Acute Study of Clinical Effectiveness of Nesiritide in Decompensated Heart Failure Adrian F. Hernandez, MD On behalf of the ASCEND-HF Committees, Investigators and Study Coordinators 1

Disclosure Information Adrian F. Hernandez, MD ASCEND-HF Trial FINANCIAL DISCLOSURE: Trial Sponsor: Scios Inc Research funding from Johnson & Johnson Honorarium from Amgen, Corthera Full listing of disclosures at dcri.org UNLABELED or UNAPPROVED USE: None

Study organization Executive Committee Sponsor Scios Inc. Chair: Rob Califf Chris O’Connor (Co-PI), Randy Starling (Co-PI) Paul Armstrong, Kenneth Dickstein, Michel Komajda, Barry Massie, John McMurray, Markku Nieminen, Jean Rouleau, Karl Swedberg, Vic Hasselblad Sponsor Scios Inc. Independent DSMB Chair: Sidney Goldstein Salim Yusuf, David DeMets, Milton Packer, John Kjekshus Clinical Event Committee Chair: John McMurray International Steering Committee North America Academic Consortium: (DCRI, C5, Jefferson, Henry Ford, Canadian VIGOUR Centre) ROW: Johnson & Johnson Global Clinical Operations Coordinating center: DCRI Adrian Hernandez, Craig Reist, Gretchen Heizer >800 Investigators and Study Coordinators at 398 Sites

Background Acute heart failure is a major health problem responsible for several million hospitalizations worldwide each year. Standard therapy has not changed since 1970s and includes diuretics and variable use of vasodilators or inotropes. In 2001, nesiritide was approved by the FDA to reduce PCWP and improve dyspnea, based on efficacy at 3 hrs. However, in 2005 two meta-analyses raised concerns regarding the risks of mortality and renal injury. Subsequently, an independent panel* was convened by Scios Inc and recommended that a clinical trial be conducted to definitively answer the question of nesiritide’s safety and efficacy. *chaired by Eugene Braunwald

Design of ASCEND-HF: Guiding principles Independent framework Pragmatic trial model Focused Efficient study design Streamlined procedures Simple follow-up Permissive enrollment criteria for broad population Meaningful outcomes Standard of care per local practice (“real world”)

Co-Primary objectives To assess whether nesiritide vs placebo, in addition to standard care provides: Reduction in rate of HF rehospitalization or all-cause mortality through Day 30 Significant improvement in self-assessed dyspnea at 6 or 24 hrs using 7-point Likert scale 60 40 20 % Subjects Markedly Better Minimally Worse Moderately Better Moderately Worse Minimally Better Markedly Worse No Change

Secondary and safety objectives Secondary endpoints: Overall well-being at 6 and 24 hours Persistent or worsening HF and all-cause mortality from randomization through discharge Number of days alive and outside of the hospital Cardiovascular rehospitalization and cardiovascular mortality Safety endpoints: All cause mortality Renal: 25% decrease in eGFR at any time from study drug initiation through Day 30 Hypotension: As reported by investigator as symptomatic or asymptomatic

Dyspnea relief at 6 and 24 hrs All-cause mortality at 180 days Study design and drug procedures Nesiritide Acute HF < 24 hrs from IV RX 24–168 hrs Rx Placebo Co-primary endpoint: Dyspnea relief at 6 and 24 hrs Co-primary endpoint: 30-day death or HF rehosp All-cause mortality at 180 days Double – blind placebo controlled IV bolus (loading dose) of 2 µg/kg nesiritide or placebo Investigator’s discretion for bolus Followed by continuous IV infusion of nesiritide 0.01 µg/kg/min or placebo for up to 7 days Usual care per investigators including diuretics and/or other therapies as needed Duration of treatment per investigator based on clinical improvement

Inclusion and exclusion criteria Key inclusion criteria Key exclusion criteria Hospitalized for ADHF <24 hrs from IV treatment Dyspnea at rest or with minimal activity 1 clinical sign: Respiratory rate ≥ 20 breaths per min Rales >1/3 bases 1 objective measure: CXR with pulmonary edema BNP ≥400 pg/mL or NT-proBNP≥1000 pg/mL Prior EF <40% within 12 months PCWP > 20 mmHg Hypotension at baseline (SBP <100 mm Hg or SBP<110 mm Hg with IV vasodilator) Significant lung disease that could interfere with interpretation of dyspnea Acute coronary syndrome Severe anemia or active bleeding Treatment with levosimendan or milrinone Unstable doses of IV vasoactive medication within 3 hours

Statistical methods Study population: modified intention-to-treat based on receiving study drug Primary analysis: Co-primary endpoints tested using Bonferroni approach Composite of HF rehospitalization and all-cause mortality tested at 0.045 significance level Dyspnea tested at 0.005 level using Hochberg method: Significant if both 6- and 24-hr assessment P values ≤0.005; or If either 6- or 24-hr assessment P values ≤0.0025 Sample size determination: Based on composite endpoint: 89% power with 7000 patients using chi-square test, assuming a placebo event rate of 14% and a relative risk reduction of 18.6% Dyspnea: >90% power to detect a 13% greater likelihood of reporting improvement for nesiritide compared to placebo

>800 Investigators and Study Coordinators Enrollment 7141 patients 30 Countries & 398 Sites Western Europe = 7% 35 sites North America = 45% 214 sites Central Europe = 14% 48 sites Asia-Pacific = 25% 62 sites Latin America = 9% 39 sites >800 Investigators and Study Coordinators

Study population Randomized (n=7141) Placebo MITT=3511 Placebo (n=3577)   Did not receive study drug (n=66) Hypotension (n=28) Exclusion criteria (n=8) Physician decision (n=6) Participant withdrew consent (n=14) Other reason (n=10) Nesiritide MITT=3496 Nesiritide (n=3564) Did not receive study drug (n=68) Hypotension (n=26) Exclusion criteria identified (n=9) Participant withdrew consent (n=16) Other reason (n=11)

Baseline characteristics Placebo (n=3511) Nesiritide (n=3496) Age (yrs) 67 (56, 76) Female (%) 34.9 33.4 Black or African American 15.0 14.7 Systolic Blood Pressure (mmHg) 124 (110, 140) 123 (110, 140) Heart rate (beats/min) 82 (72, 95) Respiratory rate (breaths/min) 24 (21,26) 23 (21, 26) Medical History (%) Ischemic heart disease 60.8 59.5 Hypertension 72.6 71.8 Atrial fibrillation 37.7 37.4 Chronic respiratory disease 16.6 16.3 Diabetes 42.9 42.3 Continuous variables as median (IQR 25th, 75th); MITT population

Baseline characteristics Placebo (n=3511) Nesiritide (n=3496) Labs/Studies LVEF <40% within 12 mths (%) 79.5 80.8 BNP (pg/mL) 989 (543, 1782) 994 (544, 1925) NT pro-BNP (pg/mL) 4461 (2123, 9217) 4508 (2076, 9174) Creatinine (mg/dL) 1.2 (1.0, 1.6) (1.0, 1.5) Pre-randomization treatment (%) Loop diuretics 95.3 94.9 Inotropes 4.4 4.3 Vasodilators 14.1 15.7 Continuous variables as median (IQR 25th, 75th); MITT population

30-day Death/HF Rehospitalization Co-Primary outcome: 30-day all-cause mortality or HF rehospitalization P=0.31 Hazard Ratio 0.93 (95% CI: 0.8,1.08) 9.4 3.6 6.0 12 10.1 4.0 6.1 10 Placebo Nesiritide 8 % 6 4 2 30-day Death/HF Rehospitalization 30-day Death HF Rehospitalization Risk Diff (95 % CI) -0.7 (-2.1; 0.7) -0.4 (-1.3; 0.5) -0.1 (-1.2; 1.0)

30 day death/HF readmission subgroups All Subjects N=6836 Baseline SBP (mmHg) < 123 ≥ 123 N=3346 N=3490 Baseline Ejection Fraction (%) <40 ≥ 40 N=4362 N=1187 Renal function- MDRD GFR (mL/min/m2) <60 ≥ 60 N=3395 N=3093 History of CAD No Yes N=3092 N=3742 History of Diabetes Mellitus N=3923 N=2913 Risk Difference <0: Favors Nesiritide; Risk Difference >0: Favors Placebo Difference (%) and 95% Confidence Interval

30 day death/HF readmission subgroups All Subjects N=6836 Inotrope Use at Randomization No Yes N=6556 N=280 Vasodilators None Any IV Vasodilators No IV Nitroglycerin IV Nitroglycerin N=5889 N=942 N=5943 N=892 Diuretics N=691 N=6145 Study Drug Bolus N=2609 N=4227 Time from Hosp to Rand (hrs) <15.5 ≥15.5 N=3426 N=3410 Risk Difference <0: Favors Nesiritide; Risk Difference >0: Favors Placebo Difference (%) and 95% Confidence Interval

Co-Primary Endpoint: 6 and 24 hour dyspnea 6 Hours 24 Hours 70 70 66.1% 3371 Nesiritide 30.4 37.8 21.2 P=0.007 8.6 68.2% 60 P=0.030 3416 Nesiritide 15.0 29.5 32.8 20.3 60 3398 Placebo 27.5 38.6 22.1 9.5 50 42.1% 44.5% 50 40 3444 Placebo 13.4 28.7 34.1 21.7 40 30 30 % Subjects 20 % Subjects 20 10 10 10 10 20 20 30 30 40 40 50 60 Markedly Better Moderately Better Minimally Better No Change Minimally Worse Moderately Worse Markedly Worse

Dyspnea at 6 and 24 Hours Odds for Marked-Moderate Improvement All Subjects N=6860 N=6769 SBP <123 ≥123 N=3369 N=3491 N=3314 N=3455 GFR <60 ≥60 N=3494 N=3121 N=3349 N=3075 Ejection Fraction <40 ≥40 N=4385 N=1186 N=4335 N=1171 CAD No Yes N=3115 N=3743 N=3082 N=3685 Diabetes N=3930 N=2930 N=3887 N=2882 OR <1: Favors Placebo; OR >1: Favors Nesiritide; Odds Ratio of Markedly/Moderately vs. Other

Dyspnea at 6 and 24 Hours Odds for Marked-Moderate Improvement All Subjects N=6860 N=6769 Inotropes No Yes N=6574 N=286 N=6481 N=288 Vasodilators None Any IV Vaso No IV Nitro IV Nitro N=5912 N=943 N=5965 N=894 N=5835 N=929 N=5886 N=882 Diuretics N=691 N=6169 N=679 N=6090 Study Medication Bolus N=2612 N=4248 N=2564 N=4205 Time from Hosp to Rand <15.5 ≥15.5 N=3428 N=3432 N=3369 N=3400 OR <1: Favors Placebo; OR >1: Favors Nesiritide; Odds Ratio of Markedly/Moderately vs. Other

Secondary endpoints Placebo (n=3511) Nesiritide (n=3496) Difference (95% CI) P-value Persistent or worsening HF or all-cause mortality through discharge 4.8% (165) 4.2% (147) -0.5 (-1.5 to 0.5) 0.30 Days alive and outside of hospital through Day 30 20.7 20.9 0.2 (-0.13 to 0.53) 0.16 CV death or CV rehosp through Day 30 11.8% (402) 10.9% (372) -0.9 (-2.4 to 0.6) 0.24 Placebo (n=3511) Nesiritide (n=3496) P-value Well Being at 6 hours* 40.3% 41.4% 0.32 Well Being at 24 hours* 63.7% 65.7% 0.02 *Combined response for moderately/markedly better

End of Treatment Creatinine Discharge or 10 day Creatinine Renal Safety Anytime Through Day 30 Placebo (n=3509) Nesiritide (n=3498) P-value >25% decrease eGFR 29.5% 31.4% 0.11 End of Treatment Creatinine Creatinine (mg/dL) Cum Dist 2 4 6 8 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Discharge or 10 day Creatinine Nesiritide Placebo

Hypotension Placebo (n=3509) Nesiritide (n=3498) Risk Difference (95% CI) P- value Any hypotension (Through Day 10/discharge) 15.3% (538) 26.6% (930) 11.3 (9.4 to 13.1) <.001 Asymptomatic Hypotension 12.4% (436) 21.4% (748) 9.0 (7.2 to 10.7) Symptomatic Hypotension 4.0% (141) 7.1% (250) 3.1 (2.1 to 4.2)

30-day mortality meta-analysis Odds Ratio (95% CI) Mills (N=163) 0.38 (0.05, 2.74) Efficacy (N=127) 1.24 (0.23, 6.59) Comparative (N=175) 1.43 (0.50, 4.09) PRECEDENT (N=147) 0.59 (0.18, 2.01) VMAC (N=498) 1.63 (0.77, 3.44) PROACTION (N=237) 6.93 (0.89, 53.91) COMBINED 30 day w/out ASCEND 1.28 (0.73, 2.25) ASCEND-HF (N=7007) 0.89 (0.69, 1.14) COMBINED with ASCEND 1.00 (0.76, 1.30) 0.1 1 10

Conclusions Nesiritide did not reduce the rate of recurrent heart failure hospitalization or death at 30 days. Nesiritide reduced dyspnea to a modest degree, consistent with previous findings but did not meet pre-specified protocol criteria for statistical significance at 6 and 24 hours. Nesiritide did not affect 30-day all cause mortality nor did it worsen renal function as had been suggested by prior meta-analyses of smaller studies.

Implications Nesiritide can now be considered a safe therapy in patients with acute heart failure. Further analysis of ASCEND-HF is likely to permit better understanding of acute heart failure and patient profiles that may potentially benefit from nesiritide. Our results from this large randomized trial emphasize both the challenges of making therapeutic decisions on inadequate evidence as well as the urgent need for large, well-conducted trials capable of informing clinical practice

Steering Committee North America: Kirkwood F. Adams Jr MD; Javed Butler, MD;Maria Rosa Costanzo, MD; Mark E. Dunlap, MD; Justin A. Ezekowitz, MBBCh, MSc; David Feldman, MD, PhD; Gregg C. Fonarow, MD; Stephen S. Gottlieb, MD, MHS; James A. Hill, MD, MS; Judd E. Hollander, MD; Jonathan G. Howlett, MD; Michael Hudson, MD; Mariell L. Jessup, MD; Serge Lepage, MD; Wayne C. Levy, MD; Naveen Pereira, MD; W.H. Wilson Tang, MD; John R. Teerlink, MD; David J. Whellan, MD; Clyde W. Yancy, MD Europe: Stefan D. Anker, MD, PhD; Dan Atar, MD; Alexander Battler, MD; Ulf Dahlstrom, MD, PhD; Aleksandras Laucevicius, MD; Marco Metra, MD; Alexander Parkhomenko, MD; Piotr Ponikowski, MD, PhD; Jindrich Spinar, MD; Svetla Torbova, MD; Filippos Triposkiadis, MD;Vyacheslav Mareev, MD; Adriaan A. Voors, MD, PhD; Faiez Zannad, MD, PhD Latin America: Rodrigo Botero, MD; Nadine Clausell, MD; Ramón Corbalán, MD; Rafael Diaz, MD; Gustavo Méndez Machedo Asia Pacific: Ping Chai, MD; Wen-Jone Chen, MD; Henry Krum, MBBS, PhD; Sanjay Mittal, MD; Byung Hee Oh, MD; Supachai Tanomsup, MD; Richard W. Troughton, MD, PhD; YueJin Yang, MD;