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B-type natriuretic peptide testing in the era of neprilysin inhibition: are the winds of change blowing?

There is nothing permanent except change.

--Heraclitus of Ephesus

The care of patients with heart failure (HF) [2] and reduced ejection fraction has traditionally included therapies resulting in inhibition of the renin-angiotensin-aldosterone system; specifically, current clinical practice guidelines articulate use of either angiotensin converting enzyme inhibitor (ACEi) or angiotensin II receptor blocker (ARB) as first-line therapy for patients with HF and left ventricular ejection fraction <40% (1). Accordingly, the use of ACEi or ARB therapy for such patients had been viewed as a foundation of HF care until recently.

In spring 2014, however, the medical community received startling news of the premature suspension of the Prospective Comparison of ARNI [Angiotensin Receptor-Neprilysin Inhibitor] with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF). The trial was suspended because of overwhelming evidence that treatment of HF and reduced ejection fraction with the combination ARB (valsartan)/neprilysin inhibitor (sacubitril) resulted in substantial reduction in a wide array of cardiovascular complications compared with the gold standard ACEi comparator, enalapril.

The magnitude of benefit from valsartan/sacubitril is hard to overstate: compared with well-dosed ACEi therapy, those treated with neprilysin inhibition in the PARADIGM-HF trial experienced 20% reduction in cardiovascular death or hospitalization for HF (P = 4.0 X [10.sup.-7]) and 16% reduction in all-cause mortality (P < 0.001) (2). Results such as those from PARADIGM-HF are a rarity in HF trials, where development of new classes of therapeutics has been challenging.

Why Inhibit Neprilysin?

Neprilysin is a neutral endopeptidase that is responsible for the degradation of several endogenous vasoactive substances, including natriuretic peptides [atrial natriuretic peptide, B-type natriuretic peptide (BNP), and C-type natriuretic peptide], bradykinin, and adrenomedullin. Given that neprilysin degrades vascular peptides, its blockade results in a net vasodilatory effect, potentiated by its combination with an ACEi or ARB; conceptually, by inhibiting neprilysin together with inhibition of the renin-angiotensin-aldosterone system, greater amelioration of neurohormonal activation, vasoconstriction, sodium retention, and ventricular remodeling would occur.

Effects of Neprilysin Inhibition on the BNP Family

In vitro studies suggest that neprilysin cleaves BNP in several regions (3). As neprilysin degrades epitopes recognized by standard immunoassays for BNP, recovery of the peptide decreases. Accordingly, neprilysin inhibition predictably results in an increase of BNP concentrations (4): in PARADIGM-HF, concentrations of plasma BNP (measured by using the Advia Centaur assay) increased on average by 50 pg/mL from a baseline median concentration of 255 pg/mL (5). After 8 months, median BNP concentrations in those treated with neprilysin inhibition were lower than at 4 weeks in PARADIGM-HF, tempting one to suggest that increased BNP concentrations eventually resolve; however, when one accounts for the simultaneous BNP reduction in enalapril-treated patients over time, it is more likely that the later decrease in BNP concentration was related to reduced BNP release (rather than neprilysin inhibition wearing off). Also, BNP values in those treated with valsartan/sacubitril (angiotensin receptor-neprilysin inhibitor) still appeared to be higher and remained modestly above baseline values.

In contrast to BNP, N-terminal pro-BNP (NT-proBNP) is invulnerable to neprilysin. Thus, reflecting the benefit of the drug, rapid and sustained reduction in NT-proBNP was seen following treatment with valsartan/sacubitril (5).

It is also known that a substantial percentage of circulating BNP and NT-proBNP detected in those with advanced HF is actually an uncleaved precursor peptide, proBNP whose detection occurs owing to cross-reactivity between epitopes in BNP and NT-proBNP.

Open Questions about BNP and Neprilysin Inhibition

Although it is known that treatment with neprilysin inhibition results in the sustained increase of BNP as determined using 1 commercially available assay, numerous questions remain.

First, does neprilysin affect BNP recovery across all assays for BNP in a similar fashion? Second, does neprilysin inhibition affect the ability to measure proBNP? Third, what does treatment with a neprilysin inhibitor do to a clinician's ability to interpret BNP concentrations?

It is in this context that Semenov and Katrukha performed a set of in vitro experiments focused on the effects of neprilysin on BNP and proBNP (6). In their work published in the April issue of Clinical Chemistry, the authors incubated BNP and proBNP with neprilysin. They used 2 separate BNP assays to measure recovery. The first was based on a capture antibody toward an epitope located in the region of amino acids 14--21 and a detection antibody recognizing an epitope in amino acid region 26--32. The second was a single epitope assay toward amino acid region 11--17. These 3 epitopes cover regions recognized by several of the most commonly used BNP assays.

Within the context of this in vitro model, the authors showed that results from the single-epitope BNP assay experienced less loss of BNP recovery than assays using capture antibodies against an epitope found between amino acids 14 and 21; both, however, demonstrated substantial decrease in BNP recovery.

In mass spectrometry analyses, the authors found that neprilysin degraded BNP in several regions, and did so in a sequential fashion, with some sites (e.g., [Met.sub.4] - [Val.sub.5] or [Arg.sub.17] - [Ile.sub.18]) affected earlier than others (e.g., [Val.sub.28] - [Leu.sub.29]). Finally, the authors demonstrated that proBNP was not degraded by neprilysin, regardless of the presence or absence of the heavy glycosylation that is known to exist in vivo.

Importantly, the results of Semenov and Katrukha suggest that use of valsartan/sacubitril may affect all currently available BNP assays, and may do so in a heterogeneous fashion: those BNP assays based on epitopes found in the more vulnerable regions of BNP (e.g., [Met.sub.4] - [Val.sub.5] or [Arg.sub.17] - [Ile.sub.18]) may report an increase in BNP different from that of assays based on less vulnerable regions.

That said, there are substantial caveats to this important possibility. First, results in vitro do not translate to what will be found in vivo; differences in timing of degradation by neprilysin between BNP regions may be meaningless outside of the test tube, so it is entirely speculative that one BNP assay will be affected more than another. Second, the effects of chronic neprilysin inhibition were not evaluated.

The Importance of Understanding Effects of Neprilysin Inhibition

Natriuretic peptide testing has revolutionized HF care by facilitating earlier and more secure diagnosis, assisting in gauging severity of HF, and representing a prognostic gold standard in the diagnosis. Furthermore, the potential use of natriuretic peptides to guide HF care of patients (with therapies adjusted with a goal to reduce natriuretic peptide concentrations) is being explored.

In those being treated with valsartan/sacubitril, the consequences of misunderstanding an increase in BNP may be significant. On the one hand, ignoring a true increase in BNP due to decompensation of HF (incorrectly assuming that it was due to neprilysin inhibition) may lead to missed opportunities to intervene and prevent hospitalization or death. On the other hand, overreacting to a neprilysin-mediated increase in BNP might result in overtitration of HF medications, with risk for treatment-related adverse events. Besides patient harm in both scenarios, it would be most undesirable for dissatisfaction to develop with natriuretic peptide testing or use of valsartan/sacubitril.

Although it is simple to suggest, as the authors do, to simply measure NT-proBNP (or proBNP) in those patients treated with valsartan/sacubitril, this may not be entirely practical, particularly in the short term. Although some may change from testing BNP to NTproBNP, it is fair to say many will not, at least initially. Clinicians must therefore be cautious in interpreting BNP results in patients being treated with valsartan/sacubitril, awaiting future studies to illuminate how neprilysin inhibition affects natriuretic peptide biology, both acutely and chronically.

The arrival of neprilysin inhibition will substantially change the landscape of the HF biomarker world. Indeed, more than a decade after the arrival of BNP testing, there remains nothing permanent in this world except for change itself. How clinicians, laboratorians, and the in vitro diagnostics industry rise to the challenge of this change is the relevant question.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: J. Januzzi, Roche Diagnostics, Critical Diagnostics, Sphingotec, Philips, Cardiorentis, Novartis, Amgen, and Boehringer-Ingelheim.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: J. Januzzi, Roche Diagnostics, Siemens Diagnostics, Singulex, and Prevencio.

Expert Testimony: None declared.

Patents: None declared.


(1.) Yancy CW, Jessup M, Bozkurt B, Butler J, Casey DE Jr, Drazner MH, et al. 2013 ACCF/ AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association taskforce on practice guidelines. J Am Coll Cardiol 2013;62:e147-239.

(2.) McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med 2014;371:993-1004.

(3.) Norman JA, Little D, Bolgar M, Di Donato G. Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass-spectrometry. Biochem Biophys Res Commun 1991;175:22-30.

(4.) Rademaker MT, Charles CJ, Espiner EA, Nicholls MG, Richards AM, Kosoglou T. Neutral endopeptidase inhibition: augmented atrial and brain natriuretic peptide, haemodynamic and natriuretic responses in ovine heart failure. Clin Sci (Lond) 1996;91:283-91.

(5.) Packer M, McMurray JJ, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, et al. Angiotensin receptor neprilysin inhibition compared with enalapril on the risk of clinical progression in surviving patients with heart failure. Circulation 2015;131:54-61.

(6.) Semenov AG, Katrukha AG. Different susceptibility of B-type natriuretic peptide (BNP) and BNP precursor to cleavage by neprilysin: the N-terminal part does matter. Clin Chem 2016;62:617-22.

James L. Januzzi, Jr. [1] *

[1] Division of Cardiology, Massachusetts General Hospital, Boston, MA.

* Address correspondence to this author at: Cardiology Division, Massachusetts General Hospital, 32 Fruit St, Yawkey 5984, Boston, MA 02114. Fax 617-643-1620; e-mail

Received February 24, 2016; accepted February 26, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.255455

[2] Nonstandard abbreviations: HF, heart failure; ACEi, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blocker; PARADIGM-HF, Prospective Comparison of ARNI with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure; BNP, B-type natriuretic peptide; NT-proBNP, N-terminal pro-BNP.
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Title Annotation:Editorials
Author:Januzzi, James L., Jr.
Publication:Clinical Chemistry
Geographic Code:1USA
Date:May 1, 2016
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