Lu Zhou,Liang Zou,Lili Sun,Hui Zhang,Wenkai Hui,Qiaogen Zou
Sacubitril–valsartan (SV) is a first-in-class inhibitor of the angiotensin II receptor and neprilysin for the treatment of chronic heart failure (HF) and hypertension. A stereoselective normal-phase high performance liquid chromatographic method was developed and validated for separation of sacubitril (SA), valsartan (VA) and their stereoisomeric impurities. Chromatography was performed using mobile phase A of n-hexane with 0.1% TFA added and mobile phase B comprising ethanol, isopropanol and TFA (80 : 20 : 0.1, v/v/v) and delivered at a flow rate of 1.0 mL min−1 on a Chiralcel OJ-H column (250 mm × 4.6 mm, 5 μm). The stereoisomers were monitored at a wavelength of 254 nm and the whole separation was achieved within 50 min. The method was validated in terms of specificity, linearity (R2 ≥ 0.998), accuracy (98.3–99.5%), precision (%RSD ≤ 1.82), limit of detection (0.06 μg mL−1 and 0.10 μg mL−1) and limit of quantification (0.2 μg mL−1 and 0.3 μg mL−1). The sample solution and mobile phase were found to be stable for at least 48 hours. The final optimized method was successfully applied to separate sacubitril–valsartan from their stereoisomers and showed characteristics of good repeatability and accuracy for quantitative determination of the stereoisomers in bulk drug.
Sacubitril/valsartan (Sac/Val) has emerged as an effective compound with myocardium-protective functions in experimental and clinical trials for heart failure. This study investigated the function of Sac/Val in hypertensive heart disease (HHD) and explored the underlying mechanism. Spontaneous hypertensive rats (SHRs) were used as an animal model of HHD. Sac/Val administration ameliorated pathological injury, fibrosis, cell apoptosis, inflammatory cytokine production, and oxidative stress in the myocardial tissues of HHD rats. Similar findings were observed in vitro, where Sac/Val treatment reduced inflammation, oxidative stress, and fibrosis-related markers in Ang II-challenged H9C2 cells. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was identified as a target of Sac/Val which was downregulated in HHD models, and CAMKK2 protein levels were restored by Sac/Val treatment. Lentiviral vector-induced CAMKK2 knockdown reduced the phosphorylation levels of AMPKα, AKT, and GSK-3β, thereby negating the treatment effects of Sac/Val. Further treatment with AICAR, an AMPK agonist, significantly reactivated the AMPK/AKT/GSK-3β cascade and alleviated inflammatory injury in both models. Collectively, this study suggests that Sac/Val ameliorates inflammation and oxidative stress in HHD by restoring the CAMKK2 protein and activating the AMPK/AKT/GSK-3β cascade.
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