Lowering of LDL-c: Novel concepts and novel promises Dr. Evan Stein University of Cincinnati Ohio, USA

 Numerous cardiovascular end point trials have confirmed more LDLc reduction results in more CVD reduction  NCEP, AHA, ACC and European guidelines continue to lower LDLc goal in high risk and even lower risk CVD patients  Growing number of statin adverse patients with limited alternatives  Special populations (e.g. FH and severe hypercholesterolemia) do not achieve even old goals Why do we need more LDL reducing drugs?

LDL Receptor Function and Life Cycle 3 3 For illustration purposes only

The Role of PCSK9 in the Regulation of LDL Receptor Expression 4 4 For illustration purposes only

Impact of an PCSK9 mAb on LDL Receptor Expression 5 5 For illustration purposes only mAb

PCSK9: Rapid Progress From Discovery to Clinic Seidah NG. Proc Natl Acad Sci USA 2003;100(3):928-33, Abifadel M. Nat Genet 2003;34(2):154-6, Maxwell KN. Proc Natl Acad Sci USA 2004;101(18):7100-5, Rashid S. Proc Natl Acad Sci USA 2005;102(15): , Lagace TA et al. JCI 2006;116: Cohen JC. N Engl J Med 2006;354(12): , Zhao Z. Am J Hum Genet 2006;79(3):514-23, Hooper AJ. Atherosclerosis 2007;193(2):445-8, Chan JC. Proc Natl Acad Sci USA 2009;106(24):9820-5; Stein et al N Engl J Med 2012;366: Stein modified from Swergold, Regeneron First publication POC in patients First patients with FH & nonFH treated with PCSK9 mAb PCSK9 LOF mutations found with 28%  LDL-C and 88%  CHD risk Humans null for PCSK9 have LDL-C ~15 mg/dL  LDL-C in mice and non- human primates treated with anti-PCSK9 mAb PCSK9 (NARC-1) discovered PCSK9 GOF mutations associated with ADH First subject treated with PCSK9 mAb Adenoviral  expression in mice PCSK9 KO mouse  LDL-C Plasma PCSK9 binds to LDLr

How effective will a mAb be? How long will effect last? Will the mAb be effective in patients treated with statins, whose LDL-R are already upregulated? As statins increase PCSK9 levels in plasma if effective will LDL-C reduction be less, the same (additive) or enhanced (synergistic) than monotherapy? In patients with heterozygous familial hypercholesterolemia (HeFH) and a defective LDL-R will PCSK9 inhibition have any significant effect? Will mAb anti-PCSK9 administration be safe and well tolerated? Inhibition of PCSK9 with Monoclonal Antibody: First in Man Studies

Phase Ib: Multidose Study Double-Blind, Randomized, Placebo-Controlled Men & women, Age 18-65; HeFH + non-FH LDL-C>100 mg/dl* Atorvastatin mg* Men & women, Age 18-65; HeFH + non-FH LDL-C>100 mg/dl* Atorvastatin mg* SC 50mg / Placebo Days Follow-up 105 days after last dose Follow-up 105 days after last dose SC 100mg / Placebo SC 150mg / Placebo 148 Dose administered Primary Endpoint incidence and severity of TEAEs Exploratory Endpoint changes in lipids and lipoproteins *An additional 150-mg arm was enrolled with no background atorvastatin and baseline LDL-C >130 mg/dL NCT Sequential Cohorts 21-day screening period Stein et al NEJM 2012; 366:

Dose Groups Stein et al NEJM 2012; 366:

ApoB & LDL-C Response Mean % Change from Baseline, Day 57 % * P < vs. Placebo † P < 0.01 vs. Placebo * * * * * * † * * * * * * Stein et al NEJM 2012; 366:

LDL-C Dose Response Atorvastatin Combo-Rx, HeFH & Non-FH Combined = Dose administered Mean % Change from Baseline 4 wks

Rosuvastatin < 40 mg Atorvastatin < 80 mg Simvastatin 20–80 mg AMG 145: Placebo, 3:1 Cohort 1 N = 8 Cohort 2 N = 8 Cohort 3 N = 8 Cohort 4 N = 8 Cohort 5 N = 8 Cohorts 1-5: Low- to moderate-dose statins, no heFH (N = 40) Rosuvastatin 40 mg Atorvastatin 80 mg AMG 145: placebo 3:1 Cohort 6: High-dose statins, no HeFH (N = 12) Rosuvastatin < 40 mg Atorvastatin < 80 mg Simvastatin 20–80 mg AMG 145: placebo 3:1 Cohort 7: Low- to moderate- dose statins, HeFH (N = 6) Study Design Men and women 18–70 years of age BMI 18–32 kg/m 2 LDL-C 70–220 mg/dL Triglycerides ≤ 400 mg/dL With or without heterozygous familial hypercholesterolemia (heFH) Blinded Dose Level Review Team safety review before dose escalation

Multiple-dose AMG 145 Reduced LDL-C up to 75% vs Placebo in Subjects on Low- to Moderate-dose Statins (Cohorts 1-5) Mean LDL-C reduction at dosing interval trough:  Up to 75% vs placebo at week 6 after 3 biweekly SC doses of AMG 145 (p < 0.001),  Up to 66% vs placebo at week 8 after 2 SC doses given every 4 weeks (p < 0.001)  Maximum reductions at week 4: –81% for biweekly dosing –80% for every-4-week dosing

Multiple-dose AMG 145 Reduced LDL-C by Up to 70% vs Placebo in Subjects on High dose Statins (Cohort 6-7) Mean LDL-C reduction in the HeFH cohort at the end of the dosing interval (Day 43):  65% vs placebo (p<0.001)  66% from baseline Maximum LDL-C reduction:  70% vs placebo (p<0.001)  73% from baseline Comparable to LDL-C reductions in dose-matched non-HeFH subjects

Treatment Emergent AEs by Treatment Group No clinically meaningful injection-site reactions. No clinically meaningful anti-drug antibodies observed. Stein et al NEJM 2012; 366:

Inhibition of PCSK9 with mAb  How effective will a mAb be? o very effective LDL-C reductions  How long will effect last? o about 2 to 4 weeks at doses tested  Will the mAb be effective in patients treated with statins, whose LDL-R are already upregulated ? o yes – no difference between diet only  As statins increase PCSK9 levels in plasma if effective will LDL-C reduction be less, the same (additive), or enhanced (synergistic) than monotherapy? o probably additive  In patients with heterozygous familial hypercholesterolemia (HeFH) and a defective LDL-R will PCSK9 inhibition have any significant effect? o yes  Will mAb anti-PCSK9 administration be safe and well tolerated? o Well tolerated in small and short term study

Additional Questions after Phase 1  Will higher doses be more effective?  Will higher doses produce longer lasting effect?  Will LDL-C effect be maintained if added to maximal dose atorvastatin?  Will initial results seen in small group of HeFH from one center be maintained in larger and more diverse HeFH population with additional LDLr defects?  Will patients with history of muscle related side effects on statins tolerate and respond to PCSK9 mAb?  Will PCSK9 mAb at higher doses, longer administration and when added to maximal dose statin still be safe and well tolerated?

Study Design LDL-C ≥ 100 mg/dL at Wk-1 while taking atorva 10, 20, or 40 mg for ≥ 6wks Placebo Q2W W-7 V1a SAR mg Q2W SAR mg Q2W SAR mg Q2W SAR mg Q4W w/alt placebo SAR mg Q4W w/alt placebo N=31 N=30 N=31 N=30 Diet* *NCEP ATP-III TLC or equivalent diet Treatment Period (12 weeks) Follow-up Period (8 weeks) W-1 V1 W0 V2 W2 V3 W4 V4 W6 V5 W8 V6 W10 V7 W12 V8 W16 V9 W20 V10 Screening Period (7 weeks) Primary Endpoint %  calculated LDL-C from baseline to week 12 Primary Endpoint %  calculated LDL-C from baseline to week 12 Secondary Endpoints %  in other lipoproteins and apolipoproteins and % patients reaching pre-specified LDL-C levels Secondary Endpoints %  in other lipoproteins and apolipoproteins and % patients reaching pre-specified LDL-C levels McKenney et al JACC 2012;59:

SAR Administered 2 weekly (Q2W) SC: Change in Calculated LDL-C from Baseline to Week 12 Mean percentage change in calculated LDL-C from baseline to weeks 2, 4, 6, 8, 10, and 12 in the modified intent-to-treat (mITT) population, by treatment group. Week 12 estimation using LOCF method. LDL-C Mean (  SE) % Change from Baseline ∆ - 8.5% ∆ % ∆ % ∆ % ∆ % ∆ - 5.1% ∆ % ∆ % McKenney et al JACC 2012;59:

SAR Administered 4 weekly (Q4W) SC: Change in Calculated LDL-C from Baseline to Week 12 Mean percentage change in calculated LDL-C from baseline to weeks 2, 4, 6, 8, 10, and 12 in the modified intent-to-treat (mITT) population, by treatment group. Week 12 estimation using LOCF method. LDL-C Mean (  SE) % Change from Baseline ∆ % ∆ % ∆ - 5.1% ∆ % ∆ % ∆ % McKenney et al JACC 2012;59:

Additional Questions after Phase 1  Will higher doses be more effective? No  Will higher doses produce longer lasting effect? The higher the dose the longer the effect  Will LDL-C effect be maintained if added to maximal dose atorvastatin?  Will initial results seen in small group of HeFH from one center be maintained in larger and more diverse HeFH population with additional LDLr defects?  Will patients with history of muscle related side effects on statins tolerate and respond to PCSK9 mAb?  Will PCSK9 mAb at higher doses, longer administration and when added to maximal dose statin still be safe and well tolerated?

Diet* Treatment Period (8 weeks) Follow-up Period (8 weeks) W-7 D-49 W-1 D-7 W0 D1 W2 D15 W4 D29 W6 D43 W8 D57 W12 D85 Screening and Run-in Period SAR 150mg SQ Q2W SAR mg Q2W + Atorvastatin 10mg (maintenance dose) SAR Placebo + Atorvastatin 80mg N=31 SAR mg Q2W + Atorvastatin 80mg N=30 N=31 Screening Visit Screening Visit Study Design * NCEP ATP-III TLC or equivalent diet N=214 Safety Population N=92 Efficacy Population mITT [LOCF] N=88 Atorvastatin 10mg W16 D113 Roth et al NEJM 2012; 367:

Change in Calculated LDL-C 2-Week Intervals from Baseline to Week 8 LDL-C Mean (  SE) % Change from Baseline BSL WK ∆ % BSL WK ∆ % BSL mg/dL WK mg/dL ∆ % BSL mg/dL WK mg/dL ∆ % BSL mg/dL WK mg/dL ∆ % * * P< vs Placebo + A80 * *P< vs PL + A80mg Roth et al NEJM 2012; 367:

Additional Questions after Phase 1  Will higher doses be more effective? No  Will higher doses produce longer lasting effect? The higher the dose the longer the effect  Will LDL-C effect be maintained if added to maximal dose atorvastatin? Yes – but appears to maximum to LDLr upregulation  Will initial results seen in small group of HeFH from one center be maintained in larger and more diverse HeFH population with additional LDLr defects?  Will patients with history of muscle related side effects on statins tolerate and respond to PCSK9 mAb?  Will PCSK9 mAb at higher doses, longer administration and when added to maximal dose statin still be safe and well tolerated?

RUTHERFORD: Study Design Population Global trial with ~55 pts per group 18−75 years, with a diagnosis of HeFH by Simon Broome criteria LDL-C > 100 mg/dL and triglycerides < 400 mg/dL At least 4 weeks of stable lipid-lowering therapy (eg, statin, ezetimibe, bile- acid sequestants, niacin) Primary endpoint: % change in LDL-C, measured by ultracentrifugation, from baseline at 12 weeks Raal et al Circulation 2012;126:

RUTHERFORD: LDLc Changes During Treatment LDL-C based on Friedewald calculation Investigational product administration Raal et al Circulation 2012;126:

Additional Questions after Phase 1  Will higher doses be more effective? No  Will higher doses produce longer lasting effect? The higher the dose the longer the effect  Will LDL-C effect be maintained if added to maximal dose atorvastatin? Yes – but appears to maximum to LDLr upregulation  Will initial results seen in small group of HeFH from one center be maintained in larger and more diverse HeFH population with additional LDLr defects? Yes  Will patients with history of muscle related side effects on statins tolerate and respond to PCSK9 mAb?  Will PCSK9 mAb at higher doses, longer administration and when added to maximal dose statin still be safe and well tolerated?

28 GAUSS: Key Entry Criteria  Statin intolerant: defined as intolerable myalgia (muscle pain, soreness, weakness, or cramps) or myopathy (myalgia plus an elevated creatine kinase level); and having symptom improvement or resolution with statin discontinuation and either unable to tolerate at least 1 statin at any dose or unable to tolerate an increase in dose above weekly maximums of rosuvastatin 35 mg, atorvastatin 70 mg, simvastatin 140 mg, pravastatin 140 mg, lovastatin 140 mg, or fluvastatin 280 mg  Elevated LDL-C: above risk-based goals recommended by the National Cholesterol Education Program (NCEP): ≥ 100 mg/dL (2.59 mmol/L) with diagnosed coronary heart disease (CHD) or risk equivalent ≥ 130 mg/dL (3.4 mmol/L) without CHD or risk equivalent and ≥ 2 risk factors, or ≥ 160 mg/dL (4.1 mmol/L) without CHD or risk equivalent and with ≤ 1 risk factor.  Background Rx: Eligible patients could receive stable doses (≥ 4 weeks before screening) of one or more of the following: statins less than or equal to the weekly maximums listed above, bile-acid sequestering resins, or plant stanols/sterols. Sullivan et al JAMA 2012;126:

Day 1 Week 8 Week 12 Week 4Week 2 Visits: 350 mg AMG 145 SC Q4W 420 mg AMG 145 SC Q4W and ezetimibe 10 mg Q4W: IP Administration (AMG 145 or placebo) Randomization 1:1:1:1:1 Screening and Placebo Run-in Period Max. 6 weeks EOS 280 mg AMG 145 SC Q4W Placebo SC Q4W and ezetimibe 10 mg Primary endpoint: Percentage change in LDL-C, by ultracentrifugation, from baseline at 12 weeks GAUSS: Study Design & Entry Criteria NCEP, National Cholesterol Education Program Sullivan et al JAMA 2012;126:

Characteristic AMG 145 Q4W AMG mg + Ezetimibe N = 30 Placebo Q4W + Ezetimibe N = mg N = mg N = mg N = 32 Sex, female, n (%) 18 (56)21 (68)20 (63) 23 (77)18 (56) Age, years, mean (SD) 62 (10)62 (9)60 (9) 62 (7) LDL-C, mg/dL, mean (SD)* 195 (48)190 (48)204 (60) 194 (60)183 (36) Free PCSK9, ng/mL, mean (SD) 383 (98)396 (129)372 (87) 379 (111)390 (91) NCEP high-risk, n (%) 14 (44)12 (39)11 (34) 10 (33)15 (47) Coronary artery disease, n (%) 3 (9)5 (16)3 (9) 6 (20)10 (31) Statins failed (muscle-related events) ≥ 1, n (%)32 (100)31 (100)32 (100)30 (100)32 (100) ≥ 2, n (%)28 (53)24 (77)23 (72)21 (70)25 (78) ≥ 3, n (%)11 (34)11 (35)12 (38)6 (20)11 (34) Worst statin-related events, any statin Myalgia, n (%) 31 (97)30 (97)29 (91) 29 (97)29 (91) Myositis, n (%) 3 (9)3 (10)2 (6) 2 (7)4 (13) Rhabdomyolysis, n (%) 0 (0.0) 1 (3) 0 (0) * LDL-C measured by ultracentrifugation. SD, standard deviation; NCEP, National Cholesterol Education Program GAUSS: Baseline Characteristics

GAUSS: % Change from Baseline in Calculated LDL-C* At All Visits * Calculated LDL-C values. Q4W, every 4 weeks; QD, daily, CI, confidence intervals Sullivan et al JAMA 2012;126:

Adverse Events, Patient Incidence, n (%) AMG 145 AMG mg + Ezetimibe 10 mg N = 30 Placebo Q4W + Ezetimibe N = mg N = mg N = mg N = 32 Treatment-emergent AEs 22 (68.8)15 (48.4)18 (56.3)20 (66.7)19 (59.4) Serious AEs* 2 (6.3)1 (3.2)1 (3.1)0 (0.0) Deaths 0 (0.0) Treatment-related AEs 8 (25.0)3 (9.7)6 (18.8)5 (16.7)7 (21.9) Muscle-related AEs Myalgia 5 (15.6)1 (3.2)1 (3.1)6 (20.0)1 (3.1) Muscle fatigue 2 (6.3)0 (0.0) 1 (3.1) Muscle spasms 1 (3.1)2 (6.5)0 (0.0) 3 (9.4) AEs leading to discontinuation 0 (0.0)1 (3.2)1 (3.1)1 (3.3)2 (6.3) Other most commonly reported AEs Nasopharyngitis 2 (6.3)2 (6.5)1 (3.1)3 (10.0)5 (15.6) Nausea 2 (6.3)1 (3.2)1 (3.1)0 (0.0)1 (3.1) Fatigue 4 (12.5)0 (0.0) 2 (6.3) GAUSS: Safety and Tolerability * Four serious adverse events were reported for AMG 145: acute pancreatitis, coronary artery disease, hip fracture, and syncope. None were considered treatment related. AE: Adverse event. Some patients experienced more than 1 AE.

Additional Questions after Phase 1  Will higher doses be more effective? No  Will higher doses produce longer lasting effect? The higher the dose the longer the effect  Will LDL-C effect be maintained if added to maximal dose atorvastatin? Yes – but appears to maximum to LDLr upregulation  Will initial results seen in small group of HeFH from one center be maintained in larger and more diverse HeFH population with additional LDLr defects? Yes  Will patients with history of muscle related side effects on statins tolerate and respond to PCSK9 mAb? Yes  Will PCSK9 mAb at higher doses, longer administration and when added to maximal dose statin still be safe and well tolerated? Although only administered for relatively short period, over 1200 patients have received mAb to PCSK9, and safety continues to be encouraging.

Inhibition of PCSK9 with fully human mAbs is a very promising, and potentially the most effective, approach to reducing LDL-C including patients: –With nonFH, HeFH and LDLr defective HoFH –On statins or diet alone and –Those unable to tolerate statins, or effective doses of statins. –Additive to all existing therapy –SC delivery every 2 or 4 weeks In large phase 2 program of 2 agents of over 1200 patients no significant adverse effects have emerged so far Phase 3 programs including 2 large CVD outcome trials are already underway with the Amgen (AMG 145) and Sanosi (alirocumab) fully human monoclonal antibodies Therapeutics Targeted to PCSK9 in Development: Conclusion

Status of Current Therapeutics Targeted to PCSK9 in Development Mode of ActionDrugCompanyPhase PCSK9 binding Monoclonal antibodies Modified binding protein (adnectin) REGN727/SAR AMG 145 RG7652 RN316 LY BMS Sanofi/Regeneron Amgen Roche/Genentech Pfizer Eli Lilly Bristol-Myers Squibb / Adnexus PCSK9 synthesis RNA interference LNA antisense * oligonucleotide RNA antisense ALN-PCS02 SPC-5001 BMS Alnylam Santaris Isis/Bristol-Myers Squibb 1 1 (terminated) Stein & Swergold. Curr Atheroscler Rep (2013) 15:310 DOI /s *LNA locked nucleic acid

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