Introduction

Recently, the novel compound sacubitril-valsartan, an angiotensin receptor neprilysin (NEP) inhibitor, was documented to improve outcomes in patients with systolic heart failure (HF).¹ The compound is a salt complex of valsartan and sacubitril, the NEP inhibitor prodrug that is processed in the liver into its active moiety,² which then interacts with a variety of vasoactive peptides of relevance in HF including enhancing natriuretic peptide (NP) system activity.² Since its approval for the treatment of chronic HF with reduced ejection fraction, a commonly raised suspicion is that treatment with this new drug may make brain, or B-type, NP (BNP) testing useless for disease monitoring of these patients because sacubitril may interfere with BNP clearance.³ Is it already justified to promote N-terminal- (NT-) proBNP as the gold standard biomarker in HF?³ In this analysis, that hasty hypothesis is critically assessed based on the pathophysiology of NP degradation, the limitations of current NP measurement technologies, and the still-limited published data on the effects of NEP inhibition on NP plasma concentrations in humans. Because the main clinical application of BNP testing currently is and most likely will remain the rule-out of clinically suspected HF, there is no major significant impairment to be expected for BNP testing in daily practice. However, some issues have to be considered when using BNP testing for disease monitoring of patients with chronic HF who are on sacubitril-valsartan treatment.

Pathophysiology of Cardiac BNP and Circulating Forms of proBNP-Derived Peptides in Humans

The family of NPs comprises 3 major forms: atrial, or A-type, NP (ANP), BNP, and C-type NP (CNP). ANP and BNP are key regulators of the body's regulation of volume and blood pressure homeostasis by stimulation of vasodilatation and natriuresis.^4-8 ANP and BNP are primarily secreted from the heart in response to increased myocardial stretch mediated by volume or pressure overload; thus, increase in NP can be regarded as markers of "cardiac stress." In contrast to ANP, only limited amounts of BNP are stored in atrial granules, so most BNP secretion requires gene transcription and subsequent peptide synthesis mostly in the ventricles. Figure 1 summarizes our current understanding of BNP synthesis and processing. The initial gene product is pre-proBNP 1-134, which undergoes removal of a 26-amino acid signal peptide in the sarcoplasmatic reticulum during translation, leading to formation of proBNP 1-108. proBNP may be glycosylated post-translation at several sites in its N-terminus to a variable degree.^9-11 Subsequently, proBNP is cleaved by prohormone convertases, such as furin and corin, to release the 76-amino acid NT portion NT-proBNP 1-76 and the biologically active C-terminal 32-amino acid molecule BNP 1-32. proBNP is secreted as well, and high concentrations have been reported in blood of patients with HF.^10,12 proBNP and NT-proBNP as well as their split products have little to no hormonal activity. Some processing of secreted proBNP (probably by furin) can occur in the blood as well. Although the exact nature and distribution of circulating proBNP-derived molecules in healthy individuals and patients with various cardiac diseases is still uncertain, it has been recognized that both BNP and NT-proBNP are modified into a mixture of various fragments in blood, although intact circulating proBNP, glycosylated NT-proBNP, and glycosylated proBNP have been observed in patients with HF as well.^8-15 Only a small portion of BNP circulates as the full-length BNP 1-32, and plasma from patients with HF mainly contains diverse degraded forms truncated from the N- and C-terminal end (e.g., BNP 3-32, 5-31, 8-31, 1-25, 1-26).¹⁵

Figure 1: Synthesis, Processing, and Circulating Forms of proBNP-Derived Peptides

The commercially available BNP and NT-proBNP routine assays are immunoassays,^8,13 and the antibodies used in BNP assay do not cross-react with NT-proBNP and vice versa. However, both BNP and NT-proBNP assays cross-react with intact proBNP. Immunoreactive BNP and NT-proBNP forms of high and low molecular mass have been described in patients; those forms still need to be better characterized using state-of-the art analytical techniques of proteomics. Depending on the antibodies used, BNP and NT-proBNP assays detect the circulating mixture of BNP and NT-proBNP degradation products to a varying extent; thus, the effects of sacubitril on BNP test results are assay dependent.^13,16 Consequently, BNP test results do not reflect the true concentration of the hormonally active BNP in the blood sample.

NP Clearance and Possible Interferences by Sacubitril

NP clearance (Figure 2) occurs by two major mechanisms: enzymatic extracellular degradation by NEP and intracellular degradation by insulin-degrading enzyme, as well as via binding to the NP receptor-C.⁶ This receptor binds all three forms of the NP family. NEP is a membrane-bound ectoenzyme that cleaves various peptide substrates including insulin beta-chain, beta-amyloid, and various peptide hormones of relevance in HF such as angiotensin II, endothelin-1, bradykinins, substance P, adrenomedullin, calcitonin gene-related peptide, and NPs.6 The combination of sacubitril with valsartan additionally inhibits the renin-angiotensin aldosterone system by blocking angiotensin 1 receptor, which is important because NEP degrades angiotensin II as well. NEP is widely expressed, including the kidneys, lungs, and vascular endothelial cells, and can be released from the cell surface yielding soluble NEP with catalytic activity.¹⁷ NEP cleaves ANP and CNP at the cysteine-phenylalanine bond and breaks the ring structure, which eliminates receptor binding. Intact human BNP, by contrast, is a much poorer substrate for NEP, and it is mainly not cleaved at this ring strucure.¹⁸ NEP inhibitors have failed to block BNP degradation by human kidney membranes.¹⁹ proBNP is resistent to NEP degradation.²⁰ It cross-reacts with commercial BNP assays and has been reported to be the major circulating form detected by BNP assays in patients with chronic HF.¹²

Figure 2: Clearance of NP With Interference of Sacubitril-Valsartan

Clinical Results: Reported Effects of Sacubitril-Valsartan Treatment on NP Concentrations

Oral NEP inhibitors elevate the NP system activity as assessed by increases in plasma and urine cyclic guanosine monophosphate (cGMP) concentrations in humans.^21,22 cGMP is the second messenger of NPs.^4,5,7 Oral sacubitril-valsartan treatment leads to a dose-dependent increase in plasma cGMP in healthy human volunteers,²¹ which provides evidence for an increase in NP system activity.²³

In PARADIGM-HF (A Multicenter, Randomized, Double-blind, Parallel Group, Active-controlled Study to Evaluate the Efficacy and Safety of LCZ696 Compared to Enalapril on Morbidity and Mortality in Patients With Chronic Heart Failure and Reduced Ejection Fraction),^1,22 plasma BNP and urinary cGMP concentrations were significantly higher in the sacubitril-valsartan group at 4 weeks and 8 months. This was suggested to reflect apparent NP system augmentation. However, the observed relative average BNP increase was small compared with cGMP increase (on average only about 10 versus 90%, respectively). By contrast, NT-proBNP was significantly lower (average decline of about 30 and 20%, respectively) in the sacubitril-valsartan group. Recently, it was reported that in the patient cohort of the PARADIGM-HF study, patients who attained a reduction of NT-proBNP <1000 ng/L had a lower subsequent rate of cardiovascular death or HF hospitalization independent of the treatment group.²⁴ A similar analysis for BNP in these patients is pending and has not yet been published.

In the earlier PARAMOUNT (Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction) trial,²⁵ in which sacubitril-valsartan was studied in patients with chronic HF and preserved ejection fraction, there was a significant and greater decline in NT-proBNP (on average about 25%) 12 weeks after treatment compared with valsartan alone. Although there was a rapid (at 4 weeks) and sustained decline in NT-proBNP, the difference between both treatment groups was no longer statistically significant at 36 weeks.²⁴ BNP was not measured in this study. In both studies, however, the reported average BNP and NT-proBNP changes, though statistically significant, were within the known relatively large week-to-week biological variation of these biomarkers.^26,27 To date, ANP and NT-proANP or CNP results have not been published in humans taking sacubitril-valsartan.

Implications of Sacubitril-Valsartan for NP Testing in Clinical Practice

Based on the available data on the effects of sacubitril-valsartan on BNP and NT-proBNP, the interpretation of NT-proBNP testing in HF appears to be unaffected by NEP inhibition. However, the relative increases in BNP plasma concentrations reported in the PARADIGM HF trial²² were also only modest (median baseline BNP value was approximately 200 ng/L to a median of about 225 ng/L in the sacubitril-valsartan arm). BNP concentrations >500 ng/L, the frequently used rule-in decision limit for HF,^28,29 were rare (75% of BNP concentrations in the sacubitril-valsartan arm were approximately 450 ng/L). The high negative predictive value for exclusion of HF for BNP testing will obviously be maintained in patients with sacubitril-valsartan treatment. After an initial modest rise, which is markedly smaller than in cases of cardiac decompensation, BNP may reach a new steady state during sacubitril-valsartan treatment and then begin to drop long term, if marked clinical improvement occurs. If one wants to use BNP for disease monitoring in these patients, it is advisable to measure BNP about 1 month after start of sacubitril-valsartan treament to obtain a new baseline value for subsequent HF monitoring. By contrast, for disease monitoring of patients with chronic HF on sacubitril-valsartan, the current concept of "the lower the NT-proBNP test result the better the prognosis" will always remain to be true. It was even suggested to use an increase in BNP to define who might respond to sacubitril-valsartan or to assess patient compliance. The potential of the BNP/NT-proBNP ratio for chronic HF monitoring with sacubitril-valsartan remains to be further investigated, and undoubtedly more clinical data are still needed.

Conclusions

• The clinical utility of BNP testing to rule out HF, which will remain its major clinical application in routine use, will not be impaired by sacubitril-valsartan treatment of patients.
• The clinical benefit of BNP or NT-proBNP testing for treatment monitoring or even therapy guidance in patients with HF including patients on sacubitril-valsartan therapy remains to be proven, and routine NP testing is currently still not recommended for this purpose.
• BNP is a poor substrate of NEP, and thus no major effects of NEP inhibition on BNP concentrations are to be expected by NEP inhibitors.
• Available data suggest that the concept "the lower the BNP test result,the better the prognosis of chronic HF patients" may no longer be true for patients during the first weeks of sacubitril-valsartan treatment due to modest BNP increases. Therefore, BNP measurement about 1 month after start of treatment is recommended to obtain a new baseline value for follow-up testing.
• The clinical interpretation of NT-proBNP test results in patients with HF is unaffected by NEP inhibition. The concept "the lower the NT-proBNP test result the better the prognosis of chronic HF" is also valid after start of treatment with sacubitril-valsartan.

References

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Keywords: Adrenomedullin, Aldosterone, Angiotensin II, Atrial Natriuretic Factor, Acute Coronary Syndrome, Blood Pressure, Calcitonin, Calcitonin Gene-Related Peptide, Cysteine, Enalapril, Endothelial Cells, Endothelin-1, Furin, Galium, Guanosine Monophosphate, Heart Failure, Systolic, Immunoassay, Insulin, Insulysin, Natriuretic Peptide, Brain, Natriuretic Peptide, C-Type, Natriuretic Peptides, Neprilysin, Peptide Fragments, Phenylalanine, Prodrugs, Proprotein Convertases, Protein Sorting Signals, Proteomics, Receptors, Angiotensin, Renin, Substance P, Transcription, Genetic, Vasodilation

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