The aim of the current study is to evaluate the antiplatelet effect of dexibuprofen in healthy volunteers in comparison with low-dose aspirin.
Healthy volunteers (n = 12) were treated in a crossover manner with 100 mg daily aspirin or with 800 mg daily dexibuprofen. Blood samples were obtained within 24 h; 3, 7, and 14 days after repeated doses; and 24 h after the last dose. In each sample, the authors measured platelet aggregation, thromboxane B2, 6-keto-prostaglandin F1alpha, and nitric oxide.
The antiplatelet effect of dexibuprofen (maximal inhibition of aggregation was 48-55% for adenosine diphosphate and 90-95% for collagen and arachidonic acid) was equal to the effect of aspirin. The main difference between the two drugs was in the degree of recovery of platelet function. The effect of aspirin persisted for 24 h after the last dose (remaining inhibition 50%, respect to the pretreatment value), whereas platelet aggregation had returned to baseline pretreatment values within 24 h after dexibuprofen was stopped.
Both aspirin and dexibuprofen inhibited platelet function with a similar intensity, but dexibuprofen exerted a reversible effect for 24 h after the last dose.
PATIENTS who interrupt chronic treatment with antiplatelet drugs (especially aspirin) aimed at preventing thrombotic events have an increased incidence of ischemic accidents related with the so-called rebound effect.1–3The reported interval between withdrawal of aspirin and thrombotic events is 14.3 days for acute cerebral events, 8.5 days for acute coronary syndromes, and 25.8 days for acute peripheral arterial syndromes.4
One set of circumstances under which antiplatelet treatment may be interrupted is preparation for surgery.5In patients scheduled to undergo surgery, antiplatelet drugs are usually discontinued 7–10 days before the procedure. Under these circumstances, the patient may be exposed to an increased risk of thrombotic events.1–4Other drugs that inhibit platelet function are nonsteroid antiinflammatory drugs (NSAIDs). However, in patients it is important to consider some limitations referring to the dose and duration of this option because an increase has been described in cardiovascular events in patients treated with selective cyclooxygenase-2 inhibitors (average duration of treatment, 39–40 days) or high doses (ibuprofen ≥ 1,200 mg/day) of nonselective cyclooxygenase inhibitors (average duration of treatment with ibuprofen, 37 days).6Despite the inhibition of platelet aggregation, it has not been demonstrated that NSAIDs exert a cardiovascular protective effect such as aspirin does.
Ibuprofen exerts its antiplatelet aggregation effects by blocking the cyclooxygenase enzyme in a reversible manner.7,8However, ibuprofen is a racemic mixture of two enantiomers, S (+)-ibuprofen—the active part—and R (−)-ibuprofen, which is inactive.9After the administration of ibuprofen, its two enantiomers undergo chiral transformation, but this process is inconsistent and is related with diminished efficacy and homogeneity of its pharmacologic effects.10,11One way to circumvent this drawback is to use the active form S (+)-ibuprofen, designated dexibuprofen for clinical use.12,13
The aim of the current study was to evaluate the antiplatelet effect of dexibuprofen in healthy volunteers and to compare this effect with aspirin at doses used in prophylaxis against thrombosis.
Materials and Methods
The participants in this study were 12 healthy volunteers (6 men, 6 women; mean age, 35 ± 3 yr) who had not taken any medication during the 15 days before their inclusion. When a volunteer gave informed consent to participate, he or she was randomly assigned with the help of a table of random numbers to receive one of the two drugs. The study was approved by the Clinical Assays Committee of the Hospital Clínico Universitario Virgen de la Victoria in Malaga, Spain.
The participants were divided into two groups. Volunteers in one group received a single dose of 100 mg plain-formulation aspirin, and those in the other group received a single dose of 400 mg of S (+)-ibuprofen (dexibuprofen). On the first day of the study, serial blood samples were obtained for the acute experiments. From days 2–14, aspirin (100 × 1 mg/day) and dexibuprofen (400 × 2 mg/day) were administered. Both drugs were administered 30 min before breakfast, and dexibuprofen was also given 30 min before dinner. The last dose of 100 mg aspirin or 400 mg dexibuprofen was taken on the morning of day 14, 2 h after a blood sample was obtained, and 24 h after this the last blood sample was obtained. In the second phase of the clinical study, after a 1-month washout period, all volunteers were crossed over to the treatment and protocol of the first phase (duration, doses, blood samples).
Blood samples were obtained from each volunteer before the drug was taken (baseline); 1, 2, 4, 8, 12, and 24 h after the first dose; 3, 7, and 14 days after repeated doses (2 h after the dose); and 24 h after the last dose. Blood samples were obtained in the morning (between 8:00 and 8:30 am) before the participants had had anything to eat or drink, and the subjects abstained from eating or drinking (except for up to 200 ml water) until after the 2-h baseline blood sample was obtained.
Aspirin was administered as 100 mg Adiro® (Bayer, Barcelona, Spain), and dexibuprofen was administered as 400 mg Seractil® (Gebro Pharma S.A., Barcelona, Spain). All reagents were from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Platelet Function Parameters.
The main indicator of platelet function was maximum intensity of platelet aggregation induced by adenosine diphosphate (ADP), collagen, or arachidonic acid in whole blood, as measured by electrical impedance14with a Chrono-Log 540S aggregometer (Chrono-Log Corp., Haverton, PA). Sodium citrate (3.8%) at a proportion of 1:10 was used as the anticoagulant for samples of whole blood. The samples were incubated for 5 min at 37°C, and then 2.5 μm ADP, 1 μg/ml collagen, or 400 μm arachidonic acid (Menarini Diagnóstica SA, Barcelona, Spain) was added. The changes in electrical impedance were recorded, and maximum intensity of aggregation was considered the maximum change in impedance 10 min after the inducing agent was added.
The production of thromboxane A2was measured as the synthesis of its metabolite thromboxane B2. We used samples of whole blood collected with 3.8% sodium citrate at 1:10 as the anticoagulant. Samples were incubated for 5 min at 37°C, then 1 μm calcium ionophore A23187 was added, and the samples were shaken for 5 min. Then, the samples were centrifuged at 10,000g for 3 min, and the supernatant was frozen at −80°C until analysis for thromboxane B2by enzyme immunoassay (Oxford Biomedical Research Inc., Oxford, MI).
Prostacyclin production was measured as the synthesis of its stable metabolite 6-keto-prostaglandin F1α. We used samples of whole blood with 3.8% sodium citrate at 1:10 as the anticoagulant. Samples were incubated for 5 min at 37°C, then 1 μm calcium ionophore A23187 was added, and the samples were shaken for 5 min. The samples were centrifuged at 10,000g for 15 min at 4°C, and samples of plasma were filtered through Ultrafree-MC microcentrifuge filters (Millipore, Gif-sur-Yvette, France) to remove hemoglobin from cell lysis. Other samples were frozen at −80°C until analysis for 6-keto-prostaglandin F1αby enzyme immunoassay (Oxford Biomedical Research Inc.).
Nitric Oxide Production.
An electrochemical method was used to quantify nitric oxide production15in neutrophil concentrates. Leukocytes were separated on a Ficoll density gradient (Hystopaque 1077 and Hystopaque 1119) and resuspended (final count: 5.2 ± 0.2 × 109leukocytes) in Ca2+physiologic saline solution that contained 140 mm NaCl, 4.6 nm KCl, 2 mm CaCl2, 1 mm MgCl2, 5 mm glucose, and 10 mm HEPES, pH 7.4. The sample was resuspended in Ca2+saline solution (95% neutrophils, 95% viability as judged by trypan blue exclusion) to a final neutrophil count of 3.4 ± 0.3 × 109neutrophils/l. The sample was then divided into aliquots, and an ISO-NO electrode for nitric oxide determinations (World Precision Instruments, Aston, Stevenage, Hertfordshire, United Kingdom) was used to measure nitric oxide production. All measurements were made at 37°C. Baseline nitric oxide production was recorded, then 1 μm calcium ionophore A23187 was added, and the increase in nitric oxide production induced by activation of constitutive or calcium-dependent nitric oxide synthase was recorded.
All data in the text, tables, and figures are the mean ± SD of the results in each group. The data were analyzed with version 12.0 of the SPSSx program for Windows (SPSS Co., Chicago, IL). Data for both the initial and crossover phases of the study were transformed into percentage values of change with respect to the baseline values. Once normal distribution of the data had been confirmed through the homogeneity of variances, the percentage values for the two treatment groups were compared with the Student t test for paired samples. Differences with P < 0.05 (two-tailed) were considered significant.
Absolute values for clinical variables and laboratory tests in the baseline blood samples (before medication) are shown in table 1. There were no significant differences in any of the data between the two groups.
Results after 24 Hours
Both aspirin and dexibuprofen significantly inhibited platelet aggregation induced in whole blood with ADP, collagen, and arachidonic acid (fig. 1). Maximal inhibition of aggregation was seen 8 h after the administration of aspirin (inhibition was 65% with collagen and 79% with arachidonic acid) and 2 h after administration when ADP was the inducer (47% inhibition). When dexibuprofen was given, maximal inhibition of aggregation was seen 2 h after administration (40% with ADP, 62% with collagen, and 69% with arachidonic acid). As table 2shows, dexibuprofen inhibited platelet aggregation after the first dose significantly earlier than aspirin. Twenty-four hours after the first dose of dexibuprofen, platelet aggregation had returned to baseline values, but after aspirin, platelet aggregation after 24 h remained inhibited almost to the extent observed after 12 h.
Platelet production of thromboxane B2showed a time-dependent decrease in both treatment groups (fig. 2). Maximum inhibition was similar in both groups (63% with aspirin and 62% with dexibuprofen), but the effect of dexibuprofen was observed earlier than aspirin (table 2). In volunteers who took aspirin, thromboxane B2production 24 h after the dose remained inhibited by 50%, whereas in the dexibuprofen group, thromboxane B2production had returned to pretreatment values by this time.
In the aspirin group, synthesis of 6-keto-prostaglandin F1αwas inhibited in a time-dependent manner, and maximal inhibition (58–60% with respect to the pretreatment value) was seen 4–8 h after administration (fig. 2). However, maximal inhibition in the dexibuprofen group (58% with respect to the pretreatment value) was seen 4 h after administration. The inhibition rates showed no significant differences between groups in the times to maximal effect, although the effect of dexibuprofen was statistically different from aspirin 6 h after administration.
When neutrophil nitric oxide production was induced via a calcium-dependent pathway (fig. 2), we found a significant increase in nitric oxide production in both groups, although the increase appeared earlier after dexibuprofen treatment than after aspirin (table 2). After 60 min, maximal increase was 148% in the aspirin group and 147% in the dexibuprofen group with respect to the pretreatment value. After 24 h, nitric oxide production in both groups had returned to baseline values.
Results after Chronic Treatment (14 Days)
Both dexibuprofen and aspirin significantly inhibited platelet aggregation induced in whole blood by ADP, collagen, and arachidonic acid, with no statistically significant differences between aspirin and dexibuprofen. Maximal inhibition of aggregation was 48–55% for ADP and 90–95% for collagen and arachidonic acid after the second dose of treatment on day 3 (fig. 3).
The inhibition of platelet production of thromboxane B2was similar (80–90%) in both treatment groups (fig. 4). However, plasma levels of 6-keto-prostaglandin F1αshowed 65–75% inhibition after aspirin administration and 80–90% inhibition after dexibuprofen (fig. 4).
Nitric oxide production increased in both groups after the first dose of treatment, but the effect 24 h after administration was significantly higher in the aspirin group.
Twenty-four hours after the last dose, all parameters in the dexibuprofen group had returned to pretreatment values, whereas in the aspirin group, they remained significantly modified (fig. 3 and 4).
To compare the antiplatelet effect of dexibuprofen with aspirin, we consider two aspects of its pharmacologic profile: (1) the similarities in the antiplatelet mechanisms and intensity of inhibition of dexibuprofen and (2) the possible reversible nature of this effect in humans. Maximal inhibition of platelet aggregation was similar to that produced by aspirin, with no significant differences after the first dose or after chronic treatment for 14 days. In quantitative terms, the intensity of inhibition of platelet aggregation with dexibuprofen was similar to that attained with low doses of aspirin. In both cases, inhibition reached approximately 50% of the maximum value 1 h after the first dose, with maximal inhibition appearing after 2–4 h.
In biochemical terms, the action of aspirin was consistent with its well-known mechanism of antiplatelet action, i.e. , irreversible inhibition of cyclooxygenase and increased nitric oxide production.7,16,17–19This finding is compatible with the mechanism of platelet aggregation induced by ADP, which results from the inhibition of adenylate cyclase activity and the decrease in intraplatelet concentrations of cyclic adenosine monophosphate, rather than the stimulation of thromboxane synthesis. The behavior of dexibuprofen was similar to that of aspirin, in that its effect was greater when collagen or arachidonic acid was used as the inducer than when ADP was used. This is unsurprising because dexibuprofen inhibits platelet cyclooxygenase activity and thus thromboxane synthesis.8
Both drugs led to cyclooxygenase inhibition as a result of the inhibition of thromboxane and prostacyclin synthesis, but the results differed somewhat after an initial dose and after treatment for 14 days. The degree of inhibition seen after the initial dose was similar with both drugs, whereas after chronic treatment, the percentage inhibition of leukocyte synthesis of prostacyclin was greater with dexibuprofen than with aspirin. The difference in behavior may reflect the inhibition by dexibuprofen of type 2 (or inducible) cyclooxygenase 2,20,21which has been shown to control, in part, prostacyclin synthesis by leukocytes.22The concentration of dexibuprofen in plasma after a single dose may be insufficient to inhibit cyclooxygenase 2, although the effect may become evident after repeated doses. The effect of aspirin on cyclooxygenase 2 is known to be one third to one fourth as potent as its effect on cyclooxygenase 1,23a difference that may account for our findings.
An imbalance in the inhibition of the thromboxane/prostacyclin ratio is considered an important point in the mechanism of the antithrombotic effect of low-dose aspirin by a higher reduction in thromboxane than in prostacyclin synthesis. From the results obtained in our study, this ratio for aspirin was 1.1 ± 0.2 with single-dose administration and 1.3 ± 0.2 with 14-day treatment, and for dexibuprofen, this ratio was 1.3 ± 0.1 and 1.2 ± 0.1, respectively. However, it is important to consider the limitation of the measurement of the synthesis of prostacyclin as plasma 6-keto-PGF1αconcentration, instead of urinary prostanoid determination; further studies must be conducted to confirm the effect of dexibuprofen on prostacyclin production.
The increase in leukocyte nitric oxide production by both aspirin and dexibuprofen is a mechanism that enhances the inhibition of platelet aggregation as a result of the inhibition of platelet cyclooxygenase. With both drugs, this mechanism stimulates leukocyte nitric oxide synthesis via the constitutive pathway (i.e. , via a calcium-dependent mechanism), and the nitric oxide thus produced further inhibits platelet functioning. In this connection, is has been shown that the effect of aspirin and ibuprofen on platelet nitric oxide synthesis is independent of cyclooxygenase and may be a result of the stimulation of constitutive nitric oxide synthase.7,16
With respect to the reversibility of its effect on platelet aggregation, our analyses of the drugs' performance after a single dose and after treatment for 14 days showed that this is where the differences between aspirin and ibuprofen were greatest. Between 12 and 24 h after the last dose of dexibuprofen, its inhibitory effect on platelet aggregation and of thromboxane and prostacyclin synthesis had reverted completely, whereas with aspirin, some of the effect persisted longer. This well-known effect of aspirin is the result of its irreversible blockage of cyclooxygenase, which must be synthesized de novo to recover baseline values. In platelets, however, blockage of cyclooxygenase is irreversible because these elements lack a nucleus and are thus unable to synthesize the enzyme de novo .24Nevertheless, prostacyclin synthesis recovered more fully than other markers of platelet inhibition after aspirin treatment, possibly because leukocytes and endothelial cells are able to rapidly synthesize cyclooxygenase.25Nitric oxide production recovers to a similar degree after treatment with both drugs, a finding that supports the notion that the effect of aspirin and ibuprofen on this activity is independent of their ability to block cyclooxygenase synthesis.7,16
Ibuprofen is a racemic mixture of S (+)-ibuprofen, which is active, and R (−)-ibuprofen, which is not. This makes it necessary to use higher doses to achieve the same effect as dexibuprofen.9Moreover, transformation of one isomer into the other after the oral intake of ibuprofen may make its effects inconsistent or hard to predict.10,11In this connection, it was recently shown that 24 h after the end of chronic ibuprofen treatment, platelet functioning measured with a platelet function analyzer (PFA-100) recovered, although no information was provided regarding the drug's net antiplatelet effect.26That study reported considerable variability between individuals in the degree of recovery of platelet function between 40 min and 24 h after the last dose of ibuprofen. We found no such variability in our volunteers, as shown by the individual time course plots of maximal intensity of inhibition of platelet aggregation in whole blood (fig. 5). Other NSAIDs (e.g. , ketorolac)27could also be considered as alternative of aspirin, but a moderate antiplatelet effect of this drug and other NSAIDs at therapeutic dosage has been demonstrated.28
The main limitations of the current study are that no participants had a major illness at the time of the study, and no clinical parameters of bleeding were measured. It is known that during the perioperative period, the thrombotic risk is markedly increased, and patients may differ because of gene polymorphisms.29In the case of patients, the side effects of dexibuprofen were not different from those usually known for ibuprofen or aspirin. At therapeutic doses, the incidence of side effects was 5–15% of the patients,30–33mainly gastrointestinal (11–13%), skin (1–1.5%), central nervous system (1–1.3%), and other minor side effects (0.9%). In these studies, racemic ibuprofen and diclofenac showed a 30% and 90%, respectively, higher incidence of adverse reactions than dexibuprofen. However, there is evidence that NSAIDs offer some additional advantages in surgical patients, mainly the reduction in pain intensity over morphine alone.34Further studies in patients are needed to confirm the current findings.
We conclude that dexibuprofen inhibits platelet function in humans through a similar mechanism with respect to aspirin, but this effect is reversible within 24 h after the last dose.
The authors thank Antonio Pino Blanes and Ana Guerrero (Technicians, Department of Pharmacology and Therapeutics, School of Medicine, University of Malaga, Malaga, Spain) for their invaluable technical assistance and Karen Shashok (Translator, Editorial Consultant, Compositor Ruiz Aznar 12 2-A, Granada, Spain) for translating parts of the manuscript into English.