NEP (neprilysin) (IC50 = 5 nM)

- 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) [3]
- 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 [3]
Brain natriuretic peptide-degrading peptides, including brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and natriuretic peptide (NP), are more abundant when sacubitril is present [2].

- 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 [1]
- 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 [3]
- 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]
Sacubitril (3, 10, and 30 mg/kg, p.o.) pretreatment raised ANP-induced plasma cGMP levels in normotensive rats by 2.4, 3.3, and 4.0 times, respectively (as compared to vehicle, 4 hours AUC) [1]. In Dahl-SS rats, sacubitril sodium (30 and 100 mg/kg, PO) has dose-dependent antihypertensive effects [1].

- 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 [3]
The rationale for the development and the Food and Drug Administration approval of LCZ-696 was based on the concept of an additive effect of the Ang II receptor blocker valsartan and the neutral endopeptidase (neprilysin) inhibitor AHU-377 for the treatment of hypertension and HF. The synergism from these drugs arises from the vasodilating effects of valsartan through its blockade of Ang II type 1 receptor and the action of natriuretic peptides atrial natriuretic peptide and B-type natriuretic peptide (BNP) by preventing their catabolism with neprilysin resulting in increase of cyclic guanosine monophosphate. This action of neprilysin is associated with increased natriuresis, diuresis, and systemic vasodilation, since these peptides have been shown to have potent diuretic, natriuretic, and vasodilating effects. In addition, it reduces the levels of N terminal pro-BNP. Therefore, administration of LCZ-696 results in significant reduction of wall stress from pressure and volume overload of the left ventricle as demonstrated by the reduction of N terminal pro-BNP, both significant constituents of hypertension and HF, and it is safe, well tolerated and is almost free of cough and angioedema [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. Finally, using telemetry, we assessed the antihypertensive effects of AHU-377 in conscious Dahl-SS and DOCA-salt models of hypertension [1].

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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 [1]
- 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]

- Oral Absorption: Sacubitril showed rapid oral absorption in rats, with Tmax of 1.5–2 hours. Absolute bioavailability was 23%, and the active metabolite LBQ657 reached Cmax within 3 hours [3]
- Metabolism: Sacubitril was rapidly hydrolyzed by esterases to LBQ657, which accounted for >90% of NEP inhibitory activity in plasma. LBQ657 had a terminal half-life of 12 hours [3]
- Excretion: Approximately 83% of the dose was excreted in bile, with 17% eliminated in urine. Less than 10% of the parent drug was detected in urine [3]

[1]. Comparative efficacy of AHU-377, a potent neprilysin inhibitor, in two rat models of volume-dependent hypertension. BMC Pharmacol 11, P33 (2011).

[2]. Sacubitril, valsartan and SARS-CoV-2. BMJ Evid Based Med. 2020 Jul 27:bmjebm-2020-111497.

[3]. Chrysant SG. Pharmacokinetic, pharmacodynamic, and antihypertensive effects of the neprilysin inhibitor LCZ-696: sacubitril/valsartan. J Am Soc Hypertens. 2017 Jul;11(7):461-468.

LCZ-696, sacubitril/valsartan, is a dual-acting molecule consisting of the angiotensin II (Ang II) receptor blocker valsartan and the neprilysin (neutral endopeptidase) inhibitor AHU-377 with significant beneficial effects in patients with hypertension and heart failure (HF). Several recent studies have demonstrated a higher effectiveness of LCZ-696 compared to valsartan in the treatment of hypertension and HF. The rationale for the development and the Food and Drug Administration approval of LCZ-696 was based on the concept of an additive effect of the Ang II receptor blocker valsartan and the neutral endopeptidase (neprilysin) inhibitor AHU-377 for the treatment of hypertension and HF. The synergism from these drugs arises from the vasodilating effects of valsartan through its blockade of Ang II type 1 receptor and the action of natriuretic peptides atrial natriuretic peptide and B-type natriuretic peptide (BNP) by preventing their catabolism with neprilysin resulting in increase of cyclic guanosine monophosphate. This action of neprilysin is associated with increased natriuresis, diuresis, and systemic vasodilation, since these peptides have been shown to have potent diuretic, natriuretic, and vasodilating effects. In addition, it reduces the levels of N terminal pro-BNP. Therefore, administration of LCZ-696 results in significant reduction of wall stress from pressure and volume overload of the left ventricle as demonstrated by the reduction of N terminal pro-BNP, both significant constituents of hypertension and HF, and it is safe, well tolerated and is almost free of cough and angioedema[3].

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- Mechanism of Action: Sacubitril acts as a prodrug that is converted to LBQ657, a selective NEP inhibitor. By blocking NEP, it prevents degradation of natriuretic peptides (ANP, BNP), leading to vasodilation, natriuresis, and inhibition of the renin-angiotensin-aldosterone system (RAAS) [3]
- Therapeutic Use: Approved in combination with valsartan (LCZ696) for the treatment of heart failure with reduced ejection fraction (HFrEF), where it reduces cardiovascular mortality and hospitalization [3]
- Clinical Pharmacology: In clinical trials, Sacubitril/valsartan (Entresto) significantly improved exercise capacity and quality of life in HFrEF patients, with a 20% lower risk of cardiovascular death compared to enalapril [3]

C24H28NNAO5

433.472598075867

433.187

C, 66.50; H, 6.51; N, 3.23; Na, 5.30; O, 18.45

149690-05-1

Sacubitril;149709-62-6;Sacubitril hemicalcium salt;1369773-39-6;Sacubitril-d4 hemicalcium salt;Sacubitril-d4;1884269-07-1; 149690-05-1 (sodium) 936623-90-4 (Valsarta + sacubitril); 936623-90-4 ; 137862-53-4

23707568

Brown solid

4.547

1

5

12

31

556

2

[Na+].O(CC)C([C@H](C)C[C@@H](CC1C=CC(C2C=CC=CC=2)=CC=1)NC(CCC(=O)[O-])=O)=O

RRTBVEJIZWGATF-JKSHRDEXSA-M

InChI=1S/C24H29NO5.Na/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);/q;+1/p-1/t17-,21+;/m1./s1

sodium 4-(((2S,4R)-1-([1,1'-biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoate

AHU377; LCZ696; AHU 377; Sacubitril sodium; 149690-05-1; AHU-377 sodium salt; Ahu-377 sodium; UNII-MOP72GEP8Z; MOP72GEP8Z; SACUBITRIL SODIUM [MI]; DTXSID90164362; 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: >10 mM

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.)