Delipidation for ACS Patients HDL Therapy Via Plasmapheresis Ron Waksman, MD Washington Hospital Center

Ron Waksman, MD DISCLOSURES Consulting Fees Grants/Contracted Research Abbott Vascular, Biotronik, Medtronic CardioVascular, Inc, Boston Scientific Corporation Grants/Contracted Research Abbott Vascular, Biotronik, Boston Scientific Corporation, The Medicines Company, GlaxoSmithKline, Schering-Plough, sanofi-aventis U.S. LLC

WHY HDL ??? Low high-density lipoprotein (HDL) cholesterol is associated with increased risk of CHD. HDL also protects low-density lipoproteins (LDL) from oxidation and inhibits expression of adhesion molecules in endothelial cells, preventing monocyte movement into the vessel wall HDL removes and transports excess cholesterol from peripheral cells to the liver for removal from the body Current drug options for increasing HDL– Cholesterol levels include the statins, fibrates, and niacin 3

Mechanism by Which HDL protects against CVD HDL-mediated efflux of cholesterol from cholesterol-loaded macrophages is one of the most extensively studied anti-atherogenic functions of HDL. The major pathway for cholesterol efflux from macrophages to HDL is by interaction of HDL with the ABCA1 transporter. The preferred acceptor for the ABCA1 transporter-mediated cholesterol efflux is poorly lipidated apoA-I or preβ-HDL. 4

Use of cholesteryl ester transfer protein inhibitors. CETP Strategies in development for increasing the function of HDL or apolipoprotein A-I and thereby reducing atherosclerotic progression Use of agents to upregulate the adenosine triphosphate-binding cassette transporter in vessel wall macrophages to increase cholesterol efflux from these cells Use of agents to stimulate endogenous apoA-I synthesis; administration of apoA-I, apoA-I Milano, apoA-I-mimetic peptides. Use of cholesteryl ester transfer protein inhibitors. CETP Delipidated HDL by apheresis. Infusion of HDL in the form of apoA-I/phospholipids complexes was associated with regression of atherosclerosis in cholesterol-fed rabbits 5

How Does HDL Therapy Work? Delipidated HDL Enhances Reverse Cholesterol Transport The major pathway for cholesterol efflux from macrophages to HDL is by interaction of HDL with the ABCA1 transporter. Liver Modification C A - I Pre-Beta HDL “Empty Dump Truck” Alpha HDL “Full Dump Truck” LDL LDL “Bad Cholesterol” Cholesterol LCAT HDL Therapy Infusion of Delipidated HDL. α HDL transports Excess Cholesterol to the liver αHDL 6 6

Plasma from African Gree monkeys underwnet selective HDL delipidation Selective delipidation of plasma HDL enhances reverse cholesterol transport in vivo Plasma from African Gree monkeys underwnet selective HDL delipidation The delipidated plasma was reinfused into five monkeys. Pre β-1-like HDL had a plasma residence of 8+6 h. Small α-HDL was converted entirely to large α-HDL Treatment with delipidated plasma tended to reduce diet –induced aortic atherosclerotic in monkeys measured by intravascular ultrasound. These findings link the conversion of small to large HDL, in vivo, to improvement in atherosclerosis Sacks, et al. J Lipid Res. 2009 . 50 : 894-907

Effect of Selective HDL delipidation on aortic atheroma in monkeys Total atheroma Volume Pre treatment Post treatment % Change Mean 5.63 5.25 -6.9% SD 1.27 1.41 11.1% Percent atheroma Volume Pre treatment Post treatment % Change Mean 31.78 30.26 -4.4% SD 4.41 5.61 14.8% Sacks, et al. J Lipid Res. 2009 . 50 : 894-907

HDL Selective Delipidation “Energized HDL” “Full Dump Truck” ~95% “Empty Dump ~5% ~80% ~20% 9 9

LIPID SCIENCE 01 TRIAL Key Trial Personnel and organization Principal Dr. Ron Waksman Investigator Associate Director of Cardiology MedStar Research Institute IVUS Dr. Neil Weissman Dr. Peter Fitzgerald Laboratories Director, IVUS Core Laboratory Director, IVUS Core Laboratory MedStar Research Institute Stanford School of Medicine ECG Core Laboratory Dr. Joseph Lindsay Director, ECG Core Laboratory MedStar Research Institute Data Safety Dr. William Wientraub Monitoring Board DSMB, Chairman Director, Christiana Center for Outcomes Research, Christiana Care Health Services Clinical Event Dr. David Cohen Committee CEC, Chairman St. Luke’s Mid American Heart Institute Data Coordinating David Hellinga Center Director, Data Center Cardiovascular Research Institute Central Laboratory Dr. Jason Umans Director, Penn Laboratories MedStar Research Institute Waksman et al. In Press in JACC 2010 10 10

Objectives The primary aim of this study was to test the safety and feasibility of autologous delipidated HDL infusions in acute coronary syndrome (ACS) patients. An exploratory aim of this study was to assess the impact on plaque volume assessed by IVUS measurements. 11 11

HDL Selective Delipidation Trial Design Patients with ACS scheduled for cardiac cath with non obstructive atheroma were randomized to HDL delipidation or control and subjected to apheresis/ reinfusion. Patients had 7 sessions each 1 week apart. IVUS performed up to 14 days from last procedure to assess atheroma volume indices. 12 12

HDL Delipidation Process Schematic Overview of the Methodology for the Selective Delipidation of HDL in Plasma Plasma Delipidated HDL Delipidation Process NOT Patient connected Uses patient’s own HDL Cholesterol removed from α-HDL to yield pre-β-HDL Delipidated plasma is reinfused into patient 13 13

Major Inclusion Criteria ≥ 18 Years And ≤ 85 Years Of Age ACS Diagnosis within the preceeding 14 days Suitable For Plasmapheresis HDL ≥ 32 mg/dL And ApoA-I Level ≥ 95 mg/dL Triglycerides < 300 mg/dL CHD In A “Target Artery” With ≥ 20% But ≤ 50% Reduction In Lumen Diameter By Visual Angiography Waksman et al. In Press in JACC 2010 14 14

Major Exclusion Criteria Elevated ST Segment MI ≤ 72 Hours Of Screening Ejection Fraction (LVEF) < 40% Hemodynamically Unstable Pregnant Or Lactating ≤ 6 Months Prior To Screening Active Liver Disease Or Hepatic Dysfunction Renal Dialysis Or Presence Of Renal Dysfunction Active Cholecystitis, Gallbladder Symptoms, or Hepatobiliary Abnormalities Insulin Dependent Diabetes Unstable Or Uncontrolled Hypertension Unstable Hypotension Cardiac Insufficiency Currently Taking Coumadin Waksman et al. In Press in JACC 2010 15 15

Study Design Patient presents with ACS with non-obstructive atheroma in ≥ 1 native coronary artery FDA Approval: 01/27/06 Trial Launch: 05/31/06 Trial Conclusion: 01/29/08 Undergo IVUS of target non-obstructive atheroma native coronary artery (n=28) Randomized (1:1) Single-Blind Control n=14 7 apheresis/reinfusion (1 week apart) HDL delipidation n=14 7 apheresis/reinfusion (1 week apart) 2 control subjects discontinued after sessions 1 & 6 Undergo repeat IVUS of target atheroma (n=26) Within 14 days after final reinfusion 16

Baseline Characteristics Variable, n (%) or median (range) Evaluated at Screening Delipidation Group n=14 Control Group Age (years) 55 (44-68) 55 (37-74) Male 10 (71.4) 12 (85.7) Diabetes 6 (42.9) 2 (14.3) History of HTN 13 (92.9) History of Hyperlipidemia Current Smokers 5 (35.7) Prior PCI 4 (28.6) Prior CABG 1 (7.4) Weight (kg) 88 (68-108) 88 (58-117) Statin Use at Randomization Non-Statin Cholesterol Lowering Medication at Randomization Waksman et al. In Press in JACC 2010 17

Delipidation Group (n=14) Lipid Profile Delipidation Group (n=14) Control Group (n=12) P value* Pre Final Visit Total Cholesterol 148.5 (106-246) 147 (90-203) 148 (121-212) 148.5 (99-221) 0.643 HDL-C 47 (33-65) 44 (34-59) 41.5 (32-82) 44 (27-86) 0.395 LDL-C 75 (45-133) 67.5 (35-120) 78 (43-113) 70.5 (40-131) 0.268 VLDL-C 20.5 (12.5-55.6) 27.5 (15.5-46) 24.2 (3-54.1) 28.3 (4.5-63.4) 0.662 ApoB 82.5 (55-123) 74 (46-105) 71 (57-100) 73 (46-116) 0.099 ApoA-1 139.1 (100.6-165.4) 129.1 (107.2-158.9) 126.4 (108.8-187.7) 124.5 (87.5-193.5) 0.554 Triglycerides 138.0 (65-446) 146.5 (66-360) 187.5 (65-505) 161.5 (64-440) 0.700 *Wilcoxon Rank Sum Test (change between groups) 18

Safety Laboratory Values Delipidation Group (n=14) Control Group (n=12) P value* Pre Final Visit Hemaglobin 14.1 (12.2-15.7) 13.8 (11.8-15.3) 14.1 (10-15.9) 13.6 (10.2-14.9) 0.315 Hematocrit 41.5 (37.5-44.7) 39.7 (37.0-43.2) 41.9 (31.3-45.9) 40.5 (31.4-43.6) 0.396 AST 27.5 (19-69) 28.5 (21-51) 24.5 (16-82) 23.5 (16-66) 0.367 ALT 38.5 (28-101) 36 (28-71) 40.5 (25-98) 35.5 (21-52) 0.777 Alkaline Phos 86 (61-132) 91 (62-129) 88 (65-163) 92.5 (60-135) 0.156 LDH 497.5 (336-708) 468.5 (354-804) 502.5 (353-696) 476.5 (367-787) 0.738 Total CPK 96.5 (31-233) 106 (40-288) 101.0 (20-314) 81.5 (20-1999#) 0.625 Potassium 4.5 (3.9-5.6) 4.4 (3.9-4.9) 4.2 (4.0-5.3) 4.3 (3.9-7.5) 0.093 *Wilcoxon Rank Sum Test (change between groups); #One patient was a body builder – CK-MB and Trops were <2xULN for this patient at final visit 19

Major Adverse Cardiac Events Variable, n (%) - ITT Delipidation Group n=14 Control Group Death Re-infarction Target Lesion Revascularization Non-Target Lesion Revascularization 1 (7.2) 2 (11.8) Unanticipated Adverse Device Effects Waksman et al. In Press in JACC 2010 20

Quantitative 2-D Gel Electrophoresis & Pre-β HDL (ELISA) Following Delipidation Waksman et al. In Press in JACC 2010 21 21

Results All reinfusion sessions were well tolerated by the patients with no adverse clinical effects. The levels of pre-β HDL in the delipidated plasma are dramatically increased as compared to the undelipidated, control plasma in a subset analysis. Associated with the pre-β HDL increase was a 5X rise in the cholesterol efflux for the delipidated plasma versus the control. Corrected 80.8% to 79.1% and 19.2% to 20.9%. (Recent correction of primary data.) Put sentence about IVUS data as its own bullet. Waksman et al. In Press in JACC 2010 22

Change in IVUS parameters, post delipidation treatments minus baseline ACS presentation Variable (mean ± SD) Delipidated Group n=14 Control Group n=12 Change in Total Atheroma Volume (mm3) -12.18 ± 36.75 2.80 ± 21.25 Change in Plaque Burden (%) -1.0 ± 4.0 0.0 ± 4.0 Change in 10 mm Most Diseased Subsegment – Atheroma Volume (mm3) -6.24 ± 17.94 -1.73 ± 11.21 Change in 10 mm Least Diseased Subsegment – Atheroma Volume (mm3) -1.10 ± 11.35 1.53 ± 11.70 Changed heading title from “Change in Atheroma Volume” to “Change in Total Atheroma Volume” Changed heading title from “Change in Most Diseased 10 mm – Atheroma Volume” to “Change in 10 mm Most Diseased Subsegment (mm3)” Changed heading title from “Change in Least Diseased 10 mm – Atheroma Volume” to “Change in 10 mm Least Diseased Subsegment (mm3)” Waksman et al. In Press in JACC 2010 23

IVUS Data Waksman et al. In Press in JACC 2010 24

Rapid Regression of Human Coronary Plaque after 5 Weekly Intravenous Injections of Recombinant rApo A-Imilano (ETC-216) *Nissen et al JAMA 2003: 290, 2292-300

Comparison of the Changes in IVUS Parameters in Lipid Sciences Selective Delipidation Trial and ApoA-I Milano Trial Variable (mean ±SD) LS-001 Trial N=14 ApoA-I Milano Trial N=36* Change in Total Atheroma Volume (mm3) -12.18 ± 36.75 -14.10 ± 39.50 Change in % Atheroma Volume (Plaque Burden) -1.0% ± 4.0% -1.1% ± 3.2% Change in Most Diseased 10 mm Subsegment, Atheroma Volume (mm3) -6.24 ± 17.94 -7.20 ± 12.60 *Nissen et al JAMA 2003: 290, 2292-300 26

Summary Pre-clinical studies have demonstrated that pre-β HDL is a key component in reverse cholesterol transport Safety and feasibility of delipidation were demonstrated Infusions are well tolerated by patients Patient compliance is excellent The PDS-2 consistently, reliably, and dramatically converts αHDL to pre-β HDL IVUS data demonstrates a numeric trend towards reduction in atheroma volumes 27 27

Conclusions In patients with ACS, serial autologous infusions of HDL delipidated plasma are well tolerated by patients, and are clinically feasible and safe. This therapy may offer a novel adjunct treatment for patients presenting with ACS, and may ultimately stabilize and regress atherosclerotic plaques. Thank You 28