Angiotensin II receptor type 1 (AT1R)

When co-transfected with Flag-AT1R-cherry, HA-β-arrestin-1, and TRPC3-GFP, HEK293 cells showed a substantial increase in the interaction of AT1R and TRPC3 with immunoprecipitated β-arrestin-1 at 100 nM of TRV120027[2]. In HEK293 cells co-transfected with AT1R, β-arrestin-1, and TRPC3, TRV120027 (100 nM) causes a rise in [Ca2+]i; however, this increase is greatly blocked by preincubation with Pyr3. TRPC3-GFP, Cherry, and HA-β-arrestin-1[2].

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Acute hormone secretion triggered by G protein-coupled receptor (GPCR) activation underlies many fundamental physiological processes. GPCR signalling is negatively regulated by β-arrestins, adaptor molecules that also activate different intracellular signalling pathways. Here we reveal that TRV120027, a β-arrestin-1-biased agonist of the angiotensin II receptor type 1 (AT1R), stimulates acute catecholamine secretion through coupling with the transient receptor potential cation channel subfamily C 3 (TRPC3). We show that TRV120027 promotes the recruitment of TRPC3 or phosphoinositide-specific phospholipase C (PLCγ) to the AT1R-β-arrestin-1 signalling complex. Replacing the C-terminal region of β-arrestin-1 with its counterpart on β-arrestin-2 or using a specific TAT-P1 peptide to block the interaction between β-arrestin-1 and PLCγ abolishes TRV120027-induced TRPC3 activation. Taken together, our results show that the GPCR-arrestin complex initiates non-desensitized signalling at the plasma membrane by coupling with ion channels. This fast communication pathway might be a common mechanism of several cellular processes.[2]

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In particular, we showed that β-arrestin-1 and β-arrestin-2 function oppositely in TRV120027-stimulated calcium increases in primary chromaffin cells (Fig. 3e,g). Whereas the β-arrestin-1 knockout abolished TRV120027-induced calcium increases that are required for catecholamine secretion, the β-arrestin-2 knockout increased intracellular calcium after TRV120027 administration. The observed phenomenon of the distinct roles of β-arrestin subtypes in TRV120027-induced calcium increases adds to the increasingly long list of physiological effects in which these two isoforms oppose each other in specific cellular contexts. To the best of our knowledge, most of the beneficial effects of TRV120027 in the heart and renin systems are mainly mediated by β-arrestin-2-mediated AT1R signalling. It's also worth to note that biased agonists of opioid receptor has been characterized to have β-arrestin subtype signalling selectivity. For agonists of AT1R, whereas the TRV120027 displayed more β-arrestin-1-biased molecular efficacy within the equi-active model analysis, the TRV120026 exhibited more β-arrestin-2-biased molecular efficacy (Supplementary Fig. 39). Therefore, future studies to identify ligands that preserve the ability to activate β-arrestin-2-biased AT1R signalling but are devoid of Gq and β arrestin-1 signalling may result in more beneficial drug candidates for treating cardiovascular diseases [2].

Furotecin plus TRV120027 (IV; 0.3 or 1.5 µg/kg per minute; infusion rate, 0.5 mL/min) decreases cardiac preload and afterload, renal and systemic vascular resistance, and left ventricular external work while raising cardiac output and renal blood flow. Dogs with experimental heart failure retain their GFR and renal excretory function [1].

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In this study, consistent with earlier studies, TRV120027 acted as an arterial vasodilator, as evidenced by the dose-dependent reduction in mean arterial pressure and systemic vascular resistance. This vasodilatory effect was rapidly reversible. In the postinfusion clearance, mean arterial pressure in both groups were similar, suggesting that the reduction of mean arterial pressure in the postinfusion clearance compared with baseline is a time effect or secondary to furosemide-induced diuresis, not a persistent effect of TRV120027. The decrease in right atrial pressure was not statistically significant, which indicates that TRV120027’s vasodilatory actions on the arterial vasculature are more prominent than its effects on the venous system. The observed reduction in pulmonary capillary wedge pressure could be because of afterload reduction or venodilation. Although not significant compared with the furosemide+vehicle group, cardiac output tended to increase compared with baseline in the furosemide+TRV120027 group. This may be secondary to improved cardiac preload and afterload, but, as in vivo and in vitro data have demonstrated, a positive effect on cardiac contractility via β-arrestin signaling may also play a role. Pulmonary vascular resistance was also decreased with addition of TRV120027, which may be of benefit to many patients with ADHF who have concomitant pulmonary arterial hypertension and right HF. Taken together, these hemodynamic data suggest that TRV120027 may help to unload the left and right heart. This is also supported by the observed decrease in ANP: ANP is secreted by the heart in response to cardiac stress, and thus a decrease can be interpreted as a neurohumoral indicator of cardiac unloading. BNP measures in this study were more variable than ANP measures, but BNP also tended to decline in the F+T group compared with the F+V group.[1]

TRV120027 increased renal blood flow compared with baseline, but this was not significant compared with the furosemide-alone group. Renal vascular resistance tended to be lower compared with furosemide alone during C2. Despite the significant reduction in renal perfusion pressure, urine flow and urinary sodium and potassium excretion were maintained throughout C2 to C4 compared with furosemide alone. GFR tended to increase in the furosemide-alone group, which could be because of its inhibition of tubular glomerular feedback, which under physiological conditions decreases GFR when sodium delivery is increased to the juxtaglomerular apparatus. Of note, increased sodium delivery to the distal nephron with long-term administration of loop diuretics can lead to hypertrophy and hyperplasia of distal nephron segments, which are insensitive to the actions of furosemide; however, the clinical importance of these changes is not well known. Addition of TRV120027 maintained GFR, despite the reduction in renal perfusion pressure, which would generally be expected to decrease hydrostatic filtration pressure in the glomerulus. However, the hydrostatic pressure promoting filtration in the glomerulus not only depends on the renal perfusion pressure but also on the differential tone of the afferent and efferent glomerular arteriole. Interestingly, angiotensin II in most situations reduces renal blood flow but maintains GFR by vasoconstricting the efferent arteriole, while having little effect on the afferent arteriole, thus increasing glomerular filtration pressure. How a β-arrestin biased AT1R ligand like TRV120027 affects afferent and efferent arteriolar tone is not specifically known. Also not known is whether TRV120027 affects podocyte function and the filtration coefficient. Of note, losartan in an ovine model of HF also maintained GFR and urinary sodium excretion, despite a reduction in renal perfusion pressure. To better characterize tubular sodium handling, we used the lithium clearance technique to assess proximal and distal fractional reabsorption of sodium. During drug administration, sodium reabsorption was not different between groups even though TRV120027 reduced renal perfusion pressure, an effect that would be expected to increase sodium reabsorption. The preserved sodium excretion can be explained with TRV120027 blocking the tubular actions of angiotensin II, which is an important mediator of increased sodium reabsorption when renal perfusion pressure is decreased. Interestingly, in the postinfusion clearance, distal fractional sodium reabsorption was significantly lower in the furosemide+TRV120027 group. This could be explained by aldosterone, plasma levels of which tended to be reduced with TRV120027 during C3. Aldosterone acts via the mineralocorticoid receptor to promote sodium reabsorption in the distal tubule. Thus, aldosterone-reducing actions of TRV120027 may be of special importance because aldosterone affects nephron segments at which loop diuretics do not act. Although the clinical impact of these findings remains to be proven, these data indicate that TRV120027 has the potential to be renal enhancing or protective. [1]

A hallmark of HF is neurohumoral activation. Increases of angiotensin II and aldosterone, if not antagonized, can promote sodium retention, vasoconstriction, and organ fibrosis, which all can contribute to the progression of HF. As seen with angiotensin receptor blockers, plasma renin activity significantly increased with addition of TRV120027. This could, in part, be due to the reduction in renal perfusion pressure but also due to antagonizing the inhibitory action of angiotensin II on renin secretion by TRV120027. Although there was a trend for angiotensin II to increase in the furosemide-alone group, we were not able to assess the angiotensin II levels in the furosemide+TRV120027 group because the angiotensin II assay cross-reacts with TRV120027. Importantly, given that aldosterone, the secretion of which is stimulated by angiotensin II, decreased with TRV120027 suggests that downstream signaling of renin was effectively blocked, which would mean that the increase in plasma renin activity with TRV120027 would be of little consequence.[1]

In summary, TRV120027 when added to furosemide unloaded the heart, preserved renal function during drug administration, and led to a more sustained diuresis postinfusion, which may be because of aldosterone suppression. These findings suggest that TRV120027 may be a promising new drug for the treatment of ADHF, and further studies are warranted [1].

Electrolytes were measured by flame photometry. Inulin was measured with the anthrone method. ANP, B-type natriuretic peptide, angiotensin II, plasma renin activity, and aldosterone were measured, as previously described. Proximal fractional reabsorption of sodium was calculated with the lithium clearance technique as follows: (1−[lithium clearance/glomerular filtration rate {GFR}])×100. Distal fractional reabsorption of sodium was calculated as: ([lithium clearance−sodium clearance]/lithium clearance)×100 [1].

Calcium measurements [2]
[Ca2+]i was measured as previously described51,52. The mouse adrenal chromaffin cells derived from WT, β-arrestin-1−/− or β-arrestin-2−/− mice were cultured for 2–4 days before the experiments. All experiments of calcium measurements using primary chromaffin or HEK293 cells were carried out at room temperature (22–25 °C). The primary cells derived from WT, β-arrestin-1−/− or β-arrestin-2−/− mice or HEK293 cells transfected with different plasmids were incubated in imaging buffer I (10 mM glucose,150 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 3 mM CaCl2, 20 mM HEPES, pH 7.4). In the Ca2+-free bath, the 3 mM CaCl2 of the imaging buffer I was replaced by 5 mM EGTA. The change of [Ca2+]i concentration was measured by an intracellular Ca2+ imaging system. Isolated mouse adrenal chromaffin cells from wild type or different knockout mice were incubated at 37 °C for 30 min in a solution containing 2 μM Fura-2/AM. We then used the F340/F380 ratio of Fura-2 intensity to monitor the intracellular calcium change challenged by a spectrum of AT1R agonists, such as Ang II (100 nM), SII (1 μM) and TRV120027 (100 nM). We measured the intracellular Ca2+ concentration in primary cells by dual-wavelength ratio-metric fluorometry. We excited the Fura-2 with alternative light between 340 and 380 nm by a monochromator-based system. The cooled charge-coupled device was used to measure the resulting fluorescence signals. We calculated the relative changes in [Ca2+]i by the ratio of F340 to F380. The electrophysiology recording was analysed using the Igor software (WaveMetrix). The statistical analysis was performed with t-test or two-way analysis of variance (ANOVA).

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Current measurement in primary chromaffin cells [2]
Adrenal chromaffin cells were isolated from 8-week-old wild-type female mice, and were cultured on coverslips used for patch-clamp experiments after 70–100 h incubation. We used the whole-cell patch-clamp to record the membrane currents of adrenal chromaffin cells derived from wild-type or different knockout mice. The extracellular buffer for these primary cell cultures contains 145 mM NaCl, 2.8 mM KCl, 10 mM HEPES, 1.0 mM MgCl2.6H2O, 2.0 mM CaCl2.2H2O, 10 mM glucose and pH 7.4 adjusted with NaOH. The K+ outward current was blocked by 50 mM Tetraethylammonium and the activation of the Na+ current was abolished by 100 nM tetrodotoxin. Electrode with a resistance of 3.9∼5.1 MΩ was filled with internal solution (145 mM CsCl, 8.0 mM Nacl, 1.0 mM Mgcl2.6H2O, 10 mM HEPES, 0.4 mM GTP and 2.0 mM Mg-ATP, which was adjusted to pH 7.3 with CsOH). We ruptured the primary cell membrane with suction when the high-resistance seal is greater than 1.5 gigohm. The leak currents of a single primary cell derived from wild-type mice or knockout mice that are greater than −30 pA were excluded from further analysis. We monitored the gap-free recording and some cell parameters, such as Ccell, Rs (series resistance), Rm (membrane resistance), to ascertain the constancy of the patch. During whole-cell recording, we used the amplifier circuitry to minimize the capacity current, and the Rs was compensated by 80% (ref. 52). Inward current in a single primary mouse chromaffin cell was induced by TRV120027 when bathed in an extracellular solution with 1 μM tetrodotoxin and 1 μM 4-AP and voltage-clamped at −60 mV. TRV120027 (final concentration, 5 μM) was added in the bath to stimulate primary cells. TheTRPC3 inhibitor Pyr3 was used in the bath at a final concentration of 5 μM. We perfused the recording chamber with standard external solution under gravity at a rate of ∼12 ml h−1. We recorded the ionic current by the data acquisition system (2012, DigiData 1322A, Axon) and an amplifier machine. We used the pClamp Version 9 software to control the command voltages. All experiments and recordings were performed at room temperature (28 °C). We use patch-clamp amplifier (2012, HEKA EPC10) and patchmaster software. Current and voltage signals of primary cells derived from wild type or knockout mice were low-pass-filtered (DC to 10 KHz) and acquired at 20 KHz. Data, graphs and current traces were analysed with the Igor software package.

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Sequential immunoprecipitation experiments [2]
HEK293 cells (generally six 100-mm culture dishes for each) co-transfected with Flag-AT1R-cherry, HA-β-arrestin-1 and TRPC3-GFP were stimulated with TRV120027 (100 nM) or control vehicle for 1 min. The plasma membrane fractions were first isolated by centrifugation at 20,000g for 1 h and then washed with PBS. The protein complexes containing Flag-AT1R were immunoprecipitated by Anti-Flag M2 agarose and then eluted with 3*Flag peptide (final concentration, 100 μg ml−1). The complexes containing Flag-AT1R were then immunoprecipitated by anti-HA beads or anti-GFP beads. TRPC3 associated with Flag-AT1R-HA-β-arrestin-1 or PLCγ1 associated with the AT1R–TRPC3 was detected by a specific antibody.

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Co-immunoprecipitation [2]
Co-immunoprecipitation was performed as previously described. HEK293 cells were co-transfected with Flag-AT1R-cherry, TRPC3-GFP (WT/Truncations) and HA-β-arrestin1 (WT/Truncations/Mutants). Forty-eight hours after transfection, the cells were starved for 12 h and then stimulated with Ang II (100 nM), TRV120055 (30 nM) or TRV120027 (100 nM) for 1 min. Subsequently, the cells were washed with cold PBS and then collected in cold lysis buffer. The cell lysates were subjected to immunoprecipitation using different antibody-conjugated beads (Flag or HA-conjugated beads), which were incubated overnight at 4 °C. Immune complexes containing AT1R or arrestin were extensively washed for at least five times with cold lysis buffer and analysed by western blotting with specific antibodies.

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BRET assay [2]
BRET assays were performed as previously described. For the measurement of β-arrestin-1 recruitment to the plasma membrane, HEK293 cells were co-transfected with plasmids encoding Flag-AT1R, Luc-β-arrestin-1, TRPC3 and Lyn-YFP. For the intermolecular BRET, HEK293 cells were co-transfected with plasmids encoding Flag-AT1R, Luc-β-arrestin-1 or Luc-β-arrestin-2 and TRPC3-YFP or TRPV1-YFP/TRPC6-YFP. For the intramolecular BRET, HEK293 cells were co-transfected with plasmids encoding Flag-AT1R, HA-β-arrestin-1 and Luc-TRPC3-YFP plasmids for 48 h. After 12 h of starvation, cells were harvested and washed at least three times with PBS, and then cells were stimulated with AngII (100 nM), TRV120027 (100 nM) or other AT1R agonists for 1 min at 37 °C. Subsequently, we incubated the transfected cells with Coelenterazine h at room temperature (final concentration, 5 μM) and two different light emissions were used for measurement (480/20 nm for luciferase and 530/20 nm for yellow fluorescent protein). All the BRET measurements were performed by a plate reader Mithras LB 940 and the signal was determined by calculating the ratio of the light intensity emitted by yellow fluorescent protein over the intensity emitted by luciferase.

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Confocal microscopy [2]
Confocal microscopy was performed as previously described26,55. HEK293 cells were co-transfected with plasmids encoding HA-β-arrestin-1, TRPC3-GFP and Flag-AT1R-cherry. The day following transfection, the cells were seeded on fibronectin-coated glass-bottom, 35-mm, at the density of 3 × 105 cells per dish. The next day, the cells were starved and then stimulated with Ang II (100 nM) or TRV120027 (100 nM) for 1 min at 37 °C. Samples were then analysed using LSM 780 laser-scanning confocal microscope.

Animal/Disease Models: Male mongrel dog (body weight, 20.5-30 kg) [1]
Doses: 0.3 or 1.5 μg/kg per minute; infusion rate, 0.5 mL/min
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: Caused dose dependence in dogs Vasodilation, increased myocardial contractility, and diminished myocardial oxygen consumption.

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Studies were performed in 2 groups of male mongrel dogs (weight, 20.5–30 kg; n=6 per group). Animals were maintained on a sodium-controlled diet (Hill’s I/d diet; Hill’s pet nutrition). Animals had a pacemaker implanted with an epicardial lead on the right ventricle, as previously described in detail.29 After at least 10 days of recovery, pacemakers were programmed to stimulate at 240 beats per minute. An acute experiment was done on day 11 of pacing. On the evening before the acute experiment, dogs were fasted and given 300 mg of lithium carbonate orally for later assessment of renal tubular function. On the day of the experiment, pacing was discontinued, and animals were anesthetized with pentobarbital and fentanyl, endotracheally intubated, and mechanically ventilated (rate, 12/min; tidal volume, 15 mL/kg body weight) with supplemental oxygen. Lines were inserted into the femoral vein for infusion of inulin, saline, and study drugs. A line was advanced via the femoral artery to measure arterial pressure and for blood sampling. A balloon-tipped, flow-directed thermodilution catheter was advanced into the pulmonary artery via the jugular vein to measure cardiac filling pressures and cardiac output. Through a left flank incision and retroperitoneal dissection, a catheter was inserted into the ureter for timed urine collections. After surgical preparation, pacing at 240 beats per minute was reintroduced. The renal artery was equipped with an electromagnetic flow probe to measure renal blood flow. A weight-adjusted inulin bolus was administered, and a continuous inulin infusion (1 mL/min) and a saline infusion (1 mL/min) were started. After 60 minutes of equilibration, a 30-minute baseline clearance (C1) was done. All clearances consisted of urine collection, hemodynamic measurements, and blood sampling midway through the clearance. After the baseline clearance, the saline infusion was stopped, and an infusion with furosemide (1 mg/kg per hour; infusion rate, 0.5 mL/min) was started. In addition, animals were randomly assigned to 1 of 2 groups. One group, F+V, received an infusion of vehicle (saline at 0.5 mL/min). In contrast, the other group, F+T, received TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH; 0.3 µg/kg per minute, diluted in saline; infusion rate, 0.5 mL/min). After a 15-minute lead-in, a second 30-minute clearance (C2) was done. After this, the F+V group continued to receive saline (0.5 mL/min), whereas the F+T group received TRV120027 at 1.5 µg/kg per minute (diluted in saline; infusion rate, 0.5 mL/min). Again, after a 15-minute lead-in, a 30-minute clearance (C3) was done. After that, furosemide and vehicle or TRV-120027 infusions were stopped and replaced with a saline infusion (1 mL/min). After a 30-minute washout period, a final 30-minute postinfusion clearance (C4) was performed. Pressures and renal blood flow were recorded digitally and analyzed offline. Cardiac output was measured by thermodilution. Renal blood flow was measured by electromagnetic flow probe (Carolina Medical Electronics, King, NC). Renal perfusion pressure was calculated as mean arterial pressure−right atrial pressure.[1]

[1]. TRV120027, a novel β-arrestin biased ligand at the angiotensin II type I receptor, unloads the heart and maintains renal function when added to furosemide in experimental heart failure.Circ Heart Fail. 2012 Sep 1;5(5):627-34. Epub 2012 Aug 13.

[2]. Arrestin-biased AT1R agonism induces acute catecholamine secretion through TRPC3 coupling.Nat Commun. 2017 Feb 9;8:14335.

TRV120027 has been used in trials studying the treatment of Heart Failure and Kidney Disease.

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Background: TRV120027 is a novel β-arrestin biased ligand of the angiotensin II type 1 receptor; it antagonizes canonical G-protein-mediated coupling while, in contrast to classical angiotensin II type 1 receptor antagonists, it engages β-arrestin-mediated signaling. Consequently, TRV120027 inhibits angiotensin II-mediated vasoconstriction while, via β-arrestin coupling, it increases cardiomyocyte contractility. We hypothesized that TRV120027 would elicit beneficial cardiorenal actions when added to furosemide in experimental heart failure.
Methods and results: Two groups of anesthetized dogs (n=6 each) with tachypacing-induced heart failure were studied. After a baseline clearance, 1 group (F+V) received furosemide (1 mg/kg per hour) plus saline for 90 minutes, whereas the other (F+T) received the same dose of furosemide plus TRV120027 (0.3 and 1.5 µg/kg per minute for 45 minutes each); 2 clearances were done during drug infusion. After a washout, a postinfusion clearance was done; *P<0.05 between groups. F+V and F+T increased diuresis and natriuresis to a similar extent during drug administration, but urine flow* and urinary sodium excretion* were higher in the postinfusion clearance with F+T. Glomerular filtration rate was preserved in both groups. Renal blood flow increased with F+T but this was not significant versus F+V. Compared with F+V, F+T decreased mean arterial pressure*, systemic* and pulmonary* vascular resistances, and atrial natriuretic peptide*. Pulmonary capillary wedge pressure* decreased to a larger extent with F+T than with F+V.
Conclusions: When added to furosemide, TRV120027, a novel β-arrestin biased angiotensin II type 1 receptor ligand, preserved furosemide-mediated natriuresis and diuresis, while reducing cardiac preload and afterload. These results provide support for TRV120027 as a promising novel therapeutic for the treatment of heart failure. [1]

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In summary, TRV120027 when added to furosemide unloaded the heart, preserved renal function during drug administration, and led to a more sustained diuresis postinfusion, which may be because of aldosterone suppression. These findings suggest that TRV120027 may be a promising new drug for the treatment of ADHF, and further studies are warranted.[1]

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Acute hormone secretion triggered by G protein-coupled receptor (GPCR) activation underlies many fundamental physiological processes. GPCR signalling is negatively regulated by β-arrestins, adaptor molecules that also activate different intracellular signalling pathways. Here we reveal that TRV120027, a β-arrestin-1-biased agonist of the angiotensin II receptor type 1 (AT1R), stimulates acute catecholamine secretion through coupling with the transient receptor potential cation channel subfamily C 3 (TRPC3). We show that TRV120027 promotes the recruitment of TRPC3 or phosphoinositide-specific phospholipase C (PLCγ) to the AT1R-β-arrestin-1 signalling complex. Replacing the C-terminal region of β-arrestin-1 with its counterpart on β-arrestin-2 or using a specific TAT-P1 peptide to block the interaction between β-arrestin-1 and PLCγ abolishes TRV120027-induced TRPC3 activation. Taken together, our results show that the GPCR-arrestin complex initiates non-desensitized signalling at the plasma membrane by coupling with ion channels. This fast communication pathway might be a common mechanism of several cellular processes. [2]

C43H67N13O10

926.073189020157

925.513

C, 55.77; H, 7.29; N, 19.66; O, 17.28

1234510-46-3

TRV-120027 TFA

3082475

H-Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH; sarcosyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L-histidyl-L-prolyl-D-alanine

GRVYIHPA; {Sar}-RVYIHP-{d-Ala}

White to off-white solid powder

-2.3

12

13

26

66

1690

8

O=C([C@H](CC1=CN=CN1)NC([C@H]([C@@H](C)CC)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H](C(C)C)NC([C@H](CCC/N=C(\N)/N)NC(CNC)=O)=O)=O)=O)=O)N1CCC[C@H]1C(N[C@@H](C(=O)O)C)=O

XIEWFECSPPTVQN-KMIMAYJXSA-N

InChI=1S/C43H67N13O10/c1-7-24(4)35(40(63)53-31(19-27-20-47-22-49-27)41(64)56-17-9-11-32(56)38(61)50-25(5)42(65)66)55-37(60)30(18-26-12-14-28(57)15-13-26)52-39(62)34(23(2)3)54-36(59)29(51-33(58)21-46-6)10-8-16-48-43(44)45/h12-15,20,22-25,29-32,34-35,46,57H,7-11,16-19,21H2,1-6H3,(H,47,49)(H,50,61)(H,51,58)(H,52,62)(H,53,63)(H,54,59)(H,55,60)(H,65,66)(H4,44,45,48)/t24-,25+,29-,30-,31-,32-,34-,35-/m0/s1

(2R)-2-[[(2S)-1-[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-(diaminomethylideneamino)-2-[[2-(methylamino)acetyl]amino]pentanoyl]amino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]propanoic acid

TRV120027; TRV 120027; TRV-120027; 1234510-46-3; trv027; TRV120,027; TRV-027; UNII-J1J4P3PQZD; TRV 027; J1J4P3PQZD; TRV-120,027

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)

H2O : ~100 mg/mL (~107.98 mM)

Note: Please refer to the "Guidelines for Dissolving Peptides" section in the 4th page of the "Instructions for use" file (upper-right section of this webpage) for how to dissolve peptides.

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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.)