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Vol. 284, Issue 3, 974-982, March 1998
Novartis Pharmaceuticals Corporation, Summit, New Jersey
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitors, by decreasing angiotensin-II production and by preventing the degradation of atrial natriuretic peptide (ANP), may be useful for the treatment of hypertension and congestive heart failure. The thiol dipeptide CGS 30440 (prodrug of CGS 30008, IC50: ACE/NEP = 19/2 nM) administered to rats (10 mg/kg p.o.) inhibited lung tissue ACE activity by 98% and 61% at 1 and 24 hr (P < .001) and inhibited the angiotensin-I pressor response by 75 to 90% for more than 6 hr. Renal tissue NEP activity was reduced by 80% at 1 hr and 73% at 24 hr (P < .001). In rats supplemented with exogenous ANP, CGS 30440 (1 mg/kg p.o.) elevated the concentration of circulating ANP (133%, P < .025) for 4 hr and increased the excretion of urine (300%, P < .001), sodium (194%, P < .025) and cyclic GMP (238%, P < .005). CGS 30440 (10 mg/kg p.o.) administered to hypertensive rats with aortic ligation between the renal arteries (mean arterial blood pressure, 209 ± 4 mm Hg) produced a 48 mm Hg blood pressure reduction (P < .001) within 4 hr. CGS 30440 given to cynomolgus monkeys at 2 mg/kg inhibited plasma ACE activity by 96% within 1 hr (P < .001), and this inhibition was maintained for 7 and 21 days in monkeys receiving the compound orally at 2.5 mg/kg b.i.d.. These studies demonstrate that CGS 30440 is an orally active agent which produces tissue ACE and NEP inhibition in rats and plasma ACE inhibition in primates and suggest that the compound may be useful in the treatment of hypertension and congestive heart failure.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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During
the past two decades ACE (EC 3.4.15.1) inhibitors have become
first-line therapeutic agents for hypertension and important adjuncts
for the treatment of CHF. Since the discovery of captopril in 1975, marked improvements in potency and duration of action have been
achieved in many newly developed ACE inhibitors. However, a large
population of patients still does not respond to these compounds and
requires the use of diuretics or other types of antihypertensive agents
(Williams, 1988
; Heber et al., 1988). Similarly, several CHF
studies suggest that ACE inhibitors are ineffective in the early stages
of the disease (Richardson et al., 1987; Anand et
al., 1990) when ANP and plasma adrenaline are increased but plasma
renin activity is not yet elevated (Bailiss et al., 1986;
Remes et al., 1991).
Since 1981 a new therapeutic approach to hypertension and cardiac
failure aimed at increasing the circulating levels of the vasodilator
and diuretic ANP has been conceived (de Bold et al., 1981).
Intravenous infusion of this peptide reduces blood pressure while
increasing urine volume and the urinary excretion of sodium and cyclic
GMP (Janssen et al., 1989). The short half-life of ANP,
however, has led to the development of inhibitors of NEP (EC 3.4.24.11)
to prevent its degradation (Roques and Beaumont, 1990). Although the
effects of NEP inhibitors in humans with hypertension are still
controversial, some authors have observed mild natriuresis and
borderline decreases in systolic and diastolic blood pressure during
short-term administration of these agents (Richards et al.,
1993a, b).
From an early stage in the development of selective ACE and NEP
inhibitors it was recognized that some compounds moderately cross-reacted with both enzymes (Roques et al., 1982; Gordon
et al., 1983; Gros et al., 1991). This finding
led to the initiation of the search for compounds able to inhibit both
ACE and NEP, and investigators at some pharmaceutical companies have
obtained effective dual inhibitors (Seymour et al., 1991;
Fournie-Zaluski et al., 1994a; French et al.,
1994; Gonzales Vera et al., 1995). In our search for a new
ACE/NEP inhibitor we found a series of dipeptide 
-thiols which are
long-acting, potent inhibitors for both enzymes and apparently possess
good bioavailability. Thus, this report describes the characterization
of the ACE and NEP inhibitory activity of the long-acting optimized
lead CGS 30440 (Chatelain et al., 1996; Fink et
al., 1996) in normal and hypertensive rats, as well as in
cynomolgus monkeys.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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CGS 30440, N-[[[1-[2(S)-Acetylthio-3-methyl-l-oxobutyl]amino]-1-cyclopentyl]
carbonyl]-O-methyl-L-tyrosine ethyl ester (fig. 1; Fink et al., 1996), is a
novel thioacetyl-containing dual inhibitor of ACE (EC 3.4.15.1) and NEP
(EC 3.4.24.11). It is a di-ester prodrug which is hydrolyzed to the
active thiol carboxylic acid CGS 30008, N-[[1-2[2(S)-mercapto-3-methyl-1-oxobutyl)amino]-1-cyclopentyl]carbonyl]-O-methyl-L-tyrosine. CGS 30440 can be synthesized in high yield in seven steps from readily
accessible starting materials. It is a white crystalline powder with a
melting point of 103 to 104°C and a solubility of 0.0516 mg/ml, pH
7.0 (Fink et al., 1996). Reference ACE inhibitors, NEP
inhibitors and dual ACE/NEP inhibitors were synthesized in house
(benazepril, candoxatril, glycopril, thiorphan, SCH 42495 and MDL
100,240) or requested from other pharmaceutical companies. Captopril
was obtained from Bristol-Myers Squibb (Princeton, NJ), enalapril from
Merck, Sharp & Dohme (Rahway, NJ) and ramipril from Hoechst-Roussel
Pharmaceuticals (Somerville, NJ).
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Inhibition of ACE and NEP in vitro.
The ACE and
NEP inhibitory activities of the test compounds initially were assessed
in vitro. The inhibitory constants (Schulz et
al., 1991) were determined from the inhibition of enzyme activity by use of at least six different concentrations of the inhibitors in
duplicate. The IC50 values (fig.
2) were derived from inhibition curves
with the nonlinear regression curve fitting program of Research
System/1 (RS/; Bolt, Beranek and Newman, Inc., Cambridge, MA) software
package on a VAX 1/750 computer equipped with VMS operating system
(Digital Equipment Corp., Marlboro, MA).
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to the stage of Nonidet-P40 extract. The
extract was frozen and stored at 
70°C until use in the enzyme
assay.
ACE activity was determined by the hydrolysis of
hippuryl-L-histidyl-L-leucine. The resulting
product, histidyl-leucine, reacts with o-phthaldialdehyde and is
measured spectrophotometrically at 360 nm (Cushman and Cheung, 1981).
The reaction mixture (250 µl) containing 300 mM NaCl, 100 mM
KH2PO4, 2 mM substrate and 35 µg partially purified ACE was incubated at 37°C for 30 min. The
reaction was terminated by the addition of 0.75 ml of 0.6 N NaOH, then
100 µl of o-phthaldialdehyde (2 mg/ml in methanol) were
added and the reaction tubes were left at room temperature for 10 min.
After adding 100 µl of 6 N HCl the tubes were centrifuged and the
optical density of the supernatant was measured at 360 nm. The optical
densities were converted to nanomoles of histidyl-leucine formed during
the 30-min incubation by comparison with a standard curve.
NEP 3.4.24.11 activity was determined by the hydrolysis of the
substrate glutaryl-Ala-Ala-Phe-2-naphthylamide by a modified procedure
of Orlowski and Wilk (1981). The incubation mixture (total volume, 125 µl) containing 4.2 µg of protein from rat kidney cortex membranes
(Maeda et al., 1983), 50 mM Tris buffer, pH 7.4 at 25°C,
500 µM substrate (final concentration) and leucine aminopeptidase M
(2.5 µg), was incubated for 10 min at 25°C. After the incubation 100 µl of fast garnet (250 µg fast garnet/ml of 10% Tween 20 in 1 M sodium acetate, pH 4.2) were added and the enzyme activity was
measured spectrophotometrically at 540 nm. One unit of NEP activity was
defined as 1 nmol of 2-naphthylamine released per minute at 25°C.
Inhibition of ACE and NEP ex vivo.
Determinations of tissue ACE and NEP were performed in the lung,
kidney, aorta and, in some experiments, the adrenals of normotensive Sprague-Dawley rats after the oral administration of the compounds or
the appropriate vehicle. The rats were sacrificed and the organs were
removed rapidly, rinsed, dissected free of adhering tissue, frozen in
liquid nitrogen and stored at 70°C until assayed. Within 15 days
the tissues were homogenized (Tekmar Tissumizer, Cincinnati, OH) and
stirred on ice for 3 hr. Aliquots were used for determinations of
enzyme activity and for protein measurements (Lowry et al., 1951). Plasma ACE was measured in blood samples obtained from monkeys,
but tissue studies were not performed in these animals. Plasma and
tissue ACE activities were determined by the methods described under
"Inhibition of ACE and NEP in Vitro."
). For the
in vitro experiments, the enzyme source was rat kidney
cortex membranes, whereas for the ex vivo studies the enzyme was from the particular tissue under study. The incubation mixture (total volume, 250 µl) contained 50 µl of tissue homogenate, 50 mM
Tris buffer, pH 7.4 at 37°C, 500 µM substrate (final concentration) and leucine aminopeptidase M (1 mg/ml). The mixture was incubated for
10 min at 37°C and 1.0 ml of fast garnet (50 µl fast garnet/ml of
10% Tween 20 in 1 M sodium acetate, pH 4.2) was added. Enzyme activity
was measured spectrophotometrically at 530 nm after 30 min at 37°C.
One unit of NEP activity was defined as 1 nmol of 2-naphthylamine
released per minute at 37°C.
Inhibition of pressor responses to angiotensin-I. The in vivo ACE inhibitory activity of CGS 30440 was studied by testing its ability to inhibit the pressor response evoked by intravenous injections of angiotensin-I. Sprague-Dawley rats (Hsd:(SD) BR, Taconic Farms, Germantown, NY) were anesthetized with methohexital sodium and instrumented with catheters in the femoral artery and vein to measure mean arterial pressure and administer angiotensin-I, respectively. The catheters were tunneled subcutaneously to exit from the lower back through a stainless steel spring and swivel system. On the next day, angiotensin-I (300 ng/kg i.v.) was injected four times at 15-min intervals to establish a reproducible control pressor response. CGS 30440 was administered orally before rechallenging with angiotensin-I at intervals ranging from 15 to 60 min during a 6-hr period. The pressor responses obtained before and after the administration of the compound were compared and the data expressed as a percent inhibition of the control response. Control animals received the vehicle alone (3% cornstarch p.o., 1 ml/kg).
Plasma ANP determinations. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were anesthetized with methohexital sodium and instrumented with catheters in the femoral artery and vein to obtain blood samples and infuse ANP, respectively. The rats were tethered with a swivel system and were allowed to recover for 24 hr before being studied in the conscious, unrestrained state. On the day of the study, all rats were infused continuously with ANP at 450 ng/kg/min i.v. for the entire 5 hr of the experiment. Sixty minutes after beginning the infusion, blood samples for base-line ANP measurements were obtained (time 0). The rats were then randomly divided into subgroups and treated orally with different doses of the ACE/NEP inhibitor or the vehicle. CGS 30440 was administered at 0.3 mgEq/kg (0.351 mg/kg, which is equivalent to 0.3 mg/kg of the active inhibitor CGS 30008), at 1 mgEq/kg (1.17 mg/kg) and at 3 mgEq/kg (3.51 mg/kg). Additional blood samples were taken 30, 60, 120, 180 and 240 min after administration of the test compound. The responses produced by each dose of CGS 30440 were expressed as a percentage of those obtained in the vehicle-treated group (3% cornstarch, 1 ml/kg).
The plasma concentrations of ANP were determined by a specific radioimmunoassay. The plasma was diluted in buffer containing 19 mM sodium phosphate monobasic and 81 mM sodium phosphate dibasic (pH 7.4), 50 mM NaCl, 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.1% NaN3. One-hundred microliters of standards [rANP(99-126)] or diluted samples were added to 100 µl of rabbit anti-rANP serum and incubated at 4°C for 16 hr. [125I]rANP (14,000 cpm) was then added to the reaction mixture which was incubated at 4°C for an additional 24 hr. Goat anti-rabbit IgG serum coupled to paramagnetic particles was added to the reaction mixture and bound [125I]rANP was pelleted by exposing the mixture to an attracting magnetic rack. The supernatant was decanted and the pellets counted in a gamma counter. All determinations were performed in duplicate.Studies of the renal effects of exogenous ANP. These studies were performed in normotensive Sprague-Dawley rats which were fasted overnight. Under amobarbital anesthesia, catheters were placed in the jugular vein for infusion and in the bladder for urine collection. Isotonic saline was infused intravenously at 25 µl/min. This solution was supplemented with amobarbital to maintain the animals under anesthesia for the entire experimental period. After obtaining base-line urine samples for two consecutive control periods, CGS 30440 or vehicle was administered orally, and 15 min thereafter ANP (300 ng/kg/min) was added to the infusion. Urine samples were collected every 15 to 30 min. Urine volume was determined gravimetrically, sodium concentration by flame photometry and cGMP concentration by use of a commercially available enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). Excretion rates for urinary sodium and cGMP were calculated by standard formulae.
Blood pressure determination. Experiments to study the effect of compounds on blood pressure were performed in outbred male Sprague-Dawley rats (Hsd:(SD)BR, Harlan Farms, Madison, WI). The animals were earmarked, housed in pending cages and given tap water and regular pelleted food ad libitum. They were maintained in an isolated room with constant temperature (74°F) and cycles of light and darkness lasting for 12 hr.
A complete aortic ligation was performed between the renal arteries (Rojo-Ortega and Genest, 1969). Sham-operated animals underwent a
similar surgical procedure by which the peritoneal cavity was opened
but the aorta remained untouched. After surgery, each animal received a
subcutaneous injection of 200,000 U of Wycillin (sterile penicillin
procaine suspension, Wyeth Laboratories, Inc., Philadelphia, PA). The
evolution of hypertension was studied in groups of aortic-ligated and
sham-operated animals sacrificed between 3 and 30 days after operation.
The elevated blood pressure reached a plateau 12 days after aortic
ligation. In this period plasma renin activity was increased
significantly, and hypertrophy and hyperplasia had developed in the
vascular system; therefore, 12-day aortic-ligated rats were used in all
the experiments. Direct blood pressure measurements were performed in
conscious rats, mildly restrained in plastic cylinders, instrumented
with an indwelling catheter (PE-50) implanted in the right carotid
artery and connected to a Gould Statham physiological pressure
transducer (Chatelain et al., 1990). This was coupled to a
transducer amplifier (Gould, model 13-4615-50), and the blood pressure
waves were displayed in a Gould 2400 S recorder, thermal writing model
(Gould Inc., Cleveland, OH). After a period of stabilization, the
compounds were administered orally and the changes in blood pressure
and heart rate were monitored for a 3- or 4-hr period. Rats receiving ANP intravenously had an additional catheter (PE-10) implanted in the
jugular vein. The responsiveness of the high blood pressure of
aortic-ligated rats to ACE inhibitors and to ANP was tested by use of
captopril at 10 mg/kg p.o. and ANP at 30 nmol/kg i.v.
Plasma ACE studies in primates.
The influence of CGS 30440 on plasma ACE was studied in trained cynomolgus monkeys (Macaca
fascicularis) housed individually and fed a standard high-protein
monkey diet. The study protocols were approved by the institutional
Animal Care and Use Committee. Blood samples (2 ml) were drawn from the
femoral vein with the animals in the fasting state and restrained in
their cages by the pole and collar technique. CGS 30440 and benazepril
were dissolved in vehicle (ethanol) and administered orally by
adsorption in banana slices. Plasma samples were maintained at 70°C
until assayed.
Statistical analysis. Values are presented as mean ± S.E.M. The significance of differences between groups were analyzed by one-way analysis of variance followed by the Dunnett's test for comparison with control when necessary. The blood pressure changes were analyzed by one-way repeated measures analysis of variance. Comparisons between compound-treated and vehicle-treated groups were performed by unpaired t tests, whereas differences with their pretreatment values were assessed by paired t test. Values of P < .05 were considered significant.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In vitro studies.
Of all the compounds tested, one
of the most potent in the -thiol series was CGS 30008 (fig. 1) with
an IC50 value of 2.2 nM for NEP and 19 nM for ACE
(fig. 2).
Ex vivo inhibition of ACE and NEP. The activities of the prodrugs of CGS 30008 were tested by measuring the inhibition of ACE and NEP activity in lung, kidney, aorta and adrenal tissue at 1 hr after oral administration of a 10 mg/kg dose. According to these criteria the most effective prodrug was CGS 30440 (fig. 1).
One hour after oral administration of CGS 30440 (10 mg/kg p.o.) lung ACE was inhibited by 98% (P < .010, fig. 3). The ACE reductions obtained in lung tissue with reference ACE inhibitors or with the dual ACE/NEP inhibitor MDL 100,240 (also administered at 10 mg/kg p.o.) are depicted in figure 3. Captopril, enalapril and MDL 100,240 inhibited lung ACE by 35, 92 and 61%, respectively, whereas benazepril and ramipril produced inhibitions amounting to 77 and 70% of control values (P < .010, fig. 3).
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Inhibition of the angiotensin-I pressor response. The intravenous administration of angiotensin-I (300 ng/kg) produced a significant but short-lived increase in mean blood pressure averaging 58.9 ± 2.5 mm Hg, (P < .001). The oral administration of CGS 30440 resulted in a statistically significant inhibition of this response within 15 min regardless of the dose tested (1, 3 and 10 mgEq/kg, P < .0001 versus the pretreatment value, fig. 5). A 70 to 90% inhibition was observed for the initial 2 hr in the three groups of animals studied. After this time and for the rest of the 6-hr period, a divergence between these groups became apparent. Animals receiving the larger dose (10 mgEq/kg) maintained a 75% inhibition for the duration of the study. Animals treated with the 3 mgEq/kg dose presented an 80% reduction of the response within 15 min, which was reduced to 46% by 6 hr. The animals receiving the 1 mgEq/kg dose were characterized by an 80 to 90% inhibition during the first hour but showed a rapid recovery of the pressor response and the inhibition was 17% at the end of the study. There was no inhibition of the pressor effect of angiotensin-I in rats treated with vehicle alone (fig. 5).
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Blood pressure studies. Twelve days after the operation the mean blood pressure of aortic-ligated rats was above the 200 mm Hg level in most of the animals. In the randomly selected group used to test CGS 30440, MAP averaged 209 ± 4 mm Hg. Oral administration of a 10 mg/kg dose produced a slow but steady decline in blood pressure which, at 1 hr, reached statistical significance from the pretreatment level (20 mm Hg, P < .010, fig. 6). The decline continued until the end of the 4-hr observation period at which time the blood pressure level was 161 ± 8 mm Hg (48 mm Hg decrease, fig. 6).
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Elevation of plasma ANP. In rats receiving an intravenous infusion of ANP the plasma concentration of this peptide averaged 5.45 ± 0.16 ng/ml (n = 6). Oral administration of CGS 30440 produced a statistically significant increase in the concentration of circulating ANP compared with values obtained in vehicle-treated controls (fig. 7). The largest dose tested in these experiments (3 mgEq/kg) produced a 172% elevation at 30 min which was sustained for the duration of the experiment and averaged 272% at 4 hr (fig. 7A). The 1 mgEq/kg dose increased plasma ANP 133% at 30 min, 182% at 2 hr and 95% at the end of the 4-hr observation period. In the group treated with the smallest dose of CGS 30440 (0.3 mgEq/kg) a slight increase was observed between the second and third hour after administration (36-66% increase, fig. 7), but this change was not statistically significant according to Dunnett's test. In rats receiving vehicle, the plasma concentration of ANP did not change throughout the study, averaging 5.55 ± 0.79 ng/ml after 4 hr (fig. 7B).
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Enhancement of the renal effects of infused ANP. The initiation of an intravenous infusion of ANP (300 ng/kg/min i.v.) to anesthetized normotensive control rats resulted in a significant increase in urinary output within 15 min, with the first significant change occurring at 30 min (a 10-fold elevation from the beginning of the ANP infusion, P < .010, fig. 8A). The enhanced urinary excretion lasted for the initial hour and declined thereafter. Normal urinary output characterized the final 2 hr of the study period despite continuation of the ANP infusion (fig. 8A). The sodium excretion profile evoked by ANP mirrored the volume change. There was a significant elevation at 30 min (P < .005), and sodium excretion was also normalized after 3 hr (fig. 8B). In addition, the urinary cGMP increase commenced within 15 min and reached significance at 45 min after the initiation of ANP infusion (P < .025) but unlike urinary volume and sodium, the excretion of the nucleotide remained elevated during the entire observation period (fig. 8C).
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Effects on plasma ACE in primates. Basal levels of plasma ACE activity in monkeys averaged 31.6 ± 2.2 nmol/min/ml (n = 7). A single oral dose of CGS 30440 at 2 mg/kg reduced plasma ACE activity to 1.34 ± 0.6 nmol/min/ml at 1 hr (96% inhibition, P < .001) and to 1.73 ± 0.7 at 3 hr (94.5% inhibition, P < .001). At 24 hr after administration, plasma ACE activity was 6.2 ± 0.7 nmol/min/ml (80% inhibition, P < .001).
Oral administration of CGS 30440 for 7 days (1 mg/kg b.i.d.) reduced ACE activity to 3.9 ± 0.6 nmol/min/ml (n = 7, P < .001). In a second group of monkeys (n = 7) in which the selective ACE inhibitor benazepril was tested (1 mg/kg b.i.d.), a similar plasma ACE inhibition was observed (3.7 ± 0.4 nmol/min/ml). This change was not statistically different from the reduction observed in those animals treated with CGS 30440. In a group of monkeys treated with CGS 30440 for 21 days (n = 8, 2.5 mg/kg b.i.d.), plasma ACE was reduced to 9.5 ± 1.3 nmol/min/ml at 18 hr after the final dose of the compound.| |
Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In previous experiments we observed that in the rat ACE activity
is the highest in the lung and NEP activity is highest in the kidney
(Chatelain et al., 1994; Odorico et al., 1995).
Therefore, these organs were used to compare the inhibitory activities
of CGS 30440 with those of other reference compounds. Selective ACE inhibitors, CGS 30440 and the dual ACE/NEP inhibitor MDL 100,240 were
administered orally to rats which were sacrificed after 1 hr. This time
was chosen because we and others have observed that nearly maximal
inhibition of tissue ACE is obtained in 1 hr with most ACE inhibitors
(Cushman et al., 1989). No attempts were made to adjust the
dose of each compound to its in vitro potency, and a uniform
10 mg/kg dose was administered. The results show that CGS 30440 is able
to inhibit lung ACE to a degree similar to the well known selective ACE
inhibitors and the dual ACE/NEP inhibitor MDL 100,240.
One hour after administration of CGS 30440, NEP activity was reduced by 80% in the kidney. Comparisons with standard NEP inhibitors and with the dual ACE/NEP inhibitors glycopril and MDL 100,240 in the tissue NEP assay are less straightforward than the comparison performed with the standards in the tissue ACE assay. This is because the optimal time of NEP inhibition in the kidney was not investigated and because the issue of tissue bioavailability, when different routes of administration are used, was not addressed. (Thiorphan was administered intravenously because it is not orally bioavailable.) Nevertheless, our results suggest that, at the 1-hr time, the renal NEP inhibitory activity of CGS 30440 is significant and, notwithstanding differences in bioavailability, as good as that of other NEP or ACE/NEP inhibitors.
From the initiation of the study of ACE inhibitors, inhibition of the pressor response to intravenous injections of angiotensin-I has been used as an indirect measure of the generalized inhibition of plasma and tissue ACE. Oral administration of CGS 30440 produced a marked inhibition of this pressor response within 15 min even when a dose as low as 1 mg/kg was used with larger doses prolonging the inhibition for more than 6 hr.
In other experiments, the oral administration of CGS 30440 also was
accompanied by an increase in the concentration of circulating ANP when
a continuous intravenous infusion of exogenous ANP was provided. The
elevation in the concentration of ANP produced by low doses of CGS
30440 was statistically significant and was sustained for more than 4 hr. Also, in anesthetized normotensive rats supplemented with exogenous
ANP, a 1 mg/kg dose of CGS 30440 significantly increased urinary
volume, cGMP and sodium excretion for more than 3.5 hr. The increase in
urinary cGMP concentration preceded the increase in sodium excretion
which in turn preceded the increase in urinary volume. This pattern of
changes is suggestive of NEP inhibition at the level of the proximal
renal tubule and may be the result of the enzyme inhibition measured
with the biochemical assay at the 1-hr time. The subject of the urinary
changes produced by NEP inhibition has been the object of intensive
investigation (Trapani et al., 1989; Webb et al.,
1989; Sybertz et al., 1989), and detailed information about
the effects of selective NEP inhibitors as well as ACE/NEP inhibitors
is available (Seymour et al., 1989, 1991; Gros et
al., 1991; French et al., 1994).
The antihypertensive effect of selective NEP inhibitors and dual
ACE/NEP inhibitors have been well characterized in the DOCA-salt rat, a
volume-dependent model of hypertension in which the renin-angiotensin system is suppressed. The high blood pressure of these animals is
responsive to NEP inhibition (Sybertz et al., 1989; Seymour et al., 1991) but unresponsive to ACE inhibition. ACE
inhibitors, on the other hand, are more effective in models in which
the renin-angiotensin system is activated. For the evaluation of CGS
30440 we used the aortic-ligated rat, a renin-dependent model of
hypertension responsive to ACE inhibitors but whose high blood pressure
is also very responsive to a challenge with exogenous ANP (Lappe
et al., 1987). Our experiments were performed in rats with
hypertension of the benign type lasting for 12 days. At this time,
plasma renin is still elevated but hypertrophy and hyperplasia have
already taken place in the cardiovascular system (Chatelain et
al., 1980; Chatelain, 1983). In these animals we confirmed the
blood pressure-lowering effects of exogenous ANP and the selective ACE
inhibitor captopril. CGS 30440 produced a blood pressure reduction
similar to that of captopril and a more sustained decrease than a large
intravenous dose of ANP, a vasodilator with a short half-life. There
was a marked difference, however, in the initiation of the
antihypertensive effect of captopril which was rapid and that of CGS
30440 which took 1 hr to reach statistical significance. The slow onset
of the blood pressure reduction in aortic-ligated rats also contrasted
with the more rapid responses evoked by CGS 30440 in some of the
parameters studied in normotensive rats. Two reasons may account for
these differences. In aortic-ligated rats we have observed a marked increase in ACE activity in the arterial wall, in the kidneys, lungs
and adrenals (experiments not described in this report). On the other
hand, CGS 30440 is a prodrug, whereas captopril is in the active form
and does not require a metabolic conversion to inhibit ACE. Thus, in
aortic-ligated animals CGS 30440 may need a longer time to achieve a
physiologically meaningful ACE inhibition in the organs or systems
involved in the maintenance of the elevated blood pressure, such as the
ischemic kidney and the vascular system.
In our experiments with cynomolgus monkeys, oral administration of CGS 30440 produced a rapid inhibition of plasma ACE, which suggests that, similar to the initial observations in the rat, the compound may also have an appropriate oral bioavailability in primates.
The importance of angiotensin-II and ACE inhibitors in hypertension is
well established. The role of ANP and NEP inhibitors in this disease,
however, is less understood; because of their recent discovery, a
significant body of clinical studies is not yet available. When tested
in humans with hypertension some NEP inhibitors have produced
significant decreases in blood pressure (Richards et al.,
1993a, c). Although the antihypertensive effect may not have been as
large as that achieved by other types of compounds, in some instances
enhanced natriuresis and cGMP excretion were observed (Richards
et al., 1993a, b). After prolonged NEP inhibition, however,
elevations in plasma renin, aldosterone and catecholamines may have
counteracted the potential beneficial effects of ANP (Richards et
al., 1993a). Some authors have concluded that NEP inhibitors have
a potent antihypertensive effect associated with an enhancement of
endogenous ANP (Ogihara et al., 1994), which may be offset
by an activation of the renin-angiotensin and sympathetic nervous
systems (Richards et al., 1993c).
The discovery of selective NEP inhibitors has created the possibility
of a new treatment for CHF with candoxatrilat having been shown to
increase diuresis and natriuresis in these patients (Northridge
et al., 1989; Good et al., 1995). Although
studies with other compounds have failed to confirm this finding,
additional but modest beneficial effects have been described (Wilkins
et al., 1993). The possibility exists that in CHF, despite a
naturally occurring increase in the concentration of circulating ANP,
the kidney does not respond to it in terms of vasodilation, diuresis and natriuresis. This unresponsiveness would impose a serious limitation to the therapeutic action of selective NEP inhibitors. Experiments in animals with CHF, however, suggest that dual ACE/NEP inhibition may offer some advantage over selective NEP inhibition. In
dogs with CHF produced by rapid ventricular pacing, ACE inhibitors potentiated both the renal hemodynamic and excretory responses to NEP
inhibitors (Margulies et al., 1991; Seymour et
al., 1993). The advantages of the dual inhibitor BMS-182657 over
selective ACE or NEP inhibition was demonstrated in hamsters with heart failure caused by genetic cardiomyopathy (Trippodo et al.,
1995). In more recent studies performed with the same dual inhibitor, the importance of the ACE inhibitory arm in the hemodynamic response and of the NEP inhibitory arm in the renal response was demonstrated in
non-human primates (Seymour et al., 1996a).
Several dual ACE/NEP inhibitors such as glycopril, alatriopril,
mixanpril and MDL 100,240 were able to decrease the activities of both
ACE and NEP in the lung and kidney of mice and rats superseding the
effects of thiorphan and selective ACE and NEP inhibitors on their
respective target enzymes. These compounds as well as BMS-182657
(Trippodo et al., 1995; Seymour et al., 1996a)
produced long-lasting inhibitions of the angiotensin-I pressor response and sustained increases in diuresis, natriuresis and cGMP excretion in
several species (Gros et al., 1991; Fournie-Zaluski et
al., 1994b; French et al., 1994). In experimental
studies with models whose high blood pressure is sensitive to either
NEP or ACE inhibitors, dual ACE/NEP inhibitors have been superior to
their selective counterparts. SQ 28,133, a dual-acting compound,
produced a blood pressure reduction in the SHR, which was not observed
with selective NEP inhibitors, as well as in the DOCA-salt rats, a
model unresponsive to selective ACE inhibitors (Seymour et
al., 1991). The antihypertensive effect of mixanpril was
demonstrated in the SHR, the DOCA-salt rat and the two-kidney, one-clip
renal hypertensive rat (Fournie-Zaluski et al., 1994b),
whereas BMS-182657 was effective in both sodium-depleted SHR, a model
sensitive to ACE inhibitors, and in DOCA-salt hypertensive rats
(Trippodo et al., 1995). These observations suggest that the
dual inhibitors may be able to suppress or ameliorate a wider spectrum
of hypertensive mechanisms than those evoked by the sole inhibition of
ACE or NEP. In this regard, it has been speculated that dual inhibition
may potentiate the action of bradykinin, substance P and brain
natriuretic peptide which are also substrates for both ACE and NEP
(Seymour et al., 1996b; Skidgel et al., 1987; Vanneste et al., 1990). Furthermore, the superiority of dual
inhibition to selective inhibition recently has been demonstrated in
humans with essential hypertension. In a group of these patients
monotherapy with the NEP inhibitor sinorphan or with captopril failed
to reduce blood pressure for a 24-hr period; however, a statistically
significant decrease was observed when both agents were combined
(Favrat et al., 1995).
The experiments performed with CGS 30440 indicate that this compound is able to produce physiological responses typical of an ACE inhibitor and of a NEP inhibitor. The vascular and renal responses evoked by CGS 30440 are long-lasting and have achieved statistical significance even at low doses. Our findings suggest that CGS 30440 has the potency and properties to merit further consideration in the treatment of both hypertension and CHF.
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Footnotes |
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Accepted for publication November 12, 1997.
Received for publication April 21, 1997.
Send reprint requests to: Ricardo E. Chatelain, M.D., Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit NJ 07901-1398.
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Abbreviations |
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ACE, angiotensin-converting enzyme (EC 3.4.15.1); ANP, atrial natriuretic peptide; CHF, congestive heart failure; DOCA, deoxycorticosterone acetate; cGMP, cyclic GMP; MAP, mean arterial blood pressure; NEP, neutral endopeptidase (EC 3.4.24.11); SHR, spontaneously hypertensive rat.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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0022-3565/98/2843-0974$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
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