ИСТИНА

Background A novel heart failure drug, EntrestoTM (LCZ696), which combines a neutral endopeptidase (neprilysin) inhibitor and an inhibitor of the angiotensin II receptor, was suggested to augment circulating natriuretic peptides concentrations, A-type and B-type natriuretic peptides (ANP and BNP), that are involved in maintaining cardiorenal homeostasis. Both ANP and BNP are synthesized in the form of the prohormones proANP and proBNP. We have previously shown that, in contrast to BNP, its precursor, proBNP, is not cleaved by neprilysin. ANP is known to be a highly unstable peptide and is inactivated by degradation into smaller fragments, mainly by neprilysin. proANP is found in the circulation along with ANP and might serve as a circulating source of ANP. Currently, the susceptibility of proANP to degradation by neprilysin is unknown. The aim of this study was to determine if proANP is susceptible to neprilysin-mediated cleavage and if plasma proANP could benefit from the inhibition of neprilysin activity by the therapy with neprilysin inhibitors (e.g. EntrestoTM). Methods: Purified human proANP (expressed in E. coli) was incubated with neprilysin (expressed in CHO cells) for different time periods and its susceptibility to neprilysin-mediated degradation was compared with synthetic ANP and BNP, as well as proBNP (expressed in E. coli). Immunoassays based on antibodies with the epitopes comprising the known sites of neprilysin cleavage of either ANP or BNP combined with mass-spectrometry (MS) analysis were employed for quantitative and qualitative analysis respectively. Results: In contrast to both ANP and BNP, proANP was not susceptible to neprilysin-mediated cleavage (similarly to proBNP) and remained intact even after prolonged incubation with the enzyme (up to 4 hours) as follows from both the results of immunoassays (where no decrease in immunological activity was observed) and MS analysis (where no degraded forms were detected). Conclusions: The current data suggests that proANP is not a substrate of neprilysin and consequently its plasma concentration should not be augmented by the inhibition of neprilysin activity. In light of this, proANP might represent a stable ANP form that is not affected by proteolytic degradation in the circulation.