Impurity of Sacubitril
2R,4S-Sacubitril targets neprilysin (NEP), an enzyme that breaks down natriuretic peptides. By inhibiting NEP, it increases the levels of natriuretic peptides, promoting vasodilation and reducing blood pressure. It is used as a reference standard in analytical method development and quality control for sacubitril-containing products.

Sacubitril was recently approved by the Food and Drug Administration for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite sacubitrilat (LBQ657) to exert its intended therapeutic effects. Thus, understanding the determinants of sacubitril activation will lead to the improvement of sacubitril pharmacotherapy. The objective of this study was to identify the enzyme(s) responsible for the activation of sacubitril, and determine the impact of genetic variation on sacubitril activation. First, an incubation study of sacubitril with human plasma and the S9 fractions of human liver, intestine, and kidney was conducted. Sacubitril was found to be activated by human liver S9 fractions only. Moreover, sacubitril activation was significantly inhibited by the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. The in vitro study of cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in human liver. The CES1 genetic variant G143E can significantly impair sacubitril activation. Therefore, CES1 genetic variants appear to be an important contributing factor to interindividual variability in sacubitril activation, and have the potential to serve as biomarkers to optimize sacubitril pharmacotherapy[1].
In vitro, 2R,4S-Sacubitril is used as a reference standard and impurity for analytical method development. Its activity as a neprilysin inhibitor is expected to be similar to that of sacubitril, but its potency may differ due to the stereochemical configuration.

In vivo, 2R,4S-Sacubitril is not administered as a therapeutic agent. As an impurity of sacubitril, its presence in pharmaceutical products is controlled to ensure product quality and safety.

In vitro assays for 2R,4S-Sacubitril are primarily analytical in nature. High-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UPLC) with UV detection is used to quantify the impurity in sacubitril drug substance and drug products. Mass spectrometry (LC-MS/MS) may also be used for identification and quantification.

Cellular assays for 2R,4S-Sacubitril are not typically performed, as it is an impurity rather than an active pharmaceutical ingredient. Its effects on neprilysin activity could be assessed in cell-based assays if needed.

In vivo animal studies for 2R,4S-Sacubitril are not typically conducted, as it is an impurity rather than an active pharmaceutical ingredient. Studies would focus on toxicological assessment of the impurity in the context of sacubitril safety evaluation.

Pharmacokinetic data for 2R,4S-Sacubitril are not available, as it is not administered as a therapeutic agent. Its PK properties would be expected to be similar to those of sacubitril if administered, but its presence in pharmaceutical preparations is typically at trace levels.

Toxicological data for 2R,4S-Sacubitril are limited. As an impurity of sacubitril, its toxicity is assessed as part of the overall safety evaluation of sacubitril drug products. At the trace levels present, it is not expected to contribute significantly to the toxicity profile.

[1]. Sacubitril Is Selectively Activated by Carboxylesterase 1 (CES1) in the Liver and the Activation Is Affected by CES1 Genetic Variation. Drug Metab Dispos. 2016 Apr;44(4):554-9.

Recently, the U.S. Food and Drug Administration approved the combination of sacubitril and valsartan for the treatment of heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) into its active metabolite, sacubitrilat (LBQ657), to exert its intended therapeutic effects. Therefore, understanding the determinants of sacubitril activation will contribute to the improvement of sacubitril pharmacotherapy. The aim of this study was to identify the enzyme responsible for sacubitril activation and to determine the impact of genetic variation on sacubitril activation. First, incubation studies of sacubitril with human plasma and S9 fractions from human liver, intestine, and kidney were conducted. It was found that only the human liver S9 fraction could activate sacubitril. Additionally, the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate significantly inhibited sacubitril activation in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. In vitro studies using cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in the human liver. The CES1 gene variant G143E significantly impairs sacubitril activation. Therefore, CES1 genetic variation appears to be an important factor contributing to interindividual variability in sacubitril activation and has the potential to serve as a biomarker for optimizing sacubitril pharmacotherapy [1].
2R,4S-Sacubitril is a stereoisomer and impurity of sacubitril, an FDA-approved neprilysin inhibitor used for heart failure. It has a molecular weight of 411.49 and formula C24H29NO5. It is used as a reference standard in analytical development and quality control for sacubitril pharmaceuticals. It is not a drug and has no clinical approval.

C24H29NO5

411.490767240524

411.204

761373-05-1

Sacubitril;149709-62-6

9978992

Colorless to light yellow ointment

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

PYNXFZCZUAOOQC-LAUBAEHRSA-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+/m0/s1

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

761373-05-1; 2R,4S-Sacubitril; (2R,4S)-5-(Biphenyl-4-yl)-4-[(3-carboxypropionyl)amino]-2-methylpentanoic acid ethyl ester; (2S,4R)-Sacubitril; 4-[[(2R,4S)-5-ethoxy-4-methyl-5-oxo-1-(4-phenylphenyl)pentan-2-yl]amino]-4-oxobutanoic acid; ahu377 isomer 2; 4-(((2R,4S)-1-([1,1'-Biphenyl]-4-yl)-5-ethoxy-4-methyl-5-oxopentan-2-yl)amino)-4-oxobutanoic acid; SCHEMBL22420467;

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 : ~100 mg/mL (~243.02 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.)