The Effect of Darapladib on Plasma Lipoprotein-Associated Phospholipase

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provided by Elsevier - Publisher Connector Journal of the American College of Cardiology Vol. 51, No. 17, 2008 © 2008 by the American College of Cardiology Foundation ISSN 0735-1097/08/$34.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2007.11.079

CLINICAL RESEARCH Clinical Trial The Effect of Darapladib on Plasma Lipoprotein-Associated Phospholipase

A2 Activity and Cardiovascular Biomarkers in Patients With Stable Coronary Heart Disease or Coronary Heart Disease Risk Equivalent The Results of a Multicenter, Randomized, Double-Blind, Placebo-Controlled Study

Emile R. Mohler III, MD, FACC,* Christie M. Ballantyne, MD, FACC,† Michael H. Davidson, MD, FACC,‡ Markolf Hanefeld, MD, PHD,§ Luis M. Ruilope, MD, PHD,ʈ Joel L. Johnson, PHARMD,¶ Andrew Zalewski, MD,¶# for the Darapladib Investigators Philadelphia, Pennsylvania; Houston, Texas; Chicago, Illinois; Dresden, Germany; Madrid, Spain; and Research Triangle Park, North Carolina

Objectives This study examined the effects of darapladib, a selective lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor, on biomarkers of cardiovascular (CV) risk.

Background Elevated Lp-PLA2 levels are associated with an increased risk of CV events. Methods Coronary heart disease (CHD) and CHD-risk equivalent patients (n ϭ 959) receiving atorvastatin (20 or 80 mg) were randomized to oral darapladib 40 mg, 80 mg, 160 mg, or placebo once daily for 12 weeks. Blood samples

were analyzed for Lp-PLA2 activity and other biomarkers. Ϯ Results Baseline low-density lipoprotein cholesterol (LDL-C) was 67 22 mg/dl. Plasma Lp-PLA2 was higher in older patients (Ն75 years), in men, in those taking atorvastatin 20 mg, at LDL-C Ն70 mg/dl or high-density lipoprotein cholesterol (HDL-C) Ͻ40 mg/dl, or in those with documented vascular disease (multivariate regression; Ͻ p 0.01). Darapladib 40, 80, and 160 mg inhibited Lp-PLA2 activity by approximately 43%, 55%, and 66% compared with placebo (p Ͻ 0.001 weeks 4 and 12). Sustained dose-dependent inhibition was noted overall in both atorvastatin groups and at different baseline LDL-C (Ն70 vs. Ͻ70 mg/dl) and HDL-C (Ͻ40 vs. Ն40 mg/dl). At 12 weeks, darapladib 160 mg decreased interleukin (IL)-6 by 12.3% (95% conﬁdence interval [CI] Ϫ22% to Ϫ1%; p ϭ 0.028) and high-sensitivity C-reactive protein (hs-CRP) by 13.0% (95% CI Ϫ28% to ϩ5%; p ϭ 0.15)

compared with placebo. The Lp-PLA2 inhibition produced no detrimental effects on platelet biomarkers

(P-selectin, CD40 ligand, urinary 11-dehydrothromboxane B2). No major safety concerns were noted.

Conclusions Darapladib produced sustained inhibition of plasma Lp-PLA2 activity in patients receiving intensive atorvastatin therapy. Changes in IL-6 and hs-CRP after 12 weeks of darapladib 160 mg suggest a possible reduction in

inﬂammatory burden. Further studies will determine whether Lp-PLA2 inhibition is associated with favorable effects on CV events. (SB-480848 in Subjects With Coronary Heart Disease; NCT00269048) (J Am Coll Cardiol 2008;51:1632–41) © 2008 by the American College of Cardiology Foundation

Residual risk of cardiovascular (CV) events persists even in patients with aggressively controlled risk factors (1). These ﬁndings suggest the presence of additional modiﬁable From the *University of Pennsylvania School of Medicine, Philadelphia, Pennsylva- nia; †Baylor College of Medicine and Methodist DeBakey Heart Center, Houston, See page 1642 Texas; ‡Radiant Research, Rush University Medical Center, Chicago, Illinois; §Center for Clinical Studies, Dresden, Germany; ʈHospital 12 de Octubre, Madrid, Spain; ¶GlaxoSmithKline, Philadelphia, Pennsylvania, and Research Triangle Park, mechanisms of the underlying atherosclerotic process. To North Carolina; and #Thomas Jefferson University, Philadelphia, Pennsylvania. Manuscript received September 26, 2007; revised manuscript received November 7, this end, atherosclerosis initiation, its progression, and the 2007, accepted November 21, 2007. transition to acute coronary syndromes (e.g., due to plaque JACC Vol. 51, No. 17, 2008 Mohler III et al. 1633

April 29, 2008:1632–41 Lp-PLA2 and Cardiovascular Biomarkers rupture) are accompanied by focal inflammation in lesion- effects of darapladib on several Abbreviations prone areas (2). In addition, elevated levels of circulating CV biomarkers of risk and safety and Acronyms were examined. coronary heart ؍ inflammatory biomarkers have been reported in several CHD populations at risk (3,4). Recent clinical trials further disease Methods confidence interval ؍ implied that achieving lower levels of high-sensitivity CI C-reactive protein (hs-CRP) in conjunction with aggressive cardiovascular ؍ Study design. The multicen- CV low-density lipoprotein cholesterol (LDL-C)–lowering ؍ ,ter, randomized, double-blind Ͻ HDL-C high-density ( 70 mg/dl) are associated with reduced progression of placebo-controlled, parallel-group lipoprotein cholesterol high-sensitivity ؍ coronary atheroma and fewer CV events (5,6). Nevertheless, study that was conducted in 110 hs-CRP statin trials offer only indirect evidence of the link between sites in 15 countries from Novem- C-reactive protein interleukin ؍ inflammation and clinical events because of the primary ber 2005 to June 2006 (a list of IL low-density ؍ benefit of concomitant lipid-lowering (7). Other treatments investigators is provided in the LDL-C that are proven to reduce CV events lack consistent anti- Online Appendix). This dose- lipoprotein cholesterol ؍ inflammatory effects in the clinical setting (e.g., aspirin or ranging study evaluated the ability Lp-PLA2 lipoprotein- angiotensin-converting enzyme inhibitors) (8). Thus, the of darapladib to produce sustained associated phospholipase A2 -platelet ؍ causal role of inflammation in the progression of atheroscle- PAF-AH inhibition of plasma Lp-PLA2 ac- rotic vascular disease and CV events remains to be proven by tivity in subjects with stable coro- activating factor- acetylhydrolase pharmacologic interventions that specifically target the in- nary heart disease (CHD) or flammatory cascade. CHD-risk equivalent receiving Lipoprotein-associated phospholipase A2 (Lp-PLA2)is concomitant atorvastatin therapy. Those subjects not currently an emerging biomarker of CV risk that is pharmacologically receiving statin therapy first received open-label atorvastatin 20 modifiable and therefore well positioned to address novel mg for 2 weeks. All eligible subjects were initially randomized mechanisms of atherosclerotic vascular disease. Lipoprotein- to double-blind atorvastatin 20 or 80 mg once daily (Fig. 1). associated phospholipase A2 is produced by inflammatory After 4 weeks, subjects who tolerated atorvastatin therapy and cells involved in atherogenesis (macrophages, T cells, mast achieved LDL-C levels of Յ115 mg/dl were then randomized cells), is predominantly bound to atherogenic lipoproteins, to concomitant administration of darapladib 40 mg, 80 mg, and accumulates in human atherosclerotic lesions (9). Al- 160 mg, or placebo once daily for 12 weeks. After discon- though this enzyme has been referred to as platelet- tinuing darapladib or placebo, subjects continued on activating factor-acetylhydrolase (PAF-AH), Lp-PLA2 ex- double-blind atorvastatin for an additional 2 weeks. Ethics hibits much broader substrate specificity (10,11). In committees approved the protocol, and all subjects provided particular, Lp-PLA2 rapidly degrades polar phospholipids written informed consent before enrollment in the study. present in oxidized LDL-C, releasing downstream products Study subjects. Subjects ages 18 to 80 years with stable such as lysophosphatidylcholine species and oxidized non- CHD or CHD-risk equivalent (defined as diabetes mellitus Ͼ esterified fatty acids (12). These products of the Lp-PLA2 requiring hypoglycemic medication; carotid stenosis 50%; reaction exhibit a wide range of pro-inflammatory and prior carotid surgery or stenting; peripheral arterial disease; pro-apoptotic effects in experimental settings (9). In this or a cluster of risk factors resulting in 10-year risk for Ͼ context, Lp-PLA2 could be proposed as the enzyme that coronary events 20% according to Framingham Risk links oxidized LDL-C with atherosclerosis progression and Score) were eligible for screening. Main exclusion criteria plaque vulnerability. This hypothesis is supported by studies were CV event or vascular procedure within the preceding 6 that suggest increased risk of CV events with elevated levels months, contraindications to double-blind atorvastatin Ͼ of circulating Lp-PLA2, the presence of high expression of therapy, serum triglycerides 400 mg/dl, elevated liver Lp-PLA2 in apoptotic macrophages in high-risk human function tests (alanine transaminase, aspartate transaminase, coronary lesions, and the ability to alter the phenotype of alkaline phosphatase, or total bilirubin Ͼ1.5 ϫ ULN), Ͼ inflammatory cells with Lp-PLA2 inhibition in vitro (13– hemoglobin subtype A1c 10%, chronic inflammatory Ͼ 18). The view of Lp-PLA2 as a pro-atherogenic enzyme, diseases, inadequately controlled hypertension ( 160 mm however, has not been uniformly accepted, because of Hg systolic or Ͼ100 mm Hg diastolic), renal disorder Ͼ contradictory results of Lp-PLA2 overexpression in preclin- (serum creatinine 2.5 mg/dl), severe congestive heart ical models of atherosclerosis, a theoretical concern that failure (New York Heart Association functional class III or Ͼ inhibition of Lp-PLA2 might lead to increase in platelet IV), and prolonged QTc interval ( 440 ms for men or activation, and conflicting data with a genetic variant of Ͼ450 ms for women).

Lp-PLA2 in Japanese subjects (9,19). Assessments. Blood assessments were carried out in the In this study, we sought to investigate whether darapladib fasting state at baseline (before randomization to darapladib

(SB-480848), a selective Lp-PLA2 inhibitor, produces sus- or placebo) and at 4 and 12 weeks of dosing with darapladib tained inhibition of plasma Lp-PLA2 activity in CV pa- or placebo. These included total cholesterol, high-density tients treated aggressively with atorvastatin. In addition, the lipoprotein-cholesterol (HDL-C), calculated LDL-C, tri- 1634 Mohler III et al. JACC Vol. 51, No. 17, 2008

Lp-PLA2 and Cardiovascular Biomarkers April 29, 2008:1632–41

Figure 1 Subject Disposition

All eligible subjects were initially randomized to double-blind atorvastatin 20 or 80 mg once daily. After 4 weeks, subjects who tolerated atorvastatin therapy and achieved low-density lipoprotein cholesterol levels of Յ115 mg/dl were then randomized to concomitant administration of darapladib 40 mg, 80 mg, 160 mg, or placebo once daily for 12 weeks. The number of subjects and reason for discontinuation are depicted in this ﬁgure. glycerides, and routine safety labs. In addition, plasma to 1.132 (equivalent to Ϯ 0.124 on the log scale), and 90%

Lp-PLA2 activity, inflammatory biomarkers, and platelet- power. With these assumptions, 196 subjects/group were related biomarkers were measured at baseline and at 4 and required, without adjustment for multiplicity or atorvastatin 12 weeks approximately 24 h after dosing with darapladib treatment group. Thus, assuming a 15% dropout rate, a or placebo. Plasma Lp-PLA2 activity and platelet-related minimum of 920 subjects (or 230/treatment group) were biomarkers were also measured 2 weeks after discontinuing planned to be randomized. darapladib or placebo. Supine vital signs were collected at Secondary end points included dose-response of darapladib baseline, 4, 8, and 12 weeks, and 2 weeks after discontinuing over log-transformed Lp–PLA2 activity and changes in darapladib or placebo; a physical examination and electro- inflammatory biomarkers and platelet-related biomarkers. Pri- cardiogram were also performed at baseline and at 4 and mary and secondary end points of changes in Lp-PLA2 activity 12 weeks. and inflammatory and platelet-related biomarkers were all

Biochemical parameters. The Lp-PLA2 activity was mea- assessed with analysis of covariance. Treatment group and sured in plasma by a colorimetric assay as previously atorvastatin doses (20 and 80 mg) were included in all described (20). The assay characteristics included intra-assay statistical models, with baseline values also included for precision of 1.7% and inter-assay precision of 4.8%. In a subset inflammatory and platelet-related biomarkers. Interactions of subjects, Lp-PLA2 activity also was measured by a for atorvastatin level by treatment group were tested at the radiometric assay with [3H]-platelet-activating factor as a 10% significance level. Correlations were assessed with substrate (21). The assay (Quest Diagnostics, Lyndhurst, Pearson product moment correlation coefficients. New Jersey) included intra-assay precision: low 3.0%, mid 4.5%, high 2.2% and interassay precision: low 6.5%, mid Results 7.4%, high 8.8%. Details regarding the analysis of the remaining biomarkers are provided online. Study population and baseline characteristics. Figure 1 Statistical analyses. The primary efficacy end point was depicts subject flow through the study. A total of 1,410 sustained inhibition of plasma Lp-PLA2 activity measured subjects were screened for eligibility, 1,082 were assigned to as the change from 4 to 12 weeks at trough levels of double-blind atorvastatin, and 964 subjects were subsequently darapladib. The primary comparison was based on 2-sided randomized to darapladib or placebo. Five of these subjects did 95% confidence intervals (CIs) for the change in log- not receive any dose of darapladib or placebo; therefore 959 transformed Lp-PLA2 activity from week 4 to week 12 for subjects were included in the analyses. The exposure to each darapladib group. Sample size was calculated with an darapladib or placebo was 81 Ϯ 13 days (mean Ϯ SD).

SD for change in log-transformed Lp-PLA2 activity values Baseline characteristics of the subject population are of 0.34 from a previous study, an equivalence limit of 0.883 presented in Table 1. At study entry, 75% of subjects were JACC Vol. 51, No. 17, 2008 Mohler III et al. 1635

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Baseline Characteristics of Study Population variable, it showed the strongest correlation with baseline Table 1 Baseline Characteristics of Study Population levels of total cholesterol (Pearson r ϭ 0.345, p Ͻ 0.0001), Demographic data LDL-C (r ϭ 0.459, p Ͻ 0.0001), and inverse correlation Age, yrs (mean)* 63 Ϯ 9 with HDL-C (r ϭϪ0.229, p Ͻ 0.0001). Baseline levels of Men/women (%) 71/29 Lp-PLA2 activity and inflammatory biomarkers exhibited Body mass index (mean, kg/m2)* 30 Ϯ 6 weak and nonsignificant correlations. Risk factors (%) Effects of darapladib on Lp-PLA2. As shown in Figure 2, Diabetes† 49 baseline Lp-PLA2 activity (colorimetric assay) was compa- Hypertension 74 rable among subjects randomized to placebo or darapladib Dyslipidemia 61 Current smoking 14 40, 80, or 160 mg. In placebo-treated subjects, Lp-PLA2 Past medical history (%) activity remained unchanged over the study duration. In Coronary heart disease 54 contrast, there was a clear dose-dependent reduction of Peripheral arterial disease 18 Lp-PLA2 activity after 4 weeks of darapladib dosing (first Stroke or transient ischemic attack 6 measurement after randomization), which was also observed No documented vascular disease‡ 39 at 12 weeks (p Ͻ 0.001 for all doses). When compared with Concomitant medications (%) placebo, the observed inhibition of Lp-PLA2 activity was Aspirin Ն75 mg, clopidogrel or ticlopidine 83 sustained at approximately 43%, 55%, and 66% for darap- Atorvastatin 100 ladib 40, 80, and 160 mg, respectively. This inhibition ACE inhibitor or ARB 68 corresponded to achieved levels of Lp-PLA2 activity (nmol/ Beta-blocker 48 min/ml) at 12 weeks as follows: placebo: 124 (95% CI 120 Thiazolidinedione 10 Baseline lipid and HbA1c values* to 128), darapladib 40 mg: 68 (95% CI 65 to 71), darapladib Total cholesterol (mg/dl) 142 Ϯ 28 80 mg: 56 (95% CI 53 to 60), and darapladib 160 mg: 43 Ϯ LDL-C (mg/dl) 67 Ϯ 22 (95% CI 40 to 45). After darapladib discontinuation (15 HDL-C (mg/dl) 50 Ϯ 13 4 days), levels of Lp-PLA2 activity returned toward baseline Triglycerides (mg/dl) 127 Ϯ 65 although they remained lower. In a subset of subjects (n ϭ HbA1c (%) 6.7 Ϯ 1.04 323), the enzyme inhibition was confirmed with radiometric Baseline biomarker values§ assay of Lp-PLA2 activity that reproduced the shape of dose Lp-PLA2 activity (nmol/min/ml) 123 (107, 145) response. As shown in Online Figure 1, excellent Pearson hs-CRP (mg/l) 1.17 (0.6, 2.5) correlations between the colorimetric and radiometric assays IL-6 (ng/l) 2.53 (1.7, 3.5) were found for the percent inhibition from baseline to week MPO (pmol/l) 556 (380, 755) 12 (r ϭ 0.97, p Ͻ 0.0001) and between achieved levels of MMP-9 (␮g/l) 499 (273, 877) ϭ Ͻ Lp-PLA2 activity at week 12 (r 0.96, p 0.0001). *Presented as arithmetic mean Ϯ SD; †denotes diabetes requiring pharmacotherapy; ‡refers to Lipoprotein-associated phospholipase A2 mass was also patients with Framingham Risk Score Ͼ20%; §presented as geometric mean (interquartile range: ϭ 25th, 75th percentile). measured in a subset of subjects (n 228). Darapladib ACE ϭ angiotensin-converting enzyme; ARB ϭ angiotensin receptor blocker; HbA1c ϭ hemoglo- reduced Lp-PLA2 mass at week 12 compared with placebo bin subtype A1c; HDL-C ϭ high-density lipoprotein cholesterol; hs-CRP ϭ high-sensitivity C-reactive Ͻ protein; IL ϭ interleukin; LDL-C ϭ low-density lipoprotein cholesterol; Lp-PLA2 ϭ lipoprotein- (p 0.001 for all doses) but without a dose response (9.6%, ϭ ϭ associated phospholipase A2; MMP-9 matrix metalloproteinase-9; MPO myeloperoxidase. 12.9%, and 9.3% reductions by darapladib 40, 80, and 160 mg, respectively). receiving statin therapy. As required by the protocol, all Lipid measurements and the effects of darapladib. Treat- subjects were assigned atorvastatin and received a high level ment with darapladib did not modify total cholesterol, of contemporary medical care. Demographic data, past LDL-C, HDL-C, or triglyceride levels at week 4 (not medical history, and concomitant treatments were balanced shown) or at week 12 as compared with placebo (Table 3). among subgroups of subjects randomized to placebo or the Hence, the observed changes in Lp-PLA2 activity levels in 3 dosing regimens of darapladib. subjects taking darapladib cannot be attributed to changes in

Baseline Lp-PLA2 activity. As shown in Table 2, baseline lipoprotein carriers of this enzyme. levels of Lp-PLA2 activity were significantly higher in older We explored whether atorvastatin dose modified the subjects, in men, in nondiabetic patients, in those with effect of darapladib on change in Lp-PLA2 activity at week LDL-C Ն70 mg/dl or HDL-C Ͻ40 mg/dl, in those 12 and found no significant interaction (p ϭ 0.60). We also receiving the lower dose of atorvastatin, and in those with explored whether baseline LDL-C or HDL-C levels influ- documented vascular disease. Diabetic subjects had lower enced inhibition of Lp-PLA2 activity over 12 weeks. There Ͻ values of Lp-PLA2 activity than nondiabetic subjects, likely was no interaction between baseline LDL-C ( 70 mg/dl owing to lower LDL-C levels (diabetic: 64 Ϯ 1 mg/dl vs. vs. Ն70 mg/dl) or HDL-C (Ͻ40 mg/dl vs. Ն40 mg/dl) and Ϯ Ͻ nondiabetic: 71 1 mg/dl, p 0.001). In the multivariate the change in Lp-PLA2 activity at 12 weeks for all darap- regression analysis, all of these variables remained statisti- ladib doses (p ϭ 0.12 and p ϭ 0.13, respectively). Within Ͻ cally significant (p 0.01) except for diabetic status. When the darapladib 160 mg group, achieved levels of Lp-PLA2 baseline Lp-PLA2 activity was examined as a continuous activity were similar at 12 weeks at different levels of 1636 Mohler III et al. JACC Vol. 51, No. 17, 2008

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Baseline Lp-PLA2 Activity in Clinical Subgroups

Table 2 Baseline Lp-PLA2 Activity in Clinical Subgroups

Lp-PLA2 (nmol/min/ml)

Variable Subgroup n Geometric Mean 95% CI p Value Age Ͻ75 yrs 857 122 120–124 0.002 Ն75 yrs 80 133 126–140 Gender Female 268 113 110–116 Ͻ0.001 Male 669 128 125–130 Body mass index Ͻ30 kg/m2 528 123 121–125 0.18 Ն30 kg/m2 409 124 121–126 Smoking No 806 123 121–125 0.08 Current 131 127 122–132 Diabetes No 436 129 126–131 Ͻ0.001 Yes 501 119 116–121 LDL-C Ͻ70 mg/dl 537 115 113–118 Ͻ0.001 Ն70 mg/dl 400 135 132–138 HDL-C Ն40 mg/dl 739 121 119–123 Ͻ0.001 Ͻ40 mg/dl 198 134 129–138 Atorvastatin 20 mg 462 130 127–133 Ͻ0.001 80 mg 475 117 114–119 Vascular disease No 336 116 113–118 Ͻ0.001 Yes 601 128 125–130

CI ϭ confidence interval; other abbreviations as in Table 1. baseline LDL-C (Ͻ70 mg/dl [n ϭ 94] vs. Ն70 mg/dl [n ϭ ground atorvastatin therapy and stable CV status of the 51]: 42 nmol/min/ml [95% CI 39 to 45 nmol/min/ml] vs. enrolled subject population. Nonetheless, treatment with 42 nmol/min/ml [95% CI 38 to 48 nmol/min/ml]; p ϭ darapladib 160 mg (n ϭ 161) produced a 20.2% (95% CI NS). Similarly, at different levels of baseline HDL-C, 160 Ϫ31% to Ϫ8%) reduction in hs-CRP at week 12 compared mg dose of darapladib produced similar achieved levels of with baseline (p ϭ 0.003; within group comparison) and a Ն ϭ Ͻ Ϫ ϩ Lp-PLA2 activity ( 40 mg/dl [n 110] vs. 40 mg/dl 13.0% (95% CI 18% to 5%) reduction from baseline [n ϭ 35]: 40 nmol/min/ml [95% CI 38 to 44 nmol/min/ compared with placebo (p ϭ 0.15; between groups compar- ml] vs. 48 nmol/min/ml [95% CI 41 to 55 nmol/min/ml]; ison) (Table 4). When response to darapladib was analyzed ϭ p NS). post hoc according to quartiles of baseline Lp-PLA2 activ- Effects of darapladib on biomarkers of CV risk. At ity, darapladib 160 mg (n ϭ 38) significantly reduced baseline, hs-CRP was low (geometric mean 1.17 mg/l hs-CRP in the highest quartile (reduction of 43.2% [95% [interquartile range 0.6 to 2.5]), owing to intensive back- CI Ϫ64% to Ϫ11%] relative to placebo [n ϭ 43] from 1.17 to 0.79 mg/l in the darapladib 160 mg group vs. 1.26 to 1.45 mg/l in the placebo group, p ϭ 0.013). In regard to interleukin-6 (IL-6), the greatest reduction was again observed with darapladib 160 mg (n ϭ 150) resulting in 21.5% (95% CI Ϫ28% to Ϫ14%) reduction from baseline (p Ͻ 0.001; within group comparison) and 12.3% (95% CI Ϫ22% to Ϫ1%) when compared with placebo (p ϭ 0.028; between groups comparison) (Table 4). When response to darapladib was analyzed post hoc according to quartiles of baseline ϭ Lp-PLA2 activity, darapladib 160 mg (n 39) reduced IL-6 in the highest quartile by 20.5% (95% CI Ϫ38% to ϩ3%) compared with placebo (n ϭ 42) from 2.62 to 2.12 ng/l versus 2.56 to 2.62 ng/l (p ϭ 0.08). These findings are not likely attributable to treatment-induced changes in body weight, because it remained stable over the course Figure 2 Plasma Lp-PLA2 Activity of the study. There were no significant changes in plasma The results are presented as geometric means with 95% confidence interval. myeloperoxidase and matrix metalloproteinase-9 in any All doses of darapladib produced highly significant inhibition of lipoprotein- treatment groups (Table 4). associated phospholipase A2 (Lp-PLA2) activity when compared with placebo at weeks 4 and 12 (p Ͻ 0.001). Theoretical concern that inhibition of Lp-PLA2 activity might adversely affect platelet activity prompted us to JACC Vol. 51, No. 17, 2008 Mohler III et al. 1637

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Effect of Darapladib on Lipid Parameters Table 3 Effect of Darapladib on Lipid Parameters

Placebo Darapladib 40 mg Darapladib 80 mg Darapladib 160 mg Total cholesterol n ϭ 184 n ϭ 167 n ϭ 164 n ϭ 162 Baseline 145 Ϯ 28 141 Ϯ 28 143 Ϯ 31 140 Ϯ 27 12 weeks 144 Ϯ 28 143 Ϯ 32 142 Ϯ 31 141 Ϯ 28 Within group comparison Ϫ0.7 (Ϫ3.2 to ϩ1.8) ϩ1.9 (Ϫ2.2 to ϩ5.9) Ϫ1.3 (Ϫ5.1 to ϩ2.6) ϩ1.3 (Ϫ1.9 to ϩ4.5) Between groups comparison N/A ϩ1.6 (Ϫ2.8 to ϩ6.1) Ϫ0.6 (Ϫ5.1 to ϩ3.9) ϩ0.9 (Ϫ3.6 to ϩ5.3) LDL-C n ϭ 181 n ϭ 166 n ϭ 159 n ϭ 160 Baseline 69 Ϯ 23 67 Ϯ 21 68 Ϯ 21 67 Ϯ 21 12 weeks 66 Ϯ 23 65 Ϯ 25 66 Ϯ 23 64 Ϯ 22 Within group comparison Ϫ3.2 (Ϫ5.2 to Ϫ1.2)* Ϫ2.6 (Ϫ5.8 to ϩ0.7) Ϫ2.6 (Ϫ5.4 to ϩ0.2) Ϫ2.4 (Ϫ5.1 to ϩ0.3) Between groups comparison N/A ϩ0.1 (Ϫ3.4 to ϩ3.6) ϩ0.6 (Ϫ3.0 to ϩ4.1) ϩ0.2 (Ϫ3.3 to ϩ3.8) HDL-C n ϭ 183 n ϭ 167 n ϭ 164 n ϭ 162 Baseline 50 Ϯ 13 50 Ϯ 13 49 Ϯ 13 49 Ϯ 13 12 weeks 50 Ϯ 12 51 Ϯ 12 48 Ϯ 12 50 Ϯ 11 Within group comparison ϩ0.04 (Ϫ1.0 to ϩ1.1) ϩ0.9 (Ϫ0.2 to ϩ1.9) Ϫ0.2 (Ϫ1.3 to ϩ1.0) ϩ1.4 (ϩ0.3 to ϩ2.5)* Between groups comparison N/A ϩ0.9 (Ϫ0.5 to ϩ2.2) Ϫ0.6 (Ϫ2.0 to ϩ0.7) ϩ1.0 (Ϫ0.3 to ϩ2.4) Triglycerides n ϭ 184 n ϭ 167 n ϭ 164 n ϭ 162 Baseline 127 Ϯ 61 118 Ϯ 48 135 Ϯ 80 126 Ϯ 70 12 weeks 142 Ϯ 65 137 Ϯ 67 145 Ϯ 98 137 Ϯ 73 Within group comparison ϩ14.2 (ϩ7.3 to ϩ21.2)* ϩ18.8 (ϩ11.4 to ϩ26.3)* ϩ10.5 (Ϫ3.2 to ϩ24.2) ϩ10.9 (ϩ2.0 to ϩ19.8)* Between groups comparison N/A 2.0 (Ϫ10.5 to ϩ14.5) Ϫ1.6 (Ϫ14.2 to ϩ11.0) Ϫ3.7 (Ϫ16.4 to ϩ8.8)

Baseline and week 12 data are presented as means Ϯ SD (mg/dl). Within group comparisons depict changes from baseline. Between group comparisons indicate changes from baseline relative to placebo. Within group comparisons (mg/dl; mean and 95% CI) were analyzed using paired t test. Between group comparisons (mg/dl; mean and 95% CI) were analyzed with analysis of covariance with baseline value, atorvastatin level, and treatment included as covariates. All subjects on concomitant atorvastatin 20 or 80 mg. Similar results were obtained comparing week 4 of treatment. *p Ͻ 0.05. Abbreviations as in Tables 1 and 2. examine the effects of darapladib on several biomarkers placebo (i.e., between groups comparison) at week 4 (not associated with enhanced platelet aggregation. There was no shown) and week 12 of treatment with darapladib (Table 5). evidence for the increase in any of the measured platelet Similarly, discontinuation of darapladib produced no signif- biomarkers when analyzed for the changes from baseline icant effect on these measurements at follow-up (not (i.e., within group comparison) or in comparison with shown). Effect of Darapladib on Inﬂammatory Biomarkers Table 4 Effect of Darapladib on Inﬂammatory Biomarkers

Placebo Darapladib 40 mg Darapladib 80 mg Darapladib 160 mg hs-CRP (mg/l) n ϭ 177 n ϭ 166 n ϭ 161 n ϭ 161 Baseline 1.09 1.14 1.03 1.28 12 weeks 1.06 1.02 1.02 1.02 Within group comparison (%) Ϫ3.3 (Ϫ15 to ϩ10) Ϫ10.5 (Ϫ22 to ϩ3) Ϫ1.5 (Ϫ17 to ϩ17) Ϫ20.2 (Ϫ31 to Ϫ8)* Between groups comparison (%) N/A Ϫ6.0 (Ϫ22 to ϩ13) Ϫ0.3 (Ϫ18 to ϩ20) Ϫ13.0 (Ϫ18 to ϩ5) IL-6 (ng/l) n ϭ 171 n ϭ 149 n ϭ 146 n ϭ 150 Baseline 2.45 2.57 2.35 2.73 12 weeks 2.28 2.17 2.17 2.14 Within group comparison (%) Ϫ6.9 (Ϫ15 to ϩ2) Ϫ15.5 (Ϫ23 to Ϫ7)* Ϫ7.3 (Ϫ16 to ϩ2) Ϫ21.5 (Ϫ28 to Ϫ14)* Between groups comparison (%) N/A Ϫ7.8 (Ϫ18 to ϩ4) Ϫ2.3 (Ϫ12 to ϩ10) Ϫ12.3 (Ϫ22 to Ϫ1)† MPO (pmol/l) n ϭ 179 n ϭ 165 n ϭ 162 n ϭ 163 Baseline 508 558 524 588 12 weeks 515 552 530 586 Within group comparison (%) 1.4 (Ϫ6toϩ10) Ϫ1.1 (Ϫ9toϩ8) 1.1 (Ϫ8toϩ11) Ϫ0.3 (Ϫ10 to ϩ10) Between groups comparison (%) N/A 1.6 (Ϫ9toϩ14) 0.9 (Ϫ10 to ϩ13) 4.7 (Ϫ6toϩ17) MMP-9 (␮g/l) n ϭ 178 n ϭ 165 n ϭ 161 n ϭ 163 Baseline 471 505 448 475 12 weeks 479 495 421 505 Within group comparison (%) 2.0 (Ϫ9toϩ13) Ϫ2(Ϫ13 to ϩ11) Ϫ6(Ϫ17 to ϩ5) 6.0 (Ϫ6toϩ20) Between groups comparison (%) N/A Ϫ0.8 (Ϫ14 to ϩ15) Ϫ9.8 (Ϫ22 to ϩ4) 4.8 (Ϫ9toϩ21)

Baseline and week 12 data are presented as geometric means. Within group comparisons depict changes from baseline. Between groups comparisons indicate changes from baseline relative to placebo. Within group comparisons were analyzed using paired t test. Between group comparisons were analyzed with analysis of covariance with baseline value, atorvastatin level, and treatment included as covariates. Values in parenthesis are 95% CI. *p Ͻ 0.05; †p ϭ 0.028. Abbreviations as in Tables 1 and 2. 1638 Mohler III et al. JACC Vol. 51, No. 17, 2008

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Effect of Darapladib on Platelet-Related Biomarkers Table 5 Effect of Darapladib on Platelet-Related Biomarkers

Placebo Darapladib 40 mg Darapladib 80 mg Darapladib 160 mg P-selectin (␮g/l) n ϭ 177 n ϭ 162 n ϭ 158 n ϭ 151 Baseline 49.21 49.11 46.80 48.54 12 weeks 47.10 47.37 44.33 46.47 Within group comparison (%) Ϫ4.3 (Ϫ9toϩ1) Ϫ3.9 (Ϫ9toϩ1) Ϫ5.0 (Ϫ11 to ϩ2) Ϫ4.3 (Ϫ9toϩ1) Between groups comparison (%) N/A ϩ1(Ϫ6toϩ8) Ϫ3(Ϫ10 to ϩ4) 0 (Ϫ7toϩ7) CD40L (pg/ml) n ϭ 174 n ϭ 162 n ϭ 158 n ϭ 159 Baseline 354.96 392.06 338.93 335.37 12 weeks 355.15 359.67 333.15 356.26 Within group comparison (%) 0 (Ϫ12 to ϩ14) Ϫ8.0 (Ϫ19 to ϩ3) Ϫ2.0 (Ϫ17 to ϩ17) ϩ6.0 (Ϫ7toϩ21) Between groups comparison (%) N/A Ϫ3.9 (Ϫ11 to ϩ14) Ϫ3.9 (Ϫ11 to ϩ14) ϩ3.4 (Ϫ13 to ϩ22) ϭ ϭ ϭ ϭ U-11-dehydro-TxB2 (ng/mmol Cr) n 168 n 158 n 158 n 154 Baseline 56.58 55.53 52.73 54.74 12 weeks 50.73 47.92 48.20 49.75 Within group comparison (%) Ϫ10.0 (Ϫ17 to Ϫ3)* Ϫ14.0 (Ϫ23 to Ϫ4)* Ϫ9.0 (Ϫ17 to ϩ0.4) Ϫ9.0 (Ϫ19 to ϩ1) Between groups comparison (%) N/A Ϫ4.5 (Ϫ16 to ϩ8) Ϫ0.6 (Ϫ12 to ϩ13) 0.2 (Ϫ12 to ϩ13)

Baseline and 12 week data are presented as geometric means. Within group comparisons depict changes from baseline. Between groups comparisons indicate changes from baseline relative to placebo. Within group comparisons were analyzed using paired t test. Between group comparisons were analyzed with analysis of covariance with baseline value, atorvastatin level, and treatment included as covariates. Values in parentheses are 95% conﬁdence interval. *p Ͻ 0.05.

CD40L ϭ CD40 ligand; Cr ϭ creatinine; U-11-dehydro-TxB2 ϭ urinary 11-dehydrothromboxane B2.

Adverse events. There were no clinically important effects in Ͻ1% (n ϭ 1) in placebo, Ͻ1% (n ϭ 2) in the darapladib on vital signs, electrocardiograms, or laboratory data in the 40 and 80 mg groups, and 1% (n ϭ 3) in the darapladib 160 darapladib groups compared with placebo. Serious adverse mg group. events were reported in 3% in the placebo (n ϭ 7) and darapladib 40 mg (n ϭ 6) and 80 mg (n ϭ 7) groups and 2% Discussion (n ϭ 5) in the darapladib 160 mg group. These events were not concentrated in any particular organ system class and Key findings. This is the first report of chronic adminis- displayed no dose-related pattern. No deaths occurred after tration of an Lp-PLA2 inhibitor, darapladib, in CHD or randomization to darapladib or placebo. The CV events CHD-risk equivalent patients in the setting of intensive were infrequent at 2% in the placebo group (n ϭ 4), Ͻ1% statin therapy. There was a significant dose-dependent and ϭ in the darapladib 40 and 80 mg groups (n 2 in both sustained reduction of Lp-PLA2 activity with darapladib. groups), and 1% in the darapladib 160 mg group (n ϭ 3). The effect was independent of baseline LDL-C or HDL-C The most commonly reported serious adverse event was levels and background atorvastatin regimen. In addition, the angina at Ͻ1% in the placebo (n ϭ 2) and darapladib 40 mg results suggested a possible anti-inflammatory effect of the (n ϭ 1) groups, none in the darapladib 80 mg group, and highest dose of darapladib treatment when applied to 1% (n ϭ 3) in the darapladib 160 mg group. patients receiving atorvastatin therapy. In regards to safety, A total of 3% (n ϭ 33) of subjects prematurely withdrew there were no adverse effects of darapladib on biomarkers of from study drug due to an adverse event; 2% (n ϭ 4) in the platelet activity and no major safety concerns emerged from placebo group, 4% (n ϭ 10) in the darapladib 40 mg group, this study. ϭ ϭ 3% (n 8) in the darapladib 80 mg group, and 5% (n 11) Relationship between Lp-PLA2, lipoproteins, and treat- in the darapladib 160 mg group. Within each organ system ment of dyslipidemia. Owing to its binding to carboxyl- class, the incidence of adverse events leading to premature terminus of human apolipoprotein B, approximately 80% of Յ withdrawal was 1% across all treatment groups with the circulating Lp-PLA2, measured as mass or activity, is exception of a 3% (n ϭ 6) incidence of premature with- associated with apolipoprotein B–containing lipoproteins drawals due to gastrointestinal events (mostly change in (22,23). The remaining Lp-PLA2 is less firmly associated feces odor or diarrhea) in the darapladib 160 mg group. A with phospholipid moiety of HDL-C and does not bind to higher incidence of odor- (mainly feces or urine) or taste- apolipoprotein A-I. Higher Lp-PLA2 mass or activity are related events was reported in all darapladib treatment found in pro-atherogenic small dense LDL-C and electro- groups (33% to 36%) compared with placebo (21%) that did negative LDL-C particles, as opposed to large buoyant not lead to frequent premature withdrawals (Յ1% incidence LDL-C and electropositive subfractions, respectively (24–26). of withdrawals due to odor- or taste-related events across all Consistent with this distribution of Lp-PLA2, our findings treatment groups). show strong correlations between Lp-PLA2 activity and No cases of rhabdomyolysis or creatine kinase elevation LDL-C and inverse relationship with HDL-C in patients Ͼ10 ϫ upper normal limit were reported. Elevation of on intensive atorvastatin treatment. Prior studies have also alanine transaminase Ն3 ϫ upper limit of normal was noted shown that various hypolipidemic drugs (e.g., statins, feno- JACC Vol. 51, No. 17, 2008 Mohler III et al. 1639

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fibrate, ezetimibe) decrease plasma Lp-PLA2 mass or activ- vations together suggested that other extra- and intracellular ity due to LDL-C lowering, without a direct effect on enzymatic pathways are involved in hydrolysis of PAF, macrophage expression of the enzyme (27,28). Comparisons independent of Lp-PLA2 activity. To this end, studies across studies are difficult, because of the use of several identified several PAF inactivating enzymes, including methods to measure Lp-PLA2 mass or activity. In general, lecithin-cholesterol acetyl transferase and unrelated phos- however, statin treatment alone (e.g., pravastatin, fluva- pholipases (e.g., group X secretory PLA2 and intracellular statin, simvastatin, atorvastatin) has been associated with PAF-AH [II]) (43,44). These findings provide mechanistic approximately 20% to 30% reduction in the measurements of explanation for the lack of detrimental effect of darapladib

Lp-PLA2 (mass or activity) in stable CV patients (29–31). on platelet biomarkers despite high levels of Lp-PLA2 Similarly, other lipid modifying drugs, such as ezetimibe and inhibition. The importance of our findings is also under- fenofibrate, modestly lower Lp-PLA2 (mass or activity) (32). scored by the fact that several biomarkers were studied, This study shows that a specific Lp-PLA inhibitor, darap- 2 including urinary 11-dehydrothromboxane B2, which is less ladib, produces substantial additional reductions in Lp- affected by pre-analytical artifacts associated with the measure- PLA2 activity when added to intensive atorvastatin therapy ments of circulating markers (45). (up to 66%). This effect was largely independent of atorva- Study limitations. There are several limitations of this statin dose and preserved in clinically relevant strata of study to be emphasized. First, the clinical relevance of the LDL-C and HDL-C values. observed Lp-PLA2 inhibition with darapladib must await Relationship between Lp-PLA2 and inflammatory bio- evidence linking Lp-PLA2 inhibition to a beneficial effect markers. Oxidatively truncated phospholipids are sub- on clinical events. Nonetheless, marked inhibition of the strates for Lp-PLA2 that promote formation of putative enzyme activity in the presence of intensive background pro-inflammatory and pro-apoptotic products (9). The statin therapy is an important pre-requisite for testing this concentration of oxidized LDL-C within human atheroma concept in high-risk CV patients. Second, our study cannot is approximately 70-fold higher than in circulation; thus, the address the effects of Lp-PLA inhibition within atheroma comparisons between Lp-PLA and other inflammatory 2 2 that is postulated to be ultimately responsible for clinical markers in plasma might not accurately reflect intralesional benefits. It is important to underscore, however, that com- inter-dependency (33). In this context, it is not surprising parable exposures of darapladib inhibit intralesional Lp- that circulating Lp-PLA shows weak correlations with 2 PLA activity in the clinical setting (46). Third, the effects other inflammatory biomarkers, as shown in this and 2 on IL-6 and hs-CRP were modest and limited to the previous studies (34,35). Nonetheless, increasing levels of highest dose of darapladib. These results might be viewed as Lp-PLA have been reported to have additive effects with 2 hypothesis-generating and will require replication in other hs-CRP or oxidized phospholipid/apolipoprotein B ratio in studies. predicting the risk of CV events (14,16,36). When assessing the effects of Lp-PLA2 inhibition on soluble inflammatory biomarkers, it is important to underscore that baseline Conclusions hs-CRP levels were lower in this study compared with other investigations using intensive atorvastatin treatment and there- Although treatment with statins has resulted in improved fore might have minimized the opportunity to observe addi- prognosis in primary and secondary prevention populations, tional effects on this biomarker (5,6,37,38). Despite these high-risk individuals continue to experience recurrent CV caveats, a significant reduction in hs-CRP was noted in the events despite adequate treatment. It is postulated that highest darapladib group (160 mg) after 12 weeks of treatment inflammatory mediators not addressed by LDL-C lowering (within group comparison). Interestingly, the darapladib 160 contribute to plaque instability and thrombotic events. The mg group also exhibited a significant reduction in IL-6 (within results of this study indicate that darapladib produces group and between groups comparisons). This finding is substantial inhibition of Lp-PLA2 activity in the presence of noteworthy, because IL-6 is a trigger for hepatic production of intensive statin therapy and suggest that such intervention hs-CRP and it is less affected by background atorvastatin might result in additional systemic anti-inflammatory ef- treatment (37,39). fects. Future studies are required to determine whether Relationship between Lp-PLA and PAF. Initial de- 2 chronic Lp-PLA inhibition will stabilize high-risk lesions scriptions of Lp-PLA focused on the ability of this enzyme 2 2 and potentially reduce CV events. to degrade PAF, a potent pro-inflammatory lipid mediator that is also implicated in platelet aggregation (10). However, responsiveness to PAF is not altered in Japanese subjects Acknowledgments with a genetic variant in Lp-PLA2 (Val276Phe) that results The authors thank all of the Darapladib Investigators (listed in in the absence of circulating enzyme (40). In addition, other the Online Appendix), their staff, and patients participating in clinical trials failed to show measurable benefit of recombi- this study. The authors also appreciate the assistance of Denise nant human Lp-PLA2 in conditions suspected to be PAF- Lawrence and Mark Dickson from GlaxoSmithKline for mediated (e.g., severe sepsis, asthma) (41,42). These obser- providing statistical analyses and programming support. 1640 Mohler III et al. JACC Vol. 51, No. 17, 2008

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