Vasopeptidase inhibitors: a new class of dual zinc metallopeptidase inhibitors for cardiorenal therapeutics

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Vasopeptidase inhibitors: a new class of dual zinc metallopeptidase inhibitors for cardiorenal therapeutics Giuseppe Molinaro*, Jean-Lucien Rouleau† and Albert Adam*‡ Vasopeptidase inhibitors are a new class of drugs that simultaneously inhibit angiotensin-I-converting enzyme and neutral endopeptidase 24.11, two metallopeptidases responsible for the breakdown of different vasoactive peptides. At least ten vasopeptidase inhibitors are in various stages of development and results obtained in preclinical and clinical studies indicate a promising future for the treatment of hypertension, heart failure and renal disease. However, like angiotensin-I-converting-enzyme inhibitors, vasopeptidase inhibitors are characterized by acute and chronic side-effects that need to be clarified. Addresses *Faculté de Pharmacie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada † Department of Cardiology, University Health Network, University of Toronto, Toronto, Ontario, M5G 2C4, Canada ‡ e-mail: [email protected] Current Opinion in Pharmacology 2002, 2:131–141 1471-4892/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ACE Ang ANP AR BNP des-Arg9-BK ECE Km kcat NEP NYHA SHR t½ tmax

angiotensin-I-converting enzyme angiotensin atrial natriuretic peptide anaphylactoid reaction brain natriuretic peptide des-arginine9-bradykinin endothelin-converting enzyme Michaelis–Menten constant turnover number neutral endopeptidase New York Heart Association spontaneously hypertensive rats half-life time to maximal plasma concentration

Introduction The term vasopeptidase is a neologism that does not correspond to any real biochemical classification of enzymes. This word results from the contraction of two words: vasoactive and peptidase. Vasopeptidase could mean any peptidase able to generate or to inactivate a vasoactive peptide. In fact, the term vasopeptidase took a more restrictive meaning when it appeared for the first time in the literature in early 1999 [1], introducing a new terminology for a new class of drugs: the vasopeptidase inhibitors. Previously known as dual metallopeptidase inhibitors, these are single molecules that, in this context, simultaneously inhibit two zinc metallopeptidases: angiotensin-I-converting enzyme (ACE) and neutral endopeptidase (NEP) 24.11. Triple vasopeptidase inhibitors, which simultaneously inhibit ACE, NEP 24.11 and endothelin-converting enzyme

(ECE), have also been synthesized and are in the preclinical stage of development [2]. In this article we first briefly review the main properties of both these metallopeptidases, NEP 24.11 and ACE, as they are relevant to this new class of drugs. Second, we discuss the potential role of the different vasoactive peptides, and mainly that of the kinins, as potential mediators of the beneficial cardiovascular actions of vasopeptidase inhibitors. Third, we review recent experimental and clinical data on the vascular, cardiac and renoprotective effects of vasopeptidase inhibitors. Finally, we discuss the potential pathophysiological mechanisms underlying the acute side-effects of vasopeptidase inhibitors. A comparative thread with ACE inhibitors, the first vasopeptidase inhibitors, will be held throughout.

Therapeutic targets of vasopeptidase inhibitors The molecular and biochemical characteristics of ACE and NEP 24.11 have been reviewed extensively elsewhere [3,4]. We summarize here the properties of these metallopeptidases as they pertain to the pharmacological activity of vasopeptidase inhibition. Angiotensin-I-converting enzyme

ACE (EC 3.4.15.1) is a widely distributed zinc metallopeptidase, located principally in the vascular endothelium of the lung. It is an ectoenzyme anchored to the plasma membrane by its C-terminal end, with the bulk of its mass branching into the lumen. It has two active catalytic sites (namely, the N- and C-domains), both exposed on the extracellular surface of the cell. In vitro, the vasopeptidase inhibitor omapatrilat inhibits both catalytic domains equally and is five times more potent than fosinoprilat, a pure ACE inhibitor, in depressing angiotensin (Ang) I hydrolysis. In vivo, omapatrilat inhibits both domains equally, whereas fosinoprilat is more specific to the N-domain [5]. Ang I was originally considered the main, if not the only, physiological substrate for ACE (Michaelis–Menten constant [Km], a measure of enzyme–substrate affinity, ≈ 16 µM). However, because of its higher affinity (Km ≈ 0.18 µM) for the nonapeptide bradykinin, ACE is now primarily considered a kininase rather than an angiotensinase [6•,7••]. In human plasma, but also at the endothelial level, ACE has been shown to be the main degrading pathway for bradykinin (reviewed in [6•,7••]). In serum, the ACE insertion/deletion (I/D) genotype determines bradykinin degradation and suggests another mechanism whereby the ACE D allele could be associated with deleterious cardiovascular effects [8]. Recently, a novel human homolog of ACE, dubbed ACE2, was identified from sequencing of a human heart-failure

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ventricle cDNA library [9]. This zinc metallo-peptidase exhibits a high degree of homology with ACE but important differences exist in its catalytic properties. For example, recombinant ACE2 hydrolyzes Ang I and desarginine9-bradykinin (des-Arg9-BK), the active metabolite of bradykinin, but surprisingly fails to hydrolyze bradykinin itself. In contrast to the seemingly ubiquitous distribution of ACE (endothelial vascular cells, lung, kidney, brain, intestine, etc.), transcripts for ACE2 are found only in the testis, heart and kidney, where it is found predominantly in the endothelium of coronary and intrarenal vessels and in the renal tubular epithelium. Moreover, its peptidase activity is not inhibited by the ACE inhibitors fosinopril and captopril, presently used to therapeutically suppress ACE activity. Until now, the inhibition, if any, of ACE2 by vasopeptidase inhibitors is undetermined (see also Update). Neutral endopeptidase 24.11

NEP 24.11 (EC 3.4.24.11, neprilysin, MME) is the prototype zinc peptidase of the M13 membrane metalloendopeptidase (MME) family. In fact, seven other related mammalian NEPlike enzymes have been identified thus far and reviewed recently [10••,11,12]. These are ECE-1 and ECE-2, which hydrolyze big endothelin to generate the vasoconstrictor endothelins; the erythrocyte cell-surface antigen KELL; PHEX, which has been associated with congenital X-linked hypophosphatemic rickets; ECEL1 (XCE or DINE), which is mainly present in the central nervous system; SEP (NL1, NEPII or MMEL1), mainly expressed in the testis; and, more recently, MMEL2 [10••,11,12]. The canonical HEXXH (His-Glu-X-X-His) motif that characterizes the zincin family is conserved within these peptidases and all extracellular domains contain the residues EXXA/GD (Glu-X-X-Ala/GlyAsp), known to be important for enzymatic activity in NEP 24.11. The pathophysiological roles of many of these enzymes, save perhaps for the ECEs, are still mostly unknown, as are their substrates, and the Ki values of the various vasopeptidase inhibitors for these NEP-24.11-like enzymes are yet to be defined. NEP 24.11 is a type II surface protein with a short membrane-proximal stalk region (~2 nm) that does not make it susceptible to release by any particular secretase. This is in contrast to ACE, a type I ectoenzyme that is colocalized on the membrane surface with its ACE secretase, itself a zincmetalloenzyme. Hence, although ACE exists primarily as a membrane-bound enzyme, a soluble form is present under normal conditions in human plasma, unlike NEP 24.11 [13]. NEP 24.11 was first identified in the brush border of the kidney epithelial cells, where it represents 4–5% of the protein content. Its immunoreactivity or activity has also been identified in cells or tissues as varied as the central nervous system, the endothelium, testis, lungs, salivary glands and bone marrow. The physiological role of NEP 24.11, however, depends on its localization and on the availability of a natural substrate. In the kidney it regulates

the natriuretic and diuretic activities of the natriuretic peptides and bradykinin. On the endothelium of the coronary bed and in rat cardiomyocyte preparations, NEP 24.11 also limits the vasodilatory properties of bradykinin. For this, NEP 24.11 is an important cardiovascular drug target. The wide distribution and broad specificity of NEP 24.11 as a peptidase, however, requires careful consideration of potential downsides to the chronic clinical use of high-affinity NEP 24.11 inhibitors. In the central nervous system it controls nociceptive effects, turning off neuropeptide signaling of substance P and the enkephalins. Furthermore, deficient degradation of brain amyloid-β peptide (Aβ), a marker for Alzheimer’s disease, by NEP 24.11 or an NEP-like enzyme may lead to biochemical and pathological deposition of Aβ1–42 in brain parenchyma [14]. NEP 24.11 may also play an important role in the development of different cancers. It inhibits androgen-insensitive prostate cancer cell migration by blocking focal adhesion kinase signaling. Recent data implicate mitogenic neuropeptides such as bombesin, endothelin-1 and neurotensin, all substrates for NEP 24.11, in cell migration that contributes to invasion and metastases of prostate cancer [15]. Also, the common acute lymphoblastic leukemia antigen CALLA (or CD10), a tumor-associated cluster differentiation antigen expressed on the surface of neutrophils and certain lymphoid progenitors, has been shown to be identical to NEP 24.11. Finally, NEP 24.11 may also exert anti-inflammatory effects, limiting the pharmacological activity of vasodilatory peptides such as the kinins, the neurokinins and adrenomedullin, which are potentially inflammatory.

Vasopeptidase inhibitors Development

In 1977, Ondetti, Rubin and Cushman [16] reported the design of potent competitive ACE inhibitors — carboxyalkanoyl and mercaptoalkaloyl derivatives of proline — which led to the release of captopril, the first ACE inhibitor, by the Squibb Pharmaceutical Company (Princeton, NJ, USA). Since that time, over a dozen ACE inhibitors have been developed and used successfully throughout the world in the treatment of different cardiovascular diseases, namely hypertension and heart failure (reviewed in [17]). Some 20 years later, Robl and colleagues [18] from the Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ, USA) reported the synthesis of mercaptoacetyl-based fused heterocyclic dipeptide mimetics as dual inhibitors of ACE and NEP 24.11. Compound 1a (omapatrilat, BMS-186716, VanlevTM), moved into the clinical phase of development for the treatment of hypertension and congestive heart failure, and is now in the most advanced stage of development of all vasopeptidase inhibitors. Other vasopeptidase inhibitors have been synthesized by a number of pharmaceutical companies and are in various stages of development (Table 1; [19]).

Vasopeptidase inhibitors Molinaro, Rouleau and Adam

Pharmacokinetics

The pharmacokinetic parameters for some vasopeptidase inhibitors have been reported. Omapatrilat has an oral bioavailability of about 30%, its protein binding is 80% and its volume of distribution is approximately 1800 L. The time to maximal plasma concentration (tmax) is about 2 hours, with a half-life (t1/2) of 14–19 hours in adults [20]. Omapatrilat is extensively metabolized and no active metabolite is found in plasma. The main route of elimination for this vasopeptidase inhibitor is urinary [21]. No dosage modification seems warranted in patients with renal impairment or undergoing dialysis, or in patients with mild-to-moderate hepatic cirrhosis. Differences in the bioavailability of omapatrilat in patients with congestive heart failure are not thought to be clinically relevant [22,23]. MDL 100240 (Aventis Pharma, USA) has an oral bioavailability of 85%, with a t1/2 estimated to be 7.5 hours. Contrary to omapatrilat, significant biliary excretion seems to occur [24]. Sampatrilat (Pfizer Inc. and Shire Pharmaceuticals Group, USA) is the third vasopeptidase inhibitor for which pharmacokinetic parameters are available. It had an originally reported bioavailability of 2% [25], but a recent reformulation by the licensee, the Shire Pharmaceuticals Group, has increased it to 20%.

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Table 1 Inhibitory potencies of vasopeptidase inhibitors under development. Drug*

Omapatrilat (Bristol-Myers Squibb, USA) BMS 182657 (Bristol-Myers Squibb, USA) Gemopatrilat (Bristol-Myers Squibb, USA) Fasidotrilat (fasidotril) (Bioproject, France) Sampatrilat (Pfizer, Shire, USA) MDL 100173 (MDL 100240) (Aventis Pharma, USA) RB105/ S21402 (mixanpril) (BP Roques, INSERM U266, France) RB 106 (BP Roques, INSERM U266, France) Z-13752A (Zambon, GlaxoSmithKline, USA) CGS 30008 (CGS 30440) (Novartis, Switzerland)

Ki (nM) NEP 24.11

ACE

8.9

6.0

6.0

12.0

305

3.6

5.1

9.8

8.0

1.2

0.11

0.08

1.7

4.2

1.6

0.35

1.8

3.2

2.2

19.0

*The prodrug is also listed (in parentheses), if applicable, and the name and location of the originator company.

In rats, the compound CGS 30440 (Novartis, Switzerland) is rapidly absorbed, with a tmax of 2 hours. The apparent t1/2 is long, estimated to be 2 weeks or more [26].

have been documented in animal and human plasma and urine but the full effects of kinin inhibition in biological fluids in vivo have yet to be fully defined.

Vasoactive pathways affected by vasopeptidase inhibitors

Ex vivo and in vivo studies

In vitro studies

Figure 1 represents the different metabolic pathways for which ACE and/or NEP 24.11 constitute a potential target for vasopeptidase inhibitors. These data result mainly from in vitro metabolic studies testing the peptidase activity of purified or recombinant enzymes on pure peptides. For some of these experiments, but not all, the values of the kinetic parameters Km and kcat/Km (turnover number/Km, a measure of substrate specificity) have been calculated and are listed in Table 2. Several metabolic effects could be expected from vasopeptidase inhibitors. By ACE inhibition, vasopeptidase inhibitors may depress Ang II synthesis from Ang I (Figure 1a); by NEP 24.11 inhibition, vasopeptidase inhibitors could prevent the degradation of atrial, brain and C-type natriuretic peptides (ANP, BNP and CNP, respectively; Figure 1b), of the endothelins (Figure 1d) and of adrenomedullin (Figure 1e; [1,27]). They also inhibit the synthesis of Ang1–7 from Ang I [28]. Finally, by dual ACE and NEP 24.11 inhibition, vasopeptidase inhibitors may increase the vasoactive properties of the kinins, mainly bradykinin (Figure 1c; [7••]). The modulating influence of vasopeptidase inhibitors on natriuretic peptides, Ang II, Ang1–7 and adrenomedullin

The actions of different vasopeptidase inhibitors have been tested in vivo, using their protective properties on the pressor response to exogenous Ang I [29,30]. The inhibitory effects of the different vasopeptidase inhibitors on Ang I were similar to those of ACE inhibitors. However, like ACE inhibitors, vasopeptidase inhibitors do not totally suppress Ang II synthesis from Ang I because other metabolic pathways, mainly serine proteases, may also transform Ang I to Ang II. In humans and animals in vivo, vasopeptidase inhibitors increase the concentration of different natriuretic peptides, of adrenomedullin and of Ang1–7 in plasma and/or urine. These actions are not observed with ACE inhibitors. Until now, however, the effects of vasopeptidase inhibitors on endogenous kinins, bradykinin and/or des-Arg9-BK have not been documented. This gap may be explained by the difficulty in quantifying kinins in biological samples and by the fact that the roles of ACE and NEP 24.11 in the metabolism of kinins are not fully understood.

Respective roles of ACE and NEP 24.11 in bradykinin metabolism Pharmacological evidence exists that pleads for a role of kinins, mainly bradykinin, in the cardiovascular effects of ACE inhibitors [31]. Theoretically, simultaneous inhibition

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Figure 1

Angiotensinogen

(a)

Renin

Vasorelaxation Blood pressure Apoptosis

Ang I

AT1

Ang II

des-Asp1-Ang I

ACE

NEP

VPi

ACE

Ang III → Ang IV Ang1–7

AT2

Vasoconstriction Blood pressure Sympathetic tone Aldosterone levels Sodium retention Cellular growth

(b) Natriuretic peptides ANP, BNP, CNP

VPi

NEP

NEP

Vasorelaxation Natriuresis? Diuresis?

Ang3–7

NPR-C

NPR-A NPR-B

ACE

Inactive fragments

VPi

Inactive fragments

VPi Vasorelaxation Blood pressure Sympathetic tone Aldosterone levels Natriuresis Diuresis

(c) HK

BK release?

Kallikrein BK B2

Kininase I B1

des-Arg9-BK

Vasorelaxation Blood pressure Ischemia Cardiac hypertrophy Natriuresis Diuresis

(d)

ACE

VPi

APP

NEP

Inactive fragments

PreproETs

(e)

ProADM

Big ETs ProADMN–20

ETs ETA and ETB SMC Vasoconstriction Blood pressure Cellular growth Antinatriuretic Sympathetic tone

PreproADM

ADM

CGRP/specific receptors?

ETB EC

Vasorelaxation Blood pressure

NEP ?

VPi

Inactive fragments

Vasorelaxation Blood pressure Natriuresis Diuresis Current Opinion in Pharmacology

Vasopeptidase inhibitors Molinaro, Rouleau and Adam

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Figure 1 legend Vasopeptidase inhibitors (VPi) are likely to modulate the effects of five vasoactive cascades. (a) By ACE inhibition, vasopeptidase inhibitors depress Ang II and Ang III synthesis from Ang I and decrease degradation of Ang1–7. By NEP 24.11 inhibition, vasopeptidase inhibitors are also likely to inhibit the synthesis of Ang1–7 from Ang I. (b) By NEP 24.11 inhibition, vasopeptidase inhibitors prevent the degradation of natriuretic peptides. (c) By dual ACE and NEP 24.11 inhibition, vasopeptidase inhibitors protect the kinins, mainly bradykinin (BK), from degradation thereby increasing their vasoactive effects. (d) By NEP 24.11 inhibition, vasopeptidase

inhibitors protect the endothelins (ETs) from degradation, thereby increasing their vasoactive effects. (e) By NEP 24.11 inhibition, vasopeptidase inhibitors prevent the degradation of adrenomedullin (ADM) and proADM NH2-terminal 20-amino-acid peptide (proADMN–20). Blue arrows and text show which receptors mediate the effects of these various vasoactive peptides. ACE and NEP 24.11, the targets of VPis, are shown within broken lines. APP, aminopeptidase P; CGRP, calcitonin gene-related peptide; EC, endothelial cell; HK, high-molecular-weight kininogen; NPR, natriuretic peptide receptor; SMC, smooth-muscle cell.

of ACE and NEP 24.11 activity would be more protective than simple inhibition of bradykinin metabolism and would increase its pharmacological impact. However, different factors must be taken into account to understand and better quantify these major effects of vasopeptidase inhibitors.

metallo-peptidases and their participation in bradykinin metabolism. Recently, Saijonmaa and colleagues [34••] reported downregulation of ACE by tumor necrosis factor α (TNFα) and interleukin-1β in cultured human endothelial cells. This dose-dependent and time-dependent downregulation was found to be mediated by the p38 mitogen-activated protein kinase (MAPK) pathway and was partly reversed by hydrocortisone.

Nature of the biological milieu

We have investigated the respective roles of ACE and NEP 24.11 in the metabolism of exogenous bradykinin (added at pmol/ml concentrations) in various preparations. Under these experimental conditions, the protective effect of vasopeptidase inhibitors on the inactivation of bradykinin depends on the nature of the biological milieu. In fact, when incubated with human plasma, omapatrilat has the same protective action on the degradation of bradykinin and des-Arg9-BK than an ACE inhibitor, and these data confirm the absence of NEP 24.11 in plasma. In the kidney, however, omapatrilat behaves mainly as a NEP 24.11 inhibitor, totally blocking bradykinin degradation in brush border membrane preparations. At this level, ACE is much less involved in bradykinin degradation [7••]. In a Langendorff perfusion model of the rat heart, both acute and chronic treatments with omapatrilat significantly decrease the rate of bradykinin degradation by the endothelium of the coronary bed and this protective effect is twice that of an ACE inhibitor at comparable doses [32]. These results confirm that, at the endothelium level, ACE is not the only metallopeptidase responsible for bradykinin metabolism, as NEP 24.11 also plays an important catabolic role. However, NEP 24.11 participation in the degradation of bradykinin becomes evident only when ACE is inhibited. This observation is easily explained by the respective Km values of ACE and NEP 24.11 for bradykinin (Table 2). Similarly, a greater protective effect of omapatrilat was measured when bradykinin was incubated with rat cardiomyocytes [33]. These results show that, like at the endothelial level, NEP 24.11 and ACE are both responsible for bradykinin inactivation at the cardiomyocyte level. The pathophysiological background

Another important factor to consider in understanding the efficiency of metallopeptidase inhibitors in different cardiovascular diseases is the influence of pathophysiological conditions on the expression of these membrane

In a model of myocardial infarction induced by ligature of the ascending coronary artery, omapatrilat decreases bradykinin metabolism both at the level of the cardiomyocyte and the endothelium, this protective effect being more significant than that of an ACE inhibitor. However, the protection afforded by both kinds of inhibitors varies with the evolution of the cardiac disease [33,35]. These results show clearly that myocardial infarction and left-ventricular hypertrophy influence the expression of both metallopeptidases. Similarly, in the human heart omapatrilat decreases bradykinin metabolism to a greater degree than the ACE Table 2 Kinetic constants for the hydrolysis of various peptides by NEP 24.11 and ACE. Peptide

Km (µM)

kcat/Km (min–1.µM–1)

92 32 6.7 62 86 121 ∼160 ∼7.5

69 159 – 41.9 43.9 14 – –

0.18 130 16 25 C-ACE: 6.8 N-ACE: 4.3

3667 – 150 9 – –

NEP 24.11 BK Substance P proADMN–20 [Met5]enkephalin [Leu5]enkephalin α-hANP hBNP-32 CNP ACE BK des-Arg9-BK Ang I Substance P Ang1–7

ADM, adrenomedullin; BK, bradykinin.

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inhibitor ramiprilat does. In this case too, the nature of the pathology influenced the effect of the vasopeptidase inhibitor. In fact, the protective action of omapatrilat was significantly stronger in dilated cardiomyopathy than in the normal heart and the effect was also significantly stronger in dilated cardiomyopathy than in ischemic cardiomyopathy ([36]; see also Update). Interestingly, insulin is also a potential regulator of ACE and NEP 24.11 activity in the cardiovascular system [37]. In a model of experimental type I diabetes induced in the rat by streptozotocin, the half-life of bradykinin degradation in a cardiomyocyte membrane preparation is not modified significantly during the time course of disease evolution. In contrast, the protective effect of an ACE inhibitor and of a vasopeptidase inhibitor on bradykinin degradation does decrease progressively. These observations could be related to reduced ACE and/or NEP 24.11 activity at the cardiac level, with subsequent overtaking of catabolism by another pathway. The treatment of affected rats with insulin for one week reverses this negative effect of diabetes on ACE activity and, mainly, on NEP 24.11 activity. Similar observations were made in the kidney. The exact mechanism by which insulin modulates the activity of both metallopeptidases remains to be elucidated. Is bradykinin a pivotal mediator of the effects of vasopeptidase inhibitors?

Recently, Dumoulin and colleagues [38] have shown that chronic treatment with omapatrilat has a greater cardioprotective effect than an ACE inhibitor used at the same dose in a rat model of total ischemia. Although both inhibitors had a similar impact on bradykinin released in the effluent, only omapatrilat increased BNP at reperfusion. Omapatrilat, but not ramipril, normalized developed-leftventricular pressure and left-ventricular diastolic pressure after ischemia. This stronger protective effect of omapatrilat was also observed when biochemical parameters were measured at reperfusion. Only the vasopeptidase inhibitor significantly inhibited the release of norepinephrine and lactate dehydrogenase. Its potentiating action on cGMP levels in the perfusate was more significant than that of the ACE inhibitor. All these beneficial effects of the vasopeptidase inhibitor were blocked not only by the bradykinin B2 receptor antagonist HOE 140 but also by a specific antagonist of BNP receptors, showing that they are mediated not only by bradykinin but also by BNP. More interestingly, BNP concentration measured in the effluent at reperfusion was significantly decreased by HOE 140. This latter observation suggests that the potentiating effect of omapatrilat on BNP could result from two different actions: a decrease of BNP degradation by NEP 24.11 and stimulation of natriuretic peptide release mediated by bradykinin via its B2 receptor.

Vasopeptidase inhibitors in hypertension Preclinical studies

Various vasopeptidase inhibitors have shown potent bloodpressure-lowering effects in animal models of hypertension,

irrespective of renin levels, in contrast with classic ACE inhibitors. In general, these antihypertensive effects are accompanied by dose-related increases of ANP and cGMP excretion, diuresis, natriuresis and decreased plasma renin activity (PRA), although some paradoxal PRA elevations have been reported (reviewed in [39••]). Interestingly, through dual ACE and NEP 24.11 inhibition vasopeptidase inhibitors also seem to afford more endothelial protection, via neurohormonal modulation, than ACE inhibitors alone. When compared with an ACE inhibitor alone, chronic treatment with a vasopeptidase inhibitor leads to greater reactivity of the aorta and mesenteric resistance arteries from Dahl salt-sensitive rats to acetylcholine. Chronic treatment with omapatrilat or captopril had comparable hypotensive effects. The protective action of omapatrilat on natriuretic peptides seems to depend on their nature and on the biological milieu, as omapatrilat increased only the plasma level of ANP. Both pharmacological agents improved the attenuated responses to acetylcholine-induced endothelium relaxation; however, only omapatrilat increased vascular cGMP content [40,41]. In stroke-prone spontaneously hypertensive rats (SHR), a malignant model of hypertension, chronic treatment with omapatrilat also improved the structure and endothelial function of mesenteric resistance arteries. This effect could be attributed to the antihypertensive action of the vasopeptidase inhibitor [42]. In both studies, however, the underlying mechanisms and mediators responsible for these functional and morphological improvements remain unclear, as the authors did not test the influence of selective receptor antagonists. Clinical studies

Clinical development programs have shown that, collectively, vasopeptidase inhibitors dose-dependently reduce systolic and diastolic blood pressures in normal and hypertensive patients, regardless of age, race or gender. In mildly-sodium-depleted healthy volunteers, omapatrilat decreased 24-hour ambulatory blood pressure [43]. In hypertensive patients, six-week treatment with omapatrilat had the greatest effect on systolic pressure, compared with equivalent doses of the ACE inhibitor lisinopril and the calcium channel blocker amlodipine (reviewed in [39••]). Omapatrilat was superior in lowering seated systolic and diastolic blood pressures when combined with the diuretic hydrochlorothiazide, compared with hypertensive patients treated with hydrochlorothiazide alone [44]. Similar hypotensive effects could also be obtained with the vasopeptidase inhibitors sampatrilat and fasidotril in essential hypertension patients [25,45]. The vasopeptidase inhibitor MDL 100240 (Aventis Pharma, USA) consistently decreased supine systolic blood pressure in healthy volunteers after high-dose or low-dose sodium intake [46]. In black hypertensive patients poorly responsive to ACE

Vasopeptidase inhibitors Molinaro, Rouleau and Adam

inhibitor monotherapy, sampatrilat significantly reduced 24-hour ambulatory blood pressure compared with lisinopril [47]. Because of the different clinical designs and dosages, it is difficult to compare the results obtained by these four vasopeptidase inhibitors in clinical trials.

Vasopeptidase inhibitors and heart failure Preclinical studies

Trippodo and colleagues [48] were the first to report comparatively on the cardiovascular effects of a dual metallopeptidase inhibitor in an experimental model of heart failure. Chronic inhibition of ACE and NEP 24.11 by omapatrilat in cardiomyopathic hamsters was more effective than ACE inhibition alone (captopril) in significantly decreasing cardiac hypertrophy and premature mortality, although both inhibitors had a similar hypotensive action [48]. These antihypertrophic effects of chronic omapatrilat treatment were confirmed recently in SHR [49]. The outcome of long-term therapy with fasidotril was assessed in rats submitted to coronary artery ligation. Fasidotril had no significant effects on arterial blood pressure and heart rate, but decreased mortality and heart hypertrophy compared with placebo [50]. In a more recent study, two-week treatment with the vasopeptidase inhibitor S21402 (INSERM, France) in SHR offered no advantage over captopril in terms of blood pressure reduction or diminution of leftventricular mass and cardiac fibrosis, but only S21402 elevated plasma ANP [51]. These results illustrate the importance of experimental models, dosages and routes of administration in the evaluation of vasopeptidase inhibitor effects, and show why drug comparisons are difficult. Clinical studies

Until now, data on the effectiveness of vasopeptidase inhibitors in patients with heart failure have been limited. The IMPRESS trial [52••] showed that although omapatrilat and lisinopril did not differ in their effects on exercise tolerance, several of the designated secondary end points were reached less frequently with omapatrilat. There was a reduction in the combined end point of mortality or hospitalization or discontinuation of medication because of worsening heart failure. Deterioration of renal function was also less frequent with omapatrilat. Omapatrilat was associated with greater improvement in New York Heart Association (NYHA) classified severity of heart failure, especially in patients with NYHA class III–IV (severe) heart failure at baseline [52••]. In a pilot study, McClean and colleagues [53] investigated arterial function in patients with heart failure. Forty-eight patients in NYHA functional class II–III (mild–moderate heart failure) with a left-ventricular ejection fraction ≤40% and in sinus rhythm were randomized to a dose-ranging (2.5, 5, 10, 20 or 40 mg) trial of omapatrilat for 12 weeks. Measurements were obtained at baseline and at 12 weeks. Decreases in systolic and mean arterial pressure were seen after 12 weeks of therapy with higher doses. Ventricular–arterial coupling was

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improved with a dose-related decrease in the augmentation index. There was no change in resting forearm blood flow between groups; however, the maximum forearm vasodilator response during reactive hyperemia increased in the high-dose groups compared with the controls [53]. Omapatrilat induced an elevation of post-dose plasma ANP levels in the high-dose groups, which is consistent with NEP 24.11 inhibition. Hence, omapatrilat produces beneficial changes in ventricular–vascular coupling and arterial function in heart failure.

Vasopeptidase inhibitors and renal function It is well-known that ACE inhibitors confer protective effects on both the glomerular filtration rate and proteinuria beyond those expected with lowering of blood pressure alone, both in diabetic and non-diabetic renal disease. Because NEP 24.11 plays an important role in the catabolism of vasoactive peptides, i.e. kinins and natriuretic peptides at the brush border of the kidney, it is expected that vasopeptidase inhibitors will offer even greater renoprotection [54]. Autoradiography studies have shown that vasopeptidase inhibitors inhibit renal ACE and NEP 24.11 activities in vivo [49,55]. In a canine model of mild heart failure produced by ventricular pacing, Chen and colleagues [56•] defined the renal actions of acute treatment with omapatrilat and fosinoprilat. Omapatrilat resulted in a greater natriuretic response than ACE inhibition alone did, in association with increases in plasma and urinary levels of cGMP and natriuretic peptides, and the glomerular filtration rate. Intrarenal administration of a natriuretic receptor antagonist attenuated the renal actions of omapatrilat [56•]. The vasopeptidase inhibitor CGS 30440 (Novartis, Switzerland) was compared with benazepril, an ACE inhibitor, in a model of chronic renal failure in rats (five-sixths nephrectomy) [57]. The results from this study indicate that CGS 30440 conferred greater renal protection than the ACE inhibition alone did. CGS 30440 caused significant reductions in proteinuria and elevated urinary cGMP, an indicator of increased intrarenal levels of ANP or bradykinin. Histological examination indicated that CGS 30440 treatment further diminished tubular dilation and proteinaceous cast formation, these effects being consistent with some of the renal actions of ANP [57]. In another study of subtotally nephrectomized rats, blood pressure was reduced in a dose-dependent manner by omapatrilat and by fosinopril [58]. Proteinuria was diminished by fosinopril and normalized by omapatrilat. Decreased glomerular filtration rates, elevated plasma urea and creatinine, glomerulosclerosis and tubulointerstitial fibrosis were ameliorated by omapatrilat and fosinopril to a similar degree [58]. The apparent superior effect of CGS 30440 over ACE inhibitors on glomerular and tubular injury, whereas omapatrilat showed no apparent advantage over ACE inhibitors, might conceivably depend on the experimental protocol

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(osmotic minipumps compared with gavage), and further stresses the difficulties in objectively comparing the therapeutic benefits of vasopeptidase inhibitors.

HOE 140, a bradykinin B2 receptor antagonist [61]. These observations, taken together, suggest possible synergy of these peptides in airway hyperresponsiveness and cough.

ACE inhibition, NEP inhibition, and dual NEP/ACE inhibition with S21402 have been compared in diabetic SHR [55]. Albuminuria in diabetic SHR was lower during treatment with both S21402 and the combination of NEP and ACE inhibition compared with vehicle. Blood pressure was better controlled with S21402 than with ACE or NEP inhibitors alone, suggesting that hypertension in type I diabetes is possibly modulated by natriuretic peptides and is thus sensitive to NEP inhibition [55].

Angioedema

Side-effects of vasopeptidase inhibitors Different chronic side-effects of vasopeptidase inhibitors reported thus far (cough, dizziness and postural hypotension) are typical of the ACE inhibitor component of these agents [39••]. Flushing seems to be more specific to vasopeptidase inhibitors and adrenomedullin could be responsible for this reaction, as its infusion in hypertensive patients was recently reported to cause facial flushing and conjunctival injection in a volunteer [59]. Of more concern is the occurrence of angioedema, which had an overall incidence of 0.5% in about 6000 patients exposed to omapatrilat. The frequency of angioedema is higher in black patients, with a 2.1% incidence [39••]. Angioedema, like cough, is not a particularity of vasopeptidase inhibitors, as these effects were first described with pure ACE inhibitors and, more recently, with AT1 receptor antagonists of Ang II. It is, however, difficult to fully understand the pathophysiological background leading to the side-effects of vasopeptidase inhibitors without giving a brief overview of the chronic and acute side-effects of pure ACE inhibitors. Cough

Cough has been reported in ACE inhibitor treatment with a frequency of roughly 10%. Attributed to bradykinin accumulation, some pharmacological evidence pleads for a combined role of kinins and tachykinins — particularly the sensory neuropeptide substance P, which is present in respiratory tract C-fibers — in the symptomatology of cough. Substance P is contained in human airway nerves, beneath and within the epithelium, matching the condensed localization of NEP 24.11, its major degrading enzyme. In guinea-pigs, inhibition of NEP 24.11 has been shown to cause cough, which was inhibited by aerosols of a selective tachykinin NK1 receptor antagonist [60]. Likewise, the antagonist inhibited the augmented cough response to aerosolized capsaicin (a C-fiber stimulant) in guinea-pigs treated long-term with an ACE inhibitor [60]. Substance P, like bradykinin, is degraded by ACE. Another paper showed that in a pig model, ACE inhibitors enhanced the cough reflex, an effect that was mediated mainly by tachykinins. Interestingly, a dose-related decrease in the cough reflex was also observed with

Angioedema is the most known acute side-effect in hypertensive and heart failure patients treated with an ACE inhibitor. It has been reported with a frequency of 0.1% to 0.2% and is higher among black patients. Besides angioedema, anaphylactoid reaction (AR) has also been noted in patients dialysed with a negatively charged membrane. Severe hypotensive reactions during blood-product transfusion and plasma pheresis have also been described [62]. Notwithstanding their rarity, these three acute side-effects are potentially life-threatening. Although plasma bradykinin concentration has been reported to increase in angioedema [63], the pathophysiological background of the acute sideeffects of ACE inhibitors remains unknown, partly because of their multifactorial nature. They could result from the combination of at least three different factors: pharmacological, pathophysiological (or physicochemical), and metabolic. The pharmacological factor is obvious: the inhibition of enzyme(s) with a metallopeptidase inhibitor, an ACE inhibitor or a vasopeptidase inhibitor. The physicochemical factor triggers bradykinin release: in AR and severe hypotensive reactions, it is the negatively charged membrane or leucoreduction filter that activates the contact system of plasma, leading to bradykinin release from highmolecular-weight kininogen. This physicochemical factor remains unknown for angioedema. The metabolic factor characterizes the patient. We have investigated this aspect in ACE-inhibitor-associated angioedema using an experimental approach developed in our lab [64]. As with AR and severe hypotensive reactions, we have found that patients who present with ACEinhibitor-associated angioedema show decreased plasma catabolism of des-Arg9-BK — the active metabolite of bradykinin, which exerts pro-inflammatory activities via its inducible B1 receptor. The slower degradation of this peptide was correlated with significantly decreased plasma aminopeptidase P activity, the main degrading metallopeptidase responsible for des-Arg9-BK inactivation, with concomitant ACE inhibitor treatment [65]. Although these findings do associate des-Arg9-BK with ACE-inhibitorinduced angioedema, they do not necessarily mean that des-Arg9-BK is the only mediator of angioedema. In fact, we have already stated that kinins could lead to the local release of tachykinins, namely substance P. Interestingly, in this regard, a decrease in dipeptidyl peptidase IV activity (a substance P degrading enzyme) has also been reported in patients who presented with angioedema [66]. Considering all this, it is conceivable that angioedema associated with ACE inhibitors and vasopeptidase inhibitors could have similar pathophysiological backgrounds. In fact, the effects of vasopeptidase inhibitors on the plasma metabolism of kinins (bradykinin and des-Arg9-BK) are

Vasopeptidase inhibitors Molinaro, Rouleau and Adam

similar, if not identical, to those of ACE inhibitors. Additionally, we did not detect any non-specific action of omapatrilat on aminopeptidase P activity and, similarly, no effect of vasopeptidase inhibitors is predictable on dipeptidyl peptidase IV, which is a serine peptidase. It is interesting to note that des-Arg9-BK is an inferior substrate for recombinant NEP 24.11, compared with bradykinin (A Adam, unpublished data). However, the higher frequencies of angioedema reported for vasopeptidase inhibitors relative to ACE inhibitors could also be explained by the pathological accumulation of substance P through NEP 24.11 inhibition. Until now, this effect has been poorly documented but it has been suggested that chronic suppression of NEP 24.11 could be responsible for the development of neurogenic inflammation [10••].

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peptides were hydrolyzed by ACE2, and in each case the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 efficiently hydrolyzed des-Arg9-BK (kcat/Km = 13.4 min–1.µm–1), but did not hydrolyze bradykinin. More interestingly, ACE2 also hydrolyzed Ang II with high catalytic efficiency (kcat/Km ≥ 105 min–1.µm–1), whereas other peptide components of the renin–angiotensin system (Ang I, Ang1–9, Ang1–7 and Ang1–5) were poorly hydrolyzed, or not at all.

Vasopeptidase inhibitors are molecules that inhibit ACE and NEP 24.11 with similar nanomolar affinity. Chronic treatment of cardiovascular disorders with vasopeptidase inhibitors would potentially afford at least two main advantages over pure ACE inhibitors. First, as shown in Figure 1, vasopeptidase inhibitors are likely to modulate the cardiovascular effects of five instead of two vasoactive cascades. Second, vasopeptidase inhibitors would also increase the number of target organs. Superior renoprotection with vasopeptidase inhibitors is expected, as NEP 24.11 is the main inactivating enzyme of both natriuretic peptides and kinins in the kidney.

The regulation of cardiac NEP 24.11 has been recently investigated in human cardiac hypertrophy and heart failure [68]. NEP 24.11 mRNA was quantified by real-time PCR in left-ventricular biopsies from patients with aortic valve stenosis and heart failure due to dilated cardiomyopathy, and from control subjects with normal systolic function. Leftventricular NEP 24.11 mRNA content was increased 3-fold in aortic valve stenosis and 4.1-fold in dilated cardiomyopathy. Myocardial NEP 24.11 enzymatic activity was increased 3.6-fold in aortic valve stenosis and 4-fold in dilated cardiomyopathy. The authors conclude that elevated cardiac NEP 24.11 activity in pressure-loaded and failing human hearts may increase the local degradation of bradykinin and natriuretic peptides. In fact, these results confirm and extend previous observations by Blais et al. [36], who observed a greater protective effect of the vasopeptidase inhibitor omapatrilat on the degradation of exogenous bradykinin incubated with cardiomyocyte membrane preparations from patients with dilated cardiomyopathy, compared with preparations from healthy human hearts.

These new pharmacological agents open new areas of research for a better understanding of the role of angiotensins, natriuretic peptides, kinins, endothelins and adrenomedullins in the cardiac and renoprotective effects of mixed ACE/NEP 24.11 inhibitors.

This further stresses the importance of the pathophysiological background on NEP 24.11 and/or ACE expression and their role in the metabolism of vasoactive peptides. These experimental observations could be important to understand the clinical effectiveness of vasopeptidase inhibitors.

Preclinical studies in experimental models of hypertension and heart failure show pharmacological advantages of vasopeptidase inhibitors and a few clinical trials demonstrate equal promise. Like pure ACE inhibitors, vasopeptidase inhibitors are characterized by chronic and acute side-effects, the frequencies of which remain to be established. Fundamental research needs to be done to clarify the role of different vasodilators and potential inflammatory mediators in the pathophysiology of these acute inflammatory reactions, particularly angioedema. Also, as the role of metallopeptidases in the metabolism of peptides is site-specific and dependent on the pathophysiological background, biochemical, cellular, tissue and animal studies are warranted to define the clinical relevance, and potential downsides, of chronic NEP 24.11 inhibition in other tissues or cell systems [10••].

Acknowledgements

Conclusions

The authors thank Guy Boileau for his critical review of the manuscript.

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