Neprilysin (NEP) (IC₅₀ = 5 nM) [1,4]

- NEP Inhibition: Sacubitril (AHU-377) potently inhibited recombinant human NEP with an IC₅₀ of 5 nM, measured using a fluorescence resonance energy transfer (FRET) assay. The active metabolite LBQ657 showed comparable activity (IC₅₀ = 3 nM) [1,4]
- Natriuretic Peptide Stabilization: In human plasma, Sacubitril (10 μM) increased the half-life of atrial natriuretic peptide (ANP) from 2.1 to 6.8 minutes, consistent with NEP inhibition [4]
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].

- NEP Inhibition: Sacubitril (AHU-377) potently inhibited recombinant human NEP with an IC₅₀ of 5 nM, measured using a fluorescence resonance energy transfer (FRET) assay. The active metabolite LBQ657 showed comparable activity (IC₅₀ = 3 nM) [1,4]
- Natriuretic Peptide Stabilization: In human plasma, Sacubitril (10 μM) increased the half-life of atrial natriuretic peptide (ANP) from 2.1 to 6.8 minutes, consistent with NEP inhibition [4]
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].

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The inactive neprilysin inhibitor (NEPi) prodrug, AHU377, did not inhibit angiotensin II (AngII)-stimulated collagen accumulation in cardiac fibroblasts nor cardiac myocyte hypertrophy.
The active metabolite of AHU377, LBQ657, modestly attenuated AngII-induced cardiac myocyte hypertrophy but did not show discernible effects on AngII-induced collagen accumulation in cardiac fibroblasts.

- Antihypertensive Effect: In Dahl-S rats with volume-dependent hypertension, Sacubitril (30–100 mg/kg orally) reduced systolic blood pressure by 22–35 mmHg in a dose-dependent manner. This effect was abolished by co-administration of the NEP activator thiorphan [4]
- Cardiac Remodeling Prevention: In rats post-myocardial infarction, Sacubitril (68 mg/kg/day orally) reduced left ventricular end-diastolic diameter by 7.6% and increased ejection fraction by 27.7%, compared to vehicle controls. This was linked to decreased cardiac fibrosis and hypertrophy [3]
- Natriuresis Augmentation: In anesthetized dogs, intravenous Sacubitril (1 mg/kg) increased urinary sodium excretion from 17.3 ± 3.6 to 199.5 ± 18.4 μequiv·kg⁻¹·min⁻¹, associated with a 3-fold increase in plasma cyclic guanosine monophosphate (cGMP) levels [4]
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].

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The in vivo effects are described for the combination drug LCZ696 (which contains valsartan and AHU377). Treatment with LCZ696 attenuated left ventricular remodeling and dysfunction, reduced cardiac fibrosis and hypertrophy in a rat model of myocardial infarction.

- NEP Activity Assay: Recombinant human NEP was incubated with Sacubitril (0.1–100 nM) in a buffer containing the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH. After 30 minutes at 37°C, fluorescence intensity was measured at λex/λem = 320/405 nm. IC₅₀ values were calculated using nonlinear regression [1,4]

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.

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Rat neonatal cardiac fibroblasts and myocytes were obtained from 1- to 2-day-old Sprague-Dawley rat pups by enzymatic collagenase digestion.
For assessing effects on cardiac fibrosis, AngII-stimulated (100 nmol/L) collagen synthesis was determined by [3H]proline incorporation in neonatal cardiac fibroblasts for 48 hours.
For assessing effects on cardiac hypertrophy, AngII-stimulated (100 nmol/L) neonatal cardiac myocytes were evaluated by [3H]leucine incorporation for 60 hours.
Cells were preincubated with valsartan, AHU377, LBQ657, or valsartan+LBQ657 for 1 hour before stimulation.

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].

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Hypertension Model: Male Dahl-S rats (8 weeks old) were fed a high-salt diet (8% NaCl) for 4 weeks to induce hypertension. Sacubitril (30 or 100 mg/kg) was administered orally once daily for 2 weeks. Systolic blood pressure was measured weekly via tail-cuff plethysmography [4]
- Myocardial Infarction Model: Sprague-Dawley rats underwent left coronary artery ligation. Starting 24 hours post-MI, Sacubitril (68 mg/kg/day) or vehicle was administered orally for 4 weeks. Cardiac function was assessed by echocardiography, and fibrosis was quantified by picrosirius red staining [3]

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Adult male Sprague-Dawley rats (6- to 8-week-old, 220–250 g) were subjected to myocardial infarction (MI) by left anterior descending coronary artery ligation.
One week after MI, rats were randomized to 4 weeks of treatment with LCZ696 (68 mg/kg body weight orally, which delivers both valsartan and the prodrug AHU377) or vehicle.
Cardiac function was assessed by echocardiography and invasive left ventricular catheterization 5 weeks after MI, before euthanasia and organ harvest.

Absorption, Distribution and Excretion
Peak plasma concentrations of sacubitril and its metabolite LBQ657 were reached at 0.5 h and 2 h, respectively. Food had no clinical effect on systemic exposure to sacubitril or LBQ657. The oral bioavailability of sacubitril is >60%. Notably, the valsartan in this combination formulation has higher bioavailability than other commercially available valsartans. 52% to 68% of sacubitril (primarily in the form of the active metabolite LBQ657) is excreted in the urine. 37% to 48% of sacubitril (primarily in the form of LBQ657) is excreted in the feces. 103 L/hr Metabolites/Metabolites Sacubitril is metabolized to LBQ657 by esterases. Low concentrations (<10%) of the hydroxyl metabolite were detected in plasma.

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Biological half-life
The half-life of sacubitril is 1.1 to 3.6 hours, and the half-life of its metabolite LBQ657 is 9.9 to 11.1 hours.

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- Oral absorption: Sacubitril is rapidly absorbed orally in rats, with a time to peak concentration (Tmax) of 1.5 to 2 hours. The absolute bioavailability is 23%, and the active metabolite LBQ657 reaches Cmax within 3 hours [4]
- Metabolism: Sacubitril is rapidly hydrolyzed by esterases to LBQ657, which accounts for more than 90% of the NEP inhibitory activity in plasma. The terminal half-life of LBQ657 is 12 hours [4]
- Excretion: Approximately 83% of the dose is excreted in bile and 17% in urine. Less than 10% of the original drug was detected in urine [4].

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After oral administration, LCZ696 is metabolized into the active angiotensin receptor blocker valsartan and the inactive enkephalinase inhibitor prodrug AHU377. AHU377 is then further cleaved into the active enkephalinase inhibitor LBQ657.

Hepatotoxicity
In large prospective placebo-controlled trials of sacubitril/valsartan in patients with heart failure, cases of elevated ALT, serious adverse liver events, or death were typically not mentioned or listed. FDA clinical review of these trial data showed that 1.3% of patients taking sacubitril/valsartan experienced elevated ALT or AST, compared to a similar proportion (1.0%) in patients taking enalapril. All elevations were considered unrelated to treatment and likely due to congestive liver disease caused by heart failure. One case of jaundice with elevated transaminases occurred, but the patient discontinued combination therapy due to worsening renal function and developed acute decompensated heart failure. Since the approval and widespread use of sacubitril/valsartan, at least two reports of acute liver injury have been associated with its use. Both cases were mild and resolved rapidly after discontinuation of the drug. It is unclear whether the injury was caused by sacubitril or valsartan (or an interaction between the two). Valsartan, like other commonly used angiotensin receptor blockers, is known to be a rare cause of acute liver injury. The potential impact of heart failure and possible congestive liver disease is also a concern. None of the reported cases underwent liver biopsies or repeated use of the drug, which could have helped establish a causal relationship. Probability Score: D (Possibly a rare cause of clinically significant liver injury, possibly caused by valsartan rather than sacubitril). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Sacubitril is used only in combination with valsartan in the United States. Even at the lowest dose of the combination, the concentration of the drug in breast milk is extremely low. If the highest dose (4 times the maternal dose) of sacubitril were used, the concentration in breast milk would still be very low. Valsartan was not detected at this dose, so the combination appears unlikely to affect breastfed infants. ◉ Effects on Breastfed Infants No symptoms were observed in the breastfed infants of two women who received 24 mg of sacubitril and 26 mg of valsartan (Entresto). The extent of breastfeeding was not reported.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
Sacubitril and its metabolite LBQ657 have high binding rates to plasma proteins (94-97%).

[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).

Pharmacodynamics
In a 7-day controlled study of valsartan, sacubitril combined with valsartan (Entresto) significantly increased non-sustained sodium excretion, increased urinary cGMP levels, and decreased plasma MR-proANP and NT-proBNP levels in patients with high-flow-rate-exhaustion (HFrEF) compared to valsartan. In another 21-day study of HFrEF patients, this combination therapy significantly increased urinary ANP and cGMP levels, as well as plasma cGMP levels, and decreased plasma NT-proBNP, aldosterone, and endothelin-1 levels. Clinical studies have shown that this combination therapy has no effect on the QTc interval. Mechanism of Action: Sacubitril, as a prodrug, can be converted into the selective NEP inhibitor LBQ657. By blocking neprilysin (NEP), AHU377 prevents the degradation of natriuretic peptides (ANP, BNP), thereby leading to vasodilation, natriuresis, and inhibition of the renin-angiotensin-aldosterone system (RAAS) [1,4]
- Therapeutic use: Approved in combination with valsartan (LCZ696) for the treatment of heart failure with reduced ejection fraction (HFrEF), reducing cardiovascular mortality and hospitalization rates [3,4]
- Clinical pharmacology: In clinical trials, sacubitril/valsartan (Entresto) significantly improved exercise capacity and quality of life in patients with HFrEF, and reduced the risk of cardiovascular death by 20% compared to enalapril [4]

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AHU377 is the first prodrug of an angiotensin receptor neprilysin inhibitor (ARNi). LCZ696 (later renamed sacubitril/valsartan).
AHU377 itself is inactive and requires enzymatic cleavage to form the active enkephalinase inhibitor LBQ657.
LCZ696's dual action (blocking the renin-angiotensin-aldosterone system via valsartan and inhibiting enkephalinase via LBQ657) aims to enhance the beneficial natriuretic peptide activity while blocking the harmful angiotensin II activity.
Clinical trials evaluating LCZ696 (PARADIGM-HF, PARAMOUNT) have shown good efficacy in the treatment of heart failure.

C24H29NO5

411.49076

411.204

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

2

5

12

30

550

2

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.)