1. Update on Anacetrapib, a Novel CETP Inhibitor
Douglas Johns, PhD, FAHA
Basic Science Lead, CETP/Anacetrapib
Merck Research Laboratories
Rahway, NJ 1

2. Douglas Johns, PhD Salary: Merck and Company, Inc.
Ownership Interest (Stocks, Stock Options, or other Ownership Interest):

3. Outline Review/update of Anacetrapib clinical programs
DEFINE
Hps3-TIMI55-REVEAL
Basic science/mechanism-of-action studies
Reverse cholesterol transport
Bulk cholesterol excretion
HDL function: Cholesterol efflux
HDL function: Anti-inflammatory effects of HDL

4. Anacetrapib clinical program

5. Cannon CP et al. N Engl J Med 2010;363 (19) online 11/17/2010
Anacetrapib treatment had robust effects on HDL-C, LDL-C, non HDL-C and Lp(a) with sustained effects over 18 months.
Anacetrapib had an acceptable side-effect profile with no effects on blood pressure, electrolytes or aldosterone.
Within the power of the study, anacetrapib did not exhibit adverse cardiovascular effects seen with a prior CETP inhibitor
The long term safety and efficacy of anacetrapib will now be tested in a large clinical outcomes trial.

6. Worldwide cardiovascular outcome study
N=30,000, anacetrapib 100mg po or placebo
Sponsor: University of Oxford Clinical Trial Service Unit
Population: Stable cardiovascular disease with high risk of recurrent events
Primary outcomes: Coronary mortality, myocardial infarction, coronary revascularization
Follow-up: minimum median follow up of at least 4 years
Progress: First patient enrolled May 2011, first patient randomized August 2011, currently enrolling in North America, Europe and Asia, enrollment is on schedule

7. Anacetrapib: basic science/ mechanism-of action studies

9. How does CETP inhibition affect HDL function?
Cholesterol efflux
and RCT
Anti-oxidant
Anti-inflammatory
Endothelial function
Improve β-cell function
Anti-thrombotic
Anti-apoptotic

10. How does CETP inhibition affect HDL function?
Cholesterol efflux
and RCT
Anti-oxidant
Anti-inflammatory
Endothelial function
Improve β-cell function
Anti-thrombotic
Anti-apoptotic

11. Comprehensive study of CETP inhibitors and cholesterol metabolism requires more than a single approach
Study cholesterol movement with CETP inhibition using “classical approaches”
Macrophage-to-feces RCT
CETP-containing rodent species (e.g hamsters)
Monitor changes in bulk cholesterol pools
High-resolution mass spectrometry
Assess HDL functionality with ex vivo methods
Macrophage to feces RCT
Bile acids

12. Macrophage to feces reverse cholesterol transport

13. Anacetrapib increases macrophage to feces reverse cholesterol transport in hamsters
Increase in macrophage-derived 3H-tracer in HDL, suggesting increased cholesterol efflux from macrophages
Increase in macrophage-derived 3H-chol and 3H-bile acids in the feces indicates net increase in macrophage-to-feces RCT
3H content in HDL
2.0
Control
***
Anacetrapib (60mg/kg)
1.5
% of injected
dose in HDL
1.0
0.5
0.0 24 48 72
Time (hours)
3H content feces
0.8
72 hours collection *
0.6 *
% injected dose
in feces
0.4
0.2
0.0
Control
Ana
Control
Ana
J Lipid Res. 2011, 52:
Cholesterol
Bile acids

14. Cholesterol excretion
Anacetrapib increases cholesterol in large HDL and promotes excretion of bulk cholesterol (no tracers)
Cholesterol in HDL subfractions
Increase in cholesterol is greatest in large HDL (HDL2)
Beyond macrophage-to-feces RCT, ANA treatment increase fecal cholesterol and bile acid concentration
These results indicate that anacetrapib is promoting movement of cholesterol out of the system
From periphery to feces (RCT)
Bulk cholesterol excretion
150
150
***
Vehicle
Vehicle ** **
Ana
100
100
Cholesterol (mg/dL) 50 50
HDL3
HDL2
Day 0
Day 14
Ana (60mg/kg)
Fecal cholesterol
Fecal cholic acid 50
100
150
200 *
29.4%
Cholesterol excretion
rate (ug/day) 5 10
19.3%
CA ( m
g/g feces)
J Lipid Res. 2011, 52:

15. HDL functionality: Cholesterol efflux
In addition to aqueous passive diffusion, cholesterol effluxes to HDL via protein-facilitated pathways
ABCG1 and SR-BI facilitate cholesterol efflux to large/alpha HDL
ABCA1 transports cholesterol to pre-beta HDL
Ex vivo methods
Apply serum from treated animals/humans and assess functional properties
Macrophage
ABCG1 α
cholesterol
SR-BI α
ABCA1
Lipid-poor apoA-I
Pre-β-HDL

16. Plasma from Anacetrapib-treated hamsters displays improved cholesterol efflux14
ABCA1
Both major pathways of cholesterol efflux (alpha-HDL, pre-beta HDL) are increased with anacetrapib treatment
↑ABCA1-mediated efflux suggests increased pre-beta HDL component
↑ABCG1, ↑SR-BI mediated efflux indicates increased efflux to αHDL
Similar observations with HDL from ANA-treated humans * 12
(% efflux) 10 8 6
Veh
Ana
SR-B1
ABCG1 5
2.0 **
*** 4
1.5 3
1.0
(% efflux)
(% efflux) 2
0.5 1
0.0
Veh
Ana
Veh
Ana
J Lipid Res. 2011, 52:

17. Plasma from Anacetrapib-treated humans displays improved cholesterol efflux
Total Cholesterol
Free Cholesterol
Cholesterol Ester
*P<0,05, significant difference vs control PEG-HDL
Increase in efflux at the same HDL-c concentration suggests an improvement in HDL “quality”
1Arterioscl Thromb Vasc Biol (7):1430-8

18. HDL from ANA-treated hamsters maintains ability to inhibit TNFα-induced inflammation in endothelial cells
VCAM-1 expression 20 40 60 80
100
120
140
160
180 **
VCAM-1 mRNA
Control
TNF a
Veh-HDL
Ana-HDL
HDL suppresses TNF-induced adhesion molecule expression1
VCAM, ICAM, E-selectin mRNA and protein
HDL from ANA-treated hamsters shows no reduction in this effect (vs vehicle)
Similar to observations with HDL from ANA-treated human1
Monocyte adhesion to endothelial cells suppressed by HDL
No reduction in this effect with ANA-treated hamster HDL vs vehicle
These results suggest that HDL functionality is improved, or not reduced with Anacetrapib treatment.
** p<0.01 vs. TNFα
* p<0.05 vs. TNFα
Monocyte adhesion 10 20 30 40 50 60 70 80 90
100
110 *
% Adherent monocyte
Control
TNF a
Veh-HDL
Ana-HDL +
1Deuel Lipid Conference poster, Napa, CA 2011
2Arterioscl Thromb Vasc Biol (7):1430-8

19. Summary
Multiple lines of evidence suggest that HDL functionality is improved or “maintained” with anacetrapib inhibition of CETP
Enhanced macrophage-to-feces reverse cholesterol transport in hamsters
Increased cholesteryl ester content in hamster HDL (enhanced maturation from HDL3 to HDL2)
Improved excretion of bulk cholesterol/bile acids in hamsters
Improvement in plasma cholesterol efflux capacity via ABCA1 (pre-beta-dependent), and via SR-BI/ABCG1
No reduction in anti-inflammatory effects of HDL