Interaction of a selective cyclooxygenase-2 inhibitor with aspirin and NO-releasing aspirin in the human gastric mucosa

In addition to inhibiting cyclooxygenase (COX)-1-derived prostanoid biosynthesis, aspirin acetylates COX-2, enabling the conversion of arachidonic acid to 15(R)-epi lipoxin A4, or aspirin-triggered lipoxin (ATL). Selective COX-2 inhibitors block ATL formation and exacerbate mucosal injury in rats treated with aspirin. In the present study, we have examined whether inhibition of COX-2 activity in healthy volunteers taking aspirin exacerbates gastric mucosal injury and if such an effect would be prevented by NCX-4016, a NO-releasing derivative of aspirin. Thirty-two volunteers were randomized to receive 2 wk of treatment with NCX-4016 (800 mg twice a day) or aspirin (100 mg once a day) alone or in combination with 200 mg of celecoxib twice a day. Mucosal damage was assessed by endoscopy. The mean mucosal injury score was 5.8 +/- 1.8 in subjects treated with aspirin and 2.4 +/- 0.7 (P < 0.01 vs. aspirin) in subjects treated with NCX-4016. Administration of celecoxib increased the injury score in volunteers treated with aspirin (9.9 +/- 1.9) but not in subjects taking NCX-4016 (1.5 +/- 0.8). Aspirin and NCX-4016 caused a comparable suppression of serum thromboxane B2 levels and increased urinary excretion of ATL. Celecoxib inhibited endotoxin-induced prostaglandin E2 generation in whole blood by approximately 80% and abolished ATL formation. These findings suggests that (i) aspirin and NCX-4016 trigger ATL formation in humans, (ii) celecoxib inhibits ATL formation and exacerbates the mucosal injury caused by low doses of aspirin, and (iii) the NO-donating moiety of NCX-4016 protects the gastric mucosa even in the presence of suppression of COX-1 and COX-2.     

Gastrointestinal safety of NO-aspirin (NCX-4016) in healthy human volunteers: a proof of concept endoscopic study

BACKGROUND AND AIMS: NCX-4016 is a nitric oxide-releasing derivative of aspirin with antiplatelet activity. The aim of this study was to investigate the effect of NCX-4016 on gastrointestinal mucosa and platelet functions in healthy human volunteers. METHODS: This was a parallel-group, double-blind, placebo-controlled study. Forty healthy subjects were randomly allocated to receive 7 days of treatment with NCX-4016 (400 and 800 mg twice daily), equimolar doses of aspirin (200 and 420 mg twice daily), or placebo. Upper endoscopies were performed before and at the end of the treatment period, and gastroduodenal lesions were graded using a predefined scoring system. Basal and posttreatment platelet aggregation in response to arachidonic acid (AA) and serum thromboxane (TX) B(2) and AA-stimulated platelet TXB(2) production were investigated. RESULTS: Mucosal endoscopic injury score on day 7 was 0.63 +/- 0.16 in the placebo group and 11.0 +/- 3.0 and 16.1 +/- 1.6 in healthy volunteers treated with 200 and 420 mg aspirin twice daily (P < 0.0001 vs. placebo). NCX-4016 was virtually devoid of gastric and duodenal toxicity, resulting in a total gastric and duodenal endoscopic score of 1.38 +/- 0.3 and 1.25 +/- 0.5 (P < 0.0001 vs. aspirin, not significant vs. placebo). NCX-4016 inhibited AA-induced platelet aggregation as well as serum TXB(2) and platelet TXB(2) generation induced by AA to the same extent as aspirin (not significant vs. aspirin). CONCLUSIONS: In this study, we have proven the concept that addition of an NO-donating moiety to aspirin results in a new chemical entity that maintains cyclooxygenase-1 and platelet inhibitory activity while nearly avoiding gastrointestinal damage.     

Gastrointestinal safety of nitric oxide-derived aspirin is related to inhibition of ICE-like cysteine proteases in rats

BACKGROUND & AIMS: Caspases, a class of cysteine proteases, modulate apoptosis. Nitric oxide (NO)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) are a new class of NSAID derivatives with reduced gastrointestinal toxicity. The aim of this study was to investigate whether cysteine endoproteases are involved in the pathogenesis of NSAID gastropathy and are target for NO-aspirin (NCX-4016). METHODS: Rats were treated orally with aspirin or equimolar doses of NCX-4016. Caspase activities were measured by fluorometric assay. Apoptosis was quantified by an enzyme-linked immunosorbent assay for histone-associated DNA, DNA ladder on agarose gel, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. A primary culture of gastric chief cells was used to investigate whether NCX-4016 modulates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathways. RESULTS: Short- and long-term (7 days) aspirin administration resulted in a time- and dose-dependent gastric injury that was associated with apoptosis and caspase up-regulation. Z-VAD.FMK, a pancaspase inhibitor, and NO donors protected from acute damage induced by aspirin. NCX-4016 spared the gastric mucosa and caused caspase inactivation by S-nitrosylation. Inhibition of tumor necrosis factor (TNF)-alpha release or activity by TAPI-2 or anti-TNF-alpha receptor monoclonal antibodies protected against mucosal damage and caspase activation. NCX-4016 protected gastric chief cells from toxicity induced by TNF-alpha by activating cGMP-dependent pathways. CONCLUSIONS: Aspirin administration leads to a TNF-alpha-dependent activation of gastric caspases. NO-aspirin spares the gastric mucosa and inhibits caspase activity through cGMP-dependent and -independent pathways.     

Efficacy and age-related effects of nitric oxide-releasing aspirin on experimental restenosis

Restenosis after percutaneous transluminal coronary angioplasty is caused by neointimal hyperplasia, which involves impairment of nitric oxide (NO)-dependent pathways, and may be further exacerbated by a concomitant aging process. We compared the effects of NO-releasing-aspirin (NCX-4016) and aspirin (ASA) on experimental restenosis in both adult and elderly rats. Moreover, to ascertain the efficacy of NCX-4016 during vascular aging, we fully characterized the release of bioactive NO by the drug. Sprague-Dawley rats aged 6 and 24 months were treated with NO releasing-aspirin (55 mg/kg) or ASA (30 mg/kg) for 7 days before and 21 days after standard carotid balloon injury. Histological examination and immunohistochemical double-staining were used to evaluate restenosis. Plasma nitrite and nitrate and S-nitrosothiols were determined by a chemiluminescence-based assay. Electron spin resonance was used for determining nitrosylhemoglobin. Treatment of aged rats with NCX-4016 was associated with increased bioactive NO, compared with ASA. NO aspirin, but not ASA, reduced experimental restenosis in old rats, an effect associated with reduced vascular smooth muscle cell proliferation. NCX-4016, but not ASA, was well tolerated and virtually devoid of gastric damage in either adult or old rats. Thus, impairment of NO-dependent mechanisms may be involved in the development of restenosis in old rats. We suggest that an NCX-4016 derivative could be an effective drug in reducing restenosis, especially in the presence of aging and/or gastrointestinal damage.     

Low gastric toxicity of nitric oxide-releasing aspirin, NCX-4016, in rats with cirrhosis and arthritis

The gastric toxic effects of aspirin (ASA) and NCX-4016, a nitric oxide (NO)-releasing ASA, were compared in normal, cirrhotic, and arthritic rats. Oral administration of ASA (100 mg/kg) produced hemorrhagic lesions on the gastric mucosa in normal rats. The gastric ulcerogenic response to ASA was significantly worsened in both cirrhotic rats induced by N-nitrosodiethylamine and in arthritic rats induced by Freund's complete adjuvant. By contrast, NCX-4016 at 190 mg/kg (a dose equimolar to 100 mg/kg of ASA) did not induce damage in normal rat stomachs but caused slight lesions in the gastric mucosa of both cirrhotic and arthritic rats. Plasma salicylate levels following administration of ASA or NCX-4016 were not different between normal, cirrhotic, and arthritic rats, although the latter drug gave significantly lower values in any group of rats as compared to the former. Acid secretion was significantly increased in both cirrhotic and arthritic rats. ASA with 150 mM HCl caused severe gastric lesions in normal rats, the degree of damage being significantly greater than that induced by ASA alone. Coadministration of NOR-3, a NO donor, significantly prevented the development of gastric lesions induced by ASA, irrespective of whether or not ASA was given together with HCl. Gastric mucosal application of ASA (100 mg/kg) for 30 min caused a marked reduction of transmucosal potential difference (PD) with a minimal effect on gastric mucosal blood flow in both normal and cirrhotic rats, while that of NCX-4016 did not cause a PD reduction and produced a marked increase in the mucosal blood flow in both groups of rats. These results suggest that gastric mucosal susceptibility to ASA-induced damage is increased in both cirrhotic and arthritic rats (the process being partly accounted for by acid hypersecretion in these animals), NCX-4016 has even less gastric toxicity in both cirrhotic and arthritic rats, and the gastric-sparing effect of NCX-4016 is due, at least partly, to an increase of gastric mucosal blood flow, mediated by NO released from this drug.     

Increased lipopolysaccharide-induced tissue factor activity and tumour necrosis factor production in monocytes after intake of aspirin: possible role of prostaglandin E2.

To determine how aspirin intake might influence lipopolysaccharide (LPS)-induced tissue factor (TF) activity and tumour necrosis factor (TNF) in human blood monocytes, we collected blood before and at various times after intake of 300 mg aspirin in 25 healthy volunteers. Aspirin intake reduced LPS-induced thromboxane B2 and PGE2 production in whole blood by 50% and 65% respectively, measured 1 h after aspirin intake. Subsequently, a 95% rise in LPS-induced TF activity in monocytes was seen as compared to a 26% rise in TNF. The rise in TF activity was maximal within 1 h after aspirin intake and no further rise was observed 3, 4 or 24 h after aspirin intake. In contrast, TF activity induced by incubating whole blood in the absence of LPS fell rapidly after the intake of aspirin. In separate experiments, a dose-dependent inhibition by PGE2 was observed in LPS-induced TF activity in monocytes. It is proposed that the increased LPS-induced TF activity and TNF production following aspirin intake may be due to suppressed PGE2 formation. The more pronounced rise in TF activity compared to TNF production may be due to an enhancement of the platelet lipoxygenase pathway that has been shown to be important for LPS-induced TF activity in monocytes.     

Effects of aspirin and NO-aspirin (NCX 4016) on platelet function and coagulation in human endotoxemia

Acetylsalicylic acid (ASA) prevents thromboembolic events by inhibiting platelet function through blocking of cyclooxygenase type 1 (COX-1). A nitroderivate of ASA, 2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)-phenyl ester (NCX 4016) was synthesized, which additionally acts through nitric oxide release. In various in vitro and animal studies NCX 4016 exhibited antithrombotic and anti-platelet properties. We used the standardized model of endotoxin infusion into human volunteers to compare the effects of NCX 4016 and ASA on platelet function and TF-induced coagulation activation. The trial consisted of two parts. In the first part, 10 healthy male volunteers were included in a randomized, open cross-over trial to find a NCX formulation with optimal tolerability and pharmacokinetic data were obtained. The second part was a randomized, double blind placebo controlled clinical trial consisting of 30 healthy male volunteers in three parallel groups (n = 10 per group). Volunteers received either NCX 4016 (800 mg b.i.d.), ASA (425 mg b.i.d.) or placebo for 7 days, before infusion of 2 ng/kg endotoxin on day 8. ASA attenuated the endotoxin-induced platelet plug formation (measured by PFA-100) significantly better than NCX 4016 and placebo (p < 0.004), while there was no difference in soluble P-selectin or VWF-levels. Urine 11-dehydro-thromboxane B₂ levels were significantly lower in the ASA and NCX 4016 groups as compared to placebo (p < 0.05). Neither ASA nor NCX 4016 significantly changed prothrombin fragment_{1 + 2}, D-Dimer or tissue factor (TF)-mRNA levels. In summary, NCX 4016 had no effect on VWF release, platelet activation as measured by soluble P-selectin or TF gene expression. NCX 4016, at the dose tested, unlike ASA, had no effect on platelet collagen/epinephrine induced plug formation under high shear rates.     

Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen.

During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression. TF expression is mediated by nuclear factor kappaB and amplified by activated platelets. TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa). Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor kappaB-dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans. Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model. Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg). Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis. Neither aspirin nor acetaminophen inhibited LPS-induced coagulation. However, LPS increased the percentage of circulating TF(+) monocytes by 2-fold. This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion. Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected. In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation. However, our study showed, for the first time, that LPS increases TF(+) monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.     

Reversal to cisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative of aspirin

Ovarian cancer is a gynecological malignancy that is commonly treated by cytoreductive surgery followed by cisplatin treatment. However, the cisplatin treatment, although successful initially, is not effective in the treatment of the recurrent disease that invariably surfaces within a few months of the initial treatment. The refractory behavior is attributed to the increased levels of cellular thiols apparently caused by the cisplatin treatment. This observation prompted us to choose a cytotoxic drug whose activity is potentiated by cellular thiols with enhanced specificity toward the thiol-rich cisplatin-resistant cells. We used NCX-4016 [2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester], a derivative of aspirin containing a nitro group that releases nitric oxide in a sustained fashion for several hours in cells and in vivo, and we studied its cytotoxic efficacy against human ovarian cancer cells (HOCCs). Cisplatin-sensitive and cisplatin-resistant (CR) HOCCs were treated with 100 microM NCX-4016 for 6 h, and/or 0.5 microg/ml cisplatin for 1 h and assayed for clonogenecity. NCX-4016 significantly reduced the surviving fractions of cisplatin-sensitive (63 +/- 6%) and CR (70 +/- 10%) HOCCs. NCX-4016 also caused a 50% reduction in the levels of cellular glutathione in CR HOCCs. Treatment of cells with NCX-4016 followed by cisplatin showed a significantly greater extent of toxicity when compared with treatment of cells with NCX-4016 or cisplatin alone. In conclusion, this study showed that NCX-4016 is a potential inhibitor of the proliferation of CR HOCCs and thus might specifically kill cisplatin-refractory cancer cells in patients with recurrent ovarian cancer.     

The lack of augmentation by aspirin of inhibition of platelet reactivity by ticlopidine

A decreased threshold for platelet activation apparently contributes to the risk of cardiovascular events, such as acute myocardial infarction. To evaluate the impact of specific agents, we characterized platelet reactivity in 9 healthy subjects before and after 5 days of ingestion of 4 commonly prescribed regimens, 81 mg of aspirin daily, 325 mg of aspirin daily, ticlopidine 250 mg twice daily, and ticlopidine plus 325 mg of aspirin daily. Platelet reactivity was assessed with (1) aggregometry induced by 4 microM adenosine diphosphate (ADP) and collagen (0.19 mg/ml) and performed in platelet-rich plasma; and (2) flow cytometric determination of ADP-induced (0.2, 0.8, and 1.5 microM) P-selectin expression in whole blood. Because anticoagulants alter platelet reactivity, results were obtained with 3 anticoagulants, citrate, enoxaparin, or corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa without effect on other coagulation factors). Ingestion of aspirin did not alter platelet activation as assessed with flow cytometry. Inhibition of the second phase of aggregation was seen with ADP-induced aggregation in platelet-rich plasma anticoagulated with citrate but not enoxaparin or CTI. Ingestion of ticlopidine led to inhibition of ADP-induced aggregation and P-selectin expression. Inhibition of platelet reactivity after the combination of aspirin and ticlopidine did not differ from ticlopidine alone. Marked interindividual variability in platelet reactivity was seen after ingestion of ticlopidine. The results indicate that assessment of effects of specific pharmacologic regimens with accurate and readily available assays of platelet reactivity may facilitate effective prophylaxis and treatment of high-risk subjects with antiplatelet regimens designed to optimally diminish platelet reactivity.     

Aspirin modifies nitric oxide synthase activity in platelets: effects of acute versus chronic aspirin treatment

OBJECTIVE: We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets. METHODS: NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration. RESULTS: Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P<0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation. CONCLUSIONS: Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.     

Chronic treatment with nitric oxide-releasing aspirin reduces plasma low-density lipoprotein oxidation and oxidative stress,arterial oxidation-specific epitopes,and atherogenesis in hypercholesterolemic mice

The effects of chronic treatment with nitric oxide-containing aspirin (NO-aspirin, NCX-4016) in comparison with regular aspirin or placebo on the development of a chronic disease such as atherosclerosis were investigated in hypercholesterolemic low-density lipoprotein (LDL)-receptor-deficient mice. Male mice were assigned randomly to receive in a volume of 10 ml/kg either placebo (n = 10), 30 mg/kg/day NO-aspirin (n = 10), or 18 mg/kg/day of regular aspirin (n = 10). After 12 weeks of treatment, the computer-assisted imaging analysis revealed that NO-aspirin reduced the aortic cumulative lesion area by 39.8 +/- 12.3% compared with that of the placebo (P < 0.001). Regular aspirin did not reduce significantly aortic lesions (-5.1 +/- 2.3%) compared with the placebo [P = 0.867, not significant (NS)]. Furthermore, NO-aspirin reduced significantly plasma LDL oxidation compared with aspirin and placebo, as shown by the significant reduction of malondialdehyde content (P < 0.001) as well as by the prolongation of lag-time (P < 0.01). Similarly, systemic oxidative stress, measured by plasma isoprostanes, was significantly reduced by treatment with NCX-4016 (P < 0.05). More importantly, mice treated with NO-aspirin revealed by immunohistochemical analysis of aortic serial sections a significant decrease in the intimal presence of oxidation-specific epitopes of oxLDL (E06 monoclonal antibody, P < 0.01), and macrophages-derived foam cells (F4/80 monoclonal antibody, P < 0.05), compared with placebo or aspirin. These data indicate that enhanced NO release by chronic treatment with the NO-containing aspirin has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic mice.     

Effect of alternate-day regular and enteric-coated aspirin on platelet aggregation, bleeding time, and thromboxane A2 levels in bleeding-time blood

The effectiveness of low-dose aspirin for primary prevention of cardiovascular mortality is being assessed among the nearly 22,000 United States physicians currently participating in the Physicians' Health Study. Because of occasional reports of gastric irritation among study participants, two enteric-coated aspirin preparations were tested as possible alternatives to regular compressed aspirin for platelet inhibition. Thirty-three volunteers were assigned randomly to one of four treatment groups: regular aspirin (325 mg), placebo, and two enteric-coated aspirin preparations (325 mg). Pills were administered every other day, duplicating the regimen used in the Physicians' Health Study. Bleeding times, platelet aggregation, and thromboxane A2 levels produced by aggregating platelets in vitro, as well as in collected bleeding-time blood, were determined. Measurements were taken before and after a single dose as well as after seven alternate-day doses. Regular and enteric-coated aspirin preparations were equally efficacious in prolonging the bleeding time, inhibiting platelet aggregation, and suppressing thromboxane A2 production. There was virtually complete suppression of thromboxane A2 production (over 99 percent), by platelets in vitro and in collected bleeding-time blood. The levels were still profoundly reduced (89 percent) 48 hours after the last dose. Enteric-coated aspirin may provide an alternative to regular aspirin in a low-dose regimen designed to inhibit platelet activity.     

Cumulative antiplatelet effect of low-dose enteric coated aspirin

Enteric coated aspirin (ECA) at doses of 325-1300 mg is an effective alternative to regular aspirin for inhibition of platelet activity while avoiding gastric irritation. The objectives of this study were to determine: (1) the lowest chronic dose of ECA providing effective inhibition of platelet activities, (2) the time course of the inhibition, and (3) the reappearance of platelet cyclo-oxygenase activity. Seven subjects were studied before and after seven daily doses of 40-325 mg ECA. Serum thromboxane (TX) B2 levels indicated that the lowest dose of ECA resulting in greater than 90% inhibition of platelet cyclo-oxygenase was 80 mg/d. Platelet aggregation and ATP release in response to collagen (1 microgram/ml) and arachidonic acid (1 mM) were abolished and bleeding times were prolonged from 6.1 +/- 1.5 min to 9.7 +/- 2.8 min (mean +/- SD, P less than 0.01). Examination of platelet cyclo-oxygenase activity on a daily basis revealed that 24 h after the first 80 mg dose serum TXB2 had decreased by approximately 60% and was suppressed by more than 90% after four doses. Recovery of platelet cyclo-oxygenase activity after a single 80 mg dose of ECA was delayed for 48-72 h indicating that aspirin reached the systemic circulation. We conclude that chronic inhibition of platelet activity may be achieved in a cumulative manner with 80 mg ECA/d.     

Effects of nitric oxide-releasing aspirin versus aspirin on restenosis in hypercholesterolemic mice

Restenosis is due to neointimal hyperplasia, which occurs in the coronary artery after percutaneous transluminal coronary angioplasty (PTCA). During restenosis, an impairment of nitric oxide (NO)-dependent pathways may occur. Concomitant hypercholesterolemia may exacerbate restenosis in patients undergoing PTCA. Here, we show that a NO-releasing aspirin derivative (NCX-4016) reduces the degree of restenosis after balloon angioplasty in low-density lipoprotein receptor-deficient mice and this effect is associated with reduced vascular smooth muscle cell (VSMC) proliferation and macrophage deposition at the site of injury. Drugs were administered following both therapeutic or preventive protocols. We demonstrate that NCX-4016 is effective both in prevention and treatment of restenosis in the presence of hypercholesterolemia. These data indicate that impairment of NO-dependent mechanisms may be involved in the development of restenosis in hypercholesterolemic mice. Although experimental models of restenosis may not reflect restenosis in humans in all details, we suggest that a NO-releasing aspirin derivative could be an effective drug in reducing restenosis following PTCA, especially in the presence of hypercholesterolemia and/or gastrointestinal damage.     

Rapidity and duration of platelet suppression by enteric-coated aspirin in healthy young men.

The recent demonstration of aspirin's ability to prevent and reduce the severity of myocardial infarction has led to a marked increase in its use and to a need for information regarding the time-course of onset and offset of its antiplatelet effect. A study of healthy men was conducted to determine (1) the rapidity of onset of inhibition of platelet aggregation in response to adenosine diphosphate, and thromboxane A2 production after chewed enteric-coated aspirin (325 mg, n = 10); and (2) the duration of platelet inhibition after cessation of enteric-coated aspirin (325 mg) every other day for 14 days (n = 10). When chewed, enteric-coated aspirin greatly inhibited platelet aggregation response to adenosine diphosphate and thromboxane A2 production within 15 minutes. Complete recovery of platelet aggregation occurred in half of the subjects by day 3, and in 80% of the subjects by day 4; the platelet response was not affected by exercise. This study demonstrates a rapid onset of aspirin's antiplatelet effect and provides information relevant for optimal timing of initiation of aspirin for acute conditions such as myocardial infarction and unstable angina, and cessation of aspirin before surgery.     

Human platelet alphaIIbeta3 integrin binding affinity and specificity of SJ874: antiplatelet efficacy versus aspirin

OBJECTIVE: To define the affinity and specificity of SJ874, a nonpeptide antiplatelet agent for platelet glycoprotein Ilb/IIIa integrin, and to determine the antiplatelet efficacy of SJ874 relative to those of glycoprotein IIbIIIa antagonists and aspirin. METHODS: Binding affinity and specificity of SJ874 for platelet glycoprotein IIb/IIIa integrin were determined using integrin-mediated binding and adhesion assays with human cells. Additionally, the antiplatelet efficacy of SJ874 was determined and compared with those of other glycoprotein IIb/IIIa antagonists and aspirin using light-transmittance and laser-scattering aggregometry. RESULTS: SJ874 inhibited aggregation of human platelets induced by 10 micromol/l adenosine diphosphate (ADP) with a concentration for half-maximal effect of 0.046 +/- 0.005 micromol/l using light-transmittance aggregometry. Using laser-scattering aggregometry, SJ874 was found to totally inhibit formation both of micro-aggregates and of macro-aggregates induced either by ADP or by epinephrine. In contrast, administration of 325 mg aspirin to normal healthy volunteers attenuated formation of macro-aggregates but not micro-aggregates. SJ874 inhibited binding of [125I]-fibrinogen to activated (by ADP, epinephrine, and arachidonic acid at concentrations of 100 micromol/l each) gel-filtered human platelets with a concentration for half-maximal effect of 0.0012 +/- 0.0005 micromol/l. SJ874 was demonstrated to associate more tightly with resting human platelets than did DMP754 [1] and slightly less tightly than did DMP802 [2]. SJ874 was demonstrated to exhibit a high degree of specificity for platelet glycoprotein IIb/IIIa (alphaIIb/beta3) integrin compared with other known integrins, including alphavbeta3, alphavbeta5, and alpha5beta1 (concentration for half-maximal effect > 100 micromol/l). CONCLUSION: SJ874 is a potent and specific platelet glycoprotein IIb/IIIa antagonist with high affinity for and tight association with human platelets. These data suggest that SJ874 might have good antiplatelet utility for inhibiting formation both of platelet micro-aggregates and of macro-aggregates of platelets and a long duration of action in humans due to its slow dissociation from human platelets.     

Comparison of increased aspirin dose versus combined aspirin plus clopidogrel therapy in patients with diabetes mellitus and coronary heart disease and impaired antiplatelet response to low-dose aspirin

The effects of therapy with aspirin 300 mg/day and with combined aspirin 100 mg/day plus clopidogrel 75 mg/day on platelet function were compared in patients with diabetes mellitus and coronary artery disease and impaired antiplatelet responses to aspirin 100 mg/day. The study population consisted of 151 outpatients with type II diabetes mellitus and coronary artery disease who were taking aspirin 100 mg/day. Of the 151 patients, a subgroup of subjects with impaired aspirin response were selected on the basis of the results of platelet aggregometry. Nonresponsiveness to aspirin was defined as mean aggregation > or =69% with 3 micromol/L adenosine diphosphate and mean aggregation > or =70% with 2 micromol/L collagen. Aspirin semiresponders were defined as meeting 1 but not both of these criteria. Nonresponders and semiresponders were randomized equally to aspirin 300 mg/day and aspirin 100 mg/day plus clopidogrel 75 mg/day, and aggregation tests were repeated after 2 weeks. Sixty of the 151 patients with diabetes (40%) were found to respond to aspirin inadequately. Platelet aggregation induced by adenosine diphosphate and collagen decreased significantly after aspirin 300 mg/day or combined therapy. Combined treatment was found to have a stronger inhibitory effect on platelet aggregation induced by adenosine diphosphate than aspirin 300 mg/day (p = 0.002). Impaired aspirin response was resolved by increasing the aspirin dose or adding clopidogrel to aspirin (p <0.0001 for each). However, desired platelet inhibition was achieved in significantly more patients by combined treatment than by aspirin 300 mg/day (p <0.05). In conclusion, aspirin 100 mg/day does not inhibit platelet function adequately in a significant number of patients with diabetes mellitus and coronary artery disease. Increasing the aspirin dose to 300 mg/day or adding clopidogrel to aspirin can provide adequate platelet inhibition in a significant number of those patients with impaired responses to low-dose aspirin.     

Indobufen inhibits tissue factor in human monocytes through a thromboxane-mediated mechanism

OBJECTIVE: To assess whether indobufen, a reversible inhibitor of platelet cyclooxygenase (Cox) activity, affects tissue factor (TF) in human monocytes and to investigate the relationship between Cox-derived products and TF. METHODS: TF was evaluated in isolated adherent monocytes, both resting and lipopolysaccharide (LPS)-stimulated, in terms of procoagulant activity, protein, and mRNA levels. The expression of TF surface antigen was determined in LPS-stimulated whole blood monocytes by flow cytometry. The levels of the stable thromboxane A2 (TxA2) metabolite, TxB2, and of prostaglandin E2 (PGE2) were measured in monocyte supernatant by immunoenzymatic techniques. Cox-1 and Cox-2 protein level, tyrosine phosphorylation, and mitogen-activated protein kinase (MAP-kinase) activation were determined by Western blot analysis. RESULTS: Indobufen prevents TF expression and activity both in isolated and in whole blood monocytes. Reduction of TxA2 synthesis, coupled with a lack of effect on PGE2 levels and prevention of ERK1/2 phosphorylation are highlighted as the mechanisms through which indobufen negatively affects TF. CONCLUSIONS: Data show that indobufen down-regulates TF in monocytes. This novel activity, coupled with the antiplatelet effect of the drug, may add benefit for its use in the management of atherothrombosis.     

Effects of very low dose daily, long-term aspirin therapy on gastric, duodenal, and rectal prostaglandin levels and on mucosal injury in healthy humans.

BACKGROUND & AIMS: The safety of low-dose daily aspirin therapy in the gastrointestinal tract is uncertain. Our objectives were to evaluate the long-term effects of very low daily aspirin doses in the gastrointestinal tract and effects on platelet-derived serum thromboxane levels in volunteers. METHODS: Subjects were randomized to receive 10 mg (n = 8), 81 mg (n = 11), or 325 mg (n = 10) aspirin daily for 3 months. Before administration of aspirin, all subjects underwent gastroduodenoscopy, and most underwent proctoscopy for assessment of mucosal injury and prostaglandin content. After 1.5 and 3 months, subjects again underwent gastroduodenoscopy and, at 3 months, another proctoscopy. RESULTS: Each aspirin dose (even 10 mg) significantly reduced gastric mucosal prostaglandin levels, to approximately 40% of the baseline value. All three doses also induced significant gastric injury, and 325 mg caused duodenal injury. Three subjects developed gastric ulcers, 1 while taking 10 mg/day of aspirin. Furthermore, aspirin at 81 mg/day and 325 mg/day (but not 10 mg/day) significantly reduced duodenal mucosal prostaglandin levels to approximately 40% of the baseline value. Only 325 mg of aspirin per day significantly reduced rectal mucosal prostaglandin levels to approximately 60% of the baseline value. Serum thromboxane levels were inhibited 62%, 90%, and 98% with 10, 81, and 325 mg of aspirin. CONCLUSIONS: The findings explain aspirin's predominant gastric toxicity and question the safety of even 10 mg of aspirin daily.