Sacubitril is selectively activated by carboxylesterase 1 (CES1) in the liver and the activation is affected by CES1 genetic variation

Sacubitril was recently approved by the FDA for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction (HFrEF). As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite LBQ657 in order 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 CES1 inhibitor bis‐(p‐nitrophenyl) phosphate (BNPP) in human liver S9. Further incubation studies with recombinant human carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) 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. Support or Funding Information Research reported in this publication was supported in part by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number 2UL1TR000433 (Hao‐Jie Zhu), the National Institute on Aging (R21AG048500) (Hao‐Jie Zhu), and the American Association of Colleges of Pharmacy (AACP) 2015 New Investigator Award (Hao‐Jie Zhu). 1Hydrolysis of sacubitril (A), enalapril (B), and fluorescein diacetate (C) by pooled human plasma, and the S9 fractions prepared from human liver (HLS9), intestine (HIS9), and kidney (HKS9). The hydrolytic products were determined after the substrates were incubated with the enzymes at 37°C for 10 min. Data are expressed as the formation rate of hydrolytic products (Mean ± S.D., n=3).2Hydrolysis of sacubitril and fluorescein diacetate by recombinant human CES1 and CES2 (A), and enzymatic kinetics of recombinant CES1‐catalyzed sacubitril hydrolysis (B). Data were presented as means ± S.D. (n=3).3Activation of sacubitril in cell S9 fractions prepared from cells stably transfected with wild type (WT) CES1 and the variant G143E. The formation of the active metabolite LBQ657 was determined after incubation of sacubitril with the S9 fractions at 37°C for 10 min. Final concentrations of sacubitril and cell S9 fractions were 200 μM and 0.05 mg/ml, respectively. Data are presented as means from three independent experiments with error bars representing S.D..4Effect of the CES1 variant G143E on sacubitril activation (left panel) and CES1 protein expression (right panel) in human liver samples. The final concentrations of HLS9 and sacubitril in the reaction system were 0.05 mg/ml and 200 μM, respectively. LBQ657 formed from sacubitril hydrolysis was determined after incubation of the substrate with individual HLS9 at 37°C for 10 min. Data are mean values of two independent experiments. Horizontal bars represent mean values in each group.