Download Effect of Oral Nimodipine on Platelet Function

10
Effect of Oral Nimodipine
on
Platelet Function
William M. Feinberg, MD, and Denise C. Bruck, AAS
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Background and Purpose: Nimodipine, a calcium antagonist, has been reported to have beneficial effects
in acute ischemic infarction. Some calcium channel antagonists have antiplatelet effects. We investigated
the effect of oral nimodipine on platelet function in healthy volunteers.
Methods: Twelve healthy volunteers (6 men and 6 women, mean age 32.9±5.6 years) took 30 mg
nimodipine every 6 hours for 24 hours, followed by a week with no medication, followed by 60 mg every 6
hours for 24 hours. Ex vivo platelet function was measured at baseline, 1 hour after the first dose at each
dosage strength, and 1 hour after the last dose at each dosage. Platelet studies included aggregation and
adenosine triphosphate release in response to collagen, epinephrine, and adenosine diphosphate;
maximal rate of primary aggregation; threshold adenosine diphosphate concentration for second-phase
aggregation; and thromboxane B2 release at threshold aggregation. The bleeding time was measured at
baseline and after the last 60-mg dose of nimodipine.
Results: No change in any platelet function study was seen with 30 mg nimodipine every 6 hours. Platelet
function studies were also unchanged after 60 mg every 6 hours, except for a slight decrease in aggregation
and adenosine triphosphate release in response to suprathreshold (10 ,uM) adenosine diphosphate
(p=O.OOl, Student's paired t test). There was no significant change in bleeding times.
Conclusions: Oral nimodipine has minimal antiplatelet activity in young, healthy subjects. (Stroke
1993;24:10-13)
KEY WORDS * calcium channel blockers * nimodipine * platelet aggregation
In recent years calcium antagonists have been proposed as therapy for cerebrovascular disease.1-8
The properties of calcium antagonists that may be
beneficial in cerebrovascular disease include vasodilation of cerebral vessels38 and reduction of Ca2+ entry
into neurons.138 Nimodipine, a dihydropyridine derivative, has been approved in the United States for
treatment of ischemia after subarachnoid hemorrhage.8'9 A beneficial effect of nimodipine has been
suggested in acute ischemic stroke, although clinical
trials have produced conflicting results.12,4,5,7,8 A recent
meta-analysis of the acute infarction trials suggested a
beneficial effect only if treatment with nimodipine was
begun within 12 hours of onset of ischemia.6
Calcium antagonists have also been noted to have
antiplatelet effects,'0-15 a property that might be important in the treatment of cerebral ischemia. The antiplatelet properties appear to vary among differing calcium antagonists.12"'415 Calcium antagonists may inhibit
ex vivo platelet aggregation in response to collagen,
epinephrine, and serotonin and cause mild prolongation
of the bleeding time'0-1316 Because nimodipine's antiplatelet activity has not been extensively studied, we
examined the effect of nimodipine on platelet function
in a group of normal subjects.
From the Department of Neurology, Arizona Health Sciences
Center, Tucson, Ariz.
Supported by a Biological Research Support Grant from the
University of Arizona College of Medicine.
Address for reprints: William M. Feinberg, MD, Department of
Neurology, Arizona Health Sciences Center, Tucson, AZ 85724.
Received June 8, 1992; final revision received September 28,
1992; accepted September 29, 1992.
Subjects and Methods
Twelve healthy volunteer subjects, 6 men and 6 women,
participated in this study. The mean+SD age was
32.9+5.6 (range, 24-44) years. The subjects were instructed not to take any other medication for 2 weeks
before the start of the study or during the 9 days of the
study. They were specifically questioned about aspirincontaining compounds and nonsteroidal anti-inflammatory drugs. Informed consent was obtained from all
subjects.
Baseline laboratory tests (chemistry panel, complete
blood count, platelet count, and pregnancy test) were used
to rule out underlying disease or pregnancy. A bleeding
time test was performed using a Simplate II device (Organon Teknika, Durham, N.C.) before ingestion of any
drug and 1 hour after the ingestion of the final dose.
During the initial study period, subjects took 30 mg
nimodipine (Miles Inc., West Haven, Conn.) every 6
hours for a 24-hour period (120 mg/day). Blood for
platelet aggregation and adenosine triphosphate (ATP)
release was drawn in the morning before the first dose
and after a low-fat breakfast (first baseline sample).
Blood was obtained 1 hour after the first dose, and again
the next morning 1 hour after ingestion of the last dose
of nimodipine.
The subjects then took no medications for 1 week,
followed by 60 mg nimodipine taken every 6 hours for a
24-hour period (240 mg/day). Blood for platelet aggregation studies and ATP release was again obtained
before the first 60-mg dose of nimodipine (second
baseline sample), 1 hour after the first dose, and 1 hour
after the final dose. A second bleeding time test was
performed 1 hour after the last 60-mg dose of
nimodipine.
Feinberg and Bruck Effect of Nimodipine on Platelets
11
TABLE 1. Percent Platelet Aggregation (Primary and Secondary Phases) in Response to Selected Agonists at
Baseline and After Oral Administration of Nimodipine
30 mg every 6 hours
Agonist
First baseline
1 hour*
24 hours*
Second baseline
Collagen
91±5
89±5
92+3
93±2
Epinephrine
73+12
79±13
77±11
84±11
ADP
1.25 ,M
57± 11
58± 13
65± 10
47+ 14
2.5 ,M
72±8
74±10
73±7
67±11
5.0 ^M
83±7
81±6
89±4
85±7
10 gM
87±5
83±5
90±3
93+4
Values (%) are mean±SE.
*Blood for platelet studies was obtained 1 hour after nimodipine dose.
tp<0.05 different from second baseline by paired t test.
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Using a 21G butterfly needle, 22.5 ml blood was drawn
into a syringe containing 2.5 ml 3.8% sodium citrate
anticoagulant. Platelet-rich plasma was prepared and
adjusted with platelet poor plasma to 2x 105/mm3 platelets. A model 660 dual-chamber platelet ionized calcium
aggregometer was used. Agonists used were thrombin,
collagen, adenosine diphosphate (ADP), and epinephrine.
Chrono-Lume reagent was used to determine the ATP
release. All reagents and the platelet aggregometer were
products of Chrono-Log (Havertown, Pa.).
For platelet aggregation studies, 450-,ul aliquots of
platelet-rich plasma were pipetted into siliconized glass
cuvettes. Individual cuvettes were allowed to warm to
37°C for at least 2 minutes but not more than 5 minutes.
Fifty microliters of Chrono-Lume was added to the
cuvette in the mixing chamber and allowed to equilibrate for 1 minute before the addition of the agonist.
Final cuvette concentrations were as follows: thrombin
1 IU/mI, collagen 2 ,ug!ml, epinephrine 10 ,M, and
ADP 1.25, 2.5, 5, and (if time and sample permitted) 10
,M. Percent aggregation and ATP release were measured at each agonist concentration. All testing was
completed within 2 hours. The maximal rate of primary
aggregation in response to 2.5 and 5 ,uM ADP was
calculated in terms of light transmittance units per
minute at the steepest part of the primary aggregation
curve, as described by Thompson and Vickers.17
Threshold for second-phase aggregation was defined as
the ADP concentration required to produce irreversible
aggregation accompanied by ATP release. ATP release
of the samples was compared with the 2-nmol standard
provided with the Chrono-Lume reagent.
Thromboxane B2 (TXB2) release was determined at
the threshold ADP concentration. After addition of the
ADP, the platelets were allowed to react for 5 minutes.
A control sample, to which no ADP was added, was
incubated under the same conditions in the other
chamber. Both supernatants were quickly obtained by
spinning in a microcentrifuge, then freezing at -20°C.
TXB2 levels were measured using an enzyme-linked
immunosorbent assay from Cayman Chemical (Ann
Arbor, Mich.), according to manufacturer's instructions.
Briefly, plastic microtiter plates were coated with mouse
monoclonal anti-rabbit immunoglobulin G. Dilutions of
the standard provided in the kit and dilutions of samples
were added to the plates. Acetylcholinesterase tracer
and thromboxane antiserum were added to the wells
and allowed to incubate overnight at 4°C. After the
60 mg every 6 hours
1 hour*
24 hours*
90±4
88±4
77±11
78±10
52± 16
64±8
82±4
90+5t
49±14
66±10
74±6
87±4
excess sample and reagents were washed out, Ellman's
reagent was added to each well and incubated to allow
color development. The color was read on a microtiter
plate reader (Titertek, McLean, Va.). Standard readings were plotted against their concentrations, and the
resultant curve was used to calculate sample concentrations. Units were picograms per milliliter and were
converted to nanograms per 10i platelets.
Platelet aggregation studies were performed in control subjects (n=3) before and after aspirin ingestion.
Aspirin abolished platelet aggregation in response to
collagen and epinephrine and second-phase aggregation
in response to ADP in all controls.
Platelet aggregation and ATP release (in response to
the above agonists) at each dosage strength of nimodipine were compared with baseline values using Student's two-tailed paired t test. Bleeding times at baseline and after 60 mg nimodipine were also compared
using Student's two-tailed paired t test. Primary aggregation rates in response to ADP were also compared
with baseline using Student's two-tailed paired t test.
Threshold ADP concentration at each dosage strength
was compared with baseline using the Wilcoxon signed
rank test. Significance was sought at the a=0.05 level.
Results
Baseline blood tests revealed no underlying disease or
pregnancy. The mean platelet count was 2.56±0.57x 105/
mm3 platelets (range, 1.51-3.72x 105/mm3; laboratory normal range, 1.5-3.5 x 105/mm3 platelets).
Percent aggregation in response to collagen, epinephrine, and ADP at each time period and nimodipine dosage
is shown in Table 1. There were no significant differences
noted at any time at 120 mg/day. At 240 mg/day, there was
a minor decrease in aggregation in response to 10 ,M
ADP after the first 60-mg dose (p=0.038) but not after 24
hours. There was no significant change in the maximum
primary aggregation rate in response to 2.5 or 5 ,uM ADP
at either nimodipine dosage.
ATP release at each time period and dosage strength
is shown in Table 2. No significant differences were
noted at a dose of 120 mg/day. ATP release in response
to 10 juM ADP at 1 hour after the first 60-mg dose of
nimodipine showed a significant decline from baseline
(0.50±0.08 nmol versus 0.28±0.05 nmol; p=0.001). At
24 hours, after the fourth 60-mg dose of nimodipine,
this appeared to recover slightly but was still signifi-
-~ *f
Stroke Vol 24, No 1 January 1993
12
TABLE 2. ATP Release in Response to Selected Agonists After Oral Administration of Nimodipine
30 mg every 6 hours
60 mg every
Agonist
First baseline
1 hour*
24 hours*
Second baseline
1 hour*
0.97±0.12
Collagen
0.95±0.10
0.90+0.10
1.29+0.26
1.17+0.14
0.53±0.12
Epinephrine
0.55±0.13
0.49+0.15
0.78+0.17
0.61 +0.09
ADP
0.11+0.03
1.25 ,M
0.13±0.05
0.16+0.09
0.11±0.08
0.13±0.11
0.26±0.08
0.18±0.05
0.29±0.11
2.5 ,M
0.26+0.11
0.26±0.09
0.32±0.07
0.37±0.08
0.37±0.07
5.0 I.M
0.43±0.08
0.36±0.05
10 gM
0.24±0.08
0.30±0.08
0.27+0.08
0.50±0.08t
0.28+0.05t
Values for ATP release (nmol) are mean± SE.
*Blood for platelet studies was obtained 1 hour after nimodipine dose.
tp=0.02 different from first baseline by paired t test.
tp<0.05 different from second baseline by paired t test.
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cantly different from baseline (0.50±0.08 nmol versus
0.29±0.06 nmol; p=0.008) (Figure 1).
Threshold ADP concentration for second-phase
platelet aggregation is shown in Figure 2. After 24 hours
of 60-mg doses of nimodipine, threshold was increased
in three subjects, decreased in one, and unchanged in
six. These differences were not statistically significant.
Threshold dose was not significantly different from
baseline at any time period or nimodipine dosage.
Levels of TXB2 are shown in Table 3. A slight
increase in TXB2 release was noted in the second versus
the first baseline samples (9.5±2.4 versus 7.5±1 1.9
ng/108 platelets; p=0.028). No other significant differences at any time period or dosage were seen.
Bleeding time after 24 hours of 60-mg doses of
nimodipine was actually slightly shorter than at baseline
(3.92±1.06 min versus 4.33±1.26 min). This difference
was not statistically significant.
Discussion
These results demonstrate no significant effect of oral
nimodipine at a dose of 120 mg/day on the ex vivo tests
of platelet function or on bleeding time. At a dose of
240 mg/day, no significant effect was seen on collagen-
10
.
6 hours
24 hours*
1.10±0.14
0.64±0.15
0.09+0.07
0.30±0.16
0.38±0.12
0.29±0.06t
epinephrine-induced platelet aggregation, threshold
for second-phase aggregation, or bleeding time. These
data are consistent with animal studies showing no
significant effect on ADP-induced aggregation by nimodipine in cat platelets.18
Only minor effects on platelet function were seen at a
dose of 240 mg/day. There was a slight decrease in
ADP-induced ATP release seen only with a suprathreshold (10 ,uM) concentration of ADP after the first
60-mg nimodipine dose when compared with the second
baseline value. Of note, the second baseline value for
ADP release showed a slight increase from the first
baseline value; if ADP release in response to 10 ,uM is
compared with the original baseline, there is no significant decrease (Table 2).
Because of the small number of patients in this study,
we may have missed a small but statistically significant
effect on platelet function. However, we do not feel that
we have missed a clinically significant effect. Clinical
trials have reported a beneficial effect of nimodipine at
a dose of 120 mg/day,2.6'7 and we saw no evidence of any
antiplatelet effect at this dose.
Several caveats apply to this study. The effect of
nimodipine ex vivo may be different from that in vivo.
or
100
10 ,uM
a
C)
C)
a)
2.i
0.8 _
80
(0
(
0
-..
0.6 _
U_--
aX
2
CO
60
(D
oq
m
0/
(CF
0
0
5.0 [M
P:
0
0.4
40
2.5 jM
0.2
20
Second
Baseline
1st dose
4th dose
FIGURE 1. Adenosine triphosphate (ATP) release (filled
circles) and percent aggregation (filled squares) in response to
10 ,M adenosine diphosphate (ADP) at baseline and after
administration of 240 mg/day nimodipine. Blood for platelet
studies was obtained 1 hour after first and fourth doses.
0 mep
1.25 jM
Second
Baseline
1st dose
4th dose
FIGURE 2. Threshold adenosine diphosphate (ADP) concentration for second-phase aggregation at baseline and after
oral administration of 240 mg/day nimodipine. Blood for
platelet studies was obtained 1 hour after first and fourth
doses.
Feinberg and Bruck Effect of Nimodipine on Platelets
13
TABLE 3. Thromboxane B2 Release at Baseline and After Oral Administration of Nimodipine
30 mg every 6 hours
60 mg every 6 hours
First baseline
1 hour*
24 hours*
Second baseline
1 hour*
24 hours*
7.5±1.9
8.0+2.3
6.4+1.8
9.5 +2.4t
6.7+2.0
7.4+2.3
Values (ng/10' platelets) are mean±SE. Thromboxane B2 release was measured in response to the threshold
adenosine diphosphate concentration for second-phase aggregation.
*Blood for platelet studies was obtained 1 hour after nimodipine dose.
tp=0.028 different from first baseline by paired t test.
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Other calcium channel blockers have been shown to
have in vivo antiplatelet effects. Verapamil and nifedipine inhibit deposition of autologous platelets onto
implanted Gore-Tex grafts in dogs.19 Verapamil infusion has also been reported to decrease circulating
platelet aggregates in patients with coronary artery
disease.20 However, in a cat global ischemia model,
nimodipine had no effect on plasma TXB2 concentrations21; this finding also supports nimodipine's lack of in
vivo effect in ischemia.
The effect of nimodipine on platelets in normal,
healthy, young control subjects may be different from
that in older patients with active vascular disease. For
instance, platelets in patients with myocardial infarction
are larger22 and show reduced sensitivity to aspirin.16
Thus, although we found no significant antiplatelet
activity in young control subjects, it is possible that an
antiplatelet effect might exist in older patients or those
with acute cerebral ischemia. Despite the negative
results of our study, it would be valuable to examine the
antiplatelet effect of nimodipine in stroke patients.
Some studies have demonstrated a synergistic effect
between calcium antagonists and aspirin." We chose to
examine the effects of nimodipine alone because several
of the major acute stroke treatment trials have specifically prohibited the use of aspirin or other antiplatelet
agents.2'7 It is possible that nimodipine may have an
additive antiplatelet effect if used with aspirin.
We studied platelet activity after 24 hours of drug
administration. Although a number of studies have demonstrated antiplatelet activity after a single dose of calcium antagonist, Jones et al23 found no antiplatelet activity
2 hours after a single dose of verapamil but found inhibition of ex vivo epinephrine-induced aggregation after 4
days of therapy. Juvela et a124 found no antiplatelet effect
during the first 5 days of intravenous nimodipine treatment in patients with subarachnoid hemorrhage but found
inhibition of platelet thromboxane release thereafter. Future studies might look at a more prolonged administration of nimodipine. The slight increase in TXB, and ATP
release in response to 10 ,uM ADP that we found on the
second baseline might suggest a rebound effect. Future
studies might also look more closely at the recovery period
after discontinuance of nimodipine.
In summary, our results show minimal antiplatelet
effect of oral nimodipine in young, healthy subjects. No
antiplatelet effect was seen at a dose of 120 mg/day, the
dose reported to have a beneficial effect in acute
stroke.67 These results do not support the hypothesis
that nimodipine exerts an effect on cerebral ischemia
through an inhibitory effect on platelet activity. These
studies should be confirmed in older persons and in
patients with acute cerebral ischemia.
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Effect of oral nimodipine on platelet function.
W M Feinberg and D C Bruck
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Stroke. 1993;24:10-13
doi: 10.1161/01.STR.24.1.10
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