ETA receptor

Ambrisentan sodium is an antagonist of the endothelin type A receptor [1]. Nrf2 is activated by ambrisentan sodium. Ambrisentan reduced hypoxia-induced BMEC leakage in comparison to vehicle control, and endothelial permeability of BMEC monolayers increased following a 24-hour exposure to hypoxia. When BMEC were transfected with siRNA targeting Nrf2 prior to treatment, these outcomes were reversed [2].

Liver hydroxyproline concentration was considerably lower in the Ambrisentan group (18.0 μg/g±6.1 μg/g versus 33.9 μg/g±13.5 μg/g liver, respectively, P=0.014) than in the control group. The ambrisentan group exhibited a significant reduction in both liver fibrosis and α-smooth muscle actin-positive areas, as determined by Sirius red staining, which indicates hepatic stellate cell activation (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 exhibited a considerable reduction in the liver RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) by 60% and 45%, respectively. In the liver, there were no appreciable differences in steatosis, inflammation, or endothelin-related mRNA expression across the groups. By lowering proollagen-1 and TIMP-1 gene expression and preventing the activation of hepatic stellate cells, ambrisentanodium slows the development of liver fibrosis. Steatosis and inflammation are unaffected by ambrisentan sodium [1].

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].

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.

[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.

Objective: To investigate the effects of ambrisentan, an endothelin A receptor antagonist, on hepatic steatosis and fibrosis in a mouse model of fatty liver. [1] Methods: Male Shionogi (FLS) FLS-ob/ob mice (12 weeks old) with fatty liver were randomly divided into two groups, one group was given ambrisentan (2.5 mg/kg, once daily, n = 8) and the other group was given water as a control (n = 5) for 4 weeks. The hepatic steatosis, fibrosis, inflammation and endothelin-related gene expression were compared between the two groups. [1] Results: The hydroxyproline content in the liver of mice in the ambrisentan group was significantly lower than that in the control group (18.0 μg/g ± 6.1 μg/g and 33.9 μg/g ± 13.5 μg/g, respectively, P = 0.014). The degree of liver fibrosis assessed by Sirius red staining and the positive area of α-smooth muscle actin (suggesting hepatic stellate cell activation) were also significantly reduced in the ambrisentan group (0.46% ± 0.18% vs 1.11% ± 0.28%, P = 0.0003; and 0.12% ± 0.08% vs 0.25% ± 0.11%, P = 0.047, respectively). In addition, the RNA expression levels of type I procollagen and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the liver of the ambrisentan group were significantly reduced by 60% and 45%, respectively. There were no significant differences in liver inflammation, steatosis and endothelin-related mRNA expression among the groups. [1] Conclusion: Ambrisentan slows the progression of liver fibrosis by inhibiting hepatic stellate cell activation and reducing the expression of type I procollagen and TIMP-1 genes. Ambrisentan has no effect on inflammation or steatosis. [1]

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Reactive oxygen species (ROS) generated during acute high-altitude exposure can lead to cerebrovascular leakage and acute mountain sickness (AMS). Nuclear factor-associated factor 2 (Nrf2) is a transcription factor that regulates the expression of more than 90% of antioxidant genes, but the use of Nrf2 activators for prophylactic treatment as an AMS therapy has not been validated. We hypothesized that prophylactic activation of the antioxidant genome using Nrf2 activators could alleviate high-altitude-induced ROS generation and cerebrovascular leakage, and that some drugs currently used to treat symptoms of acute mountain sickness (AMS) have the additional property of activating Nrf2. We screened the efficacy of commonly used AMS therapies in activating Nrf2 using a luciferase reporter gene cell system and tested their ability to reduce high-altitude cerebrovascular leakage in vivo. Compounds that showed good Nrf2 activation during screening and could reduce high-altitude cerebrovascular leakage in vivo were further used in brain microvascular endothelial cell (BMEC) experiments to determine whether they could reduce hypoxia-induced ROS generation and monolayer cell permeability. Of the nine drugs tested, only those that activated Nrf2 (Protandim, mezolaxidine, nifedipine, amlodipine, ambrisentan, and sitassentan) reduced high-altitude-induced cerebrovascular leakage in vivo, except for dexamethasone. In vitro experiments showed that activating Nrf2 in brain microvascular endothelial cells (BMEC) 24 hours before hypoxia exposure attenuated hypoxia-induced hydrogen peroxide generation and permeability. Prophylactic activation of Nrf2 effectively reduced cerebrovascular leakage caused by acute high-altitude exposure. Compared with acetazolamide, mezolaxidine may provide better protection against acute mountain sickness (AMS). In addition to its known pulmonary vasodilatory effects and prevention of high-altitude pulmonary edema, nifedipine may also have a protective effect against cerebral vascular leakage. [2]

C22H23N2NAO4

402.418796777725

400.139

1386915-48-5

Ambrisentan;177036-94-1;Ambrisentan-d10;1046116-27-1

57520499

Typically exists as solid at room temperature

0

6

7

29

481

1

CC1=CC(=NC(=N1)O[C@H](C(=O)[O-])C(C2=CC=CC=C2)(C3=CC=CC=C3)OC)C.[Na+]

GNDMILPGCDIGHE-FSRHSHDFSA-M

InChI=1S/C22H22N2O4.Na/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);/q;+1/p-1/t19-;/m1./s1

sodium;(2S)-2-(4,6-dimethylpyrimidin-2-yl)oxy-3-methoxy-3,3-diphenylpropanoate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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