ETB (endothelin receptor type B) (Ki = 16 pM)

At 19 μM for ETA and 16 PM for ETB, respectively, Ki, IRL-1620(TFA) is the most powerful and specific ETB ligand (KiETA/KiETB=120,000) [1]. With a KiETA/KiETB ratio of 1,900, IRL-1620(TFA) ET-3 is 60 times more selective for ETB receptors than it is [1].

In guinea pigs, IRL-1620 (TFA) (1-100 nM) causes tracheoconstriction. About 28 nM is the effective concentration of IRL-1620 required to cause 30% of the contraction induced by 60 mM KCI [1]. IRL-1620 (TFA) (1-100 nM) raises cytosolic Ca2+ in vascular endothelium ([Ca]E) in rat aorta and relaxes norepinephrine-stimulated tone while having minimal effect on resting muscle tone [1]. IRL-1620 (TFA) increases angiogenesis and neurogenic remodeling while improving the water maze task's acquisition (learning) and remembering (memory). Aβ-induced cognitive impairment was dramatically lessened in rats treated with IRL-1620. Comparing IRL-1620 treatment to vehicle treatment, there was a rise in the number of vessels identified with VEGF [2].

A series of C-terminal linear peptides of endothelin (ET)-1 and their Nα-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9, Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor as judged by the Ki values for ETA (1.9 μM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3. IRL 1620 (10−9–10−7 M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist. [1]

Estimation of ETB, VEGF and NGF using Western blot [2]
ETB, vascular endothelial and neuronal growth factor levels in the brain were measured via Western blotting. Animals were decapitated and the brains were flash frozen and stored at −80 °C. The tissue was homogenized in RIPA buffer (20 mM Tris–HCl pH 7.5, 120 mM NaCl, 1.0% Triton X100, 0.1% SDS, 1% sodium deoxycholate, 10% glycerol, 1 mM EDTA and 1× protease inhibitor). Proteins were isolated in solubilized form and concentrations were determined using Folin–Ciocalteu’s Reagent (Lowry et al., 1951). Protein (60 μg) was denatured in Laemmli sample buffer, resolved in 10% SDS–PAGE and transferred on a nitrocellulose membrane. The membrane was then blocked with 5% BSA (w/v) in TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20) for 30 min at room temperature. The membranes were incubated with rabbit polyclonal anti-ETB (1:1000; Abcam, Cambridge, MA, USA), anti-VEGF (1:1000) and anti-NGF antibodies (1:500) at 4 °C overnight, followed by 1.5-h incubation with goat anti-rabbit IgG, horseradish peroxidase-conjugated (HRP) secondary antibody (1:2000) at room temperature. β-Actin (1:10,000) and β-tubulin (1:2000) was used as a loading control.

Animals were randomly divided into five groups (six rats per group) (i) Sham, (ii) Aβ + Vehicle, (iii) Aβ + IRL 1620, [iv] Aβ + BQ788 (v) Aβ + BQ788 + IRL 1620. Aβ1–40 was administered intracerebroventricularly (i.c.v.) (20 μg in three equally divided doses i.e., 6.67 μg was injected three times for a total of 20-μg dose) on day 1, 7, and 14. We have used Aβ (1–40) because it is highly soluble compared to Aβ (1–42) and induces endothelial dysfunction of both cerebral and systemic blood vessels in addition to memory deficit (Weller et al., 1998, Niwa et al., 2000, Smith et al., 2004). Specific ETB receptor agonist, IRL 1620 (5 μg/kg) and specific ETB receptor antagonist, BQ788 (1 mg/kg) were administered intravenously (i.v.) on day 8. IRL 1620 was administered on day 8 three times at a dose of 5 μg/kg, i.v. at 2-h intervals between each injection. BQ788 was administered at a dose of 1-mg/kg, i.v., 15 min prior to administration of either vehicle or IRL 1620. The doses of IRL 1620 and BQ788 were based on preliminary studies and previous work conducted in our laboratory (Leonard et al., 2011, Leonard et al., 2012). [2]
IRL 1620[N-Succinyl-[Glu9, Ala11,15] endothelin 1] and BQ788 were dissolved in sterile saline and all the solutions were freshly prepared before the injections.

[1]. A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor. Biochem Biophys Res Commun. 1992 Apr 30;184(2):953-9.

[2]. Stimulation of endothelin B receptors by IRL-1620 decreases the progression of Alzheimer's disease. Neuroscience. 2015 Aug 20;301:1-11.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment that ultimately leads to death. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies in our lab have shown that stimulation of ETB receptors provide significant neuroprotection following Aβ1-40 administration. It is possible that IRL-1620 may be neuroprotective due to angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling following Aβ1-40 administration has not been established. The purpose of this study was to determine the effect of stimulation of ETB receptors by IRL-1620 on vascular and neuronal growth factors after Aβ1-40 administration. Rats were treated with Aβ1-40 (day 1, 7 and 14) in the lateral cerebral ventricles using stereotaxically implanted cannula and received three intravenous injections of IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) at 2-h interval on day 8; experiments were performed on day 15. Rats were sacrificed for estimation of brain ETB receptors, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) expression using immunofluorescence and Western blot. In the Morris swim task, amyloid-β (Aβ)-treated rats showed a significant (p<0.0001) impairment in spatial memory. Rats treated with IRL-1620 significantly (p<0.001) reduced the cognitive impairment induced by Aβ. BQ788 treatment completely blocked IRL-1620-induced improvement in cognitive impairment. IRL-1620 treatment enhanced the number of blood vessels labeled with VEGF compared to vehicle treatment. Additionally, cells showed increased (p<0.001) positive staining for NGF in IRL-1620-treated animals. ETB, VEGF and NGF protein expression significantly (p<0.001) increased in the brain of IRL-1620-treated rats as compared to vehicle. Pretreatment with BQ788 blocked the effects of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling actions of IRL-1620. Results of the present study demonstrate that IRL-1620 improves both acquisition (learning) and retention (memory) on the water maze task and enhances angiogenic and neurogenic remodeling. These findings indicate that the ETB receptor may be a novel therapeutic target for AD and other neurovascular degenerative disorders.[2]

C88H118F3N17O29

1934.97

142569-99-1 (IRL-1620)

{Suc}-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp

{Suc}-DEEAVYFAHLDIIW

White to off-white solid powder

(2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,32S,35S,38S,41S)-17-((1H-imidazol-5-yl)methyl)-2-((1H-indol-3-yl)methyl)-23-benzyl-5,8-di((S)-sec-butyl)-35,38-bis(2-carboxyethyl)-11,41-bis(carboxymethyl)-26-(4-hydroxybenzyl)-14-isobutyl-29-isopropyl-20,32-dimethyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43-tetradecaoxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaazahexatetracontanedioic acid TFA salt

IRL-1620 TFA; sovateltide TFA; sovateltidum; Endothelin-1 (8-21), succinyl-(glu(9),ala(11,15))-; Succinyl-(glu(9),ala(11,15))-endothelin-1 (8-21); Succinyl-(glutamyl(9)-alanyl(11,15))-endothelin-1 (8-21); IRL1620 TFA.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~50 mg/mL (~25.84 mM)

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.)