Download Effects of Losartan on Blood Pressure, Plasma Renin

704
Effects of Losartan on Blood Pressure, Plasma
Renin Activity, and Angiotensin II in Volunteers
Michael R. Goldberg, Wesley Tanaka, Aaron Barchowsky, Thomas E. Bradstreet,
Jacqueline McCrea, Man-Wai Lo, Edward J. McWilliams Jr., and Thorir D. Bjornsson
Losartan is an orally active, nonpeptide angiotensin II (Ang II) (site-1) receptor antagonist. We conducted
a multiple-dose study in healthy male volunteers to investigate the tolerability, blood pressure effects, and
changes in plasma renin activity (PRA) and plasma Ang II concentration associated with once-daily
administration of 100 mg losartan for a week. Subjects were studied on a standardized sodium diet
(24-hour urinary sodium excretion, 98±37 [SD] mEq per 24 hours on the placebo run-in day).
Measurements of blood pressure, heart rate, PRA, Ang II, and aldosterone were taken during a placebo
run-in day and after single and multiple (7 days) daily doses of losartan (100 mg, n = 10) or placebo (n=4).
Ang II was measured specifically by high performance liquid chromatography coupled with radioimmunoassay. In subjects given losartan, respective decreases (systolic/diastolic) from run-in in supine blood
pressure 6 hours after dosing were (mean±SD), compared with the placebo run-in day, first dose:
-8.8±9.6/-6.8±5.0, last dose: -11.6±8.9/-7.0±4.8 mm Hg (p<0.05 for all changes). At this 6-hour time
point, corresponding increases from run-in in PRA were from 1.2±0.6 to 12.0±6.3 (first dose) and 9.6±4.9
(last dose) ng angiotensin I per milliliter per hour and in Ang II were from 4.3±1.7 to 72.4±33J and
45.7± 14.1 pg/mL. All changes in PRA and Ang II were statistically significant within the losartan-treated
group, and the biochemical changes were significantly greater than those in the placebo-treated group.
The increment in Ang II was less after the last dose than after the first (p<0.05). The drug was well
tolerated by all subjects. These data indicate that, under the conditions of this study, losartan
administration (100 mg/day for eight doses over 9 days) results in treatment-related decreases in blood
pressure and increases in PRA and Ang II octapeptide. (Hypertension 1993;21:704-713)
KEY WORDS • losartan • DuP 753 • angiotensin II • plasma renin activity • receptors,
angiotensin
L
osartan (Figure 1) is a potent, orally active
nonpeptide angiotensin II (Ang II) antagonist.1"4 The drug is being investigated as therapy for hypertension and heart failure and is anticipated to be a more specific mechanism for inhibiting
the renin-angiotensin system than angiotensin converting enzyme inhibitors. Early clinical investigation
of losartan has shown single and multiple doses up to
40 mg to be well tolerated and pharmacologically
active, with doses of 10 mg or greater blocking pressor
responses to exogenous angiotensin I (Ang I) and Ang
II.5-6 Also, through blockade of Ang II receptors in the
juxtaglomerular apparatus, which inhibits renin release, losartan administration results in dose-related
increases in plasma renin activity (PRA) and immunoreactive Ang II. 5 6 Studies have also been completed
that demonstrate the tolerability of single doses up to
300 mg and more than 90% blockade of responses to
exogenous Ang II by oral doses of 80-120 mg (unpubFrom the Merck Research Laboratories (M.R.G., W.T., T.E.B.,
M.-W.L., E.J.M.), West Point, and the Division of Clinical Pharmacology (M.R.G., A.B., J.M., T.D.B.), Thomas Jefferson University, Philadelphia, Pa.
Supported by a grant from Merck Research Laboratories.
Address for correspondence: Michael R. Goldberg, MD, PhD,
Merck Research Laboratories, BL 2-7, West Point, PA 19486.
Received July 28, 1992; accepted in revised form January 13,
1993.
lished data, Du Pont Merck Pharmaceutical Corp. and
Merck Research Laboratories). In these studies, effects
of losartan on resting blood pressure and heart rate in
healthy subjects have not been readily apparent. In
addition, pharmacokinetic analyses have indicated that,
although losartan has a relatively short half-life (unpublished data, Du Pont Merck Pharmaceutical Corp. and
Merck Research Laboratories), the predominant circulating form of the drug is a carboxylic acid metabolite
(E-3174, Figure 1) that is more potent and has a longer
half-life than losartan.23 In fact, the time course of
increases in PRA and plasma Ang II concentration and
blockade of responses to exogenous angiotensin is better correlated to levels of the metabolite than to levels
of parent drug (see Reference 5 and unpublished data,
Du Pont Merck Pharmaceutical Corp. and Merck Research Laboratories).
The general objective of the present study was to
further investigate the effects of single and multiple
doses of losartan in healthy male volunteers. Of interest
were the following specific objectives: 1) confirming the
tolerability and absence of hemodynamic effects in
healthy subjects and 2) assessing changes in PRA and
Ang II concentrations after single and multiple doses,
during standardized sodium intake, using a highly specific radioimmunoassay (RIA) for Ang II coupled with
high performance liquid chromatography (HPLC).
Goldberg et al Losartan in Healthy Volunteers
705
COOH
FIGURE 1. Drawings show structures of losartan (Dup753, MK-954) and its active metabolite, E-3174.
N-N
E-3174
Losartan
(Dup-753, MK-954)
Methods
Study Design
This was a double-blind, placebo-controlled, parallel
study that was divided into three clinical phases: run-in,
first-dose, and multiple-dose (Figure 2). In each phase,
subjects were carefully studied during an inpatient
testing day when blood pressure and heart rate were
carefully monitored and blood was collected at defined
intervals for measurement of PRA, plasma Ang II
concentration, and plasma aldosterone concentration.
The first phase was a single-blind placebo run-in day
(first testing day). The second phase (next day) was a
double-blind single-dose day (second testing day) during which subjects received either a single oral 100-mg
dose of losartan (n = 10) or a single dose of placebo
(n=4), with treatment assignment according to an unbalanced, random-allocation schedule. Forty-eight
hours later, subjects entered the third, double-blind
multiple-dose phase, during which those previously alDay
Run-in
I
I Screening Visit (1-2 weeks prior)
i
•
i
•
Placebo
Diet
First-Dose
135-165 meq Na+
Double-Wind
Treatment
Losartan (N=10)
Placsbo(N=4)
70-90 meqK+
FIGURE 2. Schematic shows outline of study design. On
procedure days, subjects were sequestered on the research unit
the night before and were fasted overnight. On these days,
multiple measurements of blood pressure and heart rate were
made and blood samples collected for measurement of plasma
renin activity, plasma angiotensin II concentration, and
plasma aldosterone concentration. On the first-dose day,
subjects were randomly allocated to receive losartan, 100
mglday (n=10), orplacebo (n=4), for eight doses over 9 days.
located to receive losartan continued to take the drug,
100 mg once daily for 7 consecutive days (a total of eight
doses over 9 days), and those allocated to placebo
continued to receive placebo. The third testing day
coincided with the 24-hour period after administration
of the eighth (and last) dose of the double-blind study
drug.
For each of the three testing days, subjects were
sequestered at the clinical research unit the evening
before dosing and fasted from midnight that night until
3 hours after the dose. Subjects were fed 3 and 6.5 hours
after dosing on the testing days. Subjects remained in
the unit the night after each testing day. On other study
days, all doses of the study drug were administered
under direct observation, at the same time (±30 minutes) each day, with 250 mL water and 30 minutes
before a meal. Beginning 3 days before the run-in day
and continuing through the third testing day (a total of
13 days), subjects were provided a diet estimated to
contain 135-165 mEq sodium and 70-90 mEq potassium per 24 hours. The purpose of the diet was to
standardize sodium intake to aid in interpretation of
changes in PRA, Ang II concentration, and blood
pressure. However, we did not attempt to assure that
subjects were truly in "balance" on the diet before
testing. To monitor the diet, we made 24-hour urine
collections during the run-in day and after the first and
last dose of losartan. Respectively, on each of these
collections, urinary sodium excretion (mean±SD) in
subjects given losartan/placebo were 98 ±37/93 ±8,
66±16/46±11, and 85±21/75±23 mEq per 24 hours.
Based on these sodium excretion rates, the subjects
appear to have been moderately sodium restricted, as
indicated by the 0.7±0.6 ng Ang I per milliliter per hour
(p<0.01) increase in predose PRA from run-in to
first-dose days in the losartan group (Table 2).
Fourteen male subjects were enrolled, aged 18-31
(mean, 24.4) years and weighing 161 ±22 lb (within
±20% of ideal body weight). Subjects were in general
good health, without excessive intake of caffeine (<4
cups of coffee per day) or tobacco (<10 cigarettes per
day) and with sitting blood pressure < 135/85 mm Hg.
Before the study was begun, the protocol and consent
form were reviewed and approved by the Institutional
Review Board of Thomas Jefferson University. All
706
Hypertension
Vol 21, No 5 May 1993
subjects provided written, informed consent before enrollment into the study. Subjects were studied in two
groups of seven subjects each.
Study Procedures and Assay Methods
Blood pressure and heart rate measurements. On each
testing day, supine (average of two measurements after
5 minutes in a supine position) and standing (single
measurement after 2 minutes of standing) blood pressure and heart rate were measured at denned intervals
after dosing using a Dinamap (Critikon, Tampa, Fla.)
automated sphygmomanometer. In evaluating blood
pressure changes after losartan or placebo, the first 8
hours of each testing day were analyzed statistically.
Plasma renin activity and aldosterone assays. For measurement of PRA and aldosterone, samples were collected in precooled syringes containing ethylenediaminetetraacetate (EDTA, 1.5 mg/mL whole blood)
and quickly centrifuged at 4°C. Plasma was frozen in
1-mL aliquots at — 20°C until time of assay.
Plasma aldosterone levels were measured by solidphase RIA (Coat-a-count, Diagnostic Products, Los
Angeles, Calif.). Briefly, 0.2 mL plasma was pipetted
into antibody-impregnated tubes, followed by addition
of 1 mL 125I-aldosterone. The tubes were incubated at
37°C for 3 hours and decanted, and bound radioactivity
was measured by gamma radiation counting. Sample
values were calculated from comparison with log-logit
calibration curves created using increasing amounts of
standards (0-1,200 pg/mL). The sensitivity of the assay
was approximately 16 pg/mL.
PRA was determined by a modification of the methods described by Kodish and Katz.7-9 Plasma aliquots
(300 fiL) were added to two separate ice-cold tubes
containing 200 ju,L of 0.12 M maleate buffer, pH 6.0,
which contained the protease inhibitors phenylmethylsulfonyl fluoride (0.1 mM), soybean trypsin inhibitor (4
mg/mL), and benzamidine (1 mM). One assay tube
remained in an ice bath, and the other was placed in a
37°C bath for 2 hours. At the end of this period, 200-/xL
aliquots from each tube were transferred for RIA of
generated Ang I using previously described methods.
Specific renin activity was determined by subtracting the
values obtained in the cooled assay tube from those at
37°C. The sensitivity for the assay was approximately 10
pg Ang I, and values are reported as nanograms Ang I
per milliliter per hour.
Angiotensin II assay. Ang II concentration in plasma
was measured by modification of published methodology that couples RIA with a relatively specific antibody
for Ang II with HPLC separation of angiotensin peptides.10-12 For these measurements, blood was placed
into a prechilled tube containing (final concentrations)
enalaprilat (3.6 fiM), 1,10-phenanthroline (2.5 mM),
and K3EDTA (1.5 mg/mL), cooled thoroughly, centrifuged at 4°C, and plasma stored frozen at -20°C until
analysis. Thawed samples (2.0 mL) were spiked with
125
I-Ang II as internal standard and chromatographed
on hexane-prewashed, preconditioned 500-mg C8 disposable Bond Elute columns (Analytichem International, Harbor City, Calif.). Angiotensin peptides were
eluted with 2 x 1-mL 50% acetonitrile/50% water, containing 0.1% trifluoroacetic acid, and were dried, resuspended in 300 fiL 20% acetonitrile/0.1% trifluoroacetic
acid, and filtered.
The filtered samples were chromatographed on a
4.6x150 mm Dynamax C8 reversed-phase HPLC column (Rainin Instrument Co., Woburn, Mass.) using a
flow rate of 1 mL/min at 45°C. Angiotensin peptides
were separated with a 20-minute linear gradient from
20% to 35% acetonitrile containing 0.1% trifluoroacetic
acid adjusted to pH 4.0 with NH4OH. Retention times
(minutes) for various angiotensin peptides were: Ang I,
16.5; [Des-Asp]Ang 1,17.4; Ang II, 11.9; angiotensin III
([Des-Asp]Ang II), 13.2; Ang 11(3-8) hexapeptide,
13.5; Ang II-(4-8) pentapeptide, 10.8; and 125I-Ang II,
16. HPLC fractions (0.5 mL) were dried, reconstituted
with 200 mL buffer containing 10 mM potassium phosphate, 1 mM EDTA, 0.25 mM thimerasol, and 0.1%
(wt/vol) gelatin, pH 7.3, and an aliquot was counted for
determination of overall recovery. Ang II was quantitatively measured by competitive binding RIA using a
commercially available Ang II antisera (Amersham No.
RPN1771). The RIA reaction mixture was incubated
overnight at room temperature and treated with dextran-coated charcoal to separate bound and unbound
Ang II. The supernatent was counted, and the Ang II
values were read off a standard curve made from
solutions of known (0-96 pg per tube) Ang II concentration. Day-to-day precision for plasma control material yielded coefficient of variation values of 16% at 18.3
pg/mL (« = 15) and 13% at 5.1 pg/mL (n = 12). Crossreactivity of Ang II antisera with angiotensin peptides
was: Ang II, 100%; angiotensin III, 53%; Ang I, 0.1%;
Ang II-(3-8), 11%; and Ang II-(4-8), 15%.
Clinical tolerability assessment. The tolerability of
losartan in each subject was monitored by periodic
assessment of complete blood counts, chemistry panels,
urinalyses, and electrocardiograms. On each testing day,
24-hour urine was collected for measurement of electrolyte, protein, and creatinine excretion.
Data Analysis
In planning this study, published data on variability of
PRA and Ang II in healthy volunteers given enalapril
were used to determine sample size. Based on these
data10 and assuming a correlation, p, of 0.5 between
pretreatment and multiple-dosing values, with nine subjects completing, the study had 80% power (a=0.05,
two-tailed) to detect respective increments in PRA and
Ang II concentration of 4.5 ng Ang I per milliliter per
hour and 4.3 fmol Ang II per milliliter (approximately
4.3 pg/mL).
The general approach to the statistical analysis was to
compare measurements collected after the first and last
dose of losartan with measurements at the corresponding times after dosing on the placebo run-in day.
Multiple-dosing results were then compared with singledose results. For measurements of PRA, Ang II, and
aldosterone, all time points were of interest. For blood
pressure and heart rate, measurements during the first 8
hours after dosing on each day were analyzed, with
baseline for supine measurements on each day defined
as the average of measurements made at the time of
dosing and 15 minutes before dosing. A single measurement of standing blood pressure and heart rate, immediately before dose, was baseline for changes in standing
blood pressure and heart rate. For the purposes of
reporting the results, run-in refers to the single-blind
placebo run-in day; first dose is the period after the first
Goldberg et al
Losartan in Healthy Volunteers
707
TABLE 1. Supine Blood Pressure and Heart Responses to Losartan and Placebo
Hours
oftp»r
alter
dosing Treatment
Losartan
0
Placebo
1
Losartan
Placebo
Losartan
2
Placebo
Losartan
3
Placebo
Losartan
4
Placebo
5
Losartan
Placebo
Losartan
6
Placebo
Losartan
8
Placebo
Diastolic blood pressure (mm Hg)
Run-in
60.3 ±8.0
62.5 ±8.3
57.1±9.5§
57.0±11.3
58.2±11.0
60.3±9.5
60.7±9.6
60.8±10.8
60.0+8.2
62.5±10.1
59.9±8.4
59.5 ±7.9
60.2±7.6
62.3±11.8
59.0±9.8
61.5±6.8
First dose
1.1±4.2
-3.3±5.7
1.6±5.0
2.3±2.9
-4.2+6.7*
-2.0±2.9
-6.6±8.9||
1.5±4.7
-6.6±4.4t
-4.3±5.4
-9.9±6.0t
0.5 ±4.4
-6.8±5.0t
-0.5±4.2
-4.6±5.4||
-3.5±4.2
Systolic blood pressure (mm Hg)
Last dose
Run-in
-3.7±5.6*t
-5.9±4.3
-3.2±6.4§
-1.0±4.7
-5.6±7.7||
-0.8±7.0
-9.0±5.3*
-0.8±6.0
-9.1±5.0t
-5.8±5.1
-9.3±5.6t
-3.3±3.4
-7.0±4.8t
-0.5±5.1
-5.9±6.8||
-0.3±3.9
109.2±8.4
113.3±17.9
108.4±6.6
105.0+20.0
110.5±9.7
105.5±15.4
113.9±7.2
116.0±14.8
114.3±7.0
115.0±15.2
111.5±7.4
107.3±10.1
113.8±11.5
111.0±13.7
114.5±11.3
112.3±10.9
First dose
2.5±5.2
-4.2±4.3
-2.4±10.0
-2.3±8.0
-5.6±9.2||
-1.3±5.5
-8.5±8.6||
-7.5±2.7
-6.3+5.2*
-2.8±5.8
-9.9+8.4t
0.3±2.1
-8.8±9.6||
-1.0+6.1
-10.1±11.5||
-3.3±8.8
Heart rate (bpm)
Last dose
Run-in
First dose
-6.0±4.6+t
-11.5+5.2
-6.6±7.2||
-6.0±11.7
-10.2± 10.0||
0±5.8
-14.9+5.7*
-12.8+10.7
-10.8±5.4 + t
-7.3±5.9
-10.6±6.6*
-2.8±1.7
-11.6±8.9t
-5.0±5.7
-7.3±6.7*
-5.8±6.5
53.3±7.3
62.5 ±10.2
50.4±5.2
59.3±7.6
53.0+5.1
62.0±8.7
53.4±5.4
60.5 ±5.6
56.4±5.1
65.3±8.5
55.5±6.6
61.3±5.3
56.0±6.8
65.0±4.6
58.9±10.5
62.5±3.7
0.9±3.8
-5.9±5.4
2.1±5.5
-2.8±1.7
0.3±2.0
-4.0±12.0
0.9±8.0
-3.3±3.9
5.9±5.6t
0±4.3
5.7±7.2||
0±1.4
1.2+5.1
-1.3±7.7
3.7+11.6
6.3±8.3
Last dose
-0.3±5.7
-6.0±1.8
4.4±6.5*
-4.0±1.4
-1.6+4.1
-7.5±9.9
0.3±5.4
-0.3±2.5
3.6+4.2H
1.5±8.0
4.5+5.7H
1.5±3.0
2.2±5.9
-2.5±2.9
0.6±7.8
1.5±9.3
bpm, Beats per minute. Values are mean+SD from run-in day to first dose and last dose at corresponding times after dosing. n = 10 for
losartan (100 mg); n=4 for placebo.
*0.05</7<0.10, mean changes from run-in.
tpsO.Ol, mean changes from first to last dose (last dose measurement minus first dose measurement).
t/><0.01, mean changes from run-in.
§/><0.05, mean changes from first to last dose.
||p<0.05, mean changes from run-in.
dose of double-blind losartan or placebo; and last dose
is the period after the last dose of double-blind losartan
or placebo, i.e., the last of seven consecutive once-daily
doses, eight doses total over 9 days.
Mean changes from run-in to first and last dose and
from first to last dose were evaluated statistically for the
losartan treatment group with a two-tailed paired t
test.13 The Type I error rate was set at a=0.05. No
statistical evaluation was performed for changes within
the four subjects in the placebo treatment group.
Differences between the losartan and placebo treatment groups in mean changes were evaluated statistically
with a two-tailed pooled variance Student's t test13 or a
two-tailed Aspin-Welch separate variance t test.13 The
assumption of homogeneity of variances was evaluated
using the Folded Form F Statistic.13 The Type I error rate
was set at a=0.05 for the / tests and at a=0.10 for the
evaluation of homogeneity of variances.
Results
Blood Pressure and Heart Rate Changes
Supine and standing blood pressures were carefully
monitored to determine whether Ang II blockade would
alter blood pressure in nonhypertensive subjects. A
depressor effect was noted. Table 1 and Figure 3
summarize the changes in supine blood pressure and
heart rate from the run-in day to the corresponding time
after dosing following the first and last dose of doubleblind study drug. An average reduction of 9 mm Hg was
apparent. Statistical analysis of these changes within the
losartan group is summarized in Table 1. Because of the
small number of subjects given placebo, a within-group
analysis was not performed for this treatment. However,
statistical analysis between the two groups showed some
statistically significant (or nearly significant) differences
between treatments at the times of apparent maximal
effect: hours 3 (p<0.05 for last dose), 5 (p<0.01 for first
dose,p=0.07 for last dose), and 6 (/?<0.05 for first and
last dose) for supine diastolic blood pressure, and hour
5 (p<0.01 for first and last dose) for supine systolic
blood pressure. During the 8-hour observation period,
particularly after the first dose, supine heart rate (Figure 4) tended to increase more in the losartan than
placebo group. It was noted that, during the run-in day,
supine heart rate was stable in both groups but significantly higher in the placebo group at 1, 2, 4, and 6 hours
after single-blind placebo. Together, these data suggest
a modest blood pressure-lowering effect of losartan,
under the conditions of this study.
Similar trends to those seen for supine blood pressure
were apparent for mean standing blood pressure measurements (not shown). For example, the change in standing
blood pressure 6 hours after dosing was —14+12/—7±12
and -10±10/-6±9 mm Hg for first and last dose, respectively, in the losartan group, and 0.3±10/-l±7 and
-10±10/-l±10 mmHg at corresponding times in the
placebo group. These effects are generally similar to the
changes in supine blood pressure summarized in Table 1.
On the average, standing heart rate changes within
groups, relative to the placebo day, tended to be greater in
the losartan group than in the placebo group (Figure 4),
with effects most apparent after the first dose of losartan.
These results are consistent with the supine hemodynamic
changes described above (Table 1).
Renin-Angiotensin II-Aldosterone System Responses
Earlier reports at lower doses 56 indicated that losartan administration is associated with sustained, dose-
708
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Vol 21, No 5 May 1993
Supine Diastolic Blood Pressure
Time ( h o w after dosing)
Time (hours after dosing)
Supine Systolic Blood Pressure
T i m (hours after dosing)
Time (hows after dosing)
FIGURE 3. Line graphs show mean changes in supine diastolic (top panels) and systolic (bottom panels) blood pressures after
first (o—o) and last (•—9) dose ofplacebo (right panels) and losartan (left panels), 100 mg once daily for eight doses over 9 days.
Shown are changes (numbers in parentheses indicate reductions) in measurements from the corresponding time after dosing during
the placebo run-in day. *y<0.05 for within-group change, losartan-treated subjects only; arrows on placebo graphs indicate where
the change after losartan was significantly different (p<0.05) from that after placebo, after the first dose (filled arrow) or last dose
(open arrow). A meal was served approximately 3 hours after dose.
related increases in PRA and Ang II. However, Ang II
was measured by RIA without separation of peptides to
confirm that observed increments were in the Ang II
octapeptide. It was therefore of interest to determine
the level of stimulation of PRA after single- and multiple-dose administration of losartan using HPLC to
separate the Ang II octapeptide from Ang I and smaller
peptide fragments. Table 2 summarizes the renin-angiotensin-aldosterone system measurements during the single-blind run-in placebo day and the changes from these
measurements before ( - 1 hour) and after (6, 6.5, and
24 hours) dosing with the first and last doses of doubleblind losartan or placebo. As with the analysis of blood
pressure and heart rate, mean change is shown in
measurements on the "dosing" days from the run-in
day. Also included in the table are the differences
between first and last doses. Figure 5 depicts the
changes in supine measurements of PRA and Ang II.
With respect to both supine and standing measurements
of PRA and Ang II, clinically meaningful1415 and statistically significant (p<0.01) increases from run-in
were noted within the losartan group and between the
losartan and placebo groups. Of note, the significant
increase in predose PRA and Ang II from run-in to first
dose in the losartan group was not different from the
similar increases in the placebo group. Presumably, as
indicated above, this increment is secondary to modest
sodium restriction resulting from the diet provided to
the volunteers. Plasma aldosterone levels did not
change appreciably during the study.
Several other observations are relevant to these data.
As indicated in Table 2, the 6.5-hour renin-angiotensinaldosterone system measurements were made in samples collected after 30 minutes of ambulation. With
respect to PRA, during the run-in day, there was the
expected increase in mean activity of 1-2 ng Ang I per
milliliter per hour (approximately doubling) from supine to standing positions. As indicated by the changes
from run-in to treatment days, this absolute postural
increment in PRA was maintained during the doubleblind portion of the study. A similar postural increment
in Ang II was noted during the run-in day and after the
last dose of losartan. Six to 6.5 hours after the first dose
of losartan, the postural increment in mean Ang II
concentration was not apparent.
Six hours after dosing, PRA did not change significantly from first to last dose (-2.5±6.4 ng Ang I per
milliliter per hour,p=NS). In contrast, the mean change
in Ang II concentration was significantly less after the
last dose compared with the first dose (-26.7±35.2
pg/mL, p<0.05), suggesting some attenuation of this
aspect of the response to losartan after several days of
administration.
To investigate the relation between PRA and Ang II
during the study, we initially constructed a scatterplot of
supine Ang II concentration versus supine PRA 6 and
24 hours after single-blind placebo and after the first
and last dose of each double-blind treatment (Figure 6).
In general, this plot suggested a relation between Ang II
Goldberg et al
Losartan in Healthy Volunteers
709
Tim (horn altar doaing)
Tim* ( h o n atar
1km (hom
FIGURE 4. Line graphs show mean changes in supine (top panels) and standing (bottom panels) heart rates after first (O—O)
and last (m—») dose of placebo (right panels) and losartan (left panels), 100 mg once daily for eight doses over 9 days. Shown
are changes (numbers in parentheses indicate reductions) from the corresponding time after dosing during the placebo run-in day
(see Table 1 for statistical analysis of supine measurements). *p<0.05 for within-group change, losartan-treated subjects only;
arrows on placebo graphs indicate where the change after losartan was significantly different (p<0.05) from that after placebo,
after the first dose (filled arrow) or last dose (open arrow), bpm, Beats per minute.
and PRA. It is apparent from this plot that Ang II levels
relative to PRA in four subjects at 6 hours after the first
dose are elevated from the other points, suggesting
greater increments in Ang II for each unit of PRA. To
explore this further, we calculated the ratio of Ang II to
PRA for each of these points. The geometric mean
ratios of Ang II to PRA are summarized in Figure 7. A
statistical analysis of the change in these ratios shows
significant increases within the losartan group 6
(/?<0.05) and 24 (/?<0.01) hours after the first dose of
losartan. In one extreme, one subject had ratios of 2.9 6
hours after placebo, 16.1 after his first dose, and 10.2 6
hours after his last dose of losartan. By the last dose of
losartan, on the average, ratios of Ang II to PRA had
returned toward baseline, being significantly less 6
hours after the last dose than 6 hours after the first dose
Tolerability
As in any early study of a new drug, it was also
important to carefully evaluate subjects for untoward
consequences of study drug administration. Subject
tolerability was monitored by regular assessment of
clinical laboratory tests (complete blood count, chemistry panel, and urinalysis), by electrocardiogram, and by
observation and questioning for clinical adverse events.
Analysis of serum chemistry panels showed no significant (/?<0.05) within-group or between-group changes
in any of the measured analyses during the study (the
chemistry panel consisted of measurements of urea
nitrogen, creatinine, total bilirubin, alanine and aspartate aminotransferases, alkaline phosphatase, lactate
dehydrogenase, glucose, total protein, sodium, potassium, chloride, bicarbonate, calcium, and inorganic
phosphorous). Approximately 600 mL blood was collected during the entire 10-day study. Presumably as a
result of this blood loss, at the time of the poststudy
evaluation, hemoglobin had decreased approximately 2
g/dL and hematocrit had decreased approximately six
percentage points in subjects given either losartan
(p<0.01 within this group) or placebo. Urinalysis results showed no remarkable changes. No clinically significant changes in electrocardiographic intervals or
interpretation were noted in any subject.
Clinical adverse events were mild and nonspecific,
including one episode of loose bowel movements in one
subject, epigastric discomfort in another individual, and
several occurrences (four subjects) in which blood pressure decreases from supine to standing position were
noteworthy but asymptomatic (e.g., supine: blood pressure, 102/46 mm Hg; heart rate, 50 beats per minute; 2
minutes standing: blood pressure, 70/50 mm Hg; heart
rate, 70 beats per minute). One subject reported superficial peeling of his palms beginning during the first
several days of losartan administration. No local symptoms (such as burning, itching, or exudate) were associated with these findings, which resolved over several
weeks. However, this subject also had evidence of
710
Hypertension
Vol 21, No 5 May 1993
TABLE 2. Renin-Angiotensin-Aldosterone Responses to Losartan and Placebo
Parameter at indicated hour after dosing
Variable
Plasma renin activity
[(ng Ang I/mL)/hr]
Day
Treatment
Hour - 1
Run-in
Losartan
Placebo
1.2±0.6
1.2±0.6
Run-in to
first dose
Run-in to
last dose
First to
last dose
Losartan
Placebo
Losartan
Placebo
Losartan
Placebo
0.73±0.62*
0.43±0.61
3.1±1.9*t
-0.05±0.44
2.4±1.8*
-0.57±0.78
Run-in
Losartan
Placebo
5.0±2.0
4.0±2.4
Run-in to
first dose
Run-in to
last dose
First to
last dose
Losartan
Placebo
Losartan
Placebo
Losartan
Placebo
3.3±2.6*
1.4±1.9
13.1±7.9*t
0.09 ±2.02
9.8±7.1*t
-1.4±3.5
Run-in
Losartan
Placebo
151.1±38.7
124.6±40.7
Run-in to
first dose
Run-in to
last dose
First to
last dose
Losartan
Placebo
Losartan
Placebo
Losartan
Placebo
24.4 ±38.8*
82.9±53.0
23.2+68.7
32.8±21.7
-1.2±51.2
-50.1±52.4
Plasma angiotensin II
(pg/mL)
Plasma aldosterone
(pg/mL)
Hour 6
Hour 6.5
Mean measurement (±SD)
1.2±0.6
2.5±1.2
3.2±1.9
l.l±0.3
Mean change (±SD)
12.0±6.3*t
12.0±5.6*t
1.0±0.6
2.2±1.3
9.6±4.9*t
9.9±5.2*t
0.05 ±0.38
0.48±1.13
-2.5±6.4
-2.2±6.5
-2.7±2.3
-0.95±0.76
Mean measurement (±SD)
4.3±1.7
9.7±4.7
3.7±2.0
14.4±13.7
Mean change (±SD)
67.3±34.3*t
72.4±33.3*t
16.1 ±14.6
4.5±2.8
45.7±14.1*f
66.2±43.1*t
1.5±4.3
-3.0±8.5
-26.7±35.2§
-1.1±43.8
-3.1±5.3
-19.1+22.5
Mean measurement (±SD)
104.1 ±28.9
160.1 ±50.6
117.5±10.7
234.2+114.1
Mean change (±SD)
10.1 ±49.1
36.1 ±78.3
52.6±62.0
132.2±61.8
-10.5±15.0$
-14.7±67.0
-4.7±28.3
-3.7±41.7
-20.6±53.2
-50.9±112.1
-57.3±65.1
-135.8±63.1
Hour 24
1.5±0.8
1.6±0.9
5.1±2.3*t
0.95 ±0.37
4.1±3.2*t
0.10±0.44
-0.94 ±1.60+
-0.85±0.71
5.9±2.4
6.2+4.3
27.7±10.2*t
7.8±5.8
18.1±10.9*t
0.19±3.14
-9.6±8.8t
-8.0±8.7
137.6±49.4
168.0±77.8
82.9±44.2*
103.8±84.4
52.6±78.7t
-34.4 ±75.8
-30.3 + 64.6
-138.2+151.0
Ang I, angiotensin I. Values are mean±SD measurements on the run-in day and changes from run-in tofirstdose (day 1) and last dose
(day 9) and from first to last dose at corresponding times after dosing. n = 10 for losartan (100 mg); n=4 for placebo. Measurements at Hour
6.5 were taken after 30 minutes ambulation.
*p<0.01, mean changes from run-in or first to last dose.
tpfiO.01, losartan vs. placebo.
$0.05</?£0.10, §p<0.05, mean changes from run-in or first to last dose.
hemolysis (schistocytes, crenated red blood cells) on his
peripheral smear during the treatment period without
decreases in haptoglobin, hemoglobinuria, increases in
lactate dehydrogenase, or decreases in hemoglobin or
hematocrit (other than as expected in such a study).
These events were judged to be of uncertain relation to
administration of losartan.
Discussion
In summary, losartan was found to be sufficiently well
tolerated at 100 mg once daily to permit use of this dose
in future studies. Modest decreases of blood pressure
were noted during losartan administration, which indicate that, in healthy men, Ang II can play a role in the
maintenance of resting blood pressure. However, these
observations were made under conditions of modest
sodium restriction (based on 24-hour urinary sodium
excretion and small increases in PRA before administration of double-blind losartan or placebo). The extent
to which this study condition influenced our results
cannot be determined from this exploratory study.
Blockade of endogenous Ang II was indicated by clearly
denned increases in PRA and concomitant increases in
plasma Ang II concentration.
Effects on aldosterone concentration in blood were
not apparent, presumably because of the complex mechanisms known to control aldosterone release.16-17
Based on prior data5-6 and the known capacity of
endogenous Ang II to act on a feedback receptor on the
juxtaglomerular apparatus,18"20 it was anticipated that
losartan administration would result in an increase in
PRA and Ang II. As indicated above, both supine PRA
and plasma Ang II concentration were significantly
increased 6 and 24 hours after administration of the first
Goldberg et al
Losartan in Healthy Volunteers
711
Supine Plasma Renin Activity
RalDoM
Supine Plasma AngiotenBn Con vitiation
*
FMDOH
RntDoM
LMIDOM
LatfDoa*
FIGURE 5. Bar graphs show mean change from the corresponding time after dosing on the run-in day of plasma renin activity
(top panels) and plasma angiotensin II concentration (bottom panels) in subjects allocated to receive losartan 100 mg daily (left
panels) and placebo (right panels) for eight doses over 9 days. In each panel, changes after the first and last dose of double-blind
therapy are shown. Solid bars indicate changes from 1 hour predose on the run-in day to 1 hour before the first and last dose.
Shaded bars indicate corresponding changes 6 hours after dosing. Open bars indicate changes 24 hours after dosing. *p<0.01 vs.
change in placebo group. AI, angiotensin I.
and last 100-mg dose of losartan. The methodology
used specifically measures Ang II octapeptide, indicating that observed increments reflect changes in the
concentration of the most biologically active peptide.10"12 The magnitude of the change was greater 6
hours after dosing, compared with 24 hours. The data
also indicate that the level of stimulation of PRA and
Ang II is slightly less after the last than the first dose,
consistent with the hypothesis that the magnitude of the
initial response is related to discharge of stored renin
160
•=•120
\
a.
.£
en
c
r
Run-in, Hr 6
Run-in, Hr 24
First Dose. Hr 6
First Dose. Hr 24
Lost Dose, Hr 6
Lost Dose, Hr 24
A
A
O
•
••
FIGURE 6. Scatterplot shows supine plasma
angiotensin II and plasma renin activity measurements in subjects given losartan and placebo (inset).
80
D
••
<
from the juxtaglomerular apparatus, as has been shown
in animals given angiotensin converting enzyme inhibitors.21 This differs somewhat from prior observations.5
However, study conditions (i.e., sodium balance), losartan dose, and methodology for measurement of Ang II
also differed between the studies.
The implications of these increases in PRA and Ang
II cannot be determined from this study in healthy
volunteers. Studies in hypertensive patients are required to determine whether similar increases are noted
40
D°
o
ffl)
10
15
20
Plasma Renin Activity (ng/ml/hr)
25
30
712
Hypertension
Vol 21, No 5 May 1993
Lasa«w(N=
=10)
FIGURE 7. Line graph shows ratio of angiotensin II to
plasma renin activity (PRA) 6 and 24 hours after placebo
(Run-in) and first and last doses of double-blind study drug
(see text for statistical analysis of changes in this ratio). AI,
angiotensin I.
in this target population. Furthermore, losartan is a
selective antagonist of only a single Ang II binding
site,22-25 which mediates all known physiological effects
of angiotensin. It is not known whether unblocked
effects of increased Ang II at other binding sites are of
any clinical consequence to the safety profile and hemodynamic effects of losartan. In the absence of longlasting Ang II blockade, increases in plasma Ang II
concentration of the magnitude demonstrated would be
expected to have hemodynamic activity.14'15
In general, a relation between PRA and Ang II could
be defined; the ratio of Ang II concentration to PRA
was assumed to be an index of this relation. It was of
interest that this ratio was higher after the first dose of
losartan, when the greatest increases in Ang II were
noted, than during the run-in day and after the last dose
of losartan. The implication is that more Ang II is
produced per unit of PRA at this time than at other
times. An explanation for this observation is not readily
apparent from the data collected in this study. It is
possible that these differences are a result of greater in
vitro generation of Ang II associated with initial stimulation of renin release.10 Alternatively, several physiological hypotheses are possible, including changes in
Ang II clearance, which seem unlikely, and changes in
Ang II generation.26 One explanation is that angiotensin
converting enzyme is somehow downregulated, resulting in less-efficient conversion of Ang I to Ang II. This
seems unlikely in view of the ubiquity of the enzyme.26
Another potential control point in the system that could
be rate limiting is the amount of circulating angiotensinogen, which is under the control of both renin and
Ang II. 27 Thus, it has been demonstrated that (in
addition to consuming angiotensinogen) increases in
renin decrease the production and release of angiotensinogen, whereas Ang II stimulates its release, an effect
apparently antagonized by the peptide Ang II antagonist saralasin.27 During Ang II blockade with losartan, it
is reasonable to postulate that secondary increases in
PRA with blockade of Ang II receptors results in net
inhibition of angiotensinogen synthesis and release,
functionally downregulating the system. This hypothesis
needs to be tested in appropriate preclinical and clinical
models.
It was anticipated that Ang II blockade would result
in a decrease in plasma aldosterone concentration. This
effect was not observed. However, in the presence of
10-fold elevations of Ang II concentrations, it is reasonable to assume that the absence of changes in aldosterone concentration is consistent with Ang II blockade.14-17 Clarification of the effect of Ang II blockade on
aldosterone release awaits further study.
The tolerability of losartan for future studies in
healthy subjects and patients was investigated through
evaluation of hemodynamic effects, clinical adverse
effects, and clinical laboratory tests. Hemodynamic effects were of particular interest, as the role of Ang II in
the regulation of blood pressure in healthy volunteers
under the conditions of this study was of physiological
interest.
A modest, sustained, and persistent effect of losartan
to reduce blood pressure was noted in this study. These
changes did not result in apparent clinical symptoms.
Also noted were statistically significant increases in
standing heart rate, particularly after the first dose of
losartan, but without postural hypotension. Together,
these data suggest that, under the conditions of this
study (overnight supine rest, moderate sodium depletion, limited environmental stimulation), the blood
pressure of healthy male volunteers is somewhat dependent on endogenous Ang II.
Acknowledgment
Betty Jean Miller is acknowledged for preparation of the
manuscript.
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