Ambrisentan (formerly BSF-208075; LU-208075; BSF208075; LU208075; Letairis; Volibris; pulmonext) is a selective antagonist of the endothelin-1 type A receptor (ETA). For the treatment of pulmonary hypertension, ambrisentan has received FDA approval.

Physicochemical Properties

Molecular Formula	C22H22N2O4
Molecular Weight	378.42
Exact Mass	378.158
Elemental Analysis	C, 69.83; H, 5.86; N, 7.40; O, 16.91
CAS #	177036-94-1
Related CAS #	Ambrisentan sodium; 1386915-48-5; Ambrisentan-d10; 1046116-27-1
PubChem CID	6918493
Appearance	White to off-white solid powder
Density	1.228g/cm3
Boiling Point	551.1ºC at 760mmHg
Melting Point	>150°C (dec.)
Flash Point	287.1ºC
Vapour Pressure	5.56E-13mmHg at 25°C
Index of Refraction	1.593
LogP	3.515
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	7
Heavy Atom Count	28
Complexity	475
Defined Atom Stereocenter Count	1
SMILES	O=C([C@H](C(OC)(C1=CC=CC=C1)C2=CC=CC=C2)OC3=NC(C)=CC(C)=N3)O
InChi Key	OUJTZYPIHDYQMC-LJQANCHMSA-N
InChi Code	InChI=1S/C22H22N2O4/c1-15-14-16(2)24-21(23-15)28-19(20(25)26)22(27-3,17-10-6-4-7-11-17)18-12-8-5-9-13-18/h4-14,19H,1-3H3,(H,25,26)/t19-/m1/s1
Chemical Name	(2S)-2-(4,6-dimethylpyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoic acid
Synonyms	LU-208075; BSF-208075; BSF208075; LU208075; BSF 208075; Letairis; Volibris; LU-208075; BSF-208075; (S)-2-(4,6-Dimethylpyrimidin-2-yloxy)-3-methoxy-3,3-diphenylpropanoic acid; (S)-2-((4,6-Dimethylpyrimidin-2-yl)oxy)-3-methoxy-3,3-diphenylpropanoic acid; LU 208075; trade name Letairis; Volibris; pulmonext
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	ETA receptor
ln Vitro	In vitro activity: Ambrisentan is an antagonist of the endothelin type A receptor [1]. Nrf2 activation is induced by ambrisentan. After BMEC monolayers were exposed to hypoxia for 24 hours, endothelial permeability increased, and ambrisentan reduced hypoxia-induced BMEC leakage when compared to the vehicle control. When siRNA targeting Nrf2 is transfected into BMEC prior to treatment, these outcomes are reversed[2].
ln Vivo	The liver hydroxyproline content is significantly lower in the Ambrisentan group (18.0 μg/g±6.1 μg/g vs 33.9 μg/g±13.5 μg/g liver, respectively, P=0.014) than in the control group. The Ambrisentan group also showed significantly reduced levels of hepatic fibrosis as determined by Sirius red staining and areas positive for α-smooth muscle actin, a marker of activated hepatic stellate cells (0.46%±0.18% vs 1.11%±0.28%, respectively, P=0.0003; and 0.12%±0.08% vs 0.25%±0.11%, respectively, P=0.047). Furthermore, the Ambrisentan group exhibits a significant 60% and 45% decrease in hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) respectively. There are no appreciable differences in the groups' liver inflammation, steatosis, or endothelin-related mRNA expression. By preventing hepatic stellate cell activation and lowering procollagen-1 and TIMP-1 gene expression, ambrisentan slows the advancement of hepatic fibrosis. Ambrisentan had no effect on steatosis or inflammation[1].
Cell Assay	Cells are randomly assigned to four groups for every BMEC experiment, unless otherwise specified: (1) normoxia vehicle control (Nx-CTRL); (2) normoxia-treated; (3) hypoxia (24 h) control (Hx-CTRL); and (4) hypoxia (24 h) treated. Nrf2 activators are added 24 hours before any hypoxic exposures, as previously mentioned. Protandim (100 μg/mL), methazolamide (125 μg/mL), nifedipine (7 μg/mL), or ambrisentan (40 μg/mL) are the cell treatments. Additionally, Nrf2 siRNA is applied to a subset of cells. In these tests, siRNA is added 24 hours before medication administration. The purpose of the 24-hour hypoxia exposure for BMEC is to guarantee that the cells maintain their siRNA transfection both during the 24-hour hypoxia exposure and during the drug pre-treatment (24 hours in normoxia). On three different days (n=9), data is gathered from a minimum of three distinct cell culture preparations[2].
Animal Protocol	Mice: The experimental group consists of thirteen male FLS-ob/ob mice, weighing 42.88 g±1.74 g and aged 8 weeks. Male FLS-ob/ob mice are randomized at random to either the control (n = 5) or Ambrisentan (n = 8) group at 12 weeks or older. When a conscious animal has a gastric tube that is the right size, intragastric gavage is administered. Through the use of a gastric tube, ambrisentan (2.5 mg/kg daily) is given orally as a bolus every afternoon for four weeks. The group under control receives water treatment. The fourth week involves fasting the animals for four hours, drawing blood from the tail vein, and testing their blood glucose levels. Blood is extracted from the right ventricle and the animals are put to death after four weeks by injection with pentobarbital anesthesia. Plasma samples are kept at -80°C in a frozen state. The fat from the liver and viscera is then weighed, liquid nitrogen-snap frozen, and kept at -80°C for storage. Further liver specimens are embedded in paraffin and fixed in 10% buffered formalin for histological examination.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Ambrisentan is rapidly absorbed with peak plasma concentrations occuring around 2 hours after oral administration. Cmax and AUC increase proportionally with dose across the therapeutic dosing range. Absolute oral bioavailability of ambrisentan is unknown. Absorption is not affected by food. Ambrisentan is primarily cleared by non-renal pathways. Along with its metabolites, ambrisentan is primarily found in the feces following hepatic and/or extra-hepatic metabolism. Approximately 22% of the administered dose is recovered in the urine following oral administration with 3.3% being unchanged ambrisentan. Ambrisentan has a low distribution into red blow cells, with a mean blood:plasma ratio of 0.57 and 0.61 in males and females, respectively. The mean oral clearance of ambrisentan was found to be 38 mL/min in healthy subjects and 19 mL/min in patients with pulmonary artery hypertension. Metabolism / Metabolites Ambrisentan is a metabolized primarily by uridine 5’-diphosphate glucuronosyltransferases (UGTs) 1A9S, 2B7S,1A3S to form ambrisentan glucuronide. Ambrisentan is also metabolized to a lesser extent by CYP3A4, CYP3A5 and CYP2C19 to form 4- hydroxymethyl ambrisentan which is further glucuronidated to 4-hydroxymethyl ambrisentan glucuronide. Biological Half-Life Ambrisentan has a terminal half-life of 15 hours. It is thought that steady state is achieved after around 4 days of repeat-dosing.
Toxicity/Toxicokinetics	Hepatotoxicity Ambrisentan is associated with a low rate of serum aminotransferase elevations (0% to 3%) that in clinical trials was similar to the rate in placebo recipients. These elevations are usually mild (rarely above 3 times ULN), transient and not associated with symptoms. For these reasons, monthly monitoring of serum aminotransferase levels is no longer routinely recommended during ambrisentan therapy. There have also been no published reports of clinically apparent liver injury with jaundice associated with ambrisentan, but it has had limited general use. Other endothelin receptor antagonists (bosentan, sitaxsentan) have been linked to cases of acute liver injury, some of which have been severe. The onset of illness was usually within 1 to 6 months of starting bosentan and the enzyme pattern was typically hepatocellular or mixed. Immunoallergic features were usually not present and autoantibodies absent or present in low titer. Sitaxsentan was linked to several cases of fatal acute liver failure, for which reason it was not approved in the United States and was later withdrawn from use elsewhere. Ambrisentan has not been linked to similar cases and its chemical structure is sufficiently different to suggest lack of cross sensitivity to this complication. Likelihood score: E (unlikely cause of clinically apparent liver injury). Protein Binding Ambrisentan is 99% plasma protein bound, primarily to albumin (96.5%) and to a lesser degree alpha1-acid glycoprotein.
References	[1]. Antifibrotic effects of Ambrisentan, an endothelin-A receptor antagonist, in a non-alcoholic steatohepatitis mouse model. World J Hepatol. 2016 Aug 8;8(22):933-41. [2]. Nrf2 activation: a potential strategy for the prevention of acute mountain sickness. Free Radic Biol Med. 2013 Oct;63:264-73.
Additional Infomation	Pharmacodynamics Ambrisentan 10 mg daily had no significant effect on the QTc interval, whereas a 40 mg daily dose of ambrisentan increased mean QTc at tmax by 5 ms with an upper 95% confidence limit of 9 ms. Significant QTc prolongation is not expected in patients taking ambrisentan without concomitant metabolic inhibitors. Plasma concentrations of B-type natriuretic peptide (BNP) in patients who received ambrisentan for 12 weeks were significantly decreased. Two Phase III placebo-controlled studies demonstrated a decrease in BNP plasma concentrations by 29% in the 2.5 mg group, 30% in the 5 mg group, and 45% in the 10 mg group (p < 0.001 for each dose group) and an increase by 11% in the placebo group.

Solubility Data

Solubility (In Vitro)

DMSO: 76~100 mg/mL (200.8~264.3 mM) Water: <1 mg/mL Ethanol: 7.1~22 mg/mL (18.9~58.1 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.61 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.61 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. Solubility in Formulation 3: ≥ 0.71 mg/mL (1.88 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 7.1 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. Solubility in Formulation 4: ≥ 0.71 mg/mL (1.88 mM) 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 7.1 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well. 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. Solubility in Formulation 5: 10% EtOH + 90% Corn Oil Solubility in Formulation 6: 12.5 mg/mL (33.03 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6426 mL	13.2128 mL	26.4257 mL
	5 mM	0.5285 mL	2.6426 mL	5.2851 mL
	10 mM	0.2643 mL	1.3213 mL	2.6426 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.