Secretory Phospholipase A2: A New Risk Factor and Soon a New Target of Therapy Rabih R. Azar, MD, MSc, FACC Associate Professor of Medicine Division of Cardiology Hotel Dieu de France Saint Joseph University

Risk of CAD according to LDL and HDL

5 PROVE-ITMIRACL Schwartz et al., JAMA 2001;285:1411; Cannon et al. N Engl J Med. 2004;350:1495. Atorvastatin 80 mg Placebo RRR 16% p=.048 Death, non-fatal MI, or recurrent USA Months Atorvastatin 80 mg Months Death, MI, ACS, stroke, revascularization Pravastatin 40 mg RRR 16% p= % 20% 10% 0% High Residual Risk in ACS Despite Early Statin Use

IS LDL CHOLESTEROL THE “ONLY” PLAYER IN ATHEROSCLEROSIS?

Cholesterol distribution in CHD and non-CHD populations In spite of major advances made in the screening, detection, and management of heart disease, a major need exists for more accurate ways to predict CV risk –Approximately 50% of individuals diagnosed with coronary artery disease do not have high blood cholesterol levels –Therefore, other factors must be involved 35% of CHD occurs in people with TC considered optimal (<200mg/dL) Adapted from Castelli W. Atherosclerosis 1996 Framingham Heart Study — 26-year follow-up No CHD Total cholesterol (mg/dL) CHD

1 major risk factor 0 major risk factors 2 major risk factors 3 major risk factors 4 major risk factors 62.4% 0 to 1 major risk factor N=87,869 4 Major modifiable risk factors: hypertension, smoking, hypercholesterolemia, diabetes Traditional risk factors are a useful first step in determining who could be at risk for a coronary event Exposure to one or more CHD risk factors is also highly prevalent in individuals who do not develop clinical CHD Less than 10% of patients have 3 or 4 major risk factors Secondary testing can be used to further stratify individuals for CHD risk Prevalence of major risk factors in men with CHD Khot, et al. JAMA. 2003

LUMEN MEDIA INTIMA Stages of Atherosclerosis LDL

LUMEN MEDIA INTIMA Oxidized LDL Adhesion molecules Lyso-PC OxFA sPLA 2 Stages of Atherosclerosis

LUMEN MEDIA INTIMA Oxidized LDL Adhesion molecules Lyso-PC OxFA sPLA 2 Stages of Atherosclerosis

Cytokines Plaque formation Foam cell Monocytes Macrophage Stages of Atherosclerosis LUMEN MEDIA INTIMA Oxidized LDL Adhesion molecules Lyso-PC OxFA sPLA 2

13 What is sPLA2? Family of Ca 2+ dependent proteins Exact physiologic role in humans is not clearly known Type IIA is an acute phase protein secreted from liver in response to inflammatory cytokines Groups IIA, V, X have each been implicated in CVD All types cleave phospholipids at sn-2 position By generating intermediates acts as mediator between proximal and distal effectors of inflammation

Phosphatidylcholine (PC) Oxidatively-modified PC Lysophosphatidylcholine (Lyso-PC) Oxidized fatty acid (oxFA) Oxidation sPLA 2 Role of sPLA2 + Tselepis AD, et al. Atheroscler Suppl. 2002

lyso-PC Expression of adhesion molecules Inhibition of endothelial-derived nitric oxide Upregulation of cytokines and CD40 ligand Stimulation of macrophage proliferation Induction of MCP-1 Chemoattractant for macrophage and T-cells Cytotoxic to vascular smooth muscle cells Pro-atherogenic activities of lysophosphatidylcholine (Lyso-PC) 3. Macphee CH, et al. Biochem J Carpenter KL, et al. FEBS Lett Dada N, et al. Expert Rev Mol Diagn Quinn MT, et al. Proc Natl Acad Sci USA. 1988

Secreted Phospholipases A2: A diverse family of 9 human genes with different structural, enzymatic and functional properties. 16 sPLA2 family plays a causal role in atherosclerosis

sPLA2-IIA overexpression induces atherosclerosis in mice Transgenic overexpression of human sPLA2-IIA gene in mice induces atherosclerosis: Spontaneous atherosclerosis lesions development, even in the absence of hyperlipidemia. Elevated plasma lysoPC levels. Ivandic et al. ATVB 1999,19:1284 C57BL/6 sPLA2-IIA Tg 17

Varespladib Decreases Atherosclerosis; Synergistic with Statins in ApoE -/- Mice Monotherapy (Percent Plaque Area) 0% 5% 10% 15% 20% % Plaque Area (of Aortic Surface Area) % En Face Lesions Vehicle30 mg/kg90 mg/kg n=20 n=18 n=17 * * * p< 0.10 vs. placebo ** p< 0.05 vs. placebo † p< 0.05 vs. A-002 low dose Shaposhnik Z et al, J Lipid Res 2009;50 Fraser H et al, J Cardiovasc Pharmacol 2009;53 Combination Therapy: A Statin (Percent Plaque Area)* Placebo A mg/kg/d A mg/kg/d Pravastatin A Statin 1.5mg/kg/d A Statin 150mg/kg/d * **, † **

sPLA2 levels increased with atherosclerotic development in human plaques The extent of sPLA2s expression increased with atherosclerosis development. Kimura-Matsumoto et al., Atherosclerosis, 196 (2008) 81–91 19 sPLA2 family plays a causal role in atherosclerosis

sPLA2 Activity in stable CAD Patients PEACE Study RCT of trandolapril in 3738 patients with stable CAD and EF >40% Mean follow-up of 4.8 years Secondary events: cardiovascular death, stroke or MI. Reference: O’Donoghue M et al, American Heart Association 2009 Meeting, P Matched for age, sex, HTN, DM, smoking, prior MI, prior coronary revascularisation, apoB, apoA, eGFR, current lipid-lowering Rx. sPLA2 activity allows clinicians to predict fatal and non-fatal recurrent events after a first event Adj HR 1.19 ( ) Adj HR 1.26 ( ) Adj HR 1.56 ( ) Referent sPLA2 Activity Quartile Adjusted Hazard Ratio Q1Q2Q3Q4 CVD, MI or stroke at 5 years (P trend <0.001)

sPLA2 Activity in acute MI Patients FAST-MI Study 1036 patients with Acute Myocardial Infarction 86 cases after 6 months follow-up. Reference: Simon T et al, European Society of Cardiology 2008 Meeting, P Matched for sex, age, time, traditional CV risk factors, CRP and treatments including statins. sPLA2 activity allows clinicians to better monitor patients with MI by increasing prediction of recurrent event Adj OR 1.39 ( ) 0.82 Adj OR 2.29 ( ) Referent sPLA2 Activity and Lp-PLA2 Tertiles Adjusted Odd Ratio T1 T2T Lp-PLA2 sPLA2 Activity Death, MI or stroke at 6 months (p trend < 0.01)

sPLA2 Activity in ACS Patients GRACE Study 446 Acute Coronary Syndrome patients (38.5 % with ST elevation MI, 52.5% with Non-ST elevation MI and 9% with UA). One year cumulative incidence of death or MI: 9.63%. Reference: Mallat Z et al, J Am Coll Cardiol Oct 4;46(7): ACS Patients with high sPLA2 activity have a 4-fold risk increase of death and new or recurrent myocardial infarction

sPLA2 Activity in Asymptomatic Patients EPIC-Norfolk Study 2797 subjects (991 apparently healthy subjects with development of fatal or non-fatal coronary artery disease, 1806 matched controls). 6 years median follow-up Reference: Mallat Z et al, Arterioscler Thromb Vasc Biol. 2007, May;27(5): Matched for sex, age, enrollment time, and adjusted for diabetes, smoking, body mass index, systolic blood pressure, LDL-C, and HDL-C. After adjustment for components of the Framingham Risk Score, sPLA2 Activity allows prediction of the risk of incident CAD in asymptomatic patients on top of FRS Adj OR 1.41 ( ) Adj OR 1.70 ( ) Referent sPLA2 Activity Qartiles Adjusted Odd Ratio Q1Q2Q3Q4 Adj OR 1.38 ( ) Incident CAD at 6 years

A very high level of proof 5 studies already published (8200 patients) Relative Risk of Cardiovascular Events (adjusted odd ratio, first quartile/tertile vs last quartile/tertile) Adjusted for traditional risk factors, treatments and biomarkers when available sPLA2 Activity Acute CAD patients GRACE study (5) Primary prevention Secondary prevention 2797 asymptomatic patients EPIC-Norfolk study (6-7) Acute Coronary Events patients FAST-MI study (3-4) CHD patients KAROLA study (2) 1- O’Donoghue M et al, American Heart Association Meeting, 2009, P Koenig W et al, Eur Heart J Nov;30(22): Simon T et al, European Society of Cardiology Meeting, 2008, P Simon T et al, European Society of Cardiology Meeting, 2009, P Mallat Z et al, J Am Coll Cardiol Oct 4;46(7): Mallat Z et al, Arterioscler Thromb Vasc Biol. 2007, May;27(5): Tsimikas S et al, American Heart Association Meeting, 2008, P4998 sPLA2 activity: the first independent biomarker to allow a 2- to 4- fold CV risk increase prediction in different population types stable CHD patients PEACE study (1)

Multimarker Approach in Asymptomic Patients EPIC-Norfolk Study Mallat Z et al, Arterioscler Thromb Vasc Biol. 2007, May;27(5): Tsimikas S et al, American Heart Association 2008 Meeting, P OR=2.89 ( ) Adjusted Odd Ratio C-reactive Protein In asymptomatic patients, sPLA2 activity allows a increased prediction of CV risk: 3-fold higher risk combined with CRP 4-fold higher risk combined with oxPL/apoB100 Both matched for sex, age, enrollment time, adjusted for diabetes, smoking, BMI, SBP, LDL-C and HDL-C sPLA2 Activity oxPL/apoB Adjusted Odd Ratio OR=3.67 ( )

Effect of Ezetimibe/Atorvastatin Combination on sPLA2 in Patients With CAD or CAD Equivallent Mireille Azar, Emmanuel Valentin, Georges Badaoui, Roland Kassab, Antoine Sarkis, and Rabih Azar Am J Cardiol May 2011 (manuscript) Sponsored by Pharmaline

OBJECTIVE TO EVALUATE THE EFFECT OF ATORVASTATIN 40 mg and of ATORVASTATIN 40 mg + EZETIMIBE ON sPLA2

Slide 28 DIETARY CHOLESTEROL BILIARY SECRETION INTESTINE Excretion VLDL LDL Absorption synthesis IDL Statins Cholesterol Absorption Inhibition Endogenous Exogenous Cholesterol Absorption Inhibition for Broader Lipid Control

Slide 29 Doubling a Statin Dose Yields Only a 6% Incremental Drop in LDL-C Adapted from Knopp RH. N Engl J Med. 1999;341:498–511; Stein EA. Am J Cardiol. 2002;89(suppl):50C–57C Statin, mg Reduction of LDL-C, % Statin Rule of % drop

Slide 30 LDL-C 20% 30-45% STATIN + As high as 60% 10% 20% 30% 40% 50% MEAN LDL-C LOWERING 2,3 synthesis absorption synthesis absorption synthesis absorption 1. Assmann G, et al. J Am Coll Cardiol 2004;43(5, Suppl. 2):A445-A446; 2. Goldberg AC, et al. Mayo Clin Proc May;79(5):620-9.; 3. Davidson M et al. J Am Coll Cardiol 2002; 40: CHANGE OF SYNTHESIS AND ABSORPTION MARKERS 1 Inhibition of absorption Ezetimibe alone Inhibition of synthesis Statin alone EZETIMIBE Dual Inhibition Ezetimibe/Statin

Effect of Ezetimibe/Atorvastatin Combination on sPLA2 in Patients With CAD or CAD Equivallent -Prospective, randomized, double-blind, placebo- controlled trial -Inclusion criteria: -Patients with CAD -> 50% stenosis on angiography -MI -PCI or CABG -Patients with CAD equivalent -Diabetes requiring medications -Peripheral vascular disease -Stroke -Lipid levels were not entry criteria

Exclusion Criteria Therapy with a statin more potent than atorvastatin 20 mg/day (atorvastatin 40 or above, rosuvastatin any dose) Therapy with ezetimibe, any other cholesterol absorption inhibitor, niacine, fibrate within the last 3 months MI, CABG, PCI within the last 3 months Age > 80 years EF 2 Creatinin clearance < 30 mL/min CPK or SGPT > 2 times upper normal

Study Protocol The statin taken by the patient was stopped and replaced by atorvastatin 40 mg/day Patients were then randomized to ezetimibe 10 mg/day vs. placebo Duration of treatment 8 weeks

MEASURMENTS Standard lipid profile (Total cholesterol, VLDL, LDL, HDL) High sensitivity CRP Secretory phospholipase A2 CPK, SGPT

End-Points Primary end-point Change in sPLA2 Secondary end-points Change in hs-CRP Safety end-points Elevation of CPK or SGPT more than twice upper normal

Inclusion Criteria EzetimibePlacebon = 50 Stenosis > 50%19 (38%)23 (46%) Prior MI18 (36%)12 (24%) PCI23 (46%)15 (30%) CABG26 (52%)21 (42%) Diabetes18 (36%)21 (42%) Stroke1 (2%)1 (2%) PVD6 (12%)5 (10%) The number of inclusion criteria is superior to 100% because each patient may have more than 1 criterion that defines CAD

Baseline Characteristics Ezetimibe Placebon = 50 Age (year) Male44 (88%)41 (82%) Smoking14 (28%)12 (24%) Hyperlipidemia47 (94%)43 (86%) Hypertension35 (70%)38 (76%) Fam. Hist. CAD21 (42%)14 (28%) BMI (kg/m 2 )

Concomitant Medications EzetemibePlacebon = 50 Aspirin45 (90%)43 (86%) Clopidogrel 11 (22%)11 (22%) ACE inhb. or ARB41 (82%)34 (68%) Beta-blockers37 (74%)35 (70%) CCB8 (16%)18 (36%)* Nitrates10 (20%)11 (22%) Diuretics6 (12%)6 (12%) OAD15 (30%)17 (34%) Insulin6 (12%)6 (12%) * P = 0.02

Statin Use at Baseline 90% of patients were using a statin prior to randomization Simvastatin53% Atorvastatin30% Fluva or pravastatin7% None10%

Change in LDL P < Final LDL levels were lower in ezetimibe vs. placebo; p < % additional reduction 20% additional reduction

Change in VLDL P = 0.07 P < 0.001

P = ns

Change in Hs-CRP p = p < % reduction

Effect of Atorvastatin-Placebo on sPLA2 P = NS No Change

Reduction of sPLA2 by atorvastatin/ezetimibe combination P = % reduction

Correlation between change in sPLA2 and changes in hs-CRP p = p < 0.001

Correlation between the change in sPLA2 and in LDL p = p = NS

Variables independently associated with change in sPLA2 VariableBeta coefP -Change in CRP 0.53 < Baseline LDL BMI Diabetes Ezet/atorva (Linear Regression Model)

Safety End-Point There was no elevation of CPK or SGPT in any patient of the 2 groups

Major Findings of the Study First to demonstrate a reduction in sPLA2 with lipid lowering therapy Effect was weak: 10% reduction Low LDL level at baseline. If LDL was higher, reduction might have been more potent Cannot exclude that statins do not decrease sPLA2, because 90% of our patients were on statin at baseline and because statins decrease both LDL and hs-CRP

Summary of Results: Effects of Atorvastatin and Ezetimibe on Various Lipid Parameters LDLLarge LDL Small dense LDL Particl e size HDLVLDLOx- LDL sPLA2 More potent statin Ezetimb e The changes induced by statin are quantitative and qualitative The Changes induced by ezetimibe are only quantitative

Enhance-Study Kastelein et al., NEJM 2008, 358:

Months LDL-cholesterol ENHANCE Simva Eze-Simva Percentage change from baseline P< % incremental reduction Baseline (mg/dL) 24 months (mg/dL) Simva318 ± ± 60 Eze-Simva319 ± ± 53 Kastelein JP et al N Engl J Med 2008;358:

Slide 55 Mean cIMT during 24 months of therapy Longitudinal, repeated measures analysis ENHANCE Mean IMT (mm) Simva Eze-Simva Months P=0.88 Kastelein JP et al N Engl J Med 2008;358:

Slide 56 SANDS-Study Fleg et al., JACC 2008; 52

Slide men and women with diabetes and no CVD  40 yrs old  SBP>130, LDL>100 Standard Targets LDL-C <100; SBP <130 non-HDL-C <130 N=247 Aggressive Targets LDL-C <70; SBP <115 non-HDL-C <100 N=252 Measure CVD using carotid and cardiac ECHO at baseline 18 months and after 3 yrs intervention Primary outcome—change in CIMT SANDS Trial Design* *JAMA 2008; 299:

Slide 58 Change in IMT by Ezetimibe +/- * p<0.001 for both E + and E- compared to Standard mm

Slide 59 The results of the Study of Heart and Renal Protection (SHARP) Colin Baigent, Martin Landray on behalf of the SHARP Investigators Disclosure: SHARP was sponsored, designed, run, and analysed by the University of Oxford. Funding was received from Merck, the UK MRC, British Heart Foundation, and Australian NHMRC.

Slide 60 SHARP: Eligibility History of chronic kidney disease –not on dialysis: elevated creatinine on 2 occasions Men: ≥1.7 mg/dL (150 µmol/L) Women: ≥1.5 mg/dL (130 µmol/L) –on dialysis: haemodialysis or peritoneal dialysis Age ≥40 years No history of myocardial infarction or coronary revascularization Uncertainty: LDL-lowering treatment not definitely indicated or contraindicated

Slide 61 SHARP: Assessment of LDL- lowering

Slide Years of follow-up Proportion suffering event (%) Risk ratio 0.83 (0.74 – 0.94) Logrank 2P= Placebo Eze/simv SHARP: Major Atherosclerotic Events

Slide 63 CTT: Effects on Major Atherosclerotic Events Proportional reduction in atherosclerotic event rate (95% CI) 0% 5% 10% 15% 20% 25% 30% Statin vs control (21 trials) Mean LDL cholesterol difference between treatment groups (mg/dL) More vs Less (5 trials) SHARP 32 mg/dL

Slide 64 Proportional reduction in atherosclerotic event rate (95% CI) 0% 5% 10% 15% 20% 25% 30% Statin vs control (21 trials) Mean LDL cholesterol difference between treatment groups (mg/dL) More vs Less (5 trials) SHARP 32 mg/dL SHARP 17% risk reduction CTT: Effects on Major Atherosclerotic Events

Slide 65 Relation between proportional reduction in incidence of major coronary and vascular events and absolute LDL cholesterol reduction at 1 year Cholesterol Treatment Trialists’ (CTT) Collaborators Lancet 2005;366: SHARP

Varespladib: a New and Potent sPLA2 Inhibitor

PLASMA II 135 patients with stable CHD randomized to 8 weeks of therapy with placebo, varespladib 250 or 500 mg/day primary endpoint was change in sPLA2-IIA concentration between placebo and varespladib groups The 2 doses of varespladib lowered sPLA2 by 73% and 84% respectively (p<0.0001) compared with placebo, varespladib 500 mg reduced LDL-C by 15% (p<0.001), non-HDL-C by 15% (p<0.001), total VLDL particle concentration by 14% (p=0.022), and small VLDL particle concentration by 24% (p=0.030) Rosenson RS et al, Eur Heart J 2010, epub Nov.16

PLASMA II: Results Rosenson RS et al, Eur Heart J 2010, Epub Nov % -84% -15% -14%

69 FRANCIS-ACS Trial: Safety and Efficacy of Varespladib in Acute Coronary Syndromes 625 Patients Men/Women Unstable Angina, NSTEMI or STEMI Any one of Diabetes CRP ≥2 mg/L Met. syndrome Randomized within 96 hours of event Varespladib 500mg QD + Atorvastatin 80mg QD Placebo + Atorvastatin 80mg QD Rosenson RS et al, J Am Coll Cardiol 2010;56: Primary End Point at 8 wks  LDL-C  hs-CRP  sPLA 2 concentration Secondary End Points  Occurrence of MACEs for treatment- related trends at study completion. 6 Month Minimum Treatment Period

70 FRANCIS: Change in sPLA 2, LDL-C, and hs-CRP Median % Change From Base Line sPLA Mean % Change From Base Line LDL-C Weeks Varespladib Placebo Varespladib Placebo P< P= P= P= P=0.0071P= Median % Change From Base Line hs-CRP Varespladib Placebo Weeks P= P=0.3976P= P= P=0.0185

FRANCIS: MACE at 16 Weeks Varespladib 500 mg + Atorvastatin 80 mg Placebo + Atorvastatin 80 mg Patients Total MACE14 (4.2%)19 (6.1%) UA hospitalization5 (1.6%)9 (2.9%) Myocardial infarction2 (0.6%)4 (1.3%) Stroke1 (0.3%) Death6 (1.9%)5 (1.6%) Revascularization >60 days 0 (0%) Rosenson RS et al, J Am Coll Cardiol 2010;56:

72 Acute Coronary Syndrome Patient 1 (STEMI, NSTEMI, UA) Patient Screening Interventional procedures if necessary Patient Randomization < 96 hours Varespladib + Atorvastatin Placebo + Atorvastatin 16 week treatment 6 Months Primary Endpoint MACE Survival Status 1 As per FDA Guidance Major Adverse Coronary Events (MACE) are defined as Cardiovascular Death, Non-Fatal Myocardial Infarction, Non-Fatal Stroke, and Unstable Angina requiring urgent hospitalization Vascular Inflammation Suppression to Treat Acute Coronary Syndrome (VISTA-16)

Conclusions sPLA2 plays an important role in atherosclerosis and is an independent marker of future cardiovascular events We demonstrated for the first time that combination of statin/ezetimibe lowers sPLA2 In patients with stable CAD, increasing the potency of statin to atorvastatin 40 and adding ezetimibe is safe and results in effects that go beyond LDL lowering: decrease in small dense LDL, decrease in oxidized LDL, decrease in CRP and decrease in sPLA2 Potent and specific inhibitors of sPLA2 are currently available Whether inhibiting sPLA2 is clinically beneficial is awaiting the result of the on-going VISTA-16 trial