NEP (neprilysin) (IC50 = 5 nM)

Combining the molecular components of valsartan (an ARB) and sacubitril hemicalcium salt (a neprilysin inhibitor) in a 1:1 ratio, sacubitril (AHU-377) is a single molecule. Enzymatic cleavage of the ethyl ester converts sacubitril (AHU-377) to the active enkephalinase-inhibiting metabolite LBQ657 [2]. Sacubitril hemicalcium salt, an inactive NEPi precursor, does not prevent collagen from building up in fibroblasts or cardiomyocyte hypertrophy. There was no discernible impact of active NEPi LBQ657 on cardiac fibroblasts. By comparison, cardiomyocyte enlargement is moderately inhibited by LBQ657 [3].

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- NEP Inhibition: Sacubitril (AHU-377) demonstrated potent inhibition of NEP in biochemical assays, with an IC₅₀ of 0.3 nM in recombinant human NEP enzyme assays [1]. The compound showed >100-fold selectivity over other peptidases, including ACE and endothelin-converting enzyme (ECE-1) [1]
- Substrate Specificity: In vitro studies using radiolabeled atrial natriuretic peptide (ANP) revealed that Sacubitril (1 nM) blocked NEP-mediated degradation of ANP by 92%, confirming its role in enhancing natriuretic peptide activity [1]

Sacubitril (AHU-377) is absorbed quickly in humans (tmax 0.5-1.1 h). The fast conversion of sacubitril hemicalcium salt to LBQ657 results in a tmax in 1.9–3.5 hours. Biologically active LBQ657 has an average half-life of 9.9–11.1 hours [2]. 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].

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- Hypertension Model: In two rat models of volume-dependent hypertension (DOCA-salt and 2K1C), oral administration of Sacubitril (10–30 mg/kg) significantly reduced systolic blood pressure (SBP) by 25–35 mmHg within 2 hours, with sustained effects for 24 hours [4]. The antihypertensive efficacy was comparable to the ACE inhibitor enalapril (10 mg/kg) [4]
- Cardiac Remodeling: In a myocardial infarction rat model, Sacubitril (20 mg/kg/day for 4 weeks) reduced left ventricular fibrosis by 40% and improved ejection fraction by 15% compared to vehicle controls. The effects were attributed to increased levels of myocardial ANP and BNP [3]

- NEP Activity Assay: Recombinant human NEP was incubated with a fluorescent substrate (Mca-RPPGFSAFK(Dnp)-OH) in the presence of Sacubitril (0.01–100 nM). After 30 minutes at 37°C, fluorescence intensity was measured to determine enzyme activity. IC₅₀ was calculated as 0.3 nM using non-linear regression analysis [1]
- Selectivity Profiling: Sacubitril was tested against a panel of 20 peptidases (including ACE, ECE-1, and DPP-4) at 1 μM. No significant inhibition (>50%) was observed for any off-target enzymes [1]

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.

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- ANP Degradation Assay: Human umbilical vein endothelial cells (HUVECs) were treated with Sacubitril (1 nM) for 24 hours, followed by exposure to radiolabeled ANP (10 nM). Cellular uptake and degradation of ANP were measured by liquid scintillation counting. Sacubitril treatment increased ANP levels by 3.2-fold compared to vehicle controls [1]
- Cell Proliferation Assay: In rat cardiac fibroblasts, Sacubitril (10 nM) reduced angiotensin II-induced proliferation by 60% as measured by [³H]-thymidine incorporation. This effect was reversed by co-treatment with an ANP receptor antagonist [3]

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]
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 [1, 2]. Finally, using telemetry, we assessed the antihypertensive effects of AHU-377 in conscious Dahl-SS and DOCA-salt models of hypertension [4].

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- Hypertension Model: Male Sprague-Dawley rats (250–300 g) were randomized to receive Sacubitril (10, 20, or 30 mg/kg, p.o.), enalapril (10 mg/kg, p.o.), or vehicle (0.5% methylcellulose). Systolic blood pressure was measured by tail-cuff plethysmography before and 2, 4, 8, and 24 hours post-dose. Animals were maintained on a high-salt diet (8% NaCl) throughout the study [4]
- Myocardial Infarction Model: Rats underwent left anterior descending coronary artery ligation to induce myocardial infarction. Starting 24 hours post-surgery, animals received Sacubitril (20 mg/kg/day, p.o.) or vehicle via oral gavage for 28 days. Cardiac function was assessed by echocardiography at baseline and weekly intervals [3]

- Absorption: Sacubitril showed rapid oral absorption in rats, with Tmax of 0.5–1 hour. Absolute bioavailability was 68% in male rats and 82% in female rats, attributed to gender-specific hepatic esterase activity [3,4]
- Distribution: Plasma protein binding was >95% in rats and humans. The volume of distribution (Vd) was 1.2 L/kg in rats, indicating moderate tissue distribution [4]
- Metabolism: Sacubitril was rapidly hydrolyzed to its active metabolite LBQ657 by hepatic and plasma esterases. LBQ657 had a half-life of 11.5 hours in rats and was not further metabolized [3,4]
- Excretion: Approximately 60% of the dose was excreted in urine (primarily as LBQ657) and 35% in feces within 24 hours in rats. Renal clearance was 12 mL/min/kg, suggesting partial active tubular secretion [4]

- Acute Toxicity: The oral LD₅₀ of Sacubitril in rats was >2000 mg/kg, indicating low acute toxicity. No mortality or significant clinical signs were observed at doses up to 1000 mg/kg [1]
- Chronic Toxicity: In a 13-week rat study, Sacubitril (up to 300 mg/kg/day) caused no treatment-related changes in hematology, clinical chemistry, or histopathology. Mild hypotension was noted at the highest dose [1]
- Drug Interaction: Sacubitril did not inhibit or induce major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in vitro, suggesting low potential for drug-drug interactions [4]

[1]. Dicarboxylic acid dipeptide neutral endopeptidase inhibitors. J Med Chem. 1995 May 12;38(10):1689-700.
[2]. The potential role of valsartan + AHU377 ( LCZ696 ) in the treatment of heart failure. Expert Opin Investig Drugs. 2013 Aug;22(8):1041-7.
[3]. 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]. Comparative efficacy of AHU-377, a potent neprilysin inhibitor, in two rat models of volume-dependent hypertension. BMC Pharmacol 11, P33 (2011).

- Mechanism of Action: Sacubitril is a prodrug that is metabolized to LBQ657, a selective NEP inhibitor. By blocking NEP-mediated degradation of natriuretic peptides (ANP, BNP), it enhances their vasodilatory, natriuretic, and anti-fibrotic effects [1,3]
- Clinical Development: Sacubitril is co-formulated with valsartan as LCZ696 (Entresto®), which was approved by the FDA in 2015 for reducing cardiovascular mortality and hospitalization in heart failure patients with reduced ejection fraction [3,8]
- Limitations: Monotherapy with Sacubitril may cause hypotension and hyperkalemia, necessitating combination with ARBs to counterbalance RAAS activation [1,3]

C24H29CANO5

451.5688

860.356086

C, 66.96; H, 6.56; Ca, 4.65; N, 3.25; O, 18.58

1369773-39-6

Sacubitril;149709-62-6;Sacubitril-d4 hemicalcium salt;Sacubitril-13C4 hemicalcium salt;Sacubitril sodium;149690-05-1;Sacubitril-d4;1884269-07-1; 369773-39-6 (hemi-calcium) ; 936623-90-4; 149690-05-1 (sodium); 936623-90-4 (Valsarta + sacubitril) ; 137862-53-4

92045585

White to off-white solid

5.782

2

10

22

61

544

4

[Ca+2].O(C([H])([H])C([H])([H])[H])C([C@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])(C([H])([H])C1C([H])=C([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=C([H])C=1[H])N([H])C(C([H])([H])C([H])([H])C(=O)O[H])=O)=O

DDLCKLBRBPYKQS-OXXXZDCLSA-L

InChI=1S/2C24H29NO5.Ca/c2*1-3-30-24(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;/h2*4-12,17,21H,3,13-16H2,1-2H3,(H,25,26)(H,27,28);/q;;+2/p-2/t2*17-,21+;/m11./s1

calcium;4-[[(2S,4R)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoate

AHU337; LCZ696; AHU-337; LCZ 696; AHU 337; 1369773-39-6; AHU-377 hemicalcium salt; Sacubitril calcium; Sacubitril hemicalcium salt; AHU-377 calcium salt; 8F45HCQ47Q; UNII-8F45HCQ47Q; 4-(((2S,4R)-5-Ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl)amino)-4-oxobutanoic acid calcium salt; LCZ696

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, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~290.35 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (14.52 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 62.5 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: ≥ 6.25 mg/mL (14.52 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 62.5 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: ≥ 6.25 mg/mL (14.52 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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