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Circulation. 1995;92:825-834

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(Circulation. 1995;92:825-834.)
© 1995 American Heart Association, Inc.

Articles

Additive Effects of Combined Angiotensin-Converting Enzyme Inhibition and Angiotensin II Antagonism on Blood Pressure and Renin Release in Sodium-Depleted Normotensives

Michel Azizi, MD; Gilles Chatellier, MD, MSc; Thanh-Tam Guyene, MD; Dalia Murieta-Geoffroy, MD; Joël Ménard, MD

From Broussais Hospital Clinical Investigation Center, Assistance Publique des Hôpitaux de Paris (M.A., G.C., T.-T.G., D.M.-G., J.M.), and INSERM U 367, Paris (J.M.).

Correspondence to Dr Michel Azizi, Clinical Investigation Center, Broussais Hospital, 96 rue Didot, 75674 Paris Cedex 14, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background Angiotensin-converting enzyme (ACE) inhibitors do not decrease plasma angiotensin (Ang) II levels 24 hours after drug intake to the same extent as at peak. This intermittent partial "escape" is explained either by a renin-mediated reactive rise in plasma Ang I or by non–ACE-dependent Ang II generation. We therefore tested the hypothesis that a combination of ACE inhibition and Ang II blockade may have additive biological and hemodynamic effects.

Methods and Results In a single-dose, double-blind, randomized, four-way, crossover study, an Ang II antagonist (losartan 50 mg), an ACE inhibitor (captopril 50 mg), their combination, and matched placebos were orally administered to 12 normotensive male volunteers maintained in mild sodium depletion. When captopril 50 mg and losartan 50 mg were given alone, the magnitude of their effects on blood pressure, plasma active renin, Ang I, and aldosterone was similar, whereas the kinetics of their effects were different, reflecting differences in drug pharmacokinetics. The losartan-captopril combination completely suppressed the rise in plasma Ang II induced by losartan 2 hours after drug intake (3.3±3.6 pg/mL versus 20.3±19.1 pg/mL, respectively, P<.05). Six hours after drug intake, the losartan-captopril combination induced a significantly greater decrease in mean blood pressure than that produced by either losartan or captopril alone (73±7 mm Hg versus 79±8 mm Hg versus 81±7 mm Hg, respectively, P<.05). The maximum placebo-subtracted falls in mean blood pressure for the losartan-captopril combination, captopril 50 mg, and losartan 50 mg were 14±5 mm Hg, 10±3 mm Hg, and 9±6 mm Hg, respectively (F2.22=3.45, P<.05). The duration of the mean blood pressure fall was not prolonged by the combination. After combined losartan-captopril administration, the area under the plasma active renin versus time curve (0 to 24 hours) was significantly increased when compared with either losartan or captopril alone (6404±2961 pg · h · mL-1 versus 3105±1461 pg · h · mL-1 versus 2092±867 pg · h · mL-1, respectively, P<.05). The combination had no additive effects on plasma aldosterone decrease when compared with either losartan or captopril alone (58±17% versus 51±20% versus 53±21%, respectively, NS).

Conclusions The combined administration of a standard single oral dose of an ACE inhibitor and an Ang II antagonist to mildly sodium-depleted normal subjects (1) had a major additive effect on plasma renin rise, (2) induced an additional mean blood pressure reduction, and (3) had no additive effect on plasma aldosterone fall.


Key Words: blood pressure • angiotensin • renin


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Multiple mechanisms may contribute to the beneficial effects of angiotensin-converting enzyme (ACE) inhibition in cardiovascular therapy. The hemodynamic consequences of angiotensin (Ang) II suppression1 and bradykinin accumulation2 3 and the direct cellular effects of Ang II on heart and vascular smooth muscle cells4 5 are among the putative mechanisms explaining these beneficial effects. At first glance, the addition of two pharmacological agents that interrupt the renin-angiotensin system (RAS), such as an ACE inhibitor and an antagonist of type 1 Ang II receptors, receptors that are implicated in most of the biological effects of Ang II, is not a logical choice to increase the efficacy of a treatment based on one or the other, as shown by animal experiments.6 However, no ACE inhibitor, even when administered at the highest dose, is able to decrease plasma Ang II levels over 24 hours to a similar level to that observed a few hours after the initial dose.7 8 9 10 This "escape" is due to the reactive rise in plasma active renin and Ang I secondary to the interruption of the Ang II feedback on renin release7 8 or to the existence of other Ang II–forming enzymatic pathways.11 12

In the present study, we wanted to know if the simultaneous inhibition of ACE and blockade of type 1 Ang II receptors could achieve a more complete inhibition of the RAS. We also investigated whether the combined administration of these two drugs could blunt the type 1 Ang II receptor blockade–induced reactive rise of Ang II, which may undesirably stimulate type 2 Ang II receptors, whose functions are still unknown.4 We used a model of mild sodium depletion in normotensive volunteers, which allows the detection of falls of approximately 10 to 20 mm Hg in blood pressure when the RAS is blocked by a single administration of a RAS inhibitor 12 hours after 40 mg oral furosemide.13 Standard doses of an ACE inhibitor, captopril 50 mg, and of an Ang II antagonist, losartan 50 mg, were selected, and blood pressure, plasma renin, and aldosterone were studied for 24 hours after drug intake.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Study Design
A single-dose, double-blind, randomized, placebo-controlled, four-way, crossover study design was used. Each period was separated from its precedent by a 2-week washout interval. Treatment assignment was performed according to a random-allocation schedule. Each subject received each active drug (losartan 50 mg or captopril 50 mg separately), their combination (losartan 50 mg and captopril 50 mg), or matched placebos.

Subjects
Twelve healthy, normotensive (supine blood pressure <140/90 mm Hg) male volunteers aged 18 to 35 years and weighing 72.5±10 kg completed the study. Each subject had a medical history taken and underwent a complete physical examination and routine laboratory evaluation, including an ECG, 14 days before the study. Volunteers gave their written informed consent to receive furosemide 40 mg followed by captopril 50 mg, losartan 50 mg, their combination, or matched placebos on four separate occasions. The protocol was approved by the "Comité Consultatif de Protection des Personnes se prêtant à des Recherches Biomédicales" (PARIS-COCHIN).

Drugs
Losartan was provided by Merck Research Laboratories. Captopril, losartan, and placebo were placed in capsules identical in appearance.

Study Protocol
Before each period, all subjects were asked to refrain from smoking, drinking alcoholic beverages, and taking any medication. For each phase, subjects were instructed to arrive at the Broussais Clinical Investigation Center at 6 PM on the prestudy evening (D0), and they remained hospitalized at the center for 36 hours. To induce mild sodium depletion, subjects were given furosemide 40 mg at 9 PM on D0 and received a sodium-restricted diet (20 mmol/d) during the 36 hours of each phase. Water was given ad libitum. Between each period, volunteers were instructed to follow their regular sodium diet.

On the study day (D1), after a light, caffeine- and fat-free breakfast at 7 AM, subjects were comfortably placed in a semirecumbent position on their beds. An indwelling cannula was inserted into a brachial vein for blood sampling. At 9 AM, after 1 hour of rest in the semirecumbent position to allow equilibration of blood pressure, heart rate, and hormones, volunteers received a single oral dose of each treatment (placebo, captopril 50 mg, losartan 50 mg, or the combination of captopril 50 mg and losartan 50 mg) with 50 mL water and remained in the same position until 6 hours after drug intake (3 PM). Fluid intake throughout each study day was unrestricted, and subjects were given a light meal 6 hours and 12 hours after drug intake. All meals remained within the dietary sodium restriction. After the first meal, subjects were allowed to move from their beds. For blood pressure, heart rate, and hormonal determinations performed 12 and 24 hours after drug intake, subjects were once again placed in the semirecumbent position 1 hour before sampling.

On each study day (D1), mean blood pressure (MBP) and heart rate (average of 15 measurements performed every 2 minutes) were monitored in the semirecumbent position at defined intervals (before and 1, 2, 4, 6, 12, and 24 hours after drug intake) with an automatic blood pressure recorder (Press Mate BP 8800, Colin Co). In addition, blood pressure and heart rate were determined in the standing position (mean of three values obtained over 3 minutes) 4 hours after drug intake.

Blood was sampled before and 1, 2, 4, 6, 12, and 24 hours after drug intake for plasma active renin and aldosterone determinations. Plasma cortisol was determined before and 2, 4, 6, 12, and 24 hours after drug intake. Plasma Ang I and Ang II were determined before and 2, 4, and 24 hours after drug intake. Plasma total renin was determined before and 24 hours after drug intake.

Before oral dosing, subjects voided the 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). Urinary free aldosterone at pH 1, electrolytes, and creatinine were measured in each urinary sample.

Laboratory Methods
Heparinized tubes were used to collect blood for plasma active and total renin, ACE activity, aldosterone, and cortisol determinations. For the measurement of plasma angiotensins, blood samples (10 mL) were rapidly collected (within 10 seconds) into prechilled EDTA-K3 evacuated tubes, and 0.5 mL of an inhibitor mixture of 62.5 mmol/L EDTA, 100 µmol/L remikiren, and 100 µmol/L enalaprilat was immediately added.14 15 Blood samples were immediately centrifuged at 3500 rpm at 4°C and stored at -80°C until assay. Plasma active renin was measured by immunoradiometric assay16 17 using the two monoclonal antibodies 3E8 and 125I-4G1 in a commercially available kit (ERIA, Diagnostics Pasteur). The normal value for plasma active renin determined in 45 healthy male volunteers (aged 18 to 35 years) on a normal sodium diet and in the sitting position is 23±11 pg/mL (range, 6 to 53 pg/mL). Total renin was measured with a different pair of monoclonal antibodies, 3E8 and 125I-3-36-16.18 The difference between total renin and active renin gives the amount of inactive renin (prorenin). The normal value for plasma prorenin determined in 23 healthy male volunteers (aged 18 to 35 years) on a normal sodium diet and in the sitting position is 231±84 pg/mL (range, 116 to 454 pg/mL).

For plasma angiotensin measurements, 2 mL of plasma was extracted by solid-phase extraction chromatography with a vacuum extraction device on phenylsilylsilica columns (Bondelut PH, Analytichem) according to the method of Nussberger et al,19 and the dried extracts containing angiotensins were diluted in 500 µL of Tris HCl buffer 0.1 mol/L (pH 7.5) containing 2 g/L bovine serum albumin. The recovery of angiotensins was 98.5±3.5%.15 For the Ang I radioimmunoassay, we used a polyclonal antibody that cross-reacts 100% with des-Asp1 Ang I. For the Ang II radioimmunoassay, we used a monoclonal antibody (gift from D. Simon and B. Pau, Sanofi, Montpellier, France) that cross-reacts 190% with des-Asp1 Ang II, 100% with [Val5]Ang II (Hypertensin, CIBA-GEIGY), and less than 1% with Ang I. In both standard curves, 0.5 pg/tube can be detected, and 50% displacement of the B/B0 is achieved at 5 and 10 pg/tube for Ang I and Ang II, respectively. The within- and between-assay coefficients of variation were 6% and 13%, respectively.

Plasma ACE activity was quantified by a spectrophotometric method according to Cushman and Cheung,20 with slight modifications. To minimize dissociation of captopril from plasma ACE, blood samples were immediately centrifuged at 4°C and stored at -80°C, and plasma ACE activity was determined within 48 hours.21

Plasma and urinary free aldosterone were measured by radioimmunoassay using 125I-aldosterone as a tracer (Coat-a-count Aldosterone, Radioimmunology Behring, Diagnostic Products Corp).

Plasma cortisol was measured by radioimmunoassay with a commercially available kit (Coat-a-count Cortisol, Radioimmunology Behring).

Statistical Methods
Data were analyzed by ANOVA: The crossed factor was the subject and the within factor was treatment. Since the order of treatment 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. Analysis was performed according to the method for repeated measures proposed by Winer.22 When the F test was significant (P<.05), paired comparisons were performed using Bonferroni correction to avoid type I error due to multiple testing. Residual variance of ANOVA was taken for performing pairwise tests. The assumptions of ANOVA (homogeneity of variance and normality) were verified for each variable, and natural logarithmic transformation was applied where appropriate.

Area under the curve (AUC) versus time was calculated according to the trapezoidal rule for plasma active renin (0 to 24 hours) and aldosterone (0 to 6 hours) and for changes in MBP (0 to 24 hours).

The weighted fall in MBP during the placebo phase was calculated for each subject by AUC0-24 of the change in MBP versus time during the placebo phase divided by 24 hours. Accordingly, the average weighted fall in MBP during the placebo phase was 0.8±2.7 mm Hg. Because of the difference in the pharmacokinetic profiles between losartan and captopril, we compared the fall in MBP at the peak effect of each drug by determining the maximum placebo-subtracted fall in MBP, which was calculated for each subject and then averaged for each period. For each subject, the maximum placebo-subtracted MBP fall was calculated by subtracting the weighted placebo MBP fall from the maximum fall in MBP observed during the active periods.

The times to peak effect for MBP fall, plasma active renin rise, and plasma aldosterone decrease were determined graphically for each subject and then averaged.

Calculations were done with STATVIEW II statistical software (Apple Macintosh Abacus Concepts Inc). Data are expressed as mean±1 SD in the tables, and mean±1 SEM in the graphs. A probability value of less than .05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
For all parameters, the values measured after placebo are reported in Tables 1Down, 3Down, and 4Down. The stability of all parameters over 24 hours was consistently obtained after placebo intake, except for cortisol and aldosterone, which are influenced by circadian rhythm.


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  		Table 1. Evolution of Mean Blood Pressure, Plasma Active Renin and Prorenin Levels After Captopril, Losartan, Their Combination, and Placebo Intake in 12 Sodium-Depleted Normotensive Male Subjects


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  		Table 3. Evolution of Plasma Ang I, Ang II, ACE Levels, and Ang II/Ang I Ratio After Captopril, Losartan, Their Combination, and Placebo Intake in 12 Sodium-Depleted Normotensive Male Subjects


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  		Table 4. Evolution of Plasma Aldosterone and Cortisol Levels and Aldosterone/Cortisol Ratio After Captopril, Losartan, Their Combination, and Placebo Intake in 12 Sodium-Depleted Normotensive Male Subjects

Superimposition of a low-sodium diet after 40-mg furosemide administration induced a fall in urinary sodium and sodium/potassium ratio (<1) in the two postdose collection periods and a body weight loss of 1.1±1.0 kg (data not shown).

Blood Pressure and Heart Rate
Both captopril 50 mg and losartan 50 mg significantly decreased MBP when compared with placebo (Table 1Up). The fall in MBP was more rapid after captopril than after losartan administration (Table 2Down). Each drug decreased MBP to a similar extent, the maximum placebo-subtracted fall in MBP being 9±6 mm Hg and 10±3 mm Hg for losartan and captopril, respectively (NS, Table 2Down). No significant difference in MBP was observed between captopril and placebo 12 hours after drug intake, whereas, concurrently, MBP remained significantly lower with losartan when compared with placebo. No blood pressure effect was detected 24 hours after intake of either captopril or losartan. The combination of losartan and captopril had an additional effect on the magnitude of MBP fall (Table 1Up and Fig 1Down). At peak, the maximum placebo-subtracted fall in MBP for the losartan-captopril combination was 14±5 mm Hg (Table 2Down). The F test for this latter parameter was significant when comparing the three active periods (F2,22=3.45, P<.05). In paired comparisons, the difference between the losartan-captopril combination and each drug given separately did not reach statistical significance in the Bonferroni-adjusted tests (Table 2Down). Six hours after drug intake, the losartan-captopril combination induced a decrease in MBP that was significantly greater than that produced by each drug administered singly. Twelve hours after drug intake of the losartan-captopril combination, MBP did not significantly differ from that measured after losartan alone but remained significantly lower than that measured after captopril alone (Table 1Up). Fig 1Down shows that the kinetics of the MBP fall after the combined administration of losartan and captopril are a combination of the kinetics of each drug's fall in MBP. The fall in MBP is dependent on captopril during its first phase and on losartan during its second phase. It yields to a geometric mean time to peak for MBP fall of 5 hours, a value close to that of losartan alone (Table 2Down). The magnitude and kinetics of the additional MBP reduction induced by the losartan-captopril combination over each drug given separately is shown in Fig 2Down, which displays the average gain in blood pressure fall observed when adding either losartan to captopril or captopril to losartan. The shift from captopril's contribution to MBP fall to that of losartan occurred 4.4±2.2 hours after drug intake (Fig 2Down).


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  		Table 2. Time to Peak Effect, Value at Peak, Area Under the Curve for Maximum Placebo-Subtracted Mean Blood Pressure Fall, Plasma Active Renin, and Aldosterone



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  		Figure 1. Plots show time course evolution of mean blood pressure (MBP) and plasma active renin after losartan 50 mg (–{square}–), captopril 50 mg (–{circ}–), their combination (–{blacktriangleup}–), and placebo (–{bullet}–).



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  		Figure 2. Plots of the absolute difference in mean blood pressure (MBP) levels and plasma active renin levels between the losartan-captopril combination and losartan 50 mg (–{square}–) and captopril 50 mg (–{circ}–) given separately.

Despite this additional fall in MBP, the duration of the overall blood pressure effect was not modified by the losartan-captopril combination since, 24 hours after dosing, MBP did not significantly differ from that of placebo. The AUC0-24 of the placebo-subtracted fall in MBP did not significantly differ between the three periods (Table 2Up). Heart rate was not significantly modified by captopril, losartan, or their combination (data not shown).

The relative MBP and heart rate changes when assuming the standing position 4 hours after drug intake were similar during the four periods (data not shown). There were five mild episodes of dizziness and postural hypotension when assuming the upright position 4 hours after drug intake (captopril, one episode; losartan, one episode; combination, three episodes), which resolved in the supine position without requiring any specific treatment.

Plasma Renin Parameters
At baseline, plasma active renin and prorenin levels were elevated in comparison with values observed in healthy subjects on a normal sodium diet,15 and they did not significantly differ between the four periods (Table 1Up). The landmark of sodium depletion maintenance (and of subjects' adherence to the low-sodium diet) was the presence of persistently high levels of active renin during the 24 hours of the placebo phase (Table 1Up).

Losartan and captopril, when given separately, significantly increased plasma active renin levels when compared with placebo (Table 1Up and Fig 1Up). The 24-hour plasma active renin profile was extremely different between both drugs (Fig 1Up). Two hours after captopril intake, plasma active renin steeply increased toward a peak value (719±280% of the baseline value) and then decreased toward a value close to that of placebo 12 hours after intake. The rise in plasma active renin was more progressive after losartan intake, reaching 273±182% of the baseline value 7.5 hours after drug intake (geometric mean), slightly decreasing 12 hours after drug intake, and again staying moderately increased 24 hours after drug intake. Time to peak for plasma active renin was significantly shorter with captopril than with losartan, and maximum plasma active renin levels were significantly higher after captopril than after losartan administration (Table 2Up). AUC0-24 of plasma active renin versus time, which represents a 24-hour integrated value, did not significantly differ between captopril and losartan. The duration of plasma active renin increase was significantly shorter after captopril than after losartan administration. Twelve hours after captopril intake, plasma active renin did not significantly differ from that of placebo, whereas 24 hours after losartan intake, plasma active renin levels remained significantly higher than those observed with placebo (Table 1Up).

After intake of the losartan-captopril combination, plasma active renin abruptly increased toward a plateau 2 hours after drug intake (785±387% of the baseline value). It remained at this plateau level until 6 hours after drug intake and then decreased until the 24th hour of the experiment. The effect of the losartan-captopril combination was additive on plasma active renin levels, significantly increasing both maximum plasma active renin and AUC0-24 of plasma active renin versus time when compared with each drug given separately and significantly shortening the time to peak value when compared with losartan given alone (Table 2Up). Twenty-four hours after losartan-captopril dosing, plasma active renin levels were significantly higher than those of captopril, whereas no significant difference was observed when compared with losartan (Table 2Up). The plot of the absolute difference in plasma active renin levels between the losartan-captopril combination and each of the two drugs was drawn up for each subject to assess captopril and losartan's pharmacokinetic-pharmacodynamic contribution to the overall effect of their combination on plasma active renin (see Fig 2Up). The rapid onset of renin increase reflects captopril's contribution, and its prolongation depends on losartan's contribution. For each subject, the shift from the initial captopril contribution to the losartan terminal contribution was graphically determined (the time at which the two curves cross) and found to be not significantly different from that observed for the MBP fall (5.5±2.4 versus 4.4±2.2 hours, respectively, NS).

Twenty-four hours after drug intake, plasma prorenin levels were significantly higher during the three active phases than during the placebo phase. Losartan and captopril increased plasma prorenin levels to a similar extent, whereas prorenin was significantly higher during the losartan-captopril period (Table 1Up).

Plasma Ang I and Ang II
The plasma Ang I profile after losartan, captopril, or their combination followed the same pattern as that of plasma active renin levels (Table 3Up). Plasma Ang I levels were highly and significantly correlated to plasma active renin levels (r=.81, P=.0013, n=12). Similarly to that observed for plasma active renin levels, the losartan-captopril combination had an additive effect on plasma Ang I levels and significantly increased both the magnitude and the duration of the plasma Ang I rise when compared with each drug given separately (Table 3Up).

Whereas captopril and losartan had similar effects on plasma active renin and Ang I levels, the drugs had opposite effects on plasma Ang II levels. As expected, captopril significantly decreased, whereas losartan significantly increased, plasma Ang II levels when compared with placebo (Table 3Up and Fig 3Down). Twenty-four hours after drug intake, plasma Ang II levels returned to their baseline value after captopril administration, whereas after losartan administration they remained significantly higher than those of placebo.



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  		Figure 3. Top: Plot shows time course evolution of plasma angiotensin (Ang) II after losartan 50 mg (–{square}–), captopril 50 mg (–{circ}–), and placebo (–{bullet}–). Bottom: Time course evolution of measured (–{blacktriangleup}–) and theoretical (–{triangleup}–) plasma Ang II after losartan 50 mg and captopril 50 mg combination.

Two hours after drug intake, the losartan-captopril combination completely suppressed the rise in plasma Ang II, which would have theoretically been induced by losartan, and the plasma Ang II profile paralleled that observed after captopril given alone (Table 3Up and Fig 3Up). A partial escape to this suppressive effect of the losartan-captopril combination was observed 4 hours after drug intake, although plasma Ang II levels remained far lower than those measured after losartan administration (Table 3Up and Fig 3Up). Twenty-four hours after drug intake, no significant effect of the combination on plasma Ang II levels could be demonstrated by comparison to losartan alone.

Baseline plasma Ang II levels were highly and significantly correlated to baseline plasma active renin levels (Y=0.34X-4.54, r=.93, P<.001, n=12). This equation of the regression line of plasma Ang II can be applied to the plasma active renin levels measured after losartan-captopril intake to calculate the theoretical increase in plasma Ang II levels that would have been generated by a higher dose of losartan inducing a similar plasma active renin rise as the combination. As shown in Fig 3Up, the calculated plasma Ang II levels were much higher than those actually measured after intake of the losartan-captopril combination, the difference between these two levels quantifying the reduction of Ang II–reactive rise obtained by adding the ACE inhibitor to the Ang II antagonist instead of increasing the Ang II antagonist dosage.

In Vitro and In Vivo ACE Activity
After both losartan and placebo intake, in vitro plasma ACE activity remained remarkably constant throughout the 24 hours of the experiment. As expected, after both captopril and losartan-captopril administration, in vitro plasma ACE activity decreased rapidly toward a nadir 2 hours after drug intake and began to increase from the 4th hour after drug intake onward, reaching its baseline value 12 hours after drug intake (Table 3Up). Two hours after drug intake, captopril given either alone or in association with losartan induced 80±7% inhibition of in vitro ACE activity; 4 hours after drug intake, plasma ACE activity was still reduced by 50±12% from baseline. The plasma Ang II/Ang I ratio decreased similarly toward very low levels after either captopril or losartan-captopril administration, whereas it remained stable after losartan and placebo intake (Table 3Up and Fig 4Down). The analysis of relative changes in the plasma Ang II/Ang I ratio, which in fact represents the in vivo endothelial and plasma ACE activity, showed that 2 and 4 hours after drug intake, in vivo ACE activity was much more inhibited than the level that can be detected in vitro by Cushman's assay (reduction of 98±3% and 92±6% from baseline, respectively). Twenty-four hours after drug intake, no significant effect of captopril given alone could be detected either on in vitro plasma ACE activity or plasma Ang II/Ang I ratio (Table 3Up). After combined losartan-captopril administration, the plasma Ang II/Ang I ratio 24 hours after drug intake was still slightly and significantly lower than that of placebo (persistent reduction of 33±35% from baseline) but not that of losartan alone.



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  		Figure 4. Plots show time course evolution of in vitro plasma angiotensin-converting enzyme (ACE) activity and plasma angiotensin (Ang) II/Ang I ratio after losartan 50 mg (–{square}–), captopril 50 mg (–{circ}–), their combination (–{blacktriangleup}–), and placebo (–{bullet}–).

Plasma Cortisol, Aldosterone, and Aldosterone/Cortisol Ratio
The plasma cortisol profile followed its circadian cycle and was not influenced by any of the active drugs (Table 4Up). Plasma aldosterone followed the same circadian cycle, decreasing toward a nadir 12 hours after drug intake and thereafter increasing until the 24th hour after drug intake. To differentiate the fall in plasma aldosterone due to the blockade of the RAS from that due to its circadian cycle, we analyzed the plasma aldosterone/cortisol ratio profile. The plasma aldosterone/cortisol ratio decreased during the first 6 hours after active drug intake and increased thereafter in parallel with the increase observed during the placebo period (Table 4Up). Therefore, the influence of the RAS blockade on plasma aldosterone levels could only be studied during the first 6 hours after drug intake. During these first 6 hours, captopril and losartan significantly decreased plasma aldosterone levels when compared with placebo (Table 4Up). The time to peak for the plasma aldosterone/cortisol ratio during the captopril period was significantly shorter than that observed during the losartan period (Table 2Up). At peak, the relative fall in plasma aldosterone was the same after the two active drugs (captopril, 53±21% versus losartan, 51±20%; NS), but the AUC0-6 for plasma aldosterone versus time was significantly larger after losartan than after captopril.

Contrary to that observed for plasma active renin and Ang I levels, the losartan-captopril combination had no additive effect on the magnitude of plasma aldosterone fall (58±17%, NS; Table 2Up) or on the AUC0-6 of the plasma aldosterone fall, but the time to peak for plasma aldosterone/cortisol ratio of the losartan-captopril combination was significantly shortened when compared with that of losartan alone.

During the first collection period after drug intake, urinary free aldosterone levels significantly decreased after administration of captopril and the losartan-captopril combination when compared with placebo, and no significant effect of losartan could be detected (data not shown). No significant change in urinary free aldosterone was observed during the last two periods of urinary collection.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The main goal of the present study was to assess, in a situation of mild activation of the RAS in healthy subjects, whether the combination of a standard dose of an ACE inhibitor and of an Ang II antagonist resulted in additional effects on blood pressure, renin release, and aldosterone fall. The rationale for this approach was that the combination would neutralize the potential consequences of persistent Ang II production during ACE inhibition due to either various Ang II–forming pathways or to the renin–Ang I reactive rise.

As a prerequisite to clinical studies in patients with hypertension or congestive heart failure, we chose to investigate the biological effects of these two blockers of the RAS in a human model of mild stimulation of renin release induced by the combination of a 40-mg furosemide–induced sodium depletion followed by a 36-hour low sodium diet.13 This model induces a twofold to threefold increase in plasma active renin, aldosterone, and angiotensins and provides optimal conditions to unmask the renin dependency of blood pressure in normotensive subjects.23 These experimental conditions induce a much less important sodium depletion and stimulation of the RAS than those previously used to investigate losartan in normotensive volunteers.24

A 50-mg dose was used for both captopril and losartan, since this is the standard dose used in clinical practice either during single administration25 or chronic treatment.26 27 28 These drugs differ in their pharmacokinetic characteristics: Captopril is the active drug, with a tmax of 0.9 hours and a half-life of 1.7 hours29 ; losartan is metabolized to EXP 3174, which has a tmax and half-life values of 3 hours and 3.8 hours, respectively.30 Therefore, for each tested parameter, we compared the values at peak, the 24-hour profiles, and the AUCs for each drug and their combination.

The combined administration of 50 mg captopril and 50 mg losartan had a significant additive effect on plasma active renin and Ang I levels, a moderate additive effect on MBP fall, and no additive effect on plasma aldosterone decrease.

The most striking evidence of in vivo efficacy of ACE inhibition by captopril in the presence of type 1 Ang II receptor blockade was the complete deletion of the losartan-reactive plasma Ang II peak and the very low plasma Ang II/Ang I ratio level 2 and 4 hours after drug intake. The plasma Ang II/Ang I ratio remained significantly lower than that of placebo up to 24 hours after drug intake, indicating a slight but persistent in vivo ACE blockade that was not detected when captopril was given alone.

During combined administration, both the captopril-induced fall in plasma Ang II and the losartan-induced type 1 Ang II receptor blockade significantly increased the magnitude and duration of renin release when compared with that induced by either losartan or captopril given alone. The rise in plasma prorenin levels 24 hours after losartan-captopril intake was also significantly higher than that observed after each drug given alone. This reflects an increased renin biosynthesis secondary to renin release.31 Both AUC0-24 and peak plasma active renin levels were significantly higher after combined losartan-captopril administration than after administration of either losartan or captopril. The effect of the losartan-captopril combination on renin release could be due to the summation of either each drug's pharmacokinetic effects, their pharmacodynamic effects, or both.

This massive rise of plasma active renin increased plasma Ang I levels, which can neutralize the effect of ACE inhibition when the plasma levels of captopril start decreasing.8 This is shown by the slight increase in plasma Ang II levels, which was detected as early as 4 hours after drug intake, even though the plasma Ang II/Ang I ratio was still reduced to 92±6% of its baseline value. Plasma Ang II may also be generated through pathways other than ACE, such as chymase or other serine proteases.32

While it is known that both ACE inhibitors33 and Ang II antagonists27 elicit flat dose-response curves for blood pressure fall with concomitant steeper dose-response curves for renin rise,34 the use of losartan and captopril in combination led to an additive blood pressure fall in our study despite the major compensatory renin rise. When losartan 50 mg and captopril 50 mg were administered together, an additional MBP fall was statistically significant 6 hours after drug intake. However, the gain in the maximum placebo-subtracted MBP fall was limited (3.8±5.4 mm Hg and 4.6±7.7 mm Hg over captopril and losartan given alone, respectively). This is not surprising, since there is certainly a limitation to the renin-dependent blood pressure fall achievable in sodium-depleted normotensive subjects in the absence of side effects.23 24 The addition of captopril to losartan accelerated the onset of the blood pressure fall, whereas the addition of losartan to captopril increased the duration of the blood pressure fall, both facts reflecting each drug's specific pharmacokinetic contribution to the improvement of the overall blood pressure reduction profile induced by the losartan-captopril combination. The magnitude of the absolute difference in MBP reduction between the combination and each of its components reflects each drug's specific pharmacodynamic contribution to the additional reduction in MBP induced by the supplementary limitation of endogenous Ang II available at the vascular type 1 receptor site. The perfect symmetry of the plots obtained for the rise in active renin and the fall in MBP shown in Fig 2Up illustrates the close parallelism in the kinetics of the renin release and blood pressure changes induced by the additive effect of the combination. This parallelism strengthened the existence of an additive MBP fall, even though the placebo-subtracted MBP fall did not reach statistical difference over 24 hours when comparing the combination with either losartan or captopril.

Besides pharmacokinetic considerations, the additive MBP fall achieved by the losartan-captopril combination could be due to a more complete blockade of the RAS, as indicated by the renin rise, or a more effective blockade of the counterregulatory mechanism (mediated by the presence of Ang II bound to its receptor), as indicated by the lower plasma Ang II.

It is conceivable that captopril 50 mg and losartan 50 mg given alone are not the maximum doses that should be given to obtain a complete and long-lasting blockade of the RAS with a single drug. That the combination of both drugs at these doses may be the equivalent of an increase in the dose of one drug or the other is questionable. Experimentally, when intravenous captopril was given to normotensive, sodium-depleted rats after cumulative dosages of losartan, no additional hemodynamic effect could be detected,6 whereas in other experimental models, an additive effect was reported.35 36 In sodium-depleted guinea pigs, the combination of captopril and losartan has been shown to produce a synergistic hypotensive response.36 In normal volunteers, the 40- and 80-mg losartan doses inhibited the blood pressure response to exogenous Ang II challenges by 70% and 90%, respectively.37 According to the Hill model described by Munafo et al37 and assuming a plasma peak level of losartan's active metabolite EXP 3174 of 460 ng/mL for a 50-mg oral dose of losartan,30 it is possible to calculate that the administration of this dose leads to 80% inhibition of blood pressure response to pressor doses of exogenous Ang II. Accordingly, Doig et al24 showed that, in a human model of severe sodium depletion, the highest dose of losartan (100 mg) gave both the highest renin rise and the largest blood pressure fall.

The absence of complete RAS blockade by any dose of each drug could also be due to the compensatory dose-dependent rise in renin release,7 8 which may limit the blood pressure response to higher doses of an ACE inhibitor or an Ang II antagonist.10 24 Although the combination induces a higher renin release than each single-drug administration, ACE inhibition may favorably influence the competition between endogenous Ang II and the amount of Ang II antagonist present at the Ang II receptor level. The therapeutic advantages of chronic administration of an Ang II antagonist with an ACE inhibitor, especially in hypertensive patients, will be to overcome the limited blood pressure gain obtained by increasing the dose of each compound given alone and to limit the reactive plasma Ang II rise. A theoretical advantage is to eliminate the consequences of excessive stimulation of type 2 Ang II receptors, consequences that are still not precisely known.4

The additive MBP effect also could be due to each drug's specific pharmacodynamic effect, such as a captopril-induced vascular increase in bradykinin levels, or to a combination of all the aforementioned effects.

In contrast to the plasma active renin rise and the blood pressure fall, no additive effect was detectable on the plasma aldosterone fall. This reflects the fact that plasma aldosterone secretion is regulated through multiple pathways other than the RAS.38 The absence of additive effects on plasma aldosterone levels is reassuring as far as the existence of a risk of hypoaldosteronism and hyperkaliemia is concerned when both drugs are chronically administered to patients. With the exception of special clinical circumstances, such as renal insufficiency and hypoaldosteronism, the combined blockade of ACE and Ang II receptors is unlikely to be more dangerous than one or the other treatment given alone.

The most clinically relevant question is whether these hemodynamic results will be observed in patients with hypertension or congestive heart failure. The single-dose administration of the combination of losartan and captopril to a small number of sodium-depleted normal subjects demonstrates that the hypothesized hemodynamic and hormonal effects of the addition of one dose of the two drugs have been obtained. It remains to be demonstrated whether a more complete, 24-hour RAS blockade can be obtained with the long-term administration of these two blockers in combination than with the administration of each single drug at different dosages. During long-term administration, ACE induction is another factor that may play a role in limiting the efficacy of ACE inhibitors,39 and the combination of an ACE inhibitor and an Ang II antagonist, which does not increase plasma or tissue ACE, is a worthwhile alternative to an increase in the dosage of the ACE inhibitor.40 It must be kept in mind that even if steeper hemodynamic dose-response curves and more complete neutralization of Ang II cellular effects can be achieved by combining an ACE inhibitor with an Ang II antagonist, the price to be paid will be the addition of side effects, especially those dependent on ACE inhibition (cough), and the potential hazards of a complete RAS blockade, particularly in situations where blood pressure and renal functions are extremely renin dependent.


*    Acknowledgments
 
This work was supported by a grant from Merck Research Laboratories, West Point, Pa. The authors wish to thank the nursing staff of the Clinical Investigation Center at the Broussais Hospital (D. Ménard, RN; J. Meunier, RN; F. Soulier, RN; M. Godeau), who ran the protocol. The technical contribution of C. Dollin, who performed the assays, is also much appreciated. The authors acknowledge L. Sartori for editorial help.

Received December 5, 1994; revision received January 30, 1995; accepted February 8, 1995.


*    References
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*References
 

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