NEP (neprilysin) (IC50 = 5 nM)

The compound sacubitril (AHU-377) is a neprilysin inhibitor that is composed of the molecular moieties of valsartan (an ARB) and sacubitril (AHU-377) in a 1:1 ratio. Sacubitril (AHU-377) undergoes enzymatic cleavage of the ethyl ester to generate the active enkephalinase-inhibiting metabolite LBQ657 [2]. There is no inhibition of collagen formation in fibroblasts or cardiomyocyte hypertrophy by the inactive NEPi precursor Sacubitril (AHU-377). Active NEPi LBQ657 did not appear to have any effect on cardiac fibroblasts. Conversely, LBQ657 inhibits cardiomyocyte hypertrophy to a moderate extent [3].

ANF raised urine natriuresis in dogs treated with a vehicle from 17.3±3.6 to 199.5±18.4 pequivkglmin. The effects of sacubitril (AHU-377) were markedly amplified in the animals. Urine production is similarly increased in animals given intravenous Sacubitril (AHU-377) [1]. Sacubitril (3, 10, and 30 mg/kg, PO) pretreatment raised ANP-induced plasma cGMP levels in normotensive rats by 2.4, 3.3, and 4.0 folds, respectively (4-hour AUC compared to vehicle)[4]. In Dahl-SS rats, sacubitril (30 and 100 mg/kg, PO) has dose-dependent antihypertensive effects [4].

Cellular Cardiac Hypertrophy and Fibrosis In Vitro [3]
Rat neonatal cardiac myocytes and fibroblasts were obtained from 1- to 2-day-old Sprague–Dawley rat pups by enzymatic collagenase digestion and prepared for in vitro assays as routinely used in our laboratory.22 Cardiac myocyte hypertrophy was assessed by AngII-stimulated (100 nmol/L) neonatal cardiac myocytes with 3[H]leucine incorporation for 60 hours. AngII-stimulated (100 nmol/L) collagen synthesis was determined by 3[H]proline incorporation in neonatal cardiac fibroblasts for 48 hours. Cells were preincubated with valsartan, AHU377, LBQ657, or valsartan+LBQ657 (ARNi) for 1 hour before stimulation. Dose ranges used and NEPi to ARB ratios aimed to replicate as far as possible doses of LCZ696 used clinically. The drugs were a kind gift of Novartis, Basel, Switzerland. In addition, exogenous B-type natriuretic peptide (BNP) was added at different concentrations into the cell culture media just before AngII stimulation to assess the effect of direct augmentation of NP signaling. Experiments were repeated 2 to 4× in triplicate each time.

One week after MI, adult male Sprague-Dawley rats were randomized to treatment for 4 weeks with LCZ696 (68 mg/kg body weight perorally; MI-ARNi, n=11) or vehicle (MI-vehicle, n=6). Five weeks after MI, MI-ARNi versus MI-vehicle demonstrated lower LV end-diastolic diameter (by echocardiography; 9.7±0.2 versus 10.5±0.3 mm), higher LV ejection fraction (60±2 versus 47±5%), diastolic wall strain (0.23±0.02 versus 0.13±0.02), and circular strain (-9.8±0.5 versus -7.3±0.5%; all P<0.05). LV pressure-volume loops confirmed improved LV function. Despite similar infarct size, MI-ARNi versus MI-vehicle had lower cardiac weights (P<0.01) and markedly reduced fibrosis in peri-infarct and remote myocardium. Angiotensin II-stimulated incorporation of 3[H]leucine in cardiac myocytes and 3[H]proline in cardiac fibroblast was used to evaluate hypertrophy and fibrosis, respectively. The neprilysin inhibitor component of LCZ696, LBQ657, inhibited hypertrophy but not fibrosis. The angiotensin receptor blocker component of LCZ696, valsartan inhibited both hypertrophy and fibrosis. Dual valsartan+LBQ augmented the inhibitory effects of valsartan and the highest doses completely abrogated angiotensin II-mediated effects.[3]

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We determined the relationship between atrial natriuretic peptide (ANP) and blood pressure in anesthetized, normotensive rats. We studied the relationship between NEP inhibition and elevation of plasma cGMP evoked by ANP in the absence and presence of AHU-377, an ester prodrug of LBQ657 and a component of LCZ696. Finally, using telemetry, we assessed the antihypertensive effects of AHU-377 in conscious Dahl-SS and DOCA-salt models of hypertension [4].

[1]. Ksander GM, et al. Dicarboxylic acid dipeptide neutral endopeptidase inhibitors. J Med Chem. 1995 May 12;38(10):1689-700.
[2]. Voors AA, et al. The potential role of valsartan + AHU377 ( LCZ696 ) in the treatment of heart failure. Expert Opin Investig Drugs. 2013 Aug;22(8):1041-7.
[3]. von Lueder TG, et al. Angiotensin receptor neprilysin inhibitor LCZ696 attenuates cardiac remodeling and dysfunction after myocardial infarction by reducing cardiac fibrosis and hypertrophy. Circ Heart Fail. 2015 Jan;8(1):71-8.
[4]. Hegde, L.G., et al. Comparative efficacy of AHU-377, a potent neprilysin inhibitor, in two rat models of volume-dependent hypertension. BMC Pharmacol 11, P33 (2011).

Sacubitril is a prodrug neprilysin inhibitor used in combination with valsartan to reduce the risk of cardiovascular events in patients with chronic heart failure (NYHA Class II-IV) and reduced ejection fraction. It was approved by the FDA after being given the status of priority review for on July 7, 2015. Sacubitril's active metabolite, LBQ657 inhibits neprilysin, a neutral endopeptidase that would typically cleave natiuretic peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP). ANP and BNP are released under atrial and ventricle stress, which activate downstream receptors leading to vasodilation, natriuresis and diuresis. Under normal conditions, neprilysin breaks down other vasodilating peptides and also vasoconstrictors such as angiotensin I and II, endothelin-1 and peptide amyloid beta-protein. Inhibition of neprilysin therefore leads to reduced breakdown and increased concentration of endogenous natriuretic peptides in addition to increased levels of vasoconstricting hormones such as angiotensin II.

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Sacubitril is a neprilysn (NEP) inhibitor prodrug with natriuretic activity. Upon administration, sacubitril is metabolized by esterases to its active metabolite, sacubitrilat, which inhibits NEP, a neutral endopeptidase that cleaves natriuretic peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP), as well as certain vasoconstricting peptides including as angiotensin I and II, and endothelin-1. Additionally, sacubitrilat may inhibit NEP-mediated catabolism of certain peptide-based agents, thereby improving their in vivo stability and increasing tumor cell exposure.

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Absorption: Peak plasma concentrations of sacubitril and it's metabolite, LBQ657 are reached in 0.5 hours and 2 hours respectively. Food does not clinically affect the systemic exposure of sacubitril or LBQ657. The oral bioavailability of sacubitril is >60%. It should be noted that the valsartan found in this combination is more bioavailable than other market available valsartan.
Biological Half-Life: The half life of sacubitril is 1.1 to 3.6 hours, and the half life of it's metabolite LBQ657 is 9.9 to 11.1 hours.

C24H29NO5

411.49076

411.2045

149709-62-6

Sacubitril hemicalcium salt;1369773-39-6;Sacubitril-d4 hemicalcium salt;(2S,4S)-Sacubitril;149709-63-7;2R,4R-Sacubitril;766480-48-2;2R,4S-Sacubitril;761373-05-1;(Z)2S,4R-Sacubitril;Sacubitril sodium;149690-05-1;Sacubitril-d4;1884269-07-1;2S,4R-Sacubitril;2307668-79-5

9811834

White to light yellow solid

1.2±0.1 g/cm3

656.9±55.0 °C at 760 mmHg

351.1±31.5 °C

0.0±2.1 mmHg at 25°C

1.549

3.96

96.19

O=C(CCC(O)=O)N[C@H](CC1=CC=C(C2=CC=CC=C2)C=C1)C[C@@H](C)C(OCC)=O

PYNXFZCZUAOOQC-UTKZUKDTSA-N

InChI=1S/C24H29NO5/c1-3-30-24(29)17(2)15-21(25-22(26)13-14-23(27)28)16-18-9-11-20(12-10-18)19-7-5-4-6-8-19/h4-12,17,21H,3,13-16H2,1-2H3,(H,25,26)(H,27,28)/t17-,21+/m1/s1

4-[[(2S,4R)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoic acid

LCZ696; AHU377; LCZ 696; AHU 377; LCZ696; AHU-377; Entresto

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)

DMSO : ~75 mg/mL (~182.26 mM)
H2O : ~0.67 mg/mL (~1.63 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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