynamics or glomerular filtration rate. Big ET-1 increased the fractional excretion of sodium (from 0.5 to 1.0%). It also increased free water clearance and tended to increase the abundance of the sodium-potassium-chloride cotransporter (NKCC2) in urinary extracellular vesicles. Our protocol induced modest increases in circulating and urinary ET-1. Sodium and water excretion increased in the absence of significant hemodynamic perturbation, supporting a direct action of ET-1 on the renal tubule. Our data also suggest that sodium reabsorption is stimulated by ET-1 in the thick ascending limb and suppressed in the distal renal tubule. Fluid retention associated with ET receptor antagonist therapy may be circumvented by coprescribing potassium-sparing diuretics., (© 2017 The Authors.)

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Macitentan is the most recently approved dual endothelin-receptor antagonist (ERA) for the treatment of symptomatic pulmonary arterial hypertension. Compared to other available ERAs, it demonstrates superior receptor-binding properties, with consequently improved tissue penetration, and a longer duration of action allowing for once-daily dosing. It has a favorable adverse-effect profile, with notably no demonstrable increase in the risk of hepatotoxicity or peripheral edema, but like other ERAs, it is potentially limited by significant anemia. Phase I data have demonstrated a favorable drug-drug interaction profile and no need for dose adjustment with hepatic and renal impairment. In the pivotal SERAPHIN study, treatment of symptomatic pulmonary arterial hypertension patients with macitentan led to statistically significant improvements in functional class, exercise tolerance, and hemodynamic parameters, in addition to a reduction in morbidity in an event-driven long-term trial.

Aim: To study the mutual pharmacokinetic interactions between macitentan, an endothelin receptor antagonist, and sildenafil in healthy male subjects., Methods: In this open-label, randomized, three way crossover study, 12 healthy male subjects received the following oral treatments: A) a loading dose of 30 mg macitentan on day 1 followed by 10 mg once daily for 3 days, B) sildenafil 20 mg three times a day for 3 days and a single 20 mg dose on day 4 and C) both treatments A and B concomitantly. Plasma concentration-time profiles of macitentan and its active metabolite ACT-132577 (treatments A and C) and sildenafil and its N-desmethyl metabolite (treatments B and C) were determined on day 4 and analyzed non-compartmentally., Results: The pharmacokinetics of macitentan were not affected by sildenafil. In the presence of sildenafil Cmax and AUCτ of the metabolite ACT-132577 decreased with geometric mean ratios (90% confidence interval (CI)) of 0.82 (0.76, 0.89) and 0.85 (90% CI 0.80, 0.91), respectively. In the presence of macitentan, plasma concentrations of sildenafil were higher than during treatment with sildenafil alone, resulting in increased Cmax and AUCτ values. The respective geometric mean ratios were 1.26 (90% CI 1.07, 1.48) and 1.15 (90% CI 0.94, 1.41). The pharmacokinetics of N-desmethylsildenafil were not affected by macitentan. All treatments were well tolerated., Conclusion: A minor, not clinically relevant, pharmacokinetic interaction was observed between macitentan and sildenafil. Based on these results, no dose adjustment of either compound appears necessary during concomitant treatment with macitentan and sildenafil., (© 2014 The British Pharmacological Society.)

Aims: The aim of this study was to assess the effect of the cytochrome P450 (CYP) 3A4 and organic anion-transporting polypeptide (OATP) 1B1 inhibitor clarithromycin on the pharmacokinetics of bosentan. We also aimed to evaluate the impact of CYP2C9 and SLCO1B1 (encoding for OATP1B1) genotypes and their combination., Methods: We assessed the effect of the OATP and CYP3A inhibitor clarithromycin on bosentan pharmacokinetics at steady state and concurrently quantified changes of CYP3A activity using midazolam as a probe drug. Sixteen healthy volunteers received therapeutic doses of bosentan (125 mg twice daily) for 14 days and clarithromycin (500 mg twice daily) concomitantly for the last 4 days, and bosentan pharmacokinetics was assessed on days 1, 10 and 14., Results: Clarithromycin significantly increased bosentan area under the plasma concentration-time curve of the dosing interval 3.7-fold and peak concentration 3.8-fold in all participants irrespective of the genotype. Clarithromycin also reduced CYP3A activity (midazolam clearance) in all participants; however, these changes were not correlated to the changes of bosentan clearance., Conclusions: Clarithromycin substantially increases the exposure to bosentan, suggesting that dose reductions may be necessary., (© 2013 The British Pharmacological Society.)