ET_A receptor ( Ki = 4.7 nM ); ET_A receptor ( Ki = 95 nM )

Bosentan, a dual endothelin receptor antagonist, decreased the expression of the COL1A2 gene by reversing the transcriptional activity of Fli1 in SSc dermal fibroblasts[2]
Previous reports demonstrated that bosentan, a dual ET receptor antagonist, reverses a pro-fibrotic phenotype of SSc fibroblasts. However, the detailed mechanism by which bosentan exerts its prominent anti-fibrotic effect on SSc fibroblasts has still remained unknown. We previously demonstrated that Fli1 deficiency contributes to the establishment of the pro-fibrotic phenotype in SSc fibroblasts and imatinib mesylate, which targets the c-Abl/PKC-δ/Fli1 pathway, reverses the pro-fibrotic phenotype of these cells. Given that SSc fibroblasts are constitutively activated by autocrine stimulation of transforming growth factor- β (TGF-β), a potent inducer of ET-1, and produces an excessive amount of ET-1, autocrine ET-1 appears to be involved in the self-activation system in SSc fibroblasts. The present observation that ET-1 inactivates the transcriptional activity of Fli1 suggests that the blockade of autocrine ET-1 by bosentan reverses the pro-fibrotic phenotype of SSc fibroblasts by reactivating the transcriptional repressor activity of Fli1. To address this issue, we performed a series of experiments using cultured SSc dermal fibroblasts.[2]
Supporting the contribution of autocrine ET-1 to the activation of SSc dermal fibroblasts, exogenous ET-1 did not affect type I collagen expression (Figure 5A), whereas Bosentan suppressed the expression of type I collagen in a dose-dependent manner without any effect on cell viability in SSc dermal fibroblasts (Figure 5B and Table 1). Furthermore, the total levels and the phosphorylation levels of c-Abl and the total levels and nuclear localization of PKC-δ were decreased in SSc dermal fibroblasts treated with bosentan (Figure 5C and 5D). Consistently, Fli1 phosphorylation at threonine 312 was reduced (Figure 5E) and the occupancy of Fli1 on COL1A2 promoter was increased in SSc dermal fibroblasts treated with bosentan (Figure 5F). Importantly, bosentan did not affect the mRNA levels of the FLI1 gene in SSc dermal fibroblasts (Figure 5G). Collectively, these results indicate that autocrine ET-1 contributes to the activation of SSc dermal fibroblasts and bosentan reverses a pro-fibrotic phenotype of SSc dermal fibroblasts by increasing the DNA binding ability of Fli1.[2]

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In vitro activity: Bosentan (BOS) selectively and competitively inhibits the binding of 125I-labeled ET-1 to human placenta cells' ETB receptors and human smooth muscle cells' ETA receptors. Bosentan exhibits an in vitro binding affinity of 4.7 nM for ETA receptors on human SMC and 41 or 95 nM for ETB receptors on human SMC or placenta cells. In an in vitro 125I-labeling assay, bosentan has 67-fold higher selectivity for ETA than ETB receptors (mean IC50=7.1 vs 474.8 nM)[1].

Macitentan 30 mg/kg, when administered in addition to Bosentan 100 mg/kg, reduces mean arterial blood pressure (MAP) in hypertensive rats by an additional 19 mm Hg. Bosentan, on the other hand, does not cause an extra MAP decrease when taken in addition to Macitentan. Compared to a maximal effective dose of Bosentan, which is administered on top of Macitentan, there is no additional decrease in mean pulmonary artery pressure (MPAP) in pulmonary hypertensive rats when Macitentan 30 mg/kg is added[3].

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Bosentan increased the expression of Fli1 protein in lesional dermal fibroblasts of the BLM-induced murine model of SSc [2]
Finally, we investigated if bosentan increases the expression of Fli1 protein in lesional dermal fibroblasts of the BLM-induced SSc murine model because previous reports demonstrated that bosentan prevents the development of dermal fibrosis in this model. As we could reproduce the preventive effect of bosentan on dermal fibrosis in BLM-treated mice (Figure 6A), we carried out immunostaining for Fli1 in the skin samples taken from these mice. As shown in Figure 6B, in the absence of bosentan, the number of Fli1-positive dermal fibroblasts was much more decreased in dermal fibroblasts of BLM-treated mice than in those of PBS-treated mice. In contrast, when administered bosentan, the number of Fli1-positive dermal fibroblasts was comparable between BLM-treated mice and PBS-treated mice. Importantly, the signals of Fli1 and α-SMA, a marker of myofibroblasts, in double immunofluorescence were mutually exclusive in most of dermal fibroblasts (Figure 6C), indicating that Fli1 expression is closely related to the inactivation of dermal fibroblasts in vivo. Collectively, these results suggest that bosentan prevents the development of dermal fibrosis in the BLM-induced SSc murine model, at least partially, by increasing the expression of Fli1 protein in lesional dermal fibroblasts.

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Determination of maximal effective doses in DSS rats and observation that the maximal efficacy of macitentan is greater than that of Bosentan [3]
DSS rats developed an increase in MAP up to approximately 180 mm Hg. MAP and HR baseline values were similar between the different experimental groups. After acute oral administration, macitentan dose-dependently decreased MAP in DSS rats. The maximal effective dose of macitentan was 30 mg/kg; this dose decreased MAP by 30 ± 5 mm Hg (Table 1). The maximal effect was reached at about 24 h after administration (Tmax). Acute oral administration of bosentan dose-dependently decreased MAP in DSS rats. At the maximal effective dose of 100 mg/kg, bosentan decreased MAP by 15 ± 2 mm Hg (Table 1), with a Tmax at 6 h. No effect on HR was observed. Based on these data, the maximal effective doses of 30 mg/kg of macitentan and 100 mg/kg of bosentan were selected for the add-on study. Tmax was determined to be 6 h for bosentan and 24 h for macitentan.

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Confirmation of selection of maximal effective doses of macitentan and Bosentan using add-on protocol in DSS rats [3]
The use of maximal effective doses of macitentan and bosentan was confirmed using the add-on protocol: macitentan 30 mg/kg administered on top of macitentan 30 mg/kg did not cause an additional MAP decrease as compared to vehicle on top of macitentan (− 29 ± 2 and − 28 ± 5 mm Hg). Bosentan 100 mg/kg, administered when the maximal effect of bosentan 100 mg/kg was reached, did not induce an additional MAP decrease compared to vehicle on top of bosentan (− 13 ± 2 and − 14 ± 3 mm Hg, respectively; Fig. 2).

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Comparison between macitentan and Bosentan using add-on protocol in DSS rats [3]
Oral administration of macitentan 30 mg/kg, when the maximal effect of bosentan 100 mg/kg had been reached, decreased MAP by an additional 19 mm Hg compared to vehicle (p < 0.01) administered on top of bosentan 100 mg/kg. The maximal decrease induced by macitentan was 33 ± 4 mm Hg (Fig. 2). Conversely, bosentan, administered orally at 100 mg/kg, when the maximal effect of macitentan 30 mg/kg had been reached, did not induce an additional MAP decrease compared to vehicle administered on top of macitentan 30 mg/kg (Fig. 3).

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Determination of maximal effective doses in bleomycin-treated rats and observation that the maximal efficacy of macitentan is greater than that of Bosentan [3]
Bleomycin-treated rats developed an increase in MPAP of approximately 13 mm Hg vs. saline-instilled rats. MPAP and HR baseline values were similar between the different experimental groups. Acute oral administration of macitentan and bosentan dose-dependently decreased MPAP in bleomycin rats (Table 1) without affecting HR (data not shown). At a dose of 30 mg/kg macitentan, the maximal decrease in MPAP was 12 ± 3 mm Hg, about 24 h after administration (Tmax). At the maximal effective dose of 300 mg/kg, bosentan decreased MPAP by 7 ± 2 mm Hg (Table 1), with a Tmax around 6 h. Based on these data, the maximal effective doses of 30 mg/kg of macitentan and 300 mg/kg of bosentan were selected for the add-on study. Tmax was determined to be 6 h for bosentan and 24 h for macitentan.

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Confirmation of selection of maximal effective dose of Bosentan using add-on protocol in bleomycin-treated rats [3]
The use of maximal effective doses of macitentan and bosentan was confirmed using the add-on protocol: macitentan 30 mg/kg administered on top of macitentan 30 mg/kg did not cause an additional MPAP decrease compared to vehicle on top of macitentan (− 18 ± 2 and − 13 ± 2 mm Hg, p = 0.112). Similarly, as shown in Fig. 4, bosentan 300 mg/kg administered when the maximal effect of bosentan 300 mg/kg was reached did not cause an additional MPAP decrease compared to vehicle on top of bosentan (− 8 ± 1 and − 7 ± 1 mm Hg, respectively).

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Comparison between macitentan and Bosentan using add-on protocol in bleomycin-treated rats [3]
Oral administration of macitentan 30 mg/kg to bleomycin rats, when the maximal effect of bosentan 300 mg/kg had been reached, induced an additional MPAP decrease compared to vehicle administered on top of bosentan 300 mg/kg. The maximal decrease induced by macitentan on top of bosentan was − 11 ± 1 mm Hg (p < 0.01 vs. vehicle) (Fig. 4). Conversely, bosentan, administered orally at 300 mg/kg, when the maximal effect of macitentan 30 mg/kg had been reached, did not induce an additional MPAP decrease compared to vehicle administered on top of macitentan 30 mg/kg (Fig. 5).

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Absence of drug–drug interaction [3]
As shown in Fig. 6, administration of Bosentan did not modify the plasma concentrations of macitentan and its active metabolite ACT-132577, ruling out a modification of macitentan pharmacokinetics by bosentan during the add-on protocol.

The trypan blue exclusion test is used to assess the viability of cells. Bosentan is added to human dermal fibroblasts at the recommended concentrations (10, 20 and 40 μM). After 24 and 48 hours, cell viability is assessed. A hematocytometer is used to count both stained (dead) and unstained (viable) cells[2].

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Cell viability assay [2]
Cell viability was evaluated by the trypan blue exclusion test. Cells were treated with the indicated concentration of bosentan. Cell viability was examined at 24 and 48 hours. Stained (dead) and unstained (viable) cells were counted with a hematocytometer.

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Immunoblotting [2]
Confluent quiescent fibroblasts were serum-starved for 48 hours and harvested. In some experiments, cells were stimulated with ET-1 or bosentan for the indicated period of time before being harvested. Whole cell lysates and nuclear extracts were prepared as described previously. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gels electrophoresis and immunoblotting with the indicated primary antibodies. Bands were detected using enhanced chemiluminescent techniques. According to a series of pilot experiments, anti-Fli1 antibody and anti-phospho-Fli1 (Thr312)-specific antibody work much better in immunoblotting using nuclear extracts and whole cell lysates, respectively.

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The evaluation of COL1A2 promoter activity by RT real-time PCR [2]
Normal or SSc fibroblasts were grown to 50% confluence in 100-mm dishes, transfected with the indicated constructs along with pSV-β-galactosidase (β-GAL) using FuGENE6. After overnight incubation at 37°C, some cells were further stimulated with ET-1 or bosentan for 24 hours. The cells were harvested and CAT and β-GAL mRNA levels were determined using RT real-time PCR. Transfection efficiency was normalized by β-GAL mRNA levels. In some samples, it was confirmed that this method reproduces the results of relative promoter activity evaluated by the canonical method of CAT reporter assay using [14C]-chloramphenicol. The sequences of primers were as follows: CAT forward 5′-TTCGTCTCAGCCAATCCCTGGGTGA-3′ and reverse 5′-CCCATCGTGAAAACGGGGGCGAA-3′; β-GAL forward 5′-TCCACCTTCCCTGCGTTA-3′ and reverse 5′-AGAAGTCGGGAGGTTGCTG-3′.

Rats: Rats that are two months old—DSS and Wistar—are employed. Doses ranging from 0.1 to 100 mg/kg (Macitentan) or 3 to 600 mg/kg (Bosentan) are used to measure the pharmacological effects on heart rate (HR), mean arterial pressure (MAP), or mean pulmonary arterial pressure (MPAP), and up to 120 hours after a single gavage. 1) Macitentan is given on top of the maximum effective dose of Bosentan determined by the dose-response curve in order to assess whether Macitentan can offer greater pharmacological activity compared to Bosentan. Secondly, the maximum effective dose of Macitentan is topped off with the same dose of Bosentan. Tmax of the first tested compound is the point at which the second compound's maximal effective dose is given.

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Dose–response curves and add-on protocol [3]
First, dose–response curves of each ERA were constructed in both systemic hypertensive DSS rats and bleomycin-induced pulmonary hypertensive Wistar rats to determine the maximal effective dose and Tmax (time of observed maximal effect) of each ERA. Pharmacological effects on MAP or MPAP and HR were measured up to 120 h after a single gavage at doses ranging from 0.1 to 100 mg/kg (macitentan) or 3 to 600 mg/kg (Bosentan). To determine whether macitentan could provide superior pharmacological activity vs. bosentan, we designed a study in which: 1) macitentan was administered on top of the maximal effective dose of bosentan established by the dose–response curve as shown in Fig. 1, Fig. 2) the same dose of bosentan was administered on top of the maximal effective dose of macitentan. The maximal effective dose of the second compound was administered at Tmax of the first tested compound.

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Pharmacokinetics [3]
In order to rule out any confounding drug–drug interaction on the pharmacological effect measured, the exposure of macitentan and its active metabolite ACT-132577 was measured in the presence or absence of Bosentan in similar conditions to the add-on protocol. Vehicle or bosentan 300 mg/kg was administered to Wistar rats (n = 6/group) 6 h prior to macitentan 30 mg/kg. Plasma samples were collected at 1, 2, 3, 4, 6, 8 and 24 h after oral administration of macitentan, and quantification of macitentan and ACT-132577 was determined by liquid chromatography coupled to mass spectrometry.

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Test compounds [3]
Macitentan and Bosentan were supplied by Actelion Pharmaceuticals Ltd. Gelatin 7.5%, administered at 5 mL/kg, was used as vehicle for oral administration of the compounds by gavage.

Absorption, Distribution and Excretion
Absolute bioavailability is approximately 50% and food does not affect absorption.
Bosentan is eliminated by biliary excretion following metabolism in the liver.
18 L
4 L/h [patients with pulmonary arterial hypertension]

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Metabolism / Metabolites
Bosentan is metabolized in the liver by the cytochrome P450 enzymes CYP2C9 and CYP3A4 (and possibly CYP2C19), producing three metabolites, one of which, Ro 48-5033, is pharmacologically active and may contribute 10 to 20% to the total activity of the parent compound.
Bosentan has known human metabolites that include Hydroxy Bosentan and 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-hydroxyphenoxy)-[2,2-]bipyrimidinyl-4-yl]-benzenesulfonamide.

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Biological Half-Life
Terminal elimination half-life is about 5 hours in healthy adult subjects.

Hepatotoxicity
Bosentan is associated with elevations in serum aminotransferase levels above three times the upper limit of the normal range (ULN) in 3% to 18% of patients, averaging 7.6% using currently recommended doses. The enzyme elevations are usually self-limited and are rarely accompanied by symptoms, but can be more marked and persist and require dose reduction or discontinuation (in 3% to 4% of patients). Monthly monitoring of serum aminotransferase levels is recommended, with discontinuation for levels above 8 times the ULN or for values above 5 times the ULN that persist. There have also been rare reports of clinically apparent liver injury with jaundice associated with bosentan use. The onset of illness was usually within 1 to 6 months of starting bosentan, but cases arising during chronic therapy have also been described (Case 1). The enzyme pattern has typically been hepatocellular or mixed. Immunoallergic features are usually not present and autoantibodies are usually absent or present in low titer. Some cases have been severe and fatalities have been reported, but there have been no published reports of chronic hepatitis or vanishing bile duct syndrome attributed to bosentan. Autoimmune and immunoallergic features are usually not present.
Likelihood score: C (probable cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
A study in one patient taking bosentan during breastfeeding found very low levels in milk. Another woman breastfed her preterm newborn while taking bosentan and sildenafil with no adverse effects reported. Amounts ingested by the infant are far below doses given to treat infants and would not be expected to cause any adverse effects in breastfed infants.
◉ Effects in Breastfed Infants
A 23-year-old woman with congenital heart disease and pulmonary hypertension was treated during pregnancy with bosentan and sildenafil in unspecified dosages. These drugs and warfarin were continued postpartum. Her infant was delivered at 30 weeks by cesarean section and weighed 1.41 kg at birth. She nursed the infant in the neonatal intensive care unit for 11 weeks "with good outcome" according to the authors, but the infant died at 26 weeks from a respiratory syncytial virus infection.[2]
A woman breastfeeding her 21-month-old infant was taking 20 mg of sildenafil 3 times daily and 125 mg of bosentan twice daily to treat pulmonary arterial hypertension. The drugs were begun more than 6 months postpartum. The mother did not report any possible adverse effects, serious health problem or hospitalization of the infant in the period from birth until day 651 postpartum when the infant continued to be partially breastfed.[1]
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Greater than 98% to plasma proteins, mainly albumin.

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Toxicity Summary
Bosentan is well tolerated, and when patients receive appropriate monitoring presents a very low risk for toxicity. However, when given with cyclosporin A, bosentan’s plasma levels increased 30-fold and resulted in severe headaches, nausea, and vomiting. However, no serious adverse effects or toxicity were present in these patients. In one postmarket period, one episode of overdose by a male patient who took 10000 mg of bosentan resulted in nausea, vomiting, hypotension, blurred vision, and sweating. The patient was able to make a full recovery following adequate blood pressure support.

[1]. Bosentan: a review of its use in the management of mildly symptomatic pulmonary arterial hypertension. Am J Cardiovasc Drugs. 2009;9(5):331-50.

[2]. Bosentan reverses the pro-fibrotic phenotype of systemic sclerosis dermal fibroblasts via increasing DNA binding ability of transcription factor Fli1. Arthritis Res Ther. 2014 Apr 3;16(2):R86.

[3]. Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension. Life Sci. 2014 Nov 24;118(2):333-9.

[4]. Endothelin Regulates Porphyromonas gingivalis-Induced Production of Inflammatory Cytokines. PLoS One. 2016 Dec 28;11(12):e0167713.

Bosentan is a sulfonamide, a member of pyrimidines and a primary alcohol. It has a role as an antihypertensive agent and an endothelin receptor antagonist.
Bosentan is a dual endothelin receptor antagonist marketed under the trade name Tracleer by Actelion Pharmaceuticals. Bosentan is used to treat pulmonary hypertension by blocking the action of endothelin molecules that would otherwise promote narrowing of the blood vessels and lead to high blood pressure.
Bosentan anhydrous is an Endothelin Receptor Antagonist. The mechanism of action of bosentan anhydrous is as an Endothelin Receptor Antagonist, and Cytochrome P450 3A Inducer, and Cytochrome P450 2C9 Inducer.
Bosentan is an endothelin receptor antagonist used in the therapy of pulmonary arterial hypertension (PAH). Bosentan has been associated with serum enzyme elevations during therapy and with rare instances of clinically apparent acute liver injury.
Bosentan is a sulfonamide-derived, competitive and specific endothelin receptor antagonist with a slightly higher affinity for the endothelin A receptor than endothelin B receptor. Bosentan blocks the action of endothelin 1, an extremely potent endogenous vasoconstrictor and bronchoconstrictor, by binding to endothelin A and endothelin B receptors in the endothelium and vascular smooth muscle. Bosentan decreases both pulmonary and systemic vascular resistance and is particularly used in the treatment of pulmonary arterial hypertension.
A sulfonamide and pyrimidine derivative that acts as a dual endothelin receptor antagonist used to manage PULMONARY HYPERTENSION and SYSTEMIC SCLEROSIS.

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Drug Indication
Used in the treatment of pulmonary arterial hypertension (PAH), to improve exercise ability and to decrease the rate of clinical worsening (in patients with WHO Class III or IV symptoms).
FDA Label
Treatment of pulmonary arterial hypertension (PAH) to improve exercise capacity and symptoms in patients with WHO functional class III. Efficacy has been shown in: , , , Primary (idiopathic and familial) PAH; , PAH secondary to scleroderma without significant interstitial pulmonary disease; , PAH associated with congenital systemic-to-pulmonary shunts and Eisenmenger's physiology. , , , Some improvements have also been shown in patients with PAH WHO functional class II. , , Tracleer is also indicated to reduce the number of new digital ulcers in patients with systemic sclerosis and ongoing digital ulcer disease. ,
Treatment of pulmonary arterial hypertension (PAH) to improve exercise capacity and symptoms in patients with World Health Organization (WHO) functional class III. Efficacy has been shown in: primary (idiopathic and familial) PAH; PAH secondary to scleroderma without significant interstitial pulmonary disease; PAH associated with congenital systemic-to-pulmonary shunts and Eisenmenger's physiology. Some improvements have also been shown in patients with PAH WHO functional class II. Stayveer is also indicated to reduce the number of new digital ulcers in patients with systemic sclerosis and ongoing digital-ulcer disease.
Treatment of interstitial pulmonary fibrosis, Treatment of pulmonary arterial hypertension, Treatment of systemic sclerosis

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Mechanism of Action
Endothelin-1 (ET-1) is a neurohormone, the effects of which are mediated by binding to ET_A and ET_B receptors in the endothelium and vascular smooth muscle. It displays a slightly higher affinity towards ET_A receptors than ET_B receptors. ET-1 concentrations are elevated in plasma and lung tissue of patients with pulmonary arterial hypertension, suggesting a pathogenic role for ET-1 in this disease. Bosentan is a specific and competitive antagonist at endothelin receptor types ET_A and ET_B.

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Pharmacodynamics
Bosentan belongs to a class of drugs known as endothelin receptor antagonists (ERAs). Patients with PAH have elevated levels of endothelin, a potent blood vessel constrictor, in their plasma and lung tissue. Bosentan blocks the binding of endothelin to its receptors, thereby negating endothelin's deleterious effects.

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Bosentan (Tracleer) is an orally administered dual endothelin-1 (ET-1) receptor antagonist approved for use in patients with WHO class II (mildly symptomatic) pulmonary arterial hypertension (PAH). Oral bosentan therapy was beneficial and generally well tolerated in patients with mildly symptomatic PAH. In a well designed, placebo-controlled trial in adolescents and adults with mildly symptomatic PAH, pulmonary vascular resistance was significantly reduced with bosentan relative to placebo, but the 6-minute walk distance did not increase significantly. Similarly, pediatric patients (most of whom had mildly symptomatic PAH) in a small uncontrolled trial experienced some improvement in hemodynamic variables with bosentan, but did not experience a significant increase in exercise capacity. Adverse events associated with bosentan were consistent with those seen in other indications, with major concerns being the potential for teratogenicity and hepatotoxicity, for which regular liver function monitoring is recommended. Overall, considering the progressive nature of PAH, bosentan extends the treatment options available to patients with mildly symptomatic PAH. [1]

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Introduction: Although the pathogenesis of systemic sclerosis (SSc) still remains unknown, recent studies have demonstrated that endothelins are deeply involved in the developmental process of fibrosis and vasculopathy associated with SSc, and a dual endothelin receptor antagonist, bosentan, has a potential to serve as a disease modifying drug for this disorder. Importantly, endothelin-1 (ET-1) exerts a pro-fibrotic effect on normal dermal fibroblasts and bosentan reverses the pro-fibrotic phenotype of SSc dermal fibroblasts. The purpose of this study was to clarify the details of molecular mechanisms underlying the effects of ET-1 and bosentan on dermal fibroblasts, which have not been well studied. Methods: The mRNA levels of target genes and the expression and phosphorylation levels of target proteins were determined by reverse transcription real-time PCR and immunoblotting, respectively. Promoter assays were performed using a sequential deletion of human α2 (I) collagen (COL1A2) promoter. DNA affinity precipitation and chromatin immunoprecipitation were employed to evaluate the DNA binding ability of Fli1. Fli1 protein levels in murine skin were evaluated by immunostaining. Results: In normal fibroblasts, ET-1 activated c-Abl and protein kinase C (PKC)-δ and induced Fli1 phosphorylation at threonine 312, leading to the decreased DNA binding of Fli1, a potent repressor of the COL1A2 gene, and the increase in type I collagen expression. On the other hand, bosentan reduced the expression of c-Abl and PKC-δ, the nuclear localization of PKC-δ, and Fli1 phosphorylation, resulting in the increased DNA binding of Fli1 and the suppression of type I collagen expression in SSc fibroblasts. In bleomycin-treated mice, bosentan prevented dermal fibrosis and increased Fli1 expression in lesional dermal fibroblasts. Conclusions: ET-1 exerts a potent pro-fibrotic effect on normal fibroblasts by activating "c-Abl - PKC-δ - Fli1" pathway. Bosentan reverses the pro-fibrotic phenotype of SSc fibroblasts and prevents the development of dermal fibrosis in bleomycin-treated mice by blocking this signaling pathway. Although the efficacy of bosentan for dermal and pulmonary fibrosis is limited in SSc, the present observation definitely provides us with a useful clue to further explore the potential of the upcoming new dual endothelin receptor antagonists as disease modifying drugs for SSc. [2]

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Aims: The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine if these features translate into improved efficacy in vivo, a study was designed in which rats with either systemic or pulmonary hypertension and equipped with telemetry were given macitentan on top of maximally effective doses of another dual ETA/ETB receptor antagonist, bosentan, which does not display sustained receptor occupancy and shows less tissue distribution. Main methods: After establishing dose-response curves of both compounds in conscious, hypertensive Dahl salt-sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the maximal effective dose of bosentan. Key findings: In hypertensive rats, macitentan 30 mg/kg further decreased mean arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100 mg/kg (n=9, p<0.01 vs. vehicle). Conversely, bosentan given on top of macitentan failed to induce an additional MAP decrease. In pulmonary hypertensive rats, macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4 mm Hg on top of bosentan (n=8, p<0.01 vs. vehicle), whereas a maximal effective dose of bosentan given on top of macitentan did not cause any additional MPAP decrease. Significance: The add-on effect of macitentan on top of bosentan in two pathological models confirms that this novel compound can achieve a superior blockade of ET receptors and provides evidence for greater maximal efficacy. [3]

C27H29N5O6S

551.61

551.183

C, 58.79; H, 5.30; N, 12.70; O, 17.40; S, 5.81

147536-97-8

Bosentan-d4; 1065472-77-6; Bosentan hydrate; 157212-55-0

104865

White to off-white solid powder

1.3±0.1 g/cm3

742.3±70.0 °C at 760 mmHg

171-175 °C(lit.)

402.8±35.7 °C

0.0±2.6 mmHg at 25°C

1.607

1.15

2

11

11

39

839

0

O=S(NC1=NC(C2=NC=CC=N2)=NC(OCCO)=C1OC3=CC=CC=C3OC)(C4=CC=C(C(C)(C)C)C=C4)=O

GJPICJJJRGTNOD-UHFFFAOYSA-N

InChI=1S/C27H29N5O6S/c1-27(2,3)18-10-12-19(13-11-18)39(34,35)32-23-22(38-21-9-6-5-8-20(21)36-4)26(37-17-16-33)31-25(30-23)24-28-14-7-15-29-24/h5-15,33H,16-17H2,1-4H3,(H,30,31,32)

4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-pyrimidin-2-ylpyrimidin-4-yl]benzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.99 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (4.99 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (4.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)