http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.940887

ORIGINAL ARTICLE

The effect of prasugrel on ADP-stimulated markers of platelet activation in patients with sickle cell disease Joseph A. Jakubowski1, Chunmei Zhou1, Kenneth J. Winters1, D. Richard Lachno2, Jo Howard3, Christopher D. Payne2, Timothy Mant4, Stipo Jurcevic5, & Andrew L. Frelinger III6 Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA, 2Eli Lilly and Company, Windlesham, Surrey, UK, 3Guy’s and St. Thomas’ Hospital, London, UK, 4Quintiles Drug Research Unit at Guy’s Hospital, London, UK, 5King’s College, London, UK, and 6Division of Hematology/ Oncology, Center for Platelet Research Studies, Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

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Abstract

Keywords

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the noncrisis steady state and greater activation during vaso-occlusive crises (VOC). Prasugrel, a potent inhibitor of ADP-mediated platelet activation and aggregation, may be useful in attenuating VOC. We compared platelet responses to ADP stimulation in patients with SCD and healthy subjects before and after treatment with prasugrel. In a phase 1 study, platelet biomarker levels were assessed in 12 adult patients with SCD and 13 healthy subjects before and after 12 ± 2 days of 5.0 or 7.5 mg/day prasugrel. The following were determined in whole blood samples stimulated with 20 mM ADP: (i) percentages of monocytes and neutrophils with adherent platelets (cell–platelet aggregates); (ii) the relative number (mass) of platelets associated with each monocyte and neutrophil as reported by CD61 mean fluorescence intensity (MFI) of the monocyte–platelet and neutrophil–platelet aggregates; (iii) the percentages of platelets positive for surface expression of CD40 ligand (CD40L), P-selectin (CD62p) and activated glycoprotein IIb-IIIa (GPIIb-IIIa); and (iv) the percentages of platelets and monocyte–platelet aggregates positive for surface tissue factor (TF) expression. At baseline, there were no significant differences between cohorts in the percentages of platelets expressing activation biomarkers. Following 12 days of prasugrel administration, the percentages of platelets expressing activation biomarkers following ADP stimulation were reduced in both cohorts, and there were no significant differences between groups. Both patients with SCD and healthy subjects had significant reductions in the monocyte–platelet and neutrophil–platelet aggregate MFI and the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p50.05). Healthy subjects also had significant reductions in monocyte–platelet aggregate percentages (p ¼ 0.004), neutrophil–platelet aggregate percentages (p ¼ 0.011) and the percentage of CD40L-positive platelets (p ¼ 0.044) that were not observed in patients with SCD. Prasugrel administration to SCD patients attenuates ex vivo ADP-stimulated platelet activation as measured by the percentage of platelets positive for P-selectin and GPIIb–IIIa, thus reducing the proportion of platelets that may participate in aggregates. Furthermore, prasugrel decreases ex vivo ADP-stimulated platelet aggregation with monocytes and neutrophils as measured by the monocyte–platelet and neutrophil–platelet aggregate MFI. This implies that in the presence of prasugrel, fewer platelets adhere to monocytes and neutrophils, which may result in reducing cell–platelet aggregate size. Therefore, reduced platelet reactivity and decreased size of leukocyte–platelet aggregates suggest additional mechanisms by which prasugrel may provide benefit to patients with SCD and support further investigation of possible therapeutic benefits of prasugrel in this population.

ADP, biomarkers, P2Y12 receptor antagonist, platelets, prasugrel, sickle cell disease

Introduction Sickle cell disease (SCD) is an inherited hemoglobinopathy in which an aberrant hemoglobin structure causes red blood cells (RBC) to be rigid, misshapen and susceptible to lysis. The disease is characterised by vaso-occlusion and chronic inflammation, resulting from a complex interplay between sickled RBCs,

Correspondence: Joseph A. Jakubowski, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis 46285, IN, USA. Tel: 317-276-9036. Fax: 317-433-1996. E-mail: [email protected]

History Received 20 May 2014 Revised 24 June 2014 Accepted 30 June 2014 Published online 6 August 2014

endothelial cells, leukocytes, platelets and various plasma proteins. Patients with the disease exhibit a chronic ‘hypercoaguable’ state (increased tissue factor on circulating endothelial cells [1–4]. Prothrombin fragment F1.2 and thrombin-anti-thrombin complexes [5, 6]), with evidence of further activation during episodes of vaso-occlusive crisis (VOC); a complication resulting from ischemia and possible infarction of affected organs which may be manifested clinically as episodic localised pain [7–9]. Platelets of patients with SCD have been reported to be in a higher activation than those of healthy subjects and this heightened reactivity may further increase during VOC. The basal expression of platelet surface proteins such as CD40 ligand

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(CD40L) [4, 10, 11] and P-selectin [5] is increased on the platelets of patients with SCD and this may drive interaction between platelets, endothelial cells and inflammatory cells [5, 10], thus contributing to heterotypic aggregation. Such aggregates, along with sickled erythrocytes and leukocytes, may contribute to vessel occlusion [12]. Sickled RBC are prone to lysis, releasing ADP, a platelet agonist, and that may mediate local activation and aggregation of platelets. Markers of coagulation such as fibrin [13], tissue factor (TF) and thrombin [11, 14] have also been found to be elevated in SCD, both in the steady state and, more intensely, during episodes of VOC. Given the potential involvement of platelets in the pathophysiology of vaso-occlusion in SCD, there may be a role for anti– platelet therapies in reducing the incidence and consequences of VOC. Since erythrocyte-derived ADP may promote platelet activation and aggregation during VOC, ADP receptor antagonists could potentially reduce the frequency and severity of acute crises. Likewise, attenuating platelet activation in the steady state might reduce the degree of chronic low-level ischaemia that contributes to end-organ damage in these patients. Two studies which tested the first generation ADP-receptor antagonist ticlodipine resulted in mixed findings. In a small (N ¼ 9) 4-week study, a decrease in platelet activity was seen ex vivo, but the rate of painful crises during that time period did not change [15]. However, in a larger (N ¼ 140) placebo-controlled, 6-month study, rates of VOC were reduced significantly in those patients treated with ticlodipine [16]. Prasugrel is a third generation irreversible platelet P2Y12 ADPreceptor antagonist, which has been shown to effectively inhibit platelet activation and aggregation [17]. The pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of prasugrel have been fully described in healthy control subjects and in patients with coronary artery disease [18, 19] and were recently characterised in patients with SCD [20]. In this phase 1b, openlabel trial, a variety of platelet aggregation assays demonstrated that prasugrel reduced platelet reactivity to ADP. Using data from the same study, we have reported on unstimulated levels of cellular and soluble biomarkers of platelet activation and coagulation and found baseline elevations of biomarkers and attenuation of several of these markers following 12 days of prasugrel administration, suggesting a role for ADP as a driver of in vivo activation in SCD [21]. In the present analysis we assess the effect of prasugrel on ex vivo ADP-stimulated markers of platelet activation in patients with SCD and healthy subjects. Ex vivo stimulation of blood samples with ADP may, to some extent, reflect in vivo hemolysis and ADP release in SCD.

Methods Study design Daiichi Sankyo Co., Ltd and Eli Lilly and Company sponsored this single-centre, open-label trial completed between July 2010 and January 2011 (ClinicalTrials.gov Identifier: NCT01178099). A single 10-mg oral dose of prasugrel was given to patients with SCD and healthy controls, followed by 12 ± 2 days of either 5 mg/day (for those weighing 560 kg) or 7.5 mg/day (for those weighing 60 kg) of oral prasugrel.

Platelets, Early Online: 1–6

(within 10 years). Further details can be found in the primary publication [20]. Cellular biomarkers Venous blood samples were collected at baseline (1 hour before the first dose of prasugrel) and on Day 12, 24 hours after the last 5-mg or 7.5-mg dose of prasugrel. We compared biomarker levels between groups at baseline and at Day 12, and within groups we assessed the change in biomarker levels from baseline to Day 12 of prasugrel administration. Biomarker expression was assessed by flow cytometry within 2 hours of blood collection. To measure monocyte–platelet aggregates and TF on monocyte–platelet aggregates, whole blood samples collected into 3.2% citrate were incubated for 10 minutes with a mixture of ADP and the appropriate fluorescent antibodies (all antibodies were from BD Biosciences, Oxford, England). As previously described, monocyte and neutrophil cell populations were distinguished within whole blood on the basis of their differential expression of CD14 and light scatter properties [22–25]. The presence of monocyte–platelet and neutrophil– platelet aggregates was then determined by assessing the binding of a platelet-specific antibody, CD61, to these cells. TF present on the surface of the monocyte–platelet aggregates was measured using a CD142-specific antibody. The reactivity of platelets to ADP was evaluated by measuring activation-dependent changes in the platelet surface expression of P-selectin (CD62P), activated glycoprotein (GP) IIb-IIIa (using the activation-dependent monoclonal antibody, PAC1), CD40L (CD154) and TF (CD142) by flow cytometry after staining with antibodies specific for these activation-dependent markers [23]. Whole blood was collected into sodium citrate anti-coagulant and 5 mL was aliquoted into tubes containing ADP and fluorescent antibodies. Phosphate-Buffered Saline (Pbs) was added to adjust to 100 ml total volume in all tubes. After 10 minutes incubation at room temperature, the samples were fixed by the addition of 1 ml of 1% paraformaldehyde. Samples were maintained at room temperature until flow cytometric analysis. Platelets were identified by size, light scatter pattern and binding of a CD61specific antibody. Flow cytometric analysis was performed using a FACS Calibur Cytometer and CellQuest Pro software (both BD Biosciences, San Jose, CA). Results are presented as percent of positive cells expressing a given marker above the background control level of staining. For platelet activation markers, this related to the proportion of platelets that may participate in aggregates or inflammatory processes. Results for monocyte–platelet and neutrophil–platelet aggregates are presented as the percentages of monocytes or neutrophils with adherent platelets (e.g. the percent monocyte–platelet and neutrophil–platelet aggregates) reflecting the proportion of monocytes and neutrophils that may participate in an inflammatory response, and as the mean fluorescence intensity (MFI) of the platelet marker (CD61) per monocyte–platelet and neutrophil–platelet aggregate (e.g. monocyte–platelet aggregate CD61 MFI and neutrophil–platelet aggregate CD61 MFI), reflecting the approximate number (mass) of platelets associated with and stimulating each monocyte or neutrophil. Statistical analysis

Participants Patients were men and women with SCD genotype HbSS who were between 18 and 60 years of age and weighed between 50 and 100 kg. Patients with an occurrence of VOC within 1 month of screening were excluded. Healthy control subjects were matched for gender, weight (560 kg and 60 kg within 10 kg) and age

Values are presented as mean and standard deviation (SD) for continuous variables and as percentages for categorical variables, unless otherwise noted. A 1-sample t-test was used to analyse changes from baseline to Day 12 within each population. Monocyte–platelet aggregate CD61 MFI and neutrophil–platelet aggregate CD61 MFI data were highly skewed and so were also

Platelets in prasugrel-treated SCD patients

DOI: 10.3109/09537104.2014.940887

analysed using the non-parametric Wilcoxon signed-rank test. Biomarker levels at baseline and Day 12 were compared between patients with SCD and healthy subjects using a 2-sample t-test. A p value 50.05 was considered statistically significant for all statistical comparisons.

Results

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Demographics The 13 patients with SCD and 13 healthy subjects enrolled in this study have previously been described [20]. Due to poor venous access, one patient with SCD discontinued the study following the baseline venipuncture, immediately after the first dose of prasugrel. All healthy subjects completed the study. Healthy subjects ranged in age from 19 to 42 years with a mean of 27 years; patients with SCD ranged in age from 19 to 50 years with a mean of 29 years. All patients with SCD were of African descent and had the HbSS genotype. The majority of healthy subjects were white (69% white, 23% Black, 8% Asian). The mean weight for healthy subjects was 64.5 kg and was 63.3 kg for patients with SCD. Cellular platelet biomarkers At baseline, in vitro ADP-stimulated whole blood samples from patients with SCD and healthy subjects showed similar values for the percentage of platelets positive for surface-expressed CD40L, P-selectin, activated GPIIb-IIIa, the monocyte–platelet aggregates percentage and MFI, neutrophil–platelet aggregate percentage and MFI, the percentage of circulating platelets and monocyte– platelet aggregates positive for surface TF (p40.05 for all comparisons, Table I and Figures 1 and 2). Likewise, after 12 days of prasugrel administration, there were no significant differences between patients and healthy subjects in these biomarkers (Table I and Figures 1 and 2). Both SCD patients and healthy subjects showed significant reductions in the ADP-stimulated monocyte– platelet aggregate CD61 MFI (Figure 1C), neutrophil–platelet aggregate CD61 MFI (Figure 1D), and the percent of platelets positive for P-selectin (Figure 2B) and activated GPIIbIIIa (Figure 2C) (p50.05 for each vs. baseline). The healthy subjects also showed significant reductions in ADP-stimulated monocyte–platelet aggregates percentage (Figure 1A, p ¼ 0.004), neutrophil–platelet aggregate percentage (Figure 1B, p ¼ 0.011) and percentage CD40L-positive platelets (Figure 2A, p ¼ 0.044), whereas SCD patients showed numerical reductions in these markers which did not reach statistical significance (Figures 1A and B, and 2A). A similar statistically significant reduction in ADP-stimulated P-selectin and activated GPIIb–IIIa was observed, but no such reduction in CD40L was observed in patients with SCD when these markers were evaluated based on surface density of the marker, as reported by MFI rather than as the percentage of platelets positive for each marker (data not shown).

Discussion This clinical pharmacology study was designed to include a comparison of SCD patients with matched healthy control subjects regarding platelet reactivity to ADP and inhibition thereof by prasugrel. We found that at baseline, there were no significant differences between patients with SCD and healthy subjects in the expression of activation biomarkers by platelets following ADP-stimulation. Following 12 days of prasugrel administration, the expression of ADP-stimulated biomarkers was numerically reduced within each group (excluding tissue factor expression on platelets and monocytes), but there were no significant differences between the SCD patients and healthy

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Table I. ADP-stimulated whole blood biomarkers of platelet activation at baseline and Day 12 of prasugrel administration in healthy subjects and patients with SCD.

Biomarker

Healthy Subjects Patients with SCD (N ¼ 13)c (N ¼ 13) Mean (SD) Mean (SD) p Valueb

Monocyte–platelet aggregates (%) Baseline 81.5 (16.4) Day 12 62.1 (20.0) p Valuea 0.004

82.7 (16.8) 71.1 (18.5) 0.077

0.857 0.258

Neutrophil–platelet aggregates (%) Baseline 38.4 (18.4) Day 12 22.4 (9.2) p Valuea 0.011

34.4 (12.4) 30.7 (16.8) 0.574

0.518 0.133

Monocyte–platelet aggregates (CD61 MFI) Baseline 244.2 (167.1) 287.8 (250.1) Day 12 89.5 (34.0) 134.1 (143.7) p Valuea 0.003 0.009 Wilcoxon p Value 0.001 0.009 Neutrophil–platelet aggregates (CD61 MFI) Baseline 104.9 (45.6) 127.3 (66.4) Day 12 64.9 (13.1) 98.5 (102.0) p Valuea 0.006 0.290 Wilcoxon p Value 50.001 0.034

0.605 0.288

0.327 0.250

CD40L (% CD40L-positive platelets) Baseline 22.3 (3.5) Day 12 19.0 (5.4) p Valuea 0.044

22.0 (5.1) 20.2 (3.3) 0.171

0.870 0.522

P-Selectin (% CD62P-positive platelets) Baseline 75.7 (15.6) Day 12 32.7 (10.5) p Valuea 50.001

73.6 (14.6) 38.4 (14.2) 50.001

0.725 0.266

Activated GPIIb-IIIa (% PAC-1-positive platelets) Baseline 90.5 (7.1) 89.1 (7.6) Day 12 59.4 (13.4) 56.6 (19.0) p Valuea 50.001 50.001

0.654 0.672

Tissue factor expression on circulating platelets (% tissue factor positive platelets) Baseline 5.7 (4.3) 4.8 (3.5) 0.562 Day 12 6.0 (3.8) 5.0 (4.0) 0.506 p Valuea 0.846 0.832 Monocyte–platelet tissue factor expression (% tissue factor-positive monocyte–platelet aggregates) Baseline 5.8 (2.2) 5.6 (2.1) 0.890 Day 12 7.1 (3.4) 5.9 (1.7) 0.311 p Valuea 0.277 0.748 CD40L ¼ CD40 ligand; GPIIb-IIIa ¼ glycoprotein IIb-IIIa; N ¼ number of subjects/patients; SCD ¼ sickle cell disease; SD ¼ standard deviation. a p Values derived from 1-sample t-test. b p Values derived from 2-sample t-test. c N ¼ 13 at baseline and 11 at study end for tissue factor expression on platelets and monocytes, 12 at study end for all other biomarkers in patients with SCD.

control subjects. These latter data suggest that the PK and PD profile of prasugrel in patients with SCD is similar to that in healthy subjects. Both patients with SCD and healthy controls had significant reductions in the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p50.001). Following 12 days of prasugrel, both monocyte–platelet aggregate CD61 MFI and neutrophil platelet aggregate CD61 MFI were significantly reduced in both healthy subjects (p  0.001) and patients with SCD (both p50.05). As the MFI of the monocyte–platelet and neutrophil–platelet aggregates reflects the number or mass of platelets associated with each monocyte or neutrophil, it is implied that the size of the monocyte–platelet and neutrophil–

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p=0.004 100

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40

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Neutrophil-Platelet Aggregates p=0.011

p=0.077

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Monocyte-Platelet Aggregates (% )

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p=0.857

p=0.574

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p=0.133

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p=0.001

p=0.009

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400

200

p=0.605 p=0.288

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Day 12

Healthy

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D a y 12

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The effect of prasugrel on ADP-stimulated markers of platelet activation in patients with sickle cell disease.

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the non-crisis steady state and greater activation during vas...
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