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].

Absorption, Distribution and Excretion
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.
52% to 68% of sacubitril (primarily as the active metabolite LBQ657) is excreted in urine. 37% to 48% of sacubitril (primarily as LBQ657) is excreted in feces
103 L

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Metabolism / Metabolites
Sacubitril is metabolized to LBQ657 by esterases. A low concentration (<10%) of a hydroxyl metabolite has been identified 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 it's metabolite LBQ657 is 9.9 to 11.1 hours.

Hepatotoxicity
The large prospective, placebo-controlled trials of sacubitril-valsartan in patients with heart failure usually did not mention or list ALT elevations or severe hepatic adverse events or deaths. In the FDA clinical review of data from these trials, elevations in ALT or AST were said to arise in 1.3% of patients on sacubitril-valsartan but a similar portion of those on enalapril [1.0%], and all elevations were judged to be unrelated to therapy and likely due to congestive liver disease due to heart failure. A single case of jaundice with aminotransferase elevations occurred, but in a patient who had stopped combination therapy because of worsening renal dysfunction and who was experiencing acute decompensated heart failure. Since approval and more widespread use of sacubitril-valsartan there have been at least two reports of acute liver injury attributed to its use. Both cases were relatively mild and resolved rapidly once the drug was stopped. Whether the injury was due to sacubitril or valsartan (or an interaction of both) was not clear. Valsartan, like other commonly used angiotensin receptor blockers, is known to be a rare cause of acute liver injury. The contribution of the underlying heart failure and possible congestive hepatopathy is also an issue. The reported cases did not have a liver biopsy and were not rechallenged with the drug which might have clarified the causality.
Likelihood score: D (possible rare cause of clinically apparent liver injury, which may be due to valsartan rather than sacubitril).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Sacubitril is available in the United States only in combination with valsartan. Milk levels after the lowest dose of the combination are very low. If sacubitril milk levels of the highest dosage (4 times greater) are proportional to maternal dosage, they would still be quite low. Valsartan was undetectable at this dosage, so the combination product appears unlikely to affect the nursing infant.
◉ Effects in Breastfed Infants
Two women taking sacubitril 24 mg and valsartan 26 mg (Entresto) did not observe any symptoms in their breastfed infants. Their extent of breastfeeding was not reported.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Sacubitril and it's metabolite, LBQ657 are highly bound to plasma protein (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
n a 7-day valsartan-controlled study in patients with reduced ejection fraction (HFrEF), administration of sacubitril + valsartan (Entresto) resulted in a significant non-sustained increase in natriuresis, increased urine cGMP, and decreased plasma MR-proANP and NT-proBNP compared to valsartan. In a 21-day study in HFrEF patients, it significantly increased urine ANP and cGMP and plasma cGMP, and decreased plasma NT-proBNP, aldosterone and endothelin-1. In clinical studies, this combination had no effect on QTc interval.

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.)