What are the options left? Targeting HDL-c: What are the options left? Dr. Bryan Brewer Medstar Research Institute Washington DC, USA

Current Model of HDL Metabolism Triglycerides B-100 C-III E Triglycerides B-100 C-III E Triglycerides B-100 C-III LDLR Modification VLDL SR-BI CE CETP TG LDL LRP LPL HL ABCG1 E Triglycerides B-48 C-III LCAT HDL-M, (α3) HDL-L, (α2) HDL-VL, (α1) ABCA1 A-I HDL-VS Preβ-HDL HDL-S, (α4) Chylomicron Macrophage Arterial Wall

Targeting HDL-C: What are the Options Left I. CETP Inhibitors II. ApoA-I Inducers

Resverlogix ApoA-I Inducer: RVX 208 ASSERT Clinical Trial (n=299) Modest increases in HDL-C (8.3%) and apoA-I (5.6%) levels. Increase in larger HDL (16.2%, result of successful cholesterol efflux?) ASSURE Clinical Trial (n=310) - Effects of 6 months of RVX 208 on coronary atherosclerosis quantitated by IVUS.

Targeting HDL-C: What are the Options Left I. CETP Inhibitors II. ApoA-I Inducers III. HDL Infusions

Concept of HDL Infusion Therapy – Increase Preβ-HDL Triglycerides B-100 C-III E Triglycerides B-100 C-III E Triglycerides B-100 C-III LDLR Modification VLDL CE CETP TG SR-BI LDL LRP LPL HL ABCG1 E Triglycerides B-48 C-III LCAT HDL-VL, (α1) A-I ABCA1 LCAT HDL-L, (α2) Chylomicron LCAT HDL-M, (α3) A-I Macrophage Arterial Wall A-I Acute HDL Therapy: Key - Infusion of Preβ-HDL A-I HDL-S, (α4) HDL-VS Preβ-HDL

Acute HDL Infusion Therapy 2 Step Process for Production of Recombined ApoA-I (rHDL) Phospholipids Synthesized ApoA-I 1 243 aa rHDL A-I Clinical Trials rHDL

HDL Infusion Clinical Trial Design: 5 -7 Weekly HDL Infusions With Quantitation of Coronary Atherosclerosis in ACS Patients by Intravascular Ultrasound (IVUS) Treatment Arm Stable Hypolipidemic Drugs Control Arm Day 0 1 2 3 4 5 6 7 8 Week IVUS IVUS Treatment or Control Infusions

HDL Infusions in ACS Patients I. ApoA-I Milano

Acute HDL Infusion Therapy ApoA-I Milano 2 Step Process for Production of Recombined ApoA-I (rHDL) Phospholipids Recombinant Synthesized ApoA-I 1 243 aa rHDL A-I Clinical Trials rHDL

Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 46 Acute Coronary Patients: 5 Weekly Infusions 33 ApoA-I Milano Infusion; 11 Saline Infusions 2 4 6 8 10 14 16 - + A-I Milano* -14.10 - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Infusions in ACS Patients I. ApoA-I Milano II. CSL

Acute HDL Infusion Therapy CSL 111 2 Step Process for Production of CSL111 (ApoA-I/PL,rHDL) using Cholate Detergent Phospholipids Isolated ApoA-I from plasma 1 243 aa Cholate Detergent CSL111 (αHDL) A-I Clinical Trials CSL111 Cholate

Gel Electrophoresis of CSL111 (rHDL) Patel, S. et al. J Am Coll Cardiol 2009;53:962-971

Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 183 Acute Coronary Patients: 4 Weekly Infusions, 123 Patients Received CSL111 rHDL Infusion; 60 Patients Control Infusions 2 4 6 8 10 14 16 - + A-I Milano* -14.10 -5.34 ERASE** **Tardif JC et al. JAMA 2007;297:1675-82 - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

CSL has reformulated CSL111 and are currently developing CSL112

Phase I CSL112 (αHDL3) Infusions in Humans 4 8 12 16 20 24 Hours Cholesterol (mg/dl) Free Cholesterol Cholesteryl esters Diditchenko S et al Presented at ISA 2012 Australia

HDL Infusions in the ACS Patient I. ApoA-I Milano II. CSL III. Cerenis

Acute HDL Infusion Therapy:CER-001 2 Step Process for Production of CER-001 (ApoA-I,PL/Sphingomyelin) (αHDL) Synthesized ApoA-I 1 243 aa Phospholipids + Sphingomyelin ApoA-I/PL/ Sphingomyelin A-I Clinical Trials Cerenis-001 (αHDL)

Acute HDL Infusion Therapy Effect of Cerenis CER-001 Infusion on Plasma Lipoproteins 4 8 12 16 20 24 Hours Cholesterol (mg/dl) Free Cholesterol Cholesteryl esters

CHI SQUARE Clinical Trail IVUS Analysis of the Effect of Cerenis CER-001 on Coronary Atherosclerosis Control Arm 6 Infusions ? mg/kg n =500+ 21

HDL Infusions in ACS Patients I. ApoA-I Milano II. CSL III. Cerenis IV. HDL Therapeutics

Synthesis of Ligands for the ABCA1 Transporter 2 Step Process for Production of rHDL 1 Step Process for Synthesis of Preβ-HDL αHDL 1 Challenges of 2 Step Procedure 1. Low Yield 2. Safety 4. Cost 3. αHDL vs preβ-HDL- specificity for ABCA1 Transporter 5. Regulatory Hurdles 243 aa Synthesized ApoA-I Phospholipids Selective HDL Delipidation A-I Preβ-HDL A-I Clinical Trials Selective HDL Delipidation Clinical Trials rHDL rHDL

HDL Selective Delipidation Increase Preβ Levels by Converting αHDL to Preβ-HDL A-I A-I A-I αHDL Preβ-HDL ”Loaded Dump Truck” “Unloaded Dump Truck” 24 24

Preβ Enriched Plasma Obtained by HDL Selective Delipidation Plasma Bag 1 Plasma Collected Preβ Enriched Plasma Obtained By Selective HDL Delipidation A-I αHDL Preβ-HDL 20% 80% Plasma Bag 2 Preβ Enriched Plasma A-I A-I αHDL Preβ-HDL 95% 5% Preβ-HDL

2-D Gel Electrophoresis of Control Plasma, Sham Treated Plasma and Selective HDL Delipidated Plasma Demonstrating a Shift from aHDL to preb-HDL with Selective HDL Delipidation a HDL a HDL a HDL Preb-1 Preb-1 Preb-1 Sham Treated Plasma Selective HDL Delipidation Control Plasma Analysis performed by Dr. B. Asztalos, Tufts University Boston, MA

2-D Gel Electrophoresis of Control Plasma, Sham Treated Plasma and Selective HDL Delipidated Plasma Demonstrating a Shift from aHDL to preb-HDL with Selective HDL Delipidation a HDL a HDL a HDL Preb-1 Preb-1 Preb-1 Sham Treated Plasma Selective HDL Delipidation Control Plasma Analysis performed by Dr. B. Asztalos, Tufts University Boston, MA

Step 1 Collected ~1 Liter of Patients Plasma HDL Therapeutics Human Trial: Conducted Using Autologous Preβ-HDL Enriched Plasma Step 1 Collected ~1 Liter of Patients Plasma Step 2 Cholesterol removed from αHDL to yield preβ-HDL Step 3 Re-infused Preβ Enriched Plasma 28

HDL Selective Delipidation*** Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 28 Acute Coronary Patients: 7 Weekly Infusions, 14 Received Delipidated HDL and 14 Control Plasma Infusions 2 4 6 8 10 14 16 - + HDL Selective Delipidation*** ***Waksman R et al J. Am. Coll Card 2010;55:2727-2735C -12.10 A-I Milano* -14.10 -5.34 ERASE** **Tardif JC et al. JAMA 2007;297:1675-82 - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 - *Nissen S et al JAMA 2003;290:2292-2300.

HDL Selective Delipidation* Comparison of Change in Total Atheroma Volume in the HDL Therapeutics HDL Infusion and Saturn Trials 2 4 6 8 10 14 16 - + HDL Infusion SATURN Statin Therapy -12.10 HDL Selective Delipidation* - 6.4 - 4.4 Atorvastatin SATURN Trial** Rosuvastatin - - Change in Total Atheroma Volume (mm)3 - 12 -5.34 7 Weeks 2.2 Years - *Waksman R et al J. Am. Coll Card 2010;55:2727-2735C *Nicholls SJ et al, N Eng J Med 2011;365:2078-2087

Targeting HDL-C: What are the Options Left I. CETP Inhibitors II. ApoA-I Inducers III. HDL Infusions IV. LCAT Inducers

LCAT Targeted Therapy ACP-501 (rhLCAT) Clinical Summary ACP-501 safe and tolerable at all doses evaluated No SAE’s Rapid and substantial increase in HDL-C (43% increase from baseline) and cholesterol esterification Evidence that RCT pathway is being activated

LCAT Targeted Therapy ACP-501 (rhLCAT) I. Familial LCAT Deficiency 1. Orphan Drug Opportunity

Lipid Filled Macrophage in the Heart LCAT Therapy: Infusion of rLCAT For Treatment of Familial LCAT Deficiency Liver Intestine LDLr LCAT CETP A-I αHDL ABCG1 LDL CD36 SR-A SR-BI Lp-X Kidney (FC/PL) αHDL LCAT ABCA1 A-I αHDL Preβ-HDL Lipid Filled Macrophage in the Heart

LCAT Targeted Therapy ACP-501 (rhLCAT) I. Familial LCAT Deficiency 1. Orphan Drug Opportunity II. Infusions of rLCAT to Reduce Coronary Plaques

LCAT Therapy: Infusion of rLCAT For Treatment of Coronary Plaques Liver Intestine LDLr CETP LDL CD36 SR-A SR-BI ABCG1 A-I A-I ABCA1 LCAT A-I αHDL Preβ-HDL Lipid Filled Macrophage in the Heart αHDL Acute rLCAT Infusions

LCAT Overexpression Increased Plasma HDL and Decreased Atherosclerosis in LCAT Transgenic Rabbits Hoeg et al. Proc Natl Acad Sci USA.1996;93:11448-11453.

Take Home Messages √ √ √ √ Epidemiological and clinical trial evidence established that low HDL is an independent risk factor for CVD. √ Novel HDL therapies are under development including CETP Inhibitors, ApoA-I inducers, HDL infusions and LCAT inducers. √ HDL infusions in ACS patients were associated with marked regression of atherosclerosis and have a high probability of decreasing clinical events. √ Clinical trials will be required to definitively establish if increasing HDL will decrease clinical cardiovascular events.