Systemic and regional hemodynamic effects of zabicipril in healthy volunteers The effects of two oral doses of zabicipril, a new angiotensin converting enzyme inhibitor, on systemic (arterial pressure, heart rate, and cardiac output) hemodynamic parameters and regional (brachial, carotid and femoral arteries' diameters and flows) hemodynamic parameters and on biologic (plasmaconverting enzyme and renin activities, catecholamines, and atrial natriuretic factor) parameters were noninvasively investigated and compared with those of a placebo in a double-blind crossover study performed in six healthy male volunteers. Although it did not affect the systemic hemodynarnic parameters, zabicipril induced a strong peripheral vasodilation, significantly reducing brachial, carotid, and femoral resistances and increasing the corresponding blood flows from 3 or 4% hours to 9 hours. This vasodilation affected only the arterioles, not the large arteries, and resulted in a redistribution of cardiac output toward the three regional vascular beds. Zabicipril induced an early, potent, and long-lasting converting enzyme inhibition. Furthermore, zabicipril did not affect plasma catecholamines and atrial natriuretic factor. (CLINPWCOL THER 1992;51:308-19.)
Eric Bellissant, MD, Christian Thuillez, MD, Eric Pussard, PhD, and Jean-Franqois Giudicelli, MD Le Kremlin-Bzc&re, France
It is well established that vasodilating drugs inhomogeneously affect the different regional blood flows and vascular resistances in animals.'-6 In humans, this property has been described in patients with severe congestive heart failure7-' but, until recently, the lack of adequate noninvasive methods for measuring cardiac output has limited the number of studies in healthy volunteers. The combined use of cardiac Doppler and echocardiography and of peripheral pulsed Doppler now provides the possibility to simultaneously investigate the regional vasodilating profile and the potential cardiac output redistributing properties of a Inasmuch as angiotensin-I converting enzyme (ACE) inhibitors are widely used in the treatment of hypertension and heart failure because of their vasodilating properties, the goals of this study were (1) to characterize and quantify in healthy volunteers the regional vasodilating profile of a new ACE inhibitor, zabicipril,14 (2) to determine whether vasodilation affects both resistance arterioles and large From the Service de Pharmacologie Clinique, HBpital de Bicetre. Supported in part by 1'Institut de Recherches Internationales Servier, Courbevoie, France. Received for publication April 11, 1991; accepted Sept. 26, 1991. Reprint requests: Jean-Fran~oisGiudicelli, MD, Service de Pharmacologie Clinique, HBpital de circumflex BicCtre, 78, Rue du GCnBra1 Leclerc, 94270 Le Kremlin-Bicttre Cedex, France. 13/1/34016
conductance vessels, and (3) to establish whether a regional redistribution of cardiac output occurs. Simultaneously, the effects of zabicipril on the renin angiotensin system, plasma catecholamines, and atrial natriuretic factor were assessed, and plasma concentrations of zabiciprilat, the active metabolite of zabicipril, were determined.
METHODS Subjects Six healthy male volunteers took part in the investigation. The mean (+SEM) age of the subjects was 23.2 +- 0.5 years, mean height was 176 +- 1 cm, and mean weight was 66 +- 1 kg. Each subject gave written informed consent for the study, which was approved by the Hospital Ethical Committee (GREBB, Le Kremlin-BicCtre, France). All subjects were deemed to be healthy on the basis of a medical checkup that included a complete physical examination, measurements of arterial pressure and heart rate performed at the end of a 1-hour rest period in the supine position and 1 minute after sitting up, standard biologic blood and urinary tests, a study of the renin angiotensin system (plasma-converting enzyme and renin activities measured at the end of the rest period), and an electrocardiogram. Moreover, these subjects were nonsmokers and did not take any other medication before and during the entire period of the study.
VOLUME 5 1 NUMBER 3
Zabicipril in nomal subjects 309
Study design and treatments This was a placebo-controlled, double-blind, crossover study that included three identical sequences and was performed according to a Latin square design. At 2-week intervals and in a random order, each volunteer received oral doses of 0.5 mg zabicipril, 2.5 mg zabicipril, or a placebo. The three formulations, supplied by Laboratoires Servier (Neuilly sur Seine, France), were indistinguishable. Like most ACE inhibitors, zabicipril, or (3s)-2-[(2S)-N-[(IS)-1-carboxy3-phenylpropyl]alanyl]-2-azabicyclo[2.2.2]-octane-3carboxylic acid, 1-ethyl ester, hydrochloride, is an inactive monoacid-monoester pro-drug that is hydrolyzed in vivo into its active diacid metabolite, zabiciprilat. Investigated parameters The following clinical and biologic parameters and zabiciprilat plasma concentrations were investigated before drug administration and at regular intervals after drug intake. Systemic hemodynamic parameters. Heart rate and systolic and diastolic arterial pressures were recorded with a Roche Sentron (Bard Medical, Lombard, 111.) automatic monitor connected to a brachial cuff sphygmomanometer. For these three parameters, each reported value is the mean of four successive determinations. Mean arterial pressure (MAP) was calculated according to the formula: MAP = 1/3 Systolic arterial pressure + 213 diastolic arterial pressure
Cardiac output (CO) was determined by cardiac Doppler and echocardiography.15 As detailed previously,13 the measurement was made in two steps. First, the diameter (D) of the ascending aorta was measured at the level of the valve leaflets by use of a bidimensional echocardiograph (Diasonics DRF 600, Diasonics Inc., Les Vlis, France). Second, the mean blood flow velocity (V,) was determined at the same level by use of a pulsed Doppler system (2.25 MHz, Vingmed SD50, Vingmed Sound AIS, Horten, Norway) and the corresponding flow was instantaneously calculated according to the formula: CO =
?T
D2 V,/4
The reported value is the mean of six values that did not differ each from each other by more than 5%. Total peripheral resistance was calculated as the mean arterial pressure to cardiac output ratio. Regional hemodynamic parameters. Brachial, carotid, and femoral artery flows (BAF, CAF, and FAF)
and diameters were measured by use of a bidimensional pulsed Doppler system (Echovar Doppler pulsi, Alvar Electronic Montreuil, France) as described previously and validatedI6 and as widely used in healthy volunteers," in patients with hypertension," and in patients with congestive heart failure." Regional vascular resistances of the brachial, carotid, and femoral territories were calculated as the ratios of mean arterial pressure to the corresponding flows. Percentages of cardiac output delivered to the brachial, carotid, and femoral territories were calculated as the ratios of the corresponding flows to CO: BAF X 100/CO, CAF X 100/CO, and FAF x lOO/CO. Biologic parameters. Renin angiotensin system: plasma-converting enzyme activity was determined by spectrophotometry.l9 Converting enzyme inhibition was calculated as the percentage of variation from the pretreatment plasma-converting enzyme activity. Plasma renin activity was determined by radioimmun o a ~ s a ~ .Plasma ~' catecholamines (epinephrine and norepinephrine) were determined by HPLC." Plasma atrial natriuretic factor, after a previous extraction, was determined by radioimmunoassay.22 Drug concentrations. Plasma zabiciprilat concentrations were determined by radioimmunoassay. In brief, the principle of the method relies upon a competition between zabiciprilat and a radioactive tracer 1 2 5 ~1-1985-2, ~ or ([carboxy-1-phenyl-3-propyl-(S)amino]-2-[(hydroxy-4-phenyl)-3-propionylamino]-6hexanoyl-(S))-2-aza-2-bicyclo[2.2.2]-octanecarboxylic-3(3S) acid, hydrochloride, for anti-zabiciprilat antibodies. The method has a good reproducibility because the coefficient of variation of the measured responses relative to the concentration of the calibration standards is less than 10%. The method is specific because the pro-drug zabicipril and the glucuronid metabolites of both zabicipril and zabiciprilat do not interfere with the radioimmunoassay. The limit of detection of zabiciprilat is 0.1 ng/ml and the limit of sensitivity of the method is 0.1 nglml if the calibration range includes the 0.1 nglml zabiciprilat concentration.
Experimental protocol During each of the three experimental sequences, each volunteer arrived at the laboratory at 7 AM after eating a light breakfast (without tea, coffee, chocolate, or lipids). The subject assumed the supine position, and an indwelling catheter with an heparinized lock was immediately placed into a forearm vein of the right arm and the sphygmomanometer cuff was
3 10 Bellissant et al. placed around the left arm. The subject then rested for 90 minutes to normalize all parameters. At 8:30 AM, the control values of the systemic (heart rate, systolic and diastolic arterial pressures, and cardiac output) and regional (brachial, carotid, and femoral arteries' diameters and flows) hemodynamic parameters were recorded and blood samples were taken for the determination of the biologic parameters (plasma-converting enzyme and renin activities, epinephrine, norepinephrine, and atrial natriuretic factor) and of zabiciprilat plasma concentrations. The plasmas were immediately separated and stored at -20" C. At 9 AM, the treatment (placebo, 0.5 mg zabicipril, or 2.5 mg zabicipril) was given orally with 100 ml tap water. All the investigated parameters were repeatedly recorded at regular intervals: heart rate, systolic and diastolic arterial pressures, brachial, carotid and femoral arteries' diameters and flows, plasma-converting enzyme and renin activities, epinephrine, and norepinephrine were all investigated 1%, 3, 4% 6, 9, and 24 hours after drug intake. Two additional blood samples were taken-one at 12 hours for the determination of plasma-converting enzyme and renin activities, epinephrine, and norepinephrine, and one at 48 hours for the determination of plasma-converting enzyme activity. Cardiac output was investigated at 3, 6, and 9 hours only and atrial natriuretic factor at 3, 9, and 24 hours. Finally, zabiciprilat plasma concentrations were determined at %, 1, I%, 2, 3, 4%, 6, 9, 12, 24, and 48 hours. During the entire experiment, the volunteers were asked to remain in the supine position except for the two short 30-minute periods corresponding to the absorption of standardized light meals and occurring at 4?h and 10 hours, respectively, after drug intake. No tea, coffee, or alcoholic beverages were allowed.
Treatment of data and statistical analysis Findings are given as mean values (kSD) of the six individual values. Reported percentages of variation after zabicipril were calculated from the pretreatment values. Statistical analysis was performed with the B M D P ~ ~ statistical software (program 2V: analysis of variance and covariance with repeated measures) on an IBM PSI2 microcomputer (IBM Instruments, Inc., Greenock, Scotland). For each parameter, homogeneity of pretreatment means between the three groups of treatment was tested by a three-way ANOVA (option "Latin square design"). Analysis of treatment effects
CLIN PHARMACOL THER MARCH 1992
(i.e., variations versus corresponding pretreatment values) between the three groups during the whole experiment was performed with another three-way ANOVA (option "Two within factors, no grouping factors"). When time-treatment interaction was significant, time by time pairwise comparisons were directly performed by Newman-Keuls tests. When time-treatment interaction was not significant and treatment-effect was significant, overall followed by time by time pairwise comparisons were performed by Newman-Keuls tests. For each analysis, p values less than 0.05 were considered to be statistically significant. The following pharmacokinetic parameters were determined: zabiciprilat observed peak concentration (C,,,), time to peak concentration (t,,,), area under the curve up to 48 hours [AUC(O-48)] by use of the trapezoidal rule, and terminal elimination half-life (th) by use of a log linear regression.
RESULTS Control values Table I shows the pretreatment means (kSD) of all the investigated parameters in each group of treatment and the corresponding values of F and p. For all parameters, except brachial vascular resistance, no significant difference was observed between the three groups of treatments, indicating a good reproducibility of the subjects' status before each treatment administration. For brachial vascular resistance, there was a significant difference between the three groups, this resistance being fortuitously slightly lower in the placebo (71 + 7 mm Hg . seclml) than in the zabicipril groups (75 5 6 and 77 k 9 mm Hg - sec/ml before 0.5 and 2.5 mg, respectively). Hernodynamic effects Systemic hemodynamic effects. Compared with placebo, zabicipril, whichever the dose, did not modify mean arterial pressure (p = 0.28), heart rate (p = 0.87), cardiac output O, = 0.69), or total peripheral resistance (p = 0.48) during the 24 hours after administration of the treatments. Regional hemodynamic effects. Compared with placebo, zabicipril, whichever the dose, did not modify brachial O, = 0.99), carotid (p = 0.23), or femoral (p = 0.47) arteries' diameters during the 24 hours after administration of the treatments. Figs. 1 and 2 illustrate the kinetics of the variations of the brachial, carotid, and femoral artery flows and of the corresponding vascular resistances during the
VOLUME 51 NUMBER 3
Zabicipd in nomal subjects 3 11
Table I. Means + SD of the six individual pretreatment values observed for each investigated parameter and each of the three groups of treatments and corresponding values of F (2 and 8 degrees of freedom) and p Parameter
Zabicipril, 0.5 mg Zabicipril, 2.5 mg
Placebo
Systolic arterial pressure (mm Hg) 111 2 4 Diastolic arterial pressure (mm Hg) 65 t 5 80 t 4 Mean arterial pressure fmm Hg) Heart rate (beatslmin) 67 ? 6 CO (Llmin) 5.0 +- 0.3 Total peripheral resistance (dynes . sec/cm5) 1 2 8 7 t 109 BAF (mllmin) 71 t 14 Brachial artery diameter (mm) 4.7 2 0.3 Brachial vascular resistance (mm Hg . sectml) 71 t 18 BAF X IOOICO (%) 1.4 t 0.3 CAF (mltmin) 375 ? 28 Carotid artery diameter (mm) 5.5 t 0.3 Carotid vascular resistance (mm Hg . seclml) 13 t 2 7.5 t 0.7 CAF X lOOlCO (%) 116 ? 21 FAF (mllmin) 6.2 +- 0.3 Femoral artery diameter (mm) Femoral vascular resistance (mm Hg . seclml) 43 9 2.3 ? 0.4 FAF X 1001CO (%) Plasma-converting enzyme activity (nmollmllmin) 18 t 6 Plasma renin activity (nglL1min) 27 +- 21 58 t 20 Epinephrine (pglml) 238 t 37 Norepinephrine (pglml) 7.1 +- 2.2 Atrial natriuretic factor (fmoltml)
*
118 t 67 t 84 67 ? 4.9 t 1377t 69 t 4.7 t 75 t 1.4 t 373 ? 5.5 t 14 t 7.7 t 108 t 5.9 t 48 t 2.2 t 18 t 24 t 54 2 265 +7.1 +-
*
F,
, p Value
7 6 5 7 0.4 113 11 0.1 16 0.3 42 0.2 2 0.8 20 0.3 12 0.4 4 18 16 70 1.5
CO, Cardiac output; BAF, brachial artery flow; CAF, carotid artery flow; FAF, femoral artery flow. *Significant difference.
24 hours after each treatment administration. Compared with placebo, both doses of zabicipril increased regional flows and simultaneously decreased the corresponding resistances in the three territories with a peak effect at 6 hours. In the humeral territory, zabicipril increased significantly brachial artery flow from 3 to 9 hours while it decreased significantly brachial vascular resistance from 1Yz to 9 hours (after 0.5 mg) or 24 hours (after 2.5 mg). Both effects were dose-dependent because a significant difference was observed between the two doses at 6 hours for brachial artery flow (+39% after 0.5 mg and +64% after 2.5 mg) and from 3 to 24 hours for brachial vascular resistance (at 6 hours: -30% after 0.5 mg and -42% after 2.5 mg). In the carotid territory, zabicipril increased significantly carotid artery flow from 4% to 9 hours while it decreased significantly carotid vascular resistance from 3 to 24 hours. Although there was a trend to dose-dependency, the latter did not reach statistical significance, even at the peak effect (for carotid artery flow: + 13% after 0.5 mg and + 18% after 2.5 mg; for carotid vascular resistance: - 15% after 0.5 mg and -21% after 2.5 mg).
In the femoral territory, zabicipril increased significantly femoral artery flow from 4% to 6 hours while it decreased significantly femoral vascular resistance from 4% to 9 hours. Again, dose-dependency did not reach statistical significance, even at the peak effect (for femoral artery Bow: +35% after 0.5 mg and +49% after 2.5 mg; for femoral vascular resistance: -26% after 0.5 mg and -38% after 2.5 mg). Cardiac output redistributing properties. Fig. 3 illustrates the kinetics of the variations in regional cardiac output distribution ratios (BAF x 100IC0, CAF x 1001C0, FAF x 1001CO) during the 9 hours after each treatment administration. Compared with placebo, both doses of zabicipril induced a significant redistribution of cardiac output toward the musculocutaneous territories (i.e., the brachial and femoral vascular beds) at 6 hours. This effect was more marked, although not significantly, at 2.5 mg (+42% for BAF X 1001CO and +28% for FAF X 1001CO) than at 0.5 mg (+ 16% for BAF X 100ICO and + 12% for FAF X IOOfCO). At 9 hours, the redistribution was less important, remaining significant only in the humeral territory after 2.5 mg. In the carotid territory,
CLIN PHARMACOL THER MARCH 1992
3 12 Bellzssant et al.
- m !
I
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.
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.
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,
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, 18
.
, 21
.
, 24
ZABICIPRIL 0.5 mg ZABICIPRIL 2.5 mg
18
21
24
T l M E (hours)
TIME (hours)
Fig. 1. Kinetics of variations (A, versus pretreatment values) of flows of the brachial, carotid, and femoral arteries (in milliliters per minute) induced by placebo, 0.5 mg zabicipril, and 2.5 mg zabicipril (means SD of the six individual values).*p < 0.05 versus placebo; **p < 0.01 versus placebo.
*
CAF X 100ICO ratio, which decreased 6 and 9 hours after placebo, was maintained at its initial value after the two doses of zabicipril, and the difference with placebo was significant.
Biologic effects Renin angiotensin system. Fig. 4 illustrates the evolution with time of converting enzyme inhibition and of plasma renin activity during the 48 and 24 hours,
VOLUME 51 NUMBER 3
Zabicipril in nomal subjects
3
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, I ZABICIPRIL 2.5 mg
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Fig. 2. Kinetics of variations (A, versus pretreatment values) of brachial, carotid, and femoral vascular resistances (rnrn Hg . seclml) induced by placebo, 0.5 mg zabicipril, and 2.5 mg zabicipril (means t- SD of the six individual values). *p < 0.05 versus placebo; **p < 0.01 versus placebo.
respectively, after each treatment administration. Compared with placebo, zabicipril induced an early, dose-dependent, potent, and long-lasting converting enzyme inhibition. At both doses, this effect was sig-
nificant from 1% to 48 hours, the dose-dependency being significant only between 1% and 12 hours after drug intake. The peak effect occurred at 3 hours after 0.5 mg (-53% from the pretreatment value) and at
CLIN PHARMACOL THER MARCH 1992
3 14 Bellissant et al.
-u.*
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Eric Bellissant, MD, Christian Thuillez, MD, Eric Pussard, PhD, and Jean-Franqois Giudicelli, MD Le Kremlin-Bzc&re, France
It is well established that vasodilating drugs inhomogeneously affect the different regional blood flows and vascular resistances in animals.'-6 In humans, this property has been described in patients with severe congestive heart failure7-' but, until recently, the lack of adequate noninvasive methods for measuring cardiac output has limited the number of studies in healthy volunteers. The combined use of cardiac Doppler and echocardiography and of peripheral pulsed Doppler now provides the possibility to simultaneously investigate the regional vasodilating profile and the potential cardiac output redistributing properties of a Inasmuch as angiotensin-I converting enzyme (ACE) inhibitors are widely used in the treatment of hypertension and heart failure because of their vasodilating properties, the goals of this study were (1) to characterize and quantify in healthy volunteers the regional vasodilating profile of a new ACE inhibitor, zabicipril,14 (2) to determine whether vasodilation affects both resistance arterioles and large From the Service de Pharmacologie Clinique, HBpital de Bicetre. Supported in part by 1'Institut de Recherches Internationales Servier, Courbevoie, France. Received for publication April 11, 1991; accepted Sept. 26, 1991. Reprint requests: Jean-Fran~oisGiudicelli, MD, Service de Pharmacologie Clinique, HBpital de circumflex BicCtre, 78, Rue du GCnBra1 Leclerc, 94270 Le Kremlin-Bicttre Cedex, France. 13/1/34016
conductance vessels, and (3) to establish whether a regional redistribution of cardiac output occurs. Simultaneously, the effects of zabicipril on the renin angiotensin system, plasma catecholamines, and atrial natriuretic factor were assessed, and plasma concentrations of zabiciprilat, the active metabolite of zabicipril, were determined.
METHODS Subjects Six healthy male volunteers took part in the investigation. The mean (+SEM) age of the subjects was 23.2 +- 0.5 years, mean height was 176 +- 1 cm, and mean weight was 66 +- 1 kg. Each subject gave written informed consent for the study, which was approved by the Hospital Ethical Committee (GREBB, Le Kremlin-BicCtre, France). All subjects were deemed to be healthy on the basis of a medical checkup that included a complete physical examination, measurements of arterial pressure and heart rate performed at the end of a 1-hour rest period in the supine position and 1 minute after sitting up, standard biologic blood and urinary tests, a study of the renin angiotensin system (plasma-converting enzyme and renin activities measured at the end of the rest period), and an electrocardiogram. Moreover, these subjects were nonsmokers and did not take any other medication before and during the entire period of the study.
VOLUME 5 1 NUMBER 3
Zabicipril in nomal subjects 309
Study design and treatments This was a placebo-controlled, double-blind, crossover study that included three identical sequences and was performed according to a Latin square design. At 2-week intervals and in a random order, each volunteer received oral doses of 0.5 mg zabicipril, 2.5 mg zabicipril, or a placebo. The three formulations, supplied by Laboratoires Servier (Neuilly sur Seine, France), were indistinguishable. Like most ACE inhibitors, zabicipril, or (3s)-2-[(2S)-N-[(IS)-1-carboxy3-phenylpropyl]alanyl]-2-azabicyclo[2.2.2]-octane-3carboxylic acid, 1-ethyl ester, hydrochloride, is an inactive monoacid-monoester pro-drug that is hydrolyzed in vivo into its active diacid metabolite, zabiciprilat. Investigated parameters The following clinical and biologic parameters and zabiciprilat plasma concentrations were investigated before drug administration and at regular intervals after drug intake. Systemic hemodynamic parameters. Heart rate and systolic and diastolic arterial pressures were recorded with a Roche Sentron (Bard Medical, Lombard, 111.) automatic monitor connected to a brachial cuff sphygmomanometer. For these three parameters, each reported value is the mean of four successive determinations. Mean arterial pressure (MAP) was calculated according to the formula: MAP = 1/3 Systolic arterial pressure + 213 diastolic arterial pressure
Cardiac output (CO) was determined by cardiac Doppler and echocardiography.15 As detailed previously,13 the measurement was made in two steps. First, the diameter (D) of the ascending aorta was measured at the level of the valve leaflets by use of a bidimensional echocardiograph (Diasonics DRF 600, Diasonics Inc., Les Vlis, France). Second, the mean blood flow velocity (V,) was determined at the same level by use of a pulsed Doppler system (2.25 MHz, Vingmed SD50, Vingmed Sound AIS, Horten, Norway) and the corresponding flow was instantaneously calculated according to the formula: CO =
?T
D2 V,/4
The reported value is the mean of six values that did not differ each from each other by more than 5%. Total peripheral resistance was calculated as the mean arterial pressure to cardiac output ratio. Regional hemodynamic parameters. Brachial, carotid, and femoral artery flows (BAF, CAF, and FAF)
and diameters were measured by use of a bidimensional pulsed Doppler system (Echovar Doppler pulsi, Alvar Electronic Montreuil, France) as described previously and validatedI6 and as widely used in healthy volunteers," in patients with hypertension," and in patients with congestive heart failure." Regional vascular resistances of the brachial, carotid, and femoral territories were calculated as the ratios of mean arterial pressure to the corresponding flows. Percentages of cardiac output delivered to the brachial, carotid, and femoral territories were calculated as the ratios of the corresponding flows to CO: BAF X 100/CO, CAF X 100/CO, and FAF x lOO/CO. Biologic parameters. Renin angiotensin system: plasma-converting enzyme activity was determined by spectrophotometry.l9 Converting enzyme inhibition was calculated as the percentage of variation from the pretreatment plasma-converting enzyme activity. Plasma renin activity was determined by radioimmun o a ~ s a ~ .Plasma ~' catecholamines (epinephrine and norepinephrine) were determined by HPLC." Plasma atrial natriuretic factor, after a previous extraction, was determined by radioimmunoassay.22 Drug concentrations. Plasma zabiciprilat concentrations were determined by radioimmunoassay. In brief, the principle of the method relies upon a competition between zabiciprilat and a radioactive tracer 1 2 5 ~1-1985-2, ~ or ([carboxy-1-phenyl-3-propyl-(S)amino]-2-[(hydroxy-4-phenyl)-3-propionylamino]-6hexanoyl-(S))-2-aza-2-bicyclo[2.2.2]-octanecarboxylic-3(3S) acid, hydrochloride, for anti-zabiciprilat antibodies. The method has a good reproducibility because the coefficient of variation of the measured responses relative to the concentration of the calibration standards is less than 10%. The method is specific because the pro-drug zabicipril and the glucuronid metabolites of both zabicipril and zabiciprilat do not interfere with the radioimmunoassay. The limit of detection of zabiciprilat is 0.1 ng/ml and the limit of sensitivity of the method is 0.1 nglml if the calibration range includes the 0.1 nglml zabiciprilat concentration.
Experimental protocol During each of the three experimental sequences, each volunteer arrived at the laboratory at 7 AM after eating a light breakfast (without tea, coffee, chocolate, or lipids). The subject assumed the supine position, and an indwelling catheter with an heparinized lock was immediately placed into a forearm vein of the right arm and the sphygmomanometer cuff was
3 10 Bellissant et al. placed around the left arm. The subject then rested for 90 minutes to normalize all parameters. At 8:30 AM, the control values of the systemic (heart rate, systolic and diastolic arterial pressures, and cardiac output) and regional (brachial, carotid, and femoral arteries' diameters and flows) hemodynamic parameters were recorded and blood samples were taken for the determination of the biologic parameters (plasma-converting enzyme and renin activities, epinephrine, norepinephrine, and atrial natriuretic factor) and of zabiciprilat plasma concentrations. The plasmas were immediately separated and stored at -20" C. At 9 AM, the treatment (placebo, 0.5 mg zabicipril, or 2.5 mg zabicipril) was given orally with 100 ml tap water. All the investigated parameters were repeatedly recorded at regular intervals: heart rate, systolic and diastolic arterial pressures, brachial, carotid and femoral arteries' diameters and flows, plasma-converting enzyme and renin activities, epinephrine, and norepinephrine were all investigated 1%, 3, 4% 6, 9, and 24 hours after drug intake. Two additional blood samples were taken-one at 12 hours for the determination of plasma-converting enzyme and renin activities, epinephrine, and norepinephrine, and one at 48 hours for the determination of plasma-converting enzyme activity. Cardiac output was investigated at 3, 6, and 9 hours only and atrial natriuretic factor at 3, 9, and 24 hours. Finally, zabiciprilat plasma concentrations were determined at %, 1, I%, 2, 3, 4%, 6, 9, 12, 24, and 48 hours. During the entire experiment, the volunteers were asked to remain in the supine position except for the two short 30-minute periods corresponding to the absorption of standardized light meals and occurring at 4?h and 10 hours, respectively, after drug intake. No tea, coffee, or alcoholic beverages were allowed.
Treatment of data and statistical analysis Findings are given as mean values (kSD) of the six individual values. Reported percentages of variation after zabicipril were calculated from the pretreatment values. Statistical analysis was performed with the B M D P ~ ~ statistical software (program 2V: analysis of variance and covariance with repeated measures) on an IBM PSI2 microcomputer (IBM Instruments, Inc., Greenock, Scotland). For each parameter, homogeneity of pretreatment means between the three groups of treatment was tested by a three-way ANOVA (option "Latin square design"). Analysis of treatment effects
CLIN PHARMACOL THER MARCH 1992
(i.e., variations versus corresponding pretreatment values) between the three groups during the whole experiment was performed with another three-way ANOVA (option "Two within factors, no grouping factors"). When time-treatment interaction was significant, time by time pairwise comparisons were directly performed by Newman-Keuls tests. When time-treatment interaction was not significant and treatment-effect was significant, overall followed by time by time pairwise comparisons were performed by Newman-Keuls tests. For each analysis, p values less than 0.05 were considered to be statistically significant. The following pharmacokinetic parameters were determined: zabiciprilat observed peak concentration (C,,,), time to peak concentration (t,,,), area under the curve up to 48 hours [AUC(O-48)] by use of the trapezoidal rule, and terminal elimination half-life (th) by use of a log linear regression.
RESULTS Control values Table I shows the pretreatment means (kSD) of all the investigated parameters in each group of treatment and the corresponding values of F and p. For all parameters, except brachial vascular resistance, no significant difference was observed between the three groups of treatments, indicating a good reproducibility of the subjects' status before each treatment administration. For brachial vascular resistance, there was a significant difference between the three groups, this resistance being fortuitously slightly lower in the placebo (71 + 7 mm Hg . seclml) than in the zabicipril groups (75 5 6 and 77 k 9 mm Hg - sec/ml before 0.5 and 2.5 mg, respectively). Hernodynamic effects Systemic hemodynamic effects. Compared with placebo, zabicipril, whichever the dose, did not modify mean arterial pressure (p = 0.28), heart rate (p = 0.87), cardiac output O, = 0.69), or total peripheral resistance (p = 0.48) during the 24 hours after administration of the treatments. Regional hemodynamic effects. Compared with placebo, zabicipril, whichever the dose, did not modify brachial O, = 0.99), carotid (p = 0.23), or femoral (p = 0.47) arteries' diameters during the 24 hours after administration of the treatments. Figs. 1 and 2 illustrate the kinetics of the variations of the brachial, carotid, and femoral artery flows and of the corresponding vascular resistances during the
VOLUME 51 NUMBER 3
Zabicipd in nomal subjects 3 11
Table I. Means + SD of the six individual pretreatment values observed for each investigated parameter and each of the three groups of treatments and corresponding values of F (2 and 8 degrees of freedom) and p Parameter
Zabicipril, 0.5 mg Zabicipril, 2.5 mg
Placebo
Systolic arterial pressure (mm Hg) 111 2 4 Diastolic arterial pressure (mm Hg) 65 t 5 80 t 4 Mean arterial pressure fmm Hg) Heart rate (beatslmin) 67 ? 6 CO (Llmin) 5.0 +- 0.3 Total peripheral resistance (dynes . sec/cm5) 1 2 8 7 t 109 BAF (mllmin) 71 t 14 Brachial artery diameter (mm) 4.7 2 0.3 Brachial vascular resistance (mm Hg . sectml) 71 t 18 BAF X IOOICO (%) 1.4 t 0.3 CAF (mltmin) 375 ? 28 Carotid artery diameter (mm) 5.5 t 0.3 Carotid vascular resistance (mm Hg . seclml) 13 t 2 7.5 t 0.7 CAF X lOOlCO (%) 116 ? 21 FAF (mllmin) 6.2 +- 0.3 Femoral artery diameter (mm) Femoral vascular resistance (mm Hg . seclml) 43 9 2.3 ? 0.4 FAF X 1001CO (%) Plasma-converting enzyme activity (nmollmllmin) 18 t 6 Plasma renin activity (nglL1min) 27 +- 21 58 t 20 Epinephrine (pglml) 238 t 37 Norepinephrine (pglml) 7.1 +- 2.2 Atrial natriuretic factor (fmoltml)
*
118 t 67 t 84 67 ? 4.9 t 1377t 69 t 4.7 t 75 t 1.4 t 373 ? 5.5 t 14 t 7.7 t 108 t 5.9 t 48 t 2.2 t 18 t 24 t 54 2 265 +7.1 +-
*
F,
, p Value
7 6 5 7 0.4 113 11 0.1 16 0.3 42 0.2 2 0.8 20 0.3 12 0.4 4 18 16 70 1.5
CO, Cardiac output; BAF, brachial artery flow; CAF, carotid artery flow; FAF, femoral artery flow. *Significant difference.
24 hours after each treatment administration. Compared with placebo, both doses of zabicipril increased regional flows and simultaneously decreased the corresponding resistances in the three territories with a peak effect at 6 hours. In the humeral territory, zabicipril increased significantly brachial artery flow from 3 to 9 hours while it decreased significantly brachial vascular resistance from 1Yz to 9 hours (after 0.5 mg) or 24 hours (after 2.5 mg). Both effects were dose-dependent because a significant difference was observed between the two doses at 6 hours for brachial artery flow (+39% after 0.5 mg and +64% after 2.5 mg) and from 3 to 24 hours for brachial vascular resistance (at 6 hours: -30% after 0.5 mg and -42% after 2.5 mg). In the carotid territory, zabicipril increased significantly carotid artery flow from 4% to 9 hours while it decreased significantly carotid vascular resistance from 3 to 24 hours. Although there was a trend to dose-dependency, the latter did not reach statistical significance, even at the peak effect (for carotid artery flow: + 13% after 0.5 mg and + 18% after 2.5 mg; for carotid vascular resistance: - 15% after 0.5 mg and -21% after 2.5 mg).
In the femoral territory, zabicipril increased significantly femoral artery flow from 4% to 6 hours while it decreased significantly femoral vascular resistance from 4% to 9 hours. Again, dose-dependency did not reach statistical significance, even at the peak effect (for femoral artery Bow: +35% after 0.5 mg and +49% after 2.5 mg; for femoral vascular resistance: -26% after 0.5 mg and -38% after 2.5 mg). Cardiac output redistributing properties. Fig. 3 illustrates the kinetics of the variations in regional cardiac output distribution ratios (BAF x 100IC0, CAF x 1001C0, FAF x 1001CO) during the 9 hours after each treatment administration. Compared with placebo, both doses of zabicipril induced a significant redistribution of cardiac output toward the musculocutaneous territories (i.e., the brachial and femoral vascular beds) at 6 hours. This effect was more marked, although not significantly, at 2.5 mg (+42% for BAF X 1001CO and +28% for FAF X 1001CO) than at 0.5 mg (+ 16% for BAF X 100ICO and + 12% for FAF X IOOfCO). At 9 hours, the redistribution was less important, remaining significant only in the humeral territory after 2.5 mg. In the carotid territory,
CLIN PHARMACOL THER MARCH 1992
3 12 Bellzssant et al.
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ZABICIPRIL 0.5 mg ZABICIPRIL 2.5 mg
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Fig. 1. Kinetics of variations (A, versus pretreatment values) of flows of the brachial, carotid, and femoral arteries (in milliliters per minute) induced by placebo, 0.5 mg zabicipril, and 2.5 mg zabicipril (means SD of the six individual values).*p < 0.05 versus placebo; **p < 0.01 versus placebo.
*
CAF X 100ICO ratio, which decreased 6 and 9 hours after placebo, was maintained at its initial value after the two doses of zabicipril, and the difference with placebo was significant.
Biologic effects Renin angiotensin system. Fig. 4 illustrates the evolution with time of converting enzyme inhibition and of plasma renin activity during the 48 and 24 hours,
VOLUME 51 NUMBER 3
Zabicipril in nomal subjects
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Fig. 2. Kinetics of variations (A, versus pretreatment values) of brachial, carotid, and femoral vascular resistances (rnrn Hg . seclml) induced by placebo, 0.5 mg zabicipril, and 2.5 mg zabicipril (means t- SD of the six individual values). *p < 0.05 versus placebo; **p < 0.01 versus placebo.
respectively, after each treatment administration. Compared with placebo, zabicipril induced an early, dose-dependent, potent, and long-lasting converting enzyme inhibition. At both doses, this effect was sig-
nificant from 1% to 48 hours, the dose-dependency being significant only between 1% and 12 hours after drug intake. The peak effect occurred at 3 hours after 0.5 mg (-53% from the pretreatment value) and at
CLIN PHARMACOL THER MARCH 1992
3 14 Bellissant et al.
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