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

Based on the ETA (19 μM) and ETB (16 PM) receptors' Ki values, it can be concluded that IRL-1620 is the most selective and efficacious ligand for ETB receptors (KiETA/KiETB=120,000) [1]. Compared to ET-3 (KiETA/KiETB=1,900), IRL-1620 is 60 times more selective for ETB receptors[1].

In guinea pigs, IRL-1620 (1-100 nM) causes tracheoconstriction. The effective concentration for IRL 1620 was calculated to be 28 nM in order to cause 30% of the contraction induced by a 60 mM KCI [1]. As [Ca]E rises Tension in the rat aorta, IRL-1620 (1-100 nM) relaxes norepinephrine-stimulated muscle tone and raises cytosolic Ca2+ in the vascular endothelium ([Ca]E), having no influence on resting muscle tone [1]. IRL-1620 increases angiogenesis and neuronal remodeling while also improving learning and memory retention on a water maze task. 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.

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The in vivo efficacy of IRL-1620 was evaluated in a rat model of AD. Male Sprague-Dawley rats (4-5 months old) were used. An Alzheimer's disease-like condition was induced by intracerebroventricular (i.c.v.) administration of amyloid-β (Aβ₁₋₄₀, 20 μg total dose) in three equally divided doses (6.67 μg each) on days 1, 7, and 14 via a pre-implanted cannula. On day 8, IRL-1620 was administered intravenously (i.v.) at a dose of 5 μg/kg, three times, with a 2-hour interval between each injection. For groups receiving the ETB antagonist, BQ788 (1 mg/kg, i.v.) was administered 15 minutes prior to the first dose of IRL-1620 or vehicle. All drugs were dissolved in sterile saline and prepared fresh before injections. Behavioral testing using the Morris water maze was performed from day 15 to day 19. For biochemical and immunofluorescence analyses, animals were euthanized on day 15 without undergoing behavioral testing. [2]

[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 disease characterized by severe cognitive impairment, ultimately leading to death. Endothelin (ET) and its receptors are considered therapeutic targets for AD. Recent studies in our laboratory have shown that stimulation of ETB receptors provides significant neuroprotective effects after Aβ1-40 administration. IRL-1620 may exert its neuroprotective effects by promoting angiogenesis. However, the effect of IRL-1620 on neurovascular remodeling after Aβ1-40 administration remains unclear. This study aimed to investigate the effects of IRL-1620 stimulation of ETB receptors on angiogenesis and neuronal growth factors after Aβ1-40 administration. On days 1, 7, and 14, Aβ1-40 was injected into the lateral ventricle of rats via stereotactic cannula; on day 8, IRL-1620 (an ETB agonist) and/or BQ788 (an ETB antagonist) were administered intravenously three times every 2 hours; the experiment was conducted on day 15. Rats were sacrificed, and the expression of ETB receptor, vascular endothelial growth factor (VEGF), and nerve growth factor (NGF) in the brain was detected by immunofluorescence and Western blot. In the Morris swimming task, Aβ-treated rats exhibited significant spatial memory impairment (p<0.0001). IRL-1620 treatment significantly alleviated Aβ-induced cognitive impairment (p<0.001). BQ788 treatment completely blocked the improvement in cognitive impairment induced by IRL-1620. Compared with the vector control group, the number of VEGF-labeled vessels was significantly increased in the IRL-1620 treatment group. Furthermore, NGF-positive staining in the cells of the animals in the IRL-1620 treatment group was significantly enhanced (p<0.001). Compared with the vector control group, the expression of ETB, VEGF, and NGF proteins in the brains of rats in the IRL-1620 treatment group was significantly increased (p<0.001). Pretreatment with BQ788 blocked the effect of IRL-1620, thus confirming the role of ETB receptors in the neurovascular remodeling of IRL-1620. The results of this study indicate that IRL-1620 can improve learning and memory in the water maze task and promote angiogenesis and neurogenic remodeling. These findings suggest that ETB receptors may be a novel therapeutic target for Alzheimer's disease (AD) and other neurovascular degenerative diseases. [2]

C86H117N17O27

1820.97

1819.83

C, 56.72; H, 6.48; N, 13.08; O, 23.72

142569-99-1

IRL-1620 TFA

16130933

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

{Suc}-DEEAVYFAHLDIIW

Typically exists as solid at room temperature

1.3±0.1 g/cm3

2096.6±65.0 °C at 760 mmHg

1221.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.593

4.15

23

28

56

130

3920

16

[Suc-DEEAVYFAHLDIIW]

DXPHNGAMYPPTBJ-TZMIJSMNSA-N

InChI=1S/C86H117N17O27/c1-11-44(7)71(84(127)100-63(86(129)130)35-50-39-88-54-21-17-16-20-53(50)54)103-85(128)72(45(8)12-2)102-82(125)62(38-69(114)115)98-78(121)57(32-42(3)4)96-80(123)60(36-51-40-87-41-89-51)95-73(116)46(9)91-77(120)58(33-48-18-14-13-15-19-48)97-79(122)59(34-49-22-24-52(104)25-23-49)99-83(126)70(43(5)6)101-74(117)47(10)90-75(118)55(26-29-65(106)107)93-76(119)56(27-30-66(108)109)94-81(124)61(37-68(112)113)92-64(105)28-31-67(110)111/h13-25,39-47,55-63,70-72,88,104H,11-12,26-38H2,1-10H3,(H,87,89)(H,90,118)(H,91,120)(H,92,105)(H,93,119)(H,94,124)(H,95,116)(H,96,123)(H,97,122)(H,98,121)(H,99,126)(H,100,127)(H,101,117)(H,102,125)(H,103,128)(H,106,107)(H,108,109)(H,110,111)(H,112,113)(H,114,115)(H,129,130)/t44-,45-,46-,47-,55-,56-,57-,58-,59-,60-,61-,62-,63-,70-,71-,72-/m0/s1

(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

IRL-1620; IRL-1620; sovateltidum sovateltide

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.)