1. The role of the CETP protein in reverse cholesterol transport and atherogenesis: Is CETP good or bad? Prof. Chris Packard Western Infirmary Glasgow, United Kingdom

2. Role of CETP in HDL and LDL physiology. Natural CETP variation and CHD risk. Epidemiology of CETP. Pharmacodynamics of CETP regulation. CETP as a therapeutic target Key considerations

3. CETP in HDL physiology Role in lipoprotein metabolism LCAT LCAT Macrophage Nascent HDL ABCA1 CETP CETP - a promiscuous plasma transfer protein shuttling neutral lipid between lipoproteins. After Brewer et al. Am J Cardiol 2003; 92:10K

4. CETP in HDL physiology Role in reverse cholesterol transport CETP lacks intrinsic directionality. Metabolic gradients are maintained to promote net transfer of CE from HDL to VLDL remnants and LDL. VLDLremnants/IDL/LDL CE

5. CETP in HDL physiology Lessons from nature X CETP deficiency in humans (prevalent in Japanese) is often associated with very high HDL chol and low LDL but link with CHD risk is unclear (Barter et al ATVB (2003) 23:160-167) Rodents have no CETP. HDL is major cholesterol carrier. Natural resistance to development of atherosclerosis VLDLr/ IDL/

6. HDL subfractions and reverse cholesterol transport HDL 2 HDL 3 small, dense HDL A1:phospholipid pre  HDL 2D analysis of plasma HDL

7. CETP and the Atherogenic Lipoprotein Phenotype Raised VLDL – hypercoagulability, raised PAI1. Increased remnants – macrophage cholesterol deposition. Small, dense LDL – retention in artery wall, readily oxidised. Low HDL – impaired reverse cholesterol transport, reduced antioxidant properties. ALP type 2 diabetes, metabolic syndrome CETP central to development of ALP

8. Regulation of LDL metabolism by CETP Packard et al. J Lipid Res 2000; 41:305-317Sandhofer et al. Obesity 2006; 14:812-818

9. Origins of dyslipidemia in metabolic syndrome increased VLDL 1 plasma TG>1.5 VLDL 1  production RT=5d CETG LDL I/II HL (>15 U/l) TG CE CETP CE TG LDL III CE TG Small, dense LDL CE TG Remnants Moderate HTG Packard and Shepherd (1997) FFA Insulin X LDL I/II

10. Regulation of HDL metabolism by CETP Gut Liver LpL Chylomicrons VLDL HL CETP LCAT HDL 2 HDL 3 HDL 2 CE TG CE TG CE PL,FC PL, FA apoAI:PL pre B HDL

11. Regulation of HDL metabolism by lifestyle factors Gut Liver LpL Chylomicrons VLDL HL CETP LCAT HDL 2 HDL 3 HDL 2 CE TG CE TG CE PL,FC PL, FA apoAI:PL pre B HDL Obesity Smoking Alcohol + - +

12. LDL and HDL size in inherited CETP deficiency Bisgaier et al. J Lipid Res 1991; 32:21-33 Lack of CETP leads to extreme changes in LDL and HDL structure

13. Role of CETP in HDL and LDL physiology. Natural CETP variation and CHD risk. Epidemiology of CETP. Pharmacodynamics of CETP regulation. CETP as a therapeutic target Key considerations

14. CETP as a major gene determining lipid levels (Hepatic lipase) Teslovich et al. Nature 2010; 466:707-713

15. High-density lipoprotein subfractions in the population and CETP genotype groups Genotype CCACAATotal HDL-TG, mmol/L (n=115)0.21±0.070.20±0.060.23±0.070.21±0.06 HDL-C, mmol/L (n=219)*1.36±0.311.42±0.301.60±0.43 ‡ 1.44±0.34 HDL 2 mass, mg/100 mL (n=215)*77±3987±48112±5989±50 HDL 3 mass, mg/100 mL (n=215)266±63263±54272±65266±59 Freeman et al. (1994) ATVB 14;336-344; Dullaart et al (2008) Scan J Clin Lab Invest 68;99-105 CETP gene variation and HDL regulation Whole group (n=220) CC (n=51, 23.2%) AC (n=109, 49.5%) AA (n=60, 27.3%) ANOVA P value CETP (mg/L)2.39±0.812.96±0.962.26±0.67 *** 2.13±0.66 *** <0.001 CETP activity (nmol.L -1,s -1 )2.7 (1.9–3.9)3.4 (2.4–4.8)2.7 (1.8–3.8) *** 2.6 (1.7–3.5) *** 0.007 CETP variation in promoter region : C- common type; A - rare allele

16. CETP Genetics and CHD risk Thompson et al. JAMA 2008; 299:2777-2788 Meta-analysis of 92 studies of CETP genotype, lipid levels and CHD risk in general population. Inheritance of one ‘A’ allele in CETP gene associated with:- 9.7% lower CETP mass 8.6% lower CETP activity 4.5% higher HDLchol 2.4% higher apoA1 Odds ratio for CHD per A allele of 0.95 (0.92-0.99)

17. CETP genetics and CHD risk Influence of background statin therapy Regieli et al. Eu Heart J 2008; 29:2792-2799

18. Role of CETP in HDL and LDL physiology. Natural CETP variation and CHD risk. Epidemiology of CETP. Pharmacodynamics of CETP regulation. CETP as a therapeutic target Key considerations

19. Epidemiology of CETP Framingham study Vasan et al. Circulation 2009; 120:2414-2420 Higher CETP activity Lower risk

20. CETP mass and CHD incidence in PROVE-IT Khera et al. Am J Cardiol 2010; 106:451-456 Subjects on 40 md pravastatin / 80mg atorvastatin : Hazard ratio per SD increase in CETP mass = 0.77 (P=0.005) Interaction between CETP mass and LDL chol (P=0.007)

21. CETP: Friend or Foe ? Which pathway is quantitatively more important in reverse cholesterol transport in man, particularly in statin treated subjects ? Is CETP required to be active when LDL is low ? ? ?

22. Role of CETP in HDL and LDL physiology. Natural CETP variation and CHD risk. Epidemiology of CETP. Pharmacodynamics of CETP regulation. CETP as a therapeutic target Key considerations

23. Pharmacodynamics of CETP regulation Anacetrapib (MSD) Dalcetrapib (Roche) Cys 13 – dalcetrapib binding site Lipid tunnel CETP crystal structure Qui et al (2007) Nat Struct Mol Biol 14;106-112, Ranaletta et al (2010) J Lipid Res on line, Okamoto et al. Nature. 2000;406:203–207, Clark et al. J Lipid Res. 2006;47:537–552, Masson D. Curr Opin Invest Drugs. 2009;10:980-987, Grooth et al. Circulation. 2002;105:2159–2165, Clark et al. Arterioscler Thromb Vasc Biol. 2004;24:490–497; Krishna et al. Lancet. 2007;370:1907-1914, Stein et al. Am J Cardiol. 2009;104:82–91, Barter et al. N Engl J Med. 2007;357:2109–2122, Forrest et al. Br J Pharmacol 2008;154:1465–1473 DalcetrapibTorcetrapibAnacetrapib IC 50 9000 nmo/l50 nmol/l57 nmol/l CETP inhibition37.2%≥80%90% HDL-C increase33.9%91%129% Blood pressure increaseNoYesNo Increases aldosterone production (in vitro) NoYesNo

24. Effects of CETP regulation on plasma lipids Cannon et al. NEJM 2010; 363:2406-2415 DalcetrapibAnacetrapib HDL chol+27%+138% LDL chol-5%-40% ApoA1+13%+45% Triglyceride+1%-7% Lp(a)0-39% Robinson. Exp Opin Invest Drug 2010; 19:795-805

25. CETP deficiency and apoB metabolism FCR LDL from CETP(-) = 0.41 pools/d FCR LDL from normal = 0.60 pools/d Ikewaki et al. J Clin Invest 1995; 96:1573-1581 Lipoprotein kinetic studies in CETP deficient homozygotes LDL from CETP(-) has reduced clearance rate, reduced IDL synthesis, increased removal rate for LDL in CETP (-) despite LDL abnormality

26. Differential regulation of CETP Niesor et al. J Lipid Res 2010; 51:3443-3454 HDL 3 – HDL 2 transfer Impact on pre  HDL

27. CETP, HDL substructure and atheroprotection (after Barter et al) HDL 2 HDL 3 small,dense HDL A1:phospholipid pre  HDL Adhesion Molecule Monocyte Intim a Vessel lumen Endothelium MCP-1 Macrophage Cytokines Foam Cell HDL PROMOTE CHOLESTEROL EFFLUX HDL INHIBIT ADHESION MOLECULE EXPRESSION MODIFIED LDL HDL INHIBIT OXIDATION OF LDL LDL CETP, Hepatic lipaseLCAT, ABCA1, LpL

28. Testing the HDL paradigm Danesh et al (2009) JAMA 302:1993-2000 Males Scenario 1 – Treatment effect independent of baseline HDL. Rx given to all at a single dose. Scenario 2 – Risk reduction dependent on baseline HDL; no benefit at HDLc > 50 mg/dl. Need to measure HDL accurately. Scenario 3 – Risk reduction dependent on baseline CRP; no benefit if CRP > 3 mg/dl (low HDL linked to inflammation not reduced reverse cholesterol transport). Outcome scenarios

29. Summary -1 CETP is a major regulator of lipoprotein structure and composition. Lower CETP activity is associated with larger LDL and HDL species. Inherited deficiency of CETP in the homozygous form is linked to the appearance of very small LDL and very large HDL subfractions. Higher CETP activity may play a role in generating the dyslipidemia of metabolic syndrome. Metabolic impact of CETP variation

30. Summary - 2 Studies in general population indicate that CETP gene variants linked to lower activity are associated with reduced risk. This may not be the case in statin treated patients. Epidemiological studies demonstrate a variable association between CETP activity / mass and CHD risk. The impact of CETP modulation on atherosclerosis may depend on the metabolic context. Outcome studies are required to demonstrate clinical benefit of drugs that modify CETP activity. CETP as a target to reduce atherosclerosis