B-type natriuretic peptide precursor (BNP) and its N-terminal fragment (NT-proBNP) are widely accepted as being both useful and cost-effective biomarkers of heart failure (HF) diagnosis and therapy monitoring. However, in spite of the fact that both biomarkers are incorporated into the majority of the national and international cardiovascular therapy guidelines, our understanding of their complex nature remains far from being complete and comprehensive. Moreover, the constant development and implementation of new therapeutic agents generate new questions and challenges regarding the use of these biomarkers for HF diagnostics and the monitoring of HF therapy.
The new FDA approved heart failure (HF) drug LCZ696 (EntrestoTM, Novartis) is an example of the challenges one faces in this field. This particular drug, which combines the neprilysin inhibitor and the angiotensin II receptor inhibitor, has greatly stimulated the interest of the cardiology community in neprilysin. This ubiquitous protease is responsible for the degradation of a variety of important vasoactive peptides, including natriuretic peptides (NPs). In light of this, the beneficial effect of this new drug is expected to be achieved by inhibiting NPs degradation, increasing the level of ANP and BNP and, as a consequence, improving outcomes as well as symptoms and physical limitations of HF (1). As LCZ696 is expected to increase the circulating concentrations of BNP, it has been suggested that the clinical significance of BNP values in patients undergoing therapy with LCZ696 might be challenging or even misleading from a diagnostic perspective. However, the complex biochemistry of NPs, as well as the diversity of HF forms, means that the answer to this question is not so obvious.
Natriuretic peptides are synthesized in the form of prohormones, which have to be cleaved in order to produce bioactive compounds. A number of studies have convincingly shown that the predominant form of plasma BNP-immunoreactivity in HF patients (which is measured by commercial BNP immunoassays) is represented by its uncleaved precursor proBNP, which differs from BNP due to the presence of the 76 amino acid N-terminal extension (2-4). Therefore, the inhibition of neprilysin in HF patients might only have a pronounced effect on the level of immunoreactive BNP if proBNP is also a substrate of neprilysin.
On this basis, we investigated the degradation of proBNP (both glycosylated and non-glycosylated forms) by the action of neprilysin and tested the susceptibility of different BNP epitopes to neprilysin-dependent proteolysis. Our findings demonstrated that in contrast to BNP, the major BNP-immunoreactive form - proBNP - is not susceptible to neprilysin cleavage and remains intact even following prolonged incubation with neprilysin. In addition, we showed that in the case of BNP, the epitope 14-21, which comprises the site Arg17-Ile18 (which is known as the neprilysin cleavage site), is much more susceptible to the cleavage by neprilysin as compared to the epitope 11-17, which does not comprise this site (5).
The lack of susceptibility of proBNP to neprilysin activity suggests that the effect of neprilysin inhibitors on the measured BNP immunoreactivity should not be as significant as one might expect due to a major circulating BNP immunoreactive form, proBNP, not being degraded by neprilysin.
Furthermore, we might suggest that the effect of neprilysin inhibition on the level of immunoreactive BNP in patients treated with LCZ696 should be assay-dependent. Specifically, BNP immunoassays that utilize antibodies with epitopes comprising the site Arg17-Ile18 are expected to be more sensitive to proteolysis by neprilysin than immunoassays that utilize antibodies with other specificities. Notably, a proBNP-specific assay with no cross-reactivity to BNP might be an attractive alternative to be used along with LCZ696 therapy as proBNP (uncleaved BNP precursor, 108 amino acid residues) is not susceptible to neprilysin cleavage and as a consequence its level should not be influenced by neprilysin inhibition.
A possible rapid introduction of the LCZ696 drug in routine clinical practice has definitely presented new challenges to both ongoing and future clinical trials addressing the issues of using biomarkers to guide HF therapy. Taking into account the complexity of the NP system and the diversity of HF states, it would appear that at present no theoretical considerations can help in regard to choosing either the measurement of BNP or that indeed NT-proBNP alone should be used to follow the LCZ696 therapy.
In conclusion, we would suggest that the effect of neprilysin inhibition by new therapeutic agents (e.g. LCZ696) on the level of BNP and NT-proBNP as well as the BNP/NT-proBNP ratio should be studied in future clinical trials that use several immunoassays based on different antibodies for a better comprehension of the diagnostic and prognostic values of these biomarkers.
References
1. 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.
2. Costello-Boerrigter LC, Lapp H, Boerrigter G, Lerman A, Bufe A, Macheret F, et al. Secretion of prohormone of B-type natriuretic peptide, proBNP1-108, is increased in heart failure. JACC Heart failure 2013;1:207-12.
3. Seferian KR, Tamm NN, Semenov AG, Mukharyamova KS, Tolstaya AA, Koshkina EV, et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 2007;53:866-73.
4. Liang F, O'Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 2007;49:1071-8.
5. Semenov AG, Katrukha AG. Different susceptibility of B-type natriuretic peptide (BNP) and BNP precursor (proBNP) to cleavage by neprilysin: The N-terminal part does matter. Clin Chem 2016;62:617-22.