Pharmacodynamic effects of EV-077 in patients with diabetes mellitus and coronary artery disease on aspirin or clopidogrel monotherapy: results of an in vitro pilot investigation.

Patients with diabetes mellitus (DM) have increased propensity to generate thromboxane A2 (TXA2) and other eicosanoids which can contribute to their heightened platelet reactivity. EV-077 is a potent thromboxane receptor antagonist and thromboxane synthase inhibitor and thus represents an attractive therapy in patients with DM. However, the effects of EV-077 on pharmacodynamic (PD) profiles in patients with DM and coronary artery disease (CAD) while on antiplatelet therapy is poorly explored and represented the aim of this in vitro pilot investigation. Patients with DM and stable CAD (n = 10) on low-dose aspirin (81 mg/day) were enrolled and then switched to clopidogrel (75 mg/day) monotherapy for 7-10 days. PD assessments were conducted while on aspirin and on clopidogrel using light transmittance aggregometry following stimuli with U-46619 [TXA2 stable analogue (7 µM)], arachidonic acid [AA (1 mM)], collagen (3 µg/mL) and adenosine diphosphate [ADP (5 µM and 20 µM)] with and without in vitro EV-077. EV-077 completely inhibited U-46619-stimulated platelet aggregation (p = 0.005 for both aspirin and clopidogrel) and also showed a significant reduction of collagen-induced aggregation (aspirin p = 0.008; clopidogrel p = 0.005). EV-077 significantly reduced AA-induced platelet aggregation in clopidogrel (p = 0.009), but not aspirin (p = 0.667) treated patients. Ultimately, EV-077 significantly reduced ADP-mediated platelet aggregation in both aspirin (ADP 5 µM p = 0.012; ADP 20 µM p = 0.032) and clopidogrel (ADP 5 µM p = 0.007; ADP 20 µM p = 0.008) treated patients. In conclusion, in DM patients with CAD on aspirin or clopidogrel monotherapy, in vitro EV-077 exerts potent platelet inhibitory effects on multiple platelet signaling pathways. These data support that EV-077 has only additive platelet inhibiting effects on top of standard antiplatelet therapies. These findings warrant further investigation in ex vivo settings.

Pharmacodynamic effects of adjunctive cilostazol therapy in patients with coronary artery disease on dual antiplatelet therapy: impact of high on-treatment platelet reactivity and diabetes mellitus status.

BACKGROUND: In patients on dual antiplatelet therapy with aspirin and clopidogrel, the adjunctive use of cilostazol is associated with enhanced platelet inhibition. However, if cilostazol exerts different pharmacodynamic (PD) effects according to levels of on-treatment platelet reactivity remains unknown. This study aimed to determine the PD effects of cilostazol in patients with and without high on-clopidogrel platelet reactivity (HPR) according to diabetes mellitus (DM) status. METHODS: This is a post-hoc analysis derived from patients (n = 79) enrolled in a prospective, randomized, double-blind, double-dummy, crossover study comparing cilostazol with placebo in stable coronary artery disease patients on aspirin and clopidogrel therapy. In the present analysis, patients were divided according to the presence or absence of HPR (HPR and non-HPR). HPR was defined as a P2Y12 units (PRU) > 240 as assessed by the VerifyNow P2Y12 assay. The PD effects of cilostazol were evaluated in patients with and without HPR according to DM status. RESULTS: Significant absolute changes in PRU values were observed after adjunctive cilostazol in both HPR [53.4 (95% CI 24.7-82.1), P = 0.001] and non-HPR [40.8 (95% CI 28.7-52.8), P < 0.0001] patients. This difference was statistically significant in HPR patients with DM (P = 0.001), but not without DM (P = 0.24), and in non-HPR patients with and without DM (P = 0.0001 for both). The greatest mean reduction in PRU was observed in HPR patients with DM (72.9; 95% CI 33.7-112.0). Thrombin generation was not affected by cilostazol, irrespective of HPR status. CONCLUSION: Cilostazol reduces platelet reactivity both in HPR and non-HPR patients, although these PD effects are enhanced in HPR patients with DM. Nevertheless, larger studies are needed to better evaluate possible differential effects of cilostazol on platelet reactivity by diabetes status.

Impact of renal function on clopidogrel-induced antiplatelet effects in coronary artery disease patients without diabetes mellitus.

Diabetes mellitus (DM) is the most important predictor of chronic kidney disease (CKD), and pharmacodynamic (PD) studies have shown that DM patients with impaired renal function are characterized by reduced clopidogrel response. However, post-hoc PD studies conducted in unselected cohorts, composed of both DM and non-DM patients, have reached controversial findings on the effects of CKD on clopidogrel response, likely attributed to patient heterogeneity. The impact of renal function on clopidogrel response in non-DM patients remains unexplored and represented the aim of this prospective investigation. We conducted a prospective PD investigation in non-DM patients with and without CKD defined as an estimated glomerular filtration rate (eGFR) below or above 60 mL/min, respectively. All patients had known coronary artery disease and were on maintenance aspirin therapy. PD assessments were assessed at baseline and 2 and 24 h after a 600 mg loading dose of clopidogrel. PD assays included light transmission aggregometry (LTA) using 5 and 20 µmol ADP with and without PGE1 and flow cytometric assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) to determine the platelet reactivity index. A total of 60 patients were studied (n = 30 eGFR ≥60 mL/min; n = 30 eGFR <60 mL/min). At baseline there were no differences between groups. Following clopidogrel loading dose administration, levels of on-treatment platelet reactivity were similar between groups at 2 and 24 h as measured with LTA and VASP. Accordingly, there were no differences in rates of high on-treatment platelet reactivity between groups. In non-DM patients with CAD, the presence of impaired renal function is not associated with differences in clopidogrel-induced antiplatelet effects compared with patients with preserved renal function.

Effects of cangrelor in coronary artery disease patients with and without diabetes mellitus: an in vitro pharmacodynamic investigation.

Platelets from patients with diabetes mellitus (DM) are hyper-reactive and whether cangrelor, a potent intravenous P2Y(12) receptor blocker, has differential pharmacodynamic (PD) effects according DM status is unknown. The aim of this investigation was to evaluate the in vitro PD effects of cangrelor in coronary artery disease (CAD) patients with and without DM. This prospective study enrolled 120 clopidogrel-naïve patients with CAD on aspirin therapy. PD assessments using cangrelor (500 nmol/l) in vitro included vasodilator-stimulated phosphoprotein assay to obtain the P2Y(12) reactivity index (PRI), and multiple electrode aggregometry (MEA). In a 20 patients subgroup, dose-dependent response was assessed following exposure to escalating concentrations (baseline, 5, 50, 500 and 5,000 nmol/l); thrombin generation processes were evaluated by thromboelastography (TEG). PD data were evaluable in 103 patients. No differences in baseline PD parameters were observed in DM (n = 48) and non-DM (n = 45) subjects. Cangrelor reduced PRI values irrespective of DM status (p < 0.0001), yielding no difference in patients with and without DM (16.1 ± 12.3 vs. 16.8 ± 11.3; p = 0.346). All MEA values were significantly reduced, although this was of greater magnitude with purinergic compared to non-purinergic agonists. A trend analysis showed a dose-dependent effect on platelet inhibition, with no interaction due to DM status, whereas no significant dose-dependent effect was observed for TEG-derived parameters. Therefore, in vitro cangrelor provides potent and dose-dependent blockade of the platelet P2Y(12) receptor, with no differential effect in DM and non-DM patients. In addition, in vitro cangrelor exerts moderate inhibitory effects on non-purinergic platelet signaling pathways, without modulating platelet-derived thrombin generation processes.

A randomized study assessing the impact of cilostazol on platelet function profiles in patients with diabetes mellitus and coronary artery disease on dual antiplatelet therapy: results of the OPTIMUS-2 study.

AIMS: Patients with type 2 diabetes mellitus (T2DM) have reduced platelet inhibition compared with non-diabetics following P2Y(12) receptor blockade. Whether inhibition of P2Y(12) signalling can be enhanced by adjunctive treatment with cilostazol in T2DM patients is unknown. The aim of this pilot study was to assess the functional impact of cilostazol in T2DM patients on standard aspirin and clopidogrel treatment. METHODS AND RESULTS: This was a prospective, double-blind, double-dummy, placebo-controlled, randomized, cross-over platelet function study. T2DM patients on dual antiplatelet therapy were assigned to receive cilostazol 100 mg or placebo twice daily for 14 days and afterwards crossed-over treatment assignments for another 14 days. Platelet function was performed at three time points: at baseline, 14 days after randomization, and 14 days after treatment cross-over. The P2Y(12) reactivity index, determined through flow cytometric assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein, was the primary endpoint measure. In addition to this flow cytometric evaluation, light transmittance aggregometry and VerifyNow testing were performed. A total of 25 T2DM patients were randomized; five patients discontinued treatment due to side effects. The P2Y(12) reactivity index was significantly lower following cilostazol treatment compared with placebo (36.3 +/- 20 vs. 59.9 +/- 16%; P = 0.0002). All other P2Y(12)-specific functional assessments showed enhanced inhibition of this signalling pathway following treatment with cilostazol. CONCLUSION: Adjunctive treatment with cilostazol in T2DM patients on standard dual antiplatelet therapy enhances inhibition of platelet P2Y(12) signalling.

Randomized comparison of a high clopidogrel maintenance dose in patients with diabetes mellitus and coronary artery disease: results of the Optimizing Antiplatelet Therapy in Diabetes Mellitus (OPTIMUS) study.

BACKGROUND: After treatment with clopidogrel, patients with type 2 diabetes mellitus (T2DM) have reduced platelet inhibition compared with patients who are not diabetic. Whether platelet inhibition can be enhanced by increasing clopidogrel maintenance dosage in T2DM patients is unknown. The aim of this pilot study was to assess the functional impact of a high maintenance dose in T2DM patients with suboptimal clopidogrel-induced antiplatelet effects. METHODS AND RESULTS: T2DM patients on chronic dual antiplatelet therapy were screened to identify suboptimal clopidogrel responders. The latter were randomized to 30-day treatment with a standard (75 mg; n=20) or high (150 mg; n=20) daily maintenance dose. Platelet function was assessed at 3 time points: baseline, 30 days after randomization, and 30 days after resuming standard dosing. Platelet function parameters included adenosine diphosphate-induced (20 and 5 micromol/L) maximal and late platelet aggregation, inhibition of platelet aggregation, platelet disaggregation, and P2Y12 reactivity index. A total of 64 T2DM patients were screened to identify 40 suboptimal responders. After randomization, maximal adenosine diphosphate-induced (20 micromol/L) platelet aggregation was significantly reduced in the 150-mg group compared with the 75-mg group (P=0.002; primary end point). However, suboptimal clopidogrel response was still present in 60% of patients on the 150-mg regimen. All other platelet function parameters showed enhanced clopidogrel-induced antiplatelet effects with 150 mg, which returned to baseline values after resumption of standard dosing. CONCLUSIONS: A 150-mg maintenance dose of clopidogrel is associated with enhanced antiplatelet effects compared with 75 mg in high-risk T2DM patients. However, enhanced ex vivo platelet reactivity continues to persist, the clinical implications of which are unknown and need to be evaluated in large-scale clinical trials.

Functional impact of high clopidogrel maintenance dosing in patients undergoing elective percutaneous coronary interventions. Results of a randomized study.

The currently recommended maintenance dose of clopidogrel is often associated with inadequate platelet inhibition, suggesting the need for a higher dose. The aim of this pilot study was to assess the functional impact of a high (150 mg/day) maintenance dose of clopidogrel in patients undergoing elective percutaneous coronary intervention (PCI). This is a prospective, randomized, platelet function study which was performed in elective PCI patients assigned to treatment with either a 75 mg (n = 20) or 150 mg (n = 20) daily maintenance dose of clopidogrel for 30 days; afterwards, all patients resumed standard dosing. Platelet aggregation was performed using light transmittance aggregometry following 20 microM and 5 microM adenosine diphosphate (ADP) stimuli 30 days after randomization and 30 days after resuming standard dosing. Patients treated with 150 mg/day clopidogrel had lower 20 microM ADP-induced platelet aggregation compared to patients on 75 mg/day (52.1 +/- 9% vs. 64.0 +/- 8%; p < 0.001; primary endpoint). The dose-dependent effect was confirmed by the absolute and relative increase in platelet aggregation after resuming standard dosing (p < 0.001). No changes were observed in patients randomized to standard dosing. Parallel findings were observed following 5 microM ADP stimuli for all assessments. A broad variability in clopidogrel-induced antiplatelet effects was observed irrespective of dosing. In conclusion, a 150 mg/day maintenance dose regimen of clopidogrel is associated with reduced platelet reactivity and enhanced platelet inhibition compared to that achieved with the currently recommended 75 mg/day in patients undergoing elective PCI.

Functional effects of high clopidogrel maintenance dosing in patients with inadequate platelet inhibition on standard dose treatment.

Updated guidelines on percutaneous coronary intervention recommend increasing the dose of clopidogrel to 150 mg in high-risk patients if <50% platelet inhibition is demonstrated. However, to date, the functional impact of this recommendation has been poorly explored. The aim of this study was to assess the functional implications associated with the use of clopidogrel 150 mg/day in patients with inadequate platelet inhibition while receiving standard 75 mg/day maintenance treatment. Patients with diabetes mellitus have a higher prevalence of inadequate clopidogrel-induced antiplatelet effects and stent thrombosis compared with those without diabetes and were selected for this analysis. Platelet inhibition was assessed using the VerifyNow P2Y12 assay in patients with type 2 diabetes receiving dual-antiplatelet therapy. Patients (n = 17) with <50% platelet inhibition were treated with clopidogrel 150 mg/day for 1 month. Adenosine diphosphate-induced aggregation and the P2Y12 reactivity ratio were also assessed. Platelet function profiles were compared with that of a control group (n = 17) with >or=50% inhibition. Platelet inhibition increased from 27.1 +/- 12% to 40.6 +/- 18% in patients treated with clopidogrel 150 mg/day (p = 0.009; primary end point). All other functional measures also showed enhanced clopidogrel-induced antiplatelet effects. The degree of platelet inhibition achieved after treatment with clopidogrel 150 mg/day varied broadly, and only 35% of patients yielded a degree of platelet inhibition >or=50%. Increasing the dose in patients with inadequate response to clopidogrel did not reach the same degree of antiplatelet effects as those achieved in patients with adequate response while receiving 75 mg/day. In conclusion, the use of a 150 mg maintenance dose of clopidogrel in patients with type 2 diabetes with <50% platelet inhibition is associated with enhanced antiplatelet effects. However, the antiplatelet effects achieved are nonuniform, and a considerable number of patients persist with inadequate platelet inhibition.

Young, adult, and old rats have similar changes in mRNA expression of many skeletal genes after fracture despite delayed healing with age.

Genes active in fracture healing are not well understood. Because age slows skeletal repair, the change in gene expression between animals of differing ages may illuminate novel pathways important to this healing response. To explore this, 6-, 26-, and 52-week-old female Sprague-Dawley rats were subjected to mid-diaphyseal femoral fracture with intramedullary fixation. The fracture callus was collected at 0, 0.4 (3 days), 1, 2, 4, or 6 weeks after fracture. RNA was extracted and pooled between two animals for each sample. Three samples were done for each time point for each age for a total of 54 Affymetrix U34A GeneChip microarrays. Of the 8700 genes on each array, 3300 were scored as present. Almost all of these genes were affected by femoral fracture with either upregulation or downregulation in the 6 weeks after fracture. Upregulated genes included markers for matrix genes for both cartilage and bone, osteoblasts, osteocytes, osteoclasts, fibroblasts, and mast cells. Downregulated genes included genes related to blood cell synthesis. Nearly all genes presently associated with bone metabolism showed the same response to fracture healing regardless of the age of the animal. In conclusion, skeletal fracture led to similar changes in RNA expression for most skeletal genes despite the delay in the formation of bone to bridge the fracture gap in old rats. Defects in the healing of skeletal trauma in older rats may lie in systems not normally studied by skeletal biologists.

The effect of age on gene expression in adult and juvenile rats following femoral fracture.

OBJECTIVE: To compare mRNA gene expression during fracture healing in young and adult rats. DESIGN: Gene expression was measured at zero, 1, 2, 4, 6, 8 and 10 weeks after fracture (6 rats/age/time point) in rats at 6 and 26 weeks of age at surgery. SETTING: AAALAC-accredited vivarium of an independent academic medical center. ANIMALS: Female Sprague-Dawley rats at 6 and 26 weeks of age. INTERVENTION: An intramedullary rod was placed retrograde in the left femur, and a simple transverse closed middiaphyseal fracture was induced. MAIN OUTCOME MEASUREMENTS: mRNA gene expression was measured for 34 genes for extracellular matrix, osteoblasts, bone morphogenic protein, inflammation, cytokine, and receptor genes. RESULTS: The young rats reached radiographic union by 4 weeks after fracture, whereas the adult rats took 8 to 10 weeks to unite. All genes studied increased in mRNA expression with a peak at 1 to 2 weeks after fracture. All genes in the young rats then subsided to baseline by 4 weeks after fracture. However, during the longer period needed for radiographic union in the adult rats, only genes related to bone matrix, osteoblastic markers, angiogenesis, and the fibroblast growth factors remained significantly up-regulated at 4 and 6 weeks after fracture. Genes related to cartilage, Indian hedgehog, the bone morphogenetic proteins, and transforming growth factor-beta came to undetectable baseline values in the adult rats prior to radiographic union. CONCLUSIONS: Most stimulators of bone healing are not expressed during the later stages of fracture repair in adult rats. Other genes must control bone growth to bridge the fracture gap.

Pharmacodynamic effects of standard dose prasugrel versus high dose clopidogrel in non-diabetic obese patients with coronary artery disease.

Increased body weight is independently associated with impaired clopidogrel pharmacodynamic (PD) response. Prasugrel has more potent PD effects compared with clopidogrel, although its PD effects in obese patients are unknown. The aim of this prospective, randomised, study was to compare the PD effects of standard-dose prasugrel [60 mg loading dose (LD)/10 mg daily maintenance dose (MD)] with high-dose clopidogrel (900 mg LD/150 mg daily MD) in non-diabetic obese [body mass index (BMI) ≥30 kg/m²] patients, with coronary artery disease (CAD) on aspirin therapy. PD assessments (baseline, 2 hours post-LD and 6 ± 2 days after MD) were conducted using four platelet function assays, and the platelet reactivity index (PRI) assessed by VASP was used for sample size estimation. A total of 42 patients with a BMI of 36.42 ± 5.6 kg/m² completed the study. There were no differences in baseline PD measures between groups. At 2 hours post-LD, prasugrel was associated with lower PRI compared with clopidogrel (24.3 ± 5.5 vs 58.7 ± 5.7, p≤0.001), with consistent findings for all assays. At one-week, PRI values on prasugrel MD were lower than clopidogrel MD without reaching statistical significance (34.7 ± 5.8 vs 42.9 ± 5.8, p=0.32), with consistent findings for all assays. Accordingly, rates of high on-treatment platelet reactivity were markedly reduced after prasugrel LD, but not after MD. In conclusion, in non-diabetic obese patients with CAD, standard prasugrel dosing achieved more potent PD effects than high-dose clopidogrel in the acute phase of treatment, but this was not sustained during maintenance phase treatment. Whether an intensified prasugrel regimen is required in obese patients warrants investigation.

Impact of cigarette smoking on P2Y12 receptor binding activity before and after clopidogrel therapy in patients with coronary artery disease.

Smoking enhances the P2Y12 receptor inhibitory effects of clopidogrel. Nicotine increases P2Y12 receptor expression in platelet lysates from healthy volunteers. However, the impact of cigarette smoking on platelet P2Y12 receptor binding in clopidogrel-treated patients with coronary artery disease (CAD) is unknown. Clopidogrel-naïve patients with stable CAD (n = 20) were enrolled and stratified according to smoking status. P2Y12 receptor binding activity was determined by radioligand receptor binding prior and 24 h after a 600-mg loading dose of clopidogrel. Baseline P2Y12 receptor binding was 1.8-fold higher in smokers compared with nonsmokers. After a 600-mg loading dose of clopidogrel, smokers showed a 6.4-fold reduction in P2Y12 receptor binding indicative of marked clopidogrel-mediated blockade, while there were minimal changes among nonsmokers. Among patients with stable CAD, smokers have more P2Y12 receptor binding than nonsmokers and have a higher degree of clopidogrel-mediated platelet inhibition.

Pharmacodynamic effects of cangrelor on platelet P2Y12 receptor-mediated signaling in prasugrel-treated patients.

OBJECTIVES: The purpose of this study was to assess the in vitro P2Y12 receptor inhibitory effects of cangrelor on platelets from patients on maintenance prasugrel therapy treated with 2 reloading dose regimens. BACKGROUND: Despite its more potent and rapid antiplatelet effects compared with clopidogrel, recent studies have shown variability in prasugrel-mediated P2Y12 receptor inhibition, particularly in high-risk settings. Cangrelor is a potent intravenous P2Y12 receptor inhibitor. METHODS: A total of 60 patients with coronary artery disease on maintenance prasugrel (10 mg/day) therapy were randomized to a 30- or 60-mg reload of prasugrel. The platelet reactivity index (PRI), as assessed by whole-blood vasodilator-stimulated phosphoprotein, was measured with and without in vitro incubation of cangrelor (500 nM) at baseline, and at 1 and 4 h after reload. RESULTS: In the absence of cangrelor, prasugrel reloading reduced PRI (p < 0.001 for both doses), although a 60-mg reload had greater platelet inhibition compared with a 30-mg reload at 4 h (p = 0.001). Cangrelor was associated with a reduction in PRI values during the overall study time course in patients reloaded with 30 mg (p = 0.001) and 60 mg (p < 0.001) of prasugrel. In patients reloaded with 30 mg prasugrel, cangrelor decreased PRI at each time point (baseline, p < 0.001; 1 h, p = 0.013; 4 h, p = 0.001). In patients reloaded with 60 mg prasugrel, cangrelor decreased PRI at baseline (p < 0.001) and 1 h (p = 0.002); levels of platelet reactivity comparable to those achieved with cangrelor were observed only at 4 h (p = 0.325). The intergroup comparisons with cangrelor were not significant at any time point. CONCLUSIONS: In patients on maintenance prasugrel therapy exposed to a reloading dose (30 or 60 mg) of prasugrel, in vitro cangrelor is associated with further platelet P2Y12 receptor inhibitory effects.

Impaired responsiveness to the platelet P2Y12 receptor antagonist clopidogrel in patients with type 2 diabetes and coronary artery disease.

BACKGROUND: Several studies have shown that patients with diabetes mellitus (DM) exhibit an impaired response to clopidogrel. This may contribute to their increased risk of recurrent atherothrombotic events, despite the use of dual-antiplatelet therapy. The mechanisms for impaired clopidogrel response in DM patients have not been fully elucidated. OBJECTIVES: The aim of this study was to explore the mechanisms for impaired clopidogrel-mediated platelet inhibition in patients with DM using a comprehensive methodological approach embracing both pharmacokinetic (PK) and pharmacodynamic (PD) assessments as well as ex vivo and in vitro investigations. METHODS: Patients (DM, n = 30; non-DM, n = 30) with stable coronary artery disease taking aspirin 81 mg/day and P2Y12 antagonist naive were enrolled. Blood was collected before and at various times (0.5, 1, 2, 4, 6, and 24 h) after a 600-mg loading dose of clopidogrel. PD assessments included vasodilator-stimulated phosphoprotein, light transmission aggregometry, and VerifyNow P2Y12 ex vivo, before and after dosing and following in vitro incubation with escalating concentrations (1, 3, and 10 µM) of clopidogrel's active metabolite (Clop-AM). Exposure to Clop-AM was also determined. RESULTS: PD assessments consistently showed that during the overall 24-h study time course, residual platelet reactivity was higher in DM patients compared with non-DM patients. In vitro incubation with Clop-AM revealed altered functional status of the P2Y12 signaling pathway in DM platelets as measured by vasodilator-stimulated phosphoprotein, but not with other PD assays. Clop-AM exposure was ∼40% lower in DM patients than in non-DM patients. CONCLUSIONS: The present study suggests that among DM patients, impaired P2Y12 inhibition mediated by clopidogrel is largely attributable to attenuation of clopidogrel's PK profile. This is characterized by lower plasma levels of Clop-AM over the sampling time course in DM patients compared with non-DM patients and only modestly attributed to altered functional status of the P2Y12 signaling pathway.

Cigarette smoking and antiplatelet effects of aspirin monotherapy versus clopidogrel monotherapy in patients with atherosclerotic disease: results of a prospective pharmacodynamic study.

Smokers have a greater relative benefit of clopidogrel therapy compared with nonsmokers, likely attributed to its enhanced pharmacodynamic (PD) effects. However, to date, all PD studies have been conducted in patients on dual antiplatelet therapy with aspirin and clopidogrel, and it is unknown whether clopidogrel monotherapy can offer more effective antithrombotic effects compared with aspirin alone among smoking patients. Sixty aspirin-treated (81 mg/day) patients with vascular disease, classified as nonsmokers, light smokers, and heavy smokers according to cotinine serum levels, were enrolled. Patients were switched to clopidogrel (75 mg/day) monotherapy for 7-10 days. PD assessments were performed before and after switch by multiple electrode aggregometry (MEA) and kaolin-activated thromboelastography (TEG). Complete PD data were obtained in 57 patients (nonsmokers, n = 27; light smokers, n = 13; heavy smokers, n = 17). On treatment platelet reactivity following MEA, adenosine diphosphate (ADP) + prostaglandin E1 (PGE1) and thrombin receptor-activating peptide (TRAP) stimuli were significantly lower among heavy smokers following switch to clopidogrel. A significant inverse effect was observed with MEA arachidonic acid (ASPI), while neutral findings were shown with MEA collagen (COLL) stimulus. Thrombin and fibrin activity assessed by clot generation parameters were all nonsignificantly different but showed trends towards enhanced antithrombotic activity with clopidogrel among heavy smokers. In heavy smokers with vascular disease manifestations, clopidogrel is associated with enhanced platelet inhibitory effects, affecting purinergic and non-purinergic pathways, compared with aspirin as measured by MEA. Moreover, among smokers, clopidogrel offers trends towards enhanced effects on parameters of clot generation measured by TEG.

Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 µM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 µM: p=0.888; 3 µM: p=0.524; 10 µM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators.

Effects of pioglitazone on platelet P2Y12-mediated signalling in clopidogrel-treated patients with type 2 diabetes mellitus.

Patients with type 2 diabetes mellitus (T2DM) have impaired clopidogrel-induced antiplatelet effects, which may be in part attributed to their reduced sensitivity to insulin and consequently, results in upregulation of the P2Y12 signalling pathway. It has been hypothesised that insulin sensitising strategies may enhance clopidogrel-mediated P2Y12 inhibitory effects. The aim of this pilot pharmacodynamics (PD) study was to assess the impact of pioglitazone on clopidogrel-mediated P2Y12 inhibitory effects in patients with T2DM. This was a prospective, randomised, double-blind, placebo-controlled, cross-over PD study. Patients with T2DM and stable coronary artery disease on maintenance aspirin and clopidogrel were randomised to receive either pioglitazone 30 mg or matching placebo daily for 14 days. PD assessments were measured at baseline, 14 days after randomisation, at the end of the wash-out period, and 14 days after cross-over. The primary endpoint measure was maximal platelet aggregation (MPA) to 20 µM adenosine diphosphate (ADP) as assessed by light transmittance aggregometry (LTA). Flow cytometric analysis of vasodilator-stimulated phosphoprotein phosphorylation (VASP-PRI), and VerifyNow P2Y12 testing were also performed. A total of 15 randomised patients completed the study. MPA to 20 µM ADP (primary endpoint) was not significantly different with pioglitazone compared with placebo (49.53 ± 4.76 vs. 52.52 ± 3.89%; p = 0.594). Similarly, other PD measures did not differ significantly between the groups. In conclusion, in patients with T2DM on maintenance aspirin and clopidogrel therapy, the adjunctive use of pioglitazone does not result in enhanced inhibition of platelet P2Y12 mediated signalling.

Cigarette smoking is associated with a dose-response effect in clopidogrel-treated patients with diabetes mellitus and coronary artery disease: results of a pharmacodynamic study.

OBJECTIVES: This study sought to assess the presence of a dose-response effect of cigarette smoking and its impact on high on-treatment platelet reactivity (HPR) in patients with diabetes mellitus treated with clopidogrel. BACKGROUND: Cigarette smoking is an inducer of cytochrome P450 1A2, a hepatic enzyme involved in clopidogrel metabolism. If cigarette smoking is associated with a dose-response effect on pharmacodynamic measures in clopidogrel-treated patients is unknown. METHODS: A total of 134 type 2 diabetes mellitus patients on maintenance aspirin and clopidogrel therapy were studied. Patients were divided into 3 groups according to cotinine levels: <3 ng/ml (nonsmokers), 3 to 199 ng/ml (light smokers), and ≥ 200 ng/ml (heavy smokers). Platelet function was assessed by light transmittance aggregometry, VerifyNow P2Y12 assay (Accumetrics, San Diego, California), and vasodilator-stimulated phosphoprotein. Rates of HPR were defined using established cutoff values. RESULTS: A dose-response effect was observed for all pharmacodynamic parameters tested. Serum cotinine levels were inversely associated with platelet reactivity as assessed by light transmittance aggregometry using 5 and 20 µmol/l adenosine diphosphate (p < 0.0001 for all). Accordingly, platelet disaggregation increased with levels of serum cotinine (p < 0.0001). Similar results were found with P2Y(12) reaction units (p < 0.0001) and inhibition of platelet aggregation (p = 0.005) as defined by VerifyNow P2Y12 testing, and platelet reactivity index (p = 0.002) as assessed by vasodilator-stimulated phosphoprotein. Higher serum cotinine levels were significantly associated with lower rates of HPR, as defined according to various pharmacodynamic cutoff measures. CONCLUSIONS: Cigarette smoking is associated with a dose-response effect on clopidogrel-induced antiplatelet effects and lower rates of HPR in diabetes mellitus patients.