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

2. Douglas G. Johns, PhD
Stocks, Stock Options, other ownership interest: Merck and Company, Inc.

3. Outline Review/update of Anacetrapib clinical program
DEFINE
Hps3-TIMI55-REVEAL
Effects of anacetrapib on HDL functionality
Reverse cholesterol transport, cholesterol excretion
HDL function: Cholesterol efflux and pre-beta HDL
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 reversal phase of the DEFINE trial is being evaluated in long-term follow-up

6. Worldwide cardiovascular outcome study
N=30,000, anacetrapib 100mg po or placebo on background of atorvastatin
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
Lipid studies have been initiated in parallel (familial hypercholesterolemia)

7. Effects of anacetrapib on HDL functionality

9. DEFINE: Effects on LDL-C and HDL-C
Study Week
Baseline
Wk 6
Wk 12
Wk 18
Wk 24
Wk 30
Wk 46
Wk 62
Wk 76
HDL-C (mg/dL) (SE) 20 40 60 80
100
120
Anacetrapib
Placebo
Anacetrapib n =
776
757
718
687
647
607
572
543
Placebo n =
766
761
741
744
736
711
691
666
+138.1% increase at week 24 (p<0.001)
LDL-C
Baseline
Wk 6
Wk 12
Wk 18
Wk 24
Wk 30
Wk 46
Wk 62
Wk 76
LDL-C (mg/dL) (SE) 20 40 60 80
100
Anacetrapib
Placebo
Anacetrapib n =
804
771
716
687
646
604
568
540
Placebo n =
803
759
741
743
735
711
691
666
The point estimates are for wk 24
Study Week
-39.8% reduction at week 24 (p<0.001)
Cannon CP et al. N Engl J Med 2010;363 (19)

10. With large increases in HDL-c, how is HDL functionality affected?
Cholesterol efflux
and RCT
Anti-oxidant
Anti-inflammatory
Endothelial function
Improve β-cell function
Anti-thrombotic
Anti-apoptotic

11. 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

12. 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

13. 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:

14. 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

15. 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:

16. 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 equivalent HDL-c concentrations suggests an improvement in HDL “quality”
1Arterioscl Thromb Vasc Biol (7):1430-8

17. Evidence for increase in prebeta HDL concentration in Anacetrapib-treated hamsters
Total ApoAI
n.s.
Control human plasma (left) and hamster plasma (right) run on the same gel for size comparison
Pre-beta HDL measurements courtesy, Bela Aztalos,
Tufts University

18. HDL from anacetrapib-treated hamsters and humans shows no impairment in anti-inflammatory properties
Dyslipidemic hamsters
HDL suppresses TNF-induced adhesion molecule expression
VCAM, ICAM, E-selectin mRNA and protein
Monocyte adhesion to endothelial cells suppressed by HDL
No reduction in this effect with ANA-treated hamster HDL vs vehicle (not shown)
Whether anacetrapib improves HDL function in humans with CV disease remains to be determined
Vehicle
Ana (30mg/kg)
Humans (healthy normal)
PBO
Ana (20mg)
Ana (150mg)
Han et al. Biochim Biophys Acta doi:pii: S (12) , 2012

19. Summary
Multiple lines of evidence suggest that despite large increases in HDL cholesterol, HDL functionality is improved or “maintained” with anacetrapib inhibition of CETP
Enhanced macrophage-to-feces reverse cholesterol transport in hamsters
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
Increase in plasma pre-beta HDL concentration in anacetrapib-treated hamsters
Increase in cholesterol efflux to plasma/HDL in anacetrapib-treated human subjects
No impairment in anti-inflammatory properties of HDL
Maintained suppressive effects on adhesion molecule expression, monocyte adhesion, NF-kB signaling
Observed in hamsters and humans