Clinical Importance of CETP Modulators versus CETP Inhibitors

Clinical Importance of CETP Modulators versus CETP Inhibitors in Increasing HDL to Reduce Cardiovascular Disease EJ Niesor Ph. D Preclinical Science Leader Dalcetrapib

Eric J. Niesor, Ph. D § Salary: § Roche

CETP has Multiple Activities: Transfer Between HDL 3 and LDL and VLDL, and HDL 3 and HDL 2 HDL 3 VLDL Liu XQ, Bagdade JD. J Lipid Res. 1995; 36: 2574 -2579. HDL 2 LDL

Increasing CETP Activity Induces Pre-β-HDL Formation In Vitro CETP added (-fold) 0 2. 5 5 10 20 Lipid-poor Apo. A-I HDL 3 c (pre- -HDL) HDL 3 b HDL 3 a HDL 2 a CETP HDL 2 b Apo. A-I (RFI) 20 000 15 000 10 000 Pre- -HDL 5000 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 Fractions Dernick G et al. J Lipid Res. 2011; 52: 2323 -2331.

CETP Modulator Dalcetrapib and CETP Inhibitor Torcetrapib

Circulating Forms of Dalcetrapib and Torcetrapib: Chemical Structure and Physicochemical Properties F F O O F F NH F O SH N N O O dalcetrapib-SH torcetrapib MW: 319. 51 MW: 600. 40 F F

CETP Activity: Transfer Between HDL 3 and LDL and VLDL, and HDL 3 and HDL 2 HDL 3 VLDL Liu XQ, Bagdade JD. J Lipid Res. 1995; 36: 2574 -2579. HDL 2 LDL

Torcetrapib Prevents rh. CETP-Induced Pre- -HDL Formation in Human Plasma In Vitro: Pre- 1 -HDL Specific ELISA Pre- 1 -HDL (µg/m. L) 600 torcetrapib 0 0. 1 µM 3 µM 10 µM 500 400 300 200 100 0 0 5 10 15 20 Final CETP concentration (µg/m. L) Niesor EJ et al. J Lipid Res. 2010; 51: 3443 -3454. 25 30

Dalcetrapib Does Not Prevent rh. CETP-Induced Pre- -HDL Formation in Human Plasma In Vitro: Pre- 1 -HDL Specific ELISA Pre- 1 -HDL (µg/m. L) 600 dalcetrapib 0 0. 1 µM 3 µM 10 µM 500 400 300 200 100 0 0 5 10 15 20 Final CETP concentration (µg/m. L) Niesor EJ et al. J Lipid Res. 2010; 51: 3443 -3454. 25 30

Pre- 1 -HDL Levels in Patients Treated with Dalcetrapib: Subgroup Analyses % Change from Baseline to Month 3 and Month 12 N (placebo/ dalcetrapib) Placebocorrected change 90% CI 2 -sided P-value Overall ITT population (3 months) 26 / 34 4. 786 (-14. 33 -23. 903) 0. 6767 Pre- 1 -HDL <100 g/m. L 13 / 26 19. 417 (-9. 923 -48. 756) 0. 2702 Pre- 1 -HDL 100 g/m. L 13 / 8 -1. 575 (-51. 85 -48. 703) 0. 9564 Overall ITT population (12 months) 26 / 34 7. 526 (-15. 20 -30. 249) 0. 5814 Pre- 1 -HDL <100 g/m. L 13 / 26 26. 376 (-2. 069 -54. 820) 0. 1260 Pre- 1 -HDL 100 g/m. L 13 / 8 0. 581 (-59. 65 -60. 817) 0. 9866 Subgroup

Contribution of Intestinal ABCA 1 to HDL-C Level and Pre-β-HDL Lipidation • Intestinal ABCA 1 directly contributes to HDL biogenesis in vivo – Brunham LR et al. J Clin Invest. 2006; 116: 1052 -1062 • Origins of intestinal ABCA 1 -mediated HDL-C – Field FJ et al. J Lipid Res. 2008; 49: 2605 -2619 • Emerging roles of the intestine in control of cholesterol metabolism – Kruit JK et al. World J Gastroenterol. 2006; 12: 6429 -6439 • Intestinal cholesterol transport proteins: an update and beyond – Levy E et al. Curr Opin Lipidol. 2007; 18: 310 -318

Contribution of Intestinal Cholesterol and Phytosterol to HDL Formation ABCA 1 ABCG 5/G 8 Apo. A-I TG ACAT-2 Nascent HDLs NPC 1 L 1 ER Ezetimibe GOLGI Apical Basolateral Chylomicron Enterocyte Adapted from Kruit JK et al. World J Gastroenterol. 2006; 12: 6429 -6439.

Changes in Plasma Lipids in 22 Dalcetrapib. Treated Subjects with or without Ezetimibe dalcetrapib 3 x 300 mg ezetimibe 10 mg dalcetrapib 3 x 300 mg + ezetimibe 10 mg Change from baseline (%) 60 50 40 30 *** ** 20 *** 10 0 -10 -20 * *** *** -30 *** -40 -50 Total cholesterol * HDL-C Niesor EJ et al. Atherosclerosis. 2011; 219: 761 -767. LDL-C Triglyceride Apo. A-I Mean ± SEM; n = 22. *P < 0. 05, **P < 0. 01, ***P < 0. 001

Sterol Biomarkers of Cholesterol Homeostasis Markers of synthesis Cholesterol Lathosterol Desmosterol Markers of absorption Campesterol Sitosterol Cholestanol

Sterol/cholesterol ratio change from baseline (%) Non-Cholesterol Sterol Biomarkers of Cholesterol Homeostasis in 22 Dalcetrapib-Treated Subjects 60 40 30 20 10 0 -10 Lathosterol ß-sitosterol *** Cholestanol dalcetrapib *** 150 50 Campesterol ** 200 100 Desmosterol ezetimibe *** 0 * -50 60 50 40 30 20 10 0 -10 -20 -30 *** Niesor EJ et al. Atherosclerosis. 2011; 219: 761 -767. * dalcetrapib + ezetimibe Mean ± SEM; n = 22. *P < 0. 05, **P < 0. 01, ***P < 0. 001

Plasma Non-Cholesterol Sterols in Hamsters Given Ezetimibe, Dalcetrapib or Torcetrapib Sterol/total cholesterol (TC) ratio (% diff. from control) 400 350 Lathosterol/TC Desmosterol/TC Campesterol/TC β-sitosterol/TC Cholestanol/TC *** 300 250 200 150 100 ** *** 50 0 -50 -100 -150 *** *** *** ezetimibe Niesor EJ et al. Atherosclerosis. 2011; 219: 761 -767. dalcetrapib torcetrapib n = 6; *P < 0. 05, **P < 0. 01, ***P < 0. 001 vs control

Phytosterol Levels Correlate With HDL-C in the General Population • Plasma non-cholesterols in male distance runners and sedentary men – Sutherland WH et al. Eur J Appl Occup Physiol. 1991; 63: 119 -123 • Relationship between phytosterol levels and components of the metabolic syndrome in the PROCAM study – Assmann G et al. Eur J Cardiovasc Prev Rehabil. 2007; 14: 208 -214 • Phytosterol plasma concentration and coronary heart disease in the prospective Spanish EPIC cohort – Escurriol V et al. J Lipid Res. 2010; 51: 618 -624 • Effects of a 2 -y dietary weight-loss intervention on cholesterol metabolism in moderately obese men – Leichtle AB et al. Am J Clin Nutr. 2011; 94: 1189 -1195

Correlation Between Percent Change in Non-cholesterol Sterols and Lipoproteins HDL Atherosclerosis Treatment Study (HATS) LDL-C HDL-C 0. 32** -0. 18* Campesterol (cholesterol absorption) -0. 28*** 0. 23** β-sitosterol (cholesterol absorption) -0. 27*** 0. 27** Lathosterol (cholesterol synthesis) Patient subset from HATS (n=123, 24 month data); patients with coronary artery disease with low HDL-C treated with simvastatin and niacin ± antioxidants, or placebo. *P < 0. 05, **P < 0. 01, ***P < 0. 001 (independent of treatment) Matthan NR et al. J Lipid Res. 2003; 44: 800 -806.

Plasma Phytosterol May in Part Reflect Nascent HDL Lipidation by ABCA 1 ABCG 5/G 8 Apo. A-I TG ACAT-2 Nascent HDLs NPC 1 L 1 ER GOLGI Apical Basolateral Chylomicron Enterocyte Adapted from Kruit JK et al. World J Gastroenterol. 2006; 12: 6429 -6439.

Conclusions 1 • Raising of HDL-C with the CETP modulator dalcetrapib occurs with a simultaneous increase in trace amounts of plant sterols (campesterol and β-sitosterol) in plasma; observed both in case control and epidemiology studies in humans • In hamster, despite changes in HDL-C and LDL-C, no changes are seen in either plant sterols or sterol markers of synthesis (lathosterol and desmosterol) with the CETP inhibitor torcetrapib • Increases in plant sterols may reflect lipidation on nascent HDL (pre-β-HDL) by ABCA 1 located at the basolateral surface of enterocytes

Conclusions 2 • Modulation of CETP activity (CETP modulation) by dalcetrapib and differences with CETP inhibition can be demonstrated in vitro and in preclinical animal models • The role of CETP modulation in maintaining the HDL function(s) associated with cardiovascular protection in the clinical setting remains to be established in outcomes studies