1. Update on HDL Infusions and Atherosclerosis Regression
H. Bryan Brewer, Jr. Washington Cardiovascular Associates Medstar Heart and Cardiovascular Institute Washington Hospital Center
Washington, DC

2. Current Model of HDL and Lipoprotein Metabolism
Liver
B Asztalos Tufts University Boston, MA
aHDL
Preb-1
Control Plasma
B-100
Triglyerides
C-III E
Intestine
Cholesterol
B-100
LDLR
LPL HL
SR-BI
Cholesterol
A-I CE TG
CETP
αHDL
VLDL
LRP
LDL
Macrophage
Arterial Wall
LPL HL
ABCG1
B-48
Triglyerides
C-III E
ABCA1
LCAT
Preβ-HDL
A-I
Chylomicron

3. 2 Step Approach to the Synthesis of Preβ-HDL
2 Step Process for Production of Preβ-HDL 1
ApoA-I
243 aa
rHDL
A-I
Phospholipids
Step 1
Isolate and Purify ApoA-I
Step 2
Combine ApoA-I + Phospholipids
2 Step Process for Production of Preβ-HDL 1
ApoA-I
243 aa
rHDL
A-I
Phospholipids
Step 1
Isolate and Purify ApoA-I
Step 2
Combine ApoA-I + Phospholipids
Clinical Programs
ApoA-I Milano Esperion
Pfizer
Medicines Co
Preβ-HDL
A-I

4. ApoA-I Milano ApoA-I Milano Arg Cys 173
In apoAI milano, there is a cysteine substitution for arginine at position 173 allowing for dimerization, formation of large HDL particles that may be particularly active in RCT. It is characterized by the replacement of a single amino acid at R173C
R173S apoA-I. Carriers of the apolipoprotein A-IMilano (apoA-IM) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. Discovered by accident, the mutation was found to be present in about 3.5% of the population of Limone sul Garda, a small village in northern Italy. It has been traced to a mutation in a single man, Giovanni Pomarelli[2], who lived in the village in the 18th century and passed it on to his offspring.[3]
Figure 1eNOS expression in HDL-treated HUVECs. A, Reverse-transcription polymerase chain reaction analysis for eNOS mRNA levels in untreated cells (medium) and in cells incubated overnight with HDL (1.14 mmol/L cholesterol) isolated from control subjects ...eNOS expression in HDL-treated HUVECs. A, Reverse-transcription polymerase chain reaction analysis for eNOS mRNA levels in untreated cells (medium) and in cells incubated overnight with HDL (1.14 mmol/L cholesterol) isolated from control subjects (control HDL) or apoA-IM carriers (A-IM HDL). eNOS mRNA band intensities were normalized by GADPH values. B, Western blotting analysis of eNOS protein levels in untreated cells and in cells incubated overnight with control HDL or A-IM HDL (1.14 mmol/L cholesterol). eNOS protein band intensities were normalized by β-actin values. Results are expressed as mean±SEM of 3 separate experiments with 3 different preparations of control HDL or A-IM HDL and 2 batches of cells. *P<0.05 vs medium, #P<0.05 vs control HDL.
ApoA-I Milano
Arg Cys
173
Gomaraschi M et al. Circulation 2007;116:

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

6. HDL Infusion Therapy Update
Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial

7. ApoA-I Milano Clinical Trial : Milano Pilot Study
Primary Endpoint: Percent Atheroma Volume (PAV)
Secondary Endpoint: Total Atheroma Volume
-6.90 (-17.5,2.2) P<0.01
-4.7 (-13.,1.7) P<0.01
Placebo
MCO-216
(Entire Vessel Length)
- 2
- 4
- 6
- 8
- 10
- 12
- .20
P =0.10
P = .77
- .40
Median Change in Percent Atheroma Volume (%)
-0.5 (-1.8.,0.5) P=0.11
- .60
Median Change Volume (mm3)
- .80
-0.8 (-2.3,0.2) P<0.001
- 1
Placebo
MCO-216
- .20
Nicholls S and Nissen S AHA 2017 Presentation

8. Clinical Programs 2 Step Approach to the Synthesis of Preβ-HDL
2 Step Process for Production of Preβ-HDL
Clinical Programs
Step 1
Isolate and Purify ApoA-I
Step 2
Combine ApoA-I + Phospholipids
Medicines Co: ApoA-I Milano 1
ApoA-I
243 aa
rHDL
A-I
Phospholipids
CSL: CSL 111/112

9. 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: - -
Change in Total Atheroma Volume (mm)3 12
-5.34 -
*Nissen S et al JAMA 2003;290:

10. HDL Infusion Therapy Update
Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial
CSL Behring – AEGIS-I Trial CSL112: Safety and Efficacy Trial

11. CSL Behring AEGIS-I Trial: Clinical Infusion Therapy With CSL112
Subjects:1258 Acute Coronary Syndrome Patients
Randomized 1:1:1 2 g/A-I, 6g A-I, placebo
Stratified renal function
Trial: 4 weekly infusions of CSL 112
End Points
Primary Endpoint : Clinical Assessment of Renal and Liver Function
Secondary Endpoint : 1. Composite CV Death, non-fatal MI, stroke hospitalization for unstable angina
2. Cholesterol efflux
Results
No Significant Alteration in Liver and Kidney Function
Dose Dependent Elevated Cholesterol Efflux
Gibson CM Et al Circulation 2016;134:

12. Clinical Programs 2 Step Approach to the Synthesis of Preβ-HDL
2 Step Process for Production of Preβ-HDL
Clinical Programs
Step 1
Isolate and Purify ApoA-I
Step 2
Combine ApoA-I + Phospholipids
Medicines Co, ApoA-I Milano 1
ApoA-I
243 aa
rHDL
A-I
Phospholipids
CSL, CSL 111/112
Cerenis, CER-001

13. Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions
570 Acute Coronary Patients: 6 Weekly Infusions, Infusion Dose CER mg/kg, 6 mg/kg and 12 mg/kg 2 4 6 8 10 14 16 - +
CHI-SQUARE 3 mg/kg***
***Tardif JC et al Eur Heart J 2014;35:
-3.13
A-I Milano*
-14.10
-5.34
ERASE**
**Tardif JC et al. JAMA 2007;297: - -
Change in Total Atheroma Volume (mm)3 - 12
-5.34 -
*Nissen S et al JAMA 2003;290:

14. HDL Infusion Therapy Update
Medicines Co. – ApoA-I Milano Pilot Study: Safety and Efficacy IVUS Trial
CSL Behring – AEGIS-I Trial CSL112: Safety and Efficacy Trial
Cerenis – CER-001 CARAT: Safety and Efficacy IVUS Trial

15. Cerenis CARAT Trial: Clinical HDL Infusion Therapy With CER-001
Subjects: 292 Acute Coronary Syndrome Patients
Trial: 9 weekly infusions of 3 mg/kg CER-001
End Points
Primary Endpoint : Change in Percent Athero Volume (PAV)
Results
To Be Presented ACC
Anderson J et Cardiovasc Diagn Ther 2017;7:45-51

16. HDL Therapeutics Therapy Employs a 1 Step Process To Produce Preβ-HDL
2 Step Process for Production of rHDL
1 Step Process for Synthesis of Preβ-HDL
αHDL
Preβ-HDL
A-I
Advantages of Selective Delipidation
1. Safe
2. High Yield
4. Low Cost
3. Selective increase in preβ-HDL for Specificity for ABCA1 Transport
5. Low Regulatory Hurdles
Isolated ApoA-I
Phospholipids 1
243 aa
rHDL
A-I
Convert αHDL to Preβ-HDL by Selective HDL Delipidation
CSL, CSL111/CSL112

17. a HDL a HDL a HDL Preb-1 Preb-1 Preb-1
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

18. Acute Preβ-HDL Infusions: IVUS Clinical Trial De-risking and Feasibility Trials In ACS Patients

19. HDL Selective Delipidation****
Reduction in Atheroma Volume in Acute Coronary Syndrome Patients Following HDL Infusions 2 4 6 8 10 14 16 - +
28 Acute Coronary Patients: 7 Weekly Infusions, 14 Received Delipidated HDL and 14 Control Plasma Infusions
HDL Selective Delipidation****
****Waksman R et al J. Am. Coll Card 2010;55: C
-12.10
A-I Milano*
-14.10
-5.34
ERASE**
**Tardif JC et al. JAMA 2007;297:
CHI-SQUARE 3 mg/kg***
***Tardif JC et al Eur Heart J 2014;35:
-3.13 - -
Change in Total Atheroma Volume (mm)3 - 12
-5.34 -
*Nissen S et al JAMA 2003;290:

20. HDL Infusion Therapy Update
New Approaches to the Quantitation of Vascular Disease Following HDL Infusions

21. TVCTM: New FDA Approved Technical For Vulnerable Plaque Analysis
Coronary Artery Lesions
Distinguishes Cholesterol Filled Plaques From
Cholesterol Free Lesions
Measures Cholesterol Content of Vulnerable Plaques Before and After Therapy
NIR Scan
Cholesteryl Oleate
Cholesterol
Cholesteryl Linoleate
Collagen

22. Take Home Messages √
Selective 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 Preβ-HDL will decrease cardiovascular events.