(Hypertension. 2000;35:1226.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
In Vitro and In Vivo Inhibition of the 2 Active Sites of ACE by Omapatrilat, a Vasopeptidase Inhibitor
From Centre d’Investigations Cliniques 9201, Assistance Publique des Hôpitaux de Paris/INSERM, Hôpital Broussais, Paris, France (M.A., C.M.); and Institut National de la Santé et de la Recherche Médicale, Unité 36, Collège de France, Paris (A.M., P.C.).
Correspondence to Pr Pierre Corvol, INSERM U36, Collège de France, 3 rue d’Ulm, 75005 Paris, France. E-mail corvol{at}infobiogen.fr
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Abstract |
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Abstract—The vasopeptidase inhibitor omapatrilat inhibits both neutral endopeptidase and angiotensin-converting enzyme (ACE). The in vitro and in vivo inhibitory potency of omapatrilat and the specific ACE inhibitor fosinopril toward the 2 active sites of ACE (called N- and C-domains) was investigated with the use of 3 substrates: angiotensin I, which is equally cleaved by the 2 ACE domains; hippuryl-histidyl-leucine, specific synthetic substrate of the C-domain in high- salt conditions; and a newly synthesized specific substrate of the N-domain designed by acetylating the lysine residue of AcSDKP. In vitro, omapatrilat was 5 times more potent than fosinoprilat in inhibiting angiotensin I hydrolysis. Omapatrilat inhibited similarly both N- and C-domain hydrolysis, whereas fosinoprilat was slightly more specific for the N-domain. The in vivo selective inhibitory potency of single oral doses of 10 mg omapatrilat and 20 mg fosinopril were investigated in a double-blind, placebo-controlled, cross-over study in 9 mildly sodium-depleted normotensive subjects. In accordance with the in vitro results, fosinopril appeared to be more specific for the N-domain than the C-domain in vivo, since plasma and urine AcSDKP concentrations were significantly higher than those observed with omapatrilat. This study shows that it is possible to assess separately in vitro and in vivo the selectivity of ACE or ACE/neutral endopeptidase inhibitors. A differential selectivity may explain some peculiar properties observed with some ACE inhibitors.
Key Words: angiotensin-converting enzyme • angiotensin-converting enzyme inhibitors • AcSDKP • human
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Introduction |
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Angiotensin-converting enzyme (ACE) has 2 distinct active catalytic sites, hereafter called the N- and C-domains.1 The 2 domains of ACE have both common and different characteristics. Both sites hydrolyze angiotensin (Ang) I and bradykinin with almost the same catalytic efficiency.2 The N-domain cleaves specifically 2 physiological substrates, Ang-(1-7)3 and N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a hemoregulatory peptide.4 There is no known specific physiological substrates of the C-domain, but the C-domain activity can be assessed specifically in vitro by the use of the synthetic tripeptide hippuryl-histidyl-leucine (HHL), because its hydrolysis in high chloride concentration depends mostly on this catalytic site.1 Finally, some ACE inhibitors display different inhibitory potency toward the 2 active sites.5 For example, captopril appears to be
16 times
more efficient for blocking the N-domain than the C-domain, whereas
lisinopril is equally potent toward the 2 active
sites.6 The inhibitory potency of ACE
inhibitors toward ACE can be studied with the use of
synthetic7 and physiological
substrates8 or with the use of a competitive assay against
radiolabeled ACE inhibitors.9 In addition, it
is possible to explore the characteristics of the
inhibitors toward the 2 ACE domains separately by selecting
appropriate substrates and markers. Such studies will allow us to
specifically relate their in vitro/in vivo inhibitory
potency to their in vivo pharmacological actions. Combined neutral endopeptidase (NEP) and ACE inhibition has been proposed as a new therapeutic strategy in hypertension and congestive heart failure. Because ACE and NEP share some common catalytic mechanisms, it has been possible to design dual NEP/ACE inhibitors, also called vasopeptidase inhibitors, with Ki in the nanomolar range for both enzymes.10
Omapatrilat ({4S-[4
(R*), 7,
10aß]}-octahydro-4-[(2-mercapto-1-oxo-3-phenylpropyl)
amino]-5-oxo-7H-pyrido[2,1-b][1,3]-thiazepine-7-carboxylic
acid) is a potent, conformationally constrained peptidomimetic
vasopeptidase inhibitor with a similar nanomolar
inhibitory constant for both NEP and ACE.11 In
vivo, omapatrilat behaves as an ACE inhibitor, as shown by
the antagonism of the blood pressure response to Ang I in rats, and as
an NEP inhibitor by enhancing the natriuretic
effects of exogenous atrial natriuretic peptide in
rats.12 It has also been shown to be effective in lowering
blood pressure in different models of experimental hypertension with
high or low renin levels.12 The inhibitory
activity of omapatrilat against NEP and ACE has been recently shown in
normal subjects.13 The drug is currently evaluated in
phase III studies in hypertensive patients.14
The aims of this study were (1) to study in vitro the ACE-inhibitory constants of omapatrilat toward the N- and C- domains of ACE with the use of site-specific substrates, (2) to compare these in vitro results with the acute blockade of the 2 domains of ACE in vivo by omapatrilat in mildly sodium-depleted healthy subjects, and (3) to compare the in vitro and in vivo inhibition of ACE by omapatrilat with that of a single oral dose of a pure ACE inhibitor, fosinopril. Fosinopril was chosen as the reference compound because it has some N-domain ACE selectivity, being 6 times more N-selective than C-selective.6
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Methods |
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In Vitro Studies
Materials
Enzymes
Wild-type human somatic ACE and 2 ACE mutants containing either an N- or a C- active site were used as previously described1 : Each full-length mutant contains a single intact site; the other site was inactivated by mutation of the 2 histidine zinc ligands into lysine residues. Mutants were designed as N-domain and C-domain ACE, according to the inactivation of the other domain. Mutants were stably expressed in Chinese hamster ovary cell lines.
Peptides
HHL and Ang I were purchased from Bachem. Ang I and hippuric
acid were purchased from Sigma,
Acetyl-seryl-aspartyl-(N-acetyl)-lysyl-proline (AcSDAcKP)
and Acetyl-lysyl-proline (AcKP) were synthesized by Neosystem. The
purity of these last peptides was >90%.
In Vivo Evaluation
Subjects
Part of the in vivo data were published
previously13 and will be summarized here. In brief, 9
healthy normotensive male volunteers 18 to 35 years of age completed
the study after they gave written informed consent to participate to
the protocol. The protocol was approved by the "Comité
Consultatif de Protection des Personnes se prêtant à des
Recherches Biomédicales" (Paris-Cochin, France). The procedures
followed were in accordance with the institutional guidelines.
Study Design
A single-dose, double-blind, randomized, 3-way cross-over study
design was used. Each period was separated from the previous one by a
2-week washout interval. Treatments were assigned according to a Latin
square design. Each subject received 10 mg omapatrilat, 20 mg
fosinopril, and matched placebos on 3 separate occasions. Subjects were
instructed to arrive for each phase at the Broussais Clinical
Investigation Center at 7 PM on the prestudy evening (D0).
To induce mild sodium depletion, subjects were given 40 mg furosemide
at 9 PM on D0 and received a sodium-restricted diet
(30 mmol/d) over the 36 hours of each phase. On the study day
(D1), volunteers were given a single oral dose of the assigned
treatment at 9 AM and remained in a semirecumbent position
for 6 hours. Fluid intake throughout each study day was unrestricted.
Blood was sampled before and at 2, 4, 6, 12, and 24 hours after the
dose for plasma Ang I, Ang II, ex vivo ACE activity, and AcSDKP
determinations. Before oral dosing, subjects voided their bladder to
complete a 12-hour urine collection (from 9 PM on D0 to 9
AM on D1). Two further 12-hour urine collections were
completed after drug intake (from 9 AM to 9 PM
on D1 and from 9 PM on D1 to 9 AM on D2).
AcSDKP was determined in each urine sample.
Laboratory Methods
In Vitro Kinetic and Inhibition Studies of Recombinant ACE
Kinetic and inhibition studies of recombinant ACE and ACE
mutants were performed with the use of Ang I, HHL, and the new
synthetic peptide AcSDAcKP as substrates. Ang I is used as a substrate
equally cleaved by the N- and C-domains, whereas HHL and AcSDAcKP are
specific substrates for the C- and N-domains, respectively. The rate of
hydrolysis of all the substrates used was quantified by
high-performance liquid chromatography (HPLC)
on a Waters apparatus directed by a millennium
chromatography manager. Kinetic parameters
were calculated from Michaelis-Menten plots, and inhibitor
potency was determined by calculation of apparent
Ki values with the use of ENZFITTER
software. Plot of apparent Ki versus [S]
(substrate concentration) gives the Ki.
Kinetic Studies
The method for measuring the hydrolysis of Ang I and HHL
by ACE has been previously described.6 For measuring
N-domain activity, AcSDAcKP, a new peptide, was designed and
synthesized. The activity of the N-domain has been previously studied
by using its natural substrate, AcSDKP. However, the product of the
reaction, KP, is difficult to resolve from the injection peak by HPLC
because of its high polarity. Therefore, a more hydrophobic peptide
AcSDAcKP was designed by acetylating the lysine residue. The hydrolysis
of AcSDAcKP by the wild-type ACE and the N- and C-domains was
calculated from the production of AcKP. The hydrolysis was
performed with the use of 0.5 · 10-9
mol/L to 10 · 10-9 mol/L enzyme in
50 mmol/L HEPES, pH 7.0, 50 mmol/L NaCl, 1 mg/mL BSA, and
10 µmol/L ZnSO4. The reaction was
initiated by the addition of AcSDAcKP in a total volume of 250 µL,
and the mixture was incubated at 37°C to produce 5% to 10%
substrate hydrolysis. The reaction was stopped by the addition of 0.1%
trifluoracetic acid (final concentration). AcKP and AcSDAcKP were
resolved and quantified by reverse-phase HPLC on a 5-µm Puresil C18
column (Waters) with a gradient of increasing concentrations of
CH3CN in H2O/0.1%
trifluoracetic acid from 1% to 25% in 10 minutes and to 50% in 5
minutes, at a flow rate of 1 mL/min. Retention time was 13 and 15.6
minutes for AcKP and AcSDAcKP, respectively, with a detection at 200
nm. Initial velocities were measured over a substrate concentration
range of 10 to 2000 µmol/L.
Inhibition Studies
The inhibitory potency of omapatrilat and
fosinoprilat toward recombinant wild-type ACE was determined by
establishing dose-dependent inhibition curves at equilibrium, as
previously described.6 Inhibition of Ang I, HHL, and
AcSDAcKP hydrolysis was determined with the use of 0.1 nmol/L, 0.1
nmol/L, and 0.4 nmol/L of wild-type ACE respectively. After
preincubation with 0.05 to 2.5 nmol/L of inhibitor at
37°C for 1 hour, reactions were initiated by substrate addition at 2
different concentrations (0.5xKm and
3xKm) and performed for 5, 10, 30, and 60
minutes.
Ex Vivo Plasma ACE Activity
To determine ex vivo the separate activity of the C- and
N-domains, HHL and AcSDAcKP were used as C- and N-domain substrates,
respectively. Reactions were performed at 37°C with 10 or 20 µL of
plasma during 60 and 120 minutes, respectively. Substrate concentration
was 2.5xKm.
Plasma Ang II, Ang I, and AcSDKP Determinations
The methods used for blood samplings and for
angiotensins and AcSDKP determinations were as previously
described.15 The time course evolution of plasma and
urinary AcSDKP concentrations were used as very sensitive markers of in
vivo inhibition of the N-domain activity.16
Statistical Methods
The area under the curve (AUC) versus time was calculated
according to the trapezoidal rule and integrated from 0 to 24 hours.
Data were analyzed by ANOVA: The crossed factor was the subject
and the within factors were treatment and period. Because the order of
the treatments was randomized for each subject and a 2-week washout
period was allowed between each drug administration, it was assumed
that there were no carry-over effects. When the F test was
significant (P<0.05), paired comparisons were performed by
means of the Bonferroni correction. Residual variance of ANOVA was used
for pairwise tests. The assumptions of ANOVA (homogeneity of variance
and normality) were checked for each variable, and natural
logarithmic transformation was applied where appropriate. Calculations
were done with STATVIEW 4.01 and SUPERANOVA statistical software
packages (Abacus Concepts Inc). Data are expressed as mean±SD in the
tables and mean±SE in the graphs unless otherwise specified. A
probability value of <0.05 was considered significant.
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Results |
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Kinetic Characteristics of the Hydrolysis of AcSDAcKP, a New Specific Substrate of the N-Domain of ACE
The wild-type ACE and the N- and C-domains displayed an optimal cleavage of AcSDAcKP at pH 6.0 to 7.0 (Figure 1). The optimum chloride concentration for the hydrolysis of this substrate by the N-domain was 50 mmol/L (Figure 2) and was selected for the determination of kinetic parameters of all ACE molecules to ensure maximum selectivity for the N-domain versus the C-domain.
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).
AcKP production (nmol · mL-1 ·
min-1) was quantified as described in experimental
procedures and plotted against pH value.
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The Km values, calculated from Michaelis-Menten plots of AcSDAcKP hydrolysis by wild-type ACE and the N- and the C-domains, and the kcat values are given in Table 1. Wild-type ACE and the N-domain exhibit a similar kcat/Km value, which is 16-fold higher than that of the C-domain. The relative efficacy of the hydrolysis of this substrate by the N-domain versus the C-domain (N-domain efficacy/C-domain efficacy) is 16 and compares well with that of 50 found for the natural peptide AcSDKP.4
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In Vitro Inhibition by Omapatrilat and Fosinoprilat of the
Hydrolysis of 3 Selective ACE Substrates
The inhibitory constants of omapatrilat and
fosinoprilat toward 3 selective ACE substrates were compared under the
same conditions for each substrate (ACE concentration, pH, and NaCl
concentration). Omapatrilat was 5 times more potent
(Ki=0.06±0.05 nmol/L) than fosinoprilat
(0.37±0.24 nmol/L, data from Michaud et al6 ) in
inhibiting Ang I hydrolysis. The Ki of
omapatrilat for inhibition of HHL hydrolysis (0.45±0.28 nmol/L) was
close to that of fosinoprilat for this substrate (0.29±0.11 nmol/L,
data from Michaud et al6 ). AcSDAcKP hydrolysis was
mildly better inhibited by fosinoprilat
(Ki=0.13) than by omapatrilat
(Ki=0.31).
In Vivo Studies
No period effect was detected for any of the results, therefore
only treatment effects are reported in the text and tables.
Ex Vivo ACE Activity
Ex vivo plasma ACE activity of the N- and the C-domains decreased
rapidly after drug intake (Table 2). At
peak, omapatrilat and fosinopril induced 90±4% and 88±13%
inhibition of ex vivo ACE activity of the C-domain (HHL hydrolysis) and
88±8% and 97±3% inhibition of ex vivo ACE activity of the N-domain
(AcSDAcKP hydrolysis, NS), respectively. Twenty-four hours after the
dose was given, ex vivo ACE activity of the N- and the C-domains
differed significantly between the 2 active drugs: For the C-domain,
ACE activity was significantly lower after omapatrilat than after
fosinopril intake (0.67±0.19 versus 1.14±0.3 nmol ·
mL-1 · min-1,
respectively, P<0.05), whereas the reverse was observed for
the N-domain (10.4±6.3 versus 0.5±0.5 pmol ·
mL-1 · min-1,
respectively, P<0.05).
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Plasma and Urine AcSDKP
Plasma AcSDKP levels increased significantly after the intake of
the active treatments, whereas they remained low and stable after
placebo intake (Table 2 and Figure 3). Peak plasma AcSDKP levels and the
AUC0–24 of plasma AcSDKP versus time were higher
after fosinopril than after omapatrilat intake, but the difference
between the active drugs was not statistically significant when the
Bonferroni correction was used. Twenty-four hours after the dose was
given, plasma AcSDKP levels were significantly higher than placebo for
both active drugs, and there was no difference between the 2 active
drugs. Both active treatments also induced a much higher excretion of
AcSDKP in urine during the 2 collection periods after drug intake than
that observed after placebo intake
(F2,14=61, P<0.001, Figure 3). The 24-hour cumulative urinary AcSDKP excretion was
significantly higher after fosinopril than after omapatrilat intake
(268±60 versus 165±66 pmol/24 h, P<0.05
respectively).
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), 20 mg fosinopril (
),
and placebo (+) in 9 mildly sodium-depleted healthy subjects. Right,
12-hour urine excretion of AcSDKP after intake of 10 mg omapatrilat
(open bar), 20 mg fosinopril (closed bar), and placebo (hatched bar) in
9 mildly sodium-depleted healthy subjects (***P<0.001,
*P<0.05).
Plasma Angiotensins
Plasma angiotensin results have been previously
reported13 and will be summarized. Omapatrilat and
fosinopril induced a similar inhibition of ACE in vivo as assessed by
the changes in plasma Ang II/Ang I ratio achieving similar plasma Ang
II concentrations over a period of 24 hours.
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Discussion |
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Vasopeptidase inhibitors are a new class of antihypertensive agents characterized by their ability to block both ACE and NEP. Their inhibitory effect on each enzyme can evaluated in vitro by the use of specific substrates. The discovery of the presence of 2 active catalytic sites in ACE adds another degree of complexity in their mechanism of action, as ACE inhibitors may preferentially act on either ACE domain. The N-domain of ACE cleaves preferentially to the C-domain substrates such as AcSDKP,4 Ang-(1-7),3 and the N-terminal tripeptide LHRH.2 To a lesser extent, the C-domain hydrolyzes faster Leu5- and Met5-enkephalins17 as well as substance P.2 Whether these in vitro kinetic differences may account for some of the differences observed between different ACE inhibitors is not known. Indeed, some ACE inhibitors exhibit a preference for either domain, such as captopril6 or keto-ACE.3 It has even been possible to design a specific ACE inhibitor that blocks the N-domain 1000 times more efficiently than the C-domain activity.18 Therefore, it is important to evaluate separately the inhibitory activity of any new ACE or ACE/NEP inhibitor on the 2 ACE domains.
In this study, we report for the first time an in vitro and in vivo selective assessment of a dual NEP/ACE inhibitor, omapatrilat, and of a pure ACE inhibitor, fosinopril, in humans. Selective substrates were used in vitro and ex vivo to measure ACE inhibition on recombinant and plasma ACE, respectively. A new substrate for N-domain was designed to monitor easily the N-domain activity. The results obtained were compared with plasma and urinary AcSDKP levels, a reflection of in vivo N-domain inhibition,16 and with the Ang II/Ang I ratio, an indicator of both N-domain and C-domain activity.
Omapatrilat was found to be 5 times more efficient than fosinoprilat in inhibiting in vitro Ang I hydrolysis. It inhibited equally well in vitro the N- and C-domain activities, whereas fosinoprilat was twice more specific on the N-domain than on the C-domain of ACE.
The potency of omapatrilat and fosinopril to inhibit ACE was studied in vivo in healthy subjects. N- and C-domain ACE substrates were used to evaluate separately the ex vivo inhibitory potency of these 2 inhibitors toward the 2 ACE active sites. We used a state of mild sodium depletion in healthy subjects, which provides an experimental condition in which a 2- to 3-fold increase in plasma active renin, Ang I, and Ang II concentrations is reproducibly obtained. This approach has been used previously to investigate the additive effects of captopril and losartan because it gives optimal experimental conditions for quantifying in vivo ACE inhibition.19
In this model, we have previously shown that a single oral dose of the vasopeptidase inhibitor omapatrilat (10 mg) and of the specific ACE inhibitor fosinopril (20 mg) had a similar potency to inhibit ACE in vivo over a period of 24 hours, as assessed by changes in the plasma Ang II/Ang I ratio, giving similar plasma Ang II levels over 24 hours.13 There are, however, subtle differences between the 2 drugs. The initial inhibitory effect of omapatrilat and fosinopril for the N- and C-terminal active sites of ACE were similar, as shown by the initial changes in plasma AcSDKP and the ex vivo plasma ACE activity on the hydrolysis of HHL. In contrast, apparent dissociation from the C-domain appeared to occur significantly earlier for fosinopril than for omapatrilat: 24 hours after drug intake, the residual ex vivo ACE inhibition, assessed by HHL hydrolysis, was only 52±12% for fosinopril compared with 78±6% for omapatrilat. In addition, fosinopril appears to have higher affinity than omapatrilat for the N-domain of ACE because urinary AcSDKP concentrations were significantly higher than those observed after omapatrilat intake. These results compare well with those of Michaud et al,6 in which fosinoprilat was 6 times more efficient for inhibiting in vitro AcSDKP hydrolysis than Ang I hydrolysis. Interestingly, these differences between the enzyme-inhibiting properties of omapatrilat and fosinopril did not affect the changes in plasma Ang II levels over time because Ang I is a natural substrate equally cleaved by both domains.1 Altogether, these results show that it is possible to assess in vitro and in vivo the selectivity of an ACE inhibitor toward the 2 domains of ACE. A similar strategy has been recently used for showing in vivo the relative selectivity of captopril for the N-domain in rats.20 The possibility of assessing in vivo the 2 ACE domains in humans may help to elucidate some of the properties of ancient or new ACE inhibitors.
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Acknowledgments |
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This work was supported by a grant from BMS Laboratories, Princeton, NJ. The authors wish to thank the nursing staff of the Clinical Investigation Center at the Broussais Hospital (Danièle Ménard, Jeanne Meunier, Olivier Picart, and Michèle Godeau), who ran the protocol, and Dr Than-Tam Guyenne, who performed the angiotensin assays. The help of Dr Eric Ezan, who performed the AcSDKP determinations, is greatly acknowledged. We also want to thank Dr James Powel from BMS at Princeton for helpful discussions and review of the manuscript.
Received September 3, 1999; first decision September 30, 1999; accepted January 7, 2000.
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