ETA (IC50 = 3.4 nM); ETA (pA2 = 6.7); ETB (IC50 = 987 nM); ETB (pA2 = 5.5)

When applied to nonrecombinant cells (main human pulmonary smooth muscle cells, rat aortic smooth muscle cell line A10, and mouse fibroblast cell line 3T3), aproticentan (ACT-132577) completely inhibits the intracellular calcium rise caused by ET-1[1].

In rats, aprobicitentan (ACT-132577) has a longer half-life than its parent chemical and a volume of distribution larger than the plasma volume[1]. The range of Aprocitentan (ACT-132577) mean recovery in rat plasma is 82.6 % to 90.6%, while the range of Aprocitentan (ACT-132577) matrix effect in rat plasma is 101.4% to 115.2%[2].

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A) receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.[1]

Calibration standards and quality control samples[2]
The stock solutions of ACT-132577 (1.0 mg/mL) and diazepam (IS) (100 µg/mL) were prepared in methanol-water (50:50, v/v). The 0.25 µg/mL working standard solution of the IS was prepared from the IS stock solution by dilution with methanol; working solutions for calibration and controls were prepared from stock solutions in the same manner. All of the solutions were stored at 4°C and were brought to room temperature before use.

ACT-132577 calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were offset to range between 10-4000 ng/mL for ACT-132577 in rat plasma (10, 20, 50, 100, 200, 500, 1000, 2000 and 4000 ng/mL). Quality-control (QC) samples were prepared in the same manner as the calibration standards, in three different plasma concentrations (20, 1800, and 3600 ng/mL). The analytical standards and QC samples were stored at -20°C.

The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma-spiked ACT-132577 and IS, and a rat plasma sample.

Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of ACT-132577-to-IS were plotted against analyte concentrations. Resultant standard curves were well fitted to the equations by linear regression, with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 10-4000 ng/mL. The lower Limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves.

Stability of ACT-132577 in rat plasma were evaluated by analyzing three replicates of plasma samples at concentrations of 20 or 3600 ng/mL which were all exposed to different conditions. These results were compared with the freshly-prepared plasma samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. Freeze/thaw stability was evaluated after three complete freeze/thaw cycles (-20 to 25°C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at -20°C for 20 days. The stability of the IS (50 ng/mL) was evaluated similarly

Pharmacokinetic study[2]
Twelve Male Sprague-Dawley rats (200-220 g) were used. The ethical number of the experiment animals was wydw2013-0071. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.2 mL) were collected from the caudal vein into heparinized 1.5 mL tapered plastic centrifuge tubes at 0.0333, 0.15, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral (15 mg/kg, n=6) and intravenous (5 mg/kg, n=6) administration of macitentan, respectively. The caudal vein of rat was cleaned by 75% alcohol, after that the end of caudal vein was cut by scissors. A 1.5 mL tapered plastic centrifuge tube was used to collect the blood which dropped from the end of caudal vein by squeezing and massaging gently. The samples were immediately centrifuged at 3000 × g for 10 min. The plasma as-obtained (50 µL) was stored at -20°C until UPLC-MS/MS analysis. Plasma ACT-132577 concentration versus time data for each rat was analyzed by DAS (Drug and Statistics) software.

Absorption
The absolute oral bioavailability of apracycltan is unknown. After a single oral dose of 25 mg, the mean Cmax and AUC0-tau were approximately 1.3 mcg/mL and 23 mcg·h/mL, respectively, with Tmax between 4 and 5 hours.
Elimination Route
After a single dose of radiolabeled apracycltan, approximately 52% of the dose is excreted in the urine (0.2% unchanged) and 25% in the feces (6.8% unchanged).
Volume of Distribution
The apparent volume of distribution of apracycltan is approximately 20 L.
Clearance
The apparent clearance of apracycltan is approximately 0.3 L/h.
Protein Binding
Apracycltan is highly bound to proteins in plasma (>99%), primarily albumin. Metabolites/Metabolites
Apraciltan is primarily metabolized via N-glycosylation and non-enzymatic hydrolysis mediated by UGT1A1 and UGT2B7.
Biological Half-Life
The effective half-life of apraciltan is approximately 41 hours.
Apraciltan is well tolerated at all doses. No serious adverse events (AEs) were reported. The most common adverse event was headache. Subjects taking 100 mg once daily experienced a slight increase in body weight. Plasma concentration-time curves for apraciltan were similar after single and multiple doses, based on a 44-hour half-life, supporting the once-daily dosing regimen. Pharmacokinetics were dose-proportional after multiple doses. A 3-fold accumulation was achieved at steady state on day 8. Only minor differences in exposure were observed between healthy women and men, healthy older adults and adult subjects, and between fasting and eating states. Plasma ET-1 concentrations reflected ETB receptor antagonism and were significantly elevated at doses ≥25 mg. Time-matched electrocardiogram (ECG) parameter analysis did not indicate a drug-induced ECG effect. Exposure-response analysis showed no QTc interval prolongation at plasma concentrations up to 10 µg/mL. (Drug Des Devel Ther. 2019; 13: 949–964)

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of apraciltan during lactation. Because apraciltan binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk is likely to be low. However, its half-life is 41 hours, which may allow it to accumulate in the infant. Since there is currently no information regarding the use of apraciltan during lactation, alternative medications are recommended, especially for breastfed newborns or premature infants.
◉ Effects on Breastfed Infants
No published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information was found as of the revision date.

[1]. Pharmacology of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist. J Pharmacol Exp Ther. 2008 Dec;327(3):736-45.

[2]. Pharmacokinetic study of ACT-132577 in rat plasma by ultra performance liquid chromatography-tandem mass spectrometry. Int J Clin Exp Med. 2015 Oct 15;8(10):18420-6.

ACT-132577 belongs to the sulfonamide class of compounds, in which one amino group of the sulfonamide group is replaced by 5-(4-bromophenyl)-6-{2-[(5-bromopyrimidin-2-yl)oxy]ethoxy}pyrimidin-4-yl. It is the active metabolite of macitentan (an orphan drug for the treatment of pulmonary arterial hypertension, prepared by oxidative depropanolization). ACT-132577 has multiple functions, including antihypertensive activity, endothelin receptor antagonism, drug metabolism, and exogenous substance metabolism. It is an aromatic ether, an organic bromine compound, belonging to the pyrimidine and sulfonamide classes. Functionally, it is related to ethylene glycol. Aprocitentan is being investigated in the clinical trial NCT03541174 (a study aimed at demonstrating the efficacy of Aprocitentan in the treatment of refractory hypertension and further understanding its safety).

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Drug Indications
Treatment of hypertension.
Macitentan, also known as Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)pyrimidin-4-yl]-N'-propylaminosulfonamide], is a novel dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for tissue-targeted therapy. Macitentan was chosen based on its inhibitory potency against ET receptors and optimized physicochemical properties for high affinity to a lipophilic environment. In vivo, macitenantan is metabolized to a major, pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonize ET-1 by specifically binding to the cell membranes of cells overexpressing ET(A) and ET(B) receptors and by attenuating ET-1-induced calcium mobilization in a variety of native cell lines, with inhibitory constants in the nanomolar range. In functional studies, macitentan and ACT-132577 inhibited ET-1-induced contraction of isolated endothelin-free rat aorta (ET(A) receptor) and S6c-induced contraction of isolated rat trachea (ET(B) receptor). In a rat model of pulmonary hypertension, macitentan prevented pulmonary artery pressure elevation and right ventricular hypertrophy and significantly improved survival. In a diabetic rat model, long-term administration of macitentan reduced blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural damage). In conclusion, macitentan, with its tissue-targeting properties and dual antagonistic effect against endothelin receptors, can protect diabetic rats from end-organ damage and improve the survival rate of rats with pulmonary hypertension. These properties make macitentan a novel drug for the treatment of cardiovascular diseases associated with chronic activation of the tissue endothelin system. [1]

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It has been reported that macitentan is mainly metabolized by cytochrome P450 3A4, and its pharmacologically active metabolite ACT-132577 has a five-fold lower potency in blocking endothelin receptors than macitentan. In this study, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the determination of ACT-132577 in rat plasma. Diazepam was used as an internal standard (IS), and samples were prepared by acetonitrile precipitation. Chromatographic separation was performed on a UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) with 0.2% formic acid and methanol as the mobile phase and gradient elution. Electrospray ionization was used in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification, with target fragment ions at m/z 546.9→200.6 (ACT-132577) and m/z 285.1→193.1 (internal standard). In rat plasma, the calibration curve for ACT-132577 was linear in the range of 10–4000 ng/mL. The mean recovery of ACT-132577 in rat plasma was 82.6%–90.6%, with matrix effects ranging from 101.4% to 115.2%. The relative standard deviations (RSDs) for intra-day and inter-day precision were both less than 11%. The accuracy of the method was 96.1%–103.5%. This method has been successfully applied to pharmacokinetic studies of ACT-132577 after oral and intravenous administration of macitentan. [2]

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Apracitentan is a dual antagonist of endothelin receptors A and B, used to treat refractory hypertension. It is the active metabolite of macitentan. Approximately 10-15% of hypertensive patients suffer from refractory hypertension, defined as blood pressure that remains uncontrolled despite combined use of renin-angiotensin system blockers, calcium channel blockers, and diuretics (all at maximum tolerated doses). Patients with refractory hypertension have an increased risk of cardiovascular and renal events, and traditionally, treatment options are limited. Endothelin receptor antagonists offer a new avenue for treating refractory hypertension. In March 2024, the FDA approved aprocitentan for the treatment of hypertension that is poorly controlled with standard therapy. It is the first antihypertensive drug with a novel mechanism of action approved in nearly 40 years. Aprocitentan is an endothelin receptor antagonist. Its mechanism of action is endothelin receptor antagonism. Aprocitentan is a small molecule drug that has completed the most Phase IV clinical trials (covering all indications) and was first approved in 2024 for the treatment of hypertension, with three investigational indications. The drug has received a black box warning from the U.S. Food and Drug Administration (FDA).
Macipran Metabolites
Pharmacodynamics
Apracipitan exerts its pharmacological effects by antagonizing ETA and ETB receptors, which play a role in the pathogenesis of hypertension. In the PRECISION trial, apracipitan was superior to placebo in reducing both sitting systolic and diastolic blood pressure, with an average reduction in sitting trough blood pressure of approximately 4 mmHg greater than placebo. Most of the antihypertensive effects of apracipitan are observed within the first two weeks of treatment. Based on animal reproductive studies of other endothelin receptor antagonists, apracipitan administration during pregnancy may cause fetal harm. Pregnancy should be ruled out and effective contraception should be used before starting apracipitan treatment. Patients should monitor their pregnancy monthly and use effective contraception during treatment and for one month after discontinuation. Due to the significant risk of embryo-fetal toxicity, apracipitan is only available through the restricted program called Tryvio REMS.
Mechanism of Action
Endothelin-1 (ET-1) is the major endothelin subtype in the human cardiovascular system. Endothelin-1 (ET-1) is constitutively produced by vascular endothelial cells to maintain vascular tone and is present in a variety of other cells, including vascular smooth muscle cells, cardiomyocytes, fibroblasts, macrophages, neurons, and epithelial cells of the lungs and kidneys. ET-1 acts on two receptors located on vascular smooth muscle cells and endothelial cells: ETA and ETB, which regulate blood pressure by inducing vasoconstriction or vasodilation. ET-1 is a potent vasoconstrictor, primarily acting through interaction with the ETA receptor; under pathological conditions, ET-1 can further induce vasoconstriction through interaction with ETB2. Overexpression of ET-1 and its receptors has been confirmed in various pathological conditions, including essential hypertension, pulmonary hypertension, chronic kidney disease, and diabetes. Alprazolam is a dual endothelin receptor antagonist that inhibits the binding of ET-1 to both ETA and ETB receptors. This inhibition mitigates the hypertensive effects of ET-1 overexpression, including endothelial dysfunction, vascular hypertrophy and remodeling, sympathetic activation, and increased aldosterone synthesis.

C₁₆H₁₀D₄BR₂N₆O₄S

546.194

543.916

C, 35.18; H, 2.58; Br, 29.26; N, 15.39; O, 11.72; S, 5.87

1103522-45-7

Aprocitentan-d4

25099191

White to off-white solid powder

4.385

2

10

8

29

597

0

C1=CC(=CC=C1C2=C(N=CN=C2OCCOC3=NC=C(C=N3)Br)NS(=O)(=O)N)Br

DKULOVKANLVDEA-UHFFFAOYSA-N

InChI=1S/C16H14Br2N6O4S/c17-11-3-1-10(2-4-11)13-14(24-29(19,25)26)22-9-23-15(13)27-5-6-28-16-20-7-12(18)8-21-16/h1-4,7-9H,5-6H2,(H2,19,25,26)(H,22,23,24)

N-[5-(4-bromophenyl)-6-{2-[(5-bromopyrimidin-2-yl)oxy]ethoxy}pyrimidin-4-yl]sulfuric diamide

ACT 132577; ACT-132577; Aprocitentan; ACT132577; N-Despropyl-macitentan; Tryvio; Aprocitentan [USAN]; Macitentan metabolite m6; MZI81HV01P; Despropyl Macitentan.

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)

DMSO : ~25 mg/mL (~45.77 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.58 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 (4.58 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.)