ET-A ( Ki = 4.7 nM ); ET-B ( 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 competitively impedes the particular binding of [125 I]-labeled ET-1 on human placenta (ET-B receptors) and smooth muscle cells (ET-A receptors). On the trachea of pigs, bosentan also prevents specific ET-B ligands from binding. The selective ET-B agonist sarafotoxin S6C in the rat trachea and ET-1 in isolated rat aorta (ET-A) both induce contractions that are competitively inhibited by bosentan (pA2= 7.2 and 6.0, respectively), as is the endothelium-dependent relaxation to sarafotoxin S6C in the rabbit superior mesenteric artery (pA2= 6.7). Bosentan's specificity for ET receptors is demonstrated by the fact that it has no discernible effect on the binding of 40 other peptides, prostaglandins, ions, or neurotransmitters.[1]

Bosentan has no intrinsic agonist activity and a long half-life of action, inhibiting the presser response to big ET-1 both intravenously and orally. Additionally, bosentan blocks the presser and depressor effects of sarafotoxin S6C and ET-1. Bosentan has a pharmacological profile that makes it a potentially helpful medication for the treatment of clinical conditions linked to vasoconstriction.[1] The first oral non-peptide mixed ETA/B-receptor antagonist is called bosentan. Rats with CHF who have received long-term Bosentan treatment have significantly improved in terms of survival, hemodynamics, and cardiac remodeling. The degree to which bosentan lowers arterial blood pressure is comparable to that of an ACE inhibitor. When bosentan is administered to rats with CHF following an acute MI, it significantly lowers arterial blood pressure and has an additive effect with ACE inhibitors. In patients with congestive heart failure (CHF), both acute and long-term use of bosentan reduces peripheral and pulmonary vascular resistance while increasing cardiac output.[2]

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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 determine the viability of cells. Bosentan is applied to human dermal fibroblasts at the recommended concentrations (10, 20 and 40 μM). Viability of the cells is assessed after 24 and 48 hours. Using a hematocytometer, the number of stained (dead) and unstained (viable) cells is determined. [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. T_max 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 metabolized in the liver and excreted via bile. 18 L 4 L/h [Patients with pulmonary hypertension] Metabolism/Metabolites Bosentan is metabolized in the liver by 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% of the total activity of the parent compound. Known human metabolites of bosentan include hydroxybosentan and 4-tert-butyl-N-[6-(2-hydroxyethoxy)-5-(2-hydroxyphenoxy)-[2,2-]bipyrimidin-4-yl]-benzenesulfonamide. Biological Half-Life In healthy adults, the terminal elimination half-life is approximately 5 hours.

Hepatotoxicity
Bosentan can cause elevated serum transaminase levels exceeding three times the upper limit of normal (ULN) in 3% to 18% of patients, with an average incidence of 7.6% at the currently recommended dose. Enzyme elevations usually resolve spontaneously and are rarely accompanied by symptoms, but sometimes the elevations are significant and persistent, requiring dose reduction or discontinuation (3% to 4% of patients). Monthly monitoring of serum transaminase levels is recommended; if transaminase levels exceed eight times the ULN or remain above five times the ULN, the drug should be discontinued. Rare case reports have shown clinically significant liver injury with jaundice when using bosentan. Onset usually occurs within 1 to 6 months after starting bosentan, but cases have been reported during long-term treatment (Case 1). The enzyme profile is usually hepatocellular or mixed. Immune hypersensitivity symptoms are usually absent, and autoantibodies are usually absent or at very low titers. Some cases have been severe, with reported deaths, but there are no published reports of bosentan causing chronic hepatitis or bile duct disappearance syndrome. Autoimmune and immune hypersensitivity symptoms are generally not observed.
Probability Score: C (Possibly a cause of clinically significant liver damage).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
A study of a breastfeeding patient taking bosentan found extremely low drug concentrations in her breast milk. Another woman breastfed a premature infant while taking bosentan and sildenafil and reported no adverse reactions. The dose ingested by the infant is far below the dose expected to treat the infant, and no adverse effects are anticipated on breastfed infants.
◉ Effects on Breastfed Infants
A 23-year-old woman with congenital heart disease and pulmonary hypertension received bosentan and sildenafil during pregnancy at an unknown dose. She continued to take these medications and warfarin postpartum. Her baby was delivered by cesarean section at 30 weeks of gestation, weighing 1.41 kg. According to the author, she breastfed for 11 weeks in the neonatal intensive care unit with “good results,” but the infant died at 26 weeks of gestation from respiratory syncytial virus infection. [2]
A woman who was breastfeeding her 21-month-old infant was treated for pulmonary hypertension with 20 mg sildenafil three times a day and 125 mg bosentan twice a day. The medication was started more than 6 months postpartum. The mother did not report any possible adverse reactions, serious health problems or hospitalizations in the infant from birth to 651 days postpartum, during which time the infant continued to be partially breastfed. [1]
◉ Effects on breastfeeding and breast milk
No relevant published information was found as of the revision date.

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Protein binding
The binding rate to plasma proteins (mainly albumin) is greater than 98%.

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Toxicity overview
Bosentan is well tolerated and the risk of toxicity is extremely low when patients are under appropriate monitoring. However, when used in combination with cyclosporine A, bosentan plasma concentrations increased 30-fold, leading to severe headache, nausea, and vomiting. No serious adverse reactions or toxicities were observed in these patients. During post-marketing surveillance, one male patient overdosed on 10,000 mg of bosentan, experiencing nausea, vomiting, hypotension, blurred vision, and sweating. The patient fully recovered after adequate blood pressure support.

[1]. Pathophysiology . 2003 Sep;9(4):249-256.

[2]. J Pharmacol Exp Ther . 1994 Jul;270(1):228-35.

Bosentan hydrate is a hydrate containing the bosentan molecule. Bosentan is a sulfonamide and pyrimidine derivative used as a dual endothelin receptor antagonist to treat pulmonary arterial hypertension (PAH) and systemic sclerosis. See also: Bosentan (note moved to). Bosentan is a sulfonamide, pyrimidine, and primary alcohol compound. It is an antihypertensive drug and an endothelin receptor antagonist. Bosentan is a dual endothelin receptor antagonist marketed by Actelion Pharmaceuticals under the brand name Tracleer. Bosentan treats PAH by blocking the action of endothelin molecules, which otherwise promote vasoconstriction and lead to hypertension. Anhydrous bosentan is an endothelin receptor antagonist. The mechanism of action of anhydrous bosentan is as an endothelin receptor antagonist, a cytochrome P450 3A inducer, and a cytochrome P450 2C9 inducer. Bosentan is an endothelin receptor antagonist used to treat pulmonary arterial hypertension (PAH). Elevated serum enzymes may occur during bosentan treatment, and in rare cases, clinically significant acute liver injury may occur. Bosentan is a sulfonamide-derived competitive specific endothelin receptor antagonist with a slightly higher affinity for endothelin A receptors than for endothelin B receptors. Bosentan blocks the action of endothelin 1 (a potent endogenous vasoconstrictor and bronchodilator) by binding to endothelin A and endothelin B receptors on endothelial cells and vascular smooth muscle cells. Bosentan can reduce pulmonary vascular resistance and systemic vascular resistance, and is particularly used to treat pulmonary hypertension. A sulfonamide and pyrimidine derivative, used as a dual endothelin receptor antagonist for the treatment of pulmonary hypertension and systemic sclerosis. Drug Indications For the treatment of pulmonary arterial hypertension (PAH) to improve exercise capacity and slow the rate of clinical exacerbation (for patients with WHO grade III or IV symptoms).
FDA Label
For the treatment of pulmonary arterial hypertension (PAH) of WHO functional class III to improve exercise capacity and symptoms. Proven effective in: primary (idiopathic and familial) PAH; PAH secondary to scleroderma without significant interstitial lung disease; PAH associated with congenital systemic-pulmonary shunt and Eisenmenger syndrome. Some improvement has also been shown in patients with PAH of WHO functional class II. Tracleer is also indicated for reducing the number of new fingertip ulcers in patients with systemic sclerosis and persistent fingertip ulcers. For the treatment of pulmonary arterial hypertension (PAH) to improve exercise capacity and symptoms in patients with World Health Organization (WHO) functional class III. Proven effective in: primary (idiopathic and familial) PAH; PAH secondary to scleroderma without significant interstitial lung disease; PAH associated with congenital systemic-pulmonary shunt and Eisenmenger syndrome. Some improvement has also been shown in patients with PAH of WHO functional class II. Stayveer is also indicated for reducing the number of new fingertip ulcers in patients with systemic sclerosis and persistent fingertip ulcers.
Treatment of interstitial pulmonary fibrosis, treatment of pulmonary hypertension, treatment of systemic sclerosis
Mechanism of action
Endothelin-1 (ET-1) is a neurohormone whose action is mediated by binding to ETA and ETB receptors in endothelial cells and vascular smooth muscle. It has a slightly higher affinity for ETA receptors than for ETB receptors. Elevated ET-1 concentrations in plasma and lung tissue of patients with pulmonary hypertension suggest that ET-1 plays a role in the pathogenesis of this disease. Bosentan is a specific competitive antagonist of endothelin receptors ETA and ETB.

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Pharmacodynamics
Bosentan belongs to the class of endothelin receptor antagonists (ERAs). Endothelin levels are elevated in the plasma and lung tissue of patients with pulmonary arterial hypertension (PAH), and endothelin is a potent vasoconstrictor. Bosentan blocks the binding of endothelin to its receptors, thereby eliminating the harmful effects of endothelin.

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Bosentan (Tracleer) is an oral dual endothelin-1 (ET-1) receptor antagonist approved for the treatment of patients with WHO grade II (mild symptomatic) pulmonary arterial hypertension (PAH). Oral bosentan treatment is beneficial in patients with mild symptomatic PAH and is generally well tolerated. In a well-designed placebo-controlled trial, researchers studied adolescent and adult patients with mild symptomatic pulmonary arterial hypertension (PAH) and found that bosentan significantly reduced pulmonary vascular resistance compared to placebo, but did not significantly increase 6-minute walking distance. Similarly, in a small, uncontrolled trial, bosentan also improved hemodynamic parameters in pediatric patients, most of whom had mild symptomatic PAH, but did not significantly improve exercise capacity. Adverse effects of bosentan are consistent with those observed in other indications, with the main concern being potential teratogenicity and hepatotoxicity, and therefore regular monitoring of liver function is recommended. Overall, given the progressive nature of PAH, bosentan provides more treatment options for patients with mild symptomatic PAH. [1]

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Introduction: Although the pathogenesis of systemic sclerosis (SSc) remains poorly understood, recent studies have shown that endothelin plays an important role in the development of SSc-related fibrosis and vascular lesions, and the dual endothelin receptor antagonist bosentan holds promise as a disease-modifying agent for this disease. Importantly, endothelin-1 (ET-1) has a pro-fibrotic effect on normal dermal fibroblasts, and bosentan can reverse the pro-fibrotic phenotype of SSc dermal fibroblasts. This study aimed to elucidate the molecular mechanisms by which ET-1 and bosentan act on dermal fibroblasts, mechanisms that are currently understudied. Methods: Reverse transcription real-time PCR and Western blotting were used to detect the mRNA levels of target genes and the expression and phosphorylation levels of target proteins. Promoter analysis was performed using the human α2(I) collagen (COL1A2) promoter sequence deletion. DNA affinity precipitation and chromatin immunoprecipitation techniques were used to assess the DNA-binding capacity of Fli1. Immunostaining was used to assess the expression level of Fli1 protein in mouse skin. Results: In normal fibroblasts, ET-1 activated c-Abl and protein kinase C (PKC)-δ, and induced phosphorylation of Fli1 at threonine 312, leading to a decrease in the DNA-binding capacity of Fli1 (a potent repressor of the COL1A2 gene) and an 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 the phosphorylation level of Fli1, leading to enhanced DNA-binding capacity of Fli1 and inhibiting the expression of type I collagen in systemic sclerosis (SSc) fibroblasts. In bleomycin-treated mice, bosentan prevented dermal fibrosis and increased Fli1 expression in diseased dermal fibroblasts. Conclusion: ET-1 exerts a potent pro-fibrotic effect on normal fibroblasts by activating the c-Abl-PKC-δ-Fli1 pathway. Bosentan reverses the pro-fibrotic phenotype of SSc fibroblasts and prevents the occurrence of dermal fibrosis in bleomycin-treated mice by blocking this signaling pathway. Although the efficacy of bosentan in treating SSc dermal and pulmonary fibrosis is limited, the results of this study undoubtedly provide useful clues for further exploring the potential of upcoming novel dual endothelin receptor antagonists as disease-modifying agents for SSc. [2]

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Objective: The endothelin (ET) system is a tissue system because the production of ET isoenzymes is primarily autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties, designed to achieve more effective ET receptor blockade. To determine whether these properties translate into improved efficacy in vivo, we designed a study in which rats with systemic or pulmonary hypertension wearing telemetry devices were given macitentan in addition to a maximally effective dose of another dual ETA/ETB receptor antagonist, bosentan (which does not have sustained receptor occupancy and has less tissue distribution). Main method: After establishing dose-response curves for the two compounds in conscious, salt-sensitive Dahl hypertensive rats and bleomycin-treated pulmonary hypertension rats, macitentan was given in addition to a maximally effective dose of bosentan. Main results: In hypertensive rats, the addition of 30 mg/kg macitentan to 100 mg/kg bosentan resulted in a further reduction of mean arterial pressure (MAP) of 19 mmHg (n=9, p<0.01 compared with the solvent control group). In contrast, the addition of bosentan to macitentan did not result in a further reduction of MAP. In the rat model of pulmonary hypertension, the addition of 30 mg/kg macitentan to bosentan resulted in a further reduction of mean pulmonary artery pressure (MPAP) of 4 mmHg (n=8, p<0.01 compared with the solvent group), while the addition of the maximum effective dose of bosentan did not result in a further reduction of MPAP. Significance: The additional effects of the combination of macitentan and bosentan in the two pathological models confirm that this novel compound can block endothelin receptors more effectively and provide evidence for its higher maximum efficacy. [3]

C27H31N5O7S

569.63

569.194

157212-55-0

Bosentan; 147536-97-8

185462

White to off-white solid powder

742.3ºC at 760 mmHg

402.8ºC

3.84E-23mmHg at 25°C

5.293

3

12

11

40

839

0

SXTRWVVIEPWAKM-UHFFFAOYSA-N

InChI=1S/C27H29N5O6S.H2O/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);1H2

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

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: ≥ 2.5 mg/mL (4.39 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 2: ≥ 2.5 mg/mL (4.39 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 3: ≥ 2.5 mg/mL (4.39 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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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.39 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.5 mg/mL (4.39 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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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 (Please use freshly prepared in vivo formulations for optimal results.)