Platelet Aggregation Unchanged by Lipoprotein-Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials

et al. (2014) Platelet Aggregation Unchanged by Lipoprotein-Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials. PLoS ONE 9(1): e83094. doi:10.1371/journal.pone.0083094 Platelet Aggregation Unchanged by Lipoprotein- Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials Bonnie C. Shaddinger 0 Yanmei Xu 0 James H. Roger 0 Colin H. Macphee 0 Malcolm Handel 0 Charlotte A. Baidoo 0 Mindy Magee 0 John J. Lepore 0 Dennis L. Sprecher 0 Jeffrey S. Berger, New York University School of Medicine, United States of America 0 1 Discovery Medicine, Heart Failure Discovery Performance Unit, Metabolic Pathways and Cardiovascular Therapeutic Area, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America, 2 Discovery Biometrics, GlaxoSmithKline, King of Prussia, Pennsylvania, United States of America, 3 Research Statistics Unit , GlaxoSmithKline, Stockley Park , United Kingdom , 4 Janssen-Cilag Pty Ltd , Macquarie Park, New South Wales , Australia Background: We explored the theorized upregulation of platelet-activating factor (PAF)- mediated biologic responses following lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition using human platelet aggregation studies in an in vitro experiment and in 2 clinical trials. Methods and Results: Full platelet aggregation concentration response curves were generated in vitro to several platelet agonists in human plasma samples pretreated with rilapladib (selective Lp-PLA2 inhibitor) or vehicle. This was followed by a randomized, double-blind crossover study in healthy adult men (n = 26) employing a single-agonist dose assay of platelet aggregation, after treatment of subjects with 250 mg oral rilapladib or placebo once daily for 14 days. This study was followed by a second randomized, double-blind parallel-group trial in healthy adult men (n = 58) also treated with 250 mg oral rilapladib or placebo once daily for 14 days using a full range of 10 collagen concentrations (0-10 mg/ml) for characterizing EC50 values for platelet aggregation for each subject. Both clinical studies were conducted at the GlaxoSmithKline Medicines Research Unit in the Prince of Wales Hospital, Sydney, Australia. EC50 values derived from multiple agonist concentrations were compared and no pro-aggregant signals were observed during exposure to rilapladib in any of these platelet studies, despite Lp-PLA2 inhibition exceeding 90%. An increase in collagen-mediated aggregation was observed 3 weeks post drug termination in the crossover study (15.4% vs baseline; 95% confidence interval [CI], 3.927.0), which was not observed during the treatment phase and was not observed in the parallel-group study employing a more robust EC50 examination. Conclusions: Lp-PLA2 inhibition does not enhance platelet aggregation. Trial Registration: 1) Study 1: ClinicalTrials.gov NCT01745458 2) Study 2: ClinicalTrials.gov NCT00387257 PLOS ONE | www.plosone.org - Competing Interests: BCS, YX, JHR, CHM, CAB, MM, JJL, and DLS disclose that they are full-time employees of GlaxoSmithKline. MH was a full-time employee of GlaxoSmithKline at the time this study was completed and is currently employed by Janssen-Cilag Pty Ltd. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. Potent, selective, and orally active lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitors are in clinical development to determine their effects on reducing the risk of atherothrombosis and associated clinical sequelae (eg, acute coronary syndromes, ischemic stroke, etc) [14]. However, Lp-PLA2 was initially described as platelet-activating factor (PAF) acetylhydrolase (PAF-AH) because of its ability to hydrolyze PAF in vitro into biologically inactive lyso-PAF [57], when PAF in micromolar concentrations is added to isolated human plasma, [8]. Thus, it has been postulated that Lp-PLA2 inhibition may enhance PAFmediated biology. Based on its role in platelet function, PAF accumulation could theoretically increase platelet aggregation, potentially contributing to adverse thrombotic events in the target population for which Lp-PLA2 inhibitors are being developed [9,10]. Published evidence to date offers mixed results regarding the role of Lp-PLA2 activity in influencing PAF-mediated biology. Although relatively little direct evidence has been published [11,12], multiple studies have reported positive associations between plasma Lp-PLA2 and various disorders in which PAF has been implicated, including asthma, allergic reactions, and anaphylaxis [1318]. Conceptually, endogenous pathways for hydrolysis of PAF may potentially ameliorate PAFs otherwise inflammatory influence. As a result, understanding the relationship between Lp-PLA2 and PAF-mediated responses has been a critical step for advancing the clinical development of Lp-PLA2 inhibitors (darapladib and rilapladib) and initiating large-scale cardiovascular outcomes trials [19,20]. Both darapladib and rilapladib are potent (half maximal inhibitory concentrations of 270 pM and 230 pM, respectively) and selective Lp-PLA2 inhibitors [1]. As such, the highest clinical dose of darapladib (80 mg nonenteric coated or 160 mg enteric coated) can achieve approximately 80% inhibition of plasma LpPLA2 24 hours post dose. The top clinical dose of rilapladib (250 mg enteric coated) can attain an even greater magnitude of inhibition of around 90% at the same 24-hour postdose time point, with earlier time points demonstrating essentially complete inhibition. Therefore, rilapladib represents a more robust inquiry into the relevance of Lp-PLA2 inhibition to PAF-mediated biology and targets a more substantive physiology related to vascular disease. Thus, the purpose of the studies described herein was to explore the effects of the potent Lp-PLA2 inhibitor, rilapladib, on PAF-mediated biologic responsesin particular, platelet aggregation. The protocol for these trials and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. In Vitro Platelet Aggregation Study This study was conducted at GlaxoSmithKline, Upper Merion, PA, between March 15th and July 18th, 2005. Blood donors provided written informed consent to have their blood used for research, and this procedure was approved by the GlaxoSmithKline safety board. Platelet-rich plasma (PRP) was prepared from the blood of 10 healthy human volunteers by centrifugation at 220 g for 15 minutes (females were excluded due to possible confounding of menstrual cycles and/or estrogen therapies). After the removal of PRP, samples were centrifuged at 1500 g for 10 minutes to obtain platelet-poor plasma (PPP). Platelet-rich plasma samples were adjusted to 250,000 platelets/mL using autologous PPP. Light transmission aggregometry was performed using Chronolog aggregometers (Model 570-VS, Chronolog Corporation, Havertown, PA) (...truncated)