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Low-dose naproxen interferes with the antiplatelet effects of aspirin in healthy subjectsRecommendations to minimize the functional consequences.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 3, March 2011, pp 850–859
DOI 10.1002/art.30175
© 2011, American College of Rheumatology
Low-Dose Naproxen Interferes With the Antiplatelet
Effects of Aspirin in Healthy Subjects
Recommendations to Minimize the Functional Consequences
Paola Anzellotti,1 Marta L. Capone,1 Anita Jeyam,2 Stefania Tacconelli,1 Annalisa Bruno,1
Paola Tontodonati,1 Luigia Di Francesco,1 Linda Grossi,1 Giulia Renda,1 Gabriele Merciaro,3
Patrizia Di Gregorio,4 Thomas S. Price,2 Luis A. Garcia Rodriguez,5 and Paola Patrignani1
Objective. To investigate whether low-dose
naproxen sodium (220 mg twice a day) interferes with
aspirin’s antiplatelet effect in healthy subjects.
Methods. We performed a crossover, open-label
study in 9 healthy volunteers. They received for 6 days 3
different treatments separated by 14 days of washout:
1) naproxen 2 hours before aspirin, 2) aspirin 2 hours
before naproxen, and 3) aspirin alone. The primary end
point was the assessment of serum thromboxane B2
(TXB2) 24 hours after the administration of naproxen 2
hours before aspirin on day 6 of treatment. In 5
volunteers, the rate of recovery of TXB2 generation (up
to 72 hours after drug discontinuation) was assessed in
serum and in platelet-rich plasma stimulated with arachidonic acid (AA) or collagen.
Results. Twenty-four hours after the last dosing
on day 6 in volunteers receiving aspirin alone or aspirin
before naproxen, serum TXB2 was almost completely
inhibited (median [range] 99.1% [97.4–99.4%] and
99.1% [98.0–99.7%], respectively). Naproxen given before aspirin caused a slightly lower inhibition of serum
TXB2 (median [range] 98.0% [90.6–99.4%]) than aspirin alone (P ⴝ 0.0007) or aspirin before naproxen (P ⴝ
0.0045). All treatments produced a maximal inhibition
of AA-induced platelet aggregation. At 24 hours, compared with baseline, collagen-induced platelet aggregation was still inhibited by aspirin alone (P ⴝ 0.0003),
but not by aspirin given 2 hours before or after
naproxen. Compared with administration of aspirin
alone, the sequential administration of naproxen and
aspirin caused a significant parallel upward shift of the
regression lines describing the recovery of platelet
TXB2.
Conclusion. Sequential administration of 220 mg
naproxen twice a day and low-dose aspirin interferes
with the irreversible inhibition of platelet cyclooxygenase 1 afforded by aspirin. The interaction was smaller
when giving naproxen 2 hours after aspirin. The clinical
consequences of these 2 schedules of administration of
aspirin with naproxen remain to be studied in randomized clinical trials.
Dr. Patrignani’s work was supported by the Ministero
dell’Istruzione, dell’Università e della Ricerca (grants Fondi Ateneo
and PRIN 2006) and by the European Community Sixth Framework
Programme (Eicosanox grant LSMH-CT-2004-005033).
1
Paola Anzellotti, PhD, Marta L. Capone, PhD, Stefania
Tacconelli, PhD, Annalisa Bruno, PharmD, Paola Tontodonati,
PharmD, Luigia Di Francesco, PhD, Linda Grossi, Giulia Renda, MD,
PhD, Paola Patrignani, PhD: G. d’Annunzio University–Chieti and G.
d’Annunzio University Foundation, Chieti, Italy; 2Anita Jeyam, PhD,
Thomas S. Price, PhD: University of London, London, UK; 3Gabriele
Merciaro: SS. Annunziata Hospital, Chieti, Italy; 4Patrizia Di Gregorio, MD: SS. Annunziata Hospital, G. d’Annunzio University–Chieti,
and G. d’Annunzio University Foundation, Chieti, Italy; 5Luis A.
Garcia Rodriguez, MD: Spanish Centre for Pharmacoepidemiological
Research, Madrid, Spain.
Drs. Anzellotti and Capone contributed equally to this work.
Dr. Garcia Rodriguez has received consulting fees, speaking
fees, and/or honoraria from Bayer and AstraZeneca (less than $10,000
each).
Address correspondence to Paola Patrignani, PhD, Department of Medicine and Center of Excellence on Aging, G. d’Annunzio
University and CeSI, Via dei Vestini 31, 66100 Chieti, Italy. E-mail:
ppatrignani@unich.it.
Submitted for publication April 29, 2010; accepted in revised
form November 23, 2010.
Arthritis in general and osteoarthritis in particular are increasingly becoming global problems; however,
at this time, there is no known cure for osteoarthritis.
Most forms of treatment therefore have dealt with the
alleviation and management of chronic pain, which can
affect normal functioning and quality of life of patients.
Conventional medical treatment employs the use of
nonsteroidal antiinflammatory drugs (NSAIDs) because
850
CONSEQUENCES OF COADMINISTRATION OF NAPROXEN AND ASPIRIN
they provide unmistakable and significant health benefits in the treatment of pain and inflammation (1,2).
However, traditional NSAIDs are also associated with
increased risk of gastrointestinal (GI) and cardiovascular (CV) serious adverse events (i.e., upper GI bleeding
and nonfatal myocardial infarction [MI]) (2–6). The
safety issue of NSAIDs is considered a first priority
because they are administered to elderly persons who
are highly susceptible to their adverse effects, particularly due to GI and CV comorbidity. Both therapeutic
and adverse effects of NSAIDs are due to the inhibition
of prostanoids (7). Prostanoids play an important role in
reducing the threshold to stimulation of the peripheral
nociceptors, thereby increasing the excitability of spinal
sensory neurons, and in stimulating thermosensitive
neurons in the preoptic area of the brain (8). Further,
prostanoids mediate a broad range of homeostatic functions in GI and CV systems (9,10).
Prostanoids are produced from arachidonic acid
(AA) after its release from membrane phospholipids by
phospholipases (10,11). AA is transformed by a 2-step
reaction into prostaglandin H2 (PGH2) through the
activity of 2 different PGH synthases, named cyclooxygenase 1 (COX-1) and COX-2, which have the same
catalytic activities. In fact, COX-1 and COX-2 possess
both COX and peroxidase active sites. First, AA is
transformed to PGG2 by the COX active site, and then
the peroxidase active site reduces a hydroperoxyl in
PGG2 to a hydroxyl to form PGH2. Subsequently, PGH2
is metabolized by terminal synthases to the biologically
active prostanoids (i.e., prostacyclin [PGI2], PGD2,
PGF2␣, PGE2, and thromboxane A2 [TXA2]) (10,11).
The therapeutic effects of NSAIDs (analgesic and antiinflammatory) are due to inhibition of COX2–dependent prostanoids (mainly PGE2) (12). Thus,
selective COX-2 inhibitors (coxibs) were developed to
reduce the GI toxicity of traditional NSAIDs due, at
least in part, to the inhibition of cytoprotective prostanoids generated in the GI tract by COX-1 (2,12).
However, the use of coxibs unraveled the important role
played by COX-2–dependent prostacyclin in the CV
system (9).
Several lines of evidence suggest that the CV
hazard is also associated with traditional NSAIDs
through the same mechanism (4,9). In fact, traditional
NSAIDs, which are reversible inhibitors of COX-1 and
COX-2, profoundly affect COX-2 in the presence of a
reduction of platelet COX-1 activity (4) (i.e., ⬍95%)
that is insufficient for inhibition of platelet function (13).
Thus, most traditional NSAIDs are selective for COX-2
at therapeutic doses with respect to platelet function (4).
Naproxen is different among NSAIDs because it po-
851
tently inhibits COX-1 and has a long half-life (1), thus
affecting platelet COX-1 profoundly and persistently at
therapeutic doses (14,15). This has been proposed as
one of the mechanisms by which naproxen could have a
better CV safety profile than other traditional NSAIDs,
such as diclofenac (4,5). However, naproxen use in the
general population is not cardioprotective for 2 major
reasons. First, as a reversible inhibitor of COX-1, it is
associated with marked variability in causing complete
(⬎95%) and persistent suppression of the maximal
capacity of platelets to generate TXA2 throughout the
dosing interval (14,15), which is a fundamental requisite
for cardioprotection (16). Second, it can cause a coincident profound inhibition of the vasoprotective COX-2–
dependent PGI2 (14,15).
Due to the harmful or neutral effects of NSAIDs
on the CV system, the coadministration of low-dose
aspirin is recommended in patients with CV disease who
need NSAID therapy (traditional or coxibs) to control
arthritis symptoms (16). Low-dose aspirin is the only
NSAID that has been shown to be cardioprotective due
to its unique mechanism of action. Aspirin irreversibly
acetylates Ser529 of COX-1 and COX-2, leading to
irreversible enzyme inactivation (17). Because of its
unusual pharmacokinetics, low doses of aspirin preferentially inhibit COX-1 in circulating platelets, thereby
suppressing platelet TXA2 synthesis and attendant
thrombosis. However, the coadministration of ibuprofen
has been reported to interfere with the irreversible
inhibition of platelet COX-1 by aspirin, leaving open the
door for a potential impact on aspirin cardioprotection
(18,19).
The presence of a pharmacodynamic interaction
between aspirin and naproxen on platelet COX-1 is
more difficult to detect. In fact, naproxen binding to
COX-1 may on the one hand prevent the irreversible
acetylation of COX-1 by aspirin, while on the other hand
it may have an extended direct inhibitory effect on
COX-1 due to the long half-life of naproxen (⬃17 hours)
(1). In fact, it was shown that the extent of inhibition of
both platelet COX-1 activity and AA-induced platelet
aggregation up to 24 hours after dosing did not differ
between long-term dosing with 500 mg naproxen twice a
day administered 2 hours before or after 100 mg aspirin
and dosing with aspirin alone (20). To detect potential
interference of naproxen with irreversible inhibition of
platelet COX-1 produced by aspirin, we studied the
time-dependent recovery (up to 2 weeks) of serum
TXB2 biosynthesis after coadministration of 1 single
dose of naproxen (500 mg) and low-dose aspirin (100
mg) (20). A rapid recovery of COX-1 activity and
function was found, which is compatible with a pharma-
852
ANZELLOTTI ET AL
codynamic interaction between naproxen and aspirin
(20). A limitation of that study was that the recovery of
COX-1 activity after 1 single dose of aspirin alone was
not evaluated.
Robust evidence is still lacking regarding the
clinical consequences of the pharmacodynamic interaction between aspirin and NSAIDs. Population-based
epidemiologic studies addressing this issue need to be
large to address the potential effect modification with
individual NSAIDs, and they are always open to some
residual confounding bias (4,21–28). Very large randomized clinical trials will be necessary, but they would be
very expensive and pose major ethical dilemmas. Thus, it
is urgent to develop strategies to predict and possibly
minimize the drug–drug interaction.
We performed the present clinical study in
healthy subjects coadministered low-dose aspirin and
naproxen to address 3 questions. What is the appropriate assay to detect a pharmacodynamic interaction between aspirin and a coadministered NSAID? Can the
interference of naproxen with the irreversible inhibition
of platelet COX-1 by aspirin, previously detected with
500 mg naproxen twice a day (20), be reduced by
lowering the daily dose of naproxen? Is the potential
interaction dependent on the sequence of administration of the 2 drugs?
Naproxen is available over the counter only in a
220-mg dose. Thus, we studied the effects of its coadministration before or after aspirin versus aspirin alone
on the degree of inhibition of serum TXB2 generation (a
capacity index of platelet COX-1 activity in response to
thrombin) and platelet aggregation induced by AA or
collagen. In order to develop a suitable biochemical
assay to detect the occurrence of the pharmacodynamic
interaction between aspirin and naproxen, in a subgroup
of individuals (after discontinuing the different treatment schedules) we compared the recovery kinetics of
TXB2 generation in serum and in AA- or collagenstimulated platelet-rich plasma by assessing the slope
and y-intercept values of the least squares line using
simple linear regression analysis.
SUBJECTS AND METHODS
Study subjects. The study protocol was approved by
the Ethics Committee of G. d’Annunzio University–Chieti.
Informed consent was obtained from the 9 healthy subjects
enrolled (5 men [56%]). The subjects were ages 23–37 years,
within 30% of ideal body weight, and had unremarkable
medical histories, physical examination findings, and routine
hematologic and biochemical test results. Smokers and subjects with bleeding disorders, allergies to aspirin or any other
NSAIDs, or a history of any GI, CV, or cerebrovascular
disease were excluded. Subjects abstained from the use of
aspirin and other NSAIDs for at least 2 weeks before enrollment.
Clinical study: design, treatments, and assessment.
We performed a crossover, open-label study to evaluate the
effects of potential interactions between low-dose aspirin (100
mg/day of immediate release aspirin, not enteric coated,
Aspirinetta; Bayer) and naproxen sodium (220 mg twice a day,
Aleve; Roche), coadministered to 9 healthy subjects, on platelet thromboxane biosynthesis ex vivo and in vivo and on
platelet aggregation induced by AA (1 mM and 2 mM) and by
collagen (10 ␮g/ml) ex vivo. Subjects received for 5 days 3
different treatments separated by 14 days of washout, as
follows: 1) naproxen sodium 220 mg twice a day (at 8:00 AM
and 8:00 PM), with the first dose 2 hours before aspirin (100
mg/day at 10:00 AM); 2) 1 dose of aspirin at 8:00 AM 2 hours
before the first dose of naproxen (taken at 10:00 AM and 10:00
PM); and 3) aspirin alone (at 8:00 AM). On day 6, they received
only the morning doses (i.e., aspirin alone at 8:00 AM; naproxen at
8:00 AM [2 hours before aspirin]; or aspirin at 8:00 AM [2 hours
before naproxen]). The dosing sequences were not randomized, but all subjects were receiving the same sequence.
The sequential administration with an interval of 2
hours between the 2 study drugs was chosen to allow aspirin or
naproxen to produce their complete inhibitory effect on platelet COX-1 before the intake of the second drug. In fact, the
time to peak plasma levels of naproxen is ⬃2 hours (1,15).
Despite the fact that the major extent of platelet COX-1
acetylation by low-dose aspirin occurs in the presystemic
circulation, further decline in platelet TXB2 generation may
occur with the appearance of aspirin in blood; thus, at least
1 hour is required for plain aspirin to have a complete
inhibitory effect on circulating platelet COX-1 (29,30).
On days 1 and 6 of each sequence of treatment, the
volunteers were admitted to the clinical center at SS. Annunziata Hospital for blood and urine sample collections and the
administration of the first and the last study drugs. They were
instructed in how to take their medications. Aspirin and
naproxen were provided in 2 distinct boxes (Aspirinetta and
Aleve original packages). On the sixth day of each sequence of
treatment, they returned all boxes for compliance assessment
by pill counts. If the returned boxes were not empty, suggesting
nonadherence to the study protocol, the subjects were excluded from the study. In addition, compliance was monitored
by contacting the subjects by telephone at each time of
administration. After the washout period, new boxes were
provided to subjects. Pill count adherence was 100%, and no
enrolled subject was excluded from the study. Baseline blood
samples were collected on day 1 of each treatment schedule;
then, on day 6, in volunteers with sequential schedules, blood
samples were collected 2, 5, 12, 24, and 48 hours after the
administration of the first drug, while in volunteers taking
aspirin alone, blood samples were collected 1, 24, and 48 hours
after the last dose.
Blood samples were collected to assess inhibition of
serum TXB2 (31) and of platelet aggregation induced by AA
(1 mM and 2 mM) and collagen (10 ␮g/ml) in platelet-rich
plasma (32). Platelet aggregation induced by AA and collagen
was measured in platelet-rich plasma (32) using a Chrono-Log
platelet aggregometer, whereas immunoreactive TXB2 was measured by a previously validated radioimmunoassay technique (31).
For each pharmacologic schedule, urine samples were collected
CONSEQUENCES OF COADMINISTRATION OF NAPROXEN AND ASPIRIN
Table 1. Predrug measurements of serum TXB2, TX-M, and platelet
aggregation in the 9 healthy volunteers*
Serum TXB2, ng/ml
TX-M, pg/mg of creatinine
Platelet aggregation†
AA (1 mM)
AA (2 mM)
Collagen (10 ␮g/ml)
Aspirin
Aspirin
before
naproxen
Naproxen
before
aspirin
315 ⫾ 99
598 ⫾ 124
290 ⫾ 100
510 ⫾ 150
320 ⫾ 130
515 ⫾ 280
93 ⫾ 7
92 ⫾ 5
95 ⫾ 3
92 ⫾ 3
92 ⫾ 4
93 ⫾ 2
93 ⫾ 3
93 ⫾ 3
94 ⫾ 2
* Since predrug values (measured before each pharmacologic treatment) passed the normality test (by the Kolmogorov-Smirnov
method), the data were expressed as the mean ⫾ SD, and statistical
comparisons were made by repeated-measures analysis of variance
followed by the Student-Newman-Keuls test. TXB2 ⫽ thromboxane
B2; TX-M ⫽ 11-dehydro-TXB2; AA ⫽ arachidonic acid.
†Maximal platelet aggregation response (%).
before treatment (overnight) and on day 6 (3 sequential collections after the first study drug was administered [i.e., from 0 to
6 hours, from 6 to 12 hours, and from 12 to 24 hours]) to assess
the urinary excretion of 11-dehydro-TXB2 (TX-M), a major
enzymatic metabolite of TXB2 that is an index of TXA2
biosynthesis in vivo, mainly of platelet origin (33,34).
In a subgroup of 5 volunteers treated with naproxen
sodium 2 hours before aspirin or 2 hours after aspirin, or
treated with low-dose aspirin alone, blood samples were collected up to 72 hours after the administration of the first drug
on day 6 of treatment to assess TXB2 levels in serum and in
platelet-rich plasma in response to AA (1 mM and 2 mM) or
collagen (10 ␮g/ml). This allowed us to calculate and compare
the slope and y-intercept values of the least squares line
obtained by simple linear regression analysis of timedependent recovery of platelet TXB2 generation.
Statistical analysis. Predrug values (measured before
each treatment schedule) of serum TXB2, TX-M, and platelet
aggregation (by AA or collagen) passed the normality test (by
the Kolmogorov-Smirnov method); thus, data were expressed
as the mean ⫾ SD (Table 1), and statistical comparisons were
made by repeated-measures analysis of variance followed by
the Student-Newman-Keuls test using GraphPad Prism software. Due to heterogeneity in response to different treatment
schedules on some occasions, in particular by the sequential
administration of naproxen before aspirin, we analyzed the
pharmacologic results using nonparametric tests. The data
were expressed as the median (range) and geometric mean
(95% confidence interval [95% CI]). The primary hypothesis
was that 220 mg naproxen twice a day (with the first dose
administered 2 hours before aspirin) would interfere with the
irreversible inhibitory effect of aspirin, as assessed by the
measurement of serum TXB2 (primary end point), platelet
aggregation (secondary end point), and urinary excretion of
TX-M (secondary end point) 24 hours after the last administration of naproxen (2 hours before aspirin) on day 6.
Assuming an intersubject coefficient of variation of
25% for serum TXB2 (19), 9 subjects would allow detection of
a difference of 41% between the inhibitory effect of aspirin
alone and the inhibitory effect of its coadministration with
naproxen, with a power of 90%, on the basis of 2-tailed tests,
853
with probability values less than the Type I error rate of 0.05.
The log-transformed values of serum TXB2 and urinary TX-M
concentration and those of percent of maximal aggregation
were subject to nonparametric analysis (19,35). Mixed-effects
models with fixed effects of treatment or time plus random
intercepts for each subject were estimated by restricted maximum likelihood in /R 2.9.0/ (http://cran.r-project.org/bin/
windows/base/old/2.9.0/) by use of the function /lmer/ from
package /lme4/version0.999375-31 (http://cran.uvigo.es/web/
packages/Matrix/Matrix.pdf ). Nonparametric estimates of the
parameters and 2-tailed P values were derived by nonparametric bootstrap resampling of the residuals at each level of
the model by use of 10,000 replicates (35).
The methods used were prespecified. We compared
serum TXB2 levels (primary end point) and AA- and collageninduced platelet aggregation (secondary end point) 24 hours
(and other times) after dosing with predrug values for each
treatment schedule. Moreover, we compared values among the
3 treatment schedules at the same time points after dosing (i.e.,
24 hours and 48 hours). For TX-M, we compared the values
detected at each urine collection after dosing with predrug
values for each treatment schedule and among the 3 treatment
schedules at the same time points.
In 5 volunteers, we assessed the relationship between
the dependent variable (platelet TXB2 generation [ng/ml] in
serum and platelet-rich plasma) and time after discontinuation
Figure 1. Comparison of the degree and duration of steady-state
inhibition of cyclooxygenase 1 (COX-1) activity by administration for
6 days of naproxen sodium (220 mg twice a day) 2 hours before aspirin,
naproxen sodium 2 hours after aspirin, or low-dose aspirin alone.
Platelet COX-1 activity ex vivo (reported as the percent of inhibition
[% I]), as assessed by the measurement of serum thromboxane B2
(TXB2), was evaluated in 9 healthy subjects. Data are presented as box
plots, where the boxes represent the 25th to 75th percentiles, the lines
within the boxes represent the median, and whiskers represent the
highest and lowest values. Open symbols represent individual values.
At each time point after dosing with the 3 different treatments, serum
TXB2 was significantly reduced compared with predrug values (ⴱⴱ ⫽
P ⫽ 0.0001). § ⫽ P ⫽ 0.0007 versus aspirin alone at 24 hours. † ⫽ P ⫽
0.0045 versus aspirin before naproxen at 24 hours. # ⫽ P ⫽ 0.0011
versus aspirin alone at 48 hours. For this statistical analysis we used
mixed-effects model procedures and nonparametric bootstrap resampling technique (35). WO ⫽ washout.
854
of the different treatment schedules by linear regression
analysis using Prism software. Since the variance of the outcome of TXB2 generation was not constant across the range of
the predictor (time), we conducted linear regression (least
squares) analysis with log10 transformation of the outcomes
using Prism software. We calculated slope and y-intercept
values (when x ⫽ 0), and their 95% CIs, of the least squares
lines as well as the coefficient of determination (r2). Comparisons of slope and y-intercept values of linear regression lines
among different treatment schedules were further assessed
using Prism software. P values less than 0.05 were considered
significant.
ANZELLOTTI ET AL
RESULTS
Predrug (baseline) values of serum TXB2, urinary TX-M, and maximal platelet aggregation response
(percent) detected before the start of each treatment
schedule did not differ significantly among the 3 different occasions (Table 1). These results show that the
washout period (14 days) between treatments was long
enough to bring the inhibition back to the pretreatment
values.
The long-term administration of low-dose aspirin
Figure 2. Inhibition of platelet function ex vivo by administration for 6 days of naproxen sodium (220 mg twice a day) 2 hours before aspirin,
naproxen sodium 2 hours after aspirin, or low-dose aspirin alone. Platelet aggregation was assessed by measuring the percent of inhibition (% I).
Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and whiskers
represent the highest and lowest values. Open symbols represent individual values. A, Platelet aggregation induced by 2 mM arachidonic acid (AA).
At each time point after dosing with the 3 different treatments, platelet aggregation was significantly reduced compared with predrug values (ⴱⴱ ⫽
P ⫽ 0.0001). # ⫽ P ⫽ 0.0053 versus aspirin alone at 48 hours. B, Platelet aggregation induced by 1 mM AA. At each time point after dosing with
the 3 different treatments, platelet aggregation was significantly reduced compared with predrug values (ⴱⴱ ⫽ P ⫽ 0.0001). § ⫽ P ⫽ 0.0001 versus
aspirin before naproxen at 24 hours. # ⫽ P ⫽ 0.016 versus aspirin alone at 48 hours. † ⫽ P ⫽ 0.0003 versus aspirin alone at 24 hours. ⌽ ⫽ P ⫽
0.04 versus aspirin alone at 48 hours. C, Platelet aggregation induced by 10 ␮g/ml collagen. ⴱⴱ ⫽ P ⫽ 0.0001 versus predrug values. ⌽ ⫽ P ⫽ 0.0005
versus aspirin alone at 24 hours. † ⫽ P ⫽ 0.0013 versus aspirin alone at 48 hours. # ⫽ P ⫽ 0.0045 versus predrug values. § ⫽ P ⫽ 0.01 versus predrug
values. @ ⫽ P ⫽ 0.0005 versus aspirin alone at 48 hours. ⴱ ⫽ P ⫽ 0.0003 versus predrug values. For this statistical analysis we used mixed-effects
model procedures and nonparametric bootstrap resampling technique (35). WO ⫽ washout.
CONSEQUENCES OF COADMINISTRATION OF NAPROXEN AND ASPIRIN
caused an almost complete inhibition of platelet COX-1
activity 1, 24, and 48 hours after dosing (at 1 hour,
median 99.9% [range 99.5–100.0%], geometric mean
99.8% [95% CI 99.7–99.9%]; at 24 hours, median 99.1%
[range 97.4–99.4%], geometric mean 98.9% [95% CI
98.4–99.4%]; at 48 hours, median 95.5% [range 90.9–
98.5%], geometric mean 95.7% [95% CI 93.9–97.7%])
(P ⫽ 0.0001 versus baseline) (Figure 1). At the primary
end point, 24 hours after the administration on day 6 of
naproxen 2 hours before aspirin, serum TXB2 inhibition
(median 98.0% [range 90.6–99.4%], geometric mean
95.9% [95% CI 93.4–98.8%]) was significantly lower
than that detected after aspirin alone (P ⫽ 0.0007) or
aspirin given 2 hours before naproxen (median 99.1%
[range 98.0–99.7%], geometric mean 98.9% [95% CI
98.4–99.5%]) (P ⫽ 0.0045) (Figure 1). Forty-eight hours
after the administration of naproxen 2 hours before
aspirin, serum TXB2 inhibition (median 90.2% [range
76.8–98.2%], geometric mean 89.5% [95% CI 84.2–
95.2%]) was significantly lower than that detected after
aspirin alone (P ⫽ 0.0011), but not significantly lower
than that detected after the administration of aspirin 2
hours before naproxen (median 93.9% [range 89.3–
98.0%], geometric mean 94.0% [95% CI 92.1–96.0%])
assessed at the same time point (Figure 1). Compared
with administration of aspirin alone, administration of
aspirin 2 hours before naproxen did not cause a significant reduction of serum TXB2 both 24 hours and 48
hours after dosing (Figure 1).
At each time point after dosing with 3 different
treatments, platelet aggregation induced by AA (1 mM
and 2 mM) was significantly reduced compared with
predrug values for each pharmacologic schedule (P ⫽
0.0001) (Figures 2A and B). Compared with administration of aspirin alone, the administration of naproxen (2
hours before or after aspirin) did not significantly affect
2 mM AA–induced platelet aggregation up to 24 hours
after dosing (Figure 2A). Forty-eight hours after dosing,
a statistically significant difference in platelet function
response was only found between the administration of
naproxen before aspirin and the administration of aspirin alone (Figure 2A). However, the administration of
naproxen 2 hours after aspirin also perturbed the homogeneous suppression of 2 mM AA–induced platelet
aggregation detected in all subjects receiving aspirin
alone. In fact, in 2 of 9 subjects, a complete recovery of
platelet aggregation was detected at 48 hours (Figure
2A). Similar results were found for 1 mM AA–induced
platelet aggregation (Figure 2B).
As shown in Figure 2C, low-dose aspirin alone
caused a significant inhibition of collagen-induced platelet aggregation up to 48 hours after dosing, although
855
heterogeneity of the response was detected. In contrast,
collagen-induced platelet aggregation was rapidly recovered after the sequential administration of aspirin and
naproxen (in both directions). Twenty-four and 48 hours
after dosing with naproxen administered 2 hours before
or after aspirin, collagen-induced platelet aggregation
recovered to predrug values, and the degree of inhibition was significantly lower than that detected after
aspirin alone at the same time points (Figure 2C).
The degree and duration of steady-state inhibition of the urinary excretion of TX-M, an index of TXA2
biosynthesis in vivo (34), was evaluated after the different treatments. As shown in Figure 3, aspirin alone
profoundly reduced urinary TX-M levels. The sequential
administration of naproxen and aspirin (in both directions) did not substantially affect the inhibition caused
by aspirin alone. However, compared with administration of aspirin alone, we detected a slightly higher
inhibition in urine samples collected 6–12 hours after
dosing with aspirin given 2 hours before naproxen (P ⫽
Figure 3. Comparison of the degree and duration of steady-state
inhibition of thromboxane A2 (TXA2) biosynthesis in vivo by administration for 6 days of naproxen sodium (220 mg twice a day) 2 hours
before aspirin, naproxen sodium 2 hours after aspirin, or low-dose
aspirin alone. Urinary excretion of 11-dehydro-TXB2 (TX-M; an index
of TXA2 biosynthesis in vivo), reported as the percent of inhibition
(% I), was detected after dosing with the 3 different treatments. Data
are presented as box plots, where the boxes represent the 25th to 75th
percentiles, the lines within the boxes represent the median, and
whiskers represent the highest and lowest values. Open symbols
represent individual values. At each urine collection obtained after
dosing with the 3 different treatments, urinary excretion of TX-M was
significantly reduced compared with predrug values (ⴱⴱ ⫽ P ⫽ 0.0001).
§ ⫽ P ⫽ 0.049 versus aspirin alone at 12–24 hours. # ⫽ P ⫽ 0.037
versus aspirin alone at 6–12 hours. For this statistical analysis we used
mixed-effects model procedures and nonparametric bootstrap resampling technique (35). WO ⫽ washout.
856
ANZELLOTTI ET AL
Figure 4. Comparison of thromboxane B2 (TXB2) recovery in serum (A) or in platelet-rich
plasma stimulated with 1 mM arachidonic acid (AA) (B), 2 mM AA (C), or 10 ␮g/ml
collagen (D) up to 72 hours after discontinuation of treatment with naproxen sodium (220
mg twice a day) 2 hours before aspirin (Œ), naproxen sodium 2 hours after aspirin (f), or
low-dose aspirin alone (F) in 5 healthy volunteers. The least squares lines of log10
transformation of TXB2 values were obtained using simple linear regression analysis with
Prism software. All values presented were transformed back to the original scale.
0.037) and 12–24 hours after dosing with naproxen given
2 hours before aspirin (P ⫽ 0.049).
To gain evidence that naproxen interfered with
the irreversible inhibition of platelet COX-1 by aspirin,
in a subgroup of 5 volunteers we compared the rate of
biosynthesis of TXB2 in platelet-rich plasma stimulated
with AA and collagen and in serum up to 72 hours after
discontinuation of the different treatments by assessing
the slope and y-intercept values of the least squares lines
(using simple linear regression analysis) describing the
relationship between TXB2 biosynthesis and time (Figure 4). After discontinuation of the different treatment
schedules, the slopes of linear regression lines of timedependent recovery of TXB2 biosynthesis were not
significantly different. However, compared with administration of aspirin alone, the sequential administration
of naproxen before or after aspirin caused a significant
parallel upward shift of the regression lines (higher when
naproxen was administered 2 hours before aspirin than
in reverse order), as shown by increasing y-intercept
values (Figure 4 and Table 2).
DISCUSSION
NSAIDs are efficacious in reducing inflammation
and pain. However, several studies have shown that their
use is associated with a modest increase in CV risk
(4,6,36). Among them, naproxen has been shown to have
a better CV safety profile (5,14,36), presumably on the
basis of its pharmacodynamic and pharmacokinetic features (1,14,15) (i.e., its potent and persistent inhibitory
effect on platelet COX-1 activity [14,15] due to its long
half-life [1]). However, its use is associated with increased risk of GI bleeding and perforation (3); thus, it
is recommended to use the lowest effective dose (3). We
have recently shown that the administration of low-dose
naproxen in the absence of aspirin is associated with
marked variability in the inhibition of platelet COX-1
activity (15). Thus, like any other NSAID, it should be
coadministered with low-dose aspirin in patients with
arthritis and CV disease (16). However, the coadministration of naproxen might interfere with the irreversible
inhibition of platelet COX-1 by aspirin. We performed
the present study to shed some light on the determinants
of a pharmacodynamic interaction between low-dose
aspirin and low-dose naproxen sodium (220 mg twice a
day) in healthy subjects. In particular, we aimed to
characterize a biochemical model to detect the occurrence of this phenomenon.
Our results showed that the administration of
low-dose naproxen 2 hours before aspirin disturbed the
action of aspirin on platelet COX-1. However, 24 hours
after the last dosing on day 6, serum TXB2 inhibition
CONSEQUENCES OF COADMINISTRATION OF NAPROXEN AND ASPIRIN
857
Table 2. Recovery of thromboxane B2 biosynthesis in serum and platelet-rich plasma stimulated with AA or collagen up to 72 hours after
discontinuation of long-term dosing with aspirin alone, aspirin 2 hours before naproxen, and naproxen 2 hours before aspirin*
Aspirin
y-intercept
Serum
Platelet-rich plasma
AA (1 mM)
AA (2 mM)
Collagen (10 ␮g/ml)
95% CI
Aspirin before naproxen
2
r
P†
y-intercept
95% CI
2
r
Naproxen before aspirin
P†
y-intercept
95% CI
r2
P†
2.15‡§
1.57–2.96 0.93 ⬍0.0001
3.02§
2.35–3.90 0.89 ⬍0.0001
7.03
5.43–9.11
0.86 ⬍0.0001
0.57§¶
0.91§**
1.03§‡‡
0.23–1.38 0.76 ⬍0.0001
0.57–1.46 0.95 ⬍0.0001
0.33–3.21 0.32
0.009
0.73#
1.31††
3.73§§
0.41–1.31 0.82 ⬍0.0001
0.87–1.97 0.92 ⬍0.0001
2.80–4.98 0.88 ⬍0.0001
1.54
3.44
7.82
0.95–2.51
1.78–6.63
4.63–13.2
0.88 ⬍0.0001
0.80 ⬍0.0001
0.69 ⬍0.0001
* Y-intercept values and 95% confidence intervals (95% CIs) of the least squares lines and the coefficient of determination (r2) were calculated by
linear regression analysis using Prism software. Data were log10-transformed before linear regression analysis. Shown are the antilog of the
y-intercept (when x ⫽ 0) and its 95% CI. AA ⫽ arachidonic acid.
† Describes the probability that randomly selected points would result in a regression line.
‡ P ⫽ 0.013 versus aspirin before naproxen.
§ P ⬍ 0.0001 versus naproxen before aspirin.
¶ P ⫽ 0.028 versus aspirin before naproxen.
# P ⫽ 0.0007 versus naproxen before aspirin.
** P ⫽ 0.0025 versus aspirin before naproxen.
†† P ⫽ 0.002 versus naproxen before aspirin.
‡‡ P ⬍ 0.0001 versus aspirin before naproxen.
§§ P ⫽ 0.0004 versus naproxen before aspirin.
(the primary end point) was only slightly, even if significantly, lower than that found when naproxen was given
2 hours after aspirin or when aspirin alone was given
(Figure 1). Compared with administration of aspirin
alone, this effect did not translate into detectable alterations of AA-induced platelet aggregation (secondary
end point) up to 24 hours after the last dosing. However,
48 hours after sequential administration of naproxen
and aspirin, a heterogeneous response to AA-induced
platelet aggregation was found in association with serum
TXB2 inhibition ⬍95%. These results strengthen the
concept that serum TXB2 inhibition ⬎95% is necessary
to inhibit AA-induced platelet aggregation in all individuals (37,38) (for further details, please contact the
corresponding author). In contrast, when serum TXB2
inhibition is ⬍95%, the subjects are divided into 2
groups, one with full inhibition and the other with no
inhibition of AA-induced platelet aggregation (37,38)
(for further details, please contact the corresponding
author).
Platelet functional consequences of the disturbance of COX-1 inhibition caused by the sequential
administration of naproxen and aspirin (in both directions) were more evident when we studied platelet
aggregation induced by collagen rather than by AA
(secondary end points). This is explained by the finding
that low concentrations of TXA2 synergize with collagen
to induce full platelet aggregation (39).
In a subgroup of individuals, after discontinuing
the different treatment schedules, we compared the
recovery kinetics of TXB2 generation in serum and in
AA- or collagen-stimulated platelet-rich plasma by assessing the slope and y-intercept values of the least
squares line using simple linear regression analysis. In all
these biochemical systems, compared with administration of aspirin alone, the administration of naproxen 2
hours before or after aspirin was associated with a
significant parallel upward shift of the regression lines.
This effect was more pronounced when naproxen was
given 2 hours before aspirin. Taken together, these
results may be consistent with a reduced degree of
COX-1 acetylation by sequential dosing of naproxen and
aspirin compared with dosing with aspirin alone. Since
aspirin has a short half-life (⬃20 minutes) (17,40), the
occupation of the COX-1 active site by naproxen may
delay the binding of aspirin to COX-1, thus reducing its
capacity to cause an irreversible inhibition of platelet
TXB2 generation.
Since the interaction was less pronounced when
aspirin was administered 2 hours before naproxen than
in the reverse order, it would be reasonable to give
naproxen at least 1 hour after plain aspirin. This allows
aspirin to produce a complete inhibition of platelet
COX-1, in presystemic and systemic circulation, before
the administration of naproxen. For enteric-coated aspirin, which produces a delayed onset of COX-1 inhibition due to its slow releasing property (40), a longer
period of time would be required before the intake of
naproxen. Furthermore, it is likely that the interference
of naproxen with the irreversible inhibition of platelet
COX-1 by enteric-coated aspirin is more substantial. In
fact, it has been shown that low doses of enteric-coated
858
aspirin may be less bioavailable and thus prove insufficient in heavier patients with a large volume of distribution (38). Indeed, there are data showing that inhibition
of platelet function with 75 mg enteric-coated aspirin
daily may be incomplete in many patients with CV
disease (i.e., particularly in heavier and younger patients
and those with a history of MI) (38).
Further studies are needed to verify the possibility of varying the time-dependent sequence of administration based on, for example, the patient’s age, other
disease states, and genetic background that might affect
the drug pharmacokinetic and pharmacodynamic features. Moreover, a time course would be needed in
elderly patients at high risk of CV disease to accurately
assess such time points after dosing with different aspirin
preparations, such as plain and enteric coated.
One of the objectives of the present study was to
develop a suitable biochemical assay that could be used
in further studies of clinical pharmacology to detect the
occurrence of the pharmacodynamic interaction between aspirin and naproxen. For this purpose we compared different biomarkers of COX-1 inhibition, such as
TXB2 generation in serum and in AA- or collagenstimulated platelet-rich plasma. We showed that the
assessment of TXB2 generation in the presence of 10
␮g/ml collagen was more appropriate than the other
biomarkers to detect a pharmacodynamic interaction
between aspirin and naproxen. Thus, the assessment of
TXB2 generation in collagen-stimulated platelet-rich
plasma seems more sensitive than its assessment in
serum or AA-stimulated platelet-rich plasma for detecting small changes in COX-1 inhibition. COX-1 activity is
regulated by the concentration of AA released close to
intracellular enzyme localization and by the levels of
cellular peroxides (41). In platelets, collagen, but not
thrombin, has been shown to produce H2O2 (42), which
is able to activate COX-1 (41). Whether this collagendependent signaling may play a role in our findings will
be verified in further studies.
Our results suggest that the sequential administration of naproxen reduces the capacity of aspirin to
cause an almost complete irreversible inactivation of
COX-1. However, we showed that compared with administration of aspirin alone, the administration of aspirin 2 hours before naproxen did not significantly affect
the inhibition of platelet COX-1 activity 24 hours after
dosing. Similarly, AA-induced platelet aggregation was
profoundly inhibited up to 24 hours after dosing with
this sequential drug regimen. Also, collagen-induced
platelet aggregation was significantly inhibited up to 12
hours, and the reduction of urinary excretion of TX-M
by aspirin was not dampened. However, we detected a
ANZELLOTTI ET AL
slightly higher inhibition in urine samples collected 6–12
hours after dosing. These data may suggest a small
contribution of extraplatelet sources to TXA2 biosynthesis in vivo. Taken together, we suggest that the administration of low-dose aspirin 2 hours before low-dose
naproxen should not translate into a significant interference with aspirin cardioprotection. Few observational
studies have examined the impact of the potential interaction of concomitant use of low-dose aspirin and
NSAIDs on the risk of CV events (4,21–28). Two studies
have evaluated the clinical interaction among users of
naproxen and low-dose aspirin (4,25), one showing no
effect and the other reporting an estimate of risk compatible with a small (although not statistically significant) reduction in the cardioprotection afforded by
low-dose aspirin.
In conclusion, sequential dosing of 220 mg
naproxen twice a day and low-dose aspirin interfered
with the irreversible inhibition of platelet COX-1 afforded by aspirin. The interaction was smaller when
naproxen was given 2 hours after low-dose aspirin. The
clinical consequences of the various schedules of the
administration of naproxen with low-dose aspirin are not
yet known and remain to be studied in a randomized
clinical trial with preidentified CV end points.
ACKNOWLEDGMENTS
We thank the medical students of G. d’Annunzio
University and the personnel of the Blood Transfusion Center
of SS. Annunziata Hospital for their generous cooperation.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Patrignani had full access to all
of the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Anzellotti, Capone, Patrignani.
Acquisition of data. Anzellotti, Capone, Tacconelli, Bruno, Tontodonati, Di Francesco, Grossi, Renda, Di Gregorio.
Analysis and interpretation of data. Anzellotti, Capone, Jeyam, Tacconelli, Bruno, Merciaro, Price, Garcia Rodriguez, Patrignani.
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