Practical implications of current natriuretic peptide research

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1 Editorial review Keywords: natriuretic peptides, cardiovascular disease, vasopeptidase inhibitors Practical implications of current natriuretic peptide research Giuseppe A Sagnella Blood Pressure Unit, Physiological Medicine, St George's Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK Correspondence to: Dr Giuseppe A Sagnella Blood Pressure Unit, Physiological Medicine, St George's Hospital Medical School, Cranmer Terrace, London, SW17 0RE, UK Tel: Fax: g.sagnella@ sghms.ac.uk Accepted for publication 7th JRAAS 2000;1: Introduction The control of sodium balance by the kidney is of fundamental importance in the maintenance of cardiovascular homeostasis. Given such a vital function, renal sodium excretion is, not surprisingly, under of the influence of many interacting mechanisms including haemodynamic, physicochemical, neuronal and hormonal factors. 1 While the importance of the renin-angiotensin-aldosterone system (RAAS) in the conservation of sodium during sodium depletion had long been recognised, physiological experiments in the mid-1950s also suggested the existence of natriuretic factors. 2 Specifically, Gauer et al. provided evidence for an atrial location of a sensing mechanism influencing renal volume excretion. 3 This, in conjuction with the histological demonstration of secretory granules in atrial tissue 4 subsequently led to the discovery of atrial natriuretic peptide (ANP) in the early 1980s. 5 Since then, there has been considerable progress in our understanding of the functional significance of ANP. The cardiovascular and renal actions of ANP (Table 1),as well as the fact that the atria are strategically located to sense changes in intravascular volume, clearly attests to the importance of ANP as a major defence mechanism protecting against volume overload. 6,7 Moreover, the influence of ANP in the control of sodium excretion is strengthened by more recent experiments in transgenic mice, in that mice with ANP gene deletion and those with natriuretic peptide type A receptor knock-out displayed substantial impairment in the renal response to volume overload. 8,9 However, it is now also apparent that ANP is part of a larger family of homologous natriuretic peptides including urodilatin, an N-terminallyextended form of ANP, 10 brain natriuretic peptide (BNP), 11 C-type natriuretic peptide (CNP) 12 and D- type natriuretic peptide (DNP). 13 BNP, originally isolated from porcine brain, has a similar pattern of renal and cardiovascular actions to ANP. 14 Plasma levels of both ANP and BNP are elevated in conditions associated with high blood pressure and even higher levels are found in the plasma of those with excessive renal salt and water retention.these observations imply that these conditions are not a result of natriuretic peptide depletion and that the raised levels may represent an appropriate, although insufficient, compensatory response. Given that plasma levels of ANP and BNP are high in these conditions and given the powerful cardiovascular and renal Table 1 Major cardiovascular and renal effects of ANP. Effect ic Reduction in blood pressure Reduction in central venous pressure Steady heart rate Blood/plasma Reduction in plasma renin Reduction in aldosterone Increased haematocrit Renal Diuresis/natriuresis Mechanism of action involves Vasodilation;blood volume reduction Reduction in venous return Reduction in sympathetic drive Renal secretion Adrenal synthesis Fluid shift from intravascular space Renal haemodynamics/increased GFR Reduction in cyclic GMP-dependent distal tubular sodium reabsorption effects of these peptides, there has been intense interest in two potentially important practical aspects. The first applies to the clinical significance of the measurement of circulating levels of ANP and BNP and the second relates to the therapeutic potential of natriuretic peptides. The first part of this review examines the diagnostic and prognostic value of ANP and BNP in cardiovascular disease with particular emphasis on heart failure. Heart failure is an increasingly serious public health problem and there is a real need for relatively simple tests for the identification of patients with asymptomatic systolic dysfunction 15,16 and for prediction of outcome in those with severe heart failure. 17 Despite the recent advances in treatment, there is also a need for more efficient treatment of both hypertension 18 and of heart failure. 19 The second part of the review, therefore, explores the potential therapeutic importance of targetting the natriuretic peptides system in these conditions with particular emphasis on the newlydescribed vasopeptidase inhibitors. Structural features and peptide nomenclature Both ANP and BNP are synthesised as high molecular weight precursors.the precursor (pre-pro ANP), predicted from the analysis of the cloned human ANP gene, is 151 amino acids long. 20 This is converted to the immediate 126 amino acids long pre- 304

2 Figure 1 Schematic representation of the ANP and BNP precursors with sequence numbering defining ANP, BNP and the corresponding N-terminal proanp and N-terminal probnp. Pro-atrial natriuretic peptide (pro-anp) N-terminal pro-anp(1-98) ANP Pro-brain natriuretic peptide (pro-bnp) N-terminal pro-bnp(1-76) BNP cursor (pro-anp) after removal of the 25 residue signal sequence (Figure 1). The ANP precursor is cleaved within the C-terminal region at position to give biologically active ANP (also known as alpha-anp or as ANP(99-126)).The remaining N-terminal fragment is referred to as N-terminal proanp(1-98). Both peptides are released into the circulation. 21 Analysis of cdna clones of the BNP gene indicate that BNP is also produced as a high molecular weight precursor. 20 In the human heart, BNP is made as a 134 amino acid precursor with the first 26 amino acids possibly representing a hydrophobic signal peptide sequence. The remaining 108 amino acid probnp (1-108) appears to be cleaved at position within cardiac cells to give the biologically active 32 amino acid BNP (BNP77-108) and the N-terminal probnp(1-76) (Figure 1) and both peptides are secreted into the circulation. 22 Diagnostic and prognostic significance of plasma levels of natriuretic peptides Measurement and circulating forms The first radioimmunoassay procedures for the measurement of plasma ANP were reported not long after the structural identification of ANP. Plasma levels of ANP in normal individuals,as measured using extraction-based methods, ranged from pg/ml. 23 This wide range is possibly due to differences in the actual methods (e.g. type of extraction, antibody specificity, etc.) used in different laboratories. Radioimmunoassay procedures for the measurements of the N-terminal segment of the ANP precursor have also been developed and work on its molecular form suggest that the major circulating N-terminal form seems to be identical with proanp(1-98) (see Figure 1 for nomenclature). In plasma from normal subjects, the concentration of this peptide is considerably higher (more than 20-fold) than that of ANP. This is probably due to its slower clearance from the circulation; however, in general, there is a close association between plasma ANP and N-terminal proanp. While BNP was originally isolated from brain, immunoreactive BNP is also found in plasma and in heart tissue. 27 In the normal human heart, BNP is synthesised within both atrial and ventricular tissue, suggesting that plasma BNP originates from the heart, although secretion of BNP seems to be mainly from ventricular tissue. 28 Plasma concentrations of immunoreactive BNP in normal subjects are much lower than those of ANP (~ 5 pg/ml) but a wide range of values has been reported The N-terminal probnp form [possibly, probnp(1-76); see Figure 1 for nomenclature] has also been identified in human plasma. 34,35 The plasma levels of BNP and of the N-terminal probnp are of similar magnitude in normal subjects. However, levels of the N-terminal probnp may be up to 4-fold higher than those of BNP in subjects with heart failure, possibly reflecting the substantial augmentation in cardiac synthesis and processing of BNP. 31,36 Circulating natriuretic peptides in cardiovascular disease The observation of increased circulating ANP in manoeuvres associated with an increase in central venous pressures (supine position, volume expansion, etc.) clearly point to the importance of intraatrial pressure/stretch as a major determinant of ANP secretion. Since the early demonstration of markedly raised levels of plasma ANP in patients with congestive heart failure, it is now apparent that both plasma ANP and BNP and the corresponding N-terminal proanp and N-terminal probnp are increased in conditions associated with i) overt intravascular volume overload; ii) increased central venous pressures; and iii) left ventricular (LV) dysfunction.plasma levels are related to the magnitude of atrial or ventricular overload. This is clearly shown by comparing plasma levels of ANP and BNP in patients with increasing severity of congestive heart failure; 37 in those with hypertrophic cardiomyopathy; 38 in those with systolic dysfunction after myocardial infarction (MI) 39 and in patients with chronic renal failure with or without dialysis 29 (Figure 2).The raised plasma levels in patients with renal failure 29 are probably due to i) volume overload; ii) co-presence of intrinsic heart disease, and iii) reduced renal clearance of the peptides. Interestingly, elevated levels of plasma natriuretic peptides have also been found in patients with essential hypertension, 30,33,40 with average values in the hypertensive group about 2-3 fold higher than those in normotensive controls and, to some extent, plasma levels are related to the presence and type of LVH. 33,40 Essential hypertension is not usually associated with overt volume expansion. 41 However, an increase in central venous pressure and pulmonary wedge pressure with normal cardiac pump function has been demonstrated in patients with essential hypertension. 42 This is not due to systemic volume retention but may arise from an increased central blood volume as a consequence of systemic venous vasoconstriction. The potential practical value of plasma natriuretic peptides is based on the recognition that plasma levels of ANP and BNP are positively associated with 305

3 Figure 2 Plasma levels (logarithmic scale) of ANP (upper panel) and BNP (lower panel) in essential hypertension, 30 in congestive heart failure, 37 hypertrophic cardiomyopathy, 38 myocardial infarction 39 and renal failure. 29 central venous pressures and negatively with left ventricular ejection fraction Raised levels in the presence of LVH and diastolic dysfunction 44,47 also suggest a link with cardiac structural abnormalities and ventricular wall stress.although there are strong associations between LV function and plasma ANP or BNP, plasma BNP seems to be a better index of ventricular overload and stress.these observations suggest that plasma levels of ANP and BNP could be of value in several situations, including assessment of severity of heart failure, monitoring response to treatment in congestive heart failure, detection of hypertrophic heart disease and prediction of mortality after MI. While the identification of severe heart failure does not usually present with diagnostic problems, there has been considerable interest in the value of plasma ANP and BNP in the identification of mild heart failure and in the prognostic significance of raised plasma levels of these natriuretic peptides. Plasma natriuretic peptides and screening for mild heart failure One reason for examining the possibility that moderately raised levels of natriuretic peptides may be indicative of the presence of systolic dysfunction in the absence of overt clinical signs concerns the difficulty in assessing the presence of mild LV systolic impairment. This is especially important in patients recovering from MI, who would benefit from early treatment with ACE inhibitors. 15 Earlier work 48 found significantly raised plasma ANP in patients with LV dysfunction in the absence of overt heart failure and Lerman 49 reported raised plasma N-terminal proanp in patients with mild heart failure (NYHA Class I). However,plasma BNP may be a better marker than ANP or N-terminal proanp in the identification of mild heart failure. 50 One study, 51 performed in a select group of patients referred for coronary angiography, demonstrated that ANP, BNP and N- terminal proanp were strongly associated with indices of LV systolic dysfunction. Indeed, the relative risk of LV dysfunction was raised up to 10- fold in those with high levels of these peptides.the potential value of plasma BNP to investigate LV systolic dysfunction is also supported by recent population-based investigations. Cowie et al. 52 examined the predictive value of plasma natriuretic peptides in patients with a new primary care diagnosis of heart failure.this study found a significant increase in predictive accuracy when information on plasma BNP was combined with clinical assessment alone. A separate, population-based survey 53 confirmed that plasma BNP is significantly increased in people with LV dysfunction, in both symptomatic and asymptomatic individuals. However, the positive predictive value to identify mild heart failure at a population level was rather low. More recently, Luchner et al. 54 also evaluated plasma BNP as a marker of LV dysfunction and LVH in a populationbased sample of middle-aged subjects. The authors concluded that the measurement of plasma BNP could not be recommended for the detection of 306

4 mild impairment of LV function because of a high rate of false positives.however,and consistent with other studies, a normal value of plasma BNP was associated with a high negative predictive value. Plasma BNP therefore could provide a way to exclude those at low risk of LV dysfunction and identify those at higher risk of more severe LV dysfunction for detailed assessment of cardiac function using more specialised cardiological investigations. Prognostic significance of plasma natriuretic peptides The presence of raised plasma levels of ANP, and N-terminal proanp, has been associated with increased mortality in elderly people. 55,56 This could not be explained entirely by overt heart disease at the time of sampling, suggesting the presence of raised peptide levels even in those with intrinsic asymptomatic cardiac disease.plasma levels of BNP in elderly people (mean age 83 years) were about 3.5 times higher than those in a middle-aged group, but the raised levels in these elderly people reflected concomitant cardiac hypertrophy, diastolic dysfunction and reduced clearance from impaired renal function. 57 However, markedly higher levels of ANP and BNP are present in people after an MI and several studies have demonstrated a significant association between sustained high levels after MI and prognostic outcome. 39,61-65 While increased levels were also observed even in the absence of overt ventricular failure and of raised atrial pressures, plasma BNP levels were associated inversely with LV ejection fraction and positively with infarct size (assessed from creatine kinase and myosin light chain I). The reasons for the marked increase in plasma BNP following infarction,in the absence of overt heart failure, suggests that the raised levels may also be due to myocardial necrosis and/or increased mechanical stress on cardiac tissue subsequent to infarct expansion. The potential prognostic value of plasma BNP is further highlighted by the recent demonstration that a high plasma BNP level was a strong predictor of mortality in people with congestive heart failure. 66 Additionally, Yu and Sanderson 67 examined mortality rates in a group of individuals admitted to hospital with acute heart failure in relation to plasma BNP and other risk factors. In this prospective study, a raised plasma BNP level was a more important predictor of cardiac death than other risk factors examined, including age and LV ejection fraction; those patients with plasma BNP above median levels displayed a significantly higher one-year cardiac mortality rate (42.5% vs. 11.6%). Whether the use of plasma natriuretic peptides will substitute for other biochemical markers of infarct size, including troponin-t, remains to be seen, but a recent study 68 has gone one step further and used plasma levels of the N-terminal probnp to target treatment in heart failure. The treatment target was to reduce the plasma level of this peptide to a value comparable with that found in compensated heart failure. Selected primary and secondary endpoints of cardiovascular events were compared with those obtained with a standard clinically-guided treatment regimen over a minimun period of six months.the results demonstrated significantly fewer total cardiovascular events (death, hospital admission and deterioration of heart failure) in those treated according to plasma levels of natriuretic peptide. Changes in LV function, quality of life and renal function were similar in both groups. Despite its limitations, this study suggests that reductions in LV stress, as reflected by reduction in plasma N-terminal BNP, may have beneficial effects on myocardial survival. Which natriuretic peptide should be measured? While numerous studies suggest that ANP and BNP (or the corresponding N-terminal precursor fragments) could be used to identify LV dysfunction, the plethora of available assays and situations investigated begs the questions 1) which natriuretic peptide should be measured, and 2) what is the practical value of plasma levels of these peptides in the assessment of heart disease? Raised plasma levels of ANP and BNP, in patients with cardiovascular disease, are also usually associated with higher levels of the corresponding precursors (and N-terminal segments) and hence the plasma levels of these peptides are strongly intercorrelated. 25,26,31,33 In fact, a very strong correlation (r=0.95) between plasma levels of BNP and of the N-terminal probnp was found in patients with MI 65 and presumably either peptide could be used in this case. However, it is also apparent that differences in pathophysiology and/or differences in elimination rates may lead to subtle differences in the relative proportion of the plasma levels of each of these peptides. Therefore, which peptide to measure depends, to some extent, on the intended investigation. In view of this, it is difficult to generalise; however, current work suggests that, in the assessment of ventricular dysfunction and prediction of mortality outcome in those with severe heart failure, BNP appears to be better than N-terminal proanp or ANP. However, because the raised levels are a consequence of haemodynamic and structural abnormalities arising from diverse pathological processes (Table 2) measurement of natriuretic peptides alone is of limited value as a specific diagnostic tool.the interpretation of plasma levels is clearly not straightforward and has to be put in to the appropriate clinical context. Indeed, knowledge of renal function (or plasma creatinine at least) is essential to eliminate renal failure as a possible cause of raised plasma levels. This notwithstanding, in those suspected of heart disease a normal value of plasma natriuretic peptides, and BNP in particular, would not be consistent with overt cardiac disease; the presence of markedly increased levels of these peptides may, therefore, help to target those for subsequent detailed assessment of underlying cardiac dysfunction using more specialised procedures (e.g. 307

5 Table 2 Possible causes for the raised levels of ANP and BNP in cardiovascular diseases. Figure 3 Potential targets for pharmacological manipulation of endogenous activity of natriuretic peptides. Increased atrial pressure/distension due to volume expansion Raised LVEDP due to impaired systolic function and/or diastolic relaxation Enhanced response to cardiac growth-stimulating cytokines Reduction in peptide clearance due to renal impairment echocardiography, radionucluclide ventriculography, exercise testing, etc.). This may be especially important where the provision of these facilities are limited. Additionally, raised plasma BNP (or N-terminal probnp) is emerging as a significant predictor of cardiac damage after MI and may provide a rationale for the development of more targetted treatment. Recent introduction of more rapid immunoradiometric assay (IRMA)-based assays for plasma BNP, 70 together with the prolonged stability of BNP in blood after collection, 71 provides a step forward towards the use of the measurement of BNP on a routine basis, both at a point-of-care level within the hospital setting and in community practice where the blood may have to be sent to a distant reference laboratory.this is reinforced by the current development of an automated Triage method for the measurement of BNP 72 in whole blood, with rapidly available results (within 6 hours). Importantly, the blood levels of BNP were significantly related to the severity of congestive heart failure. Nevertheless, a definitive assessment of the value of plasma natriuretic peptides in routine clinical practice is bound to require a stricter validation of diagnostic and prognostic accuracy in relation to the spectrum of disease being investigated. This is especially relevant in those with heart disease, given the multiplicity of causes likely to lead to heart failure. Therapeutic potential of the natriuretic peptides system Since the original identification of cyclic GMP as the second messenger for the actions of ANP and the cloning of the first ANP receptor, 73 it is now apparent that there are at least three major types of receptors with different affinities for the different forms of naturally occuring natriuretic peptides. 74,75 The three natriuretic peptide receptors identified in mammalian tissues have been designated as NPR-A, NPR-B and NPR-C. Guanylate cyclase activity is an intrinsic part of NPR-A and NPR-B receptors.the type A receptor is widely distributed and appears to mediate many of the cardiovascular and renal effects of ANP and BNP, whereas the type B receptor has a much stronger affinity for CNP. The third receptor, NPR-C, is also a membrane-bound protein. This receptor has strong sequence homology with the other two natriuretic peptides in the extracellular domain, but it lacks the intracellular catalytic domains of guanylate cyclase. There is evidence suggesting that C-type receptors are coupled to the adenylate cyclase/camp signal transduction system through inhibitory guanine nucleotide regulatory protein and may be involved in the antigrowth effects of natriuretic peptides on endothelial cells. 76 However, it is now generally accepted that the NPR- C receptor seems to be of major importance in the metabolic clearance of natriuretic peptides. 77,78 These receptors, in conjuction with the potential to influence synthesis, secretion and elimination of natriuretic peptides, provide a rich variety of approaches for pharmacological intervention designed to enhance endogenous activity of natriuretic peptides (Figure 3). Some of these approaches include: Modulation of synthesis and secretion of natriuretic peptides Administration of native peptides or natriuretic peptide mimetics Prevention of intracellular degradation of cyclic GMP by inhibiting intracellular phosphodiesterases Displacement of endogenous natriuretic peptides from the clearance receptor Inhibition of proteolytic breakdown of natriuretic peptides Animal experiments have demonstrated the potential value of some of these pathways. For example, some drugs such as clonidine 79 are known to increase release of ANP and this could account for the diuretic and natriuretic effects of clonidine analogues acting on imidazoline receptors. Interestingly, inhibitors of cyclic GMP breakdown are associated with marked hypotensive and natriuretic responses. 80 Whether this is entirely through a natriuretic peptide-dependent effect or through a nitric oxide-dependent effect remains to be evaluated. Given the success of sildenefil as a target organ-specific phosphodiesterase inhibitor, further investigation of the cardiovascular and renal effects of phosphodiesterase inhibitors in relation to natriuretic peptides should be of interest. Indeed, more recently, it has been reported that prevention of cyclic GMP breakdown with a phosphodiesterase inhibitor in 308

6 dogs with heart failure was associated with improvements in cardiac haemodynamics. 81 In separate approaches, administration of natriuretic peptide analogues selective for the C-type natriuretic peptide receptor was associated with an increase in circulating ANP and an enhanced renal response. 82 Moreover,other work has explored the potential of a chimeric ANP-CNP peptide analogue (vasonatrin). 83 This peptide displays potent venodilating and natriuretic actions and mimics the actions of both diuretics and nitrates, suggesting a unique therapeutic potential. However, its action may be limited by its unusually marked arterial vasorelaxing actions. Further work on designer peptides has led to the development of analogues specific for the type A receptor. 84 The importance of this research is underscored by the prospect that natriuretic peptide analogues that are more selective for the NPR-A receptor may have an improved activity profile for the treatment of acute renal failure and acute heart failure. 84 However, to date, work in humans has been based mainly on 1) infusion of ANP and BNP; 2) neutral endopeptidase inhibitors and 3) the newly introduced vasopeptidase inhibitors. Administration of ANP and BNP Numerous studies have investigated the potential therapeutic value of infusion of native ANP in several conditions and in particular in patients with essential hypertension, congestive heart disease and renal failure. 85,86 Early studies on the effects of ANP in essential hypertension used high doses of exogenous ANP and, in general, significant natriuretic effects with relatively small reductions in blood pressure were observed. Other studies addressed the effects of low-dose and short-term infusion (up to 3 hours), to achieve plasma levels of only around two-fold over basal levels. There were significant natriuretic and diuretic effects and haemoconcentration, suggesting reductions in intravascular volume in some cases.the effects on plasma renin and aldosterone were small (or not significant). Despite these effects, however, there were no consistent reductions in blood pressure. Further work has demonstrated that effective hypotensive actions of low-dose admistration of ANP is likely to require more prolonged and sustained infusion of ANP, thereby limiting its usefulness for conditions requiring long-term treatment. 85 The renal effects of ANP and, in particular, increased GFR, inhibition of renal tubular reabsorption of sodium and chloride and redistribution of blood flow, pointed to beneficial value in the treatment of acute renal failure in critically ill subjects.this led to investigations of the potential value of ANP in patients with renal failure using anaritide, a 25-amino-acid synthetic form of ANP. 87 In this multicentre study, a continuous 24-hour infusion of anaritide (0.2 ug/kg/min) was compared with placebo on a primary efficacy endpoint of dialysis-free survival rate at 21 days. Despite a trend, treatment with anaritide was not associated with a significant improvement in the overall rate of dialysis-free survival when compared with the placebo group. However, separate investigations using urodilatin (ularitide) have reported promising results in reversing acute renal failure after organ transplantation. 88 These observations are of interest as urodilatin is a 32 amino acid extended form of ANP, characterised by the presence of an additional 4 amino acid sequence at the N-terminal site of ANP. 10 Infusion of urodilatin in humans has similar natriuretic effects as infusion of ANP; unlike ANP, urodilatin appears to be made mainly within the kidney, has not been found in blood and does not appear to be part of the circulating natriuretic peptide system. 10 Initial studies in people with congestive heart failure (CHF) investigated the effects of high dose ANP, given either as a bolus or by infusion and found an attenuation of natriuretic and diuretic responses in patients with CHF compared with healthy volunteers. 85 Nonetheless, and despite these blunted responses, it was also apparent that ANP infusion in patients with CHF decreased intracardiac pressures and increased cardiac output, thereby suggesting potential beneficial effects. By contrast, other investigations have demonstrated sustained beneficial haemodynamic effects during infusion of human BNP in subjects with CHF. 89 These effects included reductions in cardiac filling pressures and increased cardiac index associated with significant natriuresis and diuresis. In view of this, several studies have now been carried out with nesiritide, a purified human BNP produced by recombinant DNA technology. 90,91 In patients with CHF, nesiritide was associated with significant reductions in pulmonary capillary wedge pressures and systemic vascular resistance, with increases in cardiac index. This occurred in the absence of increases in heart rate. Treated patients experienced an improvement in symptoms of dyspnoea and fatigue, similar to those observed with standard intravenous therapy for heart failure consisting primarily of dobutamine or milrinone.the authors concluded that the salutary clinical and haemodynamic profile of nesiritide would be a valuable addition to the initial treatment of patients admitted to the hospital for decompensated congestive heart failure. Despite these encouraging results, however, there is some concern over the side-effects of nesiritide. 92 The most prominent of these for its use in heart failure was a dose-related hypotension. 92 A marked reduction in systemic blood pressure may impair perfusion and is likely to offset the renal effects. Administration of ANP or BNP, however, is associated with the problems intrinsic in the use of peptides (parenteral administration, rapid elimination, high commercial costs, etc.) especially in conditions likely to need long term treatment. While other routes of administering peptides, such as nasal delivery or enteric coating formulations, could be considered, considerable efforts have also been made in the development of orally active inhibitors which enhance endogenous levels by blocking the breakdown of ANP and of BNP by neutral endopeptidase (EC 24.11). 309

7 Neutral endopeptidase inhibitors Both ANP and BNP are rapidly removed from the circulation. Several tissues are involved in the clearance of these peptides.apart from removal through the type C natriuretic peptide receptors, proteolytic degradation by neutral endopeptidase is another major disposal mechanism of circulating ANP and BNP. 93 NEP cleaves the Cys105-Phe106 bond in ANP, thereby leading to a relatively inactive peptide. By contrast, BNP is cleaved at different sites within the peptide. The importance of neutral endopeptidase in the clearance of BNP in humans is unclear, but in sheep inhibition of neutral endopeptidase increased both ANP and BNP to a similar extent. 94 Neutral endopeptidase is widely distributed and the kidney is a rich source of the enzyme and, significantly in relation to the actions of natriuretic peptides, it is found within the kidney on both tubular cell membranes and in glomerular cells. 95,96 The enzyme is also found within the CNS, where it appears to be involved in the inactivation of neuropeptides. Neutral endopeptidase inhibitors, of course, are not new; as this enzyme also breaks down enkephalins, inhibitors of this enzyme such as thiorphan, sinorphan, and kelatorphan were developed about two decades ago as enkephalinase inhibitors. 96 When tested in animals, these inhibitors cause increases in plasma ANP and subtantial natriuretic effects, suggesting a potentiation of the renal effects of natriuretic peptides. The effects on blood pressure in the SHR were more variable, in that some, but not every, study demonstrated reductions in blood pressure. However, these inhibitors effectively reduced blood pressure in the DOCA-salt hypertensive rat, a model of volume-expanded hypertension. 96, 97 The effects on ANP and on the associated renal actions were confirmed in normal humans, where the major effects of the neutral endopeptidase inhibitor candoxatril were an increase in circulating ANP, a marked increase in sodium and water excretion and associated increases in both plasma and urinary cyclic GMP These observations clearly reproduce those observed after infusion of low doses of exogenous ANP, thereby suggesting that the actions of these inhibitors may be mediated through increased levels of ANP and possibly also of BNP. Despite these results suggesting an apparent enhancement of endogenous ANP, there were no substantial effects on blood pressure either in normotensives or in hypertensives Similar observations were also made when candoxatril was given on a longer term basis term in individuals with essential hypertension. 102 One possible reason for the limited effectiveness of NEP inhibitors as antihypertensive agents seems to be related to the non-specificity of the enzyme, leading to a concomitant enhancement of vasopressor peptides and angiotensin II (Ang II) in particular. 103,104 These observations suggested that NEP inhibitors might be more effective in hypertensive conditions associated with low plasma renin. In keeping with this, a significant reduction in blood pressure was observed in a study which investigated the effects of the NEP inhibitor Figure 4 ic, renal and other effects of angiotensin II (AT 1 -receptor). ic Vasoconstriction secretion Renal Increased sodium reabsorption Glomerular efferent vasoconstriction Increase in glomerular capillary hydraulic pressure Mesengial cell contraction Redistribution of renal blood flow Feedback inhibition of renin release Other effects Extracellular matrix proteins Plasminogen activator inhibitor-1 Transforming growth factor-beta Macrophage activation SCH34826 in hypertensives of African origin who are known to have low plasma renin activity. 105 Investigation of the effects of neutral endopeptidase inhibitors in patients with mild chronic heart failure using candoxatril given acutely, demonstrated some beneficial haemodynamic effects with no change in blood pressure or heart rate The spectrum of action of these agents in patients with congestive heart failure (e.g. natriuresis and diuresis in the absence of substantial activation of the RAAS, tachycardia or hypokalaemia) provide potentially important advantages over some of the existing diuretics; however, there is clearly a need for examining not only long-term effects, but also quality of life scores and toxicity. The importance of this is underscored by the results of a recent trial of ecadotril (sinorphan, the s-enantiomer of the racemate acetorphan). 110 First, patient-reported symptoms and quality-of-life scores failed to reveal any important overall symptomatic benefit. Secondly, there was a profile of adverse events and, in particular at the highest dose of ecadotril, there was a relatively high frequency of aplastic anaemia. Vasopeptidase inhibitors While NEP inhibitors increase activity of endogenous natriuretic peptides, the concomitant enhacement of the effects of vasopressor peptides, especially Ang II, is likely to offset their antihypertensive effects. Because of this, there has been considerable interest in combining NEP inhibitors with ACE inhibitors with the expectation that this combination will enhance the effects of natriuretic peptides in the presence of a reduction in Ang II. The benefits of ACE inhibitors in antagonising the effects of Ang II 111 (Figure 4) are now well established for both hypertension and for congestive heart failure. Therefore, inhibition of ACE activity to reduce Ang II and of neutral endopeptidase to enhance endogenous natriuretic peptides, should theoretically provide a powerful combination in the treatment of hypertension or congestive heart failure.this is supported by a series of experiments in animals, whilst the synergistic antihypertensive effects of combined NEP and ACE inhibition has also been demonstrated in humans

8 Figure 5 Central role of vasopeptidase inhibition for the metabolism of angiotensin II, bradykinin and natriuretic peptides. Bold arrows indicate effect of vasopeptidase inhibition on endogenous levels of angiotensin II, bradykinin and natriuretic peptides Although many compounds are highly selective inhibitors of either NEP or ACE activity, some of the available inhibitors of these enzymes displayed partial inhibition of both enzymes, setting the scene for the development of vasopeptidase inhibitors a single molecule which can inhibit both ACE and NEP. 118 Several more effective and orally active vasopeptidase inhibitors have now been developed and tested for their renal and cardiovascular effects in animal models of hypertension and heart failure These substances are potent inhibitors of both ACE and NEP in vitro with inhibitory constants in the low nanomolar range and equivalent to those of single ACE inhibitors. In animals, administration of vasopeptidase inhibitors, leads in general, to increases in circulating ANP (and BNP to some extent), urinary cyclic GMP and urinary sodium excretion, without a marked effect on potassium excretion, in association with substantial reductions in blood pressure. Moreover, the effects on blood pressure appear to be independent of plasma renin status. 123 Additionally, omapatrilat improved survival in cardiomyopathic hamsters 124 and, in a sheep model of heart failure, exhibited pronounced acute and sustained improvements in cardiac haemodynamics and renal function. 125 The haemodynamic effects of omapatrilat are consistent with those expected after activation of the natriuretic peptide system, 126 although, to some extent, the effects of neutral endopeptidase inhibitors may also involve bradykinin. Neutral endopeptidase plays a central role in the metabolism of not only natriuretic peptides and Ang II but also of bradykinin (Figure 5). Reductions in bradykinin breakdown have been demonstrated after administration of NEP inhibitors. 119,127 Since bradykinin, is also a substrate for ACE, inhibition of both ACE and neutral endopeptidase is likely to lead to an even greater enhancement of endogenous bradykinin activity. Several orally-active vasopeptidase inhibitors, including sampatrilat, MDL 100,240 and omapatrilat, have now been tested in humans. Investigations in normal subjects and in patients with essential hypertension have demonstrated significant reductions in blood pressure, together with preservation of renal haemodynamics and increased urinary sodium and volume excretion with increases in atrial natriuretic peptide and cyclic GMP excretion Omapatrilat has been tested both in subjects with hypertension and in those with congestive heart failure. Omapatrilat is a potent, orally-active, long-lasting inhibitor of both ACE and NEP. When tested in patients with hypertension, omapatrilat produced dose-related (10 to 80 mg/day) long-lasting reductions in average blood pressure The initial studies on the effects of omapatrilat in congestive heart failure displayed beneficial cardiac and renal haemodynamic effects 134,135 and these have been confirmed in a more recent study. 136 Moreover, in people with CHF, omapatrilat was associated with improvements in renal function. This is of potential importance, since renal function frequently deteriorates during the progression of chronic heart failure, and renal impairment is one of the most powerful predictors of prognosis in patients with CHF. 137 Despite these promising results, however, concerns have been expressed about the side-effect profile of omapatrilat and specifically the higher frequency of angio-oedema compared with ACE inhibitors alone. 138 These issues clearly need to be addressed further and a more detailed analysis of the risk/benefit profile of omapatrilat for the various conditions being considered is required. Nonetheless, given the potential beneficial cardiac and renal haemodynamic effects of vasopeptidase inhibitors, it is likely that this class of inhibitors may well become an important approach to the treatment of hypertension and of conditions associated with overt volume overload. Summary and conclusions Since the original discovery of ANP nearly 20 years ago and the subsequent realisation of the existence of a family of natriuretic peptides, there has been considerable progress in the elucidation of the physiological and pathophysiological significance of these peptides.this review has examined two potentially important practical aspects arising from natriuretic peptide research the significance of measurement of plasma levels of ANP and of BNP for cardiovascular disease and the therapeutic potential of targetting the natriuretic peptide system. Several situations where the measurement of plasma ANP and BNP may be of benefit in the overall assessment and prognosis of cardiac disease have been discussed. The measurement of plasma levels of these peptides appears to have limited value as a specific diagnostic tool and is unlikely to replace well-established procedures to assess cardiac function. Nevertheless, given the strong negative predictive value, the value of the measurement of plasma natriuretic peptides particularly BNPs, in people with suspected heart disease rests on the evidence that a normal value indicates a low risk of cardiac impairment. Moreover, a consistently elevated plasma level of 311

9 BNP after MI is associated with a distinctly poor prognosis. In turn, this may help to select those with high plasma levels for subsequent detailed investigation of cardiac dysfunction. This may be an important option, especially where the facilities for the more invasive cardiological procedures are not available. Intriguingly, recent research also suggests the possibility that plasma levels of natriuretic peptides may have an important role in guiding more effective therapy for heart failure. The potent cardiovascular and renal effects of ANP and BNP provide an important therapeutic potential for hypertension and for conditions associated with volume overload. A number of approaches which have been used to enhance endogenous activity of these peptides have been highlighted. The use of the native peptides ANP and BNP may well be valuable in some circumstances, such as in critically ill individuals with congestive heart failure or renal failure. 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