Neutral endopeptidase inhibition: the potential of a new therapeutic approach in cardiovascular disease evolves

The potential importance of neutral endopeptidase inhibition became obvious with the development of a new class of drugs, the so-called vasopeptidase inhibitors. These drugs were initially developed for the treatment of hypertension and congestive heart failure, although they may also be helpful in other diseases for which angiotensin-converting enzyme (ACE) inhibitors are now used [1]. Neutral endopeptidase inhibitors have a dual mechanism of action. They inhibit two metalloprotease enzymes, neutral endopeptidase and ACE, resulting in an increased availability of natriuretic peptides that exhibit vasodilatory effects and, possibly, tissue protective effects. Furthermore, the formation of angiotensin II is reduced. Since neutral endopeptidase and ACE are intimately linked to the regulation of structural and functional properties of the heart and circulation, the term vasopeptidase inhibitor has been introduced for this new class of drug. Similar to ACE, neutral endopeptidase is an endothelial cell surface metalloproteinase, which is involved in the degradation of several regulatory peptides, including the natriuretic peptides [2]. It augments vasodilatation and natriuresis through increased levels of atrial natriuretic peptide. Neutral endopeptidase is found principally in the brush-border membrane of renal tubules but also in the lungs, intestine, adrenal, brain, heart and peripheral blood vessels. Neutral endopeptidase catalyses the breakdown of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide [3]. These peptides comprise a family of peptides involved (as mentioned above) in the regulation of blood pressure and plasma volume [4]. The atrial and brain-derived natriuretic peptides are produced mainly in the myocardium in response to atrial distention. The release of C-type natriuretic peptide, which is found in the kidney, heart, lung and vascular endothelium, depends on shear stress. These peptides bind to specific, high-affinity cell-surface receptors. Natriuretic peptides exert physiological effects at several sites, resulting in vasodilation, natriuresis, diuresis, decreased aldosterone release, decreased cell growth and inhibition of the sympathetic nervous system and the renin–angiotensin–aldosterone system. C-type natriuretic peptide exerts its function predominantly in the vascular wall. It exhibits potent vasodilatory and cell-growth inhibitory properties [5]. Neutral endopeptidase also degrades other peptide hormones, such as adrenomedullin, urodilatin (a renal form of atrial natriuretic peptide) and bradykinin. Neutral endopeptidase itself appears to be well controlled by respective feedback mechanisms. It was previously demonstrated that sodium ingestion in rats governs renal NEP activity [6]. On the other hand, cGMP (cyclic guanosine-5-monophosphate), which is increased by the natriuretic peptides, stimulates neutral endopeptidase [6], suggesting a positive feedback mechanism. Since neutral endopeptidase degrades peptides with vasodilatory and renal actions, inhibition of this enzyme was assumed to be beneficial in hypertension and congestive heart failure. However, clinical experience in hypertension with mere neutral endopeptidase inhibitors were not successful [7]. Animal models have helped to explain the effects of neutral endopeptidase inhibition on blood pressure. In the deoxycorticosterone acetate-salt rat model of low-renin hypertension, an neutral endopeptidase inhibitor decreased blood pressure and increased urinary volume, atrial natriuretic peptide excretion and urinary cGMP [8]. Interestingly, this was not observed in the spontaneously hypertensive rat, a normal-renin model. In patients with congestive heart failure, neutral endopeptidase inhibition lowered cardiac filling pressures. However, neutral endopeptidase inhibitors never exhibited lasting or remarkable effects on blood pressure in hypertensive cohorts. For example, in patients given candoxatril 200 mg twice daily for 1 month, the blood pressure was unaltered. In healthy volunteers, candoxatril administration led to a rise in systolic pressure. This was associated with an increase in the concentration of endothelin [9]. One reason why neutral endopeptidase inhibitors are unable to lower blood pressure when used alone may be because inhibiting neutral endopeptidase without blocking the formation of angiotensin II is followed by pressor responses. This effect could be demonstrated in healthy volunteers [10], where the infusion of angiotensin II plus candoxatril resulted in higher angiotensin II concentrations and higher peak blood pressures compared with a control group receiving angiotensin II and placebo. Stergiou and colleagues treated hypertensive patients with a combination of candoxatril and an ACE inhibitor and induced a marked reduction in blood pressure. In a crossover study, Favrat and colleagues compared an neutral endopeptidase inhibitor (sinorphan 100 mg twice daily), captopril (25 mg twice daily), and the two drugs in combination, in patients with essential hypertension [11]. Neither agent alone produced a long-lasting fall in blood pressure, but blood pressure dropped substantially when they were combined. Hence, by inhibiting the formation of angiotensin II and potentiating the natriuretic peptide system at the same time, combined neutral endopeptidase/ACE inhibitors or vasopeptidase inhibitors reduced vasoconstriction and enhanced vasodilatation. In consequence, peripheral vascular resistance and blood pressure should decrease. Numerous preclinicial studies with vasopeptidase inhibitors documented these effects in experimental hypertension, as suggested by the studies mentioned above using two different substances [12]. Within the vessel wall, this kind of therapy may lead to a reduction of vasoconstrictor and proliferative mediators, such as angiotensin II and endothelin-1, and is also likely to increase local levels of bradykinin in addition to natriuretic peptides. There is increasing experimental evidence that neutral endopeptidase/ACE inhibition might directly protect endothelial function and reduce atheromatous vascular changes. Hence vasopeptidase inhibitors could help to improve cardiovascular prognosis. Omapatrilat is the vasopeptidase inhibitor most often studied. Clinical studies of omapatrilat in hypertension have consistently demonstrated the effectiveness of the agent in a variety of diseases [13]. Most intriguingly, in double-blind, randomized studies, omapatrilat lowered systolic blood pressure in hypertensive patients significantly and to a greater extent than ACE-inhibitors, beta-blockers and calcium-antagonists. In patients with heart failure, omapatrilat significantly improved neurohormonal and haemodynamic status. The long-term effects of omapatrilat in patients with heart failure recently were compared with those of conventional therapy in a large phase II trial (IMPRESS) [14]. The results of the study appear promising. Systolic function of the left ventricle, as assessed by the maximal exercise treadmill test, improved significantly more with omapatrilat than with ACE-inhibitors. Analysing secondary endpoints, combined cardiovascular endpoints tended to occur to a lesser extent and renal function was better preserved with ompatrilat than with ACE-inhibitors. Large clinical trials are currently in progress or already finished (OCTAVE, OVERTURE). These studies should help to define a place in therapy for agents such as omapatrilat. Eventually, it will not only be important to find a role in the treatment of hypertension, but also in the general context of cardiovascular diseases, including diabetes mellitus [15]. A decrease in endothelium-dependent vascular relaxation is a common feature in diabetes mellitus and is mainly due to an impairment in nitric oxide (NO) metabolism. Clinical and experimental studies have demonstrated that ACE inhibitors improve endothelium-dependent vasodilatation in type I diabetes. This may be explained, at least partly, by an activation of the kinin–NO pathway [16]. In non-diabetic situations, ACE is the primary enzyme responsible for the breakdown of bradykinin (1-9) into bradykinin (1-7) in the vascular wall. However, neutral endopeptidase is also involved in this catabolism, particularly during ACE inhibition. Indeed, in anaesthetized dogs, the combination of captopril and SQ-28603, a neutral endopeptidase inhibitor, synergistically enhanced the renal vasodilatation induced by exogenous bradykinin [17]. It has been suggested that endogenous bradykinin could also contribute to this effect. In one experimental study in streptozotocin-induced diabetes in rats, combined neutral endopeptidase/ACE inhibition decreased systemic peripheral vascular resistance more effectively than ACE inhibition alone, which is a finding that has also been documented in arterial hypertension. These effects were seen as linked to an increased activation of the kinin–NO pathway. It was also discussed that bradykinin could lead to NO production by activating the subgroup of the B2 receptor [18]. In this issue of the journal, Tikkanen and coworkers present data obtained from rats with streptozotocin-induced diabetes mellitus. An important finding of their study is that the similar effects on blood pressure and albuminuria were obtained not only by neutral endopeptidase/ACE inhibition, but also when neutral endopeptidase inhibition was combined with the blockade of endothelin-converting enzymes (ECE) in experimental diabetes. The greater reduction in blood pressure with the vasopeptidase inhibitor, S21402, was associated with reduced albuminuria and heart to body weight ratio. The effects of neutral endopeptidase/ACE blockade on blood pressure and heart weight are consistent with findings observed in hypertensive rat models and with former reports from the author's group concerning the diabetic spontaneously hypertensive rat. Taken together, the results confirm that combined neutral endopeptidase/ACE inhibition exhibited greater benefits in reducing blood pressure, cardiac hypertrophy and albuminuria than ACE inhibition alone [19]. Their study furthermore demonstrates that the dual neutral endopeptidase/ECE inhibitor, CGS26303, lowered blood pressure, decreased albuminuria and beneficially influenced the heart to body weight ratio in rats with diabetes. Endothelin antagonism alone did not decrease albuminuria or heart weight, so that neutral endopeptidase inhibition could have augmented the effects of endothelin blockade [20]. It seems reasonable to further pursue the putative value of neutral endopeptidase inhibition in connection with blocking the endothelin system. ECE/neutral endopeptidase might have distinct importance in the treatment of hypertension and/or diabetes which is not completely appreciated as yet. In addition, it should be noted that vasopeptidase inhibitors themselves, such as omapatrilat, might also affect ECE to some degree [21]. In 1985, it was shown that the vascular endothelium generates a peptide substance with long-acting vasoconstrictor action. This peptide was then isolated and sequenced from cultured endothelial cells and named endothelin. Three endothelin peptides have been identified, each containing 21 amino acids (named ET-1, ET-2 and ET-3) [22]. In the vasculature, ET-1 is the predominant isoform. It has many different biological actions. It is the most potent endogenous vasoconstrictor agent, has positive inotropic and chronotropic effects, presents mitogenic properties, influences homeostasis of salt and water, and stimulates the renin–angiotensin–aldosterone and sympathetic systems [23]. Various stimuli, such as vasoactive hormones, growth factors, hypoxia, shear stress, lipoproteins, free radicals, endotoxin and cyclosporin, can increase the generation of ET-1. Its production is inhibited by prostaglandins and by increasing intracellular levels of cGMP. Endothelin is produced in a variety of tissues and cells, but the expression of endothelin isoforms is tissue specific [24]. Endothelial cells are the principal sites of generation of ET-1. ET-1 is also produced by the heart, kidney, central nervous system and by human aortic vascular smooth muscle cells [25]. ET-2 is produced predominantly within the kidney and intestine and, to a lesser extent, in the myocardium, placenta, uterus and in endothelial cells. ET-2 appears to have vasoconstrictor properties similar to those of ET-1, while ET-3 shows a reduced vasoconstrictive action with respect to the other two isoforms. ET-3 has been found at high concentrations in the brain and may regulate important functions in neurones and astrocytes; it is also found in the gastrointestinal tract, in the lung and kidney, but not in endothelial cells [26]. The prepro-endothelin-1, a 212 amino acid peptide, is the initial product of the human ET-1 gene. Pro-endothelin-1 is formed after removal of a short sequence and is then cleaved to generate a 38 amino acid peptide, named big-endothelin-1, which is found in the peripheral circulation [27]. The mature endothelin-1 form is obtained by enzymatic cleavage of the pro-hormone big-endothelin-1 by the action of the endothelin-converting enzymes, a family of metalloproteases. The physiological importance of the conversion of big-endothelin-1 to endothelin-1 is due to the higher vasoconstrictor potency of ET-1 compared to big-endothelin-1 (approximately 140-fold), while prepro-endothelin-1 does not show any vasoconstrictor action. The human endothelin-converting enzyme-1 (ECE-1) is a membrane protein existing in four different isoforms which are generated from a single gene [28]. A second form of ECE (ECE-2) has been identified. ECE-2 is active only in acidic conditions and it is not expressed on the cellular plasma membrane. Two receptor subtypes, ET-A and ET-B, which mediate the biological actions of endothelins, have been identified and cloned. Both are members of the superfamily of receptors linked with guanine-nucleotide-binding (G) protein and range in size from 45 000–50 000 Da in various tissues. Endothelin receptors present the classical structure of receptors coupled to G-proteins [29]. It now appears to be evident, especially from human experiments on forearm blood flow after intra-arterial infusion of agents, that neutral endopeptidase inhibitor-induced vasoconstriction is mediated more by increased ET-1 than by angiotensin II. Remarkable haemodynamic and cardioprotective effects have been already reported with antagonists of the endothelin receptor. Specific inhibitors of the ECE inhibiting endothelin generation from its precursor, big endothelin, will have to demonstrate their full potential in the coming years [30]. If the results previously obtained with endothelin receptor antagonists can be reproduced with ECE inhibitors, and transferred to clinical medicine, endopeptidase inhibition might open new horizons in cardiovascular treatment strategies. ET-1 stimulates vasoconstriction and cell proliferation. Enzymes such as ECE, chymases and non-ECE metalloproteinases contribute to the synthesis of ET-1, which is regulated in an autocrine fashion in vascular and nonvascular cells. Endothelin ET-A receptors mediate vasoconstriction and cell proliferation, whereas ET-B receptors are involved in the clearance of ET-1, inhibition of endothelial apoptosis, release of nitric oxide and prostacyclin and inhibition of ECE-1 expression [31]. Most cardiovascular diseases, such as arterial hypertension, atherosclerosis, restenosis, heart failure, idiopathic cardiomyopathy, pulmonary hypertension and renal failure, are associated with local activation of the endothelin system. Experimental studies and preliminary clinical trials suggest that ET-1 is importantly involved in functional and structural changes of the cardiovascular system, and that many of the actions of ET-1 are mediated through pressure-independent mechanisms [32]. Endothelin antagonists and ECE inhibitors, probably in conjunction with neutral endopeptidase inhibition, might therefore become successful in the treatment of cardiovascular diseases, but these compounds are not yet so far developed as vasopeptidase inhibitors. ECE inhibition (in combination with neutral endopeptidase) blockade could be also useful in the therapy for diabetes mellitus. The importance of endothelin in the pathogenesis of diabetes mellitus may have been underappreciated until now [33]. Changes in plasma ET-1 levels have been demonstrated in experimental and human diabetes with altered endothelial function. The importance of ET-1 in diabetes-associated vascular hypertrophy was first discussed in reports demonstrating an increased release of endothelin from mesenteric vessels in diabetic rats [34]. Further reports suggested increased endothelin expression in the endothelial and adventitial layer and de novo expression of ET-1 in the media of diabetic mesenteric vessels [35]. The observed changes in endothelin expression were associated with vascular hypertrophy, increased extracellular matrix deposition, and an increased gene expression of epidermal growth factor and transforming growth factor-β 1. With respect to patients, ET-1 is seen as a marker of atherosclerotic macro- and microvascular disease in type II diabetes mellitus. The research in recent years has led to the discovery of a variety of potent selective or mixed ECE blocking substances, especially the above-mentioned ECE/neutral endopeptidase inhibitors and putative substances with triple effects on ECE/neutral endopeptidase and ACE [36]. It will be a challenging task for future research, using the already available and newly developed selective and mixed ECE-1 inhibitors with neutral endopeptidase blocking properties, to show whether the combined inhibition of more than one cardiovascular system is superior to selective inhibition and how far this will help in the development of differential therapeutic regimens (e.g. for treating hypertension and/or diabetes mellitus). We eagerly await the final results of large-scale clinical studies that will further define the role of vasopeptidase inhibitors in cardiovascular medicine.