Neprilysin Inhibitors in Heart Failure

Highlights • Neprilysin cleaves natriuretic peptides, bradykinin, adrenomedullin, substance P, angiotensin I and II, and endothelin.• In patients with very advanced HF, the downstream response to natriuretic peptides is blunted, and neprilysin inhibition does not appear to add benefit.• In post-MI patients without HF, there may not be a need for increased natriuretic peptide availability with neprilysin inhibition.• Long-term studies are needed to determine the effects of angiotensin receptor–neprilysin inhibitors on albuminuria, obesity, glycemic control, blood pressure, and cognitive function in patients with HF.

N eprilysin is a zinc-activated endopeptidase that cleaves peptides up to 40 to 50 amino acids and has a broad role in cardiovascular, renal, pulmonary, gastrointestinal, endocrine, and neurologic functions (Central Illustration). This endopeptidase was identified by unrelated investigators at different times and given different names.
Neprilysin was initially described in 1973 as a neutral proteinase in rat kidney brush border membranes. 1 A few years later, it was independently described as a brain enzyme responsible for the inactivation of enkephalin and was called enkephalinase. 2 Subsequently, in the 1980s, it was discovered that the enzyme that was identified to break down substance P and enkephalin was identical to the endopeptidase of kidney microvilli and was given the common name of endopeptidase. 3 Clinicians may be surprised to learn that that common acute lymphoblastic leukemia antigen (CALLA), an important cell surface marker for diagnosis and prognosis of acute lymphoblastic leukemia 4 ; cluster of differentiation 10 (CD10), the immunohistochemical marker correlating to a higher histologic grade, larger tumor size, metastasis, and survival rate in patients with certain solid tumors 5,6 ; and skin fibroblast elastase, implicated in skin aging and wrinkle formation, are also identical to neprilysin 7 (Table 1).
Neprilysin is widely distributed in mammalian tissues, including the renal tubules, intestine, adrenal gland, brain, endothelial cells, cardiac myocytes, lung, gut, fibroblasts, smooth muscle cells, and hematopoietic cells 8,9 (Figure 1). The highest concentrations are found in the proximal tubule of the nephrons, and its soluble form is found in the circulation, urine, and cerebrospinal fluid (CSF). Neprilysin levels are much lower in the brain than in the kidneys. Soluble neprilysin levels are elevated in patients with heart failure (HF) and are predictive of cardiovascular death and HF hospitalization in HF patients. 10 Neprilysin has putative roles in the modulation of peptides implicated in the cardiovascular system and in other systems related to amyloid deposition, opioid receptor and pain processing, gastrointestinal processes, metabolism, sperm motility, and skin aging. There are more than 50 peptide targets of neprilysin, which include vasodilatory peptides such as natriuretic peptides, bradykinin, adrenomedullin, and substance P; vasoconstrictor peptides such as angiotensin I and II, endothelin, and neurotensin; and other peptides implicated in pathways related to amyloid deposition, pain sensorium, mood, gastrointestinal processes, and metabolism, such as amyloid beta (Ab) peptide, enkephalins, endomorphins, corticotropin, neuropeptide Y, gastrin, cholecystokinin-8, somatostatin, glucagon, vasoactive intestinal peptide (VIP), and oxytocin, among others (Central Illustration).

NEPRILYSIN AS A TARGET IN CARDIOVASCULAR DISEASE
Although neprilysin has a broad role across different organ systems, its cardiovascular effects have resulted in paradigm-changing therapies in HF in the last decade. 11 Exploiting the neurohormonal benefits of natriuretic peptides has been a focus in HF since the discovery of these peptides in the 1980s. 12 Natriuretic peptides are eliminated through degradation by neprilysin and through natriuretic peptide clearance receptors. 13 Neprilysin has a high affinity for atrial natriuretic peptide (ANP) and C-type natriuretic peptide and a lower affinity for brain natriuretic peptide (BNP). 14,15 Natriuretic peptides cause vasodilation by stimulating particulate guanylate cyclase to produce cyclic guanosine monophosphate (cGMP). ANP and BNP promote natriuresis, diuresis, and vasodilation and have salutary effects of suppressing the renin-angiotensin-aldosterone (RAAS) axis, sympathetic nervous system, and, in turn, cardiac hypertrophy and fibrosis. There is evidence of increased enzymatic degradation of natriuretic peptides by increased neprilysin activity in HF. 16 In animal models of severe HF, there is a significant increase in renal neprilysin activity and neprilysin messenger RNA expression, suggesting enhanced NP degradation. 17 Cardiac neprilysin activity and messenger RNA expression are elevated in patients with HF and are related to increases in end-diastolic pressures. 16 Thus, increased enzymatic degradation of natriuretic peptides was seen as a potential target for treatment in HF.
Neprilysin also displays enzyme promiscuity by breaking down angiotensin II and, by this mechanism, can elevate blood pressure (BP). [18][19][20] In addition, neprilysin degrades bradykinin, which is a potent vasodilator, through the stimulation of endothelial nitric oxide production, is implicated in vasogenic edema, and can cause angioedema in excess.
The opposing roles of neprilysin in the degradation of both vasodilatory and vasoconstricting substrates is key to the recognition of the contrasting outcomes  Neprilysin functions along with other peptidases to also degrade enkephalins, which are endogenous opioids that are expressed throughout the nervous system and multiple organ systems. 22 Cardiac failure and hypertrophy can lead to activation of the cardiac opioid system, where enkephalins play a complex role in response to myocardial injury. 23 Higher levels of proenkephalin, a stable surrogate for enkephalin, have been associated with more advanced HF, glomerular and tubular damage, and increased mortality. 24 The questions of whether the proenkephalin levels in HF patients are markers of disease severity or are markers of maladaptive counter-regulation by an overactivated opioid system remain unanswered. 23 In the nervous system, inhibition of enkephalin catabolism has a potential therapeutic role in the management of chronic pain disorders and mood stabilization. 25  Another key neurologic role of neprilysin is in the aging brain. Alzheimer disease is marked by an abnormal accumulation of soluble and insoluble Ab content. Neprilysin breaks down Ab and is central to its elimination. 26 Mice deficient for the neprilysin gene and neprilysin knockout mice have increased Ab accumulation in the brain. 26,27 Neprilysin inhibition increases beta amyloid in wild-type and neprilysindeficient mice. 28 Polymorphisms leading to loss of function in the neprilysin gene have been associated with an increased susceptibility to Alzheimer disease, especially when associated with other enzymatic deficiencies. [29][30][31] Overexpression of neprilysin or neprilysin gene transfer reverses the Alzheimer phenotype in mouse models. 27 Ab accumulation with neprilysin inhibition has also been implicated in macular degeneration. 32 Ab levels were reduced in mouse eye tissues by intravitreally delivered neprilysin. 33 Beyond the central nervous system, neprilysin has additional neuroendocrine roles. Adrenocorticotropin, which is produced and secreted by the posterior pituitary gland and stimulates cortisol release from the adrenal cortex, is cleaved by neprilysin. Oxytocin, a neuropeptide produced by the hypothalamus and secreted by the posterior pituitary gland, also undergoes hydrolysis by neprilysin. The significance of elimination of the breakdown of these peptides is unclear.
Neprilysin also plays a potential role in obesity.
Neprilysin-deficient mice become obese under a normocaloric diet, characterized by deregulation of lipid metabolism and manifested by impaired glucose tolerance with higher blood glucose and triglyceride levels and lower high-density lipoprotein cholesterol levels. They demonstrate age-related obesity, with visceral fat accumulation and insulin resistance attributed to down-regulation of anorexigenic peptides influenced by neprilysin activity. 34 These metabolic changes have not been observed in clinical trials.
Neprilysin is also implicated in skin aging, UVinduced skin damage, wrinkle formation, and neonatal development. 7,35 Neprilysin activity is markedly enhanced in human keratinocytes and human skin fibroblasts in a pattern similar to aging and is associated with wrinkle formation and damage after exposure to UV light and irradiation exposure.
Topical neprilysin inhibitors are being tested for wrinkle prevention. 7 In early studies, the combination of neprilysin inhibitors with ACE inhibitors showed increased synergistic efficacy for BP lowering in patients with hypertension. 54 Subsequent studies showed promise with omapatrilat, a vasopeptidase inhibitor with combined neprilysin and ACE inhibition in patients with HF and hypertension. 55 In OVERTURE (Omapatrilat vs Enalapril Randomized Trial of Utility in Reducing Events), a long-term randomized study in patients with HF with reduced ejection fraction (HFrEF) and a recent HF hospital admission, omapatrilat was noninferior but not superior to enalapril.
Adverse events including HF, hypotension, and dizziness occurred similarly in both groups, with angioedema reported in 24 (0.8%) omapatrilat-and 14 (0.5%) enalapril-treated patients. 56 In the larger OCTAVE (Omapatrilat Cardiovascular Treatment vs Enalapril) trial of 25,302 patients with untreated or uncontrolled hypertension, omapatrilat reduced systolic BP 3.6 mm Hg more than enalapril and was associated with less use of adjunctive antihypertensive therapy. Overall death rates and adverse events were similar. However, angioedema was more frequent with omapatrilat than enalapril (2.17% vs 0.68%) and was associated with airway compromise. The rates of angioedema were much higher in Black individuals (5.54% for omapatrilat and 1.62% for enalapril) and in smokers (3.93% for omapatrilat and 0.81% for enalapril). 57 The lack of superiority of omapatrilat compared to enalapril in HF trials and the risk and severity of angioedema in hypertension trials forced the withdrawal of omapatrilat from consideration of approval by the U.S. Food and Drug Administration (FDA). The mechanism for the increased risk in angioedema is likely attributable to an increase in circulating bradykinins caused by inhibition of both ACE and neprilysin.
Given that all drugs in this class potentially have a high risk of angioedema, the enthusiasm to further study the combination of neprilysin inhibitors and ACE inhibitors has dissipated. With recognition that neprilysin inhibition by itself is ineffective and that the combination of neprilysin and ACE inhibition is asso-     inhibition but had no effect in nonfailing animals.
Natriuretic peptide desensitization in HF may relate, in part, to increased PDE activity, supporting a therapeutic role for PDE5 or PDE9 inhibition, especially among patients with advanced HF with blunted downstream response to natriuretic peptides. 91,92 Whether this approach can be effective in patients with advanced HF awaits further studies. Albuminuria is an independent factor for renal and cardiovascular risk and an independent predictor of prognosis in HF. 116 Whether the increase in urinary albumin with ARNi will translate into an excess risk of renal events in subjects with HF needs further exploration in longer-term trials. 105 Natriuretic peptideinduced impairment of tubular handling of other ultrafiltered proteins such as such as b2-microglobulin and free k-light chains may also need to be also be taken into consideration. 104  theoretical concern about the long-term effects of sacubitril-valsartan on cognition. Sacubitril and valsartan are highly bound to plasma proteins (94%-97%), and sacubitril is thought to cross the blood-brain barrier to a limited extent (0.28%). 70 The effects of sacubitril-valsartan on Ab concentrations in CSF and brain tissue were assessed in young cynomolgus monkeys treated with sacubitril-valsartan for 2 weeks. 121 Despite low CSF and brain penetration, CSF exposure to sacubitril was sufficient to inhibit neprilysin and resulted in an increase in the CSF levels of Ab 1-40, Ab 1-42, and total Ab. 121