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]. Okamoto T, et al. 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]. Lisk C, et al. Nrf2 activation: a potential strategy for the prevention of acute mountain sickness. Free Radic Biol Med. 2013 Oct;63:264-73

Aim: To examine the effects of the endothelin type A receptor antagonist ambrisentan on hepatic steatosis and fibrosis in a steatohepatitis mouse model.[1]
Methods: Fatty liver shionogi (FLS) FLS-ob/ob mice (male, 12 wk old) received ambrisentan (2.5 mg/kg orally per day; n = 8) or water as a control (n = 5) for 4 wk. Factors were compared between the two groups, including steatosis, fibrosis, inflammation, and endothelin-related gene expression in the liver.[1]
Results: In the ambrisentan group, hepatic hydroxyproline content was significantly lower than in the control group (18.0 μg/g ± 6.1 μg/g vs 33.9 μg/g ± 13.5 μg/g liver, respectively, P = 0.014). Hepatic fibrosis estimated by Sirius red staining and areas positive for α-smooth muscle actin, indicative of activated hepatic stellate cells, were also significantly lower in the ambrisentan group (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). Moreover, hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were significantly lower by 60% and 45%, respectively, in the ambrisentan group. Inflammation, steatosis, and endothelin-related mRNA expression in the liver were not significantly different between the groups.[1]
Conclusion: Ambrisentan attenuated the progression of hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing procollagen-1 and TIMP-1 gene expression. Ambrisentan did not affect inflammation or steatosis.[1]

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Reactive oxygen species (ROS) formed during acute high altitude exposure contribute to cerebral vascular leak and development of acute mountain sickness (AMS). Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) is a transcription factor that regulates expression of greater than 90% of antioxidant genes, but prophylactic treatment with Nrf2 activators has not yet been tested as an AMS therapy. We hypothesized that prophylactic activation of the antioxidant genome with Nrf2 activators would attenuate high-altitude-induced ROS formation and cerebral vascular leak and that some drugs currently used to treat AMS symptoms have an additional trait of Nrf2 activation. Drugs commonly used to treat AMS were screened with a luciferase reporter cell system for their effectiveness to activate Nrf2, as well as being tested for their ability to decrease high altitude cerebral vascular leak in vivo. Compounds that showed favorable results for Nrf2 activation from our screen and attenuated high altitude cerebral vascular leak in vivo were further tested in brain microvascular endothelial cells (BMECs) to determine if they attenuated hypoxia-induced ROS production and monolayer permeability. Of nine drugs tested, with the exception of dexamethasone, only drugs that showed the ability to activate Nrf2 (Protandim, methazolamide, nifedipine, amlodipine, ambrisentan, and sitaxentan) decreased high-altitude-induced cerebral vascular leak in vivo. In vitro, Nrf2 activation in BMECs before 24h hypoxia exposure attenuated hypoxic-induced hydrogen peroxide production and permeability. Prophylactic Nrf2 activation is effective at reducing brain vascular leak from acute high altitude exposures. Compared to acetazolamide, methazolamide may offer better protection against AMS. Nifedipine, in addition to its known vasodilatory activities in the lung and protection against high altitude pulmonary edema, may provide protection against brain vascular leak as well.[2]

C22H23N2NAO4

402.418796777725

400.139901

1386915-48-5

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

57520499

Typically exists as solids (or liquids in special cases) at room temperature

84.4Ų

C(C1C=CC=CC=1)(C1C=CC=CC=1)(OC)[C@@H](C(=O)O)OC1=NC(C)=CC(C)=N1.[NaH]

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.)